Plant Signaling & Behavior

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Arbuscular Mycorrhiza
Studies on the Geosiphon Symbiosis Lead to the Characterization of the
First Glomeromycotan Sugar Transporter

Arthur Schüßler, Holger Martin, David Cohen, Michael Fitz & Daniel Wipf

To cite this article: Arthur Schüßler, Holger Martin, David Cohen, Michael Fitz & Daniel
Wipf (2007) Arbuscular Mycorrhiza, Plant Signaling & Behavior, 2:5, 431-434, DOI: 10.4161/
psb.2.5.4465

To link to this article: https://doi.org/10.4161/psb.2.5.4465

Copyright © 2007 Landes Bioscience

Published online: 01 Sep 2007.

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 [Plant Signaling & Behavior 2:5, 431-434, September/October 2007]; ©2007 Landes Bioscience

Article Addendum

Arbuscular Mycorrhiza

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Studies on the Geosiphon Symbiosis Lead to the Characterization

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of the First Glomeromycotan Sugar Transporter

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Arthur Schüßler1 Abstract

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Holger Martin2 The intimate arbuscular mycorrhiza (AM) association between roots and obligate

r
symbiotic Glomeromycota (‘AM fungi’) ‘feeds’ about 80% of land plants. AM forming
David Cohen2

st
fungi supply land plants with inorganic nutrients and have an enormous impact on terres‑
Michael Fitz3 trial ecosystems. In return, AM fungi obtain up to 20% of the plant‑fixed CO2, putatively

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as monosaccharides. In a recent work we have reported the characterization of the first
Daniel Wipf3 glomeromycotan monosaccharide transporter, GpMST1, and its gene sequence. We
1Ludwig Maximilians University of Munich; Genetics, Department Biology I; discuss that AM fungi might take up sugars deriving from plant cell‑wall material. The

t
Munich, Germany GpMST1 sequence delivers valuable data for the isolation of orthologues from other AM

no
2Darmstadt University of Technology; Institute of Botany; Darmstadt, Germany
fungi and may eventually lead to the understanding of C‑flows in the AM.
3University of Bonn; Bonn, Germany

The arbuscular mycorrhiza (AM) as an outstanding terrestrial plant symbiosis directly

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*Correspondence to: Arthur Schüßler; Ludwig Maximilians University of and indirectly is a driver of most terrestrial ecosystems. It is formed by ~80% of land plants
Munich; Genetics, Department Biology I; Maria-Ward-Straße 1a; Munich and by obligate symbiotic fungi of the phylum Glomeromycota.1 The glomeromycotan

80638 Germany; Tel.: +49.89.2180.6176; Fax: +49.69.1330.3537185;
Email: arthur.schuessler@lrz.uni-muenchen.de
Original manuscript submitted: 05/23/07
Manuscript accepted: 05/23/07
Previously published online as a Plant Signaling & Behavior E-publication:
http://www.landesbioscience.com/journals/psb/article/4465
.D
fungi usually are called ‘arbuscular mycorrhizal (AM) fungi’, or ‘AMF’, and obviously play an
enormous ecological (and economical) role. Most land plants and glomeromycotan fungi
are ‘joint systems’, forming the intimate AM.2 By this fact, statements like that of the BEG
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(European Bank of Glomeromycota) committee (1993): “The study of plants without their
mycorrhizas is the study of artefacts; the majority of plants, strictly speaking, do not have
en

roots—they have mycorrhizas” were provoked. AM fungi supply the vast majority of land
plants with inorganic nutrients, mainly phosphorous, but also nitrogen, trace elements,
Key words
and water. In return, they obtain up to >20% of the photosynthetically fixed CO2 as
ci

arbuscular mycorrhiza, Geosiphon symbiosis, carbohydrates from the plants.3 It was calculated that, each year, 5 billion tons of carbon
monosaccharide transporter, hexoses are transferred from plants to fungi (and therefore partly get deposited in the soil) via the
s

AM symbiosis. AM fungi therefore represent a large sink for atmospheric CO2 on our
Addendum to: planet and play a role in C‑deposition in the soil.
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Characterization of a Carbohydrate Transporter from

Studying the AM Symbiosis is Difficult
B

Symbiotic Glomeromycotan Fungi

Schüssler A, Martin H, Cohen D, Fitz M, Wipf D The ‘AM fungi’ (Glomeromycota) are multikaryotic, asexual and obligate symbionts. Each
es

of these features makes certain molecular biological investigations difficult, the combina‑
Nature 2006; 444:933–6 tion sometimes impossible. One main problem is that, in the symbiotic phase, the fungi
PMID: 17167486
live within the plant root cells and “clean” fungal tissue cannot be harvested for e.g.,
nd

doi: 10.1038/nature05364
gene‑expression studies. Physiology and genetics of the fungal nutrient transport systems
in this life stage are largely unknown. The sequencing of the first AM fungal genome is in
progress as a milestone for obtaining basic data, however, this project is delayed. Moreover,
La

for the post‑genomic era, targeted strategies are urgently needed to characterize the fungal
transcriptome in the symbiotic stage (i.e., within the plant root). Even for Arabidopsis
thaliana, which unfortunately is a non-AM forming plant and therefore not a good model
07

for the life strategy of most plants with this respect, less then ¼ of the genes are function‑
ally characterized. Such functional characterisations will become an important topic for
future research. Regarding the uptake of the vast amounts of carbohydrates by AM fungi,
20

nothing was known about carbohydrate transporters themselves, but it was shown by
tracer studies and radiorespirometry that hexoses are taken up within the roots.4,5 The
difficulties of isolating high quality fungal mRNA from AM roots, since plant mRNA
©

is dominating, seems to be reflected by the fact that no AM fungal sugar transporter

sequence was identified, despite its key‑importance in the symbiosis.
In our study we followed an approach using the advantages of a peculiar symbi‑
osis formed by a glomeromycotan fungus (Geosiphon pyriformis) with cyanobacteria

www.landesbioscience.com Plant Signaling & Behavior 431

 Arbuscular Mycorrhiza

(Nostoc punctiforme), the unique ‘Geosiphon‑symbiosis’.6 The work

shows, which potential lies in the use of this symbiosis as a model
for the AM.7

The Geosiphon‑Symbiosis Helps to Uncover

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Mechanisms in the AM

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Why should we use the peculiar Geosiphon‑symbiosis with cyano‑

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bacteria as a model for the AM? The answer is, that the obligate
symbiotic fungal partner evolutionary and functionally is an ‘AM

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fungus’. It is phylogenetically embedded within the Archaeosporales,
one of the four main clades of the biotrophic Glomeromycota.8,9

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Therefore the ancestors of Geosiphon pyriformis putatively formed

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AM—and we have unpublished preliminary data that indicate that
G. pyriformis in parallel to the cyanobacteria symbiosis forms AM.

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The Geosiphon‑Nostoc symbiosis also shows many structural, functional
and also ecological parallels to the AM ‑ it represents an ‘AM symbiosis Figure 1. Geosiphon pyriformis bladders in liquid medium, as they were
at the fungus—cyanobacterium level’.6 Regarding the symbiotic stage of used for mRNA isolation to construct the cDNA library (see text), the bladders

t
G. pyriformis (the so called ‘bladder’, Fig. 1) it is the homologous stage shown are in average 1.5 mm in length and were harvested from cultures

to the intraradical situation in the AM, where the nutrient exchange
processes in‑between the partners take place at the symbiotic inter‑
face.10 This offers several advantages for investigating fundamental
no
on sterilized, natural substrate. All attached hyphae were cut off, and blad‑
ders were incubated in liquid nutrient solution under illumination (light/dark
cycles). All hyphae shown on the photograph were newly formed within the
five days incubation in medium, so transcripts from actively growing hyphae

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aspects like partner recognition, evolutionary aspects and nutrient
are also represented in the cDNA library. The light green bladders are
exchange mechanisms.

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formed with a different cyanobacterial strain20 than the dark green bladders,
On the gene expression level, the clue of using the Geosiphon‑ which represent the type of color found in nature.
symbiosis is that the fungal (eukaryotic) mRNA can be easily and
specifically isolated out of the symbiotic stage with poly(A) discrimi‑
ce
nating methods, which were established to reproducibly isolate pH dependence indicates that GpMST1 transport is mediated by
fungal mRNA from the symbiotic bladders. We constructed cDNA secondary active proton cotransport.
libraries suitable for yeast functional complementation from such
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mRNA11 and random sequencing of >100 cDNA clones did not Discussing C‑Transport in AM
result in any Nostoc sequences, indicating that the library is derived
Nothing was known about sugar transporter genes in the AM.
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from nearly exclusively fungal transcripts.

AM fungi seem to be restricted in their carbon supply since they
nearly exclusively take up sugars via the symbiotic interface, that
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The First Glomeromycotan Sugar Transporter

means from the photoautotrophic partner. Therefore it is conceiv‑
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As reported in Schüßler et al. 2006, to which we address this able that they might have a low number (maybe even only one?)
addendum, high‑quality fungal mRNA was isolated from symbiotic of monosaccharide transporter genes for this purpose. We can not
B

bladders and used to establish a cDNA yeast‑expression library, answer such a question yet, but our studies indicate that at least for
which then served to isolate the fungal monosaccharide transporter GpMST1 there are no close paralogs, since PCR attempts using many
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GpMST1 gene by functional complementation of a yeast hexose trans‑ different primer pairs always gave rise to GpMST1 amplicons only,
port null‑mutant.12 GpMST1 has the highest affinities for glucose with identical intron sequences.
and mannose, followed by galactose and fructose. A KM of about Generally, the description of the first Glomeromycota mono-
nd

1.2 mM was determined for glucose. Since xylose is a main constit‑ saccharide transporter and its sequence opens the field for research on
uent of plant cell walls (see discussion below), we also tested whether these key proteins, putatively being significant in the global C‑flows.
it might be taken up by GpMST1. Indeed, xylose is indicated to Isolation of orthologous genes from other AM fungi should now
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slightly compete with glucose uptake and seems to be transported. be relatively easy. Regarding GpMST1 itself, future tasks will be to
This is now supported by unpublished results showing functional isolate and characterize the promoter and one of the most important
complementation of a hexose transport null‑mutant yeast strain that steps will be answering the question about where GpMST1 is ‘doing
07

is capable of xylose metabolism. For comparison it may be noted that in its job’, by localizing the gene product with antibodies or fusion‑
Saccharomyces cerevisiae the rate of xylose transport by hexose trans‑ proteins. Some indirect evidence already indicates that GpMST1 is
porters corresponds to only 0.5% of glucose transport.13 indeed located at the symbiotic interface membrane. The membrane
20

Regarding the AM and Geosiphon symbioses, we hypothesize of the cup‑shaped symbiosome compartment in Geosiphon is derived
that GpMST1 is active at the symbiotic interface and therefore is from the fungal plasma‑membrane (by invagination) and retains the
located in the fungal symbiotic membrane. When interpreting the capability to synthesize chitin. This results in a thin cell wall layer
©

carbohydrate transport in the Geosiphon‑ and AM symbioses, it is
crucial to know whether the transport is via facilitated diffusion or an
active transport. 14C‑glucose uptake (at pH 6.5) was very sensitive to
protonophores and plasma membrane H+‑ATPase inhibitors. The strong
dependence on the presence of a proton gradient together with the
within the symbiosome, ultrastructurally appearing like an arbuscule
cell wall.10 The Geosiphon symbiosome membrane is a homologue
of the arbuscular membrane in the AM, also showing the same
function and bidirectional transport (in Geosiphon the cyanobacteria
have to receive all P, water, etc., via the fungus, they do not have

432 Plant Signaling & Behavior 2007; Vol. 2 Issue 5

 Arbuscular Mycorrhiza
direct contact to the exterior). The fungal symbiotic membranes in
the AM and Geosiphon symbioses show plasmamembrane features
—but both membranes are much derived. In the case of the mature
Geosiphon symbiosis the invaginated plasmamembrane probably has
no contact to the ‘outer’ plasmamembrane any more. Our assump‑
tion, that GpMST1 is located at the perisymbiotic membrane is
supported by the indirect evidence that no 14C glucose uptake
could be shown for G. pyriformis bladders incubated in 14C glucose
containing medium, a fact which was also observed for other AM
fungi.4 GpMST1 potentially is the first characterised member of
the ‘type’ of sugar transporters that is responsible for the transport
of billions of tons of photosynthetically fixed CO2 from plants to
AM fungi. Interestingly, the transporter sequence phylogenetically
belongs to a new clade, comprising functionally noncharacterised
putative transporter sequences, including many from parasitic fungi.
Maybe this transporter‑class is joined by some special features.
Where the Sugars Come From?
We are aware that this is still to some extend hypothetical, but if
GpMST1 indeed represents the transporter type responsible for the
monosaccharide uptake of AM fungi, what does it mean? Generally,
symbiotically derived hexoses are interpreted as to be the exclusive
energy source for glomeromycotan fungi,3,5 whereas some sugars
presumably can also be taken up in nonsymbiotic stages.14 AM fungi,
in contrast to the plant hosts, are limited in carbohydrates as energy
source. This e.g., is reflected by retaining the C‑skeleton of arginine
ce
when delivering nitrogen as NH4+ to the plant.15 Reasonably, the
fungus should save valuable carbohydrates and a reasonable uptake
mechanism would be by a non-energy‑consuming process that e.g.,
en
could be facilitated diffusion. However, it is usually suggested that
hexoses are taken up by active H+ cotransport and are derived from
sucrose that is cleaved by apoplastic acid invertase into glucose and
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fructose. If the plant acidifies the perisymbiotic space, which is indi‑
cated,16 this would also be nonenergy‑consuming for the fungus.
s
One should add at this point, that the common plant sugar transport
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form, sucrose, may also serve as C‑source in the Geosiphon‑symbiosis,
since the N. punctiforme symbiotic in Geosiphon produces high
B
amounts of sucrose for osmoregulation.17,18
Regarding sugar transport, several indications, assumptions and
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facts may be incorporated into a working hypothesis regarding the
general sugar uptake mechanisms of monosaccharides in the AM and
Geosiphon‑symbioses:
nd
• GpMST1 is probably located at the perisymbiotic membrane
(homologous to arbuscule membrane). Its characterisation indicates
that sugars are transported by means of H+‑cotransport.
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• The symbiotic interface in the AM is acidic and acidified by the
photoautotrophic partner, not by the fungus. Therefore the photoau‑
totrophic partner might regulate hexose transport rates of the fungus
07
by changing the apoplastic pH, maybe as a feedback mechanism
dependent on the nutritional status.
• The pH optimum of GpMST1 is, on the first view surprisingly,
20
only slightly acidic. This could just be an effect of the heterologous yeast
system, but it is also conceivable that this reflects the situation in the
symbiosis. An only slightly acidic pH of 6–7 would be reasonable
©
e.g., from the viewpoint, that a precipitation of the fungus‑delivered
phosphate within the perisymbiotic space would be avoided, and that
a stronger acidification by the plant could diminish sugar uptake of
the fungus, e.g., when arbuscules senesce and then get degraded by
the plant.
www.landesbioscience.com Plant Signaling & Behavior 433
• GpMST1 effectively not only transports glucose, but also the other
main cell wall constituent hexoses: mannose and galactose. Moreover
it likely transports xylose.
These points may indicate that invertase cleaved sucrose is not the
only source for the sugars taken up by AMF. H+‑ATPase activity of
.
the photoautotrophic partner might regulate the sugar supply by
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regulating acid invertase activity, the fungal transporter,19 but also
by influencing the activity of (fungal?) hydrolases degrading cell‑wall
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polysaccharides.
We therefore speculate that, in the AM as well as the AM‑like
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Geosiphon‑symbiosis, cell wall polysaccharides and glycoconjugates
could be degraded in the symbiotic space, leading to a liberation
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of glucose, mannose, and galactose. These sugars might be used in
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parallel to sucrose derived glucose and fructose. It seems that xylose
is also transported by GpMST1, and this might also hold true for
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arabinose—which would make such transporter types even interesting
candidates for e.g., biofuel production from plant‑residues, using
genetically modified S. cerevisiae strains able to metabolize pentoses.
t
In general, we suppose that the Geosiphon symbiosis can and
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should be used to uncover key mechanisms in the AM and related
symbioses, as exemplified by the characterisation of GpMST1. Such
data then can be easily used as fundamental starting points for
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detailed studies in the AM.
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References
1. Schüßler A, Schwarzott D, Walker C. A new fungal phylum, the Glomeromycota: Phylogeny
and evolution. Mycol Res 2001; 105:1413‑21.
2. Smith SE, Read DJ. Mycorrhizal Symbiosis. London: Academic Press, 1997.
3. Bago B, Pfeffer PE, Abubaker J, Jun J, Allen JW, Brouillette J, Douds DD, Lammers PJ,
Shachar-Hill Y. Carbon export from arbuscular mycorrhizal roots involves the translocation
of carbohydrate as well as lipid. Plant Physiol 2003; 131:1496‑507.
4. Pfeffer PE, Douds Jr DD, Bécard G, Shachar‑Hill Y. Carbon uptake and the metabolism
and transport of lipids in an arbuscular mycorrhiza. Plant Physiol 1999; 120:587‑98.
5. Solaiman Z, Saito M. Use of sugars by intraradical hyphae of arbuscular mycorrhizal fungi
revealed by radiorespirometry. New Phytol 1997; 136:533‑38.
6. Schüßler A, Wolf E. Geosiphon pyriformis ‑ A glomeromycotan soil fungus forming endosymbiosis
with cyanobacteria. In: Declerck S, Strullu DG, Fortin JA, eds. Soil Biology, Volume 4: In
vitro culture of mycorrhizas. Berlin Heidelberg: Springer, 2005:271‑89.
7. Schüßler A, Kluge M. Geosiphon pyriforme, an endocytosymbiosis between fungus and
cyanobacteria, and its meaning as a model system for arbuscular mycorrhizal research. In:
Hock B, ed. The Mycota IX. Berlin, Heidelberg, New York: Springer, 2001:151‑61.
8. Schüßler A. Molecular phylogeny, taxonomy, and evolution of Geosiphon pyriformis and
arbuscular mycorrhizal fungi. Plant Soil 2002; 244:75‑83.
9. James TY, Kauff F, Schoch C, Matheny B, Hofstetter V, Cox C, Celio G, Guiedan C, Fraker
E, Miadlikowska J, Lumbsch HT, Rauhut A, Reeb V, Arnold AE, Amtoft A, Stajich JE,
Hosaka K, Sung GH, Johnson D, O’Rourke B, Crockett M, Binder M, Curtis JM, Slot JC,
Wang Z, Wilson AW, Schüßler A, Longcore JE, O’Donnell K, Mozley‑Standridge S, Porter
D, Letcher PM, Powell MJ, Taylor JW, White MM, Griffith GW, Davies DR, Humber
RA, Morton JB, Sugiyama J, Rossman AY, Rogers JD, Pfister DH, Hewitt D, Hansen K,
Hambleton S, Shoemaker RA, Kohlmeyer J, Volkmann‑Kohlmeyer B, Spotts RA, Serdani
M, Crous PW, Hughes KW, Matsuura K, Langer E, Langer G, Untereiner WA, Lücking R,
Büdel B, Geiser DM, Aptroot A, Diederich P, Schmitt I, Schultz M, Yahr R, Hibbett DS,
Lutzoni F, McLaughlin DJ, Spatafora JW, Vilgalys R. Reconstructing the early evolution of
the Fungi using a six‑gene phylogeny. Nature 2006; 443:818‑22.
10. Schüßler A, Bonfante P, Schnepf E, Mollenhauer D, Kluge, M. Characterization of the
Geosiphon pyriforme symbiosome by affinity techniques: Confocal laser scanning microscopy
(CLSM) and electron microscopy. Protoplasma 1996; 190:53‑67.
11. Wipf D, Benjdia M, Rikirsch E, Zimmermann S, Tegeder M, Frommer WB. An expres‑
sion cDNA library for suppression cloning in yeast mutants, complementation of a yeast
his4 mutant and EST analysis from the symbiotic basidiomycete Hebeloma cylindrosporum.
Genome 2003; 46:177‑81.
12. Wieczorke R, Krampe S, Weierstall T, Freidel K, Hollenberg CP, Boles E. Concurrent
knock‑out of at least 20 transporter genes is required to block uptake of hexoses in
Saccharomyces cerevisiae. FEBS Lett 1999; 464:123‑8.
13. Hamacher T, Becker J, Gardonyi M, Hahn‑Hägerdal B, Boles E. Characterization of the
xylose‑transporting properties of yeast hexose transporters and their influence on xylose
utilization. Microbiol 2002; 148:2783‑88.
14. Hildebrandt U, Ouziad F, Marner FJ, Bothe H. The bacterium Paenibacillus validus stimu‑
lates growth of the arbuscular mycorrhizal fungus Glomus intraradices up to the formation
of fertile spores. FEMS Microbiol Lett 2006; 254:258‑67.
 Arbuscular Mycorrhiza

15. Govindarajulu M, Pfeffer PE, Jin H, Abubaker J, Douds DD, Allen JW, Bücking H,
Lammers PJ, Shachar‑Hill Y. Nitrogen transfer in the arbuscular mycorrhizal symbiosis.
Nature 2005; 435:819‑923.
16. Gianinazzi‑Pearson V, Arnould C, Oufattole M, Arango M, Gianinazzi S. Differential acti‑
vation of H+‑ATPase genes by an arbuscular mycorrhizal fungus in root cells of transgenic
tobacco. Planta 2000; 211:609‑13.
17. Knoblauch M. Vergleichende physiologische Charakterisierung von Nostoc punctiforme

.
und Nostoc muscorum im Hinblick auf die Symbiose von Nostoc in Geosiphon pyriforme.

te
In: Knoblauch M, ed. Master Thesis (in German). Frankfurt: University of Frankfurt,
Department of Biology, 1995:1‑70.
18. Erdman N. Organic osmoregulatory solutes in blue‑green algae. Z Pflanzenphysiol 1983;

u
110:147‑55.
19. van Kuyk PA, Jasper A, Maccabe AP, Hererro O, Ruijter GJG, Visser J. Aspergillus niger mstA

ib
encodes a high‑affinity sugar/H+ symporter which is regulated in response to extracellular
pH. Biochem J 2004; 379:375‑83.

r
20. Wolf E, Schüßler A. Phycobiliprotein fluorescence of Nostoc punctiforme changes during
the life cycle and chromatic adaptation: Characterization by spectral CLSM and spectral

st
unmixing. Plant Cell Environ 2005; 28:480‑91.

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434 Plant Signaling & Behavior 2007; Vol. 2 Issue 5