The genus Oryza, which includes rice (Oryza sativa and Oryza glaberrima) and wild relatives, is a useful genus to study leaf properties in order to identify structural features that control CO 2 access to chloroplasts, photosynthesis, water use efficiency, and drought tolerance. Traits, 26 structural and 17 functional, associated with photosynthesis and transpiration were quantified on 24 accessions (representatives of 17 species and eight genomes). Hypotheses of associations within, and between, structure, photosynthesis, and transpiration were tested. Two main clusters of positively interrelated leaf traits were identified: in the first cluster were structural features, leaf thickness (Thick leaf ), mesophyll (M) cell surface area exposed to intercellular air space per unit of leaf surface area (S mes ), and M cell size; a second group included functional traits, net photosynthetic rate, transpiration rate, M conductance to CO 2 diffusion (g m ), stomatal conductance to gas diffusion (g s ), and the g m /g s ratio. While net photosynthetic rate was positively correlated with g m , neither was significantly linked with any individual structural traits. The results suggest that changes in g m depend on covariations of multiple leaf (S mes ) and M cell (including cell wall thickness) structural traits. There was an inverse relationship between Thick leaf and transpiration rate and a significant positive association between Thick leaf and leaf transpiration efficiency. Interestingly, high g m together with high g m /g s and a low S mes /g m ratio (M resistance to CO 2 diffusion per unit of cell surface area exposed to intercellular air space) appear to be ideal for supporting leaf photosynthesis while preserving water; in addition, thick M cell walls may be beneficial for plant drought tolerance.

Summary 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.

Diffusion of CO from the leaf intercellular air space to the site of carboxylation (g ) is a potential trait for increasing net rates of CO assimilation (A ), photosynthetic efficiency, and crop productivity. Leaf anatomy plays a key role in this process; however, there are few investigations into how cell wall properties impact g and A . Online carbon isotope discrimination was used to determine g and A in Oryza sativa wild-type (WT) plants and mutants with disruptions in cell wall mixed-linkage glucan (MLG) production (CslF6 knockouts) under high- and low-light growth conditions. Cell wall thickness (T ), surface area of chloroplast exposed to intercellular air spaces (S ), leaf dry mass per area (LMA), effective porosity, and other leaf anatomical traits were also analyzed. The g of CslF6 mutants decreased by 83% relative to the WT, with c. 28% of the reduction in g explained by S . Although A /LMA and A /Chl partially explained differences in A between genotypes, the change in cell wall properties influenced the diffusivity and availability of CO . The data presented here indicate that the loss of MLG in CslF6 plants had an impact on g and demonstrate the importance of cell wall effective porosity and liquid path length on g .