Combined drought and heat stress intensifies antioxidant responses and gene expression in Persian walnut (Juglans regia L.).

Persian walnut (Juglans regia L.) frequently encounters simultaneous drought and heat stress in its natural habitat, impacting its physiology and biochemistry. Therefore, understanding the physiological and molecular responses to these combined stresses is essential for improving stress resilience in this species. The present study investigates the effects of drought stress (DS), heat stress (HS), combined drought-heat stress (DH) and control condition (CC) on the antioxidant defenses and gene expression in own-rooted walnut plants (cv. Chandler) under controlled greenhouse conditions. Combined stress significantly increased malondialdehyde and hydrogen peroxide by 2.55- and 2.05-fold, respectively, while reducing membrane stability index by 0.43-fold compared to CC. Total chlorophyll declined by 0.18- and 0.09-fold under DS and HS, respectively, with the greatest reduction (0.37-fold) under DH. Levels of phenylalanine ammonia-lyase, polyphenol oxidase, superoxide dismutase, catalase, and guaiacol peroxidase rose 2.5- to sixfold under DH compared to CC. Similarly, phenolic compounds and antioxidant capacity increased between 1.49- and 3.55-fold relative to CC. Gene expression analyses of NCED6, PPO, and GST revealed 2- to sixfold upregulation under both individual and combined stress conditions compared to control. These results highlight the crucial roles of these genes in activating antioxidant defense pathways and detoxification processes that mitigate oxidative damage in walnut under stress. Following stress relief, walnut seedlings displayed notable recovery in photosynthetic function and antioxidant activity. These findings indicate that combined drought and heat stress exerts a stronger impact than either stress alone, underscoring key adaptive mechanisms that may enhance walnut resilience to climate variability.