New Phytologist Supporting Information Regulation of anthocyanin and proanthocyanidin biosynthesis by Medicago truncatula bhlh transcription factor MtTT8 Penghui Li, Beibei Chen, Gaoyang Zhang, Longxiang Chen, Qiang Dong, Jiangqi Wen, Kirankumar S. Mysore and Jian Zhao Article acceptance date: 22 November 2015 Fig. S1 Protein sequence alignment between MtTT8 with Arabidopsis TT8 protein. Fig. S2 Phenotypes of mttt8 mutant line NF2856 with a Tnt1 insertion in MtTT8 gene. Fig. S3 Expression patterns of MtTT8 in different tissues and materials of various genetic backgrounds. Fig. S4 qrt-pcr validation of microarray data on key flavonoid biosynthetic genes. Fig. S5 Physical interaction between MtTT8, WD40 and MYB transcription factors. Fig. S6 Venn diagram of shared and unique genes differentially down-regulated among mttt8, mtpar, and mtwd40-1 mutants. Fig. S7 Subcellular localization of MtWD40-1 in nucleus. Fig. S8 Expression patterns of MtGL2 and MtEGL3 in different tissue and materials of genetic background. Fig. S9 Expression patterns of MtMYB2 in different tissues and materials of various genetic backgrounds. 1
Fig. S1 The amino acid sequence comparison between MtTT8 with Arabidopsis TT8 protein. The conserved DNA binding and domain in C-terminal end was highlighted with a box. 2
Fig. S2 Phenotypes of homozygous mttt8 mutant line NF2856 with a Tnt1 insertion in MtTT8 gene 3
Fig. S3 The expression patterns of MtTT8 in different tissues and materials of various genetic backgrounds. Data are extracted from Medicago Gene Expression Atlas (see MtGEA, http://mtgea.noble.org/v3/) 4
Fig. S4 qrt-pcr validation of microarray data on key flavonoid biosynthetic gene expression. (a) Expression of structural genes in developing seeds (12 DAP) of mttt8 mutant (MtTT8) and its segregation wild-type control (WT). (b) Expression of structural genes in leaf (2 wk old) of mttt8 mutant (MtTT8) and its segregation wild-type control (WT). (c) Expression of transcription factor genes in developing seeds (12 DAP) of mttt8 mutant (MtTT8) and its segregation wild-type control (WT). (d) Expression of transcription factor genes in leaf (2 wk old) of mttt8 mutant (MtTT8) and its segregation wild-type control (WT). 5
Fig. S5 Physical interaction between MtTT8, WD40 and MYB transcription factors. GAL activity determination by filter paper assay. MtPAR, MtWD40-1, MtLAP1, and MtTT8 in Yeast Two-Hybrid vectors pgbkt7 and pgadt7 with reading frame in fusion with the GAL4 BD and AD were tested in different combinations for their interaction on SC-Trp-Leu His. Grown yeasts were transferred to filter paper for frozen-thaw treatment and GAL activity assay with Z-buffer. Images are representatives from at least three repeat experiments. 6
Fig. S6 Venn diagram with the number of shared and unique genes differentially down-regulated among mttt8, mtpar, and mtwd40-1 mutants. Venn diagram was made with microarray data of mtpar, mttt8, and mtwd40-1 mutant seeds by using the program (http://bioinfogp. cnb.csic.es/tools/ venny/ index.html) (Oliveros, 2007). Microarray analytic data of seeds of mtpar and mtwd40-1 mutants were obtained from other sources (Pang et al., 2009; Verdier et al., 2012). 7
Fig. S7 Subcellular localization of MtWD40-1 in nucleus. The full-length EGFP (GFP) fused with MtWD40-1 (MtWD40-1-GFP) at C-terminus. MtWD40-1-GFP construct DNA was transformed into EHA105 Agrobacterium tumefaciens strains for infiltration into three-week-old Nicotiana benthamiana leaves for observation under fluorescence microscope. MtWD40-1-GFP fluorescence signal (left photo) was observed at 3-4 d after infiltration in most infected epidermal cells of tobacco leaves. The bright field photo (middle) and fluorescence field photo were merged to give right penal photo. Bar, 30 µm. 8
Fig. S8 The expression patterns of MtGL2 and MtEGL3 in different tissue and materials of genetic background. Data are extracted from Medicago Gene Expression Atlas (see MtGEA, http://mtgea.noble.org/v3/). 9
Fig. S9 The expression patterns of R2R3-type MYB transcription factor MtMYB2 in different tissues and materials of various genetic backgrounds. Data are extracted from Medicago GeneAtlas (see MtGEA, http://mtgea.noble.org/v3/). MtMYB2 expression patterns References Oliveros JC. 2007. VENNY. An interactive tool for comparing lists with Venn Diagrams. Available: http://bioinfogp.cnb.csic.es/tools/venny/index.html. Accessed 7 May 2014. Pang Y, Wenger JP, Saathoff K, Peel GJ, Wen J, Huhman D, Allen SN, Tang Y, Cheng X, Tadege M et al. 2009. A WD40 repeat protein from Medicago truncatula is necessary for tissue-specific anthocyanin and proanthocyanidin biosynthesis but not for trichome development. Plant Physiology 151: 1114 1129. 10
Verdier J, Zhao J, Torres-Jerez I, Ge S, Liu C, He X, Mysore KS, Dixon RA, Udvardi MK. 2012. MtPAR MYB transcription factor acts as an on switch for proanthocyanidin biosynthesis in Medicago truncatula. Proceedings of the National Academy of Sciences, USA 109: 1766 1771. 11