Supplemental Data. Bai et al. Plant Cell. (2012) /tpc A

Transcription

1 A B Unconserved Conserved AIF4 AIF2 AIF3 AIF1 UPB1 IBH1 Supplemental Figure 1. IBH1, UPB1 and AIFs belong to the same HLH family. (A), Part of a phylogenetic tree constructed using conserved domains shows that IBH1, UPB1 and AIFs belong to the same HLH family (The phylogenetic tree was cut from the Database for Flower Bio-engineering by CRES-T at org/ fiore/public_db/ index.shtml). (B) Sequence alignment of seven IBH1 family proteins using the PRALINE program ( pralinewww/). Color shows the degree of amino acid conservation as indicated by the scale bar. The black box is put outside the conserved AS domain. Under the sequences, the white bar indicates the basic region, the black bars indicate the helix motifs, and the black line indicates the loop region. 1

2 Cotyledon Hypocotyl Root HBI1 Supplemental Figure 2. IBH1 interacts with five homologous bhlh proteins. The left panel is the part of phylogenetic tree showing BEE and CIB family proteins (The phylogenetic tree was cut from the Database for Flower Bio-engineering by CRES-T at org/ fiore/ public_db/index.shtml). The genes labelled by red box encode IBH1-interacting bhlh factors, according to the Arabidopsis Interactome Mapping Consortium database. The right table shows the relative expression levels of corresponding genes in cotyledon, hypocotyl and root, which high expression marked by red and low expression by green color, and average expression level by yellow color (From the Database for Flower Bio-engineering by CRES-T at fiore/public_db/index.shtml). 2

3 AD BD-PRE1 +His -His AtIBH1 AS b H L H AtIBH1N AtIBH1C1 AtIBH1C2 Supplemental Figure 3. Yeast two-hybrid assays showed that IBH1 interacts with PRE1 through its HLH domain. 3

4 PP2A BEE2 BEE2L CIB1 CIBL1 CIB3 BPE BPEL CIB2 CIB4 CIB5 CIBL2 Expression ratio Supplemental Figure 4. Quantitative RT-PCR analyses of expression levels of HBI1 and its homologous genes in the HBI1-CS plants compared to wild type. Expression levels were normalized to PP2A as the internal control, and ratio between HBI1-CS and wild type was calculated for each gene. Error bars indicate SD from three biologic repeats. 4

5 Unconserved Conserved BEE2 HBI1 CIB1 AT5G48560 AT3G7340 AT1G68920 CIB5 AT1G10120 AT5G62610 AT5G50915 BPE Consistency BEE2 HBI1 CIB1 AT5G48560 AT3G7340 AT1G68920 CIB5 AT1G10120 AT5G62610 AT5G50915 BPE Consistency * * * Supplemental Figure 5. HBI1 and its homolog contain conserved basic domain to bind DNA. Alignment of HBI1 and its homolog proteins using the PRALINE program ( Color shows the degree of amino acid conservation as indicated by the scale bar. Under the sequences, the white bar marks the basic domain, the black bars indicate the helix motifs, and the black line indicates the loop region. The asterisks indicates the conserved residues of basic domain required for DNA-binding. 5

6 -4031 a b ATG Supplemental Figure 6. The gene structure diagram of EXP1 showing promoter regions analyzed for binding to HBI1. Open boxes show promoter regions, black lines show untranslated regions and introns, black boxes show coding sequences, black circles show G-box motifs and white circles show E-box motifs. The fragment used in DNA-protein pull down experiments and theregion analyzed by ChIP-qPCR are marked by lines under the promoter region marked by a and b, respectively. 6

7 Supplemental Table 1. Quantitative RT-PCR validation of the RNA-Seq data In the table, Genes RNA-Seq RT-qPCR IBH1-Ox vs Col PRE1-Ox/ IBH1-Ox vs IBH1-Ox IBH1-Ox vs Col PRE1-Ox/ IBH1-Ox vs IBH1-Ox At2g At3g At3g At3g At3g At3g At5g EXP EXP

8 Supplemental Table 2. Oligo sequences, Experiment Primer name sequence Vector construction IBH1GWF CACCACCCAAACAATGGCCTCTGCAG IBH1GWR TTTGGGAGATAAGCCATCAACG IBH1ΔBF GATTTTCAGAATTCATGCCGGTGG IBH1ΔBR GCGGAATTCTGAAAATCTTACGATCTTGTTGTC IBH1ΔASF CGCAAGCTTCGTAGAGCAGACAAAGATGAC IBH1ΔASR GCGAAGCTTCTTCCTCACGCGGTAGTTTTTTTGG T IBH1NR GCGGAATTCTGAAAATCTTACGATCTTGTTGTC IBH1C1F CACCATGAGGAGGAAGTGGAAGATATC IBH1C2F CACCGAATTCATGCCGGTGGTGGAGGAGGCGG C HBI1GWF CACCATGTTGGAAGGTCTTGTCTC HBI1GWR GTAATGAAAACCGAGGCTAGATG BEE2GWF CACCATGGACTTGTCTGTACTTGATAG BEE2GWR CTTGAGGCTGAAGAAATTGGAAAC ChIP-qPCR primers PP2A ChIP-F CGGCTTTCATGATTCCCTCT PP2A ChIP-R GCCTTAAGCTCCGTTTCCTACTT EXP1ChIPF TGTATGCGAAGCACACAAAGACATC EXP1ChIPR GATGGAGGTGCCTTAATATAAACGC EXP8ChIPF CACAACCTTCAAAGTCCCT EXP8ChIPR CAACAATGTATGCCACCG Transient assay EXP1 promoterf CACCCAGATGATAATATATGTATAATTAG EXP1 promoterr GTAAACCTGCATATAGGTAAATGAAG DNA pull down EXP1 biotinf otggaatcttgggaaaccc EXP1R GATGGAGGTGCCTTAATATAAACGC RT-qPCR HBI1RTF TGCCTGGATGCAATAAGGTCACAG HBI1RTR TGGAGCTTCGATAGATGTCGTTTGG BEE2RTF CAGTTTCAGGCTTACTTCACAGGT BEE2RTR CTTTAGCGCCGAGAGATGTGGT EXP1RTF GCTTACCGAAGGCTTACCGAAG EXP1RTR CTTGACATCGCTTGCCATCCAG EXP8RTF tcctcctcttcagcatttcgacct EXP8RTR cttgccacgactgtgtttttgagc PP2A RTF tatcggatgacgattcttcgtgcag PP2A RTR gcttggtcgactatcggaatgagag CIB1RTF aggccggattttgatatggatgac CIB1RTR ttgctgcattggattgacatgaagg CIB5RTF TGTCCACATGAACCATGAAACCG CIB5RTR CTCCACCTTCATGTCAGCTGTTG 8

9 AT1G10120 (CIBL2) RTF AT1G10120 (CIBL2) RTR BPERTF BPERTR AT1G68920 (CIB4) RTF AT1G68920 (CIB4) RTR AT3G07340 (CIB3) RTF AT3G07340 (CIB3) RTR AT5G48560 (CIB2) RTF AT5G48560 (CIB2) RTR AT5G50915 (CIBL1) RTF AT5G50915 (CIBL1) RTR AT5G62610 (BPEL) RTF AT5G62610 (BPEL) RTR At2g33230RTF At2g33230RTR At3g47250RTF At3g47250RTR At3g47350RTF At3g47350RTR At3g47680RTF At3g47680RTR At3g50330RTF At3g50330RTR At3g52680RTF At3g52680RTR At5g44440RTF At5g44440RTR At5g51630RTF At5g51630RTR ACAGTCTTGCAGAGCGGGTTAG ATCCGGGAACAAGTTCTTGAAGC ACCATTGGCGCTAGTGAAGGTG TCTGCTTCTGCTTTCCCATCACAC GCAACTGTGAACCCACAAATGGAC ACCAGCTCGTAGTTGAAGTGCATC TCCCTGGATGCAACAAGGTTACTG AAAGTCCAGCCTGGTGTTCACTG TGTGCCTGGATGCAATAAGGTTAC AATCCAGCCTGGTGTCGTTCAC TGTTCCGGGCTGTGATAAGGTAAC AGGTCGGAGCCAAAGTCATAGACC TTGCAGCGGCAAGTTGAGTTTC ACTTGCACCCGAGTTAACAACTTC GGATGGTCGAGTTCTGCAGATTG TCCTTAAGCCATGAAGGGACATTG TGGACTGCTCTGTCATCCTTTGC ACGAATCGCATAAGCGGCTTGC AAGCGATCTTCAGAGGTATTGGTG TCGACGAATCAAAGCCCACAAC CAAACCAAGCTGCACACACACC TCTTCACCGGCAGACCATTTCC TGAATCCAACACCCACCCGTTC GTTGGTGGTGAGGCATGGTTTG ACGGATTGGATGGTGTGGTCAG ACAACACCTCGCTTTCTCTCACG CGAAACACCATTCCCACATCGAG TCTTCCTCTTCTCTCCAGTAAGCC TGGGATGTCTCTCCCAGAGTTC AGCTTCACCTTCACCTTTAACTGG 9