Exploring symbiotic nitrogen fixation and assimilation in pea root nodules by in vivo 15N nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry.

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. gamma-Aminobutyric acid accumulated in the nodules during incubation, but newly synthesized (15)N gamma-aminobutyric acid seemed to be immobilized in metabolically active pea nodules, which made it NMR invisible.

Rhizobium colonization induced changes in membrane-bound and soluble hydroxyproline-rich glycoprotein composition in pea.

Abundance and distribution of plant cell surface proteins of the hydroxyproline-rich glycoprotein (HRGP) class were studied in the pea-Rhizobium symbiosis using immunoblot analysis. The MAC 265-epitope was especially abundant in pea root nodules containing nitrogen-fixing Rhizobium bacteria. A 180-kDa MAC 265-HRGP dominated in pea shoot plasma membranes, while almost no MAC 265-HRGP was detected in root plasma membranes. We show here that a major difference between the plant-derived peribacteroid membrane of the symbiosomes and the root plasma membrane was the presence of a 100-kDa MAC 265-HRGP in the former. Arabinogalactan proteins (AGPs), as recognized by the monoclonal antibodies MAC 207 and JIM 8, were not detected in the peribacteroid membrane, while two isoforms (100 and 220 kDa) were detected in shoot and root plasma membranes. Specific MAC 265-HRGP isoforms were found in the peribacteroid space fraction of the symbiosomes and thus as soluble proteins in the interface between the symbionts. The abundance of the MAC 265-epitope was much reduced in non-nitrogen-fixing nodules when this phenotype resulted from a dicarboxylate transport mutation in Rhizobium. There was no reduction in the abundance of the MAC 265-epitope in non-fixing phenotypes resulting from a mutation in the plant. The results suggest that bacterial signals related to the bacterial ability to fix nitrogen, might be responsible for the regulation of HRGP expression in root nodules.