Control of seed development in Arabidopsis thaliana by atmospheric oxygen

Seed development is known to be inhibited completely when plants are grown in oxygen concentrations below 5·1 kPa, but apart from reports of decreased seed weight little is known about embryogenesis at subambient oxygen concentrations above this critical level. Arabidopsis thaliana (L.) Heynh. plants were grown full term under continuous light in premixed atmospheres with oxygen partial pressures of 2·5, 5·1, 10·1, 16·2 and 21·3 kPa O₂, 0·035 kPa CO₂ and the balance nitrogen. Seeds were harvested for germination tests and microscopy when siliques had yellowed. Seed germination was depressed in O₂ treatments below 16·2 kPa, and seeds from plants grown in 2·5 kPa O₂ did not germinate at all. Fewer than 25% of the seeds from plants grown in 5·1 kPa oxygen germinated and most of the seedlings appeared abnormal. Light and scanning electron microscopic observation of non-germinated seeds showed that these embryos had stopped growing at different developmental stages depending upon the prevailing oxygen level. Embryos stopped growing at the heart-shaped to linear cotyledon stage in 5·1 kPa O₂, at around the curled cotyledon stage in 10·1 kPa O₂, and at the premature stage in 16·2 kPa O₂. Globular and heart-shaped embryos were observed in sectioned seeds from plants grown in 2·5 kPa O₂. Tissue degeneration caused by cell autolysis and changes in cell structure were observed in cotyledons and radicles. Transmission electron microscopy of mature seeds showed that storage substances, such as protein bodies, were reduced in subambient oxygen treatments. The results demonstrate control of embryo development by oxygen in Arabidopsis .     

Effects of flooding under hydrostatic pressure on the respiratory metabolism of germinated wheat seeds

Germinated wheat seeds ( Triticum aestivum L. cv. Barqai) that had been subjected to short hydrostatic pressure treatments (0.3–1.2 MPa) changed their normal metabolism into one which is characterized by a high ethanol production, a low O₂ consumption and a low CO₂ evolution. Alcoholic fermentation could account for ca half of the CO₂ evolved from the pressurized seeds. The level of acetaldehyde was low, though significantly higher in the pressurized seeds than in the controls. Subjection of wheat seeds to osmotic stress under aerobic conditions lowered their O₂ uptake and CO₂ evolution but did not induce ethanol production. Exposure of pressurized seeds to NaCl stress did not alter their ethanol production beyond that which had been induced by pressure. Ethanol production by pressurized seeds increased following either the addition of sucrose or by excision of the embryos from the endosperms. More electrolytes leaked into the embedding solution from pressurized seeds than from control seeds. Exogenous ethanol was toxic to wheat seeds at concentrations as low as 343 m M . The effects of hydrostatic pressure and of the consequently induced ethanol production on the mortality of flooded seeds is discussed.     

Oxygen-induced recovery from short-term nitrate inhibition of N2 fixation in white clover plants from spaced and dense stands

Nitrogenase (N₂ase; EC 1.18.6.1) activity (H₂ evolution) and root respiration (CO₂ evolution) were measured under either N₂:O₂ or Ar:O₂ gas mixtures in intact nodulated roots from white clover ( Trifolium repens L.) plants grown either as spaced or as dense stands. The short-term nitrate (5 m M ) inhibition of N₂-fixation was promoted by competition for light between clover shoots, which reduced CO₂ net assimilation rate. Oxygen-diffusion permeability of the nodule declined during nitrate treatment but after nitrate removal from the liquid medium its recovery parallelled that of nitrogenase activity. Rhizosphere pO₂ was increased from 20 to 80 kPa under N₂:O₂. A simple mono-exponential model, fitted to the nodule permeability response to pO₂, indicated NO⁻₃ induced changes in minimum and maximum nodule O₂-diffusion permeability. Peak H₂ production rates at 80 kPa O₂ and in Ar:O₂ were close to the pre-decline rates at 20 kPa O₂. At the end of the nitrate treatment, this O₂-induced recovery in nitrogenase activity reached 71 and 82%; for clover plants from spaced and dense stands, respectively. The respective roles of oxygen diffusion and phloem supply for the short-term inhibition of nitrogenase activity in nitrate-treated clovers are discussed.     

Measurement of denitrification in estuarine sediment using membrane inlet mass spectrometry

Abstract Denitrification was measured in intact sediment cores and in homogenised slurries using membrane inlet mass spectrometry. Dissolved concentrations of O₂, N₂, N₂O and CO₂ were simultaneously monitored. Using a 0.8 mm diameter needle probe, a comparison was made of the gas profiles of intact cores obtained under different conditions, i.e. with air or argon as the headspace gas and after the addition of nitrate and/or a carbon source to the sediment surface. O₂ was detectable to a depth of 1 cm under a headspace of air and the depth at which the maxima of denitrification products occurred was 1.5–2 cm. Denitrification products (N₂O, N₂) occurred in the surface layers where O₂ was above the minimum level of detectability (> 0.25 μM): diffusion of N₂ and N₂O upwards from the anoxic zone, local anaerobic microenvironments or aerobic denitrification are alternative explanations for this observation. The addition of nitrate and/or acetate increased the concentrations of N₂, N₂O and CO₂ in the sediment core. In sediment slurries, the pH, nitrate concentration, carbon source and the depth from which the sample was taken affected the rate of denitrification. Nitrogen was the sole detectable end product. Maximum denitrification occurred at pH 7.5 and at 20 mM nitrate. Denitrification was at a maximum in those slurries prepared from sections of core at 1–2 cm depth.     

Oxygen and the regulation of nitrogen fixation in legume nodules

In N₂-fixing legume nodules, O₂ is required in large amounts for aerobic respiration, yet nitrogenase, the bacterial enzyme that fixes N₂, is O₂ labile. A high rate of O₂ consumptition and a cortical barrier to gas diffusion work together to maintain a low, non-inhibitory O₂ concentration in the central, infected zone of the nodule. At this low O₂ concentration, cytosolic leghemoglobin is required to facilitate the diffusion of O₂ through the infected cell to the bacteria. The resistance of the cortical diffusion barrier is variable and is used by legume nodules to regulate the O₂ concentration in the infected cells such that it limits aerobic respiration and N₂ fixation at all times. The resistance of the diffusion barrier and therefore the degree of O₂ limitation seems to be regulated in response to changes in the O₂ concentration of the central infected zone, the supply of phloem sap to the nodule, and the rate of N assimilation into the end products of fixation.     

Production, oxidation and emission of methane in rice paddies

Abstract Production and emission of methane from submerged paddy soil was studied in laboratory rice cultures and in Italian paddy fields. Up to 80% of the CH₄ produced in the paddy soil did not reach the atmosphere but was apparently oxidized in the rhizosphere. CH₄ emission through the rice plants was inhibited by an atmosphere of pure O₂ but was stimulated by an atmosphere of pure N₂ or an atmosphere containing 5% acetylene. Gas bubbles taken from the submerged soil contained up to 60% CH₄, but only < 1% CH₄ after the bubbles had passed the soil-water interface or had entered the intercellular gas space system of the rice plants. CH₄ oxidation activities were detected in the oxic surface layer of the submerged paddy soil. Flooding the paddy soil with water containing > 0.15% sea salt (0.01% sulfate) resulted in a strong inhibition of the rates of methanogenesis and a decrease in the rates of CH₄ emission. This result explains the observation of relatively low CH₄ emission rates in rice paddy areas flooded with brackish water.     

The comparative effects of nitrogen and oxygen on the microflora of beef steaks in carbon dioxide-containing modified atmosphere vacuum skin-packaging (MA-VSP) systems

The effects of 80% oxygen–20% carbon dioxide (O₂–CO₂) and 80% nitrogen–20% carbon dioxide (N₂–CO₂) atmospheres were compared with respect to the microbial and sensory characteristics of vacuum skin-packaged grain-fed beef steaks stored at −1 and 4 °C. In both N₂–CO₂ and O₂–CO₂ atmospheres, lactobacilli were predominant over Brochothrix , pseudomonads, enterobacteria and yeasts and moulds. The results of the current investigation showed that the O₂–CO₂ atmospheres did not yield total viable counts in excess of 10⁵ cfu cm⁻² on beef steaks after 4 weeks of storage. However, the sensory analysis and thiobarbituric acid (TBA) values (as a measure of oxidative rancidity) of the products were unacceptable at this time. In contrast, the N₂–CO₂ atmospheres yielded maximum total viable counts of approximately 10⁷ cfu cm⁻² and the sensory analysis and TBA values of the product were judged to be acceptable after 4 weeks of storage at −1 °C. These results indicate that sensory effects of the product were influenced to a greater extent by the chemical effects of high concentration of O₂ on rancidity than by the high levels of lactobacilli.     

Physiological differences between strains of Tribolium castaneum selected for resistance to hypoxia and hypercarbia, and the unselected strain

Abstract. The metabolic rates, as expressed by oxygen (O₂) consumption, carbon dioxide (CO₂) production, and losses in wet and dry weights, were examined for adults of three strains of the red flour beetle Tribolium castaneum (Herbst), during exposure to two modified atmospheres (MAs). Exposure of a strain selected for resistance over twenty-one generations to an atmosphere of 65% CO₂, 20% O₂ and the balance nitrogen (N₂), termed a high carbon dioxide concentration atmosphere (HCC) and exposure of an unselected strain to HCC, showed considerable levels of aerobic metabolism during exposure. For the unselected strain water loss and mobilization of energy reserves were rapid and mortality was followed by rapid desiccation. For the HCC-resistant strain water balance was maintained and energy reserves were utilized more slowly over a prolonged period. Exposure of a strain selected for resistance over twenty-one generations to a low oxygen concentration atmosphere (LOC) of 0.5% O₂ in N₂, and an unselected strain to LOC, revealed that even at 0.5% O₂, metabolism was largely aerobic in both strains. Maintenance of water balance was not a major factor causing mortality of either strain during exposure to LOC. In air, metabolic rates of both the resistant strains were lower than that of the unselected strain.     

Protective mechanisms of nitrogenase against oxygen excess and partially-reduced oxygen intermediates

Diazotrophic systems have developed a number of strategies to protect nitrogenase (N₂ase; EC 1.18.6.1) from O₂ excess and active-oxygen species (AOS). Protection against O₂ excess is given by biochemical modifications of N₂ase, increased rates of low-efficiency respiration, temporal segregation of N₂ fixation and photosynthesis, physical barriers to O₂ diffusion, and hemoglobins. On the other hand, AOS may originate from oxidation of N₂ase components, ferredoxins, flavodoxins and hemoglobins; interaction among the AOS themselves, or between H₂O₂ and hemoglobins; and during reactions catalyzed by hydrogenase (EC 1.18.99.1), xanthine oxidase (EC 1.1.3.22) and uricase (EC 1.7.3.3). Active-oxygen species are scavenged enzymatically [superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6). peroxidase (EC 1.11.1.7), ascorbate peroxidase (EC 1.11.1.11)] or through non-enzymic reaction with low-molecular-weight compounds (ascorbate, α-tocopherol, glutathione).     

Oxygen control prevents denitrifiers and barley plant roots from directly competing for nitrate

Abstract Two denitrifying bacteria ( Pseudomonas chlororaphis and P. aureofaciens ) and a plant (barley, Hordeum vulgare ) were used to study the effect of O₂ concentration on denitrification and NO₃⁻ uptake by roots under well-defined aeration conditions. Bacterial cells in the early stationary phase were kept in a chemostat vessel with vigorous stirring and thus a uniform O₂ concentration in the solution. Both Pseudomonads lacked N₂O reductase and so total denitrification could be directly measured as N₂O production.
Denitrification decreased to 6–13% of the anaerobic rate at 0.01% O₂ saturation (0.14 μM O₂) and was totally inhibited at 0.04% O₂ saturation (0.56 μM O₂). In this well-mixed system denitrification was 10-times more oxygen sensitive than stated in earlier reports. Uptake of nitrate by plants was measured in the same system under light. The NO₃⁻ uptake rate decreased gradually from a maximum in 21% O₂-saturated medium (air saturated) to zero at 1.6% O₂ saturation (22.4 μM O₂). Owing to the very different non-overlapping oxygen requirements of the two processes, direct competition for nitrate between plant roots and denitrifying bacteria cannot occur.     

The influence of reduced photorespiration on long-term growth and development of wheat

The long-term role of photorespiration was investigated by comparing growth, development, gas exchange characteristics and mineral nutrition of a wheat crop ( Triticum aestivum L. cv. Courtot) cultivated in a culture chamber during a life cycle, either in 4% O₂ or in normal O₂ Low O₂ pressure reduced photorespiration, but CO₂ was controlled so that net photosynthesis remained the same as in the control crop. The growth and development of the low O₂ crop was slowed down. Ear appearance was 16 days late, but the rate of tillering was the same as in the control and was maintained longer so that the final number of tillers was doubled. Pigment, ribulose bisphosphate carboxylase (EC 4.1.1.39) and soluble sugar contents were similar. The response of photosynthesis to CO₂ and O₂ was not appreciably changed by the low O₂ treatment. There was almost no seed formation, and the senescence of the leaves was delayed. It appears that in non-stress conditions most of the photorespiration can be suppressed without damage to the photosynthetic apparatus. Retardation of development and inhibition of reproduction are likely due to other effects of O₂.     

Effect of oxygen tension, salinity, temperature and organic matter concentration on the growth and nitrifying activity of an estuarine strain of Nitrosomonas

Abstract The effects of O₂ tension, temperature, salt concentration and organic matter concentration on the growth and nitrifying activity of Nitrosomonas N₃ isolated from Tay Estuary sediments have been investigated. Chemostat-grown cultures were able to grow and nitrify at dissolved O₂ concentrations as low as 0.1 mg O₂· 1⁻¹ (cell population densities were 15% of those obtained in fully aerated cultures). This bacterium was sensitive to reduced temperatures as chemostat-grown cultures washed out at growth temperatures below 15°C, at dilution rates > 0.025 · h⁻¹. Batch-grown cultures of Nitrosomonas N₃ were used to study the effects of NaCl and complex organic matter concentration on nitrifying activity. Maximum rates of NH⁺₄ oxidation were recorded at NaCl concentrations of 1% w/v, whilst tryptone soya broth (TSB), nutrient broth (NB), yeast extract broth (YEB) and peptone were inhibitory at concentrations > 10 mg · 1⁻¹.     

Effects of oxygen, pH and nitrate concentration on denitrification by Pseudomonas species

Abstract The production of nitrogen-containing gases by denitrification in three organisms was examined using membrane inlet mass spectrometry. The effects of O₂ (during both growth and maintenance) and of pH, nitrate concentration and carbon source were tested in non-proliferating cell suspensions. Two strains of Pseudomonas aeruginosa were capable of co-respiration of NO₃⁻ and O₂ and, under controlled O₂ supply, gave oscillatory denitrification. Variations in culture and assay conditions affected both the rate of denitrification and the ratio of end products (N₂O:N₂). Higher rates were seen following anaerobic growth. Optimum values of pH and nitrate concentration for denitrification are given. Generally, the optimum pH was 7.0–7.5, approximately that of the growth medium. Optimum nitrate concentration was generally 20 mM.     

Aerobic nitrogen fixation is confined to a subset of cells in the non-heterocystous cyanobacterium Symploca PCC 8002

Symploca PCC 8002 Kützing is a filamentous cyanobacterium that lacks the specialized cells, known as heterocysts, that protect nitrogenase from O₂ in most aerobic N₂-fixing cyanobacteria. Nevertheless, Symploca is able to carry out N₂ fixation in the light under aerobic conditions. When cultures were grown under light/dark cycles, nitrogenase activity commenced and increased in the light phase and declined towards zero in the dark. Immunolocalization of dinitrogenase reductase in sectioned Symploca trichomes showed that the enzyme was present only in 9% of the cells. These cells lacked any obvious mechanical protection against atmospheric O₂ and their ultrastructural characteristics were similar to those of cells that did not contain any dinitrogenase reductase. The nitrogenase-containing cells possessed carboxysomes that were rich in ribulose-1,5-bisphosphate carboxylase/oxygenase and phycoerythrin, a light harvesting pigment of PS II. This indicates that these cells had a capacity for both N₂ fixation and photosynthesis. The significance of the localization pattern for dinitrogenase reductase is discussed in the context of N₂ fixation in Symploca PCC 8002.     

Effects of mycorrhizal colonization on biomass production and nitrogen fixation of black locust (Robinia pseudoacacia) seedlings grown under elevated atmospheric carbon dioxide

Interactive effects of elevated atmospheric CO₂ and arbuscular mycorrhizal (AM) fungi on biomass production and N₂ fixation were investigated using black locust ( Robinia pseudoacacia ). Seedlings were grown in growth chambers maintained at either 350 μmol mol⁻¹ or 710 μmol mol⁻¹ CO₂. Seedlings were inoculated with Rhizobium spp. and were grown with or without AM fungi. The ¹⁵N isotope dilution method was used to determine N source partitioning between N₂ fixation and inorganic fertilizer uptake. Elevated atmospheric CO₂ significantly increased the percentage of fine roots that were colonized by AM fungi. Mycorrhizal seedlings grown under elevated CO₂ had the greatest overall plant biomass production, nodulation, N and P content, and root N absorption. Additionally, elevated CO₂ levels enhanced nodule and root mass production, as well as N₂ fixation rates, of non- mycorrhizal seedlings. However, the relative response of biomass production to CO₂ enrichment was greater in non-mycorrhizal seedlings than in mycorrhizal seedlings. This study provides strong evidence that arbuscular mycorrhizal fungi play an important role in the extent to which plant nutrition of symbiotic N₂-fixing tree species is affected by enriched atmospheric CO₂.     

Kinetics of leaf oxygen uptake represent in planta activities of respiratory electron transport and terminal oxidases

We present, for the first time, the oxygen response kinetics of mitochondrial respiration measured in intact leaves (sunflower and aspen). Low O₂ concentrations in N₂ (9–1500 ppm) were preset in a flow-through gas exchange measurement system, and the decrease in O₂ concentration and the increase in CO₂ concentration as result of leaf respiration were measured by a zirconium cell O₂ analyser and infrared-absorption CO₂ analyser, respectively. The low O₂ concentrations little influenced the rate of CO₂ evolution during the 60-s exposure. The initial slope of the O₂ uptake curve on the dissolved O₂ concentration basis was relatively constant in leaves of a single species, 1.5 mm s⁻¹ in sunflower and 1.8 mm s⁻¹ in aspen. The apparent K _0.5(O₂) values ranged from 0.33 to 0.67 μ M in sunflower and from 0.33 to 1.1 μ M in aspen, mainly because of the variation of the maximum rate, V _max (leaf temperature 22°C). The initial slope of the O₂ response of respiration characterizes the catalytic efficiency of terminal oxidases, an important parameter of the respiratory machinery in leaves. The plateau of the response characterizes the activity of the mitochondrial electron transport chain and is subject to regulations in accordance with the necessity for ATP production. The relatively low oxygen conductivity of terminal oxidases means that in leaves, less than 10% of the photosynthetic oxygen can be reassimilated by mitochondria.     

Photoactivation of the latent water-oxidizing complex in photosystem II membranes isolated from dark-grown spruce seedlings

Photosystem II membranes (D-PSII) were isolated from dark-grown spruce seedlings. All major PSII proteins except the 17- and 23-kDa extrinsic proteins were present in D-PSII. O₂ evolution and Mn content in D-PSII were negligible, while PSII-donor activity showed a value comparable to that of NH₂OH-treated PSII membranes (NH₂OH-L-PSII) from light-grown seedlings. Light incubation of D-PSII with 1 m M MnCl₂, 50 m M CaCl₂ and 100 μ M DCIP at pH 5.3 resulted in activation of the latent water-oxidizing complex. Accomplishment of photoactivation of PSII membranes from dark-grown spruce seedlings clearly indicates that only ligation of Mn²⁺ to the apo-water oxidizing complex is required for expression of O₂ evolution, and that protein synthesis is not involved in the photoactivation process. There was no essential difference between 'photoactivation' of naturally Mn-free PSII membranes and 'photoreactivation' of artificially Mn-depleted PSII membranes on kinetics, pH dependence, Mn²⁺-concentration dependence. However, kinetics and pH dependence of photoactivation were appreciably different in spruce PSII membranes and in PSII membranes of angiosperms such as wheat and spinach.     

Associative cellulolysis and N2 fixation by co-cultures of Trichoderma harzianum and Clostridium butyricum: the effects of ammonium-N on these processes

Mixed cultures of the cellulolytic fungus Trichoderma harzianum with the anaerobic diazotroph Clostridium butyricum were shown to co-operatively degrade cellulose and utilize the degradation products for N₂ fixation. Cellulose degradation and N₂ fixation were stimulated by small (0.1 mg/ml) additions of (NH₄)₂SO₄. The (NH₄₂SO₄ stimulates cellulolysis thereby increasing the supply of cellulose degradation products to the diazotroph. In aerobic environments the anaerobe depends on the respiration of the aerobe to create anaerobic microsites. The N source increased O₂ uptake by the fungus increasing the number of sites suitable for the development of the anaerobe. Stimulation in the growth of T. harzianum by (NH₄₂SO₄ resulted in increased growth and N₂ fixation by Cl. butyricum.     

Effects of salicylhydroxamate on respiration, seed germination and seedling growth in Avena fatua

The effects of inhibitors of alternative respiration [salicylhydroxamate (SHAM) and propyl gallate (PG)] on germination, seedling growth and O₂ uptake in Avena fatua L. (wild oats) were studied. SHAM did not inhibit germination or O₂ uptake prior to germination. SHAM-sensitive (alternative) respiration, therefore, cannot be a pre-requisite for germination. Following germination, both chemicals inhibited seedling growth with the root being more susceptible than the shoot. SHAM concentrations that inhibited root growth by 90 to 95%, inhibited O₂ uptake of 1 cm root apices by less than 15%. While sodium azide (a cytochrome-oxidase inhibitor; 1 m M ) alone inhibited O₂ uptake by only 40 to 50%, in the simultaneous presence of SHAM (or PG), O₂ uptake was inhibited by 90 to 99%. Thus: 1) respiration of wild oat seedling root apices is predominantly cytochrome-mediated and incomplete inhibition of O₂ uptake in the presence of azide alone is due to diversion of electrons to the alternative pathway and 2) even though these roots have little alternative respiration, they maintain the capacity to support a much greater flux of electrons via this path way. SHAM and PG at concentrations (0.05 to 0.4 m M ) which inhibited O₂ uptake significantly in the presence (but not in the absence) of azide had little effect on root growth suggesting that an effect(s) other than that on respiration is involved in the inhibition of root growth at higher concentrations. The effect of SHAM on wild oat root growth is not selective as it also inhibits growth of a number of crop species.     

The Glycine-Glomus-Bradyrhizobiun symbiosis. XI. Nodule gas exchange and efficiency as a function of soil and root water status in mycorrhizal soybean

Soybean [ Glycine max (L.) Merr. cv. Hobbit] plants were inoculated with a HUP− strain of Bradyrhizobium japonicum (Nitragin 61A118) and either colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol & Gerd.) Gerd. and Trappe or fertilized with KH₂PO₄ (nonVAM). They were grown for 50 days in a growth chamber and harvested over a 4-day drought period during which available soil water decreased to 0. Nodule P concentrations and P-use efficiency declined linearly with soil and root water content during the harvest period in both VAM and nonVAM plants. Nitrogenase activity, estimated from H₂ evolution and C₂H₂ reduction data, was also a linear function of declining nodule P concentrations and CO₂-exchange rates and showed simular patterns in both treatments. Hydrogen evolution and the relative efficiency of N₂ fixation, on the other hand, reacted differently to increasing drought in VAM and nonVAM plants. Differences in the responses of nodule activity in VAM and nonVAM plants to drought are interpreted in terms of demand for nodule P and carbohydrates and of the effects of dehydration on O₂ diffusion through nodule tissue.