Partitioning of mesophyll conductance for CO2 into intercellular and cellular components using carbon isotope composition of cuticles from opposite leaf sides

We suggest a new technique for estimating the relative drawdown of CO₂ concentration (c) in the intercellular air space (IAS) across hypostomatous leaves (expressed as the ratio c_d/c_b, where the indexes d and b denote the adaxial and abaxial edges, respectively, of IAS), based on the carbon isotope composition (δ¹³C) of leaf cuticular membranes (CMs), cuticular waxes (WXs) or epicuticular waxes (EWXs) isolated from opposite leaf sides. The relative drawdown in the intracellular liquid phase (i.e., the ratio c_c/c_bd, where c_c and c_bd stand for mean CO₂ concentrations in chloroplasts and in the IAS), the fraction of intercellular resistance in the total mesophyll resistance (r_IAS/r_m), leaf thickness, and leaf mass per area (LMA) were also assessed. We show in a conceptual model that the upper (adaxial) side of a hypostomatous leaf should be enriched in ¹³C compared to the lower (abaxial) side. CM, WX, and/or EWX isolated from 40 hypostomatous C₃ species were ¹³C depleted relative to bulk leaf tissue by 2.01–2.85‰. The difference in δ¹³C between the abaxial and adaxial leaf sides (δ¹³C_AB ? ¹³C_AD, Δ_b–d), ranged from ??2.22 to +?0.71‰ (??0.09 ± 0.54‰, mean ± SD) in CM and from ??7.95 to 0.89‰ (??1.17 ± 1.40‰) in WX. In contrast, two tested amphistomatous species showed no significant Δ_b–d difference in WX. Δ_b–d correlated negatively with LMA and leaf thickness of hypostomatous leaves, which indicates that the mesophyll air space imposes a non-negligible resistance to CO₂ diffusion. δ¹³C of EWX and 30-C aldehyde in WX reveal a stronger CO₂ drawdown than bulk WX or CM. Mean values of c_d/c_b and c_c/c_bd were 0.90 ± 0.12 and 0.66 ± 0.11, respectively, across 14 investigated species in which wax was isolated and analyzed. The diffusion resistance of IAS contributed 20 ± 14% to total mesophyll resistance and reflects species-specific and environmentally-induced differences in leaf functional anatomy.