QTL 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, the contribution of integrated stomatal anatomy and physiology to wheat heat tolerance remains poorly understood, particularly under realistic field conditions and across divers germplasm. This study explored the role of stomatal anatomical and physiological traits in shaping wheat responses to heat stress. Across two years of multi-environment field trials encompassing 200 genotypes in season 1 and 50 genotypes in season 2, we examined stomatal conductance (g), anatomical traits including stomatal size and density, and the stomatal conductance operating efficiency (gse) across leaf surfaces, along with grain yield. Timely and delayed sowing treatments were used to expose key developmental stages (anthesis) to contrasting temperature regimes. Early sowing supported higher gs and gse, while delayed sowing impaired stomatal function despite similar theoretical anatomical capacity (gsmax), revealing a decoupling of structural potential and physiological performance under stress. The adaxial surface consistently exhibited higher gs, stomatal density, and gsmax than the abaxial surface, highlighting its dominant role in leaf gas exchange. Later sowing induced plastic shifts in anatomy, including smaller, denser stomata, particularly on the adaxial surface, suggesting an adaptive response to thermal stress. Significant genotypic variation was observed for gs, gse, gsmax, and stomatal anatomical traits, with moderate heritability indicating genetic control. 125 putative QTL were identified for multiple stomatal traits across environments, including several stable loci on chromosomes 2B and 5B, and numerous closely clustered QTL for anatomical traits on chromosome 7B, highlighting key genomic regions underlying stomatal anatomy. In contrast, QTL for gs and gse were fewer and season-specific, highlighting the environmentally sensitive nature of physiological stomatal regulation. 42 of the QTL identified were consistent with previously reported QTL for stomatal traits in wheat. Together, these findings elevate the role of stomatal traits, supporting an integrated breeding strategy that combines selection for favourable stomatal anatomy with efficiency physiological regulation. Incorporating traits like gse into selection frameworks may enhance yield stability and resilience in heat-prone environments, advancing the development of climate-resilient wheat ideotypes. ScopeThis manuscript examines how stomatal anatomical and physiological traits integrate to shape wheat responses to heat stress under field conditions, addressing a central challenge in understanding the roles of stomata in a warming climate. Using multi-environment field trials across two growing seasons and encompassing 200 wheat genotypes, we quantify stomatal conductance (gs), anatomical traits, and stomatal operating efficiency (gse), across adaxial and abaxial leaf surfaces, and investigate whether these are under genetic control. The study provides mechanistic insight into the dynamic regulation of stomatal function by demonstrating a decoupling between anatomical capacity and physiological performance under heat stress, alongside plastic shifts in stomatal traits across sowing times. The identification of 125 candidate QTLs, including numerous stable and co-localised QTL across seasons, supports their incorporation into breeding programs aimed at enhancing resilience and yield stability. This work aligns with the Research Topic by elucidating mechanistic underpinnings of stomatal conductance regulation, bridging stomatal biology with applied crop improvement strategies. This work is critical to improving understanding of plant water relations and carbon uptake under future climate scenarios to ensure food security in a changing climate.