LIST OF PUBLICATIONS

I ) Sumithra Kannan. Pannaga Pavan Jutur, Benjamin Dalton Carmel and Attipalli

Ramachandra Reddy (2006). Salinity-induced changes in two cuitivars of

Vigna radiata: Responses of antioxidative and proline metabolism. Plant

Growth Reguiation 50: 1 1-22

2) Ramachandra Reddy, K.V. Chaitanya, P.P. Jutur and K. Sumithra (2004).

Differential antioxidative responses to water stress among five mulberry (Morus

aiba L.) cultivars. Environmental and Experimenfal Botany 52: 33-42.

3 ) Sumithra K. and Reddy A.R. (2004). Changes in proline metabolism of cowpea

seedlings under water deficit. Journal of Planf Biology 31 (3) : 201-204.

ORIGINAL PAPER

Salinity-induced changes in two cultivars of Vigna radiata: responses of antioxidative and proline metabolism

K. Sumithra . P. P. Jutur 13. Dalton Carmel . Att~palli R. Reddy

R e ~ e ~ v e d 23 February 2005 I Accepted 13 June 2006 1 Pubhshe Springer SciencetBuslness M e d ~ a B V 2006

Abstract The Influence of increasing sallnlty stress on plant growth, antloxldant enzymes and prollne metabolism in two cultivars of Vlgna i i i l i i a r~r L (cv Pusa Bold and cv CO 4) was ~nvcst~gatcd Salt stress was Imposed on 30 days- old cultivars wlth four different concentrations of NaCl (0, 100, 200 and 300 mM) The roots and shoots of CO 4 showed greater reduction in fresh welght, dry weight and water content when com- pared to Pusa Bold with lncreaslng salt stress C!nder ra l~n~tv stress the roots and shoots of CO 4 ~ \ l u b l t ~ d higher Na K ratio than Pusa Bold The activities of reactlve oxygen species (ROS) scdvenglng enzymes and reduced glutathlone (GSH) concentratlon were found to be h~gher In the leaves of Pusa Bold than in CO 4, whereas oxldized glutathione (GSSG) concentratlon was found to be higher In the leaves of CO 4 compared lo those In Pusa Bold Our studies on oxldatlve damage in two V~gna cultivars showed lower lev- els of llp~d perovidation and H20, concentration

K Sunuthra B D Carmel Department of Blochemlstry and Molecular Biology, School of L ~ f e Sc~ences Pond~cherry University, I ' o n i l ~ ~ l i ~ ~ ~ y 605 014 I n d ~ d

P P Jutur A R Reddy (B) Department of Plant Sc~ences, School of Llfe Sciences, Unlverslty of Hyderabad, Hyderabad 500 046, Indla e mail arreddy@yahoo corn

d onllne 20 October 2006

m Pusa Bold than m CO 4 under salt stress con- dltlons Hlgh accumulation of proline and glyclne betaine under salt stress was also observed m Pusa Bold when compared to CO 4 The activltles of proline biosynthetlc enzymes were significantly high m Pusa Bold However, under salinity stress, Pusa Bold showed a greater decllne in proline dehydrogenase (ProDH) actlvlty compared to CO 4 Our data in this lnvestlgatlon demonstrate that oxidative stress plays a major role in salt-stressed Vzgna cultlvars and Pusa Bold has efficient an- tloxldative characteristics whlch could prov~de better protection agalnst oxldatlve damage in leaves under salt-stressed conditions

Keywords Antioxidant enzymes Biomass Llpid peroxidation Proline metabolism Reactlve oxygen species Salln~ty stress Vzgna radzata

Abbreviations APX Ascorbate peroxidase CAT Catalase GSH Reduced glutathione GSSG Oxidlzed glutathione GR Glutathlone reductase MDAR Monodehydroascorbate reductase POD Peroxidase ROS Reactlve oxygen specles SOD Superoxide d~smutase TBA Thiobarblturic acld

12 Plant Growth Regul (2006) 50:ll-22

Introduction

Soil salinization is one of the most serious envi- ~.onmcnial threats for plant survival and crop yield which affects 19.5% of irrigated land and 2.1 % dry land agriculture across the globe ( F A 0 2000). Salinity exerts its undesirable effects through osmotic inhibition. ionic toxicity and also by disturbing the uptake and translocation of nutritional ions (Misra and Dwivedi 2004). These cfSects can disturb the physiological and bio- chemical [unctions of the plant cell, leading to cell death (Xiong and Zhu 2002). In addition, salt stress also leads to oxidative stress through an increase in the production of reactive oxygen species (ROS).

High concentrations of salts in plants also limit [hi: uplake of CO? and results in decreased car- bon reduction by Calvin cycle, which inturn leads lo non-availability of oxidized NADP' for acceptance of electrons during photosynthesis, stimulating the formation of ROS such as super- oxide ( O;), hydrogen peroxide (H202) and hy- droxyl (OH-) radicals (Peltzer et al. 2002). The toxic effects of 0; and H202 can initiate cascade ol' rs.aclior~s resulting in generation of hydroxyl radicals and other destructive species such as lipid peroxides (Vaidyanathan et al. 2003). As a de- Sense mechanism, plants compete against these oxidative stresses by the synchronous action of various enzymatic and non-enzymatic antioxi- dants. Enzymatic antioxidants like superoxide dislnutasc (SOD), catalase (CAT), ascorbate pcroxidiisc (APX), peroxidase (POD), glutathi- one reductase (GR) and monodehydroascorbate reductase (MDAR) are the principal ROS scav- enging systems, quenching superoxide and hydrogen peroxide radicals under stressful con- ditions. :SOD dismutates superoxide radicals to hydrogen peroxide where as, CAT and peroxid- ascs arc involved in converting H 2 0 2 into water and oxygen. GR and MDAR are involved in the regeneration of ascorbate in ascorbate-glutathi- one cycle (Reddy et al. 2004a). Ascorbate and reduced glutathione (GSH) are the non-enzy- matic ROS scavenging systems which directly interact with and detoxify free radicals (Polle 2001).

During salt stress, plants adapt to osmotic stress by accumulating certain protective solutes like proline, glycine betaine, polyols, trehalose etc. (Ghoulam et al. 2002; Sakamoto and Murata 2002). Proline plays a predominant role in protecting plants from osmotic stress. In higher plants, proline is usually synthesized from glutamate via ~~-~~rroline-5-carbox~late (P5C) by two successive reactions catalyzed by ~ ' - ~ y r r o l i n e - 5-carboxylate synthetase (PSCS) and A'-pyrroline- 5-carboxylate reductase (PSCR). Proline degra- dation to P5C is caused by proline dehydrogenase (ProDH) and further to glutamate by pyrroline-5- carboxylate dehydrogenase (PSCDH) (Verbrug- gen et al. 1996). In addition to glutamate pathway, ornithine pathway is also known to be involved in proline accumulation (Charest and Chon 1990).

Vigna radiata (Mungbean) is an important summer-growing legume, grown predominanlly under semi-arid conditions of tropical and sub- tropical regions (Thomas et al. 2004). V. radiata sprouts are popular dietary products for high protein content, antioxidants, vitamins and min- erals (Randhir et al. 2004). These plants are ex- posed to varied type of environmental conditions such as drought, high temperature and salinity which could adversely affect plant growth and production. Therefore, it becomes necessary to elucidate the stress tolerance mechanisms of dif- ferent cultivars for better agronomic performance and also to breed resistant cultivars.

The aim of the present study is to determine salinity tolerance among two cultivars of V. rod- iata: Pusa Bold and CO 4 with particular refer- ence to antioxidative responses. In addition, activity of enzymes of proline metabolism and the concentrations of proline and glycine betaine under different levels of salt treatment in Pusa Bold and CO 4 were also been investigated.

Materials and methods

Plant material and treatment

Seeds of two cultivars of V. radiata (Pusa Bold and CO 4) were obtained from Tamilnadu Agricultural University (TNAU), Coimbatore, India. Plants

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Piant Growth Regui (2006) 50:ll-22 13

\ \crL. propi~pated in 25 crn clay pots containing 6 kg of air dried red soil under 12 h natural pho- toperiod irradiance of 1600-1800 pmol m-2 s-l, with dayinight temperatures of 32OCI28"C and the relative humidity between 45 and 70%. The plants were watered regularly and fertilized with ~ o a g l a n d nutrient solution (Hoagland and Arnon i t I iO). About 30-days-old-plants were subjected lo salt stress with differcnt iFaC1 concentrations (0.100,200 and 300 mM).

Plant growth

To study the growth of plants under salinity s t 1 . c ~ ~ . frcsh weight and dry weight of the plants were recorded after 72 h of NaCl treatment. To determine the fresh weight, root and shoot parts of the plants were separated and individual tissues were weighed. For dry weight determination, the parts were dried in hot air oven at 70°C for 48 h and weighed. Water content (WC) as percentage of fresh weight was calculated using the formula:

(fw-dw) x 100 fw

Measurement of ion concentration

The oven dricd root and shoot tissues (approxi- rnutci~ 70 g) were ground to fine powder and 100 mgof the powderwas transferred to adigestion flask (50 ml) containing an acid mixture of H N 0 3 and H2SO4, in the ratio 10:l (viv). The samples were digested by heating over a sand bath and then cooled. The cooled digest was diluted by adding double distilled water and made up to the desired \lo!urnc NJ' and K t ion concentrations in the acid extracts were estimated by flame photometry.

Lipid peroxidation

Lipid peroxidation rates were determined by measuring the malondialdehyde equivalents according to Hodges et al. (1909). The leaf tissue (0 .5 g) was homogenized in a mortar with 80% ethanol. The homogenate was centrifuged at 3,000 x g for 10 min at 4°C and the pellet was extracted twice with the same solvent. The

supernatants were pooled and 1 ml of rhis sample was added to a test tube with an equal volume of either a solution comprised of 20% TCA and 0.01% butylated hydroxy toluene (BHT) or a solution of 20% TCA, 0.01% BHT and 0.65% (wl v) TBA. Samples were heated at 9S°C for 25 min and cooled to room temperature. Absorbances were read at 440, 532 and 600 nm.

Estimation of H 2 0 2 concentration

HzOz concentration in the leaves of Pusa Bold and CO 4 were measured spectrophotometrically using KI as described by Alexieva et al. (2001). The reaction mixture consisted of 0.5 ml of 0.1% trichloroacetic acid (TCA), leaf extract superna- tant, 0.5 ml of 100 mM potassium-phosphate buffer and 2 ml of KI reagent (1 M KI wlv in distilled HzO). The blank probe consists of 0.1% TCA in the absence of leaf extract. The reaction was allowed for 1 h in darkness and the absor- bance was measured at 390 nm. The amount of H202 was calculated using a standard graph of known concentrations.

Extraction of ROS detoxifying enzymes

All experiments were performed at 4°C. The leaf blades (10 g) were homogenized with 50 ml of 100 mM Tris-HC1 (pH 7.5) containing 5 mM DTT, 10 mM MgC12, 1 mM EDTA and 5 mM magnesium acetate and 1.5% PVP-40. The homogenate was squeezed through four layers of cheese cloth and centrifuged at 10,000 x g for 10 min. The protein was precipitated with 75% (wl v) ammonium sulphate and spun at 30,000 x g for 30 min and the precipitate was dissolved in 50 mM Tris-HC1 buffer (pH 7.8) containing 1 mM DTT and 2 mM EDTA. The preparation was applied to a Sephadex G-25 column equili- brated with 10 mM Tris-HC1 (pH 8.0) containing 1 mM DTT, 10 mM NaHCO?, 20 mM MgC12 and 0.2 mM NADPH. The eluates were collected at room temperature and used for different assays.

Enzyme activity

Superoxide dismutase (SOD, E C 1.15.1.1) activity was determined according to Beauchamp and

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14 Plant Growth Reeul (2006) 50:ll-22

Fridovich (1971) by following the photo-reduc- tion of nitroblue tetrazolium (NET). The reaction mixture contained: 50 mM phosphate buffer (pH 7.8). 0.1 mM EDTA. 13 mM methionine, 75 LIM NBT. 7, pM riboflavin and 100 p1 of the supernatant. Riboflavin was added as the last component and the reaction was initiated by placing the tubes under two 15 W fluorescent lamps. The reaction was terminated after 10 min by removing the reaction tubes from the light source. Non-illuminated and illuminated reac- lions withour supernatant served as calibration slandards. Reaction products were measured at 560 nm.

A modified method of Luck (1974) was em- ployed for assay of catalase (CAT, EC 1.11.1.6). A volume of 50 111 of the enzyme extract was added to 3 ml of the hydrogen peroxide-phos- phate buffer (pH 7.0). The time required for the dccrcase in the absorbance (240 nm) from 0.45 to 0.40 was noted. Enzyme solution containing hydrogen peroxide-free phosphate buffer was used as control.

Glutathione reductase (GR, E C 1.6.4.2) activ- ity was determined by following the rate of NADPH oxidation as measured by decrease in 111c absorbance at 340 nm. About 1 ml of the as- say mixtui-c contained: 100 mM Tris buffer (pH 7.8), 2 mM EDTA, 50 pM NADPH, 0.5 mM GSSG and 20 p1 of the extract. The assay was initiated by the addition of NADPH at 25'C and rhe absorbance was read at 340 nm (Foyer and Halliwell 1976).

Monodchydroascorbatc reductase (MDAR, IIC 1.6.5.4) was assayed by l'oiiowing a decrease in the absorbance at 340 nm due to the oxidation of NADPH at 25'C (Schuler 1977). About 1 ml of the reaction mixture contained 50 mM HEPES- KOH (pH 7.h), 0.1 mM NADPH, 2.5 mM ascor- bare, 0.14 units ascorbate oxidase and the enzyme. The assay was initiated by the addition of NADPH and the absorbance read at 340 nm.

Estimation of GSH and GSSG

One gram of leaf tissue was homogenized with inert sand in 5 ml of 6.5% m-phosphoric acid containing 1 mM EDTA and then centrifuged at

10,000 x g' for 10 min. Reduced glutathione (GSH), oxidized glutathione (GSSG) and total glutathione concentration were assayed by the enzymatic GSSG recycling method as described by Bergmeyer et al. (1974).

Estimation of proline

The concentration of proline was estimated according to the method of Bates et al. (1973). Five gram of leaf tissue was homogenized with 30% sulphosalicylic acid and filtered through a Whatman No.1 filter paper. A volume of 2 ml of glacial acetic acid, 2 ml acid ninhydrin were added to 2 ml of filtrate and incubated for 1 h in a boiling water bath followed by cooling in ice bath. About 4 ml of toluene was then added and mixed .vigorously. The chromophore con- taining toluene was aspirated from aqueous phase and the absorbance was measured at 575 nm.

Glycine betaine

Glycine betaine concentration in the leaf extract was determined according to the method of Sto- rey and Wyn Jones (1977). Five gram of leaf tis- sue was homogenized in 25 ml of isopropyl alcohol and centrifuged at 3,000 x g for 10 min. The supernatant was dried at 40°C. The residue was washed successively twice with chloroform (20 ml) and distilled water (5 ml). The two phases of bulked washings were partitioned by centrifu- gation and the upper aqueous layer was removed. The remaining lipid layer was washed thrice wilh 20 ml of MeOH: H 2 0 (1:l). The combined aqueous layers were evaporated to dryness in a hot water bath and redissolved in distilled water (5 ml). Potassium triiodide solution (0.2 ml) was added to 1 ml of the above extract and the mix- ture was incubated for 90 min on ice bath with intermittent shaking. About 2 ml of ice cold water was added to the mixture, followed by 2 ml of 1, 2-dichloroethane. The two layers were mixed by constant stream of air bubbles for 5 min, while the temperature was maintained at 4°C. The absorbance of the lower organic layer was mea- sured at 365 nm.

Plant Growth Regui (2006) 50:ll-22 15

Extraction of enzymes of proline metabolism

XI! the opcrations were performed at 4°C. The leaf tissue (10 g) was homogenized in a pre-chilled mortar with 50 ml of extraction buffer containing 100 mM Tris-HC1 (pH 7.4), 100 mM MgS04 and 0.1 g of PVP-40. The homogenate was centrifuged at 12,000 x g for 20 min. The solution was fil- w e d off to remove the cellulose and washed thrice with extraction medium. Protein was pre- cipitated with 75% ammonium sulfate and spun at 30,000 x g for 15 min and the precipitate was dissolved in 50 mM Tris-HC1 (pH 7.8) containing 1 mM DTT and 2 mM EDTA. The preparation was applied to a sephadex G-25 column, equili- brated with 10 m M Tris-HCI (pH 8.0) which Lo~l~i~i!icd 1 mM DTT. 10 mM I\laHC03, 20 mM I\1gC12 and 0.2 mM NADPH. Eluates were col- lected and used for assaying glutamate dehydro- genase (GDH), ornithine transaminase (OT) and proline dehydrogenase (ProDH) assays. The pro- tein content in eluates was estimated according to Bradford and Hsiao (1982) with BSA as standard.

For pyrroline-5-carboxylate synthetase (PSCS) cstractioil, the leaf tissue was homogenized with extraction buffer containing 100 mM Tris-HC1 (pH 7 4 , 100 mM j3-mercaptoethanol, 10 mM MgC12 and 1 mM PMSF. The homogenate was centrifuged at 10,000 x g for 15 min. The supernatant was used for enzyme assays.

addition of 0.4 mM NADPH. The activity was measured as the rate of consumption of NADPH monitored by decrease in absorbance at 340 nm.

Ornithine transaminase activity was assayed according to Mazelis and Fowder (1969). The reaction mixture (3 ml) contained: 200 mM Tris- HC1 buffer (pH 8.0), 46.8 mM L-Ornithine, 12.5 mM a-Ketoglutaric acid and 0.2 ml of en- zyme extract. The reaction was initiated by the addition of 0.125 mM NADH. The activity was measured by following the oxidation of NADH at 340 nm.

Proline dehydrogenase activity was assayed according to Reno and Splittstoesser (1975). The reaction mixture contained: 100 mM Na2C03- NaHC03 (pH 10.3), 20 mM L-proline and 0.5 ml of enzyme extract in a final volume of 3 rnl. The reaction was initiated by the addition of 10 mM NAD. The increase in absorbance at 340 nm was measured at 32OC.

Statistical analysis

All the data in the present study are expressed as mean i SE obtained from four independent measurements. The data were subjected to anal- ysis of variance (ANOVA) with one way classifi- cation technique. Tukey's multiple range test was applied to compare the treatment means. P value of less than 0.05 was considered as significant.

Assay of proline metabolism enzymes Results

Glutamate dehydrogenase activity was assayed by the method of Kanamori et al. (1972). The reac- tion mixture contained 200 mM Tris-HC1 buffer (pH K O ) , 150 mM NH4C1, 150 mM a-ketoglutaric acid and 0.2 ml of enzyme extract in a final vol- ~lnie of 3 ml. The reaction was initiated by the addition of 3 mM NADH. The GDH activity was measured by following the oxidation of NADH at 340 nm.

Pyrroline-5-carboxylate synthetase activity was assayed as described by Garcia-Rios et al. (1997). The reaction mixture (3 ml) contained: 100 mM Tris-HC1 buffer (pH 7.2), 25 mM MgC12, 75 mM sodium glutamate, 5 mM ATP and 0.2 ml of enzyme extract. The reaction was initiated by the

Effect of salt stress on growth and Na': K' ratio

The biomass [fresh weight (fw), dry weight (dw)], water content and Naf: K' ratios in both the cultivars of V, radiata (Pusa Bold and CO 4) were studied under control and saline conditions (100, 200 and 300 mM). Table 1 shows a gradual de- crease in fw, dw and water content in the roots and shoots of both the cultivars with increasing of NaCl treatment. The roots of CO 4 showed 56% decrease in fw at 300 mM concentration, while Pusa Bold exhibited 43% decrease compared to control plants. Moreover, high salinity stress

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1 1 ; Plant Growth Reaul (2006) 50:11-22

Table 1 Effect of salt stress on fresh weight, dry weight, water content and Na': K' ratios in two Vigtzo cultivars

Cultivars NaCl Root Shoot trearment

Dry weight Water Na7:K+ Fresh Dry Waier Na+:K' ( ) t a n ) content weight weight content

(glplant) (%) (dplant) (@plant) (%)

Puh; i Bold 0 3.5 5 0.23 0.40 = 0.04 86 r 5 0.90 15.5 i 2.4 1.8 i. 0.4 88 5 0.50 100 3.2 i 0.17 0.36 i 0.03 74 i 4 1.40 13.8 I 2.3 1.6 i 0.3 78 + 6 0.67 200 2.8 i 0.33 0.30 + 0.02 56 + 3 2.30 11.4 i 1.2 1.3 r 0.2 69 t 4 0.90 300 2.0 i 0.20 0.21 z 0.03 47 i 2 4.30 10.0 i 1.1 1.1 * 0.4 55 1 3 2.20

C 0 4 0 2.7 r 0.31 0.35 5 0.04 85 i 4 1.00 11.2 = 2.0 1.4 % 0.2 87 i 5 0.70 100 2.4 i 0.22 0.30 i 0.05 53 1: 3 2.10 8.8 i 1.3 1.1 i 0.5 59 i 4 0.90 200 2.1 2 0 . 1 3 0.26i .0 .06 4 2 i 2 4.20 7 . 0 i 1.1 0 . 9 2 0 . 2 5 0 i 5 2.00 300 1.2 i 0.42 0.15 i 0.03 30 i . 1 8.50 5.5 5 1.06 0.7 * 0.06 43 i 7 5.50

30-days-old plants were subjected to salt stress for 3 days Each value represents the mean i SE obtained from four independent measurements, P < 0.05

(300 mM) resulted in 51% decrease in shoot fw of CO 4 and 35% decrease in Pusa Bold compared to control plants. There was a reduction of 47 and 57% in the root dw(s) of Pusa Bold and CO 4, rcspcctivclp with 300 mM XaC1 treatment. Simi- lar reduction in shoot dw(s) was noticed at 300 mM NaCl concentration.

The water content of roots and shoots of both the cultivars were found to be decreased with increasing salt concentrations. A lesser decrease of 45% was noticed in the roots of Pusa Bold compared to CO 4, which exhibited 65% decrease at 300 mM salt stress. Similar studies on shoot at 300 mM salt stress in CO 4 showed a significantly higher reduction in water content (51%) com- pared to Pusa Bold (37%).

Data in Table 1 also show influence of salinity stress on Na': K' ratio in the roots and shoots of Pusa Bold and CO 4 at different concentrations of N;ICI. Phc roots of CO 4 showed 8.5-fold increase

in Na': K' ratio compared to control plants, while Pusa Bold exhibited 4.8-fold increase. Data on shoots showed 7.9-fold increase in Na': K' ratio in CO 4 and increase of 4.4-fold was recorded with Pusa Bold.

Salt stress-induced lipid peroxidation and H20z levels

Table 2 shows the lipid peroxidation levels in- creased with increasing NaCl concentration in both the cultivars. At 300 mM NaCl treatment, CO 4 showed 57% higher lipid peroxidation rates compared to those in Pusa Bold. Our results also showed an increase in H202 levels with increasing salt treatment in both the cultivars. However, the accumulation of Hz02 was relatively less in Pusa Bold with 300 mM NaCl treatment, CO 4 showed 64% more accumulation compared to Pusa Bold (Table 2).

Table 2 Influence of salt stress on lipid peroxidation and Hz02 concentration in the leaves of 30-days-old V. radiaia cultivars subjected to salt stress

NaCl Pusa Bold C 0 4 treatment (mM) Lipid Hz02 Lipid Hz02

peroxidation content peroxidation content (nmoi g-' fw) ( p o l g-' f ~ v ) (nmol g-' fw) (11moI g-, f ~ v )

Each value represents the mean i SE obtained from four independent measurements, P < 0.05

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Plant Growtli Regul (2006) 50:ll-22 17

Effect of salinity on ROS scavenging enzymes

'Thc activitizs of four antioxidative enzymes (SOD. CAT, GR and MDAR) in two Vigna cultivars undcr different levels of salt treatments were assayed (Fig. la-d). Interestingly, Pusa Bold showed significantly higher activities than those in CO 4 under salt stress conditions. At 300 mM NaCi concentration, Pusa Bold showed 8.7-fold higher SOD activity compared with control plants while CO 4 showed 5.5-fold increase (Fig. la). CAT activity also increased with increasing salinity in both the cultivars (Fig. lb). Greater increase in CAT activity was observed in Pusa Bold (4.7-fold) compared to control cultivars, where as CO 4 exhibited a 2-fold increase at 300 11iM salt concentration.

Salt-induced changes in the activities of GR and MDAR in Pusa Bold and CO 4 were depicted in Fig. lc, id . There was a linear increase in GR activity with increasing concentrations of salt in both the cultivars (Fig. Ic). However, Pusa Bold showed over 6.9-fold more GR activity than controls a t .300 mM NaCl concentration while CO 4 silowed only 4.6-fold increase under similar conditions. Similarly, the activity of MDAR was

Fig. 1 Effects of salt 1500

stress on the activities of antioxidant enzymes in c

1 the leaves of two Vigna rariioto cultivars. 5 1000

sui?,jectcd to salinity stress 2 for 3 days: superoxide 2 dismutask (SOD) (a), i7 5 500 catalase (CAT) (b), n

glutathione reductase (GR) (c) and

8 monodehydroascorbate o r e d u c t a s e . ( ~ ~ ~ ~ ) (d ) . Eilch knlu; rcpresents the mi.,\!: 3 SE obtained from four independent measurements, P c 0.05

also found to be increased in both the cultivars under salt stress conditions with higher activity in Pusa Bold than in CO 4 (Fig. Id).

Salt induced changes in glutathione concentration

The changes in the levels of GSH and GSSG in response to salinity stress were studied in two V. radinta cultivars (Table 3) . There was a gradual increase in GSH levels in both salt-treated culti- vars compared to non-stressed cultivars. However, Pusa Bold showed 42% higher GSH concentra- tion, while CO 4 showed 18% increase with 300 mM NaCl treatment as compared to controls. In contrast, there was a decrease in GSSG con- centration with increasing salt stress in both the cultivars as compared to control plants. CO 4 showed 21% decrease in GSSG concentration while Pusa Bold showed 32% decrease with 300 mM NaCl treatment.

Proline and glycine betaine concentrations

The effects of salt stress on proline accumulatio~~ in two V. radiata cultivars were shown in Fig. 2a.

0- C 100 200 300

NaCl (mM)

t P u s a Bold +CO 4

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I S Plant Growth Regul (2006) 50:ll-22

Table 3 Effects of salt stress on GSH and GSSG concentration in the leaves of 30-days-old V. radiara cultlvars subjected ~ I I >tress

NaCl Pusa Bold CO 4 treatment (111 h? j GSH GSSG GSH GSSG

Conc. Conc. Conc. Conc. ( n m o l p ' Fw) (nmol g-! fw) (nmol g-' fw) (nmol g-' fw)

U 1546 ~t 16 259 + 10 1512 2 14 265 + 11 100 1689 + 18 245 i 12 I587 15 255 i 10 200 1896 i 20 214 * 9.2 1690 i: 17 234 I 8.4 300 2191 i 23 175 i 6.4 1789 i. 20 210 i 7.6

Each value represents the mean -. SE obtained from four independent measurements, P < 0.05

Fig. 2 E f l e ~ ~ \ ot salt 3 \ I K h S 011 111~

concentration of prolille (a) and glycine betaine (b) in the leaves of two cultivars of V. radiara. About 3n-days-old plants were subjected to salt stress for 3 days. Each tiilue represents the 111c~in x S E ubtained iro~li lour independent measurements, P < 0.05 C 100 200 300

NaCl (mM)

.Pus Bold C 0 4 .PUS Bold nC0 4

There was a gradual increase in proline con- celilrarion with increase in salinity in both the cultivars. Significantly higher accumulation (8.3- fold increase) of proline was observed with 300 mM NaCl in Pusa Bold compared to 5.2-fold increase in CO 4. Our results also demonstrate significant changes in glycine betaine concentra- tions with increasing salinity stress (Fig. 2b). Pusa Bold showed 56% higher increase in glycine bclaine concentration while CO 4 showed 35% increase with 300 mM NaCl treatment.

Enzymes of proline metabolism

Our data on activities of GDH, PSCS, OT and ProDH in Pusa Bold and CO 4 were shown in Fig. The activities of GDH, PSCS and OT significantly increased with progressive increase in salinity stress (Fig. 3a-c). However, Pusa Bold showed greater increase in the activities of all the three enzymes compared to CO 4 under 300 mM NaCl stress. In contrast, the activities of ProDH

significantly decreased with increasing salt stress in both the cultivars (Fig. 3d).

Discussion

Genetic variations among the crop plants pro- vides a valuable tool in the selection of cultivars with desirable traits (Misra and Dwivedi 2004). In this study, we clearly demonstrate differential responses of two Vigna cultivars subjected to salinity stress. The application of salt stress re- duced biomass yields in both the Vigna cultivars. However, the reduction in biomass, expressed on fresh and dry weight basis, was less in Pusa Bold compared to CO 4. The data indicate that Pusa Bold is relatively more salt-tolerant cultivar compared to CO 4. An interesting observation in this study was that higher ratios of Na': KT were apparent in CO 4 compared to Pusa Bold. Our results suggest that increased salt stress results in accumulation of more Na+ in the leaves of salt-

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Piant Growrh Regui (2006) 50: 11-22 19

Fig. 3 Activities of glutamate deh drogenase Y (GDH) (a). A -pyrroline- i - c . i r l x ~ \ > l a i ~ , syntheiase ( F'iCS) ib) . ornith~ne transaminase (OT) (c) and proline dehydrogenase (ProDH) (d) in two Vigtla radiatn cultivars under salinity stress. Each value represents the mean r SE obtained i'rorn lour i i~c~c~>~r?cI~111 nieasurements, P < 0.05

sensitive cultivars which might alter their func- ~ional state resulting physiological stress (Gasper ct al. 2002). A significant increase in Na': K' ratio in the two cultivars with increasing salinity ireatnlent suggests that Pusa Bold has an efficient internalization of ions by leaf cells compared to CO 4. Higher accumulation of ions like Na' may result in salt toxicity leading to cell dehydration a n d membrane disfunction. Such disturbances in ionic and osmotic balance would inhibit essential metabolic pathways leading to reduced plant growth.

Environmental stresses like salinity are known ,

to limit photosynthesis which can increase oxygen- induced, cellular damage due to increased ROS generation (Mittler 2002). Hence, any tolerance to slrcss. salt stress as studied here, should partly dcpcrid on the enhancement of antioxidant defense systems including enzymatic and non- enzymatic in plants. Accumulation of H20z in the two cultivars indicated a significant increase in leaves of CO 4 which confirms that during salt stress treatment, oxidative reactivity was elevated in CO 4 leaves leading to excessive ROS produc- lion and membrane disintegration. Our data on the

40

-- .C

8 - h

T o

; 20 E - b 5 1 0 . m

5 0

rates of lipid peroxidation actually confirm this supposition as the leaves of CO 4, which accumu- lated more HzOz, showed more lipid peroxidation with the progressive increase in salt stress (Table 2).

Antioxidative enzymes are known to protect the cell structures against the ROS generated by stress conditions (Reddy et al. 2004b). Our results indicate that under salt-stressed conditions, a significant increase in the activities of SOD, CAT, MDAR and GR was in response to oxidative stress was apparent in both cultivars. However, Pusa Bold exhibited significant increase in an- tioxidants and/or enzymes involved in ROS detoxification. ROS systems is an important index to assess the abilities of V. radiata cultivars to tolerate the stressful conditions. In the present study, increased activities of SOD in salt-treated cultivars indicate that SOD plays a crucial role in scavenging superoxide radicals during salinity stress. Interestingly, we have also observed a significantly lower accumulation of H202 in Pusa Bold than in CO 4. Therefore, the combined activities of SOD and CAT play a key role in the removal of ROS in Pusa Bold, thus minimizing

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20 Plant Growth Regul (2006) 50:11-22

Ii1.c ccllular damage caused by ROS under salt- stressed conditions (Gosset et al. 1994; Acar et al. 2001: Chaitanya et al. 2002).

Non-SOD scavenging of ROS is also of interest when it involves direcr reactions with ascorbate or GSH (Smirnoff 1995; Mittler 2002). Increased activities of DHAR and GR in response to salt sircss i n illis study suggesl that ascorbate-gluta- thione cycle also plays a crucial role as a defense system preventing oxidative damages inVigna cultivars (Asada 1999). Higher activities of MDAR in Pusa Bold under salt-stressed condi- tions indicate that the leaves of Pusa Bold could regenerate more ascorbate than those of CO 4. MDAR caLaiyzes the reduction of monodehy- droascorbatc to ascorbatc using NAD(P)H as the electron donor (Foyer and Noctor 2002; Reddy el al. 71101a). GR ia a key enzyme in ascorbate- glutathione pathway and is known to maintain higher ratios of GSHiGSSG, which are required for the regeneration of ascorbate (Broadbent ct al. 1995; Bray et al. 2000). Transgenic plants of iobacca which overcxpressed GR had elevated levels of GSH showing increased tolerance to oxidative stress in leaves. Higher activities of SOD, CAT and enzymes of ascorbate-glutathi- one cycle in salt-stressed Pusa Bold indicate that this cultivar has higher capacity for scavenging ROS, generated during salinity stress.

Prol i~~c accumulation in plants has been known LO be a stress effect rather than a cause of stress tolerance (Kumara et al. 2003). In the present study, we have reported a positive correlation between proline accumulation and salinity stress in Vigna cultivars (Fig. 2a). Proline is a low molecular-weight osmoprotectant that helps to preserve structural integrity and cellular osmotic potential within different compartments of the cell (Iyer and Caplan 1998). Higher accumulation of proline and glycine betaine in Pusa Bold than in CO 4:suggests that Pusa Bold possesses a better potential to maintain osmotic balance than CO 4 under salt stress.

Proline metabolism is a typical biochemical adaptation in living organisms subjected to stress conditions (Delauney and Verma 1993). In higher plants, proline is synthesized via both glutamic acid and ornithine pathway. Increased activities of GDH and PSCS in both the salt-treated Vigna

cultivars suggest that glutamate pathway is active in proline biosynthesis under salt-stressed condi- tions. The enhanced activity of O T in both the NaCl treated cultivars also provides an evidence for the participation of ornithine pathway to accumulate additional proline under salt stress. The enzyme ProDH is a proline degradative enzyme that converts proline to P5C in mito- chondria and further transformed into glutamate (Sanchez et al. 2002). Our results on proline metabolism clearly indicate that Pusa Bold pos- sesses better enzymatic machinery to accumulate more proline than in CO 4. Proline has also been recently reported to be an effective scavenger of ROS (Tripathi and Gaur 2004).

In conclusion, our study showed that the dif- ferences in plant biomass, NaC: K* ratios during salinity stress in two Vigna cultivars could be associated with differences in tolerance to salin- ity. Salt stress remarkably induced oxidative stress in Vigna cultivars and there were marked differences in antioxidants and antioxidant sys- tems between the two cultivars of Vigna. Our data also demonstrate that antioxidant systems could be used as a better index of oxidative stress under salt-stressed conditions. Pusa Bold, which exhib- ited lesser reduction in plant growth, higher antioxidant systems, more proline metabolism enzymes and greater accumulation of proline and glycine betaine under salt stress conditions, could be a superior cultivar for salinity tolerance.

Acknowledgements Thanks are due to Professor A . R. Muthaiah, Center for Plant Breeding and Genetics, Tamilnadu Agricultural University (TNAU), Coimbatore, India for providing the Vigna cultivars. Research Fellow- ships to KS and PPJ from Jawaharlal Nehru Memorial Fund (JNMF) are gratefully acknowledged.

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