Glutamate dehydrogenase from Pisum sativum L.

A 2–8-fold increase in the activity of glutamate dehydrogenase (GDH), accompanied by an alteration of the GDH isoenzyme pattern, was observed in detached pea shoots floated on tap water (preincubated shoots). Sugars supressed the process, whereas NH ₊ ⁴ and various metabolites as well as inhibitors of energy metabolism and protein synthesis were ineffective. The subcellular distribution pattern revealed evidence that the GDH isoenzymes are exclusively located in the mitochondrial matrix. The alterations in GDH activity occurring in preincubated shoots are restricted to the mitochondria.An experimental device suitable for studying the GDH function in isolated intact mitochondria has been established. Using [¹⁴C] citrate as the carbon source and hydrogen donor, the mitochondria synthesized considerable amounts of glutamate upon addition of NH ₊ ⁴ . The rates of glutamate formation in dependency of increasing NH ₊ ⁴ levels follow simple Michaelis-Menten kinetics. Half-saturation concentrations of NH ₊ ⁴ of 3.6±1.2 mM; 1.9±0.06 mM and 1.6±0.1 mM were calculated for the mitochondria isolated from pea shoots, roots, and preincubated shoots, respectively. The results are discussed in relation to the possible role of GDH in NH_+/4 assimilation at elevated intracellular NH_+/4 levels.Abbreviations GDH Glutamate dehydrogenase - MDH malate dehydrogenase - GOT aspartate aminotransferase - SDH succinate dehydrogenase - HEPES 4-(2-hydroxyethyl)-1-piperazineethan-sulfonic acid - BSA bovine serum albumin - TPP thiamine pyrophosphate - DNP 2,4-dinitrophenol - CCCP carbonyl cyanide m-chlorophenylhydrazone - DCPIP 2,6-dichlorophenolindophenol Dedicated to Professor Dr. Maximilian Steiner on the occasion of his 75th birthday     

Expression and regulation of the Escherichia coli glutamate dehydrogenase gene (gdh) in Rhizobium japonicum

The glutamate dehydrogenase (gdh) gene of Escherichia coli was transferred into an ammonium assimilation deficient mutant (Asm^-) of Rhizobium japonicum (CJ9) using plasmid pRP301, a broad host range derivative of RP₄. Exconjugants capable of growth on ammonia as sole N-source occurred at a frequency of 6.8×10^-6. Assimilatory GDH (NADP⁺) activity was detected in the strain carrying the E. coli gdh gene and the pattern of ammonia assimilation via GDH was similar to that of the Asm⁺ wild type strain. However, GDH mediated ammonia assimilation was not subject to regulation by l-glutamate. Nitrogenase activity was expressed ex planta in R. japonicum CJ9 harbouring the gdh gene, however, the presence of the gdh gene did not restore symbiotic effectiveness to the CJ9 Asm^- strain in nodules. The gdh plasmid was maintained in approximately 90% of the isolates recovered from soybean nodules.Abbreviations gdh glutamate dehydrogenase - Asm^- mutant ammonia assimilation deficient mutant     

The photoproduction of H2 and NH+4 fixed from N2 by a derepressed mutant of Rhodospirillum rubrum

A mutant of Rhodospirillum rubrum has been isolated, after mutagenesis with nitrosoguanidine, which is characterized by its inability to grow in the light on malate-minimal media with exogenous ammonia or alanine, poor growth on glutamine and vigorous growth on glutamate. This mutant produces low levels of a key NH⁺₄ assimilation enzyme, glutamate synthase (NADPH-dependent). It also exhibits significant derepression of nitrogenase biosynthesis in the presence of ammonia or alanine, being 15% derepressed for the former and about 70% derepressed for the latter.Some of this mutant's fixed N₂ is excreted into the medium as NH⁺₄ (1 μmol NH⁺₄ per mg cell protein in 50 h). Nitrogenase-mediated H₂ production by this strain is considerable (42 μmol H₂ per mg cell protein in 50 h), approximately twice that of the wild type assayed under similar conditions.These results demonstrate that genetic alteration of the photosynthetic N₂-fixer's NH⁺₄ assimilation system disrupts the tight coupling of N₂ fixation and NH⁺₄ assimilation normally observed in these organisms, enabling photochemical conversion steps to be utilized for the photoproduction of NH⁺₄ and H₂.     

Root respiration associated with ammonium and nitrate absorption and assimilation by barley

We examined nitrate assimilation and root gas fluxes in a wild-type barley (Hordeum vulgare L. cv Steptoe), a mutant (nar1a) deficient in NADH nitrate reductase, and a mutant (nar1a;nar7w) deficient in both NADH and NAD(P)H nitrate reductases. Estimates of in vivo nitrate assimilation from excised roots and whole plants indicated that the nar1a mutation influences assimilation only in the shoot and that exposure to NO₃⁻ induced shoot nitrate reduction more slowly than root nitrate reduction in all three genotypes. When plants that had been deprived of nitrogen for several days were exposed to ammonium, root carbon dioxide evolution and oxygen consumption increased markedly, but respiratory quotient—the ratio of carbon dioxide evolved to oxygen consumed—did not change. A shift from ammonium to nitrate nutrition stimulated root carbon dioxide evolution slightly and inhibited oxygen consumption in the wild type and nar1a mutant, but had negligible effects on root gas fluxes in the nar1a;nar7w mutant. These results indicate that, under NH₄⁺ nutrition, 14% of root carbon catabolism is coupled to NH₄⁺ absorption and assimilation and that, under NO₃⁻ nutrition, 5% of root carbon catabolism is coupled to NO₃⁻ absorption, 15% to NO₃⁻ assimilation, and 3% to NH₄⁺ assimilation. The additional energy requirements of NO₃⁻ assimilation appear to diminish root mitochondrial electron transport. Thus, the energy requirements of NH₄⁺ and NO₃⁻ absorption and assimilation constitute a significant portion of root respiration.     

Changes in nitrogen assimilation, metabolism, and growth in transgenic rice plants expressing a fungal NADP(H)-dependent glutamate dehydrogenase (gdhA)

In plants, glutamine synthetase (GS) is the enzyme that is mainly responsible for the assimilation of ammonium. Conversely, in microorganisms such as bacteria and Ascomycota, NADP(H)-dependent glutamate dehydrogenase (GDH) and GS both have important roles in ammonium assimilation. Here, we report the changes in nitrogen assimilation, metabolism, growth, and grain yield of rice plants caused by an ectopic expression of NADP(H)-GDH (gdhA) from the fungus Aspergillus niger in the cytoplasm. An investigation of the kinetic properties of purified recombinant protein showed that the fungal gdhA had 5.4–10.2 times higher V _max value and 15.9–43.1 times higher K _m value for NH₄ ⁺, compared with corresponding values for rice cytosolic GS as reported in the literature. These results suggested that the introduction of fungal GDH into rice could modify its ammonium assimilation pathway. We therefore expressed gdhA in the cytoplasm of rice plants. NADP(H)-GDH activities in the gdhA-transgenic lines were markedly higher than those in a control line. Tracer experiments by feeding with ¹⁵NH₄ ⁺ showed that the introduced gdhA, together with the endogenous GS, directly assimilated NH₄ ⁺ absorbed from the roots. Furthermore, in comparison with the control line, the transgenic lines showed an increase in dry weight and nitrogen content when sufficient nitrogen was present, but did not do so under low-nitrogen conditions. Under field condition, the transgenic line examined showed a significant increase in grain yield in comparison with the control line. These results suggest that the introduction of fungal gdhA into rice plants could lead to better growth and higher grain yield by enhancing the assimilation of ammonium.     

Nitrogen isotope composition of tomato (Lycopersicon esculentum Mill. cv. T-5) grown under ammonium or nitrate nutrition

Studies that quantify plant δ¹⁵N often assume that fractionation during nitrogen uptake and intra-plant variation in δ¹⁵N are minimal. We tested both assumptions by growing tomato (Lycopersicon esculetum Mill. cv. T-5) at NH₄⁺ or NO^?₃ concentrations typical of those found in the soil. Fractionation did not occur with uptake; whole-plant δ¹⁵N was not significantly different from source δ¹⁵ N for plants grown on either nitrogen form. No intra-plant variation in δ¹⁵N was observed for plants grown with NH⁺₄. In contrast. δ¹⁵N of leaves was as much as 5.8% greater than that of roots for plants grown with NO^?₃. The contrasting patterns of intra-plant variation are probably caused by different assimilation patterns. NH⁺₄ is assimilated immediately in the root, so organic nitrogen in the shoot and root is the product of a single assimilation event. NO^?₃ assimilation can occur in shoots and roots. Fractionation during assimilation caused the δ¹⁵N of NO^?₃ to become enriched relative to organic nitrogen; the δ¹⁵N of NO^?₃ was 11.1 and 12.9% greater than the δ¹⁵N of organic nitrogen in leaves and roots, respectively. Leaf δ¹⁵N may therefore be greater than that of roots because the NO^?₃ available for assimilation in leaves originates from a NO^?₃ pool that was previously exposed to nitrate assimilation in the root.     

The differing responses of two <Emphasis Type="Italic">Arabidopsis</Emphasis> ecotypes to ammonium are modulated by the photoperiod regime

Responses to excessive ammonium (NH₄ ⁺) were compared between two Arabidopsis ecotypes (Col-0, JA22) with respect to different photoperiods in hydroponics. In this study, we showed that external extra NH₄ ⁺ led to severe growth suppression, accumulations of free NH₄ ⁺ and amino acids and increased the activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in shoots of the two Arabidopsis ecotypes. However, the levels of free NH₄ ⁺ and total amino acids increased, whereas the activities of GS, NADH-dependent glutamate synthase and GDH decreased under the continuous light when compared with the light (16 h)–dark (8 h) cycle photoperiod. Statistical analyses suggested that strong correlations exist among the growth reduction, accumulations of free NH₄ ⁺, total amino acids and levels of GS activity in shoots under the high NH₄ ⁺ stress regardless of the photoperiod regimes. Interestingly, under the continuous light, Col-0 showed more resistant to such growth reduction and maintained about onefold higher capability of converting excess free NH₄ ⁺ into amino acids, with onefold higher GS activity induced by the external NH₄ ⁺ when compared with JA22. In contrast, these differences were abolished between Col-0 and JA22 under the light–dark cycle condition. Taken together, our results conclude that the sensitivity to NH₄ ⁺ of Col-0 and JA22 is changed between the continuous light and the light–dark cycle photoperiod, which is correlative to the alteration of the GS activity in shoots.     

γ‐Aminobutyric acid addition alleviates ammonium toxicity by limiting ammonium accumulation in rice (Oryza sativa) seedlings

Excessive use of nitrogen (N) fertilizer has increased ammonium (NH₄⁺) accumulation in many paddy soils to levels that reduce rice vegetative biomass and yield. Based on studies of NH₄⁺ toxicity in rice (Oryza sativa, Nanjing 44) seedlings cultured in agar medium, we found that NH₄⁺ concentrations above 0.75 mM inhibited the growth of rice and caused NH₄⁺ accumulation in both shoots and roots. Use of excessive NH₄⁺ also induced rhizosphere acidification and inhibited the absorption of K, Ca, Mg, Fe and Zn in rice seedlings. Under excessive NH₄⁺ conditions, exogenous γ‐aminobutyric acid (GABA) treatment limited NH₄⁺ accumulation in rice seedlings, reduced NH₄⁺ toxicity symptoms and promoted plant growth. GABA addition also reduced rhizosphere acidification and alleviated the inhibition of Ca, Mg, Fe and Zn absorption caused by excessive NH₄⁺. Furthermore, we found that the activity of glutamine synthetase/NADH‐glutamate synthase (GS; EC 6.3.1.2/NADH‐GOGAT; EC1.4.1.14) in root increased gradually as the NH₄⁺ concentration increased. However, when the concentration of NH₄⁺ is more than 3 mM, GABA treatment inhibited NH₄⁺‐induced increases in GS/NADH‐GOGAT activity. The inhibition of ammonium assimilation may restore the elongation of seminal rice roots repressed by high NH₄⁺. These results suggest that mitigation of ammonium accumulation and assimilation is essential for GABA‐dependent alleviation of ammonium toxicity in rice seedlings.     

Ammonia Assimilation in Alnus glutinosa and Glycine max: SHORT-TERM STUDIES USING [N]AMMONIUM

The pattern of assimilation of NH₄⁺ by Alnus glutinosa, a N₂-fixing, nonleguminous angiosperm, was examined. Detached nodules, roots, and nodulated roots of intact plants were exposed to ¹³NH₄⁺ for up to 15 minutes. Glutamine was the most highly labeled compound at all times; the only other compound labeled significantly was glutamate. Similar results were obtained after incubating soybean (L. merr) nodules and roots with ¹³NH₄⁺. These observations and the results of pulse-labeling and inhibitor studies with nodules of Alnus were distinctly different from those predicted for the assimilation of NH₄⁺ via glutamine synthetase and glutamate synthase and suggest that glutamate dehydrogenase may play a major role in the assimilation of exogenously supplied NH₄⁺.     

Control of symbiotic nitrogen fixation in Rhizobia regulation of NH4 assimilation

The metabolic fate of gaseous nitrogen (¹⁵N₂) fixed by free-living cultures of Rhizobia (root nodule bacteria) induced for their N₂-fixation system was followed. A majority of the fixed ¹⁵N₂ was found to be exported into the cell supernatant. For example, as much as 94% of the ¹⁵N₂ fixed by Rhizobium japonicum (soybean symbiont) was recovered as ¹⁵NH₄⁺ from the cell supernatant following alkaline diffusion. Several species of root nodule bacteria also exported large quantities of NH₄⁺ from l-histidine. Evidence is presented that overproduction and export of NH₄⁺ by free-living Rhizobia may be closely linked to the control of several key enzymes of NH₄⁺ assimilation. For instance, NH₄⁺ was found to repress glutamine synthetase whereas l-glutamate repressed glutamate synthase. Assimilation of NH₄⁺ as nitrogen source for growth of Rhizobia was inhibited by glutamate. The mechanism of regulation of NH₄⁺ production by root nodule bacteria is discussed.     

Regulation of nitrogen fixation by Rhizobia export of fixed N2 as NH4+

The metabolic fate of gaseous nitrogen (¹⁵N₂) fixed by free-living cultures of Rhizobia (root nodule bacteria) induced for their N₂-fixation system was followed. A majority of the fixed ¹⁵N₂ was found to be exported into the cell supernatant. For example, as much as 94% of the ¹⁵N₂ fixed by Rhizobium japonicum (soybean symbiont) was recovered as ¹⁵NH₄⁺ from the cell supernatant following alkaline diffusion. Several species of root nodule bacteria also exported large quantities of NH₄⁺ from l-histidine. Evidence is presented that overproduction and export of NH₄⁺ by free-living Rhizobia may be closely linked to the control of several key enzymes of NH₄⁺ assimilation. For instance, NH₄⁺ was found to repress glutamine synthetase whereas l-glutamate repressed glutamate synthase. Assimilation of NH₄⁺ as nitrogen source for growth of Rhizobia was inhibited by glutamate. The mechanism of regulation of NH₄⁺ production by root nodule bacteria is discussed.     

Uptake and Assimilation of NO(3) and NH(4) by Nitrogen-Deficient Perennial Ryegrass Turf

Assimilation of NO₃⁻ and NH₄⁺ by perennial ryegrass (Lolium perenne L.) turf, previously deprived of N for 7 days, was examined. Nitrogen uptake rate was increased up to four- to five-fold for both forms of N by N-deprivation as compared to N-sufficient controls, with the deficiency-enhanced N absorption persisting through a 48 hour uptake period. Nitrate, but not NH₄⁺, accumulated in the roots and to a lesser degree in shoots. By 48 hours, 53% of the absorbed NO₃⁻ had been reduced, whereas 97% of the NH₄⁺ had been assimilated. During the early stages (0 to 8 hours) of NO₃⁻ uptake by N-deficient turf, reduction occurred primarily in the roots. Between 8 and 16 hours, however, the site of reduction shifted to the shoots. Nitrogen form did not affect partitioning of the absorbed N between roots (40%) and shoots (60%) but did affect growth. Compared to NO₃⁻, NH₄⁺ uptake inhibited root, but not shoot, growth. Total soluble carbohydrates decreased in both roots and shoots during the uptake period, principally the result of fructan metabolism. Ammonium uptake resulted in greater total depletion of soluble carbohydrates in the root compared to NO₃⁻ uptake. The data indicate that N assimilation by ryegrass turf utilizes stored sugars but is also dependent on current photosynthate.     

Absorption of nitrate and ammonium ions by Lolium perenne from flowing solution cultures at low root temperatures

Lolium perenne L. cv. 23 (perennial ryegrass) plants were grown in flowing solution culture and acclimatized over 49 d to low root temperature (5°C) prior to treatment at root temperatures of 3, 5, 7 and 9°C for 41 d with common air temperature of 20/15°C day/night and solution pH 5·0. The effects of root temperature on growth, uptake and assimilation of N were compared with N supplied as either NH₄ or NO3 at 10 mmol m^?3. At any given temperature, the relative growth rate (RGR) of roots exceeded that of shoots, thus the root fraction (Rf) increased with time. These effects were found in plants grown with the two N sources. Plants grown at 3 and 5°C had very high dry matter contents as reflected by the fresh weight: freeze-dried weight ratio. This ratio increased sharply, especially in roots at 7 and 9°C. Expressed on a fresh weight basis, there was no major effect of root temperature on the [N] of plants receiving NHJ but at any given temperature, the [N] in plants grown with NHJ was significantly greater than in those grown with NO₃. The specific absorption rate (SAR) of NH⁺₄ was greater at all temperatures than SAR-NO₃. In plants grown with NH⁺, 3–5% of the total N was recovered as NH⁺₄, whereas in those grown with NO^?₃ the unassimilated NO^?₃ rose sharply between 7 and 9°C to become 14 and 28% of the total N in shoots and roots, respectively. The greater assimilation of NH⁺₄ lead to concentrations of insoluble reduced N (= protein) which were 125 and 20% greater, in roots and shoots, respectively, than in NO^?₃-grown plants. Plants grown with NH⁺₄ had very much greater glutamine and asparagine concentrations in both roots and shoots, although other amino acids were more similar in Concentration to those in NO^?₃ grown plants. It is concluded that slow growth at low root temperature is not caused by restriction of the absorption or assimilation of either NH⁺₄ or NO^?₃. The additional residual N (protein) in NH⁺₄ grown plants may serve as a labile store of N which could support growth when external N supply becomes deficient.     

Reassimilation of Photorespiratory Ammonium in Lotus japonicus Plants Deficient in Plastidic Glutamine Synthetase

It is well established that the plastidic isoform of glutamine synthetase (GS₂) is the enzyme in charge of photorespiratory ammonium reassimilation in plants. The metabolic events associated to photorespiratory NH₄⁺ accumulation were analyzed in a Lotus japonicus photorespiratory mutant lacking GS₂. The mutant plants accumulated high levels of NH₄⁺ when photorespiration was active, followed by a sudden drop in the levels of this compound. In this paper it was examined the possible existence of enzymatic pathways alternative to GS₂ that could account for this decline in the photorespiratory ammonium. Induction of genes encoding for cytosolic glutamine synthetase (GS₁), glutamate dehydrogenase (GDH) and asparagine synthetase (ASN) was observed in the mutant in correspondence with the diminishment of NH₄⁺. Measurements of gene expression, polypeptide levels, enzyme activity and metabolite levels were carried out in leaf samples from WT and mutant plants after different periods of time under active photorespiratory conditions. In the case of asparagine synthetase it was not possible to determine enzyme activity and polypeptide content; however, an increased asparagine content in parallel with the induction of ASN gene expression was detected in the mutant plants. This increase in asparagine levels took place concomitantly with an increase in glutamine due to the induction of cytosolic GS₁ in the mutant, thus revealing a major role of cytosolic GS₁ in the reassimilation and detoxification of photorespiratory NH₄⁺ when the plastidic GS₂ isoform is lacking. Moreover, a diminishment in glutamate levels was observed, that may be explained by the induction of NAD(H)-dependent GDH activity.     

NH4+-Excreting Azospirillum brasilense Mutants Enhance the Nitrogen Supply of a Wheat Host

Spontaneous ethylenediamine-resistant mutants of Azospirillum brasilense were selected on the basis of their excretion of NH₄⁺. Two mutants exhibited no repression of their nitrogenase enzyme systems in the presence of high (20 mM) concentrations of NH₄⁺. The nitrogenase activities of these mutants on nitrogen-free minimal medium were two to three times higher than the nitrogenase activity of the wild type. The mutants excreted substantial amounts of ammonia when they were grown either under oxygen-limiting conditions (1 kPa of O₂) or aerobically on nitrate or glutamate. The mutants grew well on glutamate as a sole nitrogen source but only poorly on NH₄Cl. Both mutants failed to incorporate [¹⁴C]methylamine. We demonstrated that nitrite ammonification occurs in the mutants. Wild-type A. brasilense, as well as the mutants, became established in the rhizospheres of axenically grown wheat plants at levels of > 10⁷ cells per g of root. The rhizosphere acetylene reduction activity was highest in the preparations containing the mutants. When plants were grown on a nitrogen-free nutritional medium, both mutants were responsible for significant increases in root and shoot dry matter compared with wild-type-treated plants or with noninoculated controls. Total plant nitrogen accumulation increased as well. When they were exposed to a ¹⁵N₂-enriched atmosphere, both A. brasilense mutants incorporated significantly higher amounts of ¹⁵N inside root and shoot material than the wild type did. The results of our nitrogen balance and ¹⁵N enrichment studies indicated that NH₄⁺-excreting A. brasilense strains potentially support the nitrogen supply of the host plants.     

PATHWAY OF AMMONIUM ASSIMILATION IN A MARINE DIATOM DETERMINED WITH THE RADIOTRACER 13N1

In unicellular algae, ammonium can be assimilated into glutamate through the action of glutamate dehydrogenase (GDH) or into glutamine through the sequential activities of glutamine synthetase and glutamate 2-oxoglutarate amidotransferase (GS-GOGAT pathway). We have shown that the first radio-labeled product of assimilation of ¹³NH₄⁺ (t_1/2= 10 min) was glutamine in the marine diatom Thalassiosira pseudonana (Hustedt). When GS-GOGAT was inhibited with methionine sulfoximine, the incorporation of radioactivity into both glutamine and glutamate was blocked, implying that the radio-labeled glutamate is formed from glutamine. Glutamine was also the first labeled product when the intracellular concentration of ammonium was elevated by preincubation with unlabeled ammonium. The results indicate that the GS-GOGAT pathway is the primary pathway for the assimilation of nitrogen in T. pseudonana.     

Root and Nodule Enzymes of Ammonia Assimilation in Two Plant-Conditioned Symbiotically Ineffective Genotypes of Alfalfa (Medicago sativa L.)

Biochemical and physiological parameters associated with nitrogen metabolism were measured in nodules and roots of glasshouse-grown clones of two symbiotically ineffective alfalfa (Medicago sativa L.) genotypes supplied with either NO₃⁻ or NH₄⁺. Significant differences were observed between genotypes for nodule soluble protein concentrations and glutamine synthetase (GS) and glutamate synthase (GOGAT) specific activities, both in untreated controls and in response to applied N. Nodule soluble protein of both genotypes declined in response to applied N, while nodule GS, GOGAT, and glutamate dehydrogenase (GDH) specific activities either decreased or remained relatively constant. In contrast, no genotype differences were observed in roots for soluble protein concentrations and GS, GOGAT, and GDH specific activities, either in untreated controls or in response to applied N. Root soluble protein levels and GS and GOGAT specific activities of N-treated plants increased 2- to 4-fold within 4 days and then decreased between days 13 and 24. Root GDH specific activity of NH₄⁺-treated plants increased steadily throughout the experiment and was 50 times greater than root GS or GOGAT specific activities by day 24.     

Assimilation of inorganic nitrogen by a mycobiont isolated from Pisonia grandis R. Br. (Nyctaginaceae) mycorrhiza

 Single isolates of a mycobiont isolated from Pisonia grandis R. Br., Pisolithus tinctorius (Pers.) Coker & Couch and Tylospora fibrillosa (Burt.) Donk were compared with regard to their relative abilities to produce key enzymes of inorganic nitrogen assimilation. Nitrate reductase (NR) activities in the P. grandis mycobiont and T. fibrillosa were significantly lower than in P. tinctorius. While specific activities for glutamate dehydrogenase (GDH) were higher in P. tinctorius than the other two fungi following NH₄ ⁺ pre-treatment, glutamine synthetase (GS) activity did not differ significantly between the three fungi. In all three fungi, specific activities for GS were significantly higher than for GDH. NR activity was expressed in all three fungi regardless of the nitrogen source in the medium, but in P. tinctorius diminished following continued exposure to either NO₃ ^–, NH₄ ⁺, glutamine or NO₃ ^– + glutamine. The data are discussed in relation to nitrogen utilisation by the P. grandis mycobiont. Accepted: 16 October 1997     

Nitrogen Assimilation in Mycorrhizas : Ammonium Assimilation in the N-Starved Ectomycorrhizal Fungus Cenococcum graniforme

Ammonium assimilation was followed in N-starved mycelia from the ectomycorrhizal Ascomycete Cenococcum graniforme. The evaluation of free amino acid pool levels after the addition of 5 millimolar NH₄⁺ indicated that the absorbed ammonium was assimilated rapidly. Post-feeding nitrogen content of amino acids was very different from the initial values. After 8 hours of NH₄⁺ feeding, glutamine accounted for the largest percentage of free amino acid nitrogen (43%). The addition of 5 millimolar methionine sulfoximine (MSX) to NH₄⁺-fed mycelia caused an inhibition of glutamine accumulation with a corresponding increase in glutamate and alanine levels.

Using ¹⁵N as a tracer, it was found that the greatest initial labeling was into glutamine and glutamate followed by aspartate, alanine, and ornithine. On inhibiting glutamine synthetase using MSX, ¹⁵N enrichment of glutamate, alanine, aspartate, and ornithine continued although labeling of glutamine was quite low. Moreover, the incorporation of ¹⁵N label in insoluble nitrogenous compounds was lower in the presence of MSX. From the composition of free amino acid pools, the ¹⁵N labeling pattern and effects of MSX, NH₄⁺ assimilation in C. graniforme mycelia appears to proceed via glutamate dehydrogenase pathway. This study also demonstrates that glutamine synthesis is an important reaction of ammonia utilization.