BEECH GENETIC RESOURCES FOR SUSTAINABLE FORESTRY IN EUROPE Transcript and protein responses in leaves of ozone-treated young European beech trees at an outdoor free air model fumigation site over two growing seasons Maren Olbrich', René Kerner’, Elke Gerstner', Gerhard Welz’, J. Barbro Winkler’, Gerhard Miiller-Starck’, Dieter Ernst! ‘Institute of Biochemical Plant Pathology, *Institute of Developmental Genetics and ‘Department of Environmental Engineering, Helmholtz Zentrum Miinchen, German Research Center for Environmental Health, 85764 Neuherberg, Germany; Section of Forest Genetics, Center of Life and Food Sciences Weihenstephan, Technische Universitat Munchen, 85354 Freising, Germany (ernst@helmholtz-muenchen.de) 1, Introduction Tropospheric ozone, one of the most abundant air pollutants, is known to be detrimental to plants, and also humans. Mainly due to anthropogenic activities, the concentration still increased during the last decades. Ozone is known as a phytotoxic pollutant and is widely regarded as a risk factor for forest trees (Fowler et al., 1999; Matyssek and Sandermann, 2003; Matyssek et al., 2008). Our interest was directed towards ozone responses of the most important deciduous tree in Central Europe, European beech (Fagus sylvatica L.). Besides the Aspen FACE, the Kranzberg Ozone Fumigation Experiment and the Kuopio Field Exposure System (Karnosky et al, 2007), this free air lysimeter study investigated, at the transcript and protein level, reliable levels of ozone over two growing seasons. 2. Experimental design The lysimeters and the area around the lysimeters were filled with forest soil in 1999. In November 2002 nursery-grown beech saplings (3-year-old) were planted into lysimeters and the surrounding area. Ozone fumigation [ambient (1x) and twice ambient (2x)] started in June 2003 until the end of the vegetation period and ended in August 2006. Of every group, ten trees were chosen as biological replicates and samples were taken during the growing season from June to October. For every biological replicate one technical replicate as dye control was carried out. For transcript analyses 3 leaves of each tree were pooled and for protein analyses 4 trees of each group were taken into consideration. 3. Microarrays Home-made microarray: Scans and primary data processing were performed with GenePixPro6.0. Further data processing was carried out with R environment (www. r-project.org) and the specific open software Bioconductor, based on R (www. bioconductor.org). 1248 ozone-responsive ESTs, 11 ESTs associated with nitrogen metabolism and 20 ESTs associated with secondary metabolism were included on this array (Olbrich et al., 2005; “The original publication is available at www.wiley. com/bw/permis.asp?ref=1435-8603 & site=1”). 85NATIONAL AGRICULTURAL RESEARCH FOUNDATION Secondary metabolism, —_fetabolism, 8.6% Unclassified, 6.9% — . Energy, 10.3% Unclear classification, 1.7% et Signal transduction, 6.9% —~ _-~ Cell growthidivision, 2.6% Transcription, 6.7% Protein synthesis, Protein destination and storage, 6.9% Intracellular traffic, 1.2% - Transporters, 5.0% 4. Results - Transcriptional changes under twice ambient ozone Heat map of identical genes in 2005 and 2006. Hierarchical clustering only for data in 2006. Values are log? ratios; white, yellow = up-regulated; red = down-regulated (n = 10) (Olbrich et al., 2009; “The original publication is available at www.springerlink. com”).BegcH GENETIC RESOURCES FOR SUSTAINABLE FORESTRY IN EUROPE o 7 | —=— = 0 . oe w | 30 £0 i § = Ao r 3 - 3g 40 710 Biscxddree Bivins etree a aye $0 a Figure 1. Number of ESTs classified into functional classes. Sampling time points are indicated (Olbrich et al., 2009; “The original publication is available at www.springerlink.com”). Proteome analyses of differentially displayed proteins upon 2x ozone in 2006 : Examples of up- and down-regulated proteins in leaves of European beech upon twice ambient ozone fumigation. Control Ozone Control Ozone Figure 2. 94 proteins were significantly up- or down-regulated in leaves of European beech upon long term treatment with twice ambient ozone (p < 0.01; n= 10). 87NATIONAL AGRICULTURAL RESEARCH FOUNDATION Figure 3. Classification of all 94 significant proteins into four different protein ratios. Nine of them were strongly accumulated (ratio>4). Figure, Classification of significantly different proteins according to different ‘average ratios between control and fumigated plants © Accumulated proteins wm Decressad proteins 1,3: LS 1,5-22-4>4 Average ratio between control and fumigated plants 88BEECH GENETIC RESOURCES FOR SUSTAINABLE FORESTRY IN EUROPE 5. Conclusions Our data demonstrated: (i) a clear transcriptional ozone response of juvenile beech. (ii) Although the expression profile was not identical in both years, a common tendency of gene expression was obvious. The more pronounced changes in 2006 may be explained by a higher stomatal conductance in 2006 compared to 2005. (iii) (iv) First proteome analyses also indicated an ozone response of beech leaves. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (SFB 607) and in part by the European Community (COST Action E52; Evoltree, 6 Framework Programme). References Fowler D, Cape JN, Coyle M, Flechard G, Kuylenstierna J, Hicks K, Derwent D, Johnson C, Stevenson D (1999). The global exposure of forests to air pollutants. Water, Air, and Soil Pollution 116: 5-32. Karnosky DF, Werner H, Holopainen T, Percy K, Oksanen T, Oksanen E, Heerdt C, Fabian P, Nagy J, Heilmann W, Cox R, Nelson N, Matyssek R (2007). Free-air exposure systems to scale up ozone research to mature trees. Plant Biology 9: 181-190. Matyssek R, Sandermann H (2003). Impact of ozone on trees: an ecophysiological perspective. Progress in Botany 64: 349-404. . Matyssek R, Sandermann H, Wieser G, Booker F, Cieslik S, Musselman R, Ernst D, (2008). The challenge of making ozone risk assessment for forest trees more mechanistic. Environmental Pollution 156, 567-582. Olbrich M, Betz G, Gerstner E, Langebartels C, Sandermann H, Ernst D (2005). Transcriptome analysis of ozone-responsive genes in leaves of European beech (Fagus sylvatica L.). Plant Biology 5: 670-676. Olbrich M, Gerstner E, Welz] G, Winkler JB, Ernst D (2009). Transcript responses in leaves of ozone-treated beech saplings at an outdoor free air model fumigation site over two growing seasons. Plant and Soil: in press, 89NATIONAL AGRICULTURAL RESEARCH FOUNDATION Secondary metabolism, —_fetabolism, 8.6% Unclassified, 6.9% — . Energy, 10.3% Unclear classification, 1.7% et Signal transduction, 6.9% —~ _-~ Cell growthidivision, 2.6% Transcription, 6.7% Protein synthesis, Protein destination and storage, 6.9% Intracellular traffic, 1.2% - Transporters, 5.0% 4. Results - Transcriptional changes under twice ambient ozone Heat map of identical genes in 2005 and 2006. Hierarchical clustering only for data in 2006. Values are log? ratios; white, yellow = up-regulated; red = down-regulated (n = 10) (Olbrich et al., 2009; “The original publication is available at www.springerlink. com”).BegcH GENETIC RESOURCES FOR SUSTAINABLE FORESTRY IN EUROPE o 7 | —=— = 0 . oe w | 30 £0 i § = Ao r 3 - 3g 40 710 Biscxddree Bivins etree a aye $0 a Figure 1. Number of ESTs classified into functional classes. Sampling time points are indicated (Olbrich et al., 2009; “The original publication is available at www.springerlink.com”). Proteome analyses of differentially displayed proteins upon 2x ozone in 2006 : Examples of up- and down-regulated proteins in leaves of European beech upon twice ambient ozone fumigation. Control Ozone Control Ozone Figure 2. 94 proteins were significantly up- or down-regulated in leaves of European beech upon long term treatment with twice ambient ozone (p < 0.01; n= 10). 87BEECH GENETIC RESOURCES FOR SUSTAINABLE FORESTRY IN EUROPE 5. Conclusions Our data demonstrated: (i) a clear transcriptional ozone response of juvenile beech. (ii) Although the expression profile was not identical in both years, a common tendency of gene expression was obvious. The more pronounced changes in 2006 may be explained by a higher stomatal conductance in 2006 compared to 2005. (iii) (iv) First proteome analyses also indicated an ozone response of beech leaves. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (SFB 607) and in part by the European Community (COST Action E52; Evoltree, 6 Framework Programme). References Fowler D, Cape JN, Coyle M, Flechard G, Kuylenstierna J, Hicks K, Derwent D, Johnson C, Stevenson D (1999). The global exposure of forests to air pollutants. Water, Air, and Soil Pollution 116: 5-32. Karnosky DF, Werner H, Holopainen T, Percy K, Oksanen T, Oksanen E, Heerdt C, Fabian P, Nagy J, Heilmann W, Cox R, Nelson N, Matyssek R (2007). Free-air exposure systems to scale up ozone research to mature trees. Plant Biology 9: 181-190. Matyssek R, Sandermann H (2003). Impact of ozone on trees: an ecophysiological perspective. Progress in Botany 64: 349-404. . Matyssek R, Sandermann H, Wieser G, Booker F, Cieslik S, Musselman R, Ernst D, (2008). The challenge of making ozone risk assessment for forest trees more mechanistic. Environmental Pollution 156, 567-582. Olbrich M, Betz G, Gerstner E, Langebartels C, Sandermann H, Ernst D (2005). Transcriptome analysis of ozone-responsive genes in leaves of European beech (Fagus sylvatica L.). Plant Biology 5: 670-676. Olbrich M, Gerstner E, Welz] G, Winkler JB, Ernst D (2009). Transcript responses in leaves of ozone-treated beech saplings at an outdoor free air model fumigation site over two growing seasons. Plant and Soil: in press, 89NATIONAL AGRICULTURAL RESEARCH FOUNDATION Figure 3. Classification of all 94 significant proteins into four different protein ratios. Nine of them were strongly accumulated (ratio>4). Figure, Classification of significantly different proteins according to different ‘average ratios between control and fumigated plants © Accumulated proteins wm Decressad proteins 1,3: LS 1,5-22-4>4 Average ratio between control and fumigated plants 88BEECH GENETIC RESOURCES FOR SUSTAINABLE FORESTRY IN EUROPE 5. Conclusions Our data demonstrated: (i) a clear transcriptional ozone response of juvenile beech. (ii) Although the expression profile was not identical in both years, a common tendency of gene expression was obvious. The more pronounced changes in 2006 may be explained by a higher stomatal conductance in 2006 compared to 2005. (iii) (iv) First proteome analyses also indicated an ozone response of beech leaves. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (SFB 607) and in part by the European Community (COST Action E52; Evoltree, 6 Framework Programme). References Fowler D, Cape JN, Coyle M, Flechard G, Kuylenstierna J, Hicks K, Derwent D, Johnson C, Stevenson D (1999). The global exposure of forests to air pollutants. Water, Air, and Soil Pollution 116: 5-32. Karnosky DF, Werner H, Holopainen T, Percy K, Oksanen T, Oksanen E, Heerdt C, Fabian P, Nagy J, Heilmann W, Cox R, Nelson N, Matyssek R (2007). Free-air exposure systems to scale up ozone research to mature trees. Plant Biology 9: 181-190. Matyssek R, Sandermann H (2003). Impact of ozone on trees: an ecophysiological perspective. Progress in Botany 64: 349-404. . Matyssek R, Sandermann H, Wieser G, Booker F, Cieslik S, Musselman R, Ernst D, (2008). The challenge of making ozone risk assessment for forest trees more mechanistic. Environmental Pollution 156, 567-582. Olbrich M, Betz G, Gerstner E, Langebartels C, Sandermann H, Ernst D (2005). Transcriptome analysis of ozone-responsive genes in leaves of European beech (Fagus sylvatica L.). Plant Biology 5: 670-676. Olbrich M, Gerstner E, Welz] G, Winkler JB, Ernst D (2009). Transcript responses in leaves of ozone-treated beech saplings at an outdoor free air model fumigation site over two growing seasons. Plant and Soil: in press, 89