Two O-methyltransferases involved in the biosynthesis of methoxypyrazines: grape-derived aroma compounds important to wine flavour Jake D. Dunlevy • Kathleen L. Soole • Michael V. Perkins • Eric G. Dennis • Robert A. Keyzers • Curtis M. Kalua • Paul K. Boss Received: 7 October 2009 / Accepted: 29 May 2010 / Published online: 23 June 2010 Ó Springer Science+Business Media B.V. 2010 Abstract 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) precur- sors. 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 associ- ated 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 com- position 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 dif- ferent viticultural and environmental factors on MP accumulation. Keywords Vitis vinifera Á O-Methyltransferase Á Methoxypyrazines Á Wine flavour Introduction The sensorial impact a consumer experiences when drinking wine is influenced by the complex mixture of volatile flavour and aroma compounds present. The volatile composition of wine is in turn determined by many vari- ables. These include viticultural parameters such as the grape variety, environmental conditions and vineyard management practices during grape production, as well as oenological variables such as yeast strain, fermentation conditions and use of oak barrels during the winemaking process. Grape variety has a major effect on wine flavour because of the ability of different varieties to synthesise a range of different aroma compounds, and the sensory attributes that are used to distinguish wines made from different varieties of grapes are known as varietal characters. One such example of a varietal character is the ‘‘vegetative’’ sensory attribute most commonly associated with Sauvignon Blanc and Cabernet Sauvignon wines (Allen et al. 1991). This vegetative character is due to the presence of the highly potent aroma compounds 3-isobutyl-2-methoxypyrazine Electronic supplementary material The online version of this article (doi:10.1007/s11103-010-9655-y) contains supplementary material, which is available to authorized users. J. D. Dunlevy Á K. L. Soole School of Biological Science, Flinders University of South Australia, GPO Box 2100, Adelaide, SA 5001, Australia M. V. Perkins Á E. G. Dennis School of Chemistry, Physics and Earth Sciences, Flinders University of South Australia, GPO Box 2100, Adelaide, SA 5001, Australia R. A. Keyzers Á C. M. Kalua Á P. K. Boss (&) CSIRO Plant Industry and Food Futures Flagship, PO Box 350, Glen Osmond, SA 5064, Australia e-mail: paul.boss@csiro.au 123 Plant Mol Biol (2010) 74:77–89 DOI 10.1007/s11103-010-9655-y