© CSIRO 2004 10.1071/FP03140 1445-4408/04/020181 Functional Plant Biology , 2004, 31, 181–194 www.publish.csiro.au/journals/fpb CSIRO PUBLISHING Physiological and morphological responses of grassland species to elevated atmospheric CO 2 concentrations in FACE-systems and natural CO 2 springs Susanna Marchi A,D , Roberto Tognetti B , Francesco Primo Vaccari A , Mario Lanini A , Mitja Kaligarič C , Francesco Miglietta A and Antonio Raschi A A Istituto di Biometeorologia, Consiglio Nazionale delle Ricerche, I-50144 Firenze, Italy. B Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Università del Molise, I-86100 Campobasso, Italy. C Department of Biology, Pedagogical Faculty, University of Maribor, SI-2000 Maribor, Slovenia. D Current address: Scuola Superiore Sant’Anna di Studi Universitari e Perfezionamento, I-56100 Pisa, Italy. Corresponding author; email: marchi@sssup.it Abstract. Stomatal density, leaf conductance and water relations can be affected by an increase in the concentration of atmospheric CO 2 , and thus affect plant productivity. However, there is uncertainty about the effects of elevated CO 2 on stomatal behaviour, water relations and plant productivity, owing to the lack of long-term experiments in representative natural ecosystems. In this work, variations in stomatal density and index, leaf water relations and plant biomass of semi-natural grassland communities were analysed under field conditions by comparing plants in three different experimental set-ups (natural CO 2 springs, plastic tunnels and mini-FACE systems). Natural degassing vents continuously expose the surrounding vegetation to truly long-term elevated CO 2 and can complement short-term manipulative experiments. Elevated CO 2 concentration effects on stomata persist in the long term, though different species growing in the same environment show species-specific responses. The general decrease in stomatal conductance after exposure to elevated CO 2 was not associated with clear changes in stomatal number on leaf surfaces. The hypothesis of long-term adaptive modifications to stomatal number and distribution of plants exposed to elevated CO 2 was not supported by these experiments on grassland communities. Elastic cell wall properties were affected to some extent by elevated CO 2 . Above-ground biomass did not vary between CO 2 treatments, leaf area index did not compensate for reduced stomatal conductance, and the root system had potentially greater soil exploration capacity. Considerable between-species variation in response to elevated CO 2 may provide a mechanism for changing competitive interactions among plant species. Keywords: biomass, leaf conductance, root density, stomatal density, water relations. Introduction The steady increase in atmospheric CO 2 concentration is well documented (Keeling et al. 1995). Stomates are integrators of all environmental factors affecting plant growth (Morison 1998), and changes in stomatal behaviour may contribute to altering ecosystem water use, carbon gain and yield in natural grasslands (Jackson et al. 1994). Generally, stomatal conductance is expected to decline in herbaceous plants with a rise in CO 2 above current levels (Field et al. 1995; Ward et al. 1999). Initial observations suggested that a reduction in stomatal density with increas- ing CO 2 concentration is a general response by plants (Woodward 1987). However, a wide range of specific stomatal responses to changes in CO 2 concentration has been reported (Amthor 1995). Indeed, experiments have shown declines, no consistent effects and even increases in stomatal density as CO 2 concentration increases (Ferris and Taylor 1994; Knapp et al. 1994; Woodward and Kelly 1995). This wide variability of responses makes the identification of mechanisms difficult because of different interspecific genetic backgrounds (Woodward et al. 2002). A major factor that could alter the response of stomata to CO 2 concentration is the degree to which they acclimate functionally and morphologically. This would be ecologically important if it either tempered or enhanced the reduction in stomatal conductance to water vapour with rising CO 2 concentration (Morison 1998). Acclimation and adjustment could have significant impacts on gas exchange and water Abbreviations used: FACE, free air CO 2 enrichment; g s , stomatal conductance; PVC, pressure–volume curve.