Regulation by Calcium-dependent protein kinases of Skaker channels expressed in guard cells
Résumé
Stomatal movements result from osmotically-driven fluxes of water, which follow massive exchanges of solutes, including K+ ions, between guard cells and surrounding tissues. These exchanges involve a set of dedicated transport systems, controlled through signalling pathways, including Ca2+-dependent ones. For example, Ca2+ signals have been reported to lead to stomatal closure through the inhibition of inward and activation of outward K+ channels. This could be mediated in particular by Ca2+-dependent kinases targeting these channels.
Here, we addressed the post-translational regulation of three K+-selective voltage-gated channels expressed in Arabidopsis guard cells (inwardly-rectifying KAT1 and KAT2, and outwardly-rectifying GORK) by Ca2+-dependent protein kinases (CPKs). Of the 34 CPK genes in
Arabidopsis thaliana, some CPK are thought to be expressed in guard cells. Seven of these CPKs were co-expressed in Xenopus oocytes with the three studied Shaker channels, and their influence on K+ transport was examined with a classical voltage-clamp technique. Pinpointed interactions were checked by on-chip phosphorylation assays on peptide arrays designed from the channel primary sequence. This enabled us to list a number of candidate phosphorylation sites and, subsequently, to design mutant channels expected to display CPK-insensitive of CPKphosphorylated phenotype. The functional characterisation of these mutant channels and the phenotype of plants overexpressing CPKs will be presented.