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.     

Increase in Nitrate Reductase Activity in Bean Leaves by Light Involves a Regulator Protein

Nitrate reductase activity, assayed either in vivo or in vitro was considerably higher in bean (Phaseolus vulgaris L.) leaves from 7-day-old light grown seedlings than those from dark grown, both in the absence as well as presence of nitrate. Cytochrome c reductase activity was however similar in both regimes, while peroxidase was lower in light than in dark. The light stimulated increase in nitrate reductase activity in leaf segments from dark grown seedlings was inhibited by cycloheximide, DNP, chloramphenicol, and sodium tungstate and was unaffected by lincomycin and DCMU. Under similar conditions, the increase in total chlorophyll was inhibited completely by cycloheximide and DNP, partially by chloramphenicol and lincomycin, and was unaffected by tungstate and DCMU. A supply of 1~5 mm reduced glutathione increased enzyme activity in the dark and also to some extent in light. The substrate induction of enzyme activity started after a lag of one hr in light or dark and continued for either 5 hr in the dark or 8 hr in light. Two proteinaceous inhibitors (Factors I and II) of nitrate reductase were isolated by ammonium sulfate precipitation and Sephadex gel filtration. The amount of Factor I was higher in the dark than in light. The amount and activity of Factor II was however, almost equal in light and dark. The inhibition of enzyme activity by these inhibitors increased with their concentration. It is proposed that light increases nitrate reductase activity by decreasing the amount of a nitrate reductase inhibitor.     

Some New Aspects of the in Vivo Assay for Nitrate Reductase in Wheat (Triticum aestivum L.) Leaves: I. REEVALUATION OF NITRATE POOL SIZES

Experiments were carried out to clarify problems encountered in measuring metabolic and storage pool sizes of nitrate in wheat leaf sections with an in vivo nitrate reductase assay. The leaf sections were from seedlings grown on 15 millimolar nitrate. Data obtained show that the cessation of nitrite accumulation, used as an index of the active nitrate pool size, could be caused by lack of anaerobiosis in the assay system, the lack of energy for nitrate reduction, or a loss of nitrate reductase activity. Availability of nitrate was never the limiting factor in this system. It is concluded that pool sizes of nitrate cannot be determined in wheat leaves with the in vivo assays employed.     

Differential effect of irradiance and nutrient nitrate on the relationship of in vivo and in vitro nitrate reductase assay in chlorophyllous tissues

Growth at increasing continuous irradiance (at high nutrient nitrate) and nutrient nitrate concentrations (at high continuous irradiance) furnished increases in the in vivo and in vitro nitrate reductase activities of corn (Zea mays L.), field peas (Pisum arvense L.), wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), and globe amaranth (Gomphrena globosa L.) leaves and of marrow (Cucurbita pepo L.) cotyledons. Ratios of in vivo to in vitro activity declined exponentially in all species with increasing nitrate reductase levels promoted by nutrient nitrate. The ratios were more nearly independent of nitrate reductase levels generated by adjusting the irradiance; major exceptions were marrow and wheat at low (1.5 klux and less) irradiances and peas throughout the irradiance range, where decreases in the ratio were accompanied by increases in in situ nitrate concentration. The ratio also increased at the highest irradiance (39.2 klux) in wheat and barley, associated with a decline of in vitro nitrate reductase. These differences in response to irradiance and nutrient nitrate indicate that the in vivo assay does not provide a simple measure of nitrate reductase but rather yields a more composite measure of nitrate reduction, possibly related both to nitrate reductase level and to the supply of reductant for in vivo activity.     

Increase in NADH-glutamate dehydrogenase activity by mercury in excised bean leaf segments

Application of Hg to excised bean leaf segments increased the glutamate dehydrogenase (NADH-GDH) activity substantially. However, specific activity of the enzyme decreased at lower concentration of Hg, and increased to lesser extent at higher concentration of Hg. Mercury supply increased the glutamate synthase (NADH-GOGAT) activity also. Mercury supply increased the NADH-GDH activity in the presence of NH4NO3, but to a lesser extent than in the absence of NH4NO3. The specific activity of the enzyme decreased considerably at lower concentration of Hg, but increased significantly at higher concentration of Hg. An increase in NADH-GOGAT activity was observed in the presence of NH4NO3, but specific activity of the enzyme decreased marginally. Increase in GDH activity due to Hg remained unaffected by the supply of sucrose, but was reduced by glutamine and glutathione and enhanced by Al. The glutamate dehydrogenase (+Hg enzyme) from mercury treated leaf segments had higher value of S0.5 for NADH than the enzyme (-Hg enzyme) from material not treated with mercury indicating that Hg binding to enzyme prevented NADH binding to the enzyme possibly at thiol groups. However, + Hg enzyme has more reactivity, as apparent Vmax value was higher for it. It has been suggested that Hg activates the NADH-GDH enzyme in the bean leaf segments by binding to thiol groups of protein and pronounced increase in activity by Hg suggests a possible role of enzyme under Hg-stress.     

Control of nitrate and nitrite assimilation by carbohydrate reserves,adenosine nucleotides and pyridine nucleotides in leaves of Zea mays L. under dark conditions

The rate of in-vivo nitrate reduction by leaf segments of Zea mays L. was found to decline during the second hour of dark anaerobic treatment. On transfer to oxygen the capacity to reduce nitrate under dark conditions was restored. These observations led to the proposal that nitrate reductase is a regulatory enzyme with ADP acting as a negative effector. The effect of ADP on the invitro activity of nitrate reductase and the changes in the in-vivo adenylate pool under dark-N₂ and dark-O₂ were investigated. It was found that ADP inhibited the activity of partially purified nitrate reductase. Similarly, the in-vivo anaerobic inhibition of nitrate reduction was associated with a build-up of ADP in the leaf tissue. Under anaerobic conditions nitrite accumulated and on transfer to oxygen the accumulated nitrite was reduced. To explain this phenomenon the following hypothesis was proposed and tested. Under anaerobic conditions the supply of reducing equivalents for nitrite reduction in the plastid becomes restricted and nitrite accumulates as a consequence. On transfer to oxygen this restriction is removed and nitrite disappears. This capacity to reduce accumulated nitrite was found to be dependent on the carbohydrate status of the leaf tissue.     

Specificity for nicotinamide adenine dinucleotide by nitrate reductase from leaves

Preliminary work revealed that nitrate reductase in crude extracts prepared from leaves of certain corn genotypes as well as soybeans could utilize NADPH as well as NADH as the electron donor. Isoelectric focusing and diethylaminoethyl cellulose chromatography confirmed previous findings that NADH and NADPH activities could not be separated, which suggests the involvement of a single enzyme. Nitrate reduction with both cofactors varies with plant species, plant age, and assay conditions. The ability of the nitrate reductase from a given genotype to utilize NADPH was associated with the amount of NADPH-phosphatase in the extract. While diethylaminoethyl cellulose chromatography of plant extracts separated nitrate reductase from the bulk (90%) of the phosphatase and caused a decrease in the NADPH activity, the residual level of phosphatase was sufficient to account for the apparent NADPH nitrate reductase activity. Addition of KH₂PO₄ and KF, inhibitors of NADPH-phosphatase activity in in vitro assays, caused a drastic reduction or abolishment of NADPH-mediated nitrate reductase activity but were without effect on NADH nitrate reductase activity. It is concluded that NADPH-nitrate reduction, in soybean and certain corn genotypes, is an artifact resulting from the conversion of NADPH to NADH by a phosphatase and that the enzyme in leaf tissue is NADH-dependent (E.C.1.6.6.1).     

Biochemical Characterization of Soybean Mutants Lacking Constitutive NADH:Nitrate Reductase

Two nitrate reductase (NR) mutants were selected for low nitrate reductase (LNR) activity by in vivo NR microassays of M₂ seedlings derived from nitrosomethylurea-mutagenized soybean (Glycine max [L.] Merr. cv Williams) seeds. The mutants (LNR-5 and LNR-6) appeared to have normal nitrate-inducible NR activity. Both mutants, however, showed decreased NR activity in vivo and in vitro compared with the wild-type. In vitro FMNH₂-dependent nitrate reduction and Cyt c reductase activity of nitrate-grown plants, and nitrogenous gas evolution during in vivo NR assays of urea-grown plants, were also decreased in the mutants. The latter observation was due to insufficient generation of nitrite substrate, rather than some inherent difference in enzyme between mutant and wild-type plants. When grown on urea, crude extracts of LNR-5 and LNR-6 lines had similar NADPH:NR activities to that of the wild type, but both mutants had very little NADH:NR activity, relative to the wild type. Blue Sepharose columns loaded with NR extract of urea-grown mutants and sequentially eluted with NADPH and NADH yielded a NADPH:NR peak only, while the wild-type yielded both NADPH: and NADH:NR peaks. Activity profiles confirmed the lack of constitutive NADH:NR in the mutants throughout development. The results provide additional support to our claim that wild-type soybean contains three NR isozymes, namely, constitutive NADPH:NR (c₁NR), constitutive NADH:NR (c₂NR), and nitrate-inducible NR (iNR).     

A Re-evaluation of the Nitrate Reductase Content of the Maize Root

The standard procedure for the in ritro extraction of nitrate reductase from the tip region (0-2 cm) of the primary root of the maize (Zea mays L.) seedling indicated an activity of the enzyme approximately 5-fold higher than that obtained with an in vivo assay. In more mature regions of the primary root the ratio of in vitro to in vivo activity was much lower and in older seedlings was less than unity. The mature root extracts had a more labile nitrate reductase and a higher level of an inactivating enzyme. The use of phenylmethylsulphonyl fluoride in the extraction medium gave only a partial protection of the nitrate reductase from the old root samples. Casein (3%) resulted in a greatly increased yield of nitrate reductase (36-fold with one sample) and a more constant in vitro-in vivo activity ratio for all root samples. With casein in the extraction medium, much higher levels of nitrate reductase were recovered from the mature root zone, and the root content of this enzyme was now shown to be quite a significant proportion of the total in the maize seedling. Casein was shown to inhibit the action of the inactivating enzyme on nitrate reductase. Evidence is also presented for a nitrate reductase inactivating enzyme in the maize scutella and leaf tissues and in the roots and shoots of pea seedlings.     

Mitochondrial Complex I Impairment and Nitrate Reductase Activity in Leaves of Nicotiana sylvestris

Loss of major mitochondrial complex I subunit Nad 7 in the leaves of CMS II mutant of Nicotiana sylvestris caused increase in both in vivo and in vitro nitrate reductase activities and the per cent activation state of NR was also higher in CMS II as against wild type. The differences suggest a possibility for export of mitochondrial NADH due to impairment of complex I, a major and high affinity sink for NADH. Loss of complex I subunit also resulted in constitutive expression of alternate oxidase (Aox) thereby providing a mechanism for continuous supply of carbon skeletons required for nitrate assimilation. The possibility of close coordination between mitochondrial redox perturbation and nitrate reduction and its further assimilation is discussed.     

Expression in Escherichia coli of Cytochrome c Reductase Activity from a Maize NADH:Nitrate Reductase Complementary DNA

A cDNA clone was isolated from a maize (Zea mays L. cv W64A×W183E) scutellum λgt11 library using maize leaf NADH:nitrate reductase Zmnr1 cDNA clone as a hybridization probe; it was designated Zmnr1S. Zmnr1S was shown to be an NADH:nitrate reductase clone by nucleotide sequencing and comparison of its deduced amino acid sequence to Zmnr1. Zmnr1S, which is 1.8 kilobases in length and contains the code for both the cytochrome b and flavin adenine dinucleotide domains of nitrate reductase, was cloned into the EcoRI site of the Escherichia coli expression vector pET5b and expressed. The cell lysate contained NADH:cytochrome c reductase activity, which is a characteristic partial activity of NADH:nitrate reductase dependent on the cytochrome b and flavin adenine dinucleotide domains. Recombinant cytochrome c reductase was purified by immunoaffinity chromatography on monoclonal antibody Zm2(69) Sepharose. The purified cytochrome c reductase, which had a major size of 43 kilodaltons, was inhibited by polyclonal antibodies for maize leaf NADH:nitrate reductase and bound these antibodies when blotted to nitrocellulose. Ultraviolet and visible spectra of oxidized and NADH-reduced recombinant cytochrome c reductase were nearly identical with those of maize leaf NADH:nitrate reductase. These two enzyme forms also had very similar kinetic properties with respect to NADH-dependent cytochrome c and ferricyanide reduction.     

Effect of multiple factor source-sink manipulation on nitrogen and carbon assimilation by soybean

The objectives of this study were to determine the effect of light enhancement and hastened reproductive development on nitrogen and dry matter accumulation by field-grown soybean (Glycine max [L.] Merr.). The impacts of photosynthate supply and reproductive development on change in the season-long profiles of in vivo leaf nitrate reductase (NR) activity and root nodule acetylene reduction (AR) activity were evaluated.

Light enhancement resulted in significant increases in dry matter accumulation, root nodule fresh weight and AR activity. Seed yield was increased in both light enhanced treatments in 1978 and in one in 1979.

Hastened flowering and seed development was accomplished through photoperiod manipulation within a single genotype. Seasonal decline in leaf NR activity was most rapid in plants entering reproductive development early. An early increase in root nodule fresh weight and AR activity was also observed in response to this treatment and was followed similarly by early decline.

The addition of high levels of soil-applied nitrogen increased leaf NR activity and delayed late season decline in NR activity for both control and early reproductive plants. Nitrate supply was therefore implicated as limiting to leaf NR activity during the decline associated with flowering and early seed development. A limited additional increase in leaf NR activity was observed in response to light enhancement plus soil-applied nitrogen. As no significant increase in leaf NR activity was observed in response to light enhancement alone, leaf nitrate supply was further implicated as more limiting to leaf NR activity than was photosynthate supply during flowering and early seed development.

     

In vivo nitrate reductase activity in dark- and light-grown sugarcane callus

The in vivo nitrate reductase activity in 8 day old dark-grown sugarcane callus was over three fold that of the light-grown callus. NADH (0.3 mM) in the reaction system, increased the in vivo nitrate reductase activity by more than two fold both in the dark- and the light-grown callus tissues. The NADH dependence of nitrate reductase activity followed Michaelian kinetics. The apparent Km values for NADH were 0.083 mM and 0.20 mM, respectively, for the dark- and the light-grown callus. In vivo nitrate reductase activity in green sugarcane leaves (field grown) was unaffected by NADH in the reaction system. Under the standard conditions of assay up to 60% of the NADH penetrated into the sugarcane callus within 2 min. No penetration of NADH into the sugarcane leaf discs was, however, recorded under identical conditions.NCL Communication No. 3454     

Purification and Characterization of NAD(P)H:Nitrate Reductase and NADH:Nitrate Reductase from Corn Roots

The nitrate reductase activity of 5-day-old whole corn roots was isolated using phosphate buffer. The relatively stable nitrate reductase extract can be separated into three fractions using affinity chromatography on blue-Sepharose. The first fraction, eluted with NADPH, reduces nearly equal amounts of nitrate with either NADPH or NADH. A subsequent elution with NADH yields a nitrate reductase which is more active with NADH as electron donor. Further elution with salt gives a nitrate reductase fraction which is active with both NADH and NADPH, but is more active with NADH. All three nitrate reductase fractions have pH optima of 7.5 and Stokes radii of about 6.0 nanometers. The NADPH-eluted enzyme has a nitrate K_m of 0.3 millimolar in the presence of NADPH, whereas the NADH-eluted enzyme has a nitrate K_m of 0.07 millimolar in the presence of NADH. The NADPH-eluted fraction appears to be similar to the NAD(P)H:nitrate reductase isolated from corn scutellum and the NADH-eluted fraction is similar to the NADH:nitrate reductases isolated from corn leaf and scutellum. The salt-eluted fraction appears to be a mixture of NAD(P)H: and NADH:nitrate reductases.     

Characteristics of a Nitrate Reductase in a Barley Mutant Deficient in NADH Nitrate Reductase

A barley (Hordeum vulgare L.) mutant, nar1a (formerly Az12), deficient in NADH nitrate reductase activity is, nevertheless, capable of growth with nitrate as the sole nitrogen source. In an attempt to identify the mechanism(s) of nitrate reduction in the mutant, nitrate reductase from nar1a was characterized to determine whether the residual activity is due to a leaky mutation or to the presence of a second nitrate reductase. The results obtained indicate that the nitrate reductase in nar1a differs from the wild-type enzyme in several important aspects. The pH optima for both the NADH and the NADPH nitrate reductase activities from nar1a were approximately pH 7.7, which is slightly greater than the pH 7.5 optimum for the NADH activity and considerably greater than the pH 6.0 to 6.5 optimum for the NADPH activity of the wild-type enzyme. The nitrate reductase from nar1a exhibits greater NADPH than NADH activity and has apparent K_m values for nitrate and NADH that are approximately 10 times greater than those of the wild-type enzyme. The nar1a nitrate reductase has apparent K_m values of 170 micromolar for NADPH and 110 micromolar for NADH. NADPH, but not NADH, inhibited the enzyme at concentrations greater than 50 micromolar.     

Oxygen Inhibition of Nitrate Reductase Biosynthesis in Detached Corn Leaves via Inhibition of Total Soluble Protein Synthesis

Detached first leaves of 3-day-old corn seedlings (Zea mays L. W64AxW183E) were incubated with nitrate in air or 100% O₂ in the light. Nitrate accumulation in the leaves was not depressed by O₂. NADH:nitrate reductase activity and enzyme protein, as measured with an enzyme-linked immunosorbent assay, increased in parallel during the 8 h nitrate treatment in air, but in O₂ the levels of enzyme activity and protein were depressed. NADH:nitrate reductase mRNA levels were the same in the air-and O₂-treated leaves. Total soluble protein levels in leaves were slightly depressed by O₂ and shifting from O₂ to an air environment increased the protein level. Incorporation of [³⁵S]methionine during nitrate treatment revealed that total soluble protein and nitrate reductase protein synthesis were both depressed by the O₂ environment relative to air, but both recovered when leaves were shifted from O₂ to air. Although O₂ accelerated inactivation of nitrate reductase in vitro, the in vivo inactivation rate appeared to be too low to account for the depressed level of nitrate reductase activity in O₂-treated leaves. We concluded that O₂ inhibition of nitrate reductase biosynthesis in detached corn leaves was largely due to inhibition of total soluble protein synthesis at the level of translation.     

Limitations on Leaf Nitrate Reductase Activity during Flowering and Podfill in Soybean

The objective of this study was to identify factors which limit leaf nitrate reductase (NR) activity as decline occurs during flowering and beginning seed development in soybean (Glycine max [L.] Merr. cv Clark). Level of NR enzyme activity, level of reductant, and availability of NO₃⁻ as substrate were evaluated for field-grown soybean from flowering through leaf senescence. Timing of reproductive development was altered within one genotype by (a) exposure of Clark to an artificially short photoperiod to hasten flowering and podfill, and (b) the use of an early flowering isoline. Nitrogen (N) was soil-applied to selected plots at 500 kilograms per hectare as an additional variable. Stem NO₃⁻ concentration and in vivo leaf NR activity were significantly correlated (R² = 0.69 with nitrate in the assay medium and 0.74 without nitrate in the medium at P = 0.001) across six combinations of reproductive and soil N-treatment. The supply of NO₃⁻ from the root to the leaf tissue was the primary limitation to leaf NR activity during flowering and podfill. Levels of NR enzyme and reductant were not limiting to leaf NR activity during this period.     

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

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

Leaf Nitrate Reductase, d-Ribulose-1,5-bisphosphate Carboxylase, and Root Nodule Development of Genetic Male-Sterile and Fertile Soybean Isolines

The objectives of this study were to determine the effect of pod and seed development on leaf chlorophyll concentration, and on activities of leaf ribulose bisphosphate carboxylase, leaf nitrate reductase, and root nodule acetylene reduction in field-grown soybean (Glycine max [L.] Merr.). Two genetic male-sterile lines and their fertile counterparts (Williams and Clark 63) were compared in both 1978 and 1979. Two additional lines (Wells × Beeson and Wells × Corsoy) were compared in 1979.

The expression of male-sterile character was nearly complete as very little outcrossing due to insect pollinators was observed. Male-sterile plants showed a delayed late season decline in leaf chlorophyll content and ribulose bisphosphate carboxylase activity when compared with fertile plants. A slight delay in the loss of in vivo leaf nitrate reductase activity was also observed for male-sterile plants. Root nodule fresh weight and acetylene reduction activity declined slightly more rapidly for fertile lines than for male-sterile lines in both years with differences significant on the last two to three sampling dates as leaf loss occurred in the control plants.

Seed development was found to increase slightly, the rate of decline of metabolic activity in fertile lines compared with that of male-sterile lines. However, pod development was not an a priori requirement for leaf and root nodule senescence. Male-sterile plants also lost photosynthetic and nitrogen metabolic competence, but at a slower rate. These results support the concept that pod and seed development does not signal monocarpic senescence per se but rather affects the rate at which senescence occurs after flowering.

     

Regulation of nitrite reductase : cell-free translation and processing

A protein with molecular mass of 67 kilodaltons is immunoprecipitated from in vitro translated products obtained from rabbit reticulocyte lysate primed with polyadenylated RNA from nitrate treated illuminated pea seedlings. This protein resembles the native nitrite reductase because of its competitive elimination when immunoprecipitation of in vitro translated products was carried out in the presence of cold unlabeled nitrite reductase or in vivo labeled pea leaf extract. This protein is of slightly higher molecular weight than that of the native nitrite reductase. Proteinaceous extracts from chloroplasts convert the in vitro product to the same molecular weight as the native peptide. The conversion appears to occur in two steps. Polyadenylated RNA from nitrate deficient plants or from nitrate-treated plants transferred to darkness do not support the synthesis of nitrite reductase. It is concluded that nitrate and light modulate the synthesis of the enzyme nitrite reductase by regulating the availability of mRNA for the enzyme.