Figure S1. DELLA Proteins Act as Positive Regulators to Mediate GA-Regulated Anthocyanin

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1 Supplemental Information Figure S1. DELLA Proteins Act as Positive Regulators to Mediate GA-Regulated Anthocyanin Biosynthesis. (A) Effect of GA on anthocyanin content in WT and ga1-3 seedlings. Mock, without GA treatment. (B and C) Anthocyanin accumulation in WT, della (rga-t2 gai-t6 rgl1-1 rgl2-1 rgl3-1), rga-24 gai-t6, Pro35S: TAP-RGAd17 and Pro35S: TAP-GAId17 seedlings. Bar in (C) = 1 mm. (D) Effect of GA on anthocyanin content in WT and ProRGA: RGAd17 seedlings. The seeds were germinated and continuously grown on the 3% sucrose-contained half MS media without (Mock) or with 10 μm GA 3 for 5 days. The transgenic seedlings with the dark green phenotype were selected for anthocyanin analysis. Fw indicate fresh weight and Error bars represent ± SD (n = 3); Letters above the error bars indicate significant differences by two-way ANOVA analysis (p<0.01, Duncan s multiple-range test). 1

2 Figure S2. The Formation and Activity of PAP1-GL3/EGL3-TTG1 Complex Are not Directly Affected by RGA. (A) Competitive binding assay of RGA and PAP1 to EGL3. The mixture of MBP-RGA and GST-PAP1 were added to immobilized His-EGL3. The precipitates were detected using Western blotting with the anti-mbp, anti-his or anti-gst antibody. The gradient indicates the increasing amount of MBP-RGA. (B and C) Dual-luciferase assays of RGA on PAP1-GL3/EGL3 activation of the DFR promoter. The reporter was cotransformed with constructs shown in (B). Relative LUC activities were normalized to the REN internal control in (C). Error bars represent ± SD (n = 4). Letters above the error bars indicate significant differences by one-way ANOVA analysis (p<0.01, Duncan s multiple-range test). (D) Dual-luciferase assays of RGA on GL1-GL3/EGL3 activation of the GL2 promoter. Error bars represent ± SD (n = 3). Asterisks indicate significant changes in Relative LUC activities between indicated groups (p<0.01, Student s t test). The LUC/REN ratio in the leaves transformed with the vector was set as 1. 2

3 Figure S3. Anthocyanin and Proanthocyanin Accumulation in Seedlings and Seed Coat of della and MYBL2OE Plants. (A and B) Anthocyanin and proanthocyanin accumulation in WT (Ler), della, MYBL2OE (Ler background) seedlings and seeds, respectively. Bar = 1 mm. 3

4 Figure S4. MYBL2 Overexpression Inhibits Anthocyanin Biosynthesis in ga1-3 Mutants. (A and B) Anthocyanin accumulation in WT, ga1-3, MYBL2OE and MYBL2OE/ga1-3 seedlings. Bar in (A) = 1 mm. Error bars represent ± SD (n = 3). Letters above the error bars (B) indicate significant differences by one-way ANOVA analysis (p<0.01, Duncan s multiple-range test). (C and D) Relative expression of MYBL2 (C) and TT8, GL3, EGL3, PAP1, DFR and ANS (D) expression in the seedlings shown in (A). The expression levels of the genes in WT seedlings were set to 1. Error bars represent ± SE (n = 3). Two biological replicates were analyzed with similar results. 4

5 Figure S5. Domains Required for the Interaction between RGA and MYBL2. (A) Domain mapping of RGA required for interaction with MYBL2 in yeast. (B) Domain mapping of MYBL2 interacting with RGA in yeast. 5

6 Figure S6. Protein-Protein Binding Assays to Detect the Interaction between DELLAs and Subunits of the MBW Complex. (A) Y2H assays of the interactions of DELLAs with TTG1, TT8 and JAZ1. TTG1 and JAZ1 were used as negative and positive controls, respectively. Transformed yeast cells were grown on LT, -LTHA, or LTHA + 10 mm 3-amino-1, 2, 4-triazole (-LTHA+3-AT) media. (B) Pull-down assays of the interaction of RGA with PAP1. JAZ1 was used as a positive control. The arrows indicate the positions of the purified GST-RGA and GST-JAZ1 (stained by CBB). (C) BiFC assays of the interactions between DELLAs and MYBL2, PAP1 or TT8. Construct pairs indicated were transfected into protoplasts isolated from leaves of 2-week-old Arabidopsis plants. TTG1 was used as a negative control. Bars = 30 μm. 6

7 Figure S7. A Putative Model of DELLA-Regulated Anthocyanin Biosynthesis through MYBL2/JAZs. The MYB-bHLH-WD40 complexes (PAP1/PAP2/MYB113/MYB114-TT8/GL3/EGL3-TTG1) are critical for anthocyanin biosynthesis under diverse conditions. MYBL2 and JAZ family proteins are well known repressors of the MBW complexes through binding to bhlh and R2R3-MYB/bHLH transcription factors, respectively. In the normal condition or in the presence of GA, the activities of MBW complexes are inhibited by MYBL2 and/or JAZ repressors. However, under stress conditions or in the absence of GA, DELLA proteins will accumulate to antagonize the function of MYBL2 and JAZ repressors through protein-protein interactions, which leads to formation of the MBW complexes (mainly represented by PAP1/TT8/TTG1), and finally activate the anthocyanin biosynthetic genes. 7

8 Figure S8. Purified Proteins Used for Competitive Binding Assays. The cdnas encoding RGA were cloned into pmal-c2x vector (MBP-tag), those encoding MYBL2, JAZ1, TT8 and EGL3 were cloned into pet28b vector (His-tag), while those encoding MYBL2, TT8, EGL3 and PAP1 were cloned into pgex-6p-3 Vector (GST-tag). These constructs were transformed into E.coli Rosetta (DE3), and protein expression was induced by 0.2 mm IPTG. The soluble MBP fusion protein was immobilized onto amylose resin, the soluble His fusion proteins were immobilized onto Ni-NTA agarose, while the soluble GST fusion proteins were immobilized onto glutathione sepharose TM 4B. The positions of purified proteins separated by SDS-PAGE are indicated with arrows (stained by CBB). 8

9 Table S1. A List of Primers Used in This Study. Primer Names Primer sequences (5'-3') Purpose MYBL2-pGWB2-F CACCATGAACAAAACCCGCCTTCGTG Overexpression MYBL2-pGWB2-R TCATCGGAATAGAAGAAGCGTTTCTTG Overexpression RGADELLA-AD-F-NdeI GGAATTCCATATGATGAAGAGAGATCATCACCAATTCC Y2H RGADELLA-AD-R-SmaI TCCCCCGGGTCACGTTGTCGTGGTGGTTGTCGTCA Y2H RGANT-AD--F-NdeI GGAATTCCATATGATGAAGAGAGATCATCACCAATTCC Y2H RGANT-AD-R-SamI TCCCCCGGGTCAGAAGTAAGTAGCCACTTTTCTCATAGCTC Y2H RGAN2-AD-F-NdeI GGAATTCCATATGATGAAGAGAGATCATCACCAATTCC Y2H RGAN2-AD-R-SmaI TCCCCCGGGTCATCTAAGCTCAAGCATCGAAGCATCGAG Y2H RGAGRAS-AD-F-NdeI GGAATTCCATATGACGGCGGCGGGTGAGTCAACTC Y2H RGAGRAS-AD-R-SmaI TCCCCCGGGTCAGTACGCCGCCGTCGAGAGTT Y2H TTG1-BD-F-EcoRI CGGAATTCATGGATAATTCAGCTCCAGATTCGTTA Y2H TTG1-BD-R-SalI ACGCGTCGACTCAAACTCTAAGGAGCTGCATTTTGTTAG Y2H TT8-BD-F-EcoRI CGGAATTCATGGATGAATCAAGTATTATTCCGGCAG Y2H TT8-BD-R-SmaI TCCCCCGGGCTATAGATTAGTATCATGTATTATGACTTGGTGG Y2H JAZ1-BD-F-NdeI GGAATTCCATATGATGTCGAGTTCTATGGAATGTTCTGAG Y2H JAZ1-BD-R-EcoRI CGGAATTCTCATATTTCAGCTGCTAAACCGAGC Y2H MYBL2-BD-F-NdeI CCCCATATGATGAACAAAACCCGCCTTCGT Y2H MYBL2-BD-R-SmaI CCCCCCGGGTCATCGGAATAGAAGAAGCG Y2H MYBL2dC-BD-F-NdeI GGAATTCCATATGATGAACAAAACCCGCCTTCGTG Y2H MYBL2dC-BD-R-EcoRI CGGAATTCTCAGGTAGAGGATTCGCTAGAGTCCTG Y2H MYBL2NT-BD-F-NdeI GGAATTCCATATGATGAACAAAACCCGCCTTCGTG Y2H MYBL2NT-BD-R-EcoRI CGGAATTCTCAGTCGATTCCCATTTTTACGAGCT Y2H MYBL2CT-BD-F-NdeI GGAATTCCATATGCCAACCAATCATCGTCTCCACCATC Y2H MYBL2CT-BD-R-EcoRI CGGAATTCTCATCGGAATAGAAGAAGCGTTTCTTG Y2H MBP-RGA-F-BamHI CGGGATCCATGAAGAGAGATCATCACCAATTCC pull down MBP-RGA-R-XbaI GCTCTAGATCAGTACGCCGCCGTCGAGAGTT pull down His-RGA-F-BamHI CGGGATCCGATGAAGAGAGATCATCACCAATTCC pull down His-RGA-R-SacI GGAGCTCGATCAGTACGCCGCCGTCGAGAG pull down His-JAZ1-F-BamHI CGGGATCCGATGTCGAGTTCTATGGAATGTTCTGAG pull down His/GST-JAZ1-R-EcoRI CGGAATTCTCATATTTCAGCTGCTAAACCGAGC pull down His-MYBL2-F-EcoRI CGGAATTCGATGAACAAAACCCGCCTTCGTG pull down His/GST-MYBL2-R-XhoI CCGCTCGAGTCATCGGAATAGAAGAAGCGTTTCTTG pull down His-PAP1-F-BamHI CGGGATCCGATGGAGGGTTCGTCCAAAGGGCTGC pull down His/GST-PAP1-R-EcoRI CGGAATTCCTAATCAAATTTCACAGTCTCTCCATCGAAA pull down 9

10 His-EGL3-F-BamHI CGGGATCCGATGGCAACCGGAGAAAACAGAAC pull down His-EGL3-R-EcoRI CGGAATTCTTAACATATCCATGCAACCCTTTGAAG pull down His-TT8-F-BamHI CGGGATCCGATGGATGAATCAAGTATTATTCCGGCAG pull down His/GST-TT8-R-EcoRI CGGAATTCCTATAGATTAGTATCATGTATTATGACTTGGTGG pull down GST-JAZ1-BamHI-F CGGGATCCATGTCGAGTTCTATGGAATGTTCTGAG pull down GST-MYBL2-SalI-F CGGAATTCGATGAACAAAACCCGCCTTCGTG pull down GST-PAP1-F-BamHI CGGGATCCATGGAGGGTTCGTCCAAAGGGCTGC pull down GST-EGL3-F-BamHI CGGGATCCATGGCAACCGGAGAAAACAGAAC pull down GST-EGL3-R-SamHI TCCCCCGGGTTAACATATCCATGCAACCCTTTGAAG pull down GST-TT8-F-BamHI CGGGATCCATGGATGAATCAAGTATTATTCCGGCAG pull down nyfp-mybl2-f CACCATGAACAAAACCCGCCTTCGTG BiFC nyfp-mybl2-r TCATCGGAATAGAAGAAGCGTTTCTTG BiFC nyfp-ttg1-f CACCATGGATAATTCAGCTCCAGATTCGTTA BiFC nyfp-ttg1-r TCAAACTCTAAGGAGCTGCATTTTGTTAG BiFC nyfp-tt8-f CACCATGGATGAATCAAGTATTATTCCGGCAG BiFC nyfp-tt8-r CTATAGATTAGTATCATGTATTATGACTTGGTGG BiFC nyfp-pap1-f CACCATGGAGGGTTCGTCCAAAGGGCTGC BiFC nyfp-pap1-r CTAATCAAATTTCACAGTCTCTCCATCGAAA BiFC cyfp-rga-f CACCATGAAGAGAGATCATCACCAATTCC BiFC cyfp-rga-r TCAGTACGCCGCCGTCGAGAGTT BiFC cyfp-gai-f CACCATGAAGAGAGATCATCATCATCATC BiFC cyfp-gai-r CTAATTGGTGGAGAGTTTCCAAGC BiFC cyfp-rgl1-f CACCATGAAGAGAGAGCACAACCACCG BiFC cyfp-rgl1-r TTATTCCACACGATTGATTCGCC BiFC Myc-MYBL2-F-SaII ACGCGTCGACATGAACAAAACCCGCCTTCGTG dual-luc, CoIP Myc-MYBL2-R-PstI AACTGCAGTCATCGGAATAGAAGAAGCGTTTCTTG dual-luc, CoIP Myc-GL1-F-XbaI GCTCTAGAATGAGAATAAGGAGAAGAGATGAAAAAG dual-luc Myc-GL1-R-SalI ACGCGTCGACCTAAAGGCAGTACTCAACATCACCAG dual-luc Myc-PAP1-F-XbaI GCTCTAGAATGGAGGGTTCGTCCAAAGGGCTGC dual-luc Myc-PAP1-R-SalI ACGCGTCGACCTAATCAAATTTCACAGTCTCTCCATCGAAA dual-luc Myc-TT8-F-SpeI GGACTAGTATGGATGAATCAAGTATTATTCCGGCAG dual-luc Myc-TT8-R-PstI AACTGCAGCTATAGATTAGTATCATGTATTATGACTTGGTGG dual-luc Myc-GL3-F-BamHI CGGGATCCATGGCTACCGGACAAAACAGAAC dual-luc Myc-GL3-R-PstI AACTGCAGTCAACAGATCCATGCAACCCTTTG dual-luc Myc-EGL3-F-BamHI CGGGATCCATGGCAACCGGAGAAAACAGAAC dual-luc Myc-EGL3-R-PstI AACTGCAGTTAACATATCCATGCAACCCTTTGAAG dual-luc 10

11 Myc-RGA-F-BamHI CGGGATCCATGAAGAGAGATCATCACCAATTCC dual-luc Myc-RGA-R-XbaI GCTCTAGATCAGTACGCCGCCGTCGAGAGTT dual-luc ProDFR-LUC-F-KpnI GGGGTACCCAATTTTGTAATAAGAAACTCCTAATTC dual-luc ProDFR-LUC-R-SpeI GGACTAGTTTTTGTGGTTATATGATAGATTGTGCTT dual-luc ProGL2-LUC-F-KpnI GGGGTACCCACGGGTAATAGATTAATGGTGAAAAG dual-luc ProGL2-LUC-R-NcoI CATGCCATGGTTTTCTTCTTAATATTCGATTTTTAATATATAC dual-luc PAP1-qRT-F TGCTGGAAGATTACCTGGTCG qrt-pcr PAP1-qRT-R TCGAGGTCGAGGCTTATAAACATTG qrt-pcr PAP2-qRT-F TCCTTCATGCCTTGGACTCAA qrt-pcr PAP2-qRT-R CAAAGTGGCCCCATGTTCA qrt-pcr TT8-qRT-F GTCAGAGGAAATGAGGCTTGGCTC qrt-pcr TT8-qRT-R GGTGTGACATGAGAAGTGTTGTTACTG qrt-pcr GL3-qRT-F AATGCCTGGACGGACGTTTG qrt-pcr GL3-qRT-R CAATCTCAACGACTCCTCCAAGG qrt-pcr EGL3-qRT-F GGAGCGTTATCCAATGGAGAAC qrt-pcr EGL3-qRT-R GTGCCAATCTCAAGGACTCCTC qrt-pcr TTG1-qRT-F CAGTCTCTGCTGATGGATCCGTTAG qrt-pcr TTG1-qRT-R GCGAACGAATATCGAGAATCACAAC qrt-pcr MYBL2-qRT-F ATCTCATCCTCAAGCTTCATGCAC qrt-pcr MYBL2-qRT-R TGTGATGGTGGAGACGATGATTG qrt-pcr DFR-qRT-F TATCTGAGGAAGGAAGCTACGATGA qrt-pcr DFR-qRT-R TTCCATTCACTGTCGGCTTTATCAC qrt-pcr ANS-qRT-F TATCAATTTGGCCTAAGACACCAAG qrt-pcr ANS-qRT-R ACCAACTTCTTTCTCTAGACGGTCA qrt-pcr UF3GT-qRT-F ATCGAATGAATCGTCAAGCATGAG qrt-pcr UF3GT-qRT-R TGAGGGATAGAGATGGTGTGGAAAG qrt-pcr ACTIN2-qRT-F ATGCCCAGAAGTCTTGTTCC qrt-pcr ACTIN2-qRT-R TGCTCATACGGTCAGCGATA qrt-pcr 11