An Investigation of the Biochemical Properties and Physiological Roles of the Arabidopsis Calmodulin-Like (CML) Genes CML15 and CML16
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Plants, as sessile organisms, require rapid and efficient signalling pathways in order to respond to environmental stimuli and to coordinate developmental programs. Stimulus-specific transient elevations in free cytosolic calcium ([Ca2+]cyt ), otherwise referred to as calcium (Ca2+) signals, are known to encode information regarding external stressors or internal developmental cues, which can be interpreted by Ca2+-binding proteins (CBPs), termed Ca2+ sensors. Though much is known about Calmodulin (CaM), the most abundant Ca2+ sensor, relatively little is known about the CaM-like family of Ca2+ sensors (CMLs), which comprise a 50-member family in Arabidopsis thaliana (Arabidopsis). In order to understand how CMLs contribute to Ca2+ signalling, it is necessary to study their biochemistry and physiological roles. In this study, two paralogous CMLs of unknown function, CML15 and CML16, were studied through a combination of approaches including protein biochemistry, gene promoter-reporter analysis, and physiological assays. Promoter-reporter analysis demonstrated that the CML16 promoter is constitutively active across a range of Arabidopsis tissues, whereas CML15 promoter activity is restricted to the stamen. Among a range of environmental conditions tested, salinity stress was observed to inhibit CML16 promoter activity. Phenotypic analysis, across a range of growth stages, of gene-knockout transgenic plants lacking either CML15 or CML16 did not reveal any differences from wild-type plants. Biochemical characterization of CML15 and CML16 indicated that these CMLs possess properties consistent with their predicted roles as Ca2+ sensors. CML15 and CML16 were observed to undergo Ca2+-dependent conformational changes that expose hydrophobic residues, likely for interaction with unknown protein targets. Furthermore, both CMLs were found to bind magnesium (Mg2+) in the absence of Ca2+, but demonstrated high affinity Ca2+ binding in the presence of Mg2+. Putative protein targets for CML15 and CML16 were isolated by yeast two-hybrid screening although the physiological relevance of these as CML effectors remains unknown. Collectively, my data reveals that these CMLs behave biochemically like typical Ca2+ sensors, possess distinct and largely non-overlapping gene expression patterns, and do not appear to be critical for Arabidopsis growth and development under typical growth conditions.