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: ABSTRACT:
Journal of Research in Ecology

Sepideh Hajbagheri1, Heavy metals are important environmental pollutants and their toxicity is
Hosein Abbaspour2, considered as a major problem, because of ecological, evolutionary, environmental
Shekoofeh Enteshari3, and nutritional effects. Many studies have shown that plants inoculated with
Saeed Habibollahi4 and mycorrhizal fungi or the use of brassinosteroids increased plants resistance to heavy
Alireza Iranbakhsh5 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
University, Iran house and hydroponic condition. The results showed that cadmium stress reduced
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
3. Biology Department, pretreated with brassinosteroid and inoculated with mycorrhizal fungi and then
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,
Iran. this plant brassinosteroid application or mycorrhizal inoculation have positive effects
on strengthening the antioxidant system in this plant against cadmium stress.
5. Professor of Biology
Department, Azad Keywords:
University, Iran Glomus mosseae, brassinosteroid, cadmium chloride, Pimpinella anisum.

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

Web Address:
http://ecologyresearch.info/
documents/EC0483.pdf
Dates:
Received: 23 July 2017 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.

Journal of Research 1033-1051| JRE | 2017 | Vol 5 | No 2

in Ecology
An International www.ecologyresearch.info
Scientific Research Journal
Hajbagheri et al., 2017
INTRODUCTION
Plants in nature are constantly exposed to the per-
sistent presence of heavy metal toxic ions and experi-
ence the damage of their cumulative toxicity. Toxic
effects of heavy metals are mainly related to these four
factors: disabling the active groups of enzymes, replac-
ing instead of essential metals, inducing structural
changes to polymers and affecting the strength and
structure of the membrane and the membrane transport
processes (Tangahu et al., 2011). Cadmium with the
symbol Cd and atomic number 48 is one of the most
toxic heavy metals. Weathering of rocks, forest fires and
volcanoes, human activities such as industrial waste
leachates, the production of artificial phosphate fertiliz-
ers are the important sources of cadmium in the environ-
ment. Cadmium accumulation in plant tissues reduced
the cell division, root growth, root hydraulic conductivi-
ty and causes growth inhibition, chlorosis and cell death
(Liu et al., 2011).
Today, a lot of research focused on the use of
growth regulators or mycorrhizal fungi in reducing the
effects of environmental stresses. In endo-mycorrhizal
coexistences, the mycorrhizal fungi play an important
role in host plants protection against the toxicity of
heavy metals. Mycorrhizal fungi change the heavy met-
al concentration through metal inactivity and the chelat-
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
ed metals through secreted compounds such as catego-
rizing the metals inside the fungal cells. The beneficial
effects of mycorrhizal on growth in terms of heavy met-
al toxicity have been reported in many plant species
(Leung et al., 2013; Sharma and Sharma, 2013; Upadh-
yaya et al., 2010). Mycorrhizal fungi regulated the bal-
ance of nutrients absorption that has been disturbed by
heavy metals and improves the toxicity of metals in
soils contaminated with heavy metals (Singh et al.,
2012). It has been reported that G. mosseae is resistant
to cadmium and plays a role in absorption, transport and
inactivity of cadmium (Upadhyaya et al., 2010).
Brassinosteroids are from steroid hormones and
a new group of plant growth regulators that are widely
distributed throughout the plant kingdom. Due to the
multiple effects of the hormone, these compounds are
known as plant hormones. This hormone stimulate the
growth and protein synthesis, increase the content of
compatible solutes, changes in transcription and expres-
sion of genes involved in stress, and the concentration
and activity of antioxidant enzymes and compounds that
all reduce the harmful effects of stress in plants in order
to maintain, and enhance the performance of the plant
(Ahammed et al., 2012; Zhang et al., 2008). The studies
have shown that the both endogenous and exogenous
brassinosteroids have an antioxidant role, and reduce the
Glomus mosseae
G.m* Br
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.,
2008). Some researchers showed that brassinosteroids
have a significant effect on plant resistance against the
accumulation of heavy metals in beans (Ali et al., 2008)
and pea (Hasan et al., 2008).
Oxidative stress caused by the accumulation of
heavy metals in plants leads to the production of reac-
tive oxygen species and oxidative damage in the cells.
One of the most important plant defense systems to con-
trol and neutralize the free radicals is to induce the syn-
thesis of antioxidant enzymes such as superoxide dis-
mutase, catalase, ascorbate peroxidase, peroxidase, pol-
yphenol oxidase and the non-enzymatic antioxidants
such as anthocyanins and phenolics. Superoxide dis-
mutase dismutation catalyzes two superoxide molecules
to H2O2 and H2O2 should detoxificate the antioxidant
defense in the next steps. Catalase enzyme breaks down
H2O2 to water and oxygen and is considered as the main
enzymes that sweep the Hydrogen Peroxide in peroxi-
somes. (Shi and Zhu, 2008). Ascorbate peroxidase en-
zymes revive hydrogen peroxide in different cell parts
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-
oxide by the oxidation of a substance. Polyphenol oxi-
dase is mono-oxygenase enzyme with copper that leads
to hydroxylation of phenols to di-phenyls and the oxida-
tion of di-phenyls (Abdul-Jaleel et al., 2009). Phenolic
compounds such as flavonoids, flavonols and phenols
are a large group of secondary plant products with only
one phenolic group. Anthocyanins are a group of water-
soluble flavonoids that are made in the biosynthesis of
flavonoids in the cytoplasm. Anthocyanin's do not only
destroy the free radicals but also prevent their produc-
tion in the plant. Antioxidant properties are the phenolic
compounds resulting from the reaction of these com-
pounds with free radicals and convert them into their
stable form electrons to these reactive radicals (Bartwal
et al., 2013).
In general, the adaptation or resistance to stress
depends on the reduction of the amount of reactive oxy-
gen species by antioxidant system. Treatment of plants
with epi-brassinolide and inoculation with mycorrhizal
fungi increases the activity of antioxidant enzymes and
phenolic compounds compared to untreated plants
which increases the resistance of plants against various
stresses such as the heavy metals (Abdul-Jaleel et al.,
2009; Ahammed et al., 2012).
Anisum with the scientific name of Pimpinella ani-
Control
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
and known as medicinal plant that contains the ingredi-
ents such as essential oils, resins, sugar compounds and
oil. Anisum has the antibacterial property and an ex-
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-
ing the resistance of P. anisum through the increase of
enzymatic and non-enzymatic antioxidants against the
cadmium chloride stress.
MATERIALS AND METHODS
This experiment was performed with three repli-
cations in completely randomized factorial design. The
applied treatments included: cadmium chloride at 0,
100, 200 and 800 ppm concentration, mycorrhizal fungi
(G. mosseae) and brassinosteroids (10-6mM).
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.
For the inoculation of seeds with G. mosseae on
Petri dishes, seeds were excluded on 5 g of biological
soil containing mycorrhizal spores of G. mosseae pre-
pared from Hamedan Organic Plant Clinic (IRAN). The
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
with distilled water. Petri dishes was put in germinator
with optical/dark conditions for 16:8 hours, at day/night
in the 23±2°C and 16±2°C temperatures respectively for
4 weeks and after wards transferred to pots (Hewitt,
1966).
Seedlings were transferred in plastic pots with
16 cm diameter and 15 cm height. In each pots, the
mixture of autoclaved perlite and coco peat was poured
with 1:3 ratio. For mycorrhizal treatments in each pot,
50 g of mycorrhizal spores soils were poured on the
surface of perlite and cocopeat mixture and seedling
was placed on this and covered with perlite and co-
copeat mixture. For each treatment, three pots were used
and so considered considered as three replicates. In each
pot, five seedlings were planted. Pots were put in green-
house with the optical condition of 14:10 light/night
hours, and 17 ± 2°C and 24 ± 2°C night/day temperature
respectively. Light intensity was 11,000 lux and humidi-
ty was 60%. For watering pots, distilled water and Long
Ashton nutrient solution were used (Hewitt, 1966).
Brassinosteroids treatments were applied after
150 days of germination when the fourth leaf of the
plants were grown and plants were sprayed with a solu-

Control
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),
MW=480.7), 3 times every 72 hours at the beginning of
the optical phase. Control plants were sprayed with dis-
tilled water. Three days after the last brassinosteroid
treatment, the cadmium chloride (100, 200 and 800 ppm
CdCl2½H2O, MW=228.34) treatment was applied three
times within 72 hour and the control plants were irrigat-
ed with distilled water. Plants were harvested 48 hours
after the last Cadmium treatment and some parameters
were studied (Hewitt, 1966).
Root mycorrhizal colonization percentage
For this measurement, Rajapakes and Miler
(1992) method was used. For root fixation using forma-
lin-acetic acid-ethanol (FAA) in the 14: 1: 1 ratio. Then
the roots were painted with fuchsine acid color and the
percentage of inoculated root was measured by using
anatomical microscope Olympus model.
Determination of protein
Total soluble protein content was measured ac-
cording to Bradford (1976) using Bovine Serum Albu-
min (BSA) as a protein standard. In each test tube to
extract leaf protein, 250 microliter phosphate buffer,
250 microliter of 1 M NaOH solution and 5 ml of Brad-
ford reagent were added. Then final volume of each
tube was made up to 6 ml. The contents of each tube
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 en-
zyme 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
3/94 mM-1cm-1 calculated on the basis of a unit (mol
hydrogen peroxide consumed per minute) per mg pro-
tein (Aebi, 1984).
Super oxide dismutase
Super oxide dismutase (SOD) was assayed by
measuring its ability to inhibit the photochemical reduc-
tion of Nitro Blue Tetrazolium (NBT). The reaction
mixture contained 50mM potassium phosphate buffer
(pH=7.5), 13 mM L-methionine, 75 μM NBT, 3 μM
riboflavin, 0.1 mM EDTA and 100 μM of enzyme ex-
tract. Riboflavin was added at the mixture. The tubes
were properly shaken and placed 30 cm below light
source from two 100 W fluorescent lamps. The reaction
was started by switching on the light and terminated
after 20 min of incubation by switching off the light.
After terminating the reaction, the tubes were covered
with black cloth to protect them from light. A non-
illuminated reaction mixture that did not develop color
served as the blank. The reaction mixture that developed
maximum color without enzyme extract were decreased
in absorbance decreased with enzyme addition. The
absorbance was recorded by the UV-visible spectropho-
tometer 1601 model at 560 nm (Gianopolitis and Ries,
1977).
Ascorbate peroxidase
Ascorbate peroxidase (APX) activity was deter-
mined by the decrease in absorbance of ascorbate at 290
nm. The reaction mixture contained 50 mM potassium
phosphate buffer (pH=7), 0.5 mM ascorbate, 1 mM
H2O2, 0.1 mM EDTA and enzyme extract. APX activi-
ty was calculated by using extinction coefficient 2.8
mM-1 cm-1 (Nakano and Asada, 1981).
Peroxidase
For this assay, 50 μl of enzyme extract, 100 mM
phosphate buffer (pH=7.0), 200 µl of 10 mM pyrogallol
and 300 μl of 5 mM H2O2 were added. After incubation,
the reaction was stopped by adding 1.0 ml of 2.5 N
H2SO4. The amount of pur-purogallin formed was esti-
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).
Polyphenoloxidase
2.8 mL of phosphate buffer at monosedic 25
mM with pH=6.8 was added to pyrogallol 10 mM and
100 ml of the enzyme extract. Enzyme activity was
measured at 420 nm using UV-visible spectrophotome-
ter 1601 based on the Orange color intensity of poor-
porogalin in times of 40 and 100 seconds after the addi-
tion of pyrogallol measured (Kar and Mishra, 1976).
Determination of non-enzymatic antioxidants
Anthocyanin
Total anthocyanin was determined according to
modified Wagner (1979) method using acidified ethanol
(ethanol: HCl 99: 1 v/v) as 0.05 g of frozen leaf was
homogenized in 2.5 ml acidified ethanol and then kept
at 25°C for 24 h in the dark. The extract was centrifuged
at 4000 g for 10 min at room temperature. The absorb-
ance of each supernatant was read at 550 nm. The ex-
tinction coefficient 33,000 (mol-1cm-1) was used to
calculate the amount of total anthocyanin and it was
expressed as μmolg-1FW (Wagner, 1979).
Phenol
0.05 g of lyophilized cells were in 3 ml of ho-
mogenized 80% methanol and 3 hours in Ben Murray at
70° C was placed. Then it was centrifuged at 9000 x g
for 15 minutes and methanol extract was separated from
the sediment. Methanol extracts to measure total phe-
nols, flavonoids and flavonoids were used.
To 50 µl of methanol extract, 500 µl Folin -
Ciocalteau solution was added. After five minutes 500
ml of 7% sodium carbonate solution was added to the
mixture and absorption at 765 nm was read after 10
minutes. Finally, total phenol content in mg/g dry
weight was measured (Singleton and Rossi, 1965).
Flavonol
For total flavonol assay 1 mL of methanolic
extract, 1 mL of aluminum chloride solution and 3 mL
of sodium acetate were mixed and absorbance was read
at 445 nm. Rutin was used as a standard for calibration
1042
curve (Akkol et al., 2008).
Flavonoid
For total flavonoid assay1 mL of methanolic
extract, 250 μL of aluminum chloride solution and 250
μL of potassium acetate were mixed and the samples
remained at room temperature for 30 min. The absorb-
ance of their action mixture was measured at 415 nm
with the UV-visible spectrophotometer 1601 model
(Akkol et al., 2008).
Statistical analysis
This experiment was performed with three repli-
cations in factorial design. Data analysis were per-
formed using SAS, version 15 and MSTAT-C statistical
software. Compared using the Duncan test at the 5%
level, and the graphs were drawn by Excel software
(Bazargan, 2017).
RESULTS
Mycorrhizal colonization in root
The results of this study showed that the roots
mycorrhizal colonization percentage decreased in 200
and 800 ppm cadmium chloride concentrations but
brassinosteroid treatment significantly increased this
parameter in all levels of cadmium chloride. The highest
percentage of root mycorrhizal colonization was in
brassinosteroid treatment and the lowest was in 800
ppm of cadmium chloride treatment (Figure 1 and 2,
Image 1-3).
Hydrogen peroxide
The results of this study showed that cadmium
chloride caused a significant increase in the amount of
hydrogen peroxide. Interactions of brassinosteroid,
G. mosseae and G. mosseae + brassinosteroid with all
levels of cadmium chloride significantly reduced this
parameter (Figure 3).
Catalase
Results of this study showed that catalase activi-
ty increased significantly in 200 and 800 cadmium chlo-
ride treatments. Treatment with brassinosteroid and
Journal of Research in Ecology (2017) 5(2): 1033–1051

G. mosseae + brassinosteroid increased significantly
this parameter. In the interactions of brassinosteroid,
G. mosseae and Glomusmosseae + brassinosteroid with
200 and 800 ppm of cadmium chloride the activity of
this enzyme significantly decreased (Figure 4).
Ascorbate peroxidase
The results of this study showed that ascorbate
peroxidase activity increased significantly in 200 ppm
cadmium chloride and G. mosseae treatments groups. In
interactions of brassinosteroid, G. mosseae and
G. mosseae + brassinosteroid with 100 and 800 ppm of
cadmium chloride the activity of this enzyme increased
significantly (Figure 5).
Superoxide dismutase
Results of this study showed that the activity of
superoxide dismutase increased significantly in the 200
and 800 ppm cadmium chloride, brassinosteroid, plants
G. mosseae and G. mosseae + brassinosteroid treat-
ments. In interactions of brassinosteroid, Glomus
mosseae, G. mosseae + brassinosteroid with all levels of
cadmium chloride activity of this enzyme significantly
decreased (Figure 6).
Polyphenol oxidase
The results of this study showed that activity of
the polyphenol oxidase enzyme increased significantly
in the 200 and 800 ppm cadmium chloride, G. mosseae
and G. mosseae + brassinosteroid. In interactions of
brassinosteroid, Glomus mosseae, G. mosseae + brassi-
nosteroid with all levels of cadmium chloride the activi-
ty of this enzyme significantly increased (Figure 7).
Peroxidase
Results of this study showed that of peroxidase
activity significantly increased in all levels of cadmium
chloride. In interaction of brassinosteroid and all levels
of cadmium chloride the activity of this enzyme signifi-
cantly increased. In interaction of G. mosseae + brassi-
nosteroid and G. mosseae with 100 and 200 ppm of cad-
mium chloride the activity of this enzyme significantly
increased (Figure 8).
Journal of Research in Ecology (2017) 5(2): 1033–1051
Hajbagheri et al., 2017
Anthocyanin
Anthocyanin content increased significantly in
interactions of G. mosseae + brassinosteroid,
G. mosseae and brassinosteroid with 0, 100 and 200
ppm of cadmium chloride (Figure 9).
Flavonoid
Flavonoid content increased significantly in
interactions of G. mosseae + brassinosteroid,
G. mosseae and brassinosteroid with 0, 100 and 200
ppm of cadmium chloride. In the other hand this param-
eter reduced significantly in all treatment group with
800 ppm cadmium chloride (Figure 10).
Flavonol and Phenol
The results of this study showed that flavonol
and phenol content increased significantly in
G. mosseae and G. mosseae + brassinosteroid. But re-
duced significantly in brassinosteroid, G. mosseae and
G. mosseae + brassinosteroid with all concentration of
cadmium chloride (Figures 11 and 12).
DISCUSSION
One of the most important indicators of mycor-
rhizal fungi activity is the colonization percentage that
is affected by several factors such as appearance and
structural properties of the root system, the quantity and
quality of root exudates, the use of fertilizers and of
high concentrations of heavy metals (Al –Karaki, 2000).
In general, the negative effects of high concentrations of
heavy metals on root colonization with mycorrhizal
fungi have been observed in the root system of plants.
For example, it has been reported that the spores of Glo-
mus intraradices fungus do not germinate in 1 mM or
10 mM cadmium concentration (Pawlowska and Char-
vat, 2004).
In this study, cadmium toxicity reduced the my-
corrhizal contamination of anisum root with G. mosseae
fungus. In mycorrhizal communities, the fungus is en-
tirely dependent on the plant growth and nutrition’s pro-
duction in the host plant. Thus, any factor which affects
1043
Hajbagheri et al., 2017
the production of carbohydrates and its transfer to the
roots can effect on the amount of contamination. Since
heavy metal stress reduced the plant growth, it can also
reduce the mycorrhizal inoculation (Asghari, 2008). In
the same reports, the mycorrhizal contamination was
reduced under cadmium toxicity in wheat plants that
inoculated with Glomus intraradices (Jamalabad and
Khara, 2008) and Cajanus cajan plant inoculated with
G. mosseae (Garg and Bhandari, 2012).
Plant hormones play an important role in
mycorrhizal symbiosis. Plant hormones or phytohor-
mones are involved in signaling pathways and regula-
tion of this symbiosis at different stages of plant growth
(Jutta, 2000). In this study, brassinosteroids has a stimu-
lating role in mycorrhizal inoculation under control and
cadmium toxicity conditions. Some experiments have
shown that brassinosteroids, gibberellin and cytokinine
are needed for the early stages of root mycorrhizal con-
tamination and are not involved in the next stages of
coexistence (Hanlon and Coenen, 2011).
The results of this study showed that the protein
content significantly decreased in 800 ppm of cadmium
chloride. Some researchers believed that protein reduc-
tion could be due to the induction of oxidative stress,
proteases activity, changes in the proteins structure and
function through denaturation and fragmentation and the
replacement of metals in the thiol groups of protein
(Rastgoo and Alemzadeh, 2011). Cadmium stress
changed protein structure or replacing a vital element
for the activities of essential enzymes and protein con-
tent of the plants. The heavy metal inhibits sulfydryl
groups of structural proteins and guides the enzymes to
incorrect folding and activity inhibitory. This compo-
nent reduced the production of proteins and stops the
growth with a disorder in nitrogen metabolism through
inhibiting the activity of enzymes such as glutamine
synthase, glutamate synthase, nitrate reductase and ni-
trate regeneration process. Ghani and Wahid (2007)
showed that some heavy metals such as lead, cobalt,
1044
cadmium, and magnesium reduced protein content in
maize, this may be due to the very close relationship
between heavy metals types and the side-chain of pro-
teins (Ghani and Wahid, 2007). Doganlar et al. (2012)
reported that the protein content reduced in high con-
centrations of manganese and nickel in Lemna and this
effect may be due to the protein degradation caused by
oxidative stress (Doganlar et al., 2012). In the sunflower
that treated with cadmium, oxidized proteins were gath-
ered in cotyledons and leaves and the protease activity
increased significantly (Pena et al., 2007).
In this study the pre-treatment of plants with
brassinosteroids, the inoculated plants with G. mosseae
and the interaction of G. mosseae and brassinosteroids,
the shoot protein increased at 200 and 800 ppm concen-
tration of cadmium chloride but the root protein content
increased at 100 and 200 ppm concentrations of cadmi-
um chloride. Studies showed that brassinosteroids have
a positive effect on the processes of DNA and RNA
modeling and protein synthesis to enhance the plant
growth. Fariduddin et al. (2009) reported that the use of
brassinosteroids increased the amount of protein in cab-
bage under copper stress (Fariduddin et al., 2009). In
Lycopersicon esculentum plants under salinity stress,
the interaction of 24-Epibrassinolide and putrescine
increased the protein content and antioxidant enzymes
and reduce the harmful effects of salinity stress (Sharma
et al., 2012).
Based on some studies insoluble glycoprotein
synthesis is one of the resistant mechanisms in mycor-
rhizal plants against heavy metals such as cadmium.
These proteins have link places with heavy metals and
have a positive role for plant protection by linking to
heavy metals. (Trotta et al., 2006). In a similar report on
wheat plant that was inoculated with Glomus intra-
radices under cadmium stress, the sugars and total pro-
teins content increased in comparison with non-
inoculated plants (Jamalabad and Khara, 2008). Also the
use of arbuscular mycorrhizal fungi in Anadenanthera
Journal of Research in Ecology (2017) 5(2): 1033–1051

colubrine increased the proteins production (Pedone-
Bonfim et al., 2012). Enteshari et al. (2012) reported
shoot and root protein content increased in mycorrhizal
sweet basil plants compared to non-mycorrhizal plants
under salinity stress conditions (Enteshari et al., 2012).
Heavy metals can be catalyzed and accelerated
reactive oxygen species. Hydrogen peroxide is the most
stable molecule of the active oxygen species. Heavy
metal ions can stimulated the metabolism of free radi-
cals and alter the concentration of H2O2 (Shaw et al.,
2004). In this study, the toxicity of cadmium increased
H2O2 levels this result was according with Belkadhi et
al., 2014 on Linum usitatissimum (Belkadhi et al.,
2014).In the other hand inoculation with mycorrhiza
(G. mosseae) or brassinosteroid treatment decreased
H2O2 content by increasing enzymatic and non-
enzymatic antioxidant parameters. In the same reports,
24-epibrassinolide in Brassica juncea under cold stress
(Kumar et al., 2010) and in mycorrhizal cucumber plant
under low temperature stress (Liu et al., 2014) H2O2
content were reduced.
The antioxidants compounds are considered as
important defensive systems in plants against oxidative
stress that induced by heavy metals (Hasan et al., 2011).
In this study, the activity of catalase, superoxide dis-
mutase, polyphenol oxidase, ascorbate peroxidase and
peroxidase, especially at high concentrations of cadmi-
um chloride increased. In the root and leaves of radish,
catalase and glutathione reductase activity increased
after 13 hours of exposure to cadmium (Vitoria et al.,
2001). Cadmium increased the superoxide dismutase
activity in sunflower (Laspina et al., 2005). In Ara-
bidopsis plant under cadmium toxicity FeSOD and
MnSOD activity were increased (Drazkiewicz et al.,
2007).
In this study brassinosteroid increased catalase,
superoxide dismutase and polyphenol oxidase activity.
The treatment of tomato with 24-epibrassinolide and 28-
Homo epibrassinolide increased the photosynthesis,
Journal of Research in Ecology (2017) 5(2): 1033–1051
Hajbagheri et al., 2017
antioxidant enzyme activities and proline content
(Hasan et al., 2011). Brassinosteroids application on
tomato plants that against cadmium photosynthesis,
antioxidant enzymes activity, transcription levels, anti-
oxidant system and secondary compounds metabolism
significantly increased these results showed that brassi-
nosteroids had positive role in the detoxification of cad-
mium stress (Ahammed et al., 2012).
In this research the activity of ascorbate peroxi-
dase, peroxidase and polyphenol oxidase increased in
inoculation of plants with G. mosseae or interaction
between mycorrhizal plants and brassinisteroid. This
study showed that inoculated plants against cadmium
toxicity were mainly related to the effect of this fungi on
the ascorbate peroxidase and polyphenol oxidase.
Glomus mosseae mycorrhiza significantly in-
creases the activity of superoxide dismutase, catalase
and peroxidase in the Cajanus cajan (Garg and
Bhandari, 2012). In inoculated tomatoes with
G. mosseae under salinity stress (Abdel latef and
Chaoxing, 2011) and pomegranates inoculated plants
with Glomus intraradices under oxidative stress under
dehydration (Bompadre et al., 2014), the activity of
superoxide dismutase, catalase, peroxidase and
ascorbate peroxidase was increased. In
Lycopersicon esculentum plants under salinity stress,
the interaction of 24-Epibrassinolide and putrescine
increase the activity of catalase, superoxide dismutase
and guaiacol peroxidase enzymes (Sharma et al., 2012).
The evidences showed that there is a relation-
ship between phenol content and polyphenol oxidase
activity in plants, when the total concentration of phenol
increased in the plants and, the polyphenol oxidase ac-
tivity also increased subsequently (Tegelberg et al.,
2004). Also in this research showed that the polyphenol
oxidase activity increased by the increase in the phenol-
ic compounds content in anisum. Likely, ascorbate pe-
roxidase activity reduction in high concentrations of
cadmium chloride is related to the inactivation of this
1045
Hajbagheri et al., 2017
enzyme by the excessive production of active oxygen
species, non-specific degradation, destruction, or bind-
ing of unnecessary heavy metals to the enzyme (Sharma
et al., 2012). Reactive oxygen species production in
cadmium stress conditions increase the lipid peroxida-
tion, caused oxidative stress and damage the antioxida-
tive enzymes (Filek et al., 2008).
Another important plant defense system to con-
trol and neutralize free radicals is inducing the synthesis
of some non-enzymatic antioxidants such as anthocya-
nin and phenolic compounds. Generally, the consistency
with stress depends on the reduction of reactive oxygen
species by the antioxidant system (Abdul-Jaleel et al.,
2009). In this study, anthocyanin content in cadmium
chloride treatments showed no significant difference,
This result cannot be attributed to the less resistance of
anisum against the of cadmium toxicity, because the
plant may use other resistant mechanisms such as oth-
er phenolic compounds likely flavonoid production. On
the other hand, in plants treated with 24-epibrassinolide
anthocyanin content increased. Brassinosteroids activat-
ed some enzymes such as phenylalanine ammonia lyase
and Chalcone Synthase (CHS) and other key enzymes in
the anthocyanin synthesis pathway (Campos et al.,
2003).
In the same report, the use of 24-epibrassinolide
in grape can increase the activity of phenylalanine am-
monia lyase enzyme or increased phenols, flavonoids,
tannins, and anthocyanin content (Xi et al., 2013). In
grapes treated with brassinosteroids, a considerable in-
crease of anthocyanin concentration was observed in
comparison with the control plant. Because of this in-
crease, the impact of brassinosteroids on structural and
regulatory genes is related to anthocyanin biosynthesis
(Luan et al., 2013). In this study, the 24-epibrassinolide
probably increased the amount of anthocyanins through
this mechanism.
In pretreated plants with mycorrhizal or
brassinosteroids and mycorrhizal fungi under the cadmi-
1046
um stress, anthocyanin content increased. It was report-
ed that the mycorrhizal inoculation with
Glomus etunicatum fungus significantly increased an-
thocyanin content in sweet basil compared to non-
mycorrhizal plants (Sharifi et al., 2011). Phenylalanine
ammonia lyase and chalcone isomerase enzymes are
involved in the anthocyanin and 3-doxyanthocyanidin (a
kind of phytoestrogen auxin), biosynthesis so that the
fungal infection or brassinosteroids severely induced
these enzyme’s gene expression (Tripathi et al., 2006). It
was reported that the inoculation of plant with mycor-
rhizal fungi increased the concentration of anthocyanin
in strawberry (Lingua et al., 2013) and lettuce (Baslam
et al., 2013) and is considered as a reliable method to
enhance the nutritional value of edible and medicinal
plants consistent with the results in the present study.
The results of this study showed that the flavo-
noids, flavonols and phenolic compound in cadmium
stress increased significantly. It seems that this plant
under cadmium stress increased phenolic compounds in
order to have good defense reactions against the stress.
Many flavonoids are an active component of secondary
metabolites from medicinal plants and their medicinal
properties. They have an important role in plants re-
sistance as physiologically active compounds, protective
factors against stress and also light absorbers (Tattini et
al., 2004). Phenolic compounds production in anisum
under cadmium stress may be due to the induction of
genes expression or the enzyme synthesis stimulation
of these compounds in the phenylpropanoids pathway.
The increase of the soluble phenolic compound content,
especially lignin precursors alongside the increase of the
cell wall thickness creates a biological barrier to pre-
vent entry of heavy metals into living cells and increases
their tolerance to these metals. These compounds direct-
ly prevent the oxidative stress by entering into the re-
ducing reactions or indirectly by chelating the cadmium.
High levels of phenolic compounds in beans have also
been reported as the answer to cadmium toxicity
Journal of Research in Ecology (2017) 5(2): 1033–1051

(Michalak, 2006)
The treatment of anisum with brassinosteroid
increased flavonoids and phenols content in low concen-
trations of cadmium chloride. One of the causes of re-
ducing the effects of oxidative stress in plants treated
with brassinosteroids is the effects of this substance on
the synthesis of substances such as flavonoids. Brassi-
nosteroids at these concentrations reduced the toxic ef-
fects of cadmium chloride by affecting the antioxidant
system. In a similar report, in Cichorium endivia L.
plants treated with brassinosteroid the antioxidant com-
pounds and total phenolics increased (Serna et al.,
2013). Brassinosteroids produce phenolic and phenol
acts as a barrier against moisture loss 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
amount of flavonoids by mycorrhizal fungi is not yet
clear, it was observed that Mycorrhizal fungi does that
by increasing enzyme activity and gene expression in a
signaling cascade (Zhang et al., 2013). Also, the role of
flavonoids of the host plant root in the regulation of
mycorrhizal symbiosis is well known. Flavonoids stim-
ulate the spore germination, mycelial growth of mycor-
rhizal fungi and root contamination by arbuscular
mycorrhizal fungi. As a result, it is certain that the in-
crease in the amount of flavonoids is observed during
the mycorrhizal symbiosis. In another report, Abbaspoor
et al. (2012) showed that the flavonoids content in pista-
chios mycorrhizal plants was higher when compared to
non- mycorrhizal plants under drought stress
(Abbaspour et al., 2012).
CONCLUSION
The results of this study showed that the induc-
Journal of Research in Ecology (2017) 5(2): 1033–1051
Hajbagheri et al., 2017
tion of the activity of catalase, superoxide dismutase,
peroxidase and polyphenol oxidase, and the amount of
flavonoids, flavonols and phenol in anisum due to cad-
mium toxicity improved the antioxidative defense sys-
tem. The treatment of anisum with brassinosteroids and
inoculation with G. mosseae improves the host plant
growth by providing more plant resistance to cadmium
toxicity stress.
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