Nitrate reductase activity and protein concentration of two populas clones

Nitrate reductase activity and protein percentage of various tree parts of two Populus clones were determined in relation to nitrate ion activity. Nitrogen was supplied as NH(4)NO(3) in a nutriculture system. Wisconsin-5 had significantly greater nitrate reductase activity than Tristis No. 1. Protein percentages of leaf plastochron index 10 leaves (tenth leaf below first leaf lamina exceeding 20 mm in length), bottom leaves, and roots in relation to nitrate ion activity were not appreciably different between clones. The nitrate reductase activity and protein percentage of Tristis No. 1 apex started to level off at the same nitrate ion activity, about 0.09 mm. In Wisconsin-5 apex protein percentage continued to increase at nitrate ion activities where nitrate reductase activity decreases sharply, suggesting that protein nitrogen was being supplied by ammonium ion. The difference in nitrate reductase activity between clones was probably due to genetically determined ability to synthesize nitrate reductase in response to nitrate ion. The expression of nitrate reductase activity was not an index of nitrogen assimilation ability but may be a useful index of growth potential when nitrate ion does not limit nitrate reductase synthesis.     

Metabolite control of squash nitrate reductase

Nitrate reductase (EC 1.6.6.1) catalyzes the pyridine nucleotide-linked reduction of nitrate to nitrite in higher plants. We have shown that in squash (Cucurbita maxima Duchesne var. Buttercup), exogenous nitrate increases nitrate reductase activity by increasing steady-state levels of nitrate reductase protein, while glutamine diminishes nitrate reductase activity both by decreasing steady-state levels of nitrate reductase protein and by decreasing cellular nitrate concentrations in plant cells. Other amino acids affect nitrate reductase similarly to glutamine; other metabolites tested including nitrate did not cause major perturbations in the synthesis of other cellular proteins. Thus, it appears that the effects of nitrate and reduced nitrogen compounds on enzymes of the nitrate assimilatory pathway are highly specific for these enzymes, and have little effect on other cellular proteins.     

Synthesis and degradation of barley nitrate reductase

Nitrate and light are known to modulate barley (Hordeum vulgare L.) nitrate reductase activity. The objective of this investigation was to determine whether barley nitrate reductase is regulated by enzyme synthesis and degradation or by an activation-inactivation mechanism. Barley seedling nitrate reductase protein (cross-reacting material) was determined by rocket immunoelectrophoresis and a qualitative immunochemical technique (western blot) during the induction and decay of nitrate reductase activity. Nitrate reductase cross-reacting material was not detected in root or shoot extracts from seedlings grown without nitrate. Low levels of nitrate reductase activity and cross-reacting material were observed in leaf extracts from plants grown on nitrate in the dark. Upon nitrate induction or transfer of nitrate-grown etiolated plants to the light, increases in nitrate reductase activity were positively correlated with increases in immunological cross-reactivity. Root and shoot nitrate reductase activity and cross-reacting material decreased when nitrate-induced seedlings were transferred to a nitrate-free nutrient solution or from light to darkness. These results indicate that barley nitrate reductase levels are regulated by de novo synthesis and protein degradation.     

Regulation of Corn Leaf Nitrate Reductase : I. Immunochemical Methods for Analysis of the Enzyme's Protein Component

NADH:nitrate reductase was extracted from corn leaves (Zea mays L. W64A × W182E) and purified on blue Sepharose. After the nitrate reductase was further purified by polyacrylamide gel electrophoresis, it was used to immunize mice and a rabbit. Western blots of crude leaf extracts were used to demonstrate monospecificity of the mouse ascitic fluids and the rabbit antiserum. The electrophoretic properties of purified corn and squash NADH:nitrate reductases in both native and denatured states were shown to be similar using western blotting with mouse ascitic fluid. The corn leaf enzyme has a 115,000 polypeptide subunit like that of squash. Western blots could detect 3 to 10 nanograms of nitrate reductase protein. But the detection of proteolytic degradation products using western blotting was inconsistent and remains to be established. An enzyme-linked immunosorbent assay (ELISA) was developed for quantifying nitrate reductase protein in the crude extracts of corn leaves. Using a standard curve based on nitrate reductase activity, the ELISA for corn nitrate reductase could detect 0.5 to 10 nanograms of nitrate reductase protein and was adequately sensitive for quantitative analysis of nitrate reductase in crude extracts of leaves even when activity levels were very low. When the ELISA was used to compare the nitrate reductase protein content of corn roots and leaves, these tissues were estimated to contain 0.24 to 0.5 and 4 to 5 micrograms nitrate reductase protein/gram root and leaf, respectively.     

Regulation of nitrate reductase in excised barley roots

When excised barley roots (Hordeum distichum L.) are appropriately pretreated, the level of nitrate reductase in the roots increases upon exposure to nitrate. Relatively low levels of nitrate (10 mum) gave maximum induction of nitrate reductase. This increase was inhibited by inhibitors of protein and RNA synthesis, indicating that de novo protein synthesis is probably involved. Induction of nitrate reductase by nitrate is partially prevented by the inclusion of ammonium, an eventual product of nitrate reduction, in the incubation medium. Under the experimental conditions used, ammonium did not inhibit the uptake of nitrate by excised barley roots. It is concluded, therefore, that ammonium, or a product of ammonium metabolism, has a direct effect on the synthesis of nitrate reductase in this tissue.     

Control by Glutamine of the Synthesis of Nitrate Reductase in Cultured Spinach Cells

Immunoblotting, using antibodies raised against electrophoreticallypure nitrate reductase, was used to study the regulation ofsynthesis of nitrate reductase in cultured spinach cells. Theextent of the loss of nitrate reductase activity that occurredwhen cultures were transferred to a glutamine-containing mediumwas correlated with the decrease in the level of cross-reactingmaterial (repression). Removal of exogenous glutamine resultedin the appearance of nitrate reductase activity as well as ofimmunoreactive protein (derepression). The activity of nitratereductase in spinach cells under "repressing" or "derepressing"conditions appears to be regulated by changes in the amountof the enzyme protein rather than by inactivation and activationof preexisting protein. (Received August 22, 1991; Accepted May 28, 1992)     

Phytochrome-mediated light regulation of nitrate reductase expression in squash cotyledons

In etiolated squash (Cucurbita maxima L.) cotyledons, nitrate-inducible NADH:nitrate reductase activity and protein were increased in darkness by red light pulses with red/far-red photoreversibility. Continuous far-red light also led to increased levels of nitrate reductase activity and protein. Poly(A)+RNA, which hybridizes to squash nitrate reductase cDNA, was also increased by light treatments. Thus, we found that after nitrate triggering, nitrate reductase expression appears to be regulated by light via phytochrome.     

Nitrate reductase in agrostemma githago comparison of the inductive effects of nitrate and cytokinin

The effect of nitrate and cytokinin on the induction of nitrate reductase (NADH-nitrate oxidoreductase, EC 1.6.6.1) in embryos of Agrostemma githago was compared. Increased enzyme levels in response to treatment with the cytokinin benzyladenine were not correlated with a general stimulation of protein synthesis or a general reduction of protein breakdown. Actinomycin D did not inhibit the formation of nitrate reductase in response to nitrate or the cytokinin. Cycloheximide and puromycin inhibited the induction by the hormone to the same extent as, or even more than, the incorporation of [¹⁴C]leucine into protein. Induction of nitrate reductase by nitrate was much less susceptible to inhibition by cycloheximide and puromycin than induction of the enzyme by benzyladenine. When induction of nitrate reductase was carried out in the presence of ²H₂O, isopycnic equilibrium centrifugation in CsCl showed that incorporation of ²H into the enzyme had occured. The increase in the buoyant density of nitrate reductase was the same whether the enzyme was induced by nitrate or by benzyladenine, indicating that at least part of the nitrate reductase molecule was newly synthesized in both instances.     

Putrescine Effect on Nitrate Reductase Activity, Organic Nitrogen, Protein, and Growth in Heavy Metal and Salinity Stressed Mustard Seedlings

Putrescine effect on nitrate reductase activity, organic nitrogen and protein contents, and plant growth under Cd or Pb (0.1 – 2 mM) and salinity (5 and 100 mM NaCl) stresses was examined in Indian mustard (Brassica juncea L. cv. RH-30) seedlings. Cd or Pb and salinity inhibited nitrate reductase activity and decreased organic nitrogen and protein contents in leaf tissue. The increased nitrate reductase activity induced by putrescine was correlated with increased organic nitrogen and protein contents and growth of plants.     

Immunological approach to the regulation of nitrate reductase in Monoraphidium braunii

The effects of different culture conditions on nitrate reductase activity and nitrate reductase protein from Monoraphidium braunii have been studied, using two different immunological techniques, rocket immunoelectrophoresis and an enzyme-linked immunosorbent assay, to determine nitrate reductase protein. The nitrogen sources ammonium and glutamine repressed nitrate reductase synthesis, while nitrite, alanine, and glutamate acted as derepressors. There was a four- to eightfold increase of nitrate reductase activity and a twofold increase of nitrate reductase protein under conditions of nitrogen starvation versus growth on nitrate. Nitrate reductase synthesis was repressed in darkness. However, when Monoraphidium was grown under heterotrophic conditions with glucose as the carbon and energy source, the synthesis of nitrate reductase was maintained. With ammonium or darkness, changes in nitrate reductase activity correlated fairly well with changes in nitrate reductase protein, indicating that in both cases loss of activity was due to repression and not to inactivation of the enzyme. Experiments using methionine sulfoximine, to inhibit ammonium assimilation, showed that ammonium per se and not a product of its metabolism was the corepressor of the enzyme. The appearance of nitrate reductase activity after transferring the cells to induction media was prevented by cycloheximide and by 6-methylpurine, although in this latter case the effect was observed only in cells preincubated with the inhibitor for 1 h before the induction period.     

Protein-carbohydrate interaction. A turbidimetric study of the interaction of concanavalin A with amylopectin and glycogen and some of their enzymic and chemical degradation products

1. RNA and protein synthesis was studied during the incubation of excised radish cotyledons in nitrate, conditions that induced nitrate reductase activity in the tissue. 2. Synthesis of total RNA and protein, as measured by the incorporation of radioactive precursor, was significantly stimulated in the presence of nitrate (compared with chloride control), but was decreased in the presence of ammonium nitrate, which induced higher enzyme activity. 3. Synthesis of RNA and protein was required for induction of enzyme activity, as determined by using the inhibitors actinomycin D, puromycin and cycloheximide. 4. On the basis of 5-fluorouracil inhibition, the synthesis of only DNA-like RNA was required for induction, but no differences, either quantitative or qualitative, were observed in DNA-like RNA synthesis in the presence or absence of induction. 5. A 100-fold purification of the nitrate reductase activity showed no increase in nitrate reductase protein, nor any increased incorporation of radioactive precursor into nitrate reductase protein in the induced versus the control system. Such results suggested that the protein synthesis required for induction may be for a protein other than nitrate reductase.     

The development of nitrate reductase in Chlorella and its repression by ammonium

Summary Chlorella vulgaris, grown with ammonium sulphate as nitrogen source, contains very little nitrate reductase activity in contrast to cells grown with potassium nitrate. When ammonium-grown cells are transferred to a nitrate medium, nitrate reductase activity increases rapidly and the increase is partially prevented by chloramphenicol and by p-fluorophenylalanine, suggesting that protein synthesis is involved. The increase in nitrate reductase activity is prevented by small quantities of ammonium; this inhibition is overcome, in part, by raising the concentration of nitrate. Although nitrate stimulates the development of nitrate reductase activity, its presence is not essential for the formation of the enzyme since this is formed when ammonium-grown cells are starved of nitrogen and when cells are grown with urea or glycine as nitrogen source. It is concluded that the formation of the enzyme is stimulated (induced) by nitrate and inhibited (repressed) by ammonium.     

Isolation and Some Properties of a 115-Kilodalton Nitrate Reductase-Inactivator Protein from Spinacia oleracea

A nitrate reductase inactivator protein in spinach leaves waspurified (90-fold). The purification involved precipitationwith ammonium sulfate, treatment at pH 4, CM-cellulose chromatog-raphyand gel filtration on a Toyopearl HW-55F column. From the ToyopearlHW-55F gel filtration step the molecular weight of the inactivatorwas estimated to be 115 kDa. The inactivator was particularly sensitive to EDTA, o-phenanthrolineand pronase. The inactivator was more stable to heat treatmentthan NADH-nitrate reductase. Incubation of purified spinachnitrate reductase with the inactivator results in a loss ofNADH-nitrate reductase and the associated partial activities,NADH-ferricyanide reductase, NADH-cytochrome c reductase, butnot in no loss in nitrate reducing activity with reduced methylviologen as the electron donor. The molecular weight of thenitrate reductase-inactivator protein complex was estimatedby gel filtration on Toyopearl HW-55F to be 460 kDa, comparedto an apparent molecular weight of 240 kDa for the untreatedcontrol estimated under the same conditions. These results indicatethat spinach nitrate reductase inactivator protein acts by bindingto nitrate reductase. The stoichiometry of binding is 2 moleculesof the inactivator protein to one dimeric molecule of nitratereductase. The action of the inactivator protein was partiallyprevented by NADH. (Received September 21, 1987; Accepted January 8, 1988)     

Regulation of Nitrate Reductase in Neurospora crassa: Stability In Vivo

Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-nitrate reductase and its related enzyme activities, NADPH-cytochrome c reductase and reduced benzyl viologen-nitrate reductase, are all induced following the transfer of ammonia-grown wild-type Neurospora mycelia to nitrate medium. After nitrate reductase is induced to the maximal level, the addition of an ammonium salt to, or the removal of nitrate from, the cultures results in a rapid inactivation of nitrate reductase and its two partial component activities. This rapid inactivation is slowed down by the protein synthesis inhibitor, cycloheximide. Experiments on the mixing of extracts in vitro rule out the presence of an inhibitor of nitrate reductase in free form in extracts containing inactivated nitrate reductase. Ammonia does not inhibit the uptake of nitrate by the mycelia. Inactivation of nitrate reductase in vivo by ammonia depends on the concentration of the ammonium salt and is not reversed by increasing the nitrate concentration of the medium. The nitrate-inducible NADPH-cytochrome c reductase activity and reduced benzyl viologen-nitrate reductase activity respectively of the nitrate-nonutilizing mutants nit-1 and nit-3 are not inactivated in vivo by the addition of an ammonium salt or the withdrawal of nitrate. This finding suggests that the integrity of the nitrate reductase complex is required for the in vivo inactivation of nitrate reductase and its associated activities.     

Regulation of nitrate reductase inRhodobacter capsulatus E1F1

The photosynthetic purple non-sulfur nitrate-assimilating bacteriumRhodobacter capsulatus E1F1 has an adaptive nitrate reductase activity inducible by either nitrate or nitrite and molybdenum traces. Nitrate reductase induction by nitrate did not occur in media with nitrate and ammonium, which showed no effect if nitrite was the inductor instead of nitrate or in the presence ofl-methionine-dl-sulfoximine (MSX) plus nitrate. In vivo, tungstate inhibited nitrate reductase activity, and this was not recovered upon addition of molybdenum unless de novo protein synthesis took place. Nitrate reductase was also repressed in nitrogen-starved cells or after the addition of azaserine to cells growing phototrophically with nitrate. Moreover, higher rates of nitrate reductase induction and nitrite excretion were found in illuminated cells grown with nitrate under air than in those grown under argon.     

Diurnal changes in nitrogen assimilation of tobacco roots.

To gain an insight into the diurnal changes of nitrogen assimilation in roots the in vitro activities of cytosolic and plasma membrane-bound nitrate reductase (EC 1.6.6.1), nitrite reductase (EC 1.7.7.1) and cytosolic and plastidic glutamine synthetase (EC 6.3.1.2) were studied. Simultaneously, changes in the contents of total protein, nitrate, nitrite, and ammonium were followed. Roots of intact tobacco plants (Nicotiana tabacum cv. Samsun) were extracted every 3 h during a diurnal cycle. Nitrate reductase, nitrite reductase and glutamine synthetase were active throughout the day-night cycle. Two temporarily distinct peaks of nitrate reductase were detected: during the day a peak of soluble nitrate reductase in the cytosol, in the dark phase a peak of plasma membrane-bound nitrate reductase in the apoplast. The total activities of nitrate reduction were similar by day and night. High activities of nitrite reductase prevented the accumulation of toxic amounts of nitrite throughout the entire diurnal cycle. The resulting ammonium was assimilated by cytosolic glutamine synthetase whose two activity peaks, one in the light period and one in the dark, closely followed those of nitrate reductase. The contribution of plastidic glutamine synthetase was negligible. These results strongly indicate that nitrate assimilation in roots takes place at similar rates day and night and is thus differently regulated from that in leaves.     

Phytochrome, nitrate movement, and induction of nitrate reductase in etiolated pea terminal buds

The role of phytochrome in the induction of nitrate reductase of etiolated field peas (Pisum arvense L.) was examined. Terminal bud nitrate concentration increased in darkness, and the increase correlated with induction of nitrate reductase following brief exposure of intact plants to red, blue, far red, and white lights. Brief light exposure of intact plants stimulated nitrate uptake and induction of nitrate reductase by terminal buds subsequently excised and incubated on nitrate solution in darkness; exposure of excised buds in contact with nitrate led to less uptake but more induction. Nitrate and nitrate reductase activity both declined during incubation with water, irrespective of light treatment. Nitrate enrichment of intact terminal buds and uptake into excised buds and increases in nitrate reductase activity were all red/far red reversible. Dimethyl sulfoxide (1%, v/v) and sugars (sucrose 0.5%, glucose 1, w/v), although stimulating nitrate uptake into excised tissue in darkness, failed to enhance nitrate reductase activity over dark controls. Phytochrome may regulate nitrate reductase via both nitrate movement and a general mechanism such as enhancement of protein synthesis.