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. HabererH. Rennenberg

Institute of Forest Botany and Tree Physiology

Chair of Tree PhysiologyFreiburg, Germany

G. Bahnweg, G. BetzH. Sandermann

GSF-National Research Center for Environment and Health

Institute for Biochemical Plant Pathology Neuherberg, Germany

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 13C is discriminated by Rubisco, discrimination by PEPC is very low

• δ13C provides information about the discrimination of 13C and hence about the internal CO2 concentration in the gaseous phase of the apoplast

stable isotopes -discrimination of 13C

CO2 ext

stomata

CO2 int substomatalcavity

chloroplasts

stomata

CO2 int

stomata

CO2 int substomatalcavity

chloroplasts

CO2 ext

CO2 ext

CO2 int

CO2 ext

gs

increase of CO2int.more negative δ13C

decrease of CO2intless negative δ13C

gs

AA

-34

-32

-30

-28

-26

-24

-22

-20

-18

-16

May June July Sept Oct

******2003

sun 1xO3 shade 1xO

3

sun 2xO3 shade 2xO

3

13C in beech leaves (bulk material)

13

C v

s V

PD

B [‰

]

May June July Sept Oct

** ****2004

stable isotopes- δ13C results

stable isotopes- δ13C related to gas exchange data

original data: Markus Löw

Photosynthetic activity (A) and stomatal conductance (gs) 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 gs and decresead A the internal CO2 concentration was equal in both ozone treatments.

• Sun and shade leaves showed the same ralationship, isotope measurements indicate higher CO2 int in the shade crown.

Photosynthetical activity should be reduced to a higher extent than between 1x and 2x O3.

1xO3/2xO3 sun/shade

A 1.4 2.3

gs 1.5 2.2

CO2 int 0.9 1.1

stable isotopes- δ15N/fractionation processes

phot

o: K

urat

oriu

m „

Bau

m d

es J

ahre

s“

assimilation in rootspreference to 14N

assimilation in mycorrhizae(retention of 15N)

transport of N-compounds

source

N-loss

species+composition

δ15N at the whole tree level

-7.02±0.6

1xO3 2xO3

-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)≈ -3 (0-25cm)+

-

stable isotopes- δ15N 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 2xO3 regime makes it necessary to develop new solutions for an enhanced uptake of N.

GENE EXPRESSION

O3

O3 / ROS

Ca 2+ K+

H2O2

H2O2

Nox

oxidative burst→surrounding cells

detoxification by ascorbate

ASC DHA

DHA

GSHGSSG

ASC

change of redox state

Ca 2+

Ca 2+ K+ stom. closure

ABA (stomatal conductance)

ROSlipid oxidationPM

AP

OP

LAS

TS

YM

PLA

ST

MAPK

jasmonic acid (lesion containment)

salicylic acid ethylen

(programmed cell death)

lignin (physical barrieres)

Ca 2+

defence and injury amplification reactions

O2

SODO2-

PR1

antioxidants- apoplastic ascorbate

June July Sept0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14***

sun 1xO3 shade 1xO

3

sun 2xO3 shade 2xO

3

*

2003

µm

ol g

-1 D

W

June July Sept

***

2004

total ascorbate in the apoplastic washing fluid of beech leaves

May June July Sept Oct0

10

20

30

40

50

60

70

80

90

100

**

*

*

sun 1xO3 shade 1xO

3

sun 2xO3 shade 2xO

3

2003

%

May June July Sept Oct

******

*

*

2004

redox state of ascorbate (reduced/total) in leaf extracts of beech

antioxidants- redox state of ascorbate

2004: MJJSO

antioxidants- redox state of glutathione

May June July Sep Oct0

5

10

15

20

25

30

35

40

May June July Sep Oct

***

*

*

2003

%

******

*

sun 1xO3 shade 1xO

3

sun 2xO3 shade 2xO

3

redox state of glutathione (GSSG/total glutathione) beech leaves

2004

2004: JJSO

second messenger molecules- salicylic acid

May June July Sep Oct0

5

10

15

20

25

30

35

40

45 ****

**

2003

µg

g-1 D

W

salicylic acid (conjugated form) in beech leaves

May June July Sep Oct

sun 1xO3 shade 1xO

3

sun 2xO3 shade 2xO

3

****

**

2004

2004: JunO

second messenger molecules- ACC (ethylen precursor)

May June July Sep Oct0

20

40

60

80

100

120******

*

*

*

*

2003

nm

ol g

-1 D

W

ACC (conjugated form) in beech leaves

May June July Sep Oct

***

*

sun 1xO3 shade 1xO

3

sun 2xO3 shade 2xO

3

2004

2004: JJ

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-methionine ethylen(cell death, senescence)

O2- H2O2 Ca 2+/O2

SIPK

DAHPS,DQDSD,CS

PAL

COMT,CAD

ZEPNCED1

ACS2

Nox SOD

yECSGSHSBCAS

PR1

SA

gene expression analyses

0

200

400

600

800

1000

1200 **

*

sun 2003

%

NCED1 ACS2 PR1 COMT

sun 2004

May June July Sep0

200

400

600

800

1000

1200

**

**

shade 2003

%

May June July Sep

transcript level 2xO3 relative to 1xO

3

*

*

shade 2004

COMT-2004: JJPR1 -2004: July

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.

5 6 7 8 9 10 11 12

4

8

12

16

200

20

40

60

80

100

120

R=0.9904

R=0.8327

CO

U

salicylic acid (cojugated form) sun crown 2003

R=0.845

R=0.983

1xO3 2xO

3

AO

T4

0

correlating significantly in leaves:

δ15N salicylic acid (conj.)

COMT

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.