Effect of salicylic acid on nitrite reductase and glutamate dehydrogenase activities in maize roots

The effects of 0.01 to 5 m M salicyclic acid on the increase in nitrite reductase or glutamate dehydrogenase activities in maize roots by nitrate or ammonium respectively, were examined. Nitrite reductase activity was inhibited by the highest concentration of the acid. The activity of NADH-glutamate dehydrogenase was stimulated slightly (but consistently) by the lowest concentration and was inhibited by higher concentrations. Total protein content was also inhibited at high concentrations. When the crude enzyme extract was stored at 25°C in light, the glutamate dehydrogenase activity in the control decreased after 4 h of incubation. Low concentrations of the acid had no effect on this decrease but higher concentration accelerated the process. The divalent cations Caz²⁺, Mn²⁺, Mg²⁺ and Zn²⁺ protected against loss of enzyme activity during storage, both in the absence and presence of the acid. The inhibitory effect of 5 m M salicylic acid on glutamate dehydrogenase activity is apparent due to interference with the activity of the enzyme rather than with its synthesis.     

Increase in glutamate synthase (NADH) activity in maize seedlings in response to nitrate and ammonium nitrogen

Roots and leaves of Zea mays L. cv. Ganga Safed-2 seedlings grown with nutrient solution containing either 10 m M KNO₃ or NH₄Cl or 5 m M NH₄NO₃ had considerably higher glutamate synthase (NADH, EC 1.4.1.14) activity than the corresponding organs from seedlings grown without any nitrogen. The supply of inorganic nitrogen for a short time, i.e. 3 h, to roots and leaves excised from seedlings grown without nitrogen also increased the enzyme activity in these organs. This increase was more pronounced with nitrate than with ammonium nitrogen. When excised roots and leaves from NH₄NO₃-grown seedlings were incubated in a minus nitrogen medium for 24 h, the enzyme activity declined considerably. This decline was inhibited to some extent by nitrogen, especially by nitrate. Inorganic nitrogen prevented similarly the decline in in vitro enzyme activity during 24 h storage at 25°C, more regularly for the root than for the leaf enzyme. The experiments demonstrate the role of inorganic nitrogen in the regulation of glutamate synthase activity.     

Effect of a constant supply of different nitrogen sources on protein and carbohydrate content and enzyme activities of Anabaena variabilis cells

The effect of the nitrogen source on carbohydrate and protein contents and on several enzymatic activities involved in the carbon and nitrogen metabolism was studied in Anabaena variabilis ATCC 29413 cells grown under a constant supply of either N, NO⁻₃ or NH⁺₄ at different concentrations. An enhancement of protein content accompanied by a parallel decrease of carbohydrates was observed with increasing NO⁻₃ or NH⁺₄ concentrations in the medium. In cultures containing 0.1 m M NO⁻₃ or 0.1 m M NH⁺₄ nitrogenase (EC 1.18.6.1) activity was 74 and 66%, respectively, of that found in N₂-grown cells. This activity was still present with 1 m M NO⁻₃ or 1 m M NH⁺₄ in the medium and even with 10 m M NO⁻₃, but it was completely inhibited by 5 m M NH⁺₄. Ferredoxin-nitrate reductase (EC 1.7.7.2) activity was detected only in NO⁻₃ grown cells and simultaneously with nitrogenase activity. Increasing concentrations of combined nitrogen in the medium, especially NH⁺₄, promoted a concomitant decline of glutamine synthetase (EC 6.3.1.2), NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42), and NAD⁺-malate dehydrogenase (EC 1.1.1.37) activities, suggesting that these enzymes play an important role in the regulation of carbon-nitrogen metabolism in cyanobacteria.     

Nitrate reductase and molybdenum cofactor in annual ryegrass as affected by salinity and nitrogen source

The influence of salinity on the activity of nitrate reductase (NR, EC 1.6.6.1) and the level of the molybdenum cofactor (MoCo) as affected by the source and concentration of nitrogen was studied in annual ryegrass ( Lolium multiflorum cv. Westerwoldicum). Plants grown in sand were irrigated with nutrient solution with an electrical conductivity of 2 or 11.2 dS m⁻¹, containing nitrogen (0.5 or 4.5 m M ) in the form of NH₄NO₃ or NaNO₃ Salinity-treated (11.2 dS m⁻¹) plants produced less biomass and more organic nitrogen while accumulating more NO⁻₃ than control plants. Increased nitrogen concentration in the irrigation solutions enhanced biomass and organic nitrogen production as well as NO⁻₃ accumulation irrespective of the electrical conductivity. Salinity inhibited shoot growth and increased shoot NR activity of plants receiving 4.5 m M NH₄NO₃ or NaNO₃. Similar effects were observed in roots of plants grown in 4.5 m M NaNO₃. Nitrate added to a complementation medium containing ryegrass MoCo and the NR apoprotein of Neurospora crassa mutant nit-1 stimulated the activity of the reconstituted NR (NADPH-nitrate reductase, EC 1.6.6.3). Increased salinity and nitrogen in the nutrient solutions caused an increase of MoCo content in roots and shoots. Similar results were observed for NR activity in the shoots. The increase of MoCo in response to salinity was more pronounced than that of NR, especially in the roots. We conclude that the pool size of MoCo in ryegrass is not constant, but varies in response to nutritional and environmental factors.     

Biochemical characterization of nitrate and nitrite reduction in the wild-type and a nitrate reductase mutant of soybean

Enzyme activities involved in nitrate assimilation were analyzed from crude leaf extracts of wild-type (cv. Williams) and mutant ( nr₁ ) soybean [ Glycine max (L.) Merr.] plants lacking constitutive nitrate reductase (NR) activity. The nr₁ soybean mutant (formerly LNR-2), had decreased NADH-NR, FMNH₂-NR and cytochrome c reductase activities, all of which were associated with the loss of constitutive NR activity. Measurement of FMNH₂-NR activity, by nitrite determination, was accurate since nitrite reductase could not use FMNH₂ as a reductant source. Nitrite reductase activity was normal in the nr₁ plant type in the presence of reduced methyl viologen. Assuming that constitutive NR is similar in structure to nitrate reductases from other plants, presence of xanthine dehydrogenase activity and loss of cytochrome c reductase activity indicated that the apoprotein and not the molybdenum cofactor had been affected in the constitutive enzyme of the mutant. Constitutive NR from urea-grown wild-type plants had 1) greater ability to use FMNH₂ as an electron donor, 2) a lower pH optimum, and 3) decreased ability to distinguish between NO₃⁻ and HCO₃⁻, compared with inducible NR from NO₃⁻-grown nr₁ plants. The presence in soybean leaves of a nitrate reductase with a pH optimum of 7.5 is contrary to previous reports and indicates that soybean is not an exception among higher plants for this activity.     

Isolation of Capsicum annuum leaf nitrate reductase and characterization of the effect of adenine nucleotides and NADH on its activity

Abstract. In the preliminary purification of Capsicum leaf nitrate reductase (EC 1.6.6.1), treatment of the crude extract on Sephadex G-25 was necessary to prevent a gelling of the extract and sedimentation of the enzyme. Its K_m values for NADH and nitrate were estimated to be 9.3 and 105mmol m⁻³ ADP and ATP gave hyperbolic competitive inhibition, with respect to NADH, while the inhibition by AMP was linear competitive. K_i values calculated were: ADP and ATP approximately lmol m⁻³ and AMP 2.3 mol m⁻³. Inhibition by ADP was not altered by reduced glutathione.
The Capsicum nitrate reduclase was very susceptible to inhibition by NADH (in the absence of nitrate) and an in vivo assay showed that the activity of the enzyme was limited by the supply of nitrate. NADH and adenine nucleotide levels measured in the Capsicum leaf were used to estimate inhibition of nitrate reductase and a prediction was made of the nitrate reductase activity at different times in the photoperiod. This was shown to follow the same trend as the measured in vivo activity of the enzyme. Changes in adenine nucleotide levels had little effect on nitrate reductase activity.     

NADH-dependent nitrate reductase from two-row barley roots: Purification, characteristics and comparison with leaf enzyme

NADH-nitrate reductase (EC 1.6.6.1) was purified 800-fold from roots of two-row barley ( Hordeum vulgare L. cv. Daisen-gold) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on TSK-gel (G3000SW). The specific activity of the purified enzyme was 6.2 μmol nitrite produced (mg protein)⁻¹ min⁻¹ at 30°C.
Besides the reduction of nitrate by NADH, the root enzyme, like leaf nitrate reductase, also catalyzed the partial activities NADH-cytochrome c reductase, NADH-ferricyanide reductase, reduced methyl viologen nitrate reductase and FMNH₂-nitrate reductase. Its molecular weight was estimated to be about 200 kDa, which is somewhat smaller than that for the leaf enzyme. A comparison of root and leaf nitrate reductases shows physiologically similar or identical properties with respect to pH optimum, requirements of electron donor, acceptor, and FAD, apparent K_m for nitrate, NADH and FAD, pH tolerance, thermal stability and response to inorganic orthophosphate. Phosphate activated root nitrate reductase at high concentration of nitrate, but was inhibitory at low concentrations, resulting in increases in apparent K_m for nitrate as well as V_max whereas it did not alter the K_m for NADH.     

Nitrate and nitrite reduction by alfalfa root nodules: Accumulation of nitrite in Rhizobium melioti bacteroids and senescence of nodules

Addition of NO⁻₃ rapidly induced senescence of root nodules in alfalfa ( Medicago sativa L. cv. Aragon). Loss of nodule dry matter began at the lowest NO⁻₃ concentration (10 m M ) but degradation of bacteroid proteins was only detected when nodules were supplied with NO⁻₃ concentrations above 20 m M .
Bacteroids from Rhizobium meliloti contained high specific activities of nitrate reductase (NR) and nitrite reductase (NiR). Both enzymes were presumably substrate-induced although substantial enzyme activities were present in the absence of NO⁻₃ Typical specific activities for soluble NR and NiR of bacteroids under NO⁻₃ free conditions were 1.2 and 1.4 μmol (mg protein)⁻¹h⁻¹, respectively. In the presence of NO⁻₃, the specific activity of NR was considerably greater than that of NiR, thus causing NO⁻₂ accumulation in bacteroids. Nitrite levels in the bacteroids were linearly correlated with specific activities of NR and NiR, indicating that NO⁻₂ is formed by bacteroid NR and that this NO⁻₂ in turn, induces bacteroid NiR. Accumulation of NO⁻₂ within bacteroids also indicates that NO⁻₂ inhibits nodule activity after feeding plants with NO⁻₃     

In vivo nitrate reductase activities in different organs of lucerne plants: Effects of combined nitrogen during first vegetative growth and after shoot harvest

Nitrate reductase (EC 1.6.6.1–3; NR) activity was evaluated in nodulated lucerne ( Medicago sativa L. cv. Europe) grown aeroponically in both the presence and absence of applied nitrogen. Determination of in vivo NR activity was done with organ pieces in 0.1 M K⁺-phosphate, pH 7.5, 0.1 M KNO₃ and 1% n -propanol. NR activity was detected in all plant parts. Leaves accounted for 40% of the whole plant activity. Root activity was as high as leaf activity. Stem NR activity accounted for 14 to 20% of the total plant activity. NR activity was also detected in symbolically dependent plants grown without combined nitrogen. Nodule NR in symbolically dependent plants accounted for 17% of the tolal plant aclivity. When nitrate was present in the nulrienl medium, NR increased 5-fold as compared lo N₂-dependenl plants. Varying levels of nitrale (1.65 to 4 m M ) had no influence on leaf or stem activities. However, root NR activity seemed to be related to the nitrale concentration in the nulrient medium. Throughoul inilial vegelative growth, in vivo NR and nitrogenase (acelylene reduction) increased simultaneously. After shoot harvest, nitrogenase (acetylene reduction) aclivity drastically decreased with reduction of photosynthate supply, whereas NR increased in all organs, especially in N₂-dependenl plants.     

Nitrate reductase activity in nodules of pea inoculated with hydrogenase positive and hydrogenase negative strains of Rhizobium leguminosarum

The nitrate reductase (NR, EC 1.6.6.1) activity in root nodules formed by hydrogenase positive (Hup⁺) and hydrogenase negative (Hup⁻) Rhizobium leguminosarum strains was examined in symbioses with the pea cultivar Alaska ( Pisum sativum L.), Rates of activity were determined by the in vivo assay in nodules from plants that were only N₂-dependent or grown in the presence of 2 m M KNO₃. The rates varied widely among strains, regardless of the Hup phenotype of the R. leguminosarum strain used for inoculation, but the overall results indicated that nodules formed by Hup⁻ strains accumulated more nitrite in the incubation medium than did those with Hup⁻ phenotypes. Total plant dry weight and reduced nitrogen content of pea plants grown in the presence of 2 m M KNO₃ and inoculated with single Hup⁺ and Hup⁻ R. leguminosarum strains were statistically different among some strains. These observations suggest that the possible advantages derived from the presence of the Hup system on whole plant growth may be counteracted by the higher rates of NR activity in the Hup⁻ strains in the R. leguminosarum -pea symbiosis.     

Time-course of the phosphinothricin effect on gas exchange and nitrate reduction in Medicago sativa

The long-time effect of phosphinothricin (PPT) on gas exchange and nitrate metabolism in intact plants of lucerne ( Medicago sativa L. cv. Aragón) was investigated. Photosynthetic CO₂ uptake, stomatal conductance, and transpiration were measured with an Infra-Red Gas Analyzer (IRGA). Under photorespiratory conditions, CO₂ uptake continuously decreased after PPT treatment. The decrease of photosynthesis led to an increase in the internal CO₂ concentration, which in turn caused stomatal closure and a reduction of transpiration rate. Nitrate reduction from plants sprayed with PPT was assayed both in vitro and in vivo. In vivo nitrate reductase was measured with and without nitrate in the infiltration medium. Both types of nitrate reductase assays indicated that the enzyme was inhibited in plants treated with PPT; however, the enzyme appeared more affected when the in vivo assay was used than when the one in vitro was applied. The nitrate reduction was pronouncedly affected after 24 h of PPT treatment, when glutamine synthetase (GS, EC 6.3.1.2.) activity and gas exchange were inhibited by more than 60%. The data suggest that the inhibition of GS leads to inhibition of photosynthesis, which, in turn, means lack of NADPH and nitrate, the substrates for nitrate reductase. The inhibition of GS also leads to a high ammonia level, which will produce a secondary inhibition of nitrate reductase activity.     

Author idex volume 36 (1986)

Abstract Nitrate reduction to ammonia by marine Vibrio species was studied in batch and continuous culture. In pH-controlled batch cultures (pH 7.4; 50 mM glucose, 20 mM KNO₃), the nitrate consumed accumulated to more than 90% as nitrite. Under these conditions, the nitrite reductase (NO⁻₂→ NH₃) was severely repressed. In pH-controlled continuous cultures of V. alginolyticus with glucose or glycerol as substrates ( D = 0.045 h⁻¹) and limiting N-source (nitrate or nitrite), nitrite reductase was significantly derepressed with cellular activities in the range of 0.7–1.2 μmol min⁻¹ (mg protein)⁻¹. The enzyme was purified close to electrophoretic homogeneity with catalytic activity concentrations of about 1800 nkat/mg protein. It catalyzed the reduction of nitrite to ammonia with dithionite-reduced viologen dyes or flavins as electron donors, had an M _r of about 50 000 (determined by gel filtration) and contained c-type heme groups (probably 4–6 per molecule).     

Nitrate reductase is inhibited in leaves of Triticum aestivum treated with high levels of copper

Copper is a potent sulfhydryl reagent which can also catalyse the generation of active oxygen. Since nitrate reductase (EC 1.6.6.1) is an SH-enzyme sensitive to oxidative environments, the relations among copper, active oxygen species and nitrate reductase (NR) activity are of interest. Foliar segments of wheat ( Triticum aestivum cv. Oasis) were floated on CuSO₄ solutions (up to 250 μ M ) for 24 h under continuous light. Copper decreased NR activity before affecting active oxygen generation as estimated by changes in oxidative parameters, including malondialdehyde, K⁺ leakage and chlorophyll degradation. Cysteine and Na-benzoate counteracted this decrease, suggesting an oxidative damage of the enzyme in leaves exposed to high copper levels. Copper-induced NR inactivation was further studied in the partially purified enzyme. Preincubation with CuSO₄ inhibited NR. Copper inhibition was reversed by subsequent incubation with EDTA, indicating that the metal bonded to key -SH groups of the enzyme. In addition, an ^˙OH-generating system (composed of CuSO₄, ascorbate and H₂O₂) irreversibly decreased the activity of purified NR to a greater extent than copper alone. Our results show that copper affects nitrogen metabolism by diminishing NR activity, involving a direct effect on key SH-groups and an indirect effect via attack by active oxygen species induced by the metal.     

Nitrate assimilation and nitrogen circulation in Austrian pine

Nitrate uptake, reduction and translocation were examined in 5-week-old Austrian pines ( Pinus nigra Arnold var. nigricans Host.) during exposure to 5 m M NaNO₃. The rate of nitrate uptake was linear during the 7 h light period. ¹⁵N-NO₃⁻ was detected in all parts of the pine except in the needles. By the 7th hour, 43% of the absorbed nitrate had been reduced, and this increased to 64% by the 24th hour. The major part of the total reduction occurred in the roots at this growth stage. Accumulation of ¹⁵N in reduced soluble and insoluble fractions was more prevalent in roots than in shoots. In the needles, the translocated nitrogen was mainly incorporated into the insoluble fraction. It is likely that most of the nitrogen from nitrate was transported from the roots to the aerial organs as organic nitrogen; however part of the upward nitrogen flux took place as nitrate ions.
An experiment in which an exposure for 24 h to 5 m M Na¹⁵NO₃ was followed by 13 days exposure to Na¹⁴NO₃ (pulse chase experiment) revealed a half time of about 1 day for depletion of root nitrate. A large part of this depletion was due to the loss of ¹⁵N-NO₃⁻ to the nutrient solution. The remaining pool of ¹⁵N-nitrate was partitioned between a metabolically inactive and an active pool. During the chase period, the simultaneous decrease of ¹⁵N-incorporation in the soluble N fraction and increase in the insoluble N fraction in different pine parts, particularly in the needles, suggested that protein synthesis occurred mostly in young tissues of the shoot and was the major sink of the newly absorbed ¹⁵N-NO₃.     

Cadmium stimulates the accumulation of salicylic acid and its putative precursors in maize (Zea mays) plants

The effect of 10, 25 and 50 µ M Cd(NO₃)₂ on the salicylic acid (SA) metabolism was investigated in young maize seedlings ( Zea mays L., hybrid Norma). Cadmium (Cd) was translocated into the leaves and induced oxidative damage, as indicated by the reduced chlorophyll content, the decreased quantum efficiency of photosystem II and the enhanced malondialdehyde (MDA) content, especially after 7 days. The activity of glutathione reductase (EC 1.6.4.2) increased from the fourth day and that of guaiacol peroxidase (EC 1.11.1.7) after 7 days of Cd stress compared with the control leaves. These effects of Cd exhibited a correlation with the concentration. Under these conditions, Cd did not affect the MDA content or the antioxidant enzyme activities in the roots. After 7 days, Cd increased the levels of free and bound forms of benzoic acid (BA), O -coumaric acid ( O -hydroxy-cinnamic) ( O- HCA) and SA in the leaves, but in the roots, only the 50 µ M rate of Cd caused changes in the free O -HCA acid and bound BA content.     

Effects of NH+4-N and NO+3-N on growth and metabolism of Sphagnum magellanicum

Photosynthetic CO₂-fixation, chlorophyll content, growth rate and nitrate reductase activity were used to examine the influence of NH⁺₄-N and NO⁻₃-N on Sphagnum magellanicum cultivated under defined conditions in phytotrons. NO⁻₃-concentrations up to 322 μ M were found to be favourable. Increased NH⁺₄ concentrations, however, resulted in growth inhibition and decreased chlorophyll content at concentrations ≧ 255 μ M ; e.g. 600 μ M NH⁺₄ caused a 20% reduction of nitrate reductase activity and net photosynthesis. For raised bog Sphagna an improved standard nutrient solution is proposed with the following ion concentrations (μ M ): 55 Na⁺; 17 K⁺; 95 NH⁺₄; 22 Ca²⁺; 22 Mg²⁺; 2 Fe³⁺; 20 Cl⁻; 100 NO⁻₃; 57 SO^2-₄; 7.4 H₂PO⁻₄; trace elements: A-Z solution (Hoagland) 50 μl 1000 ml⁻¹; pH 5.8.     

Anion uptake in maize roots: Interactions between chlorate and nitrate

Effects of nitrate, chloride and chlorate ions upon nitrate and chlorate uptake by roots of maize ( Zea mays L., cv. B73) seedlings were examined. Net nitrate uptake, ³⁶ClO₃⁻ influx and ³⁶Cl⁻ influx (the latter two in a background of 0.5 m M KNO₃) displayed similar pH profiles with optima at pH 5.5 and below. External, non-labeled chloride had little effect on the accumulation of ³⁶ClO₃⁻ (both in 5 h and 20 min uptake assays), while nitrate and chlorate had almost identical, marked inhibitory effects. Nitrate pretreatment caused an apparent induction of both ³⁶ClO₃⁻ and ¹⁵NO₃⁻ uptake activities. After 5 h of treatment in nitrate, the uptake activities of chloride- and chlorate-pretreated plants increased to that of nitrate-pretreated plants. During 6 h exposure to chlorate, ³⁶ClO₃⁻ uptake activity of nitrate-pretreated plants decreased to that of chlorate- and chloride-pretreated plants. The results support the existence of a shared nitrate/chlorate transport system in maize roots which is not inhibited by external chloride, and which is induced by nitrate, but not by chlorate or chloride. The suggestion is made that selection of chlorate-resistant mutants of maize can identify nitrate uptake as well as nitrate reductase mutants.     

Osmoregulation and role of nitrate during regrowth after cutting of ryegrass (Lolium perenne)

The osmotic role of nitrate during aftermath growth of Lolium perenne L. cv. Réveille was investigated. Plants were grown from seed in a controlled environment using a liquid medium with 1.0 m M NH₄NO₃ as nitrogen source.
Eight-week-old plants were cut 4.0 cm above the root system and then harvested over a 14-day period of regrowth on the same initial nutrient solution, except that nitrate was ¹⁵N labelled. Throughout the experimental period, nitrate storage and reduction in roots were low. In stubble and especially in leaves, nitrate accumulated during the first 6 days of regrowth whereas nitrate reduction mainly occurred after this period. Analyses of carbohydrate, chloride and potassium contents in stubble and leaves showed that the accumulation of nitrate osmotically compensated for the decrease in soluble sugars during the first 6 days of regrowth.
The cumulative osmotic potential of sugars, chloride and nitrate in differently treated plants was studied in stubble and leaves. Compared with uncut plants, the lower carbohydrate concentrations found in cut plants regrowing on 1.0 m M NH₄NO₃ were compensated for by an accumulation of nitrate. During aftermath growth on low nitrogen nutrition (0.2 m M NH₄NO₃), chloride replaced nitrate, supporting the proposed osmotic function of nitrate.
It is concluded that nitrate is involved in the osmotic adjustment of ryegrass during regrowth after cutting.     

NO2‐induced nitrate reductase activity in needles of Norway spruce (Picea abies) under laboratory and field conditions

The induction of activity of the enzyme nitrate reductase (NR, EC 1.6.6.1, 1.6.6.2) in needles of Norway spruce ( Picea abies [L.] Karst.) by nitrogen dioxide (NO₂) was studied under laboratory and field conditions. In fumigation chambers an increase in nitrate reductase activity (NRA) was detected 4 h after the start of the NO₂ treatment. During the first 2 days with 100 µg NO₂ m⁻³, NRA reached a constant level and did not change during the following 4 days. At the same level of NO₂, NRA was lower in needles from trees grown on NPK‐fertilized soil than on non‐fertilized soil. After the transfer of spruce trees from fertilized soil to NPK‐rich nutrient solution, NRA was transiently increased. This effect was assigned to root injuries causing nitrate transport to the shoot and subsequent induction of NRA. Neither trees on fertilized soil nor trees transferred to NPK‐poor nutrient solution had increased NRA unless NO₂ was provided. The NO₂ gradient in the vicinity of a highway was used to test the long‐term effect of elevated levels of NO₂ on needle NRA of potted and field‐grown spruce trees. Compared with less polluted sites, permanently increased NRAs were detected when NO₂ concentrations were above 20 µg m⁻³. Controls of field measurements some 10 years after the introduction of catalytic converters in cars showed no significant change neither in NO₂ levels nor in the decreasing NRA of spruce needles with the distance from the highway.