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Review
Blackwell Science Ltd
Research review
Carbohydrate metabolism in
ectomycorrhizas: gene expression,
monosaccharide transport and
metabolic control
Author for correspondence:
Uwe Nehls
Tel: + 49 7071 297 7657
Fax: + 49 7071 295 635
Email: [email protected]
Uwe Nehls, Sabine Mikolajewski, Elisabeth Magel and Rüdiger Hampp
Universität Tübingen, Physiologische Ökologie der Pflanzen, Auf der Morgenstelle 1, D–72076
Tübingen, Germany
Received: 30 November 2000
Accepted: 1 March 2001
Summary
Key words: Amanita muscaria,
carbon allocation, cyclic AMP,
ectomycorrhiza, gene expression,
monosaccharide transporter, Picea
abies, Populus tremula × tremuloides.
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.
© New Phytologist (2001) 150: 533–541
Introduction
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;
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Martin et al., 1987). It is commonly accepted that there is
mutual benefit to the partners, due to the exchange of plantderived 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
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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
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
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.
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Fig. 1 Outline model describing the the carbohydrate allocation
at the plant –fungus interface. G6P, glucose-6-phosphate, F6P,
fructose-6-phosphate.
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 wallbound 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-fructofuranosidefructohydrolases (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).
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 plantderived carbohydrate supporting fungal growth in ectomycorrhiza.
Generation of a strong carbohydrate sink in
ectomycorrhizas
Hexose uptake
The driving force for carbohydrate allocation to the apoplast
is consumption at the sink site. In ectomycorrhizas this is
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Fig. 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).
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).
In yeast and other saprophytic fungi, several hexose
transporter genes are present in the genome (Jennings, 1995;
Boles & Hollenberg, 1997). Monosaccharide uptake of hyphae
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Fig. 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).
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).
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Fig. 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).
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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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-Omethyl-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 hexosedependent 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).
Carbohydrates as signals that regulate fungal
gene expression in pure culture and symbiosis
Sugar-dependent gene-repression in
pure fungal culture
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.
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
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
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Fig. 5 Transcription factor binding sites within
the AmPAL promoter. Radio-labelled PCRfragments, containing different parts of the
AmPAL 5′-non coding region (promoterregion), 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).
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-methylglucose, is phosphorylated by hexokinase (but not further
metabolized), it could be concluded that monosaccharidedependent 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 sugardependent gene repression in A. muscaria, a genomic DNAfragment, containing 1.5 kbp of the AmPAL-promoter region,
was isolated and sequenced (Mikolajewski, unpublished).
Promoter fragments of approx. 150 bp in length were PCRamplified 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).
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
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Fig. 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.
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.
© New Phytologist (2001) 150: 533 – 541 www.newphytologist.com
Conclusions and future research
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
539
NPH141.fm Page 540 Monday, April 30, 2001 11:21 AM
540 Review
Research review
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
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.
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