Functional characterization of calmodulin-like protein CML3 from rice oryza sativa L.

Abstract

Previously, a large family of Calmodulin (Cam) and calmodulin-like (CML) genes has been identified in Oryza sativa L., but there is little detailed information on the functions of these proteins. Here, we examined properties of the purified recombinant protein encoded by OsCML3m (truncated OsCML3 lacking the C-terminal extension (CTE) coding sequence). The rOsCML3 and rOsCML3m proteins were found to exhibit a Ca2+-binding property and subsequent conformational change, but the ability to bind the CaM kinase II peptide was only observed for OsCML3m. Changes in their secondary structure upon Ca2+-binding measured by circular dichroism revealed that OsCML3m had a higher helical content than OsCML3. Moreover, the subcellular localization of OsCML3 and OsCML3m revealed that OsCML3 was mainly localized at the plasma membrane, whereas OsCML3m was found in the nucleus. Previously, the rice phosphatidylethanolamine (OsPEBP) and high mobility group B1 (OsHMGB1) protein were identified as two of the putative OsCML3m target proteins. By yeast two-hybrid analysis, no interaction between OsPEBP or OsHMGB1with either OsCML3 or OsCMLs (cysteine to serine mutation at prenylation site), and strong self-activation of the OsCML3m fused with GAL4 DNA-binding domain were detected. By Bimolecular Fluorescence Complementation (BiFC) analysis, the interaction of OsPEBP with OsCML3, OsCML3m or OsCML3s was not found whereas the interaction of OsHMGB1 with OsCML3, OsCML3m or OsCML3s was found in the nucleus, suggesting that HMGB1 is an OsCML3 target protein in planta. Moreover, electrophoretic mobility shift assay (EMSA) showed that, in the presence of Ca2+, OsCML3m decreased HMGB1 binding to pUC19 double stranded DNA whereas OsCML3 did not. These results indicate that the OsCML3 CTE interfere the target binding ability of OsCML3 and its interaction with OsHMGB1 inhibits the DNA-binding ability of OsHMGB1. The transcript expression analysis of CML3 in rice (Oryza sativa L.) showed that was slightly down-regulated at 1 h under salt treatment. In addition, the OsCML3 transcript level was found up-regulated at 48 h after 6-Benzylaminopurine (BA) application while slightly down-regulated at 48 h after 3-Indoleacetic acid (IAA) application. Interestingly, the transcript level of OsCML3 was significantly up-regulated to 2-3 folds at 3 h after cold stress suggesting that the OsCML3 may involve in the response to cold stress. In the OsCML3 promoter, two Putative ABREs (ABA-responsive element) and one putative LTRE (low temperature responsive element) were found. Finally, the transgenic Arabidopsis overexpressing OsCML3 or OsCML3m using Agrobacterium-mediated transformation was generated, resulting in four independent homozygous transgenic lines harboring OsCML3 or OsCML3m. Higher germination percentages of all transgenic lines at 11 °C were observed compared with those of the wild type and the control transgenic Arabidopsis transformed with the blank vector. Fresh weight and dry weight ratios of the transgenic Arabidopsis overexpressing OsCML3 or OsCML3m seedlings grown under cold stress to those under normal condition (22 °C) were mostly higher than those of the wt and the control transgenic plant. Taken together, OsCML3 probably provides a mechanism for manipulating the DNA-binding ability of OsHMGB1 in the nucleus and its C-terminal extension provides a regulatory switch. Moreover, the overexpression of OsCML3 or OsCML3m enhances cold tolerance during germination and seedling stage of Arabidopsis.