1. Identification of O-methyltransferase genes involved in the biosynthesis of 3- alkyl-2-methoxypyrazines in grapevines (Vitis vinifera L.) by Jake Dunlevy B. Biotechnology (Hons.), Flinders University A thesis submitted for the degree of Doctor of Philosophy at The Flinders University of South Australia Faculty of Science and Engineering School of Biological Sciences in collaboration with CSIRO Plant Industry Urrbrae, South Australia August, 2011 2. Table of Contents Table of Contents ........................................................................................................................................... ii Abstract ............................................................................................................................................................ v Declaration.....................................................................................................................................................vii Acknowledgements .................................................................................................................................... viii Abbreviations .................................................................................................................................................. x Chapter 1 - General introduction 1.1 Grapes and Wine .......................................................................................................................... 1 1.2 Wine flavour and aroma .............................................................................................................. 2 1.3 Methoxypyrazines ........................................................................................................................ 4 1.3.1 Identification and properties of methoxypyrazines....................................................... 4 1.3.2 Biological function of methoxypyrazines....................................................................... 5 1.3.3 Methoxypyrazines in grapes and wine ............................................................................ 7 1.3.4 Accumulation of methoxypyrazines in grapes............................................................. 12 1.3.5 Ladybird beetle as a source of MP in wine .................................................................. 21 1.3.6 Pathway of methoxypyrazine biosynthesis .................................................................. 22 1.3.7 Grapes contain a methyltransferase enzyme that methylates HPs ........................... 23 1.4 Project aims ................................................................................................................................. 27 Chapter 2 - Characterisation of two O-methyltransferases potentially involved in the final step of MP biosynthesis in berries 2.1 Introduction ................................................................................................................................. 29 2.1.1 O-methyltransferases are common to plant secondary metabolism ......................... 30 2.1.2 Plant OMTs involved in production or aroma compounds........................................ 33 2.1.3 OMTs characterized in grapevines ................................................................................ 34 2.1.4 Grapevine contains an OMT with activity against HPs ............................................. 35 2.1.5 Aims of the work presented in this chapter .................................................................. 36 2.2 Materials and Methods .............................................................................................................. 37 2.2.1 Chemicals, solutions and media ..................................................................................... 37 2.2.2 Plant material ..................................................................................................................... 39 2.2.3 RNA extraction and cDNA synthesis............................................................................ 39 2.2.4 Isolation of full length VvOMT cDNA clones ............................................................ 41 2.2.5 Phlyogenetic analysis of VvOMT encoded proteins .................................................. 41 2.2.6 Recombinant protein production .................................................................................... 41 2.2.7 SDS-PAGE and western blot analysis .......................................................................... 43 2.2.8 OMT enzyme assays ........................................................................................................ 45 2.2.9 SPME-GC-MS quantification of MPs........................................................................... 46 2.2.10 Real-time PCR analysis ................................................................................................... 48 2.2.11 Construction of VvOMT targeted hairpin constructs ................................................. 49 2.2.12 RNAi mediated silencing of VvOMT genes in grapevine hairy-roots .................... 50 2.3 Results .......................................................................................................................................... 51 2.3.1 Identification of two OMT genes encoding proteins with similarity to an enzyme with HP methylating activity .......................................................................................... 51 2.3.2 Recombinant VvOMT1 and VvOMT2 methylate HPs .............................................. 56 2.3.3 Recombinant VvOMT1 and VvOMT2 can methylate many substrates.................. 61 2.3.4 Expression of VvOMT1 coincides with MP levels in berry skin and flesh............ 62 2.3.5 RNAi silencing of VvOMTs in grapevine hair-roots ................................................. 66 2.4 Discussion.................................................................................................................................... 71 Chapter 3 - Towards mapping the gene responsible for methoxypyrazine accumulation in Cabernet Sauvignon berries 3.1 Introduction ................................................................................................................................. 76 3.1.1 Genetic diversity of cultivated grapevines ................................................................... 76 3.1.2 Genetic mapping ............................................................................................................... 77 3.1.3 Genetic mapping in grapevine ........................................................................................ 79 3.1.4 The Pinot Meunier dwarf provides a tool for rapid grapevine genetics .................. 82 3.1.5 Aims .................................................................................................................................... 84 ii 3.2 Materials and Methods .............................................................................................................. 84 3.2.1 Chemicals and solution .................................................................................................... 84 3.2.2 Plant material ..................................................................................................................... 85 3.2.3 Generation of an F2 population from a cross between CS and the PM dwarf ....... 85 3.2.4 Methoxypyrazine quantification .................................................................................... 86 3.2.5 DNA extraction and purification .................................................................................... 86 3.2.6 SNP identification and design of CAPS and dCAPS markers .................................. 87 3.2.7 Mapping using the CAPS and dCAPS markers........................................................... 87 3.3 Results .......................................................................................................................................... 88 3.3.1 Phenotypic analysis of the progeny from a CS × PM dwarf cross ........................... 88 3.3.2 Phenotypic analysis of the CS × PM dwarf F2 population........................................ 91 3.3.3 Association of IBMP accumulation with genetic markers in the PM dwarf × CS F2 population ..................................................................................................................... 93 3.3.4 IBMP-accumulation in the berries of different grapevine cultivars ......................... 94 3.3.5 Association of the IBMP-accumulating phenotype with genetic markers in the cultivar collection ............................................................................................................. 97 3.4 Discussion..................................................................................................................................101 3.4.1 Linkage mapping of the IBMP-accumulation trait in the CS × PM dwarf F2 progeny .............................................................................................................................101 3.4.2 Association mapping of the IBMP-accumulation trait in the cultivar collection 105 Chapter 4 - Characterisation of two novel O-methyltransferases found in the locus linked to methoxypyrazine accumulation in grape berries 4.1 Introduction ...............................................................................................................................109 4.2 Materials and Methods ............................................................................................................110