Dissertation (PhD)--University of Stellenbosch, 2004. / 152 Leaves printed single pages, preliminary pages i-xiv and 129 numberd pages. Includes bibliography. List of abbreviations. / ENGLISH ABSTRACT: Despite increased focus on ripening-related gene transcription in grapevine, and the large number of
ripening-related cDNAs identified from grapes in recent years, the molecular basis of processes
involved in grape berry ripening is still poorly understood. Moreover, little is known about the
mechanisms involved in the ripening-related regulation of fruit-specific genes, since the isolation
and characterisation of no ripening-related, fruit-specific promoter elements has been reported to
date. This study was aimed at the isolation and characterisation of a fruit-specific, ripeningregulated
gene from Vitis vinifera L.
In the first phase of the work, gene transcription in ripening berries of Cabernet Sauvignon (a good
quality wine cultivar) and Clairette blanche (a poor quality wine cultivar) were studied by Amplified
Fragment Length Polymorphism analysis of complementary DNA (cDNA-AFLP analysis). Total
RNA from immature (14-weeks post flowering, wpf) and mature (18-wpf) berries was used for the
analysis. A total of 1 276 cDNA fragments were visualised, of which 175 appeared to be ripening
related. Average pairwise difference of the fragments amplified from immature and mature
Clairette and Cabernet berries, suggested that ripening-related gene transcription in these two
phenotypically different cultivars is remarkably similar. Nevertheless, it was shown that seventy
percent of the 175 ripening-related cDNA fragments were cultivar-specific. It was suggested that
these differences should be targeted to identify genes related to the phenotypical differences
between the two cultivars, but also to identify genes possibly involved berry quality. Moreover, the
analysis illustrated the usefulness of cDNA-AFLPs for the analysis of ripening-related gene
transcription during grape berry ripening.
In the second phase of the work, one of the ripening-related cDNAs identified by the cDNA-AFLP
analysis, was selected for further characterisation. This work highlighted the limitation placed on
the isolation of a single specific sequence from a cDNA-AFLP gel, indicating the presence of
multiple ripening-related genes in a single band excised from a cDNA-AFLP gel. Steps to
overcome this limitation of cDNA-AFLP analysis to identify and clone a specific ripening-related
gene, were implemented. In short, the band corresponding to the particular ripening-related cDNA
was band was excised from the cDNA-AFLP polyacrylamide gel and re-amplified. Northern blot
analysis using the re-amplified, uncloned product confirmed the ripening-related transcription
demonstrated by cDNA-AFLP analysis. The re-amplified, uncloned product was then cloned.
Sequence analysis of two randomly selected candidate clones revealed two distinctly different
sequences, of which neither hybridised to messenger RNA from ripening grape berries. Furtheranalysis revealed an additional five cDNAs with terminal sequences corresponding to the selective
nucleotides of the primers used for selective amplification, in the re-amplified, uncloned product.
Of these, only two were abundantly expressed in ripening grape berries, accounting for the ripeningrelated
transcription visualised by cDNA-AFLP analysis. All seven cDNAs identified from the
particular excised band were shown to be ripening-regulated during berry development, although
most were characterised by low levels of transcription during berry ripening. One of the clones,
based on the relative high levels of the transcript and the initiation of gene transcription at the onset
of véraison (10- to 12-wpf), was identified for isolation and characterisation of the full length
coding sequence.
In the third phase of the work, it was shown that this cloned sequence corresponded to a gene
encoding a proline-rich protein (PRP) associated with ripening in Merlot and Chardonnay (mrip1,
Merlot ripening-induced protein 1). It was shown that the gene is specifically transcribed in the fruit
tissue, seed and bunchstems of grapes, from 10-wpf (véraison) to the final stages of berry ripening.
The results showed that mrip1 encodes a distinct member of the plant PRP family. Most obvious is
the central region of mrip1, which is comprised of eight consecutive repeats of 19 amino acid
residues each. In comparison with other grapevine PRPs, mrip1 revealed single amino acid
differences and deletion of one of the 19 amino acid residues repeats, all in the central region of
mrip1. In situ hybridisation studies showed that accumulation of the mrip1 transcript in the ripening
berry is limited to the mesocarp and exocarp cells of the ripening grape berry. No transcript with
high sequences similarity to mrip1 could be detected in ripening strawberry or tomato fruit. Based
on the properties and proposed function of PRPs, and the results obtained in this study, potential
applications for the use of this gene in the control of cell wall architecture in fruits, were proposed.
Furthermore, as manipulation of fruit properties in grape berries would be most important in the
later stages of ripening, mrip1 was proposed an ideal candidate gene for the isolation of a fruit- and
late-ripening-specific promoter to achieve transgene transcription in genetically modified grapevine.
The final phase of the work was dedicated to the isolation and characterisation of the mrip1
promoter element. A 5.5 kb sequence corresponding to the mrip1 5’ untranslated (UTR) flanking
region was isolated and characterised by sequence analysis. In the 2.8 kb sequence directly
upstream of the mrip1 transcription initiation site, several putative cis-acting regulatory elements
were identified. These include a spectrum of hormone-, light-, phytochrome-, sugar-and stressresponsive
elements, as well as elements implicated in tissue-specific transcription. Analysis of the
sequence further upstream (3.6 – 5.5 kb) of the mrip1 transcription initiation site (TIS), revealed the
presence of another proline-rich protein directly upstream of mrip1. Sequence identity of this
sequence (mprp2) to the mrip1 coding sequence was 88%. This information provided the first insight into the chromosomal organisation of grapevine PRPs. For functional analysis of the mrip1
promoter element, the 2.2 kb sequence directly upstream of the mrip1 TIS, was translationally fused
to the sgfpS65T reporter gene. Functionality of the mrip1:sgfpS65T fusion was verified by transient
expression in green pepper pericarp tissue, before introduction into tobacco by Agrobacteriummediated
transformation. In transgenic tobacco, transcription of the mrip1:sgfpS65T fusion was
developmentally-regulated and specific to the ovary and nectary-tissue of the developing flower.
Whilst low in immature flowers, the green fluorescent protein (GFP) rapidly accumulated to the
high level of expression visualised in the flower in full-bloom, followed by a decrease in the final
stages of ovary development. These observations suggested that the 2.2 kb mrip1 promoter is
functional and that this promoter region harbours cis-elements necessary for tissue- and
developmental-specific regulation of GFP accumulation. It furthermore suggested that the
transcriptional activation of mrip1 is mediated by developmental signals present in both grapevine
berries and tobacco flowers. Results presented, suggest that the use of tobacco as heterologous
system for the analysis of ripening-related promoters, can be more generally applied. Evidently,
characterisation of the mrip1 promoter region contributes towards a better understanding of the
regulatory mechanisms involved in non-climacteric fruit ripening, and forms a basis for future
experiments defining the cis-acting elements necessary for tissue- and cell-specific gene regulation
in fruit, more specifically in grapevine. Moreover, the mrip1 promoter is an ideal candidate for the
ripening-related, tissue-specific regulation of transgene transcription in genetically modified
grapevine. / AFRIKAANSE OPSOMMING: Ten spyte van toenemende fokus op rypwordings-verwante geentranskripsie in druiwe, en die groot
aantal rypwordings-verwante komplimentere DNA (cDNA) fragmente wat gedurende die laaste paar
jaar in druiwe geïdentifiseer is, word die molekulêre basis van prosesse betrokke by die rypwording
van die druif, steeds swak begryp. Nog te meer, is baie min bekend oor die meganismes betrokke in
the rypwordings-verwante regulering van vrugspesifieke gene, aangesien die isolering en
karakterisering van nie een rypwordings-verwante, vrugspesifieke promoter tot dusver gerapporteer
is nie. Die doel van hierdie studie was die isolering en karakterisering van ‘n vrugspesifieke,
rypwordings-verwante geen uit druiwe (Vitis vinifera L).
In die eerste fase van die werk, is geentranskripsie in rypwordende druiwekorrels van Cabernet
Sauvignon (‘n goeie kwaliteit wyn kultivar) en Clairette blanche (‘n swak kwaliteit wyn kultivar)
bestudeer deur middel van cDNA-AFLP vingerafdrukke. Totale RNA van onvolwasse (14-weke na
blom vorming) en volwasse (18-weke na blom vorming) druiwekorrels was gebruik vir die analise.
‘n Totaal van 1 276 cDNA fragmente is gevisualiseer, waarvan 175 as rypwordings-verwant
voorgekom het. Gemiddelde paarsgewyse verskille van die fragmente wat vanaf onvolwasse en
volwasse Clairette en Cabernet druiwekorrels geamplifiseer is, het aangedui dat rypwordingverwante
geentranskripsie in die twee kultivars, wat fenotipies baie van mekaar verskil,
merkwaardig soortgelyk is. Nieteenstaande, is daar gewys dat sewentig persent van die 175
rypwordings-verwante cDNA fragmente, kultivar-spesifiek is. Daar is voorgestel dat hierdie
spesifieke cDNAs verder geanaliseer word om gene betrokke by die fenotipiese verskille tussen die
twee kultivars te identifiseer; maar ook om gene te identifiseer wat moontlik by die kwaliteit van die
druiwekorrel betrokke is. Voorts, het die analise die bruikbaarheid van die cDNA-AFLP tegniek vir
die karakterisering van rypwordings-verwante geentranskripsie in rypwordende druiwekorrels,
geïllustreer.
In die tweede fase van die werk, is een van die rypwordings-verwante cDNAs wat met die cDNAAFLP
analise geïdentifiseer is, geselekteer vir verdere karakterisering. ‘n Aantal rypwordingsverwante
cDNAs is in die enkele band wat uit die cDNA-AFLP gel gesny is, geïdentfiseer. Dit het
die beperking wat geplaas word op die isolering van ‘n enkel, spesifieke cDNA uit die cDNA-AFLP
gel, beklemtoon. Stappe om hierdie beperking te oorkom, en ‘n spesifieke rypwordings-verwante
cDNA te identfiseer en te kloneer, is beskryf. In kort, die band oorstemmend met die spesifieke
rypwordings-verwante cDNA, is uit die cDNA-AFLP poli-akrielamied gel gesny en gereamplifiseer.
Noordelike klad analise waarin die ge-reamplifiseerde, ongekloneerde produk aspeiler gebruik is, het die rypwordings-verwante transkripsie soos deur cDNA-AFLP analise
aangedui, bevestig. Die ge-reamplifiseerde, ongekloneerde produk is daarna gekloneer. Nukleotied
volgorde bepaling van twee ewekansig geselekteerde kandidaat klone, het twee duidelik
verskillende cDNAs aangetoon, waarvan nie een enige hibridisering met boodskapper RNA van
rypwordende druiwekorrels getoon het nie. Verder analise het die teenwoordigheid van ‘n verder
vyf cDNAs met terminale nukleotied volgordes ooreenstemmend met die selektiewe nukleotiede
van die voorlopers wat gebruik is vir selektiewe amplifisering, aangetoon. Van hierdie, het slegs
twee hoë vlakke van geentranskripsie in rypwordende druiwekorrels getoon; heel moontlik
verteenwoordigend van die rypwordings-verwante geentranskripsie wat met die cDNA-AFLP
analise gevisualiseer is. Die studie het gewys dat al sewe cDNAs rypwordings-verwant is, alhoewel
die meeste van hierdie cDNAs baie lae vlakke van geentranskripsie tydens duiwekorrel rypwording
getoon het. Gebaseer op relatief hoë vlakke van die transkrip, en die inisiering van geen transkripsie
met die aanvang van vrugrypwording (véraison, 10- tot 12-weke na blomvorming), is een van die
cDNAs geselekteer vir isolering en karakterisering van die vollengte koderings volgorde.
In die derde fase van die werk, is dit aangetoon dat hierdie cDNA ooreenstem met ‘n geen wat vir ‘n
proline-ryke proteïen (PRP), geassosieerd met vrugrypwording in Merlot en Chardonnay, kodeer.
Hierdie geen is genoem Merlot rypwording-geïnduseerde proteïen 1 (mrip1). Die studie het verder
aangetoon dat hierdie geen spesifiek in die weefsel van druiwekorrels, saad and stammetjies van die
druiwetros getranskribeer word, vanaf 10-weke na blomvorming (véraison) tot 16-weke na
blomvorming. Resultate het aangetoon dat mrip1 vir ‘n unieke lid van die plant PRP familie kodeer.
Mees opvallend, is die sentrale gedeelte van mrip1, wat uit agt opeenvolgende herhalings van
negentien aminosure elk bestaan. In vergelyking met ander druif PRPs, toon mrip1 enkel aminosuur
verskille en ‘n delesie van een van die negentien aminosuur herhalings, alles in die sentrale gedeelte
van mrip1. In situ hibridisering het getoon dat akkumulering van die mrip1 transkrip net in selle van
die mesocarp en eksokarp van die rypwordende druif plaasvind. Geen transkip met hoë nukleotied
gelyksoortigheid aan mrip1 kon in rypwordende aarbeie of tamatie vrugte aangetoon word nie.
Gebaseer op die eienskappe en funksie van PRPs soos voorgestel in die literatuur, en die bevindinge
van hierdie studie, is potensiële toepassings vir die gebruik van die geen in die beheer van selwand
argitektuur in vrugte, voorgestel. Verder, aangesien die manipulering van vrugkwaliteit in die druif
veral belangrik is vanaf die aanvang van vrugrypwording (véraison), is daar voorgestel dat mrip1 ‘n
ideale kandidaat is vir die isolering van ‘n vrugspesifieke en rypwording-verwante promoter vir
gebruik in geneties gemodifiseerde druiwe.
Die laaste fase van die studie was gewy aan die isolering en karakterisering van die mrip1 promotor
element. ‘n 5.5 kb fragment ooreenstemmend met die mrip1 5’ ongetransleerde area is geisoleer en gekarakteriseer deur middel van nukleotied volgorde bepaling. In die 2.8 kb area direk stroomop
van die mrip1 transkripsie inisiasie punt (TIS), is verskeie moontlike cis-beherende regulatoriese
elemente geïdentifiseer. Hierdie sluit in ‘n spektrum van hormoon-, lig-, fitochroom-, suiker- en
stress-reagerende elemente, asook elemente geïmpliseer in weefselspesifieke geentranskripsie.
Analise van die area verder stroomop (3.6 – 5.5 kb) van die mrip1 TIS, het die teenwoordigheid van
‘n ander PRP direk stroomop van mrip1 getoon. Nukleotied gelyksoortigheid van hierdie geen
(MPRP2) aan die mrip1 koderingsgebied was slegs 88%. Hierdie inligting verskaf die eerste insig
in die chromosomale organisasie van druif PRPs. Vir funksionele analise van die mrip1 promotor
element, is die 2.2 kb area direk stroomop van die mrip1 TIS transkripsioneel verenig met die
sgfpS65T merker geen. Funksionaliteit van die mrip1: sgfpS65T fusie is bevestig deur middel van
kortstondige (transient) geenuitdrukking in die perikarp van groenrissie, voordat dit ingevoer is in
tabak met Agrobacterium-bemiddelde genetiese transformasie. In transgeniese tabak was
transkripsie van die mrip1:sgfpS65T fusie ontwikkelingsstadium-gereguleerd, en spesifiek in die
ovarium en heuningsakkie (nektarium) van die ontwikkelende blomme. Terwyl die vlak van
geenuitdrukking laag was in die jong blomme, het GFP baie vinnig akkumuleer tot die hoë vlakke
wat in die blomme in volle-blom gevisualiseer is. Daarna het dit weer vinnig afgeneem tydens die
finale stadiums van ovarium ontwikkeling. Hierdie waarnemings dui daarop dat die 2.2 kb mrip1
promotor element funksioneel is en dit al die nodige cis-beherende regulatoriese element bevat wat
nodig is vir weefsel- en ontwikkelingsstadium-spesifieke regulering van GFP akkumulering. Dit dui
verder daarop dat transkripsionele aktivering van mrip1 beheer word deur ontwikkelingsstadium
seine teenwoordig in beide die druif en tabakblomme. Hierdie resultate stel voor dat tabak meer
algemeen gebruik kan word as heteroloë sisteem vir die analise van rypwording-verwante
promotors. Duidelik dra die karakterisering van die mrip1 promoter element by tot ‘n beter begrip
van die regulatoriese meganismes betrokke by die rypwordingsproses van nie-klimateriese vrugte,
en vorm die basis vir toekomstige eksperimente waarin die cis-beherende regulatoriese elemente vir
vrug- en sel-spesifieke geen regulering, meer spesifiek die druif, bepaal sal word. Meer nog, is die
mrip1 promotor ‘n ideale kandidaat vir weefsel-spefieke en rypwording-verwante regulering van
transkripsie van die transgeen in geneties gemodifiseerde druiwe.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/15938 |
| Date | 12 1900 |
| Creators | Burger, Anita L. |
| Contributors | Botha, F. C., University of Stellenbosch. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xiv, 129 leaves : ill. |
| Rights | University of Stellenbosch |
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