Advance in Forestry Research November 2013, Volume 1, Issue 4, PP.45-50
Protein Analysis of Successive Continuous Generation Cuttings of Buxus Sinica Var.Parvifolia Kongshu Ji #, Mingjuan Wang, Yan Huang, Xiaofeng Wang, Ting Pan 1. Ministry of Education, Nanjing Forestry University, Nanjing, 210037, P.R. China #Email: ksji@njfu.edu.cn
Abstract Buxus sinica var. parvifolia is a rare endangered and endemic tree species in China, as well as a kind of precious material for bonsai. The experimental materials were leaves of B. sinica var. Parvifolia collected from seedling planted in 2003 and clonal stecklings of three successive generations raised in 1994, 1998 and 2000 respectively. The proteins of the materials were extracted with the 2-D Clean-Up Kit and analyzed with two dimensional gel electrophoresis. The gels were then analyzed by Image Master TM 2D Platinum 6.0 software. Four hundred and fifty-five reproducible protein spots were detected, among which forty-nine protein spots displayed differential expression. There were twenty-five, nineteen and twelve different protein spots between seedling in 2003 and steckling of generation 1994, steckling of generation1994 and 1998, and steckling of generation 1998 and 2000 respectively. Among all of them, eight proteins were up-regulated and six proteins were down-regulated. These proteins may be related to rejuvenation of Buxus sinica var. parvifolia. Keywords: B. Sinica Var. Parvifolia; Rejuvenation; Two Dimensional Gel Electrophoresis; Differential Protein
1 INTRODUCTION Buxus. sinica var. parvifolia is a rare and endangered tree species distributing in some subtropical and alpine areas of China and plays an important role in the maintenance of the landscape and ecosystem in its cliff habitats at elevations between 1200 m and 3000 m above sea level. It is an excellent material to manufacture bonsai. The successive generation cutting method is used to instruct and domesticate Buxus. sinica var. parvifolia until it completely retains its original morphological characteristics and gradually adapts to current cultivation environment (Huang, 2008). “Rejuvenation� is usually used to describe the phenomenon that tree organs or cell formations recover their juvenile characteristics, such as strong rooting ability, flourish leaves, stronger cold resistance and stronger drought resistance. Asexual rejuvenation of the tree occurs when its somatic cells receive specific exogenous stimulation or gene activation. mRNA or proteins might act as the provider of rejuvenation signals(Chen, 1996). The recent researches showed more ways about how to rejuvenate but less on its mechanism. The mechanism of rejuvenation was analyzed through studying the proteins of successive generation cuttings of Buxus sinica var. parvifolia with two-dimensional gel electrophoresis (2-DE), which also provides help for the proteome research of B. sinica var. parvifolia.
2 MATERIALS AND METHODS 2.1 Materials The leaves of B. sinica var. parvifoliawere were collected from seedling and successive generations of stecklings in Haoyu Landscaping Company, Rugao, Jiangsu Province, and China. The seedling were planted in 2003 and referred to as seedling in 2003 or seedling. The successive generations of stecklings of Buxus sinica var. parvifolia were obtained through the following method: in 1990, the first generation twigs were taken as cuttings from one original - 45 www.ivypub.org/AFR
ortet at a piedmont farm local at the Tianzhu Mountain, Anhui Province, China. Then the twigs were planted. After 10 cuttings survived, they were transplanted to the Haoyu nursery and referred to as the steckling of generation 1990 or generation 1990. In 1992, twigs were taken from generation 1990 as cuttings to make the steckling of generation 1992. By this way, stecklings of generation1994, 1998 and 2000 were obtained. From 10th to 12th June, 2008, leaves of three plants of seedling and stecklings of generation 1994, 1998 and 2000 within the same growing level were taken as the samples in four directions which were east, south, west and north, then quickly put into the ice pack and stored in -70oC in the laboratory later. For proteins extracting, 0.2 g leaves were taken from each sample.
2.2 Protein Sample Preparation Proteins were extracted with the 2-D Clean-Up Kit (Amersham Biosciences, Buckinghamshire, UK).
2.3 Proteomic Quantification Bradford method (Bradford, 1976) was improved to assess protein concentration. Firstly, a series of standard protein solutions was prepared with concentrations of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0 and 6.0 mg/mL. And 3.5 ml of coomassie brilliant blue working solution was added into them. Then, optical density (595 nm) of the standard protein solutions as well as samples was determined and the standard curves were drawn to figure out the concentration of the samples.
2.4 Two Dimensional Gel Electrophoresis 300μg of the sample proteins were rehydrated with a buffer containing 8 M urea, 2% CHAPS, 0.002% bromophenol blue, 20 mM dithiothreitol and 0.5% IPG buffer(Amersham Biosciences), on nonlinear pH 3-7 dry strips (Amersham Biosciences, Buckinghamshire, UK) for 12 h at 20oC. Isoelectric focusing was performed on Ettan IPGphor III according to the manufacturer’s instructions (Amersham Biosciences, Buckinghamshire, UK). The focusing program for 24-cm strips was as follows: 30 V for 6 h, 60 V for 6 h, 200 V for 1 h, 500 V for 1 h, 1000 V for 1 h, 4000 V for 1 h, 8000 V for 1 h and 64000 V for 1 h with an imax of 50 mA per strip. For the second dimension, SDS-PAGE, the strips were incubated in equilibration bufferⅠ (50 mM Tris-HCl, 6 M urea, 30% glyserol, 2% SDS, 0.002% bromophenol blue, 1% dithiothreitol) for 15 min at room temperature. Secondly, the strips were incubated in equilibration bufferⅡ(50m M Tris-HCl, 6M urea, 30% glyserol, 2% SDS, 0.002% bromophenol blue, 4% iodoacetamide) for 15 min at room temperature. Then the strips were loaded on to a 12% SDS-PAGE gel, and sealed with 0.5% agarose in SDS-PAGE running buffer. The strips were run at 2 W per gel for 0.5 h and thereafter at 17 W per gel in an Ettan Dalt Twelve. After this, the gels were silver-stained with lowered concentration acetic acid and formaldehyde instead of glutaraldehyde(Blum et al.,1987).
2.5 Image scan Gels were scanned by LabScan (Amersham Biosciences, Buckinghamshire, UK).
3 RESULTS There were increases in growth, drought resistance, cold resistance, rooting rate and rooting characteristics in the steklings with the raising of generation. The rooting ability of cuttings gained remarkable enhancement (p<0.01) and survival rate increased greatly from 18% to 95% under good growing conditions. If generation 1990 and generation 1994 were marked as initial generation while generation 1998 and generation 2000 were signed as successor generation, we found a very significant difference with the characteristics between the initial generation and successor generation, but not within the initial generation or successor generation. 300 µg of sample proteins extracted from leaves of seedling and stecklings of generation 1994, 1998 and 2000 were taken for 2-DE. Fig. 1 showed the 2-DE pattern of proteins from B. sinica var. parvifolia. In previous experiment of extracting proteins of leaves of B. sinica var. parvifolia, it was found that proteins of leaves mainly distributed in the pH range from 4 to 7. Thus, as for this experiment, the focusing program was on pH 3-7 NL strips, and then the - 46 www.ivypub.org/AFR
strips were loaded on a 12% SDS-PAGE gel. That was good for observing the dynamic changes in expressing graph of proteins from different material and comparing protein graphs of two different matirals in close time period. The protein graphs of seedling and generation 1994, generation 1994 and generation 1998 and generation 1998 and generation 2000 were compared in this paper.
FIG. 1 COMPARISON OF PROTEIN PATTERNS OF LEAVES OF B. SINICA VAR. PARVIFOLIA. A, B, C, D AND E SHOW THE PROTEIN PATTERNS OF CONTROL, SEEDLING, GENERATION 1994, GENERATION 1998 AND GENERATION 2000. THE NUMBERS CORRESPOND TO TABLE AND ARROWS REPRESENT DIFFERENTIALLY EXPRESSED PROTEIN SPOTS COMPARED TO THE PREVIOUS TIME POINTS. - 47 www.ivypub.org/AFR
FIG. 2 THE 3-D INTENSITY CHART OF NO.46 PROTEIN SPOT
FIG. 3 THE 3-D INTENSITY CHART OF NO.11 PROTEIN SPOT TABLE 3 PROTEIN SPOTS SHOWING SIMILAR CHANGES IN THE LEAVES OF B. SINICA VAR. PARVIFOLIA
Protein number Isoelectric point Molecular weight /KDa Generation 1994 Generation 1998 Generation 2000 5 5.84 21.1 1.1 0.3 0.8 6 3.91 30.3 0.6 1.0 0.4 11 6.03 76.2 -0.3 -1.2 -1.1 13 5.97 47.5 1.5 0.7 0.2 16 4.15 24.1 0.5 0.3 0.6 17 4.48 23.2 -2.1 -1.4 -2.0 18 4.42 24.5 0.2 1.1 0.9 21 5.59 21.5 -1.1 -4.9 -1.0 22 5.56 17.6 0.7 1.0 1.6 37 4.58 23.5 1.2 1.1 1.3 40 3.78 33.5 -1.4 -2.2 -1.4 43 5.03 22 -0.2 -2.5 -3 46 4.37 2.2 1.3 1.3 1.1 49 4.28 21.7 -0.5 -1.2 -0.6
Reference:“+”represent multiple of up-regulated,“-” represent multiple of down-regulated.
4 DISCUSSION 4.1 2-D Electrophoresis In the process of experiment, three methods of protein extraction were tried including TCA-acetone, Tris-saturated phenol and 2-D Clean-Up Kit. The results of repeated experiments showed that the better protein patterns were gained with the 2-D Clean-Up Kit method. But even when the time taken for isoelectric focusing was 2-3 hours more than theoretic time occasionally, the electrophoresis voltage of isoelectric focusing could not be increased frequently, for which might seriously affect the 2-DE results. As the leaves of B. sinica var. parvifolia contains so much salt ions and other impurities, 2-D Clean-Up Kit was used to reduce them in the preparation process of protein sample , sometimes even with second treatment for better result. Repeatability is one of the key factors in 2-DE, and the improved Bradford method has good reproducibility as well. - 48 www.ivypub.org/AFR
However, many reagents in sample preparation and solution such as reducing agents, detergents, charotropes and ampholyte could bind the coomassie brilliant blue, which made the standard curve slightly up. In other words, protein content was lower than the estimated. That was the reason why loaded samples in IEF were higher than the theoretical value. SDS-PAGE gel scanning showed 300 DPI (dot per inch)was suitable. And for bigger or smaller gel, the DPI should be adjusted on the basis of actual situation. But a reduced image would lower the image quality and decrease differentiation. In addition, an enlarged image would produce interpolated values even ending up in pixellation, which affected the determination. So one-to-one 300DPI was the optimum ratio for scan, which was applied to this experiment.
4.2 Rejuvenation Molecule Mechanism Age effect often influence asexual propagation, but with the times of successive asexual propagation increasing, age effect decreased in different degree and even disappeared. And this brings about differences within clones, however which is not sufficient to show advantages of asexual propagation. Therefore, the task on how to reduce or overcome the age effect has become hotspot. So far, some papers about rejuvenation and maturing have been published. However, they focused more on how to rejuvenate but paid less attention on its mechanism. Once people made a thorough study of rejuvenation mechanism, they can rejuvenate plants by various measures (Du, 1995). “Rejuvenation” is usually used to describe the phenomenon that tree organs or cell formations recover their juvenile characteristics (Chen, 1996). Asexual rejuvenation of tree occurs when its somatic cells receive specific exogenous stimulation or genetic activation and then recover juvenile characteristics. mRNA or proteins might act as the provider of rejuvenation signals. It has been confirmed by many studies that successive generation cuttings could lead to an effective rejuvenation of ortets (Jarvis, 1986). But so far, few papers published are about the rejuvenation of woody species caused by successive generation at the protein level. Some reports concerned with tree species rejuvenated by successive generation cuttings (Zhou and Deng, 1994; Ji et al., 1997, 1999, Huang,) focused on morphology and physiology level, which lacked of in-depth study on the relevant mechanisms. Rejuvenation is the reverse development of aging, which certainly will cause changes of some proteins (Buchanan et al., 1997; Nooden et al., 1997). In this paper, we took 300 µg proteins sample of B. sinica var. parvifolia to do 2-DE, and got 49 protein spots with differentially expressed. 14 protein spots of them were conformably expressed , among which proteins with the relative molecular mass of 2.2, 17.6, 21.2, 23.2, 24.1, 24.5, 30.3 and 47.5KD were upregulated and 21.5, 21.7, 22、23.5, 33.5 and 76.2KD were down-regulated. These proteins might be related to rejuvenation of Buxus sinica var. parvifolia by successive generation cutting. Identifying these proteins by Mass Spectrum and determining which kind of protein they belong to need further research, which might reveal the mechanism of rejuvenation thoroughly. Monteuuis(1991) found 1GKD J16 protein existing in rejuvenile wellingtonia; Huang et al.(1992) found that 36, 44, 46KD proteins were up-regulated in the rejuvenile plant, while 29KD protein was down-regulated and 34, 37KD proteins were up-regulated in mature tissues. The purpose of this study was to investigate the mechanism of Buxus sinica var. parvifolia rejuvenation at protein level and discover the proteins related with rejuvenation which may be helpful to find related genes and study the mechanism of rejuvenation of other trees. Furthermore, related proteins are not only helpful to test rejuvenation but also beneficial to research on plant transition from juvenile to mature. Certainly, it would accelerate asexual propagation of elite germplasm of B. sinica var. Parvifolia, which will help provide enough materials for bonsai.
ACKNOWLEDGMENTS Sincere appreciation to Mr Zhai Jinru who managed to bring in seedling and stecklings of Buxus sinica var. parvifolia for the domestication.
- 49 www.ivypub.org/AFR
REFERENCES [1]
Blum H, Beier H, and Gross H. “Improved silver staining of plant proteins, RNA, and DNA in Polyacrylamide gels.” Electrophoresis. 8(1987): 93-99
[2]
Bradford M.M. “A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.” Analytical Biochemistry. 72(1976):248-254
[3]
Buchanan WV, Aninsworth C. “Leaf senescence in Brassica napus: cloning of senescence- related genes by subtractive hybridization.” Plant Molecular Biology. 32(1997): 231-240
[4]
Chen Y. “A brief account of the research on forest tree rejuvenation.” World Forestry Research. 3(1996): 13-17
[5]
Du S. “Biological fundamentals of maturation and rejuvenation and tissue culture.” Journal of Inner Mongolia Forestry College. 17(1995): 15-21
[6]
Guo X. “Development of protein two dimensional electrophoresis technology.” Chinese Journal of Health Laboratory Technology. 16(2006): 638-640
[7]
Greenwood MS. “Rejuvenation of forest trees.” Plant growth regulation. 6(1998):1-12
[8]
Huang IC, Liu S, and Huang BI. “Rejuvenation of sequoia sempervirens by repeated grafting of shoot tips onto juvenile rootstocks in vitro.” Plant Physiology. 96(1992):166-173
[9]
Huang Y, Ji KS, and Zhai JR. “Relatoonship between rooting ability and endogenous phytohormone changes in successive continuous generation cuttings of Buxus sinica var. parvifolia, an endangered woody species in China.” Forestry Studies in China. 9(2007):189-197
[10] Huang Y. Genetic diversity and protection and utilization of Buxus sinica var. Parvifolia. Doctoral Dissertation of Nanjing Forestry University, 2008 [11] Jarvis BC. “Endogenous control of adventitious rooting in non-woody cuttings, In: Jackson M B (ed), New Root Formation in Plants and Cuttings.” Dordrecht: Martinus Nijhoff. 191-223,1986 [12] Ji KS, Wang ZR et al. “Study on the endogenous inhibitors in Masson pine (Pinus massoniana Lamb.) cutting.” Science Silvae Sinica. 33(1997):142-151 [13] Ji KS, Wang ZR, et al. “Cyclophysis and effect of rejuvenation with continued cottage in Pinus massoniana cutting propagation.” Journal of Zhejiang Foresty College. 16(1999):341-345 [14] Kefeli VI. Natural plant growth inhibitors and phytohormones. The Hague: Dr W Junk Publisher, 1978 [15] Monteuuis O. “Rejuvenation of a 100-year-old Sequoiadendron gigameum through in vitro meristerm culture, I. orgaogenic and morphological arguments.” Physiological Plantarum. 81(1991): 111-115 [16] Nooden LD, Guiamet JJ, and John I. “Senescence mechanisms.” Plant Physiology. 101(1997):746- 753 [17] Wiesman Z, Riov J. “Interaction of paclobutrazol and indole-3-buryric acid in relation to rooting of Mung bean (Vigna radiata) cuttings.” Plant Physiology. 92(1994): 608-612 [18] Zhou S, Deng DF. “The test of clones cutting propagation with Mexico cypress.” Hunan Forestry Science & Technology. 21(1994): 39-42
AUTHORS 1Kong-shu
A. Ji was born in 1965 in
Forestry University, Nanjing, China. In 1990 and 1996
Ninghai, Zhejiang, China. Kong-shu Ji
respectively. Now his study is mainly on genetic breeding of
obtained his B.S. of Non-wood Forest in
trees.
1987 from Zhejiang Forestry College, Zhejiagn, China and then got his M.S. of Plant Physiology and Ph.D. of Forest Genetics & Tree Breeding from Nanjing
2Ming-juan
B.Wang is a Master of Landscape Plants who was
born in 1981. 3Yan
C. Huang is a Doctor of Garden Plants born in 1980.
- 50 www.ivypub.org/AFR