Melatonin and Brassinolide Enhance Cold Tolerance in Osmanthus fragrans: Insights from Integrated Physiological and Multi-Omics Analyses

Low-temperature stress severely restricts the growth, development, and ornamental value of Osmanthus fragrans Lour. However, the molecular mechanisms by which brassinolide (BR) and melatonin (MT) alleviate low-temperature-induced damage remain unclear. Here, O. fragrans branches were exposed to low-temperature stress (5, 0, −5, −10, −15, and −20 °C for 12 h) and treated with exogenous MT (50, 100, and 200 μM) or BR (0.5, 1, and 2 μM). An integrated approach combining phenotypic observation, physiological measurements, transcriptomics, and metabolomics was employed to elucidate the regulatory mechanisms underlying BR- and MT-mediated cold tolerance. The results showed that low-temperature stress significantly increased electrolyte leakage (EL), malondialdehyde (MDA), and hydrogen peroxide (H2O2) accumulation, while reducing superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities. Compared with the control, BR and MT treatments alleviated leaf chlorosis and wilting, reduced oxidative damage, and enhanced antioxidant enzyme activities. Integrated transcriptome–metabolome analyses demonstrated that BR and MT commonly activated phenylpropanoid and flavonoid biosynthesis, thereby promoting antioxidant metabolite accumulation, while suppressing α-linolenic acid and linoleic acid metabolism associated with stress-induced lipid remodeling. Network-based transcriptomic analyses identified transcription factors, including ARF, EIL, bHLH, and GRAS, as potential regulators of cold-responsive pathways. Furthermore, BR primarily regulated hormone-responsive networks, whereas MT mainly maintained redox homeostasis and metabolic reprogramming. These findings reveal the coordinated regulatory mechanisms underlying BR- and MT-mediated cold tolerance, providing potential targets for improving cold resilience in O. fragrans.