Calcineurin B-like protein 5 (SiCBL5) in Setaria italica enhances salt tolerance by regulating Na+ homeostasis

Salinity, a major abiotic stress, reduces plant growth and severely limits agricultural productivity. Plants regulate salt uptake via calcineurin B-like proteins (CBLs). Although extensive studies of the functions of CBLs in response to salt stress have been conducted in Arabidopsis, their functions in Setaria italica are still poorly understood. The foxtail millet genome encodes seven CBLs, of which only SiCBL4 was shown to be involved in salt response. Overexpression of SiCBL5 in Arabidopsis thaliana sos3-1 mutant rescued its salt hypersensitivity phenotype, but that of other SiCBLs (SiCBL1, SiCBL2, SiCBL3, SiCBL6, and SiCBL7) did not rescue the salt hypersensitivity of the Atsos3-1 mutant. SiCBL5 harbors an N-myristoylation motif and is located in the plasma membrane. Overexpression of SiCBL5 in foxtail millet increased its salt tolerance, but its knockdown increased salt hypersensitivity. Yeast two-hybrid and firefly luciferase complementation imaging assays showed that SiCBL5 physically interacted with SiCIPK24 in vitro and in vivo. Co-overexpression of SiCBL5, SiCIPK24, and SiSOS1 in yeast conferred a high-salt-tolerance phenotype. Compared to wild-type plants under salt stress conditions, SiCBL5 overexpressors showed lower accumulations of Na+ and stronger Na+ efflux, whereas RNAi-SiCBL5 plants showed higher accumulations of Na+ and weaker Na+ efflux. These results indicate that SiCBL5 confers salt tolerance in foxtail millet by modulating Na+ homeostasis.