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 pivotal role in the formation of aromatic secondary compounds in plants. It finally leads to the formation of, for example, stilbenes, flavonoids, and lignins. Ozone effects on transcript levels of the shikimate pathway were studied in leaves of European beech saplings in the greenhouse (300 nl l -1 , 8 h/day, 30 days), and in leaves of adult beech trees at the Kranzberg Forest free-air ozone fumigation site (ambient and twice ambient ozone levels) between June and October 2004. Quantitative real-time RT-PCR (qRT-PCR) with RNA isolated from beech saplings showed a strong induction of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase3 (DAHPS3), 3-dehydroquinate dehydratase/shikimate dehydrogenase (DHQD/SD), 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS), and chorismate mutase (CM) starting at day seven from the onset of ozone treatment. In contrast, sun leaves of adult European beech showed only a weak elevation of shikimate pathway transcripts throughout the vegetation period studied. In addition, we examined lignin content and monomeric structure in leaves of European beech saplings from the greenhouse experiment. Leaves exhibiting highest amount of ozone-dependent lesions had the highest lignin content as determined according to Klason. Moreover, with increasing leaf damage, the syringyl (S) monomer content decreased, whereas the content of guaiacyl (G) and p-hydroxyphenyl (H) units increased. These results overall suggest that the composition of lignin is affected by ozone and is already regulated at early steps of the shikimate pathway.
A lysimeter study was performed to monitor effects of elevated ozone on juvenile trees of Fagus sylvatica L. as well as on the plant-soil system. During a fumigation period over almost three growing seasons, parameters related to plant growth, phenological development and physiology as well as soil functions were studied. The data analyses identified elevated ozone to delay leaf phenology at early and to accelerate it at late developmental stages, to reduce growth, some leaf nutrients (Ca, K) as well as some soluble phenolics (hydroxycinnamic acid derivatives, total flavonol glycosides). No or very weak ozone effects were found in mobile carbon pools of leaves (starch, sucrose), and other phenolic compounds (flavans). Altered gene expression related to stress and carbon cycling corresponded well with findings from leaf phenology and chemical composition analyses indicating earlier senescence and oxidative stress in leaves under elevated ozone. Conversely in the soil system, no effects of ozone were detected on soil enzyme activities, rates of litter degradation and lysimeter water balances. Despite the fact that the three reported years 2003-2005 were climatically very contrasting including a hot and dry as well as an extremely wet summer, and also mild as well as cold winters, the influence of ozone on a number of plant parameters is Water Air Soil Pollut: Focus (
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