Nitrate and ammonium influxes in soybean (Glycine max) roots: direct comparison of 13N and 15N tracing

We compared influxes and internal transport in soybean plants (Glycine max cv. Kingsoy) of labelled N from external solutions where either ammonium or nitrate was labelled with the stable isotope¹⁵N and the radioactive isotope¹³N. The objective was to see whether mass spectrometric determinations of tissue ¹⁵N content were sufficiently sensitive to measure influxes accurately over short time periods. Our findings were as follows. (1) There was a close quantitative correspondence between estimates of N influx of individual plants using ¹⁵N or ¹³N measurements with either NO_3/^? or NH₄⁺ at 4 or 2 mol^?3, respectively in the external solution. (2) Transport to the shoot of N from NO₃ absorbed over a 5–15 min period could be monitored when the external NO₃^? concentration ranged from 0–05 to 4 mol m^?3. NH₄⁺ as the N source labelled shoot tissue more slowly, and estimates of the transport between root and shoot could be made only with ¹³N. (3) Influx of NO₃^? into root tissue could be measured by ¹⁵N enrichment after 5–10 min at concentrations approaching the probable K_M of the high-affinity transport system. (4) There was some indication of isotope discrimination, especially with respect to the movement of labelled N to the shoot, when NO₃^? is the N source. For many purposes, ¹⁵N tracing can be used satisfactorily to estimate influxes of both NO₃^? and NH₄⁺ in soybean roots. Use of the short-lived radio nuclide ¹³N remains the method of choice for more refined measurements of internal distribution and assimilation.     

Restricted nitrate influx and reduction in corn seedlings exposed to ammonium

The effect of ambient ammonium (0.5 millimolar [¹⁴NH₄]₂SO₄) added to a nutrient solution containing 1.0 millimolar K¹⁵NO₃, 99 atom per cent ¹⁵N, upon [¹⁵N]nitrate assimilation and utilization of previously accumulated [¹⁴N]nitrate was investigated. Corn seedlings, 5-day-old dark-grown decapitated (experiment I) and 10-day-old light-grown intact (experiment II), which had previously been grown on K¹⁴NO₃ nutrient solution, were used. In both experiments, the presence of ambient ammonium decreased [¹⁵N]nitrate influx (20% after 6 hours) without significantly affecting the efflux of previously accumulated [¹⁴N]nitrate. In experiment I, relative reduction of [¹⁵N]nitrate (reduction as a percentage of influx) was inhibited more than was [¹⁵N]nitrate influx. Nevertheless, in experiment I, where all reduction could be assigned to the root system, the absolute inhibition of reduction during the 12 hours (13 micromoles/root) was less than the absolute inhibition in influx (24 micromoles/root). The data suggest that the influence of ammonium on [¹⁵N]nitrate influx could not be totally accounted for by the decrease in the potential driving force which resulted from restricted reduction; an additional impact on the influx process is indicated. Reduction of [¹⁵N]nitrate in experiment II after 6 hours accounted for 30 and 18% of the tissue excess ¹⁵N in the control and ammonium treatments, respectively. Relative distribution of ¹⁵N between roots and exudate (experiment I), or between roots and shoots (experiment II) was not affected by ammonium. On the other hand, the accumulation of [¹⁵N]nitrate in roots, shoots, and xylem exudate was enhanced by ammonium treatment compared to the control, whereas the accumulation of reduced ¹⁵N was inhibited.     

Initial organic products of assimilation of [N]ammonium and [N]nitrate by tobacco cells cultured on different sources of nitrogen

Glutamine is the first major organic product of assimilation of ¹³NH₄⁺ by tobacco (Nicotiana tabacum L. cv. Xanthi) cells cultured on nitrate, urea, or ammonium succinate as the sole source of nitrogen, and of ¹³NO₃⁻ by tobacco cells cultured on nitrate. The percentage of organic ¹³N in glutamate, and subsequently, alanine, increases with increasing periods of assimilation. ¹³NO₃⁻, used for the first time in a study of assimilation of nitrogen, was purified by new preparative techniques. During pulse-chase experiments, there is a decrease in the percentage of ¹³N in glutamine, and a concomitant increase in the percentage of ¹³N in glutamate and alanine. Methionine sulfoximine inhibits the incorporation of ¹³N from ¹³NH₄⁺ into glutamine more extensively than it inhibits the incorporation of ¹³N into glutamate, with cells grown on any of the three sources of nitrogen. Azaserine inhibits glutamate synthesis extensively when ¹³NH₄⁺ is fed to cells cultured on nitrate. These results indicate that the major route for assimilation of ¹³NH₄⁺ is the glutamine synthetase-glutamate synthase pathway, and that glutamate dehydrogenase also plays a role, but a minor one. Methionine sulfoximine inhibits the incorporation of ¹³N from ¹³NO₃⁻ into glutamate more strongly than it inhibits the incorporation of ¹³N into glutamine, suggesting that the assimilation of ¹³NH₄⁺ derived from ¹³NO₃⁻ may be mediated solely by the glutamine synthetase-glutamate synthase pathway.     

Estimation of nitrification rates in flooded soils

Three techniques for estimating nitrification rates in flooded soils were evaluated in short-term incubation experiments using three soils. The techniques were based on inhibition of either ammonium or nitrite oxidation and ¹³N isotope dilution. Of four inhibitors of ammonium oxidation evaluated, one (allylthiourea) was ineffective and two (2-ethynylpyridine or phenyl acetylene dissolved in ethanol) promoted immobilization of ammonium. Emulsified 2-ethynylpyridine and acetylene were equally effective inhibitors of ammonium oxidation and had little or no effect on gross rates of N mineralization and immobilization. Four inhibitors of nitrite oxidation were evaluated, but this approach was compromised by the nonspecificity of three of the compounds—potassium cyanide, 2-ethylamino-4-isopropylamino-6-methylthio-s-triazine (ametryne) and 3-(3,4-dichlorophenyl)-1-methylurea (DMU)—and by the partial effectiveness of another (potassium chlorate). Two methods based on isotope dilution gave similar estimates of nitrification rates. These rates were similar to those estimated by inhibition of ammonium oxidation in one soil but were lower in the other two soils. In the latter two soils, nitrification of labeled ammonium derived from dissimilatory nitrate reduction resulted in underestimation of nitrification rates by isotope dilution.     

Whole-system estimation of denitrification in a plains river: a comparison of two methods

Whole-system denitrification in the South Platte River was measured over a 13-month period using an open-channel N₂ method and mass-balance measurements. Concentrations of dissolved N₂ were measured with high precision by membrane-inlet mass spectrometry and estimates of denitrification were based on the mass flux of N₂, after correction for reaeration and groundwater flux. Open-channel estimates of denitrification ranged from 0 to 3.08 g N m^–2 d^–1 and the mean annual rate was 1.62 g N m^–2 d^–1, which corresponds to removal of approximately 34% of the nitrate transported by the river over a distance of 18.5 km. Over the same period of time, estimates of denitrification based on mass-balance measurements ranged from 0.29 to 5.25 g N m^–2 d^–1 and the mean annual rate was 2.11 g N m^–2 d^–1. The two methods revealed similar seasonal patterns of denitrification the highest rates were measured from late April to August and the lowest rates were in winter. Both methods provide whole-system estimates of denitrification in running waters; where reaeration rate coefficients are low and flux of groundwater is well quantified, the open-channel method has fewer sources of uncertainty and is easier to implement.     

Assimilation of 13NH 4 + by Anthoceros grown with and without symbiotic Nostoc

The pathways of assimilation of ammonium by pure cultures of symbiont-free Anthoceros punctatus L. and the reconstituted Anthoceros-Nostoc symbiotic association were determined from time-course (5–300 s) and inhibitor experiments using ¹³NH ₄ ⁺ . The major product of assimilation after all incubation times was glutamine, whether the tissues were cultured with excess ammonium or no combined nitrogen. The ¹³N in glutamine was predominantly in the amide-nitrogen position. Formation of glutamine and glutamate by Anthoceros-Nostoc was strongly inhibited by either 1mM methionine sulfoximine (MSX) or 1 mM exogenous ammonium. These data are consistent with the assimilation of ¹³NH ₄ ⁺ and formation of glutamate by the glutamine synthetase (EC 6.3.1.2)-glutamate synthase (EC 1.4.7.1) pathway in dinitrogen-grown Anthoceros-Nostoc. However, in symbiont-free Anthoceros, grown with 2.5 mM ammonium, formation of glutamine, but not glutamate, was decreased by either MSX or exogenous ammonium. These results indicate that during short incubation times ammonium is assimilated in nitrogenreplete Anthoceros by the activities of both glutamine synthetase and glutamate dehydrogenase (EC 1.4.1.2). In-vitro activities of glutamine synthetase were similar in nitrogen-replete Anthoceros and Anthoceros-Nostoc, indicating that the differences in the routes of glutamate formation were not based upon regulation of synthesis of the initial enzyme of the glutamine synthetase-glutamate synthase pathway. When symbiont-free Anthoceros was cultured for 2 d in the absence of combined nitrogen, total ¹³NH ₄ ⁺ assimilation, and glutamine and glutamate formation in the presence of inhibitors, were similar to dinitrogen-grown Anthoceros-Nostoc. The routes of immediate (within 2 min) glutamate formation and ammonium assimilation in Anthoceros were apparently determined by the intracellular levels of ammonium; at low levels the glutamine synthetase-glutamate synthase pathway was predominant, while at high levels independent activities of both glutamine synthetase and glutamate dehydrogenase were expressed.     

High-level 2H/13C/15N labeling of proteins for NMR studies

Summary The protein human carbonic anhydrase II (HCA II) has been isotopically labeled with ²H, ¹³C and ¹⁵N for high-resolution NMR assignment studies and pulse sequence development. To increase the sensitivity of several key ¹H/¹³C/¹⁵N triple-resonance correlation experiments, ²H has been incorporated into HCA II in order to decrease the rates of ¹³C and ¹H_N T₂ relaxation. NMR quantities of protein with essentially complete aliphatic ²H incorporation have been obtained by growth of E. coli in defined media containing D₂O, [1,2-¹³C₂, 99%] sodium acetate, and [¹⁵N, 99%] ammonium chloride. Complete aliphatic deuterium enrichment is optimal for ¹³C and ¹⁵N backbone NMR assignment studies, since the ¹³C and ¹H_N T₂ relaxation times and, therefore, sensitivity are maximized. In addition, complete aliphatic deuteration increases both resolution and sensitivity by eliminating the differential ²H isotopic shift observed for partially deuterated CH_nD_m moieties.     

Azolla-Anabaena Relationship : XIII. Fixation of [N]N(2)

The major radioactive products of the fixation of [¹³N]N₂ by Azolla caroliniana Willd.-Anabaena azollae Stras. were ammonium, glutamine, and glutamate, plus a small amount of alanine. Ammonium accounted for 70 and 32% of the total radioactivity recovered after fixation for 1 and 10 minutes, respectively. The presence of a substantial pool of [¹³N]N₂-derived ¹³NH₄⁺ after longer incubation periods was attributed to the spatial separation between the site of N₂-fixation (Anabaena) and a second, major site of assimilation (Azolla). Initially, glutamine was the most highly radioactive organic product formed from [¹³N]N₂, but after 10 minutes of fixation glutamate had 1.5 times more radiolabel than glutamine. These kinetics of radiolabeling, along with the effects of inhibitors of glutamine synthetase and glutamate synthase on assimilation of exogenous and [¹³N]N₂-derived ¹³NH₄⁺, indicate that ammonium assimilation occurred by the glutamate synthase cycle and that glutamate dehydrogenase played little or no role in the synthesis of glutamate by Azolla-Anabaena.     

Amino acid transporter mutants of Arabidopsis provides evidence that a non‐mycorrhizal plant acquires organic nitrogen from agricultural soil

Although organic nitrogen (N) compounds are ubiquitous in soil solutions, their potential role in plant N nutrition has been questioned. We performed a range of experiments on Arabidopsis thaliana genetically modified to enhance or reduce root uptake of amino acids. Plants lacking expression of the Lysine Histidine Transporter 1 (LHT1) displayed significantly lower contents of ¹³C and ¹⁵N label and of U‐¹³C₅,¹⁵N₂ L‐glutamine, as determined by liquid chromatography–mass spectrometry when growing in pots and supplied with dually labelled L‐glutamine compared to wild type plants and LHT1‐overexpressing plants. Slopes of regressions between accumulation of ¹³C‐labelled carbon and ¹⁵N‐labelled N were higher for LHT1‐overexpressing plants than wild type plants, while plants lacking expression of LHT1 did not display a significant regression between the two isotopes. Uptake of labelled organic N from soil tallied with that of labelled ammonium for wild type plants and LHT1‐overexpressing plants but was significantly lower for plants lacking expression of LHT1. When grown on agricultural soil plants lacking expression of LHT1 had the lowest, and plants overexpressing LHT1 the highest C/N ratios and natural δ¹⁵N abundance suggesting their dependence on different N pools. Our data show that LHT1 expression is crucial for plant uptake of organic N from soil.     

Biological role of mycorrhizal fungi on the assimilation and transportation of carbon and nitrogen to Anacamptis palustris and Anacamptis laxiflor

Fungal is a physiological trail and its understanding in the assimilation with the transfer of carbon (C) cum nitrogen (N) or (C/N) to orchid-seedlings have not been determined. Labelled stable isotopes ¹³C and ¹⁵N were used to plan the flow of C and N between orchid plants and mycorrhizal connotations in-terms of bulk transfer for C/N. This study attends to comprehend the mechanism, supporting mycorrhizal fungi which influences on orchid-seedling growth. Determined integration and transfer of C/N from amino acids (AA), ammonium nitrate (NH₄NO₃) and sugar for orchid-plant may lead to understand these mechanisms. This current study tries to estimate the importance of organic compounds as a source for C/N over the inorganic-NH₄NO₃. Generally, after begging of germination and when it is found to be associated to the nutrient resource, organic compound enhance the biomass accumulation of two orchid species. AA significantly increase the mass of ¹³C assimilated by two species. With amino acids the concentration of ¹³C in two species was greater than with NH₄NO₃ and sugar. At another phase, amount of ¹⁵N content shoots was a higher value in Anacamptis laxiflora shoots assimilated substantially additional of ¹⁵N with NH₄NO₃ plus sugar compared with ammonium nitrate only. This study showed that two terrestrial orchids species are reliant on organic compounds as a source of carbon and nitrogen more than inorganic compounds.     

Low concentrations of nitrate and ammonium stimulate nodulation and N2 fixation while inhibiting specific nodulation (nodule DW g−1 root dry weight) and specific N2 fixation (N2 fixed g−1 root dry weight) in soybean

Nitrate N is a major inhibitor of the soybean/Bradyrhizobium symbiosis in legumes and although this inhibition has been studied for many years, as yet no consensus has been reached on the specific and quantitative interactions between nitrate and ammonium supply and N₂ fixation. The effect of nitrate and ammonium supply on plant growth, nodulation and N₂ fixation capacity during the full growth cycle was investigated in both greenhouse and growth chamber experiments with three soybean genotypes. The results show that a high concentration of mineral N (10 mM), either as nitrate or ammonium or ammonium nitrate significantly suppressed nodule number, nodule dry weight and total N₂ fixed per plant of nodulated soybeans. However, lower mineral N concentrations, either 1 mM or 3.75 mM significantly enhanced nodule number, nodule dry weight and total N₂ fixed per plant, while specific nodulation (nodule dry weight g^–1 root DW, SNOD) and specific N₂ fixation (total N₂ fixed g^–1 root DW, SNF) were significantly reduced, particularly at the early vegetative growth stage V4, compared to the treatment with N₂ fixation as the only N source, in both growth chamber and greenhouse experiments. Therefore, we suggest that SNOD or SNF might be better indicators to express the suppressing effect of mineral N addition on nodule performance and N₂ fixed. Our studies also showed that ammonium alone was the more efficient N source than either ammonium nitrate or nitrate for soybean, as it resulted in higher biomass accumulation, nodule dry weight, total N accumulation and total N₂ fixed by 23, 20, 18 and 44%, respectively, compared to NO₃ ^– as the N source.     

Assimilatory nitrate uptake in Pseudomonas fluorescens studied using nitrogen-13

The mechanism of nitrate uptake for assimilation in procaryotes is not known. We used the radioactive isotope, ¹³N as NO₃ ^-, to study this process in a prevalent soil bacterium, Pseudomonas fluorescens. Cultures grown on ammonium sulfate or ammonium nitrate failed to take up labeled nitrate, indicating ammonium repressed synthesis of the assimilatory enzymes. Cultures grown on nitrite or under ammonium limitation had measurable nitrate reductase activity, indicating that the assimilatory enzymes need not be induced by nitrate. In cultures with an active nitrate reductase, the form of ¹³N internally was ammonium and amino acids; the amino acid labeling pattern indicated that ¹³NO₃ ^- was assimilated via glutamine synthetase and glutamate synthase. Cultures grown on tungstate to inactivate the reductase concentrated NO₃ ^- at least sixfold. Chlorate had no effect on nitrate transport or assimilation, nor on reduction in cell-free extracts. Ammonium inhibited nitrate uptake in cells with and without active nitrate reductases, but had no effect on cell-free nitrate reduction, indicating the site of inhibition was nitrate transport into the cytoplasm. Nitrate assimilation in cells grown on nitrate and nitrate uptake into cells grown with tungstate on nitrite both followed Michaelis-Menten kinetics with similar K _mvalues, 7 M. Both azide and cyanide inhibited nitrate assimilation. Our findings suggest that Pseudomonas fluorescens can take up nitrate via active transport and that nitrate assimilation is both inhibited and repressed by ammonium.     

Field evaluation of symbiotic nitrogen fixation by rhizobial strains using15N methodology

Summary Small differences in N₂ fixation by nodulated soybeans (Glycine max. (L.) Merr.), inoculated with various strains ofRhizobium japonicum, were assessed in field experiments using¹⁵N methodology, and compared with yields of plant dry matter and total N. Percentage of plant-N derived from atmospheric N₂ and from fertilizer, and values of %¹⁵N atom excess had lower coefficients of variation than did total N and dry matter yield. Nevertheless the precision of estimates of kg N/ha fixed were sufficient to differentiate only the extremes of the range of strains tested, and there were discrepancies between ranking of strains based on % N derived from fertilizer and on total N yield.     

Nitrogen supply and cyanide concentration influence the enrichment of nitrogen from cyanide in wheat (Triticum aestivum L.) and sorghum (Sorghum bicolor L.)

Cyanide assimilation by the β‐cyanoalanine pathway produces asparagine, aspartate and ammonium, allowing cyanide to serve as alternate or supplemental source of nitrogen. Experiments with wheat and sorghum examined the enrichment of ¹⁵N from cyanide as a function of external cyanide concentration in the presence or absence of nitrate and/or ammonium. Cyanogenic nitrogen became enriched in plant tissues following exposure to ¹⁵N‐cyanide concentrations from 5 to 200 µm , but when exposure occurred in the absence of nitrate and ammonium, ¹⁵N enrichment increased significantly in sorghum shoots at solution cyanide concentrations of ≥50 µm and in wheat roots at 200 µm cyanide. In an experiment with sorghum using ¹³C¹⁵N, there was also a significant difference in the tissue ¹³C:¹⁵N ratio, suggestive of differential metabolism and transport of carbon and nitrogen under nitrogen‐free conditions. A reciprocal ¹⁵N labelling study using KC¹⁵N and ¹⁵NH₄⁺ and wheat demonstrated an interaction between cyanide and ammonium in roots in which increasing solution ammonium concentrations decreased the enrichment from 100 µm cyanide. In contrast, with increasing solution cyanide concentrations there was an increase in the enrichment from ammonium. The results suggest increased transport and assimilation of cyanide in response to decreased nitrogen supply and perhaps to ammonium supply.     

KINETICS OF NITROGEN-15 LABELLED AMMONIUM UPTAKE BY CAULERPA CUPRESSOIDES (CHLOROPHYTA)1,2,3

The incorporation of ¹⁵NH₄⁺ as a function of time and concentration was used to estimate ammonium uptake by Caulerpa cupressoides (West) C. Agardh, taken from its habitat on the sediments of Tague Bay lagoon, St. Croix, U.S. Virgin Islands. ¹⁵N-based uptake rates followed Michaelis-Menten type saturation kinetics; the maximum uptake rate was 8.7 ± 3.0 μmol N/g dry wt·h at 26° C and the half-saturation constant was 48 ± 10 μM (X?± SE). The high half-saturatiaon constant reflects the dependence of Caulerpa on sediment pore waters as a nitrogen source. Calculations of uptake from isotope time course data were compared to estimates made from ammonium depletion. More ammonium disappeared than could be accounted for by the incorporation of ¹⁵N in Caulerpa, and isotope dilution of the ammonium pool is shown not to be responsible for underestimates of uptake, primarily because large ¹⁵N additions (40–300 μM) were used. It is suggested that either (1) a secondary ammonium sink such as wall sorption or bacterial uptake significantly influenced ammonium concentrations, or (2) ¹⁵N was lost as labelled dissolved organic nitrogen or volatilized during ¹⁵N sample preparation.     

Patterns of [13N]ammonium uptake and assimilation by Frankia HFPArl3

Nitrogen-starved cells of Frankia strain HFPArl3 incorporated [¹³N]-labeled ammonium into glutamine serine (glutamate, alanine, aspartate), after five-minute radioisotope exposures. High initial endogenous pools of glutamate were reduced, while total glutamine increased, during short term NH _inf4 ^sup+ incubation. Preincubation of cells in methionine sulfoximine (MSX) resulted in [¹³N]glutamine reduced by more than 80%, while [¹³N]glutamate and [¹³N]alanine levels increased. The results suggest that glutamine synthetase is the primary enzyme of ammonium assimilation, and that glutamate dehydrogenase and alanine dehydrogenase may also function in ammonium assimilation at low levels. Efflux of [¹³N]serine and lesser amounts of [¹³N]glutamine was detected from the Frankia cells. The identity of both Ser and Gln in the extracellular compartment was confirmed with gas chromatography/mass spectrometry. Serine efflux may be of significance in nitrogen transfer in Frankia.Abbreviations Pthr phosphothreonine - Aad -amino-adipate - MSX methionine sulfoximine     

Identification of HN-methyl NOEs in large proteins using simultaneous amide-methyl TROSY-based detection

A pair of HN-methyl NOESY experiments that are based on simultaneous TROSY-type detection of amide and methyl groups is described. The preservation of cross-peak symmetry in the simultaneous ¹H–¹⁵N/¹³CH₃ NOE spectra enables straightforward assignments of HN-methyl NOE cross-peaks in large and complex protein structures. The pulse schemes are designed to preserve the slowly decaying components of both ¹H–¹⁵N and methyl ¹³CH₃ spin-systems in the course of indirect evolution (t ₂) and acquisition period (t ₃) of 3D NOESY experiments. The methodology has been tested on {U-[¹⁵N,²H]; Ileδ1-[¹³CH₃]; Leu,Val-[¹³CH₃,¹²CD₃]}-labeled 82-kDa enzyme Malate Synthase G (MSG). A straightforward procedure that utilizes the symmetry of NOE cross-peaks in the time-shared 3D NOE data sets allows unambiguous assignments of more than 300 HN-methyl interactions in MSG from a single 3D data set providing important structural restraints for derivation of the backbone global fold.     

Acid-base regulation during ammonium assimilation in Hydrodictyon africanum

Abstract The acid-base balance during ammonium (used to mean NH ₄⁺ and/or NH₃) assimilation in Hydrodictyon africanum has been measured on cells growing with about 1 mol m^?3 ammonium at an external pH of about 6.5. Measurements made included (1) ash alkalinity (corrected for intracellular ammonium) which yields net organic negative charge, (2) the accumulation of organic N in the cells and (3) the change in extracellular H⁺ (from the pH change and the buffer capacity). These measurements showed that some 0.25 excess organic negative charge (half in the cell wall, half inside the plasmalemma) accumulates per organic N synthesized, while some 1.25H⁺ accumulate in the medium per organic N synthesized. Granted a permeability (PNH₃) of some 10^?3 cm s^?1, and a finite [NH₃] in the cytoplasm of these N-assimilating cells it is likely that most of the ammonium entering these growing cells is as NH ₄⁺. This means that most of the H ⁺ appearing in the medium must have originated from inside the cell and have been subjected to active efflux at the plasmalemma: H⁺ accumulates in the medium equivalent to any NH₃ entry by requilibration from exogenous NH ₄⁺. The cell composition (net organic negative charge, organic N content) is very similar in these ammonium-grown cells to that of NO₃⁺grown cells, suggesting that there is no action of a ‘biochemical pH stat’ during longterm assimilation of NO₃⁺in H. africanum. Short-term experiments were carried out at an external pH of 7.2 in which ammonium at various concentrations were supplied to NO₃⁺-grown cells. There was in all cases a rapid influx followed by a slower uptake; at least at the lower concentrations (less than 100 μmol dm^?3) the net influx was all attributable to NH₄⁺influx via a uniporter, probably partly short-circuited by a passive NH₃ efflux due to intrinsic membrane permeability to NH₃. The net ammonium influx was in all cases associated with H⁺ accumulation in the medium. (1.3-1.7 H ⁺ per ammonium taken up); as in the growth experiments, most of the ammonium taken up was assimilated. Determinations of cytoplasmic pH showed either no effect on, or a slight decrease in, pH during ammonium assimilation; the changes that occurred were in the direction expected for actuating a ‘pH-regulating’ change in H⁺ fluxes.     

Predicting N2O emissions from agricultural land through related soil parameters

An empirical model of nitrous oxide emission from agricultural soils has been developed. It is based on the relationship between N₂O and three soil parameters – soil mineral N (ammonium plus nitrate) content in the topsoil, soil water‐filled pore space and soil temperature – determined in a study on a fertilized grassland in 1992 and 1993. The model gave a satisfactory prediction of seasonal fluxes in other seasons when fluxes were much higher, and also from other grassland sites and from cereal and oilseed rape crops, over a wide flux range (< 1 to > 20 kg N₂O‐N ha^?1 y^?1). However, the model underestimated emissions from potato and broccoli crops; possible reasons for this are discussed. This modelling approach, based as it is on well‐established and widely used soil measurements, has the potential to provide flux estimates from a much wider range of agricultural sites than would be possible by direct measurement of N₂O emissions.     

Fixation of [13N]N2 and transfer of fixed nitrogen in the Anthoceros-Nostoc symbiotic association

The initial product of fixation of [¹³N]N₂ by pure cultures of the reconstituted symbiotic association between Anthoceros punctatus L. and Nostoc sp. strain ac 7801 was ammonium; it accounted for 75% of the total radioactivity recovered in methanolic extracts after 0.5 min and 14% after 10 min of incubation. Glutamine and glutamate were the primary organic products synthesized from [¹³N]N₂ after incubation times of 0.5–10 min. The kinetics of labeling of these two amino acids were characteristic of a precursor (glutamine) and product (glutamate) relationship. Results of inhibition experiments with methionine sulfoximine (MSX) and diazo-oxonorleucine were also consistent with the assimilation of N₂-derived NH ₄ ⁺ by Anthoceros-Nostoc through the sequential activities of glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.7.1), with little or no assimilation by glutamate dehydrogenase (EC 1.3.1.3). Isolated symbiotic Nostoc assimilated exogenous ¹³NH ₄ ⁺ into glutamine and glutamate and their formation was inhibited by MSX, indicating operation of the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway: However, relative to free-living cultures, isolated symbiotic Nostoc assimilated 80% less exogenous ammonium into glutamine and glutamate, implying that symbiotic Nostoc could assimilate only a fraction of N₂-derived NH ₄ ⁺ . This implication was tested by using Anthoceros associations reconstituted with wild-type or MSX-resistant strains of Nostoc incubated with [¹³N]N₂ in the presence of MSX. The results of these experiments indicated that, in situ, symbiotic Nostoc assimilated about 10% of the N₂-derived NH ₄ ⁺ and that NH ₄ ⁺ was made available to Anthoceros tissue where it was apparently assimilated by the GS-GOGAT pathway. Since less than 1% of the fixed N₂ was lost to the suspension medium, it appears that transfer of NH ₄ ⁺ from symbiont to host tissue was very efficient in this extracellular symbiotic association.Abbreviations DON 6-diazo-5-oxo-l-norleucine - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSX l-methionine-dl-sulfoximine