During photosynthesis, CO₂ moves from the atmosphere (C_a) surrounding the leaf to the sub‐stomatal internal cavities (C_i) through stomata, and from there to the site of carboxylation inside the chloroplast stroma (C_c) through the leaf mesophyll. The latter CO₂ diffusion component is called mesophyll conductance (g_m), and can be divided in at least three components, that is, conductance through intercellular air spaces (g_ias), through cell wall (g_w) and through the liquid phase inside cells (g_liq). A large body of evidence has accumulated in the past two decades indicating that g_m is sufficiently small as to significantly decrease C_c relative to C_i, therefore limiting photosynthesis. Moreover, g_m is not constant, and it changes among species and in response to environmental factors. In addition, there is now evidence that g_liq and, in some cases, g_w, are the main determinants of g_m. Mesophyll conductance is very dynamic, changing in response to environmental variables as rapid or even faster than stomatal conductance (i.e. within seconds to minutes). A revision of current knowledge on g_m is presented. Firstly, a historical perspective is given, highlighting the founding works and methods, followed by a re‐examination of the range of variation of g_m among plant species and functional groups, and a revision of the responses of g_m to different external (biotic and abiotic) and internal (developmental, structural and metabolic) factors. The possible physiological bases for g_m, including aquaporins and carbonic anhydrases, are discussed. Possible ecological implications for variable g_m are indicated, and the errors induced by neglecting g_m when interpreting photosynthesis and carbon isotope discrimination models are highlighted. Finally, a series of research priorities for the near future are proposed.

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.

The effects of short‐term (minutes) variations of CO₂ concentration on mesophyll conductance to CO₂ (g_m) were evaluated in six different C₃ species by simultaneous measurements of gas exchange, chlorophyll fluorescence, online carbon isotope discrimination and a novel curve‐fitting method. Depending on the species, g_m varied from five‐ to ninefold, along the range of sub‐stomatal CO₂ concentrations typically used in photosynthesis CO₂‐response curves (A_N–C_i curves; where A_N is the net photosynthetic flux and C_i is the CO₂ concentrations in the sub‐stomatal cavity), that is, 50 to 1500 µmol CO₂ mol⁻¹ air. Although the pattern was species‐dependent, g_m strongly declined at high C_i, where photosynthesis was not limited by CO₂, but by regeneration of ribulose‐1,5‐bisphosphate or triose phosphate utilization. Moreover, these changes on g_m were found to be totally independent of the velocity and direction of the C_i changes. The response of g_m to C_i resembled that of stomatal conductance (g_s), but kinetic experiments suggested that the response of g_m was actually faster than that of g_s. Transgenic tobacco plants differing in the amounts of aquaporin NtAQP1 showed different slopes of the g_m–C_i response, suggesting a possible role for aquaporins in mediating CO₂ responsiveness of g_m. The importance of these findings is discussed in terms of their effects on parameterization of A_N–C_i curves.

• Whether photosynthesis is limited during water stress and recovery because of diffusive or biochemical factors is still open to debate, and apparent contradictions appear when various studies on species with different growth forms are compared. • Ten Mediterranean species, representing different growth forms, were subjected to different levels of water stress, the most severe followed by rewatering. A quantitative limitation analysis was applied to estimate the effects of water stress on stomatal (S_L), mesophyll conductance (MC_L) and biochemical limitations (B_L). • Results confirmed a general pattern of photosynthetic response to water stress among C₃ plants when stomatal conductance (g_s) is used as a reference parameter. As g_s values decreased from a maximum to approx. 0.05 mol H₂O m⁻² s⁻¹, the total photosynthetic limitation rose from 0 to approx. 70%, and this was caused by a progressive increase of both S_L and MC_L limitations, while B_L remained negligible. When lower values of g_s were achieved (total photosynthetic limitation increased from 70 to 100%), the contribution of S_L declined, while MC_L still increased and B_L contributed significantly (20–50%) to the total limitation. • Photosynthetic recovery of severely stressed plants after rewatering showed a dominant role of MC_L, irrespective of the degree of photosynthesis recovery.

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.