Journal of Experimental Botany, Vol. 52, No. 355, pp. 223±229, February 2001
Ca2q and phosphate releases from calcified
Chara cell walls in concentrated KCl solution
Keitaro Kiyosawa1
Division of Biophysical Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka,
Osaka 560-8531, Japan
Received 24 April 2000; Accepted 11 September 2000
Abstract
2q
Ca
and Pi (inorganic phosphate) releases from
isolated calcified and uncalcified Chara cell walls
were measured with a Ca2q-selective electrode and
colorimetry, and their ionic relations were analysed
on the basis of the electroneutrality rule. The results
showed that (1) not only Ca2q but also Pi can be
released from isolated calcified Chara cell walls into
pure deionized water and 100 mM KCl solution, and
(2) the positive charge due to the Ca2q released
cannot be neutralized only by the negative charge
from the simultaneously released Pi. These findings
suggest that calcium bands of calcified Chara cell
walls are composed of mainly CaCO3 and CaHPO4
and some anions other than Pi should be released
simultaneously with the Ca2q and Pi. More Ca2q and
Pi can be solubilized from isolated Chara cell walls
in 100 mM KCl solution than in pure deionized water.
The pH value of 100 mM KCl solution in which isolated uncalcified young Chara cell walls have been
immersed is a little lower than that of pure deionized
water in which the same isolated uncalcified young
Chara cell walls have been immersed, suggesting
that some acidic substances are solubilized by
100 mM KCl. To explain this from the viewpoint of
solution chemistry, the solubilities of pure CaCO3
and pure CaHPO4 in water and 100 mM KCl solution
were measured with a Ca2q-selective electrode and
their pH values with a glass pH electrode. The conclusion reached was that the Ca2q release from isolated Chara cell walls is accompanied by the release
of Pi, CO23 and acidic substances. This suggests
that the so-called calcium bands anduor ionic relations, including ion exchange, in Chara cell walls are
chemically or physicochemically more complex than
they are currently considered to be.
1
Fax: q81 6 6850 6557. E-mail: [email protected]
ß Society for Experimental Biology 2001
Key words: Calcium band, calcium carbonate (CaCO3),
calcium hydrogenphosphate (CaHPO4), cell wall, Chara
corallina, electroneutrality rule.
Introduction
Chara internodal cells die in aqueous electrolyte solutions
such as 100 mM KCl, 10 mM MgCl2, 1.0 mM BaCl2
(Kiyosawa and Adachi, 1990), 100 mM NaCl (Katsuhara
and Tazawa, 1986; Tufariello et al., 1988), and 0.1 mM
HCl (Kiyosawa, 1990). However, adding an appropriate
amount of Ca2q (0.5±10 mM) to such aqueous electrolyte
solutions enables the internodal cells to survive for more
than a week (Katsuhara and Tazawa, 1986; Tufariello
et al., 1988; Kiyosawa and Adachi, 1990; Kiyosawa,
1993). Whittington and Smith have shown that Chara
internodal cells can survive in 100 mM NaCl, LiCl, KCl,
and RbCl solutions containing 0.1 mM Ca2q for at least
3 d (Whittington and Smith, 1992). Using the atomic
absorption method, combined with the extraction of
Ca2q from Chara cell walls by 100 mM HCl, Kiyosawa
and Adachi showed that Ca2q is released from Chara
cell walls in aqueous KCl, NaCl, MgCl2, and BaCl2
solutions, but that the addition of Ca2q reduces the
Ca2q released (Kiyosawa and Adachi, 1990).
In the present experiments, Ca2q release from isolated Chara cell walls in pure deionized water and in
100 mM KCl solution was measured with a Ca2qselective electrode. Along the surface of the Chara cell
wall grown under strong light, calcium bands, which
are believed to consist of CaCO3 crystals, are formed
(Borowitzka, 1982, 1987; Borowitzka et al., 1974;
Okazaki and Tokita, 1988).
The present study examined whether or not the Ca2q
released comes mainly from the CaCO3 forming the
224
Kiyosawa
calcium bands. To do this, the amounts of Ca2q released
from isolated Chara cell walls into pure deionized water
and aqueous 100 mM KCl solution were compared,
and their pH values were examined in relation to the
solubilities of CaCO3 in these ¯uids. The Ca2q measurements were done with a Ca2q-selective electrode and the
pH values were obtained using a glass pH-electrode.
The present paper also examines whether or not
CaHPO4, which is practically insoluble in water, but
can neutralize pure deionized water, is involved in the
calcium bands of Chara cell walls. The release of H2PO4
(Pi) from isolated Chara cell walls in pure deionized water
and in aqueous 100 mM KCl solution was measured
qualitatively and quantitatively using colorimetry. Also,
the solubilities of pure CaHPO4 in pure deionized water
and in 100 mM KCl solution were measured using a
Ca2q-selective electrode. The pH values of aqueous
CaHPO4 solutions and aqueous mixtures of CaHPO4
with 100 mM KCl were also measured with a glass
pH-electrode.
Materials and methods
Isolated cell walls of Chara corallina were used throughout the
experiments. Chara corallina was grown in buckets with several
centimetres of soil at the bottom. Illumination was provided by
one or two 20 W ¯uorescent lamps several centimetres above
the surface of the water in the laboratory. City tap water, which
was of neutral pH, was used for the culture. Internodal cells
having clear calcium bands, i.e. calci®ed Chara internodal cells
or having no visible calcium bands, i.e. uncalci®ed Chara internodal cells, were isolated from adjacent cells with scissors on
the morning of the day when the experiments were conducted.
Both ends of the internodal cells were cut with scissors and
the protoplasm was squeezed out with ®ngers in deionized
water. To remove soluble substances which might interfere with
the measurement with a Ca2q-selective electrode and colorimetry, the isolated cell walls from about 40 internodal cells
were immersed in pure deionized water for about 20 min. Next,
they were transferred into a Te¯on beaker in which either pure
deionized water or the test electrolyte solution of 25.0 ml
had been kept at 25 8C and incubated for 2 h at 25"0.5 8C
with stirring with a magnetic bar at a moderate rate. After
incubation, the cell walls were removed from the beaker with
forceps and the absorbed water was squeezed out onto sheets
of ®lter paper. The cell walls which had ®rst been immersed in
pure deionized water were immersed in test electrolyte solution
for 2 h at 25"0.5 8C with stirring with a magnetic bar at
a moderate rate. In some experiments, the isolated Chara cell
walls were directly immersed in test electrolyte solutions after
20 min immersion in pure deionized water to remove interfering
substances.
Cell walls isolated from Chara corallina grown outdoors in
the sun were also used.
The calcium concentration of pure deionized water and test
electrolyte solution in which isolated cell walls had been
incubated was measured with a Ca2q-selective combination
electrode (6583-10C, Horiba, Ltd., Kyoto, Japan) connected
to a pHumV-meter (Type F-16, Horiba, Ltd., Kyoto, Japan).
The concentrations of the aqueous electrolyte solutions were
100 mM KCl, 100 mM NaCl, 10 mM MgCl2, and 10 mM
BaCl2. Calibration curves for the relationship between the
calcium concentration [Ca2q] and the electrode potential DE of
a Ca2q-selective electrode of a combination type were obtained,
respectively, for simple aqueous CaCl2 solutions and aqueous
mixtures of CaCl2 with the electrolyte at the same concentration as the test one used for extraction of Ca2q from isolated
Chara cell walls. The Ca2q concentration of pure deionized
water and test electrolyte solutions in which isolated Chara cell
walls had been immersed, respectively, were determined by
measuring DE when a calibrated Ca2q-electrode was dipped
into them.
The pH value of pure deionized water and test electrolyte
solution in which isolated Chara cell walls had been incubated
was measured with a glass pH-combination electrode connected
to a pH meter (Type F-16, Horiba, Ltd., Kyoto, Japan) at
25"0.5 8C. Further studies on the Ca2qrelease from calci®ed or
uncalci®ed Chara cell walls and on solubilization of CaCO3
and CaHPO4 were performed using pure deionized water and
100 mM KCl solution.
Before measurements of the Ca2q concentration and pH
value of simple aqueous CaCO3, and CaHPO4 solutions or
the supernatant of their aqueous suspensions, the mixtures of
CaCO3 or CaHPO4 with pure deionized water or 100 mM KCl
solution of 1.0 dm3 had been stirred with a stirring bar at a
moderate rate for more than 2 d at 25.0"0.5 8C, and then had
been left standing for more than 2 d at the same temperature.
During the period, almost all of the insoluble CaCO3 and
CaHPO4 had precipitated except for some CaCO3 or CaHPO4
which remained ¯oating on the surface of the transparent
aqueous supernatants.
The measurements of the calcium concentration by a Ca2qselective electrode and the pH value by a pH electrode of all
of these aqueous solutions were done whilst stirring with a
magnetic bar at a moderate rate and the measured values were
printed out over 20 min as a function of time. Visual observation was conducted to decide whether or not the CaCO3 or
CaHPO4 added had completely dissolved. Their solubilities
were also determined by measuring the solubilized Ca2q
concentration as a function of the molar concentration of
CaCO3 or CaHPO4 added.
The concentration of phosphate was measured by colorimetry
(Taussky and Shorr, 1953) with some modi®cation (Kiyosawa,
1979). This method showed no interference from KCl, KNO3,
MgSO4, CaCl2, (NH4)2SO4 or TRIS pH buffer at concentrations used in usual biochemical and physiological experiments.
The pure deionized water was above 17 MV cm 1. CaCO3 of
reagent grade (99.9%) and CaHPO4.2H2O of reagent grade
(min 98.0%) were purchased from Wako Pure Chemicals
Industries, Ltd., Japan and Nakarai Tesque, Inc., Japan,
respectively.
Results
The electrode potential DE read by a pHumV meter
equipped with a Ca2q-selective electrode decreases or
increases monotonically as a function of time without
reaching constant values. One of the possible origins of
drift in electrode potential DE would be that in the
boundary potentials of a calcium selective combination
electrode. However, plotting the DE at around 15 min
after the start of measurement against the logarithm of
Calcium and phosphate release from Chara cell walls
the calcium concentration, log[Ca
curve expressed by the equation
2q
], gives a good linear
DE ˆ a log‰Ca2q Šqb
(1)
where a and b are constants. For simple aqueous
CaCl2 solutions, a is nearly equal to 2.303RTu2F; where
R, T and F are the gas constant, the absolute temperature and the Faraday constant, respectively, and about
29±30 mV at 25 8C (Fig. 1). For aqueous mixtures of
CaCl2 with an electrolyte, the constants a and b change
depending on the species and the concentration of the
electrolyte used. The constant b also varies greatly from
electrode to electrode.
After obtaining the calibration curve expressed by
Equation (1) for simple aqueous CaCl2 solutions or
aqueous mixtures of CaCl2 with the same electrolyte as
the test solution such as 100 mM KCl, it is possible to
determine the calcium concentrations [Ca2q] of samples
by measuring DE after dipping a Ca2q-selective electrode
into them. Since DE changes slightly from day to day,
calibration curves for the calcium concentration [Ca2q]
Fig. 1. Examples of the relationship between the electrode potential
DE of a Ca2q-selective electrode of a compound type and the Ca2q
concentration [Ca2q]. The solid line with open circles indicates simple
aqueous CaCl2 solutions, and the dashed line with solid circles indicates aqueous mixtures of CaCl2 with 100 mM KCl, respectively. All
measurements were done at 25"0.5 8C.
225
and the electrode potential DE expressed by Equation (1)
were obtained before every experiment.
Table 1 shows the calcium concentrations of 25.0 cm3
pure deionized water and aqueous test electrolyte solutions of 100 mM KCl, 100 mM NaCl, 10 mM MgCl2,
and 10 mM BaCl2 after the isolated calci®ed Chara cell
walls had been immersed for 2 h under stirring. The Ca2q
released from the cell walls during the 2 h changed the
pure deionized water of 25 cm3 to about 0.17±0.29 mM
Ca2q. The Ca2q released from the cell walls during 2 h of
subsequent immersion in aqueous electrolyte solutions of
25 cm3 made them 0.68±0.91 mM Ca2q. Direct immersion of the cell walls in aqueous electrolyte solutions
also had brought their calcium concentrations to
0.66±0.70 mM (Table 1).
Table 2 shows the Pi release from isolated mature
calci®ed, mature uncalci®ed and young uncalci®ed
Chara cell walls in pure deionized water and 100 mM
KCl solution together with the simultaneous Ca2q
release, and the pH values of the water and the solution.
Clearly, the table shows that (1) Pi is released from
isolated calci®ed Chara cell walls both in pure deionized
water and in 100 mM KCl solution, but little Pi is released
from uncalci®ed cell walls in either solution, (2) the
amounts of Pi and Ca2q released from cell walls in pure
deionized water during 2 h of immersion are less than
that in 100 mM KCl solution, (3) the Pi concentrations are always lower than those of the released Ca2q,
and (4) the pH values of the water and the solution
in which isolated mature Chara cell walls have been
inmersed are neutral, not weakly acidic as is the case
for pure deionized water and aqueous 100 mM KCl
solution saturated with atmospheric CO2, but (5) the pH
value of 100 mM KCl solution in which young uncalci®ed
Chara cell walls have been immersed is weakly acidic.
These observations indicate that calcium compounds,
which can dissolve very sparingly into pure deionized
water and more into 100 mM KCl solution, are rather
abundantly bound to Chara cell walls.
To examine whether or not these calcium and phosphate ions come from CaCO3 and CaHPO4, the solubilities of pure CaCO3 and CaHPO4 were measured in
Table 1. Ca 2qrelease from mature calci®ed Chara cell walls in pure deionized water (DW) and aqueous electrolyte solutions (ES)
Ca2q release in
DW (mM)
100 mM KCl
100 mM NaCl
10 mM MgCl2
10 mM BaCl2
a
0.29"0.02
±
0.25"0.01
0.17"0.03
±
0.17"0.03
±
Sequential Ca2q release in ES (mM)
Dry weight (mg)a
0.68"0.03
±
0.72"0.05
0.91"0.02
±
0.71"0.03
±
35.0"2.7
38.3
40.6"5.6
53.3"8.5
39.1"7.7
35.3"8.3
29.0"1.5
ES (mM)
(n ˆ 6)
(n ˆ 2)
(n ˆ 3)
(n ˆ 2)
±
0.70 (n ˆ 1)
±
±
0.67"0.11 (n ˆ 4)
±
0.66"0.01 (n ˆ 2)
Dry weight indicates that of the isolated Chara cell walls used.
226
Kiyosawa
both pure deionized water and 100 mM KCl, and the pH
values were also determined. These pH values were
compared with those of pure deionized water and of the
test electrolyte solution in which Chara cell walls had
been immersed for 2 h.
Figure 2 shows the Ca2q concentrations of aqueous
CaCO3 solutions and supernatants of their suspensions
measured with a Ca2q-selective electrode. The Ca2q
concentrations of aqueous CaCO3 solutions and suspensions are expressed as a function of the molar concentration when all of the CaCO3 added to 1.0 dm3 pure
deionized water or 100 mM KCl solution is assumed to
have dissolved completely.
Mixtures of CaCO3 up to about 0.28 mM in pure
deionized water become transparent when they are stirred
with a magnetic bar at a moderate speed for about 2 d.
However, at above 0.28 mM, white ¯oating material,
probably CaCO3, is ®rst observed on the surface and
if the CaCO3 concentration continues to increase, a white
precipitate, probably CaCO3, can be observed at the
bottom of a beaker which has been stirred for 2 d and
left standing for a further 2 d. The Ca2q concentrations
of the supernatant of aqueous CaCO3 suspensions are
around 0.28 mM and almost constant above this
concentration.
The Ca2q concentrations of aqueous mixtures of
CaCO3 with 100 mM KCl are plotted in the same ®gure
as the function of the apparent CaCO3 concentration.
On addition of 100 mM KCl, the saturated Ca2q concentration measured with a Ca2q-selective electrode seems
to increase up to around 0.38 mM. However, if measurement errors are taken into consideration, a more reasonable explanation would seem to be that the saturated
Ca2q concentration of CaCO3 does not change signi®cantly with the addition of 100 mM KCl. Little salt-out or
salt-in occurs in aqueous CaCO3 suspensions on addition
of 100 mM KCl.
Figure 3 shows the pH values of aqueous CaCO3
solutions and supernatants of its aqueous suspensions as
functions of the apparent CaCO3 concentration and time.
The pH value of pure deionized water saturated with
atmospheric CO2 is also shown as a function of time. The
pH values read by a pH meter change with time. The pH
value of an aqueous solution having only weak buffering
Fig. 2. Ca2q concentration of aqueous CaCO3 solutions and those of
the supernatant of aqueous suspensions, and their insigni®cant changes
on the addition of 100 mM KCl as a function of `apparent' CaCO3 concentration expressed as the molar CaCO3 concentration when all of the
CaCO3 added is assumed to dissolve. The solid line with open circles
indicates the Ca2q concentration of simple aqueous CaCO3 solutions
and those of supernatants of its aqueous suspensions, and the dashed
line with solid circles indicates the Ca2q concentration of aqueous
mixtures of CaCO3 with 100 mM KCl and those of supernatants of its
aqueous mixtures with 100 mM KCl, respectively. All measurements
were done at 25"0.5 8C.
Fig. 3. Example of the pH value of aqueous mixtures of CaCO3 with
100 mM KCl as functions of the concentration of CaCO3 and time.
Open circles and open squares indicate the pH values of aqueous
100 mM and 200 mM CaCO3 mixtures with 100 mM KCl, respectively.
Solid circles indicate the pH value of the supernatant of aqueous 700 mM
CaCO3 suspension in 100 mM KCl solution. Solid squares indicate the
pH value of pure deionized water saturated with the atmospheic CO2.
All measurements were done at 25"0.5 8C.
Table 2. Ca 2qand phosphate (Pi) releases from mature calci®ed (MCa-CW), mature uncalci®ed (MuCa-CW) and young uncalci®ed
(YuCa-CW) Chara cell walls in 100 mM KCl solution
Sample
MCa-CW
MuCa-CW
YuCa-CW
Pure deionized water
100 mM KCl solution
[Ca2q] (mM)
[Pi] (mM)
pH
[Ca2q] (mM)
[Pi] (mM)
pH
0.28"0.03
0.05"0.01
0.01"0.01
0.15"0.00
0.01"0.00
0.01"0.01
7.49"0.07
6.77"0.06
6.16"0.09
0.84"0.06
0.59"0.04
0.41"0.03
0.22"0.01
0.00"0.00
0.00"0.00
7.89"0.20
6.72"0.08
5.49"0.18
Each sample involves four experiments, i.e. n ˆ 4.
Calcium and phosphate release from Chara cell walls
227
ability measured with a pH meter was found to change
with time, not maintaining a constant equilibrium value,
but displaying scattering around a value. Thus, it is
dif®cult to determine pH values precisely (Ozeki et al.,
1998). However, the pH values of simple aqueous CaCO3
solutions read with a pH meter equipped with a glass pH
electrode change greatly as a function of time, for reasons
not yet known.
What can be said is that the pH value of pure deionized water saturated with atmospheric CO2 is around 5.4,
i.e. weakly acidic, but that of aqueous CaCO3 solutions
and supernatants of its aqueous suspensions is weakly
alkaline at all concentrations examined. At below and
above 0.28 mM, the greater the amount of CaCO3 added,
the higher is the pH of the supernatant of the aqueous
mixtures (complete data not shown). This is curious
because the Ca2q concentration of the supernatant of
aqueous CaCO3 suspensions is almost constant above
0.28 mM (cf. Fig. 2).
Figure 4 shows the Ca2q concentrations of simple
aqueous CaHPO4 solutions and supernatants of its
suspensions, and those of mixtures with 100 mM KCl
as a function of the apparent CaHPO4 concentration.
Here, `apparent' CaHPO4 concentration is that when all
of the CaHPO4 added is assumed to dissolve in 1.00 dm3
pure deionized water or 100 mM KCl solution.
The saturation concentration of CaHPO4 is about
0.37 mM in water and 0.70 mM in 100 mM KCl solution (Fig. 4). Clearly, a greater molar concentration of
CaHPO4 can be dissolved in pure deionized water than
CaCO3 and the solubility of CaHPO4 can be increased
by the addition of 100 mM KCl. In other words, salt-in
occurs in aqueous CaHPO4 suspensions by the addition
of 100 mM KCl.
The pH values of aqueous CaHPO4 solutions are
shown in Fig. 5 as a function of time. Clearly, they
change less over time than those of CaCO3. The pH
values of aqueous CaHPO4 solutions and supernatants of
its suspensions are almost neutral. The pH values of
aqueous CaHPO4 solutions and suspensions seem to be
lower than those of aqueous CaCO3 solutions and its
suspensions, but are nearly equal to those of pure
deionized water and 100 mM KCl solution in which
isolated Chara cell walls have been immersed.
Phosphate is present in Chara cell walls. Since potassium
and sodium salts of phosphate are very soluble in water
and little Kq and Naq remain bound to Chara cell walls
during short-term immersion in pure deionized water
preceding 2 h immersion in pure deionized water and
100 mM KCl solution (cf. Kiyosawa and Adachi, 1990),
the phosphate can be regarded to exist in Chara cell walls
as calcium salts which are practically insoluble in water.
One of the calcium salts of phosphate is CaHPO4.
The solubility of CaHPO4 can be increased in water
by the addition of 100 mM KCl. However, the solubility
of CaCO3 does not change signi®cantly in water on
addition of 100 mM KCl. Ca2q release from isolated
Chara cell walls can be increased in 100 mM KCl solution
(Table 1). Pi release from isolated Chara cell walls can
also be increased in 100 mM KCl solution (Table 2).
However, when the balance between the positive charge
due to cations and the negative charge due to anions
based on the electroneutrality rule was examined, the
amount of Pi released from isolated calci®ed Chara
Fig. 4. Ca2q concentration of aqueous CaHPO4 solutions and supernatants of its suspensions, and its increase by addition of 100 mM KCl.
The solid line with open circles indicates the Ca2q concentration of
simple aqueous CaHPO4 solutions and supernatants of its aqueous
suspensions, and the dashed line with solid circles indicates the Ca2q
concentration of aqueous CaHPO4 mixtures with 100 mM KCl and
those of supernatants of its suspensions in 100 mM KCl solution,
respectively.
Fig. 5. Example of the pH value of aqueous mixtures of CaHPO4 with
100 mM KCl as functions of the concentration of CaHPO4 and time.
Open circles and open squares indicate the pH value of 100 mM and
200 mM CaHPO4 mixtures with 100 mM KCl, respectively. Solid circles
indicate the pH value of the supernatant of 700 mM CaHPO4 suspension
in 100 mM KCl solution. All measurements were done at 25"0.5 8C.
Discussion
228
Kiyosawa
cell walls together with the Ca2q release is not large
enough to neutralize the positive charge due to the Ca2q.
The amount of Pi released from calci®ed Chara cell
walls averages 0.15 mEq dm 3 if the Pi is H2PO4
and 0.30 mEq dm 3 if it is HPO24 in pure deionized
water. On the other hand, the amount of Ca2q simultaneously released averages 0.56 mEq dm 3 (Table 2).
Therefore, to neutralize the excess positive charge
due to the Ca2q, some other anions amounting to
0.26 ± 0.41 mEq dm 3 should also be solubilized from
calci®ed Chara cell walls in pure deionized water. One
of these anions should be CO23 . However, the pH
value of simple aqueous CaCO3 solutions and that of
the supernatant of its aqueous suspensions are weakly
alkaline (Fig. 3).
The pH value of 100 mM KCl solution in which
isolated young uncalci®ed Chara cell walls had been
immersed was weakly acidic (Table 2) while that of
pure deionized water in which the same young uncalci®ed Chara cell walls had been immersed was neutral
(Table 2), indicating that some acidic substances can be
released by 100 mM KCl solution (Gillet et al., 1989).
This result does not deny that such acidic substances are
released from young uncalci®ed Chara cell walls even in
pure deionized water and that such acidic substances are
released from mature uncalci®ed and calci®ed Chara cell
walls more or less in pure deionized water and 100 mM
KCl solution.
These acidic substances may make pure deionized
water and 100 mM KCl solution, in which isolated Chara
cell walls have been immersed, less alkaline than that
of simple aqueous CaCO3 solutions, even bringing the
pH to neutral. Thus, anions other than CO23 which
can neutralize excess positive charge due to Ca2q over
the negative charge due to Pi are probably organic acids
(Gillet et al., 1989, 1998).
The amount of Pi released from the same Chara
cell walls in 100 mM KCl is 0.22 mEq dm 3 or
0.44 mEq dm 3 depending on whether the Pi is H2PO4 or
HPO24 . On the other hand, the released Ca2q amounts
to 1.68 mEq dm 3 in 100 mM KCl solution. Thus, some
other anions amounting to 1.24±1.46 mEq dm 3 should
be released simultaneously. These anions also seem to be
organic acids (Gillet et al., 1989, 1998) and CO23 .
Numerical values of the charges due to Ca2q, Pi and
organic acids in ionic relations including ion exchange
(MeÂtraux and Taiz, 1977; Van Cutsem and Gillet, 1981;
Gillet et al., 1989; Reid and Smith, 1992) based on the
electroneutrality rule differ among experiments using
Chara cell walls which have different amounts of calcium
bands and are of different ages (Table 2). Reid and Smith
have already shown that the amount of calcium in Chara
cell walls varies depending on the age of the cell and the
culture conditions (Reid and Smith, 1992). The content of
phosphate in calcium bands on Chara cell walls is also
thought to have much to do with its concentration in
culture media.
The pH value alone cannot show how much CO23
has been released and how much Pi has been released
from isolated Chara cell walls during the Ca2q release
in pure deionized water and 100 mM KCl solution,
respectively. Also, it is usually not very easy to quantify
the concentration of CO23 .
The pH value of pure deionized water is weakly
acidic under atmospheric conditions because CO2 in air
dissolves into it. The pH values of aqueous CaHPO4
solutions and the supernatant of its aqueous suspensions
are approximately neutral. CaHPO4 probably dissociates
to make OH in water as follows:
CaHPO4 | Ca2q qHPO24
(2)
HPO24 qH2 O |H2 PO4 qOH
(3)
CaCO3 seems to dissociate in water to make it neutral or
weakly alkaline as follows (Borowitzka, 1987; cf. Ogata,
1983):
CaCO3 | Ca2q qCO23
(4)
CO23 qH2 O | HCO3 qOH
(5)
HCO3 qHq |H2 CO3 |H2 OqCO2
(6)
Judging from the results of the present ®ndings, the
so-called calcium bands anduor ionic relations in Chara
cell walls are not chemically or physicochemically simple
(Katz, 1973; Borowitzka, 1982, 1987). This suggests that
chemical and physiological processes around and in the
cell membrane from which calcium bands are formed
inuon Chara cell walls under strong light should be more
or less modi®ed from those proposed thus far. However,
this does not necessarily deny the hypothesis that calcium bands would be formed by the simultaneous deposition of calcium and anions such as CO23 inuon the cell
wall due to localized pH changes through transmembrane transport of Hq and HCO3 (Lucas, 1975; Lucas
and Nuccitelli, 1980; Borowitzka, 1987; Fisahn et al.,
1989).
It is worth noting that Smith has suggested that the
mechanism of the formation of calcium bands on the cell
wall is not physiologically so simple (Smith, 1985a, b).
The present study seems to suggest one direction for
further studies on the mechanism of the formation of
calcium bands on Chara cell walls.
Acknowledgement
This work was supported in part by a Grant-in-Aid
(No. 10640632) for Scienti®c Research from the Ministry of
Education, Science and Culture of Japan.
Calcium and phosphate release from Chara cell walls
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