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1.
Regulation of nitrate and nitrite reduction in barley leaves 总被引:1,自引:0,他引:1
Reduction of nitrate and accumulation of nitrite were studied in barley (Hordeum vulgare L. cv. Gars Clipper ex Napier) leaf sections in the dark and in the light, under aerobic (air and mixtures of O2 and N2 ) or anaerobic (N2 ) conditions. Oxygen prevented nitrite accumulation but had no effect on accumulated or infiltrated nitrite. Most of the nitrite accumulated under dark-anaerobic conditions was in the "cytoplasmic" (the cell section between the plasma lemma and the tonoplast) fraction of the tissue. Reduction of nitrate was stimulated by 2, 4-dinitrophenol in tissue under dark-air and by 3-(3', 4'-dichlorophenyl)-l, l-dimethyl urea (DCMU) and carbonyl cyanide m -chlorophenylhydrazone (CCCP) in tissue under all environmental conditions studied. Nitrite accumulated in the light in DCMU-treated tissue under N2 or under aerobic conditions in the presence of CCCP. On its own, CCCP did not promote accumulation of nitrite in leaf sections under light-air. A model for the reduction of nitrate and nitrite is proposed. 相似文献
2.
Regulation of nitrate reductase (NR, EC 1.6.6.1) by oxygen concentration and light was studied in segments of oat ( Avena sativa L. cv. Suregrain) leaves, using the in vivo nitrate reductase assay. The activity of NR decreased after excision in either light or darkness; the addition of cycloheximide prevented this decrease. Treatments that increased tissue permeability (anoxia, Triton X-100) also increased NR activity. There was in general less NR activity in the light than in the dark and also less under aerobic (21–100% O2 ) than under anaerobic (0.3% O2 ) conditions. Treatments with antioxidants improved the activity in the light, but only at high O2 levels (21–100% O2 ).
The results suggest that NR may be regulated by inhibitory proteins synthesized in either light or darkness, by permeability changes and by light-induced oxidations that occur when O2 is present. Oxygen may control the activity by stimulating the synthesis of inhibitory proteins in the light and in the dark and by promoting oxidation of SH-groups in the light. 相似文献
The results suggest that NR may be regulated by inhibitory proteins synthesized in either light or darkness, by permeability changes and by light-induced oxidations that occur when O
3.
The NO− 3 -triggered induction of nitrate reductase (NR; EC 1.6.6.2) in the bryophyte Sphagnum magellanicum Brid. has been studied, using in vivo and in vitro assays as well as immunological methods. The time-course of induction was triphasic with maximal NR activity after 6–8 h. Results obtained from Western blots show that NR is synthesized de novo after NO− 3 application. The inhibitory effect of cycloheximide on NR induction corroborated this conclusion. Light enhanced the NO− 3 -triggered NR induction. The enzyme activity, measured in vivo, increased more than the in vitro activity. No evidence for phytochrome control of NR was found. Nitrate uptake, in contrast to NR activity, showed no lag period after NO− 3 application and, under the experimental conditions used, was not rate limiting for NR induction. Neither light nor a NO− 3 pretreatment significantly affected NO− 3 uptake. 相似文献
4.
Metabolism of nitric oxide and nitrous oxide during nitrification and denitrification in soil at different incubation conditions 总被引:1,自引:0,他引:1
Abstract NO production and consumption rates as well as N2 O accumulation rates were measured in a loamy cambisol which was incubated under different conditions (i.e. soil moisture content, addition of nitrogen fertilizer and/or glucose, aerobic or anaerobic gas phase). Inhibition of nitrification with acetylene allowed us to distinguish between nitrification and denitrification as sources of NO and N2 O. Under aerobic conditions untreated soil showed very low release of NO and N2 O but high consumption of NO. Fertilization with NH4 + or urea stimulated both NO and N2 O production by nitrification. Addition of glucose at high soil moisture contents led to increased N2 and N2 O production by denitrification, but not to increased NO production rates. Anaerobic conditions, however, stimulated both NO and N2 O production by denitrification. The production of NO and N2 O was further stimulated at low moisture contents and after addition of glucose or NO3 − . Anaerobic consumption of NO by denitrification followed Michaelis-Menten kinetics and was stimulated by addition of glucose and NO3 − . Aerobic consumption of NO followed first-order kinetics up to mixing ratios of at least 14 ppmv NO, was inhibited by autoclaving but not by acetylene, and decreased with increasing soil moisture content. The high NO-consumption activity and the effects of soil moisture on the apparent rates of anaerobic and aerobic production and consumption of NO suggest that diffusional constraints have an important influence on the release of NO, and may be a reason for the different behaviour of NO release vs N2 O release. 相似文献
5.
6.
Metabolism of nitric oxide and nitrous oxide during nitrification and denitrification in soil at different incubation conditions 总被引:4,自引:0,他引:4
Abstract NO production and consumption rates as well as N2 O accumulation rates were measured in a loamy cambisol which was incubated under different conditions (i.e. soil moisture content, addition of nitrogen fertilizer and/or glucose, aerobic or anaerobic gas phase). Inhibition of nitrification with acetylene allowed us to distinguish between nitrification and denitrification as sources of NO and N2 O. Under aerobic conditions untreated soil showed very low release of NO and N2 O but high consumption of NO. Fertilization with NH4 + or urea stimulated both NO and N2 O production by nitrification. Addition of glucose at high soil moisture contents led to increased N2 and N2 O production by denitrification, but not to increased NO production rates. Anaerobic conditions, however, stimulated both NO and N2 O production by denitrification. The production of NO and N2 O was further stimulated at low moisture contents and after addition of glucose or NO3 − . Anaerobic consumption of NO by denitrification followed Michaelis-Menten kinetics and was stimulated by addition of glucose and NO3 − . Aerobic consumption of NO followed first-order kinetics up to mixing ratios of at least 14 ppmv NO, was inhibited by autoclaving but not by acetylene, and decreased with increasing soil moisture content. The high NO-consumption activity and the effects of soil moisture on the apparent rates of anaerobic and aerobic production and consumption of NO suggest that diffusional constraints have an important influence on the release of NO, and may be a reason for the different behaviour of NO release vs N2 O release. 相似文献
7.
Measurement of denitrification in estuarine sediment using membrane inlet mass spectrometry 总被引:1,自引:0,他引:1
Abstract Denitrification was measured in intact sediment cores and in homogenised slurries using membrane inlet mass spectrometry. Dissolved concentrations of O2 , N2 , N2 O and CO2 were simultaneously monitored. Using a 0.8 mm diameter needle probe, a comparison was made of the gas profiles of intact cores obtained under different conditions, i.e. with air or argon as the headspace gas and after the addition of nitrate and/or a carbon source to the sediment surface. O2 was detectable to a depth of 1 cm under a headspace of air and the depth at which the maxima of denitrification products occurred was 1.5–2 cm. Denitrification products (N2 O, N2 ) occurred in the surface layers where O2 was above the minimum level of detectability (> 0.25 μM): diffusion of N2 and N2 O upwards from the anoxic zone, local anaerobic microenvironments or aerobic denitrification are alternative explanations for this observation. The addition of nitrate and/or acetate increased the concentrations of N2 , N2 O and CO2 in the sediment core. In sediment slurries, the pH, nitrate concentration, carbon source and the depth from which the sample was taken affected the rate of denitrification. Nitrogen was the sole detectable end product. Maximum denitrification occurred at pH 7.5 and at 20 mM nitrate. Denitrification was at a maximum in those slurries prepared from sections of core at 1–2 cm depth. 相似文献
8.
9.
Nitrate inhibits symbiotic N2 fixation and a number of hypotheses concerned with NO3 − assimilation have been suggested to explain this inhibition. These hypotheses were tested using a pea ( Pisum sativum L. cv. Juneau) with normal nitrate reductase NR; (EC 1,6,6,4) activity and two mutants of cv. Juneau, A317 and A334, with impaired NR activity. The plants were inoculated with three strains of Rhizobium leguminosarum and grown for 3 weeks in N-free medium, followed by 1 week in medium supplemented with 0, 5 or 10 m M KNO3 before harvesting. NO3 was taken up at comparable rates by the parent and the mutants and accumulated in leaf and stem tissue of the latter. Acetylene reduction rates were inhibited similarly in both the parent and mutants in the presence of KNO3 but there were differences among rhizobial strains. Starch concentration of the nodules decreased by 46% in the presence of KNO3 and there were differences among rhizobial strains but not among pea genotypes. Malate and succinate accumulated in nodules in the presence of KNO3 . These data are not consistent with the photosynthate deprivation hypothesis as a primary mechanism for NO3 inhibition of N2 fixation since NO3 affected the nodule carbohydrate composition of all three pea genotypes in a similar manner. The lack of correlation between NR activity and NO3 inhibition of N2 fixation suggests that NO3 assimilation may be only indirectly involved in the inhibition phenomenon. 相似文献
10.
Addition of 2 mM nitrite or ammonium to aerobically incubated cultures of Gloeothece rapidly inhibited N2 fixation (measured as acetylene reduction). In contrast, 2 mM nitrate inhibited N2 fixation less rapidly and less extensively, and often temporarily stimulated nitrogenase activity. The inhibitory effects of both nitrate and ammonium could be prevented by addition of 3 mM L-methionine-DL-sulphoximine, suggesting that the true inhibitor of N2 fixation was an assimilatory product of ammonium rather than either ammonium or nitrate itself. The inhibition of N2 fixation by nitrite could not, however, be prevented by addition of L-methionine-DL- sulphoximine. On the other hand, nitrite (unlike nitrate and ammonium) did not inhibit N2 fixation in cultures incubated under a gas phase lacking oxygen. These findings suggest that the mechanism whereby nitrite inhibits N2 fixation in Gloeothece differs from that of either nitrate or ammonium. The inhibitory effect of nitrite on N2 fixation did not involve reduction of nitrite to nitric oxide, though nitric oxide was a potent inhibitor of nitrogenase activity in Gloeothece . Nitrate and nitrite inhibited the synthesis of nitrogenase in Gloeothece , while ammonium not only inhibited nitrogenase synthesis but also stimulated degradation of the enzyme. In addition, all three compounds favoured the appearance of the Fe-protein of nitrogenase in its larger, presumed inactive, form. 相似文献
11.
Nitrogen fixation by Baltic cyanobacteria is adapted to the prevailing photon flux density 总被引:3,自引:0,他引:3
A. M. EVANS J. R. GALLON A. JONES M. STAAL L. J. STAL M. VILLBRANDT & T. J. WALTON 《The New phytologist》2000,147(2):285-297
N2 fixation, measured as acetylene reduction, was studied in laboratory cultures and in natural assemblages (both as a mixed population and as individually picked colonies) of the heterocystous cyanobacteria Aphanizomenon sp. and Nodularia spp. from the Baltic Sea. During a diurnal cycle of alternating light and darkness, these organisms reduced acetylene predominantly during the period of illumination, although considerable activity was also observed during the dark period. In both laboratory cultures and natural populations N2 fixation was saturated below a photon flux density of 600 μm−2 s−1 . In cyanobacterial blooms in the Baltic Sea, nitrogenase activity was mostly confined to the surface layers. Samples collected from greater depths did not possess the same capacity for acetylene reduction as samples from the surface itself, even when incubated at the photon flux density prevailing in surface waters. This suggests that, with respect to N2 fixation, Baltic cyanobacteria are adapted to the intensity of illumination that they are currently experiencing. 相似文献
12.
Nitrogenase (N2 ase; EC 1.18.6.1) activity (H2 evolution) and root respiration (CO2 evolution) were measured under either N2 :O2 or Ar:O2 gas mixtures in intact nodulated roots from white clover ( Trifolium repens L.) plants grown either as spaced or as dense stands. The short-term nitrate (5 m M ) inhibition of N2 -fixation was promoted by competition for light between clover shoots, which reduced CO2 net assimilation rate. Oxygen-diffusion permeability of the nodule declined during nitrate treatment but after nitrate removal from the liquid medium its recovery parallelled that of nitrogenase activity. Rhizosphere pO2 was increased from 20 to 80 kPa under N2 :O2 . A simple mono-exponential model, fitted to the nodule permeability response to pO2 , indicated NO− 3 induced changes in minimum and maximum nodule O2 -diffusion permeability. Peak H2 production rates at 80 kPa O2 and in Ar:O2 were close to the pre-decline rates at 20 kPa O2 . At the end of the nitrate treatment, this O2 -induced recovery in nitrogenase activity reached 71 and 82%; for clover plants from spaced and dense stands, respectively. The respective roles of oxygen diffusion and phloem supply for the short-term inhibition of nitrogenase activity in nitrate-treated clovers are discussed. 相似文献
13.
Effects of oxygen, pH and nitrate concentration on denitrification by Pseudomonas species 总被引:2,自引:0,他引:2
Abstract The production of nitrogen-containing gases by denitrification in three organisms was examined using membrane inlet mass spectrometry. The effects of O2 (during both growth and maintenance) and of pH, nitrate concentration and carbon source were tested in non-proliferating cell suspensions. Two strains of Pseudomonas aeruginosa were capable of co-respiration of NO3 − and O2 and, under controlled O2 supply, gave oscillatory denitrification. Variations in culture and assay conditions affected both the rate of denitrification and the ratio of end products (N2 O:N2 ). Higher rates were seen following anaerobic growth. Optimum values of pH and nitrate concentration for denitrification are given. Generally, the optimum pH was 7.0–7.5, approximately that of the growth medium. Optimum nitrate concentration was generally 20 mM. 相似文献
14.
Rapid modulation of nitrate reductase in leaves and roots: Indirect evidence for the involvement of protein phosphorylation/dephosphorylation 总被引:2,自引:0,他引:2
Nitrate reductase activity (NRA; NADH-nitrate reductase, E. C. 1.6.6.1) has been measured in extracts from leaves of spinach ( Spinacia oleracea L.) in response to rapid changes in illumination, or supply of CO2 or oxygen. Measured in buffers containing magnesium, NRA from leaves decreased in the dark and increased again upon illumination. It decreased also, when CO2 was removed in continuous light, and was reactivated when CO2 was added. Nitrate reductase (NR) from roots of pea ( Pisum sativum L.) was also rapidly modulated in vivo. It increased under anaerobiosis and decreased in air or pure oxygen. The half time for inactivation or reactivation in roots and leaves was 5 to 30 min.
When spinach leaves were harvested during a normal day/night cycle, extractable NRA was low during the night, and high during daytime. However, at any point of the diurnal cycle, NR could be brought to a similar maximum activity by preincubation of the desalted leaf extract with AMP and/or EDTA. Thus, the observed diurnal changes appeared to be mainly a consequence of enzyme modulation, not of protein turnover. In vivo, the reactivation of the inactivated enzyme from both leaves and roots was prevented by okadaic acid, and inhibitor of certain protein phosphatases. Artificial lowering of the ATP-levels in leaf or root tissues by anaerobiosis (dark), mannose or the uncoupler carbonyl cyanide m -chlorophenyl hydrazon (CCCP), always brought about full activation of NR.
By preincubating crude leaf or root extracts with MgATP, NR was inactivated in vitro. Partial purification from spinach leaves of two enzymes with molecular masses in the 67 kD and 100 kD range, respectively, is reported. Both participate in the ATP-dependent inactivation of NR.
Alltogether these data indicate that NR can be rapidly modulated by reversible protein phosphorylation/dephosphorylation, both in shoots and in roots. 相似文献
When spinach leaves were harvested during a normal day/night cycle, extractable NRA was low during the night, and high during daytime. However, at any point of the diurnal cycle, NR could be brought to a similar maximum activity by preincubation of the desalted leaf extract with AMP and/or EDTA. Thus, the observed diurnal changes appeared to be mainly a consequence of enzyme modulation, not of protein turnover. In vivo, the reactivation of the inactivated enzyme from both leaves and roots was prevented by okadaic acid, and inhibitor of certain protein phosphatases. Artificial lowering of the ATP-levels in leaf or root tissues by anaerobiosis (dark), mannose or the uncoupler carbonyl cyanide m -chlorophenyl hydrazon (CCCP), always brought about full activation of NR.
By preincubating crude leaf or root extracts with MgATP, NR was inactivated in vitro. Partial purification from spinach leaves of two enzymes with molecular masses in the 67 kD and 100 kD range, respectively, is reported. Both participate in the ATP-dependent inactivation of NR.
Alltogether these data indicate that NR can be rapidly modulated by reversible protein phosphorylation/dephosphorylation, both in shoots and in roots. 相似文献
15.
CHARLOTTA FREDRIKSSON GILLIAN MALIN PIRZADA J. A. SIDDIQUI & BIRGITTA BERGMAN 《The New phytologist》1998,140(3):531-538
Symploca PCC 8002 Kützing is a filamentous cyanobacterium that lacks the specialized cells, known as heterocysts, that protect nitrogenase from O2 in most aerobic N2 -fixing cyanobacteria. Nevertheless, Symploca is able to carry out N2 fixation in the light under aerobic conditions. When cultures were grown under light/dark cycles, nitrogenase activity commenced and increased in the light phase and declined towards zero in the dark. Immunolocalization of dinitrogenase reductase in sectioned Symploca trichomes showed that the enzyme was present only in 9% of the cells. These cells lacked any obvious mechanical protection against atmospheric O2 and their ultrastructural characteristics were similar to those of cells that did not contain any dinitrogenase reductase. The nitrogenase-containing cells possessed carboxysomes that were rich in ribulose-1,5-bisphosphate carboxylase/oxygenase and phycoerythrin, a light harvesting pigment of PS II. This indicates that these cells had a capacity for both N2 fixation and photosynthesis. The significance of the localization pattern for dinitrogenase reductase is discussed in the context of N2 fixation in Symploca PCC 8002. 相似文献
16.
Abstract. In the preliminary purification of Capsicum leaf nitrate reductase (EC 1.6.6.1), treatment of the crude extract on Sephadex G-25 was necessary to prevent a gelling of the extract and sedimentation of the enzyme. Its Km values for NADH and nitrate were estimated to be 9.3 and 105mmol m−3 ADP and ATP gave hyperbolic competitive inhibition, with respect to NADH, while the inhibition by AMP was linear competitive. Ki values calculated were: ADP and ATP approximately lmol m−3 and AMP 2.3 mol m−3 . Inhibition by ADP was not altered by reduced glutathione.
The Capsicum nitrate reduclase was very susceptible to inhibition by NADH (in the absence of nitrate) and an in vivo assay showed that the activity of the enzyme was limited by the supply of nitrate. NADH and adenine nucleotide levels measured in the Capsicum leaf were used to estimate inhibition of nitrate reductase and a prediction was made of the nitrate reductase activity at different times in the photoperiod. This was shown to follow the same trend as the measured in vivo activity of the enzyme. Changes in adenine nucleotide levels had little effect on nitrate reductase activity. 相似文献
The Capsicum nitrate reduclase was very susceptible to inhibition by NADH (in the absence of nitrate) and an in vivo assay showed that the activity of the enzyme was limited by the supply of nitrate. NADH and adenine nucleotide levels measured in the Capsicum leaf were used to estimate inhibition of nitrate reductase and a prediction was made of the nitrate reductase activity at different times in the photoperiod. This was shown to follow the same trend as the measured in vivo activity of the enzyme. Changes in adenine nucleotide levels had little effect on nitrate reductase activity. 相似文献
17.
Abstract A denitrifying Cytophaga was isolated from soil enriched by anaerobic incubation with glucose, sulfide (S2− ), nitrous oxide (N2 O), and acetylene (C2 H2 ). Such soil enrichments and pure cultures of the isolated Cytophaga reduced N2 O rapidly even in the presence of a normally inhibitory concentration of C2 H2 (4 kPa) providing S2− was present (8 μmol/g soil or 0.4 μmol/ml culture). Since C2 H2 inhibition of the reduction of N2 O is used as a tool in the assay of denitrification, the presence in large numbers of such a Cytophaga may influence the effectiveness of this assay especially in sulfidic environments. 相似文献
18.
The nitrate reductase (NR, EC 1.6.6.1) activity in root nodules formed by hydrogenase positive (Hup+ ) and hydrogenase negative (Hup− ) Rhizobium leguminosarum strains was examined in symbioses with the pea cultivar Alaska ( Pisum sativum L.), Rates of activity were determined by the in vivo assay in nodules from plants that were only N2 -dependent or grown in the presence of 2 m M KNO3 . The rates varied widely among strains, regardless of the Hup phenotype of the R. leguminosarum strain used for inoculation, but the overall results indicated that nodules formed by Hup− strains accumulated more nitrite in the incubation medium than did those with Hup− phenotypes. Total plant dry weight and reduced nitrogen content of pea plants grown in the presence of 2 m M KNO3 and inoculated with single Hup+ and Hup− R. leguminosarum strains were statistically different among some strains. These observations suggest that the possible advantages derived from the presence of the Hup system on whole plant growth may be counteracted by the higher rates of NR activity in the Hup− strains in the R. leguminosarum -pea symbiosis. 相似文献
19.
Metabolism of nitric oxide in soil and denitrifying bacteria 总被引:1,自引:0,他引:1
Abstract Production and consumption of NO was measured under anaerobic conditions in a slightly alkaline and an acidic soil as well as in pure cultures of denitrifying Pseudomonas aeruginosa, P. stutzeri, P. fluorescens, Paracoccus denitrificans, Azospirillum brasilense , and A. lipoferum . Growing bacterial cultures reduced nitrate and intermediately accumulated nitrite, NO, N2 O, but not NO2 . Addition of formaldehyde inhibited NO production and NO consumption. In the presence of acetylene NO was reduced to N2 O. Net NO release rates in denitrifying bacterial suspensions and in soil samples decreased hyperbolically with increasing NO up to mixing ratios of about 5 ppmv NO. This behaviour could be modelled by assuming a constant rate of NO production simultaneously with a NO consumption activity that increased with NO until V max was reached. The data allowed calculation of the gross rates ( P ) of NO production, of the rate constants ( k ), V max and K m of NO consumption, and of the NO compensation mixing ratio ( m c ). In soil, P was larger than V max resulting in net NO release even at high NO mixing ratios unless P was selectively inhibited by chlorate + chlorite or by aerobic incubation conditions. In bacteria, V max was somewhat larger than P resulting in net NO uptake at high NO mixing ratios. Both P and V max were dependent on the supply of electron donor (e.g. glucose). Both in soil (aerobic or anaerobic) and in pure culture, the K m values of NO consumption were in a similar low range of about 0.5–6.0 nM. Anaerobic soil and denitrifying bacteria exhibited m c values of 1.6–2.1 ppmv NO and 0.2–4.0 ppmv NO, respectively. 相似文献
20.
A comparison of inhibition of French-bean and soybean nitrogen fixation by nitrate, 1% oxygen or direct assimilate deprivation 总被引:1,自引:0,他引:1
Inhibition by NO− 3 of acetylene reduction in bean ( Phaseolus vulgaris L. cv. Contender) and soybean ( Glycine max L. cv. Amsoy 71) was measured in parallel with nodule carbohydrate and nitrate metabolism. In bean the onset of inhibition of C2 H2 reduction (6 h) coincided with decreased import of assimilates and a lowering of carbohydrate pools (sucrose, glucose and starch). Nitrate reductase (EC 1.6.6.1) activity was induced in all plant organs after 3 h but no nitrite was detected in the nodules. In soybean, nodule carbohydrate concentrations and import of assimilates into the nodules increased markedly between 6 to 24 h after supply of nitrate when the nitrogenase (EC 1.7.99.2) was progressively inhibited. High nitrate reductase activity was observed in the nodules and nitrites accumulated because of insufficient nitrite reductase activity. The nitrate-induced inhibition of nitrogenase was compared with the inhibition observed with low oxygen around the roots (1% O2 ) or with direct assimilate deprivation (girdling or decapitation). Soybean and bean appeared equally sensitive to these treatments as regards to acetylene reduction. The results are discussed in relation to the current hypotheses explaining nitrate-induced inhibition of dinitrogen fixation: assimilate deprivation or nitrite poisoning. Present data are in favour of the first for bean and of the second for soybean. 相似文献