A nitrate reductase inactivating enzyme from the maize root

The nitrate reductase in the mature root extract of 3-day maize (Zea mays) seedlings was relatively labile in vitro. Insoluble polyvinylpyrrolidone used in the extraction medium produced only a slight increase in the stability of the enzyme. Mixing the mature root extract with that of the root tip promoted the inactivation of nitrate reductase in the latter. The inactivating factor in the mature root was separated from nitrate reductase by (NH₄)₂SO₄ precipitation. Nitrate reductase was found in the 40% (NH₄)₂SO₄ precipitate, while the inactivating factor was largely precipitated by 40 to 55% (NH₄)₂SO₄. The latter fraction of the mature root inactivated the nitrate reductase isolated from the root tip, mature root, and scutellum. The inactivating factor, which has a Q₁₀ 15 to 25 C of 2.2, was heat labile, and hence has been designated as a nitrate reductase inactivating enzyme. The reduced flavin mononucleotide nitrate reductase was also inactivated, while an NADH cytochrome c reductase in nitrate-grown seedlings was inactivated but at a slower rate. The inactivating enzyme had no influence on the activity of nitrite reductase, glutamate dehydrogenase, xanthine oxidase, and isocitrate lyase. The activity of the nitrate reductase inactivating enzyme was not influenced by nitrate and was also found in the mature root of minus nitrate-grown seedlings.     

The distribution and characteristics of nitrate reductase and glutamate dehydrogenase in the maize seedling

In a study on 3-day maize (Zea mays) seedlings, grown on nitrate, requirements were established for the maximum extraction and optimum stabilization of nitrate reductase in vitro. With the primary root, 5 mm cysteine were required in the extraction medium, but for the scutellum, which has a high level of endogenous thiol, the use of additional thiol resulted in a reduced yield of a more labile enzyme. Activity of the root and scutella nitrate reductase was obtained with either NADH or NADPH, but that of the root enzyme with NADPH was only demonstrated in the absence of phosphate.Before leaf expansion, the nitrate reductase in the maize seedling was mainly in the scutellum. The enzyme present in the primary root was predominantly in the apical region (0-2 mm). In contrast, glutamate dehydrogenase was concentrated in the mature basal region of the root (30-60 mm). A high level of nitrate (approximately 100 mm) was required to saturate the induction of nitrate reductase in the root tip, mature root, and scutellum. The concentration of nitrate required to give half the maximum level of enzyme induced was the same for each region (29 mm).After leaf expansion, more than 90% of the nitrate reductase was in the shoot, mainly in the leaf blade, and a marked decrease occurred in the level of the enzyme in the scutellum. A large proportion of the glutamate dehydrogenase was still found in the root.     

Induction of Nitrate Reductase and Peroxidase Activity in the Seedlings of Normal and High Lysine Maize

Steady state levels of in vivo nitrate reductase activity in the endosperm, scutella, roots and shoots of maize seedlings were higher in normal maize than those in high lysine maize. Activity of peroxidase in the roots, however, was higher in the high lysine cultivar. The nitrate reductase activity increased with the supply of nitrate in all parts of the seedlings of both cultivars, although the rates of increment in the endosperm were lower than those in scutella, roots and shoots. In relation to substrate concentration, a saturation was achieved at 5 to 10 mM of nitrate except in the endosperm, where activity increased slowly up to 100 mM at least. Final levels of enzyme activity were significantly higher in the scutella of normal than in that of high lysine seedlings. In vitro enzyme activity in the roots also increased with the supply of nitrate in both cultivars, reaching maximum at 5 to 10 mM nitrate.     

Effects of a nitrate reductase inactivating enzyme and NAD(P)H on the nitrate reductase from higher plants and Neurospora.

Evidence is presented which suggests that the NAD(P)H-cytochrome c reductase component of nitrate reductase is the main site of action of the inactivating enzyme. When tested on the nitrate reductase (NADH) from the maize root and scutella, the NADH-cytochrome c reductase was inactivated at a greater rate than was the FADH2-nitrate reductase component. With the Neurospora nitrate reductase (NADPH) only the NADPH-cytochrome c reductase was inactivated. p-Chloromercuribenzoate at 50 muM, which gave almost complete inhibition of the NADH-cytochrome c reductase fraction of the maize nitrate reductase, had no marked effect on the action of the inactivating enzyme. A reversible inactivation of the maize nitrate reductase has been shown to occur during incubation with NAD(P)H. In contrast to the action of the inactivating enzyme, it is the FADH2-nitrate reductase alone which is inactivated. No inactivation of the Neurospora nitrate reductase was produced by NAD(P)H alone and also in the presence of FAD. The lack of effect of the inactivating enzyme and NAD(P)H on the FADH2-nitrate reductase of Neurospora suggests some differences in its structure or conformation from that of the maize enzyme. A low level of cyanide (0.4 mu M) markedly enhanced the action of NAD(P)H on the maize enzyme; Cyanide at a higher level (6 mu M) did give inactivation of the Neurospora nitrate reductase in the presence of NADPH and FAD. The maize nitrate reductase, when partially inactivated by NADH and cyanide, was not altered as a substrate for the inactivating enzyme. The maize root inactivating enzyme was also shown to inactivate the nitrate reductase (NADH) in the pea leaf. It had no effect on the nitrate reductase from either Pseudomonas denitrificans or Nitrobacter agilis.     

Preparative two-dimensional polyacrylamide gel electrophoresis of rat liver ribosomal proteins and determination of their amino acid compositions

A maize root fraction which inactivates nitrate reductase has been shown to have protease activity which can be measured by the hydrolysis of azocasein. This inactivating enzyme was also found to inactivate yeast tryptophan synthase. Yeast proteases A and B, which inactivate this latter enzyme, also gave a specific inactivation of the maize nitrate reductase. The maize root inactivating enzyme, like yeast protease B, degraded casein, and was inhibited by phenylmethylsulphonyl fluoride. A partially-purified yeast inhibitor prevented catalysis by the yeast proteases and maize root inactivating enzyme, but purified yeast inhibitors were without effect on the latter protein. The level of nitrate reductase-inactivating activity, and associated azocasein-degrading activity, increased with age of the maize root. Evidence was obtained for a heat stable inhibitor which maintained them in an inactive state, especially in the young root tip cells.     

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).

     

Increase in nitrate reductase activity in the presence of sucrose in bean leaf segments

The supply of sucrose to leaf segments from light-grown bean seedlings caused a substantial increase in substrate inducibility of in vivo and in vitro nitrate reductase activity but only a small increase in total protein. Cycloheximide and chloramphenicol inhibited the increase in enzyme activity by nitrate and sucrose. The in vivo decline in enzyme activity in nitrate-induced leaf segments in light and dark was protected by sucrose and nitrate. The supply of NADH also protected the decline in enzyme activity, but only in the light. In vitro stability of the extracted enzyme was, however, unaffected by sucrose. The size of the metabolic nitrate pool was also enhanced by sucrose. The experiments demonstrate that sucrose has a stimulatory effect on activity or in vivo stability ' of nitrate reductase in bean leaf segments, which is perhaps mediated through increased NADH level and/or mobilization of nitrate to the metabolic pool.     

Stability of nitrate reductase and source of its reductant in Sorghum seedlings

Pre-incubation of nitrate reductase from Sorghum seedlings with NADH increased enzyme activity by 25%. Ferricyanide had no effect. NADH protected the enzyme from inactivation during storage. Malonate inhibited in vivo nitrate reduction in Sorghum leaves by 95%. The inhibitory effect of malonate was reversed by fumarate. Sodium fluoride in the presence of phosphate also inhibited in vivo nitrate reduction by 60%. It is suggested that NADH generated via the citric acid cycle is utilized for nitrate reduction in Sorghum seedlings.     

Stabilization of nitrate reductase in maize roots by chymostatin

Nitrate reductase (NR) in maize (Zea mays cv W64A × W182E) roots has been stabilized in vitro by the addition of chymostatin to extraction buffer. Contrary to previous observations, levels of NR were higher in the mature root than in root tip sections when chymostatin was included in the extraction buffer. Two forms of NR were identified, an NADH monospecific NR found mainly in the 1cm root tip and an NAD(P)H bispecific NR found predominantly in mature regions of the root. During the first 10 days of seedling growth, NR activity in the root ranged from 50 to 80% of the activities found in the leaf (a maximum of 2.4 micromoles NO⁻₂ produced per hour per gram fresh weight was measured at 4 days).     

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.     

Effect of salicylic acid on nitrate reductase activity in maize seedlings

The effect of different concentrations of salicylic acid on total Kjeldahl nitrogen and nitrate reductase activity in the maize ( Zea mays L.) seedling was studied. The total nitrogen of the maize embryonic axis (root + shoot) from seedlings raised with 10 m M Ca(NO₃)₂ for 5 days was substantially higher than that from the control when 0.01 m M salicylic acid was supplied. As supply of high (1 m M ) concentrations of salicylic acid decreased the accumulation of organic nitrogen. The in vivo activity of nitrate reductase in the roots increased at low concentrations of salicylic acid, while high concentrations were inhibitory. The stimulative concentration of the acid protected in vivo loss of nitrate reductase activity under non-inducing conditions, whereas it had no effect on in vitro loss of enzyme. It is suggested that salicylic acid increases in vivo enzyme activity indirectly, to some extent by protecting the natural inactivation of the enzyme.     

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.     

In vivo nitrate reduction in relation to nitrate uptake, nitrate content, and in vitro nitrate reductase activity in intact barley seedlings

A study was done to relate the in vivo reduction of nitrate to nitrate uptake, nitrate accumulation, and induction of nitrate reductase activity in intact barley seedlings (Hordeum vulgare L. var. `Numar'). The characteristics of nitrate uptake in response to both time and ambient concentration of nitrate regulated reduction and accumulation. Uptake, accumulation, and in vivo reduction achieved steady state rates in 3 to 4 hours, whereas extractable (in vitro) nitrate reductase activity was still increasing at 12 hours. In vivo reduction of nitrate was better correlated exponentially than linearly over time with in vitro activity of nitrate reductase. A similar relationship occurred over increasing concentration of nitrate in the ambient solution. The results suggest that the rate of in vivo reduction of nitrate in barley seedlings may be regulated by the rate of uptake at the ambient concentrations of nitrate employed in the study.     

Influence of Glyphosate on Nitrate Reductase Activity in Soybean (Glycine max)

Experiments were conducted to determine the influence of glyphosate[N-(phosphonomethyl)glycine] on extractable nitrate reductaseactivity during light and dark growth of soybean (Glycine max)seedlings. Glyphosate (5?10^–4 M), applied via root-feedingto three-day-old etiolated seedling, significantly reduced enzymeactivity in roots (48 to 96 h) and leaves (96 h) of seedlingsplaced in the light, but had little effect on enzyme activityin cotyledons compared to enzyme levels in tissues of untreatedseedlings. During dark-growth, nitrate reductase activity increasedwith time in cotyledons of untreated seedlings (activity about85-fold less than in cotyledons of light-grown plants) but muchlower enzyme levels were found in cotyledons of glyphosate-treatedseedlings after 72 and 96 h. In leaves of dark-grown seedlings,glyphosate reduced nitrate reductase levels by 95%. Most inhibitionof extractable enzyme activity occurred in newly developingorgans (leaves and roots) which correlates well with reportsthat glyphosate is rapidly translocated to these sites. However,the fact that glyphosate inhibits growth prior to lowering enzymeactivity levels indicates a secondary effect on nitrate reductase. (Received May 18, 1984; Accepted February 12, 1985)     

Use of protein in extraction and stabilization of nitrate reductase

The in vitro instability of nitrate reductase (EC 1.6.6.1) activity from leaves of several species of higher plants was investigated. Decay of activity was exponential with time, suggesting that an enzyme-catalyzed reaction was involved. The rate of decay of nitrate reductase activity increased as leaf age increased in all species studied. Activity was relatively stable in certain genotypes of Zea mays L., but extremely unstable in others. In all genotypes of Avena sativa L. and Nicotiana tabacum L. studied, nitrate reductase was unstable. Addition of 3% (w/v) bovine serum albumin or casein to extraction media prevented or retarded the decay of nitrate reductase activity for several hours. In addition, the presence of bovine serum albumin or casein in the enzyme homogenate markedly increased nitrate reductase activity (up to 15-fold), especially in older leaf tissue.     

Nitrate reductase inactivating factor from rice seedlings

A nitrate reductase inactivating factor was found in extractsof leaf blades, leaf sheaths, and roots of rice seedlings. Thefactor was nondialyzable, precipitable with (NH₄)₂SO₄, and heatlabile. The factor from rice roots inactivated NADH nitratereductase, FMNH2 nitrate reductase, and NADH cytochrome c reductasefrom rice shoots, but had no effect on the activities of NADHdiaphorase and nitrite reductase. The factors from rice shoots,rice roots, and maize roots inactivated NADH nitrate reductaseprepared from cultured rice cells. The factor from culturedrice cells also inactivated rice shoot NADH nitrate reductase. The activity of the inactivating factor showed a diurnal changein shoots of rice seedlings grown with NO₃– medium, althoughthe fluctuation was not large compared to that of NADH nitratereductase activity. When the seedlings were placed in darkness,the activity of the factor did not change during 20 hr withNO₃– medium. However, the activity of the factor fluctuatedwith NO₃– -free medium in light; its activity startedto increase at the 8th hour after transfer. NADH nitrate reductaseactivity from rice shoots declined rapidly during the first8 hr and gradually thereafter in both types of culture. (Received August 24, 1977; )     

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.     

Nitrogen Isotope Fractionation Associated with Nitrate Reductase Activity and Uptake of NO(3) by Pearl Millet

Nitrogen isotope fractionation by Pearl Millet (Pennisetum americanum L. and P. mollissimum L.) grown on nitrate was associated with nitrate reductase activity. Fractionation was evidenced at the step of nitrate reduction when the substrate-to-enzyme ratio was high (possibly saturating for the active sites of the nitrate reductase enzyme), for instance in young seedlings having a low nitrate reductase activity or in seedlings grown on high nitrate concentration.     

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.     

Allelochemical stress produced by aqueous leachate of <Emphasis Type="Italic">Nicotiana plumbaginifolia</Emphasis> Viv.

The effects of aqueous leachate of Nicotiana plumbaginifolia Viv. on germination, seedling growth, amylase activity, sugar and starch contents of germinated seeds of maize (Zea mays L. cv. Uttam) were examined. Effects of leachate on photosynthetic pigments, protein content, activities of nitrate reductase and some antioxidants were also studied. Higher concentration of aqueous leachate of N. plumbaginifolia reduced the germination rate (GR). However, final germination percentage remained almost unaffected. Lower concentration of leachate stimulated the amylase activity and resulted in higher sugar content and GR. The increasing concentrations of leachate inhibited the conversion of starch into sugars. Allelochemicals decreased the amount of chlorophyll a, chlorophyll b, carotenoids, protein and nitrate reductase activity (NRA). The leachate of lower concentrations stimulated the activity of peroxidase but slight decrease was recorded in higher concentration. Superoxide dismutase and catalase exhibited concentration dependent increase except in seedlings treated with 100% concentration of leachate. Impairment of various metabolic activities due to leachate resulted in decreased root and shoot length.