Novel Amino Acid Changes Increase Phosphoenolpyruvate Carboxylase Output in planta

Characterization of previously reported Arabidopsis mutants with increased aluminum (Al) resistance (alr) resulting from greater release of Al-chelating malate identified three unique amino acid substitutions that each impacts PHOSPHOENOLPYRUVATE CARBOXYLASE 1, a key anaplerotic carbon fixation enzyme. Expression of mutant versions of AtPPC1 in an Arabidopsis ppc1 loss-of-function mutant increased Al resistance in association with upwards of threefold higher release of Al-chelating malate from roots. Analyses of enzyme kinetics and protein structure of the variants revealed that each amino acid position is critical to proper interplay between the allosteric regulator malate and the active site. Since these affect amino acid positions generally conserved amongst plant PPCs, they were individually introduced into maize PPCs including ZmPPC1, the isozyme responsible for the first step of C4 photosynthesis, and ZmPEP7, a key PPC in resupply of the TCA cycle via anaplerosis. This resulted in similar or greater improvements in enzyme activity and reduced regulation in vitro for both ZmPPC1 and ZmPEP7 along with increased PPC-dependent output in planta for a ZmPEP7 transgenic. Therefore, engineering these amino acid changes into plants in general could be useful for increasing carbon fixation by PPCs to turn excess CO₂ into a resource for our growing population.