Effects of air pollutants on the photosynthetic capacity of young Norway spruce trees. Response of single needle age classes during and after different treatments with O3, SO2, or NO2

Summary Potted young Norway spruce trees were exposed to different concentrations of the air pollutants ozone, sulphur dioxide and nitrogen dioxide under completely controlled environmental conditions. After the treatment, the potted trees were kept outdoors. Measurements of the maximum photosynthetic capacity (A₂₅₀₀) were performed with current-year and 1-year-old needles during and after exposure of the trees. In trees fumigated with nitrogen oxides no damage was found at the concentrations used, and the trees' ability to fix carbon dioxide was increased. Using SO₂, a rapid and marked decrease in A₂₅₀₀ was obtained within the first days of the experiment. This decrease did not continue further, but was reversed upon cessation of the fumigation. However, a clear dose-dependent decrease in A₂₅₀₀ occurred when trees were fumigated continuously with an ozone concentration of 450 nl l^–1 or more. The effect of ozone was not reversible, but continued during post-culture of the trees.     

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 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.     

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.     

Slightly elevated ozone exposure causes cell structural changes in needles and roots of Scots pine

Scots pine (Pinus sylvestris L.) seedlings were fumigated with 1.2–1.5 x ambient ozone over 2 seasons in an open-air experiment. Fumigation started in the early spring and continued into late autumn during both years. Needle and root cell structures were analyzed in the summer, autumn and early winter following the second fumigation period. Under the light microscope an increase in the intercellular space and disintegrating cells in the mesophyll tissue near the stomata and stomatal cavities were observed in the ozone-exposed needles. Darkening of chloroplast stroma, increased plastoglobulus size and decreased chloroplast size were characteristic ultrastructural changes associated with ozone exposure. In addition, less dense grouping of the chloroplasts in the needles of elevated ozone-exposed seedlings as compared to the controls (background ozone) was observed in the early winter. Fewer starch grains and an increased accumulation of tannin-like substances were detected in both mycorrhizal and uninfected roots of ozone-exposed seedlings as compared to the control seedlings. For the first time, we were able to show that the ozone-induced darkening of needle chloroplast stroma is a reversible symptom. An increased frequency of frost injury symptoms indicated that the winter hardening process was disturbed in the needles of ozone-treated seedlings.     

Isoprene decreases the concentration of nitric oxide in leaves exposed to elevated ozone

Isoprene reduces visible damage (necrosis) of leaves caused by exposure to ozone but the mechanism is not known. Here we show that in Phragmites leaves isoprene emission was stimulated after a 3-h exposure to high ozone levels. The photosynthetic apparatus of leaves in which isoprene emission was inhibited by fosmidomycin became more susceptible to damage by ozone than in isoprene-emitting leaves. Three days after ozone fumigation, the necrotic leaf area was significantly higher in isoprene-inhibited leaves than in isoprene-emitting leaves. Isoprene-inhibited leaves also accumulated high amounts of nitric oxide (NO), as detected by epifluorescence light microscopy. Our results confirm that oxidative stresses activate biosynthesis and emission of chloroplastic isoprenoid, bringing further evidence in support of an antioxidant role for these compounds. It is suggested that, in nature, the simultaneous quenching of NO and reactive oxygen species by isoprene may be a very effective mechanism to control dangerous compounds formed under abiotic stress conditions, while simultaneously attenuating the induction of the hypersensitive response leading to cellular damage and death.     

QUANTITATIVE LYSOSOMAL ENZYME ACTIVITY CHANGES IN THE NEURAL LOBE OF THE RAT FOLLOWING WATER DEPRIVATION AND LACTATION

—The activities of five lysosomal enzymes, acid phosphatase, β-glucosidase, β-glucuronidase, β-galactosidase and N-arylamidase (classified according to Marks (1970)) were measured by means of sensitive microchemical techniques in frozen-dried rat neural lobe tissue after experimental and physiological stimulation of hormone release from the hypothalamo–neurohypophysial system i.e. water deprivation (3 and 6 days), delivery and lactation (10 days). During all conditions of stimulation increases of 29 to 106 per cent were measured for lysosomal enzyme activity, expressed as mmol/ng DNA/h. With histochemical staining methods the acid phosphatase activity appears to be mainly localized in the pituicytes, but it was impossible to visualize the microchemically measured acid phosphatase activity increase within the two main compartments of the neurohypophysis, i.e. axonal endings and the neurohypophysial glial cells, the pituicytes.     

EVIDENCE FOR THE REPAIR OF OZONE-INDUCED MEMBRANE INJURY

The increase in [¹⁴C]-2-deoxy-D-glucose uptake by leaf discs 24 hr after fumigation was used as a measure of ozone injury to pinto bean leaves (Phaseolus vulgaris). This method showed that the primary leaves were most affected by ozone when plants were 10 to 12 days old, which coincided with the time of maximum leaf necrosis. However, 8-day-old plants, which had no visible leaf injury, still showed a higher uptake rate than controls, indicating that injury occurred at the cellular level. In these younger plants, uptake was shown to return to normal over a 5 day period. Moreover, the rate of this “repair” was retarded by cold or continuous darkness, enhanced by continuous light, and very markedly increased by glucose applied to the leaves. The timing of the glucose application was not critical, nor did H₂O or mannitol have an effect. The results suggest that ozone injury at the cellular level can be repaired by energy-dependent processes so that necrosis of the leaf tissue does not occur. These experiments also show that conditions and treatments after ozone exposure can alter the degree of ozone injury.     

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.     

Microscopic structure of Scots pine (<Emphasis Type="Italic">Pinus sylvestris</Emphasis> (L.)) needles during ageing and autumnal senescence

Needle ageing and senescence were studied in Scots pine (Pinus sylvestris) trees growing in natural conditions with minimal anthropogenic influence. The four existing needle generations were analyzed by light and transmission electron microscopy before and during autumnal yellowing of the oldest needle generation. The change from green to yellow occurred within less than 4 days in central Finland. The structure of the oldest needles remained largely intact as long as they were green. Increase in mitochondrion size and hypertrophy of the phloem parenchyma were the only changes, probably related to the approaching senescence. In the yellow needles, the structure of the mesophyll tissue varied from nearly intact with reduced chloroplasts and higher numbers of plastoglobuli, to totally disintegrated cells. In the disintegrated cells, peroxisomes were absent, and chloroplasts were smaller with a patchy appearance and degraded, eventually empty-looking stroma. Mitochondria were enlarged, but retained integrity until the last stages of deterioration, and lipids increased. At the light microscopic level, vacuolar volume in mesophyll cells and cavity formation in transfusion tissue increased. Ageing was characterized by increases in the vacuolar volume and cytoplasmic lipids, altered appearance of vacuolar tannin from homogenous ‘sandy’, to large spherical drops and finally to a large mass in the mesophyll, and by hypertrophy and tannin accumulation in the phloem parenchyma. Changes related to needle ageing, senescence and cell location in the mesophyll tissue were discussed relative to findings with stress by strong light, weather conditions and ozone.     

Effects of sulfur dioxide and ozone on intact leaves and isolated mesophyll cells of groundnut plants (Arachis hypogaea L.)

Difference between effects of sulfur dioxide (SO₂) and ozone (O₃) on groundnut plants (Arachis hypogaea L.) was studied by use of an exposure system of enzymatically-isolated mesophyll cells. SO₂ inhibited photosynthesis of intact groundnut leaves but induced no visible injury on leaves. SO₂ also inhibited photosynthesis of isolated mesophyll cells but did not kill the cells, suggesting that SO₂ inhibits photosynthesis by attacking rather specifically the photosynthetic apparatus in chloroplasts. O₃ inhibited photosynthesis of intact leaves and at the same time induced visible injury corresponding to the extent of photosynthesis inhibition. O₃ also inhibited photosynthesis of isolated mesophyll cells and killed the cells to the extent corresponding to photosynthesis inhibition, suggesting that O₃ inhibits photosynthesis not directly by attacking the photosynthetic apparatus but indirectly by killing cells. Since the response of intact leaves to each pollutant resembled that of isolated mesophyll cells, the difference between responses of intact leaves to both pollutants may considerably reflect that of mesophyll cells.     

OZONE SENSITIVITY OF LEAVES: RELATIONSHIP TO LEAF WATER CONTENT,GAS TRANSFER RESISTANCE,AND ANATOMICAL CHARACTERISTICS

Relative water content, resistance to gas transfer, stomatal spacing, and other characteristics of primary bean leaves were studied in relation to ozone sensitivity and injury. Cells of primary bean leaves are maximally sensitive to ozone exposure 9–10 days after germination under our experimental conditions. The stage of maximum sensitivity was not correlated with changes in stomatal number or resistance on either adaxial or abaxial leaf surfaces. It was deduced that bean leaf sensitivity was a function of more internal circumstances, and gas exchange was never the limiting factor through the developmental period studied. Changes in resistance were not significantly altered by ozone levels that produced no visible injury. After exposure to high ozone doses, a decrease in adaxial resistance occurred apparently as a result of palisade and epidermal cell lysis. Normally most gas exchange occurs through the adaxial surface. A 10 % decrease in relative water content accompanying a 60-min ozone exposure of 0.55 ppm could not be explained physiologically on the basis of cell injury as no visible leaf injury occurred.     

Contrasting effects of tropospheric ozone on five native herbs which coexist in calcareous grassland

The aim of this study was to examine the effects of increased tropospheric ozone concentrations on the growth and morphology of five native herbs commonly found to coexist in calcareous grassland in areas of Britain and continental Europe: Anthyllis vulneraria L., Cirsium acaule (L.) Scop., Festuca ovina L., Pilosella offtcinarum F. Shultz & Shultz-Bip and Lotus comiculatus L. In a chronic fumigation (mean O₃ concentration of 71 ppb (71 nl 1^?1) for 7 h d^?1 AOT40 4585 ppb-h) which lasted for 21 d, the effects of ozone were assessed using classical growth analysis. Large reductions in mean relative growth rates for shoot and root weight and root length were observed for the two legumes (Fabaceae) Lotus corniculatus and Anthyllis vulneraria, although these were only statistically significant for Lotus corniculatus. Significant reductions in specific root length (length per unit dry weight) were found for Cirsium acaule and Pilosella officinarum (Asteraceae), while for Festuca ovina (Poaceae) the allometric coefficient was reduced significantly following exposure to ozone. An acute fumigation (mean O₃ concentration of 196 ppb, 7 h) resulted in a range of visible injury, from no injury (Festuca ovina and Pilosella officinarum) through moderate levels of injury (Cirsium acaule and Lotus corniculatus) to extensive and widespread injury (Anthyllis vulneraria). Scoring of visible damage showed that this was only statistically significant for the two legumes, Lotus corniculatus and Anthyllis vulneraria. These results suggest that native herbs may differ in their sensitivity to tropospheric ozone. Both chronic and acute exposures revealed that members of the Fabaceae may be most sensitive to ozone pollution, but the study also suggests that subtle changes in root morphology occurred for members of the Asteraceae. These findings are discussed in relation to the critical levels of ozone set recently for plants and the implications of increasing tropospheric ozone for the conservation of native plant communities.     

Tropospheric ozone-induced structural changes in leaf mesophyll cell walls in grapevine plants

The structural changes in leaves of grapevine plants (Vitis vinifera L.) exposed to different ozone concentrations were investigated. Ozone fumigations were performed in open-top chambers at four different ozone levels (charcoal-filtered air (F), ambient air (N), ambient air + 25 mm³m⁻³ ozone (O-25) and ambient air + 50 mm³m⁻³ ozone (O-50)). The leaves of plants from chambers with increased ozone concentrations (O-25 and O-50) were significantly thicker than the controls (F), owing to increased thickness of the mesophyll layer. Observing O-50 leaves, it was found that the mesophyll cell wall displayed structural changes. In some places cell wall thickness increased up to 1 μm. We found callose deposits on the inner side of the cell walls of mesophyll cells. These data are in accord with the concept that the mesophyll cell wall acts as a barrier against the penetration of tropospheric ozone into the cells.     

Effect of Phosphorus Nutrition on the Cellular Distribution of Acid Phosphatase in the Leaves of Lycopersicon esculentum L.

A histochemical study using light microscopy has been made ofthe distribution of acid phosphatase (EC 3.1.3.2 [EC] ) activity intransverse sections of fully expanded leaves of Lycopersiconesculentum grown in phosphate-deficient or sufficient media.Leaf tissues were prepared by two methods and were embeddedin paraffin wax. The location of acid phosphatase activity inleaf sections was determined by trapping orthophosphate releasedfrom p-nitrophenyl phosphate with lead acetate and subsequentlyconverting the lead phosphate to optically dense lead sulphide.In leaf sections from control tissue lead sulphide depositswere larpely confined to the spongy mesophyll cells. Whereasthe staining of the palisade cells was limited and of a granularnature, the staining of the spongy mesophyll cells was heavierand coincident with the outline of the individual cells. Moreover,the minor veins were more heavily stained than the surroundingmesophyll cells. Sections of phosphorus-deficient tissues wereheavily stained in both the palisade and spongy mesophyll layersand heavy deposits of lead sulphide were present in the regionsof the minor veins. It is suggested that the enhanced acid phosphataseactivity of the mesophyll cells in fully expanded leaves couldbe involved in the remobilization of phosphate within phosphorus-deficientplants, or be part of a phosphate transporting system, concentratingthe intracellular phosphate from the limiting supply in thesolution bathing the mesophyll cells. Lycopersicon esculentum L., tomato, acid phosphatase, phosphorus nutrition     

ANATOMICAL STUDIES ON FIRST-YEAR WINTER INJURED RED SPRUCE FOLIAGE

Foliar injury to red spruce (Picea rubens Sarg.) occurred during the winter of 1989–1990. A range of damage was observed among individual trees, first-year needles on a shoot, and often within an individual needle. Anatomical comparisons of first-year foliage were made to establish patterns of necrosis due to winter injury. Mesophyll tissue was most affected by winter damage. Guard cells adjacent to injured mesophyll cells were also consistently damaged. Early evidence of injury was apparent as an off-color staining of the cytoplasm and slight plasmolysis of the protoplast. Organelle integrity was lost, and there was an extensive reduction of cytoplasmic volume. Spherical bodies of unidentified composition often appeared within the cytoplasm as subcellular integrity was lost. Severe damage resulted in complete loss of cellular structural integrity and adhesion of residual cellular contents to the cell walls. Narrow transition zones between healthy and necrotic tissues were observed, and upper surfaces tended to be more severely affected. There appeared to be a cellular specificity to winter injury within the mesophyll. Completely necrotic cells were often adjacent to apparently healthy cells, and a wide range of injury could be observed within a relatively small area of the needle in partially damaged foliage.     

3-D cell-level chlorophyll fluorescence imaging of ozone-injured sunflower leaves using a new passive light microscope system

A passive light microscope system has been developed, capable of reconstructing an extended-focus 3-D cell-level image of chlorophyll fluorescence and Phi(PSII) of intact attached leaves using a limited number of focal plane images of chlorophyll fluorescence. Using this system, the relationships between the depth of the mesophyll cells in spongy tissue and the intensity of the chlorophyll fluorescence and the Phi(PSII) were investigated in sunflower leaves exposed to 300 ppb ozone for 12 h at a PPFD of 300 micromol m(-2) s(-1) actinic light. After ozone exposure, fluorescence intensity (F) largely decreased in the cells just under the epidermal cells (within approximately 20 microm of the epidermal cells), but the sites where fluorescence intensity decreased had no relationship to the position of the stomata. By contrast, the distribution of Phi(PSII) showed no change after the ozone exposure. These findings suggest that ozone-induced inhibition occurs in the cells just under the epidermal cells by reducing the light absorption of the chloroplasts, while the operating quantum efficiency of PSII photochemistry is maintained.     

Inhalation of ozone induces DNA strand breaks and inflammation in mice

Ozone (O₃) is a well-known oxidant pollutant present in photochemical smog. Although ozone is suspected to be a respiratory carcinogen it is not regulated as a carcinogen in most countries.The genotoxic and inflammatory effects of ozone were investigated in female mice exposed to ozone for 90 min. The tail moment in bronchoalveolar lavage (BAL) cells from BALB/c mice was determined by the comet assay as a measure of DNA strand breaks. Within the first 200 min after exposure, the BAL cells from the mice exposed to 1 or 2 ppm ozone had 1.6- and 2.6-fold greater tail moments than unexposed mice. After 200 min there was no effect. It could be ruled out that the effect during the first 200 min was due to major infiltration of lymphocytes or neutrophils. Unexpectedly, ozone had no effect on the content of 8-oxo-deoxyguanosine (8-oxo-dG) in nuclear DNA or on oxidised amino acids in the lung tissue. The mRNA level of the repair enzyme ERCC1 was not increased in the lung tissue. Inflammation was measured by the cytokine mRNA level in lung homogenates. An up to 150-fold induction of interleukin-6 (IL-6) mRNA was detected in the animals exposed to 2 ppm ozone compared to the air-exposed control mice. Also at 1 ppm ozone, the IL-6 mRNA was induced. The large induction of IL-6 mRNA in the lung took place after DNA strand breaks were induced in BAL. This does not support the notion that inflammatory reactions are the cause of DNA damage. To determine whether these exposures were mutagenic, Muta™Mice were exposed to 2 ppm ozone, 90 min per day for 5 days. No treatment-related mutations could be detected in the cII transgene.These results indicate that a short episode of ozone exposure at five times the threshold limit value (TLV) in US induces lung inflammatory mediators and DNA damage in the cells in the lumen of the lung. This was not reflected by an induction of mutations in the lung of Muta™Mice.