WOX5–IAA17 feedback circuit-mediated cellular auxin response is crucial for the patterning of root stem cell niches in Arabidopsis

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(1)Published in 0ROHFXODU3ODQW

(2) ± which should be cited to refer to this work.. 809o*""'FFECBDL$JSDVJU.FEJBUFE$FMMVMBS "VYJO3FTQPOTF*T$SVDJBMGPSUIF1BUUFSOJOHPG 3PPU4UFN$FMM/JDIFTJOArabidopsis. http://doc.rero.ch. Huiyu Tiana,2, Krzysztof Wabnikb,2, Tiantian Niua, Hanbing Lic, Qianqian Yua, Stephan Pollmannd,e, Steffen Vannesteb, Willy Govaertsf, Jakub Rolčíkg, Markus Geislerh, Jiří Frimlb,i BOEZhaojun Dinga,b,1 a5IF,FZ-BCPSBUPSZPG1MBOU$FMM&OHJOFFSJOHBOE(FSNQMBTN*OOPWBUJPO .JOJTUSZPG&EVDBUJPO 4DIPPMPG-JGF4DJFODFT 4IBOEPOH6OJWFSTJUZ 4IBOEB /BOMV +JOBO $IJOB b%FQBSUNFOUPG1MBOU4ZTUFNT#JPMPHZ 7*#BOE%FQBSUNFOUPG1MBOU#JPUFDIOPMPHZBOE#JPJOGPSNBUJDT (IFOU6OJWFSTJUZ #(FOU #FMHJVN c%FQBSUNFOUPG#JPDIFNJTUSZ 6OJWFSTJUZPG.JTTPVSJ $PMVNCJB .0 64" d3VIS6OJWFSTJUÊU#PDIVN -FISTUVIMGàS1GMBO[FOQIZTJPMPHJF #PDIVN (FSNBOZ e1SFTFOUBEESFTT$FOUSPEF#JPUFDOPMPH¯BZ(FOØNJDBEF1MBOUBT 61.o*/*" $BNQVTEF.POUFHBODFEP 1P[VFMPEF"MBSDØO .BESJE 4QBJO f%FQBSUNFOUPG"QQMJFE.BUIFNBUJDTBOE$PNQVUFS4DJFODF (IFOU6OJWFSTJUZ (FOU #FMHJVN g-BCPSBUPSZPG(SPXUI3FHVMBUPST $FOUSFPGUIF3FHJPO)BOÈGPS#JPUFDIOPMPHJDBMBOE"HSJDVMUVSBM3FTFBSDIBOE'BDVMUZPG4DJFODF 1BMBDLâ6OJWFSTJUZ 0MPNPVD $[FDI3FQVCMJD h%FQBSUNFOUPG#JPMPHZ 1MBOU#JPMPHZ 6OJWFSTJUZPG'SJCPVSH 'SJCPVSH 4XJU[FSMBOE i*OTUJUVUFPG4DJFODFBOE5FDIOPMPHZ"VTUSJB *45"VTUSJB ,MPTUFSOFVCVSH "VTUSJB. ABSTRACT In plants, the patterning of stem cell-enriched meristems requires a graded auxin response maximum that emerges from the concerted action of polar auxin transport, auxin biosynthesis, auxin metabolism, and cellular auxin response machinery. However, mechanisms underlying this auxin response maximum-mediated root stem cell maintenance are not fully understood. Here, we present unexpected evidence that WUSCHEL-RELATED HOMEOBOX 5 (WOX5) transcription factor modulates expression of auxin biosynthetic genes in the quiescent center (QC) of the root and thus provides a robust mechanism for the maintenance of auxin response maximum in the root tip. This WOX5 action is balanced through the activity of indole-3-acetic acid 17 (IAA17) auxin response repressor. Our combined genetic, cell biology, and computational modeling studies revealed a previously uncharacterized feedback loop linking WOX5-mediated auxin production to IAA17-dependent repression of auxin responses. This WOX5–IAA17 feedback circuit further assures the maintenance of auxin response maximum in the root tip and thereby contributes to the maintenance of distal stem cell (DSC) populations. Our experimental studies and in silico computer simulations both demonstrate that the WOX5– IAA17 feedback circuit is essential for the maintenance of auxin gradient in the root tip and the auxin-mediated root DSC differentiation. Key words: WOX5; auxin maximum; IAA17/AXR3; root patterning; computer simulation.. INTRODUCTION. BOE TUFN DFMM EJGGFSFOUJBUJPO #FOKBNJOT BOE 4DIFSFT ; $SV[3BNJSF[FUøBM 3FDFOU UIFPSFUJDBM TUVEJFT TVHHFTU UIF QSPNJOFOU ASFGMVY MPPQIZQPUIFTJT (SJFOFJTFOFUøBM ; $SV[3BNJSF[FUøBM UIBU FOWJTJPOT QPMBS BVYJO USBOTQPSU BT B TVGGJDJFOU. %PTFEFQFOEFOU DFMMVMBS BVYJO SFTQPOTFT BSF DFOUSBM UP DFMM HSPXUIBOEDFMMGBUFTQFDJGJDBUJPOEVSJOHFNCSZPOJDBOEQPTU FNCSZPOJD QMBOU EFWFMPQNFOU ,FQJOTLJ BOE -FZTFS 5IF QSJNBSZ SPPU EFWFMPQNFOU SFRVJSFT CPUI UIF FTUBCMJTI NFOU BOE NBJOUFOBODF PG BVYJO SFTQPOTF NBYJNVN JO UIF EJTUBMSPPUUJQ 7BOOFTUFBOE'SJNM 5IJTBVYJOSFTQPOTF NBYJNVN JT EFUFSNJOFE CZ UIF DPODFSUFE BDUJPO PG QPMBS BVYJO USBOTQPSU BVYJO QSPEVDUJPO BVYJO NFUBCPMJTN -KVOH FUø BM ; 8PPEXBSE BOE #BSUFM ; 4UFQBOPWB FUø BM ; 5BPFUøBM ; 1FUFSTTPOFUøBM ; (SVOFXBMEBOE 'SJNM BOE BVYJO SFTQPOTF NBDIJOFSZ 7FSOPVY FUø BM *U JT FTTFOUJBM UP DPOUSPM DFMM EJWJTJPO DFMM FYQBOTJPO . 5PXIPNDPSSFTQPOEFODFTIPVMECFBEESFTTFE&NBJMEJOH[IBPKVO!TEV FEVDO UFM

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(D, E)%FDSFBTFEGSFF*""MFWFMTJOUIFwox5NVUBOU % BOE35SVP16IAA17mImIINVUBOUBOETJHOJGJDBOUMZJODSFBTFE*""MFWFMTPGaxr3-1NVUBOUø & (F)*ODSFBTFJOUIFGSFF*""MFWFMJO35S::WOX5-GRTFFEMJOHTHSPXOPONFEJVNXJUIøw.%&9JOEVDUJPOT (G)-POHUFSNJOEVDUJPOPG809 TFFEMJOHTHSPXOPONFEJVNXJUIøw.%&9 JOEVDFE:6$FYQSFTTJPOMFWFMUIBUXBTOPUBCTFOUVOEFSTIPSU UFSNJOEVDUJPOPG809 EBZPMETFFEMJOHTUSFBUFEXJUIøw.%&9GPSPSøI (H)*ODSFBTFEFYQSFTTJPOPG5""8&*HFOFJOaxr3-1NVUBOUBOETMJHIUMZEFDSFBTFEFYQSFTTJPOPG5""8&*JO35SVP16IAA17mImIINVUBOU DPNQBSFEUPUIFDPOUSPM 5VCVMJO JT UIF SFMBUJWF DPOUSPM GPS 21$3 BOBMZTJT &SSPS CBST NBSL TUBOEBSE FSSPST GSPN GPVS JOEFQFOEFOU CJPMPHJDBM SFQFBUT 4UVEFOUT tUFTU * Pøø øPøø . 8.

(11) METHODS Plant Materials and Growth Conditions 1VCMJTIFE USBOTHFOJD BOE NVUBOU MJOFT XFSF wox5-1, 35S::WOX5-GR 4BSLBSFUøBM WOX5::ERGFP 9VFUøBM DR5rev::GFP 'SJNM FUø BM axr3-1 NJNJDLJOH *""SFQSFTTPS 4BCBUJOJFUøBM 35SVP16IAA17mImII SFQSFTFOUJOH*""BDUJWBUPS -JFUøBM PIN3::PIN3::GFP %FMMP *PJP FUø BM 5IF wox5 axr3 EPVCMF NVUBOU XBT HFOFSBUFE CZ DSPTTJOH wox5-1 XJUI axr3-1; DR5::GUS 35S::WOX5-GRCZDSPTTJOHDR5::GUSXJUI35S::WOX5-GRBOE DR5rev::GFP/wox5 BOE DR5rev::GFP35SVP16IAA17mImII CZ DSPTTJOH DR5rev::GFP XJUI wox5-1 or 35SVP16IAA17mImII, SFTQFDUJWFMZ 5IF CQ VQTUSFBN SFHJPO GSPN UIF *"" TUBSU DPEPO XBT BNQMJGJFE BOE MJOLFE UP UIF (64 SFQPSUFE JOHBUFXBZWFDUPS1,(8'4 ,BSJNJFUøBM UPPCUBJO *""1SPNPUFS(64 SFQPSUFS DPOTUSVDU 5IF WOX5HA IAA17mImII SFQSFTFOUJOH *"" SFQSFTTPS MJOF XBT HFOFS BUFE CZ SFQMBDJOH UIF 4 QSPNPUFS PG 35S:HAIAA17mImII -JFUøBM XJUIUIFCQVQTUSFBNSFHJPOGSPNUIF 809TUBSUDPEPO4FFETXFSFTVSGBDFTUFSJMJ[FEXJUIDIMPSJOF HBTBOEJODVCBUFEGPSEBGUFSCFJOHQMBUFEPOUP.VSBTIJHF BOE 4LPPH .4 NFEJVN CFGPSF USBOTGFS UP B HSPXUI SPPN XJUIBIEBZIOJHIUSFHJNFBU¡$GPSøE. Figure 5. 4DIFNBUJDTPG809o*""'FFECBDL.FDIBOJTN. http://doc.rero.ch. #MBDL BSSPXT EFQJDU QPTJUJWF SFHVMBUJPO BOE EBTIFE BSSPXT JOEJDBUF SFQSFTTJPO NFDIBOJTN (SFFL MFUUFST DPSSFTQPOE UP TVCTFRVFOU DPN QVUFSNPEFMBTTVNQUJPOTQSFTFOUFEJOUIFNBJOUFYU. BDUJWBUFEWFSTJPOPG*""JO2$DFMMTMFEUPUIFIJHIMZQSP NPUFE BVYJO SFTQPOTFT JO 2$ BOE TVCTFRVFOUMZ FOIBODFE EJGGFSFOUJBUJPOPGSPPUTUFNDFMMT"EEJUJPOBMMZ PVSJOWFTUJHB UJPOT JOEJDBUF UIBU B NBYJNVN PG BVYJO TJHOBMJOH SFTQPOTFT JO2$JTOPUTVGGJDJFOUUPNBJOUBJOTUFNDFMMJEFOUJUJFTJUBMTP SFRVJSFTUIFSFEVDUJPOPGBVYJOSFTQPOTFTPGBEKBDFOUDPMVN VMMBSDFMMT0VSEBUBJOEJDBUFUIFQPTTJCJMJUZGPSBVYJOSFTQPOTF HSBEJFOUJOUIFSPPUUJQUIBUXPVMENPEVMBUFUIFQBUUFSOJOH PGSPPU%4$JEFOUJUZ 'JOBMMZ XFEFTJHOFEUIFDPNQVUFSNPEFMPGUIJTQSPQPTFE GFFECBDL MPPQ UIBU XBT DBQBCMF PG GBJUIGVMMZ SFQSPEVDJOH FYQFSJNFOUBMPCTFSWBUJPOT JODMVEJOHQSFEJDUJPOTPGBVYJOMFW FMT BVYJOEJTUSJCVUJPOT BOE809FYQSFTTJPOBOESPPU%4$ EJGGFSFOUJBUJPOQBUUFSOT5IJTTJNQMJGJFENPEFMUIBUDBQUVSFT UIF FTTFOUJBM GFBUVSFT PG UIF GFFECBDL SFHVMBUJPO CFUXFFO 809BOE*""XBTDBQBCMFPGQSFEJDUJOHBVYJOTJHOBMJOH NBYJNVN BOE SPPU %4$ EJGGFSFOUJBUJPO QIFOPUZQFT XIJDI XFSFPCTFSWFEFYQFSJNFOUBMMZ WOX5IBTCFFOTIPXOUPVOEFSHPUSBOTDSJQUJPOBMSFQSFT TJPO CZ UIF $-&"$3 TJHOBMJOH DBTDBEF EFSJWFE GSPN UIF DPMVNFMMBDFMMT %F4NFUFUøBM ; 4UBIMBOE4JNPO , 5IFSFGPSF JU XJMM CF JOUFSFTUJOH UP TFF IPX UIJT BOE BTZFUVOJEFOUJGJFEMBZFSTPGSFHVMBUJPODPVMECFGVSUIFSJOUF HSBUFEJOUPPVSNPEFMBOEIPXUIJTXPVMEJNQBDUPONPEFM QSFEJDUJPOTBOEVMUJNBUFMZPOPVSVOEFSTUBOEJOHPGSPPUQBU UFSOJOH QSPDFTTFT "MTP GVUVSF UFTUJOH PG UIF QPTTJCJMJUZ GPS UIF JOWPMWFNFOU PG PUIFS "69*"" QSPUFJOT BOE BTTPDJBUFE "3' USBOTDSJQUJPO GBDUPST JO UIF QSPQPTFE GFFECBDL SFHVMB UJPOXPVMETVCTUBOUJBMMZFYUFOEPVSVOEFSTUBOEJOHPGNFDIB OJTNTVOEFSMZJOHSPPUTUFNDFMMQBUUFSOJOH 5P DPODMVEF PVS DPNCJOFE FYQFSJNFOUBM BOE NPEFMJOH TUVEZTVHHFTUTUIFVOFYQFDUFEGFFECBDLMPPQUIBUNFDIBOJT UJDBMMZ MJOL 809EFQFOEFOU BVYJO QSPEVDUJPO QPMBS BVYJO USBOTQPSU BOE *""EFQFOEFOU DFMMVMBS BVYJO SFTQPOTF UP GBDJMJUBUF UIF BVYJONBYJNVN HVJEFE QBUUFSOJOH PG SPPU %4$OJDIFT*USFNBJOTUPCFUFTUFEXIFUIFSBTJNJMBSNFDIB OJTN DPVME CF JOWPMWFE JO UIF BVYJOEFQFOEFOU QBUUFSOJOH PGTIPPUT TJODF809CFMPOHTUPUIFGVODUJPOBMMZDPOTFSWFE GBNJMZPGNBKPSQMBOUTUFNDFMMSFHVMBUPST. Phenotype Analysis 4UBSDI HSBOVMFT JO SPPU UJQT XFSF TUBJOFE XJUI -VHPMT TPMV UJPO GPS oNJO NPVOUFE POUP UIF TMJEFT XJUI DIMPSBM IZESBUF BOE DIFDLFE JNNFEJBUFMZ )JTUPDIFNJDBM BOBMZTJT PG˟HMVDVSPOJEBTF (64 BDUJWJUZXBTDBSSJFEPVUCZJODVCB UJPOPGTFFEMJOHSPPUTJO(64TUBJOJOHTPMVUJPO ø./B10 CVGGFS Q)ø N. ,'F $/ N. ,'F $/ N. 9HMVDVSPOJEF BU¡$VOUJMUIFCMVFTUBJOJOHXBTWJTJCMF GPM MPXFECZUXPXBTIFTJOEJTUJMMFEXBUFSBOENPVOUJOHJO XW DIMPSBM IZESBUF BOE WW HMZDFSPM GPS NJDSPTDPQZ *NBHFBDRVJTJUJPOXBTDBSSJFEPVUXJUIBO"YJPDBN)3DBNFSB BUUBDIFEUPBO0MZNQVTNJDSPTDPQF'PSDPOGPDBMNJDSPTDPQZ JNBHFT B;FJTT-4.PS0MZNQVT'7"48DPOGPDBMTDBO OJOHNJDSPTDPQFXBTVTFE$FMMXBMMTXFSFDPVOUFSTUBJOFECZ NPVOUJOHTFFEMJOHSPPUTJOø˩.QSPQJEJVNJPEJEF. Immunodetections 5IF BOUJCPEJFT XFSF EJMVUFE BT GPMMPXT SBCCJU BOUJ1*/ SBCCJU BOUJ1*/ HFOFSPVTMZ QSPWJEFE CZ $ø-VTDIOJH BOE$ZDPOKVHBUFETFDPOEBSZBOUJNPVTF %JBOPWBBOE4JHNB . Free IAA Measurements 'PSGSFF*""DPOUFOUTBOBMZTJT BQQSPYJNBUFMZSPPUTPGSFTQFD UJWF HFOPUZQFT XFSF QPPMFE BOE BOBMZ[FE JO RVBESVQMJDBUFT BTEFTDSJCFEFBSMJFS 1FODJLFUøBM PSJUXBTRVBOUJGJFEBT EFTDSJCFE 1PMMNBOO FUø BM 'PS UIF FYUSBDUJPO PG QMBOU NFUBCPMJUFT UISFF TBNQMFT SFQMJDBUFT PG FBDI XFSF JODV CBUFEBU¡$VOEFSDPOTUBOUTIBLJOHPWFSIJONMNFUIBOPM. 9.

(12) http://doc.rero.ch Figure 6. $PNQVUFS.PEFM*OUFHSBUFT809o*""'FFECBDL.FDIBOJTNTUP1SFEJDUUIF1BUUFSOJOHPGUIF3PPUø5JQ (A–D)$PNQBUJCMFin silicoXJMEUZQFTJNVMBUJPOTPGUIFAGMVYTFQBSBUJPONPEFM 'JHVSFø# UIBUJOUFHSBUFTUIF809o*""GFFECBDLMPPQ " BOE UIFDPNQVUFSTJNVMBUJPOTPGUIFin silico wox5NVUBOU # MPTTPG*""GVODUJPO 35SVP16IAA17mImII $ BOEHBJOPG*""GVODUJPO axr3-1 % NVUBOUTUPFYQFSJNFOUBMPCTFSWBUJPOT 'JHVSFø"o% "VYJODPODFOUSBUJPOTBSFJOHSFFOBOEUIFSFMBUJWF809FYQSFTTJPOJOSFE5IFQSF EJDUFE809BDUJWJUJFTBOESFTVMUJOHTUFNDFMMMBZFST EFQJDUFECZZFMMPXUSJBOHMFTCFMPXUIF2$ DPSSFTQPOEUPUIPTFPCTFSWFEFYQFSJNFOUBMMZ 'JHVSFø$o& (E) 4UFBEZTUBUF BVYJO DPODFOUSBUJPO QSPGJMFT BMPOH MPOHJUVEJOBM TFDUJPOT UISPVHI UIF WBTDVMBS BOE DPMVNFMMB UJTTVFT GPS XJMEUZQF BOE NVUBOU TJNVMBUJPOT EJTUBODF GSPN SPPU UJQ JO wN 4UFFQ FYQPOFOUJBM BVYJO HSBEJFOUT XFSF PCTFSWFE CFUXFFO 2$ DFMMT BOE DPMVNFMMB JOJUJBMT DJO JO XJMEUZQF CMBDLBSSPXIFBE BOEaxr3-1 QVSQMFHSFFOBSSPX TJNVMBUJPOTBOEXFSFMBSHFMZBCTFOUJOUIFTJNVMBUJPOTPGwox5 CMVFBSSPX BOE 35SVP16IAA17mImII PSBOHFBSSPX NVUBOUT TJNJMBSMZUPUIPTFJOFYQFSJNFOUBMNFBTVSFNFOUT 'JHVSFø $PMPSDPEJOHJTGPSUIFSFMBUJWFBVYJO DPODFOUSBUJPOT HSFFO BOESFMBUJWF809FYQSFTTJPO SFE $PMPSTPGBSSPXTDPSSFTQPOEUPUIFBVYJOHSBEJFOUQSPGJMFT. 10.

(13) http://doc.rero.ch. TVQQMFNFOUFE XJUI ø QNPM PG <H]*"" JOUFSOBM TUBOEBSE 4VQFSOBUBOUT BOE SFTJEVBM QMBOU NBUFSJBM XFSF TFQBSBUFE CZ DFOUSJGVHBUJPO BOE UIF DFMMGSFF FYUSBDUT ESJFE VOEFS WBDVVN 3FTJEVFT XFSF EJTTPMWFE JO ø wM NFUIBOPM UP XIJDI ø wl EJFUIZM FUIFS XBT BEEFE *G OFDFTTBSZ EJTTPMWJOH XBT GPSDFE CZ VMUSBTPOJD USFBUNFOU 4POPSFY 3,4 #BOEFMJO #FSMJO (FSNBOZ 1BSUJDMFGSFF TBNQMFT XFSF MPBEFE POUP B DVTUPN NBEFNJDSPTDBMFBNJOPQSPQZMTPMJEQIBTFFYUSBDUJPODBSUSJEHF $PMVNOTXFSFXBTIFEXJUIøwMPG$)$MQSPQBOPM WW BOE*""DPOUBJOJOHGSBDUJPOTFMVUFEXJUIøwMBDJEJGJFEEJFUIZM FUIFS BDFUJDBDJE WW. &MVBUFTXFSFESJFE SFEJTTPMWFEJO øwMNFUIBOPM BOEGJOBMMZEFSJWBUJ[FECZUSFBUNFOUXJUIøwl FUIFSFBM EJB[PNFUIBOF 5IFSFBGUFS TBNQMFT XFSF USBOTGFSSFE JOUPBVUPTBNQMFSWJBMT ESJFEVOEFSBHFOUMFTUSFBNPGOJUSPHFO BOEUBLFOVQJOøwMPGDIMPSPGPSN'PS*""RVBOUJGJDBUJPO øwl PGUIFNFUIZMBUFETBNQMFTXBTJOKFDUFEJOUPUIF($o.4TZTUFN 5IF TQFDUSB XFSF SFDPSEFE PO B 7BSJBO 4BUVSO JPOUSBQ NBTTTQFDUSPNFUFSDPVQMFEXJUIB7BSJBO$1HBTDISPNB UPHSBQI 7BSJBO 8BMOVU $SFFL $" 64" $PNQPVOET XFSF TFQBSBUFECZDISPNBUPHSBQIZPOB;#GVTFETJMJDBDBQJMMBSZ DPMVNO 1IFOPNFOFY 5PSSFODF $" 64" 5IFNBTTTQFDUSPNF UFSXBTVTFEJO$*.3.QPTJUJWFJPOEFUFDUJPONPEFXJUINFUI BOPMBTUIFSFBDUBOUHBT5IFTFUUJOHGPSFOEPHFOPVT*""XBTBT GPMMPXTm/zøø<.

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(17) 5IFBNPVOUPGFOEPHFOPVT*""JOUIFTBNQMFTXBTDBMDVMBUFE GSPNUIFTJHOBMSBUJPPGUIFVOMBCFMFEPWFSUIFTUBCMFJTPUPQF MBCFMFENBTTGSBHNFOUPCTFSWFEJOUIFUXPBOBMZ[FEDIBOOFMT. CZ OVNFSJDBM DPNQVUBUJPOT PG DPVQMFE 0%& TZTUFNT XJUI BO BEBQUJWFTJ[F GJGUIPSEFS 3VOHF,VUUB NFUIPE "MM GJH VSFTXFSFQSPDFTTFEJO"EPCF*MMVTUSBUPS'JHVSFTBOE are TDSFFOTIPUT GSPN NPEFM TJNVMBUJPOT .PEFM QBSBNFUFST BSF EFTDSJCFEJO4VQQMFNFOUBM5BCMFø. SUPPLEMENTARY DATA 4VQQMFNFOUBSZ%BUBBSFBWBJMBCMFBUMolecular Plant Online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uantitative PCR Analysis. ACKNOWLEDGMENTS. 3/"XBTFYUSBDUFEXJUIUIF3/FBTZLJU 2JBHFO 1PMZ E5 D%/" XBTQSFQBSFEGSPNUPUBM3/"PGEBZPMETFFEMJOHT4VQFSTDSJQU *** SFWFSTF USBOTDSJQUJPO *OWJUSPHFO BOE RVBOUJGJDBUJPO XFSF QFSGPSNFEPOB-JHIU$ZDMFSBQQBSBUVT 3PDIF%JBHOPTUJDT XJUIUIF4:#3(SFFO*ø.BTUFSLJU 3PDIF%JBHOPTUJDT BDDPSE JOHUPUIFNBOVGBDUVSFSTJOTUSVDUJPOT"MMJOEJWJEVBMSFBDUJPOT XFSFQFSGPSNFEJOUSJQMJDBUF%BUBXFSFBOBMZ[FEXJUIR#BTF )FMMFNBOTFUøBM &YQSFTTJPOMFWFMTXFSFOPSNBMJ[FEUP UIPTFPG56#6-*/ XIJDITIPXFEOPDMFBSTZTUFNBUJDDIBOHFT JO$UWBMVF5IFQSJNFSTVTFEUPRVBOUJGZHFOFFYQSFTTJPOMFWFMT BSFEFTDSJCFEJO4VQQMFNFOUBM5BCMFTBOE. 8F BDLOPXMFEHF #FO 4DIFSFT 5PN +ø (VJMGPZMF BOE 5IPNBT -BVYTIBSJOHQVCMJTIFENBUFSJBMTBOE,FLF:J +àSHFO,MFJOF 7FIO 4FUI%BWJT BOE9V$IFOGPSDSJUJDBMSFBEJOHPGUIFNBOV TDSJQU;%øBOE,8øQMBOOFEFYQFSJNFOUT;% ,8 )5 5/ 41 BOE+3øQFSGPSNFEFYQFSJNFOUT+' ;% .( ;% ,8 47 8( )- .( BOE+'øBOBMZ[FEUIFEBUB;% ,8 BOE )5øXSPUFUIFQBQFS/PDPOGMJDUPGJOUFSFTUEFDMBSFE. REFERENCES Band, L.R., Wells, D.M., Larrieu, A., Sun, J., Middleton, A.M., French, A.P., Brunoud, G., Sato, E.M., Wilson, M.H., Péret, B., et al. 3PPUHSBWJUSPQJTNJTSFHVMBUFECZBUSBOTJFOUMBU FSBM BVYJO HSBEJFOU DPOUSPMMFE CZ B UJQQJOHQPJOU NFDIBOJTN 1SPD/BUM"DBE4DJ64"109 o. GUS Staining )JTUPDIFNJDBM˟HMVDVSPOJEBTF (64 TUBJOJOHXBTQFSGPSNFE BT EFTDSJCFE 'SJNM FUø BM PWFSOJHIU BU SPPN UFNQFS BUVSF 4FFEMJOHT NPVOUFE JO DIMPSBM IZESBUF 'MVLB XFSF BOBMZ[FEXJUIBOBVUPNBUJDWJSUVBMTMJEFTDBOOFSNJDSPTDPQF GSBNF EPU4MJEF#9NJDSPTDPQF 0MZNQVT . Benjamins, R., and Scheres, B. "VYJO UIF MPPQJOH TUBS JO QMBOUEFWFMPQNFOU"OOV3FW1MBOU#JPM59 o Benkova, E., Michniewicz, M., Sauer, M., Teichmann, T., Seifertova, D., Jurgens, G., and Friml, J. -PDBM FGGMVYEFQFOEFOU BVYJOHSBEJFOUTBTBDPNNPONPEVMFGPSQMBOUPSHBOGPSNB UJPO$FMM115 o. Computational Methods 5IF DFMMVMBS HSJE UJTTVF UFNQMBUF GPS UIF NPEFM XBT DSFBUFE XJUI UIF WFSTJPO PG 77 7FSUFY7FSUFY QSPHSBNNJOH MBO HVBHF 4NJUIFUøBM 5IFTJNVMBUJPOTXFSFQFSGPSNFE. Bennett, M.J., Marchant, A., Green, H.G., May, S.T., Ward, S.P., Millner, P.A., Walker, A.R., Schulz, B., and Feldmann, K.A.. 11.

(18) Laskowski, M., Grieneisen, V.A., Hofhuis, H., Hove, C.A., Hogeweg, P., Maree, A.F., and Scheres, B. 3PPUTZTUFN BSDIJUFDUVSFGSPNDPVQMJOHDFMMTIBQFUPBVYJOUSBOTQPSU1-P4 #JPM6 F. Arabidopsis "69 HFOF B QFSNFBTFMJLF SFHVMBUPS PG SPPUHSBWJUSPQJTN4DJFODF273 o Blilou, I., Xu, J., Wildwater, M., Willemsen, V., Paponov, I., Friml, J., Heidstra, R., Aida, M., Palme, K., and Scheres, B. 5IF 1*/ BVYJO FGGMVY GBDJMJUBUPS OFUXPSL DPOUSPMT HSPXUI BOE QBU UFSOJOHJOArabidopsisSPPUT/BUVSF433 o. Li, H., Cheng, Y., Murphy, A., Hagen, G., and Guilfoyle, T.J. $POTUJUVUJWF SFQSFTTJPO BOE BDUJWBUJPO PG BVYJO TJHOBMJOH JO Arabidopsis1MBOU1IZTJPM149 o. Brunoud, G., Wells, D.M., Oliva, M., Larrieu, A., Mirabet, V., Burrow, A.H., Beeckman, T., Kepinski, S., Traas, J., Bennett, M.J., et al. "OPWFMTFOTPSUPNBQBVYJOSFTQPOTFBOEEJTUSJCVUJPO BUIJHITQBUJPUFNQPSBMSFTPMVUJPO/BUVSF482 o. 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