Short-term effect of ammonia on nitrogenase activity of Anabaena variabilis (ATCC29413)

Abstract Intact filaments of the cyanobacterium Anabaena variabilis switch off nitrogenase activity very rapidly upon addition of NH₄Cl when incubated in an alkaline environment (pH 10.0) permitting a fast NH₃-influx into the cells. When assayed in cell-free extracts (prepared from ammonia-treated filaments), nitrogenase remains inhibited in the presence of an ATP-regenerating system. Furthermore, l -methionine- d,l -sulfoximine, an inhibitor of glutamine synthetase, added to the filaments, prevents inactivation of nitrogenase by ammonia, showing that ammonia is not the compound directly responsible for nitrogenase switch-off.     

Changes in nitrogen source modify distribution of excitation energy in the cyanobacterium Phormidium laminosum

In an attempt to clarify the interactions between the available nitrogen source and the photosystems in cyanobacteria, O₂ exchange and fluorescence emission were monitored in spheroplasts and intact cells of the non N₂-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl₁) growing on different nitrogen sources or in the absence of nitrogen. Short-term (time scale of seconds to minutes), NH⁺₄ addition to NO⁻₃-growing or N-starved cells and, to a minor extent, NO⁻₃addition to N-starved cells, induced state 2 transitions both in light and dark. Long term (time scale of days), the fluorescence yield of PSII relative to that of PSII at 77 K was higher in NO⁻₃- than in NH⁺₄ growing cells, and even higher in N-starved cells. In the dark, the plastoquinone pool was more reduced in NH⁺₄- than in NO⁻₃-growing cells. Both PSII and PSI activities and the degree of linking between both photosystems were affected in the long term, so that non-cyclic electron transport decreased in parallel to the ferredoxin requirement to assimilate each nitrogen source. Results indicate that nitrogen metabolism exerts short- and long-term control over the photosynthetic apparatus, which acclimates to the energy requirement of the available nitrogen source.     

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.     

Glutamate dehydrogenase and glutamine synthetase activities during the development of triticale grains

Glutamate dehydrogenase (GDH, EC 1.4.1.2–4) and glutamine synthetase (GS, EC 6.3.1.2) activities as well as protein content and dry matter in developing kernels of winter Triticale were determined. The relatively low level of GS activity compared to high level of NAD(P)H-dependent GDH activity during intensive filling of grains with storage compounds may indicate the participation of GDH in reductive amination of 2-oxoglutarate. The amination activity of this enzyme in all grain development phases exceeded the deaminating activity several fold. Moreover, the dynamics in the change of NAD(P)H-GDH and NAD(P)⁺-GDH activities were analysed in various tissues of the developing grains. The high amination activity of the enzyme in the seed coat, where the intensive protein synthesis occurs would also be an indication of the anabolic function of this enzyme.     

Uptake and metabolism of benzyladenine in the early stage of flower bud development in vitro in tobacco

Benzyladenine (BA) was found to regulate the number of flower buds regenerated in vitro from pedicel tissue of tobacco. Flower bud induction was particularly sensitive to BA levels in the range of 0.45 to 1.0 μ M , where a two-fold increase in concentration caused a threefold rise in the number of buds. When tissues were fed radioactive BA for 24h, only 9–12% of the counts were recovered in the original compound. The rest was present in metabolites, tentatively identified as the mono-, di- and triribotides, 7- and 9-glucosides and 9-riboside of BA. The amount of growth regulator taken up and the quantities of BA and its metabolites in the explants were all linearly related to the concentration of the medium. The internal BA concentration was ca 60% of the level in the medium after 24 h. When the concentration in the medium was raised, relatively more BA remained in the non-conjugated form. However, this change in the equilibrium between BA and the conjugates is too small to account for the steep rise in the curve representing concentration vs effect between 0.45 and 1.0 μ M .     

Effect of histamine on the development of astroglial cells in culture

The effect of histamine on different aspects of the growth of astrocytes was studied using primary cultures derived either from forebrain or from cerebellum of the rat. The influence on general growth and differentiation was monitored in terms of the activities of ornithine decarboxylase and glutamine synthetase enzymes, whereas [³H]thymidine incorporation into DNA was used as a specific index of cell proliferation. Treatment with 500 nM histamine of cells grown for 6 days in vitro, caused a time-dependent significant increase in ornithine decarboxylase activity of astrocytes from both sources. The maximum increase was observed at 4 h after histamine treatment, at that time the elevation in ornithine decarboxylase activity being about 80% and 300% over control values in the forebrain and the cerebellar astrocytes, respectively. Under similar experimental conditions, addition of histamine (500 nM) to medium resulted in a significant increase in [³H]thymidine incorporation into DNA in both types of cultures: in comparison with control, the elevation was about 45% at 48 h in forebrain astrocytes and at 24 h in cerebellar astrocytes. On the other hand, the specific activity of glutamine synthetase in cerebellar astrocytes was markedly enhanced (about 100%) by treatment with histamine (500 nM) for 4 days, but forebrain astrocytes were little affected. Addition of histamine to the culture medium produced no significant alteration in the activity of lactate dehydrogenase and protein content of either type of astroglial cells. The present findings, which support our earlier proposal that the biochemical properties of astrocytes differ between various brain regions, provide direct evidence for the involvement of histamine in the regulation of growth and development of astrocytes.     

氨和谷氨酰胺对浑球红假单胞菌（Rhodobacter sphaeroides）固氮酶...

浑球红假单胞菌菌株601具有迅速对外源氨作出“关闭”固氮酶活性的反应。氨对固氮酶的抑制作用,可被谷氨酰胺合成酶(GS)抑制剂MSX所解除。反之,加入Glu代谢抑制剂DON,可延长氨抑制的持续时间。Gln对固氮酶也有抑制作用。在脱腺苷化GS的透性细胞中,加入Gln可抑制固氮酶活性,同时,GS腺苷化状态提高。然而,氨则对透性细胞的固氮酶活性和GS腺苷化状态没有影响。     

Response of Zea mays to the Inoculation with Azospirillum on Nitrogen Metabolism under Greenhouse Conditions

The maize (Zea mays L.) plants inoculated by N₂-fixing bacterium Azospirillum showed increased activity of glutamate dehydrogenase (GDH) and glutamine synthetase (GS) in root cells free extracts over uninoculated control plants. Maximum differences in NADH-GDH activity were observed during the second and third weeks after sowing. The specific activity of GS showed a greater increase at the end of the assay. The percentage of nitrogen in leaves, root and foliage length, total fresh mass and nitrogenase activity were higher in inoculated plants than in the control ones.     

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.     

Distribution of the glutamine synthetase isozyme GSp1 in maize (Zea mays)

In maize (Zea mays L.), GSp1, the predominant GS isozyme of the developing kernel, is abundant in the pedicel and pericarp, but absent from the endosperm and embryo. Determinations of GSp1 tissue distribution in vegetative tissues have been limited thus far to root and leaves, where the isozyme is absent. However, the promoter from the gene encoding GSp1 has been shown to drive reporter gene expression not only in the maternal seed-associated tissues in transgenic maize plants, but also in the anthers, husks and pollen (Muhitch et al. 2002, Plant Sci 163: 865-872). Here we report chromatographic evidence that GSp1 resides in immature tassels, dehiscing anthers, kernel glumes, ear husks, cobs and stalks of maize plants, but not in mature, shedding pollen grains. RNA blot analysis confirmed these biochemical data. In stalks, GSp1 increased in the later stages of ear development, suggesting that it plays a role in nitrogen remobilization during grain fill.     

Activity profiles of enzymes involved in glutamine and glutamate metabolism in the apple during autumnal senescence

Seasonal changes in glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), and glutamate dehydrogenase (EC 1.4.1.3) were measured in both senescing leaf and bark tissues of ‘Golden Delicious’ apple trees (Malus domestica Borkh.). From the measured enzyme activities we attempted to estimate the in vivo catalytic potentials of the enzymes with special reference to nitrogen mobilization and conservation of senescing apple trees. The cumulative glutamine synthetase activity of leaf tissue was about three times higher than that of bark. The estimated catalytic potential of leaf glutamine synthetase was 800-fold higher than the actual protein nitrogen loss of senescing leaves. The cumulative glutamate synthase activity of bark was about six times higher than that of leaf. The estimated catalytic potential of bark glutamate synthase was 160-times higher than the actual protein nitrogen gain in that tissue. The cumulative glutamate dehydrogenase activities in leaf and bark tissue were approximately the same. However, the catalytic potential of leaf glutamate dehydrogenase was twice that of leaf glutamate synthase. It is thus concluded that the physiological role of glutamine synthetase in senescing leaf tissue is to furnish the amide(s) prior to mobilization of nitrogen to storage tissue. The higher activity of glutamate synthase in bark tissue could provide a mechanism to transform the imported amide nitrogen to amino nitrogen of glutamate for storage protein synthesis. The possible regulatory factors upon the activity of these enzymes in the tissues of senescing apple trees are discussed.     

The influence of leaf‐atmosphere NH3(g) exchange on the isotopic composition of nitrogen in plants and the atmosphere

The distribution of nitrogen isotopes in the biosphere has the potential to offer insights into the past, present and future of the nitrogen cycle, but it is challenging to unravel the processes controlling patterns of mixing and fractionation. We present a mathematical model describing a previously overlooked process: nitrogen isotope fractionation during leaf‐atmosphere NH_3(g) exchange. The model predicts that when leaf‐atmosphere exchange of NH_3(g) occurs in a closed system, the atmospheric reservoir of NH_3(g) equilibrates at a concentration equal to the ammonia compensation point and an isotopic composition 8.1‰ lighter than nitrogen in protein. In an open system, when atmospheric concentrations of NH_3(g) fall below or rise above the compensation point, protein can be isotopically enriched by net efflux of NH_3(g) or depleted by net uptake. Comparison of model output with existing measurements in the literature suggests that this process contributes to variation in the isotopic composition of nitrogen in plants as well as NH_3(g) in the atmosphere, and should be considered in future analyses of nitrogen isotope circulation. The matrix‐based modelling approach that is introduced may be useful for quantifying isotope dynamics in other complex systems that can be described by first‐order kinetics.     

Regulation of the high-affinity ammonium transporter (BnAMT1;2) in the leaves of Brassica napus by nitrogen status

Substantial concentrations of NH₄ ⁺ are found in the apoplast of the leaves of Brassica napus. Physiological studies on isolated mesophyll protoplasts with ¹⁵NH₄ ⁺ revealed the presence of a high-affinity ammonium transporter that shared physiological similarity to the high-affinity NH₄ ⁺ transporters in Arabidopsis thaliana (AtAMT1;3). PCR techniques were used to isolate a full-length clone of a B. napus homologue of AMT1 from shoot mRNA which showed 97% similarity to AtAMT1;3. The full-length cDNA when cloned into the yeast expression vector pFL61 was able to complement a yeast mutant unable to grow on media with NH₄ ⁺ as the sole nitrogen source. Regulatory studies with detached leaves revealed a stimulation of both NH₄ ⁺ uptake and expression of mRNA when the leaves were supplied with increasing concentrations of NH₄ ⁺. Withdrawal of NH₄ ⁺ supply for up to 96 h had little effect on mRNA expression or NH₄ ⁺ uptake; however, plants grown continuously at high NH₄ ⁺ levels exhibited decreased mRNA expression. BnAMT1;2mRNA expression was highest when NH₄ ⁺ was supplied directly to the leaf and lowest when either glutamine or glutamate was supplied to the leaves, which directly paralleled chloroplastic glutamine synthetase (GS2) activity in the same leaves. These results provide tentative evidence that BnAMT1;2may be regulated by similar mechanisms to GS2 in leaves.     

Glutamine synthetase activity during embryonic and larval development of the silkworm Bombyx mori L. and role of a JH analogue

Abstract. Silkworm eggs of diapause nature were chilled or treated with hydrochloric acid. Glutamine synthetase activity in such treated eggs was present soon after the treatment, whereas in non-diapause eggs it was not detectable until 24 h after the start of development. During larval life, the glutamine synthetase was found to be absent in midgut tissue. Topical application of a JH analogue resulted in extened larval duration and it reduced glutamine synthetase activity initially, but in the latter part of development the activity was higher.     

Effect of benzyladenine on development of derooted watermelon seedlings

We have examined the effects of benzyladenine (BA) on derooted watermelon ( Citrullus vulgaris Schrad., cv. Fairfax) seedlings with special attention to the cotyledons. The growth regulator was supplied either as a droplet of solution between the cotyledons (application from above) or through the hypocotyl stump (application from below). Application of BA from above stimulates several developmental parameters of cotyledons [growth, lipid breakdown, chlorophyll and carotenoid accumulation, hydroxypyruvate reductase (EC 1.1.1.81.) activity]. The stimulation is much smaller with application from below. Also the distribution of labelled BA changes according to the method of application. The bulk of the label remains in the cotyledons when BA is supplied from above and in the hypocotyl when it is supplied from below. The absolute amount of radioactivity found in the cotyledons after 24 h of treatment is approximately the same in both cases. This seems to indicate that the different effects of the two treatments depend on concentration ratios of BA (or metabolic derivates) between hypocotyl and cotyledons rather than on absolute growth regulator levels.     

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.     

Investigating the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase using lanthanide luminescence spectroscopy.

Lanthanide luminescence was used to examine the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase (GS). These studies revealed the presence of two lanthanide ion binding sites of GS of either adenylylation extrema. Individual emission decay lifetimes were obtained in both H2O and D2O solvent systems, allowing for the determination of the number of water molecules coordinated to each bound Eu3+. The results indicate that there are 4.3 +/- 0.5 and 4.6 +/- 0.5 water molecules coordinated to Eu3+ bound to the n1 site of unadenylylated enzyme, GS0, and fully adenylylated enzyme, GS12, respectively, and that there are 2.6 +/- 0.5 water molecules coordinated to Eu3+ at site n2 for both GS0 and GS12. Energy transfer measurements between the lanthanide donor-acceptor pair Eu3+ and Nd3+, obtained an intermetal distance measurement of 12.1 +/- 1.5 A. Distances between a Tb3+ ion at site n2 and tryptophan residues were also performed with the use of single-tryptophan mutant forms of E. coli GS. The dissociation constant for lanthanide ion binding to site n1 was observed to decrease from Kd = 0.35 +/- 0.09 microM for GS0 to Kd = 0.06 +/- 0.02 microM for GS12. The dissociation constant for lanthanide ion binding to site n2 remained unchanged as a function of adenylylation state; Kd = 3.8 +/- 0.9 microM and Kd = 2.6 +/- 0.7 microM for GS0 and GS12, respectively. Competition experiments indicate that Mn2+ affinity at site n1 decreases as a function of increasing adenylylation state, from Kd = 0.05 +/- 0.02 microM for GS0 to Kd = 0.35 +/- 0.09 microM for GS12. Mn2+ affinity at site n2 remains unchanged (Kd = 5.3 +/- 1.3 microM for GS0 and Kd = 4.0 +/- 1.0 microM for GS12). The observed divalent metal ion affinities, which are affected by the adenylylation state, agrees with other steady-state substrate experiments (Abell LM, Villafranca JJ, 1991, Biochemistry 30:1413-1418), supporting the hypothesis that adenylylation regulates GS by altering substrate and metal ion affinities.