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.     

Stabilization of nitrate reductase in maize roots by chymostatin

Nitrate reductase (NR) in maize (Zea mays cv W64A × W182E) roots has been stabilized in vitro by the addition of chymostatin to extraction buffer. Contrary to previous observations, levels of NR were higher in the mature root than in root tip sections when chymostatin was included in the extraction buffer. Two forms of NR were identified, an NADH monospecific NR found mainly in the 1cm root tip and an NAD(P)H bispecific NR found predominantly in mature regions of the root. During the first 10 days of seedling growth, NR activity in the root ranged from 50 to 80% of the activities found in the leaf (a maximum of 2.4 micromoles NO⁻₂ produced per hour per gram fresh weight was measured at 4 days).     

Inactivation of serine:glyoxylate and glutamate:glyoxylate aminotransferases from tobacco leaves by glyoxylate in the presence of ammonium ion

Different organs of maize seedlings are known to contain different complements of NADH and NAD(P)H nitrate reductase (NR) activity. The study of the genetic programming that gives rise to such differences can be initiated by looking for genetic variants exhibiting different patterns of distribution of the above enzymes. We demonstrate in this work that scutella of very young maize seedlings contain NADH NR almost exclusively and that this activity is gradually replaced, as the seedling ages, with NAD(P)H NR. Leaves in the seedlings contain exclusively the NADH NR activity. A genetic variant is described that contains much reduced levels of NAD(P)H NR activity but not of NADH NR activity in the scutellum. This same variant exhibits a relatively low level of NAD(P)H NR but normal NADH NR activity in seedling root tips. These observations suggest that the genetic program used to specify the scutellar complement of NR activity shares some common components with the genetic program used to determine the young root tip complement of NR activities. Parts of regenerating callus at different stages of differentiation were examined to determine when the differences in NR complement begin to appear. The same pattern of NADH NR and NAD(P)H NR activities was found in unorganized as well as in organized callus, in recognizable root-like and even in green shoot-like material, both activities being present in all these tissues. An examination of the NR complement in different organs of a number of siblings originating from a cross involving transposon Mu-containing parents and having different levels of leaf NADH NR activity shows that the leaf NADH NR activity content and the scutellum NAD(P)H NR activity content are relatively independent of each other, indicating that the genetic programs specifying the NR content of these organs are not tightly coupled, if at all.     

The distribution and characteristics of nitrate reductase and glutamate dehydrogenase in the maize seedling

In a study on 3-day maize (Zea mays) seedlings, grown on nitrate, requirements were established for the maximum extraction and optimum stabilization of nitrate reductase in vitro. With the primary root, 5 mm cysteine were required in the extraction medium, but for the scutellum, which has a high level of endogenous thiol, the use of additional thiol resulted in a reduced yield of a more labile enzyme. Activity of the root and scutella nitrate reductase was obtained with either NADH or NADPH, but that of the root enzyme with NADPH was only demonstrated in the absence of phosphate.Before leaf expansion, the nitrate reductase in the maize seedling was mainly in the scutellum. The enzyme present in the primary root was predominantly in the apical region (0-2 mm). In contrast, glutamate dehydrogenase was concentrated in the mature basal region of the root (30-60 mm). A high level of nitrate (approximately 100 mm) was required to saturate the induction of nitrate reductase in the root tip, mature root, and scutellum. The concentration of nitrate required to give half the maximum level of enzyme induced was the same for each region (29 mm).After leaf expansion, more than 90% of the nitrate reductase was in the shoot, mainly in the leaf blade, and a marked decrease occurred in the level of the enzyme in the scutellum. A large proportion of the glutamate dehydrogenase was still found in the root.     

cDNA Clones for Corn Leaf NADH:Nitrate Reductase and Chloroplast NAD(P):Glyceraldehyde-3-Phosphate Dehydrogenase : Characterization of the Clones and Analysis of the Expression of the Genes in Leaves as Influenced by Nitrate in the Light and Dark

cDNA clones were selected from a corn (Zea mays L.) leaf lambda gt11 expression library using polyclonal antibodies for corn leaf NADH:nitrate reductase. One clone, Zmnrl, had a 2.1 kilobase insert, which hybridized to a 3.2 kilobase mRNA. The deduced amino acid sequence of Zmnrl was nearly identical to peptide sequences of corn leaf NADH:nitrate reductase. Another clone, Zm6, had an insert of 1.4 kilobase, which hybridized to a 1.4 kilobase mRNA, and its sequence coded for chloroplastic NAD(P)⁺:glyceraldehyde-3-phosphate dehydrogenase based on comparisons to sequences of this enzyme from tobacco and corn. When nitrate was supplied to N-starved, etiolated corn plants, nitrate reductase, and glyceraldehyde-3-phosphate dehydrogenase mRNA levels in leaves increased in parallel. When green leaves were treated with nitrate, only nitrate reductase mRNA levels were increased. Nitrate is a specific inducer of nitrate reductase in green leaves, but appears to have a more general effect in etiolated leaves. In the dark, nitrate induced nitrate reductase expression in both etiolated and green leaves, indicating light and functional chloroplast were not required for enzyme expression.     

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.     

Effects of a nitrate reductase inactivating enzyme and NAD(P)H on the nitrate reductase from higher plants and Neurospora.

Evidence is presented which suggests that the NAD(P)H-cytochrome c reductase component of nitrate reductase is the main site of action of the inactivating enzyme. When tested on the nitrate reductase (NADH) from the maize root and scutella, the NADH-cytochrome c reductase was inactivated at a greater rate than was the FADH2-nitrate reductase component. With the Neurospora nitrate reductase (NADPH) only the NADPH-cytochrome c reductase was inactivated. p-Chloromercuribenzoate at 50 muM, which gave almost complete inhibition of the NADH-cytochrome c reductase fraction of the maize nitrate reductase, had no marked effect on the action of the inactivating enzyme. A reversible inactivation of the maize nitrate reductase has been shown to occur during incubation with NAD(P)H. In contrast to the action of the inactivating enzyme, it is the FADH2-nitrate reductase alone which is inactivated. No inactivation of the Neurospora nitrate reductase was produced by NAD(P)H alone and also in the presence of FAD. The lack of effect of the inactivating enzyme and NAD(P)H on the FADH2-nitrate reductase of Neurospora suggests some differences in its structure or conformation from that of the maize enzyme. A low level of cyanide (0.4 mu M) markedly enhanced the action of NAD(P)H on the maize enzyme; Cyanide at a higher level (6 mu M) did give inactivation of the Neurospora nitrate reductase in the presence of NADPH and FAD. The maize nitrate reductase, when partially inactivated by NADH and cyanide, was not altered as a substrate for the inactivating enzyme. The maize root inactivating enzyme was also shown to inactivate the nitrate reductase (NADH) in the pea leaf. It had no effect on the nitrate reductase from either Pseudomonas denitrificans or Nitrobacter agilis.     

Nitrate reductase induction and molecular characterization in rice (Oryza sativa L.)

Summary Barley nitrate reductase cDNA clone bNRp10 was used as a hybridization probe to screen a genomic DNA library of rice (Oryza sativa L.) cultivar M201. Two different lambda clones were isolated, subcloned to plasmids, and partially characterized. The subclone pHBH1 was tentatively identified as encoding a NADH nitrate reductase. Southern and dot blot analysis suggest that, in rice, nitrate reductase is encoded by a small gene family. Regulation of NADH nitrate reductase was investigated in rice cultivars Labelle and M201 representing the subspecies indica and japonica, respectively. In the absence of nitrate, only trace levels of nitrate reductase activity and mRNA were detected in seedling leaves. Upon addition of nitrate to seedling roots, nitrate reductase activity and mRNA increased rapidly in leaves. Nitrate reductase activity continued to increase over a 24 h period, but the mRNA accumulation peaked at about 6 h and then declined. Western blot analysis with a barley NADH nitrate reductase antiserum showed the presence of two bands of approximately 115 and 105 kDa. These protein bands were not detected in extracts of tissue grown in the absence of nitrate.     

NO参与玉米幼苗对盐胁迫的应答

以玉米幼苗为材料,研究盐胁迫下其內源NO含量、NR和NOS活性的变化;NOS专一性抑制剂L-NAME和NR非专一性抑制剂NaN3对玉米幼苗內源NO含量的影响;利用激光共聚焦显微技术观测盐胁迫下玉米幼苗根部NO含量的变化及其分布特点。结果表明,盐胁迫下玉米幼苗根尖和叶片中NO含量有猝发现象,NOS活性也随之显著提高,NR活性则显著降低;L-NAME或NaN3均可降低盐胁迫所引起的玉米幼苗NO水平的增加,L-NAME对NO含量的影响比NaN3更显著。推测,NO参与玉米幼苗对盐胁迫的应答,NOS途径是盐胁迫下玉米幼苗內源NO合成的主要途径。     

Iron Mediated Nitrate Reductase Activity in Different Parts of Young Maize Seedlings

Incubation of 5-d-old maize seedlings in the half-strength Hoagland's nutrient solution containing 10 mM KNO₃ with FeCl₃ or FeSO₄ (0.5 or 2.0 mM) caused a significant increase in nitrate reductase (NR) activity and slightly increased total protein content in root, shoot and scutellum. In case of root, NADPH:NR activity was inhibited contrary to the NADH:NR activity. In spite of NR activity, nitrate uptake was inhibited from 13 to 37 % by the iron. The results presented demonstrate an isoform specific, organ specific, and to some extent salt specific responses of NR to iron.     

Characterization and sequence of a novel nitrate reductase from barley

Summary Barley (Hordeum vulgare L.) has both NADH-specific and NAD(P)H-bispecific nitrate reductases. Genomic and cDNA clones of the NADH nitrate reductase have been sequenced. In this study, a genomic clone (pMJ4.1) of a second type of nitrate reductase was isolated from barley by homology to a partial-length NADH nitrate reductase cDNA and the sequence determined. The open reading frame encodes a polypeptide of 891 amino acids and its interrupted by two small introns. The deduced amino acid sequence has 70% identity to the barley NADH-specific nitrate reductase. The non-coding regions of the pMJ4.1 gene have low homology (ca. 40%) to the corresponding regions of the NADH nitrate reductase gene. Expression of the pMJ4.1 nitrate reductase gene is induced by nitrate in root tissues which corresponds to the induction of NAD(P)H nitrate reductase activity. The pMJ4.1 nitrate reductase gene is sufficiently different from all previously reported higher plant nitrate reductase genes to suggest that it encodes the barley NAD(P)H-bispecific nitrate reductase.Scientific Paper No. 9101-14. College of Agriculture and Home Economics Research Center, Washington State University, Research Project Nos. 0233 and 0745