Does ascorbate in the mesophyll cell walls form the first line of defence against ozone? Testing the concept using broad bean (Vicia faba L.)

Broad bean (Vicia faba L.) plants were exposed, in duplicate controlled environment chambers, to charcoal/Purafil-filtered air (CFA-grown plants) or to 75 nmol mol(-1) ozone (O(3)) for 7 h d(-1) (O(3)-grown plants) for 28 d, and then exposed to 150 nmol mol(-1) O(3 )for 8 h. The concentration of ascorbate (ASC) was determined in leaf extracellular washing fluid (apoplast) and in the residual leaf tissue (symplast) after 0, 4 and 8 h acute fumigation, and after a 16 h "recovery" period in CFA. Changes in stomatal conductance were measured in vivo in order to model pollutant uptake, while the light-saturated rate of CO(2) assimilation (A:(sat)) was recorded as an indicator of O(3)-induced intracellular damage. Measurements of A:(sat) revealed enhanced tolerance to 150 nmol mol(-1) O(3) in plants pre-exposed to the pollutant compared with equivalent plants grown in CFA, consistent with the observed reduction in pollutant uptake due to lower stomatal conductance. The concentration of ASC in the leaf apoplast (ASC(apo)) declined upon O(3)-treatment in both CFA- and O(3)-grown plants, consistent with the oxidation of ASC(apo) under O(3)-stress. Furthermore, the decline in ASC(apo) was reversible in O(3)-grown plants after a 16 h "recovery" period, but not in plants grown in CFA. No significant change in the level and/or redox state of ASC in the symplast (ASC(symp)) was observed in plants exposed to 150 nmol mol(-1) O(3), and there was no difference in the constitutive level of ASC(symp) between CFA- and O(3)-grown plants. Model calculations indicated that the reaction of O(3) with ASC(apo) in the leaves of Vicia faba is potentially sufficient to intercept a substantial proportion (30-40%) of the O(3)entering the plant under environmentally-relevant conditions. The potential role of apoplastic ASC in mediating the tolerance of leaves to O(3) is discussed.     

Over-expression of ascorbate oxidase in the apoplast of transgenic tobacco results in altered ascorbate and glutathione redox states and increased sensitivity to ozone

Transgenic tobacco ( Nicotiana tabacum L. cv. Xanthi) plants expressing cucumber ascorbate oxidase (EC.1.10.3.3) were used to examine the role of extracellular ascorbic acid in mediating tolerance to the ubiquitous air pollutant, ozone (O(3)). Three homozygous transgenic lines, chosen on the basis of a preliminary screen of AO activity in the leaves of 29 lines, revealed up to a 380-fold increase in AO activity, with expression predominantly associated with leaf cell walls. Over-expression of AO resulted in no change in the total ascorbate content recovered in apoplast washing fluid, but the redox state of ascorbate was reduced from 30% in wild-type leaves to below the threshold for detection in transgenic plants. Levels of ascorbic acid and glutathione in the symplast were not affected by AO over-expression, but the redox state of ascorbate was reduced, while that of glutathione was increased. AO over-expressing plants exposed to 100 nmol mol(-1) ozone for 7 h day(-1) exhibited a substantial increase in foliar injury, and a greater pollutant-induced reduction in both the light-saturated rate of CO(2) assimilation and the maximum in vivo rate of ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation, compared with wild-type plants. Transgenic plants also exhibited a greater decline in CO(2) assimilation rate when exposed to a brief ozone episode (300 nmol mol(-1) for 8 h). Stomatal conductance, hence O(3) uptake, was unaffected by AO over-expression. Our findings illustrate the important role played by ascorbate redox state and sub-cellular compartmentation in mediating the tolerance of plants to ozone-induced oxidative stress.     

Impacts of ozone on Plantago major: apoplastic and symplastic antioxidant status

The aim of this work was to examine the correspondence between apoplastic/symplastic antioxidant status and previously reported plant age-related shifts in the ozone (O₃) resistance of Plantago major L. Seed-grown plants were fumigated in duplicate controlled environment chambers with charcoal/Purafil®-filtered air (CFA) or CFA plus 70 nmol mol⁻¹ O₃ for 7 h d⁻¹ over a 42 d period. Measurements of stomatal conductance and antioxidants were made after 14, 28 and 42 d fumigation, on leaves at an equivalent stage of development (youngest fully expanded leaf, measured c . 9 d after emergence). Ozone exposure resulted in a similar decline in stomatal conductance across plant ages, indicating that increases in O₃ resistance with plant age were mediated through changes in the tolerance of leaf tissue rather than enhanced pollutant exclusion. Leaf apoplastic washing fluid was found to contain 'unspecific' peroxidase, ascorbate peroxidase, superoxide dismutase and ascorbate, but not glutathione and the enzymes required to facilitate the regeneration of ascorbate from its oxidized forms. A weak induction in the activity of certain symplastic antioxidants was found after 14 d O₃ fumigation, despite a lack of visible symptoms of injury, but shifts in symplastic antioxidant enzyme activity were not consistent with previously observed increases in resistance to O₃ with plant age. By contrast, changes in 'unspecific' peroxidase activity and in the small pool of ascorbate in the leaf apoplast were found to accompany age-related shifts in O₃ resistance. It is concluded that constituents of the leaf apoplast may constitute a potentially important front line defence against O₃.     

Role of Ascorbate in Detoxifying Ozone in the Apoplast of Spinach (Spinacia oleracea L.) Leaves

Both reduced and oxidized ascorbate (AA and DHA) are present in the aqueous phase of the extracellular space, the apoplast, of spinach (Spinacia oleracea L.) leaves. Fumigation with 0.3 [mu]L L-1 of ozone resulted in ozone uptake by the leaves close to 0.9 pmol cm-2 of leaf surface area s-1. Apoplastic AA was slowly oxidized by ozone. The initial decrease of apoplastic AA was <0.1 pmol cm-2 s-1. The apoplastic ratio of AA to (AA + DHA) decreased within 6 h of fumigation from 0.9 to 0.1. Initially, the concentration of (AA + DHA) did not change in the apoplast, but when fumigation was continued, DHA increased and AA remained at a very low constant level. After fumigation was discontinued, DHA decreased very slowly in the apoplast, reaching control level after 70 h. The data show that insufficient AA reached the apoplast from the cytosol to detoxify ozone in the apoplast when the ozone flux into the leaves was 0.9 pmol cm-2 s-1. The transport of DHA back into the cytosol was slower than AA transport into the apoplast. No dehydroascorbate reductase activity could be detected in the apoplast of spinach leaves. In contrast to its extracellular redox state, the intracellular redox state of AA did not change appreciably during a 24-h fumigation period. However, intracellular glutathi-one became slowly oxidized. At the beginning of fumigation, 90% of the total glutathione was reduced. Only 10% was reduced after 24-h exposure of the leaves to 0.3 [mu]L L-1 of ozone. Necrotic leaf damage started to become visible when fumigation was extended beyond a 24-h period. A close correlation between the extent of damage, on the one hand, and the AA content and the ascorbate redox state of whole leaves, on the other, was observed after 48 h of fumigation. Only the youngest leaves that contained high ascorbate concentrations did not exhibit necrotic leaf damage after 48 h.     

Factors that affect leaf extracellular ascorbic acid content and redox status

Leaf ascorbic acid content and redox status were compared in ozone-tolerant (Provider) and ozone-sensitive (S156) genotypes of snap bean ( Phaseolus vulgaris L.). Plants were grown in pots for 24 days under charcoal-filtered air (CF) conditions in open-top field chambers and then maintained as CF controls (29 nmol mol⁻¹ ozone) or exposed to elevated ozone (71 nmol mol⁻¹ ozone). Following a 10-day treatment, mature leaves of the same age were harvested early in the morning (06:00–08:00 h) or in the afternoon (13:00–15:00 h) for analysis of ascorbic acid (AA) and dehydroascorbic acid (DHA). Vacuum infiltration methods were used to separate leaf AA into apoplast and symplast fractions. The total ascorbate content [AA + DHA] of leaf tissue averaged 28% higher in Provider relative to S156, and Provider exhibited a greater capacity to maintain [AA + DHA] content under ozone stress. Apoplast [AA + DHA] content was 2-fold higher in tolerant Provider (360 nmol g⁻¹ FW maximum) relative to sensitive S156 (160 nmol g⁻¹ FW maximum) regardless of sampling period or treatment, supporting the hypothesis that extracellular AA is a factor in ozone tolerance. Apoplast [AA + DHA] levels were significantly higher in the afternoon than early morning for both genotypes, evidence for short-term regulation of extracellular ascorbate content. Total leaf ascorbate was primarily reduced with AA/[AA + DHA] ratios of 0.81–0.90. In contrast, apoplast AA/[AA + DHA] ratios were 0.01–0.60 and depended on genotype and ozone treatment. Provider exhibited a greater capacity to maintain extracellular AA/[AA + DHA] ratios under ozone stress, suggesting that ozone tolerance is associated with apoplast ascorbate redox status.     

Effects of ozone on apoplast/cytoplasm partitioning of ascorbic acid in snap bean

Apoplast/cytoplasm partitioning of ascorbic acid (AA) was examined in four genotypes of snap bean ( Phaseolus vulgaris L.) known to differ in ozone sensitivity. Plants were grown in pots under field conditions using open-top chambers to establish charcoal-filtered (CF) air (36 nmol mol⁻¹ ozone) or elevated ozone (77 nmol mol⁻¹ ozone) treatments. AA in fully expanded leaves of 36-day-old plants was separated into apoplast and cytoplasm fractions by vacuum infiltration methods using glucose 6-phosphate as a marker for cytoplasm contamination. Apoplast ascorbate levels ranged from 30 to 150 nmol g⁻¹ fresh weight. Ozone-sensitive genotypes partitioned 1–2% of total AA into the apoplast under CF conditions and up to 7% following a 7-day ozone exposure. In contrast, an ozone-tolerant genotype partitioned 3–4% of total leaf AA into the leaf apoplast in both CF and ozone-treated plants. The results suggest that genetic background and ozone stress are factors that affect AA levels in the extracellular space. For all genotypes, the fraction of AA in the oxidized form was higher in the apoplast compared to the cytoplasm, indicative of a more oxidizing environment within the cell wall.     

Impact of elevated CO(2) and O(3) on gas exchange parameters and epidermal characteristics in potato (Solanum tuberosum L.)

Potato plants (Solanum tuberosum L. cv. Bintje) were grown in open-top chambers (OTCs) under three CO(2) levels (ambient and 24 h d(-1) seasonal mean concentrations of 550 and 680 micromol mol(-1)) and two O(3) levels (ambient and a seasonal mean 8 h d(-1) concentration of 50 nmol mol(-1)). The objectives were to determine the effects of season-long exposure to these key climate change gases on gas exchange, leaf thickness and epidermal characteristics. The experimental design also provided an ideal opportunity to examine within-leaf variation in epidermal characteristics at the whole-leaf level. Stomatal and epidermal cell density and stomatal index were measured at specific locations on the youngest fully expanded leaf (centre of lamina, mid-way between tip and base) and representative whole leaves from each treatment. Effects on leaf conductance, assimilation rate and instantaneous transpiration efficiency were determined by infrared gas analysis, while anatomical characteristics were examined using a combination of leaf impressions and thin sections. Exposure to elevated CO(2) or O(3) generally increased leaf thickness, leaf area, stomatal density, and assimilation rate, but reduced leaf conductance. The irregular stomatal distribution within leaves resulted from a combination of uneven differentiation and expansion of the epidermal cells. The results are discussed with reference to sampling protocols and the need to account for within-leaf variation when examining the impact of climate change or other environmental factors on epidermal characteristics.     

Does elevated CO2 ameliorate the impact of O3 on chlorophyll content and photosynthesis in potato (Solanum tuberosum)?

This study examined the impact of season-long exposure to elevated carbon dioxide (CO2) and ozone (O3), individually and in combination, on leaf chlorophyll content and gas exchange characteristics in potato (Solanum tuberosum L. cv. Bintje). Plants grown in open-top chambers were exposed to three CO2 (ambient, 550 and 680 μmol mol-1) and two O3 treatments (ambient and elevated; 25 and 65 nmol mol-1, 8 h day-1 means, respectively) between crop emergence and maturity; plants were also grown in unchambered field plots. Non-destructive measurements of chlorophyll content and visible foliar injury were made for all treatments at 2-week intervals between 43 and 95 days after emergence. Gas exchange measurements were made for all except the intermediate 550 μmol mol-1 CO2 treatment. Season-long exposure to elevated O3 under ambient CO2 reduced chlorophyll content and induced extensive visible foliar damage, but had little effect on net assimilation rate or stomatal conductance. Elevated CO2 had no significant effect on chlorophyll content, but greatly reduced the damaging impact of O3 on chlorophyll content and visible foliar damage. Light-saturated assimilation rates for leaves grown under elevated CO2 were consistently lower when measured under either elevated or ambient CO2 than in equivalent leaves grown under ambient CO2. Analysis of CO2 response curves revealed that CO2-saturated assimilation rate, maximum rates of carboxylation and electron transport and respiration decreased with time. CO2-saturated assimilation rate was reduced by elevated O3 during the early stages of the season, while respiration was significantly greater under elevated CO2 as the crop approached maturity. The physiological origins of these responses and their implications for the performance of potato in a changing climate are discussed.     

Components of apoplastic ascorbate use in Betula pendula leaves exposed to CO2 and O3 enrichment

Here, the aim was to estimate loads imposed on the apoplastic ascorbate (ASC) pool by enzymatic and nonenzymatic reactions in Betula pendula exposed to doubled CO2 and O3 concentrations in open-top chambers. Leaf apoplastic extracts were analysed for peroxidase and oxidase activities in vitro, using different substrates. Partial loads in vivo were deduced using measured kinetic constants and substituted-enzyme catalysis approaches. Ascorbate use in O3 scavenging was calculated using measured stomatal conductances and ASC concentrations. Under elevated O3, stomatal conductance and O3 uptake were higher. O3 fluxes to the plasmalemma were levelled off by higher apoplastic ASC concentrations. The effect of CO2 enrichment on ASC concentrations under elevated O3 was minor. Under ambient O3, the ascending hierarchy of ASC users was: peroxidases, O3 scavenging, oxidases, coniferyl alcohol re-reduction. Under elevated O3, ASC use in O3 scavenging was higher than by oxidases. The redox state of ASC was not depressed by O3; there was no leaf injury. The cell wall/plasmalemma/cytosol system in birch had sufficient capacity to maintain ASC redox status in the apoplast, without necessity to restrict O3 uptake by stomatal closure.     

Ascorbate in the leaf apoplast is a factor mediating ozone resistance in Plantago major

The role of ascorbate in mediating ozone resistance was examined in Plantago major L. Seedlings of eleven populations which exhibited differential resistance to ozone were fumigated in controlled environment chambers with charcoal/Purafil®-filtered air (CFA) or CFA plus 15 nmol·mol^–1 ozone overnight rising to a maximum between 12:00–16:00 hours of 75 nmol·mol^–1 for 14 d. Measurements of ascorbate content were made on apoplastic and symplastic extracts. Populations differed in their constitutive level of ascorbate in youngest fully expanded leaves, and regression analysis revealed a significant correlation between ascorbate content in ozone-treated leaves and the ozone resistance of the populations. The relationship was stronger using apoplastic ascorbate levels than with corresponding symplastic measurements. The ascorbate content of the youngest fully expanded leaf of an ozone sensitive population was increased by foliar application of ascorbate. No significant difference in stomatal conductance was found between control and ascorbate-treated plants. Following spraying, plants were fumigated with 400 nmol·mol^–1 ozone for 7 h. In control plants, ozone exposure resulted in extensive visible leaf damage (20–70 % at the end of the fumigation period) and decreased rates of CO₂ assimilation (–57 %). However, ascorbate treatment prevented the appearance of visible injury, and ameliorated the decline in photosynthesis induced by ozone (–26 %). Modelled data estimating the extent of protection afforded by apoplastic ascorbate against ozone supported the experimental observations. The results suggested that although apoplastic ascorbate plays an important role, other factors must also contribute to the mediation of ozone resistance in P. major.     

Does nitrogen supply affect the response of wheat (Triticum aestivum cv. Hanno) to the combination of elevated CO(2) and O(3)?

Spring wheat (Triticum aestivum cv. Hanno) was grown at ambient (350 micromol mol(-1)) or elevated CO(2) (700 micromol mol(-1)) in charcoal/Purafil-filtered air (CFA <5 nmol mol(-1)) or ozone (CFA +75 nmol mol(-1) 7 h d(-1)) at three levels of N supply (1.5, 4 and 14 mM NO(-3)), to test the hypothesis that the combined impacts of elevated CO(2) and O(3) on plant growth and photosynthetic capacity are affected by nitrogen availability. Shifts in foliar N content reflected the level of N supplied, and the growth stimulation induced by elevated CO(2) was dependent on the level of N supply. At 60 d after transfer (DAT), elevated CO(2) was found to increase total biomass by 44%, 29%, 12% in plants supplied with 14, 4 and 1.5 mM NO(-3), respectively, and there was no evidence of photosynthetic acclimation to elevated CO(2) across N treatments; the maximum in vivo rate of Rubisco carboxylation (V(cmax)) was similar in plants raised at elevated and ambient CO(2). At 60 DAT, ozone exposure was found to suppress plant relative growth rate (RGR) and net photosynthesis (A) in plants supplied with 14 and 4 mM NO(-3). However, O(3) had no effect on the RGR of plants supplied with 1.5 mM NO(-3) and this effect was accompanied by a reduced impact of the pollutant on A. Elevated CO(2) counteracted the detrimental effects of O(3) (i.e. the same ozone concentration that depressed RGR and A at ambient CO(2) resulted in no significant effects when plants were raised at elevated CO(2)) at all levels of N supply and the effect was associated with a decline in O(3) uptake at the leaf level.     

Effects of elevated [CO2] and/or ozone on limitations to CO2 assimilation in soybean (Glycine max)

Soybean (Glycine max) was grown in open-top field chambers at ambient (360 mol mol^-1) or doubled [CO2] either in charcoal-filtered air (20 nmol mol^-1 [O3]) or in non-filtered air supplemented to 1,5 x ambient [O3] (70 nmol mol^-1) to determine the major limitations to assimilation under conditions of elevated [CO2] and/or [O3]. Through plant ontogeny, assimilation versus intercellular CO2 concentration (A/Ci) responses were measured to assess the limitations to assimilation imposed by the capacity for Rubisco carboxylation, RuBP regeneration, and stomatal diffusion.In the vegetative stages, no significant treatment effects of elevated [CO2] and/or [O3] were observed on Rubisco carboxylation efficiency (CE), light and CO2-saturated assimilation capacity (Amax), and chlorophyll content (Chl). However, for plants grown in elevated [CO2], the assimilation rate at growth [CO2] (A) was 60% higher than at ambient [CO2] up to the seed maturation stage, and the potential rate of assimilation by Rubisco capacity (Ap) was increased. Also in elevated [CO2]: A was 51% of Ap; the relative stomatal limitation (%Stomata) was 5%; and the relative RuBP regeneration limitation (%RuBP) was 44%. In ambient [CO2], O3 gradually decreased A per unit leaf area, but had little effect on Ap and the relative limitations to assimilation where A remained 51% of Ap, %Stomata was 27%, and %RuBP was 22%.During reproduction, CE declined for plants grown in elevated [CO2] and/or [O3]; Ap was unaffected by elevated [CO2], but was reduced by [O3] at ambient [CO2]; A increased to 72% of Ap in elevated [CO2] and/or [O3]-fumigated air; the %Stomata increased; and the %RuBP decreased, to become non significant in elevated [CO2] from the beginning of seed growth on, and in O3-fumigated air at ambient [CO2] at the seed maturation stage. The decrease in %RuBP occurred concomitantly with an increase in Amax and Chl. Significant [CO2] x [O3] interactions support the lack of an O3 effect on assimilation and its limitations at elevated [CO2] during seed maturation. These data suggest that elevated [CO2] alleviated some of the effects of O3 on photosynthesis.Keywords: CO2 by O3 interactions, elevated [CO2], O3 fumigation, Rubisco carboxylation efficiency, RuBP regeneration.      

Enhanced photosynthetic performance and growth as a consequence of decreasing mitochondrial malate dehydrogenase activity in transgenic tomato plants

Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the mitochondrial malate dehydrogenase gene in the antisense orientation and exhibiting reduced activity of this isoform of malate dehydrogenase show enhanced photosynthetic activity and aerial growth under atmospheric conditions (360 ppm CO2). In comparison to wild-type plants, carbon dioxide assimilation rates and total plant dry matter were up to 11% and 19% enhanced in the transgenics, when assessed on a whole-plant basis. Accumulation of carbohydrates and redox-related compounds such as ascorbate was also markedly elevated in the transgenics. Also increased in the transgenic plants was the capacity to use L-galactono-lactone, the terminal precursor of ascorbate biosynthesis, as a respiratory substrate. Experiments in which ascorbate was fed to isolated leaf discs also resulted in increased rates of photosynthesis providing strong indication for an ascorbate-mediated link between the energy-generating processes of respiration and photosynthesis. This report thus shows that the repression of this mitochondrially localized enzyme improves both carbon assimilation and aerial growth in a crop species.     

Amelioration of Ozone-Induced Oxidative Damage in Wheat Plants Grown under High Carbon Dioxide (Role of Antioxidant Enzymes)

O3-induced changes in growth, oxidative damage to protein, and specific activities of certain antioxidant enzymes were investigated in wheat plants (Triticum aestivum L. cv Roblin) grown under ambient or high CO2. High CO2 enhanced shoot biomass of wheat plants, whereas O3 exposure decreased shoot biomass. The shoot biomass was relatively unaffected in plants grown under a combination of high CO2 and O3. O3 exposure under ambient CO2 decreased photosynthetic pigments, soluble proteins, and ribulose-1,5-bisphosphate carboxylase/oxygenase protein and enhanced oxidative damage to proteins, but these effects were not observed in plants exposed to O3 under high CO2. O3 exposure initially enhanced the specific activities of superoxide dismutase, peroxidase, glutathione reductase, and ascorbate peroxidase irrespective of growth in ambient or high CO2. However, the specific activities decreased in plants with prolonged exposure to O3 under ambient CO2 but not in plants exposed to O3 under high CO2. Native gels revealed preferential changes in the isoform composition of superoxide dismutase, peroxidases, and ascorbate peroxidase of plants grown under a combination of high CO2 and O3. Furthermore, growth under high CO2 and O3 led to the synthesis of one new isoform of glutathione reductase. This could explain why plants grown under a combination of high CO2 and O3 are capable of resisting O3-induced damage to growth and proteins compared to plants exposed to O3 under ambient CO2.     

Paraheliotropism can protect water-stressed bean (Phaseolus vulgaris L.) plants against photoinhibition

In order to estimate the importance of leaf movements on photosynthesis in well-watered and water-stressed field grown bean cultivars (Arroz Tuscola (AT), Orfeo INIA (OI), Bayos Titan (BT), and Hallados Dorado (HD)), CO2 assimilation, leaf temperature, and capacity for the maximum quantum yield recovery, measured as Fv/Fm, were assessed. Leaf water potential was lower in water-stressed compared to control plants throughout the day. Water status determined a decrease in the CO2 assimilation and stomatal conductance as light intensity and temperature increased up to maximal intensities at midday. Both parameters were lower in stressed compared to control plants. Even though high light intensity and water-stress induced stomatal closure is regarded as a photoinhibitory condition, the recovery of variable to maximal fluorescence (Fv/Fm) after 30min of darkness was nearly constant in both water regimes. In fact, higher values were observed in OI and AT when under stress. Photochemical and non-photochemical fluorescence quenching resulted in minor changes during the day and were similar between watered and stressed plants. It is concluded that paraheliotropism, present in the four bean cultivars, efficiently protects stressed plants from photoinhibition in the field and helps maintain leaf temperatures far below the ambient temperatures, however, it may also be responsible for low CO2 assimilation rates in watered plants.     

Effects of ozone on the plasma membrane proteins in Arabidopsis thaliana (L.) leaves

The protein pattern of leaf plasma membranes from Arabidopsis thaliana (L.) Landsberg erecta was analysed in order to detect changes induced by acute short-term ozone treatment. Plasma membranes were isolated 0, 3 and 8 h after the end of a 2 h fumigation of the plants with 500 nmol mol^?1 of O₃. Proteins extracted from plasma membranes were separated by high-performance two-dimensional polyacrylamide gel electrophoresis. Eight hours after the end of fumigation, one new protein appeared and the amounts of two other proteins increased significantly. The reported study is a first step towards the identification of plasmalemma proteins altered by ozone and to a more detailed characterization of structural changes occurring in the plasma membrane after ozone exposure.     

Early production and scavenging of hydrogen peroxide in the apoplast of sunflower plants exposed to ozone

The present work set out to define the processes involved in the early O3-induced H2O2 accumulation in sunflower plants exposed to a single pulse of 150 ppb of O3 for 4 h. Hydrogen peroxide accumulation only occurred in the apoplast and this temporally coincided with the fumigation period. The inhibitor experiments suggested that both the plasma membrane-bound NAD(P)H oxidase complex and cell-wall NAD(P)H PODs contributed to H2O2 generation. To investigate the mechanisms responsible for O3-induced H2O2 accumulation further, both production and scavenging of H2O2 were investigated in the extracellular matrix after subcellular fractionation. The results indicated that H2O2 accumulation is a complex and highly regulated event requiring the time-dependent stimulation and down-regulation of differently located enzymes, some of which are involved in H2O2 generation and degradation, not only during the fumigation period but also in the subsequent recovery period in non-polluted air. Owing to the possible interplay between H2O2 and ethylene, the time-course of ethylene emission was analysed too. Ethylene was rapidly emitted following O3 exposure, but it declined to control values as early as after 4 h of exposure. The early contemporaneous detection of increased ethylene and H2O2 levels after 30 min of exposure does not allow a clear temporal relationship between these two signalling molecules to be established.     

Plasmalemma protection by the apoplast as assessed from above-zero ozone concentrations in leaf intercellular air spaces

To evaluate reactive absorption of ozone (O3) in the leaf apoplast, amphistomatous leaves of Phaseolus vulgaris L. were allowed to take up O3 through the stomata on the lower leaf surface at high rates for 3-5 min. Up to 5% of the O3 taken up diffused through the leaf and emerged from the stomata on the upper surface, suggesting above-zero O3 concentrations in the leaf intercellular air space, [O3]i. Moreover, measurements revealed that [O3]i increased during exposure to the pollutant. Time patterns of O3 fluxes through the gas phase and into the aqueous apoplast indicated that the increase in [O3]i was the result of a decrease in the diffusion-reaction conductance of the aqueous apoplast, gaq. Under an intense O3 pulse, gaq approached the value of the pure diffusional conductance within 2.5 min of exposure, suggesting the exhaustion of protective resources in the leaf apoplast. Toward the end of the exposure gaq tended to increase, suggesting either a recovery in the protective resources in the leaf apoplast and/or the induction of new defences. The possibility of estimating the degree of protection afforded by apoplast constituents and the rate of recovery of these protective systems in intact leaves using brief O3 pulses is discussed.     

Mechanisms underlying the amelioration of O3-induced damage by elevated atmospheric concentrations of CO2

There is growing evidence that rising atmospheric CO2 concentrations will reduce or prevent reductions in the growth and productivity of C3 crops attributable to ozone (O3) pollution. In this study, the role of pollutant exclusion in mediating this response was investigated through growth chamber-based investigations on leaves 4 and 7 of spring wheat (Triticum aestivum cv. Hanno). In the core experiments, plants were raised at two atmospheric CO2 concentrations (ambient [350 micro l l(-1)] or elevated CO2 [700 micro l l(-1)] under two O3 regimes (charcoal/Purafil-filtered air [<5 nl l(-1) O3] or ozone-enriched air [75 nl l(-1) 7 h d(-1)]). A subsequent experiment used an additional O3 treatment where the goal was to achieve equivalent daily O3 uptake over the life-span of leaves 4 and 7 under ambient and CO2-enriched conditions, through daily adjustment of exposures based on measured shifts in stomatal conductance. Plant growth and net CO2 assimilation were stimulated by CO2-enrichment and reduced by exposure to O3. However, the impacts of O3 decreased with plant age (i.e. leaf 7 was more resistant to O3 injury than leaf 4); a finding consistent with ontogenic shifts in the tolerance of plant tissue and/or acclimation to O3-induced oxidative stress. In the combined treatment, elevated CO2 protected against the adverse effects of O3 and reduced cumulative O3 uptake (calculated from measurements of stomatal conductance) by c. 10% and 35% over the life-span of leaves 4 and 7, respectively. Analysis of the relationship between O3 uptake and the decline in the maximum in vivo rate of Rubisco carboxylation (Vcmax) revealed the protection afforded by CO2-enrichment to be due, to a large extent, to the exclusion of the pollutant from the leaf interior (as a consequence of the decline in stomatal conductance triggered by CO2-enrichment), but there was evidence (especially from flux-response relationships constructed for leaf 4) that CO2-enrichment resulted in additional effects that alleviated the impacts of ozone-induced oxidative stress on photosynthesis.