Growth and respiration ofBradyrhizobium japonicum USDA 143 with nitrous oxide as the terminal electron acceptor

Bradyrhizobium japonicum USDA 143 grew chemoorganotrophically under anoxic conditions with exogenous N₂O as the sole terminal electron acceptor. Cell growth and dissimilatory N₂O reduction were significantly inhibited by C₂H₂ when either N₂O or N₂O plus NO ₃ ^– served as terminal electron acceptor(s). Reduction of N₂O accounted for 20% of the energy for cell growth in cultures supplied with NO ₃ ^– as the terminal electron acceptor. Nitrous oxide was produced stoichiometrically in cultures containing NO ₃ ^– and C₂H₂, but cell growth was proportionately reduced when compared with cultures supplied with an equal amount of NO ₃ ^– . Exogenous N₂O delayed the reduction of NO ₃ ^– in cultures supplied with both electron acceptors. Direct amperometric monitoring of N₂O respiration showed a specific activity of 0.082±0.004 moles N₂O/min/mg cell protein, and azide inhibited cell respiration.     

Hemoglobin expression affects ethylene production in maize cell cultures.

The formation of ethylene under different O(2) concentrations and upon addition of nitric oxide (NO) donor, sodium nitroprusside (SNP), was determined using maize (Zea mays L.) cell lines over-expressing (Hb+) or down-regulating (Hb-) hypoxically inducible (class-1) hemoglobin (Hb). Under all treatments, ethylene levels in the Hb- line were 5 to 6.5 times the levels in Hb+ and four to five times the levels in the wild type. Low oxygen partial pressures impaired ethylene formation in maize cell suspension cultures. 1-Amino-cyclopropane-1-carboxylic acid (ACC) oxidase (E.C. 1.14.17.4) mRNA levels did not vary, either between lines or between treatments. There was, however, significantly enhanced ACC oxidase (ACO) activity in the Hb- line relative to the wild type and the Hb+ line. ACO activity in the Hb- line increased under hypoxic conditions and significantly increased upon treatment with NO under normoxic conditions. The results suggest that limiting class-1 hemoglobin protein synthesis increases ethylene formation in maize suspension cells, possibly via the modulation of NO levels.     

Rhizobacteria and their potential to control <Emphasis Type="Italic">Fusarium verticillioides</Emphasis>: effect of maize bacterisation and inoculum density

Fusarium verticillioides is the most important seed transmitted pathogen that infects maize. It produces fumonisins, toxins that have potential toxicity for humans and animals. Control of F. verticillioides colonisation and systemic contamination of maize has become a priority area in food safety research. The aims of this research were (1) to characterise the maize endorhizosphere and rhizoplane inhabitant bacteria and Fusarium spp., (2) to select bacterial strains with impact on F. verticillioides growth and fumonisin B₁ production in vitro, (3) to examine the effects of bacterial inoculum levels on F. verticillioides root colonisation under greenhouse conditions. Arthrobacter spp. and Azotobacter spp. were the predominant genera isolated from maize endorhizosphere and rhizoplane at the first sampling period, whilst F. verticillioides strains showed the greatest counts at the same isolation period. All F. verticillioides strains were able to produce fumonisin B₁ in maize cultures. Arthrobacter globiformis RC5 and Azotobacter armeniacus RC2, used alone or in a mix, demonstrated important effects on F. verticillioides growth and fumonisin B₁ suppression in vitro. Only Azotobacter armeniacus RC2 significantly reduced the F. verticillioides root colonisation at 10⁶ and 10⁷ CFU g^–1 levels under greenhouse conditions.     

Nitric Oxide Induces Flowering in the Duckweed Lemna aequinoctialis Welw. (Syn. L. paucicostata Hegelm.) Under Noninductive Conditions

Nitric oxide (NO) plays diverse roles in the growth and development of plants and in their responses to various abiotic and biotic stresses. It has also been reported to repress flowering in Arabidopsis thaliana. In the present study, NO donors sodium nitroprusside (SNP), S-nitroso-N-acetyl penicillamine (SNAP), and 3-morpholinosydnonimine (SIN-1) induced flowering in Lemna aequinoctialis 6746 (a short-day strain) and in L. aequinoctialis LP₆ (a photoperiod-insensitive strain) under noninductive conditions. Nitrate and nitrite, two stable metabolites of NO, did not induce flowering. On the other hand, cyanide donors potassium ferricyanide {K₃[Fe(CN)₆]} and potassium cyanide (KCN) induced flowering in both strains under noninductive conditions. The flowering induced under a 8-h daily photoperiod regime in the short-day strain L. aequinoctialis 6746 was inhibited by NO and cyanide donors. Vegetative multiplication of both strains was adversely affected by NO and cyanide donors, irrespective of the photoperiod conditions. The observed effects of NO donors on flowering were substantially negated by NO scavengers c-PTIO [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] and methylene blue. This confirmed the role of NO in induction of flowering. The inductive effect of CN⁻ also appeared to be partly mediated through NO as NO scavengers partially negated the effect of CN⁻.     

NADH-dependent metabolism of nitric oxide in alfalfa root cultures expressing barley hemoglobin

Transgenic alfalfa (Medicago sativa L.) root cultures expressing sense and antisense barley (Hordeum vulgare L.) hemoglobin were examined for their ability to metabolize NO. Extracts from lines overexpressing hemoglobin had approximately twice the NO conversion rate of either control or antisense lines under normoxic conditions. Only the control line showed a significant increase in the rate of NO degradation when placed under anaerobic conditions. The decline in NO was dependent on the presence of reduced pyridine nucleotide, with the NADH-dependent rate being about 2.5 times faster than the NADPH-dependent rate. Most of the activity was found in the cytosolic fraction of the extracts, while only small amounts were found in the cell wall, mitochondria, and 105,000-g membrane fraction. The NADH-dependent NO conversion exhibited a broad pH optimum in the range 7–8 and a strong affinity to NADH and NADPH (K _m 3 M for both). It was sensitive to diphenylene iodonium, an inhibitor of flavoproteins. The activity was strongly reduced by applying antibodies raised against recombinant barley hemoglobin. Extracts of Escherichia coli overexpressing barley hemoglobin showed a 4-fold higher rate of NO metabolism as compared to non-transformed cells. The NADH/NAD and NADPH/NADP ratios were higher in lines underexpressing hemoglobin, indicating that the presence of hemoglobin has an effect on these ratios. They were increased under hypoxia and antimycin A treatment. Alfalfa root extracts exhibited methemoglobin reductase activity, using either cytochrome c or recombinant barley hemoglobin as substrates. There was a correspondence between NO degradation and nitrate formation. The activity was eluted from a Superose 12 column as a single peak with molecular weight of 35±4 kDa, which corresponds to the size of the hemoglobin dimer. The results are consistent with an NO dioxygenase-like activity, with hemoglobin acting in concert with a flavoprotein, to metabolize NO to nitrate utilizing NADH as the electron donor.Abbreviation Hb Hemoglobin     

Induction of apoptosis in oxygen-deprived cultures of hybridoma cells

It is now well documented that apoptosis represents the prevalent mode of cell death in hybridoma cultures. Apoptotic or programmed cell death occurs spontaneously in late exponential phase of batch cultures. Until lately, no specific triggering factors had been identified. Recently, we observed that glutamine, cystine or glucose deprivation induced apoptosis in both hybridoma and myeloma cell lines whereas accumulation of toxic metabolites induced necrotic cell death in these cells. Other triggering factors such as oxygen deprivation might also be responsible for induction of apoptosis. In the present study, induction of cell death by exposure to anoxia was examined in batch culture of the SP2/0-derived hybridoma D5 clone. The mode of cell death was studied by morphological examination of acridine orange-ethidium bromide stained cells in a 1.5 L bioreactor culture grown under anoxic conditions for 75 hours. Under such conditions, viable cell density levelled off rapidly and remained constant for 25 hours. After 45 hours of anoxia, cell viability had decreased to 30% and the dead cell population was found to be 90% apoptotic. In terms of cellular metabolism, anoxia resulted in an increase in the utilization rates of glucose and arginine, and in a decrease in the utilization rate of glutamine. The lactate production rate and the yield of lactate on glucose increased significantly while the MAb production rate decreased. These results demonstrate that glycolysis becomes the main source of energy under anoxic conditions.Cells incubated for 10 hours or less under anoxic conditions were able to recuperate almost immediately and displayed normal growth rates when reincubated in oxic conditions whereas cells incubated for 22 hours or more displayed reduced growth rates. Nonetheless, even after 22 h or 29 h of anoxia, cells reincubated in oxic conditions showed no further progression into apoptosis. Therefore, upon removal of the triggering signal, induction of apoptosis ceased.Abbreviations VNA Viable non-apoptotic cells - VA Viable apoptotic cells - NVNA Nonviable non-apoptotic or necrotic cells - NVA Nonviable apoptotic cells - CF Chromatin-free cells (late nonviable apoptotic cells) - AO Acridine orange - EB Ethidium Bromide - MAb Monoclocnal antibody - D.O. Dissolved oxygen - qMAb Specific MAb production rate (mg. (10⁹ cells)^–1.day^–1) - Specific growth rate (h^–1) - X_v Viable cell number (10⁵ cells.mL^–1) - X_t Total cell number (10⁵ cells.mL^–1) - Y_lac/glc Yield coefficient of lactate on glucose (mM lactate produced/mM glucose consumed)     

NO 2 − Efflux and its regulation in cyanobacterium Nostoc MAC

NO ₂ ^– efflux and its regulation have been studied in the cyanobacterium Nostoc MAC. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU), carbonyl cyanide-m-chlorophenyl hydrazone (CCCP), sodium azide, p-chloromercuribenzoate (PCMB), and dicyclohexylcarbodiimide (DCCD), a specific inhibitor of bacterial ATPase, inhibited the NO ₂ ^– efflux activity singificantly. No NO ₂ ^– efflux activity was observed under dark-aerobic as well as under dark-anaerobic conditions; however, the addition of ATP resulted in NO ₂ ^– efflux even under dark-aerobic condition. Maximum NO ₂ ^– efflux activity was observed when NO ₃ ^– served as the sole nitrogen source. However, NH ₄ ⁺ ions inhibited the NO ₂ ^– efflux activity when both NO ₃ ^– and NH ₄ ⁺ wer simulatneously available to the cells. The NO ₂ ^– efflux was freed from NH ₄ ⁺ repression by l-methionine-dl-sulfoximine (MSX), an irreversible inhibitor of glutamine synthetase (GS). Chloramphenicol, a protein synthesis inhibitor, inhibited the derepression of NO ₂ ^– efflux system when NH ₄ ⁺ -incubated cells were transferred to NO ₃ ^– medium. Tungstate-treated cells lacking functional NO ₃ ^– reductase but having NO ₃ ^– uptake activity also lacked NO ₂ ^– efflux activity. These results suggest that (i) NO ₂ ^– efflux in Nostoc MAC is NO ₃ ^– dependent and an energy-dependent process that can be regulated at the levels of NO ₃ ^– uptake and NO ₃ ^– reductase; (ii) NO ₂ ^– efflux system is NH ₄ ⁺ repressible; however, the product of NH ₄ ⁺ assimilation via GS is being required for repression to occur; (iii) de novo protein synthesis is required for derepression of the NO ₂ ^– efflux system; and (iv) the catalytic activity of NO ₂ ^– reductase also seems to play an important role in the regulation of NO ₂ ^– efflux system.     

Denitrification kinetics of simulated fish processing wastewater at different ratios of nitrate to biomass

Denitrification was studied in anoxic batch cultures of a simulated fish processing wastewater at 37 r C and pH 7.5, using a denitrifying enrichment culture from fishery wastewater. Different initial nitrate to biomass ratios (So/Xo) were used: nitrate and biomass varied from 7.5 to 94.7 mg NO₃-N l^–1, and from 20 to 4300 mg volatile suspended solids l^–1, respectively. The specific maximum denitrification rate (r _m) and the cell yield (Y _{X / S}) depended on the So/Xo ratio under anoxic conditions: r _m increased from 1.2 to 1584 mg NO₃-N g^–1 VSS h^–1 and Y _{X / S} decreased from 42 to 0.03 mg VSS mg^–1 NO₃-N when So/Xo varied from 5.5 10^{– 3} to 9.3 mg NO₃-N/mg VSS. Nomenclature C_{NO3 – N} nitrate concentration, mg NO₃-N l^–1 K _S saturation constant, mg NO₃-N l^–1 r _m specific maximum denitrification rate, mg NO₃-N g^–1 VSS h^–1 So initial substrate concentration, mg l^–1 t time, h TOC total organic carbon VSS volatile suspended solids x biomass concentration, g VSS l^–1 Xo initial biomass concentration, g VSS l^–1 Y _X/S substrate to biomass cell yield, mg VSS/mg N Greek symbols: _m maximum specific growth rate of the anoxic microbial population, 1 h^–1     

Microbial Processes Associated with Roots of Bulbous Rush Coated with Iron Plaques

Bulbous rush (Juncus bulbosus) is a pioneer species in acidic, iron-rich, coal mining lakes in the eastern part of Germany. Juncus roots are coated with iron plaques, and it has been suggested that microbial processes under the iron plaques might be supportive for Juncus plant growth. The objectives of this work were to enumerate the microbes involved in the turnover of iron and organic root exudates in the rhizoplane, to investigate the effect of oxygen and pH on the utilization of these exudates by the rhizobacteria, and to study the ability of the root-colonizing microbiota to reduce sulfate. Enumeration studies done at pH 3 demonstrated that 10⁶ Fe(III) reducers and 10⁷ Fe(II) oxidizers g (fresh wt root)^–1 were associated with Juncus roots. When roots were incubated in goethite-containing medium without and with supplemental glucose, Fe(II) was formed at rates approximating 1.1 mmol g (fresh wt root) ^–1 d^–1 and 3.6 mmol g (fresh wt root)^–1 d^–1 under anoxic conditions, respectively. These results suggest that a rapid microbially mediated cycling of iron occurs in the rhizosphere of Juncus roots under changing redox conditions. Most-probable-number estimates of aerobes and anaerobes capable of consuming root exudates at pH 3 were similar in the rhizosphere sediment and in Juncus roots, but numbers of aerobes were significantly higher than those of anaerobes. At pH 3, supplemental organic exudates were primarily subject to aerobic oxidation to CO₂ and not subject to fermentation. However, at pH 4.5, root exudates were also rapidly utilized under anoxic conditions. Root-associated sulfate reduction was not observed at pH 3 to 4.5 but was observed at pH 4.9. The pH increased during all root-incubation studies both under oxic and anoxic conditions. Thus, as result of the microbial turnover of organic root exudates, pH and CO₂ levels might be elevated at the root surface and favor Juncus plants to colonize acidic habitats.     

Growth and metabolism of the purple sulfur bacterium Thiocapsa roseopersicina under combined light/dark and oxic/anoxic regimens

The dominant purple sulfur bacterium of laminated sediment ecosystems in temperate environments, Thiocapsa roseopersicina, was cultivated in sulfide-limited continuous cultures (D=0.03 h^-1) subjected to various combined diel regimen of aeration and illumination in order to simulate environmental conditions in microbial mats. For comparison, cultures were grown under similar illumination regimens but continuously anoxic conditions.Bacteriochlorophyll a (BChla) and carotenoid synthesis was restricted to anoxic-dark periods and did not occur during oxic-light periods. An increase in the length of the oxic-light periods resulted in decreased pigment contents. However, phototrophic growth remained possible even at 20 h oxic-light/4 h anoxic-dark regimens. When anoxic conditions were maintained throughtout, BChla synthesis occurred both during light and dark periods.Glycogen was synthesized in the light and degraded in the dark. Calculations showed that degradation of 1/4–1/5 of the glycogen is sufficient to account for the BChla and carotenoid synthesis in the dark.The data showed that T. roseopersicina is very well adapted to cope with the combined oxygen and light regimes as they occur in microbial mats, which may explain the dominance of this bacterium in the purple layer of these sediment ecosystems.Non-standard abbreviations BChl bacteriochlorophyll - specific growth rate - D dilution rate - S_R concentration of limiting substrate in reservoir bottle     

Class-1 hemoglobin and antioxidant metabolism in alfalfa roots

In the course of nitric oxide (NO) scavenging, hemoglobin (Hb) turnover is linked to antioxidant metabolism and affects the cellular redox level. The influence of Hb presence on the ascorbate-glutathione cycle enzymes and the levels of H₂O₂ and ascorbate was investigated in alfalfa root cultures transformed to over-express (Hb+) or down-regulate (Hb–) class-1 Hb. Hb+ lines had substantially increased ascorbate levels as well as elevated monodehydroascorbate reductase and ascorbate peroxidase activities. Hb– lines showed significant increases in dehydroascorbate reductase and glutathione reductase activities. The observed changes in ascorbate and ascorbate-glutathione cycle enzymes were pronounced both at high (40 kPa) and low (3 kPa) O₂ pressures. Hb– lines had significantly reduced levels of the NO- and H₂O₂-sensitive enzyme, aconitase, as compared to Hb+ lines. This reduced activity was likely due the higher levels of NO in Hb– lines, as treatment of plant extracts with the NO-donor DEANO also affected aconitase activity. The H₂O₂ levels were not significantly different amongst the lines and showed no variation with change in oxygen partial pressure. In conclusion, the expression of class-1 Hb improves the antioxidant status through increased ascorbate levels and increased activity of enzymes involved in H₂O₂ removal.     

Oxidation of nitric oxide by a new heterotrophic Pseudomonas sp.

A new bacterial strain isolated from soil consumed nitric oxide (NO) under oxic conditions by oxidation to nitrate. Phenotypic and phylogenetic characterization of the new strain PS88 showed that it represents a previously unknown species of the genus Pseudomonas, closely related to Pseudomonas fluorescens and Pseudomonas putida. The heterotrophic, obligately aerobic strain PS88 was not able to denitrify or nitrify; however, strain PS88 oxidized NO to nitrate. NO was not reduced to nitrous oxide (N₂O). Nitrogen dioxide (NO₂) and nitrite (NO₂ ^–) as possible intermediates of NO oxidation to nitrate (NO₃ ^–) could not be detected. NO oxidation was inhibited under anoxic conditions and by high osmolarity, but not by nitrite. NO oxidation activity was inhibited by addition of formaldehyde, HgCl₂, and antimycin, and by autoclaving or disintegrating the cells, indicating that the process was enzyme-mediated. However, the mechanism remains unclear. A stepwise oxidation at a metalloenzyme and a radical mechanism are discussed. NO oxidation in strain PS88 seems to be a detoxification or a co-oxidation mechanism, rather than an energy-yielding process. Received: 15 November 1995 / Accepted: 24 February 1996     

Gaseous NO2 as a regulator for ammonia oxidation of Nitrosomonas eutropha

Cells of Nitrosomonas eutropha strain N904 that were denitrifying under anoxic conditions with hydrogen as electron donor and nitrite as electron acceptor were unable to utilize ammonium (ammonia) as an energy source. The recovery of ammonia oxidation activity was dependent on the presence of NO₂. Anaerobic ammonia oxidation activity was observed in a helium atmosphere supplemented with 25 ppm NO₂ after 20 h. Ammonia oxidation activity was detected after 2–3 days using an oxic atmosphere with 25 ppm NO₂. In contrast, ammonia consumption started after 8–9 days under oxic conditions without the addition of NO₂; in this case, small amounts of NO and NO₂ were detected and their concentrations increased with increasing ammonia oxidation activities. Hardly any ammonia oxidation was detected when nitrogen oxides were removed by intensive aeration. It would seem, therefore, that NO₂ is the master regulatory signal for ammonia oxidation in Nitrosomonas eutropha. Anaerobic ammonia oxidation activity was inhibited by the addition of NO. This inhibition was partly compensated by either increasing the NO₂ concentration or by using 2,3-dimercapto-1-propane-sulfonic acid as a NO binding substrate. DMPS was inhibitory to nitrification under oxic conditions, while increased amounts of NO or NO₂ led to increased oxidation activities.     

Chloroflexus aurantiacus and ultraviolet radiation: Implications for archean shallow-water stromatolites

The phototrophic growth ofChloroflexus aurantiacus under anoxic conditions was determined as a function of continuous UV irradiance. Cultures grown under an irradiance of 0.01 Wm^–2 exhibited a slightly depressed yield over the nonirradiated control. Yields decreased further with increasing irradiance. Inhibition was severe at an irradiance of 0.66 Wm^–2. Growth ofE. coli cultures was severely depressed at UV-C irradiances that permitted good growth ofC. aurantiacus. Low levels of Fe³⁺ provided a very effective UV absorbing screen. The apparent UV resistance ofChloroflexus and the effectiveness of iron as a UV-absorbing screen in sediments and microbial mats are suggested to be likely mechanisms of survival of early phototrophs in the Precambrian in the absence of an ozone shield.     

The role of auxiliary oxidants in maintaining redox balance during phototrophic growth of Rhodobacter capsulatus on propionate or butyrate

Phototrophic growth of Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata) under anaerobic conditions with either butyrate or propionate as carbonsource was dependent on the presence of either CO₂ or an auxiliary oxidant. NO _- ³ , N₂O, trimethylamine-N-oxide (TMAO) or dimethylsulphoxide (DMSO) were effective provided the appropriate anaerobic respiratory pathway was present. NO _- ³ was reduced extensively to NO _- ³ , TMAO to trimethylamine and DMSO to dimethylsulphide under these conditions. Analysis of culture fluids by nuclear magnetic resonance showed that two moles of TMAO or DMSO were reduced per mole of butyrate utilized and one mole of either oxidant was reduced per mole of propionate consumed. The growth rate of Rb. capsulatus on succinate or malate as carbon source was enhanced by TMAO in cultures at low light intensity but not at high light intensities. A new function for anaerobic respiration during photosynthesis is proposed: it permits reducing equivalents from reduced substrates to pass to auxiliary oxidants present in the medium. The use of CO₂ or auxiliary oxidants under phototrophic conditions may be influence by the availability of energy from light. It is suggested that the nuclear magnetic resonance methodology developed could have further applications in studies of bacterial physiology.Abbreviations DMS dimethylsulphide - DMSO dimethylsulphoxide - TMA trimethylamine - TMAO trimethylamine-N-oxide - NMR nuclear magnetic resonance     

The impact of NO inf3 sup− loading on the freshwater macrophyte Littorella uniflora: N utilization strategy in a slow-growing species from oligotrophic habitats

The decline and disappearance of Littorella uniflora from oligotrophic waters which have become eutrophic has been associated with shading or reduced CO₂ supply. However NO _inf3 ^sup– concentrations can reach very high levels (100–2000 mmol m^–3 compared with <1–3 in oligotrophic habitats). To investigate the impact of NO _inf3 ^sup– loading alone, plants were grown under three NO _inf3 ^sup– regimes (very low, near-natural and high). The interactive effects of NO _inf3 ^sup– and photon flux density (low and high regimes) on N assimilation and accumulation, CO₂ concentrating mechanisms, C₃ photosynthesis and growth were also examined. The results were unexpected. Increased NO _inf3 ^sup– supply had very little effect on photosynthetic capacity, crassulacean acid metabolism (CAM) or lacunal CO₂ concentrations ([CO₂]_i), although there was considerable plasticity with respect to light regime. In contrast, increased NO _inf3 ^sup– supply resulted in a marked accumulation of NO _inf3 ^sup– , free amino acids and soluble protein in shoots and roots (up to 25 mol m^–3, 30 mol m^–3 and 9 mg g^–1 fresh weight respectively in roots), while fresh weight and relative growth rate were reduced. Total N content even under the very low NO _inf3 ^sup– regime (1.6–2.3%) was mid-range for aquatic and terrestrial species (and 3.1–4.3% under the high NO _inf3 ^sup– regime). These findings, together with field data, suggest that L. uniflora is not growth limited by low NO _inf3 ^sup– supply in natural oligotophic habitats, due not to an efficient photosynthetic nitrogen use but to a slow growth rate, a low N requirement and to the use of storage to avoid N stress. However the increased NO _inf3 ^sup– concentrations in eutrophic environments seem likely have detrimental effects on the long-term survival of L. uniflora, possibly as a consequence of N accumulation.     

Nitrate and nitrite uptake and reduction by intact sunflower plants

Nitrogen-starved sunflower plants (Helianthus annuus L. cv. Peredovic) cannot absorb NO ₃ ^– or NO ₂ ^– upon initial exposure to these anions. Ability of the plants to take up NO ₃ ^– and NO ₂ ^– at high rates from the beginning was induced by a pretreatment with NO ₃ ^– . Nitrite also acted as inducer of the NO ₂ ^– -uptake system. The presence of cycloheximide during NO ₃ ^– -pretreatment prevented the subsequent uptake of NO ₃ ^– and NO ₂ ^– , indicating that both uptake systems are synthesized de novo when plants are exposed to NO ₃ ^– . Cycloheximide also suppressed nitrate-reductase (EC 1.6.6.1) and nitrite-reductase (EC 1.7.7.1) activities in the roots. The sulfhydryl-group reagent N-ethylmaleimide greatly inhibited the uptake of NO ₃ ^– and NO ₂ ^– . Likewise, N-ethylmaleimide promoted in vivo the inactivation of nitrate reductase without affecting nitrite-reductase activity. Rates of NO ₃ ^– and NO ₂ ^– uptake as a function of external anion concentration exhibited saturation kinetics. The calculated K_m values for NO ₃ ^– and NO ₂ ^– uptake were 45 and 23 M, respectively. Rates of NO ₃ ^– uptake were four to six times higher than NO ₃ ^– -reduction rates in roots. In contrast, NO ₂ ^– -uptake rates, found to be very similar to NO ₃ ^– -uptake rates, were much lower (about 30 times) than NO ₂ ^– -reduction rates. Removal of oxygen from the external solution drastically suppressed NO ₃ ^– and NO ₂ ^– uptake without affecting their reduction. Uptake and reduction were also differentially affected by pH. The results demonstrate that uptake of NO ₃ ^– and NO ₂ ^– into sunflower plants is mediated by energy-dependent inducible-transport systems distinguishable from the respective enzymatic reducing systems.Abbreviations CHI cycloheximide - NEM N-ethylmaleimide - NiR nitrite reductase - NR nitrate reductase - pHME p-hydroxymercuribenzoate This research was supported by grant PB86-0232 from the Dirección General de Investigatión Científica y Técnica (Spain). One of us (E.A.) thanks the Consejeria de Educación y Ciencia de la Junta de Andalucia for the tenure of a fellowship. We thank Miss G. Alcalá and Miss C. Santos for their valuable technical and secretarial assistance.     

Diurnal changes in nitric oxide emissions from conventional tillage and pasture sites in the Amazon Basin: influence of soil temperature

We have investigated a subset of restoration practices applied to a degraded pasture at Fazenda Nova Vida, a 22000 ha cattle ranch in Rond^onia, Brazil. Nitric oxide (NO) and carbon dioxide (CO₂) emissions from soils were measured in conventional tillage and current pasture sites to assess N and C losses. Mean daily NO emissions from tilled plots were at least twice those from the pasture. Nitric oxide emissions from the tilled sites showed a strong diurnal pattern, while those from the pasture sites did not. Mean daytime NO emissions from the tilled sites were 9.7 g NO-N m^–2 h^–1, while mean nighttime emissions were 29.7 g NO-N m^–2 h^–1. In the pasture sites, NO emissions were 7.6 g NO-N m^–2 h^–1 during the day, and 7.7 g NO-N m^–2 h^–1 at night. Surface soil temperature was a good inverse predictor (r ²=0.75) of NO emissions from the tilled sites. Carbon dioxide emissions from the tilled sites were generally larger than CO₂ emissions from the pasture sites. The mean CO₂ emission rate from the tilled sites was 179 mg C m^–2 h^–1, while it was 123 mg C m^–2 h^–1 from the pasture sites. There was no distinct diurnal pattern for CO₂ emissions. We found that the very high temperatures measured at the soil surface in the tillage plots, in the range of 40–45°C, reduced the rate of NO emission. The reduction in NO emissions may be because of the sensitivity of autotrophic nitrifiers to high temperatures. This study provides insights on how land-use change may alter regional NO fluxes by exposing certain microbial communities to extreme environmental conditions. Future studies of NO emissions in tropical agricultural systems where soils are bare for extend periods need to make diurnal measurements or the daily fluxes will be substantially underestimated.     

Changes in Rhodopseudomonas sphaeroides isoaccepting phenylalanyl-tRNA species during transitions from chemoheterotrophic to photoheterotrophic growth

A new iso-accepting tRNA^phe from extracts of chemoheterotrophic and photoheterotrophic cells of Rhodopseudomonas sphaeroides has been identified by both BDEAE cellulose and RPC-5 chromatography. Rechromatography of each of the tRNA^phe species in either the acylated or deacylated state shows that they migrate as single homogeneous peaks.In steady-state chemoheterotrophic cultures of R. sphaeroides tRNA _I–II ^phe account for 25–30% of the total phenylalanine accepting activity while in steadystate photoheterotrophic cultures tRNA _I–II ^phe account for no more than 10% of the total phenylalanine accepting activity.During the transition from chemoheterotrophic to photoheterotrophic growth conditions the levels of tRNA _I–II ^phe fall in an exponential manner during the first half of the intracytoplasmic membrane induction period. tRNA _I ^phe then remains at a level 10% that of its steady-state chemoheterotrophic level as long as photoheterotrophic growth conditions remain. tRNA _II ^phe , after dropping to 10% of its former chemoheterotrophic level then returns to a level 50% that of its chemoheterotrophic level as long as photoheterotrophic growth conditions remain.Abbreviations BDEAE benzoylated diethyl amino ethyl - RPC reversed phase chromatography - TCA tricholroacetic acid - ICM intracytoplasmic membrane Submitted by WDS in partial fullfilment of requirements for the M.S. degree     

The ability of several high arctic plant species to utilize nitrate nitrogen under field conditions

The ability to utilize NO _inf3 ^sup– in seven high arctic plant species from Truelove Lowland, Devon Island, Canada was investigated, using an in vivo assay of maximum potential nitrate reductase (NR) activity and applications of ¹⁵N. Plant species were selected on the basis of being characteristic of nutrient-poor and nutrient-rich habitats. In all species leaves were the dominant site of NR activity. Root NR activity was negligible in all species except Saxifraga cernua. NO _inf3 ^sup– availability per se did not appear to limit NR activity of the species typically found on nutrient-poor sites (Dryas integrifolia, Saxifraga oppositifolia, and Salix arctica), or in Cerastium alpinum, as leaf NR activities remained low, even after NO _inf3 ^sup– addition. ¹⁵NO _inf3 ^sup– uptake was limited in D. integrifolia and Salix arctica. However, the lack of field induction of NR activity in C. alpinum and Saxifraga oppositifolia was not due to restricted nitrate uptake, as ¹⁵NO _inf3 ^sup– labelled NO _inf3 ^sup– entered the roots and shoots of both species. Leaf NR activity rates were low in three of the species typical of nutrient-rich habitats (O. digyna, P. radicatum and Saxifraga cernua), sampled from a site containing low soil NO _inf3 ^sup– . Additions of NO _inf3 ^sup– significantly increased leaf NR activity in these latter species, suggesting that potential NR activity was limited by the availability of NO _inf3 ^sup– . ¹⁵N labelled NO _inf3 ^sup– was taken up by O. digyna. P. radicatum and Saxifraga cernua. Although two species (D. integrifolia and Salix arctica) showed little utilization of NO _inf3 ^sup– , we concluded that five of the seven selected high arctic plant species (C. alpinum, O. digyna, P. radicatum, Saxifraga cernua and Saxifraga oppositifolia) do have the potential to utilize NO _inf3 ^sup– as a nitrogen source under field conditions, with the highest potential to utilize NO _inf3 ^sup– occurring in three of the species typically found on fertile habitats.