UV radiation, particularly UVB, has a large photobiological influence on plants by altering both their biological and biochemical activity. While excessive UVC and UVB exposure can lead to reduced growth, unusual metabolic activity, and impaired photosynthesis, moderate levels of UV act as an environmental signal that plays a crucial role in shaping the biosynthesis and storage of secondary metabolites in plants. This research aimed to explore how exogenous melatonin and ascorbic acid influence the Trigonella foenum-graecum L. response to UVA and UVB radiation in terms of growth, physiological and biochemical characteristics, chlorophyll fluorescence, and diosgenin content. A split-plot experimental design with three replicates was implemented, where UV radiation was applied at four levels (control, UVA, UVB, and UVA + UVB) as the main factor, and stress-mitigating treatments (control, 1 mM ascorbic acid, 100 μM melatonin, and their combination) were treated as the secondary factor. The results indicated that UVB radiation decreased biomass and variable chlorophyll fluorescence, while elevating minimal fluorescence, malondialdehyde levels, electrolyte leakage, and the presence of enzymatic) ascorbate peroxidase, catalase, superoxide dismutase) and non-enzymatic (phenolics, flavonoids) antioxidants. UVA radiation had no significant impact on malondialdehyde levels, electrolyte leakage, enzymatic and nonenzymatic antioxidants, or chlorophyll fluorescence; however, it did enhance biomass and yield. Application of melatonin and ascorbic acid increased T. foenum-graecum yield and variable chlorophyll fluorescence. Their combined application reduced electrolyte leakage, minimal fluorescence, and malondialdehyde levels, particularly under UVB conditions. The study underscored the positive impact of exogenous ascorbic acid and melatonin on the total phenolic and flavonoid content of T. foenum-graecum under UVB radiation, despite decreasing the activity of ascorbate peroxidase, catalase, and superoxide dismutase enzymes. Melatonin, ascorbic acid, UVA, and UVB treatments upregulated the expression levels of genes such as SEP, SQS, CAS SSR, SMT, and C7, leading to increased diosgenin biosynthesis. The results indicated that the application of melatonin, ascorbic acid, and UV light may represent effective strategies for enhancing diosgenin synthesis in Trigonella foenum-graecum by upregulating the expression of genes involved in its biosynthetic pathway. However, further experimental validation is necessary to confirm these findings.
