Structural analysis of regulatory stress elements in OsCIPK14 the rice homologue and functional validation in wheat

Abstract

A major mechanism of stress tolerance in plants is to increase the cytosolic concentration of Ca2+ ion, a secondary messenger that mediates cellular responsiveness to the extracellular environment. The involvement of different types of Ca2+-sensing protein kinases such as CIPK in stress responses has been elucidated in different plant models. In this study, the upstream regulatory sequence of a CIPK homologue from an Egyptian rice cultivar (Oryza sativa cv Giza182) was structurally and functionally characterized and tested for its ability to drive the expression of β- glucuronidase reporter gene (uidA) in wheat. To that end, a 1275-bp DNA fragment, 49 nucleotides upstream of the putative transcription start site (TSS) of the OsCIPK14 coding sequence, referred in this study as URS-CIPK14, was cloned, sequenced and analyzed for the presence of stress-responsiveness cis-acting elements. Subsequently, the URS-CIPK14 was fused to the uidA gene with and without the Act-1 intron, cloned in pAB6 and transformed into wheat mature embryo for transient expression. Our results showed that URS-OsCIPK14 fused with the Act-1 intron was induced by NaCl and ABA, whereas URS-OsCIPK14 drove expression of uidA constitutively in the absence of Act-1 intron. Furthermore, rice wide-genome screening using URS-CIPK14 sequence obtained in this study identified another homologue on chromosome 11. Unlike URS-CIPK14, which is associated with chromosome 12, this homologue located at about 1015 nucleotides upstream from the putative TSS of OsCIPK15 coding sequence. Interestingly, the 5’-UTS in CIPK14 is longer than CIPK15 and can code for a putative short ORF that contains two Casein kinase II phosphorylation motifs (SXXD). The significance of this ORF is not known at this time and needs to be elucidated in future studies. We conclude that data from this work suggest that URS-CIPK14 can be used as a promoter candidate for high expression level under both constitutive and inducible conditions.