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Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Cellulose, a glucose polymer, is considered the most abundant fermentable polymer on earth. Agricultural waste is rich in cellulose and exploiting these renewable sources as a substrate for ethanol production can assist in producing enough bioethanol as a cost-effective replacement for currently used decreasing fossil fuels. Saccharomyces cerevisiae is an excellent fermentative organism of hexoses; however the inability of the yeast to utilize cellulose as a carbon source is a major obstruction to overcome for its use in the production of bio-ethanol. Cellobiose, the major-end product of cellulose hydrolysis, is hydrolyzed by -glucosidase or cellobiose phosphorylase, the latter having a possible metabolic advantage over -glucosidase. Recently, it has been showed that S. cerevisiae is able to transport cellobiose. The construction of a cellulolytic yeast that can transport cellobiose has the advantage that end-product inhibition of the extracellular cellulases by glucose and cellobiose is relieved. Furthermore, the extracellular glucose concentration remains low and the possibility of contamination is decreased. In this study the cellobiose phosphorylase gene, cepA, of Clostridium stercorarium was cloned and expressed under transcriptional control of the constitutive PGK1 promoter and terminator of S. cerevisiae on a multicopy episomal plasmid. The enzyme was expressed intracellulary and thus required the transport of cellobiose into the cell. The fur1 gene was disrupted for growth of the recombinant strain on complex media without the loss of the plasmid. The recombinant strain, S. cerevisiae[yCEPA], was able to sustain aerobic growth on cellobiose as sole carbon source at 30°C with Vmax = 0.07 h-1 and yielded 0.05 g biomass per gram cellobiose consumed. The recombinant enzyme had activity optima of 60°C and pH 6-7. Using Michaelis-Menten kinetics, the Km values for the colorimetric substrate p-nitrophenyl-b-D-glucopyranoside (pNPG) and cellobiose was estimated to be 1.69 and 92.85 mM respectively. Enzyme activity assays revealed that the recombinant protein was localized in the membrane fraction and no activity was present in the intracellular fraction. Due to an unfavourable codon bias in S. cerevisiae, CepA activity was very low. Permeabilized S. cerevisiae[yCEPA] cells had much higher CepA activity than whole cells indicating that the transport of cellobiose was inadequate even after one year of selection. Low activity and insufficient cellobiose transport led to an inadequate glucose supply for the yeast resulting in low biomass formation. Cellobiose utilization increased when combined with other sugars (glucose, galactose, raffinose, maltose), as compared to using cellobiose alone. This is possibly due to more ATP being available for the cell for cellobiose transport. However, no cellobiose was utilized when grown with fructose indicating catabolite repression by this sugar. To our knowledge this is the first report of a heterologously expressed cellobiose phosphorylase in yeast that conferred growth on cellobiose. Furthermore, this report also reaffirms previous data that cellobiose can be utilized intracellularly in S. cerevisiae. / AFRIKAANSE OPSOMMING: Sellulose, ‘n homopolimeer van glukose eenhede, word beskou as die volopste suiker polimeer op aarde. Landbou afval produkte het ‘n hoë sellulose inhoud en benutting van diè substraat vir bio-etanol produksie kan dien as ‘n koste-effektiewe aanvulling en/of vervanging van dalende fossielbrandstof wat tans gebruik word. Die gis, Saccharomyces cerevisiae, is ‘n uitmuntende organisme vir die fermentasie van heksose suikers, maar die onvermoë van die gis om sellulose as koolstofbron te benut is ‘n groot struikelblok in sy gebruik vir die produksie van bio-etanol. Sellobiose, die hoof eindproduk van ensiematiese hidrolise van sellulose, word afgebreek deur -glukosidase of sellobiose fosforilase. Laasgenoemde het ‘n moontlike metaboliese voordeel bo die gebruik van -glukosidase vir sellobiose hidrolise. Daar was onlangs gevind dat S. cerevisiae in staat is om sellobiose op te neem. Die konstruksie van ‘n sellulolitiese gis wat sellobiose intrasellulêr kan benut, het die voordeel dat eindproduk inhibisie van die ekstrasellulêre sellulases deur sellobiose en glukose verlig word. Verder, wanneer die omsetting van glukose vanaf sellobiose intrasellulêr plaasvind, word die ekstrasellulêre glukose konsentrasie laag gehou en die moontlikheid van kontaminasie beperk. In hierdie studie was die sellobiose fosforilase geen, cepA, van Clostridium stercorarium gekloneer en uitgedruk onder transkripsionele beheer van die konstitutiewe PGK1 promoter en termineerder van S. cerevisiae op ‘n multikopie episomale plasmied. Die ensiem is as ‘n intrasellulêre proteïen uitgedruk en het dus die opneem van die sellobiose molekuul benodig. Die disrupsie van die fur1 geen het toegelaat dat die rekombinante ras op komplekse media kon groei sonder die verlies van die plasmied. Die rekombinante ras, S. cerevisiae[yCEPA], het aërobiese groei by 30°C op sellobiose as enigste koolstofbron onderhou met mmax = 0.07 h-1 en ‘n opbrengs van 0.05 gram selle droë gewig per gram sellobiose. Die rekombinante ensiem het optima van 60°C en pH 6-7 gehad. Die K m waardes vir die kolorimetriese substraat pNPG en sellobiose was 1.69 en 92.85 mM onderskeidelik. Ondersoek van die ensiem aktiwiteit het getoon dat die rekombinante proteïen gelokaliseer was in die membraan fraksie en geen aktiwiteit was teenwoordig in die intrasellulêre fraksie nie. CepA aktiwiteit was laag as gevolg van ‘n lae kodon voorkeur in S. cerevisiae. Verder het geperforeerde S. cerevisiae[yCEPA] selle aansienlik beter CepA aktiwiteit getoon as intakte selle. Hierdie aanduiding van onvoldoende transport van sellobiose na binne in die sel tesame met die lae aktiwiteit van die CepA ensiem het gelei tot onvoldoende glukose voorraad vir die sel en min biomassa vorming. Sellobiose verbruik het toegeneem wanneer dit tesame met ander suikers (glukose, galaktose, raffinose, maltose) gemeng was, heelwaarskynlik deur die vorming van ekstra ATP’s vir die sel wat ‘n toename in sellobiose transport teweeg gebring het. Fruktose het egter kataboliet onderdrukking veroorsaak en sellobiose was nie benut nie. Sover ons kennis strek, is hierdie die eerste verslag van ‘n heteroloë sellobiose fosforilase wat in S. cerevisiae uitgedruk is en groei op sellobiose toegelaat het. Verder, bewys die studie weereens dat S. cerevisiae wel sellobiose kan opneem.

Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Soil salinity is a major limiting factor of plant and crop growth, because the absorption of water and nutrients is such a complex process while low and moderate salinity are omnipresent. Plant growth is affected negatively if a specific ion concentration exceeds its threshold and becomes toxic. The detrimental effect of soil affected by salt on crop production is increasing worldwide (Tanji, 1990). The level to which plants can tolerate high salinity levels is genetically controlled with several physiological and genetic mechanisms contributing to salt tolerance (Epstein & Rains, 1987). The most effective way of addressing the limitations of crop productivity in saline areas, is the development of salt tolerant varieties. Understanding the genetics of salt tolerance is, therefore, necessary for the development of an effective breeding strategy for salt tolerance. The department of Genetics (US) conducts a wide crosses research programme aiming to transfer genes for salt tolerance to wheat and triticale. The donor species, Thinopyrum disticum, an indigenous coastal wheat grass, adapted to high concentrations of salt, was crossed with cultivated rye (Secale cereale) in an attempt to study the genetics of salt tolerance (Marais et al., 1998). The primary goal of this study was to find molecular markers (RAPD and AFLP) which associate with chromosomes promoting salt tolerance for later attempts to transfer the genes to triticale. Seventy clones of secondary hybrids (Th disticum /4x-rye 1/2x-rye) were tested for salt tolerance and showed different levels of salt tolerance. RAPD-marker analyses were used to identify polymorphisms between salt tolerant and salt sensitive plants. Twelve RAPD primers produced clear, analyzable and repetitive polymorphic . fragments that can be used as useful markers. Different AFLP-primer combinations were tested against the genotypes of 15 clones (Marais & Marais 2001, unpublished data) and produced approximately 2000 clearly distinguishable AFLP fragments, of which 54 (3%) were polymorphic fragments. Two RAPD fragments and 4 AFLP fragments that can be used as possible markers for the presence of chromosomes that contribute to salt tolerance were identified. The interpretation of the markers was complicated by heterogeneity among plants with regard to the origin of their chromosomes and the genetic diversity of the rye genome. It is also possible that chromosome re-arrangement took place during backcrossing, which could have complicated the data. / AFRIKAANSE OPSOMMING: Versouting is een van die groot beperkende faktore op plant- en gewasgroei, omdat die opname van water en voedingstowwe so In ingewikkelde proses is en die effek van lae of matige versouting so alomteenwoordig is. Plantgroei word nadelig geaffekteer as 'n spesifieke ioonkonsentrasie sy drempelwaarde oorskry en toksies word. Die nadelige effek van soutgeaffekteerde grond op gewasproduksie, is wêreldwyd aan die toeneem (Tanji, 1990). Die vlak waartoe plante hoë konsentrasies sout kan hanteer is onder genetiese beheer met verskeie fisiologiese en genetiese meganismes wat 'n bydrae maak tot soutverdraagsaamheid (Epstein & Rains, 1987). Die mees effektiewe manier om die beperkinge op gewas produktiwiteit in versoute gebiede te oorkom, is die ontwikkeling van soutverdraagsame variëteite. Begrip van die genetika van soutverdraagsaamheid is dus noodsaaklik vir die ontwikkeling van In effektiewe telingsstrategie. Die departement Genetika (US) bedryf tans 'n wye-kruisings navorsingsprogram waarmee gepoog word om gene vir soutverdraagsaamheid na korog en koring oor te dra. Die skenkerspesie, Thinopyrum disticum, In inheemse strandkoringgras wat aangepas is by hoë konsentrasies sout, is gekruis met verboude rog (Secale cereale) in 'n poging om die oorerwing van soutverdraagsaamheid te bestudeer (Marais et al., 1998). Die hoofdoel van hierdie studie was om molekulêre merkers (RAPD en AFLP) te vind, wat assosieer met chromosome wat soutverdraagsaamheid bevorder en om nuttige merkers daar te stel vir latere pogings om die gene na korog en koring oor te dra. Ongeveer 70 klone van sekondêre hibriede (Th distichum I 4x-rog /I 2x-rog) is onderwerp aan souttoetse en het verskillende grade van soutverdraagsaamheid getoon. RAPDmerker analise is gebruik om polimorfismes te identifiseer tussen soutverdraagsame en soutsensitiewe plante. Twaalf RAPD inleiers het duidelike, ontleedbare en herhalende polimorfiese fragmente opgelewer en moontlike nuttige merkers uitgewys. Verskillende AFLP-inleier kombinasies, wat getoets is teen die genotipes van 15 klone (Marais & Marais, 2001 ongepubliseerde data) het ongeveer 2000 duidelik onderskeibare AFLP fragmente geproduseer, waarvan 54 (3%) polimorfiese fragmente was. Twee RAPD fragmente en 4 AFLP fragmente is geïdentifiseer wat as moontlike kandidaat merkers gebruik kan word vir die identifisering van chromosome wat bydra tot soutverdraagsaamheid . Die interpretasie van die merkers is bemoeilik deur heterogeniteit tussen die plante wat betref die agtergrond van chromosome wat hulle besit en die genetiese diversiteit van die rog genoom. Dit is ook moontlik dat chromosoom herrangskikking plaasgevind het tydens terugkruising, wat die data verder kon kompliseer.

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 (PhD (Genetics. Plant Biotechnology))--University of Stellenbosch, 2006. / The main aim of the work presented in this thesis was to elucidate the apparent role of pyrophosphate fructose 6-phosphate 1-phosphotransferase (PFP) in sucrose accumulation in sugarcane. PFP activity in sugarcane internodal tissue is inversely correlated to the sucrose content and positively to the water-insoluble component across varieties which differ in their capacities to accumulate sucrose. This apparent well defined and important role of PFP seems to stand in contrast to the ambiguity regarding PFP’s role in the general literature as well as the results of various transgenic studies where neither the downregulation nor the over-expression of PFP activity had a major influence on the phenotype of transgenic potato and tobacco plants. Based on this it was therefore thought that either the kinetic properties of sugarcane PFP is significantly different than that of other plant PFPs or that PFP’s role in sucrose accumulating tissues is different from that in starch accumulating tissues. In the first part of the study sugarcane PFP was therefore purified and its molecular and kinetic properties were determined. It consisted of two subunits which aggregated in dimeric, tetrameric and octameric forms depending on the presence of Fru 2,6-P2. Both the glycolytic and gluconeogenic reactions had broad pH optima and the kinetic parameters for all the substrates were comparable to that of other plant PFPs. The conclusion was therefore that sugarcane PFP’s molecular and kinetic characteristics do not differ significantly from that of other plant PFPs. The only direct way to confirm if PFP is involved in sucrose accumulation in sugarcane is to alter its levels in the same genetic background through genetic engineering. This was therefore the second focus of this study. PFP activity was successfully down-regulated in sugarcane. The transgenic plants showed no visible phenotype under greenhouse and field conditions and sucrose concentrations in their immature internodes were significantly increased. PFP activity was inversely correlated with sucrose content in the immature internodes of the transgenic lines. Both the immature and mature internodes of the transgenic plants had significantly higher fibre contents. This study suggests that PFP plays a significant role in glycolytic carbon flux in immature, metabolically active sugarcane internodal tissues. The data presented here confirm that PFP can indeed have an influence on the rate of glycolysis and carbon partitioning in these tissues. It also implies that there are no differences between the functions of PFP in starch and sucrose storing tissues and it supports the hypothesis that PFP provides additional glycolytic capacity to PFK at times of high metabolic flux in biosynthetically active tissue. This work will serve as a basis to refine future genetic manipulation strategies and could make a valuable contribution to the productivity of South African sugarcane varieties.

Thesis (PhD (Genetics))—University of Stellenbosch, 2007. / Association analysis was used to improve the efficiency of breeding sugarcane varieties for the negatively correlated traits of resistance to sugarcane smut and the eldana stalk borer. 275 RFLP and 1056 AFLP markers were scored across a population of 77 genotypes representing the genetic variation present within the SASRI breeding programme. Genetic diversity analysis did not detect significant structure within the population. Regression analysis identified 64 markers significantly associated with smut rating and 115 markers associated with eldana rating at r2 > 6.25%. Individual markers with the largest effects explained 15.9% of the phenotypic variation in smut rating and 20.2% of the variation in eldana. Five markers were significantly associated with both smut and eldana. In each case the marker effect was negatively correlated between the two traits, suggesting that they are genetically as well as phenotypically negatively correlated.

In order to understand and manipulate carbon flux to sucrose one needs to consider not only its biosynthetic pathways, but also the competing sinks for carbon in various parts of the plant and at different stages of development. The cell wall and sucrose is known to be the major sinks for carbon in young and mature tissues of sugarcane. UDP-Glucose is a central metabolite in the synthesis of both sucrose and most of the cell wall polysaccharides (including cellulose, hemicellulose and pectic polymers) and manipulation of the flux into either of the cell wall components could therefore cause an increase of flux toward one or more of the competing sinks. In the present study UDP-Glucose dehydrogenase (UGD) activity was chosen for down regulation as it catalyzes the rate limiting step in the biosynthesis of the precursors of both hemicellulose and pectin, a major competing sink for assimilated carbon. Transgenic sugarcane lines with repressed UGD activity showed significantly increased sucrose accumulation in all internodes which was highly correlated with reduced UGD activity. Sucrose phosphate synthase had increased activation which suggests an alteration in carbon flux toward sucrose. The reduction of carbon flux through UGD was compensated for by an increase in the activity of the myo-inositol oxygenation pathway (MIOP), an alternative pathway for the synthesis of cell wall matrix precursors. The increased activity of the MIOP resulted in increased total uronic acids and pentoses in the cell wall. Total cell wall glucose was also increased which is a further indication of altered carbon metabolism.

Thesis (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Plant-pathogen interactions have been intensively investigated in the last decade. This major drive towards understanding the fundamental aspects involved in plant disease resistance is propelled by the obvious agricultural and economical benefits that are intrinsically linked to disease and stress resistant plants. It is, therefore, not surprising that fundamental research in this area is not just restricted to model organisms, such as Arabidopsis and tobacco, but also extends to more traditional crop plants, such as maize, bean, soybean, apples, grapevine etc. In grapevine for instance, several genes involved in disease resistance have been isolated. One of these genes, encoding for a polygalacturonase inhibiting protein (PGIP), has been studied extensively. PGIPs are cell wall bound, contain leucine rich repeats (LRR) and are found in all dicotyledonous plants so far examined. In most cases, pgip genes occur in small multigene families and expression is often tissue specific and developmentally regulated. Up-regulation of PGIP-encoding genes typically occurs upon pathogen infection, treatment with elicitors, salicylic acid (SA), jasmonic acid (JA), cold treatment and wounding. Differential regulation and specificity have been shown to occur between members of the same multigene family. Differential regulation even extends to the utilization of separate pathways to induce pgip genes from the same family in response to a single stress stimulus. PGIPs interact with cell wall macerating polygalacturonases (PGs) that are secreted by pathogenic fungi during the infection process. The antifungal action of PGIPs is thought to depend on a dual action. The physical interaction of PGIP with PGs has an inhibitionary effect, resulting in (i) a slower fungal infection rate and (ii) the prolonged existence of long chain oligogalacturonides (OGs). These oligosaccharides are able to elicit a general plant defense response, enabling the plant to further retard or curb the spread of infection. The main objective of this study was to investigate the regulatory aspects underlying PGIP expression in grapevine. Unlike most characterized PGIP encoding genes from other dicotyledonous plant species, no evidence to support the existence of a V. vinifera PGIP multigene family could be found from either genetic or biochemical analyses. Recently, a genomic DNA fragment from Vitis vinifera cv Pinotage was pathogen interactions with regards to the fundamental processes underlying defense gene regulation. / AFRIKAANSE OPSOMMING: Die ooglopende voordele wat, vanuit 'n landboukundige én ekonomiese oogpunt, uit siekte- en stresbestande plante spruit, het gedurende die laaste dekade aanleiding gegee tot die ontwikkeling van plantpatogeen-interaksies as "n baie belangrike studieveld. Dit was dus ook te verwagte dat fundamentele navorsing in hierdie area nie net beperk gebly het tot modelorganismes soos Arabidopsis en tabak (ook natuurlik van landboukundige belang) nie, maar ook na meer tradisionele landbougewasse soos mielies, boontjies, sojaboontjies, appels, druiwe, ens. oorgevloei het. Verskeie siekteweerstands-verwante gene is byvoorbeeld al vanuit wingerd geïsoleer. Een só "n geen wat vir "n poligalakturonase-inhiberende proteïen (PGIP) kodeer, vorm deel van hierdie groep gene. Die funksie en regulering van PGIP's is baie goed bestudeer. Hierdie proteïene word normaalweg in die selwande van die meeste dikotiele plante aangetref. Leusienryke herhalings is algemeen in PGIP's en hierdie tipe van herhalings is kenmerkend van proteïene betrokke by proteïen-proteïen-interaksies. Verder word pgip-gene gewoonlik in klein multigeenfamilies aangetref, waar in die meeste gevalle die uitdrukking weefselspesifiek en die regulering spesifiek ten opsigte van die ontwikkelingsfase is. Verskeie faktore kan tot die induksie van pgip-gene lei, soos onder andere patogeen-infeksie, elisitoor-, salisiensuur-, jasmoonsuur- en kouebehandeling, asook verwonding. Differensiële regulering word in baie gevalle tussen lede van dieselfde multigeenfamilie aangetref. Hierdie differensiële regulering kan selfs bemiddel word deur onafhanklike reguleringsweë in reaksie op dieselfde induksiestimulus. PGIP's is in staat om te reageer met poligalakturonases (PGs), wat selwande afbreek en wat gedurende die infeksieproses deur swamme of fungi afgeskei word. Die effek van hierdie interaksie is tweeledig: (i) Die fisiese interaksie tussen PGIP en PG moduleer die aktiwiteit van die PG deur die ensiemaksie te inhibeer, en (ii) PGinhibisie lei tot die verhoogde stabiliteit van langketting-oligogalakturonades, molekules wat daartoe in staat is om die weerstandsrespons van plante te ontlok. Die inhibisie van die patogeen-PG's, tesame met die geïnduseerde weerstandrespons, stel die plant dan in staat om verdere infeksie te vertraag of te verhoed. Die doel van hierdie studie was om die onderliggende aspekte van PGIPregulering in wingerd te bestudeer. In teenstelling met die meeste plantspesies waar pgip-gene in klein multigeenfamilies aangetref word, is daar nie 'n pgip-multigeenfamilie in wingerd nie. Veelvuldige kopieë van In enkele pgip-geen word egter in die wingerdgenoom aangetref. Daar is onlangs in ons laboratorium In genoom-DNAfragment vanaf Vitis vinifera cv Pinotage geïsoleer wat die oopleesraam en 5'-stroomopsekwense van In PGIP-enkoderende geen (Vvpgip1) bevat. In hierdie studie is die uitdrukkingspatroon van Vvpgip1 ten opsigte van weefselspesifisiteit, korrelontwikkelingsfase, asook die effek van verskeie omgewings en patogeenverwante stres-stimuli ontleed. Die regulatoriese meganismes van Vvpgip1 bevat spesifieke in planta-ontwikkelingsfaseseine wat verder deur spesifieke faktore, insluitende omgewings- en patogeenstres, gereguleer word. In lyn hiermee is mRNS-transkripte van Vvpgip1 tot wortel- en korrelweefsels beperk, terwyl die mRNS-vlakke ook tussen verskillende korrelontwikkelingsfases wissel. Kumulatiewe uitdrukking kon waargeneem word in veráison-korrels in reaksie op verwonding en osmotiese stres. Die weefselspesifieke uitdrukkingspatroon tipies van wingerd-PGIP is in blare opgehef in reaksie op Botrytis cinerea-infeksie, verwonding, osmotiese stres, ouksien (indoolasynsuur) en salisiensuur. PGIP-uitdrukking word ook onderdruk deur In staurosporien-sensitiewe proteïenkinase, wat In goeie aanduiding is van die betrokkenheid van proteïenfosforilasie in die seintransduksiekaskade wat tot PGIPuitdrukking aanleiding gee. Die geïnduseerde PGIP-uitdrukkingsprofiel in wingerdblare kan ook nageboots word in tabak wat met die Vvpgip1-geen en -promotor getransformeer is. PG-inhibisie-eksperimente met membraan-geassosieerde proteïenekstrakte van geïnduseerde wingerdblare het ook dieselfde profiel getoon as dié van PGIP wat deur die Vvpgip1-geen geënkodeer is. Die uitdrukkingsprofiel van PGIP in die transgeniese tabakplante het ook bewys dat die promotor van die Vvpgip1-geen vir die geïnduseerde PGIP-uitdrukkingsprofiel in wingerdblare verantwoordelik is. In silica-analise van die promotorarea dui op die teenwoordigheid van verskeie cis-werkende elemente. Die kern promotor en transkripsie-aanvangsgedeelte is gevolglik eksperimenteel bepaal. Verder het uitdrukkingseksperimente met promotorfragmente verskeie dele van die promotor geïdentifiseer wat by stimulis-geassosieerde uitdrukking betrokke is. Posisioneel is hierdie fragmente in goeie konteks met die voorspelde cis-werkende elemente en kan dus die basis vorm vir verdere studies oor Vvpgip-regulering. Met hierdie studie word die eerste data verskaf waar die regulering van PGIP deur omgewingsverwante faktore verbind kan word met onwikkelingspesifieke toestande in die plant. Verder verskaf die resultate verdere bewyse vir die rol van PGIP in plant-patogeen-interaksies en lewer spesifieke bydraes tot die onderliggende prosesse wat by die regulering van siekteweerstandverwante gene betrokke is.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Increased demand of petroleum, declining fossil fuel reserves, geopolitical instability and the environmentally detrimental effects of fossil fuels have stimulated research to search for alternative sources of energy such as plant derived biofuels. The main feedstocks for production of first generation biofuels (bioethanol) are currently sucrose and starch, produced by crops such as sugarcane, sugarbeet, maize, and cassava. The use of food crop carbohydrates to produce biofuels is viewed as competing for limited agronomic resources and jeopardizing food security. Plants are also capable of storing sugars in their cell walls in the form of polysaccharides such as cellulose, hemicelluloses and pectin, however those are usually cross-linked with lignin, making their fermentation problematic, and are consequently referred to as lignocellulosics. Current technologies are not sufficient to degrade these cell wall sugars without large energy inputs, therefore making lignocellulosic biomass commercially unviable as a source of sugars for biofuel production. In the present study genes encoding for enzymes for cellulosic, hemicellulosic and starch-like polysaccharides biosynthesis were heterologously expressed to increase the amount of fermentable sugars in sugarcane. Transgenic lines heterologously expressing CsCesA, encoding a cellulose synthase from the marine invertebrate Ciona savignyi showed significant increases in their total cellulose synthase enzyme activity as well as the total cellulose content in internodal tissues. Elevation in cellulose contents was accompanied by a rise in hemicellulosic glucose content and uronic acid amounts, while total lignin was reduced in internodal tissues. Enzymatic saccharification of untreated lignocellulosic biomass of transgenic sugarcane lines had improved glucose release when exposed to cellulose hydrolyzing enzymes. Calli derived from transgenic sugarcane lines ectopically expressing galactomannan biosynthetic sequences ManS and GMGT from the cluster bean (Cyamopsis tetragonoloba) were observed to be capable of producing a galactomannan polysaccharide. However, after regeneration, transgenic sugarcane plants derived from those calli were unable to produce the polymer although the inserted genes were transcribed at the mRNA level. While the ectopic expression of Deinococcus radiodurans amylosucrase protein in the cytosol had a detrimental effect on the growth of transgenic lines (plants showed stunted growth through the 18 months growth period in greenhouse), contrastingly targeting the amylosucrase protein into the vacuole resulted in 3 months old transgenic lines which were having high maltooligosaccharide and soluble sugar (sucrose, glucose and fructose) levels in leaves. After 18 months growing in the greenhouse, the mature transgenic lines were morphologically similar to the untransformed lines and also contained comparable maltooligosaccharide and soluble sugar and starch amounts. The non-biosynthesis of galactomannan and amylose polysaccharides in the matured transgenic plants may be due to post-transcriptional protein processing and or protein instability, possibly explainable by other epigenetic mechanisms taking place to regulate gene expression in the at least allo-octaploid species of sugarcane under investigation in this study.

Dissertation (DPhil)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Xylose is the second most abundant sugar present in plant biomass. Plant biomass is the only potential renewable and sustainable source of energy available to mankind at present, especially in the production of transportation fuels. Transportation fuels such as gasoline can be blended with or completely replaced by ethanol produced exclusively from plant biomass, known as bio-ethanol. Bio-ethanol has the potential to reduce carbon emissions and also the dependence on foreign oil (mostly from the Middle East and Africa) for many countries. Bio-ethanol can be produced from both starch and cellulose present in plants, even though cellulosic ethanol has been suggested to be the more feasible option. Lignocellulose can be broken down to cellulose and hemicellulose by the hydrolytic action of acids or enzymes, which can, in turn, be broken down to monosaccharides such as hexoses and pentoses. These simple sugars can then be fermented to ethanol by microorganisms. Among the innumerable microorganisms present in nature, the yeast Saccharomyces cerevisiae is the most efficient ethanol producer on an industrial scale. Its unique ability to efficiently synthesise and tolerate alcohol has made it the ‘workhorse’ of the alcohol industry. Although S. cerevisiae has arguably a relatively wide substrate utilisation range, it cannot assimilate pentose sugars such as xylose and arabinose. Since xylose constitutes at least one-third of the sugars present in lignocellulose, the ethanol yield from fermentation using S. cerevisiae would be inefficient due to the non-utilisation of this sugar. Thus, several attempts towards xylose fermentation by S. cerevisiae have been made. Through molecular cloning methods, xylose pathway genes from the natural xylose-utilising yeast Pichia stipitis and an anaerobic fungus, Piromyces, have been cloned and expressed separately in various S. cerevisiae strains. However, recombinant S. cerevisiae strains expressing P. stipitis genes encoding xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) had poor growth on xylose and fermented this pentose sugar to xylitol. The main focus of this study was to improve xylose utilisation by a recombinant S. cerevisiae expressing the P. stipitis XYL1 and XYL2 genes under anaerobic fermentation conditions. This has been approached at three different levels: (i) by creating constitutive carbon catabolite repression mutants in the recombinant S. cerevisiae background so that a glucose-like environment is mimicked for the yeast cells during xylose fermentation; (ii) by isolating and cloning a novel xylose reductase gene from the natural xylose-degrading fungus Neurospora crassa through functional complementation in S. cerevisiae; and (iii) by random mutagenesis of a recombinant XYL1 and XYL2 expressing S. cerevisiae strain to create haploid xylose-fermenting mutant that showed an altered product profile after anaerobic xylose fermentation. From the data obtained, it has been shown that it is possible to improve the anaerobic xylose utilisation of recombinant S. cerevisiae to varying degrees using the strategies followed, although ethanol formation appears to be a highly regulated process in the cell. In summary, this work exposits three different methods of improving xylose utilisation under anaerobic conditions through manipulations at the molecular level and metabolic level. The novel S. cerevisiae strains developed and described in this study show improved xylose utilisation. These strains, in turn, could be developed further to encompass other polysaccharide degradation properties to be used in the so-called consolidated bioprocess. / AFRIKAANSE OPSOMMING: Xilose is die tweede volopste suiker wat in plantbiomassa teenwoordig is. Plantbiomassa is die enigste potensiële hernubare en volhoubare bron van energie wat tans vir die mensdom beskikbaar is, veral vir die produksie van vervoerbrandstowwe. Vervoerbrandstowwe soos petrol kan vermeng word met etanol wat uitsluitlik van plantbiomassa vervaardig is, bekend as bio-etanol, of heeltemal daardeur vervang word. Bio-etanol het die potensiaal om koolstofuitlatings te verminder en vir baie lande ook afhanklikheid op buitelandse olie (hoofsaaklik afkomstig van die Midde-Ooste en Afrika) te verminder. Bio-etanol kan vanaf beide die stysel en sellulose in plante vervaardig word, maar sellulosiese etanol word as die meer praktiese opsie beskou. Lignosellulose kan deur die hidrolitiese aksie van sure of ensieme in sellulose en hemisellulose afgebreek word en dit kan op hulle beurt weer in monosakkariede soos heksoses en pentoses afgebreek word. Hierdie eenvoudige suikers kan dan deur mikro-organismes tot etanol gegis word. Onder die tallose mikro-organismes wat in die natuur teenwoordig is, is die gis Saccharomyces cerevisiae die doeltreffendste etanolprodusent in die bedryf. Sy unieke vermoë om alkohol te vervaardig en te weerstaan het dit die werksperd van die alkoholbedryf gemaak. Hoewel S. cerevisiae ‘n taamlike breë spektrum van substrate kan benut, kan dit nie pentosesuikers soos xilose en arabinose assimileer nie. Aangesien xilose ten minste ‘n derde van die suikers wat in lignosellulose teenwoordig is, uitmaak, sou die etanolopbrengs uit gisting met S. cerevisiae onvoldoende wees omdat hierdie suiker nie benut word nie. Verskeie pogings is dus aangewend om xilosegisting deur S. cerevisiae te bewerkstellig. Deur middel van molekulêre kloneringsmetodes is gene van die xiloseweg uit ‘n gis wat xilose natuurlik benut, Pichia stipitis, en ‘n anaërobiese swam, Piromyces, afsonderlik in S. cerevisiae-rasse gekloneer en uitgedruk. ‘n Rekombinante ras wat P. stipitis- se XYL1-xilosereduktase- en XYL2-xilitoldehidrogenase gene uitdruk, het egter swak groei op xilose getoon en het dié pentosesuiker tot xilitol gegis. Die hooffokus van hierdie ondersoek was om die benutting van xilose deur ‘n rekombinante S. cerevisiae-ras wat P. stipitis se XYL1 en XYL2-gene uitdruk onder anaërobiese gistingstoestande te verbeter. Dit is op drie verskillende vlakke benader: (i) deur konstitutiewe koolstofkataboliet-onderdrukkende mutante in die rekombinante S. cerevisiae-agtergrond te skep sodat ‘n glukose-agtige omgewing tydens xilosegisting vir die gisselle nageboots word; (ii) deur ‘n nuwe xilose-reduktasegeen uit die natuurlike xilose-afbrekende swam Neurospora crassa te isoleer en deur funksionele komplementasie in S. cerevisiae te kloneer; en (iii) deur willekeurige mutagenese van die rekombinante S. cerevisiae-ras ‘n haploïede xilose-gistende mutant te skep wat ‘n gewysigde produkprofiel ná anaërobiese xilosegisting vertoon. Deur hierdie drieledige benadering te volg, is dit bewys dat dit moontlik is om die anaërobiese xilosebenutting van rekombinante S. cerevisiae-rasse in wisselende mate deur die aangepaste metodes te verbeter, hoewel etanolvorming ‘n hoogs gereguleerde proses in die sel blyk te wees. Opsommend kan gesê word dat hierdie werk drie verskillende metodes uiteensit om xilosebenutting onder anaërobiese toestande te verbeter deur manipulasies op die molekulêre en metaboliese vlak. Die nuwe S. cerevisiae-rasse wat in hierdie studie ontwikkel en beskryf word, toon verbeterde xilosebenutting. Hierdie rasse kan op hulle beurt verder ontwikkel word om ander polisakkariedafbrekende eienskappe in te sluit wat in die sogenaamde gekonsolideerde bioproses gebruik kan word.