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.

Grafting is widely applied to enhance the tolerance of some vegetables to biotic and abiotic stress. Salicylic acid (SA) is known to be involved in grafting-induced chilling tolerance in cucumber. Here, we revealed that grafting with pumpkin (Cucurbita moschata, Cm) as a rootstock improved chilling tolerance and increased the accumulation of SA, abscisic acid (ABA) and hydrogen peroxide (H₂O₂) in grafted cucumber (Cucumis sativus/Cucurbita moschata, Cs/Cm) leaves. Exogenous SA improved the chilling tolerance and increased the accumulation of ABA and H₂O₂ and the mRNA abundances of CBF1, COR47, NCED, and RBOH1. However, 2-aminoindan-2-phosphonic acid (AIP) and L-a-aminooxy-b-phenylpropionic acid (AOPP) (biosynthesis inhibitors of SA) reduced grafting-induced chilling tolerance, as well as the synthesis of ABA and H₂O₂, in cucumber leaves. ABA significantly increased endogenous H₂O₂ production and the resistance to chilling stress, as proven by the lower electrolyte leakage (EL) and chilling injury index (CI). However, application of the ABA biosynthesis inhibitors sodium tungstate (Na₂WO₄) and fluridone (Flu) abolished grafting or SA-induced H₂O₂ accumulation and chilling tolerance. SA-induced plant response to chilling stress was also eliminated by N,N′-dimethylthiourea (DMTU, an H₂O₂ scavenger). In addition, ABA-induced chilling tolerance was attenuated by DMTU and diphenyleneiodonium (DPI, an H₂O₂ inhibitor) chloride, but AIP and AOPP had little effect on the ABA-induced mitigation of chilling stress. Na₂WO₄ and Flu diminished grafting- or SA-induced H₂O₂ biosynthesis, but DMTU and DPI did not affect ABA production induced by SA under chilling stress. These results suggest that SA participated in grafting-induced chilling tolerance by stimulating the biosynthesis of ABA and H₂O₂. H₂O₂, as a downstream signaler of ABA, mediates SA-induced chilling tolerance in grafted cucumber plants.

Salicylic acid (SA) plays a role in the regulation of grafting-induced cold tolerance. However, the molecular mechanism behind it is still unknown. Here, we established that the phenylalanine ammonia-lyase (PAL) pathway-dependent elevate in SA content in grafted cucumber leaves was not only synthesized in the leaves but also transported from the roots under chilling stress. RNAi-CsPAL with low SA content as rootstock reduced SA accumulation in grafted seedling leaves while decreasing rootstock-induced cold tolerance, as evidenced by higher electrolyte leakage (EL), hydrogen peroxide (H₂O₂), and superoxide anion (O₂^·−) contents and lower expression of cold-responsive genes (CsICE1, CsDREB1A, CsDREB1B, and CsCOR47), whereas OE-CsPAL with high SA content as rootstock improved the cold tolerance of grafted plants in comparison with the wild type (WT). In addition, CsNPR1 was significantly upregulated in grafted cucumber under chilling stress, with exogenous and endogenous overexpressed SA inducing its transcriptional expression and protein stability, which exhibited higher expression in grafted plants than in self-root plants. While CsNPR1-overexpression (OE-CsNPR1) seedlings as scions were more tolerant to chilling stress than WT seedlings, CsNPR1-suppression (Anti-CsNPR1) seedlings as scions were more vulnerable to chilling stress. Notably, CsNPR1–CsICE1 interactions alleviated ROS accumulation and activated the expression of CsDREB1A, CsDREB1B, CsCOR47, CsCOR15, CsCOR413, and CsKIN1 to enhance SA-mediated chilling tolerance in grafted cucumber. Overall, our findings reveal that SA enhances chilling tolerance in grafted cucumbers via the model of the CsNPR1–CsICE1 transcriptional regulatory cascade.