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• Increasing atmospheric concentrations of phytotoxic ozone (O(3) ) can constrain growth and carbon sink strength of forest trees, potentially exacerbating global radiative forcing. Despite progress in the conceptual understanding of the impact of O(3) on plants, it is still difficult to detect response patterns at the leaf level. • Here, we employed principal component analysis (PCA) to analyse a database containing physiological leaf-level parameters of 60-yr-old Fagus sylvatica (European beech) trees. Data were collected over two climatically contrasting years under ambient and twice-ambient O(3) regimes in a free-air forest environment. • The first principal component (PC1) of the PCA was consistently responsive to O(3) and crown position within the trees over both years. Only a few of the original parameters showed an O(3) effect. PC1 was related to parameters indicative of oxidative stress signalling and changes in carbohydrate metabolism. PC1 correlated with cumulative O(3) uptake over preceding days. • PC1 represents an O(3) -responsive multivariate pattern detectable in the absence of consistently measurable O(3) effects on individual leaf-level parameters. An underlying effect of O(3) on physiological processes is indicated, providing experimental confirmation of theoretical O(3) response patterns suggested previously., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

The hypothesis was tested that O(3)-induced changes in leaf-level photosynthetic parameters have the capacity of limiting the seasonal photosynthetic carbon gain of adult beech trees. To this end, canopy-level photosynthetic carbon gain and respiratory carbon loss were assessed in European beech (Fagus sylvatica) by using a physiologically based model, integrating environmental and photosynthetic parameters. The latter were derived from leaves at various canopy positions under the ambient O(3) regime, as prevailing at the forest site (control), or under an experimental twice-ambient O(3) regime (elevated O(3)), as released through a free-air canopy O(3) fumigation system. Gross carbon gain at the canopy-level declined by 1.7%, while respiratory carbon loss increased by 4.6% under elevated O(3). As this outcome only partly accounts for the decline in stem growth, O(3)-induced changes in allocation are referred to and discussed as crucial in quantitatively linking carbon gain with stem growth., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Ground-level ozone (O(3)) has gained awareness as an agent of climate change. In this respect, key results are comprehended from a unique 8-year free-air O(3)-fumigation experiment, conducted on adult beech (Fagus sylvatica) at Kranzberg Forest (Germany). A novel canopy O(3) exposure methodology was employed that allowed whole-tree assessment in situ under twice-ambient O(3) levels. Elevated O(3) significantly weakened the C sink strength of the tree-soil system as evidenced by lowered photosynthesis and 44% reduction in whole-stem growth, but increased soil respiration. Associated effects in leaves and roots at the gene, cell and organ level varied from year to year, with drought being a crucial determinant of O(3) responsiveness. Regarding adult individuals of a late-successional tree species, empirical proof is provided first time in relation to recent modelling predictions that enhanced ground-level O(3) can substantially mitigate the C sequestration of forests in view of climate change., (Copyright 2010 Elsevier Ltd. All rights reserved.)

In this study, the effects of different light intensities either in direct sunlight or in the shade crown of adult beech (Fagus sylvatica L.) trees on delta13C and Delta18O were determined under ambient (1 x O3) and twice-ambient (2 x O3) atmospheric ozone concentrations during two consecutive years (2003 and 2004). We analysed the isotopic composition in leaf bulk, leaf cellulose, phloem and xylem material and related the results to (a) meteorological data (air temperature, T and relative humidity, RH), (b) leaf gas exchange measurements (stomatal conductance, g(s); transpiration rate, E; and maximum photosynthetic activity, A(max)) and (c) the outcome of a steady-state evaporative enrichment model. Delta13C was significantly lower in the shade than in the sun crown in all plant materials, whilst Delta18O was increased significantly in the shade than in the sun crown in bulk material and cellulose. Elevated ozone had no effect on delta13C, although Delta18O was influenced by ozone to varied degrees during single months. We observed significant seasonal changes for both parameters, especially in 2004, and also significant differences between the study years. Relating the findings to meteorological data and gas exchange parameters, we conclude that the differences in Delta18O between the sun and the shade crown were predominantly caused by the Péclet effect. This assumption was supported by the modelled Delta18O values for leaf cellulose. It was demonstrated that independent of RH, light-dependent reduction of stomatal conductance (and thus transpiration) and of A(max) can drive the pattern of Delta18O increase with the concomitant decrease of delta13C in the shade crown. The effect of doubling ozone levels on time-integrated stomatal conductance and transpiration as indicated by the combined analysis of Delta18O and delta13C was much lower than the influence caused by the light exposure.

The effects of elevated O3 on photosynthetic properties in adult beech trees (Fagus sylvatica) were investigated in relation to leaf mass per area as a measure of the gradually changing, within-canopy light availability. Leaves under elevated O3 showed decreased stomatal conductance at unchanged carboxylation capacity of Rubisco, which was consistent with enhanced delta 13C of leaf organic matter, regardless of the light environment during growth. In parallel, increased energy demand for O3 detoxification and repair was suggested under elevated O3 owing to enhanced dark respiration. Only in shade-grown leaves,light-limited photosynthesis was reduced under elevated O3, this effect being accompanied by lowered F(v)/F(m). These results suggest that chronic O3 exposure primarily caused stomatal closure to adult beech trees in the field regardless of the within-canopy light gradient. However, light limitation apparently raised the O3 sensitivity of photosynthesis and accelerated senescence in shade leaves.

The effect of free-air ozone fumigation and crown position on antioxidants were determined in old-growth spruce (Picea abies) trees in the seasonal course of two consecutive years (2003 and 2004). Levels of total ascorbate and its redox state in the apoplastic washing fluid (AWF) were increased under double ambient ozone concentrations (2xO3), whilst ascorbate concentrations in needle extracts were unchanged. Concentrations of apoplastic and symplastic ascorbate were significantly higher in 2003 compared to 2004 indicating a combined effect of the drought conditions in 2003 with enhanced ozone exposure. Elevated ozone had only weak effects on total glutathione levels in needle extracts, phloem exudates and xylem saps. Total and oxidised glutathione concentrations were higher in 2004 compared to 2003 and seemed to be more affected by enhanced ozone influx in the more humid year 2004 compared to the combined effect of elevated ozone and drought in 2003 as observed for ascorbate.

Branch-level gas exchange provided the basis for assessing ozone flux in order to derive the dose-response relationship between cumulative O3 uptake (COU) and carbon gain in the upper sun crown of adult Fagus sylvatica. Fluxes of ozone, CO2 and water vapour were monitored simultaneously by climatized branch cuvettes. The cuvettes allowed branch exposure to an ambient or twice-ambient O3 regime, while tree crowns were exposed to the same O3 regimes (twice-ambient generated by a free-air canopy O3 exposure system). COU levels higher than 20mmolm(-2) led to a pronounced decline in carbon gain under elevated O3. The limiting COU range is consistent with findings on neighbouring branches exposed to twice-ambient O3 through free-air fumigation. The cuvette approach allows to estimate O3 flux at peripheral crown positions, where boundary layers are low, yielding a meso-scale within-crown resolution of photosynthetic foliage sensitivity under whole-tree free-air O3 fumigation.

Whole-tree O3 uptake was exemplified for Picea abies, Fagus sylvatica and Larix decidua in stands at high and low altitude and contrasting water availability through sap flow measurement in tree trunks, intrinsically accounting for drought and boundary layer effects on O3 flux. O3 uptake of evergreen spruce per unit foliage area was enhanced by 100% at high relative to low elevation, whereas deciduous beech and larch showed similar uptake regardless of altitude. The responsiveness of the canopy conductance to water vapor and, as a consequence, O3 uptake to soil moisture and air humidity did not differ between species. Unifying findings at the whole-tree level will promote cause-effect based O3 risk assessment and modeling.

Investigations on sucrose and starch contents in leaves of 60-year-old beech trees ( FAGUS SYLVATICA L.) are the focus of the present study. Five trees were exposed to a twice ambient ozone regime (2 x O(3)) with a free-air canopy exposure system throughout the seasons and five trees under the prevailing ambient ozone regime served as controls (1 x O(3)). In order to examine chronic ozone (O(3)) effects, leaf samples from the sun and shade crowns of the trees were analyzed five times throughout the growing seasons in 2003 and 2004. Sucrose concentrations of leaves collected in 2004 were consistently lower than those taken in 2003, regardless of the O(3) treatment and crown position. However, the opposite was found for starch. O(3) caused a reduction of sucrose and starch contents of sun leaves in both years. Due to the fact that O(3)-responsiveness depends on the O(3) uptake through stomata during the season, all carbohydrate data were related to the cumulative O(3) uptake (COU). Little differences were found comparing sucrose and starch contents in leaves of trees grown under ambient or elevated O (3) regimes, possibly indicating the high capacity of leaves of adult beech to cope with rising O(3) exposure. Even under 2 x O(3), leaves were still able to regulate the O(3) intake by narrowing their stomata at the cost of CO(2)-uptake and sugar synthesis. In order to clarify whole-tree response patterns carbohydrate data were compared with photosynthesis, stomatal conductance and electron transport rates. In 2004 all parameters revealed a significant common response pattern to COU that indicated a reduction for all parameters under 2 x O(3).

Knowledge of responses of photosynthesis, respiration, and stomatal conductance to cumulative ozone uptake (COU) is still scarce, and this is particularly the case for adult trees. The effect of ozone (O(3)) exposure on trees was examined with 60-year-old beech trees (FAGUS SYLVATICA) at a forest site of southern Germany. Trees were exposed to the ambient O(3) regime (1 x O(3)) or an experimentally elevated twice-ambient O(3) regime (2 x O(3)). The elevated 2 x O (3) regime was provided by means of a free-air O(3) canopy exposure system. The hypotheses were tested that (1) gas exchange is negatively affected by O(3) and (2) the effects of O(3) are dose-dependent and thus the sizes of differences between treatments are positively related to COU. Gas exchange (light-saturated CO(2) uptake rate A(max), stomatal conductance g (s), maximum rate of carboxylation Vc (max), ribulose-1,5-bisphosphate turnover limited rate of photosynthesis J (max), CO(2) compensation point CP, apparent quantum yield of net CO(2) uptake AQ, carboxylation efficiency CE, day- and nighttime respiration) and chlorophyll fluorescence (electron transfer rate, ETR) were measured IN SITU on attached sun and shade leaves. Measurements were made periodically throughout the growing seasons of 2003 (an exceptionally dry year) and 2004 (a year with average rainfall). In 2004 Vc(max), J(max), and CE were lower in trees receiving 2 x O(3) compared with the ambient O(3) regime (1 x O(3)). Treatment differences in Vc (max), J (max), CE were rather small in 2004 (i.e., parameter levels were lower by 10 - 30 % in 2 x O(3) than 1 x O(3)) and not significant in 2003. In 2004 COU was positively correlated with the difference between treatments in A (max), g (s), and ETR (i.e., consistent with the dose-dependence of O(3)'s deleterious effects). However, in 2003, differences in A(max), g (s), and ETR between the two O(3) regimes were smaller at the end of the dry summer 2003 (i.e., when COU was greatest). The relationship of COU with effects on gas exchange can apparently be complex and, in fact, varied between years and within the growing season. In addition, high doses of O(3) did not always have significant effects on leaf gas exchange. In view of the key findings, both hypotheses were to be rejected.

Currently, the most important source of uncertainty in stomatal ozone flux ( FO3) modelling is the stomatal conductance ( gst) factor. Hence FO3 model accuracy will strongly depend on the gst model being implemented. In this study the recently developed semi-empirical Gst model of Dewar was coupled to the widely known biochemical photosynthesis ( An) model of Farquhar. The Gst performance of this model combination was evaluated with a 4-month time series of beech ( Fagus sylvatica L.) measurements. The Gst model was hereto optimized in two steps to a 4-day and a 8-day period. A comparison between the modelled and measured gst to O(3) (gstO3) revealed a rather good overall performance (R(2)=0.77). Errors between the model combination and the measurements are thought to be largely caused by a moderate performance of the AN model, due to poor parameterization. Two 2-day periods with distinctly differing soil and meteorological conditions were chosen to give a picture of the daily gst performance. Although instant relative differences between modelled and measured gstO3 are sometimes high, the model combination is able to simulate the rough daily courses of gstO3 and hence FO3 reasonably well. Further improvement on full parameterization of the gst model and a well-parameterized An model to be linked to are needed to draw founded conclusions about its performance. Future efforts hereto are certainly justified since the model's mechanistic nature makes it a tool able to model gst variation in space and time, O(3) effects on gst, and effective FO3.

Ozone affects adult trees significantly, but effects on stem growth are hard to prove and difficult to correlate with the primary sites of ozone damage at the leaf level. To simulate ozone effects in a mechanistic way, at a level relevant to forest stand growth, we developed a simple ozone damage and repair model (CASIROZ model) that can be implemented into mechanistic photosynthesis and growth models. The model needs to be parameterized with cuvette measurements on net photosynthesis and dark respiration. As the CASIROZ ozone sub-model calculates effects of the ozone flux, a reliable representation of stomatal conductance and therefore ozone uptake is necessary to allow implementation of the ozone sub-model. In this case study the ozone sub-model was used in the ANAFORE forest model to simulate gas exchange, growth, and allocation. A preliminary run for adult beech (FAGUS SYLVATICA) under different ozone regimes at the Kranzberg forest site (Germany) was performed. The results indicate that the model is able to represent the measured effects of ozone adequately, and to distinguish between immediate and cumulative ozone effects. The results further help to understand ozone effects by distinguishing defence from damage and repair. Finally, the model can be used to extrapolate from the short-term results of the field study to long-term effects on tree growth. The preliminary simulations for the Kranzberg beech site show that, although ozone effects on yearly growth are variable and therefore insignificant when measured in the field, they could become significant at longer timescales (above 5 years, 5 % reduction in growth). The model offers a possible explanation for the discrepancy between the significant effects on photosynthesis (10 to 30 % reductions simulated), and the minor effects on growth. This appears to be the result of the strong competition and slow growth of the Kranzberg forest, and the importance of stored carbon for the adult beech (by buffering effects on carbon gain). We finally conclude that inclusion of ozone effects into current forest growth and yield models can be an important improvement into their overall performance, especially when simulating younger and less dense forests.

Three-year-old beech (Fagus sylvatica) seedlings growing in containers were placed into the sun and shade crown of a mature beech stand exposed to ambient (1 x O(3)) and double ambient (2 x O(3)) ozone concentrations at a free-air exposure system ("Kranzberg Forst", Germany). Pigments, alpha-tocopherol, glutathione, ascorbate, and gas exchange were measured in leaves during 2003 (a drought year) and 2004 (an average year). Sun-exposed seedlings showed higher contents of antioxidants, xanthophylls, and beta-carotene and lower contents of chlorophyll, alpha-carotene, and neoxanthin than shade-exposed seedlings. In 2003 sun-exposed seedlings showed higher contents of carotenoids and total glutathione and lower net photosynthesis rates (A(max)) compared to 2004. O(3) exposure generally affected the content of chlorophyll, the xanthophyll cycle, and the intercellular CO(2) concentration (c(i)). Seedlings differed from the adjacent adult trees in most biochemical and physiological parameters investigated: Sun exposed seedlings showed higher contents of alpha-tocopherol and xanthophylls and lower contents of ascorbate, chlorophyll, neoxanthin, and alpha-carotene compared to adult trees. Shade exposed seedlings had lower contents of xanthophylls, alpha-carotene, and alpha-tocopherol than shade leaves of old-growth trees. In 2003, seedlings had higher A(max), stomatal conductance (g(s)), and c(i) under 2 x O(3) than adult trees. The results showed that shade acclimated beech seedlings are more sensitive to O(3), possibly due to a lower antioxidative capacity per O(3) uptake. We conclude that beech seedlings are uncertain surrogates for adult beech trees.

Accelerated leaf senescence is one of the harmful effects of elevated tropospheric ozone concentrations ([O(3)]) on plants. The number of studies dealing with mature forest trees is scarce however. Therefore, five 66-year-old beech trees (Fagus sylvatica L.) have been exposed to twice-ambient (2xambient) [O(3)] levels by means of a free-air canopy O(3) exposure system. During the sixth year of exposure, the hypothesis of accelerated leaf senescence in 2xambient [O(3)] compared with ambient [O(3)] trees was tested for both sun and shade leaves. Chlorophyll (chl) fluorescence was used to assess the photosynthetic quantum yield, and chl fluorescence images were processed to compare functional leaf homogeneity and the proportion of O(3)-injured leaf area (stipples) under ambient and 2xambient [O(3)] regimes. Based on the analysis of chl fluorescence images, sun leaves of both ambient and 2xambient [O(3)] trees had apparently developed typical necrotic O(3) stipples during high O(3) episodes in summer, while accelerated senescence was only observed with sun leaves of 2xambient [O(3)] trees. This latter effect was indicated along with a faster decrease of photosynthetic quantum yield, but without evidence of changes in non-photochemical quenching. Overall, treatment effects were small and varied among trees. Therefore, compared with ambient [O(3)], the consequence of the observed O(3)-induced accelerated leaf senescence for the carbon budget is likely limited.

Temperature increase and altered precipitation are facets of "Global Change", along with enhanced tropospheric ozone (O3) and CO2 levels. Both O3 and drought may curtail the probably limited capacity of "extra" carbon fixation in forest trees under a CO2-enriched atmosphere. In view of the exceptionally dry year of 2003 in Central Europe, this mini-review highlights O3/drought interactions in biochemical and ecophysiological responses of trees. Such interactions appear to vary, depending on the genotype and factorial scenarios. If O3 perturbs stomatal regulation, tolerance to both drought and persisting O3 exposure may be weakened, although drought preceding O3 stress may "harden" against O3 impact. Stomatal closure under drought may shield trees against O3 uptake and injury, which indeed was the case in 2003. However, the trees' "tuning" between O3 uptake and defence capacity is crucial in stress tolerance. Defence may be constrained due to limited carbon fixation, which results from the trade-off with O3 exclusion upon stomatal closure. Drought may cause a stronger reduction in stem growth than does ozone on an annual basis.

We characterized leaf gas exchange and antioxidative defence of two-year-old seedlings and 60-year-old trees of Fagus sylvatica exposed to ambient (1 x O3) or two-fold ambient (2 x O3) O3 concentrations (maximum of 150 ppb) in a free-air canopy exposure system throughout the growing season. Decline in photosynthesis from sun-exposed to shaded conditions was more pronounced in adult than juvenile trees. Seedling leaves and leaves in the sun-exposed canopy had higher stomatal conductance and higher internal CO2 concentrations relative to leaves of adult trees and leaves in shaded conditions. There was a weak overall depression of photosynthesis in the 2 x O3 variants across age classes and canopy positions. Pigment and tocopherol concentrations of leaves were significantly affected by canopy position and tree age, whereas differences between 1 x O3 and 2 x O3 regimes were not observed. Glutathione concentrations were significantly increased under 2 x O3 across both age classes and canopy levels. Seedlings differed from adult trees in relevant physiological and biochemical traits in ozone response. The water-soluble antioxidative systems responded most sensitively to 2 x O3 without regard of tree age or canopy position.

Abstract: Two separate objectives were considered in this study. We examined (1) internal conductance to CO2 (g i) and photosynthetic limitations in sun and shade leaves of 60-year-old Fagus sylvatica, and (2) whether free-air ozone fumigation affects g i and photosynthetic limitations. g i and photosynthetic limitations were estimated in situ from simultaneous measurements of gas exchange and chlorophyll fluorescence on attached sun and shade leaves of F. sylvatica. Trees were exposed to ambient air (1× O3) and air with twice the ambient ozone concentration (2× O3) in a free-air ozone canopy fumigation system in southern Germany (Kranzberg Forest). g i varied between 0.12 and 0.24molm−2 s−1 and decreased CO2 concentrations from intercellular spaces (C i) to chloroplastic (C c) by approximately 55μmolmol−1. The maximum rate of carboxylation (V cmax) was 22–39% lower when calculated on a C i basis compared with a C c basis. g i was approximately twice as large in sun leaves compared to shade leaves. Relationships among net photosynthesis, stomatal conductance and g i were very similar in sun and shade leaves. This proportional scaling meant that neither C i nor C c varied between sun and shade leaves. Rates of net photosynthesis and stomatal conductance were about 25% lower in the 2× O3 treatment compared with 1× O3, while V cmax was unaffected. There was no evidence that g i was affected by ozone. [Copyright &y& Elsevier]