Light dependent reduction of nitrate by pea chloroplasts in the presence of nitrate reductase and C4-dicarboxylic acids

In the presence of purified nitrate reductase (NR) and 1 mM NADH, illuminated pea chloroplasts catalysed reduction of NO₃^? to NH₃ with the concomitant evolution of O₂. The rates were slightly less than those for reduction of NO₂^? to NH₃ and O₂, evolution by chloroplasts in the absence of NR and NADH (ca 6 μg atoms N/mg Chl/hr). Illuminated chloroplasts quantitatively reduced 0.2 mM oxaloacetate (OAA) to malate. In the presence of an extrachloroplast malate-oxidizing system comprised of NAD-specific malate dehydrogenase (NAD-MDH), NAD, NR and NO₃^?, illuminated chloroplasts supported OAA-dependent reduction of NO₃^? to NH₃ with the evolution of O₂. The reaction did not proceed in the absence of any of these supplements or in the dark but malate could replace OAA. The results are consistent with the reduction of NO₃^?by reducing equivalents from H₂O involving a malate/OAA shuttle. The ratios for O₂, evolved: C₄-acid supplied and N reduced: C₄-acid supplied in certain experiments imply recycling of the C₄-acids.     

Denitrification and Assimilatory Nitrate Reduction in Aquaspirillum magnetotacticum

Aquaspirillum magnetotacticum MS-1 grew microaerobically but not anaerobically with NO₃⁻ or NH₄⁺ as the sole nitrogen source. Nevertheless, cell yields varied directly with NO₃⁻ concentration under microaerobic conditions. Products of NO₃⁻ reduction included NH₄⁺, N₂O, NO, and N₂. NO₂⁻ and NH₂OH, each toxic to cells at 0.2 mM, were not detected as products of cells growing on NO₃⁻. NO₃⁻ reduction to NH₄⁺ was completely repressed by the addition of 2 mM NH₄⁺ to the growth medium, whereas NO₃⁻ reduction to N₂O or to N₂ was not. C₂H₂ completely inhibited N₂O reduction to N₂ by growing cells. These results indicate that A. magnetotacticum is a microaerophilic denitrifier that is versatile in its nitrogen metabolism, concomitantly reducing NO₃⁻ by assimilatory and dissimilatory means. This bacterium appears to be the first described denitrifier with an absolute requirement for O₂. The process of NO₃⁻ reduction appears well adapted for avoiding accumulation of several nitrogenous intermediates that are toxic to cells.     

Denitrification, Acetylene Reduction, and Methane Metabolism in Lake Sediment Exposed to Acetylene

Samples of sediment from Lake St. George, Ontario, Canada, were incubated in the laboratory under an initially aerobic gas phase and under anaerobic conditions. In the absence of added nitrate (NO₃⁻) there was O₂-dependent production of nitrous oxide (N₂O), which was inhibited by acetylene (C₂H₂) and by nitrapyrin, suggesting that coupled nitrification-denitrification was responsible. Denitrification of added NO₃⁻ was almost as rapid under an aerobic gas phase as under anaerobic conditions. The N₂O that accumulated persisted in the presence of 0.4 atm of C₂H₂, but was gradually reduced by some sediment samples at lower C₂H₂ concentrations. Low rates of C₂H₂ reduction were observed in the dark, were maximal at 0.2 atm of C₂H₂, and were decreased in the presence of O₂, NO₃⁻, or both. High rates of light-dependent C₂H₂ reduction occurred under anaerobic conditions. Predictably, methane (CH₄) production, which occurred only under anaerobiosis, was delayed by added NO₃⁻ and inhibited by C₂H₂. Consumption of added CH₄ occurred only under aerobic conditions and was inhibited by C₂H₂.     

Growth, denitrification and nitrate ammonification of the rhizobial strain TNAU 14 in presence and absence of C2H4 and C2H2

Soil-N (NO₃ ^?) initiates as far as a threshold concentration is surpassed manifold physiological reactions on N₂-fixation. Organic N and ammonium oxidised to NO₃ ^? means oxygen depletion. Plants suffering under O₂ or infection stress start to excrete ethylene (C₂H₄). C₂H₄ widens the root intercellulars that O₂-respiration will continue. Now microbes may more easily enter the plant interior by transforming the reached methionine into C₂H₄. Surplus nitrate and C₂H₄ inhibit nodulation of leguminous plants. Excess NO₃ ^? in the nodulesphere could be diminished by N₂-fixing bacteria which in addition can denitrify or ammonify nitrate. Consequently, it was asked whether C₂H₄ interferes with the potential of N₂-fixing bacteria to reduce nitrate. The groundnut-nodule isolate TNAU 14, from which it was known that it denitrifies and ammonifies nitrate, served as inoculum of a KNO₃-mannitol-medium that was incubated under N₂-, 1% (v/v) N₂?C₂H₄-, and 1% (v/v) N₂?C₂H₂-atmosphere in the laboratory. C₂H₂ was included into the experiments because it is frequently used to quantify N₂-fixing potentials (acetylene reduction array, ARA). Gene-16S rDNA-sequencing and physiological tests revealed a high affiliation of strain TNAU 14 toRhizobium radiobacter andRhizobium tumefaciens. Strain TNAU 14 released N₂O into the bottle headspace in all treatments, surprisingly significantly less in presence of C₂H₂. Nitrate-ammonification was even completely blocked by C₂H₂. C₂H₄, in contrast rather stimulated growth, denitrification, and nitrate-ammonification of strain TNAU 14 which consumed the released NH₄ ⁺ during continuing incubation.     

Hymenolepis diminuta: Chloride fluxes and membrane potentials associated with sodium-coupled glucose transport

Glucose transport by Hymenolepis diminuta was inhibited when Cl^? in the bathing medium was replaced with acetate (C₂H₃O₂^Post?), but was unaffected when Cl^? was replaced with SCN^?. The relative effectiveness of the anions to inhibit influx of 7.4 mM Cl^? in the presence of 1 mM glucose was SCN^? > Cl^? > C₂H₃O₂^Post?. Glucose stimulated the influxes of 120 mM Cl^? and SCN^?, but had little effect on 120 mM C₂H₃O₂^Post? influx. While the diffusion rates of the anions were C₂H₃O₂^Post? > SCN^? = Cl^?, the preference of the glucose transport system for the anions was SCN^? > Cl^? > C₂H₃O₂^Post?. Efflux of Cl^? was not affected by the rate of glucose influx. Finally, microelectrode recordings of worms anesthetized with 2 mM arecoline revealed a transmembrane potential (TMP) of ?45 ± 3.6 mV (inside negative). Three to four minutes after addition of glucose (5 mM) there was a progressive hyperpolarization of the TMP to ?58 mV. A revised model of the glucose transport system that is consistent with previous observations on this organism is proposed.     

Energy-coupling mechanisms under aerobic and anaerobic conditions in autotrophically grown Pseudomonas saccharophila

The cell-free preparations from autotrophieally grown Pseudomonas saccharophila catalyzed the process of electron transport from H₂ or various other organic electron donors to either O₂ or NO₃^? with concomitant ATP generation. The respective $\text{P}\text{O}$ ratios with H₂ and NADH were 0.63 and 0.73, the respective $\text{P}\text{NO}{}_3{}^{\mathrm{?}}$ ratios were 0.57 and 0.54. In contrast, the $\text{P}\text{O}$ and $\text{P}\text{NO}{}_3{}^{\mathrm{?}}$ ratios with succinate were 0.18 and 0.11, respectively. ATP formation coupled to the oxidation of ascorbate, in the absence or presence of added N,N,N′,N′-tetramethyl-p-phenylenediamine or cytochrome c, could not be detected. Various uncouplers inhibited phosphorylation with either O₂ or NO₃^? as terminal electron acceptors without affecting the oxidation of H₂ or other substrates. The NADH oxidation at the expense of O₂ or NO₃^? reduction as well as the associated phosphorylation were inhibited by rotenone and amytal. The aerobic and anaerobic H₂ oxidation and coupled ATP synthesis, on the other hand, was unaffected by the flavoprotein inhibitors as well as by the NADH trapping system. The NADH, H₂, and succinate-linked electron transport to O₂ or NO₃^? and the associated phosphorylations were sensitive, however, to antimycin A or 2-n-nonyl-4-hydroxyquino-line-N-oxide, and cyanide or azide. The data indicated that although the phosphorylation sites 1 and II were associated with NADH oxidation by O₂ or NO₃^?, the energy conservation coupled to H₂ oxidation under aerobic or anaerobic conditions appeared to involve site II only.     

Root and nodule respiration in relation to acetylene reduction in intact nodulated peas

Inoculated pea plants (Pisum sativum L.) were grown with N-free nutrients in a controlled environment room and rates of respiratory CO₂ evolution and C₂H₂ reduction by the intact nodulated roots were determined. Experiments followed changes related to diurnal cycles, light and dark treatments, partial defoliation, aging of plants and NH₄NO₃ addition. In all experiments, changes in C₂H₂ reduction were associated with parallel changes in the respiration rate, although in all but the defoliation experiment there was a basal level of respiration which was independent of the rate of C₂H₂ reduction. In conditions which affected growth or plant size as well as C₂H₂ reduction, respiration changed by an average of 0.42 mg CO₂ (μmol C₂H₂ reduced)⁻¹. However, some treatments decreased C₂H₂ reduction without greatly changing the growth and in these conditions respiration was decreased by an average of 0.27 mg CO₂ (μmol C₂H₂ reduced)⁻¹. While this value may also include some respiration associated with other processes, it is proposed that it more closely estimates respiration directly associated with energy utilization for acetylene reduction; whereas the higher value includes respiration related to maintenance and growth processes as well.     

Dissimilatory nitrate uptake in Paracoccus denitrificans via a Δ\̃gmH+-dependent system and a nitrate-nitrite antiport system

Respiration-driven proton translocation has been studied with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing H₂ during reduction of O₂, NO^?₃, NO^?₂ or N₂O. A simplified scheme of anaerobic electron transport and associated proton translocation is shown that is consistent with the measured $\text{H}{}^{\mathrm{+}}\text{oxidant ratios}$. Furthermore, the kinetics and energetics of NO^?₃ uptake in whole cells of P. denitrificans were studied. For this purpose, we measured H₂ consumption or N₂O production after addition of NO^?₃ to a cell suspension, which indirectly gave information about uptake (and reduction) of NO^?₃. It was found that a lag phase in H₂ consumption or N₂O production appeared whenever the membrane potential was dissipated by addition of thiocyanate, carbonyl cyanide m-chlorophenylhydrazone or triphenyl-methylphosphonium bromide. However, these lag phases were not observed when NO^?₂ was present at the moment of introduction of NO^?₃. On the basis of these findings we conclude that there are two uptake systems for NO^?₃. One system is dependent on the proton-motive force and is probably used for initiation of NO^?₃ uptake. The other is an $\text{NO}{}^{\mathrm{?}}{}_3\text{NO}{}^{\mathrm{?}}{}_2$ antiport and its function is to take over NO^?₃ uptake from the first system.     

Effect of iron and phagocytosis on murine macrophage activation in vitro

Iron-exposed murine macrophages have a modified bactericidal activity as shown by previous observations. In order to assess the role of iron in macrophage activation, as measured by free radical production and by intracellular bacterial killing, murine peritoneal macrophages were cultivated in the presence of various sources of iron, human iron-saturated transferrin and ammonium ferric citrate, or iron chelators, Desferal, and human Apo-transferrin, and were infected with an enteropathogenic strain ofE. coli. The release of nitrite (NO₂ ^?), and the production of superoxide anion (O₂ ^?) and hydrogen peroxide (H₂O₂) by the phagocytes were measured and compared to the production by uninfected macrophages. The synergistic action with murine r.IFN-γ was also studied in the radical production reaction and for the bactericidal activity of macrophages. Our results show that in vitro phagocytosis ofE. coli induced elevated production of NO₂ ^? and H₂O₂ by macrophages, and that oxygen derivatives were released independently of the presence of added iron or chelator. Despite a phagocytosis-related enhancement of NO₂ ^? release, reactive nitrogen intermediates (RNI) are not directly involved in the bactericidal mechanism, as revealed by increased intracellular killing owing to RNI inhibitors. Moreover, bacterial killing may depend on oxygen derivatives, as suggested by the effect of the antioxidant sodium ascorbate leading to both a diminished H₂O₂ production and a decreased bactericidal activity of macrophages.     

Respiration and the energy requirement for nitrogen fixation in nodulated pea roots

Pisum sativum L. cv. Trapper plants were inoculated and grown in a controlled environment on N-free nutrient solution. After 4 weeks N was supplied to treatment plants as NH₄NO₃, KNO₃, or NH₄Cl and rates of C₂H₂ reduction, root + nodule respiration, and leaf photosynthesis were determined 1 week later. The increase in respiration per unit of C₂H₂ reduction was not affected by either the form of N added or the light conditions during growth, although the basal respiration rate with no C₂H₂ reduction increased with irradiance level. The mean regression coefficient from plots of respiration versus C₂H₂ reduction was 0.23 + 0.04 (P [unk] .01) mg of CO₂ (μmol of C₂H₂ reduced)⁻¹ which was very similar to the value for the coefficient of respiration associated with nitrogenase activity estimated by subtracting growth and maintenance respiration. Since the rate of N accumulation in N-free nutrient conditions was proportional to the rate of C₂H₂ reduction, it appears that the method gives a true estimate of the energy requirements for N fixation which for these conditions was equivalent to 17 grams of carbohydrate consumed per gram of N fixed.     

Denitrification by Chromobacterium violaceum

One host (Rana catesbiana)-associated and two free-living mesophilic strains of bacteria with violet pigmentation and biochemical characteristics of Chromobacterium violaceum were isolated from freshwater habitats. Cells of each freshly isolated strain and of strain ATCC 12472 (the neotype strain) grew anaerobically with glucose as the sole carbon and energy source. The major fermentation products of cells grown in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) supplemented with glucose included acetate, small amounts of propionate, lactate, and pyruvate. The final cell yield and culture growth rate of each strain cultured anaerobically in this medium increased approximately twofold with the addition of 2 mM NaNO₃. Final growth yields increased in direct proportion to the quantity of added NaNO₃ over the range of 0.5 to 5 mM. Each strain reduced NO₃⁻, producing NO₂⁻, NO, and N₂O. NO₂⁻ accumulated transiently. With 2 mM NaNO₃ in the medium, N₂O made up 85 to 98% of the N product recovered with each strain. N-oxides were recovered in the same quantity and distribution whether 0.01 atm (ca. 1 kPa) of C₂H₂ (added to block N₂O reduction) was present or not. Neither N₂ production nor gas accumulation was detected during NO₃⁻ reduction by growing cells. Cell growth in media containing 0.5 to 5 mM NaNO₂ in lieu of NaNO₃ was delayed, and although N₂O was produced by the end of growth, NO₂⁻ -containing media did not support growth to an extent greater than did medium lacking NO₃⁻ or NO₂⁻. The data indicate that C. violaceum cells ferment glucose or denitrify, terminating denitrification with the production of N₂O, and that NO₂⁻ reduction to N₂O is not coupled to growth but may serve as a detoxification mechanism. No strain detectably fixed N₂ (reduced C₂H₂).     

Physiological Ecology of Clostridium glycolicum RD-1, an Aerotolerant Acetogen Isolated from Sea Grass Roots

An anaerobic, H₂-utilizing bacterium, strain RD-1, was isolated from the highest growth-positive dilution series of a root homogenate prepared from the sea grass Halodule wrightii. Cells of RD-1 were gram-positive, spore-forming, motile rods that were linked by connecting filaments. Acetate was produced in stoichiometries indicative of an acetyl coenzyme A (acetyl-CoA) pathway-dependent metabolism when RD-1 utilized H₂-CO₂, formate, lactate, or pyruvate. Growth on sugars or ethylene glycol yielded acetate and ethanol as end products. RD-1 grew at the expense of glucose in the presence of low initial concentrations (up to 6% [vol/vol]) of O₂ in the headspace of static, horizontally incubated culture tubes; the concentration of O₂ decreased during growth in such cultures. Peroxidase, NADH oxidase, and superoxide dismutase activities were detected in the cytoplasmic fraction of cells grown in the presence of O₂. In comparison to cultures incubated under strictly anoxic conditions, acetate production decreased, higher amounts of ethanol were produced, and lactate and H₂ became significant end products when RD-1 was grown on glucose in the presence of O₂. Similarly, when RD-1 was grown on fructose in the presence of elevated salt concentrations, lower amounts of acetate and higher amounts of ethanol and H₂ were produced. When the concentration of O₂ in the headspace exceeded 1% (vol/vol), supplemental H₂ was not utilized. The 16S rRNA gene of RD-1 had a 99.7% sequence similarity to that of Clostridium glycolicum DSM 1288^T, an organism characterized as a fermentative anaerobe. Comparative experiments with C. glycolicum DSM 1288^T demonstrated that it had negligible H₂- and formate-utilizing capacities. However, carbon monoxide dehydrogenase was detected in both RD-1 and C. glycolicum DSM 1288^T. A 91.4% DNA-DNA hybridization between the genomic DNA of RD-1 and that of C. glycolicum DSM 1288^T confirmed that RD-1 was a strain of C. glycolicum. These results indicate that (i) RD-1 metabolizes certain substrates via the acetyl-CoA pathway, (ii) RD-1 can tolerate and consume limited amounts of O₂, (iii) oxic conditions favor the production of ethanol, lactate, and H₂ by RD-1, and (iv) the ability of RD-1 to cope with limited amounts of O₂ might contribute to its survival in a habitat subject to daily gradients of photosynthesis-derived O₂.     

Photosynthetic activities of isolated bundle sheath cells in relation to differing mechanisms of C-4 pathway photosynthesis.

Photosynthetic activities of bundle sheath cell strands isolated from several C₄ pathway species were examined. These included species that decarboxylate C₄ acids via either NADP-malic enzyme (Zea mays, NADP-malic enzyme-type), NAD-malic enzyme (Atriplex spongiosa and Panicum miliaceum, NAD-malic enzyme-type) or phosphoenolpyruvate carboxykinase (Chloris gayana and Panicum maximum, phosphoenolpyruvate carboxykinase-type). Preparations from each of these species fixed ¹⁴CO₂ at rates ranging between 1.2 and 3.5 μmol min^?1 mg^?1 of chlorophyll, with more than 90% of the ¹⁴C being assimilated into Calvin cycle intermediates. With added HCO₃^? the rate of light-dependent O₂ evolution ranged between 2 and 4 μmol min^?1 mg^?1 of chlorophyll for cells from NAD-malic enzyme-type and phosphoenolpyruvate carboxykinase-type species but with Z. mays cells there was no O₂ evolution detectable. Most of the ¹⁴CO₂ fixed by Z. mays cells provided with H¹⁴CO₃^? plus ribose 5-phosphate accumulated in the C-1 of 3-phosphoglycerate. However, 3-phosphoglycerate reduction was increased several fold when malate was also provided. Cells from all species rapidly decarboxylated C₄ acids under appropriate conditions, and the CO₂ released from the C-4 carboxyl was reassimilated via the Calvin cycle. Malate decarboxylation by Z. mays cells was dependent upon light and an endogenous or exogenous source of 3-phosphoglycerate. Bundle sheath cells of NAD-malic enzyme-type species rapidly decarboxylated [¹⁴C]malate when aspartate and 2-oxoglutarate were also provided, and [¹⁴C]aspartate was decarboxylated at similar rates when 2-oxoglutarate was added. Cells from phosphoenolpyruvate carboxykinase-type species decarboxylated [¹⁴C]aspartate when 2-oxoglutarate was added and they also catalyzed a slower decarboxylation of malate. Cells from NAD-malic enzyme-type and phosphoenolpyruvate carboxykinase-type species evolved O₂ in the light when C₄ acids were added. These results are discussed in relation to proposed mechanisms for photosynthetic metabolism in the bundle sheath cells of species utilizing C₄ pathway photosynthesis.     

Effect of irradiance on development of apparent nitrogen fixation and photosynthesis in soybean

Soybeans grown with 2 millimolar NO₃⁻, which optimized apparent N₂ fixation by Rhizobium symbionts, showed significantly different rates of apparent photosynthesis and C₂H₂ reduction during seedling development at two irradiances. Those physiological processes were lower for several weeks in plants grown at 1,500 microeinsteins per meter² per second than in those exposed to 700 microeinsteins per meter² per second. The irradiance-induced retardation was evident in short-term rates of apparent photosynthesis and N₂ fixation, as well as in measures of dry matter and total N accumulation. In spite of their previously inhibited development, plants grown at 1,500 microeinsteins per meter² per second were indistinguishable by day 28 from those exposed to 700 microeinsteins per meter² per second in terms of whole-shoot CO₂-exchange rate; by day 35 they were identical in terms of whole-plant C₂H₂-reduction rate. On day 38 there was no significant difference in dry weight or N content between treatments. Shifting plants between irradiance treatments on day 21 showed that the higher irradiance also had a short-term inhibitory effect on C₂H₂ reduction. The fact that 16 millimolar NO₃⁻ prevented the continuous exposure to 1,500 microeinsteins per meter² per second from inhibiting apparent photosynthesis suggested that seedlings grown on 2 millimolar NO₃⁻ with Rhizobium were N-limited. Although rates of apparent photosynthesis were similar by day 28, the additional week required to produce equal rates of apparent N₂ fixation between irradiance treatments showed that physiological adaptations of shoots, as well as photosynthesis per se, can affect root nodule activity.     

Environmental regulation of H2 utilization (3H2 exchange) among natural and laboratory populations of N2 and non-N2 fixing phytoplankton

Regulation of H₂ utilization, as monitored by the hydrogenase-mediated³H₂ exchange reaction, was examined among phytoplankton communitiesin situ and populations in culture. During a 2-year study in the Chowan River, North Carolina, at least 2 major groups of phytoplankton dominated³H₂ exchange rates. They included N₂ fixing cyanobacteria and NO₃ ^}- utilizing genera. Utilization of³H₂ by N₂ fixers was mainly dark-mediated, whereas³H₂ utilization associated with periods of NO₃ ^}- abundance revealed an increasing dependence on light. Inhibitors of N₂ fixation (C₂H₂ and NH₄ ⁺) negatively affected³H₂ utilization, substantiating previous findings that close metabolic coupling of both processes exists among N₂ fixing cyanobacteria. Conversely, NO₃ ^}- stimulated³H₂ utilization among N₂ and non-N₂ fixing genera, particularly under illuminated conditions. A variety of environmental factors were shown to control³H₂ exchange. In addition to the nitrogen sources discussed above, dissolved O₂, photosynthetically available radiation (PAR), temperature, and pH changes altered³H₂ exchange rates. It is likely that other factors not addressed here could also affect³H₂ exchange rates. At least 2 ecological benefits from H₂ utilization in natural phytoplankton can be offered. They include the simultaneous generation of adenosine triphosphate (ATP) and consumption of O₂ during the oxidation of H₂ via an oxyhydrogen or “Knallgas” reaction. Both processes could help sustain phytoplankton, and particularly cyanobacterial, bloom intensity under natural conditions when O₂ supersaturation is common in surface waters. H₂ utilization appeared to be a general feature of natural and laboratory phytoplankton populations. The magnitudes of³H₂ utilization rates were directly related to community biomass. Although it can be shown that utilization rates are controlled by specific environmental factors, the potential relationships between H₂ utilization and phytoplankton primary production remain poorly understood.     

The effect of various levels of nitrogen,potassium and phosphorus on oxidation reduction potentials in barley

The effect of nutrient solutions with certain essential elements lacking, on oxidation reduction potential (RP) of the first leaf was examined in two cultivars of barley seedlings NO₃ ^? and K⁺ decreasedRP considerably, PO ₄ ^3? did not. The effect of NO₃ ^? and K⁺ was dependent. on the illuminance. Under glasshouse conditions, the increased level of NO₃ ^? and K⁺ did not cause any change ofRP. In field conditions, the increased supply of nitrogen fertilizers resulted in a lowerRP in leaves at ear emergence and a higher yield. The use ofRP measurement in leaves for the estimation of the effect of mineral nutrition on plants is discussed.     

Oxygen stress in Salvinia natans: Interactive effects of oxygen availability and nitrogen source

The ability of Salvinia natans (L.) All. to tolerate growth in oxic, hypoxic and anoxic nutrient solutions when supplied with either NH₄⁺ or NO₃^? were studied in the laboratory to test the hypothesis that inorganic N-source affects the response of the plants to O₂ deprivation. The relative growth rate (RGR) was significantly reduced in the anoxic treatment, but in the hypoxic treatment RGR was only slightly affected. The NH₄⁺ fed plants generally had a higher shoot to root ratio than the NO₃^? fed plants, and highest in the anoxic treatment. Plants had more roots and larger leaves when supplied with NH₄⁺ as compared with NO₃^?, particularly in the oxic treatment, and root length was most affected by O₂ deprivation for NO₃^? fed plants. Cell walls in the endodermis, the bundle sheath and the cortex adjacent to endodermis developed thickened sclerenchymatous walls when deprived of O₂, and more so in plants supplied with NO₃^?. Plants lost chlorophylls, had lower rates of photosynthetic electron transport (ETR_max) and lower quantum yields (Fv/Fm ratios) when grown in anoxic solutions, and the negative effects were mildest for NO₃^? fed plants suggesting that NO₃^? may be used as an alternative e^?-acceptor in non-cyclic electron transport in the chloroplasts. Overall S. natans grew best on NH₄⁺, but it also grew well on NO₃^?, and the O₂ stress symptoms differed somewhat between NH₄⁺ fed and NO₃^? fed plants. However, because N-form itself significantly influenced morphology and cell metabolism, it was impossible to conclusively identify the role of N-form for the O₂ stress reactions. S. natans is not well-adapted to grow in O₂ deficient waters and will not tolerate completely anoxic conditions as will prevail in waters receiving high loadings of organic pollutants such as livestock wastewater.     

Variation inRhizobium leguminosarum response to short term application of NH4NO3 to nodulatedPisum sativum L.

Summary This study was conducted to determine the effect of short term application of NH₄NO₃ on nodule function and to determine whether the rhizobial isolate used was a significant factor in this effect. Pea plants were inoculated with 10 differentRhizobium leguminosarum isolates and grown for 3 weeks in N-free medium before addition of 0, 1, 2 or 5 mM NH₄NO₃ for 2 to 7 days. Acetylene reduction and leghemoglobin content decreased with increasing exposure time to NH₄NO₃ and with increasing concentration of NH₄NO₃. NH ₄ ⁺ and NO ₃ ⁻ depletion from the nutrient medium were assayed in plants exposed to 5 mM NH₄NO₃ and mean uptake rates were similar for each ion. There were significant differences among isolates in the rate of decrease of C₂H₂ reduction with increasing NH₄NO₃ concentration (C₂H₂ reduction responsiveness to NH₄NO₃) 4 and 7 days after addition of NH₄NO₃ but no differences after 2 days of exposure to NH₄NO₃. There were significant differences among isolates in NH ₄ ⁺ depletion from the nutrient medium but these differences were not correlated with the differences observed in C₂H₂ reduction. Ranking of the isolates for C₂H₂ reduction responsiveness to NH₄NO₃ applied to plants with nodules was different from that obtained when NH₄NO₃ was applied at seeding. Isolates with varying sensitivity to NH₄NO₃ may be useful tools for determining the mechanisms responsible for inhibition of symbiotic N₂ fixation by combined nitrogen. NRCC paper no. 25863.     

Microzonation of Denitrification Activity in Stream Sediments as Studied with a Combined Oxygen and Nitrous Oxide Microsensor

Microzonation of denitrification was studied in stream sediments by a combined O₂ and N₂O microsensor technique. O₂ and N₂O concentration profiles were recorded simultaneously in intact sediment cores in which C₂H₂ was added to inhibit N₂O reduction in denitrification. The N₂O profiles were used to obtain high-resolution profiles of denitrification activity and NO₃⁻ distribution in the sediments. O₂ penetrated about 1 mm into the dark-incubated sediments, and denitrification was largely restricted to a thin anoxic layer immediately below that. With 115 μM NO₃⁻ in the water phase, denitrification was limited to a narrow zone from 0.7 to 1.4 mm in depth, and total activity was 34 nmol of N cm⁻² h⁻¹. With 1,250 μM NO₃⁻ in the water, the denitrification zone was extended to a layer from 0.9 to 4.8 mm in depth, and total activity increased to 124 nmol of N cm⁻² h⁻¹. Within most of the activity zone, denitrification was not dependent on the NO₃⁻ concentration and the apparent K_m for NO₃⁻ was less than 10 μM. Denitrification was the only NO₃⁻-consuming process in the dark-incubated stream sediment. Even in the presence of C₂H₂, a significant N₂O reduction (up to 30% of the total N₂O production) occurred in the reduced, NO₃⁻-free layers below the denitrification zone. This effect must be corrected for during use of the conventional C₂H₂ inhibition technique.     

Photosynthetic energy supply for NO3− assimilation in Scenedesmus

NO₃^?-dependent O₂ in synchronous Scenedesmus obtusiusculus Chod. in the absence of CO₂ is stoichiometric with NH₄⁺ excretion, indicating a close coupling of NO₃^? reduction to non-cyclic electron flow. Also in the presence of CO₂, NO₃^? stimulates O₂ evolution as manifested by an increase in the O₂/CO₂ ratio from 0.96 to 1.11. This quotient was increased to 1.36 by addition of NO₂^?, without competitive interaction with CO₂ fixation, indicating that the capacity for non-cyclic electron transport at saturating light is non-limiting for simultaneous reduction of NO₃^? and CO₂ at high rates. During incubation with NO₃^?+ CO₂, no NH₄⁺ is released to the outer medium, whereas during incubation with NO₂^?+ CO₂, excess NH₄⁺ is formed and excreted. NO₃^? uptake is stimulated by CO₂, and this stimulation is also significant when the cellular energy metabolism is restricted by moderate concentrations of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, whereas NO₃^? uptake in the absence of CO₂ is severely inhibited by the uncoupler. Also under energy-restricted conditions NO₃^? uptake is not competitive with CO₂ fixation. Antimycin A is inhibitory for NO₃^? uptake in the absence of CO₂, and there is no enhancement of NO₃^? uptake by CO₂ in the presence of antimycin A. It is assumed that the energy demand for NO₃^? uptake is met by energy fixed as triosephosphates in the Calvin cycle. Antimycin A possibly affects the transfer of reduced triose phosphates from the chloroplast to the cytoplasm. Active carbon metabolism also seems to exert a control effect on NO₃^? assimilation, inducing complete incorporation of all NO₃^? taken up into amino acids. This control effect is not functional when NO₂^? is the nitrogen source. Active carbon metabolism thus seems to be essential both for provision of energy for NO₃^? uptake and for regulation of the process.