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.     

Nitrogen assimilation in Lolium perenne colonized by the arbuscular mycorrhizal fungus Glomus fasciculatum

To investigate nitrogen assimilation in Lolium perenne L. colonized by the arbuscular mycorrhizal (AM) fungus Glomus fasciculatum (Thax. sensu Gerd.), nitrate uptake, key enzyme activities, and ¹⁵N incorporation into free amino acids were measured. After a 4-h labelling period with [¹⁵N]nitrate, ¹⁵N content was higher in roots and shoots of AM-plants than in those of control plants. Glutamine synthetase (GS) and nitrate reductase (NR) activities were increased in shoots of AM-plants, but not in roots. More label was incorporated into amino acids in shoots of AM plants. Glutamine, glutamate, alanine and γ-aminobutyric acid were the major sinks for ¹⁵N in roots and shoots of control and AM plants. Interactions between mycorrhizal colonization, phosphate and nitrate nutrition and NR activity were investigated in plants which received different amounts of phosphate or nitrate. In shoots of control plants, NR activity was not stimulated by high levels of phosphate nutrition but was stimulated by high levels of nitrate. At 4 m M nitrate in the nutrient solution, NR activity was similar in control and AM plants. We concluded that mycorrhizal effects on nitrate assimilation are not mediated via improved phosphate nutrition, but could be due to improved nitrogen uptake and translocation.     

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.     

Traffic exposure increases natural 15N and heavy metal concentrations in mosses

Mosses have been used as biomonitors of atmospheric pollution for some years, but few studies have been carried out on the effect of NO_x emissions from traffic on moss tissue N. Eight species of moss (102 samples) growing on walls or roofs next to roads exposed to different traffic densities were collected from urban and rural sites in the UK. The shoots were sampled for total N, their stable isotope ¹⁵N/¹⁴N content (δ¹⁵N) and heavy metal content (Pb, Zn). There was a lack of correlation between tissue total N and traffic exposure, but a very good correlation between traffic exposure and tissue δ¹⁵N. Plants collected near motorways or busy urban roads had δ¹⁵N values ranging between +6 and −1‰, while in rural areas with hardly any traffic these ranged from −2 to −12‰. In a separate survey of mosses, the average δ¹⁵N of shoots from busy roadsides in London was +3.66‰, whereas from samples collected from farm buildings near poultry or cattle pens it was −7.8‰. This indicates that the two main atmospheric N sources, NO_x and NH_x, have different δ¹⁵N signatures, the former tending to be positive and the latter negative. Tissue concentrations of both Pb and Zn show a strong positive correlation with traffic exposure, with Zn in particular being greater than Pb. The results are discussed with regard to the use of moss tissue Zn as a means for monitoring or mapping pollution from vehicles, and of δ¹⁵N as an aid to distinguish between urban (NO_x) and rural (NH_x) forms of N pollution.     

Transfer of nitrogen from a tropical legume tree to an associated fodder grass via root exudation and common mycelial networks

Symbiotic dinitrogen fixation by legume trees represents a substantial N input in agroforestry systems, which may benefit the associated crops. Applying ¹⁵N labelling, we studied N transfer via common mycelial networks (CMN) and root exudation from the legume tree Gliricidia sepium to the associated fodder grass Dichantium aristatum . The plants were grown in greenhouse in shared pots in full interaction (treatment FI) or with their root systems separated with a fine mesh that allowed N transfer via CMN only (treatment MY). Tree root exudation was measured separately with hydroponics. Nitrogen transfer estimates were based on the isotopic signature of N ( δ ¹⁵N) transferred from the donor. We obtained a range for estimates by calculating transfer with δ ¹⁵N of tree roots and exudates. Nitrogen transfer was 3.7–14.0 and 0.7–2.5% of grass total N in treatments FI and MY, respectively. Root δ ¹⁵N gave the lower and exudate δ ¹⁵N the higher estimates. Transfer in FI probably occurred mainly via root exudation. Transfer in MY correlated negatively with grass root N concentration, implying that it was driven by source-sink relationships between the plants. The range of transfer estimates, depending on source δ ¹⁵N applied, indicates the need of understanding the transfer mechanisms as a basis for reliable estimates.     

Translocation of amino acids from stem nodules of Sesbania rostrata demonstrated by GC-MS in planta 15N isotope dilution

We report a novel use of the ¹⁵N dilution technique to detail the translocation of amino compounds in the legume Sesbania rostrata . The conventional ¹⁵N dilution technique follows the dilution of ¹⁵N within a labelled plant, as ¹⁴N₂ is fixed by symbiotic bacteria. In our experiments, stem-nodulated Sesbania rostrata were enriched by feeding with ¹⁵N ammonium nitrate for 2 weeks, followed by a 1 week period where the only N available to the plants was via nitrogen fixation of atmospheric N₂. We measured the composition, concentration and ¹⁵N enrichment of amino compounds in various plant tissues, both above and below the stem nodules, using GC-MS and isotopic abundance mass spectrometry techniques. Approximately 28% of the total N in the stem nodules was derived from internal plant sources. The ureides allantoic acid and allantoin were not abundant in xylem, leaf or nodule tissues. The amides asparagine and glutamine were the major export products from stem nodules although a wide range of other amino compounds are also synthesized. Amino acids within the nodules had a low level of enrichment, demonstrating that a small fraction (≈ 11%) was derived from outside the nodules, and significant cycling of N (28% of xylem N) through the root system was revealed by measurements of ¹⁵N distribution and amino acid concentrations.     

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.     

Variations in the amino acid composition of xylem sap of Betula pendula Roth. trees due to remobilization of stored N in the spring

Seasonal patterns of N translocation in the xylem sap of Betula pendula were studied, to determine whether specific amino acids were recovered in spring as a consequence of N remobilization. Seedlings were grown in sand culture and provided with ¹⁵NH₄¹⁵NO₃ (at 2·2 atom percent excess) for one growing season. The following winter dormant trees were transplanted into fresh sand and given N at natural abundance thereafter. Destructive harvests were taken during bud burst and leaf growth to determine the pattern of ¹⁵N remobilization and N uptake, along with isolation of xylem sap for analysis of their amino acid profiles and ¹⁵N enrichment by GC-MS. ¹⁵N remobilization occurred immediately following bud burst, while N derived from root uptake did not appear in the leaves until 12 d after bud burst. During N remobilization there was a 10-fold increase in the concentration of N in the xylem sap, due predominantly to increases in citrulline and glutamine. The ¹⁵N enrichment of these two amino acids demonstrated the increase in their concentration in the xylem sap following bud burst was due to N remobilization. These results are discussed in relation to measuring N remobilization and storage capacity of trees in the field.     

15N natural abundance during early and late succession in a middle-European dry acidic grassland

δ¹⁵N and total nitrogen content of above- and belowground tissues of 13 plant species from two successional stages (open pioneer community and ruderal grass stage) of a dry acidic grassland in Southern Germany were analysed, in order to evaluate whether resource use partitioning by niche separation and N input by N₂-fixing legumes are potential determinants for species coexistence and successional changes. Within each stage, plants from plots with different legume cover were compared. Soil inorganic N content, total plant biomass and δ¹⁵N values of bulk plant material were significantly lower in the pioneer stage than in the ruderal grass community. The observed δ¹⁵N differences were rather species- than site-specific. Within both stages, there were also species-specific differences in isotopic composition between above- and belowground plant dry matter. Species-specific δ¹⁵N signatures may theoretically be explained by (i) isotopic fractionation during microbial-mediated soil N transformations; (ii) isotopic fractionation during plant N uptake or fractionation during plant–mycorrhiza transfer processes; (iii) differences in metabolic pathways and isotopic fractionation within the plant; or (iv) partitioning of available N resources (or pools) among plant groups or differential use of the same resources by different species, which seems to be the most probable route in the present case. A significant influence of N₂-fixing legumes on the N balance of the surrounding plant community was not detectable. This was confirmed by the results of an independent in situ removal experiment, showing that after 3 years there were no measurable differences in the frequency distribution between plots with and without N₂-fixing legumes.     

Simulation of nitrogen assimilation by heterotrophic soil microbial biomass

Assimilation of N by heterotrophic soil microbial biomass is associated with decomposition of organic matter in the soil. The form of N assimilated can be either low molecular weight organic N released from the breakdown of organic matter (direct assimilation), or NH⁺₄ and NO⁻₃ from the soil inorganic N pool, into which mineralized organic N is released (mineralization immobilization turnover). The kinetics of C and N turnover in soil is quantifiable by means of computer simulation models. NCSOIL was constructed to represent the two assimilation schemes. The rate of N assimilation depends on the rate of C assimilation and microbial C/N ratio, thereby rendering it independent of the assimilation scheme. However, if any of the N forms is labeled, a different amount of labeled N assimilation will be simulated by the different schemes. Experimental data on inorganic N and ¹⁵N and on organic ¹⁵N dynamics in soils incubated with ¹⁵N added as NH⁺₄ or organic N were compared with data simulated by different model schemes. Direct assimilation could not account for the amount of ¹⁵N assimilated in any of the experimental treatments. The best fit of the model to experimental data was obtained for the mineralization immobilization turnover scheme when both NH⁺₄ and NO⁻₃ were assimilated, in proportion to their concentration in the soil.     

15N natural abundance in fruit bodies of different functional groups of fungi in relation to substrate utilization

Natural abundances of ¹⁵N and N concentrations of 34 fruit bodies from 24 species of ectomycorrhizal and saprophytic fungi were measured in a temperate Central European mixed forest stand. The fungi of the two life forms are known to be capable of utilizing different types of N sources (organic N compounds from the humus, inorganic N from the soil and N from litter or wood) differing by their ¹⁵N natural abundance values. Based on the two life forms and the three different N sources, four functional groups of fungi were distinguished: (1) ectomycorrhizal fungi capable of utilizing organic N from the humus; (2) ectomycorrhizal fungi known to depend on inorganic N compounds in the soil; (3) saprophytes capable of utilizing organic N from the humus; and (4) saprophytes utilizing N from dead wood or litter. Large differences were found between species in the δ¹⁵N values (−3.0 to 3.3‰) and in the N concentrations (0.84 to 6.61 mmol eq N g dw⁻¹) of the fruit bodies. In most cases fungi were more enriched in ¹⁵N than their respective bulk N source was. Fungi living in humus, and presumably having access to organic N compounds (groups 1 and 3), were significantly more enriched in ¹⁵N than fungi which are known to depend on inorganic N (e.g. Laccaria , group 2), or fungi living on litter or wood (group 4), irrespective of whether they were ectomycorrhizal or saprophytic species. Fungi living in humus had significantly higher N concentrations than fungi living on litter or wood.     

Use of different plant parts to study N2 fixation with 15N techniques in field-grown red clover (Trifolium pratense)

Red clover, Trifolium pratense L., is the dominant forage legume in Sweden and is usually harvested twice per year, once in June and once in August. Two ¹⁵N-based methods –¹⁵N isotopic dilution (ID) and ¹⁵N natural abundance (NA) – were used to study N₂ fixation from spring until first harvest in late June, from first to second harvest in late August, and from second harvest until first frost in autumn in Umeå, Sweden. The material studied comprized three neighbouring fields carrying a first year ley, a second year ley and a third year ley. For the ¹⁵N ID method, small amounts of highly enriched ¹⁵N-nitrate were added to experimental plots. The non-legumes in the plots, essentially Phleum pratense L. together with Festuca pratensis L., served as reference plants for both the ID and ¹⁵N NA measurements. Dry matter, N and ¹⁵N were separately analysed in leaves (laminae), stems (including petioles), stubble and roots. The proportion of N derived from air (pNdfa) was then calculated for each plant part and for whole plants. Estimates of the proportion of N derived from N₂ fixation (pNdfa) were always very high, usually ≥0.8. Generally, estimates of pNdfa obtained by the ID and NA methods were similar, but the ID method gave higher estimates of pNdfa than the NA method when the highest N₂ fixation levels were recorded, at the August harvest. Regression analyses suggest that estimates of pNdfa in leaves could provide useful indications of pNdfa in shoots and whole T. pratense plants, thus avoiding the need for time-consuming root analyses.     

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.     

Effects of mycorrhizal colonization on biomass production and nitrogen fixation of black locust (Robinia pseudoacacia) seedlings grown under elevated atmospheric carbon dioxide

Interactive effects of elevated atmospheric CO₂ and arbuscular mycorrhizal (AM) fungi on biomass production and N₂ fixation were investigated using black locust ( Robinia pseudoacacia ). Seedlings were grown in growth chambers maintained at either 350 μmol mol⁻¹ or 710 μmol mol⁻¹ CO₂. Seedlings were inoculated with Rhizobium spp. and were grown with or without AM fungi. The ¹⁵N isotope dilution method was used to determine N source partitioning between N₂ fixation and inorganic fertilizer uptake. Elevated atmospheric CO₂ significantly increased the percentage of fine roots that were colonized by AM fungi. Mycorrhizal seedlings grown under elevated CO₂ had the greatest overall plant biomass production, nodulation, N and P content, and root N absorption. Additionally, elevated CO₂ levels enhanced nodule and root mass production, as well as N₂ fixation rates, of non- mycorrhizal seedlings. However, the relative response of biomass production to CO₂ enrichment was greater in non-mycorrhizal seedlings than in mycorrhizal seedlings. This study provides strong evidence that arbuscular mycorrhizal fungi play an important role in the extent to which plant nutrition of symbiotic N₂-fixing tree species is affected by enriched atmospheric CO₂.     

15N enrichment as an integrator of the effects of C and N on microbial metabolism and ecosystem function

Organic carbon (C) and nitrogen (N) are essential for heterotrophic soil microorganisms, and their bioavailability strongly influences ecosystem C and N cycling. We show here that the natural ¹⁵N abundance of the soil microbial biomass is affected by both the availability of C and N and ecosystem N processing. Microbial ¹⁵N enrichment correlated negatively with the C : N ratio of the soil soluble fraction and positively with net N mineralization for ecosystems spanning semiarid, temperate and tropical climates, grassland and forests, and over four million years of ecosystem development. In addition, during soil incubation, large increases in microbial ¹⁵N enrichment corresponded to high net N mineralization rates. These results support the idea that the N isotope composition of an organism is determined by the balance between N assimilation and dissimilation. Thus, ¹⁵N enrichment of the soil microbial biomass integrates the effects of C and N availability on microbial metabolism and ecosystem processes.     

Sources of proline-nitrogen in water-stressed soybean (Glycine max). II. Fate of 15N-labelled protein

The absorption of nitrate, protein metabolism and the source of nitrogen for proline synthesis were studied in soybean ( Glycine max L. cv. Akisengoku) with ¹⁵N tracer technique under water stress conditions. The absorption of nitrate was sensitive to water stress and the flow of nitrate into the leaves completely ceased under severe stress conditions. Net protein loss from the water-stressed leaves was attributable to both a decrease in synthetic activity and a stimulation of protein degradation. Proline and asparagine accumulated extensively in the severely water-stressed plant tissues, especially in the younger green leaves. Fifty four % of the loss of leaf protein-¹⁵N during the stress period was balanced by a gain in ¹⁵N in the free amino acids, 41% being found in proline and asparagine. The increase in ¹⁵N content of the free proline was 3 times greater than the decrease in ¹⁵N content of the protein-bound proline in the leaf. The results indicate that the accumulation of proline in response to water stress was caused by enhanced synthesis and that the nitrogen source for this proline is the leaf protein. The possible association of these findings with stress tolerance is discussed.     

Elucidating the nutritional dynamics of fungi using stable isotopes

Mycorrhizal and saprotrophic (SAP) fungi are essential to terrestrial element cycling due to their uptake of mineral nutrients and decomposition of detritus. Linking these ecological roles to specific fungi is necessary to improve our understanding of global nutrient cycling, fungal ecophysiology, and forest ecology. Using discriminant analyses of nitrogen (δ¹⁵N) and carbon (δ¹³C) isotope values from 813 fungi across 23 sites, we verified collector-based categorizations as either ectomycorrhizal (ECM) or SAP in > 91% of the fungi, and provided probabilistic assignments for an additional 27 fungi of unknown ecological role. As sites ranged from boreal tundra to tropical rainforest, we were able to show that fungal δ¹³C (26 sites) and δ¹⁵N (32 sites) values could be predicted by climate or latitude as previously shown in plant and soil analyses. Fungal δ¹³C values are likely reflecting differences in C-source between ECM and SAP fungi, whereas ¹⁵N enrichment of ECM fungi relative to SAP fungi suggests that ECM fungi are consistently delivering ¹⁵N depleted N to host trees across a range of ecosystem types.     

Alteration of N nutrition in Myrica gale induces changes in nodule growth, nodule activity and amino acid composition

Changes in nodule growth and activity and in the concentrations of soluble N compounds in nodules, leaves and xylem sap under conditions of altered N nutrition in the actinorhizal plant Myrica gale L. are reported. Altering the N nutrition of symbiotic plants may alter the internal regulation of combined N which in turn may regulate nodule growth and activity. Flushing nodules daily with 100% O₂ caused a decline in amide concentration and an increase in nodule growth although plants had recovered some nitrogenase activity within 4 h of exposure to O₂. Samples of nodules, leaves and xylem sap were derivatized and amino acids identified and quantified using either reverse phase high performance liquid chromatography or gas chromatography-mass spectrometry in single ion monitoring mode. The ratio of asparagine in the nodules to that in the xylem was much higher in plants fed N (6.7 for NH⁺₄-fed and 8.3 for NO⁻₃-fed plants) than for N₂-fixing plants (2.5). Significant amounts of ¹⁵N added as ¹⁵NH⁺₄ or ¹⁵NO⁻₃ accumulated in nodules following accumulation in the shoot which is consistent with the translocation of N to the nodules via the phloem. The uptake of ¹⁵NH⁺₄ led to the synthesis and subsequent translocation of glutamine in the xylem sap. These results are discussed in terms of the feedback mechanisms that may regulate nitrogen fixation in Myrica root nodules.     

Denitrification in sediment determined from nitrogen isotope pairing

Abstract A new method for accurate and easy measurement of denitrification in sediments is presented. The water overlying intact sediment cores was enriched with ¹⁵NO₃⁻ which mixed with the ¹⁴NO₃⁻ of the natural sources of NO₃⁻. The formation by denitrification of single-labeled (¹⁴N¹⁵N) and double-labeled (¹⁵N¹⁵N) dinitrogen pairs was measured by mass spectrometry after a few hours incubation. Total denitrification including the formation of unlabeled (¹⁴N¹⁴N) dinitrogen could be calculated assuming random isotope pairing by denitrification of the uniformly mixed NO₃⁻ species. In contrast to previous approaches, by this method it is possible to measure denitrification of both NO₃⁻ diffusing from the overlying water and NO₃⁻ from nitrification within the sediment.