Journal of Research in Ecology
Journal of Research in Ecology
ISSN No: Print: 2319 –1546; Online: 2319– 1554
An International Scientific Research Journal
Original Research
The effect of mycorrhizal inoculation (Glomus mosseae) and brassinosteroid on the amount of antioxidants in Pimpinella anisum l. under cadmium stress Authors: Sepideh Hajbagheri1, Hosein Abbaspour2, Shekoofeh Enteshari3, Saeed Habibollahi4 and Alireza Iranbakhsh5
ABSTRACT: Heavy metals are important environmental pollutants and their toxicity is considered as a major problem, because of ecological, evolutionary, environmental and nutritional effects. Many studies have shown that plants inoculated with mycorrhizal fungi or the use of brassinosteroids increased plants resistance to heavy metals. In this study, the effect of mycorrhiza inoculation with Glomus mosseae and 24-epibrassinolid (10-6 µM) on Pimpinella anisum antioxidants against cadmium Institution: chloride stress (0, 100, 200 and 800 ppm) were investigated and compared in green 1. PhD Student, Azad house and hydroponic condition. The results showed that cadmium stress reduced University, Iran shoot protein content, but amount of H2O2, flavonoid, flavonol and phenolic 2. Biology Department, compound increased. The activity of catalase, superoxide dismutase, peroxidase, Azad University, Iran polyphenol oxidase increased significantly in cadmium treatments. When plants were pretreated with brassinosteroid and inoculated with mycorrhizal fungi and then 3. Biology Department, Payam Noor University, Iran treated with cadmium H2O2 have reduced some antioxidative parameters such as flavonoid and anthocyanin content, and activity of ascorbate peroxidase, peroxidase 4. Chemistry Department, and polyphenol oxidase increased significantly. Therefore, it can be concluded that in Payam Noor University, this plant brassinosteroid application or mycorrhizal inoculation have positive effects Iran. on strengthening the antioxidant system in this plant against cadmium stress. 5. Professor of Biology Keywords: Department, Azad Glomus mosseae, brassinosteroid, cadmium chloride, Pimpinella anisum. University, Iran Corresponding author: Shekoofeh Enteshari
Email ID: Article Citation: Sepideh Hajbagheri, Hosein Abbaspour, Shekoofeh Enteshari, Saeed Habibollahi and Alireza Iranbakhsh The effect of mycorrhizal inoculation (Glomus mosseae) and brassinosteroid on the amount of antioxidants in Pimpinella anisum l. under Cadmium stress Journal of Research in Ecology (2017) 5(2): 1033-1051 Dates: Received: 23 July 2017 Web Address: http://ecologyresearch.info/ documents/EC0483.pdf
Journal of Research in Ecology An International Scientific Research Journal
Accepted: 26 Aug 2017
Published: 01 Sep 2017
This article is governed by the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited.
1033-1051| JRE | 2017 | Vol 5 | No 2
www.ecologyresearch.info
Hajbagheri et al., 2017 INTRODUCTION
ed metals through secreted compounds such as catego-
Plants in nature are constantly exposed to the per-
rizing the metals inside the fungal cells. The beneficial
sistent presence of heavy metal toxic ions and experi-
effects of mycorrhizal on growth in terms of heavy met-
ence the damage of their cumulative toxicity. Toxic
al toxicity have been reported in many plant species
effects of heavy metals are mainly related to these four
(Leung et al., 2013; Sharma and Sharma, 2013; Upadh-
factors: disabling the active groups of enzymes, replac-
yaya et al., 2010). Mycorrhizal fungi regulated the bal-
ing instead of essential metals, inducing structural
ance of nutrients absorption that has been disturbed by
changes to polymers and affecting the strength and
heavy metals and improves the toxicity of metals in
structure of the membrane and the membrane transport
soils contaminated with heavy metals (Singh et al.,
processes (Tangahu et al., 2011). Cadmium with the
2012). It has been reported that G. mosseae is resistant
symbol Cd and atomic number 48 is one of the most
to cadmium and plays a role in absorption, transport and
toxic heavy metals. Weathering of rocks, forest fires and
inactivity of cadmium (Upadhyaya et al., 2010).
volcanoes, human activities such as industrial waste
Brassinosteroids are from steroid hormones and
leachates, the production of artificial phosphate fertiliz-
a new group of plant growth regulators that are widely
ers are the important sources of cadmium in the environ-
distributed throughout the plant kingdom. Due to the
ment. Cadmium accumulation in plant tissues reduced
multiple effects of the hormone, these compounds are
the cell division, root growth, root hydraulic conductivi-
known as plant hormones. This hormone stimulate the
ty and causes growth inhibition, chlorosis and cell death
growth and protein synthesis, increase the content of
(Liu et al., 2011).
compatible solutes, changes in transcription and expres-
Today, a lot of research focused on the use of
sion of genes involved in stress, and the concentration
growth regulators or mycorrhizal fungi in reducing the
and activity of antioxidant enzymes and compounds that
effects of environmental stresses. In endo-mycorrhizal
all reduce the harmful effects of stress in plants in order
coexistences, the mycorrhizal fungi play an important
to maintain, and enhance the performance of the plant
role in host plants protection against the toxicity of
(Ahammed et al., 2012; Zhang et al., 2008). The studies
heavy metals. Mycorrhizal fungi change the heavy met-
have shown that the both endogenous and exogenous
al concentration through metal inactivity and the chelat-
brassinosteroids have an antioxidant role, and reduce the Glomus mosseae G.m* Br
Figure 1. The effect of cadmium chloride and brassinosteroid on Pimpinella anisum root mycorrhizal colonization percentage by Glomus mosseae (Dis similar letters indicate significant statistical comparison of treatments based on Duncan test (p≤0.05)) 1034
Journal of Research in Ecology (2017) 5(2): 1033–1051
Hajbagheri et al., 2017 Control Brassinosteroid Glomus mosseae G.m.* Br
Figure 2. The effect of G. mosseae, brassinosteroid and cadmium on the protein content of Pimpinella anisum shoot (a) and root (b) (Dis similar letters indicate significant statistical comparison of treatments based on Duncan test (p≤0.05)) stress damage (Chandler et al., 2009; Zhang et al.,
mutase, catalase, ascorbate peroxidase, peroxidase, pol-
2008). Some researchers showed that brassinosteroids
yphenol oxidase and the non-enzymatic antioxidants
have a significant effect on plant resistance against the
such as anthocyanins and phenolics. Superoxide dis-
accumulation of heavy metals in beans (Ali et al., 2008)
mutase dismutation catalyzes two superoxide molecules
and pea (Hasan et al., 2008).
to H2O2 and H2O2 should detoxificate the antioxidant
Oxidative stress caused by the accumulation of
defense in the next steps. Catalase enzyme breaks down
heavy metals in plants leads to the production of reac-
H2O2 to water and oxygen and is considered as the main
tive oxygen species and oxidative damage in the cells.
enzymes that sweep the Hydrogen Peroxide in peroxi-
One of the most important plant defense systems to con-
somes. (Shi and Zhu, 2008). Ascorbate peroxidase en-
trol and neutralize the free radicals is to induce the syn-
zymes revive hydrogen peroxide in different cell parts
thesis of antioxidant enzymes such as superoxide dis-
in water with the help of ascorbate as an electron donor. Control Brassinosteroid Glomus mosseae G.m.* Br
Figure 3. The effect of G. mosseae, brassinosteroid and cadmium on the hydrogen peroxide content of P. anisum (Dis similar letters indicate significant statistical comparison of treatments based on Duncan test (p≤0.05)) Journal of Research in Ecology (2017) 5(2): 1033–1051
1035
Hajbagheri et al., 2017 Control Brassinosteroid Glomus mosseae G.m.* Br
Figure 4. The effect of G. mosseae, brassinosteroid and cadmium on the catalase activity of P. anisum (Dis similar letters indicate significant statistical comparison of treatments based on Duncan test (p≤0.05)) Peroxidases cause the decomposition of hydrogen per-
pounds with free radicals and convert them into their
oxide by the oxidation of a substance. Polyphenol oxi-
stable form electrons to these reactive radicals (Bartwal
dase is mono-oxygenase enzyme with copper that leads
et al., 2013).
to hydroxylation of phenols to di-phenyls and the oxida-
In general, the adaptation or resistance to stress
tion of di-phenyls (Abdul-Jaleel et al., 2009). Phenolic
depends on the reduction of the amount of reactive oxy-
compounds such as flavonoids, flavonols and phenols
gen species by antioxidant system. Treatment of plants
are a large group of secondary plant products with only
with epi-brassinolide and inoculation with mycorrhizal
one phenolic group. Anthocyanins are a group of water-
fungi increases the activity of antioxidant enzymes and
soluble flavonoids that are made in the biosynthesis of
phenolic compounds compared to untreated plants
flavonoids in the cytoplasm. Anthocyanin's do not only
which increases the resistance of plants against various
destroy the free radicals but also prevent their produc-
stresses such as the heavy metals (Abdul-Jaleel et al.,
tion in the plant. Antioxidant properties are the phenolic
2009; Ahammed et al., 2012).
compounds resulting from the reaction of these com-
Anisum with the scientific name of Pimpinella aniControl Brassinosteroid Glomus mosseae G.m.* Br
Figure 5. The effect of G. mosseae, brassinosteroid and cadmium on the ascorbate peroxidase activity of P. anisum (Dis similar letters indicate significant statistical comparison of treatments based on Duncan test (p≤0.05)) 1036
Journal of Research in Ecology (2017) 5(2): 1033–1051
Hajbagheri et al., 2017 Control Brassinosteroid Glomus mosseae G.m.* Br
Figure 6. The effect of G. mosseae, brassinosteroid and cadmium on the superoxide dismutase activity of P. anisum (Dis similar letters indicate significant statistical comparison of treatments based on Duncan test (p≤0.05)) sum L. belongs to the Apiaceae family, Apiales order
cations in completely randomized factorial design. The
and known as medicinal plant that contains the ingredi-
applied treatments included: cadmium chloride at 0,
ents such as essential oils, resins, sugar compounds and
100, 200 and 800 ppm concentration, mycorrhizal fungi
oil. Anisum has the antibacterial property and an ex-
(G. mosseae) and brassinosteroids (10-6mM).
traordinary importance in the pharmaceutical industry (Omidbeygi, 2006). The purpose of this study is to compare the role of brassinosteroids and G. mosseae inoculation in increas-
The seeds of the Pimpinella anisum L. were prepared from Pakan Bazr Company in Isfahan (IRAN), were rinsed three times by 10% sodium hypochlorite and washed with distilled water some times.
ing the resistance of P. anisum through the increase of
For the inoculation of seeds with G. mosseae on
enzymatic and non-enzymatic antioxidants against the
Petri dishes, seeds were excluded on 5 g of biological
cadmium chloride stress.
soil containing mycorrhizal spores of G. mosseae prepared from Hamedan Organic Plant Clinic (IRAN). The
MATERIALS AND METHODS This experiment was performed with three repli-
biological soil contained 15 spores per gram and 930 propagules per cubic centimeter. For non-inoculated Control Brassinosteroid Glomus mosseae G.m.* Br
Figure 7. The interaction of G. mosseae, brassinosteroid and cadmium on the polyphenol oxidase activity of P. anisum (Dis similar letters indicate significant statistical comparison of treatments based on Duncan test (p≤0.05)) Journal of Research in Ecology (2017) 5(2): 1033-1051
1037
Hajbagheri et al., 2017 Control Brassinosteroid Glomus mosseae G.m.* Br
Figure 8. The effect of G. mosseae, brassinosteroid and cadmium on the peroxidase activity of P. anisum (Dis similar letters indicate significant statistical comparison of treatments based on Duncan test (p≤0.05)) case seeds were excluded on filter paper impregnated
was placed on this and covered with perlite and co-
with distilled water. Petri dishes was put in germinator
copeat mixture. For each treatment, three pots were used
with optical/dark conditions for 16:8 hours, at day/night
and so considered considered as three replicates. In each
in the 23±2°C and 16±2°C temperatures respectively for
pot, five seedlings were planted. Pots were put in green-
4 weeks and after wards transferred to pots (Hewitt,
house with the optical condition of 14:10 light/night
1966).
hours, and 17 ± 2°C and 24 ± 2°C night/day temperature Seedlings were transferred in plastic pots with
respectively. Light intensity was 11,000 lux and humidi-
16 cm diameter and 15 cm height. In each pots, the
ty was 60%. For watering pots, distilled water and Long
mixture of autoclaved perlite and coco peat was poured
Ashton nutrient solution were used (Hewitt, 1966).
with 1:3 ratio. For mycorrhizal treatments in each pot,
Brassinosteroids treatments were applied after
50 g of mycorrhizal spores soils were poured on the
150 days of germination when the fourth leaf of the
surface of perlite and cocopeat mixture and seedling
plants were grown and plants were sprayed with a soluControl Brassinosteroid Glomus mosseae G.m.* Br
Figure 9. The effect of G. mosseae, brassinosteroid and cadmium on the anthocyanin content of P. anisum (Dis similar letters indicate significant statistical comparison of treatments based on Duncan test (p≤0.05)) 1038
Journal of Research in Ecology (2017) 5(2): 1033–1051
Hajbagheri et al., 2017 Control Brassinosteroid Glomus mosseae G.m.* Br
Figure 10. The effect of G. mosseae, brassinosteroid and cadmium on the flavonoid content of P. anisum (Dis similar letters indicate significant statistical comparison of treatments based on Duncan test (p≤0.05)) tion of 10-6 µM 24-epibrassinolide (Sigma (USA),
lin-acetic acid-ethanol (FAA) in the 14: 1: 1 ratio. Then
MW=480.7), 3 times every 72 hours at the beginning of
the roots were painted with fuchsine acid color and the
the optical phase. Control plants were sprayed with dis-
percentage of inoculated root was measured by using
tilled water. Three days after the last brassinosteroid
anatomical microscope Olympus model.
treatment, the cadmium chloride (100, 200 and 800 ppm
Determination of protein
CdCl2½H2O, MW=228.34) treatment was applied three
Total soluble protein content was measured ac-
times within 72 hour and the control plants were irrigat-
cording to Bradford (1976) using Bovine Serum Albu-
ed with distilled water. Plants were harvested 48 hours
min (BSA) as a protein standard. In each test tube to
after the last Cadmium treatment and some parameters
extract leaf protein, 250 microliter phosphate buffer,
were studied (Hewitt, 1966).
250 microliter of 1 M NaOH solution and 5 ml of Brad-
Root mycorrhizal colonization percentage
ford reagent were added. Then final volume of each
For this measurement, Rajapakes and Miler
tube was made up to 6 ml. The contents of each tube
(1992) method was used. For root fixation using forma-
were mixed in vortex and after 2 minutes the absorption Control Brassinosteroid Glomus mosseae G.m.* Br
Figure 11. The effect of G. mosseae, brassinosteroid and cadmium on flavonol content of P. anisum (Dis similar letters indicate significant statistical comparison of treatments based on Duncan test (p≤0.05)) Journal of Research in Ecology (2017) 5(2): 1033-1051
1039
Hajbagheri et al., 2017 Control Brassinosteroid Glomus mosseae G.m.* Br
Figure 12. The effect of G. mosseae, brassinosteroid and cadmium on the Phenol content of P. anisum (Dis similar letters indicate significant statistical comparison of treatments based on Duncan test (p≤0.05)) was read at a wavelength of 595 nm with UV visible
Determination of Antioxidant enzyme activity
spectrophotometer 1601 model (Bradford, 1976).
Catalase
Determination of hydrogen peroxide
Catalase (CAT) activity assay was based on
0.5 g of leaf tissue in phosphate buffer 3 ml
reduceing the amount of hydrogen peroxide at 240 nm
pH=6.8 was homogenized. The resulting extract was
wavelength for one minute. The reaction mixture con-
centrifuged for 25 min at 6000 g. To determine the
taining 100 mM potassium phosphate buffer, 15 mM
amount of hydrogen peroxide 3 ml the resulting extract
hydrogen peroxide and 100 ml enzyme extract
was removed and the 1 ml chloride titanium 0/1% in
(Extraction enzyme extract: 0.25 grams of leaf tissue
sulfuric acid at 20% (v/v) was added and the solution
were worned by liquid nitrogen and poured into eppen-
was centrifuged for 15 min at 6000 g. The absorption of
dorf 1.5 ml. Then 50 mM potassium phosphate buffer
resulting solution was read by the UV-visible spectro-
(pH = 7.5) containing 11% Triton was added to every
photometer 1601 model at 410 nm and hydrogen perox-
eppendorf 1 ml. All Extraction steps were performed on
ide content was measured according to Jana and
ice and samples were placed in the refrigerator for one
Choudhuri (1982) method.
hour. The extracts were centrifuged for 15 min at 4 °C at 15000 g. The supernatant was used to measure enzyme activity) in a final volume of 3 ml. Enzyme activi-
Figure 13. Microscopic image from the control of Pimpinella anisum root (non-mycorrhizal) (10X)
1040
Journal of Research in Ecology (2017) 5(2): 1033–1051
Hajbagheri et al., 2017
Figure 14. Microscopic image from the mycorrhizal colonization of P. anisum root (10X) ty was determined using the extinction coefficient of
maximum color without enzyme extract were decreased
3/94 mM-1cm-1 calculated on the basis of a unit (mol
in absorbance decreased with enzyme addition. The
hydrogen peroxide consumed per minute) per mg pro-
absorbance was recorded by the UV-visible spectropho-
tein (Aebi, 1984).
tometer 1601 model at 560 nm (Gianopolitis and Ries,
Super oxide dismutase
1977).
Super oxide dismutase (SOD) was assayed by
Ascorbate peroxidase
measuring its ability to inhibit the photochemical reduc-
Ascorbate peroxidase (APX) activity was deter-
tion of Nitro Blue Tetrazolium (NBT). The reaction
mined by the decrease in absorbance of ascorbate at 290
mixture contained 50mM potassium phosphate buffer
nm. The reaction mixture contained 50 mM potassium
(pH=7.5), 13 mM L-methionine, 75 μM NBT, 3 μM
phosphate buffer (pH=7), 0.5 mM ascorbate, 1 mM
riboflavin, 0.1 mM EDTA and 100 μM of enzyme ex-
H2O2, 0.1 mM EDTA and enzyme extract. APX activi-
tract. Riboflavin was added at the mixture. The tubes
ty was calculated by using extinction coefficient 2.8
were properly shaken and placed 30 cm below light
mM-1 cm-1 (Nakano and Asada, 1981).
source from two 100 W fluorescent lamps. The reaction
Peroxidase
was started by switching on the light and terminated
For this assay, 50 μl of enzyme extract, 100 mM
after 20 min of incubation by switching off the light.
phosphate buffer (pH=7.0), 200 µl of 10 mM pyrogallol
After terminating the reaction, the tubes were covered
and 300 μl of 5 mM H2O2 were added. After incubation,
with black cloth to protect them from light. A non-
the reaction was stopped by adding 1.0 ml of 2.5 N
illuminated reaction mixture that did not develop color
H2SO4. The amount of pur-purogallin formed was esti-
served as the blank. The reaction mixture that developed
mated by measuring the absorbance at 420 nm (Kar and
Figure 15. Microscopic image from the mycorrhizal colonization of Pimpinella anisum root in Glomus mosseae + brassinosteroid treatment (10X) Journal of Research in Ecology (2017) 5(2): 1033-1051
1041
Hajbagheri et al., 2017 Mishra, 1976).
curve (Akkol et al., 2008).
Polyphenoloxidase
Flavonoid
2.8 mL of phosphate buffer at monosedic 25
For total flavonoid assay1 mL of methanolic
mM with pH=6.8 was added to pyrogallol 10 mM and
extract, 250 μL of aluminum chloride solution and 250
100 ml of the enzyme extract. Enzyme activity was
μL of potassium acetate were mixed and the samples
measured at 420 nm using UV-visible spectrophotome-
remained at room temperature for 30 min. The absorb-
ter 1601 based on the Orange color intensity of poor-
ance of their action mixture was measured at 415 nm
porogalin in times of 40 and 100 seconds after the addi-
with the UV-visible spectrophotometer 1601 model
tion of pyrogallol measured (Kar and Mishra, 1976).
(Akkol et al., 2008).
Determination of non-enzymatic antioxidants
Statistical analysis
Anthocyanin
This experiment was performed with three repli-
Total anthocyanin was determined according to
cations in factorial design. Data analysis were per-
modified Wagner (1979) method using acidified ethanol
formed using SAS, version 15 and MSTAT-C statistical
(ethanol: HCl 99: 1 v/v) as 0.05 g of frozen leaf was
software. Compared using the Duncan test at the 5%
homogenized in 2.5 ml acidified ethanol and then kept
level, and the graphs were drawn by Excel software
at 25°C for 24 h in the dark. The extract was centrifuged
(Bazargan, 2017).
at 4000 g for 10 min at room temperature. The absorbance of each supernatant was read at 550 nm. The ex-
RESULTS
tinction coefficient 33,000 (mol-1cm-1) was used to
Mycorrhizal colonization in root
calculate the amount of total anthocyanin and it was
The results of this study showed that the roots
expressed as μmolg-1FW (Wagner, 1979).
mycorrhizal colonization percentage decreased in 200
Phenol
and 800 ppm cadmium chloride concentrations but 0.05 g of lyophilized cells were in 3 ml of ho-
brassinosteroid treatment significantly increased this
mogenized 80% methanol and 3 hours in Ben Murray at
parameter in all levels of cadmium chloride. The highest
70° C was placed. Then it was centrifuged at 9000 x g
percentage of root mycorrhizal colonization was in
for 15 minutes and methanol extract was separated from
brassinosteroid treatment and the lowest was in 800
the sediment. Methanol extracts to measure total phe-
ppm of cadmium chloride treatment (Figure 1 and 2,
nols, flavonoids and flavonoids were used.
Image 1-3).
To 50 µl of methanol extract, 500 µl Folin -
Hydrogen peroxide
Ciocalteau solution was added. After five minutes 500
The results of this study showed that cadmium
ml of 7% sodium carbonate solution was added to the
chloride caused a significant increase in the amount of
mixture and absorption at 765 nm was read after 10
hydrogen peroxide. Interactions of brassinosteroid,
minutes. Finally, total phenol content in mg/g dry
G. mosseae and G. mosseae + brassinosteroid with all
weight was measured (Singleton and Rossi, 1965).
levels of cadmium chloride significantly reduced this
Flavonol
parameter (Figure 3).
For total flavonol assay 1 mL of methanolic
Catalase
extract, 1 mL of aluminum chloride solution and 3 mL
Results of this study showed that catalase activi-
of sodium acetate were mixed and absorbance was read
ty increased significantly in 200 and 800 cadmium chlo-
at 445 nm. Rutin was used as a standard for calibration
ride treatments. Treatment with brassinosteroid and
1042
Journal of Research in Ecology (2017) 5(2): 1033–1051
Hajbagheri et al., 2017 G. mosseae + brassinosteroid increased significantly
Anthocyanin
this parameter. In the interactions of brassinosteroid,
Anthocyanin content increased significantly in
G. mosseae and Glomusmosseae + brassinosteroid with
interactions
200 and 800 ppm of cadmium chloride the activity of
G. mosseae and brassinosteroid with 0, 100 and 200
this enzyme significantly decreased (Figure 4).
ppm of cadmium chloride (Figure 9).
Ascorbate peroxidase
Flavonoid
The results of this study showed that ascorbate
of
G.
mosseae
+
brassinosteroid,
Flavonoid content increased significantly in
peroxidase activity increased significantly in 200 ppm
interactions
cadmium chloride and G. mosseae treatments groups. In
G. mosseae and brassinosteroid with 0, 100 and 200
interactions
and
ppm of cadmium chloride. In the other hand this param-
G. mosseae + brassinosteroid with 100 and 800 ppm of
eter reduced significantly in all treatment group with
cadmium chloride the activity of this enzyme increased
800 ppm cadmium chloride (Figure 10).
significantly (Figure 5).
Flavonol and Phenol
of
brassinosteroid,
G.
mosseae
G.
mosseae
+
brassinosteroid,
The results of this study showed that flavonol
Superoxide dismutase Results of this study showed that the activity of
of
and
phenol
content
increased
significantly
in
superoxide dismutase increased significantly in the 200
G. mosseae and G. mosseae + brassinosteroid. But re-
and 800 ppm cadmium chloride, brassinosteroid, plants
duced significantly in brassinosteroid, G. mosseae and
G. mosseae and G. mosseae + brassinosteroid treat-
G. mosseae + brassinosteroid with all concentration of
ments. In interactions of brassinosteroid, Glomus
cadmium chloride (Figures 11 and 12).
mosseae, G. mosseae + brassinosteroid with all levels of cadmium chloride activity of this enzyme significantly
DISCUSSION
decreased (Figure 6).
One of the most important indicators of mycor-
Polyphenol oxidase
rhizal fungi activity is the colonization percentage that
The results of this study showed that activity of
is affected by several factors such as appearance and
the polyphenol oxidase enzyme increased significantly
structural properties of the root system, the quantity and
in the 200 and 800 ppm cadmium chloride, G. mosseae
quality of root exudates, the use of fertilizers and of
and G. mosseae + brassinosteroid. In interactions of
high concentrations of heavy metals (Al –Karaki, 2000).
brassinosteroid, Glomus mosseae, G. mosseae + brassi-
In general, the negative effects of high concentrations of
nosteroid with all levels of cadmium chloride the activi-
heavy metals on root colonization with mycorrhizal
ty of this enzyme significantly increased (Figure 7).
fungi have been observed in the root system of plants.
Peroxidase
For example, it has been reported that the spores of Glo-
Results of this study showed that of peroxidase
mus intraradices fungus do not germinate in 1 mM or
activity significantly increased in all levels of cadmium
10 mM cadmium concentration (Pawlowska and Char-
chloride. In interaction of brassinosteroid and all levels
vat, 2004).
of cadmium chloride the activity of this enzyme signifi-
In this study, cadmium toxicity reduced the my-
cantly increased. In interaction of G. mosseae + brassi-
corrhizal contamination of anisum root with G. mosseae
nosteroid and G. mosseae with 100 and 200 ppm of cad-
fungus. In mycorrhizal communities, the fungus is en-
mium chloride the activity of this enzyme significantly
tirely dependent on the plant growth and nutrition’s pro-
increased (Figure 8).
duction in the host plant. Thus, any factor which affects
Journal of Research in Ecology (2017) 5(2): 1033–1051
1043
Hajbagheri et al., 2017 the production of carbohydrates and its transfer to the
cadmium, and magnesium reduced
roots can effect on the amount of contamination. Since
maize, this may be due to the very close relationship
heavy metal stress reduced the plant growth, it can also
between heavy metals types and the side-chain of pro-
reduce the mycorrhizal inoculation (Asghari, 2008). In
teins (Ghani and Wahid, 2007). Doganlar et al. (2012)
the same reports, the mycorrhizal contamination was
reported that the protein content reduced in high con-
reduced under cadmium toxicity in wheat plants that
centrations of manganese and nickel in Lemna and this
inoculated with Glomus intraradices (Jamalabad and
effect may be due to the protein degradation caused by
Khara, 2008) and Cajanus cajan plant inoculated with
oxidative stress (Doganlar et al., 2012). In the sunflower
G. mosseae (Garg and Bhandari, 2012).
that treated with cadmium, oxidized proteins were gath-
Plant hormones play an important role in mycorrhizal symbiosis. Plant hormones or phytohor-
protein content in
ered in cotyledons and leaves and the protease activity increased significantly (Pena et al., 2007).
mones are involved in signaling pathways and regula-
In this study the pre-treatment of plants with
tion of this symbiosis at different stages of plant growth
brassinosteroids, the inoculated plants with G. mosseae
(Jutta, 2000). In this study, brassinosteroids has a stimu-
and the interaction of G. mosseae and brassinosteroids,
lating role in mycorrhizal inoculation under control and
the shoot protein increased at 200 and 800 ppm concen-
cadmium toxicity conditions. Some experiments have
tration of cadmium chloride but the root protein content
shown that brassinosteroids, gibberellin and cytokinine
increased at 100 and 200 ppm concentrations of cadmi-
are needed for the early stages of root mycorrhizal con-
um chloride. Studies showed that brassinosteroids have
tamination and are not involved in the next stages of
a positive effect on the processes of DNA and RNA
coexistence (Hanlon and Coenen, 2011).
modeling and protein synthesis to enhance the plant
The results of this study showed that the protein
growth. Fariduddin et al. (2009) reported that the use of
content significantly decreased in 800 ppm of cadmium
brassinosteroids increased the amount of protein in cab-
chloride. Some researchers believed that protein reduc-
bage under copper stress (Fariduddin et al., 2009). In
tion could be due to the induction of oxidative stress,
Lycopersicon esculentum plants under salinity stress,
proteases activity, changes in the proteins structure and
the interaction of 24-Epibrassinolide and putrescine
function through denaturation and fragmentation and the
increased the protein content and antioxidant enzymes
replacement of metals in the thiol groups of protein
and reduce the harmful effects of salinity stress (Sharma
(Rastgoo and Alemzadeh, 2011). Cadmium stress
et al., 2012).
changed protein structure or replacing a vital element
Based on some studies insoluble glycoprotein
for the activities of essential enzymes and protein con-
synthesis is one of the resistant mechanisms in mycor-
tent of the plants. The heavy metal inhibits sulfydryl
rhizal plants against heavy metals such as cadmium.
groups of structural proteins and guides the enzymes to
These proteins have link places with heavy metals and
incorrect folding and activity inhibitory. This compo-
have a positive role for plant protection by linking to
nent reduced the production of proteins and stops the
heavy metals. (Trotta et al., 2006). In a similar report on
growth with a disorder in nitrogen metabolism through
wheat plant that was inoculated with Glomus intra-
inhibiting the activity of enzymes such as glutamine
radices under cadmium stress, the sugars and total pro-
synthase, glutamate synthase, nitrate reductase and ni-
teins content increased in comparison with non-
trate regeneration process. Ghani and Wahid (2007)
inoculated plants (Jamalabad and Khara, 2008). Also the
showed that some heavy metals such as lead, cobalt,
use of arbuscular mycorrhizal fungi in Anadenanthera
1044
Journal of Research in Ecology (2017) 5(2): 1033–1051
Hajbagheri et al., 2017 colubrine increased the proteins production (Pedone-
antioxidant enzyme activities and proline content
Bonfim et al., 2012). Enteshari et al. (2012) reported
(Hasan et al., 2011). Brassinosteroids application on
shoot and root protein content increased in mycorrhizal
tomato plants that against cadmium photosynthesis,
sweet basil plants compared to non-mycorrhizal plants
antioxidant enzymes activity, transcription levels, anti-
under salinity stress conditions (Enteshari et al., 2012).
oxidant system and secondary compounds metabolism
Heavy metals can be catalyzed and accelerated
significantly increased these results showed that brassi-
reactive oxygen species. Hydrogen peroxide is the most
nosteroids had positive role in the detoxification of cad-
stable molecule of the active oxygen species. Heavy
mium stress (Ahammed et al., 2012).
metal ions can stimulated the metabolism of free radi-
In this research the activity of ascorbate peroxi-
cals and alter the concentration of H2O2 (Shaw et al.,
dase, peroxidase and polyphenol oxidase increased in
2004). In this study, the toxicity of cadmium increased
inoculation of plants with G. mosseae or interaction
H2O2 levels this result was according with Belkadhi et
between mycorrhizal plants and brassinisteroid. This
al., 2014 on Linum usitatissimum (Belkadhi et al.,
study showed that inoculated plants against cadmium
2014).In the other hand inoculation with mycorrhiza
toxicity were mainly related to the effect of this fungi on
(G. mosseae) or brassinosteroid treatment
decreased
the ascorbate peroxidase and polyphenol oxidase.
H2O2 content by increasing enzymatic
and non-
Glomus mosseae mycorrhiza significantly in-
enzymatic antioxidant parameters. In the same reports,
creases the activity of superoxide dismutase, catalase
24-epibrassinolide in Brassica juncea under cold stress
and peroxidase in the Cajanus cajan (Garg and
(Kumar et al., 2010) and in mycorrhizal cucumber plant
Bhandari,
under low temperature stress (Liu et al., 2014) H2O2
G. mosseae under salinity stress (Abdel latef and
content were reduced.
Chaoxing, 2011) and pomegranates inoculated plants
2012).
In
inoculated
tomatoes
with
The antioxidants compounds are considered as
with Glomus intraradices under oxidative stress under
important defensive systems in plants against oxidative
dehydration (Bompadre et al., 2014), the activity of
stress that induced by heavy metals (Hasan et al., 2011).
superoxide
dismutase,
catalase,
peroxidase
and
In this study, the activity of catalase, superoxide dis-
ascorbate
peroxidase
was
increased.
In
mutase, polyphenol oxidase, ascorbate peroxidase and
Lycopersicon esculentum plants under salinity stress,
peroxidase, especially at high concentrations of cadmi-
the interaction of 24-Epibrassinolide and putrescine
um chloride increased. In the root and leaves of radish,
increase the activity of catalase, superoxide dismutase
catalase and glutathione reductase activity increased
and guaiacol peroxidase enzymes (Sharma et al., 2012).
after 13 hours of exposure to cadmium (Vitoria et al.,
The evidences showed that there is a relation-
2001). Cadmium increased the superoxide dismutase
ship between phenol content and polyphenol oxidase
activity in sunflower (Laspina et al., 2005). In Ara-
activity in plants, when the total concentration of phenol
bidopsis plant under cadmium toxicity FeSOD and
increased in the plants and, the polyphenol oxidase ac-
MnSOD activity were increased (Drazkiewicz et al.,
tivity also increased subsequently (Tegelberg et al.,
2007).
2004). Also in this research showed that the polyphenol In this study brassinosteroid increased catalase,
oxidase activity increased by the increase in the phenol-
superoxide dismutase and polyphenol oxidase activity.
ic compounds content in anisum. Likely, ascorbate pe-
The treatment of tomato with 24-epibrassinolide and 28-
roxidase activity reduction in high concentrations of
Homo epibrassinolide increased the photosynthesis,
cadmium chloride is related to the inactivation of this
Journal of Research in Ecology (2017) 5(2): 1033–1051
1045
Hajbagheri et al., 2017 enzyme by the excessive production of active oxygen
um stress, anthocyanin content increased. It was report-
species, non-specific degradation, destruction, or bind-
ed
ing of unnecessary heavy metals to the enzyme (Sharma
Glomus etunicatum fungus significantly increased an-
et al., 2012). Reactive oxygen species production in
thocyanin content in sweet basil compared to non-
cadmium stress conditions increase the lipid peroxida-
mycorrhizal plants (Sharifi et al., 2011). Phenylalanine
tion, caused oxidative stress and damage the antioxida-
ammonia lyase and chalcone isomerase enzymes are
tive enzymes (Filek et al., 2008).
involved in the anthocyanin and 3-doxyanthocyanidin (a
that
the
mycorrhizal
inoculation
with
Another important plant defense system to con-
kind of phytoestrogen auxin), biosynthesis so that the
trol and neutralize free radicals is inducing the synthesis
fungal infection or brassinosteroids severely induced
of some non-enzymatic antioxidants such as anthocya-
these enzyme’s gene expression (Tripathi et al., 2006). It
nin and phenolic compounds. Generally, the consistency
was reported that the inoculation of plant with mycor-
with stress depends on the reduction of reactive oxygen
rhizal fungi increased the concentration of anthocyanin
species by the antioxidant system (Abdul-Jaleel et al.,
in strawberry (Lingua et al., 2013) and lettuce (Baslam
2009). In this study, anthocyanin content in cadmium
et al., 2013) and is considered as a reliable method to
chloride treatments showed no significant difference,
enhance the nutritional value of edible and medicinal
This result cannot be attributed to the less resistance of
plants consistent with the results in the present study.
anisum against the of cadmium toxicity, because the
The results of this study showed that the flavo-
plant may use other resistant mechanisms such as oth-
noids, flavonols and phenolic compound in cadmium
er phenolic compounds likely flavonoid production. On
stress increased significantly. It seems that this plant
the other hand, in plants treated with 24-epibrassinolide
under cadmium stress increased phenolic compounds in
anthocyanin content increased. Brassinosteroids activat-
order to have good defense reactions against the stress.
ed some enzymes such as phenylalanine ammonia lyase
Many flavonoids are an active component of secondary
and Chalcone Synthase (CHS) and other key enzymes in
metabolites from medicinal plants and their medicinal
the anthocyanin synthesis pathway (Campos et al.,
properties. They have an important role in plants re-
2003).
sistance as physiologically active compounds, protective In the same report, the use of 24-epibrassinolide
factors against stress and also light absorbers (Tattini et
in grape can increase the activity of phenylalanine am-
al., 2004). Phenolic compounds production in anisum
monia lyase enzyme or increased phenols, flavonoids,
under cadmium stress may be due to the induction of
tannins, and anthocyanin content (Xi et al., 2013). In
genes expression or the
grapes treated with brassinosteroids, a considerable in-
of these compounds in the phenylpropanoids pathway.
crease of anthocyanin concentration was observed in
The increase of the soluble phenolic compound content,
comparison with the control plant. Because of this in-
especially lignin precursors alongside the increase of the
crease, the impact of brassinosteroids on structural and
cell wall thickness creates a biological barrier to pre-
regulatory genes is related to anthocyanin biosynthesis
vent entry of heavy metals into living cells and increases
(Luan et al., 2013). In this study, the 24-epibrassinolide
their tolerance to these metals. These compounds direct-
probably increased the amount of anthocyanins through
ly prevent the oxidative stress by entering into the re-
this mechanism.
ducing reactions or indirectly by chelating the cadmium.
enzyme synthesis stimulation
In pretreated plants with mycorrhizal or
High levels of phenolic compounds in beans have also
brassinosteroids and mycorrhizal fungi under the cadmi-
been reported as the answer to cadmium toxicity
1046
Journal of Research in Ecology (2017) 5(2): 1033–1051
Hajbagheri et al., 2017 (Michalak, 2006)
tion of the activity of catalase, superoxide dismutase,
The treatment of anisum with brassinosteroid
peroxidase and polyphenol oxidase, and the amount of
increased flavonoids and phenols content in low concen-
flavonoids, flavonols and phenol in anisum due to cad-
trations of cadmium chloride. One of the causes of re-
mium toxicity improved the antioxidative defense sys-
ducing the effects of oxidative stress in plants treated
tem. The treatment of anisum with brassinosteroids and
with brassinosteroids is the effects of this substance on
inoculation with G. mosseae improves the host plant
the synthesis of substances such as flavonoids. Brassi-
growth by providing more plant resistance to cadmium
nosteroids at these concentrations reduced the toxic ef-
toxicity stress.
fects of cadmium chloride by affecting the antioxidant system. In a similar report, in Cichorium endivia L.
REFERENCES
plants treated with brassinosteroid the antioxidant com-
Abbaspour H, Saeidi-Sar S, Afshari H and Abdel-
pounds and total phenolics increased (Serna et al.,
Wahhab MB. (2012). Tolerance of mycorrhiza infected
2013). Brassinosteroids produce phenolic and phenol
pistachio (Pistaciavera L.) seedling to drought stress
acts as a barrier against moisture loss
under glasshouse conditions. Journal Plant Physiology,
in the cell wall
and prevents the further spread of the pathogen. Also, when the photosynthetic electron transport is limited, metabolic changes may increase flavonoids that are due to the activation of metabolic pathways mediated by the phenylalanine ammonia lyase enzyme or other enzymes (Burguieres et al., 2007). Although the mechanism of increasing the
169(7): 704-709. Abdel latef AAH and Chaoxing H. (2011). Effect of arbuscular mycorrhizal fungi on growth, mineral nutrition, antioxidant enzymes activity and fruit yield of tomato grown under salinity stress. Science Horticulture, 127(3): 228-233.
amount of flavonoids by mycorrhizal fungi is not yet
Abdul-Jaleel C, Riadh K, Gopi R, Monivannan P,
clear, it was observed that Mycorrhizal fungi does that
Ines J, Al-Juburi HJ, Chang-Xing Z, Hong-Bo S and
by increasing enzyme activity and gene expression in a
Panneerselvam R. (2009). Antioxidative defense
signaling cascade (Zhang et al., 2013). Also, the role of
responses: physiological plasticity in higher plants un-
flavonoids of the host plant root in the regulation of
der abiotic constraints. Acta Physiologiae Plantarum,
mycorrhizal symbiosis is well known. Flavonoids stim-
31(3): 427-436.
ulate the spore germination, mycelial growth of mycorrhizal fungi and root contamination by arbuscular mycorrhizal fungi. As a result, it is certain that the in-
Aebi H. (1984). Catalase in vitro. Methods in Enzymology, 105: 121-126.
crease in the amount of flavonoids is observed during
Ahammed GJ, Choudhary SP, Chen S, Xia X, Shi
the mycorrhizal symbiosis. In another report, Abbaspoor
K, Zhou Y and Yu J. (2012). Role of brassinosteroids
et al. (2012) showed that the flavonoids content in pista-
in
chios mycorrhizal plants was higher when compared to
contamination-induced photosynthetic inhibition and
non-
oxidative stress in tomato. Journal of Experimental
mycorrhizal
plants
under
drought
stress
(Abbaspour et al., 2012). CONCLUSION The results of this study showed that the inducJournal of Research in Ecology (2017) 5(2): 1033–1051
alleviation
of
phenanthrene–cadmium
co-
Botany, 64(1): 199-213. Akkol EK, Goger F, Kosar M and Baser KH. (2008). Phenolic composition and biological activities of Salvia 1047
Hajbagheri et al., 2017 halophila and Salvia virgata from Turkey. Food
in pomegranate plants growing under different irrigation
Chemistry, 108(3): 942–949.
conditions. Botany, 92(3): 187-193.
Ali MB, Khatun S, Hahn EJ and Paek KY. (2008).
Bradford MM. (1976). A rapid and sensitive method
Enhancement of phenylpropanoid enzymes and lignin in
for the quantitation of microgram quantities of protein
Phalaenopsis orchid and their influence on plant accli-
utilizing the principle of protein dye binding. Analytical
matisation at different levels of photosynthetic photon
Biochemistry, 72(1-2): 248-254.
flux. Plant Growth Regulation. 49(2-3): 137-146.
Burguieres E, McCue P, Kwon YI and Shetty K.
Al-Karaki, G.N. (2000). Growth of mycorrhizal tomato
(2007). Effect of vitamin C and folic acid on seed vigor
and mineral acquisition under salt stress. Mycorrhiza
response and phenolic-linked antioxidant activity. Bio
Journal, 10(2): 51-54.
resource Technology, 98(7): 1393-1404.
Asghari HR. (2008). Vesicular_arbuscular (VA) my-
Campos AD, Ferreira AG, Hampe MMV, Antunes
corrhizae improve salinity tolerance in pre-inoculation
IF, Brancao N, Silveria EP and Osorio VA. (2003).
subterranean clover (Trifolium subterraneum) seedling.
Induction of chalcone synthase and phenylalanine am-
International Journal of Plant Production. 2(3): 243-
monia-lyase by salicylic acid and Colletotrichum linde-
256.
muthianum in common bean. Brazilian Journal of Plant
Aïcha Belkadhi, Antonio De Haro, Pilar Soen-
Physiology, 15(3): 129-134.
gas, Sara Obregon, Maria Elena Cartea, Wided
Chandler JW, Cole M, Flier A and Werr W. (2009).
Chaibi and Wahbi Djebali. (2014). Salicylic acid in-
BIM1, a bHLH protein involved in brassinosteroid sig-
creases tolerance to oxidative stress induced by hydro-
naling, controls Arabidopsis embryonic pattering via
gen peroxide accumulation in leaves of cadmium-
interaction with dornroschen and dornroschen-like.
exposed flax (Linum usitatissimum L.). Journal of Plant
Plant Molecular Biology, 69:(1-2) 57-68.
Interactions, 9(1): 647-654.
Doganlar ZB, Cakmak S and Yanik T. (2012). Metal
Bartwal A, Mall R, Lohani P, Guru SK and Arora S.
uptake and physiological changes in Lemnagibba ex-
(2013). Role of secondary metabolites and brassinoster-
posed to Manganese and Nickel. International Journal
oids in plant defense against environmental stresses.
of Biology, 4(3): 148–157.
Journal of Plant Growth Regulation, 32(1): 216–232.
Drazkiewicz M, Skorzynska-Polit E and Krupa Z.
Baslam M, Esteban R, Garcia-Plazaola J and Goi-
(2007). The redox state and activity of superoxide dis-
coechea N. (2013). Effectiveness of arbuscular mycor-
mutase classes in Arabidopsis thaliana under cadmium
rhizal fungi (AMF) for inducing the accumulation of
or copper stress. Chemosphere, 67(1): 188–193.
major carotenoids, chlorophylls and tocopherol in green and red leaf lettuces. Applied Microbiology and Biotechnology, 97(7): 11-19.
Enteshari S, Hajbagheri S and Razavizadeh R. (2012). Role of mycorrhizal fungi and salicylic acid in salinity tolerance of Ocimum basilicum L. resistance to
Bompadre JM, Vanesa AS, Laura BF, Maria D.
salinity. African Journal of Biotechnology, 11(9): 2223-
Roxana CP, Alejandro GP and Alicia GM. (2014).
2235.
Arbuscular mycorrhizal fungi alleviate oxidative stress 1048
Fariduddin Q, Yusuf M and Ahmad AH. (2009). EfJournal of Research in Ecology (2017) 5(2): 1033–1051
Hajbagheri et al., 2017 fect of 28-homobrassinolide on antioxidant capacity and photosynthesis in Brassica juncea plants exposed to different levels of copper. Environmental and Experimental Botany, 66(3): 418-424.
East Malling, England. 547p. Jamalabad KH and Khara J. (2008). The effect of arbuscular mycorrhizal fungi Glomus intraradices on some growth and physiological parameters in wheat
Filek M, Keskinen R, Hartikainen H, Szarejko I,
(cv.AZAR2)
Garg N and Bhandari P. (2012). Influence of cadmi-
Iranian Journal of Biology, 21(5): 216-230.
um stress and arbuscular mycorrhizal fungi on nodule senescence in Cajanus cajan (L.) Millsp. International Journal of Phytoremediation, 14(1): 62-74. Golam J, Xiaomin L and Chaoxing H. (2014). Arbuscular mycorrhizae improve low temperature tolerance in cucumber via alterations in H2O2 accumulation and ATPase activity. Journal of Plant Research, 127(6): 775 -785. Ghani A and Wahid A. (2007). Varietal differences for cadmium-induced seedling mortality and foliar toxicity
plants
under
cadmium
toxicity.
Janiak A, Miszalski Z and Golda A. (2008). The protective role of selenium in rape seedlings subjected to cadmium stress. Plant Physiology, 165(8): 833-844. Jana S and Choudhuri AM. (1982). Glycolate metabolism of three submerged aquatic angiosperms during aging. Aquatic Botany, 12: 345-354. Jutta LM. (2000). Hormonal Balance in Plants during Colonization by Mycorrhizal Fungi. Arbuscular Mycorrhizas: Physiology and Function, 263-285.
symptoms in mung bean (Vigna radiata). International
Kar M and Mishra D. (1976). Catalase, peroxidase,
Journal Agricultural Biology, 9(4): 555–558.
and polyphenoloxidase activities during rice leaf senes-
Gianopolitis CN and Ries SK. (1977). Superoxide
cence. Plant Physiology, 57(2): 315-319.
dismutase: occurrence in higher plants. Plant Physiolo-
Kumar M, Sirhindi, G, Bhardwaj R, Kumar S and
gy, 59: 309-314.
Jain G. (2010). Effect of exogenous H2O2 on antioxi-
Hanlon MT and Coenen C. (2011). Genetic evidence for auxin involvement in arbuscular mycorrhizal initiation. New Phytologist, 189(3): 701–709. Hasan SA, Hayat S, Ali B and Ahmad A. (2008). 28-Homobrassinolide protects chickpea (Cicer arietinum) from cadmium toxicity by stimulating antioxidants. Environmental Pollution, 151(1): 60-66. Hasan SA, Hayat S and Ahmad A. (2011). Brassinosteroids protect photosynthetic machinery against the cadmium induced oxidative stress in two tomato cultivars. Chemosphere, 84(10): 1446–1451. Hewitt EJ. (1966). Sand and water culture methods used in the study of plant nutrition. Tech. Commun. 22, Commonwealth Bureaux of Hort. and Plantation Crops, Journal of Research in Ecology (2017) 5(2): 1033–1051
dant enzymes of Brassica juncea L. seedlings in relation to
24-epibrassinolide
under
chilling
stress.
Indian Journal of Biochemistry and Biophysics, 47(6): 378-382. Laspina NV, Groppa MD, Tomaro ML and Benavides MP. (2005). Nitric oxide protects sunflower leaves
against
Cd-induced
oxidative
stress.
Plant Science, 169(2): 323-330. Leung HM, Wang ZW, Ye ZH, Yung KL, Peng XL and Cheung KC. (2013). Interactions between Arbuscular Mycorrhizae and plants in phytoremediation of metal-contaminated soils: A review. Pedosphere, 23(5): 549–563. Lingua G, Bona E, Manassero P, Marsano F, Todes1049
Hajbagheri et al., 2017 chini V, Cantamessa S, Copetta A, Dagostino G, Ga-
ed to cadmium stress. Phytochemistry, 68(8): 1139–
malero E and Berta G. (2013). Arbuscular Mycor-
1146.
rhizal fungi and plant growth-promoting Pseudomonads increases anthocyanin concentration in strawberry fruits (Fragaria x ananassavar. Selva) in conditions of reduced fertilization. International Journal of Molecular sciences, 14(8): 16207-16225. Liu GY, Zhang YX Chai TY. (2011). Phytochelatin synthase of Thlaspi caerulescens enhanced tolerance and accumulation of heavy metals when expressed in yeast and tobacco. Plant Cell Reports, 30(6): 1067– 1076.
Rajapakes S and Miler CJ. (1992). 15 Methods of studying Vesicular-arbuscular Mycorrhizal root colonization and related root physical properties. Methods in Microbiology, 24. ISBN: 0-12-521524. Rastgoo L and Alemzadeh. A. (2011). Biochemical responses of Gouan (Aeluropus littoralis) to heavy metals stress. Australian Journal of Crop Science, 5(4): 375-383. Serna M, Hernandez F, Coll F, Coll Y and Amoros
Liu A, Shuangchen C, Rui C, Dilin L, Haoran C, Luan LY, Zhang ZW, Xi ZM, Huo SS and Ma. LN. (2013). Brassinosteroids regulate anthocyanin biosynthesis in the ripening of grape berries. South African Journal of Enology and Viticulture, 34(2): 196-203. Michalak A. (2006). Phenolic compounds and their antioxidant activity in plants growing under heavy metal stress. Polish Journal of Environmental Studies, 15(4): 523-530.
A. (2013). Effects of brassinosteroid analogues on total phenols, antioxidant activity, sugars, organic acids and yield of field grown endive (Cichorium endivia L.). Journal of the Science of Food and Agriculture, 93(7): 1765-1771. Sharifi M, Mohtashamian M, Riahi H, Aghaee A and Alavi SM. (2011). The effects of vesicular-arbuscular mycorrhizal fungus Glomus etunicatum on growth and some physiological parameters of basil. Journal of Medicinal Plants, 10(38): 85-94.
Omidbeygi G. (2006). Production of medicinal plants, published by Razavi, 3: 397p.
Sharma A and Sharma H. (2013). Role of vesiculararbuscular mycorrhiza in the mycoremediation of heavy
Pawlowska T and Charvat I. (2004). Heavy-metal
toxic metals from soil. International Journal of Life
stress and developmental patterns of arbuscular mycor-
Sciences and Biotechnology Pharma Research, 2(3):
rhizal fungi. Applied and Environmental Microbiology.,
2250-3137.
70: 6643-6649.
Sharma A, Slathia S and Choudhary SP. (2012).
Pedone-Bonfim M, Lins M, Coelho I, Santana A, Silva F and Maia L. (2012). Mycorrhizal technology and phosphorus in the production of primary and secondary
metabolites
in
cebil
(Anadenanthera
colubrina (Vell.) Brenan) seedlings. Journal of the
Influence of exogenously applied epibrassinolide and putrescine on protein content, antioxidant enzymes and lipid peroxidation in Lycopersicon esculentum under salinity stress. American Journal of Plant Sciences, 3(6): 714-720.
Science of Food and Agriculture, 93(6): 1479–1484.
Shaw, B.P. Sahu, S.K. and R.K. Mishra. (2004).
Pena LB, Pasquini LA, Tomaro ML and Gallego
Heavy metal induced oxidative damage in terrestrial plants. In:
SM. (2007). 20S proteasome and accumulation of oxi-
Heavy Metal Stress in Plants. From Biomolecules to Ecosys-
dized and ubiquitinated proteins in maize leaves subject-
tems. 2nd Ed, MNV Prasad, Springer-Verlag New York, pp.
1050
Journal of Research in Ecology (2017) 5(2): 1033–1051
Hajbagheri et al., 2017 84-126.
Phytology., 2(7): 16-27.
Shi Q and Zhu Z. (2008). Effects of exogenous salicylic acid
Vitoria, A.P. Leap, J. and R.A. Azevedo. (2001). Antioxi-
on manganese toxicity, element contents and antioxidative
dant enzymes responses to cadmium in radish tissues. Phyto-
system in cucumber. Environmental and Experimental Botany,
chemistry., 57: 701–710.
63(1-3): 317-326.
Wagner GJ. (1979). Content and vacuole/extra vacuole distri-
Singh PK. (2012). Role of glomalin related soil protein pro-
bution of neutral sugars, free amino acids and anthocyanin in
duced by Arbuscular Mycorrhizal fungi: A review. Journal of
protoplasts. Journal of Plant Physiology, 64(1): 88-93.
Agricultural Science Research,
2(3): 119-125.
Singleton VL and Rossi JR. (1965). Colorimetry of total phenolics with phosphomolybdic-phosphotungestic acid reagent. American Journal of Enology and
Viticulture, 16:
Winkel-Shirley B. (2002). Biosynthesis of flavonoids and effects of stress. Current Opinion in Plant Biology, 5(3): 218223. Xi ZM, Zhang ZW, Huo SS, Luan LY, Gao X, Ma LN and Fang YL. (2013). Regulating the secondary metabolism in
144-58. Tangahu BV, Abdullah SR, Basri H, Idris M, Anuar M and Mukhlisim M. (2011). A review on heavy metals (As, Pb and Hg) uptake by plants through phytoremediation. Interna-
grape berry using exogenous 24-epibrassinolide for enhanced phenolics content and antioxidant capacity. Food Chemistry, 141(3): 3056-3065. Zhang M, Zhai Z, Tian X, Duan L and Z Li. (2008). Bras-
tional Journal of Chemical Engineering, 1-31. Tattini M, Galardi C, Pinelli P, Massai R, Remorini D and Aghati G. (2004). Different accumulation of flavenoids and hydroxyl cinnamates in leaves of Ligustrum vulgare under
sinolide alleviated the adverse effect of water deficits on photosynthesis and the antioxidant of soybean (Glycine max L.). Plant Growth Regulation., 56(3): 257-264.
excess light and drought stress. New Physiologist, 163: 547-
Zhang RQ, Zhu HH, Zhao HQ and Yao Q. (2013). Arbus-
561.
cular mycorrhizal fungal inoculation increases phenolic syn-
Tegelberg R, Julkunen-Titto R and Aphalo PJ. (2004). Red: far-red light ratio and UV-B radiation: their effect on leaf phenolics and growth of silver birch seedlings. Plant, Cell and
thesis in clover roots via hydrogen peroxide, salicylic acid and nitric oxide signaling pathways. Journal of Plant Physiology, 170(1): 74-79.
Environment, 27: 1005-1013. Tripathi BN, Mehta SK, Amar A and Gaur JP. (2006). Oxidative stress in scenedemus sp. during short - and long term exposure to Cu2+ and Zn2+. Chemosphere, 62: 538-544. Trotta A, Falaschi P, Cornara L, Minganti V, Fusconi A, Drava G and Berta G. (2006). Arbuscular mycorrhize increase the arsenic translocation factor in the as hyper accumulating fern Pteris vittate L. Chemosphere, 65: 74- 81. Turk MA, Assaf TA, Hameed KM and Al-Tawaha AM. (2006). Significance of mycorrhizae. World
Journal of Agri-
cultural Sciences, 2(1): 16-20.
Submit your articles online at ecologyresearch.info Advantages
Easy online submission Complete Peer review Affordable Charges Quick processing Extensive indexing You retain your copyright
Upadhyaya, H. Panda, S.k. Bhattacharjee, M.K. and S. Dutta. (2010). Role of arbuscularmycorrhiza in heavy metal tolerance in plants: Prospects for phytoremediation. Journal of
Journal of Research in Ecology (2017) 5(2): 1033–1051
submit@ecologyresearch.info www.ecologyresearch.info/Submit.php.
1051