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.     

Photosynthetic oxygen evolution within Sesbania rostrata stem nodules

The tropical wetland legume, Sesbania rostrata Brem. forms N₂-fixing nodules along its stem and on its roots after infection by Azorhizobium caulinodans . The N₂-fixing tissue is surrounded by a cortex of uninfected cells which, in the stem nodules (but not the root nodules), contain chloroplasts. The photosynthetic competence of these chloroplasts was assessed through a novel technique involving image analysis of chlorophyll a fluorescence. Calculation of the quantum efficiency of photosystem II (PS II) photochemistry from these images indicated that most of the chloroplasts with potential for non-cyclic photosynthetic electron transport were concentrated within the mid- and inner-cortex, close to the edge of the N₂-fixing tissue. PS II activity in the cortical cells was confirmed in vivo using O₂-specific microelectrodes which showed that the concentration of O₂ (pO₂) in the outer cortex could rise from less than 1% up to 23.4% upon increased irradiance of the nodule, but that the pO₂ of the inner cortex and infected tissue remained less than 0.0025%. Nitrogenase activity of stem nodules, as measured using a flow-through acetylene reduction assay (no H₂ evolution was evident), showed a reversible increase of 28% upon exposure of the nodules to supplemental light. This increase resembled that obtained with stem nodules upon their exposure to an external pO₂ of 40%.     

Gaseous diffusive properties of soybean nodules cultured with non-ambient pO2

Measurements of the short-term response of nodulated roots of soybean ( Glycine max L. Merr, cv. Harosoy: Bradyrhizobium japonicum USDA 16) to rapid changes in surrounding pO₂ indicate that their ability to reversibly adjust gaseous diffusive resistance is retained whether plants are cultured in rhizospheres of very low (2.8%) or very high (61.2%) pO₂. Thus the capacity for reversible short-term diffusion adjustment is additional to structural changes in the fixed diffusional barriers of nodules which allow their continued fixation of N₂ in unfavourably high or low external pO₂. Anatomical evidence, involving quantitative measurement of intercellular spaces in the cortical tissues using electron microscopy of thin sections, indicates that the major fixed diffusional barrier is a boundary layer of cells in the inner cortex which may be as small as one cell thick in nodules from 2.8% O₂ to 5 or 6 cells thick, and almost completely devoid of intercellular spaces, in those from 61.2% O₂. The data are interpreted to indicate that the variable diffusion harrier is distinct from the boundary layer and is most likely to be a property of cells and/or intercellular spaces inside the boundary layer of the nodule cortex.     

Use of the response of photosynthesis to oxygen to estimate mesophyll conductance to carbon dioxide in water-stressed soybean leaves

Methods of estimating the mesophyll conductance (g_m) to the movement of CO₂ from the substomatal airspace to the site of fixation are expensive or rely upon numerous assumptions. It is proposed that, for C₃ species, measurement of the response of photosynthesis to [O₂] at limiting [CO₂], combined with a standard biochemical model of photosynthesis, can provide an estimate of g_m. This method was used to determine whether g_m changed with [CO₂] and with water stress in soybean leaves. The value of g_m estimated using the O₂ response method agreed with values obtained using other methods. The g_m was unchanged over the tested range of substomatal [CO₂]. Water stress, which decreased stomatal conductance (g_s) by about 80%, did not affect g_m, while the model parameter V_Cmax was reduced by about 25%. Leaves with g_s reduced by about 90% had g_m values reduced by about 50%, while V_Cmax was reduced by about 64%. It is concluded that g_m in C₃ species can be conveniently estimated using the response of photosynthesis to [O₂] at limiting [CO₂], and that g_m in soybean was much less sensitive to water stress than g_s, and was somewhat less sensitive to water stress than V_Cmax.     

Oxygen Perception and O2 Toxicity in the Freshwater Ciliated Protozoon Loxodes

ABSTRACT. Loxodes reached peak abundance close to the oxic-anoxic boundary (O₂ 5% atm) in two lakes, in test tube cultures, and in glass chambers with horizontal O₂ gradients. Vertical profiles of CO₂, pH, sulfide, and Fe²⁺ in a lake were not closely related to Loxodes abundance. In a laboratory experiment, Loxodes followed a retreating source of O₂ and was repelled by a high pO₂. This behavior was sustained when cells simultaneously swam up or down gradients of both CO₂ and pH. Aggregation of cells was abolished by KCN (10^-4-10^-6 M). Sodium azide (10^-1-10^-4 M) had no effect and 2,4-DNP sharpened the aggregation. Rotenone, Antimycin A, and HOQNO had no obvious effect. Cytochrome oxidase is probably the oxygen receptor. Loxodes striatus contained low activities of superoxide dismutase and catalase. Extracellular production of superoxide (O_-²) and hydrogen peroxide (H₂O₂) were probably not responsible for the exclusion of Loxodes from water with a high pO₂. Continuous exposure of Loxodes to oxygen at normal atmospheric pressure at 10°C led to 50% mortality in 10 days. Cells left free to swim in an oxygen gradient doubled their number in the same period. Light exacerbated the toxic effects of O₂. Behavioral responses to the dissolved oxygen tension probably controlled the spatial distribution of Loxodes.     

Glycolate metabolism in cyanobacteria

A comparative analysis of glycolate excretion in 11 cyanobacteria showed that 8 strains, although grown and assayed in air, excreted glycolate. The largest quantities were excreted by the filamentous strains Plectonema boryanum 73110 and Anabaena cylindrica (Lemm). The carbon lost by excretion was at most 9% of the net fixed carbon in air for heterocystous cyanobacteria but increased (up to 60%) in some strains under a high pO₂ (0.03 kPa CO₂ in pure O₂). A. cylindrica excreted glycolate at a maximum level of 2 and 10 μmol (mg chl a )⁻¹ h⁻¹ in air and at high pO₂, respectively. The excretion continued for several hours. Increases in light intensity and pO₂ and a shift in pH from 7 to 9 increased the amount of glycolate excreted. A. cylindrica also showed the most O₂-sensitive fixation of CO₂. In vitro activity of phosphoglycolate phosphatase (EC 3.1.3.18) was found in all strains tested, with the highest activities noted for Gloeobacter violaceus 7.82 and Gloeothece 6909 and for young cultures of A. cylindrica . The lowest activities were found in Anabaena 7120 and Anacystis nidulans 625, strains excreting no or only minor quantities of glycolate.     

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.     

Effects of rapid changes in oxygen concentration on the respiration of carrot roots

Rates of CO₂ production and O₂ consumption from aged disks of carrot ( Daucus carota L.) root tissues were measured for 4 h after they were transferred from 21% to 0, 1, 2, 4 or 8% O₂ in gas mixtures. A transient peak in the rate of CO₂ production started 5 to 7 min after transfer to 2% or lower O₂ mixtures and peaked at 50 min. After the peaks in CO₂ production from the 0, 1 and 2% O₂ treatments and after the stable production from the 4 and 8% O₂ treatments, the rate of CO₂ production from all low O₂ treatments started to decline at 50 min, reaching stable rates by 160 to 240 min. Concentrations of lactate and ethanol that were significantly higher than the 21% O₂ controls had started to accumulate in disks between 10 and 50 min after exposure to atmospheres containing 2% or less O₂. Production of CO₂ started to increase 5 to 7 min after transfer to 0, 1 and 2% O₂, while the initial decline and then rise in pH and the accumulation of ethanol did not occur until 30 min after the change in atmosphere. Ethanol accumulation paralleled the increase in pH; first at 0.4 μmol g⁻¹ h⁻¹ from 30 to 60 min as the pH shifted from 5.97 to 6.11, and then at 0.08 μmol g⁻¹ h⁻¹ from 60 to 100 min as the pH stablized around 6.12. The peak at 50 min in CO₂ production roughly coincided with the shift from the rapid to the slow change in pH and ethanol accumulation.     

Accumulation of γ‐aminobutyric acid in nodulated soybean in response to drought stress

Nitrogen fixation and nodule permeability to O₂ diffusion are decreased by drought stress. Since γ‐aminobutyric acid (GABA) synthesis is rapidly stimulated by a variety of stress conditions including hypoxia, it was hypothesized that decreased O₂ availability in nodules stimulates glutamate decarboxylase (GAD) activity (EC 4.1.1.15), thereby resulting in GABA accumulation. First, the amino acid composition of xylem sap was determined in plants subjected to soil water deficits. While the xylem sap concentration of several amino acids increased when the plant was subjected to a water deficit, the greatest increase was in GABA. GABA accumulation was examined in response to stress induced by hypoxia or the addition of polyethylene glycol (PEG) to the nutrient solution. The exposure of soybean nodules to hypoxia for 6 h enhanced the GABA concentration by 6‐fold, but there was no change in GABA concentration in response to the PEG treatment. No major changes in the in vitro GAD activity were measured in nodule cytosol or bacteroids. The present data do not support the hypothesis that decreased nodule O₂ permeability and a resulting O₂ deprivation inside nodules may stimulate in vitro GAD activity and thus GABA accumulation. However, the data could indicate a possible effect of hypoxia and drought stress on the in vivo activity of GAD.     

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.     

Response of net photosynthesis in bean (Phaseolus vulgaris) leaves to the elevation of the partial pressures of oxygen and carbon dioxide

Bean ( Phaseolus vulgaris L. cv. Golden Saxa) plants were grown under low artificial light or under natural daylight. The rate of net photosynthesis (P_N) was measured at: CO₂ partial pressure, p(CO₂), of 0.03, 0.09 or 0.15 kPa; O₂ partial pressure, p(O₂), of 2, 21 or 31 kPa and at light intensities of 350 or 1000 μmol m⁻² s⁻¹ (photosynthetically active radiation). In plants which had been grown under natural light, stimulation of P_N at 21 kPa p(O₂) was found only at elevated p(CO₂) and high light. It is proposed that this phenomenon is dependent on a high capacity of the photosynthetic apparatus to regenerate ribulose 1.5-bisphosphate.     

Effects of elevated ozone on photosynthesis and stomatal conductance of two soybean varieties: a case study to assess impacts of one component of predicted global climate change

Global climatic change scenarios predict a significant increase in future tropospheric ozone (O₃) concentrations. The present investigation was done to assess the effects of elevated O₃ (70 and 100 ppb) on electron transport, carbon fixation, stomatal conductance and pigment concentrations in two tropical soybean ( Glycine max L.) varieties, PK 472 and Bragg. Plants were exposed to O₃ for 4 h·day⁻¹ from 10:00 to 14:00 from germination to maturity. Photosynthesis of both varieties were adversely affected, but the reduction was higher in PK 472 than Bragg. A comparison of chlorophyll a fluorescence kinetics with carbon fixation suggested greater sensitivity of dark reactions than light reactions of photosynthesis to O₃ stress. The O₃-induced uncoupling between photosynthesis and stomatal conductance in PK 472 suggests the reduction in photosynthesis may be attributed to a factor other than reduced stomatal conductance. An increase in internal CO₂ concentration in both O₃-treated soybean varieties compared suggests that the reduction in photosynthesis was due to damage to the photosynthetic apparatus, leading to accumulation of internal CO₂ and stomatal closure. The adverse impact of O₃ stress increased at higher O₃ concentrations in both soybean varieties leading to large reductions in photosynthesis. This study suggests that O₃-induced reductions in photosynthesis in tropical and temperate varieties are similar.     

Nitrate metabolism in soybean root nodules

The nitrate metabolism in nodules induced by Bradyrhizobium japonicum strain PJ17 on roots of soybean [ Glycine max (L.) Merr. cv. Hodgson] has been characterized by the nitrate reductase (NR; EC 1.6.6.1 and EC 1.6.6.3) activity of both partners of the symbiosis. NR activities of bacteroids and nodular cytosol were comparable and significantly higher than those of the roots. Nitrate reduction led to nitrite accumulation in root nodules, which was maximum after pod filling. The nodule had the capacity to metabolize nitrite via nitrite reductase (NiR; EC 1.6.6.4), at least in the cytosolic fraction. This activity was partly inhibited by the low content of free O₂ in the nodule. Indeed, nitrite accumulation decreased in the presence of an increased external pressure of O₂.     

Control of nitrogen and carbon metabolism in root nodules

Because legume root nodules have high rates of carbon and nitrogen metabolism, they are ideal for the study of plant physiology, biochemistry and molecular biology. Many plant enzymes involved in carbon and nitrogen assimilation have enhanced activity and enzyme protein in nodules as compared to other plant organs. For all intents and purposes the interior of the root nodule is O₂ limited. Both plant and bacterial components of effective root nodules have unique adaptive features for maximizing carbon and nitrogen metabolism in an O₂-limited environment. Plant glycolysis appears to be shunted to malic acid synthesis with further reductive synthesis to fumarate and succinate. Nodule bacteroids utilize these organic acids for the energy to fuel nitrogenase activity. Activities of the plant enzymes phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), malate dehydrogenase (MDH, EC 1.1.1.37) and aspartate aminotransferase (AAT, EC 2.6.1.1), which are very high in nodules, may mediate the flux of carbon between organic and amino acid pools. Dark CO₂ fixation via nodule PEPC can provide up to 25% of the carbon needed for malate and aspartate synthesis. At least three of the plant proteins showing enhanced expression in root nodules are O₂ regulated. Isolation of alfalfa cDNAs encoding PEPC, AAT, NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) and aldolase (EC 4.1.2.13) will offer new tools to assess molecular events controlling nodule carbon and nitrogen metabolism.     

Influence of soil O2 and CO2 on root respiration for Agave deserti

Respiration measured as CO₂ efflux was determined at various soil O₂ and CO₂ concentrations for individual, attached roots of a succulent perennial from the Sonoran Desert, Agave deserti Engelm. The respiration rate increased with increasing O₂ concentration up to about 16% O₂ for established roots and 5% O₂ for rain roots (fine branch roots on established roots induced by wetting of the soil) and then remained fairly constant up to 21% O₂. When O₂ was decreased from 21 to 0%, the respiration rates were similar to those obtained with increasing O₂ concentration. The CO₂ concentration in the root zone, which for the shallow-rooted A. deserti in the field was about 1 000 μl l^-1, did not affect root respiration at concentrations up to 2 000 μl l^-1, but higher concentrations reduced it, respiration being abolished at 20 000 μl l^-1 (2%) CO₂ for both established and rain roots. Upon lowering CO₂ to 1 000 μl l^-1 after exposure to concentrations up to 10000 μl l^-1 CO₂, inhibition of respiration was reversible. Uptake of the vital stain neutral red by root cortical cells was reduced to zero, indicating cell death, in about 4 h at 2% CO₂, substantiating the detrimental effects of high soil CO₂ concentrations on roots of A. deserti . This CO₂ response may explain why roots of desert succulents tend to occur in porous, well-aerated soils.     

Oxygen-dependent stomatal opening in Zea mays leaves. Effect of Light and carbon dioxide

The oxygen requirement for stomatal opening in maize plants ( Zea mays L. hybrid INRA 508) was studied at different CO₂ concentrations and light intensities. In the absence of CO₂, stomatal opening always required O₂, but this requirement decreased with increasing light intensity. In darkness, the lowest O₂ partial pressure needed to obtain a weak stomatal movement was about 50 Pa. This value was lowered to ca 10 Pa in light (320 μmol m⁻² s⁻¹).
On the other hand. in the absence of O₂, CO₂enabled stomatal opening to occur in the light, presumably due to the evolved photosynthetic O₂. Thus, CO₂, which generally reduced stomatal aperture, could induce stomatal movement in anoxia and light. The effect of CO₂ on stomatal opening was closely dependent on O₂ concentration and light intensity. Stomatal aperture appeared CO₂-independent at an O₂ partial pressure which was dependent on light intensity and was about 25 Pa at 320 umol m⁻² s⁻¹.
The presence of a plasmalemma oxidase, in addition to mitochondrial oxidase, might explain the differences in the O² requirement at various light intensities. The possible involvement of such a system in relation to the effect of CO₂ is discussed.     

Species-specific responses to atmospheric carbon dioxide and tropospheric ozone mediate changes in soil carbon

We repeatedly sampled the surface mineral soil (0–20 cm depth) in three northern temperate forest communities over an 11-year experimental fumigation to understand the effects of elevated carbon dioxide (CO₂) and/or elevated phyto-toxic ozone (O₃) on soil carbon (C). After 11 years, there was no significant main effect of CO₂ or O₃ on soil C. However, within the community containing only aspen ( Populus tremuloides Michx.), elevated CO₂ caused a significant decrease in soil C content. Together with the observations of increased litter inputs, this result strongly suggests accelerated decomposition under elevated CO_2. In addition, an initial reduction in the formation of new (fumigation-derived) soil C by O₃ under elevated CO₂ proved to be only a temporary effect, mirroring trends in fine root biomass. Our results contradict predictions of increased soil C under elevated CO₂ and decreased soil C under elevated O₃ and should be considered in models simulating the effects of Earth's altered atmosphere.     

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.     

Effect of modification of storage atmosphere on phospholipids and ultrastructure of cauliflower mitochondria

Differences in mitochondrial membrane composition and ultrastructure were studied after storage of cauliflower ( Brassica oleracea , L., Botrytis group) for 5 days at 25°C in air or under controlled atmospheres: 3% O₂, 21% O₂+ 15% CO₂ or 3% O₂+ 15% CO₂. In air, postharvest senescence involved a 20% decrease in mitochondrial phospholipid content. A large reduction in the relative abundance of phosphati-dylcholine (PC) and in the degree of unsaturation of PC and phosphatidyl ethanolamine (PE) was observed. However, the degree of unsaturation increased in cardiolipin (CL). Storage under 3% O₂ did not prevent phospholipid breakdown. Low O₂ prevented the relative decrease in PC observed during storage in air and the loss of linoleic acid from PC, but not from PE. This relative protection offered by the low O₂ atmosphere was lost under 3% O₂+ 15% CO₂. The high CO₂ atmospheres caused twice as much loss in phospholipids as that observed during storage in air. Extensive loss of mitochondrial protein, a marked decrease in phospholipid to protein ratio, and electron micrograph observations suggest structural alterations in the presence of high CO₂.