Direct interaction of a divergent CaM isoform and the transcription factor, MYB2, enhances salt tolerance in Arabidopsis

Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates diverse cellular functions by modulating the activity of a variety of enzymes and proteins. Plants express numerous CaM isoforms that exhibit differential activation and/or inhibition of CaM-dependent enzymes in vitro. However, the specific biological functions of plant CaM are not well known. In this study, we isolated a cDNA encoding a CaM binding transcription factor, MYB2, that regulates the expression of salt- and dehydration-responsive genes in Arabidopsis. This was achieved using a salt-inducible CaM isoform (GmCaM4) as a probe from a salt-treated Arabidopsis expression library. Using domain mapping, we identified a Ca(2+)-dependent CaM binding domain in MYB2. The specific binding of CaM to CaM binding domain was confirmed by site-directed mutagenesis, a gel mobility shift assay, split ubiquitin assay, and a competition assay using a Ca(2+)/CaM-dependent enzyme. Interestingly, the specific CaM isoform GmCaM4 enhances the DNA binding activity of AtMYB2, whereas this was inhibited by a closely related CaM isoform (GmCaM1). Overexpression of GmCaM4 in Arabidopsis up-regulates the transcription rate of AtMYB2-regulated genes, including the proline-synthesizing enzyme P5CS1 (triangle(1)-pyrroline-5-carboxylate synthetase-1), which confers salt tolerance by facilitating proline accumulation. Therefore, we suggest that a specific CaM isoform mediates salt-induced Ca(2+) signaling through the activation of an MYB transcriptional activator, thereby resulting in salt tolerance in plants.