A novel cation-dependent O-methyltransferase involved in anthocyanin methylation in grapevine.
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.
Hugueney
Philippe
P
Université de Strasbourg, UMR1131, F-67000 Strasbourg, France.
Provenzano
Sofia
S
Verriès
Clotilde
C
Ferrandino
Alessandra
A
Meudec
Emmanuelle
E
Batelli
Giorgia
G
Merdinoglu
Didier
D
Cheynier
Véronique
V
Schubert
Andrea
A
Ageorges
Agnès
A
D000595:Amino Acid Sequence; D000872:Anthocyanins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D002412:Cations / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000302:isolation & purification; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D005960:Glucosides / Q000494:pharmacology; D007700:Kinetics; D008745:Methylation / Q000187:drug effects; D008969:Molecular Sequence Data; D000073718:Pancreatitis-Associated Proteins; D011497:Protein O-Methyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology; D013379:Substrate Specificity / Q000187:drug effects; D027843:Vitis / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D000872:Anthocyanins; D002412:Cations; D018076:DNA, Complementary; D005960:Glucosides; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D011993:Recombinant Fusion Proteins; C462279:cyanidin-3-O-beta-glucopyranoside; C494120:delphinidin 3-O-glucopyranoside; D011497:Protein O-Methyltransferase