Plant Cell Physiol. 43(9): 1027–1035 (2002)
JSPP © 2002
Temporal Relationship between Cytosolic Free Ca2+ and Membrane Potential
during Hypotonic Turgor Regulation in a Brackish Water Charophyte
Lamprothamnium succinctum
Yoshiji Okazaki 1, 3, Mitsuo Ishigami 2 and Naohiko Iwasaki 1
1
2
Department of Biology, Osaka Medical College, Takatsuki, Osaka, 569-8686 Japan
Department of Biology, Faculty of Education, Shiga University, Otsu, Shiga, 520-0862 Japan
Internodal cells of a brackish water charophyte, Lamprothamnium succinctum, regulate turgor pressure in
response to changes in external osmotic pressure by modifying vacuolar concentrations of KCl. An increase in
cytosolic concentration of free Ca2+ ([Ca2+]c) is necessary
for the progress of turgor regulation induced by hypotonic
treatment. Initial changes in membrane potential and
[Ca2+]c upon hypotonic treatment were measured to examine the temporal relationship between the two parameters.
Fura-dextran (potassium salt, Mr 10,000, anionic) that had
been injected into the cytosol was used to measure [Ca2+]c.
Membrane potential and membrane conductance under a
current-clamp condition were also measured. Decrease in
external osmotic pressure by 0.16 Osm induced a simultaneous increase in [Ca2+]c with both depolarization of the
membrane and increase in the membrane conductance.
Decrease in external osmotic pressure by 0.05 Osm induced
a simultaneous increase in [Ca2+]c with membrane depolarization but the increase in membrane conductance started
later than the other two processes. There was a close temporal relationship between the increase in [Ca2+]c and membrane depolarization on the initial response of turgor regulation induced by hypotonic treatment.
Keywords: Cytoplasmic streaming — Cytosolic Ca2+ — Lamprothamnium — Membrane conductance — Membrane potential — Turgor regulation.
Abbreviations: ASW, artificial sea water; [Ca2+]c, cytosolic concentration of free Ca2+; [Ca2+]e, external concentration of Ca2+; FD,
fura-dextran; EGTA, O,O¢-bis(2-aminoethyl) ethyleneglycol-N,N,N¢,N¢tetraacetic acid; PIPES, piperazine-N-N¢-bis(2-methanesulfonic acid).
Introduction
Changes in the environmental water potential increase the
cytosolic concentration of free Ca2+ ([Ca2+]c) in plant cells as
other environmental abiotic stimuli do (recent reviews: Knight
2000, Plieth 2001). The increase in [Ca2+]c has been visualized
using a bioluminescent protein, aequorin or fluorescent dyes. A
decrease in the environmental water potential (hypertonic treat3
;
ment, drought and salinity stresses) induced an increase in
[Ca2+]c in maize root protoplasts (Lynch et al. 1989), in internodal cells of the freshwater charophyte, Nitellopsis obtusa
(Okazaki et al. 1996), in seedlings of Arabidopsis thaliana
(Knight et al. 1997, Knight et al. 1998) and in cyanobacterium
Anabena sp. (Torrecilla et al. 2001). These increases in [Ca2+]c
are considered to be an early event mediating the signal of a
decrease in environmental water potential to the cells.
On the other hand, an increase in environmental water
potential (hypotonic treatment) also induced an increase in
[Ca2+]c in internodal cells of a brackish water charophyte, Lamprothamnium succinctum (Okazaki et al. 1987), in Fucus rhizoid cells (Taylor et al. 1996), in tobacco suspension-culture
cells (Takahashi et al. 1997) and in roots of A. thaliana (Plieth
2001). Cytosolic hydration caused an increase in [Ca2+]c in
internodal cells of a freshwater charophyte (Tazawa et al. 1995,
Shimada et al. 1996). The physiological significance of the
increase of [Ca2+]c has been well established in osmoregulation
(turgor regulation) of algal cells in response to hypotonic treatment (Okazaki et al. 1987, Taylor et al. 1996).
Marine and brackish water algae with cell walls have
a mechanism of regulating their turgor pressures against
fluctuating environmental osmotic pressures (salinities) by
modifying their cellular osmotic pressures (recent reviews:
Findlay 2001, other references therein). The mechanism of the
turgor regulation upon hypotonic treatment has been studied
using salt-tolerant species of Characeae, Lamprothamnium
papulosum (Bisson and Kirst 1980), L. succinctum (Okazaki et
al. 1984) and Chara longifolia (Hoffmann and Bisson 1986).
Membrane depolarization, an increase in membrane conductance and an inhibition of cytoplasmic streaming are common
early events in the hypotonic turgor regulation in these species
(Okazaki et al. 1984, Reid et al. 1984, Okazaki and Tazawa
1986b, Hoffmann and Bisson 1990, Bisson et al. 1995, Beilby
and Shepherd 1996, Shepherd and Beilby 1999).
Calcium ion is involved in the turgor regulation upon
hypotonic treatment in these species. Internodal cells of L.
succinctum and C. longifolia regulated turgor pressure under a
normal external concentration of Ca2+ ([Ca2+]e) (4–5 mM), but
they did not under a low [Ca2+]e (0.01 mM) (Okazaki and
Tazawa 1986a, Bisson et al. 1995). Direct measurement of
[Ca2+]c with Ca2+-sensitive photoprotein, aequorin in L.
Corresponding author: E-mail, [email protected]; Fax: +81-726-84-7049.
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1028
Ca2+ and membrane potential
Fig. 1 A typical example of the changes in [Ca2+]c (closed squares)
and membrane potential (lines) in response to a decrease in external
osmotic pressure by 0.16 Osm. Internodal cells were transferred from
0.36 Osm ASW to 0.21 Osm ASW at time zero. Initial changes are
shown in expanded time scales in (B).
succinctum revealed that hypotonic treatment induced an
increase in [Ca2+]c under a normal [Ca2+]e but not under a low
[Ca2+]e (Okazaki et al. 1987). A calcium channel blocker,
nifedipine, inhibited turgor regulation (Okazaki and Tazawa
1986c, Stento et al. 2000), the membrane depolarization, the
increase in membrane conductance and the inhibition of cytoplasmic streaming (Stento et al. 2000). 45Ca2+ influx during
hypotonic treatment was also inhibited by nifedipine (Stento et
al. 2000). These results suggest a regulatory role of Ca2+ in the
hypotonic turgor regulation.
We focused our interest on initial cellular responses
immediately after completion of water influx induced by hypotonic treatment. Because simultaneous measurements of turgor
pressure, membrane potential, membrane conductance and rate
of cytoplasmic streaming in C. longifolia clearly showed that
the first event was an increase in turgor pressure, suggesting
that the change in turgor pressure may induce changes in other
parameters (Stento et al. 2000). In the present study, we
measured [Ca2+]c, membrane potential and rate of cytoplasmic
streaming using a cell into which fura-dextran (FD) had been
injected before hypotonic treatment. Simultaneous measurements of [Ca2+]c and membrane potential enabled us to see
the temporal relationship between [Ca2+]c and ion transport
across the plasmalemma because membrane potential sensitively responds to ion (charge) movements. We also compared
Fig. 2 Changes in membrane potential (closed circles), membrane
conductance (open circles) (A) and [Ca2+]c (open triangles) (B) in
response to a decrease of external osmotic pressure by 0.16 Osm.
Internodal cells were transferred from 0.36 Osm ASW to 0.21 Osm
ASW at time zero. Values of membrane conductance are shown as a
percentage of each value to the maximal value in each experiment.
Seven cells were used in (A) and 6 cells were used in (B). Bars represent ± SEM.
the changes in [Ca2+]c with the changes in membrane conductance and membrane potential through simultaneous measurements of the membrane potential and membrane conductance.
Measurements were performed under two different hypotonic
stresses since the magnitude of the hypotonic stress affected the
progress of turgor regulation (Stento et al. 2000).
Results
Steady state
The cellular osmotic pressure of intact cells preincubated
in 0.36 Osm artificial sea water (ASW) was 0.67±0.01 Osm
(number of cells = 15) and that directly after the injection of
FD was 0.67±0.01 Osm (9). The FD-injected cells were incubated for several h in 0.52 Osm ASW under dim light. The
osmotic pressure immediately before measurements of [Ca2+]c
was 0.68±0.01 Osm (9). The streaming rates of intact cells and
FD-injected cells (directly before the measurements) were
36±1 mm s–1 (20) and 23±2 mm s–1 (16), respectively. The
membrane potential and [Ca2+]c directly before hypotonic treatments were –152±5 mV (16) and 80±10 nM (16), respectively.
Ca2+ and membrane potential
1029
Fig. 4 A typical example of changes in [Ca2+]c (closed squares) and
membrane potential (lines) in response to a decrease in external
osmotic pressure by 0.05 Osm in narrower (A) and expanded time
scales (B). Internodal cells were transferred from 0.36 Osm ASW to
0.31 ASW at time zero.
Fig. 3 Relationships among [Ca2+]c, membrane potential and membrane conductance during turgor regulation in response to hypotonic
treatment. Cells were transferred from 0.36 Osm ASW to 0.21 Osm
ASW. (A) and (B) Relationship between membrane potential (A) and
membrane conductance (B) to [Ca2+]c. (C) Relationship between membrane potential and membrane conductance.
Thus the injection of FD had little effect on the cellular physiological conditions at a steady state except for reducing the
streaming rate by 40%.
Decrease in external osmotic pressure by 0.16 Osm
When internodal cells were transferred from 0.36 Osm
ASW to 0.21 Osm ASW, the cellular osmotic pressure of FDinjected cells decreased by 0.12±0.02 Osm (6) at the steady
state, while that of intact internodal cells decreased by
0.11±0.01 Osm (5). Thus injection of FD did not affect the
capacity of turgor regulation upon hypotonic treatment.
Fig. 1 shows a typical example of simultaneous measurements of [Ca2+]c (closed squares) and the membrane potential
(lines). The [Ca2+]c started to increase with a time-lag of ca.
40 s and reached a peak value ca. 100 s after the start of the
hypotonic treatment. The increase in [Ca2+]c started at the same
time as the membrane depolarization (Fig. 1B).
Fig. 2A shows changes in the average membrane potential (closed circles) and membrane conductance (open circles).
Membrane conductance immediately before hypotonic treatment and that at the peak (96 s) were 0.3±0.1 S m–2 (7) and
8.0±5.0 S m–2 (7), respectively. Since the values of membrane
conductance after hypotonic treatment showed such a large
deviation, they were normalized against maximal values in each
measurement. The membrane potential started to change at the
same time as the increase in the membrane conductance (ca.
50 s). The membrane conductance reached a peak value when
the membrane potential approached the maximal depolarized
state. Fig. 2B shows the changes in average [Ca2+]c. The [Ca2+]c
started to increase with a time-lag of ca. 50 s and reached the
peak value (460±110 nM (6)) 120 s after hypotonic treatment.
It is clear that the membrane conductance started to increase
simultaneously with the increase in [Ca2+]c and membrane
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Ca2+ and membrane potential
Fig. 5 Changes in membrane potential (closed circles), membrane
conductance (open circles) (A) and [Ca2+]c (open triangles) (B) in
response to a decrease in external osmotic pressure by 0.05 Osm.
Internodal cells were transferred from 0.36 Osm ASW to 0.31 Osm
ASW at time zero. Seven cells were used in (A) and 6 cells were used
in (B). Bars represent ± SEM.
depolarization. The membrane conductance attained a peak
value (96 s) before [Ca2+]c reached a peak value (120 s).
Fig. 3 shows the relationships between [Ca2+]c, the membrane potential and the membrane conductance. The membrane became depolarized in parallel to the increase in [Ca2+]c
at lower levels (100–200 nM) and became independent at a
higher [Ca2+]c (200–500 nM) (Fig. 3A). The increase in the
membrane conductance also paralleled the increase in [Ca2+]c
at lower levels (100–300 nM) and was maintained at a higher
[Ca2+]c (300–500 nM) (Fig. 3B). Membrane conductance increased as the membrane depolarization progressed from –150
to –90 mV (Fig. 3C).
Decrease in external osmotic pressure by 0.05 Osm
When internodal cells were transferred from 0.36 Osm
ASW to 0.31 Osm ASW, the cellular osmotic pressure in intact
cells and FD-injected cells decreased by 0.03±0.01 Osm (10)
and 0.05±0.01 Osm (5), respectively. These values amounted to
30 and 40% of the values obtained by hypotonic treatment with
0.21 Osm ASW for intact and FD-injected cells. The percentage corresponded approximately to the strength of hypotonic
treatment, since 0.05 Osm was 30% of 0.16 Osm.
Fig. 6 Relationships among [Ca2+]c, membrane potential and membrane conductance during turgor regulation in response to hypotonic
treatment. Cells were transferred from 0.36 Osm ASW to 0.31 Osm
ASW. (A) and (B) Relationship of membrane potential (A) and membrane conductance (B) to [Ca2+]c. (C) Relationship between membrane
potential and membrane conductance.
Fig. 4 shows a typical example of simultaneous measurements of [Ca2+]c (closed squares) and membrane potential
(lines). The [Ca2+]c started to increase with a time-lag of ca.
100 s and reached a peak value of ca. 250 s after the start of the
hypotonic treatment. At the time when [Ca2+]c started to
increase, the membrane began to depolarize without showing
any time separation between the two parameters (Fig. 4B) as
shown in Fig. 1B.
Fig. 5A shows the changes in the average membrane
potential (closed circles) and conductance (open circles). Values of membrane conductance were not normalized unlike that
in Fig. 2A because maximal values were not always measured
in this experiment focusing on the initial changes of the mem-
Ca2+ and membrane potential
Fig. 7 A typical example of changes in [Ca2+]c (closed circles) and
streaming rate (open circles) in response to a decrease in external
osmotic pressure by 0.16 Osm in narrower (A) and expanded (B) time
scales. The cell used was the same one as that used in Fig. 1.
brane conductance. Compared with Fig. 2A, the start of membrane depolarization was slow (90 s vs. 50 s) and the rate of
membrane depolarization at the initial phase was one-tenth
(0.2 mV s–1 vs. 2.0 mV s–1). The membrane started to depolarize (90 s) earlier than the membrane conductance started to
increase (150 s). Fig. 5B shows the changes in average [Ca2+]c.
The increase in [Ca2+]c started with a time-lag of ca. 75 s and
reached a peak value (190±60 nM (7)) of 180 s after the hypotonic treatment. The [Ca2+]c at the peak (190 nM) was 40% of
that (460 nM) under the stronger hypotonic stress (Fig. 2B).
The time-lag for the start of increase in [Ca2+]c (75 s vs. 50 s)
and time needed for reaching the peak value (180 s vs. 120 s)
were both prolonged by 50%. The timing of the increase in
[Ca2+]c and that for the start of membrane depolarization were
simultaneous as in Fig. 4B but the increase in [Ca2+]c and the
membrane depolarization preceded the increase in membrane
conductance.
Fig. 6 shows the relationships between [Ca2+]c, the membrane potential and the membrane conductance. The membrane
depolarization paralleled the increase in [Ca2+]c to 190 nM
(Fig. 6A) like that under the strong hypotonic stress (Fig. 3A),
but the membrane conductance did not change in parallel to the
increase in [Ca2+]c in the range below 150 nM (Fig. 6B). The
membrane conductance started to increase when the membrane potential became more positive than –125 mV (Fig. 6C).
1031
Fig. 8 A typical example of changes in [Ca2+]c (closed circles) and
streaming rate (open circles) in response to a decrease in external
osmotic pressure by 0.05 Osm in narrow (A) and expanded (B) time
scales. The used cell was the same one as that used in Fig. 4.
Fig. 9 Relationship between [Ca2+]c and streaming rate. The values
up to inhibition of the cytoplasmic streaming in both hypotonic treatments were used.
Streaming rate and [Ca2+]c
Fig. 7 shows a typical example of simultaneous measurements of [Ca2+]c and the streaming rate in the same cell as that
shown in Fig. 1 where the external osmotic pressure was
decreased by 0.16 Osm. Clearly the increase in [Ca2+]c pre-
Ca2+ and membrane potential
1032
ceded the decrease in the streaming rate (Fig. 7B). The start of
the membrane depolarization also preceded inhibition of the
cytoplasmic streaming (Fig. 1, 7).
Fig. 8 shows a typical example of simultaneous measurements of [Ca2+]c and the streaming rate in the same cell as that
shown in Fig. 4 where the external osmotic pressure was
decreased by 0.05 Osm. Clearly the increase in [Ca2+]c preceded the decrease in the streaming rate and the extent of the
inhibition was not as large as that under the stronger stress
(Fig. 7). The start of membrane depolarization also preceded
the inhibition of cytoplasmic streaming (Fig. 4, 8). Fig. 9
shows the relationship between [Ca2+]c and the streaming rate.
Cytoplasmic streaming was not inhibited at [Ca2+]c below
200 nM, partly inhibited at 200–400 nM and strongly inhibited
at 500 nM or more.
Discussion
[Ca2+]c in Lamprothamnium
Values of [Ca2+]c at the steady state and during hypotonic
turgor regulation could be quantified in internodal cells of L.
succinctum with a fluorescent Ca2+ indicator, FD. In previous
studies using a Ca2+-sensitive photoprotein aequorin, [Ca2+]c
could not be quantified (Okazaki et al. 1987). The [Ca2+]c at the
steady state (80 nM) were a little lower than that reported in
internodal cells of freshwater Characeae (200 nM; Plieth and
Hansen 1996). The increase in [Ca2+]c measured with FD (Fig.
1, 2B) was identical to the previous results measured with an
aequorin light emission under the same hypotonic stress; a
time-lag of ca. 60 s and attainment of peak values within 120–
240 s (Okazaki et al. 1987).
[Ca2+]c and cytoplasmic streaming
The present study clarified the in vivo relationship
between [Ca2+]c and the streaming rate in L. succinctum (Fig.
7–9). Lack of inhibition at a low [Ca2+]c (less than 200 nM) and
inhibition at high [Ca2+]c (800–1,000 nM) coincide well with
the data of plasmalemma-permeabilized cells of Nitella axilliformis (Tominaga et al. 1983) and do not coincide with data of
L. succinctum using tonoplast-free cells (incomplete inhibition
at more than 10 mM) (Okazaki and Tazawa 1986b). The cytoplasmic streaming in tonoplast-free cells of freshwater charophytes was known to be less sensitive to [Ca2+]c than that in
plasmalemma-permeabilized cells (Tominaga et al. 1983).
Streaming rate as a limited indicator of [Ca2+]c
In L. succinctum and C. longifolia, membrane depolarization was induced by hypotonic treatment even under a low
[Ca2+]e (0.01 mM) in intact cells whose cytoplasmic streaming
was not inhibited (Okazaki and Tazawa 1986a, Okazaki and
Tazawa 1986b, Bisson et al. 1995). Furthermore membrane
depolarization progressed slowly under a normal [Ca2+]e
(3.9 mM) when EGTA-injected cells of L. succinctum were
subjected to hypotonic treatment and inhibition of the cytoplas-
mic streaming was retarded (Okazaki and Iwasaki 1991). The
lack of inhibition of cytoplasmic streaming during membrane
depolarization suggests that the membrane depolarization was
induced under no increase in [Ca2+]c. However, Fig. 1, 4, 7 and
8 showed that the membrane depolarization and the increase in
[Ca2+]c preceded the inhibition of cytoplasmic streaming. Judging from the present results, a small increase in [Ca2+]c that did
not inhibit cytoplasmic streaming may have been caused in the
previous studies. The hypothesis of Ca2+-independent membrane depolarization should be reexamined by measuring
[Ca2+]c under the previous experimental conditions.
Initial response of hypotonic turgor regulation and [Ca2+]c
Simultaneous measurements of the membrane potential
and [Ca2+]c under different hypotonic stresses showed that the
initial step of membrane depolarization was very closely
related to the increase in [Ca2+]c. The start of membrane depolarization coincided with that of an increase in [Ca2+]c under a
stronger (0.16 Osm) hypotonic treatment (Fig. 1, 2) as well as
under a weaker (0.05 Osm) hypotonic treatment (Fig. 4, 5). The
close temporal relationship between the initial changes in the
membrane potential and increases in [Ca2+]c supports the idea
that an increase in influx of Ca2+ through the plasmalemma
Ca2+-permeable channels plays a role in the initial membrane
depolarization.
The initial net influxes of Ca2+ were roughly estimated to
be 30 pmol m–2 s–1 (0.16 Osm stress) and 6 pmol m–2 s–1
(0.05 Osm stress) based on the initial rates of changes in
[Ca2+]c when the cellular diameter and the cytoplasmic volume
ratio were assumed to be 320 mm and 7% (Okazaki et al. 1984),
respectively. The estimated net Ca2+-influxes were far less than
enhanced Ca2+-influx measured by 45Ca2+ in Chara longifolia
upon hypotonic treatment (18 nmol m–2 s–1; Bisson et al. 1995).
The values of net Ca2+-influx based on the rate of changes in
[Ca2+]c might be underestimated because the cytosolic capacity
for sequestering Ca2+ (Plieth and Hansen 1996) was not considered in the estimation. However, such a small influx of Ca2+
(positive charge) would contribute to the initial membrane
depolarization.
Inhibition of 45Ca2+-influx with a Ca2+ channel blocker,
nifedipine suggested the involvement of the plasmalemma
Ca2+-permeable channels in the increase in [Ca2+]c induced by
hypotonic treatment (Stento et al. 2000). Open probabilities of
the plasmalemma Ca2+-permeable channels may depend on the
magnitude of deviation of the turgor pressure from a steadystate value. The slower increase in [Ca2+]c and depression of its
peak value (40%) caused by reducing the hypotonic stress from
0.16 Osm to 0.05 Osm suggest that the influx of Ca2+ is
dependent on the magnitude of the hypotonic stress.
Stretch-activated ion channels (Beilby and Shepherd 1996,
Shepherd and Beilby 1999) and stress-induced depolarizing ion
channels (Bisson et al. 1995, Stento et al. 2000) have been proposed as being involved in triggering hypotonic turgor regulation in salt-tolerant charophytes. Stretch-activated Ca2+-
Ca2+ and membrane potential
permeable channels were suggested as being involved in Ca2+mediated hypotonic turgor regulation of Fucus rhizoid (Taylor
et al. 1996) and in rhizoid differentiation of terminal cells of
Spirogyra (Inoue et al. 2002). The membrane depolarization
with a time-lag of 50–90 s (Fig. 1, 4) suggests that the influx of
positive charges across the plasmalemma with the same timelag. If Ca2+-permeable channels are involved in causing influx
of Ca2+ in L. succinctum, the time-lag of 50–75 s for the start of
[Ca2+]c increase should represent the time needed for the Ca2+permeable channels to be activated. The increase in the turgor
pressure upon hypotonic treatment may proceed so slowly that
its effect of triggering Ca2+-permeable channels may become
evident only after a time-lag of 50–90 s. However, it was
shown that an increase in turgor pressure due to hypotonic
treatment had already ended before the membrane depolarization had started (Stento et al. 2000).
Under a weaker hypotonic condition (0.05 Osm), the
membrane conductance began to increase after both the membrane depolarization and an increase in [Ca2+]c started (Fig. 5,
6). A small influx of Ca2+ may cause membrane depolarization
and may not be detected as an increase in membrane conductance using the present method. The following increase in membrane conductance may be caused when net efflux of KCl
started. In the salt-tolerant charophyte, L. papulosum, Beilby
and Shepherd (1996) reported that, based on voltage-clamp
experiments using La3+ as a Ca2+ channel blocker, Ca2+dependent anion channels are involved in the initial increase in
membrane conductance upon hypotonic treatment. Under a
stronger hypotonic condition (0.16 Osm), membrane conductance started to increase together with the membrane depolarization and an increase in [Ca2+]c (Fig. 3). Under this condition,
the rapid increase in [Ca2+]c may cause simultaneous changes
in both membrane potential and membrane conductance.
In a patch-clamp study using a freshwater charophyte,
Chara corallina, maximal open time of Ca2+-dependent anion
channels of plasmalemma was observed when the concentration of Ca2+ on the cytoplasmic side was about 1 mM and the
clamped-membrane potential was about –80 to –100 mV
(Okihara et al. 1991). The relationship between [Ca2+]c, the
membrane conductance and the membrane potential in L. succinctum (Fig. 3, 6) can be interpreted as follows based on their
results. (1) Independence of membrane conductance at less
than 150 nM of [Ca2+]c (Fig. 6B) is explained by a low activity
of Ca2+-dependent anion channels caused by both the hyperpolarized state of membrane potential and the insensitivity to a
low [Ca2+]c. (2) When both [Ca2+]c and membrane potential
exceed the threshold values, Ca2+-dependent anion channels
begin to open and membrane conductance starts to increase.
Activation of the anion channels contributes to acceleration of
the membrane depolarization. In order to confirm this hypothesis, Ca2+- and voltage-dependency of Ca2+-dependent anion
channels need to be studied by a patch-clamp method using this
material.
In order to clarify the turgor-sensing mechanism in Lam-
1033
prothamnium in response to hypotonic treatment, further studies are necessary on the mechanisms of depolarization of the
plasmalemma and activation of Ca2+-permeable channels.
Materials and Methods
Plant materials
A brackish water charophyte, Lamprothamnium succinctum (A.
Br. in Ash.) R. D. W. was originally collected from a pond on Teba
Island on the east coast of Shikoku Island (Tokushima, Japan). It was
cultured in glass water tanks with soil and leaf mold at the bottom
under 15-h illumination per day with fluorescent lamps (about 5´10–
5
mol m–2 s–1). The culture medium was about one-third diluted modified Herbst ASW that contained 171 NaCl, 4 KCl, 4 CaCl2, 18 MgSO4,
and 20 NaHCO3 each in mM (Herbst 1904). The osmotic pressure was
0.39 Osm. The temperature of the culture room was kept at 25±1°C.
Young internodal cells near the shoot apex were isolated from
neighboring internodal cells with scissors and ligated at both cell ends
with polyester threads. The obtained cell fragments were about 3 cm in
length and about 320 mm in diameter. The ligated internodal cells were
incubated under continuous light (3´10–5 mol m–2 s–1) for more than
2 d in 0.36 Osm ASW that contained 171 NaCl, 3.6 KCl. 3.9 CaCl2,
18.3 MgSO4 and 5 HEPES each in mM. The osmotic pressure was
0.36 Osm and the pH was adjusted to 7.5 with NaOH. The internodal
cells used for measurements of [Ca2+]c were incubated in the same
medium under 12-h illumination a day (about 1.5´10–6 mol m–2 s–1) of
fluorescent lamps at 20±1.5°C for more than 2 d.
Osmotic pressures of ASWs were modified by changing a concentration of NaCl in 0.36 Osm ASW from 171 to 257 mM
(0.52 Osm), to 143 mM (0.31 Osm) and to 86 mM (0.21 Osm), respectively.
Measuring the osmotic pressure
The osmotic pressure of external medium was measured with a
vapor pressure osmometer (Type 5500; Wescor Inc., UT, U.S.A.). The
cellular osmotic pressure at steady state was measured with the turgor
balance method (Tazawa 1957).
Measuring the [Ca2+]c
The ratiometric method using FD (potassium salt, Mr 10,000,
anionic; Molecular Probes, Inc., Eugene, OR, U.S.A.) was employed
to measure [Ca2+]c (Grynkiewicz et al. 1985). The injection medium
for measuring [Ca2+]c contained 0.625 FD, 0.5 ATP, 1 MgCl2, 200
sorbitol and 10 piperazine-N-N¢-bis(2-methanesulfonic acid)] (PIPES)
each in mM. The medium pH was adjusted to 7.0 with KOH. The
method for microinjection was the same as previously reported (Okazaki and Iwasaki 1991). After measurement of cellular osmotic pressure, the internodal cell was set on an inverted microscope and the turgor pressure was reduced to ca. 0.1 MPa with increasing external
osmotic pressure of ASW by adding NaCl. Internodal cells that
showed active cytoplasmic streaming were chosen as materials. The
injection medium was sucked into the tip of an injection pipette
together with silicon oil and was injected into the cytosol with a
microinjector (IM-4A; Narishige Scientific Instrument Lab., Tokyo,
Japan). The volume of injection medium was about 4 nl. After measurement of the cellular osmotic pressure, FD-injected internodal cells
were incubated for several h under dim light at room temperature in
0.52 Osm ASW. Then they were transferred to 0.36 Osm ASW before
experiments.
After the third measurement of the cellular osmotic pressure, the
FD-injected internodal cell was fixed on a chamber whose bottom was
made of a quartz glass slide and the chamber was set on the stage of a
Ca2+ and membrane potential
1034
fluorescence microscope (OSP10-CA; Olympus Optical Co., Ltd.,
Tokyo, Japan). Red light passing through a glass filter (600 nm,
6 mmol m–2 s–1) was used as the background light. For measuring
[Ca2+]c, cytosolic FD was excited with 340 nm and 380 nm light alternatively and both intensities of emission light at 530 nm were measured by a photomultiplier. The interval time of sampling was 500 ms
and data were recorded on a hard disk.
Values for the ratio of intensities of emission light excited by
340 nm and 380 nm light (E340/E380) were converted into concentrations of free Ca2+ using Ca2+ buffers (Calcium Calibration Buffer Kit
#2 C3009; Molecular Probes, Inc.). Calibration was performed for
Ca2+ buffers containing 20 mM of FD with the concentration of free
Ca2+ of 0, 17, 38, 65, 100, 150, 225, 351, 602, 1,350 and 39,000 nM.
Calcium buffers containing no Mg2+ were used because cytosolic concentration of free Mg2+ was not known in this material.
Electrophysiology
The membrane potential was always measured with either [Ca2+]c
or membrane conductance. The membrane potential was measured
with a conventional microelectrode method using Ag/AgCl half-cells.
The tip of the reference electrode that was composed of polyethylene
tubing contained a 100 mM KCl solidified with 2% agar. The potential difference between the vacuole and the external medium was
measured with a microelectrode amplifier (MEZ-7200; Nihon Kohden
Co., Ltd., Tokyo, Japan) and recorded with a memory recorder (Memory Hicorder 8840; Hioki Co., Ltd., Nagano, Japan).
To measure the membrane conductance, pulses of constant current (duration 1 s, interval 3 s, intensity 5´10–3 A m–2 on the average)
were applied through a current-supplying glass microelectrode in
which a platinum wire was bathed in 3 M KCl. The membrane conductance was calculated from intensities of inward current pulses and
recorded responses of the membrane potential, both recorded with a
pen-writing recorder (VP6228A; Panasonic Co., Ltd., Kanagawa,
Japan).
Measuring the rate of cytoplasmic streaming
Since the rate of cytoplasmic streaming was shown to reflect
[Ca2+]c in this material (Okazaki and Tazawa 1986b), the rate of cytoplasmic streaming was simultaneously measured with [Ca2+]c and the
membrane potential. Cytoplasmic streaming was recorded on videotape and its rate was calculated on the replayed screen following
movements of fastest granules.
All data are shown as mean values ± SEM, with the number of
cells in parentheses. All measurements were performed at 20–25°C.
Acknowledgments
We thank Dr. A. Sakai (Osaka Medical College) for helping us to
establish the experimental setups and thank Dr. K. Azuma, Mr. K. Asai
(Osaka Medical College) and Dr. M. Tazawa (Fukui University Technology) for fruitful discussion throughout experiments. We are also
grateful to Dr. Y. Tominaga (Heian Jogakuin St. Agnes’ University)
and to Dr. M. Tazawa for critical reading of the manuscript
References
Beilby, M.J. and Shepherd, V.A. (1996) Turgor regulation in Lamprothamnium
papulosum. I. I/V analysis and pharmacological dissection of the hypotonic
effect. Plant Cell Environ. 19: 837–847.
Bisson, M.A., Kiegle, K., Black, D., Kiyosawa, K. and Gerber, N. (1995) The
role of calcium in turgor regulation in Chara longifolia. Plant Cell Environ.
18: 129–137.
Bisson, M.A. and Kirst, G.O. (1980) Lamprothamnium, a euryhaline charo-
phyte. I. Osmotic relations and membrane potential at steady state. J. Exp.
Bot. 31: 1223–1235.
Findlay, G.P. (2001) Membranes and the electrophysiology of turgor regulation.
Aust. J. Plant Physiol. 28: 617–634.
Grynkiewicz, G., Poenie, M. and Tsien, R.Y. (1985) A new generation of Ca2+
indicators with greatly improved fluorescent properties. J. Biol. Chem. 260:
3440–3450.
Herbst, C. (1904) Über die zur Entwickelung der Seeigellarven nothwendigen
anorganishen Stoffe, ihre Rolle und ihre Vertretbarkeit. III. Theil. Die Rolle
der nothwendigen anorganischen Stoffe. Archiv für Entwicklungsmechnik der
Organismen 17: 306–520.
Hoffmann, R. and Bisson, M.A. (1986) Chara buckellii, a euryhaline charophyte
from an unusual saline environment. I. Osmotic relations at steady state. Can.
J. Bot. 64: 1599–1605.
Hoffmann, R. and Bisson, M.A. (1990) Chara buckellii, a euryhaline charophyte
from an unusual saline environment. III. Time course of turgor regulation.
Plant Physiol. 93: 122–127.
Inoue, N., Yamada, S., Nagata, Y. and Shimmen, T. (2002) Rhizoid differentiation in Spirogyra: position sensing by terminal cells. Plant Cell Physiol. 43:
479–483.
Knight, H. (2000) Calcium signaling during abiotic stress in plants. Int. Rev.
Cytol. 195: 269–324.
Knight, H., Brandt, S. and Knight, M.R. (1998) A history of stress alters drought
calcium signalling pathways in Arabidopsis. Plant J. 16: 681–687.
Knight, H., Trewavas, A.J. and Knight, M.R. (1997) Calcium signaling in Arabidopsis thaliana responding to drought and salinity. Plant J. 12: 1067–1078.
Lynch, J., Polito, V.S. and Läuchli, A. (1989) Salinity stress increases cytoplasmic Ca activity in maize root protoplasts. Plant Physiol. 90: 1271–1274.
Okazaki, Y. and Iwasaki, N. (1991) Injection of a Ca2+-chelating agent into the
cytoplasm retards the progress of turgor regulation upon hypotonic treatment
in the alga, Lamprothamnium. Plant Cell Physiol. 32: 185–194.
Okazaki, Y., Kikuyama, M., Hiramoto, Y. and Iwasaki, N. (1996) Short-term
regulation of cytosolic Ca2+, cytosolic pH and vacuolar pH under NaCl stress
in the charophyte alga Nitellopsis obtusa. Plant Cell Environ. 19: 569–576.
Okazaki, Y., Shimmen, T. and Tazawa, M. (1984) Turgor regulation in a brackish charophyte, Lamprothamnium succinctum. II. Changes in K+, Na+ and Cl–
concentrations, membrane potential and membrane resistance during turgor
regulation. Plant Cell Physiol. 25: 573–581.
Okazaki, Y. and Tazawa, M. (1986a) Involvement of calcium ion in turgor regulation upon hypotonic treatment in Lamprothamnium succinctum. Plant Cell
Environ. 9: 185–190.
Okazaki, Y. and Tazawa, M. (1986b) Effect of calcium ion on cytoplasmic
streaming during turgor regulation in a brackish water charophyte Lamprothamnium. Plant Cell Environ. 9: 491–494.
Okazaki, Y. and Tazawa, M. (1986c) Ca2+ antagonist nifedipine inhibits turgor
regulation upon hypotonic treatment in internodal cells of Lamprothamnium.
Protoplasma 134: 65–66.
Okazaki, Y., Yoshimoto, Y., Hiramoto, Y. and Tazawa, M. (1987) Turgor regulation and cytoplasmic free Ca2+ in the alga Lamprothamnium. Protoplasma
140: 67–71.
Okihara, K., Ohkawa, T., Tsutsui, I. and Kasai, M. (1991) A Ca2+- and voltagedependent Cl– sensitive anion channel in the Chara plasmalemma: a patchclamp study. Plant Cell Physiol. 32: 593–601.
Plieth, C. (2001) Plant calcium signaling and monitoring: pros and cons and
recent experiments approaches. Protoplasma 218: 1–23.
Plieth, C. and Hansen, U.-P. (1996) Methodological aspects of pressure loading
of Fura-2 into characean cells. J. Exp. Bot. 47: 1601–1612.
Reid, R.J., Jefferies, R.L. and Pitman, M.G. (1984) Lamprothamnium, a euryhaline charophyte. IV. Membrane potential, ionic fluxes and metabolic activity
during turgor adjustment. J. Exp. Bot. 35: 925–937.
Shepherd, V.A and Beilby, M.J. (1999) The effect of an extracellular mucilage
on the response to osmotic shock in the charophyte alga Lamprothamnium
papulosum. J. Membr. Biol. 170: 229–242.
Shimada, K., Kikuyama, M. and Tazawa, M. (1996) The hydration-induced Ca2+
release is a general phenomenon in Characeae. Plant Cell Physiol. 37: 922–
928.
Stento, N.A., Gerber Ryba, N., Kiegle, E.A. and Bisson, M.A. (2000) Turgor
regulation in the salt-tolerant alga Chara longifolia. Plant Cell Environ. 23:
629–637.
Takahashi, K., Isobe, M., Knight, M.R., Trewavas, A.J. and Muto, S. (1997)
Ca2+ and membrane potential
Hypoosmotic shock induces increases in cytosolic Ca2+ in tobacco suspension-culture cells. Plant Physiol. 113: 587–594.
Taylor, A.R., Manison, N.F.H., Fernandez, C., Wood, J. and Brownlee, C.
(1996) Spatial organization of calcium signaling involved in cell volume control in the Fucus rhizoid. Plant Cell 8: 2015–2931.
Tazawa, M. (1957) Neue Methode zur Messung des osmotischen Wertes einer
Zelle. Protoplasma 48: 342–359.
Tazawa, M., Shimada, K. and Kikuyama, M. (1995) Cytoplasmic hydration triggers a transient increase in cytoplasmic Ca2+ concentration in Nitella flexilis.
1035
Plant Cell Physiol. 36: 335–340.
Tominaga, Y., Shimmen, T. and Tazawa, M. (1983) Control of cytoplasmic
streaming by extracellular Ca2+ in permeabilized Nitella cells. Protoplasma
116: 75–77.
Torrecilla, I., Leganés, F., Bonilla, I. and Fernández-Piñas, F. (2001) Calcium
transients in response to salinity and osmotic stress in the nitrogen-fixing
cyanobacterium Anabaena sp. PCC7120, expressing cytosolic apoaequrin.
Plant Cell Environ. 24: 641–648.
(Received May 2, 2002; Accepted July 9, 2002)