Insights into the Molecular Mechanisms of Litter Decomposition and Assimilation of Nitrogen by Ectomycorrhizal Fungi

Abstract

Ectomycorrhizae is the dominant type of mycorrhiza found in association with tree roots in

boreal and northern temperate forests. In this symbiosis, the fungal partner derives energy from

photosynthates provided by the host trees and in return delivers soil-derived nutrients such as

nitrogen (N). The majority of N in forest soils is embedded in recalcitrant organic matter–protein

complexes. Ectomycorrhizal fungi (EMF) are thought to have a key role in decomposing and

mobilizing nitrogen from such complexes. However, little is known about the mechanisms

governing these decomposing processes, how they are regulated by C and N availability, and the

mechanisms of organic N uptake. The work described in the thesis uses spectroscopic methods,

chemical analysis and transcriptome profiling to examine the mechanisms by which the model

ectomycorrhizal fungus Paxillus involutus degrades soil organic matter (SOM) while assimilating

the organic nitrogen from plant litter. Finally, the decomposing ability of seven other species of

EMF was examined that differ in ecology and evolutionary history.

The EMF P. involutus degraded SOM, while assimilating N, by a radical-based oxidation

involving Fenton chemistry similar to the mechanism used by saprophytic brown-rot (BR) fungi.

The key indications were the apparition of a C=O peak in the signature of cellulose, the side

chain modifications of lignin residues, and the increase in the Fe3+-reducing activity in the culture

filtrate. The set of enzymes expressed during the degradation of SOM was similar to the set of

enzymes involved in the oxidative degradation of wood by BR fungi. Secondary metabolites are

key components for Fe3+-reduction and the generation of Fenton reagent in BR oxidative

degradation of lignocellulose. The Fe3+-reducing activity of P. involutus was caused by the

pigment involutin. The saprotrophic activity of P. involutus is reduced to a radical-based

biodegradation system that efficiently disrupts the organic matter and thereby mobilizes the

entrapped N. The decomposition of plant litter and assimilation of nitrogen was triggered by the

addition of glucose while ammonium addition had minor effects. P. involutus secreted peptidase

activity, mostly contributed by aspartic peptidases while degrading proteins. The expression levels

of extracellular peptidases were regulated in parallel with transporters and enzymes involved in the

assimilation and metabolism of the released peptides and amino acids. Finally, all the examined

EMF species catalyzes oxidative degradation of complex organic components in the litter extract

with a mechanism similar to that of BR fungi. The ability to modify complex organic material by

oxidation is not restricted to rapidly-growing, long-distance exploration types of EMF, but it is

also found in slow-growing, medium- and short-distance EMF exploration types. All examined

EMF species expresses distinctively different sets oxidative enzymes to oxidize the litter material.

Thus, EMF can degrade plant litter by oxidative mechanisms similar to BR while variation in

gene expression might reflect adaptations of the decomposing mechanisms to different

environmental conditions.

Abstract (Swedish)

Popular Abstract in English

Fungi are multi-cellular eukaryotic organisms which form thread-like filaments called hyphae. They form reproductive structures called sporocarp (also known as mushrooms). Most fungi are saproptrophs, feeding on dead or decaying material while many are also parasitic, feeding on living organisms. In addition, most vascular plants are always in symbiotic association with fungi called mycorrhizae, which colonize their roots and supply essential nutrients. In boreal forests ecosystem such as the one found in Sweden, nutrients usually occurs in patches in the soil. In such nutrient-poor conditions, trees such as Pine, Spruce and Oak rely on the most abundant ectomycorrhizal fungi (EMF) (svenska: ektomykorrhiza) for acquisition of nutrients and water. In this symbiotic relationship, the branching filaments of the soil fungus encounter host root tips, forms a sheath around the root and radiate outwards into the surrounding soil and litter, forming extensive intermingling hyphal webs. It has been estimated that within one gram of humus soil hundreds of meters of EMF can be found. The mycelial network also increases the absorptive surface of the tree roots by two orders of magnitude by this symbiosis.

In EMF symbiosis, the fungal partner scavenge soil nutrients such as nitrogen and phosphorous and transfer a portion of these nutrients to their host plants. In return, the host plants supplies C mostly in the form of photosynthetic sugars. A large part of soil nitrogen is present in organic form such as proteins, nitrogen-containing sugars and complex heterocyclic compounds. This nitrogen sources usually interacts with materials present in the soil and form recalcitrant complexes that are hard to break down. The trees lack the ability to break down these complex molecules to get the nitrogen. EMF are thought to play a key role in degradation of organic matter and mobilization of nutrients from such complexes. Degradation of organic matter is well studied in wood-degrading saprotrophic fungi, such as white rot and brown-rot fungi. However, we lack the knowledge of the degradation mechanisms in EMF.

The aim of the thesis was to uncover the mechanisms by which EMF fungi degrade soil organic matter and take up nutrients. We used a model EMF, Paxillus involutus (common name, brown roll-rim or Pluggskivling (svenska)) which is one of the best studied ectomycorrhizal fungi in terms of ecology and physiology. It is also one of the most widely distributed EMF species across the Northern Hemisphere. The fungus was grown on an axenic growth system that allows measurements of chemical changes in the substrate and also the expression of enzymes during litter degradation. A combination of novel spectroscopic technique with gene expression pattern was used to understand the degrading mechanisms of the fungus. The ectomycorrhizal degradation process was compared with wood degrading brown-rot fungus degradation mechanisms.

For the first time we showed that the ectomycorrhizal fungus, P. involutus oxidatively degrades the litter material while taking up nitrogen, with a mechanism that has a chemical signature of Fenton chemistry (Fe2+ + H2O2 + H+→ Fe3+ + ●OH + H2O). In Fenton chemistry, hydroxyl radicals (●OH) are produce in the presence of Fe2+ and H2O2 and are common in brown-rot wood degradation. Spectroscopic analysis of litter extract shows radical-based oxidation patterns of polysaccharides, ployphenols and lignin matrix. We also observed an increase in Fe3+-reducing activity while the fungus was degrading plant litter extract and the activity was found to be caused by a single compound called involutin. The set of enzymes expressed by P. involutus during the degradation of plant litter material was also similar to the set of enzymes involved in the oxidative degradation of wood by brown-rot fungi. However, unlike brown-rot saprotrophs, EMF P. involutus lacks carbohydrate-degrading enzymes. Thus, the saprotrophic activity of P. involutus is reduced to a radical-based biodegradation system that efficiently disrupts the organic matter and thereby mobilizes the entrapped N. The decomposition of plant litter and assimilation of nitrogen by P. involutus was also triggered by the addition of glucose while ammonium addition had minor effects. These suggest that the C flux from the host plants can control the decomposition activity of EMF.

The EMF, P. involutus was also able to degrade proteins by secreting acidic peptidases, mostly aspartic peptidases. At transcriptome level, this peptidase activity was contributed by the expression of a large number of extracellular endo- and exopeptidases. The expression levels of these peptidases were regulated in parallel with transporters and enzymes involved in the assimilation and metabolism of the released peptides and amino acids. This is the first time a protein degradation pathway of an ectomycorrhizal fungus is described.

There are approximately 5,000-6,000 EMF species that differs extensively in their ability to take up and transfer nutrients to the host trees. Such differences have been correlated to the development of the nutrient foraging mycelium that extends from the symbiotic EMF root-tip tissue into the surrounding soil. We compared the mechanisms by which EMF of various functional groups, i.e. exploration types, degrade complex organic matter when acquiring N from forest litter material. We observed that all ectomycorrhizal fungus degrades litter extract efficiently with a consistent signature of Fenton chemistry oxidation similar to brown-rot mechanism as was observed with P. involutus. The oxidation was correlated to growth rate and nitrogen uptake. The transcriptome analyses shows that each fungal species expresses distinct sets of enzymes within similar functional categories of genes thought to be involve in oxidative degradation systems and breakdown of organic nitrogen. This reflects the adaptations of the EMF species in their degrading ability depending on different environmental conditions.

The thesis provides a step forward in understanding the degradation potential of EMF species of complex organic matter present in litter material in the natural boreal forest ecosystem. We showed that the biodegradation of lignin residues and aromatic compounds by the EMF species were comparable to that of the saprotrophic brown-rot, emphasizing the key role of EMF as potential contributor to the carbon and nitrogen cycling. The symbiotic association with the host plants helps in the removal of atmospheric CO2 as a portion of the C is allocated to the mycorrhizal fungi, which is then used to build mycelial hyphae. The EMF mycelium then degrades complex organic matter and takes up N and other nutrients leaving behind the organic C skeleton in the soil. It has been estimated that extramatrical mycelium contributes one-third of the microbial biomass and produces together with associated roots, half of the dissolved organic C in forest soils. Thus, the EMF mycelium is an important sink controlling the allocation of plant C.

• author: Shah, Firoz
• supervisor: Anders Tunlid
• opponent: Dr. Tarkka, Mika, Department of Soil Ecology, Helmholtz Centre for Environmental Research GmbH-UFZ, Halle, Germany
• organization: MEMEG; Microbial Ecology (research group); BECC: Biodiversity and Ecosystem services in a Changing Climate
• publishing date: 2014
• type: Thesis
• publication status: published
• subject: Biological Sciences
• keywords: Ectomycorrhizal fungi, Paxillus involutus, organic matter degradation, Fenton chemistry, carbon availability, N assimilation, C and N cycling, protein degradation pathway, secondary metabolites
• pages: 226 pages
• defense location: Blue Hall, Ecology Building, Sölvegatan 37, 223 62 Lund, Sweden
• defense date: 2014-05-09 10:00:00
• ISBN: 978-91-7473-937-4
• language: English
• LU publication?: yes
• id: 86ef9d3f-a0d5-43f9-8352-d9b4a2e52e2c (old id 4393306)
• date added to LUP: 2016-04-04 10:20:45
• date last changed: 2020-09-23 15:15:10