Effect of glucose on the induction of nitrate reductase in corn roots

In Zea mays L., addition of glucose to the induction medium has no effect on the induction of nitrate reductase during the initial 3 hours either in root tips (0-10 mm) or mature root sections (25-35 mm). With longer times, higher levels of enzyme activity are recovered from both root segments when glucose is present in the incubation medium. The induction in root tips is saturated by 10 mm NO(3) (-). Higher concentrations of NO(3) (-) are required for saturation in mature root sections. The response to glucose is seen over a wide range of external NO(3) (-) concentrations.Nitrate reductase activity is lost rapidly when nitrate is withdrawn from the induction medium. Additions of glucose do not prevent this loss. Additions of glucose have no effect on total uptake of NO(3) (-) by the root segments but they increase the anaerobic NO(2) (-) production in both root tips and mature root segments. This latter measurement is considered to be an estimate of an active NO(3) (-) pool in the cytoplasm. Thus the results show that glucose alters the distribution of NO(3) (-) within the root sections. This may be an important factor in controlling the in vivo stability of the enzyme or its rate of synthesis.     

Nitrate uptake and induction of nitrate reductase in excised corn roots

The characteristics of nitrate uptake and induction of nitrate reductase were studied in excised roots of corn (Zea mays L.). Upon initial exposure to nitrate, the low initial rate of nitrate uptake gradually increased until a steady uptake rate was achieved in 1 to 2 hours depending on the NO(3) (-) concentration. The pattern was observed over a wide range (0.2-5 mm) of nitrate concentrations and was independent of the accompanying cation.The nitrate uptake pattern as a function of increasing external nitrate concentrations (0.2-50 mm) followed saturation type kinetics. The reciprocal plot of the data was not linear but hyperbolic, indicating that more than one Km for nitrate uptake can be resolved from the data. This suggests the existence of either one carrier system with changing kinetic constants or the existence of dual uptake systems. The pattern of induction of nitrate reductase was coincident with the pattern of nitrate uptake as a function of time and increasing nitrate concentrations. The rate of induction of nitrate reductase was regulated by the rate of nitrate flux.Washing the roots for 2 hours enhances nitrate uptake by 2.5-fold over the nonwashed tissue. The presence of nitrate in the washing solution leads to further (3.5-fold over control) increases in the rate of nitrate uptake supporting the contention that nitrate plays a specific role in the induction of the inducible nitrate carrier independent of the washing effect.     

Synthesis and turnover of nitrate reductase in corn roots

The induction and reinduction of nitrate reductase in root tip or mature root sections show essentially a similar pattern: a lag, a period of rapid increase in enzyme activity and finally a period of relatively minor change. Both inductions are sensitive to 6-methylpurine and cycloheximide. Kinetic studies with 6-methylpurine suggest that the half-life of the messenger RNA for nitrate reductase in both sections is about 20 minutes. The rate of decay of nitrate reductase activity induced by transfer to a nitrate-free medium is slower in root tips (t½ = 3 hours) than in mature root sections (t½ = 2 hours). The enzyme from mature root sections is also less stable to mild heat treatments (27 C; 40 C) than the enzyme from root tip sections. The results indicate that factors regulating enzyme turnover show important changes as root cells mature and may be significant in determining steady state levels of the enzyme.     

Photosynthesis and the induction of nitrate reductase and nitrite reductase in bean leaves

Summary In laaves of Phaseolus vulgaris L. cv. Prelude, the light-induced increase in activity of NADH-nitrate oxidoreductase (E.C.1.6.6.2; NAR) and reduced benzylviologennitrite oxidoreductase (E.C.1.6.6.4; NIR) starts at a certain stage in the development of the chloroplasts. In leaves with completely developed chloroplasts, a higher increase in activity of NAR and NIR is observed, after induction by the addition of nitrate, in the light than in the dark. DCMU inhibits the increase in activity of the two enzymes in the light. Both in the light in the presence of DCMU, and in the dark the increase in activity reaches a higher level by the addition of sucrose.Induction of NAR, but not of NIR, can be observed in excised etiolated leaves. No induction is found in leaves of intact etiolated seedlings.The relation between photosynthetic reactions and the increase in activity of NAR and NIR is discussed. It is suggested that NADH, indirectly formed by photosynthesis, protects NAR and affects in this way the balance between synthesis and breakdown of the enzyme. The increase in activity of NIR is possibly influenced by the presence of reduced ferredoxin.Abbreviations CAP D-threo-chloramphenicol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - NAR nitrate reductase - NIR nitrite reductase     

Effect of carbon dioxide on nitrate accumulation and nitrate reductase induction in corn seedlings

Exposure of the leaf canopy of corn seedlings (Zea mays L.) to atmospheric CO₂ levels ranging from 100 to 800 μl/l decreased nitrate accumulation and nitrate reductase activity. Plants pretreated with CO₂ in the dark and maintained in an atmosphere containing 100 μl/l CO₂ accumulated 7-fold more nitrate and had 2-fold more nitrate reductase activity than plants exposed to 600 μl/l CO₂, after 5 hours of illumination. Induction of nitrate reductase activity in leaves of intact corn seedlings was related to nitrate content. Changes in soluble protein were related to in vitro nitrate reductase activity suggesting that in vitro nitrate reductase activity was a measure of in situ nitrate reduction. In longer experiments, levels of nitrate reductase and accumulation of reduced N supported the concept that less nitrate was being absorbed, translocated, and assimilated when CO₂ was high. Plants exposed to increasing CO₂ levels for 3 to 4 hours in the light had increased concentrations of malate and decreased concentrations of nitrate in the leaf tissue. Malate and nitrate concentrations in the leaf tissue of seven of eight corn genotypes grown under comparable and normal (300 μl/l CO₂) environments, were negatively correlated. Exposure of roots to increasing concentrations of potassium carbonate with or without potassium sulfate caused a progressive increase in malate concentrations in the roots. When these roots were subsequently transferred to a nitrate medium, the accumulation of nitrate was inversely related to the initial malate concentrations. These data suggest that the concentration of malate in the tissue seem to be related to the accumulation of nitrate.     

Light-modulation of nitrate reductase activity in leaves and roots of maize

The nuclear DNA content in ray cells from the 1-year-old vascular cambium of white ash ( Fraxinus americana L.) trees was determined at intervals during the annual cycle of cambial activity and dormancy by using Feulgen microspectrophotometry. By 10 September, these cells had entered dormancy in G₁ with a normal DNA distribution and a minimal average DNA content of 2.65 pg. The average amount of DNA increased to 3.51 pg by 30 November, remained at this elevated value until at least 30 March, when the cambium was still dormant, then declined to the minimum level on 1 May and 10 June, when the cells were mitotically active. The springtime decline appeared to occur both before and during cell division. Between 1 May and 10 June, the prophase (4C) and telophase (2C) DNA contents decreased significantly. The amount of nuclear DNA measured by microspectrophotometry was verified by using flow cytometry and image analysis. The results support the view that there is an annual oscillation in the nuclear genome size of shoot meristematic cells in tree species native to the northern temperate zone.     

Ammonium and amino acids as regulators of nitrate reductase in corn roots

When amino acids or ammonia are added to plant systems, the effects on the development of nitrate-dependent nitrate reductase activity are variable. In addition, amino acids added singly or as casein hydrolysate may not support a normal growth. A physiologically correct mixture of amino acids, one similar in composition to amino acids released by the endosperm, has been shown to support normal growth and protein synthesis in corn (Zea mays) embryos. In this investigation, we have used the mixture of corn amino acids to determine whether amino acids have an effect on the appearance or disappearance of nitrate reductase activity. The results show that these amino acids partially inhibit the induction of nitrate reductase in corn roots. The effect is more pronounced in mature root than in root tip sections. When glutamine and asparagine are included along with the "corn amino acid mixture," the inhibition is more severe. Amino acids or amino acid analogues added singly to the induction medium have a similar effect: i.e. when the induction of nitrate reductase is inhibited in the root tips (lysine, canavanine, azaserine, azetidine-2-carboxylic acid, dl-4-azaleucine, asparagine, and glutamine), that inhibition is more severe in mature root sections. Arginine enhanced the recovery of nitrate reductase in root tips but inhibited it in mature root sections. The effect of the amino acids is apparently on some phase of the induction processes (i.e. the uptake or distribution of nitrate or a direct effect on the synthesis of the enzyme) and not on the turnover of the enzyme.     

Correlated induction of nitrate uptake and membrane polypeptides in corn roots

Induction of corn (Zea mays L.) seedling root membrane polypeptides was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis in relation to induction of nitrate uptake. When nitrate uptake was studied using freshly harvested roots from 4-day old corn seedlings, a steady state rate of uptake was achieved after a lag of 2 to 3 hours. The plasma membrane fraction from freshly harvested roots (uninduced) and roots pretreated in 5 millimolar nitrate for 2.5 or 5 hours (induced) showed no differences in the major polypeptides with Coomassie blue staining. Autoradiography of the ³⁵S-methionine labeled proteins, however, showed four polypeptides with approximate molecular masses of 165, 95, 70, and 40 kilodaltons as being induced by both 2.5 and 5-hour pretreatment in 5 millimolar nitrate. All four polypeptides appeared to be integral membrane proteins as shown by Triton X-114 (octylphenoxypolyethoxyethanol) washing of the membrane vesicles. Autoradiography of the two-dimensional gels revealed that several additional low molecular weight proteins were induced. A 5-hour pretreatment in 5 millimolar chloride also induced several of the low molecular weight polypeptides, although a polypeptide of about 30 kilodaltons and a group of polypeptides around 40 kilodaltons appeared to be specifically induced by nitrate. The results are discussed in relation to the possibility that some of the polypeptides induced by nitrate treatment may be directly involved in nitrate transport through the plasma membrane.     

Inhibition of nitrate reductase induction by canavanine in maize roots

The induction of nitrate reductase activity in maize root tips was inhibited by canavanine and the inhibition increased with increasing concentration of canavanine between 0·1 and 1 mM. Addition of canavanine to the induced enzyme had little effect on the disappearance of the enzyme when nitrate was removed, and it is likely that the canavanine reduces the activity of the nitrate reductase by inhibiting its synthesis rather than by accelerating its breakdown.     

Comparative studies of the light modulation of nitrate reductase and sucrose-phosphate synthase activities in spinach leaves

We recently obtained evidence that the activity of spinach (Spinacia oleracea L.) leaf nitrate reductase (NR) responds rapidly and reversibly to light/dark transitions by a mechanism that is strongly correlated with protein phosphorylation. Phosphorylation of the NR protein appears to increase sensitivity to Mg²⁺ inhibition, without affecting activity in the absence of Mg²⁺. In the present study, we have compared the light/dark modulation of sucrose-phosphate synthase (SPS), also known to be regulated by protein phosphorylation, and NR activities (assayed with and without Mg²⁺) in spinach leaves. There appears to be a physiological role for both enzymes in mature source leaves (production of sucrose and amino acids for export), whereas NR is also present and activated by light in immature sink leaves. In mature leaves, there are significant diurnal changes in SPS and NR activities (assayed under selective conditions where phosphorylation status affects enzyme activity) during a normal day/night cycle. With both enzymes, activities are highest in the morning and decline as the photoperiod progresses. For SPS, diurnal changes are largely the result of phosphorylation/dephosphorylation, whereas with NR, the covalent modification is super-imposed on changes in the level of NR protein. Accumulation of end products of photosynthesis in excised illuminated leaves increased maximum NR activity, reduced its sensitivity of Mg²⁺ inhibition, and prevented the decline in activity with time in the light seen with attached leaves. In contrast, SPS was rapidly inactivated in excised leaves. Overall, NR and SPS share many common features of control but are not identical in terms of regulation in situ.     

Effect of chloramphenicol and cycloheximide on the induction of nitrate reductase and nitrite reductase in bean leaves

Summary In etiolated leaves of Phaseolus vulgaris L. cv. Prelude only low levels of NADH-nitrate oxidoreductase (E.C. 1.6.6.2; NAR) and reduced benzyl viologen-nitrite oxidoreductase (E.C. 1.6.6.4; NIR) could be detected, even in the presence of nitrate. When nitrate was available illumination of leaves of 10-day-old etiolated seedlings resulted in an induction of both NAR and NIR. In the absence of nitrate no induction of the enzymes took place, although greening of the leaves was normal. Chloramphenicol (CAP) and cycloheximide (CHI), applied at the beginning of the light period, inhibited the induction of both NAR and NIR. Administered after 24 h of illumination CHI still inhibited the induction of both enzymes whereas CAP was no longer inhibitory. The induction of NAR and NIR by nitrate in green leaves in light was inhibited by CHI but not by CAP. From these results it seems likely that both the enzymes NAR and NIR are synthesized on cytoplasmic ribosomes. Before the enzymes can be manufactured in the cytoplasm some chloroplast development is required.Abbreviations CAP chloramphenicol - CHI cycloheximide - G-6-P(-dh) glucose-6-phosphate (dehydrogenase) - NAR nitrate reductase - NIR nitrite reductase     

The role of nitrate and ammonium ions and light on the induction of nitrate reductase in maize leaves

Corn seedlings (Zea mays cv W64A × W182E) were grown hydroponically, in the presence or absence of NO₃⁻, with or without light and with NH₄Cl as the only N source. In agreement with earlier results nitrate reductase (NR) activity was found only in plants treated with both light and NO₃⁻. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer of the proteins to nitrocellulose paper and reaction with antibodies prepared against a pure NR showed that crude extracts prepared from light-grown plants had a polypeptide of approximately 116 kilodaltons (the subunit size for NR) when NO₃⁻ was present in the growth medium. Crude extracts from plants grown in the dark did not have the 116 kilodalton polypeptide, although smaller polypeptides, which reacted with NR-immunoglobulin G, were sometimes found at the gel front. When seedlings were grown on Kimpack paper or well washed sand, NR activity was again found only when the seedlings were exposed to light and NO₃⁻. Under these conditions, however, a protein of about 116 kilodaltons, which reacted with the NR antibody was present in light-grown plants whether NO₃⁻ was added to the system or not. The NR antibody cross-reacting protein was also seen in hydroponically grown plants when NH₄Cl⁻ was the only added form of nitrogen. These results indicate that the induction of an inactive NR-protein precursor in corn is mediated either by extremely low levels of NO₃⁻ or by some other unidentified factor, and that higher levels of NO₃⁻ are necessary for converting the inactive NR cross-reacting protein to a form of the enzyme capable of reducing NO₃⁻ to NO₂⁻.     

Phytochrome mediated induction of nitrate reductase activity in etiolated maize leaves

The effects of red and far-red light on the enhancement of in vitro nitrate reductase activity and on nitrate accumulation in etiolated excised maize leaves were examined. Illumination for 5 min with red light followed by a 4-h dark period caused a marked increase in nitrate reductase activity, whereas a 5-min illumination with far-red light had no effect on the enzyme activity. The effect of red light was completely reversed by a subsequent illumination with the same period of far-red light. Continuous far-red light also enhanced nitrate reductase activity. Both photoreversibility by red and far-red light and the operation of high intensity reaction under continuous far-red light indicated that the induction of nitrate reductase was mediated by phytochrome. Though nitrate accumulation was slightly enhanced by red and continuous far-red light treatments by 17% and 26% respectively, this is unlikely to account for the entire increase of nitrate reductase activity. The far-red light treatments given in water, to leaves preincubated in nitrate, enhanced nitrate reductase activity considerably over the dark control. The presence of a lag phase and inhibition of increase in enzyme activity under continuous far-red light-by tungstate and inhibitors of RNA synthesis and protein synthesis-rules out the possibility of activation of nitrate reductase and suggests de novo synthesis of the enzyme affected by phytochrome.     

Latent nitrate reductase activity is associated with the plasma membrane of corn roots

Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U₂). Analysis with marker enzymes indicated that the U₂ fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.Abbreviations L₁ first lower phase - NR nitrate reductase - NRA nitrate-reductase activity - PM plasma membrane - T:p Triton X-100 (octylphenoxy polyethoxyethanol) to protein ratio - U₂ second upper phase     

Differential regulation of nitrate reductase induction in roots and shoots of cotton plants

The induction of nitrate reductase activity in root tips of cotton (Gossypium hirsutum L.) was regulated by several amino acids and by ammonium. Glycine, glutamine, and asparagine strongly inhibited induction of activity by nitrate and also decreased growth of sterile-cultured roots on a nitrate medium. Methionine, serine, and alanine weakly inhibited induction, and 11 other amino acids had little or no effect. Ammonium also decreased induction in root tips, but was most effective only at pH 7 or higher. The optimum conditions for ammonium regulation of induction were identical to those for growth of sterile-cultured roots on ammonium as the sole nitrogen source. Aspartate and glutamate strongly stimulated induction, but several lines of evidence indicated that the mechanism of this response was different from that elicited by the other amino acids. The effects of amino acids on induction appeared to be independent of nitrate uptake.     

Effect of light and glucose on the induction of nitrate reductase and on the distribution of nitrate in etiolated barley leaves

Barley seedlings grown in the dark with 10 mm KNO(3) have low levels of nitrate reductase activity even though large amounts of No(3) (-) accumulate in the leaves. When the leaves are excised and transferred to the light, there is an increase in nitrate reductase activity both in the presence and absence of exogenous NO(3) (-). When the leaves are transferred to a glucose solution (0.05 m) but kept in the dark, induction of nitrate reductase activity occurs only when fresh NO(3) (-) is added to the system.In dark-grown leaves, there are small traces of NO(3) (-) in a "metabolic pool." Addition of glucose does not alter this distribution. Light, on the other hand, results in an appreciable accumulation of NO(3) (-) in the metabolic pool. There is a linear correlation between nitrate reductase activity and the size of the metabolic NO(3) (-) pool. Our results thus suggest that NO(3) (-) accumulates in a storage pool when seedlings are grown in continuous darkness. The transfer of this NO(3) (-) to an active metabolic pool is mediated by light but not by glucose. We believe that this transfer of NO(3) (-) leads to the induction of nitrate reductase. When NO(3) (-) is included in the medium, both light and glucose increase its incorporation into the metabolic pool. The results suggest two mechanisms for regulating the metabolic NO(3) (-) pool: (a) a transfer from the storage pool which requires light; and (b) a transfer from the external medium which requires either glucose or light.     

Comparative stability of ammonium- and nitrate-induced nitrate reductase activity in maize leaves

Ammonium sulfate (5 mM) had no effect on nitrate reductase activity during a 3 hr dark incubation, but the enzyme was increased 2.5-fold during a subsequent 24 hr incubation of the maize leaves in light. The enzyme activity induced by ammonium ion declined at a slower rate under non-inducing conditions than that induced by nitrate. The decline in ammonium stimulated enzyme activity in the dark was also slower than that with nitrate. Further. cycloheximide accelerated the dark inactivation of the ammonium-enzyme while it had no effect on the nitrate-enzyme. The experiments demonstrate that increase in nitrate reductase activity by ammonium ion is different from the action of nitrate action.     

The influence of sugars on nitrate reductase induction by exogenous nitrate or nitrite in excised pisum sativum roots

Nitrate reductase (NR) induction is enhanced by exogenously supplied sucrose in excised pea roots exposed to both exogenous nitrate and exogenous nitrite. NR synthesis is preferentially supported by sugars transported to the cells at the moment, however NR induction can take place for some time without exogenous sugar influx if roots are saturated with sugars during precultivation. Steady high NR levels are dependent on steady sugar and nitrate influxes. NR induction is low in roots precultivated for 20 h without sucrose although sugar content is still high in them. This suggests that compartmentation of sugars in the cells is of major importance during NR induction. Total nitrate content in roots exposed to nitrate is not influenced by sucrose supplied together with nitrate. Some nitrite is oxidized to nitrate in roots exposed to exogenous nitrite ; we assume that this nitrate is responsible for NR induction. Our results indicate that sugars, besides many indirect effects on NR induction, may also directly influence NR synthesis either as coinducers or as derepressors of NR synthesis. Our results further show that NR is not a product-inducible enzyme.     

Kinetic studies of the induction of nitrate reductase and cytochrome c reductase in the fungus Aspergillus nidulans

In an earlier paper (Cove, 1966) it was reported that the kinetics of appearance of nitrate reductase (NADPH–nitrate oxidoreductase, EC 1.6.6.3) on the addition of nitrate to a growing culture of Aspergillus nidulans were different in certain respects from those found for many Escherichia coli enzymes. When urea is used as an initial nitrogen source, a further difference is found: enzyme synthesis is no longer continuous. This interruption of synthesis does not appear to be due to synchronous cell division in the culture, nor to be due to accumulation of ammonia. Fluctuations in the intracellular concentration of nitrate, though appearing to be partly responsible for the discontinuity of enzyme syntheses, cannot account for all the observations. Two related hypotheses are put forward to explain this discontinuity of synthesis; each suggests that nitrate reductase is intimately concerned with its own synthesis. One possibility is that the enzyme when it is not in the form of a complex with nitrate is a co-repressor of its own synthesis, and the other that the enzyme is its own repressor.     

Intercellular localization of nitrate reductase in roots

Experiments were conducted with segments of corn roots to investigate whether nitrate reductase (NR) is compartmentalized in particular groups of cells that collectively form the root symplastic pathway. A microsurgical technique was used to separate cells of the epidermis, of the cortex, and of the stele. The presence of NR was determined using in vitro and enzyme-linked immunosorbent assays. In roots exposed to 0.2 millimolar NO₃⁻ for 20 hours, NR was detected almost exclusively in epidermal cells, even though substantial amounts of NO₃⁻ likely were being transported through cortical and steler cells during transit to the vascular system. Although NR was present in all cell groups of roots exposed to 20.0 millimolar NO₃⁻, the majority of the NR still was contained in epidermal cells. The results are consistent with previous observations indicating that limited reduction of endogenous NO₃⁻ occurs during uptake and reduction of exogenous NO₃⁻. Several mechanisms are advanced to account for the restricted capacity of cortical and stelar cells to induce NR and reduce NO₃⁻. It is postulated that (a) the biochemical system involved in the induction of NR in the cortex and stele is relatively insensitive to the presence of NO₃⁻, (b) the receptor for the NR induction response and the NR protein are associated with cell plasmalemmae and little NO₃⁻ is taken up by cells of the cortex and stele, and/or (c) NO₃⁻ is compartmentalized during transport through the symplasm, which limits exposure for induction of NR and NO₃⁻ reduction.