Genome-wide identification and characterization of SPX domain-containing proteins and their responses to phosphorus and/or nitrogen deficiency in apple (Malus domestica Borkh.)

Phosphate (Pi) and nitrate (Ni) deficiencies are the major constraints for apple, significantly impacting the fruit yield and quality. SPX domain-containing proteins play important roles in Pi homeostasis and Pi signaling pathway, as well as phosphorus (P) and nitrogen (N) nutrient balance in plants. However, SPX domain -containing proteins have not been analysed systematically in apple (Malus domestica Borkh.). In this study, a total of 25 MdSPXs were identified in M. domestica. According to phylogenetic relationships and C-terminal domains analyses, these MdSPXs were divided into four subfamilies: SPX (6), SPX-EXS (12), SPX-MFS (3), and SPX-RING (4).16 MdSPXs from these four subfamiles contain Phosphate starvation response 1-binding site (P1BS) in the promoter region. The expression profiles showed that 15 MdSPXs responded to P deficiency as well as N and P deficiencies, respectively, 13 MdSPXs responded to N deficiency in hydroponic 'M26' seedlings. The response of 11 common differentially expressed genes exhibited apparent tissue-specificity. Pi starvation response (PSR) involved in MdSPX1, MdSPX2, and MdSPX5 depended on the N availability. In the predicted protein-protein interaction network, MdSPXs interact with important components of the P signaling network such as MdPHO2, MdPHRs, MdSIZ1, and MdPHTs, etc., and components of the N signaling network such as with MdNRTs and MdNLP6, etc., in addition to several TFs such as bZIP TF MdTGA4, an important Ni-responsive TF. Overexpression of MdTGA4 changed the expression of MdSPX2, MdSPX4, MdPHO1;6, MdPHO1;7, MdPHO1;9 and MdMFS2 in the transgenic apple calluses, and these genes contain as-1, a special binding element of TGA, in the promoter region. These results preliminarily confirmed the key role of MdSPXs in Pi homeostasis and Pi signaling pathway as well as Ni signaling pathway, offered a valuable guide for integrating N-P interactive regulation pathways in apple, and provided important genetic resources for the future research on the efficient utilization of P and N nutrients in apple.