Supplementary Figure S1. Amino acid alignment of selected monocot FT-like and TFL-like sequences. Sequences were aligned using ClustalW and analyzed
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1 Supplementary Figure S1. Amino acid alignment of selected monocot FT-like and TFL-like sequences. Sequences were aligned using ClustalW and analyzed using the Geneious software. Accession numbers of the genes used for the phylogenetic analysis are as follows: onion AcFT1 (KC485348), AcFT2 (KC485349), AcFT3 (KC48535), AcFT4 (KC485351), AcFT5 (KC485352), AcFT6 (KC485353); Orchid OgFT (EU58352); barley HvFT1 (DQ1327), HvFT2 (DQ29747), HvFT3 (DQ411319), HvFT4 (DQ41132), HvFT5 (EF1222), HvTFL1 (AB447465), and HvMFT1 (AB447466); TIGR loci in rice are as follows: OsFTL1 (Os1g1194), OsFTL2 (Hd3a; Os6g632), OsFTL3 (RFT1; Os6g63), OsFTL4 (Os9g3385), OsFTL5 (Os2g3964), OsFTL6 (Os4g4113), OsFTL7 (Os12g133), OsFTL8 (Os1g159), OsFTL9 (Os1g5449), OsFTL1 (Os5g4418), OsFTL11 (Os11g1887), OsFTL12 (Os6g3594), OsFTL13 (Os2g1383), OsMFT1 (Os6g337), OsMFT2 (Os1g212), RCN1 (Os11g547), RCN2 (Os2g3295), RCN3 (Os12g559), and RCN4 (Os4g3357); ZCN1 (EU241917), ZCN2 (EU241918), ZCN3 (EU241919), ZCN4 (EU24192), ZCN5 (EU241921), ZCN6 (EU241922), ZCN7 (EU241923), ZCN8 (EU241924), ZCN9 (EU241925), ZCN1 (EU241926), ZCN11 (EU241927), ZCN12 (EU241928), ZCN14 (EU241929), ZCN15 (EU24193), ZCN16 (EU241931), ZCN17 (EU241932), ZCN18 (EU241933), ZCN19 (EU241934), ZCN2 (EU241935), ZCN21 (EU241912), ZCN24 (EU241914), and ZCN25 (EU241936).
2 a S FT1#1 35S FT1#4 35S FT1#2 35S FT1#21 35S FT2#12 35S FT2# S FT2#14 35S FT4# S FT4# b SUC2 FT1#A SUC2 FT1#2 SUC2 FT1#4 SUC2 FT1#6 SUC2 FT2#1 SUC2 FT2#5 SUC2 FT2#8 SUC2 FT4 #7 SUC2 FT4 #11 SUC2 FT4 #1 SUC2 FT4 #14 Supplementary Figure S2. Flowering time differences between AcFT1 and AcFT2 transgenes in Arabidopsis lines is not due to level of expression. of the AcFT1, AcFT2 and AcFT4 transgene driven by the 35S promoter (a) or SUC2 promoter (b) in transgenic Arabidopsis plants. The number of rosette leaves at flowering is given above each column. Note; only the low expressing 35S:AcFT4 plants were analyzed, as the other lines did not flower and produce seed that could be used for expression analysis. The expression data represents an average +/- s.e.m. of 3 individual plants, with transcripts normalized to actin
3 a b Supplementary Figure S3. Complementation of mutants with AcFT2 when expressed from 35S promoter but not from SUC2 promoter. (a) Complementation of the Arabidopsis mutant with AcFT2 expressed using either phloem specific SUC2 promoter (yellow) or the constitutive 35S promoter (blue). Flowering time, as determined by number of rosette leaves at flowering, for representative lines grown in LD. Data represents a minimum of 3 plants scored for each line +/- s.e.m. (b) Photos of 35S:AcFT4 Arabidopsis lines that did not flower (top); Photos of SUC2:AcFT4 plants showing aerial rosettes and flowers with leaf-like floral organs (bottom).
4 a b c AtTSF S FT4 #5 35S FT4 #6.7 AtAP AtFT 35S FT4 #5 35S FT4 #6 35S FT1 #2 35S FT1 #6 SUC2 FT4 #7 SUC2 FT4 #1 SUC2 FT4 #11 SUC2 FT4 #14 SUC2 FT4 #7 SUC2 FT4 #1 SUC2 FT4 #11 SUC2 FT4 #14 35S FT4 #6 35S FT4 #5 Supplementary Figure S4. Late flowering of AcFT4 is not a result of the Arabidopsis FT or TSF genes being downregulated in the leaves but of AP1 not being upregulated in the apex. levels of the Arabidopsis FT (a) and TSF (b) genes in leaves of transgenic Arabidopsis plants expressing the AcFT4 gene under the control of the 35S or SUC2 promoters. (c) of the Arabidopsis AP1 gene in dissected apical bud tissue from mutant plants and 35S:AcFT4 lines (harvested approximately when the floral transition occurs in plants) and from floral buds of AcFT1 early flowering plants. The expression data represents an average +/- s.e.m. of 3 individual plants, with transcripts normalized to actin.
5 Supplementary Figure S5. Vernalization induces flower of onion bulbs (a) Photos of onion bulbs cut in half that have either not been vernalized (left) or given a 3 month vernalization treatment (right). The vernalized bulb has developed an immature floral inflorescence (black arrow). (b) AcFT1 expression is not detected in any bulb tissue, while AcFT2 (red bars) is expressed after vernalization. The expression data represents single tissue sample with transcripts normalized to actin.
6 A Short days seedlings mature flowering B FT1 FT2 FT3 FT4 FT5 FT6 Long days FT1 FT2 FT3 FT4 FT5 FT6 Supplementary Figure S6. Expression of AcFT1-6 in onions FT expression was determined in seedlings with 3-4 leaves (young seedlings), seedlings (mature seedlings) with >1 leaves, and vernalized bulbs (flowering bulbs) grown in either a short day (a) or long day (b) photoperiod. Data represents an average +/- s.e.m of three biological replicates, with transcripts normalized toβ-tubulin.
7 .5.4 FT FT FT FT FT FT Days after shift to LD Supplementary Figure S7. Expression of AcFT1-6 after transfer from short- to long-days. Mature onion seedlings were grown under short day conditions at 2 C for 14 weeks. They were then transferred to long day conditions at 2 C and tissue samples were taken at ZT5. The data represents an average +/- s.e.m. of 3 individual plants, with transcripts normalized to β-tubulin.
8 a b c d e Supplementary Figure S8. Overexpression of AcFT4 prevents bulbing. (a) 35S:AcFT4 plants and (b) Control plants grown in a glasshouse conditions and photographed in autumn (8 May 213). (c & d) 35S:AcFT4 plants and (e) Control plant photographed in winter (14 June 213).
9 a.35.3 AtFUL b c AtSEP3 AtFT S FT1#1 35S FT1#4 35S FT1#2 35S FT1#21 35S FT2#1 35S FT2#12 35S FT2#13 35S FT2#14 Supplemental Figure S9. AcFT1 and AcFT2 have different activities in leaves when overexpressed in Arabidopsis. of the Arabidopsis meristem identity genes FUL (a) and SEP3 (b) and the FT (c) in leaves of transgenic seedlings overexpressing AcFT1 and AcFT2, along with and wild-type plants as controls. The expression data represents an average +/- s.e.m. of 3 individual plants, with transcripts normalized to actin.
10 AcFT1 to B AcFT1 to B SUC2 FT1#6 SUC2 FT2#8 SUC2 FT4 # Supplemental Figure S1. The altered activity of AcFT1 containing the B segment from AcFT2 and AcFT4 is not due to lower expression levels. of the modified AcFT1 constructs in transgenic Arabidopsis plants. The number of rosette leaves at flowering is given above each column. The expression data represents an average +/- s.e.m. of 3 individual plants, with transcripts normalized to actin.
11 Supplementary Table S1. Primers used in this study Gene Primer name # Sequence 5'-3' AcFT1 AcFT1 qfwd AAACCATCACAAATAACTCAGCA AcFT1 qrev GTTTCTCGCCCAAAGTTCG AcFT2 AcFT2 qfwd CCTTCCCAGGTTGCTCTAC AcFT2 qrev CCAATGCAAGTATTCTCGTAGACA AcFT3 AcFT3 qfwd AGGAAGTTACTAACGGGTGTGAA AcFT3 qrev CAAAGCTTGCATCTTTTGACC AcFT4 AcFT4 qfwd TGAAATAGGAGGTGTACCAAGAAT AcFT4 qrev TTCCGAAACTACCATCCATATTTG AcFT5 AcFT5 qfwd GAAATTGGAGGACGCGAC AcFT5 qrev CTTGCATCTTTTGCTTCTGGTA AcFT6 AcFT6 qfwd TCGTCAATCGATGGTTATAAATCA AcFT6 qrev TTTCCATAACTTGCATCGACTGT AcFT1 FL_AcFT1_Fwd TTACATGGCAAGAGAAAGTGACCCAT FL_AcFT1_Rev CCTATTAGTAATCCGTGTATATTCT AcFT2 FL_AcFT2_Fwd AAGGATGATGGATTCGGATCCGTTA FL_AcFT2_Rev CCATTCATCTATAAGTTCTCCTCCCA AcFT4 FL_AcFT4_Fwd CACGATGTCTTTTGATCCTTTAGTT FL_AcFT4_Rev TGTGCTAATTCTCTGATCGAAACCTT AcFT5 FL_AcFT5_Fwd TTCCATGTCAAGAGATCCTCTTGTT FL_AcFT5_Rev AGTGTCAGAGCCAGCCACTTCCT AcFT6 FL_AcFT6_Fwd ATACATGCAAGTAAAAATGTTGCGA FL_AcFT6_Rev GCAGTCAGCAAAGCCCCGAGAACCT Acβ-tubulin Acβ-tubulin_qRev TCAGTCCAGTAGGAGGAATGTCG Acβ-tubulin_qFwd GTCTTCAGAGGCAAGATGAGCAC AtFT AtFT_qFwd AGGCCTTCTCAGGTTCAAAACAAGC AtFT_qRev TGCCAAAGGTTGTTCCAGTTGTAGC AtSEP3 AtSEP3_qFwd CTAAGACTAAGGTTAGCTGATGGGTA AtSEP3_qRev ATGATGACGACCGTAGTGATCAA AtFUL AtFUL_qFwd TGCGTAACCTCCTCCAGAGAT AtFUL_qRev GTTCTACTCGTTCGTAGTGGTAGGAC AtTSF AtTSF_qFwd GTGGATCCAGATGTGCCGAGTC AtTSF_qRev GCCGGAACAATACCAACACAATACG Ac 1-SST 1-SST_RT_L_V2 AGGATGACCCTCCATCCG 1-SST_RT_R_V2 TTGCAGCATTGGGATCG At Actin At_Actin_qFwd CGCTCTTTCTTTCCAAGCTCAT At_Actin_qRev TCCTGCAAATCCAGCCTTC a q in primer name indicates it was used for quantitative RT-PCR and FL indicates primers were used to amplify full-length coding regions b start and red stop codons are indicated in green and red, respectively
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