Differential light induction of nitrate reductases in greening and photobleached soybean seedlings

Soybean (Glycine max [L.] Merr.) seeds were imbibed and germinated with or without NO₃⁻, tungstate, and norflurazon (San 9789). Norflurazon is a herbicide which causes photobleaching of chlorophyll by inhibiting carotenoid synthesis and which impairs normal chloroplast development. After 3 days in the dark, seedlings were placed in white light to induce extractable nitrate reductase activity. The induction of maximal nitrate reductase activity in greening cotyledons did not require NO₃⁻ and was not inhibited by tungstate. Induction of nitrate reductase activity in norflurazon-treated cotyledons had an absolute requirement for NO₃⁻ and was completely inhibited by tungstate. Nitrate was not detected in seeds or seedlings which had not been treated with NO₃⁻. The optimum pH for cotyledon nitrate reductase activity from norflurazon-treated seedlings was at pH 7.5, and near that for root nitrate reductase activity, whereas the optimum pH for nitrate reductase activity from greening cotyledons was pH 6.5. Induction of root nitrate reductase activity was also inhibited by tungstate and was dependent on the presence of NO₃⁻, further indicating that the isoform of nitrate reductase induced in norflurazon-treated cotyledons is the same or similar to that found in roots. Nitrate reductases with and without a NO₃⁻ requirement for light induction appear to be present in developing leaves. In vivo kinetics (light induction and dark decay rates) and in vitro kinetics (Arrhenius energies of activation and NADH:NADPH specificities) of nitrate reductases with and without a NO₃⁻ requirement for induction were quite different. K_m values for NO₃⁻ were identical for both nitrate reductases.     

Nitrate Reductase Activity in Shoots and Roots of Maize Seedlings as Affected by the Form of Nitrogen Nutrition and the pH of the Nutrient Solution

The effect of nitrogen form (NH₄-N, NH₄-N + NO₃⁻, NO₃⁻) on nitrate reductase activity in roots and shoots of maize (Zea mays L. cv INRA 508) seedlings was studied. Nitrate reductase activity in leaves was consistent with the well known fact that NO₃⁻ increases, and NH₄⁺ and amide-N decrease, nitrate reductase activity. Nitrate reductase activity in the roots, however, could not be explained by the root content of NO₃⁻, NH₄-N, and amide-N. In roots, nitrate reductase activity in vitro was correlated with the rate of nitrate reduction in vivo. Inasmuch as nitrate reduction results in the production of OH⁻ and stimulates the synthesis of organic anions, it was postulated that nitrate reductase activity of roots is stimulated by the released OH⁻ or by the synthesized organic anions rather than by nitrate itself. Addition of HCO₃⁻ to nutrient solution of maize seedlings resulted in a significant increase of the nitrate reductase activity in the roots. As HCO₃⁻, like OH⁻, increases pH and promotes the synthesis of organic anions, this provides circumstantial evidence that alkaline conditions and/or organic anions have a more direct impact on nitrate reductase activity than do NO₃⁻, NH₄-N, and amide-N.     

Early effects of salinity on nitrate assimilation in barley seedlings

The effect of NaCl and Na₂SO₄ salinity on NO₃⁻ assimilation in young barley (Hordeum vulgare L. var Numar) seedlings was studied. The induction of the NO₃⁻ transporter was affected very little; the major effect of the salts was on its activity. Both Cl⁻ and SO₄²⁻ salts severely inhibited uptake of NO₃⁻. When compared on the basis of osmolality of the uptake solutions, Cl⁻ salts were more inhibitory (15-30%) than SO₄²⁻ salts. At equal concentrations, SO₄²⁻ salts inhibited NO₃⁻ uptake 30 to 40% more than did Cl⁻ salts. The absolute concentrations of each ion seemed more important as inhibitors of NO₃⁻ uptake than did the osmolality of the uptake solutions. Both K⁺ and Na⁺ salts inhibited NO₃⁻ uptake similarly; hence, the process seemed more sensitive to anionic salinity than to cationic salinity.

Unlike NO₃⁻ uptake, NO₃⁻ reduction was not affected by salinity in short-term studies (12 hours). The rate of reduction of endogenous NO₃⁻ in leaves of seedlings grown on NaCl for 8 days decreased only 25%. Nitrate reductase activity in the salt-treated leaves also decreased 20% but its activity, determined either in vitro or by the `anaerobic' in vivo assay, was always greater than the actual in situ rate of NO₃⁻ reduction. When salts were added to the assay medium, the in vitro enzymic activity was severely inhibited; whereas the anaerobic in vivo nitrate reductase activity was affected only slightly. These results indicate that in situ nitrate reductase activity is protected from salt injury. The susceptibility to injury of the NO₃⁻ transporter, rather than that of the NO₃⁻ reduction system, may be a critical factor to plant survival during salt stress.

     

Aspects of nitrogen metabolism in the rice seedling

The effects of nitrogen source NO₃⁻ or NH₄⁺ on nitrogen metabolism during the first 2 weeks of germination of the rice seedling (Oryza sativa L., var. IR22) grown in nutrient solution containing 40 μg/ml N were studied. Total, soluble protein, and free amino N levels were higher in the NH₄⁺-grown seedling, particularly during the 1st week of germination. Asparagine accounted for most of the difference in free amino acid level, in both the root and the shoot. Nitrate and nitrite reductase activities were present mainly in the shoot and were higher in the NO₃⁻-grown seedling, whereas the activity of glutamate dehydrogenase and glutamine synthetase in the root tended to be lower than that of the NH₄⁺-grown seedling during the 1st week of germination. Glycolate oxidase and catalase activities were present mainly in the shoot. Maximum activity of the above five enzymes occurred 7 to 10 days after germination. Differences in the zymograms of nitrate reductase, glutamate dehydrogenase, and catalase were mainly between shoot and root and not from N source. Nitrite reductase bands were observed only in plants grown in plants grown in NO₃⁻.     

Nodule and Leaf Nitrate Reductases and Nitrogen Fixation in Medicago sativa L. under Water Stress

The effect of water stress on patterns of nitrate reductase activity in the leaves and nodules and on nitrogen fixation were investigated in Medicago sativa L. plants watered 1 week before drought with or without NO₃⁻. Nitrogen fixation was decreased by water stress and also inhibited strongly by the presence of NO₃⁻. During drought, leaf nitrate reductase activity (NRA) decreased significantly particularly in plants watered with NO₃⁻, while with rewatering, leaf NRA recovery was quite important especially in the NO₃⁻-watered plants. As water stress progressed, the nodular NRA increased both in plants watered with NO₃⁻ and in those without NO₃⁻ contrary to the behavior of the leaves. Beyond −15.10⁵ pascal, nodular NRA began to decrease in plants watered with NO₃⁻. This phenomenon was not observed in nodules of plants given water only.     

Characterization of the Reversible Inactivation of Ankistrodesmus braunii Nitrate Reductase by Hydroxylamine

The photoreversible nature of the regulation of nitrate reductase is one of the most interesting features of this enzyme. As well as other chemicals, NH₂OH reversibly inactivates the reduced form of nitrate reductase from Ankistrodesmus braunii. From the partial activities of the enzyme, only terminal nitrate reductase is affected by NH₂OH. To demonstrate that the terminal activity was readily inactivted by NH₂OH, the necessary reductants of the terminal part of the enzyme had to be cleared of dithionite since this compound reacts chemically with NH₂OH. Photoreduced flavins and electrochemically reduced methyl viologen sustain very effective inactivation of terminal nitrate reductase activity, even if the enzyme was previously deprived of its NADH-dehydrogenase activity. The early inhibition of nitrate reductase by NH₂OH appears to be competitive versus NO₃⁻. Since NO₃⁻, as well as cyanate, carbamyl phosphate and azide (competitive inhibitors of nitrate reductase versus NO₃⁻), protect the enzyme from NH₂OH inactivation, it is suggested that NH₂OH binds to the nitrate active site. The NH₂OH-inactivated enzyme was photoreactivated in the presence of flavins, although slower than when the enzyme was previously inactivated with CN⁻. NH₂OH and NADH concentrations required for full inactivation of nitrate reductase appear to be low enough to potentially consider this inactivation process of physiological significance.     

Nitrogen metabolism of soybeans: I. Effect of tungstate on nitrate utilization, nodulation, and growth

The effects of N source (6 mm nitrogen as NO₃⁻ or urea) and tungstate (0, 100, 200, 300, and 400 μm Na₂ WO₄) on nitrate metabolism, nodulation, and growth of soybean (Glycine max [L.] Merr.) plants were evaluated. Nitrate reductase activity and, to a lesser extent, NO₃⁻ content of leaf tissue decreased with the addition of tungstate to the nutrient growth medium. Concomitantly, nodule mass and acetylene reduction activity of NO₃⁻-grown plants increased with addition of tungstate to the nutrient solution. In contrast, nodule mass and acetylene reduction activity of urea-grown plants decreased with increased nutrient tungstate levels. The acetylene reduction activity of nodulated roots of NO₃⁻-grown plants was less than 10% of the activity of nodulated roots of urea-grown plants when no tungstate was added. At 300 and 400 μm tungstate levels, acetylene reduction activity of nodulated roots of NO₃⁻-grown plants exceeded the activity of comparable urea-grown plants.     

Comparative studies on the induction and inactivation of nitrate reductase in corn roots and leaves

A comparison of induction and inactivation of nitrate reductase and two of its component activities, namely FMNH₂-nitrate reductase and NO₃⁻-induced NADH-cytochrome c reductase, was made in roots and leaves of corn (Zea mays L. var. W64A × 182E). The three activities were induced in parallel in both tissues when NO₃⁻ was supplied. WO₄⁼ suppressed the induction of NADH- and FMNH₂-nitrate reductase activities in root tips and leaves. The NO₃⁻-induced NADH-cytochrome c reductase activity showed a normal increase in roots treated with WO₄⁼. In leaves, on the other hand, there was a marked superinduction of the NO₃⁻-induced NADH-cytochrome c reductase in the presence of WO₄⁼.     

Characteristics of Injury and Recovery of Net NO3? Transport of Barley Seedlings from Treatments of NaCl

The nature of the injury and recovery of nitrate uptake (net uptake) from NaCl stress in young barley (Hordeum vulgare L, var CM 72) seedlings was investigated. Nitrate uptake was inhibited rapidly by NaCl, within 1 minute after exposure to 200 millimolar NaCl. The duration of exposure to saline conditions determined the time of recovery of NO₃⁻ uptake from NaCl stress. Recovery was dependent on the presence of NO₃⁻ and was inhibited by cycloheximide, 6-methylpurine, and cerulenin, respective inhibitors of protein, RNA, and sterol/fatty acid synthesis. These inhibitors also prevented the induction of the NO₃⁻ uptake system in uninduced seedlings. Uninduced seedlings exhibited endogenous NO₃⁻ transport activity that appeared to be constitutive. This constitutive activity was also inhibited by NaCl. Recovery of constitutive NO₃⁻ uptake did not require the presence of NO₃⁻.     

In Vivo Determination of Parameters of Nitrate Utilization in Wheat (Triticum aestivum L.) Seedlings Grown with Low Concentration of Nitrate in the Nutrient Solution

Six genotypes of winter wheat (Triticum aestivum L.) differing in grain protein concentration were grown on a nutrient solution containing low concentrations of NO₃⁻ (2 millimolar). Total NO₃⁻ uptake varied between genotypes but was not related to grain protein content. An in vivo nitrate reductase assay was used to determine the affinity of the enzyme for NO₃⁻, and large phenotypic variations were observed. In vivo estimations of the concentration and size of the metabolic pool were variable. However, the three genotypes with the higher ratios of metabolic pool size to leaf total NO₃⁻ concentration were the high protein varieties. It is proposed that a high affinity of nitrate reductase for nitrate might be a biochemical marker for the capacity of the plant to continue assimilating NO₃⁻ for a longer period during the last stage of growth.     

Nitrate Reductase Activity in Soybeans (Glycine max [L.] Merr.): II. Energy Limitations

Growth chamber studies with soybeans (Glycine max [L.] Merr.) were designed to determine the relative limitations of NO₃⁻, NADH, and nitrate reductase (NR) per se on nitrate metabolism as affected by light and temperature. Three NR enzyme assays (+NO₃⁻in vivo, −NO₃⁻in vivo, and in vitro) were compared. NR activity decreased with all assays when plants were exposed to dark. Addition of NO₃⁻ to the in vivo NR assay medium increased activity (over that of the −NO₃⁻in vivo assay) at all sampling periods of a normal day-night sequence (14 hr-30 C day; 10 hr-20 C night), indicating that NO₃⁻ was rate-limiting. The stimulation of in vivo NR activity by NO₃⁻ was not seen in plants exposed to extended dark periods at elevated temperatures (16 hr-30 C), indicating that under those conditions, NO₃⁻ was not the limiting factor. Under the latter condition, in vitro NR activity was appreciable (19 μmol NO₂⁻ [g fresh weight, hr]⁻¹) suggesting that enzyme level per se was not the limiting factor and that reductant energy might be limiting.     

Effect of exogenous and endogenous nitrate concentration on nitrate utilization by dwarf bean

The effect of the exogenous and endogenous NO₃⁻ concentration on net uptake, influx, and efflux of NO₃⁻ and on nitrate reductase activity (NRA) in roots was studied in Phaseolus vulgaris L. cv. Witte Krombek. After exposure to NO₃⁻, an apparent induction period of about 6 hours occurred regardless of the exogenous NO₃⁻ level. A double reciprocal plot of the net uptake rate of induced plants versus exogenous NO₃⁻ concentration yielded four distinct phases, each with simple Michaelis-Menten kinetics, and separated by sharp breaks at about 45, 80, and 480 micromoles per cubic decimeter.

Influx was estimated as the accumulation of ¹⁵N after 1 hour exposure to ¹⁵NO₃⁻. The isotherms for influx and net uptake were similar and corresponded to those for alkali cations and Cl⁻. Efflux of NO₃⁻ was a constant proportion of net uptake during initial NO₃⁻ supply and increased with exogenous NO₃⁻ concentration. No efflux occurred to a NO₃⁻-free medium.

The net uptake rate was negatively correlated with the NO₃⁻ content of roots. Nitrate efflux, but not influx, was influenced by endogenous NO₃⁻. Variations between experiments, e.g. in NO₃⁻ status, affected the values of K_m and V_max in the various concentration phases. The concentrations at which phase transitions occurred, however, were constant both for influx and net uptake. The findings corroborate the contention that separate sites are responsible for uptake and transitions between phases.

Beyond 100 micromoles per cubic decimeter, root NRA was not affected by exogenous NO₃⁻ indicating that NO₃⁻ uptake was not coupled to root NRA, at least not at high concentrations.

     

Nitrate reductase activity of radish cotyledons as affected by phytohormones and different nitrogen sources

Radish (Raphanus sativus L.) seedlings pretreated with different hormones viz. kinetin, gibberellic acid and abscisic acid were subjected to different N-forms. The seedlings were treated with different concentrations of KNO₃, NH₄Cl and NH₄NO₃ and the changes in nitrate reductase activity were seen in light and dark conditions in the cotyledons. Nitrate reductase activity was affected differently by hormone application. Nitrate increased and ammonia decreased nitrate reductase activity; in both light and dark-grown seedlings KNO₃ induced more in vitro nitrate reductase activity. NH ₄ ⁺ when combined with NO ₃ ⁻ , however, could level up to some extent, with KNO₃ in light, except in kinetin. A transient response of induction of NR activity was evident with decreased levels after a certain specific ambient N-concentration, despite the presence of high N in the medium. However, the pattern of transition varied with the hormones applied. Further, hormones are found to affect induction of different isoforms of nitrate reductase by NH ₄ ⁺ and NO ₃ ⁻ . NH ₄ ⁺ induced isoform was prominently promoted by kinetin treatment in dark. The data documents a particular kind of interaction between controlling factors (light, N-source and phytohormones) which affect nitrate reductase levels.     

In Vivo Blue-Light Activation of Chlamydomonas reinhardii Nitrate Reductase

Chlamydomonas reinhardii cells, growing photoautotrophically under air, excreted to the culture medium much higher amounts of NO₂⁻ and NH₄⁺ under blue than under red light. Under similar conditions, but with NO₂⁻ as the only nitrogen source, the cells consumed NO₂⁻ and excreted NH₄⁺ at similar rates under blue and red light. In the presence of NO₃⁻ and air with 2% CO₂ (v/v), no excretion of NO₂⁻ and NH₄⁺ occurred and, moreover, if the bubbling air of the cells that were currently excreting NO₂⁻ and NH₄⁺ was enriched with 2% CO₂ (v/v), the previously excreted reduced nitrogen ions were rapidly reassimilated. The levels of total nitrate reductase and active nitrate reductase increased several times in the blue-light-irradiated cells growing on NO₃⁻ under air. When tungstate replaced molybdate in the medium (conditions that do not allow the formation of functional nitrate reductase), blue light activated most of the preformed inactive enzyme of the cells. Furthermore, nitrate reductase extracted from the cells in its inactive form was readily activated in vitro by blue light. It appears that under high irradiance (90 w m⁻²) and low CO₂ tensions, cells growing on NO₃⁻ or NO₂⁻ may not have sufficient carbon skeletons to incorporate all the photogenerated NH₄⁺. Because these cells should have high levels of reducing power, they might use NO₃⁻ or, in its absence, NO₂⁻ as terminal electron acceptors. The excretion of the products of NO₂⁻ and NH₄⁺ to the medium may provide a mechanism to control reductant level in the cells. Blue light is suggested as an important regulatory factor of this photorespiratory consumption of NO₃⁻ and possibly of the whole nitrogen metabolism in green algae.     

Nitrogenase Activity in Trifolium subterraneum L. in Relation to the Uptake of Nitrate Ions

An experiment was conducted to test the hypothesis that, when nitrogenase and nitrate reductase both contribute to the nitrogen nutrition of a nodulated legume, nitrogenase activity is inversely proportional to the rate of accumulation of organic nitrogen derived from the reduction of nitrate. Trifolium subterraneum L. plants, inoculated with Rhizobium trifolii and sown as small swards, were allowed to establish a closed canopy and steady rates of growth, dinitrogen fixation, and nitrogen accumulation. Swards were then supplied with nutrient solutions of 0, 0.5, 1.0, or 2.5 mm NO₃⁻ with a 29.69% enrichment of ¹⁵N and allowed to grow for a further 33 days. Harvests were made to measure dry weight, nitrogen accumulation, ¹⁵N accumulation, NO₃⁻ content and nitrogenase activity by acetylene reduction assay. Since the ¹⁵N of the plant organic matter could have been derived only from the NO₃⁻ of the nutrient solution, its rate of accumulation provided a measure of the rate of NO₃⁻ reduction. It was found that as this rate increased in response to external NO₃⁻ concentration the rate of nitrogenase activity decreased proportionately. It is concluded that the reduction of nitrate and the reduction of dinitrogen act in a complementary manner to supply a plant with organic nitrogen for growth.     

Variant Cell Lines of Haplopappus gracilis with Disturbed Activities of Nitrate Reductase and Nitrite Reductase

Selected variant cell lines of Haplopappus gracilis (Nutt) Gray that showed disturbed growth after transfer from an alanine medium to NO₃⁻ medium were characterized. The in vivo NO₃⁻ reductase activity (NRA) was lower in these lines than in the wild type. In vitro NRA assays suggest that decreased in vivo NRA was not caused by a lower amount of active enzyme. Cells of the variant lines revealed up to 75% lower extractable activity of NO₂⁻ reductase as compared with the wild type. This coincided with higher accumulation of NO₂⁻ by the variant than by the wild type cells after transfer from alanine medium to NO₃⁻ medium. NO₂⁻ accumulation was transient or continuous, depending on cell line, metabolic state of the cells, and light conditions.     

The occurrence of nitrate reductase in apple leaves

Nitrate reductase utilizing NADH or reduced flavin mononucleotide (FMNH₂) as electron donor was extracted from the leaves, stems and petioles, and roots of apple seedlings. Successful extraction was made possible by the use of insoluble polyvinylpyrrolidone (Polyclar AT) which forms insoluble complexes with polyphenols and tannins. The level of nitrate reductase per gram fresh weight was highest in the leaf tissue although the nitrate content of the roots was much higher than that of the leaves. Nitrite reductase activity was detected only in leaf extracts and was 4 times higher than nitrate reductase activity. Nitrate was found in all parts of young apple trees and trace amounts were also detected in mature leaves from mature trees. Nitrate reductase was induced in young leaves of apple seedlings and in mature leaves from 3 fruit-bearing varieties. An inhibitor of polyphenoloxidase, 2-mercaptobenzothiazole was used in both the inducing medium and the extracting medium in concentrations from 10⁻³ to 10⁻⁵m with no effect upon nitrate reductase activity.     

Chloride inhibition of spinach nitrate reductase

Initial rate studies of spinach (Spinacia oleracea L.) nitrate reductase showed that NADH:nitrate reductase activity was ionic strength dependent with elevated ionic concentration resulting in inhibition. In contrast, NADH:ferricyanide reductase was markedly less ionic strength dependent. At pH 7.0, NADH:nitrate reductase activity exhibited changes in the V_max and K_m for NO₃⁻ yielding V_max values of 6.1 and 4.1 micromoles NADH per minute per nanomoles heme and K_m values of 13 and 18 micromolar at ionic strengths of 50 and 200 millimolar, respectively. Control experiments in phosphate buffer (5 millimolar) yielded a single K_m of 93 micromolar. Chloride ions decreased both NADH:nitrate reductase and reduced methyl viologen:nitrate reductase activities, suggesting involvement of the Mo center. Chloride was determined to act as a linear, mixed-type inhibitor with a K_i of 15 millimolar for binding to the native enzyme and 176 millimolar for binding to the enzyme-NO₃⁻ complex. Binding of Cl⁻ to the enzyme-NO₃⁻ complex resulted in an inactive E-S-I complex. Electron paramagnetic resonance spectra showed that chloride altered the observed Mo(V) lineshape, confirming Mo as the site of interaction of chloride with nitrate reductase.     

Role of Nitrate and Nitrite in the Induction of Nitrite Reductase in Leaves of Barley Seedlings

The role of NO₃⁻ and NO₂⁻ in the induction of nitrite reductase (NiR) activity in detached leaves of 8-day-old barley (Hordeum vulgare L.) seedlings was investigated. Barley leaves contained 6 to 8 micromoles NO₂⁻/gram fresh weight × hour of endogenous NiR activity when grown in N-free solutions. Supply of both NO₂⁻ and NO₃⁻ induced the enzyme activity above the endogenous levels (5 and 10 times, respectively at 10 millimolar NO₂⁻ and NO₃⁻ over a 24 hour period). In NO₃⁻-supplied leaves, NiR induction occurred at an ambient NO₃⁻ concentration of as low as 0.05 millimolar; however, no NiR induction was found in leaves supplied with NO₂⁻ until the ambient NO₂⁻ concentration was 0.5 millimolar. Nitrate accumulated in NO₂⁻-fed leaves. The amount of NO₃⁻ accumulating in NO₂⁻-fed leaves induced similar levels of NiR as did equivalent amounts of NO₃⁻ accumulating in NO₃⁻-fed leaves. Induction of NiR in NO₂⁻-fed leaves was not seen until NO₃⁻ was detectable (30 nanomoles/gram fresh weight) in the leaves. The internal concentrations of NO₃⁻, irrespective of N source, were highly correlated with the levels of NiR induced. When the reduction of NO₃⁻ to NO₂⁻ was inhibited by WO₄²⁻, the induction of NiR was inhibited only partially. The results indicate that in barley leaves NiR is induced by NO₃⁻ directly, i.e. without being reduced to NO₂⁻, and that absorbed NO₂⁻ induces the enzyme activity indirectly after being oxidized to NO₃⁻ within the leaf.     

Nitrate Assimilation during Vegetative Regrowth of Alfalfa

Dry matter accumulation, nitrate reductase activity of various organs, nitrate accumulation, nitrogen derived from nitrate, and nitrogen content were studied during 17 days of vegetative regrowth of harvested (detopped) alfalfa (Medicago sativa L.). Seedlings were grown in the glasshouse and treated with 0, 40, and 80 kilograms N per hectare applied as K¹⁵NO₃ to determine whether reduced nitrogenase activity after shoot harvest limited vegetative regrowth. The role of nodules in reducing NO₃⁻ during this period of low nitrogenase activity was also investigated.