Evaluating the Contribution of Glutamate Dehydrogenase and the Glutamate Synthase Cycle to Ammonia Assimilation by Four Ectomycorrhizal Fungal Isolates

Abstract

Combined gas chromatography-mass spectrometry were used to evaluate the contributions of glutamate dehydrogenase (GDH) and the glutamate synthase cycle in 15N-labelled ammonium assimilation by four ectomycorrhizal fungal isolates. In all four species (Elaphomyces, Amanita, Pisolithus and Gautieria), glutamine was the major product accumulated following transfer of 14-day-old nitrogen-limited cultures to fresh medium. Label was rapidly assimilated into fungal tissue, with rates of 733 nmol g-1 FW h-1 in Pisolithus, 972 nmol g-1 FW h-1 in Amanita, 2760 nmol g-1 FW h-1 in Gautieria and 6756 nmol g-1 FW h-1 in Elaphomyces sp in the first 4 h of incubation. Incorporation of [15N]ammonium was sensitive to the inhibitory effects of both methionine sulfoximine (MSX, an inhibitor of glutamine synthetase (GS)) and albizziin (an inhibitor of glutamate synthase (GOGAT)) in three species (Amanita, Gautieria and Pisolithus) and labelling patterns were consistent with the action of the glutamate synthase cycle in ammonium assimilation. In all three species glutamine synthesis was almost totally blocked by MSX and there was no continued incorporation of 15N into glutamate. Elaphomyces displayed high levels of total incorporation of labelled ammonium in mycelium even in the presence of MSX, although incorporation into glutamine was reduced by 88%. This inhibition of GS by MSX, in addition to its partial inhibition by albizziin suggests strongly the action of glutamate synthase cycle in ammonium assimilation. The reduction in label entering glutamate under the influence of albizziin is direct evidence for the inhibition of GOGAT activity. However, MSX treatment had the effect of increasing significantly the quantity of label recovered in both glutamate and alanine. In the absence of GS inhibition there is clearly competition for ammonium which under normal physiological conditions results in assimilation through the glutamate synthase cycle. However, when GS is blocked by MSX label is able to cycle through the GDH pathway. Extra keywords: ectomycorrhiza, ammonium assimilation, glutamate synthase cycle, glutamate dehydrogenase, amino acid metabolism.