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.     

Soil 13C–15N dynamics in an N2-fixing clover system under long-term exposure to elevated atmospheric CO2

Reduced soil N availability under elevated CO₂ may limit the plant's capacity to increase photosynthesis and thus the potential for increased soil C input. Plant productivity and soil C input should be less constrained by available soil N in an N₂‐fixing system. We studied the effects of Trifolium repens (an N₂‐fixing legume) and Lolium perenne on soil N and C sequestration in response to 9 years of elevated CO₂ under FACE conditions. ¹⁵N‐labeled fertilizer was applied at a rate of 140 and 560 kg N ha^?1 yr^?1 and the CO₂ concentration was increased to 60 Pa pCO₂ using ¹³C‐depleted CO₂. The total soil C content was unaffected by elevated CO₂, species and rate of ¹⁵N fertilization. However, under elevated CO₂, the total amount of newly sequestered soil C was significantly higher under T. repens than under L. perenne. The fraction of fertilizer‐N (f_N) of the total soil N pool was significantly lower under T. repens than under L. perenne. The rate of N fertilization, but not elevated CO₂, had a significant effect on f_N values of the total soil N pool. The fractions of newly sequestered C (f_C) differed strongly among intra‐aggregate soil organic matter fractions, but were unaffected by plant species and the rate of N fertilization. Under elevated CO₂, the ratio of fertilizer‐N per unit of new C decreased under T. repens compared with L. perenne. The L. perenne system sequestered more ¹⁵N fertilizer than T. repens: 179 vs. 101 kg N ha^?1 for the low rate of N fertilization and 393 vs. 319 kg N ha^?1 for the high N‐fertilization rate. As the loss of fertilizer‐¹⁵N contributed to the ¹⁵N‐isotope dilution under T. repens, the input of fixed N into the soil could not be estimated. Although N₂ fixation was an important source of N in the T. repens system, there was no significant increase in total soil C compared with a non‐N₂‐fixing L. perenne system. This suggests that N₂ fixation and the availability of N are not the main factors controlling soil C sequestration in a T. repens system.     

15N natural abundance of vascular rainforest epiphytes: implications for nitrogen source and acquisition

The foliar natural abundance of ¹⁵N was analysed to compare the potential nitrogen sources of vascular rainforest epiphytes and associated soil-rooted trees. Leaves of epiphytes collected from six rainforest communities in Brazil, Australia and the Solomon Islands were depleted in ¹⁵N relative to the trees at each site. Epiphyte δ¹⁵N was as low as -6.4%_o, while trees were generally enriched in ¹⁵N (0.7 to 3.5%_o). These results indicate either that epiphytes use nitrogen sources depleted in ¹⁵N or that discrimination against ¹⁵N is an intrinsic function of epiphyte physiology. At three sites, epiphytes could be grouped into those having both low δ¹⁵N and low leaf-nitrogen content and those possessing both high δ¹⁵N and high leaf-nitrogen content. The second group had δ¹⁵N values in the range sometimes attributable to N₂ fixation (-2 to 0%o). There was no correlation between growth form and δ¹⁵N. It is concluded that epiphytes may utilize ¹⁵N-depleted nitrogen from atmospheric deposition and N₂ fixation.     

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.     

Australian mulga ecosystems –13C and 15N natural abundances of biota components and their ecophysiological significance

Samples of recently produced shoot material collected in winter/spring from common plant species of mulga vegetation in eastern and Western Australia were assayed for ¹³C and ¹⁵N natural abundance. ¹³C analyses showed only three of the 88 test species to exhibit C₄ metabolism and only one of seven succulent species to be in CAM mode. Non-succulent winter ephemeral C₃ species showed significantly lower mean δ¹³C values (– 28·0‰) than corresponding C₃-type herbaceous perennials, woody shrubs or trees (– 26·9, – 25·7 and – 26·2‰, respectively), suggesting lower water stress and poorer water use efficiency in carbon acquisition by the former than latter groups of taxa. Corresponding values for δ¹⁵N of the above growth and life forms lay within the range 7·5–15·5‰. δ¹⁵N of soil NH₄⁺ (mean 19·6‰) at a soft mulga site in Western Australia was considerably higher than that of NO₃^– (4·3‰). Shoot dry matter of Acacia spp. exhibited mean δ¹⁵N values (9·10 ± 0·6‰) identical to those of 37 companion non-N₂-fixing woody shrubs and trees (9·06 ± 0·5‰). These data, with no evidence of nodulation, suggested little or no input of fixed N₂ by the legumes in question. However, two acacias and two papilionoid legumes from a dune of wind-blown, heavily leached sand bordering a lake in mulga in Western Australia recorded δ¹⁵N values in the range 2·0–3·0‰ versus 6·4–10·7‰ for associated non-N₂-fixing taxa. These differences in δ¹⁵N, and prolific nodulation of the legumes, indicated symbiotic inputs of fixed N in this unusual situation. δ¹⁵N signals of lichens, termites, ants and grasshoppers from mulga of Western Australia provided evidence of N₂ fixation in certain termite colonies and by a cyanobacteria-containing species of lichen. Data are discussed in relation to earlier evidence of nitrophily and water availability constraints on nitrate utilization by mulga vegetation.     

The fate of 15N added to high Arctic tundra to mimic increased inputs of atmospheric nitrogen released from a melting snowpack

Increases in the long‐range aerial transport of reactive N species from low to high latitudes will lead to increased accumulation in the Arctic snowpack, followed by release during the early summer thaw. We followed the release of simulated snowpack N, and its subsequent fate over three growing seasons, on two contrasting high Arctic tundra types on Spitsbergen (79°N). Applications of ¹⁵N (99 atom%) at 0.1 and 0.5 g N m^?2 were made immediately after snowmelt in 2001 as either Na¹⁵NO₃ or ¹⁵NH₄Cl. These applications are approximately 1 × and 5 × the yearly atmospheric deposition rates. The vegetation at the principal experimental site was dominated by bryophytes and Salix polaris while at the second site, vegetation included bryophytes, graminoids and lichens. Audits of the applied ¹⁵N were undertaken, over two or three growing seasons, by determining the amounts of labeled N in the soil (0–3 and 3–10 cm), soil microbial biomass and different vegetation fractions. Initial partitioning of the ¹⁵N at the first sampling time showed that ～60% of the applied ¹⁵N was recovered in soil, litter and plants, regardless of N form or application rate, indicating that rapid immobilization into organic forms had occurred at both sites. Substantial incorporation of the ¹⁵N was found in the microbial biomass in the humus layer and in the bryophyte and lichen fractions. After initial partitioning there appeared to be little change in the total ¹⁵N recovered over the following two or three seasons in each of the sampled fractions, indicating highly conservative N retention. The most obvious transfer of ¹⁵N, following assimilation, was from the microbial biomass into stable forms of humus, with an apparent half‐life of just over 1 year. At the principal site the microbial biomass and vascular plants were found to immobilize the greatest proportion of ¹⁵N compared with their total N concentration. In the more diverse tundra of the second site, lichen species and graminoids competed effectively for ¹⁵NH₄‐N and ¹⁵NO₃‐N, respectively. Results suggest that Arctic tundra habitats have a considerable capacity to immobilize additional inorganic N released from the snow pack. However, with 40% of the applied ¹⁵N apparently lost there is potential for N enrichment in the surrounding fjordal systems during the spring thaw.     

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.     

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].     

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.     

Immobilization, stabilization and remobilization of nitrogen in forest soils at elevated CO2: a 15N and 13C tracer study

The fate of immobilized N in soils is one of the great uncertainties in predicting C sequestration at increased CO₂ and N deposition. In a dual isotope tracer experiment (¹³C, ¹⁵N) within a 4‐year CO₂ enrichment (+200 ppm_v) study with forest model ecosystems, we (i) quantified the effects of elevated CO₂ on the partitioning of N; (ii) traced immobilized N into physically separated pools of soil organic matter (SOM) with turnover rates known from their ¹³C signals; and (iii) estimated the remobilization and thus, the bio‐availability of newly sequestered C and N. (1) CO₂ enrichment significantly decreased NO₃^? concentrations in soil waters and export from 1.5 m deep lysimeters by 30–80%. Consequently, elevated CO₂ increased the overall retention of N in the model ecosystems. (2) About 60–80% of added ¹⁵NH₄¹⁵NO₃ were retained in soils. The clay fraction was the greatest sink for the immobilized ¹⁵N sequestering 50–60% of the total new soil N. SOM associated with clay contained only 25% of the total new soil C pool and had small C/N ratios (<13), indicating that it consists of humified organic matter with a relatively slow turn over rate. This implies that added ¹⁵N was mainly immobilized in stable mineral‐bound SOM pools. (3) Incubation of soils for 1 year showed that the remobilization of newly sequestered N was three to nine times smaller than that of newly sequestered C. Thus, inorganic inputs of N were stabilized more effectively in soils than C. Significantly less newly sequestered N was remobilized from soils previously exposed to elevated CO₂. In summary, our results show firstly that a large fraction of inorganic N inputs becomes effectively immobilized in relative stable SOM pools and secondly that elevated CO₂ can increase N retention in soils and hence it may tighten N cycling and diminish the risk of nitrate leaching to groundwater.     

Widespread foliage δ15N depletion under elevated CO2: inferences for the nitrogen cycle

Leaf ¹⁵N signature is a powerful tool that can provide an integrated assessment of the nitrogen (N) cycle and whether it is influenced by rising atmospheric CO₂ concentration. We tested the hypothesis that elevated CO₂ significantly changes foliage δ¹⁵N in a wide range of plant species and ecosystem types. This objective was achieved by determining the δ¹⁵N of foliage of 27 field‐grown plant species from six free‐air CO₂ enrichment (FACE) experiments representing desert, temperate forest, Mediterranean‐type, grassland prairie, and agricultural ecosystems. We found that within species, the δ¹⁵N of foliage produced under elevated CO₂ was significantly lower (P<0.038) compared with that of foliage grown under ambient conditions. Further analysis of foliage δ¹⁵N by life form and growth habit revealed that the CO₂ effect was consistent across all functional groups tested. The examination of two chaparral shrubs grown for 6 years under a wide range of CO₂ concentrations (25–75 Pa) also showed a significant and negative correlation between growth CO₂ and leaf δ¹⁵N. In a select number of species, we measured bulk soil δ¹⁵N at a depth of 10 cm, and found that the observed depletion of foliage δ¹⁵N in response to elevated CO₂ was unrelated to changes in the soil δ¹⁵N. While the data suggest a strong influence of elevated CO₂ on the N cycle in diverse ecosystems, the exact site(s) at which elevated CO₂ alters fractionating processes of the N cycle remains unclear. We cannot rule out the fact that the pattern of foliage δ¹⁵N responses to elevated CO₂ reported here resulted from a general drop in δ¹⁵N of the source N, caused by soil‐driven processes. There is a stronger possibility, however, that the general depletion of foliage δ¹⁵N under high CO₂ may have resulted from changes in the fractionating processes within the plant/mycorrhizal 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).     

Uptake of atmospheric 15NO2 and its incorporation into free amino acids in wheat (Triticum aestivum)

Five-week-old wheat plants were exposed, under controlled environmental conditions, to 60 nl 1^?115NO₂ or to purified air. After 48 and 96 h of exposure, leaves, stalks and roots were analysed for ¹⁵N-enrichment in α-amino nitrogen of soluble, free amino acids. In addition, the in vitro nitrate reductase (NR, EC 1.6.6.1) and nitrite reductase (NIR, EC 1.7.7.1) activities were determined in the leaves. NR activity in the leaves decreased continously during the 96-h exposure to purified air. In the leaves exposed to ¹⁵NO₂, NR activity increased within the first 24 h, then decreased, and reached the level of controls after 96 h. NiR activity in leaves exposed to purified air was almost constant during the 96-h exposure. In leaves exposed to ¹⁵NO₂, NiR activity increased within the first 48 h, then decreased, and reached the level of controls after 72 h, Exposure to ¹⁵NO₂ enhanced the total content of soluble, free amino acids in all tissues analysed. Most of this increase was attributed to Glu in the leaves and to Asn plus Gln the α-amino group of soluble, free amino acids was observed in the leaves, the lowest enrichment in the roots. The main labelled amino compounds were Glu (with 8.0%¹⁵N enrichment compared to the control), γ-aminobutyric acid (GABA; 7.9%), Ala (7.2%). Ser (6.8%), Asp (5.5%) and Gln (4.6%). Appreciable incorporation of ¹⁵ into Asn was not found. After 96 h exposure to ¹⁵NO₂ the ¹⁵N enrichment in the α-amino group of soluble, free amino acids in the leaves declined as compared to the values obtained after 48 h fumigation. The possible pathway and the time course of ¹⁵N incorporation into soluble, free amino acids from the ¹⁵NO₂ absorbed are discussed.     

Nitrogen concentration and δ15N signature of ombrotrophic Sphagnum mosses at different N deposition levels in Europe

Alteration of the global nitrogen (N) cycle because of human‐enhanced N fixation is a major concern particularly for those ecosystems that are nutrient poor by nature. Because Sphagnum‐dominated mires are exclusively fed by wet and dry atmospheric deposition, they are assumed to be very sensitive to increased atmospheric N input. We assessed the consequences of increased atmospheric N deposition on total N concentration, N retention ability, and δ¹⁵N isotopic signature of Sphagnum plants collected in 16 ombrotrophic mires across 11 European countries. The mires spanned a gradient of atmospheric N deposition from about 0.1 up to about 2 g m^?2 yr^?1. Mean N concentration in Sphagnum capitula was about 6 mg g^?1 in less polluted mires and about 13 mg g^?1 in highly N‐polluted mires. The relative difference in N concentration between capitulum and stem decreased with increasing atmospheric N deposition, suggesting a possible metabolic mechanism that reduces excessive N accumulation in the capitulum. Sphagnum plants showed lower rates of N absorption under increasing atmospheric N deposition, indicating N saturation in Sphagnum tissues. The latter probably is related to a shift from N‐limited conditions to limitation by other nutrients. The capacity of the Sphagnum layer to filter atmospheric N deposition decreased exponentially along the depositional gradient resulting in enrichment of the mire pore water with inorganic N forms (i.e., NO₃^?+NH₄⁺). Sphagnum plants had δ¹⁵N signatures ranging from about ?8‰ to about ?3‰. The isotopic signatures were rather related to the ratio of reduced to oxidized N forms in atmospheric deposition than to total amount of atmospheric N deposition, indicating that δ¹⁵N signature of Sphagnum plants can be used as an integrated measure of δ¹⁵N signature of atmospheric precipitation. Indeed, mires located in areas characterized by greater emissions of NH₃ (i.e., mainly affected by agricultural activities) had Sphagnum plants with a lower δ¹⁵N signature compared with mires located in areas dominated by NO_x emissions (i.e., mainly affected by industrial activities).     

15N natural abundance of plant and soil components of a Banksia woodland ecosystem in relation to nitrate utilization, life form, mycorrhizal status and N2-fixing abilities of component species

Studies of the variation in δ¹⁵N values for plants from a fire-prone Banksia woodland in South West Australia showed that pioneer herbaceous, non-mycorrhizal species which were active in nitrate reduction and storage, had the highest values (1.81%c). A detailed study of one such species Ptilotus polystachus demonstrated a close correspondence between the δ¹⁵N values of soil nitrate, xylem nitrate and leaf total nitrogen, suggesting an exclusive reliance on nitrate ions as nitrogen source. These pioneer species also showed a preponderance of the chloroplastic isoform of glutamine synthetase while woody species generally had higher activity associated with the cytosolic isoform. The group comprising monocotyledonous hemicryptophytes and geophytes contained species with slightly positive δ¹⁵N values and moderately active in nitrate reduction and storage. Nitrogen-fixing species had the lowest δ¹⁵N values (–0.36‰), irrespective of their apparent utilisation of nitrate. However, woody resprouter species which had low levels of nitrate reduction and storage had δ¹⁵N values which fell within the range of values obtained for the miscellaneous assemblage of N₂-fixing species. Consequently, ¹⁵N abundance values failed to distinguish N₂ fixing from non-fixing woody species, and therefore, could not be used in the ecosystem to determine the dependence of putative nitrogen fixing species on N₂ fixation. The study demonstrated complex patterns of nitrogen utilization in the ecosystem in which exploitation of different nitrogen resources related to plant life form and the physiological attributes of nitrogen assimilation by component species.     

Linking sequestration of 13C and 15N in aggregates in a pasture soil following 8 years of elevated atmospheric CO2

The influence of N availability on C sequestration under prolonged elevated CO₂ in terrestrial ecosystems remains unclear. We studied the relationships between C and N dynamics in a pasture seeded to Lolium perenne after 8 years of elevated atmospheric CO₂ concentration (FACE) conditions. Fertilizer‐¹⁵N was applied at a rate of 140 and 560 kg N ha^2?1 y^2?1 and depleted ¹³C‐CO₂ was used to increase the CO₂ concentration to 60 Pa pCO₂. The ¹³C–¹⁵N dual isotopic tracer enabled us to study the dynamics of newly sequestered C and N in the soil by aggregate size and fractions of particulate organic matter (POM), made up by intra‐aggregate POM (iPOM) and free light fraction (LF). Eight years of elevated CO₂ did not increase total C content in any of the aggregate classes or POM fractions at both rates of N application. The fraction of new C in the POM fractions also remained largely unaffected by N fertilization. Changes in the fractions of new C and new N (fertilizer‐N) under elevated CO₂ were more pronounced between POM classes than between aggregate size classes. Hence, changes in the dynamics of soil C and N cycling are easier to detect in the POM fractions than in the whole aggregates. Within N treatments, fractions of new C and N in POM classes were highly correlated with more new C and N in large POM fractions and less in the smaller POM fractions. Isotopic data show that the microaggregates were derived from the macro‐aggregates and that the C and N associated with the microaggregates turned over slower than the C and N associated with the macroaggregates. There was also isotopic evidence that N immobilized by soil microorganisms was an important source of N in the iPOM fractions. Under low N availability, 3.04 units of new C per unit of fertilizer N were sequestered in the POM fractions. Under high N availability, the ratio of new C sequestered per unit of fertilizer N was reduced to 1.47. Elevated and ambient CO₂ concentrations lead to similar ¹⁵N enrichments in the iPOM fractions under both low and high N additions, clearly showing that the SOM‐N dynamics were unaffected by prolonged elevated CO₂ concentrations.     

Effects of the ecto- and VA-mycorrhizal fungi Hydnagium carneum and Glomus clarum on the δ15N and δ13C values of Eucalyptus globulus and Ricinus communis

Glasshouse experiments with Ricinus communis showed that the presence/absence of a VA mycorrhizal fungus (Glomus clarum) changed the δ¹⁵N value of the host by as much as 2‰ when the plants were given urea (released as NH₄⁺) as their only N-source. This small change in Δ¹⁵N would create a large error in calculating sources of plant N. In particular, these results throw into doubt any models of N-cycling which assume that soil N can be treated as a single source. The correct N-source value for VAM-infected NH₄^? -using plants may be the δ¹⁵N of soil NH₄⁺+ 2‰. Treatment effects were also found in the distribution of δ¹⁵N and % N among plant organs. Plants with VAM had a lower N:P atom ratio and were larger in total biomass. Carbon discrimination (δ¹³C) was greater in the VA-infected plants. The measured effects of VAM infection suggest that for some plants the fungus may be the primary site of N assimilation. A parallel experiment with Eucalyptus globulus and the ectomycorrhizal fungus Hydnangium carneum resulted in no significant differences in any of the variables measured for this host-fungus pair when the sole N-sources were inorganic (NO₃^? and NH₄⁺ released from urea). Ectomycorrhizal fungi are diverse in their physiological behaviour, and these data should not be taken as being representative of the whole group. More work is required with other types of mycorrhiza and more complex sources of N. Future work will include a water balance to partition the effects of water use and nutrient supply in determining δ¹³C. An on-line combustion-ANCA-MS method is described for fully automated measurement of natural abundance levels of ^15/14N and ^13/12C for plant materials. This method achieves the required precision while dramatically increasing sample throughout.     

Characterizing nitrogen dynamics, retention and transport in a tropical rainforest stream using an in situ15N addition

1. This study was part of the Lotic Intersite Nitrogen eXperiment (LINX); a series of identical ¹⁵NH₄ tracer additions to streams throughout North America. ¹⁵NH₄Cl was added at tracer levels to a Puerto Rican stream for 42 days. Throughout the addition, and for several weeks afterwards, samples were collected to determine the uptake, retention and transformation pathways of nitrogen in the stream. 2. Ammonium uptake was very rapid. Nitrification was immediate, and was a very significant transformation pathway, accounting for over 50% of total NH₄ uptake. The large fraction of NH₄ uptake accounted for by nitrification (a process that provides energy to the microbes involved) suggests that energy limitation of net primary production, rather than N limitation, drives N dynamics in this stream. 3. There was a slightly increased ¹⁵N label in dissolved organic nitrogen (DON) the day after the ¹⁵NH₄ addition was stopped. This DO¹⁵N was < 0.02% of DON concentration in the stream water at the time, suggesting that nearly all of the DON found in‐stream is allochthonous, or that in‐stream DON production is very slow. 4. Leptophlebiidae and Atya appear to be selectively feeding or selectively assimilating a very highly labelled fraction of the epilithon, as the label found in the consumers became much higher than the label found in the food source. 5. A large spate (>20‐fold increase in discharge) surprisingly removed only 37% of in‐stream fine benthic organic matter (FBOM), leaves and epilithon. The fraction that was washed out travelled downstream a long distance (>220 m) or was washed onto the stream banks. 6. While uptake of ¹⁵NH₄ was very rapid, retention was low. Quebrada Bisley retained only 17.9% of the added ¹⁵N after 42 days of ¹⁵N addition. Most of this was in FBOM and epilithon. Turnover rates for these pools were about 3 weeks. The short turnover times of the primary retention pools suggest that long‐term retention (>1 month) is minimal, and is probably the result of N incorporation into shrimp biomass, which accounted for < 1% of the added ¹⁵N.     

Interactive effects of warming and increased nitrogen deposition on 15N tracer retention in a temperate old field: seasonal trends

The extent to which increased atmospheric nitrogen (N) deposition will drive changes in plant productivity and species composition over the next century will depend on how other influential global change factors, such as climate warming, affect the N retention of ecosystems. We examined the interactive effects of simulated climate warming and N deposition on the recoveries of ¹⁵N‐labeled ammonium and ¹⁵N‐labeled nitrate tracers added as a pulse to grass‐dominated, temperate old‐field plots at spring thaw. In addition to the year‐round warming treatment, a winter‐only warming treatment was applied to a set of plots to explore the contribution of this component of climate warming to the overall warming effect. By the end of the plant growing season, there was approximately twice as much ¹⁵N enrichment in the plant roots and bulk soil from ¹⁵NH₄⁺‐addition plots than from ¹⁵NO₃^?‐addition plots, but there were no effects of warming or N fertilization on ¹⁵N recovery. Over winter, approximately half of the excess ¹⁵N present in plant shoots was lost, which corresponded with large ¹⁵N losses from bulk soil in N fertilized plots and large ¹⁵N increases in bulk soil in nonfertilized plots. By the next spring, there was decreased ¹⁵N recovery in plants in response to N fertilization, which was largely offset by increases in plant ¹⁵N recovery in response to year‐round warming. However, ¹⁵N retention in bulk soil, where the major part of the ¹⁵N label was recovered, was approximately 40% higher in nonfertilized plots than in N fertilized plots. Overall, our results indicate that climate warming increases plant N sequestration in this system but this effect is overwhelmed by the overall effect of nitrogen deposition on ecosystem N losses.     

Natural abundance of 13C and 15N in C3 and C4 vegetation of southern Africa: patterns and implications

The mean annual rainfall in southern Africa is found to explain over half of the observed variance in the stable nitrogen (N) isotopic signatures of C₃ vegetation in southern Africa (r²=0.54, P<0.01). The inverse relationship between the stable N isotopic signatures of foliar samples from C₃ vegetation and long‐term southern African rainfall is found on a scale larger than previously observed. A modest relationship is found between stable carbon (C) isotopic signatures of C₃ vegetation and rainfall across the region (r²=0.20, P<0.01). No such relationship is found between stable C and N isotopic signatures of C₄ vegetation and rainfall. The explanation of the relationship between ¹⁵N in C₃ vegetation and the mean annual rainfall presented here is that nutrient availability varies inversely with water availability. This suggests that water‐limited systems in southern Africa are more open in terms of nutrient cycling and therefore the resulting natural abundance of foliar ¹⁵N in these systems is enriched. The use of this relationship may be of value to those researchers modeling both the dynamics of vegetation and biogeochemistry across this region. The use of the isotopic enrichment in C₃ vegetation as a function of rainfall may provide an insight into nutrient cycling across the semi‐arid and arid regions of southern Africa. This finding has implications for the study of global change, especially as it relates to vegetation responses to changing regional rainfall regimes over time.