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.Science, Faculty ofBotany, Department ofGraduat
