Effect of applied benzyladenine on endogenous cytokinin content during the early stages of bud development of kiwifruit

The filamentous non-N₂-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl₁) was able to utilize glutamine as the sole nitrogen source. The addition to ammonium-grown cultures of the irreversible inhibitor of glutamine synthetase activity L-methionine-D, L-sulfoximine (MSX) inhibited cell growth. Supplying glutamine to the culture restored cell growth. This re-established growth was not due to interference by glutamine of MSX uptake by the cells, since glutamine synthetase (GS, EC 6.3.1.2) activity remained completely inhibited by MSX even when glutamine was simultaneously present. Both glutamine and ammonium exerted a negative effect on nitrate reductase (NR. EC 1.7.7.2) and nitrite reductase (NiR, EC 1.7.7.1) in vivo. This negative effect was reversed by MSX. When glutamine was added to MSX-treated cells, intracellular glutamine level was high, but the activity of both reductases remained at a high level. These results suggest that the presence of the active form of glutamine synthetase is required for the in vivo prevention of nitrate assimilation caused by ammonium and glutamine.     

Methionine sulfoximine and phosphinothricin--glutamine synthetase inhibitors and activators and their herbicidal activity (A review)

Derivatives of methionine sulfoximine (MSO) and phosphinothrycin (PPT), which are analogues of glutamate, exhibit selective herbicidal activity. This effect is accounted for by impairments of nitrogen metabolism, resulting from inhibition of its key enzyme in plants, glutamine synthetase (EC 6.3.1.2). Inhibition of the enzyme causes ammoniac nitrogen to accumulate and terminates the synthesis of glutamine. Changes in the content of these two metabolites (excess ammonium and glutamine deficiency) act in a concert to cause plant death. However, low concentrations of MSO, PPT, and their metabolites produce an opposite effect: glutamine synthetase is activated, with concomitant stimulation of plant growth and productivity. The mechanisms whereby MSO and PPT affect glutamine synthetase activity are discussed in the context of nitrogen metabolism in plants.     

Methionine Sulfoximine and Phosphinothrycin: A Review of Their Herbicidal Activity and Effects on Glutamine Synthetase

Derivatives of methionine sulfoximine (MSO) and phosphinothrycin (PPT), which are analogues of glutamate, exhibit selective herbicidal activity. This effect is accounted for by impairment of nitrogen metabolism, resulting from inhibition of its key enzyme in plants, glutamine synthetase (EC 6.3.1.2). Inhibition of the enzyme causes ammoniac nitrogen to accumulate and terminates the synthesis of glutamine. Changes in the content of these two metabolites (excess ammonium and glutamine deficiency) act in concert to cause plant death. However, low concentrations of MSO, PPT, and their metabolites produce an opposite effect: glutamine synthetase is activated, with concomitant stimulation of plant growth and productivity. The mechanisms whereby MSO and PPT affect glutamine synthetase activity are discussed in the context of nitrogen metabolism in plants.     

Hairy roots of Brassica napus: I. Applied glutamine overcomes the effect of phosphinothricin treatment

Summary Hairy roots of Brassica napus (rape cv. Giant) were produced by cocultivating leaf and cotyledon explants with Agrobacterium rhizogenes strain A4T. The hairy roots grew prolifically on solid and in liquid media. Incorporation of ammonium sulphate or phosphinothricin (PPT) into the media reduced growth. PPT treatment reduced glutamine synthetase (GS) activity and increased the ammonia content of the hairy roots. We have found that PPT treatment also induces a loss of glutamine from the roots and this may influence root growth. To test this we grew hairy roots in a liquid medium containing 10 mM glutamine. This glutamine treatment overcame the PPT induced suppression of growth but also significantly increased GS activity, reduced ammonia accumulation and increased the levels of glutamate and asparagine.     

The glutamine synthetase-glutamate synthase system in Rhodopseudomonas sphaeroides

The phototrophic purple bacterium Rhodopseudomonas sphaeroides, strain 2R, can assimilate ammonium by means of glutamine synthetase and glutamate synthase. A higher activity of glutamine synthetase is displayed by cells grown in the medium with glutamate or in the atmosphere of molecular nitrogen. The activity of glutamate synthase also rises when cells grow in the atmosphere of N2. However, in contrast to glutamine synthetase, the activity of glutamate synthase does not decrease in the presence of considerable NH4+ amounts. The glutamine synthetase of R. sphaeroides is modified by adenylylation/deadenylylation. In the presence of nitrogenase in R. sphaeroides, the glutamine synthetase is found mainly in the deadenylylation state. Methionine sulfone, an inhibitor of glutamine synthetase, partly restores the activity of nitrogenase in the presence of ammonium, and prevents adenylylation of glutamine synthetase.     

Biochemical and genetic analysis of a Chlamydomonas reinhardtii mutant devoid of chloroplastic glutamine synthetase activity

A spontaneous double mutant of Chlamydomonas reinhardtii, designated ARF3, was resistant to L-methionine-S-sulfoximine (MSX), lacked chloroplastic glutamine synthetase (GS2) activity, and grew very poorly in all media tested. In segregants obtained after genetic crosses, the poor-growth phenotype was always linked to the lack of GS2 and to a diminished rate of consumption of ammonium, even under conditions where photorespiration was minimized. The ammonium permeases in mutant ARF3, however, were not altered. This indicates that, unlike in higher plants, GS2 contributes substantially to the primary assimilation of ammonia in this alga, and that its function cannot be replaced by the cytosolic glutamine synthetase. In genetic crosses, the MSX resistance and the lack of GS2 segregated independently, indicating that resistance was not due to an altered form of GS2. Received: 5 June 1998 / Accepted: 10 September 1998     

Mutants of Azospirillum brasilense resistant to methylammonium

One hundred and twenty-nine mutants of Azospirillum brasilense strain Sp6, resistant to methylammonium, were isolated. Three of the mutants were found to be able to reduce acetylene in the presence of 4 mM ammonium or 120mM methylammonium, concentrations which strongly reduced the nitrogenase activity of the parental strain. Under N₂-fixing conditions, two mutants failed to switch off nitrogenase when NH₄Cl was added. Moreover, the three mutants showed a reduced capacity to incorporate [¹⁴C]methylammonium. The level of glutamine synthetase activity found in the mutants was not reduced as compared to that of the parental strain. All of the data indicate an impairement in the mechanism of ammonium uptake by the bacterial cell.Abbreviations MEA Methylammonium - MSP minimal medium (ammonium free) - PY complete medium - GS glutamine synthetase     

Regulation of Chloroplast Development by Nitrogen Source and Growth Conditions in a Chlorella protothecoides Strain

Chlorella strain (UTEX 27) maintains optimal photosynthetic capacity when growing photoautotrophically in the presence of ammonium. Nitrate-grown photoautotrophic cells, however, show a drastic loss of chlorophyll content and ribulose-1,6-bisphosphate carboxylase/oxygenase activity, resulting in a greater than 10-fold decrease in photosynthetic capacity and growth rate. Nitrate-grown cells are not deficient in protein content, and under mixotrophic and heterotrophic conditions, the alga can utilize nitrate as well as it does ammonium. The alga metabolizes both glucose and acetate in the dark with a doubling time of 5 to 6 hours. However, its growth on acetate is inhibited by light. Ribulose-1,6-biphosphate carboxylase/oxygenase activity correlates well with photosynthetic capacity, and glucose 6-phosphate dehydrogenase and hexokinase activities are altered in a manner consistent with the availability of glucose in growing cells. The alga appears to assimilate ammonium under photoautotrophic conditions primarily via the glutamine synthetase pathway, and shows an induction of both NADH and NADPH dependent glutamate dehydrogenase pathways under mixotrophic and heterotrophic conditions. Multiple isoforms are present only for hexokinase and glucose 6-phosphate dehydrogenase. Etiolated nitrate-grown cells resume greening and increase their photosynthetic capacity after about 6 hours of incubation in the presence of ammonium under photoautotrophic conditions. Similarly, the loss of photosynthetic capacity in ammonium-grown photoautotrophic cells commence about 9 hours after their transfer to heterotrophic nitrate containing media.     

Relation between Glutamine Synthetase and Nitrogenase Activities in the Symbiotic Association between Rhizobium japonicum and Glycine max

The activity and extent of adenylylation of glutamine synthetase was examined in both free-living and bacteroid forms of Rhizobium japonicum in the presence of excess ammonia. Ammonia caused an apparent repression of glutamine synthetase in free-living R. japonicum and adenylylation of the enzyme was also increased. In contrast, neither the activity nor the extent of adenylylation of the bacteroid enzyme was consistently affected by ammonium treatment of bacteroid suspensions. Similar results were obtained after ammonium treatment of soybean plants even though nitrogenase activity was reduced markedly. We have been unable to demonstrate ammonium repression of nitrogenase activity in R. japonicum-Glycine max symbiotic association that is mediated through bacteroid glutamine synthetase. This result is in contrast to the situation in nitrogen-fixing strains of Klebsiella where a role of glutamine synthetase in the regulation of nitrogenase has been reported.     

Regulation of nitrate reductase levels in the cyanobacteria Anacystis nidulans, Anabaena sp. strain 7119, and Nostoc sp. strain 6719.

The effect of the nitrogen source on the cellular activity of ferredoxin-nitrate reductase in different cyanobacteria was examined. In the unicellular species Anacystis nidulans, nitrate reductase was repressed in the presence of ammonium but de novo enzyme synthesis took place in media containing either nitrate or not nitrogen source, indicating that nitrate was not required as an obligate inducer. Nitrate reductase in A. nidulans was freed from ammonium repression by L-methionine-D,L-sulfoximine, an irreversible inhibitor of glutamine synthetase. Ammonium-promoted repression appears therefore to be indirect; ammonium has to be metabolized through glutamine synthetase to be effective in the repression of nitrate reductase. Unlike the situation in A. nidulans, nitrate appeared to play an active role in nitrate reductase synthesis in the filamentous nitrogen-fixing strains Anabaena sp. strain 7119 and Nostoc sp. strain 6719, with ammonium acting as an antagonist with regard to nitrate.     

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.     

Evidence of covalent modification of glutamine synthetase in the purple sulfur bacterium Thiocapsa roseopersicina

Abstract It was shown that glutamine synthetase of purple sulfur bacterium Thiocapsa roseopersicina is regulated by covalent modification. This conclusion is made on the basis of results showing that: (i) incubation of cells under conditions of nitrogen deprivation in the light lead to an increase of glutamine synthetase activity; (ii) addition of ammonium to nitrogen-starved cell suspensions caused a rapid decrease of glutamine synthetase activity; (iii) inhibition of glutamine synthetase by feedback modifiers was higher in ammonium-treated cells than in those starved for a nitrogen source; (iv) treatment of purified glutamine synthetase and cell-free extracts with phosphodiesterase was accompanied by an increase of glutamine synthetase activity, indicating the cleavage of modifying residues covalently bound to glutamine synthetase molecules.     

Growth,ammonia accumulation and glutamine synthetase activity in alfalfa (Medicago sativa L.) shoots and cell cultures treated with phosphinothricin

Summary Phosphinothricin is a non-selective herbicide which inhibits glutamine synthetase (EC 6.3.1.2) activity causing an overaccumulation of ammonia in higher plants. Alfalfa (Medicago sativa L) shoot tissue and petiole-derived callus exposed to phosphinothricin show 50 and 70% reductions, respectively, in glutamine synthetase activity with a concomitant rise of 10 and 20 fold, respectively, in endogenous ammonia. The diffusibility of ammonia may limit the use of a detoxifying gene, phosphinothricin acetyltransferase, as a selectable marker for alfalfa transformation. However, the addition of up to 40 times the standard levels of ammonium nitrate to the culture media used in this study had no effect on callus growth, although glutamine synthetase activity was inhibited by 50% and endogenous ammonia increased 27 fold. Therefore, ammonia accumulation may not be the primary cause of cell death in alfalfa after exposure to phosphinothricin. It follows that diffusion of ammonia from cell to cell would not restrict the selection for phosphinothricin acetyltransferase transformed cells, thereby indicating that this enzyme could be used as a selectable marker in transformation experiments.Abbreviations PPT Phosphinothricin - PAT Phosphinothricin acetyltransferase     

Effect of L-methionine-DL-sulphoximine, a specific inhibitor of glutamine synthetase, on ammonium and nitrate metabolism in the unicellular alga Cyanidium caldarium

In the unicellular alga Cyanidium caldarium nitrate utilization is strongly inhibited by ammonium and it is resumed when ammonium has been depleted. In the presence of L-methionine-DL-sulphoximine (MSX), which prevents ammonium assimilation through a specific irreversible inhibition of glutamine synthetase, nitrate reduction is no longer inhibited by ammonium, and most of the ammonium derived from nitrate reduction is excreted into the external medium. However, in the presence of MSX, nitrate reduction to ammonium proceeds at a reduced rate (45 to 70% of the control); this is particularly marked at low nitrate concentration. It is hypothesized that either MSX or accumulating ammonium bring about decrease in the rate of nitrate entry into the cell.     

Phenotypic expression of ammonia-excreting mutants of Anabaena 7120 under nitrogen limitation

Mutants of Anabaena 7120 defective in glutamine synthetase (GS) activity were isolated following transposon mutagenesis. Mutants M11, M55 and M73 showed about 60% less GS activity in N₂-grown aerobic cultures than the wild-type strain and were resistant to the glutamate analogue l-methionine-dl-sulphoximine (MSX). These mutants had the capacity to excrete N₂-fixed ammonia continuously into the culture medium and showed an enhanced level of aerobic nitrogenase activity. The intracellular ammonium pool generated in N₂-grown cells of mutants was found to be less than that of the wild-type strain. Similarly, ammonium uptake by these mutants was 50% less in mutants compared to the wild-type, suggesting a possible role of GS in controlling this function.     

Adaptation of acrylamide producer Rhodococcus rhodochrous M8 to change in ammonium concentration in medium

The mechanism of adaptation of the acrylamide producing strain Rhodococcus rhodochrous M8 to changes in ammonium concentrations in the medium was studied. An increase in the content of ammonium in the medium changed the activity of glutamine synthetase (GS) (EC 6.3.1.2) and glutamine dehydrogenase (GD) (EC 1.4.1.4), the enzymes of ammonium assimilation, as well as the activities of enzymes responsible for nitrile utilization: nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4). This also caused inhibition of activation of GS induced by phosphodiesterase (EC 3.1.4.1). Increases in the activities of nitrile hydratase and amidase and resistance of these enzymes to ammonium were observed in mutant of R. rhodichrous resistant to phosphotricine, an inhibitor of GS. An important role of GS in the mechanism of adaptation is suggested.     

glnA mutations conferring resistance to methylammonium in Escherichia coli K12

Cells of Escherichia coli K12 were sensitive to 100 mM-methylammonium when cultured under nitrogen limitation, and resistant when grown with an excess of either NH4Cl or glutamine. Glutamine synthetase activity was required for expression of the methylammonium-sensitive phenotype. Mutants were isolated which were resistant to 100 mM-methylammonium, even when grown under nitrogen limitation. P1 bacteriophage transduction and F' complementation analysis revealed that the resistance-conferring mutations mapped either inside the glnA structural gene and/or elsewhere in the E. coli chromosome. Glutamine synthetase was purified from the wild-type and from some of the mutant strains. Strains carrying glnA-linked mutations that were solely responsible for the methylammonium-resistant phenotype yielded an altered enzyme, which was less active biosynthetically with either ammonium or methylammonium as substrate. Sensitivity to methylammonium appeared to be due to synthesis of gamma-glutamylmethylamide by glutamine synthetase, which was synthesized poorly, if at all, by mutants carrying an altered glutamine synthetase enzyme.     

The role of O2-limitation in control of nitrogenase in continuous cultures of Rhizobrium sp.

Oxygen-limited continuous cultures of the cowpea $\text{Rhizobium}$ sp. strain CB756, had high levels of nitrogenase activity, which were not significantly affected by excess ammonium ions or glutamine. When the growth-restricting O₂-limitation was partially relieved, nitrogenase was repressed and this was accompanied by increased adenylylation of glutamine synthetase. It is suggested that the restricted supply of ATP interferes with adenylylation of glutamine synthetase during O₂-limited growth, thus preventing repression of nitrogenase in the presence of excess ammonium ions.     

Regulation of flux through glutaminase and glutamine synthetase in isolated perfused rat liver

1. Glutaminase and glutamine synthetase are simultaneously active in the intact liver, resulting in an energy consuming cycling of glutamine at a rate up to 0.2 mumol per g per min. 2. An increase in portal glutamine concentration was followed by an increased flux through glutaminase, but flux through glutamine synthetase remained unchanged. Glutaminase flux was also increased by ammonium ions or glucagon; these effects were additive. 3. Glutamine synthetase flux was increased by ammonium ions, but this activation was partly overcome by increasing portal glutamine concentrations. Glutamine synthetase flux was slightly increased by glucagon at portal glutamine concentrations of about 0.2-0.3 mM, but was strongly inhibited above 0.6 mMs. 4. During experimental metabolic acidosis there was an increased net release of glutamine by the liver, being due to opposing changes of flux through glutaminase and glutamine synthetase. Conversely, an increased glutamine uptake by the liver during metabolic alkalosis was observed due to an inhibition of glutamine synthetase and an activation of glutaminase. However, the two enzyme activities respond differently depending on whether glucagon or ammonium ions are present.