Journal of Experimental Botany, Vol. 55, No. 397, pp. 731±741, March 2004
DOI: 10.1093/jxb/erh054 Advance Access publication 30 January, 2004
RESEARCH PAPER
The unusual vascular structure of the corm of Eriophorum
vaginatum: implications for ef®cient retranslocation of
nutrients
Ewa Cholewa* and Marilyn Grif®th²
Department of Biology, University of Waterloo, 200 University Ave West, Waterloo, Ontario N2L 3G1, Canada
Received 8 August 2003; Accepted 2 November 2003
Abstract
Eriophorum spp. are abundant perennial graminoids
in the Arctic tundra and boreal peatlands. Because
ecological studies indicated that some plants are
unusually productive on infertile and cold sites, the
anatomy of the overwintering corms of Eriophorum
vaginatum (L.) and Eriophorum scheuchzeri (Hoppe)
were examined to determine their involvement in
nutrient uptake and storage. Components of the
long-distance transport pathways were identi®ed
within the plants by using histochemical techniques
and transport of apoplastic and symplastic dyes. E.
scheuchzeri produced a rhizome that consisted
mainly of storage parenchyma cells within which collateral vascular bundles were centrally located and
arranged in a circle. By contrast, E. vaginatum developed a ring of horizontally arranged xylem and
phloem, in addition to axial amphivasal vascular bundles leading to the leaves, all of which were bordered
by transfer cells. As shown by the transport of ¯uorescein in the phloem and Safranine O in the xylem,
each axial bundle and adventitious root contacted
the horizontal ring of vascular tissues so that solutes
from one vascular bundle were translocated into the
vascular ring and circulated to another vascular bundle and/or to the roots. In addition, special groups of
sclereids that functioned in both phloem and xylem
transport were found at the base of the leaf traces
and within junctions of senescing roots. These sclereids were named `vascular sclerenchyma' and it was
hypothesized that they provide a moving end for the
vascular system because the corm dies progressively
from the distal end as it grows upward from the apical meristem. It was concluded that this unusual vas-
cular system of E. vaginatum is ef®cient in recycling
nutrients internally, which may account for its competitive advantage in infertile and cold sites.
Key
words:
Endodermis,
Eriophorum
scheuchzeri,
Eriophorum vaginatum, exodermis, phloem, sclereid, transfer
cell, vascular ring, vascular sclerenchyma, xylem.
Introduction
The cottongrasses Eriophorum vaginatum and E. scheuchzeri are circumpolar sedges that occupy different ecological niches in arctic and boreal regions. E. vaginatum
forms tussocks and dominates relatively infertile, permanently wet tundra, whereas E. scheuchzeri is rhizomatous
and typically inhabits more fertile ¯oodplains and other
wet sites characterized by disturbance (Mark and Chapin,
1989; McGraw and Chapin, 1989). In some sites, the
productivity of E. vaginatum is limited by nitrogen
availability (Defoliart et al., 1988), while in other sites
the net primary production is limited by low phosphate
(Thormann and Bayley, 1997).
A number of traits have been identi®ed that enhance the
competitive ability of E. vaginatum to dominate sites with
low fertility in the Arctic tundra and other peatlands. When
grown experimentally in competition with E. scheuchzeri,
E. vaginatum exhibited lower reproduction rates, slower
growth rates even when fertilized with nitrogen, higher
root:shoot ratios, lower nitrogen uptake, and higher
nitrogen use ef®ciency (McGraw and Chapin, 1989).
The phenology of the two species is also an important
factor in their relative competitive abilities. E. vaginatum
is an evergreen that forms clonal tussocks, which raise the
shoot meristems above the soil surface. Because the
* Present address: Agribiotics Ltd., 135 Turnbull Court, Cambridge, Ontario, Canada.
²
To whom correspondence should be addressed. Fax: +1 519 746 0614. E-mail: grif®[email protected]
Journal of Experimental Botany, Vol. 55, No. 397, ã Society for Experimental Biology 2004; all rights reserved
732 Cholewa and Grif®th
temperatures are warmer above the soil, E. vaginatum
initiates new growth earlier in the season and grows later
into the autumn compared with E. scheuchzeri (Mark and
Chapin, 1989). This growth strategy allows E. vaginatum
to ¯ower early in spring and then initiate the season's
vegetative growth without requiring a higher level of
mineral nutrition at any one time. In fact, ¯owering takes
place with minimal nutrient uptake because the soil is still
frozen. Leaf production is also limited in E. vaginatum. In
northern Alaska, E. vaginatum produces only 4±6 leaves
sequentially during the growing season, with each leaf
expanding at the expense of nutrients retranslocated from
an older leaf (Defoliart et al., 1988; Shaver and Laundre,
1997). With little nutrient uptake, these growth strategies
all rely on effective internal recycling of nutrient elements
and on minimal intermediate storage of nutrients in other
tissues (Shaver and Laundre, 1997).
By contrast, E. scheuchzeri is a summer-green plant
with a rhizomatous growth habit that has a shorter growing
season and must support reproductive and vegetative
growth simultaneously (Mark and Chapin, 1989). Because
more nutrients are required to support growth and development within a limited period of time, E. scheuchzeri is
restricted to sites with more fertile soils.
In this study the anatomy of E. vaginatum corms were
examined and the pathways of xylem and phloem transport
were explored. Evidence is provided that the unusual
ability of E. vaginatum to store and retranslocate nutrients
is correlated with its uniquely adapted vascular anatomy,
which differs from the closely related species E.
scheuchzeri.
Materials and methods
Plant material
Eriophorum vaginatum L. plants collected in Fairbanks, Alaska,
USA (64°51¢ N, 147°43¢ W), and E. scheuchzeri Hoppe plants
collected in Iqaluit, NU, Canada (63°45¢ N, 68°31¢ W), were divided
and planted in 10 cm pots in Turface (Plant Products, Brampton, ON,
Canada) and maintained in 2 cm of standing water in the greenhouse
in Waterloo, ON, Canada (42°18¢ N, 83°01¢ W), under natural
lighting supplemented with ¯uorescent lights to extend the daylength to 21 h.
Histochemistry and microscopy
Freehand sections were stained for lignin and suberin following the
procedure of Brundrett et al. (1988). Brie¯y, sections were stained in
0.1% (w/v) berberine hemisulphate (Sigma Chemical Co., St Louis,
MO, USA; Cat. no. 75160) in distilled water for 1 h and rinsed in
distilled water for 30 min. Sections were then counterstained by
immersion in 0.5% (w/v) aniline blue (Polysciences, Warrington,
PA, USA; Cat. no. 42755) in distilled water for 30 min, rinsed as
above, and mounted on slides in 0.1% (w/v) FeCl3 in 50% (v/v)
glycerine. When stained in this way, lignin and suberin ¯uoresced
yellow when viewed with ultraviolet light.
The presence of the lignin in the scleri®ed cells was further
con®rmed by a positive reaction with phloroglucinol-HCl (Gurr,
1965). Pectin was stained with ruthenium red according to Luft
(1971). Callose, or (1,3)-b-D-glucans, ¯uoresced yellow upon
staining with aniline blue ¯uorochrome at pH 9.0 (Stone et al.,
1984). Starch granules were visualized upon reaction with iodine/
potassium iodine (Gurr, 1965). Condensed tannins were visualized
by their reaction with 1% (w/v) 2,4-dimethoxybenzaldehyde in 95%
(v/v) ethanol mixed with 18% (v/v) HCl on the slide (Mace and
Howel, 1974).
All microscopic observations were performed using a Zeiss
Axiophot microscope (Carl Zeiss, Don Mills, Ontario, Canada)
equipped with an Osram HBO 100 W mercury lamp and optics for
epi¯uorescence. Sections were viewed with white, ultraviolet and
blue light. The ultraviolet ®lter set consisted of the G365 exciter
®lter, the FT395 chromatic beam splitter, and the OP420 barrier
®lter. The blue light set of ®lters included a BP546 exciter ®lter, a
FT580 chromatic beam splitter, and a LP590 barrier ®lter. Images
were captured on Kodak Ektachrome daylight slide ®lm, ISO 200,
then scanned and processed using Adobe Photoshop (Adobe
Systems Inc., San Jose, CA, USA).
Phloem loading
Fluorescein, the disodium salt, was dissolved in 5 mM MES buffer,
pH 6.3. Loading was performed at the beginning of the light period
by cutting the end of a mature source leaf at an oblique angle and
inserting it into a ¯uorescein-®lled 100 ml capillary. The plant was
illuminated with a tungsten lamp (100 mmol photons m±2 s±1 PAR)
for 3 h. After the loading period, the plant was dissected and sections
taken from the leaves and corm were rinsed in distilled water and
observed with blue light using the epi¯uorescent microscope. The
distribution of ¯uorescein was indicative of the presence of
functioning phloem and highlighted the long-distance pathway of
assimilates transport from leaves to roots (Grignon et al., 1989).
Xylem loading
E. vaginatum corms were separated and their roots washed in
running tap water. Roots were cut at the oblique angle under water to
prevent air embolism in their xylem elements. Once xylem tension
was relieved, roots were immersed in 1% (w/v) Safranine O solution
and allowed to transpire from 2±24 h. In some experiments, only
roots on one side of the corm were immersed in Safranine O solution,
while others were in water. At the end of loading period, plants were
removed from the loading solution, rinsed, and sectioned with a dry
razor blade to minimize tracer diffusion. Sections were mounted in
50% (v/v) glycerine and observed immediately. Safranine O stained
ligni®ed xylem elements and facilitated identi®cation of the pathway
of water and solute movement through xylem elements (Sauter,
1984; Cholewa et al., 2001; Schulte and Brooks, 2003).
Results
Anatomy of the corm
After removing all dead and living leaves and roots, the
corm of E. vaginatum appeared to be a modi®ed stem with
leaf scars forming rings around the corm at the nodes
(Fig. 1A). Longitudinal growth arising from the activity of
the apical meristem was very slow, as evidenced by the
short axis, short internodes and crowding of the appendages. As the plant grew, the end of the old corm died
progressively from the bottom, and new secondary corms
or cormels (Harris et al., 1992) grew from one or more
lateral buds (Fig. 1A, B). All leaves and adventitious roots
arose from the corm.
The corm of E. vaginatum was thickened and modi®ed
for storage. When sectioned, the major tissues of the corm
Vascular structure of Eriophorum vaginatum 733
were visible, including the epidermis, cortex, and central
vascular cylinder (Fig. 1B±O). Sectioning was dif®cult
because of the presence of many red, thick-walled,
ligni®ed cells associated with axial vascular strands within
the vascular cylinder (Fig. 1B). Cross-sections through the
mature corm with two developing cormels revealed that
the vascular cylinders of the mature corm and developing
cormels were directly connected (Fig. 1C). The majority of
parenchymal cells located within the central cylinder had
thin cellulosic primary cell walls that stained with aniline
blue (Fig. 1C). The parenchyma in both the cortex and the
vascular cylinder (Fig. 1D) stored a substantial amount of
starch, as visualized with iodine staining (Fig. 1E).
The outermost layers of the corm's cortex were modi®ed
into a protective, multi-layered exodermis, similar to that
found in many roots (Ma and Peterson, 2003). Although
Metcalfe (1971) reported that a hypodermis was absent in
E. vaginatum, staining with berberine/aniline blue revealed
the presence of suberized lamellae and Casparian bands in
the cell walls of the exodermis (Fig. 1F). The innermost
cell layer of the cortex was bordered by an endodermis,
with typical, ligni®ed, U-shaped thickenings in its cell
walls (Fig. 1G). Within the vascular cylinder enclosed by
the endodermis were numerous axial vascular bundles
where the phloem tissue was surrounded by a ring of xylem
elements. These amphivasal vascular bundles were encircled, in turn, by transfer cells, which were characterized by
thickened cell walls containing many pit®elds that made
them easy to distinguish from the storage parenchyma in
the rest of the corm (Fig. 1H).
In addition to the axial vascular bundles, there was a ring
of horizontally oriented vascular tissue adjacent to the
endodermis (Fig. 1H±L). Aniline blue staining for woundinduced callose revealed that sieve tubes of phloem were
present in the vascular ring just inside the endodermis
(Fig. 1J). Upon staining with berberine/aniline blue,
ligni®ed xylem elements with scalariform thickenings of
their cell walls were evident in the vascular ring by their
yellow ¯uorescence and were located to the interior of the
phloem (Fig. 1I). Interior to the xylem were several layers
of transfer cells.
In some parts of the corm, where the sclereids were
present in a plane of the cross-section, a different
arrangement of xylem elements of the vascular ring was
observed. It appeared that xylem elements were centripetal
to the sclereids, with some tracheids in direct contact with
the cluster of scleri®ed cells (Fig. 1M). Such a direct
connection implied that both cell types (xylem elements
and sclereids) were involved in the apoplastic longdistance transport of solutes. Another connection was
observed between the xylem and the transfer cells of the
vascular ring and xylem and transfer cells of the axial leaf
traces was observed (Fig. 1N), which would ensure
continuity of the xylem transport of solutes from vascular
ring to the leaf traces for upward delivery of solutes to the
leaves. Interestingly, xylem elements of roots were also in
direct contact with the vascular ring (Fig. 1O). It seems
that the vascular ring plays a central role in xylem
transport, being connected on one side to the roots and on
the other to the leaf traces.
Vascular sclerenchyma
The development and role of the clusters of scleri®ed cells
associated with the leaf traces of the corm of E. vaginatum
was puzzling. When compared with the surrounding cells
in the corm that were still dividing and elongating, the cell
walls of a group of sclereids began to lignify very early in
development (Fig. 2A). While the cell walls stained readily
for lignin, they did not stain with ruthenium red, thus
indicating a low amount of pectin at a stage in development where all the other tissues had primary cell walls that
were easily stained with ruthenium red (Fig. 2B). Fully
developed sclereids had massively thickened cell walls
with a small cell interior and pit canals leading to the
plasmodesmatal connections between cells (Fig. 2H).
Within the corm, the sclereids were always arranged in a
central position with respect to axial bundles, with some of
their peripheral cells in contact with a leaf trace (Fig. 2C).
In the older, distal part of the corm, clusters of such
sclereids were present in the roots (Fig. 2D). Closer
examination of the corm-root junction revealed that
sclereids ®lled the stele at the site of the lateral root
connections (Fig. 2E). To the authors' knowledge, this is
the ®rst study reporting the development of scleri®ed cells
in the steles of senescing roots.
In studies of E. vaginatum, Metcalfe (1971) identi®ed
structures on the centripetal side of vascular bundles and
named them `sclerenchyma caps'. Zee (1974) also identi®ed anatomically distinct cells associated with vascular
bundles at the nodes of bamboo culms and named them
`phloem transfer cells'. In cross-sections of bamboo nodes,
these phloem transfer cells were grouped together in a
structure associated with the vascular tissues that resembled the general structure of the cluster of sclereids in E.
vaginatum. However, the sclereids in E. vaginatum were
characterized by much thicker cell walls and were active in
both apoplastic and symplastic transport (data shown
below), so it was decided to name them `vascular
sclerenchyma'.
Phloem transport
The distribution of ¯uorescein is indicative of the presence
of functioning phloem and can be used to highlight the
long-distance pathway of symplastic assimilate transport
from leaves to roots (Grignon et al., 1989). Movement of
¯uorescein shows the existence of actively transporting
sieve tube connections (Schoning and Kollmann, 1997).
Fluorescein is ®rst taken up into the leaf apoplast, then it
accumulates in the cytosol by an ion trap mechanism
(Teskos et al., 1997) and is transferred symplastically to
734 Cholewa and Grif®th
the phloem. In these experiments, a ¯uorescein-®lled
microcapillary was used to load the dye into the cut surface
of a mature, source leaf of E. vaginatum (Fig. 3A).
Fluorescein was then detected in leaf mesophyll cells
(Fig. 3B) and in the leaf traces (Fig. 3C). Fluorescein was
transported in the phloem to the vascular cylinder of the
corm (Fig. 3D). By observing movement of the dye,
phloem connections were detected between axial vascular
bundles and the horizontal vascular ring. In addition,
vascular sclerenchyma associated with leaf traces retained
Vascular structure of Eriophorum vaginatum 735
¯uorescein even after prolonged rinsing. This indicated
that the sclerenchymal cells were symplastically connected
to the phloem of leaf traces and that the sclereids were still
alive at maturity, because only living cells with intact cell
membranes retain ¯uorescein and ¯uoresce. Although
ligni®ed cell walls may auto¯uoresce under blue light, the
intensity of the auto¯uorescence of unstained vascular
bundles shown in Fig. 3E, was very low. From these
observations, the pathway of assimilate transport in the
phloem could be de®ned. Once loaded into the phloem in
the leaves, assimilates were transported to the corm,
redistributed via the vascular ring, and delivered to the
roots and growing leaves or to the storage parenchyma.
Xylem transport
Transport of ions and water taken up by roots depends on
the dynamic co-ordination between hydraulic conductances of interconnected xylem vessels. Safranine O is
frequently used as a reliable tracer for water transport in
the xylem (Sauter, 1984; Cholewa et al., 2001; Schulte and
Brooks, 2003), so a Safranine O solution was used to
determine the pathway of water and solute transport from
E. vaginatum roots to the aerial parts of the plant (Fig. 4A±
F). When roots were allowed to take up Safranine O, it was
transported in the xylem elements of the roots and
delivered to the xylem of the vascular ring of the corm.
Safranine O was then distributed around the vascular ring
and to adjoining leaf traces (Fig. 4A). It appeared that the
delivery of water and solutes by the roots to the corm
depended on the site of root attachment to the corm. When
only some of the roots located on one side of the corm were
allowed to take up Safranine O while others were
incubated in water, Safranine O distribution was not
uniform. Under such conditions, the tracer was only
distributed to that part of the vascular ring to which the
roots submerged in Safranine O were connected. The
remaining part of vascular ring associated with roots
submerged in water did not stain with Safranine O
(Fig. 4B). Observations of the sections taken from near
the apex of the corm revealed that Safranine O was present
in discrete patches of the vascular ring from where it was
conducted upwards in some of leaf traces adjacent to the
vascular ring (Fig. 4C). Immersing roots of a mature corm
in Safranine O resulted in dye transport into young cormels
(Fig. 4D). Some of the sclereids associated with leaf traces
were stained with Safranine O. Such staining might be due
to the diffusion of Safranine O in the corm or it may
indicate that the cell walls of these sclereids are somehow
involved in apoplastic transfer of solutes (Fig. 4E). The
endodermis surrounding the vascular cylinder of the corm
effectively blocked outward diffusion of the solutes. Even
after a 24 h incubation of the roots in Safranine O, the
corm's cortex remained unstained, while Safranine O
diffused throughout the entire vascular cylinder (Fig. 4F).
These results demonstrate that the vascular ring is an
important component of the apoplastic pathway and serves
as an intermediary to distribute water and solutes to
multiple leaf traces and to the entire vascular cylinder.
Eriophorum scheuchzeri
The anatomy of aerial shoots and rhizomes of E.
scheuchzeri were examined to determine whether the
unusual vascular structures observed in E. vaginatum are
characteristic of this genus. The distinct anatomical
features present in E. vaginatum, such as the vascular
cylinder, vascular ring, endodermis, and vascular scler-
Fig. 1. Anatomy of the corm of Eriophorum vaginatum. (A) An individual corm stripped of leaf bases and lateral roots after treatment with
Safranine O. Short internodes (arrowhead) and nodes (arrow) were sites of adventitious roots and leaf attachment, respectively. The plants
reproduced vegetatively by developing new cormels (asterisk), scale bar=2 mm. (B) Unstained, longitudinal section of corm. Asterisk indicates
outgrowth of a new cormel, arrow points to lateral roots and arrowheads to masses of red sclereids associated with vascular bundles of leaf traces,
scale bar=1 mm. (C) Cross-section through mature corm with two outgrowing cormels (asterisks) stained with aniline blue and viewed with white
light, scale bar=1 mm. (D) Unstained cross-section through corm. Naturally coloured red masses of sclereids (arrowheads) were located in the
central, vascular region of the corm, which was surrounded by a constitutively aerenchymatous cortex (co), scale bar=500 mm. (E) Cross-section
of corm stained with iodine. Dark staining indicates the presence of starch in the storage parenchyma cells of the cortex (co) and within the stele,
scale bar=500 mm.(F±I) Cross-sections of corm stained with berberine/aniline blue for lignin and suberin and examined under ultraviolet light with
epi¯uorescence optics, scale bars=100 mm. (F) The outermost layers of cortex. Adjacent to the epidermis, a multilayered, suberized exodermis (ex)
protected cortical cells (co). (G) Innermost layers of cortex. Staining reveals the presence of the endodermis (en) with typical u-shaped secondary
thickenings in the cell walls. (H) Developing corm in which stain highlights ligni®ed xylem elements in axial amphivasal vascular bundles where
a ring of xylem (x) completely surrounded the phloem (ph). Arrowhead points to developing sclereids associated with one of the vascular bundles.
Arrows indicate specialized transfer cells encircling each vascular bundle. (I) Horizontally oriented xylem elements (arrowheads) within the
vascular ring located internally to the endodermis (en). (J) Cross-section of corm stained with aniline blue and examined under ultraviolet light.
Bright ¯uorescent spots present in the phloem were due to the staining of wound-induced pit callose deposited in the sieve plates of sieve tubes in
the horizontal vascular ring (arrows) and in the middle of amphivasal axial vascular bundles (arrowheads), scale bar=100 mm. (K±O) Sections
through the corm stained with berberine/aniline blue for lignin, scale bars=100 mm. (K) Peridermal section through corm. Tracheary elements of
the xylem of axial vascular bundles (arrows) were positioned in the centre of the corm whereas xylem elements of the vascular ring (arrowheads)
were positioned horizontally to the axis of the corm. (L) Longitudinal section through the corm. Cross-sectioned, ligni®ed xylem elements
(arrowheads) of horizontal vascular ring were scattered next to the endodermis (en). Arrow points to longitudinally viewed xylem of axial vascular
bundle. (M) Tangential section of the corm. Arrow points to the xylem of the axial vascular bundle. Xylem of vascular ring (arrowheads) was
concentrically arranged around group of sclereids. (N) Oblique-tangential section through the corm. Xylem of axial vascular bundles (arrow) was
connected to the xylem of vascular ring (arrowheads). Xylem of the vascular cylinder of a root (r) was visible in cross-section. (O) Oblique crosssection through the corm. Xylem of the adventitious root (r) was directly connected to the xylem of vascular ring (arrowheads).
736 Cholewa and Grif®th
enchyma were not observed in E. scheuchzeri. The aerial
part of the stem was surrounded by leaf sheaths (Fig. 5A),
and the vascular bundles formed a circle in the centre of the
stem (Fig. 5B).
The rhizome developed a multilayered exodermis, but
an endodermis was not present (Fig. 5D). The vascular
bundles were aligned with the phloem positioned outwards
and xylem inwards and some of the vascular bundles
Fig. 2. Development of vascular sclerenchyma in Eriophorum vaginatum corm. (A) Cross-section of the corm near the apex stained with
berberine/aniline blue and viewed under UV light. Developing sclereids (arrow) associated with vascular bundles (vb) were positioned towards the
centre of the corm, scale bar=500 mm. (B) Cross-section near the apex stained with ruthenium red. Red colouration revealed the presence of pectin
in thin-walled, dividing and expanding cells of the corm. The cell walls of developing sclereids (arrow) were not stained, scale bar=100 mm. (C±
F) Cross-sections of the corm stained with phloroglucinol-HCl. (C) Section taken in the middle of the corm. The cell walls of sclereids (arrows)
were ligni®ed, scale bar=500 mm. (D) Section taken at the base of corm. Ligni®ed masses of sclereids (arrows) were associated with adventitious
roots, scale bar=500 mm. (E) Higher magni®cation of corm-root junction. A mass of sclereids (arrow) developed in the centre of the root, scale
bar=100 mm. (F) Developing sclereids with enlarged cells and thickening cell walls were ligni®ed, beginning from the middle of the group, scale
bar=50 mm. (G) Unstained section through developed sclereids associated with amphivasal vascular bundle with phloem (ph) in the centre
surrounded by xylem elements (x). Massive, secondary cell walls and numerous pits were visible, scale bar=50 mm. (H) Higher magni®cation of
developed sclereids revealed that cell lumena, with pit canals (arrow), occupied a very small portion of the cells, scale bar=10 mm.
Vascular structure of Eriophorum vaginatum 737
merged together in mature regions of the rhizome (Fig. 5C),
but never completed an intact circle (Fig. 5E). Vascular
tissues were protected by suberized bundle sheath cells
with distinctive Casparian bands in their radial walls
(Fig. 5D). Lateral roots originated in the centre of the
rhizome. The xylem elements of the roots must be
connected at some point to the xylem of vascular bundles,
but their connection to other xylem elements was not
observed in this study (Fig. 5F).
Fig. 3. Fluorescein transport in the Eriophorum vaginatum phloem
visualized by yellow ¯uorescence upon irradiation with blue light. (A)
Mature third leaf of one corm was inserted in a ¯uorescein-containing
microcapillary (arrow). (B) Cross-section of a ¯uorescein-loaded leaf
showing that ¯uorescein was taken up by strands of mesophyll cells,
scale bar=500 mm. (C) Leaf sheath pulled back from the corm to show
that vascular strands (arrows) transported ¯uorescein, scale bar=100
mm. (D±E) Sections of corm taken after leaf ¯uorescein loading and
viewed under blue light. (D) An oblique section of young corm tip.
Fluorescein was located in the phloem of leaf traces (arrows) and was
unloaded into some of the parenchyma cells near apex, scale bar=500
mm. (E) Cross-section of the mature corm. Fluorescein was retained in
the phloem of axial vascular bundles (arrows), in the sclerenchyma
cells associated with vascular bundles (arrowheads), redistributed
around in horizontal vascular ring and transferred into phloem of
adventitious roots (r), scale bar=500 mm.
Discussion
Origin of the vascular ring
The vascular ring is not a common structure in the
rhizomes of the Cyperaceae. However, in an anatomical
study of Scirpus cyperinus, a genus within the Cyperaceae
considered to be closely related to Eriophorum spp.,
Plowman (1906) described `a dense plexus of transverse
and oblique ®bro-vascular strands in the surface of the
central cylinder, just inside the endodermis', but further
details of this structure are lacking in the modern literature.
The plexus was considered by Plowman (1906) chie¯y to
provide mechanical support, but he did note that `fully
90% of the branching and anastomosing of bundles in the
rhizome takes place in the super®cial plexus' and that all of
the root-strands and many of the leaf traces are attached to
it.
While the vascular ring probably does provide mechanical support to the corm, it has clearly been demonstrated that the vascular ring also functions as an
intermediary structure that promotes the ef®cient redistribution of solutes transported in either xylem or phloem
between the roots, corm and leaves (Figs 3, 4). The origin
of the vascular ring in E. vaginatum was not determined in
this study because it was not possible to obtain suf®cient
resolution with free-hand sections. The thickness of the E.
vaginatum corm indicates that a primary thickening
meristem, typical for larger monocotyledonous plants
(Esau, 1953), may be the source of the derivative cells
that form the vascular ring. It is possible that the vascular
ring differentiates from isolated procambial strands originating in a peripheral primary thickening meristem as
described in developing nodes of Zea mays (Pizzolato and
Sundberg, 2001, 2002). Alternatively, the shortened corm
axis, with nodes and internodes being very close together,
raises the possibility that curving leaf traces crowded
together may appear as a vascular ring (see Rudall, 1991,
and references therein). If this were the case, one would
expect the vascular ring to be organized similarly to the
amphivasal leaf traces. However, neither cross-sections
(Fig. 1I) nor tangential and longitudinal sections (Fig. 1K±
O) of the vascular ring revealed the presence of an
amphivasal arrangement. Moreover, cell division in the
vascular ring occurred in a different plane from the
vascular bundles. When the corm was cut in cross-section,
the axial bundles appeared as though they were cut in
cross-section, as expected (Fig. 1H, I). However, the ring
of vascular tissue (including the endodermis, phloem,
xylem, and transfer cells) appeared as though it were cut in
longitudinal section (Fig. 1H, I). When examined in a
tangential section, the vascular ring contained a network of
xylem elements that were horizontal and were easily
distinguished from axial leaf traces (Fig. 1K). These
ligni®ed xylem elements appeared to be cross-sectioned
738 Cholewa and Grif®th
Fig. 4. Safranine O transport in the Eriophorum vaginatum xylem was visualized as red colouration in the tissues. (A, B) Oblique sections of the
corm after incubation of roots in Safranine O. (A) Section taken at the base of the corm. Safranine O taken up by roots (r) was redistributed
around the horizontal vascular ring of the corm and loaded into axial vascular bundles (vb) in its vicinity while vascular bundles in the centre of
the corm remained unstained, scale bar=1 mm. (B) Section taken in the middle of the corm. Safranine O was loaded into the roots located on the
right side of the corm only, while other roots (r) were not immersed in the dye. Such loading revealed that only the part of the horizontal vascular
ring connected to the root was involved in Safranine O redistribution and its loading into axial vascular bundles (vb), scale bar=1 mm. (C, D)
Cross-section of a corm after Safranine O uptake into the roots. (C) Safranine O was observed in the xylem of the horizontal vascular ring in
discrete patches in the section taken near the apex. Axial vascular bundles adjacent to those patches conducted Safranine O to the leaves. The
remaining vascular bundles in the centre of the corm were not involved in Safranine O transport, scale bar=1 mm. (D) The mature corm
conducted Safranine O from the roots (r), redistributed it around the vascular ring, from where it was subsequently translocated to a new,
outgrowing cormel (asterisk). (E) A mass of the sclereids viewed under blue light revealed the presence of Safranine O as a pink ¯uorescence
emitted from the cell walls, scale bar=50 mm. (F) Longitudinal section of the corm after 24 h of incubation of the roots (r) in Safanine O.
Safranine O diffused throughout the centre of the old and new outgrowing corm, but its outward diffusion was prevented by the suberized
endodermis and the outer cortex (co) remained unstained, scale bar=5 mm.
when the longitudinal section of the corm was examined
(Fig. 1L).
Role of vascular sclerenchyma
The vascular sclerenchyma may play multiple roles in
initiating and maintaining the vascular system within the
corm of E. vaginatum. Gunning et al. (1970) suggested that
the role of vascular sclerenchyma in meristematic zones is
to provide a pathway in which solutes from the xylem
might be transferred to adjacent meristematic zones with
no effective vasculature. In the early stages of development of the corm (near the apex), the vascular sclerenchyma were ligni®ed before developing xylem elements
(Fig. 2A, B). Detection of lignin implies that the vascular
sclerenchyma mature earlier than the xylem because xylem
cells lignify their secondary cell walls prior to disintegra-
tion of the cytoplasm and acquisition of transport function
(Esau, 1953; Yaklich et al., 2001). Therefore, the lack of
ligni®cation indicated that the xylem elements were not yet
conductive. Because the E. vaginatum corm was already
thickened at this point, it required a substantial amount of
solutes to sustain meristematic growth and differentiation
of the tissues. The position of the vascular sclerenchyma
near the apical meristem, their early maturation (they are
ligni®ed before the xylem elements, Fig. 2), and their
ability to aid in xylem (they conducted Safranine O, Fig. 4)
and phloem (they retained ¯uorescein, Fig. 3) transport
indicate that, indeed, the vascular sclerenchyma could
ful®l the role of supplying nutrients and assimilates to the
apical meristem.
Another function of the sclereids may be mechanical
support because the cells were heavily ligni®ed early in the
Vascular structure of Eriophorum vaginatum 739
Fig. 5. Anatomy of the rhizome of E. scheucheri. (A, B) Auto¯uorescence of the cross-sections induced by UV light. (A) Section taken near the
apical meristem. Leaf bases (arrowheads) encircle the young rhizome. Arrows point to the undifferentiated pro-vascular strands, scale bar=500
mm. (B) Section taken 3 mm below section shown in (A). Differentiating vascular strands with one xylem element (arrowhead) were encircled
with suberized bundle sheath cells (arrows), scale bar=500 mm. (C±F) Cross-sections of a mature rhizome stained with berberine/aniline blue and
viewed with the ¯uorescence microscope. (C) Section taken from the middle of the rhizome. Vascular bundles (vb) were embedded in
aerenchymatous cortex (co) with large air cavities (arrows), scale bar=500 mm. (D) Details of rhizome anatomy. Multi-layered, suberized
exodermis encircled the aerenchymatous cortex with air cavities (arrows). Collateral vascular bundles with phloem (ph) and xylem (x) are
protected by suberized bundle sheath cells (arrowhead), scale bar=100 mm. (E) Section taken from the distal end of the rhizome. Vascular bundles
and their bundle sheath cells (arrow) were merged into an incomplete (arrowhead) circular structure, scale bar=100 mm. (F) Lateral root (r) and
rhizome junction. Berberine/aniline blue staining revealed spiral thickenings in the root xylem (x), scale bar=100 mm.
development of the corm. Moreover, in some parts of the
senescing corm, the vascular sclerenchyma appeared to
lose some of the thickness of their cell walls, and only the
central region of the cap reacted positively with phloroglucinol, which indicated the presence of lignin (Fig. 2F).
Although the cell wall composition is not known, observations raise the possibility that the thickened cell walls
themselves may be a form of carbohydrate storage, as has
been shown for the cell walls of the endosperm of seeds
(Reid, 1985).
As mentioned in the Introduction, E. vaginatum is a
perennial, tussock-forming plant. The tussock is composed
of many vegetatively propagated plants that continue to
grow upward while dying from the base. Thus, the living
plants form a crown upon a mass of dead material that
decomposes very slowly in the cold, wet environment.
Within each plant, the corm grows from the apical
meristem while the distal end of the corm undergoes
progressive senescence. Therefore, the plant requires the
ability to truncate the vascular system in a progressive way
so that nutrients and photoassimilates are not lost to the
dead tissues. The vascular sclerenchyma are associated
with vascular bundles both within the central cylinder and
within the stele of senescing roots. The authors propose
740 Cholewa and Grif®th
that another role of the vascular sclerenchyma in the corm
and roots is to provide a terminus to the vascular system to
reduce solute leakage and/or inhibit pathogen entry as the
distal end of the corm, as well as its associated leaves and
roots, senesces. Rather than simply block the vascular
system, these cells may aid in the recovery of nutrients and
their redistribution within the plant.
The cell walls of the vascular sclerenchyma were
coloured a dark red (Fig. 1D) or reddish-brown (Fig. 2G,
H) in unstained sections. The nature of this colour remains
to be elucidated. Seubert (1997) reported that some palm
roots have thickened, red-coloured cell walls in the
endodermis due to the presence of tannins. This is not
the case in E. vaginatum as staining for tannins with
dimethoxybenzaldehyde (Mace and Howell, 1974) was
negative in the sclereids and the presence of tannins was
detected only in vacuoles of some cortical cells (results not
shown). Another hypothesis was also explored, whether
the red colouration is due to the accumulation of iron in the
cell walls. However, cross-sections of corms were examined using energy-dispersive X-ray analysis and the
resulting spectra revealed no differences in the levels of
iron between sclereids and parenchyma cells (results not
shown).
Ecological consequences of internal recycling
E. vaginatum is competitive on cold and infertile sites with
other sedges such as Carex bigelowii (Shaver and Laundre,
1997) and E. scheuchzeri (Mark and Chapin, 1989;
McGraw and Chapin, 1989), in part because of its high
nutrient use ef®ciency. It is shown that E. vaginatum's
ability to utilize nutrients more ef®ciently most likely
results from its adaptive vascular system. The vascular ring
is an unusual and important component of the vasculature
as it interconnects the vascular tissues of the leaves and
adventitious roots. Because the vascular ring contains both
xylem and phloem, it can not only distribute newly
acquired nutrients and photoassimilates, respectively, but
also redistribute nutrients ef®ciently from senescing
organs to growing organs. Secondly, the vascular ring
has transfer cells that border not only vascular bundles, but
also parenchyma within the vascular cylinder that can
provide easily accessible storage for both mineral nutrients
and photoassimilates. Longer term storage of nutrients and
photoassimilates probably takes place in the cortical
parenchyma because transfer must occur via the endodermis. In addition, the development of scleri®ed vascular
sclerenchyma associated with both vascular bundles and
roots allows the plant to retain some of the nutrients that
may otherwise be lost during senescence of the corm and
its associated adventitious roots. Although some of the
nutrients may still leach from the senescing tissues into the
dead tissues below the plant, studies have shown that these
nutrients can be recovered from the tussock by new roots
and recirculated once again to the growing tissues
(Jonasson and Chapin, 1991).
The ability of E. vaginatum to retranslocate and store
nutrients may also improve its ability to survive additional
environmental stresses. For example, E. vaginatum rapidly
regrew and expanded its population after ®res on the
Alaskan tundra (Racine et al., 1987). In addition, E.
vaginatum was the only plant to survive and grow on areas
that were experimentally surface-saturated with crude oil
in a simulated winter spill (Collins et al., 1994; Racine,
1994). In both cases, the plant may have survived initially
because it has a slow growth rate, a vascular structure that
promotes ef®cient translocation of nutrients to growing
regions and a large storage capacity for nutrients within the
corm so that immediate nutrient uptake is not required.
Afterwards, the plant could easily expand its population
due to lower competition and increased availability of
nutrients. Because of these attributes, Racine (1994) has
recommended that E. vaginatum be planted for bioremediation of oil spills, especially in areas underlain by
permafrost where physical removal of crude oil is
undesirable. When used in combination with bacteria to
accelerate oil breakdown in situ, planting of E. vaginatum
could speed recovery by providing some vegetation cover
and below-ground biomass.
One consequence of the ef®cient storage and recirculation of nutrients within the tissues of E. vaginatum is that
minerals not required for growth may also be retained for
prolonged periods and could affect the food chain (Stoltz
and Greger, 2002). For example, high concentrations of
137
Cs have been found to persist in E. vaginatum in
ecosystems affected by contamination from the Chernobyl
accident (Jones et al., 1998a, b). The mobility and
retranslocation of 137Cs within the leaves and corms of
E. vaginatum were shown to be similar to other mobile
nutrients such as potassium and phosphate (Jones et al.,
1998a). The major routes for reducing 137Cs concentrations proved to be dilution by growth and loss through
senescing roots (Jones et al., 1998b).
Acknowledgements
This work was supported by a Discovery Grant from the Natural
Science and Engineering Research Council of Canada to MG. We
thank Heather C McIntyre, Institute of Arctic Biology, University of
Alaska-Fairbanks, Fairbanks, Alaska, USA, for providing
Eriophorum vaginatum plants for our study, and Dr Carol A
Peterson and Lynn Hoyles, Department of Biology, University of
Waterloo, Waterloo, ON, Canada, for access to the epi¯uorescent
microscope and for raising the plants, respectively.
References
Brundrett MC, Enstone DE, Peterson CA. 1988. A berberine/
aniline blue ¯uorescent staining procedure for suberin, lignin and
callose in plant tissues. Protoplasma 146, 133±142.
Vascular structure of Eriophorum vaginatum 741
Cholewa E, Vonhof MJ, Bouchard S, Peterson CA, Fenton B.
2001. The pathways of water movement in leaves modi®ed into
tents by bats. Biological Journal of the Linnean Society 72, 179±
191.
Collins CM, Racine CH, Walsh ME. 1994. The physical,
chemical, and biological effects of crude-oil spills after 15
years on a black spruce forest, interior Alaska. Arctic 47, 164±
175.
Defoliart LS, Grif®th M, Chapin FS, III, Jonasson S. 1988.
Seasonal patterns of photosynthesis and nutrient storage in
Eriophorum vaginatum L. an arctic sedge. Functional Ecology
2, 185±194.
Esau K. 1953. Plant anatomy. New York: John Wiley and Sons,
386±389.
Grignon
N,
Touraine
B,
Durand
M.
1989.
5(6)Carboxy¯uorescein as a tracer of phloem sap translocation.
American Journal of Botany 76, 871±877.
Gunning BES, Pate JS, Green LW. 1970. Transfer cells in the
vascular system of stems: taxonomy, association with nodes, and
structure. Protoplasma 71, 147±171.
Gurr E. 1965. The rational use of the dyes in biology. Baltimore:
Williams and Wilkins Co. 299.
Harris PJ, Ferguson LR, Robertson AM, McKenzie RJ, White
JB. 1992. Cell-wall histochemistry and anatomy of taro
(Colocasia esculenta). Australian Journal of Botany 40, 207±222.
Jonasson S, Chapin FS, III. 1991. Seasonal uptake and allocation
of phosphorus in Eriophorum vaginatum L. measured by labeling
with 32P. New Phytologist 118, 349±357.
Jones DR, Eason WR, Dighton J. 1998a. The partitioning of
137
Cs, in comparison to K, P, and Ca in the shoots of Eriophorum
vaginatum L. plants. Environmental Pollution 101, 437±439.
Jones DR, Eason WR, Dighton J. 1998b. Investigation of spatial
and temporal patterns of 137Cs partitioning in Eriophorum
vaginatum L. in relation to its nutrient retrieval and storage
strategy. Journal of Environmental Radioactivity 40, 271±288.
Luft JH. 1971. Ruthenium red and violet. I. Chemistry,
puri®cation, methods of use for electron microscopy and
mechanism of action. Anatomical Record 171, 347±368.
Ma F, Peterson CA. 2003. Current insights into the development,
structure, and chemistry of the endodermis and exodermis of
roots. Canadian Journal of Botany 81, 405±421.
Mace ME, Howel CR. 1974. Histochemistry and identi®cation of
condensed tannin precursors in roots of cotton seedlings.
Canadian Journal of Botany 52, 2423±2426.
Mark AF, Chapin III FS. 1989. Seasonal control over allocation to
reproduction in tussock-forming and rhizomatous species of
Eriophorum in central Alaska. Oecologia 78, 27±34.
McGraw JB, Chapin III FS. 1989. Competitive ability and
adaptation to fertile and infertile soils in two Eriophorum species.
Ecology 70, 736±749.
Metcalfe CR. 1971. Anatomy of the monocotyledons. V.
Cyperaceae. Oxford: Caledon Press, 260±261.
Pizzolato TD, Sundberg MD. 2001. Initiation of the vascular
system in the shoot of Zea mays L. (Poaceae). I. The procambial
nodal plexus. International Journal of Plant Science 162, 539±
566.
Pizzolato TD, Sundberg MD. 2002. Initiation of the vascular
system in the shoot of Zea mays L. (Poaceae). II. The procambial
leaf traces. International Journal of Plant Science 163, 353±367.
Plowman AB. 1906. The comparative anatomy and phylogeny of
the Cyperaceae. Annals of Botany XX, 1±36.
Racine CH. 1994. Long-term recovery of vegetation on 2
experimental crude-oil spills in interior Alaska black spruce
taiga. Canadian Journal of Botany 72, 1171±1177.
Racine CH, Johnson LA, Viereck LA. 1987. Patterns of
vegetation recovery after tundra ®res in northwestern Alaska.
Arctic and Alpine Research 19, 461±469.
Reid JSG. 1985. Cell wall storage carbohydrates in seeds:
biochemistry of the seed `gums' and hemicelluloses'. Advances
in Botanical Research 11, 125±155.
Rudall P. 1991. Lateral meristems and stem thickening growth in
monocotyledons. Botanical Review 57, 150±163.
Sauter JJ. 1984. Detection of embolization of vessels by double
staining technique. Journal of Plant Physiology 116, 331±342.
Schoning U, Kollmann R. 1997. Phloem translocation in
regenerating in vitro heterographs of different compatibility.
Journal of Experimental Botany 48, 289±295.
Schulte PJ, Brooks JR. 2003. Branch junctions and the ¯ow of
water through xylem in Douglas-®r and ponderosa stems. Journal
of Experimental Botany 54, 1597±1605.
Seubert E. 1997. Root anatomy of palms. I. Coryphoideae. Flora
192, 81±103.
Shaver GR, Laundre J. 1997. Exsertion, elongation, and
senescence of leaves of Eriophorum vaginatum and Carex
bigelowii in Northern Alaska. Global Change Biology 3, 146±
157.
Stoltz E, Greger M. 2002. Accumulation properties of As, Cd, Cu,
Pb, and Zn by four wetland plant species growing on submerged
mine tailings. Environmental and Experimental Botany 47, 271±
280.
Stone BA, Evans NA, Bonig I, Clarke AE. 1984. The application
of Sir¯uor, a chemically de®ned ¯uorochrome from aniline blue,
for detection of callose. Protoplasma 122, 191±195.
Teskos I, Platis F, Teskos V. 1997. Microspectrophotometric
analysis of accumulation of the ¯uorones K-fuorescein, rose
bengal, and phloxine red in living plant cells. Biotechnic and
Histochemistry 72, 304±314.
Thormann MN, Bayley SF. 1997. Above-ground plant production
and nutrient content of the vegetation in six peatlands in Alberta,
Canada. Plant Ecology 131, 1±16.
Yaklich RW, Wergin PW, Murphy CA, Erbe EF. 2001.
Anatomy of the phloem and xylem in the vascular sutures of
the soybean pod. Seed Science and Technology 29, 109±120.
Zee S-Y. 1974. Distribution of vascular transfer cells in the culm
nodes of bamboo. Canadian Journal of Botany 52, 345±347.