Identification and analysis of miRNA-mRNA regulatory networks of ZM-4 (Malus zumi) in response to salt stress

Malus zumi is an excellent saline-alkali tolerant apple rootstock resource and a valuable economic tree for greening due to its ornamental value. It is of great potential significance in the production of apples in saline-alkali areas. miRNA is a class of endogenous non-coding small RNA, which plays an important role in plant resistance to salt stress. However, the miRNA-mediated regulatory mechanisms underlying salt tolerance in Malus zumi remain unclear. In this study, the salt-tolerant apple resource Malus zumi ZM-4 and the salt-sensitive rootstock M9T337 were used as experimental materials. High-throughput sequencing was conducted on the leaves and roots of these plants treated with 75 mmol/L NaCl for 0 h and 24 h. The results showed that a total of 3280 miRNAs were identified, with 274 and 323 miRNAs were specifically expressed in ZM-4 and M9T337, respectively. Under salt stress, 299 and 654 differentially expressed miRNAs were detected in leaves and roots, targeting 994 and 1813 mRNAs, respectively. Furthermore, five (mdm-miR395j-TPS13; miR1507-y-RGA1; miR3699-z-SPL13A; novel-m0411-5p-MKK4; novel-m0641-3p/novel-m0642-3p-ALMT4) and three (miR168-y-PKS5; novel-m0474-3p-CNGC10; mdm-miR156a ∼ mdm-miR156o-SPL2/SPL6/SPL12) potential miRNA-mRNA regulatory modules were identified to response to salt stress in the leaves and roots, respectively. The qRT-PCR validation results were consistent with the RNA-seq results. A dual-luciferase assay further confirmed that mdm-miR156b negatively regulated the expression of MdSPL6. This study revealed the key regulatory network of miRNAs in the leaves and roots of ZM-4 to respond to salt stress, and the results laid the foundation for the post-transcriptional molecular regulatory mechanism of salt tolerance in ZM-4.