the integrationofdifferent developmentalcuesintheregulationofplantcelldifferentiation. epidermal differentiation. Ourfindingsshowthatfunctionalspecializationwithinatranscriptionfactorgenefamilycanfacil and cytokinins.TheserolesareconsistentwiththeirpatternofexpressiontheregionalinfluenceGAcytokinin equivalent infunction,playpartiallyredundantandessentialrolesinflorescencetrichomeinitiationitsregulation not playasignificantroleinthecytokininresponse.Collectively, GLABROUS1. Inaddition, Cytokinin-inducible is arobustsystemforstudyinghowepidermaldifferentiation is As trichomeproductionistightlyregulatedandeasilymonitored, it Perazza etal.,1998;Telfer et al.,1997;Traw andBergelson, 2003). et al.,2006;Greenboim-Wainberg etal.,2005;Kazama2004; aerial organs inmanyplantspecies (ChienandSussex,1996;Gan trichomes, whicharelarge defensive epidermalstructuresfoundon signals inplants.Inparticular, hormonelevels affect thedensityof regulators. 2006). Littleinformationisavailablesofaronthenatureofsuch downstream andcontroldistinctgenenetworks(Nemhauseret al., studies, theydeploymorespecializedregulatorsthatmayactfurther 1998). Asanotherstrategy, whichissuggestedbygeneexpression al., 2003;FuandHarberd,Penget1997;Silverstone , ethyleneandabscisicacid(Achardetal.,2006;Achard such aroleinthecontrolofplantgrowthbygibberellins(GA), the DELLA familyoftranscription factorshavebeenshowntoplay involves upstreamintegratorsofhormonesignalling;members signals.Asonestrategy, theyuseacentralizedsystemthat employ atleasttwostrategiesforthetreatmentofcompeting production ofappropriateresponses.Recentdataindicatethatplants these signalsbydownstreamregulatorsarethereforeessentialinthe pattern ofhormonesignalgenerationandtheproperintegration of growthanddevelopmentinplants.Boththespatiotemporal Phytohormones playdistinctbutoverlappingrolesintheregulation INTRODUCTION Accepted 18March 2007 2 1 KEY WORDS:Cytokinins,Epidermis,, , Trichomes, inflorescence organs, regulate trichomeinitiationin their relativeinfluenceonepidermalcellfateismodulated,weinvestigatedthemolecularmechanismsthroughwhichthey differentiation. However, bothcanstimulatetheinitiationofdefensiveepidermalstructurescalledtrichomes.To understandh act antagonisticallyinleafformationandmeristemmaintenanceGAcounteractsomeoftheeffects ofcytokininsonepiderma The effective integrationofhormonesignalsisessentialtonormalplantgrowthanddevelopment.Gibberellins(GA)cytokin Yinbo Gan regulation ofepidermalcellfate Arabidopsis Integration ofcytokininandgibberellinsignallingby (2007)doi:10.1242/dev.005017 Development 134,2073-2081 *Author forcorrespondence (e-mail:[email protected]) University ofSingapore, 117543,Singapore. Department ofBiologicalSciencesandTemasek LifeSciencesLaboratory, National CNAP, DepartmentofBiology (7),UniversityofYork, York YO105YW, UK. It isknownthatepidermaldifferentiation isregulatedbyhormone 1 , ChangLiu GIS2 ZFP8 plays aprominentroleinthecytokininresponse,whichitactsdownstreamofSPINDLY andupstreamof GIS2 transcription factorsGIS,ZFP8andGIS2inthe 2 and , HaoYu and Arabidopsis GIS2 ZFP8 , whichencodeC2H2transcriptionfactorsrelatedtoGLABROUSINFLORESCENCESTEMS(GIS). 2 and Pierre Broun mediate, like . Thecontrolbycytokininsoftrichomeproductionrequirestwogenesexpressedinlate GIS , theregulationoftrichomeinitiationbygibberellins.Bycontrast, 1, * GIS , distinct rolesin GA andcytokininresponsesduring development. production andthatthecorresponding geneshavespecializedtoplay GIS2 arefunctionallyinterchangeable activatorsoftrichome transcription factors,ZFP8and GIS2. We showthatGIS, ZFP8and requires thecollectiveaction ofGISandtwonovelrelated report thattheintegrationofcytokinin andgibberellinsignalling epidermal differentiation duringinflorescencedevelopment. We through whichgibberellinandcytokininsignallingmodulate al., 2005;Yanai etal.,2005). important inthemaintenanceofshootmeristem(Jasinski et These opposingeffects have beenfoundtobeparticularly expression thatantagoniseGA signalling(Brenneretal.,2005). Reciprocally, increasesincytokininlevelscausechangesgene (Ezura andHarberd,1995;Greenboim-Wainberg etal.,2005). the effect ofcytokinintreatments andblockcytokininsignalling signalling pathways.Forexample,gibberellinapplicationsinhibit contrasts withother, conflictingeffects ofthetwohormone The stimulationoftrichomeinitiationbybothGA andcytokinins regulates cytokininsignalling(Greenboim-Wainberg etal.,2005). counteracted bymutationsin plants causetrichomeproliferationonflowers.Thiseffect is initiation, ascytokininapplicationstoflowering (GL1) (Ganetal.,2006). regulator oftrichomeinitiationthatactsupstreamGLABROUS1 GLABROUS INFLORESCENCESTEMS(GIS),apositive organs, gibberellinsactinpartthroughthetranscriptionfactor et al.,2006;Perazza1998;Telfer etal.,1997).Ininflorescence trichome densityonleavesandstems(ChienSussex,1996;Gan and GA applicationshavebeen shown tocauseanincreasein Trichome initiationin controlled andtheroleplayedbyphytohormonesinthisprocess. ZFP8 In thepresentstudy, wehaveinvestigatedmolecularmechanisms Recent dataindicatethatcytokininscanalsostimulatetrichome and Arabidopsis GIS2 , whichencodeproteinsthatarelargely Arabidopsis SPINDLY requires gibberellinsignalling, RESEARCH ARTICLE , whichpositively Arabidopsis byGA GIS itate s on ow 2073 does ins l

DEVELOPMENT TTGTCCAAGCA-3 TGAGTAACCGCTCGATGAACTTC-3 gis basta andsulfadiazine(5.2mg/l)orpaclobutrazolerespectively. 3 primer: 5 or withagene-specific/T-DNA-specific primercombination (T-DNA AGGAGGATTGATGG-3 (50 N545674). Homozygous (SALK_045674) andalsoobtainedfromNASC(cataloguenumber 3 transforming a TGAACAG-3 5 CGTCTCT-3 analysis: following gene-specificprimersequenceswereusedforreal-time PCR at leasteightsoil-grownplantswereusedforRNA extractions.The USA) accordingtothemanufacturer’s protocol.Pooledtissuesamplesfrom Plant RNA wasextractedusingtheTRIzolreagent(Invitrogen,Carlsbad, RNA extraction,real-timeandsemi-quantitativeRT-PCR Molecular biology either 100 shoots hadreachedasizeof2-3cm.Theplantswerethensprayed with mutant andcontrolplantsweregrownonsoiluntilyounginflorescence the effect ofGA applicationsongene expression,aminimumofeight or mocksolutionsuntiltheplantswerereadyforanalysis.Formeasuring three tofourleaveshademerged andsprayedtwiceaweekwithGA3,BA respectively. Controlandmutantplantsweregrownonsoiluntilthefirst experiments thatinvolvedexogenousGA andcytokinintreatments GA3 (Sigma)andBA (6-Benzylamino-Purine,Sigma)wereusedinall Hormone treatments 3 specific primer(Spm1:5 CACCACCGACATACG-3 primers (5 mutants wereselectedusingBasta(40 was obtainedfromNASC(cataloguenumberN119489). Homozygous A transgenic linecarryingatransposonintheexonof Isolation ofnovelmutantsandconstructionmultiple trichome analysisforeachtreatment per plant.Unlessspecifiedotherwise,aminimumof20plantswasusedfor production onsepalswasevaluatedbycountingtrichomes10-15flowers was thenextrapolatedusingaverageleafareameasurements.Trichome analyses, theplantsweregrownincyclesof16hourslight(95 the Nottingham a controlifneeded.The experiments inthisstudyandtheLandsberg Plant materialandgrowth conditions MATERIALS ANDMETHODS 2074 extraction. harvested 4hours(GA)or2(BA)aftertreatmentforRNA 0.6 cm Trichome densityonthefirstandsecondcaulineleaveswasmeasuredina average internodelengthsforagivengenotype/ treatment combination. internode ofthemainstemwasextrapolatedfromthesefiguresusing cmfromthebaseofstem.Total trichomenumberonthefirst 1.5 main stemwasevaluatedbycountingtrichomeson2cmoflength, adaxial sideoftheleafblade,respectively. Trichome productiononthe was monitoredbycountingalltrichomesonthefirstinternodeor cminlength.Trichome initiationonbranchesandthethirdcaulineleaf 17 harvested fromplantswithamainstemthathadreachedapproximately cm ATGGGTTTAGGGTTCTTATCT-3 Ј Ј Ј double mutantswereselectedfromF2populationsbyselectionon -ACCGCCAACAAAACCACATT-3 ) (Tissier etal.,1999). ; , –2 GL1 ␮ gis2 second g/ml) andbyPCRusinggene-specificprimers(5 –2 RESEARCH ARTICLE , 5 area ofthemid-sectioneachleaf.Total leaftrichomeproduction Ј GIS and -GCGTGGACCGCTTGCTGCAACT-3 ␮ Ј Ј -CGACTCTCCACCGTCATTGTT-3 –1 M GA3,100 -AATCATCAAGGCAAATCCGCA-3 , 5 Ј gis gis2 , 21°C)and8hoursdark(18°C).Inflorescenceorgans were Ј ZFP8 ; Ј and 5 -TTCATGAACGTCGAATCCTTCTC-3 ARR5 Arabidopsis RNAi constructintothesemutants. Ј lines inwhich and 5 , 5 Ј gl1-1 Ј ecotype ColumbiaCol-0wasusedformost -CTGCGGATAAGTTGTCGGAGTT-3 -CTTATTTCAGTAAGAGTGTGGGGTTTTGG- Ј ␮ Ј Zfp8 -TTGCGTCCCGAGATGTTAGAT-3 zfp8 and 5 M BA oramocksolutionandtheshootswere Ј ), aloneorincombinationwithatransposon- Ј , -CCTTCGTTAAACCAAGCTCAGTATC- ga1-3 Stock Centre(NASC).Forallphenotypic was identifiedfromtheSAIL collection Ј mutants wereselectedonkanamycin -TCGGAGT TATCA CCGACGAGC-3 Ј and ϫ ; ␮ ZFP8 ZFP8 m) andbyPCRusinggene-specific Ј genotype combination. spy-3 Ј ; and 5 UBQ10 is silencedwereobtainedby , 5 erecta mutants wereobtainedfrom Ј -AAGCCGCCATTATT - Ј Ј Ј -CGCGTCGTTGATT - , 5 ). Ј ecotype wasgrownas and 5 gis gis2 GIS2 Ј -GGTTCGTAC CT- n 5 and Ј Ј Ј -TTTG AA- (SM_3_32778) and 5 -TTCTCGTA - and Ј -TGCTAC - Ј Ј Ј gis2 spy- -ACGA - ; and 5 GIS2 ␮ gis2 mol Ј Ј - ) , adjusted accordingto coding sequenceofthegene(seebelow).cDNA templateamountswerefirst was performedwiththesameprimersthatwereusedtoclonefull-length was performedover41cycles. CACCGATCAGCGAGTCT-3 CTCCCTGATCTCTCTCTTCC-3 primers wereused: primers containing constructs) orgenomicDNA (promoterfusionconstructs)using amplified frominflorescencecDNA (overexpressionandRNAi 35S:ZFP8 hygromycin-resistance gene)wasusedasthedestinationvectorfor from VIB(FlandersInteruniversityInstitute).pH2GW7(carryinga Gateway LRreaction(Invitrogen).Alldestinationvectorswereobtained before recombinationintotheappropriatedestinationvectorusing were firstinsertedintotheGatewayentryvectorpENTR-1A (Invitrogen), For theproductionofalloverexpressionandRNAiconstructs,sequences Cloning other genes(Ganetal.,2005). transcripts wereusedasaninternalcontrolfornormalizingexpressionofthe conditions wereaspreviouslydescribed(Ganetal.,2006). GATATTTTCTTGTTGATGATG-3 (BA) alsostimulate trichomeproductiononcauline leavesandstems al., 2005).We observedthatapplicationsof6-benzylaminopurine applications andmutationsinSPINDLY (Greenboim-Wainberg et Arabidopsis Cytokinins canpromotethe proliferationoftrichomeson inflorescence developmentin signalling onepidermal differentiation during Shifting influencesofgibberellin andcytokinin RESULTS the RNAiconstructs(seeabove). specific fragmentswereamplifiedusingthesameprimersasforgenerating (Gan etal.,2006).Forsynthesisofthe Non-radioactive insituhybridizationwasperformedaspreviouslydescribed In situhybridization 3 using primers5 and Bent,1998). Arabidopsis GV3101 byelectroporation. 5 CG GCCGCTTGCACAT TGGGTTTCATCA-3 5 GCCGCGAATGGA ACTAGAG GCG TAGA-3 TCGACAGCCATCCAGAGTCATA ACCA and and pB7GWIWG2(II)(Bastaresistance),respectively, for that wasfirstrestrictedwith restricted with sequences of promoter inpH2GW7afterrestrictionwith TAG AGAGAGATAAAAAGACTGGGCG-3 GAGCTCGGGGGAATGAGTCAAGAGTTC-3 promoter fragmentwasamplifiedusingtheprimers5 above. GCCGCGAATGGA ACTAGAGGCGTAGA-3 coding sequencesof for production ofoverexpressionconstructs.A modifiedstrategywastaken modified vectorfromthesameentrythatwasusedfor Ј Ј Ј -ATTCGTCGACT TCAACCTCCATTCAAACG-3 -ACTTGTCGACCAC CACATCTACGGCTTCCT-3 Semi-quantitative PCRanalysisof For theproductionof All binaryvectorconstructswereintroducedinto and 5 pGIS2 GIS2- Ј -TTAGTAGCGCTAGCGGTG GTTATGACTCTGGATGG-3 RNAi constructs;gene-specificfragmentswerefirstPCR- and constructs: a1.5kb genotypes wasperformedusingthefloraldipmethod(Clough GIS 35S:GIS2 flowers, aneffect thatisoffset bygibberellin Sac Ј -TCCTGAGAGCTCTCTCA AGTTGGCTTCGTGTG- , I, thenligatedintothevectorfragmentofpH2GW7 ZFP8 ZFP8 Sal UBQ10 GIS I and pGIS constructs; pK7GWIWG2(II)(carryingNPTII) , and overexpression: 5 ZFP8 Spe product intensitiesand promoter fusionconstructs Ј GIS2 ; Not GIS2 I, blunt-ended,thencutwith GIS2 Ј and Ј I restrictionsites.Thefollowing and 5 . Q-PCRprimerdesignandreaction GIS2 promoter fragmentwasamplified ZFP8 were thenrecombinedintothe overexpression: 5 GIS2 Ј gene expressionin -mediated transformationofall -TTGCATTGCGGCCGC TT Arabidopsis and - Ј Sac 3 were thenrecombinedas and substitutedforthe35S Ј Ј Ј Ј Ј Development 134(11) -TTCCATTGTCGA CT- . ; GIS2 ; and 5 I and ZFP8 Ј GIS2 and 5 Agrobacterium GIS2 RNA probes,gene- Ј Ј -RNAi construct: Spe -RNAi construct: -AAGATAG CG- and 5 Ј Ј and 5 -ATCTTGTC - Ј , I. Thecoding -TCAAC TG- amplification a 1.6kb ZFP8 Ј gis2 Ј -ATCTTG - -AAGCG - Sac Ј -ATTG - mutants UBQ10 -RNAi I. The strain GIS Ј - ,

DEVELOPMENT cauline leavesandbranchesin 100 wild-type plants(Fig.2A,B).AtGA concentrationsexceeding intrichomeproduction–aneffect alsoseenwith further increase, concentrations exceeding10 initiation onnormallyglabrous and GA-deficient of applyingawiderangeGA3concentrationstowild-typeplants therefore examinedindetailtheeffects onepidermaldifferentiation leaves (Ganetal.,2006;Perazza1998;Telfer etal.,1997).We contrast withtheirknownstimulationoftrichomeproductionon of-function SPY mutant (Werner etal.,2003).Similarly, wefoundthattheflowersofloss- encoding cytokininbreakdownenzymeCKX2isoverexpressed upper inflorescencestemsof that trichomeinitiationismarkedlydecreasedonflowersand limiting, butalsorequiredfortheproductionoftrichomes,wefound or paraclades(Fig.1A).Asevidencethatcytokininisnotonly and thatthiseffect increasesinintensitywithsuccessivebranches GIS cladehormonalcontrol trichomes we concludedthat cytokinin treatmentswassimilarin mutant. Astheoverallresponseofcaulineleavesandflowers to applications ontrichomeproductioninthe cytokinin response.To thisend,wetestedtheeffects ofcytokinin trichome initiation,weaskedwhetherGISalsomediatesthe interplay betweengibberellinsandcytokininsoninflorescence trichome initiation(Ganetal.,2006).Onthebasisofobserved inflorescence organs andmodulatestheregulationbyGA of The transcriptionfactorGISisrequiredfortrichomeproductionon necessary forthecytokininresponse development and,incontrasttoGIS,are trichome initiationlateininflorescence GIS homologuesZFP8andGIS2are required for inhibitory effect ofGA. inflorescence developmentisaccompaniedwithanincreasingly cytokinins, thegrowingrequirementforcytokininsignallingduring organs. ConsistentlywiththeantagonisticrolesofGA and the requirementforcytokininsislimitedtoupperinflorescence develops. GA signallingisrequired throughoutdevelopment,while play changingrolesinepidermaldifferentiation astheinflorescence (Table 1)(Greenboim-Wainberg etal.,2005). which GA signallingisincreasedand cytokininsignallinginhibited consistent withtheinflorescencetrichomephenotypeof ZFP8 the controloftrichomeproduction.We thereforeinvestigatedwhether relatedness toGISsuggestedthattheymightplayaredundantrole in (Gan etal.,2006;Tague and Goodman,1995).Moreover, their At5g06650, thatencodeproteinscloselysimilarinsequenceto GIS in which possible effects oflossfunction,wealsoproducedtransgenic plants downregulated orabolishedinthese lines(Fig.1B).To further assess that theexpressionofeither ofthesegeneswasstrongly ZFP8 start codonin this criterion.Onelinecarrieda transposon166bpupstreamofthe insertions ineitherofthetwogenesandidentifiedlinesmeeting response. We firstsearched publiccollectionsformutantscontaining process andinvestigatedtheirpossibleimplicationinthecytokinin The antagonisticeffect ofGA onflowertrichomeinitiationisin The aboveobservationsindicatedthatgibberellinsandcytokinins We previouslyidentified twogenes,At2g41940( ␮ , 139bpupstreamofthecodingregion. We foundusing RT-PCR M, wealsoobservedareductionintrichomeproductionon and At5g06650,whichwetermed ZFP8 GIS2 and ga1-3 GIS and theotheraT-DNA inthepromoterregion of GIS2 is notrequiredinthisprocess. mutants. GA applicationsrestoredtrichome spy-3 were silencedby RNAi,andselectedlines Arabidopsis ␮ are glabrous(Table 1). ga1-3 M causedadecrease,ratherthan gis ga1-3 (Fig. 2A).Theseresultswere and controlplants(Fig.1A), GIS2 plants inwhichthegene flowers, althoughGA gis , playaroleinthis loss-of-function ZFP8 spy-3 ) and , in (7) and was onlynoticeable onthethirdparaclade(Table 2).Thephenotype 3B) butonlyasmalldecreaseon branchesandcaulineleaves,which showed averystrongdecreasein trichomeproductiononflowers(Fig. PCR analysisof expression in function mutantsandRNAilines;leftpanel:RT-PCR analysisof standard error for20plants.( concentrations of6-benzylaminopurine.Values represent averagesand trichomes for12flowers.WT, wildtype. transcript levels.Sepaltrichomevaluesrepresent thetotal numberof (Fig. 3C;Table 2).Bycontrast,linesinwhich reduction intrichomedensityon uppercaulineleavesandbranches trichome phenotypeofT-DNA andRNAilines. see Table S1inthesupplementary material).We thenexaminedthe in whichtheirexpressionwassignificantlydownregulated(Fig. 1B; (termed cauline leavesofwild-type, first(bottompanel)andthird (middle panel) on sepals(toppanel)and Arabidopsis Fig. 1.Inductionoftrichomeinitiationbycytokininsinwild-type (3) , gisZFP8-R2 We observedthatZFP8lossoffunctionledtoa significant gis ZFP8-R2gis2 gis ZFP8-Rgis2 gis2 plants andGISclademutants. ZFP8 (4), and gis2 ZFP8-R1 xrsini idtp (1), expression inwild-type gis ZFP8-Rgis2 ) plantsthatwere treated withincreasing 8.Values are ratiostowildtypeofnormalized (8). gis B ) Expression of , zfp8 (5), , gis2 ZFP8-R2 gis2 mutants; rightpanel:real-time RESEARCH ARTICLE and ZFP8 ( gis gis2ZFP8-RNAi A GIS2 zfp8 ) Trichome initiation (6), and (2), was knockedout gis ZFP8-R1gis2 GIS2 gis ZFP8-R1 in loss-of- GIS2 2075 line 2

DEVELOPMENT nend.Values represent averagesandstandard error (inparentheses) for internode. Branch trichomevaluesare thetotalnumberoftrichomesforchosen Sepal trichomevaluesrepresent thenumberoftrichomes foratotaloftenflowers. overexpressors onlyproduced twocaulineleavesunderourgrowing conditions. showing asignificantdifference betweenthegenotypes are shown. ZFP8 on sepalsin trichome productionwasnearlyabolishedonthirdcaulineleavesand type plantsweresimilarinthefirstcaulineleaf,inductionof of controlplants.We foundthat,whiletheresponsesof increasing concentrationsofBA and comparedtheirresponsetothat by cytokininsoftrichomeinitiation,wetreated and supplementary material).We werealsoabletocomplementthe had asimilarimpactontrichomeinitiation(seeTable S1inthe by theinsertions,wefoundthatsilencingeitherofgenesRNAi material). Asaconfirmationthatthemutantphenotypeswerecaused production onvegetativeorgans (seeTable S2inthesupplementary 52.6(4.2) firstinternode particular, wedidnotobservesignificantdifferences intrichome of thedifferent lineswasotherwisesimilartothatofcontrols.In plants. 20 36.2(1.5) Sepals Trichome production ofcytokinin-deficientline spy-3 35S:CKX2 Wild type Genotype initiation ininflorescence organs Table 1.Influenceofcytokininsignallingontrichome 2076 obtained aminimum of30transformants witheachthe three codingregionsinthedifferent mutantbackgrounds and gis2 shown) (Ganetal.,2006). flowering andcausedtheappearance ofaerialrosettes(datanot plants, overexpressionofeitherthesegenesalsodelayed and This phenotypewasvisibleinmorethanhalfofthe30 trichomes werevisibleonflowers,inparticularcarpels(Fig. 3D). on inflorescencestemsandleaveswasincreasedectopic resembled GIS2 To assesstheleveloffunctionalredundancybetween are additive andtheireffects ontrichomeinitiation GIS induction oftrichomeinitiationbycytokininsoninflorescenceorgans. to GIS,GIS2and,alesserdegree,ZFP8arerequiredforthe inflorescence (Fig.1A).Theseobservationsindicatedthat,incontrast not significantlydifferent fromwildtypeinotherpartsofthe the cytokininresponseinthirdcaulineleaf,butwas second caulineleaves(datanotshown).ZFP8lossoffunctionaffected Figs S1,S2inthesupplementarymaterial). corresponding genesundereithernativeorconstitutivepromoters(see are similar, wefoundthat 2006). AsafirstindicationthattheactivitiesofGIS,ZFP8andGIS2 organs andectopictrichome formationonfloralorgans (Ganetal., GIS GIS2 As asecondstep,wetestedwhether thecodingregionsof To determinewhether , 35S:GIS2 leads tohighlevelsoftrichomeinitiationonallinflorescence mutants. We firstusedthe35Spromotertooverexpress the wefirstexaminedtheeffects ofoverexpressing , gis2 in awild-typebackground.We hadfoundthatoverexpressing ZFP8 and RESEARCH ARTICLE GIS2 GIS mutants byexpressingthecodingregionsof and gis2 transgenic linesthatweexamined.Asin overexpressors. Inparticular, trichomeproduction had theabilitytocomplementany ofthe (Fig. 1A)andsignificantlylesspronouncedon GIS2 ZFP8 encode functionallyequivalent 35S:ZFP8 88(.)23.4(3.9) 18.8 (1.1) . 00 75.6(6.1) 0.0 (0.0) and Average numberoftrichomes GIS2 35S:CKX2 and play aroleintheinduction 35S:GIS2 and zfp8 spindly3 Second branch, GIS plants closely gis2 and 35S:CKX2 ; onlyorgans , gis 35S:ZFP8 ZFP8 ZFP8 gis2 and wild- 35S:GIS , zfp8 GIS with zfp8 and and or , gibberellins inwild-type Fig. 2.Inflorescence trichomeinitiationinresponse to lines foreachofthecombinations. We firstconfirmed that experiment ofthe in transcriptionfactoractivities,werepeatedourcomplementation effects ofectopicexpression couldhavemaskedminordifferences shown onFig.3EandS2inthesupplementarymaterial).As the flowers tonormallevelsorhigherinallofthemutants(examples sufficient torestoretrichome productiononcaulineleaves,stemsor transgenic lines,overexpressionofeitherthethreegenes was overexpression constructs.We foundthat,inatleast40%ofthe represent averagesandstandard errors for20plants.WT, wildtype. mutants toGAapplicationsatincreasing concentrations.Values 12 flowers.( (*) Sepaltrichomevaluesrepresent thetotalnumberoftrichomesfor deficient mutants. then transformed the pGIS:GIS gis supplementary material).Complementation wasobservedinsix pGIS:GIS sequences bycomplementing the pGIS2 GIS ( pGIS had comparablelevelsofactivity tonativeregulatory ga1-3 ( (Fig. 3F)and A ) or B ) Trichome initiationoninflorescence organsofGA- ) Differential response ofGIScladeloss-of-function GIS2 and seven mutants treated withincreasing concentrationsofGA3. gis ( pGIS2 and gis pGIS2:GIS2 pGIS2:GIS2 gis2 gis2 mutant with Arabidopsis ), obtainingaminimumof40transgenic mutants usingpromoterfragmentsof respectively (seeFig.S1inthe , gis plants andGISclade pGIS:ZFP8 and ie,respectively. We lines, Development 134(11) gis2 and mutants with pGIS:GIS2 pGIS and

DEVELOPMENT (in parentheses) for20plants.RNAicontrol, transgenicplantsthatwere transformedwiththeinsert-lessRNAivector; ahconstruct.Consistentlywith theresultsofconstitutive each constructs, generatingaminimumoftentransgeniclineswith constructs andthe GIS cladehormonalcontrol trichomes idtp 85(.) 43(.) . 07 4. 26 1. 17 115.8 (1.2) 119.1 (1.8) Stem (firstinternode) 10.5(1.7) 12.7(2.1) RNAi; caul.leaf,cauline leaf;br., Thirdbr. branch. 42.5(2.6) 43.6(1.9) cm from thebase.Leaftrichomevalueswere extrapolatedusingaverageleafareas from countsobtainedwithina0.6cm of branchesormainstems.Inthecase mainstems,thevalueswere extrapolatedfromlengthsusingmeasure average internode Secondbr. Trichome production oninflorescence organs ofmutantsandRNAilinesthatare deficientinGIS,ZFP8and/orGIS2function.All 9.0(0.7) gis ZFP8-R2gis2 9.7(0.7) gis2 gis ZFP8-R1 Third caul.leaf gis2 ZFP8-R2 gis2 ZFP8-R1 54.3(2.6) gis ZFP8-R2 55.1(2.0) Secondcaul.leaf gis ZFP8-R1 gis gis2 gis2 58.5(0.9) Sepals 60.1(1.0) zfp8 gis RNAi control Wild type Genotype Table 2.InfluenceofGIS,ZFP8andGIS2oninflorescence trichomeinitiation Fig. 3.GIS,ZFP8andGIS2haveequivalentactivities. Trichome production on initiation onsepalsofwild-type(A)and ( mutants compared withwild-typestems(right).WT, wildtype. the mainstemsof background (secondbranches).( overexpression ontrichomeinitiationinwildtypeandthe 35S:ZFP8 D ) Production ofectopictrichomesoncarpelswild-type and 35S:GIS2 gis gis2 (left), 70(.) 79(.) . 04 00(.) . 00 75.5(2.9) 76.7(2.4) 0.0(0.0) 101.6(2.4) 0.0(0.0) 79.7(1.7) 0.0(0.0) 0.0(0.0) 5.6(1.2) 0.0(0.0) 2.6(0.4) 38.7(3.5) 3.0(0.5) 0.5(0.3) 3.2(0.4) 27.9(2.2) 7.7(0.6) 32.0(3.0) 27.0 (1.3) 23.9(2.0) 29.2 (1.5) 45.6(2.6) 41.3 (0.9) 32.6 (0.7) . 00 2. 22 34(.) . 00 00(.) 74.0(2.7) 75.2(2.1) 101.3(2.0) 98.4(2.1) 0.0(0.0) 0.0(0.0) 80.6(1.5) 0.0(0.0) 5.4(1.1) 0.0(0.0) 3.8(0.9) 110.1(1.8) 35.5(3.1) 0.0(0.0) 28.0(3.4) 3.4(0.4) 2.1(0.4) 6.1(0.9) 0.1(0.1) 3.8(0.5) 2.6(0.3) 41.7(2.1) 28.3(2.2) 25.7(1.8) 7.2(0.4) 31.2(2.0) 7.6(0.4) 28.8(2.6) 0.0 (0.0) 0.0 (0.0) 41.7(2.0) 2.7 (0.3) 2.3 (0.2) 59.5(1.6) 0.0 (0.0) 2.2 (0.3) plants. ( zfp8 mutant using pGIS:GIS gis (left) andwild-typecaulineleaves(right). E F ) Effects of ) Trichome branchinganddensityon gis2 Arabidopsis , pGIS2:GIS pGIS:ZFP8 gis ZFP8 and and GIS2 or flowers (B).( ( pGIS:GIS2 gis A pGIS2:ZFP8 35S:GIS , gis B ) Trichome mutant , C ) insertions (for the doubleandtriplemutantgenotypeseitherbycombiningT-DNA with apartiallyredundantroleforthethreegenes.We constructed in thecontroloftrichomeproduction. that GIS,ZFP8andGIS2havelargely equivalentactivities,atleast these cross-complementationexperimentsprovidedstrongevidence flowers (seeFig.S1inthesupplementarymaterial).Theresultsof constructs inthe branches (Fig.3E)(Ganetal.,2006), morphology of while phenotype thatwasindistinguishablefromwildtype.Interestingly, overexpression, atleastfourindependentlineshadatrichome i gis2 gis We alsofoundthatexpressionof trichome developmentalprogrammethan35S-drivenexpression. driven expressionweremoreappropriateforrestoringanormal trichomes (Fig.3F).Possibly, thetimingandlocalizationof gis pGIS:GIS2 R expression wassignificantlydownregulatedinselected supplementary material).We couldalsoverifythat seeTable S1inthe of theT-DNA insertionline(Fig.1B; mutant andlevelsof found tohaveasimilarphenotypethe We usedthisstrategybecauselinesinwhich inflorescence. and cytokinin-inducedtrichomeproductioninmostofthe ZFP8 material). Theseobservationsindicatedthat,collectively, growth anddevelopment(seeTable S2inthesupplementary double andtriplemutantswereotherwisesimilartowildtypein completely insensitivetocytokinintreatments(Fig.1A).The was alsoseeninthecytokininresponse,astriplemutant strong reductionsintrichomedensity(Table 2).Thisadditiveeffect produced inflorescenceorgans thatwereeitherglabrousorwith function werelargely additive.Inparticular, different mutants,thattheeffects ofGIS,ZFP8orGIS2 lossof 2). We found,bycomparingthetrichomephenotypesof and , The phenotypesofdoubleandtriplemutantswerealsoconsistent gis2 ZFP8-R i i2ZFP8 gis2 gis and/or 35S:ZFP8 mutants withanRNAiconstructinordertosilence GIS2 GIS gis2 plants (like and -RNAi lines(Fig.1),whichwetermed and gis2 and are absolutelyrequiredfortrichomeinitiation stem trichomes,whichproducesupernumerary i F8Rgis2, gis ZFP8-R ZFP8 ZFP8-R 35S:GIS2 GIS2 mutant restoredtrichomeproductionon 2 area. Values represent averagesandstandard error , lineinwhichthe pGIS:GIS gis expression thatwereequivalentto ) orbytransformingthe trichomeswere countedonthefirstinternode ments madeona1.5cm-longsegment 2 constructs couldnotrestorethe RESEARCH ARTICLE pGIS2:GIS respectively (Table 1;Figs1, plants) producednormal ZFP8 gis ZFP8-Rgis2 ZFP8 zfp8 GSZP gis pGIS:ZFP8 gene wassilencedby and loss-of-function is silencedwere pGIS2:ZFP8 gis gis ZFP8- , gis gis2 ZFP8 pGIS plants ZFP8 , 2077 GIS gis2 and and - . ,

DEVELOPMENT response of little effect on signalling andrequiredSPY (Fig.4).Bycontrast,thistreatmenthad transcriptional effect wasspecificallyduetovariationsincytokinin response wasabolishedinthe sensitivities of These observationsindicatedthat,inlinewiththecontrasting significant elevationin significant increaseincytokininsignalling.Theyalsocauseda response gene after BA treatment.Thestrongincrease intheexpressionofprimary analysis, developinginflorescenceorgans wereharvested2hours organs inresponsetoincreasescytokininsignalling.Forthis time PCRandthevaluesrepresent ratiosofnormalizedlevelstocontrols. grey bars:hormonetreatments. Transcript levelswere measured byreal- of 6-BA applications(100 primary response regulator transcriptional activationof trichome initiationbycytokininsproceedsinpartthrough the cytokinin signalling.Theyalsosuggestedthattheinduction of ZFP8 expression of action andultimatelytrichomeinitiation,wemeasuredthe To startdefininghowcytokininsmightmodulateZFP8andGIS2 or GIS2 induction byGAof Middle:expression of type plants. 2hoursafter6-benzylaminopurineapplications(100 plants Fig. 4.Expression of 2078 cytokinin treatments. Our resultsindicatedthat their expression isGA-inducible ZFP8 We foundthatboth treatments onthe in gibberellinresponses,wefirstexaminedtheeffect ofGA to cytokinins.To determine whetherthegenesalsoplaydistinctroles redundant transcriptionfactorsthataredivergent intheirresponse with thetrichomephenotypeof responsive toGA applicationsthanwild-typeplants.Consistently ZFP8-R gis2 leaves of cauline leaves(Fig.2B).Similarly, theflowersandthirdcauline or noincreaseintrichomeproduction afterGA treatmentonthethird ga1-3 ZFP8 and and and RESEARCH ARTICLE mutants 4hoursaftertreatment. Blackbars:mocktreatments; gis2 GIS2 GL1 GIS2 gis plants werecompletely unresponsivetoGA asthey ZFP8 ARR5 ZFP8 mutants werelesssensitiveto GA.Strikingly, gis mutant plants,noeffect on are alsodifferentially responsive toincreasesin zfp8 ZFP8 are cytokinin-inducible,butnot , participate intheGAresponse and zfp8 expression and,consistentlywiththecytokinin ZFP8, GIS2 , , ␮ indicated thathormoneapplicationsledtoa Left: expression of , gis2 GIS2 M) inthe zfp8 GIS2 ARR5 and GIS2 and GIS GIS2 and and and gis2 in inflorescence organsof spy-3 gis ZFP8-Rgis2 zfp8 , GIS and spy-3 GL1 and ZFP8 gis2 GL1 and , to cytokininapplications, GL1 mutant plants, GL1 expression intheinflorescence mutant, anindicationthatthe mutant background. Right: GL1 GL1 mutants werelocallyless in wild-typeinflorescence and GIS and in response toGAand . , transcript levels.This ZFP8 GIS2 ARR5 GIS mutant phenotypes. , GIS2 transcript levels. in response to encode partially zfp8 , ␮ GL1 Arabidopsis showed less M) towild- and GIS GIS gis , mutants, overexpressingin and genes genetic interactionsbetween downstream pathways.To testthishypothesis,weinvestigated might influencetrichomeinitiationbyactingonthesame overexpressed transcriptional regulationof both on sepalsand SPY cytokinins, wefirstexaminedgeneticinteractionsbetween To furtherinvestigatethegeneticcontroloftrichomeinitiationby upstream ofGL1 GIS2 andZFP8actdownstream ofSPYand are similarlyinduciblebyGA. GIS were tocytokinins(Fig.2B).Theaboveexperimentestablishedthat applications on cauline leavesbutthattheyproducedglabrousflowers(Table 3). trichome productionwasmostsimilarto We alsoproduced overexpressed in (Fig. 5B),aresultthatwasalsoobtainedwhen trichome initiationwasrestoredonflowersof of GA applicationsto by gibberellinsin These observationssuggestedthatthecontroloftrichomeinitiation normally inhibitory(seeFig.S4inthesupplementarymaterial). they normallystimulateinitiation,oronflowers,whereare trichome productiononlowercaulineleavesandbranches,where overexpressors. We foundthatGA applicationshadnoeffect on examined theeffect ofGA applicationson redundant genescanbesufficient forsaturatingtheresponse,we inflorescence organs toGA. they differ from GL1 inflorescence by insituhybridization. transcripts indifferent tissuesusingquantitativeRT-PCR andinthe signalling, weexaminedthe distribution of are consistentwiththeirrolesin trichomeinitiationandhormone To determinewhethertheexpressionpatternofgenes intheplant inflorescence organs ZFP8 downstream ofSPY butupstreamofthetrichomeinitiationcomplex. regulator of in increased ininflorescenceorgans of plants (datanotshown).We alsofoundthat leaves in Similarly, flowers andincreasedtrichomeproductiononbranches(Fig. 5D). functionally equivalentto treatments affect overexpression didnotinducetrichomeproductionon gis2 The similaractivitiesofGISandGIS2theeffect ofcytokinin To determinewhetherincreasingtranscriptlevelsofthese The resultsoftheseexperimentssuggestedthatGIS2isapositive On thebasistheseresults,weexaminedwhetherexogenousGA ZFP8 , . AsGIS2lossoffunctionstronglyinhibitstrichomeinitiation gl3 (Fig. 4)(Ganetal.,2006).Thisresultindicatedthat,although 5:I2gl1 35S:GIS2 ZFP8 GL1 and mutants, inparticularlatedevelopment(Fig.5E). (Fig. 5C).Conversely, aswehadseeninthecaseof zfp8 and R and and GIS2 overexpression increasedtrichomedensityoncauline GL1 GIS2 (see Fig.S3inthesupplementarymaterial),but GLABRA3 GIS2 GIS2 GIS spy-3 GL1 spy-3 ZFP8 expression andthatbothZFP8GIS2act gis2 spy-3 , aswehadpreviouslyobservedwith Arabidopsis are differentially expressed in and ga1-3 in theirresponsetocytokinins, expression suggestedthatthetworegulators are collectivelyrequiredfortheresponseof in the flowers areglabrous(Fig.5A),wefirst (see Fig.S3inthesupplementarymaterial). 5:I2gl3 35S:GIS2 and mutants stronglyinducedtheexpression ( GL3 GL3 GIS2 GIS GIS2 double mutantsandfoundthattheir spy-3 gis2 ) (Payneetal.,2000).We foundthat , inflorescence organs requiresthe , restoredtrichomeinitiationon ZFP8 and trichomeinitiationregulatory gene expression.We foundthat the maize 35S:GIS2 background. We foundthat lines wereglabrous,like ZFP8 and/or Development 134(11) gis2 5:I2spy-3 35S:GIS2 and GL1 plants butdecreased GIS2 R ZFP8 on branchesand GIS2 gene, whichis expression was ZFP8 . were found ZFP8 and gl1 35S:GIS2 and GIS2 GIS or plants ZFP8 GIS2 GIS2 was and and gl3 gl1 gis

DEVELOPMENT inflorescence; W.I., wholedevelopinginflorescence; WT, wildtype. represent ratiosofnormalizedlevelstocontrols. U.I.,upperdeveloping (right). Transcript levelswere measured byreal-time PCRandthevalues organs oftwo (left) andthird branches(right).( ( flowers of initiation onstemsof initiation regulators in was mostexpressedincaulineleaves(Fig.6B,D,E)and different levelsindifferent inflorescenceorgans. Specifically, to beexpressedatearlystagesofinflorescencedevelopmentbut GIS cladehormonalcontrol trichomes Fig. 5.Geneticinteractionsbetween leaf phenotypeof pronounced inlatercaulineleavesandflowers,withthe were consistentwiththephenotypeof in successivecaulineleaves(Fig.6E).Thesepatternsofexpression 6A,C,E). We alsofound that primary andsecondarymeristemsindevelopingflowers(Fig. expression of largely equivalent,theregulatory mechanismscontrollingthe relative influencesofZFP8andGIS2onthisprocess. of hormoneresponseduringinflorescencedevelopmentand the trichome initiation in integrating gibberellinandcytokinin signallinginthecontrolof In thisstudywehaveinvestigated themolecularmechanisms DISCUSSION development havediverged (Ganetal.,2006). D ) Effect of In summary, wefoundthat,whiletheactivityofproteinsare spy-3 R overexpression ontrichomeinitiation 35S:GIS2 (A) and GIS zfp8 gl1 , Arabidopsis . Theywerealsoconsistentwiththepatterns overexpressor lines(left)andinthe 35S:GIS2 spy-3 , Arabidopsis ZFP8 gl3 , GIS2 35S:GIS2 gl1 E ) and GL1 was expressedatincreasinglevels . We found thattheinfluenceof . ( expression ininflorescence GIS2 GIS2 A mutants (B).( , B and ) Trichome initiationon , SPY during inflorescence gis2 35S:GIS2 gl3 and trichome , whichismost C ) Trichome gis2 GIS2 gis2 flowers plants. mutant ZFP8 in the and standard error (inparentheses) for20plants.caul.leaf,caulineleaf;br.: branch mutants. SeeTable 2legendfordetailonmeasurements. Values represent averages The of activator of predominant roleinthecytokininresponse,encodesatranscriptional cytokinin signalling.Cytokinin-inducible inflorescence development,onlyZFP8andGIS2modulate regulation oftrichomeinitiationbygibberellinsthroughout and GIS2.Whileallthreetranscriptionfactorsarerequiredinthe combined actionsofGISandtworelatedtranscriptionfactors,ZFP8 the twohormoneclassesontrichomeproductionismodulatedby (first internode). Secondbr. Thirdcaul.leaf Trichome production ofdifferent inflorescence organsin Secondcaul.leaf gis2 spy-3 Sepals spy-3 gis2 Genotype Table 3.Geneticinteractionsbetween Functional specializationof hormone production. transcription factorsnotonlyupstreambutalsodownstream of GIS cladethereforehighlightsthecentralroleplayed by intheembryo(Gazzarrinietal.,2004).Theroleof the which hasbeenproposedtoregulategibberellinandabscisic acid et al.,2005;Yanai etal.,2005).ThesameholdstrueofFUSCA 3, ofthehormonebiosynthesispathways(Jasinski than downstream, cytokinin signallinginthemeristem,aslatteractupstream,rather KNOX-type transcriptionfactorsthatregulategibberellin and hormone responses,theGIScladeproteinsalsodiffer fromthe hormone signals(SunandGubler, 2004).Inmainlymodulating in aparticulartissuethanontheirspecificresponsivenessto different membersoftheclademaydependmoreontheirabundance andtherelativeinfluenceof 2003; Silverstoneetal.,2001), are post-transcriptionallyregulated(Dilletal.,2004;Sasaki a furthercontrastwithGIS,ZFP8andGIS2,theDELLA proteins (Achard etal.,2006;Achard2003;FuandHarberd,2003).In and differentiation inresponsetoavarietyofhormonesignals transcription factors,whichplayageneralroleinmodulatinggrowth respect, theGIScladediffers fromregulatorssuchastheDELLA mutant doesnotshowageneralhormoneresponsephenotype.Inthis epidermal responsestocytokininsandgibberellins,asthetriple and GIS2appeartobemainlyinthemodulationofspecific epidermal differentiation in roles inregulatingtheinfluenceofdifferent hormonesignalson Our findingsshowthatrelatedtranscriptionfactorsplaydifferent transcription factors Integration ofhormonesignallingby cytokinins onthisprocess. roles intrichomeinitiationandtheinfluenceofgibberellins inflorescence development,inawaythatisconsistentwiththeir genes havespecializedandaredifferentially regulatedduring For example,MYB transcriptionfactorWEREWOLF 1(WER)is similar differentiation programsatdifferent stagesindevelopment. appears tobeanimportantmechanism throughwhichplantsregulate specialization ofgenesencoding paralogoustranscriptionfactors in theirroleduringinflorescence development.Thefunctional proteins buthavediverged intheirresponsetophytohormones and GIS GIS , ZFP8 , ZFP8 GL1 . 00 4. 32 1. 05 37.4(3.7) 75.6(6.1) 35.9(2.5) 10.1(0.5) 14.4(1.1) 9.0(0.5) 46.8(3.2) 57.5(2.4) 0.0 (0.0) 44.9(2.4) 0.0 (0.0) 6.5 (0.5) and and that actsdownstreamofSPINDLY. Theproducts GIS2 GIS2 r agl qiaeti ucin butthe are largely equivalentinfunction, genes encodefunctionallyequivalent Arabidopsis RESEARCH ARTICLE GIS GIS2 . TherolesofGIS,ZFP8 , ZFP8 GIS2 gis2 and , spy-3 and , whichplaysa SPY and GIS2 gis2 spy-3 2079

DEVELOPMENT mere reflection of alocalbalanceingibberellin and cytokininlevels diverged, theirinfluenceontrichomeinitiationcouldbeseen asa developmental patternsof similarly toseeminglyconflicting hormonesignals(Fig.7).Asthe asitallowstheplanttorespond This mechanismisfairlyunique, modulating hormoneresponses duringinflorescencedevelopment. control maybeanadaptive mechanism thatisimportantfor domains duringleafdevelopment(Kiriketal.,2005). but 2001). Similarly, GL1andMYB23arefunctionallyinterchangeable, regulates trichomedevelopmentintheshoot(LeeandSchiefelbein, whereitregulatesepidermalhairdevelopment,whereas , however, functionally equivalenttoGL1; plants. and Arabidopsis Fig. 6.Distributionof 2080 rosette leaf;Sil,silique;St,mainstem(base). branch; CL,caulineleaf;DI,developingInflorescence; Fl,flower;RL, the expression ofthe (bottom) expression invariousplanttissues.Values are normalizedto using senseRNA.( in developingcaulineleaves(B).(C,D)Sectionsthatwere hybridized andtheepidermis(arrows, A). In thecaseofGISclade,divergence oftheirtranscriptional MYB23 ZFP8 GIS2 RESEARCH ARTICLE (B,D) probes todevelopinginflorescence organsofwild-type is strongly expressed ininflorescence meristems, floral and plant tissues. GL1 E ) Real-timePCRanalysisof UBQ10 have distinctalthoughoverlappingexpression ZFP8 GIS ( gene. 1 A-D and , ZFP8 ) Insituhybridizationof GIS2 st B, firstbranch;2 and transcripts indifferent ZFP8 GIS2 WER ZFP8 is mosthighlyexpressed expression havealso is expressedinthe (top) and nd B, second GIS2 GIS2 (A,C) GL1 trichome initiationbycytokininsisnotsolelydependentonGL1. signalling isonlysuggested,asthepossibilitythatregulation of The antagonisticrole ofSPYintheregulation byZFP8andGIS2ofGA lines: theinteractionbetweenGISandSPYwaspreviously examined. inflorescence organsinresponse toGAandcytokinins. Fig. 7.ModelofGIS,ZFP8andGIS2actionin precise mappingofhormoneinfluencewithintheplant. approaches thatintegratetheanalysisofgenenetworkswith the complete. Progressinthisfieldwillalsobenefitfromsystembiology our knowledgeoftranscriptionfactorfunctionbecomesmore and hormonalsignalsareintegratedinplantsislikelytoemerge as developmental cues.A betterunderstandingofhowdevelopmental of cellulardifferentiation and inresponsetovarioushormonaland specialization withinaplanttranscriptionfactorfamilyinthecontrol change thereforeawaitsfurtherinvestigation. and GIS2.ElucidatinghowincreasesinGISactivityinfluencephase exits, isnotjustmaskedbygeneticredundancybetweenGIS,ZFP8 gis2 and GIS2playasignificantroleinphasechange.As nature ofthe transcription factorshavesimilaractivities.Becauseofthelocalized although thesephenotypesmayjustreflectthefactthatthree indicate thatZFP8andGIS2alsoplayaroleinphasechange, heterochronic phenotypesof general roleintheregulationofphasechange(Ganetal.,2006).The suggested thatGISinfluencestrichomeinitiationaspartofamore The phenotypeof ZFP8, GIS2andtheregulation ofphasechange signals andthatthesearenotstrictlyinterdependent. diverged intheirresponsestobothdevelopmentalandhormonal different. Rather, ourdatasuggestthat inducibility ofthegenesinresponsetocytokininsareclearly overlapping locations(Ganetal.,2006)andboththeroles as transcriptsforthedifferent genesaredistributedacrosslargely within inflorescenceorgans. Thisscenarioseemsunlikely, however, http://dev.biologists.org/cgi/content/full/134/11/2073/DC1 Supplementary material forthisarticleisavailableat Supplementary material Foundation. assistance. Thisworkwasfundedby a grantfrom theGarfieldWeston overexpressing lines andKanchanThackerNickWelsby fortheirtechnical We wouldliketothankProf. OttolineLeyserforproviding seedsof In conclusion,ourresultsillustratetheimportanceoffunctional mutants gothroughnormalphasetransition,sucharole,ifit Gibberellins zfp8 and GIS gis2 overexpressors andloss-of-functionmutants GIS Trichome Initiation GL1 phenotypes, itseemsunlikelythatZFP8 35S:ZFP8 SPY ZFP8 GIS2 ? GIS and Development 134(11) , 35S:GIS2 ZFP8 Arabidopsis Cytokinins and i ZFP8-R gis lines could GIS2 Broken 35S:CKX2 have

DEVELOPMENT Greenboim-Wainberg, Y., Maymon,I.,Borochov, R.,Alvarez, J.,Olszewski, Achard, P., Vriezen, W. H.,Van DerStraeten,D.andHarberd, N.P. References GIS cladehormonalcontrol trichomes Jasinski, S.,Piazza,P., Craft,J.,Hay, A.,Woolley, L.,Rieu,I.,Phillips,A., Gazzarrini, S.,Tsuchiya, Y., Lumba,S.,Okamoto,M.andMcCourt,P. Gan, Y., Kumimoto,R.,Chang,L.,Ratcliffe, O.,Hao,Y. andBroun, P. Clough, S.J.andBent,A.F. Chien, J.C.andSussex,I.M. Brenner, W. G.,Romanov, G.A.,Kollmer, I.,Burkle,L.andSchmulling,T. Achard, P., Cheng,H.,DeGrauwe,L.,Decat,J.,Schoutteten,Moritz,T., Kirik, V., Lee,M.M.,Wester, K.,Herrmann,U.,Zheng,Z.,Oppenheimer, D., Kazama, H.,Dan,Imaseki,H.andWasteneys, G.O. Fu, X.andHarberd, N.P. Gan, Y., Filleur, S.,Rahman,A.,Gotensparre, S.andForde, B.G. Ezura, H.andHarberd, N.P. Dill, A.,Thomas,S.G.,Hu,J.,Steber, C.M.andSun,T. P. cytokinin signaling. cytokinin: theArabidopsisGAresponse inhibitorSPINDLY playsapositiverole in N., Ori,Eshed,Y. andWeiss, D. epidermal differentiation andshootmaturationinArabidopsis. GLABROUS INFLORESCENCESTEMSmodulatestheregulation bygibberellins of growth repressor function. Ethylene regulates arabidopsisdevelopmentviathemodulationofDELLA coordinate regulation ofcytokininandgibberellin activities. Hedden, P. andTsiantis, M. through thehormonesgibberellin andabscisicacid. The transcriptionfactorFUSCA3controls developmentaltiminginArabidopsis 1383-1395. Arabidopsis thaliana. Nutritional regulation ofANR1andotherroot-expressed MADS-boxgenesin -induced degradation. Arabidopsis F-boxprotein SLEEPY1targetsgibberellin signalingrepressors for Agrobacterium-mediated transformationofArabidopsisthaliana. photoperiod inArabidopsisthaliana(L.)Heynh. formation ontheadaxialandabaxialleafsurfacesbygibberellins and cascades. sensitive processes andsuggestcytokininactionthrough transcriptional thaliana identifiedbygenome-wideexpression profiling reveal novelcytokinin- (2005). Immediate-earlyanddelayedcytokininresponse genesofArabidopsis 94. responses toenvironmentally activatedphytohormonalsignals. Van DerStraeten,D.,Peng,J.andHarberd, N.P. Schiefelbein, J.andHulskamp,M. hypocotyl epidermalcells. exposure toethylenestimulatescelldivisionandaltersthefatepolarityof 1565. modulating gibberellin response. vitro shootregeneration inArabidopsisthaliana(L.)Heynh. 735-743. Plant J. 44 Plant , 314-333. Planta (2003). Auxinpromotes Arabidopsisroot growth by Plant Physiol. (1998). Floraldip:asimplifiedmethodfor (1995). Endogenousgibberellin levelsinfluencein- 222 (1996). Differential regulation oftrichome 17 (2005). KNOXactioninArabidopsisismediatedby , 1-13. , 92-102. Plant Cell Nature Plant Cell (2005). Functionaldiversificationof (2005). Cross talkbetweengibberellin and 134 421 16 , 1614-1623. , 740-743. 15 , 1392-1405. , 2816-2825. Plant Physiol. Dev. Cell (2006). Integrationofplant (2004). Transient (2004). The Planta Curr. Biol. 7 111 , 373-385. Plant Cell Science , 1321-1328. Plant J. 197 (2005). (2003). 15 , 301-305. 311 (2006). (2004). , 1560- 18 16 , 91- , , Silverstone, A.L.,Ciampaglio,C.N.andSun,T. Sasaki, A.,Itoh,H.,Gomi,K.,Ueguchi-Tanaka, M.,Ishiyama,K.,Kobayashi, Silverstone, A.L.,Jung,H.S.,Dill,A.,Kawaide,H.,Kamiya,Y. andSun,T. P. Perazza, D.,Vachon, G.andHerzog,M. Peng, J.,Carol, P., Richards, D.E.,King,K.Cowling,R.J.,Murphy, G.P. Tissier, A.F., Marillonnet,S.,Klimyuk,V., Patel,K.,Torres, M.A.,Murphy, G. Telfer, A.,Bollman,K.M.andPoethig, R. S. Tague, B.W. andGoodman,H.M. Sun, T. P. andGubler, F. Payne, C.T., Zhang,F. andLloyd,A.M. Nemhauser, J.L.,Hong,F. andChory, J. Lee, M.andSchiefelbein,J. Werner, T., Motyka,V., Laucou,V., Smets,R.,Van Onckelen,H.and Traw, M.B.andBergelson,J. Yanai, O.,Shani,E.,Dolezal,K.,Tarkowski, P., Sablowski,R.,Sandberg,G., transduction pathway. gene encodesatranscriptionalregulator repressing thegibberellin signal 299 of phosphorylatedrepressor forgibberellin signalinginanF-boxmutant. M., Jeong,D.H.,An,G.,Kitano,Ashikari,M.etal. 383. formation byUp-regulating GLABROUS1inarabidopsis. protein inArabidopsis. (2001). Repressing arepressor: gibberellin-induced rapidreduction oftheRGA Arabidopsis zincfingerprotein cDNAs. plants. 3205. pathway thatnegativelyregulates gibberellin responses. and Harberd, N.P. GL1 andTTG1. that regulates trichomedevelopmentinarabidopsisthrough interactionwith responses. regulate similarprocesses through largelynonoverlappingtranscriptional 11 transposon insertionsinArabidopsis:atoolforfunctionalgenomics. and Jones,J.D. 645-654. regulation oftrichomedistributioninArabidopsisthaliana. 1546. encode functionallyequivalentproteins inArabidopsis. 132 MYB23 andGL1genesintrichomemorphogenesisinitiation. the regulation ofshootandroot meristemactivity. multiple developmentalalterationsindicatingoppositefunctionsofcytokininsin Schmulling, T. 133 acid, andgibberellin oninductionoftrichomesinArabidopsis. biosynthesis. Samach, A.andOri,N. , 1841-1852. , 1896-1898. , 1477-1485. , 1367-1375. Annu. Rev. PlantBiol. Cell Curr. Biol. 126 (2003). Cytokinin-deficienttransgenicArabidopsisplantsshow Genetics (1999). Multipleindependentdefectivesuppressor-mutator , 467-475. (1997). TheArabidopsisGAIgenedefinesasignaling Plant Cell (2004). Molecularmechanismofgibberellin signalingin Plant Cell 15 156 (2005). ArabidopsisKNOXIproteins activatecytokinin , 1566-1571. , 1349-1362. 55 (2003). Interactiveeffects ofjasmonicacid,salicylic (2001). DevelopmentallydistinctMYBgenes , 197-223. 10 13 (1995). Characterizationofafamily , 155-169. , 1555-1566. Plant Mol.Biol. (2000). GL3encodesabHLHprotein (2006). Different planthormones (1998). Gibberellins promote trichome RESEARCH ARTICLE (1997). Phasechangeandthe (1998). TheArabidopsisRGA Plant Cell 28 Development Plant Physiol. Genes Dev. , 267-279. (2003). Accumulation Development 15 Plant Physiol. , 2532-2550. Development 11 Plant Cell 128 117 , 3194- 124 Science , 1539- , 375- 2081 ,

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