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.     

Natural 15N enrichment of amide-exporting legume nodules

The natural ¹⁵N abundance of amide-exporting nodules was compared to that of shoots in 12 plant species. Nodules were statistically less abundant in ¹⁵N than shoots in one of three cultivars of Pisum sativum L., in Vicia faba L. and in Medicago sativa L., but the ¹⁵N depletion of nodules was very samall. Nodules were statistically more abundant in ¹⁵N than shoots in Trifolium pratense L., depending on time during the growing season, Cyamopsis tetragonaloba L. Taub. and 7 Lupinus species, but the enrichment was small except for C. tetragonalova and 6 Lupinus species. Nodules of 3 Lupinus species infected with Rhizobium lupini isolated from Lupinus subcarnosa Hook, were only slightly enriched in ¹⁵N, but nodules of two of these species were substantially enriched in ¹⁵N when infected with a mix of other Rhizobium lupini strains. The third species, L. texensis Hook., was not infected by this mix of strains. Differences in ¹⁵N abundance between nodules and other tissues of amide-exporting and ureide-exporting nodules from several studies are tabulated. All ureide-exporting nodules in this tabulation are enriched in ¹⁵N. Amide-exporting nodules are considerably more variable in this regard. These results confirm that events associated with ureide synthesis or transport cannot be the sole cause of the substantial ¹⁵N enrichment seen in nodules.     

Incorporation of 15N and 14C of methionine into the mugineic acid family of phytosiderophores in iron-deficient barley roots

The role of methionine as a precursor in mugineic acid (MA) biosynthesis was studied by feeding ¹⁵N-ammonium sulfate, ¹⁴C-amino acids, and [1-¹⁴C, ¹⁵N]-methionine to iron-deficient barley roots ( Hordeum vulgare L. cv. Minorimugi), grown hydroponically. The incorporation of isotopes into amino acids was also examined. Methionine appears to be the most efficient precursor of the mugineic acid family (MAs) of phytosiderophores; homoserine was also incorporated into the MAs, but other amino acids such as glutamate, alanine, and γ-amino butyric acid did not act as precursors of MAs. Carbon-14 and ¹⁵N of methionine were incorporated into MAs. This specific incorporation of ¹⁴C and ¹⁵N indicated that the nitrogen atoms of MAs were derived from two molecules of methionine. It is suggested that deoxymugineic acid (DMA) is probably the first phytosiderophore to be synthesized on the biosynthetic pathway of MAs.     

Natural 13C and 15N abundance of field-collected fungi and their ecological implications

The natural abundance of ¹³C and ¹⁵N was measured in basidiocarps of at least 115 species in 88 genera of ectomycorrhizal, wood-decomposing and litter-decomposing fungi from Japan and Malaysia. The natural abundance of ¹³C and ¹⁵N was also measured in leaves, litter, soil and wood from three different sites. ¹⁵N and ¹³C were enriched in ectomycorrhizal and wood-decomposing fungi, respectively, relative to their substrates. Ectomycorrhizal and wood-decomposing fungi could be distinguished on the basis of their δ¹³C and δ¹⁵N signatures. Although there was high variability in the isotopic composition of fungi, the following isotope- enrichment factors (ε, mean±SD) of the fungi relative to substrates were observed:
ε_{ectomycorrhizal fungi/litter} = 6.1±0.4‰¹⁵N
ε_{ectomycorrhizal fungi/wood} = 1.4±0.8‰¹³C
ε_{wood-decomposing fungi/wood} = −0.6±0.7‰¹⁵N
ε_{wood-decomposing fungi/wood} = 3.5±0.9‰¹³C
The basis of isotope fractionation in C metabolism from wood to wood-decomposing fungus is discussed.     

Neuronal Glutamine Utilization: Pathways of Nitrogen Transfer tudied with [15N]Glutamine

Gas chromatography-mass spectrometry was used to evaluate the metabolism of [15N]glutamine in isolated rat brain synaptosomes. In the presence of 0.5 mM glutamine, synaptosomes accumulated this amino acid to a level of 25-35 nmol/mg protein at an initial rate greater than 9 nmol/min/mg of protein. The metabolism of [15N]glutamine generated 15N-labelled glutamate, aspartate, and gamma-aminobutyric acid (GABA). An efflux of both [15N]glutamate and [15N]aspartate from synaptosomes to the medium was observed. Enrichment of 15N in alanine could not be detected because of a limited pool size. Elimination of glucose from the incubation medium substantially increased the rate and amount of [15N]aspartate formed. It is concluded that: (1) With 0.5 mM external glutamine, the glutaminase reaction, and not glutamine transport, determines the rate of metabolism of this amino acid. (2) The primary route of glutamine catabolism involves aspartate aminotransferase which generates 2-oxoglutarate, a substrate for the tricarboxylic acid cycle. This reaction is greatly accelerated by the omission of glucose. (3) Glutamine has preferred access to a population of synaptosomes or to a synaptosomal compartment that generates GABA. (4) Synaptosomes maintain a constant internal level of glutamate plus aspartate of about 70-80 nmol/mg protein. As these amino acids are produced from glutamine in excess of this value, they are released into the medium. Hence synaptosomal glutamine and glutamate metabolism are tightly regulated in an interrelated manner.     

Assessing post-anthesis nitrogen uptake, distribution and utilisation in grain protein synthesis in barley (Hordeum vulgare L.) using 15N fertiliser and 15N proteinogenic and non-proteinogenic amino acids

We investigated the effects of nitrogen (N) availability during the vegetative phase on (a) post‐anthesis N uptake and (b) its translocation into ears in barley plants grown in a greenhouse at two levels of N: low (50 mg N kg^?1 sand) and optimal N supply (150 mg N kg^?1 sand). Plants in the two N treatments were fertilised with the same amount of labelled ¹⁵N [50 mg ¹⁵N kg^?1 sand at 10% ¹⁵N_exc (N_excess, i.e. N_exc, is defined as the abundance of enriched stable isotope minus the natural abundance of the isotope) applied as ¹⁵NH₄¹⁵NO₃] 10 days after anthesis (daa). In a separate experiment, the uptake and transport into ears of proteinogenic and non‐proteinogenic amino acids were studied to determine whether a relationship exists between amino acid transport into ears and their proteinogenic nature. Plants were fed with either ¹⁵N‐α‐alanine, a proteinogenic amino acid, or ¹⁵N‐α‐aminoisobutyric acid, a non‐proteinogenic amino acid. Both these amino acids were labelled at 95.6% ¹⁵N_exc. Results showed that N accumulations in stems, leaves and especially in ears were correlated with their dry matter (dm) weights. The application of 150 mg N kg^?1 sand significantly increased plant dm weight and total N accumulation in plants. During their filling period, ears absorbed N from both external (growth substrate) and internal (stored N in plants) sources. Nitrogen concentration in ears was higher in optimal N‐fed plants than in low N‐fed plants until 10 daa, but from 21 to 35 daa, differences were not detected. Conversely, ¹⁵N_exc in ears, leaves and stems was higher in low N‐fed plants than in optimal N‐fed plants. Ears acted as strong sink organ for the post‐anthesis N taken up from the soil independently of pre‐anthesis N nutrition: on average, 87% of the N taken up from the soil after anthesis was translocated and accumulated in ears. Low N‐fed plants continued to take up N from the post‐anthesis N fertiliser during the later grain‐filling period. The increase of pre‐anthesis N supply rate led to a decrease in the contribution of nitrogen derived from post‐anthesis ¹⁵N‐labelled fertiliser (N_dff) to total N in all aboveground organs, especially in ears where 44% and 22% of total N originated from post‐anthesis N uptake in low N‐fed and optimal N‐fed plants, respectively. The experiment with labelled amino acids showed that there was greater transport of proteinogenic amino acid into the ear (50% of total ¹⁵N) than non‐proteinogenic amino acid (39%). However, this transport of the non‐proteinogenic amino acids into ear suggested that the transport of N compounds from source (leaves) to sink organs (ear) might not be intrinsically regulated by their ability to be incorporated into storage protein of ears.     

Competition for tracer 15N in tussock tundra ecosystems

The objectives of this study were to assess the roles of plant species, time, and site on competition for tracer ¹⁵N (without carrier) in tussock tundra ecosystems. Six experimental sites were located in northern Alaska. After one year across the experimental sites, the recovery of ¹⁵N by litter (11.3–16.3%) and mosses (5.4–16.4%) was significantly greater than for aboveground vascular plants (2.6-5.0%). ¹⁵N recoveries by tundra vascular plants (2.6–5.0%) were low when compared to forest trees (9–25%) which suggest that competition for nitrogen is particularly severe in these cold-dominated tundra ecosystems. There were no significant differences among sites in ¹⁵N recoveries by vascular plants, by mosses, or by litter. There was a statistically significant decline in ¹⁵N recovery with time for Vaccinium vitis-idaea and Eriophorum vaginatum between the second and third year. The shallow rooted Vaccinium vitis-ideae was more highly labeled than the deep rooted Eriophorum vaginatum . Nearness to the source of the applied ¹⁵N played a critical role in competition for surface applied nitrogen.     

Improvement of the 15N dilution method for estimation of absorption of NOx by plants supplied with 15N-labelled fertilizer

To develop further the methods for estimation of NOx absorption by plants supplied with ¹⁵N-labelled fertilizer, we proposed a new calculation method, total N fixed method (TNF), and compared with the ¹⁵N dilution method and the classical mass balance method (MB).
Hydroponically grown soybean plants were supplied with ¹⁵N-labelled nitrate and exposed to 200–250 nl l⁻¹ NO₂ for 7 d. The proportions of the N derived from NO₂ to total N in exposed plants were estimated by the three methods.
The reported rates of NO₂ absorption by several plant species, estimated by the ¹⁵N dilution method, were recalculated using the TNF method. The results of the two methods were compared and showed that: (1) The ¹⁵N dilution method overestimated the content of NO₂-N in exposed plants compared with the MB method whilst the TNF method produced estimations of NO₂-N closer to those by the MB method when the plants were supplied with 5 m M nitrate. (2) The differences in estimations between the MB method and either the ¹⁵N dilution method or the TNF method increased with decreasing supply of ¹⁵N-labelled nitrate to roots.     

Acquisition and within-plant allocation of 13C and 15N in CO2-enriched Quercus robur plants

We assessed the effects of doubling atmospheric CO₂ concentration, [CO₂], on C and N allocation within pedunculate oak plants (Quercus robur L.) grown in containers under optimal water supply. A short-term dual ¹³CO₂ and ¹⁵NO₃^? labelling experiment was carried out when the plants had formed their third growing flush. The 22-week exposure to 700 μl l^?1 [CO₂] stimulated plant growth and biomass accumulation (+53% as compared with the 350 μl l^?1 [CO₂] treatment) but decreased the root/shoot biomass ratio (-23%) and specific leaf area (-18%). Moreover, there was an increase in net CO₂ assimilation rate (+37% on a leaf dry weight basis; +71% on a leaf area basis), and a decrease in both above- and below-ground CO₂ respiration rates (-32 and -26%, respectively, on a dry mass basis) under elevated [CO₂]. ¹³C acquisition, expressed on a plant mass basis or on a plant leaf area basis, was also markedly stimulated under elevated [CO₂] both after the 12-h ¹³CO₂ pulse phase and after the 60-h chase phase. Plant N content was increased under elevated CO₂ (+36%), but not enough to compensate for the increase in plant C content (+53%). Thus, the plant C/N ratio was increased (+13%) and plant N concentration was decreased (-11%). There was no effect of elevated [CO₂] on fine root-specific ¹⁵N uptake (amount of recently assimilated ¹⁵N per unit fine root dry mass), suggesting that modifications of plant N pools were merely linked to root size and not to root function. N concentration was decreased in the leaves of the first and second growing flushes and in the coarse roots, whereas it was unaffected by [CO₂] in the stem and in the actively growing organs (fine roots and leaves of the third growth flush). Furthermore, leaf N content per unit area was unaffected by [CO₂]. These results are consistent with the short-term optimization of N distribution within the plants with respect to growth and photosynthesis. Such an optimization might be achieved at the expense of the N pools in storage compartments (coarse roots, leaves of the first and second growth flushes). After the 60-h ¹³C chase phase, leaves of the first and second growth flushes were almost completely depleted in recent ¹³C under ambient [CO₂], whereas these leaves retained important amounts of recently assimilated ¹³C (carbohydrate reserves?) under elevated [CO₂].     

Nitrate fluxes in soybean seedling roots and their response to amino acids: an approach using 15N

This study was undertaken to determine which of the two NO₃^? fluxes (influx or efflux) across plasma membranes of root cells is the target of those amino acids which have been shown to inhibit net NO₃^? uptake (Muller & Touraine 1992, Journal of Experimental Botany 43 , 617–623). Parallel experiments were performed to mea-sure either the time course of ¹⁵NO₃^? release from roots of soybean seedlings previously labelled with this isotope into non-labelled solution, or the time course of ¹⁵N accumulation from labelled ¹⁵NO₃^? solution in non-labelled seedlings. Focusing on the fate of ¹⁵NO₃^? in the cytoplasmic compartment, a model is developed to describe the time courses of the accumulation and release of tracer across the plasma membranes of root cells. Both time courses can be described by the sum of an exponential plus a linear term. In our material, the linear part of the accumulation time course is obscured by the NO₃^? fluxes exiting the cytoplasm, and the curve thus appears to be quasilinear over several minutes. However, we show that the use of the net tracer accumulation rate during this time period as an estimate of NO₃^? influx does not provide accurate estimates of influx and efflux. By contrast, ¹⁵NO₃^? efflux analysis permits calculation of the unidirectional fluxes across plasma membranes of root cells and the kinetic parameters of the cytoplasmic NO₃^? pool. Under our experimental conditions, efflux accounted for 30 to 50% of influx, and the cytoplasmic NO₃^? content was found to be in the 70–400nmol g^?1 fw range. Using this methodology, the effect of amino acid accumulation on unidirectional fluxes of nitrate was then examined. Pretreatments of the seedlings with an amino acid which has been shown to inhibit net NO₃^? uptake led to concomitant decreases in net accumulation rates of ¹⁵NO₃^? and of reduced ¹⁵N in roots and total ¹⁵N in cotyledons. NO₃^? influx was markedly inhibited by these treatments, while NO₃^? efflux remained essentially unaffected, or even decreased. It is concluded that the target of the regulation of NO₃^? uptake by phloemtranslocated amino acids is the influx system.     

Prey to predator transfer of enriched 15N-contents: basic laboratory data for predation studies using 15N as marker

As an alternative to methods currently used to study predation under field conditions, we propose to mark prey with ¹⁵N, and to subsequently trace this label in the food chain. Preliminary laboratory work to develop this method is presented. The ¹⁵N‐content of polyphagous predators that have ingested ¹⁵N‐marked aphids was analysed with respect to: time after ingestion, the number of ingested ¹⁵N‐aphids, ingestion of additional non‐marked prey, and predator size. Increased ¹⁵N‐contents were detected in solid feeders [Platynus dorsalis (Pontopiddan), Coleoptera: Carabidae], as well as in fluid feeders [Erigone atra (Blackwall), Araneae: Linyphiidae] up to 11 days after ingestion. The increased ¹⁵N‐levels were constant over time from a few days after ¹⁵N‐ingestion onwards, and correlated with the number of ingested ¹⁵N‐aphids. The ingestion of additional non‐marked prey had no statistically significant influence on the predators’¹⁵N‐contents. The ¹⁵N‐contents of carabid species with varying biomasses could be compared directly. Our results are compared with literature data of other methods (e.g., ELISA).     

13C and 15N in microarthropods reveal little response of Douglas-fir ecosystems to climate change

Understanding ecosystem carbon (C) and nitrogen (N) cycling under global change requires experiments maintaining natural interactions among soil structure, soil communities, nutrient availability, and plant growth. In model Douglas-fir ecosystems maintained for five growing seasons, elevated temperature and carbon dioxide (CO₂) increased photosynthesis and increased C storage belowground but not aboveground. We hypothesized that interactions between N cycling and C fluxes through two main groups of microbes, mycorrhizal fungi (symbiotic with plants) and saprotrophic fungi (free-living), mediated ecosystem C storage. To quantify proportions of mycorrhizal and saprotrophic fungi, we measured stable isotopes in fungivorous microarthropods that efficiently censused the fungal community. Fungivorous microarthropods consumed on average 35% mycorrhizal fungi and 65% saprotrophic fungi. Elevated temperature decreased C flux through mycorrhizal fungi by 7%, whereas elevated CO₂ increased it by 4%. The dietary proportion of mycorrhizal fungi correlated across treatments with total plant biomass (n= 4, r²= 0.96, P= 0.021), but not with root biomass. This suggests that belowground allocation increased with increasing plant biomass, but that mycorrhizal fungi were stronger sinks for recent photosynthate than roots. Low N content of needles (0.8–1.1%) and A horizon soil (0.11%) coupled with high C : N ratios of A horizon soil (25–26) and litter (36–48) indicated severe N limitation. Elevated temperature treatments increased the saprotrophic decomposition of litter and lowered litter C : N ratios. Because of low N availability of this litter, its decomposition presumably increased N immobilization belowground, thereby restricting soil N availability for both mycorrhizal fungi and plant growth. Although increased photosynthesis with elevated CO₂ increased allocation of C to ectomycorrhizal fungi, it did not benefit plant N status. Most N for plants and soil storage was derived from litter decomposition. N sequestration by mycorrhizal fungi and limited N release during litter decomposition by saprotrophic fungi restricted N supply to plants, thereby constraining plant growth response to the different treatments.     

Metabolism of nitrate and ammonium in seedlings of Norway spruce (Picea abies) measured by in vivo 14N and 15N NMR spectroscopy

In vivo ¹⁵N and ¹⁴N nuclear magnetic resonance spectroscopy was used to investigate the assimilation of nitrate and ammonium in seedlings of Norway spruce (Picea abies [L.] Karst.). The main objective was to study accumulation of free NH+₄ and examine to what extent the nitrogen source affects the composition of the free amino acid pools in roots, stems and needles. NH+₄ concentrations in plants growing in the presence of 0.5–50 mM ammonium were quantified using ¹⁴N NMR. The NH+₄ values in tissues ranged from 6 to 46 μmol (g fresh weight)^?1. with highest concentrations in roots and needles. The tissue NH+₄ peaked at 5.0 mM NH+₄ in the medium. and failed to increase when NH+₄ in the medium was increased to 50 mM, indicating metabolic control of the concentration of this cation in tissues. The ¹⁴N NMR spectra were used to estimate pH of the NH+₄ storage pools. Based on the pH sensitivity of the quintet of ¹⁴NH+₄ resonance, we suggest that the pH of the ammonium storage compartments in the roots and stems should be 3.7–3.8, and in needles 3.4–3.5, representing extremely low pH values of the tissue. ¹⁵N from nitrate or ammonium was first incorporated into the amide group of glutamine and then into α-amino groups, confirming that the glutamine synthetase/ glutamate synthase cycle is the major route of nitrogen assimilation into amino acids and thus plays a role in lowering the levels of NH+₄ in the cytoplasm. NH+₄ can also be assimilated in roots in plants growing in darkness. The main ¹⁵N-labelled amino acids were glutamine. arginine and alanine. Almost no ¹⁵N signals from needles were observed. Double labelling (δN + w, wN) of arginine is consistent with the operation of the ornithine cycle, and enrichment indicates that this cycle is a major sink of newly assimilated nitrogen. Nitrogen assimilation in roots in the presence of added methionine sulphoximine and glutamate indicated the catabolic action of glutamate dehydrogenase. The ¹⁵N NMR spectra of plants grown on ¹⁵N-urea showed a marked increase in the labelling of ammonium and glutamine. indicating high urease activity. Amino acids were also quantified using high pressure liquid chromatography. Arginine was found to be an important transport form of nitrogen in the stem.     

15N stable isotope labelling of slugs (Gastropoda: Pulmonata)

Stable isotope tracers are a promising tool for investigating the ecology of terrestrial slugs, including predator‐prey relationships, migration behaviour, nutrient turnover and dietary routing. The objective of the present feasibility study was to label two economically important slug groups, Deroceras reticulatum and keeled slugs (families Limacidae and Milacidae, respectively), with the stable isotope ¹⁵N under controlled laboratory conditions. Significant isotopic enrichment in slug tissue was detected after 4 days and persisted for at least 10 days after slugs had been fed on ¹⁵N enriched food for a period of 15 days. The time course of ¹⁵N uptake into slug tissues and its relation to food consumption were well described mathematically. Estimated mean ¹⁵N assimilation efficiencies from labelled maize mixed with unlabelled wheat bran were 30% and 38%, respectively, for the species groups studied. These findings suggest that slugs can be readily and efficiently labelled and that it is feasible to devise protocols for producing large numbers of isotopically labelled slugs for use in ecological studies. A simple method is described for the collection and analysis of cutaneous mucus from individual slugs which can be used to test uniformity of isotopic labelling.