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

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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

1104 Nehls



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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).

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