Physiological and Biochemical Reactions of Olive Genotypes during Site-Relevant Ozone Exposure

Ozone (O3) is one of the major atmospheric pollutants in the Mediterranean area and negatively interacts with several biochemical and physiological processes of the plants. Olive is one of the most widely cultivated trees in the Mediterranean climates and previous studies have shown that O3 can potentially interfere with olive trees productivity. In order to test the physiological and biochemical changes induced by realistic O3 levels (100 and 50 ppb, 5 h day) in newly developed olive leaves, the effects of long term (18 months) O3 exposure were investigated on Moraiolo and Frantoio cultivars. Data on gas exchange showed that O3 treatment had significant effects on photosynthetic activity and stomatal conductance with some differences between the two cultivars. X-ray spectra obtained from guard cells of open and closed stomata confirmed the main role of K to establish the differences in the ionic state between open and closed stomata. Moreover, O3 exposure induced a decrease in K and Ca net counts with respect to the control treatment suggesting the development of an adaptive mechanism to O3 which controls stomata responses at reduced ionic levels. Biochemical assays showed that ascorbate peroxidase activity was not affected by O3 treatments in Frantoio, while it increased in Moraiolo plants exposed to 50 ppb of O3. INTRODUCTION Ozone is one of the major atmospheric pollutants in the Mediterranean basin where significant O3 concentrations (70-100 ppb) have been detected for several months of the year and in large areas, including remote sites from pollution sources (Lorenzini et al. 1994). Moreover, when O3 peak levels are not reached, lower O3 levels (50 ppb) are present as background anyway. Several biochemical and physiological processes, such as enzymes activity, photosynthesis and stomatal functions, have been observed to be negatively affected by O3 and the degree of inhibition was demonstrated to be dependent on duration of exposure, pollutant concentrations and species or cultivar tested (Matyssek et al. 1998). Olive represents one of the most widely cultivated trees in all the Mediterranean countries, and previous studies have shown that site-relevant O3 concentrations (100 ppb) during the olive growing season induced a heavy reduction in the transpiring stomatal surface and gas exchange parameters, with a different behaviour in the two cultivars (Frantoio and Moraiolo) tested (Minnocci et al. 1999). Physiological and biochemical changes induced by O3 in olive leaves during long term (18 months) exposure to realistic O3 levels (100 and 50 ppb, 5 h day) were investigated on the previously examined Moraiolo and Frantoio cultivars. MATERIALS AND METHODS Six-year-old olive plants (Olea europaea L., cultivars Frantoio and Moraiolo) were grown in 20 l pots and regularly provided with optimal water and nutrient supply. Six plants from each cultivar were treated for 18 months in chambers fumigated with Proc. 4 IS on Olive Growing Eds. C. Vitagliano & G.P. Martelli Acta Hort. 586, ISHS 2002 446 periodic (5 h d) levels of O3 (50 and 100 ppb) or with negligible O3 levels (<3 ppb). CO2 and water vapour exchange of newly-developed (ND) leaves were measured on five leaves for each plant, under saturating light conditions (800 μE m s), by an open gas-exchange system (CIRAS-1, PP-System) equipped with a Parkinson leaf chamber able to clamp single leaves. Photosynthetic activity at saturating light (Amax), stomatal conductance (Gw), transpiration rate (E), water use efficiency (WUE) and internal/ambient CO2 concentration ratio (Ci/Ca) were determined. Low-temperature scanning electron microscopy (LTSEM) and Energy-dispersive X-ray microanalysis were conduced on ND-leaves lamina samples obtained from two plants (for each cultivar and treatment). ND-leaves lamina were immediately plunge frozen in liquid nitrogen. After cryofixation, the frozen-hydrated (FH) samples were partially freeze-dried and sputter-coated with 3 nm of gold inside the preparation chamber of the SEM Cryo Unit SCU 020. Specimens were then transferred to the cold stage of the Philips SEM 515 and analysed at a temperature below -130oC. Elemental analysis of stomatal complex of FH leaf samples was performed in the SEM cold-stage equipped with an EDAX Compact Detecting Unit and spectra were processed using an EDAX DX4 energy-dispersive X-ray analysis system. Protein extraction was performed using ND-leaves lamina (1g) sampled from two plants (for each cultivar and treatment) frozen in liquid nitrogen and stored at -80oC. The frozen leaf samples were ground in liquid nitrogen and the powder was suspended in an extraction buffer of Tris/HCl 0.1M at pH 8.0 containing 1 mM of PMSF, 0,1% (p/v) of PVP, 0,5 % (v/v) of β-mercaptoethanol and 0,1% (p/v) of ascorbic acid. Extracts were centrifuged twice at 26.000xg for 20 min (4°C), to discard the cellular debris and the clarified supernatant used for determination of enzyme activity. Protein content was determined using a Bio-Rad protein assay kit and BSA as a standard. Ascorbate peroxidase (APX) activity was determined as in the protocol of Chen and Asada (1989). RESULTS AND DISCUSSION Ozone treatments had significant effects on physiological parameters of NDleaves. Gas exchange measurements highlighted a different behaviour between the two cultivars analysed. When compared to the controls, O3 treated Frantoio plants showed a pronounced reduction in Amax (Fig. 1) but not in Gw (Fig. 2), while significant differences were recorded in Ci/Ca ratio (data not shown). The high reduction of photosynthesis in Frantoio suggests a direct inhibition of the mesophyll photosynthetic capacity, which was not observed after 4 months of O3 exposure (Minnocci et al. 1999). In Moraiolo the decreases in Amax in O3 treated plants were linked to a reduced stomatal conductance at 50 ppb. The same trend as in Frantoio was observed at 100 ppb. The recovery of Amax at 100 ppb in Moraiolo apparently indicated that this cultivar was more O3-tolerant than Frantoio. However, visible negative effects (necrosis and leaf shedding) were only observed in Moraiolo (Minnocci et al. 1999) and this cultivar showed a higher growth reduction than Frantoio (Minnocci et al. 2002). This demonstrates that ranking of plant material for resistance/sensitivity to an environmental stress factor should be done taking into account several responses. Foliage is the major sink for O3 uptake. Volume changes in stomata guard cells regulate stomatal aperture on leaf surface, which in turn determines O3 uptake. X-ray spectra obtained from guard cells of open and closed stomata (Fig. 3) showed K and Ca peaks well defined above the background and demonstrated that stomatal widening was linked to the increase in K counting rates. Moreover, in guard cells of Moraiolo NDleaves, O3 exposure induced a significant decrease in K and Ca net counts in comparison to the control treatment suggesting the development of an adaptive mechanism to O3, which controls stomata responses at reduced ionic level (Minnocci et al. 2002). Ozone is a highly reactive molecule and easily produces toxic metabolites such as reactive oxygen species (ROS) after it enters into the leaf. So, prolonged O3 exposure may impair the normal defence mechanisms that protect cells from ROS. By using APX activity (Tab. 1) in ND-leaves as marker of the antioxidant defence mechanisms, it was