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. This study 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 injury was assessed by membrane damage rate, lipid peroxidation by malondialdehyde accumulation, antioxidant defense by SOD, CAT, GPX, and BPX activities, and stress-responsive regulation by qRT-PCR analysis of DREB, HSP16.9, and SOD isoforms. HSP16.9 protein accumulation was further evaluated by Western blotting. Heat stress increased membrane damage and MDA accumulation in all genotypes; however, tolerant Murov 2 and Zirva 85 showed lower oxidative membrane injury than sensitive Aran and Gyzyl bugda. Thermotolerance was associated with stronger antioxidant activation, enhanced DREB and HSP16.9 induction, and more coordinated FeSOD and MnSOD expression. The HSP16.9 protein accumulated after heat treatment, supporting its role as a stress-responsive molecular chaperone. Separate correlation analyses of tolerant and sensitive genotypes revealed stronger coordination among transcriptional, chaperone-related, and antioxidant markers in tolerant genotypes, whereas sensitive genotypes showed a more fragmented response. Microscopy further showed better preservation of chloroplast, mitochondrial, and mesophyll organization in the tolerant genotype relative to the sensitive counterpart, indicating integrated cellular protection. Together, these responses define a coordinated tolerance strategy that may guide the selection of heat-resilient wheat genotypes.