Partitioning of mesophyll conductance for CO 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 CO concentration (c) in the intercellular air space (IAS) across hypostomatous leaves (expressed as the ratio c/c, where the indexes d and b denote the adaxial and abaxial edges, respectively, of IAS), based on the carbon isotope composition (δC) 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, where c and c stand for mean CO concentrations in chloroplasts and in the IAS), the fraction of intercellular resistance in the total mesophyll resistance (r/r), 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 compared to the lower (abaxial) side. CM, WX, and/or EWX isolated from 40 hypostomatous C species were C depleted relative to bulk leaf tissue by 2.01-2.85‰. The difference in δC between the abaxial and adaxial leaf sides (δC - C, Δ), 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 difference in WX. Δ correlated negatively with LMA and leaf thickness of hypostomatous leaves, which indicates that the mesophyll air space imposes a non-negligible resistance to CO diffusion. δC of EWX and 30-C aldehyde in WX reveal a stronger CO drawdown than bulk WX or CM. Mean values of c/c and c/c 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.