PLEASE SCROLL DOWN FOR ARTICLE

This article was downloaded by: [INFLIBNET India Order]On: 19 October 2010Access details: Access Details: [subscription number 920455929]Publisher Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Communications in Soil Science and Plant AnalysisPublication details, including instructions for authors and subscription information:http://www.informaworld.com/smpp/title~content=t713597241

Responses of Components of Antioxidant System in MoongbeanGenotypes to Cadmium StressNaser Aziz Anjuma; Shahid Umara; Altaf Ahmada; Muhammad Iqbala

a Department of Botany, Faculty of Science, Hamdard University, New Delhi, India

To cite this Article Anjum, Naser Aziz , Umar, Shahid , Ahmad, Altaf and Iqbal, Muhammad(2008) 'Responses ofComponents of Antioxidant System in Moongbean Genotypes to Cadmium Stress', Communications in Soil Science andPlant Analysis, 39: 15, 2469 — 2483To link to this Article: DOI: 10.1080/00103620802292871URL: http://dx.doi.org/10.1080/00103620802292871

Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf

This article may be used for research, teaching and private study purposes. Any substantial orsystematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply ordistribution in any form to anyone is expressly forbidden.

The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae and drug dosesshould be independently verified with primary sources. The publisher shall not be liable for any loss,actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directlyor indirectly in connection with or arising out of the use of this material.

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

2469

Downloaded By: [INFLIBNET India Order] At: 05:42 19 October 2010

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).

2470 N. A. Anjum et al.

Downloaded By: [INFLIBNET India Order] At: 05:42 19 October 2010

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

Moongbean Response to Cadmium Stress 2471

Downloaded By: [INFLIBNET India Order] At: 05:42 19 October 2010

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.

2472 N. A. Anjum et al.

Downloaded By: [INFLIBNET India Order] At: 05:42 19 October 2010

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.

Moongbean Response to Cadmium Stress 2473

Downloaded By: [INFLIBNET India Order] At: 05:42 19 October 2010

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)

2474 N. A. Anjum et al.

Downloaded By: [INFLIBNET India Order] At: 05:42 19 October 2010

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

Moongbean Response to Cadmium Stress 2475

Downloaded By: [INFLIBNET India Order] At: 05:42 19 October 2010

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.)

2476 N. A. Anjum et al.

Downloaded By: [INFLIBNET India Order] At: 05:42 19 October 2010

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.)

Moongbean Response to Cadmium Stress 2477

Downloaded By: [INFLIBNET India Order] At: 05:42 19 October 2010

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

2478 N. A. Anjum et al.

Downloaded By: [INFLIBNET India Order] At: 05:42 19 October 2010

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,

Moongbean Response to Cadmium Stress 2479

Downloaded By: [INFLIBNET India Order] At: 05:42 19 October 2010

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.

2480 N. A. Anjum et al.

Downloaded By: [INFLIBNET India Order] At: 05:42 19 October 2010

REFERENCES

Aebi, H. 1984. Catalase in vitro. Methods in Enzymology 105:121–126.

Allen, R. P., R. P. Webb, and S. A. Schake. 1997. Use of transgenic plants to

study antioxidant defenses. Free Radical Biology and Medicine 23:472–479.

Anderson, M. E. 1985. Determination of glutathione and glutathione disulfides in

biological samples. Methods in Enzymology 113:548–570.

Bashir, F., Mahmooduzzafar, T. O. Siddiqi, and M. Iqbal. 2007. The

antioxidative response system in soybean plants exposed to deltamethrin, a

synthetic pyrethroid insecticide. Environmental Pollution 147:94–100.

Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of

microgram quantities of protein utilizing the principle of protein-dye binding.

Analytical Biochemistry 72:248–254.

Brisson, J. D., R. L. Peterson, J. Robb, W. Rauser, and B. E. Ellis. 1977.

Correlated phenolic histochemistry using light, transmission, and scanning

electron microscopy, with examples taken from phytophathological problems.

In Scanning electron microscopy, vol. 2: Biological applications of SEM STEM,

ed. O. Johary, 667–676. Chicago: II T Research Institute.

Chen, Y., and A. J. Huerta. 1997. Effects of sulfur nutrition on photosynthesis in

cadmium-treated barley seedlings. Journal of Plant Nutrition 20 (78): 845–856.

Chien, H. F., C. C. Lin, J. W. Wang, C. T. Chen, and C. H. Kao. 2002. Changes

in ammonium ion content and glutamine synthetase activity in rice leaves

caused by excess cadmium are a consequence of oxidative damage. Plant

Growth Regulation 36:41–47.

Cobbett, C. S. 2000a. Phytochelatins and their roles in heavy metal detoxification.

Plant Physiology 123:825–832.

Cobbett, C. S. 2000b. Phytochelatin biosynthesis and function in heavy metal

detoxification. Current Opinion in Plant Biology 3:211–216.

Dhindsa, R. H., P. Plumb-Dhindsa, and T. A. Thorpe. 1981. Leaf senescence

correlated with increased level of membrane permeability, lipid per oxidation

and decreased level of SOD and CAT. Journal of Experimental Botany 32:

93–101.

Dixit, V., V. Pandey, and R. Shyam. 2001. Differential antioxidative responses to

cadmium in roots and leaves of pea (Pisum sativum L. cv. Azad). Journal of

Experimental Botany 52:1101–1109.

Foyer, C. H., and B. Halliwell. 1976. The presence of glutathione and glutathione

reductase in chloroplasts: A proposed role in ascorbic acid metabolism. Planta

133:21–25.

Ghoshroy, S., and M. J. Nadakavukaren. 1990. Influence of cadmium on the

ultra structure of developing chloroplasts of soybean and corn. Environmental

and Experimental Botany 30 (2):187–192.

Gomez, K. A., and A. A. Gomez. 1984. Statistical procedures for agricultural

research, 2nd ed. New York: John Wiley and Sons.

Hassan, J. M., G. Shao, and G. Zhang. 2005. Influence of cadmium toxicity on

growth and antioxidant enzyme activity in rice cultivars with different grain

cadmium accumulation. Journal of Plant Nutrition 28:1259–1270.

Hendry, G. A. F., A. J. M. Baker, and C. F. Ewart. 1992. Cadmium tolerance and

toxicity, oxygen radical processes and molecular damage in cadmium-tolerant

Moongbean Response to Cadmium Stress 2481

Downloaded By: [INFLIBNET India Order] At: 05:42 19 October 2010

and cadmium-sensitive clones of Holcus lanatus. Acta Botanica Neerlandica

41:271–291.

Iannelli, M. A., F. Pietrni, L. Fiore, L. Petrilli, and A. Massacci. 2002.

Antioxidant response to cadmium in Phragmites australis plants. Plant

Physiology and Biochemistry 40:977–982.

Iqbal, M., and T. Khudsar. 2000. Heavy metal stress and forest cover: Plant

performance as affected by cadmium toxicity. In Man and forests, ed. R. K.

Kohli, H. P. Singh, S. P. Vij, K. K. Dhir, D. R. Batish, D. K. Khurana, 85–112.

Chandigarh, India: DNES, IUFRO, ISTS and Punjab University.

Khan, N. A., I. Ahmad, S. Singh, and R. Nazar. 2006. Variation in growth,

photosynthesis, and yield of five wheat cultivars exposed to cadmium stress.

World Journal of Agricultural Sciences 2 (2):223–226.

Khudsar, T., Mahmooduzzafar, and M. Iqbal. 2001. Cadmium-induced changes

in leaf epidermis, photosynthetic rate, and pigment concentrations in Cajanus

cajan. Biologia Plantarum 44:59–64.

Law, M. E., S. A. Charles, and B. Halliwell. 1983. Glutathione and ascorbic acid

in spinach (Spinacia oleracea) chloroplasts: The effect of hydrogen peroxide

and of paraquat. Biochemical Journal 210:899–903.

Leon, A. M., J. M. Palma, F. J. Corpas, M. Gomez, M. C. Romero-Puertas, D.

Chatterjee, R. M. Mateos, L. A. del Rio, and L. M. Sandalio. 2002.

Antioxidative enzymes in cultivars of pepper plants with different sensitivity

to cadmium. Plant Physiology and Biochemistry 40:813–820.

Mehindirata, S., Mahmooduzzafar, T. O. Siddiqi, and M. Iqbal. 2000. Cadmium-

induced changes in growth and structure of root and stem of Solanum

melongena L. Phytomorphology 50:243–251.

Metwally, A., V. I. Safronova, A. A. Belimov, and K. J. Dietz. 2005. Genotypic

variation of the response to cadmium toxicity in Pisum sativum L. Journal of

Experimental Botany 56:167–178.

Nakano, Y., and K. Asada. 1981. Hydrogen peroxide is scavenged by ascorbate-

specific peroxidase in spinach chloroplasts. Plant Cell Physiology 22:867–880.

Noctor, G., L. A. Gomez, H. Vanacker, and C. H. Foyer. 2002. Interactions

between biosynthesis, compartmentation, and transport in the control of

glutathione homeostasis and signaling. Journal of Experimental Botany

53:1283–1304.

Nriagu, J. O., and J. M. Pacyna. 1988. Quantitative assessment of worldwide

contamination of air, water, and soils of trace metals. Nature 333:134–139.

Olmos, E. O., J. R. Martinez-Solano, A. Piqueras, and E. Hellin. 2003. Early

steps in the oxidative burst induced by cadmium in cultured tobacco cell (BY-2

line). Journal of Experimental Botany 54:291–301.

Ozturk, L., S. Eker, and F. Ozkutlu. 2003. Effect of cadmium on growth and

concentrations of cadmium, ascorbic acid, and sulphydryl groups in durum

wheat cultivars. Turkish Journal of Agriculture and Forestry 27:161–168.

Prasad, M. N. V., P. Malec, A. Waloszek, M. Bojko, and K. Strzałka. 2001.

Physiological responses of Lemna trisulca L. (duckweed) to cadmium and

copper bioaccumulation. Plant Science 161:881–889.

Qadir, S., M. I. Qureshi, S. Javed, and M. Z. Abdin. 2004. Genotypic variation in

phytoremediation potential of Brassica juncea cultivars exposed to Cd stress.

Plant Science 167:1171–1181.

2482 N. A. Anjum et al.

Downloaded By: [INFLIBNET India Order] At: 05:42 19 October 2010

Qureshi, M. I., M. Z. Abdin, S. Qadir, and M. Iqbal. 2007. Lead-induced

oxidative stress and metabolic alterations in Cassia angustifolia Vahl. Biologia

Plantarum 51 (1):121–128.

Ramos, I., E. Esteban, J. J. Lucena, and A. Garate. 2002. Cadmium uptake and

subcellular distribution in plants of Lactica sp. Cd-Mn interaction. Plant

Science 162:761–767.

Rao, M. V. 1992. Cellular detoxification mechanisms to determine age dependent

injury in tropical plants exposed to SO2. Journal of Plant Physiology 140:

733–740.

Schutzendubel, A., P. Nikolova, C. Rudolf, and A. Polle. 2002. Cadmium and

H2O2-induced oxidative stress in Populus 6 canescens roots. Plant Physiology

and Biochemistry 40:577–584.

Schutzendubel, A., P. Schwanz, T. Teichmann, K. Gross, R. Langenfeld-Heyser,

D. L. Godbold, and A. Polle. 2001. Cadmium-induced changes in antioxidative

systems, hydrogen peroxide content, and differentiation in Scots pine roots.

Plant Physiology 127:887–898.

Slooten, L., K. Capiiau, W. Van Camp, M. Van Montagu, C. Sybesma, and D.

Inze. 1995. Factors affecting the enhancement of oxidative stress tolerance

in transgenic tobacco overexpressing Mn-SOD in the chloroplast. Plant

Physiology 107:747–750.

Wu, F. B., G. P. Zhang, and P. Dominy. 2003. Four barley genotypes respond

differently to cadmium: Lipid peroxidation and activities of antioxidant

capacity. Environmental and Experimental Botany 50:67–78.

Moongbean Response to Cadmium Stress 2483

Downloaded By: [INFLIBNET India Order] At: 05:42 19 October 2010