Introduction

Plant signalling substances and secondary metabolites, in particular phenylpropanoids not only determine growth and competition of plants, but also regulate their defence against pathogens, herbivores and abiotic stress. This project studies the role of the signalling compounds, ethylene and salicylic acid, and of phenylpropanoids in plants during growth, competition and fungal infection as well as the impact of ozone. In addition to the shikimate pathway which provides a link between primary and secondary metabolism, our emphasis is on defence genes of the phenylpropanoid and ethylene biosynthesis, as influenced by abiotic and biotic stress. European beech (Fagus sylvatica L.) is presently the main plant species that is studied at the levels of both young saplings in exposure chambers, at the GSF lysimeters and adult trees at the Kranzberg forest stand. Stress responses of beech were analyzed at the gene, protein and metabolite levels.


Results

Ozone affects shikimate pathway genes, secondary metabolites and monomeric lignin compostion in European beech (together with B2, B4)

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 all shikimate pathway genes (Fig. 1). In contrast, sun leaves of adult European beech showed only a weak elevation of the transcripts throughout the vegetation period (Fig. 2). Western blot analyses of DAHPS3 and DHQD/SD showed an increased protein level in agreement with the increased transcription levels (Fig. 3). Ozone-dependent leaf lesions appeared seven weeks after onset of ozone exposure (Fig. 4). Strongly elevated were levels of conjugates of salicylic and gentisic acids, 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 (Fig. 5). Leaves exhibiting highest amount of ozone-dependent lesions had the highest lignin content and a changed monomeric composition (Table 1). Our study clearly demonstrates an ozone-dependent reprogramming of the transcription of all shikimate pathway genes, which have effects on secondary metabolism (Betz et al. 2009, European Journal of Forest Research 128; Betz et al. 2009, Trees 23).


Table 1.
Lignin content and lignin derived monomers in ozone-exposed beech leaves (200 nl l^-1, 8 h/d, 46d). Ozone symptoms were classified as L1 = no visible lesions; L2 = single small lesions, many small lesions over the whole leaf; L3 = 50-70% lesions throughout the leaf area, leaves started to curl up. (Betz et al. 2009, European Journal of Forest Research 128)

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

The influence of ozone  on transcription levels of genes involved in biosynthesis of the stress hormone ethylene, and its precursor 1-aminocyclopropane 1-carboxylate, 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 (Fig. 6, 7). Our results demonstrate that mechanisms similar to those operating in herbaceous plants may determine beech saplings responses to ozone exposure. The findings further support the recently postulated idea that ethylene plays an important role in cell death in leaves of both saplings and adult trees (Betz et al. 2009, iForest – Biogeosciences and Forestry 2).

Following experiments will be analysed during phase IV

Microarray analysis of cDNAs obtained from beech leaves in exposure chambers (2x ambient ozone, in addition a single ozone pulse of 200 nL L^-1 for 7h was given; analysis of phenolics compounds (cooperation B5, experimental design see there).

Effects of Phytophthora citricola infection of roots of beech on gene expression and phenolic metabolites in leaves of beech saplings that were predisposed to increased CO₂ concentrations (ambient + 300 ppm)(cooperation A6, experimental design see there).

Gene expression analysis and determination of phenolic metabolites in beech saplings at the lysimeter experiments (2x ambient ozone, Phytophthora citricola infection of roots) (cooperation A6, B12, experimental design see there).

Figures:

Figure 1. qRT-PCR analysis of shikimate gene transcripts of total RNA isolated from ozone-treated leaves iof European beech. Four-year-old European beech saplings were fumigated with ozone (150-190 nl l-1, 8h/d, 83d); n= 4, ± SEM; *= increasing the ozone concentration from 150 nl l-1 to 190 nl l-1; ** = first ozone-induced leaf lesions. (Betz et al. 2009, Trees23)

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Figure 2. Ozone-induced expression of genes of the shikimate pathway in adult European beech at the Kranzberg Forest site. Trees were subjected to free-air ozone fumigation at twice-ambient conditions with a cut-off at 150 nl l-1 throughout the vegetation periods starting with the year 2000. qRT-PCR was carried out as described under Material and methods and the relative expression ratio (R) of four ozone-exposed versus four control trees was calculated according to the “Delta-delta” method (n = 4, bars indicate ± SE). 1=DAHPS1, 2=DAHPS2, 3=DAHPS3, 4=DHQS, 5=DHQD/SD, 6=SK, 7=EPSPS, 8=CS, 9=CM, 10=PR1. (Betz et al. 2009, European Journal of Forest Research 128)

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Figure 3. Western blot analysis. Accumulation of DAHPS3 (left) andc DHQD/SD (right) protein in leaves of four-year-old European beech saplings (150-190 nl l-1, 8 h/d, 83 d). (Betz et al. 2009, Trees 23)

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