The physiological importance of developmental mechanisms that enforce proper stomatal spacing in Arabidopsis thaliana

• Genetic and cell biological mechanisms that regulate stomatal development are necessary to generate an appropriate number of stomata and enforce a minimum spacing of one epidermal cell between stomata. The ability to manipulate these processes in a model plant system allows us to investigate the physiological importance of stomatal patterning and changes in density, therein testing underlying theories about stomatal biology. • Twelve Arabidopsis thaliana genotypes that have varied stomatal characteristics as a result of mutations or transgenes were analyzed in this study. Stomatal traits were used to categorize the genotypes and predict maximum stomatal conductance to water vapor (Anatomical g(smax)) for individuals. Leaf-level gas-exchange measurements determined Diffusive g(smax), net carbon assimilation (A), water-use efficiency (WUE), and stomatal responses to increasing CO₂ concentration. Genotypes with proper spacing (< 5% of stomata in clusters) achieved Diffusive g(smax) values comparable to Anatomical g(smax) across a 10-fold increase in stomatal density, while lines with patterning defects (> 19% clustering) did not. • Genotypes with clustering also had reduced A and impaired stomatal responses, while WUE was generally unaffected by patterning. • Consequently, optimal function per stoma was dependent on maintaining one epidermal cell spacing and the physiological parameters controlled by stomata were strongly correlated with Anatomical g(smax).