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

Abstract

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.