Enzymes involved in ammonia assimilation in the fungus Acremonium persicinum

Abstract Acremonium persicinum grown in batch culture with ammonium tartrate as the nitrogen source possessed an NADP⁺-dependent glutamate dehydrogenase and a glutamine synthetase. Glutamate synthase was not detected under the culture conditions used. Kinetic studies of the NADP⁺-dependent glutamate dehydrogenase at 25°C and pH 7.6 revealed an apparent K _m of 3.2 × 10⁻⁴ M for 2-oxoglutarate and an apparent K _m of 1.0 × 10⁻⁵ M for ammonium ions, with corresponding apparent V _max values of 0.089 and 0.13 μmol substrate converted/min/mg of protein, respectively. Glutamine synthetase was measured by the γ-glutamyl transferase reaction at 30°C and pH 7.55. This transferase reaction of glutamine synthetase had a higher rate at 30°C than at 25°C or 37°C.     

Impact of gaseous nitrogen deposition on plant functioning

Dry deposition of NH₃ and NO_x (NO and NO₂) can affect plant metabolism at the cellular and whole-plant level. Gaseous pollutants enter the plant mainly through the stomata, and once in the apoplast NH₃ dissolves to form NH₄⁺, whereas NO₂ dissolves to form NO₃⁻ and NO₂⁻. The latter compound can also be formed after exposure to NO. There is evidence that NH₃-N and NO_x-N can be reversibly stored in the apoplast. Temporary storage might affect processes such as absorption rate, assimilation and re-emission. Once formed, NO₃⁻ and NO₂⁻ can be reduced, and NH₄⁺ can be assimilated via the normal enzymatic pathways, nitrate reductase (NR), nitrite reductase and the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. Fumigation with low concentrations of atmospheric NH₃ increases in vitro glutamine synthetase activity, but whether this involves both or only one of the GS isoforms is still an open question. There seems to be no correlation between fumigation with low concentrations of NH₃ and in vitro GDH activity. The contribution of atmospheric NH₃ and NO₂ deposition to the N budget of the whole plant has been calculated for various atmospheric pollutant concentrations and relative growth rates ( RGRs ). It is concluded that at current ambient atmospheric N concentrations the direct impact of gaseous N uptake by foliage on plant growth is generally small.     

Translocation of NH4+ in oilseed rape plants in relation to glutamine synthetase isogene expression and activity

Translocation of NH₄⁺ was studied in relation to the expression of three glutamine synthetase (GS, EC 6.3.1.2) isogenes and total GS activity in roots and leaves of hydroponically grown oilseed rape ( Brassica napus ). The concentration of NH₄⁺ in the stem xylem sap of NO₃⁻-fed plants was 0.55–0.70 m M , which was ≈60% higher than that in plants deprived of external nitrogen for 2 days. In NH₄⁺-fed plants, xylem NH₄⁺ concentrations increased linearly both with time of exposure to NH₄⁺ and with increasing external NH₄⁺ concentration. The maximum xylem NH₄⁺ concentration was 8 m M , corresponding to 11% of the nitrogen translocated in the xylem. In the leaf apoplastic solution, the NH₄⁺ concentration increased from 0.03 m M in N-deprived plants to 0.20 m M in N-replete plants. The corresponding values for leaf tissue water were 0.33 and 1.24 m M , respectively. The addition of either NO₃⁻ or NH₄⁺ to N-starved plants induced both cytosolic gs isogene expression and GS activity in the roots. In N-replete plants, gs isogene expression and GS activity were repressed, probably due to carbon limitations, thereby protecting the roots against the excessive drainage of photosynthates. Repressed gs isogene expression and GS activity under N-replete conditions caused enhanced NH₄⁺ translocation to the shoots.     

A precise potentiometric method for determination of algal activity in an open CO2 system

Abstract. The activity of the green alga Scenedesmus obliquus was studied in simplified nutrient solutions (20 mol m₋₃ NaNO₃, 20 mol m₋₃ NH₄C1, 20 mol m₋₃ NH₄NO₃, and 20 mol m₋₃ NaCl, respectively) at 25 °C. The experiments were performed under welldefined incident photon density fluxes ranging from 10 to 200 μmol m₂ s₋₁, Light-dependent changes in pH and alkalinity (A) were followed by means of a potentiometric method using a glass electrode. In the experiments, carbon dioxide with known partial pressure was bubbled through the algal suspension, and during dark periods ul intervals of 1 h, the solution was allowed to equilibrate with the gas phase. This technique was applied to calculate equilibrium values of pH and alkalinity at regular intervals during a 12-h period. Results obtained in NaNO₃, solution show a linear increase in A with time, at each level of illumination studied. After an initial drop, A also increases in NH₄NO₃, solution in a similar way to that in NaNO₃ solution. The change in A with time was also found to increase linearly with the photon density flux studied and no saturation level could be defined. In experiments in NaCl solution, no changes in A were registered while measurements in NH₄Cl solution showed a decrease in A with time.     

Adaptation of plasma membrane H+-ATPase of rice roots to low pH as related to ammonium nutrition

The preference of paddy rice for NH₄⁺ rather than NO₃^- is associated with its tolerance to low pH since a rhizosphere acidification occurs during NH₄⁺ absorption. However, the adaptation of rice root to low pH has not been fully elucidated. This study investigated the acclimation of plasma membrane H⁺-ATPase of rice root to low pH. Rice seedlings were grown either with NH₄⁺ or NO₃^-. For both nitrogen forms, the pH value of nutrient solutions was gradually adjusted to pH 6.5 or 3.0. After 4 d cultivation, hydrolytic H⁺-ATPase activity, V _max, K _m, H⁺-pumping activity, H⁺ permeability and pH gradient across the plasma membrane were significantly higher in rice roots grown at pH 3.0 than at 6.5, irrespective of the nitrogen forms supplied. The higher activity of plasma membrane H⁺-ATPase of adapted rice roots was attributed to the increase in expression of OSA1, OSA3, OSA7, OSA8 and OSA9 genes, which resulted in an increase of H⁺-ATPase protein concentration. In conclusion, a high regulation of various plasma membrane H⁺-ATPase genes is responsible for the adaptation of rice roots to low pH. This mechanism may be partly responsible for the preference of rice plants to NH₄⁺ nutrition.     

Time-course of inorganic nitrogen uptake and incorporation by natural populations of freshwater phytoplankton

SUMMARY. 1. Time-course measurements of NH₄⁺ and NO₃⁻uptake were made on the natural phytoplankton populations in a eutrophic lake at a time when these nutrients were at their lowest annual concentration.
2. Both NH₄⁺ and NO₃⁻ uptake was increased at least five-fold during the first 5 min of incubation following near saturating pulses of these nutrients.
3. Elevated uptake was also observed following low level (∼2μg N 1⁻¹) pulses of NH₄⁺ and NO₃⁻, but substrate depletion during the first hour of incubation may have been partially responsible for this apparent enhancement.
4. Incorporation of ^I5N into TCA-insoluble material (protein) following the saturating NH₄⁺ pulse was increased less than total cellular ¹⁵N uptake, whereas no elevation of ¹⁵N incorporation into protein was observed following a saturating NO₃⁻pulse.
5. The percentage of ^I5N incorporated into protein, with respect to total cellular uptake, was ∼32% and ∼12% for NH₄⁺ and NO₃⁻, respectively, following 5 h of incubation.     

Glycolate metabolism in cyanobacteria. III. Nitrogen controls excretion and metabolism of glycolate in Anabaena cylindrica

The effect of nitrogen on excretion and metabolism of glycolate in Anabaena cylindrica (CCAP 1403/2a) was studied. Glycidate, an inhibitor of glutamate:glyoxylate aminotransferase (EC 2.6.1.4), reduced the L-methionine-DL-sulfoximine-induced NH₄⁺ release by ca 40%, while net CO₂ fixation and C₂H₂ reduction were not lowered. This indicates that at least a part of the glyoxylate synthesized in A. cylindrica is metabolized via glycine to serine. Addition of NH₄Cl or glutamate to the medium reduced the excretion of glycolate. At pH 9, under air, NH₄Cl reduced the excretion by 10–30% and under high pO2 (0.03 kPa CO₂ in O₂) by about 80–90%. At pH 7.5, under high pO₂, NH₄Cl and glulamate reduced the excretion by about 40 and 80%, respectively. Also, the presence of NH₄Cl stimulated the animation of glyoxylate under such conditions as shown by an increased glycine pool and a decreased glutamate pool. We suggest that nitrogen regulates the capacity of A. cylindrica to retain and recycle glycolate intracellularly and that glutamate serves as an amino donor in the conversion of glyoxylate to glycine.     

Effect of atmospheric ammonia on the nitrogen metabolism of Scots pine (Pinus sylvestris) needles

Four-year-old seedlings of Scots pine ( Pinus sylvestris L.) were exposed to filtered air (FA), and to FA supplemented with NH₃ (60 and 240 μg m⁻³) in controlled-environment chambers for 14 weeks. Exposure to the higher NH₃ concentration resulted in an increased activity of glutamine synthetase (GS, EC 6.3.1.2), and an increase in the concentrations of soluble proteins, total nitrogen, free amino acids and leaf pigments in the needles. The GS activity (μmol g⁻¹ fresh weight h⁻¹) in the needle extract increased to levels 69% higher than in FA and the soluble protein concentration to levels 22% higher. Total nitrogen concentration in the needles was 42% higher than in FA, while the free amino acid concentration was 300% higher, which was caused by an increase in arginine, glutamate, aspartate and glutamine. Chlorophyll a , chlorophyll b and carotenoid concentrations were 29, 38 and 11% higher, respectively. Neither the glutamate dehydrogenase (GDH, EC 1.4.1.2) activity nor the concentrations of free NH₄⁺ and glucose in the needles were affected by exposure to NH₃. After NH₃ fumigation at 240 μg m⁻³ the starch concentration decreased by 39% relative to the FA. The results indicate that the metabolism of Scots pine acclimates to concentrations of NH₃ which are 3 to 10 times higher than the average concentration in areas with intensive stock farming. The possible mechanisms underlying acclimation to NH₃ are discussed.     

Activity and composition of ammonia oxidizing bacterial communities and emission dynamics of NH3 and N2O in a compost reactor treating organic household waste

Aims:  To monitor emissions of NH₃ and N₂O during composting and link these to ammonia oxidation rates and the community structure of ammonia oxidizing bacteria (AOB).
Methods and Results:  A laboratory-scale compost reactor treating organic household waste was run for 2 months. NH₃ emissions peaked when pH started to increase. Small amounts of N₂O and CH₄ were also produced. In total, 16% and less than 1% of the initial N was lost as NH₃-N and N₂O-N respectively. The potential ammonia oxidation rate, determined by a chlorate inhibition assay, increased fourfold during the first 9 days and then remained high. Initially, both Nitrosospira and Nitrosomonas populations were detected using DGGE analysis of AOB specific 16S rRNA fragments. Only Nitrosomonas europaea was detected under thermophilic conditions, but Nitrosospira populations re-established during the cooling phase.
Conclusions:  Thermophilic conditions favoured high potential ammonia oxidation rates, suggesting that ammonia oxidation contributed to reduced NH₃ emissions. Small but significant amounts of N₂O were emitted during the thermophilic phase. The significance of different AOBs detected in the compost for ammonia oxidation is not clear.
Significance and Impact of Study:  This study shows that ammonia oxidation occurs at high temperature composting and therefore most likely reduces NH₃ emissions.     

Physiological effects of long-term exposure to low and moderate concentrations of atmospheric NH3 on poplar leaves

Abstract. Poplar shoots ( Populus euramericana L.) obtained from cuttings were exposed for 6 or 8 weeks to NH₃ concentrations of 50 and 100 μgm⁻³ or filtered air in fumigation chambers. After this exposure the rates of NH₃ uptake, transpiration, CO₂ assimilation and respiration of leaves were measured using a leaf chamber. During the long-term exposure also modulated chlorophyll fluorescence measurements were carried out to obtain information about the photosynthetic performance of individual leaves. Both fluorescence and leaf chamber measurements showed a higher photosynthetic activity of leaves exposed to 100 μg NH₃ m⁻³. These leaves showed also a larger leaf conductance and a larger uptake rate of NH₃ than leaves exposed to 50 μg m⁻³ NH₃ or filtered air. The long-term NH₃ exposure did not induce an internal resistance against NH₃ transport in the leaf, nor did it affect the leaf cuticle. So, not only at a short time exposure, but also at a long-term exposure NH₃ uptake into leaves can be calculated from data on the boundary layer and stomatal resistance for H₂O and ambient NH₃-concentration. Furthermore, the NH₃ exposure had no effect on the relation between CO₂-assimilation and stomatal conductance, indicating that NH₃ in concentrations up to 100 μg m⁻³ has no direct effect on stomatal behaviour; for example, by affecting the guard or contiguous cells of the stomata.     

Different effects of supplied ammonium on glutamine synthetase activity in mustard (Sinapis alba) and pine (Pinus sylvestris) seedlings

The activity of glutamine synthetase (GS) in mustard ( Sinapis alba L.) and Scots pine ( Pinus sylvestris L.) seedlings was used as an index to evaluate the capacity to cope with excessive ammonium supply. In these 2 species GS activity was differently affected by the application of nitrogen compounds (NH₄⁺ or NO₃⁻). Mustard seedlings older than 5 days showed a considerable increase in GS activity after NH₄⁺ or NO₃⁻ application. This response was independent of the energy flux, but GS activity in general was positively affected by light. Endogenous NH₄⁺ did not accumulate greatly after nitrogen supply. In contrast, seedlings of Scots pine accumulated NH₄⁺ in cotyledons and roots and showed no stimulation of GS activity after the application of ammonium. In addition, root growth was drastically reduced. Thus, the pine seedlings seem to have insufficient capacity to assimilate exogenously supplied ammonium. NO₃⁻, however, did not lead to any harmful effects.     

Apparent negative co-operativity and substrate inhibition in overexpressed glutamate dehydrogenase from Escherichia coli

The gene for Escherichia coli glutamate dehydrogenase (EcGDH) has been overexpressed, and a simplified purification procedure afforded greatly increased yields of c. 40 mg pure EcGDH L⁻¹ culture. EcGDH was unstable at a low concentration in plastic tubes, but stabilization measures allowed a robust kinetic characterization. Contrary to past reports, EcGDH deviates from Michaelis–Menten kinetics, exhibiting apparent mild negative co-operativity with both l -glutamate and NADP⁺, with Hill coefficients of 0.90 and 0.92, respectively. NADPH yielded simple Michaelis–Menten kinetics but both 2-oxoglutarate and NH₄⁺ showed substrate inhibition. pH optima were 9 for oxidative deamination and 8 for reductive amination.     

Cytosolic and chloroplastic glutamine synthetase of sugarbeet (Beta vulgaris) respond differently to organ ontogeny and nitrogen source

Changes in the activity and subunit composition of cytosolic glutamine synthetase (GS 1; EC 6.3.1.2) and chloroplastic GS (GS 2) were studied in response to an internal (organ ontogeny) and external signal (N source: NO₃⁻ or NH₄⁺). Maximum GS 1 activity of all organs examined was measured in the fibre roots, irrespective of the N source. The response of GS 1 to the N source was, however, organ specific. In the fibre roots, NH₄⁺ nutrition resulted in a 2- to 7-fold (based on protein or freshweight, respectively) increase of GS 1 activity compared to NO₃⁻-grown plants. In contrast to the roots, GS 1 activity in the leaf blades was 2-fold lower with NH₄⁺ nutrition, whereas only minor changes occurred in the petioles. GS 2 activity was highest in the mature and senescing leaf blade; activity was 2-fold higher with NH₄⁺ than with NO₃⁻ nutrition. Not only activity, but also subunit composition of GS 1 changed during organ ontogeny as well as in response to the N source. In contrast to GS 1, only minor changes were evident in GS 2 subunit composition, despite significant changes in GS 2 activity. Up to 5 different GS 1 subunits of ≈41–43 kDa were separated; they were identical in all organs examined. GS 2 was composed of 4 different subunits of ≈48 kDa.     

Photoactivation of the latent water-oxidizing complex in photosystem II membranes isolated from dark-grown spruce seedlings

Photosystem II membranes (D-PSII) were isolated from dark-grown spruce seedlings. All major PSII proteins except the 17- and 23-kDa extrinsic proteins were present in D-PSII. O₂ evolution and Mn content in D-PSII were negligible, while PSII-donor activity showed a value comparable to that of NH₂OH-treated PSII membranes (NH₂OH-L-PSII) from light-grown seedlings. Light incubation of D-PSII with 1 m M MnCl₂, 50 m M CaCl₂ and 100 μ M DCIP at pH 5.3 resulted in activation of the latent water-oxidizing complex. Accomplishment of photoactivation of PSII membranes from dark-grown spruce seedlings clearly indicates that only ligation of Mn²⁺ to the apo-water oxidizing complex is required for expression of O₂ evolution, and that protein synthesis is not involved in the photoactivation process. There was no essential difference between 'photoactivation' of naturally Mn-free PSII membranes and 'photoreactivation' of artificially Mn-depleted PSII membranes on kinetics, pH dependence, Mn²⁺-concentration dependence. However, kinetics and pH dependence of photoactivation were appreciably different in spruce PSII membranes and in PSII membranes of angiosperms such as wheat and spinach.     

Nitrogen regulation of erythromycin formation in Streptomyces erythreus

Abstract Erythromycin formation decreased in Streptomyces erythreus as a function of the ammonium concentration present in the medium. Total inhibition of synthesis was obtained with 100 mM NH₄Cl but medium pH and culture growth were not significantly affected. A similar effect was obtained with NH₄NO₃ or (NH₄)₂SO₄ indicating that ammonium ion probably repressed formation of antibiotic.     

Ecology of Peridinium willei and P. volzii (Dinophyceae) in Danish lakes

The distribution of Peridinium willei and P. volzii was studied in Danish lakes. Both species were confined to lakes with concentrations of Total P < 0.15 mg 1^-1, with the majority of occurrences at Total P concentration between 0.020–0.040 mg 1^-1 and concentrations of PO₄ P between detection limit and 0.040 mg 1^-1. The occurrence of the species in relation to inorganic N compounds (NH₄ N and NO₂+ NO₃ N) was significantly broader for P. willei than for P. volzii: P. willei had an almost even distribution within a wide range of NH₄ N, whereas P. volzii mainly occurred between 0.001 and 0.10 NH₄ N 1^-1. P. willei had an almost even distribution at values beween 0.005 and 0.42 mg NO₂+ NO₃ N 1^-1, whereas P. volzii mainly occurred below 0.050 mg NO₂+ NO₃ N 1¹. P. willei was found at pH values between 4.2 and 8.5, whereas P. volzii was confined to lakes with a slightly basic pH. The study confirmed the broad limits of P. willei and the much more narrow limits of P. volzii in relation to seasonal occurrence and pH, as well as an affinity of the former to ponds and lakes with a rich bottom vegetation. The study also showed, however, that the species were not as widespread and common in recent Danish lake phytoplankton as generally stated by previous authors. The use of different ecological factors to give weight to species separation is discussed. The inclusion of P. volzii in P. willei proposed by Popovsky & Phiester is not supported by the present study, as the two taxa appear to have different ecological tolerances.     

Characterization of cyclic nucleotide phosphodiesterase activity in Phaseolus vulgaris

Cyclic nucleotide phosphodiesterase (3',5'-cyclic nucleotide nucleotidohydrolase, EC 3.1.4.17) activity isolated from Phaseolus vulgaris L. cv. Limberg seedlings was partially purified and characterized by fractional (NH₄)₂SO₄ precipitation, DEAE-cellulose chromatography, chromatography on 3',5'-cAMP-agarose, gel permeation chromatography and chromatofocusing. A crude enzyme preparation, a 30–65% (NH₄)₂SO₄ pellet, showed an acidic pH optimum. The enzyme activity was stimulated by imidazole and divalent cations such as Ca²⁺, Mg²⁺ and Mn²⁺, whereas NaF, PP_i and Fe³⁺ were inhibitory. Isobutylmethylxanthine had no significant effect on the plant enzyme. An M_I of 42 000 was estimated by gel permeation high performance liquid chromatography. By chromatography on 3',5'-cAMP-agarose a phosphodiesterase was resolved that produced 5'-AMP as sole reaction product.     

Mycobiont-cyanobiont interactions during dark nitrogen fixation by the lichen Peltigera aphthosa

Acetylene reduction (nitrogenase activity) by excised cephalodia of Peltigera aphthosa Willd. slowly declined on transfer of the cephalodia from light to darkness. The decline was more rapid in the absence of CO₂ or when phosphoenolpyruvate carboxylase activity was inhibited by adding maleic acid or malonic acid. When glutamine synthetase (GS) activity was totally inhibited by adding l -methionine- dl -sulphoximine (MSX) the decline in nitrogenase activity in the absence of CO₂ still occurred. However, this loss of activity did not occur when the mycobiont was disrupted using digitonin (0.01 % w/v) and the fixed NH₄⁺ was released into the medium. The data suggest that dark CO₂ fixation by the fungus supplies carbon skeletons which remove newly fixed NH₄⁺ produced by the cyanobacterium. When such carbon skeletons are not available MH₄⁺ accumulates and inhibits nitrogenase activity even in the absence of GS activity. It is probable that NH₄⁺ and a product of GS exert independent inhibitory effects on nitrogenase activity.     

Some kinetic properties of a purified glutamine synthetase from bacteroids of Glycine max

Abstract A purified glutamine synthetase was prepared from bacteroids of Rhizobium japonicum from nodules of Glycine max . For the biosynthetic assay the K _m values (mM) were l -glutamate 12.9, NH₄Cl 8.9 and ATP 14.3. When the enzyme was assayed by the γ-glutamyltransferase reaction the K _m values (mM) were l -glutamine 11.1 and hydroxylamine 3.3 compared with 7.7 and 1.2, respectively, for the purified enzyme from Rhizobium japonicum grown in culture. The enzyme prepared from bacteroids of Glycine max was 80% adenylylated.     

l-Myo-inositol-1-phosphate synthase from lower plant groups: Partial purification and properties of the enzyme from Euglena gracilis

A survey for the enzyme L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) has been conducted among various members of the lower plant groups, mainly algac, bryophytes and fungi; some properties of the partially purified enzyme from Euglena gracilis Z . are presented. The enzyme was detected in Chloropycean algae, Marchantiales and the Basidiomycetous fungi. The enzyme from Euglena had a pH optimum at 7.5. The Km for glucose-6-P was 2.1 m M and for NAD⁺ 80 μ M . When assayed in the absence of added NAD⁺, the enzyme showed a basal activity suggesting the presence of bund NAD⁺ in the system. NH₄Cl increased the enzyme activity two-fold, altough the enzyme was inactivated by (NH₄)₂SO₄.