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UBC Theses and Dissertations

Ozone-induced signal transduction in tobacco Samuel, Marcus Abraham


A wide array of environmental signals are sensed and processed by the plant cell, resulting in specific responses that reflect the nature, intensity and duration of the input signal. One common response to many of these biotic and abiotic insults is the rapid production of "reactive oxygen species" (ROS) by the plant cells. The level of ROS accumulation can influence the cell's protective or cell death mechanisms, leading to survival or death. However, knowledge about how ROS mediate these multiple effects is still fragmentary. To dissect this complex pattern of ROS-induced responses it is experimentally advantageous to use a stressor that can generate ROS in plants on demand. One such stressor is ozone. The similarity between ozone and pathogen-induced responses, including induction of an ROS burst and cell death, makes ozone an efficient tool in probing ROS-induced signalling pathways. Since little is known about how ozone-induced redox imbalances are sensed and transmitted within the plant cell, the general objective of this study was to investigate the potential role of MAPK modules, a major eukaryotic signalling mechanism, in ozone-induced stress signalling pathways. Brief exposure to ozone leads within minutes to activation of a -46 kD ERK-type MAP kinase in tobacco. This activation process is calcium-dependent and can be blocked both by free radical quenchers and by a specific inhibitor of MEK-1 (MAPKK). Hydrogen peroxide and superoxide anion radical can substitute for ozone as the activation stimulus, and the activation process does not appear to require salicylate as an intermediary. The properties of the ozone-induced MAPK indicate that it may be SIPK (salicylate-induced protein kinase), a tobacco MAPK that is activated by a variety of stress treatments. The ability of ozone to activate SIPK indicates that this protein kinase acts as a very early transducer of redox stress signals in plant cells. Through gain-of-function and loss-of-function approaches using transgenic technology I observed that both elevation and suppression of SIPK render the plant sensitive to ROS stress. However, transgenic lines over-expressing a non-phosphorylatable version of SIPK were not ROS-sensitive. Analysis of the MAP kinase activation profiles in ROSstressed transgenic and wild type plants revealed a striking interplay between SIPK and another MAPK (Wound-Induced Protein Kinase; WIPK) in the different kinotypes. During continuous ozone exposure, abnormally prolonged activation of SIPK is seen in the SIPK-overexpression genotype, without WIPK activation, while strong and stable activation of WIPK was observed in the SIPKsuppressed lines, with concomitant accumulation of hydrogen peroxide and altered gene-induction responses. One role of activated SIPK in tobacco cells upon ROS-stimulation thus appears to be control of the inactivation of WIPK. Attempts to directly identify putative substrates for SIPK through solid-phase phosphorylation screening were unsuccessful. To my knowledge, the data presented in this thesis provide the first substantial evidence for a role of MAPKs in ozone-induced stress response and cell death pathways. The ozone-induced interplay between activated forms of SIPK and WIPK in the SIPK transgenics is the first evidence that alteration of the activity of a particular plant MAPK can lead to changes in intensity and timing of activation of another MAPK.

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