MtPEPC2 Encodes a Phosphoenolpyruvate Carboxylase Essential for Symbiotic Nitrogen Fixation in Medicago truncatula

Symbiotic nitrogen fixation (SNF) that takes place in root nodules of legumes essentially relies on the exchange of nitrogen (N) and carbon (C) metabolites between the symbiotic partners. The endosymbionts rhizobia provide ammonium to the host plants, and in return receive carbon and energy sources from the host for nitrogen fixation. In a forward genetic study, we identified FN6516 as an SNF-defective (fix^-) mutant of Medicago truncatula. Whole genome resequencing, genetic linkage analysis of an F2 segregating population, genetic complementation and gene editing results show that a plant-type PEPC, MtPEPC2, is the candidate gene. We demonstrate that MtPEPC2 expression is activated in nodules and that a high level of expression is detected at an early stage of nodule development. MtPEPC2 protein is localised in the cytoplasm of both infected and uninfected cells, but not in symbiosomes. Our work shows that a nonsense mutation in MtPEPC2 resulted in a great reduction in PEPC activities, almost complete loss of nodule nitrogen fixation activities, and defects in differentiation and/or maintenance of bacteroids. Importantly, overexpression of MtPEPC2 increased nodule nitrogenase activities.