Nitrate Reductase Enzyme Research Articles

Nitrate and ammonium differ in their impact on δ13C of plant metabolites and respired CO2 from tobacco leaves

ABSTRACT The carbon isotopic composition (δ13C) of foliage is often used as proxy for plant performance. However, the effect of vs. supply on δ13C of leaf metabolites and respired CO2 is largely unknown. We supplied tobacco plants with a gradient of to concentration ratios and determined gas exchange variables, concentrations and δ13C of tricarboxylic acid (TCA) cycle intermediates, δ13C of dark-respired CO2, and activities of key enzymes nitrate reductase, malic enzyme and phosphoenolpyruvate carboxylase. Net assimilation rate, dry biomass and concentrations of organic acids and starch decreased along the gradient. In contrast, respiration rates, concentrations of intercellular CO2, soluble sugars and amino acids increased. As decreased, activities of all measured enzymes decreased. δ13C of CO2 and organic acids closely co-varied and were more positive under supply, suggesting organic acids as potential substrates for respiration. Together with estimates of intra-molecular 13C enrichment in malate, we conclude that a change in the anaplerotic reaction of the TCA cycle possibly contributes to 13C enrichment in organic acids and respired CO2 under supply. Thus, the effect of vs. on δ13C is highly relevant, particularly if δ13C of leaf metabolites or respiration is used as proxy for plant performance.

Nitrate Reductase Nanoparticles: Synthesis and Characterization

Nanoparticles of enzyme Nitrate reductase (NaR) a soluble homodimer enzyme of ∼100 kDa polypeptide with cofactors – FAD, heme-molybdopterin (Mo-MPT) and electron donor NAD(P)H, catalyses the reduction of nitrate to nitrite has been synthesised. Nanoparticles of Nitrate reductase enzyme have been prepared by chemical desolvation method including glutaraldehyde cross-linking to form the nanoaggregate. Characterisation of NaR nanoparticles has been made by Transmission Electron Microscopy (TEM), UV-Visible Spectroscopy and by electrochemical Impedance Spectroscopic Study (EIS). TEM study revealed the size of globular aggregated was in the range of 20–30 nm. UV Visible spectroscopic studies depicted that the absorption of NaR NPS is much higher at 560 nm than that of the free enzyme, which showed maximum absorption at 540 nm. NaR NPs aggregates formed were more active, highly stable, have a higher shelf life and can be reused repeatedly. Enzyme nanoparticles with 10-100 nm dimensions and exhibit unique physical, chemical and catalytic properties due to increased surface area. Nitrate reductase nanoparticles can be used as a biochemical tool to increase protein production and grain yield by promoting amino acids production in plants. The synthesised NaR NPs are used in the fabrication of enzyme-based nanosensor in the detection of nitrates in drinking water and serum samples.

Photosynthetic efficiency and nutrient physiology of the diatom Thalassiosira pseudonana at three growth temperatures

Diatom cells utilize a variety of metabolic pathways to cope with internal energy imbalances caused by stressful environmental conditions. In this study, the model diatom species, Thalassiosira pseudonana, was grown in nutrient replete and nitrate (NO3−)- and dissolved silicate (Si)-depleted media at three growth temperatures (4, 17, 28 °C) to determine how nutrient enrichment and temperature affects diatom growth, photosynthetic efficiency, nitrate reductase (NR) enzyme activity, biogenic silica (bSiO2) deposition, and NR gene expression. Growth rates for nutrient-replete cultures were highest at 17 °C. Across all nutrient treatments, the cells grown at 17 °C had an average Fv/Fm of 0.44 ± 0.006, while the cells were grown at 4 °C and 28 °C had an average Fv/Fm of 0.37 ± 0.004 and 0.38 ± 0.01, respectively. Activity of NR was variable across treatments with no significant effect of temperature. The relative expression of the targeted NR gene was, on average, ~ 10 times higher in the 4 °C cultures and ~ 4 times higher in the 28 °C than in the 17 °C cultures, while the activity of the NR enzyme was generally highest in the cultures grown at 17 °C that were enriched with NO3−. Cells grown under nutrient-replete conditions had significantly higher bSiO2 deposition rates at 4 °C than cells grown at 17 and 28 °C. These data support the notion that cold, nutrient-replete conditions lead to increases in diatom silicification and that NR activity may be regulated downstream of mRNA transcription under specific environmental conditions.

PHYTOMEDIATED SYNTHESIS OF SILVER NANOPARTICLES AGAINST MICROBIAL PATHOGENS AND CYTOTOXICITY ON HUMAN BREAST CANCER CELLS (MCF-7)

Silver nanoparticles (AgNPs) are synthesized from the root extract of the Abutilon indicum plant. Nitrate reductase enzyme and/or other extra cellular proteins released from the extract reduce the silver nitrate to silver ions. These proteins or enzymes serve as a template for the silver nucleation sites in the development of silver nanoparticles, and also act as capping agents, preventing silver ion agglomeration. XRD analysis predicts the phase of the nanoparticles. Transmission Electron Microscope (TEM) and Scanning Electron Microscope analyses have revealed that the synthesized AgNPs are spherical in shape, with an average size of 17 nm. From the data, it is noted that the protein molecules and fatty acids present in the root extract of Abutilon indicum, play a vital role in reducing silver salts and as capping AgNPs at various concentrations. Bactericidal activity acting against the clinical pathogens was performed, and it was observed that NP inhibition is highly dependent on their size and surface. Cytotoxic studies were carried out with these synthesized silver nanoparticles using MTT assay on MCF-7 cells.

Correlation of Aerobic Exercise and High Nitrate Diet with Population of Eschericia Coli in the Digestive Tract of Liver Cirrhosis Individuals

Background: In liver cirrhosis, the population of E coli is increased. conditions such as reduced intestinal). Escherichia coli with 2 enzyme nitrate reductase (NRF and Nir) reduce nitrate to nitrite and subsequently converted to ammonia (99%) and nitric oxide (1%) in anaerobic condition. Regular aerobic exercise 2-3 times/week for 30 minutes resulted in increased 2,3-DPG which reduces the activity of E. coli to reduce nitrate to nitrite and ammonia, which only works on the anaerobic state. High Nitrate Diets lead to increased nitrate reducing bacteria such as E. coli resulting in the reduction of nitrate excess produce nitrite and ammonia in large quantities. Probiotic Lactobacillus spp. can suppress the growth of bacterial endotoxins and pathogens such as E. coli and other Enterobacteriaceae. This study aimed to determine the correlation of aerobic exercise and a high nitrate diet in gastrointestinal populations of Escherichia coli gastrointestinal tract in patient with liver cirrhosis.Method: This was a descriptive-experimental study in liver cirrhosis patients Child Pugh A/B in outpatient clinic Saiful Anwar Hospital in August 2015. Respondents were asked to fill out a questionnaire with information about the demographic data, the nitrate diet, aerobic exercise, other medical data and sanitation, and stool samples were taken for faecal culture. Eta Correlation statistical test was used to determine the correlation of aerobic exercise and a high nitrate diet high in population of E. coli. The significant difference are indicated by p 0.005.Results: A total of 36 patients diagnosed with liver cirrhosis Child Pugh A/B, 14 (39%) underwent aerobic exercise 3x /week, as many as 25 (70%) consume a high nitrate diet. There was a strong relationship between aerobic exercise and high nitrate diet with population of E. coli (Ƞ = 0.725; p 0.05).Conclusion: There was a strong relationship between aerobic exercise and high nitrate diet with a population of E. coli in liver cirrhosis. Giving probiotics in patients with liver cirrhosis suppressed the population of E. coli.

Effect of Foliar Feeding of Gluconate and EDTA Chelated Plant Nutrients on Yield, Chlorophyll Content and Nitrate Reductase Enzyme of Bt Cotton under Rainfed Ecosystem of Marathawada

An experiment was conducted to find out the “Effect of foliar feeding of Gluconate and EDTA chelated plant nutrients on yield, Chlorophyll content and Nitrate reductase enzyme activity of Bt-cotton under rainfed ecosystem of Marathawada” at Department of Soil Science and Agril Chemistry, VNMKV, Parbhani. The experiment includes sixteen treatments viz,T1-control,T2-ZnGluconate, T3-Zn EDTA, T4-Mn gluconate, T5- Mn EDTA, T6- Cu Gluconate, T7- Cu EDTA, T8- FeGluconate ,T9- Fe EDTA, T10- CaGluconate, T11- Ca EDTA, T12-MgGluconate, T13-MgEDTA, T14- Zn, Mn, Cu, Fe, Ca and Mg Gluconate, T15-Zn, Mn, Cu, Fe, Ca and Mg EDTA and T16- Govt. grade II and replicated twice. The treatments were fertilized with 120:60:60 N,P2O5 and K2O Kg ha-1 .Micronutrient sprays of gluconate and EDTA chelated plant nutrients were applied to the crop at the time of flowering i.e. at 55 DAS and second spray was applied at the time of boll development stage i.e.at 75 DAS. The treatment T2 showed more number of bolls per plant followed by treatment T3. The maximum boll weight was observed with treatment Zn gluconate. Spraying of Zn gluconate, Zn EDTA and Fe and Mg gluconate nutrients have produced more seed cotton yield. Quantitative analysis of chlorophyll was done by using DMSO as an extractant. Chlorophyll a, chlorophyll b and total chlorophyll content in leaves also influenced significantly due to different foliar feeding. The highest chlorophyll a, chlorophyll b and total chlorophyll was registered with the treatment Fe gluconate spray followed by Fe EDTA. Nitrate reductage and acid phosphate activity were improved by the application of T2 and was found to be significantly superior over control. This study was conducted under rainfed ecosystem.

Effects of boron toxicity on growth, oxidative damage, antioxidant enzymes and essential oil fingerprinting in Mentha arvensis and Cymbopogon flexuosus

BackgroundBoron (B) is an essential micronutrient and necessary to plants for their proper growth. The concentration of B in soil and plant is greatly critical for the growth and productivity. Even at the low amount available in soils, B can be extremely toxic to plants especially in semi-arid and arid environments. In the present study, the effects of high B concentrations (2.5, 5, 10, 20 and 30 mg/kg) were elucidated on two important essential oil-bearing plants; Mentha arvensis and Cymbopogon flexuosus which are aromatic and antimicrobial herbs having well-known medicinal values.ResultsApplication of different concentrations of B showed growth inhibitory effects on plant as evident by shoot and root lengths, fresh and dry weights of shoot in the studied plants. Treatments of B also reduced the total chlorophyll and carotenoid content, chlorophyll fluorescence and reduced the activities of carbonic anhydrase and nitrate reductase enzymes. Moreover, B stress considerably increased the proline content and lipid peroxide content as compared to control. The activities of antioxidant enzymes like catalase, peroxidase and superoxide dismutase were also significantly increased under B stress. The content of essential oil of M. arvensis and C. flexuosus increased at 2.5 mg/kg of B and decreased with further increase in concentrations of B.ConclusionThe findings of present work suggest that increasing concentrations of B inhibited growth and photosynthetic pigments, increased oxidative damage and activities of antioxidant enzymes; however, a mild stress of B increased essential oil production in M. arvensis and C. flexuosus plants.

СОДЕРЖАНИЕ АЗОТА В ПОЧВЕ И АКТИВНОСТЬ НИТРАТРЕДУКТАЗЫ В ЛИСТЬЯХ ОЗИМОЙ ПШЕНИЦЫ ПРИ ПРИМЕНЕНИИ АЗОТНЫХ УДОБРЕНИЙ

The purpose of the study is increasing the efficiency of using nitrogen fertilizers and its effect on nitrate-reducing capacity of the leaf apparatus. The research was conducted in 2016-2018 in the Central zone of the Samara re-gion. The predecessor is complete fellow. Svetoch winter wheat elite seed varieties were used for sowing. Seeding was carried out in the experimental fields of the laboratory «Agroecology» of the Samara state University. Nitrate and ammonium nitrogen content in soil layer of 0-30 cm depth was estimated before planting and after plant nutri-tion with nitrogen fertilizers in regard to the growth phases (tillering, elongation and heading), the content of nitro-gen, protein and nitrate reductase enzyme activity in leaves during plant development. Dynamics changes of nitro-gen content in the soil and after fertilization with nitrogen shows that during vegetation period of plants, the amount of nitrogen is sufficient for their growth. As the phenological phases of plants change, the content of nitrogen and protein in winter wheat leaves increases, and activity of the enzyme nitrate reductase lifts, which catalyzes nitrate regeneration to nitrite. The value of the activity of the enzyme nitrate reductase in leaves can serve as a criterion for evaluating the availability of plants with the nitrate form of nitrogen. In future, the activity of this enzyme can be used as an indicator of optimizing nitrogen nutrition of plants aimed at metabolic processes. The use of various nitrogen fertilizers, and to a greater extent ammonium nitrate, increased the supply of plants with nitrogen, which is later used by them in the reutilization of nitrogen from leaves to the emerging seed, thus contributing to quality of grain.

Characterization of an aerobic denitrifier Enterobacter cloacae strain HNR and its nitrate reductase gene.

Enterobacter cloacae strain HNR was found to grow well and denitrify aerobically at high NO3--N concentrations. When the concentrations of NO3--N were 200, 300 and 500 mg/L, the removal efficiencies of NO3--N were 83%, 74.5% and 75%, respectively. More importantly, the intermediates accumulation of NO2--N and NH4+-N was not obvious during the aerobic denitrification processes, resulting in a high TN removal of 82%, 74% and 70%, respectively. Meanwhile, strain HNR also presented the ability of heterotrophic nitrification. With initial NH4+-N concentrations of 20 and 80 mg/L, the NH4+-N removal efficiency reached 78% and 76%, respectively. The key nitrate reductase enzyme gene relating to denitrification was successfully amplified by polymerase chain reaction (PCR) from strain HNR, and identified it as napA, which encodings the large catalytic subunit A of periplasmic nitrate reductase (NAPA). The sequence analysis of napA indicates that NAPA is a hydrophilic, non-transmembrane protein. The existence of napA might be crucial for strain HNR to denitrify nitrate under aerobic conditions. This study showed prospect to develop novel technology for nitrogen removal by application of E. cloacae strain HNR.

Nitrate reductase rather than nitric oxide synthase activity is involved in 24-epibrassinolide-induced nitric oxide synthesis to improve tolerance to iron deficiency in strawberry (Fragaria × annassa) by up-regulating the ascorbate-glutathione cycle

Involvement of nitrate reductase (NR) and nitric oxide synthase (NOS)-like enzyme in 24-epibrassinolide (EB)-triggered nitric oxide (NO) synthesis to improve iron deficiency (ID) tolerance in strawberry plants was studied. EB was sprayed to strawberry plants every two days for two weeks. Then, the EB-treated plants were pre-treated with inhibitors of NR, tungstate, or NOS, L-NAME for 3 h. During the first three weeks, Fe was supplied as 100 μM EDTA-Fe or FeSO4 to Fe-sufficient or Fe-deficient plants, respectively. Thereafter, plants were subjected for further three weeks to control (100 μM EDTA-Fe) and Fe deficiency (ID; without Fe). ID reduced biomass, chlorophyll, and chlorophyll fluorescence, while increased oxidative stress parameters, ascorbate (AsA), glutathione (GSH), endogenous NO, and the activities of NR, NOS, and antioxidant enzymes. Pre-treatments with EB and EB + SNP improved ID tolerance of strawberry by improving leaf Fe2+, plant growth, and antioxidant enzyme activities, and causing a further elevation in AsA, GSH, NO, NR and NOS. L-NAME application reversed NOS activity, but it did not eliminate NO, however, tungstate application reversed both NR activity and NO synthesis in plants exposed to ID + EB, suggesting that NR is the main contributor of EB-induced NO synthesis to improve ID tolerance in strawberry plants.

Do nitrogen sources and molybdenum affect the nutritional quality and nitrate concentrations of hydroponic baby leaf lettuce?

Lettuce is one of the most popular vegetables, and the market niche of the baby leaf variety is expanding. The manner in which nitrogen (N) is supplied and the available concentration of the micronutrient molybdenum (Mo) affect N metabolism, with reflects on the nutritional quality of that vegetable. Here, two Mo concentrations (0.06 and 0.12mg/L) and four proportions (%) of nitrate (NO3 - )/ammonium (NH4 + )(100/0, 75/25, 50/50, and 25/75) were supplied to hydroponic baby leaf lettuce floating type to evaluate their effects on plant growth and leaf mineral and NO3 - compositions. Shoot dry mass did not differ among the different treatments, although fresh mass was lower in treatments with larger proportions of NH4 + . Higher leaf concentrations of NO3 - were observed in plants treated with 100% N-NO3 - , but they were still below tolerable limits for human health. The enzyme nitrate reductase was not found to be sensitive to the nitrogen sources or to Mo concentrations. N proportions and Mo concentrations differently affected macro- (C, N, P, K, Ca, Mg, and S) and micronutrients (Cu, Fe, Mn, Mo, and Zn) leaf concentrations. Although treatment with 100% N-NO3 - favored higher mineral concentrations in lettuce leaves, the addition of 25% N-NH4 + allowed fresh mass production with the lowest NO3 - concentrations. As such, and considering the healthy reduction of NO3 - consumption by humans and the maintenance of plant productivity, the 75/25 NO3 - /NH4 + proportion is recommended for the hydroponic cultivation of baby leaf var. Mimosa lettuce. PRACTICAL APPLICATION: We demonstrate a direct link between the constitution of nutrient solution with nitrate accumulation by hydroponic lettuce and indicate the best source of N as well as the concentration of Mn to healthy reduction of NO3 - consumption by humans and the maintenance of plant productivity.

Seasonal variation in nitrate reductase activity and protein content in response to growth in transplanted trees at Fisher forest and Lion safari of U.P.

Nitrate reductase enzyme controls the influx of nitrate ion in the cytoplasm and reduces it to nitrite for assimilation into protein. High growth and productivity (i.e. P/R more than unity) is essential for establishment of any ecosystem. Being the component of chlorophyll porphyrin and building and functional blocks of amino acids, the nitrogen plays a crucial and important role in plant metabolism. Nitrogenase complex enzymes ensure the continuous supply of nitrogenous molecules for growth and development of plant. The present study was undertaken to study the seasonal variation in nitrate reductase activity, protein content and growth in nine important tree species of Fisher forest, Etawah viz. Azadirachta indica, Bauhinia variegata, Dalbergia sissoo, Ficus bengalensis, Ficus religiosa, Ficus virens, Kigella africana, Pongamia pinnata, Tabebuia argentea. The study establishes the relationship between nitrate reductase activity, protein content and growth of transplanted trees. Nitrate reductase activity is a valuable tool for monitoring the physiological status of a plant to environmental fluctuations. Growth of undertaken trees depends on nitrate reductase activity and protein content. However, it is observed that high protein content and not the nitrate reductase activity is always responsible for growth of plant as is evident in Dalbergia sissoo.

Enzyme Mimetic Active Intermediates for Nitrate Reduction in Neutral Aqueous Media.

Nitrate is a pervasive aquatic contaminant of global environmental concern. In nature, the most effective nitrate reduction reaction (NRR) is catalyzed by nitrate reductase enzymes at neutral pH, using a highly-conserved Mo center ligated mainly by oxo and thiolate groups. Mo-based NRR catalysts mostly function in organic solvents with a low water stability. Recently, an oxo-containing molybdenum sulfide nanoparticle that serves as an NRR catalyst at neutral pH was first reported. Herein, in a nanoparticle-catalyzed NRR system a pentavalent MoV (=O)S4 species, an enzyme mimetic, served as an active intermediate for the NRR. Potentiometric titration analysis revealed that a redox synergy among MoV -S, S radicals, and MoV (=O)S4 likely play a key role in stabilizing MoV (=O)S4 , showing the importance of secondary interactions in facilitating NRR. The first identification and characterization of an oxo- and thiolate-ligated Mo intermediates pave the way to the molecular design of efficient enzyme mimetic NRR catalysts in aqueous solution.

Enzyme Mimetic Active Intermediates for Nitrate Reduction in Neutral Aqueous Media

AbstractNitrate is a pervasive aquatic contaminant of global environmental concern. In nature, the most effective nitrate reduction reaction (NRR) is catalyzed by nitrate reductase enzymes at neutral pH, using a highly‐conserved Mo center ligated mainly by oxo and thiolate groups. Mo‐based NRR catalysts mostly function in organic solvents with a low water stability. Recently, an oxo‐containing molybdenum sulfide nanoparticle that serves as an NRR catalyst at neutral pH was first reported. Herein, in a nanoparticle‐catalyzed NRR system a pentavalent MoV(=O)S4 species, an enzyme mimetic, served as an active intermediate for the NRR. Potentiometric titration analysis revealed that a redox synergy among MoV−S, S radicals, and MoV(=O)S4 likely play a key role in stabilizing MoV(=O)S4, showing the importance of secondary interactions in facilitating NRR. The first identification and characterization of an oxo‐ and thiolate‐ligated Mo intermediates pave the way to the molecular design of efficient enzyme mimetic NRR catalysts in aqueous solution.

Impact of heavy metal contamination and seasonal variations on enzyme\u2019s activity of Yamuna river soil in Delhi and NCR

Yamuna river is one of the most important rivers of India and is highly polluted. The river water and the adjacent soil are contaminated with various pollutants including heavy metals. Soil enzymes play important role in various bio-geochemical cycles and help in maintaining nutrient availability of soil. They are very sensitive toward changing environment and therefore act as a key indicator of soil health. In the present study, effects of seasonal variations and heavy metals contamination in Yamuna river water were observed on physicochemical properties and enzyme activities of soil. Soil samples were collected from ten different locations, along the course of the Yamuna river in Delhi–NCR, in three different seasons, i.e., summer, rainy and winter. These sampling sites were divided into three segments, up, middle and lower streams. The concentrations of heavy metals were found to be higher than their safe limit at all the ten sampling points. The total organic carbon content and soil respiration was significantly higher in lower stream. Dehydrogenase enzyme activity was higher in winter season, while urease and arginine deaminase enzyme activity was higher in summer season. Dehydrogenase, arginine deaminase and nitrate reductase enzyme activities were higher in lower streams, while urease enzyme shows maximum activity in the up stream. Thus, we conclude that the continuous application of Yamuna water for irrigation may degrade the soil quality.

Síntesis verde y caracterización de nanopartículas de plata de Serratia spp

The biological means of silver nanoparticles synthesis is an evolutionary step in the process of synthesizing nanoparticles to produce particles within the nano range. With the side effects of the use of chemical and physical synthesis methods yielding toxic and unstable, researches have turned to green synthesis to produce nanoparticles using the mechanism of the bacteria specifically the nitrate reductase enzyme present in most bacteria to break down silver nitrate to silver nanoparticles which are stable and reduces the cost of production. Marine bacteria plays it role by being a reservoir that can intake heavy metals and breaking down with no difficulty as a result of their harsh habitat which is rich in heavy metals. This study was done to prove that marine bacteria can synthesize silver nanoparticles and the nanoparticles are stable and has a range of size and shape through preliminary studies. Marine bacteria were isolated from marine soil samples and later identified to belong to the genus Serratia. Synthesis of the nanoparticles were done using extracellular synthesis where equal amounts of bacterial supernatant and 0.5 M silver nitrate was added and incubated under dark condition for 72 hours. 20 isolates showed positive results for synthesis which were further examined using UV-Visible Spectra analysis showing all 20 samples having a range of size and shape of nanoparticles with peaks observed between the ranges of 340 nm to 440 nm, with the sample strain AQ5-NT6 being the most potent. This study will provide fundamental data and add knowledge to the field of green synthesis of silver nanoparticles.

Biochar counteracts nitrification inhibitor DMPP-mediated negative effect on spinach (Spinacia oleracea L.) growth.

The use of nitrification inhibitors (NIs) such as 3,4-dimethylpyrazole phosphate (DMPP) has been suggested to diminish agricultural soil nitrate (NO3-) loss and increase nitrogen (N) use efficiency (NUE). However, the yield of ammonium (NH4+)-sensitive plants such as spinach (Spinacia oleracea L.) may be adversely affected by the application of NIs at high N levels and, on the other hand, the efficiency of the NIs may also be affected by soil amendments such as biochar. These two issues are still not adequately addressed. The aim of this study was to evaluate the effect of different N levels including DMPP or not in a calcareous soil with and without amendment of wheat straw biochar on spinach yield, NUE, nitrate concentration of spinach leaf, activity of enzymes nitrate reductase (NR) and nitrite reductase (NiR), and soil ammonium (NH4+) and NO3- concentration under greenhouse conditions. This experiment was carried out with different N rates factor at seven levels (un-fertilized, N0; fertilized with 50mgN kg-1 soil, N50; fertilized with 75mgN kg-1 soil, N75; fertilized with 100mgN kg-1 soil, N100; fertilized with N50+DMPP; fertilized with N75+DMPP; and fertilized with N100+DMPP) and biochar (BC) factor at two levels (0, 0%BC; and 2% (w/w), 2%BC) with six replications over a 56-day cultivation period of spinach. Results showed that the application of DMPP had no significant effect on the yield of spinach plant at low and medium levels of N (50 and 75mgN kg-1 soil), but decreased the yield of this plant at the higher level of N (100mgN kg-1 soil). However, application of BC decreased the negative effect of DMPP on spinach yield as the yield in spinach plants fertilized with N75+DMPP and N100+DMPP significantly increased. Both application of DMPP and addition of BC to soil decreased leaf NO3- concentration by 29.2% and 16.3% compared to control, respectively. Biochar compared to control decreased NR activity by 46.3%. With increasing N rate, NR and NiR activities increased, but DMPP decreased the activities of both enzymes. Biochar reduced the efficiency of DMPP as soil NH4+ concentration was higher in the treatments containing DMPP without BC at 56 days after planting. Biochar and DMPP could increase the quality of spinach plant through decreasing the leaf NO3- concentration. In general, wheat straw biochar counteracted DMPP-mediated negative effect on growth of spinach plant at high level of N by decreasing the efficiency of this inhibitor. These results provide the useful information for managing the application rate of N fertilizers including DMPP in biochar-amended soil.

Changes in metabolism, growth and nutrient uptake of Ulva fasciata (Chlorophyta) in response to nitrogen source

The form of inorganic nitrogen (N) determines the biofiltration performance of Ulva, particularly by inhibition of nitrate assimilation in the presence of ammonia. In the current study, Ulva fasciata from a biofilter of fishpond effluent was examined for its biomass production, photosynthetic activity, nutrient uptake and activity of nitrate reductase when supplied with either ammonia or nitrate as the sole nitrogen source. The effects of prior acclimation to either nitrogen source were also studied. Cultivation of Ulva with ammonia resulted in a rapid uptake of this nutrient, whereas photosynthesis, uptake of orthophosphate and C:N ratio were greater in cultures with nitrate. The transfer of Ulva from ammonia to nitrate nutrition increased carbohydrate content in the biomass. Longer culture in nitrate did not influence photosynthetic yields, growth, or N and P uptake rate by the algae, but increased both lipid and P content in biomass. Once transferred from ammonia-rich to nitrate-rich seawater, nitrate uptake by Ulva commenced immediately, although the activity of the enzyme nitrate reductase was first detected only 28 h later. Culture of Ulva in different nitrogen forms, ammonia and nitrate, may determine application of the resulting biomass in different industries, e.g., feeding, bioethanol, biofiltration, etc., considering the rapid production of protein-rich Ulva in the presence of ammonia, while yield is lower in the presence of nitrate but the biomass contains more carbohydrates and removes P from the effluent more rapidly.

Nitrate Reductase mediated synthesis of surface passivated nanogold as broad-spectrum antibacterial agent

The green synthesis of gold nanoparticles has attracted tremendous interest owing to their unique physicochemical properties and widespread applications which are primarily size-dependent. The stability, less reaction time, and use of biological resources as novel nanofactories as an alternative to conventional synthesis strategies are the main objectives of green synthesis approaches. However, to attain size-controlled synthesis from the biogenic route is still a challenge. Hence, use of nontoxic stabilizers becomes increasingly essential. Herein, we describe an emerging, simple, nonconventional approach to synthesize stable and size-controlled biogenic nanogold using cell lysate supernatant containing nitrate reductase of Bacillus licheniformis in the presence of Tween 20 and dodecanethiol respectively. The face-centered central composite design used for the optimization of gold nanoparticles (AuNPs) biosynthesis. The maximum AuNPs biosynthesis obtained using the optimized media variables, glucose (2.1 g/L), peptone (14.05 g/L), yeast extract (4.14 g/L), and potassium nitrate (3.91 g/L) was 0.769 a.u. Highly stable monodispersed nanogold of 10.4 ± 0.6 nm and 12.5 ± 0.9 nm sizes arranged in ordered self-assembly was obtained. The stability profile and kinetics of bioreduction was evaluated with respect to time, and the involvement of the nitrate reductase enzyme in bioreduction was validated by inhibitor study. The physicochemical properties of biogenic nanoparticles were characterized using multiple spectroscopy and microscopy techniques. The obtained nanogold demonstrated excellent bactericidal property against both gram-negative and gram-positive bacteria in size-dependent manner and thus could find tremendous utility in clinical, biological, and environmental applications as a broad-spectrum antibacterial agent.

Measurement of Nitrate Reductase Activity in Tomato (Solanum lycopersicum L.) Leaves Under Different Conditions.

Nitrogen is one of the crucial macronutrients essential for plant growth, development, and survival under stress conditions. Depending on cellular requirement, plants can absorb nitrogen mainly in multiple forms such as nitrate (NO3-) or ammonium (NH4+) or combination of both via efficient and highly regulated transport systems in roots. In addition, nitrogen-fixing symbiotic bacteria can fix atmospheric nitrogen in to NH4+ via highly regulated complex enzyme system and supply to the roots in nodules of several species of leguminous plants. If NO3- is a primary source, it is transported from roots and then it is rapidly converted to nitrite (NO2-) by nitrate reductase (NR) (EC 1.6.6.1) which is a critical and very important enzyme for this conversion. This key reaction is mediated by transfer of two electrons from NAD(P)H to NO3-. This occurs via the three redox centers comprised of two prosthetic groups (FAD and heme) and a MoCo cofactor. NR activity is greatly influenced by factors such as developmental stage and various stress conditions such as hypoxia, salinity and pathogen infection etc. In addition, light/dark dynamics plays crucial role in modulating NR activity. NR activity can be easily detected by measuring the conversion of NO3- to NO2- under optimized conditions. Here, we describe a detailed protocol for measuring relative NR enzyme activity of tomato crude extracts. This protocol offers an efficient and straightforward procedure to compare the NR activity of various plants under different conditions.