Constitutive Nitrate Reductase Research Articles

Sporobolomyces salmonicolor AS A TOOL FOR NITRATE REMOVAL FROM WASTEWATERS

The problem of nitrate removal from agricultural and industrial wastewaters is increasingly drawing attention since contamination more and more jeopardizes drinking water resources. The yeast Sporobolomyces salmonicolor produces a constitutive nitrate reductase and can use nitrate as the sole nitrogen source. The obtained nitrite is in turn reduced and assimilated by the yeast and converted into biomass. In the present study some biological features of a S. salmonicolor strain, relevant in relation with the problem of nitrate removal, such as temperature and pH requirements, preference towards particular carbon sources, and also availability of alternative nitrogen sources, have been studied in the light of their influence towards nitrate assimilation. Both efficiency and kinetics of nitrate assimilation were also explored. Work on real wastewater samples allowed for assessing the potential of the micro-organism for bioremediation: in particular, nitrogen-rich wastewaters were shown to be substantially depleted in their total nitrogen content, whereas noticeable amounts of protein-rich biomass were formed.

Differences in nitrogen metabolism of Faidherbia albida and other N2 -fixing tropical woody acacias reflect habitat water availability.

The activities of nitrate reductase and glutamine synthetase were evaluated in young plants of Faidherbia albida, a tropical woody legume, fed with different N sources under hydroponic conditions. Results showed that assimilation of both NO3 - and NH4 + preferentially took place in shoots. A basal amount of nitrate reductase activity was detected in shoots of plants grown with an NO3 - -free solution or placed under N2 -fixing conditions, and also in nodules of N2 -fixing plants. This strongly suggests that constitutive nitrate reductase activity is present in these organs. Analyses of the soluble nitrogenous content showed that the major form of N in the different organs was α-amino acids (particularly amides), irrespective of the N status of the culture conditions. The same result was obtained for nodulated plants grown in local sandy soil. In this case, amide-N generally accounted for more than 40% of the total soluble N. This was especially true in nodules. Ureide-N never exceeded 9% of the total soluble N and did not appear to increase with increasing nodule nitrogenase activity. Amides were also predominant in three N2 -fixing Sahelian acacias (Acacia seyal, A. nilotica and A. tortilis), showing that F. albida does not differ from Sahelian Acacia in terms of the metabolism of fixed N. However, like another Sahelian acacia growing preferentially near water (A. nilotica), F. albida can be distinguished from acacias growing strictly in arid zones (A. seyal and A. tortilis) in terms of initial growth, water and nitrate management.

Overexpression of nitrate reductase in tobacco delays drought-induced decreases in nitrate reductase activity and mRNA

Transformed (cauliflower mosaic virus 35S promoter [35S]) tobacco (Nicotiana plumbaginifolia L.) plants constitutively expressing nitrate reductase (NR) and untransformed controls were subjected to drought for 5 d. Drought-induced changes in biomass accumulation and photosynthesis were comparable in both lines of plants. After 4 d of water deprivation, a large increase in the ratio of shoot dry weight to fresh weight was observed, together with a decrease in the rate of photosynthetic CO2 assimilation. Foliar sucrose increased in both lines during water stress, but hexoses increased only in leaves from untransformed controls. Foliar NO3- decreased rapidly in both lines and was halved within 2 d of the onset of water deprivation. Total foliar amino acids decreased in leaves of both lines following water deprivation. After 4 d of water deprivation no NR activity could be detected in leaves of untransformed plants, whereas about 50% of the original activity remained in the leaves of the 35S-NR transformants. NR mRNA was much more stable than NR activity. NR mRNA abundance increased in the leaves of the 35S-NR plants and remained constant in controls for the first 3 d of drought. On the 4th d, however, NR mRNA suddenly decreased in both lines. Rehydration at d 3 caused rapid recovery (within 24 h) of 35S-NR transcripts, but no recovery was observed in the controls. The phosphorylation state of the protein was unchanged by long-term drought. There was a strong correlation between maximal extractable NR activity and ambient photosynthesis in both lines. We conclude that drought first causes increased NR protein turnover and then accelerates NR mRNA turnover. Constitutive NR expression temporarily delayed drought-induced losses in NR activity. 35S-NR expression may therefore allow more rapid recovery of N assimilation following short-term water deficit.

Modulation of carbon and nitrogen metabolism, and of nitrate reductase, in untransformed and transformed Nicotiana plumbaginifolia during CO 2 enrichment of plants grown in pots and in hydroponic culture

Transformed plants of Nicotiana plumbaginifolia Viv. constitutively expressing nitrate reductase (35S-NR) or β-glucuronidase (35S-GUS) and untransformed controls were grown for two weeks in a CO2-enriched atmosphere. Whereas CO2 enrichment (1000 μl · l−1) resulted in an increase in the carbon (C) to nitrogen (N) ratio of both the tobacco lines grown in pots with vermiculite, the C/N ratio was only slightly modified when plants were grown in hydroponic culture in high CO2 compared to those grown in air. Constitutive nitrate reductase (NR) expression per se did not change the C/N ratio of the shoots or roots. Biomass accumulation was similar in both types of plant when hydroponic or pot-grown material, grown in air or high CO2, were compared. Shoot dry matter accumulation was primarily related to the presence of stored carbohydrate (starch and sucrose) in the leaves. In the pot-grown tobacco, growth at elevated CO2 levels caused a concomitant decrease in the N content of the leaves involving losses in NO− 3 and amino acid levels. In contrast, the N content and composition were similar in all plants grown in hydroponic culture. The 35S-NR plants grown in air had higher foliar maximum extractable NR activities and increased glutamine levels (on a chlorophyll or protein basis) than the untransformed controls. These increases were maintained following CO2 enrichment when the plants were grown in hydroponic culture, suggesting that an increased flux through nitrogen assimilation was possible in the 35S-NR plants. Under CO2 enrichment the NR activation state in the leaves was similar in all plants. When the 35S-NR plants were grown in pots, however, foliar NR activity and glutamine content fell in the 35S-NR transformants to levels similar to those of the untransformed controls. The differences in NR activity between untransformed and 35S-NR leaves were much less pronounced in the hydroponic than in the pot-grown material but the difference in total extractable NR activity was more marked following CO2 enrichment. Foliar NR message levels were decreased by CO2 enrichment in all growth conditions but this was much more pronounced in pot-grown material than in that grown hydroponically. Since β-glucuronidase (GUS) activity and message levels in 35S-GUS plants grown under the same conditions of CO2 enrichment (to test the effects of CO2 enrichment on the activity of the 35S promoter) were found to be constant, we conclude that NR message turnover was specifically accelerated in the 35S-NR plants as well as in the untransformed controls as a result of CO2 enrichment. The molecular and metabolic signals involved in increased NR message and protein turnover are not known but possible effectors include NO3 −, glutamine and asparagine. We conclude that plants grown in hydroponic culture have greater access to N than those grown in pots. Regardless of the culture method, CO2 enrichment has a direct effect on NR mRNA stability.

Constitutive expression of nitrate reductase changes the regulation of nitrate and nitrite transporters in Chlamydomonas reinhardtii

SummaryThe effect of constitutive expression of nitrate reductase (NR) on the regulation pattern of nitrite and nitrate transporters (NT) has been analysed in Chlamydomonas reinhardtii. Strain Tx11‐8, bearing the chimeric Nia1 gene for NR under the control of the cabll‐1 gene promoter, showed a constitutive NR which was inactive in ammonium‐grown cells. In contrast to the wild‐type, incubation of Tx11‐8 in ammonium media containing nitrate at micromolar concentrations resulted in reactivation of inactive NR and excretion of nitrite. In addition, negligible amounts of NT gene transcripts were induced by nitrate in this strain. However, NT transcripts from Tx11‐8 cells were induced in amounts similar to those in the wild‐type, by preventing constitutive NR expression from the cabll‐1 gene promoter in the dark. Strains lacking high‐affinity NT and nitrite reductase activity and having the chimeric Nia1 gene have been constructed by genetic crosses. All these strains were sensitive to chlorate in ammonium‐containing media and excreted nitrite when nitrate was present at millimolar concentrations. Spontaneous chlorate‐resistant mutants isolated from these strains lacked constitutive NR activity though they constitutively expressed NR mRNA. The data strongly suggest that nitrate plays an important post‐transcriptional regulatory role and that NR is involved in the regulation of the nitrate/ nitrite transport activities in C. reinhardtii.

Denitrification in lucerne nodules is not involved in nitrite detoxification

Dissimilatory reduction of ionic nitrogen oxides to gaseous forms such as nitrous oxide or nitrogen can be carried out by free living or symbiotic forms of some strains of Rhizobium meliloti. In this paper we investigate whether bacteroid denitrification plays a role in the alleviation of the inhibitory effects of nitrate on nitrogen fixation both in bacteroid incubations as in whole nodules. The presence of a constitutive nitrate reductase (NR) activity in isolated bacteroids caused nitrite accumulation in the incubation medium, and acetylene reduction activity in these bacteroids was progressively inhibited, since nitrite reductase (NiR) activity was unable to reduce all the nitrite produced by NR and denitrification occurred slowly. Even nodules infiltrated with nitrate and nitrite failed to increase gaseous forms of nitrogen substantially, indicating that nitrite availability was not limiting denitrification by bacteroids. In spite of the low rates of bacteroidal denitrification, the effect of nodule denitrification on the inhibition of nitrogen fixation by nitrate in whole plants was tested. For that purpose, lucerne plants (Medicago sativa L. cv. Aragon) were inoculated with two Rhizobium meliloti strains: 102-F-65 (non denitrifying) and 102-F-51 (a highly denitrifying strain). After a seven days nitrate treatment, both strains showed the same pattern of inhibition, and it occurred before any nitrate or nitrite accumulation within the nodules could be detected. This observation, together with the lack of alleviation of the ARA inhibition in the denitrifying strain, and the limited activity of dissimilatory nitrogen reduction present in these bacteroids, indicate a role other than nitrite detoxification for denitrification in nodules under natural conditions.

Study of the Denitrifying Enzymatic System of Comamonas sp. Strain SGLY2 Under Various Aeration Conditions with a Particular View on Nitrate and Nitrite Reductases

This paper studies the effect of oxygen on the denitrifying enzymatic system of Comamonas sp. It is shown that nitrate respiration can take place in the presence of oxygen. Indeed, even if a protein synthesis inhibitor is added in the medium, immediate nitrate consumption is observed in an aerobic culture inoculated with cells that have never been subjected to nitrate. Existence of a constitutive nitrate reductase could explain this phenomenon. Moreover the nitrate and nitrite reductases are active and synthesized under aerobic conditions. The different levels of inhibition of nitrate reductase activity by respiratory inhibitors and detergent, according to the aerobic and anaerobic cultures, might suggest the existence of a double nitrate reductase enzymatic system.

Regulation of the inducible soybean nitrate reductase isoform in mutants lacking constitutive isoform(s)

In soybean, three nitrate reductase isoforms have been identified based on metabolic regulation, substrate specificity, and kinetic parameters. Two isoforms have been termed constitutive, as their activities are present without the addition exogenous nitrate to soybean seedlings. The third activity is termed inducible, as its activity is present only when soybean plants have been supplied with nitrate. The purpose of this study was to examine the regulation of the inducible nitrate reductase isoform in soybean mutants lacking one or both of the constitutive isoforms. Based on evidence obtained through measurements of enzyme activity, Western blotting, and RNA determinations, the absence of one or both of the constitutive nitrate reductase isoforms has no effect on the metabolite regulation of the inducible nitrate reductase isoform.

Nitrate reductase and glutamine synthetase activities in relation to growth and nitrogen assimilation in red oak and red ash seedlings: effects of N-forms, N concentration and light intensity

The effects of growing seedlings of red oak (Quercus rubra) and red ash (Fraxinus pennsylvanica) with Hoagland solutions containing five N-regimes, differing in the N-forms (NH4, NO3) and concentrations (High and Low), in relation to light intensity were investigated by the utilization of enzymatic markers of the N assimilation pathway, nitrate reductase (NR) and glutamine synthetase (GS). Red oak and red ash showed different patterns of N-assimilation. Red oak seedlings assimilated NO3 in low amounts in their roots and leaves, whereas red ash seedlings assimilated high amounts of NO3, mostly in the leaves. A significant amount of constitutive NR activity was found in red oak seedlings supplied with NH4 N-regime. This could be characteristic of a species adapted to soils that are poor in nitrogen. Root GS activity was lower in red oak seedlings than in red ash seedlings, indicating that the rate of NH4 assimilation differed in these two hardwood species. Low irradiance reduced growth of both hardwood species, but greatly affected the specific leaf area of red ash and reduced NO3 assimilation (when data are expressed per leaf area). Both species reacted similarly to N-regimes in terms of relative growth rate.

Isolation and characterization of transposon Tn5 mutants ofRhodobacter sphaeroidesdeficient in both nitrate and chlorate reduction.

The wild-type strain Rhodobacter sphaeroides DSM 158 is a nitrate-reducing bacterium with a periplasmic nitrate reductase. Addition of chlorate to the culture medium causes a stimulation of the phototrophic growth, indicating that this strain is able to use chlorate as an ancillary oxidant. Several mutant strains of R. sphaeroides deficient in nitrate reductase activity were obtained by transposon Tn5 mutagenesis. Mutant strain NR45 exhibited high constitutive nitrate and chlorate reductase activities and phototrophic growth was also increased by the presence of chlorate. In contrast, the stimulation of growth by chlorate was not observed in mutant strains NR8 and NR13, in which transposon Tn5 insertion causes the simultaneous loss of both nitrate and chlorate reductase activities. Tn5 insertion probably does not affect molybdenum metabolism since NR8 and NR13 mutants exhibit both xanthine dehydrogenase and nitrogenase activities. These results that a single enzyme could reduce both nitrate and chlorate in R. sphaeroides DSM 158.

Denitrifying ability of thirteen Rhizobium meliloti strains

The denitrifying ability of thirteen strains of Rhizobium meliloti was tested. Most of the strains were able to reduce nitrate to nitrous oxide or dinitrogen. However, they failed to use nitrate as electron acceptor for ATP generation or growth at low oxygen tensions. Under micro-aerobic conditions, free-living cells of R. meliloti 102-F-51 strain exhibited a constitutive nitrate reductase activity independent of the presence of nitrate. On the other hand, nitrite reductase activity was dependent not only on low levels of oxygen but also on the presence of a high nitrate concentration in the medium. Denitrification activity proceeded immediately once a threshold level of nitrite was accumulated in the medium or in cells incubated with 1mM nitrite. However, a lag period was required when cells were incubated with nitrate.

Nitrate reductase and nitrite reductase activity in free-living cells and bacteroids of Rhizobium loti

Cells of Rhizobium loti strains T1 and U226 cultured in defined medium with glutamate as the only nitrogen source and bacteroids isolated from root nodules of Lotus corniculatus, L. pedunculatus and L. tenuis did not show constitutive (non-nitrate induced) nitrate reductase activity (NRA). In contrast, nitrite reductase activity (NiRA) was present in both free-living cells and bacteroids of either strain T1 or U226. Constitutive NRA and NiRA were detected in the cytosol fraction from nodules of all three symbioses examined. An induced NRA was expressed in bacteroids after a 10 h incubation in the presence of nitrate.

Studies on the root control of non-nodulation and plant growth of non-nodulating mutants and a supernodulating mutant of soybean ( Glycine max (L.) Merr.)

Ethyl methanesulphonate mutagensis of soybean cv. Bragg led to the isolation of three non-nodulating mutants: nod49, nod772 and nod139, as well as the supernodulation mutant nts382. Mutants nod49 and nod772 are allelic to naturally-occuring mutation rj 1 while nod139 is non-allelic and represents a newly found gene conditioning non-nodulation in soybean. Using the standard reciprocal wedge grafting technique it was shown earlier that non-nodulation in mutant nod49 is root controlled and supernodulation in mutant nts382 is shoot controlled. Non-nodulation in mutants nod772 and nod139 (the newly found non-nodulation gene in soybean) is also root controlled. In addition, scions of nts382 failed to alter the nodulation phenotype when grafted onto stocks of the non-nodulating lines. Lateral roots developing from the scion of nts382 and Bragg exhibited supernodulation and the wild-type pattern of nodulation, respectively, indicating that the lesion(s) conditioning non-nodulation only affect the root in a localized manner. Physiological studies conducted on the mutants indicate that they are not altered in the assimilation of nitrogen and carbon when grown on nitrate, and a study of their nitrogen contents indicate that the anomaly in the non-nodulation mutants is clearly specific for the nodulation process. This was confirmed by assaying nitrate reductase (NR) under a range of conditions; it was observed that the non-nodulation mutants expressed inducible NR and constitutive NR activity in a manner similar to the wild type.

Constitutive nitrate reductase: a dominant conditional marker for plant genetics

SummaryWe describe the development of a counter‐selection system based on the use of an engineered nitrate reductase (NR) gene. The engineered gene consists of an NR cDNA placed under the control of the CaMV 35S promoter (35S‐NR). Seedlings and cells derived from transgenic Nicotiana plumbaginifolia plants transformed with 35S‐NR are efficiently killed by the selective agent chlorate on medium containing ammonium as the sole nitrogen source. Under these nitrate‐free conditions wild‐type plants are not affected by chlorate because the endogenous wild‐type Niagene is not expressed.

Constitutive nitrate reductase: a dominant conditional marker for plant genetics

We describe the development of a counter-selection system based on the use of an engineered nitrate reductase (NR) gene. The engineered gene consists of an NR cDNA placed under the control of the CaMV 35S promoter (35S-NR). Seedlings and cells derived from transgenic Nicotiana plumbaginifolia plants transformed with 35S-NR are efficiently killed by the selective agent chlorate on medium containing ammonium as the sole nitrogen source. Under these nitrate-free conditions wild-type plants are not affected by chlorate because the endogenous wild-type Niagene is not expressed.

Nitrate reduction and its effect on nitrogen fixation in sliced nodules of pigeon pea

Bacteroidal and cytosolic fractions of nodules of pigeon pea plants (inoculated with Rhizobium PP-11) contained a constitutive nitrate reductase which functioned with NADH, succinate or MVH as the reluctant. In bacteroids, the enzyme was associated with a particulate fraction. In contrast, MVH-nitrite reductase was located exclusively in the cytosol. In vivo nitrate reduction by sliced nodules was inhibited by dicoumarol but not by rotenone. Acetylene reduction was, however, sensitive to both these inhibitors. Acetylene reduction in nodule slices was rapidly supressed by nitrate and nitrite but not by ammonium ions. At varying concentrations of acetylene, acetylene reduction was inhibited non-competitively by nitrate. In most of the experiments, decrease in acetylene reduction was closely associated with the production of nitrite. The possible mechanism for the deleterious effect of nitrate on biological nitrogen fixation is discussed.

Constitutive nitrate reductase activity in the leguminosae

The occurrence of constitutive nitrate reductase activity (NRA) was examined in 101 species from 25 tribes in the Leguminosae. Constitutive NRA was undetectable in all species from the sub-families Caesalpiniodeae and Mimosoideae. Within the Papilionoideae it occurred in root and shoot of Lotus uliginosus (Loteae) and leaves of all members of the tribe Phaseoleae but was absent in all other plants tested. For L. uliginosus, root and shoot activity was similar in light and darkness, but for Glycine max and Phaseolus vulgaris (both Phaseoleae) leaf activity was greater in light.

Utilization of nitrate by bacteroids of Bradyrhizobium japonicum in the soybean root nodule

Bacteroids of Bradyrhizobium japonicum strain CB1809, unlike CC705, do not have a high level of constitutive nitrate reductase (NR; EC 1.7.99.4) in the soybean (Glycine max. Merr.) nodule. Ex planta both strains have a high activity of NR when cultured on 5 mM nitrate at 2% O2 (v/v). Nitrite reductase (NiR) was active in cultured cells of bradyrhizobia, but activity with succinate as electron donor was not detected in freshly-isolated bacteroids. A low activity was measured with reduced methyl viologen. When bacteroids of CC705 were incubated with nitrate there was a rapid production of nitrite which resulted in repression of NR. Subsequently when NiR was induced, nitrite was utilized and NR activity recovered. Nitrate reductase was induced in bacteroids of strain CB1809 when they were incubated in-vitro with nitrate or nitrite. Increase in NR activity was prevented by rifampicin (10 μg· ml(-1)) or chloramphenicol (50 μg·ml(-1)). Nitrite-reductase activity in bacteroids of strain CB1809 was induced in parallel with NR. When nitrate was supplied to soybeans nodulated with strain CC705, nitrite was detected in nodule extracts prepared in aqueous media and it accumulated during storage (1°C) and on further incubation at 25°C. Nitrite was not detected in nodule extracts prepared in ethanol. Thus nitrite accumulation in nodule tissue appears to occur only after maceration and although bacteroids of some strains of B. japonicum have a high level of a constitutive NR, they do not appear to reduce nitrate in the nodule because this anion does not gain access to the bacteroid zone. Soybeans nodulated with strains CC705 and CB1809 were equally sensitive to nitrate inhibition of N2 fixation.

Rhizobia in tropical legumes: Environmental factors and the reduction of nitrogen

Existing methods for measuring environmental effects on nitrogen assimilation were applied to tropical legumes. Gas-exchange data showed that optimal conditions for photosynthesis of Centrosema pubsecens and Vigna unguiculata were: a temperature of 30°C; a relative humidity of 60–80%; and a light intensity between 1000 and 1200 μE m −2 s −1. Experiments in nutrient solution demonstrated that the optimal temperature for root growth was also 30°C, while watering plants with 5–10mmol nitrate (either as KNO 3 or NH 4NO 3) promoted vegetative growth of the legumes and inhibited nodulation. Under optimal environmental conditions, the C 2H 2-reduction method gave average ratios of mol C 2H 2 reduced to mol nitrogen fixed of 3.01:1. Constitutive nitrate reductase activity was found in all nodules examined and every Bradyrhizobium species studied reduced C 2H 2 to C 2H 4 under free-living conditions. Negative correlations existed between bacteroid nitrate reductase activities and C 2H 2-reduction. Nitrate reductase of Rhizobium sp. NGR234 was partially purified and shown to be stable—perhaps explaining its high activity in old nodules. No correlation between nitrogenase and nitrate reductase activities in free-living rhizobia could be found.

Metabolite Control of Nitrate Reductase Activity in Cultured Soybean Cells

Summary Nitrogen nutritional conditions were investigated as regulators of nitrate reductase (E.C. 1.6.6.1) activity in cell cultures initiated from soybean leaves ( Glycine max var. Williams 82). Nitrate in the presence of reduced nitrogen was required for maximal cell growth and nitrate reductase activity. Cells grown with nitrate as the sole nitrogen source died after two weeks in culture, while the addition of 2 mM ammonium to the media resulted in sustained growth. When cells grown with glutamine as the sole nitrogen source were transferred to media containing nitrate, a dramatic increase in nitrate reductase activity was observed. Conversely, when cells grown with nitrate in the medium were transferred to media containing glutamine, a dramatic decay in nitrate reductase activity was observed. Finally, unlike normal leaf tissue, soybean cells in culture failed to manifest the constitutive NADPH-linked nitrate reductase activity.