|dc.description.abstract||The Arabidopsis thaliana genome contains genes encoding 10 MAP kinase kinase (MKK) and 20 Map kinase (MAPK) proteins, however the exact relationship between upstream MKK activators and downstream MAPK protein partners has not fully elucidated. In this study, the yeast two -hybrid system was used to identify protein-protein interactions between all Arabidopsis MAPKs and MKKs. In the yeast two- hybrid assay, the result from the qualitative β-galactosidase filter assay showed that 6 MAPK proteins interacted with 6 MKK proteins. Quantitative β-galactosidase liquid assay was used to confirm the interactions between MAPK and MKK in yeast.
The BiFC approach was used to test whether MKK1, MKK2 and MKK6 associated with MPK4 and MPK11 in vivo in Arabidopsis. In these assays, GFP fluorescence was observed in the protoplasts suggesting that MKK1, MKK2 and MKK6 interact with MPK4 and MPK11, respectively. However, evidence of reciprocal interaction was only seen with the combination of MKK1 and MPK11, and with MKK2 and MPK11. This contrasts with the findings seen using the yeast two-hybrid system in which reciprocal activity was seen for all combinations of MKK1, MKK2 or MKK6 with MPK4 or MPK11. Furthermore, the negative controls for BiFC (spilt GFP with no fusions partners) gave strong fluorescence, indicative of non-specific interaction between the split GFP.
Since MPK4 is known to be an important regulator of plant stress responses, MPK11 (similar in sequence to MPK4) might also be involved in such responses. Therefore, a knock-out allele of MPK11 was obtained and analysed on a molecular and physiological basis. Seed germination experiments showed that no differences between the germination of the wildtype and the mpk11 mutant by increasing levels of salt however seed germination experiments showed that seedling of mpk11 mutant are hypersensitive to ABA during germination. To aid further understanding on the function of MPK11 and the relationship between MPK11 and interacting activators, HA-epitope tagged cDNA for MPK11 was introduced into Arabidopsis Columbia Wildtype, mpk11, mkk1 and mkk2. Results from Western blot analysis indicated that several lines from each transformant produced detectable levels of MPK11-HA, thus generating tools for further analysis||en_US