QTLs for heat-induced stomatal anatomy underpin gas exchange variation in field-grown wheat

Stomata are central to leaf gas exchange, governing carbon uptake, water loss, and, ultimately, crop performance. However, how stomatal anatomy and physiology integrate to determine wheat heat tolerance under field conditions remains poorly understood. Here, we examined the contribution of stomatal anatomical and physiological traits in shaping wheat responses to heat stress. Across 2 years of multi-environment field trials encompassing 200 genotypes in season 1 and 50 genotypes in season 2, we examined stomatal conductance (g_s ), anatomical traits including stomatal size and density, and stomatal conductance operating efficiency (g_se ) across leaf surfaces, along with grain yield. Timely and delayed sowing treatments were used to expose anthesis to contrasting temperature regimes. Early sowing supported higher g_s and g_se , whereas delayed sowing impaired stomatal function despite similar theoretical anatomical capacity (g_smax ), revealing a decoupling of structural potential and physiological performance under stress. The adaxial surface consistently exhibited higher g_s , stomatal density, and g_smax than the abaxial surface, highlighting its dominant role in leaf gas exchange. Delayed sowing induced plastic anatomical shifts, including smaller, denser stomata, particularly on the adaxial surface. Significant genotypic variation and moderate heritability were observed for stomatal anatomical traits. We identified 62 putative quantitative trait loci (QTLs) across environments for multiple stomatal traits, including recurring and co-localised loci detected across seasons and times of sowing on chromosome 6B. Pleiotropic QTLs on chromosomes 1A and 2A highlight promising genomic regions. The majority of QTLs (36) were detected for the adaxial surface, positioning it as the dominant surface driving stomatal anatomical variation. A total of 21 QTLs were consistent with chromosomal regions previously reported for stomatal anatomical traits in wheat, particularly on chromosome 7A. In contrast, no QTLs were detected for stomatal physiological traits, indicating limited potential for indirect selection on these traits relative to more stable anatomical traits. Collectively, these findings demonstrate that heat stress uncouples stomatal anatomy from physiological performance in wheat and provides a comprehensive field-based characterisation of stomatal trait integration and its genetic architecture under thermal stress. This work establishes a robust foundation for informing future physiological and genetic studies of wheat heat tolerance, advancing the development of climate-resilient wheat ideotypes.