The isolation and characterisation of a developmentally-regulated gene from Vitis vinifera L. berries

Burger, Anita L. (Stellenbosch : University of Stellenbosch, 2004-12)

Dissertation (PhD)--University of Stellenbosch, 2004.

152 Leaves printed single pages, preliminary pages i-xiv and 129 numberd pages. Includes bibliography. List of abbreviations.

Thesis

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.

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