Imaging arbuscular mycorrhizal structures in living roots ...bashanfoundation.org/inmemoriam/Vierheilig-H/horstimaging.pdf · Six weeks after inoculation with G. mosseae, tobacco

Imaging arbuscular mycorrhizal structures inliving roots of Nicotiana tabacum by light,epifluorescence, and confocal laser scanningmicroscopy

Horst Vierheilig, Michael Knoblauch, Katja Juergensen, Aart J.E. van Bel,Florian M.W. Grundler, and Yves Piché

Abstract: Light and epifluorescence (blue light excitation) microscopy was used to obtain micrographs of the samesections of unstained (living roots) and stained (dead) tobacco (Nicotiana tabacumL.) roots colonized by thearbuscular mycorrhizal fungusGlomus mosseae(Nicol. & Gerd.) Gerd. & Trappe. To visualize all mycorrhizal struc-tures, roots were in situ stained with trypan blue. The metabolically active fungal tissue was determined by an in situsuccinate dehydrogenase stain. A comparison of micrographs of unstained and stained mycorrhizal tobacco roots re-vealed that (i) finely branched arbuscules do not autofluoresce, but high autofluorescence was observed in clumpedstructures of collapsed arbuscules; and (ii ) finely branched arbuscules are metabolically active, but no activity can bedetected in autofluorescent collapsed arbuscules. Confocal laser scanning microscopy was used in combination with thetwo fluorochromes 5(6)-carboxyfluorescein diacetate or 5(6)-carboxy-seminaphthorhodafluor. Both fluorochromes ad-ministered to abraded tobacco leaves are transported via the phloem to the roots. Loading plants with 5(6)-carboxyfluorescein diacetate resulted in a fluorescence of root cells with highly branched arbuscules. After loading thephloem with 5(6)-carboxy-seminaphthorhodafluor, all fungal structures in the root (from relatively thick hyphae to fin-est branches of arbuscules) were clearly visible in the intact root. The transport route of compounds from the plants toarbuscular mycorrhizal fungi is discussed.

Key words: Glomales, mycorrhiza, fluorescence, SDH, confocal, transport.

Résumé: Les auteurs ont utilisé la microscopie photonique en épifluorescence (excitation en lumière bleue) afind’obtenir des micrographies des mêmes sections de racines de tabac non-colorées (vivantes) et colorées (mortes) (Nico-tiana tabacumL.), colonisées par le champignon mycorhizien arbusculaireGlomus mosseae(Nicol. & Gerd.) Gerd. &Trappe. Afin de visualiser toutes les structures mycorhiziennes, ils ont coloré les racines in situ, au bleu trypan. Ils ontdétecté les tissus fongiques métaboliquement actifs par coloration in situ de la succinate déshydrogénase. En comparantles micrographies de racines de tabac mycorhizées colorées ou non-colorées, on observe que: i) les arbuscules finementramifiées ne sont pas autofluorescentes, mais les structures en amas formées d’arbuscules affaissées sont fortement au-tofluorescentes et ii) les arbuscules finement ramifiées sont métaboliquement actives, mais aucune activité ne se mani-feste par autofluorescence, dans les arbuscules affaissées. Les auteurs ont également utilisé la microscopie confocale aulaser en combinaison avec deux fluorochromes, le diacétate de 5(6)-carboxyfluorescéine ou le 5(6)-carboxy-seminaphtorhodafluor. Ces deux fluorochromes, appliqués sur des feuilles de tabac scarifiées, sont transportées aux ra-cines via le phloème. Lorsqu’on charge des plantes avec le diacétate de 5(6)-carboxyfluorescéine, on observe une fluo-rescence des cellules racinaires portant des arbuscules fortement ramifiées. Si on charge le phloème avec du 5(6)-carboxy-seminaphtorhodafluor, toutes les structures fongiques de la racines - des hyphes relativement épaisses auxbranches les plus fines des arbuscules - deviennent clairement visibles dans les racines intactes. On discute du sentierde transport des composés, de la plante aux champignons mycorhiziens arbusculaires.

Mots clés: Glomales, mycorhizes, fluorescence, SDH, confocal, transport.

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DOI: 10.1139/cjb-79-2-231

Received August 4, 2000. Published on the NRC Research Press website on February 15, 2001.

Y. Piché and H. Vierheilig.1 Centre de recherche en biologie forestière, Pavillon C.-E.-Marchand, Université Laval, QC GlK 7P4,Canada.K. Juergensen and F.M.W. Grundler. Institut für Phytopathologie, Christian-Albrechts-Universität Kiel, Hermann Rodewald-Strasse 9, D-24118 Kiel, Germany.M. Knoblauch and A.J.E. van Bel. Institut für Allgemeine Botanik und Pflanzenphysiologie, Justus-Liebig-Universität,Senckenbergstrasse 17, 35390 Giessen, Germany.1Corresponding author (e-mail: [email protected]).

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Introduction

Arbuscular mycorrhiza (AM) is a close symbiotic associa-tion between most vascular land plants and fungi of the classZygomycetes. The arbuscular mycorrhizal fungus (AMF)colonizes the root forming internal hyphae and highlybranched structures called arbuscules. To visualize AMF inroots a number of staining techniques has been used, givingclear images of the fungal structures (Gerdemann 1955;Phillips and Hayman 1970; Brundrett et al. 1984; Koske andGemma 1989; Nicolson 1959; Vierheilig et al. 1998;Vierheilig and Piché 1998). However, with all these tech-niques the tissue is killed, and thus, no further in vivo obser-vations of the AM association can be performed.

AMF structures, arbuscules in particular, have been re-ported to autofluoresce in living mycorrhizal roots (Ames etal. 1982; Jabaji-Hare et al. 1984; Klingner et al. 1995). Re-cently, confocal laser scanning microscopy (CLSM) imagesindicated that only collapsed arbuscules stronglyautofluoresce but not the highly branched metabolically ac-tive arbuscules (Vierheilig et al. 1999).

A method to determine the metabolically active fungal tis-sue is the succinate dehydrogenase (SDH) stain (MacDonaldand Lewis 1978). SDH, a tricarboxylic acid cycle enzyme inAMF, reacts with nitro blue tetrazolium chloride (NBT) re-sulting in insoluble formazan, which can be clearly distin-guished in roots. The SDH activity has been suggested as anindicator for the metabolic activity of AMF and has beenused in numerous studies (Ocampo and Barea 1985; Koughet al. 1987; Vierheilig and Ocampo 1989, 1991; Smith andGianinazzi-Pearson 1990; Schaffer and Peterson 1993).

By CLSM, fluorescent structures can be visualized at anextremely high resolution without mechanical sectioning(Czymmek et al. 1994). Various studies have been publishedrecently using CLSM after staining the AMF with fluoro-chromes for morphological studies of the mycosymbiont inroot sections (Melville et al. 1998; Dickson and Kolesik1999; Matsubara et al. 1999).

Fluorochromes in combination with CLSM are excellentmarkers to study in vivo transport events in plants. InArabidopsisplants, some fluorochromes (e.g., 5(6)-carboxy-fluorescein (CF) and 5(6)-carboxy-seminaphthorhodafluor(cSNARF)) administered to abraded leaves (CF is administeredas the non-fluorescent 5(6)-carboxyfluorescein diacetate(CFDA), which is cleaved to the fluorescent CF) are trans-ported via the phloem to the roots, where they move into theadjacent pericycle, endodermal, cortical, and epidermal cells(Oparka et al. 1994, 1995; Wright et al. 1996).

CF has been applied successfully to visualize the phloemunloading inArabidopsisroots into the feeding structures ofcyst nematodes (Böckenhoff et al. 1996). Because of thetranslucency of its roots,Arabidopsis thalianahas been sug-gested as a model system for microscopical in vivo studiesof plant-pathogen interactions (Wyss and Grundler 1992).Unfortunately, Arabidopsis belongs to the Brassicaceae,which do not host AMF (Harley and Harley 1987). In thisstudy we investigated the mycorrhizal host plant, tobacco.Tobacco roots are highly transparent with only a few celllayers over the central cylinder and, thus, have similar char-acteristics as found withArabidopsis.

To distinguish inactive, collapsed arbuscules from living,metabolically active arbuscules, we combined epifluores-cence (FM) and light microscopy (LM) with in situ stainingof the total fungal tissue with the dye trypan blue (Vierheiligand Piché 1998) or in situ SDH staining of viable fungal tis-sue (MacDonald and Lewis 1978). Moreover, we tested twofluorochromes for in vivo CLSM-mediated observations ofAMF structures in living roots to study possible nutrienttransport routes in the mycorrhizal symbiosis.

Materials and methods

Biological material and growing conditionsSeeds of tobacco were surface sterilized by soaking in 0.75%

sodium hypochlorite for 5 min, rinsed with tap water, and germi-nated in vermiculite. The seedlings were inoculated in a growthchamber (day:night cycle: 16 h light, 22°C : 8 h dark, 20°C; rela-tive humidity 50%) withGlomus mosseae(Nicol. & Gerd.) Gerd.& Trappe (BEG 12) in the compartment system developed by Wysset al. (1991) in a steam-sterilized (40 min, 121°C) mixture of equalparts (by volume) of sand and soil.

Differentiation of metabolically active and inactivearbuscules

Six weeks after inoculation withG. mosseae, tobacco roots wereharvested, washed with tap water, and placed on cover slides inwater for microscopical observations. Roots colonized byG. mosseaewere examined and selected by means of LM and FM,and micrographs of the same root sections with fluorescing andnon-fluorescing arbuscules were taken. Thereafter, two treatmentswere performed.(1) Localization of arbuscules: Some sections with fluorescing

and non-fluorescing arbuscules were cleared by incubation atroom temperature in KOH (10%) for about 24 h. Roots wererinsed for 15 min in tap water and then stained by incubationat room temperature in a trypan blue (0.05% w/v) – vinegar(5% acetic acid, w/v) solution for 18 h. For destaining, rootswere rinsed for 1 h in tap water. In contrast to the techniquedescribed by Vierheilig and Piché (1998), roots were notboiled. All treatments were performed at room temperature,giving equally good staining results (A. Pinior, personal com-munication). Micrographs of stained structures in the rootsection were compared with micrographs of the same sectionof the living root obtained by LM and FM before staining.

(2) Localization of metabolically active arbuscules: To determinethe viable fungal tissue, some root sections with fluorescingand non-fluorescing arbuscules were incubated overnight inNBT-succinate (Mac-Donald and Lewis 1978) without KCN(Smith and Gianinazzi-Pearson 1990). The next day, the met-abolically active tissue of the fungus could be observed dis-tinctly without clearing the roots in KOH. Micrographs ofstained structures in the root section were compared with mi-crographs of the same root section obtained by LM and FMbefore staining.

Fluorochrome applicationEight weeks after inoculation of tobacco plants with

G. mosseae, the surface of one leaf per plant was abraded withsandpaper and a CFDA (0.5 mg/mL) or a cSNARF solution(0.5 mg/mL) (Molecular Probes, Eugene, Oreg.) was applied to theabraded surface of the leaf (1–4µL). About 24 h after thefluorochrome application, excised roots were observed using CLSM.

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Microscopical studies of living tissueLM and FM observations were made using a Zeiss Axiophot mi-

croscope (Figs. 1–3) or a Reichert-Jung microscope (Figs. 4–6)equipped with a fluorescence device. Fluorescence was excited bya band-pass blue filter combination (450–495 nm). Micrographswere taken with Kodak 400 ASA film. CLSM was performed witha Leica TCS 4D (Leica, Heidelberg, Germany) at 488 nm (CF) or564 nm (cSNARF) produced by a 75-mW argon–krypton laser(Omnichrome, Chino, Calif.). The emission was observed with ahigh-magnification water immersion objective (PL APO 63×/1.20W CORR, Leica) using a FITC band-pass filter (CF) or a 590-nmlong-pass filter (cSNARF).

Results

Differentiation of metabolically active and inactivearbuscules

Figures 1–3 show the same root section observed by FM(Fig. 1), by LM before staining (Fig. 2), and after stainingthe root with trypan blue (Fig. 3). By FM, highly auto-fluorescent clumped structures could be observed in the cellof a living root (Fig. 1). In vivo observation by LM of thesame root cell revealed hyphae (Fig. 2), which did notfluoresce by FM (Fig. 1). Autofluorescence was limited tothe clumped structures, which did not fill the whole cell(Figs. 1 and 2). In a neighbouring cell a granular, non-autofluorescent structure, which seemed to fill the whole cellwas observed (Figs. 1 and 2). After in situ staining withtrypan blue (Fig. 3) in the cell with the highly auto-fluorescent structure, hyphae became apparent, whereas theclumped highly autofluorescent structure (Fig. 1) remainedunstained (Fig. 3). In the neighbouring cell after staining, thegranular, non-autofluorescent structure was revealed as ahighly branched arbuscule filling the whole root cell (Fig. 3).

Figures 4–6 present a series of micrographs analogous toFigs. 1–3. Here the metabolically active fungal tissue wasvisualized by the SDH in situ activity determination (Fig. 6).Again, cells with highly autofluorescent structures were de-tected (Fig. 4). Two cells neighbouring the cells with auto-fluorescent structures contained finely branched arbuscules(Fig. 5), which did not autofluoresce (Fig. 4). After SDHstaining, these branched non-autofluorescent arbusculescould be clearly identified as metabolically active, but struc-tures that autofluoresced were inactive (Fig. 6).

None of the structures retained their autofluorescense af-ter the trypan blue or the SDH-stain treatment.

Fluorochrome applicationAfter loading tobacco leaves with cSNARF, the fluoro-

chrome was transported via the phloem (not shown) into thecortical root cells with and without arbuscules (Figs. 7–10).The fluorescence differed between the root cells. Whereascells without arbuscules fluoresced greenish, arbusculatedcells fluoresced reddish or showed no clear fluorescence(Figs. 9 and 10). In mycorrhizal control plants, no such fluo-rescence could be detected. Interestingly, the red fluores-cence of cSNARF was also detected in intraradical hyphae(Fig. 10), in the trunk of arbuscules (Fig. 7), and in the finestbranches of arbuscules (Fig. 8). Arbuscules partially (Figs. 8and 9) or completely filled cells (Fig. 10).

Loading of the phloem with CF resulted in a differentialfluorochrome accumulation in cortical root cells reflected by

a different fluorescence depending on the presence or ab-sence of finely branched arbuscules (Fig. 11). Cells withfine granular structures, identified by LM as metabolicallyactive highly branched arbuscules (see Figs. 1–6), fluorescedas a whole (Fig. 11); however, in root cells with collapsedarbuscules, the clumped arbuscules autofluoresced but notthe rest of the root cell (Fig. 11). Fluorescence was not ob-served in cells without arbuscules. This indicates a loadingof the fluorochrome into cells with metabolically activearbuscules but not into cells with collapsed arbuscules orwithout arbuscules.

Discussion

In several FM studies the autofluorecence of AMFstructures in roots has been associated with the presence ofarbuscules (Ames et al. 1982; Jabaji-Hare et al. 1984;Klingner et al. 1995). After observing living mycorrhizalryegrass roots by CLSM, Vierheilig et al. (1999) suggestedrecently that only collapsed, clumped arbuscules auto-fluoresce but not highly ramified arbuscules with finebranches. In our study, the LM and FM images obtained be-fore and after staining the fungus supported this hyphothesisunequivocally. The collapsed, clumped arbuscules werestrongly autofluorescent in contrast to the highly branchedarbuscules. SDH staining revealed that only highly branchedbut not autofluorescing collapsed arbuscules were metaboli-cally active. This clearly demonstrates that the strong fluo-rescence observed in roots colonized by AMF can be linkedto collapsed and not to active arbuscules.

The differential fluorescent pattern of collapsed and activearbuscules might be attributed to the local synthesis ofphenolics by affected plant cells. Phenolic compounds accu-mulating in plant cells as a plant defense response againstmicroorganisms are known to fluoresce (Jahnen andHahlbrock 1988). There is some evidence that the accumula-tion of autofluorescent material can occur within the fungalcell wall (Bennett et al. 1996) and is due to the host plant’sproduction of phenolic compounds, which affects the integ-rity of the fungal cell walls (Benhamou et al. 1999). Thus,the ageing arbuscules might be trapped by accumulated phe-nolic compounds, which are responsible for the auto-fluorescence of the collapsed arbuscules.

Interestingly the induction of H2O2 synthesis in plants, theso-called oxidative burst, another powerful plant defensemechanism against microorganisms (Lamb and Dixon 1997),follows a similar pattern as the accumulation of phenolics.In root segments ofMedicago truncatulacolonized by theAMF Glomus intraradicesH2O2 accumulated in cells con-taining clumped arbuscules, but never in cells with highlybranched arbuscules (Salzer et al. 1999). As we andVierheilig et al. (1999) have shown, clumped arbuscules arecollapsed, metabolically inactive mycorrhizal structures,whereas highly branched arbuscules are the metabolicallyactive part of the fungus; thus, a different perception of thesefungal structures by the plant seems obvious. This couldmean that, whereas the active arbuscule can mask its pres-ence or block a reaction of the plant and thus in terms of ac-tivation of plant defense mechanisms remain unperceived bythe plant, the clumped, collapsed arbuscule is perceived bythe plant expressed as an activation of plant defense re-

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sponses, e.g., the accumulation of phenolics or H2O2. Thisperception possibly is due to fungal cell-wall componentsreleased by the disintegrating arbuscule eliciting plant de-fense responses.

In our study, as shown in Fig. 3, the autofluorescentclumped structures did not stain with trypan blue in contrastto the highly branched arbuscules. The compound(s) in fun-gal tissues that are stained by various dyes such as trypanblue (Phillips and Hayman 1970; Vierheilig and Piché1998), acid fuchsin (Gerdemann 1955), and ink (Vierheiliget al. 1998) are not exactly known. Chitin, a major com-pound in fungal cell walls, is thought to be at least one, ifnot the only, stained compound. Interestingly non-chitin-containing oomycetes are not stained by these dyes (e.g.,trypan blue, ink) (H. Vierheilig, unpublished results) alsohinting at chitin as the compound in fungi to which the dyesattach. Bonfante-Fasolo et al. (1990) reported that the chitincontent is reduced in collapsed arbuscules of AMF. Thiscould explain the absence of staining in the collapsedarbuscules through the reduced chitin content in this fungalstructure. This conclusion implies that only metabolicallyactive arbuscules and those that are just starting to collapse,and thus do not yet have a reduced chitin content, are clearlystained by the dyes used frequently in mycorrhizal research.Staining AMF colonized roots would thus fail to quantifycollapsed arbuscules.

Hitherto only one concept has been proven to be success-ful for the visualization of living AM structures in roots.Séjalon-Delmas et al. (1998) reported an in vivoautofluorescence of hyphae and root infection structures ofan AMF in carrot roots. The autofluorescent component,which could be observed only inGigaspora gigantea, butnot in any other AMF, was cytoplasmic and mobile in thefungal cytoplasmic streaming and, therefore, was proposedas a natural marker for studies of the dynamics of arbuscularmycorrhiza. In our study we tested two exogeneously ap-plied fluorochromes in combination with CLSM as an alter-native. In the tobacco plants used in our experiment thephloem mobility of CF and cSNARF, as reported before in

Arabidopsisplants (Oparka et al. 1994, 1995; Wright et al.1996), was confirmed. Depending on the fluorochrome, twodifferent unloading patterns in the mycorrhizal roots couldbe observed. Loading plants with CFDA resulted in an accu-mulation of CF only in root cells with highly branchedarbuscules, whereas after loading the phloem with cSNARF,all fungal structures in the root, from relatively thick hyphaeto finest branches of arbuscules, were clearly visible in theintact root. These results show that both fluorochromescould be a useful tool for in vivo studies of AMF in roots.On the other hand, by these results the question of howfluorochromes are transported into the arbusculated cell andinto the arbuscule itself arises. CF has been used to studytranslocation processes between the phloem and feedingstructures induced by root pathogenic nematodes in the vas-cular cylinder of Arabidopsis plants (Böckenhoff et al.1996). In this study it was shown that CF was specificallyunloaded from the phloem at infection sites and translocatedinto the nematode feeding structure. In a similar way ourstudy allowed us to follow the translocation pathway of thefluorochromes from the shoot via the root into thearbuscule-containing cells and eventually into the fungus. Incontrast to nematode feeding structures, which are located inthe central cylinder, the arbuscule-containing cells are exclu-sively in the cortex. Accordingly, the fluorochromes have tobe translocated out of the stelar tissue. Plasmodesmata havebeen found in stems between sieve element – companioncell complexes and parenchyma cells (Kempers et al. 1998).However, symplasmic transport was linked to physiologicalparameters depending on the developmental state of the tis-sue (Patrick and Offler 1996). Although the cortical cells aresymplastmically linked with the phloem via plasmodesmata(van Bel and Oparka 1995), their opening status and capac-ity to translocate assimilates or fluorescent markers to corti-cal cells is questionable.

Recently, Blee and Anderson (1998) hypothesized that thepresence of functional plasmodesmata at the interface be-tween the arbuscule-containing cells and neighbouring cellsmay account for the symplastic flow of carbon to the

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Fig. 1–3. Micrographs of the same root sections of tobacco roots by epifluorescence (FM) and light microscopy (LM) before (livingroots) and after staining (dead roots) with trypan blue. Scale bar = 40µm. Fig. 1. Autofluorescing structures are visible in the livingroot cell (left of asterisks) (FM). Fig. 2. In the living root cell with autofluorescing structures, hyphae and a clumped structure can beobserved (left of asterisks) (LM). In a neighbouring cell (arrow) a fine granular structure is visible, which does not autofluoresce(Fig. 1). Fig. 3. Hyphae in the root cell (left of asterisks) with autofluorescing structures (Fig. 1) are stained with trypan blue (LM). Inthe neighbouring root cell (arrow) a fully developed arbuscule is heavily stained.Figs. 4–6.Micrographs of the same root sections oftobacco roots by FM and LM before (living roots) and after staining (dead roots) with succinate dehydrogenase (SDH). Scale bar =20 µm. Fig. 4. Autofluorescing structures are indicated by asterisks (FM). Fig. 5. Fine granular structures are visible in the neighbour-ing cells of autofluorescing structures (arrows) (LM). Fig. 6. After the SDH staining fine granular structures seen in Fig. 5 can beclearly identified as metabolically active arbuscules (LM). Autofluorescent structures from Fig. 4 did not stain with SDH.Figs. 7–11.Micrographs obtained by confocal laser scanning microscopy of mycorrhizal structures in living roots after loading two differentfluorochromes on leaves of tobacco plants. Figs. 7–10. Loaded fluorochrome cSNARF (wavelength 590 nm). Fig. 7. Trunk of anarbuscule (arrowhead) filled with reddishly fluorescing cSNARF. Scale bar = 3µm. Fig. 8. Arbuscule with reddishly fluorescingcSNARF in the fine and the thicker branches. Scale bar = 10µm. Fig. 9. Arbusculated root cells (more reddishly fluorescence) andnon-arbusculated root cells (more yellowish fluorescence). Scale bar = 20µm. Fig. 10. An arbuscule with fine and thick branches,which is connected to a hypha with a reddishly fluorescing lumen. Neighbouring cells without arbuscules show a strong green fluores-cence. Scale bar = 20µm. Fig. 11. Loaded fluorochrome CFDA. The probe is cleaved in the plant cell and becomes the fluorescentCF (wavelength 488 nm). CF is accumulated in a cell (X) that showed (not shown) a fine granular structure by LM, identified beforeas a metabolically active, finely branched arbuscule. CF was not accumulated in the cell with a collapsed arbuscule (arrowhead) or incells without fungal structures. Scale bar = 30µm.



arbuscule. Thus, the presence of CF and cSNARF in thearbusculated cell seems to confirm a symplastic route fromthe phloem to the arbusculated cell. However, Böckenhoff etal. (1996) recently questioned the exclusively symplastic un-loading of CF in roots, considering an “alternative(apoplastic) step in the transfer of both CF and sucrose fromthe phloem” to nematode-infected root cells. Moreover, ithas to be kept in mind that exact loading or unloading char-acteristics of most fluorochrome dyes in plant cells are notknown.

As the site of sugar transfer from the plant to AMF is stilla matter of debate (Harrison 1999a, 1999b) we were inter-ested to know whether our translocation studies may addressthis problem. Reviewing the literature, Smith and Read(1997) suggested carbon uptake by the intercellular hyphae,whereas Blee and Anderson (1998) proposed the arbusculesas being “the key site of interchanges of carbon betweenroot cells and the hyphae of AMF”. The latter hypothesizedthat arbuscule-containing cells become a strong sink for su-crose distinct from the other cortical cells. In tobacco plants,CF was only accumulated in root cells with finely branchedarbuscules but not in root cells with autofluorescent col-lapsed arbuscules or in root cells without arbuscules. Thus,only root cells with metabolically active arbuscules appearto be sinks for CFDA. For two reasons this does not proveunequivocally that the arbuscules are the sugar uptake or-gans of AMF: (i) arbuscule-containing cells could be only asink for CF, but not for carbon; (ii ) arbuscule-containingcells might act only as specialized transport cells whichform a sink for assimilates from the phloem, subsequentlydraining them into the apoplast. In this way the assimilatescould be specifically supplied to the intercellular spacewhere they are taken up by the hyphae.

After loading the plant with cSNARF, the fluorochromewas observed in the finest branches of the arbuscule, inthicker branches, and the trunks of arbuscules, as well as inhyphae outside the arbuscule-containing cells, demonstratingclearly that the fluorochrome penetrated the fungus and notonly stained the fungal cell wall. The presence of cSNARFin the arbuscules seems to identify these structures as uptakeorgans. On the other hand, an uptake of cSNARF by the fun-gus in the intercellular space and subsequent transport intothe arbuscule cannot be excluded. Moreover, even whencSNARF is transferred from the plant directly into thearbuscule, its different characteristics from carbon do notnecessarily mean a similar transfer route for carbon.

Guttenberger (2000a, 2000b) recently reported pHchanges in the arbusculated cell. The fluorochrome cSNARFhas been described as a pH indicator (Haugland 1996); thus,the differing fluorescence we found in root cells ofcSNARF-loaded plants in absence or in presence ofarbuscules could (i) indicate differing pHs (this aspect needsfurther study; however, the aim of this work was not to de-termine the pH in root cells of mycorrhizal plants) and(ii ) be due to differing accumulation levels of the compound.

In partial contrast to our observations, Guttenberger(2000b) recently reported that, after staining mycorrhiza inliving roots with neutral red, arbuscules never fill the entirecell. In our study some arbuscules, although fully developedwith fine branches, only partially occupied the arbusculated

cell in CLSM studies, whereas other cells seemed to becompletely filled by the arbuscule.

To summarize, the parallel application of LM, FM,CLSM, and the use of fluorescence markers are promisingtools to study the functional status of AMF structures andpossible nutrient transport routes in the mycorrhizal symbio-sis. Fluorochromes with different characteristics could bepowerful tools for understanding the transfer of compoundsfrom the plant to the fungus and vice versa in a mycorrhizalassociation.

Acknowledgements

This work was supported by a grant from the Natural Sci-ences and Engineering Research Council of Canada. We ac-knowledge Dr. H. Chamberland for technical advice; Dr. J.-G. Lafontaine, both Département de Biologie, UniversitéLaval, Sainte-Foy, Que., for the use of the Reichert-Jung mi-croscope; and P. Hess, Institut für Allgemeine Botanik undPflanzenphysiologie, Giessen, Germany, for critical readingof the manuscript.

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Imaging arbuscular mycorrhizal structures in living roots ...bashanfoundation.org/inmemoriam/Vierheilig-H/horstimaging.pdf · Six weeks after inoculation with G. mosseae, tobacco