9
J[ Agronomy + Crop Science 074\ 108*116 "1999# Þ 1999 Blackwell Wissenschafts!Verlag\ Berlin ISSN 9820!1149 Division of Plant Physiolo`y\ Indian A`ricultural Research Institute\ New Delhi\ India Physiological and Biochemical Responses of Hexaploid and Tetraploid Wheat to Drought Stress V[ Chandrasekar\ R[ K[ Sairam and G[ C[ Srivastava Authors| address] Mr V[ Chandrasekar\ Dr R[ K[ Sairam and Dr G[ C[ Srivastava\ Division of Plant Physiology\ Indian Agricultural Research Institute\ New Delhi!009 901\ India With 2 _gures and 1 tables Received January 06\ 1999^ accepted April 00\ 1999 Abstract An experiment was conducted to investigate the physio! logical and biochemical responses of two hexaploids viz[\ C 295 "water stress tolerant# and Hira "water stress sus! ceptible#\ and two tetraploids\ HW 13 "Triticum dicoccum# and A 8!29!0 "Triticum durum# wheat genotypes to water stress under pot culture condition[ Water stress was imposed for a uniform period of 09 days at 49\ 59 and 69 days after sowing "DAS# and observations were recorded at 59\ 69 and 79 DAS[ Total dry matter and plant height were recorded at harvest[ Water stress caused a decline in relative water content "RWC#\ chlorophyll and carotenoid content\ membrane stability and nitrate reductase activity and increased accumulation of proline at all stages and abscisic acid "ABA# at 79 DAS in all the genotypes[ Both the tetraploids showed a lower reduction in RWC and highest ABA accumulation under water stress[ Among the hexaploids Hira showed the most decline in RWC and the lowest ABA accumulation[ The tetraploids also showed comparatively higher carotenoid content and membrane stability\ closely followed by C 295\ while Hira showed the minimum response under water stress[ Nitrate reductase activity and chlorophyll content under irrigated conditions were highest in Hira but under water stress the lowest per cent decline was observed in C 295\ followed by HW 13\ A 8!29!0\ and Hira[ Proline accumulation under water stress conditions was highest in hexaploids C 295 and Hira and lowest in tetraploids HW 13 and A 8!29!0[ Tetraploids HW 13\ followed by A 8!29!0 maintained higher plant height and total dry matter "TDM# under water stress and also showed a lower per cent decline under stress than hexaploids C 295 and Hira[ From the results it is clear that proline accumulation did not contribute to better drought tolerance of tetraploids than hexaploids[ It is also apparent that water stress tolerance is the result of the cumulative action of various physiological processes\ and all the parameters:processes may not be positively associ! ated with the drought tolerance of a particular tolerant genotype[ Key words] abscisic acid * carotenoid * chloro! phyll * membrane stability * nitrate reductase * proline * water stress * wheat U[S[ Copyright Clearance Center Code Statement] 9820Ð1149:1999:7493Ð9108 ,04[99:9 Introduction Shortage of water limits plant growth and pro! ductivity more than any other environmental factor "Boyer 0871#[ Wheat is grown all over the world in rainfall ranges of 29Ð002 cm[ Sinha et al[ "0874# stated that productivity in drought!a}ected and insu.ciently irrigated areas continues to be low[ Improvement of wheat productivity for this abiotic stress is therefore an important objective of research[ Due to their better adaptation under hot and arid regions\ tetraploid durum and dicoccum wheat "Triticum durum and Triticum dicoccum# are usually regarded as more tolerant to stress con! ditions than hexaploid wheat "Waines 0883#[ However\ the physiological basis of their stress tol! erance is not well understood[ An understanding of how plants respond to water de_cits and\ in certain instances\ are able to tolerate them should lead us eventually to ways of optimizing plant productivity in marginal environments "Smith and Gri.th 0882#[ Water stress tolerance in crop plants has been associated with various physiological factors such as levels of a certain stress hormone viz[\ ABA "Mal! donado et al[ 0866#\ which regulates stomata con! ductance\ and hence water loss under desiccation "Kriedemann et al[ l861#\ and accumulation of osmolites like proline\ manitol\ glycine betaine and soluble sugars which lower the osmotic potential of the cell sap and thus prevent the movement of water out of cell "Stewart et al[ l855\ Storey and Wyn! Jones l866#[ Besides these\ researchers have linked various physiological responses of crop plants to drought with their tolerance mechanisms such as high rela! tive water content and water potential "Clarke and McCaig l871\ Ritchie et al[ l889#\ membrane sta! bility "Kaur et al[ l877\ and Sairam et al[ l889#\

Physiological and Biochemical Responses of Hexaploid and Tetraploid Wheat to Drought Stress

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Page 1: Physiological and Biochemical Responses of Hexaploid and Tetraploid Wheat to Drought Stress

J[ Agronomy + Crop Science 074\ 108*116 "1999#Þ 1999 Blackwell Wissenschafts!Verlag\ BerlinISSN 9820!1149

Division of Plant Physiolo`y\ Indian A`ricultural Research Institute\ New Delhi\ India

Physiological and Biochemical Responses of Hexaploid and Tetraploid Wheat

to Drought Stress

V[ Chandrasekar\ R[ K[ Sairam and G[ C[ Srivastava

Authors| address] Mr V[ Chandrasekar\ Dr R[ K[ Sairam and Dr G[ C[ Srivastava\ Division of Plant Physiology\ Indian

Agricultural Research Institute\ New Delhi!009 901\ India

With 2 _gures and 1 tables

Received January 06\ 1999^ accepted April 00\ 1999

Abstract

An experiment was conducted to investigate the physio!logical and biochemical responses of two hexaploids viz[\C 295 "water stress tolerant# and Hira "water stress sus!ceptible#\ and two tetraploids\ HW 13 "Triticum dicoccum#and A 8!29!0 "Triticum durum# wheat genotypes to waterstress under pot culture condition[ Water stress wasimposed for a uniform period of 09 days at 49\ 59 and 69days after sowing "DAS# and observations were recordedat 59\ 69 and 79 DAS[ Total dry matter and plant heightwere recorded at harvest[ Water stress caused a decline inrelative water content "RWC#\ chlorophyll and carotenoidcontent\ membrane stability and nitrate reductase activityand increased accumulation of proline at all stages andabscisic acid "ABA# at 79 DAS in all the genotypes[ Boththe tetraploids showed a lower reduction in RWC andhighest ABA accumulation under water stress[ Among thehexaploids Hira showed the most decline in RWC and thelowest ABA accumulation[ The tetraploids also showedcomparatively higher carotenoid content and membranestability\ closely followed by C 295\ while Hira showed theminimum response under water stress[ Nitrate reductaseactivity and chlorophyll content under irrigated conditionswere highest in Hira but under water stress the lowest percent decline was observed in C 295\ followed by HW 13\A 8!29!0\ and Hira[ Proline accumulation under waterstress conditions was highest in hexaploids C 295 and Hiraand lowest in tetraploids HW 13 and A 8!29!0[ TetraploidsHW 13\ followed by A 8!29!0 maintained higher plantheight and total dry matter "TDM# under water stress andalso showed a lower per cent decline under stress thanhexaploids C 295 and Hira[ From the results it is clearthat proline accumulation did not contribute to betterdrought tolerance of tetraploids than hexaploids[ It is alsoapparent that water stress tolerance is the result of thecumulative action of various physiological processes\ andall the parameters:processes may not be positively associ!ated with the drought tolerance of a particular tolerantgenotype[

Key words] abscisic acid * carotenoid * chloro!phyll * membrane stability * nitrate reductase *proline * water stress * wheat

U[S[ Copyright Clearance Center Code Statement] 9820Ð1149:1999:7493Ð9108 ,04[99:9

Introduction

Shortage of water limits plant growth and pro!ductivity more than any other environmental factor"Boyer 0871#[ Wheat is grown all over the world inrainfall ranges of 29Ð002 cm[ Sinha et al[ "0874#stated that productivity in drought!a}ected andinsu.ciently irrigated areas continues to be low[Improvement of wheat productivity for this abioticstress is therefore an important objective ofresearch[ Due to their better adaptation under hotand arid regions\ tetraploid durum and dicoccumwheat "Triticum durum and Triticum dicoccum# areusually regarded as more tolerant to stress con!ditions than hexaploid wheat "Waines 0883#[However\ the physiological basis of their stress tol!erance is not well understood[ An understanding ofhow plants respond to water de_cits and\ in certaininstances\ are able to tolerate them should lead useventually to ways of optimizing plant productivityin marginal environments "Smith and Gri.th 0882#[Water stress tolerance in crop plants has beenassociated with various physiological factors suchas levels of a certain stress hormone viz[\ ABA "Mal!donado et al[ 0866#\ which regulates stomata con!ductance\ and hence water loss under desiccation"Kriedemann et al[ l861#\ and accumulation ofosmolites like proline\ manitol\ glycine betaine andsoluble sugars which lower the osmotic potential ofthe cell sap and thus prevent the movement of waterout of cell "Stewart et al[ l855\ Storey and Wyn!Jones l866#[

Besides these\ researchers have linked variousphysiological responses of crop plants to droughtwith their tolerance mechanisms such as high rela!tive water content and water potential "Clarke andMcCaig l871\ Ritchie et al[ l889#\ membrane sta!bility "Kaur et al[ l877\ and Sairam et al[ l889#\

Page 2: Physiological and Biochemical Responses of Hexaploid and Tetraploid Wheat to Drought Stress

119 Chandrasekar et al[

pigment content and stability under stress "Sairamet al[ l889\ Sairam et al[ l886:87 and Kraus et al[l884# and nitrate reductase activity "Sairam andDube l873\ Sairam et al[ l889\ He et al[ l885#[However\ reports regarding variations in thesephysiological parameters on genotypic basis or dueto ploidy levels are very rare[

The present study was therefore planned to ident!ify the parameters associated with better droughttolerance of tetroploid wheat in comparison to hex!aploid wheat with the objective of using them asselection indices in breeding programmes[

Materials and Methods

The present investigation was undertaken during the win!ter season of 0887Ð88 under pot!culture conditions[Physiological responses were studied under water stressby withholding water supply for a uniform period of 09days during preanthesis phase at 49\ 59\ and 69 days aftersowing "DAS# and samples were collected at 59\ 69 and79 DAS[ Irrigated and fully turgid plants were used asa control[ The plant material used in this experimentconsisted of 1 hexaploids "Triticum aestivum# Hira "HD0834# "drought susceptible# and C 295 "drought tolerant#\and 1 tetraploids viz[\ A 8!29!0 "Triticum durum# and HW13 "Triticum dicoccum#[

Earthen pots 29×29 cm in size were _lled with clay!loam soil and farm yard manure in 5 ] 0 ratio[ Each potwas fertilized corresponding to 019\ 59 and 59 kg ha−0 ofN\ P and K\ respectively[ After germination 3 seedlingswere retained in each pot[ Fifty pots of each genotypewere maintained in the net house[ Plants were watered asand when required to keep them fully turgid[ Recom!mended cultural practices were followed[ Samples werecollected in quadruplicate from control and stressedplants between 8[29 am to 09[29 am from the _rst fullyexpanded leaf[ The response of drought stress wasassessed in terms of relative water content "RWC#\ nitratereductase "NR# activity\ chlorophyll "Chl# and carotenoid"Car# content\ membrane stability\ and proline andabscisic acid "ABA# accumulations[

Leaf relative water content "RWC# was estimatedaccording to the method of Weatherley "0849#[ Leaf sam!ples "9[4 g# were saturated in 099ml of water for 3 h andtheir turgid weights were recorded[ Subsequently theywere packed in a butter paper bag\ oven dried at 54 >Cfor 37 h and their dry weights were recorded[ Pigments"chlorophyll and carotenoids# were extracted by the non!maceration method of Hiscox and Israelstam "0868#[ Leafsamples "9[94 g# were incubated in 4ml of dimethyl sul!foxide "DMSO# at 54 >C for 3 h[ Absorbances were re!corded at 534\ 552 and 369 nm and chlorophyll a\ b andtotal chlorophyll were calculated according to Arnon"0838# and carotenoid content according to Lichtenthalerand Wellburn "0872#[

Leaf membrane stability index "MSI# was determinedaccording to the method of Premachandra et al[ "0889#

as modi_ed by Sairam "0883#[ Leaf stripes "9[1 g# of uni!form size were taken in test tubes containing 09ml ofdouble distilled water in two sets[ Test tubes in one setwere kept at 39 >C in a water bath for 29min and electricalconductivity of the water containing the sample was mea!sured "C0# using a conductivity bridge[ Test tubes in thesecond set were incubated at 099 >C in the boiling waterbath for 04min and their electrical conductivity measuredas above "C1#[ MSI was calculated using the formulaegiven below]

MS0� ð0− "C0:C1#Ł×099[

Free proline content in the leaves was determined fol!lowing the method of Bates et al[ "0862#[ Leaf samples"9[4 g# were homogenized in 4ml of sulfosalicylic acid"2)# using a pestle and mortar\ _ltered with Whatmanno[ 0 _lter paper "Whatman International Ltd[\ Kent\UK# and the volume was made up to 09ml with sul!fosalicylic acid[ Two ml extract was taken in a test tubeand to it 1ml of glacial acetic acid and 1ml ninhydrinreagent "0[14 g ninhydrin dissolved in a bland of 29mlglacial acetic acid and 19ml ortho!phosphoric acid# wereadded[ The reaction mixture was boiled in a water bathat 099 >C for 29min[ After cooling the reaction mixture\5ml of toluene was added into it\ and after thoroughmixing\ the chromophore containing toluene was sep!arated and absorbency read at 419 nm in UV!visible spec!trophotometer\ Beckman model M 25 "CA\ USA# againsta toluene blank[

Abscisic acid in the leaf sample was estimated followingthe methods of Zeevart "0879#[ Leaf samples "1 g# col!lected 79 DAS were frozen in liquid nitrogen\ pulverizedand stored at −19 >C until used[ Frozen samples wereextracted three times with 09ml of 79) v:v acetone"79ml acetone\ 0ml glacial acetic acid and 099mg of 1\5 di!tert!butyl 3!methyl phenol in a total volume of099ml# and collected in a 099!ml volumetric ~ask[ Thetissue residue was then homogenized with a pestle andmortar with acetone "79) v:v#[ The homogenate was_ltered through a Whatman no[ 0 _lter[ The _ltrate alongwith the extracts made earlier was transferred to theboiling ~ask of rotary ~ash vacuum evaporator forremoving the acetone[ As the acetone was evaporated\the lipid soluble material was deposited on the walls ofthe boiling ~ask "round bottomed#[ This was dissolved in0) acetic acid solution and the amber coloured aqueoussolution was transferred into small vials "09ml#[ Beforeinjecting the sample into HPLC\ the samples were _lteredwith 9[34mm Millipore _lter using a 1[4ml plasticsyringe[

HPLC "Thermo!separation product\ model SpectraSystem P 1999\ CA\ USA# was used for ABA estimation[It was equipped with a variable wavelength UV!vis detec!tor and a Rheodyne injector "19ml loop#[ The columnused was mBondapak TM:C07 P:N 730:5 S N−0

"Thermo!separation products\ CA\ USA#[ The stationaryphase consisted of a Lichrosorb C!07 and the mobilephase was 0) acetic acid in 84) methanol solutionattached to the pump[ The ~ow rate was adjusted to

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110Drought Stress Responses in Wheat

1[4ml min−0 and the wavelength was set at 154 nm beforeinjecting the sample[ The instrument was set on injectionmode and 19ml of standard ABA "09 p[p[m[#:or samplesolution injected into it and then the instrument wasswitched on to operation mode[ Data on retention timeand area of the peak recorded by the integrator wascollected[ The peaks belonging to ABA were identi_edusing the data registered by the standard ABA[ The con!centration of ABA present in the sample was calculatedby comparison with the area recorded by the standardABA[

Nitrate reductase activity in leaves was measured fol!lowing the in vivo assay described by Klepper et al[ "0860#[Leaves "9[1 g# were cut into 7Ð09mm long segments andincubated in 29ml test tube containing 1[4ml each ofphosphate bu}er "9[0 M\ pH6[4# and 9[0 M potassiumnitrate solution[ The tubes were then in_ltrated using avacuum pump for about 0Ð1min\ until all the leaf seg!ments sunk below the surface of the incubation mediumand incubated in a water bath maintained at 29 >C for29min in dark[ Keeping the tubes in boiling water for1min terminated the reaction[ The tubes were cooled\9[0ml aliquot taken from them and mixed with 0ml eachof sulphanilamide "0) in 0N HCL# and N!"0\ naphthyl!ethylene diamine!dihydrochloride# "9[91) in distilledwater# and the volume was made up to 4ml with distilledwater[ Absorbency was read at 439 nm spectrophoto!metrically[ A standard curve of nitrite was prepared usingdi}erent concentrations of potassium nitrite[ All chemi!cal assays are the means of 3 replicates estimated twice[

Ten pots of each genotype were subjected to waterstress at preanthesis stage "49 DAS# for 09 days and\ afterthe expiry of the stress period\ the pots were irrigated andkept separately for yield analysis[ After maturing\ theplants were harvested from stressed and irrigated pots[Parameters like plant height and total plant biomass wereobtained for comparative study and in~uence of moisturestress on such characteristics[ Data of all the parameterswere analysed for analysis of variance by CRD "Panseand Sukhatme 0856#[

Results

Under irrigated conditions Hira maintained thehighest relative water content "RWC# at all thestages "Fig[ 0A#[ There was a signi_cant reductionin RWC under water stress in all the cultivars[ Theper cent reduction in RWC was the highest in Hira\which also showed the lowest value under waterstress at all the stages[ Tetraploid cultivars A 8!29!0 and HW 13 maintained relatively high RWC aswell as showed lower percent decline as compared tohexaploids under water stress[ Membrane stabilityindex "MSI# decreased under water stress in all thegenotypes "Fig[ 0B#[ HW 13 showed the highest MSIboth under irrigated and water stressed conditions\at all the three stages of sampling[ The per centreduction in MSI under water stress at _rst and

second stages was the lowest in HW 13 followed byA 8!29!0\ C 295 and highest in Hira[ At the thirdstage C 295\ a drought tolerant hexaploid wheat\superseded other cultivars in maintaining leastreduction in MSI\ followed by HW 13 and A 8!29!0 while Hira recorded the greatest loss in MSI understress over control[

Under irrigated condition total chlorophyll "Chl#content was highest in hexaploid cvs[ Hira and C295 as compared to tetraploids A 8!29!0 and HW13 at all the stages "Fig[ 1A#[ There was signi_cantdecrease in Chl content under water stress in all thecultivars[ C 295 followed by HW 13 maintainedhighest Chl content than Hira[ The per centreduction in Chl was highest in Hira "11[16\ 04[33and 12[28 )# at three stages and lowest in C 295at _rst and third stages\ being 09[25 and 02[01 )\respectively[ At second stage of sampling HW 13showed least reduction in Chl under water stress"4[43 )#[ Both the tetraploid genotypes were betterthan hexaploid Hira "drought susceptible genotype#in terms of per cent reduction in Chl content[

Hexaploid wheats Hira and C 295 recorded thehighest carotenoid "Car# content under irrigatedconditions "Fig[ 1B#[ However\ under water!stressconditions tetraploid "HW 13# retained more Carcontent at second and third stages\ while at the _rststage of sampling C 295 showed more Car content[The per cent reduction in Car content under waterstress was highest in Hira\ followed by C 295\ whileboth the tetraploid wheats performed better thanthe hexaploid wheats in maintaining least per centreduction in Car content at all the stages ofsampling[

The variations among the genotypes in prolinecontent under control conditions were not muchand also did not follow any pattern "Fig[ 2A#[ Therewas a steep increase in proline content in all thegenotypes when subjected to water stress[ Theincrease in proline content under water stress wasthe highest in C 295 followed by Hira\ HW 13 andleast in A 8!29!0 at all stages[

Abscisic acid "ABA# content recorded at pre!anthesis stage "79 days after sowing# increased underwater stress in all the genotypes "Table 0#[ HigherABA content was noticed in HW 13 "T[ dicoccum#under both irrigated and water!stress conditions fol!lowed by C 295\ A 8!29!0 and lowest in Hira[

Di}erences in nitrate reductase "NR# activityamong di}erent genotypes were not signi_cant atall the stages under irrigated condition "Fig[ 2B#[NR activity decreased in all the genotypes uponimposition of water stress[ C 295 showed the highest

Page 4: Physiological and Biochemical Responses of Hexaploid and Tetraploid Wheat to Drought Stress

111 Chandrasekar et al[

Fig[ 0] E}ect of water stresson "A# relative water contentand "B# membrane stabilityindex in wheat genotypes[Vertical bars indicate 2SE ofmean[ Data signi_cant atP � 9[94

Fig[ 1] E}ect of water stresson "A# chlorophyll and "B#carotenoid contents in wheatgenotypes[ Vertical bars indi!cate 2SE of mean[ Data sig!ni_cant at P � 9[94

activity and lowest per cent decline under waterstress at _rst stage followed by HW 13\ A 8!29!0and Hira[ At the second and third stages "69\ 79DAS# of sampling\ tetraploid genotype HW 13showed the least reduction in enzyme activity fol!lowed by C 295 and A 8!29!0 while Hira showedgreatest reduction under water stress[

Data on plant height and biomass yield are pre!

sented in Table 1[ Although Hira\ a triple dwarf\ hasthe lowest plant height\ it showed higher reductionin plant height\ followed by C 295\ A 8!29!0 andHW 13 in decreasing order "05[83\ 8[54\ 6[50\ 7[54 )in Hira\ C 295\ A 8!29!0 and HW 13\ respectively#[Total dry matter per plant decreased under waterstress in all the cultivars but was more pronouncedin the hexaploids[ The reduction in dry matter per

Page 5: Physiological and Biochemical Responses of Hexaploid and Tetraploid Wheat to Drought Stress

112Drought Stress Responses in Wheat

Fig[ 2] E}ect of water stresson "A# proline content and "B#nitrate reductase activity inwheat genotypes[ Verticalbars indicate 2SE of mean[Data signi_cant at P � 9[94

Table 0] E}ect of water stress on abscisic acid content "mgg−0 dry weight# in wheat genotypes

Cultivars Irrigated Stress Per cent increase

Hira 0[89 1[74 49[99C 295 2[99 3[52 43[22A 8!29!0 1[36 3[95 53[26HW 13 2[21 5[37 84[07LSD "P�9[94#Cv[ 9[064Stress 9[136Cv[× stress 9[238

Cv[] cultivar[ LSD] least signi_cant di}erence[

Table 1] E}ect of water stress on plant height and total dry matter production at harvest in wheat genotypes

Plant height "cm# Total dry matter "g plant−0#

Cultivars Irrigated Stress Per cent decrease Irrigated Stress Per cent decrease

Hira 51[99 40[49 05[83 36[64 17[04 30[99C 295 74[49 66[14 98[54 35[05 20[17 21[19A 8!29!0 81[99 74[99 96[50 36[22 24[49 13[88HW 13 75[64 68[14 97[54 38[05 27[24 10[87LSD "P�9[94#Cv[ 1[07 92[12Stress 1[41 92[62Cv[× stress 93[25 93[50

plant under water stress was the highest in Hira"30 )#\ followed by C 295 "21[1 )#\ A 8!29!0"13[8 )# and the least in HW 13 "10[87 )#[

Discussion

Wheat crop responds to water de_cit in the form ofchanges in various physiological and biochemicalprocesses[ The physiological changes observedcould be consequences of deleterious e}ects of waterde_cit on important metabolic processes as well asresponses of various defence mechanisms adaptedby the plant under drought stress[

The tetraploid genotypes showed the highestRWC as well as a lower per cent decline under stress

Page 6: Physiological and Biochemical Responses of Hexaploid and Tetraploid Wheat to Drought Stress

113 Chandrasekar et al[

at all the stages[ The results are in agreement withthe _ndings of Al!Hakimi and Mannoveux "0882#who reported high leaf water potential and RWCunder drought in tetraploids[ C 295\ a hexaploidvariety recommended for rainfed cultivationshowed higher RWC under water stress than Hira[Signi_cant di}erences in RWC:water potential intolerant and susceptible genotypes of barley "Martinet al[ 0878# and wheat "Kraus et al[ 0884# have alsobeen reported[

The highest membrane stability and minimum percent decline under water stress in tolerant genotypeHW 13 followed by A 8!29!0 and C 295 is in accord!ance with the reports of Blum and Ebercorn "0865\0870# in sorghum and wheat\ and Premachandra etal[ "0889# in maize[ Decrease in MSI re~ects theextent of lipid peroxidation caused by active oxygenspecies "Dhindsa et al[ 0870#[ The better per!formance of tetraploid genotypes under water stressin terms of MSI points to their better adaptationunder adverse conditions[ It also shows that lipidperoxidation activity is less or limited in them[

Chlorophyll maintenance is essential for photo!synthesis under drought stress[ The results showthat drought!tolerant genotypes C 295 "hexaploid#\and HW 13 and A 8!29!0 "tetraploids# showed lowerreduction in Chl content than susceptible Hira[Higher Chl content and lower per cent decreaseunder stress in tolerant genotype of wheat "Krauset al[ 0884\ Sairam et al[ 0886\ 0886:87# have alsobeen reported[

Carotenoids are responsible for the scavengingof singlet oxygen "Knox and Dodge 0874#\ hencecomparatively higher Car levels in A 8!29!0\ HW 13and C 295 demonstrates their tolerance capacity[Higher levels of Car in tolerant genotypes have alsobeen reported by Gummuluru et al[ "0878#\ Krauset al[ "0884# and Sairam et al[ "0886:87#[

Increase in proline accumulation observed underwater stress in all the four cultivars was in accord!ance with the _ndings of Baynett and Naylor "0855#in bermuda grass\ Singh et al[ "0861# in barley andKhan "0866# and Thakur and Rai "0879# in maize[The drought!resistant C 295 showed maximumaccumulation of proline content under water stressamong the four genotypes studied\ which is in linewith observations of Singh and Singh "0872# inwheat who observed the highest proline content indrought!resistant HD 0870 and the lowest in sen!sitive UP 208[ Both the tetraploid genotypes wereinferior to drought!sensitive genotype Hira in termsof proline content under water stress\ suggestingthat possibly the proline based drought tolerance

mechanism is not operative in tetraploid genotypesincluded in this study[

Stress hormone ABA regulate stomatal con!ductance in leaves and hence checks out ~ow ofwater vapours under conditions of water de_cit"Loveys and Kriedemann 0862 and Beardsell andCohen 0864#[ A tolerant genotype therefore shouldshow a higher ABA accumulation under stress[ Inthis study\ tetraploid genotype HW 13 showed thehighest increase in the content of ABA under waterstress followed by hexaploid C 295 and A 8!29!0 "tetraploid#[ The drought!susceptible hexaploidgenotype Hira showed minimum ABA contentunder water!stress conditions\ suggesting a higherstomatal conductance and water loss\ whichexplains its lowest RWC under stress[ The results arefurther in conformity with the _ndings of Larque!Saavedra and Wain "0863\ 0865#\ who reportedhigher ABA content in drought!tolerant lines ofmaize and sorghum than in drought!susceptiblelines in response to partial desiccation[

Nitrate reductase is a key enzyme regulatingnitrogen metabolism[ Its activity is adversely a}ec!ted by water stress "Plaut 0862\ Morilla et al[ 0862\Zhang et al[ 0882#[ Although the drought!sus!ceptible genotype Hira showed the highest activityunder irrigated conditions\ it also registered the gre!atest decline and showed the lowest activity underwater stress among all the genotypes[ The tolerantgenotypes of both hexaploid and tetraploids wereable to maintain higher activity under water stress[The results are in general agreement with the _nd!ings of Sairam and Dube "0873#\ Sairam et al["0889#\ Sairam "0883# and He et al[ "0885#[

The lowest per cent reduction in plant height andtotal dry matter "TDM# in both the tetraploid geno!types "HW 13 and A 8!29!0#\ followed by C 295 andmaximum reduction in Hira\ suggest that both thetetraploid genotypes HW 13 and A 8!29!0 are moretolerant to water stress than the hexaploid wheats C295 and Hira[ However\ C 295\ hexaploid genotype\was not far behind and was superior to Hira in termsof lower degree of losses experienced under waterstress[ Better drought adaptability of tetraploidsover hexaploids has also been reported by Hansonet al[ "0871#[

From the results it is apparent that tetraploidsHW 13 "T[ dicoccum#\ A 8!29!0 "T[ durum# and C 295"hexaploid# are more tolerant than Hira "hexaploid#[Stress hormone ABA\ which regulates stomataldi}usion resistance and hence transpirational losses"Beardsell and Cohen 0864#\ was highest in the twotetraploids\ followed by C 295 and lowest in Hira[

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114Drought Stress Responses in Wheat

Thus\ it is clear that higher RWC of tetraploids"T[ dicoccum and T[ durum# under stress is directlyassociated with their ABA content[ On the otherhand Hira which had the lowest ABA accumulationalso exhibited the lowest RWC[ Retention of higherwater content in tetraploids due to ABA regulatedstomatal conductance might also be responsible forthe maintenance of higher activity of essentialenzymes\ such as nitrate reductase\ which has agrowth and yield regulatory function[ Thus\ it canbe concluded that a lower per cent reduction in plantheight and biomass under conditions of water stressin tetraploids "T[ durum and T[ dicoccum# as com!pared to hexaploid genotypes C 295 "tolerant# andHira "susceptible# are possibly associated with theirbetter water retention capacity\ which may be linkedto their ABA content\ lower pigment bleaching\higher membrane stability and nitrate reductaseactivity[ C295\ a rainfed variety\ though slightlyinferior to HW 13\ was superior to Hira "drought!stress susceptible#\ as its physiological and bio!chemical activities were geared at to a higher levelthan Hira[ Proline accumulation\ which is usuallyassociated with stress tolerance\ was lower in tetra!ploids as compared to hexaploids and this could wellbe due to the absence of D!genome in tetraploids[

It is thus apparent that drought tolerance of agiven genotype is not limited to a particular physio!logical character[ A 8!29!0 "T[ durum# though wasmore tolerant than C 295 in terms of a lower percent reduction in TDM and plant height under waterstress\ it showed a higher per cent decline for chloro!phyll content and NR activity than C 295[ Thus itseems that all the parameters associated withdrought!stress tolerance may not be uniformlyfavourable in all the tolerant genotypes[ It maytherefore be useful to identify drought!tolerantgenotypes on the basis of their response to morethan one parameter\ such as RWC\ membrane sta!bility and ABA[

Zusammenfassung

Physiologische und biochemische Reaktionen vonhexaploidem und tetraploidem Weizen gegenu�berDu�rrestress

Es wurde ein Experiment durchgefu�hrt\ um die physiolo!gischen und biochemischen Reaktionen von zweihexaploiden viz[ C 295 "Du�rrestress!tolerant# und Hira"Du�rrestress!emp_ndlich# sowie zwei tetraploiden\ HW13 "T[ dicoccum# und A 8!29!0 "T[ durum# Weizentypengegenu�ber Du�rrestress in Gefa�)kulturen zu untersuchen[Du�rrestress wurde in gleichen Perioden von zehn Tagenzum 49[\ 59[ und 69[ Tag nach der Saat "DAS# angewen!

det^ die Beobachtungen wurden am 59[\ 69[ und 79[ DASdurchgefu�hrt[ Zum Zeitpunkt der Ernte wurdenGesamttrockenmasse und P~anzenho�he bestimmt[ Du�r!restress verursachte einen Ru�ckgang im relativen Wasser!gehalt "RWC#\ Chlorophyll! und Karotinoidgehalt\Membranstabilita�t und Nitratreduktaseaktivita�t underho�hte bei allen Genotypen zu allen Stadien die Akku!mulation von Prolin und Abscisinsa�ure "ABA# am 79[DAS[ Die beiden tetraploiden Sorten zeigten eine gerin!gere Reduktion in RWC und die ho�chste ABA!Akkumu!lation unter Du�rrestress[ Unter den hexaploiden zeigteHira eine maximale Abnahme in RWC und die geringsteAkkumulation von ABA[ Die Tetraploiden zeigten ver!gleichsweise ho�here Karotinoidgehalte undMembranstabilita�t\ dicht gefolgt von C 295\ wa�hrendHira eine minimale Reaktion auf Du�rrestress zeigte[Nitratreduktaseaktivita�t und Chlorophyllgehalt warenunter Bewa�sserungsbedingung am ho�chsten bei Hira\unter Du�rrestress wurde dagegen bei C 295\ gefolgt vonHW 13 und A 8!29!0 die geringste Abnahme sowie maxi!mal bei Hira beobachtet[ Die Prolinakkumulation warunter Du�rrestress am ho�chsten in der hexaploiden C 295und Hira und am geringsten in den tetraploiden HW 13und A 8!29!0[ Tetraploide HW 13 gefolgt von A 8!29!0zeigten eine gro�)ere P~anzenla�nge und Gesamttrocken!masse "TDM# unter Du�rrebedingungen^ sie zeigten einegeringere prozentuale Abnahme unter Wasserstress imVergleich zur hexaploiden C 295 und Hira[ Aus diesenErgebnissen kann abgeleitet werden\ dass die Prolinakku!mulation keine Beitrag zur Verbesserung der Du�rretole!ranz von tetraploiden im Vergleich zu hexaploiden leistet[Es ist o}ensichtlich\ dass Du�rretoleranz ein Ergebnis derkumulativen Aktion verschiedener physiologischer Pro!zesse ist und alle Parameter:Prozesse wahrscheinlichnicht positiv assoziiert mit der Du�rretoleranz einzelnertoleranter Genotypen sind[

Acknowledgements

The authors are grateful to Dr "Mrs[# Prem Dureja\ Divi!sion of Agricultural Chemicals\ for her help in ABAestimation[ V[C[ is also grateful to Indian Council ofAgricultural Research for the award of fellowship duringthe course of the study[

References

Al!Hakimi\ A[\ and P[ Mannoveux\ 0882] Morpho!physiological traits related to drought tolerance inprimitive wheats[ In] A[ B[ Damania "ed[#\ Biodiversityand Wheat Improvement\ pp[ 088*106[ AcademicPress\ New York[

Arnon\ D[ I[\ 0838] Copper enzymes in isolated chlo!roplast[ Polyphenol oxidase in Beta vul`aris[ PlantPhysiol[ 13\ 0*04[

Bates\ L[ S[\ R[ P[ Waldran\ and I[ D[ Teare\ 0862] Rapiddetermination of free proline for water stress studies[Plant Soil 28\ 194*197[

Baynett\ N[ M[\ and A[ W[ Naylor\ 0855] Amino acid

Page 8: Physiological and Biochemical Responses of Hexaploid and Tetraploid Wheat to Drought Stress

115 Chandrasekar et al[

and protein metabolism in Bermuda grass during waterstress[ Plant Physiol[ 30\ 0111*0129[

Beardsell\ M[ F[\ and D[ Cohen\ 0864] Relationshipsbetween leaf water status\ abscisic acid levels and stoma!tal resistance in maize and sorghum[ Plant Physiol[ 45\196*101[

Blum\ A[\ and A[ Ebercorn\ 0865] Genotypic response insorghum to drought stress III[ Free proline accumu!lation and drought resistance[ Crop Sci[ 10\ 133*137[

Blum\ A[\ and A[ Ebercorn\ 0870] Cell membrane sta!bility as a measure of drought and heat tolerance inwheat[ Crop Sci[ 10\ 32*36[

Boyer\ J[ S[\ 0871] Plant productivity and environment[Science 107\ 332*337[

Clarke\ J[ M[\ and T[ N[ McCaig\ 0871] Evaluation oftechniques for screening for drought resistance in wheat[Crop Sci[ 11\ 492*495[

Dhindsa\ R[ S[\ P[ Plumb!Dhindsa\ and T[ A[ Thorpe\0870] Leaf senescence] correlated with increased levelsof membrane permeability and lipid peroxidation anddecreased levels of superoxide dismutase and catalase[J[ Exp[ Bot[ 21\ 82*090[

Gummuluru\ S[\ S[ L[ A[ Hobbs\ and S[ Jana\ 0878]Physiological responses of drought tolerant anddrought susceptible wheat genotypes[ Photosynthetica12\ 368*374[

Hanson\ H[\ N[ E[ Borlaug\ and R[ G[ Anderson\ 0871]In] Wheat in the Third World\ pp[ 8*00[ WestviewPress\ Boulder\ Colorado[

He\ P[\ S[ J[ Wen\ and P[ H[ Wang\ 0885] E}ect of osmoticstress on nitrate reductase inactivating protein in wheatseedlings[ J[ South China Agric[ Univ[ 06\ 63*67[

Hiscox\ J[ D[\ and G[ F[ Israelstam\ 0868] A methodfor extraction of chloroplast from leaf tissue withoutmaceration[ Can[ J[ Bot[ 46\ 0221*0223[

Kaur\ J[\ I[ S[ Sheoran\ and H[ S[ Nainawatee\ 0877]E}ect of heat stress on photosynthesis and respiration ina wheat mutant[ In] G[ S[ Singhal "ed[#\ PhotosynthesisMolecular Biology and Bioenergitics\ pp[ 186*292[Narosa Publications House\ New Delhi[

Khan\ M[ I[\ 0866] E}ect of simulated drought on physi!ology and metabolism of maize "Zea mays L[# and Sun!~ower "Helianthus annus L[# crops[ Ph[D[ Thesis Har!yana Agricultural University\ Hissar[

Klepper\ L[\ D[ Flesher\ and R[ H[ Hageman\ 0860] Gen!eration of reduced nicotinamide adenine dinucleotidefor nitrate reduction in green leaves[ Plant Physiol[ 37\479*489[

Knox\ J[ P[\ and A[ D[ Dodge\ 0874] Singlet oxygen andplants[ Phytochemistry 13\ 778*785[

Kraus\ T[ E[\ B[ D[ McKersie\ and R[ A[ Fletcher\ 0884]Paclobutrazol induced tolerance of wheat leaves toparaquat may involve increased antioxidant enzymeactivity[ J[ Plant Physiol[ 034\ 469*465[

Kriedemann\ P[ E[\ B[ R[ Loveys\ G[ C[ Fuller\ and A[ C[Leopold\ 0861] Abscisic acid and stomatal regulation[Plant Physiol[ 38\ 731*736[

Larque!Saavedra\ A[\ and R[ L[ Wain\ 0863] Abscisicacid levels in relation to drought tolerance in varietiesby Zea mays L[ Nature 140\ 605*606[

Larque!Saavedra\ A[\ and R[ L[ Wain\ 0865] Studies onplant growth regulating substances XL II[ ABA as agenetic character related to drought tolerance[ Ann[Appl[ Biol[ 72\ 180*186[

Lichtenthaler\ H[ K[\ and W[ R[ Wellburn\ 0872] Deter!mination of total carotenoids and chlorophylls a and bof leaf extracts in di}erent solvents[ Biochem[ Soc[Trans[ 00\ 480*481[

Loveys\ B[ R[\ and P[ E[ Kriedemann\ 0862] Rapid chan!ges in abscisic acid!like inhibitors following alterationin vine leaf water potential[ Physiol[ Plant[ 17\ 365*368[

Maldonado\ C[ A[\ G[ E[ Zuniga\ L[ J[ Corcurea\ and M[Alberdi\ 0866] E}ect of water stress on frost resistanceof oat leaves[ Environ[ Exp[ Bot[ 27\ 88*096[

Martin\ M[ A[\ J[ H[ Brown\ and H[ Ferguson\ 0878]RWC and leaf water potential di}erentiated droughttolerant and drought susceptible genotypes of barley[Agron[ J[ 70\ 099*094[

Morilla\ C[ A[\ J[ S[ Boyer\ and R[ H[ Hageman\ 0862]Nitrate reductase activity and polyribosomal content ofcorn "Zea mays L[# having low leaf water potentials[Plant Physiol[ 40\ 706*713[

Panse\ V[ G[\ and P[ V[ Sukhatme\ 0856] Statisticalmethods for Agricultural Workers[ Indian Council ofAgricultural Research\ New Delhi[

Plaut\ Z[\ 0862] The e}ect of soil moisture tension andnitrogen supply to nitrate reduction and accumulationin wheat seedlings[ Plant Soil 27\ 70*83[

Premachandra\ G[ S[\ H[ Saneoka\ and S[ Ogata\ 0889]Cell membrane stability\ an indicator of drought toler!ance\ as a}ected by applied nitrogen in soyabean\ J[Agric[ Sci[ "Camb[# 004\ 52*55[

Ritchie\ S[ W[\ H[ T[ Nguyen\ and A[ S[ Holaday\ 0889]Leaf water content and gas exchange parameters of twowheat genotypes di}ering in drought resistance[ CropSci[ 29\ 094*000[

Sairam\ R[ K[\ P[ S[ Deshmukh\ D[ S[ Shukla\ and S[Ram\ 0889] Metabolic activity and grain yield undermoisture stress in wheat genotypes[ Indian J[ PlantPhysiol[ 22\ 115*120[

Sairam\ R[ K[\ and S[ D[ Dube\ 0873] E}ect of moisturestress on Nitrate reductase activity in rice in relation todrought tolerance[ Indian J[ Plant Physiol[ 16\ 153*169[

Sairam\ R[ K[\ P[ S[ Deshmukh\ and D[ S[ Shukla\ 0886]Tolerance of drought and temperature stress in relationto increased antioxidant enzyme activity in wheat[ J[Agron[ Crop Sci[ 067\ 060*066[

Sairam\ R[ K[\ 0883] E}ect of moisture stress on physio!logical activities of two contrasting wheat genotypes[Indian J[ Exp[ Biol[ 21\ 483*482[

Sairam\ R[ K[\ D[ S[ Shukla\ and D[ C[ Saxena\ 0886:87]Stress induced injury and antioxidant enzymes inrelation to drought tolerance in wheat genotypes[ Biol[Plant[ 39\ 246*253[

Singh\ K[ P[\ and K[ Singh\ 0872] In~uence of simulatedwater stress on free proline accumulation in Triticumaestivum L[ Indian J[ Plant Physiol[ 15\ 208*210[

Singh\ T[ N[\ D[ Aspinall\ and L[ G[ Paleg\ 0861] Proline

Page 9: Physiological and Biochemical Responses of Hexaploid and Tetraploid Wheat to Drought Stress

116Drought Stress Responses in Wheat

accumulation and varietal adaptability to drought inbarley] a potential metabolic measure of drought resist!ance[ Nature "New Biol[# 125\ 077*089[

Sinha\ S[ K[\ P[ K[ Aggarwal\ and R[ Khanna!Chopra\0874] Irrigation in India] a physiological and phe!nological approach to water management in graincrops[ Adv[ Irri[ 2\ 018*101[

Smith\ J[ A[ C[\ and H[ Gri.ths\ 0882] Plant responsesfrom cell to community[ In] Water De_cits\ pp[ 0*3[Bios Scienti_c Publishers Limited\ India[

Stewart\ C[ R[\ C[ J[ Morris\ and J[ F[ Thompson\ 0855]Changes in amino acid content of excised leaves duringincubation[ II[ Role of sugar in the accumulation ofproline in wilted leaves[ Plant Physiol[ 30\ 0474*0489[

Storey\ R[\ and R[ G[ Wyn!Jones\ 0866] Quartenaryammonium compounds in plants in relation to saltresistance[ Phytochemistry 05\ 336*342[

Thakur\ P[ S[\ and V[ K[ Rai\ 0879] Growth characteristic

and proline content in relation to water stress in twoZea mays L[ cultivars during dehydration[ Biol[ Plant[12\ 87*092[

Waines\ J[ G[\ 0883] High temperature stress in wildwheats and spring wheats[ Aust[ J[ Plant Physiol[ 10\694*604[

Weatherley\ P[ E[\ 0849] Studies in the water relations ofcotton plants[ I[ The _eld measurement of water de_citin leaves[ New Phytol[ 38\ 70*76[

Zeevart\ J[ A[ P[\ 0879] Changes in the levels of abscisicacid and its metabolites in excised leaf blades of Xan!thium strumarium during and after water stress[ PlantPhysiol[ 55\ 561*567[

Zhang\ J[ L[\ L[ L[ Kang\ Y[ C[ Woi\ and V[ Zheng\ 0882]E}ects of nitrogen fertilizer on physiological propertyand yield of wheat at the di}erent soil moisture[ ActaAgric Bareali Sinica 7\ 81*86[