Changes in glutamate dehydrogenase activity of Chlamydomonas reinhardtii under different trophic and stress conditions

Abstract. Under stress conditions (darkness, nitrogen starvation, high ammonium concentrations, glutamine synthetase and glutamate synthase inhibition) glutamate dehydrogenase animating activity levels of Chlamydomonas cells varied inversely to those of glutamine synthetase. Nitrogen and carbon sources also influenced glutamate dehydrogenase levels in Chlamydomonas , the highest values being found in cells cultured mixotrophically with ammonium, under which conditions glutamate dehydrogenase and glutamine synthetase levels were likewise inversely related. These facts, together with the analysis of internal fluctuations of ammonium, 2-oxoglutarate, and the amino acid pool as well as the variations of certain enzymes involved in carbon metabolism indicate that glutamate dehydrogenase animating activity is adaptative, being involved in the maintenance of intracellular levels of L-glutamate when they cannot be maintained by the GS-GOGAT cycle, and probably more connected with carbon than nitrogen metabolism.     

Nitrogen metabolism of Douglas fir and Scots pine as affected by optimal nutrition and water supply under conditions of relatively high atmospheric nitrogen deposition

Nitrogen metabolism of the needles of 40-year-old Douglas fir and Scots pine trees, growing in two forest stands on cation-poor and acidic sandy soil with a relatively high atmospheric nitrogen deposition was studied. The composition of the free amino acid (FAA) pool, the concentrations of total nitrogen and soluble protein and the activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were determined in the needles. An excessive nitrogen supply by a high atmospheric nitrogen deposition in both forest stands was indicated by the high concentrations of total nitrogen and the amino acids arginine, glutamic acid, glutamine and aspartic acid in control trees. In addition the effect of optimal nutrition and water supply (fertigation) on the needle nitrogen metabolism was evaluated. The total concentration of the FAA pool in needles of both tree species was lower in the fertigated than in the non-fertigated (control) trees, except for 1-year-old needles of Scots pine, in which the concentration after fertigation did not differ from the control. The lower total FAA concentration in the fertigated trees could be attributed to arginine, the concentration of which was on average 60% lower than in the control. Neither the concentration of soluble protein nor the activity of GS were influenced by fertigation. The activity of GDH in fertigated trees only differed significantly from the control in October. Scots pine needles had higher concentrations of protein (50%) and higher activities of GS (44%) and GDH (25%) than Douglas fir needles. Possible explanations for the lower vitality of Douglas fir compared to Scots pine are given.     

Morphogenesis and free amino acid composition of the ascomycete Sphaerostilbe repens as influenced by nitrogen and calcium

Abstract Sphaerostilbe repens utilizes nitrate and ammonium as nitrogen sources. Differentiation of mycelium into rhizomorphs and coremia was reduced in the presence of nitrate and completely inhibited in the absence of calcium. The most abundant free amino acids were, in decreasing order: alanine, glutamine, glutatomic acid, serine, aspartic acid, γ-aminobutyric acid, arginine and threonine. These compounds represented 90% of the total amino acid pool.
The free amino acid composition did not vary with cultural conditions although concentrations of individual amino acids differed. In ammonium-grown cells, γ-aminobutyric acid increased in concentration and glutamate, aspartate and alanine decreased. Calcium-deficient media reduced amino acid concentrations, especially of arginine and ornithine. Amino acid contents increased during the growth period and were higher in rhizomorphs than in vegetative mycelia.     

Over-expression of cytosolic glutamine synthetase increases photosynthesis and growth at low nitrogen concentrations.

Nitrogen, which is a major limiting nutrient for plant growth, is assimilated as ammonium by the concerted action of glutamine synthetase (GS) and glutamate synthase (GOGAT). GS catalyses the critical incorporation of inorganic ammonium into the amino acid glutamine. Two types of GS isozymes, located in the cytosol (GS1) and in the chloroplast (GS2) have been identified in plants. Tobacco (Nicotiana tabacum) transformants, over-expressing GS1 driven by the constitutive CaMV 35S promoter were analysed. GS in leaves of GS-5 and GS-8 plants was up-regulated, at the level of RNA and proteins. These transgenic plants had six times higher leaf GS activity than controls. Under optimum nitrogen fertilization conditions there was no effect of GS over-expression on photosynthesis or growth. However, under nitrogen starvation the GS transgenics had c. 70% higher shoot and c. 100% greater root dry weight as well as 50% more leaf area than low nitrogen controls. This was achieved by the maintenance of photosynthesis at rates indistinguishable from plants under high nitrogen, while photosynthesis in control plants was inhibited by 40-50% by nitrogen deprivation. It was demonstrated that manipulation of GS activity has the potential to maintain crop photosynthetic productivity while reducing nitrogen fertilization and the concomitant pollution.     

Derepression of asparaginase II during exponential growth of Saccharomyces cerevisiae on ammonium ion

The biosynthesis of asparaginase II in Saccharomyces cerevisiae is sensitive to nitrogen catabolite repression. In cell cultures growing in complete ammonia medium, asparaginase II synthesis is repressed in the early exponential phase but becomes derepressed in the midexponential phase. When amino acids such as glutamine or asparagine replace ammonium ion in the growth medium, the enzyme remains repressed into the late exponential phase. The three nitrogen compounds permit a similar rate of cell growth and are assimilated at nearly the same rate. In the early exponential phase the internal amino acid pool is larger in cells growing with glutamine or asparagine than in cells growing with ammonium sulfate as the sole source of nitrogen.     

Free amino acids and total nitrogen during shoot development in Scots pine seedlings

The concentration of free amino acids and total nitrogen was studied in needles, stems and roots of seedlings of Pinus sylvestris L. for five weeks during the second growth period ("summer"). In one group of seedlings the source/sink relation was disturbed through removal of the terminal buds. The seedlings were cultivated in artificial year-cycles in a climate chamber.
Total nitrogen increased in needles and sterns of intact seedlings in the beginning of the "summer" and decreased during shoot growth. In seedlings, from which the buds had been removed, nitrogen remained at high levels in the primary needles and accumulated in steins and roots. The results are consistent with utilization of nitrogen in older needles and in the stem during shoot elongation.
The pool of free amino acids increased in the beginning of the "summer" and decreased after bud break in primary needles, stems and roots. Arginine and glutamine, in the roots also asparagine, were the dominating amino acids (amides included). Together, these compounds (plus glutamate and aspartate) contributed about 90% of the nitrogen in the amino acid pool in all organs. In primary needles and in the stem, arginine predominated at the end of hardening (75–85% of the amino acid nitrogen). Free amino acids contributed at most ca 10% of the total nitrogen in primary needles, where the ratio of free amino acid nitrogen: total nitrogen was highest at the end of dormancy and in the early "summer". Free amino acids accumulated after bud removal in primary needles and especially in stems and roots. Glutamine became relatively more dominant than arginine in the different organs.
The observations are consistent with the role of arginine and glutamine for storage and transport of nitrogen in conifers. Because of the low concentrations of amino acid nitrogen in the primary needles, arginine is not considered a major nitrogen reserve in needles of Scots pine seedlings.     

Accumulation of Glutamine by Suspension Cultures of Symphytum officinale

A callus was induced from the veins of a leaf of Symphytum officinale, comfrey, on a medium containing the inorganic elements reported by Murashige and Skoog with addition of 3% sucrose, 0.5 mg/liter 2,4-D and 0.3~3.0 mg/liter kinetin.

Suspension cultures of this cell line obtained from the callus were shown to accumulate a large amount of L-glutamine intracellularly, The effect of growth hormones and nutrients on accumulation of the amino acid has been examined in suspension cultures. The most suitable concentrations of 2,4-D and kinetin for glutamine accumulation were 0.3 mg/liter each. The presence of potassium nitrate as a nitrogen source was beneficial for growth and ammonium nitrate stimulated the accumulation of glutamine. High levels of these nitrogen sources in the medium were required for obtaining a high level of glutamine. The concentration of glutamine accumulated reached to approximately 20% of dry cell weight when S. officinale was incubated in the medium containing 0.495 % of ammonium nitrate and 0.570% of potassium nitrate which corresponded to three times higher levels than those in a Murashige and Skoog’s medium.

Most of the amino acid was found intracellularly but a small amount was excreted into the medium in the later stages of the incubation. Addition of a cationic surfactant, cetyltrimethylammonium bromide, to the cultures caused to increase the amount of the amino acid in the culture filtrate.

The contents of free amino acids in leaves of S. officinale were compared with those in the callus. The level of glutamine in the callus was 260 times higher than that in the intact plant.     

Origin of Foliar Nitrogen and Changes in Free Amino-Acid Composition and Content of Leaves, Stubble, and Roots of Perennial Ryegrass During Re-growth after Defoliation

Nitrogen re-mobilization and changes in free amino acids werestudied as a function of time in leaves, stubble, and rootsduring ryegrass (Lolium perenne L.) re-growth. Experiments with¹⁵N labelling clearly showed that during the first days nearlyall the nitrogen in new leaves came from organic nitrogen re-mobilizedfrom roots and stubble. On the days of defoliation, stubblehad the highest content of free amino acids with 23 mg per gdry weight against 15 mg and 14 mg in leaves and roots, respectively.The major amino acids in leaves were asparagine (23% of totalcontent in free amino acids), aminobutyrate, serine, glutamine,and glutamate (between 7% and 15%) whereas in roots and stubblethe contribution of amides was high, especially asparagine (about50%). Re-growth after cutting was associated with a rapid increaseof the free amino acid content in leaves, with a progressivedecrease in roots while stubble content remained virtually unchanged.In leaves, asparagine increased from the first day of re-growth,while the aspartate level remained unchanged and glutamine increasedstrongly on the first day but decreased steadily during thenext few days of re-growth. Asparagine in stubble and rootschanged in opposite directions: in stubble it tended to increasewhereas in roots it clearly decreased. In contrast, stubbleand roots showed a similar decrease in glutamine. In these twoplant parts, as in leaves, aspartate remained at a low level.Results concerning free amino acids are discussed with referenceto nitrogen re-mobilization from source organs (stubble androots) to the sink organ (regrowing leaves). Key words: Lolium perenne L, re-growth, nitrogen, free amino acids, glutamine, asparagine     

The control of glutamine synthetase level in Lemna minor L.

Summary The specific activity of glutamine synthetase (E.C. 6.3.1.2) of Lemna minor L. is markedly reduced when either ammonium ions or glutamine are present in the growth medium. Combinations of 5 mM ammonia and 5 mM glutamic acid or 5 mM ammonia and 5 mM glutamine as nitrogen source, lead to a 4–5 fold reduction of the maximum activity measurable on 5 mM -aminobutyric acid. Analyses of the soluble pool of nitrogen indicate that the reduction in enzyme level is associated with an increase in the pool of glutamine. There is an inverse correlation between the apparent rate of synthesis of glutamine synthetase and the intracellular concentration of glutamine, and this relationship suggests that the glutamine synthetase of Lemna minor is subject to end product repression by the endogenous pool of glutamine.     

Maintenance and exchange of the aromatic amino acid pool in Escherichia coli

The pool of phenylalanine, tyrosine, and tryptophan is formed in Escherichia coli K-12 by a general aromatic transport system [Michaelis constant (K(m)) for each amino acid approximately 5 x 10(-7)m] and three further transport systems each specific for a single aromatic amino acid (K(m) for each amino acid approximately 2 x 10(-6)m, reference 3). When the external concentration of a particular aromatic amino acid is saturating for both classes of transport system, the free amino acid pool is supplied with external amino acid by both systems. Blocking the general transport system reduces the pool size by 80 to 90% but does not interfere with the supply of the amino acid to protein synthesis. If, however, the external concentration is too low to saturate specific transport, blocking general transport inhibits the incorporation of external amino acid into protein by about 75%. It is concluded that the amino acids transported by either class of transport system can be used for protein synthesis. Dilution of the external amino acid or deprivation of energy causes efflux of the aromatic pool. These results and rapid exchange observed between pool amino acid and external amino acids indicate that the aromatic pool circulates rapidly between the inside and the outside of the cell. Evidence is presented that this exchange is mediated by the aromatic transport systems. Mutation of aroP (a gene specifying general aromatic transport) inhibits exit and exchange of the small pool generated by specific transport. These findings are discussed and a simple physiological model of aromatic pool formation, and exchange, is proposed.     

Nitrogen Starvation-Induced Changes in Amino Acid and Free Ammonium Pools in <Emphasis Type="Italic">Schizophyllum commune</Emphasis> Colonies

Wood-decay fungi depend upon recycling of nitrogen-containing molecules to maintain growth in nitrogen-deficient environments. One of the pools that can support growth in these organisms is the pool of free amino acids. The free amino acid (AA) composition of Schizophyllum commune mycelium grown on the surface of nitrogen-rich (M = 6.6 mM L-asparagine) and nitrogen-poor medium (M01 = 0.06 mM L-asparagine) has been examined: When mycelium is grown on M, alanine, glutamate, and asparagine account for almost 2/3 of the amino acid pool. The free amino acid concentration is reduced by 75% for mycelium grown on the M01 medium, with alanine and glutamate predominating. In addition, free NH₄⁺ increases by 60% in nitrogen-deprived mycelia. Except for asparagine, which is absorbed by the apices, the concentration of all free amino acids is higher in the centers of M-grown, 4-day-old mycelia than in the apices. Hyphae grown to exponential growth on M and transferred to M01 for 12 h show greater free amino acid and NH₄⁺ concentrations in the apices, most likely indicating increased translocation to the apices.     

Amino Acid metabolism of pea leaves: labeling studies on utilization of amides

Short term (2-hour) incorporation of nitrogen from nitrate, glutamine, or asparagine was studied by supplying them as unlabeled (¹⁴N) tracers to growing pea (Pisum sativum L.) leaves, which were previously labeled with ¹⁵N, and then following the elimination of ¹⁵N from various amino components of the tissue. Most components had active and inactive pools. Ammonia produced from nitrate was assimilated through the amide group of glutamine. When glutamine was supplied, its nitrogen was rapidly transferred to glutamic acid, asparagine, and other products, and there was some transfer to ammonia. Nitrogen from asparagine was widely distributed into ammonia and amino compounds. There was a rapid direct transfer to glutamine, which did not appear to involve free ammonia. Alanine nitrogen could be derived directly from asparagine, probably by transamination. Homoserine was synthesized in substantial amounts from all three nitrogen sources. Homoserine appears to derive nitrogen more readily from asparagine than from free aspartic acid. A large proportion of the pool of γ-aminobutyric acid turned over, and was replenished with nitrogen from all three supplied sources.     

Analysis of glutamate homeostasis by overexpression of Fd-GOGAT gene in Arabidopsis thaliana

Glutamate plays a central role in nitrogen flow and serves as a nitrogen donor for the production of amino acids. In plants, some amino acids work as buffers: during photorespiration, ammonium derived from the conversion of glycine to serine is promptly reassimilated into glutamate by the glutamine synthetase (GS-2)/ferredoxin-dependent glutamate synthase (Fd-GOGAT) cycle. The glutamate concentration is relatively stable compared with those of other amino acids under environmental changes. The few studies dealing with glutamate homeostasis have but all used knockouts or mutants of these enzymes. Here, we generated Fd-GOGAT (GLU1)-overexpressing Arabidopsis plants to analyze changes in the amino acid pool caused by glutamate overproduction under different ammonium conditions controlled by CO₂ concentration, light intensity and nitrate concentration. Under photorespiratory conditions with sufficient ammonium supply, aspartate increased and glutamine and glycine decreased, but glutamate barely changed. Under non-photorespiratory conditions, however, glutamate and most other amino acids increased. These results suggest that the synthesized glutamate is promptly converted into other amino acids, especially aspartate. In addition, ammonium supply by photorespiration does not limit glutamate biosynthesis, but glutamine and glycine are important. This study will contribute to the understanding of glutamate homeostasis in plants.     

Physiological studies on Phymatotrichum omnivorum X. Influence of nitrogen sources on free and bound amino acid pools

The effect of nitrogen source on the free and bound amino acids of mycelium of Phymatotrichum omnivorum (Shear) Dugg was investigated. The largest free amino acid pool was present in the natural medium and the smallest in the synthetic medium. Phymatotrichum omnivorum was able to utilize different nitrogen sources with the best growth occurring with NH₄NO₃. The ratio of glycine to alanine and aspartic to glutamic was around 0.25 in the free amino acid pool and around 1 in the bound amino acid pool. The free pool of glutamic acid ranged from 5.6 % to 27.2 % depending upon the nitrogen source in the media. The free pool of alanine ranged from 35.7 % to 17.2 % in relation to the nitrogen source. Most other amino acid ratios did not vary significantly between the free amino acids and the bound amino acids.     

Nitrogen and sulphur requirements of Colletotrichum inamdarii Lal

Summary Nitrogen and sulphur requirements ofColletotrichum inamdarii Lal isolated from the leaves ofCarissa carandas L. have been studied. DL-serine, L-asparagine and L-phenylalanine have been found to be of good nitrogen source followed by potassium nitrate, calcium nitrate, magnesium nitrate, DL-alanine, ammonium nitrate, glutamic acid, ammonium sulphate, DL-valine, aspartic acid, ammonium chloride, ammonium hydrogencarbonate, L-histidine and potassium nitrite. There was no growth in the absence of nitrogen.Sporulation was excellent on calcium nitrate and sodium nitrate, Very good on DL-serine, potassium nitrate, and magnesium nitrate. Good on L-asparagine, L-phenylalanine and ammonium oxalate. Fair on DL-alanine, DL-leucine, ammonium sulphate, DL-valine, ammonium chloride and L-histidine whereas poor on glutamic acid, aspartic acid, ammonium tartarate and ammonium nitrate. Few spores were observed on ammonium hydrogencarbonate but potassium nitrite did not show any sporulation.Amongst the sulphur compounds sodium bisulphate gave the best growth and good sporulation, followed by sodium thiosulphate, magnesium sulphate, ammonium sulphate and potassium sulphate. Thiourea gave negligible growth whereas it failed to grow on zinc sulphate and potassium persulphate.     

Influence of ammonium and extracellular pH on the amino and organic acid contents of suspension culture cells of Acer pseudoplatanus

The effects of supplied ammonium and nitrate on the amino and organic acid contents and enzyme activities of cell suspension cultures of Acer pseudoplatanus L. were examined. Regardless of nitrogen source the pH of the culture medium strongly affected the malate and citrate contents of the cells; these organic acid pools declined at pH 5, but increased at pH 7 and 8. Over a period of two days, ammonium had little effect on the responses of the organic acid pool sizes to the pH of the medium. In contrast, ammonium had a strong influence on amino acid pool sizes, and this effect was dependent on the pH of the medium. At pH 5 there was no increase in cell ammonium or amino acid contents, but at higher pH values cellular ammonium content rose, accompanied by accumulation of glutamine, glutamate and asparagine. Over several days, supplied ammonium led to an increase in activity of glutamate dehydrogenase irrespective of any changes in internal ammonium and amino acid contents. If the pH of the medium was allowed to fall below pH 4 in the presence of ammonium, phosphoenolpyruvate (PEP) carboxylase activity declined to a very low value over several days; at higher pH, the activity of this enzyme, and that of NAD malic enzyme and NAD malate dehydrogenase, remained substantial irrespective of whether the nitrogen source was NH⁺₄ or NO-₃.     

Causes of conductance change in yeast cultures

The conductance change due to growth of Saccharomyces cerevisiae Y112, Zygosaccharomyces bailii M and Rhodotorula rabra NCYC 63 in culture media containing glucose, tartrate pH buffer and ammonium ions as sole nitrogen source was compared with that in a medium containing L-asparagine as sole nitrogen source. Decreases in conductance were observed in glucose-ammonium cultures of all three yeasts while little change occurred in cultures with L-asparagine as sole nitrogen source. This supports the hypothesis that the metabolic activity primarily responsible for conductance change in yeast cultures is the uptake of charged ammonium ions as nitrogen source and the reaction of protons with pH buffer compounds.
Rhodotorula rubra cultures with L-asparagine as sole carbon source caused large increases in conductance with growth. Chemical analyses of culture filtrates showed that this increase in conductance was due to use of L-asparagine as carbon source and the excretion of nitrogen surplus to biosynthetic needs as ammonium. In addition, the production of aspartate, acetate and bicarbonate contributed to the increase in conductance.     

High temperature effects on ammonium assimilation in leaves of two Festuca arundinacea cultivars with different heat susceptibility

The aim of this study was to determine the effects of high temperature stress on ammonium assimilation in leaves of two tall fescue cultivars (Festuca arundinacea), Jaguar 3 brand (J3) (heat-tolerant) and TF 66 (T6) (heat-sensitive). High temperature stress for either 10 d or 20 d, and particularly the 20 d stress, produced dramatic changes in ammonium assimilation. After 20 d of stress treatment, the accumulations of total nitrogen, nitrate, soluble protein and total free amino acid (20 amino acids) decreased in both cultivars. Moreover, the activities of main regulatory enzymes, such as nitrate reductase, glutamine synthetase (GS), NADH-dependent glutamate synthase (GOGAT), as well as Δ¹-pyrroline-5-carboxylate reductase (P5CR), also decreased in both cultivars when exposed to 20 d stress. Heat stress had little influence on ammonium accumulation in J3, but this was not the case with T6. The accumulations of nitrate, ammonium, soluble protein, and total free amino acid between the two cultivars were different. This suggests that accumulations of these nitrogen forms were associated with heat tolerance in both tall fescue cultivars. Changes of both NADH-glutamate dehydrogenase (NADH-GDH) activity and Glx (glutamine and glutamic acid) concentration in both cultivars indicated that there is an alternative system for assimilation of nitrogen through glutamate dehydrogenase (GDH) in T6 during longer high temperature stress periods. Our results provide an insight to further selection and breeding of heat-tolerant tall fescue turfgrass cultivars.     

Free amino acid, ammonium and nitrate concentrations in soil solutions of a grazed coastal marsh in relation to plant growth

Soluble free amino acids, ammonium and nitrate ions as sources of nitrogen for plant growth were measured in soils of a coastal marsh grazed by snow geese in Manitoba, Canada. Amounts of nitrogen, primarily ammonium ions, increased in the latter half of the growing season and over winter, but fell to low values early in the growing season. Free amino acid concentrations relative to ammonium concentrations were highest during the period of rapid plant growth in early summer, especially in soils in the intertidal zone, where the median ratio of amino acid nitrogen to ammonium nitrogen was 0·36 and amino acid concentrations exceeded those of ammonium ions in 24% of samples. Amino acid profiles, which were dominated by alanine, proline and glutamic acid, were similar to goose faecal profiles. In a continuous flow hydroponic experiment conducted in the field, growth of the salt‐marsh grass, Puccinellia phryganodes, on glycine was similar to growth on ammonium ions at an equivalent concentration of nitrogen. When supplies of soil inorganic nitrogen are low, amino acids represent a potentially important source of nitrogen for the re‐growth of plants grazed by geese and amino acid uptake may be as high as 57% that of ammonium ions.     

镉对满江红（Azolla imbricata）-鱼腥藻（Anabaena azollae）共生体氮代谢的影响

在实验室条件下,研究了不同浓度(0、0.01、0.05、0.1、0.5mg/L)的Cd对满江红-鱼腥藻共生体异型胞频率,固氮酶、谷氨酰氨合成酶活性以及铵态氮、游离氨基酸、可溶性蛋白、总氮含量的影响.结果表明,在整个实验期间,0.01mg/L Cd处理对上述指标均没产生显著影响,说明满江红-鱼腥藻共生体对Cd具有较强的耐性.当培养液中Cd浓度≥0.05mg/L时,随溶液中Cd浓度的增加和处理时间的推移,异型胞频率、固氮酶活性、谷氨酰氨合成酶活性、可溶性蛋白含量和总氮含量逐渐下降,而铵态氮含量在处理初期显著降低,随后迅速增加,游离氨基酸含量则逐渐增加.研究结果表明高浓度的Cd处理导致满江红-鱼腥藻共生体氮代谢的紊乱,最终造成氮素积累量的下降.