Robert L. Warner

Nitrate reductase regulation: effects of nitrate and light on nitrate reductase mRNA accumulation

SummaryRegulation of nitrate reductase mRNA by light and nitrate was studied in barley seedlings using a partial cDNA clone as a probe. Nitrate reductase mRNA was detected in roots and leaves within 40 min after supplying nitrate to the roots and reached peak accumulation at 2 h in the roots and at 12 h in the leaves. In the absence of nitrate, nitrate reductase mRNA was not detected in shoots but low levels were detected in roots. After reaching a peak, nitrate reductase mRNA declined to approximately 60% and 40% of the peak accumulation in the leaves and roots, respectively. In both roots and leaves the decline in nitrate reductase mRNA occurred while nitrate reductase activity and tissue nitrate were increasing. Light enhanced nitrate reductase mRNA accumulation but the responses of etiolated and green leaves to light were different. Seedlings grown in light and exposed to nitrate in the dark accumulated low but detectable nitrate reductase activity and mRNA. These seedlings did not respond to red, far-red, or blue light but did exhibit a strong fluence response to white light. However in etiolated seedlings, nitrate reductase mRNA increased 20-fold in response to red and blue light. These results indicate that phytochrome or another photoreceptor may facilitate induction of nitrate reductase transcription by nitrate in etiolated seedlings but not in green leaves.

Characterization of nitrate reductase-deficient barley mutants

SummaryTen nitrate reductase-deficient Hordeum vulgare mutants were characterized for NADH and FMNH2 nitrate reductase (NR), cytochrome C reductase (CR) and nitrite reductase (NiR) activities. The mutants sort into four major groups. Group I represented by mutants Az 12, Az 23, Az 29 and Az 30 have low Nr and Cr activities. Group II represented by mutants Az 13, Az 31, Az 33 and Az 34 have low NR activities but intermediate CR activities. Group III represented by mutant Az 28 has low NR activity, but above normal CR activity. Group IV represented by Az 32 has low NADH-NR, low CR, but above normal FMNH2-NR activity. All ten mutants have elevated NiR activities. None of the ten mutants were constitutive for nitrite reductase activity. Only Az 34 showed a definite high temperature sensitivity when the NADH nitrate reductase activity was compared in the 12 to 26° C range. The mutants Az 12, Az 13, Az 23, Az 28, Az 29, Az 30, Az 31, Az 32 and Az 33 are allelic and were assigned the locus designation nar1. Mutant Az 34 represents a different genetic locus designated nar2. The nar1 gene is codominant and the nar2 gene is recessive.

In vitro stability of nitrate reductase from barley leaves

Abstract Barley seedling nitrate reductase was stabilized in vitro without the use of extraneous protein by optimizing the buffer components. The extraction buffer (NRT 8.5) consists of 0.25 M Tris-HCl, pH 8.5, 3 mM DTT, 5 μM FAD, 1 μ M sodium molybdate and 1 mM EDTA. This buffer stabilizes the extracted nitrate reductase at O° and 30°, whereas the addition of extraneous protein to standard extraction buffers stabilizes the enzyme only at 0°.

Purification and partial characterization of nitrate reductase from barley leaves

NADH-nitrate reductase from barley leaves (Hordeum vulgare L. cv. Steptoe) was purified 570-fold with over 50% yield by a procedure using ammonium sulfate fractionation and blue dextran-agarose affinity chromatography. The purified enzyme had NADH-, FMNH2-, and reduced methyl viologen-nitrate reductase and NADH-cytochrome c reductase activities, but not NADPH nitrate reductase activity. The NADH-nitrate reductase has a pH optimum at 7.5 and a Michaelis constant (Km) for nitrate of 2.4 × 10−4 M. The specific activity of the purified enzyme was 8 μ mol NO2−/min/mg protein. A single protein band corresponding to reduced methyl viologen-nitrate reductase activity was observed by disc gel electrophoresis. A S20,w of 7.9 S was found by a sucrose density centrifugation and a Stokes radius of 68 A was determined by gel filtration. From these values, a molecular weight of 221 000 was estimated for the native enzyme. SDS-gel electrophoresis of the native enzyme indicated a subunit molecular weight of approximately 100 000. Thus, the barley nitrate reductase contains two subunits of the same size.

Characterization and sequence of a novel nitrate reductase from barley

SummaryBarley (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.

Expression of NADH-Specific and NAD(P)H-Bispecific Nitrate Reductase Genes in Response to Nitrate in Barley

Barley (Hordeum vulgare L.) has two, differentially regulated, nitrate reductase (NR) genes, one encoding the NADH-specific NR (Nar1) and the other encoding the NAD(P)H-bispecific NR (Nar7). Regulation of the two NR genes by nitrate was investigated in wild-type Steptoe and in an NADH-specific NR structural gene mutant (Az12). Gene-specific probes were used to estimate NADH and NAD(P)H NR mRNAs. The kinetics of induction by nitrate were similar for the two NR genes; expression was generally below the limits of detection prior to induction, reached maximum levels after 1 to 2 h of induction in roots and 4 to 8 h of induction in leaves, and then declined to steady-state levels. Derepression of the NAD(P)H NR gene in leaves of the NADH-specific NR gene mutant Az12 did not appear to be associated with changes in nitrate assimilation products or nitrate flux. Nitrate deprivation resulted in rapid decreases in NADH and NAD(P)H NR mRNAs in seedling roots and leaves and equally rapid decreases in the concentration of nitrate in the xylem sap. These results indicate that factors affecting nitrate uptake and transport could have a direct influence on NR expression in barley leaves.

Inheritance and expression of NAD(P)H nitrate reductase in barley.

SummaryNADH-specific and NAD(P)H bispecific nitrate reductases are present in barley (Hordeum vulgare L.). Wild-type leaves have only the NADH-specific enzyme while mutants with defects in the NADH nitrate reductase structural gene (nar1) have the NAD(P)H bispecific enzyme. A mutant deficient in the NAD(P)H nitrate reductase was isolated in a line (nar1a) deficient in the NADH nitrate reductase structural gene. The double mutant (nar1a;nar7w) lacks NAD(P)H nitrate reductase activity and has xanthine dehydrogenase and nitrite reductase activities similar to nar1a. NAD(P)H nitrate reductase activity in this mutant is controlled by a single codominant gene designated nar7. The nar7 locus appears to be the NAD(P)H nitrate reductase structural gene and is not closely linked to nar1. From segregating progeny of a cross between the wild type and nar1a;nar7w, a line was obtained which has the same NADH nitrate reductase activity as the wild type in both the roots and leaves but lacks NADPH nitrate reductase activity in the roots. This line is assumed to have the genotype Nar1Nar1nar7nar7. Roots of wild type seedlings have both nitrate reductases as shown by differential inactivation of the NADH and NAD(P)H nitrate reductases by a monospecific NADH-nitrate reductase antiserum. Thus, nar7 controls the NAD(P)H nitrate reductase in roots and in leaves of barley.

NADH-nitrate reductase in barley leaves: Identification and amino acid composition of subunit protein

NADH-nitrate reductase (NADH:nitrate oxidoreductase, EC 1.6.6.1) isolated from barley (Hordeum vulgate L. cv. Steptoe) leaves was identified on a native slab gel after electrophoresis by staining for reduced methyl viologen nitrate reductase activity, NO2− production and diaphorase activity. The nitrate reductase protein, when subjected to a second-dimensional SDS-polyacrylamide gel electrophoresis, migrated as a single protein band of Mr 110000. Thus, barley nitrate reductase (native molecular weight 221000) consists of two identical subunits. Preparative SDS-polyacrylamide gel electrophoresis was used to isolated highly homogeneous nitrate reductase subunit protein. Fluorograms of peptide maps indicated no homology among the nitrate reductase subunit and other polypeptides (60 kDa diaphorase, 41 kDa, 35 kDa and 25 kDa species) which are common contaminants of the affinity-purified nitrate reductase. Thus, the 60 kDa diaphorase and 41 kDa polypeptide observed under our experimental conditions are not breakdown products of the 110 kDa nitrate reductase subunit. The amino acid composition of the barley nitrate reductase subunit was similar to that reported for the nitrate reductase of Chlorella vulgaris and Ankistrodesmus braunii.

Antigenicity of nitrate reductase-deficient mutants in Hordeum vulgare L.

SummaryTen nitrate reductase (NR)-deficient mutants have been characterized for their cross-reactivity against specific barley (Hordeum vulgare L.) nitrate reductase antibodies. The rabbit antibodies raised against the purified barley wild type (cv. Steptoe) enzyme quantitatively inactivate nitrate reductase in crude extracts. All nitrate-grown (induced) mutants show positive precipitin reaction against the antiserum by Ouchterlony double diffusion test and all have the ability to neutralize antisera in a NR protection assay. Under induced growth conditions, mutants Az 12, Az 23, Az 29 and Az 30 which have low NR associated catalytic activities also have the lowest level of antigenicity; mutants Az 13, Az 31, Az 33 and Az 34 have intermediate level of both NR associated catalytic activities and antigenicity, while mutants Az 28 and Az 32 have the highest level of both NR associated catalytic activities and antigenicity. Under noninduced growth conditions, all mutants except Az 12 contain detectable but very low levels of NR antigenicity. These results support the concept that these NR-deficient mutants with various levels of NR associated catalytic activities represent different mutation events at the loci coding the NR structural components.

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

SummaryBarley 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.