Direct Interaction of a Divergent CaM Isoform and the Transcription Factor, MYB2, Enhances Salt Tolerance in Arabidopsis

Abstract

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 Ca2+-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 Ca2+/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 Gm-CaM4 in Arabidopsis up-regulates the transcription rate of AtMYB2-regulated genes, including the proline-synthesizing enzyme P5CS1 (Delta1-pyrroline-5-carboxylate synthetase-1), which confers salt tolerance by facilitating proline accumulation. Therefore, we suggest that a specific CaM isoform mediates salt-induced Ca2+ signaling through the activation of an MYB transcriptional activator, thereby resulting in salt tolerance in plants.

Identification and Molecular Properties of SUMO-Binding Proteins in Arabidopsis

“…The yeast split ubiquitin assay was performed as described previously (Laser et al, 2000;Yoo et al, 2005). Saccharomyces cerevisiae strain JD53 was used for all experiments.…”

Identification of a Calmodulin-binding NAC Protein as a Transcriptional Repressor in Arabidopsis

“…In plants, certain transcription factors of the basic helix-loop-helix family were shown to bind CaM, thus inhibiting their DNA-binding properties by masking the DNA-binding domain (13)(14)(15). In addition, it was reported that soybean CaM isoforms differentially bind to AtMYB2 and regulate its DNA-binding activity (16). Therefore, interaction of CaM with transcription factors may be a mechanism that transduces Ca 2ϩ signals to the gene expression machinery to induce specific cellular responses.…”

Raising salinity tolerant rice: recent progress and future perspectives

“…However, in Arabidopsis one of the isoforms of CaM was found to bind to a transcription factor MYB2, enhancing its DNA binding activity. Overexpression of this isoform of CaM in Arabidopsis upregulated the transcription of MYB2 regulated genes including P5CS1, a rate-limiting enzyme in proline biosynthesis (Yoo et al, 2005).…”