1. Kranzberg Forest Experiment: Variations in stable isotope ratios and contents of antioxidants in adult beech trees. Injury amplification reactions mediated by ethylene and salicylic acid and tree internal signalling of ozone stress at the gene level. S. Jehnes, M. Alexou, K. Haberer H. Rennenberg Institute of Forest Botany and Tree Physiology Chair of Tree Physiology Freiburg, Germany G. Bahnweg, G. Betz H. Sandermann GSF-National Research Center for Environment and Health Institute for Biochemical Plant Pathology Neuherberg, Germany

2. stable isotopes- theory elements exist in different isotopes if supply is ensured generally the lighter, more abundant one is incorporated to a higher extent in plants during assimilation 13 C is discriminated by Rubisco, discrimination by PEPC is very low δ 13 C provides information about the discrimination of 13 C and hence about the internal CO 2 concentration in the gaseous phase of the apoplast

3. stable isotopes -discrimination of 13 C gsgs increase of CO 2 int. more negative δ 13 C decrease of CO 2 int less negative δ 13 C gsgs AA AA

4. stable isotopes- δ 13 C results

5. stable isotopes- δ 13 C related to gas exchange data original data: Markus Löw Photosynthetic activity (A) and stomatal conductance (g s ) 2004 given in ratios between the crown positions and the ozone regimes: According to the gas exchange data Due to the combined effect of decreased g s and decresead A the internal CO 2 concentration was equal in both ozone treatments. Sun and shade leaves showed the same ralationship, isotope measurements indicate higher CO 2 int in the shade crown.  Photosynthetical activity should be reduced to a higher extent than between 1x and 2x O 3. 1xO 3 /2xO 3 sun/shade A1.42.3 gsgs 1.5 2.2 CO 2 int 0.91.1

6. stable isotopes- δ 15 N/fractionation processes photo: Kuratorium „Baum des Jahres“ assimilation in roots preference to 14 N assimilation in mycorrhizae (retention of 15 N) transport of N-compounds source N-loss species+ composition

7. δ 15 N at the whole tree level -7.02±0.6 1xO 3 2xO 3 -7.5±0.6 ** -4.45±1.6-5.96±1.2 ** leaves roots mycorrhizae soil -3.95±1.7 -4.24±1.3 ≈ -3 (0-25cm) + -

8. stable isotopes- δ 15 N hypothesis The enhanced ozone concentrations lead to a higher demand of N- compounds in the leaves. Available N is transported from roots to the leaves to a higher extent. The higher demand of N combined with a reduced N-uptake rate under the 2xO 3 regime makes it necessary to develop new solutions for an enhanced uptake of N.

9. GENE EXPRESSION O3O3 O 3 / ROS Ca 2+ K + H2O2H2O2 H2O2H2O2 Nox oxidative burst → surrounding cells detoxification by ascorbate ASCDHA GSHGSSG ASC change of redox state Ca 2+ Ca 2+ K + stom. closure ABA  (stomatal conductance) ROS lipid oxidation PM APOPLAST SYMPLAST MAPK jasmonic acid  (lesion containment) salicylic acid  ethylen  (programmed cell death) lignin  (physical barrieres) Ca 2+  defence and injury amplification reactions O2O2 SOD O 2- PR1

10. antioxidants- apoplastic ascorbate

11. antioxidants- redox state of ascorbate 2004:  MJJSO

12. antioxidants- redox state of glutathione 2004:  JJSO

13. second messenger molecules- salicylic acid 2004:  JunO

14. second messenger molecules- ACC (ethylen precursor) 2004:  JJ

15. gene expression analyses (real time PCR) shikimate pathway L-tyrosine L-tryptophane L-phenylalanine lignin (physical barriere) SA (cell death, signalling) tocopherol ABA pathway abscisic acid (stomatal conductance) glutathione synthesis glutathione (antioxidant/ Halliwell-Asada) +Gluperox+peroxides L-methionineethylen (cell death, senescence) O 2- H2O2H2O2 Ca 2+ /O 2 SIPK DAHPS,DQD SD,CS PAL COMT,CAD ZEP NCED1 ACS2 Nox SOD yECS GSHS BCAS PR1 SA

16. gene expression analyses COMT-2004:  JJ PR1 -2004:  July

17. AOT40 and COU Correlations between the data and AOT40/COU were found only in 2003. In 2004 the cumulative ozone effects turned into instantanous ones. correlating significantly in leaves: δ 15 N salicylic acid (conj.) COMT

18. Final conclusions Instantanous and cumulative ozone effects on the plants were observed on different tree levels. Due to a good defence and buffer capacity adult trees are able to cope with the enhanced ozone concentrations. Despite the lower intake of ozone in 2003 the combined ozone/drought situation lead to stronger ozone effects than in 2004. Therefore enhanced ozone concentrations can cause more severe damage under drought conditions. Both, COU and AOT40 correlated with the determined parameters. Regulation of stomatal aperture is a important mechanism to prevent ozone damages in plant organs and therefore a decisive factor for estimating consequences of ozone for plants.