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Keywords:

  • Amanita muscaria;
  • carbon allocation;
  • cyclic AMP;
  • ectomycorrhiza;
  • gene expression;
  • monosaccharide transporter;
  • Picea abies;
  • Populus tremula × tremuloides

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Impact of mycorrhiza formation on photosynthesis
  5. Carbohydrate allocation at the plant–fungus interface
  6. Generation of a strong carbohydrate sink in ectomycorrhizas
  7. Carbohydrates as signals that regulate fungal gene expression in pure culture and symbiosis
  8. Conclusions and future research
  9. Acknowledgements
  10. References

Ectomycorrhizas are mutalistic symbiotic associations formed between fine roots of higher plants, mostly trees, and a wide range of soil ascomycetes and basidiomycetes. It is commonly accepted that there is mutual benefit to the partners, due to the exchange of plant-derived carbohydrates for amino acids and nutrients supplied by the fungus. While the major concepts of mycorrhizal functioning (exchange of nutrients and metabolites) were proposed in the 1960s, their verification at the molecular level started approximately 10 years ago. This review covers concepts at the molecular level concerned with the fungal carbohydrate supply in symbiosis. We discuss: strategies used by host plants to compensate (and perhaps restrict) carbohydrate drain to the fungal partner; fungal mechanisms that generate strong monosaccharide sinks in colonized plant roots (the formation of a strong carbohydrate sink is a prerequisite for efficient fungal carbohydrate support by the plant partner); and the impact of apoplastic hexose concentration on the regulation of fungal metabolism in symbiosis, since monosaccharides not only serve as nutrients but also as a signal that regulates gene expression.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Impact of mycorrhiza formation on photosynthesis
  5. Carbohydrate allocation at the plant–fungus interface
  6. Generation of a strong carbohydrate sink in ectomycorrhizas
  7. Carbohydrates as signals that regulate fungal gene expression in pure culture and symbiosis
  8. Conclusions and future research
  9. Acknowledgements
  10. References

Ectomycorrhizas are mutalistic symbiotic associations formed between fine roots of higher plants, mostly trees, and a wide range of soil ascomycetes and basidiomycetes. The dominant trees of temperate forests, belonging to the Fagaceae and Pinaceae, are ecologically obligate ectomycorrhizal symbionts. In the symbiosis, the root and fungus no longer function independently, but form a unit with specific metabolic pathways and controlled exchange of metabolites (France & Reid, 1983; Martin et al., 1987). It is commonly accepted that there is mutual benefit to the partners, due to the exchange of plant-derived carbohydrates for amino acids and nutrients supplied by the fungus (Harley & Smith, 1983; Marschner & Dell, 1994; Smith & Read, 1997; Hampp & Schaeffer, 1999).

One of the first attempts to assay carbon flow in a mycorrhizal plant was performed by Melin & Nilsson (1957). It showed that after feeding 14C-labelled CO2 to leaves, labelled carbon appeared within one day in the hyphal mantle. Many ectomycorrhizal fungi can be cultured on synthetic media containing a single organic carbon source. With regard to symbiosis, organic compounds contained in root exudates (e.g. soluble sugars, carboxylic acids and amino acids) are of particular interest as candidates for the carbon transfer from the host to the mycorrhizal fungus (Marschner, 1995; Smith & Read, 1997; Hampp & Schaeffer, 1999). Interestingly, sucrose, which is the preferred transport sugar in most host plants, cannot be used by ectomycorrhizal (ECM) fungi investigated so far (e.g. Salzer & Hager, 1991), Laccaria bicolor being a possible exception (Tagu et al., 2000). It is thus assumed that sucrose is delivered into the apoplast at the plant–fungus interface (Hartig net) and hydrolysed via a plant-derived acid invertase (Salzer & Hager, 1991; Salzer & Hager, 1993; Schaeffer et al., 1995). The resulting hexoses are then taken up by the fungus (France & Reid, 1983; Salzer & Hager, 1991) as well as by plant root cells (Nehls et al., 2000; Wright et al., 2000).

Since the fungus forms a strong sink for carbohydrates in symbiosis, hexoses must be taken up efficiently (Chen & Hampp, 1993; Wiese et al., 2000) and quickly incorporated into fungal metabolites (e.g. trehalose, mannitol and glycogen) (Martin et al., 1987; Martin et al., 1988; Martin et al., 1998).

This review covers concepts concerned with the strategies used by host plants to compensate (and perhaps restrict) carbohydrate drain to the fungal partner, fungal mechanisms that generate strong carbohydrate sinks in colonized plant roots, and the impact of the apoplastic hexose concentration on the regulation of fungal metabolism in symbiosis.

Impact of mycorrhiza formation on photosynthesis

  1. Top of page
  2. Summary
  3. Introduction
  4. Impact of mycorrhiza formation on photosynthesis
  5. Carbohydrate allocation at the plant–fungus interface
  6. Generation of a strong carbohydrate sink in ectomycorrhizas
  7. Carbohydrates as signals that regulate fungal gene expression in pure culture and symbiosis
  8. Conclusions and future research
  9. Acknowledgements
  10. References

The production of photoassimilates is a fundamental activity of plants. Formation, storage, transport and consumption of carbohydrates are dynamic processes that are closely linked to the physiological situation. Obviously, plants are able to monitor and respond to changing sugar levels, thereby integrating external environmental conditions.

There is considerable evidence that the process of carbon assimilation by plants is mainly influenced by the strength of the sinks to which photosynthates are allocated. In general, the direction of carbon flow in the host phloem is controlled by gradients between production (source organs, such as leaves) and consumption (sink organs, such as nongreen tissues) of photoassimilates. Carbon will thus always be directed to the most active sink area in a plant and the sink strength has been shown to control the rate of photoassimilate production (Stitt, 1991; Quick & Schaffer, 1996).

Ectomycorrhizal symbiosis causes a severe carbohydrate drain: up to 30% of total photoassimilate production can be transferred to the fungal partner, enabling its maintenance and proliferation (Finlay & Söderström, 1992; Söderström, 1992). There is ample evidence that mycorrhizas direct assimilates towards the root and increase the rate of net photosynthesis of the host (Nylund & Wallander, 1989; Dosskey et al., 1990; Friedrich, 1998; Loewe et al., 2000). The importance of the fungal sink in mycorrhizal symbiosis has also been elegantly demonstrated by Lamhamedi et al. (1994), showing that growth and development of Laccaria bicolor fruitbodies is dependent on the host plant’s current rate of net photosynthesis. Fruitbody growth was clearly affected by photosynthetic photon flux densities and removal of the fruitbodies induced a rapid decrease in net photosynthesis.

One of the key steps is probably the regulation of sucrose synthesis in the leaf cytosol. This mainly occurs through fructose bisphosphatase (FBPase) and sucrose phosphate synthase (SPS). FBPase activity is inhibited by an effector metabolite, fructose 2,6-bisphosphate (F26 BP; see Stitt, 1990; Quick & Schaffer, 1996). For spruce seedlings it has been shown that the amount of this regulator is greatly decreased in source needles of mycorrhizal as opposed to nonmycorrhizal plants (Hampp et al., 1995; Loewe et al., 2000). SPS catalyses a reaction that is regarded as essentially irreversible in vivo (Stitt et al., 1987). The enzyme is subject to regulation by metabolites (glucose 6-phosphate activates, inorganic phosphate inhibits; Doehlert & Huber, 1983; Sigl & Stitt, 1990) and by protein phosphorylation (Huber et al., 1989; Loewe et al., 1996), while regulation by protein synthesis/degradation does not appear to be of significance in the short term. Phosphorylation of SPS results in deactivation (i.e. lower sensitivity toward the activator, but increased sensitivity for the inhibitor). The properties of SPS respond to mycorrhiza formation. For example, for spruce seedlings it has been shown that the phosphorylation of SPS (Loewe et al., 1996) is lower in mycorrhizal than in nonmycorrhizal source needles (Loewe et al., 2000). In addition, the affinity of this enzyme for fructose 6-phosphate was increased by mycorrhiza formation in both spruce and aspen (Loewe et al., 2000). Both observations, decreased levels of F26 BP and increased activation of SPS, are thus indicative of an increased capacity for sucrose formation in mycorrhizal plants that is in agreement with the observation of increased rates of net photosynthesis.

In addition, other plant properties have been shown to be affected by mycorrhiza formation. These include chlorophyll and carotenoid content (Vodnik & Gogala, 1994), rates of respiration, CO2 compensation point of photosynthesis, and amount of Rubisco (Martins et al., 1997).

Carbohydrate allocation at the plant–fungus interface

  1. Top of page
  2. Summary
  3. Introduction
  4. Impact of mycorrhiza formation on photosynthesis
  5. Carbohydrate allocation at the plant–fungus interface
  6. Generation of a strong carbohydrate sink in ectomycorrhizas
  7. Carbohydrates as signals that regulate fungal gene expression in pure culture and symbiosis
  8. Conclusions and future research
  9. Acknowledgements
  10. References

An attempt to assay the longitudinal distribution of soluble carbohydrates in mycorrhizas was made by Rieger et al. (1992). Sucrose, but not glucose or fructose concentrations, varied longitudinally. Sucrose levels were lowest in the central parts of a mycorrhiza (i.e. the area of most intense symbiotic interaction). Correspondingly, levels of fungus-specific compounds such as trehalose or ergosterol were increased in this area (Hampp et al., 1995). By contrast, fine roots without fungal infection did not show longitudinal variations in sugar content.

Experiments with suspension-cultured hyphae of Amanita muscaria and Hebeloma crustuliniforme (Salzer & Hager, 1991) and with protoplasts of Amanita muscaria (Chen & Hampp, 1993) indicated that these ectomycorrhizal fungi have no system for sucrose import, only for the uptake of glucose and fructose. Obviously, sucrose can be used as a carbon source by these ectomycorrhizal fungi only if it is hydrolysed by the cell wall-bound invertase of their host (Salzer & Hager, 1991; Fig. 1). In spruce cells, two wall-bound acid invertase isoforms were found, one tightly and one ionically bound. Both wall-bound isoforms were shown to function as β-D-fructofuranoside-fructohydrolases (EC 3.2.1.26) with KM values for sucrose of 15.8 mM (ionically wall-bound) and of 8.3 mM (tightly wall-bound; Salzer & Hager, 1993).

image

Figure 1. Outline model describing the the carbohydrate allocation at the plant–fungus interface. G6P, glucose-6-phosphate, F6P, fructose-6-phosphate.

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The importance of host apoplastic invertase for supplying the fungal partner with hexoses is confirmed by transformation experiments. Heterologous expression of highly active yeast invertase in roots of poplar hybrids has a profound effect on the availability of hexoses and in consequence on fungal metabolism and development after mycorrhiza formation (Guttenberger et al., 1998; Guttenberger et al., unpublished).

In addition to invertases, roots contain sucrose synthase, another sucrose-splitting enzyme (Fig. 1). A histochemical analysis showed for spruce that cell wall-bound acid invertase dominates in the root cortex, the area of symbiotic interaction, while minor activities of sucrose synthase could only be detected in the vascular bundle (Schaeffer, 1995).

From these data it appears that sucrose is the main plant-derived carbohydrate supporting fungal growth in ectomycorrhiza.

Generation of a strong carbohydrate sink in ectomycorrhizas

  1. Top of page
  2. Summary
  3. Introduction
  4. Impact of mycorrhiza formation on photosynthesis
  5. Carbohydrate allocation at the plant–fungus interface
  6. Generation of a strong carbohydrate sink in ectomycorrhizas
  7. Carbohydrates as signals that regulate fungal gene expression in pure culture and symbiosis
  8. Conclusions and future research
  9. Acknowledgements
  10. References

Hexose uptake

The driving force for carbohydrate allocation to the apoplast is consumption at the sink site. In ectomycorrhizas this is obviously influenced by the fungus. Generally, tracer studies indicate rapid translocation of 14C-labelled assimilates to the roots of ectomycorrhizal plants (Melin & Nilsson, 1957; Harley & Lewis, 1969; Smith et al., 1969), especially in young symbiotic interactions (Cairney et al., 1989).

A prerequisite for rapid uptake of monosaccharides is a membrane transport system. However, so far only one hexose transporter gene (AmMst1) has been identified from an ectomycorrhizal fungus (A. muscaria, Nehls et al., 1998). This cDNA, the first identified basidiomycete sugar transporter, encodes a protein of 520 amino acids with a molecular mass of 57 kDa with the highest sequence homology to Rco3 of Neurospora crassa (Madi et al., 1997) and to a lower extent also with Snf3 (Celenza et al., 1988) and Rgt2 (Özcan et al., 1996) from yeast.

The expression of the AmMst1 cDNA in a Saccharomyces cerevisiae strain, unable to take up hexoses, demonstrated that AmMst1 was a functional monosaccharide transporter (Wiese et al., 2000). Uptake experiments using 14C-labelled monosaccharides revealed KM values of 0.46 mM for glucose and 4.2 mM for fructose, indicating a strong preference for glucose. Glucose uptake by AmMst1 (short-term experiment, Fig. 2) in transformed yeast cells, as well as by A. muscaria hyphae (long-term experiment, Fig. 3), was strongly favoured even in the presence of a large excess of fructose (20 mM vs 1 mM). A clear preference for glucose uptake was also observed for the ectomycorrhizal ascomycete Cenococcum geophilum (Stülten et al., 1995).

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Figure 2. Kinetics of glucose and fructose uptake by yeast cells expressing AmMst1. A yeast strain expressing AmMst1 in sense orientation was incubated with increasing concentrations of glucose (circles) or fructose (triangles). Samples were taken at different times. The rate of hexose uptake was calculated from the radioactivity incorporated by yeast cells (Wiese et al., 2000).

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image

Figure 3. Inhibition of fructose uptake by Amanita muscaria in the presence of glucose. A. muscaria hyphae were grown in liquid culture containing a mixture of 14 mM fructose and 22 mM glucose as carbon sources. Glucose (circles) and fructose (squares) concentrations in the growth medium were measured at different times (Wiese et al., 2000).

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In yeast and other saprophytic fungi, several hexose transporter genes are present in the genome (Jennings, 1995; Boles & Hollenberg, 1997). Monosaccharide uptake of hyphae is thus the sum of the import properties of several transporters expressed in a given physiological situation. Nevertheless, there is evidence that in A. muscaria, in contrast to yeast, only one transporter gene is functionally expressed:

  • When AmMst1 was expressed as the only active monosaccharide transporter in yeast, the cells exhibited hexose import properties comparable to those of intact A. muscaria protoplasts (Wiese et al., 2000). Both systems also revealed the same inhibitory effect of even low glucose concentrations on fructose uptake.
  • Intensive screening of genomic DNA and mRNA using PCR and hybridization techniques did not reveal additional monosaccharide transporter genes homologous to AmMst1 in A. muscaria (Nehls et al., 1998).

This does not exclude the expression of additional monosaccharide transporters (that do not share extensive sequence homology with AmMst1) under certain conditions or their expression at quite low levels.

Hexoses are taken up from the plant–fungus interface not only by fungal hyphae but also by plant root epidermal and cortical cells. Thus, a competition for hexose uptake by plant cells might control the carbohydrate drain. A Picea abies hexose transporter cDNA (PaMst1) that encodes an open reading frame of 513 amino acids was isolated by an RT-PCR based strategy (Nehls et al., 2000). PaMst1 is expressed in the hypocotyl and in roots of P. abies seedlings, but not in needles sampled at different developmental stages (cotyledons and young needles). The transcript level of PaMst1 was similar in hypocotyl and fine roots. In addition, two putative hexose transporter gene fragments were identified from birch by RT-PCR (Wright et al., 2000). While the expression of the hexose transporter gene of Norway spruce was only slightly (approx. 30%) reduced in mycorrhizas, the transcript level of both hexose transporter genes of birch was reduced by a factor of three. Even if a reduced transcript level does not mean a decreased transporter activity, and although the number of monosaccharide transporters expressed in plant roots is not yet clear, these results suggest that plants do not increase their hexose import capacity during symbiosis.

Thus, in contrast to the plant root hexose transporters, the expression of the fungal monosaccharide transporter gene increases significantly in mycorrhizas. Since, in addition, the transcript level of the fungal (A. muscaria) monosaccharide transporter gene is much higher than that encoding the P. abies hexose transporter (U. Nehls, unpublished), the fungus represents the major carbohydrate sink in infected fine roots. It could thus be assumed that the plant does not compete for hexose import at the plant–fungus interface, and that the fungal activity determines the sink strength for carbohydrates in mycorrhizas.

Conversion of hexoses into compounds of fungal metabolism

The establishment and maintenance of a hexose gradient between the apoplast and fungal cells is necessary for the formation of a strong carbohydrate sink in ectomycorrhizas. Monosaccharides, imported by fungal hyphae, are thus either directly introduced into maintenance or growth metabolism (glycolysis, pentose phosphate shunt), or are used to build up long-term (glycogen) and intermediate (trehalose) storage pools (Martin et al., 1987; Martin et al., 1988; Martin et al., 1998). As in most organisms, the ATP-dependent phosphofructokinase is the rate-limiting step in fungal glycolysis (Kowallik et al., 1998). In A. muscaria, this enzyme is activated by fructose 2,6-bisphosphate (F26 BP; ka about 30 nM; Schaeffer et al., 1996), which is similar to the situation for the enzyme from yeast or animal cells, but different from that for plant sources. It has been shown that A. muscaria mycelia grown in the presence of high hexose concentrations as well as mycorrhizal roots have increased amounts of F26 BP (Schaeffer et al., 1996; Hoffmann et al., 1997). This could indicate increased rates of glycolysis in hyphae under elevated hexose supply (e.g. hyphae of the Hartig net; Hampp & Schaeffer, 1999).

In yeast cells, levels of F26 BP, and thus the preponderance of glycolysis over gluconeogenesis, are controlled by the formation of cyclic AMP (cAMP). Increased glucose supply causes an increase of activity of adenylate cyclase (Thevelein, 1991) and thus of the cAMP content in hyphae. cAMP activates a cAMP-dependent protein kinase (PKA) which, via phosphorylation, activates F26 BP formation while inhibiting F26 BP degradation (François et al., 1984; Thevelein, 1991; d’Enfert, 1997; RadisBaptista et al., 1998).

At least the initial steps of glucose-dependent regulation of glycolysis also exist in A. muscaria. Changes in pool sizes of cAMP have been detected in relation to glucose supply (Hoffmann et al., 1997). When suspension cultures of A. muscaria were transferred from medium containing low (1 mM) to high (40 mM) glucose concentrations, both cAMP pools as well as rates of activity of PKA increased (Fig. 4; unpublished data).

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Figure 4. Effect of glucose supply on the amount of cAMP and on the activity of cAMP-dependent protein kinase A (PKA) in Amanita muscaria. A. muscaria mycelia were pregrown for 1 wk in liquid culture containing 2 mM glucose. After addition of glucose to a final concentration of 50 mM (t = 0) mycelia were further grown for 5 h. Mycelial samples were taken at different times and PKA activity (diamonds) as well as cAMP (squares) contents of the hyphae were determined using commercial kits (Promega, MA, USA, and Amersham Pharmacia, Freiburg, Germany, respectively).

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Carbohydrates as signals that regulate fungal gene expression in pure culture and symbiosis

  1. Top of page
  2. Summary
  3. Introduction
  4. Impact of mycorrhiza formation on photosynthesis
  5. Carbohydrate allocation at the plant–fungus interface
  6. Generation of a strong carbohydrate sink in ectomycorrhizas
  7. Carbohydrates as signals that regulate fungal gene expression in pure culture and symbiosis
  8. Conclusions and future research
  9. Acknowledgements
  10. References

Sugar-dependent gene expression, controlling fungal metabolism, has been intensely investigated for saprophytic ascomycetes (Jennings, 1995). In these species the external monosaccharide concentration regulates fungal gene expression (e.g. that of monosaccharide transporters) at the transcriptional level by two different mechanisms: induction/enhancement or repression (Felenbok & Kelley, 1996; Özcan et al., 1996). Evidence for similar sugar-dependent mechanisms of regulation of gene expression was also found in the basidiomycete A. muscaria.

Sugar-dependent up-regulation of genes in pure fungal culture

In A. muscaria, the expression of the hexose transporter gene is up-regulated by a threshold response mechanism depending on the extracellular concentration of monosaccharides (Nehls et al., 1998). A. muscaria hyphae grown in the presence of glucose concentrations up to 2 mM expressed the AmMst1 gene at a basal level, while higher monosaccharide concentrations triggered a fourfold increase of the amount of the AmMst1 transcript. This upregulation could not be further enhanced by hexose concentrations of up to 100 mM. The increase of AmMst1 expression is a slow process. A transition from the basal to the maximal rate of gene expression occurred between 18 h and one day of fungal culture, in comparison to 90 min for the induction of the high affinity glucose transport system Hxt2 in yeast (Bisson & Fraenkel, 1984).

The signal that regulates the hexose-dependent, enhanced AmMst1 expression has not been identified. When mycelia precultivated in the absence of glucose were transferred to a medium containing 40 mM of the glucose analogues 3-O-methyl-glucose (taken up by fungal hyphae but not further metabolized) or 2-deoxyglucose (imported and subsequently phosphorylated by hexokinase, but not further metabolized; Allen et al., 1989; Jang & Sheen, 1994; Wiese, unpublished), AmMst1 expression was still at the basal level and not enhanced as in the presence of glucose or fructose (Nehls et al., 1998). Thus, neither the presence of glucose analogues (which are structurally much closer related to glucose than fructose) in the apoplast nor their import or phosphorylation is sufficient to trigger enhanced AmMst1 expression. Thus, hexose sensing, leading to an enhanced AmMst1 expression, is presumably performed by an intracellular (intermediate of hexose metabolism) and not an extracellular sensor. The signal for hexose-dependent upregulation of gene expression might thus be different for A. muscaria and S. cerevisiae. In yeast, membrane proteins (Snf3 and Rgt2) showing similarity to sugar transporters are involved in sensing of the external hexose concentration and trigger gene expression via a signal transduction cascade (Özcan & Johnston, 1999).

Sugar-dependent gene-repression in pure fungal culture

While AmMst1-expression is an example for sugar-dependent enhancement of gene expression in A. muscaria, a second gene (AmPAL) was identified that revealed sugar-dependent gene repression (Nehls et al., 1999). Phenylalanine ammonia lyase (PAL) is a key enzyme of secondary metabolism and thus of the production of phenolic compounds. ECM-forming fungi have been reported to use phenolic compounds for both their own protection and that of their host against bacterial or fungal attacks (Marx, 1969; Chakravarty & Unestam, 1987; Garbaye, 1991).

In A. muscaria, the transcript of AmPAL was abundant in hyphae grown at low external glucose concentrations, but exhibited a significant decrease in hyphae cultured at glucose concentrations of > 2 mM (< 1/30 of the transcript level at low glucose concentration). Mycelia precultivated in the absence of glucose and transferred to a medium containing 40 mM glucose revealed a maximal repression of AmPAL expression after 1 h of cultivation. Thus, monosaccharide dependent AmPAL repression was, in contrast to the upregulation of AmMst1, a rapid process.

Unlike AmMst1, AmPAL-expression was regulated in a hexokinase-dependent manner. When mycelia precultivated in the absence of glucose were transferred to a medium containing 40 mM of each of the glucose analogues 3-O-methyl-glucose or 2-deoxyglucose, 3-O-methyl-glucose had no effect, while 2-deoxyglucose caused the same decrease of AmPAL expression as glucose. Since 2-deoxyglucose, in contrast to 3-O-methyl-glucose, is phosphorylated by hexokinase (but not further metabolized), it could be concluded that monosaccharide-dependent AmPAL expression is regulated by sugar phosphorylation via hexokinase as sugar sensor (e.g. Sheen et al., 1999).

To identify promoter-elements responsible for the sugar-dependent gene repression in A. muscaria, a genomic DNA-fragment, containing 1.5 kbp of the AmPAL-promoter region, was isolated and sequenced (Mikolajewski, unpublished). Promoter fragments of approx. 150 bp in length were PCR-amplified and used for electrophoretic mobility shift assays (EMSA; Fisher et al., 1991, Fig. 5, unpublished data). By using protein extracts of A. muscaria hyphae grown in the presence of high glucose concentrations or in the absence of hexoses, most of the promoter fragments revealed no binding sites for transcription factors (Fig. 5,II,IV,V). Some promoter fragments, however, showed protein binding. Gel-retardation signal intensities similar for both fungal protein extracts (Fig. 5,I) revealed identical transcription factor contents in hyphae grown with or without glucose. This pattern is typical for binding sites of general transcription factors. One promoter fragment (Fig. 5,III), however, revealed protein binding exclusively for extracts isolated from fungal hyphae grown in the presence of high glucose concentrations. Since the transcript level of AmPAL is strongly reduced in the presence of high hexose concentrations, we take this as a first evidence that the sugar-dependent repression of AmPAL could be mediated by a repressor. This type of regulation of sugar-dependent gene repression would thus be similiar to that reported for yeast (Nehlin et al., 1991; Gancedo, 1998) and filamentous ascomycetes (Strauss et al., 1995; Gonzalez et al., 1997).

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Figure 5. Transcription factor binding sites within the AmPAL promoter. Radio-labelled PCR-fragments, containing different parts of the AmPAL 5′-non coding region (promoter-region), were incubated in the absence (a) or in the presence (b, c) of fungal protein extracts. Proteins were isolated from Amanita muscaria hyphae grown without glucose (b), or at high glucose concentrations (c) for 1 wk. The samples were applied to nondenaturating PAGE. PCR-fragments, where no protein is attached to (free PCR-fragments), migrate much faster than those with attached proteins (transcription factor). The first basepair 5′ of the AmPAL cDNA (Nehls et al., 2000) is named as −1. The basepairs are counted from this start-point in 3′ to 5′ orientation. AmPAL-5′-non coding region from −450 to −520 (I), −520 to −600 (II), −600 to −680 (III), −680 to −760 (IV), −760 to −900 (V).

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Sugar-dependent gene regulation in mycorrhizas

An increase of AmMst1 expression, comparable to that found in mycelia cultivated at elevated monosaccharide concentrations, was also observed in symbiotic mycelia of ectomycorrhizas (Nehls et al., 1998). It is thus assumed that both the extended lag phase for enhanced AmMst1 expression, and its threshold response to elevated monosaccharide concentrations, are monosaccharide-regulated adaptations of the ectomycorrhizal fungus to the homeostatic conditions found only at the symbiotic interface, but not in the soil.

However, not only AmMst1, but also AmPAL, was strongly expressed in entire mycorrhizas. Since both genes are differentially expressed in a hexose-dependent manner in pure culture, it could thus be concluded that in mycorrhizas the sugar-dependent regulation of both genes (as observed in pure fungal culture) is either modified by developmental events, or different in the fungal sheath and Hartig net hyphae (i.e. spatial heterogeneity in expression). To address this question, ectomycorrhizas were dissected and gene expression was investigated separately for hyphae of the fungal sheath and the Hartig net (Nehls et al., 2001). As in pure fungal culture grown on low external hexose concentrations, AmMst1 was expressed only at the basal level in hyphae of the fungal sheath. In contrast, AmPAL exhibited a high transcript level in this fungal structure. For Hartig net hyphae the opposite expression pattern was observed. As for hyphae in pure culture in the presence of high external hexose concentrations, the transcript level of AmMst1 was sixfold enhanced while the expression of AmPAL was only barely detectable.

Owing to the opposite regulation of both genes in hyphae of the fungal sheath and Hartig net, which resembles the hexose-dependent expression of these genes in pure culture, the occurrence of a hexose gradient between the apoplast of the fungus–plant interface, the Hartig net (hexose concentration above 2 mM) and the fungal sheath (lower hexose concentration) has been suggested (Nehls et al., 2001). ‘Metabolic zonation’ and ‘physiological heterogeneity’ have already been discussed as important concepts for a functional understanding of the ectomycorrhizal symbiosis (Martin et al., 1992; Cairney & Burke, 1996; Timonen & Sen, 1998). These investigations are now extended with regard to gene expression. In addition to changes induced by developmental programmes, the apoplastic hexose concentration probably generates a signal that might induce heterogeneity in fungal activities in ectomycorrhizas.

It remains to be determined how such hexose gradients could be generated and maintained in symbiotic tissues (Fig. 6). In the apoplastic compartment of the plant–fungus interface, sucrose is hydrolysed into equimolar concentrations of glucose and fructose (Salzer & Hager, 1991; Schaeffer et al., 1995). Since the hexose concentration is mainly determined by fungal activity, and A. muscaria takes up glucose preferentially until its concentration drops below 0.5 mM (Wiese et al., 2000), an increased apoplastic fructose concentration (above 2 mM) is produced in the Hartig net. This would trigger the observed hexose-dependent fungal gene expression. Fructose withdrawal from the apoplastic space presumably takes place mainly within the innermost one or two layers of the fungal sheath, since fructose uptake by A. muscaria hyphae is rather efficient when the glucose concentration is below 0.5 mM. It is thus unlikely that hexose concentrations above the threshold of about 2 mM (which would result in an increased expression of AmMst1 and a repression of AmPAL) are present in the apoplast of the majority of fungal sheath hyphae.

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Figure 6. Putative model showing the spatial distribution of hexose uptake by fungal hyphae in ectomycorrhizas. Sucrose hydrolysis in the apoplast of the Hartig net results in high glucose and fructose concentrations. In this compartment, glucose is preferentially taken up since the uptake of fructose is inhibited (by glucose concentrations above 0.5 mM). In the innermost one or two layers of the fungal sheath glucose concentration is low due to efficient uptake by fungal hyphae of the Hartig net. Thus mainly fructose is taken up. In the apoplast of other layers of the fungal sheath, glucose as well as fructose concentrations are low due to the efficient hexose uptake by hyphae of the Hartig net and the inner layers of the sheath.

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Conclusions and future research

  1. Top of page
  2. Summary
  3. Introduction
  4. Impact of mycorrhiza formation on photosynthesis
  5. Carbohydrate allocation at the plant–fungus interface
  6. Generation of a strong carbohydrate sink in ectomycorrhizas
  7. Carbohydrates as signals that regulate fungal gene expression in pure culture and symbiosis
  8. Conclusions and future research
  9. Acknowledgements
  10. References

In ectomycorrhizal symbiosis, plants increase their photosynthetic capacity to meet the enhanced carbohydrate demand caused by the fungal partner. In addition to enhanced sucrose formation in leaves, three points of control can be envisaged that could limit the carbohydrate drain from the plant: sucrose export from the phloem (which is still not completely understood), sucrose hydrolysis at the plant–fungus interface, and competition for monosaccharide uptake between plant and fungal cells. From the data obtained so far it appears that competition for hexoses does not occur at the plant–fungus interface. Furthermore, the activitiy of the host acid invertase, which generates the carbohydrates available to the fungus, does not appear to be rate-limiting – at least in Norway spruce – A. muscaria ectomycorrhizas. Thus, control of the plant carbohydrate drain could involve sucrose download from the phloem.

The generation of a strong carbohydrate sink by the fungal hyphae is a crucial process to sustain the fungal hexose supply to symbiotic tissues. Insights as to how the fungal carbohydrate sink might be generated and regulated at the physiological level are now available. Nevertheless, the regulation, and the interaction between, fungal pathways of carbohydrate metabolism are still largely unknown.

In addition to symbiosis-related developmental cues, sugar availability and partitioning probably play a key role in the regulation of fungal activities in mycorrhizal tissues. The tissue-dependent expression of hexose-regulated AmMst1 and AmPAL genes in mycorrhizas is presumably only one example of the molecular mechanisms regulating the symbiosis development. However, the occurrence of a hexose gradient in the apoplastic space of the Hartig net (and the inner layers of the fungal sheath) will require further biochemical investigations.

Identification of genes that are identified on the basis of their differential expression between mycorrhizas and the nonmycorrhizal partners has increased our understanding of the regulation cascade taking place in the symbiosis. A further step will be the identification of regulatory sequences in the promoter of these genes and the identification of transcription factors regulating their expression. Furthermore, the biological significance of the identified factors has to be ascertained by the generation of mutants.

Currently, little is known about the processes controlling movements of carbohydrates between the different fungal symbiotic compartments (e.g. hyphae of the Hartig net and those of the sheath) and the long distance transport of carbohydrates in soil hyphae.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Impact of mycorrhiza formation on photosynthesis
  5. Carbohydrate allocation at the plant–fungus interface
  6. Generation of a strong carbohydrate sink in ectomycorrhizas
  7. Carbohydrates as signals that regulate fungal gene expression in pure culture and symbiosis
  8. Conclusions and future research
  9. Acknowledgements
  10. References

We would like to thank Venetia Karidaki, Anja Müller, Margret Ecke, Andrea Bock, and Ulrike Zeissler for their skillful assistance. Work carried out in our laboratory has been supported by research grants from the Deutsche Forschungsgemeinschaft and the German Federal Ministry for Education and Research (BMBF). Helpful comments from two anonymous reviewers are acknowledged.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Impact of mycorrhiza formation on photosynthesis
  5. Carbohydrate allocation at the plant–fungus interface
  6. Generation of a strong carbohydrate sink in ectomycorrhizas
  7. Carbohydrates as signals that regulate fungal gene expression in pure culture and symbiosis
  8. Conclusions and future research
  9. Acknowledgements
  10. References
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