Research Paper Abstract: Patterns of nitrogen (N) isotope composition (δ 15 N) and total N contents were determined in leaves, fine roots, root-associated ectomycorrhizal fungi (ECM) of adult beech trees (Fagus sylvatica), and soil material under ambient (1 × O 3 ) and double ambient (2 × O 3 ) atmospheric ozone concentrations over a period of two years. From fine root to leaf material δ 15 N decreased consecutively. Under enhanced ozone concentrations total N was reduced in fine roots and δ 15 N showed a decrease in roots and leaves. In the soil and in most types of mycorrhizae, δ 15 N and total N were not altered due to ozone fumigation. The number of vital ectomycorrhizal root tips increased and the my- corrhizal community structure changed in 2 × O 3 . Simultaneous- ly, the specific rate of inorganic N-uptake by the roots was re- duced under the double ozone regime. From these results it is assumed that 2 × O 3 changes N-nutrition of the trees at the level of N-acquisition, as indicated by enhanced mycorrhizal root tip density, altered mycorrhizal species composition, and reduced specific N-uptake rates. Key words: Free-air ozone fumigation, δ 15 N, total N, net N-up- take, ectomycorrhiza. Introduction Ozone (O 3 ) is considered one of the most important phytotoxic air pollutants. It is formed by photolysis of molecular oxygen in the stratosphere and, partially, transported downwards to the troposphere (Saxe, 2002), where further predominantly anthropogenic sources of O 3 contribute to the atmospheric concentrations of this air pollutant (Krupa and Manning, 1988). Ozone is taken up by plants mainly via the stomata and decomposed rapidly in the apoplastic space of the cell wall (Laisk et al., 1989). The reaction products of ozone (ROS, reac- tive oxygen species) may cause severe damage to cellular com- ponents leading to reduction in photosynthesis and growth as well as to premature senescence (Langebartels and Kangasjär- vi, 2004). On the other hand, ROS may also act as second mes- sengers to trigger biochemical and genetic processes (Baier et al., 2005). There is a large range of ozone sensitivity among tree species that is expressed in different response patterns. The physiological responses of plants to ozone are well estab- lished and summarized in Saxe (2002). Due to enhanced re- quirements for defence and repair processes, nitrogen and car- bon demands in plants are increased under enhanced ozone concentrations (Kytöviita et al., 2001; Andersen et al., 2001). This is evident from enhanced levels of free amino acids and altered protein profiles under enhanced ozone (Schmitt and Sandermann, 1990; Dohmen et al., 1990). In addition to ozone effects at the cellular and organ level, re- sponses to the pollutant at the whole plant level, including plant internal N and C cycling, are of special interest to esti- mate ozone toxicity in the long term. The use of 15 N/ 14 N isotope ratios is an important tool to provide insight into the N cycle of plants and its connection to the carbon cycle. The ratio be- tween the two stable nitrogen isotopes 15 N and 14 N(δ 15 N, ex- pressed as the deviation from N 2 in air) varies in the biosphere due to fractionation processes on physical, chemical, and bio- logical levels (Högberg, 1997). Plants receive N from different sources. Mainly, N compounds are taken up by plants from the soil, where specific fractionation processes influence δ 15 N. Due to several factors, δ 15 N increases with soil depth. Besides the redeposition of 15 N-depleted plant N onto the soil due to litter fall (Nadelhoffer and Fry, 1994 and 1988), 15 N-enriched compounds are accumulated as a consequence of decompo- sition, nitrate leaching, and illuviation (Piccolo et al., 1996; Nadelhoffer and Fry, 1988). Additionally, the volatilization of ammonia or N 2 O, both products of decomposition and nitrifi- cation, yield 15 N-depleted gases enriching the remaining soil N (Piccolo et al., 1996; Nadelhoffer and Fry, 1994; Turner et al., 1983). Thus, the isotopic composition of plant-available N in the soil is not uniform at different sites of N uptake and for dif- ferent N sources. Epiphytes taking up N directly from the at- mosphere show lower δ 15 N than rooted plants gaining N from the soil (Hietz et al., 1999). Therefore, N sources such as gas- eous N pollutants taken up by dry and wet deposition also af- fect δ 15 N composition of plants (Heaton et al., 1997; Pearson and Stewart, 1993; Wellburn, 1990). During assimilation of inorganic NO 3 – and especially NH 4 + by plant roots of different species, 15 N is discriminated, particular- ly when N supply is high in relation to plant demand (Högberg et al., 1997, 1999; Yoneyama et al., 1991; Kohl and Shearer, 1980; Mariotti et al., 1980). 15 N fractionation processes there- Effects of Long-Term Free-Air Ozone Fumigation on δ 15 N and Total N in Fagus sylvatica and Associated Mycorrhizal Fungi K. Haberer 1 , T. Grebenc 2 , M. Alexou 1 , A. Gessler 3 , H. Kraigher 2 , and H. Rennenberg 1 1 Institute of Forest Botany and Tree Physiology, Chair of Tree Physiology, Albert Ludwigs University, Georges-Köhler-Allee 053/054, 79110 Freiburg, Germany 2 Department of Forest Physiology and Genetics, Programme Group Forest Biology, Ecology and Technology, Slovenian Forestry Institute, Vecna pot 2, 1000 Ljubljana, Slovenia 3 Institut National de la Recherche Agronomique INRA, Centre de Recherche de Nancy, 54280 Champenoux, France Received: March 3, 2006; Accepted: October 11, 2006 Plant Biol. 9 (2007): 242 – 252 © Georg Thieme Verlag KG Stuttgart · New York DOI 10.1055/s-2006-924758 ISSN 1435-8603 242