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Imaging arbuscular mycorrhizal structures in
living roots of Nicotiana tabacum by light,
epifluorescence, and confocal laser scanning
microscopy
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 same
sections of unstained (living roots) and stained (dead) tobacco (Nicotiana tabacum L.) roots colonized by the
arbuscular mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe. To visualize all mycorrhizal structures, roots were in situ stained with trypan blue. The metabolically active fungal tissue was determined by an in situ
succinate dehydrogenase stain. A comparison of micrographs of unstained and stained mycorrhizal tobacco roots revealed that (i) finely branched arbuscules do not autofluoresce, but high autofluorescence was observed in clumped
structures of collapsed arbuscules; and (ii) finely branched arbuscules are metabolically active, but no activity can be
detected in autofluorescent collapsed arbuscules. Confocal laser scanning microscopy was used in combination with the
two fluorochromes 5(6)-carboxyfluorescein diacetate or 5(6)-carboxy-seminaphthorhodafluor. Both fluorochromes administered 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 the
phloem with 5(6)-carboxy-seminaphthorhodafluor, all fungal structures in the root (from relatively thick hyphae to finest branches of arbuscules) were clearly visible in the intact root. The transport route of compounds from the plants to
arbuscular 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) afin
d’obtenir des micrographies des mêmes sections de racines de tabac non-colorées (vivantes) et colorées (mortes) (Nicotiana tabacum L.), colonisées par le champignon mycorhizien arbusculaire Glomus mosseae (Nicol. & Gerd.) Gerd. &
Trappe. Afin de visualiser toutes les structures mycorhiziennes, ils ont coloré les racines in situ, au bleu trypan. Ils ont
détecté les tissus fongiques métaboliquement actifs par coloration in situ de la succinate déshydrogénase. En comparant
les micrographies de racines de tabac mycorhizées colorées ou non-colorées, on observe que: i) les arbuscules finement
ramifiées ne sont pas autofluorescentes, mais les structures en amas formées d’arbuscules affaissées sont fortement autofluorescentes et ii) les arbuscules finement ramifiées sont métaboliquement actives, mais aucune activité ne se manifeste par autofluorescence, dans les arbuscules affaissées. Les auteurs ont également utilisé la microscopie confocale au
laser en combinaison avec deux fluorochromes, le diacétate de 5(6)-carboxyfluorescéine ou le 5(6)-carboxyseminaphtorhodafluor. Ces deux fluorochromes, appliqués sur des feuilles de tabac scarifiées, sont transportées aux racines via le phloème. Lorsqu’on charge des plantes avec le diacétate de 5(6)-carboxyfluorescéine, on observe une fluorescence 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 aux
branches les plus fines des arbuscules - deviennent clairement visibles dans les racines intactes. On discute du sentier
de transport des composés, de la plante aux champignons mycorhiziens arbusculaires.
Mots clés : Glomales, mycorhizes, fluorescence, SDH, confocal, transport.
[Traduit par la Rédaction]
Vierheilig et al.
237
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 RodewaldStrasse 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.
1
Corresponding author (e-mail: [email protected]).
Can. J. Bot. 79: 231–237 (2001)
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DOI: 10.1139/cjb-79-2-231
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Introduction
Arbuscular mycorrhiza (AM) is a close symbiotic association between most vascular land plants and fungi of the class
Zygomycetes. The arbuscular mycorrhizal fungus (AMF)
colonizes the root forming internal hyphae and highly
branched structures called arbuscules. To visualize AMF in
roots a number of staining techniques has been used, giving
clear images of the fungal structures (Gerdemann 1955;
Phillips and Hayman 1970; Brundrett et al. 1984; Koske and
Gemma 1989; Nicolson 1959; Vierheilig et al. 1998;
Vierheilig and Piché 1998). However, with all these techniques the tissue is killed, and thus, no further in vivo observations of the AM association can be performed.
AMF structures, arbuscules in particular, have been reported to autofluoresce in living mycorrhizal roots (Ames et
al. 1982; Jabaji-Hare et al. 1984; Klingner et al. 1995). Recently, confocal laser scanning microscopy (CLSM) images
indicated that only collapsed arbuscules strongly
autofluoresce but not the highly branched metabolically active arbuscules (Vierheilig et al. 1999).
A method to determine the metabolically active fungal tissue is the succinate dehydrogenase (SDH) stain (MacDonald
and Lewis 1978). SDH, a tricarboxylic acid cycle enzyme in
AMF, reacts with nitro blue tetrazolium chloride (NBT) resulting in insoluble formazan, which can be clearly distinguished in roots. The SDH activity has been suggested as an
indicator for the metabolic activity of AMF and has been
used in numerous studies (Ocampo and Barea 1985; Kough
et al. 1987; Vierheilig and Ocampo 1989, 1991; Smith and
Gianinazzi-Pearson 1990; Schaffer and Peterson 1993).
By CLSM, fluorescent structures can be visualized at an
extremely high resolution without mechanical sectioning
(Czymmek et al. 1994). Various studies have been published
recently using CLSM after staining the AMF with fluorochromes for morphological studies of the mycosymbiont in
root sections (Melville et al. 1998; Dickson and Kolesik
1999; Matsubara et al. 1999).
Fluorochromes in combination with CLSM are excellent
markers to study in vivo transport events in plants. In
Arabidopsis plants, some fluorochromes (e.g., 5(6)-carboxyfluorescein (CF) and 5(6)-carboxy-seminaphthorhodafluor
(cSNARF)) administered to abraded leaves (CF is administered
as the non-fluorescent 5(6)-carboxyfluorescein diacetate
(CFDA), which is cleaved to the fluorescent CF) are transported via the phloem to the roots, where they move into the
adjacent pericycle, endodermal, cortical, and epidermal cells
(Oparka et al. 1994, 1995; Wright et al. 1996).
CF has been applied successfully to visualize the phloem
unloading in Arabidopsis roots into the feeding structures of
cyst nematodes (Böckenhoff et al. 1996). Because of the
translucency of its roots, Arabidopsis thaliana has been suggested as a model system for microscopical in vivo studies
of plant-pathogen interactions (Wyss and Grundler 1992).
Unfortunately, Arabidopsis belongs to the Brassicaceae,
which do not host AMF (Harley and Harley 1987). In this
study we investigated the mycorrhizal host plant, tobacco.
Tobacco roots are highly transparent with only a few cell
layers over the central cylinder and, thus, have similar characteristics as found with Arabidopsis.
Can. J. Bot. Vol. 79, 2001
To distinguish inactive, collapsed arbuscules from living,
metabolically active arbuscules, we combined epifluorescence (FM) and light microscopy (LM) with in situ staining
of the total fungal tissue with the dye trypan blue (Vierheilig
and Piché 1998) or in situ SDH staining of viable fungal tissue (MacDonald and Lewis 1978). Moreover, we tested two
fluorochromes for in vivo CLSM-mediated observations of
AMF structures in living roots to study possible nutrient
transport routes in the mycorrhizal symbiosis.
Materials and methods
Biological material and growing conditions
Seeds of tobacco were surface sterilized by soaking in 0.75%
sodium hypochlorite for 5 min, rinsed with tap water, and germinated in vermiculite. The seedlings were inoculated in a growth
chamber (day:night cycle: 16 h light, 22°C : 8 h dark, 20°C; relative humidity 50%) with Glomus mosseae (Nicol. & Gerd.) Gerd.
& Trappe (BEG 12) in the compartment system developed by Wyss
et al. (1991) in a steam-sterilized (40 min, 121°C) mixture of equal
parts (by volume) of sand and soil.
Differentiation of metabolically active and inactive
arbuscules
Six weeks after inoculation with G. mosseae, tobacco roots were
harvested, washed with tap water, and placed on cover slides in
water for microscopical observations. Roots colonized by
G. mosseae were examined and selected by means of LM and FM,
and micrographs of the same root sections with fluorescing and
non-fluorescing arbuscules were taken. Thereafter, two treatments
were performed.
(1) Localization of arbuscules: Some sections with fluorescing
and non-fluorescing arbuscules were cleared by incubation at
room temperature in KOH (10%) for about 24 h. Roots were
rinsed for 15 min in tap water and then stained by incubation
at room temperature in a trypan blue (0.05% w/v) – vinegar
(5% acetic acid, w/v) solution for 18 h. For destaining, roots
were rinsed for 1 h in tap water. In contrast to the technique
described by Vierheilig and Piché (1998), roots were not
boiled. All treatments were performed at room temperature,
giving equally good staining results (A. Pinior, personal communication). Micrographs of stained structures in the root
section were compared with micrographs of the same section
of the living root obtained by LM and FM before staining.
(2) Localization of metabolically active arbuscules: To determine
the viable fungal tissue, some root sections with fluorescing
and non-fluorescing arbuscules were incubated overnight in
NBT-succinate (Mac-Donald and Lewis 1978) without KCN
(Smith and Gianinazzi-Pearson 1990). The next day, the metabolically active tissue of the fungus could be observed distinctly without clearing the roots in KOH. Micrographs of
stained structures in the root section were compared with micrographs of the same root section obtained by LM and FM
before staining.
Fluorochrome application
Eight weeks after inoculation of tobacco plants with
G. mosseae, the surface of one leaf per plant was abraded with
sandpaper and a CFDA (0.5 mg/mL) or a cSNARF solution
(0.5 mg/mL) (Molecular Probes, Eugene, Oreg.) was applied to the
abraded surface of the leaf (1–4 µL). About 24 h after the
fluorochrome application, excised roots were observed using CLSM.
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LM and FM observations were made using a Zeiss Axiophot microscope (Figs. 1–3) or a Reichert-Jung microscope (Figs. 4–6)
equipped with a fluorescence device. Fluorescence was excited by
a band-pass blue filter combination (450–495 nm). Micrographs
were taken with Kodak 400 ASA film. CLSM was performed with
a Leica TCS 4D (Leica, Heidelberg, Germany) at 488 nm (CF) or
564 nm (cSNARF) produced by a 75-mW argon–krypton laser
(Omnichrome, Chino, Calif.). The emission was observed with a
high-magnification water immersion objective (PL APO 63×/1.20
W CORR, Leica) using a FITC band-pass filter (CF) or a 590-nm
long-pass filter (cSNARF).
a different fluorescence depending on the presence or absence of finely branched arbuscules (Fig. 11). Cells with
fine granular structures, identified by LM as metabolically
active highly branched arbuscules (see Figs. 1–6), fluoresced
as a whole (Fig. 11); however, in root cells with collapsed
arbuscules, the clumped arbuscules autofluoresced but not
the rest of the root cell (Fig. 11). Fluorescence was not observed in cells without arbuscules. This indicates a loading
of the fluorochrome into cells with metabolically active
arbuscules but not into cells with collapsed arbuscules or
without arbuscules.
Results
Discussion
Differentiation of metabolically active and inactive
arbuscules
Figures 1–3 show the same root section observed by FM
(Fig. 1), by LM before staining (Fig. 2), and after staining
the root with trypan blue (Fig. 3). By FM, highly autofluorescent clumped structures could be observed in the cell
of a living root (Fig. 1). In vivo observation by LM of the
same root cell revealed hyphae (Fig. 2), which did not
fluoresce by FM (Fig. 1). Autofluorescence was limited to
the clumped structures, which did not fill the whole cell
(Figs. 1 and 2). In a neighbouring cell a granular, nonautofluorescent structure, which seemed to fill the whole cell
was observed (Figs. 1 and 2). After in situ staining with
trypan blue (Fig. 3) in the cell with the highly autofluorescent structure, hyphae became apparent, whereas the
clumped highly autofluorescent structure (Fig. 1) remained
unstained (Fig. 3). In the neighbouring cell after staining, the
granular, non-autofluorescent structure was revealed as a
highly branched arbuscule filling the whole root cell (Fig. 3).
Figures 4–6 present a series of micrographs analogous to
Figs. 1–3. Here the metabolically active fungal tissue was
visualized by the SDH in situ activity determination (Fig. 6).
Again, cells with highly autofluorescent structures were detected (Fig. 4). Two cells neighbouring the cells with autofluorescent structures contained finely branched arbuscules
(Fig. 5), which did not autofluoresce (Fig. 4). After SDH
staining, these branched non-autofluorescent arbuscules
could be clearly identified as metabolically active, but structures that autofluoresced were inactive (Fig. 6).
None of the structures retained their autofluorescense after the trypan blue or the SDH-stain treatment.
In several FM studies the autofluorecence of AMF
structures in roots has been associated with the presence of
arbuscules (Ames et al. 1982; Jabaji-Hare et al. 1984;
Klingner et al. 1995). After observing living mycorrhizal
ryegrass roots by CLSM, Vierheilig et al. (1999) suggested
recently that only collapsed, clumped arbuscules autofluoresce but not highly ramified arbuscules with fine
branches. In our study, the LM and FM images obtained before and after staining the fungus supported this hyphothesis
unequivocally. The collapsed, clumped arbuscules were
strongly autofluorescent in contrast to the highly branched
arbuscules. SDH staining revealed that only highly branched
but not autofluorescing collapsed arbuscules were metabolically active. This clearly demonstrates that the strong fluorescence observed in roots colonized by AMF can be linked
to collapsed and not to active arbuscules.
The differential fluorescent pattern of collapsed and active
arbuscules might be attributed to the local synthesis of
phenolics by affected plant cells. Phenolic compounds accumulating in plant cells as a plant defense response against
microorganisms are known to fluoresce (Jahnen and
Hahlbrock 1988). There is some evidence that the accumulation of autofluorescent material can occur within the fungal
cell wall (Bennett et al. 1996) and is due to the host plant’s
production of phenolic compounds, which affects the integrity of the fungal cell walls (Benhamou et al. 1999). Thus,
the ageing arbuscules might be trapped by accumulated phenolic compounds, which are responsible for the autofluorescence of the collapsed arbuscules.
Interestingly the induction of H2O2 synthesis in plants, the
so-called oxidative burst, another powerful plant defense
mechanism against microorganisms (Lamb and Dixon 1997),
follows a similar pattern as the accumulation of phenolics.
In root segments of Medicago truncatula colonized by the
AMF Glomus intraradices H2O2 accumulated in cells containing clumped arbuscules, but never in cells with highly
branched arbuscules (Salzer et al. 1999). As we and
Vierheilig et al. (1999) have shown, clumped arbuscules are
collapsed, metabolically inactive mycorrhizal structures,
whereas highly branched arbuscules are the metabolically
active part of the fungus; thus, a different perception of these
fungal structures by the plant seems obvious. This could
mean that, whereas the active arbuscule can mask its presence or block a reaction of the plant and thus in terms of activation of plant defense mechanisms remain unperceived by
the plant, the clumped, collapsed arbuscule is perceived by
the plant expressed as an activation of plant defense re-
Microscopical studies of living tissue
Fluorochrome application
After loading tobacco leaves with cSNARF, the fluorochrome was transported via the phloem (not shown) into the
cortical root cells with and without arbuscules (Figs. 7–10).
The fluorescence differed between the root cells. Whereas
cells without arbuscules fluoresced greenish, arbusculated
cells fluoresced reddish or showed no clear fluorescence
(Figs. 9 and 10). In mycorrhizal control plants, no such fluorescence could be detected. Interestingly, the red fluorescence of cSNARF was also detected in intraradical hyphae
(Fig. 10), in the trunk of arbuscules (Fig. 7), and in the finest
branches of arbuscules (Fig. 8). Arbuscules partially (Figs. 8
and 9) or completely filled cells (Fig. 10).
Loading of the phloem with CF resulted in a differential
fluorochrome accumulation in cortical root cells reflected by
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Fig. 1–3. Micrographs of the same root sections of tobacco roots by epifluorescence (FM) and light microscopy (LM) before (living
roots) and after staining (dead roots) with trypan blue. Scale bar = 40 µm. Fig. 1. Autofluorescing structures are visible in the living
root cell (left of asterisks) (FM). Fig. 2. In the living root cell with autofluorescing structures, hyphae and a clumped structure can be
observed (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). In
the neighbouring root cell (arrow) a fully developed arbuscule is heavily stained. Figs. 4–6. Micrographs of the same root sections of
tobacco 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 neighbouring cells of autofluorescing structures (arrows) (LM). Fig. 6. After the SDH staining fine granular structures seen in Fig. 5 can be
clearly 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 different
fluorochromes on leaves of tobacco plants. Figs. 7–10. Loaded fluorochrome cSNARF (wavelength 590 nm). Fig. 7. Trunk of an
arbuscule (arrowhead) filled with reddishly fluorescing cSNARF. Scale bar = 3 µm. Fig. 8. Arbuscule with reddishly fluorescing
cSNARF in the fine and the thicker branches. Scale bar = 10 µm. Fig. 9. Arbusculated root cells (more reddishly fluorescence) and
non-arbusculated root cells (more yellowish fluorescence). Scale bar = 20 µm. 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 fluorescence. Scale bar = 20 µm. Fig. 11. Loaded fluorochrome CFDA. The probe is cleaved in the plant cell and becomes the fluorescent
CF (wavelength 488 nm). CF is accumulated in a cell (X) that showed (not shown) a fine granular structure by LM, identified before
as a metabolically active, finely branched arbuscule. CF was not accumulated in the cell with a collapsed arbuscule (arrowhead) or in
cells without fungal structures. Scale bar = 30 µm.
sponses, e.g., the accumulation of phenolics or H2O2. This
perception possibly is due to fungal cell-wall components
released by the disintegrating arbuscule eliciting plant defense responses.
In our study, as shown in Fig. 3, the autofluorescent
clumped structures did not stain with trypan blue in contrast
to the highly branched arbuscules. The compound(s) in fungal tissues that are stained by various dyes such as trypan
blue (Phillips and Hayman 1970; Vierheilig and Piché
1998), acid fuchsin (Gerdemann 1955), and ink (Vierheilig
et al. 1998) are not exactly known. Chitin, a major compound in fungal cell walls, is thought to be at least one, if
not the only, stained compound. Interestingly non-chitincontaining oomycetes are not stained by these dyes (e.g.,
trypan blue, ink) (H. Vierheilig, unpublished results) also
hinting at chitin as the compound in fungi to which the dyes
attach. Bonfante-Fasolo et al. (1990) reported that the chitin
content is reduced in collapsed arbuscules of AMF. This
could explain the absence of staining in the collapsed
arbuscules through the reduced chitin content in this fungal
structure. This conclusion implies that only metabolically
active arbuscules and those that are just starting to collapse,
and thus do not yet have a reduced chitin content, are clearly
stained by the dyes used frequently in mycorrhizal research.
Staining AMF colonized roots would thus fail to quantify
collapsed arbuscules.
Hitherto only one concept has been proven to be successful for the visualization of living AM structures in roots.
Séjalon-Delmas et al. (1998) reported an in vivo
autofluorescence of hyphae and root infection structures of
an AMF in carrot roots. The autofluorescent component,
which could be observed only in Gigaspora gigantea, but
not in any other AMF, was cytoplasmic and mobile in the
fungal cytoplasmic streaming and, therefore, was proposed
as a natural marker for studies of the dynamics of arbuscular
mycorrhiza. In our study we tested two exogeneously applied fluorochromes in combination with CLSM as an alternative. In the tobacco plants used in our experiment the
phloem mobility of CF and cSNARF, as reported before in
Arabidopsis plants (Oparka et al. 1994, 1995; Wright et al.
1996), was confirmed. Depending on the fluorochrome, two
different unloading patterns in the mycorrhizal roots could
be observed. Loading plants with CFDA resulted in an accumulation of CF only in root cells with highly branched
arbuscules, whereas after loading the phloem with cSNARF,
all fungal structures in the root, from relatively thick hyphae
to finest branches of arbuscules, were clearly visible in the
intact root. These results show that both fluorochromes
could be a useful tool for in vivo studies of AMF in roots.
On the other hand, by these results the question of how
fluorochromes are transported into the arbusculated cell and
into the arbuscule itself arises. CF has been used to study
translocation processes between the phloem and feeding
structures induced by root pathogenic nematodes in the vascular cylinder of Arabidopsis plants (Böckenhoff et al.
1996). In this study it was shown that CF was specifically
unloaded from the phloem at infection sites and translocated
into the nematode feeding structure. In a similar way our
study allowed us to follow the translocation pathway of the
fluorochromes from the shoot via the root into the
arbuscule-containing cells and eventually into the fungus. In
contrast to nematode feeding structures, which are located in
the central cylinder, the arbuscule-containing cells are exclusively in the cortex. Accordingly, the fluorochromes have to
be translocated out of the stelar tissue. Plasmodesmata have
been found in stems between sieve element – companion
cell complexes and parenchyma cells (Kempers et al. 1998).
However, symplasmic transport was linked to physiological
parameters depending on the developmental state of the tissue (Patrick and Offler 1996). Although the cortical cells are
symplastmically linked with the phloem via plasmodesmata
(van Bel and Oparka 1995), their opening status and capacity to translocate assimilates or fluorescent markers to cortical cells is questionable.
Recently, Blee and Anderson (1998) hypothesized that the
presence of functional plasmodesmata at the interface between the arbuscule-containing cells and neighbouring cells
may account for the symplastic flow of carbon to the
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arbuscule. Thus, the presence of CF and cSNARF in the
arbusculated cell seems to confirm a symplastic route from
the phloem to the arbusculated cell. However, Böckenhoff et
al. (1996) recently questioned the exclusively symplastic unloading of CF in roots, considering an “alternative
(apoplastic) step in the transfer of both CF and sucrose from
the phloem” to nematode-infected root cells. Moreover, it
has to be kept in mind that exact loading or unloading characteristics of most fluorochrome dyes in plant cells are not
known.
As the site of sugar transfer from the plant to AMF is still
a matter of debate (Harrison 1999a, 1999b) we were interested to know whether our translocation studies may address
this problem. Reviewing the literature, Smith and Read
(1997) suggested carbon uptake by the intercellular hyphae,
whereas Blee and Anderson (1998) proposed the arbuscules
as being “the key site of interchanges of carbon between
root cells and the hyphae of AMF”. The latter hypothesized
that arbuscule-containing cells become a strong sink for sucrose distinct from the other cortical cells. In tobacco plants,
CF was only accumulated in root cells with finely branched
arbuscules but not in root cells with autofluorescent collapsed arbuscules or in root cells without arbuscules. Thus,
only root cells with metabolically active arbuscules appear
to be sinks for CFDA. For two reasons this does not prove
unequivocally that the arbuscules are the sugar uptake organs of AMF: (i) arbuscule-containing cells could be only a
sink for CF, but not for carbon; (ii) arbuscule-containing
cells might act only as specialized transport cells which
form a sink for assimilates from the phloem, subsequently
draining them into the apoplast. In this way the assimilates
could be specifically supplied to the intercellular space
where they are taken up by the hyphae.
After loading the plant with cSNARF, the fluorochrome
was observed in the finest branches of the arbuscule, in
thicker branches, and the trunks of arbuscules, as well as in
hyphae outside the arbuscule-containing cells, demonstrating
clearly that the fluorochrome penetrated the fungus and not
only stained the fungal cell wall. The presence of cSNARF
in the arbuscules seems to identify these structures as uptake
organs. On the other hand, an uptake of cSNARF by the fungus in the intercellular space and subsequent transport into
the arbuscule cannot be excluded. Moreover, even when
cSNARF is transferred from the plant directly into the
arbuscule, its different characteristics from carbon do not
necessarily mean a similar transfer route for carbon.
Guttenberger (2000a, 2000b) recently reported pH
changes in the arbusculated cell. The fluorochrome cSNARF
has been described as a pH indicator (Haugland 1996); thus,
the differing fluorescence we found in root cells of
cSNARF-loaded plants in absence or in presence of
arbuscules could (i) indicate differing pHs (this aspect needs
further study; however, the aim of this work was not to determine 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 in
living roots with neutral red, arbuscules never fill the entire
cell. In our study some arbuscules, although fully developed
with fine branches, only partially occupied the arbusculated
Can. J. Bot. Vol. 79, 2001
cell in CLSM studies, whereas other cells seemed to be
completely filled by the arbuscule.
To summarize, the parallel application of LM, FM,
CLSM, and the use of fluorescence markers are promising
tools to study the functional status of AMF structures and
possible nutrient transport routes in the mycorrhizal symbiosis. Fluorochromes with different characteristics could be
powerful tools for understanding the transfer of compounds
from the plant to the fungus and vice versa in a mycorrhizal
association.
Acknowledgements
This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. We acknowledge Dr. H. Chamberland for technical advice; Dr. J.G. Lafontaine, both Département de Biologie, Université
Laval, Sainte-Foy, Que., for the use of the Reichert-Jung microscope; and P. Hess, Institut für Allgemeine Botanik und
Pflanzenphysiologie, Giessen, Germany, for critical reading
of the manuscript.
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