WRKY REPORT

PlantCellRep(2009)28:21–30DOI10.1007/s00299-008-0614-x

GENETICTRANSFORMATIONANDHYBRIDIZATION

EnhancedheatanddroughttoleranceintransgenicriceseedlingsoverexpressingOsWRKY11underthecontrolofHSP101promoter

XiaolanWuÆYokoShirotoÆSachieKishitaniÆYukihiroItoÆKinyaToriyama

Received:18June2008/Revised:28August2008/Accepted:9September2008/Publishedonline:26September2008ÓSpringer-Verlag2008

AbstractAnOsWRKY11gene,whichencodesatran-scriptionfactorwiththeWRKYdomain,wasidenti edasoneofthegenesthatwasinducedbybothheatshockanddroughtstressesinseedlingsofrice(OryzasativaL.).TodetermineifoverexpressionofOsWRKY11confersheatanddroughttolerance,OsWRKY11cDNAwasfusedtothepromoterofHSP101ofriceandintroducedintoaricecultivarSasanishiki.OverexpressionofOsWRKY11wasinducedbyheattreatment.Afterheatpretreatment,thetransgeniclinesshowedsigni cantheatanddroughttol-erance,asindicatedbytheslowerleaf-wiltingandless-impairedsurvivalrateofgreenpartsofplants.Theyalsoshowedsigni cantdesiccationtolerance,asindicatedbytheslowerwaterlossindetachedleaves.Ourresultsindi-catethattheOsWRKY11geneplaysaroleinheatanddroughtstressresponseandtolerance,andmightbeusefulforimprovementofstresstolerance.

KeywordsDroughttoleranceÁHeattoleranceÁTransgenicriceÁWRKY

Introduction

Droughtstresshasbeenfoundtobeoneofthemajorcausesofreducedcropyield(Ozturketal.2002),andgreateffortshavebeenmadetobreeddrought-tolerantcropvarieties.Asthemostimportantworldfoodcrop,culti-vatedrice(OryzasativaL.)demandstremendousamountsofwaterduringgrowth,whichresultsinanumberofproductionchallenges.Riceisalsoapopularmodelplantforstudiesofmonocots.Improvementsinthetoleranceofcerealplantstoabioticstressareimportantwhentheef -ciencyoffoodproductionistobeincreased.Breedingoftransgenicricecultivarwithdroughttolerancecanhelpincreaseandstabilizecropyieldunderstressenvironments.Manytransgenicapproacheshavebeencarriedouttoincreasebioticandabioticstresstolerance(seeBajajandMohanty2005forareview).Onesuccessfulapproachtoincreaseabioticstresstoleranceisoverexpressionofcertainstress-inducibletranscriptionfactors,suchasthefollowing:CBF1/DREB1B,CBF2/DREB1C,CBF3/DREB1A(Thomashow1999);DREB1A(Kasugaetal.1999);DREB2A(Sakumaetal.2006);EmBP1,OSBZ8,TAF1(Zhu2002);SNAC1(Huetal.2006).However,employmentofaconstitutivepromotersuchasCaMV35Stodrivegenesfortranscriptionfactorsmaypresentsomeriskofdeleteriouseffectonplantgrowth(Romeroetal.1997;Kasugaetal.1999).OverexpressionoftheOs-DREB1Aproteinshasbeenreportedtocausegrowthretardationunderunstressedcontrolconditionsintrans-genicriceplants(Itoetal.2006).SimilarphenomenaconcerningimprovementofstresstoleranceandgrowthretardationhavebeenobservedintransgenicArabidopsisoverexpressingCBF1/DREB1B,CBF2/DREB1C,orCBF3/DREB1A(Thomashow1999;Kasugaetal.1999)andintobacco(Kasugaetal.2004).Ithasbeenreported

CommunicatedbyH.Ebinuma.

X.WuÁY.ShirotoÁY.ItoÁK.Toriyama(&)LaboratoryofEnvironmentalBiotechnology,

GraduateSchoolofAgriculturalScience,TohokuUniversity,1-1Tsutumidori-Amamiyamachi,Aoba-ku,Sendai981-8555,Japan

e-mail:torikin@bios.tohoku.ac.jp

S.Kishitani

LaboratoryofPlantBreedingandGenetics,GraduateSchoolofAgriculturalScience,

TohokuUniversity,1-1Tsutumidori-Amamiyamachi,Aoba-ku,Sendai981-8555,Japan

123

WRKY REPORT

22thatthestress-induciblerd29Apromoterinsteadoftheconstitutivepromoterminimizesthisnegativeeffectonplantgrowth(Kasugaetal.1999,2004).Itis,therefore,desirabletousestress-induciblepromoterstoproducestresstolerantplants.

Tosearchfordroughtandheat-inducibletranscriptionfactorsinrice,wepreviouslyfocusedonriceorthologsofArabidopsisgenesfortranscriptionfactorsknowntobeinducedbydroughtorheat(Sekietal.2002;Oonoetal.2003;Rizhskyetal.2004).Usinghydroponicallygrownseedlings,OsWRKY11(DDBJaccessionno.AK108745;previousnamewasWRKY16inaccessionno.AY341856)wasidenti edtoshowenhancedexpressionbyplacingtheplantsat38°Cfor1h(heattreatment),byleavingtheplantsoutofwaterfor10h(droughttreatment)andbycombinedheat/droughttreatmentfor0.5h(Shirotoetal.2004).

TheWRKYgenesencodealargegroupoftranscriptionfactors.Thereareover70WRKYgenesinArabidopsisandover100membersinrice(Wuetal.2005;ZhangandWang2005;Liuetal.2007;Ramamoorthyetal2008).Thisfamilyisde nedbyadomainof60aminoacidscontainingtheaminoacidsequenceWRKYatitsamino-terminalendandaputativezinc ngermotifatitscarboxy-terminalend.Itbindsspeci callytotheDNAsequencemotif(T)(T)TGAC(C/T),whichisknownastheWbox.WRKYgenesareknowntoparticipateinvariousdevelop-mentalandphysiologicalprograms,includingdiseaseresistance(ChenandChen2000;Dellagietal.2000;DuandChen2000;Eulgemetal.2000;Kimetal.2000;Asaietal.2002),senescence(HinderhoferandZentgraf2001;RobatzekandSomssich2001),bioticandabioticstressresponses(Yodaetal.2002;Dongetal.2003;HuangandDuman2002;Haraetal.2000;Rizhskyetal.2002,2004;Pnuelietal.2002;Sekietal.2002),andgrowthand

developmentalprocesses(Lagace

´andMatton2004;John-sonetal.2002;Sunetal.2003;Xuetal.2004).Tothebestofourknowledge,however,noinformationisavailableregardingtherelationshipbetweenoverexpressionofWRKYgenesandheatanddroughttolerance.

TodetermineifoverexpressionofOsWRKY11confersheatanddroughttolerance,OsWRKY11cDNAwasfusedtothepromoterofHSP101ofriceandintroducedintorice.ThepromoterofHSP101wasemployedinthisstudy,becausetheHSP101genehasbeenreportedtobeinducedbyheatshock(Agarwaletal.2003).Althoughthepro-moteractivityofHSP101hasnotbeenreported,itwasexpectedthatthepromoterwasstress-inducibleandcouldbeusedtominimizepossibledeleteriouseffectsofOs-WRKY11expressionunderunstressedconditions.Inthisstudy,we rstexaminedactivationofHSP101promoterbyheatanddroughtstress,andthephenotypeoftransgenicplantsoverexpressingOsWRKY11.Then,weevaluated

123

PlantCellRep(2009)28:21–30

survivalofgreenpartsofplantsafterheatanddroughttreatment,andwaterlossofdetachedaerialpartsduringdesiccation.Enhancedheatanddroughttolerancewasobserved.

Materialsandmethods

ConstructionofHSP101promoter::GUS

GenomicDNAwasextractedfromleavesofO.sativaL.cv.NipponbareusingDNeasy(Qiagen,Tokyo,Japan).Anapproximately1kb-fragmentcontainingthepromoterregionofHSP101(DDBJaccessionno.AJ316025)wasampli edfromtheDNAusingExTaq(TaKaRa-Bio,Ohtsu,Japan),thePCRenhancersystem(Invitrogen,Carlsbud,USA),andthefollowingprimers:OsHSP101p-F2primer(50-GGCCAACATGATACAAGAAACCG-30)andOsHSP101p-R1primer(50-CGTTGGTCTTGTGCGTGAAGTTG-30).Cyclingconditionswereasfollows:35cyclesat94°Cfor30s,at58°Cfor30s,andat68°Cfor30s.Theampli edfragmentsweregel-puri edusingtheMinEluteGelExtractionKit(Qiagen,USA).AHindIIIsiteandaNheIsite(italicized)wereaddedusingExTaq(Takara,Kyoto,Japan),thePCRenhancersystem(Invit-rogen,Carlsbud,USA),andOsHSP101p-Fprimer(50-AAGCTTCCTTCCGGCGATCTTGCAG-30)andOsHSP101p-Rprimer(50-GCTAGCTCCTCCTCTCCTCACACAATC-30).Theampli edfragmentwasligatedtopGEM-TEasyVectorSystems(Promega,Madison,USA).

TheHSP101promoter-GUSwastakenasaHindIII-SacIfragmentandinsertedinthesamesitesofpBI101-Ubi-Hyg,whichcontainsamaizeubiquitinpromoter(Christensenetal.1992)andahygromycinphosphotrans-ferasegeneasaselectionmarker.

ConstructionofHSP101promoter::OsWRKY11

AfulllengthcDNAofOsWRKY11inpCMVFL3(DDBJaccessionno.AK108745)wasobtainedfromtheRiceGenomeResourceCenterattheNationalInstituteofAgrobiologicalSciences,Japan(accessionno.AK108745inthericefull-lengthcDNAdatabase,KOME,http://cdna01.dna.affrc.go.jp/cDNA/).TheHSP101promoterwastakenasaEcoRIfragmentinpGEM-TEasyVectorandinsertedintothesamesitelocatedinfrontoftheOsWRKY11cDNA.TheHSP101promoter-OsWRKY11containstwoSpeIsites,onederivedfrompGEM-Teasyvectorandtheotherfromthe30endofthecDNA.TheSpeIfragmentwasinsertedintotheXbaIsiteofpBI101H,whichcontainedCaMV35Spromoter–hygromycinphos-photransferasegeneasaselectionmarker(Yokoietal.1998).

WRKY REPORT

PlantCellRep(2009)28:21–30Productionoftransgenicplants

The nalconstruct,HSP101promoter::OsWRKY11andHSP101promoter::GUS,wastransferredintoAgrobacte-riumtumefaciensstrainEHA105(Hoodetal.1993)andusedforAgrobacterium-mediatedtransformation(Yokoietal.1997).Primarytranformants,Toplants,weregrowninpots(4.5cmindiameterand5.7cmindepth)inagreenhouseat28/23°C(12/12h)undernaturaldaylengthconditions,asdescribedbyShirasawaetal.(2006).Southernblotanalysis

GenomicDNA(2lg)wasextractedfromleavesusingDNeasy(QIAGENK.K.,Japan)anddigestedwithHindIIIandelectrophoresedona0.8%agarosegel.DNAfragmentswereblottedontonylonmembrane(GEHealthcare,UK)andhybridizedwitha708-bpHPTprobelabeledwithdigoxi-genin(Roche,Switzerland)usingprimersHPT-F(50-AATGAGTTGGACCAGCAGAAG-30)andHPT-R(50-CATTCAGGTCAAACATAGGCC-30),orwithaGUSprobeusingprimersG787-F(50-TGTGAATTCGATATCTACCCGCTTCGCGTC-30)andG1809-R(50-GATGAATTCTCATTGTTTGCCTCCCTGCTG-30).Afterhybridization,blotswerewashedat65°Cin0.19SSCand0.1%SDS.GUSassay

EightindependenttransgenicplantswithHSP101pro-moter::GUSweregrowninsoilinpotsat22/18°C(12/12h)for2weeksandweresubjectedtoheatshock,cold,NaCl,ordesiccationtreatment.Forheatshocktreatment,theexcisedfullyexpandedleafblades(0.5–1cminlength),leafsheaths,androotswereplacedinwaterinmicrocentrifugetubesandincubatedinawaterbathat37,42,or45°Cfor1h.Thecontrolwassimultaneouslyplacedatroomtemperaturefor1h.Forcoldtreatment,theleavesinthemicrocentrifugetubeswereplacedinwaterat4°Cfor1h.Desiccationtreatmentwascarriedoutbyleavingtheleafsegmentsinthetubeswithneitherwaternoralidat25°Cfor1h.ForNaCltreatment,theexcisedleaveswereplacedin250mMNaClsolutioninthemicrocentrifugetubesandincubatedat25°Cfor1h.HistochemicalstainingforGUSactivitywasperformedusing5-bromo-4-chloro-3-indolyl-b-D-glucuronide(X-gluc)asachromogenicsub-strate(Jeffersonetal.1987).Tissueswerevacuumin ltratedwithX-Glucreactionbuffer(100mMsodiumphosphatebufferpH7.2,0.5mMpotassiumferrocyanide,0.5mMpotassiumferricyanide,0.1%TritonX-100and1mg/mlX-gluc)andincubatedat37°Covernight.Afterincubation,chlorophyllwasremovedfromgreentissuesbyimmersionin70%ethanol,andtissuesampleswereobservedonaNikonstereomicroscope.

23

RT-PCR

Two-week-oldseedlingsgrowninsoilat22/18°Cwereexposedtoheatpretreatmentat37/22°C(12/12h)for2weekswithwatereveryday.Forno-heatpretreatment,plantsweregrownat22/18°C.InductionofOsWRKY11expressionwasdeterminedbyRT-PCR.TotalRNAwasisolatedfromleavesusingRNeasy(Qiagen,Tokyo,Japan)accordingtothemanufacturer’sinstructions.PolyARNAwasisolatedusingtheDynabeadsmRNAPuri cationKit(Invitrogen,Carlsbud,USA).cDNAsynthesiswasaccomplishedusingSuperScriptIIIReverseTranscriptase(Invitrogen,Carlsbud,USA)andthenusedasatemplateforPCRampli cationwiththefollowingprimers:50-AAATCGTCACGCCGGTGCAAG-30and50-TCAACCTCGCTCTTGGTGAGGAA-30forOsWRKY11;50-TACAACGGTTGGCGTCGCAC-30and50-AACTTGCGCACACGGTCCAG-30fortubulinalpha-1chain(accessionno.AK069140inDDBJdatabase).PCRwascarriedoutwithExTaqpolymerase(TaKaRa-Bio,Ohtsu,Japan)for30cyclesofdenaturationfor0.5minat94°C,annealingfor0.5minat66°C,andextensionfor0.5minat72°C,followedby nalextensionfor5minat72°C.Forquan-titativeRT-PCRanalysis,PCRwasperformedusingSYBRPremixExTaqpolymerase(TaKaRa-Bio)andThermalCyclerDiceRealTimeSystemTP800(TaKaRa-Bio)for55cyclesofdenaturationfor5sat95°C,annealingfor10sat55°C,andextensionfor30sat72°C.EvaluationofheatanddroughttoleranceofseedlingsEvaluationoftoleranceagainstheatanddroughtstresswasperformedbydeterminingtheextentofrecoveryofwholeplantsfromexposureto37/22°C(12/12h)withoutawatersupplyfor2days.Priortocombinedheat/droughttreat-ment,2-week-oldseedlingsweregrownfor2weeksat37/22°Cwithawatersupply.Thentheplantswereexposedto37/22°Cfor2dayswithoutawatersupply.Theplantswerereturnedto22/18°Candgrownwithawatersupplyfor2weeks.Arialpartswereseparatedintogreenlivingpartsandnongreendeadparts.Eachdryweight(DW)wasdeterminedafterdryingat80°Cfor24h.Thepercentageofsurvivinggreenpartswascalculatedfromthefollowingformula:(DWofgreenparts/DWofgreenpartsplusdeadparts)9100.Heatanddroughttreatmentwasalsocarriedoutat37/22°Cfor2.5dayswithoutawatersupply.Survivalofplantswasobservedafter2weeksofrecovery.Evaluationofwaterlossindetachedaerialparts

Forheatpretreatment,2-week-oldseedlingsgrowninsoilat22/18°C(12/12h)wereexposedtoheatpretreatmentat37/22°Cfor2weekswithwatereveryday.Forthecontrol

123

WRKY REPORT

24conditionwithno-heatpretreatment,plantsweregrownat22/18°C.Afterthepretreatmentfor2weeks,theaerialpartsoftransgenicplantsandwildtype(WT)weredetachedandweighedimmediatelyandthenplacedonalaboratorybenchat25°Candweighedatdesignatedtimes.Detachedaerialpartswerethendriedat80°Cfor24htodetermine nalDW.Watercontentwasstandardizedasapercentagerelativetotheinitialwatercontentofaerialpartsoftheplant;itwascalculatedasfollows:[(FWi-DW)/(FW0-DW)]9100,whereFWiandFW0arefreshweightforanygivenintervalandoriginalfreshweight,respectively.FourT2plantsperlinewereusedforeachtest.Alltestsweresimultaneouslyrepeatedthreetimes.

Results

ActivationofHSP101promoterintransgenicriceplantswithHSP101promoter::GUS

Anapproximately1kb-fragmentcontainingthepromoterregionofHSP101(DDBJaccessionno.AJ316025)wasampli edfromricegenomicDNAandinsertedinpBI101-UbiHyg,whichcontainsahygromycinphosphotransferasegeneasaselectionmarker(Fig.1).TheGUSgenewasfusedtothepromoterofHSP101andintroducedintoanelitecultivarSasanishiki.Excisedleafbladesoftheeightindependenttransgenicplantsgrownat22/18°C(12/12h)weresubjectedtohistochemicalGUSassay.Incubationoftheleafsegmentsat37,42,or45°Cfor1hinducedahighlevelofGUSexpression,whilenoGUSexpressionwasdetectedatroomtemperature(Fig.2a).Bluestainingwasstrongerat42and45°C.GUSexpressionwasnotobservedinWTleafblades.StrongGUSexpressionwasalso

123

PlantCellRep(2009)28:21–30

Fig.2GUSassayofleafblades(a),leafsheathsandroots(b)intransgenicplantswithHSP101promoter::GUSwithorwithoutheatshock.StrongGUSactivity,bluestaining,wasobservedafterheatshockat45°Cfor1h(right)butnotevidentatcontrolconditionwithoutheatshock(left).Bar2mm

observedinleafsheathsandroots,whereasweakbluestainingwasobservedinthebaseofstemsatroomtem-perature.TheGUSactivityunderotherstresses,i.e.,cold(4°C),250mMNaClordroughtfor1hwasnotdetected.Thus,theheatshockactivationoftheHSP101promoterwascon rmed.

AtransgeniclinecarryingasinglecopyoftheGUSgene,whichwasrevealedbySouthernblotanalysis(datanotshown),wasselected(line#g8).T2plantshomozygousfortheintroducedGUSgenewereusedforfurtheranalysis.ProductionoftransgenicriceplantscarryingHSP101promoter::OsWRKY11

OsWRKY11cDNA(DDBJaccessionno.AK108745)wasfusedtothepromoterofHSP101ofrice(Fig.1)andintroducedintorice.Fourindependenttransgeniclines(#ox2,#ox3,#ox4,and#ox5)wereobtained.ThenumberofcopiesoftheT-DNAwasdeterminedbySouthernblotanalysis.WeusedHindIII,whichcutsonlyoncewithintheintroducedT-DNA,andprobedwiththeHPTgene(datanotshown)sothatthenumberofbandsrepresentedthecopynumber.Thetransgeniclines,#ox2and#ox3,wereidenti edtohaveasinglecopyofT-DNA,and#ox4containedtwocopiesand#ox5carriedfourcopies(Table1).

T1plantssegregatedfortheintroducedT-DNAweregrowntomaturityundernormalgrowthconditions.Threetypesofphenotypeswereobserved:dwarfwithbentleaves,normalplantlengthwithbentleavesandnormalplantlengthwithnormalleaves.Incaseofthebentleaves,theedgesofleafbladeswerebenttowardtheabaxialside,i.e.towardtheoppositedirectionobservedinthetypicalwilted

WRKY REPORT

PlantCellRep(2009)28:21–30

Table1Phenotypeofmaturetransgenicplants(T1)segregatingfortheintroducedOsWRKY11Transgenicline

CopynumberofthetransgeneTotalnumberofplantsexaminedNumberofplantswithoutthetransgeneNumberofdwarfplants(plantlength\85cm)

Numberofnormalplantlength([94cm)withbentleavesNumberofnormalplantlength([94cm)withnormalleavesNumberofplantswiththetransgene

Numberofdwarfplants(plantlength\85cm)

Numberofnormalplantlength([94cm)withbentleavesNumberofnormalplantlength([94cm)withnormalleaves

#ox21510014040

#ox31245005190163

#ox42620024202

#ox54410013102

25

WT0440040000

leaf-rollingresponse.ThenumberofplantswitheachphenotypeisshowninTable1.MostoftheOsWRKY11transgenicplantsshowthephenotypeofnormalplantlengthwithbentleaves;plantlengthwasmorethan94cm.Incontrast,#ox4showeddwarfphenotype;theplantlengthwaslessthan85cm,andtheWTplanthadaplantlengthof110–128cm.Nontransgenicplantssegregatingfromeachprimarytransformantshowedaphenotypeofnormalplantlengthwithnormalleaves.TheseresultssuggestedthatintroductionofHSP101promoter::OsWRKY11mighthavecausedsomedeleteriouseffectsonplantgrowth.TransgeniclineswithasinglecopyoftheT-DNA(i.e.,#ox2and#ox3)wereselected,andT2plantshomozygousfortheT-DNAwereusedforfurtherstudy.Atransgenicline,#g8,whichcontainedHSP101promoter::GUS,wasusedasanegativecontrol.In2-week-oldseedlingsofT2plants,plantlengthwasidenticalamong#ox2,#ox3,#g8,andWT.However,theplantlengthof#ox3wasslightlyshorterwhencomparedtothatof#ox2,#g8,andWTin4-week-oldseedlingsgrownat22/18°C(n[26,P\0.0001).Thedwarfphenotypeandbentleaveswereman-ifestedwhentheplantsweresubjectedtoheattreatmentat37/22°C(12/12h)for2weeks(Fig.3).Plantlengthof

#ox3wassigni cantlyshorterthanthatof#ox2,#g8,andWT(n[28,P\0.0001).Undernormalgrowthcondi-tions,theseedsetpercentagewas36.7±5.8for#ox2and98.0±0.8for#ox3.

InductionofOsWRKYexpressionbyheattreatmentTwo-week-oldseedlingsgrowninsoilat22/18°C(12/12h)wereexposedtoheatpretreatmentat37/22°C(12/12h)for2weekswithwatereveryday.Plantsweremaintainedat37°Cduringthedayandat22°Catnight.Forthecontrolconditionofno-heatpretreatment,plantsweregrownat22/18°C.InductionofOsWRKY11expressionwasinvestigatedinleafbladesusingRT-PCR.A164-bpbandofOsWRKY11wasdetectedin#ox2and#ox3afterheattreatment,butwasnotdetectedin#g8orWT(datanotshown).Inthecaseofno-heatpretreatment,abandofOsWRKY11washardlydetectedinthesePCRconditions.TherelativeOsWRKY11expressionlevelwasalsoinvestigatedusingreal-timequantitativeRT-PCR(Fig.4).ThevalueinFig.4indicatestherelativeexpressionlevelcomparedtothatoftubulin.TherelativeOsWRKY11expressionlevelof#ox2and#ox3withno-heattreatment(controlcondition)wasthesameasthatofWT.Incontrast,thevalueswere3.7timeshigherin#ox2and3.0timeshigherin#ox3thaninWTaftertheheatpretreatment.TheseresultsdemonstratedthatoverexpressionofWRKY11wasinducedbyheattreatment,butitwasnotdetectableunderunstressednormalconditions.Heatanddroughttoleranceofseedlings

Evaluationoftoleranceagainstcombinedheat/droughtstresswasperformedbydeterminingtheextentofrecoveryofwholeplantsfromexposureto37/22°C(12/12h)withoutawatersupplyfor2days.Priortothecombinedheat/droughttreatment,2-week-oldseedlingsweregrownfor2weeksat37/22°Cwithawatersupplyforthe

123

WRKY REPORT

26

inductionofOsWRKY11expression.Figure5ashowsthephotographsof#ox2,#ox3,andWTafterthecombinedheat/droughttreatmentfor2dayswithoutawatersupply.TheOsWRKY11transgeniclines#ox2and#ox3showaweakerleaf-wiltingphenotype.

Aftertheplantswereexposedto37/22°Cfor2dayswithoutwatersupply,theywerereturnedto22/18°Candgrownwithwatersupplyfor2weeks.Then,thepercentageofsurvivinggreenpartswasinvestigated(Fig.6).Thevalue(%)was81.7±1.9for#ox2,94.1±1.6for#ox3,whileitwas39.8±11for#g8and46.6±7.8forWT.Thedifferencewassigni cantbasedonattest(P\0.001).

Figure5bshowsthephotographsof#ox2,#ox3,andWTat2weeksofrecoveryaftercombinedheat/droughttreatmentat37/22°Cfor2.5dayswithoutawatersupply.ProlongedheatanddroughttreatmentcausedalltheWTplantstodie,whiletheOsWRKY11transgeniclines#ox2and#ox3survived.TheseresultsdemonstratedthattheOsWRKY11transgeniclines#ox2and#ox3gainedsig-ni cantheatanddroughttolerance.

Evaluationofwaterlossindetachedaerialparts

WedeterminedwhetherwaterlosswasaffectedinOsWRKY11transgenicplants#ox2and#ox3bycomparingtheratesofchangeofthefreshweightofthedetachedaerialpartsofplantsduringdehydration.Forheatpre-treatment,2-week-oldseedlingsgrowninsoilat22/18°Cwereexposedtoheatpretreatmentat37/22°Cfor2weekswithwatereveryday.Forthecontrolconditionwithno-heatpretreatment,plantsweregrownat22/18°C.Afterpre-treatmentfor2weeks,theaerialpartsofthetransgenicplantsandWTweredetachedandplacedonalaboratorybenchat25°Candweighedatdesignatedtimes.

123

PlantCellRep(2009)28:21–30

Fig.5PhotographsofOsWRKY11transgeniclines#ox2and#ox3,andWTaftercombinedheat/droughttreatmentat37/22°C(12/12h)withoutwatersupply.Two-week-oldseedlingsweregrownfor2weeksat37/22°Cwithwatersupply,andthentheplantswereexposedtocombinedheatanddroughttreatment.aThephotographsweretakenaftercombinedheat/droughttreatmentfor2days.TheOsWRKY11transgeniclines#ox2and#ox3showweakerleaf-wiltingphenotype.bThephotographsweretakenafterrecoveryat22/18°Cfor2weeksfollowingheat/droughttreatmentfor2.5days.TheOsWRKY11transgeniclines#ox2and#ox3survived,whereasWTplantsdiedafterrecoveryat22/18°Cfor2weeks.Bar5cm

ThetimecourseofwaterlossofplantswiththeheatpretreatmentisshowninFig.7aandthatofplantswiththeno-heatpretreatmentisshowninFig.7b.Inplantsofbothcases,waterlossin#ox2and#ox3wasslowerthanthatinWTor#g8.Inthecaseofplantswiththeheatpretreatment,thetime(min)atwhich20%ofwaterlosswas

achieved

WRKY REPORT

PlantCellRep(2009)28:21–30

was141±13(mean±SD)for#ox2and121±31for#ox3,whereasitwas51±13forWTand56±32for#g8.Thedifferencewassigni cantbasedonattest(P\0.01).Inthecaseofplantswiththeno-heatpre-treatment,thetime(min)atwhich20%ofwaterlosswasachievedwas69±8for#ox2and68±13for#ox3,whereasitwas34±12forWTand35±4for#g8.Thedifferencewassigni cantbasedonattest(P\0.01).Whenwecomparedtheratiobetweenthetimeof20%waterlossofplantswiththeheattreatmentandthosewiththeno-heatpretreatment,theratiowas1.5forWT,1.6for#g8,2.1for#ox2,and1.8for#ox3,indicatingthatheatpretreatmentenhanceddroughttoleranceespeciallyinthe#ox2and#ox3plants.Itisconsideredthattheheatpre-treatmentinducedtheexpressionoftheintroducedOsWRKY11inadditiontoinductionofendogenousOs-WRKY11.CrosstoleranceofheatanddroughtstresseswasimplicatedeveninWTplants.Thisexperimentwasrepe-atedtwomoretimesandreproducibilitywascon rmed(datanotshown).

Discussion

AdvantageofusingofHSP101promoter

Overexpressionoftranscriptionfactorgeneshassometimesbeenreportedtoaccompanygrowthdefectsresultinginreducedproductivity.Thisispresumablyduetoconstitu-tiveactivationofdefense-responsegenesundertheregulationofintroducedtranscriptionfactors.Suchgrowthdefectshavebeenreportedintransgenicriceplantsover-expressingOsDREB1A(Itoetal.2006)andWRKY45

27

(Shimonoetal.2007).TheWRKY45geneisoneofthebenzothiadiazole(BTH)-andsalicylicacid(SA)-inducibleWRKYgenes.GrowthretardationhasbeenobservedintransgenicriceplantswithWRKY45drivenbymaizeubiquitinpromoter(Shimonoetal.2007).Ithasbeenreportedthatgrowthconditionshaveaprofoundin uenceontheexpressionofpathogen-relatedgenesduringcon-currentWRKY45expression,whichmayinturnaffectthegrowthofWRKY45-overexpressingplants.ThisisquitesimilartoourresultsoftheappearanceofdwarfphenotypeinthetransgenicOsWRKY11lines.Dehydration-responsiveelement-bindingprotein1(DREB1)/C-repeat-bindingfactors(CBFs)areknowntospeci callyinteractwiththeDRE/CRTcis-actingelementandtocontroltheexpressionofmanystress-induciblegenesinArabidopsis.Overex-pressionoftheOsDREB1Aunderthecontrolof

CaMV35S

123

WRKY REPORT

28promoterormaizeubiquitinpromoterhasalsobeenreportedtocausegrowthretardationunderunstressedcontrolconditionsintransgenicrice(Itoetal.2006).Sometransgeniclinesshowedadwarfphenotypeevenatthereproductivestage.Kasugaetal.(1999,2004)previouslyshowedthatthestress-induciblerd29Apromoterinsteadoftheconstitutivepromoterminimizesthisnegativeeffectonplantgrowth.Itoetal.(2006)demonstratedthattheAra-bidopsisrd29Agenepromoterdidnotfunctioninriceleaves;therefore,strongstress-induciblepromotersofricewerenecessary.

OurresultsindicatedthattheHSP101promoterofriceisheat-inducible(Figs.2,4)andcanbesuccessfullyusedfortheinductionofOsWRKY11expressiontoenhanceheatanddroughttolerance.SomeleakyexpressionofOs-WRKY11,althoughitwasundetectableinleafbladesbyRT-PCR,waslikelytohaveoccurred,becausesomeplants,suchas#ox3and#ox4,showedadwarfphenotypeunderunstressednormalconditions(Table1).TheHSP101pro-motermightdriveOsWRKY11expressioninthebaseofshoots,becauseweakGUSexpressionwasobservedinthebaseofstemswithoutheattreatment(Fig.2b).ItisalsopossiblethattheCaMV35Spromoterinfrontofhygro-mycinphosphotransferasegeneaffectedtheHSP101promoteractivity.LeakyexpressionsofOsWRKY11couldalsoexplainthefactthatlessimpairedwaterlossofdetachedleaveswasobservedinplantswithoutheatpre-treatment(Fig.7).

InvolvementofOsWRKY11instresstolerance

Successfulenhancementofdroughttolerancebyoverex-pressionoftranscriptionfactorshasbeenreportedinrice.Itoetal.(2006)reportedthatoverexpressionofArabid-opsisDREB1andriceorthologOsDREB1showedimprovedtolerancetodrought,high-salt,andlow-temper-aturestressesintransgenicrice.Huetal.(2006)reportedthatoverexpressionofstress-responsivegeneSNAC(STRESS-RESPONSIVENAC1)signi cantlyenhancesdroughtresistanceintransgenicrice.Improvementofwateruseef ciencyanddroughttoleranceinricehasbeenreportedbyexpressionofHARDY,anArabidopsisdroughtandsalttolerancegeneencodinganAP2/ERF-liketran-scriptionfactor(Karabaetal.2007).Xuetal.(2008)reportedthatoverexpressionofZFP252,whichencodesaTFIIIA-typezinc ngerprotein,inriceincreasedtheamountoffreeprolineandsolublesugar,elevatedtheexpressionofstressdefensegenes,andenhancedtolerancetosaltanddroughtstresses.

WRKYproteinsconstitutealargefamilyoftranscrip-tionfactorsimplicatedinmanydifferentprocesses.WRKYgenesplayavarietyofdevelopmentalandphysiologicalrolesinplants.Themostreportedstudiesforthis123

PlantCellRep(2009)28:21–30

superfamilyofgenesaddresstheirinvolvementindiseaseresponses.OverexpressionofAtWRKY18intransgenicArabidopsisplantsresultsinenhancedexpressionofpathogenesis-relatedgenes(ChenandChen2002).EnhancementofbioticstressbyoverexpressingWRKYgeneshasbeenreported.Shimonoetal.(2007)reportedthatoverexpressionofBTH-andSA-inducibleWRKY45geneinricemarkedlyenhancedresistancetoriceblastfungus.OverexpressionofOsWRKY71geneinriceresultedinenhancedresistancetovirulentbacterialpathogensXanthomonasoryzaepv.oryzae(Xoo)13751(Liuetal.2007).However,employmentofWRKYgenestoenhanceabioticstresseshasnotyetbeenreported.

TherelationshipbetweenWRKYandabioticstressresponseshasbeenreportedforsomeplantspecies.AbarleygenecodingforWRKYprotein,Hv-WRKY38,hasbeenreportedtobeinvolvedincold-anddrought-responseinbarley(Mareetal.2004).InvolvementofanABA-inducibleWRKYgeneinabioticstresseshasrecentlybeenreportedincreosotebush(Larreatridentate)(Zouetal.2007).Qiuetal.(2004)reportedthat10of13OsWRKYgenes(OsW8-9,12–14,16,17,21–24,26,30,and45)weredifferentiallyregulatedinplantstreatedbythefourfol-lowingabioticstressfactors:NaCl,PEG,cold(4°C),andheat(42°C).Theresultingexpressionpro lesexhibitedsigni cantdifferencesinboththemannerandtimingoftheirresponsetothefourdifferentabiotictreatments.Thedifferenceofgeneexpressionpro lessuggestedthedif-ferentphysiologicalfunctionsamongtheWRKYgenes.Ourresultsinthecurrentstudyhavedemonstratedthatthetransgeniclines#ox2and#ox3showedsigni cantdesiccationtolerance,asindicatedbytheslowerwaterlossindetachedleaves(Fig.7).Afterheatpretreatment,thetransgeniclines#ox2and#ox3showedsigni cantheatanddroughttolerance,asindicatedbytheslowerleaf-wiltingandless-impairedsurvivalrateofgreenparts(Figs.5,6).OsWRKY11geneproductsmayfunctioninheatanddroughtstressresponseandtolerance.Thisisthe rstreportofenhancementofabioticstressbyoverexpressionofWRKYgenes.

Xieetal.(2005)undertookacomprehensivecomputa-tionalanalysisofricegenomicsequencesandpredictedthestructuresof81OsWRKYgenes,48ofwhicharesupportedbyfull-lengthcDNAsequences.Recently,Ramamoorthyetal.(2008)havepredicted103genesencodingWRKYtranscriptionfactorsandreportedcomprehensivetran-scriptionalpro lesoftheWRKYgenefamilyinriceundervariousabioticandphytohormonetreatments.OsWRKY11(LOC_Os01g43650)wasshowntoberelativelyhighlyexpressedinyoungandmaturepaniclesandweaklyexpressedinyoungandmatureleavesandrootsundernormalgrowthconditions.TheyreportedthatOsWRKY11wasnotinducedunderabiotic(cold,drought,andsalinity)

WRKY REPORT

PlantCellRep(2009)28:21–30orvarioushormone(ABA,IAA,gibberellicacid,methyljasmonate,andsalicylicacid)treatments.Ryuetal.(2006)carriedoutasystematicexpressionanalysisofOsWRKYgenesandidenti edthat,among45testedgenes,theexpressionof15genes,includingOsWRKY11,wasincreasedremarkablyinanincompatibleinteractionbetweenriceandriceblastfungus(Magnaporthegrisea).Theyalsosuggestedthatthesegenesarealsoinvolvedindefenseresponsetoabioticstress,suchashumidity,touchstressinducedbyspraying,andaltereddiurnalrhythmsduetoprolongeddarknessduringthepathogeninfectionpro-cedure.TheinductionofsomeOsWRKYexpressions,includingOsWRKY11,hasbeenshowntobeinducedbywoundinginclippedleaves.ItisconsideredthatOs-WRKY11isinvolvedinbioticstressresponsetodisease,aswellinasheatanddroughtresponse.Thetransgenicplants#ox2and#ox3areexpectedtogaintolerancetosuchabioticstresses.

ThemostsimilarproteintoOsWRKY11inArabidopsisisAtWRKY28encodedbyAT4G18170withasimilarityof88%intheWRKYdomainand48%inthewholeproteins.OsWRKY11containsasingleWRKYdomainwithC2H2-type(C-X4–5-C-X22–24-H-X1–2-H)zinc ngermotifandbelongstoGroupIIoftheWRKYfamily(Xieetal.2005).OsWRKY11containstheconsensuscoactivatormotif,LXXLL,whereLisLeuandXisanyaminoacid.Os-WRKY11,therefore,isexpectedtobindspeci callytotheDNAsequencemotif(T)(T)TGAC(C/T),whichisknownastheWbox,inpromoterregionsandtoactivatecertaingeneexpression.WearenowinvestigatingwhatkindsofgenesareupregulatedbyOsWRKY11.

Conclusion

Droughtandheataremajorabioticstressesoncroppro-ductionassociatedwithglobalwarming.TheHSP101promoterwasshowntobeactivatedbyheatbutnotbydroughtstress.Heatstress,however,oftenprecedesdroughtstress.TheHSP101promoter,therefore,willbeusefultoconferheatanddroughttolerancein eldcondi-tions,minimizinggrowthdefectsinunstressedconditions.OurdatasuggestthattheOsWRKY11gene,togetherwiththeHSP101promoter,holdspromisingutilityinimprovingdroughtandheattoleranceinrice.

References

AgarwalM,SahiC,Katiyar-AgarwalS,AgarwalS,YoungT,Gallie

DR,SharmaVM,GanesanK,GroverA(2003)Molecularcharacterizationofricehsp101:complementationofyeasthsp104mutationbydisaggregationofproteingranulesand

29

differentialexpressioninindicaandjaponicaricetypes.PlantMolBiol51:543–553

AsaiT,TenaG,PlotnikovaJ,WillmannMR,ChiuWL,Gomez-GomezL,BollerT,AusubelFM,SheenJ(2002)MAPkinasesignalingcascadeinArabidopsisinnateimmunity.Nature415:977–983

BajajS,MohantyA(2005)Recentadvancesinricebiotechnology–

towardsgeneticallysuperiortransgenicrice.PlantBiotechJ3:275–307

ChenC,ChenZ(2000)Isolationandcharacterizationoftwo

pathogen-andsalicylicacid-inducedgenesencodingWRKYDNA-bindingproteinsfromtobacco.PlantMolBiol42:387–396ChenC,ChenZ(2002)Potentiationofdevelopmentallyregulated

plantdefenseresponsebyAtWRKY18,apathogen-inducedArabidopsistranscriptionfactor.PlantPhysiol129:706–716ChristensenAH,SharrockRA,QuailPH(1992)Maizepolyubiquitin

genes:structure,thermalpertubationofexpressionandtranscriptsplicing,andpromoteractivityfollowingtransfertoprotoplastsbyelectroporation.PlantMolBiol18:675–689

DellagiA,HelibronnJ,AvrovaAO,MontesanoM,PalvaET,Stewart

HE,TothIK,CookeDE,LyonGD,BirchPR(2000)ApotatogeneencodingaWRKY-liketranscriptionfactorisinducedininteractionswithErwiniacarotovorasubsp.atrosepticaandPhytophthorainfestansandiscoregulatedwithclassIendoch-itinaseexpression.MolPlantMicrobeInteract13:1092–1101DongJ,ChenC,ChenZ(2003)Expressionpro lesofthe

ArabidopsisWRKYgenesuperfamilyduringplantdefenseresponse.PlantMolBiol51:21–37

DuL,ChenZ(2000)Identi cationofgenesencodingreceptor-like

proteinkinasesaspossibletargetsofpathogen-andsalicylicacid-inducedWRKYDNA-bindingproteinsinArabidopsis.PlantJ24:837–847

EulgemT,RushtonPJ,RobatzekS,SomssichIE(2000)TheWRKY

superfamilyofplanttranscriptionfactors.TrendsPlantSci5:199–206

HaraK,YagiM,KusanoT,SanoH(2000)Rapidsystemic

accumulationoftranscriptsencodingtobaccoWRKYtranscrip-tionfactoruponwounding.MolGenGenet263:30–37

HinderhoferK,ZentgrafU(2001)Identi cationofatranscription

factorspeci callyexpressedattheonsetofleafsenescence.Planta213:469–473

HoodE,GelvinSB,MelchersLS,HoekemaA(1993)New

Agrobacteriumhelperplasmidsforgenetransfertoplants.TransgenicRes2:208–218

HuH,DaiM,YaoJ,XiaoB,LiX,ZhangQ,XiongL(2006)

OverexpressingaNAM,ATAF,andCUC(NAC)transcriptionfactorenhancesdroughtresistanceandsalttoleranceinrice.ProcNatAcadSciUSA103:12987–12992

HuangT,DumanJG(2002)Cloningandcharacterizationofathermal

hysteresis(antifreeze)proteinwithDNA-bindingactivityfromwinterbittersweetnightshade,Solanumdulcamara.PlantMolBiol48:339–350

ItoY,KatsuraK,MaruyamaK,TajiT,KobayashiM,SekiM,

ShinozakiK,Yaguchi-ShinozakiK(2006)FunctionalanalysisofriceDREB1/CBF-typetranscriptionfactorsinvolvedincold-responsivegeneexpressionintransgenicrice.PlantCellPhysiol47:141–153

JeffersonRA,KavanaghTA,BevanMW(1987)GUSfusions:beta-glucuronidaseasasensitiveandversatilegenefusionmarkerinhigherplants.EMBOJ6:3901–3907

JohnsonCS,KolevskiB,SmythDS(2002)TRANSPARENTTESTA

GLABRA2,atrichomeandseedcoatdevelopmentgeneofArabidopsis,encodesaWRKYtranscriptionfactor.PlantCell14:1359–1375

KarabaA,DixitS,GrecoR,AharoniA,TrijatmikoKR,Marsch-MartinezN,KrishnanA,NatarajaKN,UdayakumarM,Pereira

123

WRKY REPORT

30

A(2007)Improvementofwateruseef ciencyinricebyexpressionofHARDY,anArabidopsisdroughtandsalttolerancegene.ProcNatlAcadSciUSA104:15270–15275

KasugaM,LiuQ,MiuraS,Yamaguchi-ShinozakiK,ShinozakiK

(1999)Improvingplantdrought,salt,andfreezingtolerancebygenetransferofasinglestress-inducibletranscriptionfactor.NatBiotechnol17:287–291

KasugaM,MiuraS,ShinozakiK,Yamaguchi-ShinozakiK(2004)A

combinationoftheArabidopsisDREB1Ageneandstress-induciblerd29Apromoterimproveddrought-andlow-temper-aturestresstoleranceintobaccobygenetransfer.PlantCellPhysiol45:346–350

KimCY,LeeSH,ParkHC,BaeCG,CheongYH,ChoiYJ,HanC,

LeeSY,LimCO,ChoMJ(2000)Identi cationofriceblastfungalelicitor-responsivegenesbydifferentialdisplayanalysis.MolPlantMicrobeInteract13:470–474

Lagace

´M,MattonDP(2004)CharacterizationofaWRKYtranscriptionfactorexpressedinlatetorpedo-stageembryosofSolanumchacoense.Planta219:185–189

LiuX,BaiX,WangX,ChuC(2007)OsWRKY71,arice

transcriptionfactor,isinvolvedinricedefenseresponse.JPlantPhysiol164:969–979

MareC,MazzucotelliE,CrosattiC,FranciaE,StancaAM,Cattivelli

L(2004)Hv-WRKY38:anewtranscriptionfactorinvolvedincold-anddrought-responseinbarley.PlantMolBiol55:399–416

OonoY,SekiM,NanjoT,NarusakaM,FujitaM,SatohR,SatouM,

SakuraiT,IshidaJ,AkiyamaK,IidaK,MaruyamaK,SatohS,Yamaguchi-ShinozakiK,ShinozakiK(2003)Monitoringexpressionpro lesofArabidopsisgeneexpressionduringrehydrationprocessafterdehydrationusingca7000full-lengthcDNAmicroarray.PlantJ34:868–887

OzturkZN,Talame

´V,DeyholosM,MichalowskiCB,GalbraithDW,GozukirmiziN,TuberosaR,BohnertHJ(2002)Monitoringlarge-scalechangesintranscriptabundanceindrought-andsalt-stressedbarley.PlantMolBiol48:551–573

PnueliL,Hallak-HerrE,RozenbergM,CohenM,GoloubinoffP,

KaplanA,MittlerR(2002)Molecularandbiochemicalmech-anismsassociatedwithdormancyanddroughttoleranceinthedesertlegumeRetamaraetam.PlantJ31:319–330

QiuY,JingS,FuJ,LiL,YuD(2004)Cloningandanalysisof

expressionpro leof13WRKYgenesinrice.ChinSciBull49:2159–2168

RamamoorthyR,JiangSY,KumarN,VenkateshPN,Ramachandran

S(2008)Comprehensivetranscriptionalpro lesoftheWRKYgenefamilyinriceundervariousabioticandphytohormonetreatments.PlantCellPhysiol49:865–879

RizhskyL,LiangH,MittlerR(2002)Thecombinedeffectofdrought

stressandheatshockongeneexpressionintobacco.PlantPhysiol130:1143–1151

RizhskyL,DavletovaS,LiangH,MittlerR(2004)Thezinc nger

proteinZat12isrequiredforcytosolicascorbateperoxidase1expressionduringoxidativestressinArabidopsis.JBiolChem279:11736–11743

RobatzekS,SomssichIE(2001)AnewmemberoftheArabidopsis

WRKYtranscriptionfactorfamily,AtWRKY6,isassociatedwithbothsenescence-anddefence-relatedprocesses.PlantJ28:123–133

RomeroC,Belle

´sJM,Vaya´JL,SerranoR,Culia´n ez-Macia`FA(1997)Expressionoftheyeasttrehalose-6-phosphatesynthasegeneintransgenictobaccoplants:pleiotropicphenotypesincludedroughttolerance.Planta201:293–297

RyuHS,HanM,LeeSK,ChoJI,RyooN,HeuS,LeeYH,BhooSE,

WangGL,HahnTR,JeonJS(2006)Acomprehensiveexpres-sionanalysisoftheWRKYgenesuperfamilyinriceplantsduringdefenseresponse.PlantCellRep25:836–847

123

PlantCellRep(2009)28:21–30

SakumaY,MaruyamaK,QinF,OsakabeY,ShinozakiK,

Yamaguchi-ShinozakiK(2006)DualfunctionofanArabidopsistranscriptionfactorDREB2Ainwater-stress-responsiveandheat-stress-responsivegeneexpression.ProcNatlAcadSciUSA103:18822–18827

SekiM,NarusakaM,IshidaJ,NanjoT,FujitaM,OonoY,KamiyaA,

NakajimaM,EnjuA,SakuraiT,SatouM,AkiyamaK,TajiT,Yamaguchi-ShinozakiK,CarninciP,KawaiJ,HayashizakiY,ShinozakiK(2002)Monitoringtheexpressionpro lesof7000Arabidopsisgenesunderdrought,coldandhigh-salinitystressesusingafull-lengthcDNAmicroarray.PlantJ31:279–292

ShimonoM,SuganoS,NakayamaA,JiangCJ,OnoK,TokiS,

TakatsujiH(2007)RiceWRKY45playsacrucialroleinbenzothiadiazole-inducibleblastresistance.PlantCell19:2064–2076

ShirasawaK,TakabeT,TakabeT,KishitaniS(2006)Accumulation

ofglycinebetaineinriceplantsthatoverexpresscholinemono-oxygenasefromspinachandevaluationoftheirtolerancetoabioticstress.AnnBot(Lond)98:565–571

ShirotoY,ToriyamaK,KishitaniS(2004)Geneexpressionanalysis

oftranscriptionfactorsinriceplantssubjectedtodrought,heatorcombinationofbothstresses.BreedRes6(Suppl2):129

SunC,PalmqvistS,OlssonH,Bore

´nM,AhlandsbergS,JanssonC(2003)AnovelWRKYtranscriptionfactor,SUSIBA2,partici-patesinsugarsignalinginbarleybybindingtothesugar-responsiveelementsoftheiso1promoter.PlantCell15:2076–2092

ThomashowMF(1999)Plantcoldacclimation:freezingtolerance

genesandregulatorymechanisms.AnnuRevPlantPhysiolPlantMolBiol50:571–599

WuKL,GuoZJ,WangHH,LiJ(2005)TheWRKYfamilyof

transcriptionfactorsinriceandArabidopsisandtheirorigins.DNARes12:9–26

XieZ,ZhangZ-L,ZouX,HuangJ,RuasP,ThompsonD,ShenQJ

(2005)AnnotationsandfunctionalanalysesofthericeWRKYGenesuperfamilyrevealpositiveandnegativeregulatorsofabscisicacidsignalinginaleuronecells.PlantPhysiol137:176–189

XuYH,WangJW,WangS,WangJY,ChenXY(2004)Character-izationofGaWRKY1,acottontranscriptionfactorthatregulatesthesesquiterpenesynthasegene(?)-d-cadinenesynthase-A.PlantPhysiol135:507–515

XuDQ,HuangJ,GuoSQ,YangX,BaoYM,TangHJ,ZhangHS

(2008)OverexpressionofaTFIIIA-typezinc ngerproteingeneZFP252enhancesdroughtandsalttoleranceinrice(OryzasativaL.).FEBSLett582:1037–1043

YodaH,OgawaM,YamaguchiY,KoizumiN,KusanoT,SanoH

(2002)Identi cationofearly-responsivegenesassociatedwiththehypersensitiveresponsetotobaccomosaicvirusandcharacterizationofaWRKY-typetranscriptionfactorintobaccoplants.MolGenGenet267:154–161

YokoiS,TsuchiyaT,ToriyamaK,HinataK(1997)Tapetum-speci c

expressionoftheOsg6Bpromoter-beta-glucuronidasegeneintransgenicrice.PlantCellRep16:363–367

YokoiS,HigashiS,KishitaniS,MurataN,ToriyamaK(1998)

IntroductionofthecDNAforArabidopsisglycerol-3-phosphateacyltransferase(GPAT)confersunsaturationoffattyacidsandchillingtoleranceofphotosynthesisonrice.MolBreed4:269–275ZhangYJ,WangLJ(2005)TheWRKYtranscriptionfactor

superfamily:itsoriginineukaryotesandexpansioninplants.BMCEvolBiol5:1–12

ZhuJK(2002)Saltanddroughtstresssignaltransductioninplants.

AnnuRevPlantBiol53:247–273

ZouX,ShenQJ,NeumanD(2007)AnABAinducibleWRKYgene

integratesresponsesofcreosotebush(Larreatridentate)toelevatedCo2andabioticstresses.PlantSci172:997–1004