Increased adaxial stomatal density is associated with greater mesophyll surface area exposed to intercellular air spaces and mesophyll conductance in diverse C 4 grasses

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