Identification et caractérisation fonctionnelle de gènes impliqués dans la voie de biosynthèse des furocoumarines chez les végétaux supérieurs

Abstract : Furanocoumarins are secondary metabolites deriving from the phenylpropanoid biosynthetic pathway. These phytoalexins are especially involved in plant defense mechanisms against insects or phytopatogenous fungi and bacteria. The molecular control of this biosynthetic pathway is still poorly understood even though it has been demonstrated since the 1960s that enzymes catalyzing the different steps are belonging to different enzymatic families. The work presented here is focused on two enzyme families: oxoglutarate dependent dioxygenases and cytochrome P450s. The first step in the furanocoumarins biosynthetic pathway is the ortho-hydroxylation of p-coumarate, which leads to the formation of umbelliferone. Based on a recent work done on Arabidopsis, we isolated six genes encoding dioxygenases from Ruta graveolens, Citrus limetta and Pastinaca sativa. The corresponding proteins share more than 58% identity with the A. thaliana féruloyle 6' Hydroxylase (F6'H). The functional characterization of these enzymes was performed in a prokaryotic heterologous expression system. Of the six enzymes, three could not be functionnaly expressed and two exhibited a similar F6'H activity as described for A. thaliana. The last enzyme has new properties not described to date. It is able to achieve both hydroxylation of féruloyle CoA and p-coumaroyle CoA. These in vitro studies were completed by a functional exploration of the protein in planta. A detailed analysis of the gene expression pattern highlighted a link with the level of umbelliferone synthesis. The function of the protein was also confirmed by an analysis of the products formed in transiently transformed Nicotiana benthamiana leaves. Cytochrome P450s catalyze 60% of the reactions of the furanocoumarin biosynthetic pathway. Therefore, a functional screening of cytochrome P450 previously identified in Ammi majus and Thapsia garganica was undertaken. The bioinformatic analyses and the changes undertaken in the procedure for expression in yeast allowed drawing hypotheses on the function of some of these P450 candidates. These exploratory and preliminary experiments allowed suggesting new hypotheses about the biosynthetic pathway
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Guilhem Vialart. Identification et caractérisation fonctionnelle de gènes impliqués dans la voie de biosynthèse des furocoumarines chez les végétaux supérieurs. Sciences agricoles. Université de Lorraine, 2012. Français. ⟨NNT : 2012LORR0390⟩. ⟨tel-01750069⟩

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