CorrespondencedbergmannstanfordeduDepartmentofBiologyStanfordUniversityStanfordCA943055020USAHowardHughesMedicalInstituteStanfordUSAFulllistofauthorinformationisavailableattheendofthearticl ID: 181002
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REVIEWOpenAccess Mechanismsofstomataldevelopment:an evolutionaryview AnneVatén 1,2 andDominiqueCBergmann 1,3* Abstract Plantdevelopmenthasasignificantpostembryonicphasethatisguidedheavilybyinteractionsbetweentheplant andtheoutsideenvironment.Thisinterplayisparticularlyevidentinthedevelopment,patternandfunctionof stomata,epidermalporesontheaerialsurfacesoflandplants.Stomatahavebeenfoundinfossilsdatingfrom morethan400millionyearsago.Strikingly,themorphologyoftheindividualstomatalcomplexislargely unchanged,butthesizes,numbersandarrangementsofstomataandtheirsurroundingcellshavediversified tremendously.Inmanyplants,stomataarisefromspecializedandtransientstem-celllikecompartmentsontheleaf. Studiesinthefloweringplant Arabidopsisthaliana haveestablishedabasicmolecularframeworkfortheacquisition ofcellfateandgenerationofcellpolarityinthesecompartments,aswellasdescribingsomeofthekeysignalsand receptorsrequiredtoproducestomatainorganizedpatternsandinenvironmentallyoptimizednumbers.Herewe presentparallelanalysesofstomataldevelopmentalpathwaysatmorphologicalandmolecularlevelsanddescribe theinnovationsmadebyparticularcladesofplants. Keywords: receptorsignaling,Cellpolarity,Asymmetriccelldivision Review Introductiontostomataandstomatalpattern Plantsconqueredlandmorethan400millionyears ago.Inthefossilrecord,theappearanceofthesepion- eerspeciesiscontemporaneouswiththeappearance ofstructuresontheirsurfacescalledstomata.Each stoma(plural,stomata)con sistsofpairedepidermal guardcells,aporebetweenthemandanairspacein thephotosyntheticmesophylltissuesubtendingit.The functionofstomataistoregulategasexchange betweentheplantanditssurroundings.Onshort timescales(minutestohours),theopeningandclosing ofthestomatalporebyturgor-drivenchangesinguard cellshapeisakeyregulatorystepinmaintainingwater andcarbondioxidebalance.Workfrommanylabora- torieshasdefinedtheintracellularsignaltransduction cascadesthatmediatechangesinporesizeinresponse tohormoneandenvironmentalsignals[1]. Thecurrentviewisthatstomataaroseonlyonceduring evolution[2].Inearlylandplants,stomataldensitywas low[3].Duringinterveningmillennia,thestomataldensity inresponsetoreducedaerialCO 2 concentration[4].The stomatalcomplexhasbeenfine-tunedbyseveralinnova- tionsincludingrecruitmentofneighboringsubsidiarycells tofacilitatestomatalopening/closing,relocationofstoma- talcomplexesunderprotectiveepidermalcellsandincorp- orationofmultipleasymmetriccelldivisionsinprecursors tocreateavarietyofstomataldistributions.Despitethe variation,thebasiccorestructurehasremainedun- changed:twoguardcellsflankthestomalpore.Innearly allspecies,twostomataareseparatedatleastbyonenon- stomatalcell,anarrangementthoughttobeessentialfor organsincludingleaves,stems,flowers,fruitsandseeds andtheydevelopgraduallyduringorgangrowthsuchthat youngorganshavefewertotalstomatathanmature organs,thoughSDoftendecreasesastheneighboringepi- dermalcellsexpandduringmaturation.Thefrequency andpositioningofstomataareorganandspecies-specific characters,butarealsoaffectedbyenvironmentalfactors. *Correspondence: dbergmann@stanford.edu 1 DepartmentofBiology,StanfordUniversity,Stanford,CA94305-5020,USA 3 HowardHughesMedicalInstitute,Stanford,USA Fulllistofauthorinformationisavailableattheendofthearticle ©2012VaténandBergmann;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited. VaténandBergmann EvoDevo 2012, 3 :11 http://www.evodevojournal.com/content/3/1/11 Paleobotanicalanalysesutilizingthefossilsoftheearlylandplantsandtheircurrentlylivingdescendantshavebeencombinedwithphylogeneticanalysestoaddresstheoriginsofstomata(Figures1and2A).Liverworts,mossesandhornwortscomprisethebryophytes,abasallandplantgroup.Liverwortsdonothavestomata;gasexchangeisfacilitatedbyepidermalairpores,structureswhosedevelopmentandmorphologydifferfromstomata.Stomataarefoundinmossesandhornworts,makingitlikelythatliverwortsdivergedfromotherbryophytesbe-foretheoriginofstomata.Intriguingly,inextantbryo-phytes,bothguardcellmorphologyandregulationofporeaperturecancloselyresemblehigherplantstomata.Inthemostrecentlyderivedplantgroup(angiosperms,orfloweringplants)thereisadedicatedepidermallineagethatproducesstomata.Indicotplants,suchastheresearchArabidopsis[9],theselineagesareinitiatedfromvarioussitesontheleaf(Figure3).Ineachlineage,acom-mittedprotodermalcellcalledthemeristemoidmothercell(MMC)dividesasymmetricallytogiverisetoalargersto-matallineagegroundcell(SLGC)andsmallermeristemoid.Themeristemoidundergoesonetothreeasymmetricdivi-sions(amplifyingdivisions)beforeitdifferentiatesintoaguardmothercell(GMC).Later,SLGCscanalsodivideasymmetricallyandproducemoremeristemoids(spacingdivisions).TheGMCdividessymmetricallytocreatetwoguardcells,andinsomespeciestheGMCsrecruitneigh-boringsubsidiarycells.Thesesubsidiarycellscanprovidemechanicalassistanceandasourceofionsrequiredforguardcellmovement.Amplifyingandspacingdivisionsandsubsidiaryrecruitmentallrequirecelltocellcommunica-tionandtogethertheycontributetopattern.Thefrequencywithwhichcellsparticipateinthesedivisiontypescanbemodifiedtoyieldtheextraordinarydiversityofstomatalpatternsseeninnature[10](Figure2B,2Dand2F).Monocotsexhibitastrongbasetotipgradientofleafdif-ferentiationwithstomata-producingcelllineagesformingatthebaseoftheleaf.AsymmetriccelldivisionsproduceGMCswithoutpriortransitthroughaself-renewingmeris-temoidstage(Figure4).ProtodermalcellsinfilesflankingtheGMCpolarizetowardstheGMCanddivideasymmet-ricallygivingrisetosubsidiarycells.Afterthis,theGMCdividestoproduceguardcellsthatexhibitanovelflattenedordumbbell-shapedmorphology(Figure2G).InmonocotslikeTradescantia,overallstomatalpatterncanberefinedwhenGMCschangefateanddifferentiateintoepidermalcells[11].Thisfatechangeisdependentondistancefromneighboringstomata,suggestinganinhibitorycommunica-tionmechanism.Giventhecurrentpatternsanddevelopmentalprocessesassociatedwithstomataoffloweringplants,whatweretheirorigins?Inthesimplifiedontogenyseeninsomemosses,stomataldevelopmentinvolvesasingleasymmet-riccelldivisiongivingrisedirectlytoaGMC(Figure4).ThisGMCmaynotdividecompletely,asseeninFunariahygrometricawheretwoguardcellnucleiareseparatedbyanincompletecellwall[12],ormaybreakthespacingrule LiverwortsHornwortsFernsLycophytesGymnospermsBasal angiosperms CharophytesFirst specialized structure for gas exchange?True stomata (Group IA and III bHLHs, EPFs)Grass stomata(Divergence of group IA bHLHs)(PAN1) 450420390380360150 Figure1Divergenceofmajorlandplantlineagesandappearanceofstomatalcharacteristics.Phylogenetictreeofextantlandplantsindicatingpositionsofmajorinnovationsintheevolutionofstomata,followingRuszalaetal.[5].Thoseinbracketsindicatepredictedappearanceofstomataldevelopmentregulatorygenes.Numbersonthex-axisrefertomultiplesofmillionsofyears.VaténandBergmannPage2of9http://www.evodevojournal.com/content/3/1/11 Figure2 Representativestomatalcomplexesandpatternsfromdifferentspecies. ( A )Scanningelectronmicrograph(SEM)ofSilurianfossil stomadisplayingcommonmorphology.Scalebar,20 m[3].( B )SEMofmoss Bryumcapillare sporangiumwithstomatavisibleonthelowerhalf. Scalebar,600 m[2].( C )SEMofmoss Bryumcapillare sporangiumstomasunkenbelowepidermalcells.Scalebar,50 m[2].( D )SEMoffern Thelypterisovata var. lindheimeri (sporophyte)leafwithstomataseparatedbypavementcells.Scalebar,10 m;s,stomata[6].( E )Leftpanel,field emissionSEMof Pinuskoraiensis (gymnosperm)stomataarrangedinrowsonneedlesurface;granularmaterialissurfacewax.Scalebar,10 m. Upperright,dewaxedstomata.Scalebar,10 m.Lowerright,dewaxedguardcells(arrows)withinanepistomatalchamber.Scalebar,2 m[7]. ( F )SEMofdicot Arabidopsisthaliana stomatalpatterninthesepal.( G )SEMofmonocot Poaannua stoma,withsubsidiarycells(sc)flankingthe narrowguardcells.Scalebar,10 m[8]. Meristemoid Mother Cell Meristemoid Guard Mother Cell Guard Cells Protodermal Cell mature satellite meristemoid Pavement Cell Spacing division Amplifying divisions SPCH FAMA MUTE SPCH SPCH SCRM1/2SCRM1/2SCRM1/2 SCRM1/2 SCRM1/2 MAPKs MAPKs MAPKs MAPKs EPF1 ER1 TMM EPF2 ER TMM STOMAGEN BASL POLAR Figure3 Stomataldevelopmentin Arabidopsis . Diagramofmajorstagesinstomataldevelopmentwithplaceofactionofthesubsetof regulatorygenesdiscussedinthisreviewnoted.Positiveregulatorsarewritteningreen,negativeregulatorsinredandpolarityregulatorsinblu e. Notallgenesknowntoregulatestomataarepresented.Theimageoftheyoungleafinthelowerrightcorneristorepresentthedispersednature ofstomatallineageinitiation.Colorcode:yellow,meristemoid;orange,guardmothercell;red,guardcell;grey,meristemoidmothercell(MMC). VaténandBergmann EvoDevo 2012, 3 :11 Page3of9 http://www.evodevojournal.com/content/3/1/11 asin Polytrichastrumformosum wherestomatasometimes formnexttoeachother.Alreadyinmosses,diversesto- matalmorphologiesareseen[2].Several(bothearly-and late-divergent)mossspeciesalsolackstomata[13].The functionofstomataintheseplantsmayalsobeunusual, forexamplethebasalgenus Sphagnum displayspseudos- tomatawhichmightfunctioninsporedesiccationrather thantypicalCO 2 acquisition[14]. Theappearanceofamplifyingdivisionsinfernsprovided novelmechanismstocontrolcellnumberaswellstomatal densityandtoproducespecializedsubsidiarycells[15,16]. Hereanepidermalcellmaygothroughoneortwoasym- metriccelldivisionsbeforeitdifferentiatesintoaGMC. Subsidiarycellsingymnosperms(forexample,pines)can arisefrommeristemoiddivisionsordivisionofprotoder- malcellsnexttostomata,orboth.In Pinusstrobus andin Pinusbanksiana ,meristemoidsdivideoncesymmetrically togenerateaGMCandasubsidiarycell[17].Thesubsid- iarycell,aswellasneighboringepidermalcells,expands inapolarfashionovertheGMC.AsaresulttheGMC, andlatertheguardcellpair,isoverlaidbyagroupofepi- dermalsubsidiarycellsofmixedoriginandinaddition,is closelyconnectedtohypodermalsubsidiarycells.Alsoin gymnosperms,webegintoseestomataincorporating someofthebiochemicalinnovationsofthisgroup (Figure2E).Forexample,subsidiarycellsdisplaythick, waterproofcuticles,andtheguardcellsbecomereinforced withlignin,acellwallpolymerthatisnotpresentin bryophytes[18]. Despitethebenefitsofstomata-mediatedgas-exchange, someplantlineageshaveloststomata.Thisissometimes facultative;forexampleamongheterophyllicspecies,two alternateleafformsaremade,dependingonwhetherthe leafissubmergedinwaterorairborne.Inthesespecies, leafsubmergenceleadstoeliminationofstomata[19]. Someparasiticplants,whosesourcesoffixedcarbonare theirhosts,mayalsoloseorinactivatetheirstomata[16]. Otherplantgroups,likethesmall,predominantlyaquatic isoetes,havemembersthathaveloststomatacompletely. TheastomatousisoetesgainCO 2 fromthesedimentvia theirextensiverootsystem[20].Isoetesperformavariant ofphotosynthesiscommonamongcacti(crassulaceanacid metabolism(CAM)),inwhichseparationofparticularbio- chemicalreactionsallow(stomatous)plantstoonlyopen stomataduringthenighttodecreasewaterloss.Useofa root-derivedcarbonsourceenabledastomatousisoetesto fixcarboncontinuouslywithoutathreatofstomata- relatedwaterloss.Ingeneral,astomatousspeciesaresmall andonlyexistinanarrowgrowthenvironment.Ithas beensuggestedthatfunctionalstomataallowplantsto developtolargersizesandtoadapttoawiderrangeof growthconditions[21]. Pathwaysforstomataldevelopmentin Arabidopsis Theregulationofstomataldevelopmentisbestunder- stoodatamolecularlevelin Arabidopsis. Here,individual cellfatetransitionsinthestomatallineagearepromoted bythreecloselyrelatedbasichelix-loop-helix(bHLH) transcriptionfactors,SPEECHLESS(SPCH),MUTEand FAMA[22-24](Figure3).ThesebHLHsareexpressedin thestomatallineage,eachinaspecificdevelopmentalwin- dow,andeachofthemisabsolutelyrequiredforstomata formation.SPCHisexpressedinsubsetofyoungepider- malcells,oftenintwoadjacentcells[24]andSPCH expressionisdynamic.Afteranasymmetriccelldivision, SPCHdisappearsfromtheSLGC,butremainsinthemer- istemoid,whichcontinuesasymmetriccelldivisions[25]. Lossof SPCH leadstoacompletelossofthestomatal lineagewhereasoverexpressionof SPCH leadstoectopic asymmetriccelldivisions[23,24,26];thusitisrequiredfor entryintothestomatallineage.MUTEisexpressedinlate meristemoidsandisrequiredforexitfromtheamplifying moss (Physcomitrella) dicot (Arabidopsis) monocot (rice) PpSMF1 SPCH MUTEFAMA SCRM1/2SCRM1/2SCRM1/2 OsFAMA OsSPCH2 OsMUTE Figure4 Comparisonofthemolecularandmorphologicalfeaturesofstomataldevelopmentin Arabidopsis andrepresentativesofthe grassesandmossesforwhichmoleculardataexist. Presentationofasimplifiedstomatallineagedisplayingonlycellidentities(inthesame colorcodesasFigure3),withtheadditionofbluetomarkthesubsidiarycellsinmonocots.Geneticregulatorsoftheprocessesareincludedat theirpointsofaction,withblacktextindicatingthatthereisdirectfunctionalevidencesupportingtheplacementandgreytextrepresenting inferencesfromcross-speciescomplementationtests.Thecurvedarrowinthedicotlineagerepresentsthecontinuedasymmetricamplifying divisionsmadebymeristemoids. VaténandBergmann EvoDevo 2012, 3 :11 Page4of9 http://www.evodevojournal.com/content/3/1/11 divisionstage,anditpromotesthemeristemoidtoGMCtransition[23,24,26].FAMAisexpressedintheGMCandinimmatureguardcells.OverexpressionofFAMAleadstoectopicformationofunpairedguardcellsindicatingthatFAMApromotesstomatalcellfatewhilerestricting(symmetric)divisions[22].ProteinsencodedbytheparalogousbHLHs,INDUCEROFCBFEXPRESSION1/SCREAM(ICE1/SCRM)andSCRM2,formheterodimerswithSPCH,MUTEandFAMAandpromoteallthreestomatalfatetransitions[27].Asemidominantscrm-Dmutantconvertstheepider-misintostomata,aphenotypeidenticaltoMUTEoverex-pression,whereasdoublemutantsofICE1/SCRMandSCRM2resemblespch[27]Interestingly,ICE1/SCRMhasbeenshowntobeinvolvedincoldstressresponse[28].Sincestomataldevelopmentisregulatedbybothenviron-mental[21]anddevelopmentalfactors[29],itispossiblethatICE1/SCRMisacross-regulatorynodewhereseveralsignalingpathwaysareintegratedtodirectstomataldevelopment.Moresignalintegrationoccursviamitogen-activatedpro-teinkinases(MAPKs)whichregulatestomataldevelopmentandstressresponsesthroughathree-stepphosphorylationcascade.MAPKkinasekinaseYODA,MAPKkinases(MKK4/5/7/9)andMAPKs(MPK3/6)areessentialfornor-malstomatalspacing[30-32].SPCHisadirecttargetofMAPK-mediatedphosphorylationandthisservestonega-tivelyregulateSPCHactivity[33].TheMAPKpathwayalsoregulatesthelaterstagesofstomataldevelopment,butthetargetshavenotbeenidentified.Morecomplexityarisesfromtherecentfindingthatsignalingintermediatesfromthesteroidhormonebrassinosteroid(BR)pathwayphos-phorylatebothYODA[34]andSPCH[35].Interestingly,BR-modulatedphosphorylationasmediatedthroughYODAandSPCHactuallyproducesoppositestomatalphe-notypes.CombinedwithotherevidencethatSPCHisdif-ferentiallymethylatedundercertainenvironmentalconditions[36],weareseeingjusthintsofthecomplexinteractionsandprecisetuningtowhichtheearlypartsofthestomatalpathwaymaybesubjected.Upstreamoftheintracellularsignallingcascades,geneticstudieshaverevealedthatstomatalspacingisregulatedbysecretedpeptidesoftheEPIDERMALPATTERNINGFACTOR-LIKE(EPFL)family[37-41],bythreeleucine-richrepeatreceptorkinases(LRR-RLKs),ERECTA(ER),ERECTA-LIKE1(ERL1)andERL2[42]andoneLRR-re-ceptor-likeprotein,TOOMANYMOUTHS(TMM)[43,44].MembersoftheER-family(ERf)arebroadlyexpressedandtheirabsenceleadstoseverestomatalover-proliferationandmispatterning,aswellaspleiotropicgrowthphenotypes,indicatingthattheyregulatemultipledevelopmentalprocesses[42,45].ERactspredominantlyasanegativeregulatorofentrydivisionswhereasERL1andERL2controllaterstages[42].TMMisexpressedintheearlystomatallineageand,thusfar,onlyrolesinsto-mataldevelopmenthavebeendescribed[44].EPF1andEPF2peptidesarestomatallineage-expressedandregulatethenumberandorientationofasymmetricdivisions[37-41].LossofeitherEPF2resultsinmorestomata,butmutantandoverexpressionphenotypesindicatethatEPF2preventsentryintothestomatallineagewhereasEPF1actslater.Theirparalogue,STOMAGEN/,bycontrast,isexpressedintheunderlyingcelllayer(mesophyll)andtravelstotheepidermistopromotestoma-taldifferentiation[46,47].EPF1,EPF2andSTOMAGENrequirethereceptorTMMforfullactivity[37-41,46,47].Surprisingly,thefunctionofthreeotherEPFLs,EPFL6/CHALLAH(CHAL),EPFL4andEPFL5,isinhibitedbythepresenceofTMM[40].AlthoughCHALwasoriginallyidentifiedbyitsstomatalphenotypeina[41],CHAL/EPFL4/EPFL5areexpressedininternaltissuesandtheirlossleadstoacompromisedgrowthphenotyperesemblinglossof[40].Thus,theylikelyrepresentligandsforERsnon-stomatalroles.TheidentificationofEPFfamilymemberswithdistinctdevelopmentalroleshasledtointerestingmodelsofhowsignalingspecificityisachievedbyusingthenon-kinasereceptorTMMtomodulateligandinteractionswiththeERfkinasesinspe-cifictissues[40].Recently,elegantbiosensorapproachesdemonstratedERandERL1primarilybindEPF2andEPF1,respectively,invitro[48]andthatinplanta,TMMcanheterodimerizewithERandERL1.Clarifyingthephysicalinteractionsandinvivoactivitiesofthefourreceptorswiththe11membersoftheEPFfamilylookstobeanexcitingfutureareaofresearchinArabidopsisHomologuesofERf,TMMandEPFLsarefoundindiversespecies,includingmonocotsandmosses,indicatingthatthepotentialforconservedsignalingsystemsexist.Todate,however,noexperimentalinformationisavailableoutsideofArabidopsisStomatallineagesinArabidopsisareestablishedbyasym-metriccelldivisions,andtheseunusualandunequaldivi-sionsinvolveseveralothernovel,plant-specific,proteins:BREAKINGOFASYMMETRYINTHESTOMATALLINEAGE(BASL)[49]andPOLARLOCALIZATIONDURINGASYMMETRICDIVISIONANDREDISTRI-BUTION(POLAR)[50].BASLdisplaysdynamicspatio-temporallocalizationinthestomatallineage.Beforeasymmetriccelldivision,BASLisdetectedinboththenu-cleusandatthecellperipherydistaltothecelldivisionplane.Afterthedivision,daughtercellsinheritBASLinamannerthatdefinestheirfate:nuclearlocalization(differ-entiationtoguardcells),peripherallocalization(differenti-ationtopavementcell),orboth(continuedasymmetriccelldivisions)[49].BASLmutantsdisplaymisorientedasymmetriccelldivisionandoverexpressionofBASLleadstoectopicoutgrowthsinthepositionswhereBASLisper-ipherallyconcentrated[49].Hence,itseemspossiblethatVaténandBergmannPage5of9http://www.evodevojournal.co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BASLcontrolsormediatescellpolarityduringasymmetriccelldivisioninthestomatallineage.POLARsharessomefeaturesoftheBASLlocalizationpattern;itisperipheralanddistaltothecelldivisionsitebeforeasymmetriccelldivisionandshowsunequalbehaviorsinthedaughters,disappearingfromthelargerdaughterandbeingupregu-latedinthesmaller,meristematic,daughter[50].AlthoughnophenotypeshavebeenascribedtolossofPOLAR,itslocalizationisdependentonBASLsuggestingthattheyactinthesamepathway[50].AdditionalrulesformonocotstomataOneofthemajordifferencesbetweendicotandmonocot(specifically,grass)stomatalpathwaysisthat,inthelatter,subsidiarycellsarerecruitedfromcellfilesflankingthesto-matallineage.ThisprocessrequiresthegenerationofahighlypolarizedcelldivisionthatisspecificallyorientedtowardtheGMC.AfterformationofaGMC(itselfformedbyasymmetricdivisionwithinthestomatallineage),neigh-boringsubsidiarymothercells(SMCs)divideasymmetric-allytoproducesmallsubsidiarycellsnexttotheGMC(Figures2Gand4).SMCpolarizationinvolveslocalizationofF-actinpatchesalongthecellwallflankingtheGMC,andnuclearmigrationtowardstheactinpatches[51].Inmaize,actinpatchesco-localizewithanLRR-RLKprotein,PANGLOSS1(PAN1)[52].Despitesharedrolesinstoma-taldevelopment,PAN1isnotinthesameLRR-kinasefamilyasERfandtherearenopolarlylocalizedLRR-kinasesimplicatedyetinArabidopsisstomataldevelop-ment.LossofPAN1leadstomislocalizationofactinandthenucleus.Thisdisruptsasymmetriccelldivisionsandresultsinabnormalsubsidiarycells[52,53].Recently,theactinregulatorsRhoofplants2(ROP2)andROP9wereshowntolocalizepolarlyinSMCsandtopromoteSMCpolarization[54].PAN1,ROP2andROP9interactandlocalizationofROP2andROP9isdependentonPAN1,butPAN1localizationisindependentofROPs.Itisat-tractivetospeculatethattheseproteinsworkinacom-monpathwaytoreceivepolaritycuesandtranslatethemintothecellularreorganizationnecessaryforSMCpolarization[54].EvolutionofstomatalregulatorsArabidopsisstomatalbHLHgenesareinstomatal-producingplantlineagesAsdescribedabove,inArabidopsis,fivebHLHgenesaremajordeterminantsoftheidentitiesandbehaviorsofdiffer-entstomatallineageprecursors.SPCH,MUTEandFAMAarefairlyrestrictedintheirexpressionpatterntosubsetsofthestomatallineagewhereasICE1/SCRMandSCRM2areexpressedthroughoutthelineageandinadditionalnon-stomatallineagecells.Whenconsideringthediversityofstomatalpatterninnature,itisinterestingtothinkabouthowtheexpression,regulationandfunction(andexistence)ofthisclassofregulatorsmaychange.Moreover,onemightaskwhethertheheterodimericpartnershipbetweenSPCH,MUTEandFAMAwithICE1/SCRMandSCRM2couldbeancientorwhetherthisisanewinnovation.ThebHLHfamilyischaracterizedbyaconservedDNAbindingregion,butthereareeasilyrecognizablesub-familieswithin.SPCHMUTEFAMAbelongtothegroupIAbHLHs[55]whosegenicintron/exonstructureandproteinC-terminiaredistinctiveenoughtoserveashighconfidencegroupcharactersthrough-outthefloweringplantsandouttoSelaginella(amodellycophyte)andPhyscomitrella(amodelmoss)[56].Dis-tinctionamongindividual1Amembersisonlyclearwithinthefloweringplants.ICE1/SCRMandSCRM2aremembersofgroupIIIandrepresentativesofthisgrouparefoundinmanycladesbacktothemosses(http://www.phytozome.net/).Intheincompletetran-scriptomeandgenomesequencesfromplantlineagespredatingtheemergenceofstomata,neithergroupIAnorgroupIIIbHLHgenesareobvious[56](CMacAlister,per-sonalcommunication).Basedonsequencescurrentlyavailable,inallcaseswheregroup1Amemberscanbedis-tinguished,thereisalsoagroupIIIbHLH,suggestingthattheirpartnershipcanbeancient.Agroup1AhomologuefromPhyscomitrellacanpartiallycomplementArabidopsismuteandfama,butnotspchmutants[56].ThesecrossspeciescomplementationresultsareinterestinginlightoftheshortenedpathwayfordevelopmentofstomatainPhyscomitrella;inthismoss,noearlyasymmetricdivisionsareevidentandinsteadasingleGMCisspecifiedandundergoesincompletecytokinesistoformtwoconnectedguardcells(Figure4).ThispathwaywouldrequireMUTE-andFAMA-likefatepromotingactivities,butnotthedivision-promotingactivityofSPCH[56].Ingrasses,thepositionsofstomataaredeterminedandfixedatearlystagesofleafdevelopmentandamplifyingdivisionsarenotpresent.Infact,onlythedifferentiation(GMCtoguardcells)stepissimilarbetweengrassesandArabidopsis(Figure4).Nonetheless,SPCHMUTEandFAMAgenescanbeidentifiedinthegenomesofmaize,riceandBrachypodium.TherehasalsobeenaduplicationSPCHintheseplants[57].Perhapsduetothedifferentstomatalontogenies,however,thericehomologuesOsSPCH1/2areexpressedveryearlyduringplantdevel-opment,possiblybeforetheproductionofstomatallineage[57].OsSPCH2mutantsinrice,do,however,havereducedstomatalnumbersandresembleweakmutantallelesofAtSPCH[57].OverexpressionofOsMUTEandOsFAMArecapitulatesoverexpressionphenotypesoftheArabidopsisgenes,indicatingthattheirGMCandguardcellidentity-promotingfunctionsareconserved.Ofthethreegenes,theonlyoneactingatastagecommontosto-mataldevelopmentinbothplantgroups(theGMCtostomatalguardcelltransition),FAMA,ismosthighlyVaténandBergmannPage6of9http://www.evodevojournal.com/content/3/1/11 conservedintermsofexpressionpatternandlossoffunc-tionphenotypesinbothriceandArabidopsis[57].NewappearanceofpolarityregulatorsHomologuesofthecellfateregulatorsandmanyofthesignalingcomponentsdiscussedaboveappearinmanyplantspecies[10,58,59].Incontrast,thetwoproteinsshowntoexhibitpolarizedlocalizationinstomatallineagecellsofArabidopsis,POLARandBASL,donot.BASLdoesnotresembleanyotherArabidopsisproteinsandonlyinthecongenericA.lyrataisthereasignificantlysimilarsequence.InArabidopsisPOLARismoderatelysimilartoanothergene(POLAR-LIKE1)andhomologuesPOLARandPOLAR-LIKE1canbefoundincloselyrelated(dicot)speciessuchaspoplar(POPTRB9IL54).Alreadyinrice,however,thesequencesimilaritybecomesrestrictedtoaverysmalldomainoftheproteins.ItisinterestingtoconsiderwhetherthisinabilitytofindsuchhomologuesisbecausethefunctionofBASLandPOLARisrequiredonlyinthedicots,orbecausesimilarfunctionsarecarriedoutbydifferentgenesandtheapparentuniquenessoftheseproteinsrepresentseitherfastsubsti-tutionratesorthattheirrolescanbeservedbyotherpro-teins.Forexample,scaffoldproteinsthatbindotherstogetherintocomplexesplayimportantrolesinpolaritygenerationinyeastandanimals,yetthesescaffoldsareoftennotwellconservedatthesequencelevelandconsistprimarilyofmultipleinteractionsurfaces.EvolutionofregulatedstomatalporeopeningAdevelopmentalapproachconcernsitselfwiththecorrectspecificationandpatternofstomata.Fromaphysiologicalpointofview,however,thebehaviorofthesefinalpro-ductsiskey.Modulationofthestomatalporeaperturedependsoncoordinatedmorphologiesoftheguardcellpairand,particularlyinthecaseofthegrasses,ontheco-ordinationofguardcellsandthespecializedsubsidiarycellsthatareobligatepartsofthestomatalcomplex[60].Forstomatalporeaperturetobeoptimizedfordailyandseasonalfluctuationsinlight,temperature,humidityandavailability,theguardcellsmustbeabletosensesuchenvironmentalfactors.Guardcellsinangiospermsappeartosensemanyofthesefactorsautonomously,andkeykinases(OST1),phosphatases(PP2C)andreceptorsforthedroughtstresshormone,ABA(PYR1)havebeenidentifiedinArabidopsis[1].RecentstudiesofCOandABAresponsivenessinnon-vascularplantshavecometodifferentconclusionsaboutwhenthesensingofthesedifferentenvironmentalcuesarose.MonitoringstomatalporeclosureinresponsetoABA,[61]concludedthatre-sponsivenesstothishormonewasanewfeatureandwasabsentinfernandlycophytespecies.Otherstudies,how-ever,provideevidencethatABAsensingmayhavearisenquiteearly.Bycross-speciescomplementation,Ruszala[5]andChater[62]showedthattheOST1homologuesfromSelaginellamoellendorffiiandPhyscomitrellapatenscouldpartiallyrestoretheabilityofArabidopsisost1stomatatorespondtoABA.Moreover,knockoutofPpOST1-1sig-nificantlyattenuatedABAresponseinP.patensstomata[62].Thedifferingconclusionsfromthesestudiescouldbeduetothedifferentrepresentativespecieschosen,ageneralcautioninevolutionarystudiesofthissystemthatisalsoechoedinthebehaviorofmaizeandricebHLHs[57].ConclusionsStomataldevelopmentinArabidopsishasbeenusedasamodelgeneticsystemfortheanalysisofcellfate,cellpo-larityandcelltocellcommunication.Thenatureofthegeneproductsidentifiedinsuchanalysis,coupledwiththelongtraditionofevaluatingthenumbersandpatternsofstomataindiverseplantsfortaxonomicpurposesmakesthissystemausefulnaturallaboratorytolookattheparal-lelevolutionofgenesanddevelopmentaltrajectories.Asthenumberofcompletedplantgenomesincreasesandtoolsforexperimentalmanipulationofnon-modelspeciesdevelop,webelievetherewillbeanexcellentopportunitytotesttherolesofcandidatecellfate-andcellsignalingfactor-encodinggenesincreatingdevelopmentaldiversity.CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.AcknowledgmentsFundingforworkonstomataintheauthorslaboratoryisprovidedbygrantsfromtheUSNationalScienceFoundation(IOS-0845521)andtheNationalInstitutesofHealth(R01GM086632).DCBisaGordonandBettyMooreFoundationinvestigatoroftheHowardHughesMedicalInstitute.AuthordetailsDepartmentofBiology,StanfordUniversity,Stanford,CA94305-5020,USA.InstituteofBiotechnology/DepartmentofBioandEnvironmentalSciences,UniversityofHelsinki,HelsinkiFIN-00014,Finland.HowardHughesMedicalInstitute,Stanford,USA.AVandDCBanddesignedthestudy,wrotethemanuscriptandpreparedthefigures.Allauthorsreadandapprovedthefinalmanuscript.Received:10April2012Accepted:12June2012Published:12June20121.SirichandraC,WasilewskaA,VladF,ValonC,LeungJ:Theguardcellasasingle-cellmodeltowardsunderstandingdroughttoleranceandabscisicacidaction.JExpBot2.LigroneR,DuckettJG,RenzagliaKS:Majortransitionsintheevolutionofearlylandplants:abryologicalperspective.AnnBot3.EdwardsD,KerpH,HassH:Stomatainearlylandplants:ananatomicalandecophysiologicalapproach.JExpBot(Suppl1):2554.FranksPJ,BeerlingDJ:MaximumleafconductancedrivenbyCO2effectsonstomatalsizeanddensityovergeologictime.ProcNatlAcadSciUSA5.RuszalaEM,BeerlingDJ,FranksPJ,ChaterC,CassonSA,GrayJE,HetheringtonAM:Landplantsacquiredactivestomatalcontrolearlyintheirevolutionaryhistory.CurrBiolVaténandBergmannPage7of9http://www.evodevojournal.com/content/3/1/11 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Submit your next manuscript to BioMed Central and take full advantage of: Convenient online submission Thorough peer review No space constraints or color gure charges Immediate publication on acceptance Inclusion in PubMed, CAS, Scopus and Google Scholar Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit VaténandBergmann EvoDevo 2012, 3 :11 Page9of9 http://www.evodevojournal.com/content/3/1/11