The tomato SlWRKY gene plays an important role in theregu

Jing-binLi,Yu-shiLuan⇑,HuiJin

SchoolofLifeScienceandBiotechnology,DalianUniversityofTechnology,Dalian116024,Chinaarticleinfoabstract

WRKY-typetranscriptionfactorsareinvolvedinmultipleaspectsofplantgrowth,developmentandstressresponses.SlWRKY,acDNAcloneencodingapolypeptideof552aminoacidsandexhibitingthestructuralfeaturesofgroupIofWRKYproteinfamily,wasisolatedfromtomato(SolanumlycopersicumL.cvZhongshuNo.4)usingthehomologouscloningmethod.Semi-quantitativeRT-PCRanalysisindi-catedthatSlWRKYwasup-regulatedbysaltanddroughttreatmentintomatoseedlings.ToinvestigatethebiologicalrolesofSlWRKY,wegeneratedtransgenictobaccosoverexpressingtheSlWRKYandana-lyzedtheirresponsestosaltanddroughtstresses.Transgenictobaccoplantsexhibitedmorevigorousgrowththanwild-typeplantsanddisplayhightolerancetosaltanddroughtstresses.Inordertomini-mizeoxidativedamage,theactivitiesofantioxidantenzymeswereincreasedbutECandtheMDAcontentweredecreasedinthetransgenictobaccoleaves.Furthermore,itwasobservedthattheSlWRKYproteinsregulatethedownstreamgenesandincreasedtheexpressionofdefense-relatedPR1andPR2genes.Theseresultsdemonstratethat,SlWRKYplaysanimportantroleinrespondingtoabioticstress.Ó2012ElsevierInc.Allrightsreserved.Articlehistory:Received17September2012Availableonline1October2012Keywords:TomatoTranscriptionfactorSlWRKYAbioticstressTobacco1.IntroductionPlantsareconstantlychallengedwithvarioustypesofabioticstresses.Tosurvivethesechallenges,plantsnotonlyhavedevel-opedelaboratemechanismstoperceiveexternalsignals,butalsoimplementedadaptiveresponseswithproperphysiologicalandmorphologicalchanges[1].Inrecentyears,researchesconcerningthemechanismsofprotectionagainsttolerancetoenvironmentalstressinplantshavebecomemoresystematic.Moreandmorestudiesthatexaminetheplants’responsestotheenvironmenthavenowfocusedonthelevelofgeneregulation.Researchontranscriptionfactorshasbecomeafocusofcurrentplantgeneresearch.Onceanexternalstimulus/signalmakescontactwithaplant,itusuallypassesthroughaseriesofsignalsandeventuallyinducestheexpressionofmanystress-responsivegenes[2,3].Amongthenumeroustranscriptionfactors,WRKYtranscriptionfactorswereanewtypeoftranscriptionfactorwhichhasbeendis-coveredinmorerecentyears.WRKYtranscriptionfactorsuper-familyisalargefamilyoftran-scriptionfactorsthatarecharacterizedbythepresenceofoneortwo60-aminoacidWRKYdomains,includingaveryhighlycon-servedWRKYGQKheptapeptideatitsN-terminuswithazinc-⇑Correspondingauthor.Fax:+8641184706365.E-mailaddress:ysluan@dlut.edu.cn(Y.-s.Luan).0006-291X/$-seefrontmatterÓ2012ElsevierInc.Allrightsreserved.http://dx.doi.org/10.1016/j.bbrc.2012.09.120finger-likemotifatitsC-terminus[4].MostofidentifiedWRKYproteinscanbindWbox(TTGAC[C/T])toregulategeneexpression.Thisfamilyofgenesiswidelydistributedamongterrestrialplants.Theyhaverapid,transientandtissue-specificexpressionfeaturesthatcouldbeinducedunderabiotic(cold,drought,andsalinity)andbiotic(pathogeny)stresses,andcouldalsoparticipateinbioticandabioticstressresponses[5].Thefactorsinvolvedinplantdefensehavebeenwidelyinvestigatedintherecentyears[6].Sofar,thereare137and89WRKYgenesidentifiedinriceandArabidopsis,respectively[7].Inrice,differentfactorshavebeenshowntoconferresistancetowardbacteria,fungiandenvironmentstress,suchasOsWRKY11[8],OsWRKY13[9]andOsWRKY89[10].Likewise,over-expressionofOsWRKY23[11]orOsWRKY45[12]resultedinenhanceddiseaseresistanceanddroughttoleranceinArabidopsis.InArabidopsis,WRKYgenesaredifferentiallyregulatedfollowingtreatmentwithanavirulentstrainofabacterialpatho-genorSA[13].Over-expressionofeitherAtWRKY25orAtWRKY33inArabidopsisincreasessalttolerance[14].Over-expressionGmWRKY13,GmWRKY21andGmWRKY54inArabidopsisresultedintheplantsbeingmoretoleranttocoldstress,droughtandsaltstress[15].TheseexamplesillustratethatWRKYfactorsformpartofthesignalingprocessesassociatedwithtranscriptionalrepro-grammingwhenplantsencounterhighsalt,heat,cold,droughtorpathogen.AlthoughthereismuchinformationimplicatingWRKYproteinsofmanyplantsinplantdefenseresponses,fewexperimentshavereportedtheuseoftomatotranscriptionfactor672J.-b.Lietal./BiochemicalandBiophysicalResearchCommunications427(2012)671–676WRKYforgeneexpressionanalysisundersalt-relatedordrought-relatedstresses.Inthisstudy,weisolatedandcharacterizedSlWRKYandexam-inedthechangesinresponsestowardvariousabioticstressesbytobaccoplantsoverexpressingSlWRKY.Physiologicalandbiochem-icalchangesofthetransgenicplantsduringstressesandputativedownstreamgeneswerealsoanalysed.Ourresultsprovideausefulreferenceforunderstandingthemolecularmechanismoftran-scriptionalregulationoftheSlWRKYgeneintomato.2.Materialsandmethods2.1.PlantmaterialsandgrowthconditionsTomato(SolanumlycopersicumL.cvZhongshuNo.4)orTobacco(NictianatabacumL.cv89)seedsweresurfacesterilizedandplacedinconicalflaskcontainingMurashigeandSkoogmediumsupple-mentedwith30g/Lsucrose,adjustedtopH5.7,andsolidifiedwith8g/Lagar.Seedlingsweregrownfor3–4weeksunderfluorescentlightfor16hat25±3°C.2.2.NucleicacidextractionandcDNAssynthesisTotalRNAwasextractedfrom6-week-oldtomatoleavesusingTRIZOLreagentaccordingtothemanufacturer’sinstructions(Taka-ra).AllRNAsamplesweresubjectedtoreverse-transcriptionforthesynthesisofthefirstcDNAstrandusingaTakaraReverseTran-scriptionKit.2.3.IsolationoftheWRKYgeneandsequenceanalysisThefull-lengthofSlWRKYwasclonedfromtomatousinghomol-ogy-basedcloningandRT-PCRmethods[16].DegeneratePCRprim-ersweredesignedbasedontheWRKYconservedcodingregionfromSolanumtuberosum(GenBankAccessionNo.ABU49723.1)andCap-sicumannuum(GenBankAccessionNo.AAO86686.1).Primerssequenceswere:WRKY-F:50-TGGMGNAARTAYGGNCARAAR-30andWRKY-R:50-GTRTGYTTNCCYTCRTANGT-30.OneoftheESTshassig-nificantsimilaritytotheMicro-Tom,databasenamedSolanumlyco-persicumcDNA,HTCinfruit(GenBankAccessionNo.AK326880).Usedapairofprimer:WRKY-F:50-GGAAGACTCTCACTCTCAGCAG-CAG-30andWRKY-R:50-TTTTTTTATATCAAATGAAAATTAT-30tofindthefull-lengthofSlWRKYasdescribedpreviously[17].TheDNAMANsoftwareandBLASTsoftwareonline(http://www.ncbi.nlm.nih.gov)wereusedtoanalyzetheDNAandproteinsequences.AconservedWRKYsuperfamilydomainwasfoundviasearchingCon-servedDomainsDatabase(CDD)(http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml).MultiplealignmentswerepreparedusingClustalX1.8.TheonlinetoolComputepI/MWprogram(http://www.expasy.org/proteomics)wasusedtopredicatethetheoreticalpI(isoelectricpoint)andMw(molecularweight)ofSlWRKYprotein.2.4.Stresstreatmentandsemi-quantitativeRT-PCRanalysisAsepticseedlingsoftomatoweregrowninMSmediumcontain-ing200mMNaClor2%polyethyleneglycol.PlantsampleswereharvestedatvarioustimesafterstresstreatmentsandfrozeninliquidnitrogenandstoredatÀ80°Cforfurtheranalysis.2.5.GenerationofSlWRKY-overexpressingtobaccoplantsThecodingregionofSlWRKYwasamplifiedwithusingforwardprimer50-TCCCCCGGGATGACTCCTGCTATGCTT-30(SmaIsequenceisunderlined)andreverseprimer50-GCGAGCTCACGGGCCAAGAAGTATC-30(SacIsequenceisunderlined),andinsertedintothepBI121vectorwithamodifiedcauliflowermosaicvirus(CaMV)35Spromoter.TheresultantrecombinantvectorpBI121-SlWRKYwastransformedintoAgrobacteriumtumefaciensstrainEHA105byafreeze–thawmethod.Singlecoloniesofthebacteriawerepickedandgrownat28°Cin50mLofliquidYEBmediumcontain-ing100mg/Lcarbenicillinand50mg/Lkanamycin.TransformationoftobaccowasperformedusingA.tumefaciensleafdiscmethod.TheputativetransformantswerescreenedusinggenomicPCRanalyses.GenomicDNAwasextractedfromtobaccoleavesusingCTABextractionbufferasdescribedpreviously[18].ForRT-PCRanalysis,totalRNAwasextractedasdescribedabove.PCRproductsweredetectedbyelectrophoresisin1%agarosegels.2.6.StresstoleranceassayofthetransgenicplantsAsepticseedlingsoftransgenictobaccoandwild-type(WT)weresubmergedinto1/2MSmediumcontaining200mMNaClor2%polyethyleneglycol.Tomakeadeepanalysisofthecontribu-tionofSlWRKYtothesaltanddroughttolerance,the4-week-oldseedlingsoftransgenic-tobaccosandWTweretransferredtothepots.Thepotwasfilledtill3/4fullwithsoilmixture(vermicu-lite/soil=1:1).Seedlingswereculturedinagreenhouse(25°C,16hlight)andirrigatedwith200mMNaClsolutionorholdingwateringfortwoweeks.Thenafter2weeks,theplantsofdroughttreatmentwererehydratedandtheirrecoverieswereassessed.Plantswereclassifiedasaliveordeadbasedontheircolor.Plantsexhibitinggreencolorin>50%oftheirtissuewerecountedassur-vivingplants.2.7.MeasurementsofEC,MDAcontent,antioxidantenzymeactivitiesandchlorophyllcontentTheelectricalconductivity(EC)wascalculatedbythemethodofLiuetal.[19].Lipidperoxidationofleaveswasmeasuredintermsofmalondialdehyde(MDA)content,asdescribedbyXuetal.[20]andexpressedaslmol/gfreshweight(FW).Thesuperoxidedis-mutase(SOD)activitywasassayedbymonitoringitsabilitytoinhibitthephotochemicalreductionofnitrobluetetrazolium(NBT).OneunitofSODwasdefinedastheamountofenzymenec-essarytoinhibitthereductionofNBTby50%.Peroxidase(POD)activitywasmeasuredusingguaiacolandH2O2assubstratesandincreaseinabsorbanceat470nmduetooxidationofguaiacolwasrecordedasdescribedpreviously[21].Thecontentofchloro-phyllwasextractedfromaknownfreshweightofleavesin80%(v/v)aqueousacetoneandspectrophotometricallydeterminedandexpressedasmg/gfreshweight[22].2.8.AnalysisofdownstreamgenesregulatedbySlWRKYgenesIntransgenicandWTtobaccos,RT-PCRwasusedtocheckforthepresenceofexpressionsofthetwodefensemarkergenes,PR1andPR2.Thegene-specificprimerswereasfollows:PR1(forwardprimer:50-CCCAAAATTCTCAACAAG-30andreverseprimer:50-TTAGTATGGACTTTCGCCTCT-30);PR2(forwardprimer:50-ATGGCTTTCTTGCAGCTGCCCTTG-30andreverseprimer:50-GAGTCCAA-AGTGTTTCTCTGTGATA-30).Primerssequencesofthehousekeepinggeneactinwere:forward:50-GTGATGGTGTGAGTCACACT-30andreverse:50-GGGAGCCAAGGCGGTGAT-30.2.9.StatisticalanalysisThedataofrootlengths,freshbiomasses,EC,MDAcontents,activitiesofantioxidantenzymesandchlorophyllcontentwereanalyzed.Valuesoncolumnsaremeansofexperimentsconductedintriplicateandstandarderrorsofmeansisindicatedinbars.Theexperimentwasindependentlyrepeatedthreetimes.DuncanJ.-b.Lietal./BiochemicalandBiophysicalResearchCommunications427(2012)671–676673multiplerangetestswereperformedbyusingone-wayanalysisofvariance(ANOVA)onSPSS17.0versionforWindowssoftwareandapvalue<0.05wasconsideredstatisticallysignificant.3.Results3.1.IdentificationofaWRKYcDNAfromtomatoThefull-lengthcDNAofSlWRKYwas2152-bp,whichcontainedanopenreadingframe(ORF)of1659-bpencoding552aminoacids,anuntranslatedregionof130-bpatthe50-endand363-bpofthenoncodingregionatthe30-end.Theestimatedmolecularmassofthepredictedproteinproductwas60347.3andanisoelectricpointof6.99.TwoWRKYDNA-bindingdomainswerefoundviasearch-ingConservedDomainsDatabase(Fig.1A).ForinvestigatingtherelationshipamongtheSlWRKYandotherWRKYproteins,ClustalX1.8wasusedtoanalyzethemultiplealignments,andtheresultsshowedthattheseavailableWRKYproteinswerefoundtopossesstwoWRKYdomains(Fig.1B).BasedonthenumberofWRKYdomainsandthefeaturesofthezincfingermotifs,WRKYfamilyaredividedintothreegroups[23].AlloftheabovedatastronglyindicatedthatSlWRKYbelongedtogroupIWRKYfamily.3.2.ConstitutiveexpressionofSlWRKYintomatoInordertoaddressthebiologicalfunctionofSlWRKY,semi-quantitativeRT-PCRwasusedtoexamineitsexpressionpatternintomato.AsshowninFig.2A,undersalttreatment,rapidaccu-mulationofSlWRKYtranscriptwasobservedafter2h,withamax-imumaccumulationafter4h,followedbyagradualdeclineafter8h.Underdroughttreatment(Fig.2B),SlWRKYtranscriptlevelincreasedonlyafter12hbutdeclinedtobackgroundlevelbeyondwithlongertreatment.IncreasesinSlWRKYexpressionlevelinresponsetosaltanddroughtstressesshownhereweresimilartotheresponsetosimilarstressesreportedforOsWRKY45[12].TheseresultsstronglysuggestthatSlWRKYisinvolvedinbasaltoleranceofsaltanddroughtstress.3.3.ConferssaltanddroughttoleranceintransgenictobaccoInordertoevaluatethefunctionalsignificance,theSlWRKYcDNAwasclonedintothebinaryvectorpBI121underthetran-scriptionalcontroloftheCaMV35SpromoterandtheresultingplasmidwasintroducedintotobaccousingtheAgrobacterium-mediatedleafdisctransformation.TransformantswerescreenedforresistancetokanamycinandthenconfirmedbyPCRanalysis.ThePCRresultsofthreerepresentativepositivetransformantsareshowninFig.2CandD.Allthreelinesshowedthepresenceofa1659-bpbandcorrespondingtosizeoftheSlWRKYcDNAwhilenobandwasdetectedinthecaseofWTtobaccos.BothtransgeniclinesandWTtobaccos(control)wereplacedinthe1/2MSmedium,containing200mMNaClor2%polyethyleneglycol.After45daysofgrowthinthesaltanddroughtstresscon-ditions,significantdifferencewasobservedinrootlengthsandfreshbiomass.ThetransgeniclinesshowedmoredevelopedrootsystemsandhigherfreshbiomasscomparedwithWT.Differenttransgeniclinesshoweddiscrepantsaltresistance(Fig.3AandB)anddroughtresistance(Fig.3CandD),andthebestonewasLine17.MDAisoneofthefinaldecompositionproductsinmembranelipidperoxidation,whichisalsotakenasanindicatoroftheextentofdamagecausedbymembranelipidperoxidation.PODandSODareubiquitousamongaerobicorganismsanditplaysaroleinreducingintracellularreactiveoxygenspeciesandtherebyFig.1.StructureandsequenceanalysisofSlWRKY.(A)CDDanalysisshowingthetwoconserveddomainsofWRKYproteins.(B)ClustalXanalysis,comparisonofdeducedaminoacidsequencesofWRKYfamilyproteinsthathavehighsequencesimilaritywithSlWRKY.TwoblackboxesabovethesequencerepresentedthehighlyconservedDNA-bindingdomain(WRKYdomain).674J.-b.Lietal./BiochemicalandBiophysicalResearchCommunications427(2012)671–676Fig.2.ExpressionanalysisofSlWRKYunderabioticstressconditions.PCRandRT-PCRanalysisoftransgenictobaccosexpressingSlWRKY.(AandB)SlWRKYexpressionlevelsinresponsetosaltanddroughtstressesweredeterminedbysemi-quantitativeRT-PCR.Expressionoftheactingenewasusedasaconstitutiveinternalcontrol.(C)PCRamplifiestheresultusingthetemplateofDNAfromtobacco.Lanes1–8:transgenictobaccoplantscontainingSlWRKY;Lane9:ddH2O;Lane10:wildtype(WT)tobacco;Lane11:pBI121-SlWRKYplasmid.(D)PositivetransgenictobaccoclonesasdetectedbyRT-PCR.Line2,Line4,Line17weretransgenictobaccoplants.M:DL2000Marker.Theproductswereseparatedbyelectrophoresisin1%agarosegels.Fig.3.RootlengthsorfreshbiomassesandbiochemicalchangesrelatedtosaltordroughtstressresponsesintobaccosexpressingSlWRKY.Therootlengthsandfreshbiomassesweremeasuredinsaltstressconditions(AandB)ordroughtstress(CandD).TheEC,MDAcontents,PODandSODactivitiesofWTandtransgenictobaccosweremeasuredundersaltstressconditions(E–H)ordroughtstress(I–L).Foreachconstruct,threelineswereexamined.Samplesmarkedwithvariouslettersweresignificantlydifferent(P<0.05)accordingtoDuncan’sMultipleRangeTest.providingaprotectiveeffectagainstcelldamage.Underthesamestressconditions,ahighreactivitymeansanincreaseintheabilitytoclearfreeradicalsandbetterprotectionfortheplants.OurresultsshowedthatECandMDAcontentweresignificantlyreducedwhereasPODandSODlevelsweresignificantlyup-regulatedinthethreetransgeniclinescomparedwiththeWTaftersalttreatments(Fig.3E–H)anddroughttreatments(Fig.3I–L),suggestingthatoverexpressionofSlWRKYintobaccoplantshelpedtoprotecttheplantsfromlipidperoxidationincurredbystresses.EC,MDAcontent,PODandSODactivityareanimportantfactorindeterminingplantgrowthanddefensecapacity[24].ExpressionofSlWRKYgeneintobaccoincreasedsaltanddroughtstresstolerance.ThesestudiesindicatedtransgenicplantscouldstaythenormalmetabolismbyaccumulatedosmoticsubstanceunderstressconditionandSlWRKYmaybeassociatedwithabioticstressresponsesignalingpathwaysandplaymultiplerolesinplants.3.4.ResponseoftobaccostosaltanddroughtstressaftertransplantingAfter2weeks,seedlingsofWTplantswerewiltingorturningyellowish,whereasthetransgenicseedlingsgrewnearlynormalandtheirleafsizewasdoubledincomparisonwiththesizebeforethetreatment(Fig.4A).Theresultsindicatedthattheoverexpres-sionofSlWRKYenhancedthesalttolerance.Afterwithholdingofwaterfor2weeks,alltransgenictobaccosandWTplantswilted.However,recoveryonnormalwateringJ.-b.Lietal./BiochemicalandBiophysicalResearchCommunications427(2012)671–676675Fig.4.Saltanddroughtstressresponsesintobaccosaftertransplanting.(A)PhenotypeoftransgeniclinesandWTundersaltstress.Transgenicplantsexhibitedbettergrowthperformance.(B)Differentphenotypesareobservedunderdroughtstress.Takerehydrationexperimentsafterdeniedwater.Althoughtransgenictobaccoleavesappearyellow,butrestoredgrowth,WTtobaccocannotrecovergrowth.(C)ExpressionanalysisofSlWRKYputativedownstreamgenes.WTtobaccoexpressionofthePR1andPR2geneswereverylowlevelsthantransgenictobaccos.ThechlorophyllcontentofWTandtransgenictobaccosweremeasuredundersaltstress(D)ordroughtstressconditions(E).Foreachconstruct,threelineswereexamined.Samplesmarkedwithvariouslettersweresignificantlydifferent(P<0.05)accordingtoDuncan’sMultipleRangeTest.wasseen,thetransgeniclineswereabletorecoverwhereasWTplantsremainedinthewiltedstage(Fig.4B).Theseresultssug-gestedthatoverexpressionofSlWRKYconferredabeneficialtraittothetransgenicplantsthatenabledtheplantstobetterwithstandtheconditionofdroughtandhelpedthemtorecoveroncethecon-ditionwasreversed.ThepossibleeffectofSlWRKYonthecontrolofdownstreamgeneexpressionsthroughtranscriptionalregulationwasalsoinvestigated.InthecaseofWTtobacco,expressionsofthePR1andPR2geneswereatverylowlevelscomparedtotransgenicto-bacco(Fig.4C).ThisagainshowedthecontributionofSlWRKYinregulatingtheexpressionlevelsofdownstreamgenes.WRKYproteinscanspecificallyrecognizesomedefense-relatedgenescontainingW-boxelementsintheirpromoters[25].There-fore,therapidincreasesintheexpressionsofWRKYgenesaregen-erallyconsideredtoplayimportantrolesinactivatingtheexpressionsofdownstreamdefense-relatedgenes,suchasPR1andPR2genes[26].OverexpressionofSlWRKYintransgenictobac-coresultedinincreasedexpressionsofdown-streamPRgenes,whicharelikelytobeinvolvedinregulatingresistancetosalt,droughtandpathogenstresses,andthiswasalsoconsistentwiththosementionedpreviously[27].Asmentionedabove,duetothepotentialactivityagainstbioticandabioticstresses,tomatoSlWRKYgenecanbeusedtoenhanceresistanceagainstbioticstress.Therefore,thefunctionsarevaluableforfurtherinvestigations.Saltanddroughtstressescanreducethephotosyntheticpig-mentsofplantsanddeclineinphotosynthesiswasseenaswellashinderingofnormaldevelopmentoftheplants.FromthedatashowninFig.4DandE,thechlorophyllcontentofWTleaveswassignificantlyreducedinresponsetosaltanddroughtstressescomparedtotransgeniclines.4.DiscussionWRKYgeneswereamongseveralfamiliesoftranscriptionfactorgenesthatarewellevidencedtohaveimportantregulatoryrolesinplantssubjectedtovarioushigh-salinityordroughtstresses[28,29].Recently,itwasalsoreportedthatoverexpressionofTaW-RKY2andTaWRKY19couldimprovethesaltanddroughtstresstol-eranceinArabidopsis[30].Inthisstudy,theup-regulatedexpressionofSlWRKYintobaccogavedirectevidence.SlWRKY,istheWRKYtranscriptionfactorisolatedformtomato.IthastwoWRKYDNA-bindingdomainsandbelongedtogroupIWRKYfamily(Fig.1).Itsexpressioncouldberapidlyaccumulatedbynotonlysalttreatmentsbutalsodroughttreatments(Fig.2AandB).TofurtherstudythefunctionsoftheSlWRKY,wetrans-formedthisgeneintotobaccoplantsanduseditintransgenicstud-iesforstress-tolerance.OverexpresssionofSlWRKYimprovedstresstoleranceintransgenictobaccoplantsasrevealedfromchangesinphysiologicalparametersincludingrootlengthsandfreshbiomass.MDAconcentrationhaswidelybeenutilizedtodif-ferentiatesalt-tolerantandsalt-sensitivecultivars[31].Itwasalsopreviouslyreportedtobedirectlyrelatedtodrought[32].Someantioxidantenzymesarealteredwhenplantsaresubjectedtostresses.Theseantioxidantshavebeentoutedasbeneficialformit-igatingtheeffectsofbioticandabioticstresses[33].ItisinterestingtonotethattheSlWRKYpromotestolerancethroughtheregulationofotherphysiologicalparameters,suchasEC,MDA,PODandSOD(Fig.3).TheantioxidantenzymesweresignificantlyhigherwhereasboththeMDAandEClevelswerelowerinstressedtrans-genicplantsthanthoseinstressedcontrolplant.Aftertransplanting,threeindependentSlWRKY-transgeniclines(2,4and17)withhighertransgeneexpressionsshowedbettergrowththantheWTplants(Fig.4AandB).Wealsoanalyzedfortheirchlorophyllproducedunderstresses(Fig.4DandE),andthehigherlevelofchlorophyllproducedbythetransgenicplantsmayprovidesomesortofprotectionagainstdamagesustainedbytheonsetofadverseenvironmentalconditions.TheseresultsindicatethatSlWRKY-overexpressingtobaccosexhibitedsaltanddroughtstresstolerance.Otherwise,thedifferentialtoleranceofthesetransgenicplantstodifferentstressesmayreflectspecificitiesofSlWRKYproteininDNAbindingandregulationofdownstreamgenes.PRgenesaredefensemarkergenesandithasbeenfoundtoincreasestresstol-erance[34].InSlWRKY-overexpressingtobaccos,thePR1andPR2expressionwasup-regulatedcomparedwiththatinWTplants(Fig.4C).ExpressionprofilesofdownstreamgenessuggestedthatSlWRKYhadaspecificmoderateaffinitywiththepromoterofPR1andPR2,anditmayimprovestresstoleranceintransgenicplantsbydirectbindingandactivatingthePRgenes.Transgenictobaccoover-expressingSlWRKYdisplayedsignifi-cantimprovementinsurvivalfollowingsaltanddroughttolerance.FurtherstudyisneededtoelucidatethemechanismsbywhichSlWRKYoperatestoenhanceresistanceagainstenvironmentalstresses.SaltanddroughtisoneofthemajorfactorstolimitthetomatoyieldinChina.ItwillbeveryinterestingandmeaningfultofurtherinvestigatetheimportantrolesofSlWRKYinresponse676J.-b.Lietal./BiochemicalandBiophysicalResearchCommunications427(2012)671–676tosaltanddroughtandtheeffectsofover-expressingSlWRKYonstresstoleranceintomato.AcknowledgmentsThisworkwassupportedbygrantsfromtheNationalNaturalScienceFoundationofChina(30972001and31171971).References[1]H.J.Bohnert,D.E.Nelson,R.G.Jensen,Adaptationstoenvironmentalstresses,PlantCell7(1995)1099–1111.[2]H.Y.Park,H.Y.Seok,D.H.Woo,S.Y.Lee,V.N.Tarte,E.H.Lee,C.H.Lee,Y.H.Moon,AtERF71/HRE2transcriptionfactormediatesosmoticstressresponseaswellashypoxiaresponseinArabidopsis,Biochem.Biophys.Res.Commun.414(2011)135–141.[3]C.W.Li,R.C.Su,C.P.Cheng,Sanjaya,S.J.You,T.H.Hsieh,T.C.Chao,M.T.Chan,TomatoRAVtranscriptionfactorisapivotalmodulatorinvolvedintheAP2/EREBP-mediateddefensepathway,PlantPhysiol.156(2011)213–227.[4]M.R.Duan,J.Nan,Y.H.Liang,P.Mao,L.Lu,L.Li,C.Wei,L.LaiL,Y.Li,X.D.Su,DNAbindingmechanismrevealedbyhighresolutioncrystalstructureofArabidopsisthalianaWRKY1protein,Nucl.AcidsRes.35(2007)1145–1154.[5]P.J.Rushton,I.E.Somssich,P.Ringler,Q.J.Shen,WRKYtranscriptionfactors,TrendsPlantSci.15(2010)247–258.[6]J.H.Lim,C.J.Park,S.U.Huh,L.M.Choi,G.J.Lee,Y.J.Kim,K.H.Peak,CapsicumannuumWRKYbtranscriptionfactorthatbindstotheCaPR-10promoterfunctionsasapositiveregulatorininnateimmunityuponTMVinfection,Biochem.Biophys.Res.Commun.411(2011)613–619.[7]H.Zhang,J.P.Jin,L.Tang,Y.Zhao,X.C.Gu,G.Gao,J.C.Luo,PlantTFDB2.0:updateandimprovementofthecomprehensiveplanttranscriptionfactordatabase,Nucl.AcidsRes.39(2011)1114–1117.[8]X.Wu,Y.Shiroto,S.Kishitani,Y.Ito,K.Toriyama,EnhancedheatanddroughttoleranceintransgenicriceseedlingsoverexpressingOsWRKY11underthecontrolofHSP101promoter,PlantCellRep.28(2009)21–30.[9]M.Cai,D.Qiu,T.Yuan,X.Ding,H.Li,L.Duan,C.Xu,X.Li,S.Wang,Identificationofnovelpathogen-responsivecis-elementsandtheirbindingproteinsinthepromoterofOsWRKY13,ageneregulatingricediseaseresistance,PlantCellEnviron.31(2008)86–96.[10]H.Wang,J.Hao,X.Chen,Z.Hao,X.Wang,YLou,Y.Peng,Z.Guo,OverexpressionofriceWRKY89enhancesultravioletBtoleranceanddiseaseresistanceinriceplants,PlantMol.Biol.65(2007)799–815.[11]S.J.Jing,X.Zhou,Y.Song,D.Q.Yu,HeterologousexpressionofOsWRKY23geneenhancespathogendefenseanddark-inducedleafsenescenceinArabidopsis,PlantGrowthRegul.58(2009)181–190.[12]Y.P.Qiu,D.Q.Yu,Over-expressionofthestress-inducedOsWRKY45enhancesdiseaseresistanceanddroughttoleranceinArabidopsis,Environ.Exp.Bot.65(2009)35–47.[13]J.Dong,C.Chen,Z.Chen,ExpressionprofilesoftheArabidopsisWRKYgenesuper-familyduringplantdefenceresponse,PlantMol.Biol.51(2003)21–37.[14]Y.Jiang,M.K.Deyholos,FunctionalcharacterizationofArabidopsisNaCl-inducibleWRKY25andWRKY33transcriptionfactorsinabioticstresses,PlantMol.Biol.69(2009)91–105.[15]Q.Y.Zhou,A.G.Tian,H.F.Zou,Z.M.Xie,G.Lei,J.Huang,C.M.Wang,H.W.Wang,J.S.Zhang,S.Y.Chen,SoybeanWRKY-typetranscriptionfactorgenes,GmWRKY13,GmWRKY21,andGmWRKY54,conferdifferentialtolerancetoabioticstressesintransgenicArabidopsisplants,PlantBiotechnol.J.6(2008)486–503.[16]H.Jin,Y.S.Luan,F.S.Luan,IsolationandcharacterizationofsalicylicacidinducedtranscriptionfactorSlWRKYfromtomato,HortScience45(2010)S302.[17]K.Aoki,K.Yano,A.Suzuki,S.Kawamura,N.Sakurai,K.Suda,A.Kurabayashi,T.Suzuki,T.Tsugane,M.Watanabe,K.Ooga,M.Torii,T.Narita,T.Shin-I,Y.Kohara,N.Yamamoto,H.Takahashi,Y.Watanabe,M.Egusa,M.Kodama,Y.Ichinose,M.Kikuchi,S.Fukushima,A.Okabe,T.Arie,Y.Sato,K.Yazawa,S.Satoh,T.Omura,H.Ezura,D.Shibata,Large-scaleanalysisoffull-lengthcDNAsfromthetomato(Solanumlycopersicum)cultivarMicro-Tom,areferencesystemfortheSolanaceaegenomics,BMCGenomics11(2010)1471–2164.[18]K.Lohman,S.Gan,M.John,R.M.Amasino,MolecularanalysisofnaturalleafsenescenceinArabidopsisthaliana,PlantPhysiol.92(1994)322–328.[19]H.P.Liu,B.H.Dong,Y.Y.Zhang,Z.P.Liu,Y.L.Liu,Relationshipbetweenosmoticstressandthelevelsoffree,conjugatedandboundpolyaminesinleavesofwheatseedlings,PlantSci.166(2004)1261–1267.[20]S.C.Xu,J.Hu,Y.P.Li,W.G.Ma,Y.Y.Zheng,S.J.Zhu,ChillingtoleranceinNicotianatabacuminducedbyseedprimingwithputrescine,J.PlantGrowthRegul.63(2011)279–290.[21]X.Y.Xu,G.X.Shi,J.Wang,L.L.Zhang,Y.N.Kang,Copper-inducedoxidativestressinAlternantheraphiloxeroidescallus,PlantCellTiss.OrganCult.106(2011)243–251.[22]A.Hegedüs,S.Erdei,G.Horváth,ComparativestudiesofH2O2detoxifyingenzymesingreenandgreeningbarleyseedlingsundercadmiumstress,PlantSci.106(2001)1085–1093.[23]T.Eulgem,P.J.Rushton,S.Robatzek,I.E.Somssich,TheWRKYsuperfamilyofplanttranscriptio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