Organ-specific changes in the activity and subunit composition of glutamine-synthetase isoforms of barley (Hordeum vulgare L.) after growth on different levels of NH+ 4

One cytosolic glutamine synthetase (GS, EC 6.3.1.2) isoform (GS 1a) was active in the germinating seeds of barley (Hordeum vulgare L.). A second cytosolic GS isoform (GS 1b) was separated from the leaves as well as the roots of 10-d-old seedlings. The chloroplastic isoform (GS 2) was present and active only in the leaves. The three GS isoforms were active in N-supplied (NH⁺ ₄ or NO ₃ ^– ) as well as in N-free-grown seedlings. This indicates (i) that a supply of nitrogen to the germinating seeds was not necessary for the induction of the GS isoforms and (ii) that no nitrogen-specific isoforms appeared during growth of seedlings with different nitrogen sources. The activity of GS, however, depended on the seedlings' nitrogen source: the specific activity was much higher in the leaves and much lower in the roots of NH⁺ ₄-grown barley than in the respective organs of NO ₃ ^– -fed or N free-grown plants. With increasing concentrations of NH⁺ ₄ (supplied hydroponically during growth), the specific activity of GS 1b increased in the leaves, but decreased in the roots. The activity of GS 2 (leaf) also increased with increasing NH⁺ ₄ supply, whereas GS 1a activity (leaf and root) was not affected. The changes in the activities of GS 1b and GS 2 were correlated with changes in the subunit compositions of the active holoenzymes: growth at increased levels of external NH⁺ ₄ resulted in an increased abundance of one of the four GS subunits, and of two of the five GS 1b subunits in the leaves. In the roots, however, the abundance of these two GS 1b subunits was decreased under the same growth conditions, indicating an organ-specific difference either in the expression of the genes coding for the respective GS 1b subunits or in the assembly of the GS 1b holoenzymes. Furthermore, growth at different levels of NH⁺ ₄ resulted in changes in the substrate affinities of the isoforms GS 1b (root and leaf) and GS 2 (leaf), presumably due to the changes in the subunit compositions of the active holoenzymes.Abbreviations FPLC fast protein liquid chromatography - GHA -glutamyl hydroxamate - GS glutamine synthetase Dr. Roger Wallsgrove's (Rothamsted Experimental Station, Harpenden, UK) generous gift of GS antiserum is greatly appreciated.     

Accumulation of ammonium ion in cadmium tolerant and sensitive cultivars of Oryza sativa

Cd-tolerant and Cd-sensitive rice cultivars were used to study the role of NH₄ ⁺ accumulation in Cd-induced toxicity. NH₄ ⁺ accumulation seems to be involved in regulating the toxicity of rice seedlings caused by CdCl₂. This conclusion was based on the observations that (a) on treatment with CdCl₂, NH₄ ⁺ content increased rapidly in the leaves of the Cd-sensitive cultivar (cv. Taichung Native 1, TN1) but not in the Cd-tolerant cultivar (cv. Tainumg 67, TNG67), (b) pretreatment with abscisic acid (ABA) enhanced Cd tolerance and reduced Cd-induced NH₄ ⁺ accumulation in TN1 seedlings, (c) exogenous application of the ABA biosynthesis inhibitor, fluridone, decreased Cd tolerance and increased NH₄ ⁺ content in leaves of TNG67, (d) exogenous application of phosphinothricin, an inhibitor of glutamine synthetase (GS), which resulted in NH₄ ⁺ accumulation in the leaves, also induced toxicity similar to Cd in TN1 seedlings. Evidence is presented to show that Cd-induced NH₄ ⁺ accumulation in TN1 leaves is attributable to a decrease in GS activity. Since Cd-treated TN1 leaves had higher glutamine and glutamate contents than control leaves, it is unlikely that glutamine (or glutamate) depletion is the mechanism which regulates Cd-induced toxicity.     

Hydrogen peroxide is required for abscisic acid-induced NH4+ accumulation in rice leaves

The role of H₂O₂ in abscisic acid (ABA)-induced NH₄⁺ accumulation in rice leaves was investigated. ABA treatment resulted in an accumulation of NH₄⁺ in rice leaves, which was preceded by a decrease in the activity of glutamine synthetase (GS) and an increase in the specific activities of protease and phenylalanine ammonia-lyase (PAL). GS, PAL, and protease seem to be the enzymes responsible for the accumulation of NH₄⁺ in ABA-treated rice leaves. Dimethylthiourea (DMTU), a chemical trap for H₂O₂, was observed to be effective in inhibiting ABA-induced accumulation of NH₄⁺ in rice leaves. Inhibitors of NADPH oxidase, diphenyleneiodonium chloride (DPI) and imidazole (IMD), and nitric oxide donor (N-tert-butyl-α-phenylnitrone, PBN), which have previously been shown to prevent ABA-induced increase in H₂O₂ contents in rice leaves, inhibited ABA-induced increase in the content of NH₄⁺. Similarly, the changes of enzymes responsible for NH₄⁺ accumulation induced by ABA were observed to be inhibited by DMTU, DPI, IMD, and PBN. Exogenous application of H₂O₂ was found to increase NH₄⁺ content, decrease GS activity, and increase protease and PAL-specific activities in rice leaves. Our results suggest that H₂O₂ is involved in ABA-induced NH₄⁺ accumulation in rice leaves.     

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.     

Effect of phosphinothricin (glufosinate) on photosynthesis and photorespiration of C3 and C4 plants

Phosphinothricin (glufosinate), an irreversible inhibitor of glutamine synthetase, causes an inhibition of photosynthesis in C₃ (Sinapis alba) and C₄ (Zea mays) plants under atmospheric conditions (400 ppm CO₂, 21% O₂). This photosynthesis inhibition is proceeding slower in C₄ leaves. Under non-photorespiratory conditions (1000 ppm CO₂, 2% O₂) there is no inhibition of photosynthesis. The inhibition of glutamine synthetase by phosphinothricin results in an accumulation of NH₄ ⁺. The NH₄ ⁺-accumulation is lower in C₄ plants than in C₃ plants. The inhibition of glutamine synthetase through phosphinothricin in mustard leaves results in a decrease in glutamine, glutamate, aspartate, asparagine, serine, and glycine. In contrast to this, a considerable increase in leucine and valine following phosphinothricin treatment is measured. With the addition of either glutamine, glutamate, aspartate, glycine or serine, photosynthesis inhibition by phosphinothricin can be reduced, although the NH₄ ⁺-accumulation is greatly increased. This indicates that NH₄ ⁺-accumulation cannot be the primary cause for photosynthesis inhibition by phosphinothricin. The investigations demonstrate the inhibition of transmination of glyoxylate to glycine in photorespiration through the total lack of amino donors. This could result in a glyoxylate accumulation inhibiting ribulose-1,5-bisphosphate-carboxylase and consequently CO₂-fixation.Abbreviations GOGAT glutamine-2-oxoglutarate-amidotransferase - GS glutamine synthetase - PPT phosphinothricin - MSO methionine sulfoximine - RuBP ribulose-1,5-bisphosphate     

Ammonium can stimulate nitrate and nitrite reductase in the absence of nitrate in Clematis vitalba

Nitrogen assimilation was studied in the deciduous, perennial climber Clematis vitalba. When solely supplied with NO₃^– in a hydroponic system, growth and N-assimilation characteristics were similar to those reported for a range of other species. When solely supplied with NH₄⁺, however, nitrate reductase (NR) activity dramatically increased in shoot tissue, and particularly leaf tissue, to up to three times the maximum level achieved in NO₃^– supplied plants. NO₃^– was not detected in plant material that had been solely supplied with NH₄⁺, there was no NO₃^– contamination of the hydroponic system, and the NH₄⁺-induced activity did not occur in tobacco or barley grown under similar conditions. Western Blot analysis revealed that the induction of NR activity, either by NO₃^– or NH₄⁺, was matched by NR and nitrite reductase protein synthesis, but this was not the case for the ammonium assimilation enzyme glutamine synthetase. Exposure of leaf disks to N revealed that NO₃^– assimilation was induced in leaves directly by NO₃^– and NH₄⁺ but not glutamine. Our results suggest that the NH₄⁺-induced potential for NO₃^– assimilation occurs when externally sourced NH₄⁺ is assimilated in the absence of any NO₃^– assimilation. These data show that the potential for nitrate assimilation in C. vitalba is induced by a nitrogenous compound in the absence of its substrate and suggest that NO₃^– assimilation in C. vitalba may have a significant role beyond the supply of reduced N for growth.     

Photoproduction of ammonium by immobilized mutant strains of Anabaena variabilis

Summary Ethylenediamine (EDA) is toxic to the cyanobacterium Anabaena variabilis and inhibits nitrogenase activity. The inhibition of nitrogenase was prevented by pretreatment of cells with l-methionine-d,l-sulphoximine (MSX). Mutant strains of Anabaena variabilis (ED81, ED92), resistant to EDA, had low levels of glutamine synthetase (GS) biosynthetic activity compared with the wild type strain. ED92 had a low level of GS protein whereas ED81 had a similar level to that of the parent strain as estimated using antibodies against GS. Both strains fixed N₂ and liberated NH₄ ⁺ into the media. Following immobilization of the mutant strains, sustained photoproduction of NH₄ ⁺ was obtained in air-lift reactors at rates of up to 50 mol NH₄ ⁺ mg chl a^–1 h^–1, which were comparable to the rates obtained when immobilized cyanobacteria were treated with MSX.Abbreviations EDA 1,2-diaminoethane (ethylenediamine) - GS glutamine synthetase - MSX l-methionine-d,l-sulphoximine     

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.     

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     

Field investigations of ammonia exchange between barley plants and the atmosphere. II. Nitrogen realiocation, free ammonium content and activities of ammonium-assimilating enzymes in different leaves

The activities of glutamine synthetase (GS) and glutamate synthase (GOGAT) in different leaves of field-grown spring barley were measured during the reproductive growth phase in 2 consecutive years. Concurrently, the contents of soluble ammonium ions and free amides in the leaves were determined. The studies were carried out to investigate the relationship between variations in these parameters and emission of NH3 from the plant foliage. GS and GOGAT activities declined very rapidly with leafage. The decline in enzyme activities was followed by an increase in soluble ammonium ions and amides in the leaf tissues. During the same period, about 75% of leaf and stem nitrogen was reallocated to the developing ear. The amount of NH₃ volatilized from the foliage during the reproductive growth phase amounted to about 1% of the reallocated nitrogen. The experimental years were characterized by very favourable conditions for grain dry matter formation and for re-utilization of nitrogen mobilized from leaves and stems. Ammonia volatilization occurring under conditions with declining GS and GOGAT activities and increasing tissue concentrations of NH₄⁺ may be useful in protecting the plant from accumulation of toxic NH₃ and NH₄⁺ concentrations in the tissues.     

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.     

NUTRIENT CONTROLS ON NITROGEN UPTAKE AND METABOLISM BY NATURAL POPULATIONS AND CULTURES OF TRICHODESMIUM (CYANOBACTERIA)

The effects of inorganic nutrient (ammonium [NH₄ ⁺ ] and nitrate [NO₃ ⁻ ]) and amino acid (glutamate [glu] and glutamine [gln]) additions on rates of N₂ fixation, N uptake, glutamine synthetase (GS) activity, and concentrations of intracellular pools of gln and glu were examined in natural and cultured populations of Trichodesmium. Additions of 1 μM glu, gln, NO₃ ⁻ , or NH₄ ⁺ did not affect short-term rates of N₂ fixation. This may be an important factor that allows for continued N₂ fixation in oligotrophic areas where recycling processes are active. N₂ fixation rates decreased when nutrients were supplied at higher concentrations (e.g. 10 μM). Uptake of combined N (NH₄ ⁺ , NO₃ ⁻ , and amino acids) by Trichodesmium was stimulated by increased concentrations. For NO₃ ⁻ , proportional increases in NO₃ ⁻ uptake and decreases in N₂ fixation were observed when additions were made to cultures before the onset of the light period. GS activity did not change much in response to the addition of NH₄ ⁺ , NO₃ ⁻ , glu, or gln. GS is necessary for N metabolism, and the bulk of this enzyme pool may be conserved. Intracellular pools of glu and gln varied in response to 10 μM additions of NH₄ ⁺ , glu, or gln. Cells incubated with NH₄ ⁺ became depleted in intracellular glu and enriched with intracellular gln. The increase in the gln/glu ratio corresponded to a decrease in the rate of N₂ fixation. Although the gln/glu ratio decreased in cells exposed to the amino acids, there was only a corresponding decrease in N₂ fixation after the gln addition. The results presented here suggest that combined N concentrations on the order of 1 μM do not affect rates of N₂ fixation and metabolism, although higher concentrations (e.g. 10 μM) can. Moreover, these effects are exerted through products of NH₄ ⁺ assimilation rather than exogenous N, as has been suggested for other species. These results may help explain how cultures of Trichodesmium are able to simultaneously fix N₂ and take up NH₄ ⁺ and how natural populations continue to fix N₂ once combined N concentrations increase within a bloom.     

Ammonia Production and Assimilation in Glutamate Synthase Mutants of Arabidopsis thaliana

Ammonia production and assimilation¹ were examined in photorespiratory mutants of Arabidopsis thaliana L. lacking ferredoxin-dependent glutamate synthase (Fd-GluS) activity. Although photosynthesis was rapidly inhibited in these mutants in normal air, NH₄⁺ continued to accumulate. The accumulation of NH₄⁺ was also seen after an initial lag of 30 minutes in 2% O₂, 350 microliters per liter of CO₂ and after 90 minutes in 2% O₂, 900 microliters per liter of CO₂. The accumulation of NH₄⁺ in normal air and low O₂ was also associated with an increase in the total pool of amino acid-N and glutamine, and a decrease in the pools of glutamate, aspartate, alanine, and serine. Upon return to dark conditions, or to 21% O₂, 1% CO₂ in the light, the NH₄⁺ which had accumulated in the leaves was reassimilated into amino acids. The addition of methionine sulfoximine (MSO) resulted in higher accumulations of NH₄⁺ in glutamate synthase mutants and prevented the reassimilation of NH₄⁺ upon return to the dark. The addition of MSO also resulted in the accumulation of NH₄⁺ in glutamate synthase mutants in the light and in 21% O₂, 1% CO₂. These results indicate that glutamine synthetase is essential for the reassimilation of photorespiratory NH₄⁺ and for primary N assimilation in the leaves and strongly suggest that glutamate dehydrogenase plays only a minimal role in the assimilation of ammonia. Levels of NADH-dependent glutamate synthase (NADH-GluS) appear to be sufficient to account for the assimilation of NH₄⁺ by a GS/NADH-GluS cycle.     

The effect of glutamine and asparagine on net NH4 + uptake in young wheat plants

Wheat plants grown during 10 days in the absence of N were pretreated with 1.0 eq m^-3 of methionine, asparagine or glutamine and/or 1.0 eq m^-3 MSX⁴ or 0.17 eq m^-3 DON. Net NH₄ ⁺ uptake was measured both in the presence or in the absence of the amino acid or enzyme inhibitor used in the pretreatment. The effect of met, asn and gln on net K⁺ uptake was also studied using K⁺-depleted plants. Changes in the contents of root free NH₄ ⁺, asn, gln and the activities of GS, PEP-carboxylase, NAD⁺-GDH and NADH-GDH were determined. Net NH₄ ⁺ uptake in gln and asn pretreated plants was markedly, and sometimes completely suppressed provided uptake was measured in the presence of the amides. On the other hand, the met pretreated plants absorbed only 35% less NH₄ ⁺ than the control. When NH₄ ⁺ uptake was measured in the absence of the amino acids, only those plants pretreated with asn showed a marked suppression of net uptake during the first 120 min. None of the 3 amino acids tested significantly inhibited K⁺ uptake. Free NH₄ ⁺ concentration in roots of N-starved plants increased after 4 h incubation with gln, asn or MSX in the absence of external NH₄ ⁺. Nevertheless, no correlation was observed between root NH₄ ⁺ concentration and the extent of net NH₄ ⁺ uptake suppression. The inhibitory effect exerted by asn decreased when it was supplied together with MSX or DON. Pretreatments with gln or asn in the absence of external NH₄ ⁺ significantly increased the level of asn in the roots, while that of gln remained unchanged. It is concluded that asn and gln specifically suppress net NH₄ ⁺ uptake in wheat, although it is not clear wether they act only from the root exterior, or through an endogenous pool exhibiting fast turn-over.Abbreviations AUR ammonium uptake rate - DON 6-diazo-5-oxo-L-norleucine - GDH glutamic dehydrogenase - GOGAT oxoglutarate- glutamine aminotransferase - GS glutamine synthetase - MSX L-methionine sulfoximine - PEP phosphoenolpyruvate - PVPP polyvinylpolypyrrolidone     

ROLE OF GLUTAMATE DEHYDROGENASE AND GLUTAMINE SYNTHETASE IN CHLORELLA VULGARIS DURING ASSIMILATION OF AMMONIUM WHEN JOINTLY IMMOBILIZED WITH THE MICROALGAE‐GROWTH‐PROMOTING BACTERIUM AZOSPIRILLUM BRASILENSE1

Enzymatic activities of glutamate dehydrogenase (GDH) and glutamine synthetase (GS) participating in the nitrogen metabolism and related ammonium absorption were assayed after the microalga Chlorella vulgaris Beij. was jointly immobilized with the microalgae‐growth‐promoting bacterium Azospirillum brasilense. At initial concentrations of 3, 6, and 10 mg · L^?1 NH₄⁺, joint immobilization enhances growth of C. vulgaris but does not affect ammonium absorption capacity of the microalga. However, at 8 mg · L^?1 NH₄⁺, joint immobilization enhanced ammonium absorption by the microalga without affecting the growth of the microalgal population. Correlations between absorption of ammonium per cell and per culture showed direct (negative and positive) linear correlations between these parameters and microalga populations at 3, 6, and 10 mg · L^?1 NH₄⁺, but not at 8 mg · L^?1 NH₄⁺, where the highest absorption of ammonium occurred. In all cultures, immobilized and jointly immobilized, having the four initial ammonium concentrations, enzymatic activities of Chlorella are affected by A. brasilense. Regardless of the initial concentration of ammonium, GS activity in C. vulgaris was always higher when jointly immobilized and determined on a per‐cell basis. When jointly immobilized, only at an initial concentration of 8 mg · L^?1 NH₄⁺ was GDH activity per cell higher.     

Growth responses of rice in ammonium-based nutrient solution with variable calcium supply

It is commonly known that calcium promotes NO₃ ^- uptake in many crop species. However, calcium enhancement of NH₄ ⁺ uptake by plants has received little attention. This study aimed to evaluate the effect of Ca supplements on NH₄ ⁺ uptake and plant growth in solution cultured rice. Supplemental Ca applied at vegetative and reproductive phases of plant ontogeny tended to stimulate NH₄ ⁺ absorption, and accordingly resulted in a better straw and grain yield. However, excessively supplied Ca (400 ppm) was detrimental to plant growth. Increases in straw and grain yield observed at Ca levels up to 300 ppm were linked to the Ca-enhanced activities of glutamine synthetase (GS), glutamate synthase (GOGAT), and ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco).     

Altered Kinetic Properties of Tyrosine-183 to Cysteine Mutation in Glutamine Synthetase of <Emphasis Type="Italic">Anabaena variabilis</Emphasis> Strain SA1 Is Responsible for Excretion of Ammonium Ion Produced by Nitrogenase

A L-methionine-D,L-sulfoximine-resistant mutant of the cyanobacterium Anabaena variabilis, strain SA1, excreted the ammonium ion generated from N₂ reduction. In order to determine the biochemical basis for the NH₄ ⁺-excretion phenotype, glutamine synthetase (GS) was purified from both the parent strain SA0 and from the mutant. GS from strain SA0 (SA0-GS) had a pH optimum of 7.5, while the pH optimum for GS from strain SA1 (SA1-GS) was 6.8. SA1-GS required Mn⁺² for optimum activity, while SA0-GS was Mg⁺² dependent. SA0-GS had the following apparent K _m values at pH 7.5: glutamate, 1.7 mM; NH₄ ⁺, 0.015 mM; ATP, 0.13 mM. The apparent K _m for substrates was significantly higher for SA1-GS at its optimum pH (glutamate, 9.2 mM; NH₄ ⁺, 12.4 mM; ATP, 0.17 mM). The amino acids alanine, aspartate, cystine, glycine, and serine inhibited SA1-GS less severely than the SA0-GS. The nucleotide sequences of glnA (encoding glutamine synthetase) from strains SA0 and SA1 were identical except for a single nucleotide substitution that resulted in a Y183C mutation in SA1-GS. The kinetic properties of SA1-GS isolated from E. coli or Klebsiella oxytoca glnA mutants carrying the A. variabilis SA1 glnA gene were also similar to SA1-GS isolated from A. variabilis strain SA1. These results show that the NH₄ ⁺-excretion phenotype of A. variabilis strain SA1 is a direct consequence of structural changes in SA1-GS induced by the Y183C mutation, which elevated the K _m values for NH₄ ⁺ and glutamate, and thus limited the assimilation of NH₄ ⁺ generated by N₂ reduction. These properties and the altered divalent cation-mediated stability of A. variabilis SA1-GS demonstrate the importance of Y183 for NH₄ ⁺ binding and metal ion coordination. Received: 3 July 2002 / Accepted: 29 July 2002     

Expression of a ferredoxin-dependent glutamate synthase gene in mesophyll and vascular cells and functions of the enzyme in ammonium assimilation in Nicotiana tabacum (L.)

GLU1 encodes the major ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in Arabidopsis thaliana (ecotype Columbia). With the aim of providing clues on the role of Fd-GOGAT, we analyzed the expression of Fd-GOGAT in tobacco (Nicotiana tabacum L. cv. Xanthi). The 5′ flanking element of GLU1 directed the expression of the uidA reporter gene in the palisade and spongy parenchyma of mesophyll, in the phloem cells of vascular tissue and in the roots of tobacco. White light, red light or sucrose induced GUS expression in the dark-grown seedlings in a pattern similar to the GLU1 mRNA accumulation in Arabidopsis. The levels of GLU2 mRNA encoding the second Fd-GOGAT and NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) were not affected by light. Both in the light and in darkness, ¹⁵NH₄⁺ was incorporated into [5−¹⁵N]glutamine and [2−¹⁵N]glutamate by glutamine synthetase (GS, EC 6.3.1.2) and Fd-GOGAT in leaf disks of transgenic tobacco expressing antisense Fd-GOGAT mRNA and in wild-type tobacco. In the light, low level of Fd-glutamate synthase limited the [2−¹⁵N]glutamate synthesis in transgenic leaf disks. The efficient dark labeling of [2−¹⁵N]glutamate in the antisense transgenic tobacco leaves indicates that the remaining Fd-GOGAT (15–20% of the wild-type activity) was not the main limiting factor in the dark ammonium assimilation. The antisense tobacco under high CO₂ contained glutamine, glutamate, asparagine and aspartate as the bulk of the nitrogen carriers in leaves (62.5%), roots (69.9%) and phloem exudates (53.2%). The levels of glutamate, asparagine and aspartate in the transgenic phloem exudates were similar to the wild-type levels while the glutamine level increased. The proportion of these amino acids remained unchanged in the roots of the transgenic plants. Expression of GLU1 in mesophyll cells implies that Fd-GOGAT assimilates photorespiratory and primary ammonium. GLU1 expression in vascular cells indicates that Fd-GOGAT provides amino acids for nitrogen translocation. The nucleotide sequence data of the GLU1 gene reported in the present study is available from GenBank with the following accession number: AY189525