A Novel Cation-DependentO-Methyltransferase Involved in Anthocyanin Methylation in Grapevine      

Abstract

(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.

Fruit skin color and the role of anthocyanin

“…There is no clear study reported on anthocyanin biosynthesis mechanism in fruit crops apart from that in apple and grape (Matus et al 2008), probably due to some genes involved in anthocyanin biosynthesis having pleiotropy and being affected by many internal and external factors. Despite this drawback, many regulatory genes have been newly identified in grape, apple and other fruit crops (Hugueney et al 2009;Cultrone et al 2010;Feng et al 2010). In this review, we describe different aspects of the gene involved in the regulation of anthocyanins and their functions in plants.…”

Flower color modification in <i>Rosa hybrida</i> by expressing the <i>S</i>-adenosylmethionine: anthocyanin 3′,5′-<i>O</i>-methyltransferase gene from <i>Torenia hybrida</i>

“…Recently, it was shown that the A3′OMT gene is encoded by MT2, the A3′5′OMT genes are encoded by MF1 and MF2, and MT1 is nonexistent (Provenzano et al 2014). A grape A3′5′OMT gene was isolated and the recombinant grape A3′5′OMT in in vitro assays was shown to catalyze the 3′-or 3′,5′-methylation of cyanidin 3-glucoside, delphinidin 3-glucoside, quercetin 3-glucoside, cyanidin, quercetin, and myricetin; however, pelargonidin 3-glucoside, catechin, and epicatechin were not suitable substrates (Hugueney et al 2009;Lucker et al 2010). Tobacco leaves that transiently expressed the genes for grape A3′5′OMT and an Arabidopsis transcriptional factor that upregulates anthocyanin biosynthesis were found to accumulate malvidin 3-rutinoside (Hugueney et al 2009).…”

Genetic Control and Evolution of Anthocyanin Methylation

“…Complementation experiments with mt2 mf1 mf2 mutants in distinct genetic backgrounds revealed remarkable differences in the substrate specificities of these Petunia spp. AMTs and a putative anthocyanin O-methyltransferase from grape (VvAOMT1; Hugueney et al, 2009). Our data provide insight into the molecular genetic mechanisms underlying the evolution of distinct anthocyanin structures.…”

“…The DIFe proteins belong to the A1 subfamily (Supplemental Fig. S1; Supplemental Table S2), which contains caffeoylCoenzyme A O-methyltransferases (CCoA-MTs) and an AMT from grape known as VvAOMT1 (Hugueney et al, 2009), also known as flavonol and anthocyanin O-methyltransferase (Lücker et al, 2010). Other (putative) flavonoid methyltransferases, such as the Arabidopsis flavanol 39methylase AtOMT1 (Saito et al, 2013), and enzymes with similar activities from Catharanthus roseus and Mentha 3 piperita, belong to the B1 and B2 groups (Lam et al, 2007;Supplemental Fig.…”

“…This suggested that MT2 and MF1 methylated only a small fraction of the cyanidin 3-rutinosides in the R78 background because they have a too low affinity/efficiency for these substrates. To test this hypothesis, we expressed in R78 the homologous grape protein VvAOMT1, which can efficiently methylate anthocyanin 3-glucosides and 3-rutinosides in vitro with a K m in the approximately 7 to 40 mM range, depending on the conditions used (Hugueney et al, 2009;Lücker et al, 2010). Four of the seven 35S:VvAOMT1 R78 transformants expressed the transgene at roughly similar levels as 35S:MT2 and 35S:MF2 (Fig.…”