NR Gene Expression Research Articles

Involvement of nitrate reductase in the ameliorating effect of 5-aminolevulinic acid on NaCl-stressed barley seedlings

Exogenous application of 5-aminolevulinic acid (ALA) is widely used to decrease the effect of different abiotic stresses in various crops while the nitrogen metabolism is a possible target of ALA in plants. We investigated the effect of exogenous ALA on growth, activity of nitrate reductase (NR; EC 1.6.6.1), synthesis of proline and ROS generation in seedlings of the salt-sensitive barley cultivar (Hordeum vulgare L. cv. Gonar). Obtained data indicated that the ALA (20, 40 and 80 mg L−1) increased elongation and leaf blade expansion of barley seedlings exposed to 150 mM NaCl as compared to control plants, which were not treated by ALA. NR gene (Nar1) expression, NR protein content and enzymatic activities increased in ALA-treated plants. ALA also induced accumulation of free proline and decreased generation of superoxide anion radicals. Overall, this study demonstrates that ALA effects on salt-stressed barley involves changes of NR activities both at transcriptional and translational levels and leads to complex physiological modifications, such as improvement of growth and antiradical defense.

Lipoteichoic acid modulates hepatic drug metabolizing enzymes by activating the Toll‐like receptor 2 signaling pathway

Background:Altered expression of drug metabolizing enzymes (DMEs) in inflammation can affect the metabolism and toxicity of drugs. It is known that inflammatory responses are induced in immune cells by Lipopolysaccahride (LPS) and Lipoteichoic acid (LTA), derived from gram −ve and gram +ve bacteria, respectively. We reported that LPS modulates gene expression of hepatic DMEs and their regulatory nuclear receptors (NRs: PXR, CAR and RXRα) by activating Toll‐like receptor (TLR) 4 signaling pathway. We hypothesize that LTA can modulate NR and DME gene expression by activating the TLR2 signaling pathway. Methods: Adult male C57BL/6 mice were i.p. injected with 6 μg/g bw LTA or saline. Livers were harvested and RNA and proteins were analyzed by real‐time PCR and western blotting, respectively.Results:LTA treatment for 2h resulted in ∼80% suppression of CAR and target DME genes, Cyp2b10, Cyp2a4 and Sultn. The NR, PXR, and the main phase 1 DME, Cyp3a11 were reduced ∼70% by 4–8 h of LTA treatment. Nuclear protein levels of RXRα were reduced in LTA‐treated mice. LTA injection induced cytokine production and cell‐signaling components in the liver. The effects of LTA on NRs, DMEs, cytokines and cell‐signaling pathways were blocked in TLR2−/− mice, indicating that the effects of LTA on hepatic genes were mediated by TLR2.Conclusions:TLR2 is involved in regulating DME gene expression during induction of inflammation by LTA.

The main origin of endogenous NO in higher non-leguminous plants

The main origin of nitric oxide (NO) production in higher non-leguminous plants is studied in this paper. NO content in the leaf of wheat ( Triticum aestivum L.), orchid ( Malaxis monophyllos L.) and aloe ( Aloe vera L.) was decreased over 90% by the treatment of heat or microwave. It indicates that NO is primarily enzymically produced in higher non-leguminous plants. Nitrate reductase (NR; EC 1.6.6.1) activity and NO content were about 74% and 56%, respectively, in wheat seedling grown in a medium lacking molybdenum. Sodium tungstate almost completely inhibited NR activity and decreased NO content near 90% in the wheat seedling extraction. Taking out inducing factors such as KNO 3 and light of NR gene expression, the NR activity was less than 30% and the NO content was less than 20% of the control in the wheat seedling. These results suggest that NO is mainly produced through NR pathway in wheat seedlings. When NaNO 2 was applied to wheat seedlings for 10 h in dark before harvest, the NO content vastly increased but dramatically decreased after treatment by heat or microwave. The provision of NaNO 2 as above and added exogenous Cu 2+ decreased both NR and nitrite reductase (NiR; EC 1.7.7.1) activity, but increased NO content. These results suggest that the conversion from NO 2 – to NO in wheat seedling is also enzymic and this process is independent of NR and NiR.

A short-term exposure of cucumber plants to rising atmospheric CO2 increases leaf carbohydrate content and enhances nitrate reductase expression and activity.

Nitrate reductase (NR; EC 1.6.6.1) is the first enzyme of the nitrate-assimilatory pathway and is regulated transcriptionally and post-translationally by several metabolic and environmental signals. To investigate whether NR is controlled by the rate of photosynthetic CO2 assimilation in cucumber (Cucumis sastivus L.), intact plants were exposed, after the dark period, to light under different atmospheric CO2 concentrations (100, 400 and 2,000 microL x L(-1)) for 2 h. The in-vivo rates of net CO2 assimilation correlated with atmospheric CO2 concentrations. The CO2-fixation rate under 2,000 microL x L(-1) CO2 was 2.4- and 5.4-fold higher than under 400 and 100 microL x L(-1), respectively. Stomatal conductances and transpiration rates were almost identical after the 2-h light period under the various CO2 concentrations tested. Increasing atmospheric CO2 concentrations caused concomitant increases in the contents of starch and soluble sugars in the leaves and a decrease in the nitrate content. The activity and activation state of NR were both higher under elevated CO2 than under low CO2. High CO2 also enhanced NR-gene expression in the leaves. Sugars were supplied via roots to intact carbohydrate-starved plants and NR mRNA levels were analysed after 7 h. Fructose markedly stimulated NR-gene transcription in both leaves and roots. It is concluded that, in cucumber plants, the rate of CO2 assimilation controls the rate of nitrate assimilation by modulation of NR expression and activity, and that sugars are presumably involved as regulatory metabolites.

Negative regulation of nitrate reductase gene expression by glutamine or asparagine accumulating in leaves of sulfur-deprived tobacco.

Tobacco (Nicotiana tabacum L.) plants were subjected to a prolonged period of sulfur-deprivation to characterize molecular and metabolic mechanisms that permit control of primary N-metabolism under these conditions. Prior to the appearance of chlorotic lesions, sulfur-deprived tobacco leaves showed a strong decrease in the sulfate content and changes in foliar enzyme activities, mRNA accumulation and amino-acid pools. The basic amino acids glutamine, asparagine and arginine accumulated in the leaves of sulfur-deprived plants, while the foliar concentrations of aspartate, glutamate, serine or alanine remained fairly unchanged. Maximal extractable nitrate reductase (NR; EC 1.6.6.1) activity decreased strongly in response to sulfur-deprivation. The decrease in maximal extractable NR activity was accompanied by a decline in NR transcripts while the mRNAs of the plastidic glutamine synthetase (EC 6.1.3.2) or the beta-subunit of the mitochondrial ATP synthase were much less affected. Nitrate first accumulated in leaves of tobacco during sulfur-deprivation but then declined. An appreciable amount of nitrate was, however, present in severely sulfur-depleted leaves. The repression of NR gene expression is, therefore, not related to the decrease in the leaf nitrate level. However, glutamine- and/or asparagine-mediated repression of NR gene transcription is a possible mechanism of control in situations when glutamine and asparagine accumulate in leaves and provides a feasible explanation for the reduction in NR activity during sulfur-deprivation. The removal of reduced nitrogen from primary metabolism by redirection and storage as arginine, asparagine or glutamine combined with the down-regulation of nitrate reduction via glutamine- and/or asparagine-mediated repression of NR gene transcription may contribute to maintaining a normal N/S balance during sulfur-deprivation and indicate that the co-ordination of N- and S-metabolism is retained under these conditions.

Coaction of light and the nitrogen substrate in controlling the expression of the tomato genes encoding nitrite reductase and nitrate reductase

Summary The co-action of light and the nitrogen source in the regulation of the expression of the genes encoding nitrite reductase (NiR; EC 1.7.7.1) or nitrate reductase (NR; EC 1.6.6.1) was investigated in the cotyledons of dark- and white light-grown tomato ( Lycopersicon esculentuni ) seedlings. Light acting at red/far-red or at blue regions of the spectrum stimulated both the NiR transcript and the NiR protein level in dark-grown tomato seedling cotyledons, and resulted in an increase in NiR activity. The light-stimulation of tomato NiR gene expression does not depend on the availability of nitrate. Thus, the mode of co-action between light and nitrate in the regulation of NiR gene expression in tomato is different than in all other plant species examined so far. In the cotyledons of tomato seedlings grown during a 16-h photoperiod in white light, nitrate is required for maximum NiR gene expression. Glutamine seems to repress NiR protein expression in green cotyledons. Taken together, our results suggest that the mode of regulation by light and nitrate of the NiR protein level in white light-grown tomato seedling cotyledons is different than during the illumination of etiolated tomato cotyledons. In etiolated and in green tomato seedling cotyledons, both light and nitrate were required for the induction of NR transcripts, NR protein and N R activity. This result confirms a synergistic coaction of both factors in the regulation of NR gene expression.

An ethylene-inducible component of signal transduction encoded by never-ripe.

The ripening-impaired tomato mutant Never-ripe (Nr) is insensitive to the plant hormone ethylene. The gene that cosegregates with the Nr locus encodes a protein with homology to the Arabidopsis ethylene receptor ETR1 but is lacking the response regulator domain found in ETR1 and related prokaryotic two-component signal transducers. A single amino acid change in the sensor domain confers ethylene insensitivity when expressed in transgenic tomato plants. Modulation of NR gene expression during fruit ripening controls response to the hormone ethylene.

The effects of deregulation of NR gene expression on growth and nitrogen metabolism ofNicotiana plumbaginifoliaplants

Transformed Nicotiana plumbaginifolia plants, constitutively expressing a chimaeric nitrate reductase gene (nia2 gene transcript fused to the constitutive CaMV 35S promoter), were cultivated in greenhouses at INRA, Versailles (from October to December 1991). They were supplied with either 12 mM or 1 mM NO 3 - , and given either a 12 h or a 24 h photoperiod. These plants exhibited a nitrate reductase activity in the leaves 25-150% higher than that of the wild-type plants. The physiological consequences of this deregulated expression were further characterized

Control of Nitrate Reductase and Nitrite Reductase Gene Expression by Light, Nitrate and a Plastidic Factor

AbstractIn the present review we consider regulation of the build‐up of the nitrate reducing apparatus (NO−3 → NH+4) in the course of light‐mediated plastidogenesis. In particular, the regulation of the appearance of nitrate reductase (NR, EC 1.6.6.1) and nitrite reductase (NIR, EC 1.7.7.1) will be treated. While both enzymes are nuclear‐encoded, NR is a cytosolic and NIR a plastidic protein. Substrate induction by nitrate, control by light (phytochrome) and dependence on a ‘plastidic factor’ are the main features of the control system.In mustard (Sinapis alba L.) seedling cotyledons the NR‐mRNA level was determined by a strong synergistic coaction of nitrate and light. Synthesis of NR (a cytosolic protein with two isoforms) correlated with the level of NR mRNA. A positive control of NR gene expression by the plastidic factor was observed. It appeared that the plant cell regulates NR gene expression as if NR would be a plastidic protein.Control of gene expression in the case of plastidic NIR in the same mustard cotyledons was conspicuously different. Whereas, in case of NR, the transcript level was determined by a synergism between light and nitrate, in the case of NIR the transcript level was controlled by light alone, with nitrate being ineffective. However, in both cases actual enzyme synthesis was controlled by nitrate, and the dependence on positive control by the plastidic factor was the same. Thus, the difference between NR and NIR gene expression appears to be a matter of transcriptional control. From a teleonomic viewpoint, the control system to establish the nitrate‐reducing apparatus appears (almost) perfect.In spinach (Spinacia oleracea L.) seedling cotyledons the NIR transcript level was unaffected by light but determined by nitrate. The strong action of light on NIR synthesis was multiplicatively superimposed on the action of nitrate, consistent with the conclusion that nitrate affects the transcript level while light controls enzyme synthesis.In tobacco (Nicotiana tabacum L., cv. Coker 176) seedlings the NIR transcript level was determined by a synergistic action between nitrate and light, i.e. both factors coact at the level of transcription. There was no effect of light on NIR synthesis in the absence of nitrate while a strong action of light on protein synthesis was observed in the presence of nitrate. The specific effect of blue light on enzyme appearance, i.e. an effect which cannot be attributed to phytochrome, was not seen at the transcript level.Thus, in tobacco a coaction of nitrate and light (phytochrome) is required to bring about a high NIR transcript level, while in mustard the NIR transcript level was determined by phytochrome alone and in spinach by nitrate alone. It is obvious that in different plants phytochrome and nitrate control NIR gene expression differently. Only the dependence on the plastidic factor appears to be the same in all cases studied so far.The conspicuous regulatory differences between spinach and tobacco offer a chance to address the crucial question of whether the NIR‐promoter from spinach fused to a reporter gene and introduced in tobacco, responds to nitrate and phytochrome in accordance with spinach (donor) or in accordance with tobacco (host). Experiments are in progress.

Regulation of transcript level and synthesis of nitrate reductase by phytochrome and nitrate in turions of Spirodela polyrhiza (L.) Schleiden

Control by light and nitrate of the appearance of nitrate reductase (NR; EC 1.6.6.1) in the turions of Spirodela polyrhiza (L.) Schieiden was investigated during the pre-germination phase, i.e. up to 120 h after the transfer from after-ripening to germination conditions. Turions are particularily suited for this type of coaction study since control of nitrate uptake by light and long-distance transport of nitrate do not play a role. Control of NR gene expression was mainly studied between 72 and 120 h after transfer to germination conditions (phase II of the pre-germination process). It was found that the effect of light on NR appearance is exerted via phytochrome. The light effect on enzyme synthesis was only observed in the presence of nitrate. On the other hand, in darkness, the NR level decreased to almost zero even in the presence of an optimum supply of nitrate. It is concluded that in phase II a coaction of light (phytochrome) and nitrate is required to bring about NR synthesis. However, when nitrate was applied to turions - following a dark incubation without nitrate - the turions responded to nitrate even in darkness with strong NR synthesis. This response was augmented by light. The highest transcript levels were observed in the presence of both factors, light and nitrate. As a single factor, light was more effective in stimulating the transcript level than nitrate. However, no correlation between the transcript level and the rate of enzyme synthesis was observed. This is consistent with the previous conclusion (Schuster et al. 1988, Planta 174, 426-432) that in higher plants control at the transcript level is only coarse and does not determine quantitatively the output at the level of enzyme protein.

Nitrate-reductase expression is under the control of a circadian rhythm and is light inducible in Nicotiana tabacum leaves

Over a 24-h light-dark cycle, the level of mRNA coding for nitrate reductase (NR; EC 1.6.6.1) in the leaves of nitrate-fed Nicotiana tabacum L. plants increased throughout the night and then decreased until it was undetectable during the day. The amount of NR protein and NR activity were two-fold higher during the day than at night. When plants were transferred to continuous light conditions for 32 h, similar variations in NR gene expression, as judged by the above three parameters, still took place in leaf tissues. On the other hand, when plants were transferred to continuous dark conditions for 32 h, the NR-mRNA level continued to display the rhythmic fluctuations, while the amount of NR protein and NR activity decreased constantly, becoming very low, and showed no rhythmic variations. After 56 h of continuous darkness, the levels of NR mRNA, protein and activity in leaves all became negligible, and light reinduced them rapidly. These results indicate the circadian rhythmicity and light dependence of NR expression.

Effect of nitrate, ammonium, light and a plastidic factor on the appearance of multiple forms of nitrate reductase in mustard (Sinapis alba L.) cotyledons

In mustard (Sinapis alba L.) cotyledons, four different forms of nitrate reductase (NR) can be separated by anion-exchange chromatography. Two of these forms (NR1 and NR2) appear in the presence of NO 3 (-) while the other two (NR3 and NR4) appear as a response to the application of NH 4 (+) as the sole nitrogen source. In the presence of NH4NO3, NR3 appears to be superimposed on nitrate-induced NR1 and NR2 while the NH 4 (+) -induced appearance of NR4 is totally abolished in the presence of equimolar amounts of NO 3 (-) . The appearance of NR1, NR2 and NR3 is strongly stimulated by red light pulses which operate via the far-red-absorbing form of phytochrome (Pfr), whereas the appearance of NR4 requires continuous light (likewise operating through pytochrome). Continuous red light is more effective in this case than continuous far-red light. Analysis of the data shows that the mode of action of phytochrome (Pfr) is the same in the case of the appearances of NR1 and NR2, whereas it is quantitatively different in the case of NR3 and totally different in that of NR4. A 'plastidic factor' has previously been postulated to be obligatorily involved in the transcriptional control of nuclear genes encoding for proteins destined for the chloroplast. Photooxidative damage of the plastid is postulated to destroy the ability of the organelle to produce this signal. If the plastids are damaged by photooxidation, the action of nitrate and phytochrome on the appearance of NR is abolished. The plant cell regulates the appearance of nitrate-induced NR, which is cytosolic, as if it were a plastidic protein. The appearance of NR3 depends on the plastidic factor in principally the same way as that of NR1 and NR2 whereas NR4 is totally independent of the plastidic factor. The data document particular kinds of interaction between controlling factors (light, nitrate, ammonium, plastidic factor) which affect gene expression in plants. These intricacies of regulation have so far not been considered in molecular studies on NR-gene expression.