Identification of O-methyltransferase genes involved in the biosynthesis of 3-alkyl-2-methoxypyrazines in grapevines (Vitis vinifera L.)

Author: Jake Dunlevy

Dunlevy, Jake, 2012 Identification of O-methyltransferase genes involved in the biosynthesis of 3-alkyl-2-methoxypyrazines in grapevines (Vitis vinifera L.), Flinders University, School of Biological Sciences

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3-alkyl-2-methoxypyrazines (MPs) are a family of potent volatile aroma compounds commonly found in various vegetables. MPs, in particular 3-isobutyl-2-methoxypyrazine (IBMP), can also be found in the grapes and subsequent wines of a small number of grapevine cultivars, including Cabernet Sauvignon and Sauvignon blanc. MPs have odour perceptions commonly described as herbaceous and green capsicum-like, which when present in wines at high concentrations are considered detrimental to wine quality. Controlling the level of MPs in wines is best achieved by manipulating the amount that accumulates in the berry, which is highly variable and influenced by numerous environmental and viticultural factors. Little is known about the biosynthesis of MPs, however a previous study has shown that in grape berries an O-methyltransferase (OMT) enzyme catalyses the final step in MP biosynthesis via the methylation of 3-alkyl-2-hydroxypyrazine (HP) precursors (Hashizume et al., 2001a). Furthermore a protein with this activity has previously been purified from the shoots of Cabernet Sauvignon and its N-terminus partially sequenced (Hashizume et al., 2001b). The aim of this project was to identify the gene encoding the OMT responsible for the final step of MP biosynthesis in grape berries. Using a candidate gene approach the gene VvOMT1 was identified showing an exact homology to the 22 amino acid sequence of the native protein purified Hashizume et al (2001b) and a second gene VvOMT2 found with close homology to VvOMT1. Kinetic analysis of recombinant VvOMT1 and VvOMT2 revealed that they each possessed methylating activity against HP substrates similar to that reported by Hashizume et al (2001b), and the expression of VvOMT1 was found to coincide with the period of MP biosynthesis in early grape development. However, RNAi mediated silencing of both VvOMT1 and VvOMT2 in grapevine hairy-roots did not result in a reduction of MPs. A mapping approach was also used to identify genes responsible for IBMP accumulation. A population of F2 progeny derived from a cross between Cabernet Sauvignon (CS) and a Pinot Meunier dwarf mutant (PM dwarf) was established and found to segregate for the trait of IBMP accumulation in young berries. Using the online genome sequence as a basis, CAPs and dCAPS markers were designed to the genomes of CS and PM, which enabled the identification of a 2.3 Mb locus that segregates with the trait of IBMP accumulation. A search of the online annotated genomic database revealed two putative OMTs, VvOMT3 and VvOMT4, located within this locus. An association mapping study directed to this locus revealed that the gene VvOMT3 is highly associated (p = 0.005) with the trait of IBMP accumulation in 91 existing grapevine cultivars. Furthermore recombinant VvOMT3 was found to have between 500 - 5,000 fold greater catalytic activity against IBHP than other VvOMTs investigated. The expression of VvOMT3 also coincided the period of MP accumulation in young grape berries and was associated with IBMP accumulation in a subset of the segregating F2 progeny. Finally the elimination of sunlight from grape bunches was found to significantly reduce the expression of VvOMT3, resulting in a reduction of IBMP levels.

Keywords: methoxypyrazine,methyltransferase,grape,aroma
Subject: Biological Sciences thesis

Thesis type: Doctor of Philosophy
Completed: 2012
School: School of Biological Sciences
Supervisor: Assoc Prof Kathleen Soole