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

Introduction

Field siteAn 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.

Horizon pH (CaCl2)

Corg Total (g kg-1)

Oh 4.2 354.3 Ah 3.2 70.0 Bv 3.9 6.0

Soil characteristics:

Sampling design

30 cm

30

cm

3 subsamples= 1 mixed probe

x 5-8

d>2m

• DNA and RNA were isolated according to Luis et al. 2004, 2005

• degenerated laccase primers were used to amplify a 140 – 200 bp laccase gene fragment between copper binding site I & II

I II III IV

DNA

Cu1F Cu2R

Cu1AF Cu2R

TTIHWHGFFQATTNWADGRAFVNQCPIAANNSFLYNFNVPGQAGTFWYHSTSIHWHGFFQSTTNWADGPAFVTQCPIMPNDSFSYEFEVPNQAGTFWYHS

TSIHWHGLEMRETPEADGVPGLTQTPIEPGATFTYRFRAY-PAGTFWYHSTSVHWHGIRQLGSLEYDGVPGVTQCPIAPGDTLTYKFQAT-QYGTTWYHS

• 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 semi-quantitative PCR

• Enzyme activity was measured using ABTS

Methods

• Sampling area homogeneously covered with beech trees (nearest distance to oak trees ≥ 7 m)

• 6 sampling dates evenly distributed through one year

• For each date, 8 plots were sampled. For each plot (20x20 cm), 3 soil subsamples were collected, mixed and stored in liquid nitrogen.

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.

0.01 substitutions/site

NJ

Russ

ulaceae

Atheliaceae

Agaricales

Boletales

Agaricales

Agaricales

Agaricales

Agaricales

Boletales

Plants

div. sp.

div. sp.

div. sp.

Laccase gene typification

Temporal laccase activity

Spatial distribution

This work is supported by the German Science foundation DFG (SPP 1090, grand Bu 941 2-1, 2-2 & 2-3)

Contact:Harald Kellner / Dr. Patricia Luis / Prof. Dr. F. Buscot University of Leipzig, Institute of Botany, Johannisallee 21, 04103 Leipzig, Germanykellner@uni-leipzig.de, luis@uni-leipzig.de

Literature:Luis et al. (2004): Diversity of laccase genes from basidiomycetes in a forest soil. Soil Biology & Biochemistry 36, 1025–1036Luis et al. (2005): A molecular method to evaluate basidiomycete laccase gene expression in forest soils. Geoderma, (Online)

T1 – March 04T2 – June 04T3 – August 04T4 – October 04T5 – January 05T6 – April 05

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).

Ect

omyc

orh

izal

Sap

rotr

oph

icS

apro

trop

hic

EC

M

O-horizon

T1 T2 T3 T4 T5 T6

lacc

ase

activ

ity -

U/g

DM

0,0

0,2

0,4

0,6

0,8

Plot 1

A-horizon

T1 T2 T3 T4 T5 T6

lacc

ase

activ

ity -

U/g

DM

0,00

0,01

0,02

0,03

0,04

0,05

sample date

T1 T2 T3 T4 T5 T6

lacc

ase

activ

ity -

U/g

DM

0,000

0,005

0,010

0,015

0,020

0,025B-horizon

Expression profile: O-horizon

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 A-horizon 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.

Tubulin 400bp

LaccaseCu1A+Cu2

150bp

140bp LaccaseCu1+Cu2

T1 T2 T3 T4 T5 T6

LaccaseCu1+Cu2

140bp200bp DNA

RNA

RNA

RNA