casiroz-workshop march/april 2005 in ljubljana group freiburg (participant 4) stable isotopes...
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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