Cooperative interactions of a subgroup of the R2R3-MYB transcription factors in controlling phenylpropanoid accumulation
Final Report Abstract
Combinatorial interactions of transcription factors (TF) often determine the activity of a target promoter, thereby increasing specificity and regulatory potential. We aimed at defining the partners of R2R3-MYB transcription factors which control, regulate, or influence the activity of the R2R3-MYB transcription factors. In this project we initiated work on four subgroups (SG) of the large R2R3-MYB transcription factor family (SG 5, 6, 7, and 15) of Arabidopsis thaliana, showing most significant homology to those MYB factors from maize and petunia that were known to regulate flavonoid biosynthesis. In the later part of the project, we concentrated on SG7. Gateway-compatible cDNA cassettes representing the complete open reading frame of all selected transcription factors (R2R3-MYB and BHLH) have been generated (with and without STOP codon). Knock-out alleles have been identified and characterized for all selected R2R3-MYB genes from various knock-out populations or from natural variations. Promoter-GUS fusions of SG 6 and 7 R2R3-MYBs have been generated and transformed into A. thaliana, giving stable transgenic lines for the analysis of detailed gene expression patterns. The selected factors have been systematically tested in transient coexpression experiments in A. thaliana protoplasts with respect to their ability to activate transcription of flavonoid biosynthesis genes as a single factors, as well as on their dependency on a cofactor (BHLH). MYB factors of SG 5, 6 and 15 showed a clear dependence on a R/B-like BHLH cofactor of SGIIIf for target gene promoter activation. Together with results from a systematic study in a yeast two-hybrid matrix, it was found that BHLH SGIIIf proteins consistently interacted with the R2R3-MYB proteins of SG6 (PAP-group) and SG5 (TT2-group). In contrast, R2R3-MYB proteins from SG15 (GL1-group) interacted with BHLH2 and BHLH12, but not with BHLH42. SG7 R2R3-MYBs were found to activate target genes independently of a BHLH cofactor in protoplast cotransfection experiments and in yeast two-hybrid assays. The genes encoding the flavonoid biosynthesis enzymes chalcone synthase, chalcone flavanone isomerase, flavanone 3-hydroxylase and flavonol synthase were identified as target genes. Metabolite analyses in SG7 (multiple) mutant lines demonstrated a tight linkage between the expression of functional SG7 R2R3-MYB proteins and the flavonol glycoside content of developing seedlings. Seedlings of a myb11 myb12 myb111 triple mutant, which genetically lack a complete SG of R2R3-MYB genes, were found to form no flavonols. Thus, we identified SG7 R2R3-MYBs as flavonol specific activator of flavonoid biosynthesis, acting in an additive manner due to their differential spatial activity.
Publications
- (2004): Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like BHLH proteins. Plant Journal 40(1): 22-34
I.M. Zimmermann, M.A. Heim, B. Weisshaar, J.F. Uhrig
- (2004): TT2, TT8, and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana. Plant Journal 39(3): 366-80
A. Baudry, M.A. Heim, B. Dubreucq, M. Caboche, B. Weisshaar, L. Lepiniec
- (2005): Differential combinatorial interactions of cis-acting elements recognized by R2R3-MYB, BZIP, and BHLH factors control light-responsive and tissue-specific activation of phenylpropanoid biosynthesis genes. Plant Molecular Biology 57(2): 155-171
U. Hartmann, M. Sagasser, F. Mehrtens, R. Stracke, B. Weisshaar
- (2005): The Arabidopsis transcription factor MYB12 is a flavonol-specific regulator of phenylpropanoid biosynthesis. Plant Physiology 138(2): 1083-1096
F. Mehrtens, H.D. Kranz, P. Bednarek, B. Weisshaar
- (2007): Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling. Plant Journal 50(4): 660-677
R. Stracke, H. Ishihara, G. Huep, A. Barsch, F. Mehrtens, K. Niehaus, B. Weisshaar
