Plant Physiol, January 2003, Vol. 131, pp. 367-378

Exploring Symbiotic Nitrogen Fixation and Assimilation in Pea Root Nodules by in Vivo ¹⁵N Nuclear Magnetic Resonance Spectroscopy and Liquid Chromatography-Mass Spectrometry¹

Risoe National Laboratory, Plant Research Department (A.M.S., H.E., L.R.) and Department of Life Sciences and Chemistry (P.E.H.), Roskilde University, Roskilde, Denmark DK-4000

Nitrogen (N) fixation and assimilation in pea (Pisum sativum) root nodules were studied by in vivo ¹⁵N nuclear magnetic resonance (NMR) by exposing detached nodules to ¹⁵N₂ via a perfusion medium, while recording a time course of spectra. In vivo ³¹P NMR spectroscopy was used to monitor the physiological state of the metabolically active nodules. The nodules were extracted after the NMR studies and analyzed for total soluble amino acid pools and ¹⁵N labeling of individual amino acids by liquid chromatography-mass spectrometry. A substantial pool of free ammonium was observed by ¹⁵N NMR to be present in metabolically active, intact nodules. The ammonium ions were located in an intracellular environment that caused a remarkable change in the in vivo ¹⁵N chemical shift. Alkalinity of the ammonium-containing compartment may explain the unusual chemical shift; thus, the observations could indicate that ammonium is located in the bacteroids. The observed ¹⁵N-labeled amino acids, glutamine/glutamate and asparagine (Asn), apparently reside in a different compartment, presumably the plant cytoplasm, because no changes in the expected in vivo ¹⁵N chemical shifts were observed. Extensive ¹⁵N labeling of Asn was observed by liquid chromatography-mass spectrometry, which is consistent with the generally accepted role of Asn as the end product of primary N assimilation in pea nodules. However, the Asn ¹⁵N amino signal was absent in in vivo ¹⁵N NMR spectra, which could be because of an unfavorable nuclear Overhauser effect. -Aminobutyric acid accumulated in the nodules during incubation, but newly synthesized ¹⁵N -aminobutyric acid seemed to be immobilized in metabolically active pea nodules, which made it NMR invisible.