Longitudinal in vivo metabolic labeling reveals tissue-specific mitochondrial proteome turnover rates and proteins selectively altered by parkin deficiency.

Our study utilizes a longitudinal isotopic metabolic labeling approach in vivo in combination with organelle fraction proteomics to address the role of parkin in mitochondrial protein turnover in mice. The use of metabolic labeling provides a method to quantitatively determine the global changes in protein half-lives whilst simultaneously assessing protein expression. Studying two diverse mitochondrial populations, we demonstrated the median half-life of brain striatal synaptic mitochondrial proteins is significantly greater than that of hepatic mitochondrial proteins (25.7 vs. 3.5 days). Furthermore, loss of parkin resulted in an overall, albeit modest, increase in both mitochondrial protein abundance and half-life. Pathway and functional analysis of our proteomics data identified both known and novel pathways affected by loss of parkin that are consistent with its role in both mitochondrial quality control and neurodegeneration. Our study therefore adds to a growing body of evidence suggesting dependence on parkin is low for basal mitophagy in vivo and provides a foundation for the investigation of novel parkin targets.

Phytotoxicity of polymetallic mine wastes from southern Tuscany and Saxony.

Restoration potential of mine wastes or approaches to improve soil conditions and to ameliorate phytotoxicity on these sites may be simulated in standardized greenhouse experiments. Plants can be cultivated side by side on materials from different origins in dilution series with defined admixtures of certain aggregates. Mine wastes used in the present study originated from Fenice Capanne (FC, Tuscany, Italy) and Altenberg (ALT, Saxony, Germany). Tailings of the Italian site contain high concentrations of lead, zinc, arsenic and sulphur while tin, wolfram, molybdenum and lithium are highly elevated in the German mine waste. We tested growth responses of five crop species and analyzed concentrations of various metals and nutrients in the shoot to evaluate the toxicity of the FC mine waste and found oilseed rape being the most and corn the least resistant crop. Interestingly, oilseed rape accumulated seven times higher levels of lead than corn without showing adverse effects on productivity. In a subsequent comparison of FC and ALT mine waste, we cultivated different species of buckwheat (Fagopyrum spec.), a fast growing genus that evolved in mountain areas and that has been shown to be tolerant to low pH and high concentrations of metals. We found that the FC mine waste was more toxic than the ALT substrate in F. tataricum and F. esculentum. However, lower admixtures of FC material (10%) resulted in stronger growth reductions than higher proportions (25%) of the mine waste which was primarily related to the slightly lower pH and higher availability of essential metals due to the admixture of sand. These results confirm the importance of managing the soil chemical and physical characteristics of wastelands and call for the development of assisted reclamation to prepare sites for regular biomass production.

Foliar nutrient and metal levels of crops in the Mount Cameroon area-reference values for plant nutrition and environmental monitoring.

The growing population number and traffic loads, increasing environmental pressures, agricultural intensification, and the establishment of Mount Cameroon National Park demand farsighted environmental management in the region and the definition of a favorable ecological status. Since plants grow in the interface between soils and the atmosphere they can be used as passive biomonitors for the environmental quality. At the same time, the accumulation of nutrients and pollutants in crops is linked to human health, so that foliar elemental levels can be used as an integrative measure for environmental pollution and impact assessment. In the present study, we collected leaf samples of plantain, cassava, cocoyam, and maize on 28 sites at the southern flanks of Mt. Cameroon and determined 20 chemical elements. Air pollution in the study area comes from biomass and waste burning mainly, but emissions from traffic and a large refinery were believed to also play a significant role. However, spatial patterns in foliar elemental concentrations reflected the geochemistry rather than specific sources of pollution. Significant differences in foliar metal and nutrient levels were observed between the four species, indicating a different demand and uptake of specific elements. The results were compared to published data on nutrient concentrations in the tested species and the so-called reference plant. The data can be used as a baseline for future studies in plant nutrition and the environmental monitoring in inner tropical regions where these crops are grown.

Exploratory study on the presence of GM oilseed rape near German oil mills.

Seed losses from imported oilseed rape (OSR) and the genetically modified (GM) admixtures therein may potentially lead to the establishment of transgenic plants and their hybridization with wild crucifers. The post-market environmental monitoring (PMEM) must therefore also address problems related to seed spillages of GM OSR. Since detailed information on imported commodity flows, GM contents, means of transport, downstream users and efficient containment of GM OSR was lacking, we performed a field study in the vicinity of large oil mills and seed processing industries at the harbours along the river Rhine. One hundred thirty-six composite samples taken from one to 20 plants per site were collected near roads, railways and waterways. Individuals or large groups of feral OSR plants were detected in all of the nine study areas, but only one plant out of 1918 tested was confirmed to be transgenic (GT73). The results suggest that a spread of herbicide tolerant GM OSR has not occurred to date. In order to confirm the absence of GM feral OSR and potentially adverse effects of GM plants in the future, we recommend monitoring feral OSR on a routine basis. We present an approach for the sampling and testing of feral OSR that is based on floristic mapping and rapid tests for the determination of herbicide tolerances.

Potential toxic effects of aircraft de-icers and wastewater samples containing these compounds.

One of the major problems of airport operation is the impact of pollution caused by runoff waters. Runoff waters at an airport may contain high concentrations of different contaminants resulting from various activities of its operation. High quantities of aircraft de-icing/anti-icing fluids are used annually at airports worldwide. Aircraft de-icers and anti-icers may have negative environmental impacts, but their effects on aquatic organisms are virtually unknown. In order to address this issue, aircraft de-icers, pavement de-icers and wastewater samples were obtained from a regional airport. To evaluate the toxicity of wastewater samples and aircraft de-icing/anti-icing fluids (ADAFs), two bio-tests were performed: the Lemna growth inhibition test according to OECD guideline 221 and the luminescent bacteria test according to ISO guideline 11348-2. In the Lemna growth inhibition test, phytotoxicity was assessed using the endpoints frond number and frond area. The luminescent bacteria test involved the marine bacterium Vibrio fischeri. The estimates of effective concentrations (EC50) values were determined using the free software R and the "drc" library. Aquatic plants and marine bacteria showed a higher sensitivity towards ADAFs than to wastewater samples. Experiments showed that aircraft de-icing/anti-icing fluids and wastewater samples were relatively more toxic towards Lemna gibba L. in comparison to V. fischeri.

Accumulation of aluminium and physiological status of tree foliage in the vicinity of a large aluminium smelter.

A pollution gradient was observed in tree foliage sampled in the vicinity of a large aluminium production facility in Patagonia (Argentina). Leaves of Eucalyptus rostrata, and Populus hybridus and different needle ages of Pinus spec. were collected and concentrations of aluminium (Al) and sulphur (S) as well as physiological parameters (chlorophyll and lipid oxidation products) were analyzed. Al and S concentrations indicate a steep pollution gradient in the study showing a relationship with the physiological parameters in particular membrane lipid oxidation products. The present study confirms that aluminium smelting results in high Al and sulphur deposition in the study area, and therefore further studies should be carried out taking into account potentially adverse effects of these compounds on human and ecosystem health.

Effects of elevated CO2 concentrations and fly ash amended soils on trace element accumulation and translocation among roots, stems and seeds of Glycine max (L.) Merr.

The carbon dioxide (CO(2)) levels of the global atmosphere and the emissions of heavy metals have risen in recent decades, and these increases are expected to produce an impact on crops and thereby affect yield and food safety. In this study, the effects of elevated CO(2) and fly ash amended soils on trace element accumulation and translocation in the root, stem and seed compartments in soybean [Glycine max (L.) Merr.] were evaluated. Soybean plants grown in fly ash (FA) amended soil (0, 1, 10, 15, and 25% FA) at two CO(2) regimes (400 and 600 ppm) in controlled environmental chambers were analyzed at the maturity stage for their trace element contents. The concentrations of Br, Co, Cu, Fe, Mn, Ni, Pb and Zn in roots, stems and seeds in soybeans were investigated and their potential risk to the health of consumers was estimated. The results showed that high levels of CO(2) and lower concentrations of FA in soils were associated with an increase in biomass. For all the elements analyzed except Pb, their accumulation in soybean plants was higher at elevated CO(2) than at ambient concentrations. In most treatments, the highest concentrations of Br, Co, Cu, Fe, Mn, and Pb were found in the roots, with a strong combined effect of elevated CO(2) and 1% of FA amended soils on Pb accumulation (above maximum permitted levels) and translocation to seeds being observed. In relation to non-carcinogenic risks, target hazard quotients (TQHs) were significant in a Chinese individual for Mn, Fe and Pb. Also, the increased health risk due to the added effects of the trace elements studied was significant for Chinese consumers. According to these results, soybean plants grown for human consumption under future conditions of elevated CO(2) and FA amended soils may represent a toxicological hazard. Therefore, more research should be carried out with respect to food consumption (plants and animals) under these conditions and their consequences for human health.

Accumulation of polycyclic aromatic hydrocarbons and trace elements in the bioindicator plants Tillandsia capillaris and Lolium multiflorum exposed at PM10 monitoring stations in Stuttgart (Germany).

The accumulation of polycyclic aromatic hydrocarbons (PAHs) in Tillandsia capillaris Ruiz and Pav. form capillaris and trace elements in T. capillaris and Lolium multiflorum (LAM) cv. Lema was assessed and evaluated in the city of Stuttgart, Germany. Several sites (urban, suburban and rural) categorized according to type and intensity of vehicular traffic were investigated. At these sites, plants of T. capillaris and standardized cultures of L. multiflorum were exposed to ambient air. Foliar concentrations of PAHs (16 priority pollutants according to US-EPA) and of the trace elements Br, Co, Cu, Fe, Mn, Ni, Pb and Zn were determined. A high level of vehicular traffic was associated with the largest concentrations of PM(10) in ambient air and with the highest contents of PAHs and heavy metals in the bioindicator plants. The results showed a similar pattern between T. capillaris and the standardized biomonitor L. multiflorum. Therefore, these results allow us to propose T. capillaris as a suitable bioindicator to assess the distribution of pollution impacts caused by PAHs and trace elements in different subtropical and tropical regions.

Effects of elevated CO2 on grain yield and quality of wheat: results from a 3-year free-air CO2 enrichment experiment.

Spring wheat (Triticum aestivum L. cv. TRISO) was grown for three consecutive seasons in a free-air carbon dioxide (CO(2)) enrichment (FACE) field experiment in order to examine the effects on crop yield and grain quality. CO(2) enrichment promoted aboveground biomass (+11.8%) and grain yield (+10.4%). However, adverse effects were predominantly observed on wholegrain quality characteristics. Although the thousand-grain weight remained unchanged, size distribution was significantly shifted towards smaller grains, which may directly relate to lower market value. Total grain protein concentration decreased significantly by 7.4% under elevated CO(2), and protein and amino acid composition were altered. Corresponding to the decline in grain protein concentration, CO(2) enrichment resulted in an overall decrease in amino acid concentrations, with greater reductions in non-essential than essential amino acids. Minerals such as potassium, molybdenum and lead increased, while manganese, iron, cadmium and silicon decreased, suggesting that adjustments of agricultural practices may be required to retain current grain quality standards. The concentration of fructose and fructan, as well as amounts per area of total and individual non-structural carbohydrates, except for starch, significantly increased in the grain. The same holds true for the amount of lipids. With regard to mixing and rheological properties of the flour, a significant increase in gluten resistance under elevated CO(2) was observed. CO(2) enrichment obviously affected grain quality characteristics that are important for consumer nutrition and health, and for industrial processing and marketing, which have to date received little attention.

High concentrations and dry deposition of reactive nitrogen species at two sites in the North China Plain.

Atmospheric concentrations of major reactive nitrogen (N(r)) species were quantified using passive samplers, denuders, and particulate samplers at Dongbeiwang and Quzhou, North China Plain (NCP) in a two-year study. Average concentrations of NH(3), NO(2), HNO(3), pNH(4)(+) and pNO(3)(-) were 12.0, 12.9, 0.6, 10.3, and 4.7 microg N m(-3) across the two sites, showing different seasonal patterns of these N(r) species. For example, the highest NH(3) concentration occurred in summer while NO(2) concentrations were greater in winter, both of which reflected impacts of N fertilization (summer) and coal-fueled home heating (winter). Based on measured N(r) concentrations and their deposition velocities taken from the literature, annual N dry deposition was up to 55 kg N ha(-1). Such high concentrations and deposition rates of N(r) species in the NCP indicate very serious air pollution from anthropogenic sources and significant atmospheric N input to crops.

Environmental monitoring of fluoride emissions using precipitation, dust, plant and soil samples.

A pollution gradient was observed in precipitation, plants and soils sampled at different locations around a fluoride producing chemical plant in Germany. In all samples the influence of emissions was discernible up to a distance of 500 m from the plant. However, fluoride concentrations in plant bioindicators (leaves of birch and black berry) and in bulk precipitation showed a more pronounced relationship with the distance from the source than fluoride concentrations in soil. Vegetables sampled in the vicinity of the plant also had elevated concentrations of fluoride, but only the consumption of larger quantities of this material would lead to exceedances of recommended daily F-intake. The present study did not indicate the existence of low phytotoxicity thresholds for fluoride in the plant species used in the study. Even at very high fluoride concentrations in leaf tissue (963 ppm) plants did not show injury due to HF. Dust sampling downwind of the chemical plant confirmed that particulate fluoride was of minor importance in the study area.

A chamberless field exposure system for ozone enrichment of short vegetation.

Only few studies have been conducted as yet which focus on the effects of rising tropospheric ozone levels on semi-natural vegetation under free-air conditions. A new technical approach was used to examine the response of calcareous grassland to ozone employing a chamberless fumigation system. Four different ozone regimes were applied (1-, 1.33-, 1.66- and 2-fold ambient air levels) with five replicates each. Ozone enrichment was carried out on circular plots of 2 m in diameter by a computer controlled exposure system. Transparent windscreens encircling each plot accelerated the mixing of ambient air and ozone released. Thus, the use of blowers could be avoided. The exposure system presented here is regarded as an appropriate technique for free-air trace gas enrichment on short vegetation avoiding microclimatic alterations known to affect plant growth and pollutant uptake. Furthermore, the chosen technical set-up was rather cost-effective. Hence, it enabled the establishment of a larger number of replications providing the basis for results of higher statistical power.

Growth and yield responses of spring wheat (Triticum aestivum L. cv. Minaret) to elevated CO2 and water limitation.

Spring wheat (Triticum aestivum L. cv. Minaret) was grown at two different CO2 concentrations (367 and 650 micromol mol(-1)) in open-top-chambers from sowing until final harvest. Furthermore two different watering treatments (well watered and water stressed) and two soil types of different fertility were used. At final harvest, which took place at growth stage 92, plants were separated into different fractions. Elevated atmospheric CO2 caused an accelerated chlorophyll-a breakdown and increased growth and yield. Total shoot biomass was enhanced by 43%, grain yield by 46% and main stem yield by 19%. Water stress also accelerated chlorophyll-a breakdown but reduced total shoot biomass by 40%, grain yield by 45%, main stem yield by 30% and thousand grain weight by 6%. On average, soil fertility altered shoot biomass by 30%, grain yield by 39% and main stem yield by 25%.

CO(2) enrichment enhances flag leaf senescence in barley due to greater grain nitrogen sink capacity.

Senescence is a highly regulated process which is under genetic control. In monocarpic plants, the onset of fruit development is the most important factor initiating the senescence process. During senescence, a large fraction of plant nutrients is reallocated away from vegetative tissues into generative tissues. Senescence may therefore be regarded as a highly effective salvage mechanism to save nutrients for the offspring. CO(2) enrichment, besides increasing growth and yield of C(3) plants, has often been shown to accelerate leaf senescence. C(3) plants grown under elevated CO(2) experience alterations in their nutrient relations. In particular their tissue nitrogen concentrations are always lower after exposure to elevated CO(2). We used a monocarpic C(3) crop - spring barley (Hordeum vulgare cv. Alexis) - grown in open-top field chambers to test the effects of CO(2) enrichment on growth and yield, on nitrogen acquisition and redistribution, and on the senescence process in flag leaves, at two applications of nitrogen fertilizer. CO(2) enrichment (650 vs. 366 µmol mol(-1)) caused an increase both in biomass and in grain yield by 38% (average of the two fertilizer applications) which was due to increased tillering. Total nitrogen uptake of the crops was not affected by CO(2) treatment but responded solely to the N supply. Nitrogen concentrations in grains and straw were significantly lower (-33 and -24%) in plants grown at elevated CO(2). Phenological development was not altered by CO(2) until anthesis. However, progress of flag leaf senescence as assessed by chlorophyll content, protein content and content of large and small subunit of RubisCO and of cytochrome b559 was enhanced under elevated CO(2) concentrations by approximately 4 days. We postulate that CO(2) enhanced flag leaf senescence in barley crops by increasing the nitrogen sink capacity of the grains.

Effects of elevated CO2, nitrogen supply and tropospheric ozone on spring wheat-II. Nutrients (N, P, K, S, Ca, Mg, Fe, Mn, Zn).

CO(2) enrichment is expected to alter leaf demand for nitrogen and phosphorus in plant species with C(3) carbon dioxide fixation pathway, thus possibly causing nutrient imbalances in the tissues and disturbance of distribution and redistribution patterns within the plants. To test the influence of CO(2) enrichment and elevated tropospheric ozone in combination with different nitrogen supply, spring wheat (Tritium aestivum L. cv. Minaret) was exposed to three levels of CO(2) (361, 523, and 639 microl litre(-1), 24 h mean from sowing to final harvest), two levels of ozone (28.4 and 51.3 nl litre(-1)) and two levels of nitrogen supply (150 and 270 kg ha(-1)) in a full-factorial design in open-top field chambers. Additional fertilization experiments (120, 210, and 330 kg N ha(-1)) were carried out at low and high CO(2) levels. Macronutrients (N, P, K, S, Ca, Mg) and three micronutrients (Mn, Fe, Zn) were analysed in samples obtained at three different developmental stages: beginning of shoot elongation, anthesis, and ripening. At each harvest, plant samples were separated into different organs (green and senescent leaves, stem sections, ears, grains). According to analyses of tissue concentrations at the beginning of shoot elongation, the plants were sufficiently equipped with nutrients. Elevated ozone levels neither affected tissue concentrations nor shoot uptake of the nutrients. CO(2) and nitrogen treatments affected nutrient uptake, distribution and redistribution in a complex manner. CO(2) enrichment increased nitrogen-use efficiency and caused a lower demand for nitrogen in green tissues which was reflected in a decrease of critical nitrogen concentrations, lower leaf nitrogen concentrations and lower nitrogen pools in the leaves. Since grain nitrogen uptake during grain filling depended completely on redistribution from vegetative pools in green tissues, grain nitrogen concentrations fell considerably with severe implications for grain quality. Ca, S, Mg and Zn in green tissues were influenced by CO(2) enrichment in a similar manner to nitrogen. Phosphorus concentrations in green tissues, on the other hand, were not, or only slightly, affected by elevated CO(2). In stems, 'dilution' of all nutrients except manganese was observed, caused by the huge accumulation of water soluble carbohydrates, mainly fructans, in these tissues under CO(2) enrichment. Whole shoot uptake was either remarkably increased (K, Mn, P, Mg), nearly unaffected (N, S, Fe, Zn) or decreased (Ca) under CO(2) enrichment. Thus, nutrient cycling in plant-soil systems is expected to be altered under CO(2) enrichment.

Effects of elevated CO2 nitrogen supply and tropospheric ozone on spring wheat. I. Growth and yield.

Spring wheat (Triticum aestivum L. cv. Minaret) was exposed to three CO(2) levels, in combination with two nitrogen fertilizer levels and two levels of tropospheric ozone, from sowing to ripening in open-top chambers. Three additional nitrogen fertilizer treatments were carried out at the lowest and the highest CO(2) level, respectively. Plants were harvested at growth stages 31, 65 and 93 and separated into up to eight fractions to gain information about biomass partitioning. CO(2) enrichment (263 microl litre(-1) above ambient levels) drastically increased biomass of organs serving as long-term carbohydrate pools. Peduncle weight increased by 92%, stem weight by 73% and flag leaf sheath weight by 59% at growth stage 65. Average increase in shoot biomass due to CO(2) enrichment amounted to 51% at growth stage 65 and 36% at final harvest. Average yield increase was 34%. Elevated nitrogen application was most effective on biomass of green tissues. Yield was increased by 30% when nitrogen application was increased from 150 to 270 kg N ha(-1). Significant interactions were observed between CO(2) enrichment and nitrogen application. Yield increase due to CO(2) ranged from 23% at 120 kg N to 47% at 330 kg N. Triticum aestivum cv. Minaret was not very responsive to ozone at 1.5 times ambient levels. 1000 grain weight was slightly decreased, which was compensated by an increased number of grains.

Influence of tyrosine residues Y705 and Y807 on the transforming potency of the v-fms oncogene product of feline sarcoma virus.

Cell transformation is characterized by overt changes in growth control and cell morphology. To study the role of tyrosine residues Y705 and Y807 of v-Fms of the McDonough strain of feline sarcoma virus in cell transformation we replaced them individually with phenylalanine residues. Cells expressing the mutant genes showed mitogenic properties similar to wild-type v-Fms transformed cells. However, the morphology of cells expressing the Y807F mutant remained the same as nontransformed cells. Four phosphoproteins of 190, 120, 55 and 50 kDa were detected in cells expressing the wild-type but were absent in cells expressing the mutant Y807F-v-fms gene.

Influence of ozone and ethylenediurea (EDU) on growth and yield of bean (Phaseolus vulgaris L.) in open-top field chambers.

Greenhouse and ambient air experiments have shown ethylene diurea (EDU) to be a strong and specific protective suppressant of ozone injury in plants. To examine how EDU affects plant responses to various ozone (O(3)) levels under controlled field conditions, Phaseolus vulgaris L. cv. Lit was treated with 150 ppm EDU every 14 days and exposed in open-top chambers to charcoal-filtered air (CF), nonfiltered air (NF) or two cf treatments with ozone added. The ozone treatments were proportional additions of one (CF1) and two (CF2) times ambient ozone levels. The mean ozone concentrations in the CF, NF, CF1 and CF2 treatments were 0.98, 14.1, 14.98 and 31.56 nl litre(-1). A two-way split plot ANOVA revealed that shoot dry weight was significantly reduced by ozone. EDU treatment was highly significant for leaf dry weight, root dry weight and shoot dry weight, but not for pod dry weight; leading to a higher biomass of EDU-treated plants. Ozone/EDU interactions were significant for root weight only, indicating that EDU reduced growth suppression by ozone. These results show that EDU action on plant biomass could be interpreted as a delay in senescence since EDU-treated plants showed a significant decreased biomass loss even in the CF treatment.

Effects of ethylenediurea and ozone on the antioxidative systems in beans (Phaseolus vulgaris L.).

To study the biochemical mechanism of EDU protection against ozone injury, peroxidase, ascorbate-dependent peroxidase, and catalase activities, and the contents of ascorbic acid, dehydroascorbic acid, malondialdehyde and soluble protein were measured in Phaseolus vulgaris L. cv. Lit exposed to ozone and ethylenediurea (EDU) in open-top chambers. Plants not treated with EDU showed foliar bronzing due to ozone, while EDU-treated plants were not affected. EDU application modified the reaction of biochemical parameters to ozone. Soluble protein content was elevated by EDU. Peroxidase activity increased with ozone exposure in untreated plants only, while ascorbate-dependent peroxidase activity was lower in EDU treated plants. Catalase activity decreased in EDU-untreated plants. The ratio of ascorbic acid to dehydroascorbic acid was significantly increased in EDU treated plants. These results suggest that EDU might induce ascorbic acid synthesis and therefore provide the plant with a very potent antioxidant. Or the content of hydrogen peroxide was reduced due to other unknown processes and caused a delay in foliar senescence, regardless of whether these processes were ozone-induced or due to natural aging processes.

Modelling stomatal responses of spring wheat (Triticum aestivum L. cv. Turbo) to ozone and different levels of water supply.

Spring wheat (Triticum aestivum L. cv. Turbo) was exposed to different levels of ozone and water supply in open-top chambers in 1991. Air was charcoal filtered (CF), non-filtered (NF) and CF plus proportional addition of ambient or twice ambient ozone (CF1, CF2). Seasonal means of O(3), taken over 24 h, were 2.3, 20.6, 17.3, and 34.5 nl litre(-1) for CF, NF, CF1 and CF2 treatments, respectively. A split-plot design was used to obtain two levels of water supply: one-half of the pots was irrigated sufficiently not to show any symptoms of drought stress; the others were exposed to low water supply and received 50% of these amounts. Using a steady-state porometer approximately 800 measurements of stomatal conductance (g(s)) were made on flag leaves from 68 to 106 days after sowing. The measurements yielded only small differences of maximum conductance between the two levels of water supply. Therefore, low water supply did not protect wheat plants against ozone injury via reduced stomatal uptake in this experiment. To describe the effects of environmental variables on the stomatal behaviour, boundary-line analysis and non-linear regression analysis were used. Besides microclimatic parameters, the ozone dose of flag leaves was introduced as an independent variable affecting stomatal aperture. A well-defined boundary line for ozone dose was found, suggesting that increasing ozone dose caused stomatal closure in wheat flag leaves. But at high ozone doses, co-acting senescence seems also responsible for the decrease in stomatal conductance. A multiplicative boundary-line model was used to predict stomatal conductance from combinations of environmental variables. In the test carried out with the measurements of stomatal conductance, the model accounted only for 40% of the variation of g(s). Generalized stomatal response patterns of the herbaceous growth form, the dependence of the variables' age and ozone dose and the lack of an important factor influencing stomatal response (water status of the plant) in the model, are suggested as explanations of the poor results of the test.