A novel cation-dependent O-methyltransferase involved in anthocyanin methylation in grapevine

Abstract

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.

Figure 1.

Comparison of grapevine AOMT with other OMTs. A, The predicted amino acid sequence of AOMT from grapevine (VvAOMT) was aligned with the amino acid sequences of PFOMT from M. crystallinum (McPFOMT) and caffeoyl-CoA-OMT from grapevine (VvCCoAOMT) using ClustalW. Residues shaded in gray indicate identical amino acids. B, Phylogenetic tree of selected OMT cDNA sequences. Type 1 OMTs include caffeic acid-OMT from grapevine (VvCOMT; accession no. AF239740) and Nicotiana tabacum (NtCOMT; AF484252), tricetin-OMT from Triticum aestivum (TaOMT2; DQ223971), flavonoid-OMTs from Catharanthus roseus (CrOMT2; AY127568) and Chrysosplenium americanum (CaOMT2; U16793), and flavonol-OMTs from Arabidopsis (AtFolOMT; U70424) and Oryza sativa (OsFolOMT1;DQ288259). Type 2 OMTs (in boldface) include caffeoyl-CoA-OMT from grapevine (VvCCoAOMT; Z54233), N. tabacum (NtCCoAOMT; U38612), and Populus trichocarpa (PtCCoAOMT; AJ224896), flavonol-OMT from O. sativa (OsFolOMT2; XM_483167), AOMT from grapevine (VvAOMT; FJ460168), PFOMT from M. crystallinum (McPFOMT; AY145521), and flavonoid-OMT from Arabidopsis (AtOMT1; AY087244). The numbers beside the branches represent bootstrap values based on 500 replicates.

Figure 2.

A and B, Accumulation of anthocyanins during Syrah (A) and Nebbiolo (B) berry development. The arrow indicates véraison, the onset of ripening. The content of each anthocyanin is expressed as equivalent (equi.) of malvidin-3-O-glucoside as pure standard. C and D, Quantitative real-time PCR expression profiling of AOMT during Syrah (C) and Nebbiolo (D) berry development. Expression values have been normalized with VvEF1α. Cy, Cyanidin; Dp, delphinidin; Mv, malvidin; Pn, peonidin; Pt, petunidin.

Figure 3.

Quantitative real-time PCR expression profiling of AOMT in various Syrah grapevine organs and in different tissues of berry at three stages of development. Expression values have been normalized with VvEF1α and expressed as relative abundance.

Figure 4.

Chemical structures of some flavonoid compounds. Listed in boldface are flavonoids assayed as substrates for AOMT. glc, Glucoside.

Figure 5.

Analysis of AOMT in vitro reaction products. HPLC/ESI-MS/DAD analysis of reaction products produced following incubation of potential substrates without (A, C, E, G, and I) or with (B, D, F, H, and J) recombinant AOMT enzyme. Reactions were carried out for 60 min in a total volume of 200 μL, with 200 μm anthocyanin or flavonol substrate, 200 μm SAM, and 5 μg of purified AOMT. Substrates were as follows: A and B, pelargonidin 3-glucoside; C and D, cyanidin 3-glucoside; E and F, delphinidin 3-glucoside; G and H, cyanidin; I and J, quercetin 3-glucoside. Anthocyanins were monitored at 520 nm and flavonols at 360 nm. Reaction products were identified according to their mass fragmentation, UV/visible absorption spectra, and retention times. The data shown are representative of three independent experiments. Pg3G, Pelargonidin 3-glucoside; Cy3G, cyanidin 3-glucoside; Pn3G, peonidin 3-glucoside; Dp3G, delphinidin 3-glucoside; Pt3G, petunidin 3-glucoside; Mv3G, malvidin 3-glucoside; Cy, cyanidin; Pn, peonidin; Q3G, quercetin 3-glucoside; Q, quercetin; IsoR3G, isorhamnetin 3-glucoside; IsoRh, isorhamnetin; P1, unknown product.

Figure 6.

Characterization of AOMT activity in planta using Agrobacterium-mediated transient transformation. Tobacco leaf sectors (200 mg fresh weight) expressing GFP (A and D) or PAP1 (B and E) or coexpressing PAP1 and AOMT (C and F) were excised 96 h after Agrobacterium-mediated transformation. Anthocyanin and flavonol contents were analyzed using HPLC-DAD. Anthocyanins were monitored at 520 nm and flavonols at 360 nm. The data shown are representative of three independent experiments. Dp3R, Delphinidin 3-O-rutinoside; Pt3R, petunidin 3-O-rutinoside; Mv3G, malvidin 3-O-glucoside; Mv3R, malvidin 3-O-rutinoside; QRH, quercetin 3-O-rutinoside O-hexoside; peak a1, delphinidin 3-O-rutinoside O-glucoside; peak a2, delphinidin 3-O-rutinoside O-hexoside; peak a3, malvidin 3-O-rutinoside O-hexoside; peaks c1, c2, and c3, isomers of caffeoyl quinic acid; peaks f1, f2, and f3, isomers of feruloyl quinic acid. MS/MS spectra of Dp3R, Mv3R, and Mv3G are shown in the middle. ABS, Absorbance; AU, absorbance unit.

Figure 7.

Subcellular localization of the AOMT-GFP fusion protein. Transient expression of nontargeted GFP (A) and the AOMT-GFP fusion protein (B) following agroinfiltration of tobacco leaves. Abaxial epidermal cell images are projections of 20- × 1-μm optical sections collected by confocal laser scanning microscopy. Bars = 20 μm. [See online article for color version of this figure.]