| Summary: | 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. === Land and Food Systems, Faculty of === Graduate
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