A rice dehydration-inducible SNF1-related protein kinase 2 phosphorylates an abscisic acid responsive element-binding factor and associates with ABA signaling

By a differential cDNA screening technique, we have isolated a dehydration-inducible gene (designated OSRK1) that encodes a 41.8 kD protein kinase of SnRK2 family from Oryza sativa. The OSRK1 transcript level was undetectable in vegetative tissues, but significantly increased by hyperosmotic stress and Abscisic acid (ABA). To determine its biochemical properties, we expressed and isolated OSRK1 and its mutants as glutathione S-transferase fusion proteins in Escherichia coli. In vitro kinase assay showed that OSRK1 can phosphorylate itself and generic substrates as well. Interestingly, OSRK1 showed strong substrate preference for rice bZIP transcription factors and uncommon cofactor requirement for Mn2+ over Mg2+. By deletion of C-terminus 73 amino acids or mutations of Ser-158 and Thr-159 to aspartic acids (Asp) in the activation loop, the activity of OSRK1 was dramatically decreased. OSRK1 can transphosphorylate the inactive deletion protein. A rice family of abscisic acid-responsive element (ABRE) binding factor, OREB1 was phosphorylated in vitro by OSRK1 at multiple sites of different functional domains. MALDI-TOF analysis identified a phosphorylation site at Ser44 of OREB1 and mutation of the residue greatly decreased the substrate specificity for OSRK1. The recognition motif for OSRK1, RQSS is highly similar to the consensus substrate sequence of AMPK/SNF1 kinase family. We further showed that OSRK1 interacts with OREB1 in a yeast two-hybrid system and co-localized to nuclei by transient expression analysis of GFP-fused protein in onion epidermis. Finally, ectopic expression of OSRK1 in transgenic tobacco resulted in a reduced sensitivity to ABA in seed germination and root elongation. These findings suggest that OSRK1 is associated with ABA signaling, possibly through the phosphorylation of ABF family in vivo. The interaction between SnRK2 family kinases and ABF transcription factors may constitute an important part of cross-talk mechanism in the stress signaling networks in plants.