Annals of Botany 107: 591 –598, 2011
doi:10.1093/aob/mcq267, available online at www.aob.oxfordjournals.org
Casparian bands occur in the periderm of Pelargonium hortorum stem and root
Chris J. Meyer and Carol A. Peterson*
Department of Biology, University of Waterloo, Waterloo, ON, Canada N2L 3G1
* For correspondence. E-mail [email protected]
Received: 15 October 2010 Returned for revision: 19 November 2010 Accepted: 29 November 2010 Published electronically: 14 January 2011
† Background and Aims Casparian bands are characteristic of the endodermis and exodermis of roots, but also
occur infrequently in other plant organs, for example stems and leaves. To date, these structures have not
been detected in phellem cells of a periderm. The aim of this study was to determine whether Casparian
bands occur in phellem cells using tests that are known to detect Casparian bands in cells that also contain
suberin lamellae. Both natural periderm and wound-induced structures were examined in shoots and roots.
† Methods Using Pelargonium hortorum as a candidate species, the following tests were conducted: (1) staining
with berberine and counterstaining with aniline blue, (2) mounting sections in concentrated sulphuric acid and (3)
investigating the permeability of the walls with berberine as an apoplastic, fluorescent tracer.
† Key Results (1) Berberine –aniline blue staining revealed a modification in the radial and transverse walls of
mature phellem cells in both stems and roots. Three days after wounding through to the cortex of stems, the
boundary zone cells ( pre-existing, living cells nearest the wound) had developed vividly stained primary
walls. By 17 d, staining of mature phellem cells of wound-induced periderm was similar to that of natural periderm. (2) Mature native phellem cells of stems resisted acid digestion. (3) Berberine was excluded from the anticlinal (radial and transverse) walls of mature phellem cells in stems and roots, and from the wound-induced
boundary zone.
† Conclusions Casparian bands are present in mature phellem cells in both stems and roots of P. hortorum. It is
proposed that Casparian bands act to retard water loss and pathogen entry through the primary cell walls of the
phellem cells, thus contributing to the main functions of the periderm.
Key words: Anatomy, apoplast, bark, Casparian bands, cork, geranium, Pelargonium hortorum, periderm,
phellem, root, stem, wound reaction.
IN T RO DU C T IO N
Casparian bands are impregnations of suberin, a polymer with
poly(aromatic) and poly(aliphatic) domains, in the intermicrofibrillar spaces of primary cell walls (Schreiber et al., 1994;
Schreiber, 1996; Zeier and Schreiber, 1997). Due to their
location, the bands essentially prevent movement of ions and
possibly also water through the cell walls (see Enstone et al.,
2003). Casparian bands are a well-known feature of roots,
where they occur in both the endodermis and the exodermis
(Evert, 2006). They have been less frequently observed in
stems and leaves (Lersten, 1997).
Casparian bands are often, but not always, associated with
suberin lamellae. These lamellae are structures that occur on
the inner face of the primary walls. In some species, the root
endodermis does not develop lamellae so that only the bands
are present. By contrast, the mestome sheaths in leaves of
grasses in the Poaceae have suberin lamellae but not
Casparian bands (Eastman et al., 1988a, b). In the periderm,
mature phellem cells characteristically possess several layers
of suberin lamellae (Sitte, 1955). To the best of our knowledge, Casparian bands have never been detected in this
tissue in any species. It is possible that their presence is
masked by the suberin lamellae.
In the current study, the presence or absence of a Casparian
band in the phellem of Pelargonium hortorum stems and roots
was determined. We used a well-established fluorescence
staining procedure that previously allowed observation of
Casparian bands in the endodermis and exodermis of roots
when suberin lamellae were also present (Brundrett et al.,
1988). In addition to other histochemical tests, berberine, a fluorescent apoplastic tracer, was used to probe the permeability
of the phellem primary walls. Development of the periderm
was followed in both normal and wounded stem tissue.
M AT E R I A L S A N D M E T H O D S
Plant material and growth conditions
Plants of Pelargonium hortorum L.H. Bailey ‘Maime Red’,
started from stem cuttings, were grown for 2.5 years in a greenhouse with natural light and humidity, and temperatures
ranging from 18 to 27 8C throughout the year. Plants were
watered with tap water as needed to maintain moist to slightly
dry soil. They were fertilized weekly with 20– 20– 20 NPK,
and monthly with 21– 7 – 7 NPK amended with 7 % Fe
chelate (Plant Products, Brampton, ON, Canada).
Experiments were conducted from August to October 2010,
by which time the older parts of both stems and roots were
invested with a well-developed periderm.
Wound periderm was induced in stems using the technique
of Biggs (1986). A 5-mm-diameter cork borer was forced into
the internodes for 1 mm, a depth sufficient to penetrate the
periderm without injuring the vascular cambium. In stems,
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Meyer & Peterson — Casparian bands in P. hortorum periderm
each internode was wounded on opposite sides so that the areas
were never axially aligned with wounds made on adjacent
internodes. The outermost layers (epidermis and mature
phellem) within the injured area were removed with fine-tipped
forceps. After 0 – 17 d, the wounded regions were removed
using a 7-mm-diameter cork borer.
Anatomy, histochemistry and permeability
The course of normal periderm development in stems was
established by sampling regions along their lengths. The development and anatomical features of both periderm types (and
associated tissues) was ascertained from freehand cross- and
longitudinal sections on which a variety of tests were performed. These were (1) staining with berberine hemisulphate
and counterstaining with aniline blue to identify Casparian
bands (Brundrett et al., 1988); (2) staining with Sudan red
7B (fat red) to identify lipids, including suberin lamellae
(Brundrett et al., 1991); (3) staining with phloroglucinol-HCl
to detect lignin (Jensen, 1962); and (4) resistance to digestion
by concentrated sulphuric acid to locate suberin polymers
(Johansen, 1940). Sections were also observed with UV light
for autofluorescence, indicative of phenolics. Results of all
staining procedures were compared with control, unstained
sections using white or UV light as appropriate.
The apoplastic permeability of stem periderms during their
development was probed with berberine, a fluorescent tracer
dye that was precipitated in place by adding potassium thiocyanate (Enstone and Peterson, 1992). Internode zones of
interest were excised and the cut ends were sealed with
molten sticky wax (Kerr Manufacturing, Mississauga, ON,
Canada). The internodes were either left intact, abraded with
a razor blade to disrupt the cuticle, cut longitudinally
through the periderm or partially peeled to remove the outermost mature cell layers (epidermis and phellem cells),
leaving the immature phellem, phellogen and phelloderm
intact. The prepared internodes were immersed in 0.1 % berberine hemisulphate for 1.5 h, rinsed briefly with water and
then treated with 0.05 M potassium thiocyanate (KCN) for
1.5 h. The internodes were rinsed with KCN prior to sectioning. Specimens were mounted in KCN and viewed with UV
light. Results were compared with both positive and negative
controls, i.e. sections stained with berberine or left unstained,
respectively.
A similar study of wound periderm development and permeability in stems was made by sampling at various times
up to 17 d post-wounding. To observe the wound reaction,
stem cores were cross-sectioned by hand and stained as
described above. Apoplastic permeability was determined
with excised internodes with sealed ends as described above.
For roots, cross-sections were cut and stained as described
above for stems. Roots were prepared for permeability tests
by grouping 10– 20 individuals together, cutting their ends
and blotting them prior to sealing with sticky wax as described
above.
Microscopy
Sections were observed with a Zeiss Axiophot epifluorescence microscope using either white or UV light (filter
set: exciter filter G 365, dichroitic mirror FT 395, and
barrier filter LP 420; Carl Zeiss, Inc.). Photomicrographs
were taken with a Q-Imaging digital camera (Retiga 2000R,
Fast 1394 Cooled Mono, 12-bit; Quorum Technologies Inc.,
Guelph, ON, Canada).
R E S U LT S
Native periderm of the stem – structure and permeability
When cross-sections of P. hortorum stem periderm were
observed with UV light, the walls of the mature phellem produced a dark blue autofluorescence (Fig. 1A). When similar
sections were stained with berberine hemisulphate and
counterstained with aniline blue, yellow– green fluorescence
characteristic of Casparian bands was evident in the radial
walls of these cells (Fig. 1B). At higher magnification, small
air spaces could be seen between individual cells (Fig. 1C).
The Casparian bands extended throughout most of the radial
wall (Fig. 1B, C). Longitudinal sections through the mature
phellem were prepared to investigate the transverse (end)
walls. In these, the transverse as well as the radial walls
stained positively for Casparian bands with berberine –
aniline blue (Fig. 1D). Further evidence for the presence of
Casparian bands was obtained by testing for acid digestion.
The cells in a mature phellem remained attached to each
other after this treatment, indicating that a Casparian band
was present within the primary walls and also extended
across the middle lamellae between the cells (Fig. 1E). In
addition to the phellem, the cuticle originally covering the epidermis also resisted acid digestion (Fig. 1E, inset). This was a
thin layer to which remnants of cuticle covering the trichomes
remained attached. Walls of the mature phellem cells stained
red with Sudan red 7B, indicating the presence of lipids
(Fig. 1F). Such staining was absent in walls of the immature
phellem, the phellogen and phelloderm (Fig. 1F). Similarly,
lignin (stained with phloroglucinol-HCl) was detected in
cells of mature but not immature phellem (Fig. 1G).
The apoplastic permeability of the phellem and its neighbouring cells was tested with external applications of berberine, a tracer that fluoresces bright yellow under UV light. In
a young stem where the cuticle was still intact, no dye
entered (Fig. 2A). In an older stem, in which mature phellem
had developed under the epidermis, the cuticle was disrupted
prior to treatment with berberine. In this case, the tracer
entered the remaining walls of the epidermis and some adjacent walls of the outermost phellem layer that had also sustained some damage (Fig. 2B). However, further penetration
of the tracer was blocked by the anticlinal walls of the intact
phellem cells (Fig. 2B). The extent to which berberine can
penetrate tissue at various depths within an organ can be
observed by making a slit along the length of the organ, thus
opening up the interior tissues to lateral application of the
tracer. When a young stem without mature periderm was
treated with berberine, the tracer moved through the walls of
the cells adjacent to the cut (Fig. 2C). These cells with permeable walls were those of the epidermis, immature
phellem, phellogen, phelloderm and cortex (Fig. 2C). When
the slit was made in an older region of the stem, one with
two layers of mature phellem cells, berberine movement
Meyer & Peterson — Casparian bands in P. hortorum periderm
593
F I G . 1. Photomicrographs of the stem periderm of P. hortorum. Specimens are in cross-sectional view unless otherwise stated. (A) Autofluorescence of mature
phellem cell walls induced by UV light. (B) Staining with berberine hemisulfate–aniline blue. Casparian bands (white arrows) fluoresced yellow–green in the
radial walls of mature phellem cells. (C) Increased magnification of the specimen in B. Casparian bands (white arrows) are deposited in the radial walls but not
in the cell corners (white arrowheads). (D) Longitudinal section of one mature phellem layer, stained with berberine hemisulfate–aniline blue. Casparian bands
(white arrows) are present in anticlinal (radial and transverse) walls. (E) The remains of acid-digested periderm. Mature phellem cells remained intact, as evidenced
in cross- and longitudinal section. (E – inset) The epidermal cuticle with trichome casings remained intact. (F) Staining with Sudan red 7B. Lipids were stained red
(black arrowheads) in mature phellem cell walls. The immature phellem cells, phellogen and phelloderm did not stain. (G) Staining with phloroglucinol-HCl. Lignin
appeared reddish purple (black arrows) in mature phellem cell walls. Abbreviations: epi, epidermis; mph, mature phellem cell; iph, immature phellem cell; pg,
phellogen; pd, phelloderm. Note that the clarity of the figure when viewed electronically may be affected by the brightness/contrast settings of the computer
screen. In addition, images obtained from fluorescence microscopy are best viewed under low ambient light. Scale bars: (A, D, E, G) ¼ 100 mm; (B, C, F) ¼ 50 mm.
through the other cell walls proceeded as usual (compare
Fig. 2C and Fig. 2D). It also moved into the tangential walls
of the mature phellem cells but did not diffuse further
through the radial walls (Fig. 2D). The same pattern of berberine permeation was seen in stems in which different parts of
the periderm had been ripped off prior to the tracer application
(Fig. 2E, F).
Wound periderm of the stem – structure and permeability
The first apparent reaction of tissue adjacent to the crushed
cells of the wound was the production of a boundary layer.
Wall modifications were first noted 2 d after wounding. On
the third day, the boundary layer was complete. After staining
with berberine – aniline blue, the walls of this first layer
became brightly fluorescent (Fig. 3A). This staining is
typical of walls with phenolics. When the same specimen
was viewed with white light, the boundary layer could be
recognized by its highly vacuolated and fairly translucent
cells (Fig. 3B). Staining with Sudan red 7B indicated the presence of lipids in the walls of this layer (Fig. 3C). At this time, 3
d after wounding, the boundary layer was impermeable to the
passage of berberine (Fig. 3D). At 7 d post-wounding, further
changes had occurred near the wound. The boundary zone had
remained intact (Fig. 3E) but a wound periderm had begun to
form centripetally and adjacent to the boundary zone (Fig. 3F).
A phellogen, phelloderm and a few immature phellem cells
were present at this time (Fig. 3E, F). By 17 d post-wounding,
some of the phellem cells had matured (Fig. 3G). These cells
had Casparian bands in their radial walls, like the cells of the
native periderm (Fig. 3G).
Root periderm structure and apoplastic permeability
The primary root of P. hortorum was composed of an outermost epidermal layer, followed by a hypodermis and central
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Meyer & Peterson — Casparian bands in P. hortorum periderm
F I G . 2. Berberine apoplastic permeability tests on native stem periderm of P. hortorum. The photomicrographs show cross-sections of the stems. (A) Intact stem
with epidermal cuticle (white arrowheads) but lacking mature phellem cells. The cuticle prevented permeation of berberine. (B) The stem was superficially
scraped to remove cuticle. Berberine, which fluoresced yellow, permeated epidermal cell walls, and the outer tangential and radial walls of the outermost
damaged phellem cell layer (yellow arrowheads). Berberine permeation was blocked by the Casparian band in the intact anticlinal walls of underlying
phellem layers. (C) Stem that lacked mature phellem cells was incised through the cortex. Berberine permeated walls of the epidermis, immature phellem
cells, phellogen, phelloderm and cortex cells (yellow arrowheads). (D) Stem with two mature phellem layers was incised through the cortex. Berberine permeation was blocked only by Casparian bands in the anticlinal walls of mature phellem cells from both the epidermal side and the cortex side (between
yellow arrowheads). (E) Stem partially stripped exposing immature phellem cells, the radial and tangential walls of some mature phellem cells, and epidermal
cells. Berberine permeated all walls of epidermal, immature phellem, phellogen, phelloderm and cortex cells (yellow arrowheads). Additionally, the dye was
detected in exposed inner and outer tangential walls of mature phellem cells (light blue arrowheads). However, berberine did not penetrate radial walls of
mature phellem cells – the location of Casparian bands. (F) Panoramic view of a stem that was partially stripped of its mature phellem layers. As in D and
E, berberine transport was prevented by Casparian bands in mature phellem cells from both the epidermal and cortex sides. The extent of lateral flow of berberine
through the epidermis is evident. Abbreviations: epi, epidermis; mph, mature phellem cell; iph, immature phellem cell; pg, phellogen; pd, phelloderm; co, cortex.
Note that the clarity of the figure when viewed electronically may be affected by the brightness/contrast settings of the computer screen. In addition, images
obtained from fluorescence microscopy are best viewed under low ambient light. Scale bars: (A, C– F) ¼ 100 mm; (B) ¼ 50 mm.
Meyer & Peterson — Casparian bands in P. hortorum periderm
595
F I G . 3. Development and permeability of the wound-induced boundary zone and periderm in the stem of P. hortorum. The photomicrographs show crosssections of the stems. (A) Panoramic view of a complete wound-induced boundary zone stained with berberine hemisulfate– aniline blue (fluoresced greenish
yellow, white arrows); 3 d after wounding. (B) Same specimen as in A, but viewed with white light. The boundary zone is evident as highly vacuolated and
fairly translucent parenchyma cells (yellow arrows). (C) Wound-induced boundary zone cells stained with Sudan red 7B for lipids (black arrowheads); 3 d
after wounding. (D) Berberine apoplastic permeability test on a 3-d-old wound-induced boundary zone. The boundary zone prevented permeation of berberine
(yellow arrowheads). (E) Wound-induced boundary zone stained with berberine– aniline blue (white arrows); 7 d after wounding. (F) Same specimen as in E, but
viewed with white light. Development of the boundary zone (yellow arrows) and wound-induced periderm is evident but phellem cells are immature. (G)
Wound-induced boundary zone plus periderm stained with berberine–aniline blue; 17 d after wounding. In boundary zone cells, all walls are modified with
Casparian band-like material (white arrows). In mature phellem cells, the radial walls stain positively for Casparian bands (white arrowheads).
Abbreviations: bz, boundary zone cell; mph, mature phellem cell; iph, immature phellem cell; pg, phellogen; pd, phelloderm; pm, phloem; xy, xylem. Note
that the clarity of the figure when viewed electronically may be affected by the brightness/contrast settings of the computer screen. In addition, images obtained
from fluorescence microscopy are best viewed under low ambient light. Scale bars: (A –F) ¼ 100 mm; (G) ¼ 50 mm.
cortex that both contained phi thickenings in their anticlinal
walls (Fig. 4A, D). These cellulosic, lignified thickenings are
known to occur in P. hortorum roots (Haas et al., 1976).
Interior to the central cortex was a single-layered endodermis,
followed by a typical eudicot stele with a pericycle, and poles
of primary phloem and xylem in a diarch arrangement (not
shown).
After development of the periderm from the pericycle, all
primary cell layers from the epidermis to the endodermis
inclusive were sloughed off. When the periderm was stained
with berberine – aniline blue, Casparian bands were detected
in the radial walls of mature phellem cells (Fig. 4B). At
higher magnification, fluorescent material was also observed
in the corners of these cells (Fig. 4C). This trait differed
from the stem periderm in which mature phellem cells had
definite intercellular air spaces at cell corners (see Fig. 1C).
When roots were stained with phloroglucinol-HCl, lignin
was detected in the phi thickenings and vessel secondary
walls (Fig. 4D). However, with this staining test, lignification
of the mature phellem cell walls was seldom seen. Roots
were also stained with Sudan red 7B, upon which mature
phellem cells stained positively for lipids (Fig. 4E, F). In
older roots, a tannin-rich region in the outermost half of the
periderm was evident. The tannins were brown in colour,
and prevented the lipids from being detected with Sudan red
7B (Fig. 4F).
Roots were also subjected to berberine apoplastic permeability tests. Tracer transport was not restricted by the
wall-modifying structures of primary cell layers. In other
words, the phi thickenings in the hypodermis and primary
cortex do not inhibit apoplastic flow (Fig. 4G). In contrast, berberine did not permeate the radial walls of the outermost
mature phellem layer (Fig. 4G, H). Interestingly, the tanninrich region in older periderms masks the UV autofluorescence
of phenolics (Fig. 4H). In ruptured areas of the periderm, berberine permeates the tangential walls of damaged phellem
cells, but the dye is still blocked by their intact radial walls
(Fig. 4I).
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Meyer & Peterson — Casparian bands in P. hortorum periderm
F I G . 4. Anatomy and permeability of primary and secondary structures in the roots of P. hortorum. Specimens are in cross-sectional view unless otherwise stated.
(A) Primary root stained with berberine hemisulfate–aniline blue. Phi thickenings (white arrows) fluoresce brightly in the radial walls of hypodermal cells.
(A – inset) Longitudinal view of hypodermal phi thickenings, located in radial and transverse walls. (B) Root periderm stained with berberine–aniline blue.
Casparian bands (white arrowheads) fluoresced yellow–green in the radial walls of mature phellem cells. However, in older phellem regions (upper part of the photomicrograph), tannin deposition masked both autofluorescence and Casparian band fluorescence except in the corners of cells. (C) Increased magnification of the
specimen in B. Casparian bands (white arrowheads) are visible at the corners of mature phellem cells. (D) Root primary and secondary tissues stained with
phloroglucinol-HCl. Lignified structures include the phi thickenings in the hypodermis and primary cortex (black arrows), and the walls of the xylem vessels.
Lignification of mature phellem walls was seldom seen. (E) Root periderm stained with Sudan red 7B. Mature phellem cells contain lipids (black arrowheads).
(F) Root periderm stained with Sudan red 7B. The inner, younger half of the mature phellem stained positively for lipids (black arrowheads). The outer, older
half of the phellem (red double-headed arrow) contains tannins (reddish brown in colour) that mask the lipid staining. (G–I) Berberine apoplastic permeability
tests. (G) Berberine, which fluoresced yellow, permeates all walls of the primary tissues, but is blocked at the first layer of mature phellem. (H) Berberine was prevented from permeating the mature phellem. Note the blue autofluorescence of the inner half of mature phellem and lack of autofluorescence in the outer half due to
masking by tannins (red double-headed arrow). (I) In a ruptured periderm, berberine permeated damaged cells and their walls. However, the dye was blocked at the
radial walls of intact phellem cells (yellow arrowheads). Abbreviations: hyp, hypodermis; co, cortex; mph, mature phellem cell; pm, phloem; xy, xylem. Note that the
clarity of the figure when viewed electronically may be affected by the brightness/contrast settings of the computer screen. In addition, images obtained from
fluorescence microscopy are best viewed under low ambient light. Scale bars: (A–C, E) ¼ 50 mm; (A – inset, D, F–I) ¼ 100 mm.
DISCUSSION
In the past, Casparian bands have been difficult to detect
when the cells of the tissue also have suberin lamellae, as
is the case in phellem cells. The autofluorescence of the
lamellae and their positive staining for phenolics and lipids
overwhelms the staining of the less conspicuous Casparian
bands in adjacent, primary walls. For this reason, Casparian
bands in the exodermis were overlooked for many decades.
However, they can be visualized by two methods, i.e.
clearing followed by staining with Chelidonium majus
extract (Peterson et al., 1982), and staining with berberine
followed by counterstaining with aniline blue (Brundrett
et al., 1988).
Casparian bands in the endodermis form a network in the
primary walls of the cells. They were isolated and described
by Priestley and North (1922), and beautifully illustrated by
Schreiber et al. (1994). Proof of a Casparian band relies on
responses to several tests (see Ma and Peterson, 2003). It
should stain positively for phenolics and lipids, resist digestion
with sulphuric acid, and provide resistance to the passage of
apoplastically moving substances. Casparian bands are typically
confined to the radial and transverse (end) walls of the cells,
although exceptions have been noted where the band extends
into the tangential walls (Seago et al., 1999; Meyer et al., 2009).
In the phellem, it is not possible to determine whether the
primary walls contain phenolics and lipids because of the
nearby suberin lamellae of the cells. However, the present
Meyer & Peterson — Casparian bands in P. hortorum periderm
work demonstrates that, by all other criteria stated above,
Casparian bands occur in mature phellem cells of
P. hortorum root and shoot and also in the wound-induced
periderm in the shoot. (Wound-induced periderm was not
tested in roots due to their narrow diameters and soil-bound
environment.) Using the berberine – aniline blue method,
Casparian bands were seen in the radial and transverse walls
of phellem cells in stems and roots. Their position correlates
with blockage of an apoplastic tracer (berberine). The
phellem cells did not separate from each other during treatment with strong acid. To the best of our knowledge, this is
the first time that Casparian bands have been identified in
stem and root periderm. There have been reports of bands in
the related ‘polyderm’ tissue (Fahn, 1990), including six
species of Lythraceae (Stevens et al., 1997; Lempe et al.,
2001). In the case of a polyderm, a pericycle-derived phellogen generates a phellem composed of alternating layers of suberized cork cells and thin-walled, non-suberized cells (Evert,
2006). The apoplastic impermeability of the polyderm of
Eucalyptus pilularis to berberine was observed by McKenzie
and Peterson (1995), but there was no correlative positive
staining of a Casparian band with berberine – aniline blue.
The Casparian bands of the phellem occupy almost the
whole extent of the radial walls. This parallels the situation
in the endodermis and exodermis of roots. In the former, the
Casparian band typically occupies only a small fraction of
the radial wall. However, if suberin lamellae form within the
cells, the band extends to fill almost the whole of these walls
(Barnabas and Peterson, 1992; McCully and Mallett, 1993).
In the exodermis, Casparian bands and suberin lamellae
form concurrently or nearly so, and here, too, the Casparian
bands occupy most of the radial and transverse walls. Thus,
the rather large Casparian bands in the phellem are to be
expected.
In wounded tissue, a boundary layer with suberized walls
forms early. Berberine tracer experiments showed that this
layer substantially reduced the permeability of the apoplast.
The early wall modifications in the boundary layer correlate
with the observed blockage of infection by the fungal pathogen Pythium ultimum beginning 2 d after wounding
P. hortorum stems (Cline and Neely, 1983).
A major function of the periderm is to prevent water loss
from the plant to its surroundings. This is an obvious requirement in shoots but could also be important for older parts of
roots near the soil surface. The suberin lamellae, located
interior to the primary walls of the phellem, are in a position
to reduce water flow through the transcellular path. As the
cells are dead at maturity, the symplastic path of transport no
longer exists. The remaining path is the apoplast located in
the primary walls of the cells. Even in living tissue, the unmodified cell walls constitute the pathway of least resistance to
water flow (Steudle and Peterson, 1998). Modifications to
the primary walls of the phellem are therefore of importance
in reducing water flow through the tissue. From previous
work it is known that the primary walls of the phellem are lignified (Godkin et al., 1983; Biggs, 1986), and this was confirmed for P. hortorum shoots and roots in the present study.
The addition of suberin, with its hydrophobic fatty acid component, to the lignin would reduce the permeability of the
primary walls to water and improve the overall function of
597
the periderm. Waxes associated with suberin are known to
be important for the hydrophobicity of the walls (Schreiber
et al., 2005). However, it would not be possible to quantify
these from the Casparian bands of the phellem due to the
large quantities of the suberin lamellae in the cells.
A second critical function of the periderm is to resist pathogen attack. According to Wood (1960), of all plant parts, suberized tissue is the most effective in this regard. The suberin
component of the primary walls and middle lamellae in the
phellem should be crucial in preventing invasion by pathogens
entering by these routes.
This is the first report of Casparian bands in the phellem of
any species, in this case P. hortorum. It would clearly be of
interest to extend this work to other species. According to
Lulai and Morgan (1992), Casparian bands are absent from
Solanum tuberosum ( potato) tuber periderm. Similarly, evidence from water permeability studies led Schönherr and
Zeigler (1980) to conclude that a Casparian band is absent
from Betula pendula phellem. We hope that any such future
research on this topic will include results obtained with the
basic methods used in the current work, i.e. staining with
berberine – aniline blue, acid digestion and apoplastic permeability tests.
ACK NOW LED GE MENTS
We are indebted to Lynn Hoyles for culturing and maintaining
the P. hortorum plants. This research was funded by a
Discovery Grant from the Natural Sciences and Engineering
Research Council of Canada to C.A.P.
L I T E R AT U R E CI T E D
Barnabas AD, Peterson CA. 1992. Development of Casparian bands and
suberin lamellae in the endodermis of onion roots. Canadian Journal of
Botany 70: 2233–2237.
Biggs AR. 1986. Phellogen regeneration in injured peach tree bark. Annals of
Botany 57: 460– 470.
Brundrett MC, Enstone DE, Peterson CA. 1988. A berberine– aniline blue
fluorescent staining procedure for suberin, lignin and callose in plant
tissue. Protoplasma 146: 133 –142.
Brundrett MC, Kendrick B, Peterson CA. 1991. Efficient lipid staining in
plant material with Sudan red 7B or Fluorol yellow 088 in polyethylene
glycol-glycerol. Biotechnic and Histochemistry 66: 111–116.
Cline MN, Neely D. 1983. The histology and histochemistry of the woundhealing process in geranium cuttings. Journal of the American Society
of Horticultural Science 108: 496 –502.
Eastman PAK, Peterson CA, Dengler NG. 1988a. Suberized bundle sheaths
in grasses (Poaceae) of different photosynthetic types. I. Anatomy, ultrastructure and histochemistry. Protoplasma 142: 92–111.
Eastman PAK, Dengler NG, Peterson CA. 1988b. Suberized bundle sheaths
in grasses (Poaceae) of different photosynthetic types. II. Apoplastic permeability. Protoplasma 142: 112 –126.
Enstone DE, Peterson CA. 1992. A rapid, fluorescence technique to probe the
permeability of the root apoplast. Canadian Journal of Botany 70:
1493– 1501.
Enstone DE, Peterson CA, Ma F. 2003. Root endodermis and exodermis:
structure, function, and responses to the environment. Journal of Plant
Growth Regulation 21: 335–351.
Evert RF. 2006. Esau’s plant anatomy. New Jersey: Wiley.
Fahn A. 1990. Plant anatomy, 4th edn. Oxford: Pergamon Press.
Godkin S, Grozdits GA, Keith C. 1983. The periderms of three North
American conifers. Part 2: fine structure. Wood Science and Technology
17: 13–30.
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Meyer & Peterson — Casparian bands in P. hortorum periderm
Haas DL, Carothers ZB, Robbins RR. 1976. Observations on the
phi-thickenings and Casparian strips in Pelargonium roots. American
Journal of Botany 63: 863– 867.
Jensen WA. 1962. Botanical histochemistry. San Francisco: W.H. Freeman.
Johansen DA. 1940. Plant microtechnique. New York: McGraw-Hill.
Lempe J, Stevens KJ, Peterson RL. 2001. Shoot responses of six Lythraceae
species to flooding. Plant Biology 3: 186–193.
Lersten NR. 1997. Occurrence of endodermis with a Casparian strip in stem
and leaf. The Botanical Review 63: 265–272.
Lulai EC, Morgan W. 1992. Histochemical probing of potato periderm with
Neutral Red: a sensitive cytofluorochrome for the hydrophobic domain of
suberin. Biotechnic & Histochemistry 67: 185–195.
Ma F, Peterson CA. 2003. Current insights into the development, structure
and chemistry of the endodermis and exodermis. Canadian Journal of
Botany 81: 405–421.
McCully ME, Mallett JE. 1993. The branch roots of Zea. 3. Vascular connections and bridges for nutrient recycling. Annals of Botany 71: 327–341.
McKenzie BEM, Peterson CA. 1995. Root browning in Pinus banksiana
Lamb. and Eucalyptus pilularis Sm. 2. Anatomy and permeability of
the cork zone. Botanica Acta 108: 138–143.
Meyer CJ, Seago JL, Peterson CA. 2009. Environmental effects on the maturation of the endodermis and multiseriate exodermis of Iris germanica
roots. Annals of Botany 103: 687– 702.
Peterson CA, Emanuel ME, Wilson C. 1982. Identification of a Casparian
band in the hypodermis of onion and corn roots. Canadian Journal of
Botany 60: 1529– 1535.
Priestley JH, North EE. 1922. Physiological studies in plant anatomy III. The
structure of the endodermis in relation to its function. New Phytologist 21:
113–139.
Schönherr J, Zeigler H. 1980. Water permeability of Betula periderm. Planta
147: 345–354.
Schreiber L. 1996. Chemical composition of Casparian strips isolated from
Clivia miniata Reg. roots: evidence for lignin. Planta 199: 596– 601.
Schreiber L, Breiner H, Riederer M, Düggelin M, Guggenheim R. 1994.
The Casparian strip of Clivia miniata Reg. roots: isolation, fine structure
and chemical nature. Botanica Acta 107: 353–361.
Schreiber L, Franke R, Hartmann K. 2005. Wax and suberin development
of native and wound periderm of potato (Solanum tuberosum L.) and its
relation to peridermal transpiration. Planta 220: 520– 530.
Seago JLJr, Peterson CA, Enstone DE, Scholey CA. 1999. Development of
the endodermis and hypodermis of Typha glauca Godr. and Typha angustifolia L. roots. Canadian Journal of Botany 77: 122– 134.
Sitte P. 1955. Der Feinbau verkorkter Zellwände. Mikroskopie (Wein) 10:
178– 200.
Steudle E, Peterson CA. 1998. How does water get through roots? Journal of
Experimental Botany 49: 775– 788.
Stevens KJ, Peterson RL, Stephenson GR. 1997. Morphological and anatomical responses of Lythrum salicaria L. ( purple loosestrife) to an
imposed water gradient. International Journal of Plant Sciences 158:
172– 183.
Wood RKS. 1960. The chemical ability to breach the host barriers. In:
Horsfall J, Dimond AE. eds. Plant pathology. An advanced treatise. II.
New York: Academic Press, 233– 272.
Zeier J, Schreiber L. 1997. Chemical composition of hypodermal and endodermal cell walls and xylem vessels isolated from Clivia miniata: identification of the biopolymers lignin and suberin. Plant Physiology 113:
1223–1231.