Nitrogen nutrition effects on δ¹³C of plant respired CO₂ are mostly caused by concurrent changes in organic acid utilisation and remobilisation

Nitrogen (N) nutrition impacts on primary carbon metabolism and can lead to changes in δ¹³C of respired CO₂. However, uncertainty remains as to whether (1) the effect of N nutrition is observed in all species, (2) N source also impacts on respired CO₂ in roots and (3) a metabolic model can be constructed to predict δ¹³C of respired CO₂ under different N sources. Here, we carried out isotopic measurements of respired CO2 and various metabolites using two species (spinach, French bean) grown under different NH₄+:NO₃− ratios. Both species showed a similar pattern, with a progressive ¹³C-depletion in leaf-respired CO₂ as the ammonium proportion increased, while δ¹³C in root-respired CO₂ showed little change. Supervised multivariate analysis showed that δ¹³C of respired CO₂ was mostly determined by organic acid (malate, citrate) metabolism, in both leaves and roots. We then took advantage of nonstationary, two-pool modelling that explained 73% of variance in δ¹³C in respired CO₂. It demonstrates the critical role of the balance between the utilisation of respiratory intermediates and the remobilisation of stored organic acids, regardless of anaplerotic bicarbonate fixation by phosphoenolpyruvate carboxylase and the organ considered.