Isotopic disequilibrium of the retro-diffusive CO2 efflux affects photosynthetic C18OO discrimination and the estimation of mesophyll conductance

Photosynthetic C¹⁸OO fractionation (Δ¹⁸O) has been used to estimate mesophyll conductance (g_m) in C₃ and C₄ species. However, this requires knowledge of the degree of isotopic equilibration (θ_r) between leaf water and the CO₂ molecules penetrating the leaf interior and re-escaping without being assimilated (F_retro). Here, we re-examine the theory of Δ¹⁸O-g_m estimation and its sensitivity to changes in the ¹⁸O composition (δ¹⁸O) of CO₂ on young and old leaves of six C₃ species. Our results showed that, when full isotopic equilibrium was assumed, g_m values were sensitive to the δ¹⁸O of the CO₂ sources. Utilizing a new isotopic mass balance model that distinguishes metabolic (R_t) and purely diffusive (F_retro) CO₂ fluxes, we found that the gross H₂O-equilibrated efflux (θ_rF_retro+R_t) represented 157±12 % (mean±SE) of net CO₂ assimilation, and was positively correlated to stomatal conductance, implying a limitation imposed by stomata. Furthermore, leaf water was not completely in equilibrium with F_retro (θ_r<1) or the CO₂ pool at the site of hydration (θ<1), which led to errors in estimated g_m. Our results have broad implications for the interpretation of Δ¹⁸O in terms of mesophyll conductance, and highlight the necessity of considering the isotopic disequilibrium of the retro-diffusive CO₂ efflux.