Recovery of nitrogen from different sources following applications to winter wheat at and after anthesis

The effects of applying nitrogen (30 or 40 kg N/ha) to wheat crops at and after anthesis, after 200 kg N/ha had already been applied to the soil during stem extension, were studied in field experiments comprising complete factorial combinations of different cultivars, fungicide applications and nitrogen treatments. Actual recoveries of late-season fertilizer nitrogen (LSFN), as indicated by N-15 studies, interacted with cultivar and fungicide treatment, and depended on nitrogen source (Urea applied as a solution to the foliage, or as ammonium nitrate applied to the soil) and year. These interactions, however, were not reflected in apparent fertilizer recoveries ((N in grain with LSFN - N in grain without LSFN)/N applied as LSFN), or in the crude protein concentration. Apparent fertilizer recovery was always lower than actual recoveries, and declined during grain filling. Fertilizer treatments with higher actual fertilizer recoveries were associated with lower net renlobilisation of non-LSFN (net remobilised N = N in above ground crop at anthesis - N in non-grain, above ground crop at harvest). LSFN also increased mineral nitrogen in the soil at harvest even when applied as a solution to the foliage. These effects are discussed in relation to potential grain N demand. (c) 2006 Elsevier B.V. All rights reserved.

Comparative growth of ectomycorrhizal fungi (Hebeloma spp.) on organic and inorganic nitrogen

In the largely organic soils in which ectomycorrhizas are commonly found, a preference for absorbing organic nitrogen over mineral forms is likely to be an advantage, especially where mineralisation rates are low. To determine rates of both independent and preferential growth of ectomycorrhizal basidiomycetes on organic and inorganic nitrogen, strains of Hebeloma were grown on nutrient agar media containing either NH4+ or glutamic acid as the sole source of nitrogen, on both single medium and split plate Petri dishes. Growth rates on the split plate Petri dishes, where the fungi had access to both nitrogen sources, were generally greater than on the single medium dishes. Growth on glutamic acid was at least equal to, and usually greater than, that on NH4+. In some cases growth on NH4+ alone appeared severely inhibited, a condition that was partially alleviated by access to glutamic acid on the split plates Petri dishes. This highlights a potential pitfall of single nitrogen source growth studies. The greater growth of most strains on glutamic acid suggests an adaptation to organic nitrogen utilisation in these strains. If this is so in soils with low mineralisation rates, direct uptake of amino acids by ectomycorrhizal plants could by-pass the bottle neck that requires mineral nitrogen to be made available for plant uptake.

Characterization of a gamma-aminobutyric acid transport system of rhizobium leguminosarum bv. viciae 3841

Spontaneous mutants of Rhizobium leguminosarum bv. viciae 3841 were isolated that grow faster than the wild type on gamma-aminobutyric acid (GABA) as the sole carbon and nitrogen source. These strains (RU1736 and RU1816) have frameshift mutations (gtsR101 and gtsR102, respectively) in a GntR-type regulator (GtsR) that result in a high rate of constitutive GABA transport. Tn5 mutagenesis and quantitative reverse transcription-PCR showed that GstR regulates expression of a large operon (pRL100242 to pRL100252) on the Sym plasmid that is required for GABA uptake. An ABC transport system, GtsABCD (for GABA transport system) (pRL100248-51), of the spermidine/putrescine family is part of this operon. GtsA is a periplasmic binding protein, GtsB and GtsC are integral membrane proteins, and GtsD is an ATP-binding subunit. Expression of gtsABCD from a lacZ promoter confirmed that it alone is responsible for high rates of GABA transport, enabling rapid growth of strain 3841 on GABA. Gts transports open-chain compounds with four or five carbon atoms with carboxyl and amino groups at, or close to, opposite termini. However, aromatic compounds with similar spacing between carboxyl and amino groups are excellent inhibitors of GABA uptake so they may also be transported. In addition to the ABC transporter, the operon contains two putative mono-oxygenases, a putative hydrolase, a putative aldehyde dehydrogenase, and a succinate semialdehyde dehydrogenase. This suggests the operon may be involved in the transport and breakdown of a more complex precursor to GABA. Gts is not expressed in pea bacteroids, and gtsB mutants are unaltered in their symbiotic phenotype, suggesting that Bra is the only GABA transport system available for amino acid cycling.

Hydroponic isotope labelling of entire plants (HILEP) for quantitative plant proteomics; an oxidative stress case study

Hydroponic isotope labelling of entire plants (HILEP) is a cost-effective method enabling metabolic labelling of whole and mature plants with a stable isotope such as N-15. By utilising hydroponic media that contain N-15 inorganic salts as the sole nitrogen source, near to 100% N-15-labelling of proteins can be achieved. In this study, it is shown that HILEP, in combination with mass spectrometry, is suitable for relative protein quantitation of seven week-old Arabidopsis plants submitted to oxidative stress. Protein extracts from pooled N-14- and N-15-hydroponically grown plants were fractionated by SDS-PAGE, digested and analysed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS). Proteins were identified and the spectra of N-14/N-15 peptide pairs were extracted using their m/z chromatographic retention time, isotopic distributions, and the m/z difference between the N-14 and N-15 peptides. Relative amounts were calculated as the ratio of the sum of the peak areas of the two distinct N-14 and N-15 peptide isotope envelopes. Using Mascot and the open source trans-proteomic pipeline (TPP), the data processing was automated for global proteome quantitation down to the isoform level by extracting isoform specific peptides. With this combination of metabolic labelling and mass spectrometry it was possible to show differential protein expression in the apoplast of plants submitted to oxidative stress. Moreover, it was possible to discriminate between differentially expressed isoforms belonging to the same protein family, such as isoforms of xylanases and pathogen-related glucanases (PR 2). (C) 2008 Elsevier Ltd. All rights reserved.

A double-blind, randomized, controlled crossover trial of glutamine supplementation in home parenteral nutrition

Objective: Studies suggest clinical benefit of glutamine-supplemented parenteral nutrition. The aim was to determine if the inclusion of 10 g of glutamine as part of the nitrogen source of home parenteral nutrition (HPN) reduces infectious complications. Subjects/Methods: Thirty-five patients on HPN were recruited and 22 completed the study. Patients were randomized to receive either standard HPN or glutamine-supplemented HPN. Patients were assessed at randomization, 3 and 6 months later then they were crossed over to the alternative HPN and reassessed at 3 and 6 months. Assessments included plasma amino acid concentrations, intestinal permeability and absorption, nutritional status, oral and parenteral intake, quality of life, routine biochemistry and haematology. Results: No difference was seen between the groups at randomization. No difference was detected between the treatment phases for infective complications (55% in the standard treatment phase and 36% in the glutamine-supplemented phase P 0.67). There were no differences in nutritional status, intestinal permeability, plasma glutamine concentrations or quality of life. Conclusion: Although limited by the sample size, the study has shown that glutamine as part of the nitrogen source of parenteral nutrition can be given to patients on HPN for 6 months without any adverse effects.

Hydroponic isotope labeling of entire plants (HILEP) for quantitative plant proteomics

Quantitative analysis by mass spectrometry (MS) is a major challenge in proteomics as the correlation between analyte concentration and signal intensity is often poor due to varying ionisation efficiencies in the presence of molecular competitors. However, relative quantitation methods that utilise differential stable isotope labelling and mass spectrometric detection are available. Many drawbacks inherent to chemical labelling methods (ICAT, iTRAQ) can be overcome by metabolic labelling with amino acids containing stable isotopes (e.g. 13C and/or 15N) in methods such as Stable Isotope Labelling with Amino acids in Cell culture (SILAC). SILAC has also been used for labelling of proteins in plant cell cultures (1) but is not suitable for whole plant labelling. Plants are usually autotrophic (fixing carbon from atmospheric CO2) and, thus, labelling with carbon isotopes becomes impractical. In addition, SILAC is expensive. Recently, Arabidopsis cell cultures were labelled with 15N in a medium containing nitrate as sole nitrogen source. This was shown to be suitable for quantifying proteins and nitrogen-containing metabolites from this cell culture (2,3). Labelling whole plants, however, offers the advantage of studying quantitatively the response to stimulation or disease of a whole multicellular organism or multi-organism systems at the molecular level. Furthermore, plant metabolism enables the use of inexpensive labelling media without introducing additional stress to the organism. And finally, hydroponics is ideal to undertake metabolic labelling under extremely well-controlled conditions. We demonstrate the suitability of metabolic 15N hydroponic isotope labelling of entire plants (HILEP) for relative quantitative proteomic analysis by mass spectrometry. To evaluate this methodology, Arabidopsis plants were grown hydroponically in 14N and 15N media and subjected to oxidative stress.

The metabolic impact of zinc oxide on porcine intestinal cells and enterotoxigenic Escherichia coli K88

Pharmacological levels of zinc oxide (ZnO) incorporated into the post-weaning piglet diet reduce the incidence of diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) K88. The mechanism for this is not understood. Here, Intestinal Porcine Epithelial Cells (IPEC) J2 were used as an in vitro model of the porcine intestine. ZnO reduced IPEC J2 viability at concentrations >= 200 mu M, and ETEC adhesion to the host cell was unaffected by ZnO. Characterisation of the metabolism of IPEC J2 cells and ETEC established the effects of ZnO treatment on the metabolic profile of both. Although 100 mu M ZnO did not inhibit growth of either host or pathogen in fully supplemented media, metabolic profiles were significantly altered. Glucose and mannose were essential energy sources for IPEC J2 cells in the presence of ZnO, as the ability to utilise other sources was compromised. The increase in specificity of requirements to support respiration in ETEC was more pronounced, in particular the need for cysteine as a nitrogen source. These findings indicate that ZnO impacts on both host cell and pathogen metabolism and may provide insight into the mechanism for diarrhoea reduction. (C) 2010 Elsevier B.V. All rights reserved.

Assessing nitrogen dynamics in European ecosystems, integrating measurement and modelling: conclusions

This contribution closes this special issue of Hydrology and Earth System Sciences concerning the assessment of nitrogen dynamics in catchments across Europe within a semi-distributed Integrated Nitrogen model for multiple source assessment in Catchments (INCA). New developments in the understanding of the factors and processes determining the concentrations and loads of nitrogen are outlined. The ability of the INCA model to simulate the hydrological and nitrogen dynamics of different European ecosystems is assessed and the results of the first scenario analyses investigating the impacts of deposition, climatic and land-use change on the nitrogen dynamics are summarised. Consideration is given as to how well the model has performed as a generic too] for describing the nitrogen dynamics of European ecosystems across Arctic, Maritime. Continental and Mediterranean climates, its role in new research initiatives and future research requirements.

Promoting microbial immobilization of soil nitrogen during restoration of abandoned agricultural fields by organic additions

Application of organic materials to soils to enhance N immobilization into microbial biomass, thereby reducing inorganic N concentrations, was studied as a management option to accelerate the reestablishment of the native vegetation on abandoned arable fields on sandy soils the Kiskunsag National Park, Hungary. Sucrose and sawdust were used at three different topographic sites over 4 years. N availability and extractable inorganic N concentrations were significantly reduced in all sites. Soil microbial biomass C and microbial biomass N increased significantly following C additions, but the microbial C to microbial N ratio remained unaffected. It is concluded that the combined application of the rapidly utilized C source (sucrose) promoted N immobilization, whereas the addition of the slowly utilized C source (sawdust) maintained the elevated microbial biomass C and microbial biomass N in the field.

Assessing nitrogen dynamics in European ecosystems, integrating measurement and modelling: conclusions

This contribution closes this special issue of Hydrology and Earth System Sciences concerning the assessment of nitrogen dynamics in catchments across Europe within a semi-distributed Integrated Nitrogen model for multiple source assessment in Catchments (INCA). New developments in the understanding of the factors and processes determining the concentrations and loads of nitrogen are outlined. The ability of the INCA model to simulate the hydrological and nitrogen dynamics of different European ecosystems is assessed and the results of the first scenario analyses investigating the impacts of deposition, climatic and land-use change on the nitrogen dynamics are summarised. Consideration is given as to how well the model has performed as a generic too] for describing the nitrogen dynamics of European ecosystems across Arctic, Maritime. Continental and Mediterranean climates, its role in new research initiatives and future research requirements.

Evaluating the risk of non-point source pollution from biosolids: integrated modelling of nutrient losses at field and catchment scales

A semi-distributed model, INCA, has been developed to determine the fate and distribution of nutrients in terrestrial and aquatic systems. The model simulates nitrogen and phosphorus processes in soils, groundwaters and river systems and can be applied in a semi-distributed manner at a range of scales. In this study, the model has been applied at field to sub-catchment to whole catchment scale to evaluate the behaviour of biosolid-derived losses of P in agricultural systems. It is shown that process-based models such as INCA, applied at a wide range of scales, reproduce field and catchment behaviour satisfactorily. The INCA model can also be used to generate generic information for risk assessment. By adjusting three key variables: biosolid application rates, the hydrological connectivity of the catchment and the initial P-status of the soils within the model, a matrix of P loss rates can be generated to evaluate the behaviour of the model and, hence, of the catchment system. The results, which indicate the sensitivity of the catchment to flow paths, to application rates and to initial soil conditions, have been incorporated into a Nutrient Export Risk Matrix (NERM).

Modelling the inorganic nitrogen behaviour in a small Mediterranean forested catchment, Fuirosos (Catalonia)

The aim of this work was to couple a nitrogen (N) sub-model to already existent hydrological lumped (LU4-N) and semi-distributed (LU4-R-N and SD4-R-N) conceptual models, to improve our understanding of the factors and processes controlling nitrogen cycling and losses in Mediterranean catchments. The N model adopted provides a simplified conceptualization of the soil nitrogen cycle considering mineralization, nitrification, immobilization, denitrification, plant uptake, and ammonium adsorption/desorption. It also includes nitrification and denitrification in the shallow perched aquifer. We included a soil moisture threshold for all the considered soil biological processes. The results suggested that all the nitrogen processes were highly influenced by the rain episodes and that soil microbial processes occurred in pulses stimulated by soil moisture increasing after rain. Our simulation highlighted the riparian zone as a possible source of nitrate, especially after the summer drought period, but it can also act as an important sink of nitrate due to denitrification, in particular during the wettest period of the year. The riparian zone was a key element to simulate the catchment nitrate behaviour. The lumped LU4-N model (which does not include the riparian zone) could not be validated, while both the semi-distributed LU4-R-N and SD4-R-N model (which include the riparian zone) gave satisfactory results for the calibration process and acceptable results for the temporal validation process.

The determination of total nitrogen and total phosphorus concentrations in freshwaters from land use, stock headage and population data: testing of a model for use in conservation and water quality management

1. Nutrient concentrations (particularly N and P) determine the extent to which water bodies are or may become eutrophic. Direct determination of nutrient content on a wide scale is labour intensive but the main sources of N and P are well known. This paper describes and tests an export coefficient model for prediction of total N and total P from: (i) land use, stock headage and human population; (ii) the export rates of N and P from these sources; and (iii) the river discharge. Such a model might be used to forecast the effects of changes in land use in the future and to hindcast past water quality to establish comparative or baseline states for the monitoring of change. 2. The model has been calibrated against observed data for 1988 and validated against sets of observed data for a sequence of earlier years in ten British catchments varying from uplands through rolling, fertile lowlands to the flat topography of East Anglia. 3. The model predicted total N and total P concentrations with high precision (95% of the variance in observed data explained). It has been used in two forms: the first on a specific catchment basis; the second for a larger natural region which contains the catchment with the assumption that all catchments within that region will be similar. Both models gave similar results with little loss of precision in the latter case. This implies that it will be possible to describe the overall pattern of nutrient export in the UK with only a fraction of the effort needed to carry out the calculations for each individual water body. 4. Comparison between land use, stock headage, population numbers and nutrient export for the ten catchments in the pre-war year of 1931, and for 1970 and 1988 show that there has been a substantial loss of rough grazing to fertilized temporary and permanent grasslands, an increase in the hectarage devoted to arable, consistent increases in the stocking of cattle and sheep and a marked movement of humans to these rural catchments. 5. All of these trends have increased the flows of nutrients with more than a doubling of both total N and total P loads during the period. On average in these rural catchments, stock wastes have been the greatest contributors to both N and P exports, with cultivation the next most important source of N and people of P. Ratios of N to P were high in 1931 and remain little changed so that, in these catchments, phosphorus continues to be the nutrient most likely to control algal crops in standing waters supplied by the rivers studied.

A comparison of models for estimating the riverine export of nitrogen from large watersheds

We evaluated the accuracy of six watershed models of nitrogen export in streams (kg km2 yr−1) developed for use in large watersheds and representing various empirical and quasi-empirical approaches described in the literature. These models differ in their methods of calibration and have varying levels of spatial resolution and process complexity, which potentially affect the accuracy (bias and precision) of the model predictions of nitrogen export and source contributions to export. Using stream monitoring data and detailed estimates of the natural and cultural sources of nitrogen for 16 watersheds in the northeastern United States (drainage sizes = 475 to 70,000 km2), we assessed the accuracy of the model predictions of total nitrogen and nitrate-nitrogen export. The model validation included the use of an error modeling technique to identify biases caused by model deficiencies in quantifying nitrogen sources and biogeochemical processes affecting the transport of nitrogen in watersheds. Most models predicted stream nitrogen export to within 50% of the measured export in a majority of the watersheds. Prediction errors were negatively correlated with cultivated land area, indicating that the watershed models tended to over predict export in less agricultural and more forested watersheds and under predict in more agricultural basins. The magnitude of these biases differed appreciably among the models. Those models having more detailed descriptions of nitrogen sources, land and water attenuation of nitrogen, and water flow paths were found to have considerably lower bias and higher precision in their predictions of nitrogen export.