Thesis (MScAgric (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The Vitis vinifera L. carotenoid cleavage dioxygenase 1 gene (VvCCD1) is a member of a structurally conserved gene family encoding enzymes that cleave multiple carotenoid substrates to form apocarotenoids. Carotenoid pigments are synthesised in the chloroplast where they are primarily involved in light harvesting and photo-protection during photosynthesis while apocarotenoids fulfill diverse roles that range from pollinator attractants to phytohormones. CCD1 cleaves carotenoids at specific double bond sites producing volatile apocarotenoids. These CCD1-derived apocarotenoids typically possess a fruity and floral aroma, thus making them desirable targets for metabolic engineering. CCD1 orthologues are highly homologous and have been isolated and characterised from a number of plant species, including Arabidopsis, tomato, rose, petunia, and grapevine. VvCCD1 is localised to the cytosol and has been shown in vitro to cleave zeaxanthin and lutein resulting in 3-hydroxy-β-ionone. Expression of VvCCD1 increases during berry ripening, peaking at véraison. Due to the impact that VvCCD1 potentially has on the flavour and aroma of grape berries and therefore wine, this study aimed to characterise the specific enzyme action as well as the biological role that this enzyme plays in grapevine. Expression of VvCCD1 in carotenoid-accumulating Escherichia coli strains demonstrated cleavage of β-carotene at the 9,10 (9’,10’) position forming β-ionone; and lycopene at the 5,6 (5’,6’) and 9,10 (9’,10’) position, forming 6-methyl-5-hepten-2-one and pseudoionone, respectively. A transgenic grapevine population with modified VvCCD1 expression was generated and genetically and metabolically characterised. The transgenic population consisted of lines in which VvCCD1 was either overexpressed or silenced. Expression analysis of stable transformants showed a 12-fold range of VvCCD1 expression relative to the wild-type. HPLC analysis of the photosynthetic pigment content of the transgenic population necessitated the development and optimisation of a method for the extraction of pigments, specifically from grapevine. A number of parameters were identified and optimised, resulting in a method that provides accurate quantification of photosynthetic pigments from grape berries and leaves. Absolute quantification of the following major photosynthetic pigments present in grapevine is now possible: chlorophyll a, chlorophyll b, lutein, -carotene, zeaxanthin, antheraxanthin, violaxanthin and neoxanthin. Data suggest that various levels of molecular control regulate carotenoid cleavage and apocarotenoid biosynthesis. The majority of lines stably transformed with a VvCCD1 overexpression cassette exhibit post-transcriptional gene silencing. Expression analysis in these lines demonstrated that, despite the additional contribution of transgene-derived VvCCD1 transcripts, the total VvCCD1 transcript levels were not significantly higher than in wild-type lines. In lines where transgenic manipulation of VvCCD1 expression was successful, subsequent analysis of carotenoids and apocarotenoids in leaf tissue showed no correlation between the measured metabolites and gene expression. The in planta action of VvCCD1 is presumably distinct from the observed in vitro activity due to the strict compartmentalisation required in photosynthetic leaf tissue preventing access of cytosolic VvCCD1 to the chloroplastic carotenoids. Future studies on reproductive organs (grape berries) from the transgenic lines generated in this study will be of great importance in further elucidation of the in planta function of VvCCD1. / AFRIKAANSE OPSOMMING: Die Vitis vinifera L. “carotenoid cleavage dioxygenase” 1 geen (VvCCD1) behoort aan ‘n geenfamilie wat struktureel gekonserveerd is en kodeer vir ensieme wat verskeie karotenoïed substrate afbreek om apokarotenoïede te vorm. Karotenoïed pigmente word in die chloroplaste gesintetiseer waar hulle primêr betrokke is by lig-insameling, sowel as beskerming tydens fotosintese, terwyl apokarotenoïede diverse funksies in die plant verrig wat strek van aantrekking van stuifmeelverspreiders tot phytohormone. CCD1 breek karotenoïede by spesifieke dubbelbindingsetels af om vlugtige apokarotenoïede te vorm. Die apokarotenoïede wat van CCD1 afkomstig is besit tipies vrugtige en blomagtige aromas wat hul gesogte teikens maak vir metaboliese manipulering. CCD1 ortoloë is hoogs homoloog en is al geїsoleer en gekarakteriseer vanuit ‘n verskeidenheid plantspesies wat Arabidopsis, tamatie, roos, petunia en wingerd insluit. VvCCD1 is in die sitosol gelokaliseer en dit is vantevore gewys dat dit beide zeaxanthin en lutein in vitro kan afbreek om 3-hidroksi-b-ionoon te vorm. Die uitdrukking van VvCCD1 vermeerder tydens korrel rypwording en bereik ‘n maksimum tydens véraison. Weens die potensieële invloed vanVvCCD1 op die geur en aroma van druiwe, en dus wyn, is hierdie studie gerig op die karakterisering van die spesifieke ensiematiese aksie, sowel as die biologiese rol van hierdie ensiem in wingerd. Uitdrukking van VvCCD1 in Escherichia coli rasse wat karotenoïede versamel het getoon dat β-karoteen by die 9,10 (9’,10’) posisie afgebreek word om β-ionoon te vorm, en likopeen by die 5,6 (5’,6’) en 9,10 (9’,10’) posisie om onderskeidelik 6-metiel-5-hepteen-2-oon en pseudo-ionoon te vorm. ‘n Transgeniese wingerd populasie is gegenereer met gewysigde VvCCD1 uitdrukking en is geneties en metabolies gekarakteriseer. Die transgeniese populasie het bestaan uit lyne waar VvCCD1 óf ooruitgedruk óf afgereguleer is. Uitdrukkings analise van die stabiele transformante het ‘n 12-voudige reeks van VvCCD1 uitdrukking getoon, relatief tot die wilde tipe. HPLC analise van die fotosintetiese-pigment inhoud van die transgeniese populasie het die ontwikkeling en optimisering van ‘n wingerd-spesifieke metode vir die ekstraksie van pigmente genoodsaak. ‘n Aantal parameters is geïdentifiseer en geoptimiseer, en het gelei tot ‘n metode wat akkurate kwantifisering van fotosintetiese pigmente in druiwe en wingerdblare kan lewer. Absolute kwantifisering van die volgende belangrike fotosintetiese pigmente aanwesig in wingerd is nou moontlik: chlorophyll a, chlorophyll b, lutein, -karoteen, zeaxantien, anteraxantien, violaxantien en neoxantien. Data dui aan dat verskeie vlakke van molekulêre beheer die afbreking van karoteen en die biosintese van apokarotenoïede reguleer. Die meerderheid van die lyne wat stabiel getransformeer is met ‘n VvCCD1 ooruitdrukkingskasset het na-transkripsioneleafregulering van die geen getoon. Uitdrukking analise van die lyne het gewys dat ten spyte van die addisionele transgeniese VvCCD1 transkripte, die totale VvCCD1 transkripvlakke nie beduidend hoër was as dié van die wilde-tipe lyne nie. In die lyne waar transgeniese manipulasie van VvCCD1 uitdrukking wel suksesvol was, het verdere analise van die karotenoïed en apokarotenoïed vlakke in blaarweefsel geen korrelasie getoon tussen die metaboliete en VvCCD1 uitdrukking nie. Die in planta aktiwiteit van VvCCD1 is vermoedelik anders as die in vitro aktiwiteit weens die streng kompartementalisering benodig in fotosintetiese blaarweefsel, wat verhoed dat die sitosoliese VvCCD1 toegang het tot die chloroplastiese karotenoïede. Toekomstige bestudering van die reproduktiewe organe (druiwe) van die transgeniese lyne wat in hierdie studie gegenereer is sal belangrik wees in die verdere verduideliking van die in planta funksie van VvCCD1.