Harnessing photosynthetic C¹⁸O¹⁶O discrimination dynamics under leaf water nonsteady state to estimate mesophyll conductance: a new, regression-based method

center dot Mesophyll conductance (g(m)) is a crucial plant trait that can significantly limit photosynthesis. Measurement of photosynthetic (COO)-O-18-O-16 discrimination (Delta O-18) has proved to be the only viable means of resolving g(m) in both C-3 and C-4 plants. However, the currently available methods to exploit Delta O-18 for g(m) estimation are error prone due to their inadequacy in constraining the degree of oxygen isotope exchange (theta) during mesophyll CO2 hydration. center dot Here, we capitalized on experimental manipulation of leaf water isotopic dynamics to establish a novel, nonsteady state, regression-based approach for simultaneous determination of g(m) and theta from online Delta O-18 measurements. center dot We demonstrated the methodological and theoretical robustness of this new Delta O-18-g(m) estimation approach and showed through measurements on several C-3 and C-4 species that this approach can serve as a benchmark method against which to identify previously-unrecognized biases of the existing Delta O-18-g(m) methods. center dot Our results highlight the unique value of this nonsteady state-based approach for contributing to ongoing efforts toward quantitative understanding of mesophyll conductance for crop yield improvement and carbon cycle modeling.