- ItemExploring multispecies interactions between wine-associated yeasts(Stellenbosch : Stellenbosch University, 2021-12) Conacher, Cleo Gertrud; Bauer, Florian; Rossouw, Debra; Blassoples-Naidoo, Rene; Stellenbosch University. Faculty of AgriSciences. Institute for Wine Biotechnology.ENGLISH ABSTRACT: The fermentation of grape must to wine is catalysed by a diverse microbial community. Yeast are primary drivers of the associated alcoholic fermentation process and have therefore garnered considerable research interest. The diversity of yeast species present during wine fermentation influences the chemical composition and related sensory properties of wine as a result of the metabolic functioning of particular yeast species in response to abiotic and biotic factors. The latter is a relatively new research field, given that microbiological science has a significant monoculture bias, and as such, there is much still to be understood about the role and mechanisms of biotic stress in wine yeast ecosystems. Moreover, while the wine yeast ecosystem was the model used in this study, there are several other yeast ecosystems of biotechnological importance, including in biofuels production, bioremediation and other food and beverage industries, that would benefit from insight into these biotic stress mechanisms. The current basis of our understanding of the molecular mechanisms of yeast interactions in the wine ecosystem is based on two-species pairings, which keeps the system interaction network uncomplicated. However, there are many more role-players in natural ecosystems, and they do not interact in a linear fashion. At the micro- and macroscopic level, the importance of these often overlooked higher-order interactions has been highlighted in other ecosystems. There is very little information on higher-order interactions in the yeast ecology field, and this must be remedied for predictive understanding of these systems. Here, we sought to address the current status quo in multispecies yeast research, by aiming to develop new tools to investigate the mechanistic basis of interaction in systems comprised of more than two species. Furthermore, the study aimed to generate a greater depth of understanding of these systems, by investigating transcriptional responses of Saccharomyces cerevisiae to co-culture in mixed-species cultures of increasing complexity. Firstly, these aims were achieved by developing a fluorescence-based multi-colour flow cytometric method for tracking of a consortium consisting of wine-associated yeast species. This involved optimizing the genetic modification of the selected environmentally isolated yeast species, followed by extensive validation to confirm the representativeness of the system as well as development of the flow cytometric protocol. This was followed by addressing the pertinent issue of reproducibility in multispecies cultures, and showing the role of the physiological state of pre-cultures in determining their growth performance in three-species and four-species consortia. Finally, to contribute to our understanding of the molecular mechanisms of interaction in non-linear yeast systems, we showed that Saccharomyces cerevisiae expresses a combination of known pair-wise as well as unique genes when grown in a three-species system. By using interactive network visualizations of the generated transcriptomic data, we were able to functionally characterize the cellular responses in more detail than has been done before in similar studies.
- ItemFunctional analysis of candidate terpenoid biosynthetic genes isolated from grapevine(Stellenbosch : Stellenbosch University, 2021-03) Bezuidenhout, Ilse-Marie; Young, Philip R.; Vivier, Melane A.; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: Terpenoids are a group of compounds found in various organisms, with diverse functions, and can be broadly grouped into primary or specialised (secondary) metabolites. This compound diversity is achieved from the universal precursors for terpenoids, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), through the activity of various enzymes. Isoprenyl diphosphate synthases (IDSs) incorporate the IPP and DMAPP precursors into compounds of varying lengths that can then serve as the substrate for terpene synthases (TPSs) to produce terpenes. Each of these steps incorporates the opportunity to further increase terpene diversity. In addition, terpenes can be modified into other products through the activity of various enzymes to increase the product range even further. Terpenoids play an important role in Vitis vinifera (grapevine), not only in the normal functioning of the plant, but also in more specialised roles involving plant–environment interactions. In terms of their economic value, terpenes, such as mono- and sesquiterpenes, contribute to wine aroma – which has supported the study of these compounds and how these metabolites are biosynthesised. Grapevine genome annotation has shown a moderately sized IDS gene family and a TPS gene family that has undergone extensive expansion. However, many of these genes have not been functionally characterised. Putative annotation based on sequences is not always accurate or does not provide a realistic indication of gene function. Functional characterisation of IDS and TPS genes involves heterologous expression followed by analysis of the produced compounds. In this study, various heterologous systems were used and evaluated with the aim of characterising grapevine genes that putatively encode IDS and TPS enzymes. In this study, the novel annotation of a eucalyptol synthase and the re-annotation of a previously characterised gene to α-thujene synthase was achieved. Various expression systems were assessed, with Saccharomyces cerevisiae deemed a viable alternative to the typical use of Escherichia coli. Different plant systems were also investigated, with Nicotiana benthamiana found to be a good option in terms of ease of use and results obtained. An in-depth study of geranylgeranyl diphosphate synthase (VviGGPS) was also conducted using an established transgenic tobacco population. The population was characterised in terms of morphology, physiology, and metabolites. The possible role of gibberellin was also determined based on the results of these analyses.
- ItemCell wall profiling of tobacco leaves and grapes in the context of Botrytis cinerea infection(Stellenbosch : Stellenbosch University, 2021-03) Weiller, Florent; Moore, John P.; Vivier, Melane A.; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: The plant cell wall has been shown to be at the centre of plant-biotic stress interactions. In the case of a pathogen encounter (e.g. fungi), the cell wall acts as the second barrier after the cuticle to stop pathogen penetration and tissue colonisation. The plant cell wall matrix composition and architecture influences the nature of resistance and susceptible responses to fungal pathogens. Botrytis cinerea, the fungus responsible for grey mold disease, causes worldwide crop losses. Understanding factors that differentiate susceptible from resistant plants is essential to develop new plant protection strategies against B. cinerea. In this study, genetically engineered plants with known resistance phenotypes, compared to the untransformed controls (with susceptible phenotypes) were used to understand the leaf cell wall changes either afforded by the transgenes introduced and/or the Botrytis infection process. The approach to investigate cell wall changes during Botrytis infection was also expanded to grapevine berry fruit. The method of choice was Comprehensive Microarray Polymer Profiling (CoMPP) technology, a high-throughput method for tracking cell wall matrix polysaccharide and protein composition and it was combined with monosaccharide profiling and various imaging tools. In the first part of this study, the leaves of nine tobacco (Nicotiana tabacum) lines: four overexpressing the known defence gene, grapevine polygalacturonase-inhibiting protein 1 (VviPGIP1), four overexpressing another known defence gene, the tobacco cinnamyl alcohol dehydrogenase 14 (NtCAD14) and the wild type SR1 were screened for cell wall compositional differences due to genetic overexpression in the absence of infection. Total lignin and monolignols were assayed using Py-GC-MS, but results showed limited variations between the different plant lines. CoMPP and GC-MS analysis for cell wall composition and monosaccharide content showed variation in homogalacturonan (HG) methylesterification patterning between the various transgenic plant lines. Arabinogalactan proteins (AGPs) and extensins increased in the majority of the VviPGIP1 lines. These results suggest that PGIP overexpression probably influences pectinmethylesterase (PME) enzyme activity and affects glycoprotein organisation. Following these results, more in-depth analysis continued during infection of PGIP plants. Infection experiments were conducted using B. cinerea on leaves of VviPGIP1 tobacco lines, compared to the wild type as control. Lesion size differences were observed from 96 hours post inoculation (hpi) with reduced lesions in the transgenic lines compared to the wild type, confirming the known resistance responses of the transgenic plants to the fungus. Cell wall alterations were followed in the 72 hpi period, showing HG degradation with RG-I signal increase, whereas the cellulose-xyloglucan network was mostly unaffected. Extensins and AGPs accumulated at and around the lesions, while distal non-infected leaves showed matrix changes from 72 hpi, with reorganisation of the cellulose/xyloglucan network and in the homogalacturonan methylesterification patterning, indicative of a systemic response. A typical Botrytis infection with cell wall depectination exposed less hemicellulose polysaccharides. In parallel, the plant seemed to build a defensive response to the infection with accumulation of defence related proteins at the lesion and in distal leaves. Unlike the wild type, the transgenic plants response was efficient in restricting Botrytis progression from 72 hpi onwards. In the last part of this study berries, at three developmental stages (veraison, post-veraison and ripe), from four Vitis vinifera cultivars (Cabernet Sauvignon, Sauvignon Blanc, Dauphine and Barlinka grape berries), were infected with B. cinerea. Ripe grapes from all four cultivars developed symptoms and showed cell wall degradation with cultivar differences were noted, while very few successful B. cinerea infections on veraison and post-veraison grapes were observed. This is in line with the generalised view that grapevine berries display ontogenic resistance in the greener developmental stages, although there are known exceptions. Cabernet Sauvignon was the least susceptible cultivar to B. cinerea and displayed a delayed lesion development in our experimental conditions. The infected grapes from all the cultivars were characterised by altered HG methylesterification patterning, extensin reorganisation, as well as glucan accumulation as the infection progressed. This work has brought new insights to existing efforts to fully characterise the role of the grapevine PGIP1 gene in plant cell wall modifications, in the presence and absence of B. cinerea infection. It provides further proof that small changes in the cell wall matrix contribute to the possible priming of plants to resist and overcome infection. Moreover, the similarities and differences observed between tobacco leaves and grape berry cell walls during Botrytis infection will help to conduct targeted experiments and to complement the existing models on plant cell wall – B. cinerea interactions.
- ItemSequence-function relationships of the Vitis vinifera L. terpene synthase (VviTPS) family towards understanding the grapevine flower volatilome(Stellenbosch : Stellenbosch University, 2020-04) Smit, Jacobus Samuel; Young, Philip R.; Vivier, Melane A.; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: Terpenes are ubiquitous to plants and represent the most diverse class of natural products. More than 50 000 terpenes have been described in nature with the enzymes involved in their biosynthesis, namely terpene synthases (TPS), facilitating some of the most complex catalytic activities observed in nature. Terpenes are hydrocarbons that are made of five carbon building blocks. The coupling and prenylation of these building blocks result in different substrates that are characteristic to the biochemical properties of the synthesised terpene. The C10 monoterpene and C15 sesquiterpene compounds are extensively studied due to their volatility. These terpene classes are often associated with the pleasant aromas emitted by flowers, fulfilling important roles as volatile attractants. Terpenes are also known to have strong antimicrobial and insecticidal activities. These metabolites are important defence compounds and are therefore also considered to be specialised metabolites due to the fitness advantage associated with their ecophysiological activities. Grapevine, Vitis vinifera L. (Vitaceae), has one of the largest TPS families, with the majority of these genes involved in mono- and sesquiterpene biosynthesis. These terpene classes are extensively studied for their organoleptic properties in grapes and wine, most often associated with aromatic wines that have floral, Muscat or pepper aromas. The genetic potential of grapevine TPS genes is largely underappreciated seeing that most studies focus on a select few terpene classes that are relevant to wine flavour and aroma. Limited studies have provided some insight into the in planta emission of grapevine terpenes. Grapevine flowers have been identified is a promising organ for terpene biosynthesis due to extensive upregulation of the TPS genes and a concomitant emission of volatile terpenes. Grapevine flowers were therefore characterised for their volatile profiles to identify chemotypic differences. This resulted in the identification of unique chemotypes, with cultivar-specific major sesquiterpene volatiles observed. The genetic factors that contribute to the volatile differences were analysed through functional and computational characterisation. This resulted in functional characterisation of numerous sesquiterpene synthases with aberrant mutations rendering more than half of the isolated genes non-functional. Furthermore, a novel sesquiterpene synthase involved in the unique chemotype of the cultivar Muscat of Alexandria was characterised. This enzyme showed a unique enzyme active site that was linked to the biosynthesis of (E)-β-farnesene. In addition to functional characterisation of genes was the annotation and computational characterisation of the TPS gene families for three diploid grapevine genomes. This allowed for new and fundamentally important insight into how this gene family differs between genotypes. The TPS gene family of grapevine is of great importance from an ecophysiological and economic perspective. By studying the genetic and chemotypic variations in multiple genotypes it was possible to characterise the grapevine TPS landscape. The insights gained from this study provided important fundamental knowledge that furthers our understanding of the complex biochemical and genetic processes involved in grapevine terpene biosynthesis.
- ItemRapid sensory profiling methods for wine : workflow optimisation for research and industry applications(Stellenbosch : Stellenbosch University, 2019-04) Brand, Jeanne; Nieuwoudt, Helene; Vivier, Melane A.; Valentin, Dominique; Næs, T.; Stellenbosch University. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.ENGLISH ABSTRACT: Descriptive sensory analysis techniques are widely used and trusted methodologies. Due to time and cost constraints, the demand for cost-effective methods for profiling is growing rapidly in food and beverage industries including the wine industry. A number of rapid methods have been tested and validated for various food products. However, further work is needed to identify and address limitations of specific rapid methods, especially reference-based methods, when evaluating complex matrices such as wine. The majority of studies employed novice consumers or trained consumers as judges. The wine industry has an advantage over most food industries with: (1) product experts who can serve as judges and (2) having an extensive lexicon in the form of aroma wheels available that can be used as check-all-that-apply (CATA) questions. The objective of this study was to identify cost-effective, rapid sensory methods that can be used for wine profiling by researchers and the wine industry alike. Furthermore, the study aimed to optimise the identified methods and to propose workflows that include sensory methods and statistical procedures suited for wine sensory analysis applications. Four rapid methods were compared to descriptive analysis (DA). The methods tested were CATA, rate-all-that-apply (RATA), Napping, and sorting. Results obtained for the rapid sensory method and DA were similar. It can therefore be concluded that rapid methods are suitable for the sensory evaluation of wine. Industry professionals can therefore be used as sensory judges, and can use a pre-determined lists of attributes as verbalisation tools. CATA and sorting provided the highest quality profiles with the best discrimination between products. Sorting highlights similarities and differences whereas CATA provides more detailed descriptions. In addition, these two methods were found to be easier than rate-all-that-apply (RATA) and Napping to use. Pivot profile (PP), a reference-based method, was validated against a CATA variant, namely frequency of attribute citation (FC). It was concluded that PP should be used with caution because the choice of pivot on the sensory space could have an influence. This method could, however, be useful when direct comparisons between samples are required, such as benchmarking. In addition to sensory method development, a number of statistical procedures were also proposed to assist with the interpretation of rapid method data. A workflow to calculate drivers of quality and a strategy to calculate confidence ellipses for PP data were developed. This study highlights the importance of selecting a fit-for-purpose method. The objective of the experiment being conducted, along with practical restrictions should be taken into account when deciding which method to use.