SummaryIn Arabidopsis thaliana, proanthocyanidins (PAs) accumulate in the innermost cell layer of the seed coat (i.e. endothelium, chalaza and micropyle). The expression of the biosynthetic genes involved relies on the transcriptional activity of R2R3-MYB and basic helix-loop-helix (bHLH) proteins which form ternary complexes ('MBW') with TRANSPARENT TESTA GLAB-RA1 (TTG1) (WD repeat protein). The identification of the direct targets and the determination of the nature and spatio-temporal activity of these MBW complexes are essential steps towards a comprehensive understanding of the transcriptional mechanisms that control flavonoid biosynthesis.In this study, various molecular, genetic and biochemical approaches were used. Here, we have demonstrated that, of the 12 studied genes of the pathway, only dihydroflavonol-4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), BANYULS (BAN), TRANSPARENT TESTA 19 (TT19), TT12 and H + -ATPase isoform 10 (AHA10) are direct targets of the MBW complexes. Interestingly, although the TT2-TT8-TTG1 complex plays the major role in developing seeds, three additional MBW complexes (i.e. MYB5-TT8-TTG1, TT2-EGL3-TTG1 and TT2-GL3-TTG1) were also shown to be involved, in a tissuespecific manner. Finally, a minimal promoter was identified for each of the target genes of the MBW complexes. Altogether, by answering fundamental questions and by demonstrating or invalidating previously made hypotheses, this study provides a new and comprehensive view of the transcriptional regulatory mechanisms controlling PA and anthocyanin biosynthesis in Arabidopsis.

SummaryTT8/bHLH042 is a key regulator of anthocyanins and proanthocyanidins (PAs) biosynthesis in Arabidopsis thaliana. TT8 transcriptional activity has been studied extensively, and relies on its ability to form, with several R2R3-MYB and TTG1 (WD-Repeat protein), different MYBbHLH-WDR (MBW) protein complexes. By contrast, little is known on how TT8 expression is itself regulated.Transcriptional regulation of TT8 expression was studied using molecular, genetic and biochemical approaches.Functional dissection of the TT8 promoter revealed its modular structure. Two modules were found to specifically drive TT8 promoter activity in PA-and anthocyanin-accumulating cells, by differentially integrating the signals issued from different regulators, in a spatio-temporal manner. Interestingly, this regulation involves at least six different MBW complexes, and an unpredicted positive feedback regulatory loop between TT8 and TTG2. Moreover, the results suggest that some putative new regulators remain to be discovered. Finally, specific cis-regulatory elements through which TT8 expression is regulated were identified and characterized.Together, these results provide a molecular model consistent with the specific and highly regulated expression of TT8. They shed new light into the transcriptional regulation of flavonoid biosynthesis and provide new clues and tools for further investigation in Arabidopsis and other plant species.

The control of growth and development of all living organisms is a complex and dynamic process that requires the harmonious expression of numerous genes. Gene expression is mainly controlled by the activity of sequence-specific DNA binding proteins called transcription factors (TFs). Amongst the various classes of eukaryotic TFs, the MYB superfamily is one of the largest and most diverse, and it has considerably expanded in the plant kingdom. R2R3-MYBs have been extensively studied over the last 15 years. However, DNA-binding specificity has been characterized for only a small subset of these proteins. Therefore, one of the remaining challenges is the exhaustive characterization of the DNA-binding specificity of all R2R3-MYB proteins. In this study, we have developed a library of Arabidopsis thaliana R2R3-MYB open reading frames, whose DNA-binding activities were assayed in vivo (yeast one-hybrid experiments) with a pool of selected cis-regulatory elements. Altogether 1904 interactions were assayed leading to the discovery of specific patterns of interactions between the various R2R3-MYB subgroups and their DNA target sequences and to the identification of key features that govern these interactions. The present work provides a comprehensive in vivo analysis of R2R3-MYB binding activities that should help in predicting new DNA motifs and identifying new putative target genes for each member of this very large family of TFs. In a broader perspective, the generated data will help to better understand how TF interact with their target DNA sequences.