Synthesis and Degradation of Barley Nitrate Reductase

Somers, David A.; Kuo, Tsung Min; Kleinhofs, A.; Warner, Robert L.; Oaks, Ann

doi:10.1104/pp.72.4.949

Cited by 129 publications

(79 citation statements)

References 24 publications

(25 reference statements)

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“…When spinach cells that had been cultured for 8 d were transferred to fresh medium, NR activity per unit cell mass increased markedly for 2 d and then declined gradually (Fig. 2), as has been reported for tobacco cells in culture (20) and for barley (15). NR activity became practically undetectable 10 d after the transfer.…”

Section: Resultsmentioning

confidence: 58%

“…NR activity became practically undetectable 10 d after the transfer. In the case of tobacco cells (20) and barley (15), evidence has been reported to indicate that the marked induction of NR activity accompanying the transfer to fresh medium is due to de novo synthesis of NR protein. In the case of nitrate-induced increase in NR activity in Chlorella cells, on the other hand, activation of an inactive form of NR protein has been observed (4,13,17).…”

Section: Resultsmentioning

confidence: 99%

“…The gradual decline of NR activity observed in spinach cells after the rapid increase was probably caused by the decrease in nitrate concentration in the medium. In fact, it has been observed in a number of higher plants that removal of nitrate from the medium results in a decrease in NR activity (2,7,15,20). Such decrease in NR activity in Neurospora crassa (1,16) and barley (15) have been reported to be caused by repression of NR synthesis and enhancement of its degradation.…”

Section: Resultsmentioning

confidence: 99%

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Regulation of Nitrate Reductase Activity in Cultured Spinach Cells as Studied by an Enzyme-Linked Immunosorbent Assay

Maki

Yamagishi²,

Sato³

et al. 1986

Plant Physiol.

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ABSTRACITAn enzyme-linked immunosorbent assay permitting the determination of nanogram quantities of nitrate reductase (NR) in cultured spinach cells has been developed and used for studies of the mechanism by which NR activity is regulated as a function of culture age. When 8-day old spinach cells were transferred to fresh medium, NR activity increased markedly in 2 days and thereafter decreased gradually until it became undetectable on the 10th day after the transfer. Determination of the amounts of NR by the immunosorbent assay indicated that the unique alteration of NR activity could be accounted for by the concomitant change in the amount of NR protein. peroxidase-conjugated donkey anti-rabbit immunoglobulin G (Amersham International). All other chemicals used were of highest quality available commercially.Preparation of Anti-NR Antibodies. NR was purified from fresh spinach leaves and subjected to PAGE under nondenaturing conditions as described previously (11) saturated (NH4)2SO4 followed by chromatography on a Toyopearl HW 55F column. Anti-NR antibodies were purified from the IgG fraction as follows. Ten polystyrene beads (4, 6 mm) were added to 2 ml of PBS containing 50 ,g of purified spinach NR and the mixture was incubated overnight at 4°C. The beads were then washed three times with 5 ml of PBS containing 0.05% (v/v) Triton X-100. The beads thus coated with NR were added to the IgG fraction (20 Mg protein/bead) and the mixture was incubated overnight at 4°C. After washing the beads three times as above, the anti-NR antibodies adsorbed were eluted from the beads with 8 M urea containing 0.5% (w/v) BSA. The eluted antibodies were subjected to gel filtration through a Sephadex G-25 column (1.5 x 30 cm) equilibrated with PBS and proteincontaining fractions were combined. The monospecificity of the antibodies was confirmed by immunoelectrophoresis (5

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Section: Resultsmentioning

confidence: 58%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Regulation of Nitrate Reductase Activity in Cultured Spinach Cells as Studied by an Enzyme-Linked Immunosorbent Assay

Maki

Yamagishi²,

Sato³

et al. 1986

Plant Physiol.

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“…Early studies on nitrate signaling demonstrated that nitrate induces de novo synthesis of nitrate reductase (NR), the first enzyme in the nitrate assimilation pathway (Zielke and Filner, 1971;Somers et al, 1983;Remmler and Campbell, 1986). Subsequent work demonstrated that nitrate induces other genes in the nitrate assimilation pathway, namely, nitrate transporters (NRTs) and nitrite reductase (NiR), as well as genes involved in energy metabolism especially in the pentose phosphate pathway (Wang et al, 2000; for review, see Redinbaugh and Campbell, 1991;Stitt, 1999).…”

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confidence: 99%

Nitrite Acts as a Transcriptome Signal at Micromolar Concentrations in Arabidopsis Roots

2007

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Nitrate serves as a potent signal to control gene expression in plants and algae, but little is known about the signaling role of nitrite, the direct product of nitrate reduction. Analysis of several nitrate-induced genes showed that nitrite increases mRNA levels as rapidly as nitrate in nitrogen-starved Arabidopsis (Arabidopsis thaliana) roots. Both nitrite and nitrate induction are apparent at concentrations as low as 100 nM. The response at low nitrite concentrations was not due to contaminating nitrate, which was present at ,1% of the nitrite concentration. High levels of ammonium (20 mM) in the growth medium suppressed induction of several genes by nitrate, but had varied effects on the nitrite response. Transcriptome analysis using 250 or 5 mM nitrate or nitrite showed that over one-half of the nitrate-induced genes, which included genes involved in nitrate and ammonium assimilation, energy production, and carbon and nitrogen metabolism responded equivalently to nitrite; however, the nitrite response was more robust and there were many genes that responded specifically to nitrite. Thus, nitrite can serve as a signal as well as if not better than nitrate.

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“…Utilization of nitrate by higher plants involves the basic processes of uptake, reduction and translocation. Nitrate reductase, the enzyme which catalyzes the rate-limiting step in nitrate assimilation, requires continued protein synthesis to maintain its activity (10,11,16,23,24). There is also evidence that uptake and translocation of nitrate are dependent on protein synthesis (9,19,26).…”

mentioning

confidence: 99%

p-Fluorophenylalanine-Induced Restriction of Ion Uptake and Assimilation by Maize Roots

Morgan¹,

Volk²,

Jackson³

1985

Plant Physiol.

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ABSTRACIRoots of decapitated maize seedlings (Zea mays L.) were exposed for 12 hours to 1.0 millimolar KNO3 (98.5 atom per cent 15N) in the presence and absence (control) of 0.1 millimolar p-fluorophenylalanine (FPA), an analog of the amino acid phenylalanine. FPA decreased nitrate uptake but had little effect on potassium uptake. In contrast, accumulation of both ions in the xylem exudate was greatly restricted. Utilization of nitrate by higher plants involves the basic processes of uptake, reduction and translocation. Nitrate reductase, the enzyme which catalyzes the rate-limiting step in nitrate assimilation, requires continued protein synthesis to maintain its activity (10,11,16,23,24). There is also evidence that uptake and translocation of nitrate are dependent on protein synthesis (9,19,26). This latter evidence, however, is based primarily on studies with inhibitors of RNA and protein synthesis, some of which are known to affect cellular processes other than protein synthesis (3, 7, 8). For example, cycloheximide has been reported to stimulate respiration and inhibit chloride uptake by beet root discs within 1 h of its application (8). Because of this lack of specificity, we have reexamined the effect of impaired protein synthesis on nitrate uptake and assimilation, using FPA,3 an analog of phenylalanine, to produce ineffective protein (18,21). Potassium fluxes were also quantified, since this cation is the common counterion associated with nitrate uptake and translocation.'Supported by National Science Foundation Grant PCM 81-18661. MATERIALS AND METHODSGrowth of Maize Seedlings. DeKalb XL-45 maize seeds (Zea mays L.) were germinated on paper towels moistened with 0.1 mM CaSO4 in an incubator maintained in darkness at 30°C and 98% RH. After 3 d, all roots except the primary roots were excised, and the seedlings were assembled into cultures of five seedlings each. The cultures were placed into aerated, nitrogenfree nutrient solution and returned to the incubator. The solution (pH 6.0) contained 0.05 mm K2S04, 0.4 mM KH2PO4, 0.25 mM MgSO4, 1.0 mm CaSO4, trace elements at 0.4 Hoagland concentration (12) and FeEDTA at 1.0 mg Fe 1-'. On the 4th d, the shoots were excised below the first node, and exudate collectors, small plastic pipet tips, were sealed to the mesocotyls with silicone grease. The cultures were placed in fresh, nitrogen-free nutrient solution and returned to the incubator. The experiment was run 20 h later, when the decapitated seedlings were 5-d old.Experimental Procedure. Just prior to the experiment, exudate was removed from the collectors, and the roots were rinsed thoroughly in 1.0 mm CaSO4 (30°C). The roots of each culture were then exposed to 250 ml of uptake solution containing 1.0 mM K'5NO3 (98.5 A % '5N) and 0 (control) or 0.1 mm FPA. Otherwise, the composition of the uptake solution was identical to that of the nitrogen-free, nutrient solution. During the 12-h exposure period, the uptake solutions (pH 6.0) were maintained at 30°C, aerated continuously, and replaced with fresh solu...

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Synthesis and Degradation of Barley Nitrate Reductase

Abstract: NR5 activity is induced by nitrate in barley (2, 24) and other higher plants (5,7,8,13,14,26)

Cited by 129 publications

References 24 publications

Regulation of Nitrate Reductase Activity in Cultured Spinach Cells as Studied by an Enzyme-Linked Immunosorbent Assay

Regulation of Nitrate Reductase Activity in Cultured Spinach Cells as Studied by an Enzyme-Linked Immunosorbent Assay

Nitrite Acts as a Transcriptome Signal at Micromolar Concentrations in Arabidopsis Roots

p-Fluorophenylalanine-Induced Restriction of Ion Uptake and Assimilation by Maize Roots

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