Thesis (MScAgric)--Stellenbosch University, 1977. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming

Thesis (MScAgric)--Stellenbosch University, 1978. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming

Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The importance of small antimicrobial peptides in the innate immune system of plants became increasingly apparent over the past decade. Antimicrobial peptides are unique and diverse molecules that are found in many tissue types in a variety of invertebrate, plant and animal species. Many of these peptides, such as plant defensins, have been found to be ubiquitous throughout the plant kingdom and have been isolated from flowers, leaves, roots, seeds, seedlings, pods, tubers and bark. The growing relevance of antimicrobial peptides (including plant defensins) in research can be largely attributed to their broad-spectrum antifungal activity. This makes them promising potential targets, both as therapeutic agents and for their use in crop protection and disease resistance. The continuing discovery of novel antimicrobial peptides has advanced the development of strategies to overexpress these genes in plants to attempt to enhance the plant’s natural ability to resist pathogenic attack. The first grapevine antifungal peptide, Vv-AMP1, was isolated and characterized and was shown to be tissue specific and developmentally regulated, being expressed only in berries at the onset of berry ripening. The peptide showed strong antifungal activity against a number of plant pathogenic fungi in vitro. In this study, the biological role of the Vv-AMP1 peptide was further investigated, both within its native host (Vitis vinifera) and under in vitro conditions against a panel of grapevine-specific pathogens. As a first step, recombinant production of Vv-AMP1 using an existing bacterial expression system was evaluated and the heterologous production of the Vv-AMP1 peptide improved. Specific optimizations targeting both production and purification of the peptide showed to improve the yield of Vv-AMP1. Steps in the production process targeted for improvement included induction conditions of peptide production by the bacterial culture as well as a number of purification steps, such as lysate preparation, binding conditions, column washing, elution conditions and thrombin protease cleavage. The optimized purification method produced up to 3 mg of pure Vv-AMP1 peptide from 1.6 L of overnight culture. While production was markedly improved, the resultant purified Vv-AMP1 proved biologically inactive and structurally unstable. This is uncharacteristic of the peptide, suggesting that an important aspect necessary for peptide activity, such as folding or the presence of specific co-factors might not be supported in this non-host prokaryotic production system. The study also entailed the characterization and evaluation of the Vv-AMP1 peptide against a panel of grapevine-specific pathogens that are culturable to sporulating cultures using in vitro antifungal assays and microscopy analysis. Vv-AMP1 showed strong inhibitory activity against all pathogens tested, inhibiting the growth of Diplodia seriata and Cylindrocarpon liriodendri by 50% at concentrations between 4.8 μg/ml and 9.6 μg/ml. Phaemoniella chlamydospora and Phomopsis viticola proved particularly sensitive, with IC50 values of 5.5 μg/ml and 4.0 μg/ml respectively. Microscopy analysis of the effect of the Vv-AMP1 peptide on P. viticola showed a severe inhibition on fungal germination and growth. The peptide did not induce morphological changes in fungal hyphae but compromises the fungal membranes, supporting the theory that the peptide induces membrane permeabilization. Functional analysis of a transgenic V. vinifera (cv. Sultana) population overexpressing Vv-AMP1 was included in this study to provide the opportunity to study the in planta role of the peptide in its native host. The genetic characterization of the putative population included confirming gene integration and copy number through PCR and Southern blot analysis as well as gene expression through northern blot analysis. A confirmed transgenic population was evaluated for improved disease resistance against Botrytis cinerea as a first test organism in an attempt to link the overexpression of the Vv-AMP1 gene to a disease resistance phenotype. Observations of lesion type, average lesion size and further statistical analysis concluded that the transgenic population showed a definite, albeit slight, improved resistance when compared to the untransformed control lines. In conclusion, the study determined that Vv-AMP1 had a strong antifungal action against grapevine-specific pathogenic fungi when tested in vitro. A definite link could be established between the overexpression of Vv-AMP1 and a mild resistance phenotype within its native host plant. The characterized transgenic population is important for further work to evaluate the in planta activity of the peptide against more grapevine pathogens such as the stem pathogens that were proven sensitive and specifically those that cannot be cultured and are obligate pathogens, such as the downy and powdery mildews. / AFRIKAANSE OPSOMMING: Die belang van klein antimikrobiese peptiede in die ingebore immuunstelsel van plante het tydens die afgelope dekade toenemend duidelik geraak. Antimikrobiese peptide is unieke en diverse molekules wat in verskeie weefseltipes in ‘n verskeidenheid van invertebraat-, plant- en dierspesies gevind word. Baie van hierdie peptiede, soos bv. “plant defensins”, word bevind om alomteenwoordig in die plantryk te wees en is reeds geïsoleer vanuit blomme, blare, wortels, sade, saailinge, peule, knolle en bas. Die toenemende belang van antimikrobiese peptiede (insluitend “plant defensins”) in navorsing kan grootliks toegeskryf word aan hul breë-spektrum antifungiese aktiwiteit. Hierdie eienskap maak hul belowende potensiële teikens, beide as terapeutiese middels asook vir gebruik in gewasbeskerming en siekteweerstand. Die voortdurende ontdekking van nuwe antimikrobiese peptiede bevorder tans die ontwikkeling van strategieë om hierdie gene in plante uit te druk in ‘n poging om die plant se natuurlike vermoeë om patogeniese aanval teen te staan te verbeter. Die eerste wingerd antifungale peptied, Vv-AMP1, is geïsoleer en gekarakteriseer as ‘n ontwikkelings-gereguleerde peptied wat slegs uitgedruk word in korrels, tydens die aanvang van bessie rypwording. Die peptied het tydens in vitro toetse sterk antifungale aktiwiteit getoon teen ‘n verskeidenheid plant-patogeniese swamme. In hierdie studie word die biologiese rol van die Vv-AMP1 peptied verder ondersoek, beide binne sy natuurlike gasheerplant, (Vitis vinifera) asook onder in vitro kondisies teen ‘n paneel van wingerd-spesifieke patogene. As ‘n beginpunt is rekombinante produksie van Vv-AMP1 met behulp van ‘n bakteriële ekspressie sisteem evalueer en die hetereloë produksie van die Vv-AMP1 peptied stelselmatig verbeter. Spesifieke optimerings het gefokus op beide die produksie en suiwering van die peptied en het die algehele opbrengs van Vv-AMP1 verhoog. Spesifieke stappe wat in die produksieproses vir verbetering geteiken is sluit beide induksietoestande van peptiedproduksie deur die bakteriële kultuur in sowel as ‘n aantal suiweringsstappe, soos lisaatvoorbereiding, bindingskondisies, kolom wasstappe, eluasie kondisies en “thrombin” protease snyding in. Die optimale suiweringsmetode het tot 3 mg suiwer Vv-AMP1 peptied opgelewer vanaf ‘n 1.6 L oornag bakteriële kultuur. Hoewel die produksie van die peptide noemenswaardig verbeter is, was die gesuiwerde Vv-AMP1 beide onaktief en struktureel onstabiel. Dit is buitengewoon vir hierdie peptied, wat daarop dui dat belangrike aspekte benodig vir antifungiese aktiwiteit, soos korrekte vou of die teenwoordigheid van spesifieke kofaktore, moontlik ontbreek in hierdie nie-gasheer prokariotiese produksiesisteem. Die studie het ook die karakterisering en evaluering van die Vv-AMP1 peptied teen 'n paneel van wingerd-spesifieke patogene wat kultureerbaar is en sporuleer, insluitend in vitro antifungale toetse en mikroskopiese analise, behels. Vv-AMP1 toon sterk inhiberende aktiwiteit teen alle patogene getoets. Dit inhibeer die groei van Diplodia seriata en Cylindrocarpon liriodendri met 50% teen konsentrasies tussen 4.8 μg/ml en 9.6 μg/ml. Phaemoniella chlamydospora en Phomopsis viticola was besonders sensitief, met IC50 waardes van 5.5 μg/ml en 4.0 μg/ml, onderskeidelik. Mikroskopiese analise van die effek van die Vv-AMP1 peptied op P. viticola het 'n ernstige inhibisie op swam ontkieming en groei aangedui. Die peptied het geen morfologiese veranderinge in swam hifes veroorsaak nie maar het wel die swam membraan beskadig. Hierdie bevinding ondersteun die teorie dat die peptied membraan permeabilisasie induseer. Funksionele analise van ‘n transgeniese V. vinifera (cv. Sultana) populasie wat die Vv-AMP1 geen ooruitdruk is by die studie ingesluit om ‘n geleentheid te bied om die in planta rol van die peptide binne sy natuurlike gasheerplant te bestudeer. Die genetiese karakterisering van die vermeende transgeniese bevolking het die bevestiging van beide geenintegrasie en kopiegetal deur PKR en Southern-klad analise ingesluit, sowel as geenuitdrukking d.m.v. noordelike-klad analise. ‘n Bevestigde transgeniese bevolking is evalueer vir potensiële verbeterde weerstand (in vergelyking met die wilde tipe) deur infeksie met Botrytis cinerea as ‘n eerste toetsorganisme in ‘n poging om ‘n weerstandbiedende fenotipe met die ooruitdrukking van Vv-AMP1 te assosieer. Waarnemings van letsel tipe, letsel grootte en verdere statistiese analise het tot die gevolgtrekking gelei dat die transgeniese bevolking ‘n definitiewe (dog geringe) verbeterde weerstand toon in vergelyking met die ongetransformeerde lyne. Ten slotte bepaal die studie dat Vv-AMP1 ‘n sterk antifungale effek teen wingerdspesifieke patogene toon tydens in vitro toetse. ‘n Definitiewe korrelasie is vasgestel tussen die ooruitdrukking van Vv-AMP1 in wingerd en ‘n weerstandsfenotipe in die transgeniese bevolking. Die gekarakteriseerde transgeniese bevolking is uiteraard belangrik vir toekomstige werk om die in planta aktiwiteit van die peptied te evalueer teen verdere wingerdpatogene soos bv. die stampatogene wat sensitief getoets het teen die peptide, asook patogene wat nie kultureerbaar is nie, insluitend verpligte patogene soos dons- en poeierskimmel.

Thesis (MScAgric)--Stellenbosch University, 1950. / No Abstract Available

Thesis (MScAgric)--Stellenbosch University, 1975. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The latest strategies in the protection of crops against microbial pathogens are rooted in harnessing the natural, highly complex defense mechanisms of plants through genetic engineering to ultimately reduce the application of chemical pesticides. This approach relies on an in-depth understanding of plant-pathogen interactions to develop reasonable strategies for plant improvement. Among the highly specialized defense mechanisms in the plant’s arsenal against pathogen attack, is the de novo production of proteinaceous antimicrobial peptides (AMPs) as part of the plant’s innate immunity. These AMPs are small, cysteine-rich peptides such as plant defensins that are known for their broad-spectrum of antifungal activity. These plant defensin peptides have been found to be present in most, if not all plant species and the defensin encoding genes are over-represented in plant genomes. Most of these defensins are generally the products of single genes, allowing the plant to deliver these molecules relatively rapidly and with minimal energetic expense to the plant. These factors contribute to establishing AMPs as excellent candidates for genetic engineering strategies in the pursuit of alternative crop protection mechanisms. The first antimicrobial peptide identified and isolated from grapevine, Vv-AMP1, was found to be developmentally regulated and exclusively expressed in berries from the onset of ripening. Recombinantly produced Vv-AMP1 showed strong antifungal activity against a wide range of plant pathogenic fungi at remarkably low peptide concentrations in vitro, however, no in planta defense phenotype could thus far be linked to this peptide. In this study, the antifungal activity of Vv-AMP1 constitutively overexpressed in its native host (Vitis vinifera) was evaluated against grapevine-specific necrotrophic and biotrophic fungi. Firstly, a hardened-off genetically characterised transgenic V. vinifera (cv. Sultana) population overexpressing Vv-AMP1 was generated and morphologically characterized. In order to evaluate the in planta functionality of Vv-AMP1 overexpressed in grapevine, this confirmed transgenic population was subjected to antifungal assays with the necrotrophic fungus, B. cinerea and the biotrophic powdery mildew fungus, Erysiphe necator. For the purpose of infection assays with a biotrophic fungus, a method for the cultivation and infection with E. necator was optimized to generate a reproducible pathosystem for this fungus on grapevine. Detached leaf assays according to the optimized method with E. necator revealed programmed cell death (PCD) associated resistance linked to overexpression of Vv-AMP1 that can be compared to that of the highly resistant grapevine species, Muscadinia rotundifolia. Contrastingly, whole-plant infection assays with B. cinerea revealed that Vv-AMP1 overexpression does not confer V. vinifera with elevated resistance against this necrotrophic fungus. An in silico analysis of the transcription of defensin-like (DEFL) genes previously identified in grapevine was included in this study. This analysis revealed putative co-expression of these DEFL genes and other genes in the grapevine genome driven by either tissue- or cultivar specific regulation or the plant’s response to biotic and abiotic stress stimuli. In conclusion, this study contributed to our knowledge regarding Vv-AMP1 and revealed an in planta defense phenotype for this defensin in grapevine. In silico analysis of the DEFL genes in grapevine further revealed conditions driving expression of these genes allowing for inferences to be made regarding the possible biological functions of DEFL peptides in grapevine. / AFRIKAANSE OPSOMMING: Die nuutste strategieë wat deel vorm van die beskerming van plant gewasse teen mikrobiese patogene het hul oorsprong in die inspanning van die natuurlike, hoogs gekompliseerde verdedigingsmeganismes van die plant deur middel van genetiese enginieurswese ten einde die gebruik van chemiese plaagdoders te verlaag. Hierdie benadering maak staat op ‘n in-diepte begrip van plant-patogeen interaksies om verstandige strategieë vir plantverbetering te kan ontwikkel. Van hierdie hoogs-gespesialiseerde verdedigingsmeganismses in die plant se arsenaal teen patogeen aanvalle sluit die de novo produksie van proteinagtige antimikrobiese peptiede (AMPs) in as deel van die plant se ingebore immuunstelsel. Hierdie AMPs is klein, sisteïen-ryke peptiede soos die plant “defensins” en is bekend vir hul breë-spektrum antifungiese aktiwiteit. Hierdie plant defensinpeptiede word aangetref in meeste, indien nie alle plant spesies nie en die defensin koderende gene word oor-verteenwoordig in plant genome. Meeste van hierdie defensins is gewoonlik die produkte van enkele gene wat die plant in staat stel om hierdie molekules relatief spoedig en met minimale energie verbruik in die plant te vorm. Hierdie faktore dra by tot die vestiging van AMPs as uitstekende kandidate vir genetiese ingenieursstrategieë as deel van die strewe na alternatiewe gewasbeskermingsmeganismes. Die eerste antimikrobiese peptied wat geïdentifiseer en geïsoleer is uit wingerd, Vv-AMP1, word beheer deur die ontwikkelingsstadium en word eksklusief uitgedruk in korrels vanaf die aanvang van rypwording. Rekombinant-geproduseerde Vv-AMP1 het sterk antifungiese aktiwiteit getoon teen ‘n wye reeks plantpatogeniese swamme teen merkwaardige lae peptied konsentrasies in vitro, alhoewel geen in planta verdedigingsfenotipe tot dusver gekoppel kon word aan hierdie peptied nie. In hierdie studie was die antifungiese aktiwiteit van Vv-AMP1 wat ooruitgedruk is in sy natuurlike gasheerplant (Vitis vinifera) ge-evalueer teen wingerd-spesifieke nekrotrofiese- en biotrofiese swamme. Eerstens is ‘n afgeharde geneties-gekarakteriseerde transgeniese V. vinifera (cv. Sultana) populasie wat Vv-AMP1 ooruitdruk gegenereer en morfologies gekarakteriseer. Om die in planta funksionaliteit van Vv-AMP1 ooruitgedruk in wingerd te evalueer is hierdie bevestigde transgeniese populasie blootgestel aan antifungiese toetse met die nekrotrofiese swam, B. cinerea en die biotrofiese swam, Erysiphe necator. Vir die doel om infeksiestudies uit te voer met ‘n biotrofiese swam is ‘n metode geoptimiseer vir die kweek en infeksies met E. necator wat gelei het tot ‘n herhaalbare patosisteem vir hierdie swam op wingerd. Blaarstudies, volgens die pas-verbeterde metode vir E. necator infeksies het ‘n geprogrammeerde seldood-geassosieërde weerstand, gekoppel aan die ooruitdrukking van Vv-AMP1 onthul, wat vergelyk kan word met dié van die hoogs-weerstandige wingerdspesie, Muscadinia rotundifolia. Hierteenoor het heel-plant infeksie studies met B. cinerea onthul dat Vv-AMP1 ooruitdrukking geen verhoogde weerstand teen dié nekrotrofiese swam aan V. vinifera bied nie. ‘n In silico analise van die transkripsie van defensin-agtige (DEFL) gene wat vroeër in wingerd geïdentifiseer is, is by hierdie studie ingesluit. Hierdie analise het vermeende gesamentlike uitdrukking van hierdie DEFL gene en ander gene in die wingerd genoom onthul wat aangedryf word deur weefsel- of kultivar-spesifieke regulering of die plant se reaksie tot biotiese en abiotiese stress stimuli. Ten slotte, hierdie resultate het bygedra tot ons kennis in verband met Vv-AMP1 en het ‘n in planta verdedigingsfenotipe vir hierdie defensin in wingerd onthul. In silico analiese van die DEFL gene in wingerd het verder toestande onthul wat die uitdrukking van hierdie gene aandryf wat ons toelaat om aannames te maak ten opsigte van die moontlike biologiese funksies van DEFL peptiede in wingerd en ondersteun die opstel en toets van hipoteses vir die rol en megansimes van aksie van die wingerd defensin familie.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: There is a growing international consumer demand for the production of lower ethanol wines. This can be attributed to various qualitative, social, economic and health concerns that are associated with high ethanol wines (Kutyna et al., 2010; Varela et al., 2012). There is continuous development and research into methods and technologies to lower the ethanol concentration in wine. However, in addition to the added cost and complexity these technologies all have various shortcomings. The development of yeast strains with lower ethanol productivity, yet desirable organoleptic and fermentation capacity, therefore remains a highly sought after research and development target in the wine industry. Biologically based approaches aim to generate yeast strains with the capacity to divert carbon from ethanol production towards targeted metabolic endpoints (Kutyna et al., 2010). This should ultimately be achieved without the production of unwanted metabolites that can negatively affect wine characteristics. In the context of these challenges, this study aimed to investigate the use of fructans as carbon sinks during fermentation to divert fructose from glycolysis and ethanol production toward intracellular fructan production by generating levan producing strains. In addition, the impact of fructan production on metabolic carbon flux during fermentation by these strains was analyzed. This was the first attempt to analyze intracellular fructan production in Saccharomyces cerevisiae under fermentative conditions with fructans acting as carbon sinks. Fructans are fructose polymers that act as storage molecules in certain plants and function as part of the extracellular matrix in microbial biofilms, and are intensively studied due to their economic interest. Here we undertook the heterologous expression of a levansucrase (LS) M1FT from Leuconostoc mesenteroides, an enzyme producing β(2-6) levan-type fructans, in the S. cerevisiae BY4742Δsuc2 strains without invertase activity (encoded by SUC2). Levansucrases indeed utilize sucrose as both fructose donor and initial polymerization substrate, and the sucrose concentration is of import to maintain transfructosylation activity of enzyme. High intracellular sucrose accumulation was achieved by the heterologous expression of either a sucrose synthase (Susy; cloned from potato) or by growing strains expressing the spinach sucrose transporter (SUT) in sucrose containing media. Endogenous sucrose synthesis was of specific interest to the overall goal of the project, which was to reroute carbon flux away from glycolysis in grape must containing only hexoses as carbon source. In addition, this approach of combining intracellular sucrose production with intracellular levan production could be used in various applications to limit the need for sucrose in media as both carbon source and LS substrate. The extracellular LS M1FT was introduced into Susy and SUT strains as either the complete gene (M1FT) or 50bp truncation (M1FTΔsp) without the predicted signal peptide. The data show that intracellular levan accumulation occurred in aerobic, but not anaerobic conditions. The data also suggest that the production of levan did not impact negatively on general yeast physiology or metabolism in these conditions. However, no significant reduction in ethanol yields were observed, suggesting that further optimisation of the expression system is required. This is the first report of levan synthesis by S. cerevisiae, and contributes towards expanding the possibilities for further industrial applications of these compounds. / AFRIKAANSE OPSOMMING: Daar is toenemende aanvraag deur wynverbruikers na laër alkohol wyne. Hierdie neiging kan toegeskryf word aan verskeie kwalitatiewe, gesondheids en sosio-ekonomiese redes wat geassosieer word met die verbruik van hoër alkohol wyne. Daar is ’n deurlopende navorsing dryf toegespits op metodes en tegnologieë om die alkohol konsentrasie van wyne te verlaag. Hierdie tegnologieë het egter, bykomstig tot koste en kompliksiteits toename, verkeie tekortkominge. Die ontwikkeling van gisrasse met verlaagde alcohol produksie, maar steeds wenslike organoleptiese en fermentasie eienskappe, bly ‘n baie gesogte navorsings en ontwikkeling teiken in die internasionale wyn industrie. Biologiese benaderings streef om gisrasse te genereer met die vermoë om koolstof weg van etanol produksie te herlei na geteikende metabolise eindpunte. Hierdie doelwit moet ook uiteindelik bereik word sonder die produksie van ongewenste metaboliete wat die wyn negatief kan affekteer. In die konteks van hierdie uitdaging, het hierdie studie gestreef om die gebruik van fruktane as ’n koolstof poel tydens fermentasie, met die doel om fruktose te herlei vanaf glikolise en etanol produksie na intrasellulêre fruktane produksie. Om hierdie doelwit te bereik, is gisrasse ontwikkel wat levaan (’n spesifieke fruktaan) produseer. Die impak van fruktaan produksie op metaboliese koolstof vloei tydens fermentasie deur hierdie gisrasse is bykomsrig ontleed. Hierdie verslag beskryf die eerste poging om intraselullêre fruktaan produksie in Saccharomyces cerevisiae te bewerkstellig, met die doel om fruktaan as ’n koolstof poel te gebruik. Fruktane is fruktose polimere wat as bergings molekules optree in sekere plante en ook funksioneer as deel van die ekstrasellulêre matriks in mikrobiese biofilms. Hierdie polimere word tans internasionaal intensief bestudeer weens hul ekonomiese belang. Hierdie studie beskryf die uitdrukking van die levaansukrase (LS) M1FT van Leuconostoc mesenteroides, wat β(2-6) levaan-tipe fruktane produseer, in S. cerevisiae BY4742Δsuc2 rasse, sonder invertase (gekodeer deur SUC2). Levaansukrases gebruik inderdaad sukrose as beide ’n fruktose donor en ook as ’n aanvanklike polimeriserings substraat. Die fruktose konsentrasie is belangrik om transfruktosilerings aktiwiteit van die ensiem te handhaaf. Hoë intrasellulêre sukrose akkumulasie was bereik deur die heteroloë uitdrukking van ’n sukrose sintase (Susy; gekloneer van aartappel) of die spinasie sukrose transporter (SUT) in media bevattende sukrose. Endogene sukrose sintese was van spesifieke belang tot die algehele doelwit om koolstof te herlei, weg van glikolise tydens fermentase van druiwe sap. Die benadering om intraselullêre sukrose produksie met levaan produksie te koppel, kan ook gebruik word in verskeie toepassings om die afhanklikheid op sukrose in die media, as substraat vir LS, te verminder. Die ekstraselullêre LS, M1FT, was as vollengte geen (M1FT) of as ’n 50bp afkapping (M1FTΔsp), sonder seinpeptied, in die Susy en SUT gisrasse uitgedruk. Die data dui aan dat die produksie van levaan nie ’n negatiewe impak het op gis fisiologie of metabolisme in die toets kondisies nie. Daar was egter geen waarbeenbare afname in etanol opbrengs nie, wat aandui dat verdere optimisering van ekspressie sisiteem benodig word. Hierdie is die eerste verslag van levaan sintese in S. cerevisiae en dra by tot die uitbreiding van moontlikhede vir indutriële toepassings van die die verbindings. / IWBT and NRF

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Proteins from various sources, including grape berry cells, yeast, bacteria and fining agents e.g. albumin and casein, have previously been identified in wine. These proteins play various critical roles in the functioning and survival of the organisms that produced them but also exhibit oenological properties, once secreted in the juice/wine. Some of them can indeed be beneficial to winemaking, by releasing aroma compounds from grape-derived precursors, or detrimental to wine quality, by causing protein haze. Yeasts contribute significantly to the protein pool during and after alcoholic fermentation. However, while the extracellular proteins of Saccharomyces cerevisiae, the main wine yeast species, have been characterised, those of non-Saccharomyces yeasts remain largely unknown, especially under winemaking conditions. Although specific extracellular enzymes released by non-Saccharomyces yeasts have been the focus of many studies in recent years, the targeted approaches used have restricted our knowledge to these specific enzymes and excluded the other secreted proteins. A more comprehensive insight into entire secretomes could improve our understanding of how yeasts survive in wine and interact with other species in mixed culture fermentations. This study aims to characterise the exo-proteome of Saccharomyces and selected non-Saccharomyces yeasts in pure and mixed cultures in a wine-like medium. Fermentation kinetics were monitored and the extracellular proteins isolated at the end of fermentation. M. pulcherrima hardly fermented whereas L. thermotolerans fermented slowly but steadily. As expected S. cerevisiae completed the fermentation rapidly. In sequential fermentations, the kinetics resembled those of the non-Saccharomyces yeasts for a period before switching to that of S. cerevisiae. This period varied from 4 to 15 days for M. pulcherrima and L. thermotolerans respectively. Visual observations of the protein content of the medium at the end of fermentation using 1D and 2D SDS-PAGE gels as well as identification of these proteins using mass fingerprinting revealed the large variety of proteins secreted and the influence of yeast interactions on each other’s secretome. The fermentation kinetics observed could partially be explained by the extent of the contribution of the different yeast to the protein content. Proteins secreted by non-Saccharomyces yeasts lowered the potential of wine to form protein haze, with both M. pulcherrima and L. thermotolerans in pure and mixed culture fermentations showing lower haze formation than S. cerevisiae. As far as we know, this is the first report on the secretome of non-Saccharomyces under winemaking condition and the influence non-Saccharomyces proteins have on the protein haze potential of wine, providing the basis for future investigations. / AFRIKAANSE OPSOMMING: Proteïene vanaf verskeie bronne (insluitend druiwe korrels, gis, bakterieë en verhelderings agente bv. albumien en kaseïen) is reeds in wyn identifiseer. Hierdie proteïene speel verskeie rolle in die funksionering en oorlewing van die organismes wat dit produseer, maar beskik ook oor wynkundige eienskappe sodra dit in die sap of wyn uitgeskei word. Hoewel sommige proteïene in wyn wel voordelig mag wees as gevolg van die vrystelling van aroma komponente vanuit druif‐voorlopers, kan dit ook nadelig wees vir wyn kwaliteit deur die troebelheid wat dit kan veroorsaak Gis dra aansienlik by tot die totale proteïen inhoud van wyn, beide gedurende asook na alkoholiese fermentasie. Alhoewel die ekstrasellulêre proteïene van Saccharomyces cerevisiae (die mees algemeen gebruikte gis vir wynmaak) reeds goed gekarakteriseer is, is die proteïene van nie-Saccharomyces giste grootliks onbekend, veral die wat tydens wynmaak vrygestel word. Gedurende die laaste paar jaar het verskeie studies gefokus op spesifieke ekstrasellulêre ensieme wat deur nie-Saccharomyces giste produseer word, maar geteikende benaderings het ons kennis beperk tot net hierdie spesifieke ensieme, en enige ander afgeskeide proteïene uitgesluit. ʼn Meer omvattende insig oor die algehele afgeskeide proteoom kan ons begrip van hoe gis in wyn oorleef en interaksies tussen gis spesies in gemengde kultuur fermentasies verbeter Hierdie studie streef om die sekretoom van Saccharomyces en geselekteerde nie-Saccharomyces giste in suiwer en gemengde kultuur fermentasies van sintetiese wyn medium te karakteriseer. Fermentasie kinetika is gemonitor en die ekstrasellulêre proteïene is teen die einde van fermentasie geïsoleer. Metschnikowia pulcherrima het swak fermenteer terwyl Lachancea thermotolerans stadig tog reëlmatig fermenteer het. Soos verwag, het S. cerevisiae vinnig tot droog fermenteer. In agtereenvolgend geïnokuleerde fermentasies is die kinetika vir ʼn tydperk soortgelyk aan die van die nie-Saccharomyces giste voordat dit oorskakel na die van S. cerevisiae. Hierdie tydperk wissel respektiewelik vanaf 4 tot 15 dae vir M. pulcherrima en L. thermotolerans. Visuele waarnemings van die proteïen-inhoud van die medium aan die einde van die gisting met behulp van 1D en 2D SDS-PAGE gels asook identifisering van hierdie proteïene met behulp van massa vingerafdrukke onthul die groot verskeidenheid proteïene wat afgeskei word, asook die invloed van die giste se interaksies op mekaar se sekretoom. Die fermentasie kinetika waargeneem kan gedeeltelik verklaar word deur die omvang van die bydrae van die verskillende gis tot die proteïen-inhoud. Proteïene wat afgeskei word deur nie-Saccharomyces giste verlaag die potensiaal van wyn om proteïen troebelheid te vorm, met beide M. pulcherrima en L. thermotolerans (in suiwer en gemengde kultuur fermentasies) wat minder troebelheid vorm as fermentasies met S. cerevisiae. Sover ons kennis strek, is hierdie die eerste verslag oor die sekretoom van nie- Saccharomyces onder wynmaak toestande en ook oor die invloed wat nie-Saccharomyces proteïene op die proteïen troebelheid van wyn het, en vorm die basis vir toekomstige navorsing. / Winetech and THRIP

Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The microclimate environment around the bunch is complex. The spatial distribution of leaves as well as bunch position and morphology impact on the path of direct radiation received by the berries. Canopy microclimate is largely determined by the meteorological conditions (air temperature, solar radiation, wind speed and direction, relative humidity, and precipitation) as well as management practices (trellis/training system, canopy height, vine spacing, row orientation, canopy management practices, irrigation and soil variability and management). The fact that the grapevine continuously responds to its environment, adds to the complexity and dynamic nature of the microclimate that the bunches experience. Field studies involving the effect of the natural bunch environment (i.e. light and temperature conditions) on berry composition, are therefore a challenge, due to the difficulties in quantifying meteorological elements such as temperature and light, which can be hugely variable. There are different sensors available to assess bunch and berry temperature and it can be deployed in different ways within the grapevine canopy, but the difficulty remains in studying the variability that exists within a bunch. This study investigated the value of available sensor technology to measure bunch/berry temperature as well as the spatial and temporal variability on a bunch. Differences in temperature on an intra-berry level were assessed whereas the impact of canopy configuration and bunch orientation on the different sensor levels was also investigated. The contribution of macro- and mesoclimate on bunch and berry temperature was addressed by measuring at two locations (Robertson and Stellenbosch). The potential long term differences in temperature within a bunch with regard to thermal accumulation are discussed. Issues around sensor placement and some technical difficulties related to the sensors are discussed. The results indicated how the effects of mesoclimate were transferred through to the different sensors. A dominating effect of the sea breeze in Stellenbosch was found. Canopy configuration/architecture affected the light regime in the canopy, consequently impacting on bunch temperature variability, specifically in Stellenbosch where a "Ballerina" trellising system was used. Bunch orientation resulted in differences in the temporal variability of bunch/berry temperature and little variability was observed in temperature within the berry. Temperatures of berries situated at the back of the bunch were judged more optimal compared to exposed berries. Direct radiation caused extreme temperatures in exposed berries, which may be detrimental to berry composition and wine quality. This emphasized the importance of the canopy (trellis/training system and management practices) in protecting the bunch from extreme conditions. The large on-bunch spatial variability, observed from measurements with the thermal imager, demonstrated the importance of sensor placement in quantifying the bunch temperature regime; this is also relevant for the future development of berry temperature modelling. Thermal accumulation through the season also illustrated the variability that existed within a bunch, suggesting a potential long term effect on the berry composition. This study proved, in conditions similar to those that may prevail in the South African wine industry, that sensor type and positioning need to be carefully considered in any viticultural/oenological study where bunch microclimate and grape temperatures are assessed. / AFRIKAANSE OPSOMMING: Die mikroklimaat omgewing rondom die tros is kompleks. Die ruimtelike verspreiding van blare sowel as trosposisie en -morfologie het 'n impak op die pad waarlangs direkte straling ontvang word deur die korrels. Lowermikroklimaat word grootliks bepaal deur die meteorologiese kondisies (lugtemperatuur, sonstraling, windspoed en -rigting, relatiewe humiditeit en reënval) sowel as bestuurspraktyke (prieel/opleistelsel, lowerhoogte, wingerdstokspasiëring, ry-oriëntasie, lowerbestuurspraktyke, besproeiing asook grondvariasie en bestuur). Die feit dat die wingerdstok voortdurend reageer op sy omgewing dra by tot die kompleksiteit en dinamiese aard van die mikroklimaat wat die trosse ervaar. Veldstudies gemoeid met die effek van die natuurlike trosomgewing (d.w.s. lig- en temperatuurkondisies) op korrelsamestelling is daarom 'n uitdaging. Die rede hiervoor is dat dit problematies is om meteorologiese elemente soos temperatuur en lig, wat baie veranderlik kan wees, te kwantifiseer. Verskillende sensors is beskikbaar waarmee tros- en korreltemperatuur bepaal kan word en dit kan op verskillende wyses binne die wingerdstoklower aangewend word. Die bestudering van die variasie wat bestaan binne 'n tros is egter steeds problematies. Hierdie studie het die waarde ondersoek van die beskikbare sensortegnologie vir die meting van tros/korreltemperatuur en die ruimtelike en tydsvariasie op 'n tros. Verskille in temperatuur op 'n intra-korrelvlak is bepaal terwyl die impak van lowerkonfigurasie en trosoriëntasie op die verskillende sensorvlakke ook ondersoek is. Die bydrae van makro- en mesoklimaat tot tros- en korreltemperatuur is ondersoek deur te meet by twee verskillende liggings (Robertson en Stellenbosch). Die potensiële langtermyn verskille in temperatuur binne-in 'n tros met betrekking tot temperatuur akkumulasie word bespreek. Kwessies rakende sensorplasing en sommige tegniese probleme wat verband hou met sensors word bespreek. Die resultate het aangedui hoedat die effekte van mesoklimaat oorgedra is na die verskillende sensors. 'n Dominerende effek van die seebries is waargeneem in Stellenbosch. Lowerkonfigurasie/argitektuur het die ligregime in die lower beïnvloed en gevolglik 'n invloed gehad op die trostemperatuur veranderlikheid. Dit was veral die geval in Stellenbosch waar 'n "Ballerina" opleistelsel gebruik is. Trosoriëntasie het gelei tot verskille in tydsvariasie van tros/ korreltemperatuur en min variasie is waargeneem in temperatuur binne die korrel. Temperature van korrels wat voorkom aan die agterkant van die tros is beoordeel as meer optimaal vergeleke met blootgestelde korrels. Direkte straling het uiterste temperature in blootgestelde korrels veroorsaak wat nadelig kan wees vir korrelsamestelling en wynkwaliteit. Hierdeur is die belang van die lower (prieel/opleistelsel en bestuurspraktyke) om die tros te beskerm teen uiterste kondisies beklemtoon. Die groot ruimtelike variasie op 'n tros, soos waargeneem in metings met die termiese kamera, het die belangrikheid van sensorplasing in die kwantifisering van die trostemperatuur regime beklemtoon. Dit is ook relevant vir die toekomstige ontwikkeling van korreltemperatuur modellering. Termiese akkumulasie gedurende die seisoen is ook geïllustreer deur die veranderlikheid wat voorkom binne 'n tros, wat dui op 'n potensiële langtermyn effek op die korrelsamestelling. Hierdie studie het bewys, in kondisies wat algemeen voorkom in die Suid-Afrikaanse wynbedryf, dat sensortipe en -plasing sorgvuldig in ag geneem moet word in enige wingerd/wynkundige studie waar trosmikroklimaat en druiftemperature bepaal word. / Winetech and the National Research Foundation for their financial support of the project

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: There is a global demand for technologies to reduce ethanol levels in wine without compromising wine quality. While several chemical and physical methods have been developed to reduce ethanol in finished wine, the target of an industrially applicable biological solution has thus far not been met. Most attempted biological strategies have focused on developing new strains of the main fermentative organism, the yeast Saccharomyces cerevisiae. Gene modification approaches have primarily focused on partially redirecting yeast carbon metabolism away from ethanol production towards glycerol production. These techniques have met with some moderate success, thus the focus of the current study was to re-direct carbon flux towards trehalose production by moderate over-expression of the TPS1 gene. This gene encodes trehalose-6-phosphate synthase, which converts glucose 6-phosphate and UDPglucose to α,α-trehalose 6-phosphate. Previous data have shown that the overproduction of trehalose restricts hexokinase activity reducing the amount of glucose that enters glycolysis. Nevertheless, preliminary TPS1 over-expression studies using multiple copy plasmids have shown some promise, but also indicated significant negative impact on the general fermentation behaviour of strains. In order to reduce such negative impacts of excessive trehalose production, a new strategy consisting in increasing the expression of TPS1 only during specific growth phases and by a relatively minor degree was investigated. Our study employed a lowcopy number episomal vector to drive moderate over-expression of the TPS1 gene in the widely used industrial strain VIN13 at different stages during fermentation. The fermentations were performed in synthetic must with sugar levels representative of those found in real grape must. This, as well as the use of an industrial yeast strain, makes it easier to relate our results to real winemaking conditions. A reduction in fermentation capacity was observed for all transformed strains and controls. Expression profiles suggest that the DUT1 promoter certainly results in increased TPS1 expression (up to 40%) during early exponential growth phase compared to the wild type strain (VIN13). TPS1 expression under the control of the GIP2 promoter region showed increased expression levels during early stationary phase (up to 60%). Chemical analysis of the yeast and the must at the end after fermentation showed an increase in trehalose production =in line with the expression data of TPS1. Importantly, glycerol production was also slightly increased, but without affecting acetic acid levels for the transformed strains. Although ethanol yield is not significantly lower in the DUT1-TPDS1 strain, s statistically significantly lower ethanol yield is observed for over-expression under the GIP2 promotor. Increasing trehalose production during stationary phase appears therefore to be a more promising approach at lowering ethanol yield and redirecting flux away from ethanol production. This controlled, growth phase specific over expression suggests a unique approach of lowering ethanol yield while not impacting on the redox balance. / AFRIKAANSE OPSOMMING: Wêreldwyd is daar ‘n aanvraag na tegnologie wat die etanol vlakke in wyn kan verminder sonder om wyngehalte te benadeel. Terwyl verskeie chemiese en fisiese metodes ontwikkel is om etanol in die finale wynproduk te verminder, is die soeke na 'n industrieel gebaseerde biologiese oplossing tot dusver nie gevind nie. Meeste biologiese strategieë fokus op die ontwikkeling van nuwe rasse van die primêre fermentatiewe organisme, naamlik Saccharomyces cerevisiae. Geen modifikasie benaderings het hoofsaaklik gefokus op die gedeeltelike kanalisering van koolstof metabolisme weg van etanol produksie na gliserol produksie. Hierdie benadering is net matiglik suksesvol, dus is ons huidige fokus om koolstof te kanaliseer na trehalose produksie deur gematigde oor-uitdrukking van die TPS1 geen. Hierdie geen kodeer vir trehalose-6-fosfaat sintase, wat glukose-6-fosfaat en UDP-glukose omskakel na α, α-trehalose-6-fosfaat. Vorige data het getoon dat die oorproduksie van trehalose hexokinase aktiwiteit beperk en die hoeveelheid glukose wat glikolise binne gaan. Voorlopige TPS1 ooruitdrukking studies met behulp van multi-kopie plasmiede toon matige sukses, maar het ook ‘n negatiewe impak op die algemene fermentasie kapasiteit van die gis. Ten einde so 'n negatiewe impak van oormatige trehalose produksie te oorkom, is 'n nuwe strategie gevolg wat bestaan uit die verhoogde uitdrukking van die TPS1 geen slegs gedurende spesifieke groei fases met baie lae vlakke van oor-uitdrukking. Ons studie gebruik 'n lae-kopie episomale vektor met matige oor-uitdrukking van die TPS1 geen in die industriële ras VIN13 op verskillende stadiums tydens fermentasie. Die fermentasie is uitgevoer in sintetiese mos met suiker vlakke verteenwoordigend van dié van werklike wyn mos. Hierdie, sowel as die gebruik van 'n industriële gisras, maak dit makliker om ons resultate te vergelyk met regte wyn fermentasie kondisies. Verlaagde fermentasie kapasiteit is waargeneem vir alle getransformeerde stamme en hul kontroles. Geen uitdrukkings profiele dui op verhoogde TPS1 uitdrukking (tot 40%) onder beheer van die DUT1 promotor gedurende die vroeë eksponensiële groeifase wanneer vergelyk word met die wilde tiepe (VIN13). TPS1 uitdrukking onder die beheer van die GIP2 promotor het verhoogde uitdrukking van tot 60% gedurende die vroeë stasionêre fase. Chemiese analise van die gis aan die einde van fermentasie dui op ‘n toename in trehalose produksie wat korreleer met die uitdrukking profiele van TPS1. Gliserol produksie is ook effens verhoog, maar sonder ‘n toename in asynsuur vlakke vir die getransformeerde rasse. Alhoewel etanol opbrengs nie aansienlik laer vir die DUT1-TPS1 ras is nie, is etanol opbrengs vir die oor-uitdrukking onder beheer van die GIP2 promotor wel laer. Toenemende trehalose produksie gedurende stasionêre fase blyk dus 'n meer belowende benadering op die verlaging van etanol opbrengs en her-kanaliseering weg van etanol produksie. Hierdie benadering met die fokus op groeifase spesifieke oor-uitdrukking dui op 'n unieke strategie vir die verlaging van etanol opbrengs sonder om die redoks balans te beinvloed. / The NRF, IWBT and Stellenbosch University for funding