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     

Seedling Nitrate Reductase Activity and Nitrate Partitioning in Maize (Zea mays L.) Strains Divergently Selected For Post-anthesis Leaf-Lamina Nitrate Reductase Activity

Two experiments were conducted to evaluate the effects of phenotypicrecurrent selection for high and low post-anthesis leaf-laminain vivo NRA on nitrate uptake, nitrate partitioning and in vitroNRA of seedling roots and leaves. In Experiment 1, intact plantsof cycle 0, 4, and 6 of the high and low NRA strains were grownon NH₄-N for 11 d, then exposed to 1.0 mol m^–3 KNO₃, andcultures sampled at 6 h and 28 h (induction and post-inductionperiods). Nitrate uptake, tissue nitrate concentration and invitro NRA were determined. The pattern of response to selectionin seedling leaf NRA was similar to that observed for in vivoNRA of field grown plants. Leaf NRA increased between 6 h and28 h. Root NRA was not affected by selection or sampling time.Treatments differed in total fresh weight but not in reductionor uptake of nitrate per unit weight, indicating a lack of correspondencebetween NRA and reduction and supporting the idea that concomitantreduction by NR is not obligatorily linked to nitrate influxin the intact plant. In Experiment 2, dark-grown plants of cycle 0, and 6 of thehigh and low NRA strains were cultured without N, detopped onday 6, transferred the following day to 0-75 mol m^–3 KNO₃and sampled at 6 h and 28 h. In contrast to Experiment 1, selectionpopulations differed in nitrate reduction and root NRA, whichby 28 h reached higher average levels than root NRA of intactplants. Translocation and reduction were inversely related amongstrains within each sampling time. The high level of translocationin detopped plants of the low NRA strain was difficult to reconcilewith its low leaf NRA level of Experiment 1. It is suggestedthat nitrate transport in detopped roots is altered relativeto the intact system in a way which permits greater NRA inductionand nitrate reduction. The results indicate that nitrate partitioningby detopped root systems should be interpreted with caution. Key words: Zea, nitrate reductase activity, nitrate uptake, nitrate reduction, nitrate partitioning, selection     

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.     

Effects of Root Temperature and Form of Nitrogen Nutrition on Nitrate Reductase Activity in Oilseed Rape (Brassica napus L.)

The effect of root temperature and form of inorganic nitrogensupply on in vitro nitrate reductase activity (NRA) was studiedin oilseed rape (Brassica napus L. cv. bien venu). Plants weregrown initially in flowing nutrient solution containing 10 µMNH₄NO₃ and then supplied with either nitrate or ammonium for15 d at root temperatures of 3, 7, 11 or 17 °C. Shoot temperatureregime was similar for all plants; 20/15 °C, day/night.Root NRA was highest when roots were grown at 3 and 7 °C.In laminae and petioles NRA was highest when roots were 11 or17 °C. The plants supplied with ammonium had much lowerlevels of NRA in roots after 5 d than the plants supplied onlywith nitrate. NRA in the laminae of plants supplied with ammoniumwas low relative to that in plants supplied with nitrate onlywhen root temperature was 11 or 17 °C. Values of the apparent activation energy (E_a) of NR, calculatedfrom the Arrhenius equation, in laminae and petioles were differentfrom roots suggesting difference in enzyme conformation. Evidencethat the temperature at which roots were growing affected E_awas equivocal. Oilseed rape, Brassica napus L., activation energy, ammonium, Arrhenius equation, nitrate, root temperature, nitrate reductase     

Nitrate Reductase Activity and Nitrite in Native Pyrenoids Purified from the Green Alga Bryopsis maxima

The native, starchless pyrenoids purified from Bryopsis maximashowed NADH-nitrate reductase [NR, EC 1.6.6.1 [EC] ] activity andcontained nitrite. The specific activity of NR was 0.024 µmolNO₂ formed per min per mg of protein. The value was 80 timesgreater than that in the crude extract of chloroplasts. Theamount of nitrite in the pyrenoids was 2.37 µmol per mgof protein, showing that nitrite was concentrated by a factorof 66 times. These results suggest a physiological role forpyrenoids in the assimilation of nitrate. (Received November 15, 1989; Accepted February 27, 1990)     

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)     

Preparation of Monoclonal Antibodies against NADH: Nitrate Reductase from the Red Alga Porphyra yezoensis

Twenty-two monoclonal antibodies were raised against the nativeform of nitrate reductase (NR) from Porphyra yezoensis, a marinered alga. All the antibodies were able to bind to NR from P.yezoensis, with resultant inhibition of full (NADH:NR) and/orpartial (NADH:FR, NADH:CR, FMNH₂:NR, and MV:NR) enzymatic activity.Fifteen of the antibodies recognized the denatured form of theenzyme. Size-exclusion gel nitration and immunoblotting of theproducts of the limited proteolysis of NR from P. yezoensisby trypsin or Staphylococcus aureus V8 protease revealed that2 out of the 15 subunit-recognizing antibodies bound to theFAD domain, 6 bound to the heme domain, and 7 bound to the Mo-pterindomain. The products of limited proteolysis of NR from P. yezoensisalso revealed that the sites of proteolytic cleavage that encompassedthe heme domain were inverted as compared to the analogous sitesin NRs of higher plants. Some of the monoclonal antibodies cross-reactedwith NRs from plants belonged to different phyla. From threeto five of the antibodies bound subunits of NR from multicellularred algae, but failed to bind NRs from unicellular red algae.Three or four of the antibodies crossreacted with NRs from higherplants, such as tobacco and spinach. One of the antibodies boundNRs from several types of plant, namely, members of Cryptophyta,Chromophyta, and Chlorophyta. All of the monoclonal antibodiesthat cross-reacted with NRs from plants other than the red algaewere specific for the Mo-pterin domain of NR from P. yezoensis. (Received May 10, 1994; Accepted September 7, 1994)     

Xylem exudation is related to nitrate assimilation pathway in detopped maize seedlings: use of nitrate reductase and glutamine synthetase inhibitors as tools

The xylem exudation of detopped 7-d-old seedlings of Zea maysL. doubled when KCI was present in the root medium comparedto seedlings maintained on water. It was further enhanced whenKCI was replaced by nitrogen compounds such as nitrate, ammoniumand glutamine. The role of the nitrate assimilation pathwayon the enhancement of xylem exudation rate was investigatedusing tungstate, an inhibitor of nitrate reductase (NR) activity,and phosphinothricin or methionine sulphoximine, inhibitorsof glutamine synthetase (GS) activity. The sap levels of NO₃^–,NH₄⁺, glutamine, and asparagine was used to ascertain the invivo inhibition of both enzymes. The tungstate effects werealso checked by measuring leaf in vitro NA activity and NR proteincontent. Xylem exudation rate of detopped seedlings fed withKNO₃ decreased when the nitrate assimilation pathway was blockedeither at the NR or at GS sites. This decrease was preventedwhen urea (acting as NH₄⁺ supply) was given simultaneously withtungstate. KNO₃ does not act directly on exudation, but throughthe involvement of NH₄⁺. The involvement of glutamine was alsoshown since GS inhibition resulted in a cancellation of theenhancing effect of KNO₃ on exudation. As change of exudationrate was not linked to change in sap osmolarity, it is assumedthat the assimilation chain could modify root water conductance.The role of glutamine was discussed. Key words: Exudation, maize, nitrate, conductance, NR, GS     

NO2 Suppression of Light-Induced Nitrate Reductase in Squash Cotyledons

NADH-nitrate reductase (NR) (EC 1.6.6.1 [EC] ) activity in the cotyledonsof squash (Cucurbita maxima Duch.) seedlings showed daily variationwhen the seedlings were subjected to an alternating light-darkcycle. When the seedlings were transferred into continuous darkness,NR activity rose at first and then decreased continuously. Irradiationafter continuous darkness induced a rapid increase in NR activity;this light induction of NR activity was inhibited completelyby fumigation with 4 ppm nitrogen dioxide (NO₂). This inhibitoryeffect of NO₂ was prominent even at 1 ppm and became more pronouncedas the concentration of NO₂ increased. NO₂ fumigation did notremarkably affect the content of reductant (NADH) in the cotyledons.The results of immunoblotting using anti-NR serum indicatedthat irradiation induced the increase in the NR-polypeptidecontent and NO₂ fumigation inhibited the increase, suggestingthat NO₂ put an inhibitory effect on the synthesis of NR inducedby irradiation. ⁴ Present address: College of Environmental Health, Azabu University,Fuchinobe, Sagamihara, Kanagawa 229, Japan ⁵ Present address: Faculty of Home Economics, Otuma Women'sUniversity, Sanban-cho, Chiyoda, Tokyo 102, Japan (Received October 21, 1987; Accepted January 13, 1988)     

Effect of Applied Nitrogen on N2 Fixation, Assimilation of Nitrate and Ammonia in Nodules of Field-grown Moong (Vigna radiata)

A field experiment was conducted to study the effect of nitrogenapplication at 15, 30 and 45 kg ha^–1 of urea at pre-flowering(PF) and pod initiation (PI) stages on the activity of nitrogenase(N₂ase), nitrate reductase (NR) and other related parametersin the nodules of moong (Vigna radiata). Nitrogen applied atPF or PI stage was found to be inhibitory to N₂ase and glutaminesynthetase (GS) activities except at 15 kg N ha^–1 whenapplied at PF in the case of N₂ase. At both the stages therewas increase in NR and glutamate dehydrogenase (GDH) activitieswith the application of nitrogen. Seed yield increased by 18per cent with the application of 15 kg N ha^–1 at PI stagewhereas nitrogen application at PF stage only increased strawyield significantly. Nitrate reductase, nitrogenase, nitrogen application, ammonia assimilation, Vigna radiata     

Effects of hydroxylamine and molybdate on the formation of nitrate reductase in the yeast Rhodotorula

NADPH-nitrate reductase (NR) and NADPH-cytochrome c reductase(CR) activities of Rhodotorula glulinis var. salinaria cellsgrown in nitrate medium supplied with hydroxylamine (0.2 mM)were respectively 1.6- and 3.1-fold higher than those of cellsgrown without hydroxylamine. NR formed in nitrate plus hydroxylaminemedium is almost totally in an inactive form which is reactivatedin vitro by K₃Fe(CN)₆. When molybdate (10^–7 M) was suppliedto this medium, total (active plus inactive) NR activity increasedfurther about twofold but CR activity somewhat decreased. Inordinary nitrate medium, such molybdate effects were small.Most of the CR derepressed (induced) excessively in the nitrateplus hydroxylamine medium had a molecular size similar to NRon the basis of Bio-Gel A 1.5 m chromatography. Some other propertiesof CR formed in this medium were the same as those of the CRmoiety of NR. Adding molybdate to the nitrate plus hydroxylamine medium aftergrowing the cells for 20 hr induced the development of NR activitywithout any increase in CR activity. This induction was completelyblocked by cycloheximide, puromycin and L-canavanine but notcompletely by 6-methylpurine. Ammonium repressed this inductionwith markedly decreasing CR activity. The roles of hydroxylamine and molybdate in the formation ofNR are discussed. (Received May 26, 1980; )     

Regulation of Nitrate Reductase in Acetabularia mediterranea

Nitrate reductase (NR) activity has been characterized on cell-freeextracts of the marine unicellular green alga, Acetabulariamediterranea. Reduced methyl viologen and bromophenol blue aswell as NAD(P)H were effective electron donors for the enzyme. NADH-NR activity increased during cell development reachingits maximum level when the cells approached their maximum length.A substantial increase was also seen in enucleated cells. Theenzyme activity was only present in the green parts of the celland became diminished as the stalks bleached in ageing cells.Under a 12 h light: 12 h darkness photoperiod, NADH-NR activityexhibited pronounced daily fluctuations reaching its maximumon the first hour of illumination (this increase was impairedby cycloheximide) and a minimum in the middle of the dark period. In N-starved algal cultures, NR was constitutively present atappreciable levels and new synthesis took place in the presenceof either NO₃ or low concentrations of NH⁺₄ . However, in thepresence of this cation the enzyme remained mostly in its inactiveform, and could be reactivated in vitro with ferrycyanide. Key words: Nitrate reductase, Acetabularia, daily rhythms, nitrate, ammonium     

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)     

Purification and Characterization of Thiol Proteinase as a Nitrate Reductase-Inactivating Factor from Leaves of Hordeum distichum L.

A thiol proteinase was purified 6,400-fold from leaves of Hordeumdistichum L. by a sequence of ammonium sulfate fractionation,gel filtration, ion exchange chromatography, hydrophobic chromatographyand chromatofocusing. This enzyme also had nitrate reductase(NR)-inactivating activity, which was associated with proteolyticactivity in almost constant proportions during the purificationprocedures. Its molecular weight was estimated as 74,000 bygel filtration, and it had an isoelectric point of 4.05 andan apparent K_m of 0.83 mg ml^–1 for azocasein. The respectiveoptimum pH for proteolytic and NR-inactivating activities were6.0 and 7.0. On electrophoresis, the proteinase gave a majorband that coincided with both activities; it also produced afaint band associated with no activity. Our purified thiol proteinase inactivated FMNH₂-NR and MVH-NRas well as NADH-NR, but it had only a slight effect on NADHcytochrome c reductase activity. This enzyme also inactivatedglutamine synthetase. (Received September 16, 1983; Accepted January 26, 1984)     

Glycolate-, Xanthine- and Paraquat-Mediated Inhibition of Nitrate Reductase in Detached Oat Leaves

Leaf segments of 8-day-old oat plants were incubated with 100µMglycolate or xanthine inthe dark or with 100µM paraquatin the light. These treatments were designed to enhance theinternalproduction of activated oxygen species and to allowus to study the possible role of such species in the regulationof the activity of NO₃-induced nitrate reductase (NR) in illuminatedgreen tissues. Each of the treatments inhibited NO₃-induced NR activity. HPMS(an inhibitor of glycolate oxidase) and allopurinol (an inhibitorof xanthine oxidase) protected NR from inactivation by glycolateand by xanthine. Free radical scavengers also protected NR frominactivation by xanthine and paraquat but they had no effecton the inhibition by glycolate. The activities of catalase andsuper oxide dismutase did not increase in response to the varioustreatments. Thus, while the production of active oxygen speciesappears to be stimulated under such conditions, the rates ofthe enzymatic destruction and "scavenging" of these speciesseem to remain unchanged. Hence, the endogenous levels of theseactive oxygen species can be expected to increase in the treatedleaves. We interpret our results as evidence that inhibition by glycolateof NR is caused by the H₂O₂ generated during the oxidation ofglycolate while the effects of xanthine and paraquat dependon the enhanced production of free radicals. It is concluded that activated oxygen species that are formedendogenously may play a role in the regulation of NR in greentissues. (Received August 12, 1991; Accepted February 17, 1992)     

Effect of Nitrogen on Nitrate Reductase Activity in the Nodules and Leaves of Summer Moong (Vigna radiata)

The relation of the in vivo nitrate reductase (NR) activityto growth period was studied in the nodules and the leaves ofthe summer moong (Vigna radiata). The maximum NR activity wasobserved 31 days after sowing (DAS) in the leaves and 28 DASin the case of the nodules. In a pot experiment, the effectof the various nitrogen concentrations, namely 0, 3, 6, 9 and12 mg kg^–1 was studied on NR activity at three growthstages. The maximum NR activity was observed at 6 mg kg^–1N during the pre-flowering stage (26 DAS). Though the noduleshave higher NR activity, its expression was limited by substrateavailability. The NR activity in the leaf could be used as anindex of NR activity in the nodules. Nitrate reductase, nitrogen, nitrate, moong, Vigna radiata     

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; )     

Possible interaction between peroxidase and NAD(P)H-dependent nitrate reductase activities of plasma membranes of corn roots

The relationship between the plasma membrane bound NAD(P)H-nitratereductase (NR) and a plasma membrane (PM)-bound peroxidase wasinvestigated using highly purified PM vesicles isolated fromcorn roots. The PM-bound NR activity was strongly enhanced byMnCl₂ and SHAM, which stimulated peroxidase activity. Sinceboth activities, the NAD(P)H-dependent NR and the peroxidasecompete for NAD(P)H as electron donor, we propose a model inwhich a product of peroxidation is able to offer electrons tothe nitrate reductase in a more reactive form with respect toNAD(P)H.Our hypothesis was confirmed by experiments in which the effectsof inhibitors of peroxidative reactions, catalase, superoxidedismutase, and ascorbate on the PM-bound NR were studied. Resultsindicate that the putative electron donor for nitrate reductioncould be a radicalic species, possibly NAD. Furthermore, sincecytochrome c decreased the activity of the plasma membrane-boundNAD(P)Hdependent NR, cytochrome b₅₅₇ might be the site of theenzyme accepting electrons from NAD. Our results indicate that the PM environment of the NR may beinvolved in the extent of the membrane associated nitrate reductionand that redox enzymes at the PM, the NAD(P)H-NR and a peroxidase-likeNADH-oxidase, can interact. Key words: Plasma membrane-bound nitrate reductase, peroxidase, Zea mays     

Stimulation of Nitrate Reductase by Light and Ammonium in Spirodela oligorrhiza

Light-enhanced nitrate reductase (NR) activity was 8 times greaterthan the dark control. Exogenous application of sucrose, glucoseand fructose increased the induction of NR in the light as wellas in the dark, whereas glycolate had no effect. DCMU [3-(3,4-dichlorophenyl)-1, 1-dimethyl urea] completely inhibited thedevelopment of NR in light. Sucrose, when added with DCMU, reversedthis inhibitory effect NR in vivo was more stable in light thanin darkness, the half-lives being 9.6 h and 6.4 h, respectively.The addition of sucrose did not change the half-life of NR ineither light or darkness. Ammonium, the end product of the inorganicnitrogen assimilatory pathway, stimulated the NR activity whereasamino acids decreased it. Key words: Spirodela oligorrhiza, nitrate reductase, ammonium, light     

Chlorophylls and Nitrate Reductase Activity in Relation to Heterosis in Coconut seedlings

The F₁ progeny of Chowghat Dwarf Orange (CDO) and West CoastTall (WCT) cross tend to be highly heterozygous with respectto the colour of petiole and seedling vigour. The study indicatedthat the greenish brown coloured vigorous plants had higherchlorophyll (a + b) content and nitrate reductase (NR) activitycompared to the orange coloured vigorous plants and the dwarfs.The greenish brown seedlings were also characterized by a largerleaf area and shoot d. wt. The results provide biochemical evidencein support of the morphological criteria being used at presentin the selection of hybrid coconut seedlings. Cocos nucifera L. (Coconut), Chowghat Dwarf Orange, West Coast Tall, heterosis, chlorophyll, nitrate reductase, leaf area, shoot dry weight