Ozone exposure induces the activation of leaf senescence-related processes and morphological and growth changes in seedlings of Mediterranean tree species

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Abstract

Four Mediterranean tree taxa, Quercus ilex subsp. ilex, Quercus ilex subsp. ballota, Olea europaea cv. vulgaris and Ceratonia siliqua, were exposed to different ozone (O3) concentrations in open top chambers (OTCs) during 2 years. Three treatments were applied: charcoal-filtered air (CF), non-filtered air (NF) and non-filtered air plus 40 ppbv of O3 (NF+). The photochemical maximal efficiency, Fv/Fm, decreased in NF+ plants during the second year of exposure, especially during the most stressful Mediterranean seasons (winter and summer). An increase of δ13C was found in three of the four studied species during the first year of exposure. This finding was only maintained in C. siliqua during the second year. Decreases in the chlorophyll content were detected during the first year of fumigations in all the species studied, but not during the second year. The NF+ treatment induced changes in foliar anatomical characteristics, especially in leaf mass per area (LMA) and spongy parenchyma thickness, which increased in some species. A reduction in N content and an increase in δ15N were found in all species during the second year when exposed in the NF+ OTCs, suggesting a change in their retranslocation pattern linked to an acceleration of leaf senescence, as also indicated by the above mentioned biochemical and anatomical foliar changes. The two Q. ilex subspecies were the most sensitive species since the changes in N concentration, δ15N, chlorophyll, leaf area, LMA and biomass occurred at ambient O3 concentrations. However, C. siliqua was the most responsive species (29% biomass reduction) when exposed to the NF+ treatment, followed by the two Q. ilex subspecies (14–20%) and O. europaea (no significant reduction). Ozone resistance of the latter species was linked to some plant traits such as chlorophyll concentrations, or spongy parenchyma thickness.

Introduction

Ozone (O3) is a phytotoxic air pollutant widely distributed in the Mediterranean region where adverse effects on different plant tree species have been reported. Probably the most studied species has been Aleppo pine (Alonso et al., 2001, Elvira et al., 1998, Peñuelas et al., 1995, Sanz et al., 2000). Recently, a screening study carried out by Inclán et al. (1999) indicated that ozone exposure induced adverse effects on the biomass of holm oak (Quercus ilex) and olive trees (Olea europaea). However, some studies have highlighted the great intraspecific variability of Mediterranean vegetation to ozone (Elvira et al., 2004, Minnocci et al., 1999). As a result, the information regarding the response of Mediterranean tree species is still scarce, increasing the uncertainties derived from the definition of O3 critical levels. Critical level is defined as the concentration of the pollutant above which adverse effects are likely to occur (UNECE, 1988). The UN-ECE Convention on Long-Range Transboundary Air Pollution has promoted the definition of ozone critical levels for different plant receptors and its activities have largely influenced current European ozone directive. The present critical level for forest tree species is defined as a 6 months AOT40 (ozone accumulated exposure over a threshold of 40 nl l−1) value of 10 ppmv·h. The suitability of this critical level is controversial since many uncertainties still remain to be resolved. Most of them are derived from the lack of experimental or field studies addressing this issue. This is especially the case for the Mediterranean area, making it difficult to establish an adequate regulatory policy for the protection of Mediterranean forests (Bussotti and Gerosa, 2002).

Due to the great diversity of tree species and populations occurring in the Mediterranean area, it would be useful to define those plant traits that could be used as indicators of their potential sensitivity to ozone exposure. Such an approach has been recently adopted by Paoletti et al. (2003) for Mediterranean vegetation, using antioxidant levels as biomarkers of this sensitivity. Although their results are controversial, since the combination of ozone exposure, drought stress and low VPD values could induce an activation of these molecules; the conceptual scheme may be still valid. But what is the key parameter is still an open issue.

The factors that determine plant sensitivity or tolerance are not clearly understood but it is thought to be related to many underlying physiological, anatomical, biochemical and environmental factors (Alonso et al., 2001, Pääkkönen et al., 1998). Ozone effects on tree biomass are the result of several processes occurring at the cellular and physiological levels. Acceleration of leaf senescence has been widely reported as one of the most characteristic processes derived from ozone exposure. These processes involve chlorophyll degradation and reductions in CO2 assimilation (Elvira et al., 1998, Zheng et al., 2002). Leaf senescence can also be characterized by reductions in N foliar levels. Therefore, carbon and nitrogen isotopic discrimination may be useful tools to describe the integrative responses of tree species to O3 exposure. The fractionation of 13C and 12C is caused by diffusion of CO2 through the leaf, boundary layer and stomata, dissolution of CO2 in the apoplastic fluid and enzymatic reactions involved in carbon fixation (Farquhar et al., 1989). In general, the processes of N loss (NH4 volatilization, nitrification, denitrification and leaching) enrich the system with the heavy isotope 15N (Peñuelas and Estiarte, 1997).

Ozone effects on leaf morphology have also been reported. For instance, the early exposure of birch to O3 resulted in a reduction in leaf size, with an increase in the density of stomata, hairs and veins (Gunthardt-Goerg et al., 1993). Similarly, the pectinaceous layer of spongy parenchyma cells was found to swell and protrude (Gunthardt-Goerg et al., 1997) following the exposure of birch to ozone. Many of these defence mechanisms are similar to those found in the schlerophyllous Mediterranean vegetation to prevent damage derived from drought, low VPD values and excessive solar radiation intensity.

This paper focuses on the response of four Mediterranean taxa presenting different physiological and anatomical traits: Quercus ilex subsp. ilex, Quercus ilex subsp. ballota, Ceratonia siliqua and Olea europaea cv. sylvestris, to assess interspecific differences in sensitivity to ozone. The response of two subspecies of Q. ilex was also studied to assess the intraspecific sensitivity to O3. The following hypotheses were tested: (1) ozone exposure enhances plant senescence-related processes; (2) plant responses in biomass are related to alterations in integrated carbon assimilation; and (3) plant traits can be associated with a differential sensitivity to ozone exposure.

Section snippets

Growth conditions and ozone treatments

Plants of Quercus ilex subsp. ilex, Quercus ilex subsp. ballota, Olea europaea cv. sylvestris, and Ceratonia siliqua were raised from seeds. One-year old uniform seedlings (approx. same height and diameter) of each species were transplanted in July 1998 into 6 dm3 containers filled with Universal substrate (peat and pine bark) with a 34% of organic material. Soil pH was 6.6, and 9 g per pot of a slow-release fertilizer (NPK 15:8:11; Osmocote plus) were supplied.

This experiment was conducted in

Ozone exposure and meteorological variables

In this experiment, AOT40 in the NF+ treatment ranged from 6142 (for the 3 months December 1998 to February 1999) to 30,147 ppbv (for the 3 months May 1999 to July 1999). In the NF treatment the AOT40 ranged between 4 ppbv and 2877, respectively, for the same periods. AOT40 was always 0 in the CF treatment (Fig. 1). Typical seasonal coastal Mediterranean trends were observed in temperature, radiation and ozone concentrations, with mild winters and warm and dry summers. Minimum temperatures

Discussion

A gradation in O3 sensitivity was found for the species involved in this experiment. Biomass responses integrate O3-induced physiological alterations and therefore they can be used as a valuable criterion to rank the sensitivity of these species to the pollutant. Both Q. ilex subspecies were the most sensitive species since detrimental effects on their biomass were detected already when exposed to ambient O3 levels, which are below the present critical level of 10000 ppbv·h for 6 months.

Acknowledgements

The authors are grateful to to M. Mendoza for his technical assistance in the OTC field. Financial support was provided by grants IMMPACTE (DURSI and DMA, Generalitat de Catalunya) and “Fundació Territori i Paisatge” from La Caixa de Catalunya. Additional financial support was obtained from grants REN2001-0003 and REN2003-04871 (MCYT, Spain) and by a CIEMAT-Spanish Ministry of Environment contract on Critical Loads and Levels.

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