Positive Autoregulation of a KNOX Gene Is Essential for Shoot Apical Meristem Maintenance in ...

15 Pages·2012·1.97 MB·English

ThePlantCell,Vol.23:4368–4381,December2011,www.plantcell.orgã2011AmericanSocietyofPlantBiologists.Allrightsreserved.
KNOX
Positive Autoregulation of a Gene Is Essential for Shoot
Apical Meristem Maintenance in Rice W OA
KatsutoshiTsuda,aYukihiroIto,bYutakaSato,candNoriKurataa,d,1
aPlant Genetics Laboratory, Genetic Strains Research Center, National Institute of Genetics, Mishima, Shizuoka 411-8540,
Japan
bGraduateSchoolofAgriculturalScience,TohokuUniversity,Aoba-ku,Sendai981-8555,Japan
cGraduateSchoolofBioagriculturalSciences,NagoyaUniversity,Chikusa,Nagoya464-8601,Japan
dDepartment of Genetics, School of Life Science, Graduate University for Advanced Studies, Mishima, Shizuoka 411-8540,
Japan
Self-maintenance of the shoot apical meristem (SAM), from which aerial organs are formed throughout the life cycle, is
crucial in plant development. Class I Knotted1-like homeobox (KNOX) genes restrict cell differentiation and play an
indispensableroleinmaintainingtheSAM.However,themechanismthatpositivelyregulatestheirexpressionisunknown.
Here,weshowthatexpressionofarice(Oryzasativa)KNOXgene,Oryzasativahomeobox1(OSH1),ispositivelyregulated
by direct autoregulation. Interestingly, loss-of-function mutants of OSH1 lose the SAM just after germination but can be
rescuedtogrowuntilreproductivedevelopmentwhentheyareregeneratedfromcallus.Doublemutantsofosh1andd6,a
loss-of-functionmutantofOSH15,failtoestablishtheSAMbothinembryogenesisandregeneration.Expressionanalysesin
thesemutantsrevealthatKNOXgeneexpressionispositivelyregulatedbythephytohormonecytokininandbyKNOXgenes
themselves.WedemonstratethatOSH1directlybindstofiveKNOXloci,includingOSH1andOSH15,throughevolutionarily
conserved cis-elements and that the positive autoregulation of OSH1 is indispensable for its own expression and SAM
maintenance.Thus,themaintenanceoftheindeterminatestatemediatedbypositiveautoregulationofaKNOXgeneisan
indispensablemechanismofself-maintenanceoftheSAM.
INTRODUCTION ISTEMLESS in Arabidopsis thaliana, lack the SAM, indicating
thatKNOXgenesareessentialfortheformationand/ormainte-
One of the more remarkable differences between animals and
nance of the SAM (Long et al., 1996; Vollbrecht et al., 2000).
plantsisthebodyplan.Whileanimalscompletetheirorganogen-
KNOX genesnegatively regulatebiosynthesisofthephytohor-
esisduringembryogenesis,plantsformasimplestructurecon-
mone gibberellin (GA), which acts in differentiated organs to
sisting of two distinctive regions at opposite poles. One is the
promote cell elongation and morphogenesis. In maize, KN1
shoot apical meristem (SAM) and the other is the root apical
directlyactivatesGA2oxidase1(GA2ox1),thegenecodingfora
meristem.Bothmeristemsareorganizedgroupsofundifferenti-
GA inactivation enzyme (Bolduc and Hake, 2009). A tobacco
ated cells responsible for postembryogenic organ formation
(Nicotiana tabacum)KNOX protein Nicotiana tabacum homeo-
(SteevesandSussex,1989).TheSAMisadome-shapedtissue
box15alsonegativelyregulatesGAproductionintheSAMby
locatedatthegrowingtipofashoot.Afterembryogenesis,lateral
directrepressionofaGAbiosyntheticenzymegene,GA20ox1
organs,suchasleavesandflowers,arecontinuouslyformedfrom
(Sakamotoetal.,2001).Anotherhormonalpathwayregulatedby
theflankoftheSAM.Branchesandstemsarealsoderivedfrom
KNOXiscytokinin(CK).CKfunctionstoactivatecelldivisionand
theSAM.Thus,theSAMisanultimatesourceofallaboveground
shootformation. InArabidopsis andrice (Oryzasativa),KNOX
organsandthereforemustbemaintainedthroughoutthelifecycle.
proteinspositivelyregulateCKproductionthroughinductionof
TheelucidationofthemechanisminvolvedinSAMmaintenanceis
theCKbiosynthesisenzyme,adenosinephosphateisopentenyl-
afundamentalissueindevelopmentalbiology.
transferase (IPT) (Jasinski et al., 2005; Yanai et al., 2005;
ClassIKnotted1-likehomeobox(KNOX)genes,whichencode
Sakamoto et al., 2006). Thus, KNOX genes are considered to
homeodomain (HD) transcription factors, play indispensable
inhibitcelldifferentiationintheSAMbydecreasingandincreas-
roles in the SAM. Loss-of-function mutants of KNOX genes,
ingtheamountofGAandCK,respectively.
suchasknotted1(kn1)inmaize(Zeamays)andSHOOTMER-
KNOXgenesareexpressedintheshootapexspecificallyand
areexcludedfromdifferentiated organs(Jacksonetal.,1994).
SeveralgenesrequiredforrepressionofKNOXgeneexpression
[email protected].
The author responsible for distribution of materials integral to the inleaveshavebeenidentified.Amongthem,theMYBtranscrip-
findingspresentedinthisarticleinaccordancewiththepolicydescribed tion factor ROUGH SHEATH2 (RS2) in maize, its Arabidopsis
in the Instructions for Authors (www.plantcell.org) is: Nori Kurata putativeorthologASYMMETRICLEAVES1(AS1),andtheplant-
([email protected]).
specificLOBfamilyproteinAS2havebeenwellstudied.Intheir
WOnlineversioncontainsWeb-onlydata.
OAOpenAccessarticlescanbeviewedonlinewithoutasubscription. loss-of-function mutants, KNOX genes are ectopically ex-
www.plantcell.org/cgi/doi/10.1105/tpc.111.090050 pressedinleaves(Timmermansetal.,1999;Tsiantisetal.,1999;
PositiveAutoregulationofaKNOXGene 4369
Byrneetal.,2000;Orietal.,2000;Semiartietal.,2001;Iwakawa sized transcript was detected, but the expression level was
etal.,2002).AS1andAS2arethoughttoformacomplexthat reduced compared with that of the wild type (Figure 1C). To
directlyrepressestranscriptionoftheKNOTTED-likegenesfrom examineOSH1proteinaccumulation,weperformedimmunoblot
Arabidopsis thaliana 1 (KNAT1)/BREVIPEDICELLUS (BP) and analysis using an anti-OSH1 antibody that specifically recog-
KNAT2(Guoetal.,2008).RS2andAS1wereshowntointeract nizestheNterminusofOSH1.Theprematurestopcodoninthe
with a chromatin remodeling protein, HIRA (Phelps-Durr et al., thirdexonpredictsatruncatedformofOSH1tobe17kD.Inthe
2005).Therefore,theseproteinsmayestablishand/ormaintain young panicle and shoot apex from wild-type plants, normal-
therepressedstateofKNOX lociviachromatin remodeling.In sizedOSH1wasdetectedataround40kD.Ontheotherhand,
addition to this, proper activities of the phytohormone auxin, neither a normal-sized nor a truncated form of OSH1 was
YABBYgenes,andPolycombgenesarealsorequiredtorepress detectedinthemutantshootapex(Figure1D).OSH1wasnot
KNOX gene expression in leaves (Kumaran et al., 2002; Katz detectedinwild-typeleavesaspreviouslydetermined(Jackson
etal.,2004;Hayetal.,2006). et al., 1994; Sentoku et al., 1999).Thus, these results indicate
Despite the critical function of KNOX genes in SAM mainte- thattheosh1-1alleleisanull.
nance, little is known of the mechanisms that activate KNOX
gene expression. In this study, we isolated a loss-of-function OSH1IsEssentialforShootMeristemMaintenanceduring
mutantofoneofthericeKNOXgenes,Oryzasativahomeobox1
theVegetativePhase
(OSH1). Genetic studies using osh1 in combination with d6 , a
loss-of-function mutant of another rice KNOX gene, OSH15, TostudythefunctionofOSH1inSAMformationandmaintenance,
revealedtheindispensableroleofKNOXgenesinSAMformation thephenotypeofosh1plantsatvariousstagesofdevelopment
andmaintenanceinrice.ExpressionlevelsofKNOXgeneswere wasobserved.Duringembryogenesis,allembryosfromhetero-
found to depend not only on CK but also on KNOX genes zygousosh1plantsinitiatedanormal-appearingSAMandthree
themselves. Furthermore, we demonstrated that OSH1 posi- embryonicleavesasinwild-typeplants(n=30).Thedefectwas
tivelyregulatestheexpressionofallfiveKNOXgenestestedina firstobservedinthematureembryoat0DAG(Figure1E),inwhich
direct manner through evolutionarily conserved cis-elements osh1hadaslightlysmallerSAMthanthewildtype.At1to3DAG,
andthatthepositiveautoregulationofOSH1isessentialforthe thedomeshapeoftheosh1mutantSAMgraduallydisappeared
expressionofitselfandSAMmaintenance.Takentogether,we withthegrowthofthethirdleafprimordia.At4DAG,osh1hada
proposethatthepositiveautoregulationofaKNOXgeneisoneof remnant bulge at the former SAM region, and after that, no
theindispensablemechanismsofself-maintenanceoftheSAM. additionalleaveswereformed.At10DAG,>90%ofosh1mutants
terminatedtheir growthwithonlythree leavesproducedduring
embryogenesis, compared with the wild type that continued to
RESULTS produce leaves (Figure 1B). However, a low frequency of osh1
plantsproducedfourto six leaves (2.5%)orone to two leaves
IsolationofaLoss-of-FunctionMutantofOSH1 (3.9%) (see Supplemental Table 1 online). In very rare cases
(<1%),osh1mutantsformedmorethansixleavesandcontinued
Inthericegenome,therearefivefunctionalclassIKNOXgenes, togrowforaboutamonth,althoughsuchplantsalsolosttheSAM
OSH1,OSH6,OSH15,OSH43,andOSH71(Sentokuetal.,1999; and terminated leaf formation eventually. Similar variability was
Sato et al., 2001). Among them, OSH15 has been studied by documented with the maize kn1 loss-of-function mutants, al-
loss-of-function analysis and was revealed to be essential for thoughthepenetranceoftheshootlessphenotypewasmuchless
internodeelongationduringthereproductivephase,butnotfor (Vollbrechtetal.,2000).Thus,theseresultsindicatethatOSH1is
SAMmaintenance(Satoetal.,1999).Toprovidefurtherinsight essentialforSAMmaintenanceaftergerminationbutnotforSAM
intothefunctionofKNOXgenes,weisolatedaloss-of-function formationduringembryogenesisinrice.
mutant of OSH1, an ortholog of maize kn1, using the Target-
InducedLocalLesionInGenomes(TILLING)systemofricethat OSH1andOSH15AreRedundantlyRequiredforSAM
we developed previously (Suzuki et al., 2008). From screening
FormationduringEmbryogenesis
1456N-methyl-N-nitrosourea(MNU)-mutagenizedM2lines,we
foundonelinethathadanucleotidesubstitutionfromGtoTat To examine the contribution of other KNOX genes to SAM
thefirstnucleotideofthethirdexon,resultinginaprematurestop formation,weobservedembryogenesisoftheosh1d6double
codon(Figure1A).Thismutantosh1allele(osh1-1)ispredictedto mutants.OSH15isexpressedinthemeristem,andd6,aloss-of-
encode a protein that lacks the HD essential for DNAbinding. functionmutantofOSH15,hasadwarfphenotype(Satoetal.,
Homozygousosh1mutantplantsterminatedgrowthsoonafter 1999;Sentokuetal.,1999).Inthewildtype,thezygotecontinued
germination(Figure1B).Weperformedacomplementationtest cell division to form a globular embryo by 3 d after pollination
ofosh1andconfirmedthatnormalmorphologywasrecovered (DAP).At4DAP,theSAMandcoleoptileprimordiumappeared
byintroducingacopyofthewild-typeOSH1(seeSupplemental asaprotrusionontheventralsideoftheembryo(Figure2A).The
Figure1online).Therefore,theosh1-1alleleislikelytobealoss- firstleafprimordiumdevelopedfromtheflankoftheSAMat5
of-functionmutantofOSH1andwasusedinfurtheranalyses. DAP(Figure2B),andthesecondandthirdleafprimordiasucces-
To investigate the effect of this mutation on OSH1 mRNA sivelyformedwithinanother3to4d(Figure2C).Subsequently,the
accumulation,weexaminedtranscriptexpressionbyRT-PCR.In embryomatured and entered dormancy(Itoh etal., 2005). Em-
themutantshootapexat10daftergermination(DAG),anormal- bryosfromplantsheterozygousforosh1andhomozygousford6
4370 ThePlantCell
Figure1. Loss-of-FunctionMutantofOSH1DoesNotMaintaintheSAMafterGermination.
(A)OSH1structureandthemutationinosh1.Graylinesindicateintronsand59and39regions.Boxesindicateexons.ConserveddomainsofOSH1are
labeledincolorasshownbelowthegenestructure.
(B)Phenotypesofwild-type(wt)(left)andosh1(right)seedlingsat10DAG.Arrowheadsindicatethepositionofeachleaf.
(C)RT-PCRanalysisforOSH1andActin(loadingcontrol)inleavesandshootapicesofwild-typeandosh1plantsat10DAG.Thisexperimentwas
performedtwotimesindependently.
(D)ImmunoblottinganalysisofOSH1proteininthewildtypeandosh1.Totalnuclearproteinextractsfrom5-mmyoungpanicles,shootapices,and
youngleafprimordiaat10DAGwereloadedineachlane.Coomassiebluestainingofmembraneafterimmunoblottingshownatabottomindicatesthat
sampleswereloadedequally.SA,shootapex;YP,youngpanicle.
(E)Shootapexsectionsofthewildtypeandosh1duringgermination.TheSAMbeforegerminationisindicatedwithablackline.
Bars=2cmin(D)and50mmin(E).
hadnovisibleabnormalitiesat3DAP(n=30).However,inabout arecoordinatelyexpressedduringshootregeneration(Gordon
one-fourthofexaminedembryosat4DAP,thecoleoptileprimor- etal.,2007).ToexaminetheimportanceofKNOXgenefunction
diumwasformednormally,buttheregionwhereSAMformation duringdenovoformationoftheSAM,weobservedthepheno-
would occur was hollow and protrusion of the SAM was not typeofshootsregeneratedfromthewildtype,osh1,d6,andtheir
observed (n = 7 out of 30 embryos; Figure 2D). Although the doublemutants.
coleoptiledevelopedasinnormalembryos,noleafprimordiawere In the wild type and d6 single mutant, normal shoots were
initiated(Figure2E).Evenafterembryomaturation,noorganswere regenerated,andnoabnormalitywasobservedatlaterstages,
observedinosh1d6embryosexceptacoleoptileandaremnant exceptforthedwarfphenotypeofd6(Satoetal.,1999).Inthe
bulgeintheformerSAMposition(Figures2Fto2H).Thus,osh1d6 case of osh1 single mutants, normal shoots were regenerated
double mutant embryos failed to form the SAM, indicating that fromcallus. Theseplants could maintain the SAM andformed
redundantfunctionsofOSH1andOSH15wereessentialforthe morethanthreeleaves,unlikegerminatedosh1mutants(Figures
initiationoftheSAM. 3Ato3C).Thephenotypeofregeneratedosh1varieddepending
on their growth conditions. When they were grown in field
ShootRegenerationfromosh1andosh1d6MutantCallus conditions,theylosttheSAMandterminatedtheirgrowthpriorto
flowering(n=56).However,almostallregeneratedosh1mutants
InArabidopsis,ithasbeenreportedthatkeytranscriptionfactors could grow until reproductive phase in light- and temperature-
fortheSAMformationandmaintenance,includingKNOXgenes, controlledgrowthchambers(n=43outof49regeneratedosh1
PositiveAutoregulationofaKNOXGene 4371
Figure2. OSH1andOSH15AreRedundantlyRequiredforSAMFormation.
(A)to(C)Sectionsofwild-type(wt)embryosat4DAP(A),5DAP(B),and10DAP(C).BlackarrowheadsindicatethepositionoftheSAM.
(D)to(F)Sectionsofosh1d6doublemutantembryosat4DAP(D),5DAP(E),and10DAP(F).WhitearrowheadsindicatethepositionwhereSAM
formationshouldbeobservedinthewildtype.
(G)Anosh1d6doublemutantat10DAG(cf.toFigure1D).
(H)Topviewof(G).
Bars=100mmin(A)to(F)and5mmin(G)and(H).
mutants).Regeneratedosh1mutantplantshadareducednumber OSH43wereexpressedconstantlyinallstagesexamined,whereas
ofspikeletswithabnormallyfusedanthers(Figures3Dto3F)and expressionofOSH6,OSH15,andOSH71waslowatthemature
werecompletelysterile.Thus,regeneratedosh1mutantsshowed embryostageandincreasedjustaftergermination(Figure4A).In
moderatephenotypescomparedwithgerminatedosh1mutants. theosh1mutant,OSH1andOSH43weresignificantlyreducedatall
Whenwetriedtoregenerateplantsfromosh1d6doublemutant stages, while expression of OSH6, OSH15, and OSH71 was
callus,onlyleaf-likestructureswereformedandnoshootformation inducedaftergermination,asnormal.Therefore,expressionofall
wasobservedateven3monthsaftertheinductionofregeneration fiveKNOXgenesislowinthematureembryoofosh1.Thisimplies
(Figure 3H). Therefore, as is the case for SAM formation during that the mutant shoot meristem temporally undergoes a state in
embryogenesis,OSH1andOSH15areredundantlyrequiredforde whichKNOXgenetranscriptsaccumulatetoverylowlevels.
novoshootformationincallus.Previously,ithasbeenreportedthat Next,weperformedexpressionanalysisduringembryogene-
Arabidopsisstmmutantscanberescuedtogrowuntilreproductive sis(Figure4B).AttheSAMformationstage,fourKNOXgenes
developmentwhentheyweretreatedwithCK(Yanaietal.,2005). exceptOSH15wereexpressedatanormallevelind6mutants.In
Because the medium for shoot regeneration contains a high osh1,asweobservedduringgermination,theexpressionlevelof
concentrationofCK,itispossiblethatCKenabledtheregenerated OSH43wasseverelyreduced,andOSH6andOSH15showed
osh1mutanttomaintaintheSAMintheabsenceofOSH1function. only slightly reduced expression, whereas OSH71 expression
However,thefailureoftheosh1d6doublemutantcallustoform wascomparabletothatofthewildtype.Ontheotherhand,in
shootsontheregenerationmediumindicatesthat,evenwithahigh osh1d6doublemutantembryos,theexpressionofallfiveKNOX
concentrationofCK,thefunctionofKNOXgenesisessentialfor geneswasreducedtoone-thirdorlessofwild-typevalues.Insitu
SAMformationand/ormaintenance. hybridizationalsoconfirmedthereductionofOSH6andOSH71
expressionintheosh1d6embryo(Figure4C).Thus,KNOXgene
ReducedExpressionofKNOXGenesinosh1andosh1d6 transcriptsbarelyaccumulatedinosh1d6embryos,whichcould
leadtothefailureoftheSAMestablishment.
Thephenotypicobservationsofosh1mutantssuggestthatthe We further performed expression analysis during de novo
contributionsofindividualriceKNOXgenestoSAMmaintenance shootformationfromcallus.Fromourpreviousstudies,wefound
varies depending on timing and environmental conditions. To thatKNOXgeneexpressionwasinducedincallusbytreatment
investigatethispossibility,weperformedexpressionanalysesof with CK (Ito et al., 2001). Consistent with this, CK treatment
KNOXgenes.Duringgerminationofwild-typeplants,OSH1and inducedtheexpressionofOSH1,OSH6,OSH15,andOSH71in
4372 ThePlantCell
Figure3. OSH1andOSH15AreRedundantlyRequiredforSAMFormationandMaintenanceduringdeNovoShootRegenerationfromCallus.
(A)Regenerated(left)andgerminated(right)seedlingsofosh1.Whitearrowheadsindicatethepositionofeachleaf.
(B)and(C)Sectionsofshootapicesoftheregeneratedwild-type(B)andosh1plants(C).ArrowheadsindicatetheSAM.
(D)Paniclesofregeneratedplantsofthewildtype(wt)(left)andosh1(right).
(E)and(F)Spikeletsformedonregeneratedplantsofthewildtype(E)andosh1(F).a,anther;p,pistil;asterisk,abnormalshortantherofosh1.
(G)and(H)Regeneratingshootfromthewildtype(G)andosh1d6(H).Arrowsindicateleaf-likestructuresformedfromosh1d6callus.
Bars=2cmin(A),50mmin(B)and(C),5cmin(D),and2mmin(E)to(H).
wild-type callus, and OSH1, OSH6, and OSH71 were also using primer pairs designed at 1-kb intervals along the OSH1
inducedtowild-typelevelsind6callus.Inosh1mutantcallus, locus as indicated in Figure 5A. A ChIP assay followed by
OSH6,OSH15,andOSH71werealsoinducedbyCK,although quantitativePCR(qPCR)revealedthatsequencesimmediately
the expression level of OSH6 was lower than in the wild type upstreamofthetranscriptionstartsiteandinthe59halfofthe
(Figure4D).ThesedatasuggestthatCKtreatmentbypassedthe thirdintronwerestronglyenrichedintheOSH1-boundchromatin
stateoflowKNOXgeneexpressioninosh1shootapicesduring fragments.Inaddition,strongenrichmentwasobtainedinsev-
de novo shoot formation and could explain the phenotype eralregionsoffourotherKNOXloci(Figures5Bto5E).Enrich-
differencebetweengerminationandregeneration. mentintheselociwasalsofoundintheir59upstreamregionsand
Intheosh1d6doublemutantcallus,notonlywasexpression thethirdintrons.TheseresultsindicatethatOSH1bindstoallfive
ofOSH1,OSH6,andOSH43reducedbutalsothatofOSH15and KNOXlociinvivointheinflorescencemeristem.
OSH71,evenafterCKtreatment(Figure4D).Thus,inductionof ToexamineifOSH1directlybindstoKNOXlociinvegetative
KNOXgenesbyCKwasimpaired,whichmayberesponsiblefor shoot meristems, we tested the enrichment in ChIP samples of
thefailureofdenovoshootformationfromcallusintheosh1d6 vegetativetissuesat1DAG.Inallhighlyenrichedregionsobserved
doublemutant. inyoungpanicles,amodestenrichmentwasalsodetectedwhen
weusedwild-typeshootapexasthesamplebutnotinthecaseof
OSH1DirectlyBindstoFiveKNOXLocithrough osh1 shoot apex and young leaves (Figure 5F). These results
EvolutionarilyConservedcis-Elements confirmedthespecificityofourChIPassayandthatOSH1bindsto
KNOXlociinvivoinallOSH1-expressingtissuesexamined.
Our observation that expression of the other KNOX genes,
OSH6, OSH43, and OSH71, was dependent on functional
OSH1BindingSitesAreEvolutionarilyConserved
OSH1andOSH15inadditiontoCKsuggestedthatKNOXgenes
mightbepositivelyautoregulated.Consistentwiththishypoth- Previously, it has been shown that KNOX proteins directly
esis, we observed increased expression of KNOX genes in activate the expression of GA2ox1 through evolutionarily con-
leaves of the 35S:OSH1 transgenic plants (see Supplemental servedTGACcoresequences(BolducandHake,2009).There-
Figure 2 online). To examine whether or not this regulation is fore,weconsideredthatifdirectbindingofOSH1isimportantfor
mediatedbyKNOXproteinsinadirectmanner,weperformed transcriptionalregulationofKNOXgenes,theTGACsequences
chromatin immunoprecipitation (ChIP) assays using an anti- should be conserved. To this end, we performed sequence
OSH1antibodythatspecificallyrecognizesOSH1.Usingyoung comparisonanalysisbetweenriceKNOXgenesandtheirputa-
panicles in which OSH1 expression is broad and strong, we tiveorthologsinPoaceaeandsearchedforTGACsequencesin
tested the enrichment of OSH1-bound DNA in ChIP samples conserved regions. We found conserved TGAC sequences in
PositiveAutoregulationofaKNOXGene 4373
Figure4. ExpressionLevelsofKNOXGenesWereReducedinoshMutants.
(A)ExpressionofKNOXgenesingerminatingembryos.Relativeexpressionlevelwascalculatedastheratiototheexpressionlevelat2DAGinthewild
type(wt)foreachgene.
(B)ExpressionofKNOXgenesintheSAMformationstageoftheembryoat5DAP.Relativeexpressionlevelwascalculatedastheratiotothe
expressionlevelinthewildtypeforeachgene.
(C)InsituhybridizationofOSH6andOSH71inwild-typeandosh1d6embryosataroundthestageofSAMformation.Bars=100mm.
(D)ExpressionofKNOXgenesincallusbeforeand1weekafterCKtreatment.Relativeexpressionlevelwascalculatedastheratiototheexpression
levelbeforeCKtreatmentinthewildtypeforeachgene.
In (A) to (C), the expression level of ubiquitin (ubq) was used as an internal control for normalization. The data represent the average of three
independentbiologicalreplicates.ErrorbarsrepresenttheSD.AsterisksindicatesignificantreductionatP<0.05comparedwiththeexpressionlevelin
thewildtype.
eachKNOXgenelocus(magentabarsinFigure5;seeSupple- TheseresultsindicatethatOSH1directlybindstothefiveKNOX
mentalFigure3online).TheOSH1locushadasingleconserved genes we examined, including OSH1 itself, via evolutionarily
sequenceinthepromoterregionandseveninthe59halfofthe conserved cis-elements and suggest that transcriptional regu-
third intron. To test whether OSH1 directly binds to these cis- lationofKNOXgenesthroughthesecis-elementshasanimpor-
elements, we performed electrophoretic mobility shift assays tantfunction.
(EMSAs)usingarecombinantproteinoftheOSH1HDfusedto
glutathioneS-transferase(GST;Figure6).Inthecaseofbinding PositiveAutoregulationofOSH1IsEssentialforIts
site1 (BS-1) in the promoter region of OSH1, GST-OSH1HD
ExpressionandSAMMaintenance
boundtowild-typeprobesbuthardlyboundtomutantprobes.In
thecompetitionassays,wild-typeBS-1coldDNAcompetedwith Our results presented so far reveal the presence of positive
radiolabeledBS-1probeforGST-OSH1HDbinding,butmutant autoregulationof OSH1. Toinvestigatetheimportance ofthe
BS-1fragmentsdidnot,confirmingthatGST-OSH1HDspecif- autoregulationfortranscriptionalregulationofOSH1,weper-
ically binds to BS-1. Similarly, GST-OSH1HD also specifically formedareporterassay.Among28transgenicplantscarryinga
bound to the other seven BS sequences in the third intron of reportergene,GFP-OSH1,inwhichgreenfluorescentprotein
OSH1.Thus,theseresultsindicatethatOSH1directlybindsto was inserted into the N terminus of an OSH1 genomic DNA
theseeightsequencesinvitro. fragment, 22 lines showed GFP fluorescence in the shoot
Several conserved TGAC sequences were also found in the meristem,afindingthatisinagreementwiththeendogenous
otherKNOXloci(seeSupplementalFigure3online).AsinOSH1, OSH1expressionpatternpreviouslyreported(Figures7Aand
theywerepreferentiallylocatedin59upstreamregionsandthe 7B; Sentoku et al., 1999). Whereas no clear difference was
third intron. Importantly, the distribution of these cis-elements observedwhenanOSH1bindingsitelocatedinthepromoter
coincided with that of highly enriched regions in ChIP assays. regionalonewasmutated(seeSupplementalFigure4online),
4374 ThePlantCell
Figure5. OSH1ProteinBindstoFiveKNOXLociinVivo.
ChIPassayusinganti-OSH1antibodyfollowedbyqPCRanalysisoftheOSH1(A),OSH6(B),OSH15(C),OSH43(D),andOSH71(E)locus.Gene
structuresareindicatedbelowthegraphs.PrimersusedinqPCRwerenamedaftertheirpositionrelativetothetranscriptionstartsiteofeachgeneand
areshownasblackbarsonthegenestructures.MagentabarsaboveandbelowthegenestructurerepresentconservedTGACsequencesintheplus
andminusstrands,respectively.ThepercentageofenrichmentrepresentstheamountofimmunoprecipitatedDNArelativetoinputDNA.Eachdata
pointistheaverageofthreeindependentbiologicalreplicates.ErrorbarsrepresenttheSD.
(A)to(E)ChIPassayfrom5-mmyoungpaniclesofthewildtype.Foranegativecontrol,controlrabbitIgGwasused.
(F)ChIPassayfromvegetativetissues.Foranegativecontrol,osh1shootapexandwild-type(wt)youngleafprimordiawereused.
PositiveAutoregulationofaKNOXGene 4375
Figure6. OSH1DirectlyBindstoConservedcis-ElementsontheOSH1LocusinVitro.
(A)OSH1bindingsites(BS)ontheOSH1locus.Graylinesindicateintronsandthe59and39regions.Boxesindicateexons.Asterisksaboveandbelow
thegenestructurerepresenteachBS,whichcontainsaconservedTGACsequence,intheplusandminusstrands,respectively.
(B)EMSAusingGST-OSH1HD.Formutantprobes(m1-m8),evolutionarilyconservedTGACsequencesweresubstitutedwithTCTC.Arrowheadsrepresent
thepositionsofshiftedprobes.GSTalonewasusedasnegativecontrol,and“comp.”representsthecompetitionassayusingwild-type(WT)coldprobes.
noneof26independenttransgeniclinesshowedGFPfluores- double mutant callus. We observed the recovery of de novo
cence, and the reporter gene expression was almost com- shootformationfromosh1d6doublemutantcallusbythewild-
pletely abolished when all OSH1 binding sites were mutated typeGFP-OSH1gene(sevenoutof58independenttransgenic
(GFP-mOSH1;Figures7Aand7C).Theseresultsindicatethat osh1 d6 calli; Figure 7D) but not by GFP-mOSH1 (none of 53
the positive autoregulation of OSH1 through evolutionarily independent transgenic osh1d6 calli; Figure 7E). The osh1d6
conserved OSH1 binding sites is essential for its proper ex- plantsrescuedbywild-typeGFP-OSH1wereabletogrowuntil
pression. the reproductive phase (Figure 7F). Thus, it was revealed that
Finally, we examined the biological importance of this auto- direct positive autoregulation of OSH1 was essential for SAM
regulation by rescue experiments using transgenic osh1 d6 formationand/ormaintenance.
4376 ThePlantCell
Figure7. PositiveAutoregulationofOSH1IsEssentialforItsExpressionandSAMMaintenance.
(A)Reportergenestructures.Graylinesindicateintronsand59and39regions.WhiteandgreenboxesindicateexonsandGFP,respectively.Inthecase
ofGFP-mOSH1,OSH1bindingsequencesweresubstitutedfromTGACtoTCTC.
(B)GFP-OSH1expressionintheshootapex.
(C)GFP-mOSH1expressionintheshootapex.
(D)Regeneratingshootsfromosh1d6calluscarryingGFP-OSH1onregenerationmedium.
(E)osh1d6calluscarryingGFP-mOSH1onregenerationmedium.Forregeneration,transgenicosh1d6calliwereculturedonregenerationmediumfor4to5weeks.
(F)Arescuedosh1d6plantcarryingGFP-OSH1grewuntilthereproductivephase.
(G)d6andwild-type(wt)plantsduringthereproductivephase.
Bars=100mmin(B)and(C),5mmin(D)and(E),and10cmin(F)and(G).
DISCUSSION is essential for its own expression and also for SAM mainte-
nance.Weproposethatmaintenanceoftheindeterminatestate
Here,weshowthatOSH1playsanindispensableroleincoop-
mediatedbypositiveautoregulationofaKNOXgeneisamech-
erationwithitsparalog,OSH15,intheestablishmentandmain-
anismforself-maintenanceoftheSAMthatensurestheunique
tenance of the SAM. Expression of KNOX genes is positively bodyplanoffloweringplants(Figure8).
regulated by the phytohormone CK and directly regulated by Regulation of cell population size and maintenance of an
OSH1 through evolutionarily conserved cis-elements. Further- indeterminate state are fundamental aspects of SAM function.
more,wedemonstratethatthepositiveautoregulationofOSH1 ContinuouscelldivisionofstemcellsatthecenteroftheSAM
PositiveAutoregulationofaKNOXGene 4377
andtheirdaughtercellsinsurroundingregionsreplenishesthe KNOXgenefamilyhaveredundantfunctionsinSAMmaintenance;
cellpopulation.Ontheotherhand,lateralorganinitiationfrom hence,itisplausiblethatnotonlyOSH1butalsoothermembersof
theflankoftheSAMconsumesthiscellpopulation.Studiesin theKNOXproteinfamilyparticipateinthisautoregulation.
Arabidopsishaveshownthatafeedbackloopbetweenamem- InArabidopsis,strongstmallelescompletelyfailtoestablish
ber of another class of homeobox genes, WUSCHEL, and the the SAMduringembryogenesis.Bycontrast,weak stmalleles
CLAVATA genes, which encode components of a signaling haveasmallerSAMthanthewildtypebutcontinueorganogen-
pathway,isessentialforhomeostasisofthestemcellpopulation esisuntilreproductivedevelopment(Clarketal.,1996;Endrizzi
(Schoofetal.,2000).Thisregulatoryfeedbackloopmaintainsthe et al., 1996). Mutations in other members of the KNOX gene
sizeofthestemcellpopulationandhasuntilnowbeentheonly family, such as KNAT1/BP and KNAT6, enhance the weak
describedmechanismforself-regulationintheSAM. phenotype of stm-2, although stm-2 knat6-1 shows additional
In our model, the stem cell population provides a source of abnormalitiesincotyledonseparation(Byrneetal.,2002;Belles-
cellsforlateralorganinitiation.AllcellsintheSAMaccumulate Boixetal.,2006).Thus,membersofArabidopsisclassIKNOX
KNOX proteins to maintain their indeterminate state (Jackson genes also have redundant functions in the SAM. It is an
et al., 1994; Sentoku et al., 1999), and KNOX proteins bind intriguingquestiontoaskwhetherKNOXgenesarealsoautor-
directlytoevolutionarilyconservedcis-elementsonKNOXlocito egulated in dicots. Given the conserved functions and similar
activate and/or maintain their expression. This autoregulation expression patterns of KNOX genes between monocots and
mayproviderobustnesstomaintainindeterminacyoftheSAM,in dicots,itispossiblethattheautoregulationisalsoconserved.
whichacomplexnetworkoftranscriptionfactorsandphytohor- Previously, rice class I KNOX genes have been categorized
mones balances stem cell activity and organogenesis (Zhao into twogroups based on theirexpression pattern in the SAM
etal.,2010).Atadefinedlocationinthemeristem,thisautoreg- (Sentokuetal.,1999).OSH1andOSH43areinthefirstgroup,
ulationisdiminishedandKNOXgenesaredownregulated. andtheyareexpressedintheentireSAMexcepttheepidermal
In our observations, mutations in both OSH15 and OSH1 layer.Ontheotherhand,expressionofasecondgroupgenes,
caused a severe reduction in the expression of other KNOX OSH6,OSH15,andOSH71,islocalizedtothebaseoftheSAM
genes and resulted in the failure of SAM establishment, which andboundariesbetweentheSAMandincipientleafprimordia.
was not the case for each single mutant. Members of KNOX Interestingly, we found that their expression profiles during
genesareexpressedinoverlappingregionsintheshootmeri- germinationalsosplitintothesetwogroups(Figure4A).Genes
stems (Jackson et al., 1994; Sentoku et al., 1999), and over- in the first group are expressed constantly before and after
expressorsofeachmembershowsimilarphenotypestothoseof germination. By contrast, the second group genes are greatly
OSH1 and OSH15 (Ito et al., 2001). In addition, it has been upregulated after germination. Therefore, these two KNOX
reported that not only KN1, but also other members of KNOX groups might also be under the control of different regulatory
protein, such as Gnarley1 and Liguleless3, directly bind to cis- mechanisms,althoughbotharepositivelyregulatedbyOSH1.
elements in the intron of GA2ox1 in maize (Bolduc and Hake, SevenOSH1bindingsitesidentifiedinthisstudyarelocatedin
2009). Considering these facts, it is likely that members of the the59halfofthethirdintronofOSH1.Thisregionalsocontainsa
clusterofsequencesthatarehighlyconservedbetweenriceand
maize (Inada et al., 2003). These conserved noncoding se-
quences (CNSs) are considered to have an important function
intherepressionofKNOXgeneexpressionbecausetransposon
insertionsanda305-bptandemduplicationintheseCNSscause
ectopic expression of KNOX genes in maize and barley
(Hordeum vulgare), respectively (Greene et al., 1994; Mu¨ller
et al., 1995). Our data reveal that these CNSs also contain
important cis-elements for positive autoregulation of OSH1.
Whetherornottherearecausalconnectionsbetweenthemu-
tationsandKNOXautoregulation,thesefactsindicatethatthis
clusterofCNSscontainsbothenhancerandsilencerelements.
In addition, there might be many unidentified cis-elements in
theseCNSs;therefore,moredetailedcis-elementanalysesare
necessarytounderstandfullythemechanismsinvolvedintran-
scriptionalregulationofKNOXgenes.
Sofar,ithasbeendebatedwhetherornotCKactsupstreamof
KNOX genes. In Arabidopsis, CK treatment enhances the ex-
pressionoftheProKNAT2:b-glucuronidasereporterintheSAM
Figure8. TheModelofaKNOXAutoregulatoryLoopforSAMMainte- (Hamant et al., 2002). By contrast, the induction of the CK
nance. biosyntheticgeneIPTinArabidopsisleavesoryoungseedlings
does not increase the expression of KNOX genes (Craft et al.,
Inthismodel,OSH1expressionispositivelyregulatedviaautoregulation.
OSH1proteindirectlyandpositivelyregulatesitsownexpressionand 2005). In this study, we observed a clear induction of KNOX
otherKNOXgenesintheSAM.KNOXgenesandCKmutuallyactivate genesbyCKduringshootregenerationfromcallus.Thisresult
eachother,creatinganotherpositiveautoregulatoryloop. stronglysuggeststhatKNOXgenesarepositivelyregulatedby