previously. co and fca-9 alleles were isolated from the Col background. Plants were

Transcription

1 Supporting Online Material Materials and Methods Plant Growth Conditions The flc-3, FRI Sf2 -Col (S1), fve-4 (S2), ld-1 (S3) and fpa (S4) lines were described previously. co and fca-9 alleles were isolated from the Col background. Plants were grown under ~100 µe.m -2.s -1 cool-white fluorescent light in long days (16 hours light / 8 hours night) at 22ºC. For vernalization, 3 day-old seedlings were transferred to 4ºC with dim light (16 hours light / 8 hours night) for 44 days. Genetic and Physical Mapping fld-1 FLC (Col background) was crossed to FLD FLC Col (Ler background; FLD FLC Col was created by introgressing the Col allele of FLC into Ler because Ler has a weak FLC allele, see reference S5). About 1,500 late-flowering F 2 from this cross were used to map the FLD locus. Plasmid Construction To construct a genomic library for complementation tests, BAC F3D20 (containing the 42-kb FLD interval) was partially digested with Sau3AI, and fragments ranging in size from 6-15 kb were cloned into ppzp212 at BamHI sites. These clones were introduced into Agrobacterium tumefaciens strain ABI, and fld-1 mutants were transformed with the entire plasmid library. To construct the FLD::GUS plasmid, a 3.6-kb FLD genomic fragment including 1.0-kb of the region 5 of the translation start codon and entire coding region sequence was translationally fused with the GUS coding sequence. The FLC::GUS translational fusion plasmid was constructed by placing the GUS coding region into the sixth exon of a genomic FLC clone (plasmid , see reference S1) at a NheI site

2 resulting in an in-frame fusion. The internal deletions of FLC genomic region were made by a PCR-mediated fusion strategy. β-glucuronidase Staining in Plants Histochemical β-glucuronidase staining was performed as described (S6). FLC::GUScontaining seedlings were stained for 3 hours, and FLD::GUS-containing seedlings were stained for 10 hours at 37ºC. ChIP Assays The chromatin immuno-precipitation experiments were performed as described by Johnson et al. (S7) using 10-day-old seedlings. The anti-hyperacetylated histone H4 and ChIP assay kit were from Upstate Biotechnology (Lake Placid, New York, USA). Each of the immunoprecipitations was performed using three independent samples. The amounts of genomic DNA immunoprecipitated were determined by quantitative PCR. The primer pair CH1 (5 CTGCGACCATGATAGATACATGAGA 3 ) and HI2 (5 TTCACTCAACAACATCATCGAGCACG 3 ) was used to amplify FLC; the primer pair used to amplify ACTIN 2/7 was described previously (S7). 29 cycles of PCR (94ºc-25s, 59ºc-35s and 72ºc-30s) were performed as separate reactions for FLC and ACTIN amplification. Aliquots of the PCR reactions were resolved by electrophoresis in 1.8% agarose gels, and transferred to nylon membranes. The membranes were probed with 32 P- labelled FLC and subsequently with ACTIN probes, exposed to phosphoimaging screens, and the signal intensity was quantified with ImageQuant software (Molecular Dynamics, Inc). The primer pair CH2 (5 GTTCTCAATTCGCTTGATTTCTAGTTTTT 3 ) and CH3 (5 GGCCCGACGAAGAAAAAGGTAGATAGGC 3 ) was used to amplify the FLC

3 transgenes close to transcription initiation point; the primer pair Kan-F (5 GCTTGGGTGGAGAGGCTATTCG 3 ) and Kan-R (5 GCCATGTGTCACGACGAGATCC 3 ) was used to amplify NPTII. Aliquots of the PCR reactions for the FLC transgene and NPTII were resolved by electrophoresis in 1.4% agarose gels, and quantified with ImageQuant software (Molecular Dynamics, Inc). Three independent T 2 transgenic lines derived from each deletion construct were used for the ChIP assays, and representative results are shown in Fig. 4E. Supporting Fig. S1. The map-based cloning of FLD. (A) Genetic map of the FLD locus. FLD was initially positioned between SSLP markers nga126 and nga162 on Chromosome 3 (S8). (B) Physical map of the FLD locus. FLD was fine-mapped to a 42- kb interval; the number of recombination events (out of approximately 3,000 chromosomes) between an indicated marker and fld-1 is given in parentheses. Rescuing clones which complemented the late-flowering phenotype of fld-1 are also shown. (C) Schematic structure of FLD. Supporting Fig. S2. Sequence alignment of FLD with human KIAA0601, human polyamine oxidase 1 (), Zea mays polyamine oxidase () and Arabidopsis polyamine oxidase (). Numbers refer to amino acid numbers; identical residues are shaded with black, while similar residues are shaded with gray. Underlined regions are the conserved SWIRM domains. The * symbols indicate the conserved residues in this domain. The symbols indicate the conserved residues involved in binding the cofactor FAD. Supporting Fig. S3. Sequence alignment of FLD with additional homologs in other species. Numbers refer to amino acid numbers; identical residues are shaded with black,

4 while similar residues are shaded with gray. The conserved chromosomal SWIRM domains are underlined, and the conserved amine oxidase domains are underlined with a broken line. Species abbreviations: At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Mm, Mus musculus; Os, Oryza sativa. GenBank accession numbers:, NP_493366;, CAE01846;, AAF49051;, XM_ Amino acid residues in do not align with the other proteins and are not shown in the figure. Supporting Fig. S4. ChIP analyses of the acetylation state of histone H4 in the FLC region. (A) Schematic structure of the FLC region. I to VI represent the regions whose acetylation states were examined by ChIP. The translation initiation point is +1. The filled boxes represent exons and open boxes represent introns. (B) ChIP analyses of histone acetylation state of FLC Region II chromatin (5 to the translation initiation point). The input is Col chromatin before immuoprecipitation; No AB refers to the control sample lacking antibody. ACTIN served as an internal control. (C) ChIP analyses of histone acetylation states of various genomic FLC regions. The input and No AB are as described in (B). The ChIP analyses of histone acetylation state of FLC Region III chromatin is shown in Fig. 3B. Supporting references S1. S. D. Michaels, R. M. Amasino, Plant Cell 11, 949 (1999). S2. fve-4 was the allele used in S. D. Michaels, R. M. Amasino, Plant Cell 13, 935 (2001). S3. I. Lee et al., Plant Cell 6, 75 (1994).

5 S4. F. M. Schomburg, D. A. Patton, D. W. Meinke, R. M. Amasino, Plant Cell 13, 1427 (2001). S5. S. D. Michaels, Y. He, K. C. Scortecci, R. M. Amasino, Proc Natl Acad Sci USA 100, (2003). S6. R. A. Jefferson, Plant Mol Biol Reporter 5, 387 (1987). S7. L. Johnson, X. Cao, S. Jacobsen, Curr Biol 12, 1360 (2002). S8. S. L. Sanda, R. M. Amasino, Plant Physiol 111, 641 (1996).

6 0.5 kb A Chr III nga126 nga162 (16.35 cm) MD1 MD2 (20.56 cm) B MD1 (3) MD4 (1) MD5 (0) MD6 (1) MD7 (3) MD2 (5) 20 kb BAC F14P13 BAC F13M14 Rescuing Clone 8 At3g10390 Rescuing Clone 5 C ATG TAG Exon I Exon II

7 MQSCESSGDSADDPLSRGLRRRGQPR MDESLANLSEDEYYSEEERNAKAEKEKKLPPPPP MVSFSAPKKR---RRGRSQRSMSSLNSLPVPNVGLLPGNSNFVSSSASSSGRFNVEVVNGSNQTVKSYPGIG QAPPEEENESEPEEPSGVEGAAFQSRLPHDRMTSQEA--ACFPDIISGPQQTQKVFLFIRNRTLQLWLDNPKIQLTFEAT DEIITINKEATTEALLALTAGFPADSLTEEEIEFGVVP-IVGGIEQVN--YILIRNHIISKWRENISSWVTKEMF ATVGPRVIVVGAGMSGISAAKRLSEAGITDLLILEAT MSTASVIIIGAGISGISAAKVLVENGVEDVLILEAT VVVIGAGLAGLAAAKALLEQGFTDVTVLEAS 113 LQQLEAPYNSDTVLVHRVHSYLERHGLINFGIYKRIK---PLPTKKTGKVIIIGSGVSGLAAARQLQSFGM-DVTLLEAR 142 LNSIPKHCSS---LLDSAYNYLVTHGYINFGIAQAIKDKFPAQSSKSS-VIIVGAGLSGLAAARQLMRFGF-KVTVLEGR 38 DHIGGRMH----KTNFAGINVELGANWVEGVNGGKMNPIWPIVNSTLKLRNFRSDFDYLAQNVYKEDGGVYDEDYVQKRI 37 DRIGGRIH----KQNFGDVPVELGAGWIAGVGGKESNPVWELA-SRFNLRTCFSDYTNARFNIYDRSGKIFPTGIASDSY 58 SHIGGRVQSVKLG----HATFELGATWIHGSHGNPIYHLAEANGL-LE-ETTDGERSVGRISLYSKNGVACYLTNHGRRI 189 DRVGGRVATFR-KGNYV---ADLGAMVVTGLGGNPMAVVSKQVNMELA-KIKQ------KCPLYEANG QAV 217 KRPGGRVYTKKMEANRVGAAADLGGSVLTGTLGNPLGIIARQLGSSLY-KVRD------KCPLYRVDG KPV 114 ELA-DSVEEMGEKLSATLHASGRDDMSILAMQRLNEHQPNGPATPVDMVVDYYKFDYEFAEPPRVTSLQNTVPLATFS KKAVDSAILKLKSLEA--QCSGQ VAEEAPSSPKTPIELAIDFILHDFEMAE VEPISTYV PKDVV---EEFSDLYNEVYNLTQEFFRH-DKPVNAESQNSVGVFTREEVRNRIRNDPDDPEATKRLKLAMIQQYLK PKEKD---EMVEQEFNRLLEATSYLSHQLDFNVLNNKHVSLGQ-ALEVVIQLQEKHVKDEQIEHWKKIVKTQEELKELLN 281 DPDVD---IKVEVAFNQLLDKASKL-RQLMGDV--SMDVSLGA-ALETFRQVSGNDVATEEM KMVNLKEKIKELHQQYKEASEVKPPRDITAEFLVKSKHRDLTALCKEYDELAETQGKLEEKLQELEANPPSDVYLSSRDR DFGDDVYFVADQRGYEAVVYYLAGQYLKTDDKSGKIVDPRLQLNKV DFGEREFLVADERGYECLLYKMAEEFLVTSH--GNILDYRLKLNQV VESCESSSHSMDEVSLSAFGEWT-----EIPGAHHIIPS--GFMRVVELLAE GIPAHVIQLGKP 405 QILDWHFANLEFANATP--LSTLSLKHWDQDDDFEFTGSHLTVRN--GYSCVPVALAE GLD---IKLNTA 336 GLFNWHLANLEYANAGL--VSKLSLAFWDQDDPYDMGGDHCFLPG--GNGRLVQALAE NVP---ILYEKT 237 VREIKYSPGGVTVKTEDNS------VYSADYVMVSASLGVLQSDL--IQFKPKLPTWKVRAIYQFDMAVYTKIFLKFPRK 213 VREVQQSRNGVVVKTEDGS------VYEANYVIVSASIGVLQSDL--LSFQPLLPRWKTEAIQKCDVMVYTKIFLKFPQC 261 VRCIHWDQASARPRGPEIE PRGVLKRQYTSF-FRPGLPTEKVAAIHRLGIGTTDKIFLEFEEP 468 VRQVRYTASGCEVIAVNTRSTSQTFIYKCDAVLCTLPLGVLKQQPPAVQFVPPLPEWKTSAVQRMGFGNLNKVVLCFDRV 399 VQTIRYGSNGVKVTAGNQ VYEGDMVLCTVPLGVLKNG--SIKFVPELPQRKLDCIKRLGFGLLNKVAMLFPYV 309 FWP EGKGREFFLYASSRRGYYGVWQEFEKQYPDANVLLVTVTDEESRRIEQQSDEQTKAEIMQVLRKM 285 FWP CGPGQEFFIYAHEQRGYFTFWQHMENAYPGSNILVVTLTNEQSKRVEAQSDQETMKEAMSVLRDM 323 FWGPECNSLQFVWEDEAESHTLTYPPELWYRKICGFDVLYPPERYGHVLSGWICGEEALVMEKCDDEAVAEICTEMLRQF 548 FWDPSVNLFGHVGSTTASRGELF----LFW------NLYKAPI-----LLALVAGEAAGIMENISDDVIVGRCLAILKGI 470 FWSTDLDTFGHLTEDPNYRGEFF----LFY------SY--APVAGGALLIALVAGEAAHKFETMPPTDAVTRVLHILRGI 377 F--PGKDVPDATDILVPRWWSDRFYKGTFSNWPVGVNRYEYDQLRAPVG F---GATIPYATDILVPRWWNNRFQRGSYSNYPMISDNQLLQNIKAPVG TGNP--NIPKPRRILRSAWGSNPYFRGSYSYTQVGSSGADVEKLAKPLPYTESSKTAHGSSTKQQPGHLFSSKCPEQPLD 613 FGSSA--VPQPKETVVSRWRADPWARGSYSYVAAGSSGNDYDLMAQPIT PGP----SIPGAPQP 538 YEPQGINVPDPLQTVCTRWGGDPFSLGSYSNVAVGASGDDYDILAESVG DG RVYFTGEHTSEHYNGYVHGAYLSGIDSAEILINCAQKKMCKYHVQGKYD RIFFTGEHTSEKFSGYVHGGYLAGIDTSKSLLEEMKQSLLLQPLLAFTESLTLTHQKPNNSQIYTNVKFIS 481 ANRGAVKPMQVLFSGEATHRKYYSTTHGALLSGQREAARLIEMYRDLFQQGT IPR LFFAGEHTIRNYPATVHGALLSGLREAGRIADQFLGAMYTLPRQATPGVPAQQSPSM R LFFAGEATTRRYPATMHGAFVTGLREAANMAQS---AKARGIRKRIDRNPSRNAHSCAILLADLFRDPDL GTS EFGS

8 32 ASAARLPFDRPTDHELAFFPELWEHKTAVEV-FLLLRNSTLATWQYNPLKECTALDVRNNVFPPFNSDLDLIQNIVHYLS FPADSLT-EEEIEFGVVPIVGGIEQVNYILIRNHIISKWRENISSWVTKEMFLNSIPKHCSS---LLDSAYNYLV FPADSLT-DEEIEAGVVSDVGGIEQVNYILIRNHLLTRWRETFNSWLAKESFATLIPPHCDH---LLNAAYSFLV 164 VFQSRLPFNKMTPNEEACFPDISRSGILGHRVFLNIRNSLLHMWVDNPKIQLSFEIALKNLPPPFDSEPSLVRRVHSFLE 199 AFQSRLPHDRMTSQEAACFPDIISGPQQTQKVFLFIRNRTLQLWLDNPKIQLTFEATLQQLEAPYNSDTVLVHRVHSYLE 111 RHGLINFGRYVRSTKISRFLVRDRRSVIVIGAGAAGISAATQLESFGFDVIVLEARNCIGGRIHSFK---SKSGEIMETG 162 THGYINFGIAQAIKDKFPAQSSK-SSVIIVGAGLSGLAAARQLMRFGFKVTVLEGRKRPGGRVYTKKMEANRVGAAADLG 174 SHGHINFGVAPAIKERIPKEPTRHNTVIVVGAGLAGLAAARQLVAFGFKVVVLEGRKRCGGRVYTKKMEGGGRSAAGDLG 244 RHGFINFGIFKRLK---PIPAKKLGKVIVIGAGISGLAVAHQLQQFGMDVIVLEARDRVGGRISTFR----KNSYIADVG 279 RHGLINFGIYKRIK---PLPIKKTGKVIIIGSGVSGLAAARQLQSFGMDVTLLEARDRVGGRVATFR----KGNYVADLG 188 GDTLRKIEDSPMATLLHQVNFEEHGVFDFTSVF-VEGRPLNEEKIHLFLDHYKSAHGALNYQAHQCEHRDDQGSFISRQQ 241 GSVLTGTLGNPLGIIARQLGSSLYKVRDKCPLYRVDGKPVDPDVDIKVEVAFNQLLDKASKLRQLMG-DVSMD--VSLGA 254 GSVLTGTFGNPLGIVAKQLGLPMHKIRDKCPLYRPDGSPVDPEVDKKVEGTFNKLLDKSSLLRASMG-DVAMD--VSLGA 317 AMVVTGVYGNPMTILSKQIGMDLVPIQQTCPLYGPDGKPVPKEKDDVIEREFNRLLESASYLSHRLDFNYAGDCPVSLGD 352 AMVVTGLGGNPMAVVSKQVNMELAKIKQKCPLYEANGQAVPKEKDEMVEQEFNRLLEATSYLSHQLDFNVLNNKPVSLGQ 267 AYENLLSMCERGTLIKYYNFCKSLETVARAREHHFNQMKQLRMTALMAENQLKKMEEEG NLEQDPVLRRSL 318 ALETFRQVSGNDVATEEMG ALETLRQTDGDLSTDQEMN ALEWIISMQEMQVMHKRGQHMQEIIATQTKIIEQRRRLKTLRDTIGKLKNEHLAMINQRKPKGTDGDLKYCYQEFNIRNT 432 ALEVVIQLQEKHVKDEQIEHWKKIVKTQEELKELLNKMVNLKEKIKELHQQYKEASEVKPPRDITA EFLVKSK 338 KRDIATSLEKFEEVADAFETADNHWQRLNEHPQAKQYMHPGSEFATFNFMLGFEEYLVGAQLEKVQFSCDSMQNKEN--G LFNWHLANLEYANAGLVSKLSLAFWDQDDPYDMGG LFNWHLANLEYANAGLLSKLSLAFWDQDDPYDMVG 477 QIKMEETISTFHDLHAEEKQMLAKLHELEQNRPSDVYLS-SRDRLILDWHFANLEFANATRLNNLSLKHWDQDDDFEFIG 505 HRDLTALCKEYDELAETQGKLEEKLQELEANPPSDVYLS-SRDRQILDWHFANLEFANATPLSTLSLKHWDQDDDFEFTG 416 VAARLTEGIAELLTQLSEKRKLDIRLKHRVLDIDY--SGFEHVLLKVQRENGDIEEMKAAFVVSTLPIGVLKKTIIADER 372 DHCFLPGGNGRLVQALAEN--VPILYEKTVQTIRYGSNGVK-VTA-GNQ------VYEGDMVLCTVPLGVLK DHCFLPGGNGRLVQSLAEN--VPIVYERTVHTIRYGGDGVQ-VVVNGGQ------VYEGDMALCTVPLGVLK HHTTVRNGYSCVPVALTEN--LDIRVNSAVKEIKYGTKGVE-VVAENLKTSNSQMTYKADLVVCTLTLGVLKVAVAHKES 584 SHLTVRNGYSCVPVALAEG--LDIKLNTAVRQVRYTASGCE-VIAVNTRSTSQTFIYKCDAVLCTLPLGVLK APT----FTPSLPDKKVEAIRNIGCGSVNKCILEFDRVFWTANGGRNQFVTVSPNIKTRGSMNIWSS---VPGSKVLCTY NGSIKFVPELPQRKLDCIKRLGFGLLNKVAMLFPYVFWSTD--LDTFGHLTEDPNYRGEFFLFYSYAPVAGGALLIAL NGGVKFVPELPQRKLDSIKRLGFGLLNKVAMLFPHVFWSTD--LDTFGHLTEDPSHRGEFFLFYSYATVAGGPLLMAL 633 QQSNTVKFDPPLPDWKQQAIKRLGFGNLNKVVLCFDRIFWDPN--ANLFGHVGSTTASRGEMFLFWS---ISSSPVLLAL 653 QQPPAVQFVPPLPEWKTSAVQRMGFGNLNKVVLCFDRVFWDPS--VNLFGHVGSTTASRGELFLFWN---LYKAPILLAL 567 IVGEEAML--ELPDDVIIQNAMINLQKAFGNNCPRAP--ISAHITRWHDDELAFGSGAFMSLRTETTSFDDVMEPLKTSD 510 VAGEAAHKFETMPPTDAVTRVLHILRGIYEPQGINVPDPLQTVCTRWGGDPFSLGSYSNVAVGASGDDYDILAESVGDG- 524 VAGEAAHNFETTPPTDAVSSVLKILRGIYEPQGIEVPDPLQSVCTRWGTDSFSLGSYSHVAVGASGDDYDILAESVGDG- 708 VAGMAANLVESVTDDIIIGRCMSVLKNIFGNTS--VPQPKETVVTRWRSDPWARGSYSYVSVGSSGSDYDLLAAPVIPPS 728 VAGEAAGIMENISDDVIVGRCLAILKGIFGSSA--VPQPKETVVSRWRADPWARGSYSYVAAGSSGNDYDLMAQPITPGP GMSRVYFAGEHTCSSYTSTIQGAWMSGARAAADISN RLFFAGEATTRRYPATMHGAFVTGLREAANMAQSAKARGIRKRIDRNPSRNAHSCAILLADLFRDPDLEFG RLFFAGEATTRRYPATMHGAFISGLREAANITLHANARAAKSKVEKGPSTNTQACAALLMDLFRQPDLEFG 786 SKDAE-GLPRLFFAGEHTIRNYPATVHGAYLSGLREAGRIADYYLGYPEGTPPDIGYSVAEAANLVSVGNVVKLRDL QRHVICHVSKLF

9 A I II +1 (ATG) III IV V VI 0.5 kb (TAG) B input No AB Col fld-3 fve-4 FRI co fca-9 ld-1 fpa FLC-II ACTIN C input No AB Col fca-9 fld-3 fve-4 ACTIN FLC-I FLC-IV FLC-V FLC-VI