Transcript p_buscot_kellner - Chair of Soil Science

Spatiotemporal diversity and activity patterns of laccase genes in a forest soil
H. Kellner, P. Luis, B. Zimdars and F. Buscot - Terrestrial Ecology, Institute of Botany, University of Leipzig, Germany
NJ
• DNA and RNA were isolated according to Luis et al.
2004, 2005
A-horizon
0,05
0,04
0,6
0,4
0,2
0,03
0,02
0,01
0,00
I
II
III
IV
0,0
T1
T2
T3
T4
T5
T6
T1
DNA
T2
Horizon
Oh
Ah
Bv
pH
(CaCl2)
4.2
3.2
3.9
Corg Total
(g kg-1)
354.3
70.0
6.0
Cu2R
T6
Plot 1
0,015
0,010
0,005
0,000
T1
Cu1F
T5
Ectomycorhizal
TSIHWHGLEMRETPEADGVPGLTQTPIEPGATFTYRFRAY-PAGTFWYHS
TSVHWHGIRQLGSLEYDGVPGVTQCPIAPGDTLTYKFQAT-QYGTTWYHS
T6
T2
T3
T4
sample date
Expression profile: O-horizon
Atheliaceae
Cu2R
• amplified products were purified, cloned and sequenced
• sequences were identified using cluster methods (PAUP
4.0) and analyzed for diversity (PC Ord 4.0)
• Expression of laccases was analyzed by semiquantitative PCR
• Enzyme activity was measured using ABTS
Spatial distribution
Sampling design
• Sampling area homogeneously covered with beech
trees (nearest distance to oak trees ≥ 7 m)
200bp
140bp
Laccase DNA
400bp
140bp
Tubulin RNA
Laccase RNA
Cu1+Cu2
150bp
Laccase RNA
Cu1+Cu2
Cu1A+Cu2
T1
T2
T3
T4
T5
T6
Laccase activity decreases with depth, whereby in general the
activity within the O-horizon is around 20 times higher as compared
to the A-horizon. The activity of each sample date shows great
variability – some spots display 5 to 10 times more activity than the
average. T1, T3 & T6 for the O-horizon and T1 & T6 for the Ahorizon displayed the highest temporal soil laccase activities.
For fungal tubulin, gene expression was found throughout the whole
year. The expression profile of laccase genes showed a different
pattern. Highest expression were detected for T3 and T5 with both
primer pairs, medium expression occurred for T1, T4 and T6 and no
expression was found for T2. Summarizing: a high laccase
expression does not correspond to high soil laccase activity – like
observed for T5 and T6.
• 6 sampling dates evenly distributed through one year
This work is supported by the German Science foundation DFG (SPP
1090, grand Bu 941 2-1, 2-2 & 2-3)
• For each date, 8 plots were sampled. For each plot
(20x20 cm), 3 soil subsamples were collected, mixed
and stored in liquid nitrogen.
d>2m
30 cm
x 5-8
30 cm
3 subsamples= 1 mixed
probe
Saprotrophic fungi detected through their laccase genes were
restricted to the upper horizons (e.g. Mycena zephirus). ECM fungi
had a broader vertical distribution. Lactarius subdulcis colonized
preferably the organic horizons (Oh), while the Russulaceae type 1
rather occupied the mineral part of the soil cores (Bv). Most detected
fungi were aggregated in small patches (<0,77 m2).
Contact:
Harald Kellner / Dr. Patricia Luis / Prof. Dr. F. Buscot
University of Leipzig, Institute of Botany, Johannisallee 21,
04103 Leipzig, Germany
[email protected], [email protected]
Literature:
Luis et al. (2004): Diversity of laccase genes from basidiomycetes in a
forest soil. Soil Biology & Biochemistry 36, 1025–1036
Luis et al. (2005): A molecular method to evaluate basidiomycete laccase
gene expression in forest soils. Geoderma, (Online)
Agaricales
ECM
Cu1AF
Boletales
Agaricales
Saprotrophic
Soil characteristics:
TTIHWHGFFQATTNWADGRAFVNQCPIAANNSFLYNFNVPGQAGTFWYHS
TSIHWHGFFQSTTNWADGPAFVTQCPIMPNDSFSYEFEVPNQAGTFWYHS
T5
0,020
laccase activity - U/g DM
An experimental station of the Institute of Ecosystem
Research (BITÖK) from the University of Bayreuth
located at 460 m above the sea level in Steigerwald /
Ebrach (49°52'26''N, 10°27'54''E). The site is covered by
a mixed stand of European beech (Fagus sylvatica L.)
and European oak (Quercus robur L.) with little ground
vegetation. The soil, Dystric Cambisol (FAO-UNESCO,
1990), is characterized by a fine moder humus layer and
a pH between 3.2 and 4.2.
T1 – March 04
T2 – June 04
T3 – August 04
T4 – October 04
T5 – January 05
T6 – April 05
T4
B-horizon
0,025
Field site
T3
Russulac eae
• degenerated laccase primers were used to amplify a 140
– 200 bp laccase gene fragment between copper binding
site I & II
O-horizon
0,8
div. sp.
Agaricales
div. sp.
Plants
div. sp.
Saprotrophic
Fungal laccases catalyze oxidation of phenolic compounds
and aromatic amines coupled to reduction of molecular
oxygen to water. Laccase can completely degrade lignins
and appear to be an important actor in SOM turnover.
Here we analyze variations of distribution and expression
of fungal genes encoding laccases in time and space.
Temporal laccase activity
laccase activity - U/g DM
Methods
laccase activity - U/g DM
Introduction
Agaricales
Boletales
Agaricales
0.01 substitutions/site
Laccase gene typification