Members of the sucrose non-fermenting related kinase Group2 (SnRK2) subclasses are implicated in both direct and indirect abscisic acid (ABA) response pathways. We have used phylogenetic, biochemical, and transient in vivo approaches to examine interactions between Triticum tauschii protein kinase 1 (TtPK1) and an interacting protein, Oryza sativa SnRK2-calcium sensor (OsSCS1). Given that TtPK1 has 100% identity with its rice ortholog, osmotic stress/ABA-activated protein kinase (OsSAPK2), we hypothesized that the SCS and TtPK1 interactions are present in both wheat and rice. Here, we show that SnRK2s are clearly divided into four pan-angiosperm clades with those in the traditionally defined Subclass II encompassing two distinct clades (OsSAPK1/2 and OsSAPK3), although OsSAPK3 lacks an Arabidopsis ortholog. We also show that SCSs are distinct from a second lineage, that we term SCSsister, and while both clades pre-date land plants, the SCSsister clade lacks Poales representatives. Our Y2H assays revealed that the removal of the OsSCS1 C-terminal region along with its N-terminal EF-hand abolished its interaction with the kinase. Using transient in planta bimolecular fluorescence complementation experiments, we demonstrate that TtPK1/OsSCS1 dimerization co-localizes with DAPI-stained nuclei and with FM4-64-stained membranes. Finally, OsSCS1- and OsSAPK2-hybridizing transcripts co-accumulate in shoots/coleoptile of drying seedlings, consistent with up-regulated kinase transcripts of PKABA1 and TtPK1. Our studies suggest that interactions between homologs of the SnRK2 and SCS lineages are broadly conserved across angiosperms and offer new directions for investigations of related proteins.

Background

Starch is of great importance to humans as a food and biomaterial, and the amount and structure of starch made in plants is determined in part by starch synthase (SS) activity. Five SS isoforms, SSI, II, III, IV and Granule Bound SSI, have been identified, each with a unique catalytic role in starch synthesis. The basic mode of action of SSs is known; however our knowledge of several aspects of SS enzymology at the structural and mechanistic level is incomplete. To gain a better understanding of the differences in SS sequences that underscore their specificity, the previously uncharacterised SSIVb from wheat was cloned and extensive bioinformatics analyses of this and other SSs sequences were done.

Results

The wheat SSIV cDNA is most similar to rice SSIVb with which it shows synteny and shares a similar exon-intron arrangement. The wheat SSIVb gene was preferentially expressed in leaf and was not regulated by a circadian clock. Phylogenetic analysis showed that in plants, SSIV is closely related to SSIII, while SSI, SSII and Granule Bound SSI clustered together and distinctions between the two groups can be made at the genetic level and included chromosomal location and intron conservation. Further, identified differences at the amino acid level in their glycosyltransferase domains, predicted secondary structures, global conformations and conserved residues might be indicative of intragroup functional associations.

Conclusion

Based on bioinformatics analysis of the catalytic region of 36 SSs and 3 glycogen synthases (GSs), it is suggested that the valine residue in the highly conserved K-X-G-G-L motif in SSIII and SSIV may be a determining feature of primer specificity of these SSs as compared to GBSSI, SSI and SSII. In GBSSI, the Ile485 residue may partially explain that enzyme's unique catalytic features. The flexible 380s Loop in the starch catalytic domain may be important in defining the specificity of action for each different SS and the G-X-G in motif VI could define SSIV and SSIII action particularly.