Abstract
Among the secondary metabolites involved in grape berry and wine quality, condensed tannins or proanthocyanidins (PAs) play a major role, especially in astringency and color stability of wine. These molecules are also involved in plant defence against biotic and abiotic stresses. Furthermore, the beneficial effects of PAs to human health are well documented. In grapevine, PAs have the distinctive feature of being esterified with gallic acid. An acylation reaction called galloylation is responsible for this modification. Studies show that the galloylation influences oenological and pharmacological properties of PAs. In the grape berry, PAs are synthesized in the early stages of development, mainly in skin and seeds. A relatively small number of enzymatic steps are required for the biosynthesis of the basic structure of these metabolites and the corresponding genes are now widely known in model plants, including in grapevine. However, the molecular mechanisms involved in the final steps, including galloylation, are only partially known. Earlier results obtained after the search of QTL influencing the composition of the grape berry, especially the galloylation ratio of PAs, and transcriptomic studies after overexpression of transcription factors that regulate PAs biosynthesis pathway, have allowed the identification of genes potentially involved in these steps. Shikimate dehydrogenase (SDH) genes were identified. These genes would intervene upstream, for the biosynthesis of gallic acid. Three identified glucosyltransferases, already characterized in the laboratory, are involved in the biosynthesis of glucose ester of gallic acid (β-glucogalline), which could serve as an intermediary for PAs galloylation. These screening methods have also helped to identify 2 serine carboxypeptidase-like acyltransferases, called glucose acyltransferases (GATs) which are capable of catalyzing the last step of galloylation: the transfer of gallic acid from β-glucogalline to PAs. The first objective of this thesis was to determine the function of the SDHs encoded by grapevine genes. Recombinant SDHs, produced heterologously in E. coli, have the capacity to generate gallic acid in vitro. Their level of expression during development and in different tissues of the berry was also established. In vitro results are supported by the metabolic profile (gallic acid, β-glucogallin and PAs) of grapevine hairy -roots transformed with a SDH gene. The second objective of this thesis was to validate the function of the GATs by transient expression in tobacco leaves and in vitro enzyme assays. The transient transformation of grapevine leaves with GATs allowed to modulate the concentration of phenolic esters and notably galloylated flavan-3-ols in planta. The study of these genes was extended to vascular plants by phylogenetic analyses which allowed to identify peptide motifs potentially involved in the studied mechanisms and reflecting the sub-functionalization of certain genes. This work has provided informations on the genetic basis and molecular mechanisms involved in the biosynthesis of gallic acid and its two-step transfer on flavan-3-ols (galloylation). New hypotheses on the intervention of different carriers and nature of transported molecules can be proposed.