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).
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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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:
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).
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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