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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

A Novel Cation-Dependent O-Methyltransferase Involved in Anthocyanin Methylation in Grapevine^{1,[C],[W],[OA]}

Université de Strasbourg, UMR1131, F–67000 Strasbourg, France (P.H., D.M.); INRA, UMR1131, F–68000 Colmar, France (P.H., D.M.); Dipartimento Colture Arboree, Università di Torino, 10095 Grugliasco, Italy (S.P., A.F., G.B., A.S.); and INRA, UMR Sciences pour l'nologie, F–34060 Montpellier, France (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.

² These authors contributed equally to the article.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Agnès Ageorges (ageorges{at}supagro.inra.fr).

^[C] Some figures in this article are displayed in color online but in black and white in the print edition.

^[W] The online version of this article contains Web-only data.

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www.plantphysiol.org/cgi/doi/10.1104/pp.109.140376

^* Corresponding author; e-mail ageorges{at}supagro.inra.fr.

Received April 24, 2009; accepted June 10, 2009; published June 12, 2009.