Characterization of the Calmodulin-Like Protein Family in <i>Chara braunii</i> and their Conserved Interaction with the Calmodulin-Binding Transcription Activator Family

Calcium sensor proteins play important roles by detecting changes in intracellular calcium and relaying that information onto downstream targets through protein-protein interaction. Very little is known about calcium sensors from plant species that predate land colonization and the evolution of embryophytes. Here, we examined the genome of the multicellular algae, Chara braunii, for orthologs to the evolutionarily conserved calcium sensor calmodulin (CaM) and for CaM-like (CML) proteins. We identified one CaM and eight CML isoforms that range in size from 16.4 to 21.3 kDa and are predicted to have between two to four calcium-binding (EF-hand) domains. Using recombinant protein, we tested whether CbCaM and CbCML1-CbCML7 possess biochemical properties of typical calcium sensors. CbCaM and the CbCMLs all displayed high-affinity calcium binding with estimated global KD,app values in the physiological µM range. In response to calcium binding, CbCaM and the CbCMLs exhibited varying degrees of increase in exposed hydrophobicity, suggesting that different calcium-induced conformational changes occur among isoforms. We found many examples of putative CaM targets encoded in the C. braunii genome and explored the ability of CbCaM and CbCMLs to interact in planta with a representative putative target, a C. braunii CaM-binding transcription factor (CbCAMTA1). CbCaM, CbCML2 and CbCML4 each associated with the C-terminal region of CbCAMTA1. Collectively, our data support the hypothesis that complex calcium signaling and sensing networks involving CaM and CMLs evolved early in the green lineage. Similarly, it seems likely that calcium-mediated regulation of transcription occurs in C. braunii via CAMTAs and is an ancient trait predating embryophytic emergence.