Activation,synthesis and turnover of nitrate reductase controlled by nitrate and ammonium in Chlorella vulgaris

Cells of Anacystis nidulans grown at 25 or 30°C were examined both by thin-section and freeze-fracture electron microscopy. Cells grown at either temperature appeared similar when fixed at the growth temperature prior to observation. When cells were chilled to near 0°C for 30 min prior to fixation, those previously grown at 25° appeared unchanged as judged by thin sectioning while those grown at 39° showed considerable morphological alteration. Freeze fracture showed particle aggregation (more pronounced in 39°-grown cells) indicating lipid-phase separation in cells chilled prior to fixation. The phase separation was totally reversed by rewarming the chilled, 25°-grown cells to their growth temperature but was only partially reversed by rewarming chilled, 39°-grown cells. These results correlate with other effects of chilling seen in Anacystis cells grown at different temperatures.     

Reversible inactivation of nitrate reductase in Chlorella vulgaris in vivo

Summary The NADH-nitrate oxidoreductase of Chlorella vulgaris has an inactive form which has previously been shown to be a cyanide complex of the reduced enzyme. This inactive enzyme can be reactivated by treatment with ferricyanide in vitro. In the present study, the activation state of the enzyme was determined after different prior in vivo programs involving environmental variations. Oxygen, nitrate, light and CO₂ all affect the in vivo inactivation of the enzyme in an interdependent manner. In general, the inactivation is stimulated by O₂ and inhibited by nitrate and CO₂. Light may stimulate or inhibit, depending on conditions. Thus, the effects of CO₂ and nitrate (inhibition of reversible inactivation) are clearly manifested only in the light. In contrast, light stimulates the inactivation in the presence of oxygen and the absence of CO₂ and nitrate. Since the inactivation of the enzyme requires HCN and NADH, and it is improbable that O₂ stimulates NADH formation, it is reasonable to conclude that HCN is formed as the result of an oxidation reaction (which is stimulated by light). The formation of HCN is probably stimulated by Mn²⁺, since the formation of reversibly-inactivated enzyme is impaired in Mn²⁺-deficient cells. The prevention of enzyme inactivation by nitrate in vivo is in keeping with previous in vitro results showing that nitrate prevents inactivation by maintaining the enzyme in the oxidized form. A stimulation of nitrate uptake by CO₂ and light could account for the effect of CO₂ (prevention of inactivation) which is seen mainly in the presence of nitrate and light. Ammonia added in the presence of nitrate has the same effect on the enzyme as removing nitrate (promotion of reversible inactivation). Ammonia added in the absence of nitrate has little extra effect. It is therefore likely that ammonia acts by preventing nitrate uptake. The uncoupler, carbonylcyanide-m-chloro-phenylhydrazone, causes enzyme inactivation because it acts as a good HCN precursor, particularly in the light. Nitrite, arsenate and dinitrophenol cause an enzyme inactivation which can not be reversed by ferricyanide in crude extracts. This suggests that there are at least two different ways in which the enzyme can be inactivated rather rapidly in vivo.     

The effect of vanadium on nitrate reductase of Chlorella vulgaris

Added vanadate ions inhibit purified nitrate reductase from Chlorella vulgaris by reacting with the enzyme in a manner rather similar to that of HCN. Thus vanadate, like HCN, forms an inactive complex with the reduced enzyme, and this inactivated enzyme can be reactivated rapidly by adding ferricyanide. The inactive vanadate enzyme complex is less stable than the inactive HCN complex, and the two can be distinguished by the fact that EDTA causes a partial reactivation of the former, but not of the latter. Vanadate can also cause an increase in HCN formation by intact Chlorella vulgaris cells. When these cells were incubated with vanadate, their nitrate reductase was reversibly inactivated, and all of this inactive enzyme could be shown to be the HCN complex rather than the vanadate complex. When HCN and vanadate are both present, the HCN-inactivated enzyme, being more stable, will be formed in preference to the vanadate-inactivated enzyme.Abbreviation EDTA ethylenediamine tetraacetate     

Physical studies on assimilatory nitrate reductase from Chlorella vulgaris.

Assimilatory nitrate reductase (EC 1.6.6.1 NADH:nitrate oxidoreductase) from Chlorella vulgaris purified by affinity chromatography was found to be homogeneous as judged by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel and by analytical ultracentrifugal techniques. The molecular weight of the intact enzyme and that of the enzyme dissociated in 6 M GuHCl, determined by sedimentation equilibrium studies, were 280,000 +/- 10,000 and 90,000 +/- 5,000, respectively. Comparable values were obtained using the S20,w value and the D20,w values in Svedberg's equation. The D20,w values were determined by laser light-scattering measurements. Active enzyme centrifugation showed that the monomer is an active species. A quantitative re-evaluation of the prosthetic groups present (FAD, heme, and molybdenum) was also made and was consistent with the conclusion that the active monomer contains three subunits as previously deduced by Solomonson et al. ((1975) J. Biol. Chem. 250, 4120). Electron micrographs showed images which corresponded to three subunits, supporting the data obtained by hydrodynamic studies. The enzyme is not cigar-shaped, as previously surmised, but has a roughly globular structure.     

185W-labelled nitrate reductase from Chlorella

By adding ¹⁸⁵W-tungstate to a Chlorella culture, it has beenpossible to incorporate this metal into the nitrate reductasecomplex. The W-labelled enzyme was completely inactive as nitratereductase, but maintained unaffected its diaphorase activity.In vivo incorporation of tungsten into the enzyme was competitivelyhindered by molybdenum. ¹ This work was supported by a grant from the Instituto de EstudiosNucleares, J.E.N., Spain. (Received July 6, 1971; )     

Induction of nitrate reductase in pumpkin seedlings

Nitrate reductase activity (NRA) was found to be induced in9-day-old pumpkin seedlings by nitrate and light. NRA was greatestin leaves and cotyledons and in vitro measurements gave highervalues than in vitro measurements. NRA was found in roots bythe in vivo method but not by the in vitro method. NRA changedwith the age of the seedling with maximum activity in 7-day-oldcotyledons and 9-day-old roots of light grown plants; and rootsof 7-day-old etiolated plants. Little activity was found inetiolated cotyledons. ¹ Present address: College of Agriculture and Animal Husbandry,P.O. Box 32, Rezaiyeh, Iran (Received September 30, 1977; )     

Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana

Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrite uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.Abbreviations EDTA ethylenediaminetetraacetic acid - FAD flavine-adenine dinucleotide - IgG immunoglobulin G - NR nitrate reductase - PM plasma membrane - TX-100 Triton X-100     

Effect of ammonium and ferricyanide on nitrate utilization by Chlorella vulgaris

It has been shown previously that added ammonium salts cause a cessation of nitrate utilization in some Chlorella species. It has also been shown that Chlorella vulgaris can form an inactivated nitrate reductase which is an HCN complex. In the present study, a comparison has been made of the rate of nitrate utilization and the rate of nitrate reductase inactivation in Chlorella vulgaris in response to the addition of ammonium salts and light-dark changes. The rate of formation of HCN-inactivated enzyme is too slow to account for the prompt inhibition of nitrate utilization caused by adding ammonium. In contrast, when nitrate utilization is inhibited by addition of ferricyanide to intact cells, the HCN-inactivated enzyme is promptly formed in vivo, and might account for the inhibition of nitrate utilization, though inhibition of nitrate uptake can not be excluded.     

The development of nitrate reductase in Chlorella and its repression by ammonium

Summary Chlorella vulgaris, grown with ammonium sulphate as nitrogen source, contains very little nitrate reductase activity in contrast to cells grown with potassium nitrate. When ammonium-grown cells are transferred to a nitrate medium, nitrate reductase activity increases rapidly and the increase is partially prevented by chloramphenicol and by p-fluorophenylalanine, suggesting that protein synthesis is involved. The increase in nitrate reductase activity is prevented by small quantities of ammonium; this inhibition is overcome, in part, by raising the concentration of nitrate. Although nitrate stimulates the development of nitrate reductase activity, its presence is not essential for the formation of the enzyme since this is formed when ammonium-grown cells are starved of nitrogen and when cells are grown with urea or glycine as nitrogen source. It is concluded that the formation of the enzyme is stimulated (induced) by nitrate and inhibited (repressed) by ammonium.     

Reversible inactivation by NADH and ADP on Chlorella fusca nitrate reductase

The active form of $\text{Chlorella fusca}$ nitrate reductase can be reversibly converted into its inactive form by reduction with NADH in the presence of ADP. Under the experimental conditions used, no inactivation occurs when nitrate is simultaneously present or when the nucleotides act isolately, the inactivating effect being maximal at a concentration of ADP (0.3 mM) equimolecular with that of NADH. The inactive enzyme thus attained can be completely reactivated by reoxidation with ferricyanide. The redox state of the pyridine nucleotide and the phosphorylation degree of the adenine nucleotide are critical for the inactivation process to ensue, since neither NAD⁺ nor AMP or ATP do exert any effect. ADP is also a powerful, although rather unspecific, protector against thermal inactivation of the NADH-diaphorase moiety of the NADH-nitrate reductase complex.     

Induction of nitrate uptake and nitrate reductase activity in trembling aspen and lodgepole pine

¹³NO₃^– influx into the roots and in vivo nitrate reductase activity (NRA) in the roots and leaves have been measured in trembling aspen (Populus tremuloides Michx.) and lodgepole pine (Pinus contorta Dougl.) seedlings after exposure to either 0·1 or 1·5 mol m^–3 NO₃^– for varying periods up to 20 d. Both NO₃^– influx and NRA were inducible in these species and, in trembling aspen, peak induction of nitrate influx and NRA were achieved within 12 h, compared to 2–4 d for influx and 4–12 d for NRA in lodgepole pine. In trembling aspen, ≈ 30% of the total ¹³N absorbed during a 10 min influx period followed by 2 min of desorption was translocated to the shoot. In lodgepole pine, by contrast, translocation of ¹³N to the shoot was undetectable during the same time period. Root NRA as well as NO₃^– influx from 0·1 mol m^–3 NO₃^– were substantially higher in trembling aspen than in lodgepole pine at all stages of NO₃^– exposure, i.e. during the uninduced, the peak induction, and steady-state stages. In order to examine whether the lower rates of NO₃^– influx and NRA were related to proportionately fewer young (unsuberized) roots in lodgepole pine, we determined these parameters in young and old (suberized) roots of this species separately. Induction of influx and NRA were initially greater in young roots but at steady-state there were only minor differences between the young and the old roots. However, even the elevated initial rates in the young roots of lodgepole pine were substantially lower than those of aspen. In pine, influx at 1·5 mol m^–3 NO₃^– was ~ 6-fold higher than at 0·1 mol m^–3 NO₃^– and appeared to be mostly via a constitutive system. By contrast, in aspen, steady-state influxes at 0·1 and 1·5 mol m^–3 were not significantly different, being similar to the rate attained by pine at only the higher [NO₃^–]. In aspen, leaf NRA was ~ 2-fold higher than that of roots. In lodgepole pine NRA of the needles was below the detection limit. These results show that trembling aspen seedlings are better adapted for NO₃^– acquisition and utilization than lodgepole pine seedlings.     

Electrochemical and kinetic analysis of electron-transfer reactions of Chlorella nitrate reductase.

Assimilatory nitrate reductase (NR) from Chlorella is homotetrameric, each subunit containing FAD, heme, and Mo-pterin in a 1:1:1 stoichiometry. Measurements of NR activity and steady-state reduction of the heme component under conditions of NADH limitation or competitive inhibition by nitrite suggested intramolecular electron transfer between heme and Mo-pterin was a rate-limiting step and provided evidence that heme is an obligate intermediate in the transfer of electrons between FAD and Mo-pterin. In addition to the physiological substrates NADH and nitrate, various redox mediators undergo reactions with one or more of the prosthetic groups. These reactions are coupled by NR to NADH oxidation or nitrate reduction. To test whether intramolecular redox reactions of NR were rate-determining, rate constants for redox reactions between NR and several chemically diverse mediators were measured by cyclic voltammetry in the presence of NADH or nitrate. Reduction of ferrocenecarboxylic acid, dichlorophenolindophenol, and cytochrome c by NADH-reduced NR was coupled to reoxidation at a glassy carbon electrode (ferrocene and dichlorophenolindophenol) or at a bis(4-pyridyl) disulfide modified gold electrode (cytochrome c), yielding rate constants of 10.5 x 10(6), 1.7 x 10(6), and 2.7 x 10(6) M-1 s-1, respectively, at pH 7. Kinetics were consistent with a second-order reaction, implying that intramolecular heme reduction by NADH and endogenous FAD was not limiting. In contrast, reduction of methyl viologen and diquat at a glassy carbon electrode, coupled to oxidation by NR and nitrate, yielded similar kinetics for the two dyes. In both cases, second-order kinetics were not obeyed, and reoxidation of dye-reduced Mo-pterin of NR by nitrate became limiting at low scan rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of nitrate reductase in auxin-induced NO synthesis

It is well known for a long time, that nitric oxide (NO) functions in variable physiological and developmental processes in plants, however the source of this signaling molecule in the diverse plant responses is very obscure.¹ Although existance of nitric oxide sythase (NOS) in plants is still questionable, LNMMA (N^G-monomethyl-L-arginine)-sensitive NO generation was observed in different plant species.^2,3 In addition, nitrate reductase (NR) is confirmed to have a major role as source of NO.^4,5 This multifaced molecule acts also in auxin-induced lateral root (LR) formation, since exogenous auxin enhanced NO levels in regions of Arabidopsis LR initiatives. Our results pointed out the involvement of nitrate reductase enzyme in auxin-induced NO formation. In this addendum, we speculate on auxin-induced NO production in lateral root primordial formation.Key words: atnoa1, indole-3-butyric acid, nia1, nia2 double mutant, nitric oxideLateral roots are formed from root pericycle cells postembryonically which process is promoted by indole-acetic acid (IAA). It was recognized that IAA share common steps with NO in the signal transduction cascade towards the auxin induced adventitious and lateral root formation.^6–8 Previously it was suggested that besides IAA, indol-3-butyric (IBA) is a true endogenous auxin in Arabidopsis, which acts in adventious and lateral root development.^9,10 Our results showed that IBA induced LR initials emitted intensive NO fluorescence in Arabidopsis. This increased level of NO was present only in the LR initials in contrast to primary root (PR) sections where it remained at the control level.In plants NO can be produced by a number of enzyme systems and non-enzymatic ways. In roots, the most likely candidates of NO synthesis are NR enzymes (cytoplasmic and plasma membrane-bounded isoenzymes, cNR and PM-NR). Recently a new type of enzyme, the PM-bounded nitrite:NO reductase (Ni:NOR) was identified as a possible source of NO in roots.¹¹ Because of the several formation potentials of NO, the identification of its source in plant tissues under different conditions is complicated. Using diverse mutants proved to be a good opportunity to investigate the possible sources of NO. In our experiments wild-type (Col-1), Atnoa1 (nitric oxide synthase associated 1 deficient) and nia1, nia2 (NR deficient) seedlings were applied in order to determine the enzymatic source of NO induced by auxin. In roots of these plants, different NO levels were measured in their control state (i.e., without IBA treatment). The NO content in Atnoa1 roots was similar to that of wild-type, while nia1, nia2 showed lower NO fluorescence than the other groups of plants. This result suggests that NR activity is needed to NO synthesis in roots. Further on, it was demonstrated that IBA induced NO generation in both the wild type and Atnoa1 root primordia, but this induction failed in the NR-deficient mutant. This reveals that the NO accumulation in root primordia induced by auxin requires NR activity. These observations were evidenced also by biochemical manner. On the one part, we applied L-NMMA, which is a specific inhibitor of mammalian NOS, on the other part, the inhibitor of NR enzyme tungstate was used and we monitored NO fluorescence in wild-type roots. The NOS inhibitor displayed no effect on NO levels neither at control state nor during auxin treatment, while tungstate inhibited NO synthesis in lateral roots and primary roots of control plants. The effect of tungstate was similar in auxin-treated roots, since application of this NR enzyme inhibitor decreased NO levels in PRs and LRs (Fig. 1).Open in a separate windowFigure 1NO fluorescence in lateral roots (white columns) and primary roots (grey columns) of control, control + 1 mM tungstate, IBA and IBA + 1 mM tungstate-treated wild-type Arabidopsis thaliana. Vertical bars are standard errors.Some speculations can be made on these results. Although more efforts are needed to make the scene clear, now we can predict that auxin somehow may induce NR isoenzymes, which produce nitrite in root cells. From this point, two further scenarios are possible: as the result of accumulated nitrite, either the NO-producing activity of NR or Ni:NOR activity are promoted, hereby NO is generated from nitrite reduction. NO formed in these two possible ways modulates the expression of certain cell cycle regulatory genes contributing to division of pericycle cells in LR primordia, as was published in tomato.¹²Nowadays research in the “NO-world” of plants is running very actively. Nevertheless, lot of more work is needed to reveal all the unknown faces of this novel multipurpose signaling molecule.