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

Abstract

Nitrogen (N) fixation and assimilation in pea (Pisum sativum) root nodules were studied by in vivo 15 N nuclear magnetic resonance (NMR) by exposing detached nodules to 15 N 2 via a perfusion medium, while recording a time course of spectra. In vivo 31 P 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 15 N labeling of individual amino acids by liquid chromatography-mass spectrometry. A substantial pool of free ammonium was observed by 15 N 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 15 N 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 15 N-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 15 N chemical shifts were observed. Extensive 15 N 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 15 N amino signal was absent in in vivo 15 N NMR spectra, which could be because of an unfavorable nuclear Overhauser effect. ␥-Aminobutyric acid accumulated in the nodules during incubation, but newly synthesized 15 N ␥-aminobutyric acid seemed to be immobilized in metabolically active pea nodules, which made it NMR invisible.Symbiotic nitrogen (N) fixation, the process whereby N 2 -fixing bacteria enter into associations with plants, provides the major source of N for the biosphere. Nitrogenase, a bacterial enzyme, catalyzes the reduction of atmospheric dinitrogen to ammonium. In rhizobia-leguminous plant symbioses, a widely accepted and simple model of N transfer from the symbiotic form of the bacterium, called a bacteroid, to the plant implies that nitrogenase-generated ammonia diffuses across the bacteroid membrane and is assimilated into amino acids in the plant compartment of the nodule tissue. However, the transport of symbiotically fixed N across the membranes surrounding the bacteroid and the form in which this occurs has been a matter of controversy.Until recently, it has been generally accepted that Rhizobium bacteroids do not assimilate ammonium into amino acids to a great extent during symbiosis, but more recent results challenge this view and suggest a possible involvement of amino acids as the form of fixed N delivered from the bacteroid to the plant (for review, see Poole and Allaway, 2000; Day et al., 2001). At present, consensus has not been reached as to whether substantial N assimilation takes place in the nodule bacteroid compartment, and new investigations of nodule N metabolism are required.The...

Characterization of a γ-Aminobutyric Acid Transport System of Rhizobium leguminosarum bv. viciae 3841

“…15 N nuclear magnetic resonance analysis on detached pea nodules showed GABA to be the second most abundant amino acid (37). This is consistent with an important role for GABA in bacteroid metabolism and a possible role in amino acid cycling.…”

“…The cytosolic concentration of GABA from soybean nodules was measured as 71 g g Ϫ1 (fresh weight), while nuclear magnetic resonance analysis of pea nodules revealed a GABA concentration of 5.72 mol g Ϫ1 (fresh weight) (37,38). High GABA concentrations have also been reported in isolated bacteroids of S. meliloti (41 nmol mg of protein Ϫ1 ) (27), suggesting it has an important role in bacteroid metabolism.…”

Pathway of γ-Aminobutyrate Metabolism in Rhizobium leguminosarum 3841 and Its Role in Symbiosis

“…viciae 3841 bacteroids was low, the high APE suggests that it turns over rapidly and therefore may play an important role in amino acid metabolism or amino acid cycling (26). It was also reported to be the most highly 15 N-labeled amino acid in pea nodules in a previous study (41), but in that study bacteroids and plant cytosol were not separated. GABA is most likely derived from the glutamate pool of the plant cytosol via ␣-decarboxylation by glutamate decarboxylase (Fig.…”

Does GABA increase the efficiency of symbiotic N2fixation in legumes?

“…5,12,20 By the aid of 15 N nuclear magnetic resonance ( 15 N NMR), GABA was determined as the second most abundant amino acid of detached pea nodules. 29 High [GABA] have been reported in bacteroids isolated from different bacterial strains including Bradyrhizobium japonicum, 30 Sinorhizobium meliloti 31 and Rhizobium leguminosarum.…”