Caratterizzazione funzionale di due Caffeoil-CoA O-metiltransferasi di Vitis vinifera L.

Methylation of oxygen, nitrogen and carbon is an ubiquitous reaction that takes place in bacteria, fungi, plants and animals. In plants, the O-methylation patterns of polyhydroxylated molecules are crucial to determining final product distribution of methylated compounds in secondary branched pathway (Zubieta et al., 2001). Plant O-methyltransferases (OMTs) form a wide group of enzymes that catalyze methylation of a wide range of substrates. Over the years OMT genes have been characterized from several plant species, and different classifications have been proposed for this wide enzyme family (Ibrahim, 1997; Lam et al., 2007). To date plant OMTs can be categorized into two major classes. Type 1 OMTs consist of homodimeric OMTs with subunit sizes of about 38 to 43 kDa, that do not require divalent cations for their activity, instead type 2 OMTs represents a group of lower molecular weight (23 to 27 kDa), cation-dependent OMTs that lack the 5 consesus sequence motifs characteristic of type 1 OMTs. Most members of this family are caffeoyl-CoA O-methyltransferase. For a lot of time we have thought that CCoAOMTs play a crucial role only in lignin biosynthesis. CCoAOMTs have been characterized from several plant spieces; in V. vinifera the only work well known to date is the article published in 1997 by Busam and collaborators. Using bioinformatic researches, we found eight genes in the grape genome that codify for putative CCoAOMTs. Thanks to the recent characterization of VvAOMT, a member of the so-called group of CCoAOMTs-like (Hugueney et al., 2009; Lücker et al., 2010), we decided to investigate deeper in the function of others OMTs found in the genome of Vitis vinifera. In order to characterize other OMTs involved in phenilpropanoid pathway, we focused our attention on two genes codifying for enzymes that are members of the so-called group of CCoAOMTs-true. To investigate the possible function of these genes, we decide to analyze their expression in different tissues and organs. The specificity of expression has shown us the involvement of these genes in the lignin pathway as described in the article of Busam. In order to evaluate their involvement in the response of biotic and abiotic stress, we performed qPCR in leaves and berries treated with Botrytis cinerea Pers. and subjected to water stress. In vitro enzymatic assays are used in order to characterize the VvCCoAOMT1 which have shown a methyltransferase activity both towards cyanidine-3-O-glucoside and caffeoyl-CoA and have confirmed the localization in the cytoplasm of this class of enzymes. VvCCoAOMT1 has a classic Michaelis-Menten kinetic, and Km values show higher affinity towards caffeoyl-CoA than cyanidine-3-O-glucoside. Also the turnover number (kcat) is higher when caffeoyl-CoA is the reaction substrate, and so this means that when VvCCoAOMT1 is saturated it transforms more molecules of caffeoyl-CoA in feruloyl-CoA than cyanidine-3-O-glucoside in peonydin-3-O-glucoside. The results show that this enzyme could play a role not only in lignin biosynthesis but also in secondary metabolism, for example methylating molecules like anthocyanins. Over the years researchers made several study about OMTs, and this work join to this group. Other experiments, for example the use of iRNA or further enzymatic assays, will be useful to confirm the role of CCoAOMTs in grape.