Plant signalling substances and secondary metabolites, in particular phenylpropanoids, not only regulate growth and competition of plants, but also determine their defence towards pathogens, herbivores and abiotic stress. Project A1 studies the role of the signalling substance, ethylene, and of phenylpropanoids under growth, competition and fungal infection as well as under the influence of ozone. In addition to the shikimate pathway, which provides a link between primary and secondary metabolism, the emphasis is on defence genes for 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 plant cell suspension cultures, young saplings, and adult trees at the Kranzberg Forest stand (Fig. 1). In addition, the project performs metabolite profiling tasks for other projects of the SFB 607.

Results

Role of ethylene in the regulation of cell death and leaf loss in ozone-exposed European beech (together with B2, B4, B5)

To test the involvement of ethylene in mediating ozone-induced cell death and leaf loss in European beech, tree seedlings were exposed to proportionally increased or decreased field ozone levels for up to 6 months (Fig. 2). Ozone treatment caused cell death and accelerated leaf loss at higher than ambient levels, but had only minor effects at ambient and no effects at subambient ozone levels.  Inhibition of ethylene biosynthesis led to reduced lesion formation whereas application of ACC accelerated ozone-induced cell death as well as leaf loss (Fig. 3-5). Similar results were obtained when adult beech trees were exposed to 2 × ozone by a whole tree free-air canopy exposure system (Kranzberg Forest). The results suggest a role of ethylene in amplifying ozone effects under field conditions in this major European broad-leaved tree species.  Taken together, ozone, once taken through stomata, initiates an amplifying programme, the oxidative cell death cycle, with ethylene as a key player which leads to cell death and accelerated leaf fall (Fig. 6). This mechanism will be incorporated into new, ecologically meaningful critical levels for ozone and quantitative risk assessment of the impact of ozone on trees (Nunn et al., 2002; Nunn et al., 2005a, b)

Transcriptome analysis of ozone-responsive genes in European beech

Suppression subtractive hybridization (SSH) was performed to isolate cDNAs representing genes that are differentially expressed in leaves of European beech upon ozone exposure. 1248 expressed sequence tags (ESTs) were obtained from 2 subtractive libraries containing early and late, respectively, ozone-responsive genes. Sequences of 1139 clones (91 %) matched to 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 in 12 functional categories according to the MIPS database (Table 1). Additionally a subtracted Phytophthora-induced cDNA library from roots is available (together with A9). The clones will be combined on one DNA-chip (Kiefer et al., 2000; Langebartels et al., 2001; Langebartels et al., 2002a; Ernst and Aarts, 2004; Matyssek et al., 2005; Olbrich et al., 2005)

Table 1.
Functional grouping of ESTs according to the MIPS classification (http://www.mips.gsf.de) obtained from the 2 SSH libraries of ozone-treated European beech leaves. (Olbrich et al. 2005, Plant Biology 7)

back to text

Light- and ozone-related effects on secondary metabolite levels (together with B2, B4)

More than 15 soluble and 10 cell wall-bound phenolics were detected in beech leaves at Kranzberg Forest. Distinct differences were found between sun and shade leaves. A typical example of such a HPLC diagram is shown in Fig. 7. (August 2002) Peak heights may differ slightly among individual trees and at different sampling dates. Compounds protective against UV-B radiation (flavonol and acylated flavonol derivatives, F and A) were almost completely absent in shade leaves. In contrast, no significant influence of ozone was detectable in both sun and shade leaves (Fig. 8; years 2001, 2003 and 2004). Levels of soluble phenolics were highest in spring but gradually decreased throughout the growing season. Cell wall-bound kaempferol derivatives were also more prominent in sun than in shade leaves, while differences between the ozone regimes were absent (Fig. 9; year 2000). On the other hand, kaempferol glycoside levels concomitantly increased suggesting deposition of the soluble fraction into cell walls during leaf ontogeny. This pattern was confirmed by data obtained from the other years (not shown). Only one newly formed compound, 3,3’,4,4’-tetramethoxy-1,1’-biphenyl, was observed, but only occurred up to a distance of 1-2 mm from typical necrotic areas as well as in the necroses themselves (Fig.10) (Bahnweg et al., 2005).

Beech leaf colonization by the endophyte Apiognomonia errabunda dramatically depends on light exposure and climatic conditions (together with A9, B2, B4)

Ozone and light effects on endophytic colonization by Apiognomonia errabunda of adult European beech trees and their putative mediation by internal defence compounds were studied at the Kranzberg Forest free-air ozone fumigation site. A. errabunda colonization was quantified by “Real-time PCR” (QPCR). A. errabunda-specific primers allowed detection without interference by DNA from European beech and several species of common plant pathogenic fungi like Mycosphaerella, Alternaria, Botrytis, and Fusarium. Colonization levels of sun and shade leaves of European beech trees exposed either to ambient or twice ambient ozone regimes were determined. Colonization was significantly higher in shade compared to sun leaves (Fig. 11,12). Ozone exhibited a small but significant inhibitory effect on fungal colonization only in young sun and shade leaves. The hot and dry summer of 2003 reduced fungal colonization dramatically, more pronounced than did ozone treatment or sun exposure. Appearance of Apiognomonia-related necroses strongly correlated with the occurrence of the stress metabolite, 3,3’,4,4’-tetramethoxybiphenyl (Fig. 10) Bahnweg et al., 2005).

Conclusions

Pathogen, light and ozone were shown to induce defence-related genes and secondary metabolites. (+I, +VIII)

Ethylene, reactive oxygen species and salicylic acid cooperate in amplifying HR-type cell death in crop plants. These compounds have for the first time been analyzed in a tree: the dose of ethylene emission correlated with necrotic leaf area. (+VIII)

New UV-B-screening pigments (kaempferol glucosides), similar to those observed in pine and spruce were identified in European beech. Foliar amounts were much higher in leaves of the sun crown in comparison to shade crowb, while 2x ambient ozone did not lead to major effects. (+VIII)

In shade leaves of European beech high amounts of Apiognomonia DNA were detected, whereas sun leaves showed drastically reduced DNA levels of this endophyte. Ozone treatment further reduced Apiognomonia DNA amounts of sun leaves in June, but not in summer and fall. (+VI)

Cooperation with SFB 607 projects A6, A7, A8, A9, A10, B2, B4, B5, B7, B12, C2, C7


Figures

Figure 1.  Schematic overview of growth and defence strategies of plants (Sandermann and Matyssek, 2004).
back to text

Figure 2. Daily time course of ozone levels (experiment 1). Ozone and climate data were recorded at the study site of Schönenbuch, Switzerland in 1990 and were used for all indoor chamber experiments. Three representative days of August 1990 are given as an example. Symbols: () 0.15 × ambient ozone; (○) ambient ozone; line: field values; (▲) 1.5 × ambient ozone; () 2 × ambient ozone. (Nunn et al. 2005, Plant Cell and Environment 28)
back to text

Figure 3. (a)–(d) Development of ozone-dependent cell death in leaves of beech seedlings exposed to 2 × ambient ozone (experiment 2). Pictures were taken 16 (a), 27 (b), 30 (c) and 33 d (d) after the beginning of the treatment. (e), (f), Necrotic stipples found in the sun-crown of adult beech trees at Kranzberg Forest in June under 2 × ambient ozone. The effect of the ethylene precursor ACC on cell death of leaves in adult beech trees exposed to 2 × ambient ozone in the field (g), Control leaf without ACC under 2 × ambient ozone (h). (Nunn et al. 2005, Plant Cell and Environment 28)
back to text

Figure 4. Correlation between cumulative ethylene emission and cell death in 4-year-old beech seedlings exposed to 1 × and 2 × ambient ozone (experiment 2). The ethylene dose was calculated as the product between the hourly ethylene emission and the time difference between the measurements. Symbols: (○) 1 × ozone; (•) 2 × ozone, r² = 0.92. (Nunn et al. 2005, Plant Cell and Environment 28)
back to text

Figure 5. Effect of ACC treatment on cell death and leaf loss of twigs of ozone-exposed (2 ×) adult beech trees at Kranzberg Forest. Results are shown from three independent control branches on the left and three ACC-treated branches on the right. The triangles indicate the dates of treatment. (Nunn et al. 2005, Plant Cell and Environment 28)
back to text



.

Figure 6. Amplification of ozone and elicitor effects in the 'oxidative cell death cycle' involving ethylene, reactive oxygen species (ROS) and NO, lipid peroxide products (LOOH) and salicylic acid (SA) (Langebartels et al., 2002 Plant Physiology and Biochemistry 40).
back to text

Figure 7. Representative HPLC diagrams of methanol extracts from shade and sun leaves of beech trees collected at Kranzberg Forest in August 2002. Slight differences in peak heights may occur with individual trees and at different sampling times.

Figure  8. Annual courses (three years) of total phenolics in sun and shade leaves of European beech at Kranzberg Forest under the influence of ambient and 2x ambient ozone.
(Bahnweg et al. 2005, Plant Biology 7)
back to text

Figure 9. Annual course (year 2000) of cell wall-bound kaempferol glycosides in sun and shade leaves of European beech at Kranzberg Forest under the influence of ambient and 2x ambient ozone. (Bahnweg et al. 2005, Plant Biology 7)
back to text

Figure 10. Shade leaf of European beech (August 2002) heavily infested by Apiognomonia errabunda and showing typical necrotic lesions, irregularly shaped and uneven in size and often delimited by leaf veins. Parallel analyses of green leaf parts, borders of necroses (approx. 2 mm) and necroses indicated that 3,3’,4,4’-tetramethoxybiphenyl occurred exclusively in heavily infected leaf parts. (Bahnweg et al. 2005, Plant Biology 7)
back to text

Figure 11. A. errabunda DNA levels (pg mg^-1 leaf fresh weight) in shade leaves of European beech at Kranzberg Forest site in 2001 through 2003. (Bahnweg et al. 2005, Plant Biology 7)
back to text

Figure 12. A. errabunda DNA levels (pg mg^-1 leaf fresh weight) in sun leaves of European beech at Kranzberg Forest site in 2001 through 2003. (Bahnweg et al. 2005, Plant Biology 7)
back to text