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Detection of sulphite in plants.

Artificially SO(2)-fumigated plant material was investigated for its sulphite content. Experiments with moderate concentrations of SO(2) (0.15-0.45 mg m(-3) lasting for 3 weeks, showed no detectable amounts of sulphite present in peas. Considering the detection limit of the method, this indicates that the steady state level in plant tissues is below 4 x 10(-5)M sulphite. Only in pea plants exposed to very high SO(2)-doses of 2.6-26mg m(-3) for 12-48 h, was sulphite recorded. We conclude from our results that sulphite dioxide, taken up by pea plants, is very effectively removed by enzymic and non-enzymic processes, and this 'detoxification' prevents accumulation of sulphite in the plant tissue, and so only very small amounts of sulphite are to be expected in SO(2)-damaged plant material. The possible role of very low sulphite concentrations in plant tissues is discussed.

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Growth and premature leaf fall in American aspen as bioindications for ozone.

Several clones of the American aspen (Populus tremuloides Michx.) differing in ozone sensitivity were fumigated continuously for 105 days with different ozone concentrations, producing different ozone doses. The suitability of the clones as possible replacements for tobacco as bioindicators in forested areas was tested. The life span of leaves of the ozone-sensitive clones was distinctly reduced by ozone. The difference to controls was significant even in the relatively clean air at Birmensdorf. Growth was diminished, too, but the decrease in height was much less pronounced than that in weight. In sensitive clones an after-effect was detectable, since the weight of new shoots (grown from reserves) decreased with increasing ozone stress in the previous year. Since the tolerant clone grew less, it is-at first sight-of decreased economic interest from the point of view of rapid productivity. In the long run, however, it might be economically interesting because it may be able to maintain productivity (although at a lower level) due to better competitive strength.

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Growth and physiological responses of yellow-poplar seedlings exposed to ozone and simulated acidic rain.

Nine-week-old, yellow-poplar (Liriodendron tulipifera L.) seedlings were exposed to ozone (O(3)) 4 h day(-1), 5 days week(-1), in combination with simulated rain (pH 3.0, 4.3, or 5.6, 1 h day(-1), 2 days week(-1), at 0.75 cm h(-1)) for 6 weeks, under controlled laboratory conditions. There was no main treatment effect of O(3) (<0.02, 0.05, 0.10, or 0.15 microlitre(-1)) on height growth or total plant biomass. However, specific leaf area increased linearly (P=0.05) with exposure of plants to increasing O(3) concentrations. Exposure of seedlings to 0.10 microl litre(-1) O(3) reduced the leaf area ratio and the stomatal conductance (S(c)), but increased the mean unit leaf rate compared to control plants. This O(3) concentration also caused S(c) to decrease more rapidly, relative to non-fumigated plants, in response to increasing vapor pressure deficit. The only main treatment effect caused by simulated rain was a linear decrease in S(c) with increasing acidity. For combined pollutant effects, O(3) applied at 0.05 or 0.10 microl litre(-1) resulted in significant (P=0.05) linear decreases in dry weights and relative growth rates (RGR) of stems and leaves, and in cumulative leaf area (LAI), as the solution pH decreased. Root to shoot ratio (RSR) decreased linearly, whereas stem RGR and LAI increased linearly in response to decreasing pH for seedlings exposed to 0.15 microl litre(-1) O(3). Seedlings treated with a pH 5.6 solution exhibited a linear decrease (P=0.05) in leaf dry weight and RGR and a linear increase (P=0.05) in RSR as O(3) concentrations increased.

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Interaction between ozone and winter stress.

In some countries, ozone (O3) is primarily a summer pollutant, but in much of Europe, elevated concentrations occur outside the growing season so perennials and over-wintering annuals may be subjected to the combined stresses of pollution, plus chilling, freezing, and winter desiccation. It is recognised that some air pollutants modify the response of plants to environmental stress, but little is known of interactions involving O3. This paper is part of a programme concerned with the effects of O3 on resistance to chilling, freezing, and winter desiccation. Pea (Pisum sativum L.) was used as a convenient model to confirm that O3 affects freezing resistance. The experiment also served as a further evaluation of the use of induced chlorophyll fluorescence kinetics to detect latent O3 injury. Two cultivars, 'Feltham First' and 'Conquest', were fumigated for 7 days, 7 h day(-1). Diffusive resistance and induced fluorescence were recorded daily during the period, then the plants were hardened at 4 degrees C day/2 degrees C night before exposure to 0, -2, -4, -6 and -8 degrees C. Ozone (0.075 ppm; 150 microg O3 m(-3)) caused stomatal closure in both cultivars, but the response was more rapid in 'Conquest'. There were also rapid effects on fluorescence kinetics, and it was concluded that FR, the rate of rise of induced fluorescence, is a useful parameter for indicating latent injury and for distinguishing between cultivars of different sensitivity. Exposure to O3 increased freezing injury and led to greater electrolyte leakage. The freezing resistance of 'Feltham First' was more affected than that of 'Conquest', probably because of the slower stomatal response to the pollutant leading to greater flux of O3 to the internal tissues. It is concluded that interactions involving pollutants and winter stress have implications for crop loss assessment. Perennials and over-wintering annuals should be exposed to the full range of environmental stresses.

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Reduction of net photosynthesis in oats after treatment with low concentrations of ozone.

Oats (Avena sativa L. cv Titus) were exposed to low concentrations of O3 in an assimilation chamber system. Net photosynthesis (net CO2 uptake), measured before and after O3 fumigation, showed significantly different responses for leaves of different age. The oldest active leaf was the most sensitive to O3. Net photosynthesis was depressed after 2 h with 0.075 ppm (150 microg m(-3)) O3. For leaves exposed to 0.150 ppm (300 microg m(-3)) O3 for 2 h, net photosynthesis was reduced significantly for 4 h, after which recovery occurred, nearly reaching the preexposure level 19 h after the exposure. Dark respiration was initially more than doubled after exposure to 0.130 ppm (260 microg m(-3)) O3. There was no visible injury after any of the experiments. The results indicate that O3 may cause crop losses through effects on photosynthesis even in Scandinavia, where a typical O3 episode lasts 1 to 2 h, and the concentration seldom exceeds 0.150 ppm.

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Analysis of air pollution plant exposure data: The soft independent modelling of class analogy (SIMCA) and partial least squares modelling with latent variable (PLS) approaches.

Air pollution plant exposure experiments sometimes involve registration of several plant variables (dependent variables) as a function of several treatment variables (independent variables). Both independent and dependent variables might be correlated, and the number of variables might exceed the number of observations. Analyzing one variable at a time gives a risk of spurious results. The multivariate statistical methods, soft independent modelling of class analogy (SIMCA) and partial least squares modelling with latent variables (PLS) allow all variables to be analyzed simultaneously. These methods are presented and applied to data from open-top chamber experiments with O3, SO2, and NO2 fumigation of three varieties of Lolium multiflorum Lam. The results demonstrate the dependence of the plant response to plant variety, plant age, climate, and pollutant dosages.

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Response of spinach and kidney bean plants to nitrogen dioxide.

Different responses of spinach and kidney bean plants to various concentrations of NO(2) in the light and in the dark were shown. Spinach is more resistant than NO(2) than kidney bean. It is not only due to its greater tolerance to NO(2)(-) accumulated in leaves, but also to its stronger ability to metabolize NO(2)(-). The injury of spinach induced by the exposure to high concentration NO(2) in the light was not caused by the accumulation of NO(2)(-), but concerned the large amount of accumulated NH(3). The primary causes of NH(3) accumulation were that the activity of nitrite reductase was not affected by the fumigation on the one hand, and the activities of glutamine synthetase and glutamate synthase were inhibited on the other.

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Ozone-induced changes in CO2 assimilation, O2 evolution and chlorophyll a fluorescence transients in barley.

Spring barley (Hordeum vulgare cv. Klaxon) plants, 9 days old, were exposed to 0.05, 0.10 or 0.15 microl litre(-1) ozone (O3) for 12 days. Fumigation was administered for 7 h between 9.00 h and 16.00 h each day. Using conventional IRGA equipment, the carbon dioxide exchange rate (CER) was shown to decrease with increasing concentration of O3 during the exposure period, falling to 60% of the control value at the highest O3 concentration. Transpiration rates and stomatal conductance showed similar trends. Light saturation curves, obtained using a leaf disc oxygen electrode, demonstrated that O3-treated leaves had lower apparent quantum yields (QY) and generally lower rates of O2 evolution at saturating light and CO2 levels. Oscillations in chlorophyll a fluorescence, normally observed in control plants, could not be detected after O3 treatment and could only be restored to some extent by feeding the phosphate sequestering agent D-mannose to the leaves.

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Acid mist and ozone effects on the leaf chemistry of two western conifer species.

Seedlings of Jeffrey pine (Pinus jeffreyi) and giant sequoia (Sequoiadendron gigantea) were more susceptible to leaf chemical changes following exposure to acid mist (pH 3.4-2.0) or acid mist/ozone combinations, than to ozone alone (0.1-0.2 microl/litre), when plants were exposed to alternating doses of these pollutants for 6-9 weeks. Under acid mist treatment, leaves exhibited higher levels of nitrogen and sulfur, two elements applied in acid mist. In addition, levels of foliar sodium, and, in the case of giant sequia, potassium, as well, increased under acid mist treatment. Iron and manganese were also mobilized, resulting in significant increases in these elements in pine, and decreases in manganese in giant sequoia foliage. The acid treatment also reduced chlorophyll b concentrations in pine, and, to a less significant extent, in giant sequoia. Calcium, magnesium, barium and strontium were differentially accumulated in giant sequoia compared to Jeffrey pine. Under acid mist treatment, all of these elements (except strontium) declined in concentration in giant sequoia, with calcium showing the most significant trend. The more extensive changes in leaf chemistry induced by acid mist are consistent with earlier observations of significant changes in spectral reflectance of these seedlings after 3 weeks of fumigation. Limited foliage samples collected from these two species in 1985 and 1986 in Sequoia/Kings Canyon National Parks in the southern Sierra Nevada do not in themselves indicate any clearcut or severe effects of ozone alone on leaf chemistry of these species, but a mild influence of nitrate-laden acid deposition, possibly in combination with ozone, is consistent with the rise in nitrogen and lignin levels in Jeffrey pine on sites observed to have moderate visible injury symptoms. No firm conclusions about effects of pollutants on leaf chemistry in these field sites is possible without further study.

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Mineral content in the soil and tree foliage.

Seedlings of fir (Abies alba Mill.) and spruce (Picea abies L. Karst.) were fumigated with SO(2), O(3) and SO(2) + O(3) in open-top chambers (OTCs) for almost 5 vegetation periods. As background stress, simulated rain of pH 4.0 was applied. Nutrient content of soil, soil solutions, and trees was investigated and balanced. In the upper partition of the soil high concentrations of exchangeable Ca(2+) were found in all chambers. The SO(2) and SO(2) + O(3) treatments led to increased Ca(2+), Mg(2+) and Mn(2+) concentrations in soil solution and the pool of exchangeable protons increased. This response was most evident in the SO(2) and SO(2) + O(3) chambers and less clear in the filtered pH 5.0 control chamber. In the SO(2) treatment increased Mn and S levels were found in the needles. Ca content in the needles showed a decreasing trend. O(3) alone had no consistent effect on needle nutrient content.

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Monoterpene patterns of different tissues and plant parts of Norway spruce (Picea abies L. Karst.).

Norway spruce saplings (Picea abies L. Karst.) were exposed for five years to controlled concentrations of ozone and/or sulphur dioxide in open-top chambers. The monoterpenes of needles, twigs (bark and wood), bark and buds harvested from these trees were qualitatively and quantitatively analysed by combined gas chromatography-mass spectrometry. Different tissues and plant parts, showed notable differences in their percentage distribution. However, comparison of the monoterpene patterns of corresponding plant material obtained from trees which had been exposed to different fumigation regimes showed no significant effects of these pollutants.

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Monoterpenes and microbial metabolites in the soil.

Volatile microbial metabolites were analysed in the soil beneath 15 H year-old Picea abies which had been fumigated for 5 consecutive years with charcoal-filtered air enriched with ozone and sulphur dioxide. Major compounds which could be attributed to the metabolism of basidiomycete fungi were oct-1-en-3-ol, oct-1-en-3-one and 1,3-octadiene. They were accompanied by other C(8)-compounds. The lower amounts of C(8)-compounds observed in the soil of the sulphur dioxide and ozone treatment chambers indicate a reduced development of basidiomycetes. The higher amounts of geosmin and 2-methylisoborneol found in the soil of the ozone treatment chamber indicate an increase in actinomycete populations. The differences of the monoterpene patterns in the particular soils were not large enough to recognize trends unequivocally.

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Performance of some growth variables.

European beech (Fagus sylvatica L.), Norway spruce (Picea abies L. Karst.) and Silver fir (Abies alba Mill.) were exposed to low concentrations of ozone (O(3)) and sulfur dioxide (SO(2)), alone and combined, and simulated acid rain (pH 4.0) in sheltered open-top chambers in Hohenheim (Southwest Germany) for almost five years. The concentrations of O(3) and SO(2) used were related to annual ambient average found in southern West Germany. Two control chambers were ventilated with charcoal filtered air and rainfall was simulated at pH 4.0 and 5.0. Because of large dense plant growth in the chambers it was only possible to measure uncompleted growth of shoots in the upper canopy. Therefore, growth analysis was restricted to this area. The treatment with acidic precipitation decreased the annual shoot growth of beech and reduced leaf surface area of those trees. Exposure to SO(2), O(3) alone and in combination resulted in further reduction of shoot length and leaf surface area. Fumigation with SO(2) and O(3) + SO(2) caused insignificant decreases of shoot length, total dry weight and needle surface area of spruce. The lateral leader shoot growth of spruce exposed to O(3) was significantly reduced only in the last year of the experiment. Growth rates of the spruce exposed to charcoal filtered air and non-acidic precipitation were reduced more than those of beech and fir. Growth variables determined for fir reflected different rates of incremental change. Exposure to O(3) resulted in the largest dry matter production of all fir groups but those exposed to charcoal filtered air and non-acidic precipitation responded with the best lateral leader shoot growth, lowest specific leaf area (SLA) and leaf area ratio (LAR) respectively indicating best metabolic efficiency. At the conclusion of this study a classification of sensitivity was developed for the tree species.

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Influence of soil substrate and ozone plus acid mist on the frost resistance of young Norway spruce.

The needles of clonal Norway spruce grown in environmental chambers on two different soils (an acidic soil 1 and a calcareous soil 2) and exposed to two levels of ozone fumigation (a low level combined with neutral mist = control, and an elevated one combined with acid mist = treatment) were analyzed for their frost hardiness. No effect of ozone was observed on either the development of frost resistance during the hardening phase or on the decrease in frost resistance during the dehardening phase. The preliminary results of Brown et al. (1987) and Barnes and Davidson (1988), which indicated that ozone treatment predisposes plants to winter injury, could thus not be confirmed. Frost resistance was, however, distinctly influenced by the content of the mineral nutrients of the soils. The pronounced K(+) deficiency of the needles of the trees growing on the neutral soil (Alps) had less effect on the development of frost resistance than did the Ca(++) and Mg(++) deficiency of the needles of the trees grown on the acidic soil 1 (Bavarian Forest). The variability of frost resistance between the different clones on soil 1 was partly attributed to fluctuations in the mineral nutrient content of the needles, rather than to a genetic predisposition.

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Surface structures and epicuticular wax composition of spruce needles after long-term treatment with ozone and acid mist.

Epicuticular wax structures and epicuticular wax composition were studied in needles of Picea abies (L.) Karst. plants after a long-term fumigation experiment. SEM observations showed aggregated as well as undamaged waxes without any relation to treatments. The chemical wax analysis revealed clone specific differences, but only one compound, the nonacosan-10-ol (C(29)-ol), was found enriched in ozone/acid mist treatments.

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Alteration of physiological parameters in needles of cloned spruce trees (Picea abies (L.) Karst.) by ozone and acid mist.

Three-year-old clonal spruce trees, kept in growth chambers, were treated with ozone and acid mist during a period of 14 months. One half of the trees were grown on an acidic sandy soil, the other half on a calcareous soil rich in carbonate. At the end of the fumigation period, carbohydrates (glucose, fructose, sucrose, raffinose, starch, glucose-1-phosphate and fructose-6-phosphate) and parameters of the energy status (ATP-, AdN-(ATP + ADP + AMP)- levels, ATP/ADP-ratios and adenylate-energy-charge-(AEC)-values) were determined in the current-year's needles. The results indicate that the metabolic status of a plant tissue is not only influenced by the nature of the air pollutants. Soil factors play an important role in metabolic changes within the plant and are thus of relevance in the manifestation of damage symptoms.

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Biochemical response of Norway spruce (Picea abies (L.) karst.) towards 14-month exposure to ozone and acid mist: part II--Effects on protein biosynthesis.

Four-year-old clonal Picea abies (L.) Karst. plants were treated with ozone (100 microm(-3) plus peaks of 130 to 360 microm(-3)) and acid mist (pH 3.0) during two vegetation periods. Pulse labelling experiments on shoots were performed with [(35)S]methionine in the second year of exposure. Extraction of soluble needle proteins in citric acid buffer of pH 2.8 revealed protein patterns on SDS polyacrylamide gels that differed from those of control needles fumigated with ambient levels of ozone (50 microg m(-3)) and mist of pH 5.6. New proteins of MW 16000 and 32000 were synthesized only in ozone-exposed needles and could not be detected in the controls.

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Interactions between Sitka spruce, the green spruce aphid, sulphur dioxide pollution and drought.

Sitka spruce trees, with and without the aphid Elatobium abietinum and/or drought treatment, were subjected to 25 nl litre(-1) of sulphur dioxide over a 2-month period. Aphids became three times as abundant on the fumigated trees if they were well watered and twice as abundant on trees from which water was withheld, compared with unfumigated controls. Growth parameters of the trees were little affected by pollution alone, but were substantially reduced by either aphids or drought. There was a significant interaction between SO(2) and aphids in a further reduction of both leader extension and root weight. Root weight was also reduced by 24% more than expected from the additive effects of the combined SO(2) and drought treatment.

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