Browsing by Author "Basson, Carin Elizabeth"

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    Analysis of intermediate carbon metabolism in strawberry plants
    (Stellenbosch : Stellenbosch University, 2008-12) Basson, Carin Elizabeth; Groenewald, J.-H.; Bauer, R.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology.
    Strawberry (Fragaria x ananassa) fruit quality is largely determined by the relative amounts of sugars and organic acids present, as well as soluble solid content. This study had three components: 1) Characterisation of cytosolic carbohydrate metabolism and carbon partitioning to sugars and organic acids in two commercial varieties, 2) analysis of transgenic strawberry fruit with increased pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase (PFP) activity and 3) analysis of transgenic strawberry fruit with increased ß-fructosidase (invertase) activity in either cytosol or apoplast. Analyses of transgenic strawberry may inform similar attempts in grape berries. Festival and Ventana, two popular commercial strawberry cultivars in South Africa, were fairly similar with respect to sugar and organic acid content. Twelve cytosolic enzymes were investigated. Temporal differences in maximum catalytic activity were observed for invertase, PFP, pyruvate kinase and ADP-glucose pyrophosphorylase (AGPase). Invertase, PFP and AGPase activity also differed between the cultivars. One enzyme, SuSy, could not be analysed effectively, due to the purification method employed. These analyses established methodology for the analysis of transgenic berries. Constructs were designed to constituitively express Giardia lamblia PFP (GL-PFP), or to express Saccharomyces cerevisiae invertase (SCI) in a fruit-specific manner. A second invertase construct was designed to target SCI to the apoplast. Strawberry (cv. Selekta) was transformed and the presence of each transgene confirmed by PCR. Untransformed Selekta was used as control in both transgenic studies. Transgenic lines were selected based on GL-PFP activity in leaves and total PFP activity in ripe fruit. Sugar and organic acid content of ripe berries with high PFP activity was determined. Although berries displayed marked changes in sugar composition, the total sugar content was similar to controls, in all except one line. Organic acid content was decreased, leading to a clear reduction in organic acid-to-sugar ratio. This points to a gluconeogenic role for PFP in strawberry fruit. Transgenic berries were screened for SCI activity. Berries containing untargeted SCI exhibited total invertase activity similar to controls and were not analysed further. Berries with apoplasttargeted SCI displayed three-fold increases in invertase activity compared to controls. Total sugar content was reduced and exhibited reduced sucrose content relative to hexoses. Despite the effect of increased invertase activity on metabolites, maximum catalytic activity of enzymes involved in cytosolic sucrose, hexose and organic acid metabolism were unchanged. Transgenic plants selected in these studies were subsequently vegetatively replicated and future work will include immature fruit.
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    Transcriptomic analysis of disease resistance responses using a tobacco-Botrytis cinerea pathosystem
    (Stellenbosch : Stellenbosch University, 2017-12) Basson, Carin Elizabeth; Vivier, Melane A.; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology.
    ENGLISH SUMMARY: Cultivation of plants for food and raw materials is regularly hampered by phytopathogens that reduce quality and yield. Necrotrophic fungi are among the most damaging pathogens, killing plant cells to complete their lifecycle. Many of these fungi release cell wall degrading enzymes (CWDE) to breach this first defensive barrier of plant cells. Some of the most important CWDE are polygalacturonases (PGs), that degrade the pectin-component of the plant cell wall and often act as virulence factors. To combat PGs, plants have evolved polygalacturonase-inhibiting proteins (PGIPs). In most cases, constitutive expression of PGIPs confer some level of resistance to pathogenic fungi. Two mechanisms have been proposed to explain PGIP-induced resistance. Firstly, they protect the cell wall from degradation through PGIP-PG inhibition interactions and secondly, they assist in activating defence signalling, by prolonging the survival of signalling molecules (oligogalacturonides, OGs) derived from cell wall degradation to activate the salicylic acid (SA) branch of defence signalling. The defence role of Vitis vinifera PGIP1 (VviPGIP1) was established using transgenic tobacco (Nicotiana tabacum) plants challenged with Botrytis cinerea, a model necrotrophic fungal phytopathogen. VviPGIP1 inhibited the two PGs that are virulence factors for B. cinerea, and reduced lesion diameter during infection assays. A subsequent characterisation of uninfected transgenic tobacco revealed wide-ranging changes in gene expression, enhanced lignin deposition and changes in hemicellulose composition, pointing to new and previously unexplored alterative functions of PGIPs in plant defence. Moreover, when the tobacco lines were infected, a more rapid accumulation of the defence hormone jasmonic acid was seen in the transgenic lines. These changes suggested that the constitutive expression of VviPGIP1 induced defence priming, a mechanism whereby plants induce slight metabolic and transcriptomic changes prior to infection, but display an enhanced defence response upon challenge. The work that formed part of this study follows on from these previous studies and had as aim to study the mechanisms that contributed to the defence phenotype observed in the tobacco lines expressing the grapevine PGIP encoding gene. The approach was to profile the molecular response of the host (tobacco) when infected by B. cinerea, contrasting the native tobacco response with that of the PGIP tobacco lines. The disease progression of B. cinerea has been extensively studied in Arabidopsis and other model host species. The first two days after infection has been established as the critical phase that determines the outcome of the interaction (susceptibility/resistance). Consequently, this period was chosen to profile transcriptional changes following B. cinerea infection of wild-type and VviPGIP1-expressing tobacco. A time-course, consisting of five sampling points (0 h, 12 h, 24 h, 36 h and 48 h), was used to profile the localised defence response and the response in distal organs. The time-course represented two day-night cycles, providing the opportunity to investigate diurnal gene expression patterns. Wild-type tobacco mounted a localised defence response that shared many elements with those of other plant hosts of B. cinerea, including the dampening of diurnal patterns and induction of antioxidant mechanisms, jasmonate and ethylene biosynthesis and secondary metabolism. In leaves distal to the infection, the diurnal patterns of gene expression were not disrupted, but genes related to anti-fungal proteins and secondary metabolite synthesis were induced. This suggested partial induced resistance (IR) had been activated by distal infection, but systemic acquired resistance (SAR) had not yet been established. Profiling of volatile secondary metabolites emphasised a strong ontogenic effect. The resistant tobacco line (expressing VviPGIP1) displayed enhanced activation of jasmonate/divinyl ether biosynthesis and repression of ethylene-responsive transcription factors. Monolignol biosynthesis was affected, and may have led to altered lignin composition. Several biological processes were affected at 24 hours after infection, reportedly a critical point during B. cinerea pathogenesis. These included enhanced activation of pterostilbene synthesis, fungitoxic SAR8.2 proteins, proteinase inhibitors and antimicrobial peptides, while oxidative stress was reduced. In terms of priming, several stress-responsive genes were more rapidly induced in the PGIP line, which also displayed an accelerated transition from source to sink metabolism. With regards to the specific role of VviPGIP1 during infection, this study represented the first untargeted transcriptomic analysis of an infected PGIP-expressing tobacco line. The enhancement of jasmonate synthesis suggested that hormone signalling may differ between VviPGIP1-expressing transgenics and plants expressing cotton or bean PGIPs. In leaves distal to the infection, where signalling molecules derived from cell wall degradation by B. cinerea would not be generated, priming- and IR-like responses were observed, further underscoring the connection of VviPGIP1 with defence priming. The recurring appearance of cell wall modification in responses to B. cinerea, and the prior analyses that found changes in hemicellulose composition, prompted a more detailed examination of the xyloglucan endotransglycosylase/hydrolase (XTH) family in tobacco. The catalytic domain and other characteristic features of XTHs were identified for predicted XTH proteins. Functional information from characterised XTHs was mapped onto homologous sequences in order to infer functions for tobacco XTHs, however, the high sequence homology of XTHs in general, and of XTHs with contrasting expression patterns, suggested that promoter analysis would be required to accurately predict functions for specific XTHs. The sequence alignments and transcriptional information generated for the XTH gene family during this study provides a useful context for studies into tobacco cell wall metabolism. This study has generated novel gene expression data for B. cinerea-infected tobacco, provided the opportunity to compare the timing and magnitude of transcriptional responses in susceptible and resistant plant lines, and to investigate the basis for PGIP-induced resistance. Further studies should consider utilising de novo sequencing to identify processes not represented on the microarray and attempt to distinguish between OG-induced responses and PGIP-induced responses. This study successfully reinforced the proposed defence priming role of VviPGIP1, not only at the site of infection, but in tissues where PGs were not active.