M.Sc. / On exposure to abiotic or biotic stresses, plants initiate a cascade of metabolic reactions, some of which lead to the biosynthesis of secondary metabolites with roles in self defense. Phytoalexins are a class of secondary metabolites synthesized de novo in response to microbial attack by activation of certain biosynthetic pathways. Cruciferae phytoalexins are all indole based with a carbon, nitrogen and sulfur containing constituent on the 3’ position of the indole ring. This common similarity of all Cruciferae phytoalexins suggests that the plants all share a common indole precursor. Camalexin is the primary phytoalexin of Arabidopsis thaliana. De novo synthesis of camalexin upon infection, as well as its antimicrobial nature supports its role in disease resistance. Evidence exists that suggests the inducible biosynthesis of camalexin involves steps of the tryptophan pathway, along with an increase in transcript and protein levels of the tryptophan pathway enzymes after microbial infection. Bacterial LPS (lipopolysaccharide) has been described as one of the microbe/pathogenassociated molecular patterns (M/PAMPs) capable of eliciting the activation of the plant innate immune system. LPS is an integral component of the cell surface of Gram-negative bacteria. It is a complex which is exposed to the external environment, and is thus involved with external interactions of the bacteria. The hypothesis investigated in this dissertation is that LPS, as a lipoglycan PAMP, results in activation of signal transduction pathways involved in defense that lead to the production of the defense metabolite, camalexin. Furthermore, that the genes CYP71B15, CYP79B2 and TSB are up-regulated in response to LPS during camalexin biosynthesis via the tryptophan pathway. To test this hypothesis, camalexin production was investigated through a combination of analytical techniques including thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography (GC), ultra pressure liquid chromatography-mass spectrometry (UPLC-MS) and fluorescence spectroscopy. Genes in the camalexin biosynthetic pathway were investigated by two-step reverse transcription polymerase chain reaction (PCR), GUS reporter gene assays and quantitative real time PCR (RT-qPCR).
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uj/uj:7137 |
| Date | 29 June 2011 |
| Creators | Beets, Caryn Ann |
| Source Sets | South African National ETD Portal |
| Detected Language | English |
| Type | Thesis |
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