CASIROZ-WorkshopMarch/April 2005 in Ljubljana

group Freiburg (participant 4)

stable isotopesascorbate and glutathione

total sugarsalicylic acid (SC 2)

gene expression analyses

overview of 2003 and 2004statistics with

correlation analyses

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 modelunivariate analyses

*= shade/sun #= 2xO3/1xO3

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-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

bivariate correlation analyses (2003+2004)

* < 0.05** < 0.01

bivariate correlation analyses (2003+2004)

0.01 0.02 0.03 0.04 0.05 0.06 0.07

0

50

100

150

200

250

300

350

SU

M0

/AO

T4

0 s

un

2xO

3

[µl l

-1 h

]

total ascorbate AWF sun 2xO3

[µmol g-1 dw]

SUM0 sun 2xO3 R2= 0.9641

AOT40 sun 2xO3 R2=0.9496

June July Sept0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

sun 1xO3 shade 1xO

3

sun 2xO3 shade 2xO

3

content of total ascorbate in the apoplastic washing fluid 2004

µm

ol g

-1 d

w

total ascorbate-apoplastic washing fluid(SUM0/AOT40)

May June July Sept Oct0

50

100

150

200

250

300

350µ

l l-1

h

SUM/AOT 2003(data received from Markus Löw)

SUM shade 1x SUM sun 1x SUM shade 2x SUM sun 2x AOT shade 1x AOT sun 1x AOT shade 2x AOT sun 2x

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

-28.5 -28.0 -27.5 -27.0 -26.5 -26.0 -25.5

0

50

100

150

200

250

300

350 SUM0 sun 2xO3 R2= -0.6934

AOT40 sun 2xO3 R2= -0.6626

SU

M0

/AO

T4

0 s

un

2xO

3

[µl l

-1 h

]

13C sun 2xO3

[permil]

-32

-30

-28

-26

-24

-22

-20May June July Sept Oct

sun 1xO3 shade 1xO

3

sun 2xO3 shade 2xO

3

13C in beech leaves (bulk material) 2004

1

3 C v

s V

PD

B [

‰]

2 4 6 8 10 12 14-31

-30

-29

-28

-27

-26

R2=0.9000

13C

200

4su

n+sh

ade

PS-rate 2004sun+shade

(data of Markus Löw)

redox state ascorbate leaves (COU)

20 25 30 35 40 45 50 55 60 65 70 75 80 85

0

2

4

6

8

10

12 R2= 0.9286

CO

U s

ha

de

1xO

3

[mm

ol m

-2]

redox state of ascorbate in leaves shade 1xO

3 [%]

May June July Sept Oct0

10

20

30

40

50

60

70

80

90

100

%

redox state of ascorbate in beech leaves 2003

sun 1xO3 shade 1xO

3

sun 2xO3 shade 2xO

3

May June July Sept Oct0

5

10

15

20

25

30

35m

mo

l m-2

COU 2003(data of Markus Löw)

COU shade 1x COU sun 1x COU shade 2x COU sun 2x

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

0

200

400

600

800

1000

1200

1400

1600

1800

1x 2x 1x 2x 1x 2x 1x 2x 1x 2x

May04 June04 July 04 Sep 04 Okt 04

nm

ol/g

r d

w

light

shade

GSSG as % of total glutathione 2004

0,00%

2,00%

4,00%

6,00%

8,00%

10,00%

12,00%

14,00%

1x 2x 1x 2x 1x 2x 1x 2x 1x 2x

May04 June04 July 04 Sep 04 Okt 04

light

shade

Total sugars in beech leaves 2004

0

100

200

300

400

500

600

700

800

1x 2x 1x 2x 1x 2x 1x 2x 1x 2x

May04 June04 July 04 Sep 04 Okt 04

µm

ol/gr

dw

light

shade

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

Parameter 2004 Ozone Exposition Ozone x Exposition

Sugars June - *** -

July - ** -

Sept. - - -

Glutathione June - *** -

July - *** -

Sept. - - -

GSSG% June * *** *

July - *** -

Sept. - ** -

SPSS Repeated measurements 2004

A.SugarsThere is a significant seasonal effect on total sugarsThe exposition effect changes within the yearThe exposition has a significant effect when the time effect is taken into consideration

B. GlutathioneThere is a significant seasonal effect on total glutathioneThe exposition effect changes within the yearThe exposition has a significant effect when counting time as wellOzone and the exposition have a combined effect on glutathione

C. GSSG(%)There is a significant seasonal effect on the oxidised part of glutathioneThe exposition effect changes within the yearOzone has a significant effect on GSSG when counting time as wellThe exposition has a significant effect when counting time as well

Effect of the year on the parameters

Sugars Glutathione GSSG%

Year

(2003-

2004)

6.7.9 Month

**

6.7.9

Month

***

6.7.9

Month

***

Year x Ozone

6.

Month

**

6.7

Month

***

6.

Month

*

Year x

Ozone x

Exposition

-

6.

Month

*

-

Table of correlations 2003-2004

2003 2003 2004 2004sugar sugar sugar sugar

korrel light shade light shadesum -0,0096503 0,76365832 0,35164955 0,91770635aot -0,03001875 0,54099845 0,33105 0,7339262cou -0,02657435 0,70833156 0,33614511 0,94382939

2003 2003 2004 2004glutathione glutathione glutathione glutathione

korrel light shade light shadesum -0,0534341 0,77989282 -0,48608367 0,36754087aot 0,07229075 0,79636826 -0,28246049 0,2478136cou -0,18123449 0,74794696 -0,62914257 0,41008818

2003 2003 2004 2004gssg gssg gssg gssg

korrel light shade light shadesum -0,1503623 -0,77966656 -0,64790577 -0,62073662aot -0,08281721 -0,67029092 -0,41934647 -0,37221661cou -0,0886992 -0,69780139 -0,76795072 -0,73944401

Comparison 2003-2004: Conclusions

1.Total sugar is in both years higher in the sun leavesOzone caused only a tendencial decrease in July 04

2.Glutathione is in both years higher in the sun leavesOzone caused only a tendencial increase in June 04

3.%GSSG is in both years higher in the shade leavesOzone 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

free salicylic acid in beech leaves (ambient O3) 2003

0%

100%

200%

300%

400%

sun shade sun shade sun shade

June July September

**

free salicylic acid in beech leaves (double O3) 2003

0%

100%

200%

300%

400%

500%

sun shade sun shade sun shade

June July September

**

total salicylic acid in beech leaves (shade crown) 2003

0%

50%

100%

150%

200%

250%

1xO3 2xO3 1xO3 2xO3 1xO3 2xO3

June July September

*

free SA in sun leaves

0

1

2

3

4

5

6

7

8

9

10

1 x O3 free 2 x O3 free 1 x O3 free 2 x O3 free

µg

SA

/g

dw

may sun

june sun

july sun

sep sun

2003 2004

* * * *

Level of free SA in sun leaves

is lower in both regimes 2004.

free SA in shade leaves

0

1

2

3

4

5

6

7

8

9

10

1 x O3 free 2 x O3 free 1 x O3 free 2 x O3 free

µg

SA

/g d

w

june shade

july shade

sep shade

2003 2004

* * *Level of free SA in shade leaves is

lower in both regimes in July and

September 2004.

total SA in sun leaves

0

5

10

15

20

25

1 x O3 total 2 x O3 total 1 x O3 total 2 x O3 total

µg

SA

/g d

wmay sun

june sun

july sun

sep sun

2003 2004

* 2004 tendecial lower level of total

SA in sun leaves.

total SA in shade leaves

0

10

20

30

40

50

60

1 x O3 total 2 x O3 total 1 x O3 total 2 x O3 total

µg

SA

/g

dw

june shade

july shade

sep shade

2003 2004

*

2004 tendetial lower level of

total SA in shade leaves

– no accumulation.

Correlation of salicylic acid with ozone dataSA in shade leaves 2003

R2 = 0,9614

R2 = 0,9121

R2 = 0,4335

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 8 9 10

COU [mmol m-2]

SA

[µg

/g d

w]

SA free

SA conj.

SA total

SA in sun leaves 2003

R2 = 0,7473

R2 = 0,8684

R2 = 0,0757

0

2

4

6

8

10

12

14

16

0 5 10 15 20 25 30 35

COU [mmol m-2]

SA

[n

g /g

dw

]

SA free

SA conj.

SA total

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 (O2- H2O2) (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 GshS

80,00

85,00

90,00

95,00

100,00

105,00

110,00

May 03 July 03 Sep. 03 May 04 June 04 July 04 Sep. 04

sun 2x/1x

shade 2x /1x

1x shade /sun

2x shade /sun

Relative expression of gene transcripts

relative expression of Glutathionperoxidase

80,00

85,00

90,00

95,00

100,00

105,00

110,00

115,00

120,00

May 03 July 03 Sep. 03 May 04 June 04 July 04 Sep. 04

sun 2x/1x

shade 2x /1x

1x shade /sun

2x shade /sun

relative expression of GamaECS

80

85

90

95

100

105

110

May 03 July 03 Sep. 03 May 04 June 04 July 04 Sep. 04

sun 2x/1x

shade 2x /1x

1x shade /sun

2x shade /sun

relative expression of NCED

80

85

90

95

100

105

110

115

120

May 03 July 03 Sep. 03 May 04 June 04 July 04 Sep. 04

sun 2x/1x

shade 2x /1x

1x shade /sun

2x shade /sun

Relative expression of gene transcripts

relative expression of MnSOD

80

85

90

95

100

105

110

May 04 June 04 July 04 Sep. 04

sun 2x/1x

shade 2x /1x

1x shade /sun

2x shade /sun

relative expression of FeSOD

80

85

90

95

100

105

110

115

120

May 04 June 04 July 04 Sep. 04

sun 2x/1x

shade 2x /1x

1x shade /sun

2x shade /sun

relative expression of CuZnSOD

80

85

90

95

100

105

110

May 04 June 04 July 04 Sep. 04

sun 2x/1x

shade 2x /1x

1x shade /sun

2x shade /sun

relative expression of SIPK

80

85

90

95

100

105

110

May 04 June 04 July 04 Sep. 04

sun 2x/1x

shade 2x /1x

1x shade /sun

2x shade /sun

relative expression of BCAS

80

85

90

95

100

105

110

May 04 June 04 July 04 Sep. 04

sun 2x/1x

shade 2x /1x

1x shade /sun

2x shade /sun

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