Some characteristics of nitrate reductase from higher plants

With respect to cofactor requirements, NADH, and FMNH₂ were equally effective as electron donors for nitrate reductase obtained from leaves of maize, marrow, and spinach, when the cofactors were supplied in optimal concentrations. The concentration of FMNH₂ required to obtain half-maximal activity was from 40- to 100-fold higher than for NADH. For maximal activity with the corn enzyme, 0.8 millimolar FMNH₂ was required. In contrast, NADPH was functional only when supplied with NADP:reductase and exogenous FMN (enzymatic generation of FMNH₂).

All attempts to separate the NADH₂- and FMNH₂-dependent nitrate reductase activities were unsuccessful and regardless of cofactor used equal activities were obtained, if cofactor concentration was optimal. Unity of NADH to FMNH₂ activities were obtained during: A) purification procedures (4 step, 30-fold); B) induction of nitrate reductase in corn seedlings with nitrate; and C) inactivation of nitrate reductase in intact or excised corn seedlings. The NADH- and FMNH₂-dependent activities were not additive.

A half-life for nitrate reductase of approximately 4 hours was estimated from the inactivation studies with excised corn seedlings. Similar half-life values were obtained when seedlings were incubated at 35° in a medium containing nitrate and cycloheximide (to inhibit protein synthesis), or when both nitrate and cycloheximide were omitted.

In those instances where NADH activity but not FMNH₂ activity was lost due to treatment (temperature, removal of sulfhydryl agents, addition of p-chloromercuribenzoate), the loss could be explained by inactivation of the sulfhydryl group (s) required for NADH activity. This was verified by reactivation with exogenous cysteine.

Based on these current findings, and previous work, it is concluded that nitrate reductase is a single moiety with the ability to utilize either NADH or FMNH₂ as cofactor. However the high concentration of FMNH₂ required for optimal activity suggests that in vivo NADH is the electron donor and that nitrate reductase in higher plants should be designated NADH:nitrate reductase (E.C. 1.6.6.1).

     

Nitrate reductase activity in corn seedlings as influenced byArthrobacter sp.

Summary Nitrate reductase activity has been assayed in corn seedlings grown in a nutrient solution containing different concentrations of nitrate and inoculated withArthrobacter sp. The enzymatic activity was greatly enhanced especially at the levels of NO₃-ions suboptimal for the maximum induction. The ability of synthetic phytohormones (IAA, IPA and GA₃) in inducting nitrate reductase activity has been also texted.     

Influences of some internal and external conditions on the induction of nitrate reductase in rice seedlings

Induction of nitrate reductase (NR) in 7-day-old rice seedlingswas depressed when endosperms were removed. NR activity in theseedlings from which endosperms were removed (deendospermedseedlings), reached a maximum 6 hr after supplying NaNO₃ andthen gradually decreased. That in intact seedlings continuedto increase for 12 hr, and then decreased fairly rapidly. Sucrose(30 mM concentration) supplied exogenously to deendospermedseedlings raised NR activity to the level of the intact seedlings.Macronutrients added exogenously did not show such an effect. NR activity in deendospermed seedlings placed in the dark wasextremely low. However, in the presence of exogenous sucrose,the activity was raised to the same level as that in the lightin the absence of exogenous sucrose. This suggests that sucrosesubstitutes for light in the induction of NR in deendospermedseedlings. Protein inhibitors suppressed NR induction when theplants were fed continuously with nitrate solution containingthe inhibitor. In cases where the plant roots were immersedin inhibitor solutions for 2 hr before transfer to nitrate solution,only chloramphenicol promoted and the others inhibited NR induction.NR induction was also suppressed by respiratory inhibitors,of which sodium azide was very potent. (Received August 25, 1976; )     

Correlation between Polyribosome Level and the Ability to Induce Nitrate Reductase in Dark-grown Corn Seedlings

Nitrate reductase can be induced in excised shoots of 3-day-old dark-grown Zea mays (var. WF9 × M14) seedlings in the absence of light. In contrast, leaves of 10-day-old dark-grown seedlings require a light treatment in order to induce enzymatic activity. Leaves of 10-day-old dark-grown seedlings contain a very low level of polyribosomes while 3-day-old shoots contain a very high level of polyribosomes. There is a gradual loss of polyribosomes from 3 to 10 days and a gradual loss of in vitro protein synthetic activity of the ribosome preparations. The loss of polyribosomes and decrease in their amino acid-incorporating activity correlate positively with the loss of ability to induce nitrate reducase activity as leaves of dark-grown corn seedlings age. These results corroborate and extend our previous results, in that light is not required for nitrate reductase induction per se in leaves of dark-grown seedlings but is required to reactivate the protein synthetic apparatus of older leaves.     

Influence of Age and Light on the Distribution and Development of Nitrate Reductase in Greening Barley Leaves

The relation between leaf age and the induction of nitrate reductase activity by continuous and intermittent light was studied with barley seedlings (Hordeum vulgare L. cv. Club Mariout). In general, nitrate reductase activity declined as the period of growth in darkness was extended beyond 5 days. Maximum activity was found near the leaf tip while activity was lowest in the morphologically youngest tissue near the base of the lamina. Increased activity was observed after continuous illumination of dark-grown seedlings for 24 hours. The increase in activity in response to light was greatly reduced when the dark pretreatment period was extended beyond 8 days. The amount of nitrate reductase activity present in the different sections of the leaf was closely related to the amount of polyribosomes present. The pattern of chlorophyll accumulation closely parallelled that of increases in nitrate reductase activity. The initial lag in the induction of nitrate reductase activity was removed by a 10-minute light treatment 6 hours before placing dark-grown barley seedlings in light. The enzyme was also induced under flashing light with various dark intervals. These induction curves closely resembled those of chlorophyll accumulation under the same conditions. The development of photosynthetic CO₂ fixation follows the same induction pattern in this system. Our results suggest that photosynthetic products may be required for the induction of significant levels of nitrate reductase activity in leaves of dark-grown seedlings, although other light effects may not be discounted.     

Nitrate reductase inactivator from Spirodela polyrhiza (L.) Schleiden

NADH:nitrate reductase (EC 1.6.6.1) activity in the crude extract from Spirodela polyrhiza was relatively labile in vitro. Inclusion of polyvinylpolypyrrolidone into the extraction medium had only a slight effect on the stability of the enzyme, whereas addition of 3 % casein, azocasein, or other proteins to the extraction medium greatly increased the nitrate reductase (NR) activity. Various protease inhibitors were tested for their ability to prevent the loss of NR activity in vitro. Iodoacetate and para-chloromercuric benzoate, the thiol-protease inhibitors, as well as pepstatin, the aspartic-protease inhibitor had no effect on stability of the nitrate reductase. EDTA had a slight stimulatory effect, whereas 5 mM o-phenantroline, another inhibitor of the metallo-proteases increased the activity of nitrate reductase. The highest enzyme activity was found in the presence of phenylmethylsulphonyl fluoride and di-isopropyl phosphorofluoridate both being serine-protease inhibitors. The protease-like inactivator was separated from Spirodela polyrhiza by ammonium sulfate fractionation and acid treatment (pH 4.0). After centrifugation the protein of inactivator in supernatant adjusted to pH 7.5 was removed. When this fraction was examined by electrophoresis in polyacrylamide which copolymerized with edestin, the protein of the nitrate reductase inactivator remained at the cathode. Fractions containing a protein of inactivator degraded casein to products soluble in trichloroacetic acid. Inhibition of the inactivator proteolytic activity by phenylmethylsulphonyl fluoride and di-isopropyl phosphorofluoridate but not by other reagents (thiol- and metallo-protease inhibitors) suggested the involvement of a serine residue at its active site. The inactivator fraction from Spirodela polyrhiza resulted in a loss of the nitrate reductase activity in crude extracts from both cucumber and corn seedlings. A biochemical nature a protein of the nitrate reductase inactivator from S. polyrhiza is discussed.     

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.     

Increase in Nitrate Reductase Activity in Bean Leaves by Light Involves a Regulator Protein

Nitrate reductase activity, assayed either in vivo or in vitro was considerably higher in bean (Phaseolus vulgaris L.) leaves from 7-day-old light grown seedlings than those from dark grown, both in the absence as well as presence of nitrate. Cytochrome c reductase activity was however similar in both regimes, while peroxidase was lower in light than in dark. The light stimulated increase in nitrate reductase activity in leaf segments from dark grown seedlings was inhibited by cycloheximide, DNP, chloramphenicol, and sodium tungstate and was unaffected by lincomycin and DCMU. Under similar conditions, the increase in total chlorophyll was inhibited completely by cycloheximide and DNP, partially by chloramphenicol and lincomycin, and was unaffected by tungstate and DCMU. A supply of 1~5 mm reduced glutathione increased enzyme activity in the dark and also to some extent in light. The substrate induction of enzyme activity started after a lag of one hr in light or dark and continued for either 5 hr in the dark or 8 hr in light. Two proteinaceous inhibitors (Factors I and II) of nitrate reductase were isolated by ammonium sulfate precipitation and Sephadex gel filtration. The amount of Factor I was higher in the dark than in light. The amount and activity of Factor II was however, almost equal in light and dark. The inhibition of enzyme activity by these inhibitors increased with their concentration. It is proposed that light increases nitrate reductase activity by decreasing the amount of a nitrate reductase inhibitor.     

Biosynthesis of Ferredoxin-Nitrite Reductase in Rice Seedlings

Changes in ferredoxin-nitrite reductase [EC 1.7.7.1 [EC] ] in etiolatedrice seedlings were followed during induction by nitrate andlight. Etiolated seedlings showed maximal induction of the enzymeactivity during greening with nitrate, while the enzyme activityin etiolated seedlings receiving nitrate in darkness increasedhalf as much as that in nitrate-treated greening plants. Theincrease in nitrite reductase activity during induction coincidedwith an increase in the content of proteins immunoprecipitatedby antibodies raised against spinach nitrite reductase. Lighthad no effect on the induction of the extractable nitrite reductasein the absence of nitrate. Poly(A)⁺-RNA extracted from nitrate-treatedgreening shoots directed the synthesis in a rabbit reticulocyte-lysateof polypeptides immunoprecipitated by spinach nitrite reductaseantibodies. One major polypeptide larger than the native enzymewas found among the translation products, suggesting that nitritereductases in greening rice shoots are synthesized as an precursorform. Analysis of two-dimensional electrophoretograms indicatedthe existence of isoforms of nitrite reductase in rice seedlingswhich had been immunoprecipitated with spinach nitrite reductaseantibodies. ¹To whom all correspondence should be sent. (Received May 15, 1987; Accepted September 7, 1987)     

Effect of carbon dioxide on nitrate accumulation and nitrate reductase induction in corn seedlings

Exposure of the leaf canopy of corn seedlings (Zea mays L.) to atmospheric CO₂ levels ranging from 100 to 800 μl/l decreased nitrate accumulation and nitrate reductase activity. Plants pretreated with CO₂ in the dark and maintained in an atmosphere containing 100 μl/l CO₂ accumulated 7-fold more nitrate and had 2-fold more nitrate reductase activity than plants exposed to 600 μl/l CO₂, after 5 hours of illumination. Induction of nitrate reductase activity in leaves of intact corn seedlings was related to nitrate content. Changes in soluble protein were related to in vitro nitrate reductase activity suggesting that in vitro nitrate reductase activity was a measure of in situ nitrate reduction. In longer experiments, levels of nitrate reductase and accumulation of reduced N supported the concept that less nitrate was being absorbed, translocated, and assimilated when CO₂ was high. Plants exposed to increasing CO₂ levels for 3 to 4 hours in the light had increased concentrations of malate and decreased concentrations of nitrate in the leaf tissue. Malate and nitrate concentrations in the leaf tissue of seven of eight corn genotypes grown under comparable and normal (300 μl/l CO₂) environments, were negatively correlated. Exposure of roots to increasing concentrations of potassium carbonate with or without potassium sulfate caused a progressive increase in malate concentrations in the roots. When these roots were subsequently transferred to a nitrate medium, the accumulation of nitrate was inversely related to the initial malate concentrations. These data suggest that the concentration of malate in the tissue seem to be related to the accumulation of nitrate.     

Effect of ammonium nutrition on the activity of nitrate reductase in the roots of apple seedlings

Active extracts of nitrate reductase were prepared from theroots of apple seedlings c.v. Granny Smith which were grownin nutrient solution under controlled enviromental conditions.The nutrient solutions contained various ratios of nitrate andammonium ions but all the treatments contained a total of 112ppm nitrogen. Maximum nitrate reductase activity in the roots was obtainedwhen plants were supplied with nitrate as the sole source ofnitrogen. Roots grown in solution containing only ammonium nitrogenhad little or no activity. When plants were supplied with bothforms of nitrogen in the nutrient solution, the presence ofammonium ions markedly lowered the activity of nitrate reductasein the roots. Plants supplied with 98 ppm nitrate nitrogen plus14 ppm ammonium nitrogen had activities only half those of plantsgrown in nitrate alone. Plants supplied with equal amounts ofammonium and nitrate nitrogen had activities less than one sixththose of plants grown in nitrate alone. (Received June 3, 1972; )     

Regulation of Chlorella nitrate reductase: control of enzyme activity and immunoreactive protein levels by ammonia

Nitrate reductase catalyzes the initial step in the conversion of nitrate to organic nitrogen and is thought to be repressed by ammonia and induced by nitrate. Induction by nitrate and repression by ammonia were studied by following changes in NADH:nitrate reductase and the associated partial activities NADH:cytochrome c reductase and methylviologenr:nitrate reductase. Immunoreactive protein was assessed by enzyme-linked immunosorbent assay and immunoblotting. Molybdenum cofactor levels were investigated using the nit-1 complementation assay as well as fluorescence of the oxidized cofactor. The results indicate that the NADH:cytochrome c reductase activity is "induced" faster than the nitrate-reducing activity and suggest that incorporation of the molybdo-pterin cofactor may be rate limiting in the expression of activity. Molybdenum cofactor levels are significantly elevated in nitrate-treated cells. Under "repressing" conditions all activities decreased at approximately the same rate. A more rapid conversion of the enzyme to a reversibly inactive form also occurred under these conditions. Changes in immunoreactive protein levels correlated most closely with NADH:cytochrome c reductase activity but appeared to increase faster during induction and decrease slightly slower during repression than the enzyme activities. Removal of exogenous ammonia results in the appearance of nitrate reducing activity, as well as immunoreactive protein (derepression). Studies using protein and RNA synthesis inhibitors indicated that de novo synthesis is required for nitrate reductase induction and were in agreement with the results of the immunoreactive studies.     

Intact plastids are required for nitrate- and light-induced accumulation of nitrate reductase activity and mRNA in squash cotyledons

Induction of nitrate reductase activity and mRNA by nitrate and light is prevented if chloroplasts are destroyed by photooxidation in norflurazon-treated squash (Cucurbita maxima L.) cotyledons. The enzyme activity and mRNA can be induced if norflurazon-treated squash seedlings are kept in low-intensity red light, which minimizes photodamage to the plastids. It is concluded that induction of nitrate reductase activity and nitrate reductase mRNA requires intact plastids. If squash seedlings grown in low-intensity red light are transferred to photooxidative white light, nitrate reductase activity accumulates during the first 12 hours after the shift and declines thereafter. Thus photodamage to the plastids and the disappearance of nitrate reductase activity and mRNA are events separable in time, and disappearance of the enzyme activity is a consequence of the damage to the plastids.     

Effect of simazine on nitrate reductase activity in corn

The nutritional and environmental parameters required for eliciting increases in the nitrogen content and growth of corn (Zea mays L.) by non-toxic levels of simazine have been established. Corn seedlings with the endosperm removed 10 days after germination, proved to be a suitable test species. The addition of simazine to the root-zone area of corn plants grown under both sub-optimal temperatures and low nitrate levels, increased the nitrogen content and dry weight of the plants by 20 to 25%. This increase was found to be associated with an effect on nitrate reductase.     

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.     

Rapid and slow modulation of nitrate reductase activity in the red macroalga <Emphasis Type="Italic">Gracilaria chilensis</Emphasis> (Gracilariales,Rhodophyta): influence of different nitrogen sources

Nitrate is one of the most important stimuli in nitrate reductase (NR) induction, while ammonium is usually an inhibitor. We evaluated the influence of nitrate, ammonium or urea as nitrogen sources on NR activity of the agarophyte Gracilaria chilensis. The addition of nitrate rapidly (2 min) induced NR activity, suggesting a fast post-translational regulation. In contrast, nitrate addition to starved algae stimulated rapid nitrate uptake without a concomitant induction of NR activity. These results show that in the absence of nitrate, NR activity is negatively affected, while the nitrate uptake system is active and ready to operate as soon as nitrate is available in the external medium, indicating that nitrate uptake and assimilation are differentially regulated. The addition of ammonium or urea as nitrogen sources stimulated NR activity after 24 h, different from that observed for other algae. However, a decrease in NR activity was observed after the third day under ammonium or urea. During the dark phase, G. chilensis NR activity was low when compared to the light phase. A light pulse of 15 min during the dark phase induced NR activity 1.5-fold suggesting also fast post-translational regulation. Nitrate reductase regulation by phosphorylation and dephosphorylation, and by protein synthesis and degradation, were evaluated using inhibitors. The results obtained for G. chilensis show a post-translational regulation as a rapid response mechanism by phosphorylation and dephosphorylation, and a slower mechanism by regulation of RNA synthesis coupled to de novo NR protein synthesis.     

Variation in the nitrate reductase of rice seedlings

Summary Nitrate reductase was induced in rice seedlings by nitrate and by chloramphenicol. During the induction period the different enzyme activities associated with nitrate reductase increased to different degrees. Nitrate induced high NADH-nitrate reductase activity and a great increase in the NADH-cytochrome c reductase activity which was associated with the nitrate reductase in a sucrose gradient. Chloramphenicol induced a nitrate reductase which had higher activity with NADPH than NADH. Chloramphenicol also induced a marked increase in NADPH-cytochrome c reductase activity as well as in NADH-cytochrome c reductase activity. Both activities were associated with the nitrate reductase in a sucrose gradient.After partial purification by sucrose gradient sedimentation or by starch gel electrophoresis, the nitrate reductase of rice induced by nitrate and chloramphenicol showed the same preference in pyridine nucleotide cofactors as was shown by the crude enzyme extracts.     

Specificity for nicotinamide adenine dinucleotide by nitrate reductase from leaves

Preliminary work revealed that nitrate reductase in crude extracts prepared from leaves of certain corn genotypes as well as soybeans could utilize NADPH as well as NADH as the electron donor. Isoelectric focusing and diethylaminoethyl cellulose chromatography confirmed previous findings that NADH and NADPH activities could not be separated, which suggests the involvement of a single enzyme. Nitrate reduction with both cofactors varies with plant species, plant age, and assay conditions. The ability of the nitrate reductase from a given genotype to utilize NADPH was associated with the amount of NADPH-phosphatase in the extract. While diethylaminoethyl cellulose chromatography of plant extracts separated nitrate reductase from the bulk (90%) of the phosphatase and caused a decrease in the NADPH activity, the residual level of phosphatase was sufficient to account for the apparent NADPH nitrate reductase activity. Addition of KH₂PO₄ and KF, inhibitors of NADPH-phosphatase activity in in vitro assays, caused a drastic reduction or abolishment of NADPH-mediated nitrate reductase activity but were without effect on NADH nitrate reductase activity. It is concluded that NADPH-nitrate reduction, in soybean and certain corn genotypes, is an artifact resulting from the conversion of NADPH to NADH by a phosphatase and that the enzyme in leaf tissue is NADH-dependent (E.C.1.6.6.1).     

Evidence for a role of calcium in nitrate assimilation in wheat seedlings

Severely Ca-deficient Triticum aestivum L. seedlings accumulated high levels of nitrite and moderate levels of nitrate and organic nitrogen, but contained unaltered levels of hydroxylamine. Nitrite accumulation was not related to molybdenum deficiency, or altered cellular pH. Nitrate reductase was decreased by Ca deficiency, apparently by repression of enzyme synthesis from accumulated nitrite and not by inhibition of enzyme activity. Nitrite reductase and NADP diaphorase activities were not affected by Ca deficiency, and Ca did not restore activity to nitrite reductase inactivated by cyanide. The results indicated that the role of Ca is in intracellular transport of nitrite and not in induction or activity of enzymes.