Journal of Experimental Botany, Vol. 52, No. 358, pp. 1051±1061, May 2001
Frequencies of plasmodesmata in Allium cepa L. roots:
implications for solute transport pathways
Fengshan Ma1 and Carol A. Peterson2
Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada
Received 21 August 2000; Accepted 27 December 2000
Abstract
Introduction
Plasmodesmatal frequencies (PFs) were analysed
in Allium cepa L. roots with a mature exodermis
(100 mm from the tip). For all interfaces within the
root, the numbers of plasmodesmata (PD) mm 2 wall
surface (Fw) were calculated from measurements of
60 walls on ultrathin sections. For tissues ranging
from the epidermis up to the stelar parenchyma, the
frequencies were also expressed as total PD numbers mm 1 root length (Fn), which is most instructive
for considering the radial transport of ions and photosynthates (because the tissues were arranged in
concentric cylinders). The Fn values were constantly
high at the interfaces of exodermis±central cortex,
central cortex±endodermis and endodermis±pericycle
(4.05 3 105, 5.13 3 105, and 5.64 3 105, respectively). If
the plasmodesmata are functional, a considerable
symplastic transport pathway exists between the
exodermis and pericycle. Two interfaces had especially low PFs: epidermis±exodermis (Fn ˆ 8.96 3 104)
and pericycle±stelar parenchyma (Fn ˆ 6.44 3 104). This
suggests that there is significant membrane transport
across the interface of epidermis±exodermis (through
short cells) and direct transfer of ions from pericycle
to protoxylem vessels. In the phloem, the highest
PF was detected at the metaphloem sieve element±
companion cell interface (Fw ˆ 0.42), and all other
interfaces had much lower PFs (around 0.10). In the
pericycle, the radial walls had a high PF (Fw ˆ 0.75), a
feature that could permit lateral circulation of solutes,
thus facilitating ion (inward) and photosynthate
(outward) delivery.
Intercellular transport between living plant cells is mediated by plasmodesmata (PD). These cytoplasmic channels
provide a low-resistance pathway that is available for a
wide range of ions and molecules (typical size exclusion
limit -1 kDa). PD are dynamic structures that can be
regulated by a number of internal and external factors
(Overall and Blackman, 1996; Ding et al., 1999). When
dealing with symplastic transport across a tissue, what
is observed is the collective, rather than the individual,
functioning of PD on that particular interface. Therefore,
the frequency of functional PD is the most important
parameter that will determine the direction, extent and
rate of symplastic transport under given conditions. Data
regarding plasmodesmatal frequencies (PFs) must be
obtained with transmission electron microscopy (TEM);
the time required for this approach has largely constrained efforts to perform large-scale surveys. Available
frequency information has been principally produced for
leaves from decades of painstaking endeavour to elucidate photosynthate transport processes (Gamalei, 1991;
Van Bel, 1993; Van Bel and Oparka, 1995; Turgeon,
1996). Also, exciting advances have been made, yet again,
in leaves, in the structural and functional regulation of
PD during macromolecule transport (Ding et al., 1999;
Lucas, 1999; Oparka and Santa Cruz, 2000). In comparison, PD in root systems have received limited attention.
In a recent study of the Arabidopsis thaliana L. root apical
meristem, a tissue-speci®c pattern of PD distribution
was observed, and dye-coupling experiments established a
correlation between this pattern and the potential for
symplastic diffusion of small molecules (Zhu et al., 1998).
For root tissues proximal to the root tip, only a very
few species have been examined and in these, PFs were
measured only for selected interfaces (Robards et al.,
1973; Robards and Jackson, 1976; Warmbrodt, 1985a, b,
Key words: Allium cepa L., phloem unloading, plasmodesmata, plasmodesmatal frequency, root, symplastic
transport, transmission electron microscopy.
1
Present address: Department of Plant Biology and Plant Biotechnology Centre, The Ohio State University, Columbus, Ohio 43210, USA.
To whom correspondence should be addressed. Fax: q1 519 746 0614. E-mail: [email protected]
Abbreviations: Fn, number of plasmodesmata mm 1 root length (at individual tissue interfaces); Ft, number of plasmodesmata mm 2 tissue interface;
Fw, number of plasmodesmata mm 2 wall surface; PD, plasmodesmata; PF, plasmodesmatal frequency; TEM, transmission electron microscopy.
2
ß Society for Experimental Biology 2001
1052
Ma and Peterson
1986a; Kurkova, 1989; Wang et al., 1995). To date, a
complete picture of symplastic connections in any root
has been lacking.
Special consideration is needed for some plants that
have an exodermis in their roots. The exodermis is an
outermost cortical layer that develops Casparian bands
(Peterson and Perumalla, 1990) and suberin lamellae
(Kroemer, 1903; Von Guttenberg, 1968). The Casparian
bands, since they are in the anticlinal walls, do not affect
the roots' symplastic transport in the radial direction. The
suberin lamellae, lying all around the cells' protoplasts,
may or may not affect the symplastic connections within
the root, according to the type of the exodermis. In the
uniform exodermis (all cells elongate), as seen in Zea
mays L. (the only species of this category examined
by TEM), suberin lamellae do not interfere with the
symplastic continuity of the layer (Wang et al., 1995).
However, in the dimorphic exodermis (with long and
short cells alternating along the axis of the root), as
in Citrus sp. (Walker et al., 1984) and Allium cepa L.
(Ma and Peterson, 2000), suberin lamella deposition in
long cells severs all their PD. Accordingly, the short cells
(without suberin lamellae) must play a paramount role in
the symplastic ¯uxes (of ions and photosynthate-derived
nutrients). In an earlier paper, an overall view of PD
relationships (but not details of frequencies) of A. cepa
roots was provided (Ma and Peterson, 2000). A closer
examination of PD distribution in the exodermis (short
cells), as well as in all other tissues, will provide further
insights into the symplastic connections in mature roots.
There are some other major issues that remain unclari®ed, one of which concerns xylem loading. Are the xylem
vessels loaded by stelar parenchyma (cells intervening
between the xylem and phloem strands) or by pericycle or
by both? (See Fig. 1 for locations of these and other cell
Fig. 1. Diagram of a cross-section of Allium cepa L. root showing cell
and tissue interfaces studied for plasmodesmatal frequencies. For the
measurement of PD mm 2 wall surface (Fw), cell walls are marked with
short, thick lines. For PD mm 1 root length (Fn), tissue interfaces are
drawn in circles of thin lines (numbered 1 through 5). The boxed area
indicates the location of Fig. 3A.
types.) Most authors have assumed that ions are transported from the cortex to the stelar parenchyma from
whence they are ®nally transferred into the vessels
(Sanderson, 1975; Stelzer et al., 1975; Robards and
Clarkson, 1976; Clarkson, 1993). This idea gained
support from experiments in which stelar parenchyma
cells proved to be capable of actively accumulating ions
from the cortex in Z. mays (LaÈuchli et al., 1971a, b,
1974a, b). However, studies on Hordeum vulgare L. roots
suggested that the pericycle might play a major role in
xylem loading (Vakhmistrov et al., 1972; Kurkova et al.,
1974; Vakhmistrov, 1981). In the latter species, the
pericycle±stelar parenchyma interface had a much lower
PF than the pericycle±endodermis interface, and it was
envisaged that the majority of ions would proceed from
the pericycle directly to the xylem vessels, rather than
through the stelar parenchyma cells (Vakhmistrov et al.,
1972; Kurkova et al., 1974; Vakhmistrov, 1981). In the
present study, the relative signi®cance of these two tissues
(stelar parenchyma and pericycle) at this critical point of
radial ion transport will be examined in Allium cepa L.
roots.
Phloem unloading in roots is another ®eld that is
poorly understood. In the root tip, symplastic transport
(phloem unloading and post-phloem transport) is intensive to sustain cell division and growth (Dick and ap Rees,
1975; Oparka et al., 1994; Zhu et al., 1998). More mature
zones (proximal to the tip) are apparently weaker sinks
for photosynthates but here all the living cells still need
a continuous supply of photosynthates for their normal
respiratory activities. Phloem unloading and post-phloem
transport of photosynthates in mature zones could be
accomplished by a symplastic pathway in several species
examined (Giaquinta et al., 1983; Warmbrodt, 1985a, b,
1986a, b). However, in A. thaliana, no symplastic phloem
unloading was observed under normal conditions of
root growth (Wright and Oparka, 1997). In the present
study, it was noted whether or not the PD associated
with the phloem were structurally normal, and what
the related PFs may imply for solute transfer.
There are several ways of expressing PFs, three of
which were used in the present study. (1) The number
of PD mm 2 wall surface (Fw). This is the basic and
most commonly used value (as in most of the studies cited
above). Fw is most useful for a comparison of cell interfaces to predict their relative capacity for symplastic
transfer and, thus, is applicable to the phloem region of
the root where the paths concerned are very short. (2) The
number of PD on a tissue interface over a unit root length
(Fn). This treatment is more instructive than the previous
one for predicting symplastic transport capacity for
tissues ranging from epidermis to pericycle. This is simply
because all the tissues (except for the central cortex) are
organized into concentric cylinders; the interface areas
of which are determined by their radii (Fig. 1). The
Frequencies of plasmodesmata in onion roots
interfaces will be traversed by both ions (in the inward
direction moving from the soil solution to the stele) and
photosynthates (in the outward direction moving from
the stele to the cortex and epidermis). (3) The number of
PD mm 2 tissue interface cylinder (Ft). The values of
Ft, which may or may not be equal to Fw (see Materials
and methods), was employed in the present study for
the purpose of comparing the present results to related
literature data that were expressed in this way.
Materials and methods
Plant materials
Bulbs of Allium cepa L. cv. Ebeneezer were planted in moist
vermiculite as described previously (Ma and Peterson, 2000). At
7±14 d, root systems were collected. Any adhering vermiculite
was gently rinsed away from the roots prior to further sampling.
Transmission electron microscopy
Root segments were excised 100 mm from the tip for TEM.
(For a full account of the technique, see Ma and Peterson, 2000.)
At this distance, the roots had a mature exodermis, i.e.
Casparian bands had developed in both short and long cells,
and the latter also had suberin lamellae. The section thickness
was set at 8 3 10 2 mm.
Calculation of tissue interface areas
Free-hand cross-sections were made 100 mm from the root tip
and photographed (using colour slide ®lms). Diameters (and thus
areas) of individual tissue interfaces were measured from the
transparencies under a dissecting microscope. The surface area
of exodermal short cells was obtained from tangential sections
of the exodermis. These data were used to calculate Fn (below).
Calculation of plasmodesmatal frequencies
PFs were calculated in three different ways. The rationale for
each was presented in the Introduction.
PD mm
2
wall ( Fw)
Calculations were performed following the formula of Gunning
(in Robards, 1976):
Fw ˆ NuwL(Tq1:5R)x
(1)
where L is the length of a given cell wall; N is the number of
PD along the wall; T is the thickness of sections, and R is the
radius of PD. The units of L, T, and R are mm. For each
interface (Fig. 1), the diameters of PD were measured from
TEM negatives (mean of 5 PD at their largest dimensions in the
longitudinal view). To obtain N and L, 60 walls were randomly
sampled from 5±30 non-serial ultrathin sections (which were
cut from 5±15 roots). On each wall, N was counted directly in
the microscope. The same wall was digitized at 3 2600 by the
program Analysis 2.0 (Soft-Imaging Service GmbH) and its
length was subsequently measured with another program,
Northern Exposure (Empix Imaging, Inc.).
1053
1
PD mm root length ( Fn )
The interfaces measured were: (1) epidermis±exodermis, (2)
exodermis±central cortex, (3) central cortex±endodermis, (4)
endodermis±pericycle, and (5) pericycle±central stele (Fig. 1). It
should be noted that this approach is not suitable for the central
cortex, because intercellular spaces had developed and the cells
were not arranged in concentric cylinders. The formula is
Fn ˆ 103 p DFw
3
(2)
2
where 10 pD is area (mm ) of the cylinder (D, diameter, in mm)
into which each tissue interface ®ts. Fw, corresponding values
obtained from formula 1.
Clearly, formula 2 was not applicable to interfaces 1 and 2
(above) since intact exodermal PD did not occur in the tangential
walls of exodermal long cells but only of short cells (Ma and
Peterson, 2000). Therefore,
Fn ˆ Asc Fw
(2-1)
where Asc is the `functional area' (i.e. occupied by short cells)
over the exodermis at the outer or inner tangential side within
a 1 mm root length (see the previous section; Fig. 1). Fw,
corresponding value obtained from formula 1.
Interface 5 is highly heterogeneous, consisting of three
sub-interfaces, i.e. pericycle±stelar parenchyma, pericycle±
companion cells, and pericycle±protophloem sieve element(s).
For the ®rst sub-interface, the Fn value was calculated from
(2-2)
Fn ˆ Asp Fw
where Asp is the outer tangential area of the stelar parenchyma
over a 1 mm root length. This value was obtained from TEM
images. (Light microscope images would not give precise results
for this particular case, as the area was very small.) Fw is the
corresponding value obtained from formula 1. For the second
and third sub-interfaces, the corresponding interface areas and
Fw values were substituted in formula 2-2.
Number of PD mm
2
tissue interface ( Ft )
The formula for this calculation is
Ft ˆ Fn uA
(3)
where A is the area of a given tissue interface over a 1 mm root
length (mm2). For interfaces 3 and 4, Ft ˆ Fw (Fig. 1) because,
for each, only one cell type occurred on either side of the
interface. Values of Ft were not estimated for the central cortex
for the reasons stated above.
Statistics
Paired comparisons t-tests were performed at a ˆ 0.05 (Sokal
and Rohlf, 1981) for the values of Fw of the phloem region and
some other interfaces (see Results). Analyses were not done for
Ft and Fn, since these values are secondary in nature (i.e. derived
from the means of Fw).
Results
Plasmodesmatal frequencies in tissues from epidermis
to stelar parenchyma
The PD at the interfaces examined were similar in diameter (ranging from 59 to 65 nm) but different in frequency. Considering that the radial symplastic path spans
between the epidermis and the stelar parenchyma, the
1054
Ma and Peterson
Table 1. Frequencies of plasmodesmata in A. cepa roots (for interfaces external to the central stele)
b
a
Fw
Fn
Ft
c
EP±EX
n.a.
EP±SC
0.21"0.16
8.96"104
2
2.77"10
n.a.
EX±CC
n.a.
SC±CC
1.03"0.44
4.05"105
1
1.68"10
n.a.
CC±CC
CC±EN
EN±PE
1.59"0.43
n.a
n.a.
0.58"0.22
5.13"105
5.83"10 1
0.70"0.19
5.64"105
7.00"10 1
PE±CP
PE±PSE
PE±SP
0.16"0.12
0.08"0.04
0.12"0.09
PE±CS d6.44"104, e1.25"104
d
2 e
PE±CS 8.96"10 , 1.74"10 2
a
Fw, PD mm 2 wall surface; Fn, PD mm 1 root length; Ft, PD mm 2 tissue interface.
CC, central cortex; CP, companion cell; CS, central stele (stele excluding pericycle); EN, endodermis; EP, epidermis; EX, exodermis; PE, pericycle;
PSE, protophloem sieve element; SP, stelar parenchyma.
c
n.a., not applicable.
d
Based on the assumption that all plasmodesmata on the PE±CS interface are functional.
e
Based on the assumption that only plasmodesmata on the PE±CS interface are functional. Both sets of data (d and e) are for the entire
PE±CS interface (see text).
b
interfaces of concern were ranked, on the basis of their Fw
values, in the following order: central cortex)short cells±
central cortex)endodermis±pericycle)central cortex±
endodermis)epidermis±short cell)pericycle±stelar parenchyma. Related data are displayed in Table 1. To
provide a clearer visual indication of PD distribution,
a plasmodesmogram (Van Bel and Oparka, 1995) was
constructed, based on the Fw values (Fig. 2).
The Fn values were rather constant in the region
from the exodermis up to the pericycle, in spite of the
uncertainty about the central cortex (Table 1). For this
latter tissue, although it is theoretically possible to obtain
an Fn value, the estimate would be misleading if applied
to any explanation of radial transport, due to the presence
of intercellular spaces and the cells' irregular arrangement. Nevertheless, the abundance of PD in the cell walls
(Fw) of the central cortex apparently renders it ef®cient
for symplastic transport. At the outermost interface of
the root, epidermis±exodermis, a very small number of PD
was observed. To provide an even sharper picture of
PD distribution along the radial path (across the concentric cylinders of tissue interfaces), this set of data was
ranked by ratios (Fig. 2). In this diagram, two different
treatments were made for interface 5 (as indicated in
Fig. 1). First, it was assumed that all PD between the
pericycle and its adjacent inner living cells are functional;
the ratios are shown in the ®rst ®le of numbers. Second,
based on the assumption that the pericycle±phloem
interface is non-functional in transferring ions, the radial
symplastic pathway is then reduced to the pericycle±
stelar parenchyma interface. In this case, the second ®le of
numbers applies.
Plasmodesmatal frequencies associated with the stele
and the endodermis
The root usually contained six phloem strands (Fig. 1).
In each, the components and their spatial relationships
were as depicted in Fig. 3A. Each strand contained 1 or 2
protophloem sieve elements, 3±5 metaphloem sieve elements and their associated companion cells. At its outer
tangential side, the phloem was in contact with the
pericycle by 1 or 2 protophloem sieve elements and 3±4
Fig. 2. Plasmodesmogram of A. cepa root: PFs from the epidermis to
the stele. The PFs (Fw, PD mm 2 wall surface) are approximately
represented by short lines that link neighbouring cells. The more the
lines, the higher the frequencies (but not drawn in perfect proportion).
The PFs associated with the phloem are not shown in this diagram (but
see Fig. 4). The dotted line between adjacent epidermal cells indicates
the presence of a few PD, but quantitative data were not obtained. This
diagram can also be used to show the overall pattern of PFs expressed
as Fn (PD mm 1 root length). The numbers on the right side are the
ratios of Fn values: the ®rst ®le is based on the assumption that all
plasmodesmata between pericycle and inner living cells are functional,
and the second ®le is based on the assumption that symplastic transport
of ions is only across the pericycle±stelar parenchyma interface (see text).
In both cases, the lowest frequency was set equal to 1.0. For the central
cortex, Fn was not calculated, but the number of PD available for
symplastic transport must be very high (assumed to be higher than the
highest known frequency among the interfaces; see text). CC, central
cortex; EN, endodermis; EP, epidermis; LC, exodermal long cells; MX,
metaxylem vessel member; PE, pericycle; PH, phloem; PX, protoxylem
vessel member; SC, exodermal short cell; SP, stelar parenchyma.
companion cells. On its ¯anks, the phloem was connected
to the stelar parenchyma by companion cells. Metaphloem
sieve elements were associated neither with the pericycle
Frequencies of plasmodesmata in onion roots
1055
Fig. 3. Structure of phloem and surrounding tissues. (A) Detail of the boxed area in Fig. 1. An overview of phloem and its relationship with
surrounding tissues. (B) Pericycle and neighbouring cells. The radial walls of pericycle cells are marked by asterisks. (C) PD between metaphloem sieve
element and companion cell. The wall was thickened. (D) Companion cells. Note thick cytoplasm with ER and mitochondria (MI). (E) Interface of
pericycle and companion cell. The PD looks normal in its structure. C, cytoplasm; CP, companion cell; EMX, early metaxylem vessel member; EN,
endodermis; IEMX, immature early metaxylem vessel member; MSE, metaphloem sieve element; PD, plasmodesma(ta); PE, pericycle; PSE,
protophloem sieve element; PX, protoxylem vessel member; SP, stelar parenchyma. Bars ˆ 50 mm (A) or 0.5 mm (B±E).
nor with the stelar parenchyma. In the phloem, companion cells had the densest cytoplasm of any cell type,
characterized by numerous mitochondria and ER pro®les
(Fig. 3B, C, D, E). Pericycle cells had denser cytoplasm
than stelar parenchyma cells (Fig. 3B). Those stelar parenchyma cells deep in the stele had a thin cytoplasm and
few organelles, comparable to immature metaxylem vessel
members.
All PD in the entire phloem region were structurally
normal (as in other areas of the root, see Ma and
Peterson, 2000) and did not appear to be damaged or
blocked (e.g. Fig. 3E). In general, the PFs (Fw) in this
region were much lower than in the tissues external to the
stele (Table 2). Within the phloem, the values were very
uniform except for the one at the metaphloem sieve
element±companion cell interface (Table 2; Fig. 4), but
1056
Ma and Peterson
Table 2. A comparison of plasmodesmatal frequencies in A. cepa roots with those in the literature
Plasmodesmatal frequencies are expressed as numbers of plasmodesmata mm
speci®ed otherwise.
Interface
Allium cepa L.
a
EP±EXb
EX±CC
CC
CC±EN
EN±EN
EN±PE
PE±PE
PE±SP
SP±MSE
SP±SP
SP±CP
PSE±PE
CP±PE
PSE±CP
CP±CP
MSE±CP
MSE±PSE
MSE±MSE
a
b
0.03
0.17
1.59
0.58
0.06
0.70
0.75
0.12
g0.1
e
0.11
0.17
0.08
0.16
0.07
0.11
0.42
0.13
0.10
2
cell or tissue interface (Fw or Ft, where applicable, see text), unless
Hordeum vulgare L.
c
1.50
d
d
0.45
0.48
0.34
0.56
0.30
0.52
0.28
0.31
0.57
0.32
0.37
0.68
0.31
0.72
e
0.37
0.32
0.75
0.54
0.25
f
Zea mays L.
f
43%
45%
12%
g
0.43
0.44
0.32
0.48
0.35
0.54
0.45
0.32
0.49
0.36
0.35
0.76
0.34
0.88
h
i
0.54
1.14
0.21b
0.22c
f
g
g
a
a, Present results. b, Peterson et al., 1978. c, Tyree, 1970. d, Warmbrodt, 1985a. e, Robards and Jackson, 1976. f, Vakhmistrov et al., 1972,
plasmodesmatal frequencies in % of the total. g, Warmbrodt, 1985b. h, Clarkson et al., 1987. i, Wang et al., 1995.
b
Abbreviations: MSE, metaphloem sieve element. For the other abbreviations, see Table 1.
c
In nodal branch roots.
d
Tyree, 1970, calculated from micrographs in Scott et al., 1956.
e
Cell association absent.
f
Cell association rare.
g
Data not available.
Fig. 4. Plasmodesmogram of A. cepa roots: PFs associated with the
phloem, endodermis and pericycle. This diagram is based on Fw values.
MX, metaxylem vessel member. (For a complete list of abbreviations,
see Fig. 3.)
this was not signi®cantly different (at a ˆ 0.05). At the
phloem±pericycle interface, the Fn value was estimated in
the following way. As seen in a cross-section of the root
(Fig. 1), the phloem was connected to about half of
the inner tangential surface area of the pericycle; this
interface area was shared between companion cells and
protophloem sieve elements at a ratio of about 4 : 1
(Fig. 3A). The Fn values for these two sub-interfaces were
estimated at 4.61 3 104 and 5.76 3 103, respectively, totalling 5.19 3 104. This number was added to that of the
pericycle±stelar parenchyma interface to express the total
Fn on the entire pericycle inner face (Table 1).
The pericycle and endodermis each exhibited unique
patterns of PD distribution. Pericycle cells had very high
PFs (Fw) in both their radial and outer tangential walls,
but signi®cantly lower PF (at a ˆ 0.05) in their inner
tangential walls (Fig. 2; Table 2). In the endodermis, the
PFs on the outer and inner tangential walls were high
and comparable to each other, but the radial walls
had an extremely low PF which was signi®cantly lower
(at a ˆ 0.05) than that of corresponding walls of pericycle
(Fig. 2, Table 2).
Discussion
The present study provided a complete assessment of PFs
that were associated with the symplastic transport, both
inward (for ions) and outward (for photosynthates), in
Allium cepa L. roots. This is the ®rst such comprehensive
treatment for any root system.
Plasmodesmatal frequencies: implications for ion
transport
Symplastic transport is made possible in A. cepa roots
by the presence of PD in all tissue interfaces. Previous
understanding of symplastic transport in the root has
been largely based on studies of the cortex (Anderson and
Reilly, 1968; Ginsburg and Ginzburg, 1970a, b; Jarvis and
Frequencies of plasmodesmata in onion roots
House, 1970; Baker, 1971; Clarkson and Sanderson, 1974;
Van Iren and Boers-Van de Sluijs, 1980). The present
results on A. cepa roots strongly support a symplastic
pathway across this tissue, since its PF (Fw) was rather
high (Table 1). In addition, this major, if not the only,
pathway is most likely to occur both near the root surface
(from short cells to central cortex) and deep in the root
(from central cortex to endodermis to pericycle); high and
constant PD numbers (Fn) were found on these interfaces
(Table 1; Fig. 2).
Short cells apparently take up ions from the apoplast,
as indicated by the following observations. As shown
in Table 1, the exodermis (through short cells) was
connected with the epidermis with a much lower PF
than with the cortex. When Ft values were compared
(2.77 3 10 2 versus 1.68 3 10 1; Table 1), there was a
6-fold difference. When Fn values (which are more instructive for examining radial transport) were compared,
the difference was also obvious (5-fold). Assuming that
a constant ¯ow of material is sustained from the soil
solution to the cortex and the PD function close to their
capacity, there should be a supplementary mechanism(s)
that makes the ¯ow possible across the epidermis±short
cells interface. It is hypothesized that ions diffuse through
the epidermal walls and enter the short cells at their
outer tangential plasma membranes. Short cells normally
display features typical of metabolically active cells
(Peterson and Enstone, 1996; Ma and Peterson, 2000).
In extreme situations, short cells exhibit elevated potential
for ion uptake by developing wall ingrowths (Atriplex
hastata L., Kramer et al., 1978; A. cepa, Wilson and
Robards, 1980). In both Citrus sp. (Walker et al., 1984)
and A. cepa (Barrowclough and Peterson, 1994, Kamula
et al., 1994), the epidermis tends to die; the movement
of ions from the apoplast to the symplast of short cells
then becomes the only pathway for ion uptake in this
zone of the root. In the Z. mays exodermis (uniform,
all cells symplastically connected to adjacent tissues),
the PFs on its outer and inner tangential walls are not
as different as in A. cepa (Table 2). Therefore, even if
the hypothesized supplementary mechanism exists in
Z. mays, it is unlikely that it plays a role as signi®cant
as in A. cepa.
The PFs of the endodermis (on its outer and inner
tangential walls) have important implications for the function of the endodermis and also of the root as a whole,
especially in ion transport. It is generally believed that
the endodermal Casparian bands divert apoplastic ¯ows
of solutes from the central cortex across the endodermal
outer tangential plasma membrane into the symplast
(of the endodermis). Supposing that all solutes in the
endodermis are then constantly moved to the pericycle
symplastically, this interface would be expected to have
a higher PF (Fw) than the outer side. In H. vulgare,
indeed, the value (Fw) is doubled (0.75 versus 0.37)
1057
(as recorded in Robards et al., 1973; see Table 2). But in
another work on the same species, the difference was
trivial (Warmbrodt, 1985a; Table 2). Due to this discrepancy in the two studies, a ®rm conclusion will require
a thorough re-examination of the system. In both A. cepa,
and Z. mays, the inner and outer tangential endodermal
walls had comparable PFs (Table 2). It is interesting
to note that in these latter species, the exodermis (with
Casparian bands) is an effective apoplastic barrier
(Peterson, 1998). It is tempting to postulate that
symplastic transport across the cortex is the principal
mechanism in both species. In A. cepa, the high PF
(Fw, Table 1) would facilitate such movement. In this
connection, since H. vulgare is a non-exodermal species
(Perumalla et al., 1990), a relatively signi®cant apoplastic
pathway across the cortex would be expected. Ideally, a
comparison of Fn values of exodermal and non-exodermal species would be more informative; unfortunately,
these values are lacking in the literature.
The PFs at the periphery of the stele are important in
considering the relative signi®cance of pericycle and stelar
parenchyma in loading the xylem. In H. vulgare roots it
was found that the pericycle was poorly connected by PD
to the internal stele tissues (Vakhmistrov et al., 1972).
They suggested that ions would move directly from the
pericycle to the xylem vessels, rather than through
the internal stele tissues (see also the last section of this
Discussion). It is inferred from a later contribution
(Vakhmistrov, 1981) that the `internal stele tissues' are
equivalent to `stelar parenchyma'. Although Warmbrodt
(Warmbrodt, 1985a) was unable to con®rm the results
of Vakhmistrov et al. (Vakhmistrov et al., 1972), the latter
authors' results were in general agreement with those
of Robards and Jackson (Robards and Jackson, 1976;
see Table 2). In further support of Vakhmistrov's idea,
a recent study of H. vulgare showed that the pericycle had
more intense expression of the plasma membrane
Hq-ATPase than the stelar parenchyma (Samuels et al.,
1992). Assuming that the PD at the pericycle±phloem
interface are non-functional in transferring ions, then the
frequency of proposed functional PD (at the pericycle±
stelar parenchyma interface) is even lower in A. cepa than
in H. vulgare (Fig. 4; Table 2). If, on the other hand,
the pericycle±phloem PD were functional, then ions could
diffuse into the phloem. However, ions in the phloem
would need to cross the companion cells±stelar parenchyma interface before entering the vessels (Figs 1, 3A);
this interface is unlikely to support a signi®cant amount
of symplastic transport in view of the extremely low PF
(Table 2). Now the major concern is how the xylem
is loaded. For A. cepa roots, there is evidence that an
active step exists from the symplast to the xylem
(for Cl ; Hodges and Vaadia, 1964), which is probably
achieved by a proton pump (Clarkson and Hanson,
1986). The exact location of the active step, however, has
1058
Ma and Peterson
not been clearly determined; it could be in the stelar
parenchyma or the pericycle or both. Traditionally, an
active role has been designated to the xylem parenchyma
(LaÈuchli et al., 1971a, b, 1974a, b; De Boer, 1999).
However, this term has frequently been used to denote a
collective of stelar parenchyma and pericycle. From the
physiological point of view, there is a need to separate
these two tissues. They are different from each other both
in their ontogeny and in their spatial relationships to
adjacent tissues, which in turn could well re¯ect their
different functional roles.
On phloem unloading and post-phloem transport of
photosynthates in mature roots
Both outside and inside the phloem, transport of photosynthates can be achieved by PD. Across the root cortex,
there is physiological evidence for this kind of transport
in several species (Dick and ap Rees, 1975; Giaquinta
et al., 1983; Fisher and Oparka, 1996). Inside the phloem,
it seems to be a common feature that companion cells are
the principal receivers of the translocated photosynthates
from the shoot through sieve tubes (Warmbrodt, 1985a, b).
These ideas gained support from the present analysis
of PFs (Fig. 4; Table 2). Yet, as to the initial steps of
the post-phloem transport (or, more speci®cally, postcompanion cell transport), variations might occur among
species. For instance, in H. vulgare (Warmbrodt, 1985a),
Z. mays (Warmbrodt, 1985b) and some other species
(Patrick and Of¯er, 1996), the preferred symplastic paths
would be from companion cells to stelar parenchyma
cells and to the pericycle, along a decreasing PF gradient.
A parallel solute concentration gradient was detected in
H. vulgare (Warmbrodt, 1986b) and Z. mays (Warmbrodt,
1987). Although A. cepa shares certain similarities with
H. vulgare (Warmbrodt, 1985a) and Z. mays (Warmbrodt,
1985b) with respect to phloem construction, no such
preference is expected on an anatomical basis in the
former since its PD were distributed approximately evenly
around the phloem (Fig. 4; Table 2).
By what pathway are photosynthates unloaded from
the phloem into the pericycle in the mature zone of
the root? Unlike the richness of literature on phloem
unloading in expanding leaves and storage organs, there
is a paucity of knowledge regarding the root (Patrick,
1990; Fisher and Oparka, 1996). In A. cepa, the interfaces
of phloem±pericycle and stelar parenchyma±pericycle
had Fn ˆ 5.19 3 104 and 1.25 3 104 PD mm 1 root, respectively. The PD appeared structurally normal (Fig. 3E).
Assuming the PD on both interfaces are functional in
transferring photosynthates to the pericycle, the combined
value (6.44 3 104, close to that at the exodermis±epidermis
interface, Table 1) would support a signi®cant amount
of symplastic transport. In the mature root zone of
A. thaliana, the PD at the interface of phloem and
surrounding cells are held closed under normal conditions; they open only during lateral primordium formation (Oparka et al., 1995) or upon application of
metabolic inhibitors, as detected by ¯uorescent tracer
dyes (Wright and Oparka, 1997). Tracer experiments
also demonstrated functional phloem isolation in stems of
several species (Hayes et al., 1985; Aloni and Peterson,
1990; Van Bel and Kempers, 1990; Van Bel and
Van Rijen, 1994). In support of these results, the
expression of the A. thaliana AtSUC2 sucrose-Hq symporter has been localized exclusively in the companion
cells of root and stem (and some other organs, Truernit
and Sauer, 1995; Stadle and Sauer, 1996). A similar
expression pattern of sucrose-Hq symporter was found
in Plantago major L. (Stadle et al., 1995). The new results
are indicative of apoplastic unloading through companion cells. If this is the case in roots, photosynthates in
the apoplast will have to enter the symplast internal to the
endodermal Casparian band (an apoplastic barrier) prior
to their outward transport. Several questions concerning
phloem unloading in roots remain to be investigated: (1)
is a sucrose Hq-symporter present in all plant roots and,
more particularly, in their mature zones? (2) is the carrier
functional and, if so, how is it regulated? and (3) how are
the PD regulated along with the carriers?
A further note on the pericycle
In addition to the involvement of PD on the tangential
walls in the radial transport as discussed above, the pericycle may also conduct ions and photosynthates across
its radial walls. Based on results obtained from H. vulgare,
Vakhmistrov suggested that this cell layer could act as
an `annular collectorudisperser' (Vakhmistrov et al., 1972;
Vakhmistrov, 1981). In brief, substances received from
the endodermis tend to move symplastically across the
radial walls (with a high PF), toward those pericycle
cells that lie near the xylem vessels (i.e. the `annular
collector' role; Fig. 5A) where the concentration of ions
is kept low by the transpiration stream. In this scenario,
it was assumed that, in the stele, little symplastic transport of ions would occur toward the vessels (see the
previous section). At the same time, the pericycle functions as an `annular disperser' (Fig. 5B) in the outward
transport of photosynthates (Kurkova et al., 1974;
Vakhmistrov, 1981). This is based on the following
consideration. Physically, the pericycle cells that are
connected with the phloem (and, to a lesser extent,
probably also with the stelar parenchyma, see above)
are able to receive substances from the phloem (if the PD
are functional). But, these substances are not directly
accessible to those pericycle cells that are in contact with
the xylem (see also Figs 1 and 4). Thus, some of the
photosynthates would move directly to the adjacent
endodermal cells, and the rest would tend to equilibrate
Frequencies of plasmodesmata in onion roots
1059
Fig. 5. Pericycle as `annular collector-disperser' (from Vakhmistrov et al., 1972, but adapted to A. cepa root structure). (A) The `annular collector'
model for ion transport. The radial walls of pericycle cells are drawn with broken lines to indicate the high PF. Possible routes and intensities of
symplastic ion transport are labelled with arrows of different sizes. Hollow arrows indicate apoplastic pathways. (B) The `annular disperser' model
for photosynthate transport. Possible routes and intensities of symplastic phloem-unloading anduor post-phloem transport are labelled with arrows
of varying sizes. See text for a discussion of various ¯ows.
across the pericycle via PD, reaching the xylem-associated
pericycle cells, and then the adjacent endodermal cells.
The present results on A. cepa roots are in favour of
this model. In Z. mays, a large number of PD was also
observed in pericycle radial walls (Warmbrodt, 1985b;
see also Table 2). Vakhmistrov's brilliant insights deserve
to be better known and research should be extended to
the examination of other species to see if this model is
generally applicable, both structurally and physiologically.
Conclusions
The ®ndings reported here show that PD connections
are present across all root tissues of A. cepa. At the
epidermis±short cells interface, both symplastic transport
and transmembrane transport are anticipated. The pericycle could play a signi®cant role in symplastic transport
of ions and photosynthates, not only directly (along
the radial path) but also indirectly (by circulation in
the tangential direction across radial walls). Symplastic
photosynthate distribution could occur in the phloem
region and beyond. These results will be useful for a functional examination of ion and photosynthate movement
across the roots of A. cepa and other species.
Acknowledgements
We thank Mr Dale Weber (University of Waterloo, Waterloo)
for his expert support with the TEM, Dr William Diehl-Jones
(University of Waterloo) for instructions on cell wall measurement, Dr Tony Robards (University of York, UK) for advice
on plasmodesmatal frequency calculation, Dr Pat Newcombe
(University of Waterloo) for statistical advice, and Ms Daryl
Enstone (University of Waterloo) for her assistance throughout
the research. The Natural Sciences and Engineering
Research Council of Canada provided funding to CAP,
and the University of Waterloo awarded Graduate Student
Scholarships to FM.
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