Browsing by Author "Du Plessis, Kari"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Analysis of antifungal resistance phenotypes in transgenic grapevines
    (Stellenbosch : Stellenbosch University, 2012-12) Du Plessis, Kari; Vivier, Melane A.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: The latest strategies in the protection of crops against microbial pathogens are rooted in harnessing the natural, highly complex defense mechanisms of plants through genetic engineering to ultimately reduce the application of chemical pesticides. This approach relies on an in-depth understanding of plant-pathogen interactions to develop reasonable strategies for plant improvement. Among the highly specialized defense mechanisms in the plant’s arsenal against pathogen attack, is the de novo production of proteinaceous antimicrobial peptides (AMPs) as part of the plant’s innate immunity. These AMPs are small, cysteine-rich peptides such as plant defensins that are known for their broad-spectrum of antifungal activity. These plant defensin peptides have been found to be present in most, if not all plant species and the defensin encoding genes are over-represented in plant genomes. Most of these defensins are generally the products of single genes, allowing the plant to deliver these molecules relatively rapidly and with minimal energetic expense to the plant. These factors contribute to establishing AMPs as excellent candidates for genetic engineering strategies in the pursuit of alternative crop protection mechanisms. The first antimicrobial peptide identified and isolated from grapevine, Vv-AMP1, was found to be developmentally regulated and exclusively expressed in berries from the onset of ripening. Recombinantly produced Vv-AMP1 showed strong antifungal activity against a wide range of plant pathogenic fungi at remarkably low peptide concentrations in vitro, however, no in planta defense phenotype could thus far be linked to this peptide. In this study, the antifungal activity of Vv-AMP1 constitutively overexpressed in its native host (Vitis vinifera) was evaluated against grapevine-specific necrotrophic and biotrophic fungi. Firstly, a hardened-off genetically characterised transgenic V. vinifera (cv. Sultana) population overexpressing Vv-AMP1 was generated and morphologically characterized. In order to evaluate the in planta functionality of Vv-AMP1 overexpressed in grapevine, this confirmed transgenic population was subjected to antifungal assays with the necrotrophic fungus, B. cinerea and the biotrophic powdery mildew fungus, Erysiphe necator. For the purpose of infection assays with a biotrophic fungus, a method for the cultivation and infection with E. necator was optimized to generate a reproducible pathosystem for this fungus on grapevine. Detached leaf assays according to the optimized method with E. necator revealed programmed cell death (PCD) associated resistance linked to overexpression of Vv-AMP1 that can be compared to that of the highly resistant grapevine species, Muscadinia rotundifolia. Contrastingly, whole-plant infection assays with B. cinerea revealed that Vv-AMP1 overexpression does not confer V. vinifera with elevated resistance against this necrotrophic fungus. An in silico analysis of the transcription of defensin-like (DEFL) genes previously identified in grapevine was included in this study. This analysis revealed putative co-expression of these DEFL genes and other genes in the grapevine genome driven by either tissue- or cultivar specific regulation or the plant’s response to biotic and abiotic stress stimuli. In conclusion, this study contributed to our knowledge regarding Vv-AMP1 and revealed an in planta defense phenotype for this defensin in grapevine. In silico analysis of the DEFL genes in grapevine further revealed conditions driving expression of these genes allowing for inferences to be made regarding the possible biological functions of DEFL peptides in grapevine.
  • Thumbnail Image
    Item
    The evaluation of the impact of microclimatic factors on grapevine berries in a vineyard setting through molecular profiling
    (Stellenbosch : Stellenbosch University, 2017-12) Du Plessis, Kari; Vivier, Melane A.; Young, Philip R.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology.
    ENGLISH SUMMARY: Grape composition is considered to be the result of the grapevine genotype, the environmental factors the grapes are exposed to and the management practices implemented during their development. However, elucidating how each of these components contributes to the outcome is notoriously difficult under field conditions due to the myriad confounding variables that grapes are influenced by. One of the viticultural management practices frequently implemented in the vineyard is the removal of leaves in the berry bunch zone in order to alter the microclimate of the developing grapes with various potentially advantageous outcomes. However, this common viticultural practice of leaf removal very rarely affects levels of light without elevating bunch temperatures as well. Moreover, definitive links between this treatment and the underlying grape molecular responses are currently lacking, particularly in cause and effect relationships. Utilizing a highly characterized Sauvignon Blanc vineyard, a leaf removal treatment was implemented according to a field-omics experimental approach, in which it was established that light exposure to the developing grapes was the predominant factor modulated by the treatment. A preceding study characterized the physical growth parameters of the developing grapes and targeted specific metabolites in order to determine how elevated light would affect grape development. The results revealed that the growth and development of the grapes were not affected by the treatment, but that specific secondary metabolites with photoprotective abilities were elevated. These results showed that the grapes acclimated to the elevated light exposure, providing the possibility to study the molecular mechanisms associated with this acclimated state in the berries. The aim of this study was therefore to explore the transcriptional responses of the developing grape berries to elevated light exposure to understand how primary metabolism and growth was maintained despite the implementation of stress mitigation strategies. The approach taken to study this transcriptional response involved RNA sequencing (RNASeq) analysis in order to generate a transcriptional snapshot of all the genes expressed in control and light exposed grapes sampled at four developmental stages throughout berry development. This analysis revealed that the green grapes implemented several photoprotective mechanisms simultaneously. Some of these mechanisms involved non-photochemical quenching and the rapid turnover of the proteins of the photosynthetic machinery, much like other foliar photosynthetic tissues, despite the profound differences in photosynthesis dynamics between these tissue types. Additionally, the genes associated with the synthesis of flavonoid compounds were significantly upregulated and these findings were further corroborated by the accumulation of high levels of flavonols that are known to have both light absorbing and antioxidant abilities. In combination, through these photoprotective mechanisms, as well as the synthesis of higher levels of carotenoids in green berries and subsequent apocarotenoids in ripe berries these grapes achieved a state of acclimation. Furthermore, the catabolism of amino acids provided energy precursors and substrates towards the redistribution of energy that contributed to the maintenance of these energetically costly stress mitigation mechanisms. To this end, green, photosynthesizing grapes maintain growth and development at all costs to protect the development and maturation of the grape seed. Therefore, when the berries achieved ripeness, the photoprotective mechanisms associated with photosynthesis had ceased and the upregulation of apocarotenoids and flavonols were no longer effectively mitigating the light stress. A subsequent investigation explored the role that grapevine heat shock factor (Hsf) genes may have played in achieving this acclimated state. The consistent upregulation of three grapevine Hsfs was established and for one of these genes, VviHsfA7a, a unique putative role in photoprotection under elevated light was identified. Furthermore, by utilizing these results, the first putative working model of the expression and regulation of the Hsfs in grapevine berries were proposed.This study further identified two groups of putative developmental stage-specific molecular biomarkers in grape berries. The first group of genes contributed to the current understanding of the underlying molecular mechanisms associated with the coordinated progression of berry development, whereas the other group of genes represented putative light-responsive molecular biomarkers that are developmentally regulated under non-stressed conditions, but that become significantly upregulated by light stress. Further investigation into the effect that the elevated light exposure may have had on the pathways associated with the synthesis of Sauvignon Blanc impact odorants was conducted. These findings provided insights into how leaf removal and elevated light exposure may lower green aroma characteristics in wine by modulating berry metabolism on a molecular level. Taken together, the findings presented in this study provided definitive insights into how light exposure effects grape berry development on a molecular level and the mechanisms that these berries implement in order to ameliorate the potentially harmful affects of light stress. This study further contributed by putting forward the first de novo assembled transcriptome for the Sauvignon Blanc grapevine genotype that can be utilized in future studies in order to draw more conclusive links between genotypic and/or treatment specific expression in grapevine.
  • Thumbnail Image
    Item
    The transcriptional responses and metabolic consequences of acclimation to elevated light exposure in grapevine berries
    (Frontiers Media, 2017) Du Plessis, Kari; Young, Philip R.; Eyeghe-Bickong, Hans A.; Vivier, Melane A.
    An increasing number of field studies that focus on grapevine berry development and ripening implement systems biology approaches; the results are highlighting not only the intricacies of the developmental programming/reprogramming that occurs, but also the complexity of how profoundly the microclimate influences the metabolism of the berry throughout the different stages of development. In a previous study we confirmed that a leaf removal treatment to Sauvignon Blanc grapes, grown in a highly characterized vineyard, primarily affected the level of light exposure to the berries throughout their development. A full transcriptomic analysis of berries from this model vineyard details the underlying molecular responses of the berries in reaction to the exposure and show how the berries acclimated to the imposing light stress. Gene expression involved in the protection of the photosynthetic machinery through rapid protein-turnover and the expression of photoprotective flavonoid compounds were most significantly affected in green berries. Overall, the transcriptome analysis showed that the berries implemented multiple stress-mitigation strategies in parallel and metabolite analysis was used to support the main findings. Combining the transcriptome data and amino acid profiling provided evidence that amino acid catabolism probably contributed to the mitigation of a likely energetic deficit created by the upregulation of (energetically) costly stress defense mechanisms. Furthermore, the rapid turnover of essential proteins involved in the maintenance of primary metabolism and growth in the photosynthetically active grapes appeared to provide precursors for the production of protective secondary metabolites such as apocarotenoids and flavonols in the ripening stages of the berries. Taken together, these results confirmed that the green grape berries responded to light stress much like other vegetative organs and were able to acclimate to the increased exposure, managing their metabolism and energy requirements to sustain the developmental cycle toward ripening. The typical metabolic consequences of leaf removal on grape berries can therefore now be linked to increased light exposure through mechanisms of photoprotection in green berries that leads toward acclimation responses that remain intact until ripening.