Journal of Experimental Botany, Vol. 52, No. 355, pp. 361±368, February 2001
Brief exposure to low-pH stress causes irreversible
damage to the growing root in Arabidopsis thaliana:
pectin±Ca interaction may play an important role in
proton rhizotoxicity
Hiroyuki Koyama1,2, Tomomi Toda2 and Tetsuo Hara
Laboratory of Plant Cell Technology, Faculty of Agriculture, Gifu University, 1-1, Yanagido,
501-1193 Gifu, Japan
Received 7 June 2000; Accepted 15 September 2000
Abstract
Introduction
The viability of Arabidopsis thaliana (strain Landsberg)
roots exposed to a low pH (4.5 or 4.7) solution that
contained 100 mM CaCl2 was examined by staining with fluorescein diacetate-propidium iodide. The
elongation zone of growing roots lost viability within
1±2 h following exposure to low pH, but non-growing
roots showed no damage under the same treatment.
Low-pH damage in growing roots was irreversible
after 1 h incubation at pH 4.5 as judged by regrowth
in growing medium at pH 5.6. Growing lateral roots
also lost viability in the same treatment, whereas
non-growing lateral roots remained viable during
and after the treatment. The low-pH damage was
ameliorated by the simultaneous application of
calcium, indicating the involvement of a calciumrequiring process in overcoming proton toxicity.
At pH 5.0, growing roots required 25 mM of calcium
to maintain elongation, and at pH 4.8 and pH 4.5 more
than 250 mM and 750 mM, respectively. The low-pH
damage was ameliorated by divalent cations in the
order of Ba2qfSr2qGCa2q)Mg2q. The monovalent
cation Kq showed no ameliorative effect, but borate
showed a strong ameliorative effect with Ca2q. These
results indicate that the primary target of proton
toxicity may be linked to a disturbance of the stability
in the pectic polysaccharide network, where calcium
plays a key role in plant roots.
Rhizotoxicity in acid soil, which involves the action of
Al3q, Hq and Mn2q, is considered to be a major
environmental stress that limits world food production
(Foy, 1984; Robson, 1988). It is important to examine
the mechanisms of metal toxicity and tolerance in plants
to improve crop productivity in acid soil. Aluminium
toxicity has been well investigated (Taylor, 1991; Delheize
and Ryan, 1995; Kochian, 1995) and mechanisms of
Al tolerance have been proposed (e.g. root exudates;
Delheize et al., 1993; Pellet et al., 1995; Ma et al., 1997;
protein alteration; Basu et al., 1994). In contrast, little
has been reported (Fawzy et al., 1954) on proton
rhizotoxicity.
A simple culture solution consisting of calcium chloride
allows the precise computation of Al speciation (Kinraide
and Parker, 1987; Kinraide et al., 1992), and is often used
to study Al toxicity (Blanca¯or et al., 1998). However,
proton toxicity is also evident in this culture solution
(Kinraide and Parker, 1990; Yokota and Ojima, 1995).
Kinraide and coworkers indicated that proton toxicity
(low pH) inhibited root elongation in several plant species
(Kinraide and Parker, 1987; Kinraide et al., 1994), and
proposed that Ca displacement by proton is part of
the toxic action of proton rhizotoxicity (Kinraide et al.,
1994). It has also been reported that root elongation
of Arabidopsis thaliana is severely inhibited by low pH
(pH 4.5±4.8) in the same culture solutions with a low
ionic strength of Ca2q (Koyama et al., 1995). Under these
experimental conditions, the growing root apex of
A. thaliana showed low viability after exposure to low
pH solution (Koyama et al., 1995) suggesting that the
Key words: Arabidopsis thaliana, calcium-requiring, low-pH
stress, root elongation, pectin network.
1
2
To whom correspondence should be addressed. Fax: q81 582932911. E-mail: [email protected]
HK and T T contributed equally to this study.
ß Society for Experimental Biology 2001
362
Koyama et al.
target of proton action may be a Ca-requiring process
in root growth, but the mechanism is still unclear.
It is well known that Ca2q ions play an important role
in plant cell growth (Schiefelbein and Somerville, 1990;
Schiefelbein et al., 1992; Bush, 1995). It was reported that
Ca-displacement by proton causes an inhibition of root
elongation in wheat, and it was suggested that Ca2q
in the apoplast is one of the major targets of proton
rhizotoxicity (Kinraide et al., 1994). Apoplastic Ca2q
affects membrane functions including membrane potentials (Mohr and Schopfer, 1995; Shimmen, 1997), as well
as the cell wall network including pectin (Matoh and
Kobayashi, 1998). To speculate on a mechanism for the
Ca2q amelioration of proton rhizotoxicity for the former,
an actual evaluation was performed using a computation model (Gouy-Chapman-System model: see Kinraide,
1998). However, the toxic effect of proton on the latter
is still unclear.
Pectic polysaccharides are one of the major constituents containing Ca2q at the polygalacturonic acid zone,
which has a possible function to stabilize the cell wall
network (Carpita and Gibeaut, 1993). Although the biological role of pectic polysaccharides has not yet been
clari®ed, another cross-linkage of pectic polysaccharides
at the rhamnogalacturonan II region, that is mediated
by borate (RG-II: Kobayashi et al., 1996; O'Neill et al.,
1996; Williams et al., 1996) has been reported. Recently,
Fleisher et al. reported tobacco cell death (lost of viability)
caused by borate deprivation (Fleisher et al., 1998). On
the other hand, the low pH of the apoplast has been
reported to stimulate pectin solubilization in tomato
fruit during development (Chun and Huber, 1998). These
results raised a question as to whether the loss of viability
in growing root tips from proton rhizotoxicity is due to
the depletion of Ca2q from the pectin network at low pH.
To test this possibility, proton rhizotoxicity was examined
in the growing root of A. thaliana, which is highly sensitive
to proton rhizotoxicity (Koyama et al., 1995). Although
the nature of Ca2q-mediated cross-linkage of pectin in
growing cells is uncertain, an `egg-box model' has been
proposed from an in vitro study (Grant et al., 1973), and
the ameliorative effects of divalent cations on proton
rhizotoxicity ®ts well in this model. Borate, which crosslinks pectin at the rhamnogalacturonan II region (O'Neill
et al., 1996; Matoh et al., 2000), also strongly ameliorated
the effect of low pH damage, suggesting that the disturbance of the pectin network may be one of the primary
targets of proton rhizotoxicity.
Materials and methods
Hydroponic culture of Arabidopsis thaliana
Arabidopsis thaliana plants were cultured according to the
method of Toda et al., which permits the measurement of
root viability while minimizing mechanical damage (Toda
et al., 1999). Arabidopsis thaliana, ecotype Landsberg, seeds
were surface-sterilized with 1% sodium hypochloride solution
for 1 min, and were then kept at 4 8C for 2 d before planting
to synchronize germination as described previously (Koyama
et al., 1995). About 100 seeds were placed on the culture
apparatus, which consisted of a nylon mesh (50 mesh per inch)
supported by a plastic photo slide mount as described previously
(Toda et al., 1999). Each apparatus was ¯oated on 100 ml
basal culture solution containing calcium chloride (100 mM)
or 1u10 strength of MGRL nutrients (Fujiwara et al., 1992).
Seedlings were grown under 12 h illumination per day
(PPDF: 150 mmol m 2 s 1) at 24±26 8C.
Measurement of root length and elongation rate
Seedlings were gently pulled from the apparatus at the designated time. Each seedling was soaked in 5 ml of test solutions
in a multi-well plate (Sumilon MS-80060, Sumitomo Bakelite,
Tokyo) and incubated at 25 8C. For the measurement of root
length, each seedling was settled on the well bottom, covered
with a micro coverslip (21 3 21 mm) and an image of the plant
was captured by a microscope video camera (Pico Scopeman,
Kenis, Tokyo, Japan). The root length was measured on a
monitor by using a multiple measure-unit (MC-300, Kenis)
and the elongation rate was calculated from the values measured
at intervals.
Test solutions used for short-term treatments
A series of simple test solutions was prepared by adding MgCl2,
CaCl2, SrCl2 (200 mM each), KCl (400 mM) or boric acid
(100 mM) to the basal test solution (100 mM CaCl2). Solutions
containing only CaCl2 or SrCl2 or MgCl2 or BaCl2 were also
prepared to determine the direct effect of divalent cations. Test
solutions containing a set of nutrients, with different ionic
strength, were prepared by a series of dilution of MGRL
nutrients. The initial pH was adjusted by adding 0.1 N HCl in
the presence of 5 mM of MES.
Measurement of root viability
Seedlings were gently transferred to 100 ml of test solutions and,
following the incubation in the test solutions, viability of root tip
cells was determined by ¯uorescein diacetate (FDA)±propidium
iodide (PI) staining or PI staining according to the method of
Jones and Senet (Jones and Senet, 1985) with minor modi®cations as described previously (Toda et al., 1999). FDA is permeable to the intact plasma membrane and is converted to
a green ¯uorescent dye, ¯uorescein, by a function of internal
esterase(s), in turn to showing green colour in viable cells. By
contrast, PI is impermeable to the intact plasma membrane.
Damaged cells having pores on the plasma membrane incorporate the dye, and generate a red ¯uorescence by forming a PI±
nucleic acid conjugate. Root staining with FDA±PI was
observed with a confocal microscope (LSM510, Carl-Zeiss,
Germany) equipped with an argon-HeNe laser. The wavelengths
for excitation and emission for ¯uorescein diacetate (FDA) were
488 nm and 505±530 nm, respectively, and those for propidium
iodide (PI) was 543 nm and 585 nm, respectively. Image processing was completed using the software supplied by the confocal microscope manufacture. For evaluation of ameliorative
effects of metals on proton rhizotoxicity, root staining with PI
was also observed with a ¯uorescent microscope (IMT-2±21RFL; Olympus, Tokyo) equipped with a dichroic mirror unit
[IMT-2-DG; Olympus] and with a density ®lter (ND6, Olympus).
Low-pH damage in Arabidopsis
Results
Short-term exposure to low pH solutions caused
damage in the apex of growing roots
The viability of root tips of seedlings was examined at
various developmental stages to determine the sensitivity
to low pH using FDA-PI staining, which showed high
sensitivity for estimating rhizotoxicity of metals (Toda
et al., 1999). After this treatment, viable cells ¯uoresce
bright green (FDA), while damaged cells ¯uoresce a bright
red colour (PI) (Fig. 2).
In the basal culture solution (100 mM CaCl2 alone:
pH 5.6), the primary roots stopped growing after 6 d
from planting (Fig. 1). In contrast, the primary roots
Fig. 1. Primary root growth of A. thaliana in basal culture solution
containing 100 mM CaCl2 (triangle) or 1u10 strength MGRL nutrient
solution (circle), and their sensitivity to low pH. The primary root at
various developmental stages was measured in length, then immersed
in a low pH solution (pH 4.5, 100 mM CaCl2) for 2 h and stained with
FDA-PI to estimate root tip viability. Closed symbols show low-pH
damage (see Fig. 2b) and open symbols show the absence of damage
(see Fig. 2a).
363
continued growing for at least 9 d in the solution supplemented with 1u10 strength of MGRL nutrients (Fig. 1).
When the roots of seedlings grown in basal culture
solution were immersed for 3 d, typical low-pH damage
was observed in root tips after exposure to the simple
solution (100 mM CaCl2 only) with low pH (pH 4.5) for
2 h (Fig. 2c). However, no damage was observed in the
roots after day 6, when primary root growth had stopped
(Fig. 2a). The primary roots, which continued growing
for at least 9 d in 1u10 strength MGRL culture solution,
were also highly sensitive to low pH (Fig. 1). Thus, the
growing primary roots seemed to be more sensitive to
low pH than non-growing roots.
As described previously, seedlings grown in medium
MGRL nutrient medium can develop healthy lateral roots
about 15 d after planting on the culture apparatus (Toda
et al., 1999). In the preliminary experiment, damage
in growing lateral roots was observed after an exposure
to low pH solution (Fig. 2d). To examine the relationship between the developmental stage of the lateral root
and low-pH sensitivity, any low-pH damage of lateral
roots that had different elongation rates during the 12 h
before low-pH treatment was examined. The percentage
of damaged lateral roots strongly increased with increasing lateral root growth rate (Fig. 3). This implies that the
low-pH damage was observed only in growing roots for
both primary and lateral roots.
Short-term low-pH stress irreversibly terminated
root elongation
Confocal images of FDA-PI ¯uorescence in roots exposed
to pH 4.5 for 30 min (Fig. 2b) showed that the pH
damage occurred not only in the epidermis but also in
internal tissues. After 2 h exposure to a low pH, the
Fig. 2. Confocal microscopic observation of A. thaliana roots exposed to low-pH stress. The primary roots of seedlings grown in basal culture solution
(a, b, c) or the lateral roots of seedlings grown in 1u10 MGRL solution (d) were immersed in a low-pH solution (pH 4.5) for 30 min (b) or 2 h (a, c, d)
and then stained with FDA±PI. (a) Primary root on day 6 in basal culture solution which had ceased growing, low pH treatment for 2 h. (b) Primary
root on day 3 in basal culture solution, low pH treatment for 30 min. (c) Primary root on day 3 in basal culture solution, low pH treatment for 2 h. (d)
Lateral root on day 15 in MGRL solution, low pH treatment for 2 h. Viable cells show green ¯uorescence while non-viable cells show red. (a) 3-D
image. (b, c, d) Confocal image. Bars indicate 100 mm.
364
Koyama et al.
central part of the root tip was also damaged (Fig. 2c, d).
To determine whether this damage caused an inhibition
of root growth, the re-growth of roots following exposure
to low pH solutions was examined. The seedlings whose
roots had been exposed to low pH solutions (100 mM
CaCl2, at pH 4.5 or 4.7) for various periods were
transferred to 1u10 MGRL medium (pH 5.6), and root
elongation during the subsequent 24 h was examined.
As shown in Fig. 4, primary root elongation was suppressed with increased exposure to a low pH. A 1 h
exposure to pH 4.5 completely suppressed subsequent
elongation, and a 2 h exposure to pH 4.7 also severely
suppressed subsequent elongation (Fig. 4). These results
clearly showed that the damage caused by brief exposure to a low pH (Fig. 2c, d) is not reversed even after
the seedlings were transferred to a non-stressed condition
(Fig. 4).
Requirement of Ca2q for maintaining root tip viability
and root growth in low pH solutions
To determine whether lowering of solution pH increases
the Ca2q-requirement for root growth, seedlings with
growing roots were soaked in solutions containing Ca2q
at various concentrations at pH 5.3, 5.0, 4.8 or 4.5, and
root elongation was measured 6 h after exposure. Root
elongation as a function of Ca2q concentration at pH 5.0
and 5.3 had sharp peaks at 25 mM, and then slightly
decreased at higher Ca2q concentrations (Fig. 5). In contrast, the growth curves for seedlings grown in solutions
at pH 4.8 and pH 4.5 showed no clear peaks. Root
elongation increased as the Ca2q concentration was
increased up to 250 mM and 750 mM, at pH 4.8 and 4.5,
respectively (Fig. 5).
When the Ca2q concentration was lower than 25 mM
root tip viability was strongly decreased within 3 h.
Following 3 h exposure to 10 mM CaCl2, about 30%
Fig. 3. Lateral root damage of A. thaliana exposed to pH 4.5 for 2 h at
various developmental stages. Y-axis shows the percentage of damaged
(showing red ¯uorescence) lateral roots. Developmental stage of lateral
roots was categorized into three classes by the root elongation during
the preceding 12 h. 1: 0±50 mm. 2: 50±150 mm. 3: )150 mm. Triplicate
analysis for each 20 seedling was performed. Means and SD values are
indicated.
of seedlings showed damage at pH 5.0 or 5.3 (data not
shown). The same level of damage was observed with
150 mM and 500 mM of calcium at pH 4.8 and 4.5,
respectively (data not shown). Thus, the Ca2q requirement of growing roots strongly increased by lowering
the pH.
Ameliorative effects of Ca, Sr, Ba, and borate on
short-term pH damage
To examine whether proton rhizotoxicity is caused by the
weakening of Ca2q-mediated cross-linkage of pectin, the
ameliorative effect of metals known to mediate crosslinkage in pectin was examined. First, the ameliorative
effect of metals in the presence of CaCl2 at pH 4.7 was
examined. During the 2 h incubation in basal test solution
(100 mM CaCl2) only 2 out of 45 roots maintained
viability in the root tip (Table 1). Under such conditions,
the addition of 200 mM CaCl2 or SrCl2 known to bind
with pectin strongly ameliorated low-pH damage. By contrast, excessive addition of Kq showed no ameliorative
Fig. 4. Primary root growth of A. thaliana after exposure to low-pH
stress. The roots of seedlings cultured for 3 d.in basal culture solution
were exposed to pH 4.5 (m) or 4.7 (k) for 30, 60 or 120 min and were
then cultured in the 1u10 strength MGRL solution at pH 5.6 for
24 h. Y-axis shows the elongation of roots after the low-pH stress.
Means "SD (nˆ7) are indicated.
Fig. 5. Primary root growth of A. thaliana in CaCl2 solutions at
different pHs. Seedlings cultured for 3 d in 1u10 strength MGRL
solution were immersed in CaCl2 solutions of various concentrations at
pH 5.3 (k), 5.0 (!), 4.8 (n) or 4.5 (h) for 6 h. Means of relative values
and SD values are indicated (nˆ7).
Low-pH damage in Arabidopsis
365
Table 1. Amelioration of low-pH damage in the root tip of A. thaliana by the simultaneous application of various metals
Period of low pH
treatment (min)
Number of seedlings with damaged roots
Control
qCa2q
qKq
qMg2q
qSr2q
qBoric acid
60
120
9.3"0.3
14.3"0.5
0.3"0.5
1.0"0.0
11.3"0.9
14.7"0.5
3.3"2.1
9.3"2.4
0.0"0.0
1.0"0.0
1.3"0.5
3.0"1.4
The roots of 15 seedlings were immersed in the basal test solution containing 100 mM of CaCl2 (control) with 200 mM of CaCl2, MgCl2 or SrCl2, or
with 400 mM of KCl or with 100 mM of borate added at pH 4.7 and then stained with PI. Means "SD from three replications are shown.
Table 2. Low-pH damage in the root tip of A. thaliana in MGRL
nutrient solution
Fig. 6. Ameliorative effects of divalent cations on short-term proton
rhizotoxicity. Groups of 20 seedlings were soaked in solutions containing CaCl2 (k), MgCl2 (!), SrCl2 (n), and BaCl2 (h) of various
concentrations at pH 4.8 for 1 h, and then stained with PI. Y-axis shows
the number of seedlings with damaged cells in the root elongation zone.
Means "SD of three replications are shown.
effect and Mg2q partially ameliorated proton rhizotoxicity. Borate strongly ameliorated low pH stress in the
presence of Ca2q. Only 9 out of 45 roots showed lowpH damage after adding 100 mM borate to the basal test
solution (Table 1).
To determine the ameliorative effect of divalent cations
more directly, root damage was examined after exposure
to solutions containing divalent cations alone. Because
high Ba2q concentrations can damage in the growing
root, moderate low-pH stress was used (1 h at pH 4.8)
in this experiment. The divalent cations, Ca2q, Sr2q and
Ba2q strongly ameliorated low-pH stress, but Mg2q
showed a very weak ameliorative effect (Fig. 6). The
ameliorative effect of Sr2q and Ba2q was higher than
that of Ca2q.
Low-pH damage in nutrient solution
To examine whether Ca2q-dependent proton rhizotoxicity would occur in a nutrient solution with a more
natural and complex compositions, the low-pH damage
was examined after exposure to MGRL nutrient solution
(1 h, pH 4.8) with a series of dilutions. Concentration of
CaCl2 in normal, 1u25 and 1u50 MGRL solutions was
2 mM, 80 mM and 40 mM, respectively. Only 1 out of
90 seedlings showed the low-pH damage during the 1 h
Strength
of medium
(dilution)
Number of seedlings with damaged roots
Control
Ca2q
1
1u25
1u50
0.0"0.0
0.3"0.5
6.7"0.5
14.7"0.5
13.3"0.5
14.0"0.9
Mg2q
0.0"0.0
0.7"0.5
5.0"2.4
Kq
0.0"0.0
0.3"0.5
4.7"1.4
The roots of 15 seedlings were immersed in a series of diluted MGRL
solution containing full set of nutrients (control), or without Ca
( Ca2q), Mg ( Mg2q) and K ( Kq) for 1 h and then stained with
PI at pH 4.8. Means "SD from three replications are shown.
incubation in normal strength and in 25 times diluted
MGRL (Table 2). By contrast, about 40% of seedlings
showed low-pH damage after exposure to 50 times diluted
MGRL solution. This value was almost similar to that
observed with the same treatment (pH 4.8 for 1 h) in
a simple culture solution containing 40 mM CaCl2 alone
(Fig. 6). Under these conditions, elimination of Ca2q
from the solutions caused about 90% damage for the
seedlings (Table 2). However, the degree of damaged
roots with Mg2q and Kq treatments was similar to
those with a control treatment (Table 2). These results
suggest Ca2q-dependent proton rhizotoxicity occurs in
nutrient solution and is comparable to that with a simple
culture solution.
Discussion
A number of studies have shown that simple culture
solutions containing only CaCl2, with a low ionic
strength, enhance proton rhizotoxicity (Koyama et al.,
1995; Yokota and Ojima, 1995; Kinraide, 1998). Using
this solution as the test medium and combining visual
measures of root tip viability to increase the sensitivity
of the technique for measuring the toxic action of proton on root elongation, it has been demonstrated that
A. thaliana displays high sensitivity to short-term proton
rhizotoxicity (Figs 2, 4; Table 1). A visual low-pH damage
was detectable within a short term, such as 1 h (Figs 2, 6),
and the results are comparable to those obtained using
366
Koyama et al.
inhibition of root elongation as an indicator for rhizotoxicity, which requires a longer term (Fig. 5). It appears
that the experimental system described here is a suitable
model system for examining proton rhizotoxicity.
Low-pH exposure caused irreversible damage in primary and lateral growing roots of A. thaliana, but not in
non-growing roots (Figs 2, 3). This observation suggests that the primary target of proton in rhizotoxicity is
the disturbance of biological functions involved in root
elongation. Higher concentrations of Ca2q were required
for maintaining root tip viability (Fig. 6) and root
elongation of A. thaliana at low pH (Fig. 5). Thus, it is
speculated that the major target of proton rhizotoxicity in
growing roots of A. thaliana seems to be Ca2q-requiring
processes involved in root elongation, which could easily
be disturbed by low pH. Using growing root hairs as
a model for studying cell elongation (tip growth) of
A. thaliana, it was reported that the Ca-gradient in the
cytosol was necessary to maintain elongation (Wymer
et al., 1997). However, internal Ca stores could support
normal root hair growth for at least an hour, even if Ca
uptake was terminated by the addition of the Ca-channel
blocker verapamil. This suggests that the internal function of Ca could be maintained during short-term
treatment, such as an hour employed in the current study
(Fig. 6). In fact, verapamil treatment, with Ca2q, showed
no effect on root tip viability (data not shown). Also,
seedlings soaked in Sr2q and Ba2q solutions, without
Ca2q, maintained normal root growth for at least 2 h
(data not shown). These results suggest that, under the
experimental conditions used, internal Ca stores supported normal root function. Thus, the low-pH damage
observed in the growing root of A. thaliana is not considered to be caused by deprivation of Ca in the symplast,
but rather in the apoplast.
The ameliorative effect of Ca2q has been investigated
for rhizotoxic metals, such as Al and Na (Kinraide, 1998).
Mechanism of Ca-amelioration has been proposed both
for the symplast and for the apoplast from the toxic
action of metals. For example, some metals, such as Al
(Kochian, 1995), inhibit Ca-uptake and Ca-homeostasis,
and therefore the addition of Ca into the toxic medium
may result in amelioration by maintaining Ca in¯ux.
Another important mechanism of Ca-amelioration,
proposed as the `Ca-displacement hypothesis', may be
involved in the apoplast. Low-pH damage under the
experimental conditions used in this study, as described
above, may be caused by Ca-deprivation from the apoplast, and thus this hypothesis was considered for
explaining the ameliorative effects of Ca2q. The Cadisplacement hypothesis for metal toxicity has been proposed for proton (Kinraide et al., 1994), Al (Reid et al.,
1995) and other ions (Cramer et al., 1985). As reported
recently (Kinraide, 1998), the change in Ca2q activity at
the action site, including the membrane surface, is one of
the major factors affecting proton rhizotoxicity. Given
the ameliorative effects of several metals other than Ca2q,
another action site for the Ca2q amelioration of proton
rhizotoxicity may be negatively charged cell wall pectin.
A series of amelioration studies on low-pH damage
supported this possibility.
Divalent cations with large ionic radii and borate
ameliorate low-pH damage (Table 1; Fig. 6). According
to the `egg-box model', divalent cations can cross-link
pectic polysaccharides through the formation of coordinate bonds with uronate residues, but monovalent and
divalent cations with a small ionic radius (such as Mg2q)
cannot. The polygalacturonic acid zone is thought to
provide an `egg-box' for stabilizing pectin (Carpita and
Gibeaut, 1993). The ameliorative effects of several cations are in agreement with this model (Table 1; Fig. 6).
Monovalent cation, Kq showed no effect and the
Ê ) showed
divalent cation, Mg2q (ionic radius: 0.66 A
2q
Ê
lower ameliorative effects than Ca (0.99 A). Both Ba2q
Ê ) and Sr2q (1.12 A
Ê ) with large ionic radii strongly
(1.34 A
ameliorated proton damage in growing roots. The
decreasing order of ameliorative effects of these ions
wBa2qfSr2qGCa2q)Mg2q (Fig. 6)x match the selectivSr
ity co-ef®cient of pectate for each ion wkCa
Mg (7.0), kMg
Ba
(9.6) and kMg (10.1)x (Haug and Smidsrùd, 1970). Under
our experimental conditions, borate, which cross-links
pectin at the rhamnogalacturonan II region (Kobayashi
et al., 1996; Williams et al., 1996), also ameliorated lowpH stress in the presence of Ca2q (Table 1). However,
borate provided no amelioration of low-pH stress when
applied without Ca2q (data not shown). This Ca2q
requirement of borate amelioration ®ts the mechanism of
cross-linkage of pectic polysaccharides with borate, which
requires Ca2q (O'Neill et al., 1996). Although the internal
effects of metals wSr affects Ca release from the internal
Ca stores (Bauer et al., 1998) and borate increases
ascorbate synthesis (Lukaszewski and Blevins 1996)x have
been reported, these results indicate that short-term
proton rhizotoxicity is caused by weakening of Camediated cross-linkage of pectin due to low pH. In the
experimental conditions used here, pectolyase treatment
(pH 5.3, 500 mM Ca2q, 0.01% pectolyase for 1 h) caused
severe damage in the root tip of growing roots (data not
shown), suggesting that the pectin network plays an
important role for root tip viability. The necessity of the
pectin network in cell growth has been reported in cell
cultures of Chenopodium album L. (Fleisher et al., 1998)
and mutant tomato (Shedletzky et al., 1990). Unfortunately, there are no reports showing that development
of the pectin network is essential for root elongation.
Further analysis is needed to test this hypothesis in
relation to the vital importance of the development of the
pectin network for root growth.
Under the experimental conditions used here, low-pH
damage was not evident in solutions containing the
Low-pH damage in Arabidopsis
normal levels of nutrients (Table 2), as similar to previous
reports (Koyama et al. 1988; Osaki et al., 1997) which
used growth as the indicator for estimating low-pH
damage. However, low-pH damage was again clear in a
50-fold diluted nutrient solution (50 times dilution) with a
low ionic strength of CaCl2 (40 mM). The degree of low
pH damage of this condition was almost similar to that in
a simple test solution containing CaCl2 alone (Table 2;
Fig. 6). Thus, it could be speculated that Ca2q-dependent
proton rhizotoxicity may occur in a solution with more
natural and complex compositions, such as a soil solution. Whether short-term proton rhizotoxicity is a serious
problem in the natural environment remains unknown.
However, similar short-term rhizotoxicity has been
observed in other plants including woody species (data
not shown). In addition, acid precipitation decreases
Ca availability in some soils (Knoepp and Swank, 1994;
Likens et al., 1997), and also occasionally lowers soil
pH below 4.7 (Radojevic and Brunei, 1997). Rapid and
irreversible rhizotoxicity associated with brief exposure
to low pH should be considered a potential risk of acid
precipitation.
Acknowledgements
We wish to thank Dr Neil S Harris at the University of
Alberta and Dr SS Kantha for critical reading of the manuscript. We wish to thank Dr H Suga of Gifu University Gene
Research Center for his kind technical support for confocal
microscopic analysis. Part of this work has been ®nancially
supported by a Grant in-aid for Scienti®c Research from the
Ministry of Education Science and Culture of Japan (for HK
10760036).
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