Hydrogen Sulfide Improves the Cold Stress Resistance through the CsARF5-CsDREB3 Module in Cucumber

As an important gas signaling molecule, hydrogen sulfide (H₂S) plays a crucial role in regulating cold tolerance. H₂S cooperates with phytohormones such as abscisic acid, ethylene, and salicylic acid to regulate the plant stress response. However, the synergistic regulation of H₂S and auxin in the plant response to cold stress has not been reported. This study showed that sodium hydrosulfide (NaHS, an H₂S donor) treatment enhanced the cold stress tolerance of cucumber seedlings and increased the level of auxin. CsARF5, a cucumber auxin response factor (ARF) gene, was isolated, and its role in regulating H₂S-mediated cold stress tolerance was described. Transgenic cucumber leaves overexpressing CsARF5 were obtained. Physiological analysis indicated that overexpression of CsARF5 enhanced the cold stress tolerance of cucumber and the regulation of the cold stress response by CsARF5 depends on H₂S. In addition, molecular assays showed that CsARF5 modulated cold stress response by directly activating the expression of the dehydration-responsive element-binding (DREB)/C-repeat binding factor (CBF) gene CsDREB3, which was identified as a positive regulator of cold stress. Taken together, the above results suggest that CsARF5 plays an important role in H₂S-mediated cold stress in cucumber. These results shed light on the molecular mechanism by which H₂S regulates cold stress response by mediating auxin signaling; this will provide insights for further studies on the molecular mechanism by which H₂S regulates cold stress. The aim of this study was to explore the molecular mechanism of H₂S regulating cold tolerance of cucumber seedlings and provide a theoretical basis for the further study of cucumber cultivation and environmental adaptability technology in winter.