Hydrogen peroxide mediates melatonin-induced chilling tolerance in cucumber seedlings

Abstract Melatonin (MT) and hydrogen peroxide (H₂O₂) are important endogenous signaling molecules that play multifaceted roles in plant responses to abiotic stress. However, the interactive mechanism by which MT and H₂O₂ regulate chilling tolerance remains unclear. Here we found that MT exhibited a positive regulatory effect on the chilling tolerance of cucumbers, with an optimum concentration of 100 µM. MT markedly enhanced RBOH1 mRNA expression, activity and endogenous H₂O₂ accumulation in cucumber seedlings. However, 1.0 mM H₂O₂ had no significant effect on mRNA levels of TDC and ASMT, the key genes for MT synthesis, and endogenous MT content. Both MT and H₂O₂ significantly decreased malondialdehyde (MDA), electrolyte leakage (EL) and chilling injury index (CI) by activating the antioxidant system, thereby alleviating chilling damage in cucumber seedlings. MT and H₂O₂ improved photosynthetic carbon assimilation, which was primarily attributed to an increase in activity, mRNA expression, and protein levels of RuBPCase and RCA. Meanwhile, MT and H₂O₂ induced the photoprotection for both PSII and PSI by enhancing the QA’s electron transport capacity and elevating protein levels of the photosystems. Moreover, MT and H₂O₂ significantly upregulated the expression of cold response genes. MT-induced chilling tolerance was attenuated by N’, N’-dimethylthiourea (DMTU), a H₂O₂specific scavenger. Whereas, the MT synthesis inhibitor (p-chlorophenylalanine, p-CPA) did not influence H₂O₂-induced chilling tolerance. The positive regulation of MT on the antioxidant system, photosynthesis and cold response gene levels were significantly attenuated in RBOH1-RNAi plants compared with WT plants. These findings suggest that H₂O₂ may functions as a downstream signaling molecule in MT-induced chilling tolerance in cucumber plants.