Mesophyll diffusion conductance to CO(2) is a key photosynthetic trait that has been studied intensively in the past years. The intention of the present review is to update knowledge of g(m), and highlight the important unknown and controversial aspects that require future work. The photosynthetic limitation imposed by mesophyll conductance is large, and under certain conditions can be the most significant photosynthetic limitation. New evidence shows that anatomical traits, such as cell wall thickness and chloroplast distribution are amongst the stronger determinants of mesophyll conductance, although rapid variations in response to environmental changes might be regulated by other factors such as aquaporin conductance. Gaps in knowledge that should be research priorities for the near future include: how different is mesophyll conductance among phylogenetically distant groups and how has it evolved? Can mesophyll conductance be uncoupled from regulation of the water path? What are the main drivers of mesophyll conductance? The need for mechanistic and phenomenological models of mesophyll conductance and its incorporation in process-based photosynthesis models is also highlighted.

Foliage photosynthetic and structural traits were studied in 15 species with a wide range of foliage anatomies to gain insight into the importance of key anatomical traits in the limitation of diffusion of CO₂ from substomatal cavities to chloroplasts. The relative importance of different anatomical traits in constraining CO₂ diffusion was evaluated using a quantitative model. Mesophyll conductance (g_m) was most strongly correlated with chloroplast exposed surface to leaf area ratio (S_c/S) and cell wall thickness (T_cw), but, depending on foliage structure, the overall importance of g_m in constraining photosynthesis and the importance of different anatomical traits in the restriction of CO₂ diffusion varied. In species with mesophytic leaves, membrane permeabilities and cytosol and stromal conductance dominated the variation in g_m. However, in species with sclerophytic leaves, g_m was mostly limited by T_cw. These results demonstrate the major role of anatomy in constraining mesophyll diffusion conductance and, consequently, in determining the variability in photosynthetic capacity among species.

Leaf age‐dependent changes in structure, nitrogen content, internal mesophyll diffusion conductance (g_m), the capacity for photosynthetic electron transport (J_max) and the maximum carboxylase activity of Rubisco (V_cmax) were investigated in mature non‐senescent leaves of Laurus nobilis L., Olea europea L. and Quercus ilex L. to test the hypothesis that the relative significance of biochemical and diffusion limitations of photosynthesis changes with leaf age. The leaf life‐span was up to 3 years in L. nobilis and O. europea and 6 years in Q. ilex. Increases in leaf age resulted in enhanced leaf dry mass per unit area (M_A), larger leaf dry to fresh mass ratio, and lower nitrogen contents per dry mass (N_M) in all species, and lower nitrogen contents per area (N_A) in L. nobilis and Q. ilex. Older leaves had lower g_m, J_max and V_cmax. Due to the age‐dependent increase in M_A, mass‐based g_m, J_max and V_cmax declined more strongly (7‐ to 10‐fold) with age than area‐based (5‐ to 7‐fold) characteristics. Diffusion conductance was positively associated with foliage photosynthetic potentials. However, this correlation was curvilinear, leading to lower ratio of chloroplastic to internal CO₂ concentration (C_c/C_i) and larger drawdown of CO₂ from leaf internal air space to chloroplasts (Δ_C) in older leaves with lower g_m. Overall the age‐dependent decreases in photosynthetic potentials were associated with decreases in N_M and in the fraction of N in photosynthetic proteins, whereas decreases in g_m were associated with increases in M_A and the fraction of cell walls. These age‐dependent modifications altered the functional scaling of foliage photosynthetic potentials with M_A, N_M, and N_A. The species primarily differed in the rate of age‐dependent modifications in foliage structural and functional characteristics, but also in the degree of age‐dependent changes in various variables. Stomatal openness was weakly associated with leaf age, but due to species differences in stomatal openness, the distribution of total diffusion limitation between stomata and mesophyll varied among species. These data collectively demonstrate that in Mediterranean evergreens, structural limitations of photosynthesis strongly interact with biochemical limitations. Age‐dependent changes in g_m and photosynthetic capacities do not occur in a co‐ordinated manner in these species such that mesophyll diffusion constraints curb photosynthesis more in older than in younger leaves.

In sclerophylls, photosynthesis is particularly strongly limited by mesophyll diffusion resistance from substomatal cavities to chloroplasts (r_m), but the controls on diffusion limits by integral leaf variables such as leaf thickness, density, and dry mass per unit area and by the individual steps along the diffusion pathway are imperfectly understood. To gain insight into the determinants of r_m in leaves with varying structure, the full CO₂ physical diffusion pathway was analysed in 32 Australian species sampled from sites contrasting in soil nutrients and rainfall, and having leaf structures from mesophytic to strongly sclerophyllous. r_m was estimated based on combined measurements of gas exchange and chlorophyll fluorescence. In addition, r_m was modelled on the basis of detailed anatomical measurements to separate the importance of different serial resistances affecting CO₂ diffusion into chloroplasts. The strongest sources of variation in r_m were S_c/S, the exposed surface area of chloroplasts per unit leaf area, and mesophyll cell wall thickness, t_cw. The strong correlation of r_m with t_cw could not be explained by cell wall thickness alone, and most likely arose from a further effect of cell wall porosity. The CO₂ drawdown from intercellular spaces to chloroplasts was positively correlated with t_cw, suggesting enhanced diffusional limitations in leaves with thicker cell walls. Leaf thickness and density were poorly correlated with S_c/S, indicating that widely varying combinations of leaf anatomical traits occur at given values of leaf integrated traits, and suggesting that detailed anatomical studies are needed to predict r_m for any given species.