Rapid and slow modulation of nitrate reductase activity in the red macroalga <Emphasis Type="Italic">Gracilaria chilensis</Emphasis> (Gracilariales,Rhodophyta): influence of different nitrogen sources

Nitrate is one of the most important stimuli in nitrate reductase (NR) induction, while ammonium is usually an inhibitor. We evaluated the influence of nitrate, ammonium or urea as nitrogen sources on NR activity of the agarophyte Gracilaria chilensis. The addition of nitrate rapidly (2 min) induced NR activity, suggesting a fast post-translational regulation. In contrast, nitrate addition to starved algae stimulated rapid nitrate uptake without a concomitant induction of NR activity. These results show that in the absence of nitrate, NR activity is negatively affected, while the nitrate uptake system is active and ready to operate as soon as nitrate is available in the external medium, indicating that nitrate uptake and assimilation are differentially regulated. The addition of ammonium or urea as nitrogen sources stimulated NR activity after 24 h, different from that observed for other algae. However, a decrease in NR activity was observed after the third day under ammonium or urea. During the dark phase, G. chilensis NR activity was low when compared to the light phase. A light pulse of 15 min during the dark phase induced NR activity 1.5-fold suggesting also fast post-translational regulation. Nitrate reductase regulation by phosphorylation and dephosphorylation, and by protein synthesis and degradation, were evaluated using inhibitors. The results obtained for G. chilensis show a post-translational regulation as a rapid response mechanism by phosphorylation and dephosphorylation, and a slower mechanism by regulation of RNA synthesis coupled to de novo NR protein synthesis.     

Substrates regulate the phosphorylation status of nitrate reductase

The effect of substrates on the phosphorylation status of nitrate reductase (NR; EC 1.6.6.1) was studied. The enzyme was obtained from the first leaf of 7-day-old oat (Avena sativa L. cv. Suregrain) plants, grown in the light. When desalted crude extracts were incubated with ATP, NR was strongly phosphorylated, as evidenced by the inhibition of the enzyme's activity in the presence of Mg²⁺. NR sensitivity to Mg²⁺ remained unchanged when 10 mM nitrate was added to crude extracts after ATP. Addition of nitrate before or simultaneously with ATP slightly decreased Mg²⁺ inhibition of NR, which was strongly diminished in the presence of 10 mM NO₃^?+ 100 µM NADH. Incubation with NADH alone did not affect the enzyme's susceptibility to Mg²⁺ inhibition. When ammonium sulfate was added to crude extracts, NR was recovered in a 0-40% saturation fraction (F1). After incubation of F1 with ATP, the sensitivity of the enzyme to Mg²⁺ inhibition remained low, but it strongly increased after mixing F1 with a 45-60% saturation fraction (F2) suggesting that also in oats an additional factor (inactivating protein, IP), which probably binds to phospho-NR, would be required to keep the phosphorylated enzyme inactive in a +Mg²⁺ medium. Addition of 10 mM NO₃^?+ 100 µM NADH together with desalted F2 did not prevent Mg²⁺ inhibition suggesting that NO₃^? did not interfere with IP binding to phospho-NR. Again, incubation of F1 with both substrates during in vitro phosphorylation kept the enzyme active after adding F2, even in the presence of Mg²⁺, After in vitro phosphorylation, NR in crude extract was hardly reactivated when incubated alone or in the presence of 10 mM NO₃^? at 30°C. On the other hand, a strong and very rapid reactivation was found when the extract was incubated with both nitrate and NADH. Microcystine, an inhibitor of types 1 and 2A phosphoprotein phosphatases, inhibited the reactivation of phospho-NR induced by the substrates. The results presented here show that the substrates could prevent NR phosphorylation and induce the enzyme's dephosphorylation, but they were effective only after their binding to the NR protein. Thereby, they seemed to affect the NR protein itself and not the phosphatase- or the kinase-proteins. It has been reported that nitrate binding to the enzyme's active site induces conformational changes in the NR protein. We propose that this conformational change would prevent NR phosphorylation, by converting the enzyme into a form in which the site recognized by the protein kinase is no longer accessible, and, simultaneously, stimulate NR dephophorylation by allowing the specific phosphatases to recognize NR.     

Latent nitrate reductase activity is associated with the plasma membrane of corn roots

Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U₂). Analysis with marker enzymes indicated that the U₂ fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.Abbreviations L₁ first lower phase - NR nitrate reductase - NRA nitrate-reductase activity - PM plasma membrane - T:p Triton X-100 (octylphenoxy polyethoxyethanol) to protein ratio - U₂ second upper phase     

The activity and stability of wheat nitrate reductase in vitro

The activity and decay characteristics of nitrate reductase from wheat (Triticum aestivum) were studied in crude, partially-purified and highly-purified preparations. The decay of nitrate reductase activity in crude extracts was due to spontaneous dissociation of the enzyme and to the effects of two decay factors, one present in the 0–30% and the other in the 50–70% saturated (NH₄)₂SO₄ fraction of a crude extract. Low rates of factor-mediated NR decay in vitro were associated with high levels of NR activity in vivo.     

Partial purification of two proteins (100 kDa and 67 kDa) cooperating in the ATP-dependent inactivation of spinach leaf nitrate reductase

Using a three-step purification procedure, two protein fractions which catalyzed the ATP-dependent in-activation of nitrate reductase (NR) were obtained from spinach (Spinacia oleracea L.) leaf extracts. Purification involved ammonium-sulfate fractionation, anion-exchange chromatography and size-exclusion chromatography. The capacity of the fractions to inactivate NR by preincubation with ATP was examined by using as target either a crude NR-ammonium sulfate precipitate or partially purified NR (ppNR). The fractions were also examined for protein-kinase activity by measuring the phosphorylation of histone III S (or casein) with-[³²P]ATP as substrate, and subsequent SDS-PAGE, autoradiography and liquid scintillation counting of cut-off histone bands. The two proteins had apparent molecular weights in the 67-kDa and 100-kDa region (termed P₆₇ and P₁₀₀, respectively). Neither P₆₇ nor P₁₀₀ alone was able to inactivate ppNR by preincubation with ATP. However, when P₁₀₀ and P₆₇ were added together to ppNR, ATP-dependent inactivation was observed, with a half-time of about 10 min. The P₆₇, but not P₁₀₀ had histone-kinase activity (casein was not phosphorylated). Using the partially purified system, various compounds were examined as possible effectors of NR inactivation. Sugar phosphates had little effect on the inactivation of NR. Addition of AMP at very high concentrations (5 mM), and removal of Mg²⁺ by excess EDTA also prevented the inactivation.Abbreviations AS ammonium sulfate - DTT dithiothreitol - NR NADH-nitrate reductase - NRA nitrate reductase activity - ppNR partially purified nitrate reductase     

Nitrate reductase activity in spheroplasts from Rhodobacter capsulatus E1F1 requires a periplasmic protein

Spheroplasts from Rhodobacter capsulatus E1F1 cells grown in nitrate maintained nitrate uptake and nitrate reductase activity only when they were illuminated under anaerobiosis in the presence of the periplasmic fraction and nitrate. The effects on nitrate uptake and nitrate reductase activity of spheroplasts were observed at low concentrations of periplasmic protein (about 50 x ml^-1). Periplasm from nitrate-grown cells was also required for nitrate reductase activity in spheroplasts isolated from ammonia-grown or diazotrophic cells which initially lacked this enzymatic activity. Both the maintenance of nitrate reductase in spheroplasts from nitrate-grown cells and the appearance of the activity in spheroplasts from diazotrophic cells were dependent on de novo protein synthesis. A periplasmic, 45-kDa protein which maintained the activity of nitrate reductase in spheroplasts was partially purified by gel filtration chromatography of periplasm obtained from nitrate-grown cells.Abbreviations NR nitrate reductase - CCCP carbonyl cyanide m-chlorophenylhydrazone - CAM chloramphenicol     

Modifications of the activity of nitrate reductase from cucumber roots

The regulatory properties of NADH-dependent nitrate reductase (NR) in desalted root extracts from hydroponically grown cucumber (Cucumis sativus L.) seedlings were examined. The lowest activity of NR was detected in extracts incubated with Mg²⁺ and ATP. An inhibitory effect of Mg-ATP was cancelled in the presence of staurosporine (the protein kinase inhibitor) and completely reversed after addition of ethylenediaminetetraacetate (EDTA) as well as AMP into reaction mixture. Reactivation of enzyme due to AMP presence, contrary to the chelator-dependent NR activation, was sensitive to microcystin LR (the protein phosphatase inhibitor). Above results indicated that the nitrate reductase in cucumber roots was regulated through reversible phosphorylation of enzyme protein. A drop in the activity of NR was also observed after incubation of enzyme at low pH. At low pH, the presence of ATP alone in the incubation medium was sufficient to inactivate NR, indicating that H⁺ can substitute the Mg²⁺ in formation of an inactive complex of enzyme. ATP-dependent inactivation of NR at low pH was prevented by staurosporine and reversed by AMP. However, AMP action was not altered by microcystin LR suggesting that in low pH the nucleotide induced reactivation of NR is not limited to the protein phosphorylation.     

Monoclonal antibodies to a higher-plant nitrate reductase: Differential inhibition of enzyme activities

A set of monoclonal antibodies has been raised against NADH-nitrate reductase (NR; EC 1.6.6.1) from spinach (Spinacea oleracea L.) leaves. Antibodies were screened by enzyme-linked immunosorbent assay and by their ability to inhibit various activities of the enzyme. The six monoclonals selected (AFRC MAC 74 to 79) are all gamma globulins; four (MAC 74 to 77) inhibit all terminal donating activities (NADH-NR; flavin mononucleotide, reduced form (FMNH₂)-NR; and methyl viologen, reduced form (MV)-NR) and two (MAC 78 and 79) inhibit the acceptor activities (NADH-NR, and NADH-cytochrome c reductase). MAC 74 to 77 inhibit the NADH-NR activity of crude extracts of a variety of species (mono- and dicotyledoneae) while MAC 78 and 79 are effective against spinach and marrow, but not oil-seed rape, cucumber, oats, wheat and barley.Abbreviations Cyt c Rase cytochrome c reductase - ELISA enzyme-linked immunosorbent assay - FAD(H₂) flavin adenine dinucleotide (reduced form) - FMN(H₂) flavin mononucleotide (reduced form) - McAb monoclonal antibody - MV methyl viologen reduced form - NR nitrate reductase     

Two differentially regulated nitrate reductases required for nitrate-dependent,microaerobic growth of Bradyrhizobium japonicum

Native PAGE of Triton x-100-solubilized membranes from Bradyrhizobium japonicum strain PJ17 grown microaerobically (2% O₂, v/v) in defined nitrate-containing medium resolved two catalytically active nitrate reductase (NR) species with apparent molecular masses of 160 kDa (NR_I) and 200 kDa (NR_II). NR_I and NR_II were also found in membranes from cells of strain PJ17 that were first grown in defined medium with glutamate and further incubated microaerobically in the presence of 5 mmol/l KNO₃. However, only NR_I was detected in cell membranes of strain PJ17 when nitrate was omitted from the microaerobic incubation medium. Four mutants unable to grow at low O₂ tension in the presence of nitrate were isolated after transposon Tn5 mutagenesis. Membranes from mutants GRF110 and GRF116 showed mainly NR_I, while the other two mutants, GRF3 and GRF4, expressed mostly NR_II. These results indicate that the ability of B. japonicum PJ17 to grow under microaerobic conditions depends upon the presence of two membrane-bound NR enzymes whose synthesis seem to be independently induced by microaerobiosis (NR_I) or by both microaerobiosis and nitrate (NR_II).Abbreviations NR Nitrate reductase - M _r Relative molecular mass - PMSF Phenylmethylsulfonyl fluoride     

Properties of nitrate reductase from seedling leaves of selected barley genotypes

Crude extracts from leaves of 6-day barley seedlings of parental genotypes (cv. Aramir and primitive line R567) and selected doubled haploid (DH) lines were not found to have significant differences in the NADH:NR activity, while considerable differences between these genotypes were shown by the NAD(P)H:NR activity. The cv. Aramir and DH lines did not differ by nitrate accumulation in the leaves. However, the primitive line R567, as compared to the remaining genotypes, was characterized by an appreciably lower ability to accumulate nitrates. In partially purified leaf extracts, significant differences in total NADH:NR activity and in distal activity dependent on methyl viologen (MV:NR) were found between the parental genotypes and selected DH lines. The studied genotypes differed also in dehydrogenase NR activity, i.e. cytochrome c reductase activity in crude extracts. In the studied genotypes, the NADH:NR activity in partially purified leaf extracts did not substantially differ by Km values for nitrates. Calculated Vmax values for NADH:NR in these genotypes were similar to total NR activity in partially purified extracts. Significant differences between the parental genotypes and selected DH lines were found in the thermal NADH:NR stability in crude and partially purified leaf extracts. From the performed studies it follows that different NR stability was one of the reasons of revealed differences in total activity and in partial NR activities in the leaf extracts between the studied genotypes of spring barley. Besides, it is suggested that varied NR gene expression in the leaves of these barley genotypes could also influence NR activity.     

Nitrate regulation of nitrate uptake and nitrate reductase expression in barley grown at different nitrate:ammonium ratios at constant relative nitrogen addition rate

Barley (Hordeum vulgare L. cv. Golf) was cultured using the relative addition rate technique, where nitrogen is added in a fixed relation to the nitrogen already bound in biomass. The relative rate of total nitrogen addition was 0.09 day^?1 (growth limiting by 35%), while the nitrate addition was varied by means of different nitrate: ammonium ratios. In 3- to 4-week-old plants, these ratios of nitrate to ammonium supported nitrate fluxes ranging from 0 to 22 μmol g^?1 root dry weight h^?1, whereas the total N flux was 21.8 ± 0.25 μmol g^?1 root dry weight h^?1 for all treatments. The external nitrate concentrations varied between 0.18 and 1.5 μM. The relative growth rate, root to total biomass dry weight ratios, as well as Kjeldahl nitrogen in roots and shoots were unaffected by the nitrate:ammonium ratio. Tissue nitrate concentration in roots were comparable in all treatments. Shoot nitrate concentration increased with increasing nitrate supply, indicating increased translocation of nitrate to the shoot. The apparent V_max for net nitrate uptake increased with increased nitrate fluxes. Uptake activity was recorded also after growth at zero nitrate addition. This activity may have been induced by the small, but detectable, nitrate concentration in the medium under these conditions. In contrast, nitrate reductase (NR) activity in roots was unaffected by different nitrate fluxes, whereas NR activity in the shoot increased with increased nitrate supply. NR-mRNA was detected in roots from all cultures and showed no significant response to the nitrate flux, corroborating the data for NR activity. The data show that an extremely low amount of nitrate is required to elicit expression of NR and uptake activity. However, the uptake system and root NR respond differentially to increased nitrate flux at constant total N nutrition. It appears that root NR expression under these conditions is additionally controlled by factors related to the total N flux or the internal N status of the root and/or plant. The method used in this study may facilitate separation of nitrate-specific responses from the nutritional effect of nitrate.     

UV-B Radiation Mediated Alterations in the Nitrate Assimilation Pathway of Crop Plants 2. Kinetic Characteristics of Nitrite Reductase

Cowpea [Vigna unguiculata (L.) Walp. cv. Co 4] seedlings were subjected to a weighted irradiance of 3.2 W m^-2 s^-1 of biologically effective ultraviolet-B radiation (UV-B, 280–320 nm) and the changes in the kinetic and other characteristics of nitrite reductase (NiR) were recorded. The activity of NiR was hampered by 19 % under UV-B irradiation compared to the control. The UV-B treated plants required higher concentrations of nitrate for the induction of NiR synthesis than the controls. The NiR activity decay kinetics showed that the UV-B treatment significantly lowers the t_1/2 of the enzyme, thereby indicating a reduced rate of enzyme turnover. The comparison of kinetic characteristics of nitrate reductase (NR) and NiR under UV-B treatment showed that NiR was not so sensitive to UV-B radiation as NR. As shown by enzyme turnover rates, NiR extracted from plants irradiated by UV-B in situ was less sensitive to UV-B radiation than the enzyme extract subjected to in vitro UV-B irradiation. Though NiR was less damaged by UV-B treatment than NR, subtle changes occurred in its kinetic characteristics. This revised version was published online in June 2006 with corrections to the Cover Date.     

Short-term modulation of nitrate reductase activity by exogenous nitrate in Nicotiana plumbaginifolia and Zea mays leaves

Maize (Zea mays L.) grown on low (0.8 mM) NO ₃ ^- , as well as untransformed and transformed Nicotiana plumbaginifolia constitutively expressing nitrate reductase (NR), was used to study the effects of NO ₃ ^- on the NR activation state. The NR activation state was determined from the relationship of total activity extracted in the presence of ethylenediaminetetracetic acid to that extracted in the presence of Mg²⁺. Light activation was observed in both maize and tobacco leaves. In the tobacco lines, NO ₃ ^- did not influence the NR activation state. In excised maize leaves, no correlation was found between the foliar NO ₃ ^- content and the NR activation state. Similarly, the NR activation state did not respond to NO ₃ ^- . Since the NR activation state determined from the degree of Mg²⁺-induced inhibition of NR activity is considered to reflect the phosphorylation state of the NR protein, the protein phosphatase inhibitor microcystin LR was used to test the importance of protein phosphorylation in the NO ₃ ^- -induced changes in NR activity. In-vivo inhibition of endogenous protein phosphatase activity by microcystin-LR decreased the level of NR activation in the light. This occurred to the same extent in the presence or absence of exogenous NO ₃ ^- . We conclude that NO ₃ ^- does not effect the NR activation state, as modulated by protein phosphorylation in either tobacco (a C₃ species) or maize (a C₄ species). The short-term regulation of NR therefore differs from the NO ₃ ^- -mediated responses observed for phosphoenolpyruvate carboxylase and sucrose phosphate synthase.Abbreviations Chl chlorophyll - MC microcystin-LR - PEP-Case phosphoenolpyruvate carboxylase - SPS sucrose-phosphate synthase We are indebted to Madeleine Provot and Nathalie Hayes for excellent technical assistance. This work was funded by EEC Biotechnology Contract No. BI02 CT93 0400, project of technical priority, Network D — Nitrogen Utilisation and Efficiency.     

茶树硝酸还原酶基因克隆及表达分析

利用茶树全器官转录组文库中硝酸还原酶(NR)的EST,通过RACE技术扩增出NR基因的cDNA,并利用实时荧光定量PCR检测了NR基因在不同茶树品种中的表达。结果表明:NR基因cDNA全长2 927bp,开放阅读框2 652bp,编码一个有884个氨基酸蛋白质,GenBank登录号为JX987133。经BlastX比对,与GenBank中登录的烟草NR相似性达到74%。茶树NR蛋白属于亲水性蛋白,可能为胞质蛋白。25个茶树品种叶片中NR表达水平差异明显,最高值是最低值的22.75倍。因NR是植物氮代谢过程中的关键限速酶,推测25个茶树品种间氮吸收利用能力存在差异。     

Nitrate-reductase expression is under the control of a circadian rhythm and is light inducible in Nicotiana tabacum leaves

Over a 24-h light-dark cycle, the level of mRNA coding for nitrate reductase (NR; EC 1.6.6.1) in the leaves of nitrate-fed Nicotiana tabacum L. plants increased throughout the night and then decreased until it was undetectable during the day. The amount of NR protein and NR activity were two-fold higher during the day than at night. When plants were transferred to continuous light conditions for 32 h, similar variations in NR gene expression, as judged by the above three parameters, still took place in leaf tissues. On the other hand, when plants were transferred to continuous dark conditions for 32 h, the NR-mRNA level continued to display the rhythmic fluctuations, while the amount of NR protein and NR activity decreased constantly, becoming very low, and showed no rhythmic variations. After 56 h of continuous darkness, the levels of NR mRNA, protein and activity in leaves all became negligible, and light reinduced them rapidly. These results indicate the circadian rhythmicity and light dependence of NR expression.     

The roles of nitrate and ammonium in the regulation of the development of nitrate reductase in Chlamydomonas reinhardii

The regulation of the development of nitrate reductase (NR) activity in Chlamydomonas reinhardii has been compared in a wild-type strain and in a mutant (nit-A) which possesses a modified nitrate reductase enzyme that is non-functional in vivo. The modified enzyme cannot use NAD(P)H as an electron donor for nitrate reduction and it differs from wild-type enzyme in that NR activity is not inactivated in vitro by incubation with NAD(P)H and small quantities of cyanide; it is inactivated when reduced benzyl viologen or flavin mononucleotide is present. After short periods of nitrogen starvation mutant organisms contain much higher levels of terminal-NR activity than do similarly treated wild-type ones. Despite the inability of the mutant to utilize nitrate, no nitrate or nitrite was found in nitrogen-starved cultures; it is therefore concluded that the appearance of NR activity is not a consequence of nitrification. After prolonged nitrogen starvation (22 h) the NR level in the mutant is low. It increases rapidly if nitrate is then added and this increase in activity does not occur in the presence of ammonium, tungstate or cycloheximide. Disappearance of preformed NR activity is stimulated by addition of tungstate and even more by addition of ammonium. The results are interpreted as evidence for a continuous turnover of NR in cells of the mutant with ammonium both stimulating NR breakdown and stopping NR synthesis. Nitrate protects the enzyme from breakdown. Reversible inactivation of NR activity is thought to play an insignificant rôle in the mutant.Abbreviations NR nitrate reductase - BV benzyl viologen     

The nitrate reductase promoter of birch directs differential reporter gene expression in tissues of transgenic tobacco

A 1535 bp promoter of the nitrate reductase gene (nia) from birch (Betula pendula) and a series of 5′ deletions were fused to the β-glucuronidase (GUS) gene and introduced into Nicotiana plumbaginifolia. In transgenic plants the NR promoter sequences directed strong GUS expression in the root epidermal hair cells, and in phloem cells of leaf and stem vascular tissue. The NR promoter confers also a significant stimulation of the GUS gene expression by nitrate. These findings might indicate that nitrate flow is one of the signals involved into tissue and cell specific expression of the NR promoter GUS fusions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nitrate reductases inEscherichia coli

Escherichia coli expresses two different membrane-bound respiratory nitrate reductases, nitrate reductase A (NRA) and nitrate reductase Z (NRZ). In this review, we compare the genetic control, biochemical properties and regulation of these two closely related enzyme systems. The two enzymes are encoded by distinct operons located within two different loci on theE. coli chromosome. ThenarGHJI operon, encoding nitrate reductaseA, is located in thechlC locus at 27 minutes, along with several functionally related genes:narK, encoding a nitrate/nitrite antiporter, and thenarXL operon, encoding a nitrate-activated, two component regulatory system. ThenarZYWV operon, encoding nitrate reductase Z, is located in thechlZ locus located at 32.5 minutes, a region which includes anarK homologue,narU, but no apparent homologue to thenarXL operon. The two membrane-bound enzymes have similar structures and biochemical properties and are capable of reducing nitrate using normal physiological substrates. The homology of the amino acid sequences of the peptides encoded by the two operons is extremely high but the intergenic regions share no related sequences. The expression of both thenarGHJI operon and thenarK gene are positively regulated by two transacting factors Fnr and NarL-Phosphate, activated respectively by anaerobiosis and nitrate, while thenarZYWV operon and thenarU gene are constitutively expressed. Nitrate reductase A, which accounts for 98% of the nitrate reductase activity when fully induced, is clearly the major respiratory nitrate reductase inE. coli while the physiological role of the constitutively expressed nitrate reductase Z remains to be defined.Abbreviations NR nitrate reductase On leave from Department of Biochemistry and Molecular Biology, The University of Texas Medical school at Houston, Houston, Texas, 77225, USA