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

Abstract

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 Mn(2+) over Mg(2+). 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.

DREB2C Interacts with ABF2, a bZIP Protein Regulating Abscisic Acid-Responsive Gene Expression, and Its Overexpression Affects Abscisic Acid Sensitivity

“…Overexpression of ABF3 or ABF4 in Arabidopsis (Arabidopsis thaliana), for instance, confers ABA hypersensitivity and drought tolerance (Kang et al, 2002;Kim et al, 2004aKim et al, , 2004c, whereas their knockout mutants are partially insensitive to ABA and susceptible to drought (Kim et al, 2004c;Finkelstein et al, 2005). Several studies indicate that the in vivo functions of ABFs/AREBs are modulated by various kinases (Choi et al, 2005;Furihata et al, 2006;Chae et al, 2007). ABFs/AREBs are highly homologous to ABI5 (Finkelstein and Lynch, 2000;Lopez-Molina and Chua, 2000), sunflower (Helianthus annuus) and Arabidopsis DPBFs (Kim et al, 1997;Kim and Thomas, 1998;Kim et al, 2002), and rice (Oryza sativa) TRAB1 (Hobo et al, 1999b).…”

Toward Understanding Molecular Mechanisms of Abiotic Stress Responses in Rice

“…Recent studies in both Arabidopsis and rice have shown that SnRK2s can be activated by both ABA and abiotic stress [15,18,36,86,87]. The activated SnRK2s phosphorylate and activate AREB/ABF/ABI5, and subsequently activate expression of AREB/ABF/ABI5 target genes [15,18,36,86].…”

Sucrose non-fermenting 1-related protein kinase 2 (SnRK2): a family of protein kinases involved in hyperosmotic stress signaling

“…Dehydration, osmotic stress and ABA all induced an OSRK1 gene identical to SAPK6 (Kobayashi et al, 2004), encoding a 41.8-kDa protein kinase belonging to the SnRK2 family from Oryza sativa. This protein kinase showed a preference for an uncommon cofactor requirement for Mn 2+ over Mg 2+ (Chae et al, 2007). Using reverse genetics approach, Fujii et al (2007) isolated single and double mutants of SnRK2.2 and SnRK2.3, and found that the double mutant was insensitive to ABA during seed germination and seedling growth indicating a role for SnRK2.2 and SnRK2.3 protein kinases in mediating ABA signaling during seed germination.…”

“…Two conserved boxes are located in the carboxy-terminal region, after the catalytic domain, of SnRK2.6/OST1/SRK2E, a member of SnRK2, the SnRK2-specific box (glutamine-303 to proline-318) and the ABAspecific box (leucine-333 to methionine-362) (Belin et al, 2006). OSRK1 represents another dehydration inducible gene (1522 bp in length, 1,095 bp of coding region, 94 bp of the 5'untranslated region, and 123 bp of 3'untranslated region) from rice (Chae et al, 2007).…”

“…Dehydration inducible SnRK2 (OSRK1) is capable of autophosphorylation, and phosphorylates MBP and histone (Chae et al, 2007). OSRK1 can also phosphorylate OREB1, a rice homolog of ABI5/ABF/ AREB subfamily of bZIP transcription factor, at multiple functional domains.…”