Methoxypyrazines (MPs) are volatile, grape-derived aroma compounds that contribute to the distinct herbaceous characters of some wines. Although the full pathway leading to MP production has not been elucidated, there is strong evidence that the final step involves the methylation of non-volatile hydroxypyrazine (HP) precursors. Two cDNA encoding O-methyltransferases (OMTs) that have homology to an enzyme previously purified and shown to catalyse the methylation of HPs were isolated from Cabernet Sauvignon. Recombinant protein from the cDNAs (VvOMT1 and VvOMT2) was produced in E. coli and activity assays demonstrated that both encode OMTs able to methylate HPs to produce MPs, however both showed greatest activity against the flavonol quercetin. VvOMT1 has higher catalytic activity against isobutyl hydroxypyrazine compared to isopropyl hydroxypyrazine, whereas the converse is true for VvOMT2. The timing of the expression of VvOMT1 in the skin and the flesh of developing Cabernet Sauvignon grape berries was associated with the period of MP accumulation in these tissues, while VvOMT2 expression was greatest in roots, which were found to contain high levels of MPs. The MP composition of these tissues also reflects the relative levels of expression of these genes and their substrate preference. The identification of genes responsible for MP production in grapevine will help in understanding the effect of different viticultural and environmental factors on MP accumulation.

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SUMMARYMethoxypyrazines are a family of potent volatile compounds of diverse biological significance. They are used by insects and plants in chemical defence, are present in many vegetables and fruit and, in particular, impart herbaceous/green/vegetal sensory attributes to wines of certain varieties, including Cabernet Sauvignon. While pathways for methoxypyrazine biosynthesis have been postulated, none of the steps have been confirmed genetically. We have used the F 2 progeny of a cross between a rapid flowering grapevine dwarf mutant, which does not produce 3-isobutyl-2-methoxypyrazine (IBMP), and Cabernet Sauvignon to identify the major locus responsible for accumulation of IBMP in unripe grape berries. Two candidate methyltransferase genes within the locus were identified and one was significantly associated with berry IBMP levels using association mapping. The enzyme encoded by this gene (VvOMT3) has high affinity for hydroxypyrazine precursors of methoxypyrazines. The gene is not expressed in the fruit of Pinot varieties, which lack IBMP, but is expressed in Cabernet Sauvignon at the time of accumulation of IBMP in the fruit. The results suggest that VvOMT3 is responsible for the final step in methoxypyrazine synthesis in grape berries and is the major determinant of IBMP production.