Nitrate Reductase Enzyme Research Articles

Biosynthesis of Silver Chloride Nanoparticles Using Bacillus subtilis MTCC 3053 and Assessment of Its Antifungal Activity

The present investigation reported the synthesis of silver chloride nanoparticles using Bacillus subtilis. The adsorption of colloidal silver chloride nanoparticles showed an intense peak at the wavelength of 400 nm after 20 hrs of biomass incubation. The size of the silver nanoparticles ranges from 20 to 60 nm which was obtained from transmission electron microscope (TEM). The X-ray diffraction (XRD) pattern confirmed the crystalline nature of the nanoparticles. The bright circular spots of selected diffraction area (SAED) pattern also confirmed the good crystalline nature of the silver chloride nanoparticles with high magnification of TEM images. The presence of nitrate reductase enzyme in the cellular membrane of B. subtilis was confirmed by sodium dodecyl (SDS) polyacrylamide gel electrophoresis and it was found that the molecular weight is 37 kDa. The possible functional groups of the reductase enzyme in B. subtilis were identified by Fourier transform infrared spectroscopy (FTIR). Finally, antifungal activity of silver chloride nanoparticle was examined against Candida albicans, Aspergillus niger, and Aspergillus flavus. We conclude that the synthesis of silver chloride nanoparticles using microorganisms is more economical and simple. The antifungal property of silver chloride nanoparticles will play a beneficial role in biomedical nanotechnology.

Nitrate reductase activity in plant species of varied spatial association with acidic soils beneath Agathis australis

Distinctly different vegetation communities occur on the acidic soils that form beneath Agathis australis compared with adjacent lower acidity soils developed under other canopy species. Globally, species that occur on acidic soils typically have inherently low activity levels of the enzyme nitrate reductase, as they predominantly use NH4-N rather than NO3-N. Using in vivo assays, I examined the nitrate reductase activity in the leaves and roots of eight plant species of varying spatial association with A. australis. Seedlings of each species were grown together in a shade house in potting soils containing saturating levels of nitrate. I did not find evidence to suggest that the species common beneath A. australis, including Leucopogon fasciculatus in the Ericaceae, had lower abilities to metabolize nitrate when available than the understorey species dominant in surrounding forest with A. australis absent. The results suggest that the reduced abundance of A. australis-associated species within the surrounding forest is unlikely to be related to their ability to use NO3-N.

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Evaluasi Penampilan dan Efek Heterosis Hasil Persilangan Beberapa Aksesi Jarak Pagar (Jatropha curcas L.)

Agronomic performance and heterosis effect of progeny are important criteria for selection of parental hybrid. The agronomic performance evaluation of Jatropha progeny which was derived from crossing of some Jatropha accessions has been carried out for those goals. The study was conducted at Agro Techno Park, Indralaya South Sumatra. The crossing combinations were Pidie x Medan, Curup x Lampung, Pidie x Lampung, Lampung x Pidie, Curup x Medan, Indralaya x Pontianak, Medan x Palembang, Curup x Pidie, and Jogya x Komering. Oil production, nitrate reductase enzyme activity, leaf chlorophyll content and relative plant growth rate were evaluated. The result showed that the relative plant growth rate could be used as early detection parameter for yield potential. Progenies of Pontianak x Indralaya, Palembang x Indralaya and Pidie x Lampung were selected as promising hybrids based on high productivity and heterosis effect. Keywords: Jatropha curcas, heterosis, relative plant growth rate, nitrate reductase activity

Differential response of rice seedlings to salt stress in relation to antioxidant enzyme activity and membrane stability index

Three rice genotypes, IR 74802, IR 73104 and IR 72593, along with FL 478 and IR 29 as resistant and susceptible controls, respectively, were subjected to 21 days' salinity stress at the seedling stage in modified yoshida solution with two salt levels (60 and 120 mM NaCl). The results indicated that there was a profound increase in proline and ascorbic acid levels, and in the activity of nitrate reductase and antioxidant enzymes, i.e. catalase, peroxidase and ascorbate peroxidase, as well as malondialdhyde and membrane stability index, which were associated with salt tolerance. Salt stress had a significant and drastic effect on all parameters when the salinity level increased to 120 mM NaCl. The increased enzyme activity was directly related to an increased membrane stability index, as in IR 72593, which is identified as the most tolerant among the genotypes tested. It is clearly confirmed that predicting tolerance at the early seedling stage is the best way to assess the salinity tolerance level by utilizing physiological parameters, especially antioxidant enzyme activities which are found to be closely associated with salinity tolerance. Physiological adaptation of the plant to NaCl salt stress resulted in enhanced activity of stress-related enzymes and low sodium uptake in tolerant genotypes.

Eficiência na absorção e utilização de nitrogênio e atividade enzimática em genótipos de milho

O objetivo desse estudo foi investigar o emprego dos componentes da eficiencia no uso de nitrogenio e das atividades das enzimas nitrato redutase e glutamina sintetase na selecao de genotipos de milho eficientes no uso de nitrogenio (N). Foram avaliados 10 genotipos de milho em estadio de V4 em condicoes de alto e baixo N. O experimento foi composto por um fatorial 2 x 10 (dois niveis de N e 10 genotipos de milho) em delineamento inteiramente casualizados com tres repeticoes. Os seguintes caracteres foram avaliados: as eficiencias na absorcao (EAbN), na utilizacao (EUtN) e no uso de N (EUsN) e as atividades das enzimas glutamina sintetase (GS) e nitrato redutase (NR). Para os caracteres que o efeito de genotipos foi significativo (p<0,05) na analise de variância por nivel de N, as medias foram comparadas por meio do teste t (p<0,05). Os ganhos indiretos na EUsN e em seus componentes com selecao sobre as atividades das enzimas NR e GS foram estimados em baixo N. Conclui-se que em alto N: a EAbN e eficiente para discriminar genotipos de milho eficientes no uso de nitrogenio; em baixo N: as EAbN e EUtN sao eficientes para discriminar genotipos de milho eficientes no uso de nitrogenio, a atividade de NR nao e um bom parâmetro fisiologico para discriminar genotipos de milho eficientes no uso de nitrogenio, e a selecao sobre atividade da enzima GS possibilita a selecao indireta de genotipos de milho eficientes no uso de nitrogenio.

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Identification of factors constraining nitrate assimilation in Lake Superior, Laurentian Great Lakes

Despite a well-documented rise in nitrate concentration over the past century, Lake Superior has retained an oligotrophic character. In part, this status results from physical attributes of the lake including low temperatures and prolonged isothermy, resulting in deep-mixing and light limitation which constrain primary production. Lake Superior is also phosphorus deficient which limits phytoplankton growth. We conducted large (20 l) volume factorial bioassay experiments to assess the influence of light and nutrients (P, Fe) on nitrate assimilation by a Lake Superior chlorophyte alga. Bioassays seeded with the chlorophyte yielded a strong response to light result- ing in the rapid depletion of nitrate. High light resulted in higher activities of the key N-assimilation enzyme nitrate reductase (NR) and increased algal biomass compared to low light treatments. NR activity was highly correlated with rates of nitrate incorporation in bioassays and field surveys suggesting that NR occupies a critical place in nitrate metabolism. In bioassays, the addition of nutrients (P, Fe) only slightly increased the rate at which nitrate became depleted. Parallel trials using a luminescent cyano- bacterial bioreporter confirmed the lack of response by added nutrients supporting light as an important factor in constraining nitrate assimilation by phytoplankton in the lake.

Antioxidant defense system responses and role of nitrate reductase in the redox balance maintenance in Bradyrhizobium japonicum strains exposed to cadmium

In this work, we evaluated the effects of cadmium (Cd) on the antioxidant defense system responses and the role of nitrate reductase (NR) in the redox balance maintenance in Bradyrhizobium japonicum strains. For that, B. japonicum USDA110 and its NR defective mutant strain (GRPA1) were used. Results showed that the addition of 10μM Cd did not modify the aerobic growth of the wild type strain while the mutant strain was strongly affected. Anaerobic growth revealed that only the parental strain was able to grow under this condition. Cd reduced drastically the NR activity in B. japonicum USDA110 and increased lipid peroxide content in both strains. Cd decreased reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio in B. japonicum USDA110 although, a significant increased was observed in the mutant GRPA1. GSH-related enzymes were induced by Cd, being more evident the increase in the mutant strain. This different behavior observed between strains suggests that NR enzyme plays an important role in the redox balance maintenance in B. japonicum USDA 110 exposed to Cd.

Nitric oxide controls nitrate and ammonium assimilation in Chlamydomonas reinhardtii

Nitrate and ammonium are major inorganic nitrogen sources for plants and algae. These compounds are assimilated by means of finely regulated processes at transcriptional and post-translational levels. In Chlamydomonas, the expression of several genes involved in high-affinity ammonium (AMT1.1, AMT1.2) and nitrate transport (NRT2.1) as well as nitrate reduction (NIA1) are downregulated by ammonium through a nitric oxide (NO)-dependent mechanism. At the post-translational level, nitrate/nitrite uptake and nitrate reductase (NR) are also inhibited by ammonium, but the mechanisms implicated in this regulation are scarcely known. In this work, the effect of NO on nitrate assimilation and the high-affinity ammonium uptake was addressed. NO inhibited the high-affinity uptake of ammonium and nitrate/nitrite, as well as the NR activity, in a reversible form. In contrast, nitrite reductase and glutamine synthetase activities were not affected. The in vivo and in vitro studies suggested that NR enzyme is inhibited by NO in a mediated process that requires the cell integrity. These data highlight a role of NO in inorganic nitrogen assimilation and suggest that this signalling molecule is an important regulator for the first steps of the pathway.

Nitric oxide signaling and homeostasis in plants: a focus on nitrate reductase and S-nitrosoglutathione reductase in stress-related responses

Studies in the last two decades have firmly established that the gaseous free radical nitric oxide (NO) is an intracellular and intercellular mediator of signal transduction pathways controlling plant growth and development, as well as plant responses to biotic and abiotic stresses. The underlying mechanisms of NO action may rely on its reactivity with different kinds of biomolecules, leading to modulation of enzymatic activities, and of gene transcription, with profound impact on metabolism and signal transduction pathways. NO homeostasis depends on the appropriate coordination of NO synthesis and degradation under different physiological conditions. The mechanisms by which NO is synthesized de novo in plants are still a matter of controversy, although in the last years, the key role of the enzyme nitrate reductase (NR) in plants NO production has been widely accepted. In addition, S-nitrosoglutathione (GSNO), which forms by spontaneous reaction of NO with glutathione, is likely a major NO reservoir and NO donor in plant cells. GSNO levels are controlled by the enzyme GSNO reductase that has emerged as the main enzyme responsible for the modulation of S-nitrosothiol pools. The number of plant processes influenced/modulated by NO has dramatically increased in the last years. This review particularly emphasizes the roles of NR and GSNOR enzymes in NO homeostasis and NO-mediated plant responses to environmental challenges.

Interactive effect of salicylic acid on some physiological features and antioxidant enzymes activity in ginger (Zingiber officinale Roscoe).

The effect of foliar salicylic acid (SA) applications (10−3 and 10−5 M) on activities of nitrate reductase, guaiacol peroxidase (POD), superoxide dismutases (SOD), catalase (CAT) and proline enzymes and physiological parameters was evaluated in two ginger varieties (Halia Bentong and Halia Bara) under greenhouse conditions. In both varieties, tested treatments generally enhanced photosynthetic rate and total dry weight. Photosynthetic rate increases were generally accompanied by increased or unchanged stomatal conductance levels, although intercellular CO2 concentrations of treated plants were typically lower than in controls. Lower SA concentrations were generally more effective in enhancing photosynthetic rate and plant growth. Exogenous application of SA increased antioxidant enzyme activities and proline content; the greatest responses were obtained in plants sprayed with 10–5 M SA, with significant increases observed in CAT (20.1%), POD (45.2%), SOD (44.1%) and proline (43.1%) activities. Increased CAT activity in leaves is naturally expected to increase photosynthetic efficiency and thus net photosynthesis by maintaining a constant CO2 supply. Our results support the idea that low SA concentrations (10–5 M) may induce nitrite reductase synthesis by mobilizing intracellular NO3− and can provide protection to nitrite reductase degradation in vivo in the absence of NO3–. Observed positive correlations among proline, SOD, CAT and POD activities in the studied varieties suggest that increased SOD activity was accompanied by increases in CAT and POD activities because of the high demands of H2O2 quenching.

A Novel Tri-Enzyme System in Combination with Laser-Driven NMR Enables Efficient Nuclear Polarization of Biomolecules in Solution

NMR is an extremely powerful, yet insensitive technique. Many available nuclear polarization methods that address sensitivity are not directly applicable to low-concentration biomolecules in liquids and are often too invasive. Photochemically induced dynamic nuclear polarization (photo-CIDNP) is no exception. It needs high-power laser irradiation, which often leads to sample degradation, and photosensitizer reduction. Here, we introduce a novel tri-enzyme system that significantly overcomes the above challenges, rendering photo-CIDNP a practically applicable technique for NMR sensitivity enhancement in solution. The specificity of the nitrate reductase (NR) enzyme is exploited to selectively in situ reoxidize the reduced photo-CIDNP dye FMNH2. At the same time, the oxygen-scavenging ability of glucose oxidase (GO) and catalase (CAT) is synergistically employed to prevent sample photodegradation. The resulting tri-enzyme system (NR-GO-CAT) enables prolonged sensitivity-enhanced data collection in 1D and 2D heteronuclear NMR, leading to the highest photo-CIDNP sensitivity enhancement (48-fold relative to SE-HSQC) achieved to date for amino acids and polypeptides in solution. NR-GO-CAT extends the concentration limit of photo-CIDNP NMR down to the low micromolar range. In addition, sensitivity (relative to the reference SE-HSQC) is found to be inversely proportional to sample concentration, paving the way for the future analysis of even more diluted samples.

P74: Novel human mitochondrial enzyme can transform nitrite into nitric oxide

Endogenous metabolism of nitrate to nitrite to nitric oxide (NO) affects numerous biological events in humans, such as blood pressure control and hypoxic vasodilation, yet the molecular mechanism of this transformation remains uncertain and controversial. Nitrate and nitrite conversion to NO are reductive processes, requiring electron and proton transfer reactions, suggesting that oxidoreductase enzymes are involved. Nitrate reduction may occur through a combination of endogenous (i.e. human enzymes) and exogenous (i.e. commensal bacteria) nitrate reductase enzymes, while nitrite reduction to nitric oxide is likely catalyzed by endogenous nitrite reductase enzymes. Several mechanisms have been investigated, yet no conclusive data are available to support a single enzyme as an indispensable component for reduction of nitrite to NO in human tissue. It is likely that different enzyme systems are active in specific tissues under differential hypoxia, pH and co-factor regulatory control. We have identified a novel human nitrite reductase enzyme, which we hypothesize may contribute to mitochondrial reduction of nitrite to NO. The mitochondria amidoxime reducing component (mARC) enzyme is widely expressed in human tissue, but has an undefined physiological function. We hypothesize that mARC catalyzes the NADH-dependent reduction of nitrite to NO. To test this hypothesis the kinetics of nitrite reduction to NO by human mARC was investigated. Recombinant mARC was generated using standard molecular biology techniques, and then isolated using metal affinity chromatography. Site directed mutagenesis was used to change the active site cysteine into alanine. Human cytochrome b5 and cytochrome b5 reductase were also isolated to determine if mARC can utilize NADH as an electron source in conjunction with these enzymes. Nitric oxide chemiluminescence spectroscopy was used to measure NO-formation rates under anaerobic conditions. Our study established that mARC can generate NO from nitrite in the presence of NADH, cytochrome b5, and cytochrome b5 reductase at pH 7.4. The maximum velocity (Vmax) of NO-formation measured was 5 nmoles NO s−1 mg−1 protein. Moreover, mutation of the putative active site cysteine residue to alanine, and substitution of tungsten for molybdenum, completely abolished enzyme activity. The kinetic data supports our hypothesis and establishes that human mARC is capable of catalyzing reduction of nitrite to NO. Moreover, these data suggest that cysteine 270 and molybdenum are important in the transformation of nitrite to NO. Disclosure Supported by institution training grant (T32).

P59: Enteric bacterial nitrate/nitrite/nitric oxide/ammonia metabolism

In recent years the salivary bacterial reduction of nitrate ions to nitrite has been recognized as an important metabolic conversion in humans. The nitrogen cycle also involves reduction of nitrogen oxides to ammonium salts (denitrification) and oxidation of ammonia into nitrites and nitrates (nitrification) by soil bacteria. Neither process has significant relevance to human health, yet extensive research in the past 40–50 years showed that most enteric bacteria are capable of catalytic conversion of nitrate to ammonia via nitrite, a short-circuit in the nitrogen cycle. The importance of this pathway and the link between commensal gut bacteria, nitrogen oxides and human health or diseases has been little studied. Here we report quantification of nitrite and ammonia production from inorganic nitrate at different oxygen levels in Escherichia coli, the best understood enteric bacteria. E.coli uses nitrate as electron acceptor during anaerobic respiration and in our experiments grew well during both anaerobic and hypoxic conditions when supplied with low millimolar nitrate. We followed nitrite and ammonia formation during bacteria stationary growth under anaerobic, hypoxic (2–10% oxygen) and oxygenated conditions up to 48 h. We have found that in the presence of nitrate, nitrite accumulates inside bacteria but is eventually excreted in the media. Concentration levels rise with decreasing oxygen supplementation. The respiratory nitrate reduction involves the membrane-bound molybdenum containing enzyme nitrate reductase and nitrite formation was inhibited by supplementation of tungsten oxide in the growth media. Nitrite accumulation correlates with ammonia production, which follows a similar course but reaches about 25% of the nitrite produced at 24 h. In similar experiments we also measured nitric oxide (NO) production by chemiluminescence and show that intact E.coli cells are able to release NO gas when supplied with nitrate; initial rates of NO production increase as the media pH is decreased. Our result indicate that nitrate is first converted to nitrite by a molybdenum containing enzyme and subsequently reduced to ammonium, possibly by the well-characterized penta-heme cytochrome c nitrite reductase. A growing number of reports are providing evidence of the tight relationship between human microbiota and health: we suggest the hypoxically-induced bacterial nitrate to ammonia reduction could be an important aspect of the mammalian nitrate/nitrite/NO metabolism. Disclosure Nothing to disclose.

Post-translational control of nitrate reductase activity responding to light and photosynthesis evolved already in the early vascular plants

Regulation of nitrate reductase (NR) by reversible phosphorylation at a conserved motif is well established in higher plants, and enables regulation of NR in response to rapid fluctuations in light intensity. This regulation is not conserved in algae NR, and we wished to test the evolutionary origin of the regulatory mechanism by physiological examination of ancient land plants. Especially a member of the lycophytes is of interest since their NR is candidate for regulation by reversible phosphorylation based on sequence analysis. We compared Selaginella kraussiana, a member of the lycophytes and earliest vascular plants, with the angiosperm Arabidopsis thaliana, and also tested the moss Physcomitrella patens. Interestingly, optimization of assay conditions revealed that S. kraussiana NR used NADH as an electron donor like A. thaliana, whereas P. patens NR activity depended on NADPH. Examination of light/darkness effects showed that S. kraussiana NR was rapidly regulated similar to A. thaliana NR when a differential (Mg2+ contra EDTA) assay was used to reveal activity state of NR. This implies that already existing NR enzyme was post-translationally activated by light in both species. Light had a positive effect also on de novo synthesis of NR in S. kraussiana, which could be shown after the plants had been exposed to a prolonged dark period (7 days). Daily variations in NR activity were mainly caused by post-translational modifications. As for angiosperms, the post-translational light activation of NR in S. kraussiana was inhibited by 3-(3,4-dichlorophenyl)-1*1-dimethylurea (DCMU), an inhibitor of photosynthesis and stomata opening. Evolutionary, a post-translational control mechanism for NR have occurred before or in parallel with development of vascular tissue in land plants, and appears to be part of a complex mechanisms for coordination of CO2 and nitrogen metabolism in these plants.

渍水对油菜苗期生长及生理特性的影响

通过盆栽试验研究了模拟渍水条件对苗期油菜（<em>Brassica napus</em> L.）中双11号生长及生理特性的影响。结果表明，渍水超过3 d就会显著抑制苗期油菜的生长，这与渍水降低油菜叶片叶绿体和光合色素含量、降低氮素吸收利用和引起过氧化胁迫有关。渍水后苗期油菜的生理恢复，无论是叶绿体和光合色素含量，还是硝酸还原酶及抗氧化酶活性的恢复均表现出一定的滞后性，而且渍水时间越长滞后期越长。此外，在苗期油菜叶片光合色素中，渍水对类胡萝卜素的影响要大于对叶绿素的影响。;A pot experiment was conducted to investigate the effects of waterlogging on the growth and physiological properties of juvenile oilseed rape (<em>Brassica napus</em> L cv. Zhongshuang No. 11). Waterlogging lasted 3 or more days significantly depressed the plant growth, which might be due to it decreased the chloroplast and photosynthetic pigment content, nitrogen uptake and utilization, and induced the oxidization of plant. The effects of waterlogging on juvenile oilseed rape growth is mainly attributed to its influence on the recovery process of physiological properties after flooding, which was prolonged with the period of waterlogging lasted, including the chloroplast and photosynthetic pigment content, nitrate reductase and antioxidant enzyme activities. Moreover, in photosynthetic pigment, the effect of waterlogging on the carotenoid content was more significant than chlorophyll content of juvenile oilseed rape.

Determination of Denitrification Genes Abundance in Environmental Samples

Diversity of microorganisms involved in the biogeochemi- cal N cycle is of fundamental interest in microbial ecology. Denitrifica- tion is a key step in the cycle by which nitrate is reduced to dinitrogen gas via the soluble nitrite and the gaseous compounds nitric oxide and nitrous oxide. The process is carried out by the sequential activity of the nitrate, nitrite, nitric oxide, and nitrous oxide reductase enzyme, respectively. The fluorescence-based quantitative real-time polymerase chain reaction (qPCR) is widely used for quantification of nucleic acids in samples obtained from numerous, diverse sources. Here, we provide a well-proven methodology for isolation of DNA from environmen- tal samples and describe relevant experimental conditions for utiliza- tion of qPCR to assay the 16S rRNA and nar/nap, nirK/nirS ,c -nor/q- nor ,a ndnos denitrification genes that encode synthesis of denitrifying enzymes. The ISO 11063 standard method and MIQUE guidelines are considered with the aim to increase experimental transparency.

Effect of methanol on winter rape seedlings

Oil seed rape seedlings which had been treated with 10-30% methanol grew faster, their yield of fresh matter exceeded that of untreated control plants by 102%, of dry matter by 80%. Although methanol did not affect the content of chlorophyll or carotene in the leaves, the overall pigment yield grew with the dry matter of one seedling. The biomass of rape leaves which had been sprayed with 10 or 20% methanol solutions was by 50-90% higher as compared to untreated plants. The activity of some enzymes (nitrate reductase and alkaline phosphatase) also increased in methanol treated plants.

Diversity Indices and Growth Parameters of Cyanobacteria from Three Lakes of Rajasthan

Twenty three cyanobacterial strains were isolated from alkaline water samples of three aquatic sites (Anasagar, Pushkar lake and Sambhar lake) from Ajmer district of Rajasthan. Microscopic examination showed one colonial, nine heterocystous and the remaining non-heterocystous filamentous forms. Genera wise distribution showed abundance of Nostoc, Phormidium and Plectonema followed by Anabaena and Westiellopsis. Microcystis and Oscillatoria were least abundant. Nostoc and Phormidium showed highest abundance in terms of CFU/mL followed by Anabaena and Plectonema. Pigments (chlorophyll, carotenoids and phycobilins) and N assimilatory enzymes (nitrate reductase and glutamine synthetase) differed significantly amongst the identified cyanobacterial strains.