Fusion genetic analysis of jasmonate-signalling mutants in Arabidopsis

Fusiongeneticanalysisofjasmonate-signallingmutantsinArabidopsisAndersB.Jensen,DoraRaventos²andJohnMundy*InstituteofMolecularBiology,UniversityofCopenhagen,ésterFarimagsgade2A,DK-1353CopenhagenK,DenmarkReceived23August2001;revised2November2001;accepted29November2001.*Forcorrespondence(fax+4535322128;e-mailmundy@biobase.dk).²Presentaddress:NovozymesA/S,Smùrmosevej25,DK-2880Bagsvñrd,Denmark.SummaryJasmonatesinduceplant-defenceresponsesandacttoregulatedefence-relatedgenesincludingpositivefeedbackofthelipoxygenase2(LOX2)geneinvolvedinjasmonatesynthesis.Toidentifyjasmonate-signallingmutants,weusedafusiongeneticstrategyinwhichthe®re¯yluciferase(FLUC)andEscherichiacolib-glucuronidase(GUS)reporterswereexpressedundercontrolofthejasmonate-responsiveLOX2promoter.Spatialandtemporalpatternsofreporterexpressionweredeterminedinitially,andrevealedthatJA-responsiveexpressionfromtheLOX2promoterrequireddenovoproteinsynthesis.Reporteractivitywasalsoinducedbytheproteinkinaseinhibitorstaurosporineandantagonizedbytheproteinphosphataseinhibitorokadaicacid.FLUCbio-imaging,RNAgel-blotanalysisandprogenyanalysesidenti®edthreerecessivemutantsthatunderexpresstheFLUCreporter,designatedjue1,2and3,aswellastworecessivemutants,designatedjoe1and2,thatoverexpressthereporter.GeneticanalysisindicatedthatreporteroverexpressioninthejoemutantsrequiresCOI.joe1respondedtoMeJAwithincreasedanthocyaninaccumulation,whilejoe2respondedwithdecreasedrootgrowthinhibition.Inaddition,reporterinductionandendogenousLOX2expressionbystaurosporinewasabsentinjoe2.Keywords:bio-imaging,kinase,jasmonate,lipoxygenase,luciferase,phosphatase.IntroductionJasmonates(JAs),includingjasmonicacid(JA)andmethyljasmonate(MeJA),aresynthesizedfromlinolenicacidviaaninducibleoctadecanoidpathway(Mueller,1997).Theyactasplanthormonesbyregulatingdevelop-mentalprocessesandresponsestoenvironmentalcues.Theseincluderootgrowth,pollendevelopment,abscis-sion,senescence,andresponsestowoundingandUVirradiation(CreelmanandMullet,1997;Farmer,1994;ReymondandFarmer,1998).Jasmonatescanbedetectedinvarioustissueswheretheymayaltergeneexpressionattranscriptionalandpost-transcriptionallevels(CreelmanandMullet,1997;WasternackandParthier,1997).SeverallinesofevidenceimplicateJAsinwound-responsivegeneexpression.First,knownwound-respon-sivegenescanbeinducedbyapplicationofexogenousJAs(ReymondandFarmer,1998).Second,levelsofJAsincreaselocallyandsystemicallyuponmechanicalwound-ing(Hildmannetal.,1992),andsystemicinductionofdefencegenesmayrequiretheactionofJAs(Lightnerã2002BlackwellScienceLtd

etal.,1993;Titarenkoetal.,1997).Third,geneticstudiesinArabidopsisandotherplantsindicatethatJAsarerequiredforprotectionagainstpathogens(McConnetal.,1997;Penninckxetal.,1996;Pieterseetal.,1998;Staswicketal.,1998).MolecularstudieshaveelucidatedaspectsofJA-signall-ingpathwaysandhowtheymayrelatetopathwaysmediatedbyabscisicacidorethylene(O'Donnelletal.,Âsetal.,19);Äa-Corte1996;Rojoetal.,1999);elicitors(Penthepolypeptidesystemin(Pearceetal.,1991);orelectricstimuli(Wildonetal.,1992).AnalysesofthepromotersofJA-responsegenesinArabidopsis,barleyandsoybeanhaveidenti®edcis-actingsequences,butlittleisknownaboutthetrans-actingfactorsthatmayrecognizethem(Masonetal.,1993;Rousteretal.,1997;Vignutellietal.,1998).However,MeJAinducestheproductionofmanysecondarymetabolites,andrecentworkhaselucidatedcis-actingsequencesandtrans-actingfactorsregulatingtheaccumulationofterpenoidindolealkaloidsin595

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Catharanthus(vanderFitsandMemelink,2000;Menkeetal.,1999).TreatmentswithpharmacologicalcompoundsindicatethatJA-responsivesignallinginArabidopsisinvolvesactivationbyaproteinphosphatase,andrepressionbyaproteinkinase(Rojoetal.,1998).GeneticstudiesinArabidopsishaveidenti®edputativeJA-signallingmutantswithreducedsensitivitytoJAs(Bergeretal.,1996;Feysetal.,1994;Staswicketal.,1992).Oneofthese,coi1,hasbeenshowntobedisruptedinanF-boxproteinthatmayrecruitregulatorsofdefenceresponsesandpollendevelopmentformodi®cationbyuibiquitination(Xieetal.,1998).Mutantsthatexhibitconstitutiveaccumulationofjasmonate-regulatedgeneshavealsobeenidenti®ed(EllisandTurner,2001;Hilpertetal.,2001;Xuetal.,2001).Interestingly,cev1exhibitsincreasedresistancetopow-derymildew,andconstitutiveaccumulationofVSPincev1wasrepressedinacev1/coi1doublemutant(EllisandTurner,2001).Incontrast,doublemutantsofcex1andcoi1maintainedthecex1phenotype,indicatingthatCEX1actsdownstreamofCOI1inaJA-signallingcascade(Xuetal.,2001).OtherworkinArabidopsishasshownthatcrosstalkoccursbetweensignallingpathwaysregulatedbyJAs,ethyleneandsalicylicacid(SA).Forexample,studiesoftheeffectofmutationsinETR1,EIN2andCOI1onexpressionofthePDF1.2defensingeneinresponsetoinfectionwithAlternariabrassicicola,orbytreatmentswithJAandethylene,suggestamodelwherebyethyleneandJAarebothrequiredforPFD.1.2expression(Penninckxetal.,1998).InteractionsorcrosstalkbetweenJA-andSA-dependentsignallingremainlessclear.Forexample,simultaneousactivationofSA-andJA-depen-dentpathwayshasbeenobservedinthecpr5andcpr6constitutivepathogen-responsemutants(Clarkeetal.,2000).OtherevidenceindicatesthatJAandSAsignallingactantagonistically.Forexample,SAinhibitsJA-respon-sivePin2mRNAaccumulationintomato(PenÄa-CorteÂsetal.,1993).RepressionmayalsobeindependentofSAbecausePDF1.2expressionwasnotJA-inducibleintheArabidopsisconstitutivepathogen-responsemutantmpk4expressingtheNahGsalicylatehydroxylasetransgene(Petersenetal.,2000).Jasmonatealsoantagonizesozone-inducedSAandhydrogenperoxideaccumulationinArabidopsis(Raoetal.,2000),andSA-dependentresponsesintobaccoandotherplants(Nikietal.,1998).WedescribehereafusiongeneticapproachtoidentifyadditionalcomponentsofJAsignalling.TransgenicArabidopsisplantswereproducedthatexpressbothEscherichiacoliuidA(GUS)and®re¯yluciferase(FLUC)reportersundercontroloftheMeJA-responsiveLOX2promoter.LOX2encodesachloroplast-targetedlipoxy-genasethoughttobeinvolvedinthebiosynthesisofJAsfromlinolenicacid(Belletal.,1995).Ifso,LOX2expressionispositivelyfedback,regulatedbyproductJAs,presum-ablyviaapromoterregulatoryelementthatisamoleculartargetofaJA-signallingpathway.SeedofthetransgenicplantsweremutagenizedandtheprogenyscreenedformutantsexhibitingalteredexpressionoftheJA-respon-sivetransgenes.Suchfusiongeneticscreensformutantsbasedonmeasurementsoftransgeneexpressionofferanalternativetootherphenotypicselections(Millaretal.,1992).Theuseofdualreporters,inthiscaseFLUCandGUS,mayalsofacilitateidenti®cationoftrans-signallingmutations.ResultsExpressionofjasmonateresponsegenesandcharacterizationoftheLOX2promoterTranscriptsofseveralArabidopsisgenessuchasLOX1and2(BellandMullet,1993),JR12and3(Titarenkoetal.,1997),thedefensinPDF1.2,andthepathogenesis-relatedgenesPR-3and4(Thommaetal.,1998)havebeenshowntoaccumulateonMeJAtreatment.Recently,micro-arrayanalysisidenti®edasetof221ArabidopsismRNAsmorethan2.5-foldinducedbyMeJAtreatment(Schenketal.,2000).ManyofthesemRNAshavealsobeenshowntoaccumulatewhenplantsareexposedtowoundingorinsectfeeding(Reymondetal.,2000)ToselectaJA-responsivepromoterforfusiongeneticanalyses,NorthernblotanalysiswasperformedwithLOX2,JR1andPDF1.2indifferentmutantbackgrounds.Nine-day-oldCol-0,ein2,ein3andcoi1seedlingsweretransferredfromplatestoliquidmediumandpre-incub-atedfor2days.MeJAwasaddedforthelast8hbeforeRNAwasextractedfromcontrolandMeJA-treatedplants.AsshowninFigure1(a),LOX2andJR1mRNAsaccumu-latedinethylene-insensitiveein2andein3tosimilarlevelsasinCol-0plants.Incontrast,PDF1.2wasnotinducibleinein2,andnoneofthethreemRNAswasinducibleincoi1.TheseresultsindicatethatLOX2andJR1aresimilarlyregulatedbyMeJA,andthattheirexpressionisdependentonCOI1,whilemutationsinanethylene-signallingpath-waydonotin¯uencetheirexpression.Incontrast,MeJAinductionofPDF1.2requiresCOI1andEIN2,suggestingthatCOI1participatesintwopathwaysorinabranchedpathwaytoregulatePDF1.2expression.LOX2,JR1andPDF1.2mRNAsaccumulatedtothesamelevelinEIN3asinCol-0wild-typeplants.Toavoidthepossibilityofisolatingethylene-signallingmutants,wethereforedecidedtousetheLOX2promoterinourgeneticscreen.A1.8kbp5¢upstreamfragmentoftheLOX2gene(Melanetal.,1993)wasfused5¢toboththeFLUC(Millaretal.,1992)andGUSreportersinasinglebinaryT-DNAvectorLOX2-FLUC/GUSandusedtostablytransformArabidopsisCol-0.HistochemicalstainingwasperformedtoanalyseãBlackwellScienceLtd,ThePlantJournal,(2002),29,595±606

Figure1.LOX2expressioninmutantsandMeJA-inducibleFLUC/GUStransgenics.(a)Northernblothybridizationof20mgtotalRNAfrom11-day-oldwild-type,ein2,ein3andcoi1-1plantletstreatedwithorwithout50mMMeJAforthelast8h.Filterswerehybridizedtogene-speci®cprobesofLOX2,JR1andPDF1.2andthe18SrDNAcontrol.(b)HistochemicallocalizationofGUSin11-day-oldsoil-grownplantlets(topleft)andin¯oralorgansof6-week-oldplants(topright).GUSaccumulationin11-day-oldplantletstreatedfor24hwith50mMMeJA(bottom).theaccumulationoftheGUSreporter(Figure1b).GUSactivitywasdetectableonlyinthehydathodesofsterile-grownseedlings(Figure1b,topleft).GUSactivitywassubsequentlydetectedduringdevelopmentinsepals,petalsandimmatureanthers,andlaterinthestyleandatthebaseofthecarpels(Figure1b,topright).Inplantsgrownundernormalgreenhouseconditions,GUSactivitycouldbedetectedaroundinfectionsites(notshown).ToanalyseGUSreporterresponsivenesstoMeJA,plantsweresprayedwith50mMMeJAandstained24hlater.YoungplantsaccumulatedhighlevelsofGUSactivityfollowingMeJAtreatmentsinthemesophyllandinvasculartissue(Figure1b,lowerpanel).OlderplantsalsorespondedtoMeJAtreatments,butaccumulatedlowerlevelsofGUSactivitythanyoungerplants.TheseresultsindicatethatregulatoryelementsmediatingMeJA-ãBlackwellScienceLtd,ThePlantJournal,(2002),29,595±606

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responsiveGUSexpressionarecontainedinthe1.8kbLOX2promoter.MeJA-inducedaccumulationofLOX2mRNArequiresdenovoproteinsynthesisSeveraltargetgenesregulatedbyjasmonatehavebeenshowntorequiredenovoproteinsynthesisfortheirinduction(Rojoetal.,1998).ToanalysewhetherdenovoproteinsynthesisisrequiredforMeJA-inducedLOX2expression,wild-typetransgenicplantswereincubatedinduplicatefor24hwith50mMMeJAwithorwithout100mMchloramphenicoland10mMcycloheximide(PSI;Figure2).Asexpected,theseinhibitorsofchloroplasticandcytosolicproteinsynthesisinhibitedFLUCexpression(Figure2a,row2).Moreover,100mMabscisicacid(ABA),whichinducesseveralJA-responsivegenes(Hildmannetal.,1992),didnotaffectFLUCactivityeitheraloneortogetherwithMeJA.Afterbioluminescenceimaging,plantsofonesamplesetwerewashedthreetimeswithMSandleftat21°Ctotesttheeffectofthetreatmentsonplantviability.Allplantssurvivedthetreatmentswithnosigni®cantnegativein¯uenceontheirgrowthestimatedafter1week.TotalRNAwasextractedfromplantsoftheothersampleset,andNorthernblotanalysisperformedwithagene-speci®cLOX2fragmentasprobe.Figure2(c)showsthatMeJA,butnotABA,inducedaccumulationofLOX2mRNA.However,MeJA-inducedLOX2mRNAaccumulationcouldnotbedetectedinplantstreatedwiththeprotein-synthesisinhibitors.ThisindicatesthatthepathwayfromMeJAtoLOX2expressionrequiresdenovosynthesisofoneormoresignallingand/ortranscriptionalcomponents.ProteinkinasesandphosphatasesmodulateLOX2expressionReversibleproteinphosphorylationmodulatesseveralhormone-signallingpathways.Northernblottinghasrevealedthatinhibitorsofproteinkinasesandphosphat-asesaffecttheaccumulationofseveralMeJA-responsivemRNAs(Rojoetal.,1998).ToquantitativelyanalysetheresponsesoftheLOX2promotertowardssimilarinhibi-tors,equalnumbersof8-day-oldplantsweretransferredfromplatestoliquidmediuminduplicate,andincubated2daysbeforetreatmentwithMeJAinthepresenceorabsenceofstaurosporineorokadaicacid.Staurosporineisabroad-range,serine±threonineproteinkinaseinhibitor(MackintoshandMackintosh,1994),whileokadaicacidinhibitsproteinphosphatasetype1(PP1)and2A(PP2A;Cohenetal.,1990).FLUCemissionwasmeasured24hafteradditionoftheinhibitors.AsshowninFigure3(a),1mMstaurosporineincreasedFLUCactivityto52%ofthelevelobservedaftertreatmentwith50mMMeJAalone.Moreover,applicationofbothMeJAandstaurosporine598AndersB.Jensenetal.

Figure2.FLUCandLOX2expressioninthepresenceofinhibitorsofproteinsynthesis.(a)Bioluminescenceofliquidculturesof11-day-oldplantletstreatedwithoutorwith50mMMeJA,50mMABAor50mMMeJAandABAintheabsence(row1)orpresence(row2)of100mMchloramphenicoland10mMcycloheximide(PSI)added30minbeforeMeJAapplication.(b)Bright-®eldimageoftheplateshownin(a).(c)Northernblotanalysisof20mgtotalRNAextractedfromtheplantsshownin(b),andhybridizedwithradiolabelledprobesofLOX2and18SrDNAcontrol.resultedin138%inductioncomparedtotreatmentswith50mMMeJAalone.ThissuggeststhataproteinkinaseactivitynegativelyregulatestranscriptionfromtheLOX2promoter.Incontrast,1±500nMokadaicacidinhibitedMeJA-responsiveFLUCactivitysuchthathalfmaximumFLUCactivity(IC50)wasobservedbetween5and10nMokadaicacid(Figure3b).Calyculin,whichalsoinhibitsPP1andPP2A(Ishiharaetal.,19),alsoinhibitedFLUCactivity,Figure3.ReversibleproteinphosphorylationmodulatesFLUCexpression.(a)FLUCactivitiesofliquidculturestreatedfor24hwithoutorwith50mMMeJA,1mMstaurosporine(Stau),oracombinationof50mMMeJAand1mMstaurosporine.(b)Plantletsgrownasin(a)andtreatedfor24hwithoutorwith50mMMeJAorcombinationsof50mMMeJAandincreasingconcentrationsofokadaicacid(OA).FLUCemissionisgivenastotalgreyvalues(TGV).althoughhigherconcentrationswereneededtoobtainthesamelevelsofinhibition(datanotshown).Asdescribedabove,allplantswerefoundtobeviableaftertheseãBlackwellScienceLtd,ThePlantJournal,(2002),29,595±606

pharmacologicaltreatments.OkadaicacidinhibitsPP2AactivitymorestronglythanPP1(IC500.1±1nM,PP2A;10±15nM,PP1;Cohenetal.,1990).Inaddition,ithasbeendeterminedthatinvivoconcentrationsofokadaicacidupto1mMonlyinhibitproteinphosphatasetype2AinArabidopsis(Rojoetal.,1998).Thissuggeststhat,asforJR1,JR2andVSP,aPP2Aactivatesandaserine±threonineproteinkinasenegativelyregulatesMeJAactivationofLOX2.ToensurethattheseinhibitorsdidnotalterFLUCtranslationorstability,thesamesetoftreatmentswereperformedontransgenicplantsexpressingtheFLUCgeneundercontroloftheconstitutiveactive35SCAMVpro-moter.Nosigni®cantin¯uenceonFLUCactivitywasobservedundertheseconditions(resultsnotshown).IsolationofmutantsalteredinLOX2andFLUCaccumulationPooledM2seedswereplatedandimagedforFLUCactivitybeforeand48hafterMeJAtreatmenttoidentifymutantswithimproperreporter-expressionlevels.Screeningatotalof40000plantsidenti®edasingleplantwithhigh,inducibleFLUCactivitybeforeandafterMeJAtreatment.Thisplantwasdesignatedjoe1(jasmonateoverexpress-ing).Similarly,21plantswereidenti®edwithhighFLUCactivitiesonlyafterMeJAtreatment.Ofthese21hyper-sensitiveplants,onlyonesurvivedtosetseed,andthiswasdesignatedjoe2.Theprogenyofjoe1andjoe2werethenrescreened(Figure4a).ThisindicatedthatMeJA-inducedFLUCactivityinjoe2wasmorethan100-foldcomparedtosome14-foldinwildtypeandjoe1.joe2maythusbeconsideredaMeJA-hypersensitivemutant.Incontrast,joe1exhibitedthesamelevelofMeJAinductionasthewildtype,andisthereforenotstrictlyhypersensitivetoMeJA.However,FLUCactivityinuntreatedjoe1wasmorethan10-foldhigherthaninuntreatedjoe2orwildtype,andthree-and13-foldhigherfollowingMeJAthanjoe2orwildtype(Figure4b).ThesamesetofimageswasalsoanalysedformutantsthatexpressedtheFLUCtransgeneatlowerlevelsthanwildtype.Of78putativelow-expressingmutants,progenyrescreeningidenti®edthreelineswithmorethan®vefoldreducedFLUCactivity(Figure4c).Theseplantsweredesignatedjue1,2and3(jasmonateunderexpressing).ToanalysethetranscriptlevelsofendogenousLOX2intheseplants,totalRNAwasextractedfrom11-day-oldplantssprayedwithoutorwith50mMMeJA24hbeforeextraction.AsshowninFigure4(d),jue1,2and3exhibitedseverelyreducedLOX2mRNAlevelscomparedwiththewildtype.Inaddition,histochemicalGUSstainingofjue1,2and3beforeandafterMeJAtreatmentsshowedthatGUSactivityaccumulatedtomuchlowerlevelsinthemutantsthaninwildtype(notshown).AsthelevelsofãBlackwellScienceLtd,ThePlantJournal,(2002),29,595±606

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activityofbothreportergenesandendogenousLOX2mRNAwereseverelyreducedinjue1,2and3,theirphenotypesmaybecausedbymutationsinpositivelyactingcomponentsofapathwaymediatingMeJA-respon-sivenessofLOX2.joe1and2werechosenforfurtheranalysis,andtheinheritanceofjoe1and2examinedinF1progenyofback-crossestothenon-mutagenizedwildtype.Bio-imagingshowedthatprogenyofbothcrossesexpressedFLUCatsimilarlevelstoF1plantsofacrossbetweenthewild-typetransgenicparentandCol-0.Theseresultsindicatethatbothjoe1and2mutantsarerecessivemutations.Inaddition,segregationanalysisoftheF2progenyfromthesecrossescon®rmedthatbothmutantsarecausedbysingle-locus,recessivemutations.AllelismwastestedbyanalysingFLUCexpressioninF1progenyofacrossbetweenjoe1andjoe2.ThisshowedthatFLUCexpressionintheheterozygousjoe1/joe2wassimilartothatobservedfornon-mutagenizedwild-typeplants,indicatingthatjoe1and2arenotallelic(notshown).Geneticmappingofjoe1andjoe2wasperformedbycrossingthemutantswithLer,andrecombinationeventsscoredusingcleaved-ampli®edpolymorphicsequenceandsimplesequence-linkedpolymorphicmarkers(BellandEcker,1994).Thisindicatedthatjoe1islocatedina12cMregionbetweenCAPSmarkersAGandRPS2onchromosome4,whereasjoe2mapstoa15cMintervalatthetopofchromosome5betweennga225andnga151.MeJAresponsivenessofjoe1and2Toanalysetheresponsesofjoe1and2todifferentlevelsofMeJA,8-day-oldplantsweretransferredfromplatestoliquidmediumandallowedtoacclimatefor2daysbeforeincreasingconcentrationsofMeJAwereaddedforthelast24h.AsseeninFigure5(a),FLUCactivitiesinjoe1and2increasedupto15-foldhigherthanwildtypeexposedto50mMMeJA.FLUCactivitiesofjoe1treatedwith0.5mMMeJAaccumulatedtohigherlevelsthanwildtypetreatedwith50mMMeJA.ThemostdramaticdifferencesintheMeJAdose-responsesofjoe1and2werefoundatconcentrationsof0.5and1mMMeJA:FLUClevelswerearoundthreefoldhigherinjoe1attheseconcentrations.Thisdifferencewasreducedathigherjasmonateconcentrations,suggestingthatjoe1ismoreresponsivethanjoe2tolowconcentra-tionsofMeJA,whilebothjoe1andjoe2exhibitsimilarsensitivitiestohigherconcentrationsofMeJA.NorthernblottingwasperformedtoexaminewhetherendogenousLOX2expressionwasalsoperturbedinjoe1and2.Plantsweretreatedasabove,exceptthatMeJAwasonlyaddedforthelast8hbeforetotalRNAwasextracted.AsshowninFigure5(b),endogenousLOX2mRNAaccu-mulatedtosigni®cantlyhigherlevelsinjoe1treatedwith1,600AndersB.Jensenetal.

10and50mMMeJAthaninwildtype.Likejoe1,joe2respondedtoincreasingconcentrationsofMeJA,exceptthattheLOX2mRNAlevelat1mMMeJAwassimilartothatinwildtype,eventhoughtheFLUClevelwassigni®cantlyhigherinjoe2thaninthewildtype.ThisdiscrepancybetweeninductionlevelsofFLUCactivityandendogenousmRNAaccumulationhasalsobeenobservedinothermutantsisolatedbyLUCbio-imaging(Raventosetal.,2000),andcouldbeduetopost-transcriptionalregulationofthedifferentmRNAs.Figure7.Phenotypiccharacterizationofjoe1and2.(a)14-day-oldwild-type(wt),joe1andjoe2plantletsgrownonMSmediumintheabsenceorpresenceof10mMMeJA.Bars,10mm.(b)Rootlengthof14-day-oldCol-0wt,joe1andjoe2plantsgrownverticallyonMSmediuminthepresenceofincreasingconcentrationsofMeJA.Errorbarsshowstandarddeviationofatleast30plantsforeachtreatment.Figure4.FLUCandLOX2expressionofjoeandjuemutants.(a)Bioluminescenceof9-day-oldjoe1,joe2andwild-type(wt)plantletsbefore(topleft)andaftertreatmentfor48hwith50mMMeJA(topright).Bright-®eldimagesofthesameplatebeforeandafterMeJAtreatment(bottom).(b)QuantitativeFLUCactivities(TGV)ofjoe1,joe2andwtplantletsfromtheplateshownin(a).(c)FLUCactivities(TGV)of9-day-oldjue1,jue2,jue3andwtplantletsbeforeandafter48htreatmentwith50mMMeJA.(d)NorthernblotanalysisofRNAextractedfromseedlingstreatedasin(c)andhybridizedwithaLOX2probe.ãBlackwellScienceLtd,ThePlantJournal,(2002),29,595±606

Figure5.MeJAdose-responsesofjoe1and2.(a)FLUCactivities(TGV)of11-day-oldplantletsofwild-type(wt),joe1andjoe2seedlingstreatedwithincreasingconcentrationsofMeJAfor24h.(b)RNAgel-blotanalysisof11-day-oldwt,joe1and2plantletstreatedfor8hwithincreasingconcentrationsofMeJA.The®lterwashybridizedtoprobesofLOX2,JR1,PDF1.2andthe18SrDNAcontrol.Transcriptlevelsoftwootherjasmonate-regulatedgenes,JR1andPDF1.2,werealsomonitored(Figure5b).JR1followedthesameexpressionpatternasLOX2,suggestingthatjoe1and2acttorepressseveralMeJAtargetgenes.ElevatedlevelsofPDF1.2werealsodetectedinjoe1and2aftertreatmentwith50mMMeJA.ThislaterdifferencecouldresultfromanincreasedPDF1.2transcriptstability,ormayindicatethatfeedbackregulationofPDF1.2isimpairedinbothmutants.ãBlackwellScienceLtd,ThePlantJournal,(2002),29,595±606

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Figure6.Responsesofjoe1and2totreatmentswithstaurosporineandokadaicacid.(a)FLUCactivities(TGV)of11-day-oldwild-type(wt),joe1andjoe2plantletstreatedfor24hwithoutorwith50mMMeJA,1mMstaurosporine(Stau)or50mMMeJAand0.5mMokadaicacid(OA).(b)RNAgel-blotanalysisof11-day-oldwt,joe1andjoe2plantletstreatedfor24hwithoutorwith50mMMeJA,1mMstauor1mMStauand50mMMeJA.The®lterwashybridizedtoprobesofLOX2,PDF1.2andthe18SrDNAcontrol.StaurosporineactivationofLOX2isblockedinjoe2ToanalysetheeffectofstaurosporineandokadaicacidonLOX2expressioninjoe1and2,FLUCactivitieswereimagedinequalnumbersof10-day-oldplantsexposedtoMeJAinthepresenceorabsenceofthetwoinhibitors.Whenstaurosporinewasaddedtothegrowthmedium,FLUCactivitiesofwildtypeandjoe1increasedsevenfoldcomparedtotheuntreatedcontrols(Figure6a).Incontrast,602AndersB.Jensenetal.

staurosporinedidnotsigni®cantlyinduceFLUCactivityinjoe2,althoughokadaicacidseverelyreducedMeJAactiv-ationofFLUCinbothjoe1and2.Okadaicacidalonehadnosigni®canteffectonthebasalFLUCactivities.TheseresultssuggestthatJOE1actsupstream,whileJOE2actsdownstream,ofaproteinkinasemodulatingLOX2mRNAaccumulation.NorthernblotanalysisofRNAfromthetreatmentsdescribedabovecon®rmedthatexpressionoftheendo-genousLOX2genewasaffectedsimilarlytotheFLUCreporter(Figure6b).Inaddition,thesame®lterwashybridizedwithagene-speci®cfragmentofPDF1.2.However,PDF1.2wasnotinducedbystaurosporineinwildtypeorinjoe1and2.ThissuggeststhatMeJAactivationofPDF1.2is,inpart,mediatedbyapathwaydifferentfromthatactivatingLOX2.Visiblephenotypesofjoe1and2Phenotypicdifferencesbetweenwild-typeandjoemutantswerenotedduringvegetativedevelopmentandwhengrownonMeJA.Whereasjoe1hadnoobviousabnorm-alitiesduringnormalgrowthinsoil,itaccumulatedhigherlevelsofanthocyaninonmediumcontainingMeJAthandidthewildtype(Figure7).joe2grewslightlymoreslowlythanwildtypeduringthe®rst6±7daysofdevelopment,althoughthisdifferencedisappearedlaterindevelopment.Inaddition,joe2seedyieldwasonly15%ofwildtype.Thisreducedfertilitywasmainlyduetoreducedpollenreleasefromthepollensacs.Growthofjoe2onMeJA-containingmediumresultedinincreasedrootinhibitioncomparedtowildtype(Figure7b).Incontrast,joe1respondedtogrowthonMeJAsimilarlytowildtype.TheseresultssuggestthatJOE1haslittleaffectonMeJA-mediatedrootinhibition,whereasitappearstoberequiredasanegativeregulatorofanthocyaninaccumulation.Incontrast,JOE2haslittleaffectonanthocyaninaccumulation,althoughitmayberequiredtorelieveMeJA-mediatedrootinhibition.Joe1and2aredependentonCOI1COI1isrequiredforMeJAregulationofLOX2(Figure1).Thelocationofjoe1and2inthejasmonate-signallingcascadecanthereforebeexaminedbytheanalysisofdoublemutantswithcoi1.However,asneitherjoe1norjoe2exhibitsvisiblephenotypicdifferencesfromwildtype,doublemutantsarenoteasilyidenti®ed.F2progenyofcoi1/joe1andcoi1/joe2wereplatedonMSmediumcontaining50mgl±1kanamycinand50mMMeJA.Undertheseconditions,18.75%oftheprogenyshouldberesist-anttokanamycinandinsensitivetoMeJAifcoi1isepistatictojoe1or2,and14.1%inthereversescenario.After12days'growthonkanamycinandMeJA,thesegregationofkanamycin-resistantandMeJA-insensitiveTablegeneration1.MeJAofcrossesinsensitivitybetweenandcoi1-FLUC1andactivitytheinjoethemutantsF2CrossTotalseedsInsensitiveExpectedFLUCac2coi1-coi1-11QQjoe1joe2130914082402572462nonenone5.86.2aFLUCactivityofinsensitiveplants.coi1homozygousseedlingswasanalysed,andtheseplantletstransferredtootherplatesandscreenedforFLUCactivity.AsshowninTable1,morethan1300seedswereplatedfromfourindependentcoi1/joe1andcoi1/joe2crosses.Thecoi1phenotypesegregatedasexpected,as18.75%(240of1309;P>0.05and257of1408;P>0.05)oftheF2progenyofthecoi1/joe1andcoi1/joe2crosseswereinsensitivetojasmonate.HighFLUCactivitywasnotdetectedinanyofthesejasmonate-insensitive,kanamycin-resistantplants,suggestingthatbothjoe1andjoe2actupstreamofCOI1inajasmonatesignal-transductionpathway.DiscussionWeareinterestedinidentifyingcomponentsregulatingtheactionofthehormonejasmonicacid.FusiongeneticscreensareastrategytoselectmutantsinJA-signallingpathwaysbasedonalterationsintheinvivoresponseofatransgenicreportersuchasFLUC.HereweusedthepromoteroftheMeJA-responsiveLOX2genefusedtotheFLUCandGUSreporterstoisolatemutantsalteredinMeJAsignalling.SeveralMeJA-responsivegenes,suchasPDF1.2,requirefunctionalCOI1andEIN2geneproductsforinductionbyMeJA(Penninckxetal.,1998).Incontrast,otherJA-responsegenessuchasVSP,JR1andJR2arehyper-inducedinethylene-insensitiveein2,ein3andetr1mutants,suggestingthatafunctionalethylenesignalpathwayisrequiredfortheirrepression(Rojoetal.,1999).WethereforecomparedtheaccumulationofLOX2,JR1andPDF1.2inein2,ein3andcoi1(Figure1).Incontrasttoapreviousreport(Rojoetal.,1999),wefoundsimilarinducedlevelsofLOX2andJR1mRNAinwild-type,ein2andein3plantsonMeJAtreatment.Whilethisdiscrepancymaybeexplainedbydifferencesinexperimentalcondi-tions,Reymondetal.(2000)alsodidnotdetectaneffectofein2onLOX2expression.Inanyevent,wefoundthatMeJAinductionofLOX2,JR1andPDF1.2requiredfunc-tionalCOI1,andPDF1.2accumulationwasnotobservedintheein2mutanttreatedwithMeJA.NormalPDF1.2levelsweredetectedinein3,suggestingthattheEIN3transcrip-tionactivatordoesnotparticipateinMeJAactivationofãBlackwellScienceLtd,ThePlantJournal,(2002),29,595±606

PDF1.2.Unfortunately,mutationsintheEILgenes(EIN3-LIKE;Chaoetal.,1997)haveyettobecharacterizedtodeterminewhethertheymaybeinvolvedinMeJAactiv-ationofPDF1.2.Nonetheless,theseresultssuggestthatCOI1mayactindifferentMeJA-signallingpathways,oneofwhichisdependentonEIN2butindependentofEIN3.AnalternativepossibilityisthatCOI1andEIN2actinabranchedpathwaytoregulatePDF1.2expression.SinceMeJA-responsiveLOX2mRNAaccumulationwasunaffectedinethylene-insensitivemutants,afusionscreenusingreporterscontrolledbytheLOX2promoterwouldbeunlikelytoidentifyethylene-signallingcomponents.Wethereforemadetransgenicplantscarryinga1.8kbpLOX2promoterfusedtotheFLUCandGUSreporters.YoungseedlingsproducedweakGUSactivityinleaftipsandhydathodesthataccumulatedhighGUSandFLUCactivi-tiesinthemesophyllandvasculatureuponMeJAtreatment(Figures1and4).ThisindicatesthattheLOX2-reportersprovidetoolstoidentifymutantscompromisedinMeJAsignalling.JasmonateresponsivenessofgenessuchasJR1,JR2andVSPrequiresdenovoproteinsynthesis(Rojoetal.,1998).WefoundthatproteinsynthesisisalsorequiredforMeJAinductionofLOX2.TofurthercharacterizeLOX2regulation,pharmacologicalstudiesrevealedthatbothFLUCactivityandLOX2mRNAaccumulationwereinducedbytreatmentswiththeserine±threonineproteinkinaseinhibitorstaurosporine(Figures3and6).Incontrasttostaurosporine,theproteinphosphataseinhibitorokadaicacidpreventedMeJA-inducedFLUCactivity(Figure3).Jasmonate-responsiveJR1,JR2andVSPmRNAaccumulationalsorequiresanactiveproteinphosphatase(Rojoetal.,1998),suggestingacommonmechanismregulatingLOX2andthosetargetgenes.Byanalogy,SAinductionoftobaccoPR-1andPR-2mRNAaccumulationisalsoactivatedbybothstaurosporineandK-252a,andisrepressedbyokadaicacidandcalyculin(Conrathetal.,1997).Thisarguesthatdifferentsignallingpathwaysregulatingpathogen-andwound-induceddefencegenesusesimilarregulatorymechanisms.Wesubsequentlyscreenedapopulationofg-irradiatedprogenyofatransgenicLOX2reporterlinebyLUCbio-imagingtoisolateputativeMeJA-signallingmutants.Thisresultedintheisolationof®vemutantsdesignatedjoe1and2andjue1,2and3.ThesemutantsexhibitedalteredexpressionofbothFLUCactivitiesandendogenousLOX2expression,indicatingtheyareprobablycausedbytrans-actingmutationsaffectingJAsignaltransduction.Interestingly,JA-hypersensitiveoroverexpressionmutantssuchasjoe1and2havenotbeenisolatedinotherrecentscreensusingJA-responsivereporters(EllisandTurner,2001;Hilpertetal.,2001).Furthercharacteriz-ationofjoe1and2revealedthattheyexhibitdifferentMeJAdose-responses.ComparisonofendogenousLOX2ãBlackwellScienceLtd,ThePlantJournal,(2002),29,595±606

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expressioninwild-type,joe1and2plantstreatedwith50mMMeJAfor24hshowedlittledifferenceinmRNAaccumulation(notshown).Inaddition,wound-inducibleLOX2expressionpeakedaround2haftermechanicalwounding,followedbyadramaticdecreaseintheexpres-sionratioat8±24h(Reymondetal.,2000).ThereforeexpressionofLOX2,JR1andPDF1.2wasassayedatearliertimepointsaftertreatmentwithvaryingMeJAconcentrations(Figure5).Undertheseconditions,LOX2mRNAaccumulatedtosigni®cantlyhigherlevelsinjoe1and2thaninwildtypetreatedwithMeJA.ExpressionofJR1followedthesamepatternasLOX2,suggestingthatJOE1andJOE2acttorepressbothLOX2andJR1expressionbyacommonmechanismTransgenicLOX2-FLUC/GUSplantsrespondedtoMeJAandstaurosporinewithincreasedFLUCactivitiesthatcouldbeinhibitedbyokadaicacid.Wethereforeanalysedtheeffectsofthesecompoundsongeneexpressioninjoe1and2.WhileFLUCactivitiesinjoe1showedsensitivitytowardsbothMeJAandstaurosporine,joe2didnotrespondtostaurosporinewithincreasedFLUCactivitiesorLOX2expression(Figure6).Thisindicatesthatthejoe2mutationmayleadtoinactivationofakinaseoritssubstrate,whilejoe1mayactpriortothephosphorylationeventinaJAsignalpathway.Inaddition,thephosphorylationeventappearstoactdownstreamofJAandnotinaconvergingpathway,becausejoe1exhibitedsimilarresponsestothewildtypetobothMeJAandstaurosporine.Phenotypiccharacterizationofjoe1and2revealedthattheyexhibitdifferentresponsestoappliedMeJA.Joe1accumulatedhigherlevelsofanthocyanin,butexhibitedthesameinhibitionofrootgrowthasthewildtype.ThisincreasedanthocyaninaccumulationcouldbeasecondarystressresponseduetoalteredJAsignalling.Incontrast,root-growthinhibitioninjoe2washypersensitivetoMeJA.Whilethisindicatesthatjoe1and2differintheirresponsestoJAindifferenttissues,differencesinthehistochemicallocalizationofGUSwerenotseeninjoe1and2(notshown).Jasmonateresponses,includingLOX2expression,aredependentonCOI1.Asexpected,JA-insensitive,kana-mycin-resistantF2progenyofcrossesbetweencoi1andjoe1or2didnotexhibitincreasedexpressionoftheLOX2-FLUC/GUSreportersinresponsetoMeJA.Inaddition,basallevelsofGUSaccumulationinthehydathodesofsuchplantsweresigni®cantlyreduced.ThisindicatesthatJOE1andJOE2actpriortoCOI1toregulateLOX2expression.Physicalmappinglocalizedjoe1toa12cMintervalonthebottomofchromosomeIVtowhichnomutantswithJA-relatedphenotypeshavebeenmapped.Incontrast,joe2mapstothesameregiononchromosomeVascev1(EllisandTurner,2001).Cev1exhibitsconstitutivelyactiveJAandethylenesignalling,aswellasenhancedresistance604AndersB.Jensenetal.

towardspathogens.Severaldifferencesbetweenthephenotypesofjoe2andcev1suggesttheyde®nedifferentlocimodulatingJAsignalling.Forexample,cev1exhibitsreducedJA-responsiveroot-growthinhibitionandanapparentrepressionofahighconstitutivebasallevelofVSP,CHI-BandPDF1byJAtreatment(EllisandTurner,2001;Figure3).Nonetheless,allelicvariabilitymightexplainthesedifferences,andwearecurrentlytestingallelismbetweencev1andjoe2.LittleisknownaboutearlystepsinjasmonateperceptionandhowJAsin¯uencedevelopmentalprocessesbycontrollingspeci®cgeneexpression.Theidenti®cationofnewresponsemutantsandtheireventualmolecularcharacterizationwillincreaseourunderstandingofhowjasmonatesaffectspeci®cgeneexpressionandtherebymediatedevelopmentalprocessesandstressresponses.ExperimentalproceduresPlantmaterialandtreatmentsWildparentaltypereferstotheplatedDenmark)online1QinecotypeCol-0.non-mutagenizedSeedsweresurface-sterilizedLOX2-FLUC/GUSandgrowthpHMurashige5.7with1%andsucroseSkoog(Sigma-Aldrich,and0.8%agar.Vallensbaek,Plateskanamycin.ofwildForSeedtype,joe1andjoe2alsocontained50mgforl±18sucrosedaysliquidwasthenvernalizedat4°Cfor4daysinthedark.undercultures,constantplatedlight,seedlingsthenwereincubatedat21°CforGermany)for48hbeforeadditiontransferredofMeJAto0.5QMSwith1%performedand/orplatings.intriplicateinhibitors.andrepeatedAllluciferasetwicefromdeterminations(Serva,Heidelberg,wereandStocksolutionsof100mindependentseedMMeJA,10mMcycloheximideethanol;100mMsolutionschloramphenicolof1mstaurosporine(Sigma)were(Sigma)preparedandin0.599%MmMokadaicSeedsacid(LifeTechnologies,Taastrup,Denmark)inDMSO.Biologicalofein2-describedResource1andCenter.ein3-1wereReporterobtainedassaysfromtheArabidopsisverticallyForroot-inhibitionpreviouslyassays,(Raventossurface-sterilizedetal.,2000).wereperformedasseedscontainingforBright-®eld1%14sucrose,daysunder0.7%constantlightonMSweremediumgrownlengtheachmeasuredimagestreatment.usingwereobtainedagarwithand0,aCCD1,5cameraor10mMandMeJA.rootMETAMORPHforsoftware,foratleast30plantsProductionofLOX2-FLUC/GUStransgenicplantsAwas5¢upstreamlibraryisolatedbyfragmentnestedlong-rangeoftheLOX2PCRgenefrom(BellDNAandofMullet,a1993)etprimeral.,1995).ofecotypeCol-0insertedintoEMBL3vectorarmsgenomic(Mundyprimer5¢-GGCTGTAGAGAACTCCTCGTCFirst-roundPCRwasperformedandusingalaarm-speci®cLOX2cDNAandLOX2U02426).5¢-CATGGTGTCCGACTTATGCCCSecond-roundPCRwasperformed(GenBankwithNostheL23968multipleprimersinglecloningandsiteanested-arm5¢primerclosetotheEMBL3sameBamAarhus,HI/productXhoDenmark)Iandofinsertedapproximately-CGACTCACTATAGGGAGCTCG.2750bpwasdigestedwithThetoyieldintoLOX2/pSK.thosesitesSequencingofpSKboth(Stratagene,strandsoncorrespondedanAppliedBiosystemsBiosystems,totheLOX2ABI5310¢upstreamcon®rmedthatthisfragmentwasprimerampli®edNaerum,fromLOX2/pSKDenmark).bylong-rangeTheputativesequencePCRLOX2(Appliedusingpromoterlinkerdigestionprimer5¢-CGCCAGGGTTTTCCCAGTCACGAC5¢-GGAAGATCTTTTTGGGTGAAGTTGAAG.andaBamaHIplasmidLOX2AfterintotranscriptionaltheBamwithHIBamsiteHI,ofthetheresultantbinary1.8T-DNAkbpfragmentvectorVIP11wasclonedasaleaderfusiontoFLUCcontainingaCaMV35Somega(MillarandGUSetal.,pea1992).RbcSTheE9fragmentterminatorwastoalsoyieldVIP11-LOX2/FLUCGUSinblunt-endfusionpBS:GUS3toyieldLOX2/GUS(Varagonaclonedetalupstream.,1992).ThisofinclonedwasintothentheexcisedNot1asanEcoRI/Not1fragmentandwasthe(BechtoldtransformedconstructdesignatedsiteofVIP11-LOX2/FLUC,resultingsegregationetal.,1993).withTthisLOX2-FLUC/GUS.constructbyvacuumArabidopsisin®ltrationCol-02seedoftransgeniclinesexhibiting3:1whichselected,expressedfortheandhomozygousbothkanamycin-resistanceFLUCTandGUSuponMeJAgeneforfurthertreatment,inVIP11,andanalyses.were3seedobtainedMutagenesisandmutantisolationBulked40T4Denmark.kradwithLOX2/FLUC/GUSacobalt60sourceseedsatwereRisùIndustrialgamma-irradiatedIrradiation,atplantscollectedallowedSeedstowereself-pollinate.dividedintoMpoolsof1200andtheresulting2seedsscreenedindependently,plates.ofScreeningbygerminatingand7000seedsfromfromeacheachofpool10poolswerewasperformed150seedsby®rstinroundmeasuring150QFLUC15mmMSmeasure9-day-oldJA-responsiveseedlingsgrownadditionalontoplatesreporterunderthatwereexpression,constantlightat21activity°C.Toincubated5inmlthe50lightmMwassprayedforMeJAplantsimagedwere48cameraforsprayedhbeforeFLUCactivitywithasecond5msetofimageswasobtained.TheanMwithluciferin,aliquid0.01%nitrogenTritoncooledX-100,andwareexposure(Universal(PrincetonImaging,Instruments,PA,NJ,USA)USA)whichwithCCDMETAMORPHautomatesimagesoft-wereSeedstransferredandprocessingtosoiland(Raventosgrowntoetal.,2000).PutativemutantsmutagenizedwerecollectedLOX2/FLUC/GUSforrescreeningmaturityinthegreenhouse.parentalandline.back-crossestothenon-RNAanalysesTotalplantsRNAwereaccordingwasisolatedtoSternfromandwild-type,transgenicandmutantandseparatedby1%formaldehydeNewtonagarose(1984).RNAsgelelectrophoresis(20mglane±1)PharmaciablottedtoHybond-Nnylonmembranes(Amershamwasprobescon®rmedBiotech,Horsholm,Denmark).EqualloadingofRNAprimerandsequenceswereampli®edbyethidiumbyPCRbromidefromgenomicstaining.orGene-speci®ccDNAswithvided18SstringencyJR1rDNAfromLOX2(GI:431257),PDF1.2(GI:4759674),in(GI:16506).JoseJ.Sanchez-Serranokindlypro-randomlyBromma,primedconditionspUC18.Hybridizationwasperformedunderhigh-DNAusingprobefragmentslabelledbyeithertwicefromSweden).independentRNAlabellingornicktranslation(Roche,experiments.gel-blothybridizationswererepeatedGeneticanalysesjoe1wereandallowedjoe2towereself,crossedandFwithLandsbergerecta.F1progeny2seedharvested.Homozygousjoe1ãBlackwellScienceLtd,ThePlantJournal,(2002),29,595±606

andsoil.2andGenomicmutantsEcker,mappingDNAwerewasperformedwasselectedextractedbyLUCimagingandtransferredtoaspreviouslyfromeachdescribedplant2weeks(Belllater,andmapped1994;ancebyanalysingRaventostheetalsegregation.,2000).TheT-DNAinsertionwasLandsbergmarkerinFseedlingsfromtheofcrossthebetweenkanamycin-resist-3joe2andandwerejoe250platedanderectaoncoi1.F1progenyofcrossesbetweenjoe1andcoi1,MS,weremediumallowedcontainingtoself-pollinate,1%andF2seedsresistantm50mgml±1kanamycin.Aftersucrose,100.7%agar,MMeJAandnewFLUCplates.tokanamycinactivityThenasdescribed4daysandafterinsensitivetoJAweretransferreddays,plantstoabove.transfertheplantswereimagedforAcknowledgementsWeÊtechnicalwishtoSanchez-Serranoassistance,thankYingJohnKaringG.TurnerandSuksawadVonvisuttikunforgrantsfortheJR1cDNA.Thisforworkcoi1seed,wassupportedandJosebyJ.ResearchtoJ.M.fromtheDanishAgriculturalandProgramCouncil(530000258),theDanishBiotechnologyVeterinary(9400347).(9602416),andtheDanishNaturalSciencesFoundationReferencesBechtold,N.,Ellis,J.andPelletier,G.(1993)InplantaAgrobacteriummediatedgenetransferbyin®ltrationofadultArabidopsisplants.C.R.Acad.Sci.Paris.LifeSci.316,1194±1199.Bell,C.J.andEcker,J.R.(1994)Assignmentof30microsatellitelocitothelinkagemapofArabidopsis.Genomics,19,137±144.Bell,E.andMullet,J.E.(1993)CharacterizationofanArabidopsislipoxygenasegeneresponsivetomethyljasmonateandwounding.PlantPhysiol.103,1133±1137.Bell,E.,Creelman,R.A.andMullet,J.E.(1995)Achloroplastlipoxygenaseisrequiredforwound-inducedjasmonicacidaccumulationinArabidopsis.Proc.NatlAcad.Sci.USA,92,8675±8679.Berger,S.,Bell,E.andMullet,J.E.(1996)Twomethyljasmonate-insensitivemutantsshowalteredexpressionofAtVspinresponsetomethyljasmonateandwounding.PlantPhysiol.111,525±531.Chao,Q.,Rothenberg,M.,Solano,R.,Roman,G.,Terzaghi,W.andEcker,J.R.(1997)ActivationoftheethylenegasresponsepathwayinArabidopsisbythenuclearproteinETHYLENE-INSENSITIVE3andrelatedproteins.Cell,,1133±1144.Clarke,J.D.,Volko,S.M.,Ledford,H.,Ausubel,F.M.andDong,X.(2000)Rolesofsalicylicacid,jasmonicacid,andethyleneincpr-inducedresistanceinArabidopsis.PlantCell,12,2175±2190.Cohen,P.,Holmes,C.F.andTsukitani,Y.(1990)Okadaicacid:anewprobeforthestudyofcellularregulation.TrendsBiochem.Sci.15,98±102.Conrath,U.,Silva,H.andKlessig,D.F.(1997)Proteindephosphorylationmediatessalicylicacid-inducedexpressionofPR-1genesintobacco.PlantJ.11,747±757.Creelman,R.A.andMullet,J.E.(1997)Oligosaccharins,brassinolides,andjasmonates:nontraditionalregulatorsofplantgrowth,development,andgeneexpression.PlantCell,9,1211±1223.Ellis.C.andTurner.J.G.(2001)TheArabidopsismutantcev1hasconstitutivelyactivejasmonateandethylenesignalpathwaysãBlackwellScienceLtd,ThePlantJournal,(2002),29,595±606

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