2-Hydroxymelatonin mitigates cold stress in Trifolium repens by modulating gene expression and metabolic pathways

Low temperature is one of the major abiotic stress that broadly constrains plant growth, survival, and overwintering capacity. It highlights an urgent need for effective regulatory strategies or stress-protective agents. Here, we demonstrate that 2-hydroxymelatonin (2-OHM), a major metabolite of melatonin (Mel), plays a key role in enhancing cold tolerance in Trifolium repens. Exogenous application of 2-OHM markedly improved plant water status, biomass accumulation, and chlorophyll retention during prolonged chilling, while significantly reduced reactive oxygen species accumulation and membrane lipid peroxidation. Compared to the protective effects of Mel towards cold, 2-OHM consistently produced stronger and sustained physiological benefits. 2-OHM enhanced osmotic adjustment by increasing the accumulation of soluble sugars, soluble proteins, and proline. Furthermore, 2-OHM reprogrammed primary metabolic pathways, particularly amino acid and carbohydrate metabolism associated with cold acclimation, as revealed by integrated transcriptomic and metabolomic analyses. Our results further revealed that TrM2H catalyzes the conversion of Mel to 2-OHM, and its expression was induced by both cold stress and exogenous Mel application. Collectively, 2-OHM is demonstrated to be an emerging agent that improves plant cold tolerance, potentially benefiting for crop production.