Transcript 06_Kraigher_Hanke

Mycorrhiza, cytokinins and abscisic acid
in the response of beech to 2xO3
Tine Grebenc, Joanna Winwood, Adrienne Pate,
Andrew O’Brien, David Hanke & Hojka Kraigher
Institute of Forestry, Slovenija, and University of Cambridge, UK
ECTOMYCORRHIZAE affect
nutrient cycling & biodiversity in forest
ecosystems Rainfall, throughfall...
Litter
Leaves, buds, flowers, fruits,
shoots, branches
Gaseous
losses &
gains
Inputs
Retranslocation in time & space
CWD
Immobilization
Common mycelial
Mineralisation
Uptake &
retranslocation of
networks
water & nutr.
Weathering of minerals
From: KRAIGHER 2003, as modif. after DIGHTON & BODDY 1998
Slovenian Forestry Institute
The mycelium of
mycorrhizal fungi:
 affects the field performance of
forest trees through:
•capture & uptake of nutrients
•protection against pathogens &
toxic elements
•extending feeder root longevity
•spatial & temporal linkages
between sinks & sources of
nutrients.
Photos by H.Kraigher
Slovenian Forestry Institute
 depends on:
•functional compatibility of
species & strain of the fungus &
the plant
•therefore identification of the
fungal partner is important.
Mycorrhiza is considered a microbial key
species in soils, which are defined as
organisms that possess important functions
in the soil ecosystem or are of human health
concern (Nielsen & Winding: Microorganisms
as indicators of soil health, 2002)
Identification, quantification,
qualification of ECM:
0.6
• Standardized soil core for sampling (270 ml,  3,5 cm,
0-18 cm deep, 2 - 5 per sampling date, 5 - 30 per plot),
cleaning, sorting of morphotypes & documenting.
• Counting (root tips above 1 mm length) and image
analyses of vital types, old un-identifiable types and
Photos by H.Kraigher
non-mycorrhizal roots.
• Anatomical and molecular identification or
characterisation of the sorted morphotypes, comparisons
to the reference material, PCR-ITS-RFLP library or
GenBank.
• Presentation (lists, tables, graphs), biodiversity
indices (Atlas, Bartha 1981), statistics (CANOCO, …) .
• Search for physiological / ecological roles
of the identified sp.
• Modelling with environmental parameters.
Mg-depo
Ca-depo
NT9.J LACSER.J
BYSATR NT13.J TOMsp2
TRIsp3 RUSOCH.J
NT12.J NT11.J
LACCAME
RUSOCH
LACPAL
CENGEO.J
NT5.J
NT6.J
CENGEO MomVl10.
NT15.J
LACACR
NT14.J
LACSUB
TOMTER
RUSMAI
NT7.J
Padav
RUSCYA
LACCsp1
FAGFUS
TRISCI
LACsp1.J
ENTNID LACSAL.J
NH3-depo NT2.J TOMfam1.
RUSsp3. NT3.J NT1.J
Right: CANOCO by T.Grebenc
Slovenian Forestry Institute
-0.8
RUSILL
TRIsp1
-1.0
1.0
Identification of types of ectomycorrhizae:
Anatomical characteristics (AGERER 1987-2004),
Molecular methods (PCR-ITS-RFLP & sequencing) (GARDES &
BRUNS 1993, as descr. in KRAIGHER & al. 1995; sequencing.: as
descr. in MARTĺN 2000)
Photos by H.Kraigher
©T.Bruns http:/
Slovenian Forestry Institute
Right: Extract from the PCR-ITS-RFLP database (containing ca 900 isolates of
fungi & types of ECM) of the Slovenian Forestry Institute (Grebenc 2005)
Results &
discussion
Response of frequently occurring species to 2xO3 fumigation
Species with > 5% of all ECM root
tips
Response to two-fold ozone
fumigation of tree canopies
Cenococcum geophilum
↑
Russula cyanoxantha
↓
Xerocomus chrysenteron
↓
Russula fellea
↑
Russula illota
↑
Russula ochroleuca
↓
Russula sp. 2
↑
The chronic ozone effect seems to change the succession stage of
ECM in the stand causing a shift of some ectomycorrhizal species
either to become more dominant (C. geophilum, R. fellea) or to
decrease in abundance and finally disappear from the treated plots (R.
cyanoxantha, Xerocomus chrysenteron & R. ochroleuca).
Slovenian Forestry Institute
Results &
discussion Cenococcum geophilum was the most common and
abundant type of ectomycorrhize
significant response to ozone treatment.
with
highly
Cenococcum geophilum (2003)
C. geophilum, magnif. 45x
700
600
Sampling time
500
Absolute number
of vital ECM
tips
400
300
200
100
May
June
July
0
1x 1x
1x 1x
O3 O3
O3 O3 2x 2x 2x
2x
O3 O3
O3 O3 2x
O3
September
Photo:P. Zeleznik
October
Treatment
Cenococcum geophilum (2004)
1000
800
600
Absolute number
of vital ECM tips
Sampling time
400
200
October
0
1x 1x
1x 1x
O3 O3
2x 2x
O3 O3
2x 2x
O3 O3
O3 O3 2x
O3
Treatment
Slovenian Forestry Institute
20 m
July
May
C. geophilum, outer mantle layer
photo T.Grebenc
Results &
discussion
Parameters describing
ectomycorrhizae and short root
dynamics
Shannon-Weaver index
Response to the fumigation
of beech crowns with twofold ozone
↑ (n.s.)
Species richness
↑
Sum of vital ectomycorrhizal root tips
↑
Old and non-mycorrhizal root tips
↑
•Shannon-Weaver index showed a non-significantly
higher diversity in 2xO3 compared to 1xO3
•Species richness showed a trend of increase in 2xO3
compared to 1xO3
•Total number of vital ectomycorrhizal and old&nonmycorrhizal root tips were significantly higher (indicating
a higher fine root turnover) for trees under 2xO3
Slovenian Forestry Institute
Conclusions
•Chronic exposure of trees to increased ozone
concentration caused changes in below-ground processes.
•The community structure of ectomycorrhizal fungi was
changed, i.e. some species became more frequent,
•although only trends in species richness indices were
observed.
•The increase in fine root turnover in increased ozone
exposure was significant.
Slovenian Forestry Institute
Root levels
2004 Roots
2003 Roots
150000
200000
1xO3 iso
2xO3 iso
2xO3 aro
1xO3 aro
150000
100000
CK content (fmol/gFM)
CK content (fmol/gFM)
250000
100000
1xO3 iso
2xO3 iso
1xO3 aro
2xO3 aro
50000
50000
0
0
5
6
7
8
Time (months)
9
10
11
5
6
7
8
Time (months)
9
10
11
Xylem Levels
iP-types 2003
Z-types 2003
250
200
1XO3
2XO3
1XO3
2XO3
150
100
Sun
Sun
Shade
Shade
50
CK content (fmoles/50ul)
CK content (fmoles/50ul)
250
200
1XO3
2XO3
1XO3
2XO3
150
100
50
0
0
6
7
8
9
10
6
7
Time (months)
8
9
10
Time (months)
iP-types 2004
Z-types 2004
250
800
700
200
600
1XO3 Sun
2XO3 Sun
1XO3 Shade
2XO3 Shade
500
400
300
200
100
CK content (fmoles/50ul)
CK content (fmoles/50ul)
Sun
Sun
Shade
Shade
150
1XO3 Sun
2XO3 Sun
1XO3 Shade
2XO3 Shade
100
50
0
0
6
7
8
Time (months)
9
10
6
7
8
-50
Time (months)
9
10
Isoprenoid Levels: 2003
2003 shade leaves
100000
100000
90000
90000
80000
80000
CK content (fmol/gFM)
CK content (fmol/gFM)
2003 sun leaves
70000
60000
50000
40000
30000
70000
60000
50000
40000
30000
20000
20000
10000
10000
0
0
20
25
30
35
40
45
20
25
30
35
Time (weeks)
Time (weeks)
1xO3
1xO3
2xO3
2xO3
40
45
Aromatic Levels: 2004
2004 shade leaves
80000
160000
70000
140000
60000
120000
CK content (fmol/gFM)
CK content (fmol/gFM)
2004 sun leaves
50000
40000
30000
20000
10000
100000
80000
60000
40000
20000
0
18
23
28
33
Time (weeks)
1xO3
2xO3
38
43
0
18
23
28
33
Time (weeks)
1xO3
2xO3
38
43
Z O-Glucoside Levels: 2004
2004 shade leaves
1800000
1000000
1600000
900000
1400000
800000
CK content (fmol/gFM)
CK content (fmol/gFM)
2004 sun leaves
1200000
1000000
800000
600000
400000
700000
600000
500000
400000
300000
200000
200000
100000
0
0
18
23
28
33
Time (weeks)
1xO3
2xO3
38
43
18
23
28
33
Time (weeks)
1xO3
2xO3
38
43
Increased cytokinin content of roots and leaf xylem
in 2xO3 accords with prediction from root growth and
mycorrhiza
Decreased leaf content does not, and suggests a higher
rate of destruction of cytokinin in leaves in 2xO3
Mean ABA content of beech leaves (nmol g-1 FW)
Changing ABA content of leaves: 2003 and 2004
70
60
50
40
30
20
10
0
June
July
September
October
70
60
50
40
30
20
10
0
May
Sun 1 x O3
June
Sun 2 x O3
July
September
Shade 1 x O3
October
Shade 2 x O3
Changes in phloem ABA concentration
12.00
ABA concentration (nmol mL-1)
10.00
2003
8.00
6.00
4.00
2.00
0.00
June
12.00
10.00
July
September
2004
8.00
6.00
4.00
2.00
0.00
June
1 x O3 Shade
July
1 x O3 Sun
September
2 x O3 Shade
2 x O3 Sun
ABA in sun and shade leaves plotted with
AOT40 and COU in 2004
Mean ABA content of leaves(nmol/g FW)
50
45
40
35
30
25
20
15
10
5
0
0
10
20
30
40
50
60
70
AOT40
30
25
20
15
10
5
0
0
5
10
15
20
25
Ozone flux
June
September
October
July
May
30
Mean ABA content of leaves(nmol/g FW)
Diurnal changes in leaf ABA content in July 2004
50
45
40
35
30
25
20
15
10
5
0
-1
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Hours from 5 AM on the first day
Sun 1 x O3
Sun 2 x O3
3
Ratio 2xO3/1xO3
2
2
1
5:00 AM
1
0
9:00 AM
11:00 AM
4:00 PM
9:00 PM
12:00 AM
05:00 (2)
Because the effect of 2xO3 on leaf ABA content
is positive, at the site of synthesis, and ABA is a
stress signal inducing protective responses,
O3-induced increases in leaf ABA are more likely
to be part of the response to damage.
By contrast, O3- induced decreases in leaf cytokinin
content are more likely to be part of the damage
and not the response to it
Acknowledgements:
Thanks to Amanda Price
and Maydelin Dorado Bermudez
for help with hormone analyses
Correlations with other measured parameters
0.35
•
Negative correlation
with stomatal
conductance in sun
leaves in 2003 and
2004.
0.30
0.25
0.20
0.15
0.10
0.05
•
•
Stronger correlation in
control leaves than in
2 x O3 leaves.
High levels of ozone
my reduce the
capacity of ABA to
close stomata.
0.00
0
10
20
30
40
50
60
70
ABA content of leaves (nmol g-1 FW )
0.3
0.25
0.2
0.15
0.1
0.05
0
0.00
10.00
20.00
30.00
40.00
50.00
-1
ABA content (nmol g FW)
1 x O3 sun
2 x O3 sun
1 x O3 shade
2 x O3 shade
60.00
Isoprenoid Levels: 2004
2004 shade leaves
2004 sun leaves
80000
100000
70000
90000
CK content (fmol/gFM)
CK content (fmol/gFM)
80000
60000
50000
40000
30000
20000
70000
60000
50000
40000
30000
20000
10000
10000
0
0
18
23
28
33
Time (weeks)
1xO3
2xO3
38
43
18
23
28
33
Time (weeks)
1xO3
2xO3
38
43
Aromatic Levels: 2003
2003 sun leaves
2003 shade leaves
250000
350000
300000
CK content (fmol/gFM)
CK content (fmol/gFM)
200000
150000
100000
250000
200000
150000
100000
50000
50000
0
0
20
25
30
35
40
45
20
25
30
35
Time (weeks)
Time (weeks)
1xO3
1xO3
2xO3
2xO3
40
45
Z O-Glucoside Levels: 2003
2003 sun leaves
2003 shade leaves
1200000
1000000
900000
1000000
800000
CK content (fmol/gFM)
CK content (fmol/gFM)
700000
800000
600000
400000
600000
500000
400000
300000
200000
200000
100000
0
0
20
25
30
35
Time (weeks)
1xO3
2xO3
40
45
20
25
30
35
Time (weeks)
1xO3
2xO3
40
45