Drought-Induced Antioxidant and Biochemical Responses in Castanea sativa Cultivars: A Mediterranean Case Study

Chestnut (Castanea sativa Mill.) is a key crop in Mediterranean regions increasingly threatened by recurrent drought stress. To investigate cultivar-specific tolerance mechanisms, we evaluated four Portuguese cultivars (Longal, Judia, Martaínha, and ColUTAD®) across four orchards with contrasting water regimes. Physiological (midday stem water potential—Ψwmid, soluble sugars, electrolyte leakage and proline) and biochemical traits (phenolics, flavonoids, catalase, peroxidase, ascorbate peroxidase and ferric reducing antioxidant power) were quantified under a natural drought gradient. Results revealed that environmental factors had a stronger influence than genetic background. Longal showed robust osmotic adjustment with high proline and soluble sugar levels, alongside stable starch reserves; Judia relied on inducible antioxidant activity, particularly peroxidase and ascorbate peroxidase; and Martaínha exhibited intermediate plasticity, whereas ColUTAD® was consistently stress-sensitive, with weaker defences and greater membrane damage. Clustering analysis confirmed that location effects outweighed cultivar differences, separating orchards into conservative strategies (better water balance, higher starch, stronger peroxidase activity) and stress-adaptive strategies (enhanced enzymatic antioxidants). Overall, resilience in chestnut is not determined by a single trait but by a synergistic network of osmotic regulation, membrane protection, and antioxidant activity. Traits such as proline accumulation, starch stability, and inducible enzyme activation emerged as reliable biochemical indicators of tolerance. These findings provide a physiological basis for selecting climate-resilient cultivars and designing site-specific management strategies, thereby supporting the sustainability of chestnut production under Mediterranean climate change scenarios.