Natural15N abundance as a method of estimating the contribution of biologically fixed nitrogen to N2-fixing systems: Potential for non-legumes

The¹⁵N abundance of plants usually closely reflects the¹⁵N abundance of their major immediate N source(s); plant-available soil N in the case of non-N₂-fixing plants and atmospheric N₂ in the case of N₂ fixing plants. The¹⁵N abundance values of these sources are usually sufficiently different from each other that a significant and systematic difference in the¹⁵N abundance between the two kinds of plants can be detected. This difference provides the basis for the natural¹⁵N abundance method of estimating the relative contribution of atmospheric N₂ to N₂-fixing plants growing in natural and agricultural settings. The natural¹⁵N abundance method has certain advantages over more conventional methods, particularly in natural ecosystems, since disturbance of the system is not required and the measurements may be made on samples dried in the field. This method has been tested mainly with legumes in agricultural settings. The tests have demonstrated the validity of this method of arriving at semi-quantitative estimates of biological N₂-fixation in these settings. More limited tests and applications have been made for legumes in natural ecosystems. An understanding of the limits and utility of this method in these systems is beginning to emerge. Examples of systematic measurements of differences in¹⁵N abundance between non-legume N₂-fixing systems and neighbouring non-fixing systems are more unusual. In principle, application of the method to estimate N₂-fixation by nodulated non-legumes, using the natural¹⁵N abundance method, is as feasible as estimating N₂-fixation by legumes. Most of the studies involving N₂-fixing non-legumes are with this type of system (e.g., Ceanothus, Chamabatia, Eleagnus, Alnus, Myrica, and so forth). Resuls of these studies are described. Applicability for associative N₂-fixation is an empirical question, the answer to which probably depends upon the degree to which fixed N goes predominantly to the plant rather than to the soil N pool. The natural¹⁵N abundance method is probably not well suited to assessing the contribution of N₂-fixation by free-living microorganisms in their natural habitat, particularly soil microorganisms.This work was supported in part by subcontracts under grants from the US National Science Foundation (DEB79-21971 and BSR821618)     

Nitrogen Transfer Within and Between Plants Through Common Mycorrhizal Networks (CMNs)

Mycorrhizae play a critical role in nutrient capture from soils. Arbuscular mycorrhizae (AM) and ectomycorrhizae (EM) are the most important mycorrhizae in agricultural and natural ecosystems. AM and EM fungi use inorganic NH₄ ⁺ and NO₃ ^?, and most EM fungi are capable of using organic nitrogen. The heavier stable isotope ¹⁵N is discriminated against during biogeochemical and biochemical processes. Differences in ¹⁵N (atom%) or δ¹⁵N (‰) provide nitrogen movement information in an experimental system. A range of 20 to 50% of one-way N-transfer has been observed from legumes to nonlegumes. Mycorrhizal fungal mycelia can extend from one plant's roots to another plant's roots to form common mycorrhizal networks (CMNs). Individual species, genera, even families of plants can be interconnected by CMNs. They are capable of facilitating nutrient uptake and flux. Nutrients such as carbon, nitrogen and phosphorus and other elements may then move via either AM or EM networks from plant to plant. Both ¹⁵N labeling and ¹⁵N natural abundance techniques have been employed to trace N movement between plants interconnected by AM or EM networks. Fine mesh (25～45 μm) has been used to separate root systems and allow only hyphal penetration and linkages but no root contact between plants. In many studies, nitrogen from N₂-fixing mycorrhizal plants transferred to non-N₂–fixing mycorrhizal plants (one-way N-transfer). In a few studies, N is also transferred from non-N₂–fixing mycorrhizal plants to N₂-fixing mycorrhizal plants (two-way N-transfer). There is controversy about whether N-transfer is direct through CMNs, or indirect through the soil. The lack of convincing data underlines the need for creative, careful experimental manipulations. Nitrogen is crucial to productivity in most terrestrial ecosystems, and there are potential benefits of management in soil-plant systems to enhance N-transfer. Thus, two-way N-transfer warrants further investigation with many species and under field conditions.     

1H and 15N NMR studies of protonation and hydrogen-bonding in the binding of trimethoprim to dihydrofolate reductase

The binding of trimethoprim and [1,3,2-amino-¹⁵N₃]-trimethoprim to Lactobacillus casei dihydrofolate reductase has been studied by ¹⁵N and ¹H NMR spectroscopy. ¹⁵N NMR spectra of the bound drug were obtained by using polarisation transfer pulse sequences. The ¹⁵N chemical shifts and ¹H-¹⁵N spin-coupling constants show unambiguously that the drug is protonated on N1 when bound to the enzyme.The N1-proton resonance in the complex has been assigned using the ¹⁵N-enriched molecule. The temperature-dependence of the linewidth of this resonance has been used to estimate the rate of exchange of this proton with the solvent: 160±10s^-1 at 313 K, with an activation energy of 75 (±9) kJ·mole^-1. This is considerably faster than the dissociation rate of the drug from this complex, demonstrating that there are local fluctuations in the structure of the complex.     

Simultaneous CT-13C and VT-15N chemical shift labelling: Application to 3D NOESY-CH3NH and 3D 13C,15N HSQC-NOESY-CH3NH

Based on the HSQC scheme, we have designed a 2D heterocorrelated experiment which combines constant time (CT) ¹³C and variable time (VT) ¹⁵N chemical shift labelling. Although applicable to all carbons, this mode is particularly suitable for simultaneous recording of methyl-carbon and nitrogen chemical shifts at high digital resolution. The methyl carbon magnetisation is in the transverse plane during the whole CT period (1/J_CC=28.6 ms). The magnetisation originating from NH protons is initially stored in the 2H_zN_z state, then prior to the VT chemical shift labelling period is converted into 2H_zN_y coherence. The VT -¹⁵N mode eliminates the effect of ¹ J _N,CO and ^1,2 J _N,CA coupling constants without the need for band-selective carbon pulses. An optional editing procedure is incorporated which eliminates signals from CH₂ groups, thus removing any potential overlap with the CH₃ signals. The CT-¹³CH₃,VT-¹⁵N HSQC building block is used to construct two 3D experiments: 3D NOESY-CH₃NH and 3D ¹³C,¹⁵N HSQC-NOESY-CH₃NH. Combined use of these experiments yields proton and heteronuclear chemical shifts for moieties experiencing NOEs with CH₃ and NH protons. These NOE interactions are resolved as a consequence of the high digital resolution in the carbon and nitrogen chemical shifts of CH₃ and NH groups, respectively. The techniques are illustrated using a double labelled sample of the CH domain from calponin.     

Evaluation of15N methods to measure nitrogen transfer from alfalfa to companion timothy

Four different methods: direct¹⁵N₂ exposure, legume leaf labeled with¹⁵N,¹⁵N dilution and total N balance were applied to assess the nitrogen transfer (NT) from alfalfa to companion timothy. Evidence of NT was obtained in all cases, which represents about 3% of total N fixed by alfalfa or 10% of N content in timothy at the first cycle of growth. All the three¹⁵N methods gave identical results, while the conventional calculation of NT from the difference of N content in timothy from mixture and monoculture resulted in an over-estimation. The advantages and disadvantages of each method as applied to field conditions are discussed.Contribution No 1158 from the Plant Research Centre.     

Losses of fertilizer-derived N from transplanted rice after heading

Microplot field experiments with ¹⁵N-labeled (NH₄)₂SO₄ were conducted to investigate the changes in the percentage of fertilizer ¹⁵N recovery (%FNR) of transplanted rice (Oryza sativa L.) with basal (4 g m^–2) and topdress (3 g m^–2) applications for two years. In 1999, %FNR from the basal ¹⁵N application decreased by 6% (0.23 g N m^–2)from heading + 2 d to maturity, and %FNR from the topdress ¹⁵N application decreased by 11% (0.32 g N m^–2)from heading – 4 d to maturity. In 2000, %FNR from the topdress ¹⁵N application decreased by 9% (0.26 g N m^–2) from heading – 1 d to maturity, whereas there was no significant decline in %FNR from the basal ¹⁵N application after heading. A decrease in %FNR occurred even when total N of rice plants showed no decrease after heading. Nitrogen harvest index of fertilizer ¹⁵N with the topdress ¹⁵N application was not significantly different from that with the basal ¹⁵N application in 1999, whereas N harvest index of fertilizer ¹⁵N with the topdress ¹⁵N application was higher than that with the basal ¹⁵N application in 2000. N harvest index of fertilizer ¹⁵N, that indicated the proportion of the ¹⁵N remobilized to panicles after heading, was substantially responsible for changes in %FNR from the basal and topdress ¹⁵N applications after heading.     

Estimation of symbiotically fixed nitrogen in field grown soybeans: An application of natural15N/14N abundance and a low level15N-tracer technique

Summary Plants from agricultural and natural upland ecosystem were investigated for¹⁵N content to evaluate the role of symbiotic N₂-fixation in the nitrogen nutrition of soybean. Increased yields and lower δ¹⁵N values of nodulating soybeansvs, non-nodulating isolines gave semi-quantitative estimates of N₂ fixation. A fairly large discrepancy was found between estimations by δ¹⁵N and by N yield at 0 kg N/ha of fertilizer. More precise estimates were made by following changes in plant δ¹⁵N when fertilizer δ¹⁵N was varied near¹⁵N natural abundance level. Clearcut linear relationships between δ¹⁵N values of whole plants and of fertilizer were obtained at 30 kg N/ha of fertilizer for three kinds of soils. In experimental field plots, nodulating soybeans obtained 13±1% of their nitrogen from fertilizer, 66±8% from N₂ fixation and 21±10% from soil nitrogen in Andosol brown soil; 30%, 16% and 54% in Andosol black soil; 7%, 77% and 16% in Alluvial soil, respectively. These values for N₂ fixation coincided with each corresponding estimation by N yield method. Other results include: 1)¹⁵N content in upland soils and plants was variable, and may reflect differences in the mode of mineralization of soil organics, and 2) nitrogen isotopic discrimination during fertilizer uptake (δ¹⁵N of plant minus fertilizer) ranged from −2.2 to +4.9‰ at 0–30 kg N/ha of fertilizer, depending on soil type and plant species. The proposed method can accurately and relatively simply establish the importance of symbiotic nitrogen fixation for soybeans growing in agricultural settings.     

Dissecting non-canonical interactions in frameshift-stimulating mRNA pseudoknots

A variety of powerful NMR experiments have been introduced over the last few years that allow for the direct identification of different combinations of donor and acceptor atoms involved in hydrogen bonds in biomolecules. This ability to directly observe tertiary structural hydrogen bonds in solution tremendously facilitates structural studies of nucleic acids. We show here that an adiabatic HNN-COSY pulse scheme permits observation and measurement of J(N,N) couplings for nitrogen sites that are separated by up to 140 ppm in a single experiment at a proton resonance frequency of 500 MHz. Crucial hydrogen bond acceptor sites in nucleic acids, such as cytidine N3 nitrogens, can be unambiguously identified even in the absence of detectable H41 and H42 amino protons using a novel triple-resonance two-dimensional experiment, denoted H5(C5C4)N3. The unambiguous identification of amino nitrogen donor and aromatic nitrogen acceptor sites associated with both major groove as well as minor groove triple base pairs reveal the details of hydrogen bonding networks that stabilize the complex architecture of frameshift-stimulating mRNA pseudoknots. Another key tertiary interaction involving a 2′-OH hydroxyl proton that donates a hydrogen bond to an aromatic nitrogen acceptor in a cis Watson–Crick/sugar edge interaction can also be directly detected using a quantitative J(H,N) ¹H,¹⁵N-HSQC experiment.     

Denitrification and total N losses from an irrigated sandy-clay loam under maize–wheat cropping system

Denitrification and total N losses were quantified from an irrigated field cropped to maize and wheat, each receiving urea at 100 kg N ha^-1. During the maize growing season (60 days), the denitrification loss measured directly by acetylene inhibition-soil cover method amounted 2.72 kg N ha^-1 whereas total N loss measured by ¹⁵N balance was 39 kg ha^-1. Most (87%) of the denitrification loss under maize occurred during the first two irrigation cycles. During the wheat growing season (150 days), the denitrification loss directly measured by acetylene inhibition-soil cover and acetylene inhibition-soil core methods was 1.14 and 3.39 kg N ha^-1, respectively in contrast to 33 kg N ha^-1 loss measured by ¹⁵N balance. Most (70-88%) of the denitrification loss under wheat occurred during the first three irrigation cycles. Soil moisture and NO ₃ ^- -N were the major factors limiting denitrification under both crops. Higher N losses measured by ¹⁵N balance than C₂H₂ inhibition method were perhaps due to underestimation of denitrification by C₂H₂ inhibition method and losses other than denitrification, most probably NH₃ volatilization.     

Resonance assignment and structural analysis of acid denatured E. coli [U-15N]-glutaredoxin 3

Virtually complete sequence specific ¹H and ¹⁵N resonance assignments are presented for acid denatured reduced E. coli glutaredoxin 3. The sequential resonance assignments of the backbone rely on the combined use of 3D F₁-decoupled ROESY-¹⁵N-HSQC and 3D ¹⁵N-HSQC-(TOCSY-NOESY)-¹⁵N-HSQC using a single uniformly ¹⁵N labelled protein sample. The sidechain resonances were assigned from a 3D TOCSY-¹⁵N-HSQC and a homonouclear TOCSY spectrum. The presented assignment strategy works in the absence of chemical exchange peaks with signals from the native conformation and without ¹³C/¹⁵N double labelling. Chemical shifts, ³J(H, NH) coupling constants and NOEs indicate extensive conformational averaging of both backbone and side chains in agreement with a random coil conformation. The only secondary structure element persisting at pH 3.5 appears to be a short helical segment comprising residues 37 to 40.Abbreviations HSQC heteronuclear single quantum coherence - NMR nuclear magnetic resonance - NOE nuclear Overhauser effect - NOESY two-dimensional NOE spectroscopy - ROE nuclear Overhauser effect in the rotating frame - ROESY two-dimensional ROE spectroscopy - TOCSY total correlation spectroscopy - TPPI time proportional phase incrementation Correspondence to: G. Otting     

C, N CP MAS and high resolution multinuclear NMR study of methyl

Summary The uptake and distribution of¹⁵NH ₄ ⁺ ,¹⁵NO ₃ ⁻ and¹⁵N₂ was studied in greenhouse-grown beans (Phaseolus vulgaris L.) with a commercial cultivar and 2 recombinant inbred backcross lines;¹⁵N was supplied in the nutrient solution at the R3 (50% bloom) stage. Plants were harvested 1, 5 and 10 days after treatment, and were separated into nodules, roots, stems, mature leaflets, immature leaflets, and flowers/fruits. All 3 lines showed rapid increases in the N content of flowers/fruits after the R3 stage. However, the percentage N in these tissues decreased after the R3 stage. One of the recombinant lines showed a greater uptake of NH ₄ ⁺ than the other 2 lines. Rates of¹⁵N₂ fixation and NO ₃ ⁻ uptake were similar for all 3 lines, N₂ fixation estimated from total N content showed the 2 recombinant lines with 24 and 34 percent greater activity than the commercial cultivar. Distribution of¹⁵N at the whole plant level was similar for all 3 lines for a similar N source.¹⁵NO ₃ ⁻ was transported first to leaflets and the label then moved into flowers/fruits. Transport of fixed N₂ was from the nodules to roots, stems and into flowers/fruits; usually less than 10 percent entered the leaflets. This indicates that N₂ fixation furnishes N directly to flowers/fruits with over 50 percent of the fixed N being deposited into flowers/fruits within 5 days after treatment.     

Comparative study of N uptake and distribution in three lines of common bean (Phaseolus vulgaris L.) at early pod filling stage

Summary The uptake and distribution of¹⁵NH ₄ ⁺ ,¹⁵NO ₃ ⁻ and¹⁵N₂ was studied in greenhouse-grown beans (Phaseolus vulgaris L.) with a commercial cultivar and 2 recombinant inbred backcross lines;¹⁵N was supplied in the nutrient solution at the R3 (50% bloom) stage. Plants were harvested 1, 5 and 10 days after treatment, and were separated into nodules, roots, stems, mature leaflets, immature leaflets, and flowers/fruits. All 3 lines showed rapid increases in the N content of flowers/fruits after the R3 stage. However, the percentage N in these tissues decreased after the R3 stage. One of the recombinant lines showed a greater uptake of NH ₄ ⁺ than the other 2 lines. Rates of¹⁵N₂ fixation and NO ₃ ⁻ uptake were similar for all 3 lines, N₂ fixation estimated from total N content showed the 2 recombinant lines with 24 and 34 percent greater activity than the commercial cultivar. Distribution of¹⁵N at the whole plant level was similar for all 3 lines for a similar N source.¹⁵NO ₃ ⁻ was transported first to leaflets and the label then moved into flowers/fruits. Transport of fixed N₂ was from the nodules to roots, stems and into flowers/fruits; usually less than 10 percent entered the leaflets. This indicates that N₂ fixation furnishes N directly to flowers/fruits with over 50 percent of the fixed N being deposited into flowers/fruits within 5 days after treatment.     

Variability in nitrogen fixation of common bean (Phaseolus vulgaris L.) intercropped with maize

Thirty one selected bean lines were evaluated in the field for ability to support N₂ fixation when intercropped with maize which received 0, 30 and 60 kg N ha^–1 as ammonium sulphate. The amount of fixed N₂ was estimated using the natural variation of ¹⁵N and wheat as the standard non-fixing crop. Nitrogen as low as 15 kg N ha^–1 at sowing suppressed nodule weight and activity (acetylene reduction activity) but not nodule number, suggesting that the main effect of mineral N was on nodule development and function. ¹⁵N data revealed a high potential of the bean genotypes to fix N₂, with the most promising ones averaging between 50–60% of seed N coming from fixation. Bean lines CNF-480, Puebla-152, Mexico-309, Negro Argel, CNF-178, Venezuela-350 and WBR22-3, WBR22-50 and WBR22-55 were ranked as good fixers.     

A bioassay of the availability of residual 15N fertilizer eight years after application to a forest soil in interior British Columbia

Although a high proportion of fertilizer N may be immobilized in organic forms in the soil, no studies have examined the long-term availability of residual fertilizer ¹⁵N in forestry situations. We investigated this by growing lodgepole pine (Pinus contorta) seedlings in surface (0–10 cm) soil sample eight years after application of ¹⁵N-urea, ¹⁵NH₄NO₃ and NH₄ ¹⁵NO₃ to lodgepole pine in interior British Columbia. After nine months of growth in the greenhouse, seedlings took up an average of 8.5% of the ¹⁵N and 4.6% of the native N per pot. Most of the mineral N in the pots without seedlings was in the form of nitrate, while pots with seedlings had very low levels of mineral N. In contrast to the greenhouse study, there was no significantuptake of ¹⁵N by trees in the field study after the first growing season, although half of the soil organic ¹⁵N was lost between one and eight years after fertilization. This indicates the need to understand the mechanisms which limit the uptake of mineral N by trees in the field, and the possible mismatch of tree demand and mineral N availability.     

Effects of CO2 and N fertilization on decomposition and N immobilization in ponderosa pine litter

Naturally senesced needles from ponderosa pine (Pinus ponderosa Dougl.), grown from seed in open-top chambers under three levels of CO₂ (350, 525 and 700 μl l^-1) and three levels of N fertilization (0, 10 and 20 g N m^-2 yr^-1), were used in a field litterbag decomposition study and in a laboratory study on potential microbial and nonmicrobial N immobilization. The litterbag studies revealed no statistically significant effects of either CO₂ or N treatment on mass loss, N concentration, or N content over a 26-month period. The laboratory study of potential ¹⁵N immobilization revealed no statistically significant effects of CO₂ or N treatment on either total or microbial immobilization. Elevated (CO₂) did have a significant negative effect on nonmicrobial immobilization, however. Natural abundance of ¹⁵N was significantly greater with elevated (CO₂) in both live and naturally senesced needles under all N treatments. This pattern combined with ¹⁵N natural abundance in soils suggests that saplings grown under elevated (CO₂) were either taking up more N from surface horizons or from a more recalcitrant soil N pool in either horizon. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nitrogen-15 labeling effectiveness of two tropical legumes

Nitrogen-15 foliar applications for the production of field-labeled plant tissues may achieve more effective labeling of plant shoot and root tissues and minimize directly labeling the soil N fraction as occurs when¹⁵ N is soil applied. Consequently, foliar-labeled plant tissues should be better suited for subsequent ¹⁵N mineralization studies. A field experiment was conducted to determine the effectiveness of ¹⁵N-labeling and the accumulation of ¹⁵N in various plant parts of two tropical legumes. Desmodium ovalifolium Guillemin and Perrottet and Pueraria phaseoloides (Roxb.) Benth., grown in 0.5 m² microplots, were labeled with foliar-applied urea containing 99 atom% ¹⁵N. Plants in each microplot received a total of 0.1698 g ¹⁵N that was applied all at once or split equally into two, three or four applications. Legume shoots and roots and soil were destructively harvested and analyzed for total ¹⁵N content. Averaged over both legumes and foliar application rates, total plant (shoots, flowers, leaf litter, and roots) recovery was approximately 79% of the ¹⁵N applied. The soil contained 3% of the ¹⁵N applied, of which 2.5 and 0.5% were in the inorganic and organic fractions, respectively. Nitrogen-15 recovery in shoots (76%) was sixty-five fold greater than in roots (1%) and about nineteen fold greater than the sum of roots and soil (4.1%), a much greater percent recovery than observed in other foliar labeling studies. Averaged over all four foliar split-application rates, ¹⁵N recovery by Desmodium shoots was greater than Pueraria. Results demonstrate that ¹⁵N foliar application to legumes is an effective method for labeling, resulting in atom% excess ¹⁵N levels and ¹⁵N recoveries comparable to those reported with the more traditional soil-labeling approach. Another advantage of this method is a nondestructive, in situ labeling method that permits separation of shoot and root residual N contribution to subsequent crops in N tracer studies.     

Relationship between intracellular pH and N metabolism in maize (Zea mays L.) roots

In vivo ³¹P nuclear magnetic resonance (NMR) was used to characterize the effect of the N form (NO₃ vs. NH₄) and the external pH (4, 6, and 8), on the intracellular pH of root tips (0–5 mm) and root segments (5–30 mm). Ammonium-grown root tips were the most sensitive to changes in the external pH. In vivo ¹⁵N NMR was used to characterize the pathway of primary ammonium assimilation in the ammonium-grown roots and to compare the activity of the apical and more-basal root parts. The kinetics of ¹⁵NH₄ ⁺ incorporation showed that primary assimilation in both root tips and root segments followed the glutamine synthetase (GS) pathway. In agreement with the reported gradient of GS along the seminal root of maize, incorporation of label into glutamine amide was more rapid in tips than in segments. It is suggested that this higher GS activity increases the endogenous proton production and thus contributes to the greater dependence of the cytoplasmic pH on the external pH in the ammonium-treated root tips.     

2D and 3D TROSY-enhanced NOESY of 15N labeled proteins

Recently, several TROSY-based experiments have been designed for backbone chemical shift assignment and measurement of the NOEs of ²H, ¹³C and ¹⁵N labeled proteins. Here, we present TROSY-enhanced NOESY experiments, namely the 2D S3E-NOESY-S3E, 3D TROSY-NOESY-S3E and S3E-NOESY-TROSY experiments. These experiments use the spin-state selective excitation method (S3E), and have the TROSY effect in all the indirectly and directly detected dimensions, and so provide optimal resolution for amide protons. The first two experiments provide an additional useful feature in that the diagonal peaks of the amide proton region are cancelled or greatly reduced, allowing clear identification of NOE cross peaks that are close to diagonal peaks.     

Changes in Canopy Processes Following Whole-Forest Canopy Nitrogen Fertilization of a Mature Spruce-Hemlock Forest

Abstract Most experimental additions of nitrogen to forest ecosystems apply the N to the forest floor, bypassing important processes taking place in the canopy, including canopy retention of N and/or conversion of N from one form to another. To quantify these processes, we carried out a large-scale experiment and determined the fate of nitrogen applied directly to a mature coniferous forest canopy in central Maine (18–20 kg N ha⁻¹ y⁻¹ as NH₄NO₃ applied as a mist using a helicopter). In 2003 and 2004 we measured NO₃ ⁻, NH₄ ⁺, and total dissolved N (TDN) in canopy throughfall (TF) and stemflow (SF) events after each of two growing season applications. Dissolved organic N (DON) was greater than 80% of the TDN under ambient inputs; however NO₃ ⁻ accounted for more than 50% of TF N in the treated plots, followed by NH₄ ⁺ (35%) and DON (15%). Although NO₃ ⁻ was slightly more efficiently retained by the canopy under ambient inputs, canopy retention of NH₄ ⁺as a percent of inputs increased markedly under fertilization. Recovery of less than 30% of the fertilizer N in TF suggested that the forest canopy retained more than 70% of the applied N (>80% when corrected for N which bypassed tree surfaces at the time of fertilizer addition). Results from plots receiving ¹⁵N enriched NO₃ ⁻ and NH₄ ⁺ confirmed bulk N estimations that more NO₃ ⁻ than NH₄ ⁺ was washed from the canopy by wet deposition. The isotope data did not show evidence of canopy nitrification, as has been reported in other spruce forests receiving much higher N inputs. Conversions of fertilizer-N to DON were observed in TF for both ¹⁵NH₄ ⁺ and ¹⁵NO₃ ⁻ additions, and occurred within days of the application. Subsequent rain events were not significantly enriched in ¹⁵N, suggesting that canopy DON formation was a rapid process related to recent N inputs to the canopy. We speculate that DON may arise from lichen and/or microbial N cycling rather than assimilation and re-release by tree tissues in this forest. Canopy retention of experimentally added N may meet and exceed calculated annual forest tree demand, although we do not know what fraction of retained N was actually physiologically assimilated by the plants. The observed retention and transformation of DIN within the canopy demonstrate that the fate and ecosystem consequences of N inputs from atmospheric deposition are likely influenced by forest canopy processes, which should be considered in N addition studies. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cyanobacterial inoculation and nitrogen fertilization in rice

During three rice-growing seasons in Uruguay, field experiments were conducted to study the contribution of cyanobacterial inoculation and chemical N fertilization to rice production. Neither grain yield nor fertilizer recovery by the plant were affected by inoculation with native cyanobacterial isolates. A low fertilizer use efficiency (around 20%) was observed when labelled (NH₄)₂SO₄ was applied at sowing. Recovery of applied ¹⁵N by the soil–plant system was 50%. Inoculation did not modify ¹⁵N uptake by the plant when the fertilizer was three-split applied either. The total N-fertilizer recovery was higher when the fertilizer was split than when applied in a single dose. Plant N-fertilizer uptake was higher when the fertilizer was applied at tillering. Uptake of ¹⁵N from cyanobacteria by rice was studied in a greenhouse pots experiment without chemical nitrogen addition. Recovery of ¹⁵N from labelled cyanobacteria by rice in greenhouse growth conditions was similar to that of partial recovery of (NH₄)₂SO₄ applied at sowing in the field. Cyanobacterial N mineralization under controlled conditions was fast as cyanobacterial N was detected in plants after 25 days. Moreover 40 days after inoculation non-planted and inoculated soil had more inorganic N than the non-inoculated one.