Relationships between nitrate level,nitrate reductase activity and anaerobic nitrite production inPisum sativum leaf tissue

Anaerobic nitrite production (thein vivo NO₃-R activity) in an incubation medium lacking exogenous nitrate but containing 0.5%n-propanol and 0.1% Triton X-100 showed higher correlation (y - ax ^b) with the level of endogenous nitrate inPisum sativum L. leaves than thein vitro nitrate reductase activity. Thein vivo NO₃-R activity correlated well with thein vitro activity up to the 50 ppm NO₃-N level of endogenous nitrate. The ratioin vivo: in vitro activity slightly decreased with increasing level of endogenous nitrate in leaf tissue.     

Effect of DCMU, Simazine and Atrazine on Nitrate Reductase Activity in Hordeum vulgare in vitro

The effect of three herbicides—DCMU (1,1-dimethyl-3- (3,4-dichlorophenyl) -urea), Simazine (2,4-bis(ethylamino)- 6-chloro-s-triazine), and Atrazine (2-chloro-4-ethylamino-6-iso-propylamino-5-triazine)—on the induction of nitrate reduc–tase and its in vivo activity was studied in detached leaves of Hordeum vulgare L. All increased both extractable nitrate reductase activity and nitrate content. The increases occurred at optimum temperatures for growth and at several concentrations of nitrate. It was also determined that the herbicides did not protect the enzyme against inactivation in vivo. Although the extractable nitrate reductase was greater, the in vivo activity of nitrate reductase was decreased in the presence of the herbicides resulting in a higher internal concentration of nitrate. Since in viva nitrate reduction is dependent upon photosynthesis it is reasonable that reduction is decreased by these known inhibitors of photosynthesis. Hence, the effect of the inhibitors on induction of nitrate reductase activity may be secondary. The higher concentration of nitrate resulting from a decreased rate of in vivo reduction in the presence of the inhibitors could conceivably be responsible for the greater corutent of nitrate reductase.     

Reassessment of the in vivo Assay for Nitrate Reductase in Leaves

The in vivo assay procedure for nitrate reductase and its dependence on the concentration of nitrate and other ions were examined. It was found that high ion concentrations led to an increased release of nitrite to the reaction media which could be interpreted as a stimulated nitrate reductase activity. This phenomenon is not an osmotic effect, since equivalent concentrations of mannitol did not lead to identical results. The effect of ions on the enhanced nitrite production was attributed to changes in cell membrane permeability rather than to a supply of substrate. This conclusion is based on several findings: (a) in in vitro assays, the rate of nitrite production was not affected by ion concentrations: (b) the stimulation of nitrite production was obtained by various ions and not only by nitrate; (c) pretreatment of alfalfa leaves with nitrate did not increase the NO₂^? release rate to the external solution; and (d) nitrate and nitrite export from leaf discs to the solution was stimulated even in discs which were enzymatically inactive. Calcium ions in the presence of KNO₃ inhibited the enhanced nitrite production, probably due to alteration of membrane stability. The effect of ions on the rate of nitrite production was reversible and the high rate of nitrite production was reduced to the control rate when discs were transferred to a solution of low concentration.     

Differential effect of tungsten on the development of endogenous and nitrate-induced nitrate reductase activities in soybean leaves

The effect of tungsten on the development of endogenous and nitrate-induced NADH- and FMNH₂-linked nitrate reductase activities in primary leaves of 10-day-old soybean (Glycine max [L.] Merr.) seedlings was studied. The seedlings were grown with or without exogenous nitrate. High levels of endogenous nitrate reductase activities developed in leaves of seedlings grown without nitrate. However, no endogenous nitrite reductase activity was detected in such seedlings. The FMNH₂-linked nitrate reductase activity was about 40% of NADH-linked activity. Tungsten had little or no effect on the development of endogenous NADH- and FMNH₂-linked nitrate reductase activities, respectively. By contrast, in nitrate-grown seedlings, tungsten only inhibited the nitrate-induced portion of NADH-linked nitrate reductase activity, whereas the FMNH₂-linked activity was inhibited completely. Tungsten had no effect on the development of nitrate-induced nitrite reductase activity. The complete inhibition of FMNH₂-linked nitrate reductase activity by tungsten in nitrate-grown plants was apparently an artifact caused by the reduction of nitrite by nitrite reductase in the assay system. The results suggest that in soybean leaves either the endogenous nitrate reductase does not require molybdenum or the molybdenum present in the seed is preferentially utilized by the enzyme complex as compared to nitrate-induced nitrate reductase.     

Effect of Elevated Temperature on Nitrite and Nitrate Reduction in Leaves and Intact Chloroplasts

Barley (Hordeum vulgare L.) leaves and intact spinach (Spinacia oleracea L.) chloroplasts were exposed to short-term heating, and the aftereffects of heat treatment on in vitro andin vivo activities of nitrate reductase and noncyclic electron transport associated with nitrite reduction were studied. Heating of leaves at temperatures above 40°C led to a monotonic decrease in nitrate reductase in vitro activity. On the contrary, the in vivo enzyme activity, assayed in intact leaf tissues after 5-min heat treatment, increased 1.5 times upon elevating the pretreatment temperature from 37 to 40°C and gradually decreased at higher temperatures. Noncyclic electron transport related to CO₂ fixation in intact chloroplasts decreased gradually after heat exposures above 39°C, unlike the electron transport to nitrite as a terminal acceptor, which was stimulated by heating of intact chloroplast suspensions in the temperature range from 33 to 40°C. The heating at higher temperatures inhibited nitrite photoreduction. It is concluded that the heating of phototrophic cells at sublethal temperatures stimulates the mobilization of inorganic nitrogen and thereby facilitates the repair of thermally induced injuries of proteinaceous cell structures. The stimulation of nitrate reductase activity in vivo at the temperature range 37–40°C provides an evidence for the increase in the availability of reductants in the cytosolic compartment of the leaf cell.     

A plasma membrane-bound enzyme of tobacco roots catalyses the formation of nitric oxide from nitrite

Purified plasma membranes (PMs) of tobacco (Nicotiana tabacum L. cv. Samsun) roots exhibited a nitrite-reducing enzyme activity that resulted in nitric oxide (NO) formation. This enzyme activity was not detected in soluble protein fractions or in PM vesicles of leaves. At the pH optimum of pH 6.0, nitrite was reduced to NO with reduced cytochrome c as electron donor at a rate comparable to the nitrate-reducing activity of root-specific succinate-dependent PM-bound nitrate reductase (PM-NR). The hitherto unknown PM-bound nitrite: NO-reductase (NI-NOR) was insensitive to cyanide and anti-NR IgG and thereby proven to be different from PM-NR. Furthermore, PM-NR and NI-NOR were separated by gel-filtration chromatography and apparent molecular masses of 310 kDa for NI-NOR and 200 kDa for PM-NR were estimated. The PM-associated NI-NOR may reduce the apoplastic nitrite produced by PM-NR in vivo and may play a role in nitrate signalling via NO formation. Received: 8 May 2000 / Accepted: 24 August 2000     

Effect of Grapevine Leafroll on the Photosynthesis of Field Grown Grapevine Plants (Vitis vinifera L. cv. Lagrein)

We have studied the effect of grapevine leafroll infection on some features of the thylakoids from field grown grapevine (Vitis vinifera L.) leaves. Changes in photosynthetic pigments, soluble proteins, ribulose‐1,5‐bisphosphate carboxylase (RuBP), nitrate reductase, photosynthetic activities and thylakoid membrane proteins were investigated. The level of total chlorophyll (Chl) and carotenoids were reduced in virus‐infected leaves. Similar results were also observed for soluble proteins and RuBP case activity. The in vivo nitrate reductase activity was significantly reduced in infected leaves. Virus infection considerably decreased leaf net photosynthetic rate (P_n), stomatal conductance (g_s) and transpiration rate (E) in grapevine leaves. When various photosynthetic activities were followed in isolated thylakoids, virus infection caused marked inhibition of whole chain and photosystem (PS) II activity while the inhibition of PSI activity was only marginal. The artificial exogenous electron donors, diphenyl carbazide and hydroxylamine (NH₂OH) significantly restored the loss of PSII activity in infected leaves. The same results were obtained when F_v/F_m was evaluated by Chl fluorescence measurements. The marked loss of PSII activity in infected leaves could be due to the loss of 47, 43, 33, 28–25, 23 and 17 kDa polypeptides. It is concluded that virus infection inactivates the donor side of PSII. This conclusion was confirmed by immunological studies showing that the content of the 33 kDa protein of the water‐splitting complex was diminished significantly in infected leaves.     

Nitrate content and nitrate reductase activity in Rumex obtusifolius L.

Summary With Rumex obtusifolius L., the influence of some environmental conditions on nitrate uptake and reduction were investigated. Nitrate concentrations of plant material were determined by HPLC, the activity of nitrate reductase by an in vivo test. As optimal incubation medium, a buffer containing 0.04 M KNO₃; 0.25 M KH²PO₄; 1.5% propanol (v/v); pH 8.0 was found. Vacuum infiltration caused an increase of enzyme activity of up to 40%.High nitrate concentrations were found in roots and leaf petioles. Nitrate reductase activity of these organs, however, was low. On the other hand, the highest nitrate reductase activity was observed in leaf laminae, which contained lowest nitrate concentrations.In leaves, nitrate content and nitrate reductase activity exhibited inverse diurnal fluctuations. During darkness, decreasing activities of the enzyme were followed by increasing nitrate concentrations, while during light the contrary was true. In petioles diurnal fluctuations in nitrate content were observed, too. No significant correlations with illumination, however, could be found.Our results prove that Rumex obtusifolius is characterized by an intensive nitrate turnover. Theoretically, internal nitrate content of the plant would be exhausted within a few hours, if a supply via the roots would be excluded.     

Aerobic nitrate and nitrite reduction in continuous cultures of Escherichia coli E4

Nitrate and nitrite was reduced by Escherichia coli E4 in a l-lactate (5 mM) limited culture in a chemostat operated at dissolved oxygen concentrations corresponding to 90–100% air saturation. Nitrate reductase and nitrite reductase activity was regulated by the growth rate, and oxygen and nitrate concentrations. At a low growth rate (0.11 h^–1) nitrate and nitrite reductase activities of 200 nmol · mg^–1 protein · min^–1 and 250 nmol · mg^–1 protein · min^–1 were measured, respectively. At a high growth rate (0.55 h^–1) both enzyme activities were considerably lower (25 and 12 nmol mg^–1 · protein · min^–1). The steady state nitrite concentration in the chemostat was controlled by the combined action of the nitrate and nitrite reductase. Both nitrate and nitrite reductase activity were inversely proportional to the growth rate. The nitrite reductase activity decreased faster with growth rate than the nitrate reductase. The chemostat biomass concentration of E. coli E4, with ammonium either solely or combined with nitrate as a source of nitrogen, remained constant throughout all growth rates and was not affected by nitrite concentrations. Contrary to batch, E. coli E4 was able to grow in continuous cultures on nitrate as the sole source of nitrogen. When cultivated with nitrate as the sole source of nitrogen the chemostat biomass concentration is related to the activity of nitrate and nitrite reductase and hence, inversely proportional to growth rate.     

Light and ethylenediamine tetraacetic acid mediated differential effects of ammonium on nitrate reductase activity in maize seedlings

Abstract Effect of ammonium on in vivo activity of nitrate reductase in roots, shoots and leaves of maize (Zea mays L.) seedlings was studied in relation to light/dark conditions and EDTA supply. Supply of 5 mM (NH₄)₂SO₄ increased the steady state level of enzyme only in leaves and in light, while it had no effect in roots and shoots and in the dark. The substrate induction of enzyme was also little affected by 1 to 10 mM (NH₄)₂SO₄ in roots and shoots. In the leaves the activity in the dark was either inhibited (minus EDTA) or stimulated (plus EDTA) by 5 to 10 mM (NH₄)₂SO₄. The activity was stimulated in the light also in the presence of EDTA at higher concentrations of ammonium. When different concentrations of ammonium were supplied without any exogenous nitrate in the light, the enzyme activity increased at low concentration and was either inhibited or unaffected at higher concentrations depending upon the tissue used. Supply of EDTA with ammonium modified its effect to some extent. It is suggested that the effect of ammonium on nitrate reductase activity depends upon the tissue used and the effective concentration of the ammonium.     

In vivo Characterization of Nitrate Reductase Activity in Cotton

The in vivo nitrate reductase activity in leaf tissue of cotton (Gossypium hirsutum L.) was characterized. Enzymatic activity was linear with time up to 60 min. The assay for nitrate reductase activity was optimized in leaf slices 400 μm wide incubated in an anaerobic system at 30°C, in a 0.02 M KNO₃ medium at pH 7.0 with 1 % propanol. In vivo activity was highest in recently matured leaves at the top of the plant. Both light and nitrate enhanced in vivo enzymatic activity. The activity was highest after 9 hours in the light and then decreased steadily for several more hours even in the presence of light. The nitrate reductase activity was more strongly correlated to the levels of NO₃-N in the culture solution than to the NO₃-N level in the tissue. The utility of this technique in nitrate reductase assay in a tissue containing large amounts of phenolic compounds is discussed.     

Some New Aspects of the in Vivo Assay for Nitrate Reductase in Wheat (Triticum aestivum L.) Leaves: I. REEVALUATION OF NITRATE POOL SIZES

Experiments were carried out to clarify problems encountered in measuring metabolic and storage pool sizes of nitrate in wheat leaf sections with an in vivo nitrate reductase assay. The leaf sections were from seedlings grown on 15 millimolar nitrate. Data obtained show that the cessation of nitrite accumulation, used as an index of the active nitrate pool size, could be caused by lack of anaerobiosis in the assay system, the lack of energy for nitrate reduction, or a loss of nitrate reductase activity. Availability of nitrate was never the limiting factor in this system. It is concluded that pool sizes of nitrate cannot be determined in wheat leaves with the in vivo assays employed.     

Optimized nitrate reductase assay predicts the rate of nitrate utilization in the halotolerant microalga Dunaliella viridis

An in situ method for measuring nitrate reductase (NR) activity in Dunaliella viridis was optimized in terms of incubation time, concentration of KNO₃, permeabilisers (1-propanol and toluene), pH, salinity, and reducing power (glucose and NADH). NR activity was measured by following nitrite production and was best assayed with 50 mM KNO₃, 1.2 mM NADH, 5% 1-propanol (v/v), at pH 8.5. The estimated half-saturation constant (K_s) for KNO₃ was 5 mM. Glucose had no effect as external reducing power source, and NADH concentrations >1.2 mM inhibited NR activity. Nitrite production was linear up to 20 min; longer incubation did not lead to higher nitrate reduction. The use of the optimized assay predicted the rate of NO ₃ ⁻ removal from the external medium by D. viridis with high degree of precision. This revised version was published online in September 2006 with corrections to the Cover Date.     

Decrease of Nitrate Reductase Activity in Spinach Leaves during a Light-Dark Transition

In leaves of spinach plants (Spinacia oleracea L.) performing CO₂ and NO₃⁻ assimilation, at the time of sudden darkening, which eliminates photosystem I-dependent nitrite reduction, only a minor temporary increase of the leaf nitrite content is observed. Because nitrate reduction does not depend on redox equivalents generated by photosystem I activity, a continuation of nitrate reduction after darkening would result in a large accumulation of nitrite in the leaves within a very short time, which is not observed. Measurements of the extractable nitrate reductase activity from spinach leaves assayed under standard conditions showed that in these leaves the nitrate reductase activity decreased during darkening to 15% of the control value with a half-time of only 2 minutes. Apparently, in these leaves nitrate reductase is very rapidly inactivated at sudden darkness avoiding an accumulation of the toxic nitrite in the cells.     

Nitrate and Nitrite Reduction in Relation to Nitrogenase Activity in Soybean Nodules and Rhizobium japonicum Bacteroids

Soybean (Glycine max L. cv Williams) seeds were sown in pots containing a 1:1 perlite-vermiculite mixture and grown under greenhouse conditions. Nodules were initiated with a nitrate reductase expressing strain of Rhizobium japonicum, USDA 110, or with nitrate reductase nonexpressing mutants (NR⁻ 108, NR⁻ 303) derived from USDA 110. Nodules initiated with either type of strain were normal in appearance and demonstrated nitrogenase activity (acetylene reduction). The in vivo nitrate reductase activity of N₂-grown nodules initiated with nitrate reductase-negative mutant strains was less than 10% of the activity shown by nodules initiated with the wild-type strain. Regardless of the bacterial strain used for inoculation, the nodule cytosol and the cell-free extracts of the leaves contained both nitrate reductase and nitrite reductase activities. The wild-type bacteroids contained nitrate reductase but not nitrite reductase activity while the bacteroids of strains NR⁻ 108 and NR⁻ 303 contained neither nitrate reductase nor nitrite reductase activities.

Addition of 20 millimolar KNO₃ to bacteroids of the wild-type strain caused a decrease in nitrogenase activity by more than 50%, but the nitrate reductase-negative strains were insensitive to nitrate. The nitrogenase activity of detached nodules initiated with the nitrate reductase-negative mutant strains was less affected by the KNO₃ treatment as compared to the wild-type strain; however, the results were less conclusive than those obtained with the isolated bacteroids.

The addition of either KNO₃ or KNO₂ to detached nodules (wild type) suspended in a semisolid agar nutrient medium caused an inhibition of nitrogenase activity of 50% and 65% as compared to the minus N controls, and provided direct evidence for a localized effect of nitrate and nitrite at the nodule level. Addition of 0.1 millimolar sucrose stimulated nitrogenase activity in the presence or absence of nitrate or nitrite. The sucrose treatment also helped to decrease the level of nitrite accumulated within the nodules.

     

Time-course of Nitrate Uptake and Nitrate Reductase Activity in Nitrogen-depleted Dwarf Bean

The rate of nitrate uptake by N-depleted French dwarf bean (Phaseolus vulgaris L. cv. Witte Krombek) increased steadily during the first 6 h after addition of NO₃ -After this initial phase the rale remained constant for many hours. Detached root systems showed the same time-course of uptake as roots of intact plants. In vivo nitrate reductase activity (NRA) was assayed with or without exogenous NO₃^- in the incubation medium and the result ing activities were denoted potential and actual level, respectively. In roots the difference between actual and potential NRA disappeared within 15 min after addition of nitrate, and NRA increased for about 15 h. Both potential and actual NRA were initially very low. In leaves, however, potential NRA was initially very high and was not affected by ambient nitrate (0.1–5 mol m^-3) for about 10 h. Actual and potential leaf NRA became equal after the same period of time. In the course of nitrate nutrition, the two nitrate reductase activities in leaves were differentially inhibited by cycloheximide (3.6 mmol m^-3) and tungstate (1 mol m^-3). We suggest that initial potential NRA reflects the activity of pre-existing enzyme, whereas actual NRA depends on enzyme assembly during NO₃^- supply. Apparent induction of nitrate uptake and most (85%) of the actual in vivo NRA occurred in the root system during the first 6 h of nitrate utilization by dwarf bean.     

Nitrate reductase-deficient mutant cell lines of Nicotiana tabacum

Summary The wild-type line and 14 nitrate reductase-deficient mutant cell lines of Nicotiana tabacum were tested for the presence of nitrate reductase partial activities, and for nitrite reductase and xanthine dehydrogenase activity. Data characterizing the electron donor specificity of nitrate reductase (EC 1.6.6.1., NADH:nitrate oxidoreductase) and nitrite reductase (EC 1.7.7.1., ferredoxin:nitrite oxidoreductase) of the wild-type line are presented. Three lines (designated cnx) simultaneously lack NADH-, FADH₂-, red. benzyl viologen-nitrate reductase, and xanthine dehydrogenase activities, but retain the nitrate reductase-associated NADH-cytochrome c reductase activity. These mutants are, therefore, interpreted to be impaired in gene functions essential for the synthesis of an active molybdenum-containing cofactor. For cnx-68 and cnx-101, the sedimentation coefficient of the defective nitrate reductase molecules does not differ from that of the wild-type enzyme (7.6S). In 11 lines (designated nia) xanthine dehydrogenase activity is unaffected, and the loss of NADH-nitrate reductase is accompanied by a loss of all partial activities, including NADH-cytochrome c reductase. However, one line (nia-95) was found to possess a partially active nitrate reductase molecule, retaining its FADH₂- and red. benzyl viologen nitrate reductase activity. It is likely that nia-95 is a mutation in the structural gene for the apoprotein. Both, the nia and cnx mutant lines exhibit nitrite reductase activity, being either nitrate-inducible or constitutive. Evidence is presented that, in Nicotiana tabacum, nitrate, without being reduced to nitrite, is an inducer of the nitrate assimilation pathway.     

Nitrate reductase activity in leaves and roots ofAlnus glutinosa (L) Gaertner

Summary Thein vivo nitrate reductase activity (NRA) was determined inAlnus glutinosa plants grown nonsymbiotically on ammonium, nitrate, a combination of both, or symbiotically with atmospheric nitrogen as the only nitrogen source. Root NRA was absent when ammonium or atmospheric nitrogen was the nitrogen source. With nitrate in the culture solution the roots showed a high NRA. However, the leaf NRA behaved quite differently: with negligible activities on all nitrogen sources except atmospheric nitrogen. The foliar NRA measured, however, is likely not due to the activity of the plant but of microbial origin. Methods commonly used to facilitate produced nitrite to leak out of the tissue, such as addition of propanol and cutting the plant material, did not increase the nitrite release from the leaves. A turbidity developed when testing the samples for nitrite which was positively correlated with the NRA. Populations of microorganisms in the phyllosphere did not differ between the nutritional treatments. Bacteria, able to grow on a low-nitrogen medium, were present on the leaves. Nitrifiers could not be detected. The bacteria on the leaves appear to produce nitrite when incubated with leaf material. Grassland Species Research Group, Publication no. 106     

Utilization of nitrate by bacteroids of Bradyrhizobium japonicum in the soybean root nodule

Bacteroids of Bradyrhizobium japonicum strain CB1809, unlike CC705, do not have a high level of constitutive nitrate reductase (NR; EC 1.7.99.4) in the soybean (Glycine max. Merr.) nodule. Ex planta both strains have a high activity of NR when cultured on 5 mM nitrate at 2% O₂ (v/v). Nitrite reductase (NiR) was active in cultured cells of bradyrhizobia, but activity with succinate as electron donor was not detected in freshly-isolated bacteroids. A low activity was measured with reduced methyl viologen. When bacteroids of CC705 were incubated with nitrate there was a rapid production of nitrite which resulted in repression of NR. Subsequently when NiR was induced, nitrite was utilized and NR activity recovered. Nitrate reductase was induced in bacteroids of strain CB1809 when they were incubated in-vitro with nitrate or nitrite. Increase in NR activity was prevented by rifampicin (10 g· ml^-1) or chloramphenicol (50 g·ml^-1). Nitrite-reductase activity in bacteroids of strain CB1809 was induced in parallel with NR. When nitrate was supplied to soybeans nodulated with strain CC705, nitrite was detected in nodule extracts prepared in aqueous media and it accumulated during storage (1°C) and on further incubation at 25°C. Nitrite was not detected in nodule extracts prepared in ethanol. Thus nitrite accumulation in nodule tissue appears to occur only after maceration and although bacteroids of some strains of B. japonicum have a high level of a constitutive NR, they do not appear to reduce nitrate in the nodule because this anion does not gain access to the bacteroid zone. Soybeans nodulated with strains CC705 and CB1809 were equally sensitive to nitrate inhibition of N₂ fixation.Abbreviations NR nitrate reductase - NiR nitrite reductase - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Regulation of nitrate and nitrite reductases in dinitrogen-fixing cyanobacteria and Nif− mutants

The effect of the nitrogen source on nitrate reductase and nitrite reductase synthesis has been studied in several filamentous dinitrogen-fixing cyanobacteria belonging to the genera Anabaena, Nostoc and Calothrix. Nitrate and nitrite uptake were also studied. High levels of both nitrate reductase and nitrite reductase were found only in the presence of nitrate or nitrite, as long as ammonium was absent from the culture medium. On the other hand, whereas nitrate uptake is an active process, two components, diffusion of nitrous acid and active transport of nitrite, appear to contribute to nitrite uptake.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - MOPS 3-(N-morpholino)propanesulfonic acid - TES N-tris(hydroxymethyl)methyl-2-aminoethane-sulfonic acid - Tricine N-tris(hydroxymethyl)methylglycine