ectomycorrhiza carbohidrate
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Journal of Experimental Botany, Vol. 59, No. 5, pp. 10971108, 2008
doi:10.1093/jxb/erm334 Advance Access publication 13 February, 2008
SPECIAL ISSUE REVIEW PAPER
Mastering ectomycorrhizal symbiosis: the impact of
carbohydrates
Uwe Nehls*
Universitat Tubingen, Botanisches Institut, Physiologische Okologie der Pflanzen, Auf der Morgenstelle 1,D-72076 Tubingen, Germany
Received 7 August 2007; Revised 20 November 2007; Accepted 30 November 2007
Abstract
Mycorrhiza formation is the consequence of a mutualistic
interaction between certain soil fungi and plant roots
that helps to overcome nutritional limitations faced by
the respective partners. In symbiosis, fungi contribute to
tree nutrition by means of mineral weathering and
mobilization of nutrients from organic matter, and obtain
plant-derived carbohydrates as a response. Support with
easily degradable carbohydrates seems to be the driving
force for fungi to undergo this type of interaction. As
a consequence, the fungal hexose uptake capacity is
strongly increased in Hartig net hyphae of the model
fungi Amanita muscaria and Laccaria bicolor. Next to
fast carbohydrate uptake and metabolism, storage
carbohydrates are of special interest. In functional A.
muscaria ectomycorrhizas, expression and activity of
proteins involved in trehalose biosynthesis is mainly
localized in hyphae of the Hartig net, indicating an
important function of trehalose in generation of a strong
carbon sink by fungal hyphae. In symbiosis, fungal
partners receive up to ;19 times more carbohydrates
from their hosts than normal leakage of the root system
would cause, resulting in a strong carbohydrate de-
mand of infected roots and, as a consequence, a more
efficient plant photosynthesis. To avoid fungal parasit-
ism, the plant seems to have developed mechanisms to
control carbohydrate drain towards the fungal partner
and link it to the fungus-derived mineral nutrition. In this
contribution, current knowledge on fungal strategies toobtain carbohydrates from its host and plant strategies
to enable, but also to control and restrict (under certain
conditions), carbon transfer are summarized.
Key words: Carbohydrate metabolism, ectomycorrhiza, fungi,
soil, symbiosis, transport.
Introduction
In contrast to arbuscular mycorrhizal fungi, which are
obligate biotrophs, ectomycorrhizal (EM) fungi as well as
their host plants are not dependent on one another under
optimized nutritional conditions.However, in natural forest ecosystems, major nutrients
(nitrogen, phosphate) are fixed in the organic layer or are
contained in micro-organisms, and lower animals and
trees have only a limited access to this resource (Harley
and Smith, 1983; Smith and Read, 1997), making them
ecologically dependent on EM fungal partners.
Although litter and humus layers of forest soils are quite
rich in complex carbohydrates (e.g. cellulose and lignin),
most EM fungi (and a large proportion of other soil
microbes) seem to be dependent on simple, readily utilizable
carbohydrates which are contained in organisms but are rarein forest soils (Wainwright, 1993). The reason for this is that
EM fungi only have a limited capability to degrade complex
carbohydrates as a carbon source compared with wood and
litter decomposers (Colpaert and van Tichelen, 1996; Read
and Perez-Moreno, 2003). In contrast to the soil, plant root
exudates can be rich in simple carbohydrates. As EM fungal
hyphae cover tree fine roots (see below), they have a direct
and privileged access to root exudates. This and the fact that
ectomycorrhizal roots gain much more carbon than non-
mycorrhizal plant roots help EM fungi to overcome
carbohydrate limitation and increase their competitive
ability in soil (Smith and Read, 1997; Leake et al., 2001).
While both the fungus and the plant can profit from theinteraction, the question of who controls the relationship
has been discussed extensively. In the past, this debate
was based on ecological observations and experimental
modulation of environmental conditions. However, with
the power of molecular biological techniques, this debate
has recently been revived (Fitter, 2006). Different aspects
* E-mail: [email protected]
The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.For Permissions, please e-mail: [email protected]
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of symbiosis are often mixed in this discussion, including:
the degree of dependency of each partner on a successful
symbiosis (reflecting amongst others the current nutri-
tional state of plant and fungus, see above), the de-
velopmental control of the partners over the establishment
of interacting structures (which is still a black box), and
the exchange of nutrients and metabolites at the plant
fungus interface. The focus of this contribution will be onthe last point and target the impact of carbohydrates,
whose acquisition is presumably the main reason for the
fungal partner to enter symbiosis.
The ectomycorrhizal fungal colony
EM fungal mycelia can comprise up to 80% of the total
fungal biomass and 30% of the microbial biomass in
forest soils (Wallander et al., 2001; Hogberg andHogberg, 2002; Wallander, 2006) and are regarded as key
elements of forest ecosystem processes (e.g. nutrient
cycling and carbon entry; for recent reviews see Readet al., 2004; Anderson and Cairney, 2007). Ectomycor-rhizal mycelial biomass varies with soil depth and host
tree species, but seems to correlate with the distribution of
tree roots in the respective soil profile (Wallander et al.,2004; Goransson et al., 2006).
Soil-growing hyphae that explore litter or mineral layers
for nutrients constitute a large part of the EM fungal
colony (Fig. 1). In contrast to fast growing, saprophytic
model fungi such as Aspergillus or Neurospora, different
parts of the EM fungal colony remain functionally
interconnected (Cairney et al., 1991). In forests, certainEM fungi are spread over areas that are often several
square metres, implying that mycelia in the field can
extend for considerable distances and persist for several
years (Dahlberg and Stenlid, 1990; Baar et al., 1994;Dahlberg, 1997; Anderson et al., 1998). Size of and
interconnectivity within the colony seem to depend on thefungal capability to establish specialized long-distance
transport hyphae (cords or rhizomorphs, Fig. 1; reviewed
by Agerer, 2001). Species that remain non-rhizomorphic
are thought to have a limited ability to explore the
surrounding soil, while those that possess highly differen-
tiated rhizomorphs are regarded as more adapted to long-
distance exploration.
When soil-growing hyphae recognize an emerging fine
root of a plant partner, they direct their growth towards it
(Martin et al., 2001) and colonize the root surface, (often)forming a sheath or mantle of hyphae, which encloses the
root and isolates it from the surrounding soil (Fig. 1;
Blasius et al., 1986). Root hairs, which are normallyformed by rhizodermal cells, are suppressed by ectomy-
corrhiza formation. After or parallel to sheath formation,
fungal hyphae grow inside the infected fine root, forming
highly branched structures in the apoplast of the rhizoder-
mis or, in the case of gymnosperms in the entire root
cortex, the so-called Hartig net (Fig. 1; Kottke and
Oberwinkler, 1986).
Both fungal networks of ectomycorrhizas (fungal sheath
and Hartig net) have different functions (Harley and
Fig. 1. Scheme of an ectomycorrhizal fungal colony (without fruit bodies). Shown is a scheme of an ectomycorrhizal fungal colony (upper part) andphotographs of the respective fungal structures (lower part, from left to right: soil-growing hyphae, rhizomorph, ectomycorrhiza).
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Smith, 1983; Kottke and Oberwinkler, 1986; Smith and
Read, 1997). The Hartig net serves as an interface
between plant and fungus where cells are adapted to the
exchange of plant-derived carbohydrates for fungus-
derived nutrients. The function of the fungal sheath is that
of an intermediate storage for (i) nutrients that are
delivered by soil-growing hyphae and are intended for the
Hartig net and (ii) carbohydrates that are taken up byhyphae of the Hartig net and are designated for transport
towards the soil-growing mycelium (Jordy et al., 1998).
Fungal carbohydrate nutrition in symbiosis
One of the first attempts to assay carbon flow in a
mycorrhizal plant was performed by Melin and Nilsson
(1957). They showed that feeding [14C]CO2 to leaves
resulted in the appearance of labelled carbon in the hyphal
mantle within 1 d. These results were confirmed by
a number of researchers with different experimental
systems later on (Lewis and Harley, 1965b; Cairneyet al., 1989; Leake et al., 2001; Wu et al., 2002). Carbonallocation to EM fungi was estimated by different
investigators to be as much as 2025% of net primary
production (Soderstrom, 1992; Hogberg and Hogberg,
2002; Hobbie, 2006).Potential carbon compounds delivered by the plant
partner in symbiosis are soluble sugars, carboxylic acids,
and amino acids. Plant cell wall compounds such as
pectin, hemicellulose, cellulose, or proteins have also been
under discussion (for reviews see Harley and Smith, 1983;
Smith and Read, 1997). However, owing to the huge rates
of carbon consumption by the fungal partner, cell wall
compounds are not likely to constitute a major carbonsource. Additionally, even though there are indications
that certain EM fungi have some cell wall-degrading
activity, the degradation rate is too slow to meet the
fungal carbohydrate demand (Trojanowski et al., 1984;Haselwandter et al., 1990; Entry et al., 1991). Whengrown under axenic conditions, which, however, do not
necessarily reflect the situation in ectomycorrhizas, the
majority of EM fungi investigated so far grow best onsimple sugars such as glucose and fructose (Palmer and
Hacskaylo, 1970; Salzer and Hager, 1991). Therefore, it
seems likely that soluble sugars and organic acids are the
best candidates for plant-derived carbohydrates for fungal
nutrition in symbiosis.It is commonly accepted that sucrose, which is excreted
by plant root cells into the common apoplast of the plant
fungus interface, is hydrolysed by a plant-derived in-vertase in EM symbiosis (Lewis and Harley, 1965a;Salzer and Hager, 1991; Rieger et al., 1992; Schaeffer
et al., 1995; Nehls, 2004). The lack of an invertase in(at least many) EM fungi (Palmer and Hacskaylo, 1970;
Salzer and Hager, 1991; Hatakeyama and Ohmasa, 2004;
Daza et al., 2006) is a profound difference fromphytopathogenic (Walters et al., 1996; Voegele et al.,2001) and also ericoid mycorrhizal fungi (Straker et al.,1992; Hughes and Mitchell, 1996). In combination with
their low cell wall-degrading activity (compared with
wood- and litter-degrading but also ericoid mycorrhizal
fungi), lack of invertase activity makes EM fungi de-
pendent on the activity of the plant partner (Smith andRead, 1997), which might be essential for the function of
this type of symbiosis.
Compared with regular root exudation, which constitutes
;35% of carbon fixed in photosynthesis (Pinton et al.,2001), up to 2025% of net photosynthesis products are
transferred towards the fungus in ectomycorrhizal symbio-
sis (Soderstrom and Read, 1987; Soderstrom, 1992; Farrar
and Jones, 2000; Hogberg and Hogberg, 2002; Hobbie,
2006). Thus, plant fine roots lose 619 times more
carbohydrates in symbiosis (Bevege et al., 1975; Cairneyet al., 1989; Wu et al., 2002). Prerequisites for theformation of a strong fungal sink are the ability of Hartig
net hyphae to take up carbohydrates speedily from thecommon apoplast and convert them into fungal metabolites.
Sugar uptake
Two hexose transporter genes from Amanita muscaria(AmMST1, Nehls et al., 1998; AmMST2, Nehls, 2004) andone hexose transporter gene from Tuber borchii (Tbhxt1;Polidori et al., 2007) have been investigated from EMfungi so far. The hexose transporter proteins of both T.borchii and A. muscaria clearly favour glucose uptakeover that of fructose. However, while the T. borchii
protein revealed a KM value of;38 lM when heterolo-gously expressed in yeast (Polidori et al., 2007), the
A. muscaria protein showed a 10 times higher KM value(460 lM; Wiese et al., 2000). Tbhxt1 expression wasfurthermore enhanced by carbohydrate starvation of Tuberhyphae, while AmMST1/2 ( A. muscaria) expression wasenhanced by increased external sugar concentrations and
in ectomycorrhizas (Fig. 2; Nehls et al., 1998; Nehls,2004). It was thus concluded that Tbhxt1 is mainly
responsible for carbohydrate uptake by soil-growing
hyphae and the reduction of sugar leakage from hyphae
(Polidori et al., 2007), while the A. muscaria proteins areresponsible for carbohydrate uptake in symbiosis (Nehls,
2004).The sequenced genomes of ascomycetes contain ;20
functional sugar transporter genes (Saccharomyces cerevi-siae, Boles and Hollenberg, 1997; Aspergillus niger, Pelet al., 2007) and those of basidiomycetes (Laccariabicolor, ectomycorrhizal: Martin et al., 2004; Coprinopsiscinerea, saprotroph, http://www.broad.mit.edu/annotation/genome/coprinus_cinereus/Home.html; Phanerochaetechrysosporium, wood decaying: Martinez et al., 2004)
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;15 putative MST genes (UNehls, unpublished data). The
expression of a subset (six out of 15) of the identified
Laccaria bicolorgenes (belonging to different transportersubgroups) is mycorrhiza enhanced, while one gene
(revealing a generally very low transcript level) is sup-
pressed in symbiosis (UNehls, unpublished data). Three of
the genes that revealed elevated transcript levels in
ectomycorrhizas were specifically induced in symbiosis,
while a further three genes also showed an enhanced
expression in fruit bodies. Together with the two
functionally characterized A. muscaria hexose transportergenes, which also showed strongly enhanced expression
upon ectomycorrhiza formation (Nehls et al., 1998; Nehls,2004), the data from L. bicolor clearly suggest anenhanced fungal sugar uptake capacity in symbiosis.
In conclusion, even when the number of, as yet
characterized, hexose transporters from EM fungi is rather
small compared with the large number of potential sugar
transporter genes in their genomes, two important func-
tions are already visible: (i) sugar uptake by soil-growing
hyphae and avoidance of monosaccharide leakage under
carbohydrate starvation; and (ii) enhanced sugar uptake in
symbiosis.
Fungal sugar metabolism
According to investigations by nuclear magnetic resonance
(NMR; Martin et al., 1998) and biochemical techniques(Hampp et al., 1995), hexoses that are imported by fungalhyphae are utilized for ATP generation, amino acid
biosynthesis, and the formation of (potential) carbohydrate
storage compounds. For the maintenance of a strong carbon
sink in symbiosis, monosaccharides have to be quickly
converted into fungal metabolites, by (i) increased carbon
flux through glycolysis and/or (ii) the generation of fungal
storage compounds.
Increased flux rates through fungal glycolysis and the
tricarboxylic acid cycle (TCA) are indicated by large-scale
expression analysis of established Pisolithus microcarpus/ Eucalyptus globulus ectomycorrhizas (Duplessis et al.,2005) and supported by investigations of enzyme activi-
ties and metabolic regulator content. The enzyme phos-
phofructokinase performs the rate-limiting step in EM
fungal glycolysis (Kowallik et al., 1998). In A. muscaria,this enzyme is activated by fructose 2,6-bisphosphate
(F26BP), and symbiotic A. muscaria hyphae have in-creased amounts of F26BP (Schaeffer et al., 1996),resulting in an increased glycolytic flux in hyphae at the
plantfungus interface. In contrast to data from Pisolithusand A. muscaria, ectomycorrhizas formed by Paxillusinvolutus showed slightly reduced expression of genesinvolved in sugar uptake and glycolysis (Le Quere et al.,2005; Wright et al., 2005). This could either indicatespecies-specific differences of EM fungi upon ectomycor-
rhiza formation or insufficient experimental data. Analysis
of gene expression up to now has covered only a limited
number of fungal genes. Furthermore, protein activity is
not necessarily reflected by gene expression. Thus,
expression data of whole pathways together with analysisof protein function and metabolite content of further EM
fungi are necessary to draw sound conclusions.
Two different pools of potential storage carbohydrates
can be distinguished in fungi: (i) short chain carbohyd-
rates such as trehalose and polyols; and (ii) the long chain
carbohydrate glycogen.
When grown in the presence of a rich carbon source but
also in functional ectomycorrhizas, EM fungi produce
Fig. 2. Increased expression of plant and fungal hexose importer genes at the plantfungus interface in functional A. muscaria/poplarectomycorrhizas. Total RNA was isolated from non-mycorrhizal poplar fine roots (FR), non-mycorrhizal fungal hyphae (H), as well as A. muscaria/poplar ectomycorrhizas (M). Expression analysis was performed by northern blot ( A. muscaria hexose transporter gene AmMST1; Nehls et al., 1998)or quantitative RT-PCR (poplar hexose transporter genes PttMST1.2, PttMST2.1, and PttMST3.1; Grunze et al., 2004; Nehls et al., 2007). Theexpression of the fungal as well as all three root-expressed poplar sugar transporter genes was enhanced in functional ectomycorrhizas, indicating anincreased hexose uptake capacity of both partners.
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a series of sugars and sugar alcohols (mannitol, arabitol,
erythritol) among which trehalose and mannitol dominate
(Table 1). Amanita muscaria growth in axenic culture onglucose as a carbon source results in an increase in
trehalose content over time, which is later continuously
depleted again under glucose starvation (Wallenda, 1996).
Furthermore, trehalose dominates in the most intense
plantfungus interaction zone of functional mycorrhizas,indicating a conversion of glucose into this compound
(Rieger et al., 1992). In accordance with this, trehalosebiosynthesis occurs mainly in hyphae of the plantfungus
interface and is strongly increased upon ectomycorrhiza
formation (Fig. 3; Fajardo Lopez et al., 2007). Together,these data indicate that in A. muscaria trehalose mightfunction as a storage carbohydrate. In contrast to
trehalose, mannitol is rarely found in A. muscaria. Onlyfruiting bodies contain larger mannitol concentrations
(Wallenda, 1996). With the exception of Hygrophoruspustulatus, trehalose is clearly dominant in substratemycelia, ectomycorrhizas, and fruit bodies of investigated
ectomycorrhizal basidiomycetes belonging to the orderAgaricales (Table 1). However, in the non-mycorrhizal
fungus Agaricus bisporus (belonging to same order),mannitol is found in higher concentrations than trehalose
in hyphae grown on straw and fruiting bodies. The
situation is even more puzzling in the order Boletales,
where in relatively closely related EM fungi sometimes
mannitol and sometimes trehalose dominates. Members of
other orders of ectomycorrhizal basidiomycetes are only
rarely investigated and here trehalose or mannitol could
dominate. For ascomycotic EM fungi the situation seems
to be clearer. Mannitol (sometimes also arabitol) is clearly
dominant in substrate mycelia and mycorrhizas, while
trehalose is rarely found (Table 1). However, in hyphae ofthe non-mycorrhizal ascomycete Cordyceps bassiana,trehalose dominates. Together with the small number
of species of ascomycotic EM fungi investigated, this
might indicate a similar situation to that in basidiomycetes.
With the exception of a few models, the functional
interpretation of published data is generally difficult for
most of the investigated EM fungi due to missing
knowledge about fungal physiology. It is further compli-
cated by the fact that trehalose (for a review see Crowe,
2007) and polyols (for a review see Solomon et al., 2007)could act as osmo-, cryo-, heat, and drought protectants
and as scavengers for radical oxygen species in certain
fungi, in addition to their postulated function in carbohyd-rate storage and distribution (see below). Thus, in future
more detailed investigations on fungal physiology to-
gether with manipulation of carbon metabolism in model
fungi are necessary.
In Lactarius, glycogen content is high during winter,declines due to strong fungal propagation until summer,
and is restored during autumn (Genet et al., 2000).In contrast, short-term (up to 3 weeks) exposure of
A. muscaria hyphae in the presence or absence of acarbon source had nearly no effect on glycogen content
(Wallenda, 1996). Nevertheless, Jordy et al. (1998)observed changes in glycogen content and localization
during ectomycorrhiza formation. While glycogen was
initially found in Hartig net hyphae, it was later (in
agreement with a potential function in long-term storage)
observed exclusively in hyphae of the fungal sheath. Theinitial occurrence of glycogen in Hartig net hyphae could,
however (like temporal starch formation in leaves), be
interpreted as a flux control mechanism. When carbohyd-
rate import into hyphae exceeds its export to other parts of
the fungal colony, long-term storage pools are filled to
ensure continuous fungal carbohydrate sink strength in
symbiosis. Together these data thus indicate a potential
function of glycogen (like starch in plants) in long-term
carbohydrate storage, which is usually only mobilized
when the short-term pools (e.g. trehalose and polyols) are
empty.
Because EM fungi commonly form an extensive
external mycelium, and soil-growing hyphae are depen-dent on carbohydrate support by mycorrhizas (Cairney
and Burke, 1996; Leake et al., 2001), long-distancetransport of carbon is of great importance for fungal
physiology. Since glycogen is stored in the cytoplasm as
large, non-mobile granules, it is rather unlikely that it
(similar to starch in plants) serves as the long-distance
carbohydrate transport form in fungi. In contrast, polyols
and trehalose are present in large quantities in fungal
hyphae and are (like sucrose in plants) quite mobile,
making them good candidates for long-distance transport
carbohydrates. However, further investigations, includ-
ing the generation of mutants in certain pathways, are
necessary to support this hypothesis.
Carbohydrates as a signal for the regulation offungal physiology in ectomycorrhizas
Sugar-dependent regulation of gene expression was in-
vestigated in functional A. muscaria ectomycorrhizasusing hexose importer genes (Nehls et al., 1998; Nehls,2004) and a phenylalanine ammonium lyase (AmPAL;
Nehls et al., 1999). In axenic culture, the expression ofthese genes is regulated by a threshold response mecha-
nism dependent on the extracellular monosaccharide
concentration (Nehls et al., 2001a). In functional ectomy-corrhizas, AmPAL was only detectable in hyphae of the
fungal sheath, while elevated hexose transporter gene
expression was exclusively observed in hyphae of theHartig net (Nehls et al., 2001a). Owing to the opposing(sugar-dependent) regulation of these genes in both
hyphal networks, hexose (glucose or fructose) concen-
trations >2 mM can be expected in the apoplast of the
root region containing the Hartig net, while lower
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concentrations are probably present in the apoplast of
hyphae of the fungal sheath (Nehls et al., 2001b).Basidiomycotic EM fungi (Salzer and Hager, 1993;
Stulten et al., 1995; Nehls et al., 2001b) and also mostinvestigated plant monosaccharide transporters (Buttner
and Sauer, 2000) strongly favour glucose over fructose
import. Since sucrose is hydrolysed at the plantfungus
interface into equimolar amounts of glucose and fructose,preferential glucose uptake would result in increased
apoplastic fructose concentrations, which could trigger
fungal physiology in symbiosis (Fig. 4; Nehls, 2004; see
below).
Metabolic zonation and physiological heterogeneity
have been discussed as important concepts for a functional
understanding of EM symbiosis (Cairney and Burke,
1996; Timonen and Sen, 1998). Differences in the
apoplastic hexose concentration at the Hartig net versus
the fungal sheath could be a signal that regulates fungalphysiological heterogeneity in ectomycorrhizas (Nehls
et al., 2001b; Nehls, 2004). To investigate the generaleffect of the external sugar concentration on A. muscariagene expression, microarray hybridization (800 tentative
genes) was performed with probes from mycelial samples
grown under axenic conditions with either 1 mM or
25 mM glucose as carbon source. For ;5% of the
investigated genes an at least 2-fold difference in gene
expression was observed (Nehls et al., 2007; UNehls,unpublished data). This indicates that (for A. muscaria)sugar-dependent regulation of fungal gene expression
caused by differences in the apoplastic hexose concentra-
tion at the plantfungus interface versus the fungal sheathmay explain some of the local adaptations of fungal
physiology in functional ectomycorrhizas.
In yeast and filamentous ascomycetes the external sugar
concentration is sensed by monosaccharide transporter-
like proteins that regulate sugar-dependent gene induction.In addition to external sugar sensors, monosaccharide-
dependent gene repression is regulated via a hexokinase-
dependent signalling pathway (Ozcan and Johnston,
1995). As in ascomycetes, hexokinase obviously acts as
Fig. 3. Expression of fungal ( A. muscaria) genes encoding proteinsinvolved in trehalose biosynthesis in sheath and Hartig net hyphae offunctional ectomycorrhizas (Fajardo Lopez et al., 2007). To comparegene expression in both hyphal networks, functional ectomycorrhizaswere dissected into fungal sheath (Sheath) and the remaining fine rootcontaining Hartig net hyphae (HN). RNA was isolated and quantitativeRT-PCR was performed from first-strand cDNA using gene-specificprimers for trehalose-6-phosphate synthetase (AmTPS), trehalose-6-phosphate phosphatase (AmTPP), and trehalose phosphorylase (AmTP).Calibration of the mRNA content was performed using gene-specificprimers of the constitutively expressed fungal reference SCIV038 (Nehlset al., 2001a).
Fig. 4. Model for sugar-regulated adaptation of fungal physiology inectomycorrhizas (modified according to Nehls et al., 2001b andreproduced by kind permission of Wiley-Blackwell Publishing Ltd.)Sucrose hydrolysis into equimolar amounts of glucose and fructosetogether with preferred glucose uptake by plant and fungal cells of theHartig net result in enhanced fructose concentrations in the correspondingapoplast. Fructose is supposed to be taken up by hyphae of the outer layerof the Hartig net and (perhaps) the first layer of the fungal sheath,resulting in a low apoplastic hexose concentration for most fungal sheathhyphae. This scenario would result in a steep hexose gradient between theapoplasts of the fungal sheath and the Hartig net, and could explain sugar-dependent fine-tuning of fungal physiology as observed for A. muscaria.
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a regulator of sugar-dependent gene repression in the EM
fungus A. muscaria (Nehls et al., 1999). However, incontrast to ascomycetes, the sugar-dependent induction of
A. muscaria sugar transporter genes seems to be regulatedby an internal sensor located either further downstream
from hexokinase in glycolysis or in storage carbohydrate
biosynthesis (Nehls et al., 1998, 2001a; Nehls, 2004).
In contrast to the situation in A. muscaria, mycorrhiza-induced sugar transporter gene expression was not regu-lated in a sugar-dependent manner in L. bicolor (UNehls,unpublished data). However, also in A. muscaria, genesinvolved in sugar metabolism (e.g. trehalose biosynthesis)
are regulated in a sugar-independent manner (Fajardo
Lopez et al., 2007). Thus, the extent of fine-tuning of EMfungal physiology by sugar regulation might be species
dependent and has to be further addressed in the future.
The host plant
McDowell et al. (2001) report that 4759% of plantphotosynthates are allocated to ectomycorrhizas, and
several authors estimate that 2025% of the net photosyn-
thesis rate is used for fungal support (Soderstrom and
Read, 1987; Soderstrom, 1992; Farrar and Jones, 2000;
Hogberg and Hogberg, 2002; Hobbie, 2006). Together
these data indicate, that (i) both the fungus and infected
roots consume similar amounts of fine root-allocated
carbohydrates and (ii) carbohydrate uptake by both
partners must be strongly increased in symbiosis. Inagreement with this, sugar transporter gene expression is
significantly enhanced in fungal hyphae (see above) and
root cells (see below, Fig. 2). In response to the huge
carbohydrate drain in symbiosis, the plant can (i) increaseits photosynthetic efficiency and (ii) control carbohydrate
loss toward the fungus to avoid fungal parasitism.
Increased host photosynthesis in EM symbiosis
A constant sugar supply towards the fungus by the host
plant is essential for mycorrhizal functioning and seems to
be tightly coupled to photosynthetic activity (Hogberg
et al., 2002; van Hees et al., 2005). Direct and indirectobservations indicate that mycorrhization can up-regulate
the net photosynthesis rate of the host plant to meet the
increased carbohydrate demand in symbiosis. Vodnik and
Gogala (1994) found increased chlorophyll and carotenoidcontents in needles of spruce seedlings mycorrhized with
different EM fungi. Mycorrhized Castanea sativa plantshave decreased respiration rates, resulting in a lower CO2compensation point and an increased amount of ribulose
bisphosphate carboxylase (Martins et al., 1997). Gasexchange measurements revealed an increased CO2fixation rate for mycorrhized Norway spruce, poplar, and
birch (Friedrich, 1998; Loewe et al., 2000; Wright et al.,
2000). Furthermore, Lamhamedi et al. (1994) demon-strated that the removal of L. bicolor fruiting bodies,which form a huge extra carbon sink generated by EM
fungi, resulted in a rapid decrease in net photosynthesis of
host plants.
Control of local carbohydrate loss by plant roots
The first hints of a control of carbon drain towards the
fungal partner in symbiosis came from fertilization experi-
ments, showing that increased soil nitrogen availability is
often associated with decreased production of extraradical
hyphae, reduced colonization intensity of fine roots, and
decreased fruiting body formation by EM fungi (Wallenda
and Kottke, 1998; Nilsson and Wallander, 2003; Nilsson
et al., 2005; Hendricks et al., 2006). Decreased fungalgrowth rates under elevated soil nitrogen content indicate
a reduction in the carbohydrate flux towards the fungus,
caused by a diminished dependency of the host plant on
the fungal partner.The prevention of a carbohydrate drain from the plant
towards the fungus can occur at several levels, including
(i) the control of sucrose export into the common apoplast
(that is still largely unknown); (ii) the control of sucrose
hydrolysis, and (iii) competition between root cells and
fungal hyphae for hexoses present in the apoplast of the
root region containing the Hartig net.
Root exudation of non-charged molecules (sucrose,
hexoses) occurs mainly passively, aided by the concentra-
tion gradient between plant cells and the apoplast. This
makes it difficult for plants to exert direct control over
many components of their C efflux (Jones and Darrah,
1996), but sugar re-import by root cells can be directly up-regulated to help alleviate stress (Jones et al., 2004). Thus,root sink strength, rates of local sucrose hydrolysis, and
hexose re-uptake by root cells are thought to regulate
fungal carbohydrate nutrition in ectomycorrhizas.
Apoplastic sucrose hydrolysis by plant-derived cell wall
acid invertases is a prerequisite for fungal carbohydrate
support in ectomycorrhizal (Salzer and Hager, 1991;
Salzer and Hager, 1993; Schaeffer et al., 1995) andarbuscular (Wright et al., 1998; Schaarschmidt et al.,2006) mycorrhiza symbiosis. While acid invertase activity
seems not to be a rate-limiting step in Medicagotruncatula (arbuscular mycorrhiza symbiosis; Schaarsch-
midt et al., 2007) and Norway spruce (ectomycorrhiza;Schaeffer et al., 1995), enzyme activity is increased uponectomycorrhizal symbiosis in Betula (Wright et al., 2000)and poplar (UNehls, unpublished data). This indicates that
invertase activity might be a checkpoint for fungal
carbohydrate support in some plants.
In A. muscaria/ P. tremula3tremuloides ectomycorrhi-zas, expression of monosaccharide importer genes of both
fungal and plant origin is strongly enhanced compared
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with non-mycorrhizal fine roots (Fig. 2). Three out of
a total of 24 potential monosaccharide transporter genes
present in the poplar genome (Tuskan et al., 2006) are up-regulated by ectomycorrhiza formation (Grunze et al.,2004; Nehls et al., 2007; UNehls, unpublished data), andone of them (PttMST3.1) is at least 10 times more highlyexpressed in roots than any other hexose transporter gene.
As a consequence, intense competition for apoplastichexoses can be supposed to occur at the plantfungus
interface. This raises the question of how the observed net
transfer of large amounts of carbohydrates towards the
fungus can occur under these conditions. A hint of how
the increased plant hexose transporter gene expression and
an efficient fungal sugar support could fit together came
from attempts to express the poplar sugar transporter
PttMST3.1 in yeast or Xenopus laevis oocytes. Asheterologous expression of the entire cDNA failed in both
experimental systems, Grunze et al. (2004) speculated thatthis transporter may also be regulated at the post-trans-
lational level (e.g. by phosphorylation). Indeed, potential
phosphorylation sites are present in the deducedPttMST3.1 protein sequence (Grunze, 2004). This feed-
back control mechanism would allow a fine-tuning of the
activation status of selected transporters in root cells as
a reaction to the amount of nutrients delivered by the
fungus. If the fungus provides sufficient nutrients, the
activity of PttMST3.1 would be shut off, while the protein
would be activated as soon as the nutrient transfer is
insufficient (Fig. 5). To test this hypothesis, transgenic
poplar plants were generated that overexpress an addi-
tional hexose importer gene under conditions that cannot
be controlled by the plant (UNehls, unpublished data). In
agreement with the assumption by Grunze et al. (2004),these transgenic plants reveal a reduced mycorrhizalinfection capability and an abnormal termination of the
symbiosis (UNehls, unpublished data).
Summary and outlook
Together with their fungal partners, root systems of EM
forest trees receive about half of the photosynthetically fixed
carbon. This carbohydrate demand is the consequence of
a plant nutritional strategy that is in large part dependent on
microbial partners. On the other hand, plant-derived carbo-
hydrate input into EM fungi is driving a microbial-based
recycling machinery which is essential for the establishment
of forest ecosystems and sustainable tree growth.
To enable a strong carbohydrate sink at the plant
fungus interface, most investigated model fungi increase
monosaccharide uptake capacity and carbohydrate flux
through glycolysis and the TCA cycle (energy production
for transport and metabolic processes). Furthermore,
increased contents of trehalose and/or mannitol in func-tional ectomycorrhizas indicate that these compounds are
potential intermediate carbohydrate stores, which are
perhaps also involved in long-distance transport of carbon
within the fungal colony. The relevance of trehalose and
polyols for fungi is, however, controversially discussed.
Only in some ascomycotic fungi (basidiomycotic fungi
are only rarely investigated) could a function for these
compounds as carbohydrate storage be confirmed by
genetic approaches. The situation is further complicated
by the fact that trehalose and mannitol could act as stress
protectants in some fungi. Thus, genetic approaches to
verify the particular function of these compounds in EM
fungi are necessary.Regulation and fine-tuning of fungal physiology in EM
fungal networks seems to be controlled by developmental
mechanisms and sugars. To what extent sugars play a role
in gene regulation seems to be dependent on the fungal
species. However, further EM fungi have to be investi-
gated to draw a more general picture.
Host trees increase their photosynthetic capacity in
symbiosis, but indications were found that they also control
and restrict carbohydrate flux towards their partners to avoid
fungal parasitism. The mechanisms by which this occurs arestill unclear, and further investigations are needed.
Acknowledgements
The author is indebted to Dr Nina Grunze and Dr Sylvia Schrey forcritical reading of the manuscript. This work was financed by theGerman Science Foundation (DFG).
References
Agerer R. 2001. Exploration types of ectomycorrhizae. Mycorrhiza11, 107114.
Fig. 5. Model of local control of sugar and nutrient transfer at theplantfungus interface. Shown are supposed sugar (hexoses) and
nutrient fluxes between plant and fungal cells at the Hartig net. Hexoses(generated from sucrose hydrolysis by plant-derived acid invertases)could be taken up by plant or fungal cells through monosaccharideimporters (circles). Fungal nutrient exporters are represented bya hexagon, and plant nutrient importers by a triangle. Inactive proteinsare shown as filled symbols, and active proteins as open symbols. Aslong as the fungus is supporting plant cells with sufficient nutrients, thefunction of plant hexose importers is mainly suppressed and a largehexose portion is imported by fungal cells. However, diminishednutrient export by fungal hyphae results in a strongly increased hexoseimport into plant cells as long as the fungus does not increase itsnutrient export again.
Mastering ectomycorrhizal symbiosis 1105
by on 7 June 2009http://jxb.oxfordjournals.orgDownloaded from
http://jxb.oxfordjournals.org/http://jxb.oxfordjournals.org/http://jxb.oxfordjournals.org/http://jxb.oxfordjournals.org/ -
8/4/2019 ectomycorrhiza carbohidrate
10/12
-
8/4/2019 ectomycorrhiza carbohidrate
11/12
Jones DL, Hodge A, Kuzyakov Y. 2004. Plant and mycorrhizalregulation of rhizodeposition. New Phytologist 163, 459480.
Jordy MN, Azemar LS, Brun A, Botton B, Pargney JC. 1998.Cytolocalization of glycogen, starch, and other insoluble poly-saccharides during ontogeny of Paxillus involutus/ Betula pendulaectomycorrhizas. New Phytologist 140, 331341.
Kitamoto Y, Gruen HE. 1976. Distribution of cellular carbohyd-rates during development of the mycelium and fruit bodies of Flammulina velutipes. Plant Physiology 58, 485491.
Koide RT, Shumway DL, Stevens CM. 2000. Soluble carbohyd-rates of red pine (Pinus resinosa) mycorrhizas and mycorrhizalfungi. Mycological Research 104, 834840.
Kottke I, Oberwinkler F. 1986. Mycorrhiza of forest treesstructure and function. Trees 1, 124.
Kowallik W, Thiemann M, Huang Y, Mutumba G,Beermann L, Broer D, Grotjohann N. 1998. Completesequence of glycolytic enzymes in the mycorrhizal basidiomy-
cete, Suillus bovinus. Zeitschrift fur Naturforschung 53, 818827.Lamhamedi MS, Godbout C, Fortin JA. 1994. Dependence of
Laccaria bicolorbasidiome development on current photosynthe-sis of Pinus strobes seedlings. Canadian Journal of ForestResearch 24, 17971804.
Leake JR, Donnelly DP, Saunders EM, Boddy L, Read DJ.2001. Rates and quantities of carbon flux to ectomycorrhizal
mycelium following14
C pulse labeling of Pinus sylvestrisseedlings: effects of litter patches and interaction with a wood-decomposer fungus. Tree Physiology 21, 7182.
Le Quere A, Wright D, Soederstroem B, Tunlid A,Johansson T. 2005. Global patterns of gene regulation associatedwith the development of ectomycorrhiza between birch (Betulapendula Roth.) and Paxillus involutus (Batsch) Fr. Molecular PlantMicrobe Interactions 18, 659673.
Lewis DH, Harley JL. 1965a. Carbohydrate physiology ofmycorrhizal roots of beech. I. Identity of endogenous sugars and
utilization of exogenous sugars. New Phytologist 64, 224237.Lewis DH, Harley JL. 1965b. Carbohydrate physiology of
mycorrhizal roots of beech. III. Movement of sugars betweenhost and fungus. New Phytologist 64, 265275.
Loewe A, Einig W, Shi L, Dizengremel P, Hampp R. 2000.
Mycorrhiza formation and elevated CO2 both increase thecapacity for sucrose synthesis in source leaves of spruce and
aspen. New Phytologist 145, 565574.Martin F, Boiffin VV, Pfeffer PE. 1998. Carbohydrate and amino
acid metabolism in the Eucalyptus globulus Pisolithus tinctoriusectomycorrhiza during glucose utilization. Plant Physiology 118,627635.
Martin F, Duplessis S, Ditengou F, Lagrange H, Voiblet C,Lapeyrie F. 2001. Developmental cross talking in the ectomycor-rhizal symbiosis: signals and communication genes. New Phytol-ogist 151, 145154.
Martin F, Ramstedt M, Soderhall K, Canet D. 1988. Carbohyd-rate and amino acid metabolism in the ectomycorrhizal ascomy-
cete Sphaerosporella brunnea during glucose utilization: a 13CNMR study. Plant Physiology 86, 935940.
Martin F, Tuskan GA, DiFazio SP, Lammers P, Newcombe G,Podila GK. 2004. Symbiotic sequencing for the Populusmesocosm. New Phytologist 161, 330335.
Martinez D, Larrondo LF, Putnam N, Gelpke MDS, Huang KM,Chapman J, Helfenbein KG, Ramaiya P, Detter JC,Larimer F. 2004. Genome sequence of the lignocellulose degrad-ing fungus Phanerochaete chrysosporium strain RP 78. Nature
Biotechnology 22, 695700.Martins A, Cisimiro A, Pais MS. 1997. Influence of mycorrhiza-
tion on physiological parameters of micropropagated Castaneasativa Mill. plants. Mycorrhiza 7, 161165.
McDowell NG, Balster NJ, Marshall JD. 2001. Below-groundcarbon allocation of Rocky Mountain Douglas-fir. CanadianJournal of Forest Research 31, 14251436.
Melin E, Nilsson H. 1957. Transport of C14
-labelled photosynthate
to the fungal associate of pine mycorrhiza. Svensk BotaniskTidskrift 51, 166186.
Nehls U. 2004. Carbohydrates and nitrogen: nutrients and signalsin ectomycorrhizas. In: Varma A, Abbott L, Werner D, Hampp R,
eds. Plant surface microbiology. Berlin: Springer Verlag,
373392.Nehls U, Bock A, Ecke M, Hampp R. 2001a. Differential
expression of hexose-regulated fungal genes within Amanitamuscaria/ Populus tremula3tremuloides ectomycorrhizas. New
Phytologist 150, 583589.Nehls U, Ecke M, Hampp R. 1999. Sugar- and nitrogen-dependent
regulation of an Amanita muscaria phenylalanine ammoniumlyase gene. Journal of Bacteriology 181, 19311933.
Nehls U, Grunze N, Willmann M, Reich M, Kuster H. 2007.Sugar for my honey: carbohydrate partitioning in ectomycorrhizal
symbiosis. Phytochemistry 68, 8291.Nehls U, Mikolajewski S, Magel E, Hampp R. 2001b. The role of
carbohydrates in ectomycorrhizal functioning: gene expression
and metabolic control. New Phytologist 150, 533541.Nehls U, Wiese J, Guttenberger M, Hampp R. 1998. Carbon
allocation in ectomycorrhizas: identification and expressionanalysis of an Amanita muscaria monosaccharide transporter. Molecular PlantMicrobe Interactions 11, 167176.
Nilsson LO, Giesler R, Baath E, Wallander H. 2005. Growth andbiomass of mycorrhizal mycelia in coniferous forests along short
natural nutrient gradients. New Phytologist 165, 613622.Nilsson LO, Wallander H. 2003. Production of external mycelium
by ectomycorrhizal fungi in a Norway spruce forest was
reduced in response to nitrogen fertilization. New Phytologist158, 409416.
O zcan S, Johnston M. 1995. Three different regulatory mecha-nisms enable yeast hexose transporter (HXT) genes to be induced
by different levels of glucose. Molecular and Cellular Biology15, 15641572.
Palmer JG, Hacskaylo E. 1970. Ectomycorrhizal fungi in pure
culture I.. Physiologia Plantarum 23, 11871197.Pel HJ, de Winde JH, Archer DB, et al . 2007. Genome
sequencing and analysis of the versatile cell factory Aspergillusniger CBS 513.88. Nature Biotechnology 25, 221231.
Pinton R, Varanini Z, Nannipieri P. 2001. The rhizosphere. NewYork: Marcel Dekker Inc.
Polidori E, Ceccaroli P, Saltarelli R, Guescini M, Menotta M,Agostini D, Palma F, Stocchi V. 2007. Hexose uptake in theplant symbiotic ascomycete Tuber borchii Vittadini: biochemicalfeatures and expression pattern of the transporter TBHXT1.Fungal Genetics and Biology 44, 187198.
Rangel-Castro JI, Danell E, Pfeffer PE. 2002. A13
C-NMR study
of exudation and storage of carbohydrates and amino acids in the
ectomycorrhizal edible mushroom Cantharellus cibarius. Myco-logia 94, 190199.
Rast D. 1965. Zur Stoffwechselphysiologischen Bedeutung vonMannit und Trehalose in Agaricus bisporus. Planta 64, 8193.
Read DJ, Leake JR, Perez-Moreno J. 2004. Mycorrhizal fungi asdrivers of ecosystem processes in heathland and boreal forest
biomes. Canadian Journal of Botany 82, 12431263.Read DJ, Perez-Moreno J. 2003. Mycorrhizas and nutrient
cycling in ecosystemsa journey towards relevance? NewPhytologist 157, 475492.
Rieger A, Guttenberger M, Hampp R. 1992. Soluble carbohyd-rates in mycorrhized and non-mycorrhized fine roots of spruce
seedlings. Zeitschrift fur Naturforschung 47, 201204.
Mastering ectomycorrhizal symbiosis 1107
by on 7 June 2009http://jxb.oxfordjournals.orgDownloaded from
http://jxb.oxfordjournals.org/http://jxb.oxfordjournals.org/http://jxb.oxfordjournals.org/http://jxb.oxfordjournals.org/ -
8/4/2019 ectomycorrhiza carbohidrate
12/12
Salzer P, Hager A. 1991. Sucrose utilization of the ectomycor-rhizal fungi Amanita muscaria and Hebeloma crustuliniformedepends on the cell wall-bound invertase activity of their host Picea abies. Botanica Acta 104, 439445.
Salzer P, Hager A. 1993. Characterization of wall-bound invertaseisoforms of Picea abies cells and regulation by ectomycorrhizalfungi. Physiologia Plantarum 88, 5259.
Schaeffer C, Johann P, Nehls U, Hampp R. 1996. Evidence foran up-regulation of the host and a down-regulation of the fungal
phosphofructokinase activity in ectomycorrhizas of Norwayspruce and fly agaric. New Phytologist 134, 697702.
Schaeffer C, Wallenda T, Guttenberger M, Hampp R. 1995.Acid invertase in mycorrhizal and non-mycorrhizal roots ofNorway spruce (Picea abies [L.] Karst.) seedlings. New Phytol-ogist 129, 417424.
Schaarschmidt S, Roitsch T, Hause B. 2006. Arbuscularmycorrhiza induces gene expression of the apoplastic invertaseLIN6 in tomato ( Lycopersicon esculentum) roots. Journal of Experimental Botany 57, 40154023.
Schaarschmidt S, Gonzalez MC, Roitsch T, Strack D,Sonnewald U, Hause B. 2007. Regulation of arbuscular mycorrh-ization by carbon. The symbiotic interaction cannot be improvedby increased carbon availability accomplished by root-specificallyenhanced invertase activity. Plant Physiology 143, 18271840.
Smith SE, Read DJ. 1997. Mycorrhizal symbiosis. London:Academic Press.
Soderstrom B. 1992. The ecological potential of the ectomycor-rhizal mycelium. In: Read DJ, Lewis DH, Fitter AH, AlexanderIJ, eds. Mycorrhizas in ecosystems. Wallingford, UK: CABInternational, 7783.
Soderstrom B, Finlay RD, Read DJ. 1988. The structure andfunction of the vegetative mycelium of ectomycorrhizal plants IV.Qualitative analysis of carbohydrate contents of myceliuminterconnecting host plants. New Phytologist 109, 163166.
Soderstrom BE, Read DJ. 1987. Respiratory activity of intact andexcised ectomycorrhizal mycelial systems growing in unsterilizedsoil. Soil Biology and Biochemistry 11, 231237.
Solomon PS, Waters ODC, Oliver RP. 2007. Decoding the mannitolenigma in filamentous fungi. Trends in Microbiology 15, 257262.
Straker CJ, Schnippenkoetter WH, Lemoine M-C. 1992.Analysis of acid invertase and comparison with acid phosphatasein the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read)Korf and Kernan. Mycorrhiza 2, 6367.
Stulten C, Kong F, X, Hampp R. 1995. Isolation and regenerationof protoplasts from the ectomycorrhizal ascomycete Cenococcumgeophilum Fr. Mycorrhiza 5, 259266.
Tibbett M, Sanders FE, Cairney JWG. 2002. Low-temperature-induced changes in trehalose, mannitol and arabitol associatedwith enhanced tolerance to freezing in ectomycorrhizal basidio-mycetes (Hebeloma spp.). Mycorrhiza 12, 249255.
Timonen S, Sen R. 1998. Heterogeneity of fungal and plantenzyme expression in intact Scots pineSuillus bovinus and Paxillus involutus mycorrhizospheres developed in natural foresthumus. New Phytologist 138, 355366.
Trojanowski J, Haider K, Hu ttermann A. 1984. Decomposition
of14
C-labelled lignin, holocellulose and lignocellulose bymycorrhizal fungi. Archives of Microbiology 139, 202206.
Tuskan GA, Difazio S, Jansson S, et al. 2006. The genome ofblack cottonwood, Populus trichocarpa (Torr. & Gray). Science313, 15961604.
van Hees P, Jones D, Finlay R, Godbold D, Lundstrom U. 2005.The carbon we do not seethe impact of low molecular weightcompounds on carbon dynamics and respiration in forest soils:a review. Soil Biology and Biochemistry 37, 113.
Vodnik D, Gogala N. 1994. Seasonal fluctuations of photosynthe-sis and its pigments in 1-year mycorrhized spruce seedlings.Mycorrhiza 4, 277281.
Voegele RT, Struck C, Hahn M, Mendgen K. 2001. The role ofhaustoria in sugar supply during infection of broad bean by the
rust fungus Uromyces fabae. Proceedings of the National Academy of Sciences, USA 98, 81338138.
Wainwright M. 1993. Oligotrophic growth of fungistress ornatural state. In: Jennings DH, ed. Stress tolerance of fungi. NewYork: Marcel Dekker, 127144.
Wallander H. 2006. External mycorrhizal myceliathe importanceof quantification in natural ecosystems. New Phytologist 171,240242.
Wallander H, Goransson H, Rosengren U. 2004. Production,standing biomass and natural abundance of
15N and
13C in
ectomycorrhizal mycelia collected at different soil depths in twoforest types. Oecologia 139, 8997.
Wallander H, Nilsson LO, Hagerberg D, Baath E. 2001.Estimation of the biomass and seasonal growth of externalmycelium of ectomycorrhizal fungi in the field. New Phytologist
151, 753760.Wallenda T. 1996. Untersuchungen zur Physiologie der Pilzpartnervon Ektomykorrhizen der Fichte ( Picea abies [L.] Karst.). Thesis,Physiologische Okologie der Pflanzen, Tubingen, Germany.
Wallenda T, Kottke I. 1998. Nitrogen deposition and ectomycor-rhizas. New Phytologist 139, 169187.
Walters DR, Cowley T, McPherson A, Marshall G,McRoberts N. 1996. Sugar transport in the light leaf spotpathogen Pyrenopeziza brassicae. FEMS Microbiology Letters143, 285289.
Wannet WJB, Hermans JHM, van der Drift C, Op denCamp HJM. 2000. HPLC detection of soluble carbohydratesinvolved in mannitol and trehalose metabolism in the ediblemushroom Agaricus bisporus. Journal of Agricultural and FoodChemistry 48, 287291.
Wiese J, Kleber R, Hampp R, Nehls U. 2000. Functionalcharacterization of the Amanita muscaria monosaccharide trans-porter AmMst1. Plant Biology 2, 15.
Wright DP, Scholes JD, Read DJ, Rolfe SA. 2000. Changes incarbon allocation and expression of carbon transporter genes inBetula pendula Roth. colonized by the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr. Plant, Cell and Environment 23,3949.
Wright DP, Read DJ, Scholes JD. 1998. Mycorrhizal sink strengthinfluences whole plant carbon balance of Trifolium repens L. Plant, Cell and Environment21, 881891.
Wright DP, Johansson T, Le Quere A, Soderstrom B, Tunlid A.2005. Spatial patterns of gene expression in the extramatricalmycelium and mycorrhizal root tips formed by the ectomycor-rhizal fungus Paxillus involutus in association with birch (Betulapendula) seedlings in soil microcosms. New Phytologist 167,
579596.Wu B, Nara K, Hogetsu T. 2002. Spatiotemporal transfer of
carbon-14-labelled photosynthate from ectomycorrhizal Pinusdensiflora seedlings to extraradical mycelia. Mycorrhiza 12,8388.
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