Doctoral Degrees (Institute for Wine Biotechnology)

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Browsing Doctoral Degrees (Institute for Wine Biotechnology) by browse.metadata.advisor "Pretorius, I. S."

Now showing 1 - 4 of 4
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    Characterisation, evaluation and use of non-Saccharomyces yeast strains isolated from vineyards and must
    (Stellenbosch : Stellenbosch University, 2004-03) Jolly, N. P. (Neil Paul); Pretorius, I. S.; Augustyn, O. P. H.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Wine is the product of a complex biological and biochemical interaction between grapes and different microorganisms (fungi, yeasts, lactic acid bacteria and acetic acid bacteria, as well as the mycoviruses and bacteriophages affecting them) in which yeasts play the most important role regarding the alcoholic (primary) fermentation. These wine-associated yeasts can be divided into Saccharomyces and non-Saccharomyces yeasts. During fermentation, there is a sequence of dominance by the various non-Saccharomyces yeasts, followed by Saccharomyces cerevisiae, which then completes the fermentation. This is especially evident in spontaneously fermenting must, which has a low initial S. cerevisiae concentration. Some non- Saccharomyces yeasts can also be found throughout the fermentation. The non- Saccharomyces presence in the fermentation can affect wine quality, either positively or negatively. A positive contribution could be especially useful to improve wines produced from grape varieties with a neutral flavour profile due to non-optimal climatic conditions and/or soil types. As part of a comprehensive South African research programme, the specific objectives of this study were: the isolation of indigenous non-Saccharomyces yeasts from vineyards and musts; the identification of these isolates; the characterisation and evaluation of predominant species under winemaking conditions; and the development of a protocol for their use in enhancing wine quality. Initially, 720 isolates representing 24 different species, were isolated from grape (vineyard) and must samples taken over three vintages from four distinctly different wine producing regions. The isolates were characterised and grouped utilising biochemical profiles and DNA karyotyping, whereupon representative isolates were identified. The yeast species that had the highest incidence of predominance in the vineyard was Kloeckera apiculafa. However, some vineyard samples were characterised by low numbers or absence of this yeast, which is not according to generally accepted norms. Other species that also predominated in a few of the vineyard samples were Candida pulcherrima, Kluyveromyces thermofolerans, Rhodotorula sp. and Zygosaccharomyces bailii. Generally, there was a greater diversity of yeasts in the processed must than from the vineyard samples. Furthermore, while each sample showed a different yeast population, no pattern linking species to climatic zone was observed. Four species i.e. Candida collieulosa, Candida pulcherrima, Candida stel/ata and Kloeckera apiculata, were found to predominate in grape must samples. Representative strains consequently received further attention during laboratory and small-scale winemaking trials. A protocol was developed whereby individual species could be used in co-inoculated fermentations with S. cerevisiae in the small-scale production of wine. An improvement in wine quality was achieved and it was found that there was a link between specific species and grape cultivar. The ability of C. pulcherrima to improve Chenin blanc wine quality was investigated further. Results over three vintages showed that the wine produced by the co-inoculated fermentation was superior to that of a reference wine (produced by S. cerevisiae only). The improvement in wine quality was not linked to increased ester content nor were the standard chemical analyses adversely affected. The effects of pH and wine production parameters i.e. 802, fermentation temperature and use of di-ammonium phosphate (DAP), on this yeast followed the same pattern as that known for S. cerevisiae. This study was successfully completed and the developed protocol can be used for the improvement of Chenin blanc wine where additional aroma and quality is needed.
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    Molecular analyses of candidate carotenoid biosynthetic genes in Vitis vinifera L.
    (Stellenbosch : Stellenbosch University, 2004-03) Young, Philip Richard; Vivier, Melane A.; Pretorius, I. S.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Plants cannot avoid stress and must therefore be capable of rapidly responding to extreme environmental changes. An inability to control and regulate the photosynthetic process during stress conditions will lead to the formation of highly reactive oxygen species that concomitantly causes photo-oxidative damage to the pigments and proteins of the photosynthetic apparatus. Since light is the primary source of energy for the photosynthetic process, it is clear that plants are continuously required to balance the light energy absorbed for the photochemical reactions against photoprotection in a dynamic way in order to survive. Carotenoids are precursors of abscisic acid, but more importantly structural components of the photosynthetic apparatus. During photosynthesis carotenoids function as accessory light-harvesting pigments, and also fulfil a photoprotective function by quenching the reactive molecules formed during conditions that saturate the photosynthetic process. Due to the importance of carotenoids to plant fitness and human health (as Vitamin A precursors) this study has attempted to isolate and characterise genes that are directly, or indirectly involved in carotenoid biosynthesis in Vitis vinifera. In total eleven full-Iength- and eight partial genes have been isolated, cloned and sequenced. These genes can be grouped into the following pathways: (i) the 1- deoxy-D-xylulose 5-phosphate (DOXP)/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (i.e. the plastidic isopentenyl diphosphate biosynthetic pathway); (ii) the mevalonate pathway (i.e. the cytosolic/mitochondrial IPP biosynthetic pathway); (iii) the carotenoid biosynthetic pathway; (iv) the abscisic acid biosynthetic pathway (as a degradation product of carotenoids); and general isoprenoid biosynthetic pathways (as precursors of carotenoids). The full-length genes (i.e. from the putative ATG to the STOP codon) of DOXP synthase (DXS), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (lytB), IPP isomerase (IPI), 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS), phytoene synthase (PSY), Iycopene ~-cyclase (LBCY), ~-carotene hydroxylase (BCH), zeaxanthin epoxidase (lEP), 9-cis-epoxy carotenoid dioxygenase (NCED), farnesyl diphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS) have been isolated from cDNA. In addition, the full-length genomic copy and putative promoters of DXS, PSY, LBCY, BCH, NCED and lEP have also been isolated from genomic DNA by the construction and screening of sub-genomic libraries. Alignments of the genomic copies of these genes to the corresponding cDNA sequences have provided useful information regarding the genomic organisation of these genes, including the intron-exon junction sites in V. vinifera. The copy number of the DXS, PSY, LBCY, BCH, NCED and lEP encoding genes in the Vitis genome have been determined. DXS, PSY, BCH and lEP are single copy genes, whereas LBCY and NCED have two and three copies, respectively. The transcriptional activity of the putative promoters of six of the isolated genes (i.e. DXS, PSY, LBCY, BCH, lEP and NCED) were tested with a transient reporter gene assay. None of the putative promoters tested showed any transcriptional activity of the reporter gene. The transcription of these genes, has however been shown using northern blot analysis and/or RT-PCR. Preliminary expression profiles for PSY, LBCY, BCH, and lEP were determined in different plant organs and the expression of these genes was generally higher in photosynthetically active tissues. The expression of these genes following different treatments (abscisic acid, NaCI and wounding) was also assayed. The functionality of five of the isolated full-length genes (IPI, GGPS, PSY, LBCY and BCH) has been shown in a bacterial colour complementation assay. In silica analysis of the predicted protein sequences of all eleven isolated genes revealed that they are conserved and share a high degree of homology to the corresponding proteins in other plant species. The sequences were further analysed for conserved domains in the protein sequences, and these proteins typically demonstrated similar domain profiles to homologues in other species (plant, bacteria and algae). The predicted protein sequences were further analysed for transit peptides, the presence of which would provide evidence for the sub-cellular localisation of the mature peptides. Since these genes are involved in biosynthetic pathways that are active in discrete organelles, the sub-cellular localisation of most of these proteins is known. The carotenoid biosynthetic genes (PSY, LBCY, BCH and ZEP), the abscisic acid biosynthetic gene, NCED, as well as the DOXP/MEP pathway genes (DXS, lytB and IPI) were all localised to the chloroplast. The mevalonate pathway gene, HMGS, was localised to both the cytosol and the mitochondria, and the general isoprenoid precursor genes, FPS and GGPS, were localised to the cytosol and the chloroplast, respectively. All these results are in agreement with the localisation of the respective pathways. In order to increase our understanding of carotenoid biosynthesis and functions in plants, we constitutively overexpressed one of the isolated genes (BCH) in the model plant, Nicotiana tabacum. Plants expressing the BCH gene in the sense orientation maintained a healthy photosynthetic rate under stress conditions that typically caused photoinhibition and photodamage in the untransformed control plants. This result was inferred using chlorophyll fluorescence and confirmed using CO2 assimilation rates and stomatal conductance. Chlorophyll fluorescence measurements indicated that the photo protective non-photochemical quenching ability of the BCH-expressing plants increased, enabling the plants to maintain photosynthesis under conditions that elicited a stress response in the untransformed control plants. An integral photosynthetic protein component, the D1 protein, was specifically protected by the additional zeaxanthin in the BCH sense plants. Plants expressing an antisense BCH proved the converse, i.e. lower levels of BCH resulted in decreased zeaxanthin levels and made the transgenic plants more susceptible to high-light induced stress. These results have shown the crucial role of carotenoids (specifically the xanthophylls) in the photoprotective mechanism in plants. The increased photoprotection provided by the BCH expressing plants suggests that the scenario in plants is not optimal and can be improved. Any improvement in the photoprotective ability of a plant will affect both the fitness and productivity of the plant as a whole and will therefore find application in a number of crop plants on a global scale. This study has resulted in the successful isolation and characterisation of genes involved in the direct, or indirect, carotenoid biosynthetic pathways. The further study and manipulation of these genes in model plants will provide useful insights into the physiological role of specific carotenoids in photosynthesis and in plants as a whole.
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    A pathogen-derived resistance strategy for the broad-spectrum control of grapevine leafroll-associated virus infection
    (Stellenbosch : Stellenbosch University, 2003-12) Freeborough, Michael-John, 1971-; Pretorius, I. S.; Burger, J. T.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Grapevine leafroll-associated virus-3 is one of ten members of the C/osteroviridae that are known to infect grapevine. Nine of these viruses are associated with grapevine leafroll disease, of which GLRaV-1 and GLRaV-3 are the most important and widespread. Members of the C/osteroviridae are unique amongst the viruses, as it is the only known family whose members encode a heat shock protein 70 kOa homolog (Hsp70h). The Hsp70h is a movement protein (MP) that is required for the active translocation of the virion structure through the plasmodesmata into adjacent cells. Broad-spectrum resistance to unrelated viruses can be obtained by a pathogen-derived resistance (POR) strategy that is based on the expression of a dysfunctional MP in plants. The Hsp70h has two distinct domains. The N-terminal two thirds of the protein is an ATPase domain and shares high homology with the ATPase domains of all Hsp70h proteins from the C/osteroviridae and Hsp70 proteins from the prokaryote and eukaryote kingdoms. Conserved amino acids are found in the ATPase domain and are required for the positioning of the ATP at the catalytic site for ATP hydrolysis. The C-terminal domain is variable and the function of this domain in the Closteroviridae is not known. In prokaryote and eukaryote Hsp70 proteins, the C-terminal domain is required for protein-protein interactions. The American NY-1 isolate of GLRaV-3 has been sequenced and POR strategies have been attempted with the coat protein, divergent coat protein and replicase genes, but not with a dysfunctional form of the hsp70h gene. In this study, double-stranded RNA was isolated from a commercial vineyard with unknown virus status, but with distinct grapevine leafroll symptoms, and from two grapevine sources of known virus status, one with mild and one with severe symptoms. The GLRaV-3 hsp70h gene was amplified by RT-PCR from the dsRNA and the gene sequence was analysed. The hsp70h gene from the three virus sources contained more than 94% nucleotide sequence homology to the NY-1 isolate and the conserved amino acids required for ATPase activity were present. The hsp70h gene isolated from GLRaV-3 from a commercial Stellenbosch vineyard showing clear leafroll symptoms was selected for further work and was subjected to site-directed mutagenesis to engineer four point mutations in the gene. These four mutations resulted in the substitution of Asn for Asp", Gly for Thr1O, Lys for Glu 174 and Asn for Asp 197. The wild type (WT) and mutated (Mut) forms of the hsp 70h genes were cloned into a bacterial expression vector. Expression of both the WT- and Mut-Hsp proteins was achieved, and the protein was expressed in the insoluble inclusion bodies. All attempts to refold and isolate active proteins from the inclusion bodies were unsuccessful. Attempts to increase the concentration of soluble protein within the expressing bacteria were unsuccessful. Due to the lack of active protein, biochemical tests on the ATPase activity of the WT- and Mut-Hsp proteins could not be conducted. The wt- and mut-hsp genes were cloned into a plant expression vector for transformation into tobacco plants. These transformations were successful and gave rise to 22 Km' and 18 Km' plants from the WT- and Mut-Hsp constructs respectively. Two plant lines, M5 and M10, transformed with the mut-hsp transgene construct, appeared to have a high level of resistance to the challenging potato X potexvirus, whereas all the other tested plants were susceptible to the challenging virus. It was thus shown that a dysfunctional form of the GLRaV-3 Hsp70h could provide resistance to an unrelated virus in tobacco.
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    Regulation of the Vitis vinifera PGIP1 gene encoding a polygalacturonase-inhibiting protein
    (Stellenbosch : Stellenbosch University, 2004-03) Joubert, Dirk Albert, 1973-; Vivier, Melane A.; Pretorius, I. S.; De Lorenzo, G.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    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.