Pathway of ammonia assimilation in illuminated and darkened Chlamydomonas reinhardii

Evidence is presented which shows that NH₃ assimilation in Chlamydomonas occurs exclusively via the glutamate synthase cycle in illuminated and darkened cells and those in which the internal level of NH₃ is elevated. This result indicates that glutamate dehydrogenase probably plays a catabolic rather than anabolic role in the N nutrition of the alga. Glutamine synthetase and glutamate dehydrogenase were characterized and their kinetic properties shown to be consistent with these proposals. It is suggested that reversible activity modulations of glutamine synthetase regulate the operation of the glutamate synthase cycle in the light but the availability of reductant and ATP limits its activity in darkened cells. The possible involvement of the two glutamate synthase enzymes in both light and dark assimilation is discussed.     

Properties of glutamate dehydrogenase from Lemna minor

Sterile cultures of Lemna minor grown in the presence of either nitrate, ammonium or amino acids failed to show significant changes in glutamate dehydrogenase (GDH) levels in response to nitrogen source. Crude and partially purified GDH preparations exhibit NADH and NADPH dependent activities. The ratio of these activities remain ca 12:1 during various treatments. Mixed substrate and product inhibition studies as well as electrophoretic behaviour suggest the existence of a single enzyme which is active in the presence of both coenzymes. GDH activity was found to be localized mainly in mitochondria. Kinetic studies revealed normal Michaelis kinetics with most substrates but showed deviations with NADPH and glutamate. A Hill-coefficient of 1.9 determined with NADPH indicates positive cooperative interactions, whereas a Hill-coefficient of 0.75 found with glutamate may be interpreted in terms of negative cooperative interactions. NADH dependent activity decreases rapidly during gel filtration whereas the NAD⁺ and NADPH activities remain unchanged. GDH preparations which have been pretreated with EDTA show almost complete loss of NADH and NAD⁺ activities. NADPH activity again remains unaffected. NAD⁺ activity is fully restored by adding Ca²⁺ or Mg²⁺, whereas the NADH activity can only be recovered by Ca²⁺ but not at all by Mg²⁺. Moderate inhibition of GDH reactions observed with various adenylates are fully reversed by adding Ca²⁺, indicating that the adenylate inhibition is due solely to the chelating properties of these compounds.     

Control of glutamate dehydrogenase from Lemna minor by divalent metal ions

The NADH and NAD⁺ dependent reactions catalyzed by glutamate dehydrogenase (GDH) from sterile cultures of Lemna minor are completely inactivated by EDTA. The activities of both reactions can be fully restored by addition of Ca²⁺ and to a lesser extent Mn²⁺, Zn²⁺, Sr²⁺ or La³⁺, whereas Mg²⁺ reactivates only the NAD⁺ dependent reaction. Activation of the NADH reaction by Ca²⁺ has been studied by using partially purified, EDTA pretreated, and Mg²⁺ saturated GDH preparations. Saturation kinetic curves with Ca²⁺ were always sigmoidal, whereas saturation plots for the 3 substrates of the aminating reaction at various fixed Ca²⁺ concentrations showed normal Michaelis kinetics. However, a pronounced substrate inhibition at low Ca²⁺ levels was found, particularly with NH₄⁺ and NADH. Product inhibition studies revealed unchanged enzyme substrate binding characteristics for NADH and 2-oxoglutarate in the Ca²⁺ free enzyme. A drastic alteration was established for the third substrate NH₄⁺. The kinetic data suggest that Ca²⁺ governs an equilibrium between a catalytically inactive (Ca²⁺ free) and an active (Ca²⁺ saturated) enzyme form. Inactivation by removal of Ca²⁺ is related to an alteration in the binding characteristics or binding sequence of the substrate NH₄⁺.     

Effect of abscisic acid on photosynthetic products of Lemna minor

Lemna minor fronds were grown on nutrient only, or nutrient plus 10^?6M abscisic acid (ABA) for 2 or 8 days. After various ¹⁴CO₂ pulse-chase time periods, the fronds were harvested and the photosynthetic products separated into acidic, lipid, residue, sugar and amino acid fractions. Compared with the control fronds, total ¹⁴C-fixation was 15% higher in the 2 day ABA-treatment and 6% lower in the 8 day ABA-treatment. This pattern was reflected in all the fractions examined, and it appeared that ABA did not alter the distribution of ¹⁴C between the photosynthetic products during the ¹⁴CO₂ pulse. During the chase, less ¹⁴C was lost from the carbohydrate fractions in the ABA-treated fronds than in the control fronds. The results indicate that the previously reported ABA-mediated increase in carbohydrate levels was a consequence of decreased degradation rather than an increase in synthesis from assimilated carbon.     

Control of methionine synthesis by lysine in Lemna minor

When Lemna minor was cultured in the presence of 0.25 mM l-lysine, the concentration of free methionine and formyl and methyl tetrahydrofolate (THFA) were decreased. l-lysine, l-homoserine, l-threonine and l-methionine at concentrations up to 8 mM did not affect N¹⁰-formyl THFA synthetase (E.C. 6.3.4.3) and N⁵,N¹⁰-methylene THFA reductase (E.C. 1.1.1.68). In contrast, serine hydroxymethyltransferase (E.C. 2.1.2.1) activity was inhibited by lysine. This inhibition gave a sigmoidal curve when plotted for a range of l-lysine or THFA concentrations. Exogenous lysine also reduced the incorporation of glycine [¹⁴C] and serine [3-¹⁴C] into free and protein methionine. Lysine, which is known to control synthesis of homocysteine in L. minor, may also regulate production of C-1 units for methionine synthesis by inhibition of serine hydroxymethyltransferase.     

Changes in the levels of enzymes involved in ammonia assimilation during the development of Phaseolus vulgaris seedlings.Effects of exogenous ammonia

Seeds of Phaseolus vulgaris L. cv. White Kidney were germinated and grown either in a nitrogen-free or in an ammonia-supplied medium. The changes in the soluble protein concentration and in the levels of glutamine synthetase (GS, EC 6.3.1.2), NADH–glutamate synthase (NADH-GOGAT, EC 1.4.1.14), ferredoxin-glutamate synthase (Fd-GOGAT, EC 1.4.7.1) and glutamate dehydrogenase (GDH, EC 1.4.1.2), both NADH- and NAD⁺-dependent, were examined in cotyledons and roots during the first 10 days after sowing. Soluble protein declined rapidly in the cotyledons and increased slightly in the roots. GS activity was initially high both in cotyledons and roots but subsequently decreased during seedling growth. Exogenous ammonia hardly affected GS activity. High levels of NADH-GOGAT were present both in cotyledons and roots during the first days of germination. The activity then gradually declined in both organs. In contrast, Fd-GOGAT in cotyledons was initially low and progressively increased with seedling development. In roots, the levels of Fd-GOGAT were higher in young than in old seedlings. Supply of ammonia to the seedlings increased the levels of NADH-GOGAT and Fd-GOGAT both in cotyledons and roots. NADH-GDH (aminating) activity gradually increased during germination. In contrast, the levels of NAD⁺-GDH (deaminating) activity were highest during the first days of germination. Exogenous ammonia did not significantly affect the activities of GDH.     

Ammonia assimilation in Corynebacterium glutamicum and a glutamate dehydrogenase-deficient mutant

In the wild-type of Corynebacterium glutamicum, the specific activity of glutamate dehydrogenase (GDH) remained constant at 1.3 U (mg protein)–1 when raising the ammonia (NH4) concentration in the growth medium from 1 to 90 mM. In contrast, the glutamine synthetase (GS) and glutamate synthase (GOGAT) activities decreased from 1.1 U (mg protein)–1 and 42 mU (mg protein)–1, respectively, to less than 10 % of these values at NH4 concentrations > 10 mM suggesting that under these conditions the GDH reaction is the primary NH4 assimilation pathway. Consistent with this suggestion, a GDH-deficient C. glutamicum mutant showed slower growth at NH4 concentrations 10 mM and, in contrast to the wild-type, did not grow in the presence of the GS inhibitor methionine sulfoximine. © Rapid Science Ltd. 1998     

Some properties of glutamine synthetase and glutamate synthase from Derxia gummosa

The incorporation of ¹⁵N into washed cells of Derxia gummosa from labelled-(NH₄)₂SO₄ and -KNO₃ respectively was inhibited by both L-methionine-DL-sulphoximine and azaserine. Glutamine synthetase purified to homogeneity from this bacterium had a molecular weight of 708 000 and was composed of 12 similar subunits each of 59 000. The enzyme assayed by γ-glutamyltransferase method had K_m values for L-glutamine and hydroxylamine of 12.5 and 1.2 mM, respectively. Optimal pH values for adenylylated and deadenylylated forms were pH 7.0 and pH 8.0, respectively. The adenylylated enzyme was deadenylylated by treatment with snake venom phosphodiesterase. The inhibitions by both glutamate and ammonia were competitive. The activity was markedly inhibited by L-methionine-DL-sulphoximine, alanine, glycine and serine and to a lesser extent by aspartate, phenylalanine and lysine. Various tri-, di- and mono-phosphate nucleotides, organic acids (pyruvate, oxalate and oxaloacetate) were also inhibitory. Glutamate synthase purified 167-fold had specific requirements for NADH, L-glutamine and 2-ketoglutarate. The K_m values for NADH, glutamine and 2-ketoglutarate were 9.6, 270 and 24 μM respectively. Optimal pH range was 7.2–8.2. The enzyme was inhibited by azaserine, methionine, aspartate, AMP, ADP and ATP.     

Regulation of ammonium assimilation in Haloferax mediterranei: Interaction between glutamine synthetase and two GlnK proteins

GlnK proteins belong to the PII superfamily of signal transduction proteins and are involved in the regulation of nitrogen metabolism. These proteins are normally encoded in an operon together with the structural gene for the ammonium transporter AmtB. Haloferax mediterranei possesses two genes encoding for GlnK, specifically, glnK₁ and glnK₂. The present study marks the first investigation of PII proteins in haloarchaea, and provides evidence for the direct interaction between glutamine synthetase and both GlnK₁ and GlnK₂. Complex formation between glutamine synthetase and the two GlnK proteins is demonstrated with pure recombinant protein samples using in vitro activity assays, gel filtration chromatography and western blotting. This protein–protein interaction increases glutamine synthetase activity in the presence of 2-oxoglutarate. Separate experiments that were carried out with GlnK₁ and GlnK₂ produced equivalent results.     

Manipulating the pathway of ammonia assimilation through genetic engineering and breeding: consequences to plant physiology and plant development

In this article we discuss the ways in which our understanding of the nature of the molecular controls of nitrogen assimilation have been increased by the use of leguminous and non-leguminous plants with modified capacities for ammonium assimilation. These modifications have been achieved through genetic engineering and breeding. An improved understanding of nitrogen assimilation will be vital if improvements in crop nitrogen use efficiency are to be made to reduce the need for excessive input of fertilisers. In this review we present an overall view of past work and more recent studies on this topic. In our work, using tobacco and Lotus as model plants, glutamine synthetase and glutamate synthase activites have been altered by stimulating or inhibiting in an organ- or tissue-specific manner the expression of the corresponding genes. The physiological impact of these genetic manipulations has been studied on plants grown under different nitrogen regimes. This revised version was published online in June 2006 with corrections to the Cover Date.     

The pathway of nitrogen assimilation in plants

The major route of nitrogen assimilation has been considered for many years to occur via the reductive amination of α-oxoglutarate, catalysed by glutamate dehydrogenase. However, recent work has shown that in most bacteria an alternative route via glutamine synthetase and glutamine: 2-oxoglutarate aminotransferase (glutamate synthase) operates under conditions of ammonia limitation. Subsequently the presence of a ferredoxin-dependent glutamate synthase in green leaves and green and blue-green algae, and a NAD(P)H and ferredoxin-dependent enzyme in roots and other non-green plant tissues, has suggested that this route may also function in most members of the plant kingdom. The only exceptions are probably the majority of the fungi, where so far most organisms studied do not appear to contain glutamate synthase. Besides the presence of the necessary enzymes there is other evidence to support the contention that the assimilation of ammonia into amino acids occurs via glutamine synthetase and glutamate synthase, and that it is unlikely that glutamate dehydrogenase plays a major role in nitrogen assimilation in bacteria or higher plants except in circumstances of ammonia excess.     

Glutamine synthetase and glutamate synthase from Sclerotinia sclerotiorum

Glutamine synthetase (GS, EC 6.3.1.2) and glutamate synthase (GOGAT, EC 1.4.1.13) were purified from Sclerotinia sclerotiorum and some of their properties studied. The GS transferase and biosynthetic activities, as well as GOGAT activity, were sensitive to feedback inhibition by amino acids and other metabolites. GS showed a marked dependence on ADP in the transferase reaction and on ATP in the Mg²⁺-dependent biosynthetic reaction. Regulation of GS activity by adenylylation/deadenylylation was demonstrated by snake venom phosphodiesterase treatment of the purified enzyme. GOGAT required NADPH as an electron donor; NADH was inactive. GOGAT was strongly inhibited by p-chloromercuribenzoate and the inhibition was reversed by cysteine. The enzyme was also markedly inhibited by o-phenanthroline, 2,2′-bipyridyl and azaserine. l-Methionine-dl-sulphoximine (MSX) and azaserine inhibited the incorporation of ¹⁵N-labelled ammonium sulphate into washed cells of S. sclerotiorum. MSX and azaserine respectively also inhibited purified GS and GOGAT activities. GDH activity was not detected in cell-extracts. Thus the GS/GOGAT pathway is the main route for the assimilation of ammonium compounds in this fungus.     

Glutamine-dependent asparagine synthetase from Lupinus luteus

Asparagine synthetase (glutamine-hydrolyzing [l-aspartate: l-glutamine amido-ligase (AMP-forming), E.C. 6.3.5.4] was purified over 500-fold from cotyledon extracts of 1-week-old yellow lupin seedlings. The enzyme was labile and required protection by high levels of thiols; glycerol and the substrates also stabilized it. The reaction products were shown to be asparagine, AMP, PPi and glutamate. The limiting K_m values were for aspartate 1·3 mM, for MgATP 0·14 mM and for glutamine 0·16 mM. Positive homotropic cooperativity was observed for MgATP only, and gel filtration studies indicated that the substrate-free enzyme (MW 160 000) associated to a dimer (MW 320 000 in the presence of MgCl₂ and ATP. The purified enzyme, which had some glutaminase activity, catalyzed an aspartate- and glutamine-independent ATP-PPi exchange reaction at a rate 5–7-fold higher than the rate of asparagine synthesis. Initial velocity studies and exchange data indicated an overall ping-pong mechanism. Compared to similar enzymes isolated from mammalian tumor cells, the lupin enzyme appears to be unique with respect to MW, reaction mechanism and regulatory properties. The allosteric properties observed suggest an important role for this enzyme in the regulation of asparagine biosynthesis.     

Effect of nitrogen starvation on ammonium assimilation by Chlamydomonas reinhardtii

In nitrogen-starved Chlamydomonas reinhardtii , wild type, strain 21 gr cells, consumption of nitrate, nitrite and ammonium may occur in the dark in the absence of an added carbon source. Consumption of ammonium in the dark was about 25% higher than in the light, while consumption of nitrate or nitrite in the dark was lower than in the light.
N starvation produced a linear increase with time in the intracellular level of glutamine synthetase (GS, EC 6.3.2.1) and glutamate synthase (NADH-GOGAT, EC 1.4.1.14 and ferredoxin-GOGAT, EC 1.4.7.1) activities in C. reinhardtii . The effect on GS₁ (3-fold) and NADH-GOGAT (4.5-fold) was higher than that on GS₂ (1.5-fold) and ferredoxin-GOGAT (1.5-fold).
Experiments with methylammonium, L-methionine-D, L-sulfoximine (MSX) and azaserine suggest that: 1) Ammonium itself decreases the intracellular levels of glutamine synthetase and ferredoxin-glutamate synthase activities; and 2) a metabolite resulting from ammonium assimilation by the alga may be a negative modulator of NADH-glutamate synthase activity.     

Effects of water stress on enzymes of ammonia assimilation in root nodules of alfalfa (Medicago sativa)

Protein content and activities of the enzymes glutamine synthetase (EC 6.3.1.2), NADH-glutamate synthase (EC 1.4.1.14), NADH-glutamate dehydrogenase (reductive amination (EC 1.4.1.2) and NAD⁺-glutamate dehydrogenase (oxidative deamination) (EC 1.4.1.2) from the plant fraction of root nodules of alfalfa ( Medicago sativa L. cv. Aragon) were determined under water stress. Only NADH-glutamate synthase activity was inhibited during drought. The results indicate that the glutamine synthetase/NADH-glutamate synthase cycle was fully operational in alfalfa nodules of control or even mildly stressed plants when N₂-fixation was not inhibited, but that the coupling between glutamine synthetase and NADH-glutamate synthase was lost as drought progressed. Patterns of glutamine synthetase and NADH-/NAD⁺-gluta-mate dehydrogenase activities reflect changes in ammonia content of nodules and/or availability of carbon substrates, and indicate that nodules maintain sufficient enzyme activity for ammonia assimilation throughout water stress.     

Antisense reduction of serine hydroxymethyltransferase results in diurnal displacement of NH4+ assimilation in leaves of Solanum tuberosum

Serine hydroxymethyltransferase (SHMT) is part of the mitochondrial enzyme complex catalysing the photorespiratory production of serine, ammonium and CO(2) from glycine. Potato plants (Solanum tuberosum cv. Solara) with antisensed SHMT were generated to investigate whether photorespiratory intermediates accumulated during light lead to nocturnal activation of the nitrogen-assimilating enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT). The transformant lines contained 70-90% less SHMT protein, and exhibited a corresponding decrease in mitochondrial SHMT activity. SHMT antisense plants displayed lower photosynthetic capacity and accumulated glycine in light. Glycine was converted to serine in the second half of the light period, while serine, ammonium and glutamine showed an inverse diurnal rhythm and reached highest values in darkness. GS/GOGAT protein levels and activities in the transgenics also reached maximum levels in darkness. The diurnal displacement of NH(4)(+) assimilation was accompanied by a change in the subunit composition of GS(2), but not GS(1). It is concluded that internal accumulation of post-photorespiratory ammonium is leading to nocturnal activation of GS/GOGAT, and that the time shift in ammonia assimilation can constitute part of a strategy to survive photorespiratory impairment.     

Allogibberic acid: An inhibitor of flowering in Lemna perpusilla

Allogibberic acid (I) has been identified as the compound responsible for the inhibition of flowering, increase in frond multiplication rate and decrease in frond size produced in Lemna perpusilla 6746 by autoclaved, unbuffered aqueous solutions of gibberellic acid (VII). 13-Deoxyallogibberic acid (IV), a product of autoclaving aq. GA₇ (VIII) solutions, also inhibits flowering in L. perpusilla and is about 10 times more active than allogibberic acid.