ABA-HypersensitiveGermination1encodesaproteinphosphatase2C,anessentialcomponentofabscisicacidsignalinginArabidopsisseedNoriyukiNishimura1,2,3,†,TomoYoshida2,3,NobutakaKitahata4,TadaoAsami4,KazuoShinozaki3andTakashiHirayama1,2,*1LaboratoryofEnvironmentalMolecularBiology,RIKENWakoInstitute,2-1Hirosawa,Wako,Saitama351-0198,Japan,2IntegratedGraduateSchoolofArtandScience,YokohamaCityUniversity,1-7-29Suehiro,Tsurumi,Yokohama230-0045,Japan,3GeneDiscoveryResearchGroup,RIKENPlantScienceCenter,1-7-22Tsurumi,Yokohama230-0045,Japan,and4LaboratoryofCellularBiochemistry,RIKENWakoInstitute,2-1Hirosawa,Wako,Saitama351-0198,JapanReceived21November2006;revised10January2007;accepted22January2007*Forcorrespondence(fax+81455087363;e-mailhirayama@gsc.riken.jp).†Presentaddress:DivisionofBiologicalSciences,CellandDevelopmentalBiologySection,CenterforMolecularGenetics,UniversityofCalifornia,SanDiego,9500GilmanDriveLaJolla,CA92093-0116,USA.SummaryThephytohormoneabscisicacid(ABA)regulatesphysiologicallyimportantstressanddevelopmentalresponsesinplants.TorevealthemechanismofresponsetoABA,weisolatedseveralnovelABA-hypersensitiveArabidopsisthalianamutants,namedahg(ABA-hypersensitivegermination).ahg1-1mutantsshowedhypersensitivitytoABA,NaCl,KCl,mannitol,glucoseandsucroseduringgerminationandpost-germinationgrowth,butdidnotdisplayanysignificantphenotypesinadultplants.ahg1-1seedsaccumulatedslightlymoreABAbeforestratificationandshowedincreasedseeddormancy.Map-basedcloningofAHG1revealedthatahg1-1hasanonsensemutationinageneencodinganovelproteinphosphatase2C(PP2C).Wepreviouslyshowedthattheahg3-1mutanthasapointmutationintheAtPP2CAgene,whichencodesanotherPP2CthathasamajorroleintheABAresponseinseeds(Yoshidaetal.,2006b).ThelevelsofAHG1mRNAwerehigherindryseedsandincreasedduringlateseedmaturation–anexpressionpatternsimilartothatofABI5.Transcriptomeanalysisrevealedthat,inABA-treatedgerminatingseeds,manyseed-specificgenesandABA-induciblegeneswerehighlyexpressedinahg1-1andahg3-1mutantscomparedwiththewild-type.DetailedanalysissuggesteddifferencesbetweenthefunctionsofAHG1andAHG3.Dozensofgeneswereexpressedmorestronglyintheahg1-1mutantthaninahg3-1.Promoter–GUSanalysesdemonstratedbothoverlappinganddistinctexpressionpatternsinseed.Inaddition,theahg1-1ahg3-1doublemutantwasmorehypersensitivethaneithermonogenicmutant.TheseresultssuggestthatAHG1hasspecificfunctionsinseeddevelopmentandgermination,sharedpartlywithAHG3.Keywords:ABAhypersensitivemutant,germination,proteinphosphatase2C,microarray,Arabidopsis.IntroductionTheplanthormoneabscisicacid(ABA)isinvolvedinimportantplantphysiologicalphenomena,includingseeddormancy,germination,growthregulation,developmentalprocessesandstomatalclosure.TounderstandtheABAresponsemechanisms,manyapproaches,includingfor-wardandreversegeneticanalysesandbiochemicalanaly-sis,havebeenutilized,andhaveallowedtheidentificationofmanycomponentsinvolvedinABAsynthesis,inABAsign-ª2007TheAuthors
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alingfromperceptiontotheregulationofgeneexpression,andinABAcatabolism(Finkelsteinetal.,2002;NambaraandMarion-Poll,2005;Yamaguchi-ShinozakiandShinozaki,2006).Theresultsobtainedfromthestudiesonthesecom-ponentsfacilitateourunderstandingoftheABAresponsemechanisms.TheABAsignalingpathwayhasbeenstudiedextensivelyfordecades.GeneticanalysisofABA-insensitiveloci935
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(Koornneefetal.,1984)revealedthatABI1andABI2proteinphosphatases2C(PP2Cs)(Leungetal.,1994,1997;Meyeretal.,1994)andthetranscriptionalregulatorsABI3,ABI4andABI5havecrucialrolesintheABAresponse(Finkelsteinetal.,1998;Giraudatetal.,1992;Lopez-MolinaandChua,2000).ThesestudiesofferedabasicviewoftheABAsignalingpathwayintermsoftheregulationofproteinmodificationandgeneexpression.TheABA-hypersensitivelociabh1,sad1,hyl1andahg2wereshowntoencodecomponentsinvolvedinRNAmetabolism,stronglysuggestthepost-transcriptionalregulationintheresponse(Hug-ouvieuxetal.,2001;LuandFedoroff,2000;Nishimuraetal.,2005;Xiongetal.,2001).RecentbiochemicalstudieshaverevealedthatFCA,anmRNA-bindingproteininvolvedinfloweringtimecontrol,andtheMg-chelataseHsubunit,functionasABAreceptors(Razemetal.,2006;Shenetal.,2006).IthasbeenpredictedthatproteinkinasesplaycrucialrolesintheABAsignalingpathway,asinothercellularresponsephenomena.AlthoughtherearemanyreportsofproteinkinasegeneswhoseexpressionismodulatedbyABA,thephysiologicalrelevanceofmostofthosekinasesremainsunclear.GeneticandbiochemicalapproacheshaverevealedthattheABA-dependentactivationofOST1/SRK2e,anSNF1-relatedkinase2,isrequiredfortheABAresponseofguardcells(Mustillietal.,2002;Yoshidaetal.,2002).RecentstudiesofSnRK2kinaseshaveindicatedthepresenceofacomplexphosphorylationnetworkorcascadeintheABAsignalingpathway(Belinetal.,2006;Kobayashietal.,2004;Yoshidaetal.,2006a).AnothertypeofSNF1-relatedkinase,SnRK3/PKS/CIPK,isalsoimplicatedintheABAresponse.TheimpairedfunctionofsomeofSnRK3s,suchasSOS2,PKS3,CIPK3andPKS18,hasbeendemonstratedtoaffecttheABAresponse(Gongetal.,2002;Guoetal.,2002;Kimetal.,2003;Ohtaetal.,2003).Inaddition,calcium-dependantproteinkinasesplaypivotalrolesintheABAresponseinguardcells(Morietal.,2006).Whereaproteinkinaseregulatesaphenomenon,acounteractingproteinphosphatasenegativelyregulatesit.ThisisthecaseintheABAsignalingpathway.SeveralPP2CshavebeenproposedasnegativeregulatorsoftheABAresponse.Severallinesofevidencesupportthispresump-tion.Theabi1-1andabi2-1mutationsconferdominantstrongABAinsensitivity,whereasintragenicrevertantmutationsofthesemutationshaveturnedouttobeloss-of-functionmutations(Gostietal.,1999;Merlotetal.,2001),up-ordownregulationoftheexpressionofsomePP2Csdecreasesorincreases,respectively,thesensitivitytoABA.(Gonzalez-Garciaetal.,2003;TahtiharjuandPalva,2001),anddisruptionmutationsofPP2CgenesconferABAhyper-sensitivity(Kuhnetal.,2006;Saezetal.,2004;Yoshidaetal.,2006b).Combinationofloss-of-functionmutationsstreng-thensthephenotypebutisnotperfectlyadditive,suggestingoverlappingfunctionofPP2Cs(Merlotetal.,2001;Saezetal.,2006).Inaddition,directinteractionsbetweenPP2CsandseveralABA-relatedcomponentssuchaskinasesandtranscriptionfactorshavebeenreported(Guoetal.,2002;Himmelbachetal.,2002;Ohtaetal.,2003;Yoshidaetal.,2006a).Arabidopsishasmorethan70PP2Cgenes(Sch-weighoferetal.,2004).PhylogeneticanalysisbasedonaminoacidsequencesindicatesthateightorninerelatedPP2Cs,includingABI1andABI2,formaclade.Amongthem,ABI1,ABI2,HAB1,HAB2andAHG3/AtPP2CAhavebeenshowntobeimplicatedinABAresponsesinguardcells(Armstrongetal.,1995;Leonhardtetal.,2004;Peietal.,1997;Saezetal.,2004)orinseeds(Kuhnetal.,2006;Leungetal.,1994,1997;Saezetal.,2004;Yoshidaetal.,2006b).Previously,wedescribedanABA-hypersensitivelocus,AHG3,encodingAtPP2CA,anddemonstratedthatdisrup-tionmutationsofthefiveABA-relatedPP2CsaffectedABAsensitivityingermination,andfurtherthatAHG3/AtPP2CAhadthestrongesteffectamongthem(Yoshidaetal.,2006b).BasedonexpressionanalysisofPP2Cgenes,wehypothes-izedthatexpressionlevelisamajordeterminantofthecontributiontotheABAresponseingermination.Ouranalysesofthemis-sensealleleofAHG3/AtPP2CAandtransgeniclineswithanabi1-1-typemutationofAHG3/AtPP2CAimpliedthattheabi1-1-typedominantmutationsarenotadominant-negativemutation.Robertetal.alsoreportedsimilarresultsbyanalyzingHAB1(Robertetal.,2006).However,todrawfurtherconclusions,informationonthenativesubstratesanddetailedbiochemicalanalysisarenecessary.Inthisstudy,wedescribeanotherABA-hypersensitivelocus,AHG1(Nishimuraetal.,2004).Theahg1-1mutantshowedABAhypersensitivityasstrongasthatofahg3-1ingerminationandpost-germinationgrowth,butnotinadultplants.WeidentifiedtheAHG1genebymap-basedcloning.Interestingly,AHG1wasAt5g51760,encodingaPP2CrelatedtoAHG3/AtPP2CA.Accordingtopublicmicroarraydatabases,AHG1hasthestrongestexpressioninseedsamongABI1-relatedPP2Cs.OurdatasupporttheviewthatPP2CshavenegativeregulatoryfunctionsintheABAresponse,andthatthereisacorrelationbetweenexpressionlevelsandthecontributiontotheABAresponseingermi-nation.DetailedcharacterizationofthemutantsandgeneexpressionanalysisrevealedbothoverlappinganddistinctfunctionsofAHG1andAHG3/AtPP2CA,suggestingelabor-ateregulationoftheABAresponseinseed.ResultsTheahg1-1mutantishypersensitivetoABAWehavepreviouslyshownthatahg1-1isanABA-hyper-sensitivemutantthatgerminatesandgrowspoorlyinthepresenceofABA(Nishimuraetal.,2004).ToassessfurtherthefunctionofAHG1intheABAresponse,weexaminedª2007TheAuthors
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theefficienciesofgermination(radicleemergence)andpost-germinationgrowth(seedlingwithexpandedgreencotyledons)inthepresenceofvariousconcentrationsofABA.Theseefficienciesintheahg1-1mutantwereappar-entlyreducedinthepresenceofexogenousABA(Figure1a,b).Time-courseassaysofgerminationandpost-germinationgrowthrevealeddelayedgerminationandpost-germinationgrowthoftheahg1-1mutantinthepres-enceof0.3lMABA(Figure1c,d).Todeterminewhethertheahg1-1phenotypeisspecifictoexogenouslyappliedABA,weexaminedtheeffectofstratificationlengthongermi-nationefficiencyintheabsenceofexogenousABA(Figure1e,f).Theahg1-1mutantshowedadramaticinhi-bitionofgerminationandpost-germinationgrowthintheabsenceofstratification,butnoeffectwhenstratifiedfor2or4days.ahg1-1seedlingsandadultplantsshowednoabnormalresponsestoABA(Nishimuraetal.,2004).Wecheckedtheeffectondroughtstresstoleranceinadultplantsgrownonsoilfor4weeksandthenleftunwatered.After10days,ahg1-1andwild-typeplantsshowedawiltedphenotype(Figure1g),whereasera1-2plantsseemedhealthierthanthewild-type,consistentwithpreviousresults(Peietal.,1998).Awaterlossassayshowednosignificantdifferencebetweenahg1-1andwild-type(Figure1h).TheseresultsindicatethatAHG1functionsmainlyduringgerminationorseeddevelopment.TodeterminewhetherornottheABAhypersensitivityandincreasedseeddormancyofahg1-1wereduetoahigherlevelofaccumulationofABA,wemeasuredtheendogenousABAcontentsofseedsandstressedplants.Dryahg1-1seedsaccumulated30%moreendogenousABAthanthewild-type.Afterstratification,theABAlevelofahg1-1seedswasreducedtoalmostthesameasthatofthewild-type(Figure2a).Two-week-oldrosetteplantsofahg1-1hadalmostthesameamountofendogenousABAasthewild-type.Uponosmoticstresstreatmentwithmannitol,theendogenousABAcontentsofbothlineswereincreaseddramaticallytothesamelevel,indicatingthattheregulationofABAproductionbyahg1-1isnormalinadultplants(Figure2b).AHG1encodesanovelproteinphosphatase2CToidentifyhowtheahg1-1mutationconferredtheenhancedsensitivitytoABA,weidentifiedtheAHG1genebymap-basedcloning.TheAHG1locuswaslocalizedbetweenmarkersAtSO191andMBK5onchromosome5(Nishimuraetal.,2004).Weanalyzed1964chromosomesofF2plantsobtainedfromanahg1-1·Lertestcross,andnarrowedtheahg1-1regiondowntoasegmentofapproximately80kbspanningthreeBACclones,K17N15,K10D11andMIO24(Figure3a).Bydeterminingthenucleotidesequencesofcandidategenesinthisregion,wefoundabasesubstitutionfromCtoTinthefirstexonofAt5g51760,whichcausedanonsensemutation,changingGln155toastopcodon.Interestingly,At5g51760hasbeenassignedasencodinganovelPP2C.ToconfirmthatthisgenewasAHG1,weexaminedthephenotypeofdisruptionmutantsofAt5g51760.Forthispurpose,weobtainedDstransposoninsertion(Ds54-0076-3)andT-DNAinsertion(SALK_095052)mutants.TheinsertionsitesofDs54-0076-3andSALK_095052wereinthefirstexonandthethirdintron,respectively.WeexaminedtheirphenotypesandfoundthatbothhadthesameABA-hyper-sensitivephenotypeingerminationandpost-germinationgrowthasahg1-1(Figure3b,Table1).Wenamedtheselinesahg1-2andahg1-3,respectively.Wealsoconstructedahg1-1transgenicplantsexpressingthewild-typeAt5g51760cDNAfusedtotheCaMV35Spromoter.AtransgeniclineharboringAt5g51760(lineL1)hadnormalpost-germinationgrowth,andacontroltransgeniclinewiththeemptyvectorshowedABAhypersensitivity(Figure3c,d).TheseresultsconfirmedthatAHG1isAt5g51760,whichencodesanovelPP2C.LineswithhigherexpressionofAt5g51760inthewild-typebackground(WT/L1)andahg1-1background(ahg1-1/L1)germinatedandgrewnormallyinthepresenceof0.8lMABA,whereasacontroltransgeniclinewiththeemptyvectorgrewpoorly(Figure3d).TheseresultssuggestthatanovelPP2C,AHG1,functionsasanegativeregulatorintheABAsignalingpathway.AHG1isexpressedduringseeddevelopmentandgermina-tionAsshowninFigure3(e),AHG1belongstoagroupconsist-ingofABI1-relatedPP2Cs.Wepreviouslydemonstratedthat,amongsuchPP2Cs,AHG3/AtPP2CAhadadominantroleinseeds,andpredictedthatitsmRNAlevelisthemajordeterminantofitscontributiontotheABAresponse,asAHG3/AtPP2CAhadthehighestmRNAlevelinseedsamongtheeightPP2Cgenesweexamined(Yoshidaetal.,2006b).Notably,AHG1isthePP2Cgenemosthighlyexpressedindryseedamongmembersofthecladeaccordingtoanextensivetranscriptomestudyofseeds(Nakabayashietal.,2005)andpublicmicroarraydata(FigureS1,TableS1).WeexaminedtheexpressionlevelsofAHG1mRNAusingsemi-quantitativeRT-PCRanalysis(Figure4a).ThemRNAlevelsofAHG1werehigherindryseedsandwerereducedtoun-detectablelevelsafterstratification.However,themRNAlevelsofAHG1apparentlyincreasedinthepresenceofABAinseedlings.Toinvestigatetheseed-specificexpressionofAHG1,wenextexaminedtheexpressionpatternofAHG1duringseeddevelopmentandmaturation.ExpressionofAHG1wasfirstdetectedat8daysafterflowering(DAF),andincreasedcontinuouslyuntil16DAF,whereastheexpres-sionofAHG3/AtPP2CAremainedsteady.Interestingly,thisdevelopment-dependentexpressionpatternofAHG1issimilartothatofABI5(Figure4b).ª2007TheAuthors
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(a)(b)(c)(d)(e)(f)(g)(h)Figure1.Theahg1-1mutantshowshypersensitivitytoABAduringgermination.(a,b)Germinationefficiencies(a)andpost-germinationgrowthefficiencies(b)ofwild-typeseeds(closedcircles)andahg1-1seeds(opencircles)inthepresenceofvariousconcentrationsofABAat(a)3daysand(b)7daysafterstratification.(c,d)Germination(c)andpost-germinationgrowthefficiencies(d)ofwild-typeseeds(closedcircles)andahg1-1seeds(opencircles)inthepresenceof0.3lMABAfor7daysafterstratification.(e,f)Germination(e)andpost-germinationgrowthefficiencies(f)ofwild-typeseeds(closedsymbols)andahg1-1seeds(opensymbols)afterstratificationfor0days(circles),2days(triangles)or4days(squares).Errorbarsindicatethestandarddeviationofthreeindependentexperiments(a-f).(g)Phenotypesofahg1-1,era1-2andwild-typeunderdroughtconditions.Four-week-oldplantsgrownonsoilwereexposedtodroughtstressbywithholdingwaterfor10days.(h)Waterlossofwild-typeplants(closedcircles)andahg1-1plants(openedcircles)expressedaspercentageofinitialfleshweightofdetachedrosetteleaves.Errorbarsindicatethestandarddeviationoftwoindependentexperiments(n=5rosetteleavesperexperiment).ª2007TheAuthors
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(a)100ABA content (ng g–1 FW)806040200(b)ABA content (ng g–1 FW)25201510504000Days40Mannitol (mM)Figure2.EndogenousABAlevelsintheahg1-1mutant.(a)EndogenousABAlevelsofwild-typeseeds(closedbars)andahg1-1seeds(openbars)stratifiedfor0or4days.(b)EndogenousABAlevelsof2-week-oldwild-typeplants(closedbars)andahg1-1plants(openbars)treatedwithwateror400mMmannitolfor4h.Errorbarsindicatethestandarddeviationofthreeindependentexperiments(a,b).ThisobservationimpliedthattheexpressionlevelsandpatternsofthesegenesmightberegulatedbyABI3,ABI4orABI5,whichareimplicatedinthecontrolofseeddevelop-mentandgermination.Toinvestigatethis,weextractedtotalRNAfromabi3-1,abi4-1,abi5-1andwild-typeseedlingsgrownonMSplateswithorwithout3.0lMABAfor3daysafterstratification.Theresultsofsemi-quantitativeRT-PCRexperimentsshowedthatthelevelsofAHG1mRNAwereslightlybutnotsignificantlydecreasedinthemutantlinescomparedwiththewild-typelinestreatedwithABA,whereasthemRNAlevelofAtEm6,whichisregulatedbyABI3andABI5,wasdramaticallydecreasedinabi3-1andabi5-1mutants(Figure4c).ThelevelsofAHG3/AtPP2CAmRNAweresimilarinthemutantlinesandwild-type.TheseresultsindicatethatexpressionofAHG1andAHG3/AtPP2CAisnotregulatedbyABI3,ABI4orABI5,despitetheirincreasedexpressioncausedbyABAtreatmentofgermina-tingseeds.Toconfirmthisidea,wecharacterizedthegeneticrela-tionshipbetweenahg1-1andtheABA-insensitivelociabi3-1,abi4-1andabi5-1.Weprepareddoublemutantlinesandexaminedtheirpost-germinationgrowthfor7daysonplatescontainingABA(Table2).Theahg1-1abi3-1andahg1-1abi5-1doublemutantsshowedanABA-insensitivephenotypesimilartothatofabi3-1orabi5-1.TheseresultsindicatethatAHG1functionsupstreamofABI3andABI5intheABAsignalingpathway.Interestingly,theahg1-1abi4-1doublemutantshowedanABA-insensitivephenotypeinthepresenceof0.3lMABAbutnotinthepresenceof3.0lMABA.TherelationshipbetweenABI4andAHG1intheABAsignalingpathwayisnotclearbuttheirrelationshipseemsweaker.ToexaminethespatialexpressionpatternsofAHG1andAHG3/AtPP2CAinmoredetail,weconstructedtransgenicplantswithrecombinantgenespossessingthepromoterregionofAHG1orAHG3/AtPP2CAfusedtoaglucuronidase(GUS)reportergene(PAHG1::GUSandPAHG3::GUS,respect-ively).WeexaminedGUShistochemicalstainingpatternsofseedsobtainedfromtransgeniclines.GUSstainingwasobservedintheseedsofbothtransgeniclines,butthestainingpatternsweredifferent.DryPAHG1::GUStransgenicseedsshowedstrongGUSactivityinthewholeembryo(Figure5a).Theactivitydecreasedtosomeextentafterimbibition,althoughthespatialpatternwasthesame(Figure5b,c).Bycontrast,PAHG3::GUSseedlingsshowedstrongGUSactivityinapicalandrootmeristemsandvascularbundles(Figure5e–g).Fourdaysafterimbibition,thestainingwasdecreasedbutmaintainedthesamespatialpattern.Beforegermination,thestainingpatternsintheseedcoat/endospermofbothlineswereverysimilar(Figure5i–n).Aftergermination,theGUSactivitiesofbothlinesdecreased;theeffectwasgreaterinthePAHG1::GUSline,(Figure5o,p).Interestingly,atthisstage,GUSactivityshowedspatialdifferentiationinthePAHG1::GUSline;GUSactivitywasstrongerinvascularbundlesandtherootcapthaninotherparts(Figure5d).Itshouldbenotedthatthestaininginseedcoat/endospermofthePAHG1::GUSlinealmostdisappeared,whilethatofthePAHG3::GUSlinewasclearlydetectable(Figure5o,p).In6-day-oldseedlings,thePAHG1::GUSlineshowedGUSactivityonlyatthebasesofthelateralrootbuds(Figure5q,r),butthePAHG3::GUSlineshowedGUSactivityinvascularbundlesandthewholeofthelateralrootbud(Figure5s,t),consistentwithapreviousreport(Chereletal.,2002).TheseresultssuggestthatAHG1andAHG3arecontrolleddifferently,andfurtherimplytheirdistinctfunctions.Incontrasttothehigherexpressioninseed,wecouldnotdetectAHG1expressioninadultplantorgans,includingrosetteandcaulineleaves,stem,flowerandroot,eveninthepresenceofABA(datanotshown).Thisisconsistentwithourobservationthattheahg1-1planthadanormaldroughtstressresponse(Figure1g,h).TheseresultsindicatethatAHG1hasseed-specificfunctions.ª2007TheAuthors
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(a)
Figure3.IdentificationofAHG1.(a)SchematicrepresentationsofmappingandstructureofAHG1.Theexon–intronorganizationofAHG1isshown.Mutationsitesareindicated.(b)ABAhypersensitivityofahg1-2andahg1-3.SeedsweresownonMSplatescontaining0.3lMABAorcontrolmediumandgrownfor7days.(c)RT-PCRanalysisofAHG1expressioninwild-type(L1,L2,L3)andahg1(L1)transgeniclinescontainingPCaMV35S-AHG1cDNAandvectorcontrollines(VC).TotalRNAwasisolatedfromseedlingsgrownonMSplatesfor10days.Thetranscriptsofthe18srRNAgenewereusedasaninternalcontrol.(d)Phenotypesofgerminatingseedsofwild-typeandahg1-1transgeniclines.SeedsweresownandgrownonMSplatescontaining0.3or0.8lMABAorcontrolmediumandgrownfor7days.(e)PhylogenetictreeofPP2CsrelatedtoAHG1.ThetreewasbasedonthealignmentofaminoacidsequencesofPP2CscalculatedwithCLUSTALX.At2g30020wasusedasanout-group.Thebarindicates0.1substitutionspersite.(b)
Table1GerminationefficienciesofT-DNAandDstransposoninsertionallelesofAHG1Percentageofgerminatedandgrownseedsa(c)
GenotypeColNosahg1-1ahg1-2ahg1-3MSb97.4Æ3.2100Æ096.9Æ0.693.6Æ4.5100Æ00.3lMABAb89.0Æ7.696.8Æ1.80Æ01.5Æ1.55.07Æ2.8(d)
Approximately50seedsofeachlineweresownonMSplatescontaining0.3lMABAorcontrolmedium.Thepost-germinationgrowthwasscoredafter7daysofincubation.bDatarepresentmeansÆstandarddeviationsofthreeindependentexperiments.a(e)
MicroarrayanalysisduringgerminationinthepresenceofABANextwetriedtoidentifythedownstreampathwaysregula-tedbyAHG1orAHG3byanalyzinggeneswhoseexpressionwasaffectedbytheahg1-1orahg3-1mutations.Wild-type,ahg1-1andahg3-1seedswerestratifiedfor4daysonnor-malMSplatesandthenincubatedfor2daysonMSplatescontaining0.5lMABA.RNAsampleswereextractedandthensubjectedtomicroarrayexperimentsusingAffymetrixATH1genechips.Analysisoftheexpressionprofileshowedthatmostgenesup-anddownregulatedbyABAinthewild-type(>93%and>94%,respectively)wereup-anddown-regulatedintheahg1-1andahg3-1mutantsaswell(Figure6a–c,TableS2).SomegenesthatwereupregulatedbyABA,suchasAtEm6andABI5,weremorehighlyex-pressedinahg1-1andahg3-1thaninthewild-type(Table3),consistentwithourpreviousresults(Nishimuraetal.,2004).Interestingly,theexpressionlevelsofseveralgeneswasdifferentiallyaffectedintheahg1-1andahg3-1mutantswhenseedsweretreatedwithABA(Table3,FigureS2,TableS3).Mostgenesthatwereupregulatedtoagreaterextentinahg1-1thaninahg3-1wereseed-specificgenes,suchasthoseforseedstorageproteins.Genesthatweredownregulatedinahg1-1weremoredivergent,andcorre-lationsintheirexpressionpatternswerenotclearwhenanalyzedintheATTED-IIdatabase(http://www.atted.bio.ti-tech.ac.jp/).However,thecommonfeatureofmorethanhalfofthesegeneswasroot-preferentialexpression.Theseª2007TheAuthors
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(a)(b)
RelationshipbetweenAHG1andAHG3/AtPP2CAToexaminethegeneticandphysiologicalrelationshipsbe-tweenAHG1andAHG3/AtPP2CAinABAsignalinginmoredetail,wecrossedahg1-1withahg3-1andobtainedadoublemutantlinefromF2progeny.Weexaminedthepost-ger-minationgrowthofthislinefor7daysonplatescontainingABA(Figure7a,b).Theefficiencyofgrowthofthedoublemutantwasremarkablyreducedinthepresenceofaslittleas0.1lMABA,showingstrongerABAhypersensitivitythantheparentalmonogeneticmutants.EvenintheabsenceofABA,theefficiencyofpost-germinationgrowthwasde-creasedmoredrasticallythanthatoftheparentalmono-geneticmutants,implyingthatthislinehaddeeperseeddormancy.Theseresultsindicateanadditiveeffectofahg1-1andahg3-1ontheABAsensitivityofseed,andfurthersug-gestthatAHG1andAHG3haveoverlappingfunctionsbutalsosomedistinctfunctions.Theahg1-1ahg3-1doublemutantdidnothaveanydetectablephenotypesintheadultstages(datanotshown).DiscussionIsolationoftheahg1-1mutantThroughanalysisofahg1-1,whichshowsincreasedsensi-tivitytoABAingerminationandpost-germinationgrowth,weshowedthatanovelPP2C,AHG1,isanimportantcom-ponentoftheABAsignalingpathwayinseed(Nishimuraetal.,2004).Thephenotypeofahg1-1issimilartothatofahg3-1:accumulationofhigherendogenousABAindryseedbutnotinimbibedseedoradultplants,andclearABAhypersensitivityingerminationbutnotinadultplants.Map-basedcloningofAHG1revealedthatAHG1isAt5g51760,whichencodesaPP2C.TheidentificationofAHG1asaPP2Cgeneprovidesconcreteconfirmationofthenegativeregu-latoryfunctionofPP2CsintheABAresponse.Moreimportantly,itunveilsthecrucialroleofanovelPP2CintheABAresponseingermination.Accordingtoagenome-widesimilarityanalysis,Arabid-opsishasatleast76genesforPP2Cs(Schweighoferetal.,2004).Sofar,onlyfivePP2Cs,namelyABI1,ABI2,HAB1,HAB2andAHG3/AtPP2CA,havebeenshowntobeinvolvedintheABAresponse.AHG1iscloselyrelatedtothesePP2Cs,andbelongstothesamecladeinthephylogenetictree,althoughatthefarthestdistance(Figure3e)(Schweighoferetal.,2004).Inthisclade,ABI1,ABI2,HAB1andHAB2formonegroup,andAHG1,AHG3,At5g59220,At2g29380andAt1g07430formtheother.ItisofinterestthatAHG1andAHG3havefunctionsrelatedtotheABAresponseinseedsandgermination,buttheotherthreePP2Cshavenot,althoughtheyareexpressedinseed(Yoshidaetal.,2006b).Sofar,wecannotfindanysignaturesintheaminoacidsequencesthatwouldexplainthedifferenceintheir(c)
Figure4.ExpressionpatternsofAHG1andAHG3/AtPP2CA.(a)RT-PCRanalysisofAHG1in(A)dryseedsand(B)seedsattheendofstratification,seedsincubatedonMSmediumfor(C)1dayor(D)3days,orseedsincubatedonMSmediumcontaining0.5lMABAfor(E)1dayor(F)3daysat22°Cafterstratification.(b)RT-PCRanalysisofAHG1,AHG3/AtPP2CA,ABI3andABI5.TotalRNAwasisolatedfromdevelopingandmaturesiliquesat6–16daysafterflowering(DAF).(c)RT-PCRanalysisofAHG1,AHG3/AtPP2CAandAtEm6inabi3-1,abi4-1,abi5-1andrespectivewild-typeseedlingsgrownonMSplatescontaining3.0lMABA(+)orcontrolmedium(–)for3daysafterstratification.Transcriptsofthe18srRNAgene(rRNA)wereusedasaninternalcontrol.Table2GerminationefficienciesofsingleanddoublemutantsPercentageofgerminatedseedsaGenotypeColLerWsabi3-1abi4-1abi5-1ahg1-1ahg1-1abi3-1ahg1-1abi4-1ahg1-1abi5-1aMSb98.0Æ1.096.7Æ2.399.0Æ0.892.4Æ3.595.6Æ2.198.1Æ2.098.5Æ1.491.6Æ2.6100Æ098.1Æ1.60.3lMABAb90.7Æ2.986.1Æ0.289.0Æ3.989.3Æ6.095.4Æ3.396.6Æ1.54.5Æ1.595.7Æ2.195.6Æ3.499.4Æ1.13.0lMABAb0Æ00.8Æ1.30Æ092.5Æ4.073.0Æ9.796.1Æ2.50Æ087.4Æ2.10Æ089.2Æ3.5Approximately50seedsofeachlineweresownonMSplatescontaining0.3or3.0lMABAorcontrolmedium.Thepost-germina-tiongrowthwasscoredafter7daysofincubation.bDatarepresentmeansÆstandarddeviationsofthreeindependentexperiments.resultsareconsistentwithourviewthatahg1-1ismoredormantthanahg3-1.Moreimportantly,theysuggestthatahg1-1andahg3-1affectgerminationdifferently.TheexpressionofAHG1seemstobeaffectedbyboththeahg1-1andahg3-1mutations.However,asahg1-1isanonsensemutation,itwillaffectthemRNAstability.ThereforetheexpressiondataforAHG1inahg1-1shouldbeconsideredcarefully.TheexpressionofotherABA-relatedPP2Cgeneswasnotaffectedbyahg1-1andahg3-1(Table3).ª2007TheAuthors
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Figure5.Analysisofspatialexpressionpatternusingpromoter–GUStransgenicplants.ImagesshowGUSstainingpatternsofthePAHG1::GUStransgenicline(a–d,i,k,m,o,q,r)orthePAHG3::GUStransgenicline(e–h,j,l,n,p,s,t).(a–h)GUSstainsofembryosorseedlings:(a,e)dryseeds,(b,f)2-day-imbibedseeds,(c,g)4-day-imbibedseeds,(d,h)2-day-oldseedlings.(i–p)GUSstainsofseedcoat/endosperm:(i,j)dryseeds,(k,l)2-day-imbibedseeds,(m,n)4-day-imbibedseeds,(o,p)2-day-oldseedlings.(q–t)GUSstainsof6-day-oldseedlings:(q,s)expandedcotyledons,(r,t)hypocotyl–rootjunctions.ª2007TheAuthors
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Asgerminationandpost-germinationgrowthareaffectedbymanyphysiologicalabnormalitiessuchasdefectsingrowth,developmentalcontrolandmetaboliccontrol,itwasexpectedtobedifficulttoidentifymutantsspecifictotheABAresponse.However,disruptionofaconsiderablenum-berofgenesrelatedtotheABAresponseturnedouttocauseanABA-hypersensitivephenotypeingermination,indica-tingthevalueofisolationandcharacterizationofmutantswiththisphenotype.TheidentificationhereofanovelPP2CinvolvedintheABAresponseinseed,bymeansofanalyzingsuchamutant,clearlydemonstratesitseffectiveness.Asneitherahg1-1norahg3-1hadaclearadultphenotype,onlysuchscreeningwouldallowustoidentifythosetwocomponents,confirmingtheimportanceoftheisolationandanalysisofABA-hypersensitivemutants.PhysiologicalfunctionofAHG1inseeddevelopmentandgerminationTheahg1-1mutation,presumablyaloss-of-functionmuta-tion,causesrecessiveABAhypersensitivityduringgermi-nation.Bycontrast,over-expressionofAHG1conferredclearABAinsensitivityduringgermination,eveninanahg1-1background.TheseobservationsstronglysuggestthattheAHG1PP2CfunctionsasanegativeregulatoroftheABAresponse,asdoAHG3/AtPP2CAandotherABI1-relatedPP2Cs.ahg1-1seedshad30%higherendogenousABA,asdidahg3-1seeds.ThisslightincreaseoftheendogenousABAlevelinseeddoesnotseemenoughtoexplainthedramaticelevationoftheABAsensitivityofthismutantinseedgerminationandpost-germinationgrowth.ThiswasconfirmedbythefactthatanABA-defectivemutant,aba2-1,didnotsuppresstheABA-hypersensitivephenotypeofahg1-1(datanotshown).Therefore,itismorelikelythatahg1-1isanABA-hypersensitivemutantratherthanasimpleABAover-productionmutant.Theenhancedsensitivityofahg1-1toABAinseedsmightactivatepositivefeedbackofABAproduction,which,inturn,increasesseeddormancyfurtherandthesensitivitytoABA.Extensiveanalysisoftheahg1-1mutantandtheAHG1generevealedthespecificfunctionofAHG1inseedsandgermination.First,althoughahg1-1hadclearABAhyper-sensitivityingerminationandpost-germinationgrowth,wecouldnotobserveanydetectablephenotypeatadultstages.Second,AHG1wasbarelyexpressedinadultplants(datanotshown).Third,ourexperimentsandpublicmicroarraydatashowedstrongexpressionofAHG1indevelopingseedanddryseed(Figures4and5,FigureS1).Previously,wepostulatedthattheexpressionlevelofPP2CmRNAsisoneofthemajordeterminantsoftheroleofPP2CinABAsignalinginseedgermination(Yoshidaetal.,2006b).Theclearcorrelationbetweentheseed-specificexpressionofAHG1andtheABA-hypersensitivephenotypeoftheahg1-1mutantisconsistentwiththisidea.(c)
Figure6.ClassificationofABAup-anddownregulatedgenesinahg1andahg3.(a,b)TotalnumberofABAupregulatedgenes(a)orABAdownregulatedgenes(b)identifiedbyAffymetrixgenechipanalysesofwild-type(Col)andahgplants.Relativesignalintensitieswerecalculatedbycomparingwithcontrolexperiments(wild-type,absenceofABA)andusedforVenndiagrams.(c)RT-PCRanalysesofgeneswithhighermRNAlevelsinahg1-1thaninahg3-1.TotalRNAwasextractedfromwild-type,ahg1-1andahg3-1seedlingsgrownfor2daysonMSplatescontaining0.5lMABA(+)orcontrolmedium(–).Transcriptsofthe18srRNAgene(rRNA)wereusedasaninternalcontrol.ABA-relatedfunctions.TodeterminethephysiologicalfunctionofeachPP2Cwillrequiredetailedexaminationonebyone.ª2007TheAuthors
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Table3.Genesaffectedmorestronglybyahg1-1thanahg3-1ingerminationinthepresenceofABAaRelativesignalintensityGeneIDahg1versusahg3bahg1versusWTbahg3versusWTb0.121.780.241.90)0.42)0.10)0.110.681.050.440.110.600.970.930.040.85)0.97)0.97)0.94)1.57)1.60)1.63)1.51)1.00)1.62)2.26)1.83)1.53)1.340.600.920.49)0.24)0.56d)0.320.010.11ABAversuscontrolc0.301.641.232.611.120.571.180.181.111.240.591.083.403.790.60)1.243.07)0.95)0.391.692.71)0.07)0.443.68)1.861.350.98)0.20)3.352.264.912.271.29)0.25d0.930.440.63Annotation2Salbuminstorageprotein2LipidtransferproteinReductase(SDR)familyprotein2Sseedstorageprotein112Sseedstorageprotein2Sseedstorageprotein3MotherofFTandTF1proteinPlantECmetallothionein-likeproteinPutativelysimilartomanganesesuperoxidedismutase(MSD1)Putative(PDF2.1)plantdefensinproteinGibberellin-regulatedprotein2(GASA2)AmidasefamilyproteinOleosinLEAproteiningroup3Short-chaindehydrogenase/reductase(SDR)familyproteinMulti-copperoxidasetypeIPhotosystemIreactioncentersubunitVChlorophylla/bbindingproteinb-fructosidase(BFRUCT4)Homeoboxleucinezipperprotein13Hydroxyproline-richglycoproteinHydroxyproline-richglycoproteinAcyl-[acyl-carrier-protein]desaturasePectinesterasefamilyproteinTerpenesynthase/cyclasefamilyproteinCER1proteinidenticaltomaizegl1homologJacalinlectinfamilyproteinLateembryogenesisabundantproteinInvertase/pectinmethylesteraseinhibitorfamilyproteinABI5AtEm6AHG1AHG3/AtPP2CAABI1ABI2HAB1HAB2Genesexpressedatahigherlevelinahg1-1At4g271504.003.91At5g547403.324.94At5g506002.652.95At4g271402.634.21At1g038802.482.28At4g271602.342.39At1g181002.302.22At2g420002.152.49At3g563502.112.77At2g02120At4g09610At5g07360At3g27660At3g53040At3g052602.061.991.651.641.641.612.322.252.162.582.371.66Genesexpressedatalowerlevelinahg1-1At4g37160)1.08)0.40At1g55670)0.680.12At1g61520)0.82)1.12At1g12240)0.860.93At1g69780)0.240.45At2g34870)0.320.47At3g02120)0.30)0.28At1g43800)0.64)1.26At5g47500)1.314.01At5g48110)0.890.22At1g02205)0.360.85At5g35940)0.891.99At5g54370)1.340.11At5g62340)1.55)1.96OthersAt2g36270At2g40170At5g51760At3g11410At4g26080At5g57050At1g72770At1g17550a0.610.540.070.360.020.070.160.011.201.430.750.20)0.55d)0.010.150.15Top15genesexpressedtohigherandlowerlevelsinahg1-1thaninahg3-1arelisted.Forgenesexpressedatalowerlevelinahg1-1,datawithlessconsistencybetweentwoindependentexperimentswereremoved.SignalvaluesofmicroarrayexperimentswereRobustMulti-ArraynormalizedandcomparedusingaffylmGUI.Thevaluesarethemeansoftwoindependentexperimentsandareshowninlog2.ThemicroarraydataweredepositedintheNCBIGEOdatabasewithaccessionnumberGSE6638.bRelativesignalintensitiesbetweenahg1-1andahg3-1(ahg1-1versusahg3-1),ahg1-1andwild-type(ahg1-1versusWT),ahg3-1andwild-type(ahg3-1versusWT).RNAwasextractedfromgerminatingseedstreatedwith0.5lMABAfor2days(seeExperimentalprocedures).cRelativesignalintensitiesbetweenwild-typeseedstreatedwith(ABA)orwithout0.5lMABA(control)for2days.dThesevalueswerededucedusingtwodatasetsofABA-treatedwild-typeseedsthatwereverydifferenteachother(seeTableS2).TheexpressionofAHG1seemstobepositivelyregu-latedbyABA,althoughABI3andABI5werenotinvolved.ThepromoterregionofAHG1hasseveralsequencessimilartoRYorABRE(ABAresponsiveelement)ciselements,andAHG1wasactivatedbyABAingerminatingseeds(Figure4a,TableS4).Inaddition,wefoundthattheexpressionpatternofAHG1wassimilartothatofABI5duringseeddevelopment.Presumably,thefunctionsofpositiveABAregulatorsandofAHG1,anegativeregula-tor,constituteanegativefeedbacklooptofine-tunetheª2007TheAuthors
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DistinctorredundantfunctionsofAHG1andAHG3/AtPP2CATheAHG1genesharesmanyfeatureswithAHG3/AtPP2CA.BothencodecloselyrelatedPP2Csandareexpressedstronglyinseed.Loss-of-functionmutationsofbothgenescausestrongABAhypersensitivityingerminationandpost-germinationgrowth.However,detailedcharacterizationofthesemutantsrevealeddifferencesinthefunctionsofthesePP2Cs,andthemutantshaveslightlydifferentphenotypes.Whencomparedwithahg3-1,theahg1-1mutanthadstrongerABAhypersensitivityinradicleemergence(ger-mination)anddeeperseeddormancy.Withoutstratifica-tion,thegerminationefficiencyofahg1-1remainedlow(Figure1f),whereasthatofahg3-1eventuallyrosetothenormallevel(Yoshidaetal.,2006b).Thesemutantsalsohaddifferentsensitivitiestosugaringermination(Nishimuraetal.,2004)(TableS5).Themicroarrayexperimentssup-portedthedifferencesinthesetwomutants,showingthatsomegeneswereaffectedmorespecificallybyahg1-1orahg3-1.Inaddition,theahg1-1ahg3-1doublemutantshowedastrongerphenotypethaneithermonogenicpar-entalmutantline,indicatingtheiradditiveeffect.Thesere-sultsstronglysuggestthatAHG1andAHG3havebothoverlappinganddistinctfunctionsintheABAresponseinseeds.Howthisdifferencecomesaboutisintriguing,becauseithasbeenpostulatedthatPP2Csarenotregulatedatthepost-translationallevel(Schweighoferetal.,2004).Themostplausibleexplanationisthedifferenceinthetem-poralandspatialexpressionpatternsandlevelsbetweenAHG1andAHG3/AtPP2CAduringseeddevelopmentandgermination.AHG3/AtPP2CAisconstitutivelyexpressedduringthisperiod.Bycontrast,AHG1wasgraduallyactivatedasseeddevelopmentprogressed(Figure4b).TheGUSactivityofthepromoter–GUStransgenicplantsindicatedthatAHG1isexpresseduniformlyintheembryo,whereasAHG3isexpressedmorestronglyinspecificpartssuchasmeristemsandvascularbundles(Figure5).Thesedifferentialexpressionsmustcontribute,atleastpartly,tothedistinctfunctionsofAHG1andAHG3/AtPP2CA.Proteinphosphatasesareusuallyconsideredtobenon-specific.However,interactionswithothercomponentscangivethespecificity(forexample,Meskieneetal.,2003;MapesandOta,2004).RecentstudiesofABI1andABI2revealedtheirphysicalinteractionswithABA-relatedcom-ponents.ABI1bindstokinasessuchasOST1/SRK2e,PKS3,PKS18andthetranscriptionalregulatorATHB6,whereasABI2bindstoSOS2,PKS3,PKS11,PKS24,fibrillinandglutathioneperoxidase(Guoetal.,2002;Himmelbachetal.,2002;Miaoetal.,2006;Ohtaetal.,2003;Yangetal.,2006;Yoshidaetal.,2006a).Inaddition,ABI1hasbeenshowntoberegulatedbyphosphatidicacidproducedbyphospholipaseD(Mishraetal.,2006;Zhangetal.,2004).(b)
Figure7.Relationshipbetweenahg1andahg3.(a)Doublemutantanalysisofahg1-1ahg3-1.Post-germinationgrowthefficienciesofwild-type(closedcircles),ahg1-1(closedtriangles),ahg3-1(closedsquares)andahg1-1ahg3-1(opencircles)seedlingsinthepresenceofvariousconcentrationsofABAat7daysafterstratification.Errorbarsindicatethestandarddeviationofthreeindependentexperiments.(b)PhenotypeofseedlingsinthepresenceofABA.SeedsweresownonMSplatescontaining0.1or0.3lMABAorcontrolmediumandgrownfor7days.ABAactionduringseeddevelopmentandtomaintainmatureseed.TheimportantrolesofAHG1inseeddevelopmentanddormancyareevidencedbythedeeperdormancyofahg1-1(Figure1).Thehigherlevelofaccu-mulationofendogenousABAindryseedsandthehigherlevelofexpressionofseed-specificgenesinABA-treatedseedsoftheahg1-1mutantlinesupportthisidea.SeveralrecentreportsshowthatendospermhasanimportantfunctionintheABAresponseinseedsandduringgermination(Lefebvreetal.,2006;Mulleretal.,2006;Penfieldetal.,2006).Inthisrespect,thestrongexpressionofAHG1andAHG3/AtPP2CAintheendospermisinter-esting(Figure5).ª2007TheAuthors
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Althoughthesedifferencesininteractionhavenotclearlyexplainedtheirdistinctfunctionsyet,theseresultssuggestthatthedistinctfunctionsareduetotheabilitytointeractwithdifferentcomponents.Forinstance,AHG3/AtPP2CAinteractswithAKT2,apotassiumtransporter(Chereletal.,2002).AnalysisoftheproteinsinteractingwithAHG1andAHG3mightoffersomecluestotheirdistinctfunc-tions.Eventhoughtheyhavedifferentsubstrateprefer-ences,theypresumablyhaveanabilitytosharethetargetsbasedontherelativelyhighsimilarityinaminoacidsequences.Takentogether,similarbiochemicalpropertiesbutdifferentspatio-temporalexpressionpatternsanddifferentpreferencesforinteractingproteinswouldgiverisetothedistinctandoverlappingfunctionsofthesePP2Cs.Theseargumentscanbeextendedtotherelation-shipsamongotherABA-relatedPP2Cs.WhenonePP2Cmalfunctions,otherPP2Cscancompensateforthedefectbutonlypartlybecauseofdifferentexpressionpatternsandtargetpreferences.OurstudiesshowtheimportanceofelucidatingthepreciserolesofeachPP2CinordertounderstandtheABAresponse.Forthispurpose,substratesmustbedetermined.Proteomicapproacheshaveledtogreatsuccessesinanalyzingthephosphorylationstatusofcellularproteins.Characterizationofphosphorylatedproteinsinahg1-1,ahg3-1andtheahg1-1ahg3-1doublemutantwouldbeaworthwhileapproach.Inaddition,despitethestrongABAhypersensitivity,theahg1-1ahg3-1doublemutantwasstillabletogerminate,suggestingtheimportantfunctionsofotherPP2Cs.ItwouldbeofinteresttoconstructtripleorquadruplePP2C-defectivemutantstorevealthecontributionofotherPP2CsandtounderstandthefunctionsofPP2CsandABAingermination.ExperimentalproceduresPlantmaterialsandgrowthconditionsArabidopsisthaliana(L.)Heynh.ecotypesColumbia(Col),Lands-bergerecta(Ler),Wassilewskija(Ws)andNossen(Nos)wereused.Plantgrowthconditionshavebeendescribedpreviously(Nishimuraetal.,2004).Theahg1-2line(54-0076-3,Nosbackground)andtheahg1-3line(SALK_095052,Colbackground)wereobtainedfromRIKENBioResourceCenterandtheArabidopsisBiologicalResourceCenter,respectively.Map-basedcloningoftheAHG1locusTheahg1-1mutantwascrossedwithLer,andF2progenywereobtained.ABA-hypersensitiveindividualswereselectedonmediumcontaining0.2lMABAandgrownonnormalmedium.IsolationofgenomicDNAandPCRconditionshavebeendescribedelsewhere(Hirayamaetal.,1999).AdditionalSSLPandCAPSmarkersweredevelopedandused(TableS6).Complementationanalysisofahg1-1Thefull-lengthAHG1cDNAfragmentwasobtainedbyPCRusinghigh-fidelityDNApolymerase(KOD-Plus,Toyobo;http://www.toyobo.co.jp/e/index.htm)withtheBamHIlinkerprimerAHG1-BamHI-F1,theKpnI/Acc65IlinkerprimerAHG1-KpnI-R1,andfirst-strandcDNAmixture(TableS7).ItwassubclonedintopBlue-scriptSK-(Stratagene;http://www.stratagene.com/),andthenucleotidesequencewasconfirmed.ThisAHG1cDNAwasre-introducedintothebinaryvectorpROK2.Agrobacteriumtume-faciensstrainGV3101wastransformedwiththeresultantplasmidandusedforinfectionofArabidopsisplantsbytheflowerdippingmethod(CloughandBent,1998).Transgeniclineswerescreenedbykanamycintoleranceinthenextgeneration.GUShistochemicalstainingForconstructionofPAHG1::GUStransgenicplants,thegenomicDNAfragmentspanningfrom2137bpupstreamto117bpdownstreamofthetranslationinitiationsiteofAHG1wasintroducedintopBI101.ForconstructionofPAHG3::GUStransgenicplants,thegenomicDNAfragmentspanningfrom1529bpupstreamto1bpupstreamofthetranslationinitiationsiteofAHG3wasintroducedintopBI101.TheresultantplasmidswereintroducedintoColwild-typeplants.BeforeGUSstaining,seedcoat/endospermandembryowereseparatedbyforceps.Thesamplesweretreatedwith90%acetoneonicefor15min.Afterwashingwith100mMNaPO4buffer(pH7.0),plantmaterialswereincubatedin100mMNaPO4buffer,10mMEDTA,2mMpotassiumferricyanide,2mMpotassiumferrocyanide,0.1%TritonX-100and0.5mgml)1X-Glucfor14hat37°C.DNAmicroarrayanalysisTotalRNAwasisolatedwithTrizolreagent(Invitrogen;http://www.invitrogen.com/)andRNAwaspurifiedusinganRNeasypurificationkit(Qiagen;http://www.qiagen.com/).cDNAsynthesis,cRNAsynthesisandhybridizationtotheAffymetrixATH1genomearraywereperformedaccordingtothemanufacturer’sinstructions(Affymetrix;http://www.affymetrix.com/).Theexperimentswereduplicatedusingdifferentlotsofseeds.MicroarraydatawereprocessedusingtheaffylmGUIpackagerunningontheRprogram(Smyth,2004).MicroarraydataweredepositedinNationalCenterforBiotechnologyInformation(NCBI)GEOdatabasewithaccessionnumberGSE6638.GerminationassaysandABAmeasurementForgerminationassays,approximately50seedsweresownonplatescontaining1·Murashige&Skoogsaltmixandvariouscon-centrationsof(Æ)-cis,trans-abscisicacid(ABA,Sigma-Aldrich;http://www.sigmaaldrich.com/).Germination(emergenceofradi-cles)andpost-germinationgrowth(greenandexpandedcotyle-dons)werescoreddailyfor7days.ExtractionandquantitativeanalysesofABAwereperformedasdescribedpreviously(Nishim-uraetal.,2005).RT-PCRanalysisTotalRNAwasisolatedwithaRNeasyplantminikit(Qiagen)orasdescribedpreviously(Nishimuraetal.,2004).AftertreatmentwithRNase-freeDNaseI(Qiagen),first-strandcDNAwassynthesizedfrom1lgtotalRNAusingtheReverTraPlusRT-PCRkit(Toyobo)withrandomhexamers,accordingtothemanufacturer’sinstructions.Toperformsemi-quantitativeRT-PCR,1/40thoftheª2007TheAuthors
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first-strandreactionmixturewasusedforPCRreactionwithgene-specificprimers(TableS7).PCRconditionswere95°Cfor90sec,then20,28or29cyclesof95°Cfor15sec,58°Cfor10secand72°Cfor60sec,followedby72°Cfor5min.DNAfragmentsforAHG1,AHG3/AtPP2CA,ABI3,ABI5andAtEm6wereamplifiedfor28PCRcycles,andthoseforthegenesidentifiedinmicroarrayexperimentswereamplifiedfor29PCRcycles.The18srRNAgenetranscriptswereamplifiedfor20PCRcycles.kinaseactivityandOST1functioninguardcells.PlantPhysiol.141,1316–1327.Cherel,I.,Michard,E.,Platet,N.,Mouline,K.,Alcon,C.,Sentenac,H.andThibaud,J.B.(2002)PhysicalandfunctionalinteractionoftheArabidopsisK+-channelAKT2andphosphataseAtPP2CA.PlantCell,14,1133–1146.Clough,S.J.andBent,A.F.(1998)Floraldip:asimplifiedmethodforAgrobacterium-mediatedtransformationofArabidopsisthaliana.PlantJ.16,735–743.Finkelstein,R.R.,Wang,M.L.,Lynch,T.J.,Rao,S.andGoodman,H.M.(1998)TheArabidopsisabscisicacidresponselocusABI4encodesanAPETALA2domainprotein.PlantCell,10,1043–1054.Finkelstein,R.R.,Gampala,S.S.andRock,C.D.(2002)Abscisicacidsignalinginseedsandseedlings.PlantCell,14,S15–S45.Giraudat,J.,Hauge,B.M.,Valon,C.,Smalle,J.,Parcy,F.andGoodman,H.M.(1992)IsolationoftheArabidopsisABI3genebypositionalcloning.PlantCell,4,1251–1261.Gong,D.,Zhang,C.,Chen,X.,Gong,Z.andZhu,J.K.(2002)ConstitutiveactivationandtransgenicevaluationofthefunctionofanArabidopsisPKSproteinkinase.J.Biol.Chem.277,42088–42096.Gonzalez-Garcia,M.P.,Rodriguez,D.,Nicolas,C.,Rodriguez,P.L.,Nicolas,G.andLorenzo,O.(2003)NegativeregulationofabscisicacidsignalingbytheFagussylvaticaFsPP2C1playsaroleinseeddormancyregulationandpromotionofseedgermination.PlantPhysiol.133,135–144.Gosti,F.,Beaudoin,N.,Serizet,C.,Webb,A.A.,Vartanian,N.andGiraudat,J.(1999)ABI1proteinphosphatase2Cisanegativeregulatorofabscisicacidsignaling.PlantCell,11,1897–1910.Guo,Y.,Xiong,L.,Song,C.P.,Gong,D.,Halfter,U.andZhu,J.K.(2002)AcalciumsensoranditsinteractingproteinkinaseareglobalregulatorsofabscisicacidsignalinginArabidopsis.Dev.Cell,3,233–244.Himmelbach,A.,Hoffmann,T.,Leube,M.,Hohener,B.andGrill,E.(2002)HomeodomainproteinATHB6isatargetoftheproteinphosphataseABI1andregulateshormoneresponsesinArabid-opsis.EMBOJ.21,3029–3038.Hirayama,T.,Kieber,J.J.,Hirayama,N.,Kogan,M.,Guzman,P.,Nourizadeh,S.,Alonso,J.M.,Dailey,W.P.,Dancis,A.andEcker,J.R.(1999)RESPONSIVE-TO-ANTAGONIST1,aMenkes/Wilsondisease-relatedcoppertransporter,isrequiredforethylenesignalinginArabidopsis.Cell,97,383–393.Hugouvieux,V.,Kwak,J.M.andSchroeder,J.I.(2001)AnmRNAcapbindingprotein,ABH1,modulatesearlyabscisicacidsignaltransductioninArabidopsis.Cell,106,477–487.Kim,K.N.,Cheong,Y.H.,Grant,J.J.,Pandey,G.K.andLuan,S.(2003)CIPK3,acalciumsensor-associatedproteinkinasethatregulatesabscisicacidandcoldsignaltransductioninArabid-opsis.PlantCell,15,411–423.Kobayashi,Y.,Yamamoto,S.,Minami,H.,Kagaya,Y.andHattori,T.(2004)Differentialactivationofthericesucrosenonfermenting1-relatedproteinkinase2familybyhyperosmoticstressandabscisicacid.PlantCell,16,1163–1177.Koornneef,M.,Reuling,G.andKarssen,C.(1984)Theisolationandcharacterizationofabscisicacid-insensitivemutantsofArabid-opsisthaliana.Physiol.Plant.61,377–383.Kuhn,J.M.,Boisson-Dernier,A.,Dizon,M.B.,Maktabi,M.H.andSchroeder,J.I.(2006)TheproteinphosphataseAtPP2CAnegat-ivelyregulatesabscisicacidsignaltransductioninArabidopsis,andeffectsofabh1onAtPP2CAmRNA.PlantPhysiol.140,127–139.Lefebvre,V.,North,H.,Frey,A.,Sotta,B.,Seo,M.,Okamoto,M.,Nambara,E.andMarion-Poll,A.(2006)FunctionalanalysisofGeneticanalysisofdoublemutantsTheahg1-1mutant(Col)wascrossedwithabi3-1(Ler),abi4-1(Col),abi5-1(Ws),aba2-1(Col)orahg3-1(Col).Doublemutantswereob-tainedfromF2progenybyuseofmutant-specificCAPSmarkers(TableS7).Thepresenceofahg1-1wasmonitoredbyPCR-basedmethods;correspondinggenomicDNAwasamplifiedusingprim-ersAHG1-SacI-F1andAHG1-R3anddigestedwiththeSacIrestric-tionenzyme.Theahg3-1fragmentwasdigestedwithBspHI,theabi3-1fragmentwithAcll,theabi4-1andabi5-1fragmentswithAvall,andtheaba2-1fragmentwithAflII.AcknowledgementsWethankDrsJulianSchroederandBeariceRoseforcriticalreadingandcommentsonthemanuscript.WearegratefultoDrsM.Oka-motoandE.Nambara,RIKENPSC,forprovidingvariouscDNAmixtures,andtotheArabidopsisBiologicalResourceCenterandRIKENBioResourceCenterforprovidingtheT-DNAinsertionlineandtheDs-transposontaggingline,respectively.ThisworkwaspartlysupportedbyaGrant-in-AidfromtheMinistryofEducation,Sports,Culture,ScienceandTechnologyofJapan,andtheRIKENpresident’sSpecialResearchGrant(toT.H.),andpartlybythePro-gramforPromotionofBasicResearchActivitiesforInnovativeBiosciences(K.S.).SupplementaryMaterialThefollowingsupplementarymaterialisavailableforthisarticleonline:FigureS1.ThepublicexpressiondataofPP2Cgenes.FigureS2.Mapmanprojectionofahg1-1affectedgenes.TableS1.PublicmicroarraydatausedforFigureS1.TableS2.Microarraydata.TableS3.Genegroupsdifferentiallyaffectedbyahg1-1andahg3-1.TableS4.NumbersofABRE,CEandRYelementsinthepromoterregions.TableS5.Germinationefficienciesofsugarsofahg1-1andahg3-2.TableS6.Oligonucleotidesusedformappinginthiswork.TableS7.Oligonucleo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Journalcompilationª2007BlackwellPublishingLtd,ThePlantJournal,(2007),50,935–949
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