Characterization of the Arabidopsis Calcineurin B-like Calcium Sensors in Environmental and Developmental Signal Transduction

Abstract

In plants, the regulatory networks that have evolved to generate the appropriate cellular responses to external and internal stimuli often include a transient increase in intracellular calcium (Ca²⁺). One ten member, plant-specific family of Ca²⁺ sensing elements, the calcineurin B-like (CBL) protein family, is thought to relay the Ca²⁺ signal to downstream targets when plants experience an abiotic stress. The purpose of this study was to uncover critical functions of four CBL proteins. CBL10 was chosen for its distinct amino acid sequence and unique genomic structure among the CBL proteins. CBL1 and CBL9, generated by segmental duplication, were chosen based on their high level of amino acid identity. CBL8 was chosen based on its sequence and genomic structure similarities to SOS3/CBL4, the founding member of the CBL family. An Arabidopsis CBL10 knock-out insertion, cbl10-1, was isolated and found to have reduced stamen elongation, leading to male sterility. The mutant also showed growth arrest in aerial portions of the plant and developed chlorosis in response to increasing salt concentrations. Alternative pre-mRNA splicing generated five CBL10.2 variants whose transcript levels were regulated by cold or salt treatment, suggesting that CBL10 is involved in normal development and plant growth during abiotic stress via tight regulation of transcript levels. Phylogenetic analysis suggests that CBL1 and CBL9 may compensate for each other in the regulation of essential functions in the single mutant backgrounds. To investigate the crucial functions of these genes, a cbl1 cbl9 double mutant was generated. When grown under drought conditions, the double mutant was less sensitive to ABA, lost more water and produced more shriveled seeds than wild-type plants suggesting that both CBL1 and CBL9 play key roles in relaying drought stress signals during vegetative and reproductive growth. Because phylogenetic analysis and amino acid sequence comparisons suggest that CBL8 may function redundantly with SOS3/CBL4, a cbl4 cbl8 double mutant was generated. The cbl4 cbl8 double mutant phenocopied the sos3-1 (cbl4) mutant during salt and ABA treatments. CBL8 promoter activity in accessory cells of trichomes and root hairs suggests that CBL8 may have a function in development of these specialized epidermal cells.