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ContentslistsavailableatScienceDirect
Journal of Plant Physiology
jou rn al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / j p l p h
Physiology
Isohydric and anisohydric strategies of wheat genotypes under osmotic stress:
Biosynthesis and function of ABA in stress responses
Ágnes Gallé
∗, Jolán Csiszár, Dániel Benyó, Gábor Laskay, Tünde Leviczky, László Erdei, Irma Tari
DepartmentofPlantBiology,UniversityofSzeged,H-6701Szeged,Középfasor52.,P.O.Box654,Hungary
a r t i c l e i n f o
Articlehistory:
Received3January2013
Receivedinrevisedform5April2013 Accepted25April2013
Available online 20 May 2013
Keywords:
Abscisicacid Anisohydricstrategy Isohydricstrategy Osmoticstress Triticumaestivum
a b s t r a c t
Changesinwaterpotential( w),stomatalconductance,abscisicacid(ABA)accumulation,expression ofthemajorgenesinvolvedinABAbiosynthesis,activitiesofabscisicaldehydeoxidase(AO,EC1.2.3.1) andantioxidantenzymeswerestudiedintwowheatcultivarswithcontrastingacclimationstrategies subjectedtomediumstrengthosmoticstress(−0.976MPa)inducedbypolyethyleneglycol(PEG6000).
BecausethebiosyntheticpathwayofABAinvolvesmultiplegeneproducts,theaimofthisstudywasto unravelhowthesegenesareregulatedinisohydricandanisohydricwheatgenotypes.Intheroottissues oftheisohydriccultivar,Triticumaestivumcv.Kobomugi,osmoticstressincreasedthetranscriptlevelsof 9-cis-epoxycarotenoiddioxygenase(NCED)gene,controllingtheratelimitingstepofABAbiosynthesis.
Moreover,thiscultivarexhibitedahigherbasalactivityandahigherinductionofaldehydeoxidaseisoen- zymes(AAO2-AAO3),responsibleforconvertingABAldehydetoABA.Itwasfoundthatthefastactivation oftheABAbiosynthesisintherootsgeneratedanenhancedABApoolintheshoot,whichbroughtabouta fasterclosureofthestomatauponincreasingosmoticstressand,asaresult,theplantscouldmaintain w
inthetissuesclosetothecontrollevel.Incontrast,theanisohydricgenotype,cv.GKÖthalom,exhibited amoderateinductionofABAbiosynthesisintheroots,leadingtothemaintenancebutnoincreaseinthe concentrationofABAonthebasisoftissuewatercontentintheleaves.Duetotheslowerresponseof theirstomatatowaterdeficit,thetissuesofcv.GKÖthalomhavetoacclimatetomuchmorenegative waterpotentialsduringincreasingosmoticstress.Adecreasedactivityofsuperoxidedismutase(SOD) wasfoundintheleavesandrootsofbothcultivarsexposedtoosmoticstress,butintherootselevated activitiesofcatalase(CAT),peroxidase(POX),glutathionereductase(GR)andglutathionetransferase (GST)weredetectedintheisohydriccultivar,suggestingthatthisgenotypewasmoresuccessfulinthe eliminationofreactiveoxygenspeciescausedbythestressconditions.
© 2013 Published by Elsevier GmbH.
Introduction
Theresponseofwheatgenotypestodroughtstresshasbeen investigatedextensivelybecausesoildroughtrepresentsamajor constraintforsuccessfulcropproduction.Plantscanreadilychange theirmetabolicand physiologicalprocesses,aswellasthemor- phologyoftheabove-groundpartsandtherootsysteminresponse towaterdeficit.
Cropplants cancopewithdrought stressbyavoiding tissue dehydration,thus, theseisohydricplants are abletokeep their
Abbreviations:ABA,abscisicacid;AO,aldehydeoxidase;CAT,catalase;GR,glu- tathionereductase;GST,glutathionetransferase;MDA,maldondialdehyde;NCED, 9-cis-epoxycarotenoiddioxygenase;PEG,polyethyleneglycol;POD,peroxidase;
SOD,superoxidedismutase;ZEP,zeaxanthinepoxidase.
∗Correspondingauthorat:Szeged6726,Középfasor52,Hungary.
Tel.:+3662544307;fax:+3662544307.
E-mailaddresses:galleagnes@gmail.com,gallea@bio.u-szeged.hu(Á.Gallé).
tissuewaterpotentialalmostunchangedbythefastclosureoftheir stomataaswellasbyalternativewatersavingmechanisms.Aniso- hydricplants,ontheotherhand,toleratesoildroughtandrespond tothedecreaseofwateravailabilityintheenvironmentbytissue dehydration(Drew,2006).
The firstreactionofplants belongingtotheformer groupis adecreaseintheirstomatalconductance,inwhichroot-to-shoot chemicalorhydraulicsignallingeventsareinvolved.Abscisicacid (ABA)isaplanthormoneinvolvedinmanyphasesofplantdevel- opmentand intheresponseofplantstovariousenvironmental stresses(Wilkinsonand Davies,2002;Blum,2011;Pantinetal., 2012).Becausemanyofthephysiologicalprocessesarecorrelated with endogenous ABA levels, theregulation of ABA biosynthe- sis has a pivotal role in the elucidation of these physiological characteristics.ABAbiosynthesisincreasesfirstintheroots,and thenthehormoneistranslocatedtotheshootviathexylemand functionsasalong-distancechemicalsignalfromtheroottothe shootduringwaterstress(ZhangandDavies,1987;Wilkinsonand Davies,2002,2010).Inadditiontothistypeofchemicalsignalling, 0176-1617/$–seefrontmatter© 2013 Published by Elsevier GmbH.
http://dx.doi.org/10.1016/j.jplph.2013.04.010
ABAcanpromotestomatalclosurebyitsindirecthydrauliceffect throughdecreasingthewaterpermeabilityoftheleafvasculartis- sues(Pantinetal.,2013).Sincetherearelargedifferencesbetween theapparentsensitivityofleafconductancetotheconcentrationof ABAinthexylem(CorreiaandPereira,1995),chemicalsignalling canaccountformorethan40–70%decreaseinthestomatalcon- ductanceasopposedtohydraulicevents(ZhangandDavies,1990;
KhalilandGrace,1993).
Identification of genes encoding enzymes involved in the biosynthesis of ABA has revealed details of the main biosyn- thetic pathways (Seo and Koshiba, 2002). Expression of 9- cis-epoxycarotenoid dioxygenase (NCED) in Arabidopsis, which represents a rate limiting step in controlling drought stress- induced ABA biosynthesis, is up-regulated significantly during droughtstress (Xiong et al.,2002).Five members of the NCED familyinArabidopsisareimplicatedinABAbiosynthesis,among themtheproductofNCED3genemakesthemajorcontributionto increaseABAlevelsleadingtotheinductionoftolerancemecha- nismsinvegetativetissues(Uranoetal.,2009;Freyetal.,2012).
Inavocado,anotherisoform,PaNCED1ishighlyexpressedinthe leavesanditsexpressionisinducedbydehydration(Chernysand Zeevaart,2000).NCEDwasthoughttobethekeyregulatoryenzyme ofABAbiosynthesis,sincethetranscriptamountofthisenzymewas directlyproportionaltotheABAcontent,anditwasinducedearly afterwaterwithdrawal(ChernysandZeevaart,2000;Tayloretal., 2000;Thompsonetal.,2007a).TheinitialaccumulationofABAup- regulatestheexpressionofothergenesinvolvedinthebiosynthesis ofABA,suchaszeaxanthinepoxidase(ZEP),abscisicaldehydeoxi- dase(AAO)andmolybdatecofactorsulfurase(MCSU)inArabidopsis.
Increasedexpressionsofthesegenesleadtoafastaccumulationof ABAinanautocatalyticprocess(Xiongetal.,2002).
The tobacco mutants impared in ZEP expression and activ- ityprovedtobeABA-deficientsandexhibitedlowerxylemABA contents during drought stress (Borel et al., 2001). The mem- bersofthealdehydeoxidase(AOEC1.2.3.1)genefamilycatalyse theoxidationofvarious aldehydesto carboxylicacids andcer- tain isoenzymes efficiently transform abscisic aldehyde toABA (Seoetal.,2000a,2000b).Differentisoenzymesexhibiteddifferent expressionlevelsinplantorgans.PsAO1andPsAO2weremainly expressedintheleavesofpeaplants,whilePsAO3wasexpressed in ageing leaves and seeds (Zdunek-Zastocka, 2008). Aldehyde oxidaseisoforms,whichuseabscisicaldehydeasaphysiological substrate,wereidentifiedinArabidopsisrosetteleaves(Seoetal., 2000a),andtwo isoforms,AO2and AO3, werealsodetectedin barleyroots(Omarovetal.,2003).
Soil droughtdecreasedstomatalconductance(gs)and w of wheatgenotypessignificantly(Guóthetal.,2009).However,other authorsdidnotfindsuchafastresponseofgstodecreasedsoil water potentials or atmospheric vapour pressure deficit (VPD) (Inoue et al., 1989). This suggests that the responses of gs to soilwater stress or VPD wererelated tothecultivars (Condon etal.,1992)ortothedegreeofthedroughtstress(Rawsonetal., 1977).TherateofABAbiosynthesis determinesthedecreasein stomatalconductance,therebythediffusionrateofCO2 intothe chloroplaststroma andthecarboxylating efficiencyof ribulose- 1,5-bis-phosphate carboxylase/oxygenase. Thus, photosynthesis, which is the mostsignificant process influencingcrop produc- tion, may also be inhibited by drought stress (Guóth et al., 2009).
Seriouswater-stresscantriggeranincreasedformationofreac- tiveoxygenspecies(ROS),suchassuperoxideradicalandhydrogen peroxide,whichcandirectlyattackmembranelipidsanddamage proteins(Navari-Izzoet al.,1994;Bartoliet al.,1999).Detoxifi- cationofROSisaccomplishedbytheantioxidantdefencesystem comprisingnonenzymaticcomponents(ascorbate,glutathione,␣- tocopherol,carotenoids)andaseriesofenzymes(Alscheretal.,
1997; Foyer and Noctor, 2005; Nikolaeva et al., 2008).One of the mostimportant antioxidant enzymes is superoxide dismu- tase(SOD),whichconvertssuperoxideradicalstothelessharmful H2O2. Hydrogen peroxide can then be scavenged by catalase (CAT)andperoxidasesandalsoindirectlybyglutathione-related enzymes,suchasglutathionereductase(GR),whichisacompo- nentoftheascorbate-glutathionecycleandreducesglutathione disulfide(GSSG)toglutathione(GSH).GSHcontributestothemain- tenance of cellularredox potential and generatesreduced GSH for other enzymatic reactionssuch as for the detoxification of harmfulmetabolitesbyglutathionetransferases(GST)(Galléetal., 2009).These detoxification processes also play important roles inthesuccessfulacclimationofplantsand canbecontrolledby ABA.
Certainenvironmentalfactors,suchasdroughtstress,induce ABAbiosynthesisprincipallythroughthetranscriptionalregulation ofABAbiosyntheticgenes.However,itmayvarynotonlybetween differentspeciesbutalsobetweendevelopmentalstagesandplant parts.
Here we present a comparativestudy betweenan isohydric andanisohydricwheatcultivarcarried outinordertoelucidate theputativerole oftissuedehydrationintheregulationofABA biosynthesis.Thepatternsofacclimationofananisohydric(cv.GK Öthalom)andanisohydricwheatgenotype(cv.Kobomugi)toPEG 6000-inducedosmoticstresswerecomparedwithspecialempha- sisontheinductionofZEP,NCEDandAO,changesinAOactivities andaccumulationofABAbothinleafandroottissues.Theeffect ofosmoticstressonwaterpotential,stomatalconductance,enzy- maticantioxidant defenceof thetwo wheatcultivarswerealso comparedtorevealthedifferencesbetweenisohydricandaniso- hydricstrategiesduringosmoticstress.
Materialsandmethods
Inourexperiments,twowheatcultivars,TriticumaestivumL.GK Öthalom,amoderncultivarwithmediumdroughttolerance,and Kobomugi,adroughttolerantlandracefromCentralAsia,weresub- jectedtoosmoticstress.Theseedlingsweregrowninplasticdishes containing10lofHoaglandsolution(5mMCa(NO3)2,5mMKNO3, 1mM KH2PO4, 2mM MgSO4, 1M Fe-EDTA, 0.048M H2BO3, 14.48M MnCl2, 0.815M ZnCl2, 0.373M CuCl2, 0.001M Na2MoO4)in Convironcabinet chambers,at 24/19◦C day/night temperature,12h/12hlightperiodandat200molm−2s−1light intensity.Onehundredplantsweregrowninonedishandthecul- turesolutionwaschangedtwiceaweek.Therootswereaerated withaquariumpump.
Osmotic stress was induced by polyethylene glycol treat- ments (PEG 6000) (Money, 1989). Increasing amounts of PEG 6000 reachingthefinal value of 400mOsm(−0.976MPa) were appliedgraduallyintheculturemediaofone-week-oldplants.The seedlingswereexposedto100mOsmPEGonday7,thenthecon- centrationwasraisedstepwiseeverysecondday,onday9–200 andonday11–400mOsm(Csiszáretal.,2012).Onthe7th,9th, 11thdayssampleswerepreparedbeforeincreasingtheosmotic potentialoftheculturesolution.Theexperimentswereperformed inthreebiologicalreplicates.
Waterstatusoftheplants
Middayleafwaterpotentials( w)weremeasuredusingapres- surechamber(PMSInstrumentCo.,Corvallis,Oregon,USA)onthe secondfullyexpandedleaves.Stomatalconductance(gs)wasdeter- minedinthemiddleoftheapicalleafletsofthesecondexpanded leavesusingasteady-stateporometer(PMR-2,PPSystems,UKand USA).
Table1
Theprimersusedforgeneexpressionanalyses.
Sequence Primers
Forward Reverse
18SrRNA GTGACGGGTGACGGAGAATT GACACTAATGCGCCCGGTAT
EF1␣subunit AACTTCACCTCCCAGGTCAT GTCACCAGCTCAGCAAACTT
AO2TC447676 ACGAGGACTAGGCGACGAA TCAACGTAGGGATCTTGTACGT
NCEDTC404702 CCTCGAAGCCCAGCACTAAT GAGAGCGAGAGGTCCAATGG
ZEPAF384103.2 GGAGTTATGAGAAGGAGAGAAAGC AAAACGACAAAGGTCCCAGA
SearchingforsequencesparticipatinginABAbiosynthesisin wheat
Wheat NCED,AAO,and ZEPsequences wereidentified using an in silico approach. Screening for wheat sequences was ini- tially performed on the DFCI-Gene Index (http://compbio.dfci.
harvard.edu/tgi/)wheatdatabaseusingpublishedplantaldehyde oxidaseand9-cis-epoxycarotenoid dioxygenasesequences from DDBJ/EMBL/GenBanksequencedatabase.ForRealTimePCRmea- surementthreesequenceswereused.Thechosenaldehydeoxidase issimilartootheraldehydeoxidase2proteins:blastxsearching resultedin aldehydeoxidase2 proteinsfromOryza sativa(Acc.
No:Q852M1.1,Exp:3e-88)andBrachypodiumdistachylon(Acc.No:
XP003557918.1,Exp:1e-92).AccordingtoDFCIGeneIndexthe sequencewasannotatedtoshowsimilaritytoaldehydeoxidase 2fromZeamays.Thechosen9-cis-epoxycarotenoiddioxygenase (NCED)sequence(TC404702)ishomologoustoHordeumvulgare NCEDaccordingtotheannotationinDFCIGeneIndex,andishighly homologoustoHvNCED1(AK36199.1,Exp:0.0)and toHvNCED2 (AK358040.1,Exp:2e-144)genesdescribedbyMillaretal.(2006) andSeileretal.(2011),respectively.Furthermore,onewheatzeax- antinexpoxidase(AF384103.2)waschosenforthemeasurements, thehomologofthissequence(AK362500.1,Exp:0.0)wasidentified bySeileretal.(2011)asZEP2inH.vulgare(Table1).
RNApurificationandexpressionanalyseswithreal-timePCR
RNAwasextractedfromrootsamplesatdifferentdevelopmen- talstages (9,11,22 days)accordingtoChomczynskiandSacchi (1987)aspublishedearlier(Galléetal.,2009).DNasedigestions wereapplied(Fermentas).FirststrandcDNAwassynthesizedusing MMLVreversetranscriptase(Fermentas).Primersweredesigned usingPrimer 3software (Rozen and Skaletsky,2000)and were synthesized in the Nucleicacid synthesis laboratory, Biological Research Centre (Szeged, Hungary). Primer pairs are shown in Table1.The expressionrateof theABS biosynthesissequences wasmonitoredbyquantitativereal-timePCR(BioRad,MJResearch) usingSYBRGreenprobes(AppliedBiosystems;Karsaietal.,2002).
Eachreactionwasrepeatedatleastthreetimes.QRT-PCRwasini- tiatedbydenaturationat95◦Cfor10minfollowedby41cyclesof denaturationat95◦Cfor15sandannealing,extensionat60◦Cfor 1min.DataanalysiswasperformedusingOpticonmonitorsoft- ware.Todeterminethespecificityofthereaction,ameltingcurve analysisoftheproductwasperformedimmediatelyafterthefinal PCRcyclebyincreasingthetemperaturefrom55◦Cto90◦C(0.2◦C 0.2s−1).18SribosomalRNAandElongationfactor1␣subunitwere usedforhighandlowcontrols(Nicotetal.,2005).Datawerenor- malizedusingtheinitialcontrolsamples(valuesofthe7-day-old seedlings).
Abscisicaldehydeoxidase(AAO,EC1.2.3.1.)activity
AAO tissue extraction and native-polyacrylamide gel elec- trophoresis (PAGE)were carriedout asdescribed by Sagi etal.
(1998). Root and shoot tissues (1g) were homogenized using
250mM Tris–HClbuffer(pH 8.5) containing1mM EDTA, 1mM 1,4-dithio-dl-threitol,5mMl-cysteine,80MNa2MoO4,10M antipain, 0.1mM phenazine methosulphate,10mM glutathione and0.03mMFAD.Thesampleswerecentrifugedat30,000×gfor 15minat4◦C.The resultingsupernatantswereused fornative PAGE.Afterthequantitationofthetotalproteincontentusingthe methodofBradford(1976),(1)5-mm-thickslabsof7.5%polyacryl- amidegelwereloadedwith100and300gofproteinsfromthe rootandshoottissueextracts,respectively.Enzymeactivitieswere determinedbyincubatingthegelsin0.2Mphosphatebuffer(pH 8)for10min,andtheninareactionmixturecontaining0.1mM phenazinemethosulphate,1mM3-(4,5-dimethylthiazolyl-2)-2,5- diphenyltetrazoliumbromidein0.1MTris–HClbuffer(pH8,5)at 25◦Cinthepresenceof,1mMindole-3-aldehyde(IAld)substrates.
Thebands wereanalysedusinga KodakEDAS-290GelAnalysis System.
DeterminationofabscisicacidbyELISA
The quantitative determination of ABA was carried out via an enzyme linked immuno-sorbent assay (ELISA) (Phytodetek- ABA, Sigma–Aldrich, St. Louis, MO). Plant tissues (1.0g) were extracted with15mlofa coldmixtureof100mMNaHCO3 and methanol(80:20,v/v)containing1mgofbutylatedhydroxytoluene in a volume of 100ml. The samples were extracted twice at 4◦Cfor24heach,and werethen evaporated.Theassayutilizes a monoclonal antibodyfor ABA, and the determination of (+)- cis-ABA(Sigma–Aldrich, St.Louis,MO) inthe plantextractwas based onthe competitivebindingof ABA and thetracer (alka- linephosphatase-labelledABA)totheantibody-coatedmicrowell.
Tracerandstandardsolutionswerepreparedfollowingtheman- ufacturer’s instructions.100lofstandardABAorplantextract andthen100lofdilutedtracerwereaddedtoeachwell.After incubationfor3hat4◦C,thewellswerewashedthreetimesby adding200lofwashsolution.Thealkalinephosphatasereaction wasstartedbytheadditionof200lofsubstratesolution.After 60minat37◦C,thereactionwasstoppedwith50lofstopreagent andtheabsorbancewasdeterminedat405nm,usingaMR4000 microplatereader(Dynatech)(Guóthetal.,2009).ABAconcentra- tionisexpressedasnmolABAin1goftissuewatercontentofthe wheatsamples.
Activityofantioxidantenzymes
Enzymeactivitiesweredeterminedbothin rootsand shoots ofthePEG-treatedandcontrolplants.0.75gofplanttissuewas homogenizedonicein3mlextractionbuffer(50mMphosphate buffer pH 7.0, containing 1mM EDTA, 1mM phenylmethylsul- fonylfluoride,PMSFand1%polyvinyl-polypyrrolidone(PVPP).The homogenatewasfilteredthroughtwolayersofcheese-clothand centrifugedfor25minat15,000×gat4◦C.Thesupernatantwas usedforenzymeactivityassays.Thehomogenizationwasrepeated twoorthreetimes,themean±SDwerecalculatedfromthedataof atleast3independentmeasurements.
Fig.1. WaterpotentialchangesinthesecondleavesofwheatcultivarsGKÖthalomandKobomugiinthefunctionoftimeafterexposureto100(onday7),200(onday9) and400mOsm(onday11)PEG6000treatment(mean±SD,n=10).Datalabelledwith*differedsignificantlyfromtheuntreatedcontrolsat*P≤0.05,**0.01or***0.001level (Student’st-test).
Superoxidedismutase (SOD,EC.1.15.1.1) activitywasdeter- mined by measuring the ability of the enzyme to inhibit the photochemicalreduction ofnitro bluetetrazolium (NBT)inthe presenceofriboflavininthelight(Dhindsaetal.,1981).Oneunit (U)ofSODwastheamountofenzymethatcauseda50%inhibition ofNBTreductionandthespecificenzymeactivitywasexpressed asUmg−1protein.
Catalase (CAT, EC. 1.11.1.6) activity was determined by the decompositionofH2O2measuredspectrophotometricallybyfol- lowingthedecreaseinabsorbanceat240nm(Upadhyayaetal., 1985).OneUequalstheamountofH2O2(inmol)decomposedin 1min.
Peroxidase(POD,EC1.11.1.7)activitywasdeterminedbymoni- toringtheincreaseinabsorbanceat470nmduringtheoxidationof guaiacol(Upadhyayaetal.,1985).Theamountofenzymeproducing 1molmin−1ofoxidizedguaiacolwasdefinedas1U.
Glutathionereductase(GR,EC1.6.4.2)activitywasdetermined by measuringthe absorbance increment at 412nm when 5,5- dithio-bis(2-nitrobenzoicacid)(DTNB)wasreducedbyglutathione (GSH),generatedfromglutathionedisulfide(GSSG)(Smithetal., 1988).Thespecificactivitywascalculatedastheamountofreduced DTNB,inmolmin−1proteinmg−1,ε420=13.6mM−1cm−1.
Glutathionetransferase(GST,EC2.5.1.18)activitywasdeter- mined spectrophotometrically by using an artificial substrate, 1-chloro-2,4-dinitrobenzene (CDNB), according to Habig et al.
(1994). The reaction was initiated by the addition of CDNB, andtheincreasein A340 wasdetermined.OneUis theamount of theenzyme producing 1mol conjugated product in 1min, ε340=9.6mM−1cm−1.The proteincontents oftheextractswere determinedbythemethodofBradford(1976).
Malondialdehydedetermination
MDAformationwasassayedbyusingthethiobarbituricacid method(Ederlietal.,1997).100mgleaftissuewashomogenized with1ml0.1% trichloroacetic acid(TCA);toavoid furtherlipid peroxidation100l4%butylhydroxytoluene(BHT)wasaddedto theextract.Aftercentrifugationat12,000×gfor20min,250lof supernatantwasmixedwith1ml0.5%thiobarbituricacidin20%
TCAandthemixturewasincubatedinboilingwaterfor30min.
Theabsorbancewasmeasuredat532nmandadjustedfornonspe- cificabsorbanceat600nm.MDAconcentrationwasestimatedby usinganextinctioncoefficientof155mM−1cm−1.
Statisticalanalysis
Significantdifferencesbetweenthecontrolandtreatedsamples preparedatthesametimepointsweredeterminedbyStudent’s
t-test.DifferenceswereconsideredsignificantifP≤0.05.Insome cases,themeanvalueswerecomparedbyDuncan’stestandthe differenceswereconsideredsignificantifP≤0.05.Statisticalanal- ysiswascarriedoutwithSigmaStat3.1.statisticalsoftware.Alldata presentedaremeans±SD.
Results Waterrelations
Physiologicalresponsestowaterdeficitweremeasuredintwo wheatcultivars exposed to400mOsmPEG6000in hydroponic culture.Waterdeficit decreasedthe relativegrowthrateof the leavesandrootsinbothcultivars(datanotshown).Thethirdleaf developedonlyafter15days,thereforewaterpotentialsandsto- matalconductivitiesweremeasuredonthedaysofsamplinginall fullyexpandedleaves.Sincesimilartendenciesweredetectablein allcases,onlythedataofthesecondleafarerepresented.Water potentialoftheleavesdecreasedsignificantlyincv.GKÖthalom duringthefirstweekofPEGexposure,butthedifferenceexhib- itedsomefluctuationsandattheendofexperiment wdeclined to−1.18MPa.Incv.Kobomugionlysmallandnon-significantdif- ferenceswerefoundinthewaterpotentialbetweenthecontrol andPEG-treatedleavesfromday11today21( w=−0.2MPa) (Fig.1).Thesedatasuggestthatcv.GKÖthalomfollowsananiso- hydricstrategy,whiletheresponseofcv.Kobomugiwascloseto isohydric.
Stomatalconductivity
In cv. Kobomugi stomata were closed in all leaves after 2 daysofexposuretoeven thelowest concentrationofPEG6000 (100mOsm, −0.245MPa). Stomatal conductivity of the leaves exposedtoosmoticstressdecreasedsignificantlyonday11and thevaluesremainedunderthecontrolleveloneverysamplingday fromthattime(Fig.2).Thetendencyofthechangeswassimilarin thefirstleaves(datanotshown).Incv.GKÖthalom,stomatadidnot respondtoeven200mOsmPEGandclosureofstomatabecamesig- nificantonlyonday13,twodaysaftertheexposureto400mOsm PEG(Fig.2).
ExpressionpatternofgenesparticipatinginABAbiosynthesis
Itwasageneralresponsethatunderdroughtstressthebiosyn- thesisofABAwasinducedfirstintherootsandthehormonewas readilytransportedinthexylemtotheleaves.Tofollowthechanges inABAbiosynthesisindifferentplantparts,thetranscriptamounts ofZEP(AF384103.2),NCED(TC404702)andAO2(TC354638)were
Fig.2.ChangesintotalstomatalconductanceofthesecondleavesofwheatcultivarsGKÖthalomandKobomugiinthefunctionoftimeafterexposureto100(onday7), 200(onday9)and400mOsm(onday11)PEG6000treatment(mean±SD,n=10).Datalabelledwith*differedsignificantlyfromtheuntreatedcontrolsat*P≤0.05,**0.01 or***0.001level(Student’st-test).
measuredinthecourseoftheacclimationprocess.Fortheselec- tionoftheNCEDandAO2sequences,thereportedaldehydeoxidase andepoxycarotenoiddioxygenasegenesofotherPoaceaespecies wereused for searching among the wheattentative consensus sequences(TC).Intheleavesthetranscriptamountofthethree chosensequencesshowedatime-dependentdecreaseinalmost everycase,bothincontrolandtreatedplants,suggestingadevelop- mentalphase-specificregulationofABAbiosynthesis.Thepattern oftranscriptabundanceinrootswasdifferentinthetwocultivars betweencontrolconditionandosmoticstress.InKobomugi,the transcriptamountsofthetwoABAbiosyntheticenzymesstudied, NCEDandAAO,wereelevatedbytheosmoticstresswhiletherewas noinductionintheexpressionofZEP.TheexpressionofNCEDwas significantlyinducedinbothcultivarsduetoPEGtreatmentand thehighesttranscriptlevelsweredetectedincv.Kobomugionday 11,twodaysafterapplying100mOsmPEG.Theincreaseobserved intherelativetranscriptlevelofNCEDondays11and13were18- and11-foldinKobomugiand7-and8-foldinGKÖthalom(Fig.3).
Aldehydeoxidaseactivities
TofollowthechangesinthesubsequentstepsofABAbiosyn- thesis during the experimental period, the activity of the AO isoenzymeswasstudiedinnon-denaturatinggels.StainingforAO activityusingindole-3-aldehyde(IAld)asasubstrateresultedin fiveisoformsintherootsofcv.Kobomugiandsixbandsincv.GK Öthalom.Inanearlierworkitwasreportedthatinthepresenceof IAldsubstratethebandofAAO1isoformcouldnotbedetectedin barley,sothebandwiththeleastmobilityinoursamplesoriginates fromtheactivityofAAO2(Omarovetal.,2003).Inourexperiments, thebasicactivitiesofAO2-3bandsweremoreintenseinthecontrol plantsandthePEG-inducedisoformsexhibitedmuchhigheractivi- tiesintheisohydricgenotypecv.Kobomugithanincv.GKÖthalom (Figs.4and5).ThreeAOisoformsweredetectableintheleavesof bothcultivarsbuttherewerenosignificantdifferencesbetween thecontrolandPEG-treatedsamplesduringthestudyperiod(data notshown).
ABAcontent
ItiswellknownthatABAsynthesisisinducedfirstintheroots andsotheABAaccumulatedintheshootisderivedmainlyfrom therootsystem.Leavesofisohydriccultivars mayhave amuch lowerwaterlossthanthoseofanisohydricplants.Similarly,pho- tosyntheticactivityandbiomassproductioncanalsobedifferently affectedinplantsbelongingtodifferentwaterstressacclimation strategies(Guóthetal.,2009).Inordertoexcludethesedifferences, ABAconcentrationswerecalculatedonthebasisoftissuewater
content.Unexpectedly,plants wereabletomaintaina constant ABAconcentrationduringosmoticstress(Fig.6).InGKÖthalom plantsthehighestABAcontentwasmeasuredintheshootonthe secondsamplingdayandasmall,butnon-significantincreasedue toosmoticstresswasdetectedintheleavesonday13,twodays afterthenutrientsolutionreachedthefinal400mOsmvalue.In therootsasignificantaccumulationofABAwasobservedonday 21.PEGtreatmentcausedanenhancedaccumulationofABAinthe leavesofcv.Kobomugionday13,butincontrasttoleaves,theABA contentoftherootsdecreasedsignificantlyonthelastsampling day.
Antioxidantenzymeactivities
Measurementofthethiobarbituratereactivecompounds(MDA content)onthe21thdayrevealedthattheosmoticstresscaused elevated level of lipid peroxidation in both cultivars, but the increasewashigherinGKÖthalomthaninKobomugiplants(Fig.7).
Theactivitiesofseveralantioxidantenzymeswerealsostudiedon thissamplingday.SODactivitydecreasedintheleavesandroots ofbothcultivarsaftertwoweeksofPEGtreatment.However,the basalactivityoftheenzymewashighinthecontrolsamplesand remainedmuchhigherduringosmoticstressintheleavesofcv.
Kobomugi.ThestresscausedaslightdecreaseinCATactivityinGK Öthalombutincreaseditincv.Kobomugi,andintherootsthese changesweresignificant.Guaiacolperoxidase(POD)activitydid notshowanysignificantchangesineitherofthetwowheatcul- tivarsafterosmotictreatmentalthoughitwashigherthaninthe controlbothintheshootsandrootsofKobomugi.GRandGSTactiv- itiesincreasedintherootsofbothgenotypesasaneffectofthePEG treatment,butthechangeswerestatisticallysignificantonlyinthe rootsofKobomugi(Fig.6).
Discussion
In nature,wateris usuallythemostlimitingfactor forplant growth.Ifplantsdonotreceiveadequaterainfallorirrigation,the resultingdrought stresscanreducegrowthmorethanallother environmentalstressescombined.Thismaybetruefortherela- tivelyancientandwell-adaptedlandracesaswellasformodern cultivars,too.
Inthepresenceofosmoticstress,waterstatusparametersare among thefirstlyaffectedphysiologicaltraits.Asseenfromthe changesinwaterpotential,thetwowheatcultivarsinvestigatedin ourexperimentsfollowdifferentstrategiestocopewithosmotic stress: GK Öthalom showedtissue dehydration, which it could tolerateduringtheacclimation,whileKobomugiprovedtobeiso- hydric. Onereasonfor thealmostuneffectedwater potentialin
Fig.3.TranscriptlevelsofAAO2(TC354638),NCED(TC404702)andZEP(AF384103.2)intheshootsandrootsofGKÖthalomandKobomugicultivars.Thetranscriptlevelin thecontrolsamplesonthefirstsamplingday(initialcontrol)wasequalledtoone.Datawerenormalizedusingthewheat18SrRNSandelongationfactor␣subunit(EF-1) ashighandlowcontrols,respectively.Statisticaldifferencescomparedtothecontrolsareindicatedby*P≤0.05,**P≤0.01,***P≤0.001.
leavesof Kobomugicould bethefast decrease in thestomatal conductance.Stomatalclosurecausedbyosmoticstressincv.GK ÖthalomdevelopedlaterthaninKobomugi,whichismanifestedin thedecreaseinthewaterpotentialinthiscultivar.
It iswellestablishedthatstomatalconductanceisincorrela- tionwiththeopeningofstomatalporesandisinhibitedbywater deficit(QuarrieandJones,1979;Quicketal.,1992;Tardieuetal., 2006).Closedstomataareanimportantmeanstoprotecttheplants fromwaterloss,butthisstrategyhasanunfavourableinfluenceon CO2 diffusionand,asaconsequence,onthephotosyntheticrate (Morgan,1984).
Droughtstressisoneoftheenvironmentalfactorsthathighly activate ABA biosynthesis. Regulation of ABA content can be achievedattranscriptionallevelespeciallybytheup-regulationof NCED.Ontheotherhand,thelevelsofAAOmRNAwereincreased bywaterstressin Arabidopsiswithnochangeintheamountof theAAOprotein(SeoandKoshiba,2002).NCEDover-expressing tomato also showed an increased ABA content. This de novo biosynthesisisresponsibleforenhancedABAlevelsinroottissues whichcontributestothecontroloftranspirationandleafexpan- sionintheshoots(Thompsonetal.,2007b;PelegandBlumwald, 2011).
Fig.4. Changesinaldehydeoxidase(AO)activitiesintherootsofwheatcultivarsGKÖthalomandKobomugiinthefunctionoftimeafterexposureto100(onday7),200 (onday9)and400mOsm(onday11)PEG6000treatment.Theactivityoftheenzymeinthegelswasdeterminedusing1mMindole-3-aldehydeasasubstrate.
Inourexperiment,theinvestigatedwheatZEP,NCEDandAAO2 genesin therootsshowedsimilar expressionpatternstothose foundinArabidopsisplants(Xiongetal.,2002).Thehighestinduc- tionwasdetectableintheNCEDtranscriptlevelsintheroots,a lowerincreasewasmeasuredinAAO2incv.KobomugiandinZEP expressioninGKÖthalomcultivar.However,theinductionofNCED andAAO2occurredearlierandwasmorepronouncedinKobomugi thaninGKÖthalom.AtthesametimethebiosynthesisofABAwas notup-regulatedintheleaves.
IthasbeensuggestedthattheinductionofABAbiosynthesisin therootsandABAtransportbythexylemfromtherootstoshootsis along-distancesignalforshoottissues,whichdeterminestherate ofstomatalclosure,thus,itisresponsiblefortheadjustmentofleaf waterstatus(WilkinsonandDavies,2002).Itwasfoundthatlocal- izationoftheAAO3gene(Koiwaietal.,2004)orNCED3andAAO3 proteinsinArabidopsisplantsprovedtobetissuespecificandthe genewasexpressedinthevascularparenchymacellsoftheroots orshoots(Endoetal.,2008),whichpermitsfastloadingofABAinto thexylemsap.InourexperimentsitseemslikelythatAAOactivity canberegulatednotonlyattranscriptionalbutalsoatthepro- teinlevelorbyadirectcontroloftheenzymeactivity.Althoughno
changesinthegeneexpressionwerefound,higherAAO2activities couldbedetectedintherootsofcv.Öthalomexposedtoosmotic stressonday13.Moreover,theactivityofAAO2-3wasenhanced verysignificantlyasaneffectoftheosmoticstressintherootsof cv.Kobomugi.
Water-stressedleavesaccumulatedlargeamountofABAand phaseicacidordihydrophaseicacid,theoxidativemetabolitesof thehormone(Seileretal.,2011).Thelattersareinactivatedforms ofABAandreducethephysiologicallyactivehormonepool(Qin andZeevaart,2002).Inaddition,allthethreecompoundscanbe convertedtoglucosylester-conjugate,thus,thesteady-stateABA levelsareunderthecontrolnotonlyofthesynthesisbutoftherates ofcatabolismandconjugation.Theclosureofstomatadependsalso ontheleafcapacitytocompartmentalizeandmetabolizeABA.
IfABAconcentrationwasexpressedontissuewatercontentit wasfoundthattheup-regulationofABAbiosynthesiscontributed rathertoamaintenancethantoanincreaseinABAconcentration.
Othercalculationmethods(ABAcontentpermgfreshordrymass) gaveverydifferentresults.Thephysiologicallyrelevantconcentra- tionofABAisbasedontheavailabilityofactivehormonemolecule forABAreceptors,inotherwordsonthecompartmentalization.In
Fig.5. Changesintherelativedensitiesofaldehydeoxidase(AO)isoenzymes(AO2,3)intheroottissuesofwheatcultivarsGKÖthalomandKobomugiinthefunctionof timeafterexposureto100(onthe7thday),200(onthe9thday)and400mOsm(onthe11thday)PEG6000treatment.(mean±SD).Datalabelledwith*differedsignificantly fromtheuntreatedcontrolsat*P≤0.05,**0.01or***0.001level.
waterstressedplantstheguardcellsrespondtosmallconcentra- tionchangesinapoplasticABAtransportedbythexylemsapfrom rootstoleaftissues.
Thus,closureofthestomatamaybeinducedbyABAwithout anychangeinbulktissueABAlevels(CornishandZeevaart,1985).
Also,thedistributionofABAinsinkandsourceleavescanshow differences,sourceleavescontinuouslyfeedyoungleaveswithABA
viathephloem(CornishandZeevaart,1984).Inthecourseofthe investigationperiod,theleavesofcv.Kobomugishowedsignifi- cantlyhigheraccumulationofABArelatedtotissuewatercontent inthestressedplants.BecausethisstronginductionofABAbiosyn- thesisoccurredintheroots,itcanresultfromaneffectivexylem transportinthiscultivar.Incv.Öthalomtherewasasmallerinduc- tionofABAbiosynthesisintheroots,butthisenabledtheplantsto
Fig.6. Changesintheabscisicacidcontent(ABA)intheleavesandrootsofwheatcultivarsGKÖthalomandKobomugiinthefunctionoftime,calculatedonthebasisoftissue watercontent,afterexposureto100(onday7),200(onday9)and400mOsm(onday11)PEG6000treatment.(mean±SD,n=3).Datalabelledwith*differedsignificantly fromtheuntreatedcontrolsat*P≤0.05,**0.01or***0.001level.
Fig.7.ChangesinMDAcontentandSOD,CAT,POD,GR,GSTactivitiesonday21intheleavesandrootsofwheatcultivarsGKÖthalomandKobomugiaftertwoweeksof PEG6000treatment.(Mean±SD).Meansdenotedbydifferentlettersindicateasignificantdifference(P<0.05,Duncantest).
maintainasteady-statehormoneconcentrationintheleaveseven duringosmoticstress.ABAlevelsinthelastsamplingdaywere higherintheroottissues,providingagoodopportunityfor the inductionofdefencemechanismsintherootsoftheanisohydric cultivar.
ItwasreportedbyShatil-Cohenetal.(2011)thatABAtrans- ported by the xylem decreased the water permeability of the vascularbundlesheathcellsandreducedtheleafhydraulicconduc- tancebydown-regulatingtheiraquaporins.Thisisinaccordance withtheresultofPantinetal.(2013)whodemonstratedthatABA promotedstomatalclosureinadualway:viaahydrauliceffectand bydirectactivationofguardcellreceptors.Thus,thestomataincv.
Kobomugileavesmayhaveahighersensitivitytosmallchangesin ABAlevels.
Thecoordinativecontrolandregulationoftheexpressionand activityofantioxidantenzymesmaybeimportantforthesurvival ofplantsduringdroughtstress(BianandJiang,2009).
Inthepresentstudy,almostallantioxidantenzymesinvesti- gated wereaffectedby osmoticstressin a differentmanner in the two wheat cultivars. In the leavesof cv.GK Öthalom, the investigated enzymesworkedat alower level(SOD)ordidnot changesignificantly(CAT,POD,GR, GST).In Kobomugithehigh basalactivityofSODdeclinedduringosmoticstress,butCAT,POD, GR and GST activities were enhancedin the leavesand, more significantly,intherootsaftertwoweeksofosmoticstress.Our results suggest that a higher activity or theinduction of these enzymesintherootsoftheisohydricgenotypecanprotectagainst oxidativedamage.Theisohydriccv.Kobomugiaccumulatedless
malondialdehydethancv.Öthalom duringosmoticstress.MDA can be regarded as a biomarker for lipid peroxidation, so the decreaseinMDAcontentindicateshigheranti-oxidativeability, whichcanreflecthigherresistancetodrought(Dhandaetal.,2004).
CATinductionhasa pivotalrole inthe defenceandadaptation inthepresenceofexcessH2O2(Vranováetal.,2002;Tarietal., 2008), and drought stress increased CATactivity in the leaves ofwheat (Lunaet al.,2005).Guaiacolperoxidases are involved not only in scavenging H2O2 but also in plant growth, devel- opment,lignification,suberization,andcross-linkingofcellwall compounds.Salt-ordrought-tolerantplantsoftenhavehigherPOD activitiesthanthesensitiveonesduringstressconditions(Wang etal.,2009;Csiszáretal.,2012).Glutathionereductaseisapartof theascorbate-glutathioneenzymesystem,convertingglutathione disulfide(GSSG)toreducedglutathione(GSH).HighGSTactivityis acommoncharacteristicofseveralcultivatedTriticumspecies,and accordingtotheliterature,thedifferencesintheGSTactivitiesare inagoodcorrelationwiththeirstresstolerance(Bartolietal.,1999;
Edwardsetal.,2000;Galléetal.,2009).TheprotectingroleofGSTs againstdifferentstresseshasbeenprovedinseveralplantspecies (Edwardsetal.,2000),andtransgenictobaccoplantsoverproduc- ingaGSTgenewithGSH-PXactivityexhibitedsignificantoxidative stresstolerance(Roxasetal.,2000).
Activationandinductionofantioxidantenzymesunderdrought stressinwheatshowstimedependenceanddependsonthesever- ityofthestress(Bartolietal.,1999).Itwasreportedearlierthat changesinH2O2contentsinapicalrootsegmentsofwheatgeno- typesexhibitedagenotype-specificpatternduringosmoticstress (Csiszáretal.,2012).Inthepresentexperiments,theroleofantiox- idantenzymeactivitiesinthestressresponsewasapparentduring moreseverewater stressconditions(onday21,oneweekafter applying400mOsmPEG) inboth cultivars.ABA-responsiveele- ments (ABRE) were found in the promoter regions of several enzymesoftheantioxidantdefence,e.g.POX(Csiszáretal.,2012), CAT(Scandalios,2005),SOD(Sakamotoetal.,1995),GR(Kaminaka etal.,1998)and in GST(Xuetal., 2002)isoenzymes,but their expressionmaybecontrolledbyH2O2 orbyABAthroughinde- pendentsignaltransductionpathways.
Beyond the putative control of ROS scavenging systems it wasfoundthatincreasedABAcontentsintomatoconstitutively expressingLeNCED1 ledtoa higher guttation rate(Iuchiet al., 2001).ThissuggeststhatABAmayfacilitatexylemloadingandthe enhancementofrootpressureundernon-transpiringconditions.
TheleakageofK±andotherinorganicionsfromthecellswasalso dependentonROSproduction(Demidchiketal.,2010)andonthe antioxidantstatusofthetissues(Tarietal.,2002).Thus,theinduc- tionofantioxidant enzymesinthe rootsof anisohydricwheat genotypemaycontroltheaccumulationofinorganicosmolytes, whichcancontributetothemaintenanceofwaterpotential.
Inconclusion,thetwocultivarsrespondeddifferentlytoosmotic stressandthesuccessfulacclimationcanbeattributedtovarious componentsinbothcultivars.Thequickstomatalclosureandthe increasedshootABAcontentsincv.Kobomugimayplayarolein themaintenanceofthewaterpotentialoftheplantsatcontrollevel.
Thestress-inducibleABAbiosynthesisandantioxidantdefencein therootsystemofKobomugicouldarisefromtheadaptationof thislandracetosemidesertenvironmentalconditions.Theclosure ofstomatainparallelwiththeABAbiosynthesisinGKÖthalomwas aresponsetoahigherosmoticstressandtheantioxidantdefence waslesspronouncedintheroots,whichledtoahigherdamageto themembranestructureintherootcells.
Acknowledgements
Theauthorsgratefullyacknowledgethefinancialsupportofthe NationalOffice forResearch and Technology ofthe Republicof
Hungary(Grant“TellerEde”,GrantNo.2006ALAP3-01435/2006) andthesupportofHungarianScientificResearchFund(OTKACNK 80988).
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