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1.
The absorption of nitrate and the activity of nitrate reductase were much lower in Ca-deficient plants of Cururbita pepo L., cv. ‘Kveta’ than in normal plants grown in complete nutrient solution for a period of 8 days. After the addition of nitrate to the nutrient medium, nitrate reductase activity in the roots of NO 3-deficient plants sharply rose during the first 6 h and then remained constant during the following 6 h; the content of endogenous NO 3 ? rose slowly and continuously. These processes were depressed in (Ca, NO 3)-deficient plants independently of the addition of Ca 2+ to the medium in the variant with NO 3 ?. Thus it seems that the whole nitrogen metabolism, i.e. both NO 3 ? absorption and the synthesis of nitrate reductase, is impaired in Ca-deficient plants. 相似文献
2.
The effect of nitrogen starvation on the NO 3-dependent induction of nitrate reductase (NR) and nitrite reductases (NIR) has been investigated in the halophilic alga Dunaliella salina. When D. salina cells previously grown in a medium with NH 4 + as the only nitrogen source (NH 4 + -cells) were transferred into NO 3 ? medium, NR was induced in the light. In contrast, when cells previously grown in N-free medium were transferred into a medium containing NO 3 ? , NR was induced in light or in darkness. Nitrate-dependent NR induction, in darkness, in D. salina cells previously grown at a photon flux density of 500 umol · m ?2 s ?1 was observed after 4 h preculture in N-free medium, whilst in cells grown at 100 umol · m ?2 s ?1 NR induction was observed after 7–8 h. An inhibitor of mRNA synthesis (6-methylpurine) did not inhibit NO 3 ? -induced NR synthesis when the cells, previously grown in NH 4 + medium, were transferred into NO 3 ? medium (at time 0 h) after 4-h-N starvation. However, when 6-methylpurine was added simultaneously with the transfer of the cells from NH 4 + to NO 3 ? medium (at time 0 h), NO 3 ? induced NR synthesis was completely inhibited. The activity of NIR decreased in N-starved cells and the addition of NO 3 ? to those cells greatly stimulated NIR activity in the light. The ability to induce NR in darkness was observed when glutamine synthetase activity reached its maximal level during N starvation. Although cells grown in NO 3 ? medium exhibited high NR activity, only 0.33% of the total NR was found in intact chloroplasts. We suggest that the ability, to induce NR in darkness is dependent on the level of N starvation, and that NR in D. salina is located in the cytosol. Light seems to play an indirect regulatory role on NO 3 ? uptake and NR induction due to the expression of NR and NO 3 ? -transporter mRNAs. 相似文献
3.
Induction of nitrate reductase (EC 1.6.6.1) activity was measured in Paul's Scarlet rose cell suspensions cultured in media containing nitrate (NO
3
-
) or urea (U) as nitrogen source, and with (+Mo) or without molybdenum (-Mo). There was a lag of 30 min during induction by NO
3
-
in +Mo cultures but no lag occurred during induction after adding Mo to NO
3
-
-Mo or to U-Mo cultures preincubated with NO
3
-
. Actinomycin D, cycloheximide, and puromycin completely blocked induction by NO
3
-
, but had no effect on the initial rate of induction by Mo. Cycloheximide and puromycin blocked induction by NO
3
-
more quickly than actinomycin D. Induction by NO
3
-
appeared to involve mRNA-dependent synthesis of apoprotein followed by rapid activation with molybdenum in intact cells independently of protein synthesis. Nitrate-induced apoprotein appeared less stable than the holoenzyme. When induced by NO
3
-
in the absence of Mo, apoprotein concentration was about half the amount of maximally induced nitrate reductase. Cycloheximide stabilised preformed nitrate reductase which disappeared steadily in the presence of puromycin. Apoprotein was not stabilised by either antimetabolite.Abbreviations Mo
molybdenum
- NO
3
-
+Mo
standard, MX 1 culture medium
- NO
3
-
-Mo
MX 1 medium purified of Mo
- NR
nitrate reductase
- PSR
Paul's Scarlet rose
- U
urea
- U+Mo
MX 1 medium with NO
3
-
replaced by urea
- U-Mo
MX 1 medium with NO
3
-
replaced by urea and also purified of Mo 相似文献
4.
Hydroxylamine added to the nutrient medium in sublethal concentrations (0.2 to 1.0 mN) enhanced NADH2 dependent glutamate dehydrogenase activity in isolated pea roots. The increase in activity depended on proteosynthesis and was lower in the presence of NO 3 ? and NH 4 + ions. The induction of nitrate reductase and of nitrite reductase was partly inhibited by sublethal hydroxylamine concentrations. 相似文献
5.
After x-ray irradiation, 13 mutants of Chlorella sorokiniana incapable of using NO 3− as N source were isolated using a pinpoint method. Using immunoprecipitation and Western blot assays, no nitrate reductase was found in five strains while in eight mutants the enzyme was detected. The latter strains contained different patterns of nitrate reductase partial reactions. All isolates were of the nia-type as indicated by the inducibility of purine hydroxylase I and by complementation of nitrate reductase activity in the Neurospora crassa mutant Nit-1. A restoration of NADP-nitrate reductase in Nit-1 was also obtained with NH 4+-grown cells indicating that Mo-cofactor is constitutive in Chlorella. Complementation experiments among the Chlorella mutants resulted in restoration of NADH-nitrate reductase activity. The characteristics of some of the Chlorella mutants are discussed in view of an improper orientation of Mo-cofactor in the residual nitrate reductase protein. 相似文献
6.
The mechanism of anaerobic reduction of NO 2? to N 2O in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans, was investigated. With ascorbate-reduced phenazine methosulfate (PMS) as the electron donor, the nitrite reductase of this photodenitrifier reduced NO 2? to NO and a trace amount of N 2O. With dithionite-reduced benzyl viologen as the electron donor, the major product of NO 2? reduction was NH 2OH, and a trace amount of N 2O was also produced. The nitrate reductase itself had no NO reductase activity with ascorbate-reduced PMS. It was concluded that the essential product of NO 2? reduction by the purified nitrite reductase is NO. Chromatophore membranes stoichiometrically produced N 2O from NO 2? with any electron donor, such as dithionite-redduced benzyl viologen, ascorbate-reduced PMS or NADH/FMN. The membranes also contrained activity of NO reduction of N 2O with either ascorbate-reduced PMS or duroquinol. The NO reductase activity with duroquinol was inhibited by antimycin A. Stoichiometric production of N 2O from N 2? also was observed in the reconstituted NO 2? reduction system which contained the cytochrome bc1 complex, cytochrome c2, the nitrite reductase and duroquinol as the electron donor. The preparation of the cytochrome bc1 complex itself contianed NO reductase activity. From these results the mechanism of NO 2? reduction to N 2O in this photodenitrifier was determined as the nitrite reductase reducing NO 2? to NO with electrons from the cytochrome bc1 complex, and NO subsequently being reduced, without release, to N 2O with electrons from the cytochrome bc1 complex by the NO reductase, which is closely associated with the complex. 相似文献
7.
Initial rate studies of spinach ( Spinacia oleracea L.) nitrate reductase showed that NADH:nitrate reductase activity was ionic strength dependent with elevated ionic concentration resulting in inhibition. In contrast, NADH:ferricyanide reductase was markedly less ionic strength dependent. At pH 7.0, NADH:nitrate reductase activity exhibited changes in the Vmax and Km for NO 3− yielding Vmax values of 6.1 and 4.1 micromoles NADH per minute per nanomoles heme and Km values of 13 and 18 micromolar at ionic strengths of 50 and 200 millimolar, respectively. Control experiments in phosphate buffer (5 millimolar) yielded a single Km of 93 micromolar. Chloride ions decreased both NADH:nitrate reductase and reduced methyl viologen:nitrate reductase activities, suggesting involvement of the Mo center. Chloride was determined to act as a linear, mixed-type inhibitor with a Ki of 15 millimolar for binding to the native enzyme and 176 millimolar for binding to the enzyme-NO 3− complex. Binding of Cl − to the enzyme-NO 3− complex resulted in an inactive E-S-I complex. Electron paramagnetic resonance spectra showed that chloride altered the observed Mo(V) lineshape, confirming Mo as the site of interaction of chloride with nitrate reductase. 相似文献
8.
Summary The assimilatory nitrate reductase of the N 2-fixing bacterium Azotobacter chroococcum has been prepared in a soluble form from cells grown with nitrate as the nitrogen source, and some of its properties (electron donors and cofactors, K
mvalues for substrates, molecular weight, inhibitors, activators, etc.) have been studied. The enzyme is of an inducible nature and can exist in two interconvertible forms, either active or inactive.Tungstate very efficiently inhibits growth of the microorganism in media with nitrate. When either nitrite or ammonia are substituted for nitrate as the nitrogen source, growth is unaffected by tungstate concentrations which otherwise completely suppress growth on nitrate. Tungstate interferes by decreasing the cellular level of nitrate reductase activity, preventing, as a consequence, utilization of nitrate. 相似文献
9.
The effect of some ammonium salts on nitrate reductase (NR) level, on in vivo nitrate reduction and on nitrate content was followed in the presence of nitrate in the medium, under changing experimental conditions, in excised Pisum sativum roots, and their effect was compared with that of KNO 3, Ca(NO 3) 2 and NaNO 3 at 15 mM NO 3 - concentration, i.e. at a concentration which considerably exceeded the level of saturation with nitrate with respect to nitrate reductase. The effect of ammonium salts on NR level is indirect and changes from a positive one to a strongly negative one which is dependent on the time of action of the salt, on the presence of other cations, on pH of the solution of the ammonium salt and on the nature of the anion of the ammonium salt. A positive effect on the enzyme level can be observed in the presence of other cations than NH 4 + at suitable concentrations of those ammonium salts, the solutions of which have their pH values in the acid region (i.e. NH 4H 2PO 4, (NH 4) 2SO 4 and NH 4NO 3). However their positive effect is independent of the presence of NH 4 + ions, and it is obviously the result of an increased concentration of H + ions. A clear-cut negative effect on NR level can be observed after 24 h in one-salt NH 4NO 3 solution where NH 4 + is not balanced with other cations and thus certainly can adversely influence many metabolic processes, and in the solutions containing neutral (pH 6.2) and dibasic ammonium phosphates in which dissolved undissociated ammonia [(NH 3). (H 2O) which can also affect many metabolic processes incl. proteosynthesis] probably has a toxic influence. The in vivo nitrate reduction is always depressed in excised pea roots in the presence of ammonium salts in the medium, regardless of the level of nitrate reductase. Under the described conditions, no relationship could be established between the enzyme level and the so-called metabolic NO 3 - pool (i.e. NO 2 - production under anaerobic conditions), nor between NR level and the total nitrate content in the roots. One-salt solutions of NaNO 3, Ca(NO 3) 2 and KNO 3 exert different effects on the level of nitrate reductase and on the content of NO 3 - in the roots, but the in vivo NO 3 - reduction shows the same trend as NR level in the roots influenced by these salts. Cl - ions, supplied in NH 4C1, depress both NR level and NO 3 - content in the roots at higher concentrations, but they do not significantly affect the in vivo nitrate reduction in comparison with other ammonium salts. These results indicate that NR level, in vivo nitrate reduction, and nitrate uptake can be regulated in pea roots independently of each other. 相似文献
10.
Growth and nitrate reductase activity were measured in Paul's Scarlet rose cell suspensions, cultured in media purified from molybdenum and containing nitrate or urea as sole nitrogen source with or without added Mo. Urea could replace nitrate to yield 80% of the fresh weight in nitrate medium. Nitrate reductase activities were compared by in vivo and in vitro assays. The latter varied due to inactivation during extraction. Compared with activities in cells in complete NO 3
- medium, activity in NO 3
--Mo cells was reduced to 30% and, in urea-grown cells, to trace amounts. Increases in nitrate reductase activity were found when NO 3
- alone was added to NO 3
- or urea+Mo cultures. In NO 3
--Mo cultures, Mo alone or with NO 3
- caused a similar increase in activity, whereas urea-Mo cultures required both NO 3
- and Mo for enzyme induction.Abbreviations FAD
flavin adenine dinucleotide
- Mo
molybdenum
- NADH
reduced nicotinamide adenine dinucleotide
- NO 3
-+Mo
standard MX 1 culture medium
- NO 3
--Mo
MX 1 medium purified of Mo and used for continuous subculture with nitrate
- NR
nitrate reductase
- PSR
Paul's Scarlet rose
- PVP
polyvinylpyrrolidone
- U
urea
- U+Mo
MX 1 medium containing urea instead of nitrate
- U-Mo
MX 1 medium containing urea instead of nitrate and also purified of Mo 相似文献
11.
Soybean ( Glycine max [L.] Merr.) seeds were imbibed and germinated with or without NO 3−, tungstate, and norflurazon (San 9789). Norflurazon is a herbicide which causes photobleaching of chlorophyll by inhibiting carotenoid synthesis and which impairs normal chloroplast development. After 3 days in the dark, seedlings were placed in white light to induce extractable nitrate reductase activity. The induction of maximal nitrate reductase activity in greening cotyledons did not require NO 3− and was not inhibited by tungstate. Induction of nitrate reductase activity in norflurazon-treated cotyledons had an absolute requirement for NO 3− and was completely inhibited by tungstate. Nitrate was not detected in seeds or seedlings which had not been treated with NO 3−. The optimum pH for cotyledon nitrate reductase activity from norflurazon-treated seedlings was at pH 7.5, and near that for root nitrate reductase activity, whereas the optimum pH for nitrate reductase activity from greening cotyledons was pH 6.5. Induction of root nitrate reductase activity was also inhibited by tungstate and was dependent on the presence of NO 3−, further indicating that the isoform of nitrate reductase induced in norflurazon-treated cotyledons is the same or similar to that found in roots. Nitrate reductases with and without a NO 3− requirement for light induction appear to be present in developing leaves. In vivo kinetics (light induction and dark decay rates) and in vitro kinetics (Arrhenius energies of activation and NADH:NADPH specificities) of nitrate reductases with and without a NO 3− requirement for induction were quite different. Km values for NO 3− were identical for both nitrate reductases. 相似文献
13.
Soil-N (NO 3 ?) initiates as far as a threshold concentration is surpassed manifold physiological reactions on N 2-fixation. Organic N and ammonium oxidised to NO 3 ? means oxygen depletion. Plants suffering under O 2 or infection stress start to excrete ethylene (C 2H 4). C 2H 4 widens the root intercellulars that O 2-respiration will continue. Now microbes may more easily enter the plant interior by transforming the reached methionine into C 2H 4. Surplus nitrate and C 2H 4 inhibit nodulation of leguminous plants. Excess NO 3 ? in the nodulesphere could be diminished by N 2-fixing bacteria which in addition can denitrify or ammonify nitrate. Consequently, it was asked whether C 2H 4 interferes with the potential of N 2-fixing bacteria to reduce nitrate. The groundnut-nodule isolate TNAU 14, from which it was known that it denitrifies and ammonifies nitrate, served as inoculum of a KNO 3-mannitol-medium that was incubated under N 2-, 1% (v/v) N 2?C 2H 4-, and 1% (v/v) N 2?C 2H 2-atmosphere in the laboratory. C 2H 2 was included into the experiments because it is frequently used to quantify N 2-fixing potentials (acetylene reduction array, ARA). Gene-16S rDNA-sequencing and physiological tests revealed a high affiliation of strain TNAU 14 to Rhizobium radiobacter and Rhizobium tumefaciens. Strain TNAU 14 released N 2O into the bottle headspace in all treatments, surprisingly significantly less in presence of C 2H 2. Nitrate-ammonification was even completely blocked by C 2H 2. C 2H 4, in contrast rather stimulated growth, denitrification, and nitrate-ammonification of strain TNAU 14 which consumed the released NH 4 + during continuing incubation. 相似文献
14.
A comparison of induction and inactivation of nitrate reductase and two of its component activities, namely FMNH 2-nitrate reductase and NO 3−-induced NADH-cytochrome c reductase, was made in roots and leaves of corn ( Zea mays L. var. W64A × 182E). The three activities were induced in parallel in both tissues when NO 3− was supplied. WO 4= suppressed the induction of NADH- and FMNH 2-nitrate reductase activities in root tips and leaves. The NO 3−-induced NADH-cytochrome c reductase activity showed a normal increase in roots treated with WO 4=. In leaves, on the other hand, there was a marked superinduction of the NO 3−-induced NADH-cytochrome c reductase in the presence of WO 4=. 相似文献
15.
Effect of nitrate on the nitrogenase (C 2H 2-reduction) activity, growth of nodule tissue accumulation of nitrate and nitrate reductase activity in 4-weeks-old nodulated peas ( Pisum sativum l.) was investigated. A relatively slow decrease of the total nitrogenase activity (μmol C 2H 4 per root per h), as compared with plants cultivated without nitrate, was due to both retardation of further growth of the nodule tissue and to a decrease of their specific nitrogenase activity (μmol C 2H 4 per g f.wt. per h). However, an absolute and pronounced decrease of both nitrogenase activities occurred only 4 or 7 d after the application of nitrate. The addition of nitrate led to its rapid accumulation in the nodule and leaf tissue with a simultaneous induction of the nitrate reductase activity. The nitrogenase activity was not completely inhibited even after a 7-d cultivation with 280 ppm NO 3 ?-N in the nutrient medium and after accumulation of up to 180 ppm NO 3 ?-N f.wt. in the nodule tissue. The results obtained indicate that the “photosynthate deprivation” reflects competition between assimilation of nitrate and fixation of dinitrogen. 相似文献
16.
Nitrate uptake and in vivo, nitrate reductase activity (NRA) in roots of Phaseolus vulgaris, L. cv. Witte Krombek were measured in nitrogen-depleted plants of varying sugar status, Variation in sugar status was achieved at the start of nitrate nutrition by excision, ringing, darkness or administration of sugars to the root medium. The shape of the apparent induction pattern of nitrate uptake was not influenced by the sugar status of the absorbing tissue. When measured after 6 h of nitrate nutrition (0.1 mol m ?3), steady state nitrate uptake and root NRA were in the order intact>dark>ringed>excised. Exogenous sucrose restored NRA in excised roots to the level of intact plants. The nitrate uptake rate of excised roots, however, was not fully restored by sucrose (0.03–300 mol m ?3). When plants were decapitated after an 18 h NO 3? pretreatment, the net uptake rate declined gradually to become negative after three hours. This decline was slowed down by exogenous fructose, whilst glucose rapidly (sometimes within 5 min) stimulated NG ?3 uptake. Presumably due to a difference in NO 3? due to a difference in NO 3? uptake, the NRA of excised roots was also higher in the presence of glucose than in the presence of fructose after 6 h of nitrate nutrition. The sugar-stimulation of, oxygen consumption as well as the release of 14CO 2 from freshly absorbed (U- 14C) sugar was the same for glucose and fructose. Therefore, we propose a glucose-specific effect on NO 3? uptake that is due to the presence of glucose rather than to its utilization in root respiration. A differential glucose-fructose effect on nitrate reductase activity independent of the effect on NO 3? uptake was not indicated. A constant level of NRA occurred in roots of NO 3? induced plants. Removal of nutrient nitrate from these plants caused an exponential NRA decay with an approximate half-life of 12 h in intact plants and 5.5 h in excised roots. The latter value was also found in roots that were excised in the presence of nitrate, indicating that the sugar status primarily determines the apparent rate of nitrate reductase decay in excised roots. 相似文献
17.
Anaerobic nitrite production (the in vivo NO 3-R activity) in an incubation medium lacking exogenous nitrate but containing 0.5% n-propanol and 0.1% Triton X-100 showed higher correlation ( y - ax
b) with the level of endogenous nitrate in Pisum sativum L. leaves than the in vitro nitrate reductase activity. The in vivo NO 3-R activity correlated well with the in vitro activity up to the 50 ppm NO 3-N level of endogenous nitrate. The ratio in vivo: in vitro activity slightly decreased with increasing level of endogenous nitrate in leaf tissue. 相似文献
18.
Nitrate reductase utilizing NADH or reduced flavin mononucleotide (FMNH 2) as electron donor was extracted from the leaves, stems and petioles, and roots of apple seedlings. Successful extraction was made possible by the use of insoluble polyvinylpyrrolidone (Polyclar AT) which forms insoluble complexes with polyphenols and tannins. The level of nitrate reductase per gram fresh weight was highest in the leaf tissue although the nitrate content of the roots was much higher than that of the leaves. Nitrite reductase activity was detected only in leaf extracts and was 4 times higher than nitrate reductase activity. Nitrate was found in all parts of young apple trees and trace amounts were also detected in mature leaves from mature trees. Nitrate reductase was induced in young leaves of apple seedlings and in mature leaves from 3 fruit-bearing varieties. An inhibitor of polyphenoloxidase, 2-mercaptobenzothiazole was used in both the inducing medium and the extracting medium in concentrations from 10 −3 to 10 −5m with no effect upon nitrate reductase activity. 相似文献
19.
Exposure of the leaf canopy of corn seedlings ( Zea mays L.) to atmospheric CO 2 levels ranging from 100 to 800 μl/l decreased nitrate accumulation and nitrate reductase activity. Plants pretreated with CO 2 in the dark and maintained in an atmosphere containing 100 μl/l CO 2 accumulated 7-fold more nitrate and had 2-fold more nitrate reductase activity than plants exposed to 600 μl/l CO 2, after 5 hours of illumination. Induction of nitrate reductase activity in leaves of intact corn seedlings was related to nitrate content. Changes in soluble protein were related to in vitro nitrate reductase activity suggesting that in vitro nitrate reductase activity was a measure of in situ nitrate reduction. In longer experiments, levels of nitrate reductase and accumulation of reduced N supported the concept that less nitrate was being absorbed, translocated, and assimilated when CO 2 was high. Plants exposed to increasing CO 2 levels for 3 to 4 hours in the light had increased concentrations of malate and decreased concentrations of nitrate in the leaf tissue. Malate and nitrate concentrations in the leaf tissue of seven of eight corn genotypes grown under comparable and normal (300 μl/l CO 2) environments, were negatively correlated. Exposure of roots to increasing concentrations of potassium carbonate with or without potassium sulfate caused a progressive increase in malate concentrations in the roots. When these roots were subsequently transferred to a nitrate medium, the accumulation of nitrate was inversely related to the initial malate concentrations. These data suggest that the concentration of malate in the tissue seem to be related to the accumulation of nitrate. 相似文献
20.
With respect to cofactor requirements, NADH, and FMNH 2 were equally effective as electron donors for nitrate reductase obtained from leaves of maize, marrow, and spinach, when the cofactors were supplied in optimal concentrations. The concentration of FMNH 2 required to obtain half-maximal activity was from 40- to 100-fold higher than for NADH. For maximal activity with the corn enzyme, 0.8 millimolar FMNH 2 was required. In contrast, NADPH was functional only when supplied with NADP:reductase and exogenous FMN (enzymatic generation of FMNH 2). All attempts to separate the NADH2- and FMNH2-dependent nitrate reductase activities were unsuccessful and regardless of cofactor used equal activities were obtained, if cofactor concentration was optimal. Unity of NADH to FMNH2 activities were obtained during: A) purification procedures (4 step, 30-fold); B) induction of nitrate reductase in corn seedlings with nitrate; and C) inactivation of nitrate reductase in intact or excised corn seedlings. The NADH- and FMNH2-dependent activities were not additive. A half-life for nitrate reductase of approximately 4 hours was estimated from the inactivation studies with excised corn seedlings. Similar half-life values were obtained when seedlings were incubated at 35° in a medium containing nitrate and cycloheximide (to inhibit protein synthesis), or when both nitrate and cycloheximide were omitted. In those instances where NADH activity but not FMNH2 activity was lost due to treatment (temperature, removal of sulfhydryl agents, addition of p-chloromercuribenzoate), the loss could be explained by inactivation of the sulfhydryl group (s) required for NADH activity. This was verified by reactivation with exogenous cysteine. Based on these current findings, and previous work, it is concluded that nitrate reductase is a single moiety with the ability to utilize either NADH or FMNH2 as cofactor. However the high concentration of FMNH2 required for optimal activity suggests that in vivo NADH is the electron donor and that nitrate reductase in higher plants should be designated NADH:nitrate reductase (E.C. 1.6.6.1). 相似文献
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