HD-Zip Proteins GL2 and HDG11 Have Redundant Functions

ThePlantCell,Vol.26:2184–2200,May2014,http://wendang.chazidian.comã2014AmericanSocietyofPlantBiologists.Allrightsreserved.

HD-ZipProteinsGL2andHDG11HaveRedundantFunctionsinArabidopsisTrichomes,andGL2ActivatesaPositiveFeedbackLoopviaMYB23

AashimaKhosla,a,bJanetM.Paper,aAllisonP.Boehler,a,1AmandaM.Bradley,aTitusR.Neumann,aandKathrinSchricka,b,c,2

aDivision

ofBiology,KansasStateUniversity,Manhattan,Kansas66506-4901

ofBiochemistryandMolecularBiophysics,KansasStateUniversity,Manhattan,Kansas66506-3702

cMolecular,Cellular,andDevelopmentalBiology,KansasStateUniversity,Manhattan,Kansas66506-4901

bDepartment

TheclassIVhomeodomainleucinezippertranscriptionfactorGLABRA2(GL2)actsinacomplexregulatorycircuitthatregulatesthedifferentiationoftrichomesinArabidopsisthaliana.WedescribeageneticinteractionwithHOMEODOMAINGLABROUS11(HDG11),previouslyidenti?edasanegativeregulatoroftrichomebranching.gl2hdg11doublemutantsdisplayenhancedtrichomecell-typedifferentiationdefects.TransgenicexpressionofHDG11usingtheGL2promoterpartiallysuppressesgl2trichomephenotypes.Viceversa,expressionofGL2underthecontrolofitsnativepromoterpartiallycomplementshdg11ectopicbranching.Sincegl2hdg11andgl2myb23doublemutantsandthetriplemutantdisplaysimilartrichomedifferentiationdefects,weinvestigatedaconnectiontotheR2R3-MYBtranscriptionfactorMYB23.WeshowthatMYB23transcriptlevelsaresigni?cantlyreducedinshootsfromgl2mutantsandthatGL2candrivetheexpressionofaMYB23-promoterfusiontogreen?uorescentprotein.Yeastone-hybrid,chromatinimmunoprecipitation,andinplantareportergeneexperimentsindicatethatanL1-boxintheMYB23promoteractsasaGL2bindingsite.Takentogether,our?ndingsrevealafunctionalredundancybetweenGL2andHDG11,twohomeodomainleucinezippertranscriptionfactorspreviouslythoughttomediateopposingfunctionsintrichomemorphogenesis.AmodelisproposedinwhichGL2transcriptlevelsaremaintainedthroughapositivefeedbackloopinvolvingGL2activationofMYB23.

INTRODUCTION

Inplants,theepidermisofleavesisderivedfromtheouterL1layeroftheshootapicalmeristem.Asleafprimordiadevelop,theepidermalcelllayerdifferentiatestoformanorganizedpatternofdistinctcelltypes,includingpavementcells,stomata,andtri-chomes.InArabidopsisthaliana,matureleaftrichomesarechar-acteristicallylarge(300to500mm)branchedhaircellswhosenucleihaveundergonemultipleroundsofendoreduplicationandarepresentontheleafsurfaceinanonrandomregulardistribution(Hülskampetal.,1994;Balkundeetal.,2010).Amongotherplantspecies,leaftrichomesarehighlyvariableandmaybebranchedorunbranched,singlecelledormulticellular,andmayfunctionasglandularorgans.Proposedfunctionsoftrichomesincludepro-tectionfromherbivoresandshieldingfromabioticenvironmentalfactorssuchasfrost,evaporation,andUVradiation(Balkundeetal.,2010;YangandYe,2013).

TranscriptionfactorsoftheclassIVhomeodomainleucine-zipper(HD-Zip)familyplaycentralrolesinthedifferentiationofthe

1Current

address:BayerCropScienceUnitedStates,KansasCity,MO

64120.

2Addresscorrespondencetokschrick@ksu.edu.

Theauthorresponsiblefordistributionofmaterialsintegraltothe?ndingspresentedinthisarticleinaccordancewiththepolicydescribedintheInstructionsforAuthors(http://wendang.chazidian.com)is:KathrinSchrick(kschrick@ksu.edu).OnlineversioncontainsWeb-onlydata.

http://wendang.chazidian.com/cgi/doi/10.1105/tpc.113.120360

epidermisduringbothembryonicandpostembryonicdevelop-ment.Thisproteinfamilyischaracterizedbyahomeodomain(HD)Nterminaltoaplant-speci?cLeuzipperhavinganinternalloop(zipper-loop-zipper[ZLZ])(alsocalledtruncatedleucinezipper[Yangetal.,2002]),followedbyasteroidogenicacuteregulatoryprotein–relatedlipidtransfer(START)domain(Schricketal.,2004).TheArabidopsisgenomeencodesatotalof16classIVHD-Zipfamilymembers,severalofwhichareknowntobeinvolvedincellfatedeterminationwithinlayer-speci?ccontexts(Nakamuraetal.,2006).

TheclassIVHD-ZiptranscriptionfactorGLABRA2(GL2)playsapivotalroleinregulatingthedifferentiationoftheepidermisinnumeroustissues,includingtheroot(DiCristinaetal.,1996)andseedcoat(Westernetal.,2001).Itsroleintrichomesisthemajorfocusofthisarticle.gl2mutantsdisplayabortedtrichomeswiththincellwallsthatlackpapillae,andsometrichomesaberrantlyexpandintheplaneofthepavementcells(Koornneefetal.,1982;Rerieetal.,1994).GL2ishighlyexpressedinmaturetrichomes(Marksetal.,2009),andlateactivityofGL2seemstoberequiredforpropertrichomedevelopment.WhileGL2isrequiredforthedifferentiationandmaintenanceoftrichomecellfate,itisdis-pensablefortrichomeinitiation.Inacurrentmodel,GL2actsasadownstreamtranscriptionfactorinaregulatorycircuittogetherwiththemitosisinhibitorSIMthatisrequiredforendoreduplication(Grebe,2012).TheinitiationoftrichomecellfateandtranscriptionalactivationofGL2andSIMinvolvesanactivatorcomplexcom-posedofaWD40proteinencodedbyTRANSPARENTTESTAGLABRA1(TTG1),anR2R3-MYBtranscriptionfactorencoded

GL2,HDG11,andMYB23inTrichomes2185

byGL1andthepartiallyredundantgeneMYB23,abasichelix-loop-helix(bHLH)proteinencodedbytwogenes,GL3andENHANCEROFGL3(EGL3),andthepossibleadditionofan-othercomponentencodedbyMYC1(Grebe,2012).Thiscom-plexiscounteractedbyinhibitoryR3MYBtranscriptionfactorsencodedbyagroupofatleastsixgenes,includingCAPRICEandTRIPTYCHON,whichdiffusebetweencells,competingwiththeR2R3-MYBproteinsforbindingtothebHLHprotein.

Basedonmutantphenotypesandexpressionpro?ling,threeotherclassIVHD-ZiptranscriptionfactorsinadditiontoGL2areimplicatedintrichomecell-typedifferentiation.HOMODOMAINGLABROUS2(HDG2)isstronglyexpressedinmaturetrichomes,similartothelevelobservedforGL2(Marksetal.,2009).Whilehdg2mutantsdisplaynoobviousdefectsinoveralltrichomecellmorphology,theirtrichomesurfacesappearmoretransparentandtheircellwallcompositionisabnormal(Marksetal.,2009).Basedonmutantanalysis,HDG11andHDG12playnegativeregulatoryrolesintrichomebranching.hdg11mutantsresultinexcessbranchingoftrichomes,aphenotypethatisenhancedincombinationwithhdg12(Nakamuraetal.,2006).Whilepromoter–b?glucuronidase(GUS)reporterlinesforbothexhibitstrongexpressionintrichomes(Nakamuraetal.,2006),tran-scriptlevelsofHDG11andHDG12arelowandnotdetectable,respectively,inmaturetrichomes(Marksetal.,2009),consistentwithexpressionbeingmostlyrestrictedtothebranchingphaseoftrichomedevelopment.

Inthisarticle,weuncoverageneticinteractionandfunctionalredundancybetweentheclassIVHD-ZiptranscriptionfactorsGL2andHDG11intrichomes.Thephenotypeofgl2hdg11doublemutantsledustoinvestigateaconnectiontotheR2R3-MYBtranscriptionfactorMYB23.MembersoftheexpansiveR2R3-MYBgenefamilyplaykeyrolesinvariousplant-speci?cprocesses,includingtheregulationofcellfateindevelopment(Strackeetal.,2001;Dubosetal.,2010).MYB23hadpreviouslybeenfoundtoactredundantlywithGL1intrichomecell-typespeci?cation(Kiriketal.,2005).Intheroot,MYB23hadbeenshowntofunctionincellfate

determinationandtobinditsownpromoterinaMYB23-mediatedpositivefeedbackloop(Kangetal.,2009).Here,weprovideevi-denceforanadditionalpositivefeedbackmechanisminvolvingGL2transcriptionalactivationofMYB23inthemaintenanceofcellfateduringtrichomemorphogenesis.

RESULTS

SequenceAnalysisofGL2andHDG11RevealsHighlyRelatedHDs

GL2andHDG11arelinkedlocionArabidopsischromosome1.Phylogeneticanalysisofthe16membersoftheclassIVHD-ZipfamilydoesnotrevealahighdegreeofrelatednessbetweenGL2andHDG11incomparisonwithotherfamilymembers(Nakamuraetal.,2006).TofurtherprobesequencesimilaritybetweenGL2andHDG11,wedividedtheirproteinsequencesintofourdistinctconserveddomains:(1)HD,(2)ZLZ,(3)START,and(4)STARTadjacentdomain(SAD)(Figures1Aand1B).TheN-terminalHDimplicatedinDNAbindingisfollowedbytheZLZplant-speci?cLeuzipperdimerizationdomainconservedamongmembersoftheclassIVfamily(Schricketal.,2004).TheSTARTdomainim-plicatedinligandbinding(PontingandAravind,1999;Schricketal.,2004)liesinthemiddle,followedbytheSADofunknownfunctionattheCterminus.Wedeterminedthepercentageaminoacididentitiesandsimilaritiesbetweenthefourdomainsofthetwoproteinsinpairwisealignmentsfromprotein–proteinBLAST(Figure1B)andfoundtheHDdomaintobemostconserved,exhibiting74and89%identityandsimilarity,respectively(Figure1C).PairwisealignmentswereusedtocomparetheHDdomainofGL2witheachofthe15othermembersoftheclassIVHD-Zipfamily(SupplementalTable1).RankingbyaminoacididentitytoGL2,HDG11placed?rst,followedbyHDG5andPROTO-DERMALFACTOR2(PDF2).AmongtheotherclassIVmemberswhosemRNAtranscriptsareexpressedinleaftrichomes

(HDG2,

Figure1.SequenceRelationshipsbetweenClassIVHD-ZipTranscriptionFactorsGL2andHDG11.

(A)DomainarrangementsoftheGL2andHDG11proteinscomposedof747and722aminoacids,respectively.

(B)Percentageaminoacid(aa)identitiesandsimilaritiesbetweenGL2andHDG11forthefourconserveddomainsandtherespectivefull-lengthproteins.TheHDdomainsexhibitthehighestdegreeofaminoacididentityandsimilarity.

(C)AminoacidalignmentoftheHDdomainsfromtheGL2andHDG11proteins,generatedwithGeneious5.4.6software(Biomatters).Greenandyellowshadingindicateidenticalandsimilarresidues,respectively.

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2186ThePlantCell

Figure2.gl2-5hdg11-1DoubleMutantsDisplayanEnhancedTrichomeDifferentiationDefect.

(A)to(D)Rosettesfrom3-week-oldArabidopsisplants.

(A)and(B)Colwild-type(A)andhdg11-1(B)plantsdisplaytrichomescoveringleafsurfaces.

(C)and(D)gl2-5plantsappearmostlyglabrousexceptfortrichomesattheleafmargins(arrows)(C),whileincomparison,gl2-5hdg11-1doublemutantsdisplayanenhancedglabrousphenotype(D).

(E)to(L)Scanningelectronmicrographsofleaves([E]to[H])andleafmargins([I]to[L])revealdetailedtrichomemorphologies.

(E)and(I)Colwild-typetrichomesexhibitthreetofourbranches.

(F)and(J)hdg11-1leavesshow?veormorebranchesforsometrichomes(arrows).

(G)and(K)gl2-5trichomesaresmallandundifferentiatedexceptatleafmargins,wherebranching(arrow)isobserved.

(H)and(L)gl2-5hdg11-1doublemutantsexhibitsmallerundifferentiatedtrichomesatleafmargins.

(M)to(T)Firstleaves([M]to[P])anddetailededgesof?rstleaves([Q]to[T]).

(M)and(Q)Colwild-typetrichomeshavethreetofourbranches.

(N)and(R)hdg11-1mutantsexhibitectopictrichomebranching(arrows).

(O)and(S)gl2-5mutantsdisplayundifferentiatedtrichomesexceptatleafedges,wheresometrichomesdisplaybranching(arrows).

GL2,HDG11,andMYB23inTrichomes2187

HDG11,andHDG12),HDG11exhibitsthehighestdegreeofaminoacididentityandsimilaritytoGL2.

DoubleMutantsforgl2andhdg11DisplayEnhancedTrichomeDifferentiationDefects

Crossesbetweengl2-5andhdg11-1plantsoftheColumbia(Col)ecotypewereperformedtoconstructhomozygousdoublemu-tants.Incomparisonwithgl2-5singlemutants,thedoublemu-tantsexhibitedenhancedtrichomedifferentiationdefects(Figure2).Trichomephenotypeswerevisualizedonrosetteleavesbystereomicroscopy(Figures2Ato2D).Leaftrichomemorpholo-gieswereobservedunderhighermagni?cationusingscanningelectronmicroscopy(Figures2Eto2T).Whiletrichomeinitiationappearsnottobealteredingl2-5singleandgl2-5hdg11-1doublemutants,outgrowthanddifferentiationofthetrichomesareaffected.Trichomesaregenerallyabortedonleavesfromgl2-5mutants,whereastrichomesattheleafmarginsaremoredifferentiatedandtypicallybranchedwithtwo(orrarelythree)branches.Quanti?cationoftrichomeoutgrowthrevealedasig-ni?cantdifferenceinthenumberofbranchedtrichomesattheleafmarginsingl2-5mutantsversusgl2-5hdg11-1doublemu-tants(Figure2U;SupplementalTable2).Weobservedectopicbranchinginhdg11-1mutants,asreportedpreviously(Nakamuraetal.,2006).On?rstleaves,trichomeswith?vebranchesormorewereveryrareinwild-typeCol(;0.4%),whilehdg11-1mutantsexhibited;13%ofthetotaltrichomeswith?veormorebranches.Incontrast,ingl2-5hdg11-1doublemutants,therewasacom-pleteabsenceofbranchedtrichomes,whilegl2-5mutantsdis-playedbranchedtrichomesamong;11%ofthetotaltrichomeson?rstleaves.TrichomesalsoformontheepidermisofsepalsfromArabidopsis?owers.Analogousdeviationsintrichomemor-phogenesisweredetectedonsepalsofhdg11-1andgl2-5mutants(SupplementalFigure1).Wild-typesepalsexhibitedunbranchedtrichomes,whilehdg11-1sepalsshowedbothunbranchedandbranchedtrichomes.gl2-5mutantsexhibitedfewerandsmallertrichomes,whilegl2-5hdg11-1mutantstypicallylackedtrichomesonsepals.

UsingtheLandsbergerecta(Ler)ecotypeasthegeneticbackground,wecrossedgl2-1andhdg11-3mutantstoproducethecorrespondingdoublemutantplants(SupplementalFigure2).Thequantitativedifferencebetweentrichomebranchingon?rstleavesforgl2-1singlemutantsversusgl2-1hdg11-3doublemutantswasmorestrikingintheLergeneticbackgroundascomparedwiththeColecotype(SupplementalFigure2MandSupplementalTable2).Wealsoconstructedgl2-5hdg11-3doublemutantsandfoundtheirtrichomecell-typedifferentiationdefectsenhancedincomparisonwithgl2-5siblings(SupplementalFigure3).

gl2hdg11TrichomeDefectsResembleThoseofgl2myb23Mutants

Previouslyitwasshownthatthetrichomedifferentiationdefectofgl2mutantsisenhancedinadoublemutantfortheColallelesgl2-4AAandmyb23-2(Kiriketal.,2005).Itwasreportedthatmyb23mutantsexhibitreducedtrichomebranchingbutnoob-viousdefectintrichomepatterning(Kiriketal.,2005)anddisplaysmallertrichomes(Lietal.,2009).Wecon?rmedthepreviouslyreportedtrichomedefectsusingthemyb23-3mutantallele(Fig-ure3).Thegl2-5myb23-3doublemutantwasconstructed,andenhancedtrichomedefectswerecon?rmedforthisnewalleliccombinationintheColecotype.Similartogl2hdg11mutants,gl2-5myb23-3mutantsexhibitedtrichomesthatweredistinctlysmallerandlessexpandedthangl2-5trichomes,andthiswasespeciallyapparentattheleafmargins(http://wendang.chazidian.comingscanningelectronmicroscopytoviewtrichomemorphologiesindetail,weobservedtrichomedifferentiationdefectsingl2-5myb23-3mutantsthatwereanalogoustothoseseeningl2hdg11mutants(Figures2and3Eto3L).Quanti?cationofthetrichomedefectson?rstleavesindicatedthatwhile;11%ofthetotaltrichomesingl2-5mutantswerebranched,noneofthetri-chomesinthegl2-5myb23-3mutantswerebranched(Figure3M;SupplementalTable2).

Constructionofthegl2hdg11myb23TripleMutantRevealsGeneticInteractions

Giventhesimilaritybetweentheleaftrichomephenotypesofgl2hdg11andgl2myb23doublemutants,weinvestigatedgeneticinteractionsbetweenhdg11andmyb23inthegl2background.Ifthehdg11andmyb23mutationsaffectthesamebiologicalpathwayintheabsenceofGL2,thenthetriplemutantgl2hdg11myb23wouldbeexpectedtodisplaysimilartrichomedifferen-tiationdefectstoeitherdoublemutant.Indeed,wefoundthatthegl2-5hdg11-1myb23-3triplemutantdisplaysatrichomephenotyperesemblingthedoublemutants(Figures3Nto3P).Quanti?cationoftrichomeson?rstleavesindicatedthatthetriplemutanthasaphenotypethatisindistinguishablefromgl2-5myb23-3(Figure3Q),andexaminationof?rstleavesbyscanningelectronmicroscopyshowsstrikingsimilaritybetweenthedoubleandtriplemutants(SupplementalFigure4).

Duringconstructionofthetriplemutant,weobservedtrichomedefectsintheF1progenyfromreciprocalcrossesbetweengl2hdg11andgl2myb23,whosegenotypewashomozygousforgl2-5andheterozygousforbothhdg11-1andmyb23-3(SupplementalFigure5).Unlikegl2mutantsthatexhibittrichomesonleafmargins,thegl2/gl2hdg11/+myb23/+plantsexhibitedenhancedtrichomedefectssimilartothoseofgl2hdg11andgl2myb23doublemu-tants(SupplementalFigures5Ato5F).Unexpectedly,wealso

Figure2.(continued).

(P)and(T)gl2-5hdg11-1doublemutantshaveundifferentiatedtrichomesthroughouttheleaf.

(U)Quanti?cationoftrichomesandtrichomebranchingon?rstleaves.FortheColwildtypeandhdg11-1,greenbarsshowthenumberoftrichomeswiththreetofourbranchesandyellowbarsindicatethenumberoftrichomeswith?veormorebranches.Forgl2andgl2hdg11,graybarsshowthenumberofunbranchedsingle-spikedtrichomeswhiletheredbarindicatesbranchedtrichomes.PositiveerrorbarsindicateSDforn$20plants.Asterisksindicatesigni?cantdifferencesintrichomebranching(two-tailedttest,P<0.0001).

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2188ThePlantCell

Figure3.EnhancedTrichomeDifferentiationDefectsinthegl2-5myb23-3DoubleMutantandthegl2hdg11myb23TripleMutant.

(A)to(D)Rosettesfrom3-week-oldplants.

(A)and(B)Colwild-type(A)andmyb23-3(B)plantsdisplaytrichomescoveringleafsurfaces.

(C)and(D)gl2-5plantsdisplaytrichomesatleafmargins(arrows)(C),whilegl2-5myb23-3doublemutantsexhibitglabrousleaves(D).

(E)to(L)Scanningelectronmicrographsof?rstleavesrevealtrichomemorphologydetails.

(E)and(I)Colwild-typetrichomesdisplaythreetofourbranches.

(F)and(J)myb23-3trichomesexhibittwotothreebranches.Arrowsmarkabnormaltrichomeswithtwobranches.

(G)and(K)gl2-5trichomesdisplaydifferentiationdefects,althoughsometrichomesatleafedgesshowbranching(arrows).

(H)and(L)gl2-5myb23-3doublemutantsexhibitsmallundifferentiatedtrichomesthatlackbranching.

(M)Quanti?cationoftrichomeson?rstleaves.ForColandmyb23-3,greenbarsindicatethenumberoftrichomeswiththreetofourbranchesandthebluebarindicatesthenumberofabnormaltrichomeswithfewerthanthreebranches.Forgl2-5andgl2-5myb23-3,graybarsshowthenumberof