Final Report Summary - GRAPENOIDS (Functional metabolites: Investigation of terpenoid biosynthesis in grapes.) The “Grapenoids” project consisted of an outgoing phase to the Wine Innovation Cluster of the University of Adelaide, in collaboration with the Australia Wine Research Institute (AWRI), and a return phase to the Department of Plant and Environmental Sciences at the University of Copenhagen. The project combined molecular expertise at the University of Copenhagen with strengths in grape and wine research at the University of Adelaide. The overall objectives of the Grapenoids project included the development of a comprehensive description of genes that are likely to be involved in the biosynthesis of volatile aroma compounds in grape berries and other plants, and the biochemical characterization of these enzymes in model systems. As an example of a compound of interest, rotundone is a recently discovered biochemical that is responsible for the distinct pepper aroma of some wine varieties, and has a profound impact on the characteristics of wine at extremely low concentrations. We hypothesized that we could identify the genes responsible for the production of rotundone, and of similar related compounds, allowing us to link grapevine genetics with wine character. During the Outgoing Phase of the project, a comprehensive analysis of gene activity during grape berry development was carried out in order to identify target genes; that is, genes that became more active during periods when aroma compounds were produced. To do this, grape berries from the variety Shiraz were monitored throughout a growing season, and samples were collected at 7-10 day intervals for RNA extraction. These samples formed the basis for the analysis of transcriptional gene activity, as well as the analysis of grape chemical content. To further aid our selection of genes for further analysis, we developed a gene co-expression database for grapevine. This database enabled us to determine genes that are commonly activated at the same time or in the same tissue, thus suggesting that co-expressed genes may be involved in the same pathways. For example, if three specific genes are required for the production of a particular compound, it is like that these three genes will be highly correlated with each other. The results of our transcriptional and co-expression analysis were published in the journal, BMC Genomics, in 2012 and 2013, respectively. The data in these publications will continued to provide an invaluable resource for grape and wine researchers in the future. The data provided by our transcriptional and gene co-expression analyses provided a basis for the selection of gene for further study. In total, 17 full-length genes were chosen for further study and isolated from Shiraz berries. These genes were transferred in to yeast, either individually or in combination, and new products synthesized by the yeast cells were detected and analysed using gas chromatography and mass spectrometry. This was based on the principle that normal yeast cells produce and narrow range of volatile compounds, and therefore any novel compounds produced by yeast cells expressing a grape gene can be linked to the function of that gene. In total, approximately 40 different volatile compounds were identified as products of enzymes coded by these grape-specific genes. Of particular interest, we identified a gene that is highly active in ripening grape berries that codes and enzyme which produces a compounds called guaiene when expressed in yeast. This gene is of interest because guaiene is the probable precursor of the previously described peppery smelling compound, rotundone. We also focused on combinatorial biosynthesis whereby multiple genes were expressed at the same time in the same yeast cells. This also yielded several novel compounds that are currently undergoing further analysis. Some genes may have differing activity when expressed in yeast than they do in their native organism. We therefore also investigated the function of several of our grapevine genes in a model plant system called Nicotiana bethamiana, a fast-growing relative of tobacco. While confirming the products seen in yeast in many cases, we also found that there appear to be plant enzymes present in tobacco that further modify the compounds created by our genes. This is of interest because it is possible that this also happens in grapes, and that the chemical compounds produced in grapes by our genes of interest are also modified in the same way. Data obtained from both the yeast and plant expression systems currently forms the basis for a manuscript under preparation that details a comprehensive list of aroma compounds or aroma precursors produced by these berry-specific enzymes.Results stemming from this project have the potential to uncover novel aroma compounds and describe their effect on the character of wine. This could have implications on viticultural practices that might be found to affect gene expression and subsequent metabolite production in grape berries immediately prior to harvest. Taking a wider perspective, the identification of novel aroma compounds could have implications for flavor and aroma based industries, including perfume companies, and could results in commercial quantities of previously unidentified metabolites.
