Foliar Responses of Olive Trees (Olea Europaea L.) under Field Exposure to Elevated CO2 Concentration

Five-year-old olive plants (cvs. Frantoio and Moraiolo) grown in large pots were exposed for seven months to ambient or high atmospheric CO2 concentration ([CO2]) in free-air CO2 enrichment facility. Exposure to elevated [CO2] enhanced net photosynthesis and decreased stomatal conductance, leading to greater water use efficiency. Stomatal density also decreased in elevated [CO2], while the ratio of intercellular to atmospheric [CO2] did not differ among leaves grown in ambient or enriched [CO2]. Cultivar differences in response to high [CO2] should be taken into account when planning future olive plantations in forecasted warmer and drier Mediterranean sites. INTRODUCTION Due to the increase of potential evapotranspiration linked to changes in global [CO2] and mean temperature at the earth surface, and to precipitation decrease at Mediterranean latitudes forecasted by General Circulation Models, Mediterranean agro-ecosystems will also have to face more severe drought conditions. Rising [CO2] has the potential to directly alter plant growth via its effects on gas exchange. Generally, assimilation rates are enhanced and stomatal conductance is reduced for plants grown under high [CO2] (Saxe et al. 1998). Stomatal adjustment in the long-term through changes in stomatal frequency may have equally significant impact on leaf conductance, thus on gas exchange and water use efficiency (Morison 1998). Morphological and physiological responses to high [CO2] may vary between species, but also between genotypes (Tognetti et al. 1999). Current predictions about the effects of rising [CO2] on woody plants are still based on data from experiments on saplings growing in chambers, rather than in the field. There has been discussion of problems associated with interpreting woody plant responses to high [CO2] when grown in manipulated environments (Saxe et al. 1998). Olive (Olea europaea L.) is an important widely cultivated evergreen tree of the Mediterranean climate, but its response to increasing [CO2] has never been studied. We examined the effects of prolonged exposure to high [CO2] on gas exchange acclimation potential of olive leaves. We compared leaves of two cvs. (Frantoio and Moraiolo) which have shown physiological and morphological differences in response to ozone fumigation (Minnocci et al. 1999). Plants were grown in conditions close to their natural environments in a free-air CO2 enrichment (FACE) facility. MATERIALS AND METHODS Two olive (Olea europaea L.) cvs., Frantoio and Moraiolo, were raised in 30-L pots for five years. On 1 March (before bud break), 1999, the plants, 10 per cv., were transported to the CO2 degassing vent of Bossoleto (Rapolano Terme, Siena, Italy), where they were constantly exposed to high [CO2]. Control plants were placed nearby. After three months, plants were randomly assigned to four FACE-system rings (see Tognetti et al. 1999): two CO2-enriched (560 μmol mol) and two at ambient [CO2] (360 μmol mol). The distance between the rings was 30 m. Each pot was manually weeded, and all plants were daily drip irrigated throughout the experiment and fertilized. During September-October gas exchange measurements (net photosynthesis, A, stomatal conductance, gs, leaf transpiration, E, and intercellular [CO2], Ci) were done on Proc. 4 IS on Olive Growing Eds. C. Vitagliano & G.P. Martelli Acta Hort. 586, ISHS 2002 450 current-year leaves of 5-year-old trees. Maximum A, gs and WUE (instantaneous water use efficiency, as A/E) were measured during sunny days with an IRGA (CIRAS-1, PP-systems), from 9 to 14 h, under saturating PAR conditions (> 1000 μmol m s). Stomatal density (SD, stomata mm) was measured on leaf portions taken from the central area of fully expanded leaves (two leaves per plant from each cv.-treatment combination) collected in mid-October. Leaf portions were immediately plunged in liquid nitrogen and then stored until observation. Frozen-hydrated leaf portions were prepared and analysed in a SEM (515, Philips) equipped with a SEM Cryo Unit (LTSEM). Slow-scan images were digitized at a resolution of 768 x 576 pixels (256 grey levels). Stomatal density of three fields per sample (500 x magnification) was determined on the recorded digital images. Stomata overlapping the margins were excluded. RESULTS Maximum A of current-year foliage responded positively to high growth [CO2] (Table 1). Increasing ambient [CO2] enhanced A by 44% in Moraiolo and by 31% in Frantoio. Maximum A was higher in Frantoio than in Moraiolo. Stomatal conductance was also significantly affected by elevated [CO2]. Foliage of plants grown in elevated [CO2] had lower gs than those in ambient [CO2], and the reduction was stronger in Frantoio. Comparison of gs at the growth [CO2] shows that increase in ambient [CO2] from 360 to 560 μmol mol led to a 24% and 37% decrease in gs for Moraiolo and Frantoio, respectively. Stomatal conductance was tendentially higher in Frantoio than in Moraiolo when plants were grown at ambient [CO2]. Generally, as a result of increased A and decreased gs, WUE increased by 73% in Moraiolo and by 94% in Frantoio when plants were grown at elevated [CO2]. Frantoio had higher WUE than Moraiolo, particularly when plants were grown in elevated [CO2]. The ratio of intercellular to atmospheric [CO2] (Ci/Ca) was unaffected by [CO2] enrichment in both cvs. (2.3% increase for Moraiolo and 1.4% decrease for Frantoio). Leaves of Moraiolo showed generally higher SD than those of Frantoio (Table 1 and Figure 1). In October, growth at elevated [CO2] had caused a consistent reduction in SD in leaves of both cvs., Frantoio (11%) and Moraiolo (9%). DISCUSSION Comparison of A at the growth [CO2] shows that high [CO2] resulted in a persistent increase in A of current-year leaves after seven months of exposure in the field. Such results are in agreement with the majority of literature reports on exposure of C3 plants to elevated [CO2] (Saxe et al. 1998). Stomatal closure in response to elevated [CO2] is a common phenomenon (Morison 1998). In our study, the decline in gs between elevated and ambient [CO2]-grown plants was mainly a result of the direct effect, short-term stomatal response to CO2. As these plants were relatively young, well-watered and supplied with nutrients this was consistent with the absence of evident photosynthetic acclimation to CO2 (Drake et al. 1997). In plants grown at full irradiance and with an adequate supply of soil water, stomatal sensitivity to CO2 may be, in fact, reduced. The close coupling of mesophyll and stomata was shown by the relative constancy of Ci/Ca which was unchanged at high [CO2] (Morison 1998). Despite elevated [CO2] reduced gs, the constancy of Ci/Ca (approximately 0.7 μmol mol) suggests that there was little or no stomatal accclimation to elevated [CO2] in these olive plants. A possible mechanism of stomatal acclimation and adjustment was through the reduction of SD in plants grown at elevated [CO2], according to the hypothesis of adaptive modifications of stomatal number (Woodward 1987). This change may have significant impact on gs and hence on gas exchange, particularly on WUE. Comparisons of olive SD responses to CO2 indicate a general decline in SD with increasing [CO2], with no evidence of consistent changes in CO2 sensitivity; as well as obeserved in other species from different climates and over the different time scale of centuries and millennia. As a result of changes in maximum A and gs, WUE increased under elevated [CO2] in both cvs., but the increase of the ratio between A and E was more remarkable in Frantoio. It