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 (g(m)) is the diffusion of CO2 from intercellular air spaces (IAS) to the first site of carboxylation in the mesophyll cells. In C-3 species, g(m) is influenced by diverse leaf structural and anatomical traits; however, little is known about traits affecting g(m) in C-4 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 C-4 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 C-4-g(m) and could be the target traits selected or modified for achieving greater g(m) and TEi in C-4 species.