Observations on the subcellular distribution of the ammonium ion in maize root tissue using in-vivo 14N-nuclear magnetic resonance spectroscopy

We show that the pH dependence of the base-catalysed exchange rate of the ammonium ion provides a basis for discriminating between the cytoplasmic and vacuolar pools of ammonium in plant tissues. In vivo, ¹⁴N-nuclear magnetic resonance spectra were recorded with and without ¹H-decoupling and information on the subcellular distribution of NH ₄ ⁺ was obtained from a lineshape analysis of the ¹H-coupled spectrum. We applied this method to maize (Zea mays L.) root tissues and found that: (i), the cytoplasmic ammonium concentration was low, which was in accord with the large activity of glutamine synthetase present in the roots; and (ii), inhibition of glutamine synthetase with methionine sulphoximine increased the cytoplasmic ammonium concentration, and led to the appearance of ammonium in the xylem sap.Abbreviations GS glutamine synthetase - MSO l-methionine sulphoximine - NMR nuclear magnetic resonance - Pi inorganic phosphate On secondment to the Department of Plant Sciences, University of Oxford.We acknowledge the financial support of the Agricultural and Food Research Council. R.B. Lee also thanks the Department of Plant Sciences, University of Oxford, for hospitality.     

Regulation of ammonium and potassium uptake by glutamine and asparagine in Pisum arvense plants

Pisum arvense plants were subjected to 5 days of nitrogen deprivation. Then, in the conditions that increased or decreased the root glutamine and asparagine pools, the uptake rates of 0.5 mM NH₄ ⁺ and 0.5 mM K⁺ were examined. The plants supplied with 1 mM glutamine or asparagine took up ammonium and potassium at rates lower than those for the control plants. The uptake rates of NH₄ ⁺ and K⁺ were not affected by 1 mM glutamate. When the plants were pre-treated with 100 μM methionine sulphoximine, an inhibitor of glutamine synthesis, the efflux of NH₄ ⁺ from roots to ambient solution was enhanced. On the other hand, exposure of plants to methionine sulphoximine led to an increase in potassium uptake rate. The addition of asparagine, glutamine or glutamate into the incubation medium caused a decline in the rate of NH₄ ⁺ uptake by plasma membrane vesicles isolated from roots of Pisum arvense, whereas on addition of methionine sulphoximine increased ammonium uptake. The results indicate that both NH₄ ⁺ and K⁺ uptake appear to be similarly affected by glutamine and asparagine status in root cells. The research was supported by grant of KBN No. 6PO4C 068 08     

Stimulation of bicarbonate incorporation by ammonium in nonphotosynthetic cell suspension cultures of Acer pseudoplatanus

Ammonium supplied to Acer cells incubated in Tricine buffer raised net dark incorporation of ¹⁴C-bicarbonate by 1.5 to 3.3 fold; this stimulation was not abolished by prior inhibition of glutamine synthetase by methionine sulphoximine. With cells in phosphate buffer, ammonium gave a smaller 1.15 to 1.3 fold stimulation which was abolished when glutamine synthetase was inhibited. Ammonium had no direct effect on the activities of phosphoenolpyruvate carboxylase and pyruvate kinase assayed in partially purified extracts or on the enzymes catalysing release of label.     

Nitrogen assimilation in Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 assimilated ammonia via a constitutive glutamine synthetase/glutamate synthase enzyme system.Glutamine synthetase had a K _m for NH ₄ ⁺ of 0.38 mM whilst the nicotinamide adenine dinucleotide linked glutamate synthase had a K _m for glutamine of 0.55 mM. R. acidophila utilized only a limited range of amino acids as sole nitrogen sources: l-alanine, glutamine and asparagine. The bacterium did not grow on glutamate as sole nitrogen source and lacked glutamate dehydrogenase. When R. acidophila was grown on l-alanine as the sole nitrogen source in the absence of N₂ low levels of a nicotinamide adenine dinucleotide linked l-alanine dehydrogenase were produced. It is concluded, therefore, that this reaction was not a significant route of ammonia assimilation in this bacterium except when glutamine synthetase was inhibited by methionine sulphoximine. In l-alanine grown cells the presence of an active alanine-glyoxylate aminotransferase and, on occasions, low levels of an alanine-oxaloacetate aminotransferase were detected. Alanine-2-oxo-glutarate aminotransferase could not be demonstrated in this bacterium.Abreviations ADH alanine dehydrogenase - GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulphoximine     

Metabolism of 15N-labelled ammonium by the ectomycorrhizal fungus Pisolithus tinctorim (Pers.) Coker & Couch

Summary Glutamine was the major product accumulated following transfer of nitrogen-limited cultures of the ectomycorrhizal fungus Pisolithus tinctorius to an ammonium medium. Experiments in which mycelium was transferred to [¹⁵N]H ₄ ⁺ showed glutamine amide was the most heavily labelled product. Assimilation of ammonium into glutamate was markedly inhibited by azaserine. The kinetics of ¹⁵N-labelling and the effects of azaserine and methionine sulphoximine on the distribution of ¹⁵N-labelled products are entirely consistent with the operation of the glutamate synthase cycle. No evidence was found for ammonium assimilation via glutamate dehydrogenase. The labelling pattern observed in mycelium treated with aminooxyacetate suggests that transamination reactions are an important source of glutamate for the synthesis of glutamine.     

Sources of ammonium in oat leaves treated with tabtoxin or methionine sulfoximine

Excised 7-day-old oat (Avena sativa L. cv. Jaycee) leaves were incubated in media containing 7.1 millimolar KNO₃ and 0.15 millimolar tabtoxin or 1 millimolar methionine sulfoximine (MSO) to investigate the sources of the observed ammonium accumulated. Tabtoxin and MSO are known inhibitors of glutamine synthetase, the first enzyme in the primary pathway of ammonium assimilation. During a 4- to 6-hour incubation, there was little net change in protein or total amino acid concentration. Alanine, aspartate/asparagine, and glutamate/glutamine decreased markedly under these treatments, whereas several other amino acids increased. Exogenous ¹⁵N from K¹⁵NO₃ was taken up and incorporated into the nitrate and ammonium fractions of leaves treated with tabtoxin or MSO. This result and the high in vitro activities of nitrate reductase indicated that reduction of nitrate was one source of the accumulated ammonium. Leaves incubated under 2% O₂ to reduce photorespiration accumulated only about 13% as much ammonium as did those under normal atmospheres. We conclude that most of the tabtoxin- or MSO-induced ammonium came from photo-respiration, and the remainder was from nitrate reduction.     

Disturbed ammonium assimilation is associated with growth inhibition of roots in rice seedlings caused by NaCl

The effects of NaCl on changes in ammonium level and enzyme activities of ammonium assimilation in roots growth of rice (Oryza sativa L.) seedlings were investigated. NaCl was effective in inhibiting root growth and stimulated the accumulation of ammonium in roots. Accumulation of ammonium in roots preceded inhibition of root growth caused by NaCl. Both effects caused by NaCl are reversible. Exogenous ammonium chloride and methionine sulfoximine (MSO), which caused ammonium accumulation in roots, inhibited root growth of rice seedlings. NaCl decreased glutamine synthetase and glutamate synthase activities in roots, but increased glutamate dehydrogenase activity. The growth inhibition of roots by NaCl or MSO could be reversed by the addition of L-glutamic acid or L-glutamine. The current results suggest that disturbance of ammonium assimilation in roots may be involved in regulating root growth reduction caused by NaCl.Abbreviations GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSO methionine sulfoximine     

Changes in intracellular and extracellular α-amino acids in Gloeothece during N2-fixation and following addition of ammonium

Changes in extracellular and intracellular free amino acids were followed during cyclic phases of N₂-fixation (acetylene reduction) by cultures of the axenic, non-heterocystous cyanobacterium Gloeothece incubated under alternating light and darkness or continuous illumination. Changes in intracellular amino acids were minor, with only arginine (increasing during N₂-fixation) and glutamate (decreasing during fixation) showing significant changes in cells incubated under 12 h light: 12 h dark. The intracellular concentration of glutamine in cultures was always very low and the value of the ratio glutamine: glutamate (GLN:GLU), used as an index of C–N status in eukaryote microbes, was consistently less than 0.05 suggesting that the cells were nitrogen-stressed. On addition of ammonium, there was a transient accumulation of intracellular glutamine, and the ratio GLN:GLU increased rapidly to a value greater than 0.5, typical of unstressed eukaryotes. In contrast to intracellular amino acids, there were significant changes in extracellular amino acids in cultures incubated under alternating light and darkness. Glycine, serine and alanine were released during the dark phase and were taken up again in the light, paralleling the diurnal pattern of nitrogenase activity (high in darkness). It is postulated that this release is usually retained in the mucilage surrounding the cells (but disturbed during even gentle filtration) and that this mucilage may constitute an extracellular vacuole.     

Light-Dependent Ammonium Ion Toxicity of Rice Leaves in Response to Phosphinothricin Treatment

Ammonium ion accumulation in detached rice leaves treated with phosphinothricin (PPT), an inhibitior of glutamine synthetase (GS), was investigated in the light and darkness. PPT treatment increased NH₄ ⁺ content and induced toxicity in rice leaves in the light but not in darkness, suggesting the importance of light in PPT-induced NH₄ ⁺ toxicity in detached rice leaves. PPT treatment in the light resulted in a decrease of activities of the cytosolic form of GS and the chloroplastic form of GS. The photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea reduced NH₄ ⁺ accumulation induced by PPT in the light. In darkness, PPT-induced NH₄ ⁺ accumulation and toxicity were observed in the presence of glucose or sucrose.     

The Effect of Methionine Sulphoximine on the Absorption of Ammonium by Maize and Barley Roots over Short Periods

Pre-treating intact, ammonium-grown plants of maize and barleywith the glutamine synthetase inhibitor, L-methionine sulphoximine(MSO), increased subsequent accumulation of ¹³N or ¹⁵N fromlabelled ammonium very markedly—up to 8-fold in extremecases. The phenonemon could not be satisfactorily explainedin terms of an effect of MSO on the cation-exchange propertiesof the root free space, because neither potassium (⁸⁶Rb⁺) norsodium absorption was enhanced by MSO pretreatment, and rapidabsorption of ammonium continued for too long in relation tothe likely free-space content of a monovalent cation. Instead,the response is seen as a consequence of the enlargement ofthe cytoplasmic and vacuolar ammonium pools that occurs whenammonium assimilation is inhibited by MSO. The results, whichincluded comparisons between short-term and longer-term ratesof ammonium absorption, provide tentative evidence of rapidfluxes of ammonium through the plasma membrane. Key words: Cytoplasm, vacuole, flux, transport     

Photorespiratory ammonium release by the cyanobacterium Anabaena cylindrica in the presence of methionine sulfoximine

A release of ammonium by non-nitrogen-fixing Anabaena cylindrica (grown on NH₄Cl) in the presence of MSX (methionine sulfoximine) and absence of any external nitrogen source was found. In the light the release was maximal at 0.2 mM MSX, a concentration which did not affect net CO₂ fixation nor the glycollate excretion, but inhibited the glutamine synthetase activity and the reassimilation of ammonium. It is suggested that the major source of the ammonium released is the photorespiratory conversion of glycine to serine as (1) the release was stimulated by increase in light intensity, (2) high CO₂ (3%) lowered the release, if not given as a longer pretreatment (as CO₂ or HCO ₃ ^- ) when a stimulation was observed, (3) glyoxylate and glutamate stimulated the release, the latter compound particularly under nitrogen-deficient conditions and (4) isonicotinic acid hydrazide caused a reduced release of ammonium. Furthermore, a substantial part of the ammonium released by N₂-fixing A. cylindrica in presence of MSX may thus originate from the glycollate pathway. The data show that in the light the glycine to serine conversion is active in cyanobacteria with a concomitant production of ammonium which is assimilated by glutamine synthetase.Abbreviations MSX L-methionine-Dl-sulfoximine - INH isonicotinic acid hydrazide - RuDP ribulose 1,5-diphosphate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - GS glutamine synthetase - GOGAT glutamate synthase - DTT Dl-dithiothreitol     

Fixation of [13N]N2 and transfer of fixed nitrogen in the Anthoceros-Nostoc symbiotic association

The initial product of fixation of [¹³N]N₂ by pure cultures of the reconstituted symbiotic association between Anthoceros punctatus L. and Nostoc sp. strain ac 7801 was ammonium; it accounted for 75% of the total radioactivity recovered in methanolic extracts after 0.5 min and 14% after 10 min of incubation. Glutamine and glutamate were the primary organic products synthesized from [¹³N]N₂ after incubation times of 0.5–10 min. The kinetics of labeling of these two amino acids were characteristic of a precursor (glutamine) and product (glutamate) relationship. Results of inhibition experiments with methionine sulfoximine (MSX) and diazo-oxonorleucine were also consistent with the assimilation of N₂-derived NH ₄ ⁺ by Anthoceros-Nostoc through the sequential activities of glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.7.1), with little or no assimilation by glutamate dehydrogenase (EC 1.3.1.3). Isolated symbiotic Nostoc assimilated exogenous ¹³NH ₄ ⁺ into glutamine and glutamate and their formation was inhibited by MSX, indicating operation of the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway: However, relative to free-living cultures, isolated symbiotic Nostoc assimilated 80% less exogenous ammonium into glutamine and glutamate, implying that symbiotic Nostoc could assimilate only a fraction of N₂-derived NH ₄ ⁺ . This implication was tested by using Anthoceros associations reconstituted with wild-type or MSX-resistant strains of Nostoc incubated with [¹³N]N₂ in the presence of MSX. The results of these experiments indicated that, in situ, symbiotic Nostoc assimilated about 10% of the N₂-derived NH ₄ ⁺ and that NH ₄ ⁺ was made available to Anthoceros tissue where it was apparently assimilated by the GS-GOGAT pathway. Since less than 1% of the fixed N₂ was lost to the suspension medium, it appears that transfer of NH ₄ ⁺ from symbiont to host tissue was very efficient in this extracellular symbiotic association.Abbreviations DON 6-diazo-5-oxo-l-norleucine - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSX l-methionine-dl-sulfoximine     

Some regulatory aspects of [14C]methylamine influx intoPisum sativum L. cv. Feltham First seedlings

[¹⁴C]Methylamine influx intoPisum sativum L. cv. Feltham First seedlings showed Michaelis-Menten-type kinetics with apparentV _max=49.2 mol·g^-1 FW·h^-1 and apparentK _m=0.51 mM. The competitive interactions between ammonium and methylamine were most obvious when biphasic kinetics were assumed with saturation of the first phase at 0.05 mM. The inhibitor constant for ammonium (K _i)=0.027 mM. When [¹⁴C]methylamine was used in trace amounts with ammonium added as substrate, the influx of tracer showed Michaelis-Menten-type kinetics with apparentV _max=3.46 mol·g^-1 FW·h^-1 and apparentK _m=0.15 mM. The initial rate of net ammonium uptake corresponded with that found when [¹⁴C]methylamine was used to trace ammonium influx. The latter was also stimulated by high pH_o and inhibited by nitrate. Ammonium pretreatment±methionine sulphoximine or glutamine pretreatment of the seedlings inhibited subsequent [¹⁴C]methylamine influx, while methylamine or asparagine pretreatment stimulated [¹⁴C]methylamine influx. There was also a stimulatory effect of prior inoculation withRhizobium. The results are discussed in terms of current models for the regulation of ammonium uptake in plants.     

Alteration of cyanobacterial glutamine synthetase activity in vivo in response to light and NH 4 +

Extractable glutamine synthetase activity of the cyanobacterium Anabaena cylindrica was reduced by approximately 50% when N₂-fixing cultures were treated with 10 mM NH ₄ ⁺ or were placed in darkness. The deactivated enzyme could be rapidly reactivated (within 5 min) by adding 40 mM 2-mercaptoethanol to the biosynthetic reaction mixture. The enzyme could also be reactivated in vivo by replacing the culture in light or by removing NH ₄ ⁺ . When the enzyme was deactivated by simultaneously adding NH ₄ ⁺ and placing the culture in darkness, reactivation occurred on reillumination and removal of NH ₄ ⁺ . The removal of NH ₄ ⁺ in darkness did not result in reactivation. On in vitro reactivation of glutamine synthetase from dark or NH ₄ ⁺ -treated cultures the maximum glutamine synthetase activity observed frequently exceeded that of glutamine synthetase extracted from untreated cultures. Anacystis nidulans showed a similar type of reversible dark deactivation to A. cylindrica but Plectonema boryanum and a Nostoc did not. With A. cylindrica, a direct positive correlation between the size of the intracellular pool of glutamate and biosynthetic glutamine synthetase activity occurred during light/dark shifts, and on treatment with NH ₄ ⁺ . The changes in activity of glutamine synthetase in A. cylindrica in response to light resemble in some respects the light modulation of enzymes of the oxidative and reductive pentose phosphate pathways noted in cyanobacteria by others.     

THE EFFECT OF METHIONINE SULPHOXIMINE ON THE INCORPORATION OF LABELLED GLUCOSE, ACETATE, PHENYLALANINE AND PROLINE INTO GLUTAMATE AND RELATED AMINO ACIDS IN THE BRAINS OF MICE

—The incorporation of radioactivity from labelled glucose, acetate, phenylalanine and proline into glutamate, aspartate and glutamine was measured in mice treated with methionine sulphoximine and in the control animals. The labelled precursors were injected and their incorporation determined before the onset of convulsions. The incorporation of radioactivity from labelled glucose into the dicarboxylic amino acids was reduced, in particular the incorporation into glutamine. The incorporation of radioactivity from labelled acetate and phenylalanine into glutamate and aspartate was increased by methionine sulphoximine, while the incorporation into glutamine was not changed very much. The labelling of glutamine, relative to glutamate, was reduced with all precursors, indicating that glutamine synthetase was inhibited in vivo by methionine sulphoximine. It is very likely that methionine sulphoximine affects many aspects of energy metabolism in brain; in particular the metabolism of glucose seems to be inhibited, while the rate of conversion of substrates other than glucose seems to be increased.     

Isolation,improvement and characterization of an ammonium excreting mutant strain of the heterocytous cyanobacterium,Anabaena variabilis PCC 7937

Besides potential applications in the agriculture field as natural nitrogen fertilizer, N₂-fixing cyanobacteria have recently gained some attentions for new applications linked to the potential production of biologically active molecules or biohydrogen. Ammonium bioproduction is also gaining attention with the potential use of microalgae in biofuels production and the concerns about the increasing needs for nitrogen substrates. This study has investigated some phenotypic traits linked to biomass production and ammonium release in multicellular cyanobacteria, Anabaena variabilis PCC 7937. It confirms that this wild-type strain has no natural ability for ammonium excretion under diazotrophic conditions. A mutant strain, A. variabilis PCC 7937-C9, was obtained after double random mutagenesis treatments with ethyl methane–sulfonate and screening in batch cultures for resistance to the effect of a glutamine synthetase inhibitor, l-methionine-d,l-sulfoximine (MSX). Although significantly characterized by shorter cell filaments, the growth parameters in photobioreactors of the mutant strain cultures were in the same range of values than those of the wild type. In the presence of MSX this strain was shown to produce extracellular ammonium, with specific rates up to 4.9 μmol NH₄⁺ mg Chl a⁻¹ h⁻¹. The efficiency of this strain, estimated by its specific rate of ammonium excretion, was shown to be improved after consecutive batch cultures with increasing concentrations of MSX. Such mutant strains are of potential use for investigating ways to improve extracellular ammonium bioproduction.     

Assimilation of exogenous and dinitrogen-derived13NH 4 + byAnabaena azollae separated fromAzolla caroliniana Willd

Anabaena azollae was isolated fromAzolla caroliniana by the gentle roller method and differential centrifugation. Incubation of suchAnabaena preparations for 10 min with [¹³N]N₂ resulted in the formation of four radioactive compounds; ammonium, glutamine, glutamate and alanine. Ammonium accounted for 66% of the total radioactivity recovered and 58% of the ammonium was in an extracellular fraction. Since essentially no extracellular¹³N-labeled organic compounds were found, it appears that ammonium is the compound most probably made available toAzolla during dinitrogen-dependent growth of the association.The kinetics of incorporation of exogenous¹³NH ₄ ⁺ into glutamine and glutamate were characteristic of a precursor (glutamine)-product (glutamate) relationship and consistent with assimilation by the glutamine synthetase-glutamate synthase pathway. The results of experiments using the glutamine synthetase inhibitor, methionine sulfoximine, the glutamate synthase inhibitor, diazo-oxonorleucine, and increasing the ammonium concentration to greater than 1 mM, provided evidence for assimilation primarily by the glutamine synthetase-glutamate synthase pathway with little or no contribution from biosynthetic glutamate dehydrogenase.While showing that N₂ fixation and NH ₄ ⁺ assimilation were not tightly coupled metabolic processes in symbioticAnabaena, these results reflect a composite picture and do not indicate the extent to which ammonium assimilatory enzymes might be regulated in filaments associated with specific stages in theAzolla-Anabaena developmental profile.Non-standard abbreviations DON 6-Diazo-5-oxo-l-norleucine - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSX l-methionine-Dl-sulfoximine     

Inhibition of glutamine synthesis induces glutamate dehydrogenase-dependent ammonia fixation into alanine in co-cultures of astrocytes and neurons

It has been previously demonstrated that ammonia exposure of neurons and astrocytes in co-culture leads to net synthesis not only of glutamine but also of alanine. The latter process involves the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT). In the present study it was investigated if the glutamine synthetase (GS) inhibitor methionine sulfoximine (MSO) would enhance alanine synthesis by blocking the GS-dependent ammonia scavenging process. Hence, co-cultures of neurons and astrocytes were incubated for 2.5 h with [U-¹³C]glucose to monitor de novo synthesis of alanine and glutamine in the absence and presence of 5.0 mM NH₄Cl and 10 mM MSO. Ammonia exposure led to increased incorporation of label but not to a significant increase in the amount of these amino acids. However, in the presence of MSO, glutamine synthesis was blocked and synthesis of alanine increased leading to an elevated content intra- as well as extracellularly of this amino acid. Treatment with MSO led to a dramatic decrease in glutamine content and increased the intracellular contents of glutamate and aspartate. The large increase in alanine during exposure to MSO underlines the importance of the GDH and ALAT biosynthetic pathway for ammonia fixation, and it points to the use of a GS inhibitor to ameliorate the brain toxicity and edema induced by hyperammonemia, events likely related to glutamine synthesis.     

Ammonia emission from young barley plants: influence of N source, light/dark cycles and inhibition of glutamine synthetase

Barley (Hordeum vulgare L. cv. Golf) plants were grown at twodifferent relative addition rates; 0.1 and 0.2 d^–1 ofnitrate. Three to five days before measurements started theplants were transferred to a nutrient solution with 2 mM nitrateor ammonium. The ammonium-grown plants showed increased ammoniumlevels in both shoots and roots and also increased ammoniumconcentrations in xylem sap. Ammonia emission measured in cuvettes connected to an automaticNH₃ monitor was close to zero for nitrate-grown plants but increasedto 0.59 and 0.88 nmol NH₃ m^–2 S^–1 for plants transferredto ammonium after growing at RA=0.2 and 0.1 d^–1, respectively.In darkness, NH₃ emission decreased together with photosynthesisand transpiration, but increased rapidly when the light wasturned on again. Addition of 0.5 mM methionine sulphoximine (MSO) to the plantscaused an almost complete inhibition of both root and shootglutamine synthetase (GS) activity after 24 h. Ammonia emissionincreased dramatically and photosynthesis and transpirationdecreased in both nitrate- and ammonium-grown plants as a resultof the GS inhibition. At the same time plant tissue and xylemsap ammonium concentrations increased, indicating the importanceof GS in controlling plant ammonium levels and thereby NH₃ emissionfrom the leaves. Key words: Hordeum vulgare, ammonia emission, ammonium, glutamine synthetase, nitrogen nutrition, photosynthesis, transpiration