WhatCanTellUsAbouttheEvolutionoftheCarpel?Traditionally,thecarpelhasbe - PDF document

WhatCanTellUsAbouttheEvolutionoftheCarpel?Traditionally,thecarpelhasbeenthoughttohaveevolvedbytheclosureofafemaleorgan,homologoustotheovule-bearingscalesofgymnosperms.Thisclosuremayhaveproceededbymeansofapartiallyclosedcupule,suchasthoseknownfromandseveralotherfossilgymnospermswhoserelation-shipstotheangiospermsareuncertain(35).Conversely,themorerecent‘‘mostlymaletheory’’(36)proposesthatthecarpelevolvedbytheclosureofamaleorgan,themicrosporophyll,aroundovulesthathaddevelopedectopically.Wehaveshownthattheancestralsequencesofarelikelytohavecontrolledcarpeldevelopmentintheancestoroftheextantangiosperms.Itwillnowbeinterestingtodiscoverwhetherorthologuesofthesegenesarealsoexpressedinthereproductivestructuresoftheangiosperms’nearestlivingrelatives,thegym-nosperms.Iforthologuesofarenotexpressedingymnospermreproductivestructures,itmaybethatthesegeneswerenewlyrecruitedtocarpeldevelopmentintheangiospermlineageandmaythereforehaveplayedimportantrolesincarpelevolution.If,bycontrast,wefindthatorthologuesofareexpressedingymnospermreproductivestructures,itwillbeinterestingtoknowwhethertheirpreciseexpressionpatternssupportamale,orafemale,originforthecarpel.WethankFranc¸oisMicheneau,FabienCarrezandMarjorieCombeofAnthiasS.A.(LesChe`res,France)forthegenerousgiftofinflorescencetissuesofandSuzanneMogliaandGe´raldMogliaofCold’Amieu,NewCaledonia,forhelpinlocatingspecimensoftrichopodaWethankPrs.PeterEndressandEdSchneiderforhelpfuldiscussions,Dr.SylvieBaudinoforhelpwithelectronmicroscopy,andallofthetechnicalstaffoftheReproductionetDe´veloppementdesPlantesLaboratory.ThisprojectwassupportedthroughaFrenchGovernmentgrant,ACIBiologieduDe´veloppementetPhysiologieInte´grative.1.Kuzoff,R.K.&Gasser,C.S.(2000)TrendsPlantSci330–336.2.Zanis,M.J.,Soltis,D.E.,Soltis,P.S.,Mathews,S.&Donoghue,M.J.(2002)Proc6848–6853.3.Qiu,Y.L.,Lee,J.,Whitlock,B.A.,Bernasconi-Quadroni,F.&Dombrovska,O.(2001)4.Goremykin,V.V.,Hirsch-Ernst,K.I.,Wolfl,S.&Hellwig,F.H.(2003)1499–1505.5.Goremykin,V.V.,Hirsch-Ernst,K.I.,Wolfl,S.&Hellwig,F.H.(2004)6.Soltis,D.E.,Albert,V.A.,Savolainen,V.,Hilu,K.,Qiu,Y.L.,Chase,M.W.,Farris,J.S.,Stefanovic,S.,Rice,D.W.,Palmer,J.D.&Soltis,P.S.(2004)TrendsPlantSci477–483.7.Stefanovic,S.,Rice,D.W.&Palmer,J.D.(2004)BMCEvol8.Endress,P.K.(2001)PlantSci9.Pelaz,S.,Ditta,G.S.,Baumann,E.,Wisman,E.&Yanofsky,M.F.(2000)200–203.10.Tandre,K.,Albert,V.A.,Sundas,A.&Engstrom,P.(1995)PlantMol69–78.11.Winter,K.U.,Becker,A.,Munster,T.,Kim,J.T.,Saedler,H.&Theissen,G.Proc12.Bowman,J.L.,Baum,S.F.,Eshed,Y.,Putterill,J.&Alvarez,J.(1999)Curr13.Ferrandiz,C.,Pelaz,S.&Yanofsky,M.F.(1999)14.Bowman,J.L.&Smyth,D.R.(1999)Development(Cambridge15.Eshed,Y.,Baum,S.F.&Bowman,J.L.(1999)199–209.16.Alvarez,J.&Smyth,D.R.(1999)Development(Cambridge17.Yamaguchi,T.,Nagasawa,N.,Kawasaki,S.,Matsuoka,M.,Nagato,Y.&Hirano,H.Y.(2004)PlantCell500–509.18.Roe,J.L.,Rivin,C.J.,Sessions,R.A.,Feldmann,K.A.&Zambryski,P.C.939–950.19.Roe,J.L.,Durfee,T.,Zupan,J.R.,Repetti,P.P.,McLean,B.G.&Zambryski,P.C.(1997)5838–5845.20.Roe,J.L.,Nemhauser,J.L.&Zambryski,P.C.(1997)PlantCell21.Han,Z.B.,Saam,J.R.,Adams,H.P.,Mango,S.E.&Schumacher,J.M.(2003)Curr22.Ehsan,H.,Reichheld,J.P.,Durfee,T.&Roe,J.L.(2004)PlantPhysiol1488–1499.23.Chang,S.,Puryear,J.&Cairney,J.(1993)PlantMol114–117.24.Rose,T.M.,Schultz,E.R.,Henikoff,J.G.,Pietrokovski,S.,McCallum,C.M.&Henikoff,S.(1998)NucleicAcidsRes1628–1635.25.Thompson,J.D.,Higgins,D.G.&Gibson,T.J.(1994)NucleicAcidsRes4673–4680.26.Galtier,N.,Gouy,M.&Gautier,C.(1996)27.Teakle,G.R.,Scutt,C.P.&GilmartinP.M.(2002)inMolecularPlantBiology,ed.Gilmartin,P.M.&Bowler,C.(OxfordUniv.Press,Oxford),Vol.2,pp.3–40.28.Scutt,C.P.,Vinauger-Douard,M.,Fourquin,C.,Ailhas,J.,Kuno,N.,Uchida,K.,Gaude,T.,Furuya,M.&Dumas,C.(2003)PlantPhysiol653–665.29.Ferrandiz,C.&Sessions,A.(2002)inArabidopsis:ALaboratoryManual,ed.Weigel,D.&Glazebrook,J.(ColdSpringHarborLab.Press,Plainview,NY),pp.195–203.30.Endress,P.K.&Igersheim,A.(2000)PlantSci31.Helliwell,C.&Waterhouse,P.(2003)289–295.32.Yamada,T.,Ito,M.&Kato,M.(2004)PlantSci33.Friis,E.M.,Pedersen,K.R.&Crane,P.R.(2001)34.Feild,T.S.,Arens,N.C.&Dawson,T.E.(2003)PlantSciS129–S142.35.Taylor,T.N.(1993)TheBiologyandEvolutionofFossilPlants(Prentice–Hall,EnglewoodCliffs,NJ).36.Frohlich,M.W.(2003)559–566.37.Saitou,N.&Nei,M.(1987)406–425.etal 2–3.5mminlength,moststronglyinthedevelopingdehiscencezones(Fig.4Kand4L).Bycontrast,expressionofhasnotbeenreportedintheantherepidermis.IsHighlyExpressedinTissuesSpecializedforBuoyancyinFlowersofaresupportedatthewater’ssurfacebyspecializedleavesthatareroundandflattenedinshape,bycomparisonwiththehighlydissected,submergedleavesofthisspecies(Fig.3Insituhybridizationdetectedstrongexpressionofinboththepedunclesofflowersandthepetiolesoffloatingleaves.Thisexpressionwaslocalizedtosinglelayersofparenchymacellssurroundingaircanalsthatruntheentirelengthofthepedunclesandpetiolesandfunctiontoprovidebuoyancytothesestructures(Fig.4PlayRolesinGynoeciumDevelopmentThatSeemtoHaveBeenConservedSincetheCommonAncestoroftheExtantAngio-Thepresentstudydemonstratesthatputativeortho-loguesofareexpressedingynoeciumtissuesinoneorotherofthespeciestrichopodainanearidenticalmanner,bothspaciallyandtemporally,tothecorre-genes.Wearguesuchsimilarexpressionpatternsinorthologousgenestobestronglyindicativeoftheconservationoffunctions.trichopodasentthetwoearliestdiverginggroupsofangiosperms(1,2).Theancestorsharedbetweenthesegroupsandtheremaininglin-eage,containing,mayberegardedasthecommonancestorofthelivingfloweringplants.Wethereforeconcludethattheancestralsequencesofarelikelytohaveplayedrolesincarpeldevelopmentintheancestoroftheextantangiospermsthataresimilaroridenticaltothepresent-dayrolesofthesegenesin.Wehavemappedourexpressiondataontoaphylogenyofsomeoftheangiospermspeciesfororthologuesareknown,showninFig.5.Ourresultsfallshortofproofofconservationofgenefunctionforthreemainreasons.First,wecannotdiscountthepossibilitythatsimilarityingeneexpressionmayhaveresultedfromtheparallelrecruitmentoforthologousgenestosimilarrolesaftertheevolutionarydivergenceoftheirrespectiveplantlineages.Sec-ond,evidenceofgeneexpressionisnotdirectfunctionalevi-dence.Suchevidencewilldependontechnicaladvancesthatallow,forexample,thedisruptionofgeneexpressionbyRNAinterference(31)inbasalangiosperms.Third,ourconclusionsarebasedonthehypothesisthatANITAangiospermsarebasalinthefloweringplantclade.Arecentlypublishedstudy(32)hasshownaputativeortho-logueofYABBY2(YAB2)tobeexpressedinadaxial,ratherthanabaxial,tissuesoflateralorgansintrichopoda.Theseauthorspostulatethataswitchinpolaritymusthaveoccurredfortheorthologueineitherthetrichopodalineages.ThepresentstudysuggeststhatsuchapolarityswitchhasnottakenplaceforatleastoneotherpairofYABBYorthologues,TheRoleofinRiceWasAcquiredAftertheDivergenceoftheMonocotLineage.,theorthologuefromrice,hasbeenshowntospecifycarpelidentityandcontrolsleafdevelopment,.Theexpressionpatternofinriceflowersisconsistentwithitsroleindeterminingcarpelidentityanddoesnotresembletheexpressionpatternsof(14)andtrichopoda.Ourresults,combinedwiththoseofYamaguchietal.(17),suggestthatthenovelfunctionsandexpressionpatternofinricewereacquiredafterthedivergenceofthemonocotlineage,asshowninFig.5.Thisandotherchangesingenefunctionmayhavebeenimportantfortheevolutionofthehighlyspecializedflowersandleavesofmonocots.TheRoleofintheDevelopmentofSecretoryTissuesWasAcquiredLaterThanItsRoleinGynoeciumDevelopment.controlsnectarydevelopmentin,inadditiontoitsroleingynoeciumdevelopment.SpecializednectariesareabsentfromtheflowersofANITAgradeangiosperms(8),suggestingthattheseorgansevolvedaftertheseparationoftheANITAcladesfromtheremaininglineage.Infemaletrichopodaflowers,thestigmasarehighlysecretory,althoughexpressionwasnotdetectedinthesetissues.Studieswithinlater-divergingangiospermgroupsmayindicatethelevelatwhichtheancestorwasrecruitedtoaroleinthedevelopmentoffloralsecretorytissues.MayPlayaRoleintheDevelopmentofStructuresSpecializedforBuoyancyThatIsMoreRecentThanItsRoleinGynoeciumWehaveshownthatishighlyexpressedincellssurroundingtheaircanalsinpetiolesandpedunclesthatfunctiontocarryfloatingleavesandflowersoftothewater’ssurface.FossilevidenceindicatesthatanaquaticgrowthhabithasbeenpresentintheNymphaealesforatleast115millionyears(33).Feildetal.(34)havearguedfromacompar-isonofbasalangiospermgroups,however,thattheancestoroftheextantangiospermswasawoody,terrestrialspecies.Ifthisinterpretationiscorrect,thepotentialroleofindevel-opmentalspecializationforbuoyancymayhavebeenacquiredsubsequentlytoitsroleingynoeciumdevelopment.ItwillbeinterestingtodiscoverwhetherorthologuesfromotherspeciesoftheNymphaealesareexpressedinbuoyancy-relatedtissues. Fig.5.Mappingofexpressioningynoeciumtissuesontothephylogenetictreeoftheangiosperms.Thephaeales)lineagesareplacedinequallybasalpositionstoreectuncertaintyintheirorderofbranching(2).etalMarch22,2005vol.102no.12PLANTBIOLOGY expressedinflowersbutnotinleavesoftrichopoda,similarlytoitsorthologue.Topreciselylocalizetheexpressionofinflowertissues,insituwereperformed,showninFig.4.Femaleflowersoftrichopoda(Fig.4)typicallycontainaperianthofseventoeighttepals,fiveseparatecarpels,andonetotwostaminodes(sterilestamens)thatmayberelicsofabisexualancestor(30).Insituhybridizationindicatedtobeexpressedspecificallyinthecarpelwallatearlytomid-developmentalstages(Fig.4).Thisexpressionseemedstrongertowardtheabaxial(outer)surfaceofthecarpelwall,mirroringtheexpressionofinthegynoecium(14).Laterinfemaleflowerdevelop-ment,awet,secretorystigmaformsateachcarpelapex(Fig.4Thesewetstigmasmaypossiblyprovidearewardforpollinatinginsects(8),inadditiontoareceptivesurfaceforpollengrains.Bythisstage,expressioncouldnolongerbedetected(resultsnotpresented).VirtualNorthernhybridizationsdemonstratedtobeexpressedinbothmaleandfemaleflowersoftrichopoda3).Maleflowerbudsoftrichopodacontainaperianthof9–11tepalsthatencloses12–21stamens.Eachstamenconsistsofafour-loculateanthersupportedonashort,widefilament(30).Expressionofinmaleflowerswaslocalizedbyinsituhybridizationtothestamenfilaments(Fig.4).Bycontrast,expressionhasnotbeenshowninthestamenfilamentsof(14),andmutantsarenotaffectedinstamendevelopment(16).IsExpressedinC.aquaticaStyleandStigmaTissues,CloselyExpressioninNorthernblothybridiza-tions(Fig.3)demonstratedexpressionofinbothinflorescenceandvegetativetissuesof,suggesting,likeitsorthologue,playsrolesinbothreproductiveandvegetativedevelopment.Flowersoftypicallycontainthreesepals,threepetals,sixstamens,andagynoeciumofthreeseparatecarpels.Insituhybridizationtoflowerbudsdemonstratedstrongexpressioninstyleandstigmatissuesofcarpelsatmidtolatedevelopmentalstages.Inbudsof3-mmlength,wasstronglyexpressedintheinternalcelllayersofthestyleandstigma(Fig.4wasexpressedinthecellssurroundingasecretion-filledcanalrunninglongitudinallythroughthestyle(Fig.4).Thisfeatureisconsideredanimportantpleisiomorphiccharacterintheangiosperms(8).Inslightlylatercarpeldevelopmentcorre-spondingtoflowerbudsof4mmlength,expressionshowedareductioninthestyleandanincreaseinthestigma(Fig.4).Thisdevelopmentalstagecorrelatedwithaphaseofrapidelongationofthestigmapapillae(Fig.4).Expressionofhasalsobeendemonstratedinthestyleandstigmaduringlaterstagesofflowerbuddevelopment(20).Expressionofingynoeciumtissuesthereforestronglyresemblesthatofitsorthologue.Inadditiontoitsexpressioninfemaletissues,insituhybridizationrevealedexpressioninstamens.Expressionofapparentintheouterepidermisoftheantherwallinbudsof Fig.4.insituhybridizationsshowingexpressionof,withaccompanyingimages.Insituhybridizationsignalsappearblueorviolet.Verydarkmaterialintepalsofisnaturalcoloration.Allgeneexpressionsignalsreferredtowerespecictoantisense-strandriboprobesandwerenotobservedbyusingnegativecontrolsense-strandriboprobesonserialsectionsfromthesametissueblocks(resultsnotpresented).()AfemaleowerofInsituhybridizationtoalongitudinalsection(l.s.)ofanfemaleowerbudshowingexpressioninthecarpelwall.()SEMofafemaleowerbudwiththeperianthremoved(stageasfor).()SEMofamaturefemaleower.(Insituhybridizationtoanl.s.ofanmaleowerbudshowingexpressioninthestamenlaments.(Insitutoanl.s.ofaowerbudshowingexpressionofinthestyleandstigma.()SEMofthegynoecium(stageasfor).(Insituhybridizationtoatransversesection(t.s.)ofa3-mm-longowerbudshowingexpressionofinthestyleandstigma.(Insitutoanl.s.ofa4-mm-longowerbudshowingexpressionofinthestigma.()SEMofthestigmaandupperstyleof(stageasfor).(Insituhybridizationtoat.s.ofaowerbudshowingexpressionofintheantherwall.()SEMofaowerbudshowingthestamens(stageasfor).()AoweranddimorphicleavesofInsituhybridizationtoat.s.ofaowerpeduncleshowingexpressionincellssurroundingaircanals.(Insituhybridizationtoanl.s.ofaoatingleafshowingexpressionofincellssurroundingaircanalsinthepetiole.a,anther;ac,aircanal;al,antherloculus;c,carpel;d,dehiscencezone;f,stamenlament;fb,oatingleafblade;o,ovary;ov,ovule;p,perianth;pd,peduncle;pe,petiole;r,receptacle;sc,stylarcanal;sm,staminode;st,stigma;su,submergedleaves;sy,style;t,tepal.etal NorthernandVirtualNorthernBlotHybridizations.Northernblotsofwerepreparedcontaining2.5gpertrackofpolyadenylatedRNAsamples.VirtualNorthernblots,whicharecDNAblotsgivingasensitivityofdetectionequivalenttopolyadenylatedRNANorthernblots,werepreparedfromtrichopodatotalRNAsamplesasdescribedbyTeakleetal.(27).Blothybridizationswereperformedbyusingradio-labeledprobescorrespondingtofull-lengthcDNAsasdescribed(28),althoughusingahybridizationbuffercontaining1%(wtbovineserumalbumen,0.2Msodiumphosphate(pH7.2),1mMEDTA,7%(wtvol)SDS,and15%(volvol)formamide.InSituinsituhybridizationtotissuesectionsofantisenseandsense(control)strandriboprobesderivedfromcDNAswasperformedasdescribedbyFerrandizandSessions(29).Insituimageswerecapturedunderbright-fieldilluminationbyusingaZeissAxiovert125invertedmicroscope.ScanningElectronMicroscopy(SEM).PlantmaterialwasfixedandstoredinFAA(3.7%formaldehyde5%aceticacid50%etha-nol).SampleswererehydratedandexaminedbyusingaHitachi(Tokyo)S800environmentalscanningelectronmicroscope.PutativeOrthologuesofAreExpressedinFlowerTissuesoftheMostEarlyDivergingGroupsofAngiosperms.RT-PCRwasusedtoamplifyPCRproductshomologoustofromflowerRNAsofthebasalangiospermstrichopoda.ThePCRproductsobtainedwereligatedintoplasmidvectorsforDNAsequencingandreexcisedforuseasprobestoscreenflowercDNAlibraries.Thepredictedaminoacidsequencesoffull-lengthcDNAsobtainedfromlibraryscreenswerealignedwithknownhomologoussequences,andphylogenetictreeswereconstructedtoinfergeneorthology-likecDNAfromtrichopoda,encodedaproteincontainingzinc-fingerandYABBYdomains,inasimilararrangementtoCRCandotherYABBYputativetranscriptionfactors,asshowninFig.1.Inphylogeneticanalyses,groupedcloselywithanditsputativeorthologues,asshowninFig.2.Thisgroupingprovedveryrobustandwasmaintainedbyusingdifferentcombinationsofaminoacidornucleicaciddatasetsanddifferentanalysismethods.wasthereforeconcludedtorepresentaputativeorthologueof.Bycontrast,afull-lengthcDNAobtainedfromgroupedmostcloselytoinphylogeneticanalyses(resultsnotpresented)andwasconcludedtonotrepresentanorthologueof.FurtherscreensofacDNAlibraryatareducedstringencyofhybridizationusingprobesidentifiedseveralfurther-likecDNAs,althoughnoneofthesegroupedwithinphylogeneticanalyses.flower-expressedcDNAswillrequirefurtherstudy.-likecDNAidentifiedfromencodedaproteincontainingacoiled-coilputativeregulatorydomain,nuclearlocalizationsignals,andaputativeserine-threonineproteinkinasedomain,therebyshowingoverallstruc-turalsimilaritytoTSL,asshowninFig.1.CabTSLdidnotcontainanN-terminalglutamine-richdomainofunknownfunc-tionthatispresentinTSL,althoughitisabsentfromTSLorthologuesofriceandmaize(accessionnumbersAC091811andAY644701).Becauseisauniquegeneinmayberegardedasitsputativeorthologue.ArelatedcDNAidentifiedfromourotherbasalangiospermtrichopoda,wasfoundtobeinterruptedbyastopcodonupstreamofitspredictedkinasedomain.Furthermore,noin-framestartcodonwaspresentupstreamofthereadingframeinthismolecule.Thesefeatures,inconsistentwiththesynthesisofanactiveTSL-likekinase,werefoundtobeconservedbetweenseveralindependentcDNAsandtheiranal-ysiswasnotcontinued.Thepresentstudyhas,therefore,iden-tifiedtwocDNAsencodingputativeCRC-andTSL-orthologousproteins,fromtrichopoda,respectively.IsExpressedAbaxiallyintheA.trichopodaCarpelWall,CloselyResemblingExpressioninVirtualNorthernblothybridizations,showninFig.3,indicatedtobe Fig.1.ComparisonsofpredictedproteindomainsinCRCandAmbCRC(andTSLandCabTSL().CC,coiled-coildomain;CAT,proteinkinasecatalyticdomain;NLS,nuclearlocalizationsignal;Q,glutamine-richregion;Y,YABBY(DNA-binding)domain;Zf,zinc-ngerdomain. Fig.2.PhylogeneticanalysisofAmbCRCwithotherpredictedYABBYpro-teins.Thetreeshownwasconstructedbytheneighborjoiningmethod(37)fromanalignmentcorrespondingtoaminoacidresidues19–55(thezinc-ngerdomain)and110–157(theYABBYdomain)ofsequencesareunderlined,andpercentagesupportfornodesin500bootstrapreplicatesareencircled.Unpublishedsequenceaccessionnumbersareasfollows:AY451399(),AY703987(),AY071845),andAF545436(wheat). Fig.3.NorthernandvirtualNorthernblothybridizationsof,andtheirorthologues.HybridizationsaretovirtualNorthernblotsofandpolyadenylatedRNANorthernblotsof.HybridizationstoacDNAencodingGAPDHfromeachspeciesareincludedtodemonstrateequivalentloadingoftracks.F,femaleowers;I,inorescences;L,leaves;M,maleowers;RL,rosetteleaves;andV,vegetativetissues(submergedleavesandstems).etalMarch22,2005vol.102no.12PLANTBIOLOGY carpelswithfewovules,aspiralphyllotaxyoffloralorgans,andaperianththatwasnotdistinctlydividedintopetalsandsepals.ThegynoeciumofArabidopsisthaliana,ahighlyevolvedeudicotspecies,comprisestwocongenitallyfusedcarpels.ThedevelopmentofthisstructureisspecifiedbytheexpressionoftheMADSboxC-functiongene,,accordingtotheABCEmodel(9).PutativeC-functionorthologuesareexpressedinreproductiveorgansofgymnospermsasinangiosperms,indicatingaspectsofC-functionactivitytohavebeenconservedsincethecommonancestoroftheseedplants(10,11).TheancientoriginoftheC-functiongenesuggeststhatevolutionarychangesinothergenesmayhavebeenresponsibleforthemorerecentoriginofthecarpel.Candidatesforthesegenesincludethemanysequencesencodingtranscriptionfactors,proteinkinases,andotherdevelopmentalregulators,whoserolesincarpeldevelopmenthavebeeninvestigatedbytheanalysisofmutants(12,13).Wehavebeguntodeterminewhichgenesofdevelopmentmayhaveconservedtheirfunctionssincetheancestorofthelivingfloweringplants.Toperformthisanalysis,wehavesearchedforputativeorthologuesofcarpeldevelop-mentgenesinrepresentativesofthetwomostprobablybasalgroupsoffloweringplants,trichopoda(Amborellales,Am-borellaceae)andCabombaaquatica(Nymphaeales,Cabom-trichopodaisascramblingdioecioustreeofhumidtropicalforests,endemictoNewCaledonia.isasmallaquaticplant,nativetoNorthernBrazil.Bothofthesespeciesshowthelikelypleisiomorphiccharactersrelatingtocarpeldevelopment(8).Forcertainclassesofcarpeldevelopmentgenesthatarethesubjectsofcontinuingstudiesinourlabora-tory,orthologyrelationshipshaveprovedcomplex,orexpressionpatternshavedifferedconsiderablybetweenbasalangiosperms.However,wepresentherethecasesofCRABSCLAW(CRC)andTOUSLED(TSL),twogenesforwhichstrongevidencehasbeenobtainedforaconservationoffunctioningynoeciumdevelopmentsincethecommonancestorofthelivingfloweringplants.encodesamemberofthesmallfamilyofplant-specificYABBYputativetranscriptionfactorsin.Itisexpressedonlyinthegynoeciumandnectariesandcontrolsthedevelopmentofthesestructures(14).mutantshaveabnor-mallywidegynoeciathatareincompletelyclosedattheapexandshowadefectincarpelfusion.interactsgeneticallywiththreedifferentclassesofgenestospecifyabaxialcellfateintheovarywall(15).Thisfunctionisconsistentwiththespecificexpressionofintheabaxialcelllayersoftheovary.mutationseliminatecarpelloidstructuresinthefirstwhorlofC-functiondoublemutants(16),suggestingthatcontrolelementsofaC-function-likepathwayinawaythatismaskedbygeneticredundancyinwild-typeplants.AputativeDROOPINGLEAF(DL),isknownfromrice(17).Thisgene,unlike,seemstoplayamajorroleinthespecificationofcarpelidentityasmutantsshowhomeoticconversionofcarpelstostamens.Consistentwiththisfunction,isexpressedinthepresumptivezone,oranlagen,intheflowermeristemfromwhichthegynoeciumdevelops.,also,controlsthedevelopmentofthemid-ribinleaves.isauniquegeneinthathaspleiotropiceffectsonflowerandleafdevelopment(18).Itencodesaserine-threonineproteinkinasecontaininganN-terminalregulatorydomaininadditiontoaC-terminalkinasedomain.Theregula-torydomainisnecessaryfortheformationofhomooligomers,uponwhichthecatalyticactivityofTSLdepends(19).Inmutants,thenumberoffloralorganprimordiainwhorlsonetothreeisreduced,althoughtheorgansthatdevelopfromthesearenotgreatlyaffected(18).Carpelfusionmaybereducedbymutations,probablyduetouncoordinatedgrowthofcarpelprimordia.Otherthanthiseffect,intervenesonlyatalatestageingynoeciumdevelopment.Inmutants,thestyleandstigmadevelopincompletely,andthegynoeciumremainsopenatitsapex(20).Thiseffectisconsistentwiththespecificexpressionofinthestyleandstigmaatlatestagesofflowerbuddevelopment.TOUSLED-likekinases(TLKs)arewidelydistributedineukaryotes,includingotherplantspecies,Caenorhabditiselegans,andmammals,suggestingthatthesemoleculesplayfundamentalbiochemicalroles.In,TLKisessentialfortranscription,anditsinactivationleadstothecompletearrestofdevelopment(21).Resultsfromvariousanimalsystems,discussedbyEhsanetal.(22),showthatTLKactivityislinkedtoDNAreplicationandthatTLKsmayparticipateintheregulationofgeneexpressionthroughchro-matinmodification.TheseauthorshavedemonstratedlinksforTSLinwithboththecellcycleandwithputativecomponentsofchromatinassembly(22),suggestingbiochemicalparallelswithanimalTLKs.Despitethesesimilarities,theeffectmutationsinremainslessseverethanthatofTLKinactivationin,suggestingsomedivergenceintherolesofTLKsbetweenplantandanimallineages.MaterialsandMethodsPlantMaterial.MaterialoftrichopodaBaill.wasfield-collectedfromlocationsnearCold’Amieu,NewCaledonia(mapIGN4825).MaterialofAubletwasobtainedfromAnthiasS.A.,LesChe`res69,France.SeedsofHeyn.Lands-ecotypewereobtainedfromtheNottinghamStockCentre,Nottingham,U.K.,andplantsweregrowntomaturityinpeat-basedcompostinagrowthchamberat20°Cunder16hlight8hdarkcycles.RNAPreparationandRT-PCR.RNAwasextractedfromtissuesoftrichopodabythemethodofChangetal.(23),andfromtissuesbyusingTRIzolreagent(Invitro-gen).PolyadenylatedRNAforuseincDNAlibraryconstructionandNorthernblottingwaspurifiedfromtotalRNAbyusingaPolyAtractkit(Promega).RT-PCRwasperformedontotalRNAsamplesoftrichopodabytheCODE-HOPmethod(24).ConservedregionsofmRNAshomologoustowereamplifiedbyusingthepartiallydegenerateprimers-TTGGACACAGTGACAGTGAAGTGYGGNCAYTGand-AGCCCAATTCTTAGCAGCAGCASWRAANGCYTC.ThoseofmRNAshomologoustowereamplifiedbyusingthepartiallydegenerateprimers5-AATAAGAAGTCTCA-GAAGATTATHCAYTAYGAand5-TTCAAAGCATTCT-GGTGGCAAATACCARTANGT.cDNALibraryConstructionandScreening.cDNAlibrarieswerepreparedbyusingaBacteriophageUni-ZapIIkit(Strategene)frompolyadenylatedRNAoffemaleflowersoftrichopodaandofinflorescencesofthatincludedflowerbudstagesuptoanthesis.BacteriophageplaquesweretransferredontonylonhybridizationmembranesandscreenedwithradiolabeledRT-PCRproductscorrespondingtofragmentsof-and-likecDNAsaccordingtoZapprototcols(Stratagene).Positivelyhybridizingbacteriophagecloneswerepurifiedthrougharoundofsecondaryscreening,andcDNAswereobtainedfromtheseinpBlueScriptIIplasmidvectorsbyexcisionusingExAssist(Stratagene)M13helperphage.MolecularPhylogeneticAnalysis.AlignmentofpredictedaminoacidsequenceswasperformedbyusingCLUSTALW(25).Phylo-genetictreeswereconstructedandbootstrappedbyusingthePHYLOWINcomputerpackage(26),allowingcomparisonofresultsusingneighborjoining,maximumlikelihood,andmaxi-mumparsimonymethods.etal Thispaperwassubmitteddirectly(TrackII)tothePNASofce.CRABSCLAWDROOPINGLEAF;TLK,TOUSLED-likekinase;SEM,scanningelectronmicroscopy.Datadeposition:ThesequencesreportedinthispaperhavebeendepositedintheEMBLdatabase[accessionnos.AJ877257(AmbCRC)andAJ877258(CabTSL)].Towhomcorrespondenceshouldbeaddressed.E-mail:charlie.scutt@ens-lyon.fr.©2005byTheNationalAcademyofSciencesoftheUSApnas.0409577102PNASMarch22,2005vol.102no.124649–4654 PLANTBIOLOGY EvidencethatCRABSCLAWconservedtheirrolesincarpeldevelopmentsincetheancestoroftheextantangiospermsFourquin*,MarionVinauger-Douard*,BrunoFogliani,ChristianDumas*,andCharlesP.Scutt**LaboratoiredeReproductionetDe´veloppementdesPlantes,Unite´MixtedeRecherche5667,CentreNationaldelaRechercheScientique,InstitutNationaldelaRechercheAgronomique,Universite´ClaudeBernard-Lyon,InstitutFe´de´ratifdeRecherche128-Biosciences-LyonGerland,46Alle´ed’Italie,EcoleNormaleSupe´rieuredeLyon,69364LyonCedex07,France;andLaboratoiredeBiologieetPhysiologieVe´ge´talesApplique´es,Universite´dela´donie,BP4477Noume´a,NewCaledoniaEditedbyJohnF.Doebley,UniversityofWisconsin,Madison,WI,andapprovedFebruary14,2005(receivedforreviewDecember21,2004)