Nitrate reductase from Dunaliella tertiolecta, purification and properties

The purification and properties of a nitrate reductase fromthe green alga Dunaliella tertiolecta are described. The enzymeis soluble, with a molecular weight greater than 500,000 andhas Km values of 0.26, 0.18, 0.10 and 0.06 m for NO₃^–,NADH, NADPH and FADH₂ respectively. Even at the highest specificactivity obtained, (0.86 µmoles NO₃^– reduced min^–1mg protein^–1) the enzyme retains the capacity to acceptelectrons from both NADH and NADPH. Unlike other nitrate reductasesit does not appear to be able to use reduced viologens as electrondonors. Its other properties are consistent with its being amolybdoflavoprotein of high molecular weight, which is alsoable to function as a cytochrome C reductase. ¹ Supported in part by the National Research Council of Canada. (Received June 18, 1972; )     

The Partitioning of Nitrate Assimilation Between Root and Shoot of a Range of Temperate Cereals and Pasture Grasses

Nitrate reductase activity (NRA, in vivo assay) and nitrate(NO^-₃) content of root and shoot and NO^-₃ and reduced nitrogencontent of xylem sap were measured in five temperate cerealssupplied with a range of NO^-₃ concentrations (0·1–20mol m^–3) and three temperate pasture grasses suppliedwith 0·5 or 5 0 mol m^–3 NO^-₃ For one cereal (Hordeumvulgare L ), in vitro NRA was also determined The effect ofexternal NO^-₃ concentration on the partitioning of NO^-₃ assimilationbetween root and shoot was assessed All measurements indicatedthat the root was the major site of NO3 assimilation in Avenasatwa L, Hordeum vulgare L, Secale cereale L, Tnticum aestivumL and x Triticosecale Wittm supplied with 0·1 to 1·0mol m^–3 NO^-₃ and that for all cereals, shoot assimilationincreased in importance as applied NO^-₃ concentration increasedfrom 1.0 to 20 mol m^–3 At 5.0–20 mol m^–3 NO3,the data indicated that the shoot played an important if notmajor role in NO^-₃ assimilation in all cereals studied Measurementson Lolium multiflorum Lam and L perenne L indicated that theroot was the main site of NO^-₃ assimilation at 0.5 mol m^–3NO^-₃ but shoot assimilation was predominant at 5.0 mol m^–3NO^-₃ Both NRA distribution data and xylem sap analysis indicatedthat shoot assimilation was predominant in Dactylis glomerataL supplied with 0.5 or 5.0 mol m^–3 NO^-₃ Avena sativa L., oats, Hordeum vulgare L., barley, Secale cereale L., rye, x Triticosecale Wittm., triticale, Triticum aestivum L., wheat, Dactylis glomerata L., cocksfoot, Lolium multiflorum Lam., Italian ryegrass, Lolium perenne L., perennial ryegrass, nitrate, nitrate assimilation, nitrate reductase activity, xylem sap     

The Apparent Induction of Nitrate Uptake by Chara corallina Cells following Pretreatment with or without Nitrate and Chlorate

Nitrate provision has been found to regulate the capacity forChara corallina cells to take up nitrate. When nitrate was suppliedto N sufficient cells maximum nitrate uptake was reached after8 h. Prolonged treatment of the cells in the absence of N alsoresulted in the apparent ability of these cells to take up nitrate.Chlorate was found to substitute partially for nitrate in the‘induction’ step. The effects on nitrate reductionwere separated from those on nitrate uptake by experiments usingtungstate. Tungstate pretreatment had no effect on NO^–₃uptake ‘induced’ by N starvation, but inhibitedNO^–₃ uptake associated with NO^–₃ pretreatment. Chloridepretreatment similarly had no effect on NO^–₃ uptake ‘induced’by N deprivation, but inhibited NO^–₃ uptake followingNO^–₃ pretreatment. The data suggest that there are atleast two mechanisms responsible for the ‘induction’of nitrate uptake by Chara cells, one associated with NO^–₃reduction and ‘induced’ by CIO^–₃ or NO^–₃and one associated with N deprivation. Key words: Nitrate, Chlorate, Chara corallina, Induction     

Effect of Plant Growth Regulators on Nitrate Utilization by Roots of Nitrogen-Depleted Dwarf Bean

We examined the effect of pretreatments (18 h at 5 µmoldm^–3) with abscisic acid, the ethylene-releasing substance‘Ethephon’, gibberellic acid, indoleacetic acid,kinetin and zeatin on nitrate uptake and in vivo nitrate reductaseactivity (NRA) in roots of nitrogen-depleted Phaseolus vulgarisL. Nitrate uptake showed an apparent induction pattern witha steady state after about 6 h, in all treatments. The nitrateuptake rate after 6 h was unaffected or at most 30% lower aftertreatments with the plant growth regulators. Gibberellic acid, kinetin and zeatin induced substantial NRAin roots in the absence of nitrate, whereas Ethephon enhancedNRA only during nitrate nutrition. Kinetin-induced NRA (Ki-NRA)was maximal after a pretreatment at 1 µmol dm^–3,and showed a lag phase of 6–8 h. Ki-NRA was additive tonitrate-induced NRA (NO^–₃-NRA) for at least 24 h, independentof the induction sequence. After full induction, Ki-NRA approximated20% of NO-₃-NRA. Abscisic acid counteracted the developmentof Ki-NRA, but not of NO^–₃-NRA. Cycloheximide and tungstatewere equally effective to suppress the development of nitratereductase activity after supply of kinetin or NO^–₃. Our data are consistent with the operation of two independentenzyme fractions (Ki-NRA and NO^–₃-NRA) with apparentlyidentical properties but with separate control mechanisms. Theabsence of major effects of plant growth regulators on the time-courseand rate of nitrate uptake suggests that exogenous regulators,and possibly endogenous phytohormones are of minor importancefor initial nitrate uptake. The differential effect of someregulators on nitrate uptake and root NRA furthermore indicatesthat the processes of uptake and reduction of NO^–₃ arenot obligatory or exclusively coupled to each other.     

The Effect of Abscisic Acid on Amino Nitrogen and Protein Content of Potato Plants in Relation to the Inhibition of Nitrate Reductase Activity

Treatment of potato plants grown in nutrient solution with 3.8µM ABA resulted in reduced soluble protein in roots andin leaves at 24 h, but not in stems. This treatment reducedin vivo nitrate reductase activity in all organs for about 48h with the most pronounced reduction occurring in the roots.Excised root and leaf segments from plants treated with ABAfor 24, 48 and 72 h absorbed significantly more ¹⁴C leucine,compared to the control but the percent incorporation into proteinwas not altered in roots. In response to ABA total free amino nitrogen in leaves was lowerat 5 and 72 h and in stems at 72 h. Amino nitrogen content ofroots was enhanced by ABA at 5, 24 and 72 h due to generallyhigher levels of aspartate, serine, glutamate, proline and ammonia.There was no consistent relationship between ABA suppressionof nitrate reductase activity and ammonia or specific aminoacid (except proline) levels in leaves and stems. The increasedfree amino nitrogen levels in response to the hormone may bethe result of impaired NO₃– reduction rather than thecause. The results of protein synthesis studies and solubleprotein content suggest that ABA inhibition of nitrate reductaseis not due to general inhibition of protein synthesis and mayinvolve specific inhibition of nitrate reductase protein synthesis. ¹ Contribution No. 684, Department of Plant Science, Universityof Manitoba.     

The Distribution of Nitrate Assimilation Between Root and Shoot in Vicia faba L.

Nitrate assimilation was examined in two cultivars (Banner Winterand Herz Freya) of Vicia faba L. supplied with a range of nitrateconcentrations. The distribution between root and shoot wasassessed. The cultivars showed responses to increased applied nitrateconcentration. Total plant dry weight and carbon content remainedconstant while shoot: root dry weight ratio, total plant nitrogen,total plant leaf area and specific leaf area (SLA) all increased.The proportion of total plant nitrate and nitrate reductase(NR) activity found in the shoot of both cultivars increasedwith applied nitrate concentrations as did NO₃^–: Kjeldahl-Nratios of xylem sap. The cultivars differed in that a greaterproportion of total plant NR activity occurred in the shootof cv. Herz Freya at all applied nitrate concentrations, andits xylem sap NO₃^–: Kjeldahl-N ratio and SLA were consistentlygreater. It is concluded that the distribution of nitrate assimilationbetween root and shoot of V. faba varies both with cultivarand with external nitrate concentration. Vicia faba L., field bean, nitrate assimilation, nitrate reductase, xylem sap analysis     

Nitrate reductase inactivating factor from rice seedlings

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

Studies of the Uptake of Nitrate in Barley: III. COMPARTMENTATION OF NO3-

Compartmental analyses of intact roots of barley (Hordeum vulgareL. cv. Klondike) plants, grown with different levels of NO₃^–(up to 1·0 mol m^–3) in the external media, wereundertaken using ¹³NO₃^–. Two additional treatments, namelysodium dodecyl sulphate (SDS) or brief exposure to high temperature,designed to investigate the identity of the three NO₃^–compartments revealed by compartmental analyses, provided supportfor the identification of the latter as corresponding to superficialsolution, apoplasm, and cytoplasm. Half-lives for exchange ofthese compartments, 3 s, 30 s, and 7 mm, were unaffected bythe level of NO₃^– provided during growth. Independentestimates of ¹³NO₃^– fluxes obtained by direct methodsagreed well with values of fluxes calculated from the compartmentalanalyses. Cytoplasmic [NO₃^–], estimated from the compartmental analyses,were in the range from 1–37 mol m^–3, and increasedwith increasing [NO₃^–] of the medium. Such values forcytoplasmic [NO₃^–] are inconsistent with an earlier proposal(Siddiqi, Glass, Ruth, and Rufty, 1990; Glass, Siddiqi, Ruth,and Rufty, 1990) of passive NO₃^– uptake in the concentrationrange above 10 mol m^–3. A model, based upon localizeddistribution of nitrate reductase activity in epidermal cells,is proposed in which the proposed passive low affinity NO uptakeat high external [NO₃^–] is restricted to epidermal cells. During loading periods with ¹³NO₃^–, significant amountsof ¹³N were translocated to the shoot. Two pools of ¹³N, onebeing the root symplasm, appear to participate in the transferof labelled N to the shoot. Key words: Barley, compartmentation, nitrate, nitrate reductase, ¹³N     

In vivo Nitrate Reductase Activity in Barley (Hordeum vulgare L.) during Ear Development

Experiments were conducted during the 1974–75 and 1975–76winter season with the barley (Hordeum vulgare L.) cultivarJyoti. From amongst the various plant parts, the flag leaf bladehad higher in vivo nitrate reductase (NR) activity than thelower two leaf blades, glumes, and grains. However, the potentialof a plant part to reduce NO₃^– is a function of its freshweight and the NR per unit fresh weight. On this basis, thesecond and third leaf blades could reduce more NO₃^– thanthe flag leaf blade. N fertilizer application resulted in enhancementof the activity of the leaf blades alone. N fertilizer appliedduring the reproductive phase was taken up and assimilated bythe various plant parts. The studies suggest that, even whenthe fertilizer is applied at optimum levels for obtaining maximumyields, the upper leaf blades have sub-optimal NR activity andthat there is a likelihood of either a preferential flow ofNO₃^– to the leaf blades or transnational barriers to NO₃^–movement to the ear.     

Effect of Nitrate, Ammonium and Some Amino Acids on Growth and Nitrate Reductase Activity in Suspension Cultures of Atropa belladonna

Growth and nitrate reductase activity (NRA) of Atropa belladonnacells were studied in medium supplemented with NaNO₃, NH₄NO₃,and amino acid precursors to tropane alkaloids. Growth and NRAwere stimulated by NH₄⁺ and by proline, by proline plus ornithine,but not by glutamate, in NO₃^–-containing medium. Testedamino acids inhibited neither utilization of inorganic nitrogennor growth. (Received September 30, 1988; Accepted August 28, 1989)     

Purification and Characterization of Assimilatory Nitrate Reductase from the Cyanobacterium Plectonema boryanum

Assimilatory nitrate reductase (NR) was solubilized by acetonetreatment from Plectonema boryanum and was purified 7,700-foldby heat treatment, ammonium sulfate fractionation and chromatographyon DEAE-Sephacel and Sephadex G-150. Purified NR had a specificactivity of 85 µmol NO₂^– formed min^–1 mg^–1protein. The enzyme retained both ferredoxin (Fd)- and methylviologen (MV)-linked NR activities throughout the purificationprocedure. Molecular weight was 80,000. The pH optimum was 10.5in the MV-assay and 8.5 when assayed with enzymatically reducedFd as the electron donor. Apparent Km values for nitrate andMV were 700 µM and 2,500µM in the MVassay and 55µM and 75 µM for nitrate and Fd in the Fd-assay.The enzyme was inhibited by thiol reagents and metal-chelatingreagents. (Received October 1, 1982; Accepted March 8, 1983)     

Nitrogen utilization by plant species from acid heathland soils: II. Growth and shoot/root partitioning of NO3- assimilation at constant low pH and varying NO3-/NH4+ ratio

The growth of four heathland species, two grasses (D. flexuosa,M. caerulea) and two dwarf shrubs (C. vulgaris, E. tetralix),was tested in solution culture at pH 4.0 with 2 mol m^–3N, varying the N0₃^–/NH₄⁺ ratio up to 40% nitrate. In addition,measurements of NRA, plant chemical composition, and biomassallocation were carried out on a complete N0₃^–/NH₄⁺ replacementseries up to 100% nitrate. With the exception of M. caerulea, the partial replacement ofNH₄⁺ by NO₃^– tended to enhance the plant's growth ratewhen compared to NH₄⁺ only. In contrast to the other species,D. flexuosa showed a very flexible response in biomass allocation:a gradual increase in the root weight ratio (RWR) with NO₃^–increasing from 0 to 100%. In the presence of NH₄⁺, grassesreduced nitrate in the shoot only; roots did not become involvedin the reduction of nitrate until zero ambient NH₄⁺. The dwarfshrubs, being species that assimilate N exclusively in theirroots, displayed an enhanced root NRA in the presence of nitrate;in contrast to the steady increase with increasing NO₃^–in Calluna roots, enzyme activity in Erica roots followed arather irregular pattern. Free nitrate accumulated in the tissuesof grasses only, and particularly in D. flexuosa. The relative uptake ratio for NO₃^– [(proportion of nitratein N uptake)/(proportion of nitrate in N supply)] was lowestin M. caerulea and highest in D. flexuosa. Whereas M. caeruleaand the dwarf shrubs always absorbed ammonium highly preferentially(relative uptake ratio for NO₃^– <0.20), D. flexuosashowed a strong preference for NO₃^– at low external nitrate(the relative uptake ratio for N0₃^– reaching a value of2.0 at 10% NO₃^–). The ecological significance of thisprominent high preference for NO₃^– at low NO₃^–/NH₄⁺ratio by D. flexuosa and its consequences for soil acidificationare briefly discussed. Key words: Ammonium, heathland lants, N0₃^–/NH₄⁺ ratio, nitrate, nitrate reductase activity, soil acidification, specific absorption rate     

Influence of abscisic acid on nitrogen partitioning, sucrose metabolism and nitrate reductase activity of chicory suspension cells

Batch suspension cultures of chicory cells (Cichorium intybusL. var. Witloof) possess a NADH-specific nitrate reductase activitythat peaks on day 3 of a 10 d growth cycle. When both nitrateand ammonium are used as nitrogen sources, chicory cells absorbnitrate irst. Ammonium uptake becomes predominant at day 3,even though NO₃^– was still present in the medium. Althoughabscisic acid impairs growth as well as ¹⁵NO₃^– uptakeand reduction, it promotes nitrate reductase activity as measuredboth in vivo and in vitro. Specific activity is 50% higher inABA-treated cells than in controls. These conflicting data maybe explained either in erms of nitrate reductase levels or bythe availability of reducing power and energy. Since NRA isgenerally controlled by the availability of the reducing power,the energy status of the cell, the adenylate nucleotide pools,were measured simultaneously with the carbohydrate levels withinthe cell and the growth medium. The energy charge was not modifiedduring the growth cycle, regardless of the rowth conditions.Yet ABA modified the intracellular carbohydrate metabolism andinhibited the acidic invertase, the sucrose synthase and thesucrose phosphate synthase activities. Modified assimilationrates of nitrate in chicory cells grown in the presence of ABA,were probably correlated to modified carbohydrate metabolismpathways leading to increased availability of reducing power,energy and C-skeletons. Key words: Abscisic acid, Cichorium intybus L, nitrate reductase, reductase, invertase, sucrose synthase, sucrose phosphate synthase     

Nitrate Effects on Pre-emergence Growth and Emergence Percentage of Wheat (Triticum aestivum L.) from Different Sowing Depths

The effects of a range of applied nitrate (NO₃^–) concentrations(0–20 mol m3) on germination and emergence percentageof Triticum aestivum L. cv. Otane were examined at 30, 60, 90and 120 mm sowing depths. Germination percentage was not affectedby either sowing depth or applied NO₃^– concentration whereasemergence percentage decreased with increased sowing depth regardlessof applied NO₃^– concentration. Nitrate did not affectemergence percentage at 30 mm sowing depth, but at 60 to 120mm depth, emergence percentage decreased sharply with an increasedapplied NO₃^– concentration of 0 to 1·0 mol m^–3then decreased only slightly with further increases in appliedNO₃^– of about 5·0 mol m^–3. Root and shoot growth, NO₃^– accumulation and nitrate reductaseactivity (NRA) of plants supplied with 0, 1·0 and 1·0mol m^–3 NO₃^– at a sowing depth of 60 mm were measuredprior to emergence. The coleoptile of all seedlings opened withinthe substrate. Prior to emergence from the substrate, shootextension growth was unaffected by additional NO₃^– butshoot fr. wt. and dry wt. were both greater at 1·0 and1·0 mol m^–3 NO₃^– than with zero NO₃^–.Root dry wt. was unaffected by NO₃^–. Nitrate concentrationand NRA in root and shoot were always low without NO₃^–.At 1·0 and 10 mol m3 NO₃^–, NO₃^– accumulatedin the root and shoot to concentrations substantially greaterthan that applied and caused the induction of NRA. Regardlessof the applied NO₃^– concentration, seedlings which failedto emerge still had substantial seed reserves one month afterplanting. Coleoptile length was substantially less for seedlingswhich did not emerge than for seedlings which emerged, but wasnot affected by NO₃^–. It is proposed that (a) decreasedemergence percentage with increased sowing depth was due tothe emergence of leaf I from the coleoptile within the substrateand (b) decreased emergence percentage with additional NO₃^–was due to the increased expansion of leaf 1 within the substrateresulting in greater folding and damage of the leaf. Key words: Triticum aestivwn L., nitrate, sowing depth, seedling growth, seedling emergence     

Regulation of Nitrate Reductase Activity by Nitrate during Light-Dark Transition in Detached Oat Leaves

In oat (Avena sativa L. cv. Suregrain) leaf segments, light-darkmodulation of nitrate reductase (NR) activity could be observedonly when segments were kept in –NO₃ conditions. We presenthere evidence that nitrate would regulate NR activity by modulatingthe phosphorylation status of the enzyme. (Received June 19, 1995; Accepted August 14, 1995)     

Nitrate Reduction by Cassava

Nitrate reductase (NR) was assayed in vivo in cassava (Manihotesculenta Crantz). Activity in the leaves ranged from 0 to 2.51µmole of NO₃^– reduced g^–1 h^–1, withno activity in the younger leaves (leaf 1 on top). NR activitywas localized in the sides and toward the tip of the lobes ofthe leaf. (Received December 10, 1985; Accepted April 8, 1986)     

Nitrate Effects on Growth of the First Four Main Stem Leaves of a Range of Temperate Cereals and Pasture Grasses

The effects of different applied NO₃^– concentrations onextension growth and final length and area of leaves 1–4of five cereals and six pasture grasses of temperate originwere examined. Increased applied NO₃^– in the range 0.1–0.5.0mol m^–3 caused decreased duration of growth but increasedgrowth rate and final length of leaves 2–4 of the cerealsAvena saliva, Hordeum vulgare, Secale cereale, x Triticosecaleand Triticum aestivum. For all cereals, increased NO₃^–resulted in increased area of leaves 1–4. Pasture grasseswere supplied either 0.5 or 50 mol m^–3 NO₃^–. Increasedapplied NO₃^– (0.5–5.0 mol m^–3) resulted indecreased duration of growth and increased growth rate and finalarea of leaves 1–4 of Bromus wiltdenowii, leaves 2–4ofFestuca arundinaceae and leaves 3 and 4 of Lolium muitiflorum.In addition, length of leaves 3 and 4 of B. witidenowii increasedwith increased NO₃^–. Increased NO₃^– resulted inincreased area of leaves 2–4 of Dactylis gtomerata andLolium perenne and leaves 3 and 4 of Phalaris aquaiica but hadno effect on extension growth of all three species. Avena sativa L, oat, Hordeum vulgare L, barley, Secale cereale L, rye, x Triticosecale Wittm, triticale, Triticum aestivum L, wheat, Bromus willdenowii Kunth, prairie grass, Dactylis gtomerata L, cocksfoot, Festuca arundinaceae Shreb, tall fescue, Lolium multijlorum Lam, Italian ryegrass, Lolium perenne L, perennial ryegrass, Phalaris aquatica L, nitrate, leaf extension, leaf expansion     

Nitrate Transport into Chara corallina Cells using 36ClO-3 as an Analogue for Nitrate: I. INTERACTION BETWEEN 36ClO-3 AND NO-3 AND CHARACTERIZATION OF 36CIO-3/NO-3 INFLUX

The use of chlorate as an analogue for NO^–₃ during nitrateuptake into Chara corallina cells has been investigated. NO^–₃inhibits ³⁶C1O^–₃ influx into Chara over the concentrationrange 0–1000 mmol m^–3. Lineweaver-Burke plots ofthe data are characteristic of competitive inhibition by NO^–3in the low concentration range (0–300 mmol m^–3 ClO^–₃)and apparent K_INO3 is 140 mmol m^–3 which is of a similarorder of magnitude as apparent K_mCIO3- 180 mmol m^–3. Athigher substrate concentrations the inhibition by NO^–₃was not characteristic of competitive or uncompetitive inhibition. ³⁶C1O^–₃/NO^–₃ influx was dependent on K⁺ and Ca²⁺in the external medium and inhibited by FCCP. NO^–₃ pretreatmentor N starvation increased subsequent ³⁶C1O^–₃/NO^–₃influx into Chara. A comparison between rates of net NO^–₃uptake and ³⁶C1O^–₃/NO^–₃ influx supported the previoushypothesis that NO^–₃ efflux is an important componentin the determination of overall uptake rates. Key words: Nitrate, Chara, ³⁶CIO^–₃