miR164c–CUC2 Module Modulates Salinity Stress Tolerance in Soybean by Regulating Abscisic Acid Signalling and Reactive Oxygen Species Scavenging Pathways

Soil salinisation-induced salinity stress severely inhibits soybean growth and development. MicroRNA164 (miR164) modulates plant responses to salinity stress; however, the underlying regulatory mechanism remains unclear. In this study, using a field salinity pond system and CRISPR/Cas9 multiplex editing, we found that gma-miR164c knockout increased salt sensitivity, while overexpression improved tolerance. A comprehensive analysis showed that the NAC transcription factor family member CUP-SHAPED COTYLEDON 2 (GmCUC2) is a target of gma-miR164c. GmCUC2 overexpression compromised plant tolerance to salinity stress. gma-miR164c mutation downregulated the expression of genes involved in the abscisic acid (ABA) signalling pathway and reactive oxygen species (ROS) scavenging system. Co-expression analysis indicated that GmCUC2 expression negatively correlated with the expression of these genes. Dual-luciferase assays showed that two regulators of ABA signalling, namely, C2-DOMAIN ABA-RELATED PROTEIN 1 (GmCAR1) and C2-DOMAIN ABA-RELATED PROTEIN 2 (GmCAR2) are targets of GmCUC2; in fact, their overexpression improves salinity stress tolerance. Additionally, GmCUC2 repressed GmCAR1 and GmCAR2 expression, whereas gma-miR164c mitigated this inhibitory effect. These findings show that gma-miR164c suppresses its target gene GmCUC2, which is a negative regulator of ABA signalling and ROS scavenging, thereby enhancing soybean salinity tolerance. This study expands relevant mechanistic insights and provides genetic resources for breeding salt-tolerant varieties.