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Ozone- and ultraviolet radiation-induced signalling in plants Miles, Godfrey Preston

Abstract

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. Treatment of the tobacco cells with PD98059, a specific inhibitor of human MEK-1 & 2, blocked ozone- and hydrogen peroxide-induced activation of MAPK⁴⁶, but not UVC-induced activation. These results indicate that the ozone- and hydrogen peroxide-induced activation of MAPK⁴⁶ is signalling through a cognate MAPKK(s) that is sensitive to PD98059, whereas UVC may utilize a MAPKK which is insensitive to PD98059. 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 oxidantinduced activation of MAPK⁴⁶ 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. To identify a possible oxidant-induced MAPKK involved in the activation of AtMPK6, a transgenic Arabidopsis loss-of-function genotype was developed using RNAi technology directed at AtMKK5, a candidate cognate MAPKK for AtMPK6. When exposed to ozone, the RNAi-MKK5 genotype showed a reduction in the activation of AtMPK3 and AtMPK6. I show that even partial silencing of MKK5 is sufficient to render the transgenic genotype highly susceptible to ozone damage, as determined by visible leaf damage and abnormally high levels of hydrogen peroxide accumulation in leaf tissue. This sensitivity to ozone was not lost over successive generations of the AtMKK5 genotype. The MKK5-RNAi genotype is more sensitive to the injurious effects of ozone than is the AtMPK6-RNAi genotype. Mastoparan (MP), a cationic, amphiphilic tetradecapeptide isolated from wasp venom, is capable of directly stimulating the guanine nucleotide exchange reaction of the α-subunit of animal heterotrimeric G proteins via a mechanism analogous to that of G protein coupled receptors (GPCR). This leads to a range of downstream events including the activation of MAPKs. I show that the induction of plant MAPK signaling by MP does not require the participation of either the Gα - or Gβ-subunits of the Arabidopsis heterotrimeric G-proteins, but is reliant on ROS, a cognate MAPKK, and an influx of extra-cellular Ca²⁺ ions. While these findings do not preclude a role for a heterotrimeric G protein in MAPK signaling, they highlight the need for caution in drawing conclusions from published experiments using MP. To gain some additional insight into the cellular changes associated with oxidant stress, and the possible role of MPK6 in regulating those changes, I used isotope-coded affinity tagging (ICAT) technology to examine ozone-induced changes in protein expression in Arabidopsis, where the proteomes of both WT and MPK6-RNAi genotypes were compared in the context of an ozone challenge. Functional classification of the proteins from ozone treated tissue that were differentially expressed in the MPK6-suppressed and WT backgrounds was conducted based on the GO ontology system (Table 5.4, p148). The majority of the 0 hr-air and 8 hr-O₃ proteins are annotated as being located in the chloroplast, while the rest are associated with the mitochondria, nucleus, and cytosol. The categories of 'other metabolic processes', electron transport, energy pathways or antioxidant-related make up the bulk ofthe 0 hr-air and 8 hr- O₃ protein entries with an assortment of other categories covering the rest of the protein entries. 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 ofthe 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.

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