HANABA TARANU Is a GATA Transcription Factor That Regulates Shoot Apical Meristem and ...

16 Pages·2004·1.03 MB·English

ThePlantCell,Vol.16,2586–2600,October2004,www.plantcell.orgª2004AmericanSocietyofPlantBiologists
HANABA TARANU Is a GATA Transcription Factor That
Regulates Shoot Apical Meristem and Flower
Development in Arabidopsis W
YuanxiangZhao,LeonardMedrano,1,2KazuakiOhashi,2,3JenniferC.Fletcher,4HaoYu,5
HajimeSakai,6andElliotM.Meyerowitz7
DivisionofBiology,CaliforniaInstituteofTechnology,Pasadena,California91125
We have isolated a new mutant, hanaba taranu (han), which affects both flower and shoot apical meristem (SAM)
developmentinArabidopsisthaliana.Mutantshavefusedsepalsandreducedorgannumbersinallfourwhorls,especiallyin
the2nd(petal)and3rd(stamen)whorls.hanmeristemscanbecomeflatterorsmallerthaninthewildtype.HANencodes
a GATA-3–like transcription factor with a single zinc finger domain. HAN is transcribed at the boundaries between the
meristemanditsnewlyinitiatedorganprimordiaandattheboundariesbetweendifferentfloralwhorls.Itisalsoexpressed
invasculartissues,developingovulesandstamens,andintheembryo.haninteractsstronglywithclavata(clv)mutations
(clv1,clv2,andclv3),resultinginhighlyfasciated SAMs,andwefind thatWUSexpression isalteredinhan mutantsfrom
early embryogenesis. In addition, HAN is ectopically expressed both in clv1 and clv3 mutants. We propose that HAN is
normallyrequiredforestablishingorganboundariesinshootsandflowersandforcontrollingthenumberandpositionof
WUS-expressing cells. Ectopic HAN expression causes growth retardation, aberrant cell division patterns, and loss of
meristemactivity,suggestingthatHANisinvolvedincontrollingcellproliferationanddifferentiation.
INTRODUCTION originateasabuttressofundifferentiatedcells(thefloralmeri-
stem) growing at the peripheral zone of the SAM, which soon
During the reproductive growth phase of Arabidopsis thaliana,
separatesitselffromtheSAM(onlyconnectstothestemthrough
flowersariseinaspiralphyllotaxisfromtheflanksoftheshoot
itspeduncle)andsequentiallygivesrisetothesepals,thepetals,
apicalmeristem(SAM),whichislocatedatthetipofthestemand
andthestamens.Eventuallytheremaininginnerfloralmeristem
encompasses a stem cell population whose descendants es-
cells differentiate into two congenitally fused carpels (Smyth
sentiallygiverisetoalloftheaerialpartsoftheplant.TheSAMcan
etal.,1990).
bedividedintothreezones:thecentralzone,whichiscomposed
Twomajorpathways,theWUSCHEL(WUS)–CLAVATA(CLV)
ofasmallnumberofslowlydividingstemcellsatthemeristem
pathwayandtheSHOOTMERISTEMLESS(STM)pathway,have
apex,theribmeristemzone,whichliesunderneaththecentral
been characterized as pivotal for meristem establishment and
zoneandgivesrisetothepithandvascularstructureofthestem,
maintenance. Both wus and stm mutants fail to initiate an
andtheperipheralzone,whichsurroundsthecentralzoneand
embryonicSAMandhaveprematureterminationofadventitious
providesfoundercellsfortheformationofnewleavesandflowers
shootandfloralmeristems(Clarketal.,1996;Lauxetal.,1996;
(reviewed in Fletcher and Meyerowitz, 2000). Floral primordia
Longetal.,1996).WUSencodesahomeodomainproteinthatis
expressedneartheboundaryofthecentralzoneandribmeristem
1Current address: Ceres Inc., 1535 Rancho Conejo Blvd., Thousand
inshootandfloralmeristemsandfunctionstopromotemeristem
Oaks,CA91320.
2Theseauthorscontributedequallytothework. activity (Mayer et al., 1998). The clv mutations (clv1, clv2, and
3Currentaddress:LaboratoryofMolecularandBiochemicalToxicology, clv3)affectbothSAMandfloralmeristemdevelopment,resulting
GraduateSchoolofPharmaceuticalSciences,TohokuUniversity,Japan. inenlargedshootapicalandfloralmeristemsaswellasincreased
4Current address: Plant Gene Expression Center, USDA/University of
floralorgannumbers(Clarketal.,1996,1997;KayesandClark,
California,Berkeley,CA94710.
5Currentaddress:DepartmentofBiologicalSciences,NationalUniver- 1998).CLV3isnormallyexpressedinthecentralzone,overlying
sityofSingapore,117543Singapore. the WUS domain (Fletcher et al., 1999), whereas CLV1 is ex-
6Currentaddress: DuPont Crop Genetics, Experimental Station E353, pressedintheribmeristemzone,embracingtheWUSdomain
Wilmington,DE19880. basallyandlaterally(Clarketal.,1997).Ithasbeensuggestedthat
7To whom correspondence should be addressed. E-mail meyerow@
CLV3,asecretedsmallprotein(Fletcheretal.,1999;Rojoetal.,
caltech.edu;fax[phone].
The author responsible for distribution of materials integral to the 2002), interacts with the CLV1/CLV2 Leu-rich repeat receptor
findingspresentedinthisarticleinaccordancewiththepolicydescribed proteinsasaligand/receptorcomplextoactivateasignaltrans-
in the Instructions for Authors (www.plantcell.org) is: Yuanxiang Zhao duction cascade that limits WUS expression (Fletcher et al.,
([email protected]).
1999;Brandetal.,2000;Schoofetal.,2000).Intheabsenceof
WOnlineversioncontainsWeb-onlydata.
Article, publication date, and citation information can be found at CLVactivity,WUSactivityincreasesandcausesaccumulation
www.plantcell.org/cgi/doi/10.1105/tpc.104.024869. of stem cells and thereby an enlarged, fasciated meristem.
ArabidopsisHANandMeristemDevelopment 2587
Conversely,ithasbeenshownthatectopicWUSexpressioncan mutationsandthatHANisrequiredfornormalcelldivisionand
induceectopicCLV3expression(Schoofetal.,2000).Ithasbeen positioningofWUS-expressingcellsintheSAM,suggestingthat
proposed, therefore, that WUS regulates its activity so as to HANalsorepresentsoneoftheextrinsicmoleculeslinkingthe
control SAM size by employing a negative feedback system morematureboundaryandvascularcellswithSAMactivities.
involvingtheCLVproteins.STMencodesamemberoftheclass1
KNOXfamilyofhomeodomainproteins(Longetal.,1996).STMis RESULTS
expressedthroughouttheSAMwhereitappearstoactthrough
downregulating the ASYMMETRIC LEAVES genes (AS1 and
hanMutantPhenotypesandHANCloning
AS2). Loss of AS1 activity restores meristem function to stm
mutants by derepressing the activities of other KNOX class Fourdifferenthanmutantalleles(han-1tohan-4)wereisolatedin
genes,suchasKNAT1(alsonamedBREVIPEDICELLUS;Venglat threedifferentmutagenesisscreensinArabidopsis.han-2was
etal.,2002)andKNAT2(Byrneetal.,2000).Morerecentlyithas isolatedinanethylmethanesulfonatemutagenesisscreen,and
beenshownthattheSTMandWUSpathwayscanacttogether the han-1, han-3, and han-4 alleles were generated in two
to confer ectopic meristematic cell fate (Gallois et al., 2002; separateAgrobacteriumtumefaciens–mediatedT-DNAscreens
Lenhardetal.,2002). (see Methods). All han mutants have similar flower defects,
Althoughmuchisknownabout geneticregulatorypathways includingfusedsepals,reducednumbersofpetalsandstamens,
within the SAM, little is known about the interactions between and, sometimes, unfused carpels. Phenotypically han-2 is the
meristematic cells and their neighboring differentiating cells. weakestallele,andhan-3isthestrongest,whereashan-4and
Nevertheless,moleculescouplingdifferentiatingcellswithmeri- han-1havesimilar,intermediateexpressivity.Mutantshomozy-
stem development have been uncovered in recent years. In gousforhan-3,unlikelineshomozygousfortheotheralleles,have
Petuniahybrida,HairyMeristem(HAM)encodesaGRASfamily fasciatedSAMs.Inaddition,thehan-3alleleshowsslightsemi-
putative transcription factor that is expressed in provascular dominance,whereastheotherhanmutationsarefullyrecessive.
tissue, but ham mutations result in early termination of SAM We will focus on the recessive mutations in this report. The
activity.Hence,HAMmayrepresentanextrinsicantidifferentia- majorityofhan-2mutantflowershavetwotofoursepals(sepals
tion factor that is required to maintain stem cells in the SAM fused together are counted as 1) in the 1st whorl, one or two
(Stuurmanetal.,2002).Additionally,lateralorganpolaritygenes, organsinthe2ndwhorl(includingpetalsaswellasfilamentous
such as Antirrhinum majus PHANTASTICA and Arabidopsis structures), four or five stamens (including filaments lacking
PHABULOSA, also appear to have positive effects on the for- anthers)inthe3rdwhorl,andtwoasymmetriccarpelsinthe4th
mation and maintenance of apical meristems (McConnell and whorl(Table1,Figures1Bto1E).Malformedorgansobserved
Barton,1998;Waitesetal.,1998).Furthermore,membersofthe includebifidstamenswithtwostalksfusedpartiallyorcompletely
NAC-domaingenefamilyexpressedattheboundariesofmeri- alongtheirlengthbutwithanthersseparated(Figure1C,arrow),
stemsandprimordia,includingthePetuniaNOAPICALMERI- filamentousstructuresbetweenwhorls(Figure1D),andstamens
STEM(Soueretal.,1996),theAntirrhinumCUPULIFORMIS(Weir withabnormalanthers.In>50%(31/54)ofthecases,thevalvesof
etal.,2004),andtheArabidopsisCUP-SHAPEDCOTYLEDON thetwofusedcarpelsarenotsymmetrical,withoneshorterthan
genesCUC1,CUC2,andCUC3(Aidaetal.,1999;Takadaetal., theotheratthebasalregion(Figure1E,arrow).Atamuchlower
2001;Vroemenetal.,2003),regulateSAMdevelopmentaswell. frequency (4/54), extra tissue is formed apically (Figure 1E,
Inthisreport,wedescribeanewgeneinArabidopsis,HANABA arrowhead).han-2homozygousplantsarepartiallyfertile.han-1
TARANU(HAN;meaningfewerfloralleavesinJapanese),thatis flowershavestrongerdefectsinallwhorlswhencomparedwith
requiredfornormalflowerandSAMdevelopment.Thisgeneis han-2flowers.Inapproximatelyhalfofhan-1flowers,allofthe
expressedintheprovasculartissuesduringembryogenesisand sepalsareeitherpartiallyorcompletelyfused(Table1,Figures1F
laterisexpressedattheboundary tissuesbetweenmeristems to 1H). Mature petals are rare and only appear in a few early
andinitiatingorganprimordiaaswellasinthevasculartissues. flowers.Themajorityofhan-1flowerslacksecondwhorlorgans.
We demonstrate that han mutations interact strongly with clv In the third whorl, organ numbers are generally decreased to
Table1. hanFlowerPhenotypesSummary
OrganNumber Sepal Petal Stamen Carpel
FlowerNumber 0 1 2 3 4 5 0 1 2 3 4 0 1 2 3 4 5 6 2 Fused Unfused
han-2 0 4 27 57 21 1 13 31 40 23 3 0 0 0 6 40 55 9 49b 54 0
110a,b (4) (10) (13) (2) (4) (6) (1)
han-1 0 53 37 13 4 0 77 24 4 2 0 0 8 12 37 32 16 1 107 91 16
107a (6) (3) (3) (11) (77) (90) (62) (4)
Thenumbersinparenthesesrepresentthetotalnumberoffilamentousstructuresinallflowersofeachcategory.
aTotalnumberofflowersexamined.
bOnly54flowerswereexaminedforcarpelnumberandfusiondefects.Ofthe54,49hadtwocarpels,1hadasinglecarpel,and4hadextracarpel
tissuespositionedapically.
2588 ThePlantCell
Figure1. hanMutantFlowerandFloralMeristemPhenotypes.
(A)Awild-typeflower.
(B)to(E)han-2homozygotes.
(F)to(H)han-1homozygotes.
(B)Ahan-2flower.
(C)Thearrowpointstothefusionoftwostamens.
(D)Filamentousstructuresinplaceofapetalorastamenareindicatedbythearrowandarrowhead,respectively.Sepalswereremoved.
(E)Carpeldefectsincludeasymmetricsiliquevalves(arrow)andextracarpeltissuepositionedattheapicalregionofthesilique(arrowhead).Siliques
shownarenotage-matched.
(F)Ahan-1inflorescence.
(G)Anearly-arisinghan-1flower.
(H)Aflowerwithsepalsallfusedintoanopensemicircle.
(I)Awild-typestage3flower.Se,sepal;FM,floralmeristem.
(J)Ahan-1stage3flower(becausehanfloralmeristemdevelopmentisgenerallydelayed,floralstageisdeterminedbasedonsepalsize).
(K)Awild-typestage6flower.St,stamens.
(L)Ahan-1stage6flower.
(M)Ahan-1inflorescenceatlatestage.Theblackarrowpointstoacarpelloidsepal,andthewhitearrowheadpointstoasepalloidcarpel.
(N)Anenlargedviewofthecarpelloidsepalindicatedin(M).Arrowpointstostigmaticpapillaeattheedgeofthesepal.
(O)Anenlargedviewofthesepal(Se)andcarpel(Ca)fusionstructurein(M).
(P)Awild-typeSAM.
(Q)Ahan-1mutantSAM.
Barsin(A)to(H)¼0.5mm;barsin(I)to(Q)¼50mm(unlessindicatedotherwise).
threeorfour,andstamenswithnormalmorphologyarerare.The smallerfloralmeristemsthanthewildtype,asshownforastage
frequencyoffilamentousstructuresoccupyingstamenpositions 3flowerinFigure1J(arrowpointstoanareaofcongenitalsepal
in han-1 flowers is much higher than in han-2 flowers. han-1 fusion). In addition, inner whorl organ primordia either fail to
homozygousplantshaveextremelylowfertility.Consistentwith developoraredelayedinappearancecomparedwiththewild
theirreducedfloralorgannumbersandsizes,han-1mutantshave typeinstage6flowers(Figures1Kand1L).
ArabidopsisHANandMeristemDevelopment 2589
Vegetativegrowthandinflorescencestructureofhanmutants two copies of a T-DNA sequence into the intron at 112 bp,
appearsfairlynormal.However,insomeofthehan-1andhan-4 followed by a deletion of 20 bp. Both a 9- and 6-kb genomic
plants,flowersthatarisetowardtheendofthereproductivestage fragmentspanningtheHANgenefullyrescuesthehanpheno-
are morphologically more deformed than early flowers, com- types(datanotshown).Databasesearchesidentified25puta-
posed either of carpels alone (sometimes subtended by leaf/ tiveGATAgenesintheArabidopsisgenome,amongwhichare
sepal-like structures), carpelloid sepal/leaf-like structures (Fig- two close HAN relatives, located on chromosomes 2 and 4
ures 1M and 1N, arrows), or chimeric organs with carpelloid (At2g18380 and At4g36620), and here named HANL1 and
character (Figures 1M, arrowhead, and 1O). In addition, later- HANL2.TheHANgeneshares46and50%sequencesimilarity
arising flowers fail to originate in a typical phyllotactic spiral. toitstwoclosehomologsHANL1andHANL2,respectively.
Perturbed floralphyllotaxy generally reflectsaSAMdefect. To
determineifsuchadefectisvisibleinhan-1mutants,bothearly
HANExpression
(postembryonicday30)andlatestage(afterpostembryonicday
40)inflorescenceSAMswereexaminedusingscanningelectron TheHANexpressionpatternwasexaminedbyinsituhybridiza-
microscopy.EarlystagehanSAMsarenormalcomparedwiththe tionusingafull-lengthcDNAprobeandaHAN-specific59-cDNA
wildtype,asalsoconfirmedbyconfocallaserscanningmicros- probe, both of which are specific to HAN (see Methods) and
copy(datanotshown).However,forthelatestagehaninflores- whichgavethesameresults.HANisexpressedinvegetativeand
cences, five out of eight SAMs appeared flattened instead of inflorescenceSAMs,axillarySAMs,floralmeristems,developing
dome-shapedasinthewildtype(Figures1Pand1Q)andinsome ovulesandstamens,vasculartissues,andintheembryo.Inthe
casesappearedsmallerandlessdistinctfromthesurrounding developingaxillarySAM,itisexpressedattheboundarybetween
initiatingorganprimordia.IntheflattenedSAMs,earlystagefloral nascentaxillarymeristemsandtheadaxialsideofleaves(Figure
organ primordia were not the usual hemispherical masses of 3A,a,arrows).ExpressioninallmatureSAMsissimilar,locatedat
undifferentiatedcellsbutwereridge-shaped.Thisindicatesthat theboundariesbetweenthecentralSAMandtheinitiatingorgan
thereisagradualtransitiontowardanaberrantSAMmorphology primordia, as well as between the neighboring initiating organ
inhanmutantsduringreproductivedevelopment. primordia(Figures3A,bandc,and3B).
TheHANgenewasclonedbybothpositionalmappingofthe Expressioninthefloralmeristemreiteratesthepatternseenin
han-1 allele and by T-DNA tagging of the han-4 allele. HAN is theSAM,withstrongexpressionattheboundariesbetweenthe
locatedonchromosome3(At3g50870),anditsfull-lengthcDNA meristematicdomeandtheinitiatingfloralorganprimordia,and
wasisolated fromaflower-specific lcDNAlibrary.Theprotein alsoattheboundariesbetweentheprimordiaofdifferentwhorls
sequencededucedfromtheopenreadingframeconsistsof295 (Figure 3A, d). Expression at the boundaries attenuates as the
aminoacids,encodedbytwoexons,andresemblesaGATA-3– organ primordia grow apart. In stage 5 flowers, expression
like protein witha single zinc finger motif (C-X -C-X -C-X -C) remainsattheboundarybetweenthecentralmeristematiccells
2 18 2
(Figure 2). In addition, there is a stretch of 14 amino acids anddifferentiatingstamenprimordia(datanotshown).Instage6
N-terminal to the zinc finger that appears highly conserved flowers, expression is the strongest at the medial ridge region
amongsomeplantGATAtranscriptionfactors.Thehan-1muta- of the carpel (Figure 3A, e). In the developing ovule, HAN is
tionresultsinacompletedeletionofthegene,startingfrom709 expressedintheinnerandouterinteguments,withsignalabsent
bpupstreamofthetranslationinitiationcodonandendingat1298 fromthenucellus(Figure3A,fandg).Inalloftheaerialtissues
bpdownstreamofthestopcodon.Themutationinhan-2results examined,includingflowers,HANisexpressedstronglyincell
inasingleaminoacidchangeatposition179,Gly(GGC)toSer typesassociatedwiththephloemtissues(arrowsinFigure3A,f
(AGC).Themutationinhan-4iscausedbytheinsertionofatleast andg).Itisexpressedstronglyinthedevelopinganthersinas
Figure2. ProteinandGeneStructure.
TheHANproteinsequencededucedfromtheopenreadingframeofthefull-lengthHANcDNA.Thepositionoftheintronisindicatedbytheopen
triangle;a14–aminoaciddomainthatishighlyconservedamongsomeplantGATAfactorsisunderlinedtwice;thezincfingerdomainisunderlined
once.Themutationinhan-1resultsindeletionofthewholegene.Themutationinhan-2changesaGly(GGC)toaSer(AGC)ataminoacidposition179
(asterisk).Themutationinhan-4resultsfromaT-DNAinsertionwithintheintron(arrow).The59junctionsequenceisflankedbytheBargeneinthe
T-DNA,whereasthe39isflankedbythe4335SpromoterintheT-DNA(neartherightborder).
2590 ThePlantCell
Figure3. HANExpressionPattern.
(A)BuddingaxillarySAMsareshownin(a).ArrowspointtotheexpressiondomainsofHANattheboundariesbetweentheSAMandtheadaxialsidesof
leaves.Lf,leaf.(b)OlderaxillarySAM.ArrowspointtotheexpressiondomainsofHANattheboundariesbetweentheSAMandthenewlyinitiatedorgan
primordia(pr,smallarrowheads).LargearrowheadindicatesthejunctionoftheHANexpressiondomainintheSAManditsvascularexpressioninthe
stem.(c)InflorescenceSAM.ArrowspointtotwostripesofHANexpressionattheboundariesbetweentheSAMandnewlyinitiatedfloralprimordia.
SmallarrowheadsindicateHANexpressioninastage2flowerattheboundariesbetweenthefloralmeristemandthesoon-to-be-specifiedsepal
primordia.LargearrowheadspointtothejunctionoftheHANexpressiondomainintheSAMandinthestem(notethattheangleofthissectionistilted
towardthereader,andthedomainindicatedbythelargearrowincludessomeoftheexpressionofHANbetweentheSAMandaprimordiumpointing
towardthereader).(d)Stage5flower.ArrowpointstotheconnectionarchofHANexpressioninthefloralmeristemanditsexpressioninthepeduncle
vasculartissue.Se,sepal.(e)Expressioninthelateralandbasalregionsofcarpelprimordiainastage6flower.(f)Earlystage12ovary(crosssection).
HANisexpressedininitiatinginnerandouterintegumentsaswellasinthevasculartissue(arrows).(g)Expressioninintegumentscontinuesinthestage
13ovary.Arrowpointstoexpressioninfuniculusvasculartissue.(h)HANexpressioninthestamensofastage8flower.lo,locule.(i)HANexpression
persists inthetapetum celllayeruntilit hasdegenerated andisabsent inmature haploid pollen. t,tapetum.(j)Eight-cellstage embryo. HAN is
expressedinallcellsoftheembryoproper.(kandl)Globularandtransitionstageembryos.HANisexpressedinthecenterfilesofcells.(mandn)Heart
andtorpedostageembryos.HANexpressionremainsinthecentercellsdestinedtobeprovasculartissues.(o)Latetorpedostage.Bars¼10mm.
(B)ExpressioninSAMinseriallongitudinal(atof)andcrosssections(gtoj).ArrowspointtoexpressionattheboundariesbetweentheSAMandnew
organprimordiaaswellasbetweenorganprimordia.ExpressionofHANintheSAMmergeswithitsexpressioninthevascularstrandsinthestem
(arrowheads)asillustratedin(k).In(k),redlinesrepresenttheexpressiondomainsofHANintheSAM,stem,anddevelopingorganprimordia(P1and
P2).ArrowheadcorrespondstotheregionsindicatedbylargearrowheadsinFigure3A(bandc).P,primordium.Bars¼10mm.
earlyasstage8flowerswhenloculesareinitiated,inthetapetum outermosttwoapicallayersthroughthetransitionandlateheart
celllayer,aswellasthemicrosporocytes(Figure3A,h).Expres- stages(Figure3A,lton).Inthetorpedostageembryo,expres-
sion persists until the tapetum layer degenerates and haploid sionisdetectedinallprovasculartissues(Figure3A,o).
pollenismature(Figure3A,i).ExpressionofHANintheembryois
detecteduniformlyintheembryoproperoftheeight-cellstage
GeneticInteractionsofhanwithMutationsAffecting
embryo(Figure3A,j).Itisthenconcentratedinthecentercells
FlowerDevelopment
of the embryo and absent from the epidermal cell layer at the
globularstage(Figure3A,k).Thisexpressionpatternpersistsin Doublemutantsofhan-1withthefloralorganidentitymutations
the center fourfiles of cells; expression isnot observed in the apetala3,pistillata,andagamousaregenerallyadditive(datanot
ArabidopsisHANandMeristemDevelopment 2591
shown).Todetermineiffloralorganidentitygenesareaffectedin numerousflowersatthetopoftheinflorescenceSAM(Figure4E).
theirexpressioninhanmutants,AP1andAP3expressionwas These double mutant flowers generally have reduced organ
examined in han-1 flowers. For both genes, expression was numbersintheouterthreewhorlscomparedwithhan-2mutant
detectedinthemutantincorrectspatialandtemporalpatterns, flowers (Figure 4F). The han-2 clv2-1 double mutants either
butsignalintensitywasrelativelylow(datanotshown).Thelower appear similar to han-2 clv1-1 mutants or lack primary shoot
expressionintensityispotentiallybecauseofasmallernumberof dominance and have several unfasciated stems terminating in
floral organ cells in han mutants. This indicates that the floral aballofcarpelloidstructures(Figure4G).han-1clv1-1andhan-1
organ identity genes act independently of HAN in controlling clv2-1 double mutants produce inflorescences more fasciated
flowerdevelopment. thanclvsinglemutants,withflowersforthemostpartcomposed
Doublemutantsofhanandclv(includingclv1,clv2,andclv3)or onlyofsepalsandsometimesunfusedcarpels(Figure4H).The
han and wus-1 were generated. Any combination of han/clv phenotypesofhan-4clvdoublemutantsaresimilartothoseof
doublemutantsresultsinincreasedinflorescencefasciationand han-1clvdoublemutants.
increased floral abnormalities as compared with either single han-1wus-1doublemutantsresemblewus-1singlemutants,
mutant. Among all clv mutants, the clv3-2 mutants show the except that they appear darker green in color and have many
strongest phenotype with regards to SAM fasciation and in- moreradial-shaped leaf-likestructures(with trichomes)arising
creasedfloralorgannumbersinallfourwhorls(Figures4Aand fromtheleafaxils(Figure4J).Noflowerswereobservedinhan-1
4B).Interactionsbetweenhan-2andclv3-2orhan-1andclv3-2 wus-1doublemutants(threeplants).Thehan-1/han-1wus-1/þ
makeforveryshortplantswithfasciatedstemsthickerthanthose mutantsarephenotypicallysimilartohan-1singlemutants(data
of clv3-2 single mutants. Early-arising flowers have elongated not shown). We further compared the number of floral organs
pedicels (Figure 4C, arrowhead) and are composed only of present in han-1/þ wus-1/wus-1 flowers versus wus-1/wus-1
reduced sepals (or sepal-like tissue) and carpels, which are flowers. The former have an average of 3.2 (61.1) sepals, 1.5
sometimesindeterminateandgeneratemanysepal-orleaf-like (61.3)petals,and0.5(60.9)stamens(includingin27%ofcases
structuresfromthebase,orontopofanundifferentiatedmass filamentousstructures)in22flowerscounted,whereasthelatter
within the carpel valves (Figure 4D). Later-arising organs are hasanaverageof3.9(61.0)sepals,3.9(60.6)petals,and1.1
either filamentous or are bracts tipped with stigmatic tissue, (60.3)stamens(nofilamentousstructures)of10flowerscounted.
arisingsimultaneouslyinlargenumbers(Figure4C,arrow).The ThePvalueforeachorgantypedifferencebasedonanunpaired
han-2clv1-1doublemutantshaveeitherafairlynormalstemor Student’sttestissmallerthan0.05,suggestingthatthediffer-
are shorter and more fasciated than clv1-1 and accumulate enceissignificant.Thissuggeststhatwhereaswusisepistaticto
Figure4. GeneticInteractionsofhanandclv.
(A)clv3-2inflorescence,topview.
(B)clv3-2flower,topview.
(C)han-1clv3-2doublemutantinflorescence.Arrowheadpointstoanearly-arisingflowerwithlongpeduncle,andarrowpointstolate-arisingbract-like
orfilamentousorgans.
(D)han-1clv3-2doublemutantflower.
(E)han-2clv1-1doublemutantinflorescence.
(F)han-2clv1-1doublemutantflower.
(G)han-2clv2-1doublemutantinflorescence.
(H)han-1clv1-1doublemutantinflorescence.
(I)wus-1singlemutantaxillaryshoot.
(J)han-1wus-1doublemutantaxillaryshoot.
Bars¼1mm.
2592 ThePlantCell
haninoveralldevelopment,flowerdevelopmentinwusmutants appearsdiffusecomparedwiththewildtype,withafewcentral
reliesonHANactivityinadose-dependentmanner. cells showing the strongest expression and surrounding cells
showing weaker expression (Figures 5E and 5G). In the floral
meristem,WUSexpressionisclearlyshiftedtoincludetheL2and
HANRegulatesWUS-ExpressingCells
L1layers(Figures5Fand5H).AsintheSAM,thesignalappears
WUSservesastheearliestknownmarkerforSAMdevelopment. diffuse,withtheborderbetweentheWUS-expressingcellsand
It is detected in the inner two cells at the apical region of the thesurroundingcellsnotassharpasisobservedinthewildtype
16-cell stage embryos, and its expression domain enlarges in or in clv3-2 mutants. In some han meristems, however, WUS
embryonicandpostembryonicSAMsofclvmutants(Mayeretal., expression appears comparable to that in the wild type (see
1998; Brand et al., 2000; Schoof et al., 2000). Because han SupplementalFigure1onlineandDiscussion).
mutationsstronglyenhanceclvSAMphenotypes,weexamined Inhan-1clv3-2doublemutants,theinflorescenceSAMregion
WUSexpressioninbothhansinglemutantsandhanclvdouble is expanded compared with clv3-2 single mutants (Figures 5K
mutants. In wild-type inflorescence meristems and floral meri- and5L).Incontrastwithitsexpressionpatternintheclv3-2single
stems, WUS is expressed in the central region of the SAM, mutant,WUSexpressionintheSAMsofthedoublemutantsis
beneaththeoutermosttwoorthreecelllayers(Figures5Aand concentrated in the L2 layer cells (Figure 5I), and in the floral
5B).Expressioninthefloralmeristemattenuatesafterstage3and meristems, expression extends upward intothe epidermal cell
isnolongerdetectableafterstage6.WUSexpressioninclv3-2 layerinsomecases(Figure5J).EctopicexpressionofWUSin
inflorescenceandfloralmeristemsexpandsoutwardalongthe han-1 or han-4 flowers all along the inflorescences is also
fasciatedmeristemsandupwardintotheL2layer(Figures5Cand observed,appearinginundifferentiatedcellsinlatestageflowers
5D).Inmostofhan-1andhan-4inflorescences,whetherinearly andinthesepals,whichinsomecasesareclearlycarpelloidas
or late stages of development, the WUS expression domain theyproduceWUS-expressingovules(datanotshown).
Figure5. HANRegulatesWUS-ExpressingCells.
(A)and(B)WUSexpressioninwild-typeinflorescenceSAMisbelowtheoutermostthree(shown)ortwo(datanotshown)layers.
(B)WUSexpressioninthewild-typefloralmeristem(FM)isbelowtheoutermosttwolayers.
(C)WUSexpressionintheclv3-2inflorescenceSAMconcentratesinthecellsbelowtheL2layerandatlowerlevelintheL2layer.
(D)WUSexpressionintheclv3-2floralmeristemexpandsintotheL2layer.
(E)and(G)WUSexpressioninmostofthehan-1inflorescenceSAMsisdiffuse.
(F)and(H)WUSexpressioninthehan-1floralmeristemsisshiftedtoincludetheL2andL1layers.
(I)WUSexpressioninthehan-1clv3-2doublemutantinflorescenceSAMisconcentratedintheL2layer.
(J)WUSexpressioninthehan-1clv3-2doublemutantfloralmeristemextendsinsomecasesintotheL1layer.
(K)Overviewofaclv3-2inflorescenceSAM.
(L)Partialviewofahan-1clv3-2doublemutantinflorescenceSAM.
Bars¼50mm.
ArabidopsisHANandMeristemDevelopment 2593
TofindouthowearlytheaberrantWUSexpressionoccursin was altered in clv1 mutants. HAN shows a similar expression
hanmutants,aswellashowearlyitsimpactonmorphological pattern in the SAM of clv1-4 mutants as in clv3-2 (data not
developmentismanifested,embryosfromhan-1/þsiliqueswere shown).However,HANisnotectopicallyexpressedintheclv2-1
examined.Morphologicaldefectscanbeobservedasearlyas SAM(datanotshown).TofindouthowearlyHANexpressionis
globular stage, when the shapes of what are likely the han-1 perturbed, we examined HAN expression in clv3-2 embryos.
mutantembryosdeviatefromthewildtype(Figures6Aand6B). Fromglobularstagetomatureembryos,theHANexpressionis
Bytheheartstage,somemutantembryosclearlyfailtoproperly similarinwild-typeandclv3-2mutants(Figures8Eto8G).This
developcotyledons,whichappearassmallstubs(Figure6D)or suggeststhatHANexpressionisalteredintheclv3-2SAMduring
are barely visible (data not shown). By the torpedo or walking postembryonicdevelopment.
stickstages,mutantembryosappearstuntedtovariousdegrees,
with a majority having thickened hypocotyls and small cotyle-
PhenotypesCausedbyEctopicExpressionofHAN
donsinnumbersrangingfromtwotofour(Figures6Fand6H).
Inwild-typeembryos,WUSisexpressedinthecentertwocells ToexaminetheeffectsofHANgain-of-function,wegenerated
atthesubepidermallayeroftheapicalregionattheglobularand transgenicplantscarrying35S:HANor35S:HAN-GR(glucocor-
transitionstages(Figure6I).Attheheartstage,WUSexpression ticoidreceptorbindingdomain;Lloydetal.,1994)transgenes.
shiftstothecentraltwocellsofthecorpus(Figure6K).Inmature Among Landsberg erecta (Ler) plants transformed with
embryos,WUSisexpressedevendeeperinthemeristemandis a35S:HANconstruct,onlyfourT1transgenicplantswerepro-
excludedfromtheL2layer(Figure6M).However,inapparenthan duced from >4 mL (;1.5 3 104) of seeds screened, which is
mutant embryos, WUS expression is observed in both L2 and substantiallylowerthanthenormaltransformationefficiencyfor
corpus cells and in more than two cells in the transition stage thebinaryvectorused(>0.05%).Ofthefour,onelookedfairly
embryo,whichcorrespondstotheenlargedsizeoftheembryo normal,buttheothersweresmallinsize,withunevenlyshaped
(Figure 6J). At the heart stage, WUS expression is likewise rosetteleavesandcaulineleavesthatgraduallyturnedpurple,
concentrated in both L2 cells and deeper cells (Figure 6L). By startingfromtheabaxialside(Figure9A).Thesmallestoneofthe
the walking stick stage, expression in some apparent han-1 threediedprematurelyaftermakingsomeminiatureflowersthat
embryos stays strong in L2 cells (Figures 6N to 6P). Aberrant did not produce seeds. The remaining two plants had smaller
WUSexpressioninearlystagehanembryossuggeststhatHANis inflorescences than the wild type (Figures 9B and 9C). Floral
normallyrequiredtocontrolthenumberofWUSexpressingcells organswereshorterinlengthwhencomparedwiththewildtype
andtocorrectlypositionthesecells. atsimilarstages.Thegynoeciumwasshortandalsoappeared
Because there isafeedback loop interaction between WUS bulky,withwavysurfacesandelongatedpedunclescompared
and CLV3 in shoot and floral meristems (Brand et al., 2000; with the wild type (Figures 9D and 9E). The scarcity of T1
Schoofetal.,2000),wealsoexaminedCLV3expressioninhan transformants suggests that constitutive overexpression of
mutantsinbothembryonicandinflorescencestages.Noobvious HANmightbedeleterious.
difference in CLV3 expression was observed between han More than 50 T1 35S:HAN-GR transgenic plants were gen-
mutant and wild-type plants during embryogenesis, but CLV3 eratedandalllookednormalintheabsenceofdexamethasone
expression becomes variable in han inflorescence meristems (DEX).Whentreatedthreetimesat1-dintervalswith10mMDEX
(Figure7).Inapproximatelyhalfofthecases,CLV3expression solution, the transgenic plants showed a distinctive morphol-
appearsfairlynormalascomparedwiththewildtype(Figures7C ogy. Young flowers opened up precociously (Figure 9F), and
and7Dversus7Aand7B).However,intheotherhalfofthecases, stem elongation was retarded. In the 2-d interval between the
CLV3 expression becomes slightly (Figure 7E, arrow) or more 2nd and 3rd treatments, the mock-treated control plants in-
widelydiffuse(Figures7Fto7H,arrows).Theeffectofhanonthe creased in height an average of 2.1 cm, whereas the DEX-
CLV3expressiondomaincouldbebecauseofthealterationof treatedtransgenicplantsonlyincreasedanaverageof0.8cm.
theWUSexpressiondomainthatoccursinearlyembryogenesis When seedlings from a homozygous T2 line were treated with
beforeCLV3expressionisinitiated. 10 mM DEX four times at 1-d intervals, starting on postgermi-
nation day 11 (solution applied via soil), plants began to show
accelerated leaf senescence after the second treatment. New
InteractionsbetweenHANandtheCLVPathway
leaves were still produced, but leaf expansion was inhibited,
TouncoverpotentialregulatoryinteractionsbetweenHANand and leaf blades were serrated, similar to the leaves of the
CLV,wealsoexaminedHANexpressioninclv3-2mutants.The 35S:HANtransgeniclines(Figure9H,arrow).Twelvedaysafter
HANexpressionpatterninclv3-2wasunchangedinalltissues the last treatment, the mock-treated control group plants had
exceptforthemeristems,whereHANwasdetectedstronglyin already bolted (Figure 9G), but approximately half of the DEX-
theL2andcorpuslayersalongtheentirefasciatedSAMaswellas treatedplantsweredead,andthesurvivingplantsremainedin
inthelatestagefloralmeristems(Figures8A,arrowheads,8B, miniatureform(Figure9H).Theminiatureplantswerestillableto
largearrow).ThisexpressionpatternmimickedtheWUSexpres- produceflowersthatgrewextremelyslowlyandneverreached
sionpatterninclv3-2mutants(Figure8C,arrowhead,8D,large normal size. These flowers had normal organ identities and
arrow), suggesting that in the absence of CLV3 activity, HAN numbers but were male sterile. When seeds from a T2 homo-
expressionisectopicallyinducedintheapicalregionoftheSAM. zygous line were germinated on DEX plates, all seedlings
Because WUS is also ectopically expressed in clv1 mutants ceased growth at the stage shown in Figure 9I and did not
(Schoofetal.,2000),wealsoassessedwhetherHANexpression develop further. Transgenic seedlings on non-DEX MS plates
2594 ThePlantCell
Figure6. hanMutantEmbryoDefectsandPerturbedWUSExpressioninhanEmbryos.
(A)Globularstagewild-typeembryo.
(B)Globularstagehan-1mutantembryo.
(C)Lateheartstagewild-typeembryo.C,cotyledon.
(D)Lateheartstagehan-1embryowithstuntedcotyledons.
(E)Walkingstickstagewild-typeembryo.
(F)Walkingstickstagehan-1embryo.
(G)Maturewild-typeembryo.
(H)Maturehan-1mutantembryowiththreecotyledons.
(I)WUSexpressioninthetransitionstagewild-typeembryoisconcentratedintwocellsinthesubepidermalL2layer.
(J)WUSexpressioninthetransitionstagehan-1embryoislocatedinmorethantwocellswithinandbeneaththeL2layer.
(K)WUSexpressionintheheartstagewild-typeembryoisshiftedtotwocentralcellsinthecorpus.
(L)WUSexpressionintheheartstagehan-1embryointheL2layer.
(M)WUSexpressioninthematurestagewild-typeembryoiscenteredbelowtheoutermosttwolayers.
(N)WUSexpressioninthematurehan-1embryo.
(O)and(P)WUSexpressionintheL2layerofmaturehan-1embryos.(P)isanenlargedviewof(N).
Bars¼50mmin(A)to(H)and10mmin(I)to(P).
ArabidopsisHANandMeristemDevelopment 2595
wasfoundintheDEX-treatedseedlings(Figure9N),suggesting
that they had already lost active SAM activity. Similarly, STM
expression is also lost in treated seedlings by day 6 (data not
shown). HAN expression in the same plants was strong and
ubiquitous (data not shown). DEX-treated 35S:HAN-GR seed-
lings had smaller cells in all cell layers of the cotyledons
comparedwithuntreatedones(datanotshown),whichexplains
at least in part why the treated plants have small cotyledon
blades.Inaddition,stomatalporesinthecotyledonsofinduced
seedlings were often composed of three or more guard cells
insteadoftwocellsasinthecontrol(Figures9Oto9Q),implying
abnormal cell division of the guard cell mother cells or in-
completedifferentiationoftheguardcells.Incontrastwiththe
cotyledoncells,therootcellsoftheinducedseedlingswerein
generallargerinsizethanthoseofthecontrolgroup(Figures9R
and 9S). Additionally, in the induced roots, cell files were not
as organized as in the control, and root caps were usually
Figure7. CLV3ExpressioninhanMutants.
(A) and (B) CLV3 expression in the wild-type inflorescence SAM and
floralmeristem(FM)isconcentratedinthecentraloutermostthreelayers,
ataboutthreetofourcellswidth.
(C) and (D) CLV3 expression appears fairly normal in some han in-
florescenceSAMsandfloralmeristems.
(E)CLV3expressionisslightlydiffuseinsomehanmutants.
(F)CLV3expressionisexpandedinsomehanfloralmeristems.
(G)and(H)CLV3expressionismarkedlydiffuseinsomehanmutants.
Arrowspointtotheregionsofdiffuseexpression.
Bars¼50mm.
grewnormally,asdidwild-typeseedlingsonDEXandnon-DEX
plates.
Stalledgrowthin35S:HAN-GRDEX-treatedtransgenicseed-
lings suggests inhibited cell proliferation/growth and possible
Figure8. HANIsEctopicallyExpressedinclv3-2Mutants.
cessationofshootandrootmeristemactivity.Toevaluatethis,
theSAMstructuresofhomozygous35S:HAN-GRseedlingsthat (A)HANexpressioninaclv3-2inflorescenceSAM(arrowheads).
were germinated on either DEX or non-DEX plates were (B)HANexpressionintheundifferentiatedcenterdomecellsinalate
examined on postgermination day 10 using scanning electron stageclv3-2flower(largearrow).SmallarrowpointstoHANexpression
microscopy.Whereasthenon-DEX-treatedcontrolSAMswere intheintegumentsofovule.
(C)WUSexpressioninaclv3-2inflorescenceSAM(arrowhead).
dome-shapedlikewild-typeSAMs(Figure9J),theDEX-treated
(D)WUSexpressionintheundifferentiatedcellsinalatestageclv3-2
SAMs either were flat (Figure 9K, arrow) or were no longer
flower (large arrow). Small arrows point to WUS expression in the
identifiable between the leaf primordia (Figure 9L, arrow).
nucellus.
Because WUS serves as a marker of active meristematic cell
(E)HANexpressioninaheartstageclv3-2embryo.
activity in the SAM during normal development, we examined (F)and(G)HANexpressionintheSAMandtherootofaclv3-2embryo
WUSexpressioninday10plantsaswellasinday6seedlings inlatetorpedostage,respectively.
germinatedonDEXplates.Inbothcases,noWUSexpression Bars¼50mm.