Brassinolide‐mediated physiological and biochemical adaptation in rye ( Secale cereale L.) under aluminum and freeze–thaw stress

BACKGROUND Environmental stresses such as extreme temperatures, salinity, drought, metal toxicity and freeze–thaw (FT) cycles severely limit crop productivity. The increasing frequency of FT events combined with aluminum (Al) toxicity, exacerbated by global warming, poses a serious threat to seedling survival, especially in early spring. Brassinolide (BL), a phytohormone, enhances plant resilience under abiotic stress, yet its role under combined FT and Al stress in rye ( Secale cereale L.) remains unexplored. RESULTS Al stress increased malondialdehyde content, indicating membrane lipid peroxidation, at the same time as reducing chlorophyll content, photosynthesis and transpiration. Combined Al + FT stress elevated intercellular CO₂ concentration and soluble sugars but lowered stomatal conductance and water‐use efficiency, revealing biochemical limitations to photosynthesis. BL alone restored stomatal conductance, lowered intercellular CO₂ and enhanced water‐use efficiency, improving CO₂ fixation. Al stress produced the highest H₂O₂ accumulation under both non‐FT and FT conditions alongside compensatory increases in ascorbate peroxidase (APX) activity, reflecting an overwhelmed antioxidant response; co‐application of BL with Al reduced H₂O₂ and moderated this APX elevation relative to Al alone. BL alone increased glutathione content above all treatments, although this benefit was partly constrained when combined with Al under FT. BL also improved shoot relative water content, photosynthetic efficiency and soluble sugar regulation, promoting osmotic adjustment and cold tolerance. CONCLUSION BL enhances rye tolerance to combined FT and Al stress by restoring photosynthetic function and modulating antioxidant responses, offering a promising approach to safeguard crop productivity in regions prone to such abiotic challenges.