Two-Source δ¹⁸O Method to Validate the CO¹⁸O-Photosynthetic Discrimination Model: Implications for Mesophyll...

Theoretical models of photosynthetic isotopic discrimination of CO₂ (¹³C and ¹⁸O) are commonly used to estimate mesophyll conductance (g _m). This requires making simplifying assumptions and assigning parameter values so that g _m can be solved for as the residual term. Uncertainties in g _m estimation occur due to measurement noise and assumptions not holding, including parameter uncertainty and model parametrization. Uncertainties in the ¹³C model have been explored previously, but there has been little testing undertaken to determine the reliability of g _m estimates from the ¹⁸O model (g _m18). In this study, we exploited the action of carbonic anhydrase in equilibrating CO₂ with leaf water and manipulated the observed photosynthetic discrimination (Δ¹⁸O) by changing the oxygen isotopic composition of the source gas CO₂ and water vapor. We developed a two-source δ¹⁸O method, whereby two measurements of Δ¹⁸O were obtained for a leaf with its gas-exchange characteristics otherwise unchanged. Measurements were performed in broad bean (Vicia faba) and Algerian oak (Quercus canariensis) in response to light and vapor pressure deficit. Despite manipulating the Δ¹⁸O by over 100‰, in most cases we observed consistency in the calculated g _m18, providing confidence in the measurements and model theory. Where there were differences in g _m18 estimates between source-gas measurements, we explored uncertainty associated with two model assumptions (the isotopic composition of water at the sites of CO₂-water exchange, and the humidity of the leaf internal airspace) and found evidence for both. Finally, we provide experimental guidelines to minimize the sensitivity of g _m18 estimates to measurement errors. The two-source δ¹⁸O method offers a flexible tool for model parameterization and provides an opportunity to refine our understanding of leaf water and CO₂ fluxes.