Thesis (MSc (Botany and Zoology. Plant Biotechnology))--University of Stellenbosch, 2005. / Sugarcane (Saccharum spp) is an important crop worldwide and is cultivated for the high level of sucrose in its mature internodes. Because of the exhaustion of the genetic potential in the commercial sugarcane germplasm conventional breeding has not lately been able to enhance sucrose content. Currently there is a concerted effort to improve culm sucrose content by genetic engineering which will require appropriate transgenes and promoters. One of the major constraints to genetic engineering of sugarcane is the lack of stable promoters required to drive tissue- or organ-specific expression of transgenes. Tissue and developmental stage specific promoters allow targeting of transgene activity and in doing so reduce the impact on non-target tissues. These promoters could also be advantageous to manipulate certain aspects of sucrose metabolism specifically in mature culm tissue. In addition, no promoters are currently freely available to the South African Sugar Industry for use in their transgenic program. The primary goal of this project was therefore to isolate a mature tissue-specific promoter for use in transgenic sugarcane plants. The approach followed was firstly, to identify an endogenous gene expressed in the desired pattern, and then to isolate the corresponding promoter from the sugarcane genome. cDNA macroarrays were initially used to identify differentially expressed sequences. The tissue specificity of potential clones was confirmed using RNA blot analysis. Two clones (c23-a and c22-a) were isolated and confirmed to be mature culm specific. Clone c22-a (putative dirigent-like protein) was selected for promoter isolation based on its culm tissue specific expression pattern and its proximity to the 5’ end of the gene. Furthermore, to confirm the activity of this promoter in the storage parenchyma cells, the exact cellular localisation of the transcript in the mature tissue was determined through in situ hybridisation. In situ hybridisation results confirmed the presence of the transcript in the parenchyma cells of mature culm tissue only. Moreover, the transcript is present in high concentrations in the parenchyma tissues surrounding the vascular bundles and parenchyma cells of the vascular complex. The selected dirigent-like gene was sequenced to allow the design of primers that could be used for the isolation of the corresponding promoter region using a long-range inverse PCR (LR-iPCR) method. Using these we have successfully isolated two highly homologous promoter regions of the dirigent like gene of respectively 1151 and 985 base pairs. In silico analyses confirmed the presence of various transcription motifs, including a TATA-box. However, experimental verification is needed to fully assess the functionality of these promoter regions. Verifying the activity of the isolated promoters through transient expression analysis proved to be problematic because of their highly mature culm specificity. Both constructs are therefore being used to obtain stable transformants in which promoter activity can be evaluated in mature internodal tissues.

A vector, called pWreckl, was constructed for the direct DNA transformation of N. tabacum protoplasts. This vector was shown to confer kanamycin resistance to transformed callus. Using derivatives of this plasmid containing various reporter genes, a protocol for direct DNA transformation by PEG-mediated uptake was established. However, all attempts to obtain transformed callus by electroporation were unsuccessful. The CAB6 gene from N. tabacum was identified as a suitable candidate for gene targeting and was further manipulated to produce constructs in pWreckl which were used in gene targeting experiments. Using a derivative of pWreckl, called BinsupF, transformed tobacco plants were generated and used as a model system for the rescue of integrated sequences by supF selection. This system was found to be capable of rescuing integrated pWreckl sequences, but at an efficiency below that which would be required for the rescue of rare gene targeting events. Therefore a second protocol was developed which used the technique of polymerase chain amplification to amplify specific integration events. Following direct DNA transformation with a pWreckl.CAB6 construct, a number of possible gene targeting events were identified and subcloned into pUC13. Partial sequencing of these clones revealed that they were not the result of homologous recombination, but arose through a combination of plasmid degradation and rearrangement which appeared to precede the actual integration event.

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Thesis (Ph. D.)--University of Alberta, 2010. / Title from pdf file main screen (viewed on April 22, 2010). A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Science, Department of Agricultural, Food and Nutritional Science, University of Alberta. Includes bibliographical references.

Thesis (MSc)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.

Thesis (PhD (Genetics. Plant Biotechnology))--University of Stellenbosch, 2007. / Plants are constantly exposed to adverse environmental conditions including variations in light intensity and the availability of water resources. These abiotic factors are expected to worsen as the changing global climate places additional daily and seasonal demands on plant growth and productivity. As plants are incapable of avoiding stress they have developed a number of mechanisms to manage and adapt to the unfavourable conditions. Carotenoids represent one of these mechanisms; with the xanthophylls (oxygenated carotenes) playing an essential role in photoprotection following exposure to excess light energy. They are also precursors to the plant hormone abscisic acid (ABA) which plays a known role in stomatal regulation and thus drought tolerance. Carotenoids have been identified as potential targets for genetic manipulation to meet the existing nutritional demands (particularly vitamin A) and to enable plants to survive the climatic variations predicted. Thorough investigations into the regulation and functioning of each carotenoid biosynthetic gene in vivo as well as the roles of their encoded proteins are prerequisite. Within the Grapevine Biotechnology Programme, a number of isoprenoid biosynthetic genes have been isolated from Vitis vinifera L. cv. Pinotage. From this vast resource two genes were chosen; namely a lycopene b-cyclase (b-LCY) and 9-cis epoxycarotenoid dioxygenase (NCED) for detailed in planta analyses to address knowledge gaps in our current understanding of carotenoid biosynthesis in general, its regulation and the roles of the two target genes in these processes. Currently, the role of b-LCY within the chloroplasts is not well known. Although the relationship between NCED overexpression, ABA levels, reduced stomatal conductance and increased tolerance to water stress has been well-established, comprehensive physiological analysis of the resulting mutants during conditions of both water availability and shortage is not well documented. To assess their in planta role, functional copies of both genes were isolated from Vitis vinifera (cv. Pinotage), characterised and independently transformed into the genome of the model plant, Arabidopsis thaliana, in the sense orientation under a constitutive promoter. In order to investigate these pertinent scientific questions and thus to evaluate the physiological role of each gene in vivo, a number of technologies were developed and/or adopted. These included a high-performance liquid chromatography method for profiling the major plant pigments in leaf tissue, a combination vapour phase extraction and electron impact-gas chromatography/mass spectrometry method for the phytohormone profiling as well as various physiological analyses including the use of chlorophyll a fluorescence to assess the photosynthetic and non-photochemical quenching (NPQ) capacities of the plants. Overexpression of grapevine b-LCY (Vvb-LCY) decreased lutein levels due to preferential partitioning of lycopene into the b-branch. This decrease was not met by an increase in either b-carotene or the xanthophyll cycle pigments implying that Vvb-LCY is not able to regulate the flow of carbon through the pathway and provides additional evidence to the fluidity of this pathway whereby pigment levels are continually balanced. The decreased lutein levels observed under low light (LL) did not compromise the plants’ ability to induce and maintain NPQ over a wide actinic light range. Vvb-LCY transgenics also had lower neoxanthin levels (and specifically the cis-isomer) under both LL and following exposure to high light (HL), which could be correlated to an increase in malondialdehyde. Although not corroborated, a novel and unexpected finding was an essential role for neoxanthin, and potentially lutein, in preventing or at least reducing lipid peroxidation under HL stress. The lower neoxanthin amounts may be due to silencing of the Arabidopsis b-LCY by the Vvb-LCY, as the former may function as a NSY paralog as NSY is not encoded for in the Arabidopsis genome. Clearly, this study has confirmed that Vvb-LCY partitions the carbon flux between the a- and b-branches, however, the catalytic action of this enzyme is dependent on the amount of substrate available and is thus not a regulatory step directing the flux within the pathway. Enzyme kinetic and detailed transcriptional analyses would confirm the above findings. Overexpression of grapevine NCED1 (VvNCED1) increased ABA concentrations, delayed seed germination and resulted in a slight to severe reduction in the overall plant growth rate. NCED cleaves the 9-cis xanthophylls regulating ABA synthesis. However, contrary to expectations, constitutive levels of this regulatory enzyme did not deplete the total and individual chlorophylls and carotenoids in well-watered plants. Instead the VvNCED1 transgenics simply exhibited a lower chloroplastic pigment complement with no concomitant effects on their photosynthetic capacity. Of particular interest, well-watered plants overexpressing the VvNCED1 gene had an increased NPQ capacity of which the thermal energy dissipation component (qE) was the most significant. It has been speculated that this NPQ is associated with the phenotype conferred by VvNCED1 overexpression and occurs independently of the xanthophyll cycle, and specifically zeaxanthin. This study confirmed that VvNCED1 functions during drought tolerance via ABA regulation of stomatal conductance. A detailed study was done to understand the plants’ response during water deficit. Typically, decreases in total and individual carotenoids and the maximum efficiency of photochemistry (Fv/Fm) as well as the relative water and soil moisture content were recorded. No changes were recorded in salicylic acid (SA) levels, while indole acetic acid (IAA) was positively correlated to ABA or vice versa. In contrast, the physiology of VvNCED1 overexpressing lines was largely unaffected, indicating that a reduced stomatal conductance protects the plants against water stress. This study has resulted in the isolation and characterisation of a carotenoid biosynthetic gene (b-LCY) and an abscisic acid synthesising gene (NCED). Significant advancements in our existing knowledge of the in planta role of both genes have been achieved. We have also reaffirmed that strict regulatory control and fluidity exists within the carotenoid biosynthetic pathway whereby individual pigment levels are constantly brought back into balance despite constitutive expression of one of the pathway gene members. These analyses provide valuable baseline information about individual genes which can be extended upon with other omic technologies in order to comprehend the full complexity involved in carotenogenesis.

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.

Dissertation (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: This study had three main goals: 1. to investigate the occurrence on the protein level of sucrose synthase (SuSy) isoforms in sugarcane sink tissue, 2. to determine the kinetic properties of these isoforms, 3. to establish the tissue localisation of SuSy in the sugarcane culm The results are summarised below: Three SuSy isoforms were obtained from leaf roll tissue. The SuSyA and SuSyB isoforms differed in terms of charge characteristics, with SuSyA not binding to an anion exchange column that bound SuSyB and SuSyC under the same conditions. Both SuSyB and SuSyC isoforms were eluted at 180 mM KCl. The SuSyA and SuSyB isoforms were present during autumn, but during winter only the SuSyC isoform could be isolated. Even though they eluted at the same salt concentration, SuSyB and SuSyC were different isoforms, because they had different kinetic parameters, as well as different immunological properties. SuSyB and SuSyC could not have been mixtures of the same isoforms, since a polyclonal antiserum against SuSyB, which inactivates native SuSyB, did not inactivate SuSyC. All three isoforms had significantly different kinetic parameters, with the SuSyA isoform also having a much lower sucrose breakdown/synthesis ratio than the other two isoforms. Therefore, at least three SuSy isoforms occur in sugarcane leaf roll tissue on the protein level. The SuSyC isoform was subsequently kinetically characterised in detail. Data showed that the enzyme employs an ordered ternary complex mechanism, with UDP binding first and UDP-glucose dissociating last. These experimentally obtained kinetic parameters were then used to extend a kinetic model of sucrose accumulation. Data show that when the experimentally determined SuSy kineticparameters were entered into the model, a 40 % increase in sucrose concentration and 7 times reduction in fructose concentration resulted. These data illustrate the pronounced physiological effects that may result from the presence of different SuSy isoforms. SuSy protein localisation data, obtained by an immunohistochemical approach, indicated that SuSy protein was present in both storage parenchyma and vascular tissue of young, intermediate, and mature internodes. SuSy enzyme activity in different parts of the internodes was similar, except for internode 3, which had much higher activity in the bottom part of the internode, possibly because growth is faster here, hence a higher demand for sucrose cleavage exists here. / AFRIKAANSE OPSOMMING: Hierdie studie het ten doel gehad: 1. om die teenwoordigheid van sukrose sintase (SuSy) isovorme in suikkerriet swelgweefsel te ondersoek 2. om die kinetiese eienskappe van hierdie isovorme te ondersoek 3. om die weefsellokalisering van SuSy in die suikerrietstingel te bepaal Die resultate word hieronder opgesom: Drie SuSy isovorme is gevind in blaarrol weefsel. Die SuSyA en SuSyB isovorme het verskil in terme van ladingseienskappe, met SuSyA wat nie aan ‘n anioonuitruilkolom gebind het nie waaraan SuSyB en SuSyC wel onder dieselfde kondisies gebind het. Beide SuSyB en SuSyC isovorme is geëlueer van die kolom teen 180 mM KCl. Die SuSyA en SuSyB isovorme was teenwoordig gedurende herfs, maar in die winter was slegs SuSyC teenwoordig. Ten spyte van die feit dat SuSyB en SuSyC teen dieselfde soutkonsentrasie geëlueer is, het hulle verskillende isovorme verteenwoordig, aangesien hulle kinetiese en immunologiese eienskappe verskil het. SuSyB en SuSyC kon nie mengsels van dieselfde isovorme gewees het nie, want ‘n poliklonale antiserum teen SuSyB, wat SuSyB geïnaktiveer het, het nie SuSyC geïnaktiveer nie. Al drie isovorme het betekenisvol verskil wat kinetiese eienskappe betref, met die SuSyA isovorm wat ook ‘n baie laer sukrose afbraak/sintese verhouding gehad het as die ander twee isovorme. Daar is dus ten minste drie SuSy isovorme teenwoordig op die proteïen vlak in suikerriet blaarrol weefsel. Die in-detail kinetiese analise van die SuSyC isovorm het getoon dat die ensiem ‘n geordende drietallige kompleks meganisme het, met UDP wat eerste bind en UDP-glukose wat laaste dissosieer. Die eksperimenteel bepaalde kinetiese parameters is toe gebruik om ‘n kinetiese model van sukrose akkumulering uit tebrei. Data het getoon dat wanneer die generiese SuSy kinetiese parameters in die oorspronklike model vervang word met die eksperimenteel bepaalde waardes, die berekende sukrose konsentrasie met ongeveer 40 % toeneem, terwyl die fruktose konsentrasie ongeveer 7 keer afneem. Hierdie resultaat toon die groot fisiologiese effek wat die uitdrukking van verskillende SuSy isovorme op suikermetabolisme kan hê. Die SuSy proteïen lokaliseringsdata, wat met ‘n immunohistochemiese benadering verkry is, het aangedui dat SuSy in beide bergingsparenchiemselle sowel as vaatweefsel teenwoordig is in jong, intermediêre en volwasse internodes. SuSy ensiemaktiwiteit in verskillende dele van die internodes was soortgelyk, behalwe in internode 3, wat baie hoër aktiwiteit gehad het in die onderste deel van die internode as bo, moontlik weens vinniger groei in hierdie deel van die internode, wat afhanklik is van afbraakprodukte van sukrose.