African Journal of
Biotechnology Volume 11 Number 29 10 April 2012 ISSN 1684-5315
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Dr Helal Ragab Moussa Bahnay, Al-bagour, Menoufia, Egypt. Dr VIPUL GOHEL Flat No. 403, Alankar Apartment, Sector 56, Gurgaon-122 002, India. Dr. Sang-Han Lee Department of Food Science & Biotechnology, Kyungpook National University Daegu 702-701, Korea. Dr. Bhaskar Dutta DoD Biotechnology High Performance Computing Software Applications Institute (BHSAI) U.S. Army Medical Research and Materiel Command 2405 Whittier Drive Frederick, MD 21702 Dr. Muhammad Akram Faculty of Eastern Medicine and Surgery, Hamdard Al-Majeed College of Eastern Medicine, Hamdard University, Karachi. Dr. M.MURUGANANDAM Departtment of Biotechnology St. Michael College of Engineering & Technology, Kalayarkoil, India. Dr. Gökhan Aydin Suleyman Demirel University, Atabey Vocational School, Isparta-Türkiye, Dr. Rajib Roychowdhury Centre for Biotechnology (CBT), Visva Bharati, West-Bengal, India. Dr.YU JUNG KIM Department of Chemistry and Biochemistry California State University, San Bernardino 5500 University Parkway San Bernardino, CA 92407
Editorial Board Dr. Takuji Ohyama Faculty of Agriculture, Niigata University
Dr. Mehdi Vasfi Marandi University of Tehran
Dr. FÜgen DURLU-ÖZKAYA Gazi Üniversity, Tourism Faculty, Dept. of Gastronomy and Culinary Art
Dr. Reza Yari Islamic Azad University, Boroujerd Branch
Dr. Zahra Tahmasebi Fard Roudehen branche, Islamic Azad University
Dr. Tarnawski Sonia University of Neuchâtel – Laboratory of Microbiology
Dr. Albert Magrí Giro Technological Centre
Dr. Ping ZHENG Zhejiang University, Hangzhou, China. Prof. Pilar Morata University of Malaga
Dr. Greg Spear Rush University Medical Center
Dr. Mousavi Khaneghah College of Applied Science and Technology-Applied Food Science, Tehran, Iran.
Prof. Pavel KALAC University of South Bohemia, Czech Republic.
Dr. Kürsat KORKMAZ Ordu University, Faculty of Agriculture, Department of Soil Science and Plant nutrition
Dr. Tugay AYAŞAN Çukurova Agricultural Research Institute, PK:01321, ADANA-TURKEY.
Dr. Shuyang Yu Asistant research scientist, Department of Microbiology, University of Iowa Address: 51 newton road, 3-730B BSB bldg.Tel：+319-3357982, Iowa City, IA, 52246, USA.
Dr. Binxing Li E-mail: Binxing.Li@hsc.utah.edu
Dr Hsiu-Chi Cheng National Cheng Kung University and Hospital.
Dr. Kgomotso P. Sibeko University of Pretoria, South Africa.
Dr. Jian Wu Harbin medical university , China.
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African Journal of Biotechnology
International Journal of Medicine and Medical Sciences Table of Content:
Volume 11
Number 29
10 April, 2012
ences ARTICLES Review Gluten proteolysis as alternative therapy for celiac patients: A mini-review Sana M’hir, Manel Ziadi, Nadia Chammem and Moktar Hamdi
7323
Research Articles GENETICS AND MOLECULAR BIOLOGY Assessment of genetic relatedness of the two Amaranthus retroflexus populations by protein and random amplified polymorphic DNA (RAPD) markers 7331 Drinic Mladenovic Snezana, Kostadinovic Marija, Ristic Danijela, Simic Milena, and Stefanovic Lidija
Polymorphism of the prolactin gene (PRL) and its relationship with milk production in American Swiss cattle 7338 Edy Alfonso, Reyna Rojas, José G. Herrera, María E. Ortega, Clemente Lemus, César Cortez, Jaime Ruiz, René Pinto and Heriberto Gómez
Expression of androgen and estrogen receptors in the testicular tissue of chickens, quails and chicken-quail hybrids 7344 Herong Liao, Xiaoling Guo, Lingling Zhou, Yan Li, Xingming Li, Du Liang, Daquan Li and Ningying Xu
Using inter simple sequence repeat (ISSR) markers to study genetic polymorphism of pistachio (Pistacia vera L.) in Algeria 7354 KEBOUR Djamila, BOUTEKRABT Ammar and MEFTI Med
rDNA internal transcribed spacer sequence analysis of Lycoris Hert. Miaohua Quan, Lijun Ou, Chaowen She, Xianjin Wu and Dongming Chen
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Table of Content:
Volume 11
Number 29
10 April, 2012
ences ARTICLES Genetic evaluation of domestic walnut cultivars trading on Korean tree markets using microsatellite markers 7366 Ho Bang Kim, Ji Hyun Jeon, A Reum Han, Yi Lee, Sung-Soo Jun, Tae-Houn Kim, GunHyoung Cho and Phun Bum Park
Full-length enriched multistage cDNA library construction covering floral bud development in Populus tomentosa 7373 Xin-Min An, Dong-Mei Wang, Ze-Liang Wang, Mei-Xia Ye and Zhi-Yi Zhang
Isolating Barley (Hordeum vulgare L.) B1 Hordein Gene Promoter and Using Sequencing Analaysis For The Identification of Conserved Regulatory Elements By Bioinformatic Tools 7378 Kobra Nalbandi, Bahram Baghban Kohnehrouz, Khalil Alami Saeed1 and Ashraf Gholizadeh
Genome shotgun sequencing and development of microsatellite markers for gerbera (Gerbera hybrida H.) by 454 GS-FLX 7388 Kyoung-In Seo, Gi-An Lee, Sang-Kun Park, Mun-Sup Yoon, Kyung-Ho Ma, Jung-Ro Lee, Yu-Mi Choi, Yeon-ju Jung and Myung-Chul Lee
PLANT AND AGRICULTURAL TECHNOLOGY Pathogenesis mechanism of Pestalotiopsis funerea toxin (Pf-toxin) on the plasmalemma of needle cells of different pine species 7397 Shujiang Li, Tianhui Zhu Hanmingyue Zhu, Shan Han, Fanglian Li, Wei Yang and Hua Yang
In vitro propagation through root-derived callus culture of Swertia chirata Buch.Ham. ex Wall. 7408 Manu Pant, Prabha Bisht and Manju P. Gusain
Cloning and characterization of functional keratin-associated protein 5-4 gene in maize 7417 Lin Yang, Feng-Ling Fu, Long-Qun Deng, Shu-Feng Zhou, Tai-Ming Yong and WanChen Li
Table of Content:
Volume 11
Number 29
10 April, 2012
ences ARTICLES Cloning and selection of reference genes for gene expression studies in Ananas comosus 7424 Jun Ma, Ye-hua He, Cheng-hou Wu, He-ping Liu and Zhong-yi Hu
ENVIRONMENTAL BIOTECHNOLOGY The use of selected purple nonsulfur bacteria to remove heavy metals and salts from sediment and water collected from contaminated areas to decrease their phytotoxicity 7434 Saijai Panwichian, Duangporn Kantachote, Banjong Wittayaweerasak and Megharaj Mallavarapu
Biofixation of carbon dioxide by Chlorococcum sp. in a photobioreactor with polytetrafluoroethene membrane sparger 7445 Xiaoli Chai, Xin Zhao, and Wang Baoying
Cloning, expression and purification of cold adapted acetate kinase from Shewanella species AS-11 Md. Abul Kashem Tang, Hiroyuki Motoshima and Keiichi Watanabe
Biodecolorization of Reactive Black 5 by laccase-mediator system Ismat Bibi and Haq Nawaz Bhatti
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Evaluation of cadmium bioaccumulation and translocation by Hopea odorata grown in a contaminated soil 7472 A. Arifin, A. Parisa, A.H. Hazandy, T. M. Mahmud, N. Junejo, A. Fatemeh, S. Mohsen, M.E. Wasli and N.M. Majid
Phenotypic diversity and plant growth promoting characteristics of Mesorhizobium species isolated from chickpea (Cicer arietinum L.) growing areas of Ethiopia 7483 Mulissa Jida and Fassil Assefa
Table of Content:
Volume 11
Number 29
10 April, 2012
ences ARTICLES INDUSTRIAL MICROBIOLOGY Isolation and screening of lactic acid bacteria, Lactococcus lactis from Clarias gariepinus (African catfish) with potential use as probiotic in aquaculture 7494 Tengku Haziyamin Tengku Abdul Hamid, Ahmed Jalal Khan, Muhammad Fauzi Jalil and Nur Shazana Azhar
Bioactive potential of symbiotic bacteria and fungi from marine sponges V. Vasanthabharathi and S. Jayalakshmi
7500
Isolation and screening of Streptomyces from soil of Tunisian oases ecosystem for nonpolyenic antifungal metabolites 7512 Lilia Fourati Ben Fguira, Samir Bejar and Lotfi Mellouli
APPLIED BIOCHEMISTRY Preliminary characterisation of the phytotoxin of sheath-blight disease of rice caused by Rhizoctonia solani 7520 Xiao-Xing Liang and Ai-Ping Zheng
MEDICAL AND PHARMACEUTICAL BIOTECHNOLOGY Changes in transforming growth factor (TGF)-β and mothers against decapentaplegic homolog (Smad) expression in chronic asthmatic rats induced by ovalbumin and aluminum hydroxide 7528 Zhu-Mei Sun, Fu-Feng Li, Peng Qian and Jie Zhao
Identification of overexpressed cytokines as serum biomarkers of hepatitis C virusinduced liver fibrosis using bead-based flexible multiple analyte profiling 7535 Shu-Lin Liu, Yang-Chih Cheng, Chun-Chao Chang, Ai-Sheng Ho, Chun-Chia Cheng, LingYun Chen, Jungshan Chang, Chia-Chi Wang
Table of Content:
Volume 11
Number 29
10 April, 2012
ences ARTICLES BIOTECHNIQUES Highly efficient in vitro adventitious shoot regeneration of Adenosma glutinosum (Linn.) Druce using leaf explants 7542 Ru-Ping TU, Jun-Yan HU, Qian-Qian JI, Guo-Hua XIA and Bing-Song ZHENG
Regeneration of plantlets from nodal and shoot tip explants of Anoectochilus elatus Lindley, an endangered terrestrial orchid 7549 N. Ahamed Sherif, J. H. Franklin Benjamin, S. Muthukrishnan, T. Senthil Kumar and M. V. Rao
Comparative toxicity study of two different synthesized silver nanoparticles on the bacteria Vibrio fischeri 7554 Ehsan Binaeian, Ali Akbar Safekordi, Hossein Attar, Reza Saber, Mohammad Javad Chaichi and Abasalt Hosseinzadeh Kolagar
ANIMAL SCIENCE Fetal neurohistopathology of chloropyrifos in mice 7565 Kausar Raees, Asmat Ullah, Tahir Abbas, Muhammad Khalid Mukhtar, Muhammad Arshad, Shafaat Yar Khan, Hafiz Muhammad Tahir and Khawaja Raees Ahmad
Prenatal and perinatal acrylamide disrupts the development of cerebrum and medulla oblongata in albino rats 7570 Allam A., Abdul-Hamid M., Zohair K, Ajarem J, Allam G and El-Ghareeb A.
Effects of additional DL-methionine in broiler starter diet on blood lipids and abdominal fat Mohammad Amiri Andi
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African Journal of Biotechnology Vol. 11(29), pp. 7323-7330, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.726 ISSN 1684–5315 © 2012 Academic Journals
Review
Gluten proteolysis as alternative therapy for celiac patients: A mini-review Sana M’hir*, Manel Ziadi, Nadia Chammem and Moktar Hamdi Laboratoire d’Ecologie et de Technologie Microbienne (LETMI), Institut National des Sciences Appliquées et de Technologie (INSAT), BP 876, 1080 Tunis, Tunisie. Accepted 19 August, 2011
Celiac disease (CD) results from damage to the small intestinal mucosa due to an inappropriate immune response to a cereal protein (wheat, rye, barley). The only treatment for CD is life-long avoidance of gluten proteins. Gluten-free products are not widely available and usually more expensive. That is why; there is an urgent need to develop an alternative therapy. Enzymatic degradation of gluten among other approaches, abolishing its immunogenic and toxigenic activities, is an attractive alternative strategy for oral therapy in CD. Several proteases following different approaches were studied. This review focuses on enzymes (microbial or vegetal) designed to digest gluten. Also, recent biotechnological procedures that use microorganisms (cell factories for enzymes) as starter culture to eliminate gluten are reviewed in this manuscript. Key words: Celiac disease, gluten, proteolytic activity, lactic acid bacteria, therapy.
INTRODUCTION Celiac disease (CD) is a chronic inflammatory disorder characterized by damage of the small intestinal mucosa caused by gluten proteins from wheat (the waterinsoluble storage proteins) and similar proteins of barley and rye in genetically susceptible subjects (Mäki and Collin, 1997; Fasano and Catassi, 2001; Di Sabatino and Corazza, 2009). The disease is characterized by severe, immune-mediated damage to the jejuna mucosa (subtotal villous atrophy), typically involves chronic diarrhea, abdominal distension, weight loss and malnutrition (Mäki and Collin, 1997; Holmes and Catassi, 1999; Green and Cellier, 2007). Gluten is a mixture of related proteins which are soluble in alcohol-water mixture (prolamins) and the glutenin which are insoluble polymers stabilized by interchain disulphide bonds (Wieser, 2007). During dough mixing, wheat flour is hydrated and as a result of the mechanical energy input discrete masses of the gluten proteins are
*Corresponding author. E-mail: sana.mhir@insat.rnu.tn. Abbreviations: CD, Celiac transglutaminase; PEP, prolyl germinating cereal proteases; lactobacillus.
disease; tTG, tissue endopeptidases; GCP, F, flavobacterium; Lb,
disrupted (Goesaert et al., 2005). The gluten proteins are transformed into a continuous cohesive visco-elastic gluten protein network. These proteins are unique and cannot even be found in cereals closely related to wheat such as barley and rye. During dough fermentation, the gluten network plays a major role in retaining the carbon dioxide. Gas retention properties in turn determine loaf volume and crumb structure of the resulting bread (Goesaert et al., 2005). Currently, CD may affect approximately 1% of the population, according to serologic population based studies (Fasano et al., 2003; Green, 2007). Such a rate establishes CD as one of the most common food intolerances (Fasano and Catassi, 2001) and its prevalence is apparently increasing (Green and Cellier, 2007). In fact, CD has been recognized in populations with high wheat consumption: wheat is one of the most consumed cereals (Tatham and Shewry, 2008). CD has a worldwide distribution, detected not only in Europe and countries populated by Europeans, but also in North Africa (Rätsch and Catassi, 2001). A high prevalence occurs in North African and Middle Eastern populations. It is not reported in black African people (Woodward, 2007). In Western Sahara, CD is a common disorder, where the prevalence was reported as 5.6% in the Saharawi childrens (Rätsch and Catassi, 2001). In Europe, Australia and North
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America, the prevalence was estimated between 0.5 and 1% (Cataldo and Montalto, 2007). In Asia, recent report from Hangzhou in China also suggested that the prevalence of adult CD may be more common in China (Freeman et al., 2010) than previously appreciated (Jiang et al., 2009). Of note, celiac disease has also been reported in immigrants to Canada from China, Japan and South Asia, particularly from the Punjab region of India (Freeman, 2010) In Tunisia, CD is frequent. Probably, the Tunisian diet, which consists mainly of bread, couscous and pasta, contains 25 to 30 g gluten daily. Gluten is introduced early in Tunisian infant’s diet, occasionally as early as the first month of life (Mankaï et al., 2006). Moreover gluten intake has increased because of its use in processed foods, especially fast foods. In addition, cereal grains are the “staple foods” of Tunisian diet population. The prevalence of celiac disease in the general population (in Tunisia) has not been previously investigated. In 2006, the prevalence of CD was 1/700 in a population of apparently healthy blood donors (Bdioui et al., 2006; kallel et al., 2009). The prevalence of celiac disease in Tunisian schoolchildren was estimated to be about 1/157; close to the European prevalence (Ben Hariz et al., 2007). The only treatment for celiac disease is a gluten-free diet. This involves elimination of the grains containing gluten, wheat, rye and barley, as well as food products and additives derived from them including bread, biscuits, cakes, pizzas, pasta, sauces and gravy (Green and Jabri, 2003). In fact, gluten is used on many foods to confer properties such as emulsification, cohesiveness, viscoelasticity and foaming (Esteller et al., 2005; Dayab et al., 2006). CD treatment also requires avoiding other glutenous products like soaps and cosmetics (which can be ingested while bathing or kissing) and preventing cross contamination of safe foods through processing and preparation (Thompson, 2008). So, total avoidance is extremely difficult. Thus, new strategies are being actively pursued to find new treatments or to eliminate noxious prolamins from cereal grains. During the last eight years, many approaches based on gluten hydrolysis in order to detoxify harmful gluten peptides were investigated. Recently, potential therapeutic maneuvers were well reviewed (Tennyson et al., 2009; Lerner, 2010). In this review, we summarize the current enzymatic strategies (microbial or vegetal) used to hydrolyze gluten.
PATHOGENESIS OF CELIAC DISEASE Gliadins and glutenins both contain disease-activating proteins (Dewar et al., 2006). After ingestion of gluten, it is degraded to multiple segments. Several gluten epitopes are immuno stimulatory; some are more active than others. An immuno dominant peptide of 33 amino acids (residues 57 to 89) identified from an α-gliadin fraction has functional properties attributable to many
proline and glutamine residues (Shan et al., 2002). Proline gives the peptide increased resistance to gastrointestinal proteolysis and causes a left-handed helical conformation, which strengthens binding with human leukocyte antigens HLA-DQ2 and HLA-DQ8 molecules on antigen-presenting cells (Woodward, 2007). Furthermore, researchers report that multiple non-HLA genes contribute to the genetic risk for CD (Zhernakova et al., 2011; Freeman et al., 2011). Additionally, glutamine residues are a preferred substrate for tissue transglutaminase-mediated deamidation, which confers an enhanced immunogenicity (Di Sabatino and Corazza, 2009). This leads to T-cell proliferation and production of cytokines, particularly - interferon that appears to perpetuate damage and uptake of antigenic gluten (Schumann et al., 2008; Bethune et al., 2009). Interestingly, those immunogenic peptides are proline (15%) and glutamine (35%) rich polypeptides that are at the base of two major steps in the celiac inflammatory cascade: 1. they confer resistance to enzymatic breakdown, since the human intestine lack prolyl endopeptidase who can readily cleave proline-rich immunestimulatory gluten peptides and 2) the glutamine rich gluten peptides are an ideal substrate for deamination by the tissue transglutaminase (tTG), an ubiquitous connective tissue enzyme (Dieterich et al., 1997). The deamination is crucial for the stability and avidity of the presented peptide in the HLA-DQ (human leukocyte antigen) groove and recognition of T-cell epitopes (Lerner, 2010). tTG is the auto antigen against which the abnormal immune response is directed to (Reif and Lerner, 2004) and two main auto antibodies: anti endomysium and anti tTG are the most useful serological markers to screen for the disease (Shamir et al., 2002). DETOXIFICATION PROTEOLYSIS
OF
GLUTEN
PEPTIDES
BY
Generally, two alternative hydrolysis philosophies exist: to hydrolyse toxic gluten peptides after ingestion, in the gastrointestinal tract (the medical approach) or to hydrolyse them prior to the gluten ingestion, and during food processing (the food technological approach) (Loponen, 2006) (Figure1). In fact, the detoxification of gluten by proteolysis is not a novel idea and neither is the use of more than one protease in an effective detoxification procedure. For instance, Messer et al. (1964) showed that crude papain (which contains several diverse proteolytic activities) could detoxify gluten, whereas one purified papain proteinase failed to detoxify it. MICROBIAL ENZYMATIC SOURCE Gluten degradation can be performed by prolyl endopeptidases (PEPs). These are proteases, found primarily in plants and microorganisms. Prolyl
M’hir et al.
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Figure 1. Two alternatives hydrolysis for gluten detoxification (ALV003 is a mixture of two glutenases; a cysteine endoprotease from germinating barley seeds (EP-B2) and a prolyl endopeptidase from Sphingomonas capsulata (SC-PEP); Asp: Aspergillus).
endopeptidases (PEPs) of microbial origin are endoproteolytic enzymes which, in contrast to human gastrointestinal protease, can readily cleave Pro-rich immunostimulatory gluten peptides (Hausch et al., 2002). This can be achieved by bacterial, or fungal enzymes that lend themselves to large-scale manufacturing (Piper et al., 2004; Stepniak et al., 2006). A prolyl-endopeptidase produced by Flavobacterium meningosepticum, showed hydrolysing effect on a 33mer peptide (the 33-mer was rich in proline: 13 residues and glutamine: 10), which is one of the most potent peptides involved in triggering the disease (Shan et al., 2002; Piper et al., 2004). The use of this endopeptidase has been proposed for an oral therapy for CD patients (Shan et al., 2002). In vivo studies with rats supported these findings, as the perfusion of PEP together with gluten peptides into the rat intestine accelerated the digestion of the gluten peptide in vivo by 50 to 100% (Piper et al., 2004). In a follow-up study, Pyle et al. (2005) showed that pre-treatment of gluten with PEP from F. meningosepticum avoided the development of fat or carbohydrate malabsorption in the majority of CD patients who ingested a low dose of a gluten supplement daily (5 g) during a challenge lasting 14 days. Similar properties (gluten detoxification) were obtained with PEP from Myxococcus xanthus and Sphingomonas capsulata (Shan et al., 2004; Gass et al., 2005) and Lactobacillus
helveticus (Chen et al., 2003). Nevertheless, some contradictory results were noticed concerning PEP from F. meningosepticum. MatysiakBudnik et al. (2004) showed that the hydrolysis of the 33mer by PEP of F. meningosepticum in CD patients was not complete and led to the release of potentially immunogenic peptides. In addition, Shan et al. (2004) and Stepaniak et al. (2006) reported that PEPs are inactivated by pepsin and acidic conditions in stomach. Therefore, Stepaniak et al. (2006) introduced the use of a new enzyme; a prolyl endopeptidase from Aspergillus niger that was stable under gastric conditions (pH 2.0), optimally active at pH 4 to 5 and is completely resistant to digestion with pepsin, and efficiently degrades gluten proteins. Also, this PEP can be used as an oral supplement to reduce gluten intake in patients (Stepaniak et al., 2006; Tennyson et al., 2009). This enzyme can be produced at low-cost at food-grade quality in an industrial setting (Edens et al., 2005).
CEREAL PROTEASE GERMINATING CEREALS)
(PROTEASES
FROM
The role of the proline and glutamine-rich storage proteins of cereals is to supply the embryo with nitrogen and amino acids during the first period of seedling
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Figure 2. Proteolysis by sourdough fermentation: a tool for making gluten free products.
development. Therefore, it is likely that endogenous cereal proteases synthesized during germination (GCP: germinating cereal proteases) would be capable of extensively hydrolyzing these proteins. Many studies have checked this approach. Hartmann et al. (2006) showed the ability of proteases, isolated from wheat, rye and barley to degrade gliadin-petides toxic for celiac patients. Results show that GCP were able to degrade intact gluten and celiac toxic peptides. These authors assumed that GCP are active in the stomach during digestion of food and also in the small intestine. Bethume et al. (2006) showed gliadin hydrolysis (the 33-mer peptide) by EP-B2, a barley cystein proteinase responsible for hydrolyzing the bulk of the hordeins during barley germination. To facilitate gluten degradation, a two-enzyme cocktail, consisting of a glutamine-specific cysteine protease derived from barley B2) and a bacterially derived PEP (from S. capsulata), was developed (Siegel et al., 2006; Gass et al., 2007). The enzyme cocktail was called ‘glutenase’. The efficacy of this two-enzyme glutenase was verified in a rat model of gastric gluten digestion. By combining two enzymes with gastric activity, it should be possible to increase the safe threshold of ingested gluten, thereby ameliorating the burden of a highly restricted diet for patients with celiac sprue. Recently, Tye-Din et al. (2010) reported that pre-treatment of gluten using glutenase (ALV003) can
abolish immune responses induced by gluten in patients (in vivo) with CD for three days.
PROTEOLYSIS BY LACTIC ACID BACTERIA AS STARTERS FOR SOURDOUGH FERMENTATION: CEREAL FOOD PROCESS Proteolysis by lactic acid bacteria has been suggested as a new tool for food processing for celiac persons (Di Cagno et al., 2002, 2004, 2008; Rizzello et al., 2006; Gobbetti et al., 2007). The potential of sourdough lactic acid bacteria as source of proteolytic enzymes was investigated during the last years. Sourdough is a mixture of flour and water that is fermented with indigenous lactic acid bacteria and yeasts (De Vuyst and Neysens, 2005). The use of sourdough as a natural leavening agent in the modern biotechnology of baked goods is increasing, largely because of the metabolic activities of lactic microflora. The use of sourdough fermentation for gluten degradation is shown in Figure 2. Lactobacilli have been shown to possess an outstanding potential in decreasing the CD-inducing effects of gluten (Rollán et al., 2005; Gobbetti et al., 2007; Corsetti and Settani, 2007). Di Cagno et al. (2002) demonstrated a considerable degradation of various Pro-
M’hir et al.
rich peptides, including the 33-mer peptide during sourdough fermentation by some lactobacilli species. This finding has been exploited to produce sourdoughs containing 30% of wheat flour and 70% of other (non-CDinducing) flours such as oat, buckwheat and millet, started with selected lactobacilli and fermented for 24 h (under specific processing conditions: long-time and semi-liquid fermentation). Following this, the mixed starter composed of Lb. alimentarius, Lb. brevis, Lb. sanfranciscensis and Lactobacillus hilgardii was shown to almost completely hydrolize gliadin fractions and consequently the resulting bread was tolerated by CD patients as shown by intestinal permeability challenge (Di Cagno et al., 2004). The type of bread was technologically suitable. The same approach as those described for sourdough wheat bread (Di Cagno et al., 2004) was adapted for pasta making. The same pool of selected sourdough lactobacilli (L. alimentarius 15M, L. brevis 14G, L. sanfranciscensis 7A and L. hilgardii 51B) was used to preferment durum wheat semolina under semi-liquid conditions (Di Cagno et al., 2005). After fermentation, the dough was freeze-dried, mixed with buckwheat flour at a ratio of 3:7, and used to produce the ‘‘fusilli’’ type Italian pasta at an industrial level. As shown by immunological analysis, the concentration of gluten decreased from 6280 to 1045 ppm (destructive efficacy 83%) in the pasta fermented with lactic acid bacteria. This value was higher than those recommended by the Codex. Two levels are distinguished by the Codex Alimentarius Commissions of the World Health Organization and the Food and Agriculture Organization of the United Nations; < 20 ppm for foods that are naturally free of gluten or < 200 ppm for foods that have been rendered gluten free (Gallagher et al., 2004). Recently, the same research team listed in the foregoing (Rizzello et al., 2007) showed that selected sourdough lactobacilli (for high and complementary proteolytic activities), in combination with fungal proteases, decreased the residual concentration of gluten (Triticum aestivum flour) below 10 ppm during food fermentation. The gluten concentration was lower than the threshold level indicated by the Codex Alimentarius Commissions of WHO and FAO for the gluten-free foods. This sourdough was fermented for 48 h at 37°C with ten lactobacili (Lb. alimentarius 15M, Lb. brevis 14G, Lb. sanfranciscensis 7A , Lb. hilgardii 51B and Lb. sanfranciscensis LS3, LS10, LS19, LS23, LS38, LS47) 9 (each strain at 10 CFU/ml of dough) and two proteases of A. niger and A. oryzae, that were routinely used for bakery applications. Proteins fractions (gliadin and glutenin) extracted from this sourdough were freeze dried and incubated with small intestine mucosa (in vitro organ culture) from six patients. None of the intestinal T-cell lines demonstrated immunoreactivity (no interferon production) on the contrary to the negative control (dough without a bacterial and enzyme inoculum). The same approach was investigated by M’hir et al.
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(2009) where a pool of selected three enterococci (each 9 strain at 10 CFU/ml of dough) (M’hir et al., 2008) and fungal proteases (R. oryzae) was used to hydrolyse wheat gluten during long-time fermentation (doughs were incubated for 48 h at 37°C). The residual gluten on sourdough started with enterococci was 1648 from 75 621 ppm (98% of the gluten was hydrolysed) as shown by R5 antibody-based sandwich and competitive enzyme-linked immunosorbent assay (ELISA). By adding fungal proteases, the residual gluten decreased to a concentration of 1106 ppm, higher than those requested by the Codex Alimentarius Commission. Fungal proteases, routinely used as bakery improvers, are indispensable to start the primary proteolysis of gluten. Polypeptides of intermediate dimensions (4 to 40 amino acids), generated from the native proteins, are the substrates for secondary proteolysis by complementary peptidases of sourdough lactobacilli (De Angelis et al., 2010; Gänzle et al., 2008; Rizzello et al., 2007). A combination of sourdough lactic acid bacteria selected for high and complementary proteolytic activities and an external addition of two fungal protease preparations were shown to hydrolyse gluten (72 h at 37°C) of durum wheat to less than 20 ppm (De Angelis et al., 2010). Durum wheat is an important food crop of the Mediterranean area, not only because of the large acreage but also for its importance in the human diet (Flagella, 2006). Durum wheat is largely used for making pasta, especially in the European and North Africa countries. Bread, burghul and couscous are also manufactured with durum wheat in several countries. Longtime fermentation of dough by selected lactic acid bacteria was also shown to be a potential tool to decrease the risk of rye contamination of gluten free products for celiac patients (De Angelis et al., 2006a; Rizzello et al., 2006). Alternatively, probiotics have been demonstrated to degrade gluten during sourdough fermentation (De Angelis et al., 2006b, 2007). In fact, as reported by Gobbetti et al. (2010) probiotics are functional microorganisms that contribute to food tolerance through their enzyme portfolio. Functional microorganisms are used in novel strategies for decreasing phenomenon of food intolerance (gluten intolerance) and allergy. The probiotic VSL#3 preparation (VSL Pharmaceuticals, Gaithesburg, MD) (ca. 450 billion cells/sachet) used containing Streptococcus thermophilus, Lb. plantarum, Lb. acidophilus, Lb. casei, Lb. delbrueckii spp. bulgaricus, Bifidobacterium breve, Bifidobacterium longum and Bifidobacterium infantis. When VSL#3 was used as a starter for bread making, it caused a marked degradation of wheat proteins. Celiac jejunal biopsies exposed to the Peptic-Tryptic digest from the dough fermented by VSL#3 did not show an increase of the infiltration of CD3+ intraepithelial lymphocytes. Loponen et al. (2007, 2009) used germinated grains (wheat or rye) as a raw material in sourdough
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Table 1. Summary of studies using proteolysis to degrade celiac peptides.
Strategy used
Protease from microorganism and/ or cereal Flavobacterium meningosepticum Myxococcus xanthus Sphingomonas capsulata Lactobacillus helveticus
Reference Shan et al. (2002) Piper et al. (2004) Gass et al. (2005) Chen et al.(2003)
Fungal protease
Aspergillus niger
Stepaniak et al. (2006)
Germinated cereal protease (GCP)
Wheat, rye and barley
Hartmann et al. (2006) and Loponen et al. (2007,2009)
Mixture: GCP and bacterial protease
GCP from barley (EP-B2) and PEP: Sphingomonas capsulata (SC-PEP)
Siegel et al. (2006), Gass et al. (2007) and Tye-Din et al. 2010
LAB used as « starter »
Lactobacillus Lactobacillus+Streptococcus+Bifidobacterium Enterococcus
Di Cagno et al. (2002, 2004, 2008), Rollàn et al. (2005) and De Angelis et al. (2006,2007) M’hir et al. (2008)
Mixture: fungal protease and bacteria used as « starter »
Lactobacillus + Po Aspergillus oryzae +Pn Aspergillus niger Enterococcus + S Rhizopus oryzae
Rizzello et al. (2007), Greco et al. (2011) and M’hir et al. (2009)
Bacterial protease
PEP, Prolyl endopeptidases; Po, purified protease from Aspergillus oryzae; Pn, purified protease from Aspergillus niger; S, supernatant containing protease of Rhizopus oryza
fermentation. The results show that prolamins, including gliadins, were extensively hydrolyzed. These examples of trends in food technology to use sourdough fermentation for hydrolysis of the cereal proteins were attractive alternatives strategies as reported by Cabrera-Chávez and Calderón de la Barca (2010). For the first time, wheat flour which was rendered gluten-free during sourdough fermentation and was shown to be not toxic after administration to CD patients. Patients showed normal values of hematology, serology and intestinal permeability (Di Cagno et al., 2010). Later, the safety of daily administration of sweet baked goods made of wheat flour extensively digested by lactobacilli and fungal proteases was evaluated within patients with CD (in vivo) for 60 days. These patients’ did not show clinical
symptoms, neither an increase of anti-TG antibodies nor a modification of the architecture or the grade of inflammation of the intestinal mucosa (Greco et al., 2011).
CONCLUSION CD involves a complex interplay between environmental, genetic and immunologic factors. Wheat gluten and related proteins lead to inflammation in the small intestine. Stress factors like gastrointestinal infections have been found to increase the risk of triggering CD. The only currently available treatment for CD is complete elimination of gluten from toxic cereals: wheat, rye and barley. They can be substituted by other grains
such as rice, corn, quinoa, amaranth, sorghum, oats without cross contamination (with toxic cereals) and buckwheat, which are found to be safe (Briani et al., 2008; Kemppainen et al., 2007; Saturni et al., 2010). Improvements of symptoms are generally seen within days to weeks after the initiation of gluten-free diet. Alternative treatments, such as oral doses of microbial endopeptidases to degrade wheat peptides are under trials. Also, sourdough degradation of gluten proteins is an option for food processing that includes fermentation. From the reported results, the gluten hydrolysis can be achieved by cereal or bacterial or fungal protease or the combination of them. Table 1 summarizes strategies that have been used for gluten hydrolysis. Fundamental studies
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(sourdough fermentation during long-time, adding selected lactic acid bacteria, fungal proteases and germinated cereal protease) have revealed several attractive targets for gluten destruction and prevention of CD. This alternative food technology may provide the option to reduce or even eliminate the harmful prolamins from cereal grains.It will be interesting to see whether any of these will become reality in the coming years.
ACKNOWLEDGEMENTS The authors thank Prof. Gobbetti M. (Italy) and Prof. Thonart P. (Belgium) for collaboration with the projects on celiac disease. Suggestions by two anonymous reviewers also helped strengthen the manuscript. This work was funded by “Ministère de l’Enseignement Supérieur, de la Technologie et de la Recherche Scientifique Tunisie”. REFERENCES Bdioui F, Sakly N, Hassine M, Saffar H (2006). Prevalence of celiac disease in Tunisian blood donors. Gastroenterol. Clin. Biol. 30: 33-36. Ben Hariz M, Kallel-Sellami M, Kallel L, Lahmer A, Halioui S, Bouraoui S, Laater A, Sliti A, Mahjoub A, Zouari B, Makni S, Maherzi A (2007). Prevalence of celiac disease in Tunisia: mass-screening study in schoolchildren. Eur. J. Gastroenterol. Hepatol. 19: 687-94. Bethune MT, Siegel M, Howles-Banerji S, Khosla C. (2009) Interferongamma released by gluten-stimulated celiac disease-specific intestinal T cells enhances the transepithelial flux of gluten peptides. J. Pharmacol. Exp. Ther. 329: 657-668. Bethune MT, Strop P, Tang Y, Sollid LM, Khosla C (2006). Heterologous expression, purification, refolding, and structuralfunctional characterization of EP-B2, a self-activating barley cysteine endoprotease. Chem. Biol. 13: 637–647. Cabrera-Chávez F, Calderón dela, Barca AM (2010). Trends in wheat technology and modification of gluten proteins for dietary treatment of coeliac disease patients. J. Cereal Sci. 52: 337-341. Cataldo F, Montalto G (2007). Celiac disease in the developing countries: a new and challenging public health problem. World J. Gastroenetrol. 13: 2153-2159. Chen YS, Christensen JE, Broadbent JR, Steele JL (2003). Identification and characterization of Lactobacillus helveticus PepO2, an endopeptidase with post-proline specificity. Appl. Environ. Microbiol. 69: 1276-1282. Corsetti A, Settani L (2007). Lactobacilli in sourdough fermentation. Food Res.Int. 40: 539-558. Dayab L, Augustinab MA, Bateybc IL, Wrigleybc CW (2006). Wheatgluten uses and industry needs. Trends Food Sci. Technol. 17: 82-90 De Angelis M, Coda R, Silano M, Minervini F, Rizzello CG, Di Cagno R, Vicentini O, De Vincenzi M, Gobbetti M (2006a). Fermentation by selected sourdough lactic acid bacteria to decrease the intolerance to rye and barley flours. J. Cereal Sci. 43: 301-314. De Angelis M, Cassone A, Rizzello CG, Gagliardi F, Minervini F, Calasso F, Di Cagno R, Francavilla R, Gobbetti M (2010). Mechanism of Degradation of Immunogenic Gluten Epitopes from Triticum turgidum L. var. durum by Sourdough Lactobacilli and Fungal Proteases. Appl. Environ. Microbiol. 76: 508 - 518. De Angelis M, Rizzello CG, Fasano A, Clemente MG, De Simone C, Silano M, De Vincenzi M, Losito I, Gobbetti M (2006b). VSL#3 probiotic preparation has the capacity to hydrolyze gliadin polypeptides responsible for celiac sprue. Biochim. Biophys. Acta 1762: 80-93. De Angelis M, Rizzello CG, Scala E, De Simone C, Farris GA, Turrini F, Gobbetti M (2007). Probiotic preparation has the capacity to hydrolyze proteins responsible for wheat allergy. J. Food Prot. 70:
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African Journal of Biotechnology Vol. 11(29), pp. 7331-7337, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.1254 ISSN 1684â&#x20AC;&#x201C;5315 Š 2012 Academic Journals
Full Length Research Paper
Assessment of genetic relatedness of the two Amaranthus retroflexus populations by protein and random amplified polymorphic DNA (RAPD) markers Drinic Mladenovic Snezana*, Kostadinovic Marija, Ristic Danijela, Simic Milena and Stefanovic Lidija Maize Research Institute Zemun Polje, Belgrade, Serbia. Accepted 19 March, 2012
Two populations of Amaranthus retroflexus with different morphology were collected from field of the Maize Research Institute Zemun Polje, Serbia. Random amplified polymorphic DNA (RAPD) and seed protein analysis were performed to study the genetic differences in two grain Amaranthus populations. The studied populations have different protein and DNA profile. A total of 171 DNA fragments were generated by 31 RAPD primers, with an average of 5.5 fragments per primer. Of these, 61.4% fragments were polymorphic among the two populations. 18 protein fraction were obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The populations differed in the four protein fractions of different molecular weight. The seed protein electrophoresis and RAPD markers are useful for genetic determination of A. retroflexus populations and identification of biotypes with atypical morphology. Key words: Amaranthus retroflexus, biotypes, molecular markers, proteins.
INTRODUCTION The genus Amaranthus, with about 70 species, is characterized with a high degree of morphological diversity and a wide spectrum of adaptability to different ecological conditions. The amaranth gene pool involve a diverse group of wild relatives and weedy species, a group of cultivated grain species, and individual landrace populations collected from unique localities. The weed species A. retroflexus L., commonly referred as pigweeds, is one of the most widespread and frequent weeds of arable land worldwide as well as in Serbia (Stanojevic et al., 1996; Vrbnicanin et al., 2008). Populations of species A.retroflexus share many features, as a tendency to mutation and hybridization, and they demostrate different reaction to acetolactate synthase inhibitors that could be related to a evolution of resistance in their population (Ferguson et al., 2001). Correct genotype identification is important to evaluate
*Corresponding author. E-mail: msnezana@mrizp.rs. Tel: +381 11 3756704. Fax.+38111 3756707.
the genetic diversity of local Amaranthus, and for efficient weed control, but is often difficult because of similar morphological characteristics among species and variation within species, so misidentification is common (Horak et al., 1994; Wax, 1995). According to Wetzel et al. (1999) 12 of 92 Amaranthus accessions that had been collected and identified by weed scientists were misidentified. Ahrens et al. (1981) found that 13 of 14 accessions that were identified as redroot pigweed were actually smooth pigweed (Amaranthus hybridus L.) or Powell amaranth (Amaranthus powellii S. Wats.). Electrophoresis analysis of seed proteins proved to be useful for distinguishing species and cultivars of Amaranthus, for describing similarity between species and for estimation of its outcrossing rate (Zheleznov et al., 1997; Drzewiecki, 2001; Juan et al., 2007). For more accurate study of genetic diversity and phylogenetic relationships between Amaranthus species, different molecular markers including random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR) and amplified fragment length polymorphism (ALFP) have been used (Xu and Sun 2001; Wassom and Tranel 2005; Lee et al.,
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2008; Ray et al., 2008, Popa et al., 2010). The RAPD is a prefferd method for identification of genotypes because it is relatively inexpensive, utilizes arbitrary primers, and randomly samples a potentially large number of loci in a less complex pattern than other polymerase reaction (PCR) based markers (Hadrys et al., 1992; Williams et al.,1993, Das et al., 2005). Transue et al. (1994) and Chan and Sun (1997) used RAPD markers for the study of evolutionary relationships among grain amaranths and their wild relatives. Popa et al. (2010) studied genetic diversity and phylogenetic relationship among six species of Amaranthus by RAPD markers and showed that there is slightly intra and inter species polymorphism. The A. retroflexus green plants with pronounced stem hairiness are widespread and frequently occurred weed in maize fields in Serbia as well as in field of Maize Research Institute, Belgrade. Recently, the plants of A. retroflexus with green plant with pronounced red pigment admixture and with sparse stem hairs were new appeared genotypes in part of same field of Maize Research Institute, Belgrade. The objective of this study was to evaluate the level of genetic differences between two populations of A. retroflexus as well as usefulnes of seed protein electrophoresis and RAPD markers for distinquishing plants with atypical morphology.
MATERIALS AND METHODS Two A. retroflexus populations, population 1 (green plant with pronounced red pigment admixture and with sparse stem hairs) and population 2 (green plant with pronounced stem hairiness) were collected from field of the Maize Reserch Institute Zemun Polje. The limited number of plant of population 1 was the reason why we collected only 12 plants from each population. DNA was extracted from leaf tissue of randomly chosen five individual plants as well as from pooled samples of all collected plants (12) of each populations by the method of Rogers and Bendich (1985). Approximately 250 mg young leaf tissue was frozen in liquid nitrogen, and ground to fine powder in a pre-cooled mortar. The ground tissue was suspended in extraction buffer (100 mM Tris-Cl pH 8.0, 1.4 m NaCl, 20 mM EDTA, 2%CTAB, and 1% PVP) and incubated 30 min at 65°C with occasional mixing by inversion, followed by chloroform extraction. After precipitation with CTAB precipitation puffer, dried samples were dissolved in TE puffer. The quality and concentration of the DNA was evaluated by viewing samples in agarose gels and by spectrophotometer analysis of 260 and 280 nm light absorption. An analysis of five randomly choosen individual plants of each populations was conducted with the nine selected RAPD primers. For futher analysis PCR amplification of bulked DNA from all 12 plants, was tested on 40 10-mers arbitrary RAPD primers (Genosys Biotechnologies, Operon Technologies) in two rounds of amplification by the modified method of Williams et al. (1990). The list of RAPD primers is shown in Table 1. RAPD reactions were done in a volume of 25 μl containing 2.5 mM MgCl2, 100 μl dNTPs, 0.2 μl of decamer primers, 2.5 U of Taq Polymerase (Fermentas, Canada), and 10 ng of template DNA. Amplifications were carried out in a PTC-100 Thermocycler (MJ Research, Waltham, MA) with the following program: an initial denaturation step at 94°C for 2 min followed by 45 cycles at 94°C for 30 s, annealing at 40°C for 1 min, and extension at 72°C for 1 min and a final cycle at 72°C for 7 min. The amplified products were separated by electrophoresis in
1.5% agarose in a 1×TBE buffer (Tris-borate 89 mM and EDTA 0.5 M pH 8.0), visualized after ethidium bromide staining and photographed under UV light. The Generuler 1kb DNA Ladder (Fermentas, Canada) was used as a standard molecular weight marker. The band of equal molecular weight and mobility generated by the same RAPD primer were considered to be individual loci. Only consistely reproducible, well resolved fragments in the size range of ~250 to 3000 bp were scored. The presence or absence of each individual band was recorded for each line of the gel representing different plant sample. Proteins were extracted from seeds of two populations according to Wang et al. (1994). Isolated proteins along with a PAGE ruler unstained protein ladder (Fermentas, Canada) were analyzed through SDS-PAGE following discontinuous method of electrophoresis (Laemmli, 1970). The gel concentration was 10%. After electrophoresis, gel was stained with coomassie brilliant blue for about 20 to 30 min and then destained in 5% methanol and 20% acetic acid until the color of the background disappeared and the electrophoretic bands were clearly visible. The differences in number, pattern and intensity of protein fractions were determined by direct observation of gels and photographs.
RESULTS In the study of the 40 primers tested, an initial screening resulted in selection of 31 decamer primers that produced clear and reproducible RAPD profiles. RAPD assays of each population were performed at least two times each, with only reproducible, amplified fragments being scored. Initially screening of the randomly chosen five plants of both populations with the nine primers showed an identical or very similar profile of amplification products within individuals of same population (Figure 1). As observed, intrapopulation polymorphism was quite low for further analysis; we used group samples (bulk DNA) per population. The RAPD data in the present study indicated that no two primers revealed identical profiles in the analyzed populations (Figure 2). RAPD primers yielded a total of 171 fragments, ranging from 250 to 3000 bp in size of which 105 fragments (61.4%) were polymorphic. The total number of polymorphic loci detected varied between primers. The primer GEN 2-80-4 gave the highest number of fragments (13), while the minimum number of fragments in the two analysed populations (2) was obtained with OPB05, OPB09, and GEN1-70-3 primers. One primer, Gen 2-80-3, was monomorphic. The average number of polymorphic fragments per primer among the two populations was 5.5. The percent polymorphism of primers ranged from 0% (GEN 2-80-3) to 88.8% (OPB01), the average was 56.7%. In OPB-01, eight bands out of nine were noted as polymorphic bands, whereas in GEN 4-70-5, six out of seven bands were polymorphic. Certain amplified bands appeared to be common to both populations, whereas others were present in one population, but absent in the other. Primers Gen-2-80-1 and OPB09, after confirmation, could be used as markers to differentiate population of A. retroflexus plants with normal morphology from
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Table 1. List of primers along with their percent of polymorphism.
Total number of fragment
Number of polmorphic fragment
CGCAGACCTC GCAGCTCCGG
8 13
5 8
Polymorphism (%) 62.5 61,5
GEN 4-70-9 GEN 4-7-5 GEN 2-80-8 GEN 1-70-5 GEN 1-80-9 GEN 2-80-1 GEN 4-70-3 GEN 2-80-10
CCGGGGTTAC CATGTCCGCC GGCCACAGCG TAGATCCGCG GCACGGTGGG GCAGCAGCCG CTGTCGGCTC CGCGAACGGC
10 7 9 4 5 4 10 5
7 6 6 2 4 1 4 3
70.0 85.7 66.6 50.0 80.0 25.0 40.0 60.0
GEN 1-70-3
ACGGTGCCTG
2
1
50.0
GEN 1-70-9/1
TGCAGCACCG
4
2
50.0
GEN 4-70-2
GGACCGACTG
5
4
80.0
GEN 2-80-3 GEN 4-70-8 GEN 4-70-7 GEN 1-80-4 GEN 1-70-1 GEN 4-70-4* GEN 1-70-9/2* GEN 2-80-7|*
ACCCGTCCCC GAGAGGGAGG CTATCGCCGC CGCCCGATCC CATCCCGAAC GGACCGCTAG GGACTCCACG GCAGGTCGCG
1 6 5 4 0 0 0 0
0 3 4 2 0 0 0 0
0.0 50.0 80.0 50.0 0.0 0.0 0.0 0.0
GEN 2-80-5*
CGAGACGGGC
0
0
0.0
GEN 2-80-6*
ACCGCCTCCC
0
0
0.0
GEN 4-70-1* GEN 1-70-4* OPB01 OPB06
GCCCCTCTTG CGCATTCCGC GTTTCGCTCC TGCTCTGCCC
0 0 9 3
0 0 8 2
0.0 0.0 88.8 66.6
OPB09 OPB 02* OPB19 OPB03 OPB05 OPB11 OPB18 OPB04 OPB07 OPB12 OPB13
TGGGGGACTC TGATCCCTGG ACCCCCGAAG CATCCCCCTG TGCGCCCTTC GTAGACCCGT CCACAGCAGT GGACTGGAGT GGTGACGCAG CCTTGACGCA TTCCCCCGCT
2 0 3 5 2 5 7 3 8 7 4
1 0 2 3 1 3 3 2 5 4 1
50.0 0.0 66.6 60.0 50.0 60.0 42.8 66.6 62.5 57.1 25.0
OPB17
AGGGAACGAG
6
3
50.0
OPB20
GGACCCTTAC
5
4
80.0
Primer
Sequence
GEN 4-70-10 GEN 2-80-4
*no clear and reproducible fragments
population with atypical morphology.Seed protein profiling of two A. retroflexus populations showed distinct polymorphism in electrophoretic banding patterns and led to detection of a total of 18 polypeptide bands of which 14
were polymorphic (Figure 3). The molecular weights of peptides ranged from 20 to 200 kDa with the presence or absence of particular band. The 22% variation in seed protein fraction was observed between the two popu-
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M
p1
p1
p1
p1
p1
p2
p2
p2
p2
p2
Figure 1. RAPD-agarose gel image of five individual plants per population with GEN 4-70-3.
Figure 1. RAPD-agarose gel image of 5 individual plants per population with GEN lations. Population 1 had3 four more bands of different molecular weight compared with population 2. Seed protein profiling using SDS-PAGE has the potential to make a distinction between plants with atypical morphology from plants with normal one.
DISCUSSION Weedy species of the genus Amaranthus have increased in frequency and severity over the past years. Identification of these weeds is difficult because of similar morphological characteristics among species and variation within species. In Serbia, A. retroflexus is an invading and economically harmful weed species. In the Maize Research Institute field, two biotypes of A. retroflexus is presented; population 1 having stem covered in sparse hairs and population 2 with notable dense stem hairs. The results of previous study of morphological traits of those two populations (Vrbnicanin et al., 2009) indicate that apart from differences in hairiness, they differ also in the anatomy of stem (stem
diameter, epidermis thickness, cortex diameter, collenchymas thickness, and central cylinder diameter) as well as leaf anatomy (mesophyll thickness). According to same authors, such differences between populations provide basis for better understanding of plant reaction in terms of herbicide uptake and translocation that are potentially connected to an evolution of resistance to herbicides in the locality Zemun Polje, Serbia. To aid in the identification of morphologically atypical genotypes, the seed proteins was analyzed by SDS PAGE electrophoresis. Seed proteins, as genetic markers, are relatively frequently applied in the study of plant genomes for distinguishing species and for describing similarity between species, but there are relatively few studies on Amaranthus (Gudu and Gupta, 1988; Gorinstein et al., 1991; Zheleznov et al., 1997; Drzewiecki, 2001; Juan et al., 2007). The protein patterns obtained from the two populations consisted of 18 fraction of which 78% was polymorphic. Some polypeptides, presented in both analyzed populations, showed â&#x20AC;&#x153;conservativenessâ&#x20AC;?, whereas some protein fractions were variable between populations. By protein patterns, population 1 with
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Figure 2. Polymorphic RAPD-agarose gel image of the two populations (p1, p2) obtained with primer OPB03, OPB05, OPB11,OPB18 and GEN 170-5.
atypical morphology clearly differs from the other population. Our results are in agreement with literature data that Amaranthus seed protein is characterized by essential molecular heterogeneity. According to Zheleznov et al. (1997), electrophoretic analysis of storage proteins is a very useful method of describing phylogenetic relationships between the ten species of Amaranthus, while Gorinstein et al. (1991) obtained very slight differences of proteins between four Amaranthus species. The use of RAPD assay to identify genetic variation is preferred over the morphological and biochemical markers since these are completely devoid of the effects of environment and the stage of the experimental material, thus making them highly reliable. Analysis with nine RAPD markers of individual plants of both popualtions revealed a realtively low polymorphism. It is possible that the primers we used amplified mostly the conserved part of genome so they show low variation.
So, for futher analysis, we used bulk DNA sample per population. This approach had positive results in the study of populations of a great number of plant species (Transue et al., 1994; Roman et al., 2003). 31 decamer primers selected for RAPD profiling of two populations produced 105 (61.4%) polymorphic and 66 monomorphic banding sites. The level of polymorphism is comparable with the results for other Amaranthus species (Mandal and Das, 2002; Popo et al, 2010). Our results indicate that RAPD markers could be useful for verifying the identity of genotypes with ambiguous or a typical morphology.
ACKNOWLEDGEMENT This study was conducted as part of a project financed by the Ministry of Education and Science of the Republic of Serbia, TR 31037.
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kDa
200 150 120 100 85 70 60 50
40
30 25 20
M
p1
p2
p1
p2
Figure 3. SDS PAGE electrophoresis of seed from the two populations.
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Vrbnicanin S, Malidza G, Stefanovic L, Elezovic I, Stankovic Kalezic R, Marisavljevic D, Radovanov Jovanovic K, Pavlovic D, Gavric M (2008). Distribution of some harmful, invasive and quarantine weeds on the territory of Serbia. Plant Doctor, 36(5): 303-312. Vrbnicanin S, Stefanovic L, Bozic D, Saric M, Radosevic R (2009). Comparative Analysis of the Anatomy of Two Populations of RedRoot Amaranth (Amaranthus retroflexus L.). Pesticides, Phytomedicine, 24: 103-112. Wang C, Bian K, Zhang H, Zhou Z, Wang J (1994). Polyacrylamide gel electrophoresis of salt soluble proteins for maize variety identification and genetic purity assessment. Seed Sci. Tech. 21(51). Wassom JJ,Tranel PJ (2005). Amplified Fragment Length Polymorphism-Based Genetic Relationships Among Weedy Amaranthus Species J. Heredity, 96(4): 410-416 Wax LM (1995). Pigweeds of the Midwest-distribution, importance,and management. Proc Iowa Integrated Crop Manage. Conf. 7: 239-242. Wetzel DK, Horak MJ, Skinner DZ (1999). Use of PCR-based molecular markers to identify weed Amaranthus species. Weed Sci. 47: 518523.
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Williams JGK, Hanafey MK, Rafalski JA, Tingey SV (1993). Genetic analysis using random amplified polymorphic DNA markers. Methods, Enzymol. 218: 704-740. Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV (1990). DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18(22): 6531-6535 Xu F, Sun M (2001). Comparative analysis of phylogenetic relationships of grain amaranths and their wild relatives (Amaranthus; Amaranthaceae) using internal transcribed spacer, amplified fragment length polymorphism, and double-primer fluorescent intersimple sequence repeat markers. Mol. Phylogenet. Evol. 21: 372-387. Zheleznov AV, Solonenko LP, Zheleznova NB (1997). Seed proteins of the wild and the cultivated Amaranthus species. Euphytica, 97: 177182.
African Journal of Biotechnology Vol. 11(29), pp. 7338-7343, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.1485 ISSN 1684–5315 © 2012 Academic Journals
Full Length Research Paper
Polymorphism of the prolactin gene (PRL) and its relationship with milk production in American Swiss cattle Edy Alfonso1, Reyna Rojas1, José G. Herrera1, María E. Ortega1, Clemente Lemus2*, César Cortez1, Jaime Ruiz3, René Pinto4 and Heriberto Gómez4 1
Colegio de Posgraduados. Recursos Genéticos y Productividad-Ganadería. Carretera México-Texcoco km 36.5. Montecillo, Estado de México. CP 56230. 2 Unidad Acadèmica de Medicina Veterinaria y Zootecnia, Posgrado CBAP-UAN. Tepic, Nayarit, México. 3 Facultad de Medicina Veterinaria y Zootecnia, UNACH. Tuxtla Gutiérrez, Chiapas, México. 4 Facultad de Ciencias Agronómicas. UNACH. Villaflores, Chiapas, México. Accepted 20 January, 2012
The modern dairy cattle breeding strategy in the Mexican tropic is to identify genes or allelic variants that can be incorporated into selection programs such as the prolactin gene (PRL) which is associated with milk production and quality. The aim of this study is to screen an American Swiss population in Chiapas, Mexico, in order to analyze the polymorphism of the prolactin gene as well as its relationship with milk production in blood samples of 417 American Swiss cattle. The genotypes were determined through the polymerase chain reaction-restriction fragments length polymorphism (PCR-RFLP) technique, using RsaI restriction endonuclease, showing a 156 bp fragment located in exon 3. Allele frequencies in the studied breed were: A = 0.8765 and B = 0.1235. The genotype frequencies of AA, AB and BB were 0.776, 0.174 and 0.026, respectively. The Chi-square indicated that genotype distributions were not in the Hardy-Weinberg equilibrium (P<0.05). The results show that animals with genotype AA had a greater milk production during lactation than genotypes AB and BB (P<0.05), with genotype BB being the one that had the lowest production (P<0.05). It was concluded that the identification of the prolactin polymorphism in this population will allow the achievement of a better efficiency in the selection of breeding animals. Key words: Brown Swiss, prolactin, polymorphism, milk, RFLP-RsaI.
INTRODUCTION American Swiss livestock has been the base of dual purpose cattle system in the Mexican tropics, as a pure breed or through crossbreeding with local genotypes, due to milk and reproductive efficiency and environment adaptation (Johnson and Vanjonak, 1976; Finch, 1986). In the Chiapas region, the introduction of sires to the local livestock populations is a common practice, being a breeding strategy. The contribution of molecular genetics to the selection procedures, identification of genes with
*Corresponding author. E-mail: drclemus@yahoo.com.mx or ealfonso@colpos.mx. Tel: 5951086161
important effects on characteristics such as milk production or its components and the results in the search of quantitative trait loci (QTLs) in several species have led to the development of selection methods assisted by molecular markers (MAS), currently considered as one of the most important tools in animal improvement (Georges et al., 1995; Grisart et al., 2002). In order to improve breeding efficiency of dual purpose cattle, nowadays farmers in Chiapas are developing programs of genes identification or allelic variants that can be incorporated into the selection programs, such as the prolactin gene (PRL), associated with milk production and quality (Brymet et al., 2005; Ghasemiet et al., 2009). Milk production is a complex phenomenon in which several
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Table 1. Distribution of samples of American Swiss cattle from six herds in Chiapas, Mexico.
Farm
Cow
Calve
1 2
107 54
67 37
3 4 5 6
46 31 10 16
14 23 5 6
Total
264
152
Sire
Total 174 91
1
1
60 55 15 22 417
genetic and hormonal factors interact. Hormones such as growth hormone, insulin, thyroxin and prolactin are involved (Collier et al., 1984), with prolactin being one of the most important in this process (Sacravarty et al., 2008). PRL participates in multiple biological functions related with reproduction, osmoregulation, tegument growth and synergism with steroids (Barendse et al., 1997). It is necessary for the initiation and maintenance of lactation; it acts at the level of mammary alveoli, promoting synthesis and secretion of proteins, lactose, lipids, and other important components of milk (Leprovost et al., 1994). It also regulates immunological functions and participates in cell differentiation and growth (Loretz and Bern, 1982). Moreover, it is an immunomodulating molecule with relevant physiological effects, being considered as a cytosine. The PRL molecule can be linked to different groups: it can be glycolized, dimerized, polymerized or hydrolyzed to originate different variants (Méndez et al., 2005). PRL secretion does not differ between high and low milk production. However, some researchers have found that it increases its metabolism and distribution between days 30 and 150 of lactation (Collier et al., 1984). The gene of bovine prolactin located in chromosome 23, is made up of five exons and four introns. There is a silent adenosine-guanine (A-G) mutation in the codon codifying amino acid 103 in exon 3 of the bovine prolactin gene. The restriction fragment length polymorphism (RFLP) technique is used to detect small alterations that happen naturally in the genome from changes due to deletions or insertions of one or more pairs of nucleotides (Lewin et al., 1992 and Skinkyté, 2005). To determine similarities among populations, the estimation of their genetic distances is necessary, referred as the difference between the gene frequencies for a specific characteristic (http://www.answer.com). A better way to represent these genetic distances is by using dendograms, tree-shaped data diagrams, which make possible a graphical overview of the relationship among the studied populations. The aim of this study was to screen Mexican Brown Swiss population in Chiapas, Mexico, in order to analyze the polymorphism of the prolactin gene through RFLP,
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determine its allele and genotype frequencies, as well as its relationship with milk production. MATERIALS AND METHODS A total of 417 blood samples were taken from 264 cows, 152 calves, and a bull. All these animals were American Swiss cattle from six milking farms in the “Frailesca” region, Chiapas, Mexico (Table 1). The blood samples were taken from the caudal vein of the animals, using vaccutainer tubes and EDTA (2.5 mg/2.5 mL blood). Samples were refrigerated at 4°C and preserved until processing. DNA extraction was done using the technique by Miller et al. (1988) in the Physiology and Molecular Biology Laboratory of the Phytopathology Program of the Colegio de Postgraduados (COLPOS), Mexico. To amplify the prolactin gene through polymerase chain reaction (PCR), a couple of specific primers were used: Forward (5'-CGA GTC CTT ATG AGC TTG ATT CTT-3') and reverse (5'-GCC TTC CAG AAG TCG TTT GTT TTC -3'). The PCR reaction mix was made up of: 11.25 μl dH2O, 2.5 μl buffer 1X, 2.5 μl MgCl2, 0.5 μl dNTPs, 0.25 μl Amplicase (Biogenic), 2.0 μl of each primer at 20 pmoles, and 4.0 μl DNA (50 ng aprox.) in a final volume of 25 μl. The reactions were run in a TECHNE TC-512 thermocycler at 30 cycles, denaturalized at 94°C/3 min, aligned at 55°C/30 s, extended at 72°C/1 min, and final extension was done at 72°C/3 min. Once the reaction was finished, 5 μl PCR products were taken and placed in agarose gel at 1% with ethidium bromide. Electrophoresis was done at 80 V for one hour. After this time the gel was placed and observed in a UV transilluminator, Model Gel-Doc 2000, BIO RAD® and analyzed with the QuantityOne 4.0.3 software. Polymorphism was obtained once the presence of a 156 pb band corresponding to the molecular weight of the prolactin gene was verified. Digestion was carried out with the Rsal enzyme. To do this, 15 of the amplified product was taken from each processed sample and placed in a 0.5 ml tube with 2 μl dH2O, 2.5 μl enzyme buffer and 5 U or 0.5 μl, with a final volume of 20 μL. This mixture was digested in an incubator (Boekel Scientific Mod. 133000) at 37°C all night. To verify digestion, 15 μl of the digestion product was taken and separated by electrophoresis in 3% agarose with SB 1X buffer (5 mM disodium borate decahydrate or 10 mM sodiun hydroxide, pH adjusted to 8.5 with boric acid) as run buffer, and ethidium bromide. The marker used was GeneRuller 50 bp DNA ladder from Fermentas®. The determination of the prolactin genotypes was based on protocols described by Udina et al. (2001) and Mitra et al. (1995), with modifications. To establish the relationship between polymorphisms in the prolactin gene and milk production, the total production was estimated per cow through a periodic sampling with 14-day interval, at fixed monthly dates for 10 months. The dates were adjusted to 305 days, using multiplicative regional fit factors (Ochoa, 1991). Data were analyzed with SAS (2002), using the following model: Yijkl = + ai + bj + ck + ijkl I = 1,2,3...t
j = 1,2,3...r k= 1,2,3...l
Where, Yijkl = mean observed values of the characteristic; (milk production); µ = general mean; ai = effect of the i-th lactation year (I = 1,….,6); bj = effect of the j-th lactation number (j = 1,…,6); ck = effect of the k-th Prl-RsaI genotype (k = AA, AB, and BB); ijkl = random error, ij ~ N(0, ²). To calculate the allele and genotype frequencies, Hardy Weinberg equilibrium, degree of heterozygocity, Shannon index, genetic distances among animal sub-populations, and construction of dendograms, the POPGENE version 1.31 software were used (Yeh et al., 1999).
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Figure 1. Amplification of prolactin gene by electrophoresis with agarose 1% and ethidium bromide. Lane 1, Marker of molecular weight of 50 bp; lanes 2 to 8, bands of 156 bp.
Figure 2. Polymorphism fragments of the gene prolactin obtained with the enzyme RsaI in agarose gel to 3% with ethidium bromide. Lane 1). Marker of molecular weight of 50 bp, 2). Lane 2, genotype AB, lanes 3 â&#x20AC;&#x201C; 7, genotype AA and lane 8, genoype BB.
RESULTS The primers used allowed PCR amplification of a 156 bp fragment, corresponding to the prolactin gene (Figure 1). The digestion of the amplified fragment with the RsaI restriction enzyme showed the presence of three genotypes: AA, which had no digestion, obtaining the 156 bp fragment; AB, with three fragments, 156, 82, and 74 bp; and BB, with two fragments, 82 and 74 bp (Figure 2). The most abundant genotype was AA with 0.776, AB 0.174, and BB 0.026 (Table 2). The allele with the highest frequency was A with 0.8765 and B 0.1235. The degree of heterozygocity was 0.196. The Shannon index was 0.3762; thus rejecting the null hypothesis of the existence of the Hardy Weinberg equilibrium (X2, P < 0.05). This could be attributed to the characteristics of management in the studied herds, because in these herds breeding is done with semen and studs brought from outside the herds, and to the frequent inclusion of young cows and embryos to improve the genetic quality of the herds. This causes an increase in the probability for mutations to
happen, as a result of constant gene combinations, besides the environment effect. Only if the population is product of a random breeding generation of the individuals of the original population, it will be in HW equilibrium for each specific locus. The genetic distances between herds 2, 3 and 4, 6 are closely related, which indicates a great similarity between the populations. Therefore the behavior observed in the prolactin gene in this study was the one expected. On the other hand, a notable genetic distance was observed between herds 1 and 5, with a value of 0.0273, and a genetic identity of 0.9731 (Table 3 and Figure 3). The relationships between herds 4 and 6 were closer at 0.9999 and 0.0001 genetic distance, and the relationship between herds 2, 3, and 6 had a value of 0.9998 genetic identity, and 0.0002 and 0.0007 genetic distance. Herd one is different from the rest of the herds since it showed a higher frequency of genotypes AB and BB. This could be a consequence of the type of crosses, or the selection done in this farm. The structure of the populations was determined with the Chi-square test,
Alfonso et al.
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Table 2. Allelic and genotypic frequencies of PRL in American Swiss cattle in Chiapas, Mexico.
American Swiss cattle in Chiapas, México Locus
PRL RFLP-RSaI
Genotypic
Frec. genotypic
Frec. allelic
Shannon Index
Chi-square test HW test
AA AB BB
0.776 0.174 0.026
(A) 0.8765 (B) 0.1235
0.3762
4.230563 Degree of freedom: 1 Probability: 0.039702
Heterozygocity (Ho)
0.1966
Table 3. Genetic distance between herds of American Swiss cattle in Chiapas, México.
Farm 1 2 3 4 5 6
1 **** 0.0074 0.0101 0.0154 0.0273 0.0128
2 0.9926 **** 0.0002 0.0014 0.0061 0.0007
3 0.9900 0.9998 **** 0.0005 0.0041 0.0002
4 0.9848 0.9986 0.9995 **** 0.0017 0.0001
5 0.9731 0.9939 0.9959 0.9983 **** 0.0027
6 0.9873 0.9993 0.9998 0.9999 0.9973 ****
Nei's identity genetics (upper diagonal) and distance genetics (lower diagonal).
Figure 3. Dendrogram of studied herds of American Swiss cattle in Chiapas, México.
Table 4. Effect prolactin genotypic on milk production in American Swiss cows in Chiapas, México.
Prolactin genotypic AA AB BB
Sample 175 32 3
Production mean 305d. (kg) 3251.57 2789.91 2603.79
showing differences among groups as a consequence of the identification of different genotypes in the studied populations. The effect of the prolactin gene in milk production showed the best mean value for the genotype -1 AA, with 3251. 57 kg L , followed by AB with 2789.91, -1 and BB with 2603.79 kgL (Table 4). There was a significant difference between genotype AA and the other two, thus determining the influence of the A allele in milk
production, which could be due to the similarity in management of the genetic improvement programs focused mainly on milk production.
DISCUSSION From the prolactin genotype variants obtained through the RFLP-PCR technique with the RsaI endonuclease, the genotype AA had the greatest frequency, which is similar to data obtained by several researchers in different regions of the world, with different breeds, and sample sizes, who had reported genotype frequencies from 0.47 to 0.96 in the breeds: Black and White, Red Pied, Jersey, Gorbatov Red, Ayrshire, Black Pied, Montebeliard, Sahiwal and Holstein Friesian (Kalashnikova et al., 2009; Ghasemi et al., 2009; Kumari et al., 2008; Brym et al., 2005; Khatami, 2005; Dybus et
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al., 2005; Alipanah et al., 2007; Skinkyté, 2005; Ripoli et al., 2003; and Udina et al., 2001). However, other authors have reported lower frequencies for genotype AA, and higher frequencies for genotype AB: Jersey (0.65), Kankrej (0.62), Gyr (0.49), and Red Sindhi (0.62) (Kumari et al., 2008). In the case of Black and White cattle, Khatami (2005) found frequencies of 0.47 which is similar to genotype AA. When analyzing the genotypes favorable for milk production, it was found that the genotype AA had the -1 best average with 3251.57 kg . This result is similar to that reported by Brym et al., (2005) for Black and White cattle, and Ghasemi et al. (2009) with Montebeliard cattle, who reported a production of 5805 L. Dybus et al. (2005) found that the genotype AA was favorable for the second and third lactations, while the genotype AB was in the first lactation in Jersey cattle, and both genotypes AA and AB were favorable in Black and White cattle. Other authors (Alipanah et al., 2008) indicated that the genotype AB in Black Pied cattle affected milk, fat and protein production, while in the case of Red Pied cattle, the genotype BB was favorable. Sacravarty et al. (2008) reported that the best genotype for milk production was BB in cows from the second to fourth lactation in Kankrej cattle from India. Chrenek et al. (1999) examined the influence of polymorphism of PRL-RsaI in Brown Swiss cattle, and found no significant differences among cows with diverse PRL genotypes. Another important characteristic related with the PRL genotypes reported by other authors is somatic cell count (SSC), related to the presentation of sub-clinic mastitis, being the genotype BB more favorable. However, it came out negative for fat content in milk in Yaroslavl cattle (Brym et al., 2005). With regard to the heterozygocity found in this study with American Swiss cattle, it was 0.196, and the population was not in Hardy Weinberg equilibrium. This heterozygocity is similar to that estimated by Skinkyté (2005), who found a heterozygocity value of 0.33 and 0.23 in Black and White and Red cattle. Ghasemi et al. (2009) reported 0.15 in Montebeliard cattle. Kalashnikova et al. (2009) observed values of 0.40 in Black Pied cattle. Brym et al. (2005) found 0.038 in Black and White, and 0.33 in Jersey cattle. Alipanah et al. (2007) reported 0.39 in Russian Red Pied cattle. Dybus et al. (2005) found 0.28 in Black and White and 0.43 in Jersey. The genetic distances found in this study, related with the effect of the gene, are notorious, mainly in populations 1 and 5, proven by the presence of genotype BB in population 1, which was absent in population 5. To this regard, Plastow et al. (2003) determined the genetic distances in pigs using RFLP through the polymorphism found in the bands, considering this technique as appropriate for this end. The differences found in genotype frequencies in different studies, related with polymorphism of the prolactin gene, can be attributed to differences in the
breeds and the reduced number of analyzed samples (n < 50), which does not allow the genotypes to be efficiently represented (Brym et al., 2005). This is not the case of this study, where the sample size was greater (n > 400). According to the results obtained in this study, it is concluded that the structure of the studied populations show similarities with regard to productive characteristics, as a consequence of the use of genetic material from the same origin. On the other hand, the determination of prolactin genotypes at an early animal’s age represents an advantage, considering that this hormone is a good candidate to be considered in programs of marker assisted selection, since it shortens the interval between generations. Finally, allele A of prolactin can be considered as a good indicator for milk production in the American Swiss breed. REFERENCES Alipanah ML, Alexandrovna K, Veladimirovich RG (2008). Kappa-casein and PRL-RsaI Genotypic Frequencies in two Russian Cattle Breeds.Department of Animal Science.University of Zabol.Zabol. Iran. 98615-538. Arch. Zootec. 57(218): 131-138. Alipanah M, Kalashnikova L, Rodionov G (2007). Association of prolactin gene variants with milk production traits in Russian Red Pied cattle. Department of Animal Science, Faculty of Agriculture, University of Zabol, I.R. Iran. All-Russian. Iran. J. Biotechnol. 5(3): 158-161. Barendse W, Vaiman D, Kemp SJ, Sugimoto Y, Armitage SM, Williams JL, Sun A (1997). Medium-Density Genetic Linkage Map of The Bovine Genome. Mamm. Genome, 8: 21-28. Brym P, Kamiñski S, Wójcik E (2005). Nucleotide sequence polymorphism within exon 4 of the bovine prolactin gene and its associations with milk performance traits. Department of Animal Genetics, University of Warmia and Mazury, Olsztyn, Poland. J. Appl. Genet. 45(2): 179-185. Chrenek P, Huba J, Oravcova M, Hetenyi L, Peskovieova D, Bulla J (1999). Genotypes of bGH and bPRL genes in relationships to milk production. Proceeding of the 50th Annual Meeting of the EAAP. Zuerich. Book of Abstracts, p. 40. Collier RJ, McNamara JP, Wallace CR, Dehoff MH (1984). A review on endocrine regulation of metabolism during lactation. J. Anim. Sci. 59: 495-510. Dybus A, Grzesiak W, Kamieniecki H, Szatkowska I, Sobek Z, Blaszczyk P, Czerniawskapia E, Zych S, Muszynska M (2005). Association of genetic variants of bovine prolactin with milk production traits of Black and White and Jersey cattle. Agriculture University of Szcecin, Departament of Rumiants Science, Poland. Arch. Tierz., Dummersstorf. 48(2): 149-156. Georges M, Nielsen D, Mackinnon M, Mishra A, Okimoto R, Pasquino AT, Sargeant LS, Sorensen A, Steele MR, Zhao X, Womack JE, Hoeschele I (1995). Mapping quantitative trait loci controlling milk production by exploiting progeny testing. Genetics, 139: 907-920. Ghasemi N, Zadehrahmani M, Rahimi G, Hafezian SH (2009). Associations between prolactin gene polymorphism and milk production in montebeliard cows.Genetic Department, Safayeh, Bouali Street, Research and Clinical Centre for Infertility, Yazd ShahidSadoughi Medical Sciences University, Yazd, Iran. Int. J. Genet. Mol. Biol. 1(3): 048-051. Grisart B, Coppieters W, Farnir F, Karim L, Ford C, Berzi P, Cambisano N, Mni M, Reid S, Simon P, Spelman R, Georges M, Snell R (2002). Positional Candidate Cloning of a QTL in Dairy Cattle: Identification of a Missense Mutation in the Bovine DGAT1 Gene with Major Effect on Milk Yield and Composition. Genome Res. 12: 222-231. Kalashnikova LA, Khabibrakhmanova YA, Tinaev ASH (2009). Effect of
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Polymorphism of Milk Protein and Hormone Genes on Milk Productivity of Black Pied Cows All-Russian Pedigree Animal Breeding Research Institute, Moscow oblast, 141212 Russia. ISSN 1068-3674, Russian Agric Sci. 35(3): 192-195. Khatami SR, Lazebny OE, Maksimenko VF, Sulimova GE (2005). Association of ADN polymorphisms of the growth hormone and prolactin genes with milk productivity in Yaroslavl and Black and White cattle. Vavilov Institute of general genetic Russian Academy of sciences, Moscow, Rusia. Russian J. Genet. 41(2): 167-173. Kumari AR, Singh KM, Soni JK, Patel RK, Chauhan JB, Sambasiva RKR (2008). Genotyping of the polymorphism within exon 3 of prolactin gene in various dairy breeds by PCR RFLP. Biotechnology, National Dairy Development Board, Anand-388 001, India. Arch. Tierz., Dummerstorf. 513: 298-299. Le Provost E, Leroux C, Martin P, Gafe P, Dijane J (1994). Prolactin gene expression in ovine and caprine mammary gland. Neuroendocrinology, 60: 305-313. Lewin HA, Schmitt K, Hubert R, Van EMJ, Arnheim N (1992). Close linkage between bovine PRL-Rsa I and BoLA-DRB3 genes: genetic mapping in cattle by single sperm typing. Genomics, 13: 44-48. Loretz CA, Bern HA (1982). Prolactin And Osmoregulation In Vertebrates. Neuroendocrinology, 35: 292-304. Méndez I, Cariño C, Díaz L (2005). La prolactina en el sistema inmunológico: aspectos de síntesis y efectos biológicos. Departamento de Biología de la Reproducción. Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán. México. Rev. Invest. Clín. 57(3): ISSN 0034-8376. Miller SA, Dykes DD, Poletsky HF (1988). A simple salting out procedure for extracting DNA from human nucleated cells. Nucl. Acid. Res. 16: p. 1215. Mitra A, Schelee P, Balakrishnan CR, Pirchner F (1995). Polymorphisms at growth-hormone and prolactin loci in Indian cattle and Buffalo. J. Anim Breed. Genet. 112: 71-74. Ochoa GP (1991). Mejoramiento genético del bovino productor de leche. Departamento de Biogenética y Bioestadística. Facultad de Medicina Veterinaria y Zootecnia. UNAM. Ciencia Veterinaria. 5: 7072.
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Plastow G, K Siggens, M Bagga, B Brugmans, H Heuven, J Peleman (2003). Utilization of AFLP for genetic distance analysis in pigs. PIC Group. University of Cambridge.CB2 1QP. UK. Arch. Zootec. 52: 157-164. Ripoli MV, Corva PM, Antonini A, De Luca JC, Rojas F, Dulout FN, Giovambattista G (2003). Asociación entre cinco genes candidatos y producción de leche en la raza criolla Saavedreña. Universidad de Córdova España. Archivos de Zootécnia, 52(197): 89-92. Sacravarty G, Vadodaria VP, Joshi CG, Brahmkshtri BP, Shah RR, Solanki JV (2008). Prolactin Gene Polymorphism and its Association With Economic Traits in Kankrej Cattle. Veterinary College, Sardarkrushinagar Dantiwda Agricultural University, Sardarkrushinagar 385 506, Dist. Banaskantha, Gujarat, India. IJDS, 61: p. 4. SAS Institute Inc. (2002). SAS/STAT User's Guide: ed. SAS Institute Inc., Cary, North Carolina. 9: p. 5. Skinkytė R, Zwierzchowski L, Riaubaitė L, Baltrėnaitė L, Miceikienė KI (2005). Distribution of allele frequencies important to milk production traits in lithuanian Black & White and Lithuanian Red cattle. Janušauskas Laboratory of Animal Genetics, Lithuanian Veterinary Academy, Tilžės g. 18,LT-47181 Kaunas, Lithuania. Veterinarijair Zootechnika. T. 31(53): ISSN 1392-2130. Udina IG, Turkova SO, Kostyuchenco MV, Levedeva LAY Sulimova GE (2001). Polymorphism of Bovine Prolactin Gene: Microsatellites, PCR-RFLP. Vavilov Institute of general Gnetics, Russian Academy of Sciences, Moscow 119991. Russian J. Genet. 374: 407-411. Translated from Genetika, 37(4): 511-516. Yeh FC, Rong-cai Y, Tim B (1999). POPGENE VERSION Microsoft Window-based Freeware for Population Genetic Analysis. University of Alberta and Centre for Int. Forest Res. 1: p. 31.
African Journal of Biotechnology Vol. 11(29), pp. 7344-7353, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.1609 ISSN 1684â&#x20AC;&#x201C;5315 Š 2012 Academic Journals
Full Length Research Paper
Expression of androgen and estrogen receptors in the testicular tissue of chickens, quails and chicken-quail hybrids Herong Liao1,2#, Xiaoling Guo1#, Lingling Zhou2, Yan Li2, Xingming Li2, Du Liang2, Daquan Li2* and Ningying Xu1* 1
College of Animal Sciences, Zhejiang University, Hangzhou 310012, China. College of Animal Science and Technology, Shihezi University, Shihezi 832003, China.
2
Accepted 30 September, 2011
36 New Roman cocks, 30 Korean male quails and 30 chicken-quail hybrids of different day-age were selected and their body weight and testes weights were measured and as well, their testes were collected. Real-time polymerase chain reaction (RT-PCR) was performed to evaluate the messenger ribonucleic acid (mRNA) expression patterns of androgen receptors (AR) and estrogen receptors (ER) genes in testicular tissue of chickens, quails and chicken-quail hybrids at different growth stages. The results show that the testes of chickens and quails grew and developed normally with body weight gain, but the testes of chicken-quail hybrids had a slower growth rate and stunted growth. Real-time PCR showed AR and ER mRNA expression patterns in testes of chickens and quails at different growth stages were similar. AR mRNA expression in chickens and quails reached a significant peak at 80 and 30 days of age, respectively and their ER gene expression showed fluctuation slightly. The AR and ER expression of chicken-quail hybrids were different from the above expression patterns; the hybrids AR gene expression showed a gradual decline and ER gene expression gradually increased. The chickenquail hybrids AR and ER gene expression was abnormal and we speculate this is an important molecular factor for the testicular dysplasia of chicken-quail hybrids. Our results show that AR gene expression was upregulated by ER gene and we suggest that the synergetic effect of AR and ER gene regulated the normal testis growth and development of chicken and quail. Key words: Chicken, quail, chicken-quail hybrid, testis, androgen receptors (AR), estrogen receptors (ER) expression.
INTRODUCTION Both chicken and quail are from the same pheasant family Phasianidae of the class Aves, order Galliformes, but they belong to Gallus gallus and Coturnix coturnix, respectively (Khosravinia et al., 2005). The crossing between chickens and quails is a typical distant hybridization. Studies show that the chicken-quail hybrids have
*Corresponding author. E-mail: nyxu@zju.edu.cn, lidaquan37@163.com. Te1: 0571-86971199, 0993-2058271. Fax: 0571-86971199, 0993-2058271. #These authors contributed equally to this work
a significant heterosis for growth rate, body size, meat quality and other production performance (Liao et al., 2008) and even have a very strong resistance to birdsribonucleic acid (RNA) tumor virus (Greenfield et al., 1986). On the other hand, there are some problems or challenges in the distant hybridization, such as the incompatibility of heterogenous germ cells, low rate of fertilization (11.5%), hybrid reproductive organs dysplasia and hybrid sterility (Wilcox and Clark, 1961; Takashima and Mizuma, 1981). It is also interesting and confusing that the chickenquail hybrids embryos had males and females before 120 h incubation and only males were found by cytological inspection after they had been hatched (Yoshihiro, 1982).
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Hence, the chicken-quail hybrids can be used as a good material for animal genetic studies. Androgen is one kind of steroid hormones in male animal body and exists mainly in the form of testosterone and dihydrotestosterone (DHT), which plays an important role in maintenance of the male signs as well as in the growth and development (Wang et al., 2009). The development and function of male sexual organs are regulated mainly by androgen and estrogen is also essential for the development and function of male reproductive organs (Ferlin et al., 2010). The physiological functions of androgen and estrogen can be achieved only through their receptors (Walters et al., 2010; BI et al., 2005). Androgen receptor gene (AR) and estrogen receptor gene (ER) are members of nuclear hormone receptor family (Heinlein and Chang, 2002; Luconi et al., 2002). AR gene mutation or deletion can cause developmental abnormalities in male reproductive organs or infertility (Brinkmann et al., 1995). The expression of ER mRNA in the gonad of the male and female chicken embryos at 26 weeks (4.5 days incubation) was found by whole-mount in situ hybridization (Elbrecht and Smith, 1992). The estrogen receptors are expressed earlier than gonadal differentiation in chick embryo development and estrogen plays a permanent role in sex differentiation in birds (Andrews et al., 1997). Transgenic experiments showed that the testes of ERα knockout male mice were significantly smaller than those of wild-type mice, and the semini-ferous epithelium was thinning, sperm count and animals mating were reduced, and fertility was decreased (Eddy et al., 1996). However, the effects of AR and ER genes in the growth and development of chicken-quail hybrid testes have not been reported. Since the success of hybridization between chickens and quails, some studies of chicken-quail hybrids were carried out, such as effects of lactate dehydrogenase and alcohol dehydrogenase on the development of hybrids embryos and individuals, molecular mapping of neuropeptide and peptide, heredity of mitochondrial mRNA and coat color (Boswell et al., 1998; Meyerhof and Haley, 1975; Le Vine and Haley, 1975; Minvielle et al., 2002; Watanabe et al., 2005). The aim of this study was to define AR and ER expression patterns in testis in different growth stages of chickens, quails and chickenquail hybrids using real time quantitative PCR, to further our understanding of the role of androgen and estrogen in chickens and quails genital development and to clarify the molecular factors for testicular dysplasia of chickenquail hybrids. MATERIALS AND METHODS Experimental animals New Roman chickens, Korean egg-type quails and chicken-quail hybrids were raised under the same conditions at Shihezi University Experiment Station. Chicken-quail hybrids were produced through the mating of New Roman cocks and Korean quail hens by artificial insemination. Six cocks were selected at the age of 20, 40, 60, 80,
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100 and 120 days respectively. Six male quails and six chickenquail hybrids were selected at the age of 10, 20, 30d, 40 and 50 days respectively. A total of 36 cocks, 30 male quails and 30 chicken-quail hybrids were chosen. The selected animals were weighed and then sacrificed. The testes were rapidly removed and weighed. They were then frozen in liquid nitrogen immediately and then stored at -80°C. Before storage, the testes from a 120-day-old cock, a 50-day-old male quail and a 50-day-old chicken-quails hybrid were photographed with a digital camera (CANON-IXUS 1000 HS, Japan) and slices with a thickness of 0.5 cm were harvested from the left and right testes at the center line. The slices were then fixed in 10% formalin solution for 24 h. After washing, dehydrating and paraffin-embedding, they were sliced to 4 um sections using paraffin slicing machine (Leica, Germany), which were then stained with hematoxylin-eosin and photographed under optical microscope (Olympus, Japan) with 40-fold magnification. Total ribonucleic transcription
acid
(RNA)
extraction
and
reverse
Total RNA was extracted using TRIZOL reagent according to the manufacture’s protocol. The integrity of total RNA was detected on 1.2% agarose denaturing formaldehyde gel. RNA concentration was measured at 260 nm using a Smart SpecTM Plus UV spectrophotometer (BIO-RAD, U.S.A). Total RNA was reverse transcribed into first strand complementary deoxyribonucleic acid (cDNA) using SYBR® PrimeScriptTM RT-PCR Kit (TaKaRa, Dalian, China). Isolation of AR and estrogen receptors ER cDNA fragment Based on the chicken AR and ER mRNA sequence (GenBank acce-
ssion Nos. NM_001040090 and AF442965, respectively), primers were designed using Primer 5.0 to amplify AR and ER cDNA fragments on quails (Table 1). The PCR products were ligated into the pGEM-T easy vector system (TaKaRa, Japan) and then transformed into competent Top10 cell. Plasmid DNA was purified and sequenced in Sangon company (Shanghai, China) using an automated ABI3730 analyzer (Applied Biosystems, CA, USA). SYBR green real-time polymerase chain reaction (PCR) analysis of expression pattern The expression of AR and ER genes were detected by iQTM5 Muhicolor real-time PCR detection system (USA). The gene expression level was quantified relative to the expression of β-actin gene (GenBank accession No. AY550069). The forward and reverse primers for AR and ER genes are listed in Table 1. Realtime PCR was performed in triplicate in 25 μl mixture containing 12.5 µ SYBR Premix Ex TaqTM (2×), 0.5 µl of forward and reverse primers, 2 μl of template cDNA, and 9.5 µl of ddH2O. The cycling condition consisted an initial cycle of 15 s at 95°C followed by 45 cycles of 10 s at 95°C (for denaturation), 10 s at 60°C (for annealing) and 20 s at 72°C (for polymerization). The expression level was calculated using double-standard curve method. The differences in means of expression level was examined using oneway analysis of variance (ANOVA) and Duncan's multiple comparison test (p<0.05).
RESULTS Growth and development of testes of chickens, quails and chicken-quail hybrids Body weights and testes (pair) weight of chickens, quails
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Table 1. Conditions of PCR and parameters of oligonucleotide primer pair.
Target gene
GenBank number
AR
NM_001040090
280 bp
ER
AF442965
253 bp
β-actin
L08165
152 bp
Sequence of primer(5′→3') F: CCAGATTTGGTCTTCAACG R: GCTGGTAAAACCGCCTA F: CCCTTCATCCATCACCACA R: ACAAGACCAGACCCCATAAT F: CCTGAACCTCTCATTGCCA R: AGAAATTGTGCGTGACATCA
PCR product
Tm 56°C 56°C 56°C
Table 2. Living weight and testis weight of cock, quail and chicken-quail hybrid (g).
Breed
Trait
Cock
Day 10
20
30
40
50
60
80
100
120
Living weight Testis weight
/ /
84.37 0.00552
/ /
327.62 0.2871
/ /
680.33 1.2357
1048.29 6.4362
1263.87 12.7252
1527.36 18.6401
Quail
Living weight Testis weight
22.41 0.0082
46.46 0.0437
8.83 1.7835
142.79 3.3572
182.48 6.6735
/ /
/ /
/ /
/ /
ChickenQuail hybrid
Living weight Testis weight
29.33 0.0065
66.73 0.0131
128.65 0.0581
286.62 0.2153
352.56 0.3626
/ /
/ /
/ /
/ /
and chicken-quail hybrids of different day-ages are listed in Table 2. Body weight and testes weight of chickens increased by 18 times and 337 times from 84.37 and 0.0552 g at 20-day-age to 1527.36 and 18.6401 g at 120day-age, respectively. Body weight and testes weight of quails increased by 8 times and 814 times from 22.41 and 0.0082 g at 10-day-age to 182.48 and 6.6735 g at 50-day-age, respectively. Body weight and testes weight of chicken-quail hybrids increased by 12 times and 56 times from 29.33 and 0.00065 g at 10-day-age to 352.56 and 0.3626 g at 50-day-age, respectively. It was indicated that testicular growth and development of chicken-quail hybrids was very slow. From the cumulative growth curves of the body weight and testis weight of chicken, quail and chicken-quail hybrids (Figure 1), it can be seen that the three cumulative growth and developmental curves of body weight of chicken, quail and chicken-quail hybrids were consistent, but their growth and developmental curves of testes were very similar only in chicken and quail. However, hybrids did not have rapid growth and developmental period. Through the comparative study of the sections of testicular tissue of three adults (Figure 2), it was discovered that the hybrid’s testicular structure was significantly abnormal, the number of sertoli cells was small, seminiferous tubules diameter was also small, and secondary spermatocyte, spermatid and sperm could not be found within the lumen, which showed significant dysplasia. The typical male testis organ was also
discovered in the external morphology of the hatched hybrid (Figure 3). The adult testis of hybrid individual was small and there was no surrounding vascular distribution. Through the above comparative study, normal testicular growth and development was found in both chicken and quails, while the testes of hybrids showed developmental deficiency and typical male sterility.
The expression of AR mRNA in the testicular tissue in cock, quail and hybrid AR mRNA expression analysis is shown in Figure 4. Figure 4 shows that the relative expression of AR mRNA was highest at the age of 80 days, and lowest at the age of 120 days; the difference was significant (P<0.01). It was highest at the age of 30 days in quails and the lowest at the age of 10 days; the difference was significant (P<0.01). It was highest at the age of 10 days in chicken-quail hybrids and the lowest at the age of 50 days; the difference was significant (P<0.01). From the expression curve figure of the testicular tissue AR gene in chicken, quails and their hybrids (Figure 4), it could be seen that the relative expression curves of the testicular tissue AR mRNA in chicken and quails were similar, which reached the peak in the adolescent (chicken at 80 day-old, quail at 30 day-old) and showed increase first followed by a decline after the peak; the expression of hybrid testis AR mRNA declined linearly from 10-day-old
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Figure 1. The growth curve of cock, quail, and hybrid cumulative body and testis weights. A, Cumulative growth curves of body weights of cock, quail and chicken-quail hybrid; B, cumulative growth curves of testis weights of cock, quail and chicken-quail hybrid.
to 50-day-old.
The expression of ER mRNA in testicular tissue in cock, quail and hybrid The results of ER mRNA expression analysis are shown in Figure 5. Figure 5 shows that the mRNA expression of chicken ER was highest at 60 days and lowest at 80 days and the difference was significant (P<0.01); it was the
highest at the age of 50 days in quails, and lowest at the age of 40 days; the difference was significant between the two (0.01<P<0.05), it was highest at the age of 50 days and lowest at the age of 10 days in chicken-quail hybrids, the difference was significant (P<0.01). The expression curves of testicular tissue ER genes in chicken, quail and their hybrids (Figure 6) showed that at the different developmental stages of chicken and quails, ER mRNA expression in testicular tissue appeared to have fluctuating sequential changes; it was the highest at
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Figure 2. Schematic diagram of testicular biopsy (40 times). A, Cock; B, male quail; C, chicken-quail hybrid; ① mesenchymal cells; ② sertoli cells; ③ seminiferous tube cavity; (The contorted seminiferous tubules caliber (μm) of cock, male quail and chicken-quail hybrid were 275.09±46.07, 328.28±30.15, 20.98±10.43, respectively. The seminiferous epithelium depth (μm) of cock, male quail and chicken-quail hybrid were 97.01±8.12, 100.89±11.02, 4.223±2.507, respectively).
the age of 20 days in chicken, and the lowest at the age of 40 days, it gradually increased from 40 to 80 days and later decreased gradually; it also decreased gradually in the age of 10 to 40 days in quail, with the lowest point at the age of 40 days and the highest point at the age of 50 days. The hybrid ER mRNA expression in testis continued to rise linearly from 10 to 50 days, and with the increase in age, its ER mRNA expression was much more than those in chicken and quail.
DISCUSSION AR gene expression in testis tissue of chicken, quail and chicken-quail hybrid Testicular development many factors, the most which is a class of development of male
and function are dependent on important of which is androgen, hormones that promote the genitals and secondary sex
characteristics and stimulate the differentiation of spermatogenic cells and the sperm generation. The biological activity of androgen is mediated by the AR. Therefore, AR is essential for the male gonadal development (Jarow and Zirkin, 2005). It was showed that AR gene mutation or deletion may cause developmental abnormalities in male reproductive organs or infertility (Carreau et al., 2003). Within puberty in rats, a higher AR levels in leydig cells can promote testosterone secretion and with luteinizing hormone (LH) together, the synthesis ability of androgen in interstitial cells of adolescent rat testis increase many times and further promote the testis development (Hardy et al., 1990). It was also found by in situ hybridization that AR mRNA expression in rat leydig cells rose shortly in adolescence and reached the highest on the 35th day after birth. It was indicated that AR gene plays an important role on the differentiation of mesenchymal cells (O’Shaughnessy et al., 2002). O’Shaughnessy et al. (2002) found AR expression disorder with age and the mesenchymal cells
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Figure 3. Testicular morphology. A, Cock; B, male quail; C, chicken-quail hybrid.
Figure 4. The expression of AR mRNA in the testicular tissue in cock, quail and chicken-quail hybrid.
development is also abnormal, only with partial function of interstitial cells, although embryonic mesenchymal cells develop normally in mice with lack of AR function. It was indicated that AR plays an important role in
promoting the normal development of stromal cells. AR expression in testicular tissue of rat, goat and human had similar time series. AR expression measured by in situ hybridization on 21, 35 and 90 days of rat increases firstly
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Figure 5. The expression of ER mRNA in testicular tissue in cock, quail, and chicken-quail hybrid.
and decrease after reaching its peak (Shan et al., 1995). The intensity of AR immunostaining of rat and goat testicular sertoli cells also increase with increasing age and reach the strongest at sexual maturity (Goyal et al., 1997). It was also found that AR immunostaining in the human adult testis cell has cyclical changes, that is, the intensity of AR immunostaining is the strongest in spermatogenesis on the 3rd stage and decrease within the 4 to 5 and 1 to 6 stage (Suárez-Quian et al., 1999). In our study, AR expression in testicular tissue of chickens and quails (Figure 4B) also showed similar time series, but the AR expression pattern of chicken-quail hybrids was different. Our results suggest that AR expression with time series regulates the normal growth and development of the testis supporting cell of cock and male quail; by contrast, the AR expression of chickenquail hybrids, which showed a linear decline and was different with that of cock and male quail, is abnormal. The abnormal AR expression in the hybrid testis reduced the function of androgen regulating sertoli cells and the growth of testicular supporting cells of hybrids was blocked, leading to testicular growth and slow development of hybrid; there wass no strong period, the testes of 50-day hybrids were undersized and showed a significant dysplasia.
ER gene expression in testis tissue of chicken, quail and chicken-quail hybrid Estrogen not only indirectly affects germ cell development in the testis seminiferous tubules, but also directly
affects spermatogenesis and inhibits the apoptosis of germ cells (Pentikainen et al., 2000; Shetty et al., 1997). Small doses of estrogen can promote spermatogenesis in minor voles. On the contrary, large doses of estrogen or the estrogen receptor antagonists is bad to testis and will lead to testicular atrophy (Gancarczyk et al., 2004). ERα expression is carries through mainly in stromal cells in the testis development of rats, mice, pigs, dogs, marmosets and humans from juvenile to adult (Zhou et al., 2002; Rago et al., 2004; Nie et al., 2002; Pelletier and El-Alfy, 2000; Sar and Welsch, 2000; Fisher et al., 1997). It was found by in situ hybridization that ER mRNA is expressed in the gonad both in the male and female chicken 26-stage embryos (4.5-day incubation). ERβ expression was found in germ cells and sertoli cells, except in leydig cells at different developmental stages of male (Carreau et al., 2003). Hence, ERβ gene directly affects germ cells in the process of testis development and spermatogenesis. Transgenic experiments showed that the testis ERα-knockouted male mouse with thinning seminiferous epithelium, reducing sperm count, reducing mating number and decreased fertility, was significantly smaller than wild-type mice (Gancarczyk et al., 2004). Our results show that the model of ER mRNA expression in testis of chicken-quail hybrids is different from that of chickens and quails. The ER mRNA expressions of hybrids increased linearly continuously and gradually out distant that of chickens and quails with days (Figure 5B). Therefore, we speculate that this model of the sustained incremental expression and higher levels of expression of ER gene is the important factor that causes azoospermia in the testicular seminiferous tubules of hybrids.
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Figure 6. The expression curves of AR and ER gene mRNA in the chicken, quail, and chicken-quail hybrid testis.
AR and ER gene in chickens, quails and chicken-quail hybrids testis tissue Female and male hormones promote male reproductive system with a synergistic effect through their receptors
(Oliveira et al., 2004; Carreau and Hess, 2010). The lack of estrogen in young male can directly affect the reproductive capacity in adult. Figure 6 shows that, the length of growth and developmental periods of chickens and quail were different, but the AR and ER expression
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patterns in their testes were similar and showed a time series. In all ages, AR expression was higher than the corresponding ER, and three had a common characteristic which was when AR expression was at the highest, ER was at the lowest point. On the contrary, ER expression was at the peak while AR tended to be at the lowest point. It demonstrated that AR and ER genes synergistically regulated the growth and development of testicular tissue. We speculate ER gene has the function of up-regulating AR gene or AR gene has the function of down-regulating ER gene. The reason could be due to the amount of androgen secretion which increased AR, whereas competition reduced estrogen secretion which led to the decrease in ER. Therefore, it is speculated that the opposite and synergistic expression pattern is the molecular regulation of normal growth patterns in chicken and quail testis. In this regulatory pattern, the testes of chicken and quails grew and developed normally, while the expression of testis AR mRNA continued to decline in the hybrids with the continuous increase in ER which is completely different from the normal regulatory patterns of cocks and male quails, showing abnormal expression, and resulting in abnormal testicular growth and development in the hybrids from chicken and quail hybridization.
Conclusion AR and ER genes both participate in the regulation of the testicular tissue growth in chicken, quails and their hybrids. Its regulation has certain time sequence. Since there are changes in the mRNA regulatory patterns of AR and ER genes in hybrid testicular tissue, an extreme type of uncoordinated abnormal expression was shown resulting in slow growth and development of hybrid testis, testicular tissue structural abnormalities and the absence of normal reproductive functions.
ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 30360071 and 30660125). ABBREVIATIONS AR, Androgen receptors; ER, estrogen receptors; RT-PCR, realtime polymerase chain reaction; mRNA, messenger ribonucleic acid; LH, luteinizing hormone. REFERENCES Andrews JE, Smith CA, Sinclair AH (1997). Sites of estrogen receptor and aromatase expression in the chicken embryo. Gen. Comp. Endocrinol. 108: l82-190. Bi XD, Chu MX, Jin HG, Fang L, Ye SC (2005). Estrogen receptor as a candidate gene for prolificacy of small tail Han sheep. Acta. Genetica.
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normal adult Leydig cell development in androgen-receptor-deficient mice. Cell Sci. 115: 3491-3496. Pelletier G, El-Alfy M (2000). Immunocytochemical: Localization of estrogen receptors α and β in the human reproductive organs. J. Clin. Endocrinol. Metabolism, 85(12): 4835-4840. Pentikainen V, Erkkila K, Suomalainen L, Parvinen M, Dunkel L (2000). Estradiolacts as a germ cell survival factor in the human testis in vitro. The J. Clin. Endocrinol. Metabolism, 85(5): 2057-2067. Rago V, Maggiolini M, Vivacqua A, Palma A, Carpion A (2004). Differential expression of estrogen receptors (ERalpha/ERbeta) in testis of mature and immature pigs. Anat. Rec. A Discov. Mol. Cell Evol. Biol. 281(2): 1234-1239. Sar M, Welsch F (2000). Oestrogen receptor alpha and beta in rat prostate and epididymis. Andrologia, 32: 295-301. Shan LX, Zhu LJ, Bardin CW, Hardy MP (1995). Quantitative analysis of androgen receptor messenger ribonucleic acid in developing Leydig cells and Sertoli cells by in situ hybridization. Endocrinology, 136(9): 3856-3862. Shetty G, Krishnamurthy H, Krishnamurthy HN, Bhatnagar S, Moudgal RN (1997). Effect of estrogen deprivation on the reproductive physiology of male and female primates. J. Steroid. Biochem. Mol. Biol. 61: 157-166. Suárez-Quian CA, Martínez-García F, Nistal M, Regadera J (1999). Androgen receptor distribution in adult human testis. J. Clin. Endocrinol. Metabolism, 84(1): 350-358. Takashima Y, Mizuma Y (1981). Studies on chicken-quail hybrids. Japanese Poultry Sci. 18: 267-272. Walters KA, Simanainen U, Handelsman DJ (2010). Molecular insights into androgen actions in male and female reproductive function from androgen receptor knockout models. Human Reprod. Update. September, 16(5): 543-558.
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African Journal of Biotechnology Vol. 11(29), pp. 7354-7360, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.2136 ISSN 1684–5315 © 2012 Academic Journals
Full Length Research Paper
Using inter simple sequence repeat (ISSR) markers to study genetic polymorphism of pistachio (Pistacia vera L.) in Algeria KEBOUR Djamila*, BOUTEKRABT Ammar and MEFTI Med Laboratory of Biotechnology, Université Saad Dahlab, Blida, Algeria. Accepted 30 March, 2012
Pistacia vera L. is a widely represented plant in Algerian semi-arid regions. It is potentially used to restore degraded ecosystems. Genetic relationships among the cultivars was assessed by using six inter simple sequence repeat (ISSR) primers. During the ISSR screening in this study, good amplification products were obtained from primers based on guanine-adenine (GA), cytosine-adenine (CA) and guanine-adenine-adenine (GAA) repeats. Primers based on cytosine-tyrosine (CT) and CAA repeats produced few large separate bands, so these primers were not selected for the final analysis (eliminated for the final analysis). This study shows that ISSR-PCR analysis is quick, reliable and produces sufficient polymorphisms for large-scale DNA fingerprinting purposes. The total of 111 bands of which 60 were polymorphic, (with 54.04%) was amplified by the six primers, an average of seven bands per primer. The total number of amplified fragments was between five to ten and the number of polymorphic fragments ranged from four to seven. The range of genetic similarity was from 0/84 to 1 and the constructed unweighted pair group method with arithmetic averages (UPGMA), dendrogram classified the tested genotypes into two main clusters. This study shows that there was low genetic diversity among the tested cultivars and the ISSR-PCR analysis produced sufficient polymorphisms for large-scale DNA fingerprinting. This study reports the first application of the ISSR technique in characterization of Algerian pistachio cultivars original from Syria. Key words: Pistacia vera L., genetic relationships, DNA extraction, ISSR, clustering.
INTRODUCTION The pistachio fruit, although grown for centuries in the Mediterranean area, was introduced in Algeria in the midtwentieth century by ACSAD (Syria). Demonstration orchards have been established in different regions to develop its culture. Its current area covers 400 ha, and spread over different climatic stages. Recognizing the potential interest for the development of many regions, the Ministry of Agriculture and relevant department have planned during the late eighties, the extension of this culture to approximately 2000 ha. This objective has been achieved due to several constraints including those linked to the nature of the case, ignorance of technology
*Corresponding author. E-mail : djakeb@hotmail.com.
and especially his leadership in safe condition experienced by our country in this period. In recent years there has been an expansion of the cultivation of pistachio through Algeria, thus meeting the development objectives of the arid and semi-arid and the conservation of soil against erosion. The genus Pistacia is a diploid (2n = 30) (Zohary, 1952; Ila et al., 2003) member of the Anacardiaceae family, contains 13 or more species, among which Pistacia vera L. (Whitehouse, 1957), produces commercially valuable edible nuts. The pistachio (P. vera. Linnaeus, 1753), is a shrub native to the Middle East. It is a dioecious tree (Kafkas et al., 2006), measuring 3 to 8 m. It is also a deciduous tree and up to 12 cm, with 3 to 7 leaflets. Its fruits have a dimension of 1 to 3.5 cm (Zohary 1952). Pistachio flowers has no petal and shows perfect dioecy
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and maturity of pistachio seedlings takes between 5 and 8 years. Female flowers have no trace of stamens and mature male flowers lack any evidence of female structures (Zohary, 1952). Therefore, there is no honey bee-attraction to facilitate indirect pollination; instead pollination is usually performed by wind. Among the nut tree crops, pistachio tree ranks sixth in the world production behind almond, walnut, Cashew, hazelnut and chestnut (Mehlenbacher, 2003). Its use is recommended for the safe guarding of the pastoral potential and restoration of degraded ecosystems. Although the number of varieties constituting the species P. vera L. is considerable, inventory and identification are facing problems of taxonomic confusion. Earlier work on classification and identification of varieties of pistachio dates back to the nineteenth century. However, Zohary (1952) was the first to use the various organs of pistachio trees (leaf, fruit and endocarp) to characterize and classify the varieties of this species. Since then, various studies on varietal identify-cation have been developed in Iran, Syria, Turkey and Italy from the combination of morphological, agronomic and phonological (Faostat, 2006). Due to the need to overcome the difficulties encountered in the morphological characterization, our study was conducted in the Laboratory of Biotechnology (Algeria) in order to undertake further studies on varietal identification based on genetic markers (molecular markers) to determine the polymorphism breeders of this species in order to find the best ecotypes adapted to contrasting environmental conditions. Since the mid 1980s, genome identification and selection has progressed rapidly with the help of polymerase chain reaction (PCR) technology. Among them, random amplified polymorphic DNA (RAPD) (Williams et al., 1990) has been the most commonly used method in pistachio cultivars characterization (Hormaza et al., 1994, 1998; Kafkas and Perl-Treves, 2002; Katsiotis et al., 2003; Golan-Gpldhirsh et al., 2004; Mirzaei et al., 2005). Amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) techniques have also been used in pistachio to study genetic relationship among Pistacia species and cultivars (Ibrahim Basha et al., 2007; Ahmadi Afzadi et al., 2007). Each marker technique has its own advantages and disadvantages. RAPD markers are very quick and easy to develop (because of the arbitrary sequence of the primers), but lack reproducibility (Virk et al., 2000). AFLP has medium reproducibility but is labour-intensive and has high operational and development costs (Hansen et al., 1998). On the other hand, microsatellites are specific and highly polymorphous (Jones et al., 1997), but they require knowledge of the genomic sequence to design specific primers and, thus, are limited primarily to economically important species. The first ISSR studies were published in 1994 and focused on cultivated species (Wolfe and Liston, 1998). These studies demonstrated the hyper
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variable nature of ISSR markers. Microsatellites or simple sequence repeats (SSRs), are polymorphic loci present in nuclear DNA and organellar DNA that consist of repeating units of 1 to 4 base pairs in length (Zietkiewicz et al., 1994). Inter simple sequence repeat (ISSRs) are semi arbitrary markers amplified by PCR in the presence of one primer complementary to a target microsatellite. Amplification in the presence of nonanchored primers also has been called microsatelliteprimed PCR (Karp et al., 1997). Each band corresponds to a DNA sequence delimited by two inverted microsatellites. Like RAPDs, ISSRs markers are quick and easy to handle, but they seem to have the reproducibility of SSR markers because of the longer length of their primers. Amplification in this technique does not require genome sequence information and leads to multilocus and highly polymorphous patterns ( Zietkiewicz et al., 1994), and involves longer (16-18 nucleotides) primers encoding microsatellite elements that amplify DNA segments Intramicrosatellite repeats (Zietkiewicz et al., 1994). ISSR is a dominant marker like RAPD (scored using presence or absence of band at a locus) but with greater robustness in repeatability and extremely high variability. The genetic variability between and within specific study mainly concerns non-coding regions of the genome that are characterized by the abundance of highly repetitive sequences within which the mutations are quite frequent. This variability has been studied by the technique of inter simple sequence repeat. ISSR is a dominant marker like RAPD, however scoring using presence or absence of band at features make ISSR better than other readily available marker systems in investigating the genetic variation among very closely related individuals and in crop cultivar classification (Fang and Roose, 1997; Nagaoka and Ogihara, 1997). Recently this marker technique has been used to detect DNA polymorphism and genetic diversity of pistachio germplasm (Kafkas et al., 2006). One aspect of this study is concerned with the determination of genetic polymorphism of a collection of P. vera L. based on genetic markers. The objectives of the study are: 1) to assess genetic diversity and relationships among some Algerian pistachio cultivars and 2) to set up and use first ISSR technique in pistachio cultivar identification in Algerian.
MATERIALS AND METHODS Plant material and DNA extraction A set of 10 P. vera L. varieties listed in Table 1 were investigated. These were chosen for their good fruit quality and are the most common genotypes in the main Algerian plantation. All the varieties recently introduced (1998) from Syria; the Arab Center for the Studies of Arid Zones and Dry lands (A.C.S.A.D) were included in the study. The plant material consisting of young leaves provided from 400 trees (one for each genotype) were randomly chosen and sampled directly from a collection maintained in culture of Pastoretum I.T.A.F.V (State institution) and others at different
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Table 1. Algerian Pistacia vera L. varieties used in this study.
Variety name Adjmi Ashouri Batouri Bayadhi Jalab ahmer Lazwardi Nab djamel Marawhi Oleimi Boundouki
Label 1 2 3 4 5 6 7 8 9 10
Origin Syria (A.C.S.A.D) Syria (A.C.S.A.D) Syria(A.C.S.A.D) Syria(A.C.S.A.D) Syria(A.C.S.A.D) Syria(A.C.S.A.D) Syria(A.C.S.A.D) Syria(A.C.S.A.D) Syria(A.C.S.A.D) Syria(A.C.S.A.D)
region of Algeria.
DNA preparation Extracts from frozen young leaves of adult trees were kept in liquid nitrogen tanks for the purpose of DNA extraction and ISSR analyses according to the protocol of cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1990) with minor modifications. After purification, DNA concentrations were determined using a Gene Quant spectrometer and its integrity was checked with agarose minigel electrophoresis according to Sambrook et al. (1989). Frozen tissue (0.5 to 0.75 g) was ground in a mortar and pestle in liquid nitrogen and homogenized in 5 ml of preheated (60°C). DNA was extracted according to the CTAB (hexadecyltrimethylammonium bromide) method of Doyle and Doyle (1987) with some modifications. Young leaf tissue (100 mg) was ground to fine powder in liquid nitrogen in 1.5 centrifuge tubes and mixed with 0.5 ml of CTAB extraction buffer (100 mM TRISHC1, 1.4 M NaC1, 20 mM EDTA, 2% CTAB, 1% PVP, 0.2% mercaptoethanol and 0.1% NaHSO3). The sample was incubated at 65°C for 1 h, mixed with an equal volume of chloroform-isoamyl alcohol (24:1) and centrifuged at 13000 rpm for 5 min in a desktop centrifuge. The aqueous phase was recovered and mixed with equal volume of isopropanol to precipitate the DNA. The nucleic acid pellet was then washed with 1 ml of 10 mM ammonium acetate in 76% ethanol, dried overnight and resuspended in 100 μL modified TE buffer (10 mM TRIS-HCl, 0. l mM EDTA). DNA was extracted separately from each individual plant. In all cases, the extracted DNA (25 ng per 20 μL reaction mix) was subjected to polymerase chain reaction (PCR) amplification. DNA quantity and quality were estimated both using an UV spectrophotometer by measuring absorbencies at A260 and A280 and 0.8% agarose gel electrophoresis by comparing band intensity with λ DNA of known concentrations. DNA was extracted separately from each individual plant. In all cases, extracted DNA (25 ng per 20 μL reaction mix) was subjected to polymerase chain reaction (PCR) amplification. DNA samples were diluted to 10 ng / μL for ISSR reactions.
Primers and ISSR assay A total of 10 primers were tested to amplify the isolated DNA. These primers are listed in Table 2, and their composition has been arbitrarily established. For PCR amplifications, a 25-μL reaction mixture was used and it contained between 20 and 30 ng of total genomic DNA (1 μL), 60 pg of primer ISSR primers (1 μL), 2.5 μl of
10X Taq DNA polymerase reaction buffer, 1.5 unit of Taq DNA polymerase (Fermentas, Lithuania) and 200 mM of each dNTPs (DNA polymerization mix, Amersham-Pharmacia, France). Amplification reactions were done in a 25 μL volume containing: 10 mM Tris-HCl, (pH 8.0), 50 mM KCl, 1.5 Mm MgCl2, 200 mM each of d'NTPs, 10 pmol of a given primer, 1 unit of Taq DNA polymerase (Fermentas, Lithuania) and 10 ng of genomic DNA.
PCR Amplification PCR was performed using ISSR and amplification reactions were carried out in an Eppendorf Master Cycler Gradient (Eppendorf Netheler-Hinz, Hamburg, Germany). The apparatus is programmed to execute the following conditions in 1 cycle: a denaturation step of 5 min at 94°C, followed by 35 cycles of 30 s at 94°C, 90 s at the annealing temperature, and 90 s at 72°C. A final extension of 72°C for 5 min was included. ISSR amplification products were analyzed by gel electrophoresis in 1.8% agarose in 1x TBE buffer, stained with ethidium bromide (0.5 μg ml-1) according to Sambrook et al. (1989) and digitally photographed under ultraviolet light at 300 nm. Reproducibility of the patterns was checked by running the reactions in duplicates.
Data analysis For Each DNA sample, ISSR bands were transformed into a binary matrix where the presence of reproducible polymorphic DNA band at particularly position on gels is scored manually as 1 (present), while a 0 (absent) denotes its absence of co-migrating fragments for all accessions. Only the clearest and strongest reproducible bands across two PCR amplification replicates were used for cluster analysis. Clearly detectable amplified ISSR ranged from 200 to 2400 bp in size. The genetic similarity metrics were constructed using Jaccard’s (Jaccard, 1908). Dendrograms were constructed by the unweighted pair-group method using arithmetic average (UPGMA) and complete linkage algorithms. In addition to cluster analysis, principal component analysis (for precise relationships between the P. vera L. varieties) was used to confirm the results of cluster analysis. The efficiency of clustering algorithms and their goodness of fit were determined based on the cophenetic correlation coefficient. Data analyses were performed by the NTSYS software version 2.2 (Rohlf, 1998).
RESULTS AND DISCUSSION This study reports the first application of the ISSR technique in pistachio characterization of Algerian varieties. This study showed that ISSR-PCR analysis is quick, reliable, produces sufficient polymorphisms for large-scale DNA fingerprinting purposes, and also showed that ISSR markers are able to reveal variability between pistachio cultivars. The results of this molecular assay in fingerprinting of the 10 pistachio genotypes are presented in Table 2. In ISSR, according to the reported results of Kafkas et al. (2006), the first ten primers were used and after initial screening, six out of these primers were eventually selected for the final analysis. A total of 111 bands were amplified by the six primers, an average of eight bands per primer of which 60 were polymorphic (54.04%). The total number of amplified fragments was
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Table 2. ISSR primer sequences used for analysis of Pistacia vera L. with primer annealing temperatures, number of bands amplified, and number of polymorphic bands amplified.
Primer
5’- 3’ sequence
1* 2* 3* 4* 5* 6* 7* 8* 9* 10* TOTAL
5`- (AG)8C -3` 5`- (GA)8T -3` 5`- (TGGA)4 -3` 5`- (CA)7AG -3` 5`- (GA)8CG-3` 5`- (ACTG)4 -3` 5`- CCAG(GT)7-3` 5`- (GACAC)4-3` 5`- (AC)8T-3` 5`- (TG)8TT-3
Annealing temperature (°C) 52 54 45 56 65 48 56 45 65 45
Total band amplified 6 7 10 10 18 10 10 11 12 7 111
Number of polymorphic band 6 5 0 9 16 2 9 2 10 1 60
Polymorphism (%) 100 71.42 0 90 89 20 90 18 83.33 14.26
60*100/111= 54.04% ; R = purines: G or A ; Y = pyrimidines: C or T.
Figure 1. Typical example of ISSR polymorphism banding patterns in a subset of Algerian Pistacia vera L. varieties using (GA)8CG primer. M, Standard molecular size marker: sizes of molecular weight markers are indicated in kb; 1, Adjmi; 2, Ashouri; 3, Batouri; 4, Bayadhi; 5, Jalab ahmer; 6, Lazwardi; 7, Nab djamel; 8, Marawhi; 9, Oleimi; 10, Boundouki.
between 6 to 18 and the number of polymorphic fragments ranged from 0 to 16. Figure 2 shows the results of amplification with primer ISSR (GA)8CG) on agarose 1.8% with 16 lanes gel tray. From the results of Ehsanpour et al. (2008), good amplification products were obtained from primers based on AC, repeats [(AC)8CG and (AC)8TA], since, primers based on CT, GT, CAG and CAA repeats produced few large separate bands which were eliminated for the final analysis. Kafkas et al. (2006) using 20 primers obtained a total of 156 bands, an average of 7.7 bands per primer, of
which 73(46.2%) were polymorphic, which is similar to our results in this study. A total of 10 primers were screened for their ability to generate consistently amplified band patterns and to access polymorphism in the tested varieties. Among these primers, only six revealed polymorphic and unambiguously scorable bands while smear or no amplified products were observed with the other primers. These six primers generated five to 10 polymorphic DNA bands with a range of 200 to 2500 bp. Typical amplified products are reported in Figure 1. The polymorphic patterns obtained suggest that the ISSR
0,96 0.96
jaccard's similarity coefficient
0.88 0,88
0,8 0.8
0.72 0,72
0.64 0,64
Figure 2. UPGMA dendrograms of 10 varieties of Pistacia vera by ISSR analysis using Jaccard’s similarity matrices.
procedure constitutes an alternative approach that is suitable to examine the P. vera's genetic diversity at the DNA level. A total of 60 polymorphic ISSR products were obtained (Table 2). The matrix has a genetic distance of 0.61 to 0.82 with a mean of 0.5688. Thus, it may be assumed that the varieties are characterized by a high degree of genetic diversity at the DNA level. The smallest distance value of 0.61 was observed between Adjmi - Lazwouardi and Ashouri - Nabdjamel varieties indicating that these ecotypes are the most similar. The maximum distance value (0.82) suggesting high divergence was detected between marawhi and JalabAhmer varieties (Table 3). Furthermore, the phenogram obtained (Figure 2) supports the varietal clustering. The cluster analysis generated a dendrogram with two main branches that clustered individuals that share the same gene pool of origin. Branch ‘A’ included all those cultivars whose genomic background is mainly from parent plants collected from Syria (cultivars Boundouki , Bayadhi and Jalab
ahmer) and cluster ‘B’ included cultivars with parents from Syria too (cultivars Adjmi, Ashouri, Batouri, Adjmi, Ashouri and Batouri). The cophenetic correlation (0.6627), a measure of the correlation between the similarity represented on the dendrograms and the actual degree of similarity, was calculated for each dendrogram. Among the different methods, the highest value (r= 0.82) was observed for UPGMA based on Jaccard’s coefficient (Table 3). The principle coordinate analysis (PCA) based on genetic similarity matrices were also used to visualize the genetic relationships among genotypes (Figure 3), and it confirmed the results of cluster analysis. The results of this study show that there is a relatively low level of genetic diversity in the studied samples, which was expected in view of the dioecius and outbreeding nature of the cultivated pistachio cultivars, as well as the high level of heterozygosity due to the crosspollinating nature of the plant established during the evolution and domestication processes, which have been conserved by the propagation of clones through
Adjmi
Oleimi
Marawhi
Nabdjamel
Lazwardi
Batouri
Ashouri
Jalabahmer
Boundouki
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Bayadhi
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Table 3. Genetic similarity among studied Pistacia vera L. based on Jaccardâ&#x20AC;&#x2122;s coefficient.
Adjmi Ashouri Batouri Bayadhi JalabAhmer Lazwardi NabDjamel Marawhi Oleimi Boundouki
Adjmi 1 0.61194 0.68657 0.64179 0.71642 0.67164 0.61194 0.68657 0.80597 0.62687
Ashouri Batouri Bayadhi JalabAhmer Lazwardi NabDjamel Marawhi
Oleimi
Boundouki
1 0.61111 0.52727 0.48333 0.53571 0.49091 0.47458 0.58333 0,43103
1 0.71429
1
1 0.53448 0.54098 0.46774 0.5 0.4375 0.6129 0.51724
1 0.71698 0.76 0.71429 0.61818 0.76364 0.63462
1 0.72222 0.64815 0.70909 0.82143 0.8
1 0.65385 0.71698 0.73684 0.67308
1 0.61111 0.72727 0.72917
1 0.72414 0.66038
1, Adjmi; 2, Ashouri; 3, Batouri; 4 , Bayadhi; 5, Jalab ahmer; 6, Lazwardi; 7, Nab djamel; 8, Marawhi; 9, Oleimi; 10, Boundouki.
Jalabahmer
1,5
1 Batouri
Lazw ardi 0,5
Nabdjamel
Component 3
Oleimi Boundouki -3
-2,5
-2
-1,5
-1
-0,5
0,5
1
1,5
-0,5 Adjmi Maraw hi -1
Bayadhi
-1,5
-2 Ashouri -2,5
-3 Component 2
Figure 3. Relationships among the pistachio genotypes revealed by principal component analysis based on ISSR genetic similarity.
vegetative reproduction. REFERENCES Ahmadi Afzadi M, Seyed Tabatabaei BE, Mohammadi SA, Tajabadipur A (2007). Comparison of genetic diversity in species and cultivars of
pistachio (Pistacia vera L.) based on amplified fragment length polymorphism marker. Iran. J. Biotechnol . 5: 147-152. Basha. AI, Padulosi S, Chabane K, Hadji-Hasan A, Dulllo E, Pagnota MA, Porceddu E (2007). Genetic diversity of Syrian pistachio (Pistacia vera L.) varieties evaluated by AFLP markers. Genet. Resour. Crop Evol. 54: 1807-1816. Doyle JJ and Doyle JL (1987). A rapid isolation procedure for small quantities of fresh leaf tissue. Phytochemistry Bulletin, 19: 11-15.
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Doyle JJ, Doyle JL (1990). Isolation of plant DNA from fresh tissue. Focus 12:13-15. Ehsanpour AA, Tavassoli M, Arab L (2008). Sex determination of P vera l. using issr markers. Malaysia Appl. Biol. 37(2): 25-28. Fang DQ, Roose ML, Krueger RR, Federici CT (1997). Fingerprinting trifoliate orange germplasm accessions with isozymes, RFLPs and inter-simple sequence repeat markers. Theor. Appl. Genet. 95: 211219. Faostat (2006). FAOSTAT Database. FAO statistics database on The World Wide Web. http://apps.fao.org (accessed December 2006). Golan-Goldhirsh A, Barazani O, Wang ZS, Khadka DK, Saunders JA, Koatiukovsky V, Rowland LJ (2004). Genetic relationships among Mediterranean Pistacia evaluated by RAPD and AFLP markers. Plant Syst. 246: 9-18. Hansen M, Hallena C, Ell T (1998). Error rates and polymorphism frequencies for three RAPD protocols. Plant Mol. Biol. Rep. 16: 139146. Hormaza JI, Pinney K, Polito VS (1998). Genetic diversity of pistachio (Pistacia vera. Anacardiaceae) germplasm based on Randomly Amplified Polymorphic DNA (RAPD) markers. Econ. Bot. 52: 78-87. Hormaza JI, Dollo L, Polito VS (1994). Identification of a RAPD marker linked to sex determination in Pistacia vera using segregant analysis. Theor. Appl. Genet. 89: 9-13. Ila Hb, Kafkas, S, Topaktas M (2003). Chromosome numbers of Four (Anacardiaceae) species. J. Hort. Sci. Biotechnol. 78: 35-38. Jaccard P (1908). Nouvelles recherches sur la distribution. florale. Bull. Soc. Vaud Sci. Nat. 44: 223-270. Jones CJ, Edwards KJ, Castrglione S, Winfield MO, Sale F, Van de Wiel C, Bredemeijer G, Buiatti M, Maestri E, Malcevshi A, Marmiroli N, Aert R, Volckaert G, Rueda J, Linacero R, Vazquez A, Karp A (1997). Reproducibility testing of RAPD, AFLP and SSR markers in plants by a network of European laboratories. Mol. Breed. 3: 381390. Kafkas S, Ozkan HBE, Acar I, Atli HS, Koyoncu S (2006). Detecting DNA polymorphism and genetic diversity in a wide germplasm: comparison of AFLP, ISSR, RAPD markers. Am. Soc. Hort. Sci. 131: 522-529. Kafkas S, Perl-Treves R (2002). Interspecific relattionships in Pistacia based on RAPD fingerprinting. Hort. Sci. 371: 168-171. Karp A, Kresovich S, Bhat KV, Ayada WG, Hodgkin T (1997). Molecular tools in plant genetic resources conservation: a guide to the technologies. IPGRI technical bulletin no 2. International Plant Gen. Res. Inst. Rome. Italy. Katsiotis A, Hagidimitriou M, Drossou A, Pontikis C, loukas M (2003). Genetic relationship among species and cultivars of Pistacia using RAPDs and AFLPs. Euphytica, 132: 279-286. Mehlenbacher SA (2003). Progress and prospects in nut breeding. Acta Hortic. 622: 57-79.
Mirzaei S, Bahar M, Sharifnabi B (2005). A phylogenetic study of Iranian wild pistachio species and some cultivars using RAPD markers. Acta Hortic. 726: 39-43 Nagaoka T, Ogihara Y (1997). Applicability of inter-simple sequence repeat polymorphisms in wheat for use as DNA markers in comparison to RAPD and RFLP. Rohlf FJ (1998). Ntsys-pc: Numerical taxonomy and multivariate analysis system. Version 2.0. Department of ecology and evolution. State University of New York. Sambrook J, Fritsch EF, Maniatis T, (1989). Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Virk PS, Zhu J, Newbury HJ, Bryan GJ, Jackson MT, Ford-Liodl BV (2000). Effectiveness of different classes of molecular markers for classifying and revealing variations in rice (Oryza sativa) germplasm. Euphytica, 112: 275-284. Whitehouse WE (1957). The pistachio nut. A new crop for the Western United States. Econ. Bot. 11: 281-321. Williams JGK, Kubelik AR, Levak KJ, Rafalski JA, Tingey SV (1990). DNA polymorphism amplification by arbitary primers is useful as genetics markers. Nucleic Acids Res. 18: 6531-6535. Wolfe AD, Liston A (1998). Contributions of the polymerase chain reaction to plant systematics. In: Molecular Systematics of Plants II: DNA Sequencing (eds Soltis DE, Soltis PS and Doyle JJ). Kluwer, N. Y. pp. 43-86. Ziekiewicz E, Rafalski A, Labuda D, (1994). Genome fingerprinting by simple sequence repeat (SSR)-anchore PCR amplification. Genomics, 20: 176-183. Zohary M (1952). Amorphological study of the genus Pistacia. Palestine J. Bot. 5: 187-196.
African Journal of Biotechnology Vol. 11(29), pp. 7361-7365, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.3380 ISSN 1684â&#x20AC;&#x201C;5315 Š 2012 Academic Journals
Full Length Research Paper
rDNA internal transcribed spacer sequence analysis of Lycoris Hert. Miaohua Quan*, Lijun Ou, Chaowen She, Xianjin Wu and Dongming Chen Department of Life Science, Huaihua University, Key Laboratory of Hunan Province for Study and Utilization of Ethnic Medicinal Plant Resources, Key Laboratory of Hunan Higher Education for Hunan-western Medicinal Plant and Ethnobotany, Huaihua, 418008, China. Accepted 8 March, 2012
The interspecific relationships of Lycoris species were studied by internal transcribed spacer (ITS) sequences. ITS fragments of 14 species were amplified, sequenced and analysed. The results showed that ITS sequences of 14 species were different from each other and the ITS lengths of 14 species were about 652 bp. The GC content of ITS2 sequences was bigger than that of ITS1. Clustering results based on ITS sequences showed that Lycoris species could be divided into three clades. The classification was basically consistent with those of karyotype and morphology. This paper suggested that the likelihood of hybrid origin of Lycoris species was supported and ITS could be used as a good molecular marker to identify plants of Lycoris. Key words: Lycoris Hert., internal transcribed spacer (ITS), molecular taxonomy, interspecific relationship.
INTRODUCTION The genus Lycoris Herb. belongs to the family Amaryllidaceae, and it is mainly distributed in China and Japan. It has about 20 species in the world and about 15 species and two varieties in China, which are mainly distributed in the south of the Yangtze River, especially in warm regions (Xu et al., 1985; Yuan et al., 2008). Lycoris spp. show that its bulb has important medicinal value and exploitation-utilization prospects with rich galantamine, lycorine and other alkaloids (Xie et al., 2007). In Lycoris, hybridization has been proved to be one of the important modes of speciation (Kurita and Hsu, 1996). And development of leaves of Lycoris does not coincide with its flowering during its growth and development. It is difficult to identify the species with only morphological features. Therefore, it is very important to identify accurately Lycoris species by means of alternative method, such as using the techniques of karyology and the molecular taxonomy. The interspecific relationships and identification of Lycoris species were performed by example cytology (Zhou et al., 2005), random amplified polymorphic DNA (RAPD) (Zhang et al., 2002) and inter
simple sequence repeat (ISSR) makers (Yuan et al., 2007). The karyotype studies on Lycoris showed that chromosomes of Lycoris were classified into three types: 1) chromosomes with constrictions in median region (M); 2) chromosomes with constrictions in terminal region (T); and 3) chromosomes with constrictions in subterminal region (ST) (Zhao et al., 2008). Some information on classification of Lycoris was provided, but the origin and relationship of Lycoris species are not clear (Ma et al., 2004). The internal transcribed spacer (ITS) region of the 18S-5.8S-26S nuclear ribosomal DNA (nrDNA) has been commonly used for phylogenetic inference in plants. Two ITS regions, ITS1 and ITS2, generally evolve more rapidly than coding regions and have been shown to be equally informative, and able to differentiate between closely related species (Baldwin, 1992; Christopher et al., 2009; Vijaykumar et al., 2010; Yan et al., 2010). In this study, the genetic polymorphisms and relationships of 14 species in Lycoris were evaluated by using ITS fragment in order to provide theoretical support for the phylogenetic relationship and identification of Lycoris resources. MATERIALS AND METHODS
*Corresponding author. E-mail: hhqmh100@163.com. Tel: +86 745 2851055
A total of 14 species of Lycoris, three individuals per species, from
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Table 1. Source of Lycoris.
S/N
Species
Chromosome number*
1 2 3 4 5 6 7
Lycoris straminea Lindl. Lycoris anhuiensis Y. Hsu & Q. J. Fan Lycoris aurea (L’ Her.) Herb. Lycoris caldwellii Traub. Lycoris chinensis Traub. Lycoris haywardii Traub. Lycoris houdyshelii Traub.
3M+5T+11ST 6M+10T 6M+10T 6M+10T+11ST 6M+10T 22ST 3M+5T+22ST
8
Lycoris incarnata Comes ex C. Sprenger
4M+3T+22ST+1m
9
Lycoris longituba Y. Hsu & Q. J. Fan
6M+10T
10
Lycoris longituba var. flava Y. Hsu & X. L. Huang
6M+10T
11
Lycoris radiata (L’ Her.) Herb.
22ST
12
Lycoris rosea Traub & Moldenke
22ST
13
Lycoris sprengeri Comes ex Baker
22ST
14
Lycoris squamigera Maxim
6M+10T+11ST
*Chromosome number (Wu et al., 2007; Zhao et al., 2008).
Yuanling, Zhongfang, Hangzhou botanical garden and Nanjing botanical garden Mem Sun Yat-sen (Table 1) were used. Each species were planted in 3 plots with size of 2 × 5 m and grown at normal fertilization and watering condition. Total genomic DNA was extracted from young leaves according to a modified DNA extraction procedure reported in Sharpe et al. (1989). ITS primers were chosen according to White (1990): 5’TCCTCCGCTT ATTGA TAT GC -3’ and 5’-GGAAGGTAAAAGTC AAGG-3’. PCR were performed in 50 μl reaction system containing 5.0 mm3 10 × PCR buffer, 1.0 mm3 10 mmol.L-1 dNTPs, 1.0 mm3 50μmol.L-1 primer, 1.5 U Taq enzyme and 40 ng template under the following conditions: 95°C denaturation for 5 min, followed by 35 cycles of 94°C denaturation for 45 s, 56°C annealing for 45 s and 72°C extension for 45 s and a final extension at 72°C for 10 min (Wu et al., 2007; Yuan et al., 2008). The PCR products were fractionated on 1% agarose gel, and the gel images were obtained with the GelLogic 100 image system. The target fragments were isolated from the agarose gel under UV radiation, reclaimed and purified with the reagent kit (Tiangel midi purification kit), and then directly sequenced. Sequence analysis was performed with Dnaman, Garli and MEGA 5.05. Unweighted pair group method with arithmetic mean (UPGMA) was used to make cluster analysis by soft MEGA 5.05 (Felsenstein, 1989; Tamura et al., 2011).
Genetic distance of Liquors The genetic distances of 14 species in Lycoris were relatively small, from 0.001 to 0.066, their average was 0.04154. Lycoris haywardii and Lycoris caldwellii, Lycoris sprengeri and Lycoris caldwellii had the largest genetic distances with 0.066. Lycoris longituba and Lycoris longituba var. flava had the shortest genetic distance with 0.001(Table 3). Phylogenetic tree with bootstrap method The phylogenetic tree constructed on the basis of the ITS sequences showed that 14 species were divided into three clades: clade I with Lycoris longituba, Lycoris longituba var. flava, Lycoris anhuiensis, Lycoris aurea and Lycoris chinensis; clade II with Lycoris radiata, Lycoris haywardii, Lycoris rosea and Lycoris sprengeri; clade III with Lycoris caldwellii, Lycoris straminea, Lycoris houdyshelii, Lycoris incarnata and Lycoris squamigera (Figure 1).
RESULTS Length and GC content of ITS sequences
DISCUSSION
The ITS lengths of 14 species were about 652 bp. The internal transcribed spacer 1 (ITS1) and internal transcribed spacer 2 (ITS2) sequences were about 235 and 254 bp, respectively. GC contents of Lycoris species changed slightly, of which ITS1 and ITS2 were 65.7 to 69.9% and 70.8 to 73.6%, respectively. The GC content of ITS2 sequences was larger than that of ITS1 sequences (Table 2).
ITS sequence had more informative sites and it was widely applied to the fields of intraspecific variation and interspecific relationships of plants in recent years. This study showed that the ITS sequences of 14 species of Lycoris were different from each other and they were divided into three branches (Figure 1), which were consistent with the analysis of chromosome number and karyotype. L. chinensis, L. aurea, L. anhuiensis, L.
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Table 2. Length and GC content of ITS sequences.
species Lycoris straminea Lindl. Lycoris anhuiensis Y. Hsu & Q. J. Fan Lycoris aurea (Lâ&#x20AC;&#x2122; Her.) Herb. Lycoris caldwellii Traub. Lycoris chinensis Traub. Lycoris haywardii Traub. Lycoris houdyshelii Traub. Lycoris incarnata Comes ex C. Sprenger Lycoris longituba Y. Hsu & Q. J. Fan Lycoris longituba var. flava Y. Hsu & X. L. Huang Lycoris radiata (Lâ&#x20AC;&#x2122; Her.) Herb. Lycoris rosea Traub & Moldenke Lycoris sprengeri Comes ex Baker Lycoris squamigera Maxim
ITS Length 652 653 653 653 652 653 652 652 653 653 653 653 653 652
Length 235 235 235 235 235 236 236 236 235 236 235 236 235 236
ITS1 GC content (%) 66.8 68.7 68.5 67.2 66.8 69.9 67.4 65.7 68.1 68.3 68.9 69.9 69.8 66.1
Length 254 254 255 255 254 254 253 253 255 254 255 254 255 253
ITS2 GC content (%) 72.0 72.4 73.0 71.0 72.0 70.8 73.6 72.8 72.2 72.0 72.2 70.8 71.0 72.8
Table 3. The pairwise distance of Lycoris.
Parameter L. straminea L. anhuiensis L. aurea L. caldwellii L. chinensis L. haywardii L. houdyshelii L. incarnata L. longituba L.longituba var. flava L. radiata L. rosea L. sprengeri L. squamigera
1 1.000 0.049 0.053 0.019 0.054 0.061 0.015 0.017 0.049 0.049 0.056 0.056 0.058 0.014
2
3
4
5
6
7
8
9
10
11
12
13
14
1.000 0.030 0.056 0.030 0.051 0.051 0.052 0.006 0.006 0.051 0.049 0.048 0.052
1.000 0.057 0.006 0.044 0.049 0.054 0.023 0.023 0.041 0.043 0.043 0.054
1.000 0.059 0.066 0.019 0.022 0.056 0.056 0.061 0.064 0.066 0.022
1.000 0.048 0.051 0.056 0.023 0.023 0.043 0.044 0.044 0.056
1.000 0.056 0.063 0.048 0.048 0.015 0.005 0.006 0.059
1.000 0.017 0.051 0.051 0.048 0.054 0.056 0.014
1.000 0.052 0.052 0.057 0.058 0.059 0.003
1.000 0.001 0.048 0.046 0.044 0.052
1.000 0.048 0.046 0.044 0.052
1.000 0.017 0.019 0.054
1.000 0.002 0.054
1.000 0.056
1.000
longituba and L. longituba var. flava were classified into clade I with the same chromosome numbers and karyotype (6M+10T). They had leaves in spring except L. aurea which had leaves in autumn. The results were also consistent with the analysis of cytology (Deng and Zhou, 2005) and comparative anatomy (Zhou et al., 2006). L. anhuiensis and L. longituba had close genetic relationship, the evidences of morphology (Zhou et al., 2005) and RAPD markers (Zhang et al., 2002) supported it. L. sprengeri, L. rosea, L. haywardii and L. radiata were classified into clade II with the same chromosome number and karyotype (22ST), and had leaves in autumn. The flower color of L. radiata and L. rosea was red, that of L. sprengeri and L. haywardii was red and blue, and these were consistent with the analysis of
morphology, cytology and molecular markers (Yuan et al., 2008; Kurita, 1987; Zhou et al., 2005). L. squamigera (6M+10T+11ST), L. incarnate (4M+3T+22ST+1m), L. houdyshelii (3M+5T+22ST), L. straminea (3M+5T+11ST) and L. caldwellii (6M+10T+11ST) were classified into clade III, their karyotype was a mix of chromosomes with constrictions in median, terminal and subterminal region (M+T+ST), but their chromosome number had big variation. Hybridization in Lycoris is a very common phenomenon. Hybrid played a key role in the formation of Lycoris species (Liu and Hsu, 1990). Although, the origin and relationship of Lycoris species are not clear, seven diploid species among them were considered to be progenitors of the other species on the basis of
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Figure 1: Phylogenetic tree of Lycoris based on ITS sequences with bootstrap method
cytological studies and hybridization results (Hsu et al., 1994; Ma et al., 2004). Bose (1963) and Kurita (1987) showed that the 2n=19 (such as L. straminea and L. albiflora) in Lycoris was diploid hybrids of 2n=16 and 2n=22, 2n=27 (such as L. caldwellii and L. squamigera) was hybrids of gametes not subtrahend of 2n=16 and normal gametes of 2n=22. From the results of this study, it is thus supposed that the species of clade III could be hybrids of diploid species of the other clades. The likelihood of hybrid origin of Lycoris species was supported.
ACKNOWLEDGEMENTS This project was supported by the Innovation Platform Foundation of the Higher Education Institutions of Hunan Province (No. 11K051) and the Planned Science and Technology Project of Hunan Province, China (No. 2009FJ2008).
REFERENCES Baldwin BG(1992). Phylogenetic utility of the internal transcribed spacers of the nuclear ribosomal DNA in plants. An example from the Compositae. Mol. Phylo. Evol. 1: 3-16. Bose S (1963). A new chromosome number and karyotype in L.radiata. Nature, 197(4873): 1229-1230. Christopher HJ, Kenneth JS, Harvey EBJ (2009). Evolutionary relationships, interisland biogeography, and molecular evolution in the Hawaiian violets (Viola: Violaceae). Am. J. Bot. 96: 2087-2099.
Deng CL, Zhou J (2005). A cladistic analysis of Lycoris (Amaryllidaceae). Bull. Bot. Res. 25(4): 393-399. Felsenstein J (1989). Phylip-phylogeny inference package. Cladistics, 5: 164-166. Hsu PS, Kurita S, Yu ZZ, Lin JZ (1994). Synopsis of the genus Lycoris (Amaryllidaceae). Sida, 16: 301-331. Kurita S (1987). Chromosome evolution in Lycoris. Proc. Jpn. Soc. Plant. Tax. 4: 8-9. Kurita S, Hsu PS (1996). Hybrid complex in Lycoris, Amaryllidaceae. Am. J. Bot. 83: p. 207. Liu Y, Hsu BS (1990). Mechanism of sterility of diploid hybrid in genus Lycoris. Acta Agric. Shanghai, 6: 27-30. Ma B, Ogawa T, Tarumoto I (2004). Genetic segregation of allozymes in selfed progenies of diploid Lycoris species (Amaryllidaceae). Sci. Rep. Grad. Sch. Agric. Biol. Sci. Osaka Pref. Univ. 56: 17-22. Sharpe PJ, Chao S, Desai S, Gale MD (1989). This isolation, characterization and application in the Triticeae of a set of wheat RFLP probes identifying each homologous chromosome arm. Theor. Appl. Gen. 78: 342-348. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28(10): 2731-2739. Vijaykumar A, Saini A, Jawali N (2010). Phylogenetic analysis of Subgenus vigna species using nuclear Ribosomal RNA ITS: Evidence of hybridization among vigna unguiculata subspecies. J. Hered. 101: 177-188. White TJ, Bruns T, Lee S, Taylor JW (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ eds. PCR protocols: a guide to methods and applications. California: Academic Press. pp. 315-322. Wu L, Lu YJ, Shi SD, Fu CX (2007). Analysis of Inter-species relationship of Lycoris by use of ITS sequence. Subtrop. Plant Sci. 36(1): 31-35. Xie J, Tan F, Feng W, Chen B (2007). Advances in studies on classification, identification,medicinal ingradients, and biotechnological application of plants in Lycoris Herb. Chin. Tradit.
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Herb. Drugs. 38(12): 1902-1905. Xu Y, Hu ZB, Huang XL, Fan GJ (1985). Flora reipublicae popularis sinicae (vol.16, book 1). Beijing: Science Press: 16-27. Yan J, Deng J, Zhou CJ, Zhong BY, Hao F (2010). Phenotypic and molecular characterization of madurella pseudomycetomatis sp. nov. a novel opportunistic fungus possibly causing black-grain mycetoma. J. Clin. Microbiol. 48: 251-257. Yuan JH, Sun S, Peng F, Feng X, Xia B (2007). Comparison between ISSR and RAPD markers in genetic diversity of plants in Lycoris Herb. Chin. Tradit. Herb. Drugs, 38(10): 1555-1561. Yuan JH, Sun S, Peng F, Feng X, Zheng YH, Xia B (2008). Genetic variations in trnL-F sequence and phylogenetic clustering of Lycoris species. Chin. J. Chin. Mater. Med. 33(13): 1523-1527. Zhang L, Cai YM, Zhu GQ, Zou HY, Huang MR, Wang MX (2002). Analysis of the inter-species relationships on Lycoris(Amaryllidaceae)by use of RAPD. Acta Genet. Sin. 29(10): 915-921.
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Zhao TR, Shi YT, Cai JG (2008). The headway of research in Lycoris. North. Hortic. 4: 65-69. Zhou SB, Luo Q, Li JH, Wang Y (2006). Comparative anatomy of leaves in 12 species of Lycoris(Amaryllidaceae). Acta Bot. Yunnanic, 28(5): 473-480. Zhou SB, Yu BQ, Luo Q, Qin WH, Wang Y (2005). Pollen morphology of Lycoris Herb. and its taxonomic significance. Acta Heroic. Sin. 32(5): 914-917.
African Journal of Biotechnology Vol. 11(29), pp. 7366-7374, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.3647 ISSN 1684â&#x20AC;&#x201C;5315 Š 2012 Academic Journals
Full Length Research Paper
Genetic evaluation of domestic walnut cultivars trading on Korean tree markets using microsatellite markers Ho Bang Kim1#*, Ji Hyun Jeon1#, A Reum Han1, Yi Lee2, Sung-Soo Jun3, Tae-Houn Kim4, GunHyoung Cho1 and Phun Bum Park5* 1
Life Sciences Research Institute, Biom edic Co., Ltd., Bucheon 420-852, Republic of Korea. Department of Industrial Plant Science and Technology, Chungbuk National University, Cheongju 361-763, Republic of Korea. 3 School of General Education, Institute for Scientifically Able Youth, Kyungwon University, Seoungnam 461-701, Republic of Korea. 4 Department of PrePharmMed, College of Natural Sciences, Duksung Women's University, Seoul 132-714, Republic of Korea. 5 Department of Bioscience and Biotechnology, University of Suwon, Hwaseong 445-743, Republic of Korea.
2
Accepted 27 January, 2012
Walnut (Juglans regia L.) is regarded as a healthy food because of its high nutritional composition and various health benefits. Although several walnut cultivars are being actively traded for domestic plantation or ornament in Korea, no particular effort has been made to evaluate genetic quality management of walnut cultivars in domestic tree markets. In this study, as an effort to evaluate the status of walnut seedling trade, we collected walnut seedlings of diverse cultivars from domestic tree markets and several locations in Korea and performed genotype analysis for the collections using microsatellite markers (76 individuals belonging to eight domestic cultivars). We used 12 markers that were previously reported to be informative and polymorphic in walnuts. The number of alleles that was detected from the collections using these markers ranged from 6 to 16, with heterozygosity values ranging from 0.03 to 0.75. Dendrogram revealed that the domestic walnut cultivars trading on tree markets could be genotypically distinguished from various foreign cultivars. However, genotyping data also showed that individual plants belonging to identical cultivars were sporadically distributed on the dendrogram, indicating that walnut cultivars trading on domestic tree markets seem to be poorly managed. Keywords: Walnut (Juglans regia L.), microsatellite markers, tree market, seedling trade, domestic cultivars, foreign cultivars, cultivar management. INTRODUCTION Walnut (Juglans regia L.), belonging to the family Juglandaceae, is a tall deciduous broadleaf tree. It is widely naturalized in temperate and tropical regions including Asia (western, Caucasus, middle and tropical) and southeastern Europe. It has various common names
*Corresponding author. E-mail: hobang@ibiomedic.co.kr, pbpark@suwon.ac.kr. Tel: +82-32-218-1515, +82-31-220-2236. Fax: +82-32-218-1517, +82-31-220-2519.
#These authors contributed equally to this work.
including Persian walnut and English walnut. It is also prized as a multipurpose species, including ornamental,food (nut and oil) for humans and wildlife, industrial materials (dyestuff and timber) and folklore medicines (United States Department of AgricultureAgricultural Research Service, Germplasm Resources Information Network; http://www.ars-grin.gov/). Since the first introduction of a walnut cultivar into Korea from China approximately 700 years ago, it has become widely cultivated in the south of Pyeongtaek, Wonju and Gangneung provinces (http://www.nature.go.kr/). In Korea, walnuts are widely used as ingredients in various food types including drinks and desserts.
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Microsatellites are sequences made up of a single sequence motif usually not more than six bases long and tandemly repeated. Other various names have been used to describe the tandemly repeated sequences, including simple sequence repeats (SSRs) and short tandem repeats (STRs) (Hancock, 1999; Ellegren, 2004). Microsatellites appear to be more or less uniformly distributed throughout eukaryotic genomes, showing high allelic diversity due to variable numbers of the tandem repeats, and are inherited in a codominant fashion (Powell et al., 1996). Various approaches have been used to explore genetic diversity and relationships among species or cultivars, including isozymes (Ouji et al., 2011), restriction fragment length polymorphism (RFLP) (Fjellstrom et al., 1994), randomly amplified polymorphic DNA (RAPD) (Ku et al., 2011) and inter-simple sequence repeat (ISSR) (Yang et al., 2007) markers. Among them, microsatellite markers are characterized by high polymorphism, reproducibility, and an easy and cost-effective manner (Powell, 1996). Microsatellite markers are now widely used for various genetic analyses including cultivar identification, gene flow and parentage analysis, genome mapping, and genetic characterization of germplasm (Ellegren, 2004). As walnut is accepted as a health-improving food because of its high nutritional composition and excellent antioxidant effects, the market size of walnut products is rapidly increasing in Korea. Although various walnut cultivars for plantation or ornament are being traded in domestic tree markets, no practical efforts have been made to evaluate genetic quality management of walnut cultivars trading on domestic tree markets. Nuclear microsatellite markers have been developed for the genus Juglans, including J. regia L. (Woeste et al., 2002; Dangl et al., 2005; Hoban et al., 2008), and used for cultivar identification, paternity analysis, gene flow, genetic variation and population structure analysis (Dangl et al., 2005; Bai et al., 2007; Bai et al., 2010; Gunn et al., 2010). In this study, we performed genotype profiling for domestic walnut cultivars using previously developed microsatellite markers for two purposes: 1) to know whether it is possible to genetically distinguish domestic walnut cultivars from foreign cultivars, and 2) to know whether genetic quality management of walnut seedlings trading on domestic tree markets is under tight control. MATERIALS AND METHODS Plant material and extraction of genomic DNA Samples were collected from 76 individuals of eight domestic cultivars of J. regia L. Domestic cultivars and the number of plants (in parentheses) used in this study are as follows: Bong Hwa (10), Bong Hwang (10), Gwang Duck (7), Geum Wang (10), Hwang Ryoung (10), Sang Chon (10), Shin Nong (11) and Yo Ryoung (8). One plant belonging to the Gwang Duck group was collected from a natural monument growing in the temple Gwang Duck. Bong Hwa
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and Sang Chon cultivars were collected from local farms located in Bong Hwa-gun and Sang Chon-myeon, respectively. Seedlings of all other plants were purchased from local tree markets. Leaf or stem tissues were ground in liquid nitrogen, and then total genomic DNA was isolated using the method of Doyle and Doyle (1987). DNA quality and quantity were determined using a 1% agarose gel (Biobasic, Inc., Canada).
Polymerase chain reaction (PCR) amplification and genotyping Genomic DNA was diluted to 25 ng/µl in nuclease-free water (Biomedic Co., Ltd., Korea). PCR was conducted using an ABI 2720 thermal cycler (Applied Biosystems, USA) in a total volume of 50 µl containing 50 ng DNA, 1 x FX Taq buffer (Biomedic Co., Ltd.), 1.5 mM MgCl2, 0.2 mM each dNTP, 0.2 µM each primer, 1.25 unit FX Taq polymerase (Biomedic Co., Ltd.). The conditions for PCR amplification were as follows: 3 min for initial denaturation at 95°C, 30 cycles of 30 s at 94°C, 30 s at 56 or 60°C, 1 min at 72°C, concluding with 1 cycle of 5 min at 72°C. PCR products were separated in a 1.5% agarose gel to check PCR amplification. PCR primer sets used in this experiment are listed in Table 1. Forward primers were labeled with a virtual dye 5'-FAM (Applied Biosystems). After PCR amplification, 0.2 µl of PCR products were mixed with 9.8 µl Hi-Di formamide (Applied Biosystems) and 0.2 µl of GeneScanTM 500 LIZ® size standard (Applied Biosystems). The mixture was denatured at 95°C for 5 min and placed on ice. The amplified fragments were separated by capillary electrophoresis on an ABI 3730xl DNA analyzer (Applied Biosystems) using a 50-cm capillary with a DS-33 install standard as a matrix. We analyzed size of alleles using GeneMapper software (version 4.0; Applied Biosystems) and calculated allele number and heterozygosity.
Data analysis We calculated the observed number of alleles, effective number of alleles, observed and expected heterozygosity, and observed genotypes using PopGene (version 1.32; Yeh and Boyle, 1997). We used PowerMarker (version 3.25; Liu and Muse, 2005) to calculate polymorphic information content (PIC) (Bostein et al., 1980). A dendrogram based on the genetic distance values was constructed using unweighted pair group method with arithmetic mean (UPGMA) algorithm (Sneath and Sokal, 1973). Genetic distance was calculated using CS Cord distance (Cavalli-Sforza and Edwards, 1967).
RESULTS AND DISCUSSION To know whether it is possible to genetically distinguish domestic walnut cultivars from foreign cultivars, we merged and analyzed genotype profiling data for 76 individuals belonging to eight domestic cultivars using eight microsatellite markers with the previous allele sizing data of 12 foreign cultivars in the United States, United Kingdom and France (Dangl et al., 2005). Amplified PCR products ranged in size from 140 to 280 base pairs (bp) (Supplementary Table 1). The number of alleles detected per locus varied from 6 (WGA332) to 16 (WGA376), with an average of 11 (Table 2). The observed hetero-zygosity (Ho) of a given locus ranged from 0.16 (WGA004) to 0.61 (WGA376), with an average over all eight loci of 0.38 (Table 2). As an approach to evaluate whether genetic
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Table 1. Twelve microsatellite markers used in this study.
Locus name
Primer sequence (5'→3')
WGA002
a
WGA004
a
WGA007
a
WGA042
a
WGA045
a
WGA069
a
WGA276
b
WGA321
b
WGA331
b
WGA332
b
WGA349
b
WGA376
b
c
FAM-GACGACGAAGGTGTACGGAT GTACGGCTCTCCTTGCAGTC
Temperature (°C)
Repeat motif
Expected amplicon d size range (bp)
56
(GA)n
169
56
(GT)n(GA)n(GA)n
228-241
56
(GA)n
222
60
(GA)n
241
60
(CT)n
233
60
(GA)n
158-182
60
(TC)n
143-192
56
(GA)n
222-245
60
(GA)n
273-277
56
(CT)n
214-225
60
(CT)n
258-274
56
(GA)n
218-254
FAM-TGTTGCATTGACCCACTTGT TAAGCCAACATGGTATGCCA FAM-CAAACAAAATCCGACCGC AAACCTCGATGAGCGAAGAA FAM-GTGGGTTCGACCGTGAAC AACTTTGCACCACATCCACA FAM-TCGTTACCACCAGCACAGAG GACATAGCGAGGGGCTAGG FAM-TTAGTTAGCAAACCCACCCG AGATGCACAGACCAACCCTC FAM-CTCACTTTCTCGGCTCTTCC GGTCTTATGTGGGCAGTCGT FAM-TCCAATCGAAACTCCAAAGG GTCCAAAGACGATGATGGA FAM-TCCCCCTGAAATCTTCTCCT CGGTGGTGTAAGGCAAATG FAM-ACGTCGTTCTGCACTCCTCT GCCACAGGAACGAGTGCT FAM-GTGGCGAAAGTTTATTTTTTGC ACAAATGCACAGCAGCAAAC FAM-GCCCTCAAAGTGATGAACGT a
TCATCCATATTTACCCCTTTCG b
Primer sequences were obtained from Woeste et al. (2002); Primer sequences were obtained from Dangl et al. (2005); cForward primers were presented first; dThe expected amplicon size range is based on Woeste et al. (2002) and Dangl et al. (2005).
quality management of walnut seedlings trading on domestic tree markets is under tight control, we performed genotyping analysis using 12 microsatellite loci (Woeste et al., 2002; Dangl et al., 2005) for 76 individuals belonging to eight domestic cultivars, which are largely cultivated at walnut farms in Korea. Table 3 summarizes characteristics of 12 microsatellite markers based on 76 individuals belonging to eight domestic cultivars. Allele sizes varied from 140 to 338 bp (Supplementary Table 2). The number of alleles ranged from 4 (WGA007) to 16 (WGA045 and WGA376) across 12 microsatellite loci, with an average of 9.58. The average Ho value was 0.36, ranging from 0.03 (WGA007) to 0.75 (WGA042) (Table 3). The average Ho values obtained from two analyses (Tables 2 and 3) was much lower than the previous value (0.59) from the analysis using 47 Persian walnut accessions (Dangl et al., 2005).
This low heterozygosity at several loci is possibly attributable to careless clonal propagation (for example, grafting to rootstocks) of some domestic cultivars during seedling production. Several markers (WGA002, WGA045, WGA331, WGA376, WGA349 and WGA069) failed to make amplification from several individuals, resulting in availability values lower than one (Tables 2 and 3). Failure of PCR amplification might be attributable to mutations on the conserved sites for PCR primers flanking the tandem repeat region, possibly signifying null alleles (Dakin and Avise, 2004). Dendrograms are an efficient means of summarizing microsatellite data and can reveal relationships, including individuals with identical genotypes. A UPGMA tree was generated based on the genetic distances between 76 individuals of eight domestic cultivars and 12 foreign cultivars, including one hybrid rootstock Paradox
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Table 2. Characteristics of 8 microsatellite markers based on 8 domestic and 12 foreign cultivars.
Locus WGA004 WGA069 WGA276 WGA321 WGA331 WGA332 WGA349 WGA376
Allele frequency 0.88 0.75 0.26 0.77 0.55 0.75 0.48 0.2
a
N 88 88 88 88 88 88 88 88
No. of Obs. 88 87 88 88 83 88 86 86
b
No. of Allele 11 13 12 9 11 6 10 16
Availability 1 0.99 1 1 0.94 1 0.98 0.98
c
He 0.23 0.43 0.82 0.4 0.65 0.41 0.72 0.87
d
Ho 0.16 0.24 0.57 0.26 0.42 0.3 0.52 0.61
e
PIC 0.23 0.42 0.79 0.38 0.61 0.37 0.69 0.85
a
N, sum of 76 individuals belonging to 8 domestic cultivars and 12 foreign cultivars analyzed previously (Dangl et al., 2005); Availability is defined as 1-Obs/N; Obs., number of observations; N, number of individuals sampled; CHe, expected heterozygosity; d Ho, observed heterozygosity; ePIC, polymorphism information content. b
Table 3. Characteristics of 12 microsatellite markers based on 8 domestic cultivars.
Locus WGA002 WGA004 WGA007 WGA042 WGA045 WGA069 WGA276 WGA321 WGA331 WGA332 WGA349 WGA376
Allele frequency 0.78 0.92 0.97 0.57 0.24 0.81 0.3 0.88 0.58 0.82 0.54 0.23
a
N 76 76 76 76 76 76 76 76 76 76 76 76
No. of Obs. 71 76 76 76 74 75 76 76 72 76 74 74
No. of allele 8 10 4 9 16 12 8 6 11 6 9 16
b
Availability 0.93 1 1 1 0.97 0.99 1 1 0.95 1 0.97 0.97
c
He 0.38 0.15 0.05 0.64 0.87 0.34 0.78 0.23 0.63 0.31 0.65 0.87
d
Ho 0.1 0.13 0.03 0.75 0.61 0.19 0.57 0.2 0.39 0.24 0.5 0.6
e
PIC 0.36 0.15 0.05 0.62 0.85 0.33 0.74 0.22 0.6 0.28 0.62 0.85
a
N, sum of 76 individuals belonging to 8 domestic cultivars; bAvailability is defined as 1 - Obs/N; Obs., number of observations; N, number of individuals sampled; CHe, expected heterozygosity; dHo, observed heterozygosity; ePIC, polymorphism information content.
'Burbank' (Juglans hindsii Ă&#x2014; J. regia) (Dangl et al., 2005). The tree clearly showed that domestic walnut cultivars are genetically different from the foreign cultivars (Figure 1). Interestingly, Lozeronne, a cultivar originating from France, showed closer genetic relationships with some individuals of domestic cultivars (especially, HR01 and SN03) than the other foreign cultivars. Several individuals belonging to Hwang Ryoung and Shin Nong cultivars possibly share pedigree with the French cultivar, Lozeronne (Figure 1). New markers need to be developed to separate Lozeronne cultivar from HR01 and SN03. Mitochondrial DNA analysis is also needed to clearly reveal the genetic relationships among Lozeronne, HR01 and SN03. Two individuals (YR02 and YR04) of Yo Ryoung cultivar were not genetically separated when we analyzed the collections with eight markers. However, we performed genotype profiling for the collections with 12 markers including WGA002, WGA007, WGA042, and WGA045, the individuals were
separated, showing a genetic distance with CS Cord value of 0.053 (Figure 2). The tree also revealed that genetic distances are widely observed even among individuals from identical domestic cultivars (Figure 1), indicating that quality management of walnut seedling trading in domestic tree markets is not under tight control. Such genetic dispersion among individuals of identical cultivars can be attributable to coarse management (for example, seedmixing) during seedling production by seed distributors. Open and cross pollination could be one explanation for the genetic dispersion, although the observed heterozygosity was not high (Tables 2 and 3). The genetic mixing among domestic walnut cultivars not only causes severe problems in the production of nuts with uniform quality, but also hinders development of walnut varieties with new traits through the conventional breeding program. Our data further suggest that microsatellite markers are a useful molecular tool for a practical management of cultivar stocks trading in tree markets.
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ParadoxBurbank Sharkey Chandler How ard Serr Sunland Cisco Franquette Placentia Marchetti Payne Lozeronne HR01 SN03 SN11 SN08 SN09 HR06 SN05 HR02 GD05 GD06 BH01 SC09 GD02 BH03 BH04 BH05 GD07 BH09 GD08 GW01 GW09 BHW07 GW04 GW05 GW03 HR09 BH10 BHW03 SC04 BHW09 GW08 HR08 YR05 BH08 GW06 BHW02 GW02 BHW05 SN01 GD04 BHW04 BHW08 BHW10 GW07 GD01 SC06 SC07 SC03 YR01 YR07 YR08 YR03 YR02 YR04 BHW06 YR06 SC05 SC10 BH02 SC01 BHW01 SC08 BH06 GW10 SN02 SC02 SN07 SN10 SN04 SN06 HR05 HR03 HR07 HR04 HR10 BH07 0.1
Figure 1. A UPGMA tree based on genetic distances between 76 individuals of 8 domestic cultivars and 12 foreign cultivars including one hybrid (Paradox Burbank), using 8 previously reported polymorphic microsatellite markers (Woeste et al., 2002; Dangl et al., 2005). YR, Yo Ryoung; BHW, Bong Hwang; GW, Geum Wang; GD, Gwang Duck; BH, Bong Hwa; SC, Sang Chon; HR, Hwang Ryoung; SN, Shin Nong.
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BH07 HR07 HR10 SN07 SN10 SN04 SN05 HR06 SC02 SN02 SN09 SN11 HR01 SN03 SN08 HR02 HR03 HR05 HR04 GW01 GW09 BHW05 GD06 SN06 GD02 SN01 BHW03 BH08 BH02 BH10 BH01 SC09 BH05 SC04 BH03 BH04 GW05 GW03 HR09 GW06 BHW02 GW02 BH09 GD01 GD07 GD08 BHW09 BHW07 GW04 GW08 HR08 YR07 YR08 YR03 YR01 YR06 YR02 YR04 BH06 GW10 SC01 BHW01 BHW06 SC05 SC03 SC10 SC06 SC08 BHW10 SC07 BHW04 GD04 BHW08 GW07 GD05 YR05 0.05
Figure 2. A UPGMA tree based on genetic distances of 76 individuals of 8 domestic cultivars, using previously reported 12 polymorphic microsatellite markers (Woeste et al., 2002; Dangl et al., 2005). YR, Yo Ryoung; BHW, Bong Hwang; GW, Geum Wang; GD, Gwang Duck; BH, Bong Hwa; SC, Sang Chon; HR, Hwang Ryoung; SN, Shin Nong. UPGMA, Unweighted pair group method with arithmetic mean.
ACKNOWLEDGMENTS This work was supported by the Industry-University Technology Cooperation Program grant, funded by Small
and Medium Business Administration (grant no. 00044103 to P. B. Park and Biomedic Co., Ltd). The authors are grateful to Dr. Chang Jae Oh (Seoul National University, Republic of Korea) for critical comments on
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the manuscript, and Ms. Karen Bird (MSU-DOE Plant Research Laboratory, Michigan State University, USA) and Mr. Chan-Hyoung Cho (Biomedic Co., Ltd., Republic of Korea) for their help in editing the manuscript. REFERENCES Bai WN, Liao WJ, Zhang DY (2010). Nuclear and chloroplast DNA phylogeography reveal two refuge areas with asymmetrical gene flow in a temperate walnut tree from East Asia. New Phytol. 188: 892-901. Bai WN, Zeng YF, Zhang DY (2007). Mating patterns and pollen dispersal in a heterodichogamous tree, Juglans mandshurica (Juglandaceae). New Phytol. 176: 699-707. Bostein D, White RL, Skolinick M, Davis RW (1980). Construction of a genetic linkage map in man using restriction fragment length polymorphism. Am. J. Hum. Genet. 32: 314-331. Cavalli-Sforza LL, Edwards AWF (1967) Phylogenetic analysis: models and estimation procedure. Am. J. Hum. Genet. 19: 233-257. Dakin EE, Avise JC (2004). Microsatellite null alleles in parentage analysis. Heredity., 93: 504-509. Dangl GS, Woeste K, Aradhya M, Koehmstedt A, Simon C, Potter D, Leslie CA, McGranahan G (2005). Characterization of 14 microsatellite markers for genetic analysis and cultivar identification of walnut. J. Am. Hort. Sci. 130: 348-354. Doyle J, Doyle J (1987). A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19: 11-15. Ellegren H (2004). Microsatellites: Simple sequences with complex evolution. Nat.ure Rev. 5: 435-445. Fejellstrom RG, Parfitt DE, McGranahan GH (1994). Genetic relationships and characterization of Persian walnut (Juglans regia L.) cultivars using restriction fragment length polymorphisms (RFLPs). J. Am. Soc. Hort. Sci. 119: 833-839. Gunn BF, Aradhya M, Salick JM, Miller AJ, Yongping Y, Lin L, Xian H (2010). Genetic variation in walnuts (Juglans regia and J. sigillata; Juglandaceae): species distinctions, human impacts, and the conservation of agrobiodiversity in Yunnan, China. Am. J. Bot. 97: 660-671. Hancock JM (1999). Microsatellite and other simple sequences: genomic context and mutational mechanisms. In Goldstein DB, Schlรถtterer C, eds, Microsatellites: Evolution and applications. Oxford University Press, New York, USA. pp.1-9. Hoban S, Anderson R, McCleary T, Schlarbaum S, Romero-Severson J (2008). Thirteen nuclear microsatellite loci for butternut (Juglans cinerea L.). Mol. Ecol. Resour. 8: 643-646.
Ku Y-B, Oh HK, Chun YJ, Cho K-H (2011). High genetic differentiation in endangered Sedum ussuriense and implications for its conservation in Korea. J. Plant Biol. 54: 262-268. Liu K, Muse SV (2005). PowerMarker: Integrated analysis environment for genetic marker data. Bioinformatics, 21: 25-32. Ouji A, Suso MJ, Rouaissi M, Abdellaaoui R, Gazzah ME (2011). Genetic diversity of nine faba bean (Vicia faba L.) populations revealed by isozyme markers. Genes Genomics, 33: 31-38. Powell W, Morgante M, Andre C, Hanafey M, Vogel J, Tingey S, Rafalski A (1996). The comparison of RFPL, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Mol. Breed. 2: 225238. Sneath PHA, Sokal RR (1973). Numerical taxanomy: The principles and practice of numerical classification. Freeman, San Francisco, USA. pp.1-573. Woeste K, Burns R, Rhodes O, Michler C (2002). Thirty polymorphic nuclear microsatellite loci from black walnut. J. Hered. 93: 58-60. Yang Y-X, Wu W, Zheng Y-L, Chen L, Liu R-J, Huang C-Y (2007). Genetic diversity and relationships among safflower (Carthamus tinctorius L.) analyzed by inter-simple sequence repeats (ISSRs). Genet. Resour. Crop. Eviron. 54: 1043-1051. Yeh FC, Boyle TJB (1997). Population genetic analysis of co-dominant and dominant markers and quantitative traits. Belgian J. Bot. 129: p. 157.
African Journal of Biotechnology Vol. 11(29), pp. 7373-7377, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB10.1826 ISSN 1684–5315 © 2012 Academic Journals
Full Length Research Paper
Full-length enriched multistage cDNA library construction covering floral bud development in Populus tomentosa Xin-Min An, Dong-Mei Wang, Ze-Liang Wang, Mei-Xia Ye and Zhi-Yi Zhang* National Engineering Laboratory for Tree Breeding; Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants, Ministry of Education; Tree and Ornamental Plant Breeding and Biotechnology Laboratory of State Forestry Administration, Beijing Forestry University, Beijing 100083, P. R. China. Accepted 12 August, 2011
Flowering involves expression of a suite of genes associated with floral development. The genome of the Chinese white poplar (Populus trichocarpa) was sequenced because of its importance as a model tree for genetic studies as well as being an economically important woody plant. However, information on expressed genes involved in poplar floral bud development is insufficient to allow annotation of genes and use of the genomic information. To isolate and characterize genes involved in flowering of Populus tomentosa, floral bud samples were collected at different developmental stages from floral bud initiation to flower maturity, and full-length enriched cDNA libraries from both male and female floral buds were constructed. The results of titer analysis showed that the titer of the female and male 5 5 primary libraries were 8.00 × 10 and 7.20 × 10 pfu/ml, respectively, and the titer of the amplified 8 8 libraries were 2.60 × 10 and 2.56 × 10 pfu/ml, respectively. The combination ratio reached 90% and the insert size was 400 to 2000 bp. The results indicated that cDNA libraries were successfully constructed. Key words: cDNA library, floral bud, flowering, Populus tomentosa.
INTRODUCTION Flowering is an important event in the life cycle of all flowering plants. Current knowledge on the molecular mechanism underlying flower induction and development mostly comes from extensive studies on the model plant Arabidopsis thaliana (Tan et al., 2007). A host of genes involved in flowering have been isolated and characterized in Arabidopsis. The model tree, poplar, differs markedly from Arabidopsis in floral traits. Poplar is a perennial tree with a long juvenile phase and lifespan (Braatne et al., 1996), and it flowers annually or seasonally during the reproductive phase after flowering for the first time (Yuceer et al., 2003). The reiterating
*Corresponding author. E-mail: zhangzy@bjfu.edu.cn. Tel: 8610-62338502. Fax: 86-10-62338502. Abbreviations: DMSO, Dimethyl sulfoxide; LD-PCR, longdistance polymerase chain reaction; dscDNA, double-strand complementary DNA; pfu, plaque-forming unit.
developmental cycles between vegetative and reproductive growth periods are also absent in Arabidopsis (Boss et al., 2004). The male and female flowers are borne on separate trees from axillary inflorescences, but occasionally, trees are monoecious. Instead of four concentric whorls of organs, poplar flowers have only two whorls, comprising a reduced perianth cup surrounding either the stamens or carpels (Boes and Strauss, 1994; Sheppard, 1997; Rottmann et al., 2000). Clearly, the molecular mechanism regulating floral induction and development in poplar is more complex than that in Arabidopsis. Chinese white poplar (Populus tomentosa Carr.) is a Chinese-native tree species in Populus section Leuce that is fast-growing and produces high-quality wood in the north of China. It currently plays an important role in forest production and forest reclamation, and will be important for biomass and biofuel use in the near future. However, the catkins produced by male and female P. tomentosa trees seriously impact on the environment in both urban and rural regions. Pollen from male catkins
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are allergenic for some individuals, while hairs produced by female catkins contribute to environmental pollution in rural and urban areas during spring and might be a potential fire risk, or even a vector for the spread of viruses. Therefore, it is important to regulate or control flowering of poplar. Several genes, such as PTLF, PTD, PtFT and PtAG, involved in poplar flowering have been identified (Brunner et al., 2000; Rottmann et al., 2000; Sheppard et al., 2000; Böhlenius et al., 2006). The complete P. trichocarpa genome has been sequenced (Tuskan et al., 2006). However, the information presently available on a limited number of genes involved in poplar flowering is insufficient to allow annotation of genes and to understand the genetic and molecular mechanisms underlying floral development in P. tomentosa. Construction and screening of a cDNA library is one of the most important methods for gene isolation, and it is also a potential approach to identify novel genes. In this investigation, full-length enriched cDNA libraries were constructed for P. tomentosa from male and female floral buds sampled at 15 developmental stages. This study will ultimately allow the isolation of genes expressed during the development of P. tomentosa floral buds, and will contribute to elucidation of the molecular mechanism regulating flowering in P. tomentosa.
MATERIALS AND METHODS Floral buds were collected from female and male clones of P. tomentosa trees, growing in the Beijing Forestry University Nursery, at 15 developmental stages from floral bud initiation to flower maturity (between June and March of the following year). The samples were frozen immediately in liquid nitrogen and stored at – 80°C until use.
Extraction and qualification of total RNAs The total RNAs from both male and female floral buds were extracted using a CTAB-based method as described previously (Chang et al., 1993). The extracted RNAs were pretreated with RQ1 DNase I (Promega) to remove genomic DNA contaminants. The concentration of total RNAs was measured using a SPEKOL 1300 spectrophotometer (Jena). A 1 μl sample of the extracted RNAs was electrophoresed on 1% agarose gel. Equal quantities of total RNAs from male and female floral buds at each developmental stage were mixed to examine the integrity of the RNAs and samples were electrophoresed as described earlier to measure the total RNA concentration. mRNAs were isolated and purified with the PolyATtract® mRNA Isolation System (Promega).
Construction of SMART cDNA libraries The purified mRNAs extracted from male and female floral buds were reverse-transcribed into corresponding single-strand cDNAs. Double-strand cDNAs (dscDNAs) were synthesized by longdistance polymerase chain reaction (LD-PCR) with 20 cycles, and 5 μl dscDNAs were analyzed by 1.2% agarose gel electrophoresis. A portion (50 µl) of the dscDNAs was digested with proteinase K and Sfi I, and fractions >200 bp in length were recovered using the QIAquick™ Gel Extraction Kit (QIAGEN). Finally, the fractions were
ligated into a λ TriplEx2 vector digested by Sfi I, and the cDNA ligation products were packaged in vitro into the λ phage using the Packagene Lambda DNA Packaging System (Promega). The reclaimed products were electrophoresed on a 1.1% agarose gel to examine cDNA quality.
Titering of primary and amplified libraries To examine the titer of the primary libraries, the packaged ligation products were diluted by 1:5, 1:10 or 1:20 with buffer. A 1 μl sample of the diluted products was combined with 200 μl Escherichia coli XL1-Blue overnight culture, and incubated at 37°C for 15 min. To the infected bacteria, 2 ml melted top agar was added, and the mixture was spread onto the surface of a 90 mm LB/MgSO4 agar plate. The plate was inverted and incubated at 37°C for 12 to 18 h until the phagocytes were visible. The phagocytes were counted and the titer of the primary library was calculated as: Titer (pfu/ml) = number of plaques × dilution factor The libraries were amplified according to routine methods. They were stored at 4°C for up to six months or in 7% DMSO (v/v) at 70°C for up to one year. Samples of 0, 5, 10 or 20 μl of 10 4 dilutions were used to test the titer of the amplified libraries following the earlier-mentioned method.
Determining the percentage of recombinant clones To investigate recombinant efficiency and sizes of inserted cDNA, we randomly selected 12 plaques from the plates of cDNA libraries and placed them in vials containing 20 μl SM for use as PCR templates. The 20 μl PCR reaction mixture was composed of 1 × PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2), 1 μl SM containing phages, 0.4 μl of each 10 μM sequencing primer (5' λTriplEx2: 5'-CTCCGAGATCTGGACGAGC-3'; T7: 5'TAATACGACTCACTATAGGGC-3'), and 1 U Taq DNA polymerase. Thermo cycling was performed at 94°C for 5 min, then 94°C for 20 s, 60°C for 20 s, and 72°C for 1 min for 30 cycles, then 72°C for 7 min and finally kept at 4°C. The PCR products were examined with 1.5% agarose gel electrophoresis.
RESULTS Isolation and quality of RNAs The respective integrity of RNAs from male and female floral bud samples was examined by agar gel electrophoresis, as shown in Figure 1. The relative brightness ratio of the two bands for 28S rRNA and 18S rRNA was close to 2:1, which indicated that the total RNAs from male and female floral buds had not been degraded. Spectrophotographic analysis gave OD260/OD280 values of 2.04 ± 0.02 and 2.00 ± 0.06 (Table 1), which showed that the RNAs were complete and of high integrity and purity, and met the requirements for cDNA synthesis.
Isolation of mRNAs and synthesis of dscDNA Integrity of mRNAs was analyzed separately for male and
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Figure 1. Integrated total RNAs from floral buds of P. tomentosa.
Table 1. Quality and estimated concentration of integrated total RNAs extracted from female and male floral buds of P. tomentosa.
RNA source Female floral buds Male floral buds
OD260 value 2.90 ± 0.01 1.96 ± 0.04
OD280 value 1.42 ± 0.02 0. 98 ± 0.01
OD260/OD280 value 2.04 ± 0.02 2.00 ± 0.06
OD, Optical density.
female floral bud RNAs. Electrophoretic analysis indicated that dispersion of mRNAs ranged from 400 bp to 5.0 kb (Figure 2A). The recovered dscDNAs were represented by an approximately 0.4 to 5.0 kb smear on the 1.1% agarose gel, indicating that the male and female dscDNAs were complete and did not comprise small fragments(Figure 2B).
Construction and quality estimation of cDNA libraries To ensure the highest-quality library was obtained from the cDNA, three parallel ligations of the cDNA and vector were performed, followed by a separate λ phage packaging reaction for each ligation. The average titer of 5 5 the primary libraries was 8.00 × 10 and 7.20 × 10 pfu/ml for male and female floral bud libraries, respectively. The primary libraries were used to produce the amplified 8 8 libraries, which had a titer of 2.60 × 10 and 2.56 × 10 pfu/ml for male and female floral buds, respectively. The results indicated that the titers of both primary and amplified libraries were relatively optimal and met the requirements of a SMART cDNA library. The size of the inserts ranged from 400 bp to 2.0 kb, and the percentage of inserted fragments exceeding 400 bp in length was approximately 100% (Figure 3).
DISCUSSION Construction and screening of a cDNA library is a common means of identifying transcriptional products of target genes. Generally, the percentage of mRNAs in a cell is only about 5%, although they are essential for
protein synthesis. Moreover, many genes show spatiotemporal expression patterns. The mRNAs transcribed during a certain period are used as the starting material to construct the library, and thus represent limited gene expression information. Therefore, a researcher major concern is whether the cDNA library is enriched with most of the genes expressed during the development of the tissue or organ and contains full-length cDNAs. In this study, male and female floral buds were collected over the course of the entire bud development and their total RNAs were mixed equally to construct the cDNA libraries, which ensured that most genes involved in flower development should be represented. The libraries will provide a valuable tool for future investigations of the regulation of flower development in P. tomentosa. Commonly used cDNA synthesis methods rely on the ability of reverse transcriptase (RT) to transcribe mRNA into single-stranded DNA in the first-strand reaction. The occurrence of RT termination before transcription is complete and is a very common problem, particularly for long mRNA sequences. In this study a SMART™ cDNA Library Construction Kit was employed to construct the cDNA libraries, which differentiated it from conventional studies. The SMART protocols are designed to enrich preferentially full-length cDNAs, and eliminate T4 DNA polymerase and adaptor ligation. Therefore, libraries constructed with a SMART cDNA protocol clearly contain a higher percentage of full-length clones and full-length cDNAs with complete 5' ends. The regulation of flowering is very important in poplar breeding. On one hand, promotion of early flowering is beneficial to shorten breeding cycles and, on the other hand, suppression of flowering will improve environmental quality by reducing production of seed hairs and
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Figure 2. Purification of mRNAs and synthesis of dscDNA. A: Isolation of mRNAs from integrated total RNAs of male and female floral buds. B: synthesis of dscDNA from male and female floral buds.
Figure 3. Identification of insert length of cDNA fragment in both female and male floral buds cDNA library. A: PCR products of 位 phages from male floral buds cDNA library; B: PCR products of 位 phages from female floral buds cDNA library (M, DL2000 marker; 1 to 12, PCR products of different 位 phages from male and female floral buds cDNA libraries).
pollen. However, current knowledge of the molecular mechanism regulating poplar flowering is still poor, although the complete poplar genome sequence has been published (Tuskan et al., 2006). Considering the complexity of gene interaction networks and the
specificity of gene expression in dioecious poplar, isolation and characterization of genes involved in flowering is essential. The enriched full-length cDNA libraries constructed from multiple samples and covering all floral-bud development stages will provide an
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alternative approach to obtain a comprehensive understanding of gene interactions involved in flowering of P. tomentosa.
ACKNOWLEDGEMENTS This work was supported by the Forestry Public Benefit Research Foundation (201004009), National High-tech R&D Program of China (2011AA100201) and National Natural Science Foundation of China (31170631). REFERENCES Boes TK, Strauss SH (1994). Floral phenology and morphology of black cottonwood, Populus trichocarpa (Salicaceae). Am. J. Bot. 81: 562567. Bรถhlenius H, Huang T, Charbonnel-Campaa L, Brunner AM, Jansson S, Strauss SH, Nilsson O (2006). CO/FT Regulatory Module Controls Timing of Flowering and Seasonal Growth Cessation in Trees. Science 312: 1040-1043. Boss PK, Bastow RM, Mylne JS, Dean C (2004). Multiple pathways in the decision to flower enabling, promoting, and resetting. Plant Cell 16(Suppl): S18-S31. Braatne JH, Rood SB, Heilman PE (1996). Life history, ecology and conservation of riparian cottonwoods in North America. In: Stettler RF, Bradshaw HD, Heilman PE and Hinckley TM (ed) Populus and its Implications for Management and Conservation, Part I, Chapter 3, NRC Res. Press, Natl. Res. Council of Canada, Ottawa, ON, Canada, pp57-85
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Brunner AM, Rottmann WH, Sheppard LA, Krutovskii K, DiFazio SP, Leonardi S, Strauss SH (2000). Structure and expression of duplicate AGAMOUS orthologues in poplar. Plant Mol. Biol. 44: 619-634. Chang S, Puryear J, Cairney J (1993). A simple and efficient method for isolating RNA from pine tree. Plant Mol. Biol. Rep. 11: 113-116. Rottmann WH, Meilan R, Sheppard LA, Brunner AM, Skinner JS, Ma C, Cheng S, Jouanin L, Pilate G, Strauss SH (2000). Diverse effects of overexpression of Leafy and PTLF, a poplar (Populus) homolog of Leafy/Floricaula, in transgenic poplar and Arabidopsis. Plant J. 22: 235-246. Sheppard LA (1997). PTD: a Populus trichocarpa gene with homology to floral homeotic transcription factors. PhD Dissertation, Oregon State Univ., Corvallis, USA. Sheppard LA, Brunner AM, Krutovskii KV, Rottmann WH, Skinner JS, Vollmer SS, Strauss SH (2000). A DEFICIENS homolog from the dioecious tree Populus trichocarpa is expressed in both female and male floral meristems of its two-whorled, unisexual flowers. Plant Physiol. 124(2): 627-640. Tan FC, Swain SM (2007). Functional characterization of AP3, SOC1 and WUS homologues from citrus (Citrus sinensis). Physiol. Plant. 131: 481-495. Tuskan GA, Difazio S, Jansson S, Bohlmann J, Grigoriev I, Hellsten U, Putnam N, Ralph S, Rombauts S, Salamov A (2006). The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313(5793): 1596-1604. Yuceer C, Land Jr SB, Kubiske ME, Harkess RL (2003). Shoot morphogenesis associated with flowering in Populus deltoides (Salicaceae). Am. J. Bot. 90: 196-206.
African Journal of Biotechnology Vol. 11(29), pp. 7378-7387, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.2875 ISSN 1684â&#x20AC;&#x201C;5315 Š 2012 Academic Journals
Full Length Research Paper
Isolating Barley (Hordeum vulgare L.) B1 Hordein Gene Promoter and Using Sequencing Analaysis For The Identification of Conserved Regulatory Elements By Bioinformatic Tools Kobra Nalbandi1, Bahram Baghban Kohnehrouz2*, Khalil Alami Saeed1 and Ashraf Gholizadeh3 1
Ramin Agricultural and Natural Resources University, Mollasani, Ahwaz, Iran. Department of Plant Breeding and Biotechnology, University of Tabriz, Tabriz, Iran. 3 Research Institute for Fundamental Sciences (RIFS), University of Tabriz, Tabriz, Iran. 2
Accepted 28 February, 2012
Gene expression is a complex multi-step process. For the efficient expression of foreign genes in plants, it is essential to optimize every step of the process for the plant machinery, which includes choosing suitable promoters. Promoters play the most important role in determining the temporal and spatial expression pattern and transcript level of a gene. Some strong constitutive promoters, such as cauliflower mosaic virus 35s promoter, are widely used in plant genetic engineering research. However, the expression levels of the foreign genes in all tissues are often unsatisfied, but some of such problems can be may solved by using a strong seed-specific promoter to restrict gene expression to the seed only. The aim of this article was to characterize a B1 hordein-specific promoter. The promoter region of B1 hordein gene was isolated from the genomic DNA of Walfajre and Alger barley by polymerase chain reaction. Sequence analysis showed that the cloned fragment B hordein promoter (BHP) - contained motifs like TATA box, (CA)n box, ACGT motif, AAAG motif, GCN4-like motif and Ebox, which constituted the seed-specific promoter activity. It was therefore concluded that B1 hordein promoters can be used to engineer and subsequently study stable seed specific gene expression in barley, and potentially to modify barley seeds through genetic engineering. Key words: Barley, Seed Specific Promoter, Motif, Hordein.
INTRODUCTION In most cereal species, the major seed storage proteins are prolamins, a complex group of alcohol-soluble polypeptides. The prolamin proteins in barley endosperm are usually termed hordein due to their high levels of proline and glutamine (Piston et al., 2004). The four main groups of hordein, namely the B, C, D and Y hordeins, account for approximately 50% of the total protein in the
*Corresponding author. Email: Bahramrouz@yahoo.com. Tel: +98-411-3392031. Fax:+98-411-3356003. Abbreviations: PCR, polymerase chain reaction; BHP,B Hordein Promoter.
mature grain. Hordeins B, C and D are specified by separate compound genetic loci on chromosome 5 designated Hor-2, Hor-1 and Hor-3, respectively (Blake et al., 1982; Jensen et al., 1980; Shewry et al., 1980, 1983). Bhordeins are sulfur-rich prolamins, which account for 70 to 80% of the total hordein fraction in barley. The genes encoding hordein seed storage proteins are under seedspecific developmental regulation. Synthesis of these proteins is regulated at the transcription level and continues until it comprises of up to 60 to 80% of the total protein in mature seeds (Evanns et al., 1984; Gatehouse et al., 1986). Promoters used in biotechnology are of different types according to the intended type of control of gene expression. Tissue-specific or development stage
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Figure 1. Schematic structure of B1 hordein gene promoter.
specific promoters could regulate the expression of a gene in specific tissue(s) or at certain stages of development. Thus, seed specific promoters provide an excellent system for studying the expression control of plant genes and are used in developing “transgenic” for improving seed quality. This study reports the isolation and charac-terization of a promoter sequence of B1 hordein in barley cultivated in Iran and presents a comparative analysis bybioinformatics tools.
MATERIALS AND METHODS Hordeum vulgare L. cv. Walfajre and Alger seeds (Kindly provided by professor M.Mogadam) were grown in plastic trays at a glass house until two to three leave stages in order to extract their total cellular genomic DNA. Escherichia coli strain DH5α, was used for cloning purposes. The pGEMT-Easy PCR cloning kit and Taq DNA polymerase along with restriction enzymes were purchased from Promega and Takara corporations, respectively. The gel and plasmid DNA extraction kits were provided from Bioneer Corporation. The sequencing of the promoters was carried out by Millegen (Labège, France).
further subcloning, respectively.
PCR amplification The total genomic DNA was used as a template for the amplification of promoters at the concentration of 50 ng/µL. The amplification program was done as the primary denaturing using 94°C for 5 min, followed by 35 cycles of 94°C for 50 s, 63°C for 50 s and 72°C for 1 min, and a final extension step at 72°C for 7 min. The amplified DNA products were separated by 0.8% agarose gel electrophoresis and extracted using the DNA gel extraction kit. The concentration of the eluted fragment was adjusted to 50 ng/µL and ligation reaction was done at 4°C for 24 h for cloning in the pGEMT-Easy vector. The E. coli competent cells were used for transformation by 5 µL of ligation reaction and the transformants were selected on white-blue test media containing antibiotic ampicillin, isopropyl β-D-1thiogalactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl-betaD-galacto-pyranoside (X-gal). The plasmid DNA was extracted from the positive colony PCR of white colonies using the extraction kit. The cloned fragments in pGEMT-Easy were confirmed by reamplification and restriction enzymes of HindIII and NcoI. The obtained clones harboring putative promoters were sequenced by a MILLGENE company and followed by in silico analysis which was done using any bioinformatics tools.
Extracting total DNA of barley The total DNA was extracted from fine powder of 24 h dark grown leaf blades by liquid nitrogen (N2) after removing midribs using cetyltrimethylammonium bromide (CTAB) extraction method as described by Saghai-Maroof (1984). Its quality and quantity were determined by 0.8% agarose gel electrophoresis and its concentration was adjusted on 50 ng/µL.
Designing the primer The sequence of H. vulgare B1 hordein gene (GeneBank accession number, X87232) consisting of a promoter and coding regions of B1 hordein gene, were retrieved from GeneBank in order to design a specific primer for the promoter region and signal peptide of B1 hordein gene (Figure 1), forward primer of 5′ATAAGCTTGTCGAGAAGAACCGTCCAC3′ and reverse primer of 5'TGCCATGGTACTTGTTGCCGCAATG 3' were designed using Primer3 Online software and their validity were confirmed by Oligo5 and NTC tools. The restriction enzyme sites of HindIII and NcoI were added to the 5′ end of the forward and reverse primers for
RESULTS AND DISCUSSION Cloning seed-specific promoter fragment ProB-Hor.A and ProB-Hor.W The promoter fragments ProB-Hor.A and ProB-Hor.W were obtained by PCR amplification via primers PF and PR, respectively. A 518-nt-long sequence of the barley B hordein promoter was PCR amplified, the result of which is shown in Figure 2. This fragment was ligated to pGEMT-Easy in order to obtain pT-ProB-Hor.W and pTProB-Hor.A sequences. The procedure to construct the pT- ProB-Hor.A and pT- ProB-Hor.W is shown in Figure 3. The cloned fragments in pGEMT-Easy were further confirmed by PCR and digested by restriction enzymes, HindIII and NcoI, as shown in Figure 4. Individual digestion with NcoI and HindIII enzymes indicated the fragment to be 3015 bp, which included the 518 bp B
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Figure 2. PCR product 1, Walfajre; 2, Alger; M, DNA marker.
hordein promoter. Double digestion with NcoI and HindIII enzymes indicated the 518 bp B hordein promoter fragment in the vector. Sequencing analyses of the seed-specific promoter fragments ProB-Hor.A and ProB-Hor.W To find regulatory elements in promoter sequences, the PLANTCARE (http://bioinformatics.psb.ugent.be/ webtools/plantcare/html) and Softberry (http://linux1.softberry.com/berry.phtml) software were applied. Analysis of the sequences of the seed-specific promoter fragments ProB-Hor.A and ProB-Hor.W revealed several kinds of seed specific promoter motifs in 518 base pairs of these cloned promoter fragments as shown in Figures 5 and 6. The comparison of the two sequences with the sequences available in NCBI indicated the differences in the motifs (Figure 7). These kinds of seed specific promoter motifs in frag-ments ProB-Hor.A and ProB-Hor.W may be responsible for its seed-specific promoter activity. The results of the identified general transcription and potential regulatory elements are summarized in Table 1. Based on these seed-specific motifs and the elements identified by the bioinformatics analysis, the isolated regions were selected as candidates for promoting gene expression in seed (Zavallo et al., 2010). The sequences important in gene expression were likely to be conserved among a group of genes with the same pattern of expression (Davidson et al., 1983). The 5â&#x20AC;˛ noncoding regions were highly similar to those of the genes
encoding B Hordein polypeptides. Bioinformatics analyses of these sequences allowed for the identification of conserved motifs. A-box motif found in Petroselinum crispum, is named CCGTCC-box in Arabidopsis thaliana, and is related to meristem specific activation (Logemann et al., 1995). AAGAA and AC-II motifs were found in Avena sativa and Phaseolus vulgaris, respectively. G-Box in Pisum sativum and A. thaliana )Persson et al., 1998), GA-motif in Helianthus annuus (Waksman G et al., 1987), GAGmotif in Spinacia oleracea (Werneke et al., 1989), I-box in Zea mays (Sheen, 1991) and MRE motif in Petroselinum crispum in the MYB binding site (Feldbrugge et al., 1997) were involved in light responsiveness. ARE motif in Z. mays is essential for the anaerobic induction (Manjunath and Sachs, 2005). ABRE motif in A. thaliana is involved in the abscisic acid (ABA) responsiveness on the rd29B gene (Yamaguchi-Shinozaki and Shinozaki, 1993). CAAT-box motif common cis-acting element in promoter and enhancer regions is often located in -80 positions, but much farther distances from the starting point and can also operate in both directions. CAAT box plays an important role in determining the efficiency of promoter (Lewin, 2009) and is found in Brassica rapa, A. thaliana, Glycine max, Petunia hybrid and H. vulgare (Shirsat et al., 1989). CGTCA-motif and TGACG-motif in Hordeum vulgare are cis-acting regulatory elements involved in the methyl jasmonate (MeJA) responsiveness (Rouster et al., 1997). GCN4-motif and Skn-1 motif Oryza sativa are required for endosperm expression (Takaiwa et al., 1991). A TATA box sequence has been found in almost all plant genes in recent studies (Mesing et al., 1983). Many eukaryotic promoters between 10 and 20% of all genes (Gershenzon and Ioshikhes.,2005) contain a TATA box (sequence TATAAA), which in turn binds a TATA binding protein and assists the formation of the RNA polymerase transcriptional complex (Smale and Kadonaga, 2003). The TATA box typically lies very close to the transcriptional start site (often within 50 bases). The TATA box tends to be surrounded by GC rich sequences. A sequence which is rich in guanidine (G) and cytidine (C) nucleotides is usually found in multiple copies in the promoter region and normally surrounds the TATA box and CAP site. A TATA box is located at -79 base pairs from the start codon. The (CA) n box-like sequence has been designated a (CA) n box located at -138 bp from the initiation sites. TATA and (CA) n boxes within both promoters identified as well as other transcriptional enhancer boxes (Forde et al., 1985). The sequence TGTAAAG, commonly named the prolamin box (p-box), was initially identified on the basis of both its highly conserved nucleotide sequence and location (-300 bp) relative to the transcription initiation site (TIS) of prolamin genes. It was recognized as a strong candidate for coordinating the expression of many seed specific expressions of many storage proteins
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Figure 3. Construction of pT- ProB-Hor.A and pT- ProB-Hor.W
Figure 4. Construction and identification of ProB-Hor.A and ProB-Hor.W fragment M, DNA marker; 1, digestion product pT- ProB-Hor.A /HindIII; 2, digestion product pT- ProB-Hor.A /NcoI; 3, double digestion product pTProB-Hor.A /HindIII/NcoI; 4, double digestion product pT- ProB-Hor.W HindIII/NcoI.
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Figure 5. Sequence analysis of B1 hordein specific promoter fragment ProB-Hor.A A, E-box; B, Dof core recognition sequence; C, GLM motif; D, (CA)n box; E: N mtif; F, TATA box; G, start codon;â&#x201D;&#x20AC;, ACGT motif.
Figure 6. Sequence analysis of B1 hordein specific promoter fragment ProB-Hor.W A, E-box; B, Dof core recognition sequence; C, GLM motif; D, (CA)n box; E, N mtif; F, TATA box; G, start codon.
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Pro-BH-Alger Pro-BH-walfajre Pro-BH-Query
GTCGAGAAGAACCGTCCACATGTAAAGCTTTAACAACCCACACAT-GATTGCAACTTAGT GTCGAGAAGAACCGTCCACGTGTAAAGCTTTAACAACCCACACAT-GATTGCAACTTAGT GTCGAGAAGAACCGTCCACATGTAAAGCTTTAACAACCCACACATTGATTGCAACTTAGT ******************* ************************* **************
Pro-BH-Alger Pro-BH-walfajre Pro-BH-Query
CCTACACAAGTTTTCCATTCTTGTTTCACGCTAACAACCTATACAAGTTTCCAAAATCAT CCTACACAAGTTTTCCATTCTTGTTTCAGGCTAACAACCTATACAAGGTTCCAAAATCAT CCTACACAAGTTTTCCATTCTTGTTTCAGGCTAACAACCTATACAAGGTTCCAAAATCAT **************************** ****************** ************
Pro-BH-Alger Pro-BH-walfajre Pro-BH-Query
TTGCAAAAGTGATGCTAGGTTGATAATTGTGTGACATGTAACGTGAATAAGGTGAGCCAT TTGCAAAAGTGATGCTAGGTTGATAATTGTGTGACATGTAAAGTGAATAAGGTGAGTCAT --GCAAAAGTGATGCTAGGTTGATAAT-GTGTGACATGTAAAGTGAATAAGGTGAGTCAT ************************* ************* ************** ***
Pro-BH-Alger Pro-BH-walfajre Pro-BH-Query
GCATACCAAACCTCGGGATTTCTGTACTTTGTGTATGATCATATGCACAACTAAAAAGCA GCATACCAAACCTCGGGATTTCTATACTTTGTGTATGATCATGTGCACAACTAAAAAGCA GCATACCAAACCTCGGGATTTCTATACTTTGTGTATGATCATATGCACAACTAAAAGGCA *********************** ****************** ************* ***
Pro-BH-Alger Pro-BH-walfajre Pro-BH-Query
ACTTTGATTATCAATTCAGAAGTAC-GCTTGTAGCTTGTGCAACCTAACACAATGTACCA ACTTTGATTATCAATTCAAAAGTAC-GCTTGTAGCTTGTGCAACCTAACACAATGTACCA ACTTTGATTATCAATTGAAAAGTACCGCTTGTAGCTTGTGCAACCTAACACAATGT-CCA **************** * ****** ****************************** ***
Pro-BH-Alger Pro-BH-walfajre Pro-BH-Query
AAAATCCGTTTGCAAAA-CATCCAAACACAATTGTTAAAGCTGATGCAAAGAA--AGAAA AAAATCCATTTGCAAAA-CATCCAAACACAATTGTTAAAGCTGATGCAAAGAA--AGAAA AAAATCCATTTGCAAAAGCATCCAAACACAATTGTTAAAGCTGTTCAAACAAACAAAGAA ******* ********* ************************* * ** ** * **
Pro-BH-Alger Pro-BH-walfajre Pro-BH-Query
GAGATGAAGCCCTGGCTACTATAAATAGGCATGTAGTATAGAGATCATCACAAGCACAAG GAGATGAAGCCCTGGCTACTATAAATAGGCATGTAGTATAGAGATCATCACAAACACAAG GAGATGAAGCC-TGGCTACTATAAATAGGCAGGTAGTATAGAGATCTACACAAGCACAAG *********** ******************* ************** ***** ******
Pro-BH-Alger Pro-BH-walfajre Pro-BH-Query
CATCAAAATCAAGAAACACTAGTTAACACCAATCCACTATGAAGACCTTCCTCATCTTTG CATCAAAACCAAGAAACACTAGTTAACACCAATCCACTATGAAGACCTTCCTCATCTTTG CATCAAAACCAAGAAACACTAGTTAACACCAATCCACTATGAAGACCTTCCTCATCTTTG ******** ***************************************************
Pro-BH-Alger Pro-BH-walfajre Pro-BH-Query
CACTCCTTGCCATTGCGGCAACAAGTACCATGGCA CACTCCTCGTCATTGCGGCAACAAGTACCATGGCA CACTCCTCGCCATTGCGGCAACAAGTACGATTGCA ******* * ****************** ** ***
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Figure7. Comparing the two sequences with sequences available in NCBI
(SSPs) because of its presence within the promoters of all zein genes in maize (Thompson and Larkins, 1989), as well as many storage protein genes from the related cereals. Prolamin box (p-box or E box), a conserved motif (5′ TGTAAAG 3′), is present in cereal storage protein gene promoters (Forde et al., 1985). In many prolamin genes, p-box and GCN4 motifs are coupled with each other with only a few nucleotides which are separating them. This tandem module is designated as the bifactorial endosperm box (Marzabel et al., 1998). Two endosperm box (E-box) and GCN4-like motifs (GLM-motif
5´GTGAGTCAT 3´) are present in these promoters (Muller et al., 1995). Ebox is also named an endosperm box and is the binding site of endosperm-specific nuclear factors named barley-prolamin-box binding factor (BPBF) and binding was prevented when the pbox motif was mutated to 5´TGTAgAc 3´(Mena et al., 1998). In the second piece of E-box in position 164, the PROB-HOR.W fragment is a cytosine to adenine mutation. ACGT motif is also a sequence motif required for seedspecific expression (Vincentz et al., 1997). In barley, nitrogen regulation is mediated by the endosperm
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Table 1. Regulatory elements in promoter sequence.
Site
Sequence
Position
Strand
12 348
+ +
-
17
+
cis-acting element involved in the abscisic acid responsiveness
(C/T)T(T/C)(C/T)(A/C)(A/C) C(A/C)A(A/C)C(C/A)(C/A)C
(C/T)T(T/C)(C/T)(A/C)(A/C) C(A/C)A(A/C)C(C/A)(C/A)C
28
+
-
ACE
ACGTGGA
-
15
-
cis-acting element involved in light responsiveness
ARE
TGGTTT
-
421
-
cis-acting regulatory element essential for the anaerobic induction
CAAT
CAAT
445 289
+
common cis-acting element in promoter and enhancer regions
CCGTCC-box
CCGTCC
CCGTCC
12
+
cis-acting regulatory element related to meristem specific activation
CGTCA-motif
CGTCA
-
483
+
cis-acting regulatory element involved in the MeJA-responsiveness
CACGTG
CACGTG
17 159
+ -
cis-acting regulatory element involved in light responsiveness
AAAGATGA AGAGATG
AAAGATGA AGAGATG
467 355
+
GTGAGTCAT
-
171
+
part of a light responsive element part of a light responsive element BF:bZIP transcription factor, cis-regulatory element involved in endosperm expression
I-box
gGATAAGGTG
gGATAAGGTG
164
+
MRE
AACCTAA
AACCTAA
280
+
ProOB-Hor.A CCGTCC
ProB-Hor.W CCGTCC GAAAGAA
ABRE
CACGTG
AC-II
A-Box AAGAA-Motif
CAAT-box
G-Box
GA-motif GAG-motif GCN4-like or GSN;hor1-box
Function Cis acting regulatory element
part of a light responsive element MYB binding site involved in light responsiveness
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Table 1. Contd.
Prolamin-box (E-box)
tgagaTGTAAAGtgaat
tgacaTGTAAAGtgaat
Site
Sequence ProOB-Hor.A
ProB-Hor.W
Skn-1_motif
GTCAT
TATA box
151
+
Conserved in cereal seed storage protein gene promoters
Position
Strand
Function
-
175 484
+
cis-acting regulatory element required for endosperm expression
TATAAATA
TATAAATA
375
+
core promoter element around -30 of transcription start, Important for recognition by RNA polymeraseII,
TGACG-motif
TGACG
-
483
-
cis-acting regulatory element involved in the MeJA-responsiveness
Unnamed__4 (CA)n ACGT motif N motif Dof core recognition sequence
CTCC CATCCAAACACAA ACGT GATGAAGCCCT AAAG
CTCC CATCCAAACACAA GATGAAGCCCT AAAG
478 315 17 357 Very frequent
+ + + + +
Seed specificity Seed development
GA-2, Arabidopsis (A. thaliana) GA-1, Arabidopsis (A. thaliana) EM1,wheat (T. aestivum) GLM1, wheat (T. aestivum) GLM2, wheat (T. aestivum)
AGAAAGAGA TGTAAAGTG GGTGAGTCAT TGTGTGACAT
AGAAAGAGA AGAAAGAAA TGTGTGACAT
350 346 155 169 146
+ + + + +
BF: BPC BF: BPC1 BF: Unknown nuclear factor BF: Unknown nuclear factor BF: Unknown nuclear factor
box via the interaction of endosperm and GCN4- like motif (Muller et al., 1995). The N motif has the consensus sequence G (A/G) TGAAGTCAT (Shewry and Halford, 2002). In these fragments, N motifs had only two nucleotide mutations that had occurred by replacing nucleotide T with C and A with C. This motif requires additional investi-gations. AAAG is Dof core recognition sequence and is so frequent that is regulated by light and tissue specific gene expression (Vicente-Carbajosa et al., 1997). The alignment between ProB-Hor.A and ProB-Hor.W showed that there were nine mismatches between the two sequences. Nucleotides 462 to 518 constituted a signal
Light regulation, tissue specific gene expression
peptide that began with one ATG codon and encoded a signal peptide (Watson. 1984). The presence of a signal peptide would be consistent with the evidence that the B hordeins were synthesized on the rough endoplasmic reticulum and were deposited in protein bodies (Matthews and Miflin., 1980; Cameron-Mills., 1980). A typical signal peptide comprises of three distinct regions: a polar N terminal end (n-region) that may have a net positive charge, a central hydrophobic core (h-region) that consists of 6Âą15 hydrophobic amino acids and a polar C-terminal (c-region) end that contains prolines and glycines (von Heijne, 1985). A signal peptide containing the consensus sequence and
proper cleavage site ensures that proteins are inserted into the endo-plasmic reticulum (ER) membrane. Mutations within the sequence immediately downstream the signal peptide affected protein processing. The nucleotide sequence of the signal peptide geno-type Alger and Walfajr into amino acid sequence was converted by vector NTI 9.1 software. The sequence of the signal peptide region of Alger genotype was compared with the signal peptide recorded by basic local alignment search tool program (BLASTP). Their similarity was in the range of 90 to 95%. The results indicate that the amino acid methionine that mutated the isoleucine in the Alger
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Query 1 MKTFLIFALLVIAATSTMA 19
Query
MKTFLIFALLVIAATST A Alger1 MKTFLIFALLVIAATSTIA
1 MKTFLIFALLAIAATSTMA 19 MKTFLIFALLAIAATST A
19
Walfajre 1 MKTFLIFALLAIAATSTIA 19
A Alger
1 MKTFLIFALLVIAATSTMA
B 19
MKTFLIFALL IAATSTMA Walfajre 1 MKTFLIFALLAIAATSTMA
n Sp.B-Hordein,Alger
19
h
c
MKTFLIFALLVIAATSTMA
Sp.B-Hordein,Walfajre MKTFLIFALLAIAATSTMA
n
h
c
D
C
Figure 8. A, Comparison of the signal peptide sequence in Alger genotype; B, comparison of the signal peptide sequence in walfajre genotype, C: Results of BLAST P signal peptide in Alger and walfajre; D, various section of the signal peptide and cleavage site is shown withâ&#x2020;&#x2018;
box via the interaction of endosperm and GCN4- like motif (Muller et al., 1995). The N motif has the consensus sequence G (A/G) TGAAGTCAT (Shewry and Halford, 2002). In these fragments, N motifs had only two nucleotide mutations that had occurred by replacing nucleotide T with C and A with C. This motif requires additional investi-gations. AAAG is Dof core recognition sequence and is so frequent that is regulated by light and tissue specific gene expression (Vicente-Carbajosa et al., 1997). The alignment between ProB-Hor.A and ProB-Hor.W showed that there were nine mismatches between the two sequences. Nucleotides 462 to 518 constituted a signal peptide that began with one ATG codon and encoded a signal peptide (Watson. 1984). The presence of a signal peptide would be consistent with the evidence that the B hordeins were synthesized on the rough endoplasmic reticulum and were deposited in protein bodies (Matthews and Miflin., 1980; Cameron-Mills., 1980). A typical signal peptide comprises of three distinct regions: a polar N terminal end (n-region) that may have a net positive charge, a central hydrophobic core (h-region) that consists of 6Âą15 hydrophobic amino acids and a polar C-terminal (c-region) end that contains prolines and glycines (von Heijne, 1985). A signal peptide containing the consensus sequence and proper cleavage site ensures that proteins are inserted into the endoplasmic reticulum (ER) membrane. Mutations within the sequence immediately downstream the signal peptide affected protein processing. The nucleotide sequence of the signal peptide geno-type Alger and Walfajr into amino acid sequence was converted by vector NTI 9.1 software. The sequence of the signal peptide region of Alger genotype was compared with the signal peptide recorded by basic local alignment search tool program (BLASTP). Their similarity was in the range of 90 to 95%. The results indicate that the amino acid methionine that mutated the isoleucine in the Alger genotype did not prevent any transfer of proteins in vacuoles (Figure 8A). The sequenceof the signal region of Alger genotype in
comparison with the signal peptide recorded by BLASTP had 95% similarity (Figure 8B). The amino acid similarity of signal peptide of two genotypes Alger and Walfajr was
95%. The comparison of DNA blast and peptide signals (Figure 8C) showed that mutations in sequences coding of 10th amino acids (leucine) did not change it because of the 3rd nucleotides changes. However, the changes in the second nucleotide in the 11th amino acid led to the change of valine in Alger to Alanin in Walfajre. Signal peptide was investigated by the online applicationof: www.cbs.dtu.dk/services/SignalP/ index.php. Cleavage site in the signal peptide of Alger th and Walfajre genotypes was located between the 13 th and 14 amino acids with probability of 97% (Figure 8D). Proteins transfer to the vacuoles was not conducted if the cleavage site was located in other areas.
Conclusion The aim of this research was to isolate one of the seed specific promoters of H. vulgare. Two barley varieties that were common in Iran were selected and their genomes were extracted using the specific designed primers for amplifying the B hordein promoter. The amplified fragment of the insert was cloned in an appropriate vector and then was transformed to E. coli. At last, for the final admission of accuracy, the cloned fragments were sent for sequencing. The results are compared with the sequences existing in data banks. The promoter region contained motifs; like TATA box, (CA) n box, ACGT motif, AAAG motif, GCN4-like motif and E-box, which constituted the seed-specific promoter activity. The results obtained from the neural network algorithm show three scores in signal peptide: the high S-score, Cscore and the Y-score. The amino acid similarity of signal peptide of two genotypes (Alger and Walfajr) was 95%.
Nalbandi et al.
REFERENCES BlakeL TK, Ullrich SE, Nilan RL (1982). Mapping of Hor-3 locus encoding D hordein in barley.Theor. Appl. Genet. 53: 367-371. Cameron-Mills V (1980). The structure and composition of protein bodies purified from barley endosperm by silica sol density gradients. Carlsberg Res. Commun. 45: 557-576. Davidson EH, Jacobs HT, Britten RJ (1983). Eukaryotic gene expression: Very short repeats and coordinate induction of genes. Nature, 301: 468-470. Evans IM, Gatehouse JA, Croy RD, Boulter D (1984). Regulation of the transcription of storage protein mRNA in nuclei isolated from developing pea (Pisum sativum) cotyledons. Planta, 160: 559-568. Feldbrugge M, Sprenger M, Hahlbrock K, Weisshaar B (1997). PcMYB1, a novel plant protein containing a DNA-binding domain with one MYB repeat, interacts in vivo with a light-regulatory promoter unit. Plant J. 11(5): 1079-1093. Forde BG, Heyworth A, Pywell J, Kreis M (1985). Nucleotide sequence of a B1 hordein gene and the identification of possible upstream regulatory elements in endosperm storage protein genes from barley, wheat and maize. Nucleic Acids Res. 13: p. 20. Gatehouse JA, Evans IM, Croy RRD, Boulter D (1986). Differential expression of genes during legume seed development. Philos Trans. R. Soc. Lond (B) Biol. Sci. 314: 367-384. Gershenzon NI, Ioshikhes IP (2005). Synergy of human Pol II core promoter elements revealed by statistical sequence analysis. Bioinformatics, 21(8): 1295-300. Jensen J, Jorgensen JH, Jensen HP, Giese H, Doll H (1980). Linkage of the hordein loci, Hor 1 and Hor 2 with the powdery mildew resistance loci MI-k and Mt-a on barley chromosome 5. Theor. Appl. Genet. 58: 27-31. Lewin B (2009).GENES VIII. Pearson Prentice Hall. p. 1030. Logemann E, Parniske M, Hahlbrock K (1995). Modes of expression and common structural features of the complete phenylalanine ammonia-lyase gene family in parsley. Proc Natl Acad. Sci. USA. 92: 5905-5909. Manjunath S, Sachs MM (2005). Molecular characterization and promoter analysis of the maize cytosolic glyceraldehyde 3-phosphate dehydrogenase gene family and its expression during anoxia. Unpublished. AC(NCBI). pp. U45858. Marzabal P, Busk PK, Ludevid M D, Torrent M (1998). The bifactorial endosperm box of g-zein gene: characterisation and function of the Pb3 and GZM cis-acting elements. Plant J. 16: 41-52. Matthews JA, Miflin BJ (1980). In vitro synthesis of barley storage proteins. Planta, 149: 262-268. Mena M, Vicente-Carbajosa JJ, Schmidt R, Carbonero P (1998). An endosperm-specific DOF protein from barley, highly conserved in wheat, binds to and activates transcription from the prolamin-box of a native B-hordein promoter in barley endosperm. Plant J. 16(1): 53-62. Messing J, Geraghty D, Heidecker G, Hu NT, Kridl J, Rubsenstein I (1983) in Genetic engineering of Plants, Kosuge, T., Meredith, C.P. and Hollaender, A. Eds., Plenum Press, New York. pp. 211-227. Muller M , Muth J R, Gallusci P, Knudsen S, Maddaloni M, Motto M, Schmitzd D, Sorensen M B, Salamini F, Von Wettstein D, Thompson RD (1995). Regulation of storage protein synthesis in cereal seeds: developmental and nutritional aspects. Plant Physiol. 145: 606-613. Persson EL, Brosche M, Strid A (1998). Cultivar differences in the sequences of chalcone synthase promoters in pea. Unpublished. AC (NCBI): pp. AF060237. Piston F, Martin A, Dorado G, Barro F (2005). Cloning and molecular characterization of B-hordeins from Hordeum chilense (Roem. et Schult). Theor. Appl. Genet. 111: 551-560. Rouster J, Leah R, Mundy J, Cameron-Mills V (1997). Identification of a methyl jasmonate responsive region in the promoter of a lipoxygenase 1 gene expressed in barley grain. Plant J. 11(3): 513-523. Saghai-Maroof MA, Soliman K, Jorgensen RA, Allard RW (1984). Ribosomal DNA spacer length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. PNAS, 81: 8014-8018.
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Sheen J (1991). Molecular mechanisms underlying the differential expression of maize pyruvate, orthophosphate dikinase genes. Plant Cell, 3(3): 225-245. Shewry PR, Faulks AJ, Pickering RA, Jones IT, Finch RA, Miflin BJ (1980). The genetic analysis of barley storage protein. Heredity, 44: 383-389. Shewry PR, Finch RA, Parmar SJ, Frankin J, Miflin BJ (1983). Chromosomal location of Hor3, a new locus governing storage proteins in barley. Heredity, 50(2): 179-189. Shewry PR, Halford NG (2002). Cereal seed storage proteins: structures, properties and role in grain utilization. J. Exp. Bot. 53(370): 947-958. Shirsat A, Wilford N, Croy R, Boulter D (1989).Sequences responsible for the tissue specific promoter activity of a pea legumin gene in tobacco. Mol. Gen. Genet. 215: 326-331. Smale T, Kadonaga T (2003). The RNA polymerase II core promoter. Ann Rev. Biochem. 72: 449-479. Takaiwa F, Oono K, Wing D, Kato A (1991). Sequence of three members and expression of a new major subfamily of glutelin genes from rice. Plant Mol. Biol. 17(4): 875-885. Thompson GA, Larkins BA (1989). Structural elements regulating zein gene expression. Bioassayse, 10: 108-113. Vincentz M, Leite A, Neshich G, Vriend G, Mattar C, Barros L, Weinberg D, De Almeida ER, De Carvalho MP, Aragao F, Gander ES (1997). ACGT and vicilin core sequences in a promoter domain required for seed-specific expression of a 2S storage protein gene are recognized by the opaque-2 regulatory protein. Plant Mol. Biol. 34: 879-889. Vicente-Carbajosa J, Moose SP, Parsons RL, Schmidt RJ (1997). A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2. Proc. Natl. Acad. Sci. USA. 94: 7685-7690. Von Heijne G (1985). Signal sequences. The limits of variation. J. Mol. Biol. 184: 99-105. Waksman G, Lebrun M, Freyssinet G (1987). Nucleotide sequence of a gene encoding sunflower ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (rbcs) Nucleic Acids Res. 15(17): 7181-7181. Watson MEE (1984). Compilation of published signal sequences Nucleic. Acids Res. 12: 5145-5164. Werneke JM, Chatfield JM, Ogren WL (1989). Alternative mRNA splicing generates the two ribulosebisphosphate carboxylase/oxygenase activase polypeptides in spinach and Arabidopsis Plant Cell, 1(8): 815-825. Yamaguchi-Shinozaki K, Shinozaki K (1993). Arabidopsis DNA encoding two desiccation-responsive rd29 genes. Plant Physiol. 101(3): 1119-1120. Zavallo D, Lopez BM, Esteban Hopp H, Heinz R (2010). Isolation and functional characterization of two novel seed-specific promoters from sunflower (Helianthus annuus L.). Plant Cell Rep. 29: 239-248.
African Journal of Biotechnology Vol. 11(29), pp. 7388-7396, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.4064 ISSN 1684–5315 © 2012 Academic Journals
Full Length Research Paper
Genome shotgun sequencing and development of microsatellite markers for gerbera (Gerbera hybrida H.) by 454 GS-FLX Kyoung-In Seo1, Gi-An Lee1, Sang-Kun Park2, Mun-Sup Yoon1, Kyung-Ho Ma1, Jung-Ro Lee1, Yu-Mi Choi1, Yeon-ju Jung1 and Myung-Chul Lee1* 1
National Agrobiodiversity Center, National Academy of Agricultural Science, RDA, 88-20, Seodun-Dong, Suwon, Gyunggi-do, 441-707, Korea. 2 Floriculture Research Division, National Institute of Horticultural and Herbal Science, RDA, Suwon, Gyunggi-do, 440706, Korea. Accepted 26 January, 2012
The objective of this research was to develop and characterize microsatellite markers for gerbera. We used shotgun sequencing with Roche 454 GS-FLX Titanium technology to identify microsatellite loci in gerbera genomic DNA (Gerbera hybrida). The total length of non-redundant sequences obtained was 22,527,019 bp, which consisted of 3,085 contigs and 28,249 singletons. We assembled 61,958 reads into 3,085 contigs, of which 114 (3.70%) contained microsatellite repeats. The average G+C content was 39.3%. Functional annotation to known sequences yielded 14.7% unigenes in the ‘Raon’ cultivar. Analysis of the gerbera genome DNA (‘Raon’) general library showed that sequences of (AT), (AG), (AAG) and (AAT) repeats appeared most often, whereas (AC), (AAC) and (ACC) were the least frequent. Primer pairs were designed for 80 loci. Only eight primer pairs produced reproducible polymorphic bands in the 28 gerbera accessions analyzed. A total of 30 alleles were identified from the eight polymorphic SSR loci, with two to eight alleles per locus (average level of 3.75). These markers will be useful for investigating genetic diversity and differentiation in gerbera. Key words: Genetic diversity, genomics, microsatellite isolation, pyrosequencing, SSRs.
INTRODUCTION Gerbera species, belonging to the family Asteraceae, are among the most popular decorative plants worldwide, with uses as garden plants, potted plants and cut flowers. Gerbera plants generally show variation in their stalks, and through hybrid breeding (they are polyploid) thin, soft and cut flower varieties have been produced (Kanwar and Kumar, 2008; Yu et al., 1999). Most of the gerbera cultivated in Korea have large flowers that are used as cut flowers. Various cut flower gerbera cultivars have different flower colors. In addition, year-round production is possible and is a common practice. Evaluations of the genetic diversity of gerbera have been conducted using random amplified polymorphic DNA (RAPD) and
*Corresponding author. E-mail: mclee@rda.go.kr. Tel: 82-31299-1887. Fax: 82-31-294-6029.
expressed sequence tag-simple sequence repeats (ESTSSR) (Mata et al., 2009; Gong and Deng, 2010). In addition, Bhatia et al. (2009) studied the effects of explant source on the genetic integrity of 45 in vitro-raised gerbera plants (Gerbera jamesonii Bolus) using 32 ISSR markers. However, microsatellite (SSRs) markers were not developed and tested for these gerbera genetic studies. Simple sequence repeats (SSR) or microsatellites have been used to examine genotypic variation and for breeding research (Eujayl et al., 2001; Ruiz et al., 2000). Furthermore, these markers are widely used in cultivar fingerprinting and molecular mapping. When compared with other types of molecular markers, SSR markers are abundant, co-dominant and have high reproducibility. In the past, microsatellite markers were developed using the enrichment method and hybridization probes. However, this method had a disadvantage: it could not detect the correct repeat sequence in the
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Table 1. Gerbera accessions analyzed using SSR markers and characteristics of gerbera flower types.
S/N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Accession name Marathon Venturi Samba Angi Liz Kopabo Amado Mirage Goldfinger Kabana Albert Diablo Esperanza Sunset Rosamatt Ma Duella Essence Salina Palazo Escada Stephane Raon Misty Red Royal Mandorin Jinno Dino
Flower type Semi-double Double Single Semi-double Semi-double Single Semi-double Double Double Double Double Semi-double Double Double Double Semi-double Semi-double Double Double Double Semi-double Semi-double Double Double Semi-double Semi-double Semi-double Double
genome because a probe that facilitated hybridization was not available. Lagercrantz et al. (1993) demonstrated that (AT)/(TA) and (CG)/(GC) probes more easily construct complementary structures than do other kinds of probes. In addition, the method was costly, labor intensive during the cloning process, and produced many redundant clones. The recent introduction of next-generation sequencing technologies, such as the 454 Genome Sequencer (GS)-FLX (Roche) which uses massively parallel pyrosequencing, may allow for more effective production of microsatellites. The GS-FLX by 454 Life Sciences depends on an oil-emulsion based PCR followed by massively parallel and individual pyrosequencing of the clonally amplified beads in a PicoTiterPlate (Pettersson et al., 2009). At present, 454 technology offers the longest read length by far, ~400 bp on the GS-FLX Titanium platform. Recently, in Khaya senegalensis, a microsatellite marker that can characterize genetic variation was developed using next-
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generation sequencing (Sexton et al., 2010). Many studies have used 454 pyrosequencing to analyze genomic DNA (Sato et al., 2010), polymerase chain reaction (PCR) amplicons (Bonnal et al., 2010), BAC DNA (Wicker et al., 2006) and cDNA (Yang et al., 2010) samples. Tangphatsornruang et al. (2009) developed polymorphic SSR markers for genotyping mungbean using genome shotgun sequencing. They reported that shotgun sequences were useful for marker development, for determining phylogenetic relationships and for gene discovery. The present study examined microsatellite sequences in gerbera genomic data and evaluated genetic relationships between gerbera cultivars. In this paper, we reported the development of micro-satellites and the characterization of gene content in gerbera (Gerbera hybrida) using cost-effective 454pyrosequencing. MATERIALS AND METHODS Plant material and DNA extraction PCR and genotyping was performed on 28 gerbera (Gerbera hybrida H.) accessions collected from the National Horticultural Research Institute (NHRI), Rural Development Administration (RDA) of Korea (Table 1). The ‘Raon’ and ‘Misty Red’ cultivars were bred at the NHRI of Korea and the rest of the cultivars were bred abroad. The scoring of flower type was based on the number of petals, such as single, double and semi-double. Among 28 accessions, only 2 accessions have single type flower and 12 other accessions were semi-double type and 14 accessions have double type. Total genomic DNA was extracted from young fresh leaves by the modified cetyltrimethylammonium bromide (CTAB) method described by Dellaporta et al. (1983). A total of 8 μg of ‘Raon’ (double petal type) DNA was used to perform shotgun sequencing using 454 GS-FLX Titanium pyrosequencing (Roche Applied Science, Mannheim, Germany).
Shotgun genomic sequencing A small/medium plate (1/8 PicoTiterPlate; PTP) was used for 454 GS-FLX Titanium pyrosequencing for samples processed by the shotgun method according to the manufacturer’s instructions. In short, the samples prepared using the shotgun method was fractionated into smaller pieces (300 to 800 bp) by nebulization. Subsequently, the fragment length distribution of the library was assessed using a gerbera DNA 7500 LabChip (Agilent Technologies, Waldbronn, Germany) to ensure that the input material was adequate. The ends of the sheared gerbera genome DNA fragments were polished by the action of T4 DNA polymerase and T4 polynucleotide kinase. Short adaptors were then ligated to the ends of each sample DNA fragment. These adaptors provide priming regions to support both amplification and nucleotide sequencing. Finally, the quality of the library was assessed with a Bioanalyzer RNA 6000 Pico LabChip (Agilent Technologies). Emulsion titration was performed with different amounts of input ssDNA. Medium-scale emPCR was conducted based on the titrated ssDNA amount. Enriched bead samples were then counted using a Z1 Coulter Counter (Beckman Coulter, Brea, CA, USA) to calculate the percent enrichment. Samples prepared using the above procedure were loaded,
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along with other reagents, into the wells of a 1/8 PTP device, and sequencing was performed with a GS FLX Titanium Reagents XLR70, a 70 x 75 mm Titanium PTP device and a Genome Sequencer FLX Instrument (454 Life Science, Roche Applied Science). Sequencing data were obtained after a 9-h run of the GS-FLX. After sequencing, sequence assembly was performed using the GS De Novo Assembler software to produce the contigs and singletons. All sequence alignments were conducted using the Basic Local Alignment Search Tool (BLAST; database: NT) (Altschul et al., 1997). Selection of SSRs and primer design Identification of SSR motifs within contig sequences and primer design were conducted using the ARGOS program (Kim, 2004). The contigs were screened for mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide repeats. The minimum number of repeats was set to four. From these contigs, we designed 80 primer pairs to amplify microsatellite regions. The primer design included the following specifications: (1) melting temperature (Tm) between 54 and 64°C; (2) GC content above 30%; (3) amplicon size of 150 to 300 bp. For each locus, forward primers were synthesized to incorporate a 5’ fluorescent label, M13 (-21)-Tag TGTAAAACGACGGCCAGT (Messing, 1983). Polymorphism analysis The M13F-tail (5′-TGTAAAACGACGGCCAGT-3′) PCR method of Schuelke (2000) was used to measure PCR product size, as described by Lee et al. (2008). PCR was performed in a 20 μl reaction mixture containing 2 μl of template genome DNA (20 ng/μl), 0.2 μl of locus-specific primer (10 pmol/μl), 0.4 μl of M13 universal primer (10 pmol/μl), 0.6 μl normal reverse primer, 2.0 μl of 10X PCR buffer (SolGent, Daejeon, Korea), 1.6 μl of dNTP (2.5 mM) and 0.2 μl h-Taq-polymerase (5 U/μl; SolGent). The 5'-M13 sequence was attached to a forward primer to incorporate a florescent dye [blue (FAM), green (NED) or yellow (HEX)] into the PCR product. PCR was performed as follows: initial denaturation at 94°C (3 min), then 30 cycles each of 94°C (30 s), 55°C (45 s) and 72°C (1 min), followed by 10 cycles of 94°C (30 s), 53°C (45 s) and 72°C (1 min), and a final extension at 72°C for 10 min (PTC-100 Thermocycler, MJ Research, Waltham, MA, USA). All denatured products were run on an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA) using the molecular size standard (35 to 500 bp) GeneScan 500 ROX (6-carboxyrhodamine). Genotypes were obtained using GeneMapper v4.0 software (Applied Biosystems). Data analysis PCR genotyping results were converted to binary form (0 = absence; 1 = presence), and similarity coefficients were calculated among all 28 accessions considered in this study with the program NTSYS, version 2.1 (Exeter Software, Setauket, NY, USA), using SIMQUAL with the Dice coefficient (Dice, 1945). The data were subjected to unweighted pair group method with arithmetic mean (UPGMA) cluster analysis using NTSYS 2.1. The number of distinct genotypes and genetic diversity were computed using GenoType/GenoDive software (Meirmans and Van Tienderen, 2004). Sequence annotation and analysis of gene content Functional annotation and major functional categories (biological process, molecular function and cellular component) were analyzed
using the BLAST2GO software suite v2.3.1 (www.blast2go.de) (Conesa and Götz, 2008). Homology searches (BLASTX and BLASTN) were performed remotely on the NCBI server through a query-friendly version of BLAST (QBLAST), following a sequential strategy.
RESULTS Sequence analysis of the gerbera genome The status yield of sequencing runs and statistics for ‘Raon’ are summarized in Table 2. We also provided sequence data for all contigs of ‘Raon’ as Supplementary File 1. The 454 GS-FLX Titanium sequencing run yielded 22.5 Mbp of a genome DNA library from ‘Raon’ (G. hybrida), including 22,527,019 reads, with an average read length of 363.6 bp and 114 contigs (3.69%) containing microsatellite repeats. The ‘Raon’ genome DNA was assembled into 3,085 contigs and 28,249 singletons. After the assembly (3,085 contigs + 28,249 singletons), the contigs had an average length of 344.03 bp and an average depth of coverage of 10.92 reads per base pair. A total of 430 large contigs (≥500 bp), with an average length of 1,046 bp, was detected for ‘Raon’. The longest contig was 25,066 bp. To better understand the general mechanisms working in the gerbera, we performed the gene ontology (GO) analysis and carried out all aligned sequences (3,085 contigs and 28,249 singletons). Functional annotation was analyzed using BLAST2GO (Conesa and Götz, 2008), which led to consistent gene annotations and the assignment of gene names, gene products and gene ontology (GO) numbers. In total, 452 unigenes were assembled, and assembled contig sequences were available. All unigenes were investigated for their association with GO terms from level 4 biological processes, molecular function and cellular components; the number of associations was 137 (30.3%), 85 (18.8%) and 56 (12.4%), respectively. Eighty-five assignments were made to molecular function ontology, with a large proportion of these functioning in DNA binding (32.7%) and cation binding (27.3%) (Figure 1). Under biological process ontology, 137 assignments were made, with a large proportion of the assignments falling into cellular macromolecule metabolic process (24%) and nucleobase, nucleoside, nucleotide and nucleic acid metabolic process (23%) categories. The top 10 contigs by number of assembled reads were aligned to the NCBI database using BLASTN (Table 3). Four had significant -3 (e-value ≤1 x 10 and a bit score >40) hits to different genes, while three aligned to Lactuca sativa cultivar Salinas chloroplast complete genome DNA and three others aligned to Guizotia abyssinica chloroplast complete genome DNA. Characteristics of the genome SSR SSR motifs found in ‘Raon’ are summarized in Figure 2.
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Table 2. Sequencing results by GS FLX Titanium.
Total bases (bp) Significant BLAST hits
22,527,019 Z
152
G+C content (%)
39.3
Length Average (bp)
363.58
Total Contig length (bp)
Z
1,061,348
Number of contigs
3,085
Mean depth of contig
10.92
Number of singletons
28,249
Percentage of reads containing microsatellites
3.69%
Sequences with amplifiable microsatellite sequences
0.29%
-3
Significant BLAST hit criteria was an e-value ≤1 × 10 and a bit score>40.
Of the 3,085 unique contigs, 114 (3.70%) contained microsatellite sequences. Among the 114 microsatellite regions, we found dinucleotide, trinucleotide and pentanucleotide repeat motifs. The frequency of the appearance of these SSRs was estimated to be one SSR for every 9.3 kb in the 1.06 Mbp sequences from the Gerbera genome. The repeat number for dinucleotides (62.3%) was higher than that for trinucleotides (36.8%). In addition, a pentamer repeat was found in one contig (0.9%). The number of repeats ranged from 4 to 33. Among the SSRs, AT/TA repeat motifs were the most common (28.9%) followed by AG/GA repeat motifs (21.1%). The most frequent trinucleotide motif was AAG/AGA/GAA (13.2%), which was closely followed by the AAT/ATA/TAA motif (7.9%).
Development of SSRs markers After screening all the contigs (n = 3,085) for SSR regions with the ARGOS PROGRAM (Kim, 2004), we identified a total of 114 SSR regions. A random set of 80 primer sets was designed which included SSR regions; these primers were used to amplify gerbera genomic regions. There were six perfect repeats having no interruptions, one imperfect repeat and one compound repeat sequence. Four of the loci: GB-GJ-002, GB-GJ049, GB-GJ-059 and GB-GJ-063, which had (AAT)4, (AT)4, (ACA)6, (AG)7, (ATC)4, and (CAT)5 repeats, respectively, were labeled with FAM (blue). Two others, GB-GJ-011 and GB-GJ-074, with (CAC)5 and (CAT)5 repeats, respectively, were labeled with HEX (yellow). The last loci, GB-GJ-021 and GB-GJ-077, which had (AAAG)4 and (TGA)6 repeats, respectively, were labeled with NED (green). GB-GJ-049 had the widest allele size
range of the eight loci (160 to 317 bp) (Table 4). When used with 28 accessions, eight primer pairs managed to amplify 30 distinct alleles, with an average of 3.75 alleles per locus.
Genetic diversity based on SSR data Genetic diversity among G. hybrida accessions was evaluated using an UPGMA model (Figure 3). Cluster analysis separated the three groups: Groups-I, II and III. Group-I accommodated the highest number of accessions (16 accessions, 57.1%) followed by Group-II (nine accessions, 32.1%). Group-III contained the lowest number of accessions (three accessions, 10.7%). As shown in Figure 3, most of the semi-double type accessions were clustered in Group-I. Group-II consisted of nine accessions, which were mainly double type; Group-III consisted of three accessions: one single type, one double type, and one semi-double type. Coefficients of similarity ranged from 0.68 to 0.96. Flower type did not differentiate clearly among gerbera accessions. The ‘Misty Red’ and ‘Raon’ accessions, which were uniquely bred in Korea (Choi et al., 2001), belonged to Groups-I and II, respectively. Our analyses of genetic distances (NTSYS) and calculations of clonal diversity (GenoType/ GenoDive) divided the G. hybrida accessions in Group-I into 14 genotypes. The Groups-II and III populations were divided into eight and three genotypes, respectively. This is because SSR markers detected multilocus genotypes within the groups. Calculations of clonal diversity (Genotype/Genodive) resulted in a Nei’s diversity index (corrected for sample size) of 0.98 for Group-I (Table 5). For Groups-II and III, clonal diversity was 0.97 and 1.00, respectively, as nine and three genotypes were identified,
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A
B
Figure 1. Gene ontology classification for gerbera according to molecular function (A) and biological process (B) using BLAST2GO with E-6 cutoff.
respectively. Total diversity over all accessions was 0.99 and average diversity within populations was 0.98. The fraction of diversity among populations, corrected Gâ&#x20AC;˛ST, was 0.01. DISCUSSION The 454 GS-FLX technology provided a very cost-
effective method for obtaining SSR sequences. This technology will accelerate research on less-studied gerbera plants. The 454 GS-FLX has been broadly used for transcriptome sequencing, whole genome sequencing, metagenomics and microbial diversity (http://454.com/ applications/index.asp). As nextgeneration sequencing, 454 GS-FLX Titanium possesses more powerful sequencing capabilities, with a read length of 400 bp and higher sequencing accuracy. In this study,
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Table 3. The top ten contigs determined by the number of assembled reads and their respective alignments.
Contig BLASTN result 1931 1954 2020 28 5 56 3831 1446 2 64
Contig Length (bp) No. of assembled reads
Lactuca sativa cultivar Salinas chloroplast, complete genome Guizotia abyssinica chloroplast, complete genome Corokia cotoneaster 26S ribosomal RNA gene, complete sequence Guizotia abyssinica chloroplast, complete genome Guizotia abyssinica chloroplast, complete genome Trachelium caeruleum chloroplast, complete genome Oryza punctata, complete sequence Oryza australiensis, complete sequence Lactuca sativa cultivar Salinas chloroplast, complete genome Lactuca sativa cultivar Salinas chloroplast, complete genome
25066 18240 6606 14762 11466 10443 1296 5698 6924 7569
916 460 319 306 253 230 226 218 150 134
Figure 2. Distribution pattern of SSRs found in sequence of the â&#x20AC;&#x2DC;Raonâ&#x20AC;&#x2122; cultivar.
G. hybrida genome DNA was sequenced using a 454 GS-FLX system, producing a total of 22,527,019 sequence reads before assembly (reads only). After assembly, 3,085 contig sequences were obtained. The GC content of all reads was 39.3%, which is within the range commonly observed for plant genomes [e.g., tomato (Zhu et al., 2008) and Arabidopsis (Arabidopsis Genome Initiative, 2000)]. Our sequencing runs yielded reads with an average length of 363.5 bp. In the present study, 3,085 contigs (about 1.1 Mb) were used in an SSR search, which produced 114 (3.69%) SSR-containing sequences, including at least four dinucleotide, trinucleotide, tetranucleotide, penta-nucleotide and hexanucleotide repeats. This is a relatively low abundance of SSRs as compared to the numbers of ESTs containing SSRs found in an earlier report: Citrus spp. 6.09%, Lactuca sativa 4.90%, Rosa spp. 8.05% and
Prunus persica 4.99% (Kumpatla and Mukhopadhyay, 2005). The occurrence of SSRs in ESTs from the gerbera transcriptome was at the rate of one SSR per 5.16 kb (Gong and Deng, 2010), whereas in this study, using genomic sequences (represented by 1.1 Mbp of gerbera genome DNA), the frequency was one SSR for every 9.3 kb. The proportion of AT/TA motifs in the SSRs generated from the gerbera genome was much higher than the proportion of (CA)/(TG) repeat motifs (Figure 2). This result is consistent with surveys of microsatellites in Jatropha curcas L. (Sato et al., 2010) and also with studies on barley (Becker and Heun, 1995), Cannabis sativa (Algharim and Almirall, 2003) and citrus (Chen et al., 2006). However, studies of Brassica napus (Uzunova and Ecke, 1999) and Phaseolus vulgaris (Benchimol et al., 2007) found greater abundances of (CA)/(TG) motifs.
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Table 4. Characteristics of polymorphic microsatellite markers isolated for gerbera.
Locus
GenBank accession number
Repeat motif
Repeat status
Primer sequence (5’→3’)
GB-GJ-002
JF343527
(AAT)4
Perfect
F: TCAGTCTTCTTTGCCGTTTC
Fluoresce nt dye
Tm (°C)
Z
Expected Product size
Gerbera size range (bp)
FAM
58
188
203-206
2
# Alleles
W
R: TGATCCACGATTTCCCAG GB-GJ-011
JF343528
(CAC)5
Perfect
F: GTGCATGGCTGTGTTCAA R: CCCAGGACAAAGTAGCCC
HEX
58
255
234-273
4
GB-GJ-021
JF343529
(AAAG)4
Perfect
F: GCCTCCGAATTTGCTTTT
NED
58
283
299-303
2
GB-GJ-049
JF343530
(AT)4, (ACA)6
Compound
F: AATTGTCCTCGACTCCGTT
FAM
58
296
160-317
8
GB-GJ-059
JF343531
(AG)7
Perfect
R: ATCCATGTAGCCAACCCC F: TGTCACTGTGTGAGAGTCGC
FAM
58
189
182-211
4
R: TTCAAGCATGAGGGCAAG
R: GGATGACGATGACGAGGA GB-GJ-063 GB-GJ-074
JF343532
(ATC)4, (CAT)5
JF343533
(CAT)5
Imperfect
F: TTTCCCCCATCATCCTTT
FAM
58
192
194-235
4
Perfect
R: GGATGACGATGACGAGGA F: TCTCAAATTGCCTTCCGA
HEX
58
283
296-299
2
NED
58
197
195-218
4
R: GGTCTATCAAGGGGCCAG GB-GJ-077
JF343534
(TGA)6
Perfect
F: GAGCAAGCCATCATCACC R: CTTGCTTTCCTGCTCCCT
Z
W
Tm, annealing temperature; #Alleles , the total number of observed alleles among the 28 accessions.
Malausa et al. (2011) reported that enriched libraries are able to design primers about three times more than shotgun library. But, the genomic library enriched method have the disadvantage of the difficulty encountered in hybridization with AT and GC probe, which makes it to easily construct the self-complementary structure than the other kinds of repeat types of DNA sequences (Ashkenazi et al., 2001; Lagercrantz et al., 1993). Malausa et al. (2011) analyzed Apis mellifera using enriched and shotgun libraries; the percentage of microsatellite motifs of AT nucleo-tides was 8 and 39%, respectively. These results were similar to that detected in our gerbera shotgun library.
The importance of microsatellites as genetic markers and their potential use in various applications, such as diversity analysis or genetic mapping for crop improvement, have been well verified in plants (Csencsics et al., 2010; Lee et al., 2009; Takundwa et al., 2010). Despite their many advantages, SSR markers had not been developed or used in gerbera. Therefore, we developed and used SSRs to assess genetic diversity in gerbera accessions. Of these SSR markers, 75% were perfect type, 12.5% were imperfect and 12.5% were compound type. In the dendrogram shown in Figure 3, all 28 G. hybrida cultivars had no clear distinction between flower types. These differences could be attributed to the low number of SSR markers employed.
of
In this study, we reported the successful development eight polymorphic markers using 454-
pyrosequencing technology. We also tested the applicability of these markers in G. hybrida. Although, only eight markers have been tested here, these microsatellite markers provide a useful tool for gerbera breeding programs. The com-prehensive dataset from this study will contribute to further genetic improvements, genomic information and gene discovery in gerbera. To our knowledge, this is the first report on microsatellite markers for gerbera (G. hybrida) and it confirms the usefulness of this kind of marker as a tool for cultivar identification and the breeding of
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Figure 3. Dendrogram showing relationships among 28 G. hybrida accessions based on UPGMA of SSR profiles.
Table 5. Calculation of clonal diversity within groups.
Group Group-I Group-II Group-III Total Total diversity Average diversity within populations Z
Number of samples 16 9 3 28
Number of genotypes 14 8 3 25
Z
Nei’s div 0.98 0.97 1.00 0.99 0.98
Nei’s div: Nei’s (1987) genetic diversity corrected for sample size.
new cultivars. ACKNOWLEDGEMENT This study was supported by a grant (Code no. PJ006825) ->PJ008368 from the National Academy of Agricultural Science, RDA, Republic of Korea.
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African Journal of Biotechnology Vol. 11(29), pp. 7397-7407, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.3843 ISSN 1684–5315 © 2012 Academic Journals
Full Length Research Paper
Pathogenesis mechanism of Pestalotiopsis funerea toxin (Pf-toxin) on the plasmalemma of needle cells of different pine species Shujiang Li1, Tianhui Zhu1* Hanmingyue Zhu2, Shan Han1, Fanglian Li1, Wei Yang1 and Hua Yang1 1
College of Forestry, Sichuan Agricultural University, Ya’an, Sichuan, China. Department of Foreign Affairs Administration, Chengdu Institute, Sichuan International Studies University, Chengdu, China.
2
Accepted 14 March, 2012
The Pf-toxin (C5H11O5N) has been genetically associated with the pathogenesis mechanism in plasmalemma cells of pine needles in previous reports. In this study, a toxin was obtained from Pestalotiopsis funerea (called Pf- toxin) by concentrating and column chromatography. Responses of the needles of eight pine species against the toxin were investigated. The O 2 production rate, malondialdehyde (MDA) content, fatty acid composition, relative conductivity, and lesion length of the needles were determined. The severest damage and lipid peroxidation were exhibited by the needle plasmalemma of Pinus massoniana, Pinus yunnanensis, and Pinus tabuliformis. Pinus elliottii and Pinus taeda followed. Pinus armandi, Pinus radiata and Pinus thunbergii came last. The resistance capability of resistant species against the Pf-toxin precedes that of susceptible species. Key words: Pestalotiopsis funerea, Pestalotia needle blight, Pinus, resistance.
INTRODUCTION Pestalotia needle blight caused by Pestalotiopsis funerea (Desm.) Stey is a common and serious disease in young pine trees. This has been the most important conifer disease in Chinese forests since 1980 (Qiu et al., 1980; Wu and Wei, 1987). To date, many pine species have been infected by this disease. Such species include Pinus massoniana Lamb., Pinus yunnanensis Franch., Pinus armandii Franch., Pinus tabulaeformis Carr., Pinus thunbergii Parl., Pinus elliottii Engelm, Pinus caribaea Morelet, Pinus taeda Linn., and Pinus latteri Mason. P. massoniana and Pinus tabuliformis are the most seriously susceptible to the diseases. Their foliage turns brown and their twigs die. Successive years of severe infection result in decreased growth, and ultimately, death. Previous studies have focused mainly on the pathogen and symptoms (Zhao and He, 1993; Huang and He, 2000; Sutarman et al., 2004), the hosts and
*Corresponding author. E-mail: zhutianhui@yahoo.cn, zhuth1227@tom.com. Tel: 086-835-2882335.
regularity (Liang et al., 2002; Jeewon et al., 2004), as well as disease control (Qiao et al., 2006; Jiang et al., 2007; Pan et al., 2010). However, the toxicity of the compounds produced by P. funerea (Pf-toxin) has only been reported by us. We have studied the cultivating conditions (Zheng and Zhu, 2006), isolation and purification (Zhu et al., 2003), as well as the structure (Zhu et al., 2005) of the Pf-toxin. Therefore, the pathogenesis mechanisms of the Pf-toxin on pines are still unknown. Pathogens damage hosts mainly producing toxins, enzymes, and/or altering the metabolism of phytohormones (Heitefuss and Williamas, 1991). Previous studies have demonstrated that the toxin destroys the structure and function of the plasmalemma, cell nucleus, mitochondria, chloroplasts, as well as ribosomes (Damann et al., 1974; Holden, 1984; Ye et al., 2000; Manning and Ciuffetti, 2005; Potrich et al., 2009). Changes in the plasmalemma permeability are an ordinary reaction of plant tissues upon toxin exposure. These changes are usually characterized by electrolyte leakage (EL) as well as depolarization and hyperpolarization of
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the membrane electric potential energy (Shah, 2005). Thus far, these effects have been reported to be exhibited by toxins such as those involved in fusariose on pineapple (Hidalgo et al., 1998), Ptr ToxA on wheat (Rasmussen et al., 2004), as well as AK-I, AK, and AM-I on pear (Park et al., 1987; Shimizu et al., 2006). In addition, Zhang et al. (2006) and Jiang et al. (2007) have suggested that microbial toxins cause increased potential differences and the eventual disruption of the host cell. However, Cahill (1996) has indicated that, in Eucalyptus marginata seedling infected by Pc-toxin, EL may be a resistance reaction and not a result of infection. Nevertheless, the Pf-toxin has been confirmed as one of the major factors in the pathogenesis of Pestalotia needle blight on pine trees (Luo and Zhu, 2002; Zhu et al., 2003, 2005). The introduction of the mature toxin into the pine needles has resulted in a typical response similar with the disease symptoms induced by the pathogen. Such symptoms include chlorosis, necrotic bands on live needles, and ultimately, death of the needles. However, the mechanisms by which the Pf-toxin acts on the plasmalemma and of lipid peroxidation have not been reported until now. Moreover, a lot of reports had claimed that a series of reactions of the plasmalemma might reflect the resistance of plants against the toxin, and then indirectly revealed their resistance level against pathogens (Lu et al., 2004; Zhen and Li, 2004; Yang et al., 2011). Although so far, the data of the relationship between Pf-toxin and pines’ resistance is still lacking. The Pf-toxin is usually removed from P. funerea by column chromatography. On this basis, the present study aimed to determine the effects of this toxin on needle cells of different pine species. The parameters evaluated were the production rate of the superoxide anion radical (O2 ), malondialdehyde (MDA), which is an indicator for lipid peroxidation, membrane fatty acid composition, relative conductivity, and lesion length in the needles of different pine species. The pathogenesis mechanism of the Pf-toxin on the plasmalemma was proposed, and the resistance of different pine species was also determined by above tests.
MATERIALS AND METHODS Isolation and purification of the Pf-toxin from culture filtrates P. funerea (Desm.) Stey (provided by the Laboratory of Forest Protection, Sichuan Agricultural University) was statically cultured in liquid potato dextrose agar at 25°C for 27 days. The culture was filtered through double gauze, and the filtrate was centrifuged at 10 000 ×g for 30 min. The supernatant was filtered through a 0.45 mm millipore filter and used as a crude toxin extract (Dubery and Smit, 1994). The crude toxin was loaded onto silica gel for column chromatography (100 to 200 mesh) with the selective phase (nbutanol: methanol: H2O = 4:1:2). The flow rate was kept constant at 2 ml·min-1. The compound was confirmed as C5H11O5N (Mw = 165) using mass spectrometry, nuclear magnetic resonance, and infrared spectroscopy (Zhu et al., 2005). The purified toxin was diluted by sterile distilled water to a concentration of 100 μg·ml-1
and was stored at 4°C.
Plant materials and toxin treatments Five-year-old pines were planted at the arboretum of Sichuan Agricultural University. These included the susceptible species P. massoniana, P. tabuliformis, and P. yunnanensis, as well as the resistant ones P. armandi, P. elliottii, P. taeda, Pinus radiata, and P. thunbergii. One-year-old needles were used for seven toxin treatments (0, 6, 12, 24, 48, 72, and 96 h) with the impregnation method (Ho et al., 1996); the clean needles from the shoots were cultured in solution containing 1 ml of 100 μg·ml-1 purified toxin in centrifuge tubes at 25°C. A control treated with sterile distilled water was used. 10 g needles were used per one treatment and each treatment was repeated five times (total 400 g needles per species). All treated needles were measured lesion lengths firstly, and then used to assay the other items.
Determination of lesion lengths in the pine needles After 0, 6, 12, 24, 48, and 96 h of toxin treatment, lesion lengths (mm) in the pine needles were measured. Measurement of the superoxide anion radical (O2-) production rate The O2- production rate was determined by the hydroxylamine oxidation method (Elstner and Heupel, 1976; Wang and Luo, 1990) with some modifications. About 0.5 g of needle samples was homogenized with 3 ml of 65 mmol·l-1 potassium phosphate buffer (pH 7.8). The solution was then centrifuged at 10 000 ×g for 15 min. Subsequently, 0.5 ml of the supernatant was mixed with 0.5 ml of 65 mmol·l-1 potassium phosphate buffer (pH 7.8) and 1 ml of 10 mmol·l-1 hydroxylamine chloride. The homogenized mixture was warmed for 20 min at 25°C. About 1 ml of 58 mmol·l-1 paminobenzene sulfonic acid and 1 ml of 7 mmol·l-1 α-naphthylamine were added. The mixture was warmed for 20 min at 25°C. About 4 ml of n-butyl alcohol was added, and the final supernatant was used for measuring the absorbance at 530 nm. A standard curve was constructed using the nitrogen dioxide radical (NO 2-) to calculate the production rate of O2-. This rate was expressed in μmol·min-1·g-1FW. Analysis of the fatty acid composition Membrane fatty acids were extracted following the procedure of Su et al. (1980) with slight modifications. About 2 g of needle samples were heated for 5 min at 100°C to inactivate enzymes. Homogenization with chloroform-methanol (1:2, v/v) followed. The homogenized mixture was centrifuged at 10 000 ×g for 10 min, and the supernatant was mixed with 2 ml of chloroform for washing. Subsequently, 2 ml of 0.76% NaCl were added. After standing and layering, the subnatant liquid was mixed with 1 ml of methanol, and was washed three times with petroleum ether (boiling temperature, Tb, range = 90 to 120°C). The thrice-washed subnatant liquids were mixed back together, and were rewashed twice with petroleum ether before removing the superstratum. The extract was vacuum dried with drops of 0.4 N KOH and 1 ml of petroleum ether (Tb range = 30 to 60°C)/benzene (1:1, v/v). After allowing the extract to stand for 15 min, distilled water was added. The mixture was allowed to stand for another 5 min. The supernatant was used in the fatty acid analyses. The analyses were performed on a gas chromatograph (HP 6890, Hewlett Packard, Avondale, PA, USA) equipped with a
Li et al.
mass selective detector (Agilent 5973, Hewlett Packard). A capillary column (60 m × 0.25 mm; BPX 70, SGE, Victoria, Australia) was used. Helium was utilized as the carrier gas (1.2 ml·min-1), and the injection volume was 1 μl. The injection was done in the splitless mode for 2 min. The oven temperature was increased from 65 to 230°C at 5°C·min-1, and was maintained for l0 min at 230°C. The temperature during both injection and detection was 230°C. The results were expressed as relative percentages of each fatty acid, which were calculated as the ratio of the surface area of the considered peak to the total area of all peaks. All analyses were made in triplicate. All chemicals used were analytical grade.
Assessment of lipid peroxidation The level of lipid peroxidation was measured by the amount of MDA, a product of unsaturated fatty acid peroxidation. The method of Heath and Packer (1968) was used with slight modifications. About 0.5 g of needle samples were homogenized in 8 ml of 10% trichloroacetic acid and the homogenate was centrifuged at 4000 ×g for 20 min. About 2 ml of 0.6% thiobarbituric acid were added to 2 ml of the supernatant. The sample was then incubated at 100°C for 20 min. The reaction was stopped by placing the reaction tubes in an ice bath. The samples were then centrifuged at 10 000 ×g for 30 min. The supernatant was removed, and the absorptions at 532, 600, and 450 nm were obtained. The concentration was calculated according to the following formula: CMDA (µmol·l-1) = 6.45 (OD532 – OD600) – 0.56OD450. The MDA content was computed according to: [MDA] (µmol·g-1FW) = CMDA × extract volume (ml) / fresh weight (g). Assessment of relative electrical conductivity Relative electrical conductivity (EL) was measured as described by Ye et al. (2000) with slight modifications. The conductivity was determined using an automatic conductivity meter (DDS-307). The initial conductivity was described as E1 with the needle sample treated by the toxin. On the other hand, E2 represented needle samples treated by sterile distilled water. The formula of the relative conductivity is: Relative conductivity (%) = (E1 – E2) / E2 × 100. Statistical analyses All data were subjected to one-way analysis of variance (ANOVA) to determine the significance of individual differences between different Pf-toxin treatments at P < 0.05 level. Significant means were compared using the least significant difference (LSD) test. All statistical analyses were conducted using the commercial SPSS statistical package (Version 13.0 for Windows, SPSS Inc., Chicago, USA).
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before 12 h, and varied afterwards among the eight pine species studied (Figure 1). For P. massoniana, P. tabuliformis, and P. yunnanensis (first group), their O2 production rates did not significantly differ from one another before 12 h. From 12 to 48 h, the rates declined and then increased thereafter. The O2 production rate of P. massoniana was significantly higher than the others. For P. elliottii and P. taeda (second group), the rates gradually increased before 48 h, and then decreased thereafter. The succeeding rates were always higher than the initial rates. The rates of P. elliottii and P. taeda were close. For P. radiata, P. thunbergii, and P. armandi (third group), the rates inconspicuously increased for the entire experiment. The rate of P. radiata was the highest in this group. Overall, the O2 production rate had the trend: first group > second group > third group.
Fatty acid components of pine needles The fatty acid components of the plasmalemma of eight pines species were cetylic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2) and linolenic (C18:3) acids (Table 1). In saturated fatty acids (SFAs), the content of C16:0 was higher than that of C18:0. In addition, C18:3 content was the highest in unsaturated fatty acids (USFAs). The content of C16:0 in the needles of P. massoniana and P. tabuliformis was the highest. In the other species, the content of C18:3 was the highest. The content changes in these components differed from one another according to the toxin treatment time. The SFA contents (C16:0 and C18:0) declined from 0 to 12 h, and then increased afterwards. The USFA contents (C18:1, C18:2 and C18:3) had the opposite trend (Figure 2). Among the eight pine species, the SFA content was the highest in P. massoniana, and was significantly different from the other species. In contrast, the SFA content was the lowest and relatively constant in P. armandi. On the other hand, the USFA content was significantly highest in P.armandi and remained constant. The USFA content rapidly declined after 12 h in P. massoniana, P. tabuliformis, P. yunnanensis, and P. elliottii. Moreover, the results of index of unsaturated fatty acids (IUFA) (Figure 3) indicated a trend similar with USFA content. The IUFA of all eight pine species decreased after Pftoxin treatment. However, the IUFA of P. armandi showed a relatively smooth change, whereas that of P. massoniana and P. tabuliformis rapidly decreased.
RESULTS
Malondialdehyde (MDA) content in pine needles
Superoxide anion radical production rate in the pine needles
Lipid peroxidation measured as an increase in MDA content is known to be a good indicator of oxidative damage to membrane lipids. In this study, the MDA content increased with the Pf-toxin treatment compared with the control. The differences were significant between the control and toxin-treated groups for all eight pine
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O2 is the mono-negatron reduction product of O2, and is the active oxygen species initially produced by an organism. The rate of O2 production gradually increased
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O2 - producing rate [µmol/(min·gFW)]
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Figure 1. O2- production rate in the plasmalemma of pine needles treated with the Pf-toxin. O2- production rate was indirectly replaced with the absorbance amount at 530 nm (A530), and A530 was converted to the concentration of [NO2-] according to the standard curve of nitrous acid colour reaction (Elstner and Heupel, 1976). O 2- production rate (µmol·min-1·g-1FW) = ([NO2-] concentration × 2 × total volume of solution) / (warmed time × fresh weight of plant tissue). Data in the same column followed by different lowercase letters indicate significant differences by the LSD test (P < 0.05, n = 5). LSD, Least significant difference.
Table 1. Fatty acid components of pine needles treated with the Pf-toxin.
P. massoniana
0 6 12 24 48 72 96
C16:0 48.15 38.75 32.56 36.26 38.71 47.14 60.03
Component (%) C18:0 C18:1 C18:2 14.01 2.25 17.22 12.02 5.00 22.00 9.03 6.45 28.05 12.00 4.08 22.28 14.51 2.34 21.33 15.08 2.06 17.25 15.56 0.50 11.55
C18:3 18.37 22.23 23.91 25.38 23.11 18.47 12.36
P. yunnanensis
0 6 12 24 48 72 96
29.89 23.00 20.80 25.60 26.04 29.11 39.19
10.01 6.40 5.33 9.91 12.13 14.55 18.36
2.54 1.59 1.89 1.26 0.67 0.35 0.33
21.38 26.51 30.85 27.45 26.12 22.56 17.08
36.18 42.50 41.13 35.78 35.04 33.43 25.04
P. taeda
0 6 12 24 48 72 96
27.54 21.59 20.16 22.20 26.14 32.97 35.99
9.61 8.13 7.09 9.05 10.79 12.35 15.50
3.49 6.87 7.97 6.89 5.36 3.38 2.04
25.86 30.32 31.25 29.78 26.67 23.09 21.69
33.50 33.09 33.53 32.08 31.04 28.21 24.78
Species
Time (h)
P. tabuliformis
0 6 12 24 48 72 96
C16:0 33.54 24.46 21.25 24.66 37.17 42.51 55.96
Component (%) C18:0 C18:1 C18:2 5.69 8.99 19.26 3.91 11.27 11.61 2.57 12.14 14.06 7.17 7.72 19.82 10.17 1.26 15.84 11.21 1.05 12.34 14.84 0.26 10.00
C18:3 32.52 48.75 49.98 40.63 35.56 32.89 18.94
P. elliottii
0 6 12 24 48 72 96
27.67 22.02 19.48 22.55 27.60 33.87 36.65
9.95 8.74 6.67 9.73 10.43 14.32 16.30
3.01 6.35 7.34 5.09 3.12 2.56 1.99
25.40 30.50 31.05 28.77 25.53 22.77 22.81
33.97 32.39 35.46 33.86 33.32 26.48 22.25
P. radiata
0 6 12 24 48 72 96
20.12 14.06 15.69 16.41 19.03 23.30 25.54
3.97 2.05 2.99 3.78 7.97 9.19 12.31
7.41 10.55 10.01 9.50 7.20 4.41 2.79
26.69 30.25 28.77 28.00 25.15 22.88 21.95
41.81 43.09 42.54 42.31 40.65 40.22 37.41
Species
Time (h)
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P. thunbergii
16.36 12.52 14.14 15.33 16.20 19.00 21.21
3.38 1.87 2.98 4.74 5.93 8.02 8.59
9.99 11.65 11.47 11.03 10.41 8.14 8.07
29.07 31.19 30.15 28.16 27.04 25.35 24.94
41.20 42.77 41.26 40.74 40.42 39.49 37.19
0 6 12 24 48 72 96
P. armandi
9.51 7.62 10.17 11.22 11.75 12.33 12.66
â&#x20AC;&#x201C; â&#x20AC;&#x201C; 0.41 0.45 0.46 0.46 0.46
P.massoniana P.taeda
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P.tabuliformis P.radiata
P.yunnanensis P.thunbergii
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48.98 49.05 48.84 48.57 48.54 48.48 48.44
Percent of USFA (%)
Percent of SFA (%)
29.17 30.06 28.12 27.67 27.31 26.95 26.74
P.elliottii P.armandi
70 a 50
12.34 13.27 12.46 12.09 11.94 11.78 11.7
A
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72
96
Figure 2. Percent saturated fatty acids (SFA) (a), and unsaturated fatty acids (USFA) (b), in the plasmalemma of pine needles treated with the Pf-toxin. The content of SFA was the sum of C16:0 and C18:0 content; the content of USFA was the sum of C18:1, C18:2 and C18:3 content. Percent SFA (%) = SFA content / total content of fatty acid; percent USFA (%) = USFA content / total content of fatty acid. Data in the same column followed by different lowercase letters indicate significant differences by the LSD test (P < 0.05, n = 5). LSD, Least significant difference.
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P.massoniana
250 a
a
200
b c
b
IUFA (%)
c cc
b c c d
c
b b
bb c b d
e 100
a
a
a
P.tabuliformis
a
P.yunnanensis
b
150
a
b
c b c c c d
b
c
P.elliottii
b
b
b
c
c d
c c
c d
c
e
P.taeda P.radiata
c
dd
d
b
c
P.thunbergii P.armandi
e e
50
0 0
6
12
24
48
72
96
Time (h) Figure 3. Index of unsaturated fatty acids (IUFA) in the plasmalemma of pine needles treated with the Pf-toxin. IUFA (%) =1Ă&#x2014;C18:1(%) + 2Ă&#x2014;C18:2(%) + 3Ă&#x2014;C18:3(%). Data in the same column followed by different lowercase letters indicate significant differences by the LSD test (P < 0.05, n = 5). LSD, Least significant difference.
species (Table 2). In the Pf-toxin treatment group, the MDA content increased from 6 to 12 h, and decreased from 12 to 24 h among all the pine species. The MDA content in P. massoniana was the highest, and was significantly different from the other pines. The MDA content in P. thunbergii was the lowest during the entire experiment. Figure 4 shows that the changes in MDA content were similar for all pines. The MDA content increased from 6 to 12 h, and peaked at 12 h. The MDA content then decreased rapidly until 24 h, except in P. massoniana (48 h). After 24 h, the changes remained constant. The increase rate was significantly highest in P. tabuliformis, followed by P. massoniana and P. yunnanensis with non-significant differences. P. elliottii, P. taeda, P. radiata, P. thunbergii, and P. armandi came last with non-significant differences.
Relative electrical conductivity (EL) in pine needles The effects of the Pf-toxin on the structure and function of the plasmalemma is usually expressed as the EL, which is measured as the relative conductivity (Figure 5). The relative conductivity indices of each pine species increased until the peak was reached during Pf-toxin treatment, and then became steady. However, the degrees of relative conductivity changed differently for each pine species. In P. massoniana, P. tabuliformis, and
P. yunnanensis, the changes in the relative conductivity were similar as the index rapidly increased from 6 to 24 h; afterwards, the high levels were maintained. On the other hand, the degrees of relative conductivity in P. elliottii and P. taeda were significantly lower than those of the aforementioned three pine species, although in a proportional manner. Moreover, the increased amplitudes of P. radiata, P. thunbergii, and P. armandi were less inconspicuous than the aforementioned five pine species. These indices increased up to 48 h of toxin treatment.
Effects of Pf-toxin on lesion length of pine needles Disease spots are the visible symptoms of pine needles infected by Pf-toxin. The lesion length is one of the criteria for determining the degree of infection. In this study, changes in the lesion lengths are shown in Figure 6. The lesion length increased with the time of toxin treatment. From 0 to 24 h, the lesion lengths sharply increased. However, from 24 to 96 h, these indices steadily increased. All the lesion lengths of the eight pine species had significant differences after 6 h of treatment time. There were three tendencies in lesion length changes. First is the high level length increase (P. massoniana, P. tabuliformis, and P. yunnanensis), second is the middle level increase (P. elliottii and P. taeda), and third is the low level increase (P. radiata, P.
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Table 2. Malondialdehyde (MDA) content (μmol·g-1FW) in the plasmalemma of pine needles.
Species
Treatment
Time (h) 24 48 Bd Ef 2.36 ± 0.03 1.60 ± 0.01 Cd Gf 2.16 ± 0.01 1.44 ± 0.03
P. massoniana
Pf-toxin Control
6 Ab 3.12 ± 0.01 Ba 3.28 ± 0.01
12 Ba 3.56 ± 0.03 Dc 2.40 ± 0.02
72 Ae 2.24 ± 0.02 Ce 2.00 ± 0.04
96 Ac 2.80 ± 0.02 Bb 2.56 ± 0.01
Pf-toxin Control
Cb
2.72 ± 0.01 Da 2.64 ± 0.01
Aa
3.60 ± 0.02 Jc 1.76 ± 0.01
Ac
2.48 ± 0.03 Hd 1.60 ± 0.01
Ad
P. tabuliformis
2.24 ± 0.03 Cb 2.00 ± 0.03
1.92 ± 0.02 Fc 1.76 ± 0.01
Ee
1.80 ± 0.04 Hd 1.60 ± 0.01
Pf-toxin Control
Eb
2.56 ± 0.03 Fa 2.48 ± 0.02
Ca
3.44 ± 0.02 Fb 2.28 ± 0.02
Cd
2.16 ± 0.01 Ec 2.00 ± 0.02
Ac
P. yunnanensis
2.24 ± 0.03 Cc 2.00 ± 0.03
1.60 ± 0.02 Ie 1.48 ± 0.02
Hf
1.76 ± 0.01 Hd 1.60 ± 0.01
Pf-toxin Control
Gb
2.16 ± 0.03 Ha 2.08 ± 0.03
Ea
2.36 ± 0.02 Gb 2.00 ± 0.03
Ed
2.00 ± 0.02 Fd 1.84 ± 0.02
Bc
P. elliottii
2.08 ± 0.02 Dc 1.92 ± 0.01
2.04 ± 0.03B Dc 1.96 ± 0.03
2.00 ± 0.01 Ec 1.96 ± 0.02
Pf-toxin Control
Id
2.00 ± 0.03 Jc 1.88 ± 0.01
Fa
2.28 ± 0.03 Ic 1.88 ± 0.03
Dbc
2.12 ± 0.04 Ea 2.00 ± 0.02
Cd
2.00 ± 0.04 Dbc 1.92 ± 0.02
Cd
P. taeda
2.00 ± 0.01 Dab 1.96 ± 0.03
2.08 ± 0.03 Da 2.00 ± 0.03
Pf-toxin Control
Kc
1.76 ± 0.01 Lb 1.68 ± 0.01
Ga
2.00 ± 0.02 Jb 1.68 ± 0.02
Fb
1.84 ± 0.04 Ga 1.76 ± 0.02
Ed
1.60 ± 0.02 Ec 1.60 ± 0.05
Fc
P. radiata
1.76 ± 0.01 Gb 1.68 ± 0.02
1.60 ± 0.03 Hc 1.60 ± 0.03
Pf-toxin Control
Lb
1.68 ± 0.02 Mb 1.60 ± 0.03
Ha
1.92 ± 0.02 Ka 1.68 ± 0.01
Hc
1.60 ± 0.01 Ic 1.52 ± 0.02
Fd
1.52 ± 0.02 Gd 1.44 ± 0.01
Jf
P. armandi
1.44 ± 0.03 Jd 1.44 ± 0.02
1.48 ± 0.02 Jd 1.44 ± 0.01
Pf-toxin Control
Mb
1.60 ± 0.02 Ma 1.60 ± 0.01
Ja
1.76 ± 0.01 Lb 1.52 ± 0.03
Ic
1.52 ± 0.03 Ib 1.52 ± 0.02
Fc
1.52 ± 0.02 Fb 1.52 ± 0.01
Kd
P. thunbergii
1.40 ± 0.01 Lc 1.36 ± 0.03
1.28 ± 0.03 Ld 1.24 ± 0.01
Ff
Ge
cd
Dd
Cc
Hd
Ie
Ke
Data in the same row followed by different lowercase letters indicate significant differences between different exposure times by the LSD test (P < 0.05, n = 5). Data in the same column followed by different capital letters indicate significant differences within pine species by the LSD test (P < 0.05, n = 5). LSD, Least significant difference.
thunbergi and P. armandi). Correlation analysis between physiological indices and lesion lengths Correlation coefficients were determined, and the results are shown in Table 3. All indices reached or exceeded significant levels. The correlation coefficients of P. thunbergii and P. armandi were also lower than the others, except for IUFA. The opposite was true for P. massoniana and P. tabuliformis. DISCUSSION Increased permeability and decreased stability are the indications that the plasmalemma of a plant cell has been exposed to a phytotoxin. These indicators accurately reveal that the plasmalemma is the initial toxin action site (Hartung, 1987; Yang et al., 2000). The results of the present study have suggested that the Pf-toxin may alter the permeability of pine needle plasmalemma and cause disease spots. These effects became more apparent with
longer treatment times. Moreover, the capability of pine species in resisting toxic effects depended on the resistance of the plasmalemma against the Pf-toxin. Zhen and Li (2004) had confirmed that Verticillium dahliae toxin made different damage degrees to cell wall and plasmalemma in different cotton species; Yang et al. (2011) had reported that Phytophthora infestans toxin had effected different reactions of three potato species; on the basis of these theories, this study has indicated that the plasmalemma of resistant pine species incurred less damaged pine needles than that of susceptible species. Therefore, we believe that the plasmalemma is the action site of the Pf-toxin. The first reaction during a phytotoxin-induced oxidative burst is believed to be the one-electron reduction of molecular oxygen to form the superoxide anion (O2 ) (Mehdy, 1994; Gechev et al., 2006). Keppler and Novacky (1987) reported that lipid peroxidation and pathogen-induced changes in membrane components were virtually the results of an O2 startup. Similarly, in this study, the rate of O2 production increased with increased toxin treatments in eight species of pines in varying degrees. Moreover, the rate of O2 production rapidly increased in
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120 a Rela tive percent of MDA content (%)
100
P.massoniana
P.tabuliformis
P.yunnanensis
P.elliottii
P.taeda
P.radiata
P.thunbergii
P.armandi
80 a
60
b
b
40 c
20 a
0
a a b a a a a 6
c
c c
c b
b
12
a b b bc
a a ac b b c
24
48
-20
a
a b aa b bb b 72
aa b b c c c 96
Time (h)
Figure 4. MDA content (relative percent) in pine needles. MDA content (relative percent, %) = [(MDA content in the toxin-treated group â&#x20AC;&#x201C; MDA content in the control) / MDA content in the control] Ă&#x2014; 100%. Data in the same column followed by different lowercase letters indicate significant differences by the LSD test (P < 0.05, n = 5). LSD, Least significant difference; MDA, malondialdehyde.
35
P.massoniana
a
Rela tive conductivity (%)
30
a b
b
b
c
25
c
P.tabuliformis
a
a
P.yunnanensis
b
c
c
P.elliottii P.taeda P.radiata
20
P.thunbergii
a 15
P.armandi
a
c
10
b
d
d
5
a
0
b aa ab ab b b 6
e e
e 12
d e ff
f
d e ff
f
24
48
d e f f
f
72
e ff
f
96
-5 Time (h) Figure 5. Relative conductivities in pine needles. Relative conductivity (%) = (E 1-E2)/E2Ă&#x2014;100; E1, the initial conductivity with the needle sample treating by toxin; E2, the initial conductivity with the needle samples treating by sterile distilled water. Data in the same column followed by different lowercase letters indicate significant differences by the LSD test (P < 0.05, n = 5). LSD, Least significant difference.
Li et al.
10
Lesion length (mm)
8
a
a
a b
b
b
b
b
b
a 6 4
c
a b b c d dc e
2 a aa 0 a a aa 0 -2
6
f
e g
d
d
d f
f
24 Time (h)
48
P.massoniana P.tabuliformis P.yunnanensis P.elliottii P.taeda P.radiata P.thunbergii P.armandi
e
e
e
h 12
c
c
d
c c
c
c
b
7405
f
96
Figure 6. Lesion lengths of pine needles. Lesion lengths (mm) in the pine needles were measured after 0, 6, 12, 24, 48, and 96 h of toxin treatment. Data in the same column followed by different lowercase letters indicate significant differences by LSD test (P<0.05, n=5). LSD, Least significant difference.
Table 3. Correlation coefficients among physiological indices and lesion lengths.
Test -
O2 producing rate MDA content IUFA Relative conductivity
P. massoniana 0.877* 0.947* 0.847* 0.946**
P. tabuliformis 0.883* 0.971** 0.850* 0.933**
Species P. P. yunnanensis elliottii 0.801* 0.765* 0.911** 0.904** 0.840* 0.840* 0.973** 0.978**
P. taeda 0.639* 0.938** 0.918** 0.985**
P. radiata 0.681* 0.770* 0.862* 0.904**
P. thunbergii 0.592* 0.759* 0.936** 0.897*
P. armandi 0.635* 0.750* 0.927** 0.897*
MDA, malondialdehyde; IUFA, index of unsaturated fatty acids. Lesion lengths in 24 h were used for correlation analysis. Data followed by different letters indicate significant differences at P < 0.05 by the LSD test. *Significant correlations among physiological indices and lesion lengths. LSD,
Least significant difference.
the initial stage of toxin treatment. This result showed that all the pines were sensitive to the toxin. However, the rate of O2 production in P. radiata, P. thunbergii and P. armandi remained stable in later stages. This stability may be due to the stronger resistance of these three species than the others. MDA is the product of lipid peroxidation and membrane damage. These phenomena result in physiological and biochemical disorders in related tissues, and could be regarded as signs of plasmalemma damage (Yuan et al., 2007). In this study, the MDA content increased sharply during Pf-toxin treatment. This result could confirm the occurrence of lipid peroxidation. However, after 12 h, the MDA content probably decreased because of the instability of MDA and the aging of cells (Ye et al., 2000). The degree of EL is also an important parameter in determining changes in plasmalemma permeability (Zhang et al., 2008). In the EL experiment, relative
conductivity initially increased, and then remained stable. These results suggest that all pine species had tolerance to the Pf-toxin, but the resistant pine species were more tolerant than the susceptible species. Fatty acids are key nutrients associated with energy production and storage as well as gene regulation (Jump, 2004). Fatty acids are also the essential components of cell membrane phospholipids (Van der Vusse et al., 1992). Kasamo et al. (1992) indicated that the phase transformation of plasmalemma occurs with difficulty, and that the plasmalemma may modulate the degree of unsaturation to improve membrane fluidity. The IUFA of all the pine species changed after Pf-toxin treatment. The SFA contents in the pine needles of P. armandi, P. thunbergii, and P. radiata were lower than in those of P. massoniana, P. tabuliformis, and P. yunnanensis. In contrast, the USFA contents had opposite trends. These results have indicated that the needles of P. massoniana,
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P. tabuliformis, and P. yunnanensis were more easily damaged by lipid peroxidation. Their unsaturated bonds were oxidized in partial fatty acids (Wang et al., 2006). The needles of P. massoniana were the most seriously damaged. Nevertheless, the changes in the SFA and USFA contents did not increase or decrease only in response to the superoxide anion radical (Ye et al., 2000). Subsequently, we found that the changes in USFA contents (C18:1, C18:2, and C18:3) were opposite to the SFA contents (C16:0 and C18:0). These results are consistent with previous studies on P. elliottii (Ye et al., 2000), Pleurotus sp. (Pedneault et al., 2007), kiwifruit (Antunes and Sfakiotakis, 2008), P. tabuliformis (Ma et al., 2010), and Spiraea sp. (Liu et al., 2011). The results of the present study have indicated that lesion lengths in pine needles rapidly increased before 24 h of toxin treatment. This finding significantly correlated with the rate of O2 production, MDA content, IUFA, and relative conductivity. Based on the study of Guo et al. (2005), we presumed that lesion lengths were visible symptoms of phytotoxin exposure. We also presumed its close relation to internal physiological indices. Conclusion In conclusion, although phytotoxins may change plasmalemma permeability and damage cell tissues, the plasmalemma can self-adjust and recover. More importantly, our data reveals the resistance capabilities of different pine species. In the present study, certain pine species (P. massoniana, P. tabuliformis, and P. yunnanensis) whose plasmalemma permeability increased and with damaged cells and tissues have less resistance capabilities, while others (P. armandi, P, elliottii, P, taeda, P, radiata, and P, thunbergii). However, the activation of toxin degradation and the relief of toxicity in resistant species remain unclear. Nevertheless, the Pftoxin could be used to select pine species resistant against pine needle blight. Further research on the permeation of the Pf-toxin into the plasmalemma and on its other molecular and cellular targets is necessary, as well as the signal transduction pathway involved in plant resistance needs to be investigated.
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African Journal of Biotechnology Vol. 11(29), pp. 7408-7416, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.1604 ISSN 1684–5315 © 2012 Academic Journals
Full Length Research Paper
In vitro propagation through root-derived callus culture of Swertia chirata Buch.-Ham. ex Wall Manu Pant1*, Prabha Bisht1 and Manju P. Gusain2 1
Tissue Culture Discipline, Botany Division, Forest Research Institute, Dehradun, Uttarakhand, 248006, India. 2 Zoology and Biotechnology Department, H.N.B. Garhwal University, Srinagar Garhwal, Uttarakhand, India. Accepted 2 August, 2011
A procedure for regeneration of complete plantlets of Swertia chirata via indirect organogenesis is described. Callus was obtained from in vitro regenerated roots on Murashige and Skoog (1962) (MS) medium supplemented with varying concentrations of 6, benzylaminopurine (BAP) and 2,4dichlorophenoxyacetic acid (2,4-D). 13.32 µM BAP in combination with 0.90 µM 2,4-D proved to be the most effective concentration for callus induction, multiplication and adventitious shoot regeneration from callus surface. The optimal hormone combination for shoot multiplication was shown to be 8.88 µM BAP, 2.85 µM indole-3 acetic acid (IAA) and 271.45 µM adenine sulphate (Ads) giving an average of 10.70 shoots after 4 weeks and 17.50 shoots after 8 weeks. Individual elongated shoots were rooted on half-strength MS medium supplemented with varying concentrations of auxins. Best rooting was obtained with 4.90 µM indole-3 butyric acid (IBA) where an average of 14.40 and 21.50 roots per shoot could be obtained after 4 and 8 weeks, respectively. In vitro raised plantlets with well developed shoots and roots were acclimatized successfully. Key words: Swertia chirata, in-vitro propagation, callus, adventitious shoots, rosette clumps.
INTRODUCTION Swertia chirata (family Gentianaceae; commonly known as chirata) is an indigenous species of temperate Himalaya, found at an altitude of 1200 to 3000 m from Kashmir to Nepal and also distributed in Bhutan, Khasi hills and Sikkim (The Wealth of India, 1976; Garg, 1987; Kirtikar and Basu, 1998; Pradhan and Badola, 2010). The species is priced for its bitter bioactive compounds viz. amarogentin, xanthones, iridoid glycosides, mangiferin and C-glucoflavones (Dalal and Shah, 1956; Asthana et al., 1991; Jensen and Schripsema, 2002; Mallikarjun et al., 2010). S. chirata can be traced through the medicinal history as a safe ethnomedicinal herb. It is used in the treatment of various ailments like chronic fever, malaria,
*Corresponding author. E-mail: himaniab@gmail.com. Abbreviations: MS, Murashige and Skoog (1962); BAP, 6, benzylaminopurine; NAA, α-naphthalene acetic acid; IBA, indole-3 butyric acid; IAA, indole-3 acetic acid; GA3, gibberellic acid; Ads, adenine sulphate; 2,4-D, 2,4-dichlorophenoxy acetic acid.
liver and stomach disorders, cold and cough, asthma and joint pains. It’s blood-purifying, antifungal and antihelmintic qualities have also been extensively exploited. Propagation of S. chirata through seeds is hampered by poor seed germination rate (only 2 to 4%), low seed viability and long gestation period (Anonymous, 1997; Rai et al., 2000; Joshi and Dhawan, 2005). This, in addition to indiscriminate extraction of the plants from the wilderness, subsequent habitat losses and lack of adequate commercial plantations have accelerated the genetic erosion of the species leading to it being characterized as critically endangered (Nayar and Shastry, 1990; Semwal et al., 2007; IUCN, 2008; Pradhan and Badola, 2010). Conventional approaches of propagation alone cannot guarantee the re-establishment and recovery of this important plant species. Consequently, the application of alternative reproducible micropropagation strategies has become inevitable for mass propagation and sustainable utilization of this age-old medicinal plant. There are only some reports on in vitro propagation of S. chirata. Ahuja et al. (2003), Chaudhuri et al. (2007),
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Koul et al. (2009) and Pant et al. (2010) have reported micropropagation of S. chirata via field-grown nodal explants, while seedling derived shoot tip explants were used in the study by Joshi and Dhawan (2007a) and Balaraju et al. (2009). Joshi and Dhawan (2007b) also performed ISSR marker analysis of genetic diversity among S. chirata genotypes of temperate Himalayan region of India. Direct shoot organogenesis from in vitro leaves and regeneration via immature seed cultures of S. chirata was reported by Chaudhuri et al. (2008, 2009). Wang et al. (2009) developed a method for in vitro shoot regeneration from leaves taken from field-grown plants. In vitro propagation of S. chirata from roots has been reported by Wawrosch et al. (1999) and Pant et al. (2010) who described direct shoot regeneration via root explants. To our knowledge, the present study would be the first report on in vitro propagation and mass multiplication of S. chirata via root segment derived callus culture.
MATERIALS AND METHODS Explant source In our earlier study on S.chirata (Pant et al., 2010), shoots were regenerated in vitro from nodal explants, further multiplied and subsequently rooted on half-strength MS medium supplemented with 4.90 µM IBA. In vitro grown roots harvested from these axenic cultures were used as explant material in the present study. Eight weeks old roots were aseptically removed from in vitro regenerated shoots and repeatedly washed in sterile distilled water to remove agar adhering to the surface. proximal, middle and distal root segments were excised from these roots and used as explants for callus induction.
Induction and development of callus Various combinations of phytohormones viz. BAP and 2,4-D were tested for their effect on callus formation on root explant. For this purpose, 10 mm sections of roots were incised and placed horizontally on agar gelled (0.7% w/v) MS medium and supplemented with 3% sucrose and different concentrations of BAP (4.44 to 22.20 µM) in combination with 2,4-D (0.90 to 4.52 µM). MS medium lacking growth regulators served as control. After 5 weeks of incubation, observations were recorded. Data on callus were scored as the percent of the root explant forming callus and degree of callusing. Response percent of in vitro root induced callus formation was calculated as: Number of explants in which callus was induced Response (%) =
×100 Total number of explants cultured
Degree of callusing = + (poor callusing); ++ (moderate callusing); +++ (good callusing); ++++ (very good callusing).
Multiplication and organogenesis)
differentiation
of
callus
(shoot
The callus obtained was transferred onto full-strength MS medium fortified with combinations of BAP (4.44 to 13.32 µM) and 2,4-D
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(0.90 to 2.26 µM). Data on organogenic response percent and multiplication rate of callus was scored after 4 weeks, while observations on number of shoots induced per callus clump and length of differentiated shoots were recorded after 5 weeks of incubation. Organogenic calli were transferred on to maintenance medium. Two parameters were used to assess organogenic response of callus as: Number of organogenic cultures Organic response of callus (%) =
×100 Total number of explants cultured
Final fresh weight of callus Multiplication rate =
×100 Initial weight of callus inoculated
In vitro shoot multiplication Regenerated shoots were used to assess the effect of growth regulators on further in vitro shoot multiplication. Full strength MS medium supplemented with varying concentrations of BAP (4.44 to 13.32 µM) alone and in combination with IAA (0.57 to 2.85 µM) and/or adjuvant Ads (271.45 µM) was tested for further multiplication of adventitious buds differentiated from root induced callus. Single shoots of size which were up to 1 cm were used for this purpose. Observations pertaining to average shoot number and average shoot length were recorded after periodic intervals of 4 and 8 weeks. Routine sub-culturing of multiple shoots was carried out at periodic interval of every 4-weeks.
In vitro rooting Experiments on in vitro rooting of in vitro raised shoots were attempted with 2 to 3 cm long shoots developed during shoot multiplication. These experiments were initiated after 180 days of sub-culturing to get maximum material for testing different hormone concentrations. Shoots were cultured on MS medium containing 2% sucrose and supplemented with auxins IAA (1.14 to 11.40 µM), IBA (0.98 to 9.80 µM) and NAA (1.07 to 10.74 µM). Single shoot was cultured for initiation of roots and observations pertaining to mean number of roots produced and mean root length were recorded after 4 and 8 weeks interval.
Medium and culture conditions The pH of medium was adjusted to 5.8 (using 1.0 N HCl or 1.0 N NaOH) before adding 0.7% agar (w/v) and sterilized by autoclaving at 15 lbs (1.8 kg/cm2) pressure at 121°C for 15 min. Cultures were incubated in culture room at 25 ± 1°C temperature and 60 to 65% relative humidity. To assess the culture conditions required for induction and multiplication of callus, explants were incubated both in complete dark conditions (16 h) and under a 16/8 h (light/dark) photoperiod with light supplied by cool-white fluorescent tubes (Philips, India) at an intensity of 2500 lux. All other experiments pertaining to in vitro shoot differentiation, shoot multiplication and in vitro rooting were carried out under a photoperiod of 16 h light and 8 h dark, maintained using photoperiodic stimulators.
Hardening and acclimatization For in vitro hardening, rooted shoots were transferred to 1/4 strength MS medium having 2% sucrose devoid of PGR for 7 days in flasks. Thereafter, they were transferred to polybags containing a
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mixture of soil : sand : manure (1:1:1), covered with perforated polythene bags and kept under agronet-shade house conditions. Acclimatized plants were later shifted to polybags filled with soil.
Statistical analysis All experiments were repeated thrice. Each treatment consisted of minimum of ten replicates. The data was analysed using analysis of variance (ANOVA) of completely randomized design (CRD) in GenStat 5 Edition 3.2 for PC/Windows NT {Copyright 1995, Lawes Agricultural Trust (Rothamsted Experimental Station)}. The significance level was determined at 5% (p ≤ 0.05), 1% (p ≤ 0.01) and 0.1% (p ≤ 0.001). Mean values of treatments were compared with least significance difference (LSD).
RESULTS Initiation and development of callus Observations after 2 to 3 weeks of incubation revealed enlargement of most of the root explants. Little incisions made on the explants resulted in the formation of callus. Callus induction occurred on all media combinations (Figure 1A). Final observations after 5 weeks showed that MS medium + 13.32 µM BAP + 0.90 µM 2,4-D proved to be most efficient in inducing caulogenic response in maximum (70%) explants with maximum degree of callusing (Figure 1B).
Callus growth organogenesis)
and
differentiation
(shoot
Callus obtained was further multiplied on MS medium fortified with different concentrations of BAP and 2,4-D (Table 1). All the treatments tried were significantly superior to control as no callus multiplication occurred in medium devoid of any plant growth regulator. The multiplied callus exhibited shoot organogenesis in 4 to 5 weeks after 2 to 3 sub-cultures. Shoots developed through organogenesis were excised and used for further in vitro shoot multiplication. This organogenic callus was sub-cultured again for callus multiplication and shoot formation after every 4 weeks. For sub-culturing initially, 50 mg of callus was inoculated for callus multiplication and shoot regeneration. Best callus multiplication rate of 11.63 folds was obtained in treatment 4 (13.32 µM BAP + 0.90 µM 2,4-D). Optimal amount of yellowish-white callus was obtained on this medium. The same media combination also gave the best organogenic response with maximum number of 8.10 shoots produced per callus clump in 100% of the callus cultured (Figure 1B). Furthermore, all cultures were maintained on MS medium supplemented with 13.32 µM BAP and 0.90 µM 2,4-D and regular sub-culturing was carried out every 4 weeks to multiply the callus and to maintain the organogenic potential of callus. Sub-culture interval of more than 5 weeks resulted in the formation of brownish
non-organogenic callus with a general decline in callus growth rate (data not shown). Organogenic callus multiplied and produced new shoots only under light conditions. Callus sub-cultured on organogenic medium and kept under dark conditions did not multiply and no shoot differentiation from callus was recorded.
In vitro shoot multiplication Adventitious shoots regenerated from callus were in the form of rosette clumps and needed elongation (Figure 1C). For this, a pulse treatment of GA3 (1.16 µM) was given and elongation was observed within 14 days. Subsequently, elongated shoots were used for in vitro multiplication (Table 2). Results in the present study showed the necessity of plant growth regulators for in vitro shoot multiplication as the shoots cultured on basal MS medium (devoid of any PGR) did not multiply and died after some time. Shoot number and length (as observed after 4 and 8 weeks) varied significantly in all the treatments tested. On BAP supplemented medium, results were significantly superior to the data recorded on medium containing Kn. Treatment 3 (8.88 µM BAP) gave an average of 6.30 shoots (1.02 cm shoot length) after 4 weeks and 9.30 shoots (1.58 cm shoot length) after 8 weeks. Comparatively, on medium fortified with Kn alone, a maximum of only 4.80 shoots (mean shoot length 0.99 cm) after 4 weeks and 7.70 shoots (mean shoot length 1.42 cm) after 8 weeks could be obtained in treatment 6 (9.29 µM Kn). Only BAP supplemented medium was used for further experiments on the effect of interactions of auxin IAA and adjuvant Ads. IAA at 2.85 µM concentration proved to be more effective in enhancing shoot multiplication rate and treatment 12 (8.88 µM BAP + 2.85 µM IAA) gave the best results of all the BAP + IAA combinations tested. Incorporation of adenine sulphate in BAP and IAA supplemented medium resulted in significant improvement in in vitro shoot multiplication. Best results with mean number of 10.70 shoots and mean shoot length of 1.89 cm after 4 weeks interval and mean number of 17.50 shoots with mean shoot length of 2.75 cm after 8 weeks period were obtained in treatment 15 (8.88 µM BAP + 2.85 µM IAA + 271.45 µM Ads) (Figure 1D). The results far exceeded the number of shoots and shoot length recorded in all other treatments. MS medium supplemented with 4.44 µM BAP was used for routine sub culturing of multiple shoots at an interval of every 4-weeks.
In vitro rooting Response to rooting was different in different concentrations of hormones. All the treatments tested in the present study varied significantly. In treatment 1 (half
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A
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B
C D
E
F
Figure 1. A) Callus induction; B) callus multiplication and shoot organogenesis; C) rosetting of adventitious shoots; D) in vitro shoot multiplication on MS + 8.88 µM BAP + 2.85 µM IAA + 271.45 µM Ads; E) in vitro regenerated plantlet; F) Ex vitro hardening.
strength MS medium without any plant growth regulator), no rooting could be obtained while all other treatments proved effective in root induction and in most of the treatments, root initiation was observed within 10 to 15 days of culture (Table 3).
Maximum rooting was recorded in treatment 12 (4.90 µM IBA) (Figure 1E). The treatment proved to be most efficient for all other treatments tested for in vitro rooting of adventitious shoots giving a maximum of 14.40 and 21.50 average number of roots (after 4 and 8 weeks
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Table 1. Effect of plant growth regulators’ interactions on callus multiplication and shoot organogenesis in S. chirata. Data were recorded after 5 weeks (50 mg callus cultured initially).
MS medium + PGR concentration (µM) Treatment
1 (Control) 2 3 4 5 6 7 8 9 10 11 Grand mean Significance LSD
BAP
2,4-D
0.00 4.44 8.88 13.32 17.76 22.20 4.44 8.88 13.32 17.76 22.20
0.00 0.90 0.90 0.90 0.90 0.90 2.26 2.26 2.26 2.26 2.26
Observation after 4 week Fresh weight of callus (mg) 000 204 468 581 525 209 377 341 357 283 203 323 *** 155.20
Nature of callus Yellow-white friable Yellow-white friable Yellow-white friable Yellow-white friable Yellow-brown friable Yellow-brown friable Brown-compact Brown-compact Brown-compact Brown-compact
Observation after 5 week
Callus multiplication rate (fold)
Organogenic response of callus (%)
Mean number of shoot per callus clump
0.00 4.08 9.36 11.63 10.49 4.18 7.53 6.82 7.13 5.66 4.06 6.45 *** 3.10
0 40 60 100 70 40 20 30 40 30 20
0.00 1.50 2.30 8.10 4.70 1.50 0.80 0.80 1.60 0.80 0.40 2.05 *** 1.76
Mean shoot length (cm) 0.00 0.13 0.32 0.72 0.35 0.12 0.08 0.12 0.23 0.31 0.14 0.23 *** 0.25
*** - Significance at 0.1%
period) with mean root length of 1.15 and 1.88 cm, respectively. Further increase in the concentration of IBA (9.80 µM) resulted in a decline in mean number of roots per shoot which is 9.60 after 4 weeks, 19.20 after 8 weeks and mean root length of 0.44 cm after 4 weeks and 1.35 cm after 8 weeks.
of soil : sand : manure (1:1:1). These were covered with perforated polythene bags in shade and maintained in agronet shade house for the next 30 days where they exhibited enhanced growth. Hardened plantlets were subsequently transferred to soil in polybags for further growth and development and later exposed to field conditions where a survival rate of over 85% was achieved (Figure 1F).
Hardening and acclimatization For in vitro hardening, rooted shoots were transferred to liquid 1/4 MS strength medium having 2% sucrose and devoid of any plant growth regulator, for 7 days in flasks. Plantlets with welldeveloped shoots and roots were subsequently transferred to polybags containing a rooting mixture
DISCUSSION In the present study, callus was induced on in vitro root segments using MS medium supplemented with 4.44 to 22.20 µM BAP and 0.90 to 4.52 µM 2,4-D. The combination of a cytokinin with an auxin has
been reported to strongly enhance callus induction in dicots (Reinert and Bajaj, 1990; George, 2008). An
auxin is generally required for the induction of callus from explants. The auxin most commonly employed to initiate callus cultures is 2,4 -D. The efficiency of 2,4-D in callus induction from root explants has also been reported in Dacus carota (Syono, 1965), Cymbidium ensifolium (Chang and Chang, 1998), Ceropegia sahyadrica (Nikam and Savant, 2007) and Clitoria ternatea (Shazad et al., 2007). In our study, combination of BAP and 2,4-D gave the best results of shoot organogenesis via callus. The efficiency of BAP in shoot induction via callus has been reported to be due to the ability of plant tissue to metabolize natural hormones more
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Table 2. Effect of plant growth regulators’ interactions on in vitro multiplication of adventitious shoots regenerated via rootderived callus of S. chirata.
MS medium + PGR concentration (µM) Treatment
Mean shoot number After 4 After 8 week week
Mean shoot length (cm) After 4 After 8 week week
BAP
Kn
IAA
Ads
1(Control)
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
2
4.44
0.00
0.00
0.00
4.40
5.50
0.98
1.27
3
8.88
0.00
0.00
0.00
6.30
9.30
1.02
1.58
4 5
13.32 0.00
0.00 4.65
0.00 0.00
0.00 0.00
4.70 3.80
6.70 6.00
1.06 0.79
1.45 1.12
6
0.00
9.29
0.00
0.00
4.80
7.70
0.87
1.39
7 8 9 10 11 12 13 14 15 16 Grand mean Significance LSD
0.00 4.44 8.88 13.32 4.44 8.88 13.32 4.44 8.88 13.32
13.95 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 1.14 1.14 1.14 2.85 2.85 2.85 2.85 2.85 2.85
0.00 0.00 0.00 0.00 0.00 0.00 0.00 271.45 271.45 271.45
4.50 4.40 5.10 5.30 4.70 6.20 6.10 7.80 10.70 6.50 5.33 *** 1.18
7.60 5.10 6.80 6.90 6.50 7.70 7.40 16.50 17.50 10.50 7.98 *** 1.83
0.99 1.09 1.11 1.21 1.61 1.68 1.55 1.79 1.89 1.34 1.19 *** 0.33
1.42 1.32 1.70 1.47 2.11 2.16 1.79 2.42 2.75 1.91 1.62 *** 0.43
***Significance at 0.1%.
Table 3. Effect of different auxins and their concentrations on rooting of in vitro regenerated adventitious shoots of S. chirata developed via indirect organogenesis.
Treatment
1/2 strength MS medium +Auxin (µM)
Average length of root (cm)
After 4 week
After 8 week
After 4 week
After 8 week
0.00 2.70
0.00 6.80
0.00 0.22
0.00 0.60
0.00
3.80
6.50
0.55
0.79
0.00
3.70
7.00
0.45
0.85
0.00 0.00 0.00 0.00 0.00 0.98 2.46 4.90 9.80
2.50 3.00 4.40 4.70 3.70 3.40 5.10 14.40 9.60 4.69 *** 2.16
4.80 9.20 7.40 9.60 9.40 5.70 8.20 21.50 19.20 8.87 *** 3.07
0.38 0.62 0.68 0.81 0.57 0.48 0.68 1.15 0.44 0.54 *** 0.24
0.86 0.88 1.05 1.28 1.09 0.85 0.98 1.88 1.35 0.96 *** 0.27
1.(Control) 2.
NAA 0.00 1.07
IAA 0.00 0.00
IBA 0.00 0.00
3.
2.69
0.00
4.
5.37
0.00
5. 6. 7. 8. 9. 10. 11. 12. 13. Grand Mean Significance LSD
10.74 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 1.14 2.85 5.70 11.40 0.00 0.00 0.00 0.00
***Significance at 0.1%.
Average number of root per shoot
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readily than artificial growth regulators or due to the ability of BAP to induce production of natural hormones such as zeatin within the tissue and thus work through natural hormone system (Sharma and Wakhlu, 2003). However, the regenerated adventitious shoots were in the form of rosette clumps (Figure 1D). GA3 is reported to be required for adventitious shoot development from callus once the meristemoids are formed (Jarret and Hasegawa, 1981). Therefore, a pulse treatment of GA3 for 1 week was given to the shoots and it was found to promote shoot elongation within 14 days. This observation is in con-firmation with previous reports which indicated that GA3 is conducive for in vitro differentiation of shoot buds and promotes shoot elongation and regeneration in works on Saussurea lappa (Arora and Bhojwani, 1989), Ficus Acacia sinuata (Vengadesan et al., 2000, 2003), Ricinus communis (Ganesh Kumari et al., 2008). The in vitro regenerated shoots were further multiplied on full-strength MS medium containing 3% sucrose and supplemented with different concentrations and combinations of plant growth regulators. Of the different treatments tested, full strength MS medium fortified with BAP (8.88 µM), IAA (2.85 µM) and Ads (271.45 µM) gave the best results of in vitro multiplication of shoots regenerated from root-derived callus. The positive role of adenine sulphate in multiplication of adventitious shoots has previously been highlighted in some species viz. Psoralea coryfolia (Saxena et al., 1997), Centella asiatica (Patra et al., 1998), Melia azedarach (Vila et al., 2005), Pentamena indicum (Sivanesan and Jeong, 2007), Musa acuminate (Kacar et al., 2010). In an earlier study on in vitro propagation of S. chirata via root segments procured from in vitro raised seedlings, Wawrosch et al. (1999) reported callusing on cytokinin (2 iP/ BAP/ Kn/ Z) supplemented MS medium. However, they optimized a procedure for shoot regeneration without any intervening callus phase by culturing of root explants on BAP (3 µM) supplemented medium and subsequent transfer on to basal MS medium (for 3 weeks). By this method, they could obtain an average of only 1.9 shoots per explant and the regenerated shoots were vitrified. Low frequency of shoot regeneration, small size and hyperhydration of shoots militates against the use of their protocol. In our study, a mean number of 8.10 healthy shoots per root explants after 4 weeks could be obtained which were further multiplied with 17.50 fold shoot multiplication in 8 weeks. In the present study, IBA at 4.90 µM concentration proved to be most efficient for rhizogenesis which was regenerated, resulting in long and healthy root system. Documented literature shows that IBA has been found to be superior for in vitro rooting of adventitious shoots in some other species such as Flacourtia jangomas (Chandra and Bhanja, 2002), Exacum travancoricum (Kannan et al., 2007), Clitoria ternatea (Shahzad et al., 2007), Lins culinaris (Omran et al., 2008), Sarcostemma
brevistigma (Thomas and Shankar, 2009), S. chirata (Koul et al., 2009) and Podophyllum hexandrum (Chakraborty et al., 2010). Contrastingly, in some species, basal MS medium devoid of any plant growth regulator proved to be the best for in vitro rooting of rootderived shoots viz. Averrhoa carambola (Kantharajah et al., 1992), Gentiana sp. (Hosokawa et al., 1996), Pothomorphe umbellate (Pereira et al., 2000), Blackstonia perfoliata (Bijelovic et al., 2004). However, in the present investigation, medium lacking any plant growth regulator proved to be completely incompetent for root induction. Wawrosch et al. (1999), in their study on S. chirata via root explants, reported callus formation on transfer of in vitro shoots to rooting medium supplemented with auxin. In contrast, in our study, callusing was not observed in any of the treatments tested and long, healthy roots developed on IBA augmented medium. Plants growing under tissue culture conditions are semi-autotrophic and susceptible to transplantation shock due to delicate root system, reduced amount of epicuticular wax and reduced or abnormal stomata (Hazarika, 2003). Therefore, the plants need to be properly acclimatized when they are transferred from in vitro conditions to soil. In the present study, in vitro hardening and acclimatization was carried out for two months before transferring them to field conditions. The eight weeks old tissue culture raised plantlets which were directly transferred to polybags containing sand : soil : FYM without hardening and acclimatization showed 0 to 5% survival rate. In contrast, over 85% plantlet survival rate was obtained when plantlets were well-acclimatized prior to transfer to pots containing soil in field conditions. In vitro hardening of ex-agar rooted plantlets was tested in culture room. Regenerated plantlets were first hardened under controlled conditions of culture room for a period of one week on 1/4 X liquid MS medium devoid of any plant growth regulator and sucrose supply was decreased to 2%. Plantlets were placed on absorbent cotton soaked in this liquid medium for supporting root systems. After a week, these plantlets were transferred to a rooting mixture of soil : sand : manure (1:1:1) and covered with perforated polythene bags in net house. Holes were made in polythene bags for gaseous exchange. For acclimatizing the plantlets, bags were withdrawn periodically and on emergence of new leaves, the polybags were completely removed. The plants maintained in net house were irrigated with tap-water. During the process, the shoots elongated, leaves turned greener and expanded. Well developed plants were subsequently shifted to polybags filled with soil at High Altitude Herbal Garden, Chakrata (Forest Research Institute) and exposed to field conditions with a good survival percentage. The present study, thus, connotes first report on in vitro production of S. chirata plants via in vitro raised roots through an intermediary callus phase. The method described is simple, reproducible, affordable and has
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been successfully used for recurrent shoot production without loss of multiplication potential in more than two years of 4-week sub-culture. Thus, this package of technology has the potential of increasing the productivity by mass scale propagation of S. chirata. High shoot multiplication rate with healthy rooting are attractive features of this study and credited by the following aspects: 1. Root explants being derived aseptically do not face the problem of contamination and are therefore ideal for germplasm exchange and cryopreservation. 2. The system will be useful for biochemical and physiological studies in relation to organ differentiation and genetic improvement studies via indirect organogenesis. REFERENCES Ahuja A, Koul S, Kaul BL, Verma NK, Kaul MK, Raina RK, Qazi GN (2003). Media compositions for faster propagation of Swertia chirayita. WO 03/045132 AL, US Patent 7238527. Anonymous (1997). Biodiversity Conservation Prioritization Project, conservation assessment and management plan (CAMP) for endemic medicinal plants in India, Central Institue of Medicinal and Aromatic Plants, Lucknow. Arora R, Bhojwani SS (1989). In vitro propagation and low temperature storage of Saussurea lappa C B Clarke, an endangered medicinal plant. Plant Cell Rep., 8: 44-47. Asthana PK, Sharma NK, Kulshreshtha DK, Chatterjee SK (1991). A xanthone from Swertia chirayita. Phytochemistry, 30: 1037-1039. Balaraju K, Agastian P, Ignacimuthu S (2009). Micropropagation of S.chirata Buch. -Hams. ex Wall.: a critically endangered medicinal herb. Acta Physiol. Plant, 31: 487-494. Bijelovic A, Rosic N, Miljus-Djukic J, Ninkovic S, Grubisic D (2004). In vitro regeneration and transformation of Blackstonia perfoliata. Biol. Plant, 48(3): 333-338. Chakraborty A, Bhattacharya D, Ghanta S, Chattopadhyay S (2010). An efficient protocol for in vitro regeneration of Podophyllum hexandrum, a critically endangered medicinal plant. Indian J. Biotech., 9: 217-220. Chandra I, Bhanja P (2002). Study of organogenesis in vitro from callus tissue of Flacourtia jangomas (Lour.) Roeusch through scanning electiron micrscopy. Curr. Sci., 83(4): 476-479. Chang C, Chang WC (1998). Plant regeneration from callus culture of Cymbidium ensifolium Var. misericors. Plant Cell Rep., 17(4): 251255. Chaudhuri RK, Pal A, Jha TB (2007). Production of genetically uniform plants from nodal explants of S.chirata Buch. - Ham. ex Wall.:- an endangered medicinal herb. In Vitro Cell. Dev. Biol. Plant, 43: 467472. Chaudhuri RK, Pal A, Jha TB (2008). Conservation of S.chirata through direct shoot multiplication from leaf explants. Plant Biotech. Rep., 2: 213-218. Chaudhuri RK, Pal A, Jha TB (2009). Regeneration and characterization of S.chirata Buch.-Ham. ex Wall. plants from immature seed cultures. Sci. Hortic., 120: 107-114. Dalal SR, Shah RC (1956). Swerchirin, a novel xanthone from S.chirata. Chem. Ind., 7: p. 664. Ganesh Kumari K, Ganesan M, Jayabalan N (2008). Somatic organogenesis and plant regeneration in Ricinus communis. Biol. Plant, 52(1): 17-25. Garg S (1987). Gentianaceae of the North West Himalaya (a revision), International Bioscience Monograph 17. Today and Tomorrowâ&#x20AC;&#x2122;s Publication Co., New Delhi, India. George EF (2008). Plant Tissue Culture Procedure- Background, in: George EF, Hall MA, Deklerk GJ (Eds), Plant Propagation by
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African Journal of Biotechnology Vol. 11(29), pp. 7417-7423, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.3494 ISSN 1684â&#x20AC;&#x201C;5315 Š 2012 Academic Journals
Full Length Research Paper
Cloning and characterization of functional keratinassociated protein 5-4 gene in maize Lin Yang, Feng-Ling Fu, Long-Qun Deng, Shu-Feng Zhou, Tai-Ming Yong and Wan-Chen Li* Maize Research Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, P.R. China. Accepted 2 March, 2012
Keratin-associated proteins (KAPs) 5-4 which belongs to keratin-associated protein (KRTAP) type 5 family has two major groups: high/ultrahigh cysteine (HS) and high glycine-tyrosine (HGT). Based on bioinformatic prediction, we experimentally cloned a fragment containing an open reading frame of 1849 bp from maize, which encodes a protein of 408 amino acids. BLAST analysis indicated that KAP5-4 is homologous to the qPE9-1 protein in rice. Conserved domains analysis predicted the presence of five domains. Real time reverse transcriptase polymerase chain reaction (RT-PCR) indicated that the expression of this gene is up-regulated in response to water-deficit stress in the root and leaf. Key words: Maize, keratin-associated protein 5-4, water-deficit stress.
INTRODUCTION In the hair cortex, hair keratins intermediate filaments are embedded in an interfilamentous matrix, which consists of hair keratin-associated proteins (KATAP, usually abbreviated as KAP) (Powell et al., 1991; Wu et al., 2008). KAP are considered to have originated independently, and to be essential for the formation of rigid hair shafts and resistant to them through their extensive disulfide bond cross-linking with the abundant cysteine residues of hair keratins or hydrophobic interactions with keratins (Rogers et al., 2006; Powell et al., 1991, 1995, Powell and Rogers, 1997). KAP were encoded by multigene families, which can be divided into two major groups: high/ ultrahigh cysteine (HS) and high glycine-tyrosine (HGT), and can be further grouped into 27 subfamilies, termed KAP1 to KAP27, based on phylogeny (Rogers et al., 2006, 2007). KAP 5-4 belongs to KAP type 5 family. Gene families in which duplications, rate variation and pseudogenization occur frequently are likely involved in functional innovation and adaptation (Hughes, 1999). Meanwhile, keratin-associated protein
*Corresponding author. E-mail: aumdyms@sicau.edu.cn. Tel: 86-835-2882526. Fax: +86-835-2882154. Abbreviations: KAP, Keratin-associated protein; KRTAP, keratin-associated protein; HS, high/ultrahigh cysteine; HGT, high glycine-tyrosine.
(KRTAP) participated in stability against stress (Hesse et al., 2004). Maize is one of the most important foodstuff plants in the world; water-deficit stress poses serious threats to its production (Jiang et al., 2009; Lai et al., 2010; Zhuang et al., 2007). There were some researches on the gene expression analysis of maize, on how the reproductive organs respond to water-deficit stress conditions. Comparative analysis of a number of studies in droughtstressed maize (Zea mays L.) reporting quantitative trait loci (QTLs) for abscisic acid concentration, root characteristics, other morpho-physiological traits (MPTs) and grain yield (GY) reveals their complex genetic basis and the influence of the genetic background and the environment on QTL effects (Tuberosa et al., 2002). However, delineating a QTL to a single gene using genetic approaches is time-consuming and technically demanding (Fridman et al., 2000, 2004). As an alternative, microarray technology is a tool for analyzing genome-wide gene expression (Schena et al., 1995). Under water-deficit stress, placenta/pedicel and endosperm differed considerably in their transcriptional responses at nine days after pollination (DAP). Of the stress-response genes, 89% were upregulated in placenta/pedicel and 82% were downregulated in endosperm (Yu and Setter, 2003). Zhuang et al. (2007, 2008) used oligo microarray to examine 57452 transcripts expression at one and seven days after water-deficit
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stress, respectively. Most of these transcripts had been previously reported to be associated with water stress. Zinselmeier et al. (2002) used oligonucleotide microarray containing 1,502 genes to examine genes expression at four days after silking and eight days after pollination in maize ear and kernel in response to water-deficit stress. This revealed 17 genes (10 upregulated, seven downregulated) that were affected by stress among these tissues and demonstrated that gene expression in the pedicel was more responsive than that in other tissues. However, the microarray technology measuring gene expression was Einsatz gewaltiger Mittel or was limited by the after points of process-time. Consequently, using the functional sequence of other species BLAST in the maize databases, understanding maize response to water-deficit stress requires a comprehensive evaluation of stress-induced changes in gene expression and is expected to advance our insight into crop improvement, and then, the successful use of KAP5-4 genes encourages us to transform and overexpress endogenous KAP5-4 genes in maize. Here, we reported the cloning and characterization of a functional KAP5-4 gene from maize, and its expression profile under drought stress. It is expected that the information obtained in such a study will help in the development of approaches to manipulate the genes to increase tolerance and improve yield of the maize crop. MATERIALS AND METHODS Plant materials, growth conditions, stress treatments and first strand synthesis The seeds of maize inbred line 18-599 were sterilized and germinated in vermiculite. At the two leaf stage, the seedlings were transplanted into a plastic mesh grid for aquaculture and grown hydroponically at 28°C for a photoperiod of 12-h light/12-h dark (illumination of 20000 lux), with relative humidity of 60 to 80% and modest aeration. The nutrient solution was replaced every three days. At the three leaf stage, identical seedlings were subjected to drought stress treatments. For drought stress treatment, 16% polyethyleneglycols (PEG) was added to the nutrient solution. At 0 (control), 1, 2, 4, 6, 8, 12, 24, 48 and 72 h after stress treatment, leaves and roots were separately sampled from three seedlings, and frozen in liquid nitrogen immediately. RNA was isolated using the Trizol reagent (INVTROGEN, USA) according to the user manual and reverse transcribed to cDNA using PrimeScript RT reagent Kit (TakaRa China).
(www.maizesequence.org).
Open reading frame cloning Primers were designed based on the predicted maize KAP5-4 gene using primer 5.0. The 1289 bp fragments of KAP5-4 gene including the predicted active sites was amplified using the forward primer (5’ CATCAGCCAGCCACCACTC 3’) and the reverse primer (5' CGAAGCCAACAGGCATCATAA 3'), respectively. PCR amplification was conducted using PrimeSTAR HS DNA Polymerase (TakaRa China) with proof reading activity. The temperature cycle was 2 min at 95°C for one cycles, 10 s at 98°C, 90 s at 68°C for 35 cycles, and 5 min at 68°C. The amplified product was purified using Universal DNA Purification Kit (TIANGEN China), dATP was added in the tail of sequences using the TaKaRa TaqTM (TakaRa China), cloned into pMD19-T vector (TakaRa China), and sequenced by ShangHai Majorbio Bio-pharm Technology Co., Ltd (China) and SinoGeno Max Co., Ltd (China). The resulted sequence was identified at DNAman software and NCBI website (http://www.ncbi.nl- m.nih.gov).
Real time RT-PCR For real-time RT-PCR, a pair of primers (KAPs5-4F and KAPs5-4R) was designed to amplify a 103 bp fragment of the maize KAP5-4 ORF. The GAPDH gene (GAPDHF and GAPDHR) was used as housekeeping control for template standardization (Table 1). The detection of amplification rates was performed using SsoFast EvaGreen Supermix (contained 2×reaction buffer with dNTPs, Sso7d-fusion polymerase, MgCl2, EvaGreen dye and stabilizers; Bio-Rad USA). Real-time PCR analysis was performed on a BioRad iCycler iQ5 Real-time PCR Systems. The annealing temperature (60 and 59°C) was used in the primers of KAPs5-4 and GAPDH. Real-time RT-PCR amplification was replicated at least three times.
RESULTS Cloning of maize KAPs5-4 gene By searching uniprot database (www.uniprot.org), a protein sequence deduced from maize encoding gene sequence B6UAE5 was found to be named KAP5-4 protein. With the specific primers designed based on the identified encoding gene sequence, a fragment of 1224 bp was amplified from the cDNA sample of maize inbred line B73 (Figure 1). The sequence of the fragment was the ORF of KAP5-4. This ORF sequence was registered at ‘maizesequence’ (www.maizesequence.org) database with accession number GRMZM2G172320.
Database searches and data acquisition The amino acid sequence of the KAP5-4 was found in uniprot database (www.uniprot.org). The maize gene, KAP5-4 was used to analyze the gene structure using maizesequence (www. maizesequence.org). Multiple sequence alignment was conducted among the deduced protein sequence and the deposited functional KAP5-4 protein sequences in NCBI protein database. Phylogenetic analysis of these sequences was carried out by uniprot database (www.uniprot.org) and DNAman software. The family domains and functional domains of the deduced protein were analyzed using uniprot database (www.uniprot.rog) and maizesequence
Deduced amino acid sequence, the domains and phylogenetic relationship of maize keratin-associated protein 5-4 The polypeptide encoded by maize KAP5-4 gene is 408 amino acids long. The amplified sequence has the highest homology (54.839%) with the protein of the qPE9-1 gene in rice. There were 328 identical positions and 82 similar positions (Figure 2). Multiple alignment
Yang et al.
Table 1. The primers of real-time PCR.
Primer KAPs5-4F KAPs5-4R GAPDHF GAPDHR
Sequences from 5â&#x20AC;&#x2122; to 3â&#x20AC;&#x2122; CCCACTAATACCGATAACGAAG CAATGGCAGGAACAGCAGA CCATCACTGCCACACAGAAAC AGGAACACGGAAGGACATACCAG
Figure 1. The fragment of the keratinassociated protein 5-4 protein in maize inbred line B73. 1, The band of the maker, 2, the band of the maize KAP54 gene.
show that the variability of the amino acids sequences were 0.323, 0.275, 0.263, 0.260 and 0.384 in Z. mays (B6U8WT), Z. mays (B6UAE5), Oryza sativa subsp. Japonica (Q67UU9), O. sativa subsp. Indica (B8Y995) and Homo sapiens (Q6L8H1), respectively (Figure 3). These illustrated that the relation was close in the compared sequences. The domains of maize KAP5-4 was forecasted by uniprot database (www.uniprot.rog) and maizesequence (www.maizesequence.org). There were five domains in the sequence of the KAP5-4 (Table 2). These implied that maize KAP5-4 has some functions which are associated with the domains.
Expression of maize keratin-associated 5-4 gene In the root, the expression of gene KAP5-4 gradually increased at 1 h and peaked at 24 h after water-deficit stress treatment, and then decreased, until a similar level at 72 h (Figure 4). In the leaf, the expression which was up-regulated gradually increased at 1 h and the first peaked at 4 h after water-deficit stress treatment, and then gradually decreased, until the second peak value was exhibited at 24 h after water-deficit stress treatment, until a lower level at 72 h (Figure 5). The expression in
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the root was higher than that in the leaf. These suggested that maize KAP5-4 contributed to the response to waterdeficit stress. DISCUSSION The length of fragment was same with the prediction online, and the amplified sequence was compatible with the searching result online. These suggested that the KAP5-4 really exist in the genome of the maize. In the inbred B73, the 2.3-billion-base sequence (the largest genetic blueprint yet worked out for any plant species) includes more than 32,000 protein-coding genes spread across maize's 10 chromosomes (Palmer et al., 2003; Whitelaw et al., 2003; Schnable et al., 2009). The complex repetition and diversity of the maize genome make it a bigger challenge to explore the maize than other plants, and maize genomic survey sequence is available for use. This facilitates the discovery of functional genes. In the five domains, the domain of the Von Willebrand Factor Type C (VWFC) emerged in the qPE9-1 of the rice. The qPE9-1 encoding keratin associated protein of 426 amino acid residues contained three VWFC domains (residues 99 to 153, 276 to 316 and 339 to 385), one transmembrane domain (residues 88 to 106), and one 4disulfide-core domain (residues 153 to 166). An additional gene controlling grain size (GS3) has been identified in rice and also carries VWFC and transmembrane domains (FAN et al., 2006), demonstrating the importance of these structures (Zhou et al., 2009; Huang et al., 2009). The domain of maize Prot_inh_BBI was same with the domain of the Triticum aestivum wali 6 protein, encoding a small protein that is related to the previously isolated wali3 and wali5 genes, whose molecular function were interrelated with water transport (Richards et al., 1994; Snowden and Gardner, 1993; Ji et al., 2010). Therefore, we forecasted that the maize KAP5-4 gene had functional resemblance with the wali6, which is related to drought. The results of the expression suggested that maize KAP5-4 participated in the response to water-deficit stress. The originally upregulated expression of maize KAP5-4 is likely to promote the content of analogous wali family genes, especially wali6, to induce stress signal transduction related genes (Snowden et al., 1993; Richards et al., 1994; Ji et al., 2010). The wali6 was against aluminum stress in the root and induced expression (Richards et al., 1994; Snowden et al., 1993). These were why the expression of KAP5-4 gene in the root was higher than that of leaf. The leaves and roots of maize seedlings had different expression profiles after PEG treatment and there was a lot of overlap between PEG- and drought-stress induced up-regulated transcripts (Zheng et al., 2004). In maize seedlings or tassels, the transcripts could be differentially expressed
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Figure 2. The result of the comparison of the amino acid sequences of the keratin-associated protein in rice with the keratin-associated protein 5-4 in maize. ORYSI, Keratin associated protein (O. sativa subsp. Indica); MAIZE: keratinassociated protein 5-4 (Z. mays).
Figure 3. Neighbor-joining tree of the keratin-associated protein 5-4 sequences of other four species. B8Y995_ORYSI, Keratin associated protein (O. sativa subsp. Indica); Q67UU9_ORYSJ, DENSE PANICLE 1 (O. sativa subsp. Japonica); B6UAE5_MAIZE, keratinassociated protein 5-4 (Z. mays); B6U7W7_MAIZE, keratin-associated protein 5-4 (Z. mays); Q6L8H1_HUMAN: keratin-associated protein 5-4 (H. sapiens).
Ya n g e t a l.
Table 2. The domains of the keratin-associated protein 5-4.
InterPro
Description
Beginning
Ending
IPR000020
Anaphylatoxin/fibulin
154
184
IPR018072
Conotoxin_a-typ_CS
170
180
IPR022353
Insulin_CS
299
313
IPR000877
Prot_inh_BBI
155
170
IPR001007
VWF_C
103
155
Figure 4. Normalized fold expression of the gene of keratin-associated protein 5-4 under water-deficit stress in root.
Figure 5. Normalized fold expression of the gene of keratin-associated protein 5-4 under water-deficit stress in leaf.
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an d u pr eg u la t ed under water- deficit stress for a long or short time (Jia et al., 2006; Zhuang et al., 2 0 07 , 2008) . Therefore, we considered that the KAP5-4 gene had response to drought stress, but the mechanism of signal transduction involving KAP5-4 gene in maize remained to be explored. The maize KAP5-4 gene has been identified, which have significantly increased expression and probably involved in water stress signaling pathway based on data analysis.
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Powell BC, Rogers GE (1997). The role of keratin proteins and their genes in the growth, structure and properties of hair. EXS. 78: 59148. Richards KD, Snowden KC, Cardner RC (1994). Wali6 and wali7 Genes lnduced by Aluminum in Wheat (Trificum aestivum 1.) Roots. Plant. Physiol. 105(4): 1455-1456 Rogers MA, Langbein L, Praetzel-Wunder S, Winter H, Schweizer J (2006). Human hair keratin-associated proteins (KAPs). Int. Rev. Cytol. 251: 209-263. Rogers MA, Winter H, Langbein L, Wollschlager A, Praetzel-WunderS, Jave-Suarez LF, Schweizer J (2007). Characterization of human KAP24.1, a cuticular hair keratin-associated protein with unusual amino-acid composition and repeat structure. J. Invest. Dermatol. 127(5): 1197-1204. Schena M, Shalon D, Davis RW, Brown PO (1995). Quantitative monitoring of gene expression patterns with a complimentary DNA microarray. Science, 270(5235): 467-470. Schnable PS, Ware D, Fulton RS, Stein JC, Wei FS, Pasternak S, Liang CZ, Zhang JW, Fulton L, Graves TA, Minx P, Reily DA, Laura Courtney L, Kruchowski SS, Tomlinson C, Strong C, Delehaunty K, Fronick C, Bill Courtney B, Rock SM, Belter E, Du FY, Kim K, Abbott RM, Cotton M, Levy A, Marchetto P, Ochoa K, Jackson SM, Gillam B, Chen WZ, Yan L, Higginbotham J, Cardenas M, Waligorski J, Applebaum E, Phelps L, Falcone J, Kanchi K, Thane T, Scimone A, Thane N, Henke J, Wang T, Ruppert J, Shah N, Rotter K, Hodges J, Ingenthron E, Cordes M, Kohlberg S, Sgro J, Delgado B, Mead K, Chinwalla A, Leonard S, Crouse K, Collura K, Kudrna D, Currie J, He R, Angelova A, Rajasekar S, Mueller T, Lomeli R, Scara G, Ko A, Delaney K, Wissotski M, Lopez G, Campos D, Braidotti M, Ashley E, Golser W, Kim H, Lee S, Lin J, Dujmic Z, Kim W, Talag J, Zuccolo A, Fan CZ, Sebastian A, Kramer M, Spiegel L, Nascimento L, Zutavern T, Miller B, Ambroise C, Muller S, Spooner W, Narechania A, Ren L, Wei S, Kumari S, Faga B, Levy MJ, McMahan L, Buren PV, Vaughn MM, Ying K, Yeh CT, Emrich SJ, Jia Y, Kalyanaraman A, Hsia AP, Barbazuk WB, Baucom RS, Brutnell TP, Carpita NC, Chaparro C, Chia JM, Deragon JM, Estill JC, Fu Y, Jeddeloh JA, Han YJ, Lee H, Li PH, Lisch DR, Liu SZ, Liu ZJ, Nagel DH, McCann MC, SanMiguel P, Myers AM, Nettleton D, Nguyen J, Penning BW, Ponnala L, Schneider KL, Schwartz DC, Sharma A, Soderlund C, Springer NM, Sun Q, Wang H, Waterman M, Westerman R, Wolfgruber TK, Yang LX, Yu Y, Zhang LF, Zhou SG, Zhu QH, Bennetzen JL, Dawe RK, Jiang JM, Jiang N, Presting GG, Wessler SR, Aluru S, Martienssen RA, Clifton SW, McCombie WR, Wing RA, Wilson RK (2009). The B73 maize genome: complexity, diversity, and dynamics. Science, 326(5956): 1112-1115. Snowden KC, Gardner RC (1993). Five genes induced by aluminum in wheat (Triticum aestivum L.) roots. Plant. Physiol. 103(3): 855-861. Whitelaw CA, Barbazuk WB, Pertea G, Chan AP, Cheung F, Lee Y, Zheng L, Heeringen S, Karamycheva S, Bennetzen JL, SanMiguel P, Lakey N, Bedell J, Yuan Y, Budiman MA, Resnick A, Van Aken S, Utterback T, Riedmuller S, Williams M, Feldblyum T, Schubert K, Beachy R, Fraser CM, Quackenbush J (2003). Enrichment of GeneCoding sequences in Maize by genome filtration. Science, 302(5653): 2118-2120. Tuberosa R, Salvi S, Sanguineti M, Landi P, Maccaferri M, Conti S (2002). Mapping QTLs regulating morphophysiological traits and yield: case studies, shortcomings and perspectives in droughtstressed maize. Ann. Bot. 89: 941-963. Wu DD, Irwin DM, Zhang YP (2008). Molecular evolution of the keratin associated protein gene family in mammals, role in the evolution of mammalian hair. BMC Evol. Biol. 8: 241-256. Yu LX, Setter TL (2003). Comparative transcriptional profiling of placenta and endosperm in developing maize kernels in response to water deficit. Plant. Physiol. 131(2): 568-582. Zhuang YL, Ren GJ, Yue GD, Li ZX, Qu X, Hou GH, Zhu Y, Zhang JR (2007). Effects of water-deficit stress on the transcriptomes of developing immature ear and tassel in maize. Plant Cell. Pept. 26(12): 2137-2147. Zhuang YL, Ren GJ, Zhu Y, Hou GH, Qu X, Li ZX, Yue GD, Zhuang JR (2008). Transcriptional profiles of immature ears and tassels in maize at early stage of water stress. Biol. Planta. 52(4): 754-758. Zheng J, Zhao JF, Tao YZ, Wang JH, Liu YJ, Fu JJ, Jun Y, Gao P,
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African Journal of Biotechnology Vol. 11(29), pp. 7424-7433, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB12.111 ISSN 1684–5315 © 2012 Academic Journals
Full Length Research Paper
Cloning and selection of reference genes for gene expression studies in Ananas comosus Jun Ma1, 2, Ye-hua He1*, Cheng-hou Wu1, He-ping Liu1 and Zhong-yi Hu1 1
Horticultural Biotechnology College, South China Agricultural University, Guangzhou, Guangdong 510642, China. 2 College of Landscape Architecture, Sichuan Agricultural University, Chengdu, Sichuan 611100, China, Accepted 9 March, 2012
Full length mRNA sequences of Ac-β-actin and Ac-gapdh, and partial mRNA sequences of Ac-18SrRNA and Ac-ubiquitin were cloned from pineapple in this study. The four genes were tested as housekeeping genes in three experimental sets. GeNorm and NormFinder analysis revealed that β-actin was the most appropriate reference gene for qPCR analysis of callus under induction conditions and in different tissue types, meanwhile, 18SrRNA was the most stable reference gene during organ development. Gapdh was the most unstable gene in all tested experimental sets. Transcript level analysis result of AcSERK1 in stressed callus normalized by β-actin and 18SrRNA further confirmed that reference genes selected in this study were suitable for transcript level analysis of pineapple. The expression pattern of AcSERK1 during somatic embryogenesis normalized by β-actin coincided with the cytological features of calluses during somatic embryogenesis. These results will enable more accurate and reliable normalization of qPCR results for transcription analysis in pineapple. Key words: Reference genes, qPCR, pineapple, geNorm, NormFinder.
INTRODUCTION Gene transcript level analysis is significant for the understanding of gene expression pattern and the revealing of gene function. It is one of the foundations of modern molecular biology. Real time PCR, Northern blotting, ribonuclease protection assay (RPA), and gene chips are the main methods for the study of gene transcript level. Quantitative real-time PCR (qPCR) is one of the most sensitive and accurate techniques for rapid quantification of gene transcript levels. However, several factors, such as the quantity of the initial material, the quality of the RNA, the efficiency of cDNA synthesis, primer performance, selection of reference genes, and the statistical analysis methods, can affect the accuracy of qPCR results in gene transcript level studies. The accuracy of qPCR results relies a lot on the adequate use of appropriate normalization techniques to compensate for the differences in sample amount, RNA isolation, reverse
*Corresponding autor. E-mail: heyehua@hotmail.com. Tel: 0086-020-85288262.
transcription efficiency and qPCR reaction. Among the normalization approaches which have been proposed, the use of reference gene has become one of the most effective and convenient methods (Baudino, 2001; Malik, 2007; Qi, 2010). Ideal reference genes should be stably expressed under different experimental parameters and conditions (Rodriguez, 2008). Many studies used potential housekeeping genes (HKGs) as reference genes without validating their suitability for normalization (Bustin, 2002). However, many studies confirmed that the transcript levels varied depending on species, tissues, developmental stages and experimental conditions (Que et al., 2009; Spinsanti et al., 2006; Infante et al., 2008; Lin and Lai, 2010). It is acknowledged that it is important and necessary to validate the expression stability of candidate reference genes in each specific experimental system before its use for normalization of the transcript level (Chari, 2010; Shen, 2010; Luo, 2010; Gresner, 2010; de Almeida, 2010). The use of inappropriate HKGs as reference genes can result in biased expression profiles (Xu, 2010). Pineapple (Ananas comosus) is one of the three most
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important tropical fruit crops in the world. It is self-sterile and in practice, its propagation depends on the suckers. However, since there are only about five suckers on a pineapple plant per year, its propagation lags behind other seed propagated crops. Somatic embryogenesis is an effective regeneration protocol for plants. We have established a somatic embryogenesis system under the induction of 2,4-dichlorophenoxyacetic acid (2,4-D) (He et al., 2007). Induction and synchronization of somatic embryos are the key traits for pineapple in vitro regeneration. Despite the morphological and anatomical studies (He et al., 2008, 2010) available, very little is known about the molecular mechanisms of somatic embryogenesis in pineapple. Studies showed that the expression variation of most somatic embryogenesis related-genes were inconspicuous (Nolan, 2003; Sharma et al., 2008). In this sense, it is very important to choose proper reference genes for gene transcript level analysis of somatic embryogenesis-related genes. So far, there is no report about the validation of the expression stability and the selection of reference genes for gene transcript level studies in pineapple. We have isolated AcSERK1 from pineapple (Ma et al., 2012). It belongs to the somatic embryogenesis receptor-like kinase (SERK) gene family. Studies confirmed that the SERKs played an important role during somatic embryogenesis and were receptors of many internal and environmental signals (Albrecht et al., 2008; Baudino et al., 2001; Huang et al., 2010, Sharma et al., 2008). It was reported that some SERKs partially mediated defense signal transduction leading to stress resistance (Hu et al., 2005; Song et al., 2008; Huang et al., 2010). We isolated and examined the expression of four commonly used control genes including β-actin, gapdh, 18SrRNA and ubquitin by the qPCR technique; actin is one of the most abundant proteins in eukaryotic cells. It is highly conserved and participates in more protein-protein interactions than any known protein (Dominguez and Holmes, 2011). Gapdh is a key enzyme in the glycolytic pathway, and it catalyzes the oxidation and subsequent phosphorylation of substrate aldehydes to acylphosphates, resulting in the production of adenosine triphosphate through the electron transport chain (Mozdziak et al., 2004). Ribosomal ribonucleic acid (rRNA) is one of the structural materials for ribosomes, which help build proteins. Ubiquitin has been suggested to play a key role in a wide variety of essential cellular functions ranging from differential regulation of gene expression to protein degradation (Monia et al., 1990). The expression stability of the four genes in three experimental sets was validated by geNorm and NormFinder programs. The results show that β-actin was the most appropriate reference gene for qPCR of pineapple callus under induction conditions and in different tissue types, meanwhile, 18SrRNA was the most stable reference gene during developmental stages. gapdh was the most unstable gene in all tested experimental sets.
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MATERIALS AND METHODS Sample collection Three sets of samples as follows were used to isolate RNA for the expression stability study of the four candidate genes; 1) callus were harvested at five, 10, 15, 20, 25, 30, 35, 40 and 45 days after cultured on 2,4-D containing medium to induce somatic embryogenesis; 2) calyxes were taken at the time points of 0, seven, 15, 30 and 60 days of flower bloom; and 3) stem, calyx, petal, stylus and ovary were harvested at the first day of flower bloom. To test the suitability of the putative reference genes, four different induction conditions were used: high temperature (40°C) treatment for 24 h, low temperature (4°C) treatment for 24 h, culturing in NaCl (3% w/v) containing medium for 24 h, culturing in ethylene (1000 mg·L-1) containing medium for 24 h, and after injury treatment for 24 h. RNA was isolated and the expression levels of AcSERK1 normalized by the selected reference genes were analyzed. All the tissue samples obtained were quick-frozen immediately in liquid nitrogen and then stored at -80°C for later use. Three biological replicates of each sample were used for RNA isolation.
Total RNA extraction, quality controls Total RNA was isolated using Plant RNAiso kit (Takara, Japan) according to the manufacturer’s instructions. To remove the contaminating genomic DNA, we treated total RNA with RNase-free DNase I (Takara, Japan). Nucleic acid concentrations were measured at 260 nm with a BioPhotometer (Eppendorf, Germany). Purity of the total RNA was determined by the A260/280 and A260/230 ratio and its integrity was tested by electrophoresis using 1% formaldehyde denaturing agarose gel.
cDNA synthesis and quality confirmation The first-strand complementary DNA (cDNA) was synthesized by reverse transcribing 2 μg of total RNA with Oligo dT Primer in a final reaction volume of 20 μl using M-MLV RTase Synthesis Kit (Takara, Japan) according to the manufacture’s instructions. The cDNA was diluted according to the ratio of 1 μl cDNA to 9 μl nuclease-free water. The primers P23/P24 of AcSERK1 (5’GAAGTT/ CCATCTTGGCCAGC-3’ and 5’ACCCAGTCC/AAGAAGCATGAC-3’), which can amplify a target with an intron from genomic DNA, were used to confirm that the cDNA samples were free of genomic DNA.
Cloning of four candidate reference genes Primers used for the isolation of the four candidate reference genes are listed in Table 1. Degenerate primers were designed to amplify the conserve fragments of these genes. Then, 5’ and 3’ sequences of these genes were amplified by rapid amplification of cDNA ends (RACE) using the 3’-Full RACE Core Set Ver.2.0 kit and 5’-Full RACE Core Set Ver.2.0 kit (Takara, Japan) according to the manufacturer’s instructions. The PCR production was purified and ligated into pMD19-T vector (Takara, Japan). The positive clones were sequenced by Sangon Co (China). Sequences were edited, aligned and analyzed using DNAMAN and CLUSTAL software tools. Specific primers were designed according to the results of the RACE to amplify the full-length cDNA sequences.
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Table 1. Primer sequences for the cloning of four candidate genes in pineapple.
Symbol
Target Conserved fragment 5’ fragment
gapdh 3’ fragment Full length
Conserved fragment 5’ fragment β-actin 3’ fragment Full length
18SrRNA
Conserve fragment
Conserved fragment ubquitin 3’ fragment
Primer name gapdhup gapdhdv gapdh5raceout gapdh5racein gapdh3raceout gapdh3racein gapdhqcup gapdhqcdv
Primer sequence (5’-3’) CATGGSSAAGATYAAGATCGG AGATGCTKGACCTGYTGTCACC AAGGGTCTTAGAATCCTTCACCT AAGGGATCATTGACGGCGACGAG TGCATTGAATGGGAAACTGACCG TACGACTATGTTCCCTGGTATTGC CTCTGTATTCTCCATCTCCATTTTCGTCTC GCCAAATCCCAATTAAACTCTGATCACTT
actinup actindv actin5raceout actin5racein actin3raceout actin3racein actinqcup actinqcdv
CAGTGGTCGTACAACTGGTAT ATCCTCCAATCCAGACACTGT ATCCCAGTTGCTGACAATGCCGTGC CTCTTGGACTGGGCCTCATCCACCC TGCGGGTATTCACGAGACCACTT TACGACTATGTTCCCTGGTATTGC GCTCTCTTAGGATCCATAAAAAATG CAATCCAGGAAATTTAGAAGCACTT
18Sup 18Sdv
CATCATTCAAATATCTGCCCTATCAA CAGCCTTGCGACCATACTCCC
ubup ubdv ub3raceout ub3racein
TTGTGTTCTCTTTTGAAGTTC CAAACCACGAGCATTCAAGTT GCCTGCGTCCTGTTCTATGAA CGTCTCGTGTTCTTTGCTGTGGGTC
Primer design According to the conserved sequences of gapdh, β-actin, 18SrRNA and ubiquitin in other plants, such as Oryza sativa, Setaria italica, Zea mays, Hordeum vulgare, Glycyrrhiza uralensis, etc., deposited at NCBI database, we designed specific primers (Table 1) for the isolation of these genes. The expression stability of the four genes was evaluated by qPCR. Primers were designed based on the cloned sequences using DNAMAN software 6.0 (Lynnon Biosoft). All oligonucleotides were synthesized by Sangon Co (China). The gene name, gene description, accession number, primer sequence, product size, PCR efficiencies, and annealing temperatures are provided in Table 2. The primers for qPCR analysis of AcSERK1 were designed based on the cDNA sequence of AcSERK1 we cloned before. Specificity of the amplifications was verified by 1.5% agarose gel electrophoresis and at the end of the PCR run, by melt curve analysis with a temperature gradient from 65 to 94°C.
qPCR analyses qPCR was performed on the Bio-rad iQ5 real time PCR System (Biorad, USA) using THUNDERBIRD SYBR qPCR Mix (Toyobo, Japan). Each amplifications was performed in a 20 μl final volume containing 10 μl of 2×SYBR Mix; 1.0 μl of 10× diluted cDNA sample (about 0.01 μg RNA); 2 μl of specific primer pair mix at 2.5 μM and 7 μl of ddH2O. Reactions were performed in triplicates for technical replicates. Amplification was carried out with the following cycling parameters: heating for 2 min at 95°C, 40 cycles of denaturation at 95°C for 10 s, annealing for 20 s at 52°C, and extension at 72°C for
35 s. Analysis of the relative gene expression data was conducted using the 2-∆∆CT method (Livak and Schmittgen, 2001). Negative control with no sample was included for each primer pair. Statistical analysis The PCR efficiency and quantification cycle (Cq) value were calculated by iQ5 System Software (Biorad). The calculated results are shown in Table 2 and Figure 3. The expression stability was evaluated by both geNorm (Vandesompele et al., 2002) and NormFinder (Andersen et al., 2004) programs. AcSERK1 mRNA expression analysis The qPCR analysis of the expression of AcSERK1 in pineapple callus under different stressful conditions was used to validate the normalization based on the reference genes we selected in this study. The qPCR primers of AcSERK1 are listed in Table 2. We also used the selected reference gene to analyze the expression pattern of AcSERK1 during somatic embryogenesis.
RESULTS Cloning of the four candidate reference genes In order to design proper primers for qPCR, we isolated
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Table 2. Primer sequences, product size, PCR efficiencies, and annealing temperatures of the candidate reference genes.
Primer sequence (5’-3’)
18S rRNA
Accession number JN129389
Product size (bp)
PCR efficiency (%)
Annealing temperature (°C)
F: ATGGTGGTGACGGGTGAC R: CAGACACTAAAGCGCCCGGTA
162
99.2
58
β-actin
HQ148720
F: CTGGCCTACGTGGCACTTGACTT R: CACTTCTGGGCAGCGGAACCTTT
135
99.3
63
gapdh
HM768296
F: CTTTAACATCATTCCCAGCAGCACT R: GGTAATCTCAAAGGCATCTTGGG
216
100.1
61
ubquitin
JN129388
F: TTGGCTGACTACAATATCCAGAAGG R: GACCCACAGCAAAGAACACGA
182
98.8
59
AcSERK1
HM236375
F: TCTTGGTGTTCCACCCCCTTGCCAG R:GGAACCAACGTACCAGACAGTGCTGC
217
99.7
63
Symbol
Figure 1. a) Confirmation of the integrity of RNA isolated; b) PCR result of primer pair P23/P24. M, Marker; 1, amplification result of genomic DNA; 2, amplification result of cDNA; 3, negative control without template.
the mRNA sequence of β-actin, gapdh, ubiquitin and 18SrRNA using the primers listed in Table 1. The NCBI accession numbers of these sequences are listed in Table 2. Ac-β-actin was composed of 1169 bp, containing an 1134 bp open reading frame. The identities of Ac-βactin to other actins were 85 to 88%. Ac-gapdh was composed of 1259 bp, containing 1014 bp open reading frame. The identities of Ac-gapdh to other gapdhs were 83 to 85%. A 835 bp fragment of Ac-18SrRNA was isolated, the identities of it to other 18SrRNAs were 97 to 99%. A 319 bp fragment of Ac-ubiquitin was isolated; the identities of Ac-ubiquitin to other ubiquitins were 90 to 95%.
PCR efficiency and amplification specificity The quality and integrity of RNA is essential in gene expression analysis. The integrity of RNA was confirmed by agarose gel electrophoresis. Only the RNA samples with A260/A280 ratio between 1.9 and 2.1, A260/A230 ratio higher than 2.0 and with integral three bands (Figure 1a) were used for further analysis. The amplification results of P23/P24 showed a belt of 1200 bp from genomic sample and an 810 bp product from cDNA sample (Figure 1b). It confirmed that the cDNA samples were free of genomic DNA. The melt curve analysis (Figure 2) showed that all the primers used in the study
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Figure 2. Melt curve of the four candidate reference genes; a) 18S rRNA, b) gapdh, c) β-actin, d) ubiquitin.
generated a single PCR product of the desired size from the various cDNAs. It indicated the good specificity of all of the primer pairs used in this study. A standard curve was created for each analyzed gene using a set of dilutions with five orders of magnitude. The significant 2 linear relationships (r >0.98) and PCR efficiency values (ranged from 98 to 101%) are listed in Table 2.
Variations in the expression of candidate reference genes Transcripts of the four candidate reference genes were detected in all the 16 samples by qPCR. The mean Cq values of three biological replicates of all the samples at same cDNA input are shown in Figure 3. Analysis of the raw expression levels in all the samples showed that the transcript levels of the four genes were significantly impacted by experimental conditions, developmental stages and tissue types. β-actin showed the lowest Cq values in all tested samples indicating the highest transcript levels. Contrarily, ubiquitin showed the highest Cq values. Some Cq values of ubiquitin were a little more than 30, but it can still be included by the standard curve. This indicated that the Cq values still obey the linearity of the PCR reaction, and it would not compromise the accuracy of the analysis. The other candidate reference genes were expressed at moderate levels. Each candidate gene displayed similar transcript levels in the respective experimental condition tested in this study. It can be assumed that all of the candidate genes were
relatively stable in the tested experimental conditions.
Analysis of gene expression stability and reference genes selection Different statistical methods based on respective principles can potentially yield discrepant results for different experimental data sets (Lin and Lai, 2010). Therefore, we compared two different statistical approaches in this study. GeNorm program calculates an average expression stability value (M) based on the average pairwise variation existing between all pairs of the candidate genes. All the M value of the four candidate genes were below the default limit of M=1.5. According to the geNorm program, which calculates an average expression stability value (M) based on the average pairwise variation existing between all pairs of the candidate genes (Vandesompele et al., 2002), the expression stability of the four candidate genes in the three experimental sets was as follows (Table 3): 1) β-actin and 18S rRNA were the most stable genes in callus under the induction of 2,4-D, from most stable (lowest M value) to least stable (highest M value): β-actin /18S rRNA > ubiquitin > gapdh; 2) in different developmental stages of calyx, from most stable to least stable: 18S rRNA / βactin > ubiquitin > gapdh; therefore, for these two situations, 18S rRNA and β-actin were the most stable genes; 3) in different tissue types, β-actin and ubiquitin were the most stable genes.
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15
0
Petal
20
25
7
Calyx
30
15
Stylus
35
40 d
30
60 d
Steam
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Ovary
Figure 3. RNA transcription levels of candidate reference genes tested, compared at same cDNA inputs, and presented as mean value of Cq in different samples. Cq values are mean of three biological replicates; a) time duration of 2,4-D induction (in days), b) developmental stages of calyx in (days), c) different organs. 2,4-D, 2,4-Dichlorophenoxyacetic acid.
The M value from the lowest to the highest is: β-actin/ ubiquitin < 18S rRNA < gapdh. Pairwise variations (Vn/Vn+1) were calculated by geNorm. The V2/3 values of the three experiment sets were all smaller than the cutoff threshold of 0.2. The results indicate that using two genes as internal controls could strongly reduce the variability, and it is sufficient for accurate normalization in pineapple (Figure 4). NormFinder (Andersen et al., 2004) ranked the candidate reference genes according to their expression variation between intergroups and intragroups. According to it, β-actin and 18S rRNA were the best normalization factors with the lowest stability values of 0.005, 0.008 and
0.013 respectively in the three experimental sets (Table 3). The expression stability order of the four candidate genes analyzed by NormFinder was coincident with the results of geNorm program. Stability of gapdh was the lowest among four candidate genes in the three experimental sets.
AcSERK1 expression Use of β-actin and 18S rRNA was evaluated as normalization genes to analyze the transcript level of AcSERK1 in pineapple callus under the following stressful condi-
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Table 3. Candidate genes ranked according to their expression stability, estimated using Normfinder and geNorm.
2,4-D induction Stability value Gene name (NormFinder) β-actin 0.005 18SrRNA 0.019 ubiquitin 0.040 gapdh 0.055 β-actin Best gene
Stability value (geNorm) 0.519 0.562 0.648 0.603 β-actin
tions: high or low temperature, ethane, NaCl, and injury. The results show that AcSERK1 expression pattern normalized by β-actin was strongly agreed with the result of 18S rRNA (Figure 5). Under NaCl-stressed conditions, the AcSERK1 transcript level was six to seven folds to that of low temperature treatment. The transcript levels under the induction of high temperature and ethane were close to those of low temperature. The similarity of AcSERK1 expression pattern normalized by β-actin and 18S rRNA confirmed that the reference genes we selected in this study are suitable for the transcript level analysis of pineapple. β-actin was used as normalization factor to analyze expression pattern of AcSERK1 during somatic embryogenesis in pineapple. The results show that there were two peaks of expression at different times (Figure 6). The first peak of expression occurred at 10 to 15 days of culture in 2,4-D containing medium, and at that time, many cells have acquired embryogenic competence. The second peak of expression is observed at 40 days of culture in 2,4-D containing medium. At this stage, there were many competent cells, meristematic cell clusters and globular embryos in the callus. These results coincided with the
Different tissue types Stability value Gene name (NormFinder β-actin 0.008 ubiquitin 0.008 18SrRNA 0.062 gapdh 0.077 β-actin Best gene
Stability value (geNorm) 0.512 0.572 0.659 0.747 β-actin
appearance of the cytological features of calluses under 2,4-D induction to form somatic embryo (He et al., 2010). The results suggest that accumulation of 2,4-D was sensed by cells and could induce the expression of AcSERK1, indicating that this gene may play a role in induction of embryogenesis of pineapple callus.
DISCUSSION The expression level of a stable reference gene should vary minimally even between different experiments (Dean et al., 2002). Our results show that each experimental condition may demand a specific set of reference genes, thereby emphasizing the importance of validating reference genes for each experimental condition. To our knowledge, this study was the first attempt to validate a set of candidate reference genes under in vitro induction conditions, in different tissue types and during developmental stages for qPCR normalization in pineapple. The expression stability analysis of the candi-date reference genes showed that there were apparent differences in the stability between the four genes in three experimental conditions. This is coincident with the view of Luo (2010), de Almeida (2010), Gresner
Developmental stages Stability value Gene name (NormFinder) 18SrRNA 0.013 β-actin 0.028 ubiquitin 0.032 gapdh 0.041 18SrRNA Best gene
Stability value (geNorm) 0.516 0.596 0.824 0.662 18SrRNA
(2010) and Shen (2010). Similar to the result of Gresner (2010), the stability order ranked by geNorm closely matched the result of NormFinder, but in Populus (Xu, 2010) and Brassica rapa L.ssp.pekinensis (Qi, 2010), the ranking result of geNorm and NormFinder was different. This indicated that the candidate reference genes expressed more stably in pineapple. It was known that β-actin is a good reference gene for its stable expression in many species (Hu et al., 2009; Luo, 2010), but not stable in others, such as rice (Jain, 2006). In this study, β-actin was the most stable gene in the tested samples. Some studies (Chari, 2010; Shen, 2010; Gresner, 2011) showed that gapdh was a good reference gene, but in pineapple, its expression level was so slow and unstable, that it was not suitable as reference gene in the tested samples, as described for water lily (Luo, 2010). It was reported that gapdh expression changed apparently with age and nutrition status (Mozdziak et al., 2003) and varied in the oocytes matured in low oxygen (Bermejo-Alvarez et al., 2010), This study with pineapple further confirmed that gene expression stability often varies with species, tissue types and developmental stages. Based on the analysis results, we used β-actin and 18S rRNA as normalization genes to analyze the AcSERK1expression under stressful conditions.
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Figure 4. Ranking of gene stability and combination of genes for normalization factor calculation evaluated by geNorm; a, b) time duration of 2,4-D induction; c, d) developmental stages of calyx; e, f) different organs; a, c and e) the M value was calculated for each gene, and the least gene was automatically excluded for the next round of calculations. b, d and f) Determination of the optimal number of reference genes for normalization was conducted. The V value defines the pairwise variation between two sequential normalization factors. 0.2 is set as the cutoff,
The high similarity of the results derived by the two normalization genes suggest that the reference genes we selected in this study was optimal for the transcription analysis of pineapple, then, β-actin was used to normalize the expression pattern of AcSERK1 during embryogenesis. The expression data obtained was reasonable. It demonstrated that AcSERK1 expression was accumu-
lated during somatic embryogenesis, and AcSERK1 expression could be effectively induced by 2,4-D. It was reported that 2,4-D was necessary and effective for the induction of somatic embryogenesis of pineapple (He et al., 2007), and SERK could be used as a mark gene for somatic embryogenesis (Baudino et al., 2001; Hecht et al., 2001). These results indicate that the analysis results
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8.00
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a
Normalized fold Expression
Normolized fold Expression
10.00
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b 6.00
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0.00 LW
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Figure 5. The expression pattern of AcSERK1 in callus under induction conditions; a) expression pattern of AcSERK1 using β-actin as reference gene; b) expression pattern of AcSERK1 using 18S rRNA as reference gene (LW, low temperature; HT, high temperature; ET, ethane; NaCl; IJ, injury).
0
5
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15
20
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30
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Figure 6. Relative expression of AcSERK1 measured using β-actin as reference gene in callus cultured on 2,4-D containing medium to induce somatic embryo.
normalized by the selected reference gene can reflect the real situation of gene expression in pineapple. The validation results will enable more accurate and reliable normalization of qPCR results for transcription analysis in pineapple.
of Agriculture (2010-G2-11) and commonweal industry scientific research project of Ministry of Agriculture (nyhyzx07-30). REFERENCES
ACKNOWLEDGEMENTS This research was supported by the Natural Science Foundation of China (30971984), Project 948 of Ministry
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African Journal of Biotechnology Vol. 11(29), pp. 7434-7444, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.3092 ISSN 1684â&#x20AC;&#x201C;5315 Š 2012 Academic Journals
Full Length Research Paper
The use of selected purple nonsulfur bacteria to remove heavy metals and salts from sediment and water collected from contaminated areas to decrease their phytotoxicity Saijai Panwichian1, Duangporn Kantachote1,3*, Banjong Wittayaweerasak2,3 and Megharaj Mallavarapu4 1
Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai 90112, Thailand. 2 Faculty of Environmental Management, Prince of Songkla University, Hat Yai 90112, Thailand. 3 National Center of Excellence for Environmental and Hazardous Waste Management- Satellite Center at Prince of Songkla University, Hat Yai 90112, Thailand. 4 Center for Environmental Risk Assessment and Remediation, University of South Australia, Mawson Lakes Boulevard, Mawson Lakes, SA 5095, Australia. Accepted 3 February, 2012
The potential of the purple nonsulfur bacteria (PNSB), NW16 and KMS24, to remove heavy metals (HMs) 2+ 2+ 2+ 2 and salts was investigated in a synthetic solution (62.63 Pb , 34.60 Cu , 58.5 Zn and 0.75 Cd mg/L) containing 3% NaCl, sediment, and water collected from contaminated post cultured shrimp ponds and seed germination of 2 plants were used to assay their plant toxicities after bioremediation. Both light 2+ 2+ metal ions (85 mg/L Ca and 160 mg/L Mg to the synthetic HMs solution) significantly decreased the HMs removal efficiency and the mixed culture gave the highest efficiency to remove HMs (removal 2+ 2+ 2+ 2+ percentages; 85 Pb , 74 Cu , 47 Zn and 28 Cd ). The best set for the treatment of contaminated water 2+ 2+ from shrimp ponds (Cu , Zn ; 0.043, 0.057 mg/L and salinity, 10.23%0) under the conditions of aerobicdark and microaerobic-light was a set of native with added mixed culture with a decrease of roughly 75, 2+ 2+ 31 and 77% for Cu , Zn and salinity, respectively. For the sediment samples, a set of native with added mixed culture also produced the highest efficiency to remove HMs (initial concentrations in 2+ 2+ 2+ 2+ mg/kg dry weight; 23.15 Pb , 15.05 Cu , 22.16 Zn and 0.29 Cd ) and salinity (0.84%0) under aerobicdark conditions with the removal percentages of HMs; 84.29, 62.52, 43.33 and 40.95, and 100% salinity. Consequently, this set produced the most effective treatment as the germination index was 34.50 and 35.29% for rice seed (Oryza sativa) and water spinach (Ipomoea aquatic) respectively in the treated water and 115.70 and 139.33% for rice and water spinach respectively in the treated sediment. Key words: Bioremediation, contaminated shrimp ponds, heavy metals, photosynthetic bacteria, salinity, seed germination index.
INTRODUCTION The increased demand for shrimp in world markets has encouraged many developing countries to enter into shrimp farming but this can have damaging effects on the
*Corresponding author. E-mail: duangporn.k@psu.ac.th. Tel: +66-74288333. Fax: +66-74446661.
local environment (Chua, 1992). The extension of shrimp farming from coastal areas to freshwater areas has affected those areas, some previously used for growing rice, fruit plantations and fisheries. Traditionally, seawater from coastal waters is directly used to rear the shrimp with no additional processes and often these coastal waters are contaminated by many kinds of pollutants including heavy metals (HMs) (Cheung and Wong, 2006;
Panwichian et al.
Visuthismajarn et al., 2005; Cheevaporn and Menasveta, 2003). In addition, water removed during shrimp pond drainage during harvesting is often directly discharged into canals and flows into other cultivated areas, together with the illegal disposal of shrimp pond sediments (Dierberg and Kiattisimkul, 1996). Consequently, this can cause serious pollution to soil in agricultural areas, especially rice-fields and vegetable crops, resulting in an accumulation of HMs, salts and chemical substances. The accumulation of HMs in agricultural soil is of increasing concern due to food safety issues and potential health risks because they cannot be biodegraded and may be leached to surface water runoff, groundwater storages, plant absorption, etc. HMs are frequently accumulated by agriculturally important crops and become concentrated in the plant tissues to produce damaging effects on the plants themselves and may also pose a health hazard to animals and humans (Mokhtar et al., 2009; Yap et al., 2004). Stress from HMs and salts can have a negative impact on processes associated with biomass production and grain yield in almost all major field grown crops and this can result in the reduction of growth rate and pigment content and low productivity (John et al., 2009). Thailand is the biggest rice exporting country; however, the development of shrimp farming in Thailand has opened the door for shrimp farming away from the coast into the paddy land, particularly in this region of southern Thailand. Hence, low rice yields and the contamination of groundwater aquifers have rendered large areas of land unsuitable for cultivation (Flaherty et al., 1999). Accumulation of HMs in rice may cause some illness such as Itai Itai by Cd (Shimbo et al., 2001). Water spinach (Ipomoea aquatica) is an herbaceous aquatic or semi-aquatic perennial plant of the tropics and subtropics. It is a fast growing plant and can be cultivated on most kinds of soils. Contamination of HMs in the water where I. aquatica is grown may cause the risk of poisoning to consumers (Gothberg et. al., 2002). There are many advantages for using bioremediation instead of physical and/or chemical processes as it is a natural process, produces harmless end products and any bioremediated soil/water can be re-used (Barker and Bryson, 2002). Bioremediation of HMs from contaminated soil and water would provide decontaminated soil/water that could be used for agriculture. However, toxicity of contaminated soil/water after bioremediation must be evaluated prior to use and bioassay has gained widespread attention over the past few decades. Seed germination assay is one of the most common techniques used to assess phytotoxicity as a rapid method due to its simplicity, sensitivity and inexpensive cost (Kapanen and Itavaara, 2001; Wang et al., 2001). In our previous studies, two purple nonsulfur bacteria (PNSB) strains, NW16 and KMS24, have proven their abilities to effectively remove HMs (Cd, Cu, Pb and Zn) containing 3% NaCl present in contaminated shrimp pond water (Panwichian
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et al., 2010a, b). Therefore, our aims in this present study were to investigate the potential of these PNSB strains to remove HMs and salts from the sediment and water collected from contaminated shrimp ponds after harvesting and to assay the phytotoxicity of the sediment and water after treatment using a seed germination index of economical plants; rice (Oryza sativa) and water spinach (Ipomoea aquatica). MATERIALS AND METHODS Preparation of heavy metal solutions The inorganic salts; CdCl2, PbCl2, CuCl2 and ZnCl2 were used for preparing stock solutions of each heavy metal (HM) ion whereas CaCl2 and MgCl2 were used for preparing light metal ions. Each metal was dissolved in deionized water (DI water) to obtain the concentration as designated and then the stock solution was sterilized using a 0.22 µm filter membrane. They were stored at 4°C until used. The concentration of HMs was analyzed using inductively coupled plasma optical emission spectroscopy (ICPOES) (PerkinElmer, Germany).
Preparation of PNSB for uptake of heavy metals Two PNSB strains, Rhodobium marinum NW16 and Rhodobacter sphaeroides KMS24, used in this study were isolated from water and sediment samples collected from shrimp ponds contaminated with HMs (Panwichian et al., 2010a). A ten percent inoculum of each active isolate was grown in Glutamate-Malate medium (GM medium) as previously described by Panwichian et al. (2010a) under microaerobic-light conditions (3000 lux). Culture broths were harvested in the log phase of growth because previously it had been established that this was the most effective time for them to remove HMs (Panwichian et al., 2010b). After centrifugation (Sorvall RC 5C Plus, Du-pont, Delaware, USA) at 9,300 x g for 15 min, the cell pellets were washed twice with 0.1% peptone water. The cell pellets were later prepared for uptake of HMs with the optimum biomass equivalent to 4.5 and 5.0 mg dried cell weight (DCW)/ml for NW16 and KMS24, respectively. In this study, mixed culture with the biomass equivalent to 2.5 mg DCW/ml of NW16 and 2.5 mg DCW/ml of KMS24 was also prepared for testing the uptake of HMs. Effect of Ca2+ and Mg2+ on the removal of heavy metals by PNSB Experiments in this study were designed based on the highest concentration of HMs and the average concentrations of Na+, Ca2+ and Mg2+ in shrimp ponds (Panwichian et al., 2010a). The mixed solution of HMs containing 0.75 mg/L Cd2+, 62.63 mg/L Pb2+; 34.60 mg/L Cu2+; 58.50 mg/L Zn2+ in 3% NaCl solution and with or without added 85 mg/L Ca2+ and 160 mg/L Mg2+ was prepared. These were used for the treatment and control sets for investigating the effects of both Ca2+ and Mg2+ on the efficiency of HM removal by both pure cultures and a mixed culture of PNSB. The sets of HMs solution without inoculating with bacterial cells were used as abiotic controls. The optimum conditions for removing HMs by each culture was adopted from one of our previous studies (Panwichian et al., 2010b) as follows; 4.5 mg DCW/ml, pH 6.0, 30°C, 30 min for strain NW16 and 5.0 mg DCW/ml, pH 5.5, 35°C, 45 min for strain KMS24. The mixed culture consisted of 2.5 mg DCW/ml of each culture and
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it was tested at both the optimal conditions for removal of HMs by strains NW16 and KMS24. Cell suspensions were shaken in an incubator at a speed of 150 rpm for 30 to 45 min under aerobic-dark conditions. Aerobic-dark conditions were also used because they provided a higher efficiency for removal of HMs when compared to microaerobic-light conditions (Panwichian et al., 2010b). Cell suspensions were centrifuged, and the remaining HMs in each supernatant was analyzed using ICP-OES.
Collection of sediment and water from contaminated shrimp ponds Post cultured shrimp ponds contaminated with the highest levels of HMs (Cu, Zn, Pb and Cd) in the areas of Ranot, Songkhla province; HuaSai, Nakhon Si Thammarat province and Pak Phayun, Pattalung province were chosen for collecting sediment while shrimp pond water was collected from post cultured contaminated shrimp ponds in the Pak Phayun district (Panwichian et al., 2010a). After shrimp harvesting, sediment sub-samples, each of about 100 g were collected from the bottom of a pond at a depth of 5 cm in two diagonal and a half points from each bank (13 subsamples/pond). Water sub-samples (13 sub-samples/pond) were collected at the time for shrimp harvesting, roughly 100 ml of water at about 50 cm below the surface water level. All sub samples of sediment and water were kept in a big ice box during transport and then at the laboratory all sub-samples were promptly mixed well to obtain one representative sample each for sediment and water. Concentrations of HMs (Cd, Pb, Cu, and Zn) were analyzed using ICP-OES. In addition, samples of sediment and water were also measured for pH, EC (electrical conductivity) and salinity as described by Panwichian et al. (2010a).
Removal of HMs and salts in the water collected from post cultured contaminated shrimp ponds The uptakes of HMs and salts by biomass of both PNSB strains were conducted under both microaerobic-light and aerobic-dark conditions due to both incubating conditions having an effect on the efficiency of HMs removal by PNSB (Panwichian et al., 2010b). The cells of NW16 and KMS24 present as a suspension of either a pure or mixed culture were added into the collected water samples that had been sterilized (autoclaving at 121°C, 15 min) and not sterilized (native set). A sterile water set without inoculation of PNSB served as an abiotic control while a single culture or a mixed culture was inoculated into a sterile set, namely a pure culture or a mixed PNSB set. In contrast to the non sterile water sets (namely a native set prepared together with NW16 or KMS24 or a mixed PNSB set with the two strains inoculated together). The uptake of HMs was investigated under optimum conditions as previously described as follows: a pH of 6.0, 30°C, 30 min for strain NW16 and a pH of 5.5, 35°C, 45 min for strain KMS24. This study of uptake of HMs by the mixed culture of NW16 and KMS24 cells was investigated at the optimum conditions of pH 5.5 and 35°C for 45 min based on the result of the previous experiment. The optimum condition of a mixed culture was also used for studying uptake of HMs in both control sets using a longer time of exposure when compared with the optimum condition for strain NW16. In these studies, removal of HMs was focused on only Cu2+ and Zn2+ as these are 2 cations in the water column of the collected shrimp ponds that exceeded the standard guidelines for aquaculture (Panwichian et al., 2010a). To follow the real situation in shrimp ponds and to test toxicity of salinity and salts, samples of treated water in each set from both incubating conditions were mixed to obtain one sample of each set.
Removal of HMs and salts in sediment collected from post cultured contaminated shrimp ponds The sediment was made into slurry using sterile DI water at a ratio of 1v:1w (10 ml DI water and 10 g fresh sediment). The uptake of HMs and salts was also investigated using the same protocol as used for the water samples. After incubation, the sediment slurry samples were centrifuged at 9,300 x g for 20 min and the loss of each HM was calculated based on the amounts of HMs in the supernatant together with the amounts determined in the pellet (sediment plus bacterial cells) at zero time and at the end of the experiment. The amounts of HMs were expressed based on the sediment dry weight as the sediment was dried in an oven at 105°C overnight until a constant weight was obtained. To monitor salinity and toxicity, treated sediment samples were prepared in a similar way with the treated water samples as previously described.
Toxicity assessment by seed germination for the sediment and water after treatment Toxicity of the water and sediment samples from post cultured contaminated shrimp ponds after treatment with PNSB; NW16, KMS24, a mixed culture in the presence or absence of native flora from the previous experiments was tested for their effects on seed germination. The plants used in this study were rice (Oryza sativa) and water spinach (Ipomoea aquatica). Results were evaluated by comparing the results among sets of treatments and control sets (abiotic control and native control). Briefly for the seed germination test, water samples from each set were filtered with a 0.45 m filter membrane to remove the organisms. 5 ml of filtered water sample without dilution was added to a 9 cm sterile Petri dish, using Whatman # 1 as a bed, and then 10 grains were placed on the bed. All Petri dishes were incubated in the dark at room temperature for 72 h. The percentages of relative seed germination (RSG), relative root elongation (RRE) and the germination index (GI) were calculated and compared with the distilled water as the control set (Hoekstra et al., 2002). Sediment samples were prepared to obtain the sediment solution by adding 25 ml of sterile DI water into 5 g wet weight of sediment and shaking overnight. Thereafter, it was centrifuged at 13,950 x g for 10 min. The supernatant as the sediment solution was filtered with a 0.45 m filter membrane and the toxicity was tested by the same method as previously described for the water samples.
Statistical analysis All experiments in this work were conducted in triplicate. Data are presented as mean and standard deviation. One way ANOVA was used to analyze statistical differences at a p-value < 0.05 and mean comparisons were performed by the Duncan’s multiple range test.
RESULTS Effect of Ca by PNSB
2+
and Mg
2+
on removal of heavy metals
As there was no loss of HMs in the abiotic control sets the loss of HMs in the sets after inoculation were caused by the presence of bacterial cells (data not shown). Results of the removal of HMs in 3% NaCl in the 2+ presence and absence of 85 mg/L Ca and 160 mg/L
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Figure 1. The heavy metals removal efficiency in the synthetic solution (Cd, 0.75 mg/L; Pb, 62.63 mg/L; Cu 34.60 mg/L; Zn, 58.50 mg/L) containing 3% NaCl in the presence or absence of 85 mg/L Ca2+ and 160 mg/L Mg2+ under aerobic-dark conditions by (A) pure culture of 4.5 mg DCW/ml of NW16 and the mixed of NW16 and KMS24 (2.5 + 2.5 mg DCW/ml) under optimum conditions for removal of HMs of NW16; pH 6.0, 30째C, 30 min and (B) pure culture of 5.0 mg DCW/ml of KMS24 and the mixed culture under optimum conditions for removal of HMs of KMS24; pH 5.5, 35째C, 45 min. Lower case letters with numbers above bars (that is, a1, b1, c1 and d1) using different letters indicate significant differences (p < 0.05).
2+
Mg by the pure cultures or a mixed culture of PNSB strains (NW16 and KMS24) under aerobic-dark conditions are shown in Figure 1. The mixed culture produced a significantly higher HM removal for all HMs than each of the pure cultures separately, both with and 2+ 2+ without the added cations (Ca and Mg ). In addition, the extra light metal ions significantly decreased the
removal of HMs by all cultures (Figure 1A and 1B). In the 2+ 2+ presence of Ca and Mg in the HMs solution containing 3% NaCl with optimum conditions for the strain NW16, 2+ 2+ 2+ the pure culture NW16 removed Pb , Cu , Zn and 2+ Cd by about 65, 56, 21 and 11%, respectively but the removal percentages of the mixed culture was roughly 78, 65, 36 and 21%, respectively (Figure 1 A). However,
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Table 1. Values of salinity and electrical conductivity (EC) in sediment and water samples from post cultured contaminated shrimp ponds before and after treatment by the selected purple nonsulfur bacteria (PNSB).
Water
Treatment
Salinity (%0)
EC (mS/cm)
Sediment Salinity (%0) EC (mS/cm)
10.23 0.78 b 9.68 0.13 c 9.02 0.06 d 7.32 0.21 e 5.17 0.13 f 6.78 0.20 e 5.04 0.13 g 2.69 0.03 g 2.36 0.05
4.68 0.34 a 4.71 0.19 ab 4.46 0.10 bc 4.21 0.28 c 3.90 0.23 d 3.29 0.25 e 2.82 0.04 f 2.50 0.01 f 2.4 0.01
0.84 0.05 b 0.59 0.05 b 0.64 0.05 c 0.27 0.02 d 0.14 0.01 e 0.35 0.01 d 0.16 0.03 f 0.01 0.00 f 0.07 0.02
a
Before treatment Abiotic Native Sterile + NW16 Native + NW16 Sterile + KMS24 Native + KMS24 Sterile + mixed culture Native + mixed culture
a
1.27 0.06 b 1.37 0.04 a 1.25 0.04 c 1.04 0.001 d 0.95 0.002 cd 1.00 0.013 e 0.72 0.002 e 0.72 0.001 e 0.68 0.001
a
a
Values in the same column with different lower case letters indicate significant differences (p < 0.05).
Table 2. The removal percentage of Cu2+ and Zn2+ in the contaminated water from post cultured shrimp ponds by the selected purple nonsulfur bacteria under microaerobic-light and aerobic-dark conditions.
Treatment
Cu
2+
% Removal 2+ (initial concentration, 0.043 mg/L) Zn (initial concentration, 0.057 mg/L) Light Dark Light Dark
Control Abiotic Native
11.87 ± 6.05 aA 10.40 ± 4.16
aA
13.42 ± 1.02 bA 11.14 ± 1.72
NW16 Sterile + NW16 Native + NW16
35.29 ± 1.03 cA 41.18 ± 1.26
bA
KMS24 Sterile + KMS24 Native + KMS24
39.41 ± 1.41 dA 45.12 ± 0.96
cA
43.23 ± 0.91 fB 48.40±0.86
Mixed culture(NW16 + KMS24) Sterile + mixed culture Native + mixed culture
65.91 ± 1.50 fA 73.78 ± 1.34
eA
71.97 ± 1.65 hA 75.92 ± 0.52
aA
7.95 ± 0.89 aA 6.44 ± 0.82
aA
aA
40.62 ± 0.97 dB 45.84 ± 1.26
cB
18.60 ± 0.83 cA 22.58 ± 1.11
bA
22.52 ± 1.06 cB 25.73 ± 0.82
eB
25.93 ± 1.04 eA 30.52 ± 1.77
dA
25.47 ± 0.74 dA 29.93 ± 1.38
gB
29.24 ± 1.77 eA 30.40 ± 2.35
eB
26.35 ± 0.72 eA 31.67 ± 1.03
7.78 ± 2.09 aA 6.41 ± 1.02
bB
cA
cA
Values in the same columns with different lower case letters indicate significant differences (p<0.05). different upper case letters in the same row indicate significant differences between light and dark conditions of each set (p < 0.05).
2+
2+
the effect of Ca and Mg had less effect on the efficiency of HMs removal by strain KMS24 itself (Figure 2+ 2+ 2+ 1 B). The removal percentages of Pb , Cu , Zn and 2+ Cd by the pure culture KMS24 with its optimum conditions containing both light metal ions were 75, 62, 27 and 15, respectively but the mixed culture performed with a higher efficiency of 85, 74, 47 and 28%, respectively (Figure 1B). Therefore, the optimum condition for HMs removal by the strain KMS24 was applied for use in further experiments in the case of the mixed culture.
Removal of HMs and salts in the water collected from post cultured contaminated shrimp ponds The water sample used in this study was a composite sample collected from various shrimp ponds contaminated with HMs as previously described and its physicochemical properties were as follows: salinity, 10.23%0; EC, 4.68 mS/cm (Table 1) and pH, 8.07. The composite sample had the following HMs in mg/L; < 2+ 2+ 2+ 2+ 0.001 Cd , < 0.005 Pb , 0.043 Cu , and 0.057 Zn . As 2+ previously stated, only the efficiency for removal of Cu
Panwichian et al.
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Table 3. The removal percentage of Cd2+, Pb2+, Cu2+, and Zn2+ in the contaminated sediment from shrimp ponds after harvesting by the selected purple nonsulfur bacteria under microaerobic-light and aerobic-dark conditions.
% Removal Treatment
2+
Cd Light
2+
(0.29 mg/kg) Dark
2+
Pb (23.15 mg/kg) Light Dark
2+
Cu (15.05 mg/kg) Light Dark
Zn (22.16 mg/kg) Light Dark
Control Abiotic Native
20.2 ± 2.54 aB 19.96 ± 3.19
18.92 ± 3.87 aA 17.76 ± 2.47
aA
21.49 ± 3.41 bA 24.31 ± 5.22
aA
19.64 ± 1.99 bA 24.31 ± 4.22
aA
20.67 ± 2.15 aA 21.09 ± 1.79
aA
20.52 ± 2.36 aA 19.53 ± 2.70
aA
17.48 ± 1.50 bA 20.57 ± 1.40
aA
19.06 ± 2.91 bA 21.67 ± 2.16
NW16 Sterile + NW16 Native + NW16
30.28 ± 0.77 cA 36.83 ± 1.08
bA
32.54 ± 0.54 cB 40.32 ± 1.07
bB
60.20 ± 1.99 cA 66.95 ± 1.41
cA
63.24 ± 0.99 dB 69.35 ± 0.72
cB
31.83 ± 0.64 cA 41.24 ± 0.68
bA
34.25 ± 0.65 cA 39.63 ± 1.09
bB
33.76 ± 0.80 eA 39.15 ± 0.98
cdA
35.63 ± 0.54 eA 40.12 ± 0.90
KMS24 Sterile + KMS24 Native + KMS24
29.96 ± 1.08 dA 39.34±0.95
bA
32.26 ± 0.49 cA 40.19 ± 1.01
bB
67.66 ± 1.27 dA 71.01 ± 1.23
cB
65.27 ± 1.08 dA 70.97 ± 1.38
cdA
35.28 ± 0.43 eA 41.72 ± 0.87
dA
39.74 ± 0.92 dB 45.89 ± 0.94
cB
30.48 ± 0.86 dA 35.40 ± 0.61
cA
34.82 ± 1.04 deB 37.75 ± 0.69
Mixed of PNSB cells cA Sterile + Mixed 36.65 ± 1.56 dA Native + Mixed 39.80 ± 0.81
39.23 ± 1.22 dB 43.33 ± 0.92
cA
76.20 ± 1.81 eA 78.48 ± 1.12
eA
81.35 ± 2.91 fB 84.29 ± 3.41
eB
57.57 ± 1.02 gA 60.30 ± 2.04
fA
59.16 ± 1.62 fA 62.52 ± 1.07
eA
32.83 ± 2.60 dA 35.48 ± 1.93
cdA
30.02 ± 2.39 eB 40.95 ± 1.05
aA
aA
dB
dB
cA
The number in each bracket is the initial concentration of heavy metal ions; values in the same columns with different lowercase letters indicate significant differences (p<0.05). different upper case letters in the same row indicate significant differences between light and dark conditions of each set (p < 0.05).
2+
and Zn was studied. The removal of HMs and the control sets is presented in Table 2. There were no significant differences found for the 2+ 2+ removal of Cu and Zn under the two incubating conditions (microaerobic- light and aerobic-dark) between the abiotic and native control sets. In contrast, most treatment sets, more HMs was removed in the aerobic-dark than the microaerobic-light conditions although the differences may be not significant for some. The pure culture of KMS24 was performed with a significantly higher effi-ciency to remove both HMs ions than strain NW16 both in the presence and absence of native flora and with both incubating conditions. The presence of the native flora in all
cases produced a significant increase in the removal of HMs. The most effective treatment for 2+ Cu was observed with a mixed culture in the presence of the native flora that removed 73.78 and 75.92% under conditions of microaerobic-light and aerobic-dark, respectively. The mixed culture 2+ had a significant increased ability to remove Cu from 48.40% for the pure KMS24 culture plus native flora to 75.92%, while the comparable 2+ increase for Zn removal was from 29.93 to 31.67%, both with dark conditions. In all treatment sets as well as in both controls, salinity significantly decreased, parti-cularly those in a set of mixed culture either with sterile or native cultures (Table 1). However, based on the EC
values, significant differences were observed only in the inoculated sets. The highest efficiency to reduce salinity and EC values (roughly 77 and 49%) were found in the mixed culture set as previously stated.
Removal of HMs and salts in the sediment collected from contaminated post cultured shrimp ponds A composite sediment sample collected from shrimp ponds contaminated with HMs as previously described had the following physicochemical properties: 1.27 mS/cm EC, 0.84‰
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Afr. J. Biotechnol.
Table 4. Germination index of rice and water spinach in sediment and water samples after treatment by the selected purple nonsulfur bacteria.
Treatment
Germination index (% GI) Rice (Oryza sativa) Water spinach (Ipomoea aquatica) Water Sediment Water Sediment
Control Abiotic Native
10.50 ± 0.59 b 15.44 ± 0.29
a
59.38 ± 1.32 b 70.04 ± 0.30
a
10.59 ± 0.59 b 13.41 ± 0.07
NW16 Sterile+NW16 Native+NW16
20.47 ± 4.44 c 23.86 ± 2.26
c
80.57 ± 7.44 c 85.39 ± 6.29
c
KMS24 Sterile + KMS24 Native + KMS24
21.46 ± 0.85 c 24.03 ± 0.66
c
79.41 ± 0.45 c 83.35 ± 2.59
c
Mixed of NW16 and KMS24 cells d Sterile + Mixed 28.33 ± 1.28 e Native + Mixed 34.50 ± 2.23
106.14 ± 6.56 e 115.70 ± 3.22
d
a
78.30 ± 1.62 b 92.38 ± 2.09
21.75 ± 2.82 c 24.41 ± 1.06
c
114.83 ± 6.86 cd 120.71 ± 4.13
22.16 ± 2.54 d 26.91 ± 2.91
c
115.34 ± 3.86 d 126.90 ± 4.61
e
121.89 ± 2.83 e 139.33 ± 9.51
30.11 ± 3.17 f 35.29 ± 1.03
a
c
c
cd
Values in the same columns with different lower case letters indicate significant differences (p < 0.05).
salinity (Table 1) and pH 6.93 (data not shown). The 2+ amounts of HMs were in mg/kg dry weight; 0.29 Cd , 2+ 2+ 2+ 23.15 Pb , 15.05 Cu , and 22.16 Zn . The removal of HMs by the pure cultures of NW16 or KMS24 or their mixed culture with sterile sediment or non sterile (native) sediment under conditions of microaerobic-light and aerobic-dark is shown in Table 3. Comparing the removal percentages between the abiotic and native control sets under both incubating conditions, there was 2+ 2+ no significant difference for Cd and Cu , but a slight significant increase with the native flora was found for 2+ 2+ Pb and Zn . In all controls, the percentage removal from the sediment samples was higher than for the water 2+. samples especially for Zn removal and this was 2+. reflected in the values for Zn removal with the other pure and mixed cultures. The presence of the native flora with the pure or the mixed culture with both culture conditions caused a significant increase in the removal of all HMs. As was observed from the results with the contaminated water, strain KMS24 was mostly slightly better than strain NW16 at removing HMs (Table 2). Also, 2+ the best removal rate was for Pb with 84.29% removed by the mixed culture plus native flora in dark-aerobic conditions while the corresponding figure with the same 2+ 2+ 2+ conditions for Cu , Cd and Zn were 62.52, 43.33 and 40.95%, respectively (Table 3). Again the results were similar to those with the water samples, that is, the sediment samples, the efficiency to remove HMs in most treatment sets were higher in the aerobic-dark conditions than in the microaerobic-light conditions. A similar trend was observed in water and sediment samples after treatment as the salinity in all treatment sets and also in
both controls were significantly reduced and again the most effective treatment was produced by a set of mixed culture either with sterile or native population (Tables 2 and 3). No significant difference was found for the EC values after treatment in a native control; however, the EC values were significantly reduced in all of the inoculated sets. As expected, the set of the mixed culture with or without native flora in the sediment samples also produced the best reduction of salinity and EC (100 and 45%), as shown in Table 1. Toxicity assessment by seed germination for the sediment and water samples after treatment The toxicity of water and sediment samples after treatment by pure cultures of NW16 or KMS24 or their mixed culture was assayed by seed germination and results are presented as a germination index in percent (% GI) with rice (Oryza sativa) and water spinach (Ipomoea aquatic) (Table 4). All samples from the contaminated water inhibited germination but the inhibition was considerably reduced after any treatment with the most significant reduction occurring after treatment with the mixed culture with the native population. Samples from the sediment before treatment also inhibited germination and again all treatments reduced this inhibition. Water contaminated with HMs after treatment was more toxic than treated sediment samples. Treatment with the mixed culture with and without native flora resulted in an increased % GI. There were no significant differences found between the sets of pure cultures (NW16 or KMS24), but together they did
Panwichian et al.
have a synergistic effect. In all cases, the presence of the native population significantly increased the germination index up to 139.3% after treatment of the sediment samples with the mixed culture plus the native population. Rice was almost always more susceptible to inhibition than water spinach (Table 4). DISCUSSION Effect of Ca by PNSB
2+
and Mg
2+
on removal of heavy metals
In this study, biosorption by biomass of the selected PNSB strains (NW16 and KMS24) was used to remove HMs from a 3% NaCl synthetic solution (in the presence 2+ 2+ or absence of the light metal ions; Ca and Mg ), and samples of water and sediment were collected from shrimp ponds contaminated with HMs. The exposure time for binding between HMs and cells was short between 30 and 45 min, so biosorption was likely to be the main mechanism for removing HMs (no energy requirement). This biosorption may require many passive processes such as adsorption, covalent bond formation, complexation, chelation, ion exchange and microprecipitation (Panwichian et al., 2010b; Ahluwalia and Goyal, 2007). However, some bioaccumulation (requiring energy) of HMs by PNSB cells was possible (Panwichian et al., 2010b). Hence, with the use of both biosorption and bioaccumulation, one explanation for the mixed culture being more efficient than a pure culture might be that different organisms might use different processes resulting in a significant increase in the total removal of HMs when incubated together. The native population may also use alternative processes and might even facilitate both biosorption and bioaccumulation as well. In general, co-ions interfere and reduce the biosorption capacity of another metal ion such as by competition to use the same processes such as competition for binding sites on the PNSB cells. In this present study, light metal + 2+ +2 ions, such as Na , Ca and Mg , were present in the synthetic solution and the experimental data showed that 2+ 2+ + Ca and Mg light bivalent cations but not Na had a big effect on the biosorption of HMs (Figure 1). This indicated that the cell surface binding exhibits a low degree of specificity and similarly charged cations will also increase competition for binding sites on the cell surface. However, the results in this present study indicate that the use of a mixed culture of PNSB cells does have the potential to remove HMs in any of the conditions of the shrimp ponds.
Removal of HMs and salts in the water and sediment samples from post culturing contaminated shrimp ponds This work was focused on investigating the possibility for
7441
removal of HMs and salts in samples collected from shrimp ponds by PNSB prior to testing their use in the field. The physicochemical properties of the composite post cultured water from contaminated shrimp ponds showed a pH of 8.07 (data not shown) and this may have an effect on the solubility of HMs as a high pH normally accelerates their precipitation (Gazso, 2001), particularly 2+ 2+ for Pb and Cd (< 5 g/L Pb and < 1 g/L for Cd ). However, the amount of Cu and Zn ions were present at 43 and 57 µg/L, respectively and they exceeded the standard guidelines for marine aquatic animal cultivation ( 8 and 50g/L for Cu and Zn) (Pollution Control Department, 2006). In addition, any HM accumulation in the food webs might have an adverse effect on human beings as previously described. Therefore, the HMs contaminated water was treated by the selected PNSB strains under microaerobic-light and aerobic-dark conditions. Fortunately, removal of HMs by PNSB cells did occur under both incubating conditions and in general, the conditions of aerobic-dark produced a better removal efficiency than microaerobic-light conditions. In general, actively growing PNSB cells have been used to treat various wastewaters including that in shrimp ponds (Watanabe et al., 2003). These results show that the application of PNSB cells to clean up water will rapidly remove HMs from both the sediment which has microaerobic-light conditions and in the water column with aerobic-dark conditions at night time. However, biosorption using the biomass of PNSB should be investigated under the conditions of aerobic-light as well, although the biosorption process did occur without requiring energy from the cells. 2+ The results in Table 2 showed that removal of Cu and 2+ Zn in an abiotic control occurred (7.78 to 13.42%) in the water tested under both incubating conditions. This 2+ 2+ indicates the adsorption of HMs, either Cu or Zn , to non living organism or inorganic and organic matters in the water. It is well recognized that adsorption can remove metals over a wider range of pH values at lower concentrations, as in this present study than can be removed by alkaline precipitation (Baker and Khalili, 2004). In addition, biopolymers produced by microorganisms also bind metals strongly (Iyer et al., 2005; Watanabe et al., 2003; Panwichian et al., 2011). However, in this present study there was no significant difference for the removal percentage of HMs by native and abiotic controls. It might be that in this case, biopolymers derived from native flora were not present. Results of this study indicated that the biomass of KMS24 might have a greater affinity for both HMs ions than the biomass of NW16 as it provided higher efficiencies under either a set of pure culture or with native flora. In addition to biosorption of HMs to cells, bioaccumulation of HMs into cells can occur as previously described. This was 2+ supported by the removal of Zn under dark condition being significantly higher using a mixed culture (26.35%) than found for NW16 (22.52%) but there was no signifi-
7442
Afr. J. Biotechnol.
significant difference with KMS24 (25.47%). This was due 2+ to the toxicity of Zn on the strain NW16 being higher than the strain KMS24 and this result was in agreement with Panwichian et al. (2011). As each culture may have some different properties for binding or uptake of HMs including HMs tolerance; thereby synergistic removal of HMs was significantly increased with the mixed culture and again with higher efficiencies being obtained in the set with normal native flora. Results in Table 3 demonstrated that contamination of HMs in the sediment collected from shrimp ponds was at acceptable levels for use as agricultural soil (≤1.5, 75, 2+ 2+ 2+ 2+ 65 and 200 mg/kg for Cd , Pb , Cu and Zn , respectively) (Pollution Control Department, 2004; HKGS, 1998) although, the sediment initially contained higher concentrations of HMs than that found in the water. The abiotic control in the sediment samples removed more HMs than found for the water samples (Tables 2 and 3) due perhaps to the sediment having more organic and inorganic matter including clay particles to bind HMs. Moreover, a higher concentration of HMs in the sediment could increase its removal efficiency as the uptake rate of the metals ions will increase along with its increasing initial concentration when the amount of adsorbent is constant (Wang and Chen, 2006). There was no 2+ significant difference found for the removal of Cd and 2+ Cu in both control sets (abiotic and native) but there 2+ 2+ was a higher significant removal of Pb and Zn found in the native control. In addition, results obtained from the pure culture sets and the culture of NW16 or KMS24 with native flora confirmed that native flora did increase the efficiency to remove HMs. As previously described, the strain KMS24 was more 2+ resistant to Zn in the water tested than strain NW16 and the experiments with the sediment samples also 2+ showed that the former strain was more resistant to Cu (Tables 2 and 3). This can be explained using the same reasoning that was previously given in the water experiment as bioaccumulation could be involved and thus a pure culture of KMS24 produced a higher 2+ efficiency to remove Cu in the sediment samples tested. Again, it is not surprising that the mixed culture of both PNSB strains had the highest efficiency to remove HMs from the sediment by a synergistic action as previously explained. Some difference was found for the removal 2+ 2+ efficiency of HMs from the synthetic solution (Pb > Cu 2+ 2+ 2+ 2+ > Zn > Cd ) and the sediment samples (Pb > Cu > 2+ 2+ Cd ≈ Zn ) (Figure 1 and Table 3). There are many factors such as organic matter, soil particles like clay in the sediment samples that can affect removal efficiency when compared with the synthetic solution and this is why a higher efficiency was observed in the sediment samples because removal was by both biosorption and also adsorption. Furthermore, those factors also had an impact on the removal of Zn or Cd from the sediment samples. No significant differences were observed in the sets of control in both incubating conditions; however, a higher
efficiency to remove HMs from contaminated water and sediment samples by PNSB cells was observed in the aerobic-dark conditions than in the microaerobic-light conditions (Tables 2 and 3). The possible reason was due to the use of live PNSB cells that acted mainly as a biosorbent for the HMs ions with only a little by bioaccumulation because the contact time was only between 30 and 45 min (Panwichian et al., 2010b). As the binding of HMs to live cells may be toxic and possibly alter their surfaces and their metabolism thus it will affect the HMs removal by cells (Panwichian et al., 2011). In this case both PNSB cells were more sensitive to HMs ions in microaerobic-light conditions than those of aerobic-dark conditions (Tables 2 and 3) and the results were in accordance with Panwichian et al. (2011). The results of this study indicated that samples of water and sediment from post cultured contaminated shrimp ponds after treatment by the selected mixed PNSB (NW16 and KMS24) in both incubating conditions showed a significant decrease in both salinity and EC values. This was supported by our previous study which reported both strains under conditions of aerobic-dark and microaerobic-light have the potential to remove sodium ion in amounts that were detected in shrimp ponds (Panwichian et al., 2010a). In addition, this study 2+ 2+ demonstrated that light metal ions (Ca and Mg ) were competitors with HMs for binding with biomass (Figure 1). It is well recognized that the main salt composition of water in shrimp ponds is NaCl, CaSO4 and MgSO4 due to the brackish water used for shrimp cultivation (Panwichian et al., 2010b). Toxicity assessment by seed germination for the sediment and water after treatment The seed germination bioassay has been documented as one of the popular techniques for investigating the phytotoxicity of HMs (Ye et al., 2002) and the germination index (% GI) is regarded as the most sensitive parameter that is able to detect low toxicity that affects root growth and seed germination (Zucconi et al., 1981). Results of the germination of rice seed (Oryza sativa) and water spinach seed (Ipomoea aquatic) in treated water and sediment samples by all treatments was significantly higher than those in both the control sets (native > abiotic) and the % GI of both plants, rice and water spinach in the treated sediment was remarkably higher than that found in the treated water samples (Table 4). Biosorption of HMs and salts by PNSB strains as a pure culture or mixed culture either with native flora did not produce a significant decrease of their toxicity to plants and this corresponded with the removal percentage of HMs and salts.The results in this study demonstrated that in all treatment sets, particularly in a set of mixed culture with/without native flora, salinity sharply decreased while there was a moderate decrease for the EC values (Table 1). This is because salinity is the saltiness or dissolved
Panwichian et al.
salt content (predominantly NaCl and followed by CaSO 4 and MgSO4) of a body of water collected from post cultured shrimp ponds whereas EC is the total amount of dissolved ions in the water. As shown in this study, it seemed to be proper to use salinity for interpreting results of phytotoxicity test. It has long been known that increased salinity has an adverse effect on plant growth including halophytes (Khan et al. 2000). Hence, in order to explain why treated sediment had less toxicity to plants, there could be a number of reasons. First, contamination of HMs in the sediment was within the acceptance values for each HMs to allow plants to grow. Secondly, the salinity of the sediment samples (0.07%0) was much less than in the water (2.36%0). Moreover, the presence of higher amounts of nutrients in the sediment may enhance plant growth and help to reduce the toxicity of HMs and salts to both plants. Conclusion 2+
2+
The presence of light metal ions, Ca and Mg , had a negative effect on the removal of HMs by biosorption. However, the use of a mixed culture of PNSB (NW16 and KMS24) demonstrated the potential for successful application for reducing HM levels. The results clearly indicated that the biosorption of HMs by the selected PNSB in the water or sediment samples collected from contaminated shrimp ponds after harvesting; particularly with the mixed culture alone or with native flora significantly decreased the toxicity of HMs and salts to plants as demonstrated by an increased GI values. However, toxicity was still found in the treated water but that could be caused by salinity. It is therefore suggested that the wastewater from post culturing shrimp ponds could be treated by bioremediation such as biosorption prior to discharge into the environment. ACKNOWLEDGEMENTS This research was granted by Office of the Higher Education Commission, Saijai Panwichian was supported by CHE PhD. Scholarship. This work was also funded by a project number SCI520001S, Prince of Songkla University. Abbreviations PNSB, Purple nonsulfur bacteria; HMs, heavy metals. REFERENCES Ahluwalia SS, Goyal D (2007). Microbial and plant derived biomass for removal of heavy metals from wastewater. Bioresource Technol. 98 : 2243-2257. Barker AV, Bryson GM (2002). Bioremediation of heavy metals and organic toxicants by composting. The Sci. World J. 2: 407-420. Baker H, Khalili F (2004). Analysis of the removal of lead (II) from aqueous solutions by adsorption onto insolubilized humic acid:
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African Journal of Biotechnology Vol. 11(29), pp. 7445-7453, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.3508 ISSN 1684â&#x20AC;&#x201C;5315 Š 2012 Academic Journals
Full Length Research Paper
Biofixation of carbon dioxide by Chlorococcum sp. in a photobioreactor with polytetrafluoroethene membrane sparger Xiaoli Chai1*, Xin Zhao1 and Wang Baoying2 1
State Key Laboratory of Pollution Control and Resource Reuse, Tongji University, Shanghai, 200092, China. 2 Shanghai Tongji Construction Co., Ltd, Shanghai 200092, P.R. China. Accepted 28 February, 2012
The greenhouse effect, caused by excessive carbon dioxide emissions, constitutes a major aspect of global warming. Biological fixation of carbon dioxide using microalgae is an effective carbon dioxide reduction technology, but its widespread implementation is limited by the poor mass transfer efficiency. In this study, Chlorococcum sp. was cultured in a photobioreactor with a polytetrafluoroethene membrane sparger (PTFE) to study CO2 biofixation and microalgae growth. Daily variations of dissolved oxygen (DO), pH and dissolved CO2 were analyzed during batch culture of Chlorococcum sp. in the photobioreactor. The culture of Chlorococcum sp. under different operating conditions, such as pH, light cycle (light:dark) and nitrate feeding, were carried out to optimize the CO 2 biofixation rate and the algal productivity. The results confirmed that the photobioreactor with a membrane sparger is an alternative option for CO2 removal from flue gas by cultivation of microalgae. Key words: Bioreactor, polytetrafluoroethene membrane sparger (PTFE) membrane sparger, Chlorococcum sp., greenhouse gas. INTRODUCTION The excessive combustion of fossil fuel constituting a major aspect of global warming causes severe environmental destruction on the earth (IPCC, 2007). Currently, more than 80% of the energy produced globally each year is generated through the combustion of fossil fuels, which is the largest single CO 2 emission source (Korre et al., 2010; Sayre, 2010). Nearly one-third of anthropogenic CO2 emission comes from combustion of fossil fuels in power plants worldwide (Mansourizadeh and Ismail, 2010). Emission trading and related CO 2 certificates have proven to be very costly for industries dependent on the burning of fossil fuels (Borkenstein et al., 2010). Biofixation of carbon dioxide can be achieved
*
Corresponding author. E-mail: xlchai@tongji.edu.cn. Tel: +8621-65981831. Fax: +86-21-65982684. Abbreviations: PTFE, Polytetrafluoroethene sparger; DO, dissolved oxygen.
membrane
through the photosynthesis of terrestrial plants as well as photosynthetic microorganisms. Microalgae has a higher photosynthetic efficiency and biomass productivity, a faster growth rate and high added-value by-products, such as biofuel and feedstocks for animal, when compared with other photosynthetic plants (Gouveia and Oliveira, 2009; Sayre, 2010). Microalgae are a feasible and effective alternative for biofixation of carbon dioxide from the flue gas. The CO2 transfer efficiency is one of the most important parameters for enhancing the CO2 biofixation rate and algal productivity in a photobioreactor culture system (Fan et al., 2007; Ryu et al., 2009). The carbon dioxide mass transfer capacity of a photobioreactor is determined by the liquid-phase mass transfer coefficient and the specific area available for mass transfer (Markl, 1977; Carvalho et al., 2006). For the purpose of mass transfer, the most frequently used approach is bubbling CO 2enriched air into the bottom of the photobioreactor with diffusers (Kumar et al., 2010). However, the associated drawbacks are a loss of CO2 to the atmosphere and poor
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mass transfer rates because of the relatively low interfacial specific surface area and the low residence times of the gas in the culture (Cheng et al., 2006; Kumar et al., 2010). Another method for CO2 mass transfer in a photobioreactor is using hollow fiber membranes. Compared with conventional gas dispersion methods, hollow fiber membranes have advantages of surprisingly high interfacial area provided by the membrane and the dramatic reduction in the amount of CO2 lost to the atmosphere (Ferreira et al., 1998; Carvalho and Malcata, 2001). However, the hollow fiber membrane systems suffer from some drawbacks, including less turbulent hydrodynamic pattern, low mixing rate and biomass settling. In this study, a photobioreactor with a polytetrafluoroethene membrane sparger (PTFE) membrane sparger, with the pore size of 0.22 μm, was specifically designed to explore the possibility of coupling CO 2 biofixation with microalgal growth. Artificial flue gas was used as feed gas with the composition of 10% CO 2, 5% O2 and 85% N2 to study the CO2 biofixation rate and growth rate by Chlorococcum sp. in a photobioreactor. Operational conditions of the photobioreactor, such as pH, light cycles, nitrate feeding were investigated to maximize the CO2 biofixation rate.
online and recorded every minute by the controlling system. Light was supplied by ten 40 W fluorescent lamps, uniformly placed outside the photobioreactor. The mixing of algal culture was achieved by using a peristaltic pump with a circulation rate of 500 ml min-1. A schematic diagram of the experimental setup is shown in Figure 1a. A membrane sparger with an internal aperture diameter of 0.22 μm that was made of PTFE was installed at the bottom of the photobioreactor. The parameters of the membrane sparger are presented in Table 1. A schematic diagram of the structure of the membrane sparger is shown in Figure 1b.
MATERIALS AND METHODS
Analysis of CO2 and O2 concentration
Strain and culture medium
The CO2 and O2 concentration in the influent gas and effluent gas was measured by a gas chromatograph (GC9160, Ouhua Technology, Shanghai) equipped with a thermal conductivity detector. A chromatography workstation (HW2000, China) combined with a data acquisition computer was connected to the gas chromatograph.
Chlorococcum sp. (FACHB-957) was provided by the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). The modified soil extract medium (SE medium) is composed of the following components (per liter): 250 mg NaNO3, 75 mg K2HPO4·3 H2O, 75 mg MgSO4·7 H2O, 25 mg CaCl2·2H2O, 175 mg KH2PO4, 25 mg NaCl, 40 ml soil extracts, 5 mg FeCl3·6H2O, 1 ml Fe-EDTA, 1 ml A5 solution and 958 ml distilled water. Soil extract solution: 200 g soil without fertilization and 1000 ml of deionized water were placed in a 2 L bottle. The bottle with 100°C water bath heating for 2 h cooled to room temperature and the procedure was repeated three times. The mixture of the bottle was left to settle and the supernatant collected and filtered. The A5 solution was composed of the following (per 100 ml): 286 mg H3BO3, 181 mg MnCl2·4H2O, 22 mg ZnSO4·7H2O, 7.9 mg CuSO4·5 H2O and 3.9 mg (NH4)6Mo7O24·4H2O.
Photobioreactor system The study of microalgae cultivation was carried out in an 8 L enclosed cylindrical glass photobioreactor with an inner diameter of 12 cm and a height of 75 cm, under the illumination of fluorescent lights. The aeration gas was passed through a bacterial gas filter prior to injection. The water was circulated through a water jacket surrounding the bubble column to control the temperature of the photobioreactor. The inflow gas was composed of 85% N 2, 5% O2 and 10% CO2 and was injected into the culture with 30 ml/min. Three probes for dissolved CO2, dissolved oxygen (DO) and pH were installed at the top of the photobioreactor. The dissolved carbon dioxide, pH and DO of the algal culture were monitored
Measurement of light intensity A light sensor (TES1332A, Taiwan) was used to measure the light intensity on the surface of the bioreactor. The average light intensity was calculated by taking the weighted average of 60 measurements, which are located at the three horizontal planes uniformly located along the vertical axis of the reactor, and 20 evenly distributed measuring points along the radial axis for each horizontal plane height of the reactor (Ryu et al., 2009). Measurement of biomass concentration The biomass concentration (g/L) was measured by analyzing the optical density of the cell suspension at an absorbance wavelength of 685 nm using a spectrophotometer (UV-Vis 2550, Shimadzu, Japan). A calibrated straight line was been obtained previously by plotting the A685 dry cell weight (g/L).
Measurement of the inorganic carbon (IC) and the total concentration of nitrogen sources (TN) The IC and TN in the culture medium were measured by a TOC/TN analyzer (TOC-VCPN, Shimadzu, Japan). The culture medium was first filtered through a cellulose acetate membrane filter (0.22 μm) before it was used for IC and TN measurements. CO2 biofixation rate and CO2 removal efficiency The total carbon content of Chlorococcum sp. was measured by an elementar analyzer (Vario EL III, German). The oxygen production efficiency, the CO2 biofixation rate and the removal efficiency can be calculated by the following equations: CO2 biofixation rate =
Xmax X 0 C 44 t 12
CO2 removal efficiency =
Yin Yout 100% Yin
(1)
(2)
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a b
Figure 1. (a) Experiment setup of the airlift photobioreactor with an online process monitoring unit. 1, Bioreactor; 2, artificial flue gas cylinder; 3, flowmeter; 4, bacterial gas filter; 5, aeration membrane sparger; 6, water jacket; 7, thermostatic bath; 8,18-peristaltic pump; 9, light source; 10, microcomputer timer; 11, DO electrode; 12, pH electrode; 13, CO2 electrode; 14, gas outlet; 15, signal translator; 16, gas chromatography; 17, computer. (b) Structure of the membrane sparger.
where, Xmax and X0 are the maximum cell concentration and the initial inoculated cell concentration (mg/L); t is the time required to reach the maximum cell concentration (d); C is the carbon content of dried biomass analyzed by the element analyzer; 44 is the molecular weight of CO 2, and Yin and Yout are the CO2 molar fractions in the inlet and outlet gas phases, respectively. The mass transfer coefficient for CO2 (KLa(CO2)) The KLa(CO2) was determined in the microalgae culture with cells by a dynamic-state method according to the method use to measure KLa(O2) (Qi and Xia , 2004 ).
(3) where, [CO2]* is the equilibrium concentration of CO2 in culture; [CO2] is the dissolved CO2 concentration; is the CO2 bioďŹ xation rate. After injecting the gas, the [CO2] is monitored continuously with a peristaltic pump to make the culture homogenous. The CO2 bioďŹ xation rate calculated as,
can be (4)
When the concentration of dissolved CO2 dropped below 1.0 mol/m3, start to inject the inflow gas to the photobioreactor, and then the [CO2] begin to increase. (5) A
straight
line
relationship
between
and
can be obtained. The negative reciprocal
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Table 1. Parameters of the membrane sparger.
Parameter Pore size (μm) Radius (cm) Height (cm)
Value 0.22 7.0 15.5
value of the slope is the KLa (CO2).
RESULTS AND DISCUSSION Characteristics of CO2 biofixation by chlorococcum sp. during batch cultivation Chlorococcum sp. was cultivated in the photobioreactor with a membrane sparger for six days. During the batch culture, after one day’s preadaption, the algae exponential growth phase was observed from the second to the fourth day, followed by the stationary growth phase. Figure 2a shows that the dissolved CO2, DO and the pH of the culture changed periodically with the illumination time and dark time during the batch cultivation. The value of dissolved CO2 coincided with the different photosynthetic efficiencies during the culture stages. The maximum and minimum values were 14.3 and 7.7 mM, respectively. The daily DO peak increased gradually with the increase of cell mass when the algae were in the exponential growth phase, and it decreased during the stationary phase. The O2 production and carbon dioxide removal were closely related to the photosynthesis intensity of the algae in the culture. On the third day, the algae in the culture reached the highest photosynthetic intensity, and a maximum DO of 135% and a minimum dissolved CO2 of 7.7 mM were obtained. The pH value increased from 5.9 to 6.6 over the course of the culture time. The gradual increase of pH during batch culture could be attributed to the consumption of IC in the photosynthetic culture. The pH value increased and reached a maximum value at the end of the daily illumination period because the IC in the culture was utilized as carbon source by the algae (Jacob-Lopes et al., 2008). The daily variations of O2 and CO2 concentration in the batch culture are shown in Figure 2b. The variations of O2 and CO2 in the outlet gas were consistent with the DO and dissolved CO2 in the culture. The O2 in the outlet increased as soon as the light was turned on at 8:00 in the morning and decreased sharply to a minimum following dark respiration at 20:00 in the evening. The CO2 concentration at the outlet decreased with the illumination time and increased gradually after the light was turned off. The O2 decline and the CO2 increase can be attributed to the extinction of photosynthesis without
light. Moreover, higher O2 concentration and lower CO2 concentration were obtained with the increase in the photosynthetic intensity at the following day. The results suggest that O2 production and CO2 removal were observed during the illumination period, indicating that operational conditions should be optimized to photosynthesis intensity in order to enhance the CO 2 removal efficiency of the photosynthetic system. Figure 2c shows the variation of CO2 removal efficiency during batch cultivation. The CO2 removal efficiency increased from 36 to 65% with the increase of cultivation time. A previous study (Chiu et al., 2008) reported a 20% CO2 reduction in a semicontinuous photobioreactor with a high-density culture of Chlorella sp. with 10% CO2 in air. The results of the present study indicate that a photobioreactor with a membrane sparger can provide a high level of dissolved CO2 and high transfer efficiency. The high value of dissolved CO2 and the long retention time sufficiently enhance the CO2 biofixation rate and thus reduce the CO2 flowing out of the photobioreactor. Hydrophobic material and microbubbles produced by the membrane sparger make the CO2 transfer from the gas phase into the liquid phase more easily in the culture. The mixing condition of the membrane sparger was improved compared with hollow fiber membranes. The relationship between KLa(CO2) and the aeration rate is shown in Table 2. The KLa(CO2) increase with the increasing aeration rate. High aeration rate contribute to low CO2 removal efficiency and high KLa(CO2). When the aeration rate was above 0.004 vvm, dissolved CO 2 could maintain above 0.68 mM. It was reported that dissolved CO2 above 0.68 mM would balance the demand for the photosynthesis of the microalgae cells (Becker, 1994). The total carbon input flux is determined by inflow rate and partial pressure of CO2 (Carvalho et al., 2006). -1 KLa(CO2) of 4-6 h was required for a plate photobioreactor to meet the limiting CO2 concentration in the liquid (Zhang et al., 2002). Regarding production cost and CO2 removal efficiency, suitable KLa and aeration rate should be determined. In this study, KLa(CO2) of -1 0.52h can satisfy the CO2 demand of the photosynthesis in the culture.
Effect of pH on the growth of microalgae and the CO2 biofixation rate In this study, the pH was controlled by automatic addition of NaOH (4 M) solution and HCl (1 M) solution. Figure 3 shows the stable growth profiles of Chlorococcum sp. under pH 5.6, 8.0 and 10.0. As shown in Figure 3, the maximum cell concentration was found in the culture at pH 8.0 with a cell concentration of 0.61 g/L after five days. The growth rate at pH 10.0 was relatively low, and the cell concentration reached 0.17 g/L after four days of cultivation.
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a
7.0
120
6.8
CO2
100
6.6
80
14
Dissolved CO2 (mM)
DO pH
140
12
6.4 pH
DO (%)
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6.2
10
40 6.0
20 0
8
5.8 0
1
2
3
4
5
6
Time (day) Time (d) O2
10
CO2
O2 Concentration (%)
9
7 8
6
7
6
CO2 Concentration (%)
8
b
5 5 00:00
08:00
16:00
00:00
08:00
16:00
00:00
Time Figure 2. (a) Variation of pH, DO and dissolved CO2 during batch cultivation; (b) daily variation of the O 2 80 CO2 concentrations during batch cultivation; (c) variation of CO2 removal efficiency during batch cultivation. DO, Dissolved oxygen.
c
70 fficiency (%)
According to an analysis of the elements in the 60at the end of cultivation, 1 g of Chlorococcum sp. cells
50 40
biomass contained approximately 0.48 g carbon in the study. The value was used to estimate the CO2
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08:00
16:00
00:00
08:00
16:00
00:00
Time
c
80
CO2 removal efficiency (%)
70 60 50 40 30 20 10 0 0
1
2
3
4
5
6
Time (day) Time (d) Figure 2. Contd.
Table 2. The relationship between KLa(CO2) and the aeration rate.
Aeration rate (vvm) 0.013 0.025 0.038
-1
(h ) 0.52 0.84 1.62
biofixation rate. The CO2 biofixation rate (mg /L /day) was calculated according to Equation 1, and the results are presented in Table 3. The highest CO2 biofixation rate was 197 mg/L/day (pH 8.0). The lowest biomass concentration and CO2 biofixation rate were observed when the pH was 10.0. The pH affects the carbon concentration mechanism (CCM) in part by acting on the enzyme ribulose-1, 5bisphosphate carboxylase. The activity of this enzyme is pH-dependent; its activity increases with an increase of pH (Jacob-Lopes et al., 2008). pH will influence the activity of ribulose-1, 5-bisphosphate carboxylase as well as the amount of inorganic carbon. Eukaryotic microalgae acquire IC from the surrounding aqueous medium to support photosynthesis (Colman et al., 2002). Most of – microalgae species take up both CO2 and HCO3 ; few
2−
can use CO3 as a carbon source. The concentration of IC increased with the culture time and exceeded 2000 mg/L at six days for a pH of 10.0. The predominant form 2− of dissolved inorganic carbon is CO3 , which is not available for microalgae to use for photosynthesis. More – than 98% of the IC is in the form of HCO3 when the pH is 8.0, while the main form of the IC is CO2 and H2CO3 when the pH is 5.6 (Clark and Flynn, 2000). This implies that microalgae at a pH of 8.0 will have a higher carbon fixation rate than they do at pH 5.6. Effect of the light cycle on the growth of microalgae and the CO2 biofixation rate The light duration during the illumination time will affect the amount of energy received by the microalgae. The amount of light energy is closely related with the productivity in terms of the biomass and the cell growth rate, which consequently determines the photoautotrophic carbon fixation capacity (Janssen et al., 2001; Jacob-Lopes et al., 2009). Figure 4 shows the growth profiles of the Chlorococcum sp. under different light cycles (day/night). Table 4 shows the CO2 biofixation rate by microalgae under the various light cycles. The maximum biomass concentrations under light cycles of 24:0, 16:8 and 12:12 were 0.76, 0.95 and 0.62 g/L,
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0.7 pH=5.6 pH=8.0 pH=10.0
Biomass Concentration (g/L)
0.6 0.5 0.4 0.3 0.2 0.1 0.0 0
1
2
3
4
5
6
7
8
Time(day) (d) Time Figure 3. Growth of Chlorococcum sp. under various pH values. Table 3. CO2 biofixation rate for various pH values.
pH 5.6 8.0 10.0
Maximum cell conc. (g/L) 0.53 0.61 0.17
Biomass concentration (g/L)
1.0
Biofixation rate (mg/L/day) 166 197 49
24:0 16:8 12:12
0.8
0.6
0.4
0.2
0.0 0
1
2
3
4
5
Time (day) Time (d) Figure 4. Growth of Chlorococcum sp. under the various light cycles.
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Table 4. CO2 biofixation rate under various light cycles.
Light cycle (light:dark) 24:0 16:8 12:12
a
Biofixation rate (mg/L/day) 252 305 199
1.4 with nitrate feeding without nitrate feeding
1.2
Biomass concentration(g/L)
Maximum cell conc. (g/L) 0.76 0.95 0.62
1.0 0.8 0.6 0.4 0.2 0.0 0
2
4
6
8
10
12
Time (day) Time (d) 60
Nitrogen concentration (mg/L) nitrogen concentration (mg/L)
b
with nitrate feeding without nitrate feeding
50 40 30 20 10 0 0
2
4
6
8
10
12
Time (day) Time (d)
Figure 5. (a) Growth of Chlorococcum sp. with and without nitrate feeding (b) Variation of nitrogen concentration with and without nitrate feeding.
respectively. The corresponding CO2 biofixation rates were 252, 305 and 199 mg/L/day, respectively. The maximum carbon dioxide fixation rate was obtained under the light cycle of 16:8. Different cell growth proďŹ les were seen as a function of the light periods. The cultures with light cycle of 16:8
(light: dark) showed similar characteristics to the algae cultivated with a continuous supply of light energy for the first 3 days. The maximum biomass concentration was observed at light cycle of 16:8. The culture with light cycle of 24:0 obtained a lower cell concentration than that with16:8 at the end of the culture. The growth profile of
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12:12 showed an adaption phase of two days before the exponential phase. The results show that supplying suitable light energy was a key factor for enhancing the growth of Chlorococcum sp. and the CO2 biofixation rate. Effect of nitrate feeding on the growth of microalgae and the CO2 biofixation rate The effect of the nitrate feeding was analyzed to confirm that nitrogen availability is the primary limiting factor for algal growth and CO2 biofixation (Jin et al., 2006; Kumar et al., 2010). The CO2 biofixation efficiency has a close relation with the growth phase of the microalgae, which declines precipitously when the cell growth phase reaches the stationary phase, followed by a death phase (Jin et al., 2006). Fig. 5a shows the growth profiles of Chlorococcum sp. with and without nitrate feeding. The nitrogen source decreased sharply from 32 mg/L to 3 mg/L in 4 days without nitrate feeding. The maximum biomass concentration and carbon biofixation rate were only 0.69 g/L and 1.1 gCO2/L, respectively. Nitrogen source was added to the culture to avoid nitrogen deficiency and maintain the nitrogen concentration above the limiting level (Fig. 5b). The biomass concentration and the carbon biofixation with nitrate feeding were 1.9 times of the values without nitrate feeding. The maximum concentration was 1.3 g/L, and the biofixation rate was 2.1 gCO2/L. In addition, the logarithm growth phase was prolonged by three days with nitrate feeding. The results suggest that photobioreactors with nitrate feeding can efficiently accelerate the CO2 biofixation and algae growth rate. Conclusion The results indicate that it is an effective way to supply CO2 by the PTFE membrane sparger during microalgal culture. The maximum CO2 removal efficiency reached 65% at the third day of the cultivation. The optimal pH and light cycle for the microalgae growth was 8.0 and 16:8. The maximum biomass concentration and the CO2 biofixation rate of light cycle 16:8 were 0.95 g/L and 305 mg/L/d, respectively. The logarithmic growth phase of the algae was prolonged by three days with nitrate feeding. The principle of this photobioreactor design will be of great interest for both algal cultivation and biofixation of CO2 from flue gas. REFERENCES Becker EW (1994). Large-scale cultivation. In Microalgae: Biotechnology and Microbiology. Becker EW, Ed., Cambridge University Press: New York. pp. 163-171. Borkenstein CG, Knoblechner J, Frühwirth H, Schagerl M (2010). Cultivation of Chlorella emersonii with flue gas derived from a cement
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plant. J. Appl. Phycol. 23: 131-135 Carvalho AP, Malcata FX (2001). Transfer of carbon dioxide within cultures of microalgae: plain bubbling versus hollow-fiber modules. Biotechnol. Prog. 17: 265-272. Carvalho AP, Meireles LA, Malcata FX (2006). Microalgal reactors: a review of enclosed system designs and performances. Biotechnol. Prog. 22: 1490-1506. Cheng LH, Zhang L, Chen HL, Gao CJ, (2006). Carbon dioxide removal from air by microalgae cultured in a membrane-photobioreactor. Sep. Purif. Technol. 50: 324-329. Chiu SY, Kao CY, Chen CH, Kuan TC, Ong SC, Lin CS (2008). Reduction of CO2 by a high-density culture of Chlorella sp. in a semicontinuous photobioreactor. Bioresour. Technol. 99: 3389-3396. Clark DR, Flynn KJ (2000). The relationship between the dissolved inorganic carbon concentration and growth rate in marine phytoplankton. Proc. R. Soc. Lond. B. 267: 953-959. Colman B, Huertas IE, Bhatti S, Dason JS (2002). The diversity of inorganic carbon acquisition mechanisms in eukaryotic microalgae. Funct. Plant Biol. 29: 261-270. Fan LH, Zhang YT, Zhang L, Tang DS, Cheng HL (2007). Optimization of carbon dioxide fixation by Chlorella vulgaris cultivated in a membrane photobioreactor. Chem. Eng. Technol. 30: 1094-1099. Ferreira BS, Fernandes HL, Reis A, Mateus Ma (1998). Microporous hollow fibres for carbon dioxide absorption: mass transfer model fitting and the supplying of carbon dioxide to microalgal cultures. J. Chem. Technol. Biotechnol. 71: 61-70. Gouveia L, Oliveira AC (2009). Microalgae as a raw material for biofuels production. J. Ind. Microbiol. Biotechnol. 36: 269-274. Intergovernmental Panel on Climate Change (IPCC) (2007). Climate Change. Synthesis Report. Fourth Assessment Report. Cambridge University Press, Cambridge, UK. Jacob-Lopes E, Lacerda LMCF, Franco TT (2008). Biomass production and carbon dioxide fixation by Aphanothece microscopica Nägeli in a bubble column photobioreactor. Biochem. Eng. J. 40: 27-34. Jacob-Lopes E, Scoparo CHG, Lacerda LMCF, Franco TT (2009). Effect of light cycles (night/day) on CO2 fixation and biomass production by microalgae in photobioreactors. Chem. Eng. Process, 48: 306-310. Janssen M, Slenders P, Tramper J, Mur LR, Wijffels RH (2001). Photosynthetic efficiency of Dunaliella tertiolecta under short light/dark cycles. Enzyme Microb. Technol. 29: 298-305. Jin HF, Lim BR, Lee K (2006). Influence of nitrate feeding on carbon dioxide fixation by microalgae. J. Environ. Sci. Health. Part A. 41: 2813-2824. Korre A, Nie ZG, Durucan S (2010). Life cycle modelling of fossil fuel power generation with post-combustion CO2 capture. Int. J. Greenhouse Gas Control. 4: 289-300. Kumar A, Ergas S, Yuan X, Sahu A, Zhang Q, Dewulf J, Malcata FX, Langenhove HV (2010). Enhanced CO2 fixation and biofuel production via microalgae: recent developments and future directions. Trends Biotechnol. 28: 371-380. Mansourizadeh A, Ismail AF (2010). A developed asymmetric PVDF hollow fiber membrane structure for CO2 absorption. Int. J. Greenhouse Gas Control. 5: 374-380. Markl H (1977). CO2 transport and photosynthetic productivity of a continuous culture of algae. Biotechnol. Bioeng. 19: 1851-1862. Qi YZ, Xia J (2004). Biological Reaction Engineering. Chemical Industry Press: Beijing, pp. 141-142. Ryu HJ, Oh KK, Kim YS (2009). Optimization of the influential factors for the improvement of CO2 utilization efficiency and CO2 mass transfer rate. J. Ind. Eng. Chem. 15: 471-475. Sayre R (2010). Microalgae: The Potential for Carbon Capture. Bioscience, 60: 722-727. Zhang K, Kurano N, Miyachi S (2002). Optimization aeration by carbon dioxide gas for microalgal production and transfer characterization in a vertical flat-plate photobioreactor. Bioprocess Biosyst. Eng. 25: 97101.
African Journal of Biotechnology Vol. 11(29), pp. 7454-7463, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.3741 ISSN 1684–5315 © 2012 Academic Journals
Full Length Research Paper
Cloning, expression and purification of cold adapted acetate kinase from Shewanella species AS-11 Md. Abul Kashem Tang1, 2*, Hiroyuki Motoshima1 and Keiichi Watanabe1, 2 1
Department of Applied Biochemistry and Food Science, Saga University, Japan. The United Graduate School of Agricultural Sciences, Kagoshima University, Japan.
2
Accepted 6 February, 2012
A psychrotrophic bacterium, Shewanella sp. AS-11 was isolated from a buccinid (shell) Neobuccinum eatoni living in the Antarctic ice-covered sea. An open reading frame of 1203 bp, coding for acetate kinase gene, called ack, was amplified, cloned into the expression vector, pETY-16b, and the enzyme was overproduced by using T7 system in Escherichia coli BL21 (DE3). After extraction of crude recombinant acetate kinase, the desired enzyme was able to be purified on a Blue Sepharose CL-6B and Super-Q affinity column chromatography. The molecular mass of the enzyme is about 86 kDa, which is associated with two monomers. In respect of pH, the enzyme was stable between 6 to 8 and maximum activity was obtained at 7.5. The purified enzyme was stable at 30°C but ligand bound enzyme was stable at 40°C. The structural comparison to mesophilic and thermophilic acetate kinases demonstrates that the psychrophilic one contains lower number of salt bridges and cation-pi interaction. So, it can be suggested that the enzyme is cold adapted with thermolabile and flexible structure. Key words: Acetate kinase, thermolabile, cold adapted, flexible, activity.
INTRODUCTION Acetate kinase plays important role for bioconversion of organic compounds to methane and annually more than billion metric tons of methane are produced from the decomposition of organic matter by anaerobic microbial consortia (Ferry, 1992), which can help to reduce environmental pollution and energy crisis. Temperature is one of the most important factors for bioconversion of organic waste materials. In most parts of our earth that are cold as well as in all over the world, a large seasonal variation is observed. In cold environment (at low temperatures), the growth of microorganisms is reduced several times; as a result, degrading rate of the organic pollutants is decreased, which ultimate end product is
*Corresponding author. E-mail: makashemsa@gmail.com watakei@cc.saga-u.ac.jp. Fax /Tel: +81 952 28 8774.
methane. The bioconversion process of recalcitrant compounds can be improved by using mixed culture containing specific cold adapted microorganisms (Kumar et al., 2011). Many environment pollutant compounds such as nitrates, hydrocarbons, aromatic compounds, cellulose, chitin, lignin, protein, heavy metals, etc. are already reduced by using psychrophilic or psychrotrophic microorganisms (Timmis and Pieper, 1999; Vazquez et al., 1995). Psychrophilic or psychrotrophic organisms are colonized in cold environment and can synthesize cold adapted enzymes. These enzymes bear special character like high catalytic activity at low temperatures, a large flexibility, etc. to adapt to the organisms in cold environment (Gerday et al., 2000; Feller et al., 1999; Chiuri et al., 2009). For this specific nature, the enzymes are offered in potential economic advantages in different biochemical sectors such as bioremediation of polluted wastes, biomass conversion, detergents industry, food
Tang et al.
processing, etc. by catalyzing the reaction at low and moderate temperatures instead of high temperature. As a result psychrophiles have received increasing attention due to their pertinence in both basic and applied research. Acetate kinase, (EC 2.7.2.1, ATP:acetate phosphotransferase) is a member of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of phosphotransferases (Buss et al., 2001), which catalyzes the phosphorylation of acetate with ATP to form acetyl phosphate. The enzymes, which belong to this group exhibit large conformational changes during catalysis and these conformational changes are easily observed by domain movement (Diao et al., 2009). Acetate kinase especially psychrophilic acetate kinase (generally, more flexible structure is formed by psychrophilic enzyme than mesophilic or thermophilic enzymes) is an ideal enzyme for effective biodegradation of organic waste materials at low temperature. Shewanella sp. AS-11 is a Gram-negative, rod-shaped and aerobic bacterium isolated from a buccinid (shell) Neobuccinum eatoni living in the Antarctic ice-covered sea. Shewanella sp. AS-11 grows most rapidly at 20°C and can grows well at 4°C but cannot grow above 30°C, being classified to psychrotroph according to Morita (1975). The amino acid sequences of the protein encoded by acetate kinase gene have been determined from the genomic DNA of Shewanella sp. AS-11 bacterium by Tanoue et al. (2010). In our present study, the acetate kinase from psychrotrophic bacterium Shewanella sp. AS-11 was cloned and expressed in Escherichia coli DH5 and BL21 (DE3), respectively. Finally, expressed enzyme was purified and partially characterized.
MATERIALS AND METHODS
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2400. The acetate kinase gene was amplified with forward (P-1) and reverse primer (P-2) 5`GTTTAACTTTAAGAAGGAGATATACCATGTCAGACAAATTAGT ACTCGT-3` and 5`AGCTTCCTTTCGGGCTTTGTTAGCAGCCTCATTACTTAGCTGT GATCAGCTTAATG-3`, respectively (bold and under lines nucleotides are homologous of pETY-16b vector, starting and stop codon, respectively) under the following reaction condition: 2 min at 94°C for initial denaturation; followed by 30 cycles of 10 s at 98°C for denaturation, 30 s at 53°C for annealing, and 1 min at 68°C for extension using KOD Plus DNA polymerase. The primer-1 was designed to include 26-nucleotide sequences homologous to that just upstream of the starting codon of pETY-16b vector (The pETY16b, E. coli-yeast shuttle vector, which was constructed by introducing a 2 micro replication origin and a selectable marker (URA3) of yeast to pET-16b) at the 5` end so that the recombinant enzymes is produced without His•Tag. The primer-2 was designed, to include 28-nucleotide sequence homologous to that just downstream of the BamHI site of pETY-16b vector at 5` end. Amplified gene was tested on 1% agarose-S gel electrophoresis.
Transformation and isolation of recombinant plasmid The cells of Saccharomyces cerevisiae were transformed according to the method of Gietz and Wood (2002). The yeast cells were suspended in 90 µl transformation mixture and cultivated. The mixture was prepared by mixing 60 µl of 50% polyethylene glycol 3500, 9 µl of 0.1 M lithium acetate, 12.5 µl of 2 mg/ml boiled SScarried DNA, 6 µl of amplified genes (100 ng) and 2.5 µl BamHI digested by pETY-16b vector (80 ng). The recombinant plasmid was isolated from the yeast cells by using QIAprep Miniprep kit (Qiagen) according to the manufacturer’s instructions. The graphical representation of the amplified acetate kinase ORF into pETY-16b by homologous recombination in yeast is shown in Figure 1. Recombinant plasmid was transferred into E. coli DH5α according to the method of Pope and Kent (1996) and the recombinant plasmid DNA was isolated. The plasmid DNA was digested with restriction enzyme, PstI and analyzed by 1% agaroseS gel electrophoresis.
Bacterial strains, plasmids, enzyme and reagents Expression and purification of acetate kinase Shewanella sp. AS-11 was isolated from a buccinid (shell) N. eatoni living in the Antarctic ice-covered sea, which was a source of cold adapted acetate kinase gene. The sources of other materials used were as follows: Deoxyribonucleic acid used as carrier DNA was obtained from Sigma-Aldrich Co. Ltd. The pET-16b vector was used from Novagen. Polymerase chain reaction (PCR) primers were purchased from Hokkaido System Science Co. Ltd. (Japan); KODplus DNA polymerase, BamHI and E. coli from TOYOBO; PstI from Wako Nippon Gene; QIAprep Miniprep kit from Qiagen; DNA ladder marker and protein marker from New England BioLabs. All other chemicals were of analytical grade for biochemical use.
PCR amplification of acetate kinase gene Genomic DNA of Shewanella species AS-11 was prepared as described by Sambrook et al. (1989) and open reading frames (ORFs) of acetate kinase gene was obtained by a PCR using genomic DNA, as template with Perkin Elmer Gene PCR system-
Recombinant plasmid DNA (isolated from E. coli DH5α) was transformed into E. coli BL21 (DE3) (Puyet A, 1987) for expression. The cells were grown at 37°C to an A600 of 0.6 to 0.9; at that time isopropyl -D-thiogalactopyranoside (IPTG) was added (final concentration, 1 mM) to the culture medium and temperature of the growth medium was shifted to 20, 25, 30 and 37°C and the cells were cultivated at the shifted temperatures for an additional 2 to 24 h for determination of optimum induction temperature and time for acetate kinase expression. Cells were harvested and the total proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12.5% gel (Laemmli, 1970). Cells (25 to 30 mg/ml) were re-suspended in 20 mM Tris-HCl buffer, pH 8.0 containing 0.5 M NaCl at 4°C and mixed with phenyl methyl sulfonyl fluoride (PMSF), freshly prepared lysozyme and sodium deoxycholate (final concentration was 1 mM, 1 mg/ml and 2.5 mg/ml, respectively). The cell suspension was incubated about 20 to 30 min with stirring at 4°C and then frozen at -80°C for 15 to 20 min. Then PMSF, DNase and RNase were added (final concentra-
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BamH1 MCS pETY-16b 8615bp
Ampr P-1 5`
3`
Ack gene, 1203 bp
P-2
2µ Ori
URA3
Digested with BamH1 PCR
P-1
Ack gene, 1203 bp
P-2
Ampr
2 µ Ori
URA3
Homologous recombination in Yeast P-1 1
Ampr
Ack gene, 1203 bp
P-2
pETY-16b-ack 9818 bp
2µ Ori
URA3
Figure 1. Graphical representation of PCR amplification and homologous recombination of acetate kinase ORF with pETY-16b. Ack is acetate kinase gene, pETY-16b-ack is pETY-16b vector contain acetate kinase gene. PCR, Polymerase chain reaction.
tion was 1 mM, 25 and 25 µg/ml, respectively) to the suspension to remove nucleic acids. The supernatant was collected and subjected to ammonium sulfate fractionation. The active fraction was collected and dialyzed against 20 mM Tris-HCl buffer containing 8 mM MgCl2, 0.5 mM dithiothreitol (DTT) and 7.5% glycerol (v/v) at pH 7.5 and applied on a Blue Sepharose CL-6B affinity column preequilibrated with same buffer. The column was washed stepwise with buffer containing 50 mM KCl, 100 mM KCl and 200 mM potassium acetate at pH 7.5. Acetate kinase was eluted with same buffer containing 1 mM ATP and 200 mM potassium acetate at pH 7.5 (Fox and Roseman, 1986). The enzyme was further purified by Super-Q column chromatography and eluted by the linear gradient with same buffer containing 0.2 M NaCl, pH 7.5. The purity of the acetate kinase was assessed by SDS-PAGE using 12.5% gel. Protein concentration was determined by the Bradford method (1976) using protein dye reagent (Bio-Rad) and bovine serum albumin was used as the standard.
Superose-6 column chromatography Superose-6 column (125 ml) was equilibrated with about 1 L of 30 mM (N-morpholino) propanesulphonic acid (MOPS)-KOH buffer pH 7.0 containing 50 mM KCl and calibration curve was prepared with Rnase A (13.7 kDa), carbonic anhydrase (29 kDa), ovalbumin (43 kDa), bovine serum albumin (67 kDa) and -amylase (200 kDa) at a flow rate 0.5 ml/min. To determine the molecular mass of acetate kinase, 0.5 ml purified sample (1.5 mg/ml) was applied onto the same column and the retention volume was measured by same buffer and same flow rate.
Enzyme activity assays The enzyme activity assay was measured by the hydroxamate Assay method, which detecting acetyl phosphate formation. The
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1
2
1 2 3 4 5 6 7
kbp
kbp
1.5
1.2
1.0
10.0 8.0 6.0
7.8 6.6
2.0 1.5
1.6
1.0 0.5
0.5 A Fig. 2.
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Figure 2. Agarose-S gel electrophoresis of the amplified DNA and recombinant plasmid digested with PstI. (A) Lane 1, marker; lane 2, products amplified with a template of Shewanella sp. AS-11 chromosomal DNA; (B) lane 1, marker; lanes 2 to 6, pETY-16b-ack; lane 7, pETY-16b.
hydroxamate assay, an adaptation of the method of Rose, Lipmann and Aceti (Rose et al., 1954; Lipman, 1944; Aceti and Ferry, 1988), utilizes the reaction of acetyl phosphate with hydroxylamine to form acetyl hydroxamate, which forms a colored complex with trivalent iron. This method was slightly modified and used to measure the acetate kinase activity. 40 mM HEPES buffer, pH 7.5 was used instead of Tris-HCl buffer and the absorbance was measured at 540 nm using a micro plate reader (Bio-Rad, model 680XR). A molar absorption coefficient of 0.46 mM -1cm-1 was used for determination of acetyl phosphate concentration (Bock et al., 1999). The specific activity (Unit/mg) is reported as µmoles of product formed/min/mg of protein. pH dependency and stability Enzyme activity was measured in the buffer containing 20 mM sodium citrate, 20 mM sodium tetra borate and 20 mM MOPS at different (2 to 11) pH at 25°C and for pH stability measurement, acetate kinase was pre-incubated at pH values ranging from 2 to 12 in the same buffer for 30 min at 25°C and then their residual activities were measured by hydroxamate assay method at pH 7.5 at 25°C. Thermostability Acetate kinase was pre-incubated at different temperatures from 20 to 80°C for 15 min in 40 mM HEPES buffer, pH 7.5, with and
without 10 mM ATP or 200 mM potassium acetate. The residual activities were measured by hydroxamate assay method at 25°C at pH 7.5. To determine the optimum temperature of acetate kinase, activity was measured at 5 to 80°C by above method.
Sequence alignment of psychrophilic, thermophilic acetate kinases
mesophilic
and
The sequence alignment, the percentages of identity of psychrophilic, mesophilic and thermophilic acetate kinases were determined by using ClustalW (1.83). The salt bridges were determined using a distance of 4.0 Å (Kumar and Nusssinov, 1999) between interacting groups with “What if web program”. The cationpi interaction was determined with CAPTURE (http://capture.caltech.edu.) (Gallivan and Dougherty, 1999).
RESULTS AND DISCUSSION Amplification of acetate kinase gene The ORFs of ack of 11 was successfully gel electrophoresis fragment contained
psychrotrophic Shewanella sp. ASamplified by PCR. The agarose-S showed that the amplified DNA 1.2 kb (Figure 2A), which was
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kDa
kDa
175.0
175.0
83.0
83.0
62.0
62.0
1
2
47.5
47.5 32.5
32.5
25.0
25.0
16.5 16.5
A
B
Figure Fig. 3.
3. SDS-PAGE analyses of the soluble and insoluble proteins from E. coli BL21 transformants after expression at different temperatures and eluting protein from Blue Sepharose CL-6B affinity column chromatography. The gel was stained with Coomassie Blue R-250. Lane 1, molecular standard markers: MBP-ß-galactosidase (175 kDa), MBP-paramyosin (83 kDa), glutamic dehydrogenase (62 kDa), aldolase (47.5 kDa), triosephosphate isomerase (32.5 kDa), ßlactoglobulin A (25 kDa) and lysozyme (16.5 kDa); lane 2 and 3, (soluble and insoluble, respectively) at 20° C; lane 4 and 5, (soluble and insoluble, respectively) at 25°C; lane 6 and 7, (soluble and insoluble, respectively) at 30°C; lane 8 and 9, (soluble and insoluble, respectively) at 37°C (A). Lane 1, marker; lane 2, recombinant acetate kinase (B). SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
consistent with the sizes predicted from the acetate kinase ORFs from Shewanella sp. AS-11.
the ORF of acetate kinase at a desired position and was named pETY-16b-ack.
Cloning of acetate kinase ORFs
Expression and purification of enzyme
BamHI digested pETY-16b vector and PCR amplified DNA fragments were introduced into S. cerevisiae for in vivo homologous recombination. Recombinant plasmids were collected and transformed into the competent cells of E. coli DH5α for propagation. The recombinant plasmids were collected from E. coli DH5α and digested with restriction endonuclease, PstI, which formed three fragments containing 7.8, 1.6 and 0.5 kb. On the other hand, the pETY-16b vector plasmid was cut by same restriction enzyme and formed three fragments containing 6.6, 1.6 and 0.5 kb (Figure 2B). The result clearly indicates that the recombinant plasmids contain
The recombinant acetate kinase was expressed in E. coli BL21 (DE3) by induction with 1 mM IPTG in the culture at A600 of 0.6 to 0.9. The cultural growth was observed after IPTG induction and the culture growth was not inhibited. The maximum amount of soluble acetate kinase was obtained by the expression for 16 h at 20°C (Figure 3A). The results indicate that the optimum bacterial growth time is 16 h for maximum expression of acetate kinase after IPTG induction at 20°C. The cell extracts were prepared after expression and subjected to ammonium sulfate fractionation. The SDS-PAGE fractions indicate that the active enzyme was precipitated with 30 to 40%
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Table 1. Purification of recombinant acetate kinase from Shewanella sp. AS-11.
Purification step Cell extract (NH4)2SO4 fractionation Blue sepharose CL-6B Super-Q
Volume (ml) 175 25 30 26
Protein (mg) 1180.0 486.0 46.8 45.1
Specific activity (U/mg) 48 105 600 605
ammonium sulfate saturation (figure not shown). After ammonium sulfate fractionation, partially purified acetate kinase was further purified by Blue Sepharose CL-6B affinity column chromatography. Enzyme was eluted with 20 mM Tris-HCl buffer pH 7.5 containing 1 mM ATP and 200 mM potassium acetate, which was in a pure state as judged by SDS-PAGE (Figure 3B); though, the absorbance spectra showed that the maximum absorbance was at 264 nm for the enzyme (figure not shown). These results indicate that the protein sample was contaminated by non-protein substances, such as nucleotides. In this regard, the protein was further purified by Super-Q column chromatography. Two eluting peaks were observed, first peak showed maximum absorbance at 260 nm and no protein band was observed by SDSPAGE as well as no acetate kinase activity was observed; whereas second peak showed maximum absorbance within 278 to 280 nm (figure not shown) and was observed a single protein band by SDS-PAGE. The purification procedure is summarized in Table 1. 45 mg purified protein was obtained from 1 L induced culture (Table 1). The molecular masses of purified enzyme have been estimated to be 86.0 ± 2.2 kDa by Superose-6 gel filtration, but the apparent molecular masses of acetate kinase was estimated 43.0 ± 1.4 kDa (Figure 3B) on SDS-PAGE, which correspond to the predicted masses (43.7 kDa) from the amino acid sequences of acetate kinase. From these results it can be suggested that acetate kinase is associated with two monomers in a native state (Fox and Roseman, 1986).
pH stability and dependency of acetate kinase The maximum activity of recombinant acetate kinase was displayed between pH 7.0 to 7.5 (Figure 4) and the enzyme retained full activities after incubation for 30 min at pH values ranging from 6 to 8 (Figure not shown). The enzyme was not stable in a broad range of pH and both acidic and basic conditions were not good for its stability and activity. From these results it can be suggested that the neutral pH is suitable for its activity and stability.
Total activity (U) 56640 51030 28080 27285
Purification fold 1.0 2.2 12.5 12.6
Recovery (%) 100 90 49 48
Thermal stability and optimum temperature of acetate kinase Thermal stability of free acetate kinase and ligand binding acetate kinase were shown in Figure 5. The free and ligand bound acetate kinase retained full activity after 15 min incubation at 30 and 40°C, respectively but completely and irreversibly inactivated at 60 and 70°C, respectively. The results indicate that the enzyme is thermolabile and ligand binding enzymes are thermally slightly more stable than free enzyme. The results are consistent with the report by Latimer and Ferry (1993). The maximum activity was expressed at 45°C and retained 27% of its maximum activity at 5°C, but, the activity was decreased abruptly at higher than the optimum temperature (Figure not shown). According to the enzyme stability curve, enzyme stability was increase when bound with substrate, which might be due to denaturation of the enzyme at higher than the optimum temperature. It can be concluded from the enzyme activity and stability curve that the enzyme is cold adapted with thermolabile. Substrate specificity Substrate specificity of the acetate kinase was tested with propionate and butyrate analogues of acetate. Activity was not detectable for the recombinant enzyme at 200 mM concentrations of propionate and butyrate, which concentration was saturated with acetate in hydroxamate assay method. The results of substrate specificity are in agreement with the previous reports on E. coli acetate kinase by Fox and Roseman (1986) and Brown and Akagi (1966). Sequence alignment of psychrophilic, mesophilic and thermophilic acetate kinases The sequence alignment of psychrophilic (Shewanella sp. AS-11), mesophilic (E. coli K-12) and thermophilic (Methanosarcina thermophila) acetate kinases is given in Figure 6. The amino acid sequence of psychrophilic
Afr. J. Biotechnol.
Relative activity (%)
120 100 80 60 40 20 0 0
2
4
pH
6
8
10
12
Figure 4. Effect of pH on recombinant acetate kinase activities. The activity was in buffer containing 20 mM sodium citrate, 20 mM sodium tetra borate and 20 Fig.assayed 4. mM MOPS at different pH values (2 to 11) by hydroxamate assay method at 25°C. MOPS, (N-morpholino)propanesulphonic acid.
105
Relative activity (%)
7460
90 75 60 45 30 15 0 20
30
40
50
60
70
80
Temperature (C) (°C) Figure 5. Thermal stability of recombinant acetate kinase. Residual activities were measured at 25°C at pH 7.5 by hydroxamate assay method after pre-incubation at different temperatures for 15 min. Free acetate kinase; ∆, acetate binding enzyme; □, ATP binding enzyme. ATP and acetate concentration was 10 mM and 200 mM, respectively.
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1
S. AS-11 E. coli M. thermophila
10
20
30
40
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50
MSDKLVLVLNCGSSSLKFAIIDAQSGDDKISGLAECFGLEDSRIKWKFNGGKNEAKLGAF 60 MSSKLVLVLNCGSSSLKFAIIDAVNGEEYLSGLAECFHLPEARIKWKMDGNKQEAALGAG 60 ---MKVLVINAGSSSLKYQLIDMTNESALAVGLCERIGIDNSIITQKKFDGKKLEKLTDL 57 ***:*.******: :** . . **.* : : :: *. * ..*: * 61
70
80
90
100
110
S. AS-11 E. coli M. thermophila
TAHREAVEFFIVNNILAEHPELA--AQIKAIGHRIVHGGEKFTRSVIITPEVIQGIEDCA 118 AAHSEALN-FIVNTILAQKPELS--AQLTAIGHRIVHGGEKYTSSVVIDESVIQGIKDAA 117 PTHKDALEEVVKALTDDEFGVIKDMGEINAVGHRVVHGGEKFTTSALYDEGVEKAIKDCF 117 .:* :*:: .: : : .::.*:***:******:* *.: * :.*:*.
S. AS-11 E. coli M. thermophila
SLAPLHNPAHLIGIRAAIASFPQLP--QVTVFDTAFHQTMPEKAYIYALPYKLYREHSIR 176 SFAPLHNPAHLIGIEEALKSFPQLKDKNVAVFDTAFHQTMPEESYLYALPYNLYKEHGIR 177 ELAPLHNPPNMMGISACAEIMPGTP--MVIVFDTAFHQTMPPYAYMYALPYDLYEKHGVR 175 .:******.:::** . :* * *********** :*:*****.**.:*.:*
S. AS-11 E. coli M. thermophila
RYGMHGTSHFYVCREAAKVLGKDVKDINVICAHLGNGASVAAIKGGKSVDTSMGLTPLEG 236 RYGAHGTSHFYVTQEAAKMLNKPVEELNIITCHLGNGGSVSAIRNGKCVDTSMGLTPLEG 237 KYGFHGTSHKYVAERAALMLGKPAEETKIITCHLGNGSSITAVEGGKSVETSMGFTPLEG 235 :** ***** ** ..** :*.* .:: ::* .*****.*::*:..**.*:****:*****
S. AS-11 E. coli M. thermophila
LVMGTRCGDLDPSIIFHLVKQLGYTLDEVNNLLNKQSGLLGISELTNDCRGIEEGYHDGH 296 LVMGTRSGDIDPAIIFHLHDTLGMSVDAINKLLTKESGLLGLTEVTSDCRYVEDNYAT-K 296 LAMGTRCGSIDPAIVPFLMEKEGLTTREIDTLMNKKSGVLGVSGLSNDFRDLDEAASKGN 295 *.****.*.:**:*: .* . * : ::.*:.*:**:**:: ::.* * ::: :
S. AS-11 E. coli M. thermophila
KGATLALDIFCYRLAKYIASYTVPLG-RLDAIIFTGGIGENSNLIREKVLNLLEIFNFKV 355 EDAKRAMDVYCHRLAKYIGAYTALMDGRLDAVVFTGGIGENAAMVRELSLGKLGVLGFEV 356 RKAELALEIFAYKVKKFIGEYSAVLN-GADAVVFTAGIGENSASIRKRILTGLDGIGIKI 354 . * *::::.::: *:*. *:. :. **::**.*****: :*: * * :.:::
S. AS-11 E. coli M. thermophila
DDILNKAARFGQQGVITQAGTPIAMVIPTNEEWVIAEDAIKLITAK--------- 401 DHERNLAARFGKSGFINKEGTRPAVVIPTNEELVIAQDASRLTA----------- 400 DDEKNK-IRGQEIDISTPDAKVRVFVIPTNEELAIARETKEIVETEVKLRSSIPV 408 *. * * : .. . .. ..******* .**.:: .:
120
130
180
240
300
360
140
190
250
310
370
150
200
210
260
270
320
330
380
160
220
280
340
170
230
290
350
390
Figure 6. Sequence alignment of acetate kinases from Shewanella sp. AS-11, Escherichia coli K-12 and Methanosarcina thermophila. Catalytic and substrate binding amino acid residues are indicated as bold and under line, respectively. Identical () and similar ( and ) residues in the sequences are indicated in the alignment.
acetate kinase shows 64 and 46% residues identity with mesophilic and thermophilic acetate kinases, respectively. The amino acid sequence identity is distributed uniformly through the sequences and mainly corresponds to catalytic residues, substrate binding residues and secondary structural elements in all acetate kinases. The catalytic residues are strictly conserved and most of the substrate binding amino acids are also conserved except acetate binding residues (Figure 6). 179 Thermophilic acetate kinase presents Phe in acetate binding pocket, which binds acetate (Buss et al., 2001; Ingram-Smith et al., 2005) but mesophilic and psychrophilic acetate kinases present Ala and Met, respectively as acetate binding residues. As listed in Table 2, the parameters derived from the primary
structure (amino acid content) of psychrophilic, mesophilic and thermophilic acetate kinases failed to reveal significant differences, which could be attributed to temperature adaptation. The numbers of Gly and Pro residues, which affect the local mobility of the chain, are not significantly altered in psychrophilic and mesophilic acetate kinases. The positively charged amino acids and negatively charged amino acids have the potential to form multiple ion pairs and hydrogen bonds. The negatively charged amino acids are least abundant in psychrophilic acetate kinase, whereas the numbers of positively charged amino acids are almost similar. By contrast, the reduced number of salt bridges and cation-pi interactions of psychrophilic acetate kinase was determined from the comparison of the model structures
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Table 2. Main structural features possibly related to temperature adaptation in psychrophilic, mesophilic and thermophilic acetate kinases.
Parameter
Psychrophilic
Mesophilic
Thermophilic
Gly content
37
36
37
Pro content
13
12
16
Positive charge amino acids
54
53
57
Negative charge amino acids Salt bridges Cation-pi interaction
44 5 2
48 13 3
58 22 3
of psychrophilic and mesophilic acetate kinases, which are consistent with the psychrophilic character.
School of Agricultural Sciences, Kagoshima University to AKT.
Conclusion
REFERENCES
Recombinant psychrophilic acetate kinase was successfully over-expressed in E. coli BL21 (DE3) cells and purified by two steps chromatography. Psychrophilic acetate kinase showed the activity at low and moderate temperatures and regained about one third of its maximum activity at 5°C. Thermo-labiality of this enzyme indicates that the enzyme bears unstable as well as flexible structure (Chiuri et al., 2009; D’Amico et al., 2001; Bentahir et al., 2000). The lower number of salt bridges and cation-pi interaction of psychrophilic acetate kinase than mesophilic and thermophilic acetate kinases clearly proves that the psychrophilic acetate kinase displays a more flexible structure, which is consistent with the thermolability. It has been observed that some weak intra molecular interactions, e.g. salt bridges, cation-pi interactions, hydrogen bond, etc. are missing in cold adapted enzymes (Siddiqui and Cavicchioli, 2006; Feller, 2003; Bentahir et al., 2000), as a result of a flexible structure that plays a crucial role in biological system and it is one of the main characteristics of cold-adapted enzymes (Rueda et al., 2007; Papaleo et al., 2007; Olufsen et al., 2006). These studies we can suggest that the recombinant acetate kinase from Shewanella sp. AS11 is quite efficiently expressed in E. coli and the enzyme is cold adapted with thermolabile and flexible structure and may be this flexibility contributes to its cold adaptation.
Aceti DJ, Ferry JG (1988). Purification and characterization of acetate kinase from acetate-grown Methanosarcina thermophila. Evidence for regulation of synthesis. J. Biol. Chem. 263: 15444-15448. Bentahir M, Feller G, Aittaleb M, Brasseur JL, Himri T, Chessa JP, Gerday C (2000). Structural, kinetic and calorimetric characterization of the cold-active phosphoglycerate kinase from the Antarctic Pseudomonas sp. TACII18. Bock AK, Glasemacher J, Schmidt R, Schönheit P (1999). Purification and characterization of two extremely thermostable enzymes, phosphate acetyltransferase and acetate kinase, from the hyperthermophilic eubacterium Thermotoga maritime. J. Bacteriol. 181: 1861-1867. J. Biol. Chem. 275(15): 11147-11153. Bradford MM (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. Brown MS, Akagi JM (1966). Purification of acetokinase from Desulfovibrio desulfuricans. J. Bacteriol. 92: 1273-1274. Buss KA, Cooper DR, Ingram-Smith C, Ferry JG, Sanders DA, Hasson MS (2001). Urkinase: Structure of acetate kinase, a member of the ASKHA superfamily of phosphotransferases. J. Bact. 183: 680-686. Chiuri R, Maiorano G, Rizzello A, Mercato LL, Cingolani R, Rinaldi R, Maffia M, Pompa PP (2009). Exploring local flexibility/rigidity in psychrophilic and mesophilic carbonic anhydrases. Biophys. J. 96: 1586-1596. D’Amico S, Gerday C, Feller G (2001). Structural determinants of cold adaptation and stability in a large protein. J. Biol. Chem. 276(28): 25791-25796. Diao J, Ma YD, Hasson MS (2009). Open and closed conformations reveal induced fit movements in butyrate kinase 2 activation. Proteins, pp. 1-12. Feller G (2003). Molecular adaptations to cold in psychrophilic enzymes. Cell Mol. Life Sci. 60: 648-662. Feller G, D’Amico D, Gerday C (1999). Thermodynamic stability of a cold-active alpha-amylase from the Antarctic bacterium Alteromonas haloplanctis. Biochemistry, 38(14): 4613-4619. Ferry JG (1992). Methane from acetate. J. Bacteriol. 174: 5489 - 5495. Fox DK, Roseman S (1986). Isolation and characterization of homogeneous acetate kinase from Salmonella typhimurium and Escherichia coli. J. Biol. Chem. 261: 13487-13497. Gallivan JP, Dougherty DA (1999). Cation- interactions in structural biology. Biochemistry, 96: 9459-9464. Gerday C, Aittaleb M, Bentahir M, Chessa JP, Claverie P, Collins T, D'Amico S, Dumont J, Garsoux G, Georlette D, Hoyoux A, Lonhienne
ACKNOWLEDGEMENT This study was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science to KW (15380074) and by Rendaistudent Supporting Program of the United Graduate
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T, Meuwis MA, Feller G (2000). Cold-adapted enzymes: from fundamentals to biotechnology. Trends Biotechnol. 18(3): 103-107. Gietz RD, Wood RA (2002). Transformation of Yeast by the Lithium Acetate/Single-Stranded carrier DNA/Polyethylene Glycol method. Methods in Enzymology, 350: 87-96. Ingram-Smith C, Gorrel A, Lawrence SH, Iyer P, Smith K, Ferry JG (2005). Characterization of the Acetate Binding Pocket in the Methanosarcina thermophila Acetate Kinase. J. Bact. 187: 23862394. Kumar SP, Ghosh M, Pulicheria KK, Rao KRSS (2011). Cold active enzymes from the marine psychrophiles, biotechnological perspective. Advanced Biotech. 10: 16-20 Kumar S, Nussinov R (1999). Salt bridge stability in monomeric proteins. J. Mol. Biol. 293: 1241-1255. Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685. Latimer MT, Ferry JG (1993). Cloning, sequence analysis, and hyperexpression of the genes encoding phosphotransacetylase and acetate kinase from Methanosarcina thermophila. J. Bact. 175: 68226829. Lipmann F (1944). Enzymatic synthesis of acetyl phosphate. J. Biol. Chem. 155: 55-70. Morita RY (1975). Psychrophilic bacteria. Bacteriol. Rev. 39: 144-167. Olufsen M, Brandsdal BO, Smalas AO (2006). Comparative unfolding studies of psychrophilic uracil DNA glycosylase: MD simulations show reduced thermal stability of the cold-adapted enzyme. J. Mol. Graph. Model. 26: 124-134. Papaleo E, Olufsen M, De Gioria L, Brandsdal BO (2007). Optimization of electrostatics as a strategy for cold-adaptation: a case study of cold- and warm-active elastases. J. Mol. Graph. Model. 6: 93-103. Pope B, Kent HM (1996). High efficiency 5 min transformation of Escherichia coli. Nucleic Acids Res. 24: 536-537. Puyet A (1987). A simple medium for rapid regeneration of Bacillus subtilis protoplasts transformed with plasmid DNA. FEMS Microbiol. Lett. 40: 1-5. Rose IA, Grunberg-Manago M, Korey SR, Ochoa S (1954). Enzymatic phosphorylation of acetate .J. Biol. Chem. 211: 737-756.
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Rueda M, Ferrer-Costa C, Meyer T, Perez A, Camps J, Hospital A, Gelpi JL, Orozco M (2007). A consensus view of protein dynamics. Proc. Natl. Acad. Sci. USA. 104: 796-801. Sambrook J, Frisch E, Maniatis T (1989). Molecular Cloning: A laboratory Manual, pp 1.74-1.85 & 2.60-2.80. 2nd Ed. Cold Spring Harbor Laboratory Press, New York. Siddiqui KS, Cavicchioli R (2006). Cold-adapted enzymes. Annu. Rev. Biochem. 75: 403-433. Tanoue J, Tang AK, Motoshima H, Watanabe K (2010). Cloning and sequenching of the gene encoding acetate kinase from Antarctic psychrotroph Shewanella sp. AS-11. GenBank: AB597986.1. Timmis KN, Pieper DH (1999). Bacteria designed for bioremediation. Trend. Biotechnol. 17: 201-204. Vazquez SC, Merino LNR, MacCormack WP, Fraile ER (1995). Protease-producing psychrotrophic bacteria isolated from Antartica. Polar Biol.15: 131-135.
African Journal of Biotechnology Vol. 11(29), pp. 7464-7471, 10 April, 2012
Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.4193 ISSN 1684–5315 © 2012 Academic Journals
Full Length Research Paper
Biodecolorization of Reactive Black 5 by laccasemediator system Ismat Bibi1and Haq Nawaz Bhatti2* 1
Department of Chemistry, Islamia University, Bahawalpur-Pakistan. Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad-38040, Pakistan.
2
Accepted 8 March, 2012
Reactive azo dyes are widely used as textile colorants, typically for cotton dyeing, due to their variety of color shades, and minimal energy consumption. In the present study, commercial laccase from Trametes versicolor was used for the biodecolorization of Reactive Black 5 (RB-5) dye using different redox mediators viz, N-hydroxybenzotriazole (HBT), 2,2′-azino-bis-(3-ethylbenzthiazoline- 6-sulfonic acid (ABTS), 2,6-dimethoxy phenol (DMP), syringaldehyde, vanillin, aceto-vanillone, p-coumaric acid and catechol. Commercial laccase alone did not show any considerable decolorization of RB-5. However, the laccase in the presence of syringaldehyde showed the strongest decolorization rate (98%), followed by vanillin (55.21%), aceto-vanillone (53.25%), ABTS (42.78%), p-coumaric acid (41.9%), DMP (39%), and catechol (36.33%); while least decolorization was observed with HBT at dye/mediator ratio of only 1:5 after 30 min. Therefore, syringaldehyde performance was evaluated at different mediator/dye ratios (1:1, 1:5 and 1:10) using commercial laccase and it was compared with that of synthetic mediator like HBT. It was found that the presence of syringaldehyde was essential for biodecolorization of RB-5. Moreover, it was observed that syringaldehyde was an effective natural redox mediator as compared to synthetic HBT. Enhanced decolorization (98%) of RB-5 by laccase was observed with 1:5 syringaldehyde and dye ratio for 30 min but maximum removal (22%) of RB-5 was recorded with HBT at 1:1 after 40 min. Thus, the study reveals that the phenolic compounds could be used as potential redox mediators for enhanced laccase-mediated decolorization of azo dyes. Key words: Reactive Black 5 (RB-5), redox mediators, laccase, biodegradation, azo dyes.
INTRODUCTION Wastewaters of the textile industries contain considerable amounts of non-fixed dyes especially azo-dyes. Reactive azo dyes are mostly used in textile dyeing due to their variety of color shades, high wet fastness profiles, ease of application, brilliant colors, minimal energy consumption, high photolytic stability, and resistance to microbial degradation. The release of such colored wastewaters in the ecosystem is a dramatic source of
*Corresponding author. E-mail: hnbhatti2005@yahoo.com, haq_nawaz@uaf.edu.pk. Fax: +92-41-9200764. Abbreviations: RB-5, Reactive Black 5; HBT, Nhydroxybenzotriazole; ABTS, 2,2′-azino-bis-(3ethylbenzthiazoline- 6-sulfonic acid; DMP, 2,6-dimethoxy phenol.
esthetic pollution, eutrophication and perturbations in thaquatic life (Lachheb et al., 2002). Most physicochemical dye removal methods, which are generally used for effluent treatment have many limitations (Balcioglu and Arslan, 2001; Ghoreishi and Haghighi, 2003). In recent years, biological decolorization method has been considered as an alternative and ecofriendly method to dye degradation and color removal (Hafiz et al., 2008; Asgher et al., 2009; Bibi et al., 2011). Dyes biodegradation is usually carried out by white rot fungi by their ligninolytic enzymes such as lignin peroxidases, manganese peroxidases, and laccases (Asgher et al., 2008). A number of white rot fungi have been explored for decolourization of various industrial dyes and treatment of dye effluent (Bhatti et al., 2008; Asgher et al., 2010; Bibi et al., 2010). Majority of these studies were carried out with fungal mycelia. One of the
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Oxygen
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Figure 1. Mediator system of laccase (Gochev and Krastanov, 2007).
major disadvantages of using fungal cultures for dyes decolourization is the accumulation of biomass, which would cost the wastewater treatment in industrial scale. To overcome this disadvantage, the application of isolated enzymes for dye decolourization has increased in recent years (Kandelbauer et al., 2004; Bibi et al., 2011). Laccases are oxidoreductases which belong to the multinuclear copper-containing oxidases and are able to decolorize and detoxify industrial dyes (Zouari-Mechichi et al., 2006). These oxidative enzymes are particularly abundant in white rot fungi and have been purified and characterized extensively from many white rot fungi (Jordaan et al., 2004). In recent years, laccase based treatment method has received much attention in the treatment of various recalcitrant pollutants because the laccase production is constitutive in most of the white rot fungi and it can be easily enhanced by chiefly available laccase inducers. Many studies have been demonstrated for dye decolourization using both crude and purified forms of laccase (Baldrian et al., 2004). However, some of the dyes cannot be oxidized, or partly oxidized by laccase, because they are too large to penetrate into the enzyme active site or have a particularly high redox potential. In recent years, some natural phenolic compounds, including syringaldehyde and acetosyringone (AS), have been described as efficient and eco-friendly laccase mediators for textile and environmental applications. Cho et al. (2007) have intensified the range of compounds that can be oxidized by these enzymes. The mechanism by which redox mediators play a role in laccase-mediated oxidation reactions is now well characterized. When a substrate is oxidized by a laccase the redox mediator forms cation radicals (short-lived intermediates) which co-oxidize the substrate. These cation radicals can be formed by two mechanisms; the redox mediator can perform either a one-
electron oxidation of the substrate to a radical cation (Xu et al., 2001) or it abstracts an H-atom from the substrate converting it into a radical (Fabbrini et al., 2002). According to Gochev and Krastanov (2007), the mechanism of laccase mediators system is given in Figure 1. The rationale behind the present study was to evaluate the potential of different naturally occurring phenolic compounds to mediate the oxidative reactions catalyzed by laccase with the aim of identifying cheaper, more competent and ecofriendly mediators for the decolo-rization of recalcitrant dyes and for other industrial and environmental applications. Also in enzymatic dye decolourization, optimization of the concentrations of redox mediator and dye is an important criterion for successful decolourization. To our knowledge, there has been no study for the optimization of dye/mediator ratio in enzymatic dye decolourization using fungal laccase. To evaluate the mediating capabilities of these compounds, we used a test based on the decolorization of Reactive Black 5 (RB-5), a recalcitrant dye that is not oxidized by commercial laccase alone. MATERIALS AND METHODS Chemicals and microorganism Reactive Black 5 (RB-5), 2,2â&#x20AC;&#x2122;-azinobis-(3-ethylbenzothiazoline-6sulfonic acid) (ABTS), syringaldehyde (SYD), N-hydroxybenzotriazole (HBT), 2,6-dimethoxy phenol (DMP), catechol, pcoumaric acid, vanillin and aceto-vanillone were purchased from Sigma-Aldrich Co. USA. Laccase (from Trametes versicolor EC: 1.10.3.2) was obtained from Fluka, USA. The Chemical structure of Reactive Black 5 dye is shown in Figure 2. Screening of redox mediators Screening experiments were carried out to investigate the effect of redox mediators on the decolorization of RB-5 dye by commercial laccase. For this purpose, 10 Âľl of T. versicolor laccase and
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Figure 2. Chemical structure of Reactive black 5 dye (RB-5) (C.I. 20505; M.W= 991).
Reactive Black 5/redox mediator at 1:5 were incubated in the citrate-phosphate buffer (pH 4.5) for 30 min in Eppen-dorf tube at 25°C. Absorbance of each sample was noted after 30 min at λmax (595 nm) of the respective dye in order to determine the percentage decolorization.
Screening of dye/ mediator ratio Decolorization experiments were carried out in 2 ml Eppen-dorf tube. Reaction mixture (1 ml) containing 100 mM citrate-phosphate buffer (pH 4.5), laccase concentration (10 µl), and dye: syringaldehyde ratio 1:1, 1:5 and 1:10 were prepared. The reaction tubes were incubated at 25°C under dark and the decolourization was monitored spectrophotometrically (Cary 3 Bio, Varian, UK) after different time intervals by recording the absorbance at the λmax of the dye. Different sets of control were also run parallel to see the effect of mediator.
UV-Vis spectral analysis RB-5 decolorization was determined by measuring the decrease in optical density of the dye at the wavelength of maximum absorbance (λmax 595 nm) in a UV–Vis spectrophotometer (Cary 3 Bio, Varian, UK) and expressed in percentage. Different control samples were also run parallel and contained the reaction mixture i) with-out enzyme (RB-5 only), ii) SYD+RB-5 and iii) HBT+RB-5. The percentage decolorization was calculated as follows: Initial absorbance − Observed absorbance % Decolorization =
× 100 Initial absorbance
All experiments were conducted in triplicates and data were presented as mean±SD.
RESULTS AND DISCUSSION Effect of various redox mediators on the decolorization of RB-5 dye by laccase of T. versicolor Laccase mediator systems are important bioremediation
agents as the rate of reactions could be enhanced in the presence of the mediators. In order to observe the effect of redox mediators on the biodecolorization of RB-5 dye, different laccase mediators (phenolic alcohols, alde-hydes, ketones, acids, etc.) were used. The effect of the natural and synthetic mediators on the decolorization of Reactive Black 5 (30 min treatment) is shown in Figure 3. The results reveal that laccase showed strongest decolorization rate (97.45%) in the presence of syringaldehyde followed by vanillin (55.21%), aceto-vanillone (53.25%), ABTS (42.78%), pcoumaric acid (41.9%), DMP (39%), and catechol (36.33%). However, least decolorization rate (5.67%) was observed with HBT at a dye/mediator ratio of 1:5 only, while laccase alone did not decolorized RB-5 dye. The specificity of mediators towards different functional groups must also be taken into account in laccase-mediator reactions. For example, laccase- ABTS is not reactive towards benzylic ethers or alkylbenzenes, but it is effective on benzyl alcohols (Baiocco et al., 2003; Cantarella et al., 2003) and oxidized HBT and VIO also seem to be more competent towards some specific groups (Soares et al., 2002). In the present study, different decolorization rates were observed depending on the type of mediator used. Finally, syringaldehyde seemed to be highly competent to mediate the oxidation of the tested dye. Recently, two different mechanisms for the oxidation of non-phenolic compounds by laccase mediator systems have been proposed: i) an electron transfer route for mediators such as ABTS and ii) a radical hydrogen atom transfer route for mediators of the -NOH- type (Baiocco et al., 2003). It is very likely that the phenoxy radicals formed during the oxidation of natural mediators by laccase act similar to the -NO·radicals from -NOH- compounds, that is, they extract a hydrogen atom from the substrate (Acunzo and Galli, 2003). Therefore, the dissociation energy of the corresponding bond should govern their reaction with the laccase mediators. The feasibility of the laccase-mediator systems in biotransformation reactions depends on redox reversibility of the radical-substrate reaction, as well as on the balance
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+R B5 La an c+ i l Ac lin +R et oB Va -5 La ni c+ llo Pne Co +R um B -5 ar ic ac id La +R c+ BCa 5 te ch ol +R B5
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Figure 3. Effect of various redox mediators on the decolorization of RB-5 dye by laccase of Trametes versicolor.
between the stability and reactivity of the mediator radical which, in addition, should not inhibit enzyme activity (Camarero et al., 2007).The two former conditions have been confirmed with syringaldehyde, since voltametric and electron paramagnetic resonance studies have shown the true reversibility of the substrate reaction (Fernandez-Sanchez et al., 2002) and the long half-life of its phenoxy radical (Marzullo et al., 1995). In contrast, the -NO路- radical from HBT inactivates laccase, and due to its high reactivity, decays rapidly to benzotriazole and other inactive compounds (Li et al., 1998). Results showed that syringaldehyde is more easily oxidized by laccase than vanillin, ABTS, HBT, DMP, catechol and aceto-vanillone due to its high redox potential. This S-type phenols also form more stable radicals due to the presence of two methoxyl groups on the aromatic ring that prevent the formation of biphenyl-type structures by radical condensation. The higher stability of syringaldehyde phenoxy radicals is due to slightly acidic aqueous media (Caldwell and Steelink, 1969) as we carried out this reaction in phosphate buffer (pH 4.5). Optimization of redox mediator/dye ratio for enhanced decolorization of RB-5 by T. versicolor laccase It was observed experimentally that the presence of syringaldehyde is essential for decolourization of RB-5
dye because laccase alone did not decolorize the dye (Figure 3). Therefore, in screening trials, different ratios of dye and redox mediator (SYD and HBT) were run to find out the most optimum ratio for accelerated decolorization of RB5 dye. For this purpose, HBT and SYD were selected to compare the effect of synthetic and natural redox mediators on the decolorization of RB-5 dye by laccase of T. versicolor. It was noted that 1:5 (which we already used in mediator screening trials) was the optimum with SYD and 1:1 with HBT but much efficient decolorization (98%) of RB-5 dye was achived at 1:5 with SYD just within 30 min (Figure 5) while in case of HBT, the rate of decolorization was very slow and after 40 min, the maximum decolorization was only 22% at 1:1 dye: mediator ratio (Figure 6). Therefore, syringaldehyde was found to be an effective natural redox mediator as compared to synthetic HBT. Figure 5 shows the effect of 1:5 dye and mediator ratio on the decolorization of RB-5 dye by laccase. It clearly shows that the rate of decolorization was increased by increasing the dye: mediator ratio and reached a maximum of up to 98% with 1:5 but by further increase in this ratio (1:10) (Figure 4), the rate of decolorization was dramatically decreased. Figure 7 clearly indicates that the extent of color removal of RB-5 dye was maximum, whereas, there is no significant removal in the presence of HBT at 1:5 dye: mediator ratio. However, in the presence of a large amount of mediator (500 渭M), the enzyme concentration exhibited a negative effect on the rate of decolorization.
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Figure 4. Decolorization of RB-5 dye by laccase with 1:10 (dye: redox mediator (SYD /HBT) ratio.
100 90
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Figure 5. Decolorization of RB-5 dye by laccase with 1:5 (dye: redox mediator (SYD /HBT) ratio.
Wong and Yu, (1999) also reported that the efficiency of laccase-mediator systems in the decolorization reaction depended principally on the mediator concentrations and laccase activity used. Some fungal laccases as well as laccase mediator systems are efficient in dye decolorization. Figure 8 shows the UV-Vis absorption spectra of RB-5 dye at different dye: mediator ratio, which
clearly indicated that at 1:5 dye: mediator ratio, the rate of color removal was maximum. Different dyes were decolorized by different laccases at different rates. The decolorization rate depends on the structure and the redox-potential of the enzyme as well as the dye structure (Soares et al., 2002; MaalejKammoun et al., 2009). Preliminary results showed that
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Figure 6. Decolorization of RB-5 dye by laccase with 1:1 (dye: redox mediator (SYD /HBT) ratio.
Lacc+RB-5+SYD
Control
Lacc+RB-5+HBT
Figure 7. Decolorization of RB-5 dye by laccase in the presence of SYD and HBT at 1:5 dye: mediator ratio.
T. versicolor laccase did not decolorize RB-5, indicating that the presence of a mediator is required. Similarly, reports from literature show that laccase alone does not
decolorize some types of textile dyes (Rodriguez et al., 2005; Hu et al., 2009). The reason might be that the redox potential of the dye is higher than that of type 1 Cu
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of the laccase or the dye could not access the type 1 Cu active site because of its steric hindrance. However, such dyes can be oxidized by laccase in the presence of some redox mediators (Rodriguez et al., 2005; Murugesan et al., 2007).
synthetic (HBT) mediators for laccase degradation of diazo dye and other recalcitrant compounds in terms of both efficiency and velocity of oxidation. REFERENCES
Conclusions In this study, biodecolorization of Reactive Black 5 by commercial laccase from T. versicolor was studied in batch mode. The biodecolorization of RB-5 was strongly affected by redox mediators. The results show that the presence of a natural mediator, syringaldehyde (SYD) was essential for the decolorization of RB-5 dye by T. versicolor laccase. The concentration of the SYD proved to be the principal factor that affected the yield of the dye decolorization. Enhanced decolorization (98%) was obtained using commercial laccase under the optimal conditions (1:5 dye: mediator ratio). It was concluded that lignin-derived phenols (such as syringaldehyde) represent ecofriendly alternatives to
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African Journal of Biotechnology Vol. 11(29), pp. 7472-7482, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.3252 ISSN 1684–5315 © 2012 Academic Journals
Full Length Research Paper
Evaluation of cadmium bioaccumulation and translocation by Hopea odorata grown in a contaminated soil A. Arifin1,2*, A. Parisa1, A.H. Hazandy1, 2, T. M. Mahmud5, N. Junejo2, A. Fatemeh3, S. Mohsen4, M.E. Wasli6 and N.M. Majid1 1
Department of Forest Production, Faculty of Forestry, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. 2 Laboratory of Sustainable Bioresource Management, Institute of Tropical Forestry and Forest Products, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. 3 Islamic Azad University, Science and Research Branch, Ahvaz, Iran. 4 Guilan University, Department of Soil Science, Iran. 5 Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. 6 Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, Sarawak, Malaysia Accepted 19 December, 2011
Cadmium (Cd) contamination has an adverse effect on soil productivity and crop production. Phytoremediation is a long term and environmental friendly technology to remediate Cadmium polluted areas. This study was conducted to evaluate the potential of Hopea adorata for remediation of soils contaminated with Cd. Plant seedlings were planted in a clayey soil spiked contaminated with Cd in the amount of 0, 25, 50, 75, 100 and 150 mg kg-1 named as; Cd0, Cd1, Cd2, Cd3, Cd4 and Cd5 for a period of five months. The highest growth performance was recorded in the control (Cd0). Cd concentrations among plant parts were in the following trend: roots>stems>leaves. In order to evaluate the potential of species selected as phytoremediator, three indicators were used namely, bioconcentration factor (BCF, the metal concentration ratio of plant roots to soil), translocation factor (TF, the metal concentration ratio of plant shoots to roots) and removal efficiency (RE, total concentrations of metal and dry biomass of plants to total loaded metal in growth media). The highest total Cd concentration (290.23 ± 13.38 mg kg-1) and Cd removal efficiency (0.81± 0.06%) were found in Cd5 and Cd1, respectively. Cd2 exhibited the maximum total dry biomass (60.88 ± 1.78 g). H. odorata showed high BCFs (>1) and low TFs (<1). It can be concluded that this species is suitable to be used in phytoremediation of Cd-contaminated. For further confirmation, an evaluation under field condition will be needed.
Key words: Phytoremediation, Hopea odorata, heavy metals, soil pollution, removal efficiency
INTRODUCTION Contamination of soils with heavy metals has an adverse effect
on soil fertility and crop production (Alkorta and Garbisu, 2001; Odoemelam and Ukpe, 2008). Agricultural and
industrial activities are the sources of heavy metals by which Cd release into the environment and leave toxic effects (Nabulo et al., 2006; He et al., 2008). Cadmium is
*Corresponding author. E-mail: arifin_soil@yahoo.com.
a non-essential element and due to its high mobility and solubility in biological systems, it is known as one of the most hazardous element (Pinto et al., 2004; Dickinson and Pulford, 2005). It is listed as one of 126 priority pollutants (Nordberg, 2009). The widespread release of cadmium has reached 22,000 t (metric ton) over the past five decades (Jadia and Fulekar, 2009). The normal con-centration of Cd in soil ranges from 0.01 to 2.0 mg kg-1; however, in urban and agricultural soils, Cd levels exceed the thresholds set in guidelines (Alloway, 1995; Sahibin et al., 2002). Con-
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ventional method for extracting heavy metals from soils such as ex situ excavation, landfill of the top contaminated soil, soil flushing and physico chemical remediation are expensive, time consuming and labor exhaustive (Manousaki et al., 2008; Danh et al., 2009; Liu et al., 2010). Therefore, these remediation techniques are not technically and financially suitable for large contaminated areas (Soleimani et al., 2010). In contrast, phytoremediation is a low cost, longer lasting, environmental friendly new promising technology. It is applied to immobilize, degrade, remove, or detoxify contaminants including metals, pesticides, hydrocarbons, and chlorinated solvents (Zhang et al., 2010). Many studies have been done on phytoremediation of contami-nated soils using weeds and leafy wild vegetables and ornamental plants, but information is lacking regarding the potential of tropical plant species to remediate Cd-contaminated soils. The suggested ideal plant for suc-cessful phytoremediation should have rapid growth rate, high biomass, an excessive root system, accumulate high concentration of heavy metals and high tolerance when expose to high concentrations of heavy metals (Garbisu and Alkorta, 2001). Use the woody plant species that grow locally near the site is feasible for this process. These species are less competitive under local conditions and will reduce the metal concentration to an acceptable level for normal plant growth (Rajakaruna et al., 2006). One of potential woody plant species is H. odorata which is a fast growing species, and can grow up to 45 m of height (Orwa et al., 2009). The objective of this study was to assess the
growth performance and the phytore-mediation potential of H. odorata to absorb Cd in Cd-contaminated soil.
MATERIALS AND METHODS This study was conducted at the greenhouse of Faculty of Forestry, Universiti Putra Malaysia (20 59' 18.24'' N latitude and 1010 42'45.45'' E longitude). The average temperature in the green house was 27, 36 and 32°C in the morning, afternoon and evening, respectively. Relative humidity was 65%. The period of study was five months from February to June 2010. Healthy seedlings of the same age and similar form were selected for every species collected from Malaysia Agriculture Research Institute (MARDI), Serdang, and Selangor. A clayey soil (munchong series) which belongs to Ultisols was used in this experiment. The soil was airdried until it could be crushed to pass through a 4mm-seive for soil growing media. Stainless sieve was used to supply a homogenous soil composite as a growing media. Seedlings were transplanted into proper plastic pots (32.0 cm height, 106.0 cm upper diameter and 69.0 lower diameters) gently without damaging the root system. A completely randomized design (CRD) was followed with six treatments replicated four times. The growth media was prepared using soil thoroughly mixed with different levels of Cd including: Cd 1 (soil; 25 ppm Cd), Cd2 (soil; 50 ppm Cd), Cd3 (soil; 75 ppm Cd), Cd4 (soil; 100 ppm Cd) and Cd5 (soil; 150 ppm Cd) and control (Cd0 =100% soil). To provide different concentrations of Cd, cadmium chloride hydrate (CdCl2. 2.5H2O) was applied. A total of 24 seedlings were used in this experiment. The basal diameter, height and number of leaves were measured every month. Soil samples
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were air dried until it could be crushed to pass through a 2 mmsieve for analysis of physico-chemical properties in the laboratory. Soil texture was determined using the pipette gravimetric method (Tan, 2005). The pH of the soil was measured in the suspension of a 1:2.5 soil: liquid mixture. The CEC and exchangeable cations (Ca, Mg, K) were determined by leaching method using 1 M ammonium acetate at pH 7. Exchangeable Al and H determined by the NaOH titration method. Available P (ppm) was extracted using Bray II with a mixture including 0.03M ammonium fluoride (NH4F) and 0.1 M hydrochloric acid (HCl). Plants were harvested after five months for determination of plant growth, biomass and heavy metal analysis. Aqua regia method was used as described by Ahmadpouret al. (2010). Cd concentration in plant parts and soil samples was determined using atomic absorption spectrometry (AAS). Total C and N were determined by dry combustion using CNS 2000 analyzer. Three indicators were used to determine the potential of four plant species for phytoremediation of Cd-contaminated soil including translocation factor (TF), bioconcentration factor (BCF) and removal efficiency (RE).
Analysis of variance (one way ANOVA) for growth, heavy metals in soils and plant parts were implemented. Duncan Multiple Range Test (DMRT) was employed to detect any significant differences (p≤0.05) among and between the treatments of growth media, growth parameters and biomass. Correlation analysis was also performed to relate total Cd concentrations in growth media with dry biomass production and total Cd concentration in plant species. All data obtained in terms of growth, biomass and heavy metals in soil and plants were analyzed using the SAS (Define SAS) program (Release 9.2).
RESULTS AND DISCUSSION Physico-chemical properties of the control media The physico-chemical properties of the control media are shown in Table 1. The soil used in this study was sandy clay of Munchong series with content, 57.88 ± 1.97% sand, 5.25 ± 0.42% silt and 36.87 ± 1.86% clay. Total N, C, P and K were 0.03 ± 0.03, 0.74 ± 0.05, 0.03 ± 0.002 and 0.1 ± 0.003%, respectively. The soil was acidic with pH 4.62 ± 0.16. This media contain 9.17 ± 1.12 mg kg-1 available phosphorous with 14.03 ± 1.77 cmolckg-1CEC and 0.3 dS m-1 EC. The concentration of exchangeable cations for K+, Mg2+ and Ca2+ were 0.005 ± 0.001, 0.004 ± 0.001 and 0.046 ± 0.005 cmolckg-1, respectively. The values of exchangeable Al and H were 0.75 ± 0.13 and 0.13 ± 0.06 cmolckg-1,
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Table 1. Selected physico-chemical properties of the control soil. Soil Property Texture Sand (%) Silt (%) Clay (%) Field capacity (%) Total N (%) Total C (%) Total P (%) Total K (%) pH (1:2.5 soil to water) Available P (mg kg-1) CEC cmolckg-1 EC dS m-1
Value Sandy clay 57.88 5.25 36.87 28.92 0.03 0.74 0.03 0.1 4.62 9.17 14.03 0.3
Exchangeable cations (cmolckg-1) K cmolckg-1 Mg cmolckg-1 Ca cmolckg-1 Al cmolckg-1 H cmolckg-1
0.005 0.004 0.046 0.75 0.13
Total heavy metal (mg kg-1) Cd Cu Zn Fe Mn
2.6 9.93 46.75 479.4 30.6
respectively. High concentrations of exchangeable Al and H were the main source of soil acidity. Judging the content of total C and N, CEC, and exchangeable bases, the plant nutrient status in this growth media was relatively low due to the acidic nature of the soil. The concentrations of Cd, Cu, Zn, Fe and Mn were 2.6 ± 0.21, 9.93 ± 0.31, 46.75 ± 4.55, 479.4 ± 22.87 and -1 30.6 ± 3 mg kg , respectively.
Cadmium concentrations in the growth media before planting and after harvest Cadmium concentration in the growth media before planting -1 ranged from 2.13 to 145.25 mg kg having the highest concentration (145.25 ± 5.36) in Cd5 as com-pared to other treatment levels and the lowest (2.13 ± 0.22) was recorded in the control (Cd0). There was a significant difference (p ≤ 0.05) among treatments in Cd concentration in the growth media before planting (Figure 1). Cd concentration in the growth media after planting was also varied under different Cd added to the -1 growth media. It ranged from 1.47 to 124.92 mg kg having
-1
the highest (124.92 ± 5.17 mg kg ) in Cd5 and the lowest -1 (1.47 ± 0.09 mg kg ) in control media. Cd concentration in the growth media decreased at harvest as compared to Cd concentration in the growth media before planting having the highest reduction (31.06%) under control media and the lowest (7.13%) in Cd3. Generally, the Cd concentration in both media before and after planting, increased with increase in the Cd concentration applied to the growth media (Figure 1). The concentration of Cd -1 in normal soil ranged from 0.01 to 2.0 mg kg (Alloway, 1995). However, Kabata-Pendiasand Pendias (1984) reported that the critical Cd level in soil is between 3 to 5 -1 mg kg . The growth performance of Hopea odorata in terms of basal stem diameter, height and number of leaves under various Cd concentrations are shown in (Figure 2 a, b, c). Significant difference (p ≤ 0.05) was observed among different Cd concentrations in basal stem diameter, plant height and number of leaves at harvest. The highest basal stem diameter (11.53 ± 0.22 mm) was recorded in control media followed by Cd2 (10.05 ± 0.17 mm) and Cd1 (9.67 ± 0.13 mm) as compared to other Cd concentrations and the lowest basal stem diameter (8.45 ± 0.29 mm) was found in Cd4. However, there was no significant difference (p≤0.05) between Cd4 and Cd5 in basal stem diameter. Control media exhibited the maximum height (91.25 ± 1.44 cm) followed by Cd2 (83.18±1.56 cm) and Cd1 (80.75 ± 1.38 cm) while the lowest (62.9 ± 0.79 cm) was recorded in Cd 5. The number of leaves ranged from 40 to 77 with the highest (77 ± 1.55) in control media followed by Cd2 (69 ± 3.52) and Cd1 (65 ± 4.5) while the lowest (40 ± 0.95) was observed in Cd5. The basal stem diameter and number of leaves increased from 9.67 to 10.05 mm and 65 to 69 when soil treated with Cd1 and Cd2, respectively. How-ever, a reduction was observed at higher Cd concentrations. Generally, the higher concentration of Cd reduced the growth parameters while an increase was observed within each Cd levels during the growth period. As described for the previous mentioned species, the growth of H. odorata was also reduced with increasing Cd concentration added to the growth media. These results reveal that H. odorata may tolerate soils con-taminated with Cd since the growth parameters increased every month indicating the normal growth, but higher levels of this non-essential element have an adverse effect on growth parameters. This result was in line with the findings of a study by Wu et al. (2009). Similar result was also obtained by Unterbrunner et al. (2007). Dry biomass of leaves, stems and roots Leaves, stems and roots dry biomass are presented in Table 2. Leaves dry biomass was significantly different (p≤0.05) among Cd concentrations. The dry biomass of leaves increased from control media (20.88 ± 0.28 g) up
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Figure 1. Change in cadmium concentrations in the growth media after cultivation of H. odorata as influenced by different Cd concentrations including 0, 25, 50, 75, 100 and 150 mg Cd kg-1 (Cd0, Cd1, Cd2, Cd3, Cd4 and Cd5). Means ± standard errors (S.E.) are shown in error bars (p ≤ 0.05).
to Cd2 (23.8 ± 1.03 g). However, the leaves dry biomass decreased at higher Cd concentrations added to the growth media where the lowest dry biomass (17.68 ± 0.93 g) was found in plants treated with Cd5. The effect of Cd concentrations on stem dry biomass was not steady. There was a significant difference (p ≤ 0.05) among Cd concentration in stems dry biomass. The highest stem dry biomass (22.03 ± 0.59 g) was observed in seedlings grown in the growth media treated with Cd2 compared to the lowest stem dry biomass (16.35 ± 0.46 g) in seedlings treated with Cd5. As shown in Table 2, the roots dry biomass was significantly different (p ≤ 0.05) among Cd concen-trations. The dry biomass of root showed the similar trend with the stem dry biomass. The roots dry biomass increased from control media (13.48 ± 0.27 g) up to Cd2 (15.05 ± 0.61 g). However, there was no significant difference (p≤0.05) among seedlings grown in control media with plants treated with Cd1 and Cd2. The roots dry biomass then decreased with increasing the Cd applied to the growth media where the lowest dry biomass (11.1 ± 0.21 g) was found in plants grown in the growth media treated with Cd5. The dry biomass among different plant parts was in the following order: leaves> stems> roots. It was observed that the dry biomass of leaves, stems and roots decreased at higher cadmium concentration in the growth media. This probably occurred due to the adverse effect of Cd on cell expansion or division, and may be via its influence on DNA, RNA or protein metabolism (Auda and Ali, 2010). Jadia and Fulekar (2008b) reported same result on alfalfa where the biomass of this species decreases the Cd concentration increased to (40 to 50 mg kg-1). However, the result obtained by Liu et al. (2010) on 40 cabbage cultivars
showed that the biomass of Liaodaqiukang, Suancaiwang and Beijingxiaoza 56 increased under Cd concentrations (1.0, 2.5 and 5.0 mg kg-1) indicating the high tolerance of these cultivars to Cd toxicity. Anget al.(2010) described that one of the way for Cd translocation in plant is detoxification mechanism of Cd from xylem and sequestration of this metal into plant tissue. Cd can be assimilated in the stem via phytocheletins PCs-complex (phytochelatins-cadmium) resulting in the reduction of the toxicity of Cd trapped into vacuole. However, Kuzovkinaet al. (2004) described that Cd as a nonessential element is known as a strong phytotoxic elements by interfering with enzymes activates, restrict-ting the DNAmediated transformation in microorganisms as well as to inhibit in symbiosis between microbes and plants.
Plant total dry biomass in response to cadmium treatments Total dry biomass was varied under different Cd concen-tration in the growth media ranging from 45.13 to 60.88 g. A significant difference (p ≤ 0.05) was observed among soil Cd treatments in total dry biomass production. The highest total dry biomass (60.88 ± 1.78 g) was recorded in Cd2 followed by Cd1 (55 ± 0.85 g) as compared to Cd5 which gave the lowest total dry biomass (45.13 ± 0.51 g) (Figure 3). The production of total dry biomass decreased with increase in the Cd concentration in the growth media, indicating the adverse effect of Cd on dry biomass production specifically at higher Cd concentrations (Chiang et al., 2006). Heavy metals such as Cd can create indirect toxicity by replacing essential elements at cation exchange areas in plant species (Jadia and Fulekar, 2009; Taiz and Zeiger, 2+ 2002). Cd in plants also can inhibit the transportation of Ca + and K and abscisic acid in guard cell (Jadia and Fulekar, 2008a ).
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Figure 2. Plant basal diameter (a), height (b) and number of leaves (c) of H. odorata at different months after planting as influenced by different Cd concentrations including 0, 25, 50, 75, 100 and 150 mg Cd kg-1 (Cd0, Cd1, Cd2, Cd3, Cd4 and Cd5). Means Âą standard errors (S.E.) are shown in error bars (pâ&#x2030;¤0.05).
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Table 2. Leaves, stems and roots dry biomass (g) of H. odorata after 5 months growth at different Cd concentrations including 0, 25, 50, 75, 100 and 150 mg Cd kg -1 (Cd0, Cd1, Cd2, Cd3, Cd4 and Cd5).
Treatment Cd0 Cd1 Cd2 Cd3 Cd4 Cd5
Leaf b 20.88±0.28 b 21.15±0.36 a 23.8±1.03 b 20.33±1.14 bc 19.08±0.82 b 17.68±0.93
Plant part Stem ab 20.18±0.05 ab 20.28±0.2 a 22.03±0.59 bc 19.28±1.58 cd 17.23±0.72 d 16.35±0.46
Root ab 13.48±0.27 ab 13.58±0.68 a 15.05±0.61 bc 13.13±0.45 cd 11.6±0.97 d 11.1±0.21
Total 80.61 55.01 60.88 52.74 47.91 45.13
Different letters within a column represent significant difference among means at a 5% level following Duncan Multiple Range Test (p≤0.05). Data with ± is mean standard error (S.E.).
Figure 3. Total dry biomass of H. odorata as influenced by different Cd concentrations including 0, 25, 50, 75, 100 and 150 mg Cd kg-1 (Cd0, Cd1, Cd2, Cd3, Cd4 and Cd5). Different letters indicate significant difference among means at a 5% level following Duncan multiple range test (p≤0.05). Means ± standard errors (S.E.) are shown in error bars (p≤0.05).
Relationship between total cadmium in growth media and plant dry biomass
(Assche and Clijsters, 1990).
Cd concentration in the growth media and total biomass of H. odorata was significantly (p ≤ 0.01) related to each other (r = -0.70). The negative correlation between these two parameters revealed that the total dry weight of species decreased with increase in the total Cd concen-tration in the growth media. Cd inhibits the biosynthesis of chlorophyll and the alleviation of chlorophyll content could result in reduction of shoot biomass (Orcutt and Nilsen, 2000). Metals cannot degrade inside the plant cells and can create toxicity when they accumulate above the threshold by destroying the cell structure and interfering with some of cytoplamsic enzymes
Cadmium concentration in various plant parts (leaves, stems and roots) The Cd concentration in different plant parts are shown in Table 3. The concentration of Cd in leaves under various Cd concentrations applied to the growth media was signi-ficantly different (p ≤ 0.05). The Cd concentration in leaves showed inconsistent trend under different cd added to the growth media. -1 The highest Cd concentration in leaves (10.31 ± 0.64 mg kg ) was recorded in leaves of seedlings grown in the growth media treated with Cd3 as compared to the lowest Concen-
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Table 3. Cd concentrations (mg kg-1) in various parts of H. odorata in different treatments including 0, 25, 50, 75, 100 and 150 mg Cd kg-1 (Cd0, Cd1, Cd2, Cd3, Cd4 and Cd5).
Treatment Cd0 Cd1 Cd2 Cd3 Cd4 Cd5
Leaf c 1.51±0.13 c 1.65±0.06 b 3.94±0.35 a 10.31±0.64 b 3.78±0.31 c 2.31±0.16
Plant part Stem e 1.30±0.08 d 12.49±0.62 d 16±0.79 c 33.21±2.23 b 68.11±1.9 a 105.99±7.83
Root c 1.71±0.06 b 110.15±7.84 b 116.55±10.55 a 169.16±8.72 a 175.4±2.03 a 181.93±11.83
Total 4.52 124.29 136.49 212.68 247.29 290.23
Different letters within a column indicate significant difference among means at a 5% level following Duncan multiple range test (p ≤ 0.05). Data with ± is mean standard error (S.E.).
tration (1.51 ± 0.13 mg kg-1) which was observed in leaves of plants grown in control media. The Cd concentration in leaves was then significantly decreased starting from Cd3 to Cd5indicating the toxicity effect of Cd at higher concentration. In the case of stem, Cd concentration increased significantly (p≤0.05) from 1.3 mg kg-1 to 105.99 mg kg-1 in stems of seedlings grown in control media and Cd5, respectively. However, there was no significant difference (p ≤ 0.05) between the Cd concentrations in the stem of seedlings grown in the growth media treated with Cd1 and Cd2. Based on the data presented in Table 3, there was significant difference (p ≤ 0.05) among different Cd concentrations applied to the growth media in the concentration of Cd in roots. The concentration of Cd in roots increased significantly with increasing the Cd concen-tration added to the growth media where the highest Cd concentration (181.93 ± 11.83 mg kg-1) was recorded in the roots of seedlings grown in the growth media contaminated with Cd5 compared to the lowest Cd concentration (1.71 ± 0.06 mg kg-1) in roots of seedlings grown in control media. Although, there was no signifi-cant difference (p≤0.05) in Cd concentration among the roots of seedlings grown in the growth media treated with Cd1 and Cd2 and media treated with Cd3, Cd4 and Cd5. The Cd concentration in various plant
parts was in the following rank; roots> stems> leaves. The concentration of Cd in root increased with an increase of concentration of this metal in the growth media. Similar result was attained by Liu et al. (2006) on the accumulation of Cd by roots and shoots of maize (Zea mays L.) where the concentration of Cd in roots and shoot of cultivars of this species increased significantly with increasing Cd levels. The uptake and accumulation of heavy metals by plant depends on the plant genotype and can be affected by physical and chemical properties of soil, and bioavailibity of heavy metals in soil (Shuhe et al., 2005). In contrast to the tested plant species in current study, there are some species such as lettuce (Lactuca sativa L.), cabbage (Brassica oleracea L.) and
tobacco (Nicotiana tabacum L.) with ability to accumulate high concentrations of Cd in leaves rather than roots (Jadia and Fulekar, 2008a).
Total cadmium concentration in the plant Total plant Cd concentration varied under different treatment levels. It ranged from 4.52 to 290.3 mg Cd kg-1. There was a significant difference (p≤0.05) among treatments in total Cd concentrations. The maximum total Cd concentration (290.3 ± 13.38 mg kg-1) was found in Cd5 followed by Cd4 (247.28 ± 2.87 mg kg-1) while the minimum (4.52 ± 0.15 mg kg-1) recorded in control media (Figure 4). It was observed that the total Cd concentration in H. odorata increased with increasing of Cd concen-tration applied to the growth media increased (Wu et al., 2009). The plant ability to accumulate metals depends on heavy metals availability in soil andthe metabolic patterns of plants (Liuet al., 2010). Iannelli et al. (2002) reported the similar result where Phragmitesaustralis plants accu-mulated most of Cd in the roots than leaves when treated with high levels of CdSo4 (50 µM) in hydroponic culture.
Cadmium removal by total plant biomass Removal efficiency based on plant biomass is defined as the total concentrations of metal and dry biomass of plants to total loaded metal in soil (Li et al., 2009). Cd removal was varied among different Cd concentration in soil. A significant difference (p≤0.05) was observed among treatment levels in Cd removal. It was in the range of 0.26 to 1.17 %. The highest Cd removal (1.17± 0.08 %) was noted in Cd1 followed by Cd2 (0.99 ± 0.11 %) as compared to the control media which gave the lowest Cd removal (0.19 ± 0.04 %) (Figure 5). The Cd removal decreased with increase in the Cd concentration added to the growth media which may be associated with the reduction of plant dry biomass at higher Cd concentration. As a strong phototoxic element to plants; Cd influences the plant growth and development reversely and ceases their life quickly due to its great solubility and high toxicity (Das et al., 1997;
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Figure 4. Total plant Cd concentration as influenced by different treatment levels including 0, 25, 50, 75, 100 and 150 mg Cd kg-1 (Cd0, Cd1, Cd2, Cd3, Cd4 and Cd5). Different letters indicate significant difference among means at a 5% level following Duncan multiple range test (p≤0.05). Means ± standard errors (S.E.) are shown in error bars (p≤0.05).
Figure 5. Total Cd removals as influenced by different treatment levels including 0, 25, 50, 75, 100 and 150 mg Cd kg-1 (Cd0, Cd1, Cd2, Cd3, Cd4 and Cd5). Different letters indicate significant difference among means at a 5% level following Duncan multiple range test (p≤0.05). Means ± standard errors (S.E.) are shown in error bars (p≤0.05).
Kuzovkina et al., 2004). Bioconcentration cadmium
and
translocation
factor
of
Bioconcentration factor (BCF) index is defined as the ratio of heavy metal concentration in plant roots to that in soil (Malik et al., 2010) whereas translocation (TF) is defined as the ratio
of heavy metal concentration in aerial parts of plant to that in roots (Karami and Shamsuddin, 2010). The BCFs were varied under different Cd con-centrations in the soil and it was in the range of 0.83 to 4.96. There was a significant difference (p≤0.05) among treatments in BCFs. The highest BCF (that is 4.96 ± 0.28) of Cd was found in Cd1 as compared to other treatment levels, while control media exhibited the lowest BCF ( 0.83 ± 0.08) (Figure 6). The
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Figure 6. Bioconcentration factor as influenced by different Cd concentrations including 0, 25, 50, 75, 100 and 150 mg Cd kg-1 (Cd0, Cd1, Cd2, Cd3, Cd4 and Cd5). Different letters indicate significant difference among means at a 5% level following Duncan Multiple Range Test (p≤0.05). Means ± standard errors (S.E.) are shown in error bars (p ≤ 0.05).
Figure 7. Translocation factor as influenced by different Cd concentrations including 0, 25, 50, 75, 100 and 150 mg Cd kg-1 (Cd0, Cd1, Cd2, Cd3, Cd4 and Cd5). Different letters indicate significant difference among means at a 5% level following Duncan Multiple Range Test (p≤0.05). Means ± standard errors (S.E.) are shown in error bars (p≤0.05).
BCFs were >1 under various treatment levels except in control media. The BCF value of Cd usually ranged from 1 to 10 (Li et al., 2006). The BCFs decreased with increase in the Cd concentration in the growth media, which may indicate the restriction in soil-root transfer at higher Cd concentrations in the soil (Justin et al., 2011). Ho et al. (2008) found that the BCFs of Pb in kenaf (Hibiscus cannabinus L.) were >1 (1.92 to 3.21) when grown in sand tailings. TFs were also varied under different Cd concentrations in
the growth media and a significant difference (p≤0.05) observed among treatments in TFs. This index was in the range of 0.13 to 1.64. Control media showed the highest TF (1.64 ± 0.05) as compared to the other treatment levels while Cd1 exhibited the lowest TF (0.13 ± 0.01) (Figure 7). It was observed that TFs increased with increase in the applied Cd concentration in the growth media. However, the TFs under different Cd levels were <1 except in control media indicating that H. odorata was unable to tanslocate Cd from the roots to the shoots
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efficiently. Baker (1981) reported that plants are classified into accumulator if heavy metal concentration ratio (shoot to root) is more than one and excluder if this ratio is less than one. A pot experiment showed that the translocation factor in five cultivars of cabbage (New Beijing 3, Saixin 5,Fengyuanxin 3, Shuishiying 91-12 and Liaodaqiukang) was <1 under various concentrations of Cd (1.0, 2.5 and 5.0 mg kg-1) (Liu et al., 2010). The translocation of Cd is often restricted due to the ability of this element to create Cd-phytochelatin complex by sequestration in the vacuole (Lux et al., 2011). Cd movement from root to shoots probably occurs within the xylem. The levels of free Cd in the symplast can be influenced highly by cellular sequestration of Cd and, therefore, it can affect the movement of Cd throughout the plants (Niu et al., 2007). Relationship between total cadmium in the growth media and plant species Correlation analysis between total Cd concentration in the growth media and in H. odorata was significantly different (p ≤ 0.01). The total Cd concentration in the growth media was significantly related to total Cd concen-tration in H. odorata (r = 0.92). This positive correlation indicated that total concentration of Cd in H. odorata increased with an increase in total concentration of this metal in the growth media. This result was in line with the result obtained by Wu et al. (2009) on poplar where Cd accumulation increased with increase in the Cd concen-tration in growth media.
Conclusion H. odorata planted in control media showed the highest production of basal diameter, plant height and number of leaves. The highest total dry biomass production was recorded in Cd2 (60.88 ± 1.78 g). The maximum total Cd concentration (290.23 ± 13.38 mg kg-1) and total Cd removal based on total dry biomass (0.81 ± 0.06%) were found in Cd5 and Cd1, respectively. Cd was highly con-centrated in roots. H. odorata exhibited the high BCF and low TF. The BCFs were >1 except in control media, whereas TFs were <1 except in control media. The maxi-mum BCF (4.96) of Cd was found in Cd1 while the highest TF (1.64) was recorded in control media. Therefore, H. odorata can be used as an excluder based on the suggestion of Baker (1981) and remediate Cd-contaminated soil in phytoremediation through phytostabilization method to prevent distribution of Cd in contaminated areas.
ACKNOWLEDGEMENTS This research was financially supported by a fundamen-
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tal Research Grant Scheme (FRGS) from the Ministry of Higher Education of Malaysia (MOHE) through the University Putra Malaysia, Malaysia (UPM). We thank Mrs. Nooeriyanie Simon, Ms. Zarina Abdul Rahman and Mr. Ariffin Abu Hassan for their kind assistance during the laboratory analysis at the laboratory of Soil Science, Faculty of Forestry and Faculty of Agriculture, Universiti Putra Malaysia.
REFERENCES Alloway BJ (1995). Heavy metals in soils: Springer. pp. 339-367. Assche F, Clijsters H (1990). Effects of metals on enzyme activity in plants. Plant, Cell Environ. 13: 195-206. Auda MA, Ali EELS (2010).Cadmium and Zinc toxicity effects on growth and mineral nutrients of carrot (Daucuscarota).Pak. J. Bot. 42: 341351. Baker AJM (1981). Accumulators and excluders-strategies in the response of plants to heavy metals. J. Plant Nutr. (United States). 3: 1-4. Chiang PN, Wang MK, Chiu CY, Chou SY (2006). Effects of cadmium amendments on low molecular weight organic acid exudates in rhizosphere soils of tobacco and sunflower. ISSN:1522-7278. Environ. Toxicol. 21: 479-488. Das P, Samantaray S, Rout G (1997). Studies on cadmium toxicity in plants: a review. Environ. Pollut. 98: 29-36. Dickinson NM, Pulford ID (2005). Cadmium phytoextraction using shortrotation coppice Salix: the evidence trail. ISSN:0160-4120. Environ. Int. 31: 609-613. Garbisu C, Alkorta I (2001). Phytoextraction: a cost-effective plantbased technology for the removal of metals from the environment. Bioresour. Technol. 77: 229-236. Iannelli MA, Pietrini F, Fiore L, Petrilli L, Massacci A (2002). Antioxidant response to cadmium in Phragmitesaustralis plants. Plant Physiol. Biochem. 40: 977-982. Jadia CD, Fulekar M (2009). Phytoremediation of heavy metals: Recent techniques. ISSN: 1684-5315. Afr. J. Biotechnol. 8: 921-928. Jadia CD, Fulekar MH (2008a). Phytoremediation: The application of vermicompost to remove zinc, cadmium, copper, nickel and lead by sunflower plant. Environ. Engr. Manage. J. 7: 547-558. Jadia CD, Fulekar MH (2008b). Phytotoxicity and remediation of heavy metals by Alfalfa (Medicago sativa) in soil-vermicompost media. ISSN: 1995-0748. Adv. Nat. Appl. Sci. 2(3): 141-151. Justin V, Majid N, Islam MM, Abdu A (2011). Assessment of heavy metal uptake and translocation in Acacia mangium for phytoremediation of cadmium contaminated soil. J. Food, Agric. Environ. 9: 588-592. Karami A, Shamsuddi ZH (2010). Phytoremediation of heavy metals with several efficiency enhancer methods. Afr. J. Biotechnol. 9: 36893698. Kuzovkina YA, Knee M, Quigley MF (2004). Cadmium and copper uptake and translocation in five willow(Salix L.) Species. ISSN:15226514. Int. J. Phytoremed. 6: 269-287. Liu DH, Wang M, Zou JH, Jiang WS (2006). Uptake and accumulation of cadmium and some nutrient ions by roots and shoots of maize (Zea maysL.). ISSN:0556-3321. Pak. J. Bot. 38: 701-709. Lux A, Martinka M, Vaculík M, White PJ (2011). Root responses to cadmium in the rhizosphere: a review. J. Exp. Bot. 62: 21-37. Malik RN, Husain SZ, Nazir I (2010). Heavy metal contamination and accumulation in soil and wild plant species from industrial area of Islamabad, Pakistan. Pak. J. Bot. 42: 291-301. Manousaki E, Kadukova J, Papadantonakis N, Kalogerakis N (2008).Phytoextraction and phytoexcretion of Cd by the leaves of
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Tamarixsmyrnensis growing on contaminated non-saline and saline soils. ISSN:0013-9351. Environ. Res. 106: 326-332. Nabulo G, Oryem-Origa H, Diamond M (2006). Assessment of lead, cadmium, and zinc contamination of roadside soils, surface films, and vegetables in Kampala City, Uganda. ISSN:0013-9351. Environ. Res. 101: 42-52. Niu ZX, Sun LN, Sun TH, Li YS, Wang H (2007).Evaluation of phytoextracting cadmium and lead by sunflower, ricinus, alfalfa and mustard in hydroponic culture. J. Environ. Sci. 19: 961-967. Odoemelam SA, Ukpe, RA (2008). Heavy meal decontamination of polluted soils using Bryophyllumpinnatum. 7: 4301-4303. Afr. J. Biotechnol. Orcutt DM, Nilsen, ET (2000). The physiology of plants under stress: soil and biotic factors: John Wiley and Sons Inc. ISBN:0471170089. pp. 326-332. Rajakaruna N, Tompkins KM, Pavicevic PG (2006). Phytoremediation: An affordable green technology for the clean-up of metalcontaminated sites in Sri Lanka. Ceylon J. Sci. (Biol. Sci.), 35: 25-39. Sahibin AR, Zulfahmi AR, Lai KM, Errol P, Talib ML (2002). Heavy metals content of soil under vegetables cultivation in Cameron highland In:Proceedings of the regional symposium on environment and natural resources 10-11th April 2002, Kuala Lumpur, Malaysia. 1: 660-667. Soleimani M, Hajabbasi MA, Afyuni M, Mirlohi A, Borggaard OK, Holm PE (2010). Effect of endophytic fungi on cadmium tolerance and bioaccumulation by Festucaarundinacea and Festucapratensis. ISSN:1522-6514. Int. J. o Phytoremed. 12: 535-549. Taiz L, Zeiger E (2002). Plant Physiology. Massachusetts : Sinauer Associates, Inc. p. 690.
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African Journal of Biotechnology Vol. 11(29), pp. 7483-7493, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.4109 ISSN 1684â&#x20AC;&#x201C;5315 Š 2012 Academic Journals
Full Length Research Paper
Phenotypic diversity and plant growth promoting characteristics of Mesorhizobium species isolated from chickpea (Cicer arietinum L.) growing areas of Ethiopia Mulissa Jida1,2* and Fassil Assefa1 1
Microbial, Cellular and Molecular Biology Program, Faculty of Life Science, Addis Ababa University, Ethiopia. 2 Biological Science Program, Faculty of Natural Science, Wollega University, Ethiopia. Accepted 16 February, 2012
Chickpea (Cicer arietinum L.) is one of the major sources of dietary protein for majority of Ethiopian population. It also maintains soil fertility through its symbiotic nitrogen-fixation in association with Mesorhizobium species. Therefore, this study was aimed at isolation, characterization and selection of symbiotically effective native chickpea nodulating rhizobia endowed with different plant growthpromoting (PGP) characteristics. Hence, phenotypic and plant growth promoting characteristics of thirty-six rhizobia isolates recovered from root nodules of chickpea grown in soils collected from different chickpea producing areas found in Central and Northern part of Ethiopia were investigated. The result of our study indicated that chickpea rhizobial isolates have shown wide diversity in their different C and N-sources utilization pattern and tolerance to salinity, high temperatures, acid and alkaline pH, heavy metals and antibiotics. Symbiotic and morphological characterization also showed a wide diversity among tested isolates. Moreover, screening for PGP characteristics indicated that 44.4% of the isolates were phosphate solubilizer while 27.8% of them were found to be indole-3-acetic acid (IAA) producer. Furthermore, 19.4% tested isolates showed antagonistic activity against Fusarium oxysporum in dual culture assay. Generally, the present study indicates that Ethiopian soils contain symbiotically effective chickpea nodulating rhizobia which are endowed with different PGP characteristics. Key words: Chickpea, Mesorhizobium, plant growth promoting, Ethiopia, symbiotic nitrogen fixation.
INTRODUCTION Chickpea (C. aeritinum L.) is one of the major food legume crops grown widely in tropics, sub-tropics and temperate regions of the world. It is also one of the principal food legumes which has been widely grown in Ethiopia over an area of 208,388.6 ha (CSA, 2011). Nutritionally chickpea seed contains 19.8 % protein and substantial amount of other nutrients (Werner, 2005). In Ethiopia, it serves as an invaluable source of dietary protein which is commonly consumed in different preparations as supplementary food. Besides, chickpea is one of the major export commodities with significant export market option amongst the field crops (Bejiga and
*Corresponding author. E-mail: mulaeabageda@gmail.com
Daba, 2006; Shiferaw and Teklewolde, 2007). In addition to nutritional quality and source of cash, chickpea restores and maintains soil fertility through its symbiotic nitrogen-fixation in association with Mesorhizobium species (Nour et al., 1994, 1995; Jarvis et al., 1997). It is capable of -1 fixing 90-180 kg N ha (Werner, 2005) and therefore, grown in rotation with major cereals such as tef (Erograstic tef), wheat (Triticum sp.), barley (Horduem vulgare) in traditional low-input agricultural system. However, its yield has remained very low (Bejiga and Daba, 2006; Keneni et al., 2011a) and thus, many research activities have been undertaken to improve chickpea cultivars with respect to their yield, tolerance to different biotic and abiotic stresses (Anbessa and Bejiga, 2002; Ahmed and Ayalew, 2006; Keneni et al., 2011a; Keneni et al., 2011b). Consequently, many improved cultivars were released by
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Ethiopian Agriculture Research Organization (EARO) (Shiferaw and Teklewolde, 2007). Nevertheless, these alone could not improve the extremely low productivity of chickpea. One of the strategies which has been given less attention was exploiting the benefits of its symbiotic nitrogen fixation by selecting effective rhizobia. Recently, some strains of rhizobial species were found to exhibit plant growth promoting (PGP) characteristics as they promoted the growth of some crops through mechanisms that are independent of biological nitrogen-fixation (Antoun et al., 1998; Peix et al., 2001; Yanni et al., 2001; Alikhani and Yakhchali, 2009). These mecha-nisms include stimulating plant growth directly either by synthesizing phytohormones such as indole-3-acetic acid (IAA) or by promoting nutrition processes such as phosphate solubilization and siderophore production, which facilitate phosphorus and iron uptake, respectively from soil. They can also stimulate growth indirectly by protecting the plant against soil-borne fungal pathogens. Several studies showed that Mesorhizobium species also exhibit such characteristics (Peix et al., 2001; Alikhani and Yakhchali, 2009; Hemissi et al., 2011). Such kind of rhizobial strains could be used as multipurpose inoculants (Jida and Assefa, 2011) for both legume and non-legume crops grown rotationally or subsequently. Thus, native rhizobial isolates must be screened for their PGP activity in addition to their excellent symbiotic effectiveness. Mesorhizobium strains naturally vary in their nitrogen fixing capacity and adaptation to prevailing environmental stresses (Zharan, 1999; Maâtallah et al., 2002; L’taief et al., 2007). Consequently, selection of symbiotically efficient rhizobial strains which are tolerant to locally prevailing stresses is highly desirable. In other parts of the world where chickpea is commonly grown several studies have been conducted on chickpea rhizobia characterization and selection of best strains for inoculant production (Maâtallah et al., 2002; L’taief et al., 2007; Küçük and Kıvanç, 2008). However, in Ethiopia most of the hither to studies were committed to breeding the host plant for adaptation to different stresses (Anbessa and Bejiga, 2002; Ahmed and Ayalew, 2006; Keneni et al., 2011a, b) and thus, there is no information about the characteristics of chickpea rhizobia. These necessitates for research activities devoted to investigate different characteristics of chickpea rhizobia isolated from producing areas of the country. Hence, this study was aimed at isolation, characterization and selection of symbiotically effective native chickpea rhizobia isolates endowed with different PGP characteristics.
MATERIALS AND METHODS
northern parts of the country with an altitude ranging from 1526 (Alamata) to 2840 (Sheno) meter above sea level (masl) and pH from moderately acidic (5.6) to alkaline (7.9) (Table 1). About 3 kg of soil samples were excavated from 15-20 cm depth from each site. A total of 36 soil samples were collected in sterile plastic bags and carefully transported to Applied Microbiology laboratory, Addis Ababa University, for further work in October, 2009. Isolation of rhizobia The rhizobia were isolated from soil samples by inducing nodulation on chickpea cultivar called ‘Ararti’ (obtained from EARO, Debra Zeit) using plant infection method (Vincent, 1970). Each soil sample was filled into surface sterilized (95% ethanol) plastic pots. Chickpea seeds were selected and surface sterilized with 95% ethanol and 3% sodium hypochlorite solutions for 10 s and 3 min, respectively. The seeds were rinsed five times with sterilized distilled water to remove traces of sterilizing chemicals and allowed to germinate on sterile water gar (1%) surface for three days at 25°C. Five pre-germinated seeds were planted on each pot. The seedlings were thinned down to three after 5 days of emergence (DAE). All pots were situated in glasshouse over the table and watered to a field capacity every three days for 60 days after planting (DAP). Sixty DAP all the plants were carefully uprooted from the pots and washed under gently flowing tap water to remove soil particles. Large and reddish nodules were separately collected from each pot on separate sterile Petri dishes and surface sterilized as described before, and crushed using alcohol flamed glass rod. Loopful of the extract was streaked on Yeast extract Mannitol Agar (YEMA) containing 0.0025% (w/v) Congo red (Vincent, 1970). The components of YEMA g/L: 0.5 K2HPO4, 0.2 MgSO4, 0.1 NaCl, 10 Mannitol, 0.5 Yeast extract, 15 Agar (Vincent, 1970). All the plates were incubated at 28°C for 4 to 6 days. From each plate, single typical rhizobia colony were picked and transferred to test tubes which contain sterile Yeast extract Mannitol Broth (YEMB) (Vincent, 1970). The test tubes were incubated at room temperature on a gyratory shaker at 120 revolution (r) minute (m)–1 for 3 days and purified by re-streaking on new YEMA plates for growth. The pure cultures were further confirmed by presumptive tests such as gram reaction using KOH test as described by Gregorson (1978) and growth on Peptone Glucose Agar (PGA) (Somasegaran and Hoben, 1994). Pure isolates were then preserved on YEMA slants containing 0.3% CaCO3 stored at 4°C for short-term storage (Vincent, 1970) and in glycerol (50% v/v) at -20°C for long-term storage. All the rhizobial isolates were designated as CR1-50(C: Chickpea; R: Rhizobia) (Table 1).
Rhizobial inoculum preparation Rhizobial isolates were grown in YEMB on a gyratory shaker at room temperature and 120 r m-1 for 72 h. The suspension was centrifuged in sterile plastic tubes (10 ml) at 5000 r m-1 for 10 min. The pellets were re-suspended in normal saline (0.90% w/v of NaCl) solution to give a final concentration of 108 CFU/ml using the viable plate count method and optical density measurement by spectrophotometer at 540 nm. All inoculums were prepared like this unless otherwise stated.
Study sites and soil samples collection
Authentication and preliminary symbiotic characterization of the isolates
Soil samples were collected from chickpea grown farmer’s field found in Showa, Gonder, Gojam, Wallo and Tirgay areas of Ethiopia (Figure 1). The areas are distributed in central and
All rhizobial isolates were authenticated and characterized symbiotically by re-inoculating on their host plant on sand culture. About 3 kg of washed and autoclave sterilized sand was placed in
Jida and Assefa
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Figure 1. Soil sampling sites.
plastic pots (3 kg capacity). The chickpea cultivar called ‘Ararti’ seeds were surface sterilized and germinated as described before. The germinated seedlings were flooded with each rhizobial culture adjusted 109 cells per seed for 1 h on separate autoclave sterilized plates. Five inoculated seedlings were transferred to each pot which was later thinned down to three after 5 DAE. The pots were irrigated with nitrogen free plant nutrient solution as described by Somasegaran and Hoben (1994). Uninoculated but nitrogen-fertilized pots were included as positive (TN) control and uninoculated and non fertilized (T0) pots as negative control. The experiment was statistically laid out with three replications using randomized block design. As control, each block contained two pots (T0 and TN) with uninoculated seedlings. Plants were supplied with water every three days and once a week with a nitrogen-free nutrient solution (Somasegaran and Hoben, 1994). Furthermore, TN control received weekly 0.05% (w/v) KNO 3 as nitrogen source weekly. Sixty DAP all seedlings were carefully uprooted, nodule number counted, nodule dry weight and shoot dry weight were measured after drying at 70°C for 48 h to a constant weight. Relative symbiotic effectiveness of each isolate was calculated by using the formula (100 × inoculated plant shoot dry weight/ N-fertilized plant shoot dry weight) of Gibson (1987).
Colony morphology, growth and biochemical characteristics Colony morphology was evaluated by streaking a loop of the initial inoculum on YEMA plates and allowing the isolates to grow at 28°C for 5 days (Vincent, 1970; Sinclair and Eaglesham, 1984). Growth rate of the isolates was assessed by inoculating 20 µl of the inoculum into YEMB test tubes incubated in a gyratory shaker at 120 r m–1, by measuring the optical density at 540 nm using spectrophotometer every 6 h and spread plating 0.1 ml diluted culture on YEMA plates. The generation time (GT) was calculated from the logarithmic phase of growth curve as described by Somasegaran and Hoben (1994). Acid or alkali production test was carried out by growing isolates on YEMA medium containing Bromothymol blue (BTB)( Somasegaran and Hoben, 1994). Physiological characteristics All tests, except C and N-source assimilation were carried out on YEMA plates. Petri dishes containing defined medium were inoculated with 20 µl of the inoculum. After 5 days of incubation at 28°C, bacterial growth was compared to the controls. All tests were
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Table 1. Chickpea rhizobila isolates, site of isolation, growth and colony characteristics.
Isolate
Isolation site
pH of isolation site
Altitude of isolation site (masl)
Colony diameter( mm)
GT (h)
1 2 3 4 5 6
CR01 CR02 CR03 CR04 CR05 CR06
Fiche Alem Tena Debra Selam Sandafa Galessa Chirameda
6.8 7.1 6.0 6.0 7.6 6.6
2748 1637 1896 2554 2017 1747
LMM LWM LMM LCM LWM LWM
3.2 2.4 3.4 3.2 2.7 4.5
4.8 4.8 5.0 5.1 4.5 2.8
7
CR08
Ginchi
6.6
2378
LMM
2.3
4.7
8
CR09
Goro
9 10 11 12 13 14 15 16 17
CR11 CR12 CR14 CR16 CR18 CR19 CR20 CR21 CR23
Angut Michael Maksagnt Chole Olankomi Sirnka Dibulko Lalibela Yetinora Gurura
6.6
1832
LMM
3.2
5.2
6.3 6.4 6.3 6.6 6.9 6.8 7.4 6.4 7.0
1850 1978 2612 2378 1843 1992 2138 2437 1906
LWM LWM LWM LMM LWM LWM LMM LMM LMM
2.6 2.3 4.3 3.3 2.5 3.7 3.7 3.7 3.1
4.9 4.6 3.2 4.4 4.1 4.7 4.8 4.1 4.7
18
CR24
Fogera
6.4
1931
LMM
2.8
5.1
19 20 21 22 23 24 25 26 27 28
CR25
Alshin
6.7
2082
LWLM
2.2
5.0
CR28 CR29 CR31 CR32 CR34 CR36 CR37 CR38 CR39
Bilbila Amber Asgori Aja Woldya Alamata Robe Itacha Tikana
6.6 6.3 6.6 6.8 6.6 7.9 7.8 6.8 7.2
2069 2454 2078 2023 2074 1526 1658 2134 1942
SWLM LMM LWhLM LMM SWLM LWM LWhLM LWM LWhLM
1.9 4.0 2.2 3.2 1.9 3.3 3.7 2.3 2.0
5.4 2.8 4.7 4.4 5.2 4.5 5.2 4.8 5.2
29
CR40
Ilala
7.6
1924
LWLM
2.0
5.3
30
CR42
Goha Tsion
6.2
2517
LMM
2.0
4.2
31 32 33 34 35 36
CR44 CR45 CR46 CR47 CR48 CR50
Ambo Obbi Mojo Sheno Teji Debra Libanos
6.7 6.6 6.8 5.6 6.7 7.3
2170 2108 1774 2840 2065 2594
LWM SWLM LMM LCM LWM LMM
2.5 1.5 2.1 2.2 2.4 2.3
3.8 4.8 5.4 4.6 4.5 4.8
S/N
Colony characteristics
LCM: Large, creamy, mucoid; LWM: Large, watery, mucoid; LMLM: Large, milky, less mucoid; LWLM: Large watery, less mucoid; LWhLM: Large, white less mucoid; SWLM: Small watery less mucoid; GT: Generation time.
carried out in triplicates. Salt, pH and temperature tolerance Salt tolerance of the isolates was determined on YEMA plates
containing 0 to 5% (w/v) NaCl concentrations. Tolerance to extreme pH was tested on YEMA medium set at different pH (4.5 to 10) values using 1 NHCl and 1N NaOH. Temperature tolerance was evaluated on YEMA plates inoculated as described above and incubated at temperatures from 4 to 40째C.
Jida and Assefa
Intrinsic antibiotic and heavy metal resistance This intrinsic antibiotic and heavy metal resistance was determined on solid YEMA medium containing the following filter sterilized antibiotics or heavy metals (µg.ml –1 ): Ampicillin (5 and 10), chloramphenicol (5 and 10), erythromycin (5 and 10), nalidixic acid (5 and 10), streptomycin (10 and 50), neomycin (5 and 10) and tetracycline (5 and10); AlK(SO4)3.12H2O (10, 25), K2Cr2O7 (50), CoCl2 (10), CuCl2.2H2O (10), HgCl2 (5), MnCl2 (50, 75), NiSO4 (10), Pb(CH3COO)2 (10), and ZnCl2 (50).
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for 72 h (Bric et al., 1991) to determine their IAA production ability. Supernatant of the strains were collected by centrifugation at 10,000 rounds m-1 for 15 min and 2 ml supernatant of each was transferred separately to a fresh tube to which 100 µl of 10 mM O-phosphoric acid and 4 ml of Salkowaski reagent (1 ml of 0.5 mM FeCl3 in 35% HClO4) were added. Mixtures were incubated at room temperature for 25 min and observed for the development of pink color. Data analysis
Utilization of different C and N-sources Different carbohydrates were added as described by Amarger et al. (1997) at final concentration of 1 gL-1 to the basal medium containing (gL-1): K2HPO4, 1; KH2PO4, 1; FeCl3. 6H2O, 0.01; MgSO4.7H2O, 0.2; CaCl2, 0.1; NH4(SO4)2, 1; and 15 g of agar. The following filter sterilized sole carbon sources were added after autoclaving: Citrate, D-sorbitol, D-mannose, D-maltose, D-galactose, D-arabinose, gluconate, raffinose, xylose, dulcitol, cellobiose, anoditol, inulin, aesculin, trehalose and inositol. D-mannitol, D-glucose, α-lactose, D-fructose, glycerol, αcellulose, sucrose, starch, tartarate, gelatin and dextrin were added before autoclaving. Filter sterilized L-tryptophan, methionine, L-tyrosine, leucine, L-asparagine, DL-β-phenylalanine, L-arginine, glutamic acid, Llysine, analanine, serine, glycine, thiamine, niacin and riboflavin were used as sole nitrogen source for isolates by adding a final concentration of 0.5 gL-1 to the above basal medium from which NH4 (SO4)2 was omitted and mannitol was added after autoclaving (Amarger et al., 1997). All inoculated palates were incubated at 28°C and results were observed after 5 days.
All experiments were set in triplicate and the data is average of three. Symbiotic data was analyzed by ANOVA and the treatment means were compared following Duncan’s test (DT) by using SPSS (V.17). Correlations among some parameters were checked by using linear regression analysis.
RESULTS AND DISCUSSION A total of 36 chickpea rhizobial isolates were recovered from as many sampling sites from Central and Northern Ethiopia with altitude ranging from 1526 (Alamata) to 2840 masl (Sheno), and soil pH from moderately acidic (pH 5.6) to slightly alkaline (pH 7.9) (Table 1). The distribution pattern showed that most of the sample sites were from mid altitude with 1750 to 2500 masl and mildly acidic soil pH of 6.0 to 6.9. Symbiotic characteristics
Determination of PGP Properties of the Isolates Phosphate solubilization Phosphate solubilizing ability of the isolates was determined using Pikovaskaya agar plates (Pikovskaya, 1948) spot inoculated with 20 µl of the inoculum. After incubation at 28°C for 5 days, formation of a clear zone around the spot was recorded and solubilization index (SI) was calculated as described by (Edi-Premono et al., 1996) for positive isolates. Screening for in vitro antagonistic activity against Fusarium oxysporum The in vitro mycelial growth inhibition of F. oxysporum f.sp. ciceri (obtained from EARO, Debra Zeit) by the rhizobial isolates was tested using the dual culture technique as described by Landa et al. (1997). Twenty microliter (20 µl) of each isolates inoculum was equidistantly spotted on the margins of YEMA plates amended with sucrose (0.5%) and incubated at 28°C for 24 h. A 4 to 5 mm diameter agar disc from Potato Dextrose Agar (PDA) cultures of the fungal pathogens was placed at the centre of the YEMA plate for each bacterial isolate and incubated at 28°C for 5 days. The radii of the fungal colony towards and away from the bacterial colony were measured and the presence of clear inhibition zone between the bacterial isolates and growing fungi was considered as positive.
lAA production Exponentially grown cultures of each isolates were incubated separately on broth medium supplemented with 5 mM L-tryptophan
All tested isolates of chickpea rhizobia showed great variation in their capacity to induce the formation of nodule on the host plant root under glasshouse con-ditions. The mean nodule number per plant varied from 41 to 79 which were induced by isolate CR14 and CR08, respectively (Table 2). In general, 83% of the isolates induced the formation of more than 50 nodule number on the roots of their host indicating that chickpea rhizobial isolates from Ethiopian soils are the most infective strains. The highest nodule dry weight was recorded for isolate CR32, that is, 120 mg/plant whereas the lowest was 56 mg which was recorded for isolate CR02 and CR50 (Table 2). Similarly, all tested chickpea rhizobia exhibited high diversity in their capacity to fix atmospheric nitrogen and thus shoot dry matter accumulation. In comparison with TN control which represents 100% level of shoot dry matter and T0 control which represents 19.1%, all isolates showed shoot dry matter yield ranging from 0.60 to 1.36 g per plant (Table 2). The relative effectiveness, which is expressed in percent of TN control showed that isolates CR06, CR38, CR45 and CR47 were the most efficient with more than 80% dry matter yield while CR01, CR08 and CR40 were the least efficient with 44.1% relative effectiveness (Table 2). These isolates were also particularly most infective and highly effective and hence must be taken into consideration for chickpea inoculation trail at different zones under Ethiopian soil and climatic conditions. It has been demonstrated that inoculation of selected rhizobial inoculants on chickpea has beneficial effect on yield (Romdhane et al., 2009). Overall, the mean comparison showed several over-lapping
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Table 2. PGP and Symbiotic characteristics of chickpea nodulating rhizobial isolates.
Treatment (rhizobial isolates) CR01 CR02 CR03 CR04 CR05 CR06 CR08 CR09 CR11 CR12 CR14 CR16 CR18 CR19 CR20 CR21 CR23 CR24 CR25 CR28 CR29 CR31 CR32 CR34 CR36 CR37 CR38 CR39 CR40 CR42 CR44 CR45 CR46 CR47 CR48 CR50 T0 TN
Nodule Number/plant ± SE i
45.8±3.0 b-i 57.3±3.2 h-j 49.3±1.9 d-j 51.8±3.2 b-i 58.3±2.6 b-i 58. 7±2.5 a 79.3±3.2 b-h 60.8±1.6 b-h 61.2±2.5 ij 43.3±1.2 j 41.2±1.5 ij 43.8±1.4 b-g 60.8±2.8 e-j 51.2±1.6 b-h 60.51.0 c-j 54.5±3.0 b-h 59.3±2.5 a-g 66.0±2.0 b-g 62.5±2.0 a-e 64.8±2.5 h-j 49.0±3.1 a-c 68.7±3.5 ab 70.2±1.8 e-j 51.0±1.5 h-j 46.2±2.9 b-g 62.0±4.5 a-f 64.5±1.6 b-g 62.5±1.0 a-d 67.0±4.8 b-g 63.5±3.8 b-h 61.3±5.9 b-g 63.0±1.2 b-g 62.0±2.8 ab 71.0±1.2 e-j 50.8±2.0 b-i 57.3±3.2 -
Nodule dry weight (mg)/plant ± SE b
78±8 b 56±5 b 75±2 b 69±8 a 114±3 b 92±20 a 115±9 a 101±7 a 99±2 b 80±9 b 76±4 a 116±15 82±9b 92±4b a 99±2 b 89±10 b 75±5 a 96±4 a 102±6 a 97±2 b 95±9 a 105±6 a 120±3 b 77±6 a 98±7 a 100±20 a 104±30 a 99±30 b 74±8 b 90±6 b 96±1 b 94±3 b 75±6 a 100±3 b 95±7 b 56±5 -
Shoot dry weight(g)/plant ± SE
Relative effectiveness (%)
P.S. activit y(S.I.)
IAA produc tion
In vitro inhibition of F. oxysporum
44.1 66.9 50.7 59.6 60.3 83.1 44.1 63.2 55.1 46.3 67.6 64.7 72.8 71.3 75.7 52.9 59.6 52.9 69.8 58.8 58.8 72.1 76.5 59.6 65.4 51.5 100 59.6 44.1 59.6 55.9 89.7 51.5 81.6 62.5 55.1 19.1 100
1.2 1.14 1.23 1.15 1.25 1.21 1.13 1.23 1.12 1.3 1.13 1.17 1.2 1.21 1.15 -
+ + + + + + + + + + -
+ + + + + + + -
k
0.60±0.02 c-i 0.91±0.02 i-k 0.69±0.03 d-k 0.81±0.05 d-k 0.82±0.1 bc 1.13±0.04 k 0.60±0.02 c-k 0.86±0.08 fg-k 0.75±0.01 jk 0.63±0.03 c-i 0.92±0.03 c-k 0.88±0.04 c-f 0.99±0.05 c-i 0.97±0.04 c-e 1.03±0.01 g-k 0.72±0.04 d-k 0.81±0.04 g-k 0.72±0.01 cg-i 0.95±0.09 d-k 0. 80±0.05 d-j 0.80±0.03 c-g 0.98±0.01 c-e 1.04±0.02 d 0.81±0.01 -k c-j 0.89±0.05 h-k 0.70±0.07 a 1.36±0.12 d-k 0.81±0.12 k 0.60±0.01 d-k 0.81±0.02 e-k 0.76±0.04 b 1.22±0.06 h-k 0.70±0.01 b d 1.11±0.07 c-k 0.85±0.05 f-k 0.75±0.06 l 0.26±0.004 a 1.36±0.03
Numbers in the same column followed by the same letter do not differ significantly at p= 0.05 by DT; +: The character present; -: The character absent; SE: Standard error.
groups for both shoot and nodule dry matter yield and nodule numbers (Table 2). In this study, a correlation between the increase of dry matter and the number or the dry weight of
was not found to be statistically significant (p>0.05). Similarly, Maâtallah et al. (2002) has also reported that there was no positive correlation between nodules
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the increase of shoot dry matter or the number and the dry weight of nodules. Dudeja et al. (1981) demonstrated that the dry matter yield was rather correlated with the nodule leghaemogblobin concen-tration than with the number or the dry weight of nodules.
Growth characteristics and colony morphology Based on their mean generation times, 88.9% of the isolates were moderately slow growers with generation time (GT) 4.1 to 5.4 h, whereas only 11.1% of the isolate were found to be fast grower with GT 2.8 to 3.8 h (Table 1). Previous studies indicated that Mesorhizobium ciceri (Nour et al., 1994) and Mesorhizobium mediterraneum (Nour et al., 1995), which are the specific chickpea symbionts are moderately slow growing rhizobia. All isolates formed colony with circular shape, entire margin, milky-to-watery translucent to creamy and white opaque features with different level of mucus production. Most isolates (89%) exhibited copious production of mucus while the remaining isolates showed less mucus production (Table 1). The colony diameter of the isolates was ranged from 1.5 to 4.5 mm. Out of all isolates only 8% of them had colony diameter of less than 2 mm indicating that all isolates could be able to form medium to large colony on YEMA surfaces at optimum pH of growth media (6.7) and incubation temperature 28°C after 5 days of incubation (Table 1). Such characteristics are wide spread among rhizobia (Jordan, 1984). All isolates changed the color of YEMA supplemented with BTB to yellow indicating that they are acid producers (Vincent, 1970). The CR absorption test also indicated that none of the isolates absorbed CR in YEMA plates; this is distinctive character of rhizobia with only few exceptions (Somasegaran and Hoben, 1994). On the other hand, none of the tested isolates manage to grow on PGA plates. In addition, all tested isolates were KOH test positive indicating that they were Gram negative. These indicate that all isolates were not contaminant rather rhizobia (Vincent, 1970; Somasegaran and Hoben, 1994).
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among chickpea rhizobial isolates with regard to growth in relation to pH of the medium. There might be a relation between pH of origin of isolates and their acid and alkaline pH tolerance. However, in this study such kind of correlation was not statistically significant. Moreover, metal (Al and Mn) toxicity tolerance of isolates was tested at pH 5.0 and 19% of them were found to be tolerant to very low concentration of Al while about 45% of them tolerated both high and low concentrations of Mn at pH 5.0 (Table 3). Since most Ethiopian soils are acidic like any other tropical soils where associated metal toxicity problem is expected to avail such characteristics of these isolates are very desirable to use them as inoculants on acidic soils. One of the environmental stresses which pose a significant constraint to chickpea and other legume crops production in Ethiopia is soil acidity (Tilaye, 1985). It has been demonstrated that acidity limits nodulation and the nitrogen fixation (Graham, 1992); hence, rhizobia strains tolerant to acidity and associated metal toxicity might be used to improve this crop yield when inoculated on acidic soil. Temperature tolerance Temperature conditions have a great effect on rhizobial growth and symbiotic performance (Zahran, 1999). As shown in Table 3, maximum growth of all tested isolates was obtained between 20 and 30°C. Below and above these values, the percentage of isolates that grew decreased to reach 27.8% at 10°C and 42% at 35°C. None of the tested isolates could be able to tolerate and grow at 4°C while17% of them could grow at 40°C. It has been reported that some chickpea rhizobia were thermotolerant which could be able to grow at temperature of 40°C (Maâtallah et al., 2002; Küçük and Kıvanç, 2008). Increased temperature optima of these isolates may be beneficial for its application in temperature stressed conditions as symbiotic performance of different rhizobial strains under temperature stress has been correlated with their ability to grow in pure culture at elevated temperature (Hungria et al., 2000). Though correlation between climatic region of origin area of each isolates and tolerance to low or high temperature was not carried out in this study, their temperature tolerance might be related to their origin soil temperature.
Physiological characteristics Tolerance of acidic and alkaline pH As shown in Table 3, the chickpea nodulating rhizobia isolated from different Ethiopian soils exhibited a wide diversity in their different pH tolerance. All tested isolates grew well in moderately acidic pH (5.5) to neutral pH and slightly alkaline pH (8.0) (Table 3). Some isolates exhibited an acid tolerant character since 31 and 56% of them grew at pH 4.5 and 5, respectively. Similarly, some isolates showed alkaline tolerant character as 25 and 22% of the isolates grew at pH 9.5 and 10, respectively. Several studies (Nour et al., 1994; Maâtallah et al., 2002; Küçük and Kıvanç, 2008) have also indicated that chickpea rhizobia exhibit moderately acidic and alkaline pH tolerance characteristics. It is interesting to note that large variation was observed
Salt tolerance The data in Table 3 showed that chickpea nodulating rhizobia exhibited high diversity in their salt tolerance. The salt inhibitory concentrations tolerance varied among the strains. In our study, high tolerance to sodium chloride (NaCl) was observed since 75% of the tested rhizobia could grow well with 1% NaCl. However, at higher concentrations the percentage of tolerant isolate decreased with increasing salt concentration as only 11.1% of the isolates tolerated 5% NaCl. Similarly, earlier studies (Maâtallah et al., 2002; L’taief et al., 2007) observed that chickpea rhizobia also exhibited a wide variation in their salt tolerance, even among isolates from the same site. Though it is often believed that saline soils naturally select strains more tolerant to salinity, such kind of correlation was not carried out in our study.
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Table 3. Physiological characteristics of chickpea nodulating rhizobial isolates.
Isolate CR01 CR02 CR03 CR04 CR05 CR06 CR08 CR09 CR11 CR12 CR14 CR16 CR18 CR19 CR20 CR21 CR23 CR24 CR25 CR28 CR29 CR31 CR32 CR34 CR36 CR37 CR38 CR39 CR40 CR42 CR44 CR45 CR46 CR47 CR48 CR50
pH tolerated 5.0-10 4.5-8.0 5.5-10.0 5.0-10 4.5-8.0 5.0-10 5.5-8.0 4.5-8.0 5.5-8.0 5.0-9.5 4.5-9.0 5.0-9.0 4.5-8.0 4.5-8.0 5.5-8.5 4.5-8.0 4.5-8.0 4.5-8.0 5.5-10.0 5.5-8.0 4.5-8.5 4.5-8.0 4.5-9.0 5.5-8.0 5.5-8.5 5.5-8.0 5.5-8.5 4.5-8.0 5.0-10 5.5-8.5 5.0-10 4.5-8.0 5.5-8.0 5.5-8.0 5.0-10 5.5-8.0
NaCl % tolerated 1 5 3 5 5 1 0.5 1 1 2 1 4 1 2 0.5 1 0.5 1 5 0.5 2 1 1 0.5 1 1 0.5 2 3 1 1 0.5 1 1 2 0.5
Temperature tolerated 15-35 15-35 15-40 15-35 15-35 10-30 15-35 15-30 15-30 15-35 15-30 15-35 10-30 10-30 15-30 15-35 20-30 15-40 15-40 10-35 15-30 20-30 10-30 10-30 15-30 15-30 20-30 10-30 15-35 15-30 15-40 15-35 20-30 10-30 15-40 15-30
C-sources% utilized 67 56 78 93 93 82 82 56 56 56 85 59 48 52 67 56 59 52 85 63 52 82 59 67 59 59 48 56 78 63 78 56 59 82 70 37
N-sources% utilized 93 87 80 93 87 93 87 87 87 87 87 87 73 80 87 87 80 87 87 87 87 80 80 87 87 87 80 87 87 87 87 80 80 80 100 87
IAR pattern Na, Ch Na, Ch,Er Er Na, Ch,Am,St St,Ch,Ne,Nal St,Na, Ch,Am,Er Ch,St,Er Er,Na Na,Er Er, Na Na,Er,St Na,Er,Ne Na,Er Na, Er Na, Er,Ch Na, Er Ch, Na, Er Ch, Er Na,Er Er,Na,Ch Na, Er Na,Er Na,Er Er Na,Er Na,Er Na,Er Na,Er Na,Er Na,Er Na,Er Na -
Heavy metal resistance Mn, Cr,Zn Mn,Cr Mn,Al,Cr,Zn Mn,Cr Mn,Al,Cr,Zn Cr Cr,Mn Cr Mn, Cr,Pb Mn,Cr, Zn Mn,Cr,Zn Mn,Cr Mn,Cr Cr Pb,Mn,Cr,Zn Mn, Cr Cr Al,Mn,Cr Cr Mn,Cr Cr Cr Mn,Al,Cr Cr Mn, Cr Pb Mn,Al,Cr,Zn -
Am: Ampicillin; Na: Naldixic acid; Er: Erythromycin; Ch: Chloramphenicol; St: Streptomycin; Ne: Neomycin; -: Absent.
Intrinsic antibiotics and heavy metals resistance The evaluation of intrinsic resistance to antibiotics of chickpea rhizobia showed that 47.2 and 75% of the tested isolates exhibited high resistance to nalidixic acid and erythromycin, respectively (Table 3). In the presence of ampicillin, Chloroamphincol, neomycin or streptomycin only 5.6 to 30.6% (according to antibiotic and their concentrations) of isolates
were resistant. All tested isolates were found to be sensitive to low concentration of tetracycline. Several studies (Maâtallah et al., 2002; Küçük and Kıvanç, 2008) also observed great variation among chickpea rhizobia with respect to their intrinsic antibiotics resistance pattern (IAR). The IAR can be used for the identification of rhizobial strains that occupy nodules in studies designed to evaluate the ecological competitiveness (Kremer and Peterson, 1982). In addition, the pattern of antibiotics
Jida and Assefa
resistance has been used to identify diversity among strains of rhizobia (Somasegaran and Hoben, 1994). Consequently, it could be used as supplementary diagnostic character for different rhizobial strains (Amarger et al., 1997).
A comparable pattern was also observed with heavy metals resistance (Table 3). More than 16.7 and 69.4% of isolates showed good tolerance to Zinc and Chromium, respectively. Only 11% of the isolates were found to be resistant to Lead. None of the isolates exhibited an intrinsic resistance to the remaining heavy metals indicating that they are highly inhibitory to the isolates. Previous studies also reported that some Mesorhizobium species were found to be tolerant to few heavy metals (Maâtallah et al., 2002; Küçük and Kıvanç, 2008). Though metals concentrations in soils origin of the isolates were not determined, their metal toxicity tole-rance might be related to their adaptation at their soil of isolation sites. In fact, this requires further investigation. The heavy metal resistance traits of the rhizobial isolates would be used as invaluable positive markers during genetic studies (Küçük and Kıvanç, 2008). The high level of Zinc and Chromium resistance suggest that these metals could be used as selective agents for some Mesorhizobium strains (Sinclair and Eaglesham, 1984; Küçük and Kıvanç, 2008).
C and N-sources utilization Most of the chickpea rhizobia strains were able to catabolize a large variety of carbon substrates (Table 3). All tested strains grew on D-glucose, D-mannitol, galactose, maltose, lactose, raffinose, cellobiose and sucrose. All tested isolates were unable to utilize citrate as sole source of carbon. Graham and Parker (1964) found that utilization of citrate as sole sources of carbon was restricted to slow-growing bradyrhizobia. As reported by earlier studies (Graham and Parker, 1964; Sadowsky et al., 1983), fast-growing rhizobia were able to grow on a large variety of carbon substrates whereas slow-growing rhizobia were more limited in their ability to use diverse carbon sources. However, our result shows that the majority of tested slow-growing chickpea rhizobia were able to use a broad range of carbohydrates. This is in line with the result of other studies (Nour et al., 1994; Matalah et al., 2002; L’taief et al., 2007). It is very interesting to notice that the types of carbohydrates utilized also varied among chickpea rhizobia. Such characteristics are usually used as diagnostic features for root nodule bacteria (Küçük and Kıvanç, 2008; Hungria et al., 2001). Chickpea rhizobia also exhibited diversity in utilizing different amino acids and vitamins as sole N-sources (Table 3). Methionine, tyrosine, thiamine and riboflavin were utilized by all isolates whereas niacin was least preferred N-source by most isolates. Except pheny-lalanine and glycine, most isolates metabolized the remaining N-sources tested. This is in line with the previous studies (Amarger et al., 1997; Küçük and Kıvanç, 2008). The ability of isolates to utilize wide range of N-sources would give more ecological competence in the soil and it is also one of the desirable characteristics for isolates selected for
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field studies. Furthermore, utilization of different compounds by strains, as nitrogen sources is one of the most useful traits for their differentiation (Hungria et al., 2001).
PGP properties of the isolates Chickpea nodulating rhizoia showed PGP characteristics such as auxin production, inorganic phosphate solubilization, and F. oxysporum inhibition under in vitro conditions (Table 2). Out of all tested isolates 27.8% of them were found to be auxin producer while only 44.4% were insoluble inorganic phosphate solubilizer with solubilization index ranging from 1.3 to 1.12. Whether present in the rhizosphere as molecules from bacterial saprophytes or present in plant tissues as products released by endophytes, auxin can massively proliferate root hair production (Yanni et al., 2001) and thus enhance the root’s absorptive capacity and nutrient uptake of the crops (Dakora, 2003). Besides, inoculation of plants with phosphate solubilizing bacteria often stimulates plant growth by increasing phosphorus uptake (Chabot et al., 1996). Thus, this ability makes them more important as multi-purpose inoculants for this crop production (Jida and Assefa, 2011). Furthermore, 19.4% tested chickpea nodulating rhizobia were found to inhibit the growth of F. oxysporum in dual culture assay. It has also been documented that chickpea rhizobial strains showed a strong inhibitory effect against other fungal pathogen of chickpea such as Ascocyhta rabiei (Küçük and Kıvanç, 2008) and Rhizoctonia solani (Hemissi et al., 2011) under in vitro conditions. Consequently, these strains could be used as biocontrol agents against root rot and wilt causing fungal pathogens of chickpea. Nevertheless, further investigations need to be carried out on the mechanism of inhibition and evaluation of their activity under glasshouse and field conditions.
Several studies showed that different strains of Mesorhizobium species are endowed with PGP characteristics (Antoun et al., 1998; Alikhani and Yakhchali, 2009; Etesami et al., 2009; Hemissi et al., 2011). When there is no legume host rhizobia enjoy saprophytic life. They become attracted to the roots of non-legume crops and nourish root exudates in the rhizosphere (Dowling and Broughton, 1986). Consequently, rhizobia which have PGP characteristics would increase the yield of non-legume crops which could be grown in rotation or mixed cropping with legumes (Antoun et al., 1998; Alikhani and Yakhchali, 2009; Etesami et al., 2009). Thus, root colonization pattern and PGP activity of such isolates when inoculated to wheat, tef and barley which are commonly grown in rotation with chickpea under Ethiopian conditions needs to be investigated. Based on the result of our study the following isolates CR06, CR18, CR19, CR20, CR31, CR32, CR38, CR45 and CR47 are highly recommended for field test and
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ecological competitiveness studies under different Ethiopian soil and climatic conditions (Table 3), since these isolates have exhibited interesting features such as wide range of C and N-sources utilization, tolerance of acidic pH, metal toxicity and antibiotics. In addition, they exhibited PGP features such as auxin production, F. oxysporum growth inhibition, inorganic phosphate solu-bilization, and highly effective nitrogen fixation. In general, from the present study, it can be concluded that Ethiopian soils harbors highly efficient nitrogen-fixing chickpea nodulating rhizobia which are diverse in their morphological, physiological, symbiotic and PGP characteristics. In addition to Mesorhizobium ciceri (Nour et al., 1994) and M. mediterraneum (Nour et al., 1995) some bacterial species have been described to nodulate chickpea with different symbiotic efficiency (Laranjo et al., 2004; Rivas et al., 2007). However, during this study methods used for characterizing and distinguishing rhizobial strains were morphological, physiological and symbiotic. These traditional methods of rhizobial characterization frequently fail to identify strains to a species level. Hence, this study should be corroborated by polymerase chain reaction (PCR) based molecular methods such as restriction fragment length polymorphism (RFLP), rapid amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and 16S rRNA sequence analysis to evaluate their genotypic diversity and identify the isolates to the species level.
ACKNOWLEDEGEMENTS The authors are very much grateful to Addis Ababa University office of Research and Graduate Studies for financial support, Dr. Negussie Tadesse, Dr. Million Eshete and Ato Erese, EARO, Debra Zeit for gently providing chickpea seeds and F. oxysporum strains and Ato Tesfaye Muluneh, Wollega University for preparation of sample sites map. REFERENCES Ahmed S, Melkamu A (2006). Chickpea, lentil, grass pea, fenugreek and lupine disease research in Ethiopia. . In: kemal et al (eds) Food and forage legumes of Ethiopia progress and prospects. ICARDA, Addis Ababa, Ethiopia, pp. 215-220. Alikhani HA, Yakhchali B (2009). Potential of Iranian rhizobial strains as plant growth-promoting rhizobacteria (PGPR) and effects of selected strains on growth characteristics wheat, corn, and alfalfa. Desert, 14: 27-35. Amarger N, Macheret V, Laguerre G (1997). Rhizobium gallicum sp. Nov. and Rhizobium giardinii sp. Nov. from Phaseolus vulgaris. Int. Syst. Bactriol. 47: 996-1006. Anbessa Y, Bejiga G (2002). Evaluation of Ethiopian chickpea landraces for tolerance to drought. Genet. Resour. Crop Evol. 49: 557-564. Antoun H, Beauchamp CJ, Goussard N, Chabot R, Lalnde R (1998). Potential of Rhizobium and Bradyrhizobium species as plant growth promoting rhizobacteria on non-legumes: effect on radishes (Raphanus sativus L.). Plant Soil, 204: 57-67. Bejiga G, Daba K (2006). Breeding chickpea for wide adaptation.
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Environ. Microbiol. 43: 636-642. Küçük C, Kıvanç M (2008). Preliminary characterization of Rhizobium strains isolated from chickpea nodules. Afr. J. Biotechnol. 7: 772-775 L’taief B, Sifi B, Gtari M, Zaman-Allah M, Lachaal M (2007). Phenotypic and molecular characterization of chickpea rhizobia isolated from different areas of Tunisia. Can. J. Microbiol. 53: 427-434. Landa BB, Hervas A, Bethiol W, Jimenez-Diaz RM (1997). Antagonistic activity of bacteria from the chickpea rhizosphere against Fusarium oxysporum f.sp.ciceris. Phytoparasitica, 25: 305-318. Laranjo M, Machado J, Young JPW, Oliveira S (2004). High diversity of chickpea Mesorhizobium species isolated in a Portuguese agricultural region. FEMS Microbiol. Ecol. 48: 101-107. Maâtallah J, Berraho EB, Sanjuan J, Lluch C (2002). Phenotypic characterization of rhizobia isolated from chickpea (Cicer arietinum) growing in Moroccan soils. Agronomie, 22: 321-329. Nour SM, Cleyet-Maret JC, Normand P, Fernandez MP (1995). Genomic heterogeneity of strains nodulating chikpea (Cicer arietinum L.) and description of Rhizobium mediterraneum sp. nov. Int. J. Syst. Bacteriol. 45: 640-648. Nour SM, Fernandez MP, Normand P, Cleyet-Maret JC (1994). Rhizobium ciceri sp. nov. consisting of strains that nodulate chikpea (Cicer arietinum L). Int. J. Syst. Bacteriol. 44: 511-522. Peix A, Rivas-Boyero AA, Mateos PF, Rodriguez-Barrueco C, Martõanez-Molina E, Velazquez E (2001). Growth promotion of chickpea and barley by a phosphate solubilizing strain of Mesorhizobium mediterraneum under growth chamber conditions. Soil Biol. Biochem. 33: 103-110. Pikovskaya RI (1948). Mobilization of phosphorus in soil in connection with the vital activity of some microbial species. Mikrobiologiya, 17: 362-370. Rivas R, Laranjo M, Mateos PF, Oliveira S, Martinez-Molina E, Velazquez E (2007). Strains of Mesorhizobium amorphae and Mesorhizobium tianshanense, carrying symbiotic genes of common chickpea endosymbiotic species, constitute a novel biovar (ciceri) capable of nodulating Cicer arietinum. Lett. Appl. Microbiol. 44: 412418. Romdhane SB, Aouani ME, Trabelsi M, De Lajudie P, Mhamdi R (2009). Selection of High Nitrogen-Fixing Rhizobia Nodulating Chickpea (Cicer arietinum) for Semi-Arid Tunisia. J. Agron. Crop. Sci. 194: 413-420.
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Sadowsky MJ, Keyser HH, Bohlool BB (1983). Biochemical Characterization of fast- and slow-growing rhizobia that nodulate Soybeans. Int. J. Syst. Bacteriol. 33: 716-722. Shiferaw B, Teklewold H (2007). Structure and functioning of chickpea markets in Ethiopia: Evidence based on analyses of value chains linking smallholders and markets. Improving Productivity and Market Success (IPMS) of Ethiopian Farmers Project Working Paper 6. ILRI (International Livestock Research Institute), Nairobi, Kenya. p. 63. Sinclair MJ, Eaglesham ARJ (1984). Intrinsic antibiotic resistance in relation to colony morphology in three populations of West African cowpea rhizobia. Soil Biol. Biochem. 16: 247-251. Somasegaran P, Hoben HJ (1994). Handbook for Rhizobia methods in Legume-Rhizobium Technology, springer Verlag, New York, USA. Tilaye A (1985). Faba bean in Ethiopia. FABIS News letter, 12: 3-4. Vincent JM (1970). A manual for the practical study of root-nodule bacteria, IBP handbook 15. Blackwell Scientific Publications, Oxford. Werner D (2005). Production and biological nitrogen fixation of tropical legumes. In: Werner D and Newton W E. (eds) Nitrogen Fixation in Agriculture, Forestry, Ecology, and the Environment, Springer, Netherlands, pp. 1-13. Yanni YG, Rizk RY, El-Fattah FKA, Squartini A, Corich V, Giacomini A, De Bruijn F, Rademaker J, Maya-Flores J, Ostrom P, VegaHernandez M, Hollingsworth RI, Eustoquio M , Mateos P, Velazquez E, Wopereis J, Triplett E, Umali-Garcia M, Anarna JA, Rolfe BG, Ladha JK, Hill J, Mujoo R, Ng PK, Dazzo FB (2001). The beneficial plant growth-promoting association of Rhizobium leguminosarum bv. trifolii with rice roots. Aust. J. Plant Physiol. 28: 845-870. Zahran HH (1999). Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. Microbiol. Mol. Biol. Rev. 63: 968-989.
African Journal of Biotechnology Vol. 11(29), pp. 7494-7499, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.3864 ISSN 1684â&#x20AC;&#x201C;5315 Š 2012 Academic Journals
Full Length Research Paper
Isolation and screening of lactic acid bacteria, Lactococcus lactis from Clarias gariepinus (African catfish) with potential use as probiotic in aquaculture Tengku Haziyamin Tengku Abdul Hamid*, Ahmed Jalal Khan, Muhammad Fauzi Jalil and Nur Shazana Azhar Department of Biotechnology, Kulliyyah of Science, International Islamic University Malaysia, Kuantan Campus, Jalan Istana, Bandar Indera Mahkota, 25200, Kuantan, Pahang, Malaysia. Accepted 27 January, 2012
In aquaculture probiotic feeding could play a crucial role in developing microbial control strategies, since disease outbreaks are recognized as important constraints to aquaculture production and the fear of antibiotic resistance. In this study, lactic acid bacteria (LAB) strains from the intestinal tissue of African catfish Clarias griepinus were successfully screened, characterized and identified. The isolates (S1#8, S1#9, S1#10, S1#18, S1#19 S1#20 and S1#21) shared common morphological characteristics of LAB such as non-sporulating, Gram positive cocci or cocco-bacilli shape. These were the dominating morphology found in the catfish, as compared to other types of LABs reported to be found in freshwater fishes. All of the 35 isolates have the ability to utilize lactose as part of their metabolism process and showed negative reactions towards catalase test. These isolates were also tested for antimicrobial activities using disc diffusion assay against indicator Salmonella typhimurium and Escherichia coli. Based on the partial 16S rRNA sequences, the selected LAB isolates belonged to a member of Lactococcus lactis with 98% DNA similarity. This strain can be used as probiotic in aquaculture feeding. Key words: Clarias gariepinus, Lactococcus lactis, lactic acid bacteria (LAB), disc diffusion, bacteriocins.
INTRODUCTION Research on lactic acid bacteria (LAB) has advanced greatly since the last decade due to its important roles in many diverse areas of food biotechnology, nutrition, health and safety. LABs are regarded as the major group of probiotic bacteria (Collins et al., 1998; Nousiainen and Setala, 1993). The use of probiotics in aquaculture is rather recent. Aquaculture is one of the fastest growing sectors in global food production; and Asia presently contributes to almost 90% of the total production (Rana, 1997). The African catfish (Clarias gariepinus) originating from Africa has been introduced and commercially
*Corresponding author. E-mail: haziyamin@iium.edu.my. Tel: +609-571-6400. Ext: 2834. Fax: +609-571-5781. Abbreviation: LAB, Lactic acid bacteria.
cultured in Europe, South America and South East Asian countries (Marimuthu et al., 2010; Pillay and Kutty, 2005). It is an attractive freshwater fish species in Malaysia and other places due to its resistance to diseases and ability to grow at fast rate (Marimuthu et al., 2010). However, in Asia-Pacific region, aquaculture based disease outbreaks are the main productivity constraint that indirectly have impact on the socio-economy and sustainability of the producing community. Fish diseases are the major problem in fish farming industry, and bacterial infections are considered to be the major cause of mortality in fish (Gomez-Gil et al., 2000). In recent decade, prevention and control of the diseases have led to a substantial increase in the use of therapeutic medicines such as antibiotic. However, the use of antimicrobial agents as preventive measure posts significant risks since the evolution of antimicrobial resistance among pathogenic
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bacteria (Sahu et al., 2008). Until recently, it has not been generally accepted that lactic acid bacteria are part of the native microbial flora in fish intestines (Pilet et al., 1995). Thus, supplementation of such bacteria in fish feed was regarded as futile. Nevertheless, LAB isolated from fishes inhibited the growth of fish pathogens such as Vibrio anguillarum and Aeromonas salmonicida (Gildberg et al., 1995). There-fore, increased understanding of probiotic use of LAB would lead to the development of natural antibiotic and reduce the dependency on chemical or drug uses in aquaculture (Subasinghe, 1997). The progress made in genetic and biochemical analyses of LAB is exploited to improve the aquaculture products so that it will be more acceptable to the consumers. Obviously, there is a need to search for LAB strain suitable for use as probiotic in aquaculture. The main strategy in the use of probiotics is to isolate intestinal bacteria with favourable properties from mature animals and incorporate these bacteria into the feeding of young and growing animals. This is due to the fact that these bacteria often produce bacteriocins and other chemical compounds that could inhibit the growth of pathogenic bacteria. Therefore, this project aims to screen and identify potential LAB from C. gariepinus, an African catfish in order to exploit them for further uses in aquaculture feeding. MATERIALS AND METHODS Digestive tracts from two catfishes (labeled as samples 1 and 2) were sterilely dissected and washed. The fish intestines were cut, weighed and homogenized in Man Rogossa Sharp (MRS) agar and vortexed. These were serially diluted (from 10 -1 to 10-6 for two samples, respectively) and were pour plated on MRS agar. The plates were incubated anaerobically, at 30°C for 48 h. Single colonies developed were picked up for morphological, lactose utilization, gram reaction, catalase ad antimicrobial test, and further, genomic extraction for rRNA sequencing. In lactose utilization test, the selected colonies were then streaked onto nutrient agar NA with the addition of lactose containing 0.005 g/L bromo-cresol purple, as a pH indicator dye. The plates were incubated at 30°C for 24 h. Isolates that were able to utilize lactose and produce acid were differentiated by the change of media color from violet to yellow. Catalase activity was tested by adding a drop of 30% hydrogen peroxide solution onto the cell smears. Positive reaction would be seen as bubbles or froths generated from the colonies, indicating a rapid production of oxygen gas. Only isolates that showed negative reaction were subjected to further identification test. The antagonistic activity of the isolated LAB against Salmonella typhimurium and Escherichia coli were determined by using agar disc diffusion. LAB isolate was propagated in MRS broth medium and incubated anaerobically at 30°C for 48 h. Cell propagated in MRS broth was pipette onto 5 mm diameter filter paper disc (diameter of 5 mm, Whatman No.1) and dried for 10 min. About 500 µl of indicator organism, (either S. typhimurium or E. coli) was spread on the plates. The test was performed in triplicates. Then, the plates were incubated at 37°C for 24 h and the zone of inhibitions formed surrounding the disc was observed. Following overnight incubation at 30°C in 10 ml MRS broth, genomic extraction was carried out using DNeasy Blood and Tissue
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kit according the manufacturer protocol (QIAgen, USA). A pair of 16S rRNA primer was designed and synthesized based on the published 16S rRNA sequence of LAB (Edwards et al., 1989). A forward primer (pA 5’- AGA GTT TGA TCC TGG CTC AG -3’) and a reverse primer (pE 5’- CCG TCA ATT CCT TTG AGT TT -3’) were purchased from 1st Base (Malaysia) Sdn. Bhd. The amplifications were performed with initial denaturation at 94°C for 4 min, and 29 cycles of denaturation at 94°C for 2 min; annealing at 55°C for 1 min and extension at 72°C. All DNA templates used were approximately 5 ng DNA. Finally, the amplified polymerase chain reaction (PCR) products were analysed in a 1.0 (w/v) % agarose gel in 1x TAE buffer at 90 V for 65 min; and gels were visualized by using gel documentation system (Alpha Imager). The amplified DNA fragments were purified and sent to 1st Base for automated DNA sequencing (1st Base Malaysia, Sdn Bhd). The homology of the sequences were analyzed using the BLASTN programs and compared with the Genbank database at National Center for Biotechnology Information (NCBI) accessible on-line at http://www.ncbi.nlm.nih.gov/ Genbank/
RESULTS AND DISCUSSION About 60 isolates were selected for further lactic acid bacteria identification. Out of the 60 isolates, 11 isolates from sample 1 and 19 isolates from sample 2 were found to be positive in lactose utilization test and these isolates were mostly identified to be Gram positive cocci or coccobacilli (Figures 1b and c). In lactose utilization test, most isolates were able to convert lactose into lactic acid indicated by changes in the pH of the media color from purple to yellow (Figure 1a). All isolates showed negative catalase test (Table 1) due to the absence of catalase enzyme and therefore unable to decompose H2O2. By disc diffusion assay, a few of the isolated LAB bacteria demonstrated a clear bacteriocidal effect against Gram negative pathogens S. typhimurium and E. coli (Table 1). Only isolates S1#1, S2#8 and S2#25 showed inhibitory zones of at least 10 mm diameter on S. typhimurium. Isolates S1#9, S1#10, S1#18, S1#19 and S1#20 showed inhibition of at least 10 mm size on E. coli indicator bacteria (Figure 2). The inhibitory action of LAB is mainly due to accumulation of main primary meta-bolites such as lactic and acetic acids, ethanol, carbon dioxide; or antimicrobial compounds such as formic, benzoic and acids, hydrogen peroxide, diacetyl, acetoin and bacteriocins. LAB has also shown to possess inhibitory activities due to the bactericidal effect of protease sensitive bacteriocins (Jack et al., 1995). By producing these compounds, probiotic microorganisms could survive the adverse condition in gastrointestinal tract (El-Naggar, 2004). Further test using the super-natant solutions, isolates S1#8, S1#9, S1#10, S1#18, S1#19 and S1#20 gave inhibition of variable in size (between 5.6 to 10.6 mm) on S. typhimurium. Super-natant from isolates S1#8, S1# 9, S1# 10 and S1#19 gave inhibition of sizes (between 8.0 to 9.3 mm) on E. coli. These results are also shown in Table 3. From Figures 3a and b, bands of 1.5 kb in size corres-
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a)
b)
c)
Figure 1. a) Colonies producing lactic acid lowered the pH causing the purple colour to change to yellow in nutrient agar media containing lactose and bromo-cresol, a pH indicating dye. b) Gram staining observation under light microscope (Nikon, 1000x magnification) showed that all 30 isolates were Gram-positve cocci and c) were in cocco-bacilli shape.
Table 1. Gram staining characteristics of the examined LAB. Isolate
Gram Stain
Shape
S1 #8 S1 #9 S1 #10 S1 #12 S1 #16 S1 #18 S1 #19 S1 #20 S1 #21 S1 #22 S1 #23 S2 #1 S2 #2 S2 #3 S2 #4 S2 #5 S2 #6 S2 #7 S2 #8 S2 #9 S2 #10 S2 #11 S2 #12 S2 #17 S2 #20 S2 #21 S2 #22 S2 #23 S2 #24 S2 #25
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
Coccus Coccobacillus Coccus Coccus Coccus Coccus Coccus Coccus Coccus Coccus Coccus Coccus Coccus Coccus Coccus Coccus Coccobacillus Coccus Coccobacillus Coccus Coccus Coccus Coccus Coccus Coccus Coccus Coccus Coccus Coccus Coccus
*â&#x20AC;&#x2DC;+â&#x20AC;&#x2122; Indicates at least 10 mm diameter inhibition.
Catalase (+) or (-)
Lactose utilisation
-
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
Antagonist test* S. thyphi E. coli + + ++ + + + + -
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Figure 2. Positive antagonistic effect of LAB colonies against S. typhimurium and killing of the bacterial cells. Arrow shows zone of inhibition. Table 2. NCBI blast results with L. lactis strain in Genebank database that show high percentage homology with rRNA sequences of LAB strain from catfish.
No. S1#8 S1#9 S1#10 S1#18 S1#19 S1#21 S2# 8 S2#25
Highest hit Lactococcus lactis subsp. lactis Lactoc Lactococcus lactis subsp. lactis Lactococcus lactis subsp. lactis Lactococcus lactis subsp. lactis Lactococcus lactis subsp. lactis Lactococcus lactis subsp. lactis Lactococcus lactis subsp. lactis Lactococcus lactis subsp. lactis
ponding to the expected size of the 16S rRNA genes were amplified. While the DNA sequences from the other strains failed to be amplified, partial ribosomal DNA sequences of S1#8, S1#9, S1#10, S1#18, S1#19 S1#20 and S1#21 showed high homology (>97%) as compared to the 16S rRNA sequences from Genebank database. Table 2 summarizes these results and all isolates show the highest homology with Lactococcus lactis. These results are in accordance with the results obtained in the morphological and biochemical tests carried out (Table 1). However, report on the presence of Lactoccoccus sp. in freshwater fishes has been very few (Maugin and Novel, 1994, Nair and Surendran, 2005). Unlike Carnobacterium or Vagococcus, the species from other LAB groups are considered to be uncommon in aquatic environments (Stiles and Holzapfel, 1997; Ringø and Gatesoupe, 1998). Even though the presence
E Value 0 0 1e-132 0 5e-172 0 0 0
Percentage homology (%) 99 99 99 98 100 98 97 99
of LAB in Lactobacillus or lactococcus family in freshwater fishes is rather ‘atypical’, there were reports on the presences of this type of LAB in freshwater fishes and prawns. Among LAB, L. lactis represents one of the important species, widely known for the production of nisin. Nisin is a ribosomally synthesized antimicrobial peptide used for the preservation of canned foods and dairy products (Delves-Broughton et al., 1996). Inhibitory
activities (Table 3) from the supernatant of the LAB strains toward the two indicator strains could have originated from the soluble protein, however further confirmation were required to confirm its presence. Since the streptococcal infection has also become a problem in fishes, the L. lactis can be a good candidate for antagonistic strain for streptococcal infection.
Studies were carried out to enhance the use of L. lactis as a probiotic in aquaculture. For instance, the presence of L. lactis were shown to promote the growth rate of rotifers
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a 1
2
b 3
M
M
1
2
3
4
5
6
Figure 3. a) Lane M, Gene Ruler™ DNA Ladder Mix (Fermentas); lane 1, 5 µl of PCR amplified product from genomic DNA for S1#21; lane 2, S2 #8; lane 3, S2#25. b, Lane M, Gene Ruler™ DNA Ladder Mix (Fermentas); lane 1, 5 µl of PCR amplified product from genomic DNA for S1#8; lane 2, S1# 9; lane 3, S1#10; lane 4, S1#18; lane 5, S1#19; lane 6, S1#21. The control sample showed no amplification (result not shown).
Table 3. Inhibitory activities of supernatant from LAB strain against S. typhimurium and E. coli.
Cell free supernatant sample S1# 9 S1#10 S1#18 S1#19 S1#21 S2#25
Salmonella typhimurium 10.6 5.6 7.6 9.4 9.3 8.0
Escherichia coli 9.3 8.0 7.6 8.0 -
* in +/- 0.1 mm
(Harzevili, 1998; Planas et al., 2004) and inhibit the fish pathogen, Aeromonas hydrphila in tilapia (Zhou et al., 2010). Conclusion From this study, LAB strains from the intestinal tissue of African catfish Clarias garepinus were successfully screened, characterized and identified. These isolates shared common morphological characteristics of LAB such as non-sporulating, Gram positive cocci or coccobacilli shape. These were the dominating morphologies found in the African catfish, as compared to other reported LABs commonly found in freshwater fishes. All the 35 isolates have the ability to utilize lactose as part of their metabolism process and showed negative reactions towards catalase test. Some of these strains have the capability to inhibit the pathogenic strains S. typhimurium and E. coli which are common human pathogen. Based
on partial 16S rRNA sequences, the majority of the identified species (S1#9, S1#10, S1#18, S1#19 S2#8 and S1#21) belonged to the family L. lactis, a common strain in food industries which is however rare in aquaculture. Based on this features, this isolate could potentially be a useful probiotic strain for African catfish feeding or for other related formulation in aquaculture feeding. REFERENCES Collins JK, Thornton G, Sullivan GO (1998). Selection of probiotic strains for human applications. Int. J. Syst. Bacteriol. 8: 487-490. Delves-Broughton J, Blackburn P, Evans R J, Hugenholtz J (1996). Applications of the bacteriocin, nisin. A Van Leeuw J. Microb. 69: 193-202. Edwards U, Rogall T, Blocker H, Emde M, Bottger EC (1989). Isolation and direct complete nucleotide determination of entire genes. Nucleic Acid Res. 17: 7843-7853. El-Naggar MYM (2004). Comparative study of probiotic culture to control the growth of Escherichia Coli O157: H7 and Salmonella Typhimurium. Biotechnology, 3: 173-180. Gildberg A, Johansen A, Bøgwald J (1995). Growth and survival of
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Atlantic salmon (Salmo salar) fry given diets supplemented with fish protein hydrolysate and lactic acid bacteria during a challenge trial with Aeromonas salmonicida. Aquaculture, 138(1-4): 23-34. Gomez-Gil B, Roque A, Turnbull JF (2000). The use and selection of probiotic bacteria for use in the culture of larval aquatic organisms. Aquaculture, 19: 259-270. Harzevili ARS, Van Duffel H, Dhert P, Swings J, Sorgeloos P (1998). Use of a potential probiotic Lactococcus lactis AR21 strain for the enhancement of growth in the rotifer Brachionus plicatilis (Muller). Aquacult. Res. 29(6): 411-417. Pillay TVR, Kutty MN (2005). Aquaculture: Principles and Practices. Blackwell. pp. 373-384. Jack RW, Tagg JR, Ray B (1995). Bacteriocin of Gram-Positive Bacteria. Microbiol. Rev. 59(2): 171-200. Marimuthu K, Cheen AC, Muralikrishnan S, Kumar D (2010). Effect of Different Feeding Frequency on the Growth and Survival of African Catfish (Clarias Gariepinus) Fingerlings. Adv. Environ. Biol. 4(2): 187193. Maugin S, Novel G (1994). Characterization of lactic acid bacteria isolated from seafood. J. Appl. Bacteriol. 76: 616-625. Nair PS, Surendran PK (2005). Biochemical Characterization of Lactic Acid Bacteria Isolated From Fish And Prawn, J. Cult. Collect. 4: 4852. Nousiainen J, Setala J (1993). Lactic acid bacteria as animal probiotics. In Salminen, S. and von Wright A (eds), Lactic Acid Bacteria. Marcel Dekker, New York. pp. 315-356. Pilet MF, Dousset X, Barr´e R, Novel G, Desmazeaud M, Piard JC (1995). Evidence for two bacteriocins produced by Carnobacterium piscicola and Carnobacterium diveregens isolated from fish and active against Listeria monocytogenes. J. Food Prot. 58: 256-262.
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Planas M, Vázquez JA, Marqués J, Pérez-Lomba R, González MP, Murado M (2004). Enhancement of rotifer (Brachionus plicatilis) growth by using terrestrial lactic acid bacteria. Aquaculture, 240: 313329. Rana KJ (1997). Review of the State of the World Aquaculture. FAO Fisheries circular Rome, Italy: Food and Agriculture Organization of the United Nations; 1997. Status of global production and production trends. 886: 3-16. Ringø E, Gatesoupe FJ (1998). Lactic acid bacteria in fish: a review. Aquaculture, 160: 177-203 Sahu MK, Swarnakumar NS, Sivakumar K, Thangaradjou T, Kannan L (2008). Probiotics in aquaculture: importance and future perspectives. Indian J. Microbiol. 48: 299-308. Stiles ME, Holzapfel WH (1997). Lactic acid bacteria of foods and their current taxonomy. Int. J. Food Microbiol. 36: 1-29. Subasinghe R (1997). Fish health and quarantine, In Review of the State of the World Aquaculture. FAO Fisheries circular Food and Agriculture Organization of the United Nations, Rome, Italy. 886: 4549. Zhou X, Wang Y, Yao J, Li W (2010). Inhibition ability of probiotic, Lactococcus lactis, against A. hydrophila and study of its immunostimulatory effect in tilapia (Oreochromis niloticus). Int. J. Eng. Sci. Technol. 2(7): 73-80.
African Journal of Biotechnology Vol. 11(29), pp. 7500-7511, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.1378 ISSN 1684â&#x20AC;&#x201C;5315 Š 2012 Academic Journals
Full Length Research Paper
Bioactive potential of symbiotic bacteria and fungi from marine sponges V. Vasanthabharathi* and S. Jayalakshmi Faculty of Marine Sciences, Annamalai University, Parangipettai 608502, Tamil Nadu, India. Accepted 1 September, 2011
Marine sponges are rich in microbial biota. In this study, totally four sponges namely Callyspongia diffusa, Hyattella Cribriformis, Sigmadocia carnosa, Spongia officininalis Var ceylonensis were collected and their associated bacteria and fungi were isolated. Among the microbes isolated, Pseudomonas fluorescens and Penicillium citrinum were isolated from C. diffusa which showed broad range of activity against tested pathogens. This study demonstrates that the culturable fraction of bacteria and fungi from the sponges were diverse and appears to possess much potential as a source for the discovery of new medically relevant biological active agents. Key words: Sponges, antibacterial activity, antifungal activity.
INTRODUCTION Coral reefs are the most diverse marine ecosystems. Marine sponges are benthic animals found in the wide range of marine environments. The species diversity of sponges is superior in the tropical coral reef environments, while the sponges would be one of the most to-be-studied groups among reef fauna. Indeed, the sponges are often ignored by divers and naturalists. Encountering mobile animals, such as fish, turtles, mammals, rays, and even sharks, and looking at colorful corals draw much of their attraction, but sponges are quite interesting animals, in that the origins of their diversity in species, and the morphologies on the ecological significance as members of tropical coral reef habitats can be questioned. Many marine sponges contain dense, highly diverse microbial communities. More than 10 bacterial phyla (including Proteobacteria, Actinobacteria, Nitrospira, Chloroflexi, lanctomycetes, Cyanobacteria, Acidobacteria) have been found in sponges, as well as both major lineages of Archaea and a range of unicellular eukaryotes such as diatoms and dinoflagellates. Collectively, these organisms exhibit an enormous diversity of metabolic traits of potential use to the host, including nitrification, photosynthesis, anaerobic metabolism and secondary metabolite production.
*Corresponding author. E-mail: bharathigene@rediffmail.com.
However, in most cases the exact nature of the interactions between sponges and microbes remains a mystery, and to an individual sponge a given microorganism could represent a potential food source, a pathogen, a parasite or a symbiont. Microbial associates of sponges gained significance as source of bioactive compounds. Remarkable similarity was found between those compounds isolated predo-minantly from sponges and those found in terrestrial organism of entirely different taxa. It is hypothesized that symbiotic marine microorganism harbored by sponges are the original producers of these bioactive compounds (Proksch et al., 2002; Zhang et al., 2005). The bacterial association with marine sponges has been well known for a long time and several investi-gations have explored this association using different approaches. It has been reported that in some sponge species as much as 40% of animal biomass is attributed to bacteria, which exceeds the bacterial population of seawater by 2 orders of magnitude. Sponges are, however, not only rich in bacteria but also known to harbor fungi irrespective of the nature of sponge-fungi associations. Sponge-derived fungal cultures have repeatedly been shown to be interesting sources of new bioactive secondary metabolites pre-viously unknown from terrestrial strains of the same species. Unusual fungal metabolites include hortein, a new polyketide from the fungus Hortaea werneckii isolated from the sponge Aplysina aerophoba, new
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A- Callyspongia diffusa
C- Spongia officinalis var. ceylonensis
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B- Sigmadocia carnosa
D- Hyattella cribriformis
Figure1. Sponges from the gulf of mannar, Southeast coast of India analyzed in this study.
anthraquinone and betaenone derivatives as well as new çpyrones from the fungus Microsphaeropsis sp., also obtained from A. aerophoba, new spiciferone derivatives from the fungus Drechslera hawaiiensis derived from the sponge Callyspongia aerizusa (Jadulco et al., 2001) and new xestodecalactones produced by the fungus Penicillium, montanense, which was isolated from the sponge Xestospongia exigua (Proksch et al., 2002; Bringmann and Lang, 2003). Bioactive substances from sponge associated microorganisms have shown anticancer, antibacterial,antifungal, antiviral, antiprotozoal, anthelmintic, anti-inflammatory, imunosuppressive, neuro suppressive, and antifouling
activities. In this research, sponge associated bacteria, fungi and their anti microbial potential has been studied. MATERIALS AND METHODS Collection of samples Sponges were collected from the gulf of mannar, Southeast coast of India (Lat 9°5’ N; Long 79°5’ E).The sponge sample soon after collection was transferred to a sterile polyethylene bag and transported under frozen condition to the laboratory for the isolation of associated microbes. On reaching the laboratory, the
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Figure 2. Phylogenetic tree view of P. fluroscence- BCPBMS-1.
invertebrate was thawed and cut aseptically into small pieces (2 Ă&#x2014; 2 cm) using a sterile scalpel. The pieces were freed from adhering particles by vortexing twice for 20 seconds with 2 ml of sterile
seawater. The seawater was decanted, which was once again replaced with sterile seawater with continued vortexing between washings.
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Isolation of bacteria associated with marine sponge The sponge sample soon after collection was transferred to a sterile polyethylene bag and transported at 4°C to the laboratory for the isolation of associated microbes. On reaching the laboratory, the invertebrate was brought to room temperature and cut aseptically into small pieces (2 × 2 cm) using a sterile dissection razor. The pieces were freed from adhering particles by vortexing twice for 20 sec with 2 ml of sterile seawater. The seawater was decanted, which was once again replaced with sterile seawater with continued vortexing between washings. Finally, sample in sterile seawater was homogenized using sterilized mortar and pestle in a Laminar flow hood. The homogenate was serially diluted up to 10−6 dilutions and then spread plated on 50% Nutrient agar plates. The plates were incubated at room temperature for 24 to 48 h. The media composition is as follows: Peptone, 5.00 g; sodium chloride, 5.00 g; beef extract, 1.50 g; yeast extract, 1.50 g; agar, 15.00 g; 50% of seawater, 1000 ml; pH, 7.0 to 7.4. Colonies were selected on the basis of varying colony morphology and pure cultures were maintained in the same medium in slants at 4°C for further study.
Isolation of fungi associated with marine sponges One gram of sponge sample was mixed in 9 and 99 ml sterile water blank, respectively. This suspension was serially diluted up to 10 -4. 1 ml of the diluted sample was taken from 10-3 and 10-4 dilutions and was pour plated with 15 to 20 ml potato dextrose agar (PDA) and incubated at room temperature (28 ± 2°C) for 5 days. The PDA composition is as follows: Potato infusion, 200 g; dextrose, 20 g; agar, 15 g; 50% of seawater, 1000 ml; pH, 7.0 to 7.2. To eliminate the bacterial contamination of 8 ml of 1%, streptomycin was added to 1 L of the sterilized medium.
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tested for the antibacterial activity. Bacterial pathogens such as Escherichia coli, Proteus mirabilus, Salmonella typhi, Salmonella paratyphi, Vibrio cholera, Klebsiella oxytoca, Klebsiella pneumonia, Staphylococcus aureus, Lactobacillus vulgaris and spreaded on Muller Hinton agar plates. Then wells were made and 50 µl culture of each strain was inoculated in to separate well. Antagonistic activity was detected after an incubation of 24 to 48 h at 35°C. The presence of zone of clearance on agar plates was used as an indicator for the antibacterial activity. The strain which showed the maximum zone of clearance was chosen for further study. The presence of zone of clearance on agar plates was used as an indicator of bioactive potential of the strain (Portrait et al., 1999).
Antagonistic assay for bacterial and fungal strains against fungal pathogens Antagonistic activity of isolated bacterial and fungal strains were tested for their anti fungal activity (Geels and Schipper, 1983) against selected fungal pathogens such as Alternaria alternata, Botrytis cinerea, Cercospora theae, Fusarium udum, Fusarium xysporum, Macrophomina phaseolina , Poria hypolateritia, Phomopsis thae and Rhizoctonia solani, Initial screening for in vitro antagonistic activity was tested against fungal strains on PDA agar plates. Wells were made and 50 µl culture of each strain was inoculated in to separate well. Antagonistic activity was detected after an incubation of 24 to 48 h at 35°C. The presence of zone of clearance on agar plates was used as an indicator of bioactive nature of the strain.
Identification of bacteria All associated bacterial strains which were selected based on morphology were identified biochemically. For the most potential strain in addition to biochemical study, 16S Ribosomal ribonucleic acid rRNA partial sequencing also done. Morphological characters were observed under a phase contrast microscope and all the organisms were biochemically identified up to the species level by following bergey’s manual of determinative bacteriology (Buchanan et al., 1974).
Cultivation of bacterial isolates for screening The isolated bacteria were sub cultured on nutrient agar plates and incubated at 28 ± 2°C for two days. A loopfull of the bacterial culture was transferred in to Nutrient broth and incubated on a shaker at 30°C for 48 h. At the end of incubation period, broth cultures were used for screening.
Cultivation of fungi isolates for screening The isolated fungi were sub cultured on Potato dextrose agar plates and incubated at 28 ± 2°C for two days. A loopfull of the fungal culture from the plate was inoculated into 10 ml of potato dextrose broth prepared in sterile 50% aged sea water and incubated on a shaker at 30°C for 2 to 4 days. At the end of incubation period, broth cultures were used for screening.
Screening for antimicrobial activity Antagonistic assay for bacterial and fungi against bacterial pathogens Antagonistic assay was done by an agar-well diffusion method under aerobic conditions. Isolated bacterial and fungal strains were
Identification of bacteria by 16S rRNA partial sequencing The genomic DNA extracted from the marine sponges associated potent strain was PCR amplified for 16S rRNA genes using the universal bacterial primers Eubac 27F (5' - AGA GTT TGA TCM TGG CTC AG - 3') and 1492R (5' - GGT TAC CTT GTT ACG ACT T-3'). This primer combination amplifies a 1500 bp 16S rDNA fragment (Weisburg et al., 1991). Amplification reaction was performed in a 0.2 ml optical-grade PCR tube. 50 nanogram of DNA extract was added to a final volume of 50 µl of PCR reaction mixture containing 1.5 mM MgCl 2, 1X Reaction buffer (without MgCl2) (Fermentas), 200 µM of each dNTPs (Fermentas), 100 pM of each primer and 1.25U Taq DNA polymerase (Fermentas). PCR was performed in an automated thermal cycler (Lark Research Model L125 +, India) with an initial denaturation at 95°C for 5 min. followed by 30 cycles of 95°C for 30 s (denaturation), 52°C for 45 s (annealing), 72°C for 90 s (extension) and 72°C for 10 min (final extension). Polymerase chain reaction (PCR) product was run on 1% agarose in TAE buffer [40 mM Tris, 20 mM Acetic acid, 1mM EDTA (pH8.0)] to confirm that the right product (1500 bp) was formed. The PCR product was purified using the QIAGEN PCR purification kit for sequencing and further analysis. The partial 16S rRNA gene sequencing was done using Perkin Elmer Applied biosystems (ABI) and ABI Prism software was used to align the
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sequence and compare the sequences retrieved by the queries generated by BLAST of GenBank database. Phylogenetic analysis was performed with the MEGA 4.0 program (molecular evolutionary genetics analysis, version 4.0). The tree topologies were evaluated by bootstrap analyses based on 1,000 replicates and phylogenetic trees were inferred using the neighbour-joining method and submitted to NCBI GenBank accession number: 1428145 HQ907732.
Identification of fungi The sponge associated fungi were identified up to species level by referring standard mycological books and manuals (Gilman, 1959, 1998; Ellis, 1971, 1976; Subramanian, 1971; Kohlmeyer and Kohlmeyer, 1979; Ellis and Ellis, 1985). Fungal pathogens were gotten from CAS in botany, Madras University, Chennai.
RESULTS Density of microbes associated with sponges Bacterial density The sponges viz., C. diffusa, H. cribriformis, S. carnosa and S. officininalis Var ceylonensis were analysed for associated bacterial and fungal population. In C. diffusa, bacterial density was in the range of 7.68 × 103 to 1.1 × 107 CFU/g, whereas in the other three species, which are H. Cribriformis, S. carnosa and S. officininalis Var ceylonensis, 3 the bacterial density was found to be 13 × 103 to 1.6 × 107 CFU/g, 6.77 × 103 to 1.5 × 107 CFU/g and 2.69 × 103 to 1.4 × 07 CFU/g, respectively (Figure1). Fungal density In the sponge, C. diffusa fungal density was in the range of 1.6 × 102 to 6.1 × 103 CFU/g, whereas 1.5 × 102 to 8.2 × 103 CFU/g of fungal count was observed in H. Cribriformis. In S. carnosa, it was 1.8 × 102 to 5.0 × 103 CFU/g and in S. officininalis Var ceylonensis it varied from 1.9 × 102 to 7.0 × 103 CFU/g and 2 × 102 to 6.2 × 103 CFU/g (Figure1). Identification of potential bacterial strains strain by 16 s r – DNA sequencing In this study, phylogenetic tree revealed that P. fluroscenceBCPBMS-1 (bioactive compound producing bacteria from marine sponge) was isolated from marine sponge C. diffusa (Figure2).
Anti bacterial activity of sponge associated bacteria In this study, among the C.diffusa associated bacteria, maximum (17 mm) anti bacterial activity was observed against S. paratyphi by P. fluorescens (Table 1). Among
the H. crobriformis associated bacteria, maximum anti bacterial activity were observed with C. freundi (11mm) against P. mirabilis, N. mucosa (11 mm), S. aureus and L. plantrum (11mm), S. paratyphi with 11 mm zone of clearance, respectively (Table 2). Among S. carnosa associated bacteria, maximum activity was observed with B. marscencs against S. typhi of about 13 mm (Table 3). B. subtilis which was associated with S. officininalis Var ceylonensis showed maximum of about 13 mm activity against P. mirabilis (Table 4).
Anti fungal activity of sponge associated bacteria In this study, among the C. diffusa associated bacteria, maximum anti fungal activity was observed against P. hypolateritia (14mm) by P. fluorescens (Table 5). Among the H. crobriformis associated bacteria, maximum anti fungal activity was observed with C. frundi (15mm) against F. oxysporum (Table 6). Among S. carnosa associated bacteria, maximum activity was observed with B. marscencs against F. udum of about 13 mm (Table 7). B.subtilis which was associated with S. officininalis Var ceylonensis showed maximum of about 13 mm activity against P. thae (Table 8).
Antibacterial activity of sponge associated fungi In this study, among the C. diffusa associated fungi, maximum (18 mm) anti bacterial activity was observed against K. pnemoniae by P. citrinum (Table 9). Among the H. crobriformis associated fungi, maximum anti bacterial activity were observed with P. citrinum (11 mm) against V.cholareae (Table 10). Among S. carnosa associated bacteria, maximum activity was observed with A. niger against S. typhi of about 6 mm (Table 11). Penicilllium spp. which was associated with S. officininalis Var ceylonensis showed maximum of about 13 mm activity against S. aureus (Table 12).
Anti fungal activity of sponge associated fungi In this study, among the C. diffusa associated fungi, maximum (11 mm) anti fungal activity was observed against P. hypolateritia (14 mm) by P. citrinum (Table 13). Among the H. crobriformis associated bacteria, maximum anti fungal activity was observed with P. citrinum (12 mm) against R. solani (Table 14). Among S. carnosa associated bacteria, maximum activity was observed with A. flavus against M. phaseolina of about 6 mm (Table 15). A. niger which was associated with S. officininalis Var ceylonensis showed maximum of about 15 mm activity against M. phaseolina (Table 16).
Vasanthabharathi and Jayalakshmi
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Table 1. Antibacterial activity of C. diffusa associated bacteria.
Associated bacteria
S. aureus
S. typhi
S. paratyphi
Pathogens tested (zone of clearance in mm) E. K. P. L. K. oxytoca mirabilis vulgaris pneumoniae coli
V. cholerae
A. tumefaciens
A. faecalis
-
4
1
5
-
3
3
5
1
1
A. hydrophila
8
5
3
-
-
-
5
3
6
2
B. licheniformis B. subtilis
5 2
5 5
7 5
1 3
4 4
6 7
6 6
3 7
5 9
8
P. aeruginosa
12
11
11
7
8
4
4
-
2
12
P.fluroscence P. putida
10 10
11 6
17 6
9 8
12 9
5 -
16 5
11 6
8 6
14 8
Table 2. Anti bacterial activity of associated bacteria from H. cribriformis.
Associated bacteria
Pathogens tested (zone of clearance in mm) S. aureus
S. Typhi
S. paratyphi
K. oxytoca
E. coli
P. mirabilis
L. vulgaris
K. pneumoniae
V. cholerae
A. tumefaciens
B. cereus B. megaterium B. subtilis
5 4
5 -
4 7 6
5 3 6
3 2 5
3 2 4
3 1 -
2 -
2 2
6 5
C. freundii
4
2
2
2
1
11
6
6
-
4
N. mucosa
11
5
-
-
-
4
1
-
1
2
P. putida L. plantarum
10 2
4 -
3 11
3 5
7 4
8 -
5 -
6 9
7 -
9 -
Table 3. Anti bacterial activity of associated bacteria of S. carnosa.
Associated bacteria
Pathogens tested (zone of clearance in mm) E. K. P. L. K. oxytoca mirabilis vulgaris pneumonia coli
B. cereus B. macerans
10 4
S. Typhi 2 13
B. megaterium
8
5
3
-
B. brivis L. casei
5 6
3 3
3 9
2 5
S. marcescens
1
2
7
6
P. aeruginosa P. putida
11 7
6 2
2 3
6
S. aureus
S. paratyphi 11
5
DISCUSSION Marine bacteria have been recognized as an important and untapped resource for novel bioactive compounds. The chemical compounds of marine microorganisms are not well known as terrestrial counterparts. However, in the last decade
4 11
5 3
3 -
V. cholerae 2 3
-
-
5
3
6
3 4
1 9
1 1
1
3
1
4
-
-
-
9
7
4 6
10 -
3 3
11 10
2 7
8 2
4 -
A. tumefaciens 1 -
several bioactive compounds have been isolated from marine bacteria and are new resources for the development of medically useful compounds (Donia and Haman, 2003; Anand et al., 2006). Antibacterial activity among marine bacteria is a well-known pheno-menon and has been demonstrated in a number of studies (Isnansetyo and Kamei, 2003; Uzair et al.,
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Afr. J. Biotechnol.
Table 4. Antibacterial activity of S. officininalis Var ceylonensis associated bacteria.
Associated bacteria B. brevis B. subtilis B. megaterium V. parahaemolyticus E. coli
S. aureus 8 1 3 3 -
S. typhi 5 12 3 3 -
S. paratyphi 3 11 4 1 -
Pathogens tested (zone of clearance in mm) K. E. P. L. K. oxytoca coli mirabilis vulgaris pneumoniae 3 11 4 7
3 3 -
13 12 6
5 12 4 5 1
3 7 3 1
V. cholerae 6 5 3 -
A. tumefaciens 2 -
Table 5. Antifungal activity of C. diffusa associated bacteria.
Pathogens tested (zone of clearance in mm) Associated bactria
F. oxysporum
B. cinerea
A. alternata
R. solani
F. udam
M. phaseolina
P. hypolateritia
C. theae
P thae
A. faecalis
11
5
7
9
10
16
6
7
9
A. hydrophila B. licheniformis B. subtilis P. aeruginosa P. fluroscence P. putida
12 8 5 16 14
11 8 7 9 11 11
8 1 9 12 11 1
9 7 6 -
8 7 13 9 8
9 11 3 9 4
10 10 14 7
4 9 6 -
5 11 6 -
Table 6. Anti fungal activity of associated bacteria from H. Cribriformis.
Pathogens tested (zone of clearance in mm) Associated bacteria
B. cereus B. megaterium B. subtilis C. freundii N. mucosa P. putida L. plantarum V. cholarea
B.
F. oxysporum
cinerea
A. alternata
R. solani
F. udam
M. phaseolina
P. hypolateritia
C. theae
P. thae
12 14 15 6 -
2 11 10 11 -
7 10 10 2 8
9 7 2 7 -
5 -
11 9 7 4 -
2 3 7 3 3
3 -
8 2 -
C. theae 7
P. thae 7
Table 7. Anti fungal activity of associated bacteria from S. carnosa.
Associated bacteria B. cereus B. macerans B. megaterium
Pathogens tested (zone of clearance in mm) F. R. B. cinerea A. oxysporum alternata solani 3 4 9 7 1 1 6 -
F. udam 13 -
M. phaseolina -
P. hypolateritia 8 5 -
Vasanthabharathi and Jayalakshmi
7507
Table 7. Continued
B. brivis L. acidophilus S. marcescens P. aeruginosa P. putida
2 -
5 2 3
3 -
-
1 -
6 -
5 9 4
9 1 -
6 -
Table 8. Antifungal activity of S. officininalis Var ceylonensis associated bacteria.
Pathogens tested (zone of clearance in mm) Associated bacteria
B. brevis B. subtilis B. megaterium V. parahaemolyticus E. coli L. fermentum
B.
F. oxysporum
cinerea
A. alternata
R. solani
F. udam
M. phaseolina
P. hypolateritia
C. theae
P. thae
7 11 2 12
1 9
4 10 3 1 -
8 2 7 -
8 9 -
1 4 3 6
7 1 -
5
3 13 1
Table 9. Antibacterial activity of C. diffusa associated Fungi.
Associated fungi A. flavus A. flavipes A. niger P. citrinum Trichoderma sp.
S. aureus 1 4 8 13 3
S. typhi 1 6 2 1
S. paratyphi 1 6 7 5
Pathogens tested (zone of clearance in mm) K. E. P. L. K. pneumonia oxytoca coli mirabilis vulgaris 5 12 -
4 4 5 -
1 5 -
1 2 -
1 3 18 -
V. cholerae 2 5 -
A. tumefaciens
V. cholerae 11 3
A. tumefaciens
3 -
Table 10. Anti bacterial activity of associated fungi from H. cribriformi.
Associated fungi A. flavus A. fumigatus A. niger A. terreus P. citrinum Trichoderma sp.
S. aureus 3 5 10 -
S. typhi 1 4 7 4
S. paratyphi 3 -
Pathogens tested (zone of clearance in mm) K. E. P. L. K. oxytoca coli mirabilis vulgaris pneumoniae 3 7 5 4 4 5 2 7 1 7 3 -
2006). In this study among the associated strains of different sponges P. fluorescens and Penicillium cirtinum which were isolated
1 1 2
C. diffusa showed activity against all bacterial and fungal pathogens tested. Hence, they were selected for further study. from
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Afr. J. Biotechnol.
Table 11. Anti bacterial activity of associated fungi from S. carnosa.
Associated fungi A. niger A. flavus Fusarium sp.
S. aureus 4 -
S. typhi 6 1 -
Pathogens tested (zone of clearance in mm) S. K. E. P. L. K. pneumoniae paratyphi oxytoca coli mirabilis vulgaris 2 1 2 4 5 -
V. cholerae -
Table 12. Antibacterial of S. officininalis Var ceylonensis associated fungi.
Associated fungi A. fumigatus A. niger A. terreus Penicillium spp. T. viride
S. aureus 7 10 13 -
S. typhi 3 9 -
S. paratyphi 3 5 5
Pathogens tested (zone of clearance in mm) K. E. L. K. P. oxytoca coli mirabilis vulgaris pneumoniae 4 3 5 8 1 1 7 11 3 1
V. cholerae 2 7 -
A. tumefaciens 1 -
Table 13. Antifungal activity of C. diffusa associated fungi.
Pathogens tested (zone of clearance in mm) Associated fungi
B.
F. oxysporum
cinerea
A. alternata
A. flavus
9
8
A. niger P. citrinum Trichoderma sp.
3 4 9
8 8
R. solani
F. udam
M. phaseolina
P. hypolateritia
C. theae
P. thae
7
-
-
7
6
-
9
2 6 6
3 8 7
7 -
8 6 -
2 11 6
2 -
1 1 -
In recent years, fluorescent Pseudomonades have drawn attention worldwide because of the production of secondary metabolites such as antibiotics, enzymes and phyto hormones (Isnansetyo and Kamei, 2003). The extract of Pseudomonas sp. PB2 associated with a sponge, S. domuncula, exhibited antiangiogenic, hemo-lytic, antimicrobial, and cytotoxic activities (Thakur et al., 2001). Marine isolates of Pseudomonas spp. are found in diverse ecosystems, including coastal regions, the deep sea, and also in extreme environments like halophilic and thermophilic conditions. Marine Pseudomonas spp. were also reported in bacterioplankton in seawater, in asso-ciation with other marine organisms, and in sea sediment. The production of marine secondary meta-bolites can be viewed in an ecological context (Engel et al., 2002). Thus, the diversity of Pseudomonas isolated from a wide range of marine ecosystems suggested that these organisms may produce novel and diverse bioactive substances. Thakur et al. (2001) isolated two marine Pseudomonas sp.
(strains PB1 and PB2) from a S. domuncula sponge which exhibited anti bacterial activity. Anand et al. (2006) also screened for antibiotic-producing marine bacteria associated with sponges from the coastal waters of southeast India and isolated a bacterium, strain SC11, which was closely related to Pseudomonas based on the 16S rDNA sequences. There are only few reports of marine Pseudomonas sp. compared to terrestrial species that produce bioactive metabolites (Kaneko et al., 2000).However, some bioactive substances with novel bio-logical activities and mechanisms have been extracted from marine isolates of Pseudomonas, and some of these metabolites have antimicrobial properties. Freiberg et al. (2006) evaluated the in vivo efficiency of Moiramide B and some of its synthetic derivatives using a S. aureus sepsis model in mice. These pyrrolidinedione derivatives exhibited antibacterial activity with minimum inhibitory concentrations (MICs) of 0.01 to 8, 0.25 to 32, and 16 to 64 Âľg/ml against S. aureus 133, S. pneumoniae G9A, and E. coli, respectively. When evaluated in a murine model of S. aureus
Vasanthabharathi and Jayalakshmi
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Table 14. Anti fungal activity of associated fungi from H. cribriformis.
Pathogens tested (zone of clearance in mm) Associated fungi
B.
F. oxysporum
cinerea
A. alternata
R. solani
F. udam
M. phaseolina
P. hypolateritia
C. theae
P. thae
5 4 3 6 10
5 1 1 11 3
3 6 3 8
2 3 12 -
2 5 4
7 3 3 1 7 2
2 2 1 1 3 8 -
1 2 1 -
8 1 8 -
Acremonium sp. A. alternata A. flavus A. fumigatus A. niger A. terreus P. citrinum Trichoderma sp.
Table 16. Antifungal activity of S. officininalis Var ceylonensis associated fungi.
Pathogens tested (zone of clearance in mm) Associated fungi A. fumigatus A. niger A. terreus Penicillium spp. T. viride
B.
F. oxysporum
cinerea
A alternata
R. solani
F. udam
M. phaseolina
P. hypolateritia
C. theae
P. thae
F. oxysporum
2 2 2
1 7 1
7 11 9 7
8 10 1 -
1 9 4 3 5
15 1 8
7 9 11 11
4 2 3 -
1 1 1 6
-
sepsis, two of the moiramide B derivatives also showed in vivo activity comparable to linezolid, an antibiotic that is used currently. These reports indicate that antibiotics produced by marine isolates of P. fluroscence may be potential lead compounds in the search for new classes of antibiotics to treat bacterial infections. Pseudomonas sp. 1531-E7 isolated from a sponge, Homophymia sp., produced quinolones (2-undecyl-4-quinolone, 2-undecen-18-yl-4-quinolone, 2-nonyl-4-quinolone and 2-onyl-4hydroxyquinoline N-oxide) anti-Plasmodium falsifarum activity was exhibited by2-undecyl-4-quinolone, 2-undecen-18-yl-4quinolone, and2-nonyl-4-quinolone. Cytotoxicity to KB cells is noticedfor 2-undecen-18-yl-4-quinolone and 2-nonyl-4- hydroxyquinoline N-oxide. In addition, 2-undecyl-4-quinolonev and 2nonyl-4-hydroxyquinoline N-oxide are active against HIV-1 and S. aureus (Bultel et al., 1999). Wratten et al. (1977) isolated the antibiotic-producing Pseudomonas sp. 102-3 from a seawater sample from a La Jolla, California tide pool. The bacterium inhibits the growth of Vibrio anguillarum, V. harveyi, S. aureus, and C. albicans and produced three antibacterial compounds namely 4hydroxybenzaldehyde, 2-n-heptyl-4-quinolinol, and 2-n-pentyl-4quinolinol. Uzair et al. (2006) reported a marine Pseudomonas sp. CMG1030 which had antimicrobial activity. This orga-nism was originally identified as P. aeruginosa, but the strain identification
was revised as P. stutzeri CMG1030, which produced a novel antibacterial compound zafrin (Uzair et al., 2008). Kim et al. (2007) reported that P. fluroscence HAK-13, has algal-lytic activity against Heterosigma akashiwo
(Raphidophyceae), Alexandrium tamarense and Cochlodinium polykrikoides, but it was inactive against Gymnodinium catenatum. The substance responsible for the activity was proteinaceous compound that localizes to the cytoplasmic membrane of the bacterium. Barotolerant marine Pseudomonas sp. BT1 was isolated in deep water (4,418 m) in a Japanese ocean trench (Kaneko et al., 2000). Marine Pseudomonas strain I to 2, isolated from estuary water, had anti-Vibrio activity (Chythanya et al., 2002).The antibacterial activity was evaluated against the following shrimp pathogenic vibrios like V. harveyi, V. fluvialis, V. parahaemolyticus, V. damsel and V. vulnificus. The active substance was found to be non-proteinaceous substance that was soluble in chloroform. The chloroform extract from the bacterium was active against V. harveyi at the concentration 20 Âľg/ml, but there was no toxic effect found on shrimp larvae even up to 50 Âľg/ml. This suggested that the substance can be used to control pathogenic marine Vibrio. However, Pseudomonas sp. I to 2 was nonpathogenic to shrimp larvae, the bacterium could be used as a biocontrol agent against vibriosis in marine aquaculture. The fungal kingdom includes many species with unique
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Afr. J. Biotechnol.
and unusual biochemical pathways (Keller et al., 2005). The production of secondary metabolites in fungi is a complex process often coupled with morphological development (Calvo et al., 2002). Secondary metabolites often have obscure or unknown functions in organisms but have considerable importance for mankind due to their broad range of useful antibiotic, pharmaceutical as well as toxic activities (Yu and Keller, 2005). The products of these pathways include important pharma-ceuticals such as penicillin (Pelaez, 2005), cyclosporin (Bentley et al., 1997) and statins (Demain, 1999; Demain, 2006), as well as potent poisons including aflatoxins (example, aflatoxin B1) and trichothecenes (Keller et al., 2005). Citrinin (Calvo et al., 2002) was first isolated from Penicillium citrinum before world war II; subsequently, it was identified in over a dozen species of Penicillium and several species of Aspergillus (Bennett and Klich, 2003). Citrinin had also been isolated from Monascus ruber and Monascus purpureus, that is, industrial species used to produce red pigments (Blanc et al., 1995). Citrinin is bactericidal against Gram-positive bacteria (Vilar et al., 1999). Aspergillus isolates showed an inhibitory activity mainly against S. aureus, one E. coli isolate and S. albus, while Penicillium isolates were effective mainly against B. subtilis, S. albus, S. aureus and S. pyogenes. Previously, Penicillium isolates from dry-cured ham had shown wide antibiotic effects when tested against both bacteria and yeast, similarly they observed a high sensitivity in E. coli, B. subtilis and S. aureus isolates, while S. marcescens displayed a weak sensitivity (Huerta et al., 1987). Larrondo and Calvo (1990) observed that P. oxalicum was having a broader spectrum of activity against S. aureus, B. subtilis, B. cereus, P. mirabilis and Candida albicans. P. chrysogenum E01-10/3 strain was cultured from a sample of the Mediterranean sponge Ircinia fasciculate was shown to be capable for production of polyketides with pharmacologically interesting features (Bringmann and Lang, 2003). Sponge derived fungi only recently received broader interest in the natural products chemists’ community as producers of new and biologically active metabolites (Konig and Wright, 1996; Pietra, 1997; Biabani and Laatsch, 1998). Penicillium is a large anamorphic (asexual state) ascomycetous fungal genus with wide-spread occurrence in most of the environments. This genus comprises more than 200 described species and many are common soil inhabitants, as well as food borne contaminants or food ingredients used in the preparation of cheese and sausages (Pitt, 2000 and Frisvad and Samson, 2004). Penicillium species produce a much diversified array of active secondary metabolites, including antibacterial (Rancic et al., 2006; Lucas et al., 2007), antifungal substances (Nicoletti et al., 2007), immunosuppressants, cholesterol-lowering agents (Kwon et al., 2002), and also potent mycotoxins (Frisvad and Samson, 2004). Thousands of Penicillium isolates have probably been screened in bio prospecting programs since the discovery of
penicillin, and new bioactive metabolites continue to be discovered from these fungi nowadays (Larsen et al., 2007; Ge et al., 2008; Takahashi and Lucas, 2008), indicating their current importance as sources of high amounts of novel bioactive molecules to be used by pharmaceutical industry. P. cirtinum produces a variety of beneficial metabolites act against certain pathogens. It is already known for producing mycotoxin citrinin and cellulose digesting enzymes like cellulase and endoglucanase, as well as xylanase. Citrinin is produced by some Penicillium, Aspergillus and Monascus species. Pitt (2002) indicated that production of citrinin has been reported from at least 22 Penicillium species. Citrinin producing strains include P. citrinum, P. verrucosum (Frisvad and Thrane, 2002; Pitt and Hocking, 1997; Pitt, 2002), P. expansum (Vinas et al., 1993), A. terreus (Frisvad and Thrane, 2002), Monascus ruber and M. purpureus (Blanc et al., 1995; Hajjaj et al., 1999; Xu et al., 1999). El-Kassas and Khairy, (2009) reported the biological control of opportunistic pathogen ic marine fungi Fusarium solani by C. salina. Diby et al. (2005) concluded that P. pseudoalcaligenes MSP-538 which was obtained from the salty soils of the coastal agricultural belt of south coast of India, was an effective biocontrol agent against X. oryzae which is the bacterial blight pathogen of rice.
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African Journal of Biotechnology Vol. 11(29), pp. 7512-7519, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.2180 ISSN 1684â&#x20AC;&#x201C;5315 Š 2012 Academic Journals
Full Length Research Paper
Isolation and screening of Streptomyces from soil of Tunisian oases ecosystem for nonpolyenic antifungal metabolites Lilia Fourati Ben Fguira*, Samir Bejar and Lotfi Mellouli Laboratory of Microorganisms and Biomolecules, Centre of Biotechnology of Sfax, Road of Sidi-Mansour Km 6, P.O. Box 1177, 3018 Sfax, Tunisia. Accepted 5 October, 2011
The purpose of this study was to screen isolates of Streptomyces producing nonpolyenic antifungals. This choice was made to limit the problem of rediscovery of well-known antifungal families, especially polyenic antifungals. 68 Streptomyces strains were isolated from the soil sample collected from Tunisian oases ecosystem. These strains were tested for their capacity to produce active compounds using the diffusion method against two bacteria: Escherichia coli ATCC 8739, Micrococcus luteus LB 14110; two filamentous fungi: Verticillium dahliae and Fusarium sp. and two yeasts: Candida tropicalis R2 CIP203 and Candida albicans ATCC 2019. Among these isolates, 40 strains (58.82%) showed antibacterial activity, 18 strains (26.47%) showed antifungal activity, while 12 strains (17.64%) exhibited a broad-spectrum activity against all tested indicator cells. The production of nonpolyenic antifungal metabolites by promising isolates was investigated using their antibacterial activity and ergosterol inhibition as well as the UV-vis spectra of their corresponding active extracts. The obtained results showed that 13 isolates (19.11%) produced nonpolyenic antifungal activity. These data indicate the richness of the Tunisian oases ecosystem in actinomycetes bacteria producing active compounds. This fact, may partly explain the resistance phenomena of the Tunisian oasis palms against some phytopathogen fungus such as Fusarium oxysporum sp. albidenis (bayoud). Key words: Soil of Tunisian oases ecosystem, Streptomyces, nonpolyenic antifungal activity.
INTRODUCTION Despite the long list of currently available antibiotics in the market, antifungal antibiotics are a very small but significant group of drugs that have important roles in several fields: human and animal therapy, agriculture for protection of plant, food industries and treatment of wood. Only a limited number of antifungal agents are currently available for the treatment of lifethreatening fungal infections (Vicente et al., 2003). The need for new, safe and more effective antifungals is a major challenge to the pharmaceutical industry today, especially with the increase in opportunistic infections in the immunocom-promised host. However, many compounds (polyenes in particular) cannot be used because of their toxicity, while
*Corresponding author: E-mail: Tel/Fax: 00 216 74 870 451.
fourati.lilia@yahoo.com.
most of them are of interest in animal therapy, agricultural industry. These antifungal agents show some limitations, such as the significant nephro-toxicity of amphotericin B (Georgopapadakou and Walsh, 1994). The toxicity of present antifungal therapy is due to the biochemical similarity between fungal pathogens and infected hosts (all eukaryotes). The search for a new, safer, broad-spectrum antifungal antibiotic with greater potency has been progressing slowly (Gupte et al., 2002). The development of new antifungal agents, preferably naturally occurring with novel mechanisms of action, is an urgent medical need. Soil, in particular, is an intensively exploited ecological niche, the inhabitant of which produces many useful biologically active natural products, including clinically important antibiotics. The species belonging to the genus Streptomyces constitute
Fguira et al.
50% of the total population of soil actinomycetes (Williams et al; 1983), and 75 to 80% of the commercially and medicinally useful antibiotics have been derived from this genus (Miyadoh, 1993). In the course of screening for new antifungal antibiotics, several studies are oriented towards isolation of new Streptomyces species from different habitats (Ouhdouch et al., 2001; Hilali et al., 2002; Lemriss et al., 2003; Thakur et al., 2007). Fusarium oxysporum sp. albidenis (Foa) fungus (Fernandez et al., 1998; El Hadrami et al., 1997) has caused destruction of a large number of palms in the oases of Algeria and Morocco but not for those in Tunisia. This surprising fact could be due to the physico-chemical characteristics of Tunisian oases soil, and/or to the presence of antagonistic microorganisms which might inhibit Foa development and dissemination. Hence, the screening of Streptomyces from rhizospheric soil of Tunisian oases ecosystem, presented a special attention to explore the potentialities of the diverse micro flora of this region. The present research was undertaken with an aim of highlighting the presence of actinomycetes, espe-cially the genus Streptomyces, from different protected Tunisian oases soil, and express results obtained in the search for nonpolyenic antifungal metabolites produced by these strains.
MATERIALS AND METHODS Sample selection and microbial strains Sample was collected from the Tunisian oases ecosystem: rhizosphere soil of date palm ("Deglet Nour"). This sample was taken after removing approximately 20 cm of the soil surface and it was placed in sterile polyethylene bags, closed tightly and stored in the refrigerator at 4°C until use. The target strains used for screening antimicrobial activity were procured from microbial type culture collection: Bacterial strains Escherichia coli ATCC 8739 (Gram-negative bacteria) and Micrococcus luteus LB 14110 (Gram-positive bacteria), were used as indicator microorganisms for antibacterial activity assays. Antifungal activity was determined against Verticillium dahliae, Fusarium sp., Candida albicans ATCC 2091 and Candida tropicalis R2 CIP203 and amphotericin B-nystatin resistant (amphotericin B and nystatin are polyenic antifungal).
Isolation of Streptomyces One gram of the sample soil was suspended in 100 ml of physiological water (NaCl 9 g/L) then incubated in an orbital shaker incubation at 30°C with shaking at 200 rpm for 30 min. Mixture was allowed to settle, and serial dilutions up to 10 -5 were prepared using sterile physiological water and agitated with the vortex at maximum speed. An aliquot of 0.1 ml of each dilution was taken and spread evenly over the surface of Streptomyces isolation agar (5 g/L glucose; 4 g/L sodium propionate; 2 g/L casein; 0.5 g/L K 2HPO4; 0.5 g/L MgSO4.7H2O; 200 ml sterile soil extract (equal volumes of soil and distilled water were mixed overnight and filtered after sterilization at 120°C for 15 min), pH 7.2 and 20 g/L agar). The medium was then added to 5 µg/ml ampicillin and 50 µg/ml nystatin or cycloheximid to inhibit bacteria and fungal contamination,
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respectively. Plates were incubated at 30°C and monitored after 3, 5 and 7 days. Streptomyces colonies were recognized on the basis of morphology by height microscopy (G × 10). The isolates were identified morphologically as genus Streptomyces by comparing the morphology of spore bearing hyphae with entire spore chain as described in Bergey’s Manuel (Holt et al., 1994). Finally, representative colonies were selected and streaked on new plates of Streptomyces isolation agar medium. Agar plates were then inoculated with the strains and incubated at 30°C until good growth was observed. The isolated strains were conserved at 4°C for two months, and in a freezer at -80°C in the presence glycerol (15%, v/v) for a longer period. Submerge culture conditions Isolates that showed activity against test organisms in agar medium were grown in the submerged culture in 250 ml flasks containing 50 ml of Bennett medium (1 g/L beef extract, 10 glucose, 2 g/L peptone, 1 g/L yeast extract). A 2 cm2 piece of agar from each seven-day-old culture grown on Streptomyces isolation agar medium was used to inoculate the flasks. These cultures were grown in rotary shaker at 200 rpm, 30°C, for seven days. The resulting culture broths (approximately 50 ml), obtained following growth of each isolate in the culture media were separated from the mycelium by centrifugation at 900 rpm for 15 min. The supernatant, sterilized by filtration, was used for extracellular antimicrobial activity by agar well diffusion with one of the test organisms. More also, for the determination of antibacterial activities, indicator microorganisms were grown overnight in LB medium at 30°C for M. luteus LB 14110 and at 37°C for E. coli ATCC 8739, then diluted 1:100 in LB medium and incubated for 5 h under constant agitation of 200 rpm at the appropriate temperature. Furthermore, for antifungal activity determination, C. tropicalis R2 CIP203 and C. albicans ATCC 2091 were grown in YP10 medium (10 g/L yeast extract, 10 g/L peptone, 100 g/L glucose, 15 ml of 2 g/L adenine solution) at 30°C for 24 h in an orbital incubator with shaking at 200 rpm. In addition, V. dahliae and Fusarium sp. were grown in potato dextrose agar (PDA) for 7 days at 30°C. Spores were collected in sterile distilled water then adjusted to a spore density of approximately 104 spores/ml. Biological assay of antimicrobial activity To isolate new actinomycete strains producing antimicrobial activities, we used the solid media bioassay test against M. luteus LB 14110, E. coli ATCC 8739, V. dahliae, Fusarium sp., C. albicans ATCC 2091 and C. tropicalis R2 CIP203. In solid medium, the antimicrobial activity was determined by the plate diffusion method (Bauer et al., 1966). After incubation of the selected strains for 7 days at the appropriate growth temperature, an agar disk (10 mm in diameter) was recuperated and placed in LB plates covered by 3 ml of top agar containing 50 μL of a 5 h culture of M. luteus LB 14110 or E. coli ATCC 8739 test strains. Plates were first kept in a refrigerator (4°C) for at least 2 h to allow the diffusion of any antibiotics produced, then incubated overnight at 30°C for M. luteus and at 37°C for E. coli. For antifungal activity determination, plates containing Sabouraud agar medium were covered with 3 ml of top agar containing 100 μL of spore suspension already prepared from V. dahliae or Fusarium sp. or F. oxysporum sp. albidenis (Foa) and by 50 μL of C. tropicalis R2 CIP203 or C. albicans ATCC 2091 culture. After 2 h at 4°C, the plates were incubated at 30°C. The antimicrobial activity was observed after 24 h for bacteria and 48 h for fungi and yeast.
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Moreover, in liquid medium, a paper disk was impregnated with 50 μL of the corresponding sample and then laid on the surface of an agar plate containing 3 ml of top agar seeded by 40 μL of a 5-h old culture of one of the bacteria used for antibacterial tests: M. luteus LB 14110, E. coli ATCC 8739 and by 50 μL of C. tropicalis R2 CIP203 or C. albicans ATCC 2091 culture or 100 μL of spore suspension of V. dahliae or Fusarium sp. for antifungal activities. After 2 h at 4°C, plates containing M. luteus, C. tropicalis R2 CIP203, V. dahliae and Fusarium sp. were incubated at 30°C and those inoculated with E. coli at 37°C, all overnight except V. dahliae and Fusarium sp. for 48 h.
Organic crude extracts preparation chromatography (TLC) analysis
and
thin
layer
Crude antimicrobial compound was recovered from the culture filtrate of each active isolate by solvent extraction with ethyl acetate. Ethyl acetate was added to the filtrate in the ratio 1:1 (v/v) and shaken vigorously for 20 min. The organic layers were collected and the organic solvent was evaporated to dryness in a vacuum evaporator at 40°C to obtain a gummy crude extract which was recuperated in 0.5 ml of ethyl acetate and assayed against indicator microorganisms. For TLC analysis of the crude extract of the isolate TN80, a small drop of a sample was spotted onto TLC plate (Silica gel 60 F254) with a capillary and dried; the spotting process was repeated by superimposing more drops on the original spot for obtaining appropriate quantity (2 to 5 µg) of the sample on the plate. The TLC plate was developed with a CH 2Cl2/5% MeOH solvent system, and was sprayed with anisaldehyde/sulphuric acid.
("Deglet Nour"). All Streptomyces were isolated at mesophilic temperatures (25 to 37°C). These results are in agreement with other authors (Williams et al., 1989; Awad et al., 2009) who found that most species of Streptomyces were isolated at mesophilic temperature. With the exception of some Streptomyces that can adapt to wide pH range (Thakur et al., 2007), Streptomyces are usually neutrophiles and cultivated in medium of pH 7.0 to 7.5 at temperatures of 25 to 37°C for 7 days. Furthermore, these strains were isolated on the Streptomyces isolation agar medium. This medium seems to be the most specific and sensitive for Streptomyces, since it contains sodium propionate which acts as an antifungal agent to inhibit the fungal conta-mination and glucose, which most Streptomyces use as a carbon source. In addition, its transparency facilitates colony observation. Several authors rightly consider that the use of antibiotics is an essential precaution in the isolation of Streptomyces (Kitouni et al., 2005; Errakhi et al., 2009). On the other hand, ampicillin (5 µg/ml) was added to the medium to inhibit bacterial contamination. The addition of antifungal agents to the isolation medium suppresses the growth of fungal species on the plates. For this purpose either cycloheximid (50 µg/ml) or nystatin (50 µg/ml) were used. These results were therefore anticipated because several studies have shown the importance of the constituents of the screen-ing medium under which the producing microorganisms were cultivated (Iwai and Omura, 1992).
Screening for antifungal polyenic and nonpolyenic metabolites In order to determine the effect of active compounds from selected Streptomyces isolates on the ergosterol present in the fungal cell membrane, ergosterol was used as the reversal agent to test for its ability to reverse the inhibition of C. albicans by antibiotic (Ouhdouch et al., 2001). Sabouraud agar plates with 50 mg/ml ergosterol were prepared along with a control without ergosterol. The plates were seeded with C. albicans and ergosterol inhibition was tested by disc diffusion method. Sterile filter paper discs were impregnated with 50 µL organic crude extract suspension, dried and placed onto plates previously seeded with test microorganisms. The plates were incubated at the appropriate growth temperature of the indicator microorganisms for 24 h and then observed for the inhibition zone. More also, the absorption spectrum of active extracts in methanol were recorded in the region (200 to 440 nm) by using a UV-vis spectrophotometer and compared with those of known polyenic antifungal.
RESULTS AND DISCUSSION Isolation of Streptomyces This study was undertaken with the aim of highlighting the presence of Streptomyces in Tunisian oases ecosystem and selecting the strains with antibacterial and antifungal activity. Using the selective medium and the cultivation conditions previously described, a total of 68 different Streptomyces isolates were obtained from a soil sample collected from the Tunisian oases ecosystem: rhizosphere soil of date palm
Characterization of Streptomyces isolates All isolates grew on a range of agar medium showing morphology typical of Streptomyces (Locci, 1989), since the colonies were shown growing, aerobic, chalky, folded and with aerial and substrate mycelium of different colours. In addition, all colonies possessed an earthy odour. The cultural characteristics (pigment production) and morphological characteristics (colour series) of the 68 Streptomyces isolates are presented in Table 1. All of these isolates fitted the genus description as previously reported (Williams et al., 1943, 1983). The colour of the substrate mycelium and aerial spore mass also varied. Streptomyces isolates were categorized into nine colour series according to the colour of their mature sporulated aerial mycelium, with gray and white colour series being the most abundant (Table 1). Members of the gray series were found to represent 30.8% of the total number of isolates; however, the lowest occurrence was noted for the orange, violet and red series (2.9% for each). The highest occurrence of isolates of the gray series was in agreement with that earlier reported (Ndond and Semu, 2000; Barakate et al., 2002). Nevertheless, Thakur (2007) in his study on distribution of Streptomyces in forest areas reported that the white colour class dominated (46%). Table 1 also shows that of the 68 Streptomyces isolates, 13 isolates (19.11%) produced melanin and 30 isolates (44.11%) produced soluble pigment. These percentages
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Table 1. Morphological and cultural characteristics of the sixty-eight Streptomyces isolates.
Parameter Number of isolate
Gray 21 (30.8)
Pigment production Melanin Soluble
5 12
White 16 (23.5)
Yellow 10 (14.7)
Green 3 (4.4)
2 4
1 7
0 2
Colour series Cream Pink 7 (10.2) 5 (7.3)
3 3
1 1
Orange 2 (2.9)
Violet 2 (2.9)
Red 2 (2.9)
Total 68 (100)
0 0
0 1
1 0
13 (19.11) 30 (44.11)
Numbers in parentheses represent the percentage out of the total isolates.
were less than that obtained by Ndonde (2000) and comparable to that obtained by Thakur (2007). The production of melanoid pigments varied in all the series with the exception of the strains grouped in the orange series, where none produced melanoid pigments. The differences in colour of the aerial mycelium of the isolates as well as those of the pigments they produce may be an indication of the diversity of Streptomyces isolates in the site investigated.
Antimicrobial activity In order to assess whether Streptomyces isolated from the rhizospheric soil of date palm ("Deglet Nour") from Tunisian oasis may be useful in screening for natural bioactive compounds, the antimicrobial activity of the isolates was determined (Table 2). Our result indicate that 67% of isolates were active against one or more of the test organisms. This percentage was higher than as describe by many authors studying the activity of soil actinomycetes (Saadoun et al., 1998; Barakate et al., 2002). However, others reported almost an equal percentage (Ndond and Semu, 2002). Data in Table 2 also showed that the percentage of active isolates varied within each colour series. The most active compounds producing isolates belonged to the yellow and red colour series (100% for each) and only 85.7% of cream isolates were active against one or more of the test organisms. Barakate et al. (2002) while studying the antimicrobial activity of isolates from Moroccan habitats soil found that the higher percentage of active isolates was found in white and red ones. Arai (1976) however, reported that most active species of Streptomyces were found in the gray and yellow series of no chromogenic type and no antibiotic-producing species were described in the white and green series of chromogenic type. Moreover, almost 22 of Streptomyces isolates (32.35%) did not show any antibiotic activity towards the test organisms using Bennettâ&#x20AC;&#x2122;s agar as medium of production, although, it is probable that they produced other useful compounds for which they were not screened in this study. In fact, it should be noted that production of secondary metabolites by microorganisms is often connected with and influence by primary metabolism. The composition and concentrations of the constituents of the media are closely
linked with the metabolic capacities of the producing organism and greatly influence the biosynthesis of the bioactive molecules (Elleuch et al., 2010). Porter (1971) stated that probably all Streptomyces possessed some antimicrobial proprieties if proper conditions were taken into consideration during culturing of these organisms for purposes of assessing their antibiotic production.
In Table 2, antibacterial activity was observed in 40 isolates (58.82%), 18 isolates (26.47%) exhibited antifungal activity, while only twelve isolates of Streptomyces (17.64%) showed the both activity. In previous studies, it was shown that the isolation rate of Streptomyces with antimicrobial activity is higher than 40% (Lemriss et al., 2003) and in others less than 10% (Jiang and Xu, 1996). These results confirm that the Streptomyces are able to produce a wide variety of antifungal activity. The comparison of the antimicrobial activity between all the colour classes against the tested organisms showed that isolates in the gray and yellow series displayed the highest antibiosis against the bacteria organism tested; while no activity was noted in the red and orange series against this test organism. Isolates in the yellow series were found to be most active against fungi. These differences in percentage of antimicrobial activity may imply that the investigated Streptomyces isolates belonged to different species or to the same, but they produced different bioactive compounds. In fact, we notice that in the same colour class, most of the isolates showed different activity spectrum. Furthermore, results of antimicrobial activity expressed in terms of the diameter of inhibition zone seem to confirm this hypothesis (Table 3). The inhibition zone of antimicrobial activity of Streptomyces isolates against the test microorganisms is different; some isolates exhibited a strong activity (>30 mm) especially against Grampositive bacteria, but the most active isolates show a moderate antimicrobial activity (<20 mm). The highest percentage of active isolates was obtained against M. luteus (55.88%); while the lowest percentage was exhibited against E. coli (19.11%). The antifungal activity of Streptomyces strains against Fusarium sp. and V. dahliae was almost equal (26.47%). Many authors
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Table 2. Antimicrobial activity of the sixty-eight Streptomyces isolates.
Parameter Number of isolate Number of active isolate Number of active isolates against bacteria Number of active isolates against fungi a ATB + ATF
Colour series Gray 21 12 (57.1) 12 1 1
White 16 9 (56.2) 8 1 1
Yellow 10 10 (100) 10 8 6
Green 3 1 (33.3) 1 0 0
Cream 7 6 (85.7) 5 1 0
Pink 5 3 (60) 3 3 3
Orange 2 1 (50) 0 1 0
Violet 2 1 (50) 1 1 1
Red 2 2 (100) 0 2 0
Total 68 (100) 46 (67.64) 40 (58.82) 18 (26.47) 12 (17.64)
Numbers in parentheses represent the percentage out of the total isolates. aATB, Antibacterial activity; AFA, antifungal activity.
also that the Streptomyces isolates appear to be highly active against Gram-positive bacteria (Barakate et al., 2002; Thakur et al., 2007). However, the percentage of isolates active against fungi seems to be lower than that reported previously in Streptomyces screening. The antifungal activity of isolates was deter-mined by the agar diffusion method against Fusarium sp. and V. dahliae. This plant pathogen is a causal agent of verticillium wilts of tomato, potato and olivetree. F. oxysporum sp. albidenis (Foa) (El Hadrami et al., 1997; Fernandez et al., 1998) an other plant pathogen, has caused destruction of a large number of palms in the oases of Algeria and Morocco, but not to those in Tunisia. In the course of study for antimicrobial activity against F. oxysporum sp. albidenis, five different isolates of Streptomyces were selected for their strong antifungal activity using those crude extracts. According to the Table 3, these isolates nominated, TN80, TN84, TN86, TN87 and TN93, were categorized into five colour series yellow, red, orange, pink and cream, respectively. As indicated in Table 4 however, three isolates (TN84, TN86 and TN93) did not showed any antibacterial activities but the two isolates (TN80 and TN87) presented an antibacterial activities against Gram positive (M. Luteus LB 14110).
More also, according to the data in Table 4, the TN80 strain showed the strongest antifungal activities against both fungi, F. oxysporum sp. albidenis and Fusarium sp. TLC silica gel analysis of the active crude extract of the culture of the TN80 showed three dark bands having respectively the following Rf values: B1 = 0.4; B2 = 0.53 and B3 = 0.63 (Figure 1). Hu et al. (2006) reported that in sulphuric acid reaction in TLC analysis, macrolides appear generally dark. Taking into account this finding and in conside-ration of our results, we can assume the hypothesis that the strain TN80 produces simulta-neously three different macrolide molecules. Nevertheless, due to their complex structure and high molecular weight, macrolide antibiotics, are not able to penetrate the cell walls of most Gramnegative bacteria (Mellouli et al., 2003). There-fore, we can explain the inhibitory activity against the Gramnegative bacterium (E. coli ATCC 8739), of the strain TN80 (in solid and liquid media), by the fact that TN80 isolate secret at least one other active compound, beside the three macrolide molecules that inhibits the growth of Gram-negative bacteria.
Detection of polyenic and nonpolyenic antifungal activity As shown in Table 2, among the 68 isolates, 18
strains (26.47%) showed antifungal activity against at least one of the tested fungi, while 13 (19.11%) out of these 18 isolates inhibited the C. tropicalis R2, an amphotericin B-nystatin resistant strain. The production of nonpolyenic antifungal substances by the eighteen isolates having antifungal activities was investigated using several criteria: antibacterial activity, ergosterol inhibition and UV-vis spectra of active extracts (Ouhdouch et al., 2001; Lemriss et al., 2003). Ergosterol present in fungal cell membrane has a very high affinity towards polyene antibiotics. Polyene drugs form complexes with ergosterol, which open channels in the fungal membrane that cause leakage of critical intracellular constituents and subsequent cell death. This behaviour is exploited in a detection method developed to identify the presence of
polyene class of antibiotics (Bastide et al., 1986). Interpretation of resultants are as follows: reduced zone in size in presence of ergosterol-polyene type of antibiotic present and no reduced zone in presence of ergosterol-polyene type of antibiotic absent (Table 5).
The use of spectroscopy to distinguish polyenic from nonpolyenic substances were also used by several authors (Thakur et al., 2007; Saadoun et al., 2009). The UV spectral data for the ethyl acetate extract of selected active fermented broth are shown in Table 5. According to this table,
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Table 3. Inhibition zones of antimicrobial activity of Streptomyces isolates against test microorganisms.
Test microorganism
<20 9 26 10 13
Escherichia coli Micrococcus luteus Fusarium sp. Verticillium dahliae
Inhibition zone 20 to 30 3 9 7 4
>30 1 3 1 1
Number of active isolate 13 (19.11) 38 (55.88) 18 (26.47) 18 (26.47)
Numbers in parentheses represent the percentage out of the total isolates.
Table 4. Inhibition zones of antifungal activity of Streptomyces isolates against Fusarium oxysporum sp. albidenis (Foa) and Fusarium sp.
Isolates (colours) TN80 (yellow) TN87 (pink) TN86 (orange) TN84 (red) TN93 (cream
ATB
a
+ + -
Inhibition zones (mm) of antifungal activity against F. oxysporum sp. albidenis Fusarium sp. 45 60 25 30 20 28 14 20 11 16
a
Antibacterial activity against M. Luteus.
B3 B2 B1
Figure 1. TLC analysis of the crude extract of TN80 isolate after treatment with anisaldehyde/H2SO4 solution.
thirteen isolates (TN80, TN84, TN86, TN87, TN93, TN210, TN211, TN212, TN223, TN224, TN226, TN236 and TN245)
appeared promising because of activity against E. coli (cell membrane without sterols) and no marked inhibition of
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Table 5. Results of screening for nonpolyenic antifungal producing Streptomyces.
Ergosterolâ&#x20AC;&#x2122;s effect against b C. albicans ATCC 2091 With Without
Antibacterial a Activity
Activity against C. tropicalis R2
TN80
+
+
54
55
NP
TN84
+
+
32
32
NP
TN86
+
+
37
38
NP
TN87
+
+
42
44
NP
TN93
+
+
26
25
NP
TN97
-
-
0
20
P
TN201
-
-
0
22
P
TN210
+
+
13
12
NP
TN211
+
+
19
17
NP
TN212
+
+
22
22
NP
TN223
+
+
14
12
NP
TN224
+
+
16
18
NP
TN226
+
+
38
36
NP
TN236
+
+
12
17
NP
TN244
-
-
0
18
P
TN245
+
+
20
21
NP
TN246
-
-
0
13
P
TN247 c Reference
-
-
0 4
20 15
P P
Isolates
UV-vis spectral
a
Activity against E. coli ; c Amphotericin B ; NP: nonpolyenic, P: polyenic; b inhibition zone in mm in Sabouraud agar medium with or without 50 mg/ml of ergosterol.
antifungal activity by exogen ergosterol (target of polyenic antifungal compounds). In addition, maximum absorbance peaks range between 200 and 440 nm and the characteristics of absorption peaks indicate a highly polyene nature. The broad-spectrum of activity shown in these isolates is possibly due to the production of different compounds. The metabolites produced by those thirteen isolates did not show a UV-vis spectrum characteristic of a polyenic structure. Of particular interest, these thirteen isolates (19.11%) apparently do not synthesize polyene-like substances. Similarly, Lemriss et al. (2003) when screening for nonpolyenic antifungal metabolites in clinical isolates of actinomycetes, found that only five Streptomyces (4.54%) exhibited nonpolyenic antifungal activity. In our study, it was shown that the isolation rate of Streptomyces with non polyenic antifungal activity was higher than 11% (Thakur et al., 2007) and more than 15% (Ouhdouch et al., 2001). More also, the Thirteen (19.11%) isolates (TN80, TN84, TN86, TN87, TN93, TN210, TN211, TN212, TN223, TN224, TN226, TN236 and TN245) have been selected for addition studies. The identification of these selected isolates, as well as the isolation, purification and the structural elucidation of corresponding active compounds are under investigation.
Conclusion The southern region of Tunisia is characterized by the date palm oases, which constitute the principal financial resources of oases population. In this region, the major palm plantations are marked by prevalence of the elite variety "Deglet Nour". This variety is highly susceptible to the fungus Fusarium oxysporum sp. albidenis known as 'bayoud', which destroys a high number of palms in the oases of Algeria and Morocco, but unexpectedly not in those of Tunisia. Reasons for this surprising fact are still unknown, but may have an origin in antagonistic microorganisms as we assumed. The investigation of bacteria producing activity against fungi especially against 'bayoud' may therefore provide a significant contribution to understand the resistance phenomena of the Tunisia oasis palms and also an important ecological and economical contribution.
In this study, sixty-eight Streptomyces strains were isolated from Tunisian oases ecosystem. Among these isolates, forty strains (58.82%) showed antibacterial activity, eighteen strains (26.47%) showed antifungal activities; among them thirteen promising isolates (19.11%) seems to produce nonpolyenic antifungal activities. This high level of isolates producing
Fguira et al.
antifungal and nonpolyenic active compounds, compared to previous works (Ouhdouch et al., 2001; Lemriss et al., 2003; Thakur et al., 2007), may explain the protected areas of Tunisian oases ecosystem against the phyto-pathogen fungus such as F. oxysporum sp. albidenis. Equally, this Tunisian oases ecosystem represents a potential source for the discovery of new nonpolyenic antifungal compounds.
ACKNOWLEDGEMENTS This work was supported by the Tunisian Government (Contract Programme CBS-LMB). We are grateful to Prof. M. Barakate (Laboratory of Microbiology, Faculty of Sciences Semlalia, Marrakech-Morocco) for Fusarium oxysporum sp. albidenis test realisation. REFERENCES Arai T, Kuroda S, Mikami Y (1976). Classification of actinomycetes with reference to antibiotic production. In Actinomycete: the Boundary Microorganisms, ed. Aria T. pp. 261-276. Tokyo and Singapore: Topan Company, Ltd. ISBN QK 604.A7. Awad HM, El-Sahed KYI, El-Nakkadi AM (2009). Isolation, screening and identification of newly isolated soil Streptomyces (Streptomyces sp. NRC-35) for b-Lactamase inhibitor production. World Appl. Sci. J. 7 (5): 637-646. Barakate M, Ouhdouch Y, Oufdou KH, Beaulieu C (2002). Characterization of rhizospheric soil Streptomyces from Moroccan habitats and their antimicrobial activities. World J. Microb. Biot. 18: 49-54. Bastide A, De-Méo M, Andriantsoa M, Laget M (1986). Isolement et sélection de souches d’actinomycètes productrices de substances antifongiques de structure non-polyènique. World J. Microb. Biot. 2 (4): 453-466. Bauer AW, Kirby WM, Sherris JC, Turk M (1966). Antibiotic susceptibility testing by standard single disk method. Am. J. Clin. Pathol. 45: 493-496. El Hadrami I, Ramos T, El bellaj M, El Idrissi-Tourane A, Macheix JJ (1997). A sinapic derivative as an induced defence compound of date palm against Fusarium oxysporum f. sp. albedinis, the agent causing Bayoud disease. J. Phytopathol. 145: 329-333. Elleuch L, Shaaban M, Smaoui S, Mellouli L, Karray-Rebai I, Fourati Ben Fguira L, Shaaban KA, Laatsch H (2010). Bioactive Secondary Metabolites from a New Terrestrial Streptomyces sp. TN262. Appl. Biochem. Biotechnol. 162: 579-593. Errakhi R, Lebrihi A, Barakate M (2009). In vitro and in vivo antagonism of actinomycetes isolated from Maroccan rhizospherical soils against Sclerotium rolfsii : a causal agent of root rot on sugar beet (Beta vulgaris L.). J. Appl. Microbiol. 107: 672-681. Fernandez D, Ouinten M, Tantaoui A, Geiger JP, Daboussi MJ, Langin T (1998). Fot 1 insertions in the Fusarium oxysporum f. sp. Albedinis genome provide diagnostic PCR targets for detection of the date palm pathogen. Appl. Environ. Microbiol. 164: 633-636. Georgopapadakou NH, Walsh TJ (1994). Human mycoses: drugs and targets for emerging pathogens. Science, 264: 371-373. Gupte M, Kulkarni P, Ganguli BN (2002). Antifungal antibiotics. Appl. Microbiol. Biotechnol. (58): 46-57. Hilali L, Khattabi A, Nssaralah N, Malki A, Finance C (2002). Isolement des nouvelles souches d’actinomycètes productrices de substances antifongiques à partir du milieu naturel Marocain. Rev. Biol. Biotech. 2(1): 49-53.
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Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST (1994). Bergey’s manual of determinative bacteriology. Williams and Wilkins, Baltimore. Hu CQ, Zou WB, Hu WS, Ma XK, Yang MZ, Zhou SL, Sheng JF, Li Y, Cheng SH, Xue J (2006). Establishment of a Fast Chemical Identification System for screening of counterfeit drugs of macrolide antibiotics. J. Pharmaceut. Biomed. Anal. 40: 68-74. Iwai Y, Omura S (1992). Cultural conditions for screening of new antibiotics. J. Antibiot. 34: 123-41. Jiang CL, Xu L H (1996). Diversity of aquatic actinomycetes in lakes of the middle plateau, Yunnan; China. Appl. Environ. Microbiol. 62: 249253. Kitouni M, Boudemagh A, Oulmi L, Reghioua S, Boughachiche F, Zerizer H, Hamdiken H, Couble A, Mouniee D, Boulahrouf A, Boiron P (2005). Isolation of actinomycetes producing bioactive substances from water, soil and tree bark samples of the north-east of Algeria. J. Mycol. Med. 15: 45-51. Lemriss S, Laurent F, Couble A, Casoli E, Lancelin JM, SaintpierreBonaccio D, Rifai S, Fassouane A, Boiron P (2003). Screening of nonpolyenic antifungal metabolites produced by clinical isolates of actinomycetes. Can. J. Microbiol. 49(11): 669-674. Locci R (1989). Streptomycetes and related genera. In: Williams ST, Sharpe ME, Holt JG, editors. Bergey’s manual of systematic bacteriology. Baltimore, Williams and Wilkins. pp. 2451-2493. Mellouli L, Ben Ameur-Mehdi R, Sioud S, Salem M, Bejar S (2003). Isolation, purification and partial characterization of antibacterial activities produced by a new isolated Streptomyces sp. US24 strain. Res. Microbiol. 154: 345-352. Miyadoh S (1993). Research on antibiotic screening in Japan over the last decade: A producing microorganisms approach. Actinomycetologica, 7(2): 100-106. Ndond RD, Semu E (2000). Preliminary characterization of some Streptomyces species from four Tanzanian soils and their antimicrobial potential against selected plant and animal pathogenic bacteria. World J. microb. Biot. 16: 595-599. Ouhdouch Y, Barakate M, Finance C (2001). Actinomycetes of Moroccan habitats: Isolation and screening for antifungal activities. Eur. J. Soil. Biol. 37: 69-74. Porter JN (1971). Prevalence and distribution of antibiotic-production actinomycetes. Adv. Appl. Microbiol. 14: 561-564. Saadoun I, AL-Momani F, Ababneh Q, Bonjar S (2009). Comparative UV-spectra of fermented cultural extract of antifungal-active Streptomyces isolates recovered from different ecological habitats. Curr. Trend Biotechnol. Pharm. 3(2): 155-161. Saadoun I, Mohammadi MJ, Al-Momani F, Meqdam M (1998). Diversity of soil Streptomyces in northen Jordan. Actinomycetes, 9(3): 5260. Thakur D, Yadav A, Gogoi BK, Bora TC (2007). Isolation and screening of Streptomyces in soil of protected forest areas from the states of Assam and Tripura, India, for antimicrobial metabolites. J. Mycol. Med. 17: 242-249. Vicente MF, Basilio A, Cabello A, Pela’ez F (2003). Microbial natural products as a source of antifungal. Clin. Microbiol. Infect. 9: 15-32. Williams ST, Goodfellow M, Alderson G (1989). Genus Streptomyces Waksman and Henrici, In: Williams ST, Sharpe ME, Holt JG editors, Bergey’s manual of systematic bacteriology. Baltimore, Williams and Wilkins, 4: 2452-2492. Williams ST, Goodfellow M, Alderson G, Wllington EM, Sneath PH, Sacki MJ (1983). Numerical classification of Streptomyces and related genera. J. Gen. Microbiol. 129: 1747-1813. Williams ST, Goodfellow M, Alderson G (1943). Genus Streptomyces Waksman and Henrici, 339AL. In Bergey’s Manual of Systematic Bacteriology. Baltimore, Williams and Wilkins Company, 4: 24522492.
African Journal of Biotechnology Vol. 11(29), pp. 7520-7527, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB12.120 ISSN 1684–5315 © 2012 Academic Journals
Full Length Research Paper
Preliminary characterisation of the phytotoxin of sheath-blight disease of rice caused by Rhizoctonia solani Xiao-Xing Liang and Ai-Ping Zheng* Rice Research Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China. Accepted 19 March, 2012
The culture filtrates of Rhizoctonia solani was significantly toxic to rice leaves, being similar to that caused by R. solani. The culture filtrate from R. solani was extracted using ethyl acetate and isolated by silica gel column chromatography, which showed significantly high toxicity. The toxin was thermally stable even after incubation at 121°C for up to 30 min. A pH of 2.0 was helpful for pathogenesis; after 12 h, yellow lesions were found. The solvent system ethyl acetate/methanol (10:1, v/v) was the optimal solvent system used for preparative thin-layer chromatography. Eight fractions were chosen to be collected by silica gel column chromatography, but only three spots were obtained in the thin-layer chromatography analysis. The second fraction was found to be the one with the highest activity and the toxin appeared to be mainly present in this fraction. The ultraviolet and infrared spectral data indicated that lactone, ketone and benzene groups existed in the culture filtrate. This report is the first describing the preliminary characterisation of phytotoxins from an R. solani culture filtrate; it briefly presents the isolation, purification and bioassay method for the phytotoxin produced by R. solani. Key words: Richard's medium, crude extracts, bioassay, thin-layer chromatography, silica gel column chromatography.
INTRODUCTION Rice sheath blight caused by Rhizoctonia solani is a serious disease in all rice-growing countries (Roy, 1993). Symptoms of the disease include greenish gray elliptical or oval-shaped spots with yellow margins mostly found on leaf sheaths; however, at times, leaf blades are also infected (Vidhyasekaran et al., 1997; Huang et al., 2009). Similar symptoms were produced by the culture filtrates of R. solani and a strong correlation was found between rice cultivar sensitivity to R. solani and R. solani culture filtrates, suggesting the possible involvement of phytotoxins in lesion development (Vidhyasekaran et al., 1986, 1992). The phytotoxic metabolites of several types of R. solani have been partly isolated or structurally characterised ( Akoi et al., 1963; Mandava et al., 1980; Ramalingam, 1986; Lakpale et al., 1996; Vidhyasekaran
*Corresponding 02886290903.
author.
E-mail:
aipingzh@163.com.
Fax:
et al., 1997; Betancourt et al., 2000; Chen et al., 2001). All these structurally characterised toxins are low-molecularweight secondary compounds with diverse chemical structures. Several workers (Chen, 1958; Akoi et al., 1963; Frank,and Fancis 1976; Kenning and Hanchey, 1980; Iacobellis and Devay 1987; Chen et al., 2001) found that R. solani isolated from rice produced non-host-specific toxins which were phenyl acetate and its derivatives; and caused symptoms normally associated with leaf blight on a variety of plants. Other workers (Vidhyasekaran et al., 1997), using hosts and non-hosts of the pathogen, showed that toxins produced by R. solani were host-specific; the host-specific toxin from the rice sheath-blight pathogen induced the characteristic symptoms of the disease only on hosts of the
pathogen and they were carbohydrates. However, the phytotoxins produced by different types of R. solani may be different. The objectives of this study were to isolate, purify and characterise the phytotoxin produced by R. solani.
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MATERIALS AND METHODS
Adsorption of toxins from culture filtrates by active carbon
Fungi and media
Following the method of Vidhyasekaran et al. (1997), activated carbon (60 g/l) was added into the culture filtrate (50 ml), chilled overnight at 4°C, and then filtered through two Whatman No. 1 filter papers. The filtrate was then evaporated to dryness at 50°C, dissolved in 50 ml of sterile distilled water and bioassayed as previously described. The carbon remaining in the filter paper was washed with 50 ml of hot methanol (50°C), shaken (80 rpm) for 30 min and filtered; this step was repeated once. The methanol was evaporated at 50°C and the residue was dissolved in 50 ml of sterile distilled water and bioassayed as afore stated.
R. solani (Anastomosis Group IA) was obtained from Sichuang Agricultural University; all the other chemicals and reagents used in the experiment were purchased from Kelong Reagents Company (Chengdu, China). The media used in the experiments were as follows: potato sugar agar (PSA; potato, 200 g; sugar, 20 g; agar, 15 g; and distilled water, 1,000 ml; pH 7.0), potato sucrose broth (PSB; potato, 200 g; sucrose, 20 g; distilled water, 1,000 ml; pH 7.0), Richard's medium (potassium nitrate, 10 g; potassium dihydrogen phosphate, 5 g; magnesium sulfate·7H 2O, 2.5 g; ferric sulfate, 0.25 g; sugar, 50 g; and distilled water, 1,000 ml; pH 7.0).
Solvent extraction Fungal cultures and toxin production Cultures of a virulent isolate (Anastomosis Group IA) of R. solani were grown on PSA plates at 25°C for seven days. Further, 6-cmdiameter mycelial plugs from PSA cultures were transferred into a 150-ml flask containing 50 ml of PSB. The cultures were then incubated at 28°C on a rotary shaker (180 rpm) for two days and then stored at 4°C. For production of toxin, four mycelium groups from PSB cultures were transferred into a 500-ml flask containing 200 ml of Richard's medium; R. solani produces toxins in this type of medium (Chen, 1992; Xu et al., 2004; Lu et al., 2005). The cultures were then incubated under stationary conditions at 28°C on artificial vibration once a day for 15 to 20 days. This method was followed by Xu et al. (2004). Culture filtrates were obtained by passing the liquid through four layers of cheesecloth and Whatman No. 1 filter paper. Unless otherwise stated, the Richard-mediumcultured filtrate was used for all subsequent tests.
The extraction followed the procedure described by Vikrant et al. (2006), with minor modifications. The culture filtrate (50 ml) was extracted three times with half volumes of ether, benzene, ethyl acetate or chloroform using a separatory funnel. Both water and the solvent fractions were evaporated to dryness at 50°C. The residues were dissolved in 50 ml of sterile distilled water and their toxicity was measured using the leaf-necrosis assay. Sterile distilled water and the culture filtrate served as controls. Effective concentration in the bioassay Culture filtrates were evaporated to dryness at 50°C; diluted to 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50%; and bioassayed as previously done. Sterile distilled water and culture filtrate served as controls. The minimum concentration of toxins required to induce symptoms was regarded as the dilution endpoint assay and this concentration was used for all further leaf assays.
Leaf-necrosis assay and data analysis Effect of heat, strong acid and strong alkali on toxin activity The biological activities of crude and processed extracts were determined by the leaf-necrosis assay on a partial resistant cultivar (9311), and a susceptible cultivar (Lemont). Briefly, for each treatment, 10 leaves at the three-leaf stage were detached, the abaxial surface was lightly wounded by pricking gently with a sterilised needle and a 30-μl solution was applied to the wound site (Song et al., 1993; Zheng et al., 2010). The leaves were then incubated in a moist chamber at 25°C for three days and the lesion area around each wound was assessed. Each bioassay was repeated three times for the five plants; leaves treated with sterile distilled water and un-inoculated media served as controls. The data of leaf-necrosis assay were subjected to analysis of variance, and when significant treatment differences were found, means were compared by the test of least significant difference (LSD, P<0.5). Bioactivity of soluble macromolecular compounds obtained from culture filtrates In this test, 50 ml of culture filtrate was first concentrated to half the original volume in vacuo at 40°C. An equal volume of methanol or acetone was added to the culture filtrate and stored overnight at 4°C. Precipitates were collected by centrifugation at 8,500 rpm for 10 min. The precipitates were then dissolved in 50 ml of sterile distilled water and the bioactivity was assessed by the leaf-necrosis assay, as previously described. The supernatant was evaporated to dryness at 50°C, dissolved in 50 ml of sterile distilled water and bioassayed similarly.
The culture filtrates were subjected to temperatures of 60, 100 or 121°C for 30 min in a water bath (or autoclaved) and bioassayed as previously done. Sterile distilled water and untreated culture filtrate served as controls. To test whether the toxin is sensitive to pH, the pH of the culture filtrates was adjusted to 2.0, 7.0 and 12.0 using 12 mol/l hydrochloric acid and 1 mol/l potassium hydroxide; incubated for 30 min and the pH was then adjusted to 7.0. The culture filtrates of pH 2.0 to 7.0 (from 2.0 adjusted to 7.0); 7.0; and 12.0 to 7.0 (from 12.0 adjusted to 7.0) were used for the bioassay; every treatment was repeated three times. Sterile distilled water and the untreated culture filtrate served as controls. Isolation, purification and characterisation of the phytotoxin from culture filtrate The culture filtrates were acidified to pH 2.5 with 12 M hydrochloric acid and extracted three times with an equal volume of ethyl acetate. The combined extracts were concentrated to a small volume in vacuo at 40°C and then washed three times with equal volumes of 5% aqueous sodium bicarbonate. The aqueous layers were acidified to pH 2.5 with 12 M hydrochloric acid and extracted three times with ethyl acetate. This method of Iacobellis and Devay (1987) was followed, with little modifications. The combined extracts were evaporated to dryness in vacuo at 40°C. The residue was obtained and dissolved in 1 ml methanol for analysis by thin-layer chromatography (TLC). Preparative analytical TLC plates, coated
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Culture filtrate
Figure 1. Necrosis lesions caused by R. solani culture filtrate on wounded rice leaves of cultivar 9311. All treated leaves were incubated at 25°C for 3 days; when the pH of culture filtrate was adjusted to 2.0, necrosis was also found and the rice leaf was turned to be yellow; sterile distilled water served as control and no necrosis was found.
with a GF-254 fluorescent silica gel (5 × 10 cm, Qingdao, China) and spotted with the samples were developed separately in solvent systems containing ethyl acetate/methanol (1:1 to 20:1, v/v). The spots on the TLC plates obtained using the solvent aforementioned were marked under ultraviolet (UV) light at 254 nm and the Rf value of each spot was recorded. The solvent system that could yield clear and unattached spots on the TLC plates was used to purify the crude toxin by elution in a silica gel column.
the fraction that had spots on the TLC plate was assessed by detaching the leaves of six species of plants: rice, corn, soybean, rape, Chinese radish and Chinese cabbage. Each bioassay was repeated three times for the six plants; the leaves treated with sterile distilled water and un-inoculated media served as controls. Identification of the biologically active compound was conducted using infrared (IR; NEXUS 670, American Electric Company, United States) and UV spectroscopy (Beckman DU-70, Germany). The IR and UV spectra were recorded in KBr and ethanol, respectively.
Silica gel column chromatography and infrared spectrum The crude toxin was dissolved in 2 ml of methanol and applied to a 15 × 500 mm column filled with silica gel (200 to 300 mesh, Qingdao, China), which had been activated at 150°C overnight and pre-equilibrated with methanol. The dissolved toxin was added on top of the column and the column was eluted using a linear gradient with the following eluant strength: 100% ethyl acetate, methanol/ethyl acetate (1:10, 1:5, 1:1, 5:1 and 10:1; v/v) and 100% methanol. Elution was carried out at the rate of 0.25 ml min -1. 2 ml fractions were collected, analysed by TLC on GF254 using ethyl acetate/methanol (10:1. v/v) and visualised by UV light (254 nm);
RESULTS Toxin production characterisation
and
preliminary
in
vitro
The culture filtrates of R. solani grown on Richard’s liquid media can induce leaf necrosis (Figure 1); this result is consistent with that of Robert et al. (1984). Necrosis was found in both resistant and susceptible cultivars; however, the area with necrosis was smaller in resistant cultivars than
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Toxin
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lesions on the tested rice leaves. Hence, all subsequent tests were conducted with Richard’s medium filtrates. Preliminary characterisation of the toxic activity was carried out in vitro before toxin isolation. No toxicity was detected in the precipitates, which were obtained after the addition of methanol or acetone (Table 1). When the crude toxin was treated with active carbon, toxicity could be detected, both in the solution and the absorbed fraction; however, the absorbed fraction showed higher toxicity, showing that a portion of the toxin could be absorbed by the active carbon, whereas others could not (Table 1). The toxic components were found only in the solvent fraction after the culture filtrates were partitioned with ethyl acetate or chloroform; and in the water fraction, after partitioning with petroleum ether or ether. After partitioning with benzene, the toxins were detected in both water and the solvent fractions (Table 1). Because the ethyl acetate extract showed significantly higher toxicity than the one from chloroform, ethyl acetate was chosen for extraction of the toxins from the culture filtrate. The toxic activity in the culture filtrates was thermally stable even after incubation at 121°C for up to 30 min, indicating that they were highly thermostable. Moreover, a pH of 2.0 was helpful to the pathogenesis process. After 12 h, yellow lesions were found on the treated leaves (Figure1). The other symptoms were found after 24 h and there was no necrotic area on the controls, which suggested that low pH helped the toxins to infect rice (Iacobellis,and Devay 1987; Jayaraman et al., 2010). The necrotic region was isolated to prove the relationship between phytotoxin concentration and necrotic lesion development. The effective concentration causing serious leaf necrosis was 40%; concentrations less than 40% could cause only slight necrosis or not at all and concentrations more than 40% could not increase the necrotic region. Isolation and purification of R. solani toxin
Figure 2. Necrotic lesions produced by R. solani and R. solani-toxins on wounded rice leaves of cultivar 9311 (susceptible). Leaves were inoculated with fungal plugs 4 mm in diameter or treated with 10 drop of R. Solani toxins. All treated leaves were incubated at 25°C for 2 days.
that in susceptible ones. The symptoms produced in rice leaves by the culture filtrates of R. solani were similar to those produced by pathogenic R. solani (Figure 2); moreover, a strong correlation was foundbetween ricecultivar sensitivity to R. solani and sensitivity to R. solani culture filtrates, suggesting the possible involvement of phytotoxins in lesion develop-ment. However, the filtrate from 20-day-old Richard's medium cultures, which produced an average lesion size of 27 mm2, was significantly toxic to rice leaves. Filtrates from un-inoculated media caused no
To obtain a sufficient amount of the bioactive compound, the mycelium of R. solani was grown in one litre of Richard’s medium for 15 to 20 days. Extraction with ethyl acetate gave a light yellow powder (289.8 mg) with high phytotoxic activity after being dissolved in sterile distilled water and used in the leaf-necrosis assay. Spots were obtained with the solvent system ethyl acetate/methanol (10:1, v/v), wherein the Rf value was 0.42. This optimal solvent system was used for preparative TLC. The Rf value of the isolated toxin was 0.6, which showed that the isolated toxin has a small polarity, but not all the toxins in the isolate. Eight fractions were chosen to be collected in the silica gel column chromatography, whereas in the fractionation by TLC, three spots were obtained with the solvent system ethyl acetate/methanol (10:1, v/v) (Figure 3). The second, fifth, and sixth fractions produced average lesion sizes of 5, 2 and 1 mm2, respectively, in the rice leaf–necrosis assay (Table 2). The second fraction was found to be the one with the highest activity (P>0.5), which showed that the toxin is mainly in this
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Table 1. Phytotoxicity of crude toxin, isolated from culture filtrates of Rhizoctonia solani after three days, during a leaf-necrosis assay on the leaves of rice cultivar 9311.
Experiment
Treatment Methanol
1
Acetone
Fraction Water fraction Precipitate Water fraction Precipitate
Culture filtrate(control)
2
Active carbon
Ether
3
Benzene Ethyl acetate Chloroform
bc
Non adsorbed fraction Adsorbed fraction
5.4 b 12.1 a 18.1
Water fraction Solvent fraction Water fraction Solvent fraction Water fraction Solvent fraction Water fraction Solvent fraction Water fraction Solvent fraction
2.1 d 0 c 2.4 d 0 c 1.8 cd 1.4 d 0 b 8.6 d 0 c 3.2 a 18.1
Culture filtrate(control)
Petroleum ether
2
Leaf-necrosis assay (mm ) a 16.1 b 0 a 15.7 b 0 a 18.1
Culture filtrate(control)
c
Column means followed by the same letter(s) are not significantly different (P â&#x2030;Ľ 0.05) by DMRT. Culture filtrates from R. solani cultures grown for 20 days in Richard's medium served as controls.
Figure 3. Eight fractions of Silica gel column chromatography were analyzed by TLC. The solvent system was ethyl acetate/methanol, [10:1, (v/v)]; only three pots were found, and the second fraction had the highest bio-activity.
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Table 2. The results of eight fractions of silica gel column chromatography.
Fraction 1 2 3 4 5 6 7 8
Rf value 0 0.71 0 0 0.57 0.43 0 0
2
Leaf-necrosis assay (mm ) d 0 a 5 d 0 d 0 b 2 c 1 d 0 d 0
Column means followed by the same letter(s) are not significantly different (P ≥ 0.05) by DMRT.
fraction. Leaves of corn, soybean, rape, Chinese radish, and Chinese cabbage showed no necrosis. When the concentration of the fraction was increased, there was no necrosis in the leaves, however, necrosis was evidently found in rice leaves, suggesting that the toxins of R. solani are hostspecific in nature. UV (EtOH) λmax (nm) (logε) was 227.2 (2.27) and 296.73 –1 (3.575); IR (KBr, cm ): 3383.67 (–OH); 2933.05 (–CH2–); 2886.46 and 1386.54 (–CH3); 1767.61 and 1724.08 (lactone, ketone or carboxylic acid); 1643.94 (benzene C=C); and 1254.46, 1145.10 and 918.17 (C–O–C) (Figure 4). The absorption bands of the UV spectrum suggest that the toxin that was purified and had a higher absorption at 296.73 nm was the compound we had analysed. The IR spectrum showed bands characteristic of the lactone, ketone (1767.61, 1724.08) and benzene (1643.94) groups. Compared with the information from the database, the presence of lactone, ketone and benzene groups in R. solani culture filtrates was confirmed.
DISCUSSION Phytotoxins produced by fungi are often released into the artificial medium in very low amounts, causing difficulties in their isolation and purification; hence, we prepare larger amounts of the growth medium to cultivate the pathogen so that we may obtain a greater yield of the compounds (Strobel, 1982; Strange, 2007). In this study, R. solani cultivates grown in Richard’s medium for 20 days yielded more than 298.8 mg/l of crude toxins. When the fungus was incubated for 25 days, there was a reduction in the overall toxic activity, which is probably due to the degradation of the toxin in the culture medium. R. solani toxin is heat stable because the culture filtrate still showed high toxicity after exposure to a temperature of 121°C for 30 min (Chen, 1992). Solvent extraction to recover organic compounds with novel activity has been considered one of the most effective methods to isolate phytotoxins from other fungal metabolites (Vikrant, 2006). We used ethyl acetate to extract the R. solani toxin from the culture filtrate. Variable phytotoxicity of different batches of culture filtrates was observed in this study and this might be related to the age of the cultures, because toxin production of R. solani isolates was reduced after
transferring for several generations. However, the major phytotoxin, R. solani toxin, which was the most phytotoxic compound observed in the culture filtrate, was purified in this study by a combination of two chromatography steps after extraction with ethyl acetate. Although the chemical structures of the phytotoxins produced by several types of R. solani are known, the physical characteristics of the R. solani toxin differ from the previously described phytotoxins produced by other types of R. solani. Preliminary characterisation has shown that the partial R. solani toxin is an acidic compound containing phenolic hydroxyl groups (Chen, 1958; Akoi et al., 1963; Iacobellis and Devay 1987; Chen et al., 2001). Full structural characterisation and identification of R. solani toxin is currently in progress. The activity of phytotoxins in terms of symptom production on rice leaves was further substantiated in dosage–response curves, wherein increased concentrations of the purified phytotoxin caused increased severity of symptoms. A high concentration of the pure phytotoxin caused severe leaf-blight symptoms similar to those produced by R. solani in the later stages of infection. This implies that the R. solani toxin may be involved in the virulence of R. solani on rice leaves (Robert, 1984; Jiang, 2008). A more-intensive study of the ability of resistant rice cultivars to withstand the effects of the toxin and of the vulnerability of susceptible rice cultivars may shed light on the mechanism of symptom development in infected plants at the macroscopic, cellular and molecular levels.
Conclusion The R. solani toxin was isolated and showed significantly high toxicity. The toxin was thermally stable even after incubation at 121°C for up to 30 min. A pH of 2.0 was helpful for pathogenesis; after 12 h, yellow lesions were found. The solvent system ethyl acetate/methanol (10:1,
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Figure 4. Infrared spectrum (IR spectrum) used to conduct the biologically active compound obtained from silica gel column chromatography. The IR spectra were recorded in KBr.
v/v) was the optimal solvent system used for pre-parative thin-layer chromatography. Eight fractions were collected by silica gel column chromato-graphy, but only three spots were obtained in the thin-layer chromatography analysis. The second fraction was found to be the one with the highest activity and the toxin appeared to be mainly present in this fraction. The ultraviolet and infrared spectral data indicated that lactone, ketone and benzene groups existed in the culture filtrate. REFERENCES Akoi H, Sassa T, Tamura T (1963). Phytotoxic metabolites of Rhizoctonia solani. Nature, 200: p. 575. Betancourt O, Ciampi L (2000). Contribution to the study and control of Rhizoctonia solani. Extraction and bioassay of phenylacetic acid phytotoxicity produced in vitro by R. solani AG-3(J). Phytopathol. 35(2): 119-125. Chen HG, Wang YZ, Chen LF, Shi JM (2001). Secondary Metabolites of Rhizoctonia cerealis Vander Hoeven (in Chinese). Jiangsu J. Agric. Sci. 17(3): 167-171.
Chen J, Yao JM, Liu WZ, Song ZH (1992). Preliminary study on toxin of rhizoctonia solani in rice sheath blight. J. Shenyang Agric. Univ. 23(1): 19-22. Chen Y (1958). Studies on metabolic products of Hypochnussasakii Shirai. Isolation of p-hydroxy-phenylacetic acid and its physiological activity. B. Agric. Chem. Soc. Jpn. 22: 136-142. Frank JA, Fancis SK (1976). The effect of a Rhizoctonia solani phytotoxin on potatoes. Can. J. Bot. 54: 2536-2540. Huang WW (2009). Preliminary study on the extraction of crude toxin of rhizoctonia solani and its activity. J. Mal. Anhui Agric. Sci. 37(27): 13342-13345. Iacobellis NS, Devay JE (1987). Studies on pathogenesis of rhizoctonia solani in beans: an evaluation of the possible roles of phenylacetic acid and its hydroxy derivatives as phytotoxins. Physiol. Mol. Plant, 30: 421-432. Jayaraman J, Ranganathan B, Ramalingam R, Subbaratnam M, Rethinasamy V (2010). Oxalic acid-induced resistance to Rhizoctonia solani in rice is associated with induction of phenolics, peroxidase and pathogenesis-related proteins. J. Plant Interact. 5(2): 147-157. Jing SJ, Qiang S, Zhu YZ, Dong YF (2008). Isolation and phytotoxicity of a metabolite from curvularia eragrostidis and characterisation of its modes of action. Assoc. Appl. Biol. 152: 103-111. Kenning LA, Hanchey P (1980). Ultrastructure of lesion formation in Rhizoctonia-infected bean hypocotyls. Phytopathol.70: 998-1004.
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Lakpale N, Rajiv K, Khare N (1996). Studies on toxin produced by Rhizoctonia solani causing sheath blight of rice. Indian J. Mycol. Plant P. 26(3): 263-265. Lu NH, Xu RF, Wu LM, Zhang DF (2005). Effects of Different Media on the Growth Reproduction and pathogenicity of Rhizoctonia Cerealis Varder Hoeven. Chinese Agric. Sci. B. 21(2): 262-263. Mandava NB, Orellana RG, Warthen DJ (1980). Phytotoxins in Rhizoctonia solani: isolation and biological activity of m-hydroxy and m-methoxyphenylacetic acid. J. Agri. Food Chem. 28(1): 71-75. Ramalingam P (1986). Involvement of Rhizoctonia solani toxins in phenylalanine ammonia lyase and tyrosine ammonia lyase activities of rice tissues. Curr. Sci. (India). 55(3): 157-159. Roy AK (1993). Sheath blight of rice. Indian Phytopathol. 46: 197-205. Xu JY, Zhang HD, Zhang H, Tong YH, Xu Y, Chen XJ, Ji ZL (2004). Toxin Produced by Rhizoctonia solani and Its Relationship with Path0genicity of the Fungus. J. Yangzhou Univ. 25(2): 61-64. Robert P, Scheffer, Robert S, Livingston (1984). Host-selective Toxins and their Role in Plant Diseases, Science, 223(4631): 17-21. Song HS, Lim SM, Clark JM (1993). Purification and partial characterization of a host-specific pathotoxin from culture filtrates of septoria glycines. J. Physiol. Biochem. 83(6): 659-661. Strange RN (2007). Phytotoxins produced by microbial plant pathogens. Nat. Prod. Rep. 24: 127-144. Strobel GA (1982). Phytotoxins. Annu. Rev. Biochem. 51: 309-333.
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Vidhyasekaran P, Borromeo ES, Mew TW (1986). Host-specific toxin production by Helminthosporium oryzae. Phytopathol. 76: 261-266. Vidhyasekaran P, Borromeo ES, Mew TW (1992). Helmin-thosporium oryzae toxin suppresses phenol metabolism in rice plants and aids pathogen colonization. Physiol. Mol. Plant, 41: 307-315. Vidhyasekaran P, RubyPonmalar T, Samiyappan R, Velazhahan R, Vimala R, Ramanathan A, Paranidharan V, Muthukrishnan S (1997). Host-Specific Toxin Production by Rhizoctonia solani, the Rice Sheath Blight Pathogen. Am. Phytopathol. Soc. 87: 1258-1263. Vikrant P, Verma KK, Rajak RC, Pandey AK (2006). Characterization of a phytotoxin from Phoma herbarum for management of Parthenium hysterophorus L. J. Phytopathol. 154: 461-468. Zheng L, Lv R, Huangn J, Jiang D, Hsiang T (2010). Isolation, purification, and biological activity of a phytotoxin produced by Stemphylium solani. Plant Dis. 94: 1231-1237.
African Journal of Biotechnology Vol. 11(29), pp. 7528-7534, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.2959 ISSN 1684–5315 © 2012 Academic Journals
Full Length Research Paper
Changes in transforming growth factor (TGF)-β and mothers against decapentaplegic homolog (Smad) expression in chronic asthmatic rats induced by ovalbumin and aluminum hydroxide Zhu-Mei Sun, Fu-Feng Li*, Peng Qian and Jie Zhao Basic Medical Department, Traditional Chinese Medicine of Shanghai University, NO1200, Cailun Road, Pudong district, Shanghai 201203, China. Accepted 14 March, 2012
The aim of this study was to investigate the transforming growth factor (TGF)-β1/mothers against decapentaplegic homolog (Smad) signaling pathway associated with airway reconstruction in chronic asthmatic rats by studying mRNA and protein expression. Twenty-four (24) Sprague-Dawley rats were randomly divided into two groups: a normal control group treated with 0.9% saline and an OVE+ALU group treated with a mixture of 10% ovalbumin and 10% aluminum hydroxide. Hematoxylin-eosin (HE) staining was used to observe the pathomorphological changes, and the locations of TGF-β and Smad 2, 4 and 7 were determined by immunohistochemistry. The mRNA expression of TGF-β and Smad 2, 4 and 7 was determined by real time-polymerase chain reaction (RT-PCR). The protein expression of TGF-β was determined by enzyme-linked immunosorbent assay (ELISA), whereas that of Smad 2, 4 and 7 was determined by western blotting. HE staining revealed that the OVE+ALU group displayed obvious signs of bleeding and expansion of lung alveolar and inflammatory cells. Immunofluorescence showed that TGF-β and Smad 2, 4 and 7 proteins were located in the bronchial wall or alveolar wall. RT-PCR showed that mRNA expression of Smad 2 was significantly higher in the OVE+ALU group than in the control group (P < 0.05) and that mRNA expression of Smad 7 was significantly lower in the OVE+ALU group than in the control group (P < 0.05). There was no significant difference in Smad 4 mRNA expression between the two groups (P > 0.05). Western blotting showed that TGF-β1 and Smad 2 protein expression was significantly higher in the OVE+ALU group than in the control group (P < 0.05), whereas, in contrast, Smad 4 and 7 protein expression was significantly lower in the OVE+ALU group than in the control group (P < 0.05). The process of chronic asthma rats, TGF-β and Smad 2, 4 and 7 expressions were changeable, thus the TGF-β/Smad signaling pathway may play a role in chronic asthma. Key words: Chronic asthma, Smad 2, 4 and 7, real time-polymerase chain reaction (RT-PCR).
INTRODUCTION Bronchial asthma is a chronic inflammatory syndrome
*Corresponding author. E-mail: fufeng_lee@hotmail.com. Abbreviation: TGF, Transforming growth factor.
that is highly prevalent worldwide, affecting approximately 300 million individuals of all ages (World Health Organization, 2007). The incidence of chronic asthma has increased in recent years due to environmental pollution caused by car exhausts and lifestyle changes. The concept of asthma control includes clinical and functional manifestations, such as symptoms, nocturnal awakenings, use
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of rescue medication, activity limitation, and pulmonary function (Global Initiative for Asthma, 2009). Asthma has multiple negative influences on the patient’s quality of life. Although, majority of asthma patients can obtain the targeted level of control, some patients will not achieve control even with the best therapy (Nathan, et al., 2004). Patients who do not reach an acceptable level of control with the use of reliever medication plus two or more controllers can be considered to have difficult-to-treat asthma (Wenzel, 2005). Asthma is a chronic complex inflammatory airway disorder characterized by variable degrees of recurring symptoms of airflow obstruction and bronchial hyper responsiveness (National Institute of Health, 2007), and airway reconstruction is one of the pathophysiological characteristics of chronic asthma. The performances of the airway re-established include thickening of smooth muscle and the airway basal surface, deposition of extracellular matrix, infiltration of inflammatory cells, overgrowth of glandular organs and hypertrophy, which can lead to airway hyper-reactivity, irreversible airflow blockage, etc. Remodeling processes in asthma result from highly complex and poorly defined interactions between inflammatory and resident structural cells (James, 2005). Therefore, the identification of the mole-cular pathways involved in the crosstalk between these cells is a prerequisite for the development of novel therapy to control airway remodeling. Previous studies (Chu et al., 1998) have demonstrated that the transforming growth factor (TGF)-β/ mothers against decapentaplegic homolog (Smad) signaling path-way is very
important in airway re-established in chronic asthma. TGF-β is a founding member of the TGF-β super family, including activins inhibiting growth and differen-tiation factors, and bone morphogenetic proteins. TGF-β1 and its isoforms (TGF-β2 and TGF-β3) are synthesized by a variety of cells including all cell types, and are secreted as latent precursors (latent TGF-β1) complexed with latent TGF-β binding proteins (LTBP) (Wang et al., 2005; Roberts, 1998). Active TGF-β then binds its recep-tors and functions to exert its biological and pathological activities via Smad-dependent and independent signaling pathways (Derynck and Zhang, 2003). The Smad complexes then accumulate in the nucleus to regulate target gene expression cooperatively with other transcription factors in a cell context-dependent manner (Feng and Derynck, 2005; Siegel and Massague, 2003; Moustakas et al., 2001). The phosphorylated Smad 2 and 3 then bind to Smad 4 and form the Smad complex, which translocate into the nucleus and regulates the target gene transcription, including Smad 7. Smad 7 is an inhibitory Smad that functions to block Smad 2/3 activation by degrading the TβRI (Hui, 2011). This study aimed to determine whether airway reconstruction involves changes in the TGF-β/Smad signaling pathway.
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MATERIALS AND METHODS Experimental animals Specific pathogen-free Sprague-Dawley (SPF SD) male rats, weighing 180 to 200 g were obtained from the Experimental Animal Center of the Chinese Academy of Science (Shanghai, China). Experimental protocol Twenty-four (24) healthy SPF SD male rats were randomly divided into two groups: a normal control group (n = 12) and an OVE+ALU experimental group (n = 12). Each rat in the control group received subcutaneous injections of 1 ml of 0.9% saline every day for two weeks, whereas the rats in the OVE+ALU group were injected with 1 ml of a solution of ovalbumin and aluminum hydroxide dissolved in 0.9% saline (ova:alu:saline = 1:1:8, v/v). After two weeks, all the rats were sprayed with 2% ovalbumin aerosol in a sealed container every other day for two weeks to induce asthma. Except for the dead ones, all the rats were anesthetized with ether.
Histological examination Lung tissues taken from each rat were fixed in 15% buffered paraformaldehyde and dehydrated in a graded alcohol series. The specimens were then embedded in paraffin blocks and cut into 5 μm-thick sections. The sections were stained with hematoxylineosin (HE). Pathological examinations were performed by a pathologist who was blinded to the rats’ treatment assignment. Immunohistochemical analysis Fresh lung tissues taken from rats anesthetized with ether were embedded in buffer containing 4% paraformaldehyde, 20% sucrose and 30% sucrose. The specimens were then cut into 25 μm-thick cryostat sections and mounted on Superfrost plus slides or gelatincoated slides. The slides were stored at -20°C until needed. Before staining, slides were warmed at room temperature for 30 min and fixed in ice-cold acetone for 5 min, followed by air-drying for 30 min. The following antibodies were used: monoclonal anti-TGF-β antibody (1:1000 in phosphate buffered saline (PBS)), monoclonal anti-human Smad 7 antibody (1:1000), monoclonal rabbit antiSmad 2 antibody and monoclonal rabbit anti-Smad 4 antibody (1:1000). After reacting with the secondary peroxidase-conjugated antibody and washed in PBS, the antibody complex was visualized using 3,3-diaminobenzidine. The specimens were observed under a regular light microscope. Reverse transcriptase polymerase chain reaction Total RNA was isolated using a high pure RNA isolation kit according to the manufacturer’s instructions. Contaminating DNA was removed by treating the samples with RNase-free DNase. One hundred micrograms of total RNA was reverse-transcribed and subjected to polymerase chain reaction (PCR) with an initial step of 94°C for 2 min followed by 40 cycles of 94°C for 15 s, 58°C for 30 s and 72°C for 30 s, and a final extension at 72°C for 10 min. The following primers were used: rat TGF-β forward, 5′TACAGGGCTTTCGCTTCAGT-3′ and reverse, 5′-TGGTTGTAGAGGGCAAGGAC-3′; Smad 2 forward, 5′-ATACCCACTCCATTCCAG-3′ and reverse, 5′-CACTATCACTTAGGCACTGG-3′; Smad 4 forward, 5′-AGGTGGCTGGTCGGAAAG-3′ and reverse, 5′-
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A
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Figure 1. Histological profiles of lung tissue in rats (200). A, Control rats; B, OVE+ALU rats.
TTGGTGGATGTTGGATGGTT-3′; Smad 7 forward, 5′-GGCATACTGGGAGGAGAAGA-3′ and reverse, 5′-CTGTTGAAGATGACCTCCTCCAGC-3′; glyceraldehyde 3-phosphate dehydrogenase (GADPH) forward, 5′-CCGTAAAGACCTCTATGCCAACA-3′ and reverse, 5′-GGACTCATCGTACTCCTGCT-3′. All samples were subjected to reverse transcriptase (RT)-PCR along with the housekeeping gene GAPDH as an internal standard, which was amplified using the following primer sequences: GAPDH forward, 5′-CCGTAAAGACCTCTATGCCAACA-3′ and reverse, 5′GGACTCATCGTACTCCTGCT-3′. Reaction specificity was confirmed by gel electrophoresis of products after RT-PCR and melting curve analysis. Ratios for Smad/GAPDH mRNA were calculated for each sample and expressed as the mean ± standard deviation (SD). Western blotting Total protein was extracted from lung tissues and quantified using a bicinchoninic acid protein concentration assay kit. Samples (50 μg) were fractionated on a 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) separating gel using a Bio-Rad electrophoresis apparatus (Bio-Rad Laboratories, Hercules, CA, USA). After transferring proteins onto nitrocellulose membrane, the blots were probed with a mouse monoclonal antibody to Smad 2 (CST, 1:1000 dilution), a mouse monoclonal antibody to Smad 4 (R&D,1:1000 dilution) and a mouse monoclonal antibody to Smad 7 (Santa Cruz,1:1000 dilution). The secondary antibody was goat anti-mouse IgG (1:1000), which was diluted in PBS containing 1% fetal calf serum. GAPDH was used as an internal control. Statistical analysis Data were calculated as the mean ± SD and the groups were compared using one-way analysis of variance (ANOVA). Statistical significance was set at P < 0.05.
asthma on lung tissue using HE staining. The histology of the HE-stained tissues showed a marked alteration in the experimental group as compared to the control group (Figure 1).
Immunohistochemistry of lung tissues in chronic asthmatic rats Figure 2 shows the immunohistochemistry of TGF-β and Smads in the lung tissues of chronic asthmatic rats. mRNA expression of TGF-β and Smads in chronic asthma mRNA expression of TGF-β1 and Smad 2 were significantly increased in the experimental group when compared with the control group (P < 0.05). There was no significant difference in Smad 4 mRNA expression between the control group and the experimental group. mRNA expression of Smad 7 decreased significantly in the experimental group when compared with the control group (P < 0.05) (Table 1 and Figure 3). TGF-β and Smad protein expression in chronic asthma Expression of TGF-β in lung tissue increased significantly in the experimental group, as measured by enzyme-linked immunosorbent assay (ELISA) (P < 0.05). Expression of Smad 2, 4 and 7 in lung tissue increased significantly in the experimental group as detected by western blotting (Table 2).
RESULTS DISCUSSION Histology of lung tissues in chronic asthmatic rats
We examined the effects of OVE+ALU-induced chronic
The TGF-β family of cytokines encompasses a large number of versatile poly-peptide growth factors regulating
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Figure 2. Immunohistochemistry of TGF-β and Smads in the lung tissues of chronic asthmatic rats. A, Smad 2 (control group); B, Smad 2 (OVE+ALU group); C, Smad 4 (control group); D, Smad 4 (OVE+ALU group); E, Smad 7 (control group); F, Smad 7 (OVE+ALU group); G, TGF-β (normal group); H, TGF-β (OVE+ALU group). The results show that TGF-β and Smad 2, 4, and 7 were all expressed on the bronchial or alveolar wall. All groups are different about fluorescence intensity. TGF, Transforming growth factor; Smad, mothers against decapentaplegic homolog.
a multitude of cellular processes in almost every aspect of cellular function. TGF-β is a widely acknowledged multifunctional factor that can regulate cell growth, differentiation, apoptosis and synthesis of extracellular matrix (ECM) (Zhang et al., 2009). TGF-β mediates biological effects through the TGF-β1/Smad signaling
pathway. Much has been known about the TGF-β signaling pathway since the discovery of Smads 15 years ago; remarkable progress has been made during the past few years in revealing details of different regulatory mechanisms that afford a full range of control of the biological functions of the TGF-β (Ying and Zhang, 2011).
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Table 1. Expression of TGF-β1 and Smad 2, 4 and 7 mRNA (mean ± SD, n = 6).
Group Normal control Experimental
TGF-β/GAPDH 0.50 ± 0.31 1.09 ± 0.31*
Smad 2/GAPDH 0.74 ± 0.22 1.30 ± 0.13*
Smad 4/GAPDH 0.81 ± 0.22 0.77 ± 0.20
Smad 7/GAPDH 1.05 ± 0.09 0.55 ± 0.23*
TGF, Transforming growth factor; Smad, mothers against decapentaplegic homolog; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Figure 3. mRNA expression of TGF-β and Smads in chronic asthma in the lung tissues of chronic asthmatic rats. A, TGFβ1; B, Smad 2; C, Smad 4 control group; D, Smad 7 OVE+ALU group; E, GAPDH; lanes 1, 3, 5 and 7 (normal control group); 2 lanes, 4, 6, 8, 9 (OVE+ALU group). TGF, Transforming growth factor; Smad, mothers against decapentaplegic homolog; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Cell surface receptors of TGFβ signaling are mainly classified into two subtypes: type I (TGFβRI) and type II (TGFβRII). Smad-dependent TGFβ signaling from cytoplasm to nucleus are primarily three Smad\isoforms in the Smad family, that is, Smad 2, 3 and 4. The binding of ligands to TGFβRII leads (TGFβRI) to phosphorylated Smad 2 and 3 which then bind to Smad 4 forming a trimeric complex and translocate into the nucleus. In the nucleus, the Smad trimeric complex binds the Smad binding element (SBE) of target genes, regulating expression of TGFβ response genes directly or through recruiting other co-factors (co-activators or co-repressors) to target genes (Feng and Derynck, 2005; Liu and Feng, 2010). A third class of Smads, negatively regulate TGF-β and bone morphogenetic protein (BMP) signaling by
competing with R-Smads for binding to TβRI or targeting receptors to proteasomal degradation, therefore, are named the inhibitory Smads (I-Smads) (Imamura et al., 1997; Hayashi et al., 1997; Ebisawa et al., 2001; Kavsak et al., 2000). Previous studies have demonstrated that there are three types of Smads: receptor-regulated (Smad 1, 2, 3, 5 and 8), inhibitory (Smad 6 and 7) and universal (Smad 4) types (Jiang et al., 2008; Massague and Wotton, 2000). In this study, we examined the effects of OVE+ALUinduced chronic asthma on lung tissue using HE staining, histology of the HE-stained tissues showed a marked alteration in the experimental group as compared to the control group, that it means the model rats are success which means ovalbumin and aluminum hydroxide can induce the rat’s bronchial asthma.
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Table 2. Expression of TGF-β1 and Smad 2, 4 and 7 (mean ± SD, n = 6).
Group Normal control Experimental
TGF-β1 (ng/ml) 14.5 ± 2.2 22.0 ± 2.1*
Smad 2/NADPH 33.52 ± 1.05 57.94 ± 33.02*
Smad 4/NADPH 166.41 ± 15.29 125.59 ± 47.58*
Smad 7/NADPH 102.29 ± 5.0 43.87 ± 5.19*
TGF, Transforming growth factor; Smad, mothers against decapentaplegic homolog; NADPH, Nicotinamide adenine dinucleotide phosphate.
In this study, we determined mRNA expression of TGFβ1, Smad 2, 4 and 7. The results show that TGF-β1 and Smad 2 increased significantly in the experimental group when compared with the control group. There was no significant difference in Smad 4 mRNA expression between the control group and the experimental group. mRNA expression of Smad 7 was significantly decreased in the experimental group when compared with the control group. TGF-β and Smad protein expression in chronic asthma were detected by ELISA and western blotting. Protein expression of TGF-β in lung tissue increased significantly in the experimental group. Protein expression of Smad 2, 4 and 7 in lung tissue increased significantly in the experimental group. We showed that the TGF-β1/Smad signaling pathway was altered in the airway reconstruction in chronic asthma. Through the investigation of gene and protein expression of TGF-β1 and Smad 2, 4 and 7, we showed that TGF-β1 protein and mRNA were both significantly increased, thus indicating that TGF-β1 was activated. During TGF-β signal transduction, TGF-β receptor is initially activated. TGF-β and its receptor specifically recognize the Smad subgroup known as receptoractivated Smads [R-Smads (Smad 2 and 3)] (Liu et al., 2010). R-Smads are subsequently activated to form a complex consisting of R-Smads and Smad 4, the latter of which is a co-Smad. The Smads transcriptional complex translocate to the nucleus, where it binds to a certain domain of the target gene and causes gene expression, for example, in the genes it involved collagen production. In chronic asthma, up-regulation of TGF-β may activate the TGF-β receptor, which specifically recognizes the Smad subgroup. The TGF-β receptor then modulates the high expression of Smad 2, which coincides with the receptor-regulate. Excess collagen production can cause alveolar expansion verily, thus, leading to the occurrence of asthma. The role of Smad 7 in the TGF-β/Smad signaling pathway is the inhibition of R-Smads phosphorylation and the blocking of signal transduction, thus, terminating gene expression. In chronic asthma, Smad 7 expression is down-regulated; this causes the balance to be upset in order to promote the TGF-β/Smad, thus, signaling a pathway that can induce proliferation. Smad 4 forms a complex consisting of R-Smads in the TGF-β/Smad signaling pathway. In chronic asthma,
Smad 4 gene expression was not altered but protein expression was down-regulated. Further research is needed to investigate this observation. REFERENCES Chu HW, Halliday TL, Martin RJ, Martin RJ, LeungD, Szefler SJ, Wenzel SE (1998). Collagen deposition in large airways may not differentiate severe asthma from milder forms of the disease. Am. J. Rerpir. Crit. Care Med. 158(6): 1936-1944. Derynck R, Zhang YE (2003). Smad-dependent and Smad-independent pathways in TGFbeta family signalling. Nature, 425: 577-584. Ebisawa T, Fukuchi M, Murakami G, Chiba T, Tanaka K, Imamura T, Miyazono K (2001). Smurf1 interacts with transforming growth factorbeta type I receptor through Smad7 and induces receptor degradation. J. Biol. Chem. 276: 12477-12480. Feng XH, Derynck R (2005). Specificity and versatility in tgf-beta signaling through Smads. Annu. Rev. Cell. Dev. Biol. 21: 659-693. Global Initiative for Asthma (2009).Global Strategy for Asthma Management and Prevention. Bethesda: National Institutes of Health. Hayashi H, Abdollah S, Qiu Y, Cai J, Xu YY, Grinnell BW, Richardson MA,Topper JN, Gimbrone MA, Wrana JL, Falb D (1997). the MADrelated protein Smad7 associates with the TGFbeta receptor and functions as an antagonist of TGFbeta signaling. Cell. 89 (7): 11651173. Hui Yao L (2011). Diverse Roles of TGF-β/Smads in Renal Fibrosis and Inflammation. Intl. J. Biol. Sci. 7(7): 1056-1067. Imamura T, Takase M, Nishihara A, Oeda E, Hanai J, Kawabata M, Miyazonok (1997). Smad6 inhibits signalling by the TGF-beta superfamily. Nature, 389(6651): 622-626. James A (2005) .Airway remodeling in asthma. Curr. Opin. Pulm. Med. 11: 1-6. Jiang YC, Shen H, Dai FY (2008). Smad-mediated TGF-I3 signal transduction. Sericulture Communications, 28(3): 20-23. Kavsak P, Rasmussen RK, Causing CG, Bonni S, Zhu H, Thomsen GH, Wrana JL (2000). Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation. Mol. Cell. 6: 1365-1375. Liu N, Zhang PQ, Wang LY (2010). Renal failure Xiezhuo pill on renal interstitial fibrosis in rat TG F-13/smads signal transduction pathway. J. Med. 38(4): 22-24. Liu T, Feng XH (2010). Regulation of TGF-beta signalling by protein phosphatases. Biochem. J. 430(2): 191-198. Massague J, Wotton D (2000). Transcriptional control by the TGF13/Smad signaling system. EMBO, 19(8): 1745-1748. Moustakas A, Souchelnytskyi S, Heldin CH (2001). Smad regulation in TGF-beta signal transduction. J. Cell. Sci. 114: 4359-4369. Nathan RA, Sorkness CA, Kosinski M, Schatz M, Schat M, Li JT, Marcus P, Murray JJ, Pendergraft TB (2004). Development the asthma control test: a survey for assessing asthma control. J. Allergy. Clin. Immunol. 113(1): 59-65. Roberts AB (1998). Molecular and cell biology of TGF-beta. Miner Electrolyte Metab. 24: 111-9. Siegel PM, Massague J (2003). Cytostatic and apoptotic actions of
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TGF-beta in homeostasis and cancer. Nat. Rev. Cancer, 3: 807-821. US Department of Health and Human Services, National Institute of Health, National Heart, Lung, and Blood Institute National Asthma Education and Prevention Program (2007). Expert Panel Report 3: Guidelines for the Diagnosis and management of Asthma. http://www.nhlbi.nih.gov/guidelines/asthma/asthgdln.pdf. Wang W, Koka V, Lan HY (2005). Transforming growth factor-beta and Smad signalling in kidney diseases. Nephrology (Carlton), 10(1): 4856. Wenzel S (2005). Severe asthma in adults. Am. J. Respiratory Crit. Care Med. 172(2): 149-160. World Health Organization (2007). Global Surveillance, Prevention and
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African Journal of Biotechnology Vol. 11(29), pp. 7535-7541, 29 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.3738 ISSN 1684–5315 © 2012 Academic Journals
Full Length Research Paper
Identification of overexpressed cytokines as serum biomarkers of hepatitis C virus-induced liver fibrosis using bead-based flexible multiple analyte profiling Shu-Lin Liu1#, Yang-Chih Cheng2#, Chun-Chao Chang2,3, Ai-Sheng Ho4, Chun-Chia Cheng5,6, Ling-Yun Chen1, Jungshan Chang6* and Chia-Chi Wang7* 1
Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan. Division of Gastroenterology and Hepatology, Department of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan. 3 Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University Hospital,Taipei, Taiwan. 4 Division of Gastroenterology, Cheng Hsin General Hospital, Taipei, Taiwan. 5 Institute of Nuclear Energy Research, Atomic Energy Council, Taoyuan, Taiwan. 6 Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan. 7 Division of Hepatology, Buddhist Tzu Chi General Hospital, Taipei branchand School of Medicine, Tzu Chi University, Hualien, Taiwan. 2
Accepted 1 March, 2012
Hepatic inflammation is the stimulator to activate hepatic stellate cells (HSCs) and triggers fibrogenesis. Cytokines are produced during liver inflammation and maybe considered as liver fibrosis biomarker. The aim of this study was to investigate whether cytokines can be used as reliable biomarkers of liver fibrosis using flexible multianalyte profiling (xMAP). A total of 61 chronic hepatitis C patients with different severity of liver fibrosis were enrolled. Liver biopsy was used as standard to assess the severity of fibrosis according to METAVIR classification. Afterward, 15 samples from healthy controls were analyzed and totally 50 cytokines were screened using flexible multi-analyte profiling to discover differential biomarkers. Finally, levels of protein expressions of individual stages of liver fibrosis were measured. In histological examination, the necroinflammatory score (histology activity index, HAI) was increased from F1 to F4 stage in hepatitis C virus (HCV) infected patients, indicating that inflammation was accompanied with the progression of liver fibrosis. Using flexible multi-analyte profiling, four serum cytokines, including IFN-α2 (p=0.023), GRO-α (p=0.013), SCF (p=0.047) and SDF-1α (p=0.024), were identified under antibody specific recognition and elevated with HAI score. This study reveals the relationship between cytokines and liver fibrosis, and demonstrated that IFN-α2, GRO-α, SCF and SDF-1α may be used as biomarkers to predict liver fibrosis. The overexpressed cytokines may play a role in the progression of liver fibrosis and deserves further investigation. Key words: Cytokine, flexible multi-analyte profiling, hepatitis C virus, liver fibrosis.
INTRODUCTION Reliable biomarkers of liver fibrosis are important to
*Corresponding author. E-mail: uld888@yahoo.com.tw, js.chang@tmu.edu.tw. Tel.: 886 2 66289779, +886 2 27361661 Ext. 2335,3423. Fax 886 2 66289009, +886 2 23778620. #These authors contributed equally to this work.
grade the severity of liver disease in clinical practice. Although liver biopsy followed by histological examination is still the gold standard to diagnose liver fibrosis (Afdhal and Nunes, 2004), it is necessary to develop some other methods to improve diagnostic accuracy and reduce the disadvantages of biopsy such as invasive character and small sample size (Regev et al., 2002; Thampanitchawong and Piratvisuth, 1999). Nowadays,
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noninvasive diagnostic techniques such as ultrasonography, computed tomography and magnetic resonance imaging have been used to detect the morphological changes in the hepatic parenchyma for advanced fibrosis (Hirata et al., 2001). As a reliable predictor, FibroScan has been used recently to diagnose liver fibrosis (Zhu et al., 2011). Due to the low cost and ready availability, ultrasonography is the most popular technique to detect liver disease. However, ultrasonography is highly-operator dependent and liver echogenicity can not accurately differentiate hepatic fibrosis from steatosis (Bataller and Brenner, 2005). Therefore, noninvasive serological biomarkers become one of the solutions for complementing the diagnosis of liver fibrosis, indicating that the development of reliable noninvasive biomarkers for assessing hepatic fibrosis in therapeutic purpose is urgent. Approximately 70% of the patients infected with heap-titis C virus (HCV) become chronic carriers (Marcellin, 1999) and chronic inflammation could lead to fibrosis, liver cirrhosis or even hepatocellular carcinoma. Liver fibrosis is the process of repair when liver is injured or in inflammation (Marcellin, 1999). In liver tissue, hepatic stellate cells (HSCs) are the major source of extracellular matrix proteins (ECMs) excretion in facing constant inflammation and eventually triggers fibrogenesis (Eng and Friedman, 2000; Friedman, 2000, 2003; Marcellin, 1999). Furthermore, cytokines that participate in the inflammatory process (Marra, 2002) are secreted into blood. For example, the level of interferon gamma inducible protein 10 (IP-10), increases in the serum of HCV-infected patients, and has significant correlations with poor response to anti-viral therapy (Reiberger et al., 2008). Therefore, serum cytokines may be used as biomarkers of hepatic inflammation or fibrosis. Currently, many biomarkers have been reported to be correlated with liver fibrosis, including alpha-2 macroglobulin, vitamin D binding protein, apolipoprotein AI (Ho et al., 2010), tissue inhibitor of metalloproteinases-1 (TIMP-1), hyaluronic acid (HA), N-terminal propeptide of type III procollagen (PIIINP), and YKL-40 (Bataller and Brenner, 2005; Johansen et al., 2000; Poynard et al., 2002). Moreover, several biomarker panels such as FibroTest, Forns index, AST-to-platelet ratio index (APRI) and GlycoCirrhoTest have been reported to stage fibrosis (Callewaert et al., 2004; Le Calvez et al., 2004; Rossi et al., 2003; Thabut et al., 2003). In the past, the discovery of serological biomarkers was limited due to the abundant amount of albumin and IgG in serum, which would decrease the accuracy in analytical experiments. Therefore, the low abundant proteins such as cytokines in serum have not been analyzed. Regardless of two dimension electrophoresis (Gangadharan et al., 2007; White et al., 2007) or other existing technologies, analyzing proteins at micro- or pico-level quantity in serum has been difficult. Using specific antibodies to detect the known proteins based on flexible multi-analyte profiling (xMAP) technology may be
another solution to detect the low-abundant serum proteins. In this study, noninvasive serological biomarkers were discovered using Bio-Plex suspension array system based on xMAP technique. This method provided a broad screening for different proteins using specific antibody (Yurkovetsky et al., 2007). Due to the high reproducibility of robust assay and high precision of immunoassay, we used commercial cytokines 27- and 23-panels to search for putative serum biomarkers of liver fibrosis. Less total assay time, fewer progressive steps and smaller sample volume were needed using xMAP compared to conventional enzyme-linked immunosorbent assay (ELISA). It also provided multiple analyses at the same time and had higher reproducibility because the result was the mean value after multiple readings (Chowdhury et al., 2009; dupont et al., 2005; Prabhakar et al., 2002). By this way, com-parison of 50 cytokines between normal and disease group were completed, and candidate biomarkers were selected. This study aimed to investigate whether overexpressed cytokines could be used as serological biomarkers of liver fibrosis in patients with chronic hepatitis C. MATERIALS AND METHODS Serum sample Serum samples (n = 61) from HCV-infected patients were obtained in Cheng Hsin general hospital in Taiwan (approval No. 97016). Firstly the blood samples were allowed to clot for 30 min at 4°C and then centrifuged at 1400 × g at 4°C. Collected serum was frozen at -80°C for store. The patients were diagnosed by a pathologist using hepatic biopsy procedure and followed in subsequent histological examinations. The serum samples were used to determine the stages of hepatic fibrosis according to METAVIR classification, including F1, F2, F3 and F4. On the other hand, we analyzed three individual HCV-infected samples in every stage of liver fibrosis and compared results to three healthy controls; liver biopsy in control group was not performed due to ethical issues. Cytokine standard preparation The cytokine 23-plex panel or 27-plex panel (Bio-Rad) were used in this study. Each 128 μL of cytokine stocks were added with 72 μL of serum standard diluent and continued to make four-fold serial dilution to eight standard samples totally. Flexible multi-analyte profiling The 25 μL of the serum samples was diluted with 75 μl of the appropriate human serum sample diluent (Bio-Rad). We used BioPlexTM Human Cytokine 27-Plex and 23-Plex Panel to search for putative biomarkers of hepatic fibrosis in serum. The experiment followed the instruction manual described briefly as below. First, 50 μL of anti-cytokine conjugated beads was incubated with 50 μL of diluted standard or sample in each well of a 96-well filter plate at room temperature for exactly 1 h on a shaker with 300 rpm. After removing the solution and washing, we added 25 μL of detective antibody conjugated with biotin to each well and incubate for 30 min
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Table 1. Characteristic of 61 HCV-infected liver fibrosis patients
Characteristic Gender (n) BMI AST (IU/L) ALT (IU/L) WBC (1000/μl) Plt (1000/μl)
F1 (n = 20) Female: 11 Male: 9 26.0±0.8 60.5±6.8 143.6±18.2 6.5±0.3 221.6±15.7
Stages of liver fibrosis F2 ( n = 29) F3 (n = 12) Female: 11 Female: 5 Male: 18 Male: 7 23.6±1.1 24.5±0.9 111.0±40.9 88.7±11.5 131.1±17.5 141.2±21.9 5.7±0.3 6.2±0.4 189.5±7.1 178.6±13.22
F4 (n = 29) Female: 17 Male: 12 25.8±0.8 112.5±8.3 143.0±13.8 5.0±0.3 113.9±8.6
P value (ANOVA)
NS NS NS 0.016 <0.001
BMI, Body mass index; AST, aspartate aminotransferase; ALT, alanine transaminase; WBC, white blood cell; Pit, platelet; NS: non-significant.
on a shaker with 200 rpm. Then 50 μL of streptavidin-Phycoerythrin (PE) was added in each well and incubated for 10 min on a shaker. Afterward, each well was added with 125 μL of assay buffer and it was ready for the measurement. A black was necessary for the measurement and all the incubations were performed in dark.
increased with the progression of liver fibrosis (p = 0.003, Figure 1B), indicating inflammation was accompanied with the progression of liver fibrosis. Discovery of cytokine biomarker of liver fibrosis
Statistical analysis After fluorescent measurement by Bio-Plex 200 suspension array system, we used five-parameter logistic regression algorithms (5PL) to calculate the standard curve. This regression is commonly used in immunoassays and provides a large range of quantification than linear regression analysis. The concentrations of measured cytokine were calculated using Bio-Plex manager 4.1 software. The p value was calculated using one-way analysis of variance (ANOVA), and value less than 0.05 was considered to be statistically significant.
RESULTS Patient profiles A total of 61 patients with chronic hepatitis C were enrolled. The stage of liver fibrosis was determined by histological findings. The clinical characteristics of the samples were investigated by measuring several indi-cators, including aspartate aminotransferase (AST), alanine transaminase (ALT), white blood cells (WBCs), and platelet. The conventional indicators of liver function, AST and ALT, were not associated with the stage of liver fibrosis (Table 1). However, we previously demonstrate that AST and ALT increased in thioacetamide-induced liver fibrosis animal model as compared to healthy controls (Liu et al., 2011). Moreover, the levels of AST (> 60.5 IU/L) and ALT (> 131.1 IU/L) indeed were higher than healthy ones, demonstrating that AST and ALT were the markers of liver disease. WBC and platelet were decreased in advanced liver fibrosis (both p < 0.05) as previously reported (Wai et al., 2003). The APRI index was increased from F1 to F4 stage (p < 0.0001, Figure 1A). Moreover, histology activity index (HAI) score was
To identify the putative cytokine biomarkers of liver fibrosis in this study, xMAP was used. A total of 15 serum samples from HCV-infected patients’ triplicate in each stage of liver fibrosis and healthy controls were analyzed, which were selected in accordance with the level of HAI index in Figure 1B. Moreover, the samples were collected before the patients received any clinical treatment, such as anti-viral therapy, and liver transplantation. The total protein concentration was equivalent in each stage of the serum samples (data not shown). In the xMAP analysis, we totally analyzed 50 available cytokines using commercial 23- and 27-Plex panel (Bio-Rad). Due to the fact that the beads were stained with two different fluorescent dyes and formed 100 different types, it allowed us to analyze the protein level at the same time. After xMAP analysis, we finally found that four cytokines were increased gradually in serum from normal controls and patients with F1 to F4 stage of liver fibrosis, including interferon-α2 (IFN-α2), growth-related oncogene-α (GRO-α), stem cell factor (SCF), and stromal cell derived factor-1α (SDF-1α) (Figure 2). Other measured cytokines were irregular with the stages of liver fibrosis (data not shown).
ROC curves of four serum fibrosis indices We used receiver operating characteristic (ROC) analysis to distinguish normal/F1 from F2 to F4. The area under curve (AUC) of serum fibrosis indices in ROC curves are shown in Table 2, which demonstrated that these four liver fibrosis markers had acceptable discrimination (all > 0.8, p < 0.05) for diagnosing liver fibrosis. Moreover, ROC analysis was also used to determine the sensitivity and 1-specificity of the assay in detecting the severity of liver fibrosis. On this account of taking higher sensitivity and specificity, the
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Figure 1. APRI and HAI levels in the stages of liver fibrosis. (A) APRI index was used to evaluate the enrolled patients with hepatitis C virus-induced liver fibrosis. The result shows that APRI increased gradually from F1 to F4, indicating APRI is a reliable index for predicting liver fibrosis. (B) HAI was also performed to measure the necroinflmmatory levels of the tissues with liver fibrosis, which demonstrated that HAI increased from F1 to F4, indicating inflammation was accompanied with liver fibrosis. AST, aspartate aminotransferase; Plt, platelet; HAI, histology activity index. APRI, AST-to-platelet ratio index.
the cut-off points of the four indices were selected and shown in Table 2.
DISCUSSION This study aimed to widely screen serum cytokines and evaluate their relationship with liver fibrosis using xMAP. Furthermore, four cytokines including IFN-α2, GRO-α, SCF and SDF-1α were identified to elevate in serum in parallel with severity of fibrosis in chronic hepatitis C patients. In addition, the concentrations of these cytokines were measured as references to predict the stage of liver fibrosis in patients with chronic hepatitis C. Clinically, ELISA is a useful tool for determining serum antibody concentrations and is usually performed for disease diagnosis. However, Thierry’s group reported that the results obtained by xMAP assay were not consistant with the data derived from ELISAs and a constant conversion factor (Reijn et al., 2007). Therefore, the absolute concentrations of the four candidate proteins may be different between these two assays and specific reference value must be defined for each type of analysis (Elshal and McCoy, 2006; Ray et al., 2005). For clinical application, it is necessary to measure the reference values for ELISA or xMAP assay; the later one can measure multi-markers at once and could be used to replace conventional ELISA assay in the future. To our knowledge, xMAP assay was used to simultaneously evaluate the cytokine expressions in HCVinfected patients after anti-viral treatment (Wan et al., 2009). In this study, we used xMAP to evaluate the 50 kinds of
cytokines in HCV-infected liver fibrosis. For the searched results, the level of SDF-1α was found to be increased in liver disease such as fibrosis or cirrhosis (Horani et al., 2007; Itoh et al., 1994; Panasiuk et al., 2007; Radaeva et al., 2006; Wald et al., 2004; Wald et al., 2007). However, IFN-α2 and GRO-α were reported to be associated with the stage of liver fibrosis for the first time. IFN-α2 was prioritized to treat chronic hepatitis B and C in many clinical trials (Buti and Esteban, 2005; Cooksley, 2005). Furthermore, interferon plus ribavirin has become the standard of care for the treatment of chronic hepatitis C. On the other hand, GRO-α (also called CXCL1), a chemokine of C-X-C family, significantly increased in liver tissue of alcoholic hepatitis (Maltby et al., 1996). To the best of our knowledge, this is the first report that IFN-α2, SCF and GRO-α increase in liver fibrosis, thus could be used as putative biomarkers for predicting the severity of liver fibrosis. In particular, GRO-α, expressed by macrophages, neutrophils and epithelial cells, with neutrophil chemoattractant activity involved in the accumulation of neutrophils, and thus mediated liver injury and inflammation (Ramaiah and Jaeschke, 2007). Different cytokines are secreted during inflammatory process under many causes of liver injury such as ethanol abuse, viral infection, cholestasis or metabolic syndrome; these four cytokines were confirmed to be specifically associated with the stage of liver fibrosis in histological examination in this study. However, further studies are still needed to reveal the role of the four discovered cytokines in the progression of liver fibrosis. In this study, we also found that other cytokines, including interleukin (IL)-2ra, IL-3, IL-12, IL-16, IL-18, Cutaneous T cell-attracting chemokine (CTACK),
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Figure 2. The overexpressed cytokine biomarkers of HCV-infected liver fibrosis. Totally we discovered four candidate biomarkers with increase trend from normal to advanced hepatic fibrosis by using box-whisker plot, including IFN-α2, SCF, GRO-α and SDF-1α. There were 15 samples analyzed, including the samples from normal controls (n=3), F1 (n=3), F2 (n=3), F3 (n=3) to F4 (n=3). IFN-α2, interferon-α2; GRO-α, growth-related oncogene-α; SCF, stem cell factor; SDF-1α, stromal cell derived factor-1α.
hepatocyte growth factor (HGF), intercellular adhesion molecule-1 (ICAM-1), leukemia inhibitory factor (LIF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor-related apoptosis inducing ligand (TRAIL), vascular cell adhesion molecule-1 (VCAM-1), IP-10, increased in HCV-infected liver fibrosis (F2 to F4) compared to normal or early stage (data not shown). In which, the elevated IL-18 was consistent with the previous study which revealed that higher serum level of IL-18 in HCV infection (Chattergoon et al., 2011). Unfortunately, the level of IL-18 did not
gradually increase with the stages of liver fibrosis (p>0.05, data not shown). Moreover, the elevated IP-10 level was consistent with the study reported previously for predicting the response of HCV viral load, the hepatic inflammatory activity, and fibrotic stage (Reiberger et al., 2008). Those cytokines seem to associate with the HCV infection or hepatic inflammation. Take together, serological biomarkers are one of the solutions to diagnose liver fibrosis in hepatitis C patients in order to decrease the use of invasive liver biopsy. Observably, the cytokines found in this study are in low abundance (<1
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Table 2. ROC curve of the four liver fibrosis indexes.
Index
AUC
Cut-off point (pg/ml)
Sensitivity
1-Specificity
p
IFN-α2
0.907
56.30
0.778
0.167
0.010
GRO-α
0.889
46.45
0.667
0.167
0.013
SCF
0.907
28.95
0.778
0.167
0.010
SDF-1α
0.889
152.65
0.667
0.167
0.013
The cut-off point was selected to distinguish Normal/F1 (n=6) from F2 to F4 (n=9). AUC, Area under curve.
ng/mL). Due to the inference from highly abundant albumin and IgG in serum, the detection of some valuable yet less abundant proteins like cytokines that present in pico or micro levels requires highly sensitive technique. In this study, we found four putative biomarkers of liver fibrosis, including IFN-α2, SCF, GRO-α and SDF-1α, using flexible multi-analyte profiling and measured the protein concentrations in each stage of liver fibrosis. In conclusion, this study was to assess the association of cytokines with liver fibrosis using xMAP. Four cytokines, IFN-α2, GRO-α, SCF and SDF-1α, were found to be increased in serum in parallel with the severity of liver fibrosis in patients with chronic hepatitis C, suggesting they could be used as markers for predicting the severity of liver fibrosis and reduce the usage of invasive liver biopsy in clinical practice. However, the role of these cytokines in the progression of liver fibrosis needs further study to confirm. Competing interests The authors declare that they have no competing interests. Authors’ contributions Shu-Lin Liu: Experimemtal design; Yang-Chih Cheng: Patient's screening and experimemtal design; Chun-Chia Cheng: Experimental operation and article writing; AiSheng Ho: Patient's screening, tissue's collection, serum centrifugation and transportation, co-leader of the project, Jungshan Chang: Experimental design and corresponding author; Ling-Yun Chen: Manuscript revision; Chia-Chi Wang: Patient's screening and data analysis.
ACKNOWLEDGEMENTS This work was supported in part by National Science Council of Republic of China (grant number NSC963111-P-042A-004-Y and NSC 99-2314-B-038-001-MY3),
the Taipei Medical University Hospital, Taipei Medical University (grant number 99TMU-TMUH-06) and Cheng Hsin Rehabilitation Medical Center. Abbreviations HSCs, Hepatic stellate cells; xMAP, flexible multi-analyte profiling; HAI, histology activity index; HCV, hepatitis C virus. REFERENCES Afdhal NH, Nunes D (2004). Evaluation of liver fibrosis: a concise review. Am. J. Gastroenterol. (99): 1160-1174. Bataller R, Brenner DA (2005). Liver fibrosis. J. Clin. Invest. (115): 209218. Buti M, Esteban R (2005). Drugs in development for hepatitis B. Drugs (65): 1451-1460. Callewaert N, Van Vlierberghe H, Van Hecke A, Laroy W, Delanghe J, Contreras R (2004). Noninvasive diagnosis of liver cirrhosis using DNA sequencer-based total serum protein glycomics. Nat. Med. (10): 429-434. Chattergoon MA, Levine JS, Latanich R, Osburn WO, Thomas DL, Cox AL (2011). High plasma interleukin-18 levels mark the acute phase of hepatitis C virus infection. J. Infect. Dis. (204): 1730-1740. Chowdhury F, Williams A, Johnson P (2009). Validation and comparison of two multiplex technologies, Luminex((R)) and Mesoscale Discovery, for human cytokine profiling. J. Immunol. Methods, (340): 55-64. Cooksley WG (2005). Peginterferon-alpha 2a for the treatment of hepatitis B infection. Expert Opin. Pharmacother. (6): 1373-1380. Dupont NC, Wang K, Wadhwa PD, Culhane JF, Nelson EL (2005). Validation and comparison of luminex multiplex cytokine analysis kits with ELISA: determinations of a panel of nine cytokines in clinical sample culture supernatants. J. Reprod. Immunol. (66): 175-191. Elshal MF, McCoy JP (2006). Multiplex bead array assays: performance evaluation and comparison of sensitivity to ELISA. Methods, (38): 317-323. Eng FJ, Friedman SL (2000). Fibrogenesis I. New insights into hepatic stellate cell activation: the simple becomes complex. Am. J. Physiol. Gastrointest. Liver Physiol (279): G7-G11. Friedman SL (2000). Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J. Biol. Chem. (275): 2247-2250. Friedman SL (2003). Liver fibrosis -- from bench to bedside. J. Hepatol. (38 Suppl 1): S38-53. Gangadharan B, Antrobus R, Dwek RA, Zitzmann N (2007). Novel serum biomarker candidates for liver fibrosis in hepatitis C patients. Clin. Chem. (53): 1792-1799. Hirata M, Akbar SM, Horiike N, Onji M (2001). Noninvasive diagnosis of the degree of hepatic fibrosis using ultrasonography in patients with
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African Journal of Biotechnology Vol. 11(29), pp. 7542-7548, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.3466 ISSN 1684–5315 © 2012 Academic Journals
Full Length Research Paper
Highly efficient in vitro adventitious shoot regeneration of Adenosma glutinosum (Linn.) Druce using leaf explants Ru-Ping TU1,2, Jun-Yan HU1,2, Qian-Qian JI1,2, Guo-Hua XIA1,2* and Bing-Song ZHENG1,3* 1
School of Forestry and Biotechnology, Zhejiang Agriculture and Forestry University, Lin'an, Zhejiang, 311300, China. Basic Experiment Teaching Center of Forestry, Zhejiang Agriculture and Forestry University, Lin’an, Zhejiang 311300, China. 3 Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, Linan, Zhejiang 311300, China. 2
Accepted 18 January, 2012
Adenosma glutinosum (Linn.) Druce is an important aromatic plant, but no information is available regarding its regeneration, callus induction and proliferation from leaf explants. In this study, an in vitro shoot regeneration procedure was developed for native A. glutinosum using leaf explants. Callus induction and shoots regeneration from leaf explants was evaluated on Murashige and Skoog (MS) media supplemented with combinations of 6-benzylaminopurine (6-BA) and α-naphthaleneacetic acid (NAA). Callus induction in all 16 treatments exceeded 95%, and the highest adventitious shoot number per callus (7.22 shoots per explant) was obtained when leaf explants were cultured on MS medium -1 -1 supplemented with 0.5 mg• L 6-BA, 0.1 mg• L NAA, 3% sucrose and 0.72% agar. The highest shoots strengthening were obtained when adventitious buds were cultured on half-strength MS medium -1 -1 supplemented with 0.3 mg• L NAA, 3% sucrose, 1.0 g• L active carbon and 0.72% agar. The highest total root number (45.2) and root length (43.3 cm) were obtained when adventitious buds were cultured -1 −1 on half-strength MS medium supplemented with 0.0 mg• L NAA, 3% sucrose, 1.0 g L active carbon 2 3 and 0.72% agar, while the highest total root surface area (4.1 cm ) and total root volume (114.1 mm ) were obtained when adventitious buds were cultured on half-strength MS medium supplemented with -1 -1 0.5 mg• L NAA, 3% sucrose, 1.0 g• L active carbon and 0.72% agar. The efficient plant regeneration system developed here will be helpful for rapid micropropagation and further genetic improvement in A. glutinosum. Key words: Adenoma glutinous, plant growth regulator, plant regeneration.
INTRODUCTION Adenosma is a genus of aromatic and ornamental perennial plants of Scrophulariaceae in the South and
*
Corresponding author. E-mail: zjfc_ghxia@126.com; bszheng@zafu.edu.cn. Tel: +0086(0)571 63732761. Fax: 86571-63740809. Abbreviations: MS, Murashige and Skoog; 6-BA, 6benzylaminopurine; NAA, α-naphthaleneacetic acid; AC, activated charcoal.
South-East Asia and Oceania, which include about 15 species. In southern China, there are four species including Adenosma glutinosum, A. indianum, A. javanicum and A. retusilobum (Hong et al., 1998). Adenosma contains eucalyptol, β-bisabolene, limoneneand various volatile oils (Wang and Wei, 2008) which play important role in pharmacological areas that are active against many diseases, especially rheumatism, stasis eliminating and detumescence analgesic (Ji and Pu, 1985; Liang and Zhong, 2005; Wu et al., 2010). Micropropagation offers the potential to produce
Tu et al.
millions of clonal individuals through tissue culture via induction of morphogenesis from various plant tissues. This method is commonly employed for the mass propagation of plant species (Glowacka et al., 2010). To date, tissue culture of several aromatic plants has been reported with an emphasis on embryogenic callus formation, growth, and regeneration of Pogostemon cablin (Paul et al., 2010), Tagetes patula (Qi et al., 2011), Eucalyptus (Deepika et al., 2011), Rosmarinus officinalis (Deng et al., 2008), Adenosma buchneroides (Xu et al., 2008) and A. glutinosum (Liu et al., 2010). Qi et al. (2011) reported that callus induction from anther explants depended on genotype, temperature pretreatment, plant growth regulators, light regimes and sucrose concentration in T. patula. The highest callus induction rate and regeneration frequency of line 21605 was obtained when inflorescence buds were stored at 4°C for 4 days, and anthers with microspores at the mid to late uninucleate stage were cultured on Murashige and Skoog (MS) basal medium containing 6-benzylaminopurine (BA, 2.2 μM) and αnaphthaleneacetic acid (NAA, 1.82 or 2.7 μM). Frequencies of callus induction and shoot regeneration were 100 and 70.5%, respectively with the whole regeneration procedure completed in 40 days under light. Deepika et al. (2011) also compared eight Eucalyptus genotypes for callus induction and shoot regeneration potential and concluded that browning of callus tissue and surrounding culture media is a common obstacle limiting regeneration of shoots in eucalypts and an optimum protocol for the generation of transgenic plants of clonal Eucalyptus genotypes was developed. Furthermore, Deng et al. (2008) studied the effects of different content of 6-BA and NAA on shoot tip culture using shoot tip from R. officinalis as explants and found that MS + 6-BA 1.0 mg• L-1 + NAA 0.02 mg• L-1 favored shoot tip culture and 1/2 MS + IAA 0.3 to 0.4 mg• L-1 was fit for root growth. There are relatively few reports on callus induction and plant regeneration in A. glutinosum. In addition, Liu et al. (2010) established a regeneration procedure of A. glutinosum using shoot tips and found that the cultural system for adventitious buds proliferation of A. glutinosum is the MS medium with 0.25 mg• L-1 of BA and 0.5 mg• L-1 of indole-3-butyric acid (IBA). However, it induced low shoot regeneration and required very long culture periods. Due to the small number of tissue culture studies in this species, embryogenic callus formation, callus growth, and plant regeneration in A. glutinosum are not well understood. Moreover, when considering the development of high-throughput trans-formation systems, an efficient and reliable regeneration system is needed for successful industrial production and also genetic transformation. Hence, the objective of the present study was to examine tissue culture propagation systems and determine suitable optimum growth conditions and culture medium for callus induction for A. glutinosum; we examined both establishments of advent- tious buds and adventitious roots.
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MATERIALS AND METHODS Young A. glutinosum plants (2 to 3 cm long) used as explants were obtained from the botanical garden of the Zhejiang A and F University, Zhejiang, China. These plants were washed under running tap water followed by soaking in 10% liquid dishwasher detergent (P&G Co. Ltd, Cincinnati, OH) for 5 min. Thereafter, the plants were rinsed in distilled water for 8 to 10 min. They were followed by surface sterilization using mercuric chloride (0.1% w/v) in laminar flow cabinet and rinsed with sterilized distilled water 3 times. The apical shoots and axillary bud with internode were excised from the sterilized plants, cut to 8 mm and placed vertically into half strength of MS medium (Murashige and Skoog, 1962) containing various concentrations of 6-benzylaminopurine (6-BA) and α-naphthaleneacetic acid (NAA), with 3% (w/v) sucrose, 1.0 g• L-1 active carbon and 0.72% Type-A agar (Sigma Chemical Co., St. Louis, Mo., USA). Three weeks after initial culture, leaves enlarged enough to be used as leaf explants, were selected for regeneration experiment (Figure 1a). The pH of the medium was adjusted to 5.7 prior to addition of agar and was autoclaved at 105 kPa at 121°C for 20 min. All cultures were kept in growth chambers at 25 ± 2°C with 16-h photoperiod and a photon fluence of about 30 to 40 μmol m−2 s−1. Callus induction and adventitious buds from callus Leaf-derived callus were cultured on MS medium for inducing callus or adventitious buds supplemented with 6-BA at 0.3, 0.5, 1.0 or 2.0 mg• L-1, NAA at 0.1, 0.3, 0.5 and 1.0 mg• L-1 in completely randomized design. The percentage of callus induction, the percentage of adventitious bud induction and the average number of adventitious buds per callus were observed. Three leaf explants were cultured per flask, and each experiment was repeated three times with 10 culture flasks per treatment. Shoots strengthening and roots induction After adventitious shoots reached about 2.0 cm in length with 2-pair of leaves, they were excised from the callus and placed in flasks on the half strength of MS medium containing various concentrations of NAA at 0.0, 0.1, 0.3, 0.5 or 1.0 mg• L-1 in combination with activated charcoal (AC) at 1.0 g• L-1. Five shoots were cultured in each flask, and each experiment was repeated three times with 18 culture flasks per treatment. Individual roots were dissected according to the branching order, starting from the distal end of the root system that was numbered as the first order and then increased sequentially with each branch from the first order to higher order roots. Following dissection, fine root samples were scanned by the Win-RHIZO system to analyze total root length (L), total root surface area (SA), total root volume (V) and total root number (R) per plantlet. Ten roots were scanned, and each treatment was repeated three times. Greenhouse acclimatization The effects of three planting media on ex vitro plantlet acclimatization were studied. Plantlets with well-developed root systems (about 5 cm long) and shoots (6 to 8 cm long) were removed from the flasks and transplanted into plastic pots containing pre-watered mixtures. Three different mixtures were used, including common turf (Zhejiang Hongyue Seeds Co., Ltd, Zhejiang, China), Hawita turf (Hawita Gruppe GmbH, AWITA GRUPPE GmbH, Vechta, Germany) and medium containing a 1:1
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a
b
c
d
e
f
Figure 1. In vitro adventitious shoot regeneration of Adenosma glutinosum (Linn.) Druce using leaf explants. (a) Shoot sprout on stem explants, bar =1 cm; (b) callus induction from leaf explants, bar =0.5 cm; (c) adventitious buds from callus, bar =1 cm; (d) shoots strengthening and roots induction, bar =2 cm; (e) transplant and acclimatization, bar = 5 cm; (f) flowering plant after acclimatization (90 days), bar =5 cm.
(v/v) mixture of common turf: perlite (Zhejiang Hongyue Seeds Co., Ltd, Zhejiang, China). The plants were transferred to a greenhouse at 25 ± 2°C, with a photon fluence of 800 μmol m−2 s−1, humidity levels ranging from 70 to 98% and a 16-h photoperiod. Plantlets were watered at 2-day intervals. After 2 weeks of acclimatization, plantlets were fertilized weekly with 100 mg• L-1 fertilizer containing a 15:15:15 of nitrogen, phosphate and potassium. The percentage plantlet survival was recorded at 30 days after transplanting. Each
treatment consisted of 50 plantlets in triplicate.
Statistical analysis Callus induction was calculated using the following formula: (number of induced callus / total number of leaf explants) × 100%. Adventitious shoot number per callus was calculated using the
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Table 1. Effects of different plant growth regulator combinations on the induction of callus and the differentiation of adventitious shoots form the leaf explants of Adenoma glutinosum. -1
6-BA (mg•L ) 0.3 0.3 0.3 0.3 0.5 0.5 0.5 0.5 1.0 1.0 1.0 1.0 2.0 2.0 2.0 2.0
-1
NAA (mg•L ) 0.1 0.3 0.5 1.0 0.1 0.3 0.5 1.0 0.1 0.3 0.5 1.0 0.1 0.3 0.5 1.0
Number of inoculated 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90
Callus induction (%) a 97.5 ± 3.0 a 97.8 ± 3.9 a 100.0 ± 0.0 a 95.6 ± 5.1 a 98.9 ± 1.9 a 100.0 ± 0.0 a 98.9 ± 1.9 a 100.0 ± 0.0 a 97.8 ± 1.9 a 100.0 ± 0.0 a 98.9 ± 1.9 a 98.9 ± 1.9 a 100.0 ± 0.0 a 100.0 ± 0.0 a 100.0 ± 0.00 a 100.0 ± 0.0
Adventitious shoot number per callus bcd 4.5 ± 0.1 b 5.3 ± 0.5 g 2.5 ± 0.7 h 0.8 ± 0.6 a 7.2 ± 1.0 bc 5.0 ± 0.9 fg 2.9 ± 0.5 fg 2.9 ± 0.2 bcd 4.4 ± 0.5 def 3.9 ± 0.4 efg 3.2 ± 0.1 h 1.4 ± 0.6 bcd 4.8 ± 0.9 cde 4.2 ± 0.1 efg 3.2 ± 0.4 h 0.7 ± 0.1
Values followed by different letters within a column are significantly different at P<0.05. Data of callus induction and adventitious shoots number per callus were obtained after 15 and 30 days culturing, respectively. BA, Benzylamino purine; NAA, naphthalene acetic acid.
following formula: (total number of adventitious shoots / total number of callus). Rooting percentage was calculated as (number of explants with rooting / total number of explants) × 100%. Percentage data were converted to relative proportions, arcsine transformed, and then analyzed for significant differences. The data were analyzed with DPS v2.0 for Windows (Microsoft Corp., Redmond, WA) using two-way analysis of variance with replications for different plant growth regulator combinations on callus induction and adventitious shoot number per callus, and means were separated using Duncan’s multiple range test at P = 0.05. In the final analysis, all data was expressed as the mean of replicate ± standard error (SE).
RESULTS Callus induction and adventitious shoots from callus In all treatments in which callus was produced, callus initiated first on the cut edge of the petiole and 4 days later on the cut blade surface. Callus was loosely packed, yellowish to green and friable (Figure 1b). There were no significant differences in callus induction between all treatments. Callus induction in all treatments exceeded 95%, and the percentage reached 100% in the most treatments with relatively high concentration of 6-BA (0.5, 1.0 and 2.0 mg• L-1). All the treatments with 2.0 mg• L-1 6-BA resulted in 100% callus formation in leaf explants (Table 1). The callus in leaf explants differentiated into meristemoid in 2 weeks under different treatments (Figure 1c). Adventitious shoots were visible on the cut edge of the petiole in 2 to 3 weeks, and they were also visible on cut
blade surface in 4 weeks. Variation in shooting response was observed due to exogenous level of plant growth regulator in the medium. Two-way analysis of variance showed that NAA, 6BA and their interaction had obvious significant effect on adventitious shoot number per callus (P<0.01) (Table 2), while for callus induction, they had non-significance (data not shown). There was also a significant difference in average adventitious shoots number per callus observed. NAA was an important plant growth regulator for promoting shoot regeneration of leaf explants in this experiment. As the concentration of NAA 1 increased (0.1 to 1.0 mg• L- ) under any fixed 6-BA concentration, shoot number per leaf explant tended to decrease (Table 1 and Figure 2). When the concentration of 1 NAA reached 1.0 mg• L- , fewer buds and shoots were produced from leaf explants. However, the treatment of 1 1 combination of 6-BA (0.5 mg• L- ) and NAA (0.1 mg• L- ) resulted in the highest adventitious shoot number per callus nearly 100% (data not shown) and reached 7.2 shoots per explants (Figure 2e).
Shoots strengthening and rooting Shoots about 2.0 cm long with 4 leaves (2 pairs) were excised and transferred to shoots strengthening and rooting medium. Leaf explants initiated roots and axillary bud produced in 5 to 7 days, and developed a cluster with well branched root system within 20 days (Tables 3 and 4; Figure d). 100% rooting frequency was recorded on all cultured explants of all treatments containing different concentration of NAA. NAA at concentration of 0.3 mg• L-1 resulted in the best strengthening effect. The average number of shoots
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Table 2. Two-way analysis of variance with replications for different plant growth regulator combinations on adventitious shoot number per callus.
Source of variation NAA factors 6-BA factors NAA Ă&#x2014; 6-BA interaction Error Total
SS 78.4 20.2 23.4 1.7 123.7
df 3 3 9 32 47
MS 26.1 6.7 2.6 0.1
F 478.6 123.3 47.5
P-value 1.2 E-26 1.2 E-17 5.3 E-16
6-BA, 6-Benzylamino purine; NAA, naphthalene acetic acid; SS, sum of squares; df, degree of freedom; MS, mean of squares.
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
Figure 2. Effects of different plant growth regulator combinations on the induction of callus and the differentiation of adventitious shoots form the leaf explants of Adenoma glutinosum. The ordinal letters a to p match the treatments of Table 1 in series; photos were taken after 30 days culturing. Bottle diameter = 9 cm.
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Table 3. Effects of different concentration of NAA on shoots strengthening and roots induction of Adenoma glutinosum.
NAA -1 (mg•L ) 0.0 0.1 0.3 0.5 1.0
Average number of shoots per cluster
Average number of leaves per cluster
c
a
2.6 ± 0.3 b 3.6 ± 0.6 a 4.6 ± 0.3 b 3.7 ± 0.6 c 2.0 ± 0.2
Average height of cluster (cm)
Frequency of rooting (%)
bc
10.2 ± 0.6 a 10.3 ± 0.3 a 10.5 ± 0.5 a 10.4 ± 0.2 b 9.1 ± 0.7
3.9 ± 0.4 ab 4.3 ± 0.4 a 4.6 ± 0.2 c 3.7 ± 0.2 bc 3.9 ± 0.5
100.0 100.0 100.0 100.0 100.0
Values followed by different letters within a column are significantly different at P<0.05; Data were obtained after 20 days culturing.
Table 4. Effects of different concentration of NAA on root characteristics of regenerate plantlet of Adenoma glutinosum.
NAA -1 (mg•L ) 0.0 0.1 0.3 0.5 1.0
Total root length (cm)
Total root surface area 2 (cm )
a
43.3 ± 2.3 bc 39.0 ± 1.7 bc 39.2 ± 1.4 bc 35.8 ± 2.1 c 36.3 ± 2.6
bc
3.6 ± 0.2 d 2.6 ± 0.1 c 3.1 ± 0.2 a 4.1 ± 0.2 c 3.1 ± 0.1
Total root volume 3 (mm ) b
48.8 ± 3.2 c 30.4 ± 2.6 b 42.9 ± 3.1 a 114.1 ± 5.8 b 48.0 ± 2.4
Total root number a 45.2 ± 6.4 b 22.5 ± 3.4 c 11.9 ± 2.0 b 21.2 ± 3.1 c 14.5 ± 2.2
Values followed by different letters within a column are significantly different at P<0.05; Data were obtained after 20 days culturing.
Table 5. Survival rate of seedlings grown on different supporting mixtures after 30 days.
Transplanting condition Common turf Hawita turf Mixture media
Survival rate after 20 day of transplanting (%) a 91.8 ± 4.2 a 94.2 ± 4.5 a 96.2 ± 5.3
Values followed by different letters within a column are significantly different at P<0.05; Data were obtained after 20 d culturing.
and leaves per cluster reached 4.6 and 10.5, respectively
and the average height reached 4.6 cm. For the effects of different concentration of NAA on root characteristics, MS medium without NAA treatment resulted in the optimum total root length (L) and total roots number (R) per regenerated plantlet, reached 43.3 cm and 45.2, respectively. However, for the total root surface area (SA) and total root volume (V) per regenerated plantlet, the optimum concentration of NAA 1 was 0.5 mg• L- (Table 4).
Greenhouse acclimatization The micropropagated plantlets were uniform and grew vigorously after 30 days of transplanting, with high plantlet survival percentages in greenhouse. In addition, no morphological abnormalities and variations were found when compared with the control. In all three media, plantlet survival percentage was more than 90% (Table 5 and Figure e). Ninety days later, plantlets grew well and flowered (Figure f). All the regenerated plantlets were successfully acclimatized and transferred to soil and can be used for conservation and ornamental purposes.
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DISCUSSION
REFERENCES
Supplement of various plant growth regulators in the medium at appropriate level may have effect on organogenesis and produce various types of cells. The ratio of auxin to cytokinin plays a pivotal role in plant growth and development. In this study, we determined the effects of different combinations of auxin and cytokinin on regeneration from leaf explants of A. glutinosum. Explants cultured on different concentrations of plant growth regulators showed various morphogenic regeneration responses. Regeneration of plants was affected by exogenous and endogenous plant growth regulator,
Deepika R, Veale A, Ma C, Strauss SH, Myburg AA (2011). Optimization of a plant regeneration and genetic transformation protocol for Eucalyptus clonal genotypes. BMC Proceed. 5(7): 132. Deng MH, Wen JF, Zhao K (2008). Shoot tip culture for Rosmarinus officinalis. North Horticult. 10: 158-160. Glowacka K, Jezowski S, Kaczmarek Z (2010). The effects of genotype, inflorescence developmental stage and induction medium on callus induction and plant regeneration in two Miscanthus species. Plant Cell, Tiss. Org. Cult. 102: 79-86. Hong DY, Yang HB, Jin CL, Holmgren NH (1998). Adenosma R. Brown (Scrophulariaceae). In: Wu ZY, Raven PH. (eds), Flora of China. Science Press, Miss. Bot. Gard. Press. pp. 18. Ji XD, Pu QL (1985). Studies on the components of the essential oil from Adenosma indianum (Lour.) Merr. Acta Bot. Sin. 27(1): 80-83. Kumar N, Vijay Anand KG, Reddy MP (2010). In vitro plant regeneration of non-toxic Jatropha curcas L.: direct shoot organogenesis from cotyledonary petiole explants. J. Crop Sci. Biotech. 13(3): 189-194. Liang QC, Zhong M (2005). Zhuang folk Medicine of China. Guangxi Nationalities Publishing House, Nanning. pp. 188. Liu CH, Lun X, Xia GH (2010). Tissue culture and rapid propagation of Adenosma glutinosum (L.) Druce. Plant Physiol. Commun. 46(6): 601-602. Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497. Paul A, Thapa G, Basu A, Mazumdar P, Kalita MC, Sahoo L (2010). Rapid plant regeneration, analysis of genetic fidelity and essential aromatic oil content of micropropagated plants of Patchouli, Pogostemon cablin (Blanco) Benth. – An industrially important aromatic plant. Ind. Crop. Prod. 32: 366-374. Poovaiah CR, Weller SC, Jenks MA (2006). In vitro adventitious shoot regeneration of native spearmint using internodal explants. Hortscience. 41(2): 414-417. Qi Y, Ye Y, Bao M (2011). Establishment of plant regeneration system from anther culture of Tagetes patula. Afr. J. Biotechnol. 10(75): 17332-17338. Raghu AV, Unnikrishnan K, Geetha SP, Martin G, Balachandran I (2011). Plant regeneration and production of embelin from organogenic and embryogenic callus cultures of Embelia ribes Burm. f.—a vulnerable medicinal plant. In Vitro Cell. Dev. Biol. Plant. 47: 506-515. Skoog F, Miller CO (1957). Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp. Soc. Exp. Biol. 11: 118-131. Wang CC, Wei G (2008). GC-MS Analysis of volatile oil in Adenosma glutinosum (Linn.) Druce. Chinese J. Inf. on TCM 15(2): 36-37. Wang XH, Gao ZH, Wang YZ, Bressan RA, Weller SC, Xia L (2009). Highly efficient in vitro adventitious shoot regeneration of peppermint (Mentha × piperita L.) using internodal explants. In Vitro Cell. Dev. Biol. Plant. 45: 435-440. Wu HE, Liang CY, Li YH, Huang XQ, Zhu XY (2010). GC-MS analysis of chemical constituents of the essential oil from Adenosma indianum (Lour.) Merr. by different extraction methods. Chin. J. Pharm. Anal. 30(10): 1941-1946. Xu Y, Cheng BQ, Yu Z, Ding JK (2008). A preliminary study on the new perfume plant Adenosma buchneroides Bonati. In: Proceed. Seventh Perfume Plant Congress, pp. 26-29. Zhou HC, Li M, Zhao X, Fan XC, Guo AG (2010). Plant regeneration from in vitro leaves of the peach rootstock ‘Nemaguard’ (Prunus persica × P. davidiana). Plant Cell, Tiss. Org. Cult. 101: 79-87.
particularly auxins and cytokinins, the explant type and culture conditions (Skoog and Miller, 1957). Cytokinin have been reported to be good plant growth regulators for shoot induction from leaf explants when cultured on MS medium (Poovaiah et al., 2006; Wang et al., 2009; Kumar et al., 2010), while Zhou et al. (2010) and Raghu et al. (2011) reported that supplement of auxin in combination with cytokinin resulted in more number of shoot emergence. Callus induction was higher than 95% in all treatments in the leaf explants of A. glutinosum. The highest adventitious shoot number per callus was observed in leaf explants cultured on 1 media containing combinations of 0.5 mg• L- 6-BA and 0.1 mg• 1 L- NAA. This was in agreement with the previous studies on micropropagation in the other aromatic plants: P. cablin (Paul et al., 2010), T. patula (Qi et al., 2011), Eucalyptus (Deepika et al., 2011), R. officinalis (Deng et al., 2008), and A. glutinosum (Liu et al., 2010). For P. cablin, Paul et al. (2010) reported that the highest number of shoots was obtained from leaf explants on MS medium containing 2.5 μM benzylaminopurine (BAP) and 0.5 μM NAA. Qi et al. (2011) reported that the highest callus induction rate of T. patula were obtained in explants cultured on MS medium containing 2.2 μM BA and 1.82 or 2.7 μM NAA. In 1 addition, Deng et al. (2008) found that MS + 6-BA 1.0 mg• L- + 1 NAA 0.02 mg• L- was favorable for shoot tip culture. However, Liu et al. (2010) found that the cultural system for adventitious buds proliferation of A. glutinosum is the MS medium with 0.25 1 1 mg• L- of BA and 0.5 mg• L- of IBA using shoot tips.
Through this study of tissue culture in A. glutinosum, methods for the induction of callus, adventitious bud and root proliferation, and plant regeneration were established. This new, highly efficient plant regeneration system would be helpful for industrial production and development of a genetic transformation protocol for A. glutinosum. ACKNOWLEDGEMENTS This work was supported by the Department of Science and Technology of Zhejiang Province (2008C12019), the National Natural Science Foundation of China (31070604), the new-shoot talents program of Zhejiang province (No. 100204) and the students’ research training program of Zhejiang Agriculture and Forestry University (No. 100212). We would like to thank the reviewers for their helpful comments and proposals on the manuscript.
African Journal of Biotechnology Vol. 11(29), pp. 7549-7553, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.3684 ISSN 1684â&#x20AC;&#x201C;5315 Š 2012 Academic Journals
Full Length Research Paper
Regeneration of plantlets from nodal and shoot tip explants of Anoectochilus elatus Lindley, an endangered terrestrial orchid N. Ahamed Sherif1*, J. H. Franklin Benjamin1, S. Muthukrishnan1, T. Senthil Kumar2 and M. V. Rao1 1
Department of Plant Science, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India. Department of Industry-University Collaboration, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India.
2
Accepted 2 March, 2012
Anoectochilus elatus Lindley is an endangered terrestrial orchid. A procedure for the regeneration of complete plantlets of A. elatus Lindley through node and shoot tip explants resulted directly in shoots when cultured on a full strength Murashige and Skoog (1962) medium supplemented with cytokinins at different concentrations. An average number of shoots per explant is 3 in shoot tip and 4 in node, respectively. The best shoot proliferation was observed in 3.0 mg/l 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ) and the mean shoot length in 3.5 mg/l 6-furfurylaminopurine (KIN) (node) and 0.01 mg/l (shoot tip). Hundred percent rooting was achieved with the regenerated shoots in the same medium with the addition of 0.3 g/l activated charcoal (AC). Plantlets with well developed leaves and roots were transplanted to pots filled with a mixture of coconut coir, activated charcoal, commercial fertilizers (3:1:1) and acclimatized before been transferred to the greenhouse.
Key words: Anoectochilus elatus, activated charcoal, regeneration, rooting.
INTRODUCTION Orchidaceae is amongst the most diverse family of the flowering plants consisting of 35,000 species under 880 genera (Chugh et al., 2009) occurring widely in the humid tropical forests of India, Sri Lanka, South Asia, South and Central America and Mexico. Orchids belong to the highest level of organization and are distinguished by the complicated biology of reproduction with long developmental cycle (Vakhrameeva et al., 1991). The genus Anoectochilus consists of approximately 40 species belonging to a group of terrestrial orchids commonly known as Jewel orchids due to their attractive foliage, distributed throughout Southeast Asia, New Caledonia and Hawaii (Ket et al., 2004). Anoectochilus elatus Lindley is an endangered orchid
*Corresponding author. E-mail: mvrao_456@yahoo.co.in. Abbreviations: MS, Murashige and Skoog; BA, 6-benzyl adenine; TDZ, 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea; KIN, 6furfurylaminopurine; AC, activated charcoal.
endemic to Tamil Nadu and Kerala. It is a terrestrial herb found in the forest floors rich in humus in the evergreen forest at an altitude of 4265.091 ft. A. elatus prefers a lot of air and light but grows away from sunshine. It was listed endangered in the recent Conservation Assessment and Management Plan (CAMP) Workshop Report for Endemic Orchids of the Western Ghats (Kumar et al., 2001). IUCN (2001) categorizes a species as endangered (EN) when it meets any of the criteria AE. The extent of occurrence of this species (IUCN Criteria B1) is about 10 to 5000 sq. km and the area of occupancy is 10 to 500 sq. km (IUCN Criteria B2). The number of subpopulation range from 10 to 50 which shows highly fragmented nature. Mature individuals in all the population are estimated to be less than 250 (IUCN Criteria D). There is continuing decline in the number of location or subpopulation as a conse-quence of decrease in its habitat (more than 20% in the last 10 to 20 years and more than 20% decline is predicted in the next 10 years); fragmented populations with lesser mature indivi-
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duals leading to demographic instability; human interference. Based on the above data, A. elatus has been listed as endangered. A. elatus being endemic to the Tamil Nadu and Kerala is one of the few orchids or Western Ghats having an extended distribution in Eastern Ghats. A long felt need for the conservation of this species is to undertake proper survey and analyze its genetic relationship of species (Kumar et al., 2001). Several species have been used in Chinese folk medicine and used for hypertension, lung and liver diseases (Liang et al., 1990; Kan, 1986; Zheng et al., 1996). This genus is conventionally propagated by seed; however, the germination rate is very low and nowadays this orchid is under threat of extinction due to over collection from natural resources (Belitsky and Bersenev, 1999). Plant tissue culture technology has been successfully used for the commercial production of pathogen-free plants (Debergh and Maene, 1981) and to conserve the germplasm of rare and endangered species (Fay, 1992). This paper describes the propagation of A. elatus by using nodal and shoot tip explants by means of micropropagation.
agar in sterile distilled water and were transferred to paper cups (0.98 in diameter) containing coconut coir, activated charcoal, commercial fertilizer in the ratio of 3:1:1 under controlled growth chamber conditions of 78.8 ± 35.60°F, 16 h photoperiod, 80 to 85% relative humidity and 60 μmol m-2 s-1 light intensity. The potted plants were irrigated with MS basal salt solutions every 4 days for 3 weeks and the plants were then transferred to glass house. Culture conditions The pH of the medium was adjusted to 5.7 with 0.1 N NaOH or 0.1 N HCl after addition of the growth regulators, prior to the addition of 0.7% agar (Himedia, India). The medium was autoclaved at 249.80°F for 30 min. All the cultures were maintained in sterilized culture room at 78.80 ± 35.60°F, under 16/8 h light regime provided by cool white fluorescent light (60 μmol m -2 s-1 light intensity) and with 55 to 60% relative humidity. Data collection and analysis Experiments were set up in completely randomized design and repeated three times. Each treatment had seven replications for shoot proliferation and eight replications for rooting. Observations on the number of shoots, shoot height, root and root length were recorded after 4 months of culture. Data were subjected to Duncan’s multiple range tests (DMRT) (Duncan, 1955).
MATERIALS AND METHODS
RESULTS AND DISCUSSION The plants of A. elatus were collected from Pambar Shola in the Western Ghats of Tamil Nadu, India and maintained in community pots in the glass house of Bharathidasan University, Tiruchirappalli under controlled conditions (temperature 78.8 ± 35.60°F and RH 70%). Explants were collected from these plants after stabilization (Figure 1a).
Surface sterilization Shoot tip (1 to 3 mm in length) and nodal parts (0.20 to 0.28 in length) were washed under running tap water for 45 min and surface sterilized with 70% ethanol for 30 s, then rinsed 5 times with sterile distilled water and treated with 5% sodium hypochlorite for 5 min, then again rinsed with sterile distilled water. Finally, the explants were treated with 0.1% HgCl 2 for 2 min and then washed thoroughly in sterile distilled water.
Influence of cytokinins After surface sterilization, the explants were inoculated on MS (Murashige and Skoog, 1962) medium supplemented with various concentrations (0.01to 3.5 mg/l) of cytokinins (BA, TDZ and KIN) individually for shoot bud proliferation. The medium was supplemented with 3% (w/v) sucrose and solidifying agent 0.7% agar (Bacterial grade, Himedia, India).
Rooting and transplantation of the regenerated plantlet The individual regenerated shoots were transferred to rooting medium consisting of MS medium and activated charcoal (50 mg/l to 2.5 g/l). The rooted healthy plantlets were washed off adhering
In general, the type, concentration and combination of cytokinins will play an important role during in vitro propagation of many orchid species (Arditti and Ernst, 1993). MS medium is widely used in tissue culture technology for the growth of orchid seedlings. Nodal and shoot tip explants of A. elatus cultured on MS medium without phytohormones supplement developed 1 to 3 shoots within 4 months of culture (Table 1). In this medium supplemented with BA, TDZ and KIN, shoot bud differentiations occurred within 20 days of culture and were free of any intervening callus or protocorm like body formation. Among cytokinins, 3.0 mg/l TDZ developed an average of 2.86 to 3.71 shoots per explants and highest mean shoot length was observed in 3.5 mg/l KIN (node) and 0.01 mg/l (shoot tip), respectively (Figure 1b, c and Table 1). KIN is the most common cytokinins, it does not affect the growth of nodal explants; 3.5 mg/l KIN developed excellent growth as compared to the other two cytokinins. However, increasing the concentrations of KIN produced lower proliferation rates and diminutive growth in shoot tip explants of A. elatus. Level of cytokinin, which is too high in shoot tip cultures results to small shoots which typically fail to elongate. TDZ is widely used for micropropagation of several plants including many orchids because of its incredible ability to induce organogenesis (Ernst, 1994; Nayak et al., 1997). Shoots developed on a TDZ containing medium had slow growth and morphological abnormalities as well as small leaves as compared to KIN. This may be due to the physiological condition of the explants or TDZ failure with its high cytokinin activity (Huetteman and Preece, 1993). Addition of BA had slight effect on proliferation rate as compared to the control. Different concentrations of AC were used (without phytohormones) with the aim of
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Figure 1. Micropropagation of A. elatus Lindley. a) Habitant; b and c) Shoot proliferation on MS + BA 2.5 mg/l; d) Rooting on MS + AC 0.3 g/l; e) Pre-hardening under culture room condition; f) Hardened plants in green house.
stimulating in vitro root development. Full strength MS medium supplemented with 0.3 g/l AC gave the maximum rooting response with an average of 4.25 Âą 0.49 roots per microshoot and the mean root length of 0.87 Âą 0.14. Young roots are very sensitive, white in color surrounded by root hair; rooted plants showed vigorous growth and produced more expanded leaves than the
control (Table 2). The positive effect of AC may be attributed to establishment of darkened environment, adsorption of inhibitory substances and the release of growth promoting substances or adsorption by AC (Pan and Staden, 1998). The tissue culture raised plants are heterotrophic in their mode of nutrition as they grow on medium rich in
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Table 1. Effect of BA, KIN and TDZ on in vitro shoot proliferation of A.elatus after four months of culture.
Cytokinins concentration (mg/l) Control
Number of Shoots/explant Shoot tip (Mean±SE) Node (Mean±SE) ab ab 1.20±0.33 2.23±0.55
BA 0.01 0.05
0.86±0.26 abcd 1.71±0.29
d
1.86±0.88 ab 1.00±0.00
0.1 0.5 1.0 1.5
2.14±0.51 abcd 1.71±0.29 ab 2.71±1.06 abcd 2.14±0.34
abcd
2.0 2.5 3.0
1.86±0.14 bcd 1.14±0.26 abcd 2.00±0.72
abcd
1.71±0.42 ab 1.43±0.29 ab 2.00±0.57
3.5
1.86±0.34
abcd
2.00±0.43
Shoot length/Explant Shoot tip (Mean±SE) Node (Mean±SE) bcde bc 1.59±0.35 2.25±0.44
ab
1.88±0.43 bc 2.93±0.38
1.14±0.14 ab 1.14±0.14 ab 1.43±0.20 ab 1.86±0.26
ab
ab
2.27±0.23 abcd 2.59±0.19 abcde 1.78±0.46
ab
abcde
2.28±0.62 abc 3.38±0.36
bc
2.18±0.20 abcd 2.59±0.29 abcde 2.19±0.16 abcde 2.11±0.23
abcde
3.18±0.39 abc 2.95±0.40 bc 2.64±0.37 bc 2.46±0.34
abcde
3.35±0.34 abc 3.49±0.65 abc 3.43±0.30
abc
abc
abc
2.64±0.46
abcde
2.80±0.22 c 2.07±0.20
2.72±0.42
bc
TDZ 0.01 0.05
1.71±0.42 abc 2.57±0.37
abcd
2.14±0.40 ab 2.43±0.75
ab
1.83±0.16 bcde 1.65±0.38
0.1 0.5 1.0 1.5
2.88±0.61 abcd 1.67±0.21 abcd 2.43±0.81 abcd 1.29±0.18
abcd
1.86±0.14 ab 1.14±0.26 ab 1.14±0.26 ab 1.71±0.52
ab
1.02±0.09 cde 1.40±0.28 de 1.37±0.24 de 1.32±0.24
e
2.16±0.27 c 1.88±0.33 bc 2.39±0.28 bc 2.45±0.30
2.0 2.5 3.0
2.14±0.34 abcd 2.14±0.55 a 2.86±0.73
abcd
1.29±0.29 ab 1.14±0.14 a 3.71±0.97
ab
1.61±0.34 abcde 1.93±0.19 abcd 2.41±0.28
bcde
2.54±0.26 bc 2.50±0.34 c 1.83±0.46
3.5
2.57±0.29
abc
2.29±0.75
ab
2.05±0.41
abcde
2.84±0.30
KIN 0.01
2.00±0.76
abcd
1.43±0.43
ab
d
ab
3.01±0.54
a
abcde
abc
bc
bc
abc
2.10±0.46
c
abc
0.05 0.1 0.5 1.0 1.5
0.86±0.26 bcd 1.14±0.14 abcd 1.57±0.61 abcd 1.43±0.29 bcd 1.14±0.14
1.14±0.67 ab 1.00±0.00 ab 1.14±0.14 ab 1.00±0.00 ab 1.00±0.00
2.17±0.93 abcde 2.26±0.26 abcde 1.88±0.32 abcde 2.31±0.33 abcde 2.01±0.16
3.41±1.01 bc 2.75±0.27 bc 2.23±0.48 abc 2.85±0.12 bc 2.38±0.38
2.0 2.5 3.0
1.14±0.14 abcd 1.29±0.47 abcd 1.57±0.37
bcd
1.00±0.00 ab 1.00±0.00 ab 1.86±0.94
ab
2.31±0.16 abcde 2.33±0.17 abcde 2.30±0.70
abcde
2.24±0.38 c 1.85±0.40 ab 3.85±0.80
cd
1.00±0.30
ab
2.63±0.70
abcd
4.43±1.17
3.5
1.00±0.38
bc
a
1
Means with different letters within columns are significantly different according to Duncan's multiple range tests at 0.05% level. 2All the cultures were maintained in sterilized culture room at 78.80 ± 35.60°F, under 16/8 h light regime provided by cool white fluorescent light (60 μmol m-2 s-1 light intensity). BA, 6-Benzyl adenine; TDZ, 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea; KIN, 6-furfurylaminopurine.
minerals and sugar and thus, cannot withstand the environmental conditions without proper hardening and
acclimatization. For hardening, the in vitro healthy plantlets were transferred to autoclaved paper cups containing coconut
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Table 2. Effect of activated charcoal concentrations on growth of A.elatus cultured on MS medium for four months.
Activated charcoal (g/l) Control 0.05 0.10 0.20 0.30 0.40 0.50 1.00 1.50 2.00 2.50
Number of day taken to induce root 12-15 12-15 13-16 12-16 12-15 10-15 12-15 10-15 13-19 16-26 16-24
Number of roots/microshoot (Mean±SE) c 2.13±0.52 c 1.88±0.79 c 2.12±0.52 ab 2.88±0.23 a 4.25±0.49 a 4.00±0.87 ab 3.00±0.63 ab 3.50±0.46 ab 3.25±0.25 ab 3.00±0.50 c 2.00±0.26
Average root length (cm) (Mean±SE) abc 1.60±0.33 bc 1.15±0.45 bc 1.20±0.30 ab 2.01±0.26 a 2.21±0.35 a 2.20±0.28 abc 1.43±0.35 bc 1.23±0.18 c 0.95±0.15 bc 1.11±0.11 c 0.98±0.20
Rooting (%) 87.50 87.50 50.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00
1
Means with different letters within columns are significantly different according to Duncan's multiple range tests at 0.05% level. 2All the cultures were maintained in sterilized culture room at 78.80 ± 35.60°F, under 16/8 h light regime provided by cool white fluorescent light (60 μmol m-2 s-1 light intensity).
coir, AC, and commercial fertilizers (3:1:1). These plantlets were irrigated with half-strength MS medium without organics and maintained in culture room for 4 to 5 weeks (Figure 1e). This was followed by transfer to growth chamber for three weeks at relative humidity of 80 to 90% and 68 ± 75.2°F temperatures. Plants were then shifted to community pots
containing the same mixture and maintained in the green house where a survival rate of over 80% was recorded (Figure 1f). They are easy to grow, preferring high humidity, low temperature, moist and well drained soil. For proper growth and development, maintenance of continued optimum temperature of 75.2 to 82.4°F and high humidity of 80 to 85% is essential for this species. This successful protocol is suitable for large scale propagation as well as ex-situ conservation of this endangered terrestrial orchid. REFERENCES Arditti J, Ernst R (1993). In “Micropropagation of Orchids”. J. Wiley and Sons Inc., New York. pp. 682. Belitsky I, Bersenev VNA (1999). Jewel Orchids. Magazine Am. Orchid. Soc. pp. 33-37. Debergh PC, Maene LJ (1981). A scheme for commercial propagation of ornamental plants by tissue culture. Sci Hortic. 14: 335-345. Duncan DB (1955). Multiple range and multiple f-tests. Biometrics, 11: 1-42 Ernst R (1994). Effects of Thidiazuron on in vitro propagation of Phalaenopsis and Doritaenopsis (Orchidaceae). Plant. Cell. Tissue Org. 39: 273-275. Fay MF (1992). Conservation of rare and endangered plants using in vitro methods. In vitro Cell. Dev. Biol. 28: 1-4.
Huetteman CA, Preece JE (1993). Thidiazuron: a potent cytokinin for woody plant tissue culture. Plant Cell. Tissue Org. Cult. 33: 105-119. Kan WS (1986). Pharmaceutical botany. National Research Institute of Chinese Medicine, Taipei, Taiwan, p. 647. Ket NV, Hahn EJ, Park SY, Chaharabarty D, Paek KY (2004). Micropropagation of an endangered orchid Anoectochilus formosanus. Biol. Plant. 48(3): 339-344. Kumar SC, Shetty BV, Bennet SSR, Ananda Rao T, Sanjay Molur S (2001). Endemic Orchids of the Western Ghats. Conservation Assessment and Management Plan (CAMP) Workshop Report. Liang WL, Chen RC, Chiang YJ, Su CH, Yang LL, Yen KY (1990). Study on Anoectochilus species. Formosan Sci. 43: 47-58. Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. pp. 473-497. Nayak NR, Patnaik S, Rath SP (1997). Direct shoot regeneration from foliar explants of an epiphytic orchid Acampe praemorsa (Roxb.) Blatter and McCann. Plant Cell Rep. 16: 583-586. Pan MJ, Van Staden J (1998). The use of Charcoal in in vitro culture - A review. Plant Growth Regul. 26: 155-163. Samira C, Satyakam Guha I, Usha R (2009). Micropropagation of orchids: A review on the potential of different explants. Sci. Hortic. 122: 507-520. Vakhrameeva MG, Denisova, LV, Nikitina SV and Samsonov SK (1991). Orkhidei nashei strany (orchids of our lands). Moscow: Nauka. 13: 27-39. Zheng C, Huang YZ, Ji LF (1996). Pharmacognostical studies on Jin Xian Lian. Zhongcaoyao, 27: 169-172.
African Journal of Biotechnology Vol. 11(29), pp. 7554-7564, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.4050 ISSN 1684â&#x20AC;&#x201C;5315 Š 2012 Academic Journals
Full Length Research Paper
Comparative toxicity study of two different synthesized silver nanoparticles on the bacteria Vibrio fischeri Ehsan Binaeian1, Ali Akbar Safekordi1, Hossein Attar1*, Reza Saber2, Mohammad Javad Chaichi 3 and Abasalt Hosseinzadeh Kolagar 4 1
Department of Chemical Engineering, Science and Research Branch, Islamic Azad University, Poonak Square, Tehran, Iran. 2 Research Center for Science and Technology in Medicine (RCSTIM), Imam Khomini Hospital, Tehran, Iran. 3 Department of Chemistry, University of Mazandaran, Babolsar, Iran. 4 Faculty of Science, University of Mazandaran, Babolsar, Iran. Accepted 1 March, 2012
A comparative evaluation of acute toxicity of synthesized nano-silvers using two different procedures (biological and chemical reduction methods) and effect of silver ions on the bacteria Vibrio fischeri were investigated. The bacterial light inhibition test as a toxicological endpoint was used by applying of a homemade luminometer. To compare the toxicity effects as a quantitative parameter, after 5 min and 30 min exposure times, nominal effective concentration (EC) of chemicals and susceptibility constant (Zvalue) were calculated. After 5 and 30 min contact times, the EC50 values of two silver nanoparticles and the EC20 values were about similar. The EC values of nanoparticles were larger than those of the silver ions. The susceptibilities of V. fischeri (L/mg) to the silver ions were greater than those of the nano silvers. According to the EC and Z values, the toxicity of silvers decreased in the following order: silver ions >> silver nanoparticles from chemical reduction method ~ silver nanoparticles from biological method. Key words: Vibrio fischeri, toxicity, luminometer, bioluminescence, silver nano particles.
INTRODUCTION Nanotechnology plays an important role in many key technologies of the new millennium. The application of nanoscale and nano-structure materials within the range of 1 to 100 nanometres is an emerging area of nanoscience and nanotechnology. Nanomaterials may provide solutions to technological and environmental challenges in the areas of solar energy conversion, catalysis, medicine and water treatment (Sharma et al., 2007; Mandal et al., 2006). The noble metals especially gold and silver, due to their innumerable applications in different branches such as catalysis, photonics, photography, and more importantly in the field of medicine as antimicrobial factors, have drawn much attention to themselves (Nel et al., 2006; Barnard, 2006).
*Corresponding author. E-mail: attar.h@srbiau.ac.ir. Tel: +9821-44869748. Fax: +98-21-44869744.
In addition, colloidal silver is of particular interest because of distinctive properties, such as good conductivity, chemical stability, catalytic and antibacterial activity (Sarkar et al., 2007). Silver nanoparticles have many important applications which include, spectrally selective coating for solar energy absorption and intercalation material for electrical batteries, as optical receptors, polarizing filters, catalysts in chemical reaction, biolabelling and as antimicrobial agents (Ahmad et al., 2003). Silver nanoparticles and different silver-based materials containing ionic silver or metallic silver are already being commercialized for their antimicrobial activity (Wijnhoven et al., 2009; Gao et al., 2009). This antibacterial property was used in many interesting applications such as coatings on medical apparatus (Stevens et al., 2009), and assisting in fabrics (Benn and Westerhoff 2008). Such toxicity is useful for these applications; however, it also proposes eco-toxicological problems upon release (Simon-Deckers et al., 2008).
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Table 1. Nutrient media for solid cultures. Sea water agar (Twin pack).
Part A Peptic digest of animal tissue Yeast extract Beef extract agar
-1
Standard formula (gL ) 5 5 3 15
Subsequently, it is very important to understand the grade of danger regarding silver-based nanoparticles to the environment. Several mechanisms for toxic effects of silver colloid particles have been hypothesized. The main mechanism of toxicity of silver nanoparticles, results from the release of Ag ions from nanoparticles which lead to oxidation stress (OS), damage of lipids, carbohydrates, proteins and DNA (Asharani et al., 2008; Choi and Hu 2008). Some studies were conducted on the potential eco-toxicity of nano sized silver with crustaceans such as Daphnia magna and various bacterial strains. D. magna, Daphnia pulex and Ceriodaphnia dubia are approved test models for effluent release into the environment by the Environmental Protection Agency and other regulatory agencies (ASTM Standard E1193 -97, 2004; EPA, 2002) because of their sensitivity towards potential pollutants, such as metal ion species. Toxicity study of Ag and Au nanoparticles on D. magna were conducted by Li et al. (2010). The toxicity of all nanoparticles tested was found to be dose and composition dependent. The concentration of Au nanoparticles that killed 50% of the test organisms (LC50) ranged from 65 to 75 mg/L. In addition, three different sized Ag nanoparticles were studied to analyze the toxicological effects of particle size on D. magna. Changing the ratio between silver nitrate and sodium citrate during Ag NP synthesis led to an increase in the average particle size produced; however, no difference in toxicity was observed once the overall Ag was corrected using flame atomic absorption spectrometry results. Toxicity ranged from 3 to 4 Îźg/L for all three sets of Ag nanoparticles tested (Li et al., 2010). The antibacterial effect of silver nanoparticles on Escherichia coli was investigated by Sondi and Salopek Sondi (Sondi et al., 2004). The test results based on the TEM analysis and proteomic research showed that the silver nanoparticles interacted with elements of the bacterial membrane causing structural change, dis-sipation of the proton motive force, and eventually cell death (Lok et al., 2006). Different from silver nanoparticles, studies about silver ions have been performed for a long time and have been shown to interact with cytoplasmic components and nucleic acids, to inhibit chain enzymes of respiratory, and to interfere with penetrability of membrane (Russell and Hugo, 1994).
Part B Sodium chloride Potassium chloride Magnesium chloride Magnesium sulphate.7H2O Calcium chloride
-1
Standard formula (gL ) 24.0 0.7 5.3 7.0 0.1
The antimicrobial activities of silver nanopowder on three microorganisms were reported (Lee et al., 2009). As an initial step in investigating, the potential eco-toxicity to the environment and biological species, a quantitative parameter was applied to compare the susceptibilities of three microbes, and morphological changes were analyzed. In general, the susceptibilities decreased in the following order: E. coli > Bacillus subtilis > Saccharomyces cerevisiae. In this paper, the toxicity effects of two types of synthesized nano silvers and silver ions were tested using Gram-negative bioluminescence bacteria Vibrio fischeri. Toxicity effects of silver nanoparticles and silver ions to V. fischeri were compared with the application of two numerical values (Z and EC). MATERIALS AND METHODS Materials V. fischeri strain (PTCC 1693) and E. coli strain (PTCC 1763) were bought from Iranian Research Organization for Science and Technology (IROST). All components of culture medium containing sodium chloride [7647-14-5], potassium chloride [7447-40-7], sodium bicarbonate [144-55-8], calcium chloride dehydrate [1003504-8], agar [9002-18-0], peptone [91079-40-2], magnesium sulphate hepta-hydrate [10034-99-8], magnesium sulphate [7487-88-9], magnesium chloride hexa-hydrate [7791-18-6], calcium chloride [10043-52-4], magnesium chloride [7786-30-3] and sodium hydroxide [1310-73-2], hydrochloric acid [7647-01-0], silver nitrate [7761-88-8], 3-aminophthalahydrazide(luminol) [521-31-3], hydrogen peroxide[7722-84-1], copper sulphate penta-hydrate [7758-99-8] and sodium borohydride [16940-66-2] were purchased from E. Merck (Germany). Preparation of microbes V. fischeri To ensure the best quality of luminescent bacteria with maintainable viability, the bacteria can be inoculated and maintained in culture medium. Although different cultures can be used, the following culture mediums allow greatest luminescence, growth and stability that are practical for the tests. In this way, two basic growth media were examined: 1. Sea water agar (twin pack) (Table 1). 2. Sea water agar (Table 2). The first media was used for solid cultures and the second one for
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Table 2. Nutrient media for liquid cultures (DSMZ medium 246: sea water agar).
Media Sea water agar Beef extract peptone agar Tap water * Sea water
Amount 10 g 10 g 20 g 250 ml 750 ml
*Artificial sea water NaCl KCl CaCl2×2H2O MgCl2×6H2O NaHCO3 MgSO4×7H2O Distilled Water
28.13 g 0.77 g 1.60 g 4.80 g 0.11 g 3.50 g 1000 ml
Figure 1. Bioluminescence of bacteria Vibrio fischeri in liquid culture (a) and solid culture (b).
liquid cultures. The bioluminescence of V. fischeri in sea water agar culture (solid media) and liquid culture is shown in Figure 1. Solid cultures were retained in incubator at 18°C. After inoculation of liquid medium by luminous V. fischeri from solid culture, the liquid culture was incubated for 48 h at 18°C in an orbital shaker at 120 rpm (Claudia et al., 2003).
E. coli Culture medium of Muller-Hinton-Broth was prepared according to the conditions provided from the factory. Fifteen gram (15 g) MullerHinton-Broth's medium was dissolved in 500 ml Erlenmeyer flasks containing 500 ml deionized water and stirred forcefully. pH 7.3 ±
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Figure 2. Solutions of silver nitrate (1 mM) before (a) and after (b) reduction by NaBH4 (presence of synthesized silver nanoparticles).
0.2 was controlled by the addition of 1 M NaOH or 1 M HCl. After 20 min autoclave, E. coli was cultivated for a period of 1 day in MullerHinton-Broth's medium at temperature of 37ﾂｰC in incubator. After 1 day, bacterium suspension was prepared and was made ready for subsequent experiments.
possibility of silver nanoparticles production (Figure 3). In order to prove the existence of nanoparticles, product efficiency and their size, UV-VIS spectroscopy, transmission electron microscopy (TEM) were used. The wavelength of maximum peak (lmax) at 5ﾂｰC and pH of 5.5 is about 415 nm which indicates the presence of silver nanoparticles. We measured the size of about 10 NPs, and the mean size of the silver nanoparticles was estimated as 80 nm.
Preparation of silver nanoparticles by chemical reduction method Atomic absorption Aqueous solutions of AgNO3 (1 mM) and NaBH4 (2 mM) were prepared with deionized distilled water. NaBH 4 solution (15 ml) was ice cooled and stirred vigorously with the help of a magnetic stirrer. AgNO3 solution (5 ml) was then added rapidly. The color of the solution slowly turned yellowish, indicating the reduction of the silver ions (Figure 2). The prepared suspension was stored at 5ﾂｰC. The quality of nanoparticles was found to be stable for several weeks (12 to 16 weeks). Transmission electron micrographs (TEM) at 200 kV and UV窶天IS spectra were then recorded to characterize the prepared Ag NPs. The wavelength of maximum peak (lmax) is about 400 nm, which indicates the presence of silver nanoparticles. We measured the size of about 100 NPs and the mean size of the silver nanoparticles was estimated as 10 nm.
To measure the amounts of ionic silvers in silver nanoparticle suspensions, an atomic absorption spectrophotometer (Thermo Spectra AA M5) was used. In order to separate the Ag NP, the nanoparticle suspensions were centrifuged (Eppendorf, Minispin). The separation was done by centrifuging 2.2 ml of silver colloid solution, running at 12000 rpm for 15 min (Li et al., 2010). Then 2 ml of supernatant was gathered. The supernatant was then analyzed by AA and compared to the original silver colloid samples. For calibration, fresh silver standard solutions were generated in the concentration of 20 ppm. The stock solution was serially diluted to produce 10, 5.0, 1.0, 0.5 and 0.1 mg/L of Ag ion solutions.
Assay procedure and data analysis Preparation of silver nanoparticles by biological method Prepared E. coli bacterial suspension was filtered by means of a 0.22m (micron) filter. One milliter (1 ml) obtained filtrate was added to the container containing 100 ml AgNO3 (1 mM). After about few minutes (about 20 min), colourless solution of silver nitrate in container turns into brown colour. This colour change indicates
Flash assay is a test that inhibits V. fischeri luminescence and was done by homemade luminometer. One milliter (1 ml) volume of V. fischeri suspension was placed in 4.5 ml cuvette, and 1 ml volume of silver nanoparticle or silver nitrate solution at various concentrations were added. When 1 ml of bacterial suspension was mixed with 1 ml of silver nanoparticles or silver ions, the resulting
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Figure 3. Solutions of silver nitrate (1 mM) before (a) and after (b) exposure to the culture filtrate of E. coli and reduction of silver ions (presence of synthesized silver nanoparticles).
concentration was half of the initial concentration in the final 2 ml of mixed solution. A 100 ppm silver ions stock solution was prepared by dissolving 15.74 mg of AgNO3 powder in 100 ml deionized distillated water. The concentration of both type of silver nanoparticles in stock suspensions were 120 ppm. The decrease of bacterial luminescence (INH%) due to addition of test samples was calculated after 5 and 30 min, as follows: INH% = 100 – [ITT / (IT0 × KF)] × 100; KF = ICT / IC0 Where, KF is the correction factor based on control; IC0 and IT0 are the initial luminescene intensities of control and test samples. IC T and ITT are the luminescence intensities of the control and test samples after 'T' minute contact time. EC50 and EC20 values are the concentrations of nanoparticles or silver ions (mg/L) causing 50 and 20% decrease in bioluminescence after 'T' minute exposure time. Various exposure times have been used (e.g., 5, 15, 30 and 45 min) (Heinlaan et al., 2008; Mortimer et al., 2008; Parvez et al., 2006; Froehner et al., 2000; Fulladosa et al., 2005; Lappalaineh et al., 2001). In this study, 5 and 30 min were selected as contact times. Three independent assays were carried out in each test. The data for percentage inhibition obtained in each experiment were converted to gamma values, where: Gamma = % inhibition / (100 - % inhibition) Gamma values were plotted against their corresponding chemical concentrations, after first converting all data to natural logs (Ln), to generate Ln gamma / Ln concentration curves for each chemical. Values falling within the 1 to 99% inhibition range were used to fit a straight line to the Ln-transformed data by linear regression, and the resulting equations used to calculate the EC 20 and EC50. Apparatus Bioluminescence detection was carried out by a homemade luminometer equipped with a model R-446 photomultiplier (PMT) (Hamamatsu, Japan). The luminometer connected to a personal computer via a suitable interface (Micropars, Tehran, Iran) as shown in Figure 4. Experiments were done in cuvette of 100 mm2 cross sectional area and 45 mm height at 25°C. Intensity of
bioluminescence was recorded as a function of time; the time resolution of the luminometer was 0.01 s. Calibration of device was performed by the luminol chemiluminescence reaction (Skoog et al., 1998). The luminol chemiluminescence reaction is one of the most effective systems that generate chemiluminescence. The linear range of concentrations and responses (calibration graph) are shown in Figure 5.
RESULTS AND DISCUSSION Characterization of Ag nanoparticles Ag NP suspensions, synthesized by two different procedures were analyzed by applying absorption spectroscopy (UV-Vis, Cecil CE5501) and transmission electron microscopy (TEM, JEOL JEM 2010 electron microscopy, UIC). UV–VIS absorbance spectrometry is one of the first methods for the analysis of silver colloids since they are plasmonic in nature (Hayat, 1989; Chumanov et al., 1996). The oscillation of the conduction electrons, that is, the plasmon, is very sensitive to changes in particle size, dielectric constant of the medium, and aggregation state of the particles. Therefore, freshly synthesized Ag NPs were analyzed between the wavelengths of 390 to 450 nm. According to absorption spectroscopy of silver nanoparticles synthe-sized by chemical reduction method, the observed wavelength of maximum peak (lmax) was about 400 nm which was indicative of the presence of silver nanoparticles (Figure 6a). TEM results for the Ag NP samples showed about 10 nm of nanoparticles size (Figure 7a). The silver nanoparticles synthesized by biological method, were characterized by UV–VIS absor-bance spectrometry. The maximum peak (lmax) of about 415 nm was recorded (Figure 6b). Transmission electron microscopy (TEM) shows and confirms that silver nanoparticles are at nano-size (about 80 nm). TEM images of the produced nanoparticles are shown in Figure 7b. Flame atomic absorption spectro-metry (AAS) was used to determine whether excess silver ions (Ag+)
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Figure 4. Schematic representation of homemade luminometer for measuring of bioluminescence.
Figure 5. Correlation diagram (calibration diagram) for Chemiluminescence emission intensity as a function of luminol concentration. The all reagent concentrations are: CuSO4.5H2O (6 Ă&#x2014; 10 -3 M, 0.1 ml), Hydrogen peroxide (10%, 0.1 ml), water (1 ml) and varying concentrations of luminol solution in NaOH (0.1 M): (1) 1.09 ppm, (2) 1.36 ppm, (3) 1.63 ppm, (4) 1.9 ppm, (5) 2.18 ppm, (6) 2.45 ppm, (7) 2.72 ppm, (8) 3 ppm.
remained after reduction of silver nitrate. It was determined that for both types of the Ag NPs, very little silver was lost
during synthesis. When NPs were separated out of the stock solutions by centrifugation and the supernatants
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Figure 6. Absorption spectra (UV-VIS spectra) of silver nanoparticles synthesized by chemical reduction method (a) and biological method (b).
Figure 7. TEM images of silver nanoparticles synthesized by chemical reduction method (a) and biological method (b).
measured using AAS, no [Ag] was detectable in the supernatant, suggesting that all of the bioavailable Ag was in a NP form and was not in a silver ion form.
Toxicity effect of silver nanoparticles and silver ions on bacteria V. fischeri Comparison of EC values The toxic effects of exposure to Ag nanoparticles were
tested using bacteria V. fischeri, after contact times of 5 and 30 min. Also, viabilities in the presence of silver ions in aqueous silver nitrate solution were examined under the same experimental procedure. The results are shown in Figure 8a to c, Tables 3 and 4. As seen in Tables 3 and 4, after 5 and 30 min exposure times, the calculated EC values of nanoparticles are nearly similar. On the other hand, according to Figure 8a to b, both type of silver nanoparticles exhibited about the same toxicity effects (light inhibition%). TEM images (Figure 7a to b) showed a difference in the size of synthesized
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Figure 8. Concentration â&#x20AC;&#x201C; effect relationships of suspensions of Ag nanoparticles, synthesized by chemical reduction method (a), biological method (b) and silver ions (c) for bacteria V. fischeri.
nanoparticles. Therefore, it can be said that in this study, toxicity of nanoparticles did not correlate to the particle size exactly. As shown in Figure 8a to b, the concentrations of silver nanoparticles in contact with V. fischeri, killed more bacteria after 30 min than 5 min and led to higher light inhibition (INH%) and higher toxicity. In other word, they need a few times to diffuse to the cells
and degrade lipids, carbohydrates, proteins and DNA. To determine the toxicity effects of the silver ions in comparison with silver nanoparticles, the toxicity of silver ions were tested. Silver ions strongly affected the viability of V. fischeri with 50% inhibition of bioluminescence after 5 and 30 min contact times at about 15.9 and 1.85 ppm, respectively. Silver ions may affect V. fischeri cells by
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Figure 8. Contd.
Table 3. Effective concentrations (EC) for three chemicals that result in 20 and 50% inhibition in test system that measure bioluminescence of V. fischeri. The EC50 and EC20 values were calculated after 5 min exposure time at 25째C. The EC values have been obtained from linear regression equation of concentration/response curves of the form.
Chemical Nano Ag (biological method) Nano Ag (chemical reduction method) + Ag ions (Ag )
EC50 (ppm) 47.220 45.077 15.890
SD 2.330 2.18 0.813
EC20 (ppm) 17.800 15.310 4.830
SD 0.490 0.740 0.460
m 1.426 1.284 1.164
C - 5.493 - 4.889 - 3.221
r 0.963 0.971 0.978
Ln y = m Ln x + c, where y is the value of gamma [gamma = %inhibition / (100 - % inhibition)], x is the concentration and m and c are the slope and intercept, respectively. The individual values for the slope and intercept for each chemical may be used to calculate any desired gamma value and thus any required EC.
Table 4. Effective concentrations (EC) for three chemicals that result in 20 and 50% inhibition in test system that measure bioluminescence of Vibrio fischeri. The EC50 and EC20 values were calculated after 30 min exposure time at 25째C.
Chemical Nano Ag (Biological method) Nano Ag (Chemical reduction method) + Ag ions (Ag )
EC50 (ppm) 34.550 29.320 1.856
inactivating proteins through combining with the -SH group (Barnard, 2006) and interacting with respiratory chains, as believed to occur for heavy metals (Katherine and Allen 2005). According to Table 3, the EC20 and EC50 values of silver ions after 5 min were about 3.5 and 2.9 fold lower than those of both silver nanoparticles. The EC20 and EC50 values of silver ions after 30 min (Table 4), in comparison with Ag nanoparticles decreased about 5.7 and 17 times, respectively. These results indicate that Ag ions obtained from AgNO3 in water solution were always more toxic than that of both synthesized silver nanoparticles.
SD 5.670 2.220 0.007
EC20(ppm) 5.410 3.970 0.824
SD 1.550 0.860 0.005
m 0.765 0.693 1.712
C -2.690 -2.343 -1.063
r 0.949 0.929 0.957
Comparison of Z values To compare the toxicity effects of silver nanoparticles and silver ions on bacteria V. fischeri as another quantitative parameter, the susceptibility has been used in this work (Yoon et al., 2007). For the assessment of toxicity effects of silver materials, the susceptibility constant Z (L/mg) can be used and is defined by the following equation: Z = - ln (ITt/IT0)/C Where, ITt is the bioluminescence of V. fischeri after
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Table 5. Susceptibilities (Z- values) of bacteria V. fischeri.
Parameter Average Z – values (L/mg)
Time (Min) 5 30
C1* 0.032 0.192
SD 0.009 0.050
C2* 0.032 0.182
SD 0.011 0.056
†
C 0.067 0.430
SD 0.020 0.120
Z = - ln (ITt/IT0)/C. Concentration of both C* and C† ranging from 0.66 to 6.66 ppm were used for calculations. C1*= Concentration of silver nanoparticles from chemical reduction method (ppm); C2*= Concentration of silver nanoparticles from biological method (ppm); C† = Concentration of silver ions not that of nitrate ions in AgNO3 solution (ppm).
exposure to silver nanoparticles and silver ions; IT0 is the bioluminescence of V. fischeri in the absence of silver materials, and C is the concentration of the silvers (ppm). Using the Z value, a given C value, and the exposure time, the survival fraction of bacteria can be predicted (as function of light). A higher Z value shows that the micro-organisms are more sensitive to the materials, signifying that the materials are more toxic to the bacteria. The calculated susceptibilities are shown in Table 5. C* and C† represent the concentrations of silver nanoparticles in the suspensions and the concentration of silver ions in the silver nitrate solution, respectively. To estimate the Z values, a range of concentrations from 0.66 to 6.66 ppm for both C * and C† were used. As shown in Table 5, the susceptibilities increase with increasing of the exposure time from 5 to 30 min. Based on the C* concentrations and for exposure times of 5 and 30 min, the suscep-tibilities of V. fischeri were not much different, indicating that the toxic effects of two types of nanoparticles on V. fischeri are comparable (Figure 8a to b). For silver nanoparticles synthesized by chemical reduction method, the average Z value of V. fischeri increased to 0.1630 (600%) from 5 to 30 min. For biological synthesized silver nanoparticles, Z values increased to 0.1501 (570%). During 30 min contact time, the susceptibilities of V. fischeri to both silver nanoparticles increased. It may be possible that synthesized silver nanoparticles showed acute toxicity. As shown in Table 5, based on the C† con-centration, the average Z value of V. fischeri increased to 0.363 (641.8%) from 5 to 30 min. The susceptibilities of V. fischeri to silver ions were higher than two types of silver nanoparticles, indicating that V. fischeri was more sensitive to the silver ions. When comparing the results from the C*and C† concentrations, the susceptibilities of V. fischeri based on C† were larger than those of C*, then silver ions were more toxic than both type of synthesized silver nanoparticles. Diffusivity of silver ions to the bacterial cells is more than that of the Ag NPs. Conclusion In this study, we reported the toxicity of silver ions and silver nanoparticles on bacteria V. fischeri. According to the EC and Z values, the toxicity decreased in the following order: Silver ions >> silver nanoparticles from chemical reduction method ~ silver nanoparticles from biological method whereas, the effective concentrations (EC50 and
EC20) of materials after 30 min exposure times were less than 5 min and the susceptibility constants were greater at the vice versa direction. It can be concluded that the bacteria V. fischeri is so sensitive to toxicity effects of chemicals and can be used as a biosensor for rapid and low-cost detection of acute toxicity of nanomaterials. ACKNOWLEDGEMENT The authors gratefully acknowledge supports from the Cellular and Molecular Center of Science Faculty, University of Mazandaran, Babolsar, Iran. REFERENCES Ahmad A, Mukherjee P, Senapati S, Mandal D, Khan MI, Kumar R, Sastry M (2003). Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium oxysporum. Colloids. Surf. B: Biointerf. 28: 313- 321. Asharani PV, Low Kah Mun G, Hande MP, Valiyaveettil S (2008). Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACS Nano. 3: 279-290. ASTM (2004). Standard Guide for Conducting Daphnia magna LifeCycle Toxicity Tests. West Conshohocken PA: ASTM International. Standard E, 1193-1197 Barnard AS (2006). Nanoharzards knowledge is our first defence. Nat. Mater. 5: 245-248. Benn TM, Westerhoff P (2008). Nanoparticle silver released into water from commercially available sock fabrics. Environ Sci. Technol. 42: 4133-4139. Choi O, Hu ZQ (2008). Size dependent and reactive oxygen species related nanosilver toxicity to nitrifying bacteria. Environ. Sci. Technol. 42: 4583-4588. Chumanov G, Sokolov K, Cotton TM (1996). Unusual extinction spectra of nanometer-sized silver particles arranged in two-dimensional arrays. J Phys. Chem. 100: 5166-5168. Claudia L, UE, Mark, Peter G, Edward GR (2003). The Vibrio fischeri quorum-sensing systems Ain and Lux sequentially induce luminescence gene expression and are important for persistence in the squid host. Mol. Microbiol. 50: 319-331. EPA (2002). Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms. Ed Agency U S E P. Froehner K, Backhaus, Grimme LH (2000). Bioassay with Vibrio fischeri for the assessment of delayed toxicity. Chemosphere, 40: 821-828. Fulladosa E, Murat JC, Villaescusa (2005). Study on the toxicity of binary equitoxic mixtures of metals using the luminescent bacteria Vibrio fischeri as a biological target. Chemosphere, 58: 551-557. Gao J, Youn S, Hovsepyan A, Llaneza VL, Wang Y, Bitton G, Bonzongo JCJ (2009). Dispersion and toxicity of selected manufactured nanomaterials in natural river water samples: effects of water chemical composition. Environ Sci. Technol. 43: 3322-3328.
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Hayat MA (1989). Colloidal gold: principles, methods and applications. Academic. San Diego. Heinlaan M, Ivask A, Blinova I, Dubourguier HC, Kahru A (2008). Toxicity of nanosized and bulk ZnO, CuO and TiO2 to bacteria Vibrio fischeri and crustaceans Daphnia magna and Thamnocephalus platyurus. Chemosphere, 71: 1308-1316. Katherine BH, Allen JB (2005). Interaction of silver (I) ions with the respiratory chain of Escherichia coli: an electrochemical and scanning electrochemical microscopy study of the antimicrobial mechanism of micromolar Ag+. Biochemistry, 44: 13214-13223. Lappalaineh J, Juvonen R, Nurmi J, Karp M (2001). Automated color correction method for Vibrio fischeri toxicity test. Comparison of standard and kinetic assays. Chemosphere, 45: 635-641. Lee S, Lee J, Kim K, Sim SJ, Bock GM, Yi J, Lee J (2009). Eco-toxicity of Commercial Silver Nanopowders to Bacterial and Yeast Strains. Biotechnol. Bioprocess Eng. 14: 490-495. Li T, Albee B, Alemayehu M, Diaz R, Ingham L, Kamal S, Rodriguez M, Bishnoi SW (2010). Comparative toxicity study of Ag, Au, and Agâ&#x20AC;&#x201C;Au bimetallic nanoparticles on Daphnia magna. Anal. Bioanal. Chem. 398: 689-700. Lok, C N, Ho CM, Chen R, He QY, Yu WY, Sun H, Tam PK-H, Chiu JF, Che CM (2006). Proteomic analysis of the mode of antibacterial action of silver nanoparticles. J. Proteome Res. 5: 916-924. Mandal D, Bolander ME, Mukhopadhyay D, Sarkar G, Mukherjee P (2006). The use of microorganisms for the formation of metal nanoparticles and their application. Appl. Microbiol. Biotechnol. 69: 485-491. Mortimer M, Kasemets K, Heinlaan M, Kurvet I, Kahru A (2008). High throughput kinetic Vibrio fischeri bioluminescence inhibition assay for study of toxic effects of nanoparticles. Toxicology in Vitro. 22: 14121417. Nel, Xia T, Madler L, Li N (2006). Toxic potential of materials at the nanolevel. Science, 311: 622-627. Parvez S, Venkataraman C, Mukherji S (2006). A review on advantages of implementing luminescence inhibition test (Vibrio fischeri) for acute toxicity prediction of chemicals. Environ. Int. 32: 265-268.
Russell AD, Hugo WB (1994). Antimicrobial activityand action of silver. Prog. Med. Chem. 31: 351-370. Sarkar S, Jana AD, Samanta SK, Mostafa G (2007). Facile synthesis of silver nano particles with highly efficient anti-microbial property. Polyhedron, 26: 4419- 4428 Sharma VK, Yngard RA, Lin Y (2009). Silver nanoparticles: Green synthesis and their antimicrobial activities. Adv. Colloid. Interf. Sci. 145: 83- 89 Simon-Deckers A, Brun E, Gouget B, Carriere M, Sicard-Roselli C (2008). Impact of gold nanoparticles combined to X-ray irradiation on bacteria. Gold Bull. 41: 187-194. Skoog DA, Holler FJ, Nieman TA (1998). Principles of Instrumental Analysis. Saundres College Press. Fort Worth. USA. Sondi I, Salopek-Sondi B (2004). Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria. J. Colloid Interface Sci. 275: 177-182. Stevens KNJ, Crespo-Biel O, van den Bosch EEM, Dias AA, Knetsch MLW, Aldenhoff YBJ, van der Veen FH, Maessen JG, Stobberingh EE, Koole LH (2009). The relationship between the antimicrobial effect of catheter coatings containing silver nanoparticles and the coagulation of contacting blood. Biomaterials, 30: 3682-3690. Wijnhoven SWP, Peijnenburg W, Herberts CA, Hagens WI, Oomen AG, Heugens EHW, Roszek B, Bisschops J, Gosens I, Van de Meent D, Dekkers S, De Jong WH, Van Zijverden M, Sips A, Geertsma RE (2009). Nano-silver-a review of available data and knowledge gaps in human and environmental risk assessment. Nanotoxicology, 3: 109178. Yoon, K Y, Byeon JH, Park JH, Hwang J (2007). Susceptibility constants of Escherichia coli and Bacillus subtilis to silver and copper nanoparticles. Sci. Total Environ. 373: 572-575.
African Journal of Biotechnology Vol. 11(29), pp. 7565-7569, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.3726 ISSN 1684â&#x20AC;&#x201C;5315 Š 2012 Academic Journals
Full Length Research Paper
Fetal neurohistopathology of chloropyrifos in mice Kausar Raees1, Asmat Ullah2, Tahir Abbas2, Muhammad Khalid Mukhtar3, Muhammad Arshad3, Shafaat Yar Khan3, Hafiz Muhammad Tahir3 and Khawaja Raees Ahmad3* 1
Government Degree College (for Women), Farooq Colony, Sargodha Pakistan. 2 Department of Zoology, University of The Punjab, Lahore, Pakistan. 3 Department of Biological Sciences, University of Sargodha, Sargodha, Pakistan. Accepted 14 February, 2012
Chlorpyrifos (CPF) was tested for feto-neuro-histopathological manifestations on fetal central nervous system (CNS) in mice at 3 maternally sub-toxic oral doses - 0, 9 and 18 mg/kg. Each dose group was further categorized as: single (gestation day (GD) 6) and triple exposures (9, and 12 respectively). Fetuses were exteriorized on GD18. No obvious signs of toxicity were seen in the dams at these exposures. Mean fetal weight showed a dose (9 and 18 mg/kg) and exposure (single and triple) dependent decrease compared to that of the 0 mg/kg group while the litter size remained unaffected. The neurohistopathological abnormalities include vacuolations of the medullary region along with cortical lesions in CNS in 9 and 18 mg/kg groups on triple exposure only. These neurohistopathological manifestations were considered as the indicatives of neuroglial cells necrosis apoptosis. Our findings suggest that gestational exposure of CPF at motherly safe dose levels in mice induce neuroglial cells apoptosis in fetal CNS. Key words: Chlorpyrifos, neuroteratology, neurotoxicity, neurohistopathology.
INTRODUCTION Chlorpyrifos (CPF) [O, O-diethyl-O-(3, 5, 6-trichloro-2pyridyl) - phosphorothioate], an extensively used organophosphate (OP) insecticide, was tested for its effects on litter size, fetal weight and neurohistopahological abnormalities in developing mice. Although, due to its exposure risks in children, use of CPF in domestic sector was restricted in 2000 in US (EPA, 2000), it is still being used extensively worldwide (Tian et al., 2005; Dowagro 2006) especially in developing countries like Pakistan. Metabolism of CPF, in vivo, releases CPF-oxon- a very potent cholinesterase inhibitor (Cometa et al., 2007). Timchalk et al. (2006) demonstrated age-dependent variations in capability to detoxify environmental chemicals both in humans and animals. Thus, it is not surprising to see that young animals are more sensitive to OP insecticides exposure
*Corresponding author. E-mail: +92483740088, +923347511223.
karees@uos.edu.pk.
Tel:
Abbreviations: CPF, Chlorpyrifos; CNS, central nervous system; GD, gestation day.
than adults (Pope and Liu, 1997; Moser and Padilla, 1998). This greater sensitivity to the toxic effects of OP insecticides in young animals has been related to their partial incompetence in detoxification (Mortensen et al., 1998; Atterberry et al., 1997; Vidair, 2004). An in vitro study showed that OP insecticides and their oxons particularly target astroglial cell proliferation (Guizzetti et al., 2005); thus, it seems logical to speculate that OP insecticides like CPF may accumulate and inflict severe toxicity during the prenatal development and early postnatal life than adulthood. Thus, repeated sub-toxic low dose exposures of CPF particularly target developing brain during the critical period of cell division causing abnormalities at cellular and synaptic levels (Whitney et al., 1995). Most of the animal studies conducted so far have been restricted to the analysis of the feto-morphic teratology of CPF exposure, typically from the stand point of dose response model (Farag et al., 2003; Tian et al., 2005; Akhtar et al., 2006). There is a dearth of available literature reporting the in vivo neurohistopathology caused by low dose CPF exposure in fetal life. In present study, we investigated the fetal neurohistopathological manifestations of CPF at
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1400
*
*
1000
*
876.9
1006.9
1320.3
400
1116.6
600
1206.9
800
1323.7
Mean Fetal Weight (mg)
1200
200 p<0.0005
p<0.00001
0 0mg0 /kg
9 /kg 9mg
Single
0mg0 /kg
18 (mg/kg) 18mg /kg
…Exposures …
9 /kg 9mg
18 (mg/kg) 18mg /kg
Triple
Figure 1. Effect of chlorpyrifos on mean fetal weight {p values: level of significance at two exposure levels; bars indicate standard error of the means and * indicate post hoc significant difference from control (p< 0.05).
the maternally sub-toxic doses of exposure. MATERIALS AND METHODS
carefully removed, weighed and fixed in Bouin’s solution for 48 h and finally transferred in 70% alcohol for storage and further study. Mean litter size and fetal weights / litter were obtained for each group and analyzed statistically.
Animals Histology A total of 90 pregnant females, of the Swiss Webster strain of albino laboratory mice Mus musculus were used in this research work. They were kept in 12” 18” 12” wire-mesh covered steel cages under 12 h light-dark cycles. Room temperature was maintained at 232°C. Food and water were provided ad-libitum. Males were caged with estrus females for 2 to 3 h. Thereafter, the dams were observed for the presence of vaginal plug as a confirmatory sign for successful coitus. Each mated female was parted from the male and the day of coitus was marked as day 0 of gestation.
Thirty randomly selected fetuses (one from each litter) of the three experimental groups were processed for wax embedding and microtomy. The serial sections (5 µ thick) were stained with Haematoxylin and Eosin for the neurohistopathological studies. Digital photomicrographs (40 and 100) of the selected sections were obtained to report histopathological signs.
RESULTS Mean litter size and fetal weight
Experimental groups and dose regimen Bred females were randomly divided into 3 groups of 30 animals each, named as 0, 9 and 18 mg/kg groups. Each group was further divided into 2 subgroups of 15 animals each; respectively named as single [exposure on GD6] and multiple [exposure on GD6, 9, 12] exposure subgroups. The dose dilutions of CPF were made in corn oil. The animals in 0 mg/kg received pure corn oil in every treatment. The doses were delivered orally to the experimental animals by gavage.
There were no significant variations in mean litter size among the experimental groups. Mean fetal weight showed a dose (9 and 18 mg/kg) (p< 0.0005) and exposure (single and triple) related significant (p< 0.0001) decrease. Post-hoc analysis of the data indicate a significant (p< 0.05) difference between 0 and 18 mg/kg groups at single exposure and 0 mg/kg group to that of the 9 and 18 mg/kg groups at triple exposure (Figure 1). Neurohistopathology
Recovery and processing of the fetuses Gravid uteri were exteriorized through a median incision on the abdominal wall from the euthanized dams on GD18. Fetuses were
Vacuolations in the medullary region (an indication of neuroglial cells apoptosis) and cortical lesions(indicative of the damaged tracts), in CNS were seen in 8/15 fetuses processed from 18
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Figure 2. A1: selected section through the spinal chord of the fetus in 0 mg/kg triple exposure group (40×); A2: a closer look of the selected portion of A1 (100×); B1: selected section through the spinal chord of the fetus in 18mg/kg triple exposure group (40×); B2: a closer look of the selected portion of B1 (100×); a: neurocoele; b: cortical tracts; c:medullary vacuolations; d: cortical lesions.
mg/kg group at triple exposure; whereas 3/15 fetuses from 9 mg/kg group at triple exposure showed these signs. In 9 and 18 mg/kg single exposure sub-groups and 0 mg/kg group, no signs of medullary vacuolations or cortical lesions were seen (Figure 2).
DISCUSSION Chlorpyrifos is a very potent neurotoxic agent (Ehrich et al., 2004); in rats, the gestational exposure to CPF causes persistent inhibition of brain acetylcholinesterase and suppression of choline-acetyltransferase (Richardson and Chambers, 2003). Results obtained from rat embryo cultures indicated that it particularly targets brain development (Roy et al., 1998). It has also been claimed that CPF promotes neurobehavioral abnormalities throughout the embryonic and neonatal development (Roy et al., 2005). The histopathological outcomes of prenatal CPF exposure obtained in this study are well in line with these developmental neurotoxicological studies. Comparative analysis of the mean fetal weight in 0 mg/kg group to that of the 9 and 18 mg/kg groups at single and triple exposures clearly indicates that in utero CPF exposure caused dose and exposure-dependent growth retardation. In this context, it was reported earlier that levels of CPF in umbilical cord plasma are inversely proportional to the
neonatal length and birth weight in humans (Whyatt et al., 2004). The ability of CPF to interfere in the critical cellular processes such as cell division (mediated through inhibition of DNA replication), differentiation, gene expression and regulation are now well documented (Qiao et al., 2001). Whitney et al. (1995) pointed out that CPF inhibit DNA synthesis within a few hours of systemic administration to neonatal rats. Similarly, Samarawickrema et al. (2008) concluded that low dose chronic exposure to CPF leads to an increased oxidative stress and high DNA fragmentation in the fetuses. Moreover, CPF has been claimed to be a proven neuro-toxic agent in young animals as it alters the replication and differentiation of neurons and bring about alterations in synaptic transmission independent to that of choli-nergic stimulation. Induced oxidative stress of the insecticide exposure seems to play a key role in these neurotoxic outcomes (Saulsbury et al., 2009). Recently, it has been reported that CPF can induce apoptosis in well differentiated cells like human T cells (Li et al., 2009). Continued administration of CPF to neonatal rats is found to elicit a shortfall in cell numbers throughout the brain (Slotkin, 1999). Chlorpyrifos has also been found to interfere in the development of axonal projections thus logically affecting cell differentiation (Das and Barone, 1999; Li and Casida, 1998). The most critical effects involve the ability of CPF to interfere with the function of nuclear transcription factors
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that control cellular fate, including their expression, phosphorylation and capacity to bind on their DNA promoter recognition sites (Crumpton et al., 2000; Garcia et al., 2001; Schuh et al., 2002). Slotkin and Seidler (2007) have pointed out that CPF induces major transcriptional alternations in the genes controlling neural cell growth, development of glia and myelin, neural cell differentiation, cAMP-related cell signaling, oxidative stress, excitotoxicity and apoptosis. In the same context, Buratti et al. (2006) suggested that organophosphorothionate pesticides such as CPF induced neuro-developmental effects due to their in situ bioactivation by fetal enzymes. Caughlan et al. (2004) reports that independently from its proven neurotoxic effects mediated through inhibition of cholinesterase, CPF exposure may lead to apoptosis in the nervous system. Vacuolations, the medullary wide hollow spaces and the cortical lesions in the present study are respectively considered as the signs of the neuro-glial cells apoptosis and axonodendronal/synaptotoxicity (Figure 2). These observations provide an in vivo indication of the similar previous findings (Buratti et al., 2006; Caughlan et al., 2004; Das and Barone, 1999; Li and Casida, 1998; Slotkin and Seidler, 2007; Samarawickrema et al., 2008; Saulsbury et al., 2009; Slotkin, 1999; Whitney et al., 1995). The neurohistopathological defects observed in this study seem to be the logical outcomes of the afore cited broad range of developmental toxic effects of CPF. Chlorpyrifos and particularly CPF-oxon (produced by partial metabolism in maternal liver) readily cross the placental and blood-brain barriers (Parran et al., 2005). The developing fetal brains are more vulnerable and deficient in detoxification of these chemicals (Vidair, 2004; Timchalk et al., 2006). Thus, during the course of co-gestational triple exposures, CPF and CPFoxon might have stayed longer in the fetal tissues, particularly brain (Guizzetti et al., 2005; Whitney et al., 1995), thereby causing huge oxidative stress. This oxidative stress must have been the most logical cause of apoptosis of the neuroglial cells (Franco et al., 2009; Verma et al., 2007; Yu et al., 2008). In this connection, an in-depth study of the estimation of CPF and CPF-oxon concentrations in the developing fetal brains is recommended.
Conclusion On the basis of the results obtained in this study, it is concluded that the developmental exposure of CPF brings about several neurohistopathological changes; while the dose and exposure (single and triple) dependent decrease in the fetal weight is seemingly the out comes of the general growth retardation induced by the gestational CPF exposure.
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African Journal of Biotechnology Vol. 11(29), pp. 7570-7578, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.182 ISSN 1684–5315 © 2012 Academic Journals
Full Length Research Paper
Prenatal and perinatal acrylamide disrupts the development of cerebrum and medulla oblongata in albino rats Allam A.1*, Abdul-Hamid M.2, Zohair K3,4, Ajarem J1, Allam G2,5 and El-Ghareeb A.6 1
Department of Zoology, Faculty of Science, King Saud University, Saudi Arabia. 2 Department of Zoology, Faculty of Science, Beni-suef University, Egypt. 3 Cell Biology Department, National Research Center, Dokki, Cairo, Egypt. 4 Department of Pharmacology, College of Pharmacy, King Saud University, Saudi Arabia. 5 Department of Microbiology, College of Medicine and Medical Sciences, Taif University, Taif, Saudi Arabia. 6 Department of Zoology, Faculty of Science, Cairo University, Egypt. Accepted 23 March, 2012
Acrylamide is known to cause neurotoxicity in experimental animals and humans. The literature on its neurotoxic effect in adult animals is huge, but the effect of acrylamide on the embryonic and postnatal development is relatively less understood. The present study examined its effects on the development of oxidative stress of cerebrum and medulla oblongata in albino rats. Acrylamide was orally administered to non-anesthetized pregnant females by gastric intubation 10 mg/kg/day. The animals were divided into 3 groups as follows: group A - newborn from control animals, group B - newborns from mothers treated with acrylamide from day 7 (D7) of gestation till birth (prenatal intoxicated group), group C - newborns from mothers treated with acrylamide from D7 of gestation till D28 after birth (perinatally intoxicated group). Acrylamide administered either prenatally or perinatally was shown to induce significant increase of thiobarbituric acid-reactive substances (TBARS) and oxidative stress (significant reductions in glutathione (GSH), total thiols, superoxide dismutase (SOD), and peroxidase activities) in the developing cerebrum and medulla oblongata. The results of this study showed that prenatal and perinatal acrylamide or its metabolites disrupts the biochemical machinery, cause oxidative stress and induce structural changes in the developing rat cerebrum and medulla oblongata.
Key words: Acrylamide, postnatal development, cerebrum, medulla oblongata, oxidative stress.
INTRODUCTION Acrylamide, a β-unsaturated amide, is an industrial chemical used as polyacrylamide in the filtration and flocculation process in the mining, paper mill, water treatment and waste processing industries (Friedman, 2003; Doerge et al., 2008). In vivo, acrylamide is metabolized by cytochrome P450 (CYP450) to a reactive epoxide, Glycidamide, that reacts with GSH and other vital cellular nucelophiles possessing –SH, –NH2 or –OH groups and forms glutathione S-conjugates, which is the initial step in the biotransformation of electrophiles into
*Corresponding author. E-mail: allam1081981@yahoo.com.
mercapturic acids (Dixit et al., 1980, 1981, 1982,1984; Miller et al., 1982; Calleman et al., 1990; Awad et al., 1998; Sumner et al.,1992; Dybing and Sanner 2003). Acrylamide’s reaction with sulfhydryl groups of proteins is facilitated by glutathione-S-transferase (GST) to form Nacetyl-S-(3-amino-3-oxopropyl) cysteine that accounts for approximately 70% of the urinary metabolites in rats (Sumner et al., 1997). Metabolic studies have shown the interaction of acrylamide with CYP450 and GST in rat brain and erythrocyte (Dixit et al., 1980, 1981, 1982, 1984; Mukhtar et al., 1981; Das et al., 1982). Acrylamide has been shown to inhibit GST activity that further promotes the metabolism acrylamide to glycidamide by the CYP450 pathway (Dixit
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et al., 1981, 1984). Srivastava et al. (1983) has reported induction of thiobarbituric acid-reactive substances (TBARS) in different tissues and suggested that increased TBARS is a consequence of depletion of glutathione (GSH). Thiol groups are important reducing agents required for the activity of many enzymes. They are vital cellular anti-oxidants and protect mammalian cells from free radicals (Yousef and ElDemerdash, 2006; Allam et al., 2010). Glutathione is the principal endogenous thiol that acts as a redox buffer in our body (Tong et al., 2004). Acrylamide causes oxidative stress and this effect was increased at higher doses (Yousef and El-Demerdash, 2006; Zhu et al., 2008). The delicate balance between the production and the catabolism of oxidants is critical for maintenance of the biological functions (Sridevi et al., 1998; Takahashi et al., 2008). Acrylamide is a well documented neurotoxicant that produces axonal pathology both in the central and peripheral nervous system (Spencer and Schaumburg, 1977a, b, 1979; Cavanagh, 1982; LoPachin, 2004). Most of these studies with acrylamide were done using adult rodents, and not much information is available on its effect on developing brain. During maturation of the brain, there is an extensive reorganization of neuronal structures, and exposure to neurotoxicant such as acry-lamide would likely affect the expression of proteins, enzymes, lipids and nucleic acids. It has been reported that the cerebrum at birth in albino rat is in the state of immaturity and its histogenesis and morphogenesis mainly occurs postnatally (Jacobsen, 1970). The present study aimed to show the effect of acrylamide exposure during gestation on GSH, total thiols levels, superoxide dismutase and peroxidase activities in cerebrum and medulla oblongata of the newborn rats. MATERIALS AND METHODS Chemicals Acrylamide (99% pure) and other chemicals were purchased from Sigma chemical Company (St Louis, MO, USA). All other chemicals used were of analytical grade. Animal dosing schedule A total of sixty albino rats was used for this study (Rattus norvegicus). Forty five mature virgin females and 15 mature males weighing 140 to 150 g were purchased from the Organization for Vaccine and Biological Preparations, Helwan Laboratory Farms, Egypt. Animals were marked, housed 4 per cage and fed standard rodent pellet diet manufactured by the Egyptian Company for Oil and Soap, Cairo, Egypt. Animals were given tap water ad libitum. Daily examination of vaginal smear of each virgin female was carried out to determine the estrous cycle. Estrous females exhibited the presence of cornified cells in vaginal smear. Mating was done by overnight housing of 2 pro-esterous females with one male in separate cages. The presence of sperm in the vaginal smear was determined at day zero (D0) of gestation. Acrylamide was dissolved in distilled water and administered orally to nonwas dissolved in distilled water and administered orally to non-
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anesthetized pregnant rats by gastric intubation at a dose of 10 mg/kg/day. The pregnant mothers were labeled into 3 groups as follows: group A - pregnant rats which were given saline (control); group B pregnant rats which were administered acrylamide from day 7 (D7) of gestation till birth (prenatal intoxication) and group C - pregnant rats which were administered acrylamide from D7 of gestation till D28 after birth (perinatal intoxication). Postnatal investigations The newborns were investigated every day and the following notes were recorded in each group: a) The daily weights of 6 newborns from each group; b) The time of fur appearing; c) The time of ear opening; d) The time of eye opening. Biochemical assays Pups from each group were sacrificed by decapitation at D7, D14, D21 and D28. Cerebrum and medulla oblongata were dissected and 0.25 g tissue from each was homogenized in 3 ml of cold saline. The homogenate was centrifuged at 10,000 g for 10 min at 4°C and the clear supernatant were collected in a microfuge tube (0.5 ml each) and stored at -40°C until used.
Lipid peroxidation (TBARS) Lipid peroxidation was determined by assaying thiobarbituric acidreactive substances (TBARS) according to the method of Preuss et al. (1998). Briefly, 1.0 ml supernatant was precipitated with 2 ml 7.5 % trichloroacetic acid and centrifuged at 1,000 g for 10 min. Clear supernatant was mixed with 1 ml 0.70% thiobarbituric acid, incubated at 80°C and the absorbance measured at 532 nm. Tetramethoxypropane was used as the standard. Glutathione (GSH) assay Glutathione content was determined according to the procedure of Beutler et al. (1963) with some modification. Briefly, 0.20 ml of tissue supernatant was mixed with 1.5 ml precipitating solution containing 1.67% glacial metaphosphoric acid, 0.20% Na-EDTA and 30% NaCl. The mixture was allowed to stand for 5 min at room temperature and centrifuged at 1,000 g for 5 min. One milliliter clear supernatant was mixed with 4 ml 0.30 M Na2HPO4 and 0.50 ml DTNB reagent (40 mg 5,5’dithiobis-2-nitrobenzoic acid dissolved in 1% sodium citrate). A blank was similarly prepared in which 0.20 ml water was used instead of the tissue supernatant. The absorbance of the color was measure at 412 nm in a spectrophotometer. Total thiol determination Total thiol was determined according to the method of Koster et al. (1986). Briefly, 50 μl of the supernatant and 0.75 ml 0.1M phosphate buffer (pH 7.4) was mixed with 0.20 ml Ellman’s reagent (2 mM 5,5’dithiobis-2-nitrobenzoic acid), and incubated for 5 min at 37°C. A blank was similarly prepared in which 50 μl water was used instead of the tissue supernatant. The absorbance of the color was measure at 412 nm in a spectrophotometer.
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Table 1. External features appearance in newborn rats.
Features Fur appearing Ear opening Eye opening
Group A D9 D12-13 D14-15
Superoxide dismutase (SOD) assay Superoxide dismutase activity was assayed according to the method of Marklund and Marklund (1974). Briefly, 1.0 ml supernatant was mixed with 0.10 ml of tris/EDTA buffer pH 8.0 and 0.05 ml of 10 mM pyrogallol (freshly prepared). Control was prepared by adding 1.0 ml of water instead of the tissue extract. The difference in the absorbance of the color at 430 nm at 0 min, and after 10 min was recorded to calculate the enzyme activity.
Group B D11-12 D15 D16-17
Group C D12-13 D15 D16-17
mean weights of the newborns of all experimental groups varied between D1 and D28 (Table 2). The newborns of group B suffered from a marked decrease in the body weight gain, where the newborns of group C showed more noticeable decrease in their body weight gain.
Oxidative stress Thiobarbituric acid-reactive substances (TBARS)
Peroxidase activity Peroxidase activity was determined according to the method of Kar and Mishra (1976). Briefly, 1.0 ml supernatant was mixed with 3.0 ml of 0.01 M phosphate- buffered saline (pH 6.8), 315 μl of 2% pyrogallol, 154 μl H2O2 and incubated for 15 min at 25°C. The reaction was stopped by the addition of 0.50 ml of 5% H 2SO4 and the absorbance was recorded at 420 nm. Peroxidase activity was expressed as the amount of purpurogallin formed per unit absorbance.
Statistical analysis The Statistical Package for the Social Sciences (SPSS for windows version 11.0; SPSS Inc, Chicago) was used for the statistical analyses. Comparative analyses were conducted by using the general linear models procedure (SPSS, Inc). Also, the data were analyzed using one-way and two-way analysis of variance (MANOVA) followed by LSD computations to compare various groups with each other. Results were expressed as mean S.D. The level of significance was expressed as P<0.05 and highly significant at P<0.01 (Rao and Blane, 1995).
RESULTS General developmental observations The newborns in group B suffered from prenatal acry-lamide exposure while newborns of group C suffered from perinatal exposure to acrylamide and its toxic product intrauterine for 14 days and postnatal through milk from their mothers for 28 days. Signs of acrylamide toxicity were observed postnatally on the treated mothers represented by ataxia, splayed hind limb, weakness of hind-limb muscles and finally, paralysis causing alteration in maternal behavior; so, their newborns suffered from bad lactation and consequently malnutrition especially in group C. At birth, the newborns of all groups were hairless. The time of fur appearing, ear and eye opening was retarded in groups B and C (Table 1). The
In control newborns, the cerebral and medulla lipid peroxidation decreased with age development. Administration of acrylamide induced a pronounced increase in lipid peroxidation content at the investigated ages with its maximal value in group C. Administration of acrylamide increased TBARS significantly at all the ages in both groups (Tables 3 and 5). One way MANOVA of both organs lipid peroxidation data showed highly significant effect between groups (p<0.001). Two way MANOVA showed highly significant effect induced by the treatment and time (p<0.001) and treatment-time interaction also produced a significant change (p<0.05) for prenatal-intoxicated newborns. Treatment and treatmenttime interaction had a very highly significant effect (p<0.001) in the perinatal group, but time had a non-significant effect (p>0.05).
GSH content Acrylamide treatment produced marked decrease in GSH content of cerebrum and medulla oblongata in groups B and C throughout the experiment as compared to the control group (Tables 3 and 5). In perinatal group, the loss of GSH was consistently more than the prenatal group at all ages. One way MANOVA of GSH content in both organs showed a high significant depletion (p<0.001) in both prenatal and perinatal groups. Two way MANOVA indi-cated that acrylamide treatment induced a very highly significant change (p<0.001), whereas time and treatment-time interaction had non-significant effect on GSH content (p>0.05). Total thiol content Data in Table 1 illustrate marked changes in total thiol with age in the control and acrylamide treated groups.
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Table 2. Changes of body weights in newborn rats.
Days 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Group A 6.31±0.12 7.4±0.26 8.33±0.19 9.15±0.15 9.58±0.28 10.65±0.23 12.13±0.16 13.57±0.1 14.27±0.13 16.1±0.15 17.23±0.13 17.33±0.07 18.25±0.09 19.35±0.15 20.35±0.08 21.60±0.15 23.67±0.36 23.97±0.36 25.22±0.51 25.8±0.64 27.75±0.60 28.47±0.52 31.42±0.45 34.52±0.35 36.07±0.37 38.45±0.57 39.65±0.62 43.17±0.99
Group B 5.08±0.13*** 5.15±0.08*** 5.53±0.16*** 5.57±0.11*** 5.8±0.13*** 6±0.21*** 6.83±0.27*** 6.85±0.41*** 9.23±0.68*** 10.9±0.336*** 10.47±0.63*** 10.77±0.41*** 12.27±0.81*** 11.58±0.72*** 13.28±0.68*** 15.38±0.42*** 16.93±0.76** 17.36±0.78*** 19.73±0.40*** 20.68±0.69*** 20.9±0.38*** 21.2±0.51*** 22.85±0.71*** 23.68±0.99*** 23.55±0.77*** 24.65±0.99*** 25.87±77*** 26.62±0.62***
Group C 3.88±0.11*** 4.47±0.50** 5.27±0.38** 5.83±0.53*** 5.48±0.29*** 6.23±0.40*** 6.18±0.27*** 6.75±0.31*** 7.13±0.38*** 7.9±0.30*** 9.33±0.77*** 9.85±0.80*** 9.27±0.65*** 9.33±0.67*** 10.72±0.60*** 10.39±0.34*** 12.93±0.61*** 11.3±0.56*** 11.63±0.60*** 12.47±0.70*** 14.18±0.50*** 16.48±0.47*** 20.12±0.95*** 20.97±0.87*** 21.55±1.13*** 22.40±1.12*** 23.25±0.64*** 24.21±1.11***
Data are expressed as a mean ± S.E. (N =6); values significantly compare to the control newborns; p*≤0.05, p**≤0.01and p***≤0.001.
Total thiol content of cerebrum and medulla oblongata was equally reduced in both prenatally and perinatally intoxicated groups compared to the control group. These reductions in total thiol content were highly significant (Tables 3 and 5). One way MANOVA of total thiol data showed a highly significant (p<0.001) difference between groups through-out the experiment. Two way analysis displayed a highly significant effect (p<0.001) of treatment and time on total thiol content in group B, while treatment-time interaction had a highly significant effect (p<0.01). In group C, the effect of treatment time and their interaction was very highly significant (p<0.001).
groups exhibited a highly significant inhibition of peroxidase activity in both treated groups (Tables 4 and 6). The maximal inhibition of peroxidase activity was observed in group C. One way MANOVA of cerebral and medulla peroxidase activity showed highly significant difference between control and acrylamide-treated groups throughout the experiment. Two way MANOVA of the data of prenatal group showed significant effect of treatment time and treatment-time interaction (P<0.001). In group C, the effect of treatment was highly significant (P<0.001), while the effect of time and treatment-time interaction was non-significant (P>0.05). SOD activity
Peroxidase activity Peroxidase activity of cerebrum and medulla oblongata showed fluctuations in the control group. Acrylamide-treated
SOD exhibited a very highly significant (P<0.001) decreased activity in cerebrum and medulla oblongata of intoxicated groups as compared to the normal one at all investigated ages (Tables 4 and 6); this decrease was more pronounced as the
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Table 3. Effect of acrylamide administration on TBARS, GSH and total thiol contents in cerebrum of developing newborn rats.
Parameter
Time
D7
D14
D21
Group Normal Group B (prenatal) Group C (perinatally)
24.67 ± 3.10 cd 27.07 ± 1.96 a 40.10 ± 3.05
e
19.37 ± 1.60 c 27.33 ± 2.22 b 31.47 ± 2.81
a
GSH (nmol/gm)
Normal Group B (prenatal) Group C (perinatally)
57.51 ± 6.06 c 36.21 ± 3.30 d 28.22 ± 3.14
54.31 ± 2.86 c 34.07 ± 5.22 d 27.16 ± 1.74
Total thiol (mol/gm)
Normal Group B (prenatal) Group C (perinatally)
1.67 ± 0.07 de 1.00 ± 0.23 ef 0.94 ± 0.19
TBARS (nmol/ 100 mg)
c
D28
f
19.05± 1.20 c 27.56 ± 2.23 c 27.83 ± 1.01
f
18.74 ± 1.33 cd 25.45 ± 2.67 cd 26.29 ± 2.14
ab
51.12 ± 2.86 c 38.27 ± 5.981 d 23.96 ± 3.91
b
50.59 ± 2.41 c 34.61 ± 3.14 d 23.64 ± 4.33
b
1.87 ± 0.14 de 0.99 ± 0.13 f 0.81 ± 0.11
a
2.24 ± 0.09 d 1.15 ± 0.18 ef 0.84 ± 0.12
f
b
a
2.35 ± 0.14 bc 1.70 ± 0.18 ef 0.89 ± 0.16
Data are expressed as mean ±S.D. (N = 6); means which share the same superscript are not significantly different; significance level = 0.05.
Table 4. Effect of acrylamide administration on peroxidase and SOD activities in cerebrum of developing newborn rats.
Parameter
Peroxidase (U/gm)
SOD (U/gm)
Time Group Normal Group B (prenatal) Group C (perinatally)
44.61±1.42 b 37.85±0.87 cd 34.71±1.43
Normal Group B (prenatal) Group C (perinatally)
6.34±1.19 de 3.22±0.86 cde 3.40±0.44
D7
D14 a
b
D21 a
44.68±1.30 cd 35.08±0.72 d 33.55±2.58 b
6.72±1.53 cde 3.68±0.48 e 3.02±0.42
D28 a
a
44.13±1.341 b 38.87±1.59 c 35.75±1.15
43.03±2.00 b 38.49±1.28 c 35.86±1.55
a
9.48±2.16 cde 4.00±0.53 cde 3.75±0.38
10.70±2.08 c 4.59±0.63 cd 4.51±0.65
a
Data are expressed as mean ±S.D. (N = 6); means which share the same superscript are not significantly different; significance level = 0.05.
experimental period extended in group C. Otherwise, in normal and treated newborns, cerebral and medulla SOD activities changed with age development. One way MANOVA of the data showed significant effect (p<0.001) on cerebral and medulla SOD activities between groups throughout the experiment while two way MANOVA of cerebellar SOD activity revealed highly significant (p<0.001) effect of treatment time and their interaction in group B. In group C, the effect of treatment was also highly significant (p<0.001), while the effect of time and treatmenttime interaction was significant (p<0.05).
DISCUSSION The present study was designed to investigate the teratogenicity of acrylamide on the development of body weights, cerebrum and medulla oblongata in newborn rats at different conditions of maternal acrylamide expo-sure. Acrylamide and its metabolic products glycidamide readily pass through the placenta due to its solubility in water
(Sorgel et al., 2002) and is distributed in many fetus tissues during gestation (Marlowe et al., 1986; Sumner et al., 2001). Also, acrylamide passes through the mother's milk to its newborns during lactation (Sorgel et al., 2002; Takahashi et al., 2008). Acrylamide leads to bad lactation which results from bad maternal behaviors and consequently leads to postnatal newborns mal-nutrition (Frieda and William, 1999; Shaheed et al., 2006; Allam et al., 2010). In this study, fur appeared at D9 in normal newborn rats, while its appearance was delayed in the treated groups which is in agreement with Gold et al. (2000) who reported that acrylamide causes growth retardation. This retardation resulted from growth and protein deficiencies due to the malnutrition during the development (Abdul-Hamid et al., 2007). In the present study, normal new-borns, ear opening were detected at D12 to 13. Smart and Dobbing (1971) detected similar results in newborn rats. In treated groups, ear opening was delayed (at
D15). This retardation showed that acrylamide exposure impairs organogenesis as mentioned by Marlowe et al. (1986). The eye opening occurred at D14 to 15 in group A, this result was also observed by Bolles and Woods
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Table 5. Effect of acrylamide administration on the development of TBARS, GSH and total thiol contents in medulla oblongata of developing newborn rats.
Time Group Normal Group B (prenatal) Group C (perinatally)
18.94 ± 2.42 de 23.01 ± 1.55 ab 27.72 ± 0.69
GSH (nmol/gm)
Normal Group B (prenatal) Group C (perinatally)
99.84 ± 11.10 efg 74.28 ± 6.46 fg 67.09 ± 15.15
Total thiol (mol/gm)
Normal Group B (prenatal) Group C (perinatally)
2.82 ± 0.62 de 1.20 ± 0.14 ef 1.00 ± 0.11
Parameter
TBARS (nmol/100 mg)
D7
D14 gh
D21 ghi
17.46± 1.04 ef 21.56 ± 2.31 cd 24.59 ± 1.51
ab
99.04 ± 5.80 bcd 91.86 ±18.54 g 63.90 ± 7.21
a
2.86 ± 0.17 c 1.73 ± 0.19 de 1.25 ± 0.15
18.70 ± 2.28 bc 25.52 ± 4.34 a 29.07 ± 1.38
abc
ab
D28
105.43 ± 10 cde 87.06 ± 14.02 fg 67.89 ± 8.66
hi
16.42 ± 0.61 efg 20.62 ± 1.66 fg 19.93 ± 1.76
abc
110.22 ± 16.32 cde 89.46 ± 17.59 def 82.27 ± 14.97
a
3.12 ± 0.17 c 1.75 ± 0.28 f 0.89 ± 0.19
i
a
b
2.52 ± 0.31 d 1.42 ± 0.27 de 1.31 ± 0.20
Data are expressed as mean ±S.D. (N = 6); means which share the same superscript are not significantly different; significance level = 0.05.
Table 6. Effect of acrylamide administration on the development of peroxidase and SOD activities in medulla oblongata of developing newborn rats.
Parameter
Peroxidase (U/gm)
SOD (U/gm)
Time Group Normal Group B (prenatal) Group C (perinatally)
68.63± 1.25 de 56.06 ± 3.38 f 51.83 ± 1.70
a
71.17 ± 3.14 bc 59.11 ± 1.82 ef 53.28 ± 3.84
a
71.44± 1.91 b 60.97 ± 3.38 cd 57.16 ± 1.46
a
71.46 ± 1.20 b 60.42 ± 2.99 bcd 58.95 ± 1.84
Normal Group B (prenatal) Group C (perinatally)
23.01 ± 3.22 c 15.96 ± 4.46 fg 10.01 ± 2.94
a
19.37 ± 3.33 cd 14.39 ± 2.52 g 6.39 ± 0.77
b
20.78 ± 1.88 de 12.83 ±1.69 g 6.38 ± 0.64
ab
21.34 ± 3.01 cd 14.78 ± 2.99 fg 7.35 ± 1.25
D7
D14
D21
D28 a
ab
Data are expressed as mean ±S.D. (N = 6); means which share the same superscript are not significantly different; significance level = 0.05.
(1964), while it was detected in groups B and C at D16 to 17. This retardation in treated groups is in agreement with Sumner et al. (2001) who mentioned that acrylamide causes developmental alterations. Body weight loss of the newborns in treated groups was observed. Chatterjea and Shinde (2002) and Garey et al. (2005) recoded these reductions in weights. The main reason for this prenatal weight reduction resulted from intrauterine acrylamide exposures that lead to growth deficiency of the developing fetus (Tyl et al., 2000a, b). Newborns body weight was the most sensitive indicator of developmental toxicity (Wise et al., 1995). In the treated groups, the acrylamide affects the function of mammary glands because it leads to prolactin reduction in animals; thus, it impairs lactation (Uphouse et al., 1982; Frieda and William, 1999). So, there were nutria-tional deprivations and consequently, the newborns body weight loss was noticed. Frieda and William (1999) showed that the main reason for this postnatal weight reduction in treated newborns was
because they suffered from alterations in maternal behaviors caused by acrylamide as well as a decrease in food and water consumption and lactation index. The maternal acrylamide exposure during gestation and lactation periods in the present study produced a pronounced increase of oxidative stress and a marked suppression of the antioxidant defence system in the cerebrum and medulla oblongata of the newborn rats. The TBARS level was markedly elevated, while GSH and total thiol content were enormously depleted. Moreover, the activity of antioxidant enzymes; SOD and peroxidase, were also depressed in the treated groups. The highest deterioration of these variables appeared in group C due to the perinatally acrylamide intoxication. In normal new-borns, oxidative stress changed with age progress due to maturation and differentiation of the newborns tissues. Yousef and El-Demerdash (2006) indicated that rats that received oral acrylamide exhibited marked elevation of lipid peroxidation. Bhadauria et al. (2002) and Uličná et al. (2003)
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also noticed that the toxicity induce a reduction in GSH level. Induction in the levels of TBARS in different tissues is in agreement with the finding of Srivastava et
al., (1983) who suggested that enhancement of TBARS is a consequence of depletion of GSH to certain critical levels. The increase in TBARS level observed in the present study is parallel to the decrease in GSH concentration in cerebrum and medulla oblongata of the treated newborns, where GSH as a powerful reducing agent can interrupt the radical chain production of lipid peroxidation. Leedle and Aust (1990) emphasized that peroxidation of membrane phospholipids is related to an inadequate GSH concentration. TBARS of polyunsaturated lipids has been implicated in a variety of diseased states (Ghosh et al., 1994). The delicate balance between the production and catabolism of oxidants is critical for maintenance of the biological functions (Sridevi et al., 1998; Zhu et al., 2008). Glutathione is one of the essential compounds for the maintenance of the cell integrity because of its reducing properties and participation in cell metabolism (Conklin, 2000). Alterations in the ratio of GSH and oxidized glutathione have been used as indicators of oxidative stress and/or diseases in humans and laboratory animals (Gohil et al., 1988). In states of oxidative stress, GSH is converted into oxidized (GSSG) form and is depleted, leading to lipid peroxidation. Therefore, the role of GSH as a reasonable marker for the evaluation of oxidative stress is important (Recknagel et al., 1991). To prevent lipid peroxidation, it is very important to maintain the level of GSH. GSSG is reduced to GSH by glutathione reductase (GR), which is NADPH dependent. Accordingly, the reduction of GR level due to the interaction of free radicals and lipid peroxides formed by acrylamide with the sulfhydryl (SH) group present at the active site of the enzyme, which in turn prevents the enzyme from participating in the reaction, results in decreasing GSH (Gerson and Shaikh, 1984; Recknagel et al., 1991; Yousef and El-Demerdash, 2006). Furthermore, glutathione-S-transferase (GST) catalyzes the conjugation of the thiol functional groups of glutathione to electrophylic xenobiotics and results in increase solubility. The xenobiotic-GSH conjugate is then either eliminated or converted to mercapturic acid (Rao et al., 2006). Acrylamide reacts with glutathione in interacting with vital cellular nucelophiles possessing –SH, –NH2 or –OH and forms glutathione S-conjugates, which is the initial step in the biotransformation of electrophiles into mercapturic acids (Awad et al., 1998). Decreasing GSH content in the tissues with the increase of acrylamide concentration could be due to increased formation of S-conjugates between acrylamide and GSH. Thiol groups are required for the activity of many biological important proteins. They are also important reducing agents and cellular antioxi-
antioxidants (Yousef and El-Demerdash, 2006). Meng et al. (2001) found that acrylamide markedly depleted the protein thiols as reported in the present study. In addition, Yousef and El-Demerdash (2006) reported a depletion of GSH content, peroxidase and SOD activity in acrylamide intoxicated rats. Ohta et al., (1995) have also reported that the toxicity reduced the activities of SOD. The increase in the activity of SOD could be to combat free radical generation during acrylamide toxicity (Sridevi et al., 1998). The antioxidant enzymes SOD and peroxidases constitute a mutually supportive team of defence against reactive oxygen species (Tabatabaie and Floyd, 1994; Bandhopadhay et al., 1999). The decrease in the activity of SOD and peroxidase in the newborns of acrylamide-treated groups may be due to the increased TBARS or inactivation of the enzymes by cross-linking with malondialdehyde. Van de Casteele et al. (2003) showed a markedly increased malondialdehyde level and decreased SOD activity. This will cause an increased accumulation of both superoxide and hydrogen peroxide radicals, which could further stimulate TBARS (Rister and Bachner, 1976; Rajesh and Latha, 2004). The peroxidase assayed in this study using pyrogallol as a substrate, may be myeloperoxidase and eosinophil peroxidase (heme peroxidase) rather than glutathione peroxidase, which is highly specific for glutathione and cannot oxidize any other substrates (Shigeoka et al., 1991). Conclusion In conclusion, acrylamide adversely affected the development of the cerebrum and medulla oblongata of the newborns of pregnant mothers that were exposed to acrylamide during gestation and lactation periods. ACKNOWLEDGEMENTS This project was supported by King Saud University, Deanship of Scientific Research, College of Science Research Center, King Saud University, Saudi Arabia. REFERENCES Abdul-Hamid M, Allam AA, Hussein MB (2007). Effect of ethanol administration during gestation on the cerebral cortex and spinal cord of albino rat newborns and on the development of their sensorimotor reflexes. Egypt. J. Zool. 48: 137-162. Allam AA, El-Ghareeb AW, Abdul-Hamid M, Bakery AE, Gad M, Sabri M (2010). Effect of prenatal and perinatal acrylamide on the biochemical and morphological changes in liver of developing albino rat. Arch. Toxicol. 84(2):129-41. Awad ME, Abdel-Rahman MS, Hassan SA (1998). Acrylamide toxicity in isolated rat hepatocytes. Toxicology, 12: 699-704. Bandhopadhay U, Das D, Banerjee KR (1999). Reactive oxygen species: Oxidative damage and pathogenesis. Curr. Sci. 77: 658-665.
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African Journal of Biotechnology Vol. 11(29), pp. 7579-7581, 10 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.1500 ISSN 1684–5315 © 2012 Academic Journals
Short Communication
Effects of additional DL-methionine in broiler starter diet on blood lipids and abdominal fat Mohammad Amiri Andi Department of Animal Sciences, Faculty of Agriculture, Sanandaj Branch, Islamic Azad University, Sanandaj, Iran. Email: m-amiriandi@iausdj.ac.ir. Accepted 24 October, 2011
In a completely randomized design, the study was undertaken to investigate the effects of additional DLmethionine (Met) in starter diet of broilers on blood lipids (21 days of age) and abdominal fat (42 days of age). 288 days-old Ross 308 hybrid chicks were divided into 16 groups for the starting period. Dietary treatments were divided into four levels of sulfur amino acids (SAA), [0.7(control), 0.8 (NRC), 0.9 and 1.0% diet] with graded levels of Met (0, 0.1, 0.2 and 0.3% diet). At 21 days of age, chicks receiving 0.2% Met had lower (p≤0.05) serum total cholesterol (TC) than the control (98.93 vs. 113.75 mg/dl, respectively). Serum high density lipoprotein (HDL) in chicks that received 0.3% Met was significantly (p≤0.05) higher than that of the control (80.60 versus 63.45 mg/dl, respectively). In chicks fed 0.2 and 0.3% Met, serum low density lipoprotein (LDL) were lower (p≤0.05) than for chicks that received 0 and 0.1% Met containing diet (17.25 and 13.33 versus. 24.15 and 25.00 mg/dl, respectively). Serum triglyceride (TG) and very low density lipoprotein (VLDL) in the control group was significantly (p≤0.05) greater than that of the other groups (136.60 versus 72.45, 66.53 and 54.27 mg/dl for TG; 26.15 vs. 14.57, 13.33 and 10.60 mg/dl for VLDL, respectively). Consequently, In 42 days of age, 0.2% Met in the starter diet had significantly (p≤0.05) reduced abdominal fat than the control (1.58 versus 2.5%, respectively). Based on the results, it seems that additional Met in broiler starter diet can increase good cholesterol (HDL) in blood level and reduce abdominal fat.
Key words: Methionine, broilers, blood lipids, abdominal fat and starter period.
INTRODUCTION Severe selection for higher live weight gain in broiler parent/breeder production industries resulted in heavy broilers with greater body and abdominal fat. This subject not only reduces carcass quality of broilers, but also influences human health. On the other hand, producers desire to sell leaner carcasses with attention to the market demands for meat instead of live weight presently. Then, methods for reducing body or abdominal fat of new broilers had many advantages. DL-Methionine (Met) is an
Abbreviations: Met, DL-Methionine; SAA, sulfur amino acids; TC, total cholesterol; HDL, high density lipoprotein; LDL, low density lipoprotein; TG, triglyceride; VLDL, very low density lipoprotein; A. fat, abdominal fat; ME, metabolizable energy; LW, live weight; LWG, live weight gain; FI, feed intake; FCR, feed conversion ratio; HSL, hormone-sensitive lipase; CP, crud protein.
essential amino acid for poultry (NRC, 1994) and it plays an important function in lipid metabolism as a methyl group donor and act as a lipotropic agent (Pesti et al., 1979). Met supplementation in growing chick is a common practice (Neto et al., 2000; Swick et al., 1990). In many investigations, supplemental Met increased blood HDL or good cholesterol and decreased TG and abdominal fat (Kalbande et al., 2009; Mersmann, 1998; Zhan et al., 2006). Therefore, the purpose of this study was to investigate the effects of additional Met in broiler starter diet on blood lipoproteins, TG and abdominal fat. MATERIALS AND METHODS 288 Ross unsexed day old 308 strain chicks were purchased from a commercial hatchery and were randomly divided into four groups with four replicates (1.5 × 1.0 m, floor pen) in each treatment on equal body weight basis having 18 chicks. Chicks were fed corn
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Table 1. Composition of the basal diet (0 to 21 days of age).
Ingredient Corn Soybean meal Sunflower oil DCP Oyster Salt Vitamin/mineral premix ≠
% Diet 59.18 35.97 1.25 1.42 1.26 0.42 0.5
Calculated composition ≠≠ ME (kcal/kg diet) CP CF Ca P (available) Na Lys Met+Cys
2900 20.84 2.7 0.9 0.41 0.18 1.12 0.7
Analyzed composition CP Ca P (total) Met (HPLC method)
20.52 0.92 0.52 0.38
≠ Provided per kg of diet: vitamin A, 8000 IU; cholecalciferol, 2000 ICU; vitamin E, 30 mg; manadione, 2 mg; riboflavin, 5.5 mg; pantothenic acid, 13 mg; niacin, 36 mg; choline, 500 mg; vitamin B12, 0.02 mg; folic acid, 0.5 mg; thiamin, 1 mg; pyridoxine, 2.2 mg; biotin, 0.05 mg; ethoxiquin, 125 mg; Mn, 65 mg; Fe, 55 mg; Cu, 6 mg; Zn; 55 mg; ≠≠ Based on NRC, 1994.
soybean meal based diet (with 1.25% sunflower oil) as per standards of nutritional requirement recommended by NRC (1994). Ingredients and chemical composition of the basal diet are presented in Table 1. Finisher diet was similar for all groups, with 3000 kcal/kg metabolizable energy (ME) and 17.5% crude protein (CP). All the groups were subjected to similar management and nutritional regimens except the levels of SAA given to them. Graded levels of Met were 0, 0.1, 0.2 and 0.3% of diet that create SAA levels of 0.7, 0.8, 0.9 and 1.0% of starter diet. At day 21, two birds from each replicate were randomly selected for blood samples collection to estimate the serum TG and lipoproteins. After blood collection, samples were placed in a room temperature and then serum was separated and centrifuged at 3000 rpm for 15 min in a centrifuge machine. Serum samples were frozen and stored at 20°C, and analyzed subsequently. Serum TC, TG and HDL were analyzed by using appropriate laboratory kits (Friedewald et al., 1972; Gordon et al., 1977; Gowenlock et al., 1988). VLDL was calculated from TG by dividing the factor by 5. The LDL was calculated by using the equation: (LDL= TC-HDL-VLDL). At 42 days of age, eight birds per treatment were sacrificed and animal fat was separated and weighed.
Statistical analysis All data were analyzed using the one way ANOVA procedure of SAS (SAS Institute Inc, 1996) for analysis of variance. Significant differences among the treatments were identified at 5% level by Duncan's multiple range tests.
RESULTS AND DISCUSSION The effects of experimental treatments on blood serum lipids and broiler performance are shown in Table 2. At 21 days of age, chicks that received 0.2% Met had lower (p≤0.05) serum total cholesterol than the control (98.93 versus 113.75 mg/dl, respectively). Serum HDL in chicks that received 0.3% Met was significantly (p≤0.05) higher than that of the control (80.60 versus 63.45 mg/dl, respectively). In chicks fed 0.2 and 0.3% Met, serum LDL were lower (p≤0.05) than 0 and 0.1% Met in their diet (17.25 and 13.33 versus 24.15 and 25.00 mg/dl,
respectively). Serum TG and VLDL in the control chicks was significantly (p≤0.05) greater than those of the other groups (136.60 versus 72.45, 66.53 and 54.27 mg/dl for TG and 26.15 versus 14.57, 13.33 and 10.60 mg/dl for VLDL, respectively). In 42 days of age, 0.2% Met in the starter diet had significantly (p≤0.05) lower abdominal fat than the control (1.58 versus 2.5%, respectively). Experi-mental treatments had no effects on live weight gain (LWG), feed intake (FI) and feed conversion ratio (FCR). In this investigation, additional Met in broiler starter diet lowered serum TG level which is in agreement with the results of other reports (Kalbande et al., 2009; Fowler, 1996; Roth and Milstein, 1957; Zhan et al., 2006). Res-earchers demonstrated that Met results in higher levels of hormonesensitive lipase (HSL) in adipose tissue which decrease TG (Mersmann, 1998; Zhan et al., 2006). This is a reason for the reduction of abdominal fat in Met supplemented broilers. Based on the findings of this study, it seems that, higher concentration of SAA in diet of chickens resulted in the lowering of serum
TC. The findings of this investigation can be well correlated to those reported by Kalbande et al. (2009) and Roth and Milstein (1957) that dietary Met insufficiency may induce fatty liver thereby increasing total serum cholesterol. There are not sufficient researches about the effects of SAA in broiler diet on blood lipoproteins but in numerous studies, the relationships between low HDL levels and the risk of atherosclerotic disease have been shown. In this study, additional Met in broiler starter diet resulted in high level of HDL and low levels of VLDL and LDL. These findings are in agreement with Oda et al. (1991) in rat. They postulated that dietary Met increase the secretion rate of HDL cholesterol from the liver Conclusion Based on the results of this investigation, it seems that additional Met in broiler starter diet can increase good HDL in broiler blood and reduce abdominal fat. There is need for further investigations for the measurement of liver enzymes in this
subject.
Andi
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Table 2. Effects of additional methionine in broiler starter diet on serum lipids (21 days of old), performance and abdominal fat (42 days of age) 1.
Trait TG (mg/d)l TC (mg/dl) LDL (mg/dl) VLDL (mg/dl) HDL (mg/dl) A.fat (%carcass) LW (g) LWG (g/d FI (g/d) FCR (g/g)
0.0 a 136.60 ±20.96 a 113.75 ±3.95 a 24.15 ±2.00 a 26.15 ±4.85 b 63.45 ±2.55 a 2.50 ±0.21 2132.0±84.6 48.29±1.73 95.73±2.39 1.99±0.05
Additional methionine (%diet) 0.1 0.2 b b 72.45 ±11.26 66.53 ±11.81 ab b 108.14 ±4.28 98.93 ±2.50 a b 25.00 ±1.52 17.25 ±0.85 b b 14.57 ±2.36 13.33 ±2.37 ab ab 68.57 ±2.84 67.35 ±3.55 ab b 2.10 ±0.23 1.58 ±0.31 2151.2±57.5 2228.5±16.7 48.76±1.07 51.01±0.62 96.50±2.49 96.44±1.18 1.98±0.04 1.92±0.04
0.3 b 54.27 ±4.77 ab 104.53 ±3.3 b 13.33 ±1.20 b 10.60 ±0.86 a 80.60 ±7.48 ab 1.90 ±0.31 2185.0±41.1 49.77±0.72 95.73±2.02 1.89±0.06
1
Data expressed as means ± SE. Means in each row with different superscript are significantly different (p≤0.05).
a-b
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