Levels Of Nitrate Reductase Activity Research Articles

Elevated Nitrate Preference Over Ammonium in Aquatic Plants by Nitrogen Loadings in a City River

AbstractAquatic plants are the dominant biota in retaining nitrogen (N) pollutants discharged into river ecosystems, but the sensitivity of plant N pools and N assimilation to riverine N loadings, which is essential for evaluating river biogeochemistry and functions, remains poorly understood. Here, concentrations and stable isotopes of N in river water and aquatic plants were measured along the Nanming River in Guiyang of southwestern China. Total N (TN) contents of aquatic plants increased, but the increments decreased with river N concentrations. The δ15N of plant TN decreased with sewage N loadings along the river, which revealed a distinct shift of plant N preference from ammonium to nitrate. Higher levels of nitrate reductase activity in plants at the downstream river section supported the elevated nitrate preference and assimilation, which might explain the decreased nitrate concentrations observed in the river water of the downstream section. These findings contributed to understanding riverine N biogeochemical processes and their response to N loadings, which can help trace sewage N pollution and its ecological effects on river ecosystems.

Relationship of nitrogen use and nitrate reductase activity by sugarcane cultivars

Intrinsic genetic potential of each cultivar of sugarcane may determine the absorption intensity and nitrogen assimilation. It is possible to occur a lower expression of the productive potential by limitations related to low nitrate reductase (NR) activity, since this enzyme is "key" of the N metabolism. The objectives were to compare cultivars of sugarcane as the level of NR activity and its relationship to productive and nutritional variables. The experiment was conducted under condition of a greenhouse, where were grown ten sugarcane cultivars in pots of 4 dm3, filled with sand and vermiculite plus nutrients as indicated for the crop. The variables evaluated were: plant height, NR activity, dry matter (shoot and roots), N content and N accumulation and N uptake and utilization efficiency, moreover, the correlation between the variables was evaluated. The results showed that NR activity varies with the cultivar and further, that NR activity in sugarcane leaves does not correlate with the N uptake efficiency, but is positively correlated with N content and negatively with N utilization efficiency. The NR activity is not a good physiological parameter to discriminate N efficient use sugarcane genotypes

Early Root Transcriptomic Changes in Wheat Seedlings Colonized by Trichoderma harzianum Under Different Inorganic Nitrogen Supplies.

Wheat is one of the most important crops worldwide. The use of plant growth promoting microorganisms, such as those of the genus Trichoderma, constitutes an alternative to chemical fertilizers, since they are cheaper and are not detrimental to the environment. However, the interaction between Trichoderma and wheat plants has been scarcely studied, at least at a molecular level. In the present work, a microarray approach was used to study the early transcriptomic changes induced in wheat roots by Trichoderma harzianum, applied alone or in combination with different concentrations of calcium nitrate [Ca(NO3)2], which was last used as nitrogen (N) source. Our results show that T. harzianum causes larger transcriptomic changes than Ca(NO3)2 in wheat roots, and such changes are different when plants are challenged with Trichoderma alone or treated with a combination of T. harzianum and Ca(NO3)2. Overall, T. harzianum activates the expression of defense-related genes at early stages of the interaction with the roots, while this fungus reduces the expression of genes related to plant growth and development. Moreover, the current study in wheat roots, subjected to the different T. harzianum and Ca(NO3)2 combinations, reveals that the number of transcriptomic changes was higher when compared against those caused by the different Ca(NO3)2 concentrations than when it was compared against those caused by T. harzianum. N metabolism gene expression changes were in agreement with the levels of nitrate reductase activity measured in plants from Trichoderma plus Ca(NO3)2 conditions. Results were also concordant with plant phenotypes, which showed reduced growth at early interaction stages when inoculated with T. harzianum or with its combination with Ca(NO3)2 at the lowest dosage. These results were in a good agreement with the recognized role of Trichoderma as an inducer of plant defense.

Nitrate reductase activity in high-mountain plants: a test across species, growth form and habitat type

For many terrestrial plants, nitrate is the most important form of available soil nitrogen for growth. However, many plant species, which grow on acidic, ammonium-dominated soils, exhibit a constantly low level of nitrate reductase activity (NRA). Little is known about NRA in high-mountain vascular plants in similar conditions. We tested the hypothesis that high-mountain vascular plants in acidic and ammonium-dominated habitats have low levels of NRA. Twenty-six plant species of the families Asteraceae, Ericaceae, Poaceae, Polygonaceae, Salicaceae, Pinaceae, Ranunculaceae, Woodsiaceae, Cyperaceae, Juncaceae, Rosaceae and Urticaceae representing different growth forms were investigated in seven native and anthropogenic habitats of subalpine and alpine belts of the Karkonosze (Hercynian middle-mountains, Central Europe), with respect to leaf NRA and mineral nitrogen forms in soil. NRA was measured by an in vivo assay for the first time directly in the field using portable water bath. An NRA study of vascular plants from high mountains is presented for the first time, and most of the studied subalpine and alpine species were subjected to field measurements for the first time ever. Differences among species, families, growth forms and habitats for NRA were found. These differences reflected mainly the taxonomical position and in part ecological preferences. PERMANOVA analysis confirmed that variance component showed that enzyme activities were mostly explained by plant species and habitat. Overall, the subalpine and alpine species from their native habitats are characterized by very low and low ability for nitrate metabolism. We also compared NRA with Ellenberg’s N values for the studied plants. Using a regression equation, N values of some species were calculated for the first time and corrected for the others.

Nitrate assimilation pathway (NAP): role of structural (nit) and transporter (ntr1) genes in Fusarium oxysporum f.sp. lycopersici growth and pathogenicity.

The tomato pathogen Fusarium oxysporum f.sp. lycopersici possesses the capability to use nitrate as the only nitrogen source under aerobic and anaerobic conditions and to activate virulence-related functions when cultivated in the presence of nitrate, but not in ammonium. The genome of F. oxysporum f.sp. lycopersici encodes three paralogs nitrate reductase (NR) genes (nit1, nit2 and nit3) and one predicted ortholog of the Aspergillus nidulans high-affinity nitrate/nitrite transporters NtrA and NtrB, named ntr1. We set out to clarify the role of nit1, nit2, nit3 and ntr1 genes in nitrate assimilation and in the virulence of F. oxysporum f.sp. lycopersici. Quantitative RT-PCR analysis revealed that only nit1, nit2 and ntr1 are expressed at significant levels during growth in nitrate as the only nitrogen source. Targeted deletion of nit1 and ntr1, but not of nit2 or nit3, severely impaired growth of F. oxysporum on nitrate as nitrogen source, indicating that Nit1 and Ntr1 proteins are involved in nitrate assimilation by the fungus; biochemical analysis of nit mutants indicated that Nit1 and Nit2 enzymes contribute to about 50 and 30% of the total NR activity, respectively. In addition, a spontaneous chlorate-resistant mutant derived from F. oxysporum 4287, denoted NitFG, was characterized, showing inabilityto grow in nitrate under aerobic and anaerobic conditions and low levels of NR activity, in spite of itsincreased transcription levels of nit1 and nit2genes. Tomato plant infection assays showed that NitFG and ∆ntr1 mutants induced an earlier death in tomato plants, whereas the single mutants ∆nit1, ∆nit2 and ∆nit1∆nit2 double mutant showed a mortality rate similar to the wild-type strain. Taken together, these results indicate that the Nit1 and Ntr1 proteins are important for nitrate assimilation of F. oxysporum f.sp. lycopersici incubated under aerobic and anaerobic conditions and that this metabolic process is not essential for the virulence of the fungus. These observations open new questions about the role of Nit1, Nit2, and Nit3 proteins in other routes of nitrate metabolism in this pathogenic fungus and in the possible regulatory role that can be exerted by the AreA protein in these routes.

The Effects of Dose Rhizoctonia Binucleat (BNR) and Phosphorus to Nitrate Reductase Activity (NRA) and Chlorophyll of Vanilla Seedling (Vanilla planifolia Andrews)

<p>Vanilla (<em>Vanilla planifolia Andrews</em>) is one of the important exported commodities in Indonesia. Indonesia is one of top five major vanilla exporters in the world, that produce the high quality of Indonesian vanilla with high vanillin content (2.75%). The aims of this research were to determine the effects of dose binukleat Rhizoctonia (BNR) and phosphorus as well as the interaction of the nitrate reductase activity (NRA) and chlorophyll of the vanilla seedling (<em>Vanilla planifolia Andrew</em>). Method in this research used completely randomized factorial design, by involving two factors (dose of BNR inoculation and Phosphor). The first factor is without inoculation and inoculation BNR (M<sub>0</sub>, M<sub>1</sub>, M<sub>2</sub>, M<sub>3</sub>) wich consists of (0,5, 10, 15) g/polybag, the second factor is the dose of phosphorus fertilizer (P<sub>0</sub>, P<sub>1</sub>, P<sub>2</sub>, P<sub>3</sub>) which consists of (0, 3, 6, 9) g/polibag. The results showed that the inoculation dose of BNR and doses of phosphorus not significant and lower levels of NRA and chlorophyll while the interaction dose of BNR and phosphorus significantly and increase levels of NRA and chlorophyll of vanilla seedling. Nitrate Reductase Activity and chlorophyll has important role in metabolism process as a plant growth indicator.</p><p><strong>How to Cite</strong></p><p>Haryuni, H., & Dewi, T. S. K. (2016). The Effects of Dose Rhizoctonia Binucleat (BNR) and Phosphorus to Nitrate Reductase Activity (NRA) and Chlorophyll of Vanilla Seedling (<em>Vanilla planifolia Andrews</em>). <em>Biosaintifika: Journal of Biology & Biology Education</em>, 8(2), 141-147.</p>

Nitrate reductase in detached embryos may serve as a marker of the preharvest tolerance of wheat seeds

We have observed an easily determinable parameter indicative of genetic pre-harvest sprouting (PHS) tolerance – the presence of an endosperm factor, presumably ABA, capable of inhibiting nitrate reductase (NR) induction in the embryo in the presence of NO3. This finding has importance not only for the early rapid screening of PHS tolerant of cereal cultivars but may also be an important tool to determine the mechanism of NR inhibition either by genetic repression or by post-translational down regulation of NR activity. In this work we sought a simplerelationship between ABA content and NR activity level that we assumed to be closely related to PHS susceptibility. Studied the relationship between ABA content, dry weight and ability to germinate in seeds of examined wheat varieties resistant to preharvest sprouting Lutescens 70, and unstable – Novosibirskaya 67. The data obtained are shown that the level of ABA reaching its maximum to 40-45 days . The maximum content of ABA for both varieties accounted for 40 days after flowering, when the grains reach maximum wet weight in the future, as the ripening hormone levels quickly decreased with the decrease in fresh weight of grain . The level of ABA in embryos unstable to pre-harvest sprouting wheat Novosibirskaya 67 ranged from 20 to 40% below the level of the hormone in the grains resistant Lutescens 70wheat. The results showed a significant increase in the ability to germinate, since it is a phase 40-45 DAP. Reduction of endogenous ABA content in grains at the final stage of maturation, with a relatively high percentage of germination, can be explained by the need and ensuring to the start of the germination process of the seed.

Nitrate reductase activity (NRA) in the invasive alien Fallopia japonica: seasonal variation, differences among habitats types, and comparison with native species

Nitrate reductase activity (NRA) was studied in the invasive alien plant <em>F. japonica</em> (Japanese knotweed) during the vegetation season and among natural, semi-natural, and human-made habitats and compared with NRA in selected native species. NRA was measured directly in the field from the beginning of May until the beginning of October. NRA was much higher than in the plant’s native range, i.e., East Asia, and showed a high degree of variation over time with the highest values being reached at the stage of fast vegetative growth and at the beginning of fruiting. NRA was highest on dumping sites probably due to the high nitrogen input into soils and near traffic and the emission of NO<span><em><sub>x</sub></em></span> by vehicles. A comparison of the enzyme activity in four selected native plant species indicated that NRA in <em>F. japonica</em> was the highest with the exception of <em>Urtica dioica</em>, which exhibited a similar activity of the enzyme. A detailed comparison with this species showed that differences between these species on particular dates were influenced by differences in the phenology of both plants. The initial results that were obtained suggest that nitrogen pollution in an environment can contribute to habitat invasibility and a high level of NRA, which in addition to the many plant traits that are commonly accepted as characteristic of invasiveness features, may be an important factor that enhances invasion success.

Ammonium and potassium effect on nitrate assimilation in cucumber seedlings

The effect of ammonium present in the induction medium toghether with nitrate on the activity of nitrate reductase (NR), nitrite reductase (NiR), glutamic acid dehydrogenase (GDH) and absorption and accumulation of NO<sub>3</sub><sup>-</sup> in cucumber seedlings were investigated. Maximum NR and NiR activity in the cotyledons was observed when seedlings were supplied with KNO<sub>3</sub> as the sole source of nitrogen. When plants were supplied with NH<sub>4</sub>NO<sub>3</sub> the presence of NH<sub>4</sub><sup>+</sup> in the induction medium repressed by about 50 per cent the activity of both reductases in the cotyledons. Addition of K<sup>+</sup> to this medium abolished completely the inhibitory effect of NH<sub>4</sub><sup>+</sup>. The effect of K<sup>+</sup> cannot be replaced by that Na+ ions. On the other hand, ammonium has no effect on the level of NR activity in roots, while NiR was almost completely repressed. Under the experimental conditions ammonium, in the presence of nitrates, decreased the activity of GDH, but this diminution did not occur when the plants were supplied with K<sup>+</sup> simultaneously. It has found that NH<sub>4</sub><sup>+</sup> ions reduced NO<sub>3</sub><sup>-</sup> absorption but at the same time, the ratio of NO<sub>3</sub>- absorbed to that reduced was increased more than twice. The presumable mechanism of these phenomena is discussed.

Involvement of heme synthesis in the growth stimulation of maize seedlings by 5‐aminolevulinic acid

The growth of maize seedlings was stimulated by shoot‐applied 5‐aminolevulinic acid 2 days after treatment at 90 and 120 μmol L−1. The effects of 5‐aminolevulinic acid on the activity of nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.7.7.1) and the chlorophyll, ammonium, heme and total free amino acid content were investigated by using maize seedlings to clarify the involvement of nitrogen metabolism and heme synthesis in the growth stimulation. 5‐Aminolevulinic acid increased the level of nitrate reductase activity at 90 μmol L−1 and the ammonium and heme content at 90 and 120 μmol L−1 2 days after treatment. The total amino acid content increased by 90 and 150 μmol L−1 5‐aminolevulinic acid 2 and 3 days after treatment, respectively. However, no significant change was observed in the activity of nitrite reductase or the chlorophyll content after the 5‐aminolevulinic acid treatment. These results suggest that the enhancement of nitrogen metabolism by nitrate reductase activation is involved in the 5‐aminolevulinic acid‐induced stimulation of maize seedling growth. The activation of nitrate reductase might be related to an increase in the heme content following the 5‐aminolevulinic acid treatment.

Responses of nitrogen metabolism to copper stress in Luffa cylindrica roots

Abstract Pot experiments were performed to investigate the responses of nitrogen metabolism to copper stress in Luffa cylindrica roots. Four treatments were used, in which varying copper concentrations (25, 50, 75 and 100 μM) were added to MS medium. The fresh weights of the roots decreased gradually with the increasing copper concentrations. At the lower concentrations, the ammonium and nitrite levels increased compared with the control, but the nitrate concentrations significantly decreased with 75 and 100 μM copper. Compared with the control, nitrate reductase activity levels gradually increased with increasing copper concentrations of up to 50 μM, and nitrite reductase activity levels significantly decreased. Copper stress led to variable increases in the activities of glutamine synthetase and glutamate synthase compared with the controls. The NADH- glutamate dehydrogenase (NADH-GDH) and NAD-glutamate dehydrogenase (NAD-GDH) activities were affected by the copper treatments, but that of NAD-GDH was significantly reduced by 100 μM copper. The activities of alanine aminotransferase (AlaAT) and aspartate aminotransferase (AspAT) showed varying changes following the copper treatments. The present results indicate that Luffa cylindrica shows altered activities of enzymes associated with nitrogen metabolism during copper stress, enabling it to monitor and adapt to changes in its N status and supply, thereby minimizing the harmful effects of the stress.

Nitric Oxide Content in Wheat Leaves and Its Relation to Programmed Cell Death of Main Stem and Tillers Under Different Nitrogen Levels

Nitric oxide (NO) is a key signaling molecule in different physiological processes of plants, including programmed cell death (PCD). PCD of tillers plays an important role in surviving which are major components of grain yield. PCD was triggered in wheat leaves of main stem and tillers by NO content under different nitrogen treatments. In wheat, NO could be synthesized endogenously by nitrate reductase (NR). As an inducible enzyme, NR activity was closely related to substrate concentration. Therefore, different nitrogen levels would change NR activity and NO production. The objective of this study was to determine the effects of NR activity, NO production, and the correlation between them on different tillers growth, development, senescence, and kernel protein content under different nitrogen levels. Field-experiments were conducted in 2009–2011 growing seasons, using two wheat cultivars with different spike-types. Results showed that for main stem and primary tillers, NR activity and NO content reached high level at heading stage, while for secondary tiller, the level of NR activity was low, but NO content was high in the present research. The NO synthesis depending on NR activity in wheat leaves was significant in the early growing stage, but the NO synthesis weakened with the progress of growing period. NO was related to the senescence of wheat leaves, but PCD was more sensitive to marked changes of NO content than NO content itself. N application had marked influence on the aging process of primary tiller, while had little influence on that of main stem and secondary tiller. Moreover, N fertilizer application could increase spike rate and protein content of primary tiller by N fertilizer application.

METHOD OF MEASUREMENT OF NITRATE REDUCTASE ACTIVITY IN FIELD CONDITIONS

For the last three decades the interest in biomonitoring and ecological studies has been rapidly growing. Therefore, it was necessary develop of new methods of analysis for biochemical parameters which allow to quantify biological response of investigated organisms for environmental factors. The main goal of this paper demonstrates optimal conditions for enzyme kinetics analysis conducted in the field in situ. Nitrate reductase activity is typically assayed in vivo by measuring nitrite production in tissue which has been vacuum infiltrated with buffered nitrate solution. For this study a nitrate reductase assay was adapted from a number of studies with own modifications of authors. Leaves of examined plants were collected from the investigated plots and immediately placed into test tubes with buffer solution (potassium phosphate dibasic containing 0.6% propanol-1) and evacuated in 0.33 atm. for 10 minutes. Then, known amount of potassium nitrate was added, and the solution sample was analyzed in order to obtain a background level of nitrite. The foliage samples were incubated for 2 hours at 20 °C in darkness. Following this procedure, they were given the most optimal conditions for reaction stability. After incubation the amount of synthesized nitrite was determined colorimetrically using sulfanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride, measured at 540 nm. The foliage samples were oven-dried to obtain dry mass. The level of nitrate reductase activity was calculated as the amount of nitrite produced in nmol per gram of dry mass of foliage tissue per hour. The result obtained during the research demonstrate the changes of nitrate reductase dynamics according to change of incubation parameters. Dynamics of enzyme activity with changes of solution pH and incubation temperature was presented. Installation for conducting infiltration process and construction of in cubation chamber is also described in this paper.

POMIAR AKTYWNOŚCI REDUKTAZY AZOTANOWEJ W WARUNKACH TERENOWYCH – METODYKA

During recent three decades interest for biomonitoring and ecological studies was rapidly growing. Therefore was necessary develop of new methods of analysis biochemical parameters whose allow quantify biological response of investigated organisms for environmen- tal factors. The main goal of this paper demonstrates optimal conditions for enzyme kinetics analysis conducted in the field in situ. Nitrate reductase activity is typically assayed in vivo by measuring nitrite production in tis- sue which has been vacuum infiltrated with buffered nitrate solution. For this study a nitrate reductase assay was adapted from a number of studies with own modifications of authors. Leaves of examined plants were collected on investigated plots and immediately placed into test tubes with buffer solution (potassium phosphate dibasic containing 0.6% propanol-1) and evacuated in 0.33 atm. for 10 minutes. Then known amount of potassium nitrate was added, and the solution sample was analyzed in order to obtain a background level of nitrite. The foliage samples were incubated for 2 hours at 20 °C in darkness. Follow this procedure have given the most optimal conditions for reaction stability. After incubation the amount of synthesized nitrite was determined colorimetrically using sulfanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride, measured at 540 nm. The foliage samples were oven-dried to obtain their dry mass. Level of nitrate reductase activity was calculated as the amount of nitrite produced in nmol per gram of dry mass of foliage tissue per hour. The result obtained during these research demonstrate the changes of nitrate reductase dynamics according to change of incubation parameters. Dynamics of enzyme activity with changes of solution pH and incubation temperature was presented. Installation for conducting infiltration process and construction of incubation cham - ber is also described in this paper.

Nitrate accumulation, growth and leaf quality of spinach beet (Beta vulgaris Linn.) as affected by NPK fertilization with special reference to potassium

The present study was carried out at the Herbal Garden of Jamia Hamdard, New Delhi on Pusa Bharti variety of Spinach Beet (Beta vulgaris Linn.) to understand the effect of potassium with other fertilizers on nitrate accumulation, growth and quality of the leaves. Potassium application along with other fertilizers increased leaf area, per cent dry weight, Chlorophyll Content Index (CCI) and Nitrate Reductase Activity (NRA), but decreased nitrate content. Effect of 2:1 ratio of N and K was found statistically at par with 1:1 ratio of N and K. Fertilizer treatments without potassium had lower level of NRA and higher nitrate content in the leaves as compared to other applications. Application of FYM along with NPK fertilizers increased leaf area, per cent dry weight, CCI and NRA, whereas it decreases nitrate content. As higher NRA and lower nitrate content was recorded in treatments with potassium, application of K is desirable to reduce health hazards related with nitrate toxicity. Based on the results of the study, application of 1:1 ratio of N and K fertilizer and FYM is recommended. Keywords: Spinach beet, potassium, nitrate toxicity, nitrate reductase, manure; fertilizers, NPK.

Main centers of nitrate and nitrite reduction in young and nodulated yellow lupine (Lupinus luteus)

Nitrate and nitrite reduction centers in non-nodulated and symbiotic yellow lupine were analyzed. In young seedlings, nitrate was exclusively accumulated in roots, which also was shown as the main nitrate reduction center. In contrast, leaves were shown to play a key role in nitrite reduction. A similar distribution of nitrate reductase (NR) and nitrite reductase was found in nodulated plants. However, in field conditions characterized by low nitrate content, a disproportionately high level of NR activity in nodules was also observed during all stages of symbiotic growth. This feature was confirmed in nitrate-fed hydroponic cultures. Nodule NR activity was one order of magnitude higher than in roots, in spite of the small stored nitrate pool found inside nodules. This suggests that nodule NR activity had been induced not by nitrate itself but indirectly. Since bacteroids were shown to be responsible for the vast majority of nodule NR activity, the plausible explanation of this effect seems to be a dissimilatory nature of rhizobial NR. Considering that environmental nitrate could cause hypoxia inside nodules, this is the proposed way of the observed nodule NR induction.

Nitrogen nutrition of Salvinia natans: Effects of inorganic nitrogen form on growth, morphology, nitrate reductase activity and uptake kinetics of ammonium and nitrate

In this study we assessed the growth, morphological responses, and N uptake kinetics of Salvinia natans when supplied with nitrogen as NO 3 −, NH 4 +, or both at equimolar concentrations (500 μM). Plants supplied with only NO 3 − had lower growth rates (0.17 ± 0.01 g g −1 d −1), shorter roots, smaller leaves with less chlorophyll than plants supplied with NH 4 + alone or in combination with NO 3 − (RGR = 0.28 ± 0.01 g g −1 d −1). Ammonium was the preferred form of N taken up. The maximal rate of NH 4 + uptake ( V max) was 6–14 times higher than the maximal uptake rate of NO 3 − and the minimum concentration for uptake ( C min) was lower for NH 4 + than for NO 3 −. Plants supplied with NO 3 − had elevated nitrate reductase activity (NRA) particularly in the roots showing that NO 3 − was primarily reduced in the roots, but NRA levels were generally low (<4 μmol NO 2 − g −1 DW h −1). Under natural growth conditions NH 4 + is probably the main N source for S. natans, but plants probably also exploit NO 3 − when NH 4 + concentrations are low. This is suggested based on the observation that the plants maintain high NRA in the roots at relatively high NH 4 + levels in the water, even though the uptake capacity for NO 3 − is reduced under these conditions.

Effects of PII Deficiency on Expression of the Genes Involved in Ammonium Utilization in the Cyanobacterium Synechocystis sp. Strain PCC 6803

The Synechocystis sp. strain PCC 6803 mutant deficient in PII protein (the glnB gene product) was found to express glutamine synthetase activity at levels several times higher than the wild-type strain. There was no significant difference in nitrate reductase activity levels between the two strains, and the nitrite reductase levels were somewhat lower in the mutant than in the wild-type strain. The higher glutamine synthetase activity in the mutant was ascribed to higher expression levels of the glutamine synthetase genes (glnA and glnN), which belong to the regulon controlled by NtcA, a Crp-family transcription regulator. Examination of the effects of PII deficiency on other NtcA-regulated genes revealed that the transcript levels of amt1 (encoding an ammonium permease) and gifB (encoding an inhibitor of glutamine synthetase) were increased, whereas that of gifA (a homolog of gifB, encoding another glutamine synthetase inhibitor) was decreased, with those of nirA, nrtC, icd, sigE (rpoD2-V), nblA and ntcA being unaffected. Unlike the Synechococcus elongatus strain PCC 7942, induction or repression of the NtcA-regulated genes proceeded normally in the PII-deficient mutant upon nitrogen depletion. The altered steady-state expression levels of glnA, glnN, amt1, gifA and gifB in the PII-deficient mutant suggested that Synechocystis sp. strain PCC 6803 has a mechanism for regulation of the subset of the NtcA-regulated genes related directly to ammonium assimilation.

Involvement of NarK1 and NarK2 Proteins in Transport of Nitrate and Nitrite in the Denitrifying Bacterium Pseudomonas aeruginosa PAO1

Two transmembrane proteins were tentatively classified as NarK1 and NarK2 in the Pseudomonas genome project and hypothesized to play an important physiological role in nitrate/nitrite transport in Pseudomonas aeruginosa. The narK1 and narK2 genes are located in a cluster along with the structural genes for the nitrate reductase complex. Our studies indicate that the transcription of all these genes is initiated from a single promoter and that the gene complex narK1K2GHJI constitutes an operon. Utilizing an isogenic narK1 mutant, a narK2 mutant, and a narK1K2 double mutant, we explored their effect on growth under denitrifying conditions. While the DeltanarK1::Gm mutant was only slightly affected in its ability to grow under denitrification conditions, both the DeltanarK2::Gm and DeltanarK1K2::Gm mutants were found to be severely restricted in nitrate-dependent, anaerobic growth. All three strains demonstrated wild-type levels of nitrate reductase activity. Nitrate uptake by whole-cell suspensions demonstrated both the DeltanarK2::Gm and DeltanarK1K2::Gm mutants to have very low yet different nitrate uptake rates, while the DeltanarK1::Gm mutant exhibited wild-type levels of nitrate uptake. Finally, Escherichia coli narK rescued both the DeltanarK2::Gm and DeltanarK1K2::Gm mutants with respect to anaerobic respiratory growth. Our results indicate that only the NarK2 protein is required as a nitrate/nitrite transporter by Pseudomonas aeruginosa under denitrifying conditions.

Nitrate reductase activity in leaves and stems of tanner grass (Brachiaria radicans Napper)

Tanner grass (Brachiaria radicans Napper) is a forage plant that is adapted to well-drained soils or wetlands, and responds well to nitrogen (N) fertilization. The assimilation of N involves the nitrate reductase (NR) enzyme, and its activity seems to be dependent on N supply. Molybdenum (Mo) is also important because it is a cofactor of NR. In this study, the variables of an in vivo assay were optimized for measuring nitrate reductase activity (NRA) in the leaves and stem tissues. This method was used to evaluate NO3- metabolism in plants fertilized with NaNO3, NH4Cl or urea, in association with or without application of H2MoO4, aiming to provide guidelines for N management of this species. The best conditions to determine NRA involved the incubation of 300 mg of tissues in a medium composed of 200 mmol dm3 phosphate buffer (pH 7.4), 60 mmol dm3 KNO3, 10 cm³ dm3 n-butanol, 0.1 cm³ dm3 detergent (triton-X-100®), under vacuum and in the dark for a period of 60 to 100 minutes. Leaves showed NRA levels two to three times higher than stems. Although there were some interactions between treatments, stem fresh weight and NRA were not affected by N sources. Plants fertilized with NaNO3 showed the best growth and NRA values when compared with NH4Cl and urea, which had, respectively, the lowest and intermediate scores. The application of Mo in the absence of N improved NRA and did not affect leaf and stalk growth. In the presence of N, the Mo levels applied limited leaf NRA and plant development.