| Plant cells respond to a myriad of environmental stresses, including oxidant-generating agents such as ozone and ultra-violet radiation, by mobilizing a complex of cellular defenses. This response is made possible by an integration of signalling networks that mediate the perception of, and response to, these environmental stresses. However, the signalling networks linking perception of these various stress-related perturbations with the ultimate defense responses remain largely undefined.; Exposure of tobacco suspension-cultured cells to ozone, UVC [254nm, monochromatic] or hydrogen peroxide was found to induce the rapid activation of a specific 46 kDa mitogen-activated protein kinase (MAPK46) (salicylic acid-induced protein kinase). Oxidant activation of MAPK46 is transient, calcium-dependent, and can be suppressed by pre-treatment with free radical traps. The observed response is not restricted to tobacco, since oxidant treatment of plants such as Arabidopsis, poplar, spruce and Physcomitrella also resulted in rapid activation of a similar protein kinase.; To explore where these oxidants might initiate this signal response, suramin, a non-membrane permeable reagent that interferes with membrane receptor-mediated signalling in mammalian cells, was employed. Pretreatment of tobacco suspension-cultured cells with suramin strongly attenuated the oxidant-induced activation of MAPK 46 in a concentration-dependent manner, indicating that reactive oxygen species (ROS) signalling to the MAPK cascade may be initiated in large part at the cell membrane, perhaps through oxidative activation of membrane receptors.; An Arabidopsis AtMPK6-RNAi, loss-of-function genotype was constructed in order to better understand the role of this MAPK in cellular responses to oxidant signalling. When exposed to ozone, the RNAi-AtMPK6 genotype displayed more intense and prolonged AtMPK3 activation, as detected by Western blotting, when compared to WT, indicating that AtMPK6 is somehow involved in the regulation of this second MAPK in an oxidant background. The RNAi-AtMPK6 genotype was also found to be more sensitive than the WT to ozone fumigation, over a 24-hour period of continuous exposure, as evidenced by visible leaf damage and leaf-localized hydrogen peroxide accumulation.; The data presented in this thesis provide evidence for the ability of oxidants (ozone & UVC) to activate MAPK signalling in plants, and demonstrate that the initial events leading to this activation originate at the cell membrane, possibly through a receptor-based mechanism. Further, I present one of the first large-scale proteomic studies directed at plant signal transduction. The data from this study will help us to better understand the involvement of MPK6 in the overall response to oxidative stress. (Abstract shortened by UMI.)... |
