CASIROZ-Workshop March/April 2005 in Ljubljana group Freiburg (participant 4) stable isotopes ascorbate and glutathione total sugar salicylic acid (SC 2) gene expression analyses overview of 2003 and 2004 statistics with correlation analyses

assessments δ 13C: assessment of Rubisco activity and relative activity changes between Rubisco and PEPC Δ18O: stomatal conductance, relative humidity of the air 14N/15N: N-uptake, N-assimilation, fractionation processes during transport, interaction with mycorrhiza transport velocity C/N changes of the whole plant : seasonal/annual time scale ascorbate/glutathione antioxidative status of the plant, changes in total amounts and redox status: diurnal and seasonal patterns total sugar in different plant material cycling of carbohydrates (together with analyses of starch, leaf transpiration, xylem mass flow) salicylic acid mediating injury amplification reactions gene expression analyses tree-internal signaling of ozone stress and the regulation of defence reactions at the gene level

SPSS-analyses Statistical analyses of the data 2003 and 2004 (stable isotopes, ascorbate) univariate analyses repeated measurements bivariate correlation analyses of the data 2003 and 2004 with ozone SUM0 AOT40 COU stomatal conductance

general linear model univariate analyses *= shade/sun #= 2xO 3 /1xO 3

general linear model-repeated measurements A= change of parameter in the course of the year B= the influence of the O3-regime on the parameter is changing in the course of the year (time/O3-regime interaction) C= the influence of the exposition on the parameter is changing in the course of the year (time/exposition interaction) D= the influence of the O3-regime on the parameter is different under different exposition sites in the course of the year and/or the influence of the exposition on the parameter is different under different O3-regime in the course of the year (time/exposition/O3-regime interaction) E= significant differences between 1x O3 and 2x O3 in the course of the year (including the time effect) F= significant differences between sun and shade crown in the course of the year (including the time effect).

GLM- comparison between 2003 and 2004

GLM-univariate analyses and repeated measurements Strong differences between sun and shade crown in most of the analysed parameters Differences between the O 3 -regimes were observed predominantly in  15 N,  18 O, total C and total N values In 2003 the differences between the exposition sites and the O 3 regimes were more obvious than in 2004  drought situation in 2003 caused intensive stress situation Calculations of the „repeated measurements“ confirm the results of the univariate analysis, if significances were obvious for more than one months

bivariate correlation analyses ( ) * < 0.05 ** < 0.01

bivariate correlation analyses ( )

total ascorbate-apoplastic washing fluid (SUM0/AOT40)

 13 C in leaves (bulk material) SUM0/AOT40/PS-rate

C/N ratio leaves (SUM0/AOT40/COU)

redox state ascorbate leaves (COU)

bivariate correlation analyses ( ) cellulose

bivariate correlation analyses ( ) fine roots

correlation analyses the determined parameters showed different correlation coefficients when fitted to SUM0, AOT 40 or COU generally SUM0 seems to show a better correlation than AOT40 stomatal conductance not summed up did show only few correlations to the determined parameters.

Total glutathione beech leaves 2004

GSSG as % of total glutathione 2004

Total sugars in beech leaves 2004

SPSS Univariate Analysis -The exposition plays the most important role -The parameter most affected is GSSG Parameter2004OzoneExpositionOzone x Exposition SugarsJune-***- July-**- Sept.--- GlutathioneJune-***- July-***- Sept.--- GSSG%June***** July-***- Sept.-**-

SPSS Repeated measurements 2004 A.Sugars There is a significant seasonal effect on total sugars The exposition effect changes within the year The exposition has a significant effect when the time effect is taken into consideration B. Glutathione There is a significant seasonal effect on total glutathione The exposition effect changes within the year The exposition has a significant effect when counting time as well Ozone and the exposition have a combined effect on glutathione C. GSSG(%) There is a significant seasonal effect on the oxidised part of glutathione The exposition effect changes within the year Ozone has a significant effect on GSSG when counting time as well The exposition has a significant effect when counting time as well

Effect of the year on the parameters SugarsGlutathioneGSSG% Year ( ) Month ** Month *** Month *** Year x Ozone 6. Month ** 6.7 Month *** 6. Month * Year x Ozone x Exposition - 6. Month * -

Table of correlations

Comparison : Conclusions 1.Total sugar is in both years higher in the sun leaves Ozone caused only a tendencial decrease in July 04 2.Glutathione is in both years higher in the sun leaves Ozone caused only a tendencial increase in June 04 3.%GSSG is in both years higher in the shade leaves Ozone causes a tendencial increase in every month, especially in the shade leaves

Analysis of the salicylic acid level SAR (systemic acquired resistance) induced Phytoalexins (anti-microbial compounds) lignification increased Chitinases, ß-1,3 Glucanase (PR-proteins) part of ozon induced cell death program (oxidative burst) Free SA – active form – second messenger Conjugated SA – inactive form – pool/overflow

Level of free SA in sun leaves is lower in both regimes Level of free SA in shade leaves is lower in both regimes in July and September 2004.

2004 tendecial lower level of total SA in sun leaves tendetial lower level of total SA in shade leaves – no accumulation.

Correlation of salicylic acid with ozone data

Molecular Approach transcriptional expression level of genes connected to tree-internal stress signaling and regulation of defense reactions. quantitative real-time PCR used as method. Method “in silico“ – design of degenerated primers using alignments of known mRNA sequences (NCBI, Multalign...) amplifying with RT-PCR using beech RNA/cDNA sequencing of plasmid cDNA  design of specific beech-RNA primer real-time PCR- analysis of samples cloning in a plasmid

Expression analysis :  NCED1, ZEP (ABA - stress tolerance)  Superoxiddismutases (O 2 -  H 2 O 2 ) (Cu/Zn-SOD; FeSOD, MnSOD)   -ECS, GSH-S, Glutathione peroxidase (glutathion synthesis - defense)  SIPK (SA-inducing MAPKinase)  BCAS (Beta-cyanoalanine synthase - Detoxification from cyanide resulting from ET production )

Relative expression of gene transcripts

what is left to do? analyses of the remaining samples isotopes:  18 O in liquid samples of 2004; core samples (harvested spring 2005) salicylic acid diurnal samples 2004, container plant samples interpretation of results of sugar analyses and tracer techniques for sugar translocation at the whole plant level additional co-work with other participating groups partner 1: combining results of stable isotopes with transpiration, PS-rate, stomatal conductance, WUE partner 2: combining results of stable isotopes with activities of carbon fixing enzymes subcontractor 1: information about micrometeorological data (rH, O 3 data) subcontractor 2: comparing data of ACC and gene expression analyses partner 5: information about cytokinins (regulation N-uptake), comparing results of ABA- analyses with gene expression analyses partner 6: interaction roots of trees-mycorrhiza (regarding  15 N, N-assimilation) partner 8: comparing data of antioxidants adult-young trees