The effect of oxygen on nitrate and nitrite assimilation in leaves of Zea mays L. under dark conditions

The assimilation of nitrate under dark-N₂ and dark-O₂ conditions in Zea mays leaf tissue was investigated using colourimetric and ¹⁵N techniques for the determination of organic and inorganic nitrogen. Studies using ¹⁵N indicated that nitrate was assimilated under dark conditions. However, the rate of nitrate assimilation in the dark was only 28% of the rate under non-saturating light conditions. No nitrite accumulated under dark aerobiosis, even though nitrate reduction occurred under these conditions. The pattern of nitrite accumulation in leaf tissue in response to dark-N₂ conditions consisted of three phases: an initial lag phase, followed by a period of rapid nitrite accumulation and finally a phase during which the rate of nitrite accumulation declined. After a 1-h period of dark-anaerobiosis, both nitrate reduction and nitrite accumulation declined considerably. However, when O₂ was supplied, nitrate reduction was stimulated and the accumulated nitrite was rapidly reduced. Anaerobic conditions stimulated nitrate reduction in leaf tissue after a period of dark-aerobic pretreatment.     

Effect of aerobic and anaerobic conditions on the in vivo nitrate reductase assay in spinach leaves

¹⁵N-labelled nitrate was used to show that nitrate reduction by leaf discs in darkness was suppressed by oxygen, whereas nitrite present within the cell could be reduced under aerobic dark conditions. In other experiments, unlabelled nitrite, allowed to accumulate in the tissue during the dark anaerobic reduction of nitrate was shown by chemical analysis to be metabolised during a subsequent dark aerobic period. Leaves of intact plants resembled incubated leaf discs in accumulating nitrite under anaerobic conditions. Nitrate, n-propanol and several respiratory inhibitors or uncouplers partly reversed the inhibitory effect of oxygen on nitrate reduction in leaf discs in the dark. Of these nitrate and propanol acted synergistically. Reversal was usually associated with inhibition of respiration but some concentrations of 2,4-dinitrophenol (DNP) and ioxynil reversed inhibition without affecting respiratory rates. Respiratory inhibitors and uncouplers stimulated nitrate reduction in the anaerobic in vivo assay i.e. in conditions where the respiratory process is non-functional. Freezing and thawing leaf discs diminished but did not eliminate the sensitivity of nitrate reduction to oxygen inhibition.Abbreviations DNP 2,4-dinitrophenol - HOQNO 8-hydroxyquinoline-N-oxide - DCPIP 2,6-dichlorophenolindophenol - CCCP Carbonyl cyanide m-chlorophenylhydrazone - TES N-tris(hydroxymethyl)methyl-2-amino ethanesulphonic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Regulation of nitrate reduction in wheat leaves

Wheat leaves exposed to 710 nm monochromatic light, when only photosystem 1 operates, reduced small but significant amount of nitrate to nitrite. This could be due to partial inhibition of mitochondrial oxidation of NADH, brought about by cyclic photo-phosphorylation. Under dark aerobic conditions, citric acid cycle intermediates only slightly stimulated nitrate reduction. Under dark anaerobic conditions, when maximum reduction of nitrate occurred, the time course showed a 1:1 stoichiometry between nitrite and CO₂. It is suggested that for maximum reduction of nitrate under physiological conditions, CO₂ fixation and export of ATP via triose phosphate shuttle is essential.     

Anaerobic expression of nitric oxide reductase from denitrifying Pseudomonas stutzeri

NO reductase synthesis was investigated immunochemically and by activity assays in cells of Pseudomonas stutzeri ZoBell grown in continuous culture at discrete aeration levels, or in O₂-limited batch cultures supplemented with N oxides as respiratory substrate. Under aerobic conditions, NO reductase was not expressed in P. stutzeri. Oxygen limitation in combination with the presence of nitrate or nitrite derepressed NO reductase synthesis. On transition from aerobic to anaerobic conditions in continuous culture, NO reductase was synthesized below 3% air saturation and reached maximum expression under anaerobic conditions. By use of mutant strains defective in nitrate respiration or nitrite respiration, the inducing effect of individual N oxides on NO reductase synthesis could be discriminated. Nitrite caused definite, concentration-dependent induction, while nitrate promoted moderate enzyme synthesis or amplified effects of nitrite. Exogenous nitric oxide (NO) in concentrations 25 M induced trace amounts of NO reductase; in higher concentrations it arrested cell growth. Nitrite reductase or NO reductase were not detected immunochemically under these conditions. NO generated as an intermediate appeared not to induce NO reductase significantly. Antiserum raised against the P. stutzeri NO reductase showed crossreaction with cell extracts from P. stutzeri JM300, but not with several other denitrifying pseudomonads or Paracoccus denitrificans.     

Nitrogenase activity and nitrate respiration inAzospirillum spp.

The interaction between nitrate respiration and nitrogen fixation inAzospirillum lipoferum andA. brasilense was studied. All strains examined were capable of nitrogen fixation (acetylene reduction) under conditions of severe oxygen limitation in the presence of nitrate. A lag phase of about 1 h was observed for both nitrate reduction and nitrogenase activity corresponding to the period of induction of the dissimilatory nitrate reductase. Nitrogenase activity ceased when nitrate was exhausted suggesting that the reduction of nitrate to nitrite, rather than denitrification (the further reduction of nitrite to gas) is coupled to nitrogen fixation. The addition of nitrate to nitrate reductase negative mutants (nr^-) ofAzospirillum did not stimulate nitrogenase activity. Under oxygen-limited conditionsA. brasilense andA. lipoferum were also shown to reduce nitrate to ammonia, which accumulated in the medium. Both species, including strains ofA. brasilense which do not possess a dissimilatory nitrite reductase (nir^-) were also capable of reducing nitrous oxide to N₂.     

Some effects of nitrate abundance and starvation on metabolism and accumulation of nitrogen in barley (Hordeum vulgare L. cv Sonja)

Nitrate and nitrite reductases were both induced by adding three concentrations of nitrate to the nutrient supply of nitrate-starved barley seedlings. Enzyme induction was not proportional to the amount of nitrate introduced. Glutamine synthetase also increased above a high endogenous activity but the increase did not differ significantly between any of the three nitrate treatments. Nitrate accumulated rapidly in leaves of plants given 4.0 mM or 0.5 mM nitrate but not with 0.1 mM nitrate. In all treatments, amino acids in leaves increased for 2 d, chiefly attributable to glutamine, then declined. Transferring plants from the three nitrate treatments to nitrate-free nutrient produced an immediate decline in nitrate reductase but nitrite reductase continued to increase for 2 d, before declining. Glutamine-synthetase activity was not affected by withdrawal of nitrate, nor did nitrate withdrawal retard plant growth during the 9-d period of the experiment. The disparity between accumulated nitrate and nitrate-reducing capacity and the rapid decrease in leaf nitrate when nutrient nitrate supply was removed, indicated the presence of a nitrate-storage pool that could be called upon to maintain amino-acid production in times of nitrogen starvation.Abbreviations GS glutamine synthetase - NR nitrate reductase - NiR nitrite reductase     

Light and Dark Controls of Nitrate Reduction in Wheat (Triticum aestivum L.) Protoplasts

Protoplasts were isolated from the leaves of nitrate-cultured wheat (Triticum aestivum L. var. Frederick) seedlings. When incubated in the dark, protoplasts accumulated nitrite under anaerobic, but not under aerobic, conditions. The assimilation of [¹⁵N]nitrite by protoplasts was strictly light-dependent, and no loss of nitrite from the assay medium was observed under dark aerobic conditions. Therefore, the absence of nitrite accumulation under dark aerobic conditions was the result of an O₂ inhibition of nitrate reduction and not a stimulation of nitrite reduction. In the presence of antimycin A, protoplasts accumulated nitrite under dark aerobic conditions. The oxygen inhibition of nitrate reduction was apparently due to a competition between nitrate reduction and dark respiration for cytoplasmic-reducing equivalents.     

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.     

Utilization of nitrate byBeggiatoa alba

Beggiatoa alba B18LD utilizes both nitrate and nitrite as sole nitrogen sources, although nitrite was toxic above 1 mM.B. alba coupledin vivo acetate oxidation, but not sulfide oxidation, with nitrate and nitrite reduction.B. alba could not, however, grow anaerobically with nitrate as the sole electron acceptor. Furthermore, the incorporation of acetate into macromolecules under anaerobic conditions with nitrate as the sole electron acceptor was less 10% of the incorporation with oxygen as the electron acceptor. The product of nitrate reduction byB. alba was ammonia; N₂ or N₂O were not produced. The nitrate reductase activity inB. alba was soluble and it utilized reduced flavins or methyl viologen and dithionite as electron donors. Pyrimidine nucleotides were not used as in vitro electron donors, either alone or with flavins in coupled assays. TheB. alba nitrate reductase activity was competitively inhibited with chlorate and was only mildly inhibited by azide and cyanide. Nitrate was not required for induction of theB. alba nitrate reductase, and neither oxygen nor ammonia repressed its activity. Thus,B. alba nitrate reductase appears to be an assimilatory nitrate reductase with unusual regulatory properties.Non-standard abbreviations MV Methyl viologen - DT dithionite - GS glutamine synthetase - GOGAT glutamine 2-oxoglutarate aminotransferase - PPO 2-diphenyloxazole - POPOP 1,4-(bis)-[2-(5-phenyloxazolyl)] benzene - TCA trichloroacetic acid - CCCP carbonylcyanidem-chlorophenylhydrazone - FCCP carbonylcyanidep-trifluoromethoxyphenylhydrazone - TTFA thenoyltrifluoroacetone - PHEN 1,10-phenanthroline - HOQNO 2-heptyl 4-hydroxyquinoline-n-oxide - 8HQ 8-hydroxyquinoline     

Light and dark assimilation of nitrate in plants

Abstract. Heterotrophic assimilation of nitrate in roots and leaves in darkness is closely linked with the oxidative pentose phosphate pathway. The supply of glucose-6-phosphate to roots and chloroplasts in leaves in darkness is essential for assimilation of nitrite into amino acids. When green leaves are exposed to light, the key enzyme, glucoses-phosphate dehydrogenase, is inhibited by reduction with thioredoxin. Hence the dark nitrate assimilatory pathway is inhibited under photoautotrophic conditions and replaced by regulatory reactions functioning in light. On account of direct photo-synthetic reduction of nitrite in chloroplasts and availability of excess NADH for nitrate reduclase, the rate of nitrate assimilation is extremely rapid in light. Under dark anaerobic conditions also nitrate is equally rapidly reduced to nitrite on account of abolition of competition for NADH between nitrate reductase and mitochondrial oxidation.     

Heterotrophic nitrification and aerobic denitrification inAlcaligenes faecalis strain TUD

Heterotrophic nitrification and aerobic and anaerobic denitrification byAlcaligenes faecalis strain TUD were studied in continuous cultures under various environmental conditions. Both nitrification and denitrification activities increased with the dilution rate. At dissolved oxygen concentrations above 46% air saturation, hydroxylamine, nitrite and nitrate accumulated, indicating that both the nitrification and denitrification were less efficient. The overall nitrification activity was, however, essentially unaffected by the oxygen concentration. The nitrification rate increased with increasing ammonia concentration, but was lower in the presence of nitrate or nitrite. When present, hydroxylamine, was nitrified preferentially. Relatively low concentrations of acetate caused substrate inhibition (K_I=109 M acetate). Denitrifying or assimilatory nitrate reductases were not detected, and the copper nitrite reductase, rather than cytochrome cd, was present. Thiosulphate (a potential inhibitor of heterotrophic nitrification) was oxidized byA. faecalis strain TUD, with a maximum oxygen uptake rate of 140–170nmol O₂·min^-1·mg prot^-1. Comparison of the behaviour ofA. faecalis TUD with that of other bacteria capable of heterotrophic nitrification and aerobic denitrification established that the response of these organisms to environmental parameters is not uniform. Similarities were found in their responses to dissolved oxygen concentrations, growth rate and ammonia concentration. However, they differed in their responses to externally supplied nitrite and nitrate.     

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 g· ml^-1) or chloramphenicol (50 g·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.Abbreviations NR nitrate reductase - NiR nitrite reductase - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Root contribution to nitrate reduction in barley seedlings (Hordeum vulgare L.)

Summary The leaf and root nitrate reductase activities were measured in 7 day-old barley seedlings by anoxic nitrite accumulation in darkness, during 48h after the transfer from a N-starved medium to a 1.5 mM K¹⁵NO₃ medium. Thisin situ nitrate reduction was compared with the¹⁵N incorporation in the reduced N fraction of the whole seedlings.The nitrate reduction integrated fromin situ measurements was lower than the reduced¹⁵N accumulation. The rootin situ nitrate reductase activity seemed to account for only the third of the real root nitrate reduction, which may have been responsible for the overall underestimation. This discrepancy was partly explained by the ability of the root to reduce nitrite in an anoxic environment.These results suggest that, after correction of thein situ estimation of the nitrate reduction. the roots contribute to about 50% of the total assimilation.     

NarK enhances nitrate uptake and nitrite excretion in Escherichia coli.

narK mutants of Escherichia coli produce wild-type levels of nitrate reductase but, unlike the wild-type strain, do not accumulate nitrite when grown anaerobically on a glucose-nitrate medium. Comparison of the rates of nitrate and nitrite metabolism in cultures growing anaerobically on glucose-nitrate medium revealed that a narK mutant reduced nitrate at a rate only slightly slower than that in the NarK+ parental strain. Although the specific activities of nitrate reductase and nitrite reductase were similar in the two strains, the parental strain accumulated nitrite in the medium in almost stoichiometric amounts before it was further reduced, while the narK mutant did not accumulate nitrite in the medium but apparently reduced it as rapidly as it was formed. Under conditions in which nitrite reductase was not produced, the narK mutant excreted the nitrite formed from nitrate into the medium; however, the rate of reduction of nitrate to nitrite was significantly slower than that of the parental strain or that which occurred when nitrite reductase was present. These results demonstrate that E. coli is capable of taking up nitrate and excreting nitrite in the absence of a functional NarK protein; however, in growing cells, a functional NarK promotes a more rapid rate of anaerobic nitrate reduction and the continuous excretion of the nitrite formed. Based on the kinetics of nitrate reduction and of nitrite reduction and excretion in growing cultures and in washed cell suspensions, it is proposed that the narK gene encodes a nitrate/nitrite antiporter which facilitates anaerobic nitrate respiration by coupling the excretion of nitrite to nitrate uptake. The failure of nitrate to suppress the reduction of trimethylamine N-oxide in narK mutants was not due to a change in the level of trimethylamine N-oxide reductase but apparently resulted from a relative decrease in the rate of anaerobic nitrate reduction caused by the loss of the antiporter system.     

Main centers of nitrate and nitrite reduction in young and nodulated yellow lupine (<Emphasis Type="Italic">Lupinus luteus</Emphasis>)

Nitrate and nitrite reduction centers in non-nodulated and symbiotic yellow lupine were analyzed. In young seedlings, nitrate was exclusively accumulated in roots, which also was shown as the main nitrate reduction center. In contrast, leaves were shown to play a key role in nitrite reduction. A similar distribution of nitrate reductase (NR) and nitrite reductase was found in nodulated plants. However, in field conditions characterized by low nitrate content, a disproportionately high level of NR activity in nodules was also observed during all stages of symbiotic growth. This feature was confirmed in nitrate-fed hydroponic cultures. Nodule NR activity was one order of magnitude higher than in roots, in spite of the small stored nitrate pool found inside nodules. This suggests that nodule NR activity had been induced not by nitrate itself but indirectly. Since bacteroids were shown to be responsible for the vast majority of nodule NR activity, the plausible explanation of this effect seems to be a dissimilatory nature of rhizobial NR. Considering that environmental nitrate could cause hypoxia inside nodules, this is the proposed way of the observed nodule NR induction.     

Selection and organisation of denitrifying electron-transfer pathways in Paracoccus denitrificans

(1) Under anaerobic conditions the respiratory chain in cells of Paracoccus denitrificans, from late exponential cultures grown anaerobically with nitrate as electron acceptor and succinate as carbon source, has been shown to reduce added nitrate via nitrite and nitrous oxide to nitrogen without any accumulation of these intermediates. (2) Addition of nitrous oxide to cells reducing nitrate strongly inhibited the latter reaction. The inhibition was reversed by preventing electron flow to nitrous oxide with either antimycin or acetylene. Electron flow to nitrous oxide thus resembles electron flow to oxygen in its inhibitory effect on nitrate reduction. In contrast, addition of nitrite to an anaerobic suspension of cells reducing nitrate resulted in a stimulation of nitrate reductase activity. Usually, addition of nitrite also partially overcame the inhibitory effect of nitrous oxide on nitrate reduction. The reason why added nitrous oxide, but not nitrite, inhibits nitrate reduction is suggested to be related to the higher reductase activity of the cells for nitrous oxide compared with nitrite. Explanations for the unexpected stimulation of nitrate reduction by nitrite in the presence or absence of added nitrous oxide are considered. (3) Nitrous oxide reductase was shown to be a periplasmic protein that competed with nitrite reductase for electrons from reduced cytochrome c. Added nitrous oxide strongly inhibited the reduction of added nitrite. (4) Nitrite reductase activity of cells was strongly inhibited by oxygen in the presence of physiological reductants, but nitrite reduction did occur in the presence of oxygen when isoascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine was the reductant. It is concluded that competition for available electrons by two oxidases, cytochrome aa₃ and cytochrome o, severely restricted electron flow to the nitrite reductase (cytochrome cd). For this reason it is unlikely that the oxidase activity of this cytochrome is ever functional in cells. (5) The mechanism by which electron flow to oxygen or nitrous oxide inhibits nitrate reduction in cells has been investigated. It is argued that relatively small changes in the extent of reduction of ubiquinone, or of another component of the respiratory chain with similar redox potential, critically determine the capacity for reducing nitrate. The argument is based on: (i) the response of an anthroyloxystearic acid fluorescent probe that is sensitive to changes in the oxidation state of ubiquinone; (ii) consideration of the total rates of electron flow through ubiquinone both in the presence of oxygen and in the presence of nitrate under anaerobic conditions; (iii) use of relative extents of oxidation of b-type cytochromes as an indicator of ubiquinone redox state, especially the finding that b-type cytochrome of the antimycin-sensitive part of the respiratory chain is more oxidised in the presence of added nitrous oxide, which inhibits nitrate reduction, than in the presence of added nitrite which does not inhibit. Arguments against b- or c-type cytochromes themselves controlling nitrate reduction are given. (6) In principle, control on nitrate reduction could be exerted either upon electron flow or upon the movement of nitrate to the active site of its reductase. The observations that inverted membrane vesicles and detergent-treated cells reduced nitrate and oxygen simultaneously at a range of total rates of electron flow are taken to support the latter mechanism. The failure of an additional reductant, durohydroquinone, to activate nitrate reduction under aerobic conditions in the presence of succinate is also evidence that it is not an inadequate supply of electrons that prevents the functioning of nitrate reductase under aerobic conditions. (7) In inverted membrane vesicles the division of electron flow between nitrate and oxygen is determined by a competition mechanism, in contrast to cells. This change in behaviour upon converting cells to vesicles cannot be attributed to loss of cytochrome c, and therefore of oxidase activity, from the vesicles because a similar change in behaviour was seen with vesicles prepared from cells of a cytochrome c-deficient mutant.     

Regulation of nitrate and nitrite reduction in barley leaves

Reduction of nitrate and accumulation of nitrite were studied in barley (Hordeum vulgare L. cv. Gars Clipper ex Napier) leaf sections in the dark and in the light, under aerobic (air and mixtures of O₂ and N₂) or anaerobic (N₂) conditions. Oxygen prevented nitrite accumulation but had no effect on accumulated or infiltrated nitrite. Most of the nitrite accumulated under dark-anaerobic conditions was in the "cytoplasmic" (the cell section between the plasma lemma and the tonoplast) fraction of the tissue. Reduction of nitrate was stimulated by 2, 4-dinitrophenol in tissue under dark-air and by 3-(3', 4'-dichlorophenyl)-l, l-dimethyl urea (DCMU) and carbonyl cyanide m -chlorophenylhydrazone (CCCP) in tissue under all environmental conditions studied. Nitrite accumulated in the light in DCMU-treated tissue under N₂ or under aerobic conditions in the presence of CCCP. On its own, CCCP did not promote accumulation of nitrite in leaf sections under light-air. A model for the reduction of nitrate and nitrite is proposed.     

nir1, a conditional-lethal mutation in barley causing a defect in nitrite reduction

Summary Eleven green individuals were isolated when 95000 M₂ plants of barley (Hordeum vulgare L.), mutagenised with azide in the M₁, were screened for nitrite accumulation in their leaves after nitrate treatment in the light. The selected plants were maintained in aerated liquid culture solution containing glutamine as sole nitrogen source. Not all plants survived to flowering and some others that did were not fertile. One of the selected plants, STA3999, from the cultivar Tweed could be crossed to the wild-type cultivar and analysis of the F₂ progeny showed that leaf nitrite accumulation was due to a recessive mutation in a single nuclear gene, which has been designated Nir1. The homozygous nir1 mutant could be maintained to flowering in liquid culture with either glutamine or ammonium as sole nitrogen source, but died within 14 days after transfer to compost. The nitrite reductase cross-reacting material seen in nitrate-treated wild-type plants could not be detected in either the leaf or the root of the homozygous nir1 mutant. Nitrite reductase activity, measured with dithionite-reduced methyl viologen as electron donor, of the nitrate-treated homozygous nir1 mutant was much reduced but NADH-nitrate reductase activity was elevated compared to wild-type plants. We conclude that the Nir1 locus determines the formation of nitrite reductase apoprotein in both the leaf and root of barley and speculate that it represents either the nitrite reductase apoprotein gene locus or, less likely, a regulatory locus whose product is required for the synthesis of nitrite reductase, but not nitrate reductase. Elevation of NADH-nitrate reductase activity in the nir1 mutant suggests a regulatory perturbation in the expression of the Narl gene.     

Subcellular and immunocytochemical localization of the enzymes involved in ammonia assimilation in mesophyll and bundle-sheath cells of maize leaves

The cellular localization of the enzymes involved in primary nitrogen assimilation was investigated following separation of mesophyll protoplasts and bundle-sheath cells of maize (Zea mays L.) leaves. Determination of the enzymatic activities in the two types of cell revealed that nitrate and nitrite reductase are principally located in the mesophyll cells whereas glutamine synthetase (GS) and ferredoxin-dependent glutamate synthase (Fd-GOGAT) are present in both tissues with a preferential location in the bundle-sheath strands. In order to confirm the results obtained by this conventional biochemical method we have used an in-situ immunofluorescence technique to unambiguously localize GS and Fd-GOGAT at the cellular level. Thin-sectioned maize leaves treated with specific GS and Fd-GOGAT antisera followed by conjugation with fluorescein-isothiocyanate-labelled sheep anti-rabbit immunoglobulins clearly show that GS is equally distributed within the leaf whereas Fd-GOGAT is mostly present in the chloroplasts of the bundle-sheath cells. The cellular localization of nitrate reductase, nitrite reductase, GS-2 and Fd-GOGAT in maize leaf cell types strongly indicates that primary nitrogen assimilation functions in the mesophyll cells while photorespiratory nitrogen recycling is restricted to the bundle-sheath cells.     

Anaerobic energy metabolism of the sulfur-reducing bacterium “Spirillum” 5175 during dissimilatory nitrate reduction to ammonia

In a batch culture experiment the microaerophilic Campylobacter-like bacterium “Spirillum” 5175 derived its energy for growth from the reduction of nitrate to nitrite and nitrite to ammonia. Hereby, formate served as electron donor, acetate as carbon source, and l-cysteine as sulfur source. Nitrite was quantitatively accumulated in the medium during the reduction of nitrate; reduction of nitrite began only after nitrate was exhausted from the medium. The molar growth yield per mol formate consumed, Y_m, was 2.4g/mol for the reduction of nitrate to nitrite and 2.0 g/mol for the conversion of nitrite to ammonia. The gain of ATP per mol of oxidized formate was 20% higher for the reduction of nitrate to nitrite, compared to the reduction of nitrite to ammonia. With succinate as carbon source and nitrite as electron acceptor, Y_m was 3.2g/mol formate, i.e. 60% higher than with acetate as carbon source. No significant amount of nitrous oxide or dinitrogen was produced during growth with nitrate or nitrite both in the presence or absence of acetylene. No growth on nitrous oxide was found. The hexaheme c nitrite reductase of “Spirillum” 5175 was an inducible enzyme. It was present in cells cultivated with nitrate or nitrite as electron acceptor. It was absent in cells grown with fumarate, but appeared in high concentration in “Spirillum” 5175 grown on elemental sulfur. Furthermore, the dissimilatory enzymes nitrate reductase and hexaheme c nitrite reductase were localized in the periplasmic part of the cytoplasmic membrane.