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Functional characterization of a gain-of-function mutant of AtMKK9 in Arabidopsis thaliana Cluis, Corinne Pamela

Abstract

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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