NH₄ ⁺ assimilation in the ectomycorrhizal basidiomycete Laccaria bicolor (Maire) Orton, a ¹⁵ N-NMR study

Nuclear magnetic resonance spectroscopy was used to monitor ¹⁵ NH₄ ⁺ assimilation and amino acid biosynthesis in the ectomycorrhizal basidiomycete Laccaria bicolor (Maire) Orton. (strain S238). In mycelium growing rapidly on ¹⁵ NH₄ ⁺ , [amido-¹⁵ N]glutamine was the major ¹⁵ N-labelled species. When ¹⁵ N-labelled mycelium was transferred into medium containing ¹⁴ NH₄ ⁺ , the resonance for [amino-¹⁵ N]glutamine decreased with a half-life of about 3.0 h, whereas the resonance for [amino-¹⁵ N]glutamine remained unchanged. Such behaviour is consistent with glutamine synthetase (GS) being the major route of ¹⁵ NH₄ ⁺ assimilation. However, the higher accumulation of [¹⁵ N]alanine observed in the presence of the GS inhibitor, methionine sulfoximine, indicated that a part of the glutamate pool was formed by the glutamate dehydrogenase (GDH) pathway. When the mycelium was in stationary phase (i.e. low extracellular NH₄ ⁺ ), the intramolecular ¹⁵ N labelling of glutamine suggested that the GDH and GS pathways were simultaneously assimilating NH₄ ⁺ . The N supply and the growth stage, therefore, influence the expression of the activities of GDH and GS. The current isotopic data identify other fates of absorbed ¹⁵ N: glutamate decarboxylation gives rise to γ-aminobutyrate; transamination between glutamate and pyruvate yields alanine; and arginine accumulates. It is concluded that GS is the main pathway of primary assimilation of NH₄ ⁺ in L. bicolor, but GDH may also contribute significantly to this process.