Browsing by Author "Smit, Samuel Jacobus"

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    Comparative (within species) genomics of the vitis vinifera L. terpene synthase family to explore the impact of genotypic variation using phased diploid genomes
    (Frontiers Media, 2020) Smit, Samuel Jacobus; Vivier, Melane Alethea; Young, Philip Richard
    The Vitis vinifera L. terpene synthase (VviTPS) family was comprehensively annotated on the phased diploid genomes of three closely related cultivars: Cabernet Sauvignon, Carménère and Chardonnay. VviTPS gene regions were grouped to chromosomes, with the haplotig assemblies used to identify allelic variants. Functional predictions of the VviTPS subfamilies were performed using enzyme active site phylogenies resulting in the putative identification of the initial substrate and cyclization mechanism of VviTPS enzymes. Subsequent groupings into conserved catalytic mechanisms was coupled with an analysis of cultivar-specific gene duplications, resulting in the identification of conserved and unique VviTPS clusters. These findings are presented as a collection of interactive networks where any VviTPS of interest can be queried through BLAST, allowing for a rapid identification of VviTPS-subfamily, enzyme mechanism and degree of connectivity (i.e., extent of duplication). The comparative genomic analyses presented expands our understanding of the VviTPS family and provides numerous new gene models from three diploid genomes.
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    Grapevine terpenoids and their contribution to the flower volatilome
    (Stellenbosch : Stellenbosch University, 2016-03) Smit, Samuel Jacobus; Young, Philip R.; Vivier, Melane A.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Terpenoids represent the largest group of plant natural products and show tremendous diversity in chemical structure and bioactivity. This diversity arises from the action of terpene synthases (TPS), the key enzymes that accept prenylated diposhpate precursors as substrates and convert them to mono-, sesqui- and diterpenoid hydrocarbons. The promiscuity of TPSs results in a multitude of products that readily undergo further enzymatic and non-enzymatic modifications/rearrangements. The grapevine (Vitis vinifera) genome contains an overrepresented TPS-encoding gene family with extensive gene duplications. Domestication of grapevine and selection of diverse cultivars resulted in plants with varying capacities to produce terpenoids, as is evident in wines from so-called aromatic varieties. Grapevine TPSs (VviTPS) are, therefore, largely studied for their role in modulating the flavour and aroma profiles of wines from aromatic (for example, Gewürztraminer and Muscat) or spicy (Shiraz) cultivars. These aromas are largely contributed by monoterpenes that impart floral aromas, while the sesquiterpene lactone rotundone has been linked to a spicy/peppery aroma. These results suggest that cultivar differences, in terms of TPS metabolism, are present, yet limited knowledge on the biological in planta function of grapevine terpenoids exists. In other plant species the terpenoids show important ecological functions in specialised metabolic processes that contribute to plant fitness and/or adaptability, for example, in mediating defence/stress responses or as important infochemcials for attraction. Grapevine flowers show promise for exploring these in planta roles due to increased transcriptional activity and volatile emissions in some cultivars. In silico characterisation of the grapevine TPS gene family revealed that VviTPS expression was upregulated in flower developmental stages. In general, it was found that sesqui-TPS expressed at inflorescence development while mono-TPS expressed during flowering. The complexity of grapevine terpenoid metabolism was explored through co-expression analysis. Genes identified through expression networks revealed candidate genes encoding for enzymes that potentially modify terpenoids. These enzymes included cytochrome P450s, glycosidases and glycosyltransferases, all with reported functions that modify terpenoids in terms of bioactivity, solubility and volatility. Grapevine flower terpenoids were characterised for diverse wine cultivars by means of chemical analytical methods and showed remarkable differences. All cultivars produced sesquiterpenes as major volatiles with valencene, 7-epi-α-selinene, farnesenes and β-caryophyllene presence and absence affecting the volatilome of the different cultivars. The results presented show that the different cultivars differ in their capacity to produce certain terpenoids. Functional characterisation of putative TPS-encoding genes in a heterologous yeast expression system was utilised to demonstrate that cultivar-specific mutations affect VViTPS functionality. Aberrant mutations resulting in premature stop codons and/or altered protein structures affecting the catalytic site of the TPS were prevalent. A novel gene encoding for a E-β-farnesene synthase was isolated for the cultivar Muscat D'Alexandria and was functionally linked to the cultivar's unique volatilome which was dominated by E-β- farnesene (~60%). The results reported here suggest that grapevine flowers have unique TPSencoding genes as a result of independent cultivar selection pressures that influence their terpenoid volatilome. The overrepresented grapevine TPS family is of great biological and economic importance for cultivar-specific traits. Multiple levels of both transcriptional and post-transcriptional regulation allows for great diversity in terpenoid metabolism. By studying specific organ/developmental stages it was shown that unique TPS-encoding genes are involved cultivar-specific terpenoid metabolism.
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    Linking terpene synthases to sesquiterpene metabolism in grapevine flowers
    (Frontiers Media, 2019) Smit, Samuel Jacobus; Vivier, Melane Alethea; Young, Philip Richard
    Grapevine (Vitis vinifera L.) terpene synthases (VviTPS) are responsible for the biosynthesis of terpenic volatiles. Volatile profiling of nine commercial wine cultivars showed unique cultivar-specific variation in volatile terpenes emitted from grapevine flowers. The flower chemotypes of three divergent cultivars, Muscat of Alexandria, Sauvignon Blanc and Shiraz were subsequently investigated at two flower developmental stages (EL-18 and -26). The cultivars displayed unique flower sesquiterpene compositions that changed during flower organogenesis and the profiles were dominated by either (E)-β-farnesene, (E,E)-α-farnesene or (+)-valencene. In silico remapping of microarray probes to VviTPS gene models allowed for a meta-analysis of VviTPS expression patterns in the grape gene atlas to identify genes that could regulate terpene biosynthesis in flowers. Selected sesquiterpene synthase genes were isolated and functionally characterized in three cultivars. Genotypic differences that could be linked to the function of a targeted gene model resulted in the isolation of a novel and cultivar-specific single product sesquiterpene synthase from Muscat of Alexandria flowers (VvivMATPS10), synthesizing (E)-β-farnesene as its major volatile. Furthermore, we identified structural variations (SNPs, InDels and splice variations) in the characterized VviTPS genes that potentially impact enzyme function and/or volatile sesquiterpene production in a cultivar-specific manner.