Plant ammonium sensitivity is associated with the external pH adaptation, repertoire of nitrogen transporters, and nitrogen requirement.

Modern crops exhibit diverse sensitivities to ammonium as the primary nitrogen source, influenced by environmental factors such as external pH and nutrient availability. Despite its significance, there is currently no systematic classification of plant species based on their ammonium sensitivity. This study conducts a meta-analysis of 50 plant species and presents a new classification method based on the comparison of fresh biomass obtained under ammonium and nitrate nutrition. The classification uses the natural logarithm of biomass ratio as the size effect indicator of ammonium sensitivity. This numerical parameter is associated with critical factors for nitrogen demand and form preference, such as Ellenberg indicators and the repertoire of nitrogen transporters for ammonium and nitrate uptake. Finally, a comparative analysis of the developmental and metabolic responses, including hormonal balance, is conducted in two species with divergent ammonium sensitivity values in the classification. Results indicate that nitrate has a key counteracting role of ammonium toxicity in species with a higher abundance of genes encoding NRT2-type proteins and fewer of the AMT2-type proteins. Additionally, the study confirms the reliability of the phytohormone balance and methylglyoxal content as indicators for anticipating ammonium toxicity.

Evaluation of a crop rotation with biological inhibition potential to avoid N2O emissions in comparison with synthetic nitrification inhibition.

Agriculture has increased the release of reactive nitrogen to the environment due to crops' low nitrogen-use efficiency (NUE) after the application of nitrogen-fertilisers. Practices like the use of stabilized-fertilisers with nitrification inhibitors such as DMPP (3,4-dimethylpyrazole phosphate) have been adopted to reduce nitrogen losses. Otherwise, cover crops can be used in crop-rotation-strategies to reduce soil nitrogen pollution and benefit the following culture. Sorghum (Sorghum bicolor) could be a good candidate as it is drought tolerant and its culture can reduce nitrogen losses derived from nitrification because it exudates biological nitrification inhibitors (BNIs). This work aimed to evaluate the effect of fallow-wheat and sorghum cover crop-wheat rotations on N2O emissions and the grain yield of winter wheat crop. In addition, the suitability of DMPP addition was also analyzed. The use of sorghum as a cover crop might not be a suitable option to mitigate nitrogen losses in the subsequent crop. Although sorghum-wheat rotation was able to reduce 22% the abundance of amoA, it presented an increment of 77% in cumulative N2O emissions compared to fallow-wheat rotation, which was probably related to a greater abundance of heterotrophic-denitrification genes. On the other hand, the application of DMPP avoided the growth of ammonia-oxidizing bacteria and maintained the N2O emissions at the levels of unfertilized-soils in both rotations. As a conclusion, the use of DMPP would be recommendable regardless of the rotation since it maintains NH4+ in the soil for longer and mitigates the impact of the crop residues on nitrogen soil dynamics.

Could ammonium nutrition increase plant C-sink strength under elevated CO2 conditions?

Atmospheric carbon dioxide (CO2) is increasing, and this affects plant photosynthesis and biomass production. Under elevated CO2 conditions (eCO2), plants need to cope with an unbalanced carbon-to-nitrogen ratio (C/N) due to a limited C sink strength and/or the reported constrains in leaf N. Here, we present a physiological and metabolic analysis of ammonium (NH4+)-tolerant pea plants (Pisum sativum L., cv. snap pea) grown hydroponically with moderate or high NH4+ concentrations (2.5 or 10 mM), and under two atmospheric CO2 concentrations (400 and 800 ppm). We found that the photosynthetic efficiency of the NH4+ tolerant pea plants remain intact under eCO2 thanks to the capacity of the plants to maintain the foliar N status (N content and total soluble proteins), and the higher C-skeleton requirements for NH4+ assimilation. The capacity of pea plants grown at 800 ppm to promote the C allocation into mobile pools of sugar (mainly sucrose and glucose) instead of starch contributed to balancing plant C/N. Our results also support previous observations: plants exposed to eCO2 and NH4+ nutrition can increase of stomatal conductance. Considering the C and N source-sink balance of our plants, we call for exploring a novel trait, combining NH4+ tolerant plants with a proper NH4+ nutrition management, as a way for a better exploitation of eCO2 in C3 crops.

Short-Term Exposure to High Atmospheric Vapor Pressure Deficit (VPD) Severely Impacts Durum Wheat Carbon and Nitrogen Metabolism in the Absence of Edaphic Water Stress.

Low atmospheric relative humidity (RH) accompanied by elevated air temperature and decreased precipitation are environmental challenges that wheat production will face in future decades. These changes to the atmosphere are causing increases in air vapor pressure deficit (VPD) and low soil water availability during certain periods of the wheat-growing season. The main objective of this study was to analyze the physiological, metabolic, and transcriptional response of carbon (C) and nitrogen (N) metabolism of wheat (Triticum durum cv. Sula) to increases in VPD and soil water stress conditions, either alone or in combination. Plants were first grown in well-watered conditions and near-ambient temperature and RH in temperature-gradient greenhouses until anthesis, and they were then subjected to two different water regimes well-watered (WW) and water-stressed (WS), i.e., watered at 50% of the control for one week, followed by two VPD levels (low, 1.01/0.36 KPa and high, 2.27/0.62 KPa; day/night) for five additional days. Both VPD and soil water content had an important impact on water status and the plant physiological apparatus. While high VPD and water stress-induced stomatal closure affected photosynthetic rates, in the case of plants watered at 50%, high VPD also caused a direct impairment of the RuBisCO large subunit, RuBisCO activase and the electron transport rate. Regarding N metabolism, the gene expression, nitrite reductase (NIR) and transport levels detected in young leaves, as well as determinations of the δ15N and amino acid profiles (arginine, leucine, tryptophan, aspartic acid, and serine) indicated activation of N metabolism and final transport of nitrate to leaves and photosynthesizing cells. On the other hand, under low VPD conditions, a positive effect was only observed on gene expression related to the final step of nitrate supply to photosynthesizing cells, whereas the amount of 15N supplied to the roots that reached the leaves decreased. Such an effect would suggest an impaired N remobilization from other organs to young leaves under water stress conditions and low VPD.

A Multi-Species Analysis Defines Anaplerotic Enzymes and Amides as Metabolic Markers for Ammonium Nutrition.

Nitrate and ammonium are the main nitrogen sources in agricultural soils. In the last decade, ammonium (NH4 +), a double-sided metabolite, has attracted considerable attention by researchers. Its ubiquitous presence in plant metabolism and its metabolic energy economy for being assimilated contrast with its toxicity when present in high amounts in the external medium. Plant species can adopt different strategies to maintain NH4 + homeostasis, as the maximization of its compartmentalization and assimilation in organic compounds, primarily as amino acids and proteins. In the present study, we report an integrative metabolic response to ammonium nutrition of seven plant species, belonging to four different families: Gramineae (ryegrass, wheat, Brachypodium distachyon), Leguminosae (clover), Solanaceae (tomato), and Brassicaceae (oilseed rape, Arabidopsis thaliana). We use principal component analysis (PCA) and correlations among metabolic and biochemical data from 40 experimental conditions to understand the whole-plant response. The nature of main amino acids is analyzed among species, under the hypothesis that those Asn-accumulating species will show a better response to ammonium nutrition. Given the provision of carbon (C) skeletons is crucial for promotion of the nitrogen assimilation, the role of different anaplerotic enzymes is discussed in relation to ammonium nutrition at a whole-plant level. Among these enzymes, isocitrate dehydrogenase (ICDH) shows to be a good candidate to increase nitrogen assimilation in plants. Overall, metabolic adaptation of different carbon anaplerotic activities is linked with the preference to synthesize Asn or Gln in their organs. Lastly, glutamate dehydrogenase (GDH) reveals as an important enzyme to surpass C limitation during ammonium assimilation in roots, with a disparate collaboration of glutamine synthetase (GS).

Multi-omic and physiologic approach to understand Lotus japonicus response upon exposure to 3,4 dimethylpyrazole phosphate nitrification inhibitor.

Nitrogen fertilization is a major force in global greenhouse gases emissions and causes environmental contamination through nitrate leaching. The use of nitrification inhibitors has been proven successful to mitigate these effects. However, there is an increasing concern about the undesired effects that their potential persistence in the soil or accumulation in plants may provoke. In this study, we first exposed Lotus japonicus plants to high amounts of 3,4 dimethylpyrazole phosphate (DMPP) and 2-(N-3,4-dimethyl-1H-pyrazol-1-yl) succinic acid isomeric mixture (DMPSA) nitrification inhibitors. Exposure to doses higher than 1 mg·L-1 provoked DMPP accumulation mostly in the aerial part, while DMPSA was only detected from 10 mg·L-1 and nearly no translocation. To evaluate the effect that DMPP accumulation in leaves may provoke on plant performance we combined a transcriptome, proteome, and physiological analysis in plants treated with 10 mg/ L of DMPP. This treatment provoked changes in the expression of 229 genes and 59 proteins. Overall, we evidence that when DMPP accumulates in leaves it induces stress responses, notably provoking changes in cell redox balance, hormone signaling, protein synthesis and turnover and carbon and nitrogen metabolism.

Nitrogen isotope signature evidences ammonium deprotonation as a common transport mechanism for the AMT-Mep-Rh protein superfamily.

Ammonium is an important nitrogen (N) source for living organisms, a key metabolite for pH control, and a potent cytotoxic compound. Ammonium is transported by the widespread AMT-Mep-Rh membrane proteins, and despite their significance in physiological processes, the nature of substrate translocation (NH3/NH4+) by the distinct members of this family is still a matter of controversy. Using Saccharomyces cerevisiae cells expressing representative AMT-Mep-Rh ammonium carriers and taking advantage of the natural chemical-physical property of the N isotopic signature linked to NH4+/NH3 conversion, this study shows that only cells expressing AMT-Mep-Rh proteins were depleted in 15N relative to 14N when compared to the external ammonium source. We observed 15N depletion over a wide range of external pH, indicating its independence of NH3 formation in solution. On the basis of inhibitor studies, ammonium transport by nonspecific cation channels did not show isotope fractionation but competition with K+. We propose that kinetic N isotope fractionation is a common feature of AMT-Mep-Rh-type proteins, which favor 14N over 15N, owing to the dissociation of NH4+ into NH3 + H+ in the protein, leading to 15N depletion in the cell and allowing NH3 passage or NH3/H+ cotransport. This deprotonation mechanism explains these proteins' essential functions in environments under a low NH4+/K+ ratio, allowing organisms to specifically scavenge NH4+. We show that 15N isotope fractionation may be used in vivo not only to determine the molecular species being transported by ammonium transport proteins, but also to track ammonium toxicity and associated amino acids excretion.

3,4-Dimethylpyrazole phosphate and 2-(N-3,4-dimethyl-1H-pyrazol-1-yl) succinic acid isomeric mixture nitrification inhibitors: Quantification in plant tissues and toxicity assays.

Nitrification inhibitors are used to maintain ammonium available in the soil for longer periods while reducing nitrate leaching and N2O emission. In this work we evaluated the potential toxicity effects of 3,4-dimethylpyrazole phosphate (DMPP) and 2-(N-3,4-dimethyl-1H-pyrazol-1-yl) succinic acid isomeric mixture (DMPSA) nitrification inhibitors. In order to determine the potential plant capacity to take up and translocate these inhibitors, we grew clover plants in hydroponic conditions and we developed a novel methodology for extracting DMPP and DMPSA that we quantified by HPLC. In addition, we also did toxicity bioassays: seed germination and Vibrio fischeri test. When clover was exposed to high amounts of nitrification inhibitors, plants accumulated DMPP, predominantly in leaves, and also DMPSA that preferentially accumulated in roots. These inhibitors did not provoke phytotoxicity at the equivalent of the maximum amount estimated in agriculture (0.5mg/kg soil). DMPP only provoked detrimental effects in plants at very high dose (100mg/kg soil). Interestingly, DMPSA was innocuous.

Leaves play a central role in the adaptation of nitrogen and sulfur metabolism to ammonium nutrition in oilseed rape (Brassica napus).

BACKGROUND: The coordination between nitrogen (N) and sulfur (S) assimilation is required to suitably provide plants with organic compounds essential for their development and growth. The N source induces the adaptation of many metabolic processes in plants; however, there is scarce information about the influence that it may exert on the functioning of S metabolism. The aim of this work was to provide an overview of N and S metabolism in oilseed rape (Brassica napus) when exposed to different N sources. To do so, plants were grown in hydroponic conditions with nitrate or ammonium as N source at two concentrations (0.5 and 1 mM). RESULTS: Metabolic changes mainly occurred in leaves, where ammonium caused the up-regulation of enzymes involved in the primary assimilation of N and a general increase in the concentration of N-compounds (NH4+, amino acids and proteins). Similarly, the activity of key enzymes of primary S assimilation and the content of S-compounds (glutathione and glucosinolates) were also higher in leaves of ammonium-fed plants. Interestingly, sulfate level was lower in leaves of ammonium-fed plants, which was accompanied by the down-regulation of SULTR1 transporters gene expression. CONCLUSIONS: The results highlight the impact of the N source on different steps of N and S metabolism in oilseed rape, notably inducing N and S assimilation in leaves, and put forward the potential of N source management to modulate the synthesis of compounds with biotechnological interest, such as glucosinolates.

Overexpression of a pine Dof transcription factor in hybrid poplars: A comparative study in trees growing under controlled and natural conditions.

In this work, the role of the pine transcriptional regulator Dof 5 in carbon and nitrogen metabolism has been examined in poplar trees. The overexpression of the gene and potential effects on growth and biomass production were compared between trees growing in a growth chamber under controlled conditions and trees growing in a field trial during two growth seasons. Ten-week-old transgenic poplars exhibited higher growth than untransformed controls and exhibited enhanced capacity for inorganic nitrogen uptake in the form of nitrate. Furthermore, the transgenic trees accumulated significantly more carbohydrates such as glucose, fructose, sucrose and starch. Lignin content increased in the basal part of the stem likely due to the thicker stem of the transformed plants. The enhanced levels of lignin were correlated with higher expression of the PAL1 and GS1.3 genes, which encode key enzymes involved in the phenylalanine deamination required for lignin biosynthesis. However, the results in the field trial experiment diverged from those observed in the chamber system. The lines overexpressing PpDof5 showed attenuated growth during the two growing seasons and no modification of carbon or nitrogen metabolism. These results were not associated with a decrease in the expression of the transgene, but they can be ascribed to the nitrogen available in the field soil compared to that available for growth under controlled conditions. This work highlights the paramount importance of testing transgenic lines in field trials.

Meat waste as feedstock for home composting: Effects on the process and quality of compost.

Home composting is a powerful tool, which is spreading in different parts of the world, to reduce the generation of municipal waste. However, there is debate concerning the appropriateness, in terms of domestic hygiene and safety, of keeping a composter bin in the household deputed to kitchen waste of animal origin, such as meat or fish scraps and pet droppings. The purpose of our work was to study how the addition of meat scraps to household waste influences the composting process and the quality of the final compost obtained. We compared four raw material mixtures, characterized by a different combination of vegetable and meat waste and different ratios of woody bulking agent. Changes in temperature, mass and volume, phenotypic microbial diversity (by Biolog™) and organic matter humification were determined during the process. At the end of the experiment, the four composts were weighed and characterized by physicochemical analysis. In addition, the presence of viable weed seeds was investigated and a germination bioassay was carried out to determine the level of phytotoxicity. Finally, the levels of pathogens (Escherichia coli and Salmonella spp.) were also determined in the final compost. Here we show that the presence of meat waste as raw feedstock for composting in bins can improve the activity of the process, the physicochemical characteristics and maturity of the compost obtained, without significantly affecting its salinity, pH and phytotoxicity. Pathogen levels were low, showing that they can be controlled by an intensive management and proper handling of the composter bins.

Quantitative proteomics reveals the importance of nitrogen source to control glucosinolate metabolism in Arabidopsis thaliana and Brassica oleracea.

Accessing different nitrogen (N) sources involves a profound adaptation of plant metabolism. In this study, a quantitative proteomic approach was used to further understand how the model plant Arabidopsis thaliana adjusts to different N sources when grown exclusively under nitrate or ammonium nutrition. Proteome data evidenced that glucosinolate metabolism was differentially regulated by the N source and that both TGG1 and TGG2 myrosinases were more abundant under ammonium nutrition, which is generally considered to be a stressful situation. Moreover, Arabidopsis plants displayed glucosinolate accumulation and induced myrosinase activity under ammonium nutrition. Interestingly, these results were also confirmed in the economically important crop broccoli (Brassica oleracea var. italica). Moreover, these metabolic changes were correlated in Arabidopsis with the differential expression of genes from the aliphatic glucosinolate metabolic pathway. This study underlines the importance of nitrogen nutrition and the potential of using ammonium as the N source in order to stimulate glucosinolate metabolism, which may have important applications not only in terms of reducing pesticide use, but also for increasing plants' nutritional value.

Root-shoot interactions explain the reduction of leaf mineral content in Arabidopsis plants grown under elevated [CO2 ] conditions.

Although shoot N depletion in plants exposed to elevated [CO2 ] has already been reported on several occasions, some uncertainty remains about the mechanisms involved. This study illustrates (1) the importance of characterizing root-shoot interactions and (2) the physiological, biochemical and gene expression mechanisms adopted by nitrate-fed Arabidopsis thaliana plants grown under elevated [CO2 ]. Elevated [CO2 ] increases biomass and photosynthetic rates; nevertheless, the decline in total soluble protein, Rubisco and leaf N concentrations revealed a general decrease in leaf N availability. A transcriptomic approach (conducted at the root and shoot level) revealed that exposure to 800 ppm [CO2 ] induced the expression of genes involved in the transport of nitrate and mineral elements. Leaf N and mineral status revealed that N assimilation into proteins was constrained under elevated [CO2 ]. Moreover, this study also highlights how elevated [CO2 ] induced the reorganization of nitrate assimilation between tissues; root nitrogen assimilation was favored over leaf assimilation to offset the decline in nitrogen metabolism in the leaves of plants exposed to elevated [CO2 ].

Root and shoot performance of Arabidopsis thaliana exposed to elevated CO2: A physiologic, metabolic and transcriptomic response.

The responsiveness of C3 plants to raised atmospheric [CO2] levels has been frequently described as constrained by photosynthetic downregulation. The main goal of the current study was to characterize the shoot-root relationship and its implications in plant responsiveness under elevated [CO2] conditions. For this purpose, Arabidopsis thaliana plants were exposed to elevated [CO2] (800ppm versus 400ppm [CO2]) and fertilized with a mixed (NH4NO3) nitrogen source. Plant growth, physiology, metabolite and transcriptomic characterizations were carried out at the root and shoot levels. Plant growth under elevated [CO2] conditions was doubled due to increased photosynthetic rates and gas exchange measurements revealed that these plants maintain higher photosynthetic rates over extended periods of time. This positive response of photosynthetic rates to elevated [CO2] was caused by the maintenance of leaf protein and Rubisco concentrations at control levels alongside enhanced energy efficiency. The increased levels of leaf carbohydrates, organic acids and amino acids supported the augmented respiration rates of plants under elevated [CO2]. A transcriptomic analysis allowed the identification of photoassimilate allocation and remobilization as fundamental process used by the plants to maintain the outstanding photosynthetic performance. Moreover, based on the relationship between plant carbon status and hormone functioning, the transcriptomic analyses provided an explanation of why phenology accelerates under elevated [CO2] conditions.

Leaf δ(15)N as a physiological indicator of the responsiveness of N2-fixing alfalfa plants to elevated [CO2], temperature and low water availability.

The natural (15)N/(14)N isotope composition (δ(15)N) of a tissue is a consequence of its N source and N physiological mechanisms in response to the environment. It could potentially be used as a tracer of N metabolism in plants under changing environmental conditions, where primary N metabolism may be complex, and losses and gains of N fluctuate over time. In order to test the utility of δ(15)N as an indicator of plant N status in N2-fixing plants grown under various environmental conditions, alfalfa (Medicago sativa L.) plants were subjected to distinct conditions of [CO2] (400 vs. 700 µmol mol(-1)), temperature (ambient vs. ambient +4°C) and water availability (fully watered vs. water deficiency-WD). As expected, increased [CO2] and temperature stimulated photosynthetic rates and plant growth, whereas these parameters were negatively affected by WD. The determination of δ(15)N in leaves, stems, roots, and nodules showed that leaves were the most representative organs of the plant response to increased [CO2] and WD. Depletion of heavier N isotopes in plants grown under higher [CO2] and WD conditions reflected decreased transpiration rates, but could also be related to a higher N demand in leaves, as suggested by the decreased leaf N and total soluble protein (TSP) contents detected at 700 µmol mol(-1) [CO2] and WD conditions. In summary, leaf δ(15)N provides relevant information integrating parameters which condition plant responsiveness (e.g., photosynthesis, TSP, N demand, and water transpiration) to environmental conditions.

Changes in the C/N balance caused by increasing external ammonium concentrations are driven by carbon and energy availabilities during ammonium nutrition in pea plants: the key roles of asparagine synthetase and anaplerotic enzymes.

An understanding of the mechanisms underlying ammonium (NH(4)(+)) toxicity in plants requires prior knowledge of the metabolic uses for nitrogen (N) and carbon (C). We have recently shown that pea plants grown at high NH(4)(+) concentrations suffer an energy deficiency associated with a disruption of ionic homeostasis. Furthermore, these plants are unable to adequately regulate internal NH4(+) levels and the cell-charge balance associated with cation uptake. Herein we show a role for an extra-C application in the regulation of C-N metabolism in NH(4)(+) -fed plants. Thus, pea plants (Pisum sativum) were grown at a range of NH(4)(+) concentrations as sole N source, and two light intensities were applied to vary the C supply to the plants. Control plants grown at high NH(4)(+) concentration triggered a toxicity response with the characteristic pattern of C-starvation conditions. This toxicity response resulted in the redistribution of N from amino acids, mostly asparagine, and lower C/N ratios. The C/N imbalance at high NH(4)(+) concentration under control conditions induced a strong activation of root C metabolism and the upregulation of anaplerotic enzymes to provide C intermediates for the tricarboxylic acid cycle. A high light intensity partially reverted these C-starvation symptoms by providing higher C availability to the plants. The extra-C contributed to a lower C4/C5 amino acid ratio while maintaining the relative contents of some minor amino acids involved in key pathways regulating the C/N status of the plants unchanged. C availability can therefore be considered to be a determinant factor in the tolerance/sensitivity mechanisms to NH(4)(+) nutrition in plants.

The physiological implications of urease inhibitors on N metabolism during germination of Pisum sativum and Spinacea oleracea seeds.

The development of new nitrogen fertilizers is necessary to optimize crop production whilst improving the environmental aspects arising from the use of nitrogenous fertilization as a cultural practice. The use of urease inhibitors aims to improve the efficiency of urea as a nitrogen fertilizer by preventing its loss from the soil as ammonia. However, although the action of urease inhibitors is aimed at the urease activity in soil, their availability for the plant may affect its urease activity. The aim of this work was therefore to evaluate the effect of two urease inhibitors, namely acetohydroxamic acid (AHA) and N-(n-butyl) thiophosphoric triamide (NBPT), on the germination of pea and spinach seeds. The results obtained show that urease inhibitors do not affect the germination process to any significant degree, with the only process affected being imbibition in spinach, thus also suggesting different urease activities for both plants. Our findings therefore suggest an activity other than the previously reported urolytic activity for urease in spinach. Furthermore, of the two inhibitors tested, NBPT was found to be the most effective at inhibiting urease activity, especially in pea seedlings.

Expression and localization of a Rhizobium-derived cambialistic superoxide dismutase in pea (Pisum sativum) nodules subjected to oxidative stress.

Two phylogenetically unrelated superoxide dismutase (SOD) families, i.e., CuZnSOD (copper and zinc SOD) and FeMn-CamSOD (iron, manganese, or cambialistic SOD), eliminate superoxide radicals in different locations within the plant cell. CuZnSOD are located within the cytosol and plastids, while the second family of SOD, which are considered to be of bacterial origin, are usually located within organelles, such as mitochondria. We have used the reactive oxygen species-producer methylviologen (MV) to study SOD isozymes in the indeterminate nodules on pea (Pisum sativum). MV caused severe effects on nodule physiology and structure and also resulted in an increase in SOD activity. Purification and N-terminal analysis identified CamSOD from the Rhizobium leguminosarum endosymbiont as one of the most active SOD in response to the oxidative stress. Fractionation of cell extracts and immunogold labeling confirmed that the CamSOD was present in both the bacteroids and the cytosol (including the nuclei, plastids, and mitochondria) of the N-fixing cells, and also within the uninfected cortical and interstitial cells. These findings, together with previous reports of the occurrence of FeSOD in determinate nodules, indicate that FeMnCamSOD have specific functions in legumes, some of which may be related to signaling between plant and bacterial symbionts, but the occurrence of one or more particular isozymes depends upon the nodule type.

Depletion of the heaviest stable N isotope is associated with NH4+/NH3 toxicity in NH4+-fed plants.

BACKGROUND: In plants, nitrate (NO3-) nutrition gives rise to a natural N isotopic signature (δ15N), which correlates with the δ15N of the N source. However, little is known about the relationship between the δ15N of the N source and the 14N/15N fractionation in plants under ammonium (NH4+) nutrition. When NH4+ is the major N source, the two forms, NH4+ and NH3, are present in the nutrient solution. There is a 1.025 thermodynamic isotope effect between NH3 (g) and NH4+ (aq) which drives to a different δ15N. Nine plant species with different NH4+-sensitivities were cultured hydroponically with NO3- or NH4+ as the sole N sources, and plant growth and δ15N were determined. Short-term NH4+/NH3 uptake experiments at pH 6.0 and 9.0 (which favours NH3 form) were carried out in order to support and substantiate our hypothesis. N source fractionation throughout the whole plant was interpreted on the basis of the relative transport of NH4+ and NH3. RESULTS: Several NO3--fed plants were consistently enriched in 15N, whereas plants under NH4+ nutrition were depleted of 15N. It was shown that more sensitive plants to NH4+ toxicity were the most depleted in 15N. In parallel, N-deficient pea and spinach plants fed with 15NH4+ showed an increased level of NH3 uptake at alkaline pH that was related to the 15N depletion of the plant. Tolerant to NH4+ pea plants or sensitive spinach plants showed similar trend on 15N depletion while slight differences in the time kinetics were observed during the initial stages. The use of RbNO3 as control discarded that the differences observed arise from pH detrimental effects. CONCLUSIONS: This article proposes that the negative values of δ15N in NH4+-fed plants are originated from NH3 uptake by plants. Moreover, this depletion of the heavier N isotope is proportional to the NH4+/NH3 toxicity in plants species. Therefore, we hypothesise that the low affinity transport system for NH4+ may have two components: one that transports N in the molecular form and is associated with fractionation and another that transports N in the ionic form and is not associated with fractionation.

Short term physiological implications of NBPT application on the N metabolism of Pisum sativum and Spinacea oleracea.

The application of urease inhibitors in conjunction with urea fertilizers as a means of reducing N loss due to ammonia volatilization requires an in-depth study of the physiological effects of these inhibitors on plants. The aim of this study was to determine how the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) affects N metabolism in pea and spinach. Plants were cultivated in pure hydroponic culture with urea as the sole N source. After 2 weeks of growth for pea, and 3 weeks for spinach, half of the plants received NBPT in their nutrient solution. Urease activity, urea and ammonium content, free amino acid composition and soluble protein were determined in leaves and roots at days 0, 1, 2, 4, 7 and 9, and the NBPT content in these tissues was determined 48h after inhibitor application. The results suggest that the effects of NBPT on spinach and pea urease activity differ, with pea being most affected by this treatment, and that the NBPT absorbed by the plant caused a clear inhibition of the urease activity in pea leaf and roots. The high urea concentration observed in leaves was associated with the development of necrotic leaf margins, and was further evidence of NBPT inhibition in these plants. A decrease in the ammonium content in roots, where N assimilation mainly takes place, was also observed. Consequently, total amino acid contents were drastically reduced upon NBPT treatment, indicating a strong alteration of the N metabolism. Furthermore, the amino acid profile showed that amidic amino acids were major components of the reduced pool of amino acids. In contrast, NBPT was absorbed to a much lesser degree by spinach plants than pea plants (35% less) and did not produce a clear inhibition of urease activity in this species.