Three nitrate reductase activities in Alcaligenes eutrophus

Three nitrate reductase activities were detected in Alcaligenes eutrophus strain H16 by physiological and mutant analysis. The first (NAS) was subject to repression by ammonia and not affected by oxygen indicating a nitrate assimilatory function. The second (NAR) membrane-bound activity was only formed in the absence of oxygen and was insensitive to ammonia repression indicating a nitrate respiratory function. The third (NAP) activity of potential respiratory function occurred in the soluble fraction of cells grown to the stationary phase of growth. In contrast to NAR and NAS, expression of NAP did not require nitrate for induction and was independent of the rpoN gene product. Genes for the three reductases map at different loci. NAR and NAS are chromosomally encoded whereas NAP is a megaplasmid-borne activity in A. eutrophus.     

Short-term modulation of nitrate reductase activity by exogenous nitrate in Nicotiana plumbaginifolia and Zea mays leaves

Maize (Zea mays L.) grown on low (0.8 mM) NO ₃ ^- , as well as untransformed and transformed Nicotiana plumbaginifolia constitutively expressing nitrate reductase (NR), was used to study the effects of NO ₃ ^- on the NR activation state. The NR activation state was determined from the relationship of total activity extracted in the presence of ethylenediaminetetracetic acid to that extracted in the presence of Mg²⁺. Light activation was observed in both maize and tobacco leaves. In the tobacco lines, NO ₃ ^- did not influence the NR activation state. In excised maize leaves, no correlation was found between the foliar NO ₃ ^- content and the NR activation state. Similarly, the NR activation state did not respond to NO ₃ ^- . Since the NR activation state determined from the degree of Mg²⁺-induced inhibition of NR activity is considered to reflect the phosphorylation state of the NR protein, the protein phosphatase inhibitor microcystin LR was used to test the importance of protein phosphorylation in the NO ₃ ^- -induced changes in NR activity. In-vivo inhibition of endogenous protein phosphatase activity by microcystin-LR decreased the level of NR activation in the light. This occurred to the same extent in the presence or absence of exogenous NO ₃ ^- . We conclude that NO ₃ ^- does not effect the NR activation state, as modulated by protein phosphorylation in either tobacco (a C₃ species) or maize (a C₄ species). The short-term regulation of NR therefore differs from the NO ₃ ^- -mediated responses observed for phosphoenolpyruvate carboxylase and sucrose phosphate synthase.Abbreviations Chl chlorophyll - MC microcystin-LR - PEP-Case phosphoenolpyruvate carboxylase - SPS sucrose-phosphate synthase We are indebted to Madeleine Provot and Nathalie Hayes for excellent technical assistance. This work was funded by EEC Biotechnology Contract No. BI02 CT93 0400, project of technical priority, Network D — Nitrogen Utilisation and Efficiency.     

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     

A comparison of the high-active and low-active form of nitrate reductase in synchronous Chlorella sorokiniana

Chlorella sorokiniana possesses two forms of nitrate reductase (EC 1.6.6.1.). One with low activity is present in cells at the end of the light-dark cycle, the other with high activity is present after 1 h of illumination. The two forms can be distinguished by gel electrophoresis, isopycnic centrifugation, assay of the partial reactions and their sensitivity to antibodies, respectively. These differences are discussed with respect to an effect of intracellular nitrate on the activation of nitrate reductase.Abbreviations NAR nitrate reductase - FMN flavine mononucleotide - MV methylviologen     

The activation state of nitrate reductase is not always correlated with total nitrate reductase activity in leaves

The relation between nitrate reductase (NR; EC 1.6.6.1) activity, activation state and NR protein in leaves of barley (Hordeum vulgare L.) seedlings was investigated. Maximum NR activity (NRA_max) and NR protein content (Western blotting) were modified by growing plants hydroponically at low (0.3 mM) or high (10 mM) nitrate supply. In addition, plants were kept under short-day (8 h light/16 h dark) or long-day (16 h light/8 h dark) conditions in order to manipulate the concentration of nitrate stored in the leaves during the dark phase, and the concentrations of sugars and amino acids accumulated during the light phase, which are potential signalling compounds. Plants were also grown under phosphate deficiency in order to modify their glucose-6-phosphate content. In high-nitrate/long-day conditions, NRA_max and NR protein were almost constant during the whole light period. Low-nitrate/long-day plants had only about 30% of the NRA_max and NR protein of high-nitrate plants. In low-nitrate/long-day plants, NRA_max and NR protein decreased strongly during the second half of the light phase. The decrease was preceded by a strong decrease in the leaf nitrate content. Short daylength generally led to higher nitrate concentrations in leaves. Under short-day/low-nitrate conditions, NRA_max was slightly higher than under long-day conditions and remained almost constant during the day. This correlated with maintenance of higher nitrate concentrations during the short light period. The NR activation state in the light was very similar in high-nitrate and low-nitrate plants, but dark inactivation was twice as high in the high-nitrate plants. Thus, the low NRA_max in low-nitrate/long-day plants was slightly compensated by a higher activation state of NR. Such a partial compensation of a low NR_max by a higher dark activation state was not observed with phosphate-depleted plants. Total leaf concentrations of sugars, of glutamine and glutamate and of glucose-6-phosphate did not correlate with the NR activation state nor with NRA_max. Received: 24 March 1999 / Accepted: 31 May 1999     

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

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

Sources of reducing power for nitrate reduction in spinach leaves

The possible source of NADH, the energy donor for nitrate reductase (EC 1.6.6.1), has been studied using an in vivo assay involving freezing the material (leaves of Spinacea oleracea L.) in liquid nitrogen in order to render the tissue permeable to added substrates. Glycolysis and the pentose phosphate pathway were capable of generating NADH through glyceraldehyde-3-phosphate dehydrogenase. Malate and isocitrate were also capable of generating NADH white other organic acids tested were not, including glycolate which was ineffective even under anaerobic conditions.     

A cyanobacterial narB gene encodes a ferredoxin-dependent nitrate reductase

The narB gene from the cyanobacterium Synechococcus sp. PCC 7942 was cloned downstream from the LacI-regulated promoter P_trc in the Escherichia coli vector pTrc99A, rendering plasmid pCSLM1. Addition of isopropyl--D-thiogalactoside to E. coli (pCSLM1) resulted in the parallel expression of a 76 kDa polypeptide and a nitrate reductase activity with properties identical to those known for nitrate reductase isolated from Synechococcus cells. As is the case for nitrate reductase from Synechococcus cells, either reduced methyl viologen or reduced ferredoxin could be used as an electron donor for the reduction of nitrate catalyzed by E. coli (pCSLM1) extracts. This data shows that narB is a cyanobacterial structural gene for nitrate reductase.     

Screening of monoclonal antibodies to scarce and labile enzymes: A functional immunoassay for isolating monoclonal antibodies to NADPH:nitrate reductase

A functional immunoassay, that has proved very useful, is described for screening and identifying monoclonal antibodies (McAbs) against scarce and labile enzymes. This method does not require purified enzyme or antigen and it has been successfully applied to isolate three hybridomas secreting McAbs to NADPH:nitrate reductase from the chloronema cells of the mossFunaria hygrometrica. Briefly, the protocol involves: adsorption of murine antibodies from hybridoma supernatants by rabbit antimouse IgG antibody pre-adsorbed toStaphylococcus aureus cells (SAC), reaction with crude extract for 15 min, sedimentation of the SAC complex by centrifugation and measurement of residual enzymatic activity in the supernatant. A depletion indicates the presence of antibodies that bind to the active enzyme. The method is rapid, sensitive and versatile enough to be used to isolate McAbs with exquisite specificities. The three isolated McAbs recognized nitrate reductase protein in a conformation-independent and/or a conformation-dependent manner.     

In-situ localization of nitrate reductase in maize roots

The localization of nitrate reductase (NR; EC 1.6.6.2) in cells of root tissues ofZea mays L. (W64A W182L) was determined using post-embedding immunogold labeling at the electron-microscopy level and using silver enhancement of the colloidal-gold signal for light microscopy. Nitrate reductase is located in the cytoplasm of root epidermal and cortical cells, and in the cells of the parenchyma and pericycle within the vascular cylinder. A weaker signal was also obtained in parenchymal cells of the pith lying next to the xylem. A positive signal for NR protein was seen in the chloroplast fraction of maize leaves and in the plastid fraction of roots. This signal was lost when affinity-purified antibodies were used. Sections of Lowicryl-embedded tissue were found to be suitable for the localization of the non-abundant NR protein when adequate controls and signal-enhancement procedures were used.Abbreviations IgG immunoglobulin G - NR nitrate reductase - PEPCase phosphoenolpyruvate carboxylase This research was funded by Natural Sciences and Engineering Research Council (NSERC) of Canada grants ISE0125461 (AO), OGP0106265 (JSG) and an NSERC Visiting Scientist Award to E.F.     

Relationships between external nitrate availability,nitrate uptake and expression of nitrate reductase in roots of barley grown in N-limited split-root cultures

Despite the large number of studies of nitrate metabolism in plants, it remains undetermined to what extent this key plant system is controlled by overall plant N nutrition on the one hand, and by the nitrate ion itself on the other hand. To investigate these questions, V _max for nitrate uptake (high-affinity range), and nitrate reductase (NR) mRNA and activity, were measured in roots of N-limited barley (Hordeum vulgare L. cv. Golf) grown under conditions of constant relative addition of nitrate, with the seminal roots split between two culture compartments. The total amount of nitrate added per unit time (0.09·d^-1) was distributed between the two root parts (subroots) in ratios of 1000, 982, 955, 9010, 8020, and 5050. These nitrate-addition ratios resulted in nitrate fluxes ranging from 0 to 23 mol nitrate·g^-1 DW root·h^-1, while the external nitrate concentrations varied between 0 and 1.2 M. The apparent V _max for net nitrate uptake showed saturation-type responses to nitrate flux maintained during preceding growth. The flux resulting in half-maximal induction of nitrate uptake was approximately 4 mol nitrate·g^-1 DW root·h^-1, corresponding to an external nitrate concentration of 0.7 M. The activity of NR and levels of NR mRNA did not saturate within the range of nitrate fluxes studied. None of the parameters studied saturated with respect to the steady-state external nitrate concentration. At the zero nitrate addition — the 0%-root — initial uptake activity as determined in short-term ¹⁵N-labelling experiments was insignificant, and NR activity and NR mRNA were not detectable. However, nitrate uptake was rapidly induced, showing that the 0%-root had retained the capacity to respond to nitrate. These results suggest that local nitrate availability has a significant impact on the nitrate uptake and reducing systems of a split-root part when the total plant nitrate nutrition is held constant and limiting.Abbreviation NR nitrate reductase This work was supported by the Lars Hierta Memory Foundation, the Royal Swedish Academy of Sciences, and by the Swedish Natural Science Research Council via project grants (to C.-M.L. and B.I.) and visiting scientist grant (to W.H.C.). We thank Mrs. Ellen Campbell for technical advice, and Mrs. Judith V. Purves, Long Ashton Research Station, Long Ashton, UK, for analyses of ¹⁵N-labelling in tissue samples.     

The roles of nitrate and ammonium in the regulation of the development of nitrate reductase in Chlamydomonas reinhardii

The regulation of the development of nitrate reductase (NR) activity in Chlamydomonas reinhardii has been compared in a wild-type strain and in a mutant (nit-A) which possesses a modified nitrate reductase enzyme that is non-functional in vivo. The modified enzyme cannot use NAD(P)H as an electron donor for nitrate reduction and it differs from wild-type enzyme in that NR activity is not inactivated in vitro by incubation with NAD(P)H and small quantities of cyanide; it is inactivated when reduced benzyl viologen or flavin mononucleotide is present. After short periods of nitrogen starvation mutant organisms contain much higher levels of terminal-NR activity than do similarly treated wild-type ones. Despite the inability of the mutant to utilize nitrate, no nitrate or nitrite was found in nitrogen-starved cultures; it is therefore concluded that the appearance of NR activity is not a consequence of nitrification. After prolonged nitrogen starvation (22 h) the NR level in the mutant is low. It increases rapidly if nitrate is then added and this increase in activity does not occur in the presence of ammonium, tungstate or cycloheximide. Disappearance of preformed NR activity is stimulated by addition of tungstate and even more by addition of ammonium. The results are interpreted as evidence for a continuous turnover of NR in cells of the mutant with ammonium both stimulating NR breakdown and stopping NR synthesis. Nitrate protects the enzyme from breakdown. Reversible inactivation of NR activity is thought to play an insignificant rôle in the mutant.Abbreviations NR nitrate reductase - BV benzyl viologen     

Characterization of a chlorate-hypersensitive,high nitrate reductase Arabidopsis thaliana mutant

Summary A population of A. thaliana, produced by self-fertilization of ethylmethane sulfonate treated plants, was exposed to chlorate in the watering solution, and plants showing early susceptibility symptoms were rescued. Among the progeny lines of these plants five were shown to be repeatably chlorate-hypersusceptible. One of these lines (designated C-4) possessed elevated activity of nitrate reductase (NR). The NR activity of mutant C-4 was higher than that of normal plants throughout the life cycle. Nitrite reductase and glutamine synthetase activities of C-4 were normal, as were chlorate uptake rate and tissue nitrate content. The elevated NR activity apparently was responsible for the chlorate hypersusceptibility of C-4. Inheritance studies of NR indicated that the elevated activity of C-4 was probably controlled by a single recessive allele.     

Nitrite activation of nitrate reductase in higher plants

Comparative studies of nitrate-activated nitrate reductase (NR-NO₂) and nitrate-induced nitrate reductase (NR-NO₃) (EC 1.6.6.2) indicate that the enzymes differ in structure, heat stability, and pH dependence, but have the same cofactor requirment. NR-NO₂ developes in barley (Hordeum vulgare L. var. Dvir) seedlings as NR-NO₃ disappears. A transition from the active to the inactive form of nitrate reductase takes place. Nitrite seems to activate the inactive form of the enzyme.     

Monoclonal antibodies to surface glycoconjugates in Chlamydomonas eugametos recognize strain-specific O-methyl sugars

Monoclonal antibodies are described that are directed against cell surface components of the unicellular green alga Chlamydomonas eugametos. These antibodies recognize strain-specific epitopes which occur at the surface of vegetative and gametic cells. Two different groups of epitopes are distinguished that are never detectable together in one clonal cell culture. Evidence is presented showing that the antigenicity of cell surface molecules is a consequence of the presence of particular O-methylated sugars. Monoclonal antibodies reacting with one group of epitopes were studied in more detail, and immunoprecipitation and Western-blot studies showed that these epitopes can be arranged into four classes. The use of these monoclonal antibodies as strain-specific markers in light- and electron-microscopical techniques is illustrated.Abbreviations ELISA enzyme-linked immunosorbent assay - mt ^+/- mating type plus or minus - PAS periodic acid Schiff - Mab monoclonal antibody - PBS phosphate-buffered saline     

Isolation and characterisation of a strain of Pseudomonas putida that can express a periplasmic nitrate reductase

A strain of Pseudomonas putida that can express a nitrate reductase that is located in the periplasmic compartment was isolated from freshwater. The enzyme was active in vivo during arginine fermentation and at the onset of oxygen limitation in batch cultures. The activity of the enzyme increased the yield of bacteria following fermentative growth under anoxic conditions with arginine, but nitrate reduction did not support growth on nonfermentable carbon substrates under anoxic conditions. Cells expressing the periplasmic nitrate reductase were capable of reducing nitrate in the presence of oxygen. Nitrate reduction under oxic conditions was clearly coupled to a respiratory electron transport chain because: (1) the process was sensitive to the respiratory inhibitors rotenone and 2-n-heptyl-4-hydroxyquinoline N-oxide, and (2) membrane-bound and periplasmic cytochromes were involved. This is the first report of the presence of a periplasmic nitrate reductase in a member of the proteobacteria.     

Evidence for the nitrate-dependent spatial regulation of the nitrate reductase gene in chicory roots

Young chicory plants (Cichorium intybus L. var. Witloof) show a tenfold higher nitrate reductase NR activity in roots compared to leaves. Northern analysis revealed, besides the nitrate inducibility of the nitrate reductase gene (nia), a higher level of expression in the roots. By modifying the external nitrate concentration the NR activity in the leaves remained negligible whereas a maximal activity was observed in the roots when grown in the presence of 5 mM nitrate. Surprisingly, variation of the external nitrate concentration induced changes in the spatial regulation of nia within the root. In-situ hybridization mainly localized nia mRNA in the cortical cells of roots grown at low nitrate concentrations (0.2 mM). At high nitrate concentrations (5 mM), nia mRNA was more abundant in the vascular tissues. The root apex revealed a strong signal under both conditions. The isolation and characterization of the NR structural gene from chicory is also presented. Southern blot analysis revealed the presence of a single nia gene per haploid genome of chicory.