Calcineurin B-like proteins in rice can bind with calcium ion and associate with the Arabidopsis CIPK family members

Arabidopsis calcineurine B-like proteins (AtCBLs) are known to sense Ca²⁺ signals induced by a variety of extracellular stimuli such as cold, drought, low K⁺ concentration, and high salinity. The Ca²⁺-bound AtCBLs then associate with and activate a group of serine/threonine protein kinases (CIPKs) to transmit the signals further downstream. In this study, we isolated three rice cDNA clones (OsCBL1, 3, and 5), which respectively encode a polypeptide very similar to the AtCBL family. Deduced amino acid sequence analysis revealed that each OsCBL contains canonical EF-hand Ca²⁺-binding motifs. In fact, our electrophoresis mobility shift assays showed that both OsCBL1 and OsCBL3 proteins purified from Escherichia coli via the GST-fusion expression system can bind with Ca²⁺ in vitro. Using the yeast two-hybrid system and pull-down assays, we also demonstrated that OsCBLs, just like AtCBLs, can interact with the Arabidopsis CIPKs. In addition, deletion analyses indicated that the C-terminal nonkinase domain of the CIPK family is responsible for the interaction with OsCBLs. Taken together, these findings strongly suggest that OsCBLs are very likely to be the rice orthologs of AtCBLs, which may be involved in the various stress responses.