Browsing Doctoral Degrees (Viticulture and Oenology) by Title
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- ItemAn anatomical and experimental study on changes induced by Meloidogyne hapla Chitwood, 1949 in Vitis roots(Stellenbosch : Stellenbosch University, 1971) Joubert, D. J. (Daniel Jakobus); Orffer, C. J.; De Vos, M. P.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology and Institute for Wine Biotechnology .ENGLISH ABSTRACT: The object of this anatomical study was to collect scientific data on the effect of Metoidogyne hapta Chitwood, 1949, on the roots of the following grapevine cultivars viz: Vitis vinifePa L. cvs. Steen and White French and the root-stocks, Jacquez, 1202 C, 99 R, Salt.Creek and Dogridge. These cultivars differed widely in their resistance to M. hapta attacks. In the roots of Steen, White French, Jacquez and 1202 C the formation of multinucleate syncytia by the destruction of the walls of groups of cells often occurred. In Salt Creek, Dogridge and 99 R roots, syncytia were observed in the stele only. The formation o,f abnormal xylem as a result of nematodal activities was a common occurrence. In the roots of these latter three cultivars, M. hapta could not complete its life cycle. Salt Creek, Dogridge, 99 R and often Steen formed a wound periderm which prevented the nematodes from reaching the xylem. Histological changes were often induced in advance of the invading nematodes.
- ItemAspects of sucrose metabolism in transgenic tobacco(Stellenbosch : Stellenbosch University, 2004-12) Champanis, Reinette; Vivier, Melane A.; Botha, F. C.; Pretorius, I. S.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: In most plants the efficiency of sucrose production and the systemic distribution thereof are the major determinants of growth, development and yield. The factors governing sugar partitioning co-ordinate its distribution in response to intrinsic and environmental signals. These factors include sugar transporters and invertases as well as metabolites, including sucrose and glucose, which function as signalling molecules to modulate gene expression. The genetic transformation of plants and the subsequent development of transgenic lines with disturbed sugar metabolism have made an unprecedented impact on the study of sugar translocation and -partitioning. For instance, the transformation of plants with a yeast-derived invertase targeted to different subcellular compartments has led to the elucidation of several key aspects of sugar metabolism, including phloem loading mechanisms, the regulation of photosynthesis by sugars, the importance of sugar-metabolism compartmentation with regards to sucrose biosynthesis, storage and distribution, as well as the role of cell-wall invertase in phloem unloading and sink strength. In this study, a similar strategy of transgenic plant analysis was employed to expand our insight into the regulation of sugar partitioning. The yeast-invertase Suc2 gene, from Saccharomyces cere visiae , was overexpressed in either the cytosol, vacuole or apoplast of transgenic tobacco plants. These transgenic lines displayed varying increases in invertase activity, altered sugar levels and consequently disturbed sink-source interactions and sugar partitioning. Transgenic lines overproducing the yeast-derived invertase in either the vacuole (Vac-Inv) or apoplast (Apo-Inv) were utilised to analyse the effect of the altered sugar levels in sink and source organs on the expression of sugar transporters, as well as the endogenous cell wall invertase and inhibitors in these plants. Transcript levels of the sucrose transporter NtSUT1 and hexose transporter NtMST1 encoding genes increased significantly in the source leaves and roots of Vac-Inv lines, whereas increased NtMst1 transcript levels were also detected in the roots of Apo-Inv lines. The increased mRNA levels could be correlated to the altered invertase activities and sugar levels in these tissues. It is concluded that NtSUT1 and NtMST1 are differentially regulated by sucrose and/or hexose content on a transcriptional level. Furthermore, the regulatory effect of the altered sugar levels on transporter expression depended on the subcellular compartment in which the yeast invertase was expressed. It would seem that the subcellular compartmentation of sugar metabolism is also fundamental to the regulation of sugar partitioning. The transcription levels of the endogenous cell wall invertase (CWt) and cell wall invertase inhibitor (Cwi-Inh) genes were examined in the various tissues of Apo-Inv and Vac-Inv lines at both the vegetative and flowering growth stages. In comparison with the control lines, the various tissues of the Apo-Inv and Vac-Inv lines displayed altered Cwi and Cwi-Inh expression levels, depending on the sink-source status and growth stage. However, no obvious correlation between the Cwi and Cwi-Inh expression levels and soluble sugar content of these tissues was found. It is suggested that the post-transcriptional and post-translation control of these proteins by sugars might play an important role in their regulation. Analysis of the Cwi:Cwi-lnh mRNA ratio and growth observations of the various tissues of control as well as Apo-Inv and Vac-Inv lines indicated that this transcription ratio could be an accurate indicator of the sink strength of sink organs. In addition, the influence of sink-source interactions on sugar partitioning was investigated. Reciprocal grafting between Apo-Inv and control lines resulted in scions with an altered sucrose metabolism in either the sink or source organs. These scions were subjected to biomass distribution, soluble sugar quantification and C4C]- radiolabelling experiments. The latter revealed an unaltered state of sugar partitioning from the above-ground tissues of the Apo/GUS scions and a significant shift in sugar partitioning towards the roots of the GUS/Apo scions in comparison to the control GUS/GUS scions. Phenotypic changes, opposite to those observed in Apo-Inv lines expressing the heterologous invertase in both sink and source organs, could initially be observed in the GUS/Apo and Apo/GUS scions. However, no significant differences in phenotype or biomass distribution could be observed between the mature GUS/Apo, Apo/GUS and GUS/GUS scions seven weeks postgrafting. This inconsistency between phenotype and sugar partitioning might be explained by an increase in the respiration rate of the tissues as supported by the soluble sugar content. These results highlight the complexity and adaptability of sucrose metabolism and sugar partitioning. In addition, it confirms that sugar partitioning can be modulated by sink-source interactions and emphasise the importance of invertases in the regulation of sugar partitioning through its ability to alter sink strength. This study forms part of the rapidly expanding initiative to unravel the control mechanisms of sugar partitioning. The results obtained in this study confirmed again that the introduction and expression of a single heterologous gene in transgenic plants could provide significant insight into the regulation of this process. It was shown here that the expression of sugar transporters is closely regulated by sugar levels and therefore fulfils a vital function in sugar sensing and consequently the regulation of sugar partitioning. The data presented in this study also demonstrated the intricate and flexible nature of the relationship that exists between sugar metabolism, partitioning and growth phenomena.
- ItemBerry tannin structure and phenolics evolution in cv. Cabernet Sauvignon (Vitis vinifera L.) : effect of light and temperature(Stellenbosch : Stellenbosch University, 2015-12) Blancquaert, Erna; Deloire, A. J.; Oberholster, A.; Ricardo da Silva, J. M.; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology.ENGLISH ABSTRACT: This study investigated grape flavonoid (proanthocyandins, flavonols and anthocyanins) accumulation and composition in grape seeds and skins from Cabernet Sauvignon (Vitis vinifera L.) under altered light intensities and temperatures, within the bunch zone in the Stellenbosch Wine of Origin District. Furthermore, the study examined the link between wine sensory properties and the harvest date. This study was conducted in 2010/2011 and 2011/2012 and comprised of two main treatments with altered bunch microclimates in both seasons: no lateral shoot or leaf removal in the bunch zone (STD) and leaf removal in the bunch zone (LRW). The leaves were removed just after flowering on the western side of the canopy at the fruiting zone level (±35–40 cm above the cordon). Furthermore, to study the effect of change in light quality and quantity on fruit growth and composition, supplementary treatments were applied. In 2010/2011, a UV-B reducing sheet was added on the western side of the canopy to the STD (STD-UV-B) and LRW (LRW-UV-B) treatments. During the 2011/2012 season two types of UV-B reducing sheets were installed on both sides of the canopy to exclude the effect that the row direction can have on grape development. The latter resulted in the following treatments: LR (-UV-B, 2xOp50) and LR (-UV-B, 2xUHI). The accumulated thermal time varied between the treatments and within a season. The 2010/2011 season had a higher accumulated thermal time than the 2011/2012 season. There was a significant difference in the photosynthetic active radiation (PAR) (p≤0.001) among the treatments indicating that the applied treatment were successful in creating variation in the amount of sunlight intercepted in the bunch zone. There were no significant differences in berry weights in 2010/2011, but a significant difference were observed in 2011/2012 (p≤0.001). Light and temperature had little effect on grape seed flavan-3-ol monomer and dimer concentration and content. Seed development after flowering potentially influenced light quality and quantity which impacted the seed number and affected flavan-3-ol concentration and content. Grape skin flavan-3-ol concentration and content differed significantly among the treatments in 2010/2011, but not in 2011/2012. Generally, the seasonal impact was larger than those of the different treatments on flavonoid concentration and content during ripening resulting in significant differences among the treatments at harvest in the 2010/2011 season. However, treatment did not have a significant effect on either concentration or composition of a compound. Grape seed and skin (terminal and extension subunit) composition were influenced by the seasonal impact, rather than the treatment in both seasons. Moreover, the structural characteristics such as the percentage galloylation (%G), percentage prodelphinidins (%P), mean degree of polymerization (mDP) and average molecular mass (avMM) were influenced by seasonal variation. The accumulation of flavonols was higher in the exposed treatments and low in treatments with UV-B reducing sheets. This indicates that flavonol synthesis is highly dependent on UV-B radiation. The accumulation of anthocyanins commenced at véraison and had two distinct patterns of accumulation in the respective seasons. The 2010/2011 season was characterised by a higher anthocyanin concentration and content compared to the cooler 2011/2012 season. Grapes were harvested sequentially based on the sugar loading model at the fresh fruit stage (four treatments) and pre-mature (control treatment only) in 2010/2011. In the 2011/2012 season four treatments were harvested at the mature fruit stage. Identified aromas in the respective wines corresponded to the sugar loading model profile. Wine tannin, anthocyanin and flavonol concentrations were the highest in the LRW treatment in 2011/2012. Mouthfeel properties (adhesiveness, coarse, puckery – in and after expectoration) were rated higher in the treatments which were exposed to high light intensities in both seasons. This research denotes the complex nature of flavonoid biosynthesis and composition. Therefore further research is needed to elucidate impact of the functioning of individual genes in the phenylproanoid and flavonoid pathways which have an influence on the final concentration, content and composition of flavonoids at harvest.
- ItemBydrae tot die kennis omtrent die fisiologie, morfologie en sistematiek van die Apiculatus giste(Stellenbosch : Stellenbosch University, 1932) Niehaus, Chas. J. G.; Von der Heide, C.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology and Institute for Wine Biotechnology .No Abstract Available
- ItemCharacterising sensory interactions between volatile phenols and other taint-causing compounds in South African red wines(Stellenbosch : Stellenbosch University, 2019-04) McKay, Marianne; Buica, Astrid; Bauer, Florian; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology.ENGLISH ABSTRACT: South African red wine competes for limited market space, and must maintain consistent, excellent quality. One of the important modalities in assessing the quality and typicality of wine is odour perception (Hopfer et al., 2015). Most investigations quantify aroma compounds by chemical/analytical means and compare levels to odour detection thresholds (ODTs) provided by scientific literature. If malodourous compounds are present at concentrations above their ODTs, they may be considered a threat to wine quality as they exhibit odour activity values >1 (Louw et al., 2010; Prida & Chatonnet, 2010). A review of the literature reveals that studies in wine can use inappropriate ODTs for work carried out in a new wine matrix, and matrix effects on the activity and perception of a compound are often ignored. To properly scientifically evaluate the effect of any compound to wine aroma, formal sensory evaluation in the study matrix is essential (Villamor & Ross, 2013; Perry & Hayes, 2016). Certain volatile phenols (VPs) can contribute to a continuum of smoke-taint related off-flavours including ‘burnt’, ‘bretty’, ‘smoky’, and ‘ashy’ attributes in wine (Jiranek, 2011; Kennison et al., 2011) at higher levels but are generally accepted as being benign to wine aroma at subthreshold levels (Boidron et al., 1988; Prida & Chatonnet, 2010). Useful sensory studies on VPs in wine have been carried out (Simpson et al., 1986; Boidron et al., 1988; Chatonnet et al., 1992; Kennison et al., 2008; Petrozziello et al., 2014), but at supra-threshold levels, and the effect of combinations of subthreshold levels of VPs is not taken into account. The main aims of this research were to characterise the sensory contribution of specific VPs at low levels to red wine odour, and to assess their effect on the perception of attributes through interactions with themselves and other compounds known to be involved in specific wine offflavours. Chapter 2 addresses key knowledge and gaps within the literature around origin, perception and odour detection threshold of VPs associated with specific off-flavours (including ‘smoke’ and ‘ashiness’), and previous studies concerning interaction of aroma compounds which may have relevance to the current study. Chapter 3 investigated trends within the chemical (GC-MS) and sensory (Descriptive Analysis) results for twelve commercial samples of smoke-affected wine. Associations between negative attributes and bushfire events prior to harvest were found. Results also showed that certain sensory effects could have resulted from combinations of subthreshold levels of VPs. The need arose to formally test perceptual interactions at subthreshold levels to see if various effects could be explained. It was thus decided to investigate effects of three VPs: guaiacol, ortho-cresol, 4-ethylphenol, and two compounds associated with certain off-flavours in wine, viz. 3- isobutyl-2- methoxypyrazine (IBMP) and 2,4,6-trichloroanisole (TCA). The work was carried out in partially de-aromatised Shiraz, and it was necessary to establish if ODTs provided in the literature were appropriate for this matrix. Formal sensory work is known to be complex, time-consuming and expensive, particularly in establishing ODTs, and thus a pragmatic sensory approach to the work is outlined in Chapter 4. Chapters 5 and 6 address the perceptual effects of combinations of two, three, four and five offflavour compounds on red wine aroma, which has not been conducted on this scale before. The results of this DA sensory study showed olfactory opposition between clean controls, wines spiked with single compounds (generally fruity and sweet-associated), and wines spiked with complex combinations of VPs and IBMP (linked to negative attributes). Chapter 7 demonstrates another pragmatic approach, using projective mapping (PM) with a large sample size (n=18). Comparable results to the DA interaction study for four compounds in red wine were shown. Chapter 8 investigates effects of combinations of two VPs on four cultivars in order to establish whether there were perceptual olfactory effects that were common to all cultivars, or whether the matrices responded differently from an aroma perception perspective, and shows that samples spiked with combinations of VPs and IBMP show consistently negative olfactory attributes that are independent of cultivar. This research contributes to the sensorial and chemical characterization of selected VPs in red wines, and shows that subthreshold levels of VPs in combination with very low levels of IBMP and TCA can lead to olfactory interactions that cause various olfactory effects, some of them negative. This may help inform winemaking decisions, particularly when dealing with smokeaffected grapes, and/or cultivars that naturally have higher levels of methoxypyrazines, like Merlot and Cabernet Sauvignon. This study also emphasises the importance of understanding effects of VPs on wine aroma, and escalating awareness and sensitivity to these issues in the wine industry.
- ItemCharacterization and evaluation of glucose oxidase activity in recombinant Saccharomyces cerevisiae strains(Stellenbosch : Stellenbosch University, 2010-03) Malherbe, Daniel Francois; Van Rensburg, P.; Pretorius, I. S.; Du Toit, M.; Divol, Benoit; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology, 2010.ENGLISH ABSTRACT: Popular wine styles prepared from fully-ripened, more mature grapes are characterized by intense fruitiness and varietal flavors. However, lengthy maturation of grapes in the vineyard does not only translate into higher flavor intensity but also into higher sugar levels, which, in turn, leads to wines with higher concentrations of alcohol. Excessive alcohol levels can compromise wine flavor and render wine unbalanced. This, along with health issues and anti-social behavior linked to high-risk alcohol consumption patterns, stricter legislation and increased tax rates associated with high-alcohol wines, have increased demand for wines with reduced alcohol concentrations, without loss of the intense fruity aromas. Although low-alcohol wines can be made using physical post-fermentation processes, such approaches are often expensive and can impact adversely on wine flavor. As an alternative strategy, yeast strains are being developed by several research groups to convert some of the grape sugars into metabolites other than ethanol. Based on promising results from previous preliminary work, this study focused on the development of an industrial Saccharomyces cerevisiae wine strain producing glucose oxidase (GOX; b-D-glucose:oxygen oxidoreductase, EC 220.127.116.11). GOX oxidizes b-D-glucose to D-glucono-d-lactone and gluconic acid (GA) extracellularly, thus preventing its entry into glycolysis, thereby diverting a portion of the sugar carbon away from ethanol. The GOX-encoding gene from the foodgrade fungus, Aspergillus niger was used to construct three cassettes (GOX1, GOX2 and GOX2LOX). In these gene cassettes, the A. niger GOX gene was placed under the regulation of the S. cerevisiae phosphoglycerate-kinase-1 gene promoter (PGK1P) and terminator (PGK1T ). To facilitate secretion, in GOX1 the yeast mating pheromone-factor a secretion signal (MFa1S) was fused to the GOX gene, and in GOX2 the native A. niger secretion signal of GOX was used. These gene cassettes were each integrated into the genome of two laboratory yeast strains (BY4742 and S1278b) and one industrial wine yeast strain (VIN13). An additional integration cassette, designated GOX2LOX, was constructed to knock out the IME1 gene in S. cerevisiae. In GOX2LOX, GOX2 was fused to a loxP cassette. VIN13-D1 was obtained by integrating a single copy of GOX2LOX into the IME1 locus. To generate an asporogenic, GOX-producing wine yeast, VIN13-D2 was created by sporulation, micromanipulation and re-diploidisation of VIN13-D1. Comparative analysis indicated that (i) GOX2 resulted in higher levels of extracellular glucose oxidase activity than GOX1; and that (ii) the levels of secreted glucose oxidase activity in the wine yeast transformants were sufficiently high to conduct follow-up small-scale wine fermentation trials. The wine yeast transformant, VIN13-D1 was evaluated under red and white experimental winemaking conditions. Results from this work indicated that glucose oxidase was produced and secreted by VIN13-D1 that dominated the fermentation to the end, but also that the enzyme was not highly active under the evaluated winemaking conditions. Consequently, no significant decrease in ethanol concentrations was observed in the wine made from VIN13-D1 when compared to that from VIN13. Wine samples were analyzed by Fourier transform-middle infrared spectrometry (FT-MIR) to determine the chemical composition and Gas chromatography with a flame ionization detector (GC-FID) to evaluate the concentrations of aroma compounds. The levels of gluconic acid were determined by enzymatic assays. Multivariate data analysis (PCA and PLS1-discrim) was applied to highlight significant differences between the wines made by VIN13 (wild-type) and VIN13- D1. Chemometric projections of the score plots for all results allowed insight into all significant variation up to three principal components (PCA) or PLS components, which showed very clearly that GA is a key factor in evaluating the effect of GOX in VIN13-D1 fermentation with regard to VIN13 fermentations. The VIN13- D1 effect manifestations were best shown on PLS1-discrim score plots that revealed that, of the restricted variable subsets the FT-MIR-compounds and GC-compounds yielded better results, with the GC-compounds displaying greater discriminability between cultivars and VIN13 / VIN13-D1. It can be concluded from these results that the greatest influence of VIN13-D1 produced wines could be observed in the aroma components, but, because there were also discriminability effects discernable in the FT-MIR-compounds, thus the flavor components were also affected. The activity of GOX in grape juice was further investigated in controlled small scale fermentations performed in a bio-reactor. It was confirmed that GOX is active under aerobic conditions, inactive under anaerobic conditions, and can be activated instantly when an anaerobic culture is switched to aerobic conditions (simulated micro-oxygenation). These fermentations showed that glucose oxidase is active in grape juice, and that oxygen play a key-role in the enzyme’s activation. Finally, it was shown with the help of a simplified model, that under ideal conditions, GOX secreted from VIN13-D1, can be employed to reduce the ethanol by a predefined concentration for the production of low alcohol wines. This work gives more insight into how to employ a GOX-producing wine yeast during winemaking and strongly suggests the use of micro-oxygenation to activate the enzyme in order to reduce available glucose, thereby diverting a portion of the sugar carbon away from ethanol production.
- ItemCharacterization of melatonin production and physiological functions in yeast(Stellenbosch : Stellenbosch University, 2020-03) Motlhalamme, Thato; Bauer, Florian; Prior, Bernard; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology.ENGLISH ABSTRACT: Melatonin is a molecule that is found in all living organisms with numerous functions such as the regulation of circadian rhythms in animals and growth stimulation in plants. Studies suggest that melatonin acts as an antioxidant in all living organisms. Its biosynthetic pathways and biological functions are characterised in plants and animals but very little is known about these processes in microorganisms. Yeasts have been found to synthesise melatonin under various conditions such as fermentation, starvation and aerobic growth, but production patterns were found to be inconsistent. The purpose of this study was to investigate the physiological functions of melatonin in yeast in more detail by evaluating a large number of growth conditions to determine conditions that would elicit consistent melatonin production. The study used Saccharomyces cerevisiae as a model organism to study the impact of melatonin on cellular physiology and identify biosynthesis- and melatonin-responsive genes. None of the conditions investigated in this study resulted in consistent melatonin production. When detected, concentrations of melatonin were very low (ng/107 cells) and varied greatly between biological repeats. In plants and animals, melatonin production oscillates in response to diurnal cycles; however, this oscillatory pattern was not observed in the current study. An analysis of the pathway intermediates suggest that multiple enzyme reactions may be involved in the synthesis of melatonin in yeast and that yeast appears not to possess a dedicated synthesis pathway. The absence of orthologs of the enzymes involved in the biosynthetic pathway in yeast supports this conclusion. The chaotic production pattern in yeast suggests that melatonin may be a product of non-specific enzymatic reactions or overflow metabolism. This study took a different approach to evaluating the response of cultures to oxidative stress by conducting experiments in continuous culture conditions instead of batch culture. Over time, the number of differentially expressed genes decreased more rapidly together with yeast recovery from stress in melatonin treated cultures compared to melatonin untreated cultures. Transcriptomic analysis of S. cerevisiae treated with melatonin pre- and post-H2O2 induced oxidative stress suggests that it does not act through any specific stress-responsive pathway and its activity could not be linked to any specific genetic interaction or regulation. However, in the absence of stress, exogenous melatonin enhanced the expression of sulphate assimilation pathway genes. This pathway leads to the formation of methionine and cysteine which are involved in the production of glutathione, and the response therefore may prime cells for subsequent stress. When S. cerevisiae was stressed with various reactive oxygen species generating stressors, melatonin supplementation improved the survival of the cultures in a similar manner to other antioxidants, by increasing the expression of several genes that support the general antioxidant response.
- ItemCharacterization of transgenic grapevine ectopically expressing plant defensin peptides(Stellenbosch : Stellenbosch University, 2019-12) Barkhuizen, Helmien; Vivier, Melane A.; Rautenbach, Marina; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology.ENGLISH ABSTRACT: Grapevine (Vitis vinifera L.) is one of the most important and widely grown food crops in the world. The cultivation and commercial production of this crop has, however, became highly dependent on the use of pesticides. One of the strategies to limit the use of chemicals is to harness the species natural defence mechanisms. This strategy requires the understanding of these plant defence mechanisms. Among the highly specialized defence mechanism of plants is the production of specific antimicrobial peptides, called plant defensins. These peptides are small, basic, positively charged and cysteine-rich with a potent broad range of antimicrobial activity. The plant defensins form a vital part of the innate immune system of plants and are widely distributed throughout the plant kingdom. Several plant defensins have been isolated and predominantly characterized for their in vitro antifungal activity. However, other biological activities, such as heavy metal tolerance, ion channel blocking, α-amylase and protease inhibition and modulators of growth and development have also been attributed to these peptides. Some well-studied defensins have been described in literature in terms of their three-dimentional structure, antimicrobial in vitro functions/activities, their mode(s) of action, as well as their applications. Limited information, however, is available on their broader potential impacts on plant growth and development and more specific, non-defence related stress-mitigating functions within their host plants. Furthermore, little information exists on grapevine plant defensins. Although 79 defensin-like genes (DEFL) have been identified in the reference genome, only four grapevine plant defensins have been isolated and characterised to date for potential antifungal activities. The goal of this study was therefore to evaluate potential in planta/in vivo functions of plant defensins in grapevine. In this study functional characterisation studies were performed on plant defensins overexpressed in grapevine. Genotypical screens were conducted on uncharacterised transgenic populations of two V. vinifera cultivars (Sultana and Red Globe), overexpressing three different defensin peptides (Heliophilia coronopifolia antifungal peptides 1 and 4 (Hc-AFP1, Hc-AFP4) and Raphanus sativus antifungal peptide 2 (Rs-AFP2)) to assess transgene integration and expression. These analyses revealed unique transgenic lines for the transgenic populations expressing the plant defensins Hc-AFP1 and Rs-AFP2, with the majority of these lines expressing the transgene. Although the presence of the transgene was confirmed for the transgenic V. vinifera (cv. Sultana) Hc-AFP4 and V. vinifera (cv. Red Globe) Hc-AFP4 lines, they did not exhibit any transgene expression and was not included in the growth, or biotic and abiotic stress phenotypical analyses. A previously characterised population of Vvi-AMP1 overexpressed in Sultana was also included in this study. Two in silico approaches were used to contextualise the functional characterisation studies. The first was to compare the different peptides in terms of their sequence similarities, as well as deduced structural features. The Rs-AFP2 peptide’s crystal structure has been resolved and the structure-function in silico analyses made use of the data available for this peptide. The Hc-AFP1, Hc-AFP4 and Rs-AFP2 peptides showed more similarities in sequence and structure compared to the Vvi-AMP1 defensin peptide. The majority of the sequence and structural differences between Hc-AFP1, Hc-AFP4 and Rs-AFP2 and the Vvi-AMP1 peptide resided in the conserved λ-core motif that is known to be important in determining the antifungal activities of plant defensin peptides. The second in silico approach was to use existing gene expression data in grapevine to evaluate where (in which organs and tissues) and under which (stressful) conditions grapevine defensin genes show differential expression patterns. Using the Corvina gene atlas, it was shown that under normal non-stressed conditions, DEFL genes were expressed in all grapevine organs and tissues at various developmental stages. Furthermore, using available microarray data, it was found that some defensins responded to biotic stress such as B. cinerea infection, although the Vvi-AMPs did not respond. Similarly, strong upregulation was found in response to Planococcus ficus (mealybug) infestation and in response to abiotic stress such as locally applied heat stress and leaf dehydration, with Vvi-AMP1 showing the strongest upregulation to the latter. Guided by the results of the in silico gene expression analysis, the in vivo functions of plant defensin peptides in grapevine were evaluated by analysis of the transgenic grapevines, overexpressing plant defensin peptides Hc-AFP1, Rs-AFP2 and Vvi-AMP1 in terms of growth, as well as biotic and abiotic stress. From these phenotypical observations it was evident that genotypical background (Sultana versus Red Globe) had a strong effect on several of the phenotypes observed. Furthermore, some of the observed phenotypes were peptide-specific, whereas in most other instances all peptides caused the same type of response to a particular stress, but with varying strength of the response, or some differences in mechanism. In terms of growth, the transgenic populations only showed mild phenotypes and no overt stunting or abnormalities were observed. Some plant lines, however, showed slower growth and root inhibition in vivo and these observed growth alterations were possibly a result of higher metabolic load on the plants due to the overexpression of the peptides, an aspect that deserves further study. The transgenic populations were evaluated for their in vivo functions towards biotic stress through evaluating their defence phenotypes against two fungal pathogens, namely the necrotrophic fungus, Botrytis cinerea and the biotrophic fungus Erysiphe necator, as well as the insect pest, Planococcus ficus (mealybug). None of the transgenic plant lines displayed a resistant defence phenotype towards B. cinerea, whereas all the transgenic lines showed enhanced resistance towards the biotrophic powdery mildew fungus through an increased penetration resistance mechanism. Some plant lines also displayed programmed cell death (PCD) associated resistance, especially the transgenic plants that contained the Rs-AFP2 construct. PCD is associated with the mechanism of action of the Rs-AFP2 peptide. All tested plant lines also showed promising results towards the soft scale insect P. ficus, all reducing the infestation significantly, making this the first report of an in planta anti-insect activity of plant defensins Hc-AFP1, Rs-AFP2 and Vvi-AMP1. The transgenic populations were also evaluated for their in vivo functions towards abiotic stress by subjecting plants to an active drying experiment and evaluating their intrinsic water use efficiencies (WUE). The majority of the plant lines all demonstrated an increase in intrinsic WUE, but the Red Globe Rs-AFP2 plant line showed a decrease in intrinsic WUE, which can at least partly possibly be linked to the reduced growth parameters demonstrated for this plant line, specifically the reduction in root growth. In conclusion, this study contributed to the current understanding of how plant defensins function in vivo, confirming growth impacts, antifungal activities, anti-insect activity and a role in water stress management. Furthermore, we gained a vital insight into the in vivo functions of grapevine plant defensins.
- ItemA chemometric approach to investigating South African wine behaviour using chemical and sensory markers(Stellenbosch : Stellenbosch University, 2021-03) Mafata, Mpho; Buica, Astrid; Brand, Jeanne; Medvedovici, Andrei V.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology.ENGLISH ABSTRACT: The aim of this dissertation was to demonstrate the value of comprehensive narratives and elucidate critical steps in data handling in Oenology, while highlighting some common misconceptions and misinterpretations related to the process. This compilation was a journey through different stages of dealing with oenological data, with increasing complexity in both the strategies and the techniques used (sensory, chemistry, and statistics). To achieve this aim, different strategies and multivariate tools were used under two prime objectives. Firstly, several multivariate descriptive approaches were used to investigate two oenological problems and lay out the contextual foundations for the statistics-focused work (Chapters 3 and 5). Secondly, in increasing levels of complexity, statistical strategies of constructing comprehensive data fusion as well as pattern recognition models were investigated (Chapters 4 and 6). A comprehensive literature review (Chapter 2) examined and addressed common misconceptions in the different stages of data handling Oenology. The first oenological problem, described in Chapter 3, investigated the evolution of the sensory perception of aroma, as well as the antioxidant-related parameters and volatile compound composition of Sauvignon Blanc and Chenin Blanc wines stored under different conditions and durations. The study applied an appropriate sensory method for this research question, namely, Pivot©Profiling. The study was able to show the evolution of Sauvignon Blanc from ‘fruity’ and ‘herbaceous’ and of Chenin Blanc from ‘fruity’ and ‘tropical’ both towards ‘toasted’, ‘oak’, and ‘honey’ attributes. Chemically, the volatile composition did not show any trends. However, wines stored at higher temperatures for longer periods had relatively higher UV-Vis absorbance, colour density as well as higher b* (yellow) values and lower clarity in terms of L* index, compared to the control. The second oenological problem, described in Chapter 5, investigated the typicality of South African old vine Chenin Blanc perceptually and conceptually using a typicality rating and a flexible sorting task. The sensory methodology followed published strategies for investigating typicality. This study did not find a unique sensory space of the old vine Chenin Blanc due to a lack of perceptual consensus among the industry professionals for the wines included in the study. However, it did find that the industry professionals had unified ideas about the attributes of an ideal old vine Chenin Blanc wine. The first of the statistics-focused studies, described in Chapter 4, explored data fusion at low and mid-level using principal component analysis - PCA (low and mid-level) and multiple factor analysis - MFA (mid-level). The study looked at data pre-processing and matrix compatibility, which are important data handling stages for data fusion. Like the contextual chapters (Chapter 3 and 5), and keeping with the aim of this compilation, this chapter gave a detailed descriptive narrative of the data handling. Through detailed examination of the process, the study found that MFA was the most appropriate data fusion strategy. The second statistics-focused study, described in Chapter 6, continued to exploit the multiple advantages of multiblock approach of MFA. Additionally, this chapter showed the reliability of fuzzy k-means clustering compared to agglomerative hierarchical clustering (AHC).
- ItemThe colour and phenolic content of Robertson Red grape cultivars : distribution, correlation with wines and analyses(Stellenbosch : Stellenbosch University, 2013-03) Van der Merwe, Hanneli; Du Toit, Wessel J.; Nieuwoudt, Helene; De Beer, D.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology.ENGLISH ABSTRACT: South African red wine is often acknowledged world wide as being full bodied and deep in colour. This is often the result of high temperatures that is experienced during the important growth stages of grapes especially post véraison. In the Robertson area in South Africa however, temperatures often exceeds the range for optimal anthocyanin development during these growth stages. The distinction between grapes being technologically ripe and being ripe on a phenolic level is also accepted as an important determining factor for the perfect time to pick grapes. In co-operative wineries such as Robertson Winery (RW) where grapes are delivered from a large area and different producers, it is difficult to individualise grape blocks when it comes to ripeness level in terms of sugar or phenolic ripeness. In most circumstances a generalised set of parameters for deeming grapes ripe or acceptable for delivery is the best substitute. The levels of these parameters are based on research literature that is available for the area as well as data collected through years of maintaining the vineyards of that area. The grape parameters that are currently being used by RW for ripeness and quality are pH, titratable acidity (TA) and sugar level. In recent years RW in conjunction with the Department of Viticulture and Oenology, Stellenbosch University, decided to investigate more parameters to determine the quality of grapes at the time of harvest. Most importantly for the grape growers this quality is connected to a price point and therefore compensation. Two important quality parameters of red wine are the red colour and mouth feel of wine. Anthocyanin and tannins are respectively connected to these two quality attributes and are both widely accepted as quality indicators. Wine with high anthocyanin and tannin content often originates from grapes with a high colour and phenolic profile. The existence of a correlation between grape and wine anthocyanin and tannin content is therefore the basis of attempting to use these parameters in the grape to predict end wine’s colour and phenolic quantity. Determination of anthocyanin and tannin content of grapes has already become part of some private owned wineries’ standard set of determinations. However, sample preparations, extractions and consumables needed are all factors that need to be reduced to make the measurement and therefore the use of these parameters more viable in a co-operative cellar laboratory, where large volumes of grapes are received during harvest. The first objective of this work was to determine the levels of anthocyanin and tannin in red grapes from different vineyard blocks from the producers of RW from three successive vintages. This would give insight as to what can be seen as a low and high anthocyanin and tannin content for grapes received at the cellar. For this purpose, blocks of the most important red wine cultivars for RW was selected and analysed for these compounds. The ranges and average levels of anthocyanin and tannin content were determined using measurement techniques that could be used by any winery. The average mono flavanol and total colour level of the grapes were found to be lower than those often reported in literature, with total grape flavanols being higher. However, a wide range of values for these compounds were found that correlated with those found in other studies. The possible reasons for differences in levels of occurrence of these compounds were discussed and mostly pertain to differences in cultivar, micro climatic and season. The second objective was to determine the correlation between levels of colour and phenolic compounds in grapes and their corresponding wines. Such correlations will form the foundation for the use of phenolic content to predict the colour and phenolic potential of the wine and possibly wine quality as well. When the grape and wine colour and phenolic data were correlated for all seasons and cultivars inclusive it was found that grape and wine colour showed better correlations than for instance total phenols and tannins. This was especially true for total colour pigments in red grapes, measured with HPLC, when correlated with certain spectrophotometric analysis of wine colour. Cultivar and season as well as the synergism between the two were further investigated for its role in affecting correlations. When these relationships were further differentiated by season and by cultivar the resulting correlations varied. This work contributed a great deal of information to support the use of grape colour and phenolic compounds for the prediction of end wine colour and phenolic composition. The third objective was to investigate near infrared spectroscopy (FT-NIR) as a viable option to rapidly measured anthocyanins, tannins and total phenolics in red grapes. If proven successfully, this could be employed by a large cellar such as RW. FT-NIR has been used with success on grape extracts and in this instance the focus was to establish a calibration on the grape homogenate itself. Preliminary results showed that FT-NIR could be applied for the use of determination of anthocyanin and tannin levels in red grapes originating from RW. The prediction of total phenols was not found to be as accurate, but this could also be due to the reference method that was used. This work brought some interesting, practical information not only of importance for RW, but all wineries that are concerned with improving the basis on which grape quality is determined. The use of aerial data mapping for indicating areas regarding important grape colour and phenolic parameters was used in this study and is a very visual way of showing the distribution of certain ripeness parameters over a large area. Correlations between the grape and wines of such a large amount of red grape blocks for a specific area have not also been reported in South Africa before. The use of FT-NIR to determine anthocyanins and tannin concentrations in grape homogenates is also novel for its use in South African wineries. This work may assist grape and wine producers as well as analysts on the phenolic and colour profile of grapes and wines from RW.
- ItemComparative 'omic' profiling of industrial wine yeast strains(Stellenbosch : Stellenbosch University, 2009-12) Rossouw, D.; Bauer, Florian; University of Stellenbosch. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.The main goal of this project was to elucidate the underlying genetic factors responsible for the different fermentation phenotypes and physiological adaptations of industrial wine yeast strains. To address this problem an ‘omic’ approach was pursued: Five industrial wine yeast strains, namely VIN13, EC1118, BM45, 285 and DV10, were subjected to transcriptional, proteomic and exometabolomic profiling during alcoholic fermentation in simulated wine-making conditions. The aim was to evaluate and integrate the various layers of data in order to obtain a clearer picture of the genetic regulation and metabolism of wine yeast strains under anaerobic fermentative conditions. The five strains were also characterized in terms of their adhesion/flocculation phenotypes, tolerance to various stresses and survival under conditions of nutrient starvation. Transcriptional profiles for the entire yeast genome were obtained for three crucial stages during fermentation, namely the exponential growth phase (day 2), early stationary phase (day 5) and late stationary phase (day 14). Analysis of changes in gene expression profiles during the course of fermentation provided valuable insights into the genetic changes that occur as the yeast adapt to changing conditions during fermentation. Comparison of differentially expressed transcripts between strains also enabled the identification of genetic factors responsible for differences in the metabolism of these strains, and paved the way for genetic engineering of strains with directed modifications in key areas. In particular, the integration of exo-metabolite profiles and gene expression data for the strains enabled the construction of statistical models with a strong predictive capability which was validated experimentally. Proteomic analysis enabled correlations to be made between relative transcript abundance and protein levels for approximately 450 gene and protein pairs per analysis. The alignment of transcriptome and proteome data was very accurate for interstrain comparisons. For intrastrain comparisons, there was almost no correlation between trends in protein and transcript levels, except in certain functional categories such as metabolism. The data also provide interesting insights into molecular evolutionary mechanisms that underlie the phenotypic diversity of wine yeast strains. Overall, the systems biology approach to the study of yeast metabolism during alcoholic fermentation opened up new avenues for hypothesis-driven research and targeted engineering strategies for the genetic enhancement/ modification of wine yeast for commercial applications.
- ItemConsumer attitudes and sensory perceptions of wine : a South African cross-cultural study(Stellenbosch : Stellenbosch University, 2018-03) Weightman, Carla Jayne; Nieuwoudt, Helene; Bauer, Florian; Terblanche, Nic; Valentin, Dominique; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: The wine consumer landscape has changed in South Africa since 1994. Historically, some population groups had more access and greater cultural affinity for the product “wine”, but such historical patterns are rapidly changing, particularly in urban areas. South Africa has a low per capita wine consumption, but industrial research has shown that there is potential for growth. The wine industry is therefore aiming to become more consumer-driven, in order to increase domestic wine consumption. Consequently, the need for consumer research has increased. Only limited published data is available on South African wine consumers and their consumption habits. The general aim of this study was therefore to do a broad exploration of current wine consumers’ perceptions of wine. This study had a few objectives, firstly to use focus groups to obtain a better understanding of South African wine consumers from different ethnic backgrounds (Black African and Caucasian), with a focus on perceptions, style preferences, context of enjoyment, purchase and consumption patterns, and journeys towards wine consumption. Secondly to develop a questionnaire in order to investigate motivations for wine consumption on a larger scale. Thirdly to compare consumers’, trained assessors, and industry professionals’ sensory perceptions of wine. Lastly to look at the influence of cultivar name on consumer liking. The results of the focus groups showed that the predominant differences occurred between male and female consumers. With regards to ethnicity, this study did not reflect a large distinction between the different ethnic groups. Overall, the wine choice questionnaire showed motivations for drinking wine between the consumer groups to be similar. The social, sensory appeal and ethical concern factors were the three most important motivational factors for South African wine consumers. The industry professionals, trained assessors, and consumers gave very similar answers and distinguished between the sample set of wines in a similar fashion. Consumers that participated in this study mostly described themselves as novices and only somewhat knowledgeable. For them, knowing the cultivar name did not significantly change the way they described the samples, nor did it significantly alter their liking. Even though the samples were all different cultivars made in different styles, on average they were all received positively. This is the first study to focus on understanding the changed landscape of SA wine consumers. The knowledge gained made a significant contribution towards a better understanding of South African consumers’ perceptions of wine, and also highlights directions for future studies.
- ItemThe control of cellular adhesion of Saccharomyces cerevisiae by the FLO gene regulator Mss11p(Stellenbosch : University of Stellenbosch, 2010-03) Bester, Michael Christiaan; Bauer, Florian; University of Stellenbosch. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: The yeast Saccharomyces cerevisiae senses change within its environment and responds through specific adaptive cellular programmes, in particular by modifying gene expression. Many adaptive changes affect the physico-chemical properties of the cell wall, and several mechanisms that specifically affect the expression levels of genes that encode for cell wall components have been described previously. Cell wall modification directly impacts on general cell wall properties and cell-cell and cell-surface interactions. Many of these properties have been directly linked to families of cell wall proteins referred to as adhesins. In particular members of the Flocculation (FLO) gene family have been shown to play a crucial role in adhesion phenotypes. Flo11p functions in a variety of phenotypes including agar invasion, plastic adhesion and the formation of pseudohyphae, “flor” and “mats”, whereas Flo1p appears to control flocculation. The regulation of FLO11 expression is well documented and is mainly controlled by the mitogen activated protein kinase (MAPK) and cyclic AMP protein kinase A (cAMP-PKA) signalling cascades. Genetic analysis shows that Mss11p acts downstream and is central to these pathways, and furthermore interacts with the cAMP-PKA component Flo8p to activate transcription. In this study we further explore additional gene targets of Flo8p and Mss11p, as well as their regulation and their impact on cell wall characteristics and associated adhesion phenotypes. Our analysis shows that Mss11p is also required for FLO1 expression, and functions together with Flo8p to control many Flo-dependent adhesion phenotypes. Genome-wide gene expression analysis further reveals that altered Mss11p levels leads to the change in the expression of various cell membrane and cell wall genes, notably AQY2 and members of the DAN and TIR gene families. Further genetic analysis indicates that adhesion phenotypes display an almost exclusive dependence on FLO gene expression. We also demonstrate that these phenotypes require Flo10p and are thus dependent on the specific balance of Flo proteins in the cell wall. The analysis of signalling deletion mutants show that regulation of FLO10 shares signalling components with FLO11, but that the two genes are differentially regulated. Unlike FLO11, FLO10 transcription also does not display an absolute requirement for Mss11p but rather for the MAPK component Ste12p. Whole genome expression analysis were also performed on strains with altered levels of Flo8p which were compared with the above mentioned transcriptome data set. This analysis shows that Flo8p and Mss11p co-regulate the FLO genes, as well as AQY2 and TIR3, but also have significant unique gene targets. The combination of transcriptome data with current information concerning transcription factor (TF) interaction networks reveals the importance of network interaction between Cin5p, Flo8p, Mga1p and Mss11p. From these data we constructed a TF interaction model in which Flo8p acts as the predominantly activating TF component, whereas Mss11p function as a target hub TF, possibly as a mediator- or polymerase II holo-enzyme component. Finally we provide a first report on “mat” formation by an industrial wine yeast strain, and show that by adjusting FLO11 expression in this strain we are able to significantly change this phenotypic behaviour.
- ItemCross species identification of genetic factors that determine fruit surface characteristics(Stellenbosch : Stellenbosch University, 2015-12) Lashbrooke, Justin Graham; Asaph, Aharoni; Costa, Fabrizio; Vivier, Melane A.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: The plant cuticle is a typically waxy layer that covers the entire above ground part of higher plants and performs a number of important roles in vegetative organs and during fruit development and ripening, including protection from a range of abiotic and biotic stresses. This complex hydrophobic layer consists of a cutin matrix of predominantly fatty acids embedded with long chain waxes, synthesized in (and secreted from) the epidermal cells. Suberin, an aliphatic polymer which is higher in phenolic content than cutin and less elastic, may be formed due to wounding and/or cuticle damage. While the synthesis and transport of cutin and wax have been investigated for a number of decades, there are still steps in these pathways that are yet to be elucidated. In the case of suberin biosynthesis, much less has been described. The understanding of the regulatory mechanisms for these processes has only recently received attention, and has already proven to be an area of significant interest for plant scientists, particularly with regard to the interaction between the regulation of epidermal cell identity and cuticle development. In terms of fruit production the cuticle can be linked to many quality parameters, especially postharvest storage. This is particularly true for apple, a crop dependent on a long shelf life. Apple is also susceptible to the formation of a cuticle failure disorder known as russet. Apple russet results from micro-cracking of the cuticle and the formation of a corky suberized layer. During normal growth suberin can be found in potato tubers, roots, bark, and seed coat. However, on most fruit, suberin is typically an undesirable consumer trait, and in the case of apples can have a negative impact on the postharvest storage. In this work, the current understanding of plant cuticles is built upon with particular emphasis on fleshy fruit cuticle assembly and the regulation of the biosynthetic pathways generating its constituents. Research goals focused on identifying and characterizing genetic factors involved in fruit surface formation, particularly with regard to cuticle biosynthesis. Further attention is paid to understanding the regulatory mechanisms of cuticular pathways, including the emerging concept of co-regulation of epidermal cell differentiation and cuticle biosynthesis. Tomato and apple were the species in which these investigations were focused. This was due to the fact that tomato is seen as a model species for fruit surface biology research, while the applied aspects of apple surface research have far reaching impact. To examine the relationship between epidermal cell development and cuticle assembly in the context of fruit surface the tomato SlMIXTA-like gene was investigated. MIXTA/MIXTA-like proteins, initially described in snapdragon petals, are regulators of epidermal cell differentiation. In an effort to understand these processes in fruit, tomato was transgenically silenced for SlMIXTA-like expression. Plants displayed defects in the patterning of conical epidermal cells of fruit, and also showed altered postharvest water loss and resistance to pathogens. Transcriptome and cuticular lipids profiling, coupled with comprehensive microscopy, revealed significant modifications to cuticle assembly and suggested SlMIXTA-like to regulate cutin biosynthesis. Candidate genes acting downstream of SlMIXTA-like included oxidases, transferases and transporters involved in cutin synthesis and assembly. As part of a larger regulatory network of epidermal cell patterning and L1-layer identity, it was found that SlMIXTA-like acts downstream of the cutin biosynthesis regulator SlSHN3 and possibly co-operates with homeodomain-leucine zipper IV transcription factors. Hence, SlMIXTA-like is a positive regulator of both cuticle and conical epidermal cell formation in tomato fruit, acting as a mediator of the tight association between fruit cutin polymer formation, cuticle assembly and epidermal cell patterning. While russeting may occur in apples after cuticle damage, it is also a heritable trait, and therefore is to some extent under genetic control. In order to identify genetic factors controlling cuticle biosynthesis in apple (and thus preventing russet), a QTLmapping survey was performed on a full-sib population. Two genomic regions located on chromosome 2 and 15 that could be associated with russeting were identified. Apples with compromised cuticles were identified through a novel and high throughput tensile analysis of the skin, while histological analysis confirmed cuticle failure in a subset of the progeny. Additional genomic investigation of the determined QTL regions identified a set of underlying genes involved in cuticle biosynthesis. Candidate gene expression profiling by qRT-PCR on a subset of the progeny highlighted the specific expression pattern of a SHN1/WIN1 transcription factor (termed MdSHN3) on chromosome 15. The MdSHN3 transcription factor displayed extremely low expression in lines with improper cuticle formation suggesting it to be a fundamental regulator of cuticle biosynthesis in apple fruit, and thus necessary for the prevention of suberized fruit surfaces (russet). In an effort to gain a greater understanding of the mechanisms underlying suberin biosynthesis in fruit, and in plants in general, transgenic tomato were generated with compromised cutin formation. This was achieved via the transcriptional silencing of SlDCR, an orthologue to AtDCR which was previously identified as a key step in cutin biosynthesis in Arabidopsis. Silencing of this BAHD acyltransferase resulted in an almost total elimination of the major monomer from the fruit cutin (C16-9,10-dihydroxy fatty acid), and the plants developed fruit with a suberized surface. This provided an excellent opportunity for transcriptome and chemical characterization of the suberization process in fleshy fruit. In parallel an apple clone that developed a russeted fruit surface was identified, and characterized. A large scale comparative transcriptomic analysis of these tomato and apple mutants was performed, generating a list of candidate genes for suberin deposition. Increasing the comparison to include data mined from literature resulted in the elucidation of a multi-species gene expression signature for suberin biosynthesis, and allowed for the identification and characterization of novel genetic elements, including those involved in the regulation of suberin formation and its deposition. Of these genetic elements, MYB107, was demonstrated to be a positive regulator of suberin accumulation in Arabidopsis seed coat. In totality this study has produced a greater understanding of genetic mechanisms governing cuticle biosynthesis, particularly in developing fruit. This was achieved primarily through the functional characterization of regulatory elements (MIXTA-like, SHN3 and MYB107 transcription factors) controlling the synthesis of cutin and suberin matrices. Additionally the DCR enzyme was demonstrated to be a crucial step in fruit cutin biosynthesis. Finally the intricate relationship between epidermal cell development and cuticle biosynthesis has been further highlighted.
- ItemThe development of yeasts for the optimal production of flavor-active esters and higher alcohols in wine and distillates(Stellenbosch : Stellenbosch University, 2004-12) Lilly, Mariska; Pretorius, I. S.; Bauer, Florian; Lambrechts, M. G.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: Yeasts produce a broad range of aroma-active volatile esters and higher alcohols during alcoholic fermentation. Some of these esters and higher alcohols are important for the fruity flavors and therefore the final quality of wine and other fermented beverages. Esters are produced and hydrolyzed by alcohol acetyltransferases and esterases, respectively. In yeast, ester-synthesizing activities are represented by two alcohol acetyltransferases encoded by the ATFI and ATF2 genes, and by an ethanol hexanoyl transferase encoded by the EHTI gene. Atfl p and Atf2p appear responsible for the production of ethyl acetate and isoamyl acetate, while Ehtl p synthesizes ethyl hexanoate from ethanol and hexanoyl-CoA. Although a fair amount of information is available regarding the ATF 1 gene, limited information is available on the remaining alcohol acetyltransferases. Only two genes that code for esterases have been identified in yeast, namely lAHI and TIPI. It has also been shown that the balance between alcohol acetyltransferases and esterases is important for the net rate of ester accumulation. Higher alcohols are synthesized from the a-keto-acids in the branched-chain amino acid metabolic pathway by decarboxylation and reduction. The transamination of the amino acid to the respective a-keto-acid is catalyzed by mitochondrial and cytosolic branched-chain amino acid transferases, which are encoded by the BATI and BAT2 genes, respectively. In recent years, a strong scientific and industrial interest in the metabolism of flavoractive compounds has emerged, but information regarding the roles of specific enzymes and the physiological relevance of their metabolism remains limited. The aim of this project was to investigate the physiological and metabolic consequences of changes in the expression levels of some of the key enzymes involved in aroma compound production. The consequences of these changes on the chemical composition and the fermentation bouquet of wines and distillates were also investigated. The first part of the section on the results in this dissertation reports on the role and relative importance of the Saccharomyces cerevisiae enzymes involved in ester metabolism, namely Atflp, Atf2p, Ehtlp, Iahlp and Tiplp. The corresponding genes were overexpressed in a laboratory strain of S. cerevisiae, BY4742, and in a widely used commercial wine yeast strain, VIN13. Table wine and base wines for distillation were prepared with these VIN13 transformed strains. The ester concentrations and aroma profiles of the wines and distillates were analyzed and compared. The data indicated that the overexpression of ATF 1 and ATF2 increased the concentrations of ethyl acetate, isoamyl acetate, 2-pheylethyl acetate and ethyl caproate, while the overexpression of JAHI resulted in a significant decrease in the concentrations of ethyl acetate, isoamyl acetate, hexyl acetate and 2-phenylethyl acetate. The overexpression of EHTI resulted in a marked increase in the concentrations of ethyl caproate, ethyl caprylate and ethyl caprate, while the overexpression of TJP1 did not decrease the concentrations of any of the esters. In most cases, there was a correlation between the increase in esters and the decrease in higher alcohols. The data suggest that yeast balances the amount of different esters produced through alcohol acetyltransferases and esterases, and that, in some cases, these enzymes appear to overlap in function and/or influence each other's activity. In the second part of the results section, the consequences of the deletion and the overexpression of two genes, BATl and BAT2, which encode transaminases that contribute to the metabolism of higher alcohols, were investigated. The genes were both disrupted in a S. cerevisiae BY4742, and overexpressed in both this laboratory strain and in the VIN13 wine yeast strain. The effects of these modifications on the general physiology of the corresponding yeast strains and on higher alcohol metabolism were assessed in a range of growth conditions, including aerobic and anaerobic growth conditions, in the presence of glucose or raffinose as sole carbon source and growth in the presence of various concentrations of amino acids. Table wine and base wines for distillation were prepared with the modified industrial strains and the concentrations of the higher alcohols and the aroma profiles of the wine and distillates were analyzed and compared. Batl deletion seemed to be lethal under the conditions that were created, and therefore only the bat2!:!.strain, together with the BATI and BAT2 overexpression strains, were investigated. These modifications did not appear to significantly affect the general physiology of the strains. The results obtained indicated that the overexpression of BATI increased the concentrations of isoamyl alcohol and isoamyl acetate, and, to a lesser extent, the concentrations of isobutanol and isobutyric acid. The overexpression of the BAT2 gene resulted in a substantial increase in the levels of isobutanol, isobutyric acid and propionic acid production, and a modest increase in the level of propanol and isovaleric acid. Interestingly, the overexpression of BAT2 led to a decrease in isoamyl alcohol and isoamyl acetate concentrations. Sensory analyses indicated that the wines and distillates produced with the strains in which the BATl and BAT2 genes were overexpressed had more fruity characteristics (peach and apricot aromas) than the wines produced by the wild-type strains. This study offers new prospects for the development of wine yeast starter strains with optimized ester and higher alcohol-producing capability that could assist winemakers in their efforts to consistently produce wine to definable specifications and styles and a predetermined flavor profile.
- ItemDirected evolution of wine-related lactic acid bacteria and characterisation of evolved strains(Stellenbosch : Stellenbosch University, 2020-03) Tenyane, Seipati Precious; Bauer, Florian; Du Toit, M.; Rossouw, D.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology.ENGLISH ABSTRACT: Microorganisms form part of complex ecological networks, governed by either metabolic, physical or molecular processes that have positive, neutral or negative effects on microbial interactions. Understanding microbial interactions provides the opportunity to control and manipulate microbes for different biotechnological and industrial applications. For example, the production of beverages such as wine shows how microbial interactions can be controlled and manipulated to achieve desired outcomes. One example is the deliberate inoculation of lactic acid bacteria (LAB) such as Oenococcus oeni or Lactobacillus plantarum to inhibit the growth of spoilage bacteria by depleting available carbon sources such as L-malic acid in a process known as malolactic fermentation (MLF). Indeed, wine provides a good model to study microbial interactions because grape must is inhabited by multiple species of filamentous fungi, yeast, acetic acid bacteria (AAB) and LAB in an anthropogenic and relatively controlled environment. In this study, I investigated the impact of the interaction between the wine yeast Saccharomyces cerevisiae and the LAB L. plantarum. Briefly, the impact of the yeast on the evolution of the bacteria was evaluated after 50 and 100 generations first phenotypically, followed by a genome-wide analysis to identify genetic targets of evolution. A serial transfer method was used for the directed evolution (DE) experiments, introducing bottlenecks and fluctuation between nutrient rich and poor environments after each transfer. This strategy results in a ‘feast-and-famine’ regime, which results in conflicting selective pressures, resembling what normally occurs in dynamic natural environments, which was important here to generate robust and resilient bacteria. Additionally, two yeast strains were used to investigate whether microbial interactions result in yeast-specific adaptations or generic adaptations. Therefore, the yeast strains were kept constant by discarding the yeast at the end of each DE cycle and re-inoculating the mother culture at the start of each DE cycle. The data show yeast strain-specific phenotypes for isolates evolved for 50 generations. Genome-wide analysis showed that broadly targeted pathways are peptidoglycan biosynthesis and degradation, nucleic acid processing, and carbohydrate transport and metabolism in isolates evolved for 50 and 100 generations. These data show that yeast-driven DE results in yeast-specific phenotypic variations and high genetic diversity, but also in convergent evolution over time. The results obtained in this study suggest that yeast drive the evolution of bacteria by dominating the metabolic landscape, showing that strong competitive interactions promote positive selection in mixed species communities, and weak competitive interactions results in no adaptation. This work enriches our understanding of yeast-bacteria interactions over time. Moreover, an isolate that is superior to the parent strain in terms of growth and MLF was obtained, showing potential as a starter culture for winemaking.
- ItemThe effect of enzymatic processing on banana juice and wine(Stellenbosch : Stellenbosch University, 2008-12) Byarugaba-Bazirake, George William; Van Rensburg, Pierre; Kyamuhangire, William; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.Although bananas are widely grown worldwide in many tropical and a few subtropical countries, banana beverages are still among the fruit beverages processed by use of rudimentary methods such as the use of feet or/and spear grass to extract juice. Because banana juice and beer remained on a home made basis, there is a research drive to come up with modern technologies to more effectively process bananas and to make acceptable banana juices and wines. One of the main hindrances in the production of highly desirable beverages is the pectinaceous nature of the banana fruit, which makes juice extraction and clarification very difficult. Commercial enzyme applications seem to be the major way forward in solving processing problems in order to improve banana juice and wine quality. The particular pectinolytic enzymes that were selected for this study are Rapidase CB, Rapidase TF, Rapidase X-press and OE-Lallzyme. In addition this study, investigate the applicability of recombinant yeast strains with pectinolytic, xylanolytic, glucanolytic and amylolytic activities in degrading the banana polysaccharides (pectin, xylan, glucan starch) for juice and wine extraction and product clarification. The overall objective of this research was to improve banana juice and wine by enzymatic processing techniques and to improve alcoholic fermentation and to produce limpid and shelf-stable products of clarified juice and wine. The focus was on applying the selected commercial enzyme preparations specifically for the production of better clarified banana juice and wine. This is because the turbid banana juice and beer, which contain suspended solids that are characterised by a very intense banana flavour, require a holistic approach to address challenges and opportunities in order to process pure banana beverages with desirable organoleptic qualities. The specific objectives of applying commercial enzymes in the processing of banana juice and wine, comparing with grape winemaking practices, use of recombinant yeast and analyses of various parameters in the juices and wines made have enabled generation of information that could be of help to prospective banana juice and wine processors. The research findings obtained could be used to establish a pilot plant or small-scale industry in the banana processing beverages producing large quantities,and finally the overall objective of obtaining limpid and shelf stable products would be achieved.
- ItemThe effect of oxygen on the composition and microbiology of red wine(Stellenbosch : University of Stellenbosch, 2006-03) Du Toit, Wessel Johannes; Du Toit, M.; Marais, Jeannine; Pretorius, I. S.; Lonvaud-Funel, A.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Viticulture and Oenology.The winemaking process involves different complex chemical and biochemical reactions, which include those of oxygen (O2). Oxygen can come into contact with the wine through various winemaking procedures and can be used by the winemaker to enhance the quality of red wine. In wine, the main substrates for oxidation are phenolic molecules, which form quinones. These can influence the sensory characteristics of the wine. O2 can be used in fresh must to remove oxidisable phenolic molecules through a process called hyper-oxidation and can also be added to fermenting must to enhance the fermentation performance of yeast. Controlled O2 additions during ageing can lead to the wine’s colour being increased and the astringency of the wine decreased. This is due to the formation of acetaldehyde from the oxidation of ethanol, which induces the polymerisation of tannin and anthocyanin molecules. The addition of too much O2 to wine can, however, lead to unwanted over-oxidation, with certain off-odours being formed. It can also enhance the growth of unwanted spoilage microorganisms, such as Brettanomyces and acetic acid bacteria. Although research on O2 in wine was started many years ago, many questions still remain. These include the general effect of O2 on the sensory and phenolic profile of red wine especially and the microbiology of wine during ageing. An effective way of measuring oxidation, especially in red wine must also be developed. In the first part of this study, the effects of O2 and sulfur dioxide (SO2) additions on a strain of Brettanomyces bruxellensis (also known as Dekkera bruxellensis) and Acetobacter pasteurianus were investigated. Epifluorescence microscopy and plating revealed that the A. pasteurianus strain went into a viable but non-culturable state in the wine after prolonged storage under relative anaerobic conditions. This state, however, could be negated with successive increases in culturability by the addition of O2, as would happen during the transfer of wine when air is introduced. The A. pasteurianus strain was also relatively resistant to SO2, but the B. bruxellensis strain was more sensitive to SO2. A short exposure time to molecular SO2 drastically decreased the culturability of the B. bruxellensis strain, but bound SO2 had no effect on the culturability or viability of either of the two types of microorganisms. Oxygen addition to the B. bruxellensis strain also led to a drastic increase in viability and culturability. It is thus clear that SO2 and O2 management in the cellar is of critical importance for the winemaker to produce wines that have not been spoiled by Brettanomyces or acetic acid bacteria. This study should contribute to the understanding of the factors responsible for the growth and survival of Brettanomyces and acetic acid bacteria in wine, but it should be kept in mind that only one strain of each microorganism was used. This should be expanded in future to include more strains that occur in wine. The second part of this study investigated the effect of micro-oxygenation on four different South African red wines. It was found that the micro-oxygenation led to an increase in the colour density and SO2 resistant pigments of the two wines in which micro-oxygenation was started just after the completion of malolactic fermentation. In one of these wines, a tasting panel preferred the micro-oxygenation treated wines to the control. In the other two red wines, in which the micro-oxygenation was started seven months after the completion of malolactic fermentation, very little colour increase was observed. One of these two wines was also matured in an oak barrel, where the change in phenolic composition was on par with the treated wines. A prolonged period of micro-oxygenation, however, led to this wine obtaining an oxidised, over-aged character. Micro-oxygenation and maturation in an oak barrel also enhanced the survival of acetic acid bacteria and Brettanomyces in this wine. Micro-oxygenation can hence be used by the wine producer on young red wines to enhance the quality of the wine, but should be applied with care in older red wines. Future research into micro-oxygenation should focus on whether it can simulate an oak barrel. More research into the effect of micro-oxygenation on the sensory profile of the wine is needed. As mentioned, the addition of O2 can lead to oxidative degradation of wine. The brown colour in wine is often used as an indication of oxidation, but oxidative aromas can be perceived before a drastic increase in the brown colour has been observed in red wine. The third part of this study was to assess the possible use of Fourier Transform Infrared Spectroscopy (FTIR) to measure the progression of oxidation in Pinotage red wines. Three wines were used in this study and clear separation between the control and aerated wines was observed by using Principle Component Analysis (PCA). Sensory analysis of these wines confirmed this observation, with a reduction especially in berry fruit and coffee characters and an increase first in potato skin and then acetaldehyde aroma characters as the oxidation progressed. PCA analysis also revealed that in certain wines the visible spectrum of light did not indicate the progression of oxidation as sensitively as with the use of FTIR. This also correlated with the inability of the panel to observe a drastic colour change. FTIR should be further investigated as a possible means of monitoring oxidation in wine and this study should be expanded to wines made from other cultivars as well.
- ItemEffect of varying levels of nitrogen, potassium and calcium nutrition on table grape vine physiology and berry quality(Stellenbosch : Stellenbosch University, 2012-03) Raath, P. J. ( Pieter Johannes); Hunter, J. J.; Conradie, W. J.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology.ENGLISH ABSTRACT: A lack of defects is required for successful table grape marketing, which pre-suppose optimal vine performance, berry development and post-harvest quality. The supply of mineral nutrients affects vine development, physiology and berry quality. Despite a vast amount of research conducted over decades, there remain many unresolved issues regarding table grape vine nutrition to ensure optimal table grape quality and shelve-life. Unjustified fertilisation practices often include excessive applications of nitrogen (N), potassium (K) and calcium (Ca). A four-year field trial was therefore conducted on a sandy soil in the Paarl district of South Africa, using grafted on Ramsey, and trained to a gable trellis system. Nitrogen, potassium and calcium were applied, singular or in combination, at rates up to 300% the calculated annual nutritional requirement. The effect of these excessive applications on table grape performance under typical South African cultivation conditions was investigated for Vitis vinifera L. cv. Prime Seedless, a very early seedless table cultivar that is produced with minimum berry diameter of 18mm, with special reference to 1) vegetative growth, 2) expression of grapevine nutrient availability through foliar analyses, 3) berry nutrient accumulation patterns of this early cultivar, 4) manipulation of berry nutrient content through soil and bunch directed applications and 5) the effect of berry nutrient content on its quality. No definite vegetative growth responses (expressed as shoot length, leaf surface area and shoot mass) and leaf chlorophyll content differences were obtained for all the treatments. These results were obtained in a vineyard on a sandy soil where excessive N fertilisation caused a reduction of soil pH to detrimentally low levels and where the excessive N, K and Ca applications reduced mutual concentrations and that of Mg, in the soil. A lack of stimulation in vegetative growth may therefore be ascribed to the combined negative effect of these excessive applications on soil pH and vine nutrition. Although the N content of petioles was higher for treatments where N was applied, consistent significant increases in petiole N with N fertilisation were not observed. Petiole N concentration showed a decreasing trend throughout the season. Petiole K concentrations were significantly increased by the K fertilisation at all phenological stages. None of the K fertilisation treatments, however, succeeded to raise petiole K concentrations above the accepted maximum norms and petiole K concentration at a specific sampling stage varied significantly between the four seasons. A general decrease in petiole K concentration was found for all seasons. Calcium fertilisation did not increase soil Ca content, resulting in a lack of differences in petiole Ca concentrations between treatments. An increase in petiole Ca concentration towards harvest was obtained. Correlations between petiole nutrient concentration and berry mineral content at harvest were poor. The only way of knowing the mineral content of berries would seem to be by measuring it directly instead of deducing it from the results of leaf or petiole analyses. The dynamics of berry growth impacted on berry nutrient concentration. Early rapid berry growth, predominantly due to cell division and cell growth, was associated with the most rapid decreases in N, P and Ca concentration. Due to mobility of K and Mg in the plant, that exceeds other nutrients, the decrease in concentration of these two mineral elements was not as pronounced as that of the others. Nutrient accumulation was most rapid during the pre-véraison period, but only Ca showed a definite termination during the early ripening period. The continued inflow of N, P, K and Mg, albeit at slower rates immediately after véraison, should be taken into consideration when fertilisation is applied. As a table grape, total accumulation of each nutrient in Prime Seedless berries also far exceeded that of other cultivars studied thus far. A particular difference is that the berry flesh:skin ratio is much higher than that of previously studied cultivars, leading to higher levels of nutrient accumulation in the flesh. Slightly larger berry size was obtained for N applications and is ascribed to slight increases in early vegetative growth, allowing a better response to GA3 treatments. The use of GA3 for berry enlargement is also considered the reason why K fertilisation, resulting in increased berry K levels, did not affect berry size, as is often found for wine grapes. Higher available NO3 - in the soil on account of excessive N applications resulted in higher levels of berry N, despite sub-optimal soil pH regimes that were created by these treatments. Berry K concentration and content were increased by K fertilisation. Rapid vine K uptake and translocation to the berries seem to negate the reduced vine nutritional status as observed in petioles for situations of over-fertilisation with N. Berry Ca levels were not increased by Ca fertilisation or by bunch applied Ca. The rapid rates of berry growth, together with low rates of berry Ca uptake and Ca uptake that terminates at the onset of ripening, are assumed to be the main reasons for this result. Low levels of decay as well as a lack of consistently increased decay were obtained for N containing treatments. Nitrogen levels in the berries above which their susceptibility to fungal infection is increased, should be established. Information on specific N compounds that may lead to more susceptibility is required. Potentially increased berry browning on account of high rates of K fertilisation needs to be further investigated; indications that this may occur were observed. Neither soil applied Ca nor bunch applied Ca improved berry quality, although Ca treatments seemed to reduce decay during the only season that significant differences were obtained. The negative effect of excessive fertilisation on soil chemistry of sandy soils has again been highlighted by this study. This annuls the fertilisation, leading to inefficient fertilisation and a lack of the desired responses. As indicator of vine nutrient availability, petiole analysis, was proven unreliable and should be evaluated in parallel with soil analyses, taking seasonal variation into consideration. The danger of being only guided by published norms for leaf nutrient concentrations when establishing fertilisation practices has again been highlighted by this study. This research indicated that for a very early cultivar like Prime Seedless, nutrient accumulation dynamics can already start to change during the pre-véraison period in some seasons. This is due to different edaphic and climatic conditions as well as berry size, which leads to much higher flesh:skin ratios. Future research on table grapes would need to develop an understanding of the various factors and dynamics that determine berry nutrient concentration and accumulation of early ripening, large berry sized, seedless table grape cultivars.
- ItemElucidating the metabolic pathways responsible for higher alcohol production in Saccharomyces cerevisiae(Stellenbosch : University of Stellenbosch, 2011-03) Styger, Gustav; Bauer, Florian; University of Stellenbosch. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: Alcoholic fermentation, and especially wine fermentation, is one of the most ancient microbiological processes utilized by man. Yeast of the species Saccharomyces cerevisiae are usually responsible for most of the fermentative activity, and many data sets clearly demonstrate the important impact of this species on the quality and character of the final product. However, many aspects of the genetic and metabolic processes that take place during alcoholic fermentation remain poorly understood, including the metabolic processes that impact on aroma and flavour of the fermentation product. To contribute to our understanding of these processes, this study took two approaches: In a first part, the initial aim had been to compare two techniques of transcriptome analysis, DNA oligo-microarrays and Serial Analysis of Gene Expression (SAGE), for their suitability to assess wine fermentation gene expression changes, and in particular to assess their potential to, in combination, provide combined quantitative and qualitative data for mRNA levels. The SAGE methodology however failed to produce conclusive data, and only the results of the microarray data are shown in this dissertation. These results provide a comprehensive overview of the transcriptomic changes during model wine fermentation, and serve as a reference database for the following experiments and for future studies using different fermentation conditions or genetically modified yeast. In a second part of the study, a screen to identify genes that impact on the formation of various important volatile aroma compounds including esters, fatty acids and higher alcohols is presented. Indeed, while the metabolic network that leads to the formation of these compounds is reasonably well mapped, surprisingly little is known about specific enzymes involved in specific reactions, the genetic regulation of the network and the physiological roles of individual pathways within the network. Various factors that directly or indirectly affect and regulate the network have been proposed in the past, but little conclusive evidence has been provided. To gain a better understanding of the regulations and physiological role of this network, we took a functional genomics approach by screening a subset of the EUROSCARF strain deletion library, and in particular genes encoding decarboxylases, dehydrogenases and reductases. Thus, ten genes whose deletion impacted most significantly on the aroma production network and higher alcohol formation were selected. Over-expression and single and multiple deletions of the selected genes were used to genetically assess their contribution to aroma production and to the Ehrlich pathway. The results demonstrate the sensitivity of the pathway to cellular redox homeostasis, strongly suggest direct roles for Thi3p, Aad6p and Hom2p, and highlight the important role of Bat2p in controlling the flux through the pathway.