Responses of Components of Antioxidant System in Moongbean Genotypes to Cadmium Stress
Transcript of Responses of Components of Antioxidant System in Moongbean Genotypes to Cadmium Stress
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Responses of Componentsof Antioxidant System inMoongbean Genotypes toCadmium StressNaser Aziz Anjum a , Shahid Umar a , Altaf Ahmad a
& Muhammad Iqbal aa Department of Botany, Faculty of Science,Hamdard University, New Delhi, IndiaVersion of record first published: 28 Aug 2008.
To cite this article: Naser Aziz Anjum , Shahid Umar , Altaf Ahmad & MuhammadIqbal (2008): Responses of Components of Antioxidant System in MoongbeanGenotypes to Cadmium Stress, Communications in Soil Science and Plant Analysis,39:15-16, 2469-2483
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Responses of Components of Antioxidant System inMoongbean Genotypes to Cadmium Stress
Naser Aziz Anjum, Shahid Umar, Altaf Ahmad, and Muhammad Iqbal
Department of Botany, Faculty of Science, Hamdard University,
New Delhi, India
Abstract: Four genotypes (Pusa 9531, Pusa 9072, Pusa Vishal, PS-16) of
moongbean [Vigna radiata (L.) Wilczek] grown in earthen pots were treated with
cadmium at 0, 25, 50, and 100 mg kg21 soil. Cadmium tolerance (CdT), the
ability of a plant to maintain growth at high levels of cadmium (Cd), was
calculated as the ratio of dry-matter production in the untreated and the Cd-
treated soils. The moongbean genotypes showed a differential response to Cd
concentrations; Pusa 9531 was identified as Cd tolerant, whereas PS 16 was
Cd susceptible. To find out the physiological basis of these differences, we
investigated the possible role of antioxidant (enzymatic and nonenzymatic)
defense systems. Activities of superoxide dismutase (EC 1.15.1.1), catalase (EC
1.11.1.6), ascorbate peroxidase (EC 1.11.1.11), and glutathione reductase
(EC 1.6.4.2) and the amounts of ascorbate and glutathione were monitored in
the Cd-tolerant and Cd-sensitive moongbean genotypes. The results revealed
the presence of a strong antioxidant defense system in the Cd-tolerant genotype
(Pusa 9531) for providing adequate protection against oxidative stress caused
by Cd.
Keywords: Antioxidative defense system, cadmium tolerance index, Vigna radiata
Received 27 March 2007, Accepted 1 October 2007
Address correspondence to Shahid Umar, Department of Botany, Faculty of
Science, Hamdard University, New Delhi, 110 062, India. E-mail: s_umar9@
hotmail.com
Communications in Soil Science and Plant Analysis, 39: 2469–2483, 2008
Copyright # Taylor & Francis Group, LLC
ISSN 0010-3624 print/1532-2416 online
DOI: 10.1080/00103620802292871
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INTRODUCTION
Plants are normally exposed to a wide range of environmental stress,
which limits their growth and development. Natural as well as the
agricultural soil may have toxic levels of heavy metals due to mining,
smelting, waste disposal, and agricultural practices (such as excessive
use of fertilizers). Cadmium (Cd), which is discharged into the soil at
the rates of 22,000 tons per year globally (Nriagu and Pacyna 1988), is
one of the most toxic nonessential and mobile metallic elements found
in soils (Prasad et al. 2001; Mehindirata et al. 2000; Qadir et al. 2004;
Khudsar, Mahmooduzzafar, and Iqbal 2001). Cadmium damages root
tips, reduces nutrient and water uptake, and impairs photosynthesis,
thus inhibiting the growth of the plants (Iqbal and Khudsar 2000;
Ghoshroy and Nadakavukaren 1990). Cadmium becomes toxic to
plants through irreversible changes to protein conformation by forming
metal thiolate bonds and through alteration of the cell wall and
membrane permeability by binding to nucleophilic groups (Ramos et al.
2002).
Cadmium produces oxidative stress possibly by generating free
radicals and reactive oxygen species (ROS) (Hendry, Baker, and Ewart
1992). These species react with lipids, proteins, pigments, and nucleic
acids and cause lipid peroxidation and membrane damage. Plants cope
with the oxidative stress by using a battery of antioxidant components
composed of enzymes such as superoxide dismutase (SOD), ascorbate
peroxidase (APX), glutathione reductase (GR), peroxidase (POX),
catalase (CAT), and the low-molecular-weight antioxidants such as
ascorbic acid and glutathione (Schutzendubel et al. 2002; Olmos et al.
2003; Bashir et al. 2007; Qureshi et al. 2007). Treatment with Cd results in
cellular oxidative damage or lipid peroxidation (Chien et al. 2002; Dixit,
Pandey, and Shyam 2001). It can either inhibit or stimulate the activities
of antioxidant enzymes (Leon et al. 2002; Iannelli et al. 2002).
Identification of genotype(s) that can withstand the Cd toxicity would
be beneficial in areas with high Cd levels.
Differential genotypic responses to Cd stress have been studied in
crop plants such as barley (Wu, Zhang, and Dominy 2003), wheat
(Ozturk, Eker, and Ozkutlu 2003), rice (Hassan, Shao, and Zhang 2005),
and Brassica sp. (Qadir et al. 2004). However, such information with
reference to legumes is sparse. The present study examines the behavior
of ROS-scavenging enzymes (SOD, CAT, APX, and GR) and low-
molecular-mass antioxidants (GSH and AsA) in four genotypes (Pusa
9531, Pusa 9072, Pusa Vishal, and PS-16) of moongbean [Vigna radiata
(L.) Wilczek]. Our research objectives were (a) to identify the Cd-tolerant
moongbean genotype(s) and (b) to understand the physiological
mechanisms that confer Cd tolerance in those genotype(s).
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MATERIALS AND METHODS
Experimental Materials, Experimental Procedure, and Soil Characteristics
Moongbean genotypes (Pusa 9531, Pusa 9072, Pusa Vishal, PS-16) were
grown in earthen pots (120 6 120) filled with 10 kg of soil. The soil used
was sandy loam in texture, with a pH of 7.8 and an electrical conductivity
(EC) of 0.38 dsm. The soil contained 0.43% organic carbon (C),
70 mg kg21 available potassium (K), 5 mg kg21 available sulfur (S), and
0.15% extractable calcium chloride (CaCl2). Cadmium was applied as
cadmium chloride (CdCl2) to the containers at the rates of 0, 25, 50, and
100 mg Cd kg21 soil, further identified as T0, T1, T2, and T3, respectively.
The 0 mg Cd kg21 treatment (T0) served as control. Cadmium was
applied once, 20 days after sowing (DAS), by adding 250 mL of the
different CdCl2 solutions to the soil. Nitrogen (N), phosphorus (P), and
K were applied at rates of 120, 30, and 80 mg kg21 soil, respectively, in
the form of urea, singlesuperphosphate, and muriate of potash (KCl) at
the time of sowing. Three plants per pot were maintained until 30 DAS.
Pots were kept in the greenhouse of the Department of Botany, Hamdard
University, New Delhi, India, under semicontrolled condition. A
polythene plastic film was used to thwart the effects of rainfall, which
allowed the transmittance of 90% of visible wavelength (400–700 nm)
light under natural day and night conditions with a day/night
temperature 25/20 ¡ 4 uC and relative humidity of 70 ¡ 5%. The
experiments were set up in a completely randomized design with three
replicates.
Cadmium Content and Cd-Tolerance Index (Cd-TI)
Moongbean genotypes varied highly in their ability to accumulate Cd in
leaves, and the magnitude of Cd accumulation was treatment dependent
(Table 1). The Cd-tolerance index was calculated using the data on dry-
matter accumulation obtained with 100 mg Cd kg21 soil treatment and
the control (Table 2), as used by Khan et al. (2006), and expressed in
percentage as Cd-TI (%) 5 [dry-matter yield with highest Cd treatment /
dry-matter yield with control] 6 100.
Biomass and Leaf Area Measurements
The visual foliar symptoms were recorded every day. The leaf area of the
whole plant was measured for each treatment with the help of portable
leaf area meter (Model LI COR 3000A, Li-COR, Lincoln, Neb.). For the
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dry-matter measurements, plants were harvested in four replicates,
weighed, and dried in a hot-air oven at 65 ¡ 2 uC for 48 h. Thereafter,
their dry weight (DW) was determined on a digital balance by the unitary
method and expressed in g plant21.
Estimating Antioxidant Metabolites and Enzyme Assay
For estimation of antioxidants (enzymatic and nonenzymatic), leaves
were collected from four plants of each treatment in the morning (9–11
a.m.), washed with distilled water, and dried. The blot-dried leaf material
(0.5 g) was homogenized in 0.1 M phosphate buffer (pH 7.5) with 0.5 mM
ethylenediaminetetraacetic acid (EDTA) and 1 mM ascorbic acid (for
superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase
(APX), and glutathione reductase (GR) activity) at 4 uC. The
homogenate was centrifuged at 15,000 rpm for 20 min, and the super-
natant fraction was used for all enzyme assays. Total homogenized
Table 1. Values of the whole plant dry matter and the cadmium tolerance index
(Cd-TI) of four moongbean genotypes grown for 10 days in Cd+ and Cd2
(control) soil conditions
Genotypes Dry matter (g)
Control Cd+ Cd-TI (%)
Pusa 9531 1.82 1.25 90.10
Pusa Vishal 1.67 1.00 59.88
Pusa 9072 1.75 1.10 62.86
PS-16 1.56 0.85 54.48
Note. Values represent the means of four replicates.
Table 2. Cadmium content of leaves of the control and the Cd-treated Vigna
radiata (L.) Wilczek genotypes at 30 days after sowing
Genotypes Cadmium content (mg g21 DW)a
T0 T1 T2 T3
Pusa 9531 0.002 0.17 0.63 0.99
Pusa Vishal 0.001 0.29 0.83 1.13
Pusa 9072 0.002 0.21 0.70 1.05
PS-16 0.001 0.35 0.88 1.20
aT0, T1, T2, and T3 represent Cd treatment at the rate of 0, 25, 50 and
100 mg Cd kg21 soil, respectively.
Note. Values represent the means of three replications.
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solution (0.5 mL) was used for protein determination. The soluble protein
content was estimated in leaves by the method of Bradford (1976) using
bovine serum albumin (BSA, Merck, Germany) as the standard. Based
on the amount of protein per volume of the homogenized solution, the
following enzymes were assayed in volume containing a known protein
concentration to calculate the specific activities of the enzymes.
Estimation of Total and Oxidized Ascorbate
Estimation of total ascorbate was done by the method of Law, Charles,
and Halliwell (1983). Fresh leaf material (0.5 g) was ground in extraction
buffer (2 mL) and centrifuged at 10,000 rpm for 10 min. The supernatant
was collected and used for the assay immediately. The amount, calculated
in relation to fresh weight (FW), was expressed as nmol g21 FW.
Estimation of Total Glutathione
Estimation of total glutathione was done by the method of Anderson
et al. (1985). Standard curve for calculations was prepared from reduced
glutathione (GSH), covering a range of 10–100 nmol. The amount of
total GSH was expressed as nmol g21 FW.
Measurement of Superoxide (EC 1.15.1.1) Activity
The superoxide dismutase (EC 1.15.1.1) activity was estimated at
wavelength of 560 nm on a UV-vis spectrophotometer (Model Lambda
Bio 20, Perkin-Elmer, Waltham, Mass., USA) following the method
described by Dhindsa, Plumb-Dhindsa, and Thorpe (1981). One enzyme
unit was defined as the amount of enzyme that produced a 50% inhibition
of nitroblue tetrazolium (NBT) reduction during the process of assay.
The SOD activity was expressed as EU mg21 protein.
Measurement of Ascorbate Peroxidase (EC 1.11.1.11) Activity
The activity of ascorbate peroxidase (EC 1.11.1.11) was estimated by the
method used by Nakano and Asada (1981). The APX activity was
calculated by using extinction coefficient (e) 2.8 mM cm21 and expressed
in enzyme units (EU) mg21 protein. One unit of enzyme determines the
amount necessary to decompose 1 mmol of the substrate consumed per
min at 25 uC.
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Measurement of Catalase (EC 1.11.1.16) Activity
The activity of catalase (EC 11.11.11.6) in the leaves was determined by
the method of Aebi (1984). The enzyme activity was calculated by using
extinction coefficient (e), 0.036 mM cm21, and expressed in enzyme units
(mg21 protein). One unit of enzyme determines the amount necessary to
decompose 1 mmol of hydrogen peroxide (H2O2) per min at 25 uC.
Measurement of Glutathione Reductase (EC 1.6.4.2) Activity
Glutathione reductase (EC 1.6.4.2) activity was determined by the
method of Foyer and Halliwell (1976) modified by Rao (1992). The
activity was calculated by using extinction coefficient (e), 6.2 mM cm21,
and expressed in enzyme units mg21 protein. One unit of enzyme
determines the amount necessary to decompose 1 mmol of nicotinamide
adenine dinucleotide phosphate (NADPH) per min at 25 uC.
Determination of Cd in Leaves
For the determination of Cd, the fine powder of dried leaves (0.1 g) was
acid digested by sulfuric acid (H2SO4)–nitric acid (HNO3), 2:1 v/v. The
volumes of the digested samples were made equal by adding Mili Q water
and filtered with Whatman’s filter paper. The content of Cd in digested
samples was estimated by atomic absorption spectrometer fitted with
wall-type graphite tubes (AAS ZEEnit 65, Analytikjena, Germany).
The significance of differences was analyzed for all the parameters
studied, using the analysis of variance (ANOVA) technique as described
by Gomez and Gomez (1984).
RESULTS
Plant Growth and Development
Plant growth was reduced with each of the Cd treatments, with the
magnitude of growth reduction depending on Cd concentration. The
visible symptoms of Cd toxicity in the form of characteristic reddish
brown spots on primary and secondary leaves, and as the blackening,
browning, burning, and weakening of stems and leaves were observed at
the highest Cd concentration (100 mg Cd kg21 soil). Visual toxicity
symptoms were more prominent in genotypes PS-16 and Pusa Vishal
than in Pusa 9531 and Pusa 9072. Cd TI varied between 54.48% (PS-16)
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and 90.10% (Pusa 9531) (Table 1). Moongbean genotypes varied highly
in their ability to accumulate Cd in leaves, and the magnitude of Cd
accumulation in leaves was treatment dependent (Table 2).
The dry-matter production decreased significantly with each Cd
treatment in all four moongbean genotypes (Figure 1A), with the
decrease being treatment dependent. The maximum decrease in dry-
matter production was observed in PS-16 (21–45%) followed by Pusa
Vishal (15–40%), and the minimum was noted in Pusa 9531 (9–31%),
followed by Pusa 9072 (7–36%), as compared with the control treatment.
The leaf area also decreased in all the genotypes with all the Cd
treatments (Figure 1B), and the decrease was treatment dependent. The
decrease was maximum (7–45%) in genotype PS-16, followed by Pusa
Vishal (6–26%), and minimum (2–19%) in Pusa 9531, followed by Pusa
9072 (5–21%), as compared with the control treatment.
Antioxidant Metabolites
The GSH content also declined, showing a maximum decrease in PS-16
and Pusa Vishal (18–37% and 14–30%) and a minimum in Pusa 9531 and
Pusa 9072 (7–20% and 9–26%) (Figure 1C). Ascorbic acid content
decreased significantly in all the genotypes with increasing Cd
concentrations (Figure 1D). A significant decrease in AsA content was
observed in all the moongbean genotypes with each Cd treatment.
Maximum decline took place in PS-16 and Pusa Vishal (39–70% and 36–
69%) and the minimum was in genotypes Pusa 9531 and Pusa 9072 (26–
55% and 31–65%), respectively, over the control treatment (Figure 1D).
The maximum decline was seen in PS-16 (24–50%), followed by Pusa
Vishal (22–49%), whereas the minimum was in Pusa 9531 (16–43%),
followed by Pusa 9072 (19–44%), with respect to the control treatment.
The decrease in the AsA and GSH contents may be regarded as evidence
of a decline in the availability of reducing equivalents required for
regeneration of important antioxidants to scavenge the Cd-induced ROS.
Antioxidant Enzymes
The SOD activity decreased with T1 in all the genotypes, but increased
with T2 and T3. The maximum increase was observed in Pusa 9531 (31–
56%) and Pusa 9072 (26–49%) and the minimum in Pusa Vishal (10–44%)
and PS-16 (6–43%), over the respective controls (Figure 2A).
APX activity increased in all the genotypes with all the treatments
(Figure 2B) in a dose-dependent manner. The extent of increase was
maximum in genotypes Pusa 9531 (16–95%) and Pusa 9072 (12–64%) and
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Figure 1. Bar diagrams showing (A) dry-matter accumulation, (B) leaf area, (C)
GSH content, and (D) AsA content in leaves of the control and the Cd-treated Vigna
radiata (L.) Wilczek genotypes as observed at 30 days after sowing. Values represent
the means ¡ SE of three independent observations. (T0, T1, T2, and T3 represent Cd
treatment at the rate of 0, 25, 50, and 100 mg Cd kg21 soil, respectively.)
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Figure 2. Bar diagrams showing the activities of (A) superoxide dismutase, (B)
ascorbate peroxidase, (C) glutathione reductase, and (D) catalase in leaves of the
control and the Cd-treated Vigna radiata (L.) Wilczek genotypes as observed at
30 days after sowing. Values represent the means ¡ SE of three independent
observations. (T0, T1, T2, and T3 represent Cd treatment at the rate of 0, 25, 50,
and 100 mg Cd kg21 soil, respectively.)
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minimum in Pusa Vishal (6–40%) and PS-16 (5–39%), compared with the
control treatment.
GR activity increased significantly with increasing Cd concentrations
(Figure 2C). The maximum increase occurred for Pusa 9531 (54–103%)
followed by Pusa 9072 (25–92%), and the minimum occurred in Pusa
Vishal (20–89%) and PS-16 (16–68%), as compared with the control
treatment.
Increased CAT activity was detected with T1 and T3 in all the
genotypes. However, the activity decreased with T2, except in Pusa
Vishal. This decline with T2 treatment was maximum in genotype Pusa
9531 (2%) and minimum in Pusa 9072 (1%). On the other hand, the
increase with T1 and T3 was maximum in genotypes Pusa 9072 (8–17%)
and Pusa 9531 (5–20%) and minimum in genotypes Pusa Vishal (6–11%)
and PS-16 (5–8%), as compared to their respective controls (Figure 2D).
DISCUSSION
Plant tolerance to toxicity, calculated on the basis of growth (determined
by dry weight) of the whole plant, has long been an extensively used
parameter for assessing genotypic variation in tolerance to heavy metals
(Metwally et al. 2005; Khan et al. 2006). This study has demonstrated a
large variability in tolerance to Cd toxicity (CdT) among moongbean
genotypes. Pusa 9531 has been identified as a highly Cd-tolerant
genotype, and PS-16 was the most Cd-susceptible genotype of moon-
gbean. Observations indicate that Cd stress adversely affected the plant
antioxidant system in all the moongbean genotypes by inducing depletion
of the reducing equivalents.
In the present study, the intensity of color spots on the leaves,
induced overnight by the application of different Cd concentrations, was
dose-dependent, as observed in some earlier works. This could be due to
changes in metabolism of phenols and phenol-like compounds (Brisson et
al. 1977); however, the origin of color spots and the relation between
stimulation of phenolics and chasings in regulation parameters in the Cd-
treated plants require further experimental evidence.
Cadmium treatments altered the lower leaf area significantly. It was
suggested earlier that high concentrations of Cd cause premature
senescence in plants exposed for long periods (Iqbal and Khudsar
2000). The cell volume, as well as the rate of cell division, may be reduced
under heavy metal stress, thus providing a lesser area for photosynthesis
(Iqbal and Khudsar 2000; Ghoshroy and Nadakavukaren 1990; Chen
and Huerta 1997).
Plant growth, measured in terms of dry-matter production, was
significantly reduced. It was noted that Cd-treated plants produced more
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or less the same number of leaves, but a reduced leaf area occurred
wherever Cd treatments were applied. The leaves of plants treated with
high concentrations of Cd may have a direct effect on cell wall
components, causing chemical and physical changes and resulting in
more elasticity despite smaller cells (Chen and Huerta 1997).
To mitigate and repair the damage caused by free radicals, plants
have evolved complex antioxidant (both enzymatic and nonenzymatic)
systems. The primary constituents of the system include antioxidant
enzymes such as superoxide dismutase, catalases, and peroxidases and
free radical scavengers such as carotenoids, ascorbate, tocopherols, and
oxidized/reduced glutathione (Hassan, Shao, and Zhang 2005). The
present study has shown enhancement in SOD activity in all the Cd-
exposed genotypes, with the activity being more pronounced in Pusa 9531
and Pusa 9072. This may indicate an efficient resistant potential of these
genotypes against the Cd toxicity. Increased SOD activity has been
noticed in several instances for heavy-metal-stressed plant species
(Qureshi et al. 2007; Hassan, Shao, and Zhang 2005; Qadir et al.
2004). This could be due to a de novo synthesis of the enzymatic proteins
(Slooten et al. 1995; Allen, Webb, and Schake 1997).
APX, a hydrogen-peroxide-scavenging enzyme specific to plants and
algae, has a role in the protection of chloroplasts and other cell
constituents from the damage by H2O2 and its by-products. In the
present study, the APX activity was found to increase with increase in Cd
stress (Figure 2B). H2O2, produced in abundance through rapid
dismutation of superoxides by SOD, is efficiently converted into water
and oxygen molecules by APX activity using AsA as a reductant (Qureshi
et al. 2007; Qadir et al. 2004).
Alterations in the nonenzymatic cellular antioxidants (viz., AsA,
DHA, and glutathione) under Cd-induced oxidative stress are well
documented. The AsA content was reduced in all the Cd-exposed
moongbean genotypes. The activities of APX and SOD may be
correlated with the reductions in the total AsA content; greater APX
activity needed more substrate and hence consumed more AsA as an
electron donor, thus causing a decline in AsA concentration. Similar
variations in the AsA content have been reported for Cd-exposed Scots
pine (Schutzendubel et al. 2001) and Brassica genotypes (Qadir et al.
2004). The reduction in the total AsA content may again be correlated to
a decline in recycling of the reduced forms of ascorbic acid (DHA and
MDHA by DHAR and MDHAR, respectively) in the chloroplasts.
Glutathione acts as a disulphide reductant to protect thiol (-SH)
groups on enzymes, regenerates ascorbate, and reacts with singlet oxygen
and hydroxyl radicals (Noctor et al. 2002). It also participates in the
regeneration of ascorbate (AsA) from dehydro-ascorbate using the
enzyme dehydro-ascorbate reductase (E.C. 7.8.5.1). In such reactions,
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GSH is oxidized to glutathione disulphide (GSSG) and may be
regenerated by glutathione reductase (GR) in a NADPH-dependent
reaction. A number of reports are available regarding the depletion of
GSH content in various plants under Cd stress (Dixit, Pandey, and
Shyam 2001; Metwally et al. 2005). In the present study, both Cd-tolerant
(Pusa 9531) and Cd-susceptible (PS-16) genotypes showed a decline
of reduced glutathione (GSH) when exposed to Cd. However, the
decline was greater in Cd-exposed PS-16 genotype showing its high
sensitivity to Cd stress and drastic disturbances in the GSH-regeneration
mechanism.
GR activity maintained one of the important hydrophilic anti-
oxidants, the GSH. Another explanation of the high GR activity in the
present work may be that as a component of ascorbate–glutahione cycle
(AGC), GSH maintains the normal operation of AGC at a high rate to
detoxify ROS; it is also essential to keep GSH in a reduced form prior to
its incorporation into the synthesis of phytochelatins (PCs) (Cobbett
2000a, 2000b).
The increased expression of the ROS-scavenging enzymes (SOD,
CAT, APX, and GR) and the low-molecular-mass antioxidants (GSH
and AsA) in Cd-exposed plants might confer extra protection against
Cd stress, as observed earlier with other species (Wu, Zhang, and
Dominy 2003; Hassan, Shao, and Zhang 2005; Ozturk, Eker, and
Ozkutlu 2003).
CONCLUSIONS
In conclusion, the moongbean plants grown with CdCl2 may have a
concentration-dependent oxidative stress in leaves. Genotype Pusa 9531
possesses an efficient antioxidant defense system, which may provide
better protection against the Cd-induced oxidative stress. Further
investigation at the subcellular and molecular levels should provide a
deeper mechanistic insight of defense against Cd toxicity in the
moongbean genotypes.
ACKNOWLEDGMENTS
We thank Dr. Naresh Chandra and Dr. J. L. Tickoo, of Pulses Breeding
Section in the Division of Genetics, Indian Agricultural Research
Institute, New Delhi, for providing seeds of moongbean genotypes. The
research fellowship (DSW/HNF-18/2006) received from Hamdard
National Foundation (HNF), New Delhi, India, by N. A. Anjum is
gratefully acknowledged.
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