Masters Degrees (Genetics)

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    Is AtGSTF2 involved in lumichrome perception and transport?
    (Stellenbosch : Stellenbosch University, 2024-03) Warren, Kyle; Hills, Paul Norman; Stellenbosch University. Faculty of AgriSciences. Department of Genetics & Institute of Plant Biotechnology.
    ENGLISH ABSTRACT: A number of exudates from plant growth-promoting rhizobacteria (PGPR) have been increasingly studied and used as an alternative way of promoting plant health and growth. One such exudate is lumichrome, a signal molecule which has been shown to elicit a growth response across multiple species. Recent studies have identified some of the molecular mechanisms which underpin this growth response, however, to date it remains unknown how lumichrome is perceived and transported in planta. It was proposed that the Arabidopsis Glutathione S-Transferase (GST) AtGSTF2 may act as either a receptor or a transporter of lumichrome, as a previous study had found lumichrome was able to bind to AtGSTF2 with high affinity when the Arabidopsis gene was overexpressed in Escherichia coli. Consequently, AtGSTF2 overexpressor (OE) and knockout (KO) lines were generated in Arabidopsis thaliana to test whether these lines would show enhancements or reductions in the growth effects of lumichrome, and to gain additional insight into the role of AtGSTF2 in Arabidopsis. It was determined that overexpression of AtGSTF2 did not affect the growth response of treated plants to lumichrome, although it was found that the OE plants had increased overall plant growth and differences in a number of physiological responses in comparison to untreated wild-type plants. Previous studies had shown no obvious phenotypic effects from AtGSTF2 overexpression under normal growth conditions, although the plants had increased tolerance to phenolic stress; however, this study showed that overexpression of AtGSTF2 resulted in an increase in both fresh and dry biomass, an increase in the number of leaves, increased starch and sugar levels, as well as higher stomatal conductance and transpiration rates than wild-type plants. However, in both OE lines generated, lumichrome treatment reduced plant biomass, whilst leaf number was also reduced following lumichrome treatment in one of the two OE lines. RT-qPCR analysis showed decreased expression of AtGSTF2 in wild-type plants following lumichrome treatment, however, increased expression compared to wild-type controls was seen in growth-related genes XTH9 and CYCA1 and the jasmonate-related gene JAL34. However, lumichrome-treatment of OE plants resulted in significant differences in expression patterns for the lumichrome- responsive genes studied compared to its effects on the transcripts in wild-type plants, as the overexpression of AtGSTF2 appeared to decrease ACO1 and CYCA1 expression. Overexpression of AtGSTF2 increased plant growth in comparison to the wild-type plants, suggesting that AtGSTF2 plays a critical role in development. In summary, the evidence from this study revealed no correlations between lumichrome-related growth or gene expression responses and AtGSTF2 expression, suggesting that AtGSTF2 is most likely not involved in either lumichrome perception or transport. Despite being one of the most widely studied phi-class GSTs, little is known about the functions of AtGSTF2. This study proposes that AtGSTF2 plays a more important role in plant growth than previously thought, and it is recommended that future studies should focus on the role of AtGST2 in plant growth and development.
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    Investigating starch from transgenic potato plants lacking starch branching enzyme I and/or glucan, water dikinase 1
    (Stellenbosch : Stellenbosch University, 2024-03) Gouws, Nina Ellen; Lloyd, James; Van der Vyver, Christell; Stellenbosch University. Faculty of AgriSciences. Department of Genetics & Institute of Plant Biotechnology.
    ENGLISH ABSTRACT: Starch is the principal storage carbohydrate in plants where it is found in granules in plastids. It is the major calorie in the human diet and is an important feedstock in various industries. To improve the usefulness of native starch for different industrial applications, it must first undergo expensive chemical, physical or enzymatic treatments. Understanding starch biosynthesis can help to introduce modifications in planta with the aim of reducing such postharvest costs. Starch consists of two polymers, amylose and amylopectin, which are synthesised by multiple enzymes and are found in differing ratios where amylopectin normally constitutes the majority of the granule. Starch synthases elongate α-1,4 linked glucose chains to produce amylose. Starch branching enzymes can introduce α-1,6 branchpoints to form amylopectin which can be phosphorylated by glucan, water dikinases. There seems to be a relationship between the branching structure and phosphate incorporation as repression of STARCH BRANCHING ENZYME genes leads to increased granule bound phosphate. In this project, STARCH BRANCHING ENZYME I (SBEI) and GLUCAN, WATER DIKINASE 1 (GWD1) were repressed individually or simultaneously in potato using RNAi constructs. Repression of these enzymes resulted in changes to the amylose and phosphate contents. These alterations influenced granule morphology and gelling properties, but not freeze-thaw stability or starch digestibility. Rapid viscoamylography demonstrated that repression of SBEI increased phosphate content and peak viscosity. Starch pastes from SBEI transgenic plants also had improved paste clarity. GWD1 repression resulted in a decrease in phosphate content and peak viscosity. Simultaneous repression of SBEI/GWD1 resulted in higher amylose content and altered granule morphology with some multilobed starch granules being present. Despite the increase in amylose, a carbohydrate more resistant to digestion, no differences in digestibility of purified starch were found. This study adds to what is known about starch biosynthesis in potato tubers and demonstrates that starch can be modified in planta by targeting starch biosynthetic genes. Whether these changes ultimately result in improvement to end-products still needs to be investigated.
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    Investigation of the DMR6 susceptibility genes in grapevine for improving phytoplasma resistance through CRISPR/Cas9 technology
    (Stellenbosch : Stellenbosch University, 2024-03) Holm, Clara Cornelia; Burger, Johan; Campa, Manuela; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Vi6culture is considered one of the most important crop industries worldwide but is con6nually placed under pressure from a vast array of pathogens. Phytoplasma diseases are a cri6cal challenge for the grapevine industry and cause considerable yield losses. Several breeding strategies aim to improve grapevine disease resistance through the introgression of resistance genes, or through the inac6va6on of suscep6bility (S) genes, using genome edi6ng. Recent advancements of genome edi6ng tools such as CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) allows for the targeted inac6va6on of important host S factors that play a role during pathogen infec6on. The Downy Mildew Resistant 6 (DMR6) genes were ini6ally described in Arabidopsis thaliana as a suscep6bility factor for bacterial and oomycete pathogens. Subsequent inac6va6on of these genes resulted in broad range pathogen resistance in various crops such as tomato, leBuce, cucumber, pepper, and citrus. Furthermore, there is evidence that the VviDMR6 genes play a similar role in grapevine. In fact, most recently the inac6va6on of the VviDMR6 genes lead to enhanced downy mildew resistance in grapevine. However, it’s precise role during pathogenesis remains unclear. To this end, this study aimed to func6onally characterize the grapevine DMR6 genes, with par6cular focus on their involvement in plant defense. Gene co- expression networks highlighted the involvement of VviDMR6.1 in defense, while VviDMR6.2 was associated with developmental pathways. Gene expression analyses across different grapevine 6ssues revealed that VviDMR6.1 was predominantly expressed in the roots. Furthermore, to beBer understand the role of VviDMR6 during pathogenesis, in vitro grown grapevine was treated with salicylic acid (SA) to examine its response in different 6ssue types. Both VviDMR6 genes were induced upon SA treatment across different 6ssues. Addi6onally, GUS-reporter assays were used to further inves6gate the 6ssue specificity of the VviDMR6 genes. This entailed the iden6fica6on and isola6on of the puta6ve promoter regions of VviDMR6.1 and VviDMR6.2 from Chardonnay. In silico analyses unveiled core promoter elements and puta6ve regulatory mo6fs associated with stress responsiveness within both VviDMR6 promoters. Subsequently, two promoterDMR6::GUS vectors were constructed consis6ng of the respec6ve promoters fused to GUS, and were subsequently introduced into A. thaliana. The proDMR6.1::GUS transgenic lines showed dis6nct promoter ac6vity in the roots of fully grown plants and demonstrated clear induc6on upon SA exposure. Finally, two previously designed CRISPR/Cas9 constructs containing two targets for VviDMR6.1 and a single target for VviDMR6.2 were introduced to rootstock cul6var Richter 110 embryogenic callus through Agrobacterium-mediated transforma6on. Successful edi6ng of VviDMR6.1 was achieved, with a muta6on frequency of 43%. Inference of CRISPR Edits (ICE) analysis revealed small indels, primarily 2-bp dele6ons, in the VviDMR6.1 edited lines. Notably, a single transgenic line demonstrated complete edi6ng, achieving 100% edi6ng efficiency. This contributes to the advancement of genome edi6ng tools for enhancing disease resistance in grapevine and yielded the first CRISPR/Cas9-edited grapevine on the African con6nent.
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    Identification of early indicators of CTV induced stem pitting to aid the selection of CTV cross protection sources.
    (Stellenbosch : Stellenbosch University, 2024-03) Van Wyk, Luka; Maree, H. J. ; Bester, Rachelle; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Citrus tristeza virus (CTV) is the most impactful viral pathogen of citrus, having caused the death of more than 100 million citrus trees. CTV elicits several disease syndromes, but continues to affect citrus production through stem pitting disease phenotypes of varying severity. These phenotypes reduce citrus tree productivity and result in smaller, unmarketable fruit. Stem pitting is incompletely characterised, and the mechanisms underlying its induction remain elusive. This poses limitations regarding our understanding of CTV-induced stem pitting and the management thereof. This study aimed to better understand the plant-pathogen interactions underlying stem pitting in CTV-infected citrus over the course of disease onset from an untargeted perspective. To this end, high-throughput sequencing and ultra-performance liquid chromatography-mass spectrometry were used to profile the transcriptome and metabolome of ‘Mexican’ lime and ‘Duncan’ grapefruit plants infected with T3-KB, T68, and RB isolates of CTV. This profiling was carried out prior to infection, early post-infection, and after symptom development, with the goal of identifying determinants of severe CTV-induced stem pitting. It was shown that secondary metabolite profiles of ‘Mexican’ lime plants were able to clearly discern between CTV infections at ten months post-infection. These profiles indicated a common dysregulation of carbohydrate metabolism over disease onset and provided a putative identification of the furcatin molecule as a major driver of differentiation. Similarly, differential gene expression analysis implicated a carbohydrate metabolism gene, along with seven others, as significantly different in severely pitted, T3-KB infected ‘Mexican’ lime plants. These genes were consistently upregulated over the course of disease onset. This provided a candidate metabolic pathway for further study and eight candidate genes to be validated as determinants of CTV-induced stem pitting severity. The characterization of hypersensitive response and systemic acquired resistance in citrus due to CTV infection was also discussed. It was speculated that determinants of stem pitting severity may lie earlier in the infection progression of CTV within the hypersensitive response of citrus. Carbohydrate metabolism was also putatively associated as a factor of systemic acquired resistance in response to CTV infection. This emphasized the importance of the temporal component of citrus’ response to CTV infection that has only been studied in a limited capacity thus far. The utility of complementary transcriptomic and metabolomic analyses was also demonstrated, and the findings therefrom contribute to the understanding of plant responses to CTV over time.
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    Identification and characterisation of a drought-responsive galactinol synthase in wild rocket (Diplotaxis tenuifolia)
    (Stellenbosch : Stellenbosch University, 2023-12) Xonti, Alukhanyo; Peters, Shaun W.; Loedolff, Bianke; Stellenbosch University. Faculty of AgriSciences. Department of Genetics & Institute of Plant Biotechnology.
    ENGLISH ABSTRACT: The raffinose family of oligosaccharides (RFOs) are sucrosyl-galactosides that occur uniquely in the plant kingdom. The elucidated functional roles attributed to RFOs include serving as carbohydrate transporters, storage reserves and acting as protective agents against biotic and abiotic stress effects. Galactinol synthase (GolS; EC 2.4.1.123) is the key biocatalyst of the RFO biosynthetic pathway and initiates the synthesis of the precursor galactinol (Gol) with the transfer of a galactosyl moiety from UDP-galactose (UDP-Gal) to myo-inositol (Ino). RFO biosynthesis involves the sequential addition of activated galactosyl moieties from Gol to sucrose (Suc) with the enzymes raffinose synthase (RafS; EC 2.4.1.82) and stachyose synthase (StaS; EC 2.4.1.67) to produce raffinose (Raf) and stachyose (Sta) plus higher order RFOs respectively. The coupled transcriptional upregulation of GolS and subsequent RFO accumulation during water deficit stress has been well-studied in several plants; however, these functional dynamics have not been explored in wild rocket (Diplotaxis tenuifolia). The focus of the current study was to isolate and functionally characterise a putative DtGolS1 and investigate its transcriptional regulation and subsequent water-soluble carbohydrates (WSC) profile change in D. tenuifolia leaves under mild water deficit stress. With the heterologous expression of DtGolS1 in E. coli (DH5α), it was demonstrated that the extracts synthesized galactinol in vitro. The predicted DtGolS1 amino acid sequence exhibited GolS hallmarks from other plant species including the C-terminal APSAA pentapeptide and a serine phosphorylating site. Transcriptional analyses also indicated that DtGolS1 is sensitive to water deficit stress as transcript levels observed an upregulation for the most stressed rocket plants. Based on the current findings, the identification of a promising DtGolS1 candidate gene for sugar metabolic manipulation in improving water stress tolerance in wild rocket was revealed. In addition, owing to the prebiotic properties of RFOs, a greater understanding of RFO metabolism particularly in salad greens such as wild rocket could provide insight into biofortification strategies leading to the production of sustainable salad crops.