Increased adaxial stomatal density is associated with greater mesophyll surface area exposed to intercellular air spaces and mesophyll conductance in diverse C₄ grasses

• Mesophyll conductance (g m) is the diffusion of CO₂ from intercellular air spaces (IAS) to the first site of carboxylation in the mesophyll cells. In C₃ species, g m is influenced by diverse leaf structural and anatomical traits; however, little is known about traits affecting g m in C₄ species. • To address this knowledge gap, we used online oxygen isotope discrimination measurements to estimate g m and microscopy techniques to measure leaf structural and anatomical traits potentially related to g m in 18 C₄ grasses. • In this study, g m scaled positively with photosynthesis and intrinsic water-use efficiency (TEi), but not with stomatal conductance. Also, g m was not determined by a single trait but was positively correlated with adaxial stomatal densities (SDada), stomatal ratio (SR), mesophyll surface area exposed to IAS (S mes) and leaf thickness. However, g m was not related to abaxial stomatal densities (SDaba) and mesophyll cell wall thickness (T CW). • Our study suggests that greater SDada and SR increased g m by increasing S mes and creating additional parallel pathways for CO₂ diffusion inside mesophyll cells. Thus, SDada, SR and S mes are important determinants of C₄-g m and could be the target traits selected or modified for achieving greater g m and TEi in C₄ species.