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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The role of glutamine synthetase, glutamate synthase and glutamate dehydrogenase in ammonia assimilation by the mycorrhizal fungus Pisolithus tinctorius G.R. Stewart J.L. Kershaw Departement of Biology (Darwin), University College London, Gower St., London WC1 E, U.K. roots appears to be the Introduction 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 have nitrogen appears to be the most important shown that some yeasts are capable of for increasing forest productivity. Nitrogen utilising the glutamate synthase cycle for is 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 for important inorganic nitrogen ectomycorrhizal fungus Cenococcum gra- assimilation. There is evidence that ecto- niforme the GDH pathway was the primary mycorrhizae (ECM) stimulate ammonia route of ammonia incorporation (Genetet uptake by woody plants. The fungal part- et al., 1984). ner contributes nitrogen to the tree root in two ways: by translocation of nitrogenous compounds from the soil N-pool to the root, and by conversion of absorbed N into forms more easily utilised by the root. Stu- Materials andl Methods dies of the assimilation of nitrogen by pure cultures of ECM fungi provide the basis for investigation 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 at acids glutamate and glutamine, which was 25°C. then donate nitrogen to other metabolites. The route of ammonia assimilation found Mycelia were harvested daily following the in both mycorrhizal and non-mycorrhizal commencement of vegetative growth (d 4) and
assayed 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 and NADH-dependent GDH activity (Lea, 1985). After 17 or 18 d growth, the nitrogen-starved were transferred to flasks of fresh mycelia 1/2MMN 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 assays tor, or d) aminooxyacetate (0.2 mM), an inhibi- tor of aminotransferase enzymes. After 2, 4, 6 or 8 h in the fresh medium, mycelia were ex- NAD-dependent GDH activity was found tracted with sulphosalicylic acid solution (0.1 M). The supernatant was assayed for amino negligible. NADP-dependent GDH to be
and glutamine pools after 2 h in activity was detected and found to be rela- glutamate tively constant throughout the period of the fresh medium (Fig. 3a). Glutamate growth (4-14 d) (Fig. 2b). GS activity was levels remained constant after 2 h but the generally higher during the initial period of glutamine concentration continued to rapid growth (5-10 d) and decreased increase up to 6 h indicating glutamine as thereafter with ammonium concentration the primary product of assimilated ammo- nia. (Fig. 2a). When GS activity was inhibited by MSX Ammonia assimilation (Fig. 3b), glutamine concentration failed to increase as in the control samples. An ini- tial small increase in glutamine concentra- All extractswere found to contain signifi- tion was probably due to a lag in GS inhi- cant amounts of arginine, which is thought bition by MSX. The increase in the to play a major role in nitrogen storage. glutamate pool appeared to indicate assi- Arginine was the most abundant amino milation of ammonia into glutamate by acid in the nitrogen-starved mycelia (0.8 GDH activity. !mol/g fresh weight). Free amino acid pool sizes of glutamate and glutamine Inhibition of glutamate synthase by aza- were 0.38 pmol/g and 0.19 !mol/g fresh serine blocked the transfer of amide nitro- weight, respectively, in the N-starved gen from glutumate to glutamine (Fig. 3c). mycelia. The size of the glutamate pool did not increase over 8 h, thus there was no Rapid ammonia assimilation was shown incorporation of ammonia into glutamate in the controls by marked increase in the
Discussion and Conclusions by GDH. After 4 h the glutamine concen- tration peaked and remained stable, indi- cating feedback control of GS. Enzyme assays showed that the ECM fun- gus P. tinctorius was capable of ammonia Inhibition of aminotransferases (Fig. 3d) assimilation by both GS and GDH activi- led to an accumulation of glutamate after 6 h, showing glutamate to be important in ties, and that both pathways were opera- tive during the period of high ammonia the donation of nitrogen for the anabolism of nitrogenous metabolites. availability.
Ammonia was assimilated primarily into References the amide of glutamine by the activity of Genetet I., Martin F. & Stewart G.S. (1984) GS, and transferred to glutamate-amino Nitrogen assimilation in mycorrhizas. Plant by glutamate synthase activity. Inhibition PhysioL 76, 395-399 of GS and glutamate synthase blocked the Johnson B. & Brown C.M. (1974) Enzymes of ammonia assimilation in Schizosaccharomyces synthesis of glutamine and glutamate, re- spp and in Saccharomycodes ludwigii. J. Gen. spectively. When GS was inhibited, some Microbiol. 85, 1 Ei9-172 glutamine was synthesised initially, and Lea P.J. (1985) In: Techniques in Bioproductivi- this may have accounted for the increase ty and Photosynthesis. (Coombs J., Hall D.O., Long S.P. & Scurlock J.M.O., eds.), Pergamon in glutamate concentration in this experi- Press, Oxford, pp. 173-187 ment, rather than GDH activity. P. tincto- Roon R.R., Even H.L. & Latimore E. (1974) rius appeared to assimilate ammonia via synthase: properties of the reduced Glutamate the glutamate synthase cycle, with no NAD-dependent enzyme from Saccharomyces significant role played by GDH. cerevisiae. J. B!iCteriol. 118, 89-95