Pathways of Nitrogen Metabolism in Nodules of Alfalfa (Medicago sativa L.)

Exposure of intact alfalfa nodules to ¹⁵N₂ showed that in bacteroids the greatest flow of ¹⁵N was to NH₃. Label was also detected in glutamic acid, aspartic acid, and asparagine (Glu, Asp and Asn), but at far lower levels. In the host plant cytosols, more ¹⁵N was incorporated into Asn than into other compounds. Detached nodules were also used to study the metabolic pathway of N assimilation after exposure to ¹⁵N₂ or vacuum infiltration with (¹⁵NH₄)₂SO₄ in the presence or absence of different inhibitors of nitrogen assimilation: methionine sulfoximine (MSO), azaserine (AZA), or amino-oxyacetate (AOA). Treatment with MSO, an inhibitor of glutamine synthetase (GS), inhibited the flow of the label to glutamine (Gln)-amide, resulting in subsequently decreased label in Asnamide. Aza, which inhibits the formation of Glu from Gln by glutamate synthase (GOGAT), enhanced the labeling of the amide groups of both Gln and Asn, while that of Asn-amino decreased. When AOA was used to block the transamination reaction very little label was found in Asp and Asn-amino. The results are consistent with the role of GS/GOGAT in the cytosol for the assimilation of NH₃ produced by N₂ fixation in the bacteroids of alfalfa nodules. Asn, a major nitrogen transport compound in alfalfa, is mainly synthesized by a Gln-dependent amidation of Asp, according to feeding experiments using the ¹⁵N-labeled amide group of glutamine. Data from ¹⁵NH₄⁺ feeding support some direct amidation of Asp to form Asn.