NRT1.1-Related NH4 + Toxicity Is Associated with a Disturbed Balance between NH4 + Uptake and Assimilation.

A high concentration of ammonium (NH4 +) as the sole source of nitrogen in the growth medium often is toxic to plants. The nitrate transporter NRT1.1 is involved in mediating the effects of NH4 + toxicity; however, the mechanism remains undefined. In this study, wild-type Arabidopsis (Arabidopsis thaliana Columbia-0 [Col-0]) and NRT1.1 mutants (chl1-1 and chl1-5) were grown hydroponically in NH4NO3 and (NH4)2SO4 media to assess the function of NRT1.1 in NH4 + stress responses. All the plants grew normally in medium containing mixed nitrogen sources, but Col-0 displayed more chlorosis and lower biomass and photosynthesis than the NRT1.1 mutants in (NH4)2SO4 medium. Grafting experiments between Col-0 and chl1-5 further confirmed that NH4 + toxicity is influenced by NRT1.1. In (NH4)2SO4 medium, NRT1.1 induced the expression of NH4 + transporters, increasing NH4 + uptake. Additionally, the activities of glutamine synthetase and glutamate synthetase in roots of Col-0 plants decreased and soluble sugar accumulated significantly, whereas pyruvate kinase-mediated glycolysis was not affected, all of which contributed to NH4 + accumulation. By contrast, the NRT1.1 mutants showed reduced NH4 + accumulation and enhanced NH4 + assimilation through glutamine synthetase, glutamate synthetase, and glutamate dehydrogenase. Moreover, the up-regulation of genes involved in ethylene synthesis and senescence in Col-0 plants treated with (NH4)2SO4 suggests that ethylene is involved in NH4 + toxicity responses. This study showed that NH4 + toxicity is related to a nitrate-independent signaling function of NRT1.1 in Arabidopsis, characterized by enhanced NH4 + accumulation and altered NH4 + metabolism, which stimulates ethylene synthesis, leading to plant senescence.

Effects of the ammonium loading rate on nitrite-oxidizing activity during nitrification at a high dose of inorganic carbon.

In this study, the effects of the ammonium loading rate (ALR) and inorganic carbon loading rate (ILR) on the nitrification performance and composition of a nitrifying bacterial community were investigated in a moving bed biofilm reactor, using poly(vinyl alcohol) (PVA) sponge cubes as a supporting carrier. Between the two ALRs of 0.36 and 2.16 kg-N m-1 d-1, stable partial nitritation was achieved at the higher ALR. Inorganic carbon was dosed at high levels: 33.1, 22.0, 16.4, 11.0, and 5.4 times the theoretical amount. Nonetheless, nitrification efficiency was not affected by the ILR at the two ALRs. Quantitative PCR analysis of ammonia- and nitrite-oxidizing bacteria revealed that ALR is an important determinant of partial nitritation by accumulating ammonia-oxidizing bacteria in the nitrification system. In comparison, two nitrite-oxidizing bacterial genera (Nitrobacter and Nitrospira) showed almost the same relative abundance at various ALRs and ILRs. Terminal restriction fragment length polymorphism targeting the gene of ammonia monooxygenase subunit A revealed that Nitrosomonas europaea dominated under all conditions.

The logistic growth of duckweed (Lemna minor) and kinetics of ammonium uptake.

Mathematical models have been developed to describe nitrogen uptake and duckweed growth experimentally to study the kinetics of ammonium uptake under various concentrations. The kinetics of duckweed ammonium uptake was investigated using the modified depletion method after plants were grown for two weeks at different ammonium concentrations (0.5-14 mg/L) in the culture medium. The maximum uptake rate and Michaelis-Menten constant for ammonium were estimated as 0.082 mg/(g fresh weight x h) and 1.877 mg/L, respectively. Duckweed growth was assessed when supplied at different total nitrogen (TN) concentrations (1-5 mg/L) in the culture medium. The results showed that the intrinsic growth rate was from 0.22 to 0.26 d(-1), and TN concentrations had no significant influence on the duckweed growth rate.

The family of ammonium transporters (AMT) in Sorghum bicolor: two AMT members are induced locally, but not systemically in roots colonized by arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal (AM) fungi contribute to plant nitrogen (N) acquisition. Recent studies demonstrated the transport of N in the form of ammonium during AM symbiosis. Here, we hypothesize that induction of specific ammonium transporter (AMT) genes in Sorghum bicolor during AM colonization might play a key role in the functionality of the symbiosis. For the first time, combining a split-root experiment and microdissection technology, we were able to assess the precise expression pattern of two AM-inducible AMTs, SbAMT3;1 and SbAMT4. Immunolocalization was used to localize the protein of SbAMT3;1. The expression of SbAMT3;1 and SbAMT4 was greatly induced locally in root cells containing arbuscules and in adjacent cells. However, a split-root experiment revealed that this induction was not systemic. By contrast, a strictly AM-induced phosphate transporter (SbPt11) was expressed systemically in the split-root experiment. However, a gradient of expression was apparent. Immunolocalization analyses demonstrated that SbAMT3;1 was present only in cells containing developing arbuscules. Our results show that the SbAMT3;1 and SbAMT4 genes are expressed in root cortical cells, which makes them ready to accommodate arbuscules, a process of considerable importance in view of the short life span of arbuscules. Additionally, SbAMT3;1 might play an important role in N transfer during AM symbiosis.

Evidence that synergism between potassium and nitrate enhances the alleviation of ammonium toxicity in rice seedling roots.

Ammonium toxicity in plants is considered a global phenomenon, but the primary mechanisms remain poorly characterized. Here, we show that although the addition of potassium or nitrate partially alleviated the inhibition of rice seedling root growth caused by ammonium toxicity, the combination of potassium and nitrate clearly improved the alleviation, probably via some synergistic mechanisms. The combined treatment with potassium and nitrate led to significantly improved alleviation effects on root biomass, root length, and embryonic crown root number. The aberrant cell morphology and the rhizosphere acidification level caused by ammonium toxicity, recovered only by the combined treatment. RNA sequencing analysis and weighted gene correlation network analysis (WGCNA) revealed that the transcriptional response generated from the combined treatment involved cellulose synthesis, auxin, and gibberellin metabolism. Our results point out that potassium and nitrate combined treatment effectively promotes cell wall formation in rice, and thus, effectively alleviates ammonium toxicity.

Interactive effect of temperature, acidification and ammonium enrichment on the seagrass Cymodocea nodosa.

Global (e.g. climate change) and local factors (e.g. nutrient enrichment) act together in nature strongly hammering coastal ecosystems, where seagrasses play a critical ecological role. This experiment explores the combined effects of warming, acidification and ammonium enrichment on the seagrass Cymodocea nodosa under a full factorial mesocosm design. Warming increased plant production but at the expense of reducing carbon reserves. Meanwhile, acidification had not effects on plant production but increased slightly carbon reserves, while a slight stimulation of net production and a slight decrease on carbon reserves under ammonium supply were recorded. When all the factors were combined together improved the production and carbon reserves of Cymodocea nodosa, indicating that acidification improved ammonium assimilation and buffered the enhanced respiration promoted by temperature. Therefore, it could indicate that this temperate species may benefit under the simulated future scenarios, but indirect effects (e.g. herbivory, mechanical stress, etc.) may counteract this balance.

Preformulation investigation and challenges; salt formation, salt disproportionation and hepatic recirculation.

A compound, which is a selective peroxisome proliferator activated receptor (PPAR) agonist, was investigated. The aim of the presented studies was to evaluate the potential of the further development of the compound. Fundamental physicochemical properties and stability of the compound were characterized in solution by liquid chromatography and NMR and in solid-state by various techniques. The drug itself is a lipophilic acid with tendency to form aggregates in solution. The neutral form was only obtained in amorphous form with a glass-transition temperature of approximately 0°C. The intrinsic solubility at room temperature was determined to 0.03mg/mL. Chemical stability studies of the compound in aqueous solutions showed good stability for at least two weeks at room temperature, except at pH1, where a slight degradation was already observed after one day. The chemical stability in the amorphous solid-state was investigated during a period of three months. At 25°C/60% relative humidity (RH) and 40°C/75% RH no significant degradation was observed. At 80°C, however, some degradation was observed after four weeks and approximately 3% after three months. In an accelerated photostability study, degradation of approximately 4% was observed. Attempts to identify a crystalline form of the neutral compound were unsuccessful, however, salt formation with tert-butylamine, resulted in crystalline material. Results from stability tests of the presented crystalline salt form indicated improved chemical stability at conditions whereas the amorphous neutral form degraded. However, the salt form of the drug dissociated under certain conditions. The drug was administered both per oral and intravenously, as amorphous nanoparticles, to conscious dogs. Plasma profiles showed curves with secondary absorption peaks, indicating hepatic recirculation following both administration routes. A similar behavior was observed in rats after oral administration of a pH-adjusted solution. The observed double peaks in plasma exposure and the dissociation tendency of the salt form, were properties that contributed to make further development of the candidate drug challenging. Options for development of solid dosage forms of both amorphous and crystalline material of the compound are discussed.

Preparation of the chitosan grafted poly (quaternary ammonium)/Fe3O4 nanoparticles and its adsorption performance for food yellow 3.

Chitosan and its derivatives can be used to modify magnetic materials to promote the adsorption properties of the magnetic materials and avoid the weakness of chitosan and its derivatives. In the present study, chitosan grafted poly(trimethyl allyl ammonium chloride) (CTS-g-PTMAAC) was prepared by graft copolymerization; then it was coated on the surfaces of the sodium citrate coated Fe3O4 nanoparticles (SC-Fe3O4) to prepare a novel composite CTS-g-PTMAAC/SC-Fe3O4 magnetic nanoparticles, with which possesses abundant surface positive charges. The structure and properties of the CTS-g-PTMAAC/SC-Fe3O4 composite magnetic nanoparticles were characterized by FTIR, TEM, VSM, and zeta potential. The dye adsorption characteristics of the CTS-g-PTMAAC/SC-Fe3O4 nanoparticles were determined using the food yellow 3 aqueous solutions as a model food effluent. Effect of pH of the dye solution on the adsorption of food yellow 3 was determined and compared with N-2-hydroxylpropyl trimethyl ammonium chloride chitosan coated sodium citrate-Fe3O4 (CTS-g-HTCC/SC-Fe3O4) composite magnetic nanoparticles. The adsorption kinetics, adsorption isotherms, adsorption thermodynamics, and desorption and reusability of the magnetic nanoparticles were investigated.

Adaptation to rhizosphere acidification is a necessary prerequisite for wheat (Triticum aestivum L.) seedling resistance to ammonium stress.

Because soil acidification accompanies ammonium (NH4+) stress, the tolerance of higher plants to ammonium is associated with their adaptation to root medium acidification. However, the underlying mechanisms of this adaptation have not been fully elucidated. The objective of this study was thus to elucidate the effect of rhizosphere pH on NH4+ tolerance in different winter wheat cultivars (Triticum aestivum L.). Hydroponic experiments were carried out on two wheat cultivars: AK58 (an NH4+-sensitive cultivar) and XM25 (an NH4+-tolerant cultivar). Four pH levels resembling acidified (4.0, 5.0, 6.0 and 7.0) were tested and 5 mM NH4+ nitrogen (AN) was used as a stress treatment, with 5 mM nitrate nitrogen used as a control. The addition of AN led to a severe reduction in biomass and an increase in free NH4+, amino acids, and the activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in the shoots and roots of the two wheat cultivars. Further decreases in growth medium pH led to further increases in free NH4+, but decreases in total amino acids and the activities of GS and NADH-dependent glutamate synthase (NADH-GDH). However, there was less of an increase in free NH4+ and less of a reduction in the activities of GS and NADH-GDH in the cultivar XM25 compared with AK58. In addition, total soluble sugar content and the root-to-shoot soluble sugar ratio were also decreased by AN treatment, except in the shoots of XM25. Decreasing pH resulted in lower root-to-shoot soluble sugar ratios with greater reductions in the AK58 cultivar. These results indicate that wheat growth was inhibited significantly by the addition of NH4+ combined with low pH. Low medium pH reduced the capacity for nitrogen assimilation and interrupted carbohydrate transport between the shoot and root. The NH4+-tolerant cultivar XM25 was better adapted to low rhizosphere pH due to its increased capacity for assimilating NH4+ efficiently and thereby avoiding toxic levels of intracellular NH4+ at low medium pH.

Mechanisms of ammonium assimilation by Chlorella vulgaris F1068: Isotope fractionation and proteomic approaches.

Removal of ammonium (NH4(+)-N) by microalgae has evoked interest in wastewater treatment, however, the detailed mechanisms of ammonium assimilation remain mysterious. This study investigated the effects of NH4(+)-N concentration on the removal and biotransformation efficiency by Chlorella vulgaris F1068, and explored the mechanisms by (15)N isotope fractionation and proteome approaches. The results showed NH4(+)-N was efficiently removed (84.8%) by F1068 at 10mgL(-1) of NH4(+)-N. The isotope enrichment factor (ε=-2.37±0.08‰) of (15)N isotope fractionation revealed 47.6% biotransformation at above condition, while 7.0% biotransformation at 4mgL(-1) of NH4(+)-N (ε=-1.63±0.06‰). This was due to the different expression of glutamine synthetase, a key enzyme in ammonium assimilation, which was up-regulated 6.4-fold at proteome level and 18.0-fold at transcription level. The results will provide a better mechanistic understanding of ammonium assimilation by microalgae and this green technology is expected to reduce the burden of NH4(+)-N removal for municipal sewage treatment plants.

Absorption of unlabeled reduced iron of small particle size from a commercial source. A method to predict absorption of unlabeled iron compounds in humans

The absorption of a commercial brand of small-particle reduced iron was evaluated in 10 normal subjects. For each subject, the hemoglobin incorporation method was used to measure the true absorption of 60 mg of iron from either ferrous sulfate or ferric ammonium citrate. The iron tolerance test (ITT) was also studied for these two compounds and for reduced iron. This procedure consisted of measuring the area under the curve of plasma iron elevations at specified times for 6 hours, or the peak plasma iron, corrected by the plasma iron disappearance rate obtained from measuring plasma iron at specified times for 4 hours after the slow intravenous injection of 0.4 mg of iron as ferric citrate. Only the ITT was used to measure the absorption of 60 mg of reduced iron. Reference dose iron ascorbate absorption was measured in each subject. The absorption of ferric ammonium citrate and reduced iron was expressed as percent of dose and also as absorption percent of that of ferrous sulfate. Mean geometric "true absorptions" were 39.0 for reference dose, 10.4 for FeSO4 and 2.4 for ferric ammonium citrate. The later was 23 that of FeSO4. By ITT the mean geometric absorptions were 7.9, 3.7 and 3.2 for FeSO4, ferric ammonium citrate and reduced iron respectively, or 47 and 41 of that of FeSO4. We propose that the true absorption of the commercial brand of reduced iron tested was 20 that of FeSO4 based on the relation between the ITT results of reduced iron and the ITT and true absorption values of ferric ammonium citrate in relation to FeSO4. The use of this method for measuring absorption of unlabeled iron compounds is discussed.