MAPK-associated proteins in Arabidopsis thaliana

Sammanfattning: Abstract Mitogen-Activated Protein Kinases (MAPKs) are signalling proteins that are highly conserved among eukaryotes. They are involved in many biological pathways including cell differentiation and response to different stress situations. The MAPK cascade is composed of three signalling kinases, MAP3K, MAP2K and MAPK, which phosphorylate each other in a sequential manner. The different MAPK substrates are regulated by phosphorylation on serine or threonine. This thesis presents a new method for purification of phosphoproteins from a complex protein extract. Subsequent studies using this method identified AtPHOS34 to be phosphorylated upon biotic stress. The phosphorylation site of AtPHOS34 was also determined. Several Arabidopsis thaliana MPK3/6 in vitro substrates were identified by a peptide and bioinformatics screen and the predicted phosphorylation sites were confirmed with kinase assays using mutated and wild-type proteins. MAPK Substrate 80 (here denoted MKS80) was characterised as an in planta MAPK substrate and in detail analyses of MKS80 linked its function to stomatal patterning and regulation of carbohydrate balance. The MKS80 interaction partner ?-glucan phosphorylase (PHS2) was established by a yeast two-hybrid screen, in vitro pull-down and in planta co-immunoprecipitation assays. PHS2 catalyses the reversible reaction between glucose-1-phosphate and highly branched soluble heteroglycans and was shown to be important in senescence. Biochemical analyses revealed a stable level of PHS2 and light-dependent accumulation of MKS80. Arabidopsis over-expressing wild-type MKS80 had a lower starch content, whereas plants over-expressing the phosphomimetic form of MKS80 died at the seedling stage, clearly demonstrating the importance of this phosphorylation. In plants over-expressing MKS80, the anthocyanin level was lowered in different stress situations. Slower senescence was detected in detached leaves over-expressing MKS80, indicating a change in metabolism. A system for delayed senescence can be of interest for the agricultural industry.