Kinetic variation in grass phosphoenolpyruvate carboxylases provides opportunity to enhance C 4 photosynthetic efficiency

The high rates of photosynthesis and the carbon-concentrating mechanism (CCM) in C plants are initiated by the enzyme phosphoenolpyruvate (PEP) carboxylase (PEPC). The flow of inorganic carbon into the CCM of C plants is driven by PEPC's affinity for bicarbonate (K ), which can be rate limiting when atmospheric CO availability is restricted due to low stomatal conductance. We hypothesize that natural variation in K across C plants is driven by specific amino acid substitutions to impact rates of C photosynthesis under environments such as drought that restrict stomatal conductance. To test this hypothesis, we measured K from 20 C grasses to compare kinetic properties with specific amino acid substitutions. There was nearly a twofold range in K across these C grasses (24.3 ± 1.5 to 46.3 ± 2.4 μm), which significantly impacts modeled rates of C photosynthesis. Additionally, molecular engineering of a low-HCO affinity PEPC identified key domains that confer variation in K . This study advances our understanding of PEPC kinetics and builds the foundation for engineering increased-HCO affinity and C photosynthetic efficiency in important C crops.