Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.7.1.1 (nitrate reductase)
3,728 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Although nitrate reductase (NR. EC 1.6.6.1) is thought to control the rate of nitrate assimilation, mutants with 40-45% of wildtype (WT) NR activity (NRA) grow as fast as the WT. We have investigated how tobacco (Nicotiana tabacum L. cv. Gatersleben) mutants with one or two instead of four functional nia genes compensate. (i) The nia transcript was higher in the leaves of the mutants. However, the diurnal rhythm was retained in the mutants, with a maximum at the end of the night and a strong decline during the photoperiod. (ii) Nitrate reductase protein and NRA rose to a maximum after 3-4 h light in WT leaves, and then decreased by 50-60% during the second part of the photoperiod and the first part of the night. Leaves of mutants contained 40-60% less NR protein and NRA after 3-4 h illumination, but NR did not decrease during the photoperiod. At the end of the photoperiod the WT and the mutants contained similar levels of NR protein and NRA. (iii) Darkening led to a rapid inactivation of NR in the WT and the mutants. However, in the mutants, this inactivation was reversed after 1-3 h darkness. Calyculin A prevented this reversal. When magnesium was included in the assay to distinguish between the active and inactive forms of NR, mutants contained 50% more activity than the WT during the night. Conversion of [15N]-nitrate to organic compounds in leaves in the first 6 h of the night was 60% faster in the mutants than in the WT. (iv) Growth of WT plants in enhanced carbon dioxide prevented the decline of NRA during the second part of the photoperiod, and led to reactivation of NR in the dark. (v) Increased stability of NR in the light and reversal of dark-inactivation correlated with decreased levels of glutamine in the leaves. When glutamine was supplied to detached leaves it accelerated the breakdown of NR, and led to inactivation of NR, even in the light. (vi) Diurnal changes were also investigated in roots. In the WT, the amount of nia transcript rose to a maximum after 4 h illumination and then gradually decreased. The amplitude of the changes in transcript amount was smaller in roots than in leaves, and there were no diurnal changes in NRA. In mutants, nia transcript levels were high through the photoperiod and the first part of the night. The NRA was 50% lower during the day but rose during the night to an activity almost as high as in the WT. The rate of [15N]-nitrate assimilation in the roots of the mutants resembled that in the WT during the first 6 h of the night. (vii) Diurnal changes were also compared in Nia30(145) transformants with very low NRA, and in nitrate-deficient WT plants. Both sets of plants had similar low growth rates. Nitrate reductase did not show a diurnal rhythm in leaves or roots of Nia30(145), the leaves contained very low glutamine, and NR did not inactivate in the dark. Nitrate-deficient WT plants were watered each day with 0.2 mM nitrate. After watering, there was a small peak of nia transcript NR protein and NRA and, slightly later, a transient increase of glutamine and other amino acids in the leaves. During the night glutamine was low, and NR did not inactivate. In the roots, there was a very marked increase of nitrate, nia transcript and NRA 2-3 h after the daily watering with 0.2 mM nitrate. (viii) It is concluded that WT plants have excess capacity for nitrate assimilation. They only utilise this potential capacity for a short time each day, and then down-regulate nitrate assimilation in response, depending on the conditions, to accumulation of the products of nitrate assimilation or exhaustion of external nitrate. Genotypes with a lower capacity for nitrate assimilation compensate by increasing expression of NR and weakening the feedback regulation, to allow assimilation to continue for a longer period each day.
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PMID:Tobacco mutants with a decreased number of functional nia genes compensate by modifying the diurnal regulation of transcription, post-translational modification and turnover of nitrate reductase. 943 79

The regulation by glutamine of the leaf transcript level corresponding to the Arabidopsis thaliana (L.) Heynh. nitrate reductase gene nia2 was examined using a novel approach: we took advantage of the ability of a ferredoxin-dependent glutamate synthase-deficient gluS mutant of A. thaliana to accumulate glutamine in the leaves when illuminated under conditions that favour photorespiration. The accumulation of glutamine in gluS mutant leaves and the concomitant decline in the leaf glutamate pool were not correlated with a reduction in the foliar nia2 transcript level. This result indicates that glutamine may not exert a negative control of the leaf nia2 transcript pool. The pattern of diurnal nia2 mRNA oscillation did not change upon illumination of the gluS mutant in air, although the leaf glutamine level remained high during the diurnal cycle. The amplitude of the diurnal fluctuation in nia2 transcript abundance, therefore, does not seem to depend on the size of the leaf glutamine pool (which normally fluctuates in opposite phase). This result also appears to argue against a role of glutamine as an effective repressor of nia2 transcript accumulation. The application of a solution containing 100 mM glutamine to the roots of A. thaliana resulted in an increase in the leaf glutamine level and in a decrease in the leaf nia2 transcript level. Net CO2 uptake and chlorophyll fluorescence quenching by attached leaves of A. thaliana were determined as a control of the physiological status of the plants and remained unaffected by the glutamine treatment. However, there was a decrease in the foliar nitrate level. The negative effect on the nia2 transcript pool exerted by exogeneous glutamine may, therefore, be explained as a result of the down-regulation of nitrate-uptake permeases in the roots by glutamine.
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PMID:Regulation of nitrate reductase transcript levels by glutamine accumulating in the leaves of a ferredoxin-dependent glutamate synthase-deficient gluS mutant of Arabidopsis thaliana, and by glutamine provided via the roots. 982 86

Diurnal changes in carbohydrates and nitrate reductase (NR) activity were compared in tobacco (Nicotiana tabacum. L.cv. Gatersleben) plants growing in a long (18 h light/6 h dark) and a short (6 h light/18 h dark) day growth regime, or after short-term changes in the light regime. In long-day-grown plants, source leaves contained high levels of sugars throughout the light and dark periods. In short-day-grown plants, levels of sucrose and reducing sugars were very low at the end of the night and, although they rose during the light period, remained much lower than in long days and declined to very low levels again by the middle of the night. Starch accumulated more rapidly in short-day-than long-day-grown plants. Starch was completely remobilised during the night in short days, but not in long days. A single short day/long night cycle sufficed to stimulate starch accumulation during the following light period. In long-day-grown plants, the Nia transcript level was high at the end of the night, decreased during the day, and recovered gradually during the night. In short-day-grown plants, the Nia transcript level was relatively low at the end of the night, decreased to very low levels at the end of the light period, increased to a marked maximum in the middle of the night, and decreased during the last 5 h of the dark period. In long-day-grown plants, NR activity in source leaves rose by 2- to 3-fold in the first part of the light period and decreased in the second part of the light period. In short-day-grown plants, NR activity was low at the end of the night, and only increased slightly after illumination. Dark inactivation of source-leaf NR was partially reversed in long-day-grown plants, but not in short day-grown plants. In both growth regimes, mutants with one instead of four functional copies of the Nia gene had a 60% reduction in maximum NR activity in the source leaves, compared to wild-type plants. The diurnal changes in NR activity were almost completely suppressed in the mutants in long days, whereas the mutants showed similar or slightly larger diurnal changes than wild-type plants in short days. When short-day-grown plants were transferred to long-day conditions for 3 d, NR activity and the diurnal changes in NR activity resembled those in long-day-grown plants. Phloem export from source leaves of short-day-grown plants was partially inhibited by applying a cold-girdle for one light and dark cycle. The resulting increase in leaf sugar was accompanied by an marked increase in the Nia transcript level and a 2-fold increase in NR activity at the end of the dark period. When wild-type plants were subjected to a single short day/long night cycle of increasing severity, NR activity in source leaves at the end of the night decreased when the endogenous sugars declined below about 3 mumol hexose (g FW)-1. In sink leaves in short-day conditions, sugars were higher and the light-induced rise in NR activity was much larger than in source leaves on the same plants. The source leaves of wild-type plants in short-day conditions contained very high levels of nitrate, very low levels of glutamine, low levels of total amino acids, and lower protein and chlorophyll, compared to long-day-grown plants. Plants grown in short days had relatively high levels of glutamate and aspartate, and extremely low levels of most of the minor amino acids in their source leaves at the end of the night. Illumination led to a decrease in glutamate and an increase in the minor amino acids. A single short day/long night cycle led to an increase in glutamate, and a large decrease in the minor acids at the end of the dark period, and reillumination led to a decrease in glutamate and an increase in the minor amino acids. It is proposed that sugar-mediated control of Nia expression and NR activity overrides regulation by nitrogenous compounds when sugars are in short supply, resulting in a severe inhibition of nitrate assimilation. It is also proposed that su
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PMID:Growth of tobacco in short-day conditions leads to high starch, low sugars, altered diurnal changes in the Nia transcript and low nitrate reductase activity, and inhibition of amino acid synthesis. 995 17

The gene areA-GF, a homologue of the major nitrogen regulatory genes nit-2, areA, nre and NUT1 of Neurospora crassa, Aspergillus nidulans, Penicillium chrysogenum and Magnaporthe grisea, respectively, was cloned from the gibberellin (GA)-producing rice pathogen Gibberella fujikuroi. areA-GF encodes a protein of 972 amino acid residues which contains a single putative zinc finger DNA-binding domain that is at least 98% identical to the zinc finger domains of the homologous fungal proteins. The areA-GF gene has been shown to be functional in N. crassa by heterologous complementation of a RIP induced nit-2 mutant. The transformation rate was nearly as high as in a homologous complementation control. Transformants were able to utilize nitrate and expressed a normally regulated nitrate reductase activity. To generate areA-GF- mutants, gene replacement experiments were performed using a linearized replacement vector carrying the hygromycin B phosphotransferase (hph) gene. The replacement of the zinc finger by the hygromycin cassette resulted in transformants which were unable to utilize nitrogen sources other than ammonium and glutamine, and gave significantly reduced gibberellin production yields. Complementation of such a mutant with the wild-type gene led to the full recovery of gibberellin production.
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PMID:Isolation, characterization and disruption of the areA nitrogen regulatory gene of Gibberella fujikuroi. 1007 Dec 16

Far-Western overlays of soluble extracts of cauliflower revealed many proteins that bound to digoxygenin (DIG)-labelled 14-3-3 proteins. Binding to DIG-14-3-3s was prevented by prior dephosphorylation of the extract proteins or by competition with 14-3-3-binding phosphopeptides, indicating that the 14-3-3 proteins bind to phosphorylated sites. The proteins that bound to the DIG-14-3-3s were also immunoprecipitated from extracts with anti-14-3-3 antibodies, demonstrating that they were bound to endogenous plant 14-3-3 proteins. 14-3-3-binding proteins were purified from cauliflower extracts, in sufficient quantity for amino acid sequence analysis, by affinity chromatography on immobilised 14-3-3 proteins and specific elution with a 14-3-3-binding phosphopeptide. Purified 14-3-3-binding proteins included sucrose-phosphate synthase, trehalose-6-phosphate synthase, glutamine synthetases, a protein (LIM17) that has been implicated in early floral development, an approximately 20 kDa protein whose mRNA is induced by NaCl, and a calcium-dependent protein kinase that was capable of phosphorylating and rendering nitrate reductase (NR) sensitive to inhibition by 14-3-3 proteins. In contrast to the phosphorylated NR-14-3-3 complex which is activated by dissociation with 14-3-3-binding phosphopeptides, the total sugar-phosphate synthase activity in plant extracts was inhibited by up to 40% by a 14-3-3-binding phosphopeptide and the phosphopeptide-inhibited activity was reactivated by adding excess 14-3-3 proteins. Thus, 14-3-3 proteins are implicated in regulating several aspects of primary N and C metabolism. The procedures described here will be valuable for determining how the phosphorylation and 14-3-3-binding status of defined target proteins change in response to extracellular stimuli.
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PMID:Phosphorylation-dependent interactions between enzymes of plant metabolism and 14-3-3 proteins. 1034 39

The relation between nitrate reductase (NR; EC 1.6.6.1) activity, activation state and NR protein in leaves of barley (Hordeum vulgare L.) seedlings was investigated. Maximum NR activity (NRA(max)) and NR protein content (Western blotting) were modified by growing plants hydroponically at low (0.3 mM) or high (10 mM) nitrate supply. In addition, plants were kept under short-day (8 h light/16 h dark) or long-day (16 h light/8 h dark) conditions in order to manipulate the concentration of nitrate stored in the leaves during the dark phase, and the concentrations of sugars and amino acids accumulated during the light phase, which are potential signalling compounds. Plants were also grown under phosphate deficiency in order to modify their glucose-6-phosphate content. In high-nitrate/long-day conditions, NRA(max) and NR protein were almost constant during the whole light period. Low-nitrate/long-day plants had only about 30% of the NRA(max) and NR protein of high-nitrate plants. In low-nitrate/long-day plants, NRA(max) and NR protein decreased strongly during the second half of the light phase. The decrease was preceded by a strong decrease in the leaf nitrate content. Short daylength generally led to higher nitrate concentrations in leaves. Under short-day/low-nitrate conditions, NRA(max) was slightly higher than under long-day conditions and remained almost constant during the day. This correlated with maintenance of higher nitrate concentrations during the short light period. The NR activation state in the light was very similar in high-nitrate and low-nitrate plants, but dark inactivation was twice as high in the high-nitrate plants. Thus, the low NRA(max) in low-nitrate/long-day plants was slightly compensated by a higher activation state of NR. Such a partial compensation of a low NR(max) by a higher dark activation state was not observed with phosphate-depleted plants. Total leaf concentrations of sugars, of glutamine and glutamate and of glucose-6-phosphate did not correlate with the NR activation state nor with NRA(max).
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PMID:The activation state of nitrate reductase is not always correlated with total nitrate reductase activity in leaves 1055 Jun 27

The NAD(P)H nitrate reductase (NR) from Chlamydomonas reinhardtii is encoded by the structural gene Nia1. Numerous data from the literature indicate that this enzyme is submitted to complex regulation mechanisms involving multiple controls at transcriptional and post-transcriptional levels. To specifically investigate the regulation of the Nia1 gene at the transcriptional level, NR+ and NR- transformed cells harbouring the Nia1:Ars construct (Nia1 promoter fused to the arylsulfatase (ARS)-encoding Ars reporter gene) were cultivated under various experimental conditions and the ARS activities were recorded. ARS levels were very low in cells grown in the presence of NH4Cl and dramatically increased on agar medium deprived of any nitrogen source or containing nitrate, nitrite, urea, arginine or glutamine. Compared to nitrogen-free medium, a slight positive effect of nitrate in the NR+ strain and a significant negative effect of nitrite in both NR+ and NR- strains were observed. The ARS activities were high in the light and very low in the dark or in the light in the presence of DCMU, indicating that Nia1 transcription is strikingly dependent on photosynthetic activity. Acetate used as a carbon source in the dark did not substitute for light in stimulating Nia1:Ars expression. Inactivation of NR by tungstate treatment of the NR+ strain resulted in a dramatic increase of ARS level suggesting that in Chlamydomonas, like in higher plants, active NR negatively regulates the transcription of the NR structural gene. Deleting the major part of the Nia1 leader sequence still present in the chimeric gene resulted in a decrease of ARS level but did not modify the regulation pattern.
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PMID:Transcriptional regulation of the Nia1 gene encoding nitrate reductase in Chlamydomonas reinhardtii: effects of various environmental factors on the expression of a reporter gene under the control of the Nia1 promoter. 1064 29

To investigate the regulation of HvNRT2, genes that encode high-affinity NO(3)(-) transporters in barley (Hordeum vulgare) roots, seedlings were treated with 10 mM NO(3)(-) in the presence or absence of amino acids (aspartate, asparagine, glutamate [Glu], and glutamine [Gln]), NH(4)(+), and/or inhibitors of N assimilation. Although all amino acids decreased high-affinity (13)NO(3)(-) influx and HvNRT2 transcript abundance, there was substantial interconversion of administered amino acids, making it impossible to determine which amino acid(s) were responsible for the observed effects. To clarify the role of individual amino acids, plants were separately treated with tungstate, methionine sulfoximine, or azaserine (inhibitors of nitrate reductase, Gln synthetase, and Glu synthase, respectively). Tungstate increased the HvNRT2 transcript by 20% to 30% and decreased NO(3)(-) influx by 50%, indicating that NO(3)(-) itself does not regulate transcript abundance, but may exert post-transcriptional effects. Experiments with methionine sulfoximine suggested that NH(4)(+) may down-regulate HvNRT2 gene expression and high-affinity NO(3)(-) influx by effects operating at the transcriptional and post-transcriptional levels. Azaserine decreased HvNRT2 transcript levels and NO(3)(-) influx by 97% and 95%, respectively, while decreasing Glu and increasing Gln levels. This suggests that Gln (and not Glu) is responsible for down-regulating HvNRT2 expression, although it does not preclude a contributory effect of other amino acids.
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PMID:Regulation of high-affinity nitrate transporter genes and high-affinity nitrate influx by nitrogen pools in roots of barley. 1080 47

Untransformed maize and tobacco plants and tobacco plants constitutively expressing nitrate reductase were grown with sufficient NO(3)- to support maximal growth. Four days prior to treatment the tobacco plants were deprived of nitrogen. Excised maize leaves and tobacco leaf discs were fed with either 40 mM KNO(3) or 40 mM KCl (control) in the light. Phosphoenolpyruvate (PEP) carboxylase (Case) activity was measured at 0.3 mM and 3 mM PEP. The light- induced increase in PEPCase V(max) was greater in maize than tobacco. Furthermore light decreased malate sensitivity in maize (which was N-replete) but not in N-deficient tobacco. NO(3)- treatment increased PEPCase V:(max) values in both species and decreased the sensitivity to inhibition by malate, but effects of NO(3)- were much more pronounced in tobacco than maize. PEPCase kinase activity was, however, greater in maize leaves NO(3)- than in the Cl(-)-treated controls, suggesting that it is responsive to leaf nitrogen supply. A correlation between foliar glutamine content and PEPCase activity was observed. It is concluded that PEPCase is sensitive to N metabolites which favour increased flow through the anapleurotic pathway in both C(3) and C(4) plants.
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PMID:Short-term nitrogen-induced modulation of phosphoenolpyruvate carboxylase in tobacco and maize leaves. 1094 47

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.
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PMID:Negative regulation of nitrate reductase gene expression by glutamine or asparagine accumulating in leaves of sulfur-deprived tobacco. 1103 May 59


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