UBC Theses and Dissertations
Functional characterization of a gain-of-function mutant of AtMKK9 in Arabidopsis thaliana Cluis, Corinne Pamela
The relatively small number of MAPKKs encoded in the Arabidopsis genome suggests that this particular class of kinases acts as a point of convergence within the plant's 'integration of external stimuli and their transduction to elicit biological responses. In an effort to gain information about the function of the MAPKK, AtMKK9 , in Arabidopsis, I have characterized several aspects of the phenotype of DEX:CA-MKK9-FLAG transgenic plants, which express an inducible constitutively active version of AtMKK9, CA-MKK9. I have found that CA-MKK9 expression can control the production of ethylene by activating a downstream MAPK, AtMPK6, which is known to promote the stabilization of ethylene biosynthesis enzymes. CA-MKK9 induction was correlated with an increase in AtMPK6 activity in planta, and was rapidly followed by production of a burst of ethylene in the induced plant tissues. I hypothesized that CA-MKK9 directly activates AtMPK6, and demonstrated that a recombinant version of CA-MKK9 was capable of phosphorylating AtMPK6 in vitro. In addition, the production of the hormone was abolished when C A - MKK9was expressed in a mpk6 knock-out background, thus proving that AtMPK6 is required for CA-MKK9 -mediated ethylene biosynthesis. I have also confirmed preliminary data from our laboratory suggesting that CA-MKK9 plays a role in oxidative programmed cell death. The necrotic lesions induced by CA-MKK9 were still observed in the mpk6 background, indicating that programmed cell death was triggered by CA-MKK9 activity independently of AtMPK6 activity and of ethylene overproduction. In addition, in order to investigate short-term transcriptional events triggered by CA-MKK9, I attempted to capture the transcriptional profile of DEX:CA-MKK9-FLAG plants using two-channel oligonucleotide microarrays. The CA-MKK9 - affected genes included a number of genes involved in the octadecanoid pathway, and their promoters were enriched in ABRE-like elements. However, my attempts to validate the microarray results using additional biological replicates and quantitative real-time PCR revealed that the majority of these early-response microarray results were apparently false positives, indicating that the microarray experiment was probably inappropriately constructed for capturing early transcriptional responses to CA-MKK9 using the dexamethasone-inducible system.
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