Heat tolerance mechanisms in bread wheat: Insights from flag leaves and spike tissues

• Heat stress during ear peep reduces wheat grain numbers. • Prolonged heat stress during filling reduces grain weight. • Heat-tolerant genotypes keep photosynthesis, pollen viability, and redox steady. • Flag leaf heat tolerance relies on photosynthesis and redox proteins. • Spike fertility relies on carbohydrate metabolism and redox proteins. Heat stress significantly limits global bread wheat ( Triticum aestivum L.) productivity. This study investigated the mechanisms underlying heat tolerance by comparing physiological responses, yield components, and proteomic profiles in flag leaves and spike tissues of two heat-tolerant (RAJ3765-T, HD2932-T) and two susceptible (HD2329-S, HD2733-S) wheat genotypes under short-term (32 °C for 5 days) and long-term (32 °C until maturity) heat stress at ear peep (Zadoks’ stage 51). Short-term heat stress significantly reduced grain yield (6.16–42.78 %), primarily by decreasing grain number per plant (27.79–57.73 %), while long-term heat stress reduced thousand grain weight (10.55–27.33 %). Tolerant genotypes (RAJ3765-T, HD2932-T) maintained higher grain yields by preserving photosynthesis, membrane stability ( r = 0.88, p ≤ 0.05), pollen viability ( r = 0.74, p ≤ 0.05) and chlorophyll content ( r = 0.82, p ≤ 0.05) while preventing excessive reactive oxygen species (ROS) accumulation ( r =–0.83, p ≤ 0.05). These genotypes also sustained higher above-ground biomass and harvest index under both the heat conditions, whereas grain protein content increased across all genotypes (8.91–15.47 %). Proteomic analysis identified 31 and 60 differentially abundant proteins in flag leaves and spikes, respectively. Key proteins associated with heat tolerance in flag leaves were involved in photosynthesis, amino acid metabolism, and chromatin organization, while those linked to susceptibility were related to carbohydrate metabolism, methylation, chromatin and cell wall organization, and solute transport. Disrupted redox homeostasis was a typical heat susceptibility response in both spikes and flag leaves. Co-expression analysis revealed protein networks associated with redox homeostasis and chlorophyll biosynthesis, which significantly correlated with grain yield, offering novel biomarkers to breeding heat-tolerant wheat varieties.