Suppression subtractive hybridization (SSH) was performed to isolate cDNAs representing genes that are differentially expressed in leaves of Fagus sylvatica upon ozone exposure. 1248 expressed sequence tags (ESTs) were obtained from 2 subtractive libraries containing early and late ozone-responsive genes. Sequences of 1139 clones (91 %) matched the EBI/NCBI database entries. For 578 clones, no putative function could be assigned. Most abundant transcripts were O-methyltransferases, representing 7 % of all sequenced clones. ESTs were organized into 12 functional categories according to the MIPS database. Among them, 12 % (early)/15 % (late) were associated with disease and defence, 19/11 % with cell structure, 4/10 % with signal transduction, and 9/6 % with transcription. The expression pattern of selected ESTs (ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit [rbcS], WRKY-type transcription factor, ultraviolet-B-repressible protein, aquaporine, glutathione S-transferase, catalase, caffeic acid O-methyltransferase, and pathogenesis-related protein 1 [PR1]) was analysed by quantitative real-time RT-PCR (qRT-PCR) which confirmed changed transcript levels upon ozone treatment of European beech saplings. The ESTs characterized will contribute to a better understanding of forest tree genomics and also to a comparison of ozone-responsive genes in woody and herbaceous plants.

The shikimate pathway plays a central role in the formation of aromatic intermediates in the production of stilbenes, flavonoids and lignins. Ozone effects on the levels of transcripts in this pathway were studied in saplings of European beech. Complementary DNA (cDNA) clones of all genes of this pathway were isolated, and quantitative real-time RT-PCR (qRT-PCR) using RNA isolated from leaves of ozone-treated saplings showed a strong induction of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase 1 (DAHPS1), DAHPS3, 3-dehydroquinate dehydratase/ shikimate dehydrogenase (DHQD/SD), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and chorismate mutase (CM) transcripts. In contrast, DAHPS2, 3-dehydroquinate synthase (DHQS), shikimate kinase (SK), and chorismate synthase (CS) transcripts were only weakly induced. Earliest induction could be observed after 2 days of ozone treatment for DAHPS1, SK, EPSPS and CM. The coordinated regulation was evident for 3-5 weeks after the onset of ozone fumigation, and increased transcript levels were still detectable after another 7 weeks. Western blot analyses of DAHPS3 and DHQD/SD showed an increased protein level in agreement with the increased transcription levels. Ozone-dependent leaf lesions appeared 7 weeks after onset of ozone exposure. Strongly elevated were levels of conjugates of salicylic (SA) and gentisic acids (GA), either derived directly from chorismate, the key product of the shikimate pathway, or via phenylalanine, cinnamic, and benzoic acids. Concentrations of cell wall-bound phenolic compounds increased in both control and ozone-treated saplings with the latter showing slightly higher levels. Interestingly, however, this increase of cell wall-bound phenolics was accompanied by a decrease of soluble phenolics, which may indicate their deposition into the cell wall.

The still most abundant air pollutant, tropospheric ozone, leads to severe oxidative stress in plants.

Analyses of different plant stressors are often based on gene expression studies. Quantitative real-time RT-PCR (qRT-PCR) is the most sensitive method for the detection of low abundance transcripts. However, a critical point to note is the selection of housekeeping genes as an internal control. Many so-called 'housekeeping genes' are often affected by different stress factors and may not be suitable for use as an internal reference. We tested six housekeeping genes of European beech by qRT-PCR using the Sybr Green PCR kit. Specific primers were designed for 18S rRNA, actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH1, GAPDH2), α-tubulin, and ubiquitin-like protein. Beech saplings were treated with increased concentrations of either ozone or CO 2 . In parallel, the expression of these genes was analyzed upon pathogen infection with Phytophthora citricola. To test the applicability of these genes as internal controls under realistic outdoor conditions, sun and shade leaves of 60-year-old trees were used for comparison. The regulation of all genes was tested using a linear mixed-effect model of the R-system. Results from independent experiments showed that the only gene not affected by any treatment was actin. The expression of the other housekeeping genes varied more or less with the degree of stress applied. These results highlight the importance of undergoing an individual selection of internal control genes for different experimental conditions.

© iForest -Biogeosciences and Forestry IntroductionEuropean beech is the most abundant broadleaf tree in Germany (Schütt et al. 1992). It is also of major importance for the European forest industry and was therefore chosen as an experimental species for our studies, to obtain deeper molecular insights into the potentially detrimental effects of ozone on European broadleaf forest ecosystems. Plants have long been known to exhibit responses to ozone exposure resembling the hypersensitive response following pathogen attack. Ozone has been qualified as an abiotic elicitor of plant defence reactions (Sandermann et al. 1998). These responses include phytoalexin production and synthesis of pathogenesis-related (PR) proteins, accompanied by the formation of various aromatic metabolites like lignins and flavonoids .Another well-known plant response to ozone exposure is the stimulation of ethylene biosynthesis (Tuomainen et al. 1997, Rao et al. 2002, Langebartels & Kangasjärvi 2004. Activation of ethylene biosynthesis is one of the fastest ozone-dependent biochemical responses so far observed in herbaceous plants, occurring within 5 hours, or even more rapidly (Schlagnhaufer et al. 1997, Nakajima et al. 2001, Moeder et al. 2002. This response was similarly rapid in birch (Vahala et al. 2003) and in an ozone-sensitive poplar clone (Diara et al. 2005). ACC synthase (ACS) and ACC oxidase (ACO), the two enzymes of ethylene biosynthesis, are encoded by gene families in herbaceous plants; several ACS and ACO isoforms have been reported to be induced by ozone (Nakajima et al. 2001, Moeder et al. 2002. In ozone-sensitive as well as ozone-tolerant birch clones, ozoneinduced accumulation of ACS and ACO transcripts was found (Vahala et al. 2003). Similarly, ozone fumigation of beech saplings (180-200 nl L -1 ) showed enhanced transcript levels of ACS2 and ACO1 (Nunn et al. 2005a). We therefore extended our study by reporting the cloning of ACS2 and ACO2, and analysing over a period of 3 month an ozone-induced expression of the ethylene biosynthesis genes (ACS1, ACS2, ACO1, ACO2), the accumulation of the ethylene precursor free ACC and conjugated ACC, and the emission of the end product of this pathway, ethylene, using beech saplings. Materials and methods Plant material and growth conditionsThree-year-old European beech saplings (Fagus sylvatica L., provenance 81024; Schlegel Baumschulen, Riedlingen, Germany) were planted in 14-L pots filled with natural forest soil (site Höglwald, Bavaria, Germany -Betz et al. 2009b). Saplings were maintained during winter under a wooden pergola. Before bud burst, the plants were treated with Promanal ® (Neudorff, Emmerthal, Germany), to prevent insect infestation. In spring the 4-year-old saplings were transferred to climate-controlled cabinets (39 m 2 ) of a greenhouse covered with UV-permeable glass sheets (http://www.helmholtz-muen chen.de/eus/index.php -Olbrich et al. , Betz et al. 2009a. Each cabinet contained 8 tables (2 m 2 per table) and 12 saplings were placed on a single table. The sap...