Grapevine terpenoids and their contribution to the flower volatilome

Thesis (MScAgric)--Stellenbosch University, 2016.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.AFRIKAANSE OPSOMMING: Terpene verteenwoordig een van die grootste natuurlike metaboliete en toon ongelooflike diversiteit in terme van chemiese struktuur en aktiwiteit. Hierdie diversiteit kan toegedien word aan die terpeen sintase (TPS) ensieme wat prenieldifosfaat voorlopers omskakel na die onderskeie substrate wat lei tot mono-, di- en seskwiterpene. TPSe het die vermoë om sonder onderskeid op te tree en kan dus enkele of menige terpene produseer vanaf ‘n substraat wat elk verander kan word deur beide ensiematies en nie-ensiematiese prosesse. Die wingerd (Vitis vinifera) genoom toon ‘n enorme TPS geen familie met uitgebreide duplisering van gene. Inburgering van wingerd het gelei tot ‘n diverse versameling van kultivars, elk met ‘n unieke vermoë om terpene to produseer, wat lei tot die onderskeid van die sogenaamde aromatiese kultivars. Om hierdie rede word wingerd TPSe (VviTPS) grootliks bestudeer vir hul vermoë om by te dra tot die blom (bv. Gewürztraminer en Muscat) of peper (bv. Shiraz) geur in wyn. Hierdie onderskeie geure kan toegdien word aan die mono- en sesquiterpene, onderskeidelik. Dit stel voor dat daar kultivar verskille bestaan in terme van terpeen metabolisme. Die in planta rol van wingerd terpene is egter nog nie wel bekend nie. In ander plante is dit al gewys dat die terpene belangrike ekologiese funksies verrig as sekondêre metaboliete wat lei tot ‘n verhoogde vermoë vir aanpassing, byvoorbeeld vir beskerming/verdediging of insek aanloking. Wingerde blomme lyk belowend om die begenoemde in planta funksies te ondersoek weens verhoogde geen transkripsie en vrystelling van vlugtige metaboliete wat al getoon is in sekere kultivars. In silico karakterisering van die wingerd TPS geen familie het gewys dat VviTPS uitdrukking toeneem tydens blomvorming. Oor die algemeen lyk dit asof die seskwi-TPS gene uitgedruk word tydens die vroëe blomvorming stadiums terwyl die mono-TPS gene tydens die blom tydperk uitgedruk word. Die metaboliese prosesse betrokke by terpeen vorming was ondersoek deur middel van mede-uitdrukking ontleding. Dit het gelei tot die identifikasie van kandidaat gene wat lei tot byvoorbeeld sitokroom P450 en suiker hidrolase of transferase ensieme wat terpene kan modifiseer. Hierdie ensieme is bekend vir hul vermoë om die aktiwiteit en/of oplosbaarhied van terpene te verander. ‘n Versameling van wingerd kultivars was gekarakteriseer in terme van hul vlugtige terpene. Die nege kultivars geanaliseer het elk ‘n unieke terpeen profiel getoon met valencene, 7-epi-α-selinene, farnesene en β-caryophyllene as kenmerkende seskwiterpene. Die kultivars het verskil in terme van die konsentrasie en teenwoordigheid/afwesigheid van terpene wat dus toon dat kultivars verskil in hul vermoë om terpene to produseer. Belowende TPS gene was gevolglik geïsoleer en uitgedruk in ‘n heteroloë gis sisteem. Karakterisering van die gene het getoon dat mutasies spesifiek tot sekere kultivars verantwoordelik is vir ‘n verskil in ensiem funksie. Hierdie mutasies het gelei to ‘n vroëe stop kodon en/of ‘n verandering in die proteïen se aktiewe setel of struktuur. ‘n Unieke geen wat kodeer vir ‘n E-β-farnesene sintase geen was geïsoleer van die kultivar Muscat D'Alexandria en kon gekoppel word aan die unieke vlugtige profiel (E-β-farnesene as hoof produk) van hierdie kultivar. Dit is dus moontlik dat unieke TPS gene voor geselekteer is tydens inburgering en gevolglik gelei het tot ‘n veranderde vermoë om terpene to produseer. Die groot terpeen familie van wingerd is dus vir beide biologiese en ekonomiese doeleindes van belang in terme van unieke kultivar eienskappe. Die verskillende aspekte van geen reguleering en metaboliet modifikasie verleen dus aan wingerd die vermoë om menige terpene te produseer. Deur te kyk na speifieke orgaan/ontwikkelings stadia is daar getoon die TPS gene spesifiek tot ‘n kultivar verantwoordelik is vir uiteenlopende metaboliese moontlikhede