Large‐scale quantification of stomatal patterning in barley leaves overexpressing epidermal patterning factor 1 reveals differential stomatal density between the adaxial and abaxial surfaces and spatial heterogeneity that impact stomatal function

Stomatal density varies spatially over the leaf surface and between abaxial and adaxial leaf surfaces, with distribution greatly influencing plant photosynthesis and water use. However, methodological limitations have prevented quantification of spatial heterogeneity and its consequences for gaseous exchange in monocot crops. Here we introduce a simple and rapid method to image and quantify stomatal patterning over large (18 cm²) leaf areas in situ. We used this approach to assess spatial variation across the adaxial and abaxial surfaces in barley (Hordeum vulgare L.) wild-type (WT) plants and mutants overexpressing the epidermal patterning factor 1 (EPF1). Analysing over a million stomata revealed significantly greater stomatal densities on the adaxial surface and towards the leaf tip in both genotypes. Overexpression of EPF1, however, differentially reduced stomatal densities on the two surfaces, while also increasing spatial variability, particularly on the abaxial surface, compared to WT. The uneven stomatal distribution proved crucial to separate simultaneous gas exchange measurements on the two surfaces, with impacts on both photosynthetic carbon gain and water use efficiency. Knowledge of the relationship between stomatal patterning and gaseous function is critical for the development of future crops with improved performance.