Integrated Assessment of Physiological, Molecular and Ultrastructural Responses to Heat Stress in Wheat

Heat stress severely constrains wheat productivity, yet the mechanisms underlying thermotolerance remain incompletely understood. In this study, we integrated physiological, biochemical, molecular, and ultrastructural analyses to characterize heat-stress responses in four bread wheat (Triticum aestivum L.) genotypes contrasting in heat tolerance. Membrane thermotolerance was assessed using electrolyte leakage and membrane damage rate under increasing temperature stress, while antioxidant defense was evaluated by measuring the activities of superoxide dismutase, catalase, guaiacol peroxidase, and benzidine peroxidase. Gene expression responses were analyzed by qRT-PCR for DREB, HSP16.9, and compartment-specific SOD isoforms, while HSP16.9 accumulation was validated at the protein level by Western blotting. Heat stress induced progressive membrane destabilization in all genotypes. However, the tolerant genotypes Murov 2 and Zirva 85 maintained greater membrane stability than the sensitive genotypes Aran and Gyzyl bugda. This physiological advantage was accompanied by stronger activation of antioxidant defenses, enhanced induction of DREB and HSP16.9, and a more coordinated expression pattern of FeSOD and MnSOD, indicating integrated redox regulation across chloroplastic and mitochondrial compartments. HSP16.9 protein was undetectable under control conditions but accumulated after heat treatment, confirming its stress-inducible nature and supporting its role in heat-responsive proteostasis. Correlation analysis revealed a coordinated response module linking DREB, HSP16.9, MnSOD, total SOD, BPX, CAT, and GPX. Microscopy further showed that Murov 2 preserved chloroplast, mitochondrial, and mesophyll organization more effectively than Aran under heat stress. Collectively, these findings demonstrate that wheat thermotolerance is governed by the coordinated protection of membranes, redox homeostasis, molecular chaperone systems, and organelle structure, providing potential physiological and molecular targets for breeding heat-resilient wheat cultivars.