Nitrite Reductase mRNA Research Articles

Nitrite reductase (nii2) gene has a positive role in nitrogen metabolism in tobacco

Nitrite reductase (NiR) is an enzyme that can reduce the concentration of nitrite. Nitrite is one of the precursors of tobacco-specific nitrosamines (TSNA), which are potentially carcinogenic products and exist only in tobacco and tobacco products. In this study, we analyzed the over expression of ferredoxin-nitrite reductase nii2 gene (EC 1.7.7.1) in tobacco to determine the reduction of nitrate and nitrite in response to its increase in the plants. Accordingly, the Ntnii2 gene was cloned from tobacco based on the information from its wild-type (WT) homologous genes and it contained 1764 bp encoding the polypeptides of 587 amino acids. This gene was inserted into plant expression vector pBI121 and Agrobacterium-mediated transformation results showed 23 transgenic tobacco lines through kanamycin resistance screening. After PCR-based screening, Ntnii2 gene was confirmed to be successfully transcript and inherited 16 offspring’s and 8 lines were selected for further generation. The NiR mRNA levels, in vivo and in vitro NR activities were significantly higher in those selected eight transgenic lines than its counterpart WT and approximately 30% increase NiR activity in some transgenic lines. The nitrate content showed nearly 46% reductions. Although the nitrite contents were not considerably differed from that of non-transformed plants, most of the transgenic lines showed low nitrite level than wild-type plants. Therefore, transgenic plants with low level of nitrate and nitrite were produced. Key words: Nitrite reductase, over expression, nitrate, nitrite, tobacco-specific nitrosamines (TSNA).

Differential expression of the nitrite reductase gene family in tobacco as revealed by quantitative competitive RT-PCR.

Tobacco (Nicotiana tabacum L. cv. Xanthi XHFD8) possesses four nitrite reductase (NiR) genes: nii1, nii2, nii3, and nii4. Their differential expression in leaves and roots was investigated by quantitative competitive RT-PCR using gene-specific primer pairs. These results appear to contradict existing views on the expression of these NiR genes: (i) the mRNA of each of the four NiR genes was distinguishable both in leaves and roots; (ii) nitrate treatment increased nii1 and nii3 mRNA in leaves and roots by at least 4-fold (at least 5-fold in nii2 and nii4 mRNA); and (iii) the steady-state levels of nii1 and nii3 mRNA were almost the same in leaves (6-7 x 10(5) and about 3 x 10(6) copies microg(-1) of total RNA before and after nitrate treatment, respectively) and in roots (3-4 x 10(4) and 3-6 x 10(5) copies microg(-1) of total RNA before and after nitrate treatment, respectively). Very similar relationships were obtained for the steady-state levels of nii2 and nii4 mRNA in roots (2-4 x 10(5) and 8 x 10(6) copies microg(-1) of total RNA before and after nitrate treatment, respectively), and in leaves (5-9 x 10(4) and 4 x 10(5) copies microg(-1) of total RNA before and after nitrate treatment, respectively). These results demonstrate that nii1 and nii3 transcripts are a dominating, but not exclusive, NiR mRNA in leaves, and the same is true for nii2 and nii4 transcripts in roots.

Short communication: emission of nitrous oxide (N2O) from transgenic tobacco expressing antisense NiR mRNA

The emission of N2 and N2O from intact transgenic tobacco (clone 271) expressing antisense nitrite reductase (NiR) mRNA, and wild-type plants grown aseptically, on NO3-, NO2- or NH4+ -containing medium was investigated. 15N contents of gas sampled from gas-sealed pots, in which the plants were grown on 15N-containing medium, were analyzed by gas chromato- graphy and mass spectrometry (GC-MS). No emission of N2 was detected in either of the gas samples from plant clone 271 or the wild-type grown on NO3--containing medium. N2O emission from clone 271 grown on NO3--containing medium was detected, but not from the wild-type plants. The N2O emission rate of clone 271 was 106 ng N2O mg-1 incorporated N week-1 and the N2O emission was inhibited by tungstate (a nitrate reductase inhibitor). No emission of N2O was found from clone 271 or wild-type plants grown on medium containing NH4+. Emission of N2O also was detected from clone 271 grown on NO2--containing medium and its emission rate increased with increasing NO2- levels in plants. We speculate that NO3- is reduced to NO2- and that a part of NO2- is metabolized to N2O in clone 271.

A novel autophosphorylation mediated regulation of nitrite reductase in Candida utilis

The assimilatory nitrite reductase catalyses the conversion of nitrite to ammonia. The enzyme from Candida utilis has been previously purified to homogeneity and shown to be a heterodimer consisting of 58 kDa and 66 kDa subunits. The enzyme has also been shown to be induced by nitrate and repressed by ammonium ions. The levels of nitrite reductase mRNA, its protein and the enzyme activity were modulated together indicating that the primary level of regulation of this enzyme existed at the transcriptional level. Here we report that the 58 kDa and 66 kDa subunits of the enzyme were differentially phosphorylated under the induced and repressed conditions, indicating a second level of regulation. The highly phosphorylated 66 kDa subunit was shown to be dephosphorylated by calf intestinal alkaline phosphatase. The enzymatic activity associated with the native enzyme also decreased due to the dephosphorylation. Each of the subunits could undergo autophosphorylation at serine/threonine residues as demonstrated by thin layer chromatography and recognition by antibodies to phosphoamino acids. The presence of similar phosphorylated subunits under in vivo conditions has also been demonstrated. A model has been proposed to explain the post-translational regulation of the enzyme.

In vitro and in vivo regulation of assimilatory nitrite reductase from Candida utilis.

The nitrate assimilation pathway in Candida utilis, as in other assimilatory organisms, is mediated by two enzymes: nitrate reductase and nitrite reductase. Purified nitrite reductase has been shown to be a heterodimer consisting of 58- and 66-kDa subunits. In the present study, nitrite reductase was found to be capable of utilising both NADH and NADPH as electron donors. FAD, which is an essential coenzyme, stabilised the enzyme during the purification process. The enzyme was modified by cysteine modifiers, and the inactivation could be reversed by thiol reagents. One cysteine was demonstrated to be essential for the enzymatic activity. In vitro, the enzyme was inactivated by ammonium salts, the end product of the pathway, proving that the enzyme is assimilatory in function. In vivo, the enzyme was induced by nitrate and repressed by ammonium ions. During induction and repression, the levels of nitrite reductase mRNA, protein, and enzyme activity were modulated together, which indicated that the primary level of regulation of this enzyme was at the transcriptional level. When the enzyme was incubated with ammonium salts in vitro or when the enzyme was assayed in cells grown with the same salts as the source of nitrogen, the residual enzymatic activities were similar. Thus, a study of the in vitro inactivation can give a clue to understanding the mechanism of in vivo regulation of nitrite reductase in Candida utilis.

Nitrite reductase expression is regulated at the post‐transcriptional level by the nitrogen source in Nicotiana plumbaginifolia and Arabidopsis thaliana

Higher plant nitrite reductase (NiR) is a monomeric chloroplastic protein catalysing the reduction of nitrite, the product of nitrate reduction, to ammonium. The expression of this enzyme is controlled at the transcriptional level by light and by the nitrogen source. In order to study the post-transcriptional regulation of NiR, Nicotiana plumbaginifolia and Arabidopsis thaliana were transformed with a chimaeric NiR construct containing the tobacco leaf NiR1 coding sequence driven by the CaMV 35S RNA promoter. Transformed plants did not show any phenotypic difference when compared with the wild-type, although they overexpressed NiR activity in the leaves. When these plants were grown in vitro on media containing either nitrate or ammonium as sole nitrogen source, NiR mRNA derived from transgene expression was constitutively expressed, whereas NiR activity and protein level were strongly reduced on ammonium-containing medium. These results suggest that, together with transcriptional control, post-transcriptional regulation by the nitrogen source is operating on NiR expression. This post-transcriptional regulation of tobacco leaf NiR1 expression was observed not only in the closely related species N. plumbaginifolia but also in the more distant species A. thaliana.

The expression of the tobacco genes encoding plastidic glutamine synthetase or ferredoxin-dependent glutamate synthase does not depend on the rate of nitrate reduction, and is unaffected by suppression of photorespiration

The expression of the tobacco genes encoding the ammonium-assimilating enzymes plastidic glutamine synthetase (GS-2) or ferredoxin-dependent glutamate synthase (Fd-GOGAT) was examined in relation to the rates of two major ammonium-producing processes in leaves, i.e. nitrate reduction and photorespiration. Impairment of nitrate reduction induced by the application of tungstate, a molybdate analogue inhibiting nitrate reductase (NR), resulted in an increase in the NR and the nitrite reductase (NiR) mRNA pools. It is shown that this effect could in part be reversed by the application of ammonium. These results support negative regulation by nitrate-derived N-metabolites of both the NR and NiR mRNA abundance. Both the GS-2 and the Fd-GOGAT transcript level and the corresponding enzymatic activities remained largely unaffected by impairment of nitrate reduction, suggesting that both GS-2 and Fd-GOGAT gene expression are not under the control by end products of nitrogen assimilation. Growth of tobacco at a CO 2 partial pressure sufficiently high to suppress photorespiration (300 Pa CO 2 ) had no effect on either the GS-2 or Fd-GOGAT transcript levels. It is concluded that the rate of photo-respiratory ammonium production does not exert a metabolic control on GS-2 or Fd-GOGAT gene expression.

Dynamics of denitrification activity of Paracoccus denitrificans in continuous culture during aerobic-anaerobic changes.

Induction and repression of denitrification activity were studied in a continuous culture of Paracoccus denitrificans during changes from aerobic to anaerobic growth conditions and vice versa. The denitrification activity of the cells was monitored by measuring the formation of denitrification products (nitrite, nitric oxide, nitrous oxide, and dinitrogen), individual mRNA levels for the nitrate, nitrite, and nitrous oxide reductases, and the concentration of the nitrite reductase enzyme with polyclonal antibodies against the cd1-type nitrite reductase. On a change from aerobic to anaerobic respiration, the culture entered an unstable transition phase during which the denitrification pathway became induced. The onset of this phase was formed by a 15- to 45-fold increase of the mRNA levels for the individual denitrification enzymes. All mRNAs accumulated during a short period, after which their overall concentration declined to reach a stable value slightly higher than that observed under aerobic steady-state conditions. Interestingly, the first mRNAs to be formed were those for nitrate and nitrous oxide reductase. The nitrite reductase mRNA appeared significantly later, suggesting different modes of regulation for the three genes. Unlike the mRNA levels, the level of the nitrite reductase protein increased slowly during the anaerobic period, reaching a stable value about 30 h after the switch. All denitrification intermediates could be observed transiently, but when the new anaerobic steady state was reached, dinitrogen was the main product. When the anaerobic cultures were switched back to aerobic respiration, denitrification of the cells stopped at once, although sufficient nitrite reductase was still present. We could observe that the mRNA levels for the individual denitrification enzymes decreased slightly to their aerobic, uninduced levels. The nitrite reductase protein was not actively degraded during the aerobic period.

Isolation and Characterization of Nitrate Reductase-Deficient Mutants of Cultured Spinach Cells: Biochemical, Immunological and mRNA Analysis

Nitrate reductase (NR)-deficient mutants of cultured spinach cells were selected by chlorate resistance. Two mutants (12F and I-1 cell lines) were isolated and analyzed for activities, protein levels, and the expression of mRNAs of NR and nitrite reductase (NiR). When mutant cells were transferred to a medium containing 20 mM KNO 3 and 20 mM NH 4 NO 3 as a nitrogen source, the 12F cell line had no detectable NR activity or NR protein, but the NR mRNA was expressed clearly in contrast with the data of activity and protein levels. The NiR activity and protein were still induced and the NiR mRNA was also expressed clearly. In the I-1 cell line, however, the NR activity and protein were still present at low levels, and the NiR activity and protein were no longer inducible. Extremely low levels of the NR and NiR mRNAs were found in the I-1 cell line. These results suggest that the 12F and I-1 cell lines are structural gene mutant and regulatory gene mutant, respectively.

Inhibition of tobacco nitrite reductase activity by expression of antisense RNA

A tobacco nitrite reductase (NiR) cDNA and its corresponding gene were isolated from cDNA and genomic libraries. An NiR antisense mRNA was expressed in transgenic tobacco under the control of a double 35S promoter. Transformants were obtained on a medium containing ammonium as the sole source of nitrogen. One plant growing normally on ammonium but displaying drastically reduced development and chlorotic leaves when grown on nitrate as the sole source of nitrogen was studied further. This plant accumulated nitrite fivefold over wild-type level and showed reduced amounts of ammonium (11% wild-type level), glutamine (19%), and total protein (8%). NiR mRNA and activity were below detectable levels. Under these conditions, nitrate reductase (NR) activity and mRNA were overexpressed, suggesting that N-metabolites resulting from nitrate reduction are responsible for the repression of the expression of the NR gene, independently from the presence or absence of a functional NR protein.

Effect of Chlorate Treatment on Nitrate Reductase and Nitrite Reductase Gene Expression in Arabidopsis thaliana

The herbicide chlorate has been used extensively to isolate mutants that are defective in nitrate reduction. Chlorate is a substrate for the enzyme nitrate reductase (NR), which reduces chlorate to the toxic chlorite. Because NR is a substrate (NO(3) (-))-inducible enzyme, we investigated the possibility that chlorate may also act as an inducer. Irrigation of ammonia-grown Arabidopsis plants with chlorate leads to an increase in NR mRNA in the leaves. No such increase was observed for nitrite reductase mRNA following chlorate treatment; thus, the effect seems to be specific to NR. The increase in NR mRNA did not depend on the presence of wild-type levels of NR activity or molybdenum-cofactor, as a molybdenum-cofactor mutant with low levels of NR activity displayed the same increase in NR mRNA following chlorate treatment. Even though NR mRNA levels were found to increase after chlorate treatment, no increase in NR protein was detected and the level of NR activity dropped. The lack of increase in NR protein was not due to inactivation of the cells' translational machinery, as pulse labeling experiments demonstrated that total protein synthesis was unaffected by the chlorate treatment during the time course of the experiment. Chlorate-treated plants still retain the capacity to make functional NR because NR activity could be restored by irrigating the chlorate-treated plants with nitrate. The low levels of NR protein and activity may be due to inactivation of NR by chlorite, leading to rapid degradation of the enzyme. Thus, chlorate treatment stimulates NR gene expression in Arabidopsis that is manifested only at the mRNA level and not at the protein or activity level.

Effect of Light/Dark Cycles on Expression of Nitrate Assimilatory Genes in Maize Shoots and Roots

The level of nitrate reductase (NR) and nitrite reductase (NiR) varied in both shoot and root tissue from nitrate-fed Zea mays L. grown under a 16-hour light/8-hour dark regime over a 10-day period postgermination, with peak activity occurring in days 5 to 6. To study the effect of different light regimes on NR and NiR enzyme activity and mRNA levels, 6-day-old plants were grown in the presence of continuous KNO(3) (10 millimolar). Both shoot NRA and mRNA varied considerably, peaking 4 to 8 hours into the light period. Upon transferring plants to continuous light, the amplitude of the peaks increased, and the peaks moved closer together. In continuous darkness, no NR mRNA or NR enzyme activity could be detected by 8 hours and 12 hours, respectively. In either a light/dark or continuous light regime, root NRA and mRNA did not vary substantially. However, when plants were placed in continuous darkness, both declined steadily in the roots, although some remained after 48 hours. Although there was no obvious cycling of NiR enzyme activity in shoot tissue, changes in mRNA mimicked those seen for NR mRNA. The expression of NR and NiR genes is affected by the light regime adopted, but light does not have a direct effect on the expression of these genes.

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.

Transient Accumulation of Nitrite Reductase mRNA in Maize following the Addition of Nitrate

Expression of the gene coding for nitrite reductase (NiR) is induced upon the addition of nitrate. We have analyzed this induction process in hydroponically grown maize (Zea mays L.) seedlings where the level of nitrate in the medium can be easily manipulated. There is a rapid induction of NiR mRNA upon addition of nitrate, increasing first in the roots and then in the leaves. The rapidity of the response depends on the nitrate concentration and the growth medium. However, the general pattern of expression is the same: the mRNA level increases, reaches a maximum, and then decreases, despite the fact that the nitrate concentration in the medium remains constant. This decline in mRNA level can be quite rapid, particularly in root tissue. If the nitrate is given as a pulse, the mRNA levels decrease even more rapidly. It is clear that the NiR mRNA is short-lived, with a half-life in the roots of less than 30 minutes. The NiR protein level, on the other hand, increases gradually somewhat after the increase in mRNA and remains at high levels at least for 24 hours after the addition of nitrate.

Molecular Cloning of Complementary DNA Encoding Maize Nitrite Reductase

Complementary DNA has been isolated that codes for maize nitrite reductase (NiR) by using the corresponding spinach gene (E Back et al. 1988 Mol Gen Genet 212:20-26) as a heterologous probe. The sequences of the complementary DNAs from the two species are 66% homologous while the deduced amino acid sequences are 86% similar when analogous amino acids are included. A high percentage of the differences in the DNA sequences is due to the extremely strong bias in the corn gene to have a G/C base in the third codon position with 559/569 codons ending in a G or C. Using a hydroponic system, maize seedlings grown in the absence of an exogenous nitrogen source were induced with nitrate or nitrite. Nitrate stimulated a rapid induction of the NiR mRNA in both roots and leaves. There is also a considerable induction of this gene in roots upon the addition of nitrite, although under the conditions used the final mRNA level was not as high as when nitrate was the inducer. There is a small but detectable level of NiR mRNA in leaves prior to induction, but no constitutive NiR mRNA can be seen in the roots. Analysis of genomic DNA supports the notion that there are at least two NiR genes in maize.