Browsing by Author "Bezuidenhout, Ilse-Marie"

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    Functional analysis of candidate terpenoid biosynthetic genes isolated from grapevine
    (Stellenbosch : Stellenbosch University, 2021-03) Bezuidenhout, Ilse-Marie; Young, Philip R.; Vivier, Melane A.; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Terpenoids are a group of compounds found in various organisms, with diverse functions, and can be broadly grouped into primary or specialised (secondary) metabolites. This compound diversity is achieved from the universal precursors for terpenoids, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), through the activity of various enzymes. Isoprenyl diphosphate synthases (IDSs) incorporate the IPP and DMAPP precursors into compounds of varying lengths that can then serve as the substrate for terpene synthases (TPSs) to produce terpenes. Each of these steps incorporates the opportunity to further increase terpene diversity. In addition, terpenes can be modified into other products through the activity of various enzymes to increase the product range even further. Terpenoids play an important role in Vitis vinifera (grapevine), not only in the normal functioning of the plant, but also in more specialised roles involving plant–environment interactions. In terms of their economic value, terpenes, such as mono- and sesquiterpenes, contribute to wine aroma – which has supported the study of these compounds and how these metabolites are biosynthesised. Grapevine genome annotation has shown a moderately sized IDS gene family and a TPS gene family that has undergone extensive expansion. However, many of these genes have not been functionally characterised. Putative annotation based on sequences is not always accurate or does not provide a realistic indication of gene function. Functional characterisation of IDS and TPS genes involves heterologous expression followed by analysis of the produced compounds. In this study, various heterologous systems were used and evaluated with the aim of characterising grapevine genes that putatively encode IDS and TPS enzymes. In this study, the novel annotation of a eucalyptol synthase and the re-annotation of a previously characterised gene to α-thujene synthase was achieved. Various expression systems were assessed, with Saccharomyces cerevisiae deemed a viable alternative to the typical use of Escherichia coli. Different plant systems were also investigated, with Nicotiana benthamiana found to be a good option in terms of ease of use and results obtained. An in-depth study of geranylgeranyl diphosphate synthase (VviGGPS) was also conducted using an established transgenic tobacco population. The population was characterised in terms of morphology, physiology, and metabolites. The possible role of gibberellin was also determined based on the results of these analyses.