Apoplasmic barriers and their significance in the exodermis and sheath of Eucalyptus...

RESEARCH New Phytol. (2000), 145, 333–346

Apoplasmic barriers and their significance

in the exodermis and sheath of Eucalyptus

pilularis–Pisolithus tinctorius

ectomycorrhizas

PETER A. VESK", ANNE E. ASHFORD#*, ANNE-LAURE

MARKOVINA" WILLIAM G. ALLAWAY"

"School of Biological Sciences, The University of Sydney, NSW 2006, Australia

#School of Biological Science, The University of New South Wales, Sydney, NSW 2052,

Australia

Received 17 June 1999; accepted 19 October 1999

The apoplasmic permeability of ectomycorrhizal roots of intact Eucalyptus pilularis seedlings infected with

Pisolithus tinctorius on aseptic agar plates was examined using the nonbinding fluorochrome 8-hydroxypyrene-

1,3,6-trisulphonate and lanthanum ions in conjunction with anhydrous freeze substitution and dry sectioning.

Most mycorrhizas formed in the air above the agar surface, and in these the sheath rapidly became nonwettable

and impermeable to the fluorochrome but was nevertheless permeable to lanthanum ions. In a few mycorrhizas

which developed in contact with the agar the sheath remained permeable to both tracers when fully developed.

This increased hydrophobicity of the sheath in mycorrhizas in the air above the agar surface might be explained

by deposition of hydrophobins, but nevertheless it still allows an apoplasmic pathway for radial movement of ions.

Regardless of their sheath permeation both apoplasmic tracers were always found throughout the Hartig net and

were arrested at the Casparian bands and suberin lamellae of the exodermis. It is concluded that the fluorochrome

must have moved longitudinally along the Hartig net which is a region of higher permeability than the sheath.

Casparian bands in the exodermis of ectomycorrhizal roots have similar properties to those in nonmycorrhizal

roots in excluding solutes and their exclusion of lanthanum ions indicates that they are not permeable to ions. The

data do not support the concept of a totally sealed apoplasmic exchange compartment, but the differential

permeability suggests that the sheath might allow radial transfer of ions but block loss of sugars and organic

molecules of similar size.

Key words: apoplasmic permeability, Casparian band, exodermis, ectomycorrhiza, freeze substitution,

hydrophobins, lanthanum, PTS.

Early physiological experiments tended to focus on

the ability of excised ectomycorrhizal roots to absorb

ions compared with nonmycorrhizal roots of the

same species (Harley, 1969). This approach treated

ectomycorrhizal roots essentially as uptake struc-

tures. However the ectomycorrhizal roots of many

associations are connected to an extraradical my-

celium which can grow out into the soil for

considerable distances and form structures of great

complexity (Read, 1991). This mycelium is now

*Author for correspondence (fax 61 2 9385 1558; e-mail

a.ashford!unsw.edu.au).

considered to play a crucial role in nutrient and

water acquisition and in mobilization of organic

resources. Nutrients acquired by the hyphal tips are

transported back to the mycorrhizal sheath which is

now viewed more as a storage and exchange

structure, or food base, than as a nutrient-absorbing

organ (Smith & Read, 1997). One of the many pieces

of evidence which agree with this view is the

discovery that the fluorochrome calcofluor would not

penetrate the sheath of two types of ectomycorrhiza,

those formed on Eucalyptus pilularis by Pisolithus

tinctorius and those found on Pisonia grandis,

suggesting that the sheath forms an impermeable

barrier to this fluorochrome (Ashford et al., 1988,

1989). Since the mycorrhizal interface in E. pilu-

334 RESEARCH P. A. Vesk et al.

laris–P. tinctorius mycorrhizas is sandwiched be-

tween an exodermis and an impermeable sheath, this

led to the hypothesis that there is a sealed apoplasmic

compartment at the interface between partners. The

benefits of the isolation of the exchange interface are

obvious, and were discussed in Ashford et al. (1989).

The low apoplasmic permeability of the sheath in

these two types of mycorrhizas led Ashford et al.

(1989) to suggest that ion transport across the sheath

is essentially symplasmic, and that ectomycorrhizal

roots are exchange rather than uptake structures.

The apoplasmic permeability of the sheath was

assessed in these earlier experiments by applying

calcofluor to excised roots with their cut ends sealed

with sticky wax, and determining the distribution of

calcofluor in hand sections following a short wash. It

was presumed that calcofluor was retained by

electrostatic binding in the plant and fungal walls to

which it had access, but there are problems in using

this approach because the results depend on dye

binding as well as permeability. Dye molecules

might be lost because they were not bound, they

might be redistributed and preferentially bound to

more electronegative sites, or bound dye might

impede the further progress of other dye molecules

through the walls (Canny, 1986; Canny & McCully,

1986). An approach which avoids these possibilities

is to allow low molecular weight fluorochromes that

do not bind to cell walls to permeate tissue, then to

retain them in situ by anhydrous freeze substitution.

Their distribution can then be determined by

fluorescence microscopy in dry-cut sections. This

approach works well for tracing pathways of rela-

tively high permeability in the apoplast of higher

plants (Canny, 1986, 1988; Canny & McCully,

1986). It has also been used in fungi to evaluate

changes in permeability of the rind during matu-

ration of sclerotia of Sclerotinia minor (Young &

Ashford, 1992, 1996). Behrmann & Heyser (1992)

evaluated the apoplasmic permeability of Pinus

sylvestris–Suillus bovinus ectomycorrhizas by the

latter approach. They treated the mycorrhizas of

intact seedlings with the anionic tracer sulpho-

rhodamine G for 20 h and examined the distribution

of sulphorhodamine in sections of anhydrously

processed tissue. They found, in contrast to the

results with E. pilularis–P. tinctorius and Pisonia,

that the sheath of their Pinus sylvestris–Suillus

bovinus mycorrhizas was permeable and that sulpho-

rhodamine G penetrated as far as the endodermis.

There are several possible explanations for the

differences between the two sets of results : that

calcofluor had been lost from the walls of the sheath

and cortex in the eucalypt mycorrhizas during the

wash period giving a false negative result ; that the

two types of mycorrhiza are permeable to different

degrees; or that experimental conditions and state of

the mycorrhizal roots differed. It is clearly important

to revisit the issue of the permeability of E.

pilularis–P. tinctorius mycorrhizas, using tracers that

do not bind to walls, and anhydrous processing

techniques. It is also desirable to apply fluoro-

chromes to undisturbed mycorrhizal root systems of

intact seedlings since leakiness due to damage is

minimized, and water and solutes might enter via

bulk flow rather than diffusion (see Moon et al.,

1986; Aloni et al., 1998).

Here we describe experiments where the fluores-

cent tracer 8-hydroxypyrene-1,3,6-trisulphonate

(PTS) has been added to otherwise undisturbed

whole mycorrhizal root systems of seedlings of E.

pilularis produced on agar and inoculated with P.

tinctorius for various lengths of time. Following

treatment, the ectomycorrhizas are anhydrously

freeze-substituted, dry-cut, and examined byfluores-

cence microscopy. Also described are parallel experi-

ments using lanthanum (La) as an apoplasmic tracer,

in conjunction with anhydrous freeze substitution,

to clarify further the permeability of the eucalypt–

Pisolithus sheath. Since La is believed to permeate

wall spaces of diameter as small as 2 nm (Evert et al.,

1985), the data better indicate where permeability

barriers to ions occur in the mycorrhizas. Problems

of La redistribution during tissue processing, which

would confound the results, are avoided by not using

chemical fixation and conventional embedding

methods. As a further check, frozen-hydrated speci-

mens were also analysed directly in the SEM after

planing in the cryoultramicrotome, eliminating all

other tissue preparation steps. The potential of

ectomycorrhizal roots (as distinct from the extra-

radical hyphae) for ion uptake, and whether they

have a direct role as uptake structures or are merely

structures for exchange, are considered. Our findings

are discussed in relation to the observed upregulation

of hydrophobin genes that occurs during sheath

proliferation.

Pure cultures of Pisolithus tinctorius (Pers.) Coker &

Couch, (isolate 024, a culture reisolated by D. A.

Orlovich and J. W. G. Cairney from a mycorrhiza

that had been synthesized axenically with isolate DI-

15) were used in all experiments. Stock cultures were

maintained on Modified Melin-Norkrans (MMN)

agar medium (Marx, 1969) at 24°C, and subcultured

every 8 wk. Plugs of inoculum (8 mm) were cut from

the edges of actively growing colonies and trans-

ferred to fresh agar plates to produce 3-wk-old

mycelium for induction of ectomycorrhizas (Piche! &Fortin, 1982). Seeds of Eucalyptus pilularis Smith

from Royston Petrie Seeds, Kenthurst, Australia

were surface-sterilized by soaking in 70% ethanol

with 0.5% Teepol for 10 min followed by sodium

hypochlorite solution (4% available chlorine) for a

further 25 min with agitation. After three rinses in

RESEARCH Exodermis and sheath impermeability in ectomycorrhizas 335

sterile deionised water, seeds were germinated on

0.4% water agar at 24°C in the dark (Ashford et al.,

1989). Mycorrhizas were synthesized using a

modified version of ‘simple system’ plates

(Duddridge, 1986; Peterson & Chakravarty, 1991).

The rims of the bases and lids of sterile 90¬14 mm

plastic Petri dishes were notched with a hot needle

and the dishes filled with 15 ml of 0.8% water agar.

Seedlings 5–10 d old with 1–2 cm-long main roots

producing first order lateral roots were aseptically

transferred to these plates, their shoots protruding

through the notch and held in place by sealing the

notch with sterile Vaseline petroleum jelly. Two or

three plugs of inoculum were aseptically placed

around the root system. Each plate was watered with

1 ml of sterile nutrient solution before being sealed

with Parafilm. The nutrient solution contained the

following (quantities in millimoles per litre) : ammo-

nium 2.8, boron 1.7, calcium 0.33, chloride 4.3,

copper 0.033, ferric iron 8.7, magnesium 0.45,

molybdenum 0.014, manganese 0.33, potassium 6.0,

phosphate 4.1, sodium 0.30, sulphate 0.94, and zinc

0.13. The plates were incubated vertically inside a

Perspex chamber (Wong & Fortin, 1989) at approx.

23°C. Aluminium foil was placed over the bottom

half of the Petri dishes to reduce exposure of roots to

light. The seedlings received a photon flux density of

approx. 200 µmol m−# s−" in the visible range for 13 h

each day, and were watered as necessary with sterile

deionized water. The bottom and sides of the

Perspex chamber were lined with filter paper and

sprayed weekly with deionized water. The growth

chamber was cleaned routinely with 70% ethanol.

Fluorochrome experiments

Twenty-four dishes containing 3–10-wk-old healthy,

growing mycorrhizas were allocated randomly to

four fluorescent-tracer exposures: control, 15 min, 3

h and 24 h. The dishes were placed in a randomized

Latin-square arrangement with light at photon flux

density of c. 54 µmol m−# s−" from fluorescent tubes

for the whole of the short treatments and for 8 h of

the long treatment. Each treatment dish was gently

filled with 0.1% w}v 8-hydroxypyrene-1,3,6-trisul-

phonate (PTS) solution through a hole of approx. 6

mm diameter made with a soldering iron in the lid

about 1 cm from the edge of the dish and the seedling

stem. Typically, 35 ml of solution was required to

flood the root system completely. The hole was

sealed with Parafilm and the dish replaced under the

lights. After the appropriate time the shoots were cut

off outside the dish, the root systems left intact for 5

min to allow transpiration flow to cease and the

solution was gently removed. Mycorrhizas were

grasped with fine forceps, cut off with fine scissors

under the dissecting microscope, and plunge-frozen

in liquid propane cooled to near freezing point with

liquid N#, using a home-made free-fall plunging

device (G. C. Cox, pers. comm.). To avoid wounding

effects and pseudoreplication (Hurlbert, 1984), only

one mycorrhiza was taken from each plant. Samples

were freeze-substituted in tetrahydrofuran (Pa/ lsga/ rdet al., 1994) over molecular sieve for 3 d at ®80°C,

graduallywarmed to®30°Cand left for 24h,warmed

to 6°C and left for a further 1 h, then transferred to

a glove box flushed with dry N#

gas (Fitzgerald &

Allaway, 1991; Orlovich & Ashford, 1995). In the

dry box they were infiltrated over molecular sieve

with Spurr’s resin (Spurr, 1969) in 1-day steps of

30, 60, 90 and 100% at 23°C. Samples were flat-

embedded and polymerized at 60°C for 48 h and the

blocks were stored in a desiccator until sectioning.

Transverse sections 2.5 µm thick were cut with dry

glass knives in an air-conditioned room, placed on

dry glass slides pre-coatedwith chrome alum}gelatin,

and flattened by firm pressure using a pencil eraser

and cellophane (Canny & McCully, 1986). The

sections were mounted in nonfluorescent immersion

oil under a coverslip, sealed with nail polish and

stored in a desiccator in the dark until microscopy.

They were examined using a Zeiss Axiophot micro-

scope (Zeiss Goettingen, Germany) with bright-

field, Nomarski differential interference contrast,

epifluorescence using 365 nm excitation, 395 nm

dichroic mirror and 420 nm barrier filters, and

photographed on Kodak Select 100 or 400 ISO

colour-transparency film. Colours are described in

the text according to Kornerup & Wanscher (1978).

Laboratories were air-conditioned at 21–25°C.

Lanthanum treatment and X-ray microanalysis

Procedures were identical to those used for fluoro-

chrome tracing, with the following exception. Six

plates with healthy growing mycorrhizas 5–6 months

after inoculation were gently filled with 1% w}v

lanthanum nitrate solution (31 mM La(NO$)$) and

left for 24 h (of which 8 h were illuminated at c. 54

µmol m−# s−") at c. 24°C. After anhydrous freeze

substitution as described above, transverse sections

0.5 µm thick were cut with dry glass knives in an air-

conditioned room, placed on a parlodion- and

carbon-coated nickel slot grid, and covered with

another parlodion film. They were stored in a

desiccator until electron microscopy, which usually

took place within a few hours. These dry-sectioned

unstained specimens were examined at 120 kV in a

Philips (Eindhoven, The Netherlands) CM12 Scan-

ning Transmission Electron Microscope (STEM)

equipped with an Edax ultrathin-window energy-

dispersive X-ray detector and Edax DX4 analyser,

using a Be low background specimen holder. Images

were recorded in bright-field STEM mode in a

512¬400 pixel array. Energy-dispersive X-ray

microanalyses of individual areas of interest were

carried out with reduced raster sizes for 100 s. X-ray

dot maps were made by defining a 170 eV energy


window around the characteristic wavelength of each

element of interest and recording X-ray counts at

each point in a dwell time of 1000 ms in a 100¬128

pixel array, or 200 ms in a 200¬256 pixel array.

Background maps were collected from a window

between 4.09 and 4.33 keV.

Frozen-hydrated specimens

In a further experiment, frozen-hydrated specimens

were examined in SEM (after Romeo, 1996). Single

mycorrhizas (24-h La treatment and controls) were

placed on a brass block which was then immersed in

liquid N#for 45 s and the mycorrhiza fractured with

a cooled razor blade. Alternatively, mycorrhizas

were placed on aluminium pins, plunged in liquid N#

with hand-held forceps, planed with glass knives

with a Reichert-Jung FC-S cryoultramicrotome

(Leica, Vienna, Austria), then mounted in a brass

block under liquid N#. At all steps between oper-

ations the specimens, pins and brass blocks were

kept submerged in liquid N#in a covered, insulated

container. Each chilled block with specimen was

transferred as quickly as possible to the specimen

chamber (which was pumped down immediately) of

a Philips 505 SEM fitted with an Edax windowless

energy-dispersive X-ray detector and Edax DX4

analyser. The specimens remained frozen through-

out examination, which lasted up to 40 min, before

they started to freeze-dry rapidly and observation

halted. Images were recorded on Ilford FP4 roll

film. Energy dispersive X-ray microanalyses were

carried out by rastered 100-s scans of individual

areas with an accelerating voltage of 10 or 15 kV.

Fluorochrome localizations

Mycorrhizal roots showed a structure typical of this

association, with a well developed sheath and a

Hartig net restricted to the epidermis. There was a

distinct pattern of autofluorescence, and careful

comparison of treatments with controls was necess-

ary to differentiate this from PTS fluorescence. Fig.

1a shows a control section. The sheath and Hartig

net autofluoresced a high-intensity greyish yellow

while most of the root cell walls showed light-blue

autofluorescence, highest in intensity in the xylem

(not illustrated) and also seen in exodermal and other

cortical cell walls in this section. Autofluorescence of

the epidermal cell walls was a low-intensity greyish

yellow and so autofluorescence in the Hartig net was

dominated by that of the fungus, and similar to the

sheath. In the exodermis, the outer tangential walls

and outer part of the radial walls autofluoresced

yellowish white. As a result there was a distinct

difference in autofluorescence between the Hartig

net and outer tangential walls of the exodermis (Fig.

1a).

All 15-min treatments showed PTS fluorescence

only in the walls of free external hyphae and of

hyphae at the surface of the sheath (Fig. 1b). No

samples showed deeper localization of the fluoro-

chrome. The PTS fluorescence (high-intensity vivid

blue) was readily distinguished from the greyish-

yellow autofluorescence in the sheath, and also from

the light-blue and yellowish-white autofluorescence

of the root cells (Fig. 1b).

Mycorrhizas exposed to PTS solution for 3 or 24

h showed variable fluorochrome localization in the

sheath. This variability was not correlated with the

duration of treatment, but differed according to

the state of development of the mycorrhiza and to

whether or not it was surrounded by agar. In most of

the mycorrhizas exposed to PTS solution for 3 or 24

h, PTS fluorescence (high-intensity vivid blue) was

detected in the outermost sheath hyphae and in the

Hartig net and innermost sheath, but not throughout

the intervening sheath region (Fig. 1c). These

mycorrhizaswere fully developed andhad a cemented

sheath which autofluoresced yellow. Fluorochrome

in the Hartig net region was clearly restricted to the

apoplast (Fig. 1c,d) and often extended along the

interface between plant and fungal cells in the radial

walls of the epidermis and the outer tangential wall

of the exodermis (Fig. 1d). There was a sharp cut-off

of PTS fluorescence at the radial walls of the

exodermis, which show whitish autofluorescence

(Fig. 1c,d).

Fluorochrome was not detected inside the exo-

dermal Casparian band in mature regions of the

mycorrhizas (Fig. 1c,d). However, in specimens

sectioned close to the mycorrhizal tip, fluorochrome

was observed in walls of the layer of cortical cells

internal to the exodermis, suggesting that the

Casparian band had not fully developed in this

region (Fig. 1e). This PTS fluorescence was of lesser

intensity than elsewhere in the section. In some cases

there was patchy PTS fluorescence in the sheath

(Fig. 1e,f). The sheath shown in Fig. 1e was thin,

with loosely arranged but cemented hyphae. Never-

theless, only the yellow autofluorescence was present

in the middle part of the sheath, and the vivid blue

PTS fluorescence was restricted to the outer sheath,

Hartig net and innermost sheath. This pattern was

typical, and rarely was PTS fluorescence seen

spanning the sheath to connect the inner and outer

regions as in Fig. 1f. In the latter case the cemented

region of the sheath was quite thin and Hartig net

development was not complete.

In a few cases, PTS fluorescence in the mycorrhizal

sheath was more extensive. This was much rarer

than the severely restricted permeation as already

described. Fig. 1g shows one example (3-h treatment)

of a mycorrhiza from the agar surface in which PTS

fluorescence was present throughout the entire

sheath and Hartig net as far as the exodermis. The

PTS fluorescence was located in the cell walls of the


(a)

s

ep

ex

c

(b)

ep

s

(e)

ep

ex

c

s

(d)

ep

ex

s

(c)

s

ep

ex

(f)

s

ep

(g)

epex

c

s

✻

Fig. 1. Fluorescence microscopy of transverse sections of mycorrhizas : ep, epidermis; ex, exodermis; c, the

next layer of cortex; s, sheath. (a) Autofluorescence in a control specimen. Bar, 20 µm. (b) PTS fluorescence

(vivid blue) after a 15-min treatment is restricted to the walls of the outermost sheath hyphae. Bar, 20 µm. (c)

24-h PTS treatment. Vivid-blue PTS fluorescence is seen in the outer sheath hyphal walls, Hartig net and

innermost sheath, but the intervening sheath region shows only the yellow autofluorescence. Bar, 20 µm. (d)

24-h PTS treatment. Hartig net region at higher magnification. Bar, 10 µm. (e) 3-h PTS treatment. Specimen

sectioned close to the mycorrhizal tip. There are small areas of PTS fluorescence at the inner angles of

exodermal cells (arrows), and in the intervening inner walls of the exodermis. Bar, 20 µm. (f) 3-h PTS

treatment. Patches of alternating PTS fluorescence and autofluorescence in the sheath of a specimen sectioned

close to the mycorrhizal tip. Bar, 10 µm. (g) 3-h PTS treatment. Mycorrhiza of unusually high permeability,

from the agar surface. Walls of all the sheath hyphae and extra-radical hyphae show vivid-blue PTS

fluorescence. Adhering agar is also strongly PTS-fluorescent (*). Bar, 20 µm.

fungal sheath and to a lesser extent in the in-

tercellular spaces but not in the lumen of the fungal

cells. PTS fluorescence was also detected throughout

the adhering agar (Fig. 1g). From the exodermis

inwards, the walls showed autofluorescence identical

with that in the controls (Fig. 1g).


(a)

ex

ex

ep

ep

(b) La

(d) P(c) K

(f) La(e)

ex

ex

ep

ep

S

S

Fig. 2. Lanthanum-treated, freeze-substituted, dry-sectioned material : ep, epidermis; ex, exodermis; s, sheath.

Bars 5 µm. (a) Bright-field STEM image. (b) Lanthanum X-ray image of the same area. La is in the apoplast

of inner sheath, Hartig net and outer tangential walls of exodermis, but is not seen in radial or inner walls of

exodermis or further into the cortex, nor in cytoplasm or vacuoles. (c) Potassium X-ray image. K shows clearly

in apoplast of all fungal and root cells, and in the cell contents. There is a gap in the localization corresponding

to the position of the exodermal Casparian band (arrow), with a narrow strip of K on either side of it

presumably in the cytoplasm of exodermal cells. (d) Phosphorus X-ray image resembles that for K. (e) Bright-

field STEM image of another specimen with better developed Hartig net. (f) Lanthanum X-ray image. La is

in the apoplast of inner sheath, Hartig net and outer tangential walls of exodermis, terminating abruptly at the

outer part of the radial wall of the exodermis.

Lanthanum localizations

Lanthanum was typically restricted to the apoplast

(Fig. 2a,b), in clear distinction from K, which

occurred within fungal and root cells as well as in the

apoplast (Fig. 2c). Potassium and P signals were

strongest at the periphery of the plant cells, and

throughout the fungal cells, especially the vacuoles


(a)

ep

ep

ex

(b) La

(c) (d) PK

(e) (f) BgdS

s

Fig. 3. Part of the Hartig net region in lanthanum-treated, freeze-substituted, dry-sectioned material : ep,

epidermis; ex, exodermis; s, sheath. Bars 1 µm. (a) Bright-field STEM image. Fungal vacuoles of the Hartig

net, and five small aggregated particles inside the vacuole of the lower epidermal cell are electron-opaque. (b)

Lanthanum X-ray image: La is in Hartig net and inner sheath apoplast. La localization is apoplasmic, with the

exception of the five small intravacuolar particles in the epidermal cell. Diffuse scatter over the whole picture

is background and is not due to La. (c) Potassium X-ray image. K shows clearly in the apoplast of all fungal

and root cells, and in cell contents. Note the very high concentrations in Hartig net fungal vacuoles and in the

five small particles in the epidermal vacuole, and in the fungal cytoplasm and exodermal vacuole. (d)

Phosphorus X-ray image. Very high concentrations of P in Hartig net vacuoles, closely correlated with K. The

localization of both P and K fills the whole fungal vacuole profile and is not in discrete granules except for the

five small particles in (e). (e) Sulphur X-ray image. High concentraions of S in Hartig net vacuoles, correlated

with P and K. (f) Background image shows only faint outlines of Hartig net and the five small intra-vacuolar

epidermal particles, indicating that the X-ray map localizations are not due to increased mass.


La

(b)(a)(a)

ep

ex

ex

Fig. 4. Close-up of the boundary between epidermal, ep, and exodermal, ex, cells in lanthanum-treated, freeze-

substituted, dry-sectioned material. Bars 1 µm. (a) Bright-field STEM image. Electron-opaque line (arrow) in

the wall between the two exodermal cells indicates the Casparian band. Note: exodermal cell walls are more

electron-lucent than those of the epidermis or Hartig net. (b) Lanthanum X-ray map. La is present in the

epidermal walls, Hartig net apoplast, and triangular intercellular areas between epidermal and exodermal cells.

La is not detectable in the exodermal radial wall, in inverse correlation with the position of the Casparian band,

nor in the electron-lucent wall regions of the exodermal cells.

(a)

(b)

(c)

(d)

X-ray energy (keV)

Fig. 5. X-ray spectra of individual regions in lanthanum-treated, freeze-substituted, dry-sectioned material. (a)

Interface between Hartig net and exodermis: La peaks are well defined, and S and K peaks are visible; Cl is

presumed to be from the resin, and Ni from the support grid. (b) Outer corner of an exodermal cell : La is

discernible, as are K and P. (c) Inner corner of an exodermal cell : La is not detected but P and K peaks are

visible. (d) Electron-opaque material inside an exodermal cell : P and K are abundant, but La is not evident.

(Fig. 2c,d). The localization of La was similar

whether the Hartig net was well developed (Fig. 2e,f)

or not (Fig. 2a,b) : Lanthanum was commonly found

in the apoplast of the inner sheath and Hartig net as

well as in the walls of the root epidermis and the

outer wall of the exodermis. There was a sharp cut-

off of La part-way along the radial walls of the

exodermis (Fig. 2b,f), at a position indicated as the

Casparian band by a narrow gap in the localization of

K and P in the wall (Fig. 2c,d). The fungal vacuoles

showed strong and uniform K and P signals, and

weaker but also uniform S (Fig. 3a–e). The K image

along the cell peripheries in the epidermis pre-

sumably includes both cytoplasm and wall (Fig. 3c).

Potassium and S were readily detectable in the

region corresponding to the cytoplasm of the fungal


(a) (b)

ep

s

La

Fig. 6. Sheath region of lanthanum-treated, freeze-substituted, dry-sectioned material : ep, epidermis; s,

sheath. Bars 5 µm. (a) Bright-field STEM image. (b) Lanthanum X-ray image. La occurs throughout the

apoplast of the sheath, in extramatrical hyphae, other extramatrical material, the Hartig net apoplast, in

intercellular spaces in the sheath, as well as in the fungal cell walls.

(a) (b)

s

exep

c

s

h

s

s

h

m

ex

ep

Fig. 7. Cryo-SEM images of frozen-hydrated, lanthanum-treated mycorrhiza. Specimens have been planed in

the cryoultramicrotome before being put into the microscope. (a) Low-magnification image of a slightly oblique

longitudinally planed mycorrhiza shows extraradical hyphae, h; well formed sheath, s ; root apical meristem

region, m; exodermal cells, ex; and epidermal cells, ep. Bar, 100 µm. (b) Higher magnification shows sheath,

s ; epidermis, ep; well developed Hartig net, exodermis, ex; and the next layer of cortex, c. Bar, 25 µm.

cells, and there was also a distinct but weak signal for

P in this region (Fig. 3c–e). In two specimens, a few

small granules inside a plant cell showed La and

these also gave signals for K, P, S (Fig. 3a–f) and Cl.

In no other case was there any detectable La inside

the plasma membrane of any cell, either fungal or

plant. The background signal was almost uniform,

with only slightly increased intensity in areas of high

tissue density (Fig. 3f). Close examination of the

exodermis showed the presence of La in the outer

part of the outer-tangential wall but not in the inner

part of this wall (Fig. 4a,b), and in triangular

intercellular areas between epidermal and exodermal

cells (Fig. 4b) but not in the exodermal radial wall.


(a)

(b)

(c)

(d)

(e)

X-ray energy (keV)

Fig. 8. X-ray spectra of individual regions in planed lanthanum-treated, frozen-hydrated material. (a) Outer

part of the sheath: P and K are the most prominent peaks, with smaller peaks for Na, S and Cl, and clearly

defined La peaks. Cu and Fe are presumed to be in the block on which the specimen was mounted. (b) Root

epidermal wall : La is detectable, and Na, Mg, P, S, Cl and K are prominent. (c) Contents of an exodermal cell

(probably vacuole) : La is barely discernible. (d) Cortical cell inside the exodermis: typically, La is not detected.

(e) Cortical cell inside the exodermis near the mycorrhizal tip showing a rare occurrence of La in the cortex.

The bright-field image shows an electron-opaque

region in the radial walls where the La signal was not

detected (Fig. 4a), corresponding to the location of

the Casparian band. The electron-lucence of the

exodermal walls (Fig. 4a), and the absence of La

from them (Fig. 4a,b), are consistent with suberi-

zation in these walls.

Spectra of individual regions showed well defined

La peaks in the interface between the Hartig net and

exodermis, and S and K peaks could also be

discerned (Fig. 5a). It is presumed that Cl is from the

resin, which was not chloride-free, and Ni from the

support grid. At the outer corner of an exodermal

cell, La, K and P were present (Fig. 5b). At an inner

corner of an exodermal cell, however, La was not

detected, although the P and K peaks remained (Fig.

5c). Electron-opaque material inside an exodermal

cell did not show La, but P and K peaks were clearly

present (Fig. 5d): this was the typical situation inside

the cells. In view of the impermeability to PTS of

the outer and middle regions of the sheath we

examined these regions very carefully for La. All

samples, including aerial ectomycorrhizas and re-

gardless of sheath development, showed La through-

out the sheath (Fig. 6a,b).

Localizations in frozen-hydrated specimens

Frozen-hydrated specimens showed the structure of

the mycorrhizas clearly, with extraradical hyphae,

sheath and Hartig net plainly discernible (Fig. 7a,b).

The resolution of analysis on these bulk specimens

did not permit a restricted location of analysis, in

particular precluding distinction between extra-

cellular material and cytoplasm. Monte Carlo mod-

elling, assuming solid oxygen (a conservative ap-

proximation of ice) and density 1 g cm−$, suggested

that at 15 kV, as in Fig. 8, La X-rays could be

generated within a volume of up to 2.5 µm depth and

2 µm lateral radius, and that the P interaction volume

would be slightly larger. When the raster area for

analysis was on the outer part of the sheath, P and K


were the most prominent peaks, with smaller peaks

for Na, S and Cl, and clearly defined La peaks (Fig.

8a). Copper and Fe were presumed to be in the block

on which the specimen was mounted; La was also

clearly detectable in analyses with the raster area on

the root epidermal wall (Fig. 8b), but was only

marginally discernible when the raster was on the

contents (probably vacuole) of an exodermal cell

(Fig. 8c). In a cell of the cortex inside the exodermis,

La was not detected (Fig. 8d), although in two

specimens regions near the mycorrhizal tip did show

La in the cortex (Fig. 8e).

The results are consistent with our earlier obser-

vations for Eucalyptus and Pisonia mycorrhizas that

the fungal sheath of mature mycorrhizas is im-

permeable to fluorochromes. PTS is a much better

tracer than calcofluor in that it does not rely on

attraction to cell walls for tissue retention, but

requires anhydrous processing for maintenance at

the sites to which it has permeated. There is now a

body of evidence indicating that the anhydrous

freeze-substitution techniques employed here pre-

vent loss and redistribution of water-soluble fluoro-

chromes including PTS (Canny & McCully, 1986;

Fitzgerald & Allaway, 1991). The general agreement

of the localizations in frozen-hydrated material

presented here, although necessarily at lower res-

olution because of the several µm$ interaction

volume, is consistent with this.

One cannot assume that walls impermeable to

PTS are also impermeable to inorganic ions and

water. Lanthanum is a much better indicator of

permeability to ions and has been used as an ion

tracer in a number of investigations. However, most

studies have followed application of lanthanum

nitrate (La(NO$)$) with chemical fixation and}or

processing techniques where conditions were not

anhydrous. In these cases La might only have been

maintained in situ by electrostatic binding and could

have been lost, displaced or redistributed during

processing and sectioning: therefore the results have

little credibility in determining barriers to ion

permeability. The use of anhydrous techniques

avoids these problems and there is now good

evidence that ions are retained in situ by the

procedure (Pa/ lsga/ rd et al., 1994; Orlovich & Ash-

ford, 1995). To our knowledge ours is the only study

in which anhydrous processing rather than a chemi-

cal fixation has been used with La to trace apoplasmic

pathways. Use of PTS, which is anionic, and La,

which is cationic, in conjunction with freeze sub-

stitution, allows us to exclude electrostatic binding

as the primary reason for the distribution obtained in

the apoplast of these mycorrhizas.

The use of intact mycorrhizal seedlings minimizes

root disturbance and damage, thereby reducing the

potential for transient leakages (Moon et al., 1986).

The plants were capable of transpiration throughout

the experimental treatments, with potential for flow

of solution in the apoplast as well as for diffusion

(Canny, 1995; Aloni et al., 1998). In this respect

these experiments resemble a field situation more

closely than previous ones using excised roots with

sticky-wax-sealed cut ends. Restriction of sampling

to one mycorrhiza per plant also ensures that there is

no leakage into cut surfaces which might affect

results if a series of samples were taken from the

same plant. Lanthanum is known to be toxic and

might have physiological effects even at the low

concentration used here (Lazzaro & Thomson,

1992). This toxicity might account for the only two

instances of intracellular La observed in the plant

cells. The data, however, do not support extensive

‘endocytosis ’ of the tracer as commonly reported in

studies where chemical fixation has been used

(Robards & Robb, 1974; Evert et al., 1985) but are

more consistent with the occasional presence of

damaged cells than with uptake by endocytosis. It

should also be noted that the effects of La on ion

transporters might have influenced the distribution

of other ions (Lewis & Spalding, 1998).

The presence of an exodermis with Casparian

bands and suberized lamellae is well established for

eucalypt mycorrhizas, but it has been assumed that

these are barriers to apoplasmic continuity only by

extrapolation to results with nonmycorrhizal euca-

lypt roots (Ashford et al., 1989). Most evidence from

the literature suggests that the Casparian bands in

the exodermis of nonmycorrhizal roots are im-

permeable to ions, although views differ on their

permeability to water (Peterson, 1987; Peterson et

al., 1993; Varney et al., 1993; Canny & Huang,

1994; Peterson & Cholewa, 1998; Steudle & Peter-

son, 1998; Zimmermann & Steudle, 1998). It has

been shown here that the exodermal Casparian

bands are a barrier to penetration of both tracers into

the cortex of all the mycorrhizas, regardless of the

degree of permeation of PTS or La into the sheath.

The exclusion of La indicates that any pores in the

Casparian bands in the exodermis of the mycorrhizas

are small enough to exclude ions. Similarly, ex-

clusion is observed for the suberin lamellae, La

being restricted to the outer region of the outer

tangential wall of the exodermis. Only in very young

parts of the root, where there is reason to expect that

Casparian bands would not yet be fully developed,

did the apoplasmic tracers penetrate more deeply.

This supports the literature and establishes that the

exodermal Casparian bands in mycorrhizal roots

have a similarly low permeability to those of

nonmycorrhizal root systems and to the tight

junctions of animal cells (Madara, 1988). The finding

that the Casparian bands of the exodermis are a

barrier to both PTS and La$+, contrasts with the

findings of Zimmermann & Steudle (1998) that the


exodermis of corn roots allows PTS leakage. It is

consistent with their suggestion that the leakage of

PTS into the stele which they observed might be via

sites of emergence of lateral roots which breach the

exodermis. In ectomycorrhizas the apical meristem

is small and cells differentiate much closer to the

apex than in nonmycorrhizal roots. This is also true

of the exodermis, suggesting that the exodermal

apoplasmic barrier might operate much closer to the

apex in mycorrhizas than in nonmycorrhizal roots.

Mycorrhizas in contact with the wet agar surface

remained permeable to PTS while those developed

in air excluded this fluorochrome even at an early

stage. Fluorochrome was excluded from the in-

tercellular spaces and cementing material and from

the cell walls of individual hyphae in the outer sheath

whether cemented or not. The walls of these outer

sheath hyphae not only show reduced permeability,

but also tend to exclude solution from the sheath as

a whole, presumably by effects of surface tension.

Thus, mycorrhizas developed in air rapidly become

hydrophobic and nonwettable well before sheath

development is complete and the sheath is cemented,

whereas those submerged in the agar or touching its

wet surface do not. The most likely explanation for

this lies in the deposition of hydrophobins in the

walls of the sheath hyphae, providing a reason for the

upregulation of hydrophobin genes known to occur

early in sheath development and during sheath

proliferation in Eucalyptus globulus–Pisolithus tinc-

torius mycorrhizas (Martin & Tagu, 1995; Tagu &

Martin, 1995, 1996; Tagu et al., 1996, 1998).

Hydrophobins would be expected to form a hydro-

phobic layer on walls in contact with air, but to

diffuse away as monomers where walls are in contact

with water or, in this case, agar (Wessels, 1993, 1994,

1996, 1999). The higher concentrations of S found in

the vacuoles of the Hartig net hyphae, than in

vacuoles of free-living P. tinctorius mycelium

(Orlovich & Ashford, 1993; Cole et al., 1998) would

provide the S necessary for the large amounts of

cysteine required in hydrophobin production.

Thus, the observation that the aerial mycorrhizas

impermeable to PTS are permeable to La is

fascinating. The data imply that the sheath apoplast

is not totally impermeable as we formerly thought,

but that there are limits on the size of molecules

which can move across. The outer sheath is less

permeable than unmodified hyphal or plant cell

walls, but more permeable than the Casparian bands.

This should not come as a surprise. It has long been

known that tight junctions, which occlude the

apoplasmic spaces in animal systems, are selectively

permeable, and their permeability to small molecules

and La ions appears to differ greatly between

different tissues (Madara, 1988; Anderson & Van

Itallie, 1995; Chau & Lu, 1996). Although the two

are not equated in a structural sense, there is a

parallel with the sheath apoplasmic barrier which

might allow ions to move across but restrict larger

organic molecules such as those exchanged across

the interface, so that they remain trapped. A

mycorrhizal root submerged in a watery medium for

some time might therefore still be able to take up

ions via the apoplast, although it is generally

nonwettable and not very permeable.

Our long-term observations suggest that eucalypt

ectomycorrhizas tend to form best and most fre-

quently in the air above the agar. In growth pouches

they do not form in any liquid in the bottom of the

pouch. They are often found in air pockets in soil in

the wild, or at the edge of pots in the glasshouse, an

observation also made for other ectomycorrhizas in

the field and in microcosms (Francis & Read, 1994).

Such mycorrhizas formed in air are invariably

nonwettable and water-repellent, and when placed in

a liquid they float and trap air. Attention has been

drawn to the extensiveness of the extraradical

mycelium and its importance in nutrient acquisition

for a range of ectomycorrhizal associations, including

those with European isolates of P. tinctorius (Read,

1984, 1991, 1993; Smith & Read, 1997). Read has

proposed that individual ectomycorrhizal roots have

little role in nutrient uptake and considers that they

act as a food base for mycelial outgrowth and also as

structures of storage and exchange. Taking this view

further, eucalypt mycorrhizal roots, though sub-

terranean, can be viewed as aerial structures that

have more in common with fungal fruiting bodies

than with plant roots, primarily structures of storage

and exchange where the interface between partners,

supplied via uptake and symplasmic transport from

the connected extraradical mycelium (and the plant),

must be protected by a relatively impervious sheath.

Indeed, the large amount of closely packed fungal

tissue in the mycorrhizal sheath suggests a high

demand for oxygen which would not be easily

satisfied in a submerged structure with water-filled

intercellular spaces. A high requirement for oxygen

is to be anticipated during sheath expansion, for

active uptake of solutes into hyphae (Harley, 1969)

and for efficient nutrient transfer at the symbiont

interface. Retention of air-filled intercellular space

and the general nonwettability of the structure as a

whole would greatly enhance the capacity for gas

exchange, providing a clear role for hydrophobins

early in ectomycorrhizal root development, similar

to that seen in fruiting body development (Wessels,

1992; Wessels et al., 1995; Lugones et al., 1999).

However, hydrophobins are not the only substances

which might modify permeability of walls and the

extracellular matrix (Kershaw & Talbot, 1998;

Wo$ sten et al., 1999), and the possibility that other

substances could contribute to the impermeability of

the sheath in mycorrhizas should not be discounted.

It is important to emphasize that the isolate of P.

tinctorius used in this study is strongly hydrophobic.

Differences in hydrophobicity between mycorrhizas


formed with either hydrophilic or hydrophobic fungi

could explain the discrepancy in results between

different groups on permeability of different mycor-

rhizas (Behrmann & Heyser, 1992) and on expression

of ectomycorrhizins during mycorrhizal synthesis

(Guttenberger, 1994). Unestam (1991) found that

mycorrhizal mantles formed by 14 of 20 fungal

species were highly hydrophobic, although the other

six were hydrophilic. Extramatrical hyphae and

cords of fungi which produce a hydrophobic mantle

were themselves hydrophobic, except for hyphal tips

in contact with a soaked substrate (Unestam, 1991;

Unestam & Sun, 1995). Mycorrhizas produced by

the group of hydrophilic fungi might not have

upregulated expression of hydrophobin genes, could

produce lower amounts of hydrophobins, might be

more permeable, and might exhibit more direct

uptake from the environment. This is an avenue for

further study.

The permeability of the Hartig net contrasts with

that of the sheath. One explanation for the frequency

of cases where the Hartig net and inner sheath

contain significant amounts of PTS, whereas the

bulk of the sheath contains little or none, could be

that there is significant radial flow with exclusion of

this tracer, but not of water, at the exodermis. This

could concentrate tracer in the apoplast of the Hartig

net region as a sump (Canny, 1990; Varney &

Canny, 1993). We think this an unlikely explanation

here, since those sheaths that are permeable show no

obvious gradients, whereas most sheaths show

discontinuities in PTS distribution. In the latter, the

pattern of fluorescence often suggests that PTS

moves outwards from the Hartig net into some inner

parts of the sheath, whereas the material of the outer

and middle sheath is impermeable. Our data are

better explained by movement of PTS longitudinally

within the apoplast of the Hartig net from more

permeable regions, such as young or wet parts of

mycorrhizas, to hydrophobic regions. This is more

likely to be important in mycorrhizal roots of whole

transpiring plants, where mass flow of solution as

well as diffusion might occur. In the sealed excised

roots used previously (Ashford et al., 1988, 1989)

movement would certainly be restricted to diffusion.

The Casparian bands in the radial walls of the

exodermis are clearly impermeable to ions, but the

sheath has variable and differential permeability. We

can therefore now rephrase the hypothesis presented

by Ashford et al. (1989): there is an isolated shared

apoplasmic compartment at the interface between

partners, although it is not as tightly sealed on the

outside as we previously thought.

We thank the Electron Microscope Unit at The University

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and the Australian Research Council for research grants.

W. G. Allaway thanks Professor A. R. Hardham and the

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