In folio isotopic tracing demonstrates that nitrogen assimilation into glutamate is mostly independent from current CO2 assimilation in illuminated leaves of Brassica napus

P>Nitrogen assimilation in leaves requires primary NH2 acceptors that, in turn, originate from primary carbon metabolism. Respiratory metabolism is believed to provide such acceptors (such as 2-oxoglutarate), so that day respiration is commonly seen as a cornerstone for nitrogen assimilation into glutamate in illuminated leaves. However, both glycolysis and day respiratory CO2 evolution are known to be inhibited by light, thereby compromising the input of recent photosynthetic carbon for glutamate production. In this study, we carried out isotopic labelling experiments with 13CO(2) and 15N-ammonium nitrate on detached leaves of rapeseed (Brassica napus), and performed 13C- and 15N-nuclear magnetic resonance analyses. Our results indicated that the production of 13C-glutamate and 13C-glutamine under a 13CO(2) atmosphere was very weak, whereas 13C-glutamate and 13C-glutamine appeared in both the subsequent dark period and the next light period under a 12CO(2) atmosphere. Consistently, the analysis of heteronuclear (13C-15N) interactions within molecules indicated that most 15N-glutamate and 15N-glutamine molecules were not 13C labelled after 13C/15N double labelling. That is, recent carbon atoms (i.e. 13C) were hardly incorporated into glutamate, but new glutamate molecules were synthesized, as evidenced by 15N incorporation. We conclude that the remobilization of night-stored molecules plays a significant role in providing 2-oxoglutarate for glutamate synthesis in illuminated rapeseed leaves, and therefore the natural day : night cycle seems critical for nitrogen assimilation.