Genome-wide analysis of the apple CaCA superfamily reveals that MdCAX proteins are involved in the abiotic stress response as calcium transporters.

Background: Calcium (Ca) plays an important role in plant growth and development, and the maintenance of calcium homeostasis is necessary for the survival of all plant species. Ca/H exchangers (CAXs) are a subgroup of the CaCA (Ca/cation antiporter) superfamily. In general, CAX proteins mediate cytosolic Ca entry into vacuoles to prevent excessive accumulation of Ca in the cytosol. The CaCA superfamily has been identified and characterised in many plant species; however, characterisation of the CaCA superfamily and functional study of apple CAX proteins have yet to be conducted in apple (Malus × domestica Borkh.).

Results: Here, we identified 21 CaCA family proteins in apple for the first time. Phylogenetic and gene structure analysis, as well as prediction of conserved motifs, suggested that these proteins could be classified into four groups: CAX, CCX, NCL, and MHX. Expression analysis showed that the 10 MdCAX genes we cloned strongly responded to calcium and abiotic stress treatments. Collinearity analysis and characterisation of calcium transport capacity resulted in the identification of a pair of segmental duplication genes: MdCAX3L-1 and MdCAX3L-2; MdCAX3L-2 showed strong calcium transport capacity, whereas MdCAX3L-1 showed no calcium transport capacity. Yeast two-hybrid (Y2H) assays showed that these two proteins could interact with each other. The high sequence similarity (94.6%) makes them a good model for studying the crucial residues and structural basis of the calcium transport of CAX proteins. Prediction of the protein interaction network revealed several proteins that may interact with CAX proteins and play important roles in plant stress responses, such as SOS2, CXIP1, MHX, NRAMP3, and MTP8.

Conclusions: Our analysis indicated that MdCAX proteins have strong calcium transport capacity and are involved in the abiotic stress response in apple. These findings provide new insight and rich resources for future studies of MdCAX proteins in apple.