Báo cáo lâm nghiệp: The role of glutamine synthetase, glutamate synthase and glutamate dehydrogenase in ammonia assimilation by the mycorrhizal fungus Pisolithus tinctorius

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Báo cáo lâm nghiệp: " The role of glutamine synthetase, glutamate synthase and glutamate dehydrogenase in ammonia assimilation by the mycorrhizal fungus Pisolithus tinctorius"The role of glutamine synthetase, glutamate synthaseand glutamate dehydrogenase in ammonia assimilationby the mycorrhizal fungus Pisolithus tinctorius G.R. StewartJ.L. KershawDepartement of Biology (Darwin), University College London, Gower St., London WC1 E, U.K. roots appears to be theIntroduction glutamate syn- thase cycle, whereas ammonia assimila- tion in fungi is generally held to occur via the glutamate dehydrogenase (GDH)Of the major nutrients required by trees, pathway (Fig. 1Previous studies havenitrogen appears to be the most important shown that some yeasts are capable offor increasing forest productivity. Nitrogen utilising the glutamate synthase cycle foris obtained from inorganic forms present ammonia assimilation (Roon et aG, 1974;in the soil solution, and thus the root is an Johnson and Brown, 1974), but in the centre forimportant inorganic nitrogen ectomycorrhizal fungus Cenococcum gra-assimilation. There is evidence that ecto- niforme the GDH pathway was the primarymycorrhizae (ECM) stimulate ammonia route of ammonia incorporation (Genetetuptake by woody plants. The fungal part- et al., 1984).ner contributes nitrogen to the tree root intwo ways: by translocation of nitrogenouscompounds from the soil N-pool to theroot, and by conversion of absorbed N intoforms more easily utilised by the root. Stu- Materials andl Methodsdies of the assimilation of nitrogen by purecultures of ECM fungi provide the basis forinvestigation of fungal-based nitrogen Pure cultures of Pisolithus tinctorius, an ecto-metabolism within the ECM. mycorrhizal ba.sidiomycete, were grown for 18 d in half-strength modified Melin-Norkrans In most fungi and higher plants, inorgan- medium (1/2MI1AN) containing 1 mM ammo-ic nitrogen is assimilated into the amino nium. Ammonium concentration in the flasks effectively 0 after 12 d of static growth atacids glutamate and glutamine, which was 25°C.then donate nitrogen to other metabolites.The route of ammonia assimilation found Mycelia were harvested daily following thein both mycorrhizal and non-mycorrhizal commencement of vegetative growth (d 4) andassayed for glutamine synthetase (GS) activity acids by separation of the o-phthaldialdehyde derivatives on a reverse-phase HPLC column.by the biosynthetic assay, and for NADPH andNADH-dependent GDH activity (Lea, 1985). After 17 or 18 d growth, the nitrogen-starved were transferred to flasks of freshmycelia1/2MMN medium containing: a) no inhibitors Results(control), or b) methionine sulphoximine (MSX)(1 mM), an irreversible inhibitor of GS, or c)azaserine (1 mM), a glutamate synthase inhibi- Enzyme assaystor, or d) aminooxyacetate (0.2 mM), an inhibi-tor of aminotransferase enzymes. After 2, 4, 6or 8 h in the fresh medium, mycelia were ex- NAD-dependent GDH activity was foundtracted with sulphosalicylic acid solution (0.1M). The supernatant was assayed for amino negligible. NADP-dependent GDH to be and glutamine pools after 2 h inactivity was detected and found to be rela- glutamatetively constant throughout the period of the fresh medium (Fig. 3a). Glutamategrowth (4-14 d) (Fig. 2b). GS activity was levels remained constant after 2 h but thegenerally higher during the initial period of glutamine concentration continued torapid growth (5-10 d) and decreased increase up to 6 h indicating glutamine asthereafter with ammonium concentration the primary product of assimila ...