Co-Regulation of PySLD5 by miRNA-lncRNA Enhances Cold Tolerance in Poplar Under Overwintering Freeze-Thaw Stress

Freeze-thaw injury is a major cause of winter mortality in woody plants; however, the molecular mechanisms linking freeze-thaw stress to DNA damage and repair remain poorly defined. Here, we investigated the physiological thresholds of freeze-thaw injury in poplar and identified key regulatory components that enhance cold tolerance through improved DNA damage repair. Field temperature monitoring and differential scanning calorimetry revealed an effective freeze-thaw threshold of approximately 12°C, beyond which cumulative intracellular damage occurs despite the absence of extreme low temperatures. Integrated lncRNA, miRNA and mRNA sequencing demonstrated coordinated regulation of a DNA replication gene, PySLD5, by two long non-coding RNAs (MSTRG.19225.8 and MSTRG.19233.11) and the microRNA ptc-miR6476a. Functional assays, including pull-down, dual-luciferase and structural modelling, validated direct interactions among these RNAs and PySLD5. Overexpression of PySLD5 conferred enhanced cold tolerance, reduced electrolyte leakage and lower DNA fragmentation after freeze-thaw stress, whereas knockout lines showed severe cold sensitivity, disease susceptibility and reduced survival. Comet assays confirmed that repeated freeze-thaw cycles caused cumulative DNA damage. Together, these findings support a DNA damage accumulation model in which coordinated RNA regulation of PySLD5 promotes DNA repair, stabilizes replication forks and enhances overwintering survival.