(C.V., E.M., V.C., A.A.)Anthocyanins are major pigments in colored grape (Vitis vinifera) berries, and most of them are monomethoxylated or dimethoxylated. We report here the functional characterization of an anthocyanin O-methyltransferase (AOMT) from grapevine. The expression pattern in two cultivars with different anthocyanin methylation profiles (Syrah and Nebbiolo) showed a peak at start ripening (véraison), when the concentrations of all methylated anthocyanins begin to increase. The purified recombinant AOMT protein was active on both anthocyanins and flavonols in vitro, with K m in the micromolar range, and was dependent on divalent cations for activity. AOMT showed a preference for 3#,5# methylation when a 3#,4#,5# hydroxylated anthocyanin substrate was tested. In order to assess its in planta activity, we performed transient expression of AOMT in tobacco (Nicotiana benthamiana) leaves expressing the Production of Anthocyanin Pigment1 (PAP1) transcription factor from Arabidopsis (Arabidopsis thaliana). PAP1 expression in leaves induced the accumulation of the nonmethylated anthocyanin delphinidin 3-rutinoside. The coexpression of PAP1 and AOMT resulted in an accumulation of malvidin 3-rutinoside. We also showed that AOMT localized exclusively in the cytoplasm of tobacco leaf cells. These results demonstrate the ability of this enzyme to methylate anthocyanins both in vitro and in vivo, indicating that AOMT plays a major role in anthocyanin biosynthesis in grape berries.

Anthocyanins are a chemically diverse class of secondary metabolites that color most flowers and fruits. They consist of three aromatic rings that can be substituted with hydroxyl, sugar, acyl, and methyl groups in a variety of patterns depending on the plant species. To understand how such chemical diversity evolved, we isolated and characterized METHYLATION AT THREE2 (MT2) and the two METHYLATION AT FIVE (MF) loci from Petunia spp., which direct anthocyanin methylation in petals. The proteins encoded by MT2 and the duplicated MF1 and MF2 genes and a putative grape (Vitis vinifera) homolog Anthocyanin O-Methyltransferase1 (VvAOMT1) are highly similar to and apparently evolved from caffeoyl-Coenzyme A O-methyltransferases by relatively small alterations in the active site. Transgenic experiments showed that the Petunia spp. and grape enzymes have remarkably different substrate specificities, which explains part of the structural anthocyanin diversity in both species. Most strikingly, VvAOMT1 expression resulted in the accumulation of novel anthocyanins that are normally not found in Petunia spp., revealing how alterations in the last reaction can reshuffle the pathway and affect (normally) preceding decoration steps in an unanticipated way. Our data show how variations in gene expression patterns, loss-of-function mutations, and alterations in substrate specificities all contributed to the anthocyanins' structural diversity.

SummaryPetunia mutants (Petunia hybrida) with blue flowers defined a novel vacuolar proton pump consisting of two interacting P-ATPases, PH1 and PH5, that hyper-acidify the vacuoles of petal cells. PH5 is similar to plasma membrane H + P 3A -ATPase, whereas PH1 is the only known eukaryoticP 3B -ATPase. As there were no indications that this tonoplast pump is widespread in plants, we investigated the distribution and evolution of PH1 and PH5. We combined database mining and phylogenetic and synteny analyses of PH1-and PH5-like proteins from all kingdoms with functional analyses (mutant complementation and intracellular localization) of homologs from diverse angiosperms.We identified functional PH1 and PH5 homologs in divergent angiosperms. PH5 homologs evolved from plasma membrane P 3A -ATPases, acquiring an N-terminal tonoplast-sorting sequence and new cellular function before angiosperms appeared. PH1 is widespread among seed plants and related proteins are found in some groups of bacteria and fungi and in one moss, but is absent in most algae, suggesting that its evolution involved several cases of gene loss and possibly horizontal transfer events.The distribution of PH1 and PH5 in the plant kingdom suggests that vacuolar acidification by P-ATPases appeared in gymnosperms before flowers. This implies that, next to flower color determination, vacuolar hyper-acidification is required for yet unknown processes.