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

Abstract

Abstract 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 to15N2 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 observed15N-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 vivo15N chemical shifts were observed. Extensive15N 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. γ-Aminobutyric acid accumulated in the nodules during incubation, but newly synthesized 15N γ-aminobutyric acid seemed to be immobilized in metabolically active pea nodules, which made it NMR invisible.