Evidence for Symplastic Involvement in the Radial
Movement of Calcium in Onion Roots1
Ewa Cholewa2 and Carol A. Peterson*
Department of Biology, University of Waterloo, Waterloo, Ontario, Canada, N2L G1 (C.A.)
The pathway of Ca21 movement from the soil solution into the stele of the root is not known with certainty despite
a considerable body of literature on the subject. Does this ion cross an intact, mature exodermis and endodermis? If so, is its
movement through these layers primarily apoplastic or symplastic? These questions were addressed using onion (Allium cepa)
adventitious roots lacking laterals. Radioactive Ca21 applied to the root tip was not transported to the remainder of the plant,
indicating that this ion cannot be supplied to the shoot through this region where the exodermis and endodermis are
immature. A more mature zone, in which the endodermal Casparian band was present, delivered 2.67 nmol of Ca21 mm1
treated root length d1 to the transpiration stream, demonstrating that the ion had moved through an intact endodermis.
Farther from the root tip, a third zone in which Casparian bands were present in the exodermis as well as the endodermis
delivered 0.87 nmol Ca21 mm1 root length d1 to the transpiration stream, proving that the ion had moved through an
unbroken exodermis. Compartmental elution analyses indicated that Ca21 had not diffused through the Casparian bands of
the exodermis, and inhibitor studies using La31 and vanadate (VO43) pointed to a major involvement of the symplast in the
radial transport of Ca21 through the endodermis. It was concluded that in onion roots, the radial movement of Ca21 through
the exodermis and endodermis is primarily symplastic.
The pathway whereby Ca21 moves radially from
the soil solution to the tracheary elements of a root
has long been a subject of debate (for reviews, see
Clarkson, 1991; White, 2001; White and Broadley,
2003). To reach the root stele (the location of the
tracheary elements), an ion must pass through the
epidermis, exodermis (if present), central cortex, and
endodermis. Because in typical roots the walls of the
epidermis and central cortex cells are permeable to
ions, the current debate concerns the passage of ions
through the endodermis and exodermis.
One idea is that the Ca21 delivered to the xylem may
move predominantly or entirely apoplastically. This
has been considered by a variety of authors (White,
1998; McLaughlin and Wimmer, 1999; White et al.,
2000; White, 2001, White and Broadley, 2003). In some
studies, the amount of Ca21 translocated was positively correlated with the transpiration rate, as would
be expected if its movement were apoplastic (Hylmö,
1953; Bell and Biddulph, 1963; Lazeroff and Pitman,
1966). The lack of selectivity between Ca21, Ba21, and
Sr21 uptake by roots has also been cited as evidence for
the presence of an apoplastic radial pathway for Ca21
(Bowen and Dymond, 1956; White, 2001). The argu1
This work was supported by a Discovery Grant from the
Natural Sciences and Engineering Research Council of Canada to
C.A.P.
2
Present address: BIOS Agriculture, 2111 Lakeshore Road, P.O.
Box 187, Ste-Anne-de-Bellevue, QC, Canada, H9X 3V9.
* Corresponding author; e-mail [email protected]; fax 519–
746–0614.
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.103.035287.
ment against this view is that Casparian bands of the
endodermis and exodermis have proved impermeable
to several ions (de Rufz de Lavison, 1910; Baker, 1971;
Robards and Robb, 1972; Peterson, 1987; Peterson et al.,
1993) including Ca21 (Singh and Jacobson, 1977, Kuhn
et al., 2000, Bückling et al., 2002) within the limits of
the tests employed.
A second idea is that the Casparian bands in the
endodermis and exodermis are circumvented by
a symplastic step. In this case, the overall radial
movement of Ca21 to the stele would show features of
symplastic transport. In support of this idea, there are
some reports that Ca21 movement to the shoot is not
correlated with the speed of the transpiration stream
(Atkinson et al., 1992; Jarvis and House, 1970). Drew
and Biddulph (1971) found that the passage of Ca21
into the transpiration stream of bean (Phaseolus
vulgaris) was reduced by cold temperatures and
several metabolic inhibitors. Also, when the bathing
solution contained a relatively low concentration of
Ca21, the ion was concentrated in the transpiration
stream. In a correlative study of Ca21 movement into
the stele of marrow (Cucurbita pepo) roots, HarrisonMurray and Clarkson (1973) found that the entry of
Ca21 into the stele was inversely correlated with
suberin lamella development in the endodermis. Their
interpretation of the results was that Ca21 moved
through the endodermis symplastically, entering the
cytoplasm by means of Ca21 channels located in the
membranes lining the outer tangential walls of
the endodermal cells, and exiting the cytoplasm by
means of Ca21-ATPases located on the membranes
lining the inner tangential walls of these cells. Suberin
lamellae would interfere with these changes of
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Cholewa and Peterson
compartment, inhibiting Ca21 movement to the stele.
(For a full discussion, see Clarkson, 1991.) The counterargument to a path involving a symplastic step(s) is
that a radial flow of Ca21 would make it difficult for
the cytoplasm to maintain its normal low concentration of the ion (Flowers and Yeo, 1992). However,
Clarkson (1984) pointed out that appropriate cytosolic
concentrations could be maintained by rapid fluxes
through the endodermal cells. After reviewing the
evidence on the subject, White and Broadley (2003)
state ‘‘Although the relative contributions of the
apoplastic and symplastic pathways to the delivery
of Ca to the xylem are unknown, it is likely that
a functional separation of apoplastic Ca21 fluxes (for
transfer to the shoot) and symplastic Ca21 fluxes (for
cell signaling) would enable the root to fulfill the
demand of the shoot for Ca without compromising
intracellular [Ca21]cyt signals.’’
A third idea, one that reconciles the opposing views
on the pathway (apoplastic versus symplastic) of
radial Ca21 movement through the endodermis, is
that entry into the stele occurs near the root tip where
the endodermis is immature (Flowers and Yeo, 1992).
Additional points of entry in more mature zones would
be through discontinuities in the Casparian bands
associated with the growth of lateral root primordia
(Ferguson, 1979; Peterson and Moon 1993, White and
Broadley, 2003).
In this study, the passage of Ca21 from the soil
solution into the transpiration stream of onion (Allium
cepa) roots was studied with a view to answering two
questions: (1) Does this ion move radially through an
intact exodermis and endodermis, or is the ion
provided to the plant by the root tip (a region devoid
of Casparian bands)? (2) If the ion does pass through
an intact exodermis and/or endodermis, is its pathway predominantly apoplastic or symplastic? Young,
adventitious roots sprouted from bulbs were chosen
for this study because these roots lack laterals, the
formation and emergence of which leads to discontinuities in the Casparian bands of the endodermis and
exodermis, respectively (Esau, 1940; Peterson and
Moon, 1993). Thus, all areas except for the root tip
have a complete Casparian band in the endodermis,
and the exodermal Casparian band is also complete in
older root regions. To answer the first question, 45Ca21
was applied to discrete zones of roots and the amounts
moved to the xylem were estimated from radioactivity
in all parts of the plants proximal to the treated areas.
To answer the second question, different methods
proved appropriate for the exodermis and endodermis. For the former, the permeability of the Casparian
band to Ca21 was tested by compartmental elution,
a technique that can be used to measure the quantities
of ions in various compartments of plant cells (Cram,
1968; Jeschke 1982; Peterson 1987; Maclon et al., 1990).
The permeability of the exodermal Casparian bands to
Ca21 was tested by measuring the amount of the ion
eluted from the cell wall compartments of ‘‘intact’’
segments with sealed ends and ‘‘dissected’’ segments
that had been longitudinally bisected and had their
steles removed. If exodermal Casparian bands do not
restrict the apoplastic diffusion of Ca21, the amounts
eluted from the cell walls in intact and dissected
segments should be the same. If, however, these bands
do restrict apoplastic diffusion, the amount of Ca21
eluted from the cell wall compartment in the intact
segments will be substantially smaller than that from
the dissected ones. For the endodermis, the effects of
La31 (an inhibitor of Ca21 channels) and VO43 (an
inhibitor of Ca21-ATPases) on the movement of Ca21
to the stele were tested in a system where exudate was
collected from excised roots. If the radial movement is
entirely through the apoplast, the inhibitors will have
no effect on ion delivery to the stele. However, if
movement is proceeding with a symplastic step
(which necessitates the ion moving into and out of
the endodermal cells through their membranes), the
amount of Ca21 in the stele should be substantially
reduced. This will be true even if membrane effects
other than inhibition of Ca21 channels and Ca 21ATPases are elicited by the inhibitors.
RESULTS
Sites of Ca21 Uptake and Axial Transport Along
Onion Roots
Adventitious roots sprouted from onion bulbs developed uniformly when grown in the favorable
conditions provided. Such roots were without laterals,
either emerged or in the form of primordia. The roots
were divided into three anatomical zones of interest
with reference to the maturation of the endodermis,
early metaxylem vessels, and exodermis (Fig. 1). The
tip zone was the apical 5 mm of the root where
functional xylem and Casparian bands were absent.
The young zone, 15 to 40 mm from the tip, had
functional xylem and Casparian bands in the endodermis. The old zone, which began 100 mm from the
root tip and extended to the root base, possessed
functional xylem, and Casparian bands in all cells and
suberin lamellae in most cells of both the endodermis
and exodermis. Thus, passage cells were present in
both of the latter layers. For the experiments, the tip or
the central regions of the young and old zones were
sealed into a treatment chamber (Fig. 2).
When 1 mM 45Ca21 was applied to the tip zone, no
measurable radioactivity was detected in the rest of
the plant (Table I). During the 24 h treatment time, the
root tips had grown an average of 3 mm, which
resulted in a final length of 8 mm being treated. In
some cases, Casparian bands had formed in the endodermis in the proximal part of the treated zone, but the
early metaxylem was always immature. Otherwise,
the anatomy of the relevant structures did not change
during treatment. When the young zone was treated,
radioactivity was detected in the plant parts proximal
but not distal to the treated site. The amount of 45Ca21
extracted from the plant was equivalent to a delivery
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Radial Movement of Calcium in Onion Roots
a triple exponential function (Fig. 4) with average halftimes ranging from 47 to 66 s, 5.6 to 7.5 min, and 102
to 210 min corresponding to wall, cytoplasm, and
vacuole, respectively (although the identity of the last
compartment is not known with any certainty; Table II
elution at 228C). The amount of Ca21 in the wall
compartment of the intact segments (2.0 mmol g1
segment fresh weight [fw]) was substantially smaller
than that in the dissected segments (8.8 mmol g1
segment fw), indicating that the exodermal Casparian
bands are not freely permeable to the ion (Table II, Fig.
3). Identification of the cell wall compartment, critical
for this study, was validated by results of elutions at
48C. Here the efflux from the wall compartment was
unaffected (average half-time of 63 s), but that from the
next (cytoplasmic) compartment could not be distinguished from the slowest (vacuolar) compartment
(Table II). The half-time of elution from the latter
combined compartment was not significantly different
from that of the vacuole at 228C in either whole or
dissected segments (Table II).
Effect of Inhibitors on Ca21 Transport to the Stele
Figure 1. Median longitudinal section of an onion root. Major
developmental landmarks are indicated, as well as zones of interest
in this study. Not drawn to scale. A to C, Cross sections of onion root
stained with berberine-aniline blue and viewed with UV light. A,
Section taken from the old zone of the root (150 mm from tip).
Casparian bands and suberin lamellae (asterisks) are present in the
exodermis and Casparian bands (arrowheads) and suberin lamellae in
the endodermis. Late metaxylem vessels are mature and lignified. B,
Section taken from young zone of the root (30 mm from root tip).
Casparian bands (arrowheads) are present in endodermis only. Early
metaxylem vessels are mature. C, Section taken from tip zone of the
root (5 mm from tip). No Casparian bands were detected; xylem vessels
are immature. Bars ¼ 500 mm.
to the transpiration stream of 2.67 nmol mm1 treated
root length (Table I). Treatment of the old zone resulted
in transport of 0.87 nmol mm1 treated root length
(Table I). As was the case for the young zone, 45Ca21
movement from the old zone was only in an upward
direction (i.e. toward the shoot). No positive correlation between the amount of the ion transported and
transpiration rate was evident (Table I).
Impermeability of Exodermal Casparian Bands to Ca21
The data from the compartmental elution of 45Ca21
from both intact and dissected segments (Fig. 3) fitted
In the young zone of the root, treatment with
inhibitors substantially reduced the flux of Ca21 to
the stele, as measured by the amount of the ion
appearing in the root exudate. Addition of 1 mM La31
to the treatment chamber (Fig. 5) resulted in 73% less
Ca21 in the root exudate compared to the control.
Similarly, prior treatment of the cells in the stele with
VO43 fed into the transpiration stream resulted in
a 73% inhibition of Ca21 translocation (Table III).
Neither inhibitor affected the volumes of exudates
produced by the roots (Table III).
Figure 2. Sectional diagram of the apparatus used to apply 45Ca21 to
discrete zones of onion root. An entire onion with its numerous
adventitious roots (only two of which are shown) was placed in
a large tray so that the leaves projected into the atmosphere. One
root was sealed into a multi-chambered plexiglass box. Flanking (f)
and bordering (b) chambers were filled with 1 mM Ca SO4, and the
treatment chamber (t) with radioactive Ca21 (see text). The cover
and sides of the large tray were lined with wet paper towels, and
the untreated roots were covered with moist vermiculite (not
shown).
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Cholewa and Peterson
Table I. Translocation of 45Ca21 from developmentally different
zones of onion roots, and transpiration rates
Distance from
Root Tip mm
Transpiration
g water lost
during 24 h
Ca21 Translocation
nmol mm1 treated
root length
0–8 (tip)
15–30 (young)
[100 (old)
13.5 6 5.8
8.7 6 0.9
18.1 6 4.3
0.0
2.67 6 0.74*
0.87 6 0.36*
The total length of root tip (8 mm) at the end of experiment was
considered in calculations. *, statistically significant differences in the
two values analyzed (n ¼ 4, 6 SD, Student’s t test, P ¼ 0.06).
DISCUSSION
Onion proved to be an ideal species for testing the
permeability of the endo- and exodermis to Ca21 as
the roots did not develop laterals and, therefore, the
Casparian bands of these layers, once formed, remained intact. Moreover, these roots did not develop
root hairs, facilitating a clear distinction of the wall
compartment in compartmental elution experiments.
Sites of Ca21 Uptake and Axial Transport Along
Onion Roots
When 45Ca21 was applied to the root tip, the only
location in the root not invested with at least one
complete Casparian band, no measurable amount was
transported away in the transpiration stream. Perhaps
this is not surprising as this zone lacked mature xylem
vessels. The general apoplastic impermeability of the
tip noted in other studies (Lüttge and Weigl, 1962;
Rasmussen, 1968; Wierzbicka, 1986; Enstone and
Peterson, 1992; Cholewa and Peterson, 2001) also
applies to Ca21 ions since there was no detectable
movement through parenchyma to the point where
the xylem elements were functional. Enstone and
Peterson (1992) postulated that the apoplastic impermeability of the root apex may be a consequence of an
absence of sufficiently large intermicrofibrillar channels in the walls of the cells to allow tracer movement.
In view of the fact that the endodermis matures closer
to the tip than the xylem (Peterson and Lefcourt, 1990),
one would expect that the root tip would not supply
Ca21 to the transpiration stream. On the contrary,
Huang et al. (1993) found that Ca21 transport could
occur from the tip zone in wheat (Triticum aestivum).
However, it is unknown whether or not some of the
tracheary elements were mature in the treated zone.
In young zones of onion roots (between 15 and
40 mm from the root tip), where the endodermis and
early metaxylem vessels were mature (Fig. 1), radioactive Ca21 moved to the shoot. In fact, the largest
amount was transported from this zone (Table I). In
this region, no suberin lamellae are as yet developed in
endodermal cells (Barnabas and Peterson, 1992) so that
all cells comprising this layer would be available for
import of Ca21 into the symplast and export again on
the stele side of the Casparian bands. Robards et al.
(1973) correlated the anatomy of barley (Hordeum
vulgare) roots with Ca21 (using 85Sr as a tracer) transport to the stele and found that the zone of maximum
uptake was 20 mm from the root tip. As in onion, this
is in the zone in barley where the endodermis is
mature but suberin lamellae have not yet formed. The
maximum amount of Ca21 that entered the stele in
barley was 0.13 nmol mm1 treated root length
(calculated from Robards et al., 1973, Fig. 29; where
a maximum of 260 pmol of Ca21 mm3 root was
reported, and assuming 0.4 mm root radius from Aloni
et al., 1998, Fig. 13). This is much less than the average
value of 2.7 nmol mm1 in onion. Clearly, movement
through an intact endodermis in onion did not result
in less ion delivery to the stele than in barley roots
where lateral root primordia were presumably developing.
In the most mature (old) zone of the root (greater
than 100 mm from the root tip), Casparian bands were
present in the endodermis as well as in the exodermis
(Fig. 1). Despite the presence of two sets of Casparian
bands on the radial path, this zone was capable of
transferring Ca21 to the stele (Table I). This ion did,
therefore, pass through an intact, mature exo- and
endodermis. The less efficient radial transport in the
old zone (compared to that of the young zone) could
be a consequence of the presence of suberin lamellae
in both the endo- and exodermis, as similar results
were previously obtained with marrow and barley
(Harrison-Murray and Clarkson, 1973; Robards et al.,
1973; Ferguson and Clarkson, 1976).
From the above comparison of Ca21 transport
through various root zones it is clear that, in this study,
the ion does not enter the stele apoplastically at the root
tip where Casparian bands are not yet formed, nor
apoplastically in older regions through discontinuities
Figure 3. Cross sections of onion roots used for compartmental elution.
Shading indicates hypothetical areas from which Ca21 could be eluted
from the walls. A, Section from a whole segment with wax-capped
ends. Elution occurs through the epidermis. Left half of root, assuming
the exodermal Casparian band is permeable to Ca21; right half of root,
assuming the exodermal Casparian band is impermeable to Ca21. B,
Section from a dissected segment, i.e. one that had been bisected and
had its stele removed. Elution occurs through the epidermis and cortex.
For the diagram, it is assumed that Ca21 is not eluted from the anticlinal
walls of the exodermis.
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Radial Movement of Calcium in Onion Roots
in the Casparian bands of the endodermis and exodermis brought about by lateral root production.
Rather, Ca21 moved through an intact endodermis
and exodermis on its way to the stele.
In all zones tested, there was a consistent lack of
Ca21 transport toward the root tip, supporting the
general view that Ca21 is immobile in the phloem and
is not circulated within plants (Marschner, 1995). Most
previous reports agree with the exclusive basipetal
translocation of this ion, e.g. in onion (Maclon, 1975)
and maize (Zea mays, Marschner and Richter, 1973).
But in one study (Huang et al., 1993), Ca21 transport
toward the tip of wheat roots and its redistribution to
other nontreated seminal roots was detected.
Pathway of Ca21 Movement Through the Exodermis
Figure 4. Graphed results from one replicate of a typical elution
experiment (whole segments). Beginning with graph A, the times of the
elution steps on the abscissa (linear scale) are plotted against a log
(ordinate) scale of the dpm from the 45Ca21 that would have been
present in the segments at the times indicated. The line of best fit was
calculated for the linear part of the curve. The amount of Ca21 indicated
by the intercept of the line on the ordinate at time 0 (indicating the
amount of the ion in the most slowly-eluting compartment) was
subtracted from all higher values to obtain the set of data plotted in B.
Again, a line of best fit to the linear portion of the curve was obtained,
and the value at the ordinate (the amount of the ion in the second most
slowly-eluting compartment) was subtracted from the remaining values
to produce data for graph C. In graph C, the straight line of best fit
includes the point on the ordinate, indicating that this is the fastesteluting compartment of the root. The half-times of elution from the
three compartments are obtained from the slopes of the straight lines.
The elution of Ca21 from onion root segments was resolved into three
compartments: A, vacuole (or other slowly eluting compartment), B,
cytoplasm, C, cell wall.
The technique of compartmental elution was used to
discern the path of Ca21 as it moved through the
mature exodermis, a layer with Casparian bands near
the root surface. With this technique, it is imperative to
identify the compartment of interest (i.e. the wall)
correctly. In this study, when 45Ca21 was eluted from
either intact or dissected segments, three compartments were distinguished. The fastest eluting of these
was identified as the cell wall based on a comparison
of its half-time (about 1 min) with literature data
(Drew and Biddulph, 1971; Maclon, 1975; Rygiewicz
et al., 1984; Peterson, 1987; Rauser, 1987; White et al.,
1992; DiTomaso et al., 1993; Devienne et al., 1994;
Kronzucker et al., 1995a, 1995b) and its lack of change
during elution at cold temperature. A surface film,
which would have eluted more rapidly than the wall
compartment, was not evident. Perhaps the Ca21 in
such a film had associated with the wall. The second
fastest eluting compartment could be unequivocally
identified as the cytoplasm based on its half-time of
elution and extreme sensitivity to cold temperature.
The third compartment may have been the vacuole or
some other very slowly eluting compartment. The wall
compartment was identified with certainty, but it
included both the water free space and the Donnan
free space. Most of the Ca21 (99.6%) in this compartment was in the Donnan free space (calculations not
shown).
Having identified and characterized the wall compartment, it was possible to test the hypothesis
presented in the introduction, namely that a smaller
amount of Ca21 eluted from the walls of the intact
segments with sealed ends compared to the amount
eluted from the dissected segments would indicate
that the Casparian band is not freely permeable to
Ca21. (It is necessary to assume that the Donnan free
space of the epidermal and outer exodermal walls is
the same as that of the walls of the central cortex.)
These results were, in fact, obtained in experiments
run at 228C and 48C (Table II). The amounts of Ca21 in
the wall compartments based on weight (as reported
in Table II) can be corrected for sample volumes by
considering that the length of the intact segments
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Cholewa and Peterson
Table II. Amounts of Ca21 present in the compartments and their half-times of elution from intact
and dissected segments taken from an old zone of onion roots
Compartment
(Elution Temperature)
Wall
(228C)
(48C)
Cytoplasm
(228C)
(48C)
Vacuole
(228C)
(48C)
Whole Segments
Ca21 mmol g1 fw
Dissected Segments
Half-time
Ca21 mmol g1 fw
Half-time
2.0 6 0.4*
1.9 6 0.1y
66 6 10 s
69 6 14 s
8.8 6 2.8*
7.7 6 1.0y
47 6 6 s
57 6 10 s
3.1 6 1.2
ND
7.5 6 0.8 min
ND
4.1 6 0.9
ND
5.6 6 0.9 min
ND
3.4 6 0.7
3.6 6 0.5
210 6 30 min
350 6 60 min
3.2 6 0.9
4.4 6 0.6
102 6 30 min
250 6 61 min
The difference between the amounts of Ca21 eluted from the cell wall compartment from whole
segments compared to dissected segments is statistically significant from elutions at 228C (*, n ¼ 7, 6SD,
Student’s t test, P ¼ 0.004) and from elutions at 48C (y, n ¼ 3, 6SD, Student’s t test, P ¼ 0.01). ND denotes
compartment not detected.
treated was 2.0 cm (not 2.5 cm), and that the steles
were removed from the dissected segments. Then the
average ratio of the amount of Ca21 eluted from intact
to dissected segments at 228C is 1:3.3, and is near the
range of those obtained earlier (1:3.9 to 1:6.3) by
Peterson (1987) for SO42 in onion roots. By a process
of elimination, we are forced to conclude that the
movement of Ca21 through the onion root exodermis
occurs primarily in the symplast.
Pathway of Ca21 Movement Through the Endodermis
The experiments designed to discover how Ca21
moved through the endodermis on its way to the stele
entailed measuring the amounts of this ion in root
exudates, a method that has been used previously. For
example, barley roots actively exuded externally
applied 45Ca21 from young (30 mm from tip) and
mature (60 mm from tip) zones (Halperin et al., 1997).
For onion, the ability of detopped roots to remain
metabolically active and produce exudates for 19 h has
been demonstrated by Hodges and Vaadia (1964). In
this study, onion roots exuded steadily for at least 17 h
after excision (data not shown), and the volume of
exudate was not influenced by treating the roots with
either of the inhibitors employed (Table III).
In this series of experiments, distinguishing a predominantly apoplastic pathway from a symplastic one
rests on the effect of inhibitors of membrane function
on delivery of Ca21 to the root stele. If the entire route
is apoplastic, the inhibitors should have no effect. If it
is symplastic, however, a sharp reduction in the
amount of Ca21 delivered is expected. For this study,
the inhibitors were chosen to give maximum effects on
the delivery of Ca21 to the stele. According to the
model of Clarkson (1991), which is based on known
mechanisms of Ca21 passage through membranes (see
Sanders et al., 1999), entry into the cytoplasm of the
endodermis is mediated by Ca21 channels located in
plasmalemmae on the cortical side of the Casparian
band, and exit from the cytoplasm is through
Ca21-ATPases in the plasmalemmae on the stele side
of the Casparian band. (The same explanation could
be advanced to explain a symplastic step through
the exodermis.) In this study, La31 was chosen as an
inhibitor of Ca21 channels. La31 is an antagonist of
Ca21 influx into cells, and does not penetrate the
plasmalemma (DuPont and Leonard, 1977; Lonergan
and Williamson, 1988; Perdue et al., 1988). La31, a very
potent Ca21 channel blocker, has been used at
a concentration of 1 mM (as in this study) to abolish
Ca21 increases in mesophyll tissue of tobacco leaves
(Moyen et al., 1998). In maize root plasmalemma
vesicles, the class of Ca21 channels that is inhibited by
La31 mediates 70% of the Ca21 influx; the remaining
30% is inhibited by Gd31 but not La31 (Marshall et al.,
1994). It seems that La31-sensitive channels may be
exclusive Ca21 transporters in wheat roots (Huang
et al., 1994; Sasaki et al., 1994). In addition to the classes
of channels described above, others are present in
roots (for reviews, see White, 1998; Demidchik et al.,
2002); however, their involvement in radial Ca21
transport has not been investigated. To inhibit
Ca21-ATPases, the inhibitor VO43 was selected. A
number of plasmalemma Ca21-ATPases from different
plant species have been characterized (reviewed in
Bush, 1995; Sze et al., 2000). They belong to the P-type
ATPase family, i.e. they form a phosphorylated intermediate during the catalytic cycle. VO43 inhibits all
these Ca21-ATPases (Sze, 1985; Serano, 1990; Sze et al.,
2000).
Treatment of onion roots with either La31 or VO43
substantially reduced the amount of Ca21 in the root
exudate (Table III), providing unequivocal evidence
that the majority if not all the Ca21 delivered to the
stele passed through membranes. Thus, at least one
symplastic step in the radial path is indicated.
Treatment of onion roots with La31 resulted in a 73%
inhibition of Ca21 transport to the stele (Table III). This
incomplete inhibition may reflect the complex mechanism of Ca21 transport by roots. It is apparent that
other classes of Ca21 channels might be present in the
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Radial Movement of Calcium in Onion Roots
Figure 5. Sectional diagrams of plexiglass boxes (also shown in Fig. 2)
set up to test the effect of inhibitors on calcium delivery to the stele. Five
excised roots (one of which is shown) are sealed into a box so that the
cut (proximal) ends protrude into one of the flanking chambers (f). Both
flanking (f) and bordering (b) chambers are filled with 1 mM CaSO4.
Exudates from the cut ends of the five roots enter the same flanking
chamber. A, Treatment with La31. The treatment chamber (t) is filled
with radioactive Ca21 and 1mM LaCl3. B, Root segment pretreated with
Na3VO4 (see text). The distal end of the root (in the flanking chamber to
the left in the diagram) is sealed shut; the treatment chamber (t) is filled
with a solution containing radioactive Ca21. Control data were
generated from root segments set up as in A and B but without the
inhibitors.
plasmalemmae of roots (White and Davenport, 2002).
For example, Marshall et al. (1994) provided evidence
for the presence of La31-insensitive Ca21channels in
maize roots. It is likely that onion roots possess such
channels in their plasmalemmae. VO43, a well-known
inhibitor of P-type ATPases, was also effective in
reducing the amount of Ca21 movement into onion
roots. This ion is not toxic to the roots, since treatment
of onion roots with 1 mM VO43 for 6 h induced
a reversible inhibition of root growth (Navas and
Gracia-Herdugo, 1988). The observed 73% inhibition
of Ca21 exudation upon treatment of onion roots with
VO43 (in this study, Table III) strongly supports the
hypothesis that P-type Ca21 pumps are involved in
release of Ca21 into the stelar apoplast.
vesicles were involved in the radial transport of apoplastic solutes, one would expect that such dyes
applied to the outside of the root would be carried
through the endodermis and exodermis and be visible
in the more centrally located tissues. This does not
occur (Peterson et al., 1982). Is it possible that cytosolic
levels of free Ca21 can remain low in the endodermal
and exodermal cells as required by its role as a second messenger? White (1998) calculated that the
Ca21-ATPase content would need to be greater than
the average total protein content of the root plasma
membrane to supply sufficient Ca21 to the shoot. Such
a problem would be even more severe for onion roots,
since the measured flux of Ca21 at the endodermis is
greater than in White’s model (calculations not
shown). The problem of Ca21 transport across the
onion exodermis is even more serious, as this layer is
dimorphic (Kroemer, 1903) so that only the membranes of the short cells can be considered. These
occupy 13% of the surface area of the layer (Ma and
Peterson, 2001). A consideration of the protein
contents of the endodermal and exodermal membranes, and the observed radial transport of Ca21 will
be the subject of a subsequent paper.
It should be noted that this work concerns the
endodermis in two stages of development, i.e. with
Casparian bands (in the young zone) and with the
addition of suberin lamellae in most but not all cells (in
the old zone). Similarly, passage cells were always
present in the exodermis. The question of Ca21
movement through an endodermis (or exodermis)
with tertiary walls and without passage cells could not
be approached with this experimental material. It is
most likely that in this study, Ca21 movement into the
old zone was predominantly apoplastic in the epidermis and central cortex and was through the cytoplasms of only the passage cells of the exodermis and
endodermis. Such symplastic steps would not involve
Ca21 transport through plasmodesmata.
The results of this study clearly show that in onion
roots, most or all of the Ca21 that enters the root stele
moves through the symplast at some point(s). Due to
the impermeability of the endodermal Casparian band
to ions (see introduction) and of the exodermal
Movement of Ca21 through the Symplast
The results of this study support the model of
Clarkson (1991) in which a symplastic step through the
endodermis occurs during radial Ca21 transport in
roots. His model can now be extended to include the
exodermis. The mechanism(s) of Ca21 movement
through the symplast of such layers, however, are
not clear. Transport of apoplastic fluid across the
cytoplasm of onion root cells by means of endocytotic
vesicles was ruled out by earlier studies. Such vesicles occurred in the cells of the central cortex but
these emptied their contents into the cells’ vacuoles
(Cholewa and Peterson, 2001). Further, if endocytotic
Table III. Effects of La31 and VaO432 on root exudation and
Ca21 translocation from young zones of onion roots
Ca21 in Exudate
nmol mm1 root
Exudate Volume
mL mm1 root
Control
1 La31
% Inhibition
2.01 6 0.84
0.54 6 0.12
73
1.2 6 0.5*
1.3 6 0.4*
Control
1 Vanadate
% Inhibition
1.92 6 1.97
0.51 6 0.08
73
1.1 6 0.4*
1.1 6 0.4*
45
Ca21 translocation was measured in exudates after 17 h (n ¼ 3,
6SD, Student’s t test, P ¼ 0.05, *, no significant difference).
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Cholewa and Peterson
Casparian bands to Ca21 (this study), these layers
should be included in any symplastic path.
bulb used for each replicate of each experiment were examined for unemerged
lateral primordia by the clearing method of Hackett and Steward (1969).
Compartmental Elution
MATERIALS AND METHODS
Plant Material
Adventitious roots of onion (Allium cepa L. cv Ebeneezer) were generated
from bulbs kept for 12 d in vermiculite-filled pots in a greenhouse. The longest
roots measured 16 cm and none had reached the bottoms of the pots. The
bulbs were near the end of their storage time (6 months after harvest) and
sprouted leaves as well as roots.
Treatment of Various Root Zones
Zones to be treated with radioactive Ca21 were established by following
the pattern of maturation of the endodermis, exodermis, and early metaxylem
in the roots (Fig. 1) using the methods of Brundrett et al. (1988, 1991) and
Peterson and Steudle (1993). With these techniques, Casparian bands, suberin
lamellae, and functional xylem elements, respectively, were identified (Fig. 1).
Radioactive Ca21 in the form of a chloride salt (45CaCl2) was applied to
specific zones of the root (Fig. 2). An entire onion with its intact root system
was placed in a tray that had been lined with wet, absorbent paper. The root
to be treated was carefully positioned in a plexiglass box divided by partitions
to create a 15-mm-long treatment chamber, two narrow bordering chambers to
trap any accidental leakage from the treatment chamber, and two flanking
chambers to hold the adjacent parts of the root (Fig. 2). The root was sealed
into place with a mixture of 90% lanolin and 10% wax (previously heated
while mixed; Barrowclough et al., 2000), and the bounding and flanking
chambers were filled with 1 mM CaSO4 solution. To begin the treatment, 1 mM
CaSO4 labeled with 45CaCl2 (3.7 3 104 Bq ml1) was carefully pipetted into
the treatment chamber. The remaining roots (outside the plexiglass box) were
sprayed with 1 mM CaSO4 and covered with wet vermiculite. Then a tray lid,
also lined with wet, absorbent paper was set into place so that only the leaves
projected into the atmosphere. To promote transpiration, continuous light was
supplied from a Tungston, General Electric (Fairfield, CT) bulb at an intensity
of 100 mmol cm2 s1. The assembly was weighed before and after the
experiment to determine the amount of water lost by transpiration.
After 24 h, the radioactive solution was removed and the solutions in the
chambers on each side of the treatment chamber were probed for radioactivity
to monitor leakage. To measure the length of the treated segment precisely, the
incubation chamber was filled with an aqueous solution of Toluidine blue O
(0.05% w/v) for 5 min. After removal of dye, the experimental root was
measured and divided into parts, i.e. the treated segment, the distal (apical)
section, and the proximal (basal) section. All these parts, in addition to the
bulb and the leaves, were heated at 5008C for 17 h, cooled, and the resulting
ash made up to 5 mL with 0.2 N HCl. A similarly processed, entire root from an
untreated plant was used as a control. Radioactivity (b-emissions) in each
plant part was measured with a liquid scintillation counter (Searle Analytic,
model Mark III) after addition of scintillation liquid (Ecolite, ICN, Irvine, CA).
Four replicates were included in each experiment. Data from the plant parts
external to the treated root area were pooled and analyzed by Student’s t test
(2 tails, 3 types – two samples unequal variance).
Preparation of Root Segments for
Compartmental Elution
Segments used to determine the permeability of the exodermal Casparian
band were prepared according to the procedure of Peterson (1987). All root
parts were taken at least 10 cm from the root tip, a location where the
exodermis had a Casparian band. One set of 10 root segments was prepared
for each replicate of each treatment. For ‘‘intact’’ specimens, 2.5-cm-long pieces
were removed and their ends sealed with sticky wax (Kerr, Toronto) so that
2 cm of the root would be exposed to the treatment solution. ‘‘Dissected’’
segments were made by bisecting 2-cm-long pieces and removing their steles
with the aid of fine forceps and a dissecting microscope (Fig. 3). To ensure that
the expected maturation of the exodermis had occurred in the segments used
for elution experiments, freehand cross sections were cut from the adjacent
areas of the roots and assessed for the presence or absence of an exodermal
Casparian band (procedure of Brundrett et al., 1988). Three roots from each
Labeling and elution were performed following the method of Peterson
(1987). Segments to be loaded with 45Ca21 were incubated for 17 h in 25 mL of
an aerated solution of 1 mM CaSO4, adjusted to pH 5.5 with dilute HCl, and
amended with 45CaCl2 (final specific activity ¼ 3.7 3 104 Bq mole1). At the
end of this period, the segments were gently removed from the bathing
solution, blotted with absorbent tissue, and immediately transferred into
plastic vials fitted with plastic micromesh at the bottom end and an aeration
tube. Each vial contained 10 segments (intact or dissected). The vials (and the
segments they contained) were immersed in a series of elution solutions
containing 10 mL unlabeled 1 mM CaSO4, adjusted to pH 5.5. Between each
change, vials were lifted just above the solution and rinsed with 1 mL of
unlabeled solution to minimize error due to ullage. Transfers to fresh washout
solutions were carried out at the following time intervals (min): 0.5, 1, 1.5, 2, 3,
4, 5, 7, 9, 11, 15, 20, 25, 30, 40, 60, 90, 120, 150, 180, 240, 300, and 360. At the end
of the elution series, the wax caps were removed from the sealed segments,
and the batches of segments were weighed. Any remaining 45Ca21 was
extracted by a 20-min treatment with 0.2 N HCl for 20 min. This solution was
made up to 5 mL with water; then scintillation liquid (Ecolite, ICN, catalog no.
882475) was added to achieve a total volume of 20 mL. From each of the
washout solutions, a 1-mL aliquot was mixed with scintillation fluid to a total
volume of 6 mL. Samples were assessed for radioactivity as described above.
Each reading was corrected for the background present in a root from an onion
that had not been exposed to 45Ca21.
Each comparison (i.e. intact versus dissected) was replicated at least three
times. Data were analyzed as previously described (Peterson, 1987). To avoid
any error owing to subjectivity, a maximization of R2 (square of the sample
correlation coefficient, using Microsoft Excel, Redmond, WA) for linear
regression (Rygiewicz et al., 1984) was used as the criterion for determining
data points to be included in each regression line. Values for the half-time of
elution and the amount of Ca21 in each compartment were obtained using
Microsoft Excel.
To verify the identity of the cell wall compartment, three replicates of an
experiment were eluted at 48C. For this trial, intact and dissected segments
were initially incubated in the 1 mM radioactive Ca21 treatment solution at
228C for 17 h. At the end of this labeling period, segments were blotted and
then eluted (as described above) at low temperature.
Treatment with Inhibitors
To distinguish between Ca21 transport in the apoplast versus the symplast
of the endodermis, young zones of the root (with Casparian bands in the
endodermis but not in the immature exodermis) were tested. The effects of
La31 and VO43 on the amounts of Ca21 in root exudates were assessed.
For each replicate of a La31experiment, 10 roots were excised from one
onion bulb. Each root was cut obliquely under water to obtain the distal 6 cm
(i.e. the part of the root that included the tip). Five of the roots were sealed into
a plexiglass box as described above so that a 15-mm length in the central
region of the young zone could be treated (Fig. 5). Bordering and flanking
chambers were filled with 1 mM CaSO4 and a treatment solution of 1 mM LaCl3
and radioactive Ca21 (as described above) was added to the treatment
chamber. The remaining five roots were sealed into a second chamber and
treated in the same fashion but without LaCl3.
The membranes of the cells in the stele (including the membranes of the
stele side of the endodermis) were exposed to VO43 by introducing a solution
of sodium orthovanadate (Na3VO4, Aldrich, St. Louis) into the transpiration
stream. The inhibitor solution was prepared according to O’Neill and
Spanswick (1984). In short, a 5-mM stock solution was made by dissolving
Na3VO4 in distilled water at 388C for 3 h. The pH was adjusted to 7.2 with dry
MES (Sigma, St. Louis) and this colorless solution stored in a sealed bottle at
room temperature. For the experiments, aliquots were diluted with distilled
water to 1 mM VO43 and a drop of 1% aq Safranine 0 was added to color to the
solution red. To introduce this mixture into the transpiration stream, the
terminal 1.5 cm of five roots was excised and the cut ends of the roots, still
attached to the plant, were placed in the solution. The plant was allowed to
transpire (under the conditions described above) for 2 h, after which the
treated roots were removed from the bulb, their proximal ends cut (as
described above) and inspected for red coloration. In those roots in which the
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Radial Movement of Calcium in Onion Roots
treatment solution had passed through the segment in the xylem, the distal cut
ends were sealed with sticky wax (Kerr). The segments were immediately
sealed into the treatment chamber and exposed to radioactive Ca21 as
described above. Control roots were treated in the same way but without
Na3VO4. At the conclusion of the experiments (17 h of exudation), the
radioactivity in the solutions from the bounding and flanking chambers was
determined as described above, exudates from the five roots having been
pooled in one of the flanking chambers.
The potential effects of both inhibitors on the process of root exudation
were tested. In the case of La31, roots were excised from an onion bulb as
described above, and the proximal (cut) end of each root was inserted into
a 100-mL microcapillary tube and sealed in place with sticky wax (Kerr). The
entire root piece was immersed in a solution of 1 mM CaSO4 and 1 mM LaCl3. In
the case of VO43, the inhibitor was introduced into the root xylem as
described above. Roots were then severed from the bulb and sealed at the
distal ends with sticky wax. The proximal ends of the roots pieces were then
sealed into 100-mL microcapillaries (as described above) and the roots
immersed in 1mM CaSO4. Control (untreated) and inhibitor-treated roots were
allowed to exude for 17 h, after which the volumes of the exudates were
calculated from the lengths of fluid in the microcapillaries.
ACKNOWLEDGMENTS
We thank Dr. Alan Bown (Brock University, ON, Canada) and Dr. Martin
Canny (Australian National University, Canberra, Australia) for critically
reading the manuscript, Dr. Wilfried Rauser (University of Guelph, ON,
Canada) for helpful discussions, Dr. Jack Carlson (University of Waterloo,
ON, Canada) for use of the scintillation counter, and Tasneem Mohamed
(University of Waterloo, ON, Canada) for drafting Figures 2, 3, and 5.
Received October 23, 2003; returned for revision January 15, 2004; accepted
January 20, 2004.
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