The Shoot Stem Cell Niche in Angiosperms: Expression Patterns of

13 Pages·2006·1.21 MB·English

The Shoot Stem Cell Niche in Angiosperms: Expression Patterns of WUS
Orthologues in Rice and Maize Imply Major Modifications in the
Course of Mono- and Dicot Evolution
Judith Nardmann and Wolfgang Werr
Institut fu¨rEntwicklungsbiologie, Ko¨ln, Germany
InArabidopsis,stemcellhomeostasisintheshootapicalmeristem(SAM)iscontrolledbyafeedbackloopbetweenWUS
andCLVfunctions.WehaveidentifiedWUSorthologuesinmaizeandricebyadetailedphylogeneticanalysisoftheWOX
gene family and subsequent cloning. A single WUS orthologue is present in the rice genome (OsWUS), whereas the
allotetraploid maize genomecontains 2WUS paralogues(ZmWUS1 andZmWUS2).
NoneoftheisolatedgrassWUSorthologuesdisplaysanorganizingcenter–typeexpressionpatterninthevegetative
SAMasinArabidopsis.Incontrast,thegrass-specificexpressionpatternsrelatetothespecificationofnewphytomers Do
consistentwiththetranscriptionalexpressionpatternsofTD1andFON1(CLV1orthologuesofmaizeandrice,respec- w
n
tively).Moreover,thegrassWUSandCLV1orthologuesarecoexpressedinallreproductivemeristems,wherefasciation lo
a
andsupernumeraryfloralorgansoccurintd1orfon1loss-of-functionmutants.TheexpressionpatternsofWUSortho- de
lwoigthuecshiannbgoetshingrCasLsVsp1esciigensacloinmgp,ahraevdewoictchutrhroedsedoufridnigcoatnsgimiopsplyerthmatemvoaljuotriocnhaanngdersaiinseWdUouSbftusnacbtoiount,thwehuicnhiqaureenceosrsreolfattehde d fro
m
WUS/CLVantagonism in the maintenance of the shootstem cell nicheingrasses. h
ttp
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Introduction et al. 2005) and applicable to maize (Whipple et al. 2004) ://a
c
despite the different architecture of the grass flower. a
Angiosperms, flowering plants, comprise the largest d
group of plants today with more than 235,000 species in Intheshootapex,amechanismregulatingthebalance em
betweenindeterminate(meristemmaintenance)anddetermi- ic
over300families,withfossilrecordssuggestingthatmany .o
nate growth (leaf development) also appears to be ancient. u
ofthemodernfamiliesandgeneramayalreadyhaveexisted p
75 MYA. Angiosperms are considered as a monophyletic KNOX (knotted1-like homeobox) genes are expressed in a .co
m
group, a true clade, comprising 2 classes—the monocoty- meristem-specificmannerandaredownregulatedinprimor- /m
ledons and the dicotyledons—which diverged over 150 dial cells (Vollbrecht et al. 1993) by the activity of MYB be
MigiYnAre(laWtiivkesttroomtheantrdemKeenndriocuks2n0u0m1)b.eTrhoefmspoencoipehsysluegtigceostrs- oSrHthEoAloTgHue2s, (APRHPA:NATSAYSMTIMCEAT:RIWCaLitEesAVeEtS1a,l.RO1U9G98H; /article
thatancestralangiospermgenomeswerepronetorapiddi- Timmermans et al. 1999; Tsiantis et al. 1999; Byrne et al. -a
b
vmearisziefic(aZteioanminaytsheL.c)oiusrsaesopfeceivaolleuxtiaomnp.lTehbeeceavuosleutiiotnwoasf 2K0N0O0)X, –rAecRePntplyathrwevaiyewisendotinonlPyiaczoznaseervtedalb.e(t2w0e0e5n).moTnhois- strac
forced by human selection through domestication over anddicotsbutwasalsoindependentlyrecruitedintomicro- t/23
the last 6,000–10,000 years (reviewed in Doebley 2004). phyll development in lycophytes (Harrison et al. 2005). In /12
Maize still hybridizes with its ancestral wild teosinte rela- contrast,therearemajordifferencesinotheraspectsofleaf /24
9
tives Z. mays. ssp parviglumis or ssp. mexicana, and pio- development between mono- and dicots. Two paralogous 2
/9
neering genetic analyses led to the identification of few loci in maize NARROW SHEATH1/NARROW SHEATH2 7
4
major quantitative trait loci (QTL) that account for major (NS1/2) encode homologues to the Arabidopsis PRESSED 43
6
changes in the plant architecture of maize compared with FLOWER (PRS) gene (Nardmann et al. 2004). NS1/2 and b
y
the wild species such as the maize female inflorescence, PRS have maintained their marginal expression domain in g
u
theearbeinguniqueintheplantkingdom.SomeQTLshave lateralorganprimordia.Theprsmutantphenotype,however, e
s
been resolved into individual genes often encoding tran- ismostlyrestrictedtolateralsepalsoftheflowerinArabidop- t o
n
scriptionfactorssuchasteosintebranched1(tb1;Doebley sis,whereasthemaizehomologuescontributetothespecifi- 2
0
etal.1997),teosinteglumearchitecture1(tga1;Wangetal. cationofalateralleafdomainattheflankoftheshootapical N
2005), and zfl2 (Z. mays FLORICAULA/LEAFY2), a can- meristem(SAM)duringthevegetativephase(Scanlon2000). ov
e
didate gene for a QTL controlling ear rank differences Some remarkable differences exist between meristems m
b
betweTehnemgraoiszsemanodrptheoolsoingtieca(lBcohmanbgleiessbeatseadl.o2n0s0i3n)g.leloci osifs,dtihcoetSsAanMdmapopneoacrosttso.Ibned3icloatysepreedc,iewsiitnhcalutduinnigcaAcraobmidporips-- er 20
1
raise the question how common are regulatory networks ing 2 clonal layers (L1 1 L2) and the corpus commonly 8
elaboratedinthedicotmodelspeciesArabidopsisinmono- designated as the L3 layer (Szymkowiak and Sussex 1996;
cotssuchasmaize.ComparativeapproachesintheMADS EvansandBarton1997).Incontrast,monocotssuchasmaize
box gene family have provided some evidence that basic have only 1 histologically apparent single tunica layer (L1)
principles of the ABC model of flower development such andtheinnercorpus(Abbeetal.1951;Steffensen1968).Ra-
as B-function genes are conserved in angiosperms (Zahn dially,intheArabidopsisSAM,thecentralstemcellzonecan
bedistinguishedfromtheperipheralzone,wherecellsbegin
Key words: WUSCHEL orthologues, maize, rice, angiosperm to differentiate. Vegetative leaves in Arabidopsis originate
evolution.
from few founder cells specified in a spiral phyllotaxy in
E-mail:[email protected].
theperipheralzone(IrishandSussex1992),whereasincon-
Mol.Biol.Evol.23(12):2492–2504.2006 trast,themaizeleafmaybetracedbacktoapproximately200
doi:10.1093/molbev/msl125
AdvanceAccesspublicationSeptember20,2006 leaf founder cells (Poethig 1984), which are recruited from
(cid:1)TheAuthor2006.PublishedbyOxfordUniversityPressonbehalfof
theSocietyforMolecularBiologyandEvolution.Allrightsreserved.
Forpermissions,pleasee-mail:[email protected]
WUSCHELOrthologuesinMaizeandRice 2493
the whole circumference of the shoot apex and give rise to Materials and Methods
an alternate phyllotaxy. Cloning of ZmWUS1 and ZmWUS2
ThemaintenanceofpluripotentstemcellsintheSAM
To amplify the homeobox of the maize WUS ortho-
of Arabidopsis depends on the WUSCHEL/CLAVATA
logues, polymerase chain reaction (PCR) was performed on
feedbackloop.TheWUSCHEL(WUS)homeoboxgenepro-
genomicDNAofRscm2maizeusingtheprimerpairZmHD1
vides a key function for the maintenance of stem cell ho-
(5#-TGGACICCIACIACIGARCARAT-3#)andZmHD2(5#-
meostasis in the SAM (Mayer et al. 1998). WUS acts cell
GCYTTRTGRTTYTGRAACCARTARAA-3#).Isolationof
autonomously and gene expression is restricted to a few
totalRNAfollowedtheprotocolofChomczynskiandSacchi
cellsintheL3layerconsideredtobethestemcell–organizing
(1987), and rapid amplification of the 3#-cDNA ends was
center (OC). Whereas WUS promotes stem cell fate, it is
then performed on RNA of Rscm2 female inflorescences
antagonized by CLAVATA (CLV) signaling (Brand et al.
withprimerscorrespondingtothehomeoboxusingtheFirst-
2000).Actinginasinglepathway,3CLVgenes(CLV1–3)
ChoiceRLM-RACEKit(Ambion,Austin,TX)accordingto D
encode a heterodimeric transmembrane receptor kinase o
the manufacturer’s protocol; 3#-RACE was performed with w
(sCpoLnVd1in/2g;pCollayrpkeepttiadl.e1l9ig9a7n;dJe(oCnLgVe3t;alF.l1e9tc9h9e)raentdailt.s1c9o9rr9e)-. p3r#i)mearnsdZmZWmUWSaU(S5b#-CT(5C#T-GAACCTAACAGGGACTTCGGCAGGGGCGATTACAC-- nloade
WUS activity is restricted via CLV signaling via a yet un- d
knownsignaltransductionpathway(Brandetal.2000). G3#A-eAndCs-3o#f).2Andaiflyfesriesnotfgtehnees3#w-ReAnCaEmepdrodZumcWtsUreSv1eaalnedd from
BommTheertreectenalt.cl2o0n0in5g) oafnTdHFICLOKRTAALSSOELRGDAWNARNFU1M(TBDE1R; ZusmeWd:UZS2m.WFoUr5S#1-aRA(5C#E-C,pAriAmTeCrsGspGeAciAfiCcfToCrZGmTWGUTCS1CwAeGre- https
(FON1;Suzakietal.2004),CLV1orthologuesfrommaize CATCAC-3#) and ZmWUS1b (5#GAACAACCTGAA- ://a
andrice,respectively,andofFASCIATEDEAR2(FAE2),a GAAGGCGACAACGAG-3#). 5#-RACE for ZmWUS2 cad
CLV2 orthologue from maize (Taguchi-Shiobara et al. e
was performed with the primers ZmWUS2a (5#-CCAT- m
2m0o0n1o)c,ohtas.sTsuheggeexspterdessthioant CpaLtVternsisgnoaflitnhgeseisgceonnesseravsewdeilnl T(5A#-TCCAGTTGGTTACGCTGCACCTGGACCAGGAGGAAAAGGC-A3#T)GaTnCdTZCm-3W#)U.S2b ic.oup
as the phenotypes of corresponding loss-of-function mu- .c
AllPCRproductswereclonedintopCRIITOPO(Invi- o
tants, however, imply major modifications in the control m
ofmeristemsizeingrasses.InArabidopsis,CLV1transcrip- trogen,Carlsbad,CA)andsequenced.ZmWUS2wasmapped /m
to chromosome 10, between markers bnlg1450 and asg19b, b
tionisconfinedtotheL2andL3layersoftheSAMthrough- usingtheDNAKitof94IBMLines(http://www.maizemap. e/a
toruatnsthceripptlsanatrelinfeotcdyectleec(tCedlairnktheteavle.g1e9t9at7i)v.eInSAcoMntbrausttf,oTuDnd1 org/dna_kits.htm). rticle
-a
in early leaf primordia (Bommert et al. 2005). During the Computational and Database Analysis bs
reproductivephase,however,TD1istranscribedthroughout tra
the inflorescence meristem (IM), the descending spikelet- Analysis of DNA and protein sequences was per- ct/2
pairmeristems(SPMs),spikeletmeristems(SMs),andfloral formedusingtheWisconsinGCGsoftwarepackageversion 3/1
meristem(FMs).Duringlaterstagesoffloraldevelopment, 7.0 (University of Wisconsin Genetics Computer Group). 2/2
TD1transcriptsaccumulateindifferentiatedorganssuchas Homology searches were performed using TBlastN at 49
glumes, lemma, and palea, reminiscent of the expression National Center for Biotechnology Information (http:// 2/9
patterninleafprimordiaduringvegetativephase(Bommert www.ncbi.nlm.nih.gov/blast/), DNA Data Bank Japan 74
4
et al. 2005). Similarly, the rice orthologue FON1 is tran- (http://www.ddbj.nig.ac.jp), or the Plant Genome Data- 3
6
scribedthroughouttheFMbutalsoindifferentiatedlateral base (http://plantgdb.org) with default parameters. Multi- b
y
floral organs (Suzaki et al. 2004). ple sequence alignment was performed using ClustalW g
u
AsCLV1signalingisthoughttorestrictWUSactivity, (http://www.ebi.ac.uk/clustalw) and BOXSHADE (http:// es
thedifferencesinthetranscriptionpatternsofCLV1ortho- searchlauncher.bcm.tmc.edu/multi-align/multi-align.html). t on
logues between maize and rice or Arabidopsis raised the The PHYLIP (http://evolution.genetics.washington.edu/ 2
0
questionwhethertheexpressionpatternofWUSorthologues phylip.html) program was used for phylogenetic and mo- N
o
ingrasseswasalsoconserved.WUSisthefoundingmember lecularevolutionaryanalysesbasedonthemaximumlikeli- ve
of the so-called WOX (WUS homeobox) gene family en- hoodmethod.Sequencesselectedforthephylogenetictree mb
coded by 15 genes in the Arabidopsis genome (Haecker infigure1Aareasfollows(accessionnumbersinparenthe- er 2
et al. 2004). Here, we describe the identification of WUS ses): ZmNS1 (AJ536578); ZmNS2 (AJ472083), and Os- 01
orthologues in maize and rice by a detailed phylogenetic WOX5 (Q8WOF1); AtWOX1–AtWOX14 as published 8
comparisonoftheWUS/WOXgenefamilybetweenthedi- recently (Haecker et al. 2004). Accession numbers for
cots Arabidopsis thaliana and Populus trichocarpa and 2 the remaining homeodomains (HDs) are listed in table 1.
monocot species Oryza sativa and Z. mays. The allotetra- Accession numbers of the genes encoding the proteins
ploidmaizegenomecontains2WUSparalogues(ZmWUS1 for the sequence alignment are ZmWUS1 (AM234744),
andZmWUS2),whereasasingleWUSorthologueispresent ZmWUS2 (AM234745), OsWUS (AB218894), ROA
inthesmallerricegenome(OsWUS).ConsistentwithTD1/ (AY162209), TER (AF481951), PtWUS (AM234747),
FON1expressiondata,theexpressionpatternsofWUSor- and AtWUS (At2g17950).
thologues in both grass species imply that major changes
In situ Hybridization
duringangiospermevolutionhaveoccurredandraisedoubts
about the uniqueness of the WUS/CLV antagonism in the For nonradioactive in situ hybridization, samples
maintenance of the shoot stem cell niche in grasses. were prepared following the protocol of Jackson (1991).
2494 NardmannandWerr
For sections of maize embryos, kernels were trimmed on branch encode HDs with thetypicalextra tyrosine residue
both sides of the embryo axis for better penetration of intheloopbetweenhelix1andhelix2(fig.1B).Theamino
the formaldehyde fixative and the wax solution. Paraffin- acid similarity between ZmWUS1 or ZmWUS2 and At-
embedded tissue was sectioned by the use of the Leica WUS of Arabidopsis is 83% or 85%, respectively, and
RM 2145 rotary microtome, and 7-lm sections were used the similarity is higher when compared with OsWUS
for hybridization. Probes for in situ hybridization were (97%ineachcase).Wealsoincluded2recentlyidentified
clonedeitherinsenseorantisenseorientationtotheT7pro- WUSorthologues,ROA(ROSULATA;Kiefferetal.2006)
moter and then used as a template for synthesis of digoxi- andTER(TERMINATOR;Stuurmanetal.2002),fromthe
genin-labeled RNA probes by T7 RNA polymerase as dicot species Antirrhinum majus and Petunia hybrida, re-
described(Bradleyetal.1993).Insituprobesforthegrass spectively, in this alignment. Even between the dicot spe-
WUSgeneswerechosentocontainsequencesdownstream cies,thereislittlesequenceconservationbetweentheWUS
of the homeobox and amplified via PCR: ZmWUS1 pos. orthologuesoutsidetheHDapartfromafewshortmotifs:
D
294–951 of accession AM234744, ZmWUS2 pos. 267– an acidic domain, the WUS box of unknown function o
w
978ofaccessionAM234745,OsWUSpos.435–870ofac- (Haecker etal.2004),andan ethylene-responsiveelement nlo
cessionAB218894.InsituprobesforTD1andFON1were binding factor-associated amphiphilic repression (EAR)– ad
e
usedaspublished(Suzakietal.2004;Bommertetal.2005). like domain (Ohta et al. 2001). The EAR-like domain is d
The probe for KN1 corresponded to the mRNA sequence of only found in WUS and its orthologues ROA and TER fro
m
accession AY312169 (Vollbrecht et al. 1993). andnotinothermembersoftheWOXfamilyinArabidop- h
sis. The presenceof this domainin ZmWUS1,ZmWUS2, ttp
s
Light Microscopy and Image Processing and OsWUS, therefore, is further evidence for their being ://a
ImagesweretakenusinganAxioskopmicroscopeequip- WUS orthologues in these monocot species. ca
d
Wewerenotabletoidentifypolymorphismsincoding e
pedwithanAxiocamcamera(Zeiss,Oberkochen,Germany). m
PictureswereprocessedusingAdobePhotoshopversion7.0. oZrmWimUmSe1diinatdeiffueprsetnrteammappanindgpdoopwunlasttrioeanmsansdeqwueernectehseroe-f ic.ou
p
Results fore unable to map the gene so far. The map position of .co
ZmWUS2residesinaregiononchromosome10,whichis m
WUS Orthologues in Rice and Maize duplicatedonchromosome2.Theduplicationofthemaize /m
b
WhereasmembersoftheWUS/WOXgenefamilyfrom genome is also evident in the WOX2, 5, or 13 branches, e/a
rice could be identified from the genome sequence (Goff where 2 maize paralogues group with single orthologues rtic
et al. 2002), the maize family members had to be isolated in Oryza, Arabidopsis, or Populus (fig. 1A). Additionally, le
-a
using a combinatorial approach: firstly, reverse transcrip- theduplicatednarrowsheath1and2loci,whichencodepa- b
s
tase (RT)–PCR was performed on cDNAs prepared from raloguesofPRSinArabidopsis,supportthisgenomedupli- tra
c
immature embryos, roots, shoots, and inflorescences with cation and indicate conserved gene functions, although t/2
degenerate primers designed to amplify the conserved adaptedtograss-ordicot-specificdevelopmentalprograms 3/1
HD-encoding sequences of WUS/WOX relatives (Haecker (MatsumotoandOkada2001;Nardmannetal.2004).Other 2/2
et al. 2004). Secondly, the available maize genome se- significantaspectsofthephylogenyarethatorthologuesof 49
2
quences were screened for WUS/WOX HD-encoding se- AtWOX1, 6, 7, 8, 10, and 14 are absent in the 2 grasses, /9
7
quences. In total, 16 members of the WUS/WOX gene whereasorthologuesofAtWOX1,6,and7arefoundinthe 4
4
familywere identified andsubjected tophylogenetic anal- seconddicotgenome(P.trichocarpa).Notably,orthologues 3
6
ysesbasedontheHDaminoacidsequence(fig.1A).Inad- ofAtWOX8,10,and14arealsoabsentfromthePopulusge- b
y
dition to the identified rice members, the newly identified nome,andforAtWOX10andAtWOX14,noexpressionwas gu
e
maize(Zm:Zeamays)genes,andthefoundingArabidopsis detected in Arabidopsis (Haecker et al. 2004). In contrast, s
thaliana (At) family members, we included theWOX HD however,thetranscriptionalactivityofallmaizegenesexcept t on
sequences from a second dicot Populus trichocarpa (Pt). ZmWOX2Bcouldbeconfirmedbysplicedintron–spanning 2
0
Similar to the rice genome (Goff et al. 2002), the populus RT–PCRamplicons(datanotshown). N
o
genome has been fully sequenced (http://genome.jgi-psf. ve
m
org/poptr1_1/poptr1_1.home.html) and therefore should OsWUS, the Single Rice WUS Orthologue Marks Leaf be
providethefullWOXgenecomplement.Theresultingphy- Margins and Is Transiently Expressed in the SAM r 2
logenetictreeunambiguouslyreveals2WUSparaloguesto 01
8
existinmaize,ZmWUS1andZmWUS2,andasingleortho- As phylogenetic analyses revealed a single WUS ho-
logue in rice, OsWUS. All 3 monocot WUS genes group mologueinrice,OsWUS,RNAinsituhybridizationswere
within1uniquesubbranchtogetherwiththeir2dicotortho- performed in young rice seedlings, which uncovered re-
logues, AtWUS and PtWUS. Only members of this WUS markable differences between the expression patterns of
!
FIG.1.—WUSCHEL/WOXgenesinmaizeandrice.(A)PhylogenetictreebasedonHDsequenceinformationofnewlyisolatedZ.mays(Zm)WUS/
WOXrelativesincomparisontotherelativesencodedbytheArabidopsisthaliana(At),Oryzasativa(Os),andP.trichocarpa(Pt)genomes.(B)Protein
sequencecomparisonofArabidopsisWUS(AtWUS)withitsorthologueinPopulus(PtWUS),Antirrhinum(ROA),Petunia(TER),the2maizeWUS
paralogues,ZmWUS1orZmWUS2,andthesinglericeWUSorthologue(OsWUS).TheextratyrosineresiduespecificfortheWUSHDisindicatedbyan
arrow.ThepositionsoftheHD,theWUSboxes,andtheEAR-likedomainareindicatedbygraylines.
WUSCHELOrthologuesinMaizeandRice 2495
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2496 NardmannandWerr
Table1 maxima dynamically differed between opposing flanks of
Accession Numbersof WOXHDs the apex (data not shown). Moreover, consecutive (7 lm
thick) transverse sections showed that the height of this
Name Accession Name Accession
ofHD Number ofHD Number ZmWUS1domaininthecenteroftheapexchangesrelative
to the apical tip in the course of development, as does the
ZmWOX2A AM234767 OsWOX9 AM234752
ZmWOX2B AM234778 OsWOX9C AM234753 relativeheightofP1andP0(fig.3E).Thevegetativemaize
ZmWOX4 AM234768 OsWOX11/12 AM234754 phytomercomprisesaleafattachedtoanode,aninternode,
ZmWOX5A AM234769 OsWOX13 AM234755 and an axillary bud. Although located close to the midrib
ZmWOX5B AM234770 PtWOX1A AM234756
position of the subtending phytomer, the axillary meri-
ZmWOX9A AM234771 PtWOX1B AM234757
stem clonally relates to the internode above. The lateral
ZmWOX9B AM234772 PtWOX2 AM234758
ZmWOX9C AM234773 PtWOX4 AM234759 ZmWUS1maxima,therefore,coincideeitherwiththemid-
ZmWOX11/12A AM234774 PtWOX5/7A AM234765 rib positionsoftheemerging leafprimordia,P andP ,or
1 0 D
ZmWOX11/12B AM234775 PtWOX5/7B AM234766 withthepositionsoftheaxillarymeristemsattheopposite o
ZZmmWWOOXX1133AB AAMM223344777767 PPttWWOOXX69 AAMM223344776601 face of the apex. wnlo
OsNS AM234748 PtWOX11/12A AM234763 ToelucidatethepositionofthelateralZmWUS1max- ad
e
OsWOX2 AM234749 PtWOX11/12B AM234764 ima, we performed side-by-side in situ hybridization d
OsWOX4 AM234750 PtWOX13 AM234762 experimentswithaKnotted1(KN1)probe(Vollbrechtetal. fro
m
1993).TheZmWUS1transcriptiondomainsattheheightof h
the P and P phytomers in the shoot apex are shown in ttp
dOinospyWsoiUusnS(gManlaedyaeftrhpeortsimealoe.sr1dt9ai9ba8liw)s.hitOehdsaWfopUrrSothnteorauWnnsUccerSdipgtpserneaefreeirndeneAtcerecatfbeoid-r fiwgituhre0th3eE,KwNh11icphroablseo. dTehpeicPts1asdejcatcioennst sienctfiiognusrehy3bEridshizoewd s://acad
a downregulation of KN1 expression at the left flank of e
lateralleafmargins.Depictedinfigure2A1Bareleafpri- m
the SAM corresponding with the midrib of the emerging ic
mareoredviiadPen1,tPat2,thaendvePry3,taipndofhPighaensdtOPsWlatUerSaltrmanasrcgriinpst.lIenvtehles P1leaf.ZmWUS1activityisabsentintheseprimordialcells .oup
2 3 butoverlapswiththeKN1expressioninacentraldomainof .c
center of the shoot apex, where WUS is exclusively ex- the apex. However, in a deeper section, ZmWUS1 expres- om
pressedintheArabidopsisSAM,OsWUStranscriptswere sionextendstowardtheflankoftheSAMoppositetotheP /m
1 b
undetectable in the majority of consecutive sections (total midrib. Therefore, ZmWUS1 transcripts are absent in e/a
15)throughthericeSAM.However,inasmallnumberof foundercellsoftheP1leafsheath/bladebutaretranscribed rtic
consecutivesections(5outof15),OsWUSexpressionwas in cells of the prospective node/internode in the center of le
also detected in the center of the vegetative SAM in 1–2 -a
the shoot apex and in the subtending axillary meristem, b
s
mseacytiobnesbaetstthexephlaeiingehdt boyf athteraPns0iepnhtyetoxmpreerss(ifiogn.o2fAO)s.WTUhiSs which is located opposite to the P1 midrib. Consequently, trac
theexpressionmaximaattheflanksoftheapex(seefig.3D) t/2
intheshootapexincontrasttothestableOC-typeexpres- relatetotheformationofaxillarymeristemsandnottothe 3/1
sionpatternofWUSinArabidopsis.Duringlaterstagesof specificationofleaffoundercellsattheprospectivemidrib 2/2
phytomerdevelopment,OsWUSisexpressedattheabaxial position, and subsequently, the ZmWUS1 expression do- 49
face of emerging axillary meristems (fig. 2C). 2
main is confined to the abaxial face of the axillary bud /9
Thesedifferencesinexpressionpatternsandtheiden- 7
(fig. 3G). 4
tification of 2 WUS ortholgues in maize, ZmWUS1 and Insummary,theZmWUS1transcriptiondomaininthe 43
6
ZmWUS2, raised questions concerning the expression do- center of the shoot apex initiates as a disc-shaped expres- b
y
mains of the maize paralogues and whether there are siondomainattheheightofthenewP phytomers,whichis g
0 u
grass-specific alterations in the WUS expression domains e
reminiscentofthetransientexpressionofthesingleOsWUS s
compared with Arabidopsis. The expression patterns of orthologueinthecenterofthericeapex.Theexpressionof t on
the maize ZmWUS1 and ZmWUS2 differ considerably ZmWUS1inthesecellseitherpersistsorisreactivateddur- 2
0
and will be described separately below. ing subsequent phytomer development, shifting to deeper N
o
layersoftheSAM.Thetranscriptiondomainextendslater- ve
m
The ZmWUS1 Pattern in the Seedling SAM Is Dynamic ally to the position of axillary meristems in P1 (fig. 3F). be
Transcription ceases thereafter, except for activity in axil- r 2
TranscriptionalactivityofZmWUS1wasnotdetected lary meristems (fig. 3G). Similar to the transient OsWUS 01
8
earlier than the coleoptilar stage embryo. ZmWUS1 was expressioninthericeSAM,thedynamicZmWUS1expres-
transcribedinafewcellsunderlyingtheemergingcoleop- sionpatterninthemaizeshootapexdoesnotreflectthesta-
tile(fig.3A1B)inadomaininitiallyincludingtheL1layer ble OC-type expression domain of WUS in Arabidopsis.
but later extending to subtending cell layers (fig. 3A–C). However, ZmWUS1 expression appears to be correlated
Expression then ceased after the initiation of the second with the initiation of leaf phytomers and associated with
embryonic leaf, even though the maize embryo develops theinitiationoftheSAMintheembryoortheestablishment
additional4leavespriortoseeddormancy.ZmWUS1tran- of axillary meristems at the flank of the vegetative apex.
scriptionalactivitywasagaindetectedintheseedlingSAM
aftergermination,althoughthisexpressionwasweakinthe
ZmWUS2 Is Activated in the P Leaf Primordium
centeroftheapexwithtranscriptmaximadetectableatlat- 1
eralpositionsoftheapex(fig.3D).Multipleseries oflon- The in situ hybridization results obtained with the
gitudinal and transverse sections showed that transcript ZmWUS2 probe were in striking contrast to the dynamic
WUSCHELOrthologuesinMaizeandRice 2497
FIG.2.—TranscriptionpatternofOsWUS.(A1B)TransversesectionsthroughthericeplumulehybridizedwiththeOsWUSprobe.Highesttran-
scriptlevelsarefoundintheleafmarginsasisobservedforZmWUS2activity(seefig.4B).NotethatOsWUStranscriptsarepresentattheheightofP in
0
theSAMcenter(A)andabsentinthenextdeepersection(B).(C)LongitudinalsectionthroughthericeplumuleshowingOsWUSexpressionattheabaxial D
faceoftheaxillarybud. ow
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lo
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ZmWUS1 expression pattern in the maize shoot apex. Al- clusiveexpressionpatterncomparedwiththatofKN1inP d
though RT–PCR experiments indicated a low ZmWUS2 and P (fig. 4G). Therefore, the ZmWUS1 expression do0- fro
1 m
transcript level in the embryo, this was not substantiated main, which is located within the KN1 domain in P0 or h
on a cellular level. The median longitudinal section de- P (compare fig. 3E), hardly overlaps with transcriptional ttp
1 s
picted in figure 4A shows 2 points of ZmWUS2 transcrip- activityofTD1.However,theTD1expressionpatternsig- ://a
tional activity at the flank of the apex positioned at the nificantlyoverlapswiththatofZmWUS2inP1,bothinthe cad
height of the P primordium. Both longitudinal and trans- outer celllayersoftheapexandinprospectivelateral leaf e
1 m
versesectionsindicatethatexpressionextendsintodetached domains (compare fig. 4F with 4A). Especially striking ic
leafprimordia,whereZmWUS2transcriptionexertsaprefer- is the coexpression of TD1 and ZmWUS2 in lateral leaf .ou
p
enceforlateralleafdomainswithamaximumofexpression margins during subsequent leaf development. .c
o
atthemarginaltip(fig.4B),whereastranscriptionisunde- AsimilartranscriptionpatternisobservedforFON1in m
tectable in the SAM. In consequence, ZmWUS2 is tran- thericeseedlingapex:FON1transcriptsareabsentfromthe /m
b
scribed in an expression pattern similar to that of the apicaltipandinthecentralribzoneoftheSAMbut,similar e/a
single OsWUS orthologue in young leaf primordia, which to TD1, are found in outer cell layers of the apex at the rtic
also exhibits a maximum inlateralleafmargins.As moni- height of the new phytomer (fig. 4H–J). This expression le
-a
tored by cyclinB gene expression (data not shown), the patterncontradictsthatinitiallypublishedforthericeseed- b
s
ZmWUS2expressionpatterninfullydetachedP2orP3leaf lingSAM(Suzakietal.2004).Thisdiscrepancymayreside trac
primordiashowedasignificantcorrelationwithongoingcell in median versus lateral sections (see complete series in t/2
divisions in lateral leaf domains or growth zones residing SupplementaryMaterialonline), buttheFON1expression 3/1
along the proximal–distal axis (Sylvester et al. 1990). dataarehighlyconsistentandfullyagreewiththoseofTD1, 2/2
Notably,ZmWUS2transcriptsaswellasthoseofZmWUS1 the maize orthologue. Both are consistent with transcrip- 49
2
and OsWUS were detected at the position of axillary mer- tional activity of grass CLV1 orthologues in peripheral /9
7
istemsandwereconfinedtotheabaxialfaceoftheaxillary layers of the shoot apex, in prospective primordial cells 4
4
bud (fig. 4C). butnotincentraldomainsoftheSAM.Accordingtotheir 3
6
transcriptionpatterns,neitherFON1norTD1aretherefore b
y
WUSExpressionPatternsinComparisontotheExpression prone tocontrolpulsesofOsWUS activity orthedynamic gue
ZmWUS1expressioninthecenterofthevegetativeapexin s
DomainsofCLV1OrthologuesinMaizeandRice rice or maize, respectively. t on
The numberof stem cells inthe Arabidopsis SAM is ThericeFON1expressiondataalsosubstantiateapos- 20
controlledthroughCLVsignaling.BecauseTD1andFON1 sibleantagonismwithOsWUSinleafmarginsbecausesim- N
o
havebeenidentifiedinmaizeandrice,respectively,(Suzaki ilar to TD1/ZmWUS2, the rice CLV1 orthologue FON1 is ve
m
etal.2004;Bommertetal.2005)tobeorthologuesofCLV1 expressedatthemarginsofleafprimordia(fig.4J).Inboth b
e
inArabidopsis,wethereforecomparedtheTD1andFON1 grass species, WUS and CLV1 orthologues are therefore r 2
expressionpatternsduringthevegetativephasewiththose preferentially transcribed in lateral domains of young leaf 01
8
of the ZmWUS1/ZmWUS2 paralogues or the single rice primordia, which at this early stage of development still
orthologue OsWUS. have to expand laterally to enclose the apex. Later in de-
Inmaize,longitudinalandtransversesectionsthrough velopment in both grass species, the transcription patterns
theseedlingplumuleshowTD1transcriptsinaring-shaped ofCLV1andWUSorthologueshavenotdivergedbutcon-
expression domain in outer cell layers of the SAM and in sistentlyfocustoleafmargins,whichmaybebestexplained
lateraldomainsoftheP leaf(fig.4D1E).Inolderleaves, by corecruitment.
1
transcripts wereconfined tothelateral leaf margins asob-
servedforZmWUS2(comparefig.4Bwith4F).Inlongitu-
The Expression Patterns of CLV1 and WUS Orthologues
dinal sections, the highest ring of the TD1 expression
in Grasses Merge during Reproductive Development
domain corresponds to the positions of the P phytomer
0
(fig. 4D). From side-by-side comparisons with KN1, it is The architecture of the maize inflorescences is com-
evidentthatTD1transcriptsaredetectedinamutuallyex- plex, and SPM and SM emerge before lastly FMs are
2498 NardmannandWerr
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FIG.3.—ZmWUS1transcriptionpatterns.(A)Schematicdrawingofmaizeembryostages.Fromlefttoright:earlytransitionstage,coleoptilarstage,and
leafstage1.ZmWUS1transcriptionbeginsduringthecoleoptilarstage(B)andshiftsfromtheoutertunicalayerstotheinnercorpusduringleafstage1(C).(D)
Longitudinalsectionsthrough1seedlingapex.Thepositionsoftheindividualsectionsfront(I),median(II),andback(III)areindicatedintheschematic
drawingdepictedin(E).NotetherelativeheightoftheZmWUS1expressiondomaininP (I,front)andP (III,back).(E)Side-by-sidesectionshybridizedwith
0 1
theKN1orZmWUS1probesattheheightoftheP orP phytomers.Relativepositionsareindicatedbythecorrespondingnumbersintheschematicdrawingto
0 1
theleft.NoteKN1downregulationonthemidribside(leftinsection7)incontrasttoitsoverlapwithZmWUS1activityattherightflank(section8)inthe
prospectiveaxillarymeristemofP.(F)CyclingZmWUS1activityinthevegetativeapex.Fromlefttoright,transcriptionstartsinP inasmalldomain
1 0
centralwithintheSAM,shiftingtodeeperlayers.ExpressionthenspreadslaterallytowardtheprospectiveaxillarymeristeminP beforeactivityisregained
1
inthenextphytomer(nowP).(G)ZmWUS1transcriptionattheabaxialfaceofanaxillarybud.axm:axillarymeristem;mr:midrib.
0
WUSCHELOrthologuesinMaizeandRice 2499
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FIG.4.—TranscriptionpatternoftheZmWUS2paralogueandcomparisonwiththeexpressionoftheCLV1orthologuesTD1andFON1intheveg- 74
etativegrassapex.(A)Longitudinalmediansectionthroughthevegetativemaizeapex.ZmWUS2isexpressedinperipheralcelllayersattheheightoftheP 4
1 3
primordium.(B)TransversesectionthroughtheSAMattheheightofP showingtheabsenceofZmWUS2transcriptsinternallywithintheapexand 6
0 b
seixdpereosfstihoeneilnabPo1r.aNteodteaxthilelasrtyrobnugdp(rleeffte)r,etnhceesiingnleaalfatmthaergriingshtosfiPde2,oaflrtehaedaypbexegcionrnriensgpoinndthsetouappyeorumngaerrgainxiollfaPry1.b(uCd).Z(Dm)WLUonSg2ittruadnisncarlipseticotnioanttthhreouabgahxtihael y gu
maizeSAMshowingTD1transcriptsinP0andP1.(E)TransversesectionthroughP0showingthering-shapedTD1expressiondomainattheSAMperiphery es
andthepreferenceinlateraldomainsofP1.(F)TD1marginalpatterninolderleafprimordia.(G)MutuallyexclusivetranscriptionpatternsofTD1(left) t o
andKN1(right)inP0(top)andP1(bottom),assummarizedintheschematicdrawingtotheright.(H1I)Longitudinalandtransversesectionsthrough n 2
thericeapexshowingFON1transcriptsattheperipheryoftheSAM.(J)TransversesectionthroughalateP showingtheFON1preferenceinleaf 0
margins. 1 N
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b
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r 2
initiated(fig.5A);secondaryaxesinthemaletasselaredue L1 layer similar to the shift of WUS expression from the 01
8
totheactivityofearlybranchmeristems(BM).Bothmaize L3layerintheIMtotheL2layerintheFMinArabidopsis
WUS paralogues show meristem-specific expression dur- (Mayer et al. 1998). The expression pattern, therefore, re-
ing the reproductive phase (fig. 5B–G); however, signifi- flects histological differences between1 or2 tunica layers
cant differences exist between ZmWUS1 and ZmWUS2. inmaizeandArabidopsismeristems,respectively.Laterin
Although active in axillary meristem anlagen (see fig. female flower development, ZmWUS1 expression was
3G), no ZmWUS1 expression was detected in the mature detectedintheoutercelllayersofthegynoeciumintheup-
IMofthemaletasselorthefemaleear(fig.5D).ZmWUS1 perfloretandstillsimultaneouslyinthecenterofthelower
transcriptswerefoundinthedescendingSPM,SM,andFM floret(fig.5C),whichsubsequentlyabortsinthefemaleear
(fig. 5B 1 C). In SPMs, SMs, and early FMs, ZmWUS1 inflorescence.
transcriptionisconfinedtoasmallOC-typecentraldomain In contrast, ZmWUS2 transcripts are found in the IM
(fig.5B)suchasisobservedintheearlyembryo.Inthelate (fig.5G)oftenwithaslightpreferencefortheL1layerand
FM, the ZmWUS1 expression domain extends toward the with mRNA being evenly distributed through tunica and
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FIG.5.—TranscriptionalactivityofmaizeZmWUSparaloguesandthericeOsWUSorthologueduringthereproductivephase.(A)Typesofmeristems u
p
inthemaizeear:IM,SPM,SM,andupper/lowerfloralmeristem(UFM/LFM).(B)ZmWUS1transcriptionpatterninSPM,SM,andearlyFM.(C)UFM .c
andLFMshowingZmWUS1transcriptsinouterlayersintheolderUFM,whereastranscriptsarestillcentralwithintheLFM,whichwillabort.(D)No om
ZmWUS1expressionisdetectableintheIM.(E)LaterallongitudinalsectionshowingtheZmWUS2expressionpatternintheSPM,SM,orFM.Differ- /m
encesinstainingintensityareduetotwistingofseedrows.(F)Close-upofaFMshowingZmWUS2expressionthroughouttheUFMandsubtendingthe b
e
LFMbutalsotranscriptsinthedevelopingearlystamen.(G)MedianlongitudinalsectionshowingZmWUS2expressionintheIMwithaslightL1layer /a
preference.(H1I)OsWUSexpressionpatterninthericeprimarybranchmeristem(PBM)orFM,respectively.NotetheL1layerpreferenceinthePBM rtic
relativetoFM.St:stamen;ig/og:inner/outerglume. le
-a
b
s
tra
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corpusofdescendingSPMs,SMs,orFMs(fig.5E1F).This Discussion t/2
patternofZmWUS2expressioncloselyresemblesthatofTD1 The Expression Patterns of Rice and Maize WUS 3/1
(Bommertetal.2005),reflectingsomeL1layerpreference Orthologues Do Not Support a Stable OC-Type 2/2
in the IM and the transcriptional activity throughout SPM, Expression Domain in the Grass SAM 49
2
SM,orFM.WiththeexceptionoftheIM,wheretranscripts BasedonthephylogenyoftheWOXgenefamily,or- /97
areabsent,ZmWUS1isthereforealwaysexpressedinasub- 4
thologues totheArabidopsisWUS genewereidentifiedin 4
population of cells alsoexpressingZmWUS2. 3
SimilarresultswereobtainedforOsWUSandFON1ex- maizeandrice.NoneoftheisolatedgrassWUSorthologues 6 b
pression in rice despite the different architecture of the displays a transcription pattern in the vegetative SAM y g
u
which resembles the stable OC-type expression domain e
panicle.Thericeinflorescenceisbranchedsimilarlytothe s
tassel of maize. Differing from the maize tassel, however, of WUS in Arabidopsis. In contrast, their expression pat- t on
themain-axismeristem(IM)doesnotproduceanyspikelets terns relate to the specification of new phytomers, which 2
0
butabortsafterhavinginitiated10ormoreprimarybranch has 2 aspects in the grass culm. The first aspect is the re- N
meristems. These lateral branches produce numerous so- cruitment of leaf founder cells from the whole circumfer- ove
ence of the SAM before the leaf primordium detaches m
called spikelets that develop into single bisexual flowers b
from the shoot apex. The second aspect is that growth of e
(FM) or secondary branch meristems (Itoh et al. 2005). r 2
OsWUS is expressed in rice BMs (fig. 5H) and, as for theculmdependsonthecoordinatedelongationofthede- 01
ZmWUS2 in the maize IM, exhibits some preference for tached leaf sheath and the internode enclosed (Abbe et al. 8
theouterL1layer.TheL1preferenceislostintheFMwhere 1951;Steffensen1968).Essentially,thesinglericeOsWUS
OsWUS expression was detected uniformly through the orthologue shares an expression pattern with its 2 maize
meristem(fig.5I),redolentoftheZmWUS2expressionpat- paralogues. However, the different expression patterns of
terninSPMs,SMs,orFMs.Onthetranscriptionallevel,the the 2 paralogues in maize identified distinct functions in
OsWUSexpressionpatternalwaysoverlapswithFON1tran- theapex.ZmWUS1isexpressedincellsoftheprospective
scription,beingactiveinalllayersoftheIMs,BMs,andFMs nodal/internodaltissueinthecenteroftheSAMandshifts
in the rice inflorescence (Suzaki et al. 2004). Consistently to deeper layers with elaboration of the new phytomer,
andincontrasttoduringthevegetativephase,theexpression whereas ZmWUS2 is transcribed in cells recruited for leaf
ofWUSortho/paraloguesinriceormaize,therefore,ismer- primordiaandhasamaximuminbasallateralleafmargins.
istemspecificduringthereproductivephaseandcoincides ZmWUS2thereforesharesexpressioninleafprimordiaand
withtheexpressiondomainsofgrassCLV1orthologues. thepreferenceinlateralleafmarginswithOsWUS,whereas
WUSCHELOrthologuesinMaizeandRice 2501
ZmWUS1mayrelatetothepulsingOsWUStranscriptionin erence for the L1 layer in the IM, whereas transcriptional
theSAMcenter.TheexpressionpatternsofthemaizeWUS activityintheSPM,SM,andFMisconstitutivethroughout
paralogues therefore support the hypothesis that gene du- themeristems.Incontrast,ZmWUS1transcriptsareabsent
plications are preserved by subfunctionalization (Lynch intheearortasselIMbutmarkasmallOC-typedomainin
andForce2000).Afterduplication,mutationshaveresulted SPM, SM, and early FM before the expression spreads to
inareductionintheirjointexpressionlevelsandpatternsof thelargerZmWUS2/TD1coexpressiondomainduringlater
activity compared with that of the single ancestral WUS stages of floral development. Concerning the absence of
gene, which formerly may have been expressed similarly ZmWUS1activityintheIM,itshouldbeborneinmindthat
to OsWUS, the single WUS orthologue in rice. bothmaizeparaloguesarecoexpressedduringearlystages
ThedifferentexpressionpatternsofgrassWUSortho- ofaxillarymeristemdevelopmentasisobservedduringthe
loguescomparedwiththatofWUSinArabidopsisacquire initiationofSPMs,SMs,andFMs.Althoughindeterminate,
significanceduetothecorrelatedchangesintheexpression the IMs of ear or tassel are elaborated meristems, and
D
patternsofthegrassCLV1orthologues,TD1andFON1in ZmWUS2activitymay besufficient for theirmaintenance. o
w
maizeandrice, respectively (Suzaki etal.2004; Bommert Similartothatinmaize,FON1andOsWUSarecoexpressed nlo
etal.2005).Bothorthologuesaretranscribedattheflankof inBMsandFMsofthericepanicle.WUSandCLV1ortho- ad
e
theSAMincellsthathavebeenrecruitedintoleafprimor- logues of maize and rice are therefore coexpressed in all d
dia but not in the center of the SAM where ZmWUS1 and reproductivemeristems,wherefasciationandsupernumer- fro
m
OsWUS are transiently expressed. TD1 transcription ini- ary floral organs in td1 or fon1 loss-of-function mutants h
tiates at P and then overlaps with ZmWUS2 expression provide evidence for meristem enlargement (Suzaki et al. ttp
0 s
intheP1phytomerandlateralleafdomainsafterthedetach- 2004; Bommert et al. 2005). ://a
mentoftheprimordium.TheactivationofZmWUS2inP1 Conforming to the stem cell concept in Arabidopsis, cad
within the TD1 expression domain suggests that pluripo- excessiveWUSactivitycouldexplainthemeristemenlarge- e
m
tency is acquired de novo in cells already determined for mentinclvmutants(Schoofetal.2000;WaitesandSimon ic
theprimordialfate.TheOsWUSandFON1expressionpat- 2000). However, differences in the expression patterns of .ou
p
terns in rice leaf primordia conform to this assumption as maize WUS paralogues could not be detected in the td1 .c
o
wellastotheassumptionthatthegrassCLV1orthologues mutantbackground(datanotshown),whereasWUSexpres- m
TD1andFON1acttoantagonizeZmWUS2orOsWUSac- sion shifts from the L3 to the L2 layer in Arabidopsis /m
b
tivityinprospectiveprimordialcells.Laterinleafdevelop- (Schoof et al. 2000). This may relate to differences in e/a
ment,ZmWUS2andTD1inmaizeandOsWUSandFON1 theshootmeristemarchitecturebetweenmaizeandArabi- rtic
inricearecotranscribedinleafmargins.Themarginalpat- dopsis. Whereas the OC-type, WUS expression domain in le
-a
ternsofWUSandCLV1orthologuesinmaizeandriceare the Arabidopsis SAM subtends a 2-layered tunica, maize b
s
reminiscent of the narrow sheath1 and narrow sheath2 meristems possess only a single tunica layer (Abbe et al. tra
c
(NS1/NS2)geneexpressionpatternsinmaize,encodingpa- 1951; Steffensen 1968). High levels of TD1 or ZmWUS2 t/2
raloguesofthePRSgeneinArabidopsis(Nardmannetal. transcriptaredetectedinthissingletunicalayerofthema- 3/1
2004). Maize ns1/ns2 double mutants are affected in the ture IM, and both genes are cotranscribed throughout all 2/2
development of the basal lateral domains of leaves, and layers of the descending SPMs, SMs, or FMs. ZmWUS2 49
2
thesimilarityintranscriptionpatternsuggestthatZmWUS2 transcription, therefore, is not restricted to a subdomain /9
7
and OsWUS may contribute to leaf development and be of the TD1 expression domain in reproductive meristems 4
4
antagonized by TD1 and FON1, respectively. butoverlaps with TD1expression andsharesitstranscrip- 3
6
TherecentidentificationofROSULATA(ROA),which tion specificity with IM L1 layer or leaf margins. A pref- b
y
encodestheWUSorthologueinA.majus,alsoconfirmsthat erence for the tunica layer and the leaf margin suggests g
u
e
in dicots, ROA is not only involved in SAM maintenance thatanextracellularligandmustbepreferentiallyperceived s
but also acts outside the SAM (Kieffer etal. 2006). Inthe from subtending cell layers, in contrast to Arabidopsis t on
roamutant,thepetiolesofleavesarefrequentlyventralized SAM, where CLV3 is presented top-down from the L1/ 2
0
or appear radially symmetrical andthe leaf blades are less L2 tunica to the L3 layer. N
o
expandedthaninwildtype.Theroamutantsalsohaveshort TheexpressionpatternsofTD1andFON1duringthe ve
m
internodes, which are comparable to deficiencies in the reproductivephaseandthemutantphenotypessuggestthat b
e
elongation of internodes in wus-1 mutant inflorescences they may control activity of grass WUS orthologues in re- r 2
inArabidopsis.WUSfunctionsindicotsthereforecontrib- productivemeristems.Byanalogy,thisassumptionalsoim- 01
8
utetotheelongationoftheshootaxis,whichcouldrelateto pliesthatTD1andFON1mayrestrictZmWUS2andOsWUS
ZmWUS1 transcriptional activity within the SAM during activity in cells recruited for leaf primordia, where the ex-
development of the maize leaf phytomer. pression domains overlap during the vegetative phase.
Whereas the Arabidopsis leaf can be traced back to a few
foundercellswithintheapex(IrishandSussex1992),cells
Grass WUS Orthologues Display a Meristematic
forthegrassleafarerecruitedfromthewholecircumference
Expression Pattern during Reproductive Growth
oftheSAM(Poethig1984).Thestrikinglydifferentexpres-
In contrast to the vegetative phase, ZmWUS1, sion patterns of grass WUS/CLV1 orthologues relative to
ZmWUS2, and OsWUS are expressed in meristems during those in Arabidopsis during the vegetative phase therefore
the reproductive phase. The expression patterns of may relate to fundamentally different developmental pro-
ZmWUS2 and TD1 considerably overlap in all meristem gramsandthepeculiararchitectureofthegrassculm.How-
typesofthemaleorfemaleinflorescenceandexertapref- ever,themaizeorricevegetativeexpressionpatternssignify