849DEVELOPMENT AND STEM CELLS RESEARCH ARTICLE

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Development 138, 849-859 (2011) doi:10.1242/dev.057331© 2011. Published by The Company of Biologists Ltd

1Molecular Genetics Department, Centre for Research in Agricultural GenomicsCSIC-IRTA-UAB, 08013 Barcelona, Spain. 2Department of Plant Systems Biology, VIBB-9052 Gent, Belgium. 3Department of Plant Biotechnology and Genetics, GhentUniversity, 9052 Gent, Belgium. 4Fundación Instituto Leloir and IIBBA-CONICET,C1405BWE, Buenos Aires, Argentina.

*Author for correspondence (ana.cano@cid.csic.es)

Accepted 24 December 2010

SUMMARYBrassinosteroids (BRs) play crucial roles in plant growth and development. Previous studies have shown that BRs promote cellelongation in vegetative organs in several plant species, but their contribution to meristem homeostasis remains unexplored. Ouranalyses report that both loss- and gain-of-function BR-related mutants in Arabidopsis thaliana have reduced meristem size,indicating that balanced BR signalling is needed for the optimal root growth. In the BR-insensitive bri1-116 mutant, theexpression pattern of the cell division markers CYCB1;1, ICK2/KRP2 and KNOLLE revealed that a decreased mitotic activityaccounts for the reduced meristem size; accordingly, this defect could be overcome by the overexpression of CYCD3;1. The activityof the quiescent centre (QC) was low in the short roots of bri1-116, as reported by cell type-specific markers and differentiationphenotypes of distal stem cells. Conversely, plants treated with the most active BR, brassinolide, or mutants with enhanced BRsignalling, such as bes1-D, show a premature cell cycle exit that results in early differentiation of meristematic cells, which alsonegatively influence meristem size and overall root growth. In the stem cell niche, BRs promote the QC renewal anddifferentiation of distal stem cells. Together, our results provide evidence that BRs play a regulatory role in the control of cell-cycle progression and differentiation in the Arabidopsis root meristem.

KEY WORDS: Brassinosteroids, Root, Meristem, Cell division, Quiescent centre, Stem cells, Columella, Cell elongation

Brassinosteroids control meristem size by promoting cellcycle progression in Arabidopsis rootsMary-Paz González-García1, Josep Vilarrasa-Blasi1, Miroslava Zhiponova2,3, Fanchon Divol1,Santiago Mora-García1,4, Eugenia Russinova2,3 and Ana I. Caño-Delgado1,*

DEV

ELOPM

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Development ePress online publication date 26 January 2011

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MATERIALS AND METHODSPlant lines and growth conditions!"#$%&'()%)*+,#-%#.# 6X9='Z2>-.9'+%0'#.'P*&48/#%R<'6P*&R<='/%$B()*4.19A*'%5*#1'2$*,>"2'5%)#%/#&#,>:',-2'$H)>E< 84,%.,:'*)#(#.%&&>'#.'_.B-2#8R;6_.R;=' /%$B()*4.1' 6 #̀.' 2,' %&9:' ;<<;=:' +%0' #.,)*()20021' #.,*' ,-2' P*&R<2$*,>"29' A-2' 95:>R.4&&' 84,%., $"%>E>>I 6X#' %.1' P-*)>:' EFFL=' %.1' %,)%.0(2.#$'&#.2'*52)2?")200#.('95:>EBL/ 6d)#21)#$-02.'2,'%&9:';<<<H'I%.(2,'%&9:';<<E='+2)2'4021'%0'2?%8"&20'*3'#.02.0#,#5#,>',*'NM0'%.1'*3'2.-%.$21NM'0#(.%&&#.(:')20"2$,#52&>9'b,-2)'&#.20'4021'+2)2e'(7A79>J>MBF6 6P*&f.RP%)8*.%' 2,' %&9:' EFFF=:' (:7?KD?5/KMBF6 6D2' g2>&12)' 2,' %&9:' ;<<E=:' %7A7<CJ>R*52)2?")200#.('&#.2'6M#*4R[-%8&#$-#'2,'%&9:'EFFF=:'7A79>J>MBL/6\/21%RAf8%0' 2,' %&9:' ;<<F=:' ?;4001EBL/ 6g*&B2)' 2,' %&9:' ;<<E=:(84NOMBL/ 6K%)B%)'2,'%&9:';<<L=:'P7KOM7L/: P7QIMBF6R*P7>QKMBF6%.1'P7>SQMBF6:'(675MBL/*6K%/%,#.#'2,'%&9:'EFFF=: (!B0QKMBL/ 6^%+>2,'%&9:';<<J=:')T"EQ 6d4B%B#'2,'%&9:'EFFO=:'%.1'U'VOE> 6K%)B%)'2,'%&9:';<<L=9K2210'+2)2'04)3%$2'0,2)#&#C21'#.'GJh'0*1#48'->"*$-&*)#,2:'52).%&#C21'3*)'L;-*4)0'%,'WiP'#.'1%)B.200'%.1'()*+.'*.'52),#$%&&>'*)#2.,21'"&%,20'$*.,%#.#.(E! S4)%0-#(2'%.1'KB**('6SK='0%&,'8#?,4)2:'Eh'04$)*02'%.1'<9Oh'%(%)'#.,-2'%/02.$2'*)'")202.$2'*3'1#332)2.,'$*.$2.,)%,#*.0'*3'NX'6P;OZWObYHI%B*:b0%B%:'j%"%.=9'd*)'0-*),R,2)8',)2%,82.,0'"&%.,0'+2)2',)2%,21'+#,-'E' S'NX3*)'W'-*4)0:'O'-*4)0'%.1';W'-*4)09'@&%,20'+2)2'#.$4/%,21'%,';;iP'%.1'L<h-48#1#,>'4.12)'&*.(R1%>'$*.1#,#*.0'6EY'-*4)0'&#(-,]O'-*4)0'1%)B=9

Confocal microscopy!' dg' E<<<' $*.3*$%&' 8#$)*0$*"2' 6b&>8"40:' A*B>*:' j%"%.=' +%0' 4021,-)*4(-*4,',-2'0,41>9'M**,0'+2)2'0,%#.21'#.'E<' (]8&'")*"#1#48'#*1#12'6@T=3*)';RJ'8#.4,20:')#.021:'8*4.,21'#.'1Z;b:'%.1'5#04%&#C21'%3,2)'2?$#,%,#*.'/>%'[)]!)'WOOR.8'&%02)'&#.29'@T'%.1'7d@'+2)2'12,2$,21'+#,-'%'/%.1R"%00'JL<RYL<'.8'3#&,2)'%.1'J<<RJWJ'.8'3#&,2):')20"2$,#52&>9'd*)',-2'>2&&*+'%.1'$>%.3&4*)20$2.,' ")*,2#.0' 6`d@' %.1' Pd@=R,%((21' )2"*),2)0:' ,-2' 2?$#,%,#*.+%52&2.(,-0'+2)2'WOO'.8'%.1'W<J'.8:'%.1'3&4*)20$2.$2'+%0'$*&&2$,21'#.',-2)%.(20'*3'WFGRJGY'.8'6)2.12)21'#.'()22.='%.1'WY<RJ<<'.8:')20"2$,#52&>9K,%)$-'()%.4&20'#.'$*&482&&%'$2&&0'+2)2'5#04%&#C21'/>'%'8*1#3#21'@0241*RK$-#33'68@K=R@T'0,%#.#.('82,-*1'6A)42).#,'2,'%&9:';<<O=9'D#5#1#.('$2&&0'#.7A79>J>MBL/ %.1'?;4001MBL/ "&%.,0'+2)2'8%.4%&&>' $*4.,21' 3)*8$*.3*$%&'0,%$B09'T8%(20'+2)2'")*$20021'+#,-',-2'b&>8"40'dg'0*3,+%)2'%.1%0028/&21'+#,-'@-*,*0-*"'PK'6!1*/2'K>0,280:'K%.'j*02:'P!:'\K!=9

Statistical analysis of root measurementsM**,'&2.(,-:'$2&&'&2.(,-'%.1'.48/2)'*3'82)#0,28'$2&&0'#.'!"#$%&'()%) )**,0+2)2' %0020021' #.' %,' &2%0,' ,-)22' #.12"2.12.,' 2?"2)#82.,09' M**,0' +2)20$%..21' %.1' )**,' &2.(,-' 82%04)21' +#,-' T8%(2j' 0*3,+%)26-,,"e]])0/9#.3*9.#-9(*5]#Q]=9' d*)' $*8"%)#0*.0:' K,412.,k0' +R,20,0' +2)2"2)3*)821'#.'%&&'$%020'6022'A%/&2'KE'#.',-2'04""&282.,%)>'8%,2)#%&=9P2&&'&2.(,-'+%0'82%04)21'%&*.('0#.(&2'2"#12)8%&'$2&&'3#&209'A-2'.48/2)

*3'2"#12)8%&'$2&&0'#.'#.1#5#14%&'$2&&'3#&20'+%0'4021',*'(%4(2',-2'82)#0,280#C29'A-2'82)#0,28%,#$'C*.2'+%0'123#.21'%0',-2')2(#*.'*3'#0*1#%82,)#$'$2&&03)*8',-2'VP'4"',*',-2'$2&&',-%,'+%0',+#$2',-2'&2.(,-'*3',-2'#8821#%,2&>")2$21#.('$2&&9'd*)'1*02R)20"*.02'%00%>0:',-2'%52)%(2'1#0,%.$2'3)*8',-2'VP,*'2%$-'#.1#5#14%&'$2&&'+%0'$*8"4,21'%0'%'34.$,#*.'*3' ,-2'$2&&'.48/2)9X#.2%)')2()200#*.0'+2)2'%""&#21',*',-2'%52)%(2'1#0,%.$2'3)*8',-2'VP'%0'%34.$,#*.'*3'$2&&'.48/2)'3*)'/*,-',-2'82)#0,28%,#$'%.1',-2'2&*.(%,#*.'C*.29P*.02U42.,&>:',-2'0&*"2'*3',-2'&#.2%)')2(#*.0'*3',-2'$4)520')23&2$,21',-2%52)%(2'$2&&'&2.(,-'#.'2%$-'C*.29

Quantification of cell division in !-glucuronidase (GUS)-stainedrootsd*)'!R(&4$4)*.#1%02'67\K='12,2$,#*.:'YR1%>R*&1'0221&#.(0'+2)2'#882)021*.' #$2R$*&1'F<h' 65]5=' %$2,*.2:' #.$4/%,21';<'8#.4,20'*.' #$2:' )#.021' #.1Z;b:'#.3#&,)%,21'+#,-'E<<'8S'0*1#48'"-*0"-%,2'/4332)'6"Z'L9;=:'E<'8S0*1#48'_DA!:'<9Eh'A)#,*.'lRE<<:'E'8(]8&'JR/)*8*RWR$-&*)*RGR#.1*&>&R!RDR(&4$4)*.#12' 6l(&4$H'D4$-23%:'Z%%)&28:'A-2'^2,-2)&%.10=:'E<'8S"*,%00#48'32))*$>%.#12'%.1'"*,%00#48'32))#$>%.#12:'%.1'#.$4/%,21'%,'GLiP3*)'G'-*4)09'K%8"&20'+2)2')#.021',-)22',#820'#.'1Z;b:',)2%,21'+#,-'L<h2,-%.*&:'%.1'$&2%)21'#.'$-&*)%&'->1)%,29'K,%#.21')**,0'+2)2'5#04%&#C21'+#,-%.'!?#*@-*,' 6m2#00:' j2.%:'72)8%.>='8#$)*0$*"29'D#(#,%&' #8%(20'+2)2%$U4#)21'+#,-'%'D@L<'6b&>8"40='$%82)%9'A-2'.48/2)'*3'7\KR0,%#.21$2&&0'#.',-2')**,0'*3'(2.*,>"20'$%))>#.('(7A79>J>MBF6 +%0')2$*)121'+#,-%.'!?#*@-*,'6m2#00='8#$)*0$*"29

ImmunohistochemistryI-*&2R8*4.,'#884.*&*$%&#C%,#*.'#.'!"#$%&'()%) )**,0'+%0'$%))#21'*4,'%0120$)#/21'6K%42)'2,'%&9:';<<Y='+#,-'8#.*)'8*1#3#$%,#*.09'd*)'IblJe7d@:"*&>$&*.%&' %.,#R7d@' T(7' %.1' !&2?%' d&4*)' WOO' $*.Q4(%,2' 6Ee;<<=6T.5#,)*(2.:'P%)&0/%1:'P!:'\K!='+2)2'40219'A*'12,2$,'$2&&'"&%,20'3*)821#.' 1#5#1#.(' $2&&0:' +2' 4021' %.,#R[^bXX_' ")#8%)>' %.,#/*1#20' 6EeE<<<=6g*&B2)'2,'%&9:';<<E='%.1'!&2?%JJJR$*4"&21'%.,#R)%//#,'02$*.1%)>'%.,#/*1>6T.5#,)*(2.=9' K&#120' +2)2' 8*4.,21' #.' W":YR1#%8#1#.*R;R"-2.>&#.1*&26D!@T='g2$,%0-#2&1'821#48'6g2$,*)'X%/*)%,*)#20:'N4)&#.(%82:'P!:'\K!=%.1'"&%$21'%,'WiP9'D!@T'%.1'!&2?%WOO'0#(.%&0'+2)2'%$U4#)21'02U42.,#%&&>#.,*' 02"%)%,2'$-%..2&0e' 3*)'!&2?%JJJ:' 0%8"&20'+2)2'2?$#,21'%,'JJF'.8%.1' 3&4*)20$2.$2' 12,2$,21' #.' ,-2' )%.(2' JJ<RY<<' .8H' 3*)' D!@T:' ,-22?$#,%,#*.'+%52&2.(,-'+%0'W<J'.8'%.1'3&4*)20$2.$2'$*&&2$,21'#.',-2')%.(2W;JRWLJ'.89

Quantitative real-time PCRA*,%&' M^!' 3)*8' )**,' ,#"0' +%0' 2?,)%$,21' +#,-' @&%.,' M^2%0>' S#.#' [#,6V#%(2.:'Z#&12.:'72)8%.>=:'D^!'$*.,%8#.%,#*.0'+2)2')28*521'+#,-',-2D^!R3)22'[#,' 6!8/#*.:'!40,#.:'Al:'\K!=' %.1' $D^!'+%0' 0>.,-20#C21+#,-'K4"2)K$)#",' TTT'M252)02'A)%.0$)#",%02'6T.5#,)*(2.=:'%&&'%$$*)1#.(' ,*,-2'8%.43%$,4)2)k0'#.0,)4$,#*.09'b&#(*.4$&2*,#120'+2)2'120#(.21'+#,-',-2@)#82)' _?")200' K*3,+%)2' 6!""&#21' N#*0>0,280' />' X#32' A2$-.*&*(#20:P%)&0/%1:'P!:'\K!=9'!,G(;W;J<'3*)+%)1'J"RPAA77A77AAA777A7R7A7RG" %.1' )252)02' J"R!P7!A!!7!7!!!P!P!7!!!PRG"H!,G(EE;Y<' 3*)+%)1' J"RA7!APA7AAAP7!7PP77APRG" %.1' )252)02J"R!!!P!AAPAA7PAPAPA!APAA7PPRG"9@PM'")*14$,0'+2)2'12,2$,21'+#,-',-2'K`NM'7)22.'T'S%0,2)'6M*$-2

D#%(.*0,#$0:'S%..-2#8:'72)8%.>=9'X#(-,P>$&2)WO<'0*3,+%)2'E9J9<')2&2%026M*$-2'D#%(.*0,#$0='+%0'4021',*'$%&$4&%,2')2&%,#52'$-%.(2'#.'2?")200#*.&252&0:'+#,-',-)22',2$-.#$%&')2"&#$%,209'S2&,#.('$4)520'%.%&>020'%,',-2'2.1*3' ,-2' ")*$200' %.1' n.*' ,28"&%,2' $*.,)*&0k' +2)2' $%))#21' *4,' ,*' 2.04)2")*14$,R0"2$#3#$'%8"&#3#$%,#*.'+#,-*4,'")#82)R1#82)'%),#3%$,09'A*'25%&4%,2(2.*8#$'D^!'$*.,%8#.%,#*.:'%'$*.,)*&')2%$,#*.'+%0')4.'+#,-*4,')252)02,)%.0$)#",%029'A-2'2?")200#*.'*3'!,G(;W;J<'+%0'4021'%0'%.'2.1*(2.*40$*.,)*&'4.12)'252)>'$*.1#,#*.9

RESEARCH ARTICLE Development 138 (5)

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RESULTSOptimal BR signalling is required to control rootmeristem sizeM**,'()*+,-'#0'12,2)8#.21'/>',-2'/%&%.$2'/2,+22.'$2&&'1#5#0#*.%.1'$2&&'2&*.(%,#*.'6N2280,2)'%.1'N%0B#.:'EFFO=9'A*'%00200',-2')*&2*3'NM0'#.')**,'()*+,-:'+2'3#)0,'$*8"%)21',-2')**,'&2.(,-'*3'84,%.,0+#,-' %&,2)21' NM' 0#(.%&&#.(' />' 40#.(' $"%>E>>I 84,%.,0' 6X#' %.1P-*)>:'EFFL=:',)%.0(2.#$'&#.20'*52)2?")200#.('%'()22.'3&4*)20$2.,")*,2#.'67d@=R,%((21'52)0#*.'*3'95:> 6d)#21)#$-02.'2,'%&9:';<<<HI%.('2,'%&9:';<<E='%.1'$H)>E< "&%.,0'$%))>#.('%'(%#.R*3R34.$,#*.84,%,#*.',-%,'2.-%.$20'NM'0#(.%&&#.('6 #̀.'2,'%&9:';<<;=9'!'(2.2)%&,)2.1' +%0' */02)521' 3*)' %&&' NMR)2&%,21' 84,%.,0' ,-%,' 1#0"&%>210-*),2)')**,0',-%.',-*02'*3',-2'+#&1',>"2'P*&R<'6d#(9'E!:d=9^2?,:'+2'0,41#21',-2'$*.,)#/4,#*.'*3'NM0',*',-2'2&*.(%,#*.'*3

)**,'$2&&09'M**,'2"#12)8%&'$2&&0'#.',-2'1#332)2.,#%,#*.'C*.2:'+-2)2$2&&0'-%52')2%$-21',-2#)'3#.%&'0#C2'*3'95:>EBL/R*52)2?")200#.("&%.,0'o;EYpL' 8'60919=:'/q<9<<Er'%.1'$H)>E< o;GLpG' 8'60919=:/q<9<<Er' +2)2' &*.(2)' ,-%.' ,-*02' *3' ,-2' +#&1' ,>"2' oEGFp;' 860919=r:'+-2)2%0'$"%>E>>I 84,%.,'$2&&0'3%#&21',*'2&*.(%,2'")*"2)&>oFEpO' 8' 60919=:' /q<9<<Er' 6022' d#(9' K;' #.' ,-2' 04""&282.,%)>

8%,2)#%&=9' A-202' )204&,0' %)2' #.' %()2282.,' +#,-' ")25#*40*/02)5%,#*.0'0-*+#.(',-%,'NM0'#.14$2'$2&&'2&*.(%,#*.'#.',-2')**,6KC2B2)20'2,'%&9:'EFFYH'Sc00#('2,'%&9:';<<GH'S*4$-2&'2,'%&9:';<<Y=9I2'%&0*'20,#8%,21',-2'$*.,)#/4,#*.'*3'$2&&'1#5#0#*.',*'82)#0,28

0#C2'/>'$*4.,#.(',-2'.48/2)'*3'#0*1#%82,)#$'2"#12)8%&'$2&&09'A-#0.48/2)'+%0'0#(.#3#$%.,&>')214$21'#.'$"%>E>>I 84,%.,0'oEFpE'60919=:/q<9<<Er'6d#(9'EP:7='$*8"%)21'+#,-',-%,'#.',-2'+#&1',>"2'oGGp;60919=r'6d#(9'EN:7=9'T.'%11#,#*.:',-2'0-*),')**,0'*3'95:>EBL/ "&%.,0-%1'%'0&#(-,&>')214$21'.48/2)'*3'82)#0,28%,#$'$2&&0'o;Op;'60919=:/q<9<<Er'6d#(9'ED:7=9'A-#0')214$,#*.'+%0'8*)2'")*.*4.$21'#.',-2(%#.R*3R34.$,#*.'$H)>E< 84,%.,0'o;;pE'60919=: /q<9<<Er'$2&&0'6d#(9E_:7:Z=9'A-2')214$21'.48/2)'*3'82)#0,28%,#$'$2&&0'#.'/*,-'&*00R%.1'(%#.R*3R34.$,#*.'84,%.,0')252%&0',-2'#8"*),%.$2'*3'%'/%&%.$21NM'0#(.%&&#.('#.',-2'$*.,)*&'*3',-2'82)#0,28'0#C29

BR effects on root meristem size are dosedependentA*'0,41>',-2'2332$,0'*3'NM0'*.')**,'()*+,-:'+2',)2%,21'+#&1R,>"2P*&R<'0221&#.(0'+#,-'NX'$*.$2.,)%,#*.0'#.',-2')%.(2')2"*),21'3*),-2'1#)2$,'/#.1#.('*3'NX',*',-2'95:>R&#B2')2$2",*)'3%8#&>'6[#.*0-#,%

851RESEARCH ARTICLEBrassinosteroids and root development

Fig. 1. Alterations in BRs signalling result in smallerroot apical meristems. (A) Phenotype of 6-day-old wild-type Col-0 (WT), bri1-116, a transgenic lineoverexpressing BRI1-GFP (Friedrichsen et al., 2000; Wanget al., 2001) and bes1-D seedlings (Yin et al., 2002). Scalebar: 10 mm. (B-E) Confocal images of 6-day-old wild-type(B), bri1-116 (C), BRI1-GFP (D) and bes1-D (E) rootsstained with PI. Arrows indicate the boundary betweenthe proximal meristem and the elongation zone of theroot. Scale bars: 100 m. (F) Root-length measurementsof bri1-116, BRI1-GFP and bes1-D seedlings comparedwith the wild type (for statistical analysis, see Table S1 inthe supplementary material). Values represent the meanof 80 measurements ±s.d. (G) Meristem cell number inbri1-116, BRI1-GFP and bes1-D seedlings compared withthe Col-0 (wild type) at 4 or 6 days after germination(asterisks indicate significant differences from Col-0 foreach day) (for statistical analysis, see Table S1 insupplementary material). Values represent the mean of 34measurements ±s.d. (H) Spatial profile of epidermal QCdistance ( m) versus number of cells from the QC of 6-day-old bri1-116, BRI1-GFP, bes1-D and wild-type roots.Epidermal cells were counted from the QC up to theappearance of the first root hair. All BR-related mutantsanalysed exhibited a premature exit from the meristemzone (black arrowhead) and early entry into thedifferentiation zone (grey arrowhead). Values representthe mean of 5 measurements ±s.d.

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852

2,' %&9:' ;<<J=9' NX' ")*8*,21' )**,' ()*+,-' *.&>' %,' 52)>' &*+$*.$2.,)%,#*.0'6 <9<W'.S:'/q<9<J='#.'YR1%>R*&1'0221&#.(0'6d#(9;!:7='6022'A%/&2'KE'#.',-2'04""&282.,%)>'8%,2)#%&='6Sc00#('2,'%&9:;<<G=H' NX' $*.$2.,)%,#*.0' -#(-2)' ,-%.' <9<W' .S' #.-#/#,21' )**,()*+,-'%.1'82)#0,28'0#C2'6d#(9';NRd:7:ZH'/q<9<<E=9'NMR#.14$21)**,'0-*),2.#.('+%0'$*4"&21',*'%')214$,#*.'#.'82)#0,28'0#C2')%,-2),-%.' #.' $2&&' 2&*.(%,#*.' 6d#(9' ;7:ZH' /q<9<<E=9' A-2' .48/2)' *382)#0,28%,#$'$2&&0'#.'YR1%>R*&1')**,0',)2%,21'+#,-'W'.S'NX'+%0)214$21'sY<h'+-2.'$*8"%)21'+#,-'4.,)2%,21')**,0'6d#(9';N:d:ZH/q<9<<E=9'!'1*02R)20"*.02'%.%&>0#0')252%&21',-%,'NM0'")*8*,21,-2'2?#,'*3'$2&&0'3)*8',-2'82)#0,28%,#$')2(#*.:'%.1'#.1#$%,20',-%,NM0'")*8*,2'$2&&'2&*.(%,#*.'%,',-2'82)#0,28%,#$'C*.2'*3',-2')**,6d#(9' ;T=9' A-202' )204&,0' $*.$4)' +#,-' *4)' (2.2,#$' %.%&>0#0' %.104""*),' ,-%,'NM' 0#(.%&&#.(' #0' )2U4#)21' ,*'8%#.,%#.' .*)8%&' )**,()*+,-')%,20',-)*4(-',-2'$*.,)*&'*3',-2')**,'82)#0,28'0#C29

BRs are required for proper cell-cycle progressionin the root meristemA*'#.520,#(%,2'+->'%&,2)21'NM'0#(.%&&#.('+%0'12,)#82.,%&'3*)',-2)**,'82)#0,28'0#C2:'+2'0,41#21',-2'2332$,0'*3'NM0'*.',-2'$2&&'$>$&2/>'%.%&>C#.(',-2'2?")200#*.'*3',-2'DR/*?'(7A79>J>MBF6 )2"*),2)6P*&f.RP%)8*.%'2,'%&9:'EFFF='#.'NMR)2&%,21'84,%.,0'%.1'#.'NXR,)2%,21'"&%.,09'(7A79>J>MBF6 %&&*+0',-2'5#04%&#C%,#*.'*3'$2&&0'%,

,-2'7;RS'"-%02'*3',-2'$2&&'$>$&29'A-2'.48/2)'*3'7\KR0,%#.21'$2&&0+%0'0#(.#3#$%.,&>')214$21'#.'NXR,)2%,21'"&%.,0'oLp;'60919=: /q<9<<Er6d#(9'GN='%.1'#.'$H)>E< 84,%.,0'oYpE'60919=: /q<9<<Er'6d#(9'GP=+-2.'$*8"%)21'+#,-',-2'+#&1',>"2'oGWpJ'60919=r'6d#(9'G!:_=9'NXR,)2%,21' "&%.,0' 2?-#/#,21' %' 1*02R12"2.12.,' )214$,#*.' *3' 7\KR"*0#,#52' $2&&0H' 0#8#&%)' )204&,0' +2)2' */,%#.21' +#,-' ,-27A79>J>MBL/*)2"*),2)'6022'd#(9'KG'#.'04""&282.,%)>'8%,2)#%&=9'T.$"%>E>>I 84,%.,0:'%'0&#(-,' #.$)2%02'#.',-2'2?")200#*.'&252&0'+%0*/02)521'3*)',-2'(7A79>J>MBF6*)2"*),2) 6d#(9'GD:_='6/q<9<J=:+-2)2%0'$"%>E>>IJ7A79>J>MBL/ )**,0'0-*+21'%')214$,#*.'#.',*,%&7d@R8%)B21' $2&&0' 6022' d#(9' KG' #.' ,-2' 04""&282.,%)>'8%,2)#%&H/q<9<<E=9' A-202' 1#332)2.$20'8%>' /2' %,,)#/4,21' ,*' ,-2' 3%$,' ,-%,7A79>J>eBL/ #0' 8%#.&>' 2?")20021' #.' "2)#"-2)%&' $2&&' &%>2)0:+-2)2%0'(7A79>J>MBF6*#0'2?")20021'#.'%&&')**,',#00420'6022'd#(9KG'#.',-2'04""&282.,%)>'8%,2)#%&=9I2')2%0*.21',-%,',-2'%&,2)%,#*.0'#.'7A79>J> 2?")200#*.'#.',-2

$"%>E>>I*/%$B()*4.1'$*4&1')23&2$,'%'0&*+2)'")*()200#*.'%.1]*)'%12&%>21' 7;]S' ,)%.0#,#*.' *3' ,-2' $2&&' $>$&29' A*' $*))*/*)%,2' ,-#0->"*,-20#0:'%'.48/2)'*3'#.12"2.12.,'2?"2)#82.,0'+2)2'$%))#21'*4,9A-2'$>,*B#.20#0R0"2$#3#$'0>.,%?#.'[^bXX_')2U4#)21'3*)',-2'SR"-%02'")*()200#*.'+%0'4021'%0'%'8%)B2)'3*)'$2&&'1#5#0#*.'"&%.206g*&B2)'2,'%&9:';<<E=9'\0#.('%.,#R[^bXX_'%.,#/*1#20:',-2'.48/2)*3'$2&&'"&%,20'+%0'0#(.#3#$%.,&>')214$21'#.'YR1%>R*&1'"&%.,0',)2%,21

RESEARCH ARTICLE Development 138 (5)

Fig. 2. Dose-dependent effects of BRs in rootgrowth and meristem size. (A) Phenotype of 6-day-old wild-type Col-0 plants from left to rightcontrol (CTL) and treated with 0.004 nM, 0.04 nM,0.4 nM and 4 nM BL. Scale bars: 10 mm.(B-F) Effect of exogenously applied BL on rootgrowth. Images illustrate longitudinal medianconfocal images of 6-day-old primary Col-0 rootstreated with the indicated amounts of BL. Arrowsindicate the boundary between the proximalmeristem and the elongation zone of the root.Scale bar: 100 m. (G) Root length of Col-0seedlings grown in increasing BL concentrations.Values represent the mean of 30 measurements±s.d. Concentrations of at least 0.4 nM BL werefound to be sufficient to repress root growth (forstatistical analysis, see Table S1 in supplementarymaterial). (H) Meristem cell number of Col-0 rootsgrown in increasing BL concentrations for 8 daysafter germination. Values represent the mean of 80measurements ±s.d. (I) Spatial profile of cellelongation in 6-day-old Col-0 roots treated withdifferent BL concentrations. Epidermal cells werecounted from the QC up to the appearance of thefirst root hair. Values represent the mean of 5measurements ±s.d.

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+#,-' W' .S' NX' 6d#(9' G7=' 6/q<9<E=:' %.1' #.' $H)>E< 6d#(9' GZ=6/q<9<J='%.1'$"%>E>>I )**,0'6d#(9'GT='6/q<9<<E='$*8"%)21'+#,-',-2+#&1' ,>"2' 6d#(9' Gd:j=9' K#8#&%)' )204&,0' +2)2' */,%#.21' +#,-' %,)%.0&%,#*.%&'?;4001MBL/*&#.2'6022'd#(9'KG'#.',-2'04""&282.,%)>8%,2)#%&=:' 34),-2)' 128*.0,)%,#.(' ,-%,' /*,-' &*00R' %.1' (%#.R*3R34.$,#*.' NMR)2&%,21' 84,%.,0' 2?-#/#,' $2&&' 1#5#0#*.' 1232$,0' ,-%,.2(%,#52&>'#8"%$,'*.',-2'82)#0,28'0#C29^2?,:' %' )2"*),2)' 3*)' ,-2' "&%.,R0"2$#3#$' $2&&' $>$&2' #.-#/#,*)

(:7?KD?5/KMBF6 6D2'g2>&12)'2,'%&9:';<<E='+%0'%.%&>C21'#.'"&%.,0+#,-'%&,2)21'NM'0#(.%&&#.(9'A-2'2?")200#*.'*3'(:7?KD?5/KMBF6+%0'1)%8%,#$%&&>')214$21'#.'"&%.,0'+#,-'#.$)2%021'NM'&252&0'6#929'W.S'NXR,)2%,21'"&%.,0='6d#(9'G[:X=:'$H)>E< 6d#(9'GS='%.1'#.'95:>EBL/R*52)2?")200#.('"&%.,0'6d#(9'G^=:'+-2)2%0'#,'+%0'#.$)2%021'#.$"%>E>>I )**,0'6d#(9'Gb=9N2$%402',-2'%$$484&%,#*.'*3'(:7?KD?5/KMBF6 #.',-2'$"%>E>>I

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BRI1 signalling controls QC identity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d#(9' JH' 022' d#(0' KW' %.1' KJ' #.' ,-204""&282.,%)>' 8%,2)#%&=9' K"2$#3#$%&&>:' ,-2' 2?")200#*.' *3(84NOMBL/ 6d#(9'J!R7=:'(!B0QKMBL/*6d#(9'JZ:T=:'(675MBL/6d#(9'Jj:[=:'P7KOM7L/*6d#(9'JX:S='%.1'P7>QKMBF6 6d#(9'J^:b=+%0'2.-%.$21'/>'$*.,#.4*40'%""&#$%,#*.'*3'W'.S'NX9'P*.52)02&>:,-2'2?")200#*.'*3'P7QIMBF6*6d#(9'J@:V= %.1 P7>SQMBF6*6d#(9JM:K=' +%0' )214$21:' +-2)2%0' ,-2' )2"*),2)' 340#*.0' 3*)' ,-2' ,-)22/012345! 6/02='3%8#&>'828/2)0'67%&#.-%'2,'%&9:';<<L='1#1'.*,0-*+'0#(.#3#$%.,'$-%.(20'#.')20"*.02',*',-2'NX',)2%,82.,'6022'd#(9KO'#.',-2'04""&282.,%)>'8%,2)#%&=9

853RESEARCH ARTICLEBrassinosteroids and root development

Fig. 3. Regulation of cell-cycle progression byBRI1 signalling in the root meristem.(A-D) pCYCB1;1:GUS expression in 6-day-old roots.(A) pCYCB1;1:GUS (control; CTL), (B) pCYCB1;1:GUSplants treated with 4 nM BL, (C) F3 roots of bes1-D;pCYCB1;1:GUS homozygous plants and (D) F3 rootsof bri1-116; pCYCB1;1:GUS homozygous plants.(E) Number of GUS-stained cells in the meristem ofpCYCB1;1:GUS (P<0.001) not treated or treated with4 nM BL, in bes1-D;pCYCB1;1:GUS (P<0.001) andbri1-116;pCYCB1;1:GUS (P<0.05) seedlings.Asterisks indicate a statistically significant differencefrom the wild-type control. Values represent themean of 25 measurements ±s.d. (F-I) Whole-mountimmunofluorescence with anti-KNOLLE antibodies(Volker et al., 2001) in untreated (F) or 4-nM BL-treated Col-0 (G), and in bes1-D (H) and bri1-116 (I)roots. Nuclei were counterstained with DAPI (blue).Scale bar: 100 m. (J) Reduced number of cell platesin 4-nM BL-treated Col-0 (P<0.01), and in bes1-D(P<0.05) and bri1-116 (P<0.001) roots. Asterisksindicate a statistically significant difference from theuntreated Col-0 control. Values represent the meanof 10 measurements ±s.d. (K-O) pICK2/KRP2:GUS(CTL) expression in 6-day-old roots untreated (K),treated with 4 nM BL (L), and bes1-D;pICK2/KRP2:GUS (M), BRI1-GFP;pICK2/KRP2:GUS(N) and bri1-116;pICK2/KRP2:GUS homozygousplants (O). Arrowheads indicate the boundarybetween the proximal meristem and the elongationzone of the root. Scale bar: 100 m.

DEV

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854

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

RESEARCH ARTICLE Development 138 (5)

Fig. 4. Rescue of the phenotype of bri1-116mutants in the root meristem and columella cellsby overexpression of CYCD3;1. (A-D) Phenotype of6-day-old Col-0 (wild type) (A), CYCD3;1OE (B), bri1-116 (C) and double bri1-116;CYCD3;1OE (D) roots.Arrowheads indicate the boundary between themeristematic and elongation zones of the root.(E) Meristem cell number in the roots of 6-day-oldCYCD3;1OE, bri1-116 and bri1-116;CYCD3;1OE plantscompared with the wild type Col-0. Asterisk indicatessignificant differences compared with the wild type(P<0.01) (a) and with bri1-116 (P<0.01) (b). Valuesrepresent the mean of 30 measurements ±s.d.(F-I) Confocal images of 6-day-old Col-0 (F),CYCD3;1OE (G), bri1-116 (H) and bri1-116;CYCD3;1OE (I) primary roots stained with PI.Asterisks indicate the layers of CSCs. Scale bar:100 m.

DEV

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BRs restrain root growth by controlling thenormal cell cycle progression in the root meristemA*'1%,2:',-2'$*.,)#/4,#*.'*3'NM0',*'"&%.,'()*+,-'-%0'/22.'8*0,&>/%021'*.',-2#)'%/#&#,>',*'")*8*,2'$2&&'2?"%.0#*.'*3'0-**,'*)(%.06S%.1%5%:'EFOO=9'A-2'1+%)321'"-2.*,>"2'*3'NMR123#$#2.,'84,%.,0-%0' 0*' 3%)'/22.' #.,2)")2,21'%0' %' 3%#&4)2' ,*' $%))>'*4,'")*"2)' $2&&2&*.(%,#*.'6P&*402'2,'%&9:'EFFYH'X#'2,'%&9:'EFFYH' #̀.'2,'%&9:';<<;=9T.1221:',-2')214$21'$2&&'0#C2'*/02)521'#.',-2'0-*),'$"%>E>>I )**,0:1232$,#52'#.',-2'")#.$#"%&'NM')2$2",*):'04""*),0',-2'.*,#*.',-%,'NM0"&%>' %.' #8"*),%.,' )*&2' #.' $2&&' 2&*.(%,#*.' 14)#.(' ")#8%)>' )**,()*+,-9'b4)'1%,%'0-*+#.(',-%,'"&%.,0'+#,-'#.$)2%021'NM'0#(.%&&#.(%&0*' 2?-#/#,' )214$21' )**,' ()*+,-' #.1#$%,2' ,-%,' NMTER821#%,21")*8*,#*.'*3'$2&&'2&*.(%,#*.'6Sc00#('2,'%&9:';<<;H'D2'7)%4+2'2,%&9:';<<JH'A*.('2,'%&9:';<<F='#0'.*,'0433#$#2.,',*'")*14$2'&*.(')**,09A-2' )214$,#*.' #.' $2&&' .48/2)0' #.' ,-2' 82)#0,28' *3' NMR)2&%,2184,%.,0'#8"&#20',-2'2?#0,2.$2'*3'%11#,#*.%&'82$-%.#080'+-2)2/>NM0')2(4&%,2')**,'()*+,-:'04$-'%0'$2&&'1#5#0#*.'%.1'1#332)2.,#%,#*.9

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855RESEARCH ARTICLEBrassinosteroids and root development

Fig. 5. Regulation of the expression of QC-specificmarkers by BRs. (A-E) Confocal images of primary rootsexpressing pWOX5:GFP in the QC cells. Six-day-old rootsuntreated (A) or treated with 0.004 nM BL (B), 0.04 nM BL(C), 0.4 nM BL (D) and 4 nM BL (E). Roots werecounterstained with PI. (F) Number of cells expressing thepWOX5:GFP marker in roots untreated or treated with0.004 nM BL (P<0.05), 0.04 nM BL (P<0.001), 0.4 nM BL(P<0.01) and 4 nM BL (P<0.01). Values represent the meanof 15 measurements ±s.d. Asterisks indicate a statisticallysignificant difference from the wild-type control.(G) Quantitative reverse-transcription PCR levels of theWOX5 gene in wild-type Col-0 and plants grown for 6 dayswith 4 nM BL (P<0.001). Values represent the mean of 3measurements ±s.d. Asterisks indicate a statisticallysignificant difference from the wild-type control.(H-S) Expression patterns of QC markers pAGL42:GFP (H,I)and pSCR:GFP (J,K), and of promoter-trap lines QC25:CFP(L,M), QC142:GUS (N,O), QC46:GUS (P,Q) and QC184:GUS(R,S) in 6-day-old roots untreated or treated with 4 nM BL.Arrowheads indicate QC cells. Scale bars: 100 m.

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RESEARCH ARTICLE Development 138 (5)

Fig. 6. Expression analysis ofpWOX5:GFP in response to shortBL treatments and in BR-relatedmutant backgrounds.(A-H) Longitudinal view of 6-day-oldroot meristems expressingpWOX5;GFP without treatment (A,E)and treated with 1 M BL for 4 hours(B,F), 8 hours (C,G) and 24 hours(D,H). Roots were counterstained withPI. (I-P) pWOX5:GFP expression inwild-type (I,M), bri1-116 (J,N), BRI1-GFP (K,O) and bes1-D (L,P) 6-day-oldroots. Scale bar: 50 m. (Q) Numberof cells expressing the pWOX5:GFPmarker in the different mutantbackgrounds bri1-116 (P<0.05), BRI1-GFP (P<0.001) and bes1-D (P<0.001)compared with the control line (WT)(n 9). Asterisk indicates a statisticallysignificant difference betweenmutants and the wild type. Valuesrepresent the mean of 12measurements ±s.d.

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857RESEARCH ARTICLEBrassinosteroids and root development

Fig. 7. Differentiation of distal columella stem cellspromoted by BRs. (A-F) Whole-mountimmunofluorescence with anti-GFP antibodies used to labelthe QC cells of 6-day-old pWOX5:GFP plants (A-C) andpWOX5:GFP plants treated with 4 nM BL (D-F). Confocalimages corresponding to Alexa488 fluorescence (A,D), DAPIstaining (B,E) and combined DAPI+GFP (C,F). Arrowheadsindicate the position of the QC cells in the root.(G-J) pSCR:GFP expression in the roots of Col-0 (G), bri1-116(H), bes1-D (I) and Col-0 plants treated with 4 nM BL (J).Scale bar: 50 m. Arrowheads indicate the position of theQC cells in the root. (K-N) mPS-PI-stained root tips of Col-0(K), bri1-116 (L), bes1-D (M) and Col-0 treated with 4 nM BL(N) (green arrowheads indicate cells with QC identity).(O-R) mPS-PI-stained root tips of scr-4 (O), bri1-116;scr-4,(P), wox5-1 (Q) and bri1-116;wox5-1 (R). (S) Quantitativeanalysis of the effects of BRs in CSC differentiation.Frequency distribution of the number of cell layers is givenbetween the QC and the first differentiated columella cellsthat contain starch granules. (T) CSC differentiationphenotypes in roots with altered BR signalling. QC,quiescent centre; CSC, columella stem cells; CDC, columelladifferentiated cells.

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AcknowledgementsWe thank P. Benfey and B. Scheres for sharing published seeds, G. Jürgens foranti-KNOLLE antiserum, M. Ibañes for data analysis advice, J. Sánchez-Matellánfor drawing the root cartoons, A. Gañez and N. Samper for technical support,C. Gutierrez, M. A. Moreno-Risueño and J. Long for comments on themanuscript, and M. De Cock for help in preparing it. This work was supportedby grants of the Spanish Ministry of Education and Science (‘Ramón y Cajal’contract, BIO2005/01447, BIO2008/00505) and by a HSFPO award(CDA2004/007) to A.I.C.-D. M.G.-G. is the recipient of a ‘Juan de la Cierva’postdoctoral contract, J.V.-B. is funded by PhD fellowship from the Generalitatde Catalunya and M.Z. is indebted to the Belgian Science Policy (BELSPO) for afellowship for non-European Union Researchers.

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.057331/-/DC1

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RESEARCH ARTICLE Development 138 (5)

Fig. 8. Model for BR action during root meristem growth.(A) Normal cell-cycle progression at the root apex controlled by the BRsignalling pathway that maintains a balance between self-renewal ofstem cells and differentiation and elongation rates of their daughtercells. (B) Graphical representation summarizing the dose-dependenteffects of BRs in root development at three levels: cell cycle progression(top), CSC differentiation (middle) and root/meristem size (bottom).Reduced BRI1 signalling (as in bri1-116 mutants) decreases both cellelongation and entry into the M phase, leading to the production ofshorter meristems and, hence, a partial loss in the renewal activity ofdistal stem cells and a reduced supply of undifferentiated CSCs. Thecombination of a premature cell cycle exit with accelerated celldifferentiation in bes1-D and BL-treated plants are also detrimental tomeristem size and root growth. (C) At the stem cell niche, BRs playexclusive roles in QC renewal and stem cell differentiation upstream ofknown regulators of stem cell dynamics such as WOX5.

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859RESEARCH ARTICLEBrassinosteroids and root development

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