Influence of Nitrate and Ammonium Nutrition on the Uptake, Assimilation, and Distribution of Nutrients in Ricinus communis

Ricinus communis L. plants were grown in nutrient solutions in which N was supplied as NO₃⁻ or NH₄⁺, the solutions being maintained at pH 5.5. In NO₃⁻-fed plants excess nutrient anion over cation uptake was equivalent to net OH⁻ efflux, and the total charge from NO₃⁻ and SO₄²⁻ reduction equated to the sum of organic anion accumulation plus net OH⁻ efflux. In NH₄⁺-fed plants a large H⁺ efflux was recorded in close agreement with excess cation over anion uptake. This H⁺ efflux equated to the sum of net cation (NH₄⁺ minus SO₄²⁻) assimilation plus organic anion accumulation. In vivo nitrate reductase assays revealed that the roots may have the capacity to reduce just under half of the total NO₃⁻ that is taken up and reduced in NO₃⁻-fed plants. Organic anion concentration in these plants was much higher in the shoots than in the roots. In NH₄⁺-fed plants absorbed NH₄⁺ was almost exclusively assimilated in the roots. These plants were considerably lower in organic anions than NO₃⁻-fed plants, but had equal concentrations in shoots and roots. Xylem and phloem saps were collected from plants exposed to both N sources and analyzed for all major contributing ionic and nitrogenous compounds. The results obtained were used to assist in interpreting the ion uptake, assimilation, and accumulation data in terms of shoot/root pH regulation and cycling of nutrients.     

Human SLC4A11 Is a Novel NH3/H+ Co-transporter

SLC4A11 has been proposed to be an electrogenic membrane transporter, permeable to Na⁺, H⁺ (OH⁻), bicarbonate, borate, and NH₄⁺. Recent studies indicate, however, that neither bicarbonate or borate is a substrate. Here, we examined potential NH₄⁺, Na⁺, and H⁺ contributions to electrogenic ion transport through SLC4A11 stably expressed in Na⁺/H⁺ exchanger-deficient PS120 fibroblasts. Inward currents observed during exposure to NH₄Cl were determined by the [NH₃]_o, not [NH₄⁺]_o, and current amplitudes varied with the [H⁺] gradient. These currents were relatively unaffected by removal of Na⁺, K⁺, or Cl⁻ from the bath but could be reduced by inclusion of NH₄Cl in the pipette solution. Bath pH changes alone did not generate significant currents through SLC4A11, except immediately following exposure to NH₄Cl. Reversal potential shifts in response to changing [NH₃]_o and pH_o suggested an NH₃/H⁺-coupled transport mode for SLC4A11. Proton flux through SLC4A11 in the absence of ammonia was relatively small, suggesting that ammonia transport is of more physiological relevance. Methylammonia produced currents similar to NH₃ but with reduced amplitude. Estimated stoichiometry of SLC4A11 transport was 1:2 (NH₃/H⁺). NH₃-dependent currents were insensitive to 10 μm ethyl-isopropyl amiloride or 100 μm 4,4′- diisothiocyanatostilbene-2,2′-disulfonic acid. We propose that SLC4A11 is an NH₃/2H⁺ co-transporter exhibiting unique characteristics.     

Effect of Ionic Composition of Suspending Solution on Virus Adsorption by a Soil Column

The effect of various electrolytes on the adsorption of poliovirus was measured in 250-cm-long soil columns with ceramic samplers at different depths. Viruses suspended in deionized water moved much farther through the soil than those suspended in tap water, whereas movement in sewage water was intermediate. The salt content of the tap water and sewage water promoted virus adsorption, but evidently the organic compounds in sewage retarded adsorption. When viruses were suspended in chloride solutions of K⁺, Na⁺, Ca⁺, and Mg²⁺, virus adsorption increased as the cation concentration and valence increased. The depth of virus penetration was related to the ionic strength of the solutions. Virus penetration data for NO₃⁻, SO₄²⁻, and H₂PO₄⁻ salts of K⁺, Na⁺, and Ca²⁺ indicated that other anions were more effective than Cl⁻ in promoting virus adsorption. Also, NH₄⁺ was more effective than other cations in limiting the penetration depth of viruses. It seems that ions composed of radicals are more effective than ions composed of single atoms in promoting virus adsorption. Al³⁺ was the most effective ion in limiting virus penetration, probably owing to flocculation of the viruses. Adding AlCl₃ concentrations to secondary sewage effluent to provide an Al³⁺ concentration of 0.1 mM reduced the virus penetration depth to 40 cm. These studies show that the ionic composition of the suspending solutions must be considered in predicting virus penetration depths, and it may be practical to add low concentrations of a flocculating agent such as AlCl₃ to sewage water to limit virus movement through very porous soils.     

The Uptake of NO3?, NO2?, and NH4+ by Intact Wheat (Triticum aestivum) Seedlings : I. Induction and Kinetics of Transport Systems

The inducibility and kinetics of the NO₃⁻, NO₂⁻, and NH₄⁺ transporters in roots of wheat seedlings (Triticum aestivum cv Yercora Rojo) were characterized using precise methods approaching constant analysis of the substrate solutions. A microcomputer-controlled automated high performance liquid chromatography system was used to determine the depletion of each N species (initially at 1 millimolar) from complete nutrient solutions. Uptake rate analyses were performed using computerized curve-fitting techniques. More precise estimates were obtained for the time required for and the extent of the induction of each transporter. Up to 10 and 6 hours, respectively, were required to achieve apparent full induction of the NO₃⁻ and NO₂⁻ transporters. Evidence for substrate inducibility of the NH₄⁺ transporters requiring 5 hours is presented. The transport of NO₃⁻ was mediated by a dual system (or dual phasic), whereas only single systems were found for transport of NO₂⁻ and NH₄⁺. The K_m values for NO₃⁻, NO₂⁻, and NH₄⁺ were, respectively, 0.027, 0.054, and 0.05 millimolar. The K_m for mechanism II of NO₃⁻ transport could not be defined in this study as it exhibited only apparent first order kinetics up to 1 millimolar.     

Effect of ammonium on nitrate utilization by roots of dwarf bean

The effect of exogenous NH₄⁺ on NO₃⁻ uptake and in vivo NO₃⁻ reductase activity (NRA) in roots of Phaseolus vulgaris L. cv Witte Krombek was studied before, during, and after the apparent induction of root NRA and NO₃⁻ uptake. Pretreatment with NH₄Cl (0.15-50 millimolar) affected neither the time pattern nor the steady state rate of NO₃⁻ uptake.

When NH₄⁺ was given at the start of NO₃⁻ nutrition, the time pattern of NO₃⁻ uptake was the same as in plants receiving no NH₄⁺. After 6 hours, however, the NO₃⁻ uptake rate (NUR) and root NRA were inhibited by NH₄⁺ to a maximum of 45% and 60%, respectively.

The response of the NUR of NO₃⁻-induced plants depended on the NH₄Cl concentration. Below 1 millimolar NH₄⁺, the NUR declined immediately and some restoration occurred in the second hour. In the third hour, the NUR became constant. In contrast, NH₄⁺ at 2 millimolar and above caused a rapid and transient stimulation of NO₃⁻ uptake, followed again by a decrease in the first, a recovery in the second, and a steady state in the third hour. Maximal inhibition of steady state NUR was 50%. With NO₃⁻-induced plants, root NRA responded less and more slowly to NH₄⁺ than did NUR.

Methionine sulfoximine and azaserine, inhibitors of glutamine synthetase and glutamate synthase, respectively, relieved the NH₄⁺ inhibition of the NUR of NO₃⁻-induced plants. We conclude that repression of the NUR by NH₄⁺ depends on NH₄⁺ assimilation. The repression by NH₄⁺ was least at the lowest and highest NH₄⁺ levels tested (0.04 and 25 millimolar).

     

The influence of ammonium and chloride on potassium and nitrate absorption by barley roots depends on time of exposure and cultivar

Net uptakes of K⁺ and NO₃⁻ were monitored simultaneously and continuously for two barley (Hordeum vulgare) cultivars, Prato and Olli. The cultivars had similar rates of net K⁺ and NO₃⁻ uptake in the absence of NH₄⁺ or Cl⁻. Long-term exposure (over 6 hours) to media which contained equimolar mixtures of NH₄⁺, K⁺, Cl⁻, or NO₃⁻ affected the cultivars very differently: (a) the presence of NH₄⁺ as NH₄Cl stimulated net NO₃⁻ uptake in Prato barley but inhibited net NO₃⁻ uptake in Olli barley; (b) Cl⁻ inhibited net NO₃⁻ uptake in Prato but had little effect in Olli; and (c) NH₄⁺ as (NH₄)₂SO₄ inhibited net K⁺ uptake in Prato but had little effect in Olli. Moreover, the immediate response to the addition of an ion often varied significantly from the long-term response; for example, the addition of Cl⁻ initially inhibited net K⁺ uptake in Olli barley but, after a 4 hour exposure, it was stimulatory. For both cultivars, net NH₄⁺ and Cl⁻ uptake did not change significantly with time after these ions were added to the nutrient medium. These data indicate that, even within one species, there is a high degree of genotypic variation in the control of nutrient absorption.     

Nitrate Reductase Activity in Shoots and Roots of Maize Seedlings as Affected by the Form of Nitrogen Nutrition and the pH of the Nutrient Solution

The effect of nitrogen form (NH₄-N, NH₄-N + NO₃⁻, NO₃⁻) on nitrate reductase activity in roots and shoots of maize (Zea mays L. cv INRA 508) seedlings was studied. Nitrate reductase activity in leaves was consistent with the well known fact that NO₃⁻ increases, and NH₄⁺ and amide-N decrease, nitrate reductase activity. Nitrate reductase activity in the roots, however, could not be explained by the root content of NO₃⁻, NH₄-N, and amide-N. In roots, nitrate reductase activity in vitro was correlated with the rate of nitrate reduction in vivo. Inasmuch as nitrate reduction results in the production of OH⁻ and stimulates the synthesis of organic anions, it was postulated that nitrate reductase activity of roots is stimulated by the released OH⁻ or by the synthesized organic anions rather than by nitrate itself. Addition of HCO₃⁻ to nutrient solution of maize seedlings resulted in a significant increase of the nitrate reductase activity in the roots. As HCO₃⁻, like OH⁻, increases pH and promotes the synthesis of organic anions, this provides circumstantial evidence that alkaline conditions and/or organic anions have a more direct impact on nitrate reductase activity than do NO₃⁻, NH₄-N, and amide-N.     

Growth and Nitrogen Uptake Kinetics in Cultured Prorocentrum donghaiense

We compared growth kinetics of Prorocentrum donghaiense cultures on different nitrogen (N) compounds including nitrate (NO₃ ⁻), ammonium (NH₄ ⁺), urea, glutamic acid (glu), dialanine (diala) and cyanate. P. donghaiense exhibited standard Monod-type growth kinetics over a range of N concentraions (0.5–500 μmol N L⁻¹ for NO₃ ⁻ and NH₄ ⁺, 0.5–50 μmol N L⁻¹ for urea, 0.5–100 μmol N L⁻¹ for glu and cyanate, and 0.5–200 μmol N L⁻¹ for diala) for all of the N compounds tested. Cultures grown on glu and urea had the highest maximum growth rates (μ_m, 1.51±0.06 d⁻¹ and 1.50±0.05 d⁻¹, respectively). However, cultures grown on cyanate, NO₃ ⁻, and NH₄ ⁺ had lower half saturation constants (K_μ, 0.28–0.51 μmol N L⁻¹). N uptake kinetics were measured in NO₃ ⁻-deplete and -replete batch cultures of P. donghaiense. In NO₃ ⁻-deplete batch cultures, P. donghaiense exhibited Michaelis-Menten type uptake kinetics for NO₃ ⁻, NH₄ ⁺, urea and algal amino acids; uptake was saturated at or below 50 μmol N L⁻¹. In NO₃ ⁻-replete batch cultures, NH₄ ⁺, urea, and algal amino acid uptake kinetics were similar to those measured in NO₃ ⁻-deplete batch cultures. Together, our results demonstrate that P. donghaiense can grow well on a variety of N sources, and exhibits similar uptake kinetics under both nutrient replete and deplete conditions. This may be an important factor facilitating their growth during bloom initiation and development in N-enriched estuaries where many algae compete for bioavailable N and the nutrient environment changes as a result of algal growth.     

Short Term Studies of Nitrate Uptake into Barley Plants Using Ion-Specific Electrodes and ClO(3): II. Regulation of NO(3) Efflux by NH(4)

The influence of NH₄⁺, in the external medium, on fluxes of NO₃⁻ and K⁺ were investigated using barley (Hordeum vulgare cv Betzes) plants. NH₄⁺ was without effect on NO₃⁻ (³⁶ClO₃⁻) influx whereas inhibition of net uptake appeared to be a function of previous NO₃⁻ provision. Plants grown at 10 micromolar NO₃⁻ were sensitive to external NH₄⁺ when uptake was measured in 100 micromolar NO₃⁻. By contrast, NO₃⁻ uptake (from 100 micromolar NO₃⁻) by plants previously grown at this concentration was not reduced by NH₄⁺ treatment. Plants pretreated for 2 days with 5 millimolar NO₃⁻ showed net efflux of NO₃⁻ when roots were transferred to 100 micromolar NO₃⁻. This efflux was stimulated in the presence of NH₄⁺. NH₄⁺ also stimulated NO₃⁻ efflux from plants pretreated with relatively low nitrate concentrations. It is proposed that short term effects on net uptake of NO₃⁻ occur via effects upon efflux. By contrast to the situation for NO₃⁻, net K⁺ uptake and influx of ³⁶Rb⁺-labeled K⁺ was inhibited by NH₄⁺ regardless of the nutrient history of the plants. Inhibition of net K⁺ uptake reached its maximum value within 2 minutes of NH₄⁺ addition. It is concluded that the latter ion exerts a direct effect upon K⁺ influx.     

Effects of Feedstock and Pyrolysis Temperature on Biochar Adsorption of Ammonium and Nitrate

Biochar produced by pyrolysis of biomass can be used to counter nitrogen (N) pollution. The present study investigated the effects of feedstock and temperature on characteristics of biochars and their adsorption ability for ammonium N (NH₄ ⁺-N) and nitrate N (NO₃ ⁻-N). Twelve biochars were produced from wheat-straw (W-BC), corn-straw (C-BC) and peanut-shell (P-BC) at pyrolysis temperatures of 400, 500, 600 and 700°C. Biochar physical and chemical properties were determined and the biochars were used for N sorption experiments. The results showed that biochar yield and contents of N, hydrogen and oxygen decreased as pyrolysis temperature increased from 400°C to 700°C, whereas contents of ash, pH and carbon increased with greater pyrolysis temperature. All biochars could sorb substantial amounts of NH₄ ⁺-N, and the sorption characteristics were well fitted to the Freundlich isotherm model. The ability of biochars to adsorb NH₄ ⁺-N followed: C-BC>P-BC>W-BC, and the adsorption amount decreased with higher pyrolysis temperature. The ability of C-BC to sorb NH₄ ⁺-N was the highest because it had the largest cation exchange capacity (CEC) among all biochars (e.g., C-BC400 with a CEC of 38.3 cmol kg⁻¹ adsorbed 2.3 mg NH₄ ⁺-N g⁻¹ in solutions with 50 mg NH₄ ⁺ L⁻¹). Compared with NH₄ ⁺-N, none of NO₃ ⁻-N was adsorbed to biochars at different NO₃ ⁻ concentrations. Instead, some NO₃ ⁻-N was even released from the biochar materials. We conclude that biochars can be used under conditions where NH₄ ⁺-N (or NH₃) pollution is a concern, but further research is needed in terms of applying biochars to reduce NO₃ ⁻-N pollution.     

Simulated Nitrogen Deposition Reduces CH4 Uptake and Increases N2O Emission from a Subtropical Plantation Forest Soil in Southern China

To date, few studies are conducted to quantify the effects of reduced ammonium (NH₄ ⁺) and oxidized nitrate (NO₃ ⁻) on soil CH₄ uptake and N₂O emission in the subtropical forests. In this study, NH₄Cl and NaNO₃ fertilizers were applied at three rates: 0, 40 and 120 kg N ha⁻¹ yr⁻¹. Soil CH₄ and N₂O fluxes were determined twice a week using the static chamber technique and gas chromatography. Soil temperature and moisture were simultaneously measured. Soil dissolved N concentration in 0–20 cm depth was measured weekly to examine the regulation to soil CH₄ and N₂O fluxes. Our results showed that one year of N addition did not affect soil temperature, soil moisture, soil total dissolved N (TDN) and NH₄ ⁺-N concentrations, but high levels of applied NH₄Cl and NaNO₃ fertilizers significantly increased soil NO₃ ⁻-N concentration by 124% and 157%, respectively. Nitrogen addition tended to inhibit soil CH₄ uptake, but significantly promoted soil N₂O emission by 403% to 762%. Furthermore, NH₄ ⁺-N fertilizer application had a stronger inhibition to soil CH₄ uptake and a stronger promotion to soil N₂O emission than NO₃ ⁻-N application. Also, both soil CH₄ and N₂O fluxes were driven by soil temperature and moisture, but soil inorganic N availability was a key integrator of soil CH₄ uptake and N₂O emission. These results suggest that the subtropical plantation soil sensitively responses to atmospheric N deposition, and inorganic N rather than organic N is the regulator to soil CH₄ uptake and N₂O emission.     

Influence of Protein Synthesis on NO(3) Reduction, NH(4) Accumulation, and Amide Synthesis in Suspension Cultures of Paul's Scarlet Rose

Changes in the concentrations of NH₄⁺ and amides during the growth of suspension cultures of rose (Rosa cv. Paul's Scarlet) cells were examined. When cells were grown in medium possessing only NO₃⁻ as a nitrogen source, the concentrations of NH₄⁺ and amides increased to 4.0 × 10⁻¹ and 5.9 micromoles per gram fresh weight, respectively. The amounts of both constituents declined during the later stages of growth. When a trace amount of NH₄⁺ was added to the NO₃⁻ base starting medium, the concentration of NH₄⁺ in the cells was increased to 7.0 × 10⁻¹ micromoles per gram fresh weight.     

Nitrogen Uptake by Wheat Seedlings,Interactive Effects of Four Nitrogen Sources: NO3?, NO2?, NH4+, and Urea

The net influx (uptake) rates of NO₃⁻, NH₄⁺, NO₂⁻, and urea into roots of wheat (Triticum aestivum cv Yecora Rojo) seedlings from complete nutrient solutions containing all four compounds were monitored simultaneously. Although urea uptake was too slow to monitor, its presence had major inhibitory effects on the uptake of each of the other compounds. Rates of NO₃⁻, NH₄⁺, and NO₂⁻ uptake depended in a complex fashion on the concentration of all four N compounds. Equations were developed which describe the uptake rates of each of the compounds, and of total N, as functions of concentrations of all N sources. Contour plots of the results show the interactions over the range of concentrations employed. The coefficients of these equations provide quantitative values for evaluating primary and interactive effects of each compound on N uptake.     

In vitro shoot growth of Brugmansia × candida Pers

The objective of this study was to improve the growth of in vitro shoot cultures of Brugmansia × candida ‘Creamsickle’. Several mineral nutrient experiments were conducted to determine the effect of NH₄⁺, NO₃⁻, K⁺, FeSO₄/EDTA, ZnSO₄, MnSO₄, and CuSO₄ on quality, leaf width and length, size and weight of shoot mass, and shoot number. The experiment to determine the levels of NH₄⁺, NO₃⁻, and K⁺, was conducted as a 2-component NH₄⁺: K⁺ mixture crossed by [NO₃⁻] and resulted in an experimental design free of ion confounding and capable of separating the effects of proportion and concentration. The results of the NH₄⁺-K⁺-NO₃⁻ experiment revealed a region in the design space where growth was significantly improved; the region generally had lower total nitrogen and lower NH₄⁺:K⁺ ratios than MS medium. The experiments to determine the appropriate levels of Fe, Zn, Mn, and Cu were conducted at six log levels ranging from 0 to 1 mM. Of the four metal salts tested, MnSO₄ had the least effect on in vitro shoot growth and its concentration was reduced from 0.1 mM (MS level) to 0.001 mM. CuSO₄ had large effects on in vitro shoot growth and was increased from 0.0001 mM to 0.001 mM. A 2-level factorial of NH₄⁺-K⁺-NO₃⁻, FeSO₄/EDTA, and ZnSO₄ was conducted and several formulations identified for their improvements of quality and growth. In addition to the changes to MnSO₄ and CuSO₄, these formulations were characterized by lower levels of NH₄⁺, K⁺, NO₃⁻ and Zn, and higher levels of FeSO₄/EDTA. Overall, several nutrient formulations were identified as superior to MS medium for growth of in vitro shoot cultures of B. ‘Creamsickle’.     

Growth and Nitrogen Uptake Characteristics Reveal Outbreak Mechanism of the Opportunistic Macroalga Gracilaria tenuistipitata

Macroalgae has bloomed in the brackish lake of Shenzhen Bay, China continuously from 2010 to 2014. Gracilaria tenuistipitata was identified as the causative macroalgal species. The aim of this study was to explore the outbreak mechanism of G. tenuistipitata, by studying the effects of salinity and nitrogen sources on growth, and the different nitrogen sources uptake characteristic. Our experimental design was based on environmental conditions observed in the bloom areas, and these main factors were simulated in the laboratory. Results showed that salinity 12 to 20 ‰ was suitable for G. tenuistipitata growth. When the nitrogen sources'' (NH₄ ⁺, NO₃ ⁻) concentrations reached 40 µM or above, the growth rate of G. tenuistipitata was significantly higher. Algal biomass was higher (approximately 1.4 times) when cultured with NH₄ ⁺ than that with NO₃ ⁻ addition. Coincidentally, macroalgal bloom formed during times of moderate salinity (∼12 ‰) and high nitrogen conditions. The NH₄ ⁺ and NO₃ ⁻ uptake characteristic was studied to understand the potential mechanism of G. tenuistipitata bloom. NH₄ ⁺ uptake was best described by a linear, rate-unsaturated response, with the slope decreasing with time intervals. In contrast, NO₃ ⁻ uptake followed a rate-saturating mechanism best described by the Michaelis-Menten model, with kinetic parameters V_max = 37.2 µM g⁻¹ DM h⁻¹ and K_s = 61.5 µM. Further, based on the isotope ¹⁵N tracer method, we found that ¹⁵N from NH₄ ⁺ accumulated faster and reached an atom% twice than that of ¹⁵N from NO₃ ⁻, suggesting when both NH₄ ⁺ and NO₃ ⁻ were available, NH₄ ⁺ was assimilated more rapidly. The results of the present study indicate that in the estuarine environment, the combination of moderate salinity with high ammonium may stimulate bloom formation.     

Ammonium as a Driving Force of Plant Diversity and Ecosystem Functioning: Observations Based on 5 Years' Manipulation of N Dose and Form in a Mediterranean Ecosystem

Enhanced nitrogen (N) availability is one of the main drivers of biodiversity loss and degradation of ecosystem functions. However, in very nutrient-poor ecosystems, enhanced N input can, in the short-term, promote diversity. Mediterranean Basin ecosystems are nutrient-limited biodiversity hotspots, but no information is available on their medium- or long-term responses to enhanced N input. Since 2007, we have been manipulating the form and dose of available N in a Mediterranean Basin maquis in south-western Europe that has low ambient N deposition (<4 kg N ha⁻¹ yr⁻¹) and low soil N content (0.1%). N availability was modified by the addition of 40 kg N ha⁻¹ yr⁻¹ as a 1∶1 NH₄Cl to (NH₄)₂SO₄ mixture, and 40 and 80 kg N ha⁻¹ yr⁻¹ as NH₄NO₃. Over the following 5 years, the impacts on plant composition and diversity (richness and evenness) and some ecosystem characteristics (soil extractable N and organic matter, aboveground biomass and % of bare soil) were assessed. Plant species richness increased with enhanced N input and was more related to ammonium than to nitrate. Exposure to 40 kg NH₄ ⁺-N ha⁻¹ yr⁻¹ (alone and with nitrate) enhanced plant richness, but did not increase aboveground biomass; soil extractable N even increased under 80 kg NH₄NO₃-N ha⁻¹ yr⁻¹ and the % of bare soil increased under 40 kg NH₄ ⁺-N ha⁻¹ yr⁻¹. The treatment containing less ammonium, 40 kg NH₄NO₃-N ha⁻¹ yr⁻¹, did not enhance plant diversity but promoted aboveground biomass and reduced the % of bare soil. Data suggest that enhanced NH_y availability affects the structure of the maquis, which may promote soil erosion and N leakage, whereas enhanced NO_x availability leads to biomass accumulation which may increase the fire risk. These observations are relevant for land use management in biodiverse and fragmented ecosystems such as the maquis, especially in conservation areas.     

A N and N Nuclear Magnetic Resonance Study of Nitrogen Metabolism in Shoot-Forming Cultures of White Spruce (Picea glauca) Buds

Nitrogen-14 and nitrogen-15 nuclear magnetic resonance (NMR) spectra were recorded for freshly dissected buds of Picea glauca and for buds grown for 3, 6 and 9 weeks on shoot-forming medium. Resonances for Glu (and other αNH₂ groups), Pro, Ala, and the side chain groups in Gln, Arg, Orn, and γ-aminobutyric acid could be detected in in vivo¹⁵N NMR spectra. Peaks for α-amino groups, Pro, NO₃⁻ and NH₄⁺ could also be identified in ¹⁴N NMR spectra. Perfusion experiments performed for up to 20 hours in the NMR spectrometer showed that ¹⁵N-labeled NH₄⁺ and NO₃⁻ are first incorporated into the amide group of Gln and then in the αNH₂ pool. Subsequently, it also emerges in Ala and Arg. These data suggest that the glutamine synthetase/ glutamate synthase pathway functions under these conditions. The assimilation of NH₄⁺ is much faster than that of NO₃⁻. Consequently after 10 days of growth more than 70% of the newly synthesized internal free amino acid pool derives its nitrogen from NH₄⁺ rather than NO₃⁻. If NH₄⁺ is omitted from the medium, no NO₃⁻ is taken up during 9 weeks and the buds support limited growth by utilizing their endogenous amino acid pools. It is concluded that NH₄⁺ and NO₃⁻ are both required for the induction of nitrate- and nitrite reductase.     

In Vivo Blue-Light Activation of Chlamydomonas reinhardii Nitrate Reductase

Chlamydomonas reinhardii cells, growing photoautotrophically under air, excreted to the culture medium much higher amounts of NO₂⁻ and NH₄⁺ under blue than under red light. Under similar conditions, but with NO₂⁻ as the only nitrogen source, the cells consumed NO₂⁻ and excreted NH₄⁺ at similar rates under blue and red light. In the presence of NO₃⁻ and air with 2% CO₂ (v/v), no excretion of NO₂⁻ and NH₄⁺ occurred and, moreover, if the bubbling air of the cells that were currently excreting NO₂⁻ and NH₄⁺ was enriched with 2% CO₂ (v/v), the previously excreted reduced nitrogen ions were rapidly reassimilated. The levels of total nitrate reductase and active nitrate reductase increased several times in the blue-light-irradiated cells growing on NO₃⁻ under air. When tungstate replaced molybdate in the medium (conditions that do not allow the formation of functional nitrate reductase), blue light activated most of the preformed inactive enzyme of the cells. Furthermore, nitrate reductase extracted from the cells in its inactive form was readily activated in vitro by blue light. It appears that under high irradiance (90 w m⁻²) and low CO₂ tensions, cells growing on NO₃⁻ or NO₂⁻ may not have sufficient carbon skeletons to incorporate all the photogenerated NH₄⁺. Because these cells should have high levels of reducing power, they might use NO₃⁻ or, in its absence, NO₂⁻ as terminal electron acceptors. The excretion of the products of NO₂⁻ and NH₄⁺ to the medium may provide a mechanism to control reductant level in the cells. Blue light is suggested as an important regulatory factor of this photorespiratory consumption of NO₃⁻ and possibly of the whole nitrogen metabolism in green algae.     

Dissimilatory Reduction of NO2− to NH4+ and N2O by a Soil Citrobacter sp.

Dissimilatory reduction of NO₂⁻ to N₂O and NH₄⁺ by a soil Citrobacter sp. was studied in an attempt to elucidate the physiological and ecological significance of N₂O production by this mechanism. In batch cultures with defined media, NO₂⁻ reduction to NH₄⁺ was favored by high glucose and low NO₃⁻ concentrations. Nitrous oxide production was greatest at high glucose and intermediate NO₃⁻ concentrations. With succinate as the energy source, little or no NO₂⁻ was reduced to NH₄⁺ but N₂O was produced. Resting cell suspensions reduced NO₂⁻ simultaneously to N₂O and free extracellular NH₄⁺. Chloramphenicol prevented the induction of N₂O-producing activity. The K_m for NO₂⁻ reduction to N₂O was estimated to be 0.9 mM NO₂⁻, yet the apparent K_m for overall NO₂⁻ reduction was considerably lower, no greater than 0.04 mM NO₂⁻. Activities for N₂O and NH₄⁺ production increased markedly after depletion of NO₃⁻ from the media. Amendment with NO₃⁻ inhibited N₂O and NH₄⁺ production by molybdate-grown cells but not by tungstate-grown cells. Sulfite inhibited production of NH₄⁺ but not of N₂O. In a related experiment, three Escherichia coli mutants lacking NADH-dependent nitrite reductase produced N₂O at rates equal to the wild type. These observations suggest that N₂O is produced enzymatically but not by the same enzyme system responsible for dissimilatory reduction of NO₂⁻ to NH₄⁺.     

Identification of Heterotrophic Nitrification in a Sierran Forest Soil

A potential for heterotrophic nitrification was identified in soil from a mature conifer forest and from a clear-cut site. Potential rates of NO₂⁻ production were determined separately from those of NO₃⁻ by using acetylene to block autotrophic NH₄⁺ oxidation and chlorate to block NO₂⁻ oxidation to NO₃⁻ in soil slurries. Rates of NO₂⁻ production were similar in soil from the forest and the clear-cut site and were strongly inhibited by acetylene. The rate of NO₃⁻ production was much greater than that of NO₂⁻ production, and NO₃⁻ production was not significantly affected by acetylene or chlorate. Nitrate production was partially inhibited by cycloheximide, but was not significantly reduced by streptomycin. Neither the addition of ammonium nor the addition of peptone stimulated NO₃⁻ production. ¹⁵N labeling of the NH₄⁺ pool demonstrated that NO₃⁻ was not coming from NH₄⁺. The potential for heterotrophic nitrification in these forest soils was greater than that for autotrophic nitrification.