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Statistical Analyses of Stable Isotopes, Ascorbate, and Gene Expression in Beech Leaves

This workshop presentation provides an overview of statistical analyses conducted on data from 2003 and 2004, including stable isotopes, ascorbate, glutathione, total sugars, salicylic acid, and gene expression in beech leaves. The analyses include univariate, repeated measurements, and bivariate correlation analyses.

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Statistical Analyses of Stable Isotopes, Ascorbate, and Gene Expression in Beech Leaves

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  1. CASIROZ-WorkshopMarch/April 2005 in Ljubljanagroup 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

  2. 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 SPSS-analyses

  3. general linear modelunivariate analyses *= shade/sun #= 2xO3/1xO3

  4. 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).

  5. GLM-univariate analyses and repeated measurements • Strong differences between sun and shade crown in most of the analysed parameters • Differences between the O3-regimes were observed predominantly in  15N,  18O, total C and total N values • In 2003 the differences between the exposition sites and the O3 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

  6. bivariate correlation analyses (2003+2004) * < 0.05** < 0.01

  7. bivariate correlation analyses (2003+2004)

  8. total ascorbate-apoplastic washing fluid(SUM0/AOT40)

  9. 13C in leaves (bulk material)SUM0/AOT40/PS-rate

  10. redox state ascorbate leaves (COU)

  11. 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.

  12. Total glutathione beech leaves 2004

  13. GSSG as % of total glutathione 2004

  14. Total sugars in beech leaves 2004

  15. SPSS Univariate Analysis-The exposition plays the most important role-The parameter most affected is GSSG

  16. 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

  17. Effect of the year on the parameters

  18. Table of correlations 2003-2004

  19. Comparison 2003-2004: 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

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

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

  22. 2004 tendetial lower level of total SA in shade leaves – no accumulation. 2004 tendecial lower level of total SA in sun leaves.

  23. Correlation of salicylic acid with ozone data

  24. Method • “in silico“ – design of degenerated primers using alignments of • known mRNA sequences (NCBI, Multalign...) • amplifying with RT-PCR using beech RNA/cDNA • cloning in a plasmid • sequencing of plasmid cDNA  design of specific beech-RNA primer • real-time PCR- analysis of samples 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.

  25. Superoxiddismutases (O2-  H2O2) (Cu/Zn-SOD; FeSOD, MnSOD) • -ECS,GSH-S,Glutathione peroxidase (glutathion synthesis - defense) Expression analysis : • NCED1, ZEP(ABA - stress tolerance) • SIPK (SA-inducing MAPKinase) • BCAS (Beta-cyanoalanine synthase - Detoxification from cyanide resulting from ET production)

  26. Relative expression of gene transcripts

  27. Relative expression of gene transcripts

  28. what is left to do? • analyses of the remaining samples • isotopes: 18O 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, O3 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 15N, N-assimilation) • partner 8: comparing data of antioxidants adult-young trees

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