Regulation des Shikimatstoffwechsels der europ&auml;ischen Buche (<em>Fagus sylvatica</em> L.) unter dem Einflu&szlig; von Ozon.

n dieser Arbeit wurde die Regulation des Shikimatstoffwechsels bei der europ&auml;ischen Buche unter dem Einflu&szlig; von Ozon untersucht. F&uuml;r jedes der sieben Enzyme des Shikimatstoffwechsels wurden putative cDNA-Sequenzen ermittelt und f&uuml;r die Genexpressionsstudien verwendet. Im Verlauf mehrerer Expositionsversuche mit 3- bis 4-j&auml;hrigen Buchen wurde eine deutliche ozonbedingte Induktion einzelner Gene des Shikimatstoffwechsels beobachtet. Durch Westernblot-Analysen wurden diese Ergebnisse f&uuml;r ausgew&auml;hlte Enzyme auch auf Proteinebene &uuml;berpr&uuml;ft. Dar&uuml;ber hinaus wurden ozoninduzierte subtraktive cDNA-Banken erstellt und die Akkumulation einzelner Sekund&auml;rmetabolite und Signalsubstanzen untersucht. Die Befunde dieser Arbeit sprechen f&uuml;r eine stre&szlig;bedingte Umsteuerung zwischen Prim&auml;r- und Sekund&auml;rstoffwechsel in der europ&auml;ischen Buche unter dem Einflu&szlig; von Ozon

Effects of abiotic stress on gene transcription in European beech: Ozone affects ethylene biosynthesis in saplings of <em>Fagus sylvatica</em> L..

The influence of ozone (150-190 nl L-1; 8h/d) on transcription levels of genes involved in the biosynthesis of the stress hormone ethylene, and its precursor 1-aminocyclopropane-1-carboxylate (ACC), was analysed in leaves of European beech saplings. Ozone-induced leaf lesions appeared 7 weeks after onset of ozone exposure. Cell lesion formation was preceded by persistent increases in ethylene emission, in the level of its malonylated precursor ACC, and in the transcript levels of specific ACC synthase 1 (ACS1), ACS2, ACC oxidase 1 (ACO1), and ACO2. Our results demonstrate that mechanisms similar to those operating in herbaceous plants may determine beech saplings responses to ozone exposure

Ozone affects shikimate pathway transcripts and monomeric lignin composition in European beech (Fagus sylvatica L.).

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

Ozone affects shikimate pathway genes and secondary metabolites in saplings of European beech (Fagus sylvatica L.) grown under greenhouse conditions.

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