Strack Et Al. Mycorrhiza-Review J. Chem. Ecol. 29, 1955-1979

7/30/2019 Strack Et Al. Mycorrhiza-Review J. Chem. Ecol. 29, 1955-1979...

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Journal of Chemical Ecology, Vol. 29, No. 9, September 2003 (C 2003)

REVIEW PAPER

ARBUSCULAR MYCORRHIZA: BIOLOGICAL, CHEMICAL,

AND MOLECULAR ASPECTS

DIETER STRACK,1, THOMAS FESTER,1 BETTINA HAUSE,1

WILLIBALD SCHLIEMANN,1 and MICHAEL H. WALTER1

1Leibniz-Institut f ur Pflanzenbiochemie

Abteilung Sekund arstoffwechsel

Weinberg 3, D-06120 Halle (Saale), Germany

(Received April 16, 2003; accepted May 18, 2003)

AbstractMycorrhizas are the most important mutualistic symbioses on earth.The most prevalent type are the arbuscular mycorrhizas (AMs) that develop be-

tween roots of most terrestrial plants and fungal species of the Zygomycota. The

AM fungi are able to grow into the root cortex forming intercellular hyphae from

which highly branched structures, arbuscules, originate within cortex cells. The

arbuscules are responsible for nutrient exchange between the host and the sym-

biont, transporting carbohydrates from the plant to the fungus and mineral nutri-

ents, especially phosphate, and water from the fungus to the plant. Plants adapt

their phosphate uptake to the interaction with the AM fungus by synthesis of spe-

cific phosphate transporters. Colonization of root cells induces dramatic changes

in the cytoplasmic organization: vacuole fragmentation, transformation of the

plasma membrane to a periarbuscular membrane coveringthe arbuscule, increase

of the cytoplasm volume and numbers of cell organelles, as well as movement of

the nucleus into a central position. The plastids form a dense network covering

the symbiotic interface. In some of these changes, microtubules are most likely

involved. With regard to the molecular crosstalk between the two organisms, a

number of phytohormones (cytokinins, abscisic acid, jasmonate) as well as vari-

ous secondary metabolites have been examined: (i) Jasmonates occur at elevated

level, which is accompanied by cell-specific expression of genes involved in jas-

monate biosynthesis that might be linked to strong carbohydrate sink function of

AM roots and induced defense reactions; (ii) apocarotenoids (derivatives of my-

corradicin and glycosylated cyclohexenones) accumulate in most mycorrhizal

To whom correspondence should be addressed. E-mail: [email protected]

1955

0098-0331/03/0900-1955/0 C 2003 Plenum Publishing Corporation


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1956 STRACK, FESTER, HAUSE, SCHLIEMANN, AND WALTER

rootsexamined so far. Their biosynthesis via the nonmevalonate methylerythritol

phosphate (MEP) pathway has been studied resulting in new insights into AM-

specific gene expression and biosynthesis of secondary isoprenoids.

Key WordsArbuscular mycorrhiza, (apo)carotenoids, chemical interaction,ecology, MEP pathway, mutualism, phosphate transporters, secondary metabo-lism, symbiosis.

INTRODUCTION

More than 90% of terrestrial plants are associated with root-colonizing fungi, estab-

lishing a permanent and intimate mutualistic symbiosis, called mycorrhiza. Several

types of mycorrhizas exist, defined by plant/fungus combination and the symbiotic

structure. The endotrophic arbuscular mycorrhiza (AM) is the most common type,

occurring in about 80% of plant species. The AM fungi are represented by more

than 150 species of the Zygomycota included in the Glomales (Morton and Benny,

1990). Recent work on the phylogeny of AM fungi provided a basis for a new

systematics (Schuler et al., 2001; Schuler, 2002), removing these fungi from the

polyphyletic Zygomycota and placing them into a new monophyletic phylum, the

Glomeromycota.

There is a disagreement about usage of the term symbiosis, originally de-

fined in 1869 by Heinrich Anton de Bary as an intimate, outcome-independent

interaction between different species, ranging from parasitism to mutualism. Later,

especially in Europe, the term symbiosis was used to mean only mutually bene-

ficial association of organisms (=mutualism). In this review, the term symbiosis

is used in its original meaning.

The term mycorrhiza was coined in 1885 by Bernhardt Frank by recog-

nizing special structures in tree roots. The term means fungus root, later char-

acterized as ectomycorrhiza. Frank not only described its morphology but also

inferred its physiological role (Frank, 1888). Arbuscular mycorrhiza (AM) re-

placed the earlier term vesiculararbuscular mycorrhiza (VAM) because not all

endomycorrhizas of this type develop vesicles, but all form arbuscules.

There are two major types of mycorrhizas, AMs and ectomycorrhizas. The

latter evolved as a more recent symbiosis of woody trees and shrubs with ecto-

mycorrhizal fungi. The plant hosts of AM fungi are mostly angiosperms, some

gymnosperms, pteridophytes, lycopods, and mosses (Smith and Read, 1997). The

physiological interactions of lower plant mycorrhizas, however, are poorly under-

stood. With regard to the systematic distribution of AMs in higher plants, it is still

an open question how some nonhost plants, e.g., members of the Brassicaceae,

Caryophyllaceae, Chenopodiaceae, or Urticaceae, resist mycorrhizal colonization

(Vierheilig et al., 1994, 1996). The nonmycorrhizal state of nonhost plants mightbe a derived trait. It might be the outcome of specialization regarding, e.g., the

plant habitat (Fitter and Moyersoen, 1996).


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ARBUSCULAR MYCORRHIZA 1957

According to the fossil record and molecular data, the origin of the AM sym-

biosis goes back at least to the Ordovician, 450500 million years ago (Remy

et al., 1994; Redecker et al., 2000). It is assumed that this symbiosis aided plants

during their land colonization in the acquisition of water and minerals, especially

phosphate (Simon et al., 1993). At the time of land colonization, the first bryophyte-

like plants appeared in terrestrial environments. Today, a number of bryophytes

and pteridophytes are still capable of forming AMs (Read et al., 2000; Schuler,

2000).

In contrast to AMs, ectomycorrhizas (Tagu et al., 2002) and the more special-

ized ericoid mycorrhizas (Perotto et al., 2002) evolved later in evolution and are of

polyphyletic origin (Fitter and Moyersoen, 1996). These symbioses are especially

adapted to habitats rich in organic material that did not exist on earth when the

AM symbiosis developed.

Interesting questions to be addressed are on the selective forces that led to the

mutualistic coexistence of the two partners. Whereas the macrobiont (phytobiont)

can live without AM fungi, although suffering in nutrient- and water-deficient

soils, the microbiont (mycobiont) became dependent on plant roots and developed

towards an obligatory biotrophic life cycle. Another challenging problem is the

complexity of field situations, where many plant and fungal species coexist with

many different soil organisms in different ecosystems.

ECOLOGY

In accordance with the evolutionary history, AM symbioses can be found in

almost all ecosystems. They have been described from deserts (Corkidi and Rincon,

1997; Dalpe et al., 2000; Titus et al., 2002), tropical rainforests (Brundrett et al.,

1999; Guadarrama and Alvarez-Sanchez, 1999; Siqueira and Saggin-Junior, 2001;

Zhao et al., 2001; Gaur and Adholeya, 2002), aquatic environments (Khan, 1993),

as well as from ecosystems with strong saline (Carvalho et al., 2001; Sengupta and

Chaudhuri, 2002), sodic or gypsum soils (Landwehr et al., 2002). The relatively

low number of plants colonized by AM fungi in some arctic and antarctic habitats

seems to be due to a lack of suitable vectors for fungal spores rather than to other

causes (Allen, 1996).

In addition to the global distribution of AM symbioses, there is large func-

tional diversity as well. Whereas most AM symbioses are mutualistic, a growing

number of nonphotosynthetic plants are described, which are receiving a large por-

tion of their nutrients from AM fungi (Imhof, 1999; Yamato, 2001), resembling

the functioning of orchid mycorrhizas (Rasmussen, 2002). In some cases, these

mycotrophic plants are living epiparasitically on other plants using the hyphae of

their fungal partner for the transfer of nutrients (Bidartondo et al., 2002). On theother hand, AM fungi may become parasitic themselves in relation to their host

plant under special circumstances (Allen, 1996).


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Regarding plant communities, there is a large number of possible conse-

quences of AM colonization (Francis and Read, 1994). Interactions between plant

and fungal communities rely primarily on the fact that a given plant or fungus

prefers some symbiotic partners and neglects others. In addition, the benefits ob-

tained from the symbiotic partner are dependent on the actual partner and on various

other conditions. Even the direct transfer of nutrients from one plant to another

via fungal hyphae has been discussed (Francis et al., 1986). Within ecosystems,

a network of feedback dynamics based on these interactions between fungal and

plant populations has to be taken into account (Bever, 2002). These dynamics

may be important to maintain diversity within plant communities. Sanders et al.

(1996) have stressed the importance of fungal diversity for the ecological impact

of the AM symbiosis, and van der Heijden et al. (1998) have shown in a case

study that fungal and plant population diversity are directly correlated to each

other. In general, the results from single ecological studies regarding ecology of

AM symbiosis are highly dependent on the local situation (Hartnett and Wilson,

2002). As the main possible consequences, Koide and Dickie (2002) have sin-

gled out (i) increased plant reproduction, (ii) stable plant populations because

positive mycorrhizal effects might inversely correlate with population density,

(iii) favoring of the most robust individuals by AM fungi, and (iv) patchy dis-

tribution of mycorrhizal areas due to the spread of colonization starting from

mycorrhizal plants.

Apart from supplying plants with phosphate and other nutrients, further bene-

ficial effects have been described for AM fungi. The symbiosis has a positive effect

regarding plant water potential especially for plants under drought stress (Auge,

2001). AM colonized plants show a significant degree of bioprotection against

various pathogens (Cordier et al., 1996; Dugassa et al., 1996; Bdker et al., 1998;

Vaast et al., 1998; Slezack et al., 2000; Elsen et al., 2001). In addition, positive

effects of AM fungi on soil structure have been described. As a consequence, the

AM symbiosis is regarded as a key component of sustainable agriculture (Beth-

lenfalvay and Lindermann, 1992; Jeffries and Barea, 2001), whereas under con-

ventional agricultural conditions, AM fungi seem to be only of minor importance

(Ryan and Graham, 2002). Mader et al. (2000) have compared conventional and

organicbiological agricultural systems directly. They found stronger mycorrhizal

colonization for the organicbiological system and preliminary evidence for a par-

tial compensation for the disadvantages of the organicbiological system by the

AM fungi. Apart from agricultural systems (Kiers et al., 2002), the application of

AM fungi is tested for the revegetation of desertified areas (Saito and Marumoto,

2002) and in cultivation of micropropagated plantlets (Yano-Melo et al., 1999). In

this context, major technological problems are the form of application of the AM

inoculum (Saito and Marumoto, 2002) and the combinations of AM inoculum withother microorganisms that are beneficial for plant growth (Vazquez et al., 2000,

2001; Vassilev et al., 2001).


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FIG. 1. Morphology of the life cycle stages of an AM fungus, Glomus intraradices in a

maize root: A, germinating spore; B, appressorium; C, intercellular hyphae; D, arbuscule;

E, intercellular vesicle; F, extraradical spores. The fungal structures are visible after staining

with trypan blue (Phillips and Hayman, 1970).

COLONIZATION AND MORPHOLOGICAL CHANGES

Steps in root colonization by AM fungi are shown in Figure 1. The pro-

cess starts with germination (hyphal growth) of fungal spores, followed by poorly

understood events. Subsequently, appressoria are formed from which the fungus

penetrates the root surface and colonizes the intercellular space of the root cor-

tex. On the fungal side, nonaggressive cell wall-lytic enzymes become active,

and both the plant root cells and the fungus change their gene expression pat-

tern and morphology. The hyphae penetrate the cell walls and develop within

the cortex cells tree-like structures, called arbuscules, by repeated dichotomous

branching. In some cases, intercellular storage organs, lipid-rich vesicles, and

finally extraradical spores are formed, which may enter another colonization pro-

cess. Fungal root colonization is under control of the plant aiming at a morpho-logical and functional compatibility of the two partners (Bonfante and Perotto,

1995).


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The key feature of AMs is the arbuscule, a highly branched haustorium-like

structure within root cortex cells, responsible for nutrient exchange. However, the

arbuscules represent a dead-end in the growth of AM fungi (Bonfante and Perotto,

1995) and they finally senesce and collapse after 410 days of symbiosis (Sanders

et al., 1977), possibly caused by the continuously stressful environment of the host

cortex cell (Harley and Smith, 1983).

Colonization of root cortex cells by AM fungi has been shown to induce

dramatic changes in the cytoplasmic organization and morphology of root cells.

The fungal arbuscule occupies a major portion of the plant cell volume but is still

separated from the cell protoplast by the host plasma membrane. This membrane

completely surrounds the arbuscule and forms a periarbuscular membrane, leading

to a two- to fourfold increase in the plasma membranes surface. The resulting

space between the plant protoplast and the fungus develops into an apoplastic

compartment that represents the symbiotic interface (Bonfante and Perotto, 1995).

Figure 2 shows some arbuscule branches surrounded by the plama membrane and

the interfacial matrix in a colonized maize root cortex cell.

Formation of arbuscules is accompanied by alterations in morphology of

the host cell: the central vacuole is fragmented, the volume of cytoplasm and

number of cell organelles increase significantly, and the nucleus moves into a cen-

tral position and undergoes hypertrophy (Balestrini et al., 1994). The nucleus of

arbusculated cells (arbuscule-harboring cells) is characterized by enhanced fluo-

rochrome accessibility, increased nuclease sensitivity, and chromatin dispersion,

all together reflecting a greater transcriptional activity of the plant genome in

the colonized cells (Gianinazzi-Pearson, 1996). The host cytoplasm and cell or-

ganelles proliferate around the branching hyphae. The number of plastids in colo-

nized cortex cells increases (Bonfante and Perotto, 1995) and networks are formed

covering the arbuscules (Fester et al., 2001; Hans, 2003). The plastids in these net-

works are connected to each other by so-called stromules (stroma-filled tubules)

(Kohler and Hanson, 2000). In addition, the nucleus is surrounded by plastids

forming octopus-, millipede-like or ring-shaped structures. In cells that do

not contain fungal structures, the plastids are distributed anisotropically through-

out the cytoplasm, similar to cortex cells of nonmycorrhizal roots. The formation

of those dense plastid network-covering arbuscules indicates a dramatically in-

tensified metabolism in these host cells. Figure 3 shows confocal laser scanning

micrographs of plastid networks in AM roots, labelled by green fluorescent protein

(GFP) in transgenic tobacco roots (Fester et al., 2001) and by immunodetection of

the plastid-located 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) in

maize roots (Hans, 2003). This enzyme catalyzes the first committed step of the

plastid-located methylerythritol phosphate (MEP) pathway (see below), stimulated

in AM roots.According to their role in a variety of cellular functions in plants, it has been

shown that microtubules are involved in changes of host cell morphology and


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FIG. 2. Electron micrograph of an arbusculated cortex cell of a mycorrhizal maize root (bar,

0.5 m). The graph shows sections of several arbuscule branches (arb) surrounded by the

periarbuscular membrane (pam) and the interface (if). Note also some of the proliferated

organelles (m, mitochondrion; pt, plastid) and the fragmented vacuole (v). The root was

fixed by high-pressure freeze fixation and embedded in methacrylate. Ultrathin-sections

(90 nm) were stained with uranyl acetate/lead citrate and observed with an EM 900 trans-

mission electron microscope (Zeiss, Oberkochen, Germany). Micrograph courtesy of Diana

Schmidt.

cytoplasmic architecture. Plant cytoskeletal components respond to the penetration

of a symbiotic fungus with the reorganization of microtubules and microfilaments.

However, an understanding of the role played by the cytoskeleton in formation

and function of mycorrhizas has been hampered by technical difficulties in han-

dling mycorrhizal tissues. Recently, application of improved labelling techniques,

suitable for both the plant and the fungal symbiont, combined with either epi-

fluorescence microscopy or laser scanning confocal microscopy, gave interesting

results (Timonen and Peterson, 2002). They show extensive remodelling of the mi-

crotubular cytoskeleton from the early stages of arbuscule development until the

arbuscule senesces and collapses (Genre and Bonfante, 1997, 1998). Four typesof microtubule patterns were observed in arbusculated cells: (i) long bundles of

microtubules crossing the cytoplasm among the arbuscule branches and passing


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FIG.3.C

onfocallaserscanningmicrographsofplastidnetworksin

AM

roots,colonizedbyGlom

usintraradices:A,fluorescen

ceofthe

green-flu

orescentproteintargetedtoth

eplastidcompartmentintransgenictobaccoroots;B,immu

nolocalizationofDXRinarbusculated

rootcortexcellsofmaize.Theplastid-

locatedDXRproteinismainlydetectedbythegreenfluorescencearoundthenucleus(rin

g-shaped

structure)andinthenetworkaroundan

activearbuscule.Thepresenc

eofthearbusculeisindicated

byautofluorescentsignals(red)around

andwithinthenetworkstructure.Thelo

wercellinAandtheupperrightcellinBshowdisintegrating

arbuscules(increasedautofluorescence)

withonly

fewplastidsleftexhibitinggreen-fluorescentprotein(A)or

labelledDXR(B).


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through the arbuscule; (ii) short microtubules connecting fine arbuscule branches

or connecting arbuscule branches either to the cortical region of the cell or to the cell

nucleus; (iii) bundles of microtubules in the periphery (cortical region) of the host

cell and along the hyphal trunk; and (iv) perinuclear bundles of microtubules. In

addition, it was found recently that cortex cells adjacent to the arbusculated cells or

to the intercellular hyphae reorganize their microtubules as well (Blancaflor et al.,

2001). This indicates a molecular dialogue between the symbionts prior to fungal

penetration of the plant cell wall and an active role of the plant cytoskeleton in

mycorrhization rather than passive reaction to the physical pressure created by the

fungus invaginating the cell plasma membrane. The alterations of the microtubular

network are consistent with the identification of a mycorrhiza-inducible -tubulin

gene in maize. Expression studies of corresponding promoter::GUSfusions in to-

bacco indicated that this gene is induced specifically in cells in which arbuscules

are developing (Bonfante et al., 1996).

Besides the possible function of the cytoskeleton in reorganizing the cell for

the accommodation of the arbuscule, the cytoskeleton might also be involved in

developing the periarbuscular membrane. This membrane, although originating

from the plant plasma membrane, shows differences in some of its properties rela-

tive to the membrane around the periphery of the cell. In particular, high activities

of H+-ATPases and phosphate transporters were shown to be located specifically

in that membrane (Gianinazzi-Pearson et al., 1991; Rausch et al., 2001; Harrison

et al., 2002; Paszkowski et al., 2002). Recently, a plasma membrane H+-ATPase

gene from Medicago truncatula has been described for the first time that shows

arbuscule-specific induced expression in mycorrhizal tissue (Krajinski et al., 2002).

The interface compartment that develops between the plant and the fungus is

continuous with the peripheral plant cell wall (Bonfante and Perotto, 1995). Al-

though the fibrillar interface differs from the peripheral plant cell wall in structure,

its components reflect the composition of the wall of the host cell that is being

invaded. By immunocytological approaches, the presence of pectins, xyloglucans,

nonesterified polygalacturonans, arabinogalactans, and hydroxyproline-rich gly-

coproteins within the symbiotic interface was documented (Balestrini et al., 1994;

Perotto et al., 1994; Bonfante and Perotto, 1995). This mixture of primary plant

cell wall components indicates that the arbusculated plant cells have maintained

their abilities to synthesize and secrete cell wall material. That this material does

not assemble further to build up a secondary wall might be the result of lytic ac-

tivities of the fungus (Peretto et al., 1995). When the arbuscule begins to senesce,

the fibrillar material encapsulates the collapsed fungal structures that are then de-

graded completely by the plant cell. Subsequently, the cells regain their original

morphology (Jacquelinet-Jeanmougin et al., 1987) and are able to allow another

arbuscule formation.Some processes of AM establishment are known to be mediated by phyto-

hormones on the plant side, as suggested by application experiments (Barker and


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Tagu, 2000). The levels of cytokinins are higher in shoots and roots of mycorrhizal

plants compared to nonmycorrhizal ones (Allen et al., 1980). A possible role of

abscisic acid was suggested from the fact that its level increases in AM roots

(Danneberg et al., 1992; Bothe et al., 1994). Jasmonic acid applied exogenously

promotes colonization and development of mycorrhizal structures (Regvar et al.,

1996). The observed endogenous rise of jasmonates in barley roots correlating

with myorrhization, however, is more indicative for a role in AM (Hause et al.,

2002). The rise in jasmonates is accompanied by the expression of genes encoding

for an enzyme involved in jasmonate biosynthesis, allene oxide synthase (AOS),

and of a jasmonate-induced protein, JIP23. In situ hybridization and immunocy-

tochemical analyses revealed that expression of the corresponding genes occurred

cell-specifically within arbusculated root cells. Since jasmonate levels increased

after the initial step of the plantfungus interaction, the development of AM may

cause expression of jasmonate biosynthetic genes and finally elevate jasmonate

levels. The induction of jasmonate biosynthesis could be linked to the stronger car-

bohydrate sink function of mycorrhizal roots compared to nonmycorrhizal ones.

Taking into account that jasmonate-induced genes are involved in various defense

responses (Wasternack and Hause, 2002), higher endogenous jasmonate levels

could help mycorrhizal roots to become more resistant to secondary infection

and/or other stresses. It might also indicate plant control of the invading AM fungus.

CHEMISTRY

Despite increasing efforts in research on metabolic changes, virtually nothing

is known about signalling compounds or about the role of secondary metabolites

in the establishment and maintenance of a functioning AM. It is known that com-

pounds from secondary metabolism play a significant role in the interaction be-

tween plants and their biotic and abiotic environment (Harborne, 1993), and it may

be assumed that secondary metabolites play an important role in mycorrhizal sym-

bioses as well. It has been shown that flavonoids occurring in plant roots promote

spore germination of AM fungi (Gianinazzi-Pearson et al., 1989; Tsai and Phillips,

1991). Root exudates of phosphate-deficient white clover plants were more active

in enhancing hyphal elongation ofGlomus fasciculatus spores than exudates from

phosphate-fertilized plants (Elias and Safir, 1987). The level of the phytoalexin

medicarpin is reduced in mycorrhizal Medicago truncatula roots (Harrison and

Dixon, 1993). Roots and root exudates from phosphate-deficient parsley contain

additional compounds that are absent in phosphate-fertilized controls and mycor-

rhizal plants (Franken and Gnadinger, 1994), but the nature and function of these

compounds is unknown. Mycorrhization ofCitrus jambhiri led to an induced ac-

cumulation of leaf sesquiterpenoid volatiles (Nemec and Lund, 1990).It was shown in 1924 by F. R. Jones that mycorrhizal roots of many plants de-

velop a yellow coloration based on accumulation of the so-called yellow pigment.


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This phenomenon has been taken as an indicator to estimate the degree of myc-

orrhization by the naked eye (Daft and Nicholson, 1969; Fyson and Oaks, 1992)

or by colorimetric measurement of root extracts (Becker and Gerdemann, 1977;

Schmitz et al., 1991). The component containing the chromophore of the yel-

low pigment was isolated from mycorrhizal maize roots and identified (as its

dimethyl derivative) by NMR spectroscopy and MS as all-E-4,9-dimethyldodeca-

2,4,6,8,10-pentanedioic acid, named mycorradicin (Klingner et al., 1995a). Be-

sides maize, other gramineous plants (wheat, barley, millet) show the same pattern

of pigment formation (Klingner et al., 1995b). It was later shown in a screening

of 58 species from 36 different plant families that mycorradicin as part of the

yellow pigment is present in mycorrhizal roots of all Liliopsida analyzed and of

a considerable number of Rosopsida (Fester et al., 2002a). In addition, chemical

analysis of the yellow pigment indicated that mycorradicin is the core struc-

ture of a mixture of various oligo- or polyesters with glycosylated cyclohexenone

derivatives. In mycorrhizal maize plants, these esters were localized in vacuolar

hydrophobic droplets (Fester et al., 2002a).

Because of structural similarities of mycorradicin with crocetin (C20-polyene)

and azafrin (C27-apocarotenoid), it was speculated that it derives from a C40-

carotenoid precursor by splitting off two C13-units (Klingner et al., 1995a,b). At the

same time, studies on changes in secondary metabolites in roots of cereals (wheat,

barley, rye, oat) colonized by the AM fungus Glomus intraradices resulted in the

structure elucidation of a mycorrhiza-induced glycosylated cyclohexenone deriva-

tive [blumenol C 9-O-(2-O--glucuronosyl)--glucoside], called blumenin (1)

(Maier et al., 1995). The level of blumenin was found to be directly correlated with

the degree of root mycorrhization. Table 1 depicts the structure of blumenin and

lists all other cyclohexenone derivatives identified so far from mycorrhizal roots

(see below).

It was assumed (Walter et al., 2000) that the aglycone of blumenin, blumenol

C, may be another carotenoid degradation product along with mycorradicin. Thus,

cleavage at the 9,10(9,10)-positions of a C40-carotenoid should lead to mycor-

radicin and the cyclohexenone derivative. Further detailed investigation of mycor-

rhizal barley roots revealed in addition to blumenin the presence of closely related

cyclohexenone derivatives [7,8-dehydroblumenin (2) and 13-hydroxyblumenol

9-O--glucoside (3)], which showed an AM fungus-induced continuous accumu-

lation, whereas putrescine and agmatine amides of 4-coumaric and ferulic acids

increased only transiently in early stages of the root-fungus interaction (Peipp

et al., 1997).

The occurrence of compounds 13 in cereal mycorrhizas initiated a study

on their distribution within the Poaceae. After inoculation of various members of

the Poaceae with Glomus intraradices, HPLC analyses of root extracts revealedmarked changes in the patterns of UV-detectable metabolites along with accu-

mulation of these cyclohexenone derivatives. The latter occur most often in the


12/25


TABLE1

.STRUCTURESOFGLYCOSYLATEDCYCLOHEXENONEDERIV

ATIVESISOLATEDFROM

MYC

ORRHIZALROOTSOFVARIOUS

PLANTS

Compoun

d

R1

R2

R3

Occurrence

Refere

nce

Structuresche

me

1a

GlcUA(1

2)Glc-

CH3

CH3

Hv

b,Ta,Sc,As

Maieretal.,1

995

Zm

Vierheiligetal.,2000

2c

GlcUA(1

2)Glc-

CH3

CH3

Hv

Peippetal.,1

997

3

Glc-

CH2OH

CH3

Hv

Peippetal.,1

997

Hv,Ta,Sc,As

Maieretal.,1

997

Nt,Nr,Le

Maieretal.,2

000

4d

Glc(1

6)Glc-

CH2OH

CH3

Nt,Nr

Maieretal.,1

999,2000

Hv,Ta,Zm


Walteretal.,

2000

5

Glc(1

6)Glc-

COOH

CH3

Nt,Nr

Maieretal.,2

000

Hv,Ta,Zm


6

Glc-

COOH

CH3

Nt,Nr

Maieretal.,2

000

7

Glc(1

6)Glc-

CH3

CH3

Nt,Nr

Maieretal.,2

000

8

Glc(1

6)(Glc1

2)Glc-

CH3

CH3

Nt,Nr

Maieretal.,2

000

9

Glc-

CH3

COOH

Le

Maieretal.,2

000

10e

Glc(1

4)Glc-

CH3

CH3

Zm

Festeretal.,2002a

aBlumenin(inallcompoundslisted:Glc=

-glucose;GlcUA=-glucuronate).

bAs=Avenasativa;Hv=Hordeum

vulga

re;Le=Lycopersiconesculentum;Nr=Nicotianarustica;Nt=

Nicotianatabacum;Sc=Secalecereale;

Ta=Tri

ticumaestivum;Zm=Zeamays.

c7,8-Dehydroblumenin;7,8present,absentinallothercompoundslisted.

dNicoblumin.

eHydrolysisproductoftheyellowpigment.


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tribes Poeae, Triticeae, and Aveneae (Maier et al., 1997). To evaluate the AM-

specific formation of cyclohexenone derivatives, some inoculations of barley roots

with pathogens (Gaeumannomyces graminis and Drechslera sp.) or an endophyte

(Fusarium sp.) were performed. These, as well as treatments with abiotic stressors

(heat, cold, high intensities of light, heavy metals, and drought), did not induce

the formation of cyclohexenone derivatives (Maier et al., 1997). Because of the

occurrence of both C13 and C14 apocarotenoids (cyclohexenone derivatives and

mycorradicin) in mycorrhizal roots, their formation might be similar to that of ab-

scisic acid, involving a dioxygenase-catalyzed cleavage of a carotenoid precursor

(Maier et al., 1998; Walter et al., 2000).

One of the first arguments for a carotenoid origin of cyclohexenone deriva-

tives derived from NMR spectroscopic analysis of blumenin (1)after[13C]glucose-

tracing experiments, indicating a mevalonate-independent biosynthesis of the cy-

lohexenone derivative (Maier et al., 1998). Figure 4 shows the 13C-labelling pattern

of blumenol C, after feeding [1-13C]glucose to barley roots. For comparison, po-

tential labelling of mycorradicin via the MEP pathway and isopentenyldiphosphate

(IPP) synthesized via the mevalonate pathway are shown.

The assumption that the AM-specific isoprenoids are apocarotenoids was

recently supported by a study showing that carotenoid biosynthesis is strongly

stimulated in AM roots, at least partially at the transcriptional level (Fester et al.,

2002b). Tobacco plants transformed with a phytoene desaturase promoter::GUS

construct showed a cell-specific induction of the phytoene desaturase promoter

activity in arbusculated root cells.

The role of the cyclohexenone derivatives in mycorrhizal symbiosis is un-

known, although there is some indication that they might be involved in control

of mycorrhization. Exogenously applied blumenin strongly inhibits colonization

and formation of arbuscules in the early stages of mycorrhiza formation in barley

(Fester et al., 1999). Inoculation of barley, wheat, and maize with different AM

fungi (Glomus mosseae, G. intraradices, and Gigaspora rosea) led to the accu-

mulation of1, 4, 5, and another, yet unidentified, cyclohexenone derivative in all

plant/fungi associations. The relation of all compounds to each other was quantita-

tively different, but qualitatively identical, indicating no fungus-specific induction

of selected compounds (Vierheilig et al., 2000).

In addition to the well-characterized AM fungus-induced accumulation of

cyclohexenone derivatives in gramineous plants, a set of similar compounds was

found in roots of tobacco and tomato (Solanaceae) after colonization with Glomus

intraradices (Maier et al., 1999, 2000). In the major compound, named nicoblumin

(4), the aglycone of 3 (13-hydroxyblumenol C) is connected at the C-9 hydroxyl

group with a 1,6-linked diglucose (gentiobiose). Both in mycorrhizal roots of

two tobacco species (Nicotiana tabacum, N. rustica) and in tomato (Lycopersiconesculentum), 3 was found to accumulate. The aglycone of two further tobacco

cyclohexenone derivatives is formed when the hydroxymethyl group at C-5 in 3 is


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15/25


oxidized to a carboxyl group. This structure accumulates as 9- O-gentiobioside (5)

and 9-O-glucoside (6). Additional strongly induced major compounds in tobacco

are the gentiobiosides of blumenol C occurring as S- andR-isomers at C-9 (7),

whereas a minor component is a triglucosyl derivative of blumenol C (8). In tomato

roots, an additional 9-O-glucoside (9) bearing a carboxyl group at C-1 occurs, but

the relative configurations of the asymmetric sites (C-1, C-6, and C-9) could not

be etablished from NMR spectroscopic data. Further comparative studies of cyclo-

hexenone derivatives in mycorrhizal maize and wheat roots revealed the presence

of six compounds from which only the 9-R,S-isomers of blumenin (1), nicoblumin

(4), and 5 were tentatively identified by comparison of HPLC retention times with

those of reference compounds (Walter et al., 2000). By alkaline hydrolysis of the

yellow pigment purified from mycorrhizal maize roots, besides mycorradicin, an

isomer pair of a cyclohexenone derivative was liberated, which was identified by

NMR spectroscopy as blumenol C 9- O--cellobioside (10) (Fester et al., 2002a),

assumed to form esterification products (oligo- or polyesters) with mycorradicin,

establishing part of the yellow pigment.

MOLECULAR BIOLOGY

Traditionally, levels of metabolites, proteins, or transcripts of known identity

have been tested for being affected by a certain stimulus. In the case of the interac-

tion of plant roots with a symbiotic partner, mineral nutrient transport proteins are

obvious choices. Recently, nontargeted approaches, particularly at the transcript

FIG. 4. Biosynthesis of isopentenyl diphosphate (IPP) via the MEP and mevalonate path-

ways leading to various terpenoids (Rohmer, 1999; Rodriguez-Concepcion and Boronat,

2002). The DXS- and DXR-catalyzed reactions are pointed out in this review. The path-

ways show the 13C-labelling patterns of IPP and blumenol C, the aglycone of blumenin, that

accumulated in AM fungus inoculated barley roots after feeding of [1-13C]glucose (Maier

et al., 1998). For a comparison, potential 13C-labelling of mycorradicin via the MEP path-

way and of IPP via the mevalonate pathway are shown (parts of the chart have been adapted

from Lichtenthaler et al., 1997; Rohmer, 1999). The 13C enrichment in blumenol C deter-

mined by 13C NMR spectroscopy is indicated by dots. The precursor carotenoid (shaded

in grey) is still elusive. Mycorradicin (R=H) is the core structure of the yellow pig-

ment (R= glycosylated cyclohexenone derivatives) (Fester et al., 2002a). Abbreviations:

Ac-CoA, acetyl coenzyme A; AcAc-CoA, acetoacetyl-CoA; DHAP, dihydroxyacetone

phosphate; DXP, 1-deoxy-D-xylulose 5-phosphate; DXR, 1-deoxy-D-xylulose 5-phosphate

reductoisomerase; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; GAP, glyceraldehyde

3-phosphate; HMG-CoA,-hydroxy--methylglutaryl-CoA; IPP, isopentenyl diphosphate;

MEP, 2-C-methyl-D-erythritol 4-phosphate; MVA-5PP, mevalonate 5-diphosphate; TPP,

thiamine diphosphate.


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level, have become popular and have now also been applied to mycorrhizal sys-

tems (Lapopin and Franken, 2000; Franken and Requena, 2001; Stougaard, 2001).

Whole AM root transcriptomes (total mRNA transcripts at a specific stage of my-

corrhization) can be captured in cDNA libraries. Partial sequencing of all or of

a fraction of clones in the library then results in so-called expressed sequence

tags (ESTs) (Journet et al., 2002). A biochemical function can often be attributed

to specific ESTs or to the proteins they encode by finding sequences with high

sequence similarity and a known function in DNA databases. If done on a suffi-

ciently large scale, this approach can provide an, albeit still fragmentary, overview

of metabolic activities during the mycorrhizal symbiosis. Analysis of changes

in the levels of specific transcripts by RNA blot, real time PCR, or DNA array

methods can identify transcripts and genes and, thus, areas of metabolic activity

up- or downregulated during mycorrhization. Subtractive methods (subtraction

of nonmycorrhizal transcripts from the population of mycorrhiza-regulated and

mycorrhiza-unaffected transcripts) can reveal mycorrhiza-affected transcripts al-

ready at the cDNA cloning stage (Voiblet et al., 2001).

Diploid and autogamous plants with a small genome are particularly suitable

for such approaches. Unfortunately, the prime model plant Arabidopsis thaliana,

a member of the Brassicaceae, is not a host for AM fungi. However, extensive

efforts of groups in the United States, France, and Germany on the model legume

plant Medicago truncatula, which easily accommodates various symbiotic part-

ners, have now led to the availability of more than 100,000 ESTs in databases from

various Medicago truncatula tissues (Journet et al., 2002). These include control

roots or roots colonized by AM fungi or other mutualistic and pathogenic microbes.

This resource now even allows various in-silico (computer-based) analyses, e.g.,

to carry out electronic Northern experiments by comparing the frequency of spe-

cific ESTs (representing transcript levels) from defined tissues and environmental

circumstances in the database. Unfortunately, fungal sequences with known iden-

tity are poorly or not at all represented in databases.

Another valuable resource are specific mutants of host plants, which are

blocked at certain stages of the AM fungal colonization. In Medicago truncatula,

many of these mutants are also disturbed in the accommodation of Rhizobium

bacteria forming nodules for nitrogen fixation. A recent example is the identifi-

cation of SYMRK, a receptor-like kinase required for both fungal and bacterial

recognition (Stracke et al., 2002). Whereas this shows the existence of at least

one common signalling component for the recognition of two very different sym-

bionts, characterization of other mutants argues for additional SYM-independent

signalling pathways (Kistner and Parniske, 2002).

One of the plants main benefits from the AM symbiosis is improvement of

phosphate uptake. Recent molecular studies of various phosphate transporters havedemonstrated how plants can adapt their phosphate uptake to the interaction with

the AM fungus. Specific phosphate transporters with different properties compared


17/25


to those known so far are expressed in the arbusculated root cells. It is assumed

that they are located at the periarbuscular membrane where they are involved in the

acquisition of phosphate supplied by the fungus (Gianinazzi-Pearson et al., 1991;

Rausch et al., 2001; Harrison et al., 2002; Paszkowski et al., 2002). Phosphate

transporters active in nonmycorrhizal roots are downregulated. Interestingly, the

AM-specific transporter OsPT11 from rice is not an ortholog of the potato trans-

porter StPT3. Thus, this feature may have evolved several times independently

(Paszkowski et al., 2002). The molecular mechanisms of phosphate transport in

plants are described in a recent review (Rausch and Bucher, 2002).

Isotopic labelling and NMR spectroscopy contributed significantly to our

understanding of the physiology of AM symbiosis (Douds et al., 2000; Pfeffer

et al., 2001). It was shown that glucose and fructose are effectively taken up by

the fungus within the root and are metabolized to yield mainly trehalose and

lipids (Wright et al., 1998; Pfeffer et al., 1999). The lipids are then translocated

to the extraradical mycelium, translocated within AM fungal colonies, and are

recirculated throughout the fungus (Bago et al., 2002). Carbon flux and gene

expression studies indicate that the glyoxylate cycle is central to the flow of carbon

in the AM symbiosis (Lammers et al., 2001).

The first molecular studies on plant secondary metabolites in AM roots were

carried out on phenylpropanoid metabolism in alfalfa and soybean. In alfalfa, in-

creases in transcription levels of phenylalanine ammonia-lyase and chalcone iso-

merase were observed (Volpin et al., 1994). In soybean roots, however, the level

of chalcone isomerase decreased (Lambais and Mehdy, 1993). In Medicago trun-

catula and Phaseolus vulgaris, transcript levels of phenylalanine ammonia-lyase

and chalcone synthase increased (Harrison and Dixon, 1993; Blee and Anderson,

1996), whereas isoflavone reductase transcript formation was suppressed in Med-

icago truncatula (Harrison and Dixon, 1993). The latter correlated with a reduced

accumulation of the phytoalexin medicarpin.

As discussed above, the biosynthesis of apocarotenoids is known to pro-

ceed via the plastid-located nonmevalonate route, which is now commonly called

MEP pathway (Rodriguez-Concepcion and Boronat, 2002). Two key steps of the

MEP pathway, catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and

1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) (Figure 4), were inves-

tigated for changes in transcript levels upon AM formation. Levels of both DXS

and DXR transcripts were considerably higher in AM fungus-colonized roots of

various cereals compared to nonmycorrhizal roots (Walter et al., 2000). In a subse-

quent work with Medicago truncatula, two distantly related DXStranscripts were

identified, which both specified functional DXS proteins (Walter et al., 2002). Only

DXS2 exhibited elevated transcript levels in mycorrhizal roots, whereas DXS1 re-

mained unaffected at a very low level during this interaction. Conversely, DXS1was highly expressed in aerial photosynthetic tissues, where DXS2 transcripts

were low. Additional experiments with nonmycorrhizal and mycorrhizal maize


18/25


and tomato plants corroborated these results. They indicate that there might be

a division of labor between DXS1, expressed for primary functions, such as sup-

ply of chloroplast components, andDXS2 whose expression profiles correlate with

formation of secondary compounds, such as (apo)carotenoids in mycorrhizal roots

(Walter et al., 2002).

The diversification of DXS enzymes and genes from the isopentenyl diphos-

phate-generating MEP pathway and their specific involvement in primary and

secondary products came as a surprise. Previously, assignment of enzymes to

primary and secondary isoprenoid formation was known only for a later step in

this pathway, namely the one catalyzed by various terpene synthases (Trapp and

Croteau, 2001). DXS2 genes and their promoters are currently being isolated in

our laboratory to identify regulatory sequences responsible for the differential,

and particularly the mycorrhiza-dependent, expression. The subsequent step in the

MEP pathway, catalyzed by DXR (Figure 4), does not appear to be diversified,

since a single DXR transcript species accumulates in leaves and mycorrhizal roots

(Hans, 2003).

Finally, production of the AM-specific apocarotenoids is catalyzed by dioxy-

genases. The first of these enzymes, characterized from the pathway leading to the

C15 apocarotenoid abscisic acid, was the vp14 of maize (Tan et al., 1997). A related

carotenoid-cleaving dioxygenase (CCD), generating C14 and C13 apocarotenoids,

was identified later (Schwartz et al., 2001). This enzyme could be involved in the

generation of AM-specific mycorradicin and cyclohexenone derivatives. Unpub-

lished results from our work with maize and Medicago truncatula indicate that a

CCD transcript is mycorrhiza-regulated in roots (Hans, 2003). Taken together, the

AM-specific isoprenoid metabolism and apocarotenoid formation raise attractive

questions to study both the evolution of plant secondary metabolism and its role

in ecological interactions.

SUMMARY

Mutualistic symbioses of mycorrhizas are crucial in the ecology and phys-

iology of terrestrial plants and are most effective in supporting plants to cope

with various environmental stressors, such as nutrient- and water-deficient soils

or pathogenic infection. With some recent success in mycorrhiza research on the

metabolic and genetic levels, we are beginning to understand the complexity of

the chemical dialogue of the two partners. We expect that the growing interest in

mycorrhiza research and availability of new analytical techniques and molecular

genetic approaches will lead in the near future to new insights into the strategies

of plants and fungi to develop mutualistic symbiotic associations. In addition,

understanding the mechanisms that prevent mycorrhizal colonization in nonhostspecies will help to elucidate the molecular interactions responsible for a successful

establishment of mycorrhizal symbioses.


19/25


AcknowledgmentsTheinvestigations at Hallehave been supportedby the Deutsche Forschungs-

gemeinschaft in Bonn (Research Focus Programme 1084: Molecular Basics of Mycorrhizal Sym-

bioses) and Fonds der Chemischen Industrie in Frankfurt. We thank John T. Romeo (Tampa) for

encouraging us to write this review and T. Hartmann (Braunschweig) for comments on the manuscript.

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