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.     

Senescence in oat leaf segments under hypobaric conditions

The physiological effects of storing plants under hypobaric conditions were studied using oat ( Avena sativa L. cv. Victory) leaf segments as a test system. The segments from seven day old plants were floated on water and stored in darkness at 12°C, 1.6 kPa or at 25°C, 6 kPa. Low temperature or hypobaric conditions delayed senescence, whereas the combination arrested the syndrome at an early stage. One of the effects of low pressure was to force the stomata open. The hormones abscisic acid and kinetin, which affect the stomatal aperture and also senescence, did not show any effect in hypobarically stored plant material. The stomata were forced open in darkness when the pressure was lower than 77 kPa and opening time was 8 h. The senescence syndrome in hypobarically stored segments developed similar to those treated with kinetin at 101 kPa.     

Reversible inactivation of maize leaf nitrate reductase

Preincubation of maize leaves crude extracts with NADH resulted in a progressive accumulation of nitrite which mimicked a rapid and lineal activation of nitrate reductase. Nevertheless, in partially purified preparations it was found that preincubation at pH 8.8 with NADH promoted a gradual inactivation of nitrate reductase. At pH 7.5, the enzyme was not inactivated by the presence of NADH alone, but, with the simultaneous presence of a low concentration of cyanide, a fast inactivation took place. The NADH-cyanide-inactivated nitrate reductase remained inactive after removing the excess of NADH and cyanide by filtration through Sephadex G-25. However, it could be readily reactivated by incubation with ferricyanide or by a short exposure to light in the presence of FAD. Prolonged irradiation caused a progressive inactivation of the photoreactivated enzyme.     

Regulation of ethylene biosynthesis during senescence of oat leaf segments

The evolution of endogenous ethylene, the conversion of 1-aminocylopropane-1-car-boxylic acid (ACC) to ethylene and the amounts of ACC (free and conjugated) have been followed during the senescence of oat ( Avena sativa L. cv. Victory) leaf segments. During the first three days of incubation of leaf segments in darkness, endogenous ethylene evolution and ACC-dependent ethylene production displayed a close relationship, both showing an increase followed by a decrease to the basal rate. However, unlike ethylene production, the level of ACC increased during the five days of incubation in the dark without any decline. It is concluded that ACC synthesis does not limit ethylene production, at least in the last stages of leaf senescence when ethylene production markedly decreased. The level of conjugated ACC increased and reached a plateau already at the first day of incubation. Yet, at the progressive stages of senescence, when the level af ACC gradually increased, no further conjugation of ACC could be detected. Thus, conjugation of ACC cannot account for ethylene drop at the last stages of oat leaf senescence.     

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.     

Galactose inhibition of auxin-induced cell elongation in oat coleoptile segments

Auxin-induced cell elongation in oat coleoptile segments was inhibited by galactose; removal of galactose restored growth. Galactose did not appear to affect the following factors which modify cell elongation: auxin uptake, auxin metabolism, osmotic concentration of cell sap, uptake of tritium-labeled water, auxin-induced wall loosening as measured by a decrease in the minimum stress-relaxation time and auxininduced glucan degradation. Galactose markedly prevented incorporation of [¹⁴C]-glucose into cellulosic and non-cellulosic fractions of the cell wall. It was concluded that galactose inhibited auxin-induced long-term elongation of oat coleoptile segments by interfering with cell wall synthesis.     

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.     

Photooxidative inhibition of nitrate reductase during chilling

The effect of a temperature close to the freezing point (chilling) on the nitrate reductase system of leaf discs of Cucumis sativus L. cv. Kleine Groene Scherpe was determined in the absence and presence of light. The capacity of leaf discs in the light (250 μE m⁻²s⁻¹) at 20°C to increase in vivo and in vitro nitrate reductase activity, was unaffected by chilling pretreatment in the dark, but 4 h of chilling pretreatment in the light (250 μE m⁻²s⁻¹) decreased the capacity to less than 50% of the unchilled control. The chilling inhibition of the capacity to increase nitrate reductase activity was of a photooxidative nature since it only occurred in the presence of light and oxygen. Plants grown at a low light intensity (65 μE m⁻²s⁻¹) lost 95% of their capacity to increase nitrate reductase activity, while plants grown at 195 μE m⁻²s⁻¹ retained 80% of their nitrate reducing capacity after 6 h chilling pretreatment in the 250 μE m⁻²s⁻¹ light. Previously induced nitrate reductase activity was also affected by light during chilling. A lag phase of 7 h preceded a fast phase of decrease in activity. Both in vivo and in vitro activity decreased to 15% of the control value after 18 h of chilling in the light. It is concluded that the induction mechanism of nitrate reductase is primarily affected by photooxidation during chilling. The decrease in nitrate reductase activity is attributed to a decrease in the amount of activity enzyme.     

Regulation of nitrate reductase in detached oat leaves by light and oxygen

Regulation of nitrate reductase (NR, EC 1.6.6.1) by oxygen concentration and light was studied in segments of oat ( Avena sativa L. cv. Suregrain) leaves, using the in vivo nitrate reductase assay. The activity of NR decreased after excision in either light or darkness; the addition of cycloheximide prevented this decrease. Treatments that increased tissue permeability (anoxia, Triton X-100) also increased NR activity. There was in general less NR activity in the light than in the dark and also less under aerobic (21–100% O₂) than under anaerobic (0.3% O₂) conditions. Treatments with antioxidants improved the activity in the light, but only at high O₂ levels (21–100% O₂).
The results suggest that NR may be regulated by inhibitory proteins synthesized in either light or darkness, by permeability changes and by light-induced oxidations that occur when O₂ is present. Oxygen may control the activity by stimulating the synthesis of inhibitory proteins in the light and in the dark and by promoting oxidation of SH-groups in the light.     

Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana

Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrite uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.Abbreviations EDTA ethylenediaminetetraacetic acid - FAD flavine-adenine dinucleotide - IgG immunoglobulin G - NR nitrate reductase - PM plasma membrane - TX-100 Triton X-100     

Kinetics of substrate inhibition of periplasmic nitrate reductase

Periplasmic nitrate reductase catalyzes the reduction of nitrate into nitrite using a mononuclear molybdenum cofactor that has nearly the same structure in all enzymes of the DMSO reductase family. In previous electrochemical investigations, we found that the enzyme exists in several inactive states, some of which may have been previously isolated and mistaken for catalytic intermediates. In particular, the enzyme slowly and reversibly inactivates when exposed to high concentrations of nitrate. Here, we study the kinetics of substrate inhibition and its dependence on electrode potential and substrate concentration to learn about the properties of the active and inactive forms of the enzyme. We conclude that the substrate-inhibited enzyme never significantly accumulates in the EPR-active Mo(+ V) state. This conclusion is relevant to spectroscopic investigations where attempts are made to trap a Mo(+ V) catalytic intermediate using high concentrations of nitrate.     

Synthesis and degradation of nitrate reductase in Escherichia coli.

The biosynthesis, insertion, and in vivo stability of nitrate reductase were examined by following the amount of labeled enzyme present in both membranes and cytoplasm at varying times after a short pulse of radioactive sulfate. Nitrate reductase levels were measured by autoradiography of immunoprecipitated material after fractionation on sodium dodecyl sulfate-polyacrylamide gels. These experiments demonstrated that subunits A and B were synthesized in the cytoplasm and subsequently inserted into membranes. The insertion of these subunits was dependent upon the synthesis of another protein, and the rate of synthesis of this protein determined the rate of insertion of subunits A and B. The nitrate reductase produced by the chlA mutant was inserted into membranes in the normal fashion, whereas the nitrate reductase produced by the chlC and chlE mutants was poorly incorporated. The nitrate reductase in the wild type was completely stable in vivo under inducing or noninducing conditions, whereas in the chlC and chlE mutants nitrate reductase was degraded extensively in both the cytoplasm and membranes, even under inducing conditions. Under similar conditions, nitrate reductase was stable in the chlA mutant.     

Effect of hydrogen peroxide on nitrate reductase activity in detached oat leaves in darkness

Nitrate reductase (NR, EC 1.6.6.1) is sensitive to O₂ concentration, and therefore it was of interest to study the action of H₂O₂, a normal substance in plant metabolism, on NR activity in segments of 7-, 14- and 17-day-old leaves of oat (Avena sativa L. ev. Suregrain). After 4 h of treatment in the dark, H₂O₂ decreased NR activity as measured with the in vivo assay. The effect was stronger in 14- and 17- than in 7-day-old leaves. Vacuum infiltration of cysteine did not prevent this decrease. When NR was determined with the in vitro assay, H₂O₂ did not seem to affect the activity after the 4 h treatment. but NR decreased when crude extracts prepared from untreated 14-day-old leaves were incubated directly with H₂O₂. This effect was prevented by addition of cysteine, ascorbate or reduced glutathione to the extracts. In order to study the possibility that low activity of the system for defense against oxidations could account for the age-dependent response of NR to H₂O₂ in the in vivo test, activities of catalase, ascorbate peroxidase and glutathione reductase were measured during leaf development and after a 4-h treatment with H₂O₂ in the dark. No clear correlation was found between the activities of those enzymes and changes in in vivo NR activity caused by H₂O₂. The results suggest that H₂O₂ might affect NR both directly by oxidizing SH-groups and indirectly by decreasing reductant availability as a result of NADH oxidation. The age-dependent response of NR to H₂O₂ treatment could also be explained in terms of decreased NADH availability in the tissues due to decreased NADH synthesis and/or increased degradation.     

Light-dark changes in cytosolic nitrate pools depend on nitrate reductase activity in Arabidopsis leaf cells

Several different cellular processes determine the size of the metabolically available nitrate pool in the cytoplasm. These processes include not only ion fluxes across the plasma membrane and tonoplast but also assimilation by the activity of nitrate reductase (NR). In roots, the maintenance of cytosolic nitrate activity during periods of nitrate starvation and resupply (M. van der Leij, S.J. Smith, A.J. Miller [1998] Planta 205: 64-72; R.-G. Zhen, H.-W. Koyro, R.A. Leigh, A.D. Tomos, A.J. Miller [1991] Planta 185: 356-361) suggests that this pool is regulated. Under nitrate-replete conditions vacuolar nitrate is a membrane-bound store that can release nitrate to the cytoplasm; after depletion of cytosolic nitrate, tonoplast transporters would serve to restore this pool. To study the role of assimilation, specifically the activity of NR in regulating the size of the cytosolic nitrate pool, we have compared wild-type and mutant plants. In leaf mesophyll cells, light-to-dark transitions increase cytosolic nitrate activity (1.5-2.8 mm), and these changes were reversed by dark-to-light transitions. Such changes were not observed in nia1nia2 NR-deficient plants indicating that this change in cytosolic nitrate activity was dependent on the presence of functional NR. Furthermore, in the dark, the steady-state cytosolic nitrate activities were not statistically different between the two types of plant, indicating that NR has little role in determining resting levels of nitrate. Epidermal cells of both wild type and NR mutants had cytosolic nitrate activities that were not significantly different from mesophyll cells in the dark and were unaltered by dark-to-light transitions. We propose that the NR-dependent changes in cytosolic nitrate provide a cellular mechanism for the diurnal changes in vacuolar nitrate storage, and the results are discussed in terms of the possible signaling role of cytosolic nitrate.     

Inhibition of wheat leaf nitrate reductase activity by phenolic compounds

Phenolic acids inhibited the activities of wheat leaf nitrate reductase depending on phenolic structure and concentration. Possible conformational change(s) in the enzyme induced by hydrogen bonding and/or hydrophobic interactions might be the cause of the enzyme inhibition. NADH:cytochrome C reductase partial activity was unaffected, which indicates that terminal nitrate-reducing domain of NR may be the site of polyphenol binding.     

Monoclonal antibodies identify multiple epitopes on maize leaf nitrate reductase

Nine hybridoma cell lines secreting antibodies against the maize leaf nitrate reductase have been distinguished by reciprocal competition for binding to the antigenic site. Inhibition of enzymatic activities, and western blots of native enzyme and denatured subunits revealed different behaviors of individual antibodies towards the antigen. Two classes of monoclonal antibodies are inhibitory of NADH and methyl viologen nitrate reductase activities, but only one affects also NADH cytochrome c reductase activity. The associated epitopes are sensitive to antigen conformation. Among the 4 other classes, one is specific for the native conformation of the molecule, another binds more strongly to the denatured antigen, and two recognize equally well the two forms.     

Comparative studies of diurnal variations of nitrate reductase activity in wheat, oat and barley

Diurnal variations of in vitro and in vivo (intact tissue assay) nitrate reductase (EC 1.6.6.1) activity and stability were examined in leaves of wheat ( Triticum aestivum L. cv. Runar), oat ( Avcna saliva L. cv. Mustang) and barley ( Hordeum vulgure L. cv. Agneta and cv. Gunillu). Nitrate reductase activity was generally higher for wheat than for oat and barley. However, the diurnal variations of nitrate reductase activity and stability were principally the same for all species, e.g. the high activity during the photoperiod was associated with low stability. All species showed a rapid (30-60 min) increase in the in vitro and in vivo activity when the light was switched on. When light was switched off the in vitro activity decreased rapidly whereas decrease in in vivo activity was slower. These experiments support the hypothesis that an activation/ deactivation mechanism is involved in the regulation of diurnal variations in nitrate reductase activity. Red light enhanced nitrate reductase activity in etiolated wheat and barley leaves. In green leaves, however, the daily increase in nitrate reductase activity was not induced by a brief red light treatment. Indications of different regulation mechanisms for the diurnal variations of nitrate reductase activity among the cereals were not found.     

Regulation of nitrate reductase and phosphoenolpyruvate carboxylase activities in barley leaf protoplasts

Barley leaf protoplasts were incubated in light or darkness in the presence of various inhibitors, metabolites or weak acids/bases. Nitrate reductase (NR) and phosphoenolpyruvate carboxylase (PEPCase) were rapidly extracted from the protoplasts and assayed under sub-optimal conditions, i.e. in the presence of Mg²⁺ and malate, respectively. Under these conditions changes in activities are thought to reflect changes in the phosphorylation states of the enzymes. The NR was activated by illumination to 90% of its maximal activity within 10 min. Photosynthetic electron transport appeared necessary for light activation of NR since activation was inhibited by the photosynthetic electron-transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and, additionally, an electron acceptor (HCO ₃ ^- ) was required. The PEPCase was also activated by light. However, this activation was not prevented by DCMU or lack of HCO ₃ ^- . Loading of protoplasts in the dark with a weak acid resulted in activation of both NR and PEPCase. For NR, full activation was completed within 5 min, whereas for PEPCase a slower, modest activation continued for at least 40 min. Incubation of protoplasts with a weak base also gave activation of PEPCase, but not of NR. On the contrary, base loading counteracted light activation of NR. Since several treatments tested resulted in the modulation of either NR or PEPCase activity, but not both, signal transduction cascades leading to changes in activities appear to be very different for the two enzymes.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron) - DMO 5,5-dimethyl-2,4 oxazolidinedione - NR nitrate reductase - PEPCase Phosphoenolpyruvate carboxylase This work was supported by the Norwegian Research Council by a Grant to C.L: L.H.S. was supported by the Biotechnology and Biological Sciences Research Council.