Nitrate Reductase mRNA Research Articles

Intact Plastids Are Required for Nitrate- and Light-Induced Accumulation of Nitrate Reductase Activity and mRNA in Squash Cotyledons

Induction of nitrate reductase activity and mRNA by nitrate and light is prevented if chloroplasts are destroyed by photooxidation in norflurazon-treated squash (Cucurbita maxima L.) cotyledons. The enzyme activity and mRNA can be induced if norflurazon-treated squash seedlings are kept in low-intensity red light, which minimizes photodamage to the plastids. It is concluded that induction of nitrate reductase activity and nitrate reductase mRNA requires intact plastids. If squash seedlings grown in low-intensity red light are transferred to photooxidative white light, nitrate reductase activity accumulates during the first 12 hours after the shift and declines thereafter. Thus photodamage to the plastids and the disappearance of nitrate reductase activity and mRNA are events separable in time, and disappearance of the enzyme activity is a consequence of the damage to the plastids.

Functional complementation of tobacco and Nicotiana plumbaginifolia nitrate reductase deficient mutants by transformation with the wild-type alleles of the tobacco structural genes

The homeologous nitrate reductase (NR) structural genes from tobacco were used to complement nitrate reductase deficient mutants from tobacco and Nicotiana plumbaginifolia. A plasmid conferring kanamycin resistance and lambda genomic clones carrying the tobacco wild-type alleles of the genes were co-electroporated in protoplasts of the tobacco mutant. Among 266 plants regenerated from kanamycin resistant colonies, 3 were able to grow permanently on a medium containing nitrate as sole nitrogen source. One of these three plants was further characterized. The ability to grow on nitrate was transmitted as a new single Mendelian dominant marker linked to kanamycin resistance. Molecular analysis of this clone confirmed the integration of a copy of the wild-type allele, and the synthesis at a low level of an active NR. This NR activity is sufficient to regulate both exogenous wild-type and endogenous mutated alleles of the genes at the transcriptional level. A N. plumbaginifolia mutant carrying a mutation impairing NR mRNA production was transformed by Agrobacterium mediated transfer of the wild-type tobacco nia-2 gene cloned into a binary vector. Similarly, kanamycin resistant calli were tested for their ability to grow on nitrate. Among 70 kanamycin resistant transformants, 7 were restored for nitrate assimilation. Molecular analysis revealed the integration of the tobacco gene, and the synthesis at a low level of the NR mRNA and of a nitrate inducible active NR.

Nitrate Reductase mRNA Regulation in Nicotiana plumbaginifolia Nitrate Reductase-Deficient Mutants.

Light and substrate regulation of nitrate reductase (NR) expression were compared in wild type and mutant lines of Nicotiana plumbaginifolia. Mutants affected in the NR structural gene (nia) or in the biosynthesis of the NR molybdenum cofactor (cnx) were examined. nia mutants expressing a defective apoenzyme, as well as cnx mutants, overexpressed NR mRNA, whereas nia mutants devoid of detectable NR protein had reduced or undetectable NR mRNA levels. Diurnal fluctuations of NR mRNA were specifically abolished in nia and cnx mutants, suggesting that the integrity of NR catalytic activity is required for the expression of diurnal oscillations. Unlike some fungal mutants, the nia and cnx mutants examined retained nitrate inducibility of NR expression. The possibility of autogenous control of NR expression in higher plants is discussed.

An unusually rapid light-induced nitrate reductase mRNA pulse and circadian oscillations.

We have investigated the effect of light on nitrate reductase at the mRNA, proteins, and activity levels and found an unusually rapid light-induced release of nitrate reductase mRNA in the form of a sharp pulse

Oxygen Inhibition of Nitrate Reductase Biosynthesis in Detached Corn Leaves via Inhibition of Total Soluble Protein Synthesis

Detached first leaves of 3-day-old corn seedlings (Zea mays L. W64AxW183E) were incubated with nitrate in air or 100% O(2) in the light. Nitrate accumulation in the leaves was not depressed by O(2). NADH:nitrate reductase activity and enzyme protein, as measured with an enzyme-linked immunosorbent assay, increased in parallel during the 8 h nitrate treatment in air, but in O(2) the levels of enzyme activity and protein were depressed. NADH:nitrate reductase mRNA levels were the same in the air-and O(2)-treated leaves. Total soluble protein levels in leaves were slightly depressed by O(2) and shifting from O(2) to an air environment increased the protein level. Incorporation of [(35)S]methionine during nitrate treatment revealed that total soluble protein and nitrate reductase protein synthesis were both depressed by the O(2) environment relative to air, but both recovered when leaves were shifted from O(2) to air. Although O(2) accelerated inactivation of nitrate reductase in vitro, the in vivo inactivation rate appeared to be too low to account for the depressed level of nitrate reductase activity in O(2)-treated leaves. We concluded that O(2) inhibition of nitrate reductase biosynthesis in detached corn leaves was largely due to inhibition of total soluble protein synthesis at the level of translation.

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

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.

Nitrate Effects on Nitrate Reductase Activity and Nitrite Reductase mRNA Levels in Maize Suspension Cultures

Nitrate reductase (NR) activity and nitrite reductase (NiR) mRNA levels were monitored in Black Mexican Sweet maize (Zea mays L.) suspension cultures after the addition of nitrate. Maximal induction occurred with 20 millimolar nitrate and within 2 hours. Both NR and NiR mRNA were transiently induced with levels decreasing after the 2 hours despite the continued presence of nitrate in the medium. Neither ammonia nor chlorate prevented the induction of NR. Furthermore, removal of nitrate, followed by its readdition 22 to 48 hours later, did not result in reinduction of activity or message. NR was synthesized de novo, since cycloheximide completely blocked its induction. Cycloheximide had no effect on the induction of NiR mRNA or on the transient nature of its induction. These results are similar to those reported previously for maize seedlings.

CDNA Clones for Corn Leaf NADH:Nitrate Reductase and Chloroplast NAD(P)+:Glyceraldehyde-3-Phosphate Dehydrogenase

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.

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

Regulation 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.

Regulation by ammonium of nitrate and nitrite assimilation in Chlamydomonas reinhardtii

The inhibitor of mRNA synthesis, 6-methylpurine, inhibited nitrate reductase derepression in either ammonium-grown or methylammonium-treated wild-type cells of Chlamydomonas reinhardtii, but not in nitrogen-starved cells. In contrast, 6-methylpurine did not inhibit nitrate reductase synthesis in the methylammonium-resistant mutant 2170 ( ma-1) either grown on ammonium, treated with methylammonium or nitrogen starved, but did inhibit the continuous synthesis of nitrate reductase, which required the presence of nitrate in the media. In both wild-type and mutant 2170 grown on ammonium and transferred to nitrate media, cycloheximide immediately prevented nitrate reductase derepression when added either at the beginning or at different times of induction treatment. Unlike wild-type cells, mutant 2170 was able to take up either nitrate or nitrite simultaneously with ammonium in whose presence nitrate and nitrite reductases were synthesized. However, synthesis of nitrate reductase was progressively inhibited in the mutant cells when the intracellular ammonium levels were raised as a result of an increase in either the external pH or the extracellular ammonium concentrations. The results rule out the existence of maturase-like proteins in Chlamydomonas and indicate that ammonium has a double effect on the regulation of nitrate reductase synthesis: (a) it prevents nitrate reductase mRNA production; and (b) it controls negatively the expression of this mRNA.

Expression of Leaf Nitrate Reductase Genes from Tomato and Tobacco in Relation to Light-Dark Regimes and Nitrate Supply

The influence of light-dark cycles and nitrate supply on nitrate reductase (NR) mRNA levels was studied in two plant species, tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum) using specific NR DNA probes. In the same series of experiments, changes in the levels of NR protein (NRP) by enzyme-linked immunosorbent assay and changes in the level of NADH-nitrate reductase activity (NRA) were also followed. During a light-dark cycle, it was found that in both tomato and tobacco, NR mRNA accumulation increased rapidly during the dark period and reached a maximum at the beginning of the day, while NRP reached a peak 2 and 4 hours after mRNA peaked, for tomato and tobacco, respectively. At the end of the day, the amount of mRNA was decreased by a factor of at least 100 compared to sunrise in both species. These results demonstrate that light is involved, although probably not directly, in the regulation of the NR gene expression at the mRNA level. The peak of NRA in tobacco coincided with the peak in NR mRNA accumulation (i.e. sunrise), whereas in tomato the peak of NRA was approximately 5 to 6 hours after sunrise. There is no obvious correlation between NRP and NRA levels during the day. In nitrogen starvation experiments, a rapid decrease of NRP and NRA was detected, while NR mRNA levels were not significantly altered. Upon nitrate replenishment, nitrogen-starved plants accumulated NR mRNA rapidly. These results suggest that the availability of nitrogen affects the expression of NR activity at the transcriptional as well as at the post-transcriptional levels.

Cloning of DNA fragments complementary to tobacco nitrate reductase mRNA and encoding epitopes common to the nitrate reductases from higher plants

SummaryMessenger RNAs encoding the nitrate reductase apoenzyme from tobacco can be translated in a cell-free system. Poly(A)+ mRNA fractions from the 23-32 S area of a sucrose gradient were used to build a cDNA library in the expression vector λgt11 with an efficiency of cloning of approximately 104 recombinants/ng mRNA. Recombinant clones were screened with a rabbit polyclonal antibody directed against the corn nitrate reductase, which cross reacts specifically with the nitrate reductases from dicotyledons. Among 240000 recombinant plaques, eight clones were isolated containing inserts of sizes ranging from 1.6 kb to 2.1 kb and sharing sequence homologies. Seven of these clones contained a common internal 1.6 kb EcoRI fragment. The identity of these clones was confirmed as follows. A fusion protein of 170 kDa inducible by IPTG and recognized by the rabbit nitrate reductase antibody was expressed by a lysogen derived from one of the recombinants. The antibodies binding the fused protein were eluted and shown to be inhibitory to the catalytic activity of tobacco nitrate reductase. Two monoclonal antibodies directed against nitrate reductase were also able to bind the hybrid protein. The 1.6 kb EcoRI fragment was sequenced by the method of Sanger. The open reading frame corresponding to a translational fusion with the β-galactosidase coding sequence of the vector shared strong homology at the amino acid level with the heme-binding domain of proteins of the cytochrome b5 superfamily and with human erythrocyte cytochrome b5 reductase. When the 1.6 kb EcoRI fragment was used as a probe for Northern blot experiments a signal corresponding to a 3.5 kb RNA was detected in tobacco and in Nicotiana plumbaginifolia mRNA preparations but no cross-hybridization with corn mRNAs was detected. The probe hybridized with low copy number sequences in genomic blots of tobacco DNA.

Evidence for the transcriptional control of nitrate reductase in Candida nitratophila from in vitro translation studies.

In vivo labelling and in vitro translation studies were used to study the regulation of the synthesis of nitrate reductase in the yeast Candida nitratophila. These studies showed that synthesis of the enzyme subunit took place when ammonium-grown cells were nitrogen-starved and this was stimulated by subsequent addition of nitrate. Ammonium-grown cultures did not contain mRNA that could be translated into the nitrate reductase subunit in an in vitro system. Nitrate reductase mRNA could be extracted from nitrogen-starved and nitrate cultures. Synthesis of the enzyme is apparently controlled at the level of transcription in this yeast.

Nitrate reductase from squash: cDNA cloning and nitrate regulation

The assimilation of nitrate in plants involves the reduction of nitrate to ammonia in two steps. The first step requires nitrate reductase, a nitrate-inducible enzyme. When seedlings of squash (Cucurbita maxima L.) were treated with nitrate, both nitrate reductase activity and protein were induced in the cotyledons. Poly(A)(+) RNA was prepared from cotyledons of nitrate-treated seedlings and was used to construct a lambdagt11 cDNA library. Using antibodies from mice immunized against purified nitrate reductase from squash, a recombinant lambda phage was isolated that encoded part of the nitrate reductase mRNA. The antigens produced by the recombinant phage were used to affinity purify anti-nitrate reductase antibody from ascites fluid of immunized mice. The purified antibody bound to nitrate reductase protein on immunoblots and immunoprecipitated the enzyme from squash protein extracts. The cDNA insert (1.2 kilobases) hybridized to a 3.2-kilobase RNA that was 120-fold more abundant in nitrate-induced cotyledons compared with the uninduced tissue.

Cloning and nitrate induction of nitrate reductase mRNA

Nitrate is the major source of nitrogen taken from the soil by higher plants but requires reduction to ammonia prior to incorporation into amino acids. The first enzyme in the reducing pathway is a nitrate-inducible enzyme, nitrate reductase (EC 1.6.6.1). A specific polyclonal antiserum raised against purified barley nitrate reductase has been used to immunoprecipitate in vivo labeled protein and in vitro translation products, demonstrating that nitrate induction increases nitrate reductase protein and translatable mRNA. A partial cDNA clone for barley nitrate reductase has been isolated and identified by hybrid-selected translation. RNA blot-hybridization analysis shows that nitrate induction also causes a marked increase in the steady-state level of nitrate reductase mRNA.