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Location and quantification of phosphorus and other
elements in fully hydrated, soil-grown arbuscular
mycorrhizas: a cryo-analytical scanning electron
microscopy study
Blackwell Publishing Ltd.
M. H. Ryan1,3, M. E. McCully1 and C. X. Huang1,2
1
CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia; 2Research School of Biological Sciences, Australian National University, Canberra,
ACT 0200, Australia; 3Present address; School of Plant Biology MO81, The University of Western Australia, 35 Stirling Highway, Crawley WA 6009, Australia
Summary
Author for correspondence:
Megan Ryan
Tel: +61 8 93802208
Fax: +61 8 93801140
Email: [email protected]
Received: 10 April 2003
Accepted: 24 June 2003
doi: 10.1046/j.1469-8137.2003.00884.x
• Concentrations of phosphorus (P), potassium (K), magnesium (Mg) and calcium
(Ca) were determined in situ in fully hydrated arbuscular mycorrhizas by cryoanalytical scanning electron microscopy. The field- and glasshouse-grown plants
(subterranean and white clovers, field pea and leek) were colonized by indigenous
mycorrhizal fungi.
• The [P] in intraradical hyphae was generally 60–170 mM, although up to 600 mM
was recorded, and formed strong linear relationships with [K], up to 350 mM, and
[Mg], up to 175 mM. Little Ca was detected. The turgid branches of young arbuscules
contained 30–50 mM P, up to 100 mM K and little Mg. Collapsing arbuscule branches
and clumped arbuscules had greatly elevated Ca (30–250 mM), but otherwise differed little from young arbuscule branches in elemental concentration.
• The [P] was low or undetectable in 86% of uncolonized cortical cell vacuoles, but
was generally elevated in vacuoles surrounding an arbuscule and in the liquid surrounding hyphae in intercellular spaces.
• Our results suggest that both young arbuscules and intercellular hyphae are
sites for P-transfer, that Mg2+ and K+ are probably balancing cations for P anions in
hyphae, and that host cells may limit arbuscule lifespan through deposition of material rich in Ca.
Key words: arbuscular mycorrhizal fungi (AMF), cryo-scanning electron microscopy,
root intercellular spaces, root cell vacuoles, potassium, calcium, magnesium,
phosphorus, host defence response, X-ray microanalysis.
© New Phytologist (2003) 160: 429–441
Introduction
Current knowledge of the structure of arbuscular mycorrhizas
is strongly based on a small number of early microscopy
studies conducted primarily during the 1970s and early
1980s (Cox & Sanders, 1974; Kinden & Brown, 1975a,b,c,
1976; Holley & Peterson, 1979; Walker & Powell, 1979;
Gianinazzi-Pearson et al., 1981; Scannerini & BonfanteFasolo, 1983; Toth & Miller, 1984). Observations reported
in these studies, along with those from a few more recent
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structural studies (Bonfante & Perotto, 1995; Dickson
& Kolesik, 1999; Dickson & Smith, 2001; Vierheilig
et al., 2001), underpin interpretation of all research into
arbuscular mycorrhizas (see also Smith & Read, 1997).
While development of methods for more direct assessment
of mycorrhizal functioning has been rapid and diverse
(e.g. molecular techniques, plant mutants that block hyphal
development and axenic cultures), it has been difficult
to mesh together the findings from morphological and
functional studies. This is partly because the methods used
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in the structural studies, while yielding much important
architectural information, have obscured essential features of
the living, functioning tissues by the extraction of all water
and water- and solvent-soluble materials. As a consequence,
many basic aspects of mycorrhizas are still primarily subjects
of speculation. For example, while the ability of arbuscular
mycorrhizal fungi (AMF) to deliver phosphorus (P) to plants
is well appreciated, and considered the primary benefit of the
symbiosis, the actual location and method of P-transfer
remains unclear.
It is generally believed that the transfer of P to the plant
occurs at the interface between root cortical cell cytosol and
specialized, but relatively short-lived, fungal structures termed
arbuscules (Smith & Smith, 1990; Gianinazzi-Pearson et al.,
1991; Smith & Read, 1997; Smith et al., 2001). One of these
convoluted, multibranched structures may develop in individual cortical cells after the cell wall is breached by either a
large hypha growing directly from cell to cell (intracellular
hypha), or by a branch from a hypha growing through intercellular spaces (intercellular hypha). Although the arbuscule
may eventually fill much of the cell volume, it remains outside
the cell plasma membrane, which invaginates around each
small branch of the extending arbuscule. The large area of fungal/root-cell interface that is thereby formed is certainly consistent with the arbuscule being an especially efficient location
for P-transfer and much evidence supports this scenario
(Smith et al., 2001). However, the role of arbuscules in Ptransfer remains to be proven definitively (Smith et al., 2001)
and it is not possible to rule out other locations, such as intracellular hyphal coils (Dickson & Kolesik, 1999) or intercellular spaces colonized by hyphae.
As pointed out by Smith & Read (1997) all fungal structures within the root lie within the root apoplast and all transfer of nutrients between fungus and host plant is in either
direction across the apoplast. However, the apoplastic environments surrounding intercellular and intracellular fungal
components – termed by Smith & Read (1997) as intercellular
and intracellular apoplast, respectively – are likely to be quite
different and these differences may be very important for
nutrient transfer.
The nature of the intercellular space apoplast has not been
investigated in mycorrhizal roots. In the cortex of maize roots
not colonized by AMF, Canny & Huang (1993) used cryoscanning electron microscopy (cryo-SEM) to show that some
intercellular spaces were liquid-filled, while others were gasfilled. This raises the question of whether the intercellular
hyphae of AMF within roots are bathed in liquid or lie within
gas-filled space.
The conventional preparative methods that have been used
to examine AMF within roots by high-resolution optical or
electron microscopy remove all water from tissues, making it
impossible to detect liquid-filled regions, or to observe fungal
structures and host tissues in their fully turgid, chemically
unaltered state. Plant tissues, fast frozen and cryo-planed for
cryo-SEM, remain fully hydrated and retain both their inherent cell shapes and the contents of liquid-filled spaces, which
are then easily distinguished from gas-filled spaces. Combined
with quantitative X-ray microanalysis these preparations may
be used to determine accurately the elemental concentrations
within cells and tissues, including liquid in intercellular space
apoplast. (For further information on the use of analytical
cryo-SEM, see McCully et al., 2000.)
In this paper we present the results of an analytical cryoSEM study of roots, mostly from field-grown plants, colonized by indigenous AMF. As well as providing a new view of
the morphology of the symbiosis, we present the first data on
local P, potassium (K), calcium (Ca) and magnesium (Mg)
concentrations in fully hydrated arbuscular mycorrhizas.
Materials and Methods
AMF-colonized roots
Roots colonized by indigenous AMF were collected from
subterranean clover (Trifolium subterraneum L.), white clover
(Trifolium repens L.), leek (Allium porrum L.) and field pea
(Pisum sativa L.). The subterranean clover was grown in a
well-lit glasshouse maintained at 20°C/30°C night/day
in field-collected soil from Junee, Australia (34°53′ S,
147°35′ E). The soil contained 26 mg kg−1 of extractable P as
determined following Colwell (1963), 74 mg/kg of mineral
nitrogen (N) and 2.1% organic carbon, while the pH was 5.5
(CaCl2). Plants were watered daily and sampled three weeks
after sowing. The white clover and leek were grown in a
garden in Canberra, Australia (35°15′ S, 149°8′ E), in soil with
29 mg kg−1 extractable P, 34 mg kg−1 of mineral N, 3.2%
organic carbon and a pH of 4.7. Plants were watered regularly
and sampled in December when the leeks were soon to
commence flowering and the clover was in flower. The field
peas were grown in an agricultural field in Canberra in soil
with 22 mg kg−1 extractable P, 153 mg kg−1 of mineral N,
1.7% organic carbon and a pH of 5.5. The field peas received
no water other than rainfall and were sampled in April, at the
commencement of flowering. Fertilizer was not applied to any
of the plants.
Tissue preparation, cryo-SEM and X-ray microanalysis
Field-grown plants were carefully excavated so that their roots
remained in situ in a ball of soil and quickly brought to the
nearby laboratory. Glasshouse-grown plants were brought in
their pots. Individual axile roots were gently separated from
the soil, shaken to remove excess soil and briefly washed. Each
axile root was lifted from the water and held vertically so that
fine branch roots drooped vertically and clung along the larger
diameter root. Excess water was removed by gently wiping the
root between thumb and forefinger, this also helped align the
branch roots along the main axis. Each root with the aligned
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centration in m. (See McCully et al. (2000) for details of
the analysis and quantification methods, and hints for maximizing the reliability of the results.) The lower limit of reliable
detection of elements by the X-ray microanalysis was considered to be 10 m and data that fell from 0 to 10.0 m
were ascribed a notional value of 5 m.
Experimental design and data analysis
Fig. 1 Calibration graph for [P] used to convert standard ratios
obtained by X-ray analyses to concentrations (see details in McCully
et al., 2000). Measurements from frozen standard solutions
(ordinate) of K3PO4 were planed, etched, coated and analysed under
identical conditions to those used for the tissue samples. The abscissa
shows measured values of the standard ratio (% ratio × 1000/live
time) derived from means of two replicate measurements of 10
spectra from each of the standard solutions. The line is a linear
regression of standard ratio on concentration (y = 0.5037x + 0.3046;
r2 = 0.9988). Similar calibrations used for quantifying [K], [Mg] and
[Ca] were also strong with r2 = 0.9983 for [K], r2 = 0.9964 for [Mg]
and r2 = 0.9931 for [Ca].
branches was immediately frozen by plunging into liquid
nitrogen (LN2). Frozen roots were severed under LN2 and
either prepared immediately for observation in the cryo-SEM
or stored in vials in a cryostore.
Segments of frozen root, 2 mm long, were cut under LN2
and quickly affixed to stubs with low-temperature Tissue Tek
(Miles Inc., Elkhart, IA, USA), transferred to a cryomicrotome
under LN2 and planed at −90°C to a smooth transverse face
with glass and diamond knives. Each planed face included not
only a transverse profile of the central larger-diameter root but
also of the fine branch roots that were aligned along it. Planed
specimens were transferred under LN2 to a cryo-transfer unit
(Oxford Instruments, Eynsham, UK) and hence to the stage
of the cryo-SEM (JEOL 6400, JEOL Ltd., Tokyo, Japan).
Frost was removed by brief, carefully controlled gentle etching
at −90°C and the specimen then cooled to −170°C and coated
with evaporated, high purity aluminium. (For further details
of preparation methods see Huang et al. (1994) and McCully
et al. (2000)). Images were recorded at 10–15 kV to a digital
recorder (ImageSlave, OED Pty Ltd., Hornsby, Australia).
Fungal structures and host root cortical tissue were analysed with a Link eXL system (Oxford Instruments) using the
Be window. Spectral data for P, K, Ca and Mg were converted
to elemental concentrations using frozen standards prepared
exactly as the specimens (Fig. 1). The analysis does not distinguish between ions and elements in insoluble form and we
express concentrations of elements in all structures as a con-
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One transverse root face from each of seven subterranean
clover roots, seven white clover roots, two leek roots and two
field pea roots, in each case taken from at least two plants,
were examined. In each root face, numerous measurements
were made of elemental concentrations in a number of
structures:
(1) Hyphae present in intercellular spaces (intercellular
hyphae) and /or hyphae present inside cells (intracellular
hyphae).
(2) Arbuscule trunk hyphae. These were distinguished from
intracellular hyphae by being present in the midst of smaller
arbuscule branches.
(3) Arbuscules. When branches were well defined, and large
enough, the analysis was conducted inside individual
branches. In other arbuscules numerous hyphae, and presumably any immediately surrounding host cell material, were
analysed together. Arbuscules were divided into three development stages, consistent with the lifecycle and morphology
described by Kinden & Brown (1975b,c, 1976) and Toth &
Miller (1984). These were termed ‘young’, ‘collapsing’ and
‘clumped’. Young arbuscules had numerous well-defined, turgid branches. In collapsing arbuscules some fungal walls
appeared to be breaking down, although this was not necessarily apparent throughout the arbuscule. In clumped arbuscules, no individual branches remained and the arbuscule
consisted of a clump of relatively smooth material.
(4) The vacuole of uncolonized cortical cells. The remainder
of the cytoplasm and the nucleus in the highly vacuolated cortical cells were too narrow in the transverse profiles to be accurately analysed.
(5) The vacuole of cortical cells containing an arbuscule. In
some cases arbuscules filled cells so completely that this measurement was not possible.
(6) The liquid, or other substances, in uncolonized intercellular spaces.
(7) The liquid, or other substances, surrounding intercellular
hyphae. Intercellular spaces generally presented a relatively
small surface area in the transverse tissue faces, depending on
where the planed surface intersected the space. Analyses were
only done where the area of the liquid was large enough to
ensure the analysis did not include input from hyphae in the
space or surrounding cells. Such areas were relatively rare.
Unless otherwise noted, results are only presented from metabolically active cortical tissues, arbitrarily defined as those
with cortical cell vacuole [K] > 20 m.
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Fig. 2 Cryoplaned transverse face of a white clover (Trifolium
repens) root, viewed by cryo-scanning electron microscopy,
showing arbuscular mycorrhizal fungal structures in some cells and
intercellular spaces of the mid and inner cortex (e.g. cells marked with
an asterisk). The fine white lines or dots in the cortical cell vacuoles
are solutes sequestered during freezing. It is from preparations like
these that Figs 3 –5 have been taken. Bar, 50 µm.
Statistical examination of the elemental concentration data
was complicated by the variable number of analyses available
in the seven categories described, and by the small number of
analyses available in some instances, especially for the leek and
field pea. Hence, data for some structures are presented only
for the two clovers, or are combined across all hosts. Data are
generally presented as mean ± SD. All data analysis was conducted using the SPSS software  version 7.101
(SPSS, Chicago, IL, USA). No data were excluded from the
data sets presented unless specifically mentioned.
Results
Structural features
Within the planed, transverse faces of uncolonized root
cortical cells, only the frozen cell vacuole contents, with lines
or dots of sequestered solutes, could always be distinguished.
The profiles of the cytoplasm and the nucleus were too narrow
to be confidently identified. Colonization by AMF was
most commonly found in the mid and inner cortex (Fig. 2).
Intercellular hyphae were often present, and in cells colonized
by AMF, intracellular hyphae, arbuscules and fungal vesicles
could be identified, and appeared surrounded by the host cell
vacuole (Figs 2–5).
The contents of hyphae showed a more structured appearance than host cell vacuoles (Fig. 3, insets i and ii) and the
contents of some hyphae appeared denser than others (Fig. 3).
Intercellular hyphae were generally 2–7 µm in diameter, usually with one hypha in each intercellular space, although occasionally two or three were observed (Fig. 3). In subterranean
clover, leek and field pea the transverse faces of the intercellular hyphae were usually circular, leaving some liquid-filled
space surrounding them in the usually triangular profiles of
the intercellular spaces (Fig. 3, inset i). In all four species some
intercellular spaces without hyphae were also liquid-filled,
while others were gas-filled. In some white clover roots, intercellular hyphae appeared to be surrounded by expanded gel
(Fig. 3). In white clover the large-diameter, often irregularly
shaped, intercellular hyphae were frequently closely adpressed
to all surrounding cell walls and sometimes appeared to have
pushed these walls inwards (Fig. 2, and Fig. 3, main figure
and inset ii). Intracellular hyphae were observed commonly
only in white clover and leek and were 2.5–6 µm diameter
and always circular in transverse profile (Fig. 3).
Arbuscules at the three development stages defined earlier
were present in the roots. Young arbuscules had numerous,
turgid, distinct branchlets ranging from 0.6 µm to 2.9 µm
diameter and usually filled a large volume of the colonized
cortical cell (Figs 2–4). Within young arbuscules there were
often one or two much larger diameter hyphae that were
assumed to be trunk-hyphae. Collapsing arbuscules ranged
from those that had turgid, distinct branches together with
areas where some branches had coalesced, to those in which
coalesced branches had formed into loosely connected, frequently rather ropey, strands (Figs 3 and 4). In places these
strands were quite homogeneous in appearance while in other
places they showed a meshwork of aggregated, collapsed
branches (Fig. 3). Where the clumped branches had been
planed to expose a suitable section, they often appeared to be
encased in a clearly defined outer shell (short thick arrows
Fig. 3). Collapsing arbuscules occupied a decreasing volume
of the root cell as the degree of collapse increased. Clumped
arbuscules had no distinct branches remaining and consisted
of a single relatively smooth clump that appeared homogeneous, except for very thin remains of the original fungal walls
and the distinct, thin outer shell (Fig. 3, cell labelled C).
Clumped arbuscules were centrally located in the host cell and
occupied relatively little of the cell volume. The transverse
faces of white clover roots often contained all three stages of
arbuscule development (Fig. 3). In subterranean clover, while
young and collapsing arbuscules were observed in metabolically active roots (Fig. 4), clumped arbuscules were observed
only in metabolically inactive roots.
The fungal vesicles observed all had a smooth, homogeneous appearance in section except for a well-defined outer rim
(Fig. 5). Because of the low numbers of vesicles in the material
examined we are unable to describe their development definitively. The evidence we have, however, suggests that they are
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Fig. 3 Detail of fungal structures in roots of white clover (Trifolium repens; main figure and inset ii) and subterranean clover (Trifolium
subterraneum; inset i). Cells labelled A, B, and C contain arbuscules classified as young, collapsing, and clumped, respectively. A distinct surface
layer surrounds clumping regions of arbuscules (short thick arrows). Cells labelled D contain transversely planed profiles of intracellular hyphae,
while many intercellular spaces also contain hyphae (H). Intercellular hyphae in white clover often appear to be pushing inwards the walls of
surrounding host cells (long arrows). They are also sometimes of irregular shape, as in inset (ii) where a portion of a large intercellular hyphae
is seen in tangential view. Intercellular spaces (S) contain liquid contents that are either electron nonemissive (dark) or lighter, (the latter
suggesting expanded mucilage). Such content can be observed in spaces where planed profiles of hyphae are also present (indicated with
asterisk). Cells labelled E contain no fungal structures in the planed profile. Preparation as in Fig. 2. Bar, 10 µm.
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Fig. 4 Detail and elemental concentrations of
arbuscules in a subterranean clover (Trifolium
subterraneum) root. The arrows point to
regions that were analysed: A, transverse
profile of a small branch of a young arbuscule
(34 mM P, 120 mM K, 26 mM Ca, < 10 mM
Mg); B, arbuscule branches just beginning
to coalesce (45 mM P, 96 mM K, 65 mM
Ca, < 10 mM Mg); C, vacuole in cell
containing a young arbuscule (< 10 mM P,
Mg & Ca, 76 mM K); D, intercellular hypha
(204 mM P, 188 mM K, 29 mM Ca, 43 mM
Mg); and E, vacuole of cell with no fungal
structures present in the planed face
(< 10 mM P and Ca, 98 mM K, 14 mM Mg).
The lower limit of reliable detection of the
elements was considered to be 10 mM.
Preparation as in Fig. 2. Bar, 10 µm.
Fig. 5 A fungal vesicle (V) in a subterranean clover (Trifolium
subterraneum) root. The contents of the vesicle appear
homogeneous, it is surrounded by a clearly defined rim, and it
appears to have expanded to fill much of the cell in which it is
contained. Preparation as in Fig. 2. Bar, 10 µm.
initiated within a cortical cell no earlier than when arbuscules
in surrounding cells begin to collapse, and then expand to fill
the cell and ultimately kill it so that the vesicle becomes essentially extracellular in the root tissue.
Elemental analyses
Intraradical hyphae had the highest [P], averaging 60–
170 m (Figs 4 and 6), and reaching 600 m in one white
clover root where all hyphae had > 350 m. Hyphal [P] was
much higher in the two clovers than the leek and field pea.
Two hyphae in one field pea root contained no P but high [K]
and were excluded from data analyses. Arbuscules averaged
30–50 m P in all four hosts (Fig. 6). The [P] in cortical cell
vacuoles was much lower than in fungal tissues (generally
< 25 m) and tended to be slightly higher in vacuoles
surrounding arbuscules than in vacuoles of cells that appeared
uncolonized (Fig. 6). In subterranean clover, 58% of cells
containing an arbuscule in the planed face had a detectable
concentration of P (> 10 m) in the vacuole (n = 19) compared
with 14% of cells where no fungal structures were present in
the planed face (n = 14). The [K] ranged from 20 to 350 m
and was higher in intraradical hyphae than in arbuscules or
cortical cell vacuoles (Fig. 6). The [Ca] tended to be highest
in arbuscules, while [Mg] was generally highest in intraradical
hyphae (Fig. 6).
Hyphal [P] showed a strong linear correlation with both
[K] and [Mg], but not [Ca] (Fig. 7). If P is assumed to be
present primarily as a monovalent anion, it is balanced in a
near 1 : 1 relationship with K+ and Mg2+ combined (e.g. the
analysis at D in Fig. 4, Fig. 8). In subterranean clover arbuscules (Fig. 9), trunk hyphae had similar profiles of [P], [K],
[Ca] and [Mg] to neighbouring intercellular hyphae, but
slightly higher [K] and much higher [P] and [Mg] than finer
arbuscule branches. Indeed, Mg was rarely present in arbuscule fine branches (e.g. the analysis at A in Fig. 4, Fig. 9).
The data on elemental concentrations in arbuscules and
surrounding cortical cell vacuoles presented in Fig. 6 were
further investigated by division according to arbuscule development stage (Fig. 10). An adequate number of analyses were
available only for the two clovers and for subterranean clover,
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Fig. 6 Concentrations of P, K, Ca and Mg in
intraradical hyphae (IRH), arbuscules of any
development stage (Arb), the vacuole of
arbuscule-containing root cortex cells (VacC)
and the vacuole of root cortex cells where no
fungal structures were present in the planed
face (VacU): (a) subterranean clover
(Trifolium subterraneum) (IRH n = 28, Arb
n = 24, VacC n = 19, VacU n = 14); (b) white
clover (Trifolium repens) (IRH n = 29, Arb
n = 22, VacC n = 17, VacU n = 7); (c) leek
(Allium porrum) (IRH n = 7, Arb n = 5, VacC
n = 5, VacU n = 3); and (d) field pea (Pisum
sativum) (IRH n = 9, Arb n = 5, VacC n = 4,
VacU n = 4) (mean ± SD). As the lower limit
of reliable detection of the elements was
considered to be 10 mM, lower measurements
were ascribed a value of 5 mM. Results from
intercellular and intracellular hyphae did not
differ and were combined.
analyses of clumped arbuscules were available only from
metabolically inactive roots. The [P] in arbuscules remained
relatively stable as they senesced, especially in white clover.
While arbuscule [K] was 50% lower in clumped arbuscules,
host vacuolar [K] remained relatively stable as arbuscules
senesced. [Ca] was greatly elevated in the coalescing branches
of collapsing arbuscules, even when branches were only slightly
collapsed (e.g. Figure 4B), but was not elevated in turgid
branches in the same arbuscule (Fig. 10). [Ca] remained high
in clumped arbuscules. [Ca] was particularly high in white
clover, where up to 250 m was recorded. There was no
relationship between [Ca] and [P] in collapsing or clumped
arbuscules (r 2 = 0.006). One white clover clumped arbuscule
registered no Ca, but was included in analyses. Host cell vacuoles did not exhibit elevated [Ca] (Fig. 10); also elevated in
the coalescing branches of collapsing and clumped white clover arbuscules was [Mg] (Fig. 10). In field pea, two clumped
arbuscules were analysed and contained 11 m and 20 m of
P, with 36 and 58 m of Ca.
In liquid-filled intercellular spaces not colonized by AMF,
P was generally not detectable (Fig. 11). However when an
intercellular space contained a hypha, [P] in surrounding liquid, or gel-like substance, was often 10–30 m and reached
up to 100 m. There was no relationship between [P] in individual hyphae and [P] in fluid in the surrounding intercellular
space (r 2 = 0.001). Since [K] did not vary between colonized
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and uncolonized intercellular spaces it is unlikely that the
enhanced P in colonized spaces resulted from inclusion of a
portion of undetected underlying hypha within the volume
analysed.
Discussion
Fungal morphology
The morphology of mycorrhizas observed in our study is
generally consistent with other reports. Arbuscules are known
to collapse first in their finest branches, eventually forming
smooth clumps that are finally absorbed by the cell, which
itself remains viable and may even be recolonized (Kinden
& Brown, 1975a,b,c, 1976; Dickson & Smith, 2001). The
smooth texture of the frozen-planed vesicle contents is
suggestive of lipids, consistent with the histochemical
results of Nemec (1981) that indicated a high content of
neutral lipid. However, the diameter of arbuscule branches
is generally reported as 1 µm (Kinden & Brown, 1975c; Toth
& Miller, 1984), which is lower than in the present study,
probably because the tissues observed in the earlier studies
were completely dehydrated during preparation. Our values
are more likely to provide a realistic measure, as freezing
would be expected to increase the volume of watery liquid up
to about 9%, which, when translated to linear measurements,
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Fig. 8 Charge balance of P with Mg and K in the intraradical hyphae
of subterranean clover (Trifolium subterraneum), white clover
(Trifolium repens), leek (Allium porrum) and field pea (Pisum
sativum), assuming that P was a monovalent anion and Mg and K
were present as Mg2+ and K+ (r2 = 0.85). The dashed line represents
the 1 : 1 ratio.
Phosphorus concentrations in arbuscules and hyphae
Fig. 7 The [P] in intraradical hyphae of subterranean clover
(Trifolium subterraneum, filled circles), white clover (Trifolium
repens, open circles), leek (Allium porrum, filled triangles) and field
pea (Pisum sativum, open triangles); relationship with (a) [K]
(r2 = 0.73 all data, r2 = 0.88 white clover only); (b) [Ca] (r2 = 0.0003
all data, r2 = 0.005 white clover only); and (c) [Mg] (r2 = 0.81 all data,
r2 = 0.89 white clover only). As the lower limit of reliable detection of
the elements was considered to be 10 mM, lower measurements
were ascribed a value of 5 mM.
means a maximum increase of only around 2%. Thus, while
dimensions of fungal structures preserved by rapid freezing
should be close to those of their native state, conventional
preparative methods can produce marked alterations through
loss of turgor, total dehydration and chemical changes that
affect cell wall integrity.
Our results confirm and extend those of earlier X-ray
microanalytical studies that showed significant amounts
of P in intraradical hyphae and /or arbuscules of arbuscular
mycorrhizas (Schoknecht & Hattingh, 1976; Walker &
Powell, 1979; White & Brown, 1979; Cox et al., 1980). To
our knowledge, our results provide the first in situ quantitative
measurements of P in these mycorrhizas. All earlier studies
used preparative methods that would not reliably retain ions
or soluble compounds and most did not use standards for
quantification. Cox et al. (1980) using preparations from
which soluble P would have been lost, determined that the P
in insoluble polyphosphate granules would result in a P
concentration about 2.4 × 10−4 g cm−3 (i.e. 8 m) in fungal
cytoplasm in intercellular hyphae, approximately an order of
magnitude lower than found in the present study. The few
available values determined using other techniques are 34–
203 m in extraradical and intraradical hyphae (calculated by
Smith et al., 2001 from Solaiman et al., 1999) and 338 m in
extraradical hyphae (Nielsen et al., 2002).
Phosphorus transfer in arbuscules
A significant role for young arbuscules in P transfer is
consistent with the lower [P] in arbuscule fine branches
compared with trunks and intercellular hyphae, and the
greater proportion of cortical cell vacuoles which contained
a detectable [P] when an arbuscule was present. Root cell
vacuoles are known to be a P reservoir to allow maintenance
of cytosol P; however, P in both the cytosol and vacuoles may
generally be maintained well below 10 m (Lee & Ratcliffe,
1993). Thus, the relatively small increase in [P] in cortical cell
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Research
vacuoles when an arbuscule was present (Fig. 6) probably
reflects changes in vacuolar P being below the level reliably
detected by the X-ray microanalysis and, presumably, the
rapid movement of P through the root to the vascular tissue.
A technique capable of resolving concentrations < 10 m is
required to gain further insights into P-transfer in arbuscules.
The maintenance of P in clumped arbuscules (Fig. 10)
indicates the fungi did not, as has been hypothesized
elsewhere (Kinden & Brown, 1976; Walker & Powell, 1979;
Dickson & Smith, 2001), withdraw a significant amount of
P from the senescing arbuscule. The host cell could presumably use the P remaining in clumped arbuscules if they were
gradually digested (Kinden & Brown, 1976).
Phosphorus and carbon transfer in intercellular spaces
Fig. 9 Concentrations of P, K, Ca and Mg in intercellular hyphae, and
the trunk hyphae and fine branches of young arbuscules in
subterranean clover (Trifolium subterraneum) roots (n = 5, mean ± SD).
Arbuscules were paired with the closest intercellular hypha. As the
lower limit of reliable detection of the elements was considered to be
10 mM, lower measurements were ascribed a value of 5 mM.
© New Phytologist (2003) 160: 429 – 441 www.newphytologist.com
This study provides the first evidence that the intercellular
hyphae of AMF are generally surrounded by liquid and/or
gel and that [P] in this medium can be enhanced adjacent to
the hyphae. The presence of gel possibly accounts for the
particulate material occasionally seen in conventional electron
micrographs of intercellular spaces containing hyphae (e.g.
Figure 17 in Holley & Peterson, 1979). It seems most likely
that the P we detected in the intercellular spaces containing
hyphae was released locally from the hyphae and not from
adjacent root cells because where profiles of liquid-filled
intercellular spaces did not include a hypha (e.g. Figure 3,
inset i) P content was always low (Fig. 11), even when
surrounding cells contained arbuscules. Whether root cells
bordering colonized intercellular spaces can readily take up
intercellular P (Gianinazzi-Pearson et al., 1991) and whether
P transfer is possible, or perhaps enhanced, when intercellular
hyphae and cell walls are tightly adpressed to each other, are
unknown.
Arbuscular mycorrhizas with Paris-type morphology do
not form intercellular hyphae, instead consisting of only
intracellular structures. Since P-uptake and growth of the host
plant may be enhanced by Paris-type mycorrhizas (Cavagnaro
et al., 2003), it appears that P-transfer in arbuscular mycorrhizas is by no means confined to intercellular spaces and there
is a role, and perhaps a dominant one, for intracellular structures such as arbuscules or hyphal coils, if present.
The presence of liquid in intercellular spaces also raises the
possibility of carbon transfer in these spaces. Intercellular
spaces in plant tissues are preferred sites for colonization
by endophytic bacteria (Hecht-Buchholz, 1998; McCully,
2001). Except in sugarcane, where these spaces contain
sucrose solution, little is known about the presence of carbon
compounds in intercellular space liquid, but there are indications that such compounds are present and thus available to
colonizing microbes (see review by McCully, 2001). The
existence of mutant plants that block the formation of
arbuscules, but allow spread of intercellular hyphae strongly
suggests that mycorrhizal hyphae can access some carbon
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Fig. 10 Concentrations of P, K, Ca and Mg in
arbuscules and surrounding root cortical cell
vacuoles in cells with young, collapsing or
clumped arbuscules in: (a) subterranean
clover (Trifolium subterraneum) (n = 3–9);
and (b) white clover (Trifolium repens)
(n = 4 –12) (mean ± SD). In collapsing
arbuscules, data are presented from both
hyphae that appeared turgid and coalescing
hyphae of relatively smooth appearance. As
the lower limit of reliable detection of the
elements was considered to be 10 mM, lower
measurements were ascribed a value of
5 mM. No clumped arbuscules were observed
in roots of subterranean clover that contained
young arbuscules, however, clumped
arbuscules were frequently found in roots
where the majority of cells appeared
metabolically inactive and analyses of these
are included and marked with asterisks.
compounds from intercellular spaces (Smith & Read, 1997).
For example, when Gigaspora margarita colonized the Ljsym4-1
mutant of Lotus japonicus, the fungus was only very infrequently able to penetrate cell walls to form arbuscules (9% of
colonization contained arbuscules), but 8% of root length was
still colonized by intercellular hyphae (Novero et al., 2002).
In the wild type, 31.4% of root length was colonized,
presumably reflecting greater carbon transfer due to a greater
proportion of arbuscules (73% of colonization contained
arbuscules). Thus, while it appears transfer of P and carbon
compounds can occur via intercellular hyphae, for carbon
compounds at least, transfer may be slower than via arbuscules, with their far greater ratio of surface area to volume.
Arbuscules do represent a large energy investment by the
fungus and in some situations it may be more efficient for
host and fungus to rely on transfer via intra- or inter-cellular
hyphae. For example, in the long-lived, slow growing roots of
many plant species in natural ecosystems, AMF may persist as
intercellular hyphae for a number of years following the deterioration of arbuscules formed when roots were young (e.g.
Smilacina racemosa; Brundrett & Kendrick, 1990a,b). Interestingly, however, a rapid and substantial P benefit in presence
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Research
Fig. 11 [P] and [K] in intercellular hyphae (n = 22), surrounding
intercellular spaces (n = 22) and uncolonized intercellular spaces
(n = 11) (mean ± SD). As colonized intercellular spaces with
adequate area to analyse were rare, results from subterranean clover
(Trifolium subterraneum), white clover (Trifolium repens) and leek
(Allium porrum) are combined. Most colonized intercellular spaces
were liquid-filled, although a few in white clover contained a
substance of denser appearance (Fig. 3). As the lower limit of reliable
detection of the elements was considered to be 10 mM, lower
measurements were ascribed a value of 5 mM. Overall, 66% of
colonized intercellular spaces and 18% of uncolonized intercellular
spaces contained a detectable concentration of P.
of only intraradical hyphae (unspecified whether intra- and
inter-cellular) has been reported for Salix repens with less than
5% of root length colonized (van der Heijden, 2001).
Potassium and magnesium concentrations in fungal
hyphae
One of the most consistent findings of this study was the very
strong linear relationship of [P] with [K] and [Mg] in the
intraradical hyphae (Figs 7–9). Clearly, K and Mg are the
balancing cations for the predominant P forms in the fungal
hyphae. However, we can only speculate about the exact form,
and even the solubility, of these elements. While some soluble
P in hyphae of AMF may be in the form of polyphosphate,
a linear polymer of orthophosphate, polyphosphate may
© New Phytologist (2003) 160: 429 – 441 www.newphytologist.com
only represent a small proportion of the P present (Solaiman
et al., 1999). Polyphosphate has a lower osmotic impact than
equivalent amounts of orthophosphate and may bind strongly
to cations including Mg2+ (Cramer & Davis, 1984).
Significantly, Mg has not been detected in the hyphae of
arbuscular mycorrhizal fungi in earlier studies using X-ray
analysis. Instead, relatively large Ca peaks suggested that this
element formed the major balancing cation (Schoknecht &
Hattingh, 1976; Walker & Powell, 1979; White & Brown,
1979; Cox et al., 1980). Similar evidence for high Ca concentrations that would appear to balance P in ectomycorrhizas is
common in the earlier literature, but this Ca accumulation
has been shown definitively by Orlovich & Ashford (1993)
and Bücking & Heyser (1999) to be an artefact resulting from
the use of preparative methods that allow migration of soluble
components before analysis. Our finding of high [Mg] but low
[Ca] in the fungal hyphae is similar to the results of Bücking
& Heyser (1999) in an ectomycorrhiza, suggesting that the
failure of earlier studies of ectomycorrhizas and arbuscular
mycorrhizas to detect Mg rather than Ca, is because mobile
Ca was immobilized by the formation of insoluble P compounds and the Mg was lost.
The high [K] in intraradical hyphae and arbuscule trunk
hyphae is indicative of high turgor pressure. High turgor
pressure would aid hyphae to push through cell walls and
invaginate the host cell plasma membrane. In white clover,
high turgor may enable intercellular hyphae to enlarge intercellular spaces.
Calcium and arbuscule senescence
High [Ca] in collapsing and clumped arbuscules has not been
previously reported. As stores of Ca were not detected in host
cell vacuoles or fungal hyphae, the origin of the Ca remains to
be elucidated. Arbuscule collapse is thought to coincide with
loss of metabolic activity in arbuscule branches (Vierheilig et al.,
2001), an increase in host metabolic activity (Kinden & Brown,
1976) and, possibly, the recognition by the host cell of the
fungus as an invader (Salzer et al., 1999; Vierheilig et al., 2001).
It is possible that the Ca may be contained in, or infiltrated
from, the host-derived encasement material observed in this
study (Fig. 3) and by Kinden & Brown (1976) and Cox &
Sanders (1974) to be deposited as fungal walls fragment
and collapse. Scannerini & Bonfante-Fasolo (1979) showed
that this encasement material was heavily stained with ruthenium red, strongly suggesting a high acidic polysaccharide
component. Calcium would be expected to bind to such
material. Such a scenario is suggestive of a host defence
response. Calcium is well known to mediate plant responses
to stress, including fungal pathogens through changes in
cytosol [Ca], although these fluctuations are in the nanomolar
range (Xu & Heath, 1998). High [Ca] is also present in cell
wall appositions (papillae) which are deposited to provide a
physical barrier to fungal pathogens (Bonello et al., 1991).
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440 Research
Deposition of Ca would presumably seriously disrupt
functioning of the specialized host plasma membrane that
surrounds arbuscules.
Overall, our results are consistent with the arbuscule lifecycle being strongly delimited by the host cell, a conclusion
supported by the finding of hydrogen peroxide synthesis in
host cells containing senescing arbuscules (Salzer et al., 1999).
However, the trigger for such control is unknown. The length
of arbuscule lifecycle has been reported to range from around
8 d in fast-growing annual crops (onion, bean, tomato;
Alexander et al., 1989) to several months in plants in natural
ecosystems where fine arbuscule branches collapse very slowly
(Brundrett & Kendrick, 1990a).
Conclusions
Our results indicate the considerable potential of cryomicroscopy and quantitative X-ray microanalysis of fully
hydrated tissues to provide unique new insights into the
functioning of arbuscular mycorrhizas by revealing for the
first time the concentrations and precise locations of specific
elements within the root and fungal structures. The striking
finding that the distribution patterns of P, Mg, K and Ca were
similar in mycorrhizal associations of four different host
plants, plants grown in three soil types and under widely
different conditions, and, presumably, colonized by three
different populations of AMF, now suggests that such
distribution may play a basic role in the functioning of the
mycorrhizal association.
We anticipate that further analyses of mycorrhizal associations using the cryo-analytical technique will add additional
valuable insights into this complex fungal-host plant relationship and complement the increasingly sophisticated molecular studies now being conducted.
Acknowledgements
The project was financed by the Grains Research and
Development Corporation. We thank Glen Ryan for help
with processing the micrographs.
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