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 (delta C-13) 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 C-13 compared to the lower (abaxial) side. CM, WX, and/or EWX isolated from 40 hypostomatous C-3 species were C-13 depleted relative to bulk leaf tissue by 2.01-2.85 parts per thousand. The difference in delta C-13 between the abaxial and adaxial leaf sides (delta C-13(AB)-C-13(AD), Delta(b-d)), ranged from -2.22 to +0.71 parts per thousand (-0.09 +/- 0.54 parts per thousand, mean +/- SD) in CM and from -7.95 to 0.89 parts per thousand (-1.17 +/- 1.40 parts per thousand) in WX. In contrast, two tested amphistomatous species showed no significant Delta(b-d) difference in WX. Delta(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. delta C-13 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.Web of Science1411513