Ozone uptake and its effect on photosynthetic parameters of two tobacco cultivars with contrasting ozone sensitivity

Leaf discs of the ozone tolerant tobacco (Nicotiana tabacum L.) cv. Bel B and of the ozone sensitive cv. Bel W3, were exposed to an acute ozone fumigation (300 ppb) for 3 h. We measured ozone uptake by leaves and physiological characteristics before, during and after the treatment, in order to determine if the different O₃ sensitivity was correlated to the leaf uptake. In the tolerant cv. Bel B, O₃ uptake was high during the first 2 h of ozone exposure and then decreased. In the sensitive cv. Bel W3, the rate of O₃ uptake decreased constantly during ozone fumigation. The estimated cumulative uptake over the treatment time was higher (200 ± 30 μmol m^–2) in Bel B than in Bel W3 (130 ± 12 μmol m^–2). Thus, the ozone sensitivity was not correlated with ozone uptake. Stomatal conductance and photosynthesis were significantly inhibited during the fumigation in both cultivars. However, these reductions were strong and irreversible in the cv. Bel W3, while in the cv. Bel B both parameters recovered in the post-fumigation period. Thus, ozone tolerance may be related to a sustained capacity of recovery. There was no linear correlation between ozone uptake and photosynthesis reduction, but a threshold of ozone uptake was found after which photosynthesis was substantially impaired. This threshold may or may not be reached under the same external ozone level, indicating that the AOT40 may not be a sufficiently accurate index for the detection of ozone damage in plants.     

Effects of short-term ozone fumigation on tobacco plants: response of the scavenging system and expression of the glutathione reductase

The role that the constituents of the ascorbate–glutathione cycle play in the mechanism of contrasting ozone sensitivities was examined in mature and old tobacco leaves after acute ozone-fumigation (150 p.p.b., 5 h). Levels of the enzyme activities associated with the detoxifying system were lower in ozone-sensitive Bel W3 control plants than in unfumigated ozone-tolerant Bel B plants. In particular, the endogenous activities of ascorbate peroxidase (APX) and glutathione reductase (GR), and the metabolites ascorbic acid (AA) and reduced glutathione (GSH) were more abundant in Bel B than Bel W3 control plants. These results suggest that the higher tolerance of Bel B to O₃ is associated with a greater initial content of the antioxidant enzymes or metabolites. Only in the mature leaves of the ozone-tolerant Bel B cv. did fumigation trigger activation of APX and, weakly, of dehydroascorbate reductase (DHAR). The activity of these enzymes was significantly lower after ozone treatment in both mature and old leaves of Bel W3 than in control plants. Fumigation had little effect on the ascorbate content. Its main effects on the glutathione pool were that it boosted the oxidized form and lowered the reduced form, particularly in mature Bel W3 leaves. Extractable GR activity remained unchanged in both Bel B and Bel W3 immediately after fumigation, but increased slightly 24 h later, particularly in mature leaves of Bel W3. Exposure to O₃ caused a sharp decline in chloroplastic GR mRNA levels in both cultivars. However, as Western blot analysis failed to detect any major changes in GR protein content at this time, the protein must be highly stable. There is therefore a good correlation between tolerance to O₃ and high endogenous levels of antioxidant metabolites such as AA and GSH in tobacco. In addition, the degree of inducibility of the system discriminates the two cultivars investigated.     

Salicylic acid modulates ozone-induced hypersensitive cell death in tobacco plants

Ozone-tolerant Bel B and ozone-sensitive Bel W3 tobacco cultivars were subjected to acute ozone fumigation (200 p.p.b. for 3 h) and the subcellular localization of H₂O₂ was then studied. H₂O₂ accumulated on the cell walls and plasma membrane of both cultivars but the accumulation pattern differed greatly. H₂O₂ production was high in both cultivars immediately after fumigation, but, in the tolerant Bel B cultivar, after 7 h was only detected in some spongy cells adjacent to epidermal cells. Instead, in the sensitive Bel W3 cultivar, accumulation was still abundant in the cell walls of palisade, spongy and epidermal cells at this time. Significant changes in apoplastic ascorbate pool were noted in both cultivars in the first hours after fumigation. As the reduced ascorbate content remained unchanged, the marked increase in total ascorbate must have originated from the striking increase in dehydroascorbate, particularly in the ozone-sensitive Bel W3. Exposure of plants to ozone resulted in a marked transient increase in both free and conjugated salicylic acid (SA) as well as an increase in the activity of benzoic acid 2-hydroxylase which catalyses SA biosynthesis. SA induction differed greatly in the two cultivars, in that: (1) SA accumulation was far greater in the ozone-sensitive Bel W3 cv. and (2) the maximum SA peak was delayed in Bel W3 and observed only 7 h after fumigation ended. These results suggest that a high SA content, as documented in the ozone-sensitive Bel W3 cultivar, could trigger the production of ROS with subsequent SA-mediated cell-death.     

Different responses of two tobacco cultivars and their cell suspension cultures to quercinin,a novel elicitin from Phytophthora quercina

In this study we describe the response of two tobacco cultivars (Nicotiana tabacum L. cv. Bel B and Bel W3) and their cell suspension cultures to quercinin, a novel elicitin produced by the oak pathogen Phytophthora quercina. N-terminal sequencing of the purified protein proved that it belongs to the basic β-elicitins with threonine on position 13. Both tobacco leaves and cells of the cultivar Bel W3 showed hypersensitive cell death after quercinin treatment. Leaves of Bel B also developed quercinin-induced necrosis but higher concentrations of quercinin were necessary as compared to Bel W3. Also Bel B cells showed cell death induction only at the highest quercinin concentration (20 nM). In cell suspension experiments we also measured the quercinin-induced oxidative burst, which occurred in both cultivars. H₂O₂ production in Bel B increased with increasing quercinin concentration and was inhibited only at the highest elicitin concentration (20 nM) whereas the oxidative burst in Bel W3 was completely abolished by 5 nM quercinin. Furthermore we demonstrated that neither H₂O₂ nor superoxide were responsible for cell death induction since neither the inhibitor diphenyleneiodonium (DPI) nor the enzymes catalase (CAT) and superoxide dismutase (SOD) influenced the hypersensitive reaction (HR) in Bel W3 cells. Due to the different response of Bel W3 and Bel B towards the P. quercina elicitin, our system represents an interesting tool to elucidate signaling pathways in tobacco leading to hypersensitive cell death.     

Biochemical Plant Responses to Ozone : III. Activation of the Defense-Related Proteins beta-1,3-Glucanase and Chitinase in Tobacco Leaves

A single pulse of O₃ (0.15 microliter per liter, 5 hours) induced β-1,3-glucanase and chitinase activities in O₃-sensitive and -tolerant tobacco (Nicotiana tabacum L.) cultivars. In the O₃-sensitive cultivar Bel W3, the response was rapid (maximum after 5 to 10 hours) and was far more pronounced for β-1,3-glucanase (40- to 75-fold) than for chitinase (4-fold). In the O₃-tolerant cultivar Bel B, β-1,3-glucanase was induced up to 30-fold and chitinase up to 3-fold under O₃ concentrations that did not lead to visible damage. Northern blot hybridization showed a marked increase in β-1,3-glucanase mRNA in cultivar Bel W3 between 3 and 24 hours following O₃ treatment, a transient induction in cultivar Bel B, and no change in control plants. The induction of β-1,3-glucanase and chitinase activities following O₃ treatment occurred within the leaf cells and was not found in the intercellular wash fluids. In addition, O₃ treatment increased the amount of the β-1,3-glucan callose, which accumulated predominantly around the necrotic spots in cultivar Bel W3. The results demonstrate that near-ambient O₃ levels can induce pathogenesis-related proteins and may thereby alter the disposition of plants toward pathogen attack.     

Ozone impact on the photosynthetic apparatus and the protective role of polyamines

One of the primary plant mechanisms protecting leaf cells against enhanced atmospheric ozone is the accumulation of polyamines, generally observed as an increase in putrescine level, and in particular its bound form to thylakoid membranes. Ozone-sensitive plants of tobacco (cultivar Bel W3) in contrast to ozone-tolerant Bel B, are not able to increase their endogenous thylakoid membrane-bound putrescine when they are exposed to an atmosphere with enhanced ozone concentration, resulting in reduction of their photosynthetic rates and consequently reduction in plant biomass formation. In comparison to the tolerant cultivar Bel B, a prolongation of ozone exposure thus can lead to typical visible symptoms (necrotic spots) in leaves of the sensitive plant. Exogenously manipulated increase of the cellular putrescine levels of the ozone-sensitive Bel W3 is sufficient to revert these effects, whereas a reduction in endogenous putrescine levels of the tolerant cultivar Bel B renders them sensitive to ozone treatment. The results of this work reveal a regulator role for polyamines in adaptation of the photosynthetic apparatus and consequently to its protection in an environment polluted by ozone.     

Illustration of the Genetic Differences in Ozone Sensitivity Between the Varieties Nicotiana tabacum Bel W3 and Bel B Using Various Plant Systems

The ozone-sensitive tobacco variety Bel W₃ was compared with the tolerant cv. Bel B using amphidiploid and amphihaploid genotypes of both. In search of the first genotypical differences, their reaction to acute ozone treatments was investigated with systems of decreasing degree of complexity: whole plants, grown under field, greenhouse and sterile conditions, excised tissues, calli, CCP, MCP and subcellular reactions. It was common to all systems that a fumigation, which clearly exceeds the threshold of the most tolerant material, led to equal reactions in all genotypes in respect of visible injury and membrane leaching. With whole plants and leaf discs growing conditions were found to influence the ozone threshold more than the genotype. Because the most resistant field-grown plants vary widely in their reaction, only sterile or greenhouse grown genotypes were compared. With the exception of whole sterile plants (no genotypical threshold differences), amphihaploids were more susceptible to ozone than their respective amphidiploids as to threshold and sensitivity spectra in all systems investigated. Higher ozone thresholds were detected for Bel B in all systems with one exception: MCP exhibited a lower threshold but also a lower degree of damage in the first buffer range of the sensitivity spectrum than those of Bel W₃. Post-fumigation starch accumulation in mesophyll chloroplasts was the most prominent subcellular ozone reaction.     

Biochemical plant responses to ozone : I. Differential induction of polyamine and ethylene biosynthesis in tobacco

Polyamine metabolism was examined in tobacco (Nicotiana tabacum L.) exposed to a single ozone treatment (5 or 7 hours) and then postcultivated in pollutant-free air. The levels of free and conjugated putrescine were rapidly increased in the ozone-tolerant cultivar Bel B and remained high for 3 days. This accumulation was preceded by a transient rise of l-arginine decar-boxylase (ADC, EC 4.1.1.19) activity. The ozone-sensitive cultivar Bel W3 showed a rapid production of ethylene and high levels of 1-aminocyclopropane-1-carboxylic acid after 1 to 2 hours of exposure. Induction of putrescine levels and ADC activity was weak in this cultivar and was observed when necrotic lesions developed. Leaf injury occurred in both lines when the molar ratio of putrescine to 1-aminocyclopropane-1-carboxylic acid or ethylene fell short of a certain threshold value. Monocaffeoyl-putrescine, an effective scavenger for oxyradicals, was detected in the apo-plastic fluid of the leaves of cv Bel B and increased upon exposure to ozone. This extracellular localization could allow scavenging of ozone-derived oxyradicals at the first site of their generation. Induction of either polyamine or ethylene pathways may represent a control mechanism for inhibition or promotion of lesion formation and thereby contribute to the disposition of plants for ozone tolerance.     

O2- activates leaf injury, ethylene and salicylic acid synthesis, and the expression of O3-induced genes in O3-exposed tobacco

O3 is the major component of photochemical oxidants and gives rise to visible injuries on plant leaves. In O3-exposed plants, O2- is produced before the formation of the injury, but the role that O2- plays in plant response to 03 exposure is still unknown. To clarify its role, we observed the behavior of plants during O3 exposure after pretreatment with tiron, which is an O2- scavenger. When tiron-pretreated tobacco cv. Bel W3 was exposed to O3, leaf damage was attenuated. In O3-exposed tobacco, tiron inhibited increases in the levels of ethylene and salicylic acid, which promote leaf injury. Tiron pretreatment also suppressed increases in the expression of O3-induced genes. These results suggest that O2- is involved in many plant responses induced by O3 exposure. Bel B, a tobacco cultivar that is genetically related to Bel W3, is reported to be more resistant to O3 than Bel W3, but the reason for this difference is unclear. We investigated the differences between the responses of Bel B and tiron-pretreated Bel W3 to O3 exposure, and we discuss the reasons for the resistance to O3 by comparing the phenotype of Bel B with that of tiron-pretreated Bel W3.     

Control of CO2 fixation during the induction period. The role of thiol-mediated enzyme activation in the alga,Dunaliella

(1) Illumination of the unicellular green alga, Dunaliella, produced a 2–3-fold enhancement of ATPase activity in subsequently lysed algae. Using the inhibitor, tentoxin, it was shown that this light-induced activity, but not the light-independent activity, was attributable to the chloroplast coupling factor, CF₁. (1) A 4–5-fold increase in fructose-1,6-bisphosphatase activity was measured in Dunaliella lysed subsequent to illumination. (3) Experiments with methyl viologen demonstrated that both light-induced CF₁-ATPase and fructose-1,6-bisphosphatase activities were due to thiol-modulation of the enzymes by the algal thioredoxin system. (4) The light-induced increase in fructose-1,6-bisphosphatase activity could be simulated by incubation of intact algae in the dark with dithiothreitol. This thiol-induced increase in enzyme activity was accompanied by a decrease in the induction period of CO₂-dependent O₂ evolution upon subsequent measurement. (5) The kinetics of induction of both enzyme activities were very similar to the kinetics of induction of CO₂-dependent O₂ evolution in Dunaliella. As the light intensity was increased to 180 W · m² the steady-state enzyme activities increased in parallel with the rate of CO₂-dependent O₂ evolution. (6) The results are consistent with the imposition of a kinetic restraint on CO₂ fixation by the extent of enzyme activation under certain conditions in Dunaliella.     

Ozone-induced changes of mRNA levels of β-1,3-glucanase,chitinase and ‘pathogenesis-related’ protein 1b in tobacco plants

Treatment of the ozone-sensitive tobacco cultivar Bel W3 with an ozone pulse (0.15 l/l, 5 h) markedly increased the mRNA level of basic -1,3-glucanase and to a lower degree that of basic chitinase. The increase of -1,3-glucanase mRNA level occurred within 1 h and showed a transient maximum. Seventeen hours after ozone treatment, the -1,3-glucanase mRNA level decreased to lower values. The increase of basic chitinase mRNA level was delayed and was less pronounced than that of -1,3-glucanase mRNA. Cultivar Bel B showed only a small increase of -1,3-glucanase mRNA level after the same ozone treatment, whereas its basic chitinase mRNA was more strongly induced. Prolonged ozone treatment for 2 days of tobacco Bel W3 led to a persistent level of -1,3-glucanase and basic chitinase mRNAs, as well as to an increase of acidic chitinase and pathogenesis-related (PR) 1b mRNA levels. The results indicate that genes so far considered to code for PR proteins may also be involved in the plant response to oxidative stress.     

Activation of an oxidative burst is a general feature of sensitive plants exposed to the air pollutant ozone

Ozone exposure stimulates an oxidative burst in leaves of sensitive plants, resulting in the generation and accumulation of hydrogen peroxide (H₂O₂) in tobacco and tomato, and superoxide (O₂^–?) together with H₂O₂ in Arabidopsis accessions. Accumulation of these reactive oxygen species (ROS) preceded the induction of cell death, and both responses co‐occurred spatially in the periveinal regions of the leaves. Re‐current ozone exposure of the sensitive tobacco cv. Bel W3 in closed chambers or in the field led to an enlargement of existing lesions by priming the border cells for H₂O₂ accumulation. Open top chamber experiments with native herbaceous plants in the field showed that Malva sylvestris L. accumulates O₂^–? at those sites that later exhibit plant cell death. Blocking of ROS accumulation markedly reduced ozone‐induced cell death in tomato, Arabidopsis and M. sylvestris. It is concluded that ozone triggers an in planta generation and accumulation of H₂O₂ and/or O₂^–? depending on the species, accession and cultivar, and that both these reactive oxygen species are involved in the induction of cell death in sensitive crop and native plants.     

Photosynthesis of two poplar clones contrasting in O3 sensitivity

 Two clones of poplar known for their phenomenological difference in response to ozone were fumigated with 150 nl l^–1 of ozone for 5 h. In both clones the treatment significantly reduced the light-saturated rate of CO₂ uptake of recently mature leaves and this was accompanied by a decrease in stomatal conductance. Intercellular CO₂ concentration of the resistant clone increased following the fumigation. After 20 h of recovery, photosynthesis recovered completely only in the resistant clone. Electrolyte leakage of leaf disks increased in both clones to indicate damage to membranes; after the recovery time this parameter only reached values of the control in the resistant clone. The photochemical efficiency of PSII slightly decreased in the resistant clone. In the other clone, the treatment caused a decline of all chlorophyll fluorescence parameters and only some of them returned to normal values after the recovery time. The physiological response appears to be different in the two clones. In the resistant one, the most probable mechanism involved in the photosynthetic reduction was a regulatory reduction in CO₂ fixation. Also data obtained by the solute leakage indicate that in the resistant clone repair mechanisms play a role. The reduction of photosynthesis observed in the sensitive clone is related both to strong stomatal closure and to an impairment in fluorescence parameters. These alterations can indicate a general disruption at the membrane level as confirmed by the solute leakage data. Received: 30 June 1997 / Accepted: 3 September 1997     

Effects of elevated CO2 and O3 on the rate and duration of grain growth and harvest index in spring wheat (Triticum aestivum L.)

Wheat (Triticum aestivum L.) cv. Minaret was grown in open-top chambers (OTCs) in 1995 and 1996 under three carbon dioxide (CO₂) and two ozone (O₃) levels. Plants were harvested regularly between anthesis and maturity to examine the rate of grain growth (dG/dt; mg d^–1) and the rate of increase in harvest index (dHI/dt;% d^–1). The duration of grain filling was not affected by elevated CO₂ or O₃, but was 12 days shorter in 1995, when the daily mean temperature was over 3 °C higher than in 1996. Season-long exposure to elevated CO₂ (680 μmol mol^–1) significantly increased the rate of grain growth in both years and mean grain weight at maturity (MGW) was up to 11% higher than in the chambered ambient air control (chAA; 383 μmol mol^–1). However, the increase in final yield obtained under elevated CO₂ relative to the chAA control in 1996 resulted primarily from a 27% increase in grain number per unit ground area. dG/dt was significantly reduced by elevated O₃ under ambient CO₂ conditions in 1995, but final grain yield was not affected because of a concurrent increase in grain number. Neither dG/dt nor dHI/dt were affected by the higher mean O₃ concentrations applied in 1996 (77 vs. 66 nmol mol^–1); the differing effects of O₃ on grain growth in 1995 and 1996 observed in both the ambient and elevated CO₂ treatments may reflect the contrasting temperature environments experienced. Grain yield was nevetheless reduced under elevated O₃ in 1996, primarily because of a substantial decrease in grain number. The data obtained show that, although exposure to elevated CO₂ and O₃ individually or in combination may affect both dG/dt and dHI/dt, the presence of elevated CO₂ does not protect against substantial O₃-induced yield losses resulting from its direct deleterious impact on reproductive processes. The implications of these results for food production under future climatic conditions are considered.     

Effect of ozone on respiration of adult Sitophilus oryzae (L.), Tribolium castaneum (Herbst) and Rhyzopertha dominica (F.)

The effect of ozone on the respiration of three species of adult stored-product Coleoptera was tested in an air-tight flask. Sitophilus oryzae (L.), Rhyzopertha dominica (F.) and Tribolium castaneum (Herbst) adults were exposed to atmosphere containing 0.1, 0.2 or 0.4 μg/ml initial ozone at 23–25 °C and 50% r.h. Carbon dioxide (CO₂) production reflected the respiration rates of insects and was determined with a gas chromatograph (GC). The experiments showed that the effects of ozone on respiration had two distinct phases. Phase 1 involved a lower respiration rate of the adult stored-product Coleoptera under ozone atmosphere and reflected the need for insects to reduce ozone toxicity. After 1 h, CO₂ production of S. oryzae was 3.19, 2.63, 2.27 and 1.99 μl/mg for the ozone concentration of 0, 0.1, 0.2 and 0.4 μg/ml, respectively. The results also showed that there were decreases in the rate of respiration in R. dominica and T. castaneum with an increase in ozone concentration. During phase 2, respiration of S. oryzae, R. dominica, and T. castaneum adults treated with ozone increased as the ozone degraded to oxygen. After 7 h, the effect of ozone on CO₂ production, relative to the control, changed from a decrease to an increase. The findings in relation to control strategies were discussed.     

Effect of tropospheric ozone on morphological characteristics of Nicotiana tabacum L., Phaseolus vulgaris L. and Petunia×Hybrida L. in ambient air conditions

The cumulative ozone effect on morphological parameters (visible leaf injury, plant height and leaf growth, number of bean pods, petunia flowers and stalks) was examined in this study. Well-known ozonesensitive (Bel W3) and ozone-resistant (Bel B) tobacco cultivars as well as bean cv. Nerina and petunia cv. White cascade, both recognized as ozone sensitive, were used in the experiment. Investigations were carried out at two exposure sites varying in tropospheric ozone levels. Ozone negatively affected the leaf growth of both tobacco cultivars and bean. A negative relation was also found for ozone concentration and tobacco plant height. Number of petunia flowers and stalks and bean pods was positively correlated with ozone concentration. This could have been connected with earlier plant maturation due to faster generative development of plants in ozone-stress conditions.     

The influence of elevated CO2 and O3 on fine roots and mycorrhizas of naturally growing young Scots pine trees during three exposure years

Young Scots pine trees naturally established at a pine heath were exposed to two concentrations of CO₂ (ambient and doubled ambient) and two O₃ regimes (ambient and doubled ambient) and their combination in open-top field chambers during growing seasons 1994, 1995 and 1996 (late May to 15 September). Filtered ozone treatment and chamberless control trees were also included in the treatment comparisons. Root ingrowth cores were inserted to the undisturbed soil below the branch projection of each tree at the beginning of the fumigation period in 1994 and were harvested at the end of the fumigation periods in 1995 and 1996. Root biomasses were determined from different soil layers in the ingrowth cores, and the infection levels of different mycorrhizal types were calculated. Elevated O₃ and CO₂ did not have significant effects on the biomass production of Scots pine coarse (Ø > 2 mm) or fine roots (Ø < 2 mm) and roots of grasses and dwarf shrubs. Elevated O₃ caused a transient stimulation, observable in 1995, in the proportion of tuber-like mycorrhizas, total mycorrhizas and total short roots but this stimulation disappeared during the last study year. Elevated CO₂ did not enhance carbon allocation to root growth or mycorrhiza formation, although a diminishing trend in the mycorrhiza formation was observed. In the combination treatment increased CO₂ inhibited the transient stimulating effect of ozone, and a significant increase of old mycorrhizas was observed. Our conclusion is that doubled CO₂ is not able to increase carbon allocation to growth of fine roots or mycorrhizas in nutrient poor forest sites and realistically elevated ozone does not cause a measurable limitation to roots within a period of three exposure years.     

The effects of enhanced ozone and enhanced carbon dioxide concentrations on biomass, pigments and antioxidative enzymes in spruce needles (Picea abies L.)

During one growing period, 5-year-old spruce trees (Picea abies L., Karst.) were exposed in environmental chambers to elevated concentrations of carbon dioxide (750 cm³ m^?3) and ozone (008 cm³ m^?3) as single variables or in combination. Control concentrations of the gases were 350cm³ m^?3CO₂ and 0.02 cm³ m ^?3 ozone. To investigate whether an elevated CO₂ concentration can prevent adverse ozone effects by reducing oxidative stress, the activities of the protective enzymes superoxide dismutase, catalase and peroxidase were determined. Furthermore, shoot biomass, pigment and protein contents of two needle age classes were investigated. Ozone caused pigment reduction and visible injury in the previous year's needles and growth reduction in the current year's shoots. In the presence of elevated concentrations of ozone and CO₂, growth reduction in the current year's shoots was prevented, but emergence of visible damage in the previous year's needles was only delayed and pigment reduction was still found. Elevated concentrations of ozone or CO₂ as single variables caused a significant reduction in the activities of superoxide dismutase and catalase in the current year's needles. Minimum activities of superoxide dismutase and catalase and decreased peroxidase activities were found in both needle age classes from spruce trees grown at enhanced concentrations of both CO₂ and ozone. These results suggest a reduced tolerance to oxidative stress in spruce trees under conditions of elevated concentrations of both CO₂ and ozone.