Integrated physiological and multi-omics analyses identify a SmWRKY4-SmAPX2 regulatory module associated with cold...

The northward cultivation of Sapindus mukorossi Gaertn. has long been constrained by winter low temperatures, making it important to clarify the mechanisms underlying cold adaptation for germplasm utilization and molecular improvement. In this study, previously screened cold-tolerant (CT) and cold-sensitive (CS) material groups were used to examine the basis of cold adaptation through phenotypic observation, anatomical analysis, physiological and biochemical measurements, transcriptomic and metabolomic profiling, weighted gene co-expression network analysis (WGCNA), functional validation, and transcriptional regulatory assays. Compared with CS, CT maintained higher branch structural stability, showed less cellular damage, and exhibited a stronger capacity to sustain reactive oxygen species (ROS) scavenging and redox homeostasis under low-temperature stress. Integrated multi-omics analyses further showed that these differences were associated with coordinated reprogramming of sugar and amino acid metabolism, together with changes in ascorbate-glutathione (AsA-GSH)-related processes. On this basis, SmAPX2 was selected as a key candidate gene involved in antioxidant-mediated cold tolerance. Functional analyses showed that SmAPX2 increased ascorbate peroxidase (APX) activity and reduced the accumulation of H₂O₂, malondialdehyde (MDA), relative electrolyte conductivity (REC), and O₂·⁻ under low-temperature conditions, indicating that it contributes to cold tolerance by enhancing ROS detoxification and alleviating membrane damage. Further analyses showed that SmWRKY4 bound to the SmAPX2 promoter and enhanced its transcriptional activity, with the proximal W-box contributing substantially to SmWRKY4-mediated promoter activation. These findings suggest that the SmWRKY4-SmAPX2 regulatory module may participate in cold adaptation-associated antioxidant responses in S. mukorossi.