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.     

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.     

Tansley Review No. 95: 15N natural abundance in soil–plant systems

The following citations were erroneously omitted from the 'References':
Groffman PM, Zak DR, Christensen S, Mosier A, Tiedje JM. 1993. Early spring nitrogen dynamics in a temperate forest landscape. Ecology 74 : 1579–1585.
Handley LL, Brendel O, Scrimgeour CM, Schmidt S, Raven JA, Turnbull MH, Stewart GR. 1996. The ¹⁵N natural abundance patterns of field-collected fungi from three kinds of ecosystems. Rapid Communications in Mass Spectrometry 10 : 974–978.
Handley LL, Daft MJ, Wilson J, Scrimgeour CM, Ingleby K, Sattar, MA. 1993. 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. Plant, Cell and Environment 16 : 375–382.
Handley LL, Odee D, Scrimgeour CM. 1994. δ¹⁵N and δ¹³C patterns in savanna vegetation: dependence on water availability and disturbance. Functional Ecology 8 : 306–314.
Handley LL, Raven JH. 1992. The use of natural abundance of nitrogen isotopes in plant physiology and ecology: commissioned review. Plant, Cell and Environment 15 : 965–985.
Handley LL, Scrimgeour CM. 1997. Terrestrial plant ecology and ¹⁵N natural abundance: the present limits to interpretation for uncultivated systems with original data from a Scottish old field. Advances in Ecological Research 27 : 133–212.
Hansen AP, Pate JS. 1987. Evaluation of the ¹⁵N natural abundance method and xylem sap analysis for assessing N₂ fixation of understorey legumes in jarrah ( Eucalyptus marginata Donn ex Sm.) forest in S.W. Australia. Journal of Experimental Botany 38 : 1446–1458.
New Phytologist apologizes unreservedly to all authors of the above papers for this error.     

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.     

Symbiotic N2 fixation in a high Alpine grassland: effects of four growing seasons of elevated CO2

1. Increasing carbon dioxide concentration (E: 680 μl CO₂ litre^–1 vs ambient, A: 355 μl CO₂ litre^–1) around late-successional Alpine sedge communities of the Swiss Central Alps (2450 m) for four growing seasons (1992–1995) had no detectable effect on symbiotic N₂ fixation in Trifolium alpinum —the sole N₂-fixing plant species in these communities (74 ± 30 mg N m^–2 year^–1, A and E plots pooled).
2. This result is based on data collected in the fourth growing season showing that elevated CO₂ had no effect on Trifolium above-ground biomass (4·4 ± 1·7 g m^–2, A and E plots pooled, n = 24) or N content per unit land area (124 ± 51 mg N m^–2, A and E pooled), or on the percentage of N Trifolium derived from the atmosphere through symbiotic N₂ fixation (%Ndfa: 61·0 ± 4·1 across A and E plots) estimated using the ¹⁵N dilution method.
3. Thus, it appears that N inputs to this ecosystem via symbiotic N₂ fixation will not be dramatically affected in the foreseeable future even as atmospheric CO₂ continues to rise.     

Habitat use and trophic position determine mercury concentration in the straight fin barb Barbus paludinosus, a small fish species in Lake Awassa, Ethiopia

The diet, habitat use and mercury concentration of the small fish species, the straight fin barb Barbus paludinosus , were studied in Lake Awassa, Ethiopia, for a period of 1 year from February 2003 to January 2004. Stable isotope signatures of nitrogen and carbon in different total length ( L _T) classes were used to determine trophic positions and organic carbon sources, respectively. Barbus paludinosus mainly occupied the protected benthic habitats (littoral and profundal) of the lake. The δ¹³C values were in the range from −24 to −19‰, indicating that the carbon source for B. paludinosus was benthic, as well. Small individuals (≤ 60 mm L _T) mainly preyed upon ostracods, intermediate sizes (60–100 mm) on aquatic insects and gastropods, while a tiny cyprinodont fish Aplocheilichthys antinorii dominated the diet of large individuals (100–160 mm). The progressively increase in δ¹⁵N with increasing L _T also indicated a diet shift towards piscivory in larger individuals. The mercury concentration ranging from 0·02 to 0·74 mg kg⁻¹ wet mass (wm), was unexpectedly high in this small species, and was significantly positively related to L _T, as well as to δ¹⁵N. Some large individuals had mercury concentrations < 0·1 mg kg⁻¹ wm, and low δ¹⁵N, indicating substantial variations in diet between individuals of same size. The study suggests that other piscivorous species which include B. paludinosus in their diet may have a high mercury intake risk.     

Effects of temperature and fertilization on nitrogen cycling and community composition of an urban lawn

We examined the influence of temperature and management practices on the nitrogen (N) cycling of turfgrass, the largest irrigated crop in the United States. We measured nitrous oxide (N₂O) fluxes, and plant and soil N content and isotopic composition with a manipulative experiment of temperature and fertilizer application. Infrared lamps were used to increase surface temperature by 3.5±1.3 °C on average and control and heated plots were split into high and low fertilizer treatments. The N₂O fluxes increased following fertilizer application and were also directly related to soil moisture. There was a positive effect of warming on N₂O fluxes. Soils in the heated plots were enriched in nitrogen isotope ratio ( δ ¹⁵N) relative to control plots, consistent with greater gaseous losses of N. For all treatments, C₄ plant C/N ratio was negatively correlated with plant δ ¹⁵N, suggesting that low leaf N was associated with the use of isotopically depleted N sources such as mineralized organic matter. A significant and unexpected result was a large, rapid increase in the proportion of C₄ plants in the heated plots relative to control plots, as measured by the carbon isotope ratio ( δ ¹³C) of total harvested aboveground biomass. The C₄ plant biomass was dominated by crabgrass, a common weed in C₃ fescue lawns. Our results suggest that an increase in temperature caused by climate change as well as the urban heat island effect may result in increases in N₂O emissions from fertilized urban lawns. In addition, warming may exacerbate weed invasions, which may require more intensive management, e.g. herbicide application, to manage species composition.     

Plant growth-altering effects of Azospirillum brasilense and Bacillus C–11–25 on two wheat cultivars

The effect of Azospirillum brasilense Cd, Bacillus C–11–25, indole acetic acid, gibberellic acid and cytokinin on plant growth characteristics of two wheat ( Triticum aestivum L. emend Thell) cultivars was studied under laboratory and greenhouse conditions. Responses of wheat plants to bacterial inoculation were similar to those caused by the addition of gibberellic acid in growth pouches. Chester and Fielder wheat varieties differed in responses to the bacteria and hormone additions. When added to growth pouches, bacterial culture filtrates and dead bacterial cells caused plant growth responses similar to those caused by the addition of live cells. Bacteria and hormone additions resulted in increased permeability of Fielder wheat to ¹⁵Nlabelled nitrate, and decreased nitrate permeability of Chester wheat. Bacterial inoculation of soil in pots caused ¹⁵N isotope dilution in Fielder but not in Chester wheat. Hormone addition to pots caused isotope dilution in Chester wheat. It appeared that genetic differences between cultivars affected plant growth responses. The accuracy of estimates of N₂ fixation by associative bacteria based on ¹⁵N isotope dilution calculations may be reduced if control plants differ in plant response to these bacteria.     

Nitrogen and carbon isotope ratios in termites: an indicator of trophic habit along the gradient from wood-feeding to soil-feeding

1. Nitrogen and carbon stable-isotope ratios (δ¹⁵N and δ¹³C) of body tissues, mound/nest materials and dietary substrates were determined in termite species with differing trophic habits, sampled from the Mbalmayo Forest Reserve, southern Cameroon.
2. δ¹⁵N of termite tissues was enriched gradually along a spectrum of species representing a trophic gradient from wood- to soil-feeding. Species that could be identified from their general biology and from gut content analysis as feeding on well-rotted wood or as wood/soil interface feeders showed δ¹⁵N intermediate between sound-wood-feeders and soil-feeders. It is proposed that δ¹⁵N is therefore a possible indicator of the functional position of species in the humification process. Differences in δ¹³C were also observed between wood-feeding and soil-feeding forms.
3. High values of δ¹⁵N in soil-feeding termites suggest that nitrogen fixation is of little importance in these species.
4. A wide range of isotope effects (the difference in isotope ratios between termites and their diet) was observed for both nitrogen (Δδ¹⁵N = –1.6 to + 8.8‰) and carbon (Δδ¹³C = –2.2 to + 3.0‰), which suggests a diversity of nutrient acquisition mechanisms within termites and diverse relationships between termites and their intestinal micro-organisms.     

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.     

Rate of Glutamate Synthesis from Leucine in Rat Brain Measured In Vivo by 15N NMR

Abstract: The rate of glutamate synthesis from leucine by the branched-chain aminotransferase was measured in rat brain in vivo at steady state. The rats were fed exclusively by intravenous infusion of a nutrient solution containing [¹⁵N]leucine. The rate of glutamate synthesis from leucine, determined from the rate of increase of brain [¹⁵N]glutamate measured by ¹⁵N NMR and the ¹⁵N enrichments of brain and blood leucine analyzed by gas chromatography-mass spectrometry, was 0.7–1.8 µmol/g/h at a steady-state brain leucine concentration of 0.25 µmol/g. A comparison of the observed fractional ¹⁵N enrichments of brain leucine (0.42 ± 0.03) and glutamate (0.21 ± 0.015) showed that leucine provides ∼50% of glutamate nitrogen under our experimental condition. From the observed rate (0.7–1.8 µmol/g) and the known K _m of the branched-chain aminotransferase for leucine (1.2 m M ), the rate of glutamate synthesis from leucine at physiological brain leucine concentration (0.11 µmol/g) was estimated to be 0.35–0.9 µmol/g/h, with leucine providing ∼25% of glutamate nitrogen. The results strongly suggest that plasma leucine from dietary source, transported into the brain, is an important external source of nitrogen for replenishment of brain glutamate in vivo. Implications of the results for treatment of maple-syrup urine disease patients with leucine-restricted diet are discussed.     

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.     

Elevated atmospheric CO2 does not affect per se the preference for symbiotic nitrogen as opposed to mineral nitrogen of Trifolium repens L.

The objective of this investigation was to examine the effect of an elevated atmospheric CO₂ partial pressure ( p CO₂) on the N-sink strength and performance of symbiotic N₂ fixation in Trifolium repens L. cv. Milkanova. After initial growth under ambient p CO₂ in a nitrogen-free nutrient solution, T. repens in the exponential growth stage was exposed to ambient and elevated p CO₂ (35 and 60 Pa) and two levels of mineral N (N-free and 7·5 mol m^–3 N) for 36 d in single pots filled with silica sand in growth chambers. Elevated p CO₂ evoked a significant increase in biomass production from day 12 after the start of CO₂ enrichment. For plants supplied with 7·5 mol m^–3 N, the relative contribution of symbiotically fixed N (%N_sym) as opposed to N assimilated from mineral sources (¹⁵N-isotope-dilution method), dropped to 40%. However, in the presence of this high level of mineral N, %N_sym was unaffected by atmospheric p CO₂ over the entire experimental period. In plants fully dependent on N₂ fixation, the increase in N yield reflects a stimulation of symbiotic N₂ fixation that was the result of the formation of more nodules rather than of higher specific N₂ fixation. These results are discussed with regard to physiological processes governing symbiotic N₂ fixation and to the response of symbiotic N₂ fixation to elevated p CO₂ in field-grown T. repens .     

Photosynthetic 14CO2 fixation and [15N]-ammonia assimilation during UV-B radiation of Lithodesmium variabile

Uptake of [¹⁵N]-ammonia was more sensitive to UV-B exposure than the total ¹⁴CO₂ fixation rate of Lithodesmium variabile Takano. Short-term UV-B radiation (15 min) had practically no effect on the kinetics of [¹⁵N]-ammonia, whereas there was an effect on [¹⁴C]-bicarbonate uptake rate. A significant reduction was found after 30 and 60 min UV-B stress. The time course of photosynthetic uptake of ¹⁵NH₄Cl at several wavelengths was markedly depressed at shorter wavelengths (irradiation with WG 280). A short-term (11 min) exposure to ultraviolet radiation had no influence on the [¹⁴C]-labeled photosynthetic products. However, the [¹⁵N]-label of several amino acids and the ratio of [¹⁵N]-glutamine to [¹⁵N]-glutamic acid varied after irradiation with different ultraviolet wavebands. The results are discussed with reference to UV damage to the key enzymes of nitrogen metabolism.     

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.     

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.     

Fixation of molecular nitrogen by Methanosarcina barkeri

Abstract Methanosarcina barkeri cells were observed in ammonia-free anaerobic acetate enrichments for sulfate-reducing bacteria. The capacity of Methanosarcina to grow diazotrophically was proved with a pure culture in mineral media with methanol. The cell yields with N₂ or NH₄⁺ ions as nitrogen source were 2.2 g and 6.1 g dry weight, respectively, per mol of methanol. Growth experiments with ¹⁵N₂ revealed that 84% of the cell nitrogen was derived from N₂. Acetylene was highly toxic to Methanosarcina and only reduced at concentrations lower than 100 μmol dissolved per 1 of medium. Assimilation of N₂ and reduction of acetylene were inhibited by NH₄⁺ ions. The experiments show that N₂ fixation occurs not only in eubacteria but also in archaebacteria. The ecological significance of diazotrophic growth of Methanosarcina is discussed.     

Stable isotopes of carbon and nitrogen as indicators of diet and trophic structure of the fish community in a shallow hypereutrophic lake

Stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotopes were employed to elucidate energy flows and trophic interactions in Lake Apopka, a hypereutrophic lake in central Florida, U.S.A. Isotope compositions of lake biota ranged from −27·1 to −3·0‰ for δ¹³C, and from 3·7 to 13·9‰ for δ¹⁵N. The food web was based primarily on plankton production with diatoms, Microcystis and zooplankton dominating the diet of fish. Carbon isotope evidence showed that pico- and nano-phytoplankton were not a direct carbon source for fish, but were important to zooplankton. δ¹⁵N mass balance estimates indicated that planktivorous fish obtained 48–85% of their diets from zooplankton. The ∼3‰ range of δ¹⁵N in gizzard shad reflected increasing dependence on zooplankton as fish grew whereas the positive relationship between total length and δ¹⁵N of largemouth bass reflected increasing predation on larger planktivorous fish with growth. The broad ranges of δ¹³C (−25·9 to −9·5‰) and δ¹⁵N (5·8 to 14·4‰) of blue tilapia were indicators of diet diversity. Two presumed omnivores (brown bullhead and white catfish) and piscivores (black crappie, largemouth bass and Florida gar) were found to depend on planktivorous fish. However, stable isotope data revealed no trophic links between blue tilapia, an abundant fish in the near-shore area, and piscivores.     

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.     

The Eocene Arctic Azolla bloom: environmental conditions, productivity and carbon drawdown

Enormous quantities of the free-floating freshwater fern Azolla grew and reproduced in situ in the Arctic Ocean during the middle Eocene, as was demonstrated by microscopic analysis of microlaminated sediments recovered from the Lomonosov Ridge during Integrated Ocean Drilling Program (IODP) Expedition 302. The timing of the Azolla phase (~48.5 Ma) coincides with the earliest signs of onset of the transition from a greenhouse towards the modern icehouse Earth. The sustained growth of Azolla , currently ranking among the fastest growing plants on Earth, in a major anoxic oceanic basin may have contributed to decreasing atmospheric p CO₂ levels via burial of Azolla -derived organic matter. The consequences of these enormous Azolla blooms for regional and global nutrient and carbon cycles are still largely unknown. Cultivation experiments have been set up to investigate the influence of elevated p CO₂ on Azolla growth, showing a marked increase in Azolla productivity under elevated (760 and 1910 ppm) p CO₂ conditions. The combined results of organic carbon, sulphur, nitrogen content and ¹⁵N and ¹³C measurements of sediments from the Azolla interval illustrate the potential contribution of nitrogen fixation in a euxinic stratified Eocene Arctic. Flux calculations were used to quantitatively reconstruct the potential storage of carbon (0.9–3.5 10¹⁸ gC) in the Arctic during the Azolla interval. It is estimated that storing 0.9 10¹⁸ to 3.5 10¹⁸ g carbon would result in a 55 to 470 ppm drawdown of p CO₂ under Eocene conditions, indicating that the Arctic Azolla blooms may have had a significant effect on global atmospheric p CO₂ levels through enhanced burial of organic matter.