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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/4370 |
| Date | 03 1900 |
| Creators | Lashbrooke, Justin Graham |
| Contributors | Young, Philip R., Vivier, Melane A., University of Stellenbosch. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 102 p. : ill. |
| Rights | University of Stellenbosch |
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