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.     

Extracellular superoxide production, viability and redox poise in response to desiccation in recalcitrant Castanea sativa seeds

Reactive oxygen species (ROS) are implicated in seed death following dehydration in desiccation-intolerant 'recalcitrant' seeds. However, it is unknown if and how ROS are produced in the apoplast and if they play a role in stress signalling during desiccation. We studied intracellular damage and extracellular superoxide (O₂^·−) production upon desiccation in Castanea sativa seeds, mechanisms of O₂^·− production and the effect of exogenously supplied ROS. A transient increase in extracellular O₂^·− production by the embryonic axes preceded significant desiccation-induced viability loss. Thereafter, progressively more oxidizing intracellular conditions, as indicated by a significant shift in glutathione half-cell reduction potential, accompanied cell and axis death, coinciding with the disruption of nuclear membranes. Most hydrogen peroxide (H₂O₂)-dependent O₂^·− production was found in a cell wall fraction that contained extracellular peroxidases (ECPOX) with molecular masses of ∼50 kDa. Cinnamic acid was identified as a potential reductant required for ECPOX-mediated O₂^·− production. H₂O₂, applied exogenously to mimic the transient ROS burst at the onset of desiccation, counteracted viability loss of sub-lethally desiccation-stressed seeds and of excised embryonic axes grown in tissue culture. Hence, extracellular ROS produced by embryonic axes appear to be important signalling components involved in wound response, regeneration and growth.     

Differential response of four tree species to ozone-induced acceleration of foliar senescence

Ozone (O₃)-induced accelerated senescence of leaves was measured in four tree species: black cherry ( Prunus serotina ), hybrid poplar ( Populus maximowizii x trichocarpa , clone 245), northern red oak ( Quercus rubra ) and sugar maple ( Acer saccharum ). Seedlings or ramets of the four species were subjected to chronic O₃ exposures and designated leaves harvested periodically from emergence to senescence. Gas exchange was analysed, and concentrations of total soluble protein and ribulose-1,5-bisphosphate carboxylase/oxygenase were measured as indicators of leaf senescence. Total antioxidant potential and ascorbate peroxidase and glutathione reductase activities also were determined. Black cherry and hybrid poplar exhibited O₃-induced accelerated leaf senescence, whereas sugar maple and northern red oak did not. When the O₃ effects were related to cumulative uptake of the gas, black cherry was the most sensitive of the four species. Although hybrid poplar exhibited similar symptoms of O₃-induced accelerated senescence after the same exposure period as did black cherry, this species took up much greater quantities of O₃ to achieve the same response. The O₃-induced increase in glutathione reductase activity in hybrid poplar was consistent with the capacity of this species to take up high concentrations of the gas. Relative tolerance of northern red oak and sugar maple could be explained only in part by lower cumulative O₃ uptake and lower rate of uptake. Sugar maple had the highest antioxidant potential of all four species, which may have contributed to O₃ tolerance of this species. Ascorbate peroxidase activity, when expressed on a fresh weight basis, could not account for differential sensitivity among the four species.     

Ozone-induced oxidative stress: Mechanisms of action and reaction

In this review we explore several models which might explain ozone (O₃)-induced injury to plant foliage. Ozone enters the cell through the wall and plasma membrane where active oxygen species are generated. If the concentration of O₃ is very high, unregulated cell death will occur. Alternatively, the active oxygen species, or succeeding reaction products, may serve as elicitors of regulated plant responses. These regulated responses include the induction of ethylene which could serve as a primary signal for—or a facilitator of—subsequent responses. The role of regulated suppression of photosynthetic genes and induction of chitinases and β-1,3-glucanase in programmed cell death is explored. Induction of antioxidants, enzymes of lignification and glutathione- S -transferase are discussed in the context of O₃-induced cell repair or cell protection. A second model is postulated to explain induction of accelerated foliar senescence by low levels of O₃. The notion that O₃-induced elicitation of responses in the nucleus might lead to increased oxidative stress in the chloroplast is considered as a mechanism for accelerating the rate of degradation of ribulose-1,5-bisphosphate car-boxylase/oxygenase (Rubisco). The mechanisms by which O₃ induces loss of Rubisco, and the relationship to accelerated foliar senescence are 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.     

A novel device detects a rapid ozone-induced transient stomatal closure in intact Arabidopsis and its absence in abi2 mutant

To follow stomatal responses to ozone (O₃) in different Arabidopsis lines, we constructed a rapid-response O₃ exposure/gas-exchange measurement device consisting of eight through-flow whole-rosette cuvettes. To separate rosette from roots and growth substrate, plant is grown through an agar-filled hole in a polished glass plate fixed on the pot. Following insertion of the plant, the plate forms air-tight bottom surface of the cuvette; thus the rosette is enclosed without touching it during any phase of the insertion of the plant to the cuvette. The device allows monitoring rapid responses in the stomatal function. For example, an acute exposure to 150 ppb O₃ decreased stomatal conductance to 60–70% of its initial value within 9–12 min. Thereafter, the conductance regained its preexposure value within further 30–40 min in spite of the continuing O₃ exposure. The transient decrease was absent in the abscisic acid-insensitive mutant abi2 defective in a class 2C protein phosphatase. This provides an in vivo confirmation that the early transient decrease in stomatal conductance is not a result of physical damage by the reactive oxygen species (ROS) formed from O₃ breakdown but reflects the biological action of ROS, transduced through a signalling cascade. Thus, the apparatus will be helpful in specifying complex molecular and genetic interactions in rapid responses in guard cells in vivo.     

Induction of proteins during phage reactivation induced by UV irradiation, oxygen and peroxide in Bacteroides fragilis

Abstract Phage reactivation systems in Bacteroides fragilis were induced by far-UV irradiation, O₂ and H₂O₂. These three treatments also induced the synthesis of 3, 6, and 4 protein bands, respectively, which were easily detectable by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Two proteins with apparent M _r s of approx. 90 000 and 70 000 were induced by all three treatments. Caffeine completely inhibited UV- and O₂-induced phage reactivation and prevented the synthesis of the M _r 90 000 and M _r 70 000 proteins. The results suggest that these two proteins may be involved in phage reactivation processes induced by UV, O₂ and H₂O₂ in B. fragilis .     

EDU and ozone protection: Foliar glycerolipids and steryl lipids in snapbean exposed to O3

Effects of the antiozonant EDU, N-[2-(2-oxo-1-imidazolidinyl) ethyl]-N'-phenylurea, on the content and composition of foliar lipids in snapbean ( Phaseolus vulgaris L. cv. Bush Blue Lake 290) before and after a single, acute ozone (O₃) exposure were assessed. Pretreatment with EDU conferred protection against O₃-induced necrosis and losses of glycerolipids and chlorophyll. Systemic treatment of snapbean plants with EDU did not significantly alter membrane lipids in the first trifoliate leaf. Leaves of untreated controls had lost ca 50% of both galacto- (GL) and phospholipids (PL) by the end of a 3 h exposure to 0.4 μl l⁻¹ O₃. A decline in the ratio of mono- to di-galactosyldiacylglycerol (MGDG/DGDG) was associated with the loss of GL, and a decline in the ratio of linoleic to linolenic acid (18:2/18:3) was associated with the loss of PL in untreated controls. EDU-treated plants showed no significant loss of foliar GL and PL. The MGDG/DGDG ratio declined only slightly, and the 18:2/18:3 ratio in PL increased during O₃ exposure of EDU-treated seedlings. The level of total membrane sterols, including free sterols (FS), acylated steryl glycosides (ASG) and steryl glycosides (SG), did not change during O₃ exposure of either treated or untreated plants. However, in the controls the proportions of ASG and SG increased at the expense of FS, and the ratio of stigmasterol/sitolsterol increased in FS and SG. In EDU-treated plants, a relatively small increase in SG was offset by a decrease in FS, and there was no change in the stigmasterol/sitosterol ratio in ASG, SG or FS. The results indicate that EDU may confer tolerance to O₃ through induction of enzyme systems involved in the elimination of activated oxygen species and free radicals.     

Hydrogen Production by Rumen Holotrich Protozoa: Effects of Oxygen and Implications for Metabolic Control by In Situ Conditions

Experiments with washed suspensions of holotrich protozoa (Isotricha spp. and Dasytricha ruminantium ) showed that both organisms have an efficient 0,-scavenging capability (apparent K_m values 2.3 and 0.3 μM, respectively). Reversible inhibition of H₂, production increased almost linearly with increasing O₂ up to 1.5 μM; higher levels of O₂ gave irreversible inhibition. In situ determinations of H, CH₄, O₂, and CO₂, in ovine rumen liquor, using a membrane inlet mass spectrometer probe, indicated that O₂, was present before feeding at 1-1.5 μM and decreased to undetectable levels (<0.25 μM) within 25 min after feeding. A transient increase in O₂. concentration after feeding occurred only in defaunated animals and resulted in suppression of CH₄ and CO₂ production. The presence of washed holotrich protozoa decreases the O₂ sensitivity of CH₄ production by suspensions of a cultured methanogenic bacterium Methanosarcina barkeri . It is concluded that holotrich protozoa play a role in ruminal O₂ utilization as well as in the production of fermentation end products (especially short-chain volatile fatty acids) utilized by the ruminant and H, utilized by methanogenic bacteria. These hydrogenosome-containing protozoa thus both control patterns of fermentation by influencing O₂ levels, and are themselves regulated by the low ambient O₂ concentrations they experience in the rumen.     

Nitric oxide reduces Cu toxicity and Cu-induced NH4+ accumulation in rice leaves

Nitric oxide (NO) is a highly reactive, membrane-permeable free radical, which has recently emerged as an important antioxidant. Here we investigated the protective effect of NO against the toxicity and NH₄⁺ accumulation in rice leaves caused by excess CuSO₄ (10 mmol L⁻¹). It was found that free radical scavengers (sodium benzoate, thiourea, and reduced glutathione) reduced the toxicity and NH₄⁺ accumulation in rice leaves caused by excess CuSO₄. NO donor sodium nitroprusside (SNP) was also effective in reducing CuSO₄-induced toxicity and NH₄⁺ accumulation in rice leaves. The protective effect of SNP on the toxicity and NH₄⁺ accumulation can be reversed by 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethyl- imidazoline-1-oxyl-3-oxide, a NO scavenger, suggesting that the protective effect of SNP is attributable to NO released. Results obtained in the present study suggest that reduction of CuSO₄-induced toxicity and NH₄⁺ accumulation by SNP is most likely mediated through its ability to scavenge active oxygen species.     

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.     

The presence of oxygen in rumen liquor and its effects on methanogenesis

In situ measurement of O₂ in the rumen liquor of cows, sheep and goats using a membrane-covered O₂ electrode revealed the presence of up to 1630 nmol/l O₂; O₂ became undetectable immediately after feeding of animals. The effects of O₂ on H₂ production and methanogenesis in samples of rumen liquor were investigated using a mass spectrometer fitted with a membrane inlet system. Methanogenesis was totally and irreversibly inhibited after short term exposure (about 10 min) to 5 KPa (0·05 atm) O₂; H₂ production was unaffected. Glucose additions produced rapid transient increases in H₂ levels and increased O₂ uptake.     

Ozone induced emissions of biogenic VOC from tobacco: relationships between ozone uptake and emission of LOX products

Volatile organic compound (VOC) emissions from tobacco ( Nicotiana tabacum L. var. Bel W3) plants exposed to ozone (O₃) were investigated using proton-transfer-reaction mass-spectrometry (PTR-MS) and gas chromatography mass-spectrometry (GC-MS) to find a quantitative reference for plants' responses to O₃ stress. O₃ exposures to illuminated plants induced post-exposure VOC emission bursts. The lag time for the onset of volatile C₆ emissions produced within the octadecanoid pathway was found to be inversely proportional to O₃ uptake, or more precisely, to the O₃ flux density into the plants. In cases of short O₃ pulses of identical duration the total amount of these emitted C₆ VOC was related to the O₃ flux density into the plants, and not to ozone concentrations or dose–response relationships such as AOT 40 values. Approximately one C₆ product was emitted per five O₃ molecules taken up by the plant. A threshold flux density of O₃ inducing emissions of C₆ products was found to be (1.6 ± 0.7) × 10⁻⁸ mol m⁻² s⁻¹.     

Aluminium Inhibits Muscarinic Agonist-Induced Inositol 1,4,5-Trisphosphate Production and Calcium Mobilization in Permeabilized SH-SY5Y Human Neuroblastoma Cells

Abstract: The effects of aluminium (as Al³⁺) on carbachol-induced inositol 1,4,5-trisphosphate (lnsP₃) production arid Ca²⁺ mobilisation were assessed in electropermeabilised human SH-SY5Y neuroblastoma cells. Al³⁺ had no effect on lnsP₃-induced Ca²⁺ release but appreciably reduced carbachol-induced Ca²⁺ release (lC₅₀ of ∼90 μ M ). Aβ³⁺ also inhibited lnsP₃ production (lC₆₀ of ∼15 μ M ). Dimethyl hydroxypyridin-4-one, a potent Al³⁺ chelator (K₅= 31), at 100 μ M was able to abort and reverse the effects of Al³⁺ on both Ca²⁺ release and lnsP₃ production. These data suggest that, in permeabilised cells, the effect of Al³⁺ on the phosphoinositide-mediated signalling pathway is at the level of phosphatidylinositol 4,5-bisphosphate hydrolysis. This may reflect interference with receptor-G protein-phospholipase C coupling or an interaction with phosphatidylinositol 4,5-bisphosphate.     

Azide-stimulated oxygen consumption by the green alga Selenastrum minutum

Dark O₂ consumption by the green alga Selenastrum minutum was sensitive to inhibition by the cytochrome pathway respiration inhibitor cyanide in the absence of an alternative oxidase inhibitor, consistent with previous work that suggested that this alga lacks alternative oxidase capacity. In contrast, addition of low concentrations of the cytochrome pathway inhibitor azide (50–750 μ M ) resulted in a stimulation of dark O₂ consumption, while higher concentrations of azide (1–2 m M ) partially inhibited O₂ consumption. Measurements of changes in cellular levels of pyruvate, malate and pyridine nucleotides upon cyanide addition were consistent with the absence of alternative oxidase capacity, and suggested that cyanide inhibition of O₂ consumption was not due to nonspecific effects of cyanide. Addition of salicylhydroxamic acid (SHAM) also resulted in an increase in the rate of O₂ consumption. Both azide- and SHAM-stimulated O₂ consumption were sensitive to inhibition by 50 m M ascorbate or by cyanide. However, the ubiquinone analogs chloroquine and quinacrine specifically inhibited azide-stimulated O₂ consumption, with only minor effects on SHAM-stimulated O₂ consumption. These results suggest that azide-stimulated O₂ consumption was not mediated by the previously characterized SHAM-stimulated oxidase, and are consistent with the possibility that azide-stimulated O₂ consumption is mediated by a plasma membrane redox system.     

Mycelial growth and ochratoxin A production by Aspergillus section Nigri on simulated grape medium in modified atmospheres

Aims:  To evaluate the impact of modified atmosphere packaging on in vitro growth of Aspergillus carbonarius and Aspergillus niger , and possible effects on ochratoxin A (OTA) biosynthesis.
Methods and Results:  Ochratoxigenic isolates belonging to the species A. carbonarius and A. niger were grown on a synthetic grapejuice medium (SNM) and packaged in combinations of controlled O₂ (1% and 5%) and CO₂ levels (0% and 15%), and in air as a control. Colony diameters were recorded every 3 days up to 21 days, and OTA was analysed after 7, 14 and 21 days. The greatest reductions in mycelial growth rate were observed at 1% O₂ followed by 1% O₂/15% CO₂, whereas 5% O₂ stimulated the growth of all isolates. OTA production by A. carbonarius and A. niger isolates was minimized at 1% O₂/15% CO₂ and 1% O₂, respectively, after 7 days of incubation. Maximal OTA accumulation after 7 days was observed for all isolates in the control pack and at 5% O₂.
Conclusions:  Of the atmospheres tested, only 1% O₂ combined with 15% CO₂ consistently reduced fungal growth and OTA synthesis by A. carbonarius and A. niger .
Significance and Impact of the Study:  Storage under modified atmospheres is unlikely to be suitable as the sole method for OTA minimization and grape preservation; other inhibitory factors are necessary.     

Effects of propylene and oxygen on the ethylene-producing system of apples

Hypobaric conditions and treatments with ethylene and the ethylene analogue propylene were used to investigate effects of oxygen and elhylene on 1-aminocyclopropane-1-carboxylic acid (ACC) content, ACC synthase activity and ethylene production of apples ( Malus sylveslris Mill. cv. Golden Delicious). Prcclimacteric apples were stored in air at 6.6 kPa (reduced pressure); 6.6 kPa ventilated with pure O₂; 6.6 kPa ventilated with 2600 μl 1⁻¹ C₂H₄; and in air at 101.3 kPa (atmospheric pressure) for 4 months at 4°C. No ACC synthase activity was detectable in apples stored at 6.6 kPa, whereas ACC synthase activity was induced in apples stored at 6.6 kPa and ventilated with either O₂ or C₂H₄. In a further experiment, preclimacteric apples were stored for 14 days either in air at 20 kPa or at 20 kPa ventilated with pure O₂. Both treatments were supplied with 58 500 μl 1⁻¹ propylene from day 0 to day 9 or from day 9 to day 12. Ethylene production of apples treated with propylene from day 0 to day 9 increased earlier than ethylene production of untreated apples. Propylene treatment from day 9 to day 12 did not stimulate ethylene production. Ethylene and propylene induced and stimulated extractable ACC synthase activity and ACC formation of apples. Oxygen enhanced this effect. The results also suggest inhibition of in vivo ACC synthase activity by propylene.     

Atmospheric nitrogenous compounds and ozone – is NOx fixation by plants a possible solution?

Air quality thresholds for O₃ for the protection of human health and vegetation set by the European Union (EU) have been exceeded in Europe regularly in the 1990s. Because target reductions for oxides of nitrogen (NO_x) set for the year 2000 are unlikely to be achieved, these O₃ exceedances are likely to continue into the next millenium. Improvements of plant tolerance towards O₃ are being investigated but very little work has been done to explore NO_x tolerance and plant acclimation to NO₂ and NO. However, it is clear that within the populations of some plant species there is wide variation, and some individuals can fix NO_x and use the nitrogen directly from the atmosphere, rather than rely upon, for example, root uptake of nitrate. It is possible that individuals capable of fixing NO_x could be selected for a range of species, and genotypes with high rates of uptake could be of value as crops or for forestation in polluted areas (e.g. landscaping in the vicinity of motorways) to reduce tropospheric concentrations of NO_x significantly and also to decrease the potential for O₃ production.