| Summary: | M.Sc. === in the current study the effect of the addition of methyl Jasmonate (MeJA), chitosan, a cell wall elicitor prepared from Phytophthora nicotlanae to tobacco cells and the subsequent defense responses elicited in these cells were Investigated. The defense responses investigated can be divided into three categories according to the time scale whereby resistance responses in plant cells are induced: early events which included the analysis of lipid peroxidation, the induction of lipoxygenase (L0)0 enzyme activity as well as the changes in phosphoprotein profiles; intermediate to later responses which included investigations of peroxidase (POD) activity, lignin content, phytoalexin content and phenolic content and also late responses which included studies of pathogenesis-related proteins (PR) and 13-1,3-giucanase activity. An approach also followed in this study was the addition of MeJA to tobacco cells for 24 h followed by the addition of either the cell wall elicitor or chitosan as a secondary elicitors, to investigate possible preconditioning or sensitisation by MeJA. Results obtained in this study revealed the time and concentration dependent accumulation of phytoalexins (secondary metabolites) when MeJA was added to tobacco cells and an optimal concentration of MeJA to use in further studies was determined as 1 mM. MeJA was the most effective inducer of lipid peroxidation (22 fold induction), a response observable after 2 h of exposure to MeJA. Conditioning with MeJA, followed by both chitosan (19 fold induction) and elicitor (25 fold induction) led to an earlier accumulation as well as significant increases in the levels of malondialdehyde, the product of lipid peroxidation. LOX enzyme activity was significantly increased by the addition of MeJA (6 fold Induction), chitosan (4 fold induction) and elicitor (3.8 fold induction). Conditioning with .MeJA, followed by both chitosan (3.3 fold induction) and elicitor (3.9 fold Induction) also led to noteworthy increases in enzyme activity. Analysis of the phosphoprotein profiles do not reveal the accumulation of phosphorylated proteins when MeJA was added to cells and very little accumulation of such proteins when chitosan was added. Phosphorylated proteins could be observed in cells treated with elicitor and In the cases where conditioning with MeJA, followed by secondary elicitation with either chitosan or elicitor, was studied, the differential induction of phosphorylated cellular proteins could also be observed. No significant induction of POD activity could be observed under any of the conditions, except for a possible slight increase in POD activity starting at 16 - 24 h after the elicitor had been added and a more definite increase after 24 h which was sustained up to 48 h after the addition of MeJA. PAGE of peroxidase, followed by activity staining revealed the presence of a slow migrating anionic peroxidase as well as a fast migrating peroxidase. Conditioning with MeJA, followed by secondary elicitation with both chitosan and elicitor revealed enhanced POD activity as well increased induction of a fast migrating anionic peroxidase on PAGE gels. MeJA was a more effective inducer of elevated levels of lignin content than the elicitor or chitosan and the addition of MeJA to tobacco cells led to a 2.2 fold increase in the lignin content, a response observed after 24 h and sustained up to 48 h. Chitosan as secondary elicitor did not lead to any increase in lignin content, but the cell wall elicitor as secondary agent significantly increased the lignin content after 40 - 48 h. Analysis of phenolic content did not show any significant increases In the total soluble phenolics when the agents were used on their own and only the phenolic content of the MeJA-conditioned cells, followed by the addition of chitosan showed a slight increase. In this case, the HPLC analysis of the phenolics also revealed a shift In the profiles for phenolics. SDS-PAGE of PR proteins revealed the induction of constitutive as well as new proteins when MeJA and elicitor, but not chitosan were used as elicitation agents. However, In the MeJA-pretreated cells addition of both chitosan and elicitor led to increased accumulation of PR proteins with molecular masses ranging from 6 - 70 kDa. Results from the i3-1,3-glucanase activity assay indicate a strong induction (4-5 fold) when MeJA and elicitor (4 fold), but not when chitosan was added to cells. Conditioning effects were revealed when both chitosan (3 fold induction) and elicitor (2.5 fold induction) were used as secondary elicitors. The increases in intensities of bands with molecular masses ranging from 31- 35 kDa observed on SOS-PAGE gels where chitosan and elicitor were added as secondary agents corresponded in a time dependent manner with the increased levels obtained in thep-1,3-glucanase activity assay.
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