The Assimilation of Nitrogen from Ammonium Salts and Nitrate by Fungi

1. The assimilation of inorganic nitrogen by Scopulariopsis brevicaulisand some physiologically similar species has been studied. Theirfailure to assimilate completely from ammonium sulphate hasbeen shown to be due to the fall in pH of the medium inducedby the initial uptake of ammonia.
2. Complete assimilation ofammonia takes place in the presenceof the neutral salts ofeach of thirteen organic acids investigated.The organic acidsact primarily through their buffering effectwhich preventsor slows down the fall in pH. They are not specificallyrequiredfor ammonia assimilation by these fungi and can beeffectivelyreplaced by certain inorganic buffers.
3. The influence of severalexternal factors on the rate of assimilationof ammonia, nitrate,and nitrite has been studied in S. brevicaulis.In correspondingconditions the mycelium assimilates ammoniamore rapidly thannitrate over a wide range of conditions.
4. Ammonia, even invery low concentration, completely suppressesnitrate assimilationwhen both sources of nitrogen are presenttogether. Nitrite,however, is assimilated simultaneously withammonia. It is thereforeconcluded that ammonia blocks the reductionof nitrate to nitriteby the fungus.
5. The suppression of nitrate assimilation inthe presence of ammoniais common to many mould fungi besidesS. brevicaulis, and isbelieved to have adaptive significancein natural habitats.
6. The nitrate-reducing and assimilatingsystem is formed, evenwhen S. brevicaulis is grown in completeabsence of nitrate(ammonia medium with organic acid). It comesinto action rapidlywhen the inhibiting effect of ammonia isremoved. Similarly,nitrate-grown mycelium is capable of assimilatingammonia atmaximal rate without any adaptive lag.

     

Effect of iron supply on the activities of the nitrate-reducing system from Chlorella

Summary In Chlorella, as in most photosynthetic organisms, the reduction of nitrate to ammonia proceeds sequentially in two independent and well characterized steps, catalyzed by the enzymes of the nitrate-reducing system: 1. the reduction of nitrate to nitrite by the flavomolybdoprotein NADH-nitrate reductase, and 2. the reduction of nitrite to ammonia by the ironprotein ferredoxin-nitrite reductase. In this communication, it is shown that, in Chlorella, the cellular level of nitrite reductase activity specifically increases in response to the iron content of the culture medium. By contrast, the activity of nitrate reductase is apparently not affected by the concentration of iron in the nutrient solution under the same conditions.     

The regulation of activity of the enzymes involved in the assimilation of nitrate by higher plants

1. Possible mechanisms regulating the activities of three enzymes involved in nitrate assimilation, nitrate reductase, nitrite reductase and glutamate dehydrogenase, were studied in radish cotyledons. 2. Nitrate-reductase and nitrite-reductase activities are low in nitrogen-deficient cotyledons, and are induced by their substrates. 3. Glutamate dehydrogenase is present regardless of the nitrogen status, and the enzyme can be increased only slightly by long-term growth on ammonia. 4. Although nitrate is the best inducer of nitrate reductase, lower levels of induction are also obtained with nitrite and ammonia. The experiments did not distinguish between direct or indirect induction by these two molecules. 5. Nitrite reductase is induced by nitrite and only indirectly by nitrate. 6. The induction of both nitrate reductase and nitrite reductase is prevented by the inhibitors actinomycin D, puromycin and cycloheximide, indicating a requirement for the synthesis of RNA and protein. 7. The decay of nitrate reductase, determined after inhibition of protein synthesis, is slower than the synthesis of the enzyme. Nitrite reductase is much more stable than nitrate reductase. 8. The synthesis of nitrate reductase is not repressed by ammonia, but is repressed by growth on a nitrite medium. 9. There is no inhibition of nitrate reductase, nitrite reductase or glutamate dehydrogenase by the normal end products of assimilation, but cyanate is a fairly specific inhibitor of nitrate reductase.     

Nitrogen Metabolism of Lemna minor. II. Enzymes of Nitrate Assimilation and Some Aspects of Their Regulation

In L. minor grown in sterile culture, the primary enzymes of nitrate assimilation, nitrate reductase (NR), nitrite reductase (NiR) and glutamate dehydrogenase (GDH) change in response to nitrogen source. NR and NiR levels are low when grown on amino acids (hydrolyzed casein) or ammonia; both enzymes are rapidly induced on addition of nitrate, while addition of nitrite induces NiR only. Ammonia represses the nitrate induced synthesis of both NR and NiR.NADH dependent GDH activity is low when grown on amino acids and high when grown on nitrate or ammonia, but the activities of NADPH dependent GDH and Alanine dehydro-genase (AIDH) are much less affected by nitrogen source. NADH-GDH and AIDH are induced by ammonia, and it is suggested that these enzymes are involved in primary nitrogen assimilation.     

Diurnal changes in nitrogen assimilation of tobacco roots.

To gain an insight into the diurnal changes of nitrogen assimilation in roots the in vitro activities of cytosolic and plasma membrane-bound nitrate reductase (EC 1.6.6.1), nitrite reductase (EC 1.7.7.1) and cytosolic and plastidic glutamine synthetase (EC 6.3.1.2) were studied. Simultaneously, changes in the contents of total protein, nitrate, nitrite, and ammonium were followed. Roots of intact tobacco plants (Nicotiana tabacum cv. Samsun) were extracted every 3 h during a diurnal cycle. Nitrate reductase, nitrite reductase and glutamine synthetase were active throughout the day-night cycle. Two temporarily distinct peaks of nitrate reductase were detected: during the day a peak of soluble nitrate reductase in the cytosol, in the dark phase a peak of plasma membrane-bound nitrate reductase in the apoplast. The total activities of nitrate reduction were similar by day and night. High activities of nitrite reductase prevented the accumulation of toxic amounts of nitrite throughout the entire diurnal cycle. The resulting ammonium was assimilated by cytosolic glutamine synthetase whose two activity peaks, one in the light period and one in the dark, closely followed those of nitrate reductase. The contribution of plastidic glutamine synthetase was negligible. These results strongly indicate that nitrate assimilation in roots takes place at similar rates day and night and is thus differently regulated from that in leaves.     

Molecular Components of Nitrate and Nitrite Efflux in Yeast

Some eukaryotes, such as plant and fungi, are capable of utilizing nitrate as the sole nitrogen source. Once transported into the cell, nitrate is reduced to ammonium by the consecutive action of nitrate and nitrite reductase. How nitrate assimilation is balanced with nitrate and nitrite efflux is unknown, as are the proteins involved. The nitrate assimilatory yeast Hansenula polymorpha was used as a model to dissect these efflux systems. We identified the sulfite transporters Ssu1 and Ssu2 as effective nitrate exporters, Ssu2 being quantitatively more important, and we characterize the Nar1 protein as a nitrate/nitrite exporter. The use of strains lacking either SSU2 or NAR1 along with the nitrate reductase gene YNR1 showed that nitrate reductase activity is not required for net nitrate uptake. Growth test experiments indicated that Ssu2 and Nar1 exporters allow yeast to cope with nitrite toxicity. We also have shown that the well-known Saccharomyces cerevisiae sulfite efflux permease Ssu1 is also able to excrete nitrite and nitrate. These results characterize for the first time essential components of the nitrate/nitrite efflux system and their impact on net nitrate uptake and its regulation.     

Nitrite reduction in Veillonella alcalescens.

Nitrite reduction was examined in Veillonella alcalescens C-1, and obligate anaerobe with an ATP-yielding nitrate-reducing system. Hydrogen donors for nitrite reduction included hydrosulfite, hydrogen gas, and pyruvate, but not pyridine nucleotides, in the presnece or absence of flavins. Pyruvate-linked nitrite reduction was not inhibited by 4,4,4-trifluoro-1-(2-thienyl) 1,3-butanedione, dicoumarol, or 2-heptyl-4-hydroxy-quinoline-N-oxide. The noninvolvement of membrane-bound factors was supported by the fact that 100% of pyruvate-linked activity remained in the soluble fraction after fractionation of crude extracts by ultracentrifugation. Using DEAE-cellulose column chromatography, however, the participation of ferredoxin in nitrite reduction was demonstrated. The product of nitrite reduction appeared to be ammonia, as determined from H2-to-NO2- ratios. Nitrite reductase was induced by nitrate or nitrite and was repressed by increased levels of reduced nitrogenous compounds.     

Regulation of the nitrate-reducing system enzymes in wild-type and mutant strains of Chlamydomonas reinhardii

Summary Six mutant strains (301, 102, 203, 104, 305, and 307) affected in their nitrate assimilation capability and their corresponding parental wild-type strains (6145c and 21gr) from Chlamydomonas reinhardii have been studied on different nitrogen sources with respect to NAD(P)H-nitrate reductase and its associated activities (NAD(P)H-cytochrome c reductase and reduced benzyl viologen-nitrate reductase) and to nitrite reductase activity. The mutant strains lack NAD(P)H-nitrate reductase activity in all the nitrogen sources. Mutants 301, 102, 104, and 307 have only NAD(P)H-cytochrome c reductase activity whereas mutant 305 solely has reduced benzyl viologen-nitrate reductase activity. Both activities are repressible by ammonia but, in contrast to the nitrate reductase complex of wild-type strains, require neither nitrate nor nitrite for their induction. Moreover, the enzyme from mutant 305 is always obtained in active form whereas nitrate reductase from wild-types needs to be reactivated previously with ferricyanide to be fully detected. Wild-type strains and mutants 301, 102, 104, and 307, when properly induced, exhibit an NAD(P)H-cytochrome c reductase distinguishable electrophoretically from contitutive diaphorases as a rapidly migrating band. Nitrite reductase from wild-type and mutant strains is also repressible by ammonia and does not require nitrate or nitrite for its synthesis. These facts are explained in terms of a regulation of nitrate reductase synthesis by the enzyme itself.     

Decrease of Nitrate Reductase Activity in Spinach Leaves during a Light-Dark Transition

In leaves of spinach plants (Spinacia oleracea L.) performing CO₂ and NO₃⁻ assimilation, at the time of sudden darkening, which eliminates photosystem I-dependent nitrite reduction, only a minor temporary increase of the leaf nitrite content is observed. Because nitrate reduction does not depend on redox equivalents generated by photosystem I activity, a continuation of nitrate reduction after darkening would result in a large accumulation of nitrite in the leaves within a very short time, which is not observed. Measurements of the extractable nitrate reductase activity from spinach leaves assayed under standard conditions showed that in these leaves the nitrate reductase activity decreased during darkening to 15% of the control value with a half-time of only 2 minutes. Apparently, in these leaves nitrate reductase is very rapidly inactivated at sudden darkness avoiding an accumulation of the toxic nitrite in the cells.     

Nitrate transport system in Neurospora crassa

Nitrate uptake in Neurospora crassa has been investigated under various conditions of nitrogen nutrition by measuring the rate of disappearance of nitrate from the medium and by determining mycelial nitrate accumulation. The nitrate transport system is induced by either nitrate or nitrite, but is not present in mycelia grown on ammonia or Casamino Acids. The appearance of nitrate uptake activity is prevented by cycloheximide, puromycin, or 6-methyl purine. The induced nitrate transport system displays a K_m for nitrate of 0.25 mM. Nitrate uptake is inhibited by metabolic poisons such as 2,4-dinitrophenol, cyanide, and antimycin A. Furthermore, mycelia can concentrate nitrate 50-fold. Ammonia and nitrite are non-competitive inhibitors with respect to nitrate, with K_i values of 0.13 and 0.17 mM, respectively. Ammonia does not repress the formation of the nitrate transport system. In contrast, the nitrate uptake system is repressed by Casamino Acids. All amino acids individually prevent nitrate accumulation, with the exception of methionine, glutamine, and alanine. The influence of nitrate reduction and the nitrate reductase protein on nitrate transport was investigated in wild-type Neurospora lacking a functional nitrate reductase and in nitrate non-utilizing mutants, nit-1, nit-2, and nit-3. These mycelia contain an inducible nitrate transport system which displays the same characteristics as those found in the wild-type mycelia having the functional nitrate reductase. These findings suggest that nitrate transport is not dependent upon nitrate reduction and that these two processes are separate events in the assimilation of nitrate.     

Mode of action of natural inactivator proteins from corn and rice on a purified assimilatory nitrate reductase

The molecular basis for the action of two natural inactivator proteins, isolated from rice and corn, on a purified assimilatory nitrate reductase has been examined by several physical techniques. Incubation of purified Chlorella nitrate reductase with either rice inactivator protein or corn inactivator protein results in a loss of NADH:nitrate reductase and the associated partial activity, NADH:cytochrome c reductase, but no loss in nitrate-reducing activity with reduced methyl viologen as the electron donor. The molecular weight of the reduced methyl viologen:nitrate reductase species, determined by sedimentation equilibrium in the Beckman airfuge after complete inactivation with rice inactivator protein or with corn inactivator protein, was 595,000 and 283,000, respectively, compared to a molecular weight of 376,000 for the untreated control determined under the same conditions. Two protein peaks were observed after molecular-sieve chromatography on Sephacryl S-300 of nitrate reductase inactivated by corn inactivator protein. The Stokes radii of these fragments were 68 and 24 Å, compared to a value of 81 Å for untreated nitrate reductase. The large fragment contained molybdenum and heme but no flavin, and had nitrate-reducing activity with reduced methyl viologen as electron donor. The small fragment contained FAD but had no NADH:cytochrome c reductase or nitrate-reducing activities. Molecular weights determined by sodium dodecyl sulfate-gel electrophoresis were 67,000 and 28,000 for the large and small fragments, respectively, compared to a subunit molecular weight of 99,000 determined for the untreated control. No change in subunit molecular weight of nitrate reductase after inactivation by rice inactivator protein was observed. These results indicate that rice inactivator protein acts by binding to nitrate reductase. The stoichiometry of binding is 1–2 molecules of rice inactivator protein to one tetrameric molecule of nitrate reductase. Corn inactivator protein, in contrast, acts by cleavage of a M_r 30,000 fragment from nitrate reductase which is associated with FAD. The remaining fragment is a tetramer of M_r 70,000 subunits which retains nitrate-reducing activity and contains molybdenum and heme but has no NADH:dehydrogenase activity. The action of rice inactivator protein was partially prevented by NADH and completely prevented by a combination of NADH and cyanide, while the action of corn inactivator protein was not significantly affected by these effectors.     

Nitrogen metabolism in tomato seedlings

Tomato seedlings absorbed increasing amounts of nitrate-N. The total uptake was doubled as the concentration of nitrate was quadruplicated. NO₃?N absorption seemed to be accompanied by efflux of OH^? ions which shift the pH of the media to the alkaline side. A minor fraction of the absorbed nitrate accumulated in the tissues while the major part was assimilated into peptides and proteins. The dry matter gain was by the end of experiment relatively higher than the control samples raised on nitrogen-free nutrient solution. Nitrate assimilation seemed to involve its reduction down to ammonia level. Since neither nitrite nor ammonia was recovered in the tissue-medium system, it was postulated that the rate of reduction was slower than the rate of product assimilation. The first step in nitrate reduction (nitrate→nitrate) appeared to be limiting while further reduction steps occurred rapidly and accompanied by simultaneous assimilation of ammonia. The enzyme responsible for the first step of nitrate reduction,i.e., nitrate reductase, was extracted from tomato shoots and roots. The activity in root extract amounted to about 30% of that of the shoot. This may suggest the localization of nitrate reduction in the leaves and realizes the relation between nitrate metabolism and photosynthesis.     

Induction of ferredoxin-NADP+ oxidoreductase and ferredoxin synthesis in pea root plastids during nitrate assimilation

A 14.5 kDa protein with antigenic components in common with pea leaf ferredoxin was detected on transblots of the soluble proteins of pea root plastids. The amount of this protein was found to increase during the induction of nitrate assimilation in pea roots, reaching a maximal level at 8–12 h. Concurrent with this, a fourfold increase in NADPH-dependent ferredoxin-NADP⁺ oxidoreductase (FNR) activity was observed corresponding to an increase in the amount of this protein detected immunologically on transblots using a leaf FNR antibody. These changes were not observed in plastids from roots of plants grown on ammonia or depleted of nitrogen. It is suggested that in addition to the already well reported induction by nitrate of nitrate reductase and nitrite reductase, there is a co-induction of a plastid located ferredoxin and FNR. Both these proteins are necessary for the transfer of reductant generated by the oxidative pentose phosphate pathway to nitrite reductase.     

Regulation of Chlorella nitrate reductase: control of enzyme activity and immunoreactive protein levels by ammonia

Nitrate reductase catalyzes the initial step in the conversion of nitrate to organic nitrogen and is thought to be repressed by ammonia and induced by nitrate. Induction by nitrate and repression by ammonia were studied by following changes in NADH:nitrate reductase and the associated partial activities NADH:cytochrome c reductase and methylviologenr:nitrate reductase. Immunoreactive protein was assessed by enzyme-linked immunosorbent assay and immunoblotting. Molybdenum cofactor levels were investigated using the nit-1 complementation assay as well as fluorescence of the oxidized cofactor. The results indicate that the NADH:cytochrome c reductase activity is "induced" faster than the nitrate-reducing activity and suggest that incorporation of the molybdo-pterin cofactor may be rate limiting in the expression of activity. Molybdenum cofactor levels are significantly elevated in nitrate-treated cells. Under "repressing" conditions all activities decreased at approximately the same rate. A more rapid conversion of the enzyme to a reversibly inactive form also occurred under these conditions. Changes in immunoreactive protein levels correlated most closely with NADH:cytochrome c reductase activity but appeared to increase faster during induction and decrease slightly slower during repression than the enzyme activities. Removal of exogenous ammonia results in the appearance of nitrate reducing activity, as well as immunoreactive protein (derepression). Studies using protein and RNA synthesis inhibitors indicated that de novo synthesis is required for nitrate reductase induction and were in agreement with the results of the immunoreactive studies.     

Immunological approach to the regulation of nitrate reductase in Monoraphidium braunii

The effects of different culture conditions on nitrate reductase activity and nitrate reductase protein from Monoraphidium braunii have been studied, using two different immunological techniques, rocket immunoelectrophoresis and an enzyme-linked immunosorbent assay, to determine nitrate reductase protein. The nitrogen sources ammonium and glutamine repressed nitrate reductase synthesis, while nitrite, alanine, and glutamate acted as derepressors. There was a four- to eightfold increase of nitrate reductase activity and a twofold increase of nitrate reductase protein under conditions of nitrogen starvation versus growth on nitrate. Nitrate reductase synthesis was repressed in darkness. However, when Monoraphidium was grown under heterotrophic conditions with glucose as the carbon and energy source, the synthesis of nitrate reductase was maintained. With ammonium or darkness, changes in nitrate reductase activity correlated fairly well with changes in nitrate reductase protein, indicating that in both cases loss of activity was due to repression and not to inactivation of the enzyme. Experiments using methionine sulfoximine, to inhibit ammonium assimilation, showed that ammonium per se and not a product of its metabolism was the corepressor of the enzyme. The appearance of nitrate reductase activity after transferring the cells to induction media was prevented by cycloheximide and by 6-methylpurine, although in this latter case the effect was observed only in cells preincubated with the inhibitor for 1 h before the induction period.     

Nitrate reduction in citrus tree leaves

Summary A nitrate-reducing enzyme system is active in citrus tree leaf fragments. The system is inactivated in disrupted and damaged cells of leaf macerate. The measurement of nitrite as an indication of nitrate reductase activity is some times misleading because of its rapid disappearance due to high nitrite reductase activity.Contribution from the National and University Institute of Agriculture, Rehovot, Israel. 1964 Series, No. 710-E.     

Regulation of the level of yeasts citrate synthase by oxygen availability

Summary The dark and light reduction of nitrate and nitrite by cell-free preparations of the blue-green algaAnacystis nidulans has been investigated. The three following methods have been successfully applied to the preparation of active particulate fractions from the alga cells: (a) shaking with glass beads, (b) lysozyme treatment and lysis of the resulting protoplasts, and (c) sonication. The two enzymes of the nitrate-reducing system-namely, nitrate reductase and nitrite reductase-are firmly bound to the isolated pigment-containing particles, and can be easily solubilized by prolonging the vibration or sonication time.Both enzymes-whether solubilized or bound to the particles-depend on reduced ferredoxin as the immediate electron donor. In its presence, the alga particles catalyze the gradual photoreduction of nitrate to nitrite and ammonia, a process that can thus be considered as one of the most simple and relevant examples of Photosynthesis. Some of the properties of nitrate reductase have been studied. Nitrate reductase as well as nitrite reductase are adaptive enzymes repressed by ammonia.An invited article.     

植物中硝酸还原酶和亚硝酸还原酶的作用

植物通过硝酸盐同化途径以硝酸盐和氨的形式吸收氮元素。硝酸盐的同化是一个受到严格控制的过程,其中两个先后参加反应的酶——硝酸还原酶(NR)和亚硝酸还原酶(NiR)对初级氮的同化起主要调控。在高等植物中,NR和NiR基因的转录及转录后加工受到各种内在和外在因素的影响,翻译后调控是消除亚硝酸盐积累的重要机制。随着分子生物学技术的发展,可以更容易地通过突变体和转基因方式来研究NR和NiR基因的调控。     

Chlorate resistant mutants of Pseudomonas stutzeri affected in respiratory and assimilatory nitrate utilization and expression of cytochrome cd1

Abstract Chlorate-resistant mutants were generated by random insertion of the transposon Tn5 into genomic DNA of Pseudomonas stutzeri ZoBell strain and selected for loss of nitrate respiration (Nar phenotype). The mutants were differentiated by restriction-fragment analysis, by assaying for nitrate assimilation and for molybdenum co-factor activity, and by the amount of respiratory nitrate reductase. Two mutants, lacking both nitrate respiration and nitrate assimilation, over-produced an inactive nitrate reductase but synthesized in the presence of nitrate only a reduced amount of respiratory nitrite reductase (cytochrome cd ₁). Expression of cytochrome cd ₁ in these mutants was specifically induced by nitrate, suggesting a sensor system for this substrate.