COMPARISONS OF NITRATE UPTAKE, STORAGE, AND REDUCTION IN MARINE DIATOMS AND FLAGELLATES

Diatoms, but not flagellates, have been shown to increase rates of nitrogen release after a shift from a low growth irradiance to a much higher experimental irradiance. We compared NO₃ ^? uptake kinetics, internal inorganic nitrogen storage, and the temperature dependence of the NO₃ ^? reduction enzymes, nitrate (NR) and nitrite reductase (NiR), in nitrogen‐replete cultures of 3 diatoms (Chaetoceros sp., Skeletonema costatum, Thalassiosira weissflogii) and 3 flagellates (Dunaliella tertiolecta, Pavlova lutheri, Prorocentrum minimum) to provide insight into the differences in nitrogen release patterns observed between these species. At NO₃ ^? concentrations <40 μmol‐N·L ^{? 1}, all the diatom species and the dinoflagellate P. minimum exhibited saturating kinetics, whereas the other flagellates, D. tertiolecta and P. lutheri, did not saturate, leading to very high estimated K _s values. Above ～60 μmol‐N·L ^{? 1}, NO₃ ^? uptake rates of all species tested continued to increase in a linear fashion. Rates of NO₃ ^? uptake at 40 μmol‐N·L ^{? 1}, normalized to cellular nitrogen, carbon, cell number, and surface area, were generally greater for diatoms than flagellates. Diatoms stored significant amounts of NO₃ ^? internally, whereas the flagellate species stored significant amounts of NH₄ ⁺ . Half‐saturation concentrations for NR and NiR were similar between all species, but diatoms had significantly lower temperature optima for NR and NiR than did the flagellates tested in most cases. Relative to calculated biosynthetic demands, diatoms were found to have greater NO₃ ^? uptake and NO₃ ^? reduction rates than flagellates. This enhanced capacity for NO₃ ^? uptake and reduction along with the lower optimum temperature for enzyme activity could explain differences in nitrogen release patterns between diatoms and flagellates after an increase in irradiance.     

Reduction, assimilation and transport of N in normal and gibberellin-deficient tomato plants

A fast-growing normal and a slow-growing gibberellin-deficient mutant of Lycopersicon esculentum (L.) Mill. cv. Moneymaker were used to test the hypothesis that slow-growing plants reduce NO₃^? in the root to a greater extent than do fast-growing plants. Plants that reduce NO₃^? in the root may grow more slowly due to the higher energetic and carbon costs associated with root-based NO₃^? reduction compared to photosynthetically driven shoot NO₃^? reduction. The plants were grown hydroponically with a complete nutrient solution containing 10 mM NO₃^? and the biomass production, gas exchange characteristics, root respiratory O₂ consumption, nitrate reductase activity and translocation of N in the xylem were measured. The gibberellin-deficient mutants accumulated more total N unit^?1 dry weight than did the faster-growing normal plants. There were no significant differences between the genotypes in the rates of photosynthesis expressed on a leaf dry weight basis. The plants differed in the proportion of photosynthetic carbon available to growth due to a greater proportion of daily photo-synthate production being consumed by respiration in the slow-growing genotype. This difference in allocation of carbon was associated with differences in the specific leaf area and specific root length. In addition, a greater leaf weight ratio in the fast-growing than in the slow-growing plants indicates a greater investment of carbon into biomass supporting photosynthetic production in the former. We did not find differences in the activity or distribution of nitrate reductase or in the N composition of the xylem sap between the genotypes. We thus conclude that the growth rate was determined by the efficiency of carbon partitioning and that the site of NO₃^? reduction and assimilation was not related to the growth rate of these plants.     

Effects of non-steady-state iron limitation on nitrogen assimilatory enzymes in the marine diatom thalassiosira weissflogii (BACILLARIOPHYCEAE)

Since the recognition of iron‐limited high nitrate (or nutrient) low chlorophyll (HNLC) regions of the ocean, low iron availability has been hypothesized to limit the assimilation of nitrate by diatoms. To determine the influence of non‐steady‐state iron availability on nitrogen assimilatory enzymes, cultures of Thalassiosira weissflogii (Grunow) Fryxell et Hasle were grown under iron‐limited and iron‐replete conditions using artificial seawater medium. Iron‐limited cultures suffered from decreased efficiency of PSII as indicated by the DCMU‐induced variable fluorescence signal (F_v/F_m). Under iron‐replete conditions, in vitro nitrate reductase (NR) activity was rate limiting to nitrogen assimilation and in vitro nitrite reductase (NiR) activity was 50‐fold higher. Under iron limitation, cultures excreted up to 100 fmol NO₂^?·cell^?1·d^?1 (about 10% of incorporated N) and NiR activities declined by 50‐fold while internal NO₂^? pools remained relatively constant. Activities of both NR and NiR remained in excess of nitrogen incorporation rates throughout iron‐limited growth. One possible explanation is that the supply of photosynthetically derived reductant to NiR may be responsible for the limitation of nitrogen assimilation at the NO₂^? reduction step. Urease activity showed no response to iron limitation. Carbon:nitrogen ratios were equivalent in both iron conditions, indicating that, relative to carbon, nitrogen was assimilated at similar rates whether iron was limiting growth or not. We hypothesize that, diatoms in HNLC regions are not deficient in their ability to assimilate nitrate when they are iron limited. Rather, it appears that diatoms are limited in their ability to process photons within the photosynthetic electron transport chain which results in nitrite reduction becoming the rate‐limiting step in nitrogenassimilation.     

NITRATE REDUCTASE MUTANTS IN EUDORINA ELEGANS (CHLOROPHYCEAE)1

Three nitrate reductase mutants were independently isolated and characterized in the colonial alga, Eudorina elegans Ehrenberg. nar-1 is a leaky mutant, deficient in the production of nitrate reductase. nar-2 and nar-3 both lack the ability to produce nitrate reductase. However, nar-2 grows and nar-3 does not grow when hypoxanthine is the sole nitrogen source. The specific activity of the next enzyme, in the pathway, nitrite reductase is increased in nar-3 when compared to wild-type, nar-1 and nar-2.     

Assimilatory nitrate uptake in Pseudomonas fluorescens studied using nitrogen-13

The mechanism of nitrate uptake for assimilation in procaryotes is not known. We used the radioactive isotope, ¹³N as NO₃ ^-, to study this process in a prevalent soil bacterium, Pseudomonas fluorescens. Cultures grown on ammonium sulfate or ammonium nitrate failed to take up labeled nitrate, indicating ammonium repressed synthesis of the assimilatory enzymes. Cultures grown on nitrite or under ammonium limitation had measurable nitrate reductase activity, indicating that the assimilatory enzymes need not be induced by nitrate. In cultures with an active nitrate reductase, the form of ¹³N internally was ammonium and amino acids; the amino acid labeling pattern indicated that ¹³NO₃ ^- was assimilated via glutamine synthetase and glutamate synthase. Cultures grown on tungstate to inactivate the reductase concentrated NO₃ ^- at least sixfold. Chlorate had no effect on nitrate transport or assimilation, nor on reduction in cell-free extracts. Ammonium inhibited nitrate uptake in cells with and without active nitrate reductases, but had no effect on cell-free nitrate reduction, indicating the site of inhibition was nitrate transport into the cytoplasm. Nitrate assimilation in cells grown on nitrate and nitrate uptake into cells grown with tungstate on nitrite both followed Michaelis-Menten kinetics with similar K _mvalues, 7 M. Both azide and cyanide inhibited nitrate assimilation. Our findings suggest that Pseudomonas fluorescens can take up nitrate via active transport and that nitrate assimilation is both inhibited and repressed by ammonium.     

Mechanism of nitrite reduction to nitrous oxide in a photodenitrifier,Rhodopseudomonas sphaeroide forma sp. denitrificans

The mechanism of anaerobic reduction of NO₂^? to N₂O in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans, was investigated. With ascorbate-reduced phenazine methosulfate (PMS) as the electron donor, the nitrite reductase of this photodenitrifier reduced NO₂^? to NO and a trace amount of N₂O. With dithionite-reduced benzyl viologen as the electron donor, the major product of NO₂^? reduction was NH₂OH, and a trace amount of N₂O was also produced. The nitrate reductase itself had no NO reductase activity with ascorbate-reduced PMS. It was concluded that the essential product of NO₂^? reduction by the purified nitrite reductase is NO. Chromatophore membranes stoichiometrically produced N₂O from NO₂^? with any electron donor, such as dithionite-redduced benzyl viologen, ascorbate-reduced PMS or NADH/FMN. The membranes also contrained activity of NO reduction of N₂O with either ascorbate-reduced PMS or duroquinol. The NO reductase activity with duroquinol was inhibited by antimycin A. Stoichiometric production of N₂O from N₂^? also was observed in the reconstituted NO₂^? reduction system which contained the cytochrome bc₁ complex, cytochrome c₂, the nitrite reductase and duroquinol as the electron donor. The preparation of the cytochrome bc₁ complex itself contianed NO reductase activity. From these results the mechanism of NO₂^? reduction to N₂O in this photodenitrifier was determined as the nitrite reductase reducing NO₂^? to NO with electrons from the cytochrome bc₁ complex, and NO subsequently being reduced, without release, to N₂O with electrons from the cytochrome bc₁ complex by the NO reductase, which is closely associated with the complex.     

Ammonium can stimulate nitrate and nitrite reductase in the absence of nitrate in Clematis vitalba

Nitrogen assimilation was studied in the deciduous, perennial climber Clematis vitalba. When solely supplied with NO₃^– in a hydroponic system, growth and N-assimilation characteristics were similar to those reported for a range of other species. When solely supplied with NH₄⁺, however, nitrate reductase (NR) activity dramatically increased in shoot tissue, and particularly leaf tissue, to up to three times the maximum level achieved in NO₃^– supplied plants. NO₃^– was not detected in plant material that had been solely supplied with NH₄⁺, there was no NO₃^– contamination of the hydroponic system, and the NH₄⁺-induced activity did not occur in tobacco or barley grown under similar conditions. Western Blot analysis revealed that the induction of NR activity, either by NO₃^– or NH₄⁺, was matched by NR and nitrite reductase protein synthesis, but this was not the case for the ammonium assimilation enzyme glutamine synthetase. Exposure of leaf disks to N revealed that NO₃^– assimilation was induced in leaves directly by NO₃^– and NH₄⁺ but not glutamine. Our results suggest that the NH₄⁺-induced potential for NO₃^– assimilation occurs when externally sourced NH₄⁺ is assimilated in the absence of any NO₃^– assimilation. These data show that the potential for nitrate assimilation in C. vitalba is induced by a nitrogenous compound in the absence of its substrate and suggest that NO₃^– assimilation in C. vitalba may have a significant role beyond the supply of reduced N for growth.     

Fate of nitrate during initial nitrate utilization by nitrogen-depleted dwarf bean

The fate of nitrate and nitrogen-15 was followed during the apparent induction phase (6h) for nitrate uptake by N-depleted dwarf bean (Phaseolus vulgaris L. ev. Witte Krombek). Experiments were done with intact plants and with detached root systems. Qualitatively and quantitatively, xylem exudation from detached roots was a bad estimate of the export of NO^?₃ or NO^?₃-¹⁵N from roots of intact plants. In vivo nitrate reductase activity (NRA) agreed well with in situ reduction, calculated as the difference between uptake and accumulation in whole plants, provided NRA was assayed with merely endogenous nitrate as substrate (‘actual’ NRA). The majority (75%) of the entering nitrate remained unmetabolized. Both nitrate reduction and nitrate accumulation occurred predominantly in the root system. Some (< 25%) of the root-reduced nitrate-N was translocated to the shoot. Nitrate uptake occurred against the concentration gradient between medium and root cells, and probably against the gradient of the electro-chemical potential of nitrate. Part of the energy expended for NO^?₃ absorption came from the tops, since decapitation and ringing at the stem base restricted nitrate uptake.     

THE INABILITY OF THE TEXAS “BROWN TIDE” ALGA TO USE NITRATE AND THE ROLE OF NITROGEN IN THE INITIATION OF A PERSISTENT BLOOM OF THIS ORGANISM1

A planktonic alga similar in general morphology and pigments to Aureococcus anophagefferens Hargraves and Sieburth has caused persistent and ecologically damaging blooms along the south Texas coast. Experiments using 100 μM NO₃^?, NO₂^?, and NH₄⁺ demonstrated that the alga could not use NO₃^? for growth but could use NO₂^? and NH₄⁺. Doubling iron or trace metal concentrations did not permit growth on NO₃^?. Chemical composition data for cultures grown in excess NO₃^? or NH₄⁺, respectively, were as follows: N·cell^?1 (0.88 vs. 1.3 pg), C:N ratio (25:1 vs. 6.4:1), C:chlorophyll a (chl a) (560:1 vs. 44:1), and chl a·cell^?1 (0.033 vs. 0.16 pg). These data imply that cells supplied with NO₃^? were N-starved. Culture addition of 10 mM final concentration chlorate (a nitrate analog) did not affect the Texas isolate while NO₃^? utilizing A. anophagefferens was lysed, suggesting that the NO₃^? reductase of the Texas isolate is nonfunctional. Rates of primary productivity determined during a dense bloom indicated that light-saturated growth rates were ca. 0.45 d^?1, which is similar to maximum rates determined in laboratory experiments (0.58 d^?1± 0.16). However, chemical composition data were consistent with the growth rate of these cells being limited by N availability (C:N 28, C:chl a 176, chl a·cell^?1 0.019). Calculations based on a mass balance for nitrogen suggest that the bloom was triggered by an input of ca. 69 μM NH₄⁺ that resulted from an extensive die-off of benthos and fish.     

Role of sugars in nitrate utilization by roots of dwarf bean

Nitrate uptake and in vivo, nitrate reductase activity (NRA) in roots of Phaseolus vulgaris, L. cv. Witte Krombek were measured in nitrogen-depleted plants of varying sugar status, Variation in sugar status was achieved at the start of nitrate nutrition by excision, ringing, darkness or administration of sugars to the root medium. The shape of the apparent induction pattern of nitrate uptake was not influenced by the sugar status of the absorbing tissue. When measured after 6 h of nitrate nutrition (0.1 mol m^?3), steady state nitrate uptake and root NRA were in the order intact>dark>ringed>excised. Exogenous sucrose restored NRA in excised roots to the level of intact plants. The nitrate uptake rate of excised roots, however, was not fully restored by sucrose (0.03–300 mol m^?3). When plants were decapitated after an 18 h NO₃^? pretreatment, the net uptake rate declined gradually to become negative after three hours. This decline was slowed down by exogenous fructose, whilst glucose rapidly (sometimes within 5 min) stimulated NG^?₃ uptake. Presumably due to a difference in NO₃^? due to a difference in NO₃^? uptake, the NRA of excised roots was also higher in the presence of glucose than in the presence of fructose after 6 h of nitrate nutrition. The sugar-stimulation of, oxygen consumption as well as the release of ¹⁴CO₂ from freshly absorbed (U-¹⁴C) sugar was the same for glucose and fructose. Therefore, we propose a glucose-specific effect on NO₃^? uptake that is due to the presence of glucose rather than to its utilization in root respiration. A differential glucose-fructose effect on nitrate reductase activity independent of the effect on NO₃^? uptake was not indicated. A constant level of NRA occurred in roots of NO₃^? induced plants. Removal of nutrient nitrate from these plants caused an exponential NRA decay with an approximate half-life of 12 h in intact plants and 5.5 h in excised roots. The latter value was also found in roots that were excised in the presence of nitrate, indicating that the sugar status primarily determines the apparent rate of nitrate reductase decay in excised roots.     

Nitrogen Fixation and Nitrogen Assimilation in a Temperate Saline Ecosystem

As nitrogen is known to be a limiting factor for plant growth, we were interested in the relationship between soil microbial activity and the nitrogen assimilation of 5 different halophytes from 4 saline sites near the lake “Neusiedlersee”, Austria. The following were studied between May and October 1985: nitrogen fixation (¹⁵N₂ and acetylene reduction): N-mineralization; several soil characteristics and in vivo nitrate reductase activity of roots and shoots of these plants. NO^?₃, org. N- and carboxylate contents of both roots and shoots, as well as the effect of NO^?₃-fertilization on the amounts of these substances, were determined on plants growing in the field during a 3-day period in September 1985. Fertilization led to a decrease in acetylene reduction activity at most sites, and an increase in the nitrate reductase activity of the shoots of all plants. Overall, carboxylate and organic nitrogen contents of these halophytes did not change in response to fertilization. Only in the roots of Aster tripolium and Atriplex hastata was there a marked increase in the nitrate reductase activity in response to fertilization. Species growing at the same site, such as Plantago maritima and Lepidium crassifolium showed contrasting levels of assimilatory activity. Apparent low rates of ammonification and nitrification were detected in soils from the 4 sites. The results are discussed in relation to the nitrogen and carbon economies of the microorganisms and plants.     

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     

Reduction of N-oxides and sulfoxide by the same terminal reductase inProteus mirabilis

Proteus mirabilis can grow anaerobically on the fermentable substrate, glucose. When the glucose medium was supplemented with an electron acceptor, growth doubled. However, the organism failed to grow anaerobically on the oxidizable substrate glycerol unless the medium was supplemented with an external electron acceptor. Dimethyl sulfoxide (DMSO), trimethylamine N-oxide (TMAO), nicotinamide N-oxide (NAMO), and nitrate (NO₃) can serve this function. Cell-free extracts ofP. mirabilis can reduce these compounds in the presence of various electron donors. In order to determine whether the same or different terminal reductase(s) are involved in the reduction of these compounds, we isolated mutants unable to grow on glycerol/DMSO medium. When these mutants were tested on glycerol medium containing TMAO, NAMO, and NO₃ as electron acceptors, it was found that there were two groups. Group I mutants were unable to grow with DMSO, TMAO, and NAMO, while their growth was unaffected with NO₃. Group II mutants were unable to grow on any electron acceptor including NO₃. Enzyme assays using reduced benzyl viologen with both groups of mutants were in agreement with growth studies. On the basis of these results, we conclude that the same terminal reductase is involved in the reduction of DMSO, TMAO, and NAMO (group I) and that the additional loss of NO₃ reductase in group II mutants is probably owing to a defect in the synthesis or insertion of molybdenum cofactor.     

The influence of enriched rhizosphere CO2 on N uptake and metabolism in wild-type and NR-deficient barley plants

Positive influences of high concentrations of dissolved inorganic carbon (DIC) in the growth medium of salinity-stressed plants are associated with carbon assimilation through phosphoenolpyruvate carboxylase (PEPc) activity in roots; and also in salinity-stressed tomato plants, enriched CO₂ in the rhizosphere increases NO^?₃uptake. In the present study, wild-type and nitrate reductase-deficient plants of barley (Hordeum vulgare L. cv. Steptoe) were used to determine whether the influence of enriched CO₂ on NO^?₃ uptake and metabolism is dependent on the activity of nitrate reductase (NR) in the plant. Plants grown in NH₄⁺and aerated with ambient air, were transferred to either NO₃^? or NH₄⁺ solutions and aerated with air containing between 0 and 6 500 μmol mol^?1 CO₂. Nitrogen uptake and tissue concentrations of NO₃^? and NH₄⁺ were measured as well as activities of NR and PEPc. The uptake of NO^?₃ by the wild-type was increased by increasing CO₂. This was associated with increased in vitro NR activity, but increased uptake of NO₃^? was found also in the NR-deficient genotype when exposed to high CO₂ concentrations; so that the influence of CO₂ on NO₃^? uptake was independent of the reduction of NO₃^? and assimilation into amino acids. The increase in uptake of NO₃^? in wild-type plants with enriched CO₂ was the same at pH 7 as at pH 5, indicating that the relative abundance of HCO₃^? or CO₂ in the medium did not influence NO₃^? uptake. Uptake of NH₄⁺ was decreased by enriched CO₂ in a pH (5 or 7) independent fashion. Thus NO₃^? and NH⁺₄ uptakes are influenced by the CO₂ component of DIC independently of anaplerotic carbon provision for amino acid synthesis, and CO₂ may directly affect the uptake of NO₃^? and NH₄⁺ in ways unrelated to the NR activity in the tissue.     

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.     

Aerobic nitrate and nitrite reduction in continuous cultures of Escherichia coli E4

Nitrate and nitrite was reduced by Escherichia coli E4 in a l-lactate (5 mM) limited culture in a chemostat operated at dissolved oxygen concentrations corresponding to 90–100% air saturation. Nitrate reductase and nitrite reductase activity was regulated by the growth rate, and oxygen and nitrate concentrations. At a low growth rate (0.11 h^–1) nitrate and nitrite reductase activities of 200 nmol · mg^–1 protein · min^–1 and 250 nmol · mg^–1 protein · min^–1 were measured, respectively. At a high growth rate (0.55 h^–1) both enzyme activities were considerably lower (25 and 12 nmol mg^–1 · protein · min^–1). The steady state nitrite concentration in the chemostat was controlled by the combined action of the nitrate and nitrite reductase. Both nitrate and nitrite reductase activity were inversely proportional to the growth rate. The nitrite reductase activity decreased faster with growth rate than the nitrate reductase. The chemostat biomass concentration of E. coli E4, with ammonium either solely or combined with nitrate as a source of nitrogen, remained constant throughout all growth rates and was not affected by nitrite concentrations. Contrary to batch, E. coli E4 was able to grow in continuous cultures on nitrate as the sole source of nitrogen. When cultivated with nitrate as the sole source of nitrogen the chemostat biomass concentration is related to the activity of nitrate and nitrite reductase and hence, inversely proportional to growth rate.     

Iodate Reduction by Shewanella oneidensis Does Not Involve Nitrate Reductase

Microbial iodate (IO₃^?) reduction is a major component of iodine biogeochemical cycling and is the basis of alternative strategies for remediation of iodine-contaminated environments. The molecular mechanism of microbial IO₃^? reduction, however, is not well understood. In several microorganisms displaying IO₃^? and nitrate (NO₃^?) reduction activities, NO₃^? reductase is postulated to reduce IO₃^? as alternate electron acceptor. In the present study, whole genome analyses of 25 NO₃^?-reducing Shewanella strains identified various combinations of genes encoding one assimilatory (cytoplasmic Nas) and three dissimilatory (membrane-associated Nar and periplasmic Napα and Napβ) NO₃^? reductases. Shewanella oneidensis was the only Shewanella strain whose genome encoded a single NO₃^? reductase (Napβ). Terminal electron acceptor competition experiments in S. oneidensis batch cultures amended with both NO₃^? and IO₃^? demonstrated that neither NO₃^? nor IO₃^? reduction activities were competitively inhibited by the presence of the competing electron acceptor. The lack of involvement of S. oneidensis Napβ in IO₃^? reduction was confirmed via phenotypic analysis of an in-frame gene deletion mutant lacking napβA (encoding the NO₃^?-reducing NapβA catalytic subunit). S. oneidensis ΔnapβA was unable to reduce NO₃^?, yet reduced IO₃^? at rates higher than the wild-type strain. Thus, NapβA is required for dissimilatory NO₃^? reduction by S. oneidensis, while neither the assimilatory (Nas) nor dissimilatory (Napα, Napβ, and Nar) NO₃^? reductases are required for IO₃^? reduction. These findings provide the first genetic evidence that IO₃^? reduction by S. oneidensis does not involve nitrate reductase and indicate that S. oneidensis reduces IO₃^? via an as yet undiscovered enzymatic mechanism.     

Nitrate assimilation in leaf blades of wheat of different age

A field experiment on wheat (Triticum aestivum L.) ev. Shera grown at 120 kg N ha^?1 was conducted. Half of the dose of fertilizer N was applied at the pre-sowing stage and the other half when the seedlings were one month old. The leaf blades were examined for their NO₃^? content and NO₃^? assimilatory activity at various stages of growth and development. Soil nitrate level at 50 cm depth was determined throughout the wheat growing season in terms of cencentration (μg/ml) and total amount (kg ha^?1). The upper leaf blades were examined for their capacity to assimilate NO₃^?. Highly significant correlation between NR (nitrate reductase) activity and NO₃^? content in the leaf blades. NR activity and soil NO₃^?, and between soil NO₃^? and leaf blade NO₃^? was observed. Findings on low soil NO₃^? status during the reproductive phase and the capacity of the upper leaf blades to assimilate additional amounts of NO₃^?, point to the need for developing a programme of soil fertilizer application whereby all the leaf blades can utilize the NO₃^? optimally and thus result in greater N harvest.     

Absorption of Atmospheric NO2 by Spruce (Picea abies) Trees. III. Interaction with Nitrate Reductase Activity in the Needles and Phloem Transport

In order to compare the effects of excess pedospheric and atmospheric nitrogen supply on nitrate reductase activity (NR. EC 1.6.6.1) excised spruce branches were exposed to nitrate solutions or were fumigated with NO₂. Immersion of spruce branches in 6 mM nitrate caused an increase in NR activity by a factor of 14 or 19 in current-year and in one-year-old needles, respectively, as compared to controls incubated in tap water. Exposure to 65 nl I^?1 NO₂ increased NR activity by a factor of 1.5 in current-year needles and by a factor of 2.5 in one-year-old needles as compared to non-fumigated controls. Addition of cycloheximide (0.17 μM) or puromycin (200 μM) to the incubation solution prevented the induction of NR activity from both nitrate and NO₂ exposure. This finding indicates that induction of NR activity by both atmospheric NO₂ or increased nitrate supply of the needles is both caused by de-novo synthesis of NR protein. The increase in NR activity in needles of branches still attached to the tree as a consequence of exposure to 65 nl I^?1 NO₂ was found to be a transient phenomenon. The increase persisted for several days only and was no longer observed after one week of sustained NO₂ exposure. An interruption of phloem transport by girdling, applied subsequent to the induction of NR activity by atmospheric NO₂, prevented the decrease in NR activity. Apparently, export out of the exposed needles and phloem transport within the stem are involved in the regulation of NR activity upon NO₂ exposure.