Divergence and reciprocity in signaling through closely-related oxidative stress-activated MAPKs Gregory Lampard*, Godfrey Miles*, Juergen Ehlting, Nathalie Mattheus, Yuelin Zhang and Brian Ellis Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada, V6T 1Z3 Acknowledgements Funding for this project was provided by Natural Sciences and Engineering Research Council of Canada (NSERC), and Genome Canada. GL is also the recipient of a UBC Killam Postgraduate Fellowship. * These researchers contributed equally to this study Abstract MAPK signaling modules play central roles in many essential plant processes including plant development, cytokinesis, stress management and programmed cell death. Two of the most well-characterized plant MAPKs are AtMPK3 and AtMPK6, along with their respective tobacco orthologues, WIPK and SIPK. This pair of evolutionarily related MAPKs is known to be activated by discrete and overlapping stimuli, including abiotic and biotic stresses. Ozone, in particular, is known to activate both AtMPK3 and AtMPK6. Ozone-treated transgenic tobacco plants in which SIPK signaling has been suppressed by RNAi show both hyper- and prolonged activation of WIPK (Samuel et al, 2002), and an analogous response occurs in AtMPK6-suppressed Arabidopsis plants. Some reciprocity of action is evident, since elimination of AtMPK3 signaling leads to a similar hyper- and prolonged activation of AtMPK6 in ozone-treated plants. In order to identify the discrete and common roles played by AtMPK3 and AtMPK6 in ozone response signaling, a series of microarray experiments were conducted using a full transcriptome Arabidopsis 70-mer oligo microarray. The transcriptional responses of wild-type seedlings were compared independently with AtMPK3 knock-out or AtMPK6 knock-out Arabidopsis seedlings exposed to ozone (500 ppb) for two hours. Transcriptional responses could be categorized into four classes: Those unique to AtMPK3 signaling, those unique to AtMPK6 signaling, those associated with both AtMPK3 and AtMPK6 signaling, and those reciprocally controlled by AtMPK3 and AtMPK6. The microarray results were independently validated by real-time PCR analysis of a subset of genes from each class, using additional biological replicates. Response of Arabidopsis to acute ozone stress The wild-type response of Arabidopsis (“Columbia” ecotype) to acute ozone fumigation was characterized using full transcriptome 70-mer oligo microarrays and cDNA derived from three-week old plants exposed to 500 ppb ozone for two hours. A total of 2840 genes showed at least two-fold differential expression (p ≤ 0.05). Up-regulated genes included antioxidant machinery, ethylene-related genes, ABA-responsive genes, auxin- and jasmonic acid-biosynthetic genes (Table 1). Down-regulated genes included energy metabolism associated, chloroplast-targeted, CDPKs and disease resistance genes (Table 1). Table 1: Top 30 up- and down-regulated genes in the response of three week old WT Col-0 plants to fumigation with 500 ppb ozone for two-hours. Response of an AtMPK3-deletion line to acute ozone stress Elimination of AtMPK3 leads to prolonged and hyper-activation of AtMPK6. The effect of eliminating AtMPK3 on the transcriptional response of Arabidopsis to acute ozone fumigation was characterized using aforementioned microarrays and comparing expression profiles of three week old AtMPK3 and WT Col-0 plants. Relative to the WT ozone response, only 91 genes showed greater than two-fold altered expression profiles (p ≤ 0.05; Table 2). A clear pattern could not be discerned amongst these genes. However, several plant defensin proteins (PDFs), which have been identified as jasmonate-responsive, showed abnormal transcript profiles. Table 2: 16 up-regulated and top-30 down-regulated genes in the AtMPK3 line relative to WT Col-0 upon exposure to 500 ppb ozone for two-hours Table3: Top 30 up- and down-regulated genes in the AtMPK6 line relative to WT Col-0 upon exposure to 500 ppb ozone for two-hours Response of an AtMPK6-deletion line to acute ozone stress Elimination of AtMPK6 leads to prolonged and hyper-activation of AtMPK3. The effect of eliminating AtMPK6 on the transcriptional response of Arabidopsis to acute ozone fumigation was characterized using aforementioned microarrays and comparing expression profiles of three week old AtMPK6 and WT Col-0 plants. Relative to the WT ozone response, only 168 genes showed greater than two-fold altered expression profiles (p ≤ 0.05; Table 3). The major class of genes showing abnormal transcriptional regulation in the AtMPK6 line were associated with stress-related metabolism. However, several protein kinases (4.5% of altered genes) and transcription factors (4.2%) were also found to be affected. Synergy and reciprocity in AtMPK3 and AtMPK6 signaling in response to ozone Only three genes showed synergistic expression patterns in response to ozone when either AtMPK3 or AtMPK6 signaling is eliminated (Table 4; all microarray data were validated by real-time PCR using cDNA derived from three independent biological replicates). Contrary to the reciprocity in AtMPK3 and AtMPK6 activation at the post-translational level, no downstream transcriptional reciprocity was observed upon the elimination of AtMPK3 or AtMPK6 signaling in response to ozone. Table 4: Genes responding in a similar fashion upon the elimination of either AtMPK3 or AtMPK6 signaling in response to ozone. The characteristic response pattern for each gene in the wild-type two-hour ozone response is illustrated as a reference point Discussion A large number of genes (2840) are differentially expressed in WT tissue exposed to ozone (500ppb) for two hours Elimination of AtMPK3 or AtMPK6 signaling affects only a very small proportion of these genes (3.2% and 5.9% respectively) Reciprocity between AtMPK3 and AtMPK6 in downstream transcriptional responses is not apparent Very little synergy between AtMPK3 and AtMPK6 in downstream transcriptional responses is evident Reciprocity and synergy in AtMPK3 and AtMPK6 signaling may only be significant at the post-translational level JA-related genes are differentially regulated in the AtMPK3 and AtMPK6 lines during the ozone response Octadecanoid biosynthetic genes are differentially regulated in the AtMPK3 line during the ozone response AtMPK3 signaling may positively regulate JA biosynthesis JA is known to protect plants from ozone-mediated cell death JA-related genes are incorrectly regulated in the AtMPK6 line during the response to exogenous MeJA PR1a regulation is also affected in the ΔAtMPK6 line AtMPK6 signaling may function antagonistically to JA Association of AtMPK3/6 signaling with octadecanoid signaling Both jasmonic acid biosynthetic and JA-responsive genes showed altered transcriptional responses in the AtMPK3 and AtMPK6 lines in response to ozone. These differentials were validated by real-time PCR using cDNA derived from three independent biological replicates. Furthermore, the response of these genes to methyl jasmonate treatment in WT Col-0, MPK3 and MPK6 lines was examined (Figure 1). Three-week old plants were exposed to methyl jasmonate for two hours and gene expression was analyzed using cDNA prepared from three biological replicates. Figure 1: Response pattern of jasmonic acid (JA) biosynthetic and responsive genes to methyl jasmonate treatment. Plants were exposed to methyl jasmonate for two hours and gene expression profiles were collected using cDNA derived from three biological replicates. The response pattern of PR-1 is also affected in the AtMPK6 line; this is consistent with the known role of AtMPK6 in salicylic acid (SA) and ethylene (ET) signaling and is consistent with a model in which JA signaling functions antagonistically to SA and ET signaling. Conclusions While AtMPK3 and AtMPK6 appear to be reciprocally regulated at the post-translational level during the early ozone response, there is no reciprocity in the transcriptional responses controlled by AtMPK3/6 Elimination of AtMPK3 or AtMPK6 impacts relatively few genes during the ozone response Major role of AtMPK3 and AtMPK6 signaling may be at the post-translational level Since silencing of either AtMPK3 or AtMPK6 renders plants more ozone sensitive, these kinases may help regulate the cellular response to oxidant stress primarily at the level of the “stress hormones” - JA, SA and ET