A

tsrwmiflcp©

K

1

nmdaAetd

c

atPd

0d

Environmental and Experimental Botany 60 (2007) 276–283

Acclimation to drought stress generates oxidative stress tolerance indrought-resistant than -susceptible wheat cultivar under field conditions

Renu Khanna-Chopra ∗, Devarshi S. SeloteStress Physiology and Biochemistry Laboratory, Water Technology Centre, Indian Agricultural Research Institute, New Delhi 110012, India

Received 15 October 2006; accepted 30 November 2006

bstract

Wheat crop may experience water deficit cycles during their life cycle, which induces oxidative stress. Present study was conducted to evaluatehe role of oxidative stress management in the leaves of two wheat (Triticum aestivum L.) cultivars, C306 (drought-resistant) and Moti (drought-usceptible), when subjected directly to severe water stress (non-acclimated) or to water stress cycles of increasing intensity with an intermittentewatering (drought-acclimation). Mild water stress during vegetative growth enabled C306 to acclimatize better than Moti during subsequentater stress of severe nature during post-anthesis period. The drought-acclimated C306 leaves maintained favourable water relations and lowerembrane injury due to low H2O2 accumulation than non-acclimated C306 plants during severe water stress. This is due to systematic increase

n the activity of H2O2 scavenging enzymes particularly APX and POX and maintenance of ascorbate and glutathione redox pool by efficientunctioning of GR enzyme in the drought-acclimated C306 plants. In contrast, both acclimated as well as non-acclimated Moti plants exhibited

oss in turgor potential, high H2O2 levels and poor antioxidant enzyme response leading to enhanced membrane damage during severe water stressonditions. Hence, present study shows that genotypic differences in drought tolerance could be, at least in part, attributed to the ability of wheatlants to acclimate and induce antioxidant defense under water deficit conditions.

2006 Published by Elsevier B.V.

um

tiTaaocei(

eywords: Antioxidant defense; Water stress; Oxidative stress; Triticum aestiv

. Introduction

Drought stress limits plant growth and crop productivity sig-ificantly. However in certain tolerant/adaptable crop plantsorphological and metabolic changes occur in response to

rought, which contribute towards adaptation to such unavoid-ble environmental constraints (Sinha et al., 1982; Blum, 1996).mong crop plants, wheat (Triticum aestivum), which often

xperiences water-limited conditions, is an attractive study sys-em because of the natural genetic variation in traits related to

rought tolerance (Loggini et al., 1999).

Drought stress invariably leads to oxidative stress in the plantell due to higher leakage of electrons towards O2 during pho-

Abbreviations: AsA, ascorbate; APX, ascorbate peroxidase; DAS, daysfter sowing; DHA, dehydroascorbate; CAT, catalase; GR, glutathione reduc-ase; GSH, glutathione; GSSG, oxidized glutathione; H2O2, hydrogen peroxide;OX, guaiacol peroxidase; MSI, membrane stability index; SOD, superoxideismutase; ROS, reactive oxygen species; RWC, relative water content∗ Corresponding author. Tel.: +91 11 25846012; fax: +91 11 25843830.

E-mail address: renu wtc@rediffmail.com (R. Khanna-Chopra).

p1gppslacsa

098-8472/$ – see front matter © 2006 Published by Elsevier B.V.oi:10.1016/j.envexpbot.2006.11.004

osynthetic and respiratory processes leading to enhancementn reactive oxygen species (ROS) generation (Asada, 1999).he ROS such as O2

−, H2O2 and •OH radicals, can directlyttack membrane lipids, inactivate metabolic enzymes and dam-ge the nucleic acids leading to cell death (Mittler, 2002). Duringptimal conditions, the balance between ROS formation andonsumption is tightly controlled by an array of antioxidantnzymes and redox metabolites (Noctor and Foyer, 1998). Thisncludes superoxide dismutase (SOD, EC 1.15.1.1), catalaseCAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11),eroxidases (POX, EC 1.11.1.7), glutathione reductase (GR, EC.6.4.2) and redox metabolites such as ascorbate (AsA) andlutathione (GSH). The AsA–GSH, APX, and GR are the com-onents of “ascorbate–glutathione (AsA–GSH) cycle”, whichrovides an efficient protection against lethal ROS in all theub-cellular organelles of the plant cell (Moller, 2001). Theevel of AsA–GSH and the activities of antioxidant enzymes

re generally increased during abiotic stress conditions andorrelate with enhanced cellular protection. During droughttress, the plant water relations play a key role in the activationnd/or modulation of antioxidant defense mechanism (Menconi

tal and Experimental Botany 60 (2007) 276–283 277

e2

tmgattfittin(tstddios

2

2

rwA222psFao5

ts(iiti((o1drec

Fig. 1. Schematic diagram showing the water stress treatment to two wheat (T.aestivum L.) cultivars, C306 (drought resistant) and Moti (drought susceptible).A group of plants of each cultivar were subjected to acclimation treatment (A)i.e., two cycles of water stress, first of mild intensity mild (S1) during vegetativegrowth period (A1) and subsequently a second one of severe intensity (S2) duringpost-anthesis period (A2). Rewatering (R) period of 48 h terminates each stressceo

Iasgmttp

1twamotfw

2

mEFpiwsIrmd

2

R. Khanna-Chopra, D.S. Selote / Environmen

t al., 1995; Srivalli et al., 2003; Selote and Khanna-Chopra,004)

The response of resistant versus susceptible wheat cultivarso water deficit-induced oxidative stress and antioxidant defense

anagement, at a particular growth stage and under controlledrowth conditions has been reported in the literature (Loggini etl., 1999; Sgherri et al., 2000; Lascano et al., 2001). However, tohe best of our knowledge their response has not been evaluatedo multiple cycles of drought stress across growth stages undereld conditions. The present study was an effort to understand

he response of wheat cultivars differing in drought resistanceo successive water stress cycles of increasing intensity withntermittent recovery periods. Two wheat (T. aestivum) cultivars,amely C306 (drought-resistant) and Moti (drought-susceptible)Sinha et al., 1986) were subjected to drought-acclimation i.e.,wo consecutive water stress-recovery cycles of increasing inten-ity during vegetative stage to post-anthesis stage of plants. Bothhe cultivars were also exposed directly to severe water stressuring post-anthesis period (non-acclimated). The response ofrought-acclimated and non-acclimated plants was character-zed with reference to leaf water status, membrane stability,xidative stress and antioxidant defense management duringtress and recovery phases.

. Materials and methods

.1. Plant material and experimental conditions

Two wheat (T. aestivum L.) cultivars, namely C306 (droughtesistant) and Moti (drought susceptible) (Sinha et al., 1986)ere grown in the field of Water Technology Centre, Indiangricultural Research Institute, New Delhi (77◦12′E, 28◦40′N,28.6 m.s.l.). The seeds were sown on 22 November 2001, inm × 2 m sized plots, in rows 20 cm apart and plant density of00 plants m−2. Randomized complete block design (RCBD)attern was adopted for the experiment. Total 18 plots wereown with nine plots per cultivar and three plots per treatment.ertilizer was applied at the rate of 100:60:60 kg ha−1 N:P:Ks split dose, first at 15 days after sowing (DAS) at the ratef 50:60:60 kg ha−1 N:P:K and second at 74 DAS at the rate of0:0:0 kg ha−1 N:P:K.

Each cultivar were grouped into three sets and subjectedo water stress treatments (Fig. 1). Water stress treatment wastarted at 34 DAS. Control plants (C) were given six irrigationsat 15, 33, 54, 76, 94 and 108 DAS) from the date of sow-ng to maturity. Drought-acclimated plants (A) received fourrrigations (at 15, 33, 76 and 108 DAS) and were exposed towo successive stress-recovery cycles, first cycle (S1) of mildntensity (A1) followed by second cycle (S2) of severe intensityA2) and each stress cycle was terminated by 48 h rewateringR). The non-acclimated plants (D), with no previous historyf water stress, received five irrigations (at 15, 33, 54, 76 and08 DAS) and were directly exposed to severe water stress con-

itions during second stress cycle period (S2) followed by 48 hewatering (R). The first stress cycle (S1) was given during veg-tative growth phase between 34 and 76 DAS and second stressycle (S2) during post-anthesis period between 79 and 108 DAS.

(

w

ycle. Another group of plants (non-acclimated) of each genotype were made toxperience directly single water stress of severe intensity (D) followed by 48 hf rewatering (R) period. DAS indicates days after sowing.

n control plants, anthesis (50%) occurred at 90 DAS in Motind 96 DAS in C306 cultivar. However the duration to anthe-is was shortened by 2–3 days due to water stress in both theenotypes. Total rainfall received by the crop from sowing toaturity was 2.6 cm. Rainfall was meager and evapotranspira-

ion rate (ET) was higher (data not shown), which helped inhe development of severe water stress between anthesis andost-anthesis periods.

Sampling was done around midday between 11:00 and2:00 h from the top fully emerged young leaves from con-rol and stressed/rewatered plants for quantifying the plantater relations, membrane stability (or injury), H2O2 level and

ntioxidant defense components. Quantification of H2O2 andembrane stability was performed immediately after sampling

f the tissue For the antioxidant metabolites and enzyme assays,he leaves were cut into small pieces, weighed 0.2 g in replicates,rozen in liquid nitrogen and stored at −80 ◦C. Three replicatesere maintained for all the measurements.

.2. Leaf water relations

Leaf water potential (ψw) of control and stressed plants waseasured using pressure chamber (Model 3005, Soil Moisturequipment Corp., Santa Barbara, CA) (Scholander et al., 1964).or osmotic potential (ψs) measurement, same leaves werelaced in a glass vial and kept in −20 ◦C for 24 h. After thaw-ng at room temperature (about 15 min), cell sap was expressedith a gentle hand press, and the osmotic potential was mea-

ured using vapour pressure osmometer (Model 5500, Wescornc., Logan, UT) (Moinuddin and Khanna-Chopra, 2004). Theelative water content (RWC) was measured by following theethod of Barrs and Weatherly (1962). Turgor potential (P) was

erived by subtracting the value of ψs from ψw.

.3. H2O2 concentration and membrane stability index

MSI)

For the measurement of H2O2 content, leaf samples (0.2 g)ere homogenized with liquid nitrogen and suspended in chilled

2 tal a

5HDSat8LFoeubdOiawdfrM

2

2

ht(cwasbtbab(azsbAm3(vta(A

2

s

wo(odoTbocAqgnaaosGd2giG

2

(tlwd

3

3

itcRcscptm(eam

78 R. Khanna-Chopra, D.S. Selote / Environmen

% trichloroacetic acid and centrifuged at 30,000 × g for 10 min.2O2 was measured after passing the supernatant through aowex anion exchange resin (1× 8–400, Sigma–Aldrich Co.,t. Louis, USA) to remove the coloured compounds (Warmnd Laties, 1982). The reaction mixture contained 50 �l ofest solution, 50 �l luminol in 0.2 M NH4OH (pH 9.5) and00 �l 0.2 M NH4OH in 1.0 ml test tube, which were placed inuminoskan TL Plus luminometer (Labsystems Inc., Helsinki,inland). Chemiluminescence was initiated by the additionf 100 �l 0.5 mM K3Fe(CN)6 in 0.2 M NH4OH and photonsmitted were counted over 5 s. H2O2 content was determinedsing a calibration curve. Membrane stability was estimatedy measuring the leakage of electrolytes (conductivity) due toamaged cell membrane according to Shanahan et al. (1990).ne gram of leaf material (10 mm × 10 mm pieces) was taken

n 10 mL of double distilled water in glass vials and keptt 10 ◦C for 24 h with shaking. The initial conductivity (C1)as recorded after bringing sample to 25 ◦C by using con-uctivity meter. The samples were then autoclaved at 0.1 MPaor 10 min, cooled to 25 ◦C and final conductivity (C2) wasecorded. Membrane stability index (MSI) was calculated as

SI = [1 − (C1/C2)] × 100.

.4. Antioxidant enzymes and metabolites assay

.4.1. Antioxidant enzymes assayFrozen leaf samples were ground in liquid nitrogen and

omogenized in 50 mM sodium phosphate buffer (pH 7.0) con-aining 2 mM EDTA and 5 mM �-mercaptoethanol and 4%w/v) polyvinylpyrrolidine-40 (PVP-40). The homogenate wasentrifuged at 30,000 × g for 30 min at 4 ◦C. The supernatantas used for antioxidant enzyme (SOD, CAT, POX, APX,

nd GR) analysis. For ascorbate peroxidase (APX) activity,eparate extraction was carried out with the above-mentioneduffer containing additional 5 mM ascorbate in order to pro-ect APX activity. Protein content was determined as describedy Lowry et al. (1951). Total superoxide dismutase (SOD)ctivity was measured spectrophotometrically based on inhi-ition in the photochemical reduction of nitroblue tetrazoliumBeauchamp and Fridovich, 1971). One unit of SOD is defineds the amount of enzyme that inhibited the nitroblue tetra-olium (NBT) reduction by 50%. APX activity was determinedpectrophotometrically by monitoring the decrease in ascor-ate at A290 (ε= 2.8 mM−1 cm−1) as described by Nakano andsada (1981). Glutathione reductase (GR) was estimated byonitoring the oxidation of NADPH (ε= 6.22 mM−1 cm−1) at

40 nm according to Schaedle and Bassham (1977). PeroxidasePOX) activity was determined at 470 nm by its ability to con-ert guaiacol to tetraguaiacol (ε= 26.6 mM−1 cm−1) accordingo the method of Chance and Maehly (1955). Catalase (CAT)ctivity was measured by following the reduction of H2O2ε= 39.4 mM−1 cm−1) at 240 nm according to the method ofebi (1984).

.4.2. Ascorbate and glutathione measurementTotal ascorbate (AsA plus DHA) and ascorbate (AsA) mea-

urement was based on the reduction of ferric to ferrous ion

l(Co

nd Experimental Botany 60 (2007) 276–283

ith ascorbic acid in acid solution followed by the formationf the red chelate between ferrous ion and bathophenanthrolineWang et al., 1991). 0.2 g leaves was homogenized in 3.0 mlf cold 5% trichloroacetic acid containing 4% polyvinylpyrroli-one (PVP-40). The homogenate was filtered through four layersf cheesecloth and centrifuged at 16,000 × g for 10 min at 4 ◦C.he supernatant was used for total ascorbate and reduced ascor-ate (AsA) assay. Total AsA was determined through reductionf DHA to AsA by DTT. Dehydroascorbate (DHA) was cal-ulated from the difference of total ascorbate and AsA values.

standard curve in the range of 0–10 �mol AsA was used foruantification. Total glutathione (GSH plus GSSG) and oxidizedlutathione (GSSG) were determined by the 5,5′-dithio-bis-itrobenzoic acid (DTNB)-GR recycling procedure (Loggini etl., 1999). 0.2 g leaves was homogenized in ice-cold 5% sulphos-licylic acid. The homogenate was filtered through four layersf cheesecloth and centrifuged at 10,000 × g for 10 min. Theupernatant was used for the GSSG and total glutathione assay.SSG was determined from the sample after 2-vinylpyridineerivatization of GSH. Changes in absorbance due to the 5-thio-nitrobenzene (TNB) formation was measured at A412 and the

lutathione content was calculated using a standard curve rang-ng from 0 to 50 nmol. GSH was determined after subtraction ofSSG value from the total glutathione value.

.5. Statistical analysis

The results are expressed as means with standard error±S.E.). The significant difference (at P < 0.05) between con-rol and stressed/recovered samples for plant water relations,eaf growth, H2O2, MSI, antioxidant metabolites and enzymesas evaluated by analysis of variance (ANOVA). ANOVA wasone by using CIMMYT-MSTAT (Version 1.00/EM 1988).

. Results

.1. Leaf water relations under stress

Exposure to mild water stress (S1) resulted in minor declinen water relations i.e., water potential (ψw), solute potential (ψs),urgor potential (P) and RWC in both drought-tolerant and sus-eptible wheat genotypes as compared to controls (Table 1).ewatering led to complete recovery of leaf water relations toontrol levels in both the wheat genotypes. Subsequent watertress of severe nature during post-anthesis period caused signifi-ant decline in water relation parameters in Moti acclimated (A2)lants as compared to C306 acclimated (A2) plants with respecto their control (P < 0.05). The drought-acclimated (A2) C306

aintained higher turgor potential and RWC than acclimatedA2) Moti plants under severe water stress (Table 1). Directxposure of severe water stress resulted in loss of turgor in non-cclimated (D) Moti plants while non-acclimated C306 leavesaintained positive turgor. Rewatering resulted in recovery of

eaf water relation parameters to control levels in the acclimatedA2) plants of both the wheat genotypes and non-acclimated306 plants. The recovery was poor in non-acclimated (D) plantsf Moti (Table 1).

R. Khanna-Chopra, D.S. Selote / Environmental and Experimental Botany 60 (2007) 276–283 279

Tabl

e1

Eff

ect

ofac

clim

atio

nan

ddr

ough

tst

ress

(non

-acc

limat

ion)

trea

tmen

tson

leaf

wat

erpo

tent

ial

(ψw

),so

lute

pote

ntia

l(ψ

s),t

urgo

rpo

tent

ial

(P)

and

rela

tive

wat

erco

nten

t(R

WC

)in

the

two

whe

at(T

.ae

stiv

umL

.)cu

ltiva

rsdi

ffer

ing

indr

ough

tres

ista

nce

Firs

tstr

ess

cycl

eR

ewat

erin

g(4

8h)

Seco

ndst

ress

cycl

eR

ewat

erin

g(4

8h)

C1

A1

C1

RC

2A

2D

C2

AR

DR

ψw

(−M

Pa)

Mot

i1.

40±

0.06

1.67

±0.

03*

1.57

±0.

031.

57±

0.09

2.13

±0.

072.

77±

0.09

*2.

97±

0.10

*2.

13±

0.09

2.27

±0.

032.

37±

0.07

C30

61.

40±

0.10

1.63

±0.

09*

1.50

±0.

131.

53±

0.07

1.93

±0.

072.

59±

0.09

*2.

70±

0.12

*−1

.97

±0.

132.

03±

0.03

2.07

±0.

13

ψs

(−M

Pa)

Mot

i1.

88±

0.07

1.98

±0.

101.

95±

0.08

2.03

±0.

122.

39±

0.10

2.87

±0.

12*

3.00

±0.

14*

2.39

±0.

032.

44±

0.06

2.49

±0.

09C

306

1.91

±0.

052.

03±

0.07

1.96

±0.

031.

98±

0.05

2.28

±0.

102.

86±

0.11

*2.

86±

0.12

*2.

31±

0.08

*2.

33±

0.12

*2.

34±

0.10

P(M

Pa)

Mot

i0.

48±

0.03

0.31

±0.

040.

48±

0.02

0.46

±0.

040.

26±

0.04

0.10

±0.

05*

0.04

±0.

02*

0.25

±0.

040.

17±

0.04

*0.

12±

0.05

*

C30

60.

51±

0.05

0.40

±0.

030.

46±

0.04

0.45

±0.

030.

35±

0.02

0.27

±0.

02*

0.16

±0.

04*

0.36

±0.

030.

30±

0.05

0.28

±0.

06

RW

C(%

)M

oti

87.9

±2.

5478

.9±

1.50

*87

.6±

2.08

87.4

±1.

3775

.6±

1.52

65.1

±1.

19*

58.7

±2.

30*

74.2

±2.

1370

.9±

2.80

67.4

±3.

56*

C30

687

.9±

1.20

82.8

±1.

75*

87.7

±1.

4688

.0±

1.05

79.7

±1.

8074

.1±

1.61

*69

.5±

2.95

*79

.0±

1.60

76.8

±2.

4476

.2±

3.20

Mild

stre

ssoc

curr

edat

76da

yaf

ter

sow

ing

(DA

S)i.e

.,pr

e-an

thes

isph

ase,

whe

reas

seve

rest

ress

occu

rred

at10

8D

AS

i.e.,

post

-ant

hesi

spe

riod

.Val

ues

are

mea

n±

S.E

.(n

=3)

.C:c

ontr

ol,A

:acc

limat

edw

ater

stre

ss,

D:n

on-a

cclim

ated

wat

erst

ress

,and

R:r

ewat

erin

g.W

ater

stre

sstr

eatm

entl

egen

dsar

eas

inFi

g.1.

*Si

gnifi

cant

lydi

ffer

entf

rom

the

cont

rola

tP<

0.05

.

Fig. 2. (a and b) Hydrogen peroxide (H2O2), and (c and d) membrane stabilityindex in the leaves of two wheat genotypes subjected to acclimation and droughtsFs

3

sb(esiRl(rM

3

3

etacHaDai(aAAqa(

tress (non-acclimation) treatments. Water stress treatment legends are as inig. 1. Control (�), acclimated water stress (©), and non-acclimated watertress (�). Vertical bars indicated ±S.E. (n = 3). DW indicates dry weight.

.2. H2O2 accumulation and membrane injury

During mild water stress, acclimated (A1) C306 leaveshowed less H2O2 accumulation and maintained higher mem-rane stability as compared to acclimated (A1) Moti leavesFig. 2). During subsequent water stress of severe intensity,xcept drought-acclimated (A2) C306, all other plants exhibitedignificant enhancement (P < 0.05) in H2O2 levels and reductionn membrane stability as compared to control plants (Fig. 2).ewatering resulted in complete recovery, in terms of H2O2

evels and membrane injury to control level in both acclimatedA2) and non-acclimated (D) plants of C306 while only partialecovery was observed in both acclimated and non-acclimated

oti leaves after rewatering (Fig. 2).

.3. Antioxidant defense response

.3.1. Antioxidant enzyme activitiesIn control (unstressed) plants the activities of antioxidant

nzymes (except SOD) declined from vegetative to reproduc-ive growth phase in drought-susceptible cultivar Moti, wheres reverse of this trend was observed in the drought-resistantultivar C306 (Figs. 3 and 4). This coincided with increase in2O2 levels and decline in membrane stability in Moti leaves

s compared to C306 leaves under control conditions (Fig. 2).uring mild water stress, significant increase in the activity of

ntioxidant enzyme SOD, CAT, POX, and GR was observedn acclimated (A1) Moti leaves (P < 0.05). Similarly, acclimatedA1) C306 leaves showed enhanced activity of SOD, APX, CATnd GR (Figs. 3 and 4). Rewatering resulted in an increase inPX activity in Moti leaves. In contrast, C306 maintained higher

PX and CAT activity after rewatering (Figs. 3 and 4). Subse-uent severe water stress resulted in significant increase in SODctivity (P < 0.05) in the acclimated (A2) and non-acclimatedD) Moti leaves. However in both treatments activity of APX

280 R. Khanna-Chopra, D.S. Selote / Environmental a

Fig. 3. Activity of antioxidant enzymes: (a and b) superoxide dismutase (SOD),(c and d) ascorbate peroxidase (APX), and (e and f) catalase (CAT) in theleaves of two wheat cultivars subjected to acclimation and drought stress (non-a(b

adia

Fass(D

aS(r(tl(

3

otMia(ciaAp(reduced AsA contents and AsA/DHA ratio in comparison tocontrol plants (P < 0.05) (Fig. 5). In contrast, both acclimatedand non-acclimated Moti leaves showed significant reduction inthe total and reduced AsA contents and AsA/DHA ratio than

cclimation) treatments. Water stress treatment legends are as in Fig. 1. Control

�), acclimated water stress (©), and non-acclimated water stress (�). Verticalars indicated ±S.E. (n = 3). DW indicates dry weight.

nd POX exhibited no change, while CAT and GR activityeclined (Figs. 3 and 4). Non-acclimated (D) C306 leaves exhib-ted enhancement in SOD activity and reduction in APX, CATnd POX activities and showed no response in GR. In contrast,

ig. 4. Activity of antioxidant enzymes: (a and b) guaiacol peroxidase (POX),nd (c and d) glutathione reductase (GR) in the leaves of two wheat cultivarsubjected to acclimation and drought stress (non-acclimation) treatments. Watertress treatment legends are as in Fig. 1. Control (�), acclimated water stress©), and non-acclimated water stress (�). Vertical bars indicated ±S.E. (n = 3).W indicates dry weight.

Fabdas

nd Experimental Botany 60 (2007) 276–283

cclimated (A2) C306 leaves exhibited significant increase inOD, APX, CAT, POX and GR activity as compared to controlP < 0.05) (Figs. 3 and 4). Rewatering resulted in significant up-egulation all antioxidant enzyme activities in non-acclimatedD) C306 leaves except SOD (P < 0.05) (Figs. 3 and 4). In con-rast, rewatering had no effect on antioxidant enzymes in theeaves of both acclimated as well as non-acclimated Moti plantsFigs. 3 and 4).

.3.2. Total and reduced ascorbate (AsA)During mild water stress, significant decline (P < 0.05) was

bserved in the reduced ascorbate (AsA) content and reducedo oxidized ascorbate (AsA/DHA) ratio in the acclimated (A1)

oti leaves. In contrast, acclimated (A1) C306 leaves exhib-ted an increase in total and reduced AsA content with nopparent change in AsA/DHA ratio during mild water stressFig. 5). Rewatering resulted in increase in AsA/DHA ratio toontrol level in Moti leaves. Subsequent water stress of severentensity did not result in any significant change in the totalnd reduced AsA content but exhibited significant reduction insA/DHA ratio in the acclimated (A2) plants of C306 as com-ared to control (P < 0.05) (Fig. 5). However, non-acclimatedD) plants of C306 exhibited significant decline in total and

ig. 5. Changes in the (a and b) total ascorbate content, (c and d) reducedscorbate (AsA) content, and (e and f) ratio of reduced (AsA) to oxidized ascor-ate (DHA) in the leaves of two wheat cultivars subjected to acclimation androught stress (non-acclimation) treatments. Water stress treatment legends ares in Fig. 1. Control (�), acclimated water stress (©), and non-acclimated watertress (�)). Vertical bars indicated ±S.E. (n = 3). DW indicates dry weight.

R. Khanna-Chopra, D.S. Selote / Environmental an

Fig. 6. Changes in the (a and b) total glutathione content, (c and d) reducedglutathione (GSH) content, and (e and f) ratio of reduced (GSH) to oxidizedglutathione (GSSG) in the leaves of two wheat cultivars subjected to acclimationaaw

cRlw(

3

c(SdG(lldapp

4

tal

etafapDmtndwctuw

ditctpDmraddc

esaMsiiCiret(gdtTma

ls

nd drought stress (non-acclimation) treatments. Water stress treatment legendsre as in Fig. 1. Control (�), acclimated water stress (©), and non-acclimatedater stress (�)). Vertical bars indicated ±S.E. (n = 3). DW indicates dry weight.

ontrol plants under severe water stress conditions (P < 0.05).ewatering resulted in regeneration of AsA pool to control

evels in non-acclimated (D) C306 plants while no recoveryas observed in acclimated and non-acclimated Moti plants

Fig. 5).

.3.3. Total and reduced glutathione (GSH)No significant change in total and reduced glutathione (GSH)

ontents and GSH/GSSG ratio was observed in both acclimatedA1) Moti and C306 leaves during mild water-stress (Fig. 6).ubsequent severe water stress imposition resulted in significantecline (P < 0.05) in the total glutathione, GSH contents andSH/GSSG ratio in both acclimated (A2) and non-acclimated

D) Moti leaves with the former exhibiting less reduction than theatter. Acclimated C306 leaves exhibited less decrease in GSHevel and GSH/GSSG ratio than non-acclimated C306 plantsuring severe water stress (Fig. 6). Both acclimated and non-cclimated plants of C306 exhibited complete recovery of GSHool after rewatering, whereas acclimated Moti plants showedoor recovery of GSH pool (Fig. 6).

. Discussion

The response of resistant and susceptible wheat genotypeso water deficit-induced oxidative stress and antioxidant man-gement, at a particular growth stage has been reported in theiterature (Loggini et al., 1999; Sgherri et al., 2000; Lascano

s(tI

d Experimental Botany 60 (2007) 276–283 281

t al., 2001). However their response has not been evaluatedo multiple cycles of drought stress with intermittent recoverycross growth stages under field conditions. It is well known thatreezing tolerance is an inducible, genetically determined char-cter. To achieve the full genetic potential of freezing tolerance,lants must be cold acclimated (Guy, 1990; Thomashow, 1999).rought tolerance of crop plants is also a genetically deter-ined character but interaction with environment determines

he expression of the plant traits. Lascano et al. (2001) observedo clear correlation between water-stress tolerance and antioxi-ant system behaviour between drought-tolerant and susceptibleheat cultivars under field conditions. The present study showed

lear difference in the participation of antioxidant defense sys-em in the drought tolerance of wheat (T. aestivum L.) cultivarsnder field conditions when subjected to drought stress with andithout acclimation treatment.In the present study, during mild and severe water stress, the

rought-resistant cv. C306 maintained better leaf water relationsn terms of turgor potential (P) and RWC as compared to suscep-ible cv. Moti under both acclimated as well as non-acclimatedonditions (Table 1). Maintenance of favourable plant water rela-ions is vital for the development of drought resistance in croplants (Blum, 1996; Lilley and Ludlow, 1996; Passioura, 2002).uring control (unstressed) conditions, increase in H2O2 accu-ulation and decrease in MSI was observed from vegetative to

eproductive growth phase in drought-susceptible cultivar Motis compared to drought-tolerant cultivar C306 under field con-itions (Fig. 2). This might be due to the higher activity of H2O2etoxifying enzymes such as APX, CAT and POX in C306 asompared to Moti plants (Figs. 3 and 4).

The exposure of water deficit of severe nature led to differ-ntial H2O2 accumulation, membrane damage (MSI) and ROScavenging response in the acclimated and non-acclimated C306nd Moti plants. Under severe water deficit, acclimated (A2)oti plants and non-acclimated (D) plants of both the cultivars

howed enhanced SOD activity without concomitant increasen H2O2 scavenging APX leading to increase in H2O2 levelsn these plants (Figs. 2 and 3). In contrast, acclimated (A2)306 plants showed less increase in H2O2 level due to lower

nduction in SOD and higher APX activity. Differential O2•−

adical generation under drought stress in susceptible and tol-rant wheat cultivars has been linked to lipid composition ofhylakoidal membrane and thylakoidal electron transport rateQuartacci et al., 1994; Sgherri et al., 1996). Our results sug-ested that maintenance of favourable water relations in therought-acclimated (A2) C306 plants might have contributedowards the regulation of ROS generation (Selote et al., 2004).here is need to understand the biochemical and molecularechanisms underlying the regulation of ROS generation under

cclimated conditions.Both drought-acclimated (A2) and non-acclimated (D) Moti

eaves as well as non-acclimated (D) C306 leaves exhibitedignificantly higher membrane damage due to severe water

tress as compared to drought-acclimated (A2) C306 plantsFig. 2). This is due to systematic increase in H2O2 levels dueo lack of increase in H2O2 scavenging APX, CAT and POX.n contrast, drought-acclimated C306 plants showed increase

2 tal a

ilactwtaRwIlFamifi(taAcalis2

iHtAlet(eoaa

cmittwnoit

A

l(

R

AA

B

B

B

C

G

L

L

L

L

M

M

M

M

M

M

N

N

P

Q

S

S

S

S

82 R. Khanna-Chopra, D.S. Selote / Environmen

n overall enzyme activities (Figs. 3 and 4). We have observedess membrane lipid peroxidation in cultivar C306 in drought-cclimated as compared to non-acclimated plants under potulture conditions (Selote et al., 2004). During cold-acclimationreatment, no significant difference in the antioxidant defenseas observed between frost-resistant and -sensitive wheat geno-

ypes but cold-acclimated seedlings exhibited enhanced POXctivity in both leaf and root tissues (Scebba et al., 1999).ecently higher POX activity has been shown to be associatedith higher water retention in leaves (Mercado et al., 2004).

n consistence with this, the drought-acclimated (A2) C306eaves also showed higher RWC and POX activity (Table 1,ig. 4). In sunflower seedlings an increase in GSH levels andn induction of ascorbate–glutathione cycle enzymes duringild water stress minimized the oxidative damage, but decrease

n AsA, GSH content and AsA–GSH cycle enzymes intensi-ed oxidative processes during severe water stress conditionsSgherri and Navari-Izzo, 1995). Lesser oxidative damage inhe tolerant wheat cultivar during osmotic stress has beenttributed to higher AsA and GSH content, and induction ofsA–GSH cycle enzymes (Lascano et al., 2001). The sus-

eptibility of an Arabidopsis mutant with low ascorbic acidnd transgenic Arabidopsis plants with less than 5% wild-typeeaf GSH content to oxidative damage further highlighted theirmportance in oxidative stress management under environmentaltress conditions (Xiang et al., 2001; Munne-Bosch and Alegre,002).

In the present study, susceptibility of Moti leaves dur-ng severe water stress was evident from the failure in

2O2 management (Figs. 3 and 4) and by drastic oxida-ion of ascorbate–glutathione pool and significant reduction insA/DHA and GSH/GSSG ratio (Figs. 5 and 6). The high H2O2

evel and/or oxidation of ascorbate pool might have inhibitoryffect on antioxidant enzymes particularly APX and GR inhe susceptible Moti leaves and in non-acclimated C306 leavesShigeoka et al., 2002). Drought-tolerant cv. C306 exhibited anxcellent recovery capacity than susceptible cv. Moti in termsf membrane stability (Fig. 2) due to down-regulation of SODnd up-regulation of H2O2 scavenging enzymes (Figs. 3 and 4)nd regeneration of ascorbate–glutathione pool (Figs. 5 and 6).

In conclusion, genotypic differences in drought toleranceould be, at least in part, attributed to the ability of plants to accli-ate and induce antioxidant defense under severe water stress

n wheat. Drought-resistant wheat genotype acclimated betterhan susceptible genotype by maintaining higher water rela-ions, low ROS accumulation and oxidative damage by inducingell-coordinated antioxidant defense, which included mainte-ance of ascorbate–glutathione redox pool and up-regulationf all the studied antioxidant enzymes. Drought-acclimationnduced oxidative stress resistance is thus mostly dependent onhe genetic potential of the genotypes.

cknowledgement

This research was supported by the grants of National Fel-ow (16-15) project of Indian Council of Agricultural ResearchICAR), New Delhi, to Dr. Renu Khanna-Chopra.

S

nd Experimental Botany 60 (2007) 276–283

eferences

ebi, H., 1984. Catalase in vitro. Meth. Enzymol. 105, 121–126.sada, K., 1999. The water–water cycle in chloroplasts: scavenging of active

oxygen and dissipation of excess photons. Annu. Rev. Plant Physiol. PlantMol. Biol. 50, 601–639.

arrs, H.D., Weatherly, P.E., 1962. A re-examination of the relative turgid-ity technique for estimating water deficits in leaves. Aust. J. Biol. Sci. 15,413–428.

eauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assay andan assay applicable to acrylamide gels. Anal. Biochem. 44, 276–287.

lum, A., 1996. Crop responses to drought and the interpretation of adaptation.Plant Growth Regul. 20, 135–148.

hance, B., Maehly, A.C., 1955. Assay of catalase and peroxidases. Meth.Enzymol. 2, 764–775.

uy, C.L., 1990. Cold-acclimation and freezing stress tolerance: role of proteinmetabolism. Annu. Rev. Plant Physiol. Plant Mol. Biol. 41, 187–223.

ascano, H.R., Antonicelli, G.E., Luna, C.M., Melchiorre, M.N., Gomez, L.D.,Racca, R.W., Trippi, V.S., Casano, L.M., 2001. Antioxidant system responseof different wheat cultivars under drought: field and in vitro studies. Aust.J. Plant Physiol. 28, 1095–1102.

illey, J.M., Ludlow, M.M., 1996. Expression of osmotic adjustment and dehy-dration tolerance in diverse rice lines. Field Crops Res. 48, 185–197.

oggini, B., Scartazza, A., Brugnoli, E., Navari-Izzo, F., 1999. Antioxidantdefense system, pigment composition, and photosynthetic efficiency in twowheat cultivars subjected to drought. Plant Physiol. 119, 1091–1099.

owry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, H.J., 1951. Protein mea-surement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275.

enconi, M., Sgherri, C.L.M., Pinzino, C., Navari-Izzo, F., 1995. Activatedoxygen production and detoxification in wheat plants subjected to a waterdeficit programme. J. Exp. Bot. 46, 1123–1130.

ercado, J.A., Matas, A.J., Heredia, A., Valpuesta, V., Quesada, M., 2004.Changes in the water binding characteristics of the cell walls from trans-genic Nicotiana tabacum leaves with enhanced levels of peroxidase activity.Physiol. Plant 122, 504–512.

ittler, R., 2002. Oxidative stress, antioxidants and stress tolerance. TrendsPlant Sci. 7, 405–410.

oinuddin, Khanna-Chopra, R., 2004. Osmotic adjustment in chickpea in rela-tion to seed yield and yield parameters. Crop Sci. 44, 449–455.

oller, I.M., 2001. Plant mitochondria and oxidative stress: electron transport,NADPH turnover, and metabolism of reactive oxygen species. Annu. Rev.Plant Physiol. Plant Mol. Biol. 52, 561–591.

unne-Bosch, S., Alegre, L., 2002. Interplay between ascorbic acid andlipophilic antioxidant defenses in chloroplast of water-stressed Arabidopsisplants. FEBS Lett. 524, 145–148.

akano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbatespecific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867–880.

octor, G., Foyer, C.H., 1998. Ascorbate and glutathione: keeping active oxygenunder control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 249–279.

assioura, J.B., 2002. Environmental plant biology and crop improvement.Funct. Plant Biol. 29, 537–546.

uartacci, M.F., Sgherri, C.L.M., Pinzino, C., Navari-Izzo, F., 1994. Superoxideradical production in wheat plants differently sensitive to drought. Proc. R.Soc. Edinb. 102B, 287–290.

cebba, F., Sebastiani, L., Vitagliano, C., 1999. Protective enzymes against acti-vated oxygen species in wheat (Triticum aestivum L.) seedlings: responsesto cold acclimation. J. Plant Physiol. 155, 762–768.

chaedle, M., Bassham, J.A., 1977. Chloroplast glutathione reductase. PlantPhysiol. 59, 1011–1012.

cholander, P.E., Hammel, H.T., Hemmingsen, E.E., Bradstreet, E.D., 1964.Hydrostatic pressure and osmotic potential in leaves of mangroves and otherplants. Proc. Natl. Acad. Sci. U.S.A. 52, 119–125.

elote, D.S., Khanna-Chopra, R., 2004. Drought-induced spikelet sterility is

associated with an inefficient antioxidant defense in rice panicles. Physiol.Plant 121, 462–471.

elote, D.S., Bharti, S., Khanna-Chopra, R., 2004. Drought acclimation reducesO2

•− and lipid peroxidation in wheat seedlings. Biochem. Biophys. Res.Commun. 314, 724–729.

tal an

S

S

S

S

S

S

S

S

T

W

W

R. Khanna-Chopra, D.S. Selote / Environmen

gherri, C.L., Navari-Izzo, M.F., 1995. Sunflower seedlings subjected to increas-ing water deficit stress: oxidative stress and defense mechanisms. Physiol.Plant 93, 25–30.

gherri, C.L.M., Pinzino, C., Navari-Izzo, F., 1996. Sunflower seedlings sub-jected to increasing stress by water deficit: changes in O2

•− productionrelated to the composition of thylakoid membranes. Physiol. Plant 96,446–452.

gherri, C.L.M., Maffei, M., Navari-Izzo, F., 2000. Antioxidative enzymes inwheat subjected to increasing water deficit and rewatering. J. Plant Physiol.157, 273–279.

hanahan, J.F., Edwards, I.B., Quick, J.S., Fenwick, J.R., 1990. Membranethermostability and heat tolerance of spring wheat. Crop Sci. 30, 247–251.

higeoka, S., Ishikawa, T., Tamoi, M., Miyagawa, Y., Takeda, T., Yabuta, Y.,Yoshimura, K., 2002. Regulation and function of ascorbate peroxidase isoen-

zymes. J. Exp. Bot. 53, 1305–1319.

inha, S.K., Aggarwal, P.K., Chaturvedi, G.S., Singh, A.K., Kailasnathan,K.K., 1986. Performance of wheat and triticale cultivars in a variablesoil–water environment. I. Grain yield stability. Field Crops Res. 13, 289–299.

X

d Experimental Botany 60 (2007) 276–283 283

inha, S.K., Khanna-Chopra, R., Aggarwal, P.K., Chaturvedi, G.S., Koundal,K.R., 1982. Effect of drought on shoot growth: significance of metabolismto growth and yield. In: Drought Resistance in Crop with Emphasis on Rice.IRRI, Manila, Philippines, pp. 153–169.

rivalli, B., Sharma, G., Khanna-Chopra, R., 2003. Antioxidative defence sys-tem in an upland rice cultivar subjected to increasing intensity of water stressfollowed by recovery. Physiol. Plant 119, 503–512.

homashow, M.F., 1999. Plant cold acclimation: freezing tolerance genes andregulatory mechanisms. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50,571–599.

ang, S.Y., Jiao, H.J., Faust, M., 1991. Changes in ascorbate, glutathione, andrelated enzyme activities during thidiazuron-induced bud break of apple.Physiol. Plant 82, 231–236.

arm, E., Laties, G.G., 1982. Quantification of hydrogen peroxide in plant

extracts by the chemiluminescence reaction with luminol. Phytochemistry21, 827–831.

iang, C., Werner, B.L., Christensen, E.M., Oliver, D.J., 2001. The biologi-cal functions of glutathione revisited in Arabidopsis transgenic plants withaltered glutathione levels. Plant Physiol. 126, 564–574.