Uninfected cell-specific enzymes coordinate carbon supply and nitrogen assimilation in Medicago truncatula nodules.

Abstract

In legume root nodules, rhizobia invade host cells to form symbiosomes that drive atmospheric nitrogen fixation. Although the metabolic roles of infected cells (ICs) are well established, the contributions of adjacent uninfected cells (UCs) have remained largely unexplored. Here, through forward genetics methods, we identify DEBINO4, a phosphoenolpyruvate carboxylase (PEPC) uniquely expressed in UCs, as a pivotal regulator of carbon metabolism essential for sustaining symbiosome function and nitrogen assimilation. DEBINO4-deficient mutants display premature nodule senescence characterized by nonviable symbiosomes in the fixation zone and disrupted carbon and nitrogen metabolic profiles. The nodule-specific PEPC kinases (PPCKs), which are probably involved in DEBINO4 activation, are required to preserve symbiosome integrity, while Glutamine Synthetase 1a (GS1a), also restricted to UCs, is critical for ammonium assimilation and maintaining differentiated symbiosomes. Comprehensive analysis of metabolism-related genes further reveals that UCs execute specialized, stage-specific functions during nitrogen fixation. Collectively, our findings underscore the importance of cell-type-specific metabolic networks in orchestrating successful symbiosis and provide a framework for understanding how distinct nodule cell populations coordinate carbon and nitrogen metabolism to support efficient nitrogen fixation.