Abstract

Legumes form symbioses with nitrogen-fixing bacteria, well studied metabolically but less so in terms of respiration. Symbiotic nitrogen fixation demands high respiratory ATP and carbon skeletons, linking nitrogen assimilation and both NADH- and ATP-dependent process to mitochondrial respiration. The plant mitochondrial electron transport chain contains two terminal oxidases that differentially fractionate against 18O, providing estimations in vivo of the energy efficiency of respiration. The regulation of N2 fixation by plant respiratory parameters remains unknown. To investigate the regulatory interactions of these two metabolic processes, we tested the effect of different plant N status and sources on respiratory parameters and nutrition in Lotus japonicus. Plants were grown with two levels of KNO3 fertilization (5 mM and 25 mM) and with the N2 fixing symbiotic bacteria Mesorhizobium loti, which induced the formation of root nodules (NP). Additionally, we characterized roots containing non-fixing nodules by growing plants that display spontaneous nodule formation (snf) (SNF). We evaluated the natural abundances of 13C and 15N, and 18O discrimination during respiration in leaves and roots using isotope-ratio mass spectrometry. NADH and nutrient content were measured using ultra-performance liquid chromatography and inductively coupled plasma spectrometry. We observed that cytochrome c oxidase activity was higher in nodulated roots capable of nitrogen fixation than in plants fertilized with high availability of nitrate, and that nitrogen status strongly associates to respiratory parameters. These findings highlight the role of cytochrome c oxidase in meeting the carbon and energy demands of symbiotic nitrogen fixation. © 2025 The Authors