The involvement of glutamate dehydrogenase and glutamine synthetase in ammonia assimilation by the rapidly growing ectomycorrhizal ascomycete, Cenococcum geophilum Fr.

Abstract

SUMMARYIn order to detern-iine signifieance oi ti-ie glutamate cieiiycirogenase pathvvav-aiui a glutamine svnthetase/glutamate synthase cycle in NH,^assimilation, we followed a t-itit-i-iher of different metholic parameters (nutrient uptake, free amino acid pools, NH|*-indueed glutamine acetimuhnion) and ''"N incorporation into an-iino acids of rapidly growing Ceiioeorciiiii geophiluin. .Arginine was a major free amino aeid in C. neopliiliiiti during its entire growth period, C. /^eophihiiii synthesized and aeeumulated very iarge an-iout-its of gliitan-iine at the heginning of tiie rapid phase of growth in low nitrogen mediun-i, ciuring the wiioie growth period in high nitrogen medium, anti immediately after addition of NH,', Therefore, the accumulation of a large an-iount of glutatnine took place when the external ammoniut-i-i concentration was high. The current data identify four pathways of N n-ietaholism in rapidly growing C. geophiluiir. (1) glutamine synthesis, involving transfer of N to hoth an-iino and amido moieties;(2) glutamate formation; (.3) transamination with pyruvate to yield aianine; (4) transamination with oxaioaeetate to yield asparate. The higher accumulation oi glutan-iate anti related ammo acitis (alanine and asparlate) in the presence of the glutamine svnthetase inhii>itor methionine sulpiioximine indicates that giutamate, the precursor of glutamine, was formed hy a pathway insensitive to methionine sulphoximine, the glutan-iate dehydrogenase pathway. Up to 40",, of the assimilated '''N terminated in the at-i-iido-N of glutamine. These data are eonsistent with a piv-otal role for glutan-iine synthetase activity and indicates that the primary assimilation of Nll,^ in rapidly growing C. geopliiliiiii is brought ahout hy coiKurrent activitv-of the (il)ll ai-iti (IS pathways. The patiiwav-of primary assimilation of NH ,* hy ('. geophituiii in the rapid phase of growth therefore differs fron-i those operating in the stationary phase of growth where N Hux through (JDH is higher than the flux through the GS pathway.

Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon–nitrogen interface

“…The biosynthetic role of fungal NADP-GDH is well supported by genetic (Fincham, 1962;Kinghorn & Pateman, 1973;Fincham et al, 2000), physiological (Martin et al, 1988;Kusnan et al, 1989;Schwartz et al, 1991) and kinetic (Hudson & Daniel, 1993) data. The kinetic constants obtained for the various substrates of the A. niger NADP-GDH (Table 2) are consistent with its role in ammonium assimilation.…”

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

“…-Nancy). The vegetative mycelium was cultivated in liquid cultures on Pachlewski's medium as described previously (Martin et al, 1988). The growth medium contained 2-5 mM diammonium tartrate as N source and glucose as carbon source.…”

“…There is evidence that primary incorporation of ^'''NH4^ in the ectomycorrhizal ascomycete Cenococcum geophilum (Genetet et al, 1984;Martin, 1985;Martin et al, 1988) and the basidiomycete Hebeloma sp. (Chalot et al, 1991) is brought about by the GDH pathway.…”

“…Despite growing circumstantial evidence based on enzymatic studies (Dell et al, 1989;Ahmad et al, 1990;Botton & Dell, 1994) and ^»N labelling experiments (Martin, 1985;Martin et al, 1988;Kershaw & Stewart, 1992), the regulation of pathways of N assimilation in ectomycorrhizal fungi remains puzzling. There is evidence that primary incorporation of ^'''NH4^ in the ectomycorrhizal ascomycete Cenococcum geophilum (Genetet et al, 1984;Martin, 1985;Martin et al, 1988) and the basidiomycete Hebeloma sp.…”

L'assimilation de l'azote minéral chez les ectomycorhizes

“…Chez Cenococcum geophilum en phase exponentielle de croissance, Ia voie GS predomine puisque les deux tiers de l'ammonium assimile sont incorpores dans Ia fonction amide de Ia glutamine (Martin et al, 1988). En revanche, Ia voie GDII devient majoritaire en phase stationnaire de croissance (Gcnetet et al, 1984).…”