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1.
Nitrogen assimilation in three nitrate reductase (NR) mutants of soybean ( Glycine max L. Merr. cv Williams) was studied in the growth chamber and in the field. These mutants, LNR-2, LNR-3, and LNR-4, lack the non-NO 3−-inducible or constitutive fraction of leaf NR activity found in wild-type plants, but this had no effect on the concentration of nitrogen accumulated when grown on NO 3− in the growth chamber. Dry weight accumulation of two of the mutants (LNR-3 and LNR-4) was decreased relative to LNR-2 and wild type. In the field, LNR-2 had dry weights and nitrogen concentrations similar to the wild type at 34 and 61 days after planting, and at maturity. Acetylene reduction activities were also similar at 61 days. 相似文献
2.
The effects of N source (6 m m nitrogen as NO 3− or urea) and tungstate (0, 100, 200, 300, and 400 μ m Na 2 WO 4) on nitrate metabolism, nodulation, and growth of soybean ( Glycine max [L.] Merr.) plants were evaluated. Nitrate reductase activity and, to a lesser extent, NO 3− content of leaf tissue decreased with the addition of tungstate to the nutrient growth medium. Concomitantly, nodule mass and acetylene reduction activity of NO 3−-grown plants increased with addition of tungstate to the nutrient solution. In contrast, nodule mass and acetylene reduction activity of urea-grown plants decreased with increased nutrient tungstate levels. The acetylene reduction activity of nodulated roots of NO 3−-grown plants was less than 10% of the activity of nodulated roots of urea-grown plants when no tungstate was added. At 300 and 400 μ m tungstate levels, acetylene reduction activity of nodulated roots of NO 3−-grown plants exceeded the activity of comparable urea-grown plants. 相似文献
3.
Two nitrate reductase (NR) mutants were selected for low nitrate reductase (LNR) activity by in vivo NR microassays of M 2 seedlings derived from nitrosomethylurea-mutagenized soybean ( Glycine max [L.] Merr. cv Williams) seeds. The mutants (LNR-5 and LNR-6) appeared to have normal nitrate-inducible NR activity. Both mutants, however, showed decreased NR activity in vivo and in vitro compared with the wild-type. In vitro FMNH 2-dependent nitrate reduction and Cyt c reductase activity of nitrate-grown plants, and nitrogenous gas evolution during in vivo NR assays of urea-grown plants, were also decreased in the mutants. The latter observation was due to insufficient generation of nitrite substrate, rather than some inherent difference in enzyme between mutant and wild-type plants. When grown on urea, crude extracts of LNR-5 and LNR-6 lines had similar NADPH:NR activities to that of the wild type, but both mutants had very little NADH:NR activity, relative to the wild type. Blue Sepharose columns loaded with NR extract of urea-grown mutants and sequentially eluted with NADPH and NADH yielded a NADPH:NR peak only, while the wild-type yielded both NADPH: and NADH:NR peaks. Activity profiles confirmed the lack of constitutive NADH:NR in the mutants throughout development. The results provide additional support to our claim that wild-type soybean contains three NR isozymes, namely, constitutive NADPH:NR (c 1NR), constitutive NADH:NR (c 2NR), and nitrate-inducible NR (iNR). 相似文献
4.
Soybean ( Glycine max [L.] Merr.) leaves have been shown to contain three forms of nitrate reductase (NR). Two of the forms, which are present in leaves of wild-type (cv. Williams) plants grown in the absence of NO 3−, are termed constitutive and designated c 1NR and c 2NR. The third form, which is present in NO 3−-grown mutant (nr 1) plants lacking the constitutive forms, is termed inducible and designated iNR. Samples of c 1NR, c 2NR, and iNR obtained from appropriately treated plants were analyzed for the presence of partial activities, response to inhibitors, and ability to complement a barley NR which lacks the molybdenum cofactor (MoCo) but is otherwise active. The three forms were similar to most assimilatory NR enzymes in that they (a) exhibited NADH-cytochrome c reductase, reduced flavin mononucleotide-NR, and reduced methyl viologen-NR partial activities; (b) were inhibited by p-hydroxymercuribenzoate at the site of initial electron transport through each enzyme; (c) were more inhibited by CN− in their reduced enzyme state as compared with their oxidized state; and (d) complemented a MoCo-defective NR (e.g. contained cofactors with characteristics similar to the MoCo found in barley NR and commercial xanthine oxidase). However, among themselves, they showed dissimilarities in their response to treatment with HCO3− and CN−, and in their absolute ability to complement the barley NR. The site of effect for these treatments was the terminal cofactor-containing portion of each enzyme. This indicated that, although a terminal cofactor (presumably a MoCo) was present in each form, structural or conformational differences existed in the terminal cofactor-protein complex of each form. 相似文献
5.
The comparative induction of nitrate reductase (NR) by ambient NO 3− and NO 2− as a function of influx, reduction (as NR was induced) and accumulation in detached leaves of 8-day-old barley ( Hordeum valgare L.) seedlings was determined. The dynamic interaction of NO 3− influx, reduction and accumulation on NR induction was shown. The activity of NR, as it was induced, influenced its further induction by affecting the internal concentration of NO 3−. As the ambient concentration of NO 3− increased, the relative influences imposed by influx and reduction on NO 3− accumulation changed with influx becoming a more predominant regulant. Significant levels of NO 3− accumulated in NO 2−-fed leaves. When the leaves were supplied cycloheximide or tungstate along with NO 2−, about 60% more NO 3− accumulated in the leaves than in the absence of the inhibitors. In NO 3−-supplied leaves NR induction was observed at an ambient concentration of as low as 0.02 m m. No NR induction occurred in leaves supplied with NO 2− until the ambient NO 2− concentration was 0.5 m m. In fact, NR induction from NO 2− solutions was not seen until NO 3− was detected in the leaves. The amount of NO 3− accumulating in NO 2−-fed leaves induced similar levels of NR as did equivalent amounts of NO 3− accumulating from NO 3−-fed leaves. In all cases the internal concentration of NO 3−, but not NO 2−, was highly correlated with the amount of NR induced. The evidence indicated that NO 3− was a more likely inducer of NR than was NO 2−. 相似文献
6.
Corn seedlings ( Zea mays cv W64A × W182E) were grown hydroponically, in the presence or absence of NO 3−, with or without light and with NH 4Cl as the only N source. In agreement with earlier results nitrate reductase (NR) activity was found only in plants treated with both light and NO 3−. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer of the proteins to nitrocellulose paper and reaction with antibodies prepared against a pure NR showed that crude extracts prepared from light-grown plants had a polypeptide of approximately 116 kilodaltons (the subunit size for NR) when NO 3− was present in the growth medium. Crude extracts from plants grown in the dark did not have the 116 kilodalton polypeptide, although smaller polypeptides, which reacted with NR-immunoglobulin G, were sometimes found at the gel front. When seedlings were grown on Kimpack paper or well washed sand, NR activity was again found only when the seedlings were exposed to light and NO 3−. Under these conditions, however, a protein of about 116 kilodaltons, which reacted with the NR antibody was present in light-grown plants whether NO 3− was added to the system or not. The NR antibody cross-reacting protein was also seen in hydroponically grown plants when NH 4Cl − was the only added form of nitrogen. These results indicate that the induction of an inactive NR-protein precursor in corn is mediated either by extremely low levels of NO 3− or by some other unidentified factor, and that higher levels of NO 3− are necessary for converting the inactive NR cross-reacting protein to a form of the enzyme capable of reducing NO 3− to NO 2−. 相似文献
7.
Growth chamber studies with soybeans ( Glycine max [L.] Merr.) were designed to determine the relative limitations of NO 3−, NADH, and nitrate reductase (NR) per se on nitrate metabolism as affected by light and temperature. Three NR enzyme assays (+NO 3−in vivo, −NO 3−in vivo, and in vitro) were compared. NR activity decreased with all assays when plants were exposed to dark. Addition of NO 3− to the in vivo NR assay medium increased activity (over that of the −NO 3−in vivo assay) at all sampling periods of a normal day-night sequence (14 hr-30 C day; 10 hr-20 C night), indicating that NO 3− was rate-limiting. The stimulation of in vivo NR activity by NO 3− was not seen in plants exposed to extended dark periods at elevated temperatures (16 hr-30 C), indicating that under those conditions, NO 3− was not the limiting factor. Under the latter condition, in vitro NR activity was appreciable (19 μmol NO 2− [g fresh weight, hr] −1) suggesting that enzyme level per se was not the limiting factor and that reductant energy might be limiting. 相似文献
8.
The effect of water stress on patterns of nitrate reductase activity in the leaves and nodules and on nitrogen fixation were investigated in Medicago sativa L. plants watered 1 week before drought with or without NO 3−. Nitrogen fixation was decreased by water stress and also inhibited strongly by the presence of NO 3−. During drought, leaf nitrate reductase activity (NRA) decreased significantly particularly in plants watered with NO 3−, while with rewatering, leaf NRA recovery was quite important especially in the NO 3−-watered plants. As water stress progressed, the nodular NRA increased both in plants watered with NO 3− and in those without NO 3− contrary to the behavior of the leaves. Beyond −15.10 5 pascal, nodular NRA began to decrease in plants watered with NO 3−. This phenomenon was not observed in nodules of plants given water only. 相似文献
9.
The nr 1 soybean ( Glycine max [L.] Merr.) mutant does not contain the two constitutive nitrate reductases, one of which is responsible for enzymic conversion of nitrite to NO x (NO + NO 2). It was tested for possible nonenzymic NO x formation and evolution because of known chemical reactions between NO 2− and plant metabolites and the instability of nitrous acid. It did not evolve NO x during the in vivo NR assay, but intact leaves did evolve small amounts of NO x under dark, anaerobic conditions. Experiments were conducted to compare NO 3− reduction, NO 2− accumulation, and the NO x evolution processes of the wild type (cv Williams) and the nr 1 mutant. In vivo NR assays showed that wild-type leaves had three times more NO 3− reducing capacity than the nr 1 mutant. NO x evolution from intact, anerobic nr 1 leaves was approximately 10 to 20% that from wild-type leaves. Nitrite content of the nr 1 mutant leaves was usually higher than wild type due to low NO x evolution. Lag times and threshold NO 2− concentrations for NO x evolution were similar for the two genotypes. While only 1 to 2% of NO x from wild type is NO 2, the nr 1 mutant evolved 15 to 30% NO 2. The kinetic patterns of NO x evolution with time weré completely different for the mutant and wild type. Comparisons of light and heat treatments also gave very different results. It is generally accepted that the NO x evolution by wild type is primarily an enzymic conversion of NO 2− to NO. However, this report concludes that NO x evolution by the nr 1 mutant was due to nonenzymic, chemical reactions between plant metabolites and accumulated NO 2− and/or decomposition of nitrous acid. Nonenzymic NO x evolution probably also occurs in wild type to a degree but could be easily masked by high rates of the enzymic process. 相似文献
10.
The effects of nitrogen source NO 3− or NH 4+ on nitrogen metabolism during the first 2 weeks of germination of the rice seedling ( Oryza sativa L., var. IR22) grown in nutrient solution containing 40 μg/ml N were studied. Total, soluble protein, and free amino N levels were higher in the NH 4+-grown seedling, particularly during the 1st week of germination. Asparagine accounted for most of the difference in free amino acid level, in both the root and the shoot. Nitrate and nitrite reductase activities were present mainly in the shoot and were higher in the NO 3−-grown seedling, whereas the activity of glutamate dehydrogenase and glutamine synthetase in the root tended to be lower than that of the NH 4+-grown seedling during the 1st week of germination. Glycolate oxidase and catalase activities were present mainly in the shoot. Maximum activity of the above five enzymes occurred 7 to 10 days after germination. Differences in the zymograms of nitrate reductase, glutamate dehydrogenase, and catalase were mainly between shoot and root and not from N source. Nitrite reductase bands were observed only in plants grown in plants grown in NO 3−. 相似文献
11.
Enzyme activities involved in nitrate assimilation were analyzed from crude leaf extracts of wild-type (cv. Williams) and mutant ( nr 1 ) soybean [ Glycine max (L.) Merr.] plants lacking constitutive nitrate reductase (NR) activity. The nr 1 soybean mutant (formerly LNR-2), had decreased NADH-NR, FMNH 2-NR and cytochrome c reductase activities, all of which were associated with the loss of constitutive NR activity. Measurement of FMNH 2-NR activity, by nitrite determination, was accurate since nitrite reductase could not use FMNH 2 as a reductant source. Nitrite reductase activity was normal in the nr 1 plant type in the presence of reduced methyl viologen. Assuming that constitutive NR is similar in structure to nitrate reductases from other plants, presence of xanthine dehydrogenase activity and loss of cytochrome c reductase activity indicated that the apoprotein and not the molybdenum cofactor had been affected in the constitutive enzyme of the mutant. Constitutive NR from urea-grown wild-type plants had 1) greater ability to use FMNH 2 as an electron donor, 2) a lower pH optimum, and 3) decreased ability to distinguish between NO 3− and HCO 3−, compared with inducible NR from NO 3−-grown nr 1 plants. The presence in soybean leaves of a nitrate reductase with a pH optimum of 7.5 is contrary to previous reports and indicates that soybean is not an exception among higher plants for this activity. 相似文献
12.
The role of NO 3− and NO 2− in the induction of nitrite reductase (NiR) activity in detached leaves of 8-day-old barley ( Hordeum vulgare L.) seedlings was investigated. Barley leaves contained 6 to 8 micromoles NO 2−/gram fresh weight × hour of endogenous NiR activity when grown in N-free solutions. Supply of both NO 2− and NO 3− induced the enzyme activity above the endogenous levels (5 and 10 times, respectively at 10 millimolar NO 2− and NO 3− over a 24 hour period). In NO 3−-supplied leaves, NiR induction occurred at an ambient NO 3− concentration of as low as 0.05 millimolar; however, no NiR induction was found in leaves supplied with NO 2− until the ambient NO 2− concentration was 0.5 millimolar. Nitrate accumulated in NO 2−-fed leaves. The amount of NO 3− accumulating in NO 2−-fed leaves induced similar levels of NiR as did equivalent amounts of NO 3− accumulating in NO 3−-fed leaves. Induction of NiR in NO 2−-fed leaves was not seen until NO 3− was detectable (30 nanomoles/gram fresh weight) in the leaves. The internal concentrations of NO 3−, irrespective of N source, were highly correlated with the levels of NiR induced. When the reduction of NO 3− to NO 2− was inhibited by WO 42−, the induction of NiR was inhibited only partially. The results indicate that in barley leaves NiR is induced by NO 3− directly, i.e. without being reduced to NO 2−, and that absorbed NO 2− induces the enzyme activity indirectly after being oxidized to NO 3− within the leaf. 相似文献
13.
The objective of this experiment was to elucidate the manner in which N metabolism is influenced by S nutrition. Maize ( Zea mays L.) seedlings supplied with Hoagland solution minus SO 42− exhibited S deficiency symptoms 12 days after emergence. Prior to development of these symptoms, a decline in leaf blade nitrate reductase (NR, EC 1.6.6.1) activity was observed in S-deprived seedlings compared to normal seedlings. Twelve days after emergence, in vitro NR activity was diminished 50% compared to normal seedlings. Glutamine synthetase (EC 6.3.1.2) and NAD-glutamate dehydrogenase (EC 1.4.1.2) activities were less severely affected (19 and 13%, respectively, at day 12). NADP-glutamate dehydrogenase (EC 1.4.1.4) activity and leaf blade fresh weight were not altered by S deprivation. Concentrations of soluble protein and chlorophyll ( a and b) in leaf blades were reduced 18 and 25%, respectively, at day 12. A significantly higher concentration of NO 3−-N was observed for leaf blade and stem (culms, leaf sheaths, and unfurled leaves) fractions (46 and 31%, respectively) in S-deprived plants. In contrast to the other parameters measured, NR activity in S-deprived seedlings could be readily restored to the normal level by addition of SO 42−. The apparent preferential effect of S deprivation on NR activity could be causally related to the observed changes in NO 3−-N and soluble protein concentration. 相似文献
14.
Membrane associated nitrate reductase (NR) was detected in plasma membrane (PM) fractions isolated by aqueous two-phase partitioning from barley ( Hordeum vulgare L. var CM 72) roots. The PM associated NR was not removed by washing vesicles with 500 millimolar NaCl and 1 millimolar EDTA and represented up to 4% of the total root NR activity. PM associated NR was stimulated up to 20-fold by Triton X-100 whereas soluble NR was only increased 1.7-fold. The latency was a function of the solubilization of NR from the membrane. NR, solubilized from the PM fraction by Triton X-100 was inactivated by antiserum to Chlorella sorokiniana NR. Anti-NR immunoglobulin G fragments purified from the anti-NR serum inhibited NO 3− uptake by more than 90% but had no effect on NO 2− uptake. The inhibitory effect was only partially reversible; uptake recovered to 50% of the control after thorough rinsing of roots. Preimmune serum immunoglobulin G fragments inhibited NO 3− uptake 36% but the effect was completely reversible by rinsing. Intact NR antiserum had no effect on NO 3− uptake. The results present the possibility that NO 3− uptake and NO 3− reduction in the PM of barley roots may be related. 相似文献
15.
Experiments were conducted with segments of corn roots to investigate whether nitrate reductase (NR) is compartmentalized in particular groups of cells that collectively form the root symplastic pathway. A microsurgical technique was used to separate cells of the epidermis, of the cortex, and of the stele. The presence of NR was determined using in vitro and enzyme-linked immunosorbent assays. In roots exposed to 0.2 millimolar NO 3− for 20 hours, NR was detected almost exclusively in epidermal cells, even though substantial amounts of NO 3− likely were being transported through cortical and steler cells during transit to the vascular system. Although NR was present in all cell groups of roots exposed to 20.0 millimolar NO 3−, the majority of the NR still was contained in epidermal cells. The results are consistent with previous observations indicating that limited reduction of endogenous NO 3− occurs during uptake and reduction of exogenous NO 3−. Several mechanisms are advanced to account for the restricted capacity of cortical and stelar cells to induce NR and reduce NO 3−. It is postulated that (a) the biochemical system involved in the induction of NR in the cortex and stele is relatively insensitive to the presence of NO 3−, (b) the receptor for the NR induction response and the NR protein are associated with cell plasmalemmae and little NO 3− is taken up by cells of the cortex and stele, and/or (c) NO 3− is compartmentalized during transport through the symplasm, which limits exposure for induction of NR and NO 3− reduction. 相似文献
16.
Phosphate-limited chemostat cultures were used to study cell growth and N assimilation in Anabaena flos-aquae under various N sources to determine the relative energetic costs associated with the assimilation of NH 3, NO 3−, or N 2. Expressed as a function of relative growth rate, steady state cellular P contents and PO 4 assimilation rates did not vary with N-source. However, N-source did alter the maximal PO 4-limited growth rate achieved by the cultures: the NO 3− and N 2 cultures attained only 97 and 80%, respectively, of the maximal growth rate of the NH 3 grown cells. Cellular biomass and C contents did not vary with growth rate, but changed with N source. The NO 3−-grown cells were the smallest (627 ± 34 micromoles C · 10 −9 cells), while NH 3-grown cells were largest (900 ± 44 micromoles C · 10 −9 cells) and N 2-fixing cells were intermediate (726 ± 48 micromoles C · 10 −9 cells) in size. In the NO 3−-and N 2-grown cultures, N content per cell was only 57 and 63%, respectively, of that in the NH 3-grown cells. Heterocysts were absent in NH 3-grown cultures but were present in both the N 2 and NO 3− cultures. In the NO 3−-grown cultures C 2H 2 reduction was detected only at high growth rates, where it was estimated to account for a maximum of 6% of the N assimilated. In the N 2-fixing cultures the acetylene:N 2 ratio varied from 3.4:1 at lower growth rates to 3.0:1 at growth rates approaching maximal. Compared with NH3, the assimilation of NO3− and N2 resulted either in a decrease in cellular C (NO3− and N2 cultures) or in a lower maximal growth rate (N2 culture only). The observed changes in cell C content were used to calculate the net cost (in electron pair equivalents) associated with growth on NO3− or N2 compared with NH3. 相似文献
17.
In vivo NO 3− reduction in roots and shoots of intact barley ( Hordeum vulgare L. var Numar) seedlings was estimated in light and darkness. Seedlings were placed in darkness for 24 hours to make them carbohydrate-deficient. During darkness, the leaves lost 75% of their soluble carbohydrates, whereas the roots lost only 15%. Detached leaves from these plants reduced only 7% of the NO 3− absorbed in darkness. By contrast, detached roots from the seedlings reduced the same proportion of absorbed NO 3−, as did roots from normal light-grown plants. The rate of NO 3− reduction in the roots accounted for that found in the intact dark-treated carbohydrate-deficient seedlings. The rates of NO 3− reduction in roots of intact plants were the same for approximately 12 hours, both in light and darkness, after which the NO 3− reduction rate in roots of plants placed in darkness slowly declined. In the dark, approximately 40% of the NO 3− reduction occurred in the roots, whereas in light only 20% of the total NO 3− reduction occurred in roots. A lesser proportion was reduced in roots because the leaves reduced more nitrate in light than in darkness. 相似文献
18.
13NO 3− was used to investigate patterns of NO 3− influx into roots of barley plants ( Hordeum vulgare L. cv Klondike) previously grown with (`induced') or without (`uninduced') a source of external NO 3− ([NO 3−] 0). In both induced and uninduced plants, 13NO 3− influx was biphasic in the range from 0.005 to 50 moles per cubic meter [NO 3−] 0. In the low concentration range (<1 mole per cubic meter for induced plants and <0.3 mole per cubic meter for uninduced plants), influx was saturable and Vmax and Km values for influx either increased or decreased according to NO 3− pretreatment. By contrast, 13NO 3− influx in the high concentration range revealed a strictly linear concentration dependence. These fluxes appeared to be mediated by a constitutive, rather than an inducible, transport system. 相似文献
19.
Transformed Nicotiana plumbaginifolia plants with constitutive expression of nitrate reductase (NR) activity were grown at different levels of nitrogen nutrition. The gradients in foliar NO
3
–
content and maximum extractable NR activity observed with leaf order on the shoot, from base to apex, were much decreased as a result of N-deficiency in both the transformed plants and wild type controls grown under identical conditions. Constitutive expression of NR did not influence the foliar protein and chlorophyll contents under any circumstances. A reciprocal relationship between the observed maximal extractable NR activity of the leaves and their NO
3
–
content was observed in plants grown in nitrogen replete conditions at low irradiance (170 mol photons·m –2 ·s –1). This relationship disappeared at higher irradiance (450 mol photons·m –2·S –1) because the maximal extractable NR activity in the leaves of the wild type plants in these conditions increased to a level that was similar to, or greater than that found in constitutive NR-expressors. Much more NO
3
–
accumulated in the leaves of plants grown at 450 mol photons·m –2·s –1 than in those grown at 170 mol photons·m –2·s –1 in N-replete conditions. The foliar NO
3
–
level and maximal NR activity decreased with the imposition of N-deficiency in all plant types such that after prolonged exposure to nitrogen depletion very little NO
3
–
was found in the leaves and NR activity had decreased to almost zero. The activity of NR decreased under conditions of nitrogen deficiency. This regulation is multifactoral since there is no regulation of NR gene expression by NO
3
–
in the constitutive NR-expressors. We conclude that the NR protein is specifically targetted for destruction under nitrogen deficiency. Consequently, constitutive expression of NR activity does not benefit the plant in terms of increased biomass production in conditions of limiting nitrogen.Abbreviations Chl
chlorophyll
- N
nitrogen
- NR
NADH-nitrate reductase
- WT
wild type 相似文献
20.
The nature of the injury and recovery of nitrate uptake (net uptake) from NaCl stress in young barley ( Hordeum vulgare L, var CM 72) seedlings was investigated. Nitrate uptake was inhibited rapidly by NaCl, within 1 minute after exposure to 200 millimolar NaCl. The duration of exposure to saline conditions determined the time of recovery of NO 3− uptake from NaCl stress. Recovery was dependent on the presence of NO 3− and was inhibited by cycloheximide, 6-methylpurine, and cerulenin, respective inhibitors of protein, RNA, and sterol/fatty acid synthesis. These inhibitors also prevented the induction of the NO 3− uptake system in uninduced seedlings. Uninduced seedlings exhibited endogenous NO 3− transport activity that appeared to be constitutive. This constitutive activity was also inhibited by NaCl. Recovery of constitutive NO 3− uptake did not require the presence of NO 3−. 相似文献
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