A calcium sensor calcineurin B-like 9 negatively regulates cold tolerance via calcium signaling in Arabidopsis thaliana.

Calcineurin B-like protein 9 (CBL9) plays important roles in response to ABA, K deprivation in plants. However, whether CBL9 modulates plant adaptation to low-temperature stress is elusive. In this study, we demonstrated that the cbl9 mutants increased freezing tolerance under both cold-acclimating and nonacclimating conditions in Arabidopsis. Cold-induced changes of cytosolic free calcium concentration ([Ca]) were then monitored by aequorin-expressed Arabidopsis plants. The results showed that the cold-triggered increases in [Ca] levels in cbl9 mutants were clearly higher than those in wild type (WT) plants, while cold-affected changes in free calcium concentration within cytosolic microdomains adjacent to the vacuolar membrane ([Ca]) in cbl9 mutants were similar to those in WT plants. In addition, treatments of seedlings with Ca chelator ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and Ca channel blocker lanthanum chloride markedly inhibit changes of [Ca] in cbl9 mutants, while the inhibition of calcium release by lithium chloride from intracellular pools demonstrated consistent suppression of [Ca] in cbl9 mutants and WT plants. Together, these results indicate that CBL9 negatively modulates cold tolerance through decreasing [Ca] in Arabidopsis.