Isoenzymes of Glutamine Synthetase in Roots of Pea (Pisum sativum L. cv Little Marvel) and Alfalfa (Medicago media Pers. cv Saranac)

Cell organelles have been isolated from protoplast lysates and total homogenates obtained from root tips of Pisum sativum L. (cv Little Marvel) and Medicago media Pers. (cv Saranac) grown in hydroponics with nitrate nutrient solutions. Density-gradient and differential centrifugation procedures have been used to prepare mitochondria-and plastid-enriched fractions in which glutamine synthetase (GS) activity was estimated. Even when purified protoplasts were gently ruptured, significant breakage of plastids occurred during preparation as shown by the high proportion of nitrite reductase recovered in the soluble fraction. Of the total GS activity recovered, up to 20% was associated with the plastid fraction, depending on the source of plant material and the GS assay utilized; when corrected for recovery of the plastid marker nitrite reductase, it was calculated that 15 to 57% of alfalfa and 14 to 64% of pea root GS was located in the plastids. A true biosynthetic assay in which glutamine production was monitored by high performance liquid chromatography was devised to estimate the physiological significance of the transferase and the semibiosynthetic assays currently used for activity measurements. When compared with the true and semibiosynthetic assays, the transferase assay for GS appeared to underestimate the root plastid enzyme. Root plastid GS was partially purified by ion-exchange chromatography, and results show that the isoenzyme found in root plastids is different from chloroplastic or cytosolic GS.     

The location of nitrite reductase and other enzymes related to amino Acid biosynthesis in the plastids of root and leaves

Density gradient separation of plastids from leaf and root tissue was carried out. The distribution in the gradients of the activity of the following enzymes was determined: nitrite reductase, glutamine synthetase, acetolactate synthetase, aspartate aminotransferase, catalase, cytochrome oxidase, and triosephosphate isomerase. The distribution of chlorophyll was followed in gradients from leaf tissue. The presence of plastids that have retained their stroma enzymes was denoted by a peak of triosephosphate isomerase activity. Coincidental with this peak were bands of nitrite reductase, acetolactate synthetase, glutamine synthetase, and aspartate aminotransferase activity. The results suggest that most, if not all, the nitrite reductase and acetolactate synthetase activity of the cell is in the plastids. The plastids were found to contain only part of the total glutamine synthetase, aspartate aminotransferase, and triosephosphate dehydrogenase activity in the cell. Some evidence was obtained for low levels of glutamate dehydrogenase activity in chloroplasts.     

Carbon and nitrogen metabolism in the seagrass, Zostera marina L.: Environmental control of enzymes involved in carbon allocation and nitrogen assimilation

This study experimentally examined influences of environmental variables on the activities of key enzymes involved in carbon and nitrogen metabolism of the submersed marine angiosperm, Zostera marina L. Nitrate reductase activity in leaf tissue was correlated with both water-column nitrate concentrations and leaf sucrose levels. Under elevated nitrate, shoot nitrate reductase activity increased in both light and dark periods if carbohydrate reserves were available. When water-column nitrate was low, glutamine synthetase activity in leaf tissue increased with environmental ammonium. In contrast, glutamine synthetase activity in belowground tissues was statistically related to both nitrate and temperature. At the optimal growth temperature for this species (ca. 25 °C), increased water-column nitrate promoted an increase in glutamine synthetase activity of belowground tissues. As temperatures diverged from the optimum, this nitrate effect on glutamine synthetase was no longer evident. Activities of both sucrose synthase and sucrose-P synthase were directly correlated with temperature. Sucrose-P synthase activity also was correlated with salinity, and sucrose synthase activity was statistically related to tissue ammonium. Overall, the enzymatic responses that were observed indicate a tight coupling between carbon and nitrogen metabolism that is strongly influenced by prevailing environmental conditions, especially temperature, salinity, and environmental nutrient levels.     

Nitrogen Starvation and the Regulation of Glutamine Synthetase in Agmenellum quadruplicatum

The level of glutamine synthetase activity in Agmenellum quadruplicatum strain PR-6 was dependent on the nitrogen source used for growth and on the nutritional status of the cells. During exponential growth, glutamine synthetase activity was low in cells grown on ammonia, urea, or nitrate. During the transition from nitrogen replete to nitrogen starved growth, glutamine synthetase activity began to rise. With ammonia as a nitrogen source, glutamine synthetase activity as determined in whole cells increased from 1 nanomole per minute per milliliter during exponential growth to 22 nanomoles per minute per milliliter during severe nitrogen starvation. In cells grown on nitrate the increase was from 5 to 39 nanomoles per minute per milliliter, and in cells grown on urea the increase was from 4 to 31 nanomoles per minute per milliliter.     

Appearance of nitrate reductase, nitrite reductase and glutamine synthetase in different organs of the Scots pine (Pinus sylvestris) seedling as affected by light, nitrate and ammonium

Appearance of nitrate reductase (NR, EC 1.6.6.1–3), nitrite reductase (NiR, EC 1.7.7.1) and glutamine synthetase (GS, EC 6.3.1.2) under the control of nitrate, ammonium and light was studied in roots, hypocotyls and needles (cotyledonary whorl) of the Scots pine ( Pinus sylvestris L.) seedling. It was found that appearance of NiR was mainly controlled by nitrate whereas appearance of GS was strongly controlled by light. In principle, the NR activity level showed the same dependency on nitrate and light as that of NiR. In the root, both nitrate and ammonium had a stimulatory effect on GS activity whereas in the whorl the induction was minor. The level of NiR (NR) activity is high in the root and hypocotyl and low in the cotyledonary whorl, whereas the GS activity level per organ increases strongly from the root to the whorl. Thus, in any particular organ the operation of the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle is not closely connected to the operation of the nitrate reduction pathway. The strong control of GS/GOGAT by light and the minor sensitivity to induction by nitrate or ammonium indicate a major role of the GS/GOGAT cycle in reassimilation of endogeniously generated ammonium.     

Localization of Nitrogen-Assimilating Enzymes in the Chloroplast of Chlamydomonas reinhardtii

The specific activities of nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, and glutamate dehydrogenase were determined in intact protoplasts and intact chloroplasts from Chlamydomonas reinhardtii. After correction for contamination, the data were used to calculate the portion of each enzyme in the algal chloroplast. The chloroplast of C. reinhardtii contained all enzyme activities for nitrogen assimilation, except nitrate reductase, which could not be detected in this organelle. Glutamate synthase (NADH- and ferredoxin-dependent) and glutamate dehydrogenase were located exclusively in the chloroplast, while for nitrite reductase and glutamine synthetase an extraplastidic activity of about 20 and 60%, respectively, was measured. Cells grown on ammonium, instead of nitrate as nitrogen source, had a higher total cellular activity of the NADH-dependent glutamate synthase (+95%) and glutamate dehydrogenase (+33%) but less activity of glutamine synthetase (−10%). No activity of nitrate reductase could be detected in ammonium-grown cells. The distribution of nitrogen-assimilating enzymes among the chloroplast and the rest of the cell did not differ significantly between nitrate-grown and ammonium-grown cells. Only the plastidic portion of the glutamine synthetase increased to about 80% in cells grown on ammonium (compared to about 40% in cells grown on nitrate).     

Metabolism of Urtica dioica as dependent on the supply of mineral nutrients

Plants of Urtica dioica L., a very nitrophilous species, were grown in a nutrient solution containing either high (100%) or low (2%) nutrient supply. Part of these plants were subjected to a sudden switch from 100% to 2% or vice versa. Plant weight, sugar and organic nitrogen (both soluble and insoluble) and nitrate content were measured during growth. The activities of two nitrogen assimilating enzymes, nitrate reductase (NR) and glutamine synthetase (GS) were determined.
Growth of Urtica dioica was retarded at low nutrient supply. Root growth was limited by another factor than nitrogen. This was shown by a higher protein content. In the first period after a switch from 100% to 2%, redistribution of nitrogen from shoot to root could be demonstrated, and leakage from the root into the nutrient solution. It is suggested that in these conditions GS in the root reacted to this downward flux. Comparison with earlier findings on the less nitrophilous Plantago lanceolata showed that at 100% nutrient supply a correlation occurs between nitrate reduction and glutamine synthetase activity in that plant part which exported reduced nitrogen: the root in P. lanceolata and the shoot in U. dioica. In the importing plant part, glutamine synthetase was influenced by nitrate reduction as well as by imported reduced nitrogen.     

Effects of aerobic versus anoxic conditions on glutamine synthetase activity in eelgrass (Zostera marina L.) roots: regulation of ammonium assimilation potential

Root glutamine synthetase (GS; EC 6.3.1.2) activity was measured daily (0 to 4 days) for eelgrass (Zostera marina L.) plants held under continuous darkness rooted in sediments, continuous darkness without sediments, continuous light without sediments, and control light/dark cycle (Control L/D). Roots experiencing prolonged aerobiosis exhibited lower activity in vitro than controls, whereas roots experiencing prolonged anoxia exhibited increased activity. Plants held in darkness without sediments had activity intermediate between controls and anoxic roots. One-hour pretreatment of root extracts with ATP slightly reduced in vitro glutamine synthetase activity, whereas pretreatment with ADP and AMP increased activity ≈50%. While glutamine synthetase activity increased with higher adenylate energy charge (AEC) in the reaction mixture, pretreatment of enzyme extracts at high adenylate energy charges decreased subsequent activity relative to pretreatment at lower energy charges. One-hour pretreatment with l-alanine (Ala) had little effect on enzyme activity. Pretreatment with l-glutamine (Gln), l-glutamate (Glu), and γ-amino butyric acid (GABA) increased activity ≈75%. Incubation of excised roots under anoxic conditions for 24 h nearly doubled enzyme activity. However, addition of cycloheximide to anoxic root incubations lessened or prevented the increase in activity. It appears that enhanced glutamine synthetase activity following periods of root anoxia results from interactions with metabolites that fluctuate between aerobic and anoxic conditions, particularly adenylates, and from de novo synthesis of glutamine synthetase or some other protein synthesis-dependent process.     

Inorganic nitrogen assimilation in plants of Austrlian rainforest communities

In a study of the plant communities of two Australian rainforests, it was found that pioner species had high levels of nitrate reductase (EC 1.6.6.1) and were predominantly leaf nitrate assimilators. Under- and over-storey species had low levels of shoot and root nitrate reductase activity, and many of them showed little capacity for nitrate reduction even when nitrate ions were freely available. Although closed-forest species have lower levels of nitrate reductase than those of gaps and forest margins, their total nitrogen contents were similar, suggesting the former utilize nitrogen sources other than nitrate ions. Glutamine synthetase (EC 6.3.1.2) was present in the leaves of all species examined. In the leaves of pioneer species the chloroplastic isoform of glutamine synthetase predominted, while in most of the species typical of closed-forest the cytosolic isoform accounted for at least 40% of total leaf activity. Low levels of chloroplastic glutamine synthetase were correlated with a low capacity for leaf nitrate reduction, and both are characteristic of many species that regenerate and grow for some time in shade. Low levels of chloroplastic glutamine synthetase imply that, in some of these woody plants, photorespiratory ammonia is re-assimilated via cytosolic glutamine synthetase.     

Glutamine transport and feedback regulation of nitrate reductase activity in barley roots leads to changes in cytosolic nitrate pools

The size of tissue amino acid pools in plants may indicate nitrogen status and provide a signal that can regulate nitrate uptake and assimilation. The effects of treating barley roots with glutamine have been examined, first to identify the transport system for the uptake of the amino acid and then to measure root NR activity and cellular pools of nitrate. Treating N replete roots with glutamine elicited a change in the cell membrane potential and the size of this response was concentration dependent. In addition, the size of the electrical change depended on the previous exposures of the root to glutamine and was lost after a few cycles of treatment. Whole root tissue pools of glutamine and phenylalanine increased when roots were incubated in a nutrient solution containing 10 mM nitrate and 1 mM glutamine. Treating roots with 1 mM glutamine increased cytosolic nitrate activity from 3 mM to 7 mM and this change peaked after 2 h of treatment. Parallel measurements of root nitrate reductase activity during treatment with 1 mM glutamine showed a decrease. These measurements provide evidence for feedback regulation on NR activity that result in changes in cytosolic nitrate activity. After 6 h in glutamine both root NR activity and cytosolic nitrate activity returned to pretreatment values, while tissue concentrations of glutamine and phenylalanine remained elevated. The data are discussed in terms of the mechanisms that are most likely to be responsible for the changes in cytosolic nitrate.     

Glutamine synthetase regulation by energy charge in sunflower roots

Energy charge [(ATP) + ½ (ADP)]/[(ATP) + (ADP) + (AMP)] and glutamine synthetase activity (transferase reaction) of roots increase in a near congruent manner when decotyledonized sunflower plants (Helianthus annuus L. var. Mammoth Russian) are grown in nitrate for 9 days. Replacement of nitrate with ammonium for the final 2 days leads to a higher energy charge and increased enzyme activity. Similar correlations occur when nitrate plants are placed on a zero nitrogen regimen and when they are subjected to continuous darkness. A rank order correlation of 0.72 is obtained for all data. Control concepts such as adenylylation-deadenylylation and ammonium inhibition of enzyme synthesis are not supported by the data. Energy charge-enzyme activity plots support the view that glutamine synthetase of sunflower roots is subject to control by end products of glutamine metabolism. Alanine appears to exert a modulating effect on the regulation of glutamine synthetase by energy charge.     

Action of Inhibitors of Ammonia Assimilation on Amino Acid Metabolism in Hordeum vulgare L. (cv Golden Promise)

Barley (Hordeum vulgare L. cv Golden Promise) plants were grown in a continuous culture system in which the root and shoot ammonia and amino acid levels were constant over a 6-hour experimental period. Methionine sulfoximine (MSO), 1 millimolarity when added to the culture medium, caused a total inactivation of root glutamine synthetase with little effect on the shoot enzyme. Root ammonia levels increased and glutamine levels decreased, irrespective of whether the plants were grown in 1 millimolar nitrate or 1 millimolar ammonia. Levels of glutamate, aspartate, serine, threonine, and asparagine all increased. There was little alteration in the amino acid and ammonia levels in the shoot, suggesting that MSO is not rapidly transported.

The addition of azaserine (25 micrograms per milliliter) to nitrate-grown plants caused a rapid increase in root ammonia, glutamine, and serine levels with a corresponding decrease in glutamate, aspartate, and alanine. Glutamine levels also increased in the shoot.

The in vivo effect of MSO and azaserine was as would be predicted by their known in vitro inhibitory action if the glutamine synthetase/glutamate synthase pathway of ammonia assimilation was in operation.

     

Behaviour of nodulatedPisum sativum L. under short-term nitrate stress conditions

Nitrate (20 mM) applied to the root medium of 28-day-old nodulated pea plants (Pisum sativum L., cv. Jupiter) immediately retarded nodule growth and inhibited root nodulation. Acetylene-reducing and H₂-evolving nitrogenase activities were also significantly inhibited. The inhibitory effect of nitrate on nodule respiration was less pronounced while the respiration of roots was increased after the addition of nitrate. The levels of cytosol glutamine synthetase and nitrate reductase in nodule cytosol were permanently decreased from the 4th day after nitrate application. These results indicate that the inhibitory effect of high nitrate concentration on whole nodule metabolism is nonspecific in nature.     

o-Phosphotyrosyl glutamine synthetase: modification of the nucleotide ligation site of adenylylated glutamine synthetase

The nucleotide ligation site of adenylylated glutamine synthetase, which contains a unique tyrosyl residue linked through a phosphodiester bond to 5'-AMP, was studied by digestion with three hydrolytic enzymes. The products on micrococcal nuclease digestion were adenosine and o-phosphotyrosyl glutamine synthetase. The Km for this macromolecular substrate with the nuclease was 40 microM, at pH 8.9. The glutamine synthetase activity was not affected by deadenosylation with the nuclease, in contrast to SVPDE digestion, with which the glutamine synthetase activity was markedly increased. The Km for the native adenylylated glutamine synthetase with the SVPDE was 36 microM, i.e., similar to that for the nuclease. When the isolated o-phosphotyrosyl enzyme was incubated with alkaline phosphatase at pH 7.2, the glutamine synthetase activity rapidly increased to the same level as that of the SVPDE treated enzyme. Furthermore, kinetic properties of the o-phosphotyrosyl glutamine synthetase were compared with those of the adenylylated enzyme. The optimum pH, apparent Km for each of three substrates, glutamate, ATP, and NH3, and Vmax were in good agreement, as to either Mg2+- or Mn2+-dependent biosynthetic activity. From these results we can conclude that the regulation of glutamine synthetase activity simply requires the phosphorylation of the tyrosyl residue in each subunit, without recourse to adenylylation.     

Properties of the Bacillus licheniformis A5 glutamine synthetase purified from cells grown in the presence of ammonia or nitrate.

The glutamine synthetase from Bacillus licheniformis A5 was purified by using a combination of polyethylene glycol precipitation and chromatography on Bio-Gel A 1.5m. The resulting preparation was judged to be homogeneous by the criteria of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, equilibrium analytical ultracentrifugation, and electron microscopic analysis. The enzyme is a dodecamer with a molecular weight of approximately 616,000, and its subunit molecular weight is 51,000. Under optimal assay conditions (pH 6.6, 37 degrees C) apparent Km values for glutamate, ammonia, and manganese.adenosine 5'-triphosphate (1:1 ratio) were 3.6, 0.4, and 0.9 mM, respectively. Glutamine synthetase activity was inhibited approximately 50% by the addition of 5 mM glutamine, alanine, glycine, serine, alpha-ketoglutarate, carbamyl phosphate, adenosine 5'-diphosphate, or inosine 5'-triphosphate to the standard glutamine synthetase assay system, whereas 5 mM adenosine 5'-monophosphate or pyrophosphate caused approximately 90% inhibition of enzyme activity. Phosphorylribosyl pyrophosphate at 5 mM enhanced activity approximately 60%. We were unable to detect any physical or kinetic differences in the properties of the enzyme when it was purified from cells grown in the presence of ammonia or nitrate as sole nitrogen source. The data indicate that B. licheniformis A5 contains one species of glutamine synthetase whose catalytic activity is not regulated by a covalent modification system.     

Ontogenetic Variation of Nitrogenase, Nitrate Reductase, and Glutamine Synthetase Activities in Oryza sativa

The relationship between the rates of nitrogenase, nitrate reductase, and glutamine synthetase activities, and plant ontogeny in rice (Oryza sativa L.), cultivar `M9', grown in salt marsh sediment with and without nitrate treatment was studied. In both treatments, nitrogenase activity measured as the immediate linear rate of acetylene reduction by bacteria associated with the roots varied with plant age. In control plants, the nitrogenase activity developed during the vegetative stage, peaked during early reproductive growth and then declined. The application of 10 kilograms N per hectare as KNO₃ once every 2 weeks delayed the development of and decreased the nitrogenase activity. The nitrogenase activity in both treatments developed as leaf nitrate reductase activity declined. The per cent nitrogen of roots was negatively correlated with the rates of acetylene reduction during the life cycles of control and nitrate-treated plants. This suggests that the concentration of combined nitrogen in the plants controlled the development and rate of root-associated nitrogenase activity. During reproductive growth, no nitrate reductase activity was detected in the roots from either treatment. In control plants, the patterns of nitrogenase activity and glutamine synthetase activity in the roots were similar. Thus, rice roots have the potential to assimilate ammonia while fixing N₂. During the vegetative and early reproductive stages of growth, the development of maximal rates of nitrogenase activity coincided with an increase of total nitrogen of the plants in both treatments.     

Correlation of nitrogen metabolism with biosurfactant production by Pseudomonas aeruginosa

A direct relationship between increased glutamine synthetase activity and enhanced biosurfactant production was found in Pseudomonas aeruginosa grown in nitrate and Proteose Peptone media. A chloramphenicol-tolerant strain showed a twofold increase in biosurfactant production and glutamine synthetase activity. Increased ammonium and glutamine concentrations repressed both phenomena.     

Correlation of nitrogen metabolism with biosurfactant production by Pseudomonas aeruginosa.

A direct relationship between increased glutamine synthetase activity and enhanced biosurfactant production was found in Pseudomonas aeruginosa grown in nitrate and Proteose Peptone media. A chloramphenicol-tolerant strain showed a twofold increase in biosurfactant production and glutamine synthetase activity. Increased ammonium and glutamine concentrations repressed both phenomena.     

Developmental formation of glutamine synthetase in greening pumpkin cotyledons and its subcellular localization

During the germination of pumpkin (Cucurbita sp. Amakuri Nankin) seeds in dark, the activity of glutamine synthetase in cotyledons gradually increased, reaching a maximum at 5 to 6 days. A measurable enhancement (about 4-fold) of the enzyme activity occurred when the seedlings were exposed to continuous illumination from day 4 up to day 8. Glutamine synthetase activity was detectable only in the cytosolic fraction in the etiolated cotyledons, whereas it was found both in the cytosolic and chloroplast fractions in the green cotyledons. The two isoenzymes of glutamine synthetase have been separated by DEAE-cellulose column chromatography of extracts from the green cotyledons. These data indicate that during the greening process the chloroplastic glutamine synthetase is newly synthesized. The roles of cytosolic and chloroplastic glutamine synthetase in germinating pumpkin cotyledons concerning assimilation of NH₃ are discussed.     

Prolonged root hypoxia effects on enzymes involved in nitrogen assimilation pathway in tomato plants

In order to investigate the effects of root hypoxia (1–2% oxygen) on the nitrogen (N) metabolism of tomato plants (Solanum lycopersicum L. cv. Micro-Tom), a range of N compounds and N-assimilating enzymes were performed on roots and leaves of plants submitted to root hypoxia at the second leaf stage for three weeks. Obtained results showed that root hypoxia led to a significant decrease in dry weight (DW) production and nitrate content in roots and leaves. Conversely, shoot to root DW ratio and nitrite content were significantly increased. Contrary to that in leaves, glutamine synthetase activity was significantly enhanced in roots. The activities of nitrate and nitrite reductase were enhanced in roots as well as leaves. The higher increase in the NH₄⁺ content and in the protease activities in roots and leaves of hypoxically treated plants coincide with a greater decrease in soluble protein contents. Taken together, these results suggest that root hypoxia leaded to higher protein degradation. The hypoxia-induced increase in the aminating glutamate dehydrogenase activity may be considered as an alternative N assimilation pathway involved in detoxifying the NH₄⁺, accumulated under hypoxic conditions. With respect to hypoxic stress, the distinct sensitivity of the enzymes involved in N assimilation is discussed.Key words: tomato, hypoxia, nitrogen, glutamine synthetase, protease, glutamate dehydrogenase