Discrimination in the dark. Resolving the interplay between metabolic and physical constraints to phosphoenolpyruvate carboxylase activity during the crassulacean acid metabolism cycle

A model defining carbon isotope discrimination (Δ¹³C) for crassulacean acid metabolism (CAM) plants was experimentally validated using Kalanchoe daigremontiana. Simultaneous measurements of gas exchange and instantaneous CO₂ discrimination (for ¹³C and ¹⁸O) were made from late photoperiod (phase IV of CAM), throughout the dark period (phase I), and into the light (phase II). Measurements of CO₂ response curves throughout the dark period revealed changing phosphoenolpyruvate carboxylase (PEPC) capacity. These systematic changes in PEPC capacity were tracked by net CO₂ uptake, stomatal conductance, and online Δ¹³C signal; all declined at the start of the dark period, then increased to a maximum 2 h before dawn. Measurements of Δ¹³C were higher than predicted from the ratio of intercellular to external CO₂ (p_i/p_a) and fractionation associated with CO₂ hydration and PEPC carboxylations alone, such that the dark period mesophyll conductance, g_i, was 0.044 mol m ^-2 s^-1 bar^-1. A higher estimate of g _i (0.085 mol m^-2 s^-1 bar^-1) was needed to account for the modeled and measured Δ¹⁸O discrimination throughout the dark period. The differences in estimates of g_i from the two isotope measurements, and an offset of -5.5‰ between the ¹⁸O content of source and transpired water, suggest spatial variations in either CO₂ diffusion path length and/or carbonic anhydrase activity, either within individual cells or across a succulent leaf. Our measurements support the model predictions to show that internal CO₂ diffusion limitations within CAM leaves increase Δ¹³C discrimination during nighttime CO₂ fixation while reducing Δ¹³C during phase IV. When evaluating the phylogenetic distribution of CAM, carbon isotope composition will reflect these diffusive limitations as well as relative contributions from C₃ and C₄ biochemistry.