Characterization of Tn5 mutants deficient in dissimilatory nitrite reduction in Pseudomonas sp. strain G-179, which contains a copper nitrite reductase.

Tn5 was used to generate mutants that were deficient in the dissimilatory reduction of nitrite for Pseudomonas sp. strain G-179, which contains a copper nitrite reductase. Three types of mutants were isolated. The first type showed a lack of growth on nitrate, nitrite, and nitrous oxide. The second type grew on nitrate and nitrous oxide but not on nitrite (Nir-). The two mutants of this type accumulated nitrite, showed no nitrite reductase activity, and had no detectable nitrite reductase protein bands in a Western blot (immunoblot). Tn5 insertions in these two mutants were clustered in the same region and were within the structural gene for nitrite reductase. The third type of mutant grew on nitrate but not on nitrite or nitrous oxide (N2O). The mutant of this type accumulated significant amounts of nitrite, NO, and N2O during anaerobic growth on nitrate and showed a slower growth rate than the wild type. Diethyldithiocarbamic acid, which inhibited nitrite reductase activity in the wild type, did not affect NO reductase activity, indicating that nitrite reductase did not participate in NO reduction. NO reductase activity in Nir- mutants was lower than that in the wild type when the strains were grown on nitrate but was the same as that in the wild type when the strains were grown on nitrous oxide. These results suggest that the reduction of NO and N2O was carried out by two distinct processes and that mutations affecting nitrite reduction resulted in reduced NO reductase activity following anaerobic growth with nitrate.     

Effects of Nitrate Nutrition on Nitrogen Metabolism in Cassava

Two experiments were conducted independently with plants of cassava (Manihot esculenta Crantz) growing in sand with nutrient solutions with four nitrate concentrations (0.5, 3, 6 or 12 mM). In leaves, nitrate-N was undetectable at the low nitrate applications; total-N, ammonium-N, amino acid-N, reduced-N and insoluble-N all increased linearly, while soluble proteins did it curvilinearly, with increasing nitrate supply. In contrast, soluble-N did not respond to N treatments. Total-N and soluble proteins, but not nitrate-N or ammonium-N, were much higher in leaves than in roots. Plants grown under severe N deficiency accumulated ammonium-N and amino acid-N in their roots. Further, plants were exposed to either 3 or 12 mM nitrate-N, and leaf activities of key N-assimilating enzymes were evaluated. Activities of nitrate reductase, glutamine synthetase, glutamate synthase and glutamate dehydrogenase were considerably lower in low nitrate supply than in high one. Despite the low nitrate reductase activity, cassava leaves showed an ability to maintain a large proportion of N in soluble proteins.     

Impact of nitrate supply in C and N assimilation in the parasitic plant Striga hermonthica (Del.) Benth (Scrophulariaceae) and its host Sorghum bicolor L

The threshold of tolerance for nitrate of the parasitic weed Striga hermonthica (Del.) Benth and the host plant Sorghum bicolor L. was determined by estimating the impact of increasing nitrate loads on plant growth and various parameters of C and N assimilation. Nitrate supply improved chlorophyll (Chl) content and photosystem II (PSII) photochemistry of infected S. bicolor that, in comparison to S. hermonthica, displayed a low imbalance between C and N assimilation when nitrate was supplied up to 1500 mg N per plant. Indeed, nitrate supplies increased strongly the leaf N:C ratio of the parasite. The higher nitrate load induced strong accumulation of nitrate, nitrite and ammonium, and consequently the death of S. hermonthica. Nevertheless, lower nitrate loads (up to 500 mg N per S. bicolor in this study) promoted leaf expansion, PSII photochemistry and N metabolism of S. hermonthica mature (M) plants, as attested by the significant rise in soluble protein and free amino-acid contents. Following these N supplies, the nitrate tolerance of S. hermonthica was correlated with an increase in PSII activity and a high incorporation of N excess into asparagine. This confirmed the central role of asparagine in the N metabolism of S. hermonthica, although this detoxification pathway was insufficient to limit ammonium accumulation under higher nitrate loads.     

Dependence of nitrate-induced oxalate accumulation on nitrate reduction in rice leaves

Oxalate, a common constituent in many plants, is known to play important functional roles in plants. However, excess levels of oxalate in edible parts of plants adversely affect their quality as food. Understanding the regulatory mechanism in plants, particularly in food crops, is of both scientific and practical significance. While a number of studies have shown that nitrate can efficiently induce oxalate accumulation in plants, how it elicits such an effect is not well understood. This study aimed to gain a further insight into the mechanism underlying the nitrate-induced oxalate accumulation. Nitrate-N efficiently caused oxalate accumulation in rice leaves, depending on the nitrate concentrations and treatment time. In contrast, same nitrogen molar levels of the other N forms such as nitrite, ammonium, glutamate and urea either had no effect on the accumulation or even reduced the oxalate level. When glutamate, glutamine, asparate and asparagine were added into the nutrient solution that already contained saturating concentration of nitrate, both oxalate levels and NR activity were correspondingly decreased. In all of these modes of treatment, the change in NR activity was positively paralleled to that in oxalate levels. For a further confirmation, we generated the transgenic rice plants with a NR interference gene introduced. The result further demonstrated that in the transgenic plants, unlike in wild-type plants, oxalate was no longer able to accumulate in response to the nitrate treatment even though the endogenous nitrate levels were substantially elevated. Taken together, our results suggest that the nitrate-induced oxalate accumulation in rice leaves is dependent on the NR-catalyzed nitrate reduction, rather than on nitrate itself or nitrite reduction or its downstream metabolites.     

Sulfur starvation and restoration affect nitrate uptake and assimilation in rapeseed

We analyzed the effect of omission of sulfur (S) from the nutrient solution and then restoration of S-source on the uptake and assimilation of nitrate in rapeseed. Incubation in nutrient solution without S for 1–6 days led to decline in uptake of nitrate, activities, and expression levels of nitrate reductase (NR) and glutamine synthetase (GS). The nitrite reductase (NiR) and glutamate synthase (GOGAT) activities were not considerably affected. There was significant enhancement in nitrate content and decline in sulfate content. Evaluation of amino acid profile under S-starvation conditions showed two- to fourfold enhancement in the contents of arginine, asparagine and O-acetyl-l-serine (OAS), whereas the contents of cysteine and methionine were reduced heavily. When the S-starved plants were subjected to restoration of S for 1, 3, 5, and 7 days, activities and expression levels of NR and GS recovered within the fifth and seventh days of restoration, respectively. Exogenous supply of metabolites (arginine, asparagine, cysteine, glutamine, OAS, and methionine) also affected the uptake and assimilation of nitrate, with a maximum for OAS. These results corroborate the tight interconnection of S-nutrition with nitrate assimilation and that OAS plays a major role in this regulation. The study must be helpful in developing a nutrient-management technology for optimization of crop productivity.     

Interactions Between Nitrogen and Manganese Nutrition of Barley Genotypes Differing in Manganese Efficiency

Ammonium-fed plants may acidify the rhizosphere and thus increaseavailability of Mn in calcareous alkaline soils. The importanceof N nutrition in the differential expression of tolerance toMn deficiency among cereal genotypes is not yet clear. Two factorialexperiments testing effects of the NH₄-N/NO₃-N ratio and Mnfertilization on growth of barley genotypes differing in toleranceto Mn deficiency were conducted in two calcareous alkaline soilsin pots in a controlled environment. In the soil containing80% CaCO₃at pH 8.5, better root and shoot growth and highershoot Mn concentrations were achieved with nitrate supply, especiallyat lower rates of Mn fertilization. The Mn-efficient genotypeWeeah (tolerant of Mn deficiency) achieved better root and shootgrowth than Mn-inefficient Galleon barley (sensitive to Mn deficiency)regardless of experimental treatment. Fertilization with Mndid not influence total N concentration in barley roots andshoots. In the soil containing 5% CaCO₃at pH 7.8, ammonium-fedplants had better root and shoot growth and, at shoot Mn concentrationsabove the critical level, Mn-inefficient Galleon performed betterthan Mn-efficient Weeah barley. It appears that differentialexpression of Mn efficiency among barley genotypes is not associatedwith differences in Mn availability expected to be producedby differential rhizosphere acidification as a response to differentforms of N supply. There is an apparent preference of locallyselected barley genotypes for nitrate nutrition when grown onthe highly calcareous alkaline soils of southern Australia. Ammonium; calcareous soil; Hordeum vulgare ; manganese; nitrate; nitrogen form; nutrient efficiency; rhizosphere     

Physiology and enzymology involved in denitrification by Shewanella putrefaciens

Nitrate reduction to N2O was investigated in batch cultures of Shewanella putrefaciens MR-1, MR-4, and MR-7. All three strains reduced nitrate to nitrite to N2O, and this reduction was coupled to growth, whereas ammonium accumulation was very low (0 to 1 micromol liter-1). All S. putrefaciens isolates were also capable of reducing nitrate aerobically; under anaerobic conditions, nitrite levels were three- to sixfold higher than those found under oxic conditions. Nitrate reductase activities (31 to 60 micromol of nitrite min-1 mg of protein-1) detected in intact cells of S. putrefaciens were equal to or higher than those seen in Escherichia coli LE 392. Km values for nitrate reduction ranged from 12 mM for MR-1 to 1.3 mM for MR-4 with benzyl viologen as an artifical electron donor. Nitrate and nitrite reductase activities in cell-free preparations were demonstrated in native gels by using reduced benzyl viologen. Detergent treatment of crude and membrane extracts suggested that the nitrate reductases of MR-1 and MR-4 are membrane bound. When the nitrate reductase in MR-1 was partially purified, three subunits (90, 70, and 55 kDa) were detected in denaturing gels. The nitrite reductase of MR-1 is also membrane bound and appeared as a 60-kDa band in sodium dodecyl sulfate-polyacrylamide gels after partial purification.     

Physiology and enzymology involved in denitrification by Shewanella putrefaciens.

Nitrate reduction to N2O was investigated in batch cultures of Shewanella putrefaciens MR-1, MR-4, and MR-7. All three strains reduced nitrate to nitrite to N2O, and this reduction was coupled to growth, whereas ammonium accumulation was very low (0 to 1 micromol liter-1). All S. putrefaciens isolates were also capable of reducing nitrate aerobically; under anaerobic conditions, nitrite levels were three- to sixfold higher than those found under oxic conditions. Nitrate reductase activities (31 to 60 micromol of nitrite min-1 mg of protein-1) detected in intact cells of S. putrefaciens were equal to or higher than those seen in Escherichia coli LE 392. Km values for nitrate reduction ranged from 12 mM for MR-1 to 1.3 mM for MR-4 with benzyl viologen as an artifical electron donor. Nitrate and nitrite reductase activities in cell-free preparations were demonstrated in native gels by using reduced benzyl viologen. Detergent treatment of crude and membrane extracts suggested that the nitrate reductases of MR-1 and MR-4 are membrane bound. When the nitrate reductase in MR-1 was partially purified, three subunits (90, 70, and 55 kDa) were detected in denaturing gels. The nitrite reductase of MR-1 is also membrane bound and appeared as a 60-kDa band in sodium dodecyl sulfate-polyacrylamide gels after partial purification.     

Respiratory nitrate ammonification by Shewanella oneidensis MR-1

Anaerobic cultures of Shewanella oneidensis MR-1 grown with nitrate as the sole electron acceptor exhibited sequential reduction of nitrate to nitrite and then to ammonium. Little dinitrogen and nitrous oxide were detected, and no growth occurred on nitrous oxide. A mutant with the napA gene encoding periplasmic nitrate reductase deleted could not respire or assimilate nitrate and did not express nitrate reductase activity, confirming that the NapA enzyme is the sole nitrate reductase. Hence, S. oneidensis MR-1 conducts respiratory nitrate ammonification, also termed dissimilatory nitrate reduction to ammonium, but not respiratory denitrification.     

Molecular biology, genetics and regulation of nitrite reduction in higher plants

Nitrite reductase (ferredoxin:nitrite oxidoreductase, EC 1.6.6.1) carries out the six-electron reduction of nitrite to ammonium ions in the chloroplasts/plastids of higher plants. The complete or partial nucleotide sequences of a number of nitrite reductase apoprotein genes or cDNAs have been determined. Deduced amino acid sequence comparisons have identified conserved regions, one of which probably is involved in binding the sirohaem/4Fe4S centre and another in binding the electron donor, reduced ferredoxin. The nitrite reductase apoprotein is encoded by the nuclear DNA and is synthesised as a precursor carrying an N-terminal extension, the transit peptide, which acts to target the protein to, and within, the chloroplast/plastid. In those plants examined the number of nitrite reductase apoprotein genes per haploid genome ranges from one (barley, spinach) to four ( Nicotiana tabacum ). Mutants defective in the nitrite reductase apoprotein gene have been isolated in barley. During plastidogenesis in etiolated plants, synthesis of nitrite reductase is regulated by nitrate, light (phytochrome), and an uncharacterised 'plastidic factor' produced by functional chloroplasts. In leaves of green, white-light-grown plants up-regulation of nitrite reductase synthesis is achieved via nitrate and light and down-regulation by a nitrogenous end-product of nitrate assimilation, perhaps glutamine. A role for phytochrome has not been demonstrated in green, light-grown plants. Light regulation of nitrite reductase genes is related more closely to that of photosynthetic genes than to the nitrate reductase gene. In roots of green, white-light-grown plants nitrate alone is able to bring about synthesis of nitrite reductase, suggesting that the root may possess a mechanism that compensates for the light requirement seen in the leaf.     

不同小麦品种氮效率与氮吸收对氮素供应的响应及生理机制

以具有典型特征的不同氮效率小麦品种为材料,研究了低氮和高氮条件下小麦的生物学性状、生理参数和氮同化代谢酶活性.结果表明:低氮条件下,不同氮效率小麦品种根系干质量、茎叶干质量、植株氮累积量基本上为氮高效品种>中效品种>低效品种.低氮条件下,氮吸收高效品种（冀97-6360）的根系活跃吸附面积、TTC还原力、叶片硝酸还原酶活性和叶片NO₃^-含量最大;生理高效品种(石新5418)具有较高的叶片亚硝酸还原酶活性和谷氨酰胺合成酶活性,较低的植株全氮含量、叶片NO₃^-含量和硝酸还原酶活性.低氮条件下植株氮利用效率与氮吸收系数显著相关.不同小麦品种在高氮条件下的生物学性状、生理参数和氮同化代谢酶活性与低氮条件下不尽一致.     

Nitrate Reductase in Barley Roots under Sterile, Low Oxygen Conditions

Levels of nitrate reductase activity (EC 1.9.6.1.) as high as 11 μmoles nitrite produced/hour gram fresh weight were found in barley (Hordeum vulgare cv. Compana) roots grown under low oxygen conditions. Roots of plants given identical treatment under sterile conditions did not develop the high levels of nitrate reductase activity. The results suggest that the buildup of particulate, reduced viologen-utilizing nitrate reductase reported in barley roots may be caused by bacterial contamination. The nitrate reductase activity in roots grown under low oxygen conditions was not specific for reduced nicotinamide adenine dinucleotide like the assimilatory nitrate reductase (EC 1.6.6.1.) normally found in aerated plant roots.     

Assimilatory nitrate uptake in Pseudomonas fluorescens studied using nitrogen-13

The mechanism of nitrate uptake for assimilation in procaryotes is not known. We used the radioactive isotope, ¹³N as NO₃ ^-, to study this process in a prevalent soil bacterium, Pseudomonas fluorescens. Cultures grown on ammonium sulfate or ammonium nitrate failed to take up labeled nitrate, indicating ammonium repressed synthesis of the assimilatory enzymes. Cultures grown on nitrite or under ammonium limitation had measurable nitrate reductase activity, indicating that the assimilatory enzymes need not be induced by nitrate. In cultures with an active nitrate reductase, the form of ¹³N internally was ammonium and amino acids; the amino acid labeling pattern indicated that ¹³NO₃ ^- was assimilated via glutamine synthetase and glutamate synthase. Cultures grown on tungstate to inactivate the reductase concentrated NO₃ ^- at least sixfold. Chlorate had no effect on nitrate transport or assimilation, nor on reduction in cell-free extracts. Ammonium inhibited nitrate uptake in cells with and without active nitrate reductases, but had no effect on cell-free nitrate reduction, indicating the site of inhibition was nitrate transport into the cytoplasm. Nitrate assimilation in cells grown on nitrate and nitrate uptake into cells grown with tungstate on nitrite both followed Michaelis-Menten kinetics with similar K _mvalues, 7 M. Both azide and cyanide inhibited nitrate assimilation. Our findings suggest that Pseudomonas fluorescens can take up nitrate via active transport and that nitrate assimilation is both inhibited and repressed by ammonium.     

Differential effect of tungsten on the development of endogenous and nitrate-induced nitrate reductase activities in soybean leaves

The effect of tungsten on the development of endogenous and nitrate-induced NADH- and FMNH₂-linked nitrate reductase activities in primary leaves of 10-day-old soybean (Glycine max [L.] Merr.) seedlings was studied. The seedlings were grown with or without exogenous nitrate. High levels of endogenous nitrate reductase activities developed in leaves of seedlings grown without nitrate. However, no endogenous nitrite reductase activity was detected in such seedlings. The FMNH₂-linked nitrate reductase activity was about 40% of NADH-linked activity. Tungsten had little or no effect on the development of endogenous NADH- and FMNH₂-linked nitrate reductase activities, respectively. By contrast, in nitrate-grown seedlings, tungsten only inhibited the nitrate-induced portion of NADH-linked nitrate reductase activity, whereas the FMNH₂-linked activity was inhibited completely. Tungsten had no effect on the development of nitrate-induced nitrite reductase activity. The complete inhibition of FMNH₂-linked nitrate reductase activity by tungsten in nitrate-grown plants was apparently an artifact caused by the reduction of nitrite by nitrite reductase in the assay system. The results suggest that in soybean leaves either the endogenous nitrate reductase does not require molybdenum or the molybdenum present in the seed is preferentially utilized by the enzyme complex as compared to nitrate-induced nitrate reductase.     

Effects of seed nickel reserves or externally supplied nickel on the growth,nitrogen metabolites and nitrogen use efficiency of urea- or nitrate-fed soybean

Background and aims

Nickel (Ni) has a critical role in the urea metabolism of plants. This study investigated the impact of seed Ni content along with external Ni supply on the growth, various nitrogen (N) metabolites and N use efficiency (NUE) of soybean plants.

Methods

Soybean plants raised from Ni-poor or Ni-rich seeds were grown in nutrient solution with or without external Ni supply and fed with either urea or nitrate as the sole N source. The changes in growth, leaf chlorophyll levels, Ni and N concentrations of different plant parts, tissue accumulation of various N metabolites and N uptake of soybean as well as NUE and its components were examined.

Results

Nickel starvation reduced the shoot biomass of urea-fed plants by 25 % and the leaf chlorophyll levels by up to 35 %, but nitrate-fed plants were unaffected. Visual toxicity symptoms were not observed in urea-fed plants. Under urea supply, Ni-deficient plants had lower levels of total N, protein and free amino acids in various organs. Root uptake of urea was severely depressed in Ni-deprived plants. Availability of Ni did not have any effect on the NUE of nitrate-fed plants, whereas its deficiency reduced the NUE of urea-fed plants by 30 %. The growth and N nutritional status of urea-fed soybean were significantly improved by high seed Ni reserves as well as external Ni supply.

Conclusion

Adequate Ni supply is required for maximizing the growth, root uptake of urea and NUE of urea-fed plants. Seed Ni reserves contribute significantly to the Ni and thus N nutritional status of soybean.     

Reduced nicotinamide–adenine dinucleotide–nitrite reductase from Azotobacter chroococcum

1. The assimilatory nitrite reductase of the N(2)-fixing bacterium Azotobacter chroococcum was prepared in a soluble form from cells grown aerobically with nitrate as the nitrogen source, and some of its properties have been studied. 2. The enzyme is a FAD-dependent metalloprotein (mol.wt. about 67000), which stoicheiometrically catalyses the direct reduction of nitrite to NH(3) with NADH as the electron donor. 3. NADH-nitrite reductase can exist in two either active or inactive interconvertible forms. Inactivation in vitro can be achieved by preincubation with NADH. Nitrite can specifically protect the enzyme against this inactivation and reverse the process once it has occurred. 4. A. chroococcum nitrite reductase is an adaptive enzyme whose formation depends on the presence of either nitrate or nitrite in the nutrient solution. 5. Tungstate inhibits growth of the microorganism very efficiently, by competition with molybdate, when nitrate is the nitrogen source, but does not interfere when nitrite or NH(3) is substituted for nitrate. The addition of tungstate to the culture media results in the loss of nitrate reductase activity but does not affect nitrite reductase.     

Expression of a bacterial asparagine synthetase gene in oilseed rape (Brassica napus) and its effect on traits related to nitrogen efficiency

The investigation and improvement of nitrogen efficiency in oilseed rape ( Brassica napus L.) are important issues in rapeseed breeding. The objective of this study was to modify ammonium assimilation in transgenic rapeseed plants through the expression of the Escherichia coli asparagine synthetase (AsnA, E.C. 6.3.1.1) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and to study its influence on amino acid composition in leaves and on seed traits related to nitrogen efficiency. In regenerated transgenic plants, the 37 kDa AsnA protein was detected by Western blot analysis, but was lacking in untransformed control plants of cv. Drakkar. In the transformants, in vitro asparagine synthetase activities ranged from 105 to 185 nmol asparagine mg⁻¹ protein h⁻¹, whereas, in untransformed control plants, only negligible asparagine synthetase activities of up to 5 nmol asparagine mg⁻¹ protein h⁻¹ were found. Despite these significant activities, no changes in the amino acid composition in the leaves or in the phloem of transgenic plants were detectable. In a pot experiment, two transgenic lines expressing the prokaryotic asparagine synthetase clearly performed inferiorly to control plants at limiting nitrogen (N) fertilizer supply. Although the seed N content was increased, the seed yield and the seed N yield were reduced, which was interpreted as an increased nitrate assimilation leading, at limiting N supply, to a reduced seed yield and seed N yield. At high N fertilizer supply, the differences were less pronounced for one transgenic line, whereas the other showed a higher seed N yield and an improved nitrogen harvest index. The results show that the expression of the E. coli asnA gene in oilseed rape could be of advantage at high N supply, but not at limiting N fertilizer supply.