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In 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 GLABRA1 (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 tissue‐specific 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.

Sugar has only recently been identified as a key player in triggering bud outgrowth, while hormonal control of bud outgrowth is already well established. To get a better understanding of sugar control, the present study investigated how sugar availability modulates the hormonal network during bud outgrowth in Rosa hybrida. Other plant models, for which mutants are available, were used when necessary. Buds were grown in vitro to manipulate available sugars. The temporal patterns of the hormonal regulatory network were assessed in parallel with bud outgrowth dynamics. Sucrose determined bud entrance into sustained growth in a concentration-dependent manner. Sustained growth was accompanied by sustained auxin production in buds, and sustained auxin export in a DR5::GUS-expressing pea line. Several events occurred ahead of sucrose-stimulated bud outgrowth. Sucrose upregulated early auxin synthesis genes (RhTAR1, RhYUC1) and the auxin efflux carrier gene RhPIN1, and promoted PIN1 abundance at the plasma membrane in a pPIN1::PIN1-GFP-expressing tomato line. Sucrose downregulated both RwMAX2, involved in the strigolactone-transduction pathway, and RhBRC1, a repressor of branching, at an early stage. The presence of sucrose also increased stem cytokinin content, but sucrose-promoted bud outgrowth was not related to that pathway. In these processes, several non-metabolizable sucrose analogues induced sustained bud outgrowth in R. hybrida, Pisum sativum, and Arabidopsis thaliana, suggesting that sucrose was involved in a signalling pathway. In conclusion, we identified potential hormonal candidates for bud outgrowth control by sugar. They are central to future investigations aimed at disentangling the processes that underlie regulation of bud outgrowth by sugar.

Large-scale analysis of transcription factorcis-acting element interactions in plants, or the dissection of complex transcriptional regulatory mechanisms, requires rapid, robust and reliable systems for the quantification of gene expression. Here, we describe a new system for transient expression analysis of transcription factors, which takes advantage of the fast and easy production and transfection of Physcomitrella patens protoplasts, coupled to flow cytometry quantification of a fluorescent protein (green fluorescent protein). Two small-sized and high-copy Gateway (R) vectors were specifically designed, although standard binary vectors can also be employed. As a proof of concept, the regulation of BANYULS (BAN), a key structural gene involved in proanthocyanidin biosynthesis in Arabidopsis thaliana seeds, was used. In P. patens, BAN expression is activated by a complex composed of three proteins (TT2/AtMYB123, TT8/bHLH042 and TTG1), and is inhibited by MYBL2, a transcriptional repressor, as in Arabidopsis. Using this approach, two new regulatory sequences that are necessary and sufficient for specific BAN expression in proanthocyanidin-accumulating cells were identified. This one hybrid-like plant system was successfully employed to quantitatively assess the transcriptional activity of four regulatory proteins, and to identify their target recognition sites on the BAN promoter.

Bud burst is a crucial factor in plant architecture and is strongly induced by light. In Rosa sp., this light effect was correlated with the growth of axillary buds and RwMAX1 and RwMAX2 expression within buds. In this paper, we investigated whether strigolactone pathway is involved in the regulation of axillary bud in response to light intensity. Hence, young roses were subjected to two contrasting light intensity regimes: high/high and high/low. The phenotype was characterized in both conditions and the expression of RwMAX1 and RwMAX2 genes was measured in the basal, middle and apical parts of rose primary branch. Light treatments showed a strong impact on axillary bud. The percentage of bud burst was severely reduced in the treatment high/low compared to the treatment high/high in all branch parts. In addition, the expression of RwMAX1 and RwMAX2 was strongly inhibited by high/high light regime and was conversely correlated with the rate of bud burst. In in vitro-grown axillary buds supplied with sucrose, glucose and fructose, RwMAX1 expression was significantly stimulated whereas that of RwMAX2 was significantly inhibited. Our results suggest that although RwMAX1 and RwMAX2 expression can be regulated by light, this expression does not explain the ability of bud burst.