EVALUATING THE CONTRIBUTION OF GLUTAMATE-DEHYDROGENASE AND THE GLUTAMATE SYNTHASE CYCLE TO AMMONIA ASSIMILATION BY 4 ECTOMYCORRHIZAL FUNGALISOLATES

Combined gas chromatography-mass spectrometry were used to evaluate th e contributions of glutamate dehydrogenase (GDH) and the glutamate syn thase cycle in N-15-labelled ammonium assimilation by four ectomycorrh izal fungal isolates. In all four species (Elaphomyces, Amanita, Pisol ithus and Gautieria), glutamine was the major product accumulated foll owing 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 [N-15 ]ammonium was sensitive to the inhibitory effects of both methionine s ulfoximine (MSX, an inhibitor of glutamine synthetase (GS)) and albizz iin (an inhibitor of glutamate synthase (GOGAT)) in three species (Ama nita, Gautieria and Pisolithus) and labelling patterns were consistent with the action of the glutamate synthase cycle in ammonium assimilat ion. In all three species glutamine synthesis was almost totally block ed by MSX and there was no continued incorporation of N-15 into glutam ate. Elaphomyces displayed high levels of total incorporation of label led ammonium in mycelium even in the presence of MSX, although incorpo ration 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 in hibition 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 c ycle through the GDH pathway.