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.

Effects of abiotic stress on gene transcription in European beech: ozone affects ethylene biosynthesis in saplings of Fagus sylvatica L.

“…Thus, 4-year-old beech saplings can be classified as ozone-sensitive. This idea is further supported by the absence of free SA in sensitive birch clones (Vahala et al 2003), as well as in beech saplings (Betz et al 2009b). ACO1 and ACO2 were strongly induced at the onset of ozone treatment (150-190 nL L -1 ), but decreased upon continuous exposure (Fig.…”

“…The saplings were cultivated until full leaf development and were kept at a relative humidity of 70-80% and temperatures of 22-25 °C during day hours (6:00 to 20:00) and 17-20 °C during the night (20:00 to 6:00). At the beginning of June, saplings were exposed to ozone for 8 h/d (150 nl L -1 ) or pollutant-free air, and after 13 d the ozone concentration was increased to 190 nl L -1 for a further 70 d (Betz et al 2009b). For pollutant treatments ozone was added to conditioned air by mass flow controllers and injected into the cabinet by a fan (1180 m 3 h -1…”

“…Three-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.…”

Atmospheric change alters foliar quality of host trees and performance of two outbreak insect species

“…Elevated O 3 can alter the enzymatic activity of the shikimic acid pathway, enhancing the production of a number of phenolic compounds involved in stress responses (Close and McArthur 2002;Cabané et al 2004). High molecular mass polyphenolics, such as condensed tannins, have been reported to be effective antioxidants (Hagerman et al 1997), and increases in phenolic compounds under elevated O 3 may be a stress-defensive antioxidant response (Heath 2008;Betz et al 2009). Also, increased lignin accumulation in poplar under elevated O 3 is suggested to provide a structural barrier and/or antioxidant activity to reactive oxygen species, thereby increasing tolerance to O 3 (Cabané et al 2004).…”

Impacts of Elevated Atmospheric CO2 and O3 on Forests: Phytochemistry, Trophic Interactions, and Ecosystem Dynamics

“…3). Ozone also functions as a general abiotic elicitor of defense signaling pathways, particularly of phenolic compounds (Kangasjärvi et al 1994;Heath 2008;Betz et al 2009a). The two mechanisms may, of course, interact, as reductions in carbohydrate precursors for the shikimic acid pathway may constrain induced defensive responses.…”