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In the mutualistic ectomycorrhizal symbiosis, the nutritional relationships between the plant–fungus partners rely on a bidirectional flux of nutrients. The mycobiont hyphal networks radiating into the soil and litter absorb soil nutrients that are translocated throughout strands and rhizomorphs to the host root. The absorption, translocation and assimilation of mineral ions by hyphae require carbon skeletons, ATP and reducing power, as NAD(P)H, which are generated by carbohydrate oxidative pathways. Although ectomycorrhizal fungi are facultative saprotrophs, the analysis of the Laccaria bicolor genome has revealed that this ectomycorrhizal basidiomycete is poorly adapted for efficient degradation of soil carbon-rich lignocellulose, which likely reflects a reliance on host-supplied photoassimilates. However, several species of ectomycorrhizal fungi show a stronger saprophytic ability (Koide et al., 2008). Up to 30% of these assimilates, mainly as sucrose, can be transferred to the associated fungus (Finlay & Söderström, 1992). Sucrose downloaded into the symbiotic apoplastic interface is then hydrolysed into fructose and glucose via the action of the plant sucrose invertase (Nehls et al., 2007). The resulting glucose and fructose are actively taken up by the fungal hyphae where they feed the carbohydrate metabolism, leading to the synthesis of trehalose, polyols and other storage compounds (glycogen, fatty acids) (Martin et al., 1998). Carbohydrate catabolism also provides energy for hyphal growth and supplies carbon skeleton to other metabolisms (notably the amino acid biosynthesis). Storage carbohydrates fulfil multiple functions in ectomycorrhizas; they not only constitute a source of carbon and energy but also protect mycorrhiza against a variety of environmental stresses such as desiccation and frost (Elbein et al., 2003). Furthermore, the conversion of host hexoses into fungus-specific storage carbohydrates, such as polyols and trehalose, creates a strong driving force for plant carbon allocation to symbiotic tissues (Martin et al., 1998; Nehls et al., 2001; López et al., 2007). Polyols may be the compatible solutes responsible for generating the hydrostatic pressure used by the hyphae to break the root surface and penetrates between epidermal cells to initiate the Hartig net (Martin et al., 1998). Both mannitol and trehalose play a key role in the regulation of glucose metabolism and carbon storage (Wiemken, 2007), but biosynthesis and degradation pathways of these carbohydrates have not been comprehensively described in ectomycorrhizal fungi and it remains to be determined whether they are fully operational.
There is evidence that the development and functioning of ectomycorrhizal symbiosis bring about dramatic modification of carbon metabolism in the host roots and in the mycobiont forming the mutualistic association (Martin et al., 1987; Hampp & Schaeffer, 1995; Martin et al., 1998). The utilization patterns of [1–13C]glucose by Eucalyptus globulus seedlings and Pisolithus microcarpus mycelium was influenced by mycorrhizal colonization, with a greater allocation of carbon to short chain polyols, arabitol and erythritol and to trehalose in the mycelium and a suppression of sucrose synthesis in colonized roots (Martin et al., 1998). It appears that fungal metabolism dominates the assimilation of exogenous carbohydrates into symbiotic tissues. Several P. microcarpus transcripts coding for enzymes involved in the glycolysis, tricarboxylic acid (TCA) cycle and the mitochondrial electron transport chain were upregulated in symbiotic tissues 7–12 d after contact (Duplessis et al., 2005), confirming a general stimulation of the glucose respiration pathways. Transcript profiling confirmed this shift in carbon metabolism in the Paxillus involutus–Betula pendula ectomycorrhiza (Johansson et al., 2004; Le Quéréet al., 2005).
So far, the primary carbohydrate metabolism of an ectomycorrhizal fungus has not been characterized at a genome scale and it is not known if symbiotic fungi have gained or lost specific pathways compared with saprotrophic fungi. Here, we characterize the complete set of genes encoding enzymes involved in primary carbohydrate metabolism in the recently sequenced L. bicolor genome (Martin et al., 2008). This includes cataloguing predicted carbohydrate metabolism proteins, surveying their level of transcripts in various tissues and conducting phylogenetic analyses on enzymes of trehalose and mannitol metabolism.
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The extramatrical hyphae of L. bicolor may have a significant saprotrophic ability, as revealed by the abundance of proteases, glucanases and carbohydrate-active enzymes acting on animal and bacterial polysaccharides in its genome (Cullen, 2008; Martin et al., 2008). However, L. bicolor has only a single gene encoding an endoglucanase with a cellulose-binding domain, and no genes for exocellobiohydrolases. There is also little evidence of the oxidative systems necessary for lignin degradation, such as lignin-depolymerizing peroxidases. The hyphae forming the Hartig net in colonized roots are likely biotrophic and rely on the host sucrose for their carbon metabolism. Carbohydrate exchanges between plant roots and L. bicolor mycelium is the cornerstone of the mycorrhizal symbiosis. Interestingly, enzymatic activities measurements and NMR analyses performed on various ectomycorrhizal fungi suggested that the primary carbohydrate metabolism of these symbionts does not differ from the one of nonsymbiotic fungal species (Martin et al., 1985; Ramstedt et al., 1989; Martin et al., 1998; Bago et al., 1999; Rangel-Castro et al., 2002). This is confirmed by the present annotation of L. bicolor genome: all the common glycolytic and storage pathways have been identified and seem to be functional as they are all transcribed. The evolution toward mycorrhizal symbiosis did not lead to the loss or to the expansion of gene families involved in the primary carbon metabolism as it is often observed in obligatory symbiosis (Moran, 2007).
The recent sequencing of the genomes of five basidiomycetes (C. neoformans, Loftus et al., 2005; C. cinerea, http://www.broad.mit.edu/annotation/genome/coprinus_cinereus/Home.html; P. chrysosporium, Martinez et al., 2004; U. maydis, Kämper et al., 2006; and L. bicolor , Martin et al., 2008) allowed better characterization of trehalose and mannitol metabolism. Trehalose and mannitol are the main carbohydrates accumulated in fungi, including ectomycorrhizal species, where they can contribute up to 30% of the mycelium dry weight (Martin et al., 1985; Ramstedt et al., 1989; Martin et al., 1998; Stoop & Mooibroek, 1998; Parrou et al., 2005). Both metabolites can serve as a storehouse of glucose and for synthesis of cellular components. Mannitol is also involved in osmotic stabilization of hyphae and may play an important role in the recycling of reductants (NADPH and NADP). By contrast, trehalose could act in fungi as a stabilizer of cellular membranes and proteins. Our genome analysis provides new insights into trehalose and mannitol metabolism in L. bicolor. For trehalose metabolism, we showed that trehalose phosphorylase (TP), often described as a secondary enzyme present in a limited number of fungi (Elbein et al., 2003; Parrou et al., 2005; Avonce et al., 2006), is found in all sequenced genomes. Homologues are present in A. muscaria (López et al., 2007), P. chrysosporium, C. cinerea, C. neoformans, A. fumigatus, Magnaporthe grisea and N. crassa genomes (Fig. 3). By contrast, no homologue was found either in the basidiomycete U. maydis or in the ascomycete S. cerevisiae. It remains to be determined whether the hydrolytic activity of the TP is reversible. While the degradation activity has been demonstrated (Kitamoto et al., 1998; Han et al., 2003), its anabolic activity has only been established in vitro (Saito et al., 1998; Wannet et al., 1998). However, recent observations indicated that the enzyme could also work in this way in vivo (Han et al., 2003; López et al., 2007).
Our genomic survey also provided new insights on acid trehalase classification. Parrou et al. (2005) established that acid trehalases can be clustered into two groups depending on the presence of a signal peptide or an N-terminal transmembrane domain. A third category was established for trehalases from M. grisea, N. crassa and Gibberella zeae that harboured a noncanonical structure with dual characteristics of both neutral and acid trehalases. The acid trehalase of L. bicolor belongs to this latter category. This class of extracellular enzymes may contain many acid trehalases from filamentous fungi as it was identified in the genome of L. bicolor S238N, and in all the sequenced genomes of filamentous fungi.
In basidiomycetes, mannitol synthesis is thought to occur through MtDH. Indeed, no M1PDH activity has ever been measured in any basidiomycetes (Hult et al., 1980). Two genes have been annotated as encoding M1PDH enzymes in C. neoformans. But the enzymatic activities of the corresponding proteins have not been measured. Orthologues of these genes are present in all the sequenced basidiomycetous genome, including L. bicolor. However, they are more closely related to alcohol dehydrogenase than to mannitol dehydrogenase according to the phylogenetic analysis (Fig. 2). Furthermore, the two MDR transcripts from L. bicolor are transcribed in free-living mycelium, while no mannitol was detected in hyphae by NMR. Conversely, MtDH transcript was barely detectable in free-living mycelium. Together, these results suggest that these genes do not encode for M1PDH.
The ectomycorrhizal symbiosis leads to dramatic changes in carbon metabolism in the mycobiont forming the association (Martin et al., 1987, 1998; Hampp & Schaeffer, 1995; López et al., 2007). Trehalose, mannitol and various small polyols have been reported to accumulate during mycorrhiza formation (Ineichen & Wiemken, 1992; Martin et al., 1998; Nehls et al., 2001). This shift in fungal metabolism was correlated with an alteration of the transcription of genes encoding proteins involved in glucose respiratory pathways (Voiblet et al., 2001; Johansson et al., 2004; Duplessis et al., 2005). A single gene encoding hexokinase (HK) was found upregulated whatever the basidiomycetous species analysed (e.g. P. microcarpus and P. involutus). In L. bicolor, glucokinase- and hexokinase-encoding genes showed a weak increased transcription in both poplar and Douglas fir mycorrhizas. In S. cerevisiae, the HXK2 gene, encoding for a hexokinase, plays a pivotal role in the control of the expression of genes encoding enzymes of primary carbon metabolism, including its own transcription (Moreno & Herrero, 2002). The ectomycorrhizal hexokinase may also participate in carbon metabolism regulation during the symbiosis establishment, as already suggested in the ascomycete Tuber borchii (Ceccaroli et al., 1999). This ectomycorrhizal fungus harbours three distinct enzymatic forms of hexokinases that are differentially expressed during mycelium growth.
Another striking alteration in L. bicolor carbohydrate metabolism is the upregulation of all the genes encoding proteins of the trehalose synthase complex in symbiotic tissues, indicating that the accumulation of trehalose in L. bicolor mycorrhizas is controlled at the transcriptional level. By contrast, we observed the repression of the genes encoding trehalose phosphorylase and neutral trehalase, and a strong upregulation of mannitol dehydrogenase genes in fruiting body. This suggests that a metabolic shift is likely to occur during L. bicolor fruiting body formation. However, mannitol was not detected in the fruiting bodies of L. bicolor using natural abundance 13C NMR (Fig. 5). This suggests that if mannitol synthesis occurs the turnover of the polyol pool is so high that it does not accumulate. In the ectomycorrhizal ascomycetous fungi, C. geophilum and S. brunnea, the synthesized mannitol is immediately consumed, as demonstrated by the high isotopic scrambling observed in 13C-NMR experiment (Martin et al., 1985; Ramstedt et al., 1989). Although trehalose and mannitol are the most commonly carbohydrate accumulated in fungi, patterns of accumulation of these compounds differ greatly between species of ectomycorrhizal fungi: in C. geophilum (Martin et al., 1985), T. borchii (Ceccaroli et al., 2003) and Pisolithus tinctorius (Martin et al., 1998), the main carbohydrate detected by NMR in free-living mycelium is mannitol. Conversely, L. bicolor and Piloderma croceum accumulate only trehalose (Ramstedt et al., 1989, present study), while both trehalose and mannitol were found in Cantharellus cibarius mycelium (Rangel-Castro et al., 2002). The cause of these various metabolic patterns remains to be determined.
The present in silico metabolic reconstruction of the central carbon metabolism in L. bicolor showed that the carbohydrate metabolism in this symbiotic fungus does not differ from saprophytic fungi and that ectomycorrhiza formation induces a carbon metabolic shift that is controlled at the transcriptional level.