Grain number is a critical yield component in wheat, and its stability is increasingly threatened by heat stress due to global warming. Short-term heat events, lasting hours or days, frequently coincide with key developmental stages, compromising floret fertility. While the impacts of heat stress on wheat grain settings have been extensively studied at plant and canopy scales, floret-level responses remain poorly understood.

Wheat's asynchronous development among spikes, spikelets, and florets suggests positional variability in heat stress susceptibility, as pollen response to heat is highly stage-specific. To address this, we conducted four experiments to investigate floret fertility responses to 5-day heat stress at 36°C during different developmental stages. A novel quantitative model was developed and validated to characterize and predict developmental gradients among florets within a plant. Additionally, we introduced the "cohort concept," grouping florets by shared developmental stages, to better elucidate their collective responses to stress.

Our findings reveal that floret developmental gradients are key drivers of grain number sensitivity to heat stress. By accounting for the variability in floret-level susceptibility, this study highlights the importance of incorporating floret-scale developmental dynamics into heat stress assessments for wheat and other cereals and also provides new insights for breeding new genotypes with enhanced heat resilience in wheat.