2024年4月20日发(作者：)

ThePlantCell,Vol.23:2143–2154,June2011,ãhtsreserved.

TheCOP1OrthologPPSRegulatestheJuvenile–Adultand

Vegetative–ReproductivePhaseChangesinRice

CW

NobuhiroTanaka,

a

HironoriItoh,

b

NaokiSentoku,

b

MikikoKojima,

c

HitoshiSakakibara,

c

TakeshiIzawa,

b

Jun-IchiItoh,

a

andYasuoNagato

a,1

a

Graduate

b

National

SchoolofAgriculturalandLifeSciences,UniversityofTokyo,Tokyo113-8657,Japan

InstituteofAgrobiologicalSciences,Tsukuba,Ibaraki305-8602,Japan

c

RIKENPlantScienceCenter,Tsurumi,Yokohama230-0045,Japan

Becauseplantreproductivedevelopmentoccursonlyinadultplants,thejuvenile-to-adultphasechangeisanindispensable

tifiedtwoallelicmutants,peterpansyndrome-1(pps-1)andpps-2,thatprolongthe

juvenilephaseinrice(Oryzasativa)andshowedthatricePPSisanorthologofArabidopsisthalianaCONSTITUTIVE

-1mutantexhibitsdelayedexpressionofmiR156andmiR172andthesuppressionofGA

biosyntheticgenes,reducingtheGA

3

eofitsprolongedjuvenilephase,thepps-1mutant

flowersearly,andthisisassociatedwithderepressionofRAP1Bexpressioninpps-1plantsindependentlyoftheHd1-Hd3a/

tronglyexpressedinthefourthandfifthleaves,suggestingthatitregulatestheonset

litytoregulatethevegetativephase

changeandthetimeoffloweringsuggeststhatricePPSacquirednovelfunctionsduringtheevolutionofrice/monocots.

INTRODUCTION

Thelifecycleofhigherplantshasthreemutuallydistinctdevel-

opmentalstages:theembryogenetic,vegetative,andreproduc-

etativestagecanbefurtherdividedintothe

juvenileandadultphases,whicharedistinguishedbymany

morphologicalandphysiologicaldifferencesbothinwoody

plants,suchasEnglishivy(Hederahelix)andEucalyptus

occidentalis(Poethig,1990;Jayaetal.,2010),whichexhibit

obviousheteroblasty,andinherbaceousplants,whichexhibit

differentmorphologicalandphysiologicalcharacteristicsinthe

juvenileandadultphases(LawsonandPoethig,1995;Telfer

etal.,1997;Asaietal.,2002).Importantly,becauseplantscan

initiatereproductivegrowthunderappropriateenvironmental

conditionsonlyduringtheadultphase(Simpsonetal.,1999;

Poethig,2003),thejuvenile-to-adultphasechange(alsoknown

asthevegetativephasechange)hasacriticalroleinplant

ghthemechanismsunderlyingthevegeta-

tivephasechangeremainlargelyunknown,recentstudiesusing

heterochronicmutantshaverevealedthatmicroRNAs(miRNAs)

playasignificantroleinthephasechange.

Arabidopsisthalianarosetteleavesareroundedwithsmooth

leafmarginsinthejuvenilephaseandarelong,ovate,and

enileandadultleavesalso

correspondencetoanagato@.

Theauthorresponsiblefordistributionofmaterialsintegraltothe

findingspresentedinthisarticleinaccordancewiththepolicydescribed

intheInstructionsforAuthors()is:YasuoNagato

(anagato@).

C

Somefiguresinthisarticlearedisplayedincoloronlinebutinblack

andwhiteintheprintedition.

W

OnlineversioncontainsWeb-onlydata.

/cgi/doi/10.1105/tpc.111.083436

1

Address

differconspicuouslyintheirpatternsoftrichomedistribution;

trichomesarefoundonlyontheadaxialsideofjuvenileleavesbut

arefoundonbothsidesofadultleaves(Telferetal.,1997).These

phenotypicmarkershavebeenusedtoidentifyArabidopsis

mutationscausingaprecociousphasechange,manyofwhich

ons

inZIPPY,SUPPRESSOROFGENESILENCING3,andRNA-

DEPENDENTPOLYMERASE6,whicharerequiredforpost-

transcriptionalsilencing,areassociatedwiththepresenceof

trichomesontheabaxialsidesofjuvenileleaves(Hunteretal.,

2003;Peragineetal.,2004).Inthesquintmutant,whichforms

abaxialtrichomesonearlyleaves,theactivityofmicroRNA156

(miR156)isdecreasedbecausetheARGONAUTE1proteinis

misfolded,resultingintheenhancedexpressionofthetarget

genes,SQUAMOSAPROMOTERBINDINGPROTEIN-LIKE

(SPL)familygenesencodingtranscriptionfactors(Smithetal.,

2009).Tenofthe16SPLgenesinArabidopsisaredownregulated

bymiR156b(Schwabetal.,2005),andthespl9andspl15T-DNA

insertionmutantsexhibitprolongedjuvenilephases(Schwarz

etal.,2008).Conversely,overexpressionofSPL3/4/5lackingthe

miR156targetsitecausesaprecociousphasechange(Wuand

Poethig,2006).ThesefindingsindicatethatsmallRNAsplayan

importantroleinthevegetativephasechangeinArabidopsis.

Inmaize(Zeamays),juvenileleaveshaveepicuticularwaxand

noepidermalhair,andadultleaveshaveepidermalhairbutno

wax(LawsonandPoethig,1995).AsinArabidopsis,miRNAsare

inantCorn-

grass1(Cg1)mutantencodesmiR156,leadingtooverexpression

ofmiR156andprolongationofthejuvenilephase(Chucketal.,

2007).Theglossy15(gl15)mutantshortensthejuvenilephasein

themaizeepidermis(MooseandSisco,1994);GL15isanAP2-

likegeneharboringtwoAP2domains(MooseandSisco,1996)

thataretargetsofmiR172(Lauteretal.,2005).Intheearly

2144ThePlantCell

vegetativestage,transcriptionofmiR156farexceedsthatof

miR172,whereaslaterinthevegetativestage,theinverse

patternisseen:transcriptionofmiR172farexceedsthatof

miR156(Lauteretal.,2005;Chucketal.,2007).InversemiR156

andmiR172expressionpatternsarealsoobservedinArabidop-

sis(AukermanandSakai,2003;WuandPoethig,2006).

InarecentArabidopsisstudy(Wuetal.,2009),miR172ex-

pressionwasshowntooccurdownstreamofSPL9andSPL10

expression,,miR156and

miR172maybekeygenesinthejuvenile–adultphasechange.

ThetargetsofmiR156andmiR172areknown,butlittleisknown

abouttheirupstreamgenes.

Gibberellin(GA)isawell-establishedregulatoryphytohormone

ofthejuvenile–idopsis,mutationsin

genesfunctioninginthebiosynthesisofandresponsetoGA

prolongthejuvenilephase(Telferetal.,1997;TelferandPoethig,

1998);themostseveremutation,ga1-3,causestheplanttofailto

developadultleaves(Telferetal.,1997;TelferandPoethig,

1998).Furthermore,GAtreatmentpromotesthetransitionto

adultphaseinbothArabidopsisandmaize(EvansandPoethig,

1995;Schwarzetal.,2008).

AlthoughGAisknowntopromotethevegetativephase

change,theotherupstreamanddownstreamfactorscontrolling

r,becausethe

juvenile–adultphasechangeaffectsalargenumberoftraits,itis

safetoassumethatalargenumberofgenes,includingthose

encodingmiRNAs,ehen-

siveunderstandingofthephasechangewillrequiretheidenti-

ficationandfunctionalcharacterizationofmoreoftherelevant

genes.

Todate,moleculargeneticstudiesofthevegetativephase

changehavebeenconcentratedmainlyintwospecies,Arabi-

r,rice(Oryzasativa)alsohasmany

morphologicalandphysiologicaltraitsthatdifferbetweenthe

raitsincludethesizeofthe

shootapicalmeristem(SAM),sizeandshapeofleafblades,

presenceorabsenceofmidribs,vascularorientationinthestem,

node–internodedifferentiation,andphotosyntheticrate(Itoh

etal.,2005).Onlyonemutationaffectingthericevegetative

phasechange,mori1,hasbeenreportedtodate(Asaietal.,

2002).Althoughthismutanthasbeencharacterizedasperpet-

uallymaintainingthejuvenilephase(Asaietal.,2002),thecausal

genehasnotyetbeencloned.

Inthisstudy,weattemptedtoidentifyadditionalricemutants

ult,weisolated

thepeterpansyndrome(pps)mutant,whichexhibitsaprolonged

juvenilephaseandearlyflonalcloningrevealed

thatPPSisanorthologofArabidopsisCONSTITUTIVEPHOTO-

MORPHOGENIC1(COP1).

RESULTS

VegetativePhenotypesofppsMutant

Weidentifiedonerecessivericemutantwithadwarf,dark-green

phenotype(Figure1A).Thedwarfphenotypewasmaintained

untilthefleadetailedexaminationofthe

juvenile/adultmarkertraits(describedbelow)ofthismutant

revealedthatithasaprolongedjuvenilephase,wenamedthis

mutantpeterpansyndrome-1(pps-1).Subsequently,weiden-

tifiedanadditionalallelicmutant,pps-2

,thatexhibitedaneven

s-2seedscouldnot

germinate,andonly

;

5%ofseedscouldgerminateonnutrient

mediumcontainingsucrosebutdiedinaweek(Figure1B).Thus,

ouranalyseswereperformedprimarilyonpps-1.

Oftheconsiderablenumberofmorphologicalandphysiolog-

icaltraitsknowntodifferbetweenthejuvenileandadultphasesin

rice(Itohetal.,2005),wefirved

thattheratioofleafbladelengthtowidthrapidlyincreaseswith

elevationofleafposition(Figure1C);theleaf-bladelength:width

ratioforthesecondleafbladeis

;

10:1,whereasthatofthefifth

leafblade,whichismuchmoreslender,isgreaterthan50:

contrast,norapidincreaseofthelength:widthratiowithelevation

ofleafpositionwasobservedinthepps-1mutant;theratioof

eventheeighthleafbladeofthemutantwascomparabletothat

ofthethirdleafofwild-typeplants(Figure1C).

Wealsoexaminedtheleafbladesofthepps-1mutantforthe

presenceofamidrib,

wild-typeplants,midribswererarelyobservedinsecondleaves

(Figure1D).Inthirdandfourthleaves,midribswereobservedto

coverapproximatelyone-halfofthetotallengthfromleafbaseto

leaftip(

;

40and60%ofthetotallength,respectively;Figures1E

and1I).Inthehigherleaves,midribscoveredatleast75%ofthe

lengthoftheblade(Figure1I).Bycontrast,second,third,and

fourthpps-1leavesexhibitedalmostnomidrib(Figures1Fand

1I),andmidribformationinfifthleafwas

;

20%ofthetotallength

(Figures1Gand1I).Eveninthesixthpps-1leaf,midriblength

wascomparabletothatofawild-typethirdleaf(Figures1Hand

1I).Thus,secondtofourthpps-1leavesarestructurallysimilar

towild-typesecondleaves,andfifthtosixthpps-1leavesare

ggeststhatthe

pps-1sixthleafisintermediatebetweenjuvenileandadult,sug-

gestingthatpps-1hasmorejuvenileleaves.

Anexaminationofothermorphologicaltraitssupportedthis

ly,SAMsenlarge

graduallyduringdevelopment(Figure1J),butthepps-1SAM

remainedunchangedinsizeuntilatleastthefifth-leafstage

(Figure1J).Furthermore,thepps-1mutantdisplayeddifferent

node–ly,below

theinsertionofthefourthleaf,thevascularbundlesareirregular

inorientation,andnonode–internodedifferentiationisevident,

whereasabovethefourthleaf,thestemhasobviousnodes

(Figure1K);inthemutant,ontheotherhand,thevascular

orientationremainedirregularuptothesixthleafinsertion,and

nonodedifferentiationwasobservableonthestemuntilthe

insertionoftheseventhleaf(Figure1L).Therefore,thestem

structureofthepps-1mutantbecomesadultabovethesixthor

seventhleafinsertionandisalsoconsistentwithaprolonged

juvenilephase.

Thephysiologicaltraitsassociatedwiththephasechangein

thepps-1mutantwerealsoconsistentwithaprolongedjuvenile

mple,inwild-typericeplants,photosynthesisis

reducedinjuvenileleaves(secondleaves)andmuchmore

quicklyinfourthandhigherleaves(Figure1M).Bycontrast,

photosynthesisoccurredslowlyeveninthefourththrougheighth