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Plant, Cell and Environment (2008) 31, 1575–1591
doi: 10.1111/j.1365-3040.2008.01866.x
Mechano-perception in Chara cells: the influence of
salinity and calcium on touch-activated receptor
potentials, action potentials and ion transport
VIRGINIA A. SHEPHERD1, MARY J. BEILBY1, SABAH A.S. AL KHAZAALY1 & TERUO SHIMMEN2
1
Department of Biophysics, School of Physics, The University of New South Wales, Sydney, New South Wales 2052, Australia
and 2Department of Life Science, Graduate School of Life Science, University of Hyogo, Harima Science City, Hyogo
678-1297, Japan
ABSTRACT
This paper investigates the impact of increased salinity on
touch-induced receptor and action potentials of Chara
internodal cells. We resolved underlying changes in ion
transport by current/voltage analysis. In a saline medium
with a low Ca2+ ion concentration [(Ca2+)ext], the cell background conductance significantly increased and proton
pump currents declined to negligible levels, depolarizing
the membrane potential difference (PD) to the excitation
threshold [action potential (AP)threshold]. The onset of spontaneous repetitive action potentials further depolarized the
PD, activating K+ outward rectifying (KOR) channels. K+
efflux was then sustained and irrevocable, and cells were
desensitized to touch. However, when [Ca2+]ext was high, the
background conductance increased to a lesser extent and
proton pump currents were stimulated, establishing a PD
narrowly negative to APthreshold. Cells did not spontaneously
fire, but became hypersensitive to touch. Even slight touch
stimulus induced an action potential and further repetitive
firing. The duration of each excitation was extended when
[Ca2+]ext was low. Cell viability was prolonged in the absence
of touch stimulus. Chara cells eventually depolarize and die
in the saline media, but touch-stimulated and spontaneous
excitation accelerates the process in a Ca2+-dependent
manner. Our results have broad implications for understanding the interactions between mechano-perception and
salinity stress in plants.
Key-words: excitation; plant mechano-perception; proton
pump; salt stress.
INTRODUCTION
The sometimes-dramatic responses of plants to touch have
intrigued biologists for over a century. A voluminous literature is devoted to the well-known thigmonastic behaviours
of Mimosa, the Venus’s Flytrap Dionaea, the bladderwort
Aldrovanda (Sibaoka 1991; Shimmen 2001) and the triggerplant Stylidium (Hill & Findlay 1981; Findlay 1984). In
Correspondence: V. A. Shepherd. Fax: +61 2 9385 6060; e-mail:
[email protected]
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd
recent years, it has become clear that the capacity for perceiving and responding to mechanical stimuli is also fundamental to subtle plant behaviours including responses to
gravity, temperature, environmental osmolarity, and turgorcontrolled growth and development (reviewed by Fasano,
Massa & Gilroy 2002; Jaffe, Leopold & Staples 2002;
Baluska et al. 2003; Telewski 2006; Haswell 2007; Pickard
2007).
It is thought that mechano-perception in plant cells commences with membrane deformation, whether this occurs
directly, or indirectly, via transmembrane protein tethers
coupled to the cytoskeleton and the cell wall (reviewed by
Fasano et al. 2002). Specifically, force perception translates
into the activity of mechano-sensory Ca2+ ion channels,
modulated in a multitude of ways accounting for multiple
types of response (Pickard 2007). Mechano-perception
rests upon Ca2+-dependent second messenger systems, and
cytoplasmic Ca2+ is elevated following different kinds of
mechanical stimulus (Haley et al. 1995). In Trewavas’ model
(Trewavas 1999a,b), each kind of stimulus elicits a unique
Ca2+ signal with distinct topology.
The electrophysiology of mechano-perception is difficult
to study in small cells of complex tissues. Giant internodal
cells of the Charales have been widely used to research
mechano-perception at the cellular level.
Embryophytes (land plants) are often viewed as being
synonymous with the entire plant kingdom. Phylogenetically, the Charales are the extant sister group to all embryophytes, with which they share some fundamental
biochemical and physiological processes (McCourt, Delwiche & Karol 2004). These processes include aspects of
excitability, membrane transport (Tazawa & Shimmen
2001) and mechano-perception (Shimmen 2001).
In giant cells of the Charales, mechanical stimuli producing compressive or decompressive mechanical stress on the
cell wall deform (Iwabuchi, Kaneko & Kikuyama 2005) or
alter the tension (Iwabuchi, Kaneko & Kikuyama 2008) of
the cell membrane, activating mechano-sensory ion channels. These channels transduce the touch stimulus into an
electrical signal or receptor potential (Shimmen 1996,
1997a,b,c). The receptor potential is a small depolarization brought about by mechano-sensory Ca2+ channels at
1575
1576 V. A. Shepherd et al.
the plasma membrane. The Ca2+ released stimulates Ca2+activated Cl- channels (Kaneko et al. 2005), producing a Clefflux (Shimmen 1996, 1997a,b,c). The receptor potential
increases in amplitude and/or duration more or less incrementally with increasing magnitude of stimulus (Kishimoto
1968; Shimmen 1996; Kaneko et al. 2005) until a critical
threshold voltage initiates an action potential (reviewed by
Beilby 2007). In contrast to the action potential, the receptor potential does not propagate intercellularly.
An action potential involves a significant elevation of
cytoplasmic Ca2+ from 200 to 700 nm (Plieth & Hansen
1996). The elevated Ca2+ activates Cl- efflux via Ca2+activated Cl- channels, further depolarizing the cell and
activating outwardly directed voltage-dependent K+ channels (reviewed, Beilby 2007). Under normal conditions, the
action potential has a characteristic threshold and form, and
propagates from cell to cell (Beilby 2007). The result is an
efflux of Cl- and K+ (Oda 1976; Beilby 1984, 2007; Wayne
1994), water efflux and transient turgor reduction (Zimmermann & Beckers 1978), and cell contraction (Oda & Linstead 1975). The motif of elevated cytoplasmic Ca2+, efflux
of Cl- and K+, and turgor loss and contractility, is elaborated
into turgor-based osmotic machinery that enables a wide
repertoire of mechano-responses in land plants (Hill &
Findlay 1981).
We established that cell turgor pressure modulates
mechano-perception in Chara internodal cells (Shepherd,
Shimmen & Beilby 2001; Shepherd, Beilby & Shimmen
2002). The critical receptor potential, which triggers an
action potential, occurs with weaker stimulus when turgor
pressure is decreased (Shepherd et al. 2001, 2002). Only
the receptor potential is affected. The voltage threshold for
initiating an action potential is unchanged (Staves & Wayne
1993; Shepherd et al. 2001). As discussed earlier, the receptor potential is the electrophysiological manifestation of
mechano-perception. Furthermore, it is a change in turgor
pressure, rather than its absolute magnitude, that elicits
sensitization to mechanical stimulus (Shepherd et al. 2001).
The ability to regulate cell volume and/or turgor pressure
is fundamental to the lives of cells. Action potentials probably evolved from osmo-regulatory processes of ancient
bacteria inhabiting environments of variable salinity (Gradmann & Mummert 1980). The responses of charophyte cells
to changes in environmental osmolarity and salinity involve
mechano-perception (Shepherd et al. 2002). This may
extend to other plant cells. For example, Arabidopsis leaf
cells respond to hypotonic or hypertonic media with Ca2+
transients, mediated by stretch-activated (or mechanosensory) channels (Hayashi et al. 2006), and increased
salinity also elevates cytoplasmic Ca2+ in Arabidopsis cells
(Knight, Trewavas & Knight 1997).
Considering that Chara cells become increasingly sensitive to mechanical stimulation as turgor decreases (Shepherd et al. 2001), it is probable that mechano-perception
plays a role in their responses to increased environmental
salinity. Broadly speaking, an increase in environmental
salinity can impact on plant cells in two ways: firstly, through
osmotic reduction of cell turgor pressure, essentially a
mechanical stimulus, and secondly, through the increased
influx of Na+, which may then compete with and displace K+
from carboxylate groups of cell proteins such as actin.These
impacts are coupled in salt-sensitive Chara cells. When
turgor pressure is reduced in the presence of NaCl, the
magnitude of Na+ influx increases (Davenport, Reid &
Smith 1996).
In this paper, we set out to compare and contrast the
effects of turgor pressure reduction and elevated environmental salinity on mechano-perception in Chara cells.
Because elevated extracellular Ca2+ ion concentrations
reduce the negative impact of high salinity in a wide range
of plant cells (reviewed by Cramer 2002), including Chara
(Hoffmann, Tufariello & Bisson 1989; Whittington & Smith
1992), we conducted our experiments in saline media
including either high or low Ca2+ concentrations. Elevated
extracellular Ca2+ diminishes both Na+ influx via nonselective cation channels (NSCCs), and Na+-induced K+
efflux through outward rectifier channels in Arabidopsis
(Shabala et al. 2006). We employed the powerful techniques
of current–voltage (I/V) analysis and mathematical modelling (Beilby & Walker 1996) to identify changes in ion transporters induced both by turgor pressure reduction and by
saline media.
Plant physiological experiments frequently focus on a
single environmental factor, such as salinity, or mechanical
stimulus, when both factors are present in the natural world.
Giant charophyte cells are unique experimental materials
that enable investigation of the electrophysiological
phenomena associated with mechano-perception under
conditions of high salinity. Cells of embryophytes and
charophytes respond to mechanical stimulus with a similar
electrophysiological modus of receptor and action potentials (Shimmen 2001). Our results may have broad implications for understanding the interactions between
mechano-perception and salinity stress in plant cells.
MATERIALS AND METHODS
Cell preparation
Chara corallina was cultured in Japan as described previously (Mimura & Shimmen 1994). Chara australis Brown
(Garcia & Chivas 2006) was collected from Little Bay,
Sydney, New South Wales, Australia, and was planted in
aquaria containing a handful of autoclaved garden soil, a
handful of rotting leaves and rainwater. C. australis was
cultured under equal numbers of Sylvania Gro-Lux fluorescent tubes (Sylvania Australasia Pty. Ltd., Lisarow,
New South Wales, Australia) and cool white fluorescent
tubes, providing a photosynthetically active radiation of
80 mmol m-2 s-1, on a cycle of 10 h light and 14 h darkness.
The C. corallina internodal cells used in mechanostimulus experiments were between 45 and 53 mm long.
Cells were cut from vigorous plants and were kept in artificial pond water (APW; Table 1a) in a growth cabinet for at
least a week before experiments. Experiments were conducted at room temperature (28 °C).
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1575–1591
Mechano-perception in Chara cells 1577
Chemical constituents
Solution acronym
KCl (mm)
NaCl (mm)
CaCl2 (mm)
(a)
APW
180sorb
100 KCl
100 Na/1.0 Ca
100 Na/0.1 Ca
0.1
0.1
100.0
0.1
0.1
–
100.0
100.0
(b)
APW
90sorb
50 Na/1.0 Ca
50 Na/0.1 Ca
0.1
0.1
0.1
0.1
1.0
1.0
50.0
50.0
0.1
0.1
1.0
0.1
(c)
APW
180sorb
100 Na/10 Ca
100 Na/1.0 Ca
100 Na/0.1 Ca
0.1
0.1
0.1
0.1
0.1
1.0
1.0
100.0
100.0
100.0
0.1
0.1
10.0
1.0
0.1
1.0
1.0
0.1
0.1
–
1.0
0.1
Sorbitol (mm)
Table 1. Chemical composition and
acronyms for media used in (a)
mechano-perception experiments,
(b) voltage-clamp experiments and (c)
cell viability tests
–
180.0
–
–
–
–
90.0
–
–
–
180.0
–
–
–
The pH of all the solutions was 7.0, maintained by the addition of 5.0 mm HEPES-Tris.
APW, artificial pond water; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulforic acid.
The C. australis internodal cells used in voltage-clamp
experiments were about 10–15 mm in length. Sub-apical
internodal cells were cut from healthy plants and were left
to recover in APW for at least 3 d. Experiments were conducted at room temperature (26 °C). C. australis internodal
cells were also used in cell viability tests.
Experimental protocols
We used three experimental protocols: (1) mechanostimulation, (2) I/V measurements and (3) cell viability tests.
Mechano-stimulation experiments
We measured and compared responses with mechanostimulus in the media listed in Table 1a.
Figure 1 shows the experimental protocols. Firstly,
internodal cells were placed into a Perspex chamber (inhouse), with two compartments (A and B) connected by a
groove in the Perspex. The compartments A and B were
electrically isolated using petroleum jelly. Both compartments initially contained APW, in which cells remained for
at least 30 min.
Mechano-stimulus dislodges impaled microelectrodes
and damages the cells, and we used extracellular electrodes
to record difference potentials (Shimmen 1996). A difference potential is the difference in electrical potential
between the two electrically isolated ends A and B of the
cell. The membrane potential difference (PD) at each end
of the cell is similar in APW, and hence EA - EB is initially
close to zero.
The electrical PD between compartments A and B
(EA - EB) was measured using two 3 m KCl-agar electrodes
(EA and EB) connected to Ag/AgCl wire and inserted into
compartments A and B, respectively. Signals from the electrodes were amplified using a differential microelectrode
amplifier (MEZ7101; Nihon Kohden,Tokyo, Japan), and the
signal EA - EB (the difference potential) was recorded using
a chart recorder (VP-6521A; National, Tokyo, Japan) with
variable speed and sensitivity.
We used the K+ anesthesia technique to measure cell PDs
(Shimmen, Kikuyama & Tazawa 1976). The 180sorb solution was introduced into both compartments to reduce
turgor pressure (Fig. 1a). The 180sorb in compartment B
was then exchanged for an isotonic solution of 100 KCl
(Fig. 1b). Because the PD is close to zero in 100 KCl,
EA - EB gives the membrane PD of the part of the cell in
compartment A.
Cells were mechanically stimulated using the device
constructed by Shimmen (1996). Briefly, a small polyacrylate stimulator (dimensions, 5 ¥ 8 ¥ 1 mm) was suspended
adjacent to the cell flank in compartment A. The stimulator was impacted by a thin glass rod (weight, 1.28 g)
released from increasing heights (0.5–6 cm). The device
delivers a series of reproducible mechanical stimuli (f0.5,
f1, f2, f3 and f4) of increasing energy (see Shepherd et al.
2001). Stimulations were applied until an AP was induced.
We avoided summation of the receptor potentials (Kishimoto 1968) by leaving 4–5 min between stimulations.
Cells were allowed to recover for at least an hour following an AP.
In separate experiments, we mechanically stimulated
cells in 100 Na/1.0 Ca and in 100 Na/0.1 Ca (Fig. 1c). The
180sorb in compartment A was exchanged for either 100
Na/1.0 Ca or 100 Na/0.1 Ca, and the cell was left for 60 min
to stabilize Ca2+/Na+ exchange in the cell wall.
In one group of experiments, we compared mechanoresponses of the same cells in 100 Na/1.0 Ca and in 100
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1575–1591
1578 V. A. Shepherd et al.
(a)
Stimulator
180sorb
180sorb
Reduction of cell turgor
pressure
A
EA – EB
B
Difference PD
(b)
100KCl
180sorb
+
K anesthesia. 100KCl
introduced to chamber B for
measuring cell PD in A
A
EA – E B
B
(PD in A)
(c)
100KCl
100Na/0.1Ca OR
100Na/1.0Ca
100Na/0.1Ca OR
100Na/1.0Ca introduced to
chamber A for ~45 min
mechano-stimulus
A
E A – EB
B
(d)
100KCl
100Na/0.1Ca
Cells surviving mechanostimulus in 100Na/1.0Ca
transferred into100Na/0.1Ca:
further mechano-stimulus
A
EA – EB
B
Na/0.1 Ca. Cells stimulated in 100 Na/1.0 Ca were left to
recover for 60 min, and the 100 Na/1.0 Ca was exchanged
for 100 Na/0.1 Ca. Cells were mechanically stimulated
30–45 min later (Fig. 1d).
We recorded the cell resting membrane PDs, the peak
amplitudes of receptor potentials, the occurrence of touchinduced action potentials, and PD oscillations.
In a control experiment, we subjected a group of cells to
the protocol of repetitive solution changes and mechanostimulus, using only APW. As found previously (Shepherd
et al. 2001), these procedures had only slight effect on the
mechano-responses.
Figure 1. Diagrammatic representation
of the protocols for mechano-sensing
experiments. (a) Cell placed in
two-compartment chamber. Both
chambers filled with 180sorb solution
to reduce cell turgor pressure. (b) The
100 KCl solution introduced into
compartment B eliminates the membrane
PD in this region, enabling measurement
of cell PD. (c) Either 100 Na/1.0 Ca or
100 Na/0.1 Ca is introduced into chamber
A. After 45 min. cells are mechanically
stimulated. (d) Cells that survived
mechano-stimulus in 100 Na/1.0 Ca now
exposed to 100 Na/0.1 Ca, so that
responses can be com pared. PD,
potential difference.
The I/V measurements
Cells did not survive voltage-clamping protocols in 100 mm
NaCl solutions, regardless of the Ca2+ concentration. We
reduced the NaCl concentration to 50 mm, containing
0.1 mm Ca2+ or 1.0 mm Ca2+. This increased the activity of
Ca2+ from 0.58 (in 100 mm NaCl) to 0.65 in 50 mm NaCl
(Butler 1968). We reduced cell turgor pressure using isotonic 90sorb. Table 1b lists the chemical composition of the
media.
The voltage-clamping procedure was as previously
described (Beilby & Shepherd 1996). Cells were
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1575–1591
Mechano-perception in Chara cells 1579
compartment-clamped, with a microelectrode located in the
cytoplasm or vacuole. The plasma membrane I/V characteristics dominate because of the high tonoplast conductance
(Beilby 1990). We measured I/V characteristics using a
bipolar staircase voltage command, with pulses of width
between 60 and 100 ms, separated by 120–250 ms, at the
resting PD.
The order and time course of solution changes mimicked
those in mechano-stimulus experiments. We first obtained
I/V characteristics of cells in APW and in 90sorb. We then
measured I/V characteristics of cells transferred into either
50 Na/1.0 Ca or 50 Na/0.1 Ca. We also compared I/V characteristics of the same cells in 50 Na/1.0 Ca and 50 Na/0.1
Ca.
PDs in these media were not significantly different. As
reported previously (Shepherd et al. 2001), cells were sensitized to mechano-stimulus when turgor pressure was
reduced. In 180sorb, cells responded to given stimuli with
larger amplitude receptor potentials than in APW. Stimuli
between f1 and f4 (modal value f4) induced action potentials in APW. Stimuli between f0.5 and f3 (modal value f0.5)
induced action potentials in 180sorb.
In both APW and 180sorb, the amplitude and duration of
receptor potentials increased incrementally with increased
stimulus energy, until an action potential was induced
(Fig. 2a,b; Table 3a,b).
Mechano-responses in 100 Na/1.0 Ca
Modelling and analysis of I/V data
We resolved the total current into contributions made by
parallel populations of ion transporters. We modelled the
electrogenic proton pump at the plasma membrane using
the two-state Hansen, Gradmann, Sanders and Slayman
(HGSS) model (Hansen et al. 1981). We fitted the inward
and outward rectifiers using the Goldmann, Hodgkin and
Katz (GHK) model, supplemented by the Boltzmann distribution (Amtmann & Sanders 1999). We used an empirical model of the ‘background current’, with a reversal PD
of -100 mV and a PD-independent conductance (Beilby &
Shepherd 2006a; Al Khazaaly & Beilby 2007). Channels
passing the ‘background current’ are the probable equivalent of NSCCs (non-selective cation permeable channels)
found in land plants (Beilby & Shepherd 2001).
Cell viability tests
We assessed cell viability from the rate of cytoplasmic
streaming in control cells, where mechano-stimulus was
avoided. Cells cut from healthy plants were left in APW for
1 week. Ten cells were transferred into each of the media
(Table 1c). The media were replaced every second day, and
care was taken to avoid mechano-stimulus. We estimated
the rate of cytoplasmic streaming as the time taken for
cytoplasmic particles to traverse a 32¥ field of view. The
number of viable cells was counted daily over 7 d. Dead
cells were removed.
RESULTS
Figure 2 shows excerpts from chart records of cell PDs,
and cell mechano-responses, in 180sorb, 100 Na/1.0 Ca and
100 Na/0.1 Ca. Table 1 shows the composition of the media
and explains the acronyms used for each. Table 2 shows
the mean resting membrane PDs of cells in these media.
Table 3a,b summarizes mechano-responses of cells in all
experiments.
Mechano-responses in APW and 180sorb
The hyperpolarized PDs of cells in both APW and 180sorb
(Table 2) were characteristic of the ‘pump state’. Resting
Cells began depolarizing immediately in isotonic 100 Na/
1.0 Ca, reaching a mean PD of -123 ⫾ 21 mV after 60 min
(Tables 2 and 3a; Fig. 2c), which was significantly different
to that in APW (P0 = 0.00). Small magnitude PD oscillations
observed in APW and 180sorb increased in amplitude and
period in all cells (Fig. 2c). These oscillations are not clearly
visible at the scale of reproduction of the figure. We will
describe such oscillations in detail in a forthcoming paper.
Most cells (3, 4, 6 and 7 in Table 3a) became more
sensitive to mechano-stimulus in 100 Na/1.0 Ca (Fig. 2c,
Table 3a). Cells 1 and 5 did not decrease their sensitivity to
stimulus. However, the limited resolution of the apparatus
did not enable us to determine if the sensitivity of these
cells had increased. Because 100 Na/1.0 Ca and 180sorb
were isotonic media, the increased sensitivity of most cells
results from increased NaCl concentration. Cell 2 alone
decreased its sensitivity to mechano-stimulus.
Initially, all cells recovered the resting PD following the
touch-induced action potential. However, all cells (with the
exception of 7) then began to depolarize after 45–60 s
(Fig. 2d). When the cell PD approached a mean value of
-90 ⫾ 23 mV (n = 6, limits: -60 to -113 mV), the cells
entered a series of spontaneous repetitive action potentials
(Fig. 2d). The duration and form of these action potentials
was unique to each cell and repeated over a period of up to
60 min. At their conclusion, cells gradually hyperpolarized
to a mean PD of -118.2 ⫾ 9.5 mV (Table 2). This PD
remained stable for a further 60 min. Cell 7 alone did not
depolarize further from its post-action potential PD of
-135 mV, neither did it begin repetitive firing.
Mechano-responses of the same cells in
100 Na/0.1 Ca
After 60 min in 100 Na/0.1 Ca, cells depolarized further to
-82 ⫾ 43 mV (Tables 2 and 3a; Fig. 2e). The amplitudes of
small-scale resting PD oscillations increased to between 5
and 10 mV (Fig. 2e) with occasional spikes of approximately 25 mV (not shown). We distinguished three types of
electrophysiological behaviour prior to mechano-stimulus
(Table 3a): (1) cells 1 and 7 fired a single spontaneous action
potential. Cell 1 subsequently remained much depolarized.
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1575–1591
1580 V. A. Shepherd et al.
Cell 7 recovered its PD for a few seconds, then depolarized
to zero PD and ceased streaming, and died; (2) cells 2 and 3
spontaneously entered repetitive action potentials, and then
depolarized significantly; and (3) cells 4, 5 and 6 retained a
similar PD to that in 100 Na/1.0 Ca (Fig. 2e).
Initiation of spontaneous action potentials was a markedly different behaviour. Spontaneous action potentials
had only followed touch-induced action potentials in
100 Na/1.0 Ca.
Responses to mechano-stimulus differed from those in
100 Na/1.0 Ca. Most cells (1, 2, 3 and 6 in Table 3a)
decreased their sensitivity to mechano-stimulus and
responded to given stimuli with smaller amplitude receptor
potentials. Of these, only cells 2 and 6, with PDs of -81 and
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1575–1591
Mechano-perception in Chara cells 1581
Figure 2. Responses to mechanical stimulation of a Chara cell in 180sorb, 100 Na/1.0 Ca and 100 Na/0.1 Ca. The composition of the
media is shown in Table 1. Results (a–f) are from the same cell throughout. Vertical bars indicate where the chart recorder speed was
slowed, and the time scale is in minutes. (a) Cell difference potentials (see Fig. 1a), with turgor pressure reduced in 180sorb. The cell
responds to increasing mechanical stimuli f1 and f2 with receptor potentials; small, transient depolarizations. Stimulus f3 induces an action
potential (AP). The asterisk (*) symbol marks the threshold receptor potential that initiates the AP. The AP appears biphasic because a
touch-induced AP occurred in both ends of the cell. Note that the difference potential does not necessarily show the true amplitude of
the response. (b) Cell membrane potential differences (PDs), measured by K+ anesthesia, with turgor pressure reduced in 180sorb (see
Fig. 1b). The resting membrane PD was -225 mV. As described previously, the cell responds to mechanical stimuli f1 and f2 with receptor
potentials of increasing amplitude but similar duration (9 mV and 35 s for f1, and 18 mV and 35 s for f2), and stimulus f3 induces an AP.
The touch-induced AP appears monophasic because cell part B (see Fig. 1b) has zero PD. The cell recovers its resting PD following the
touch-induced AP. (c) Cell membrane PD in isotonic 100 Na/1.0 Ca. The y-axis is located 20 min after introducing 100 Na/1.0 Ca. The cell
began depolarizing immediately (not shown), reaching a stable PD of -137 mV after 45 min. Small-scale PD oscillations (within vertical
bars) were measured with the chart speed set to a time scale of minutes. These oscillations are not clearly visible at the scale of
reproduction of the figure. We will describe them in detail in a forthcoming paper. Oscillation amplitudes were between 2 and 5 mV, and
their duration, approximately 1 min. These amplitudes and periods were larger than small-scale oscillations in 180sorb (not shown). The
cell increased its sensitivity to mechanical stimulus, in comparison with its responses in 180sorb. Stimulus f0.5 provoked a receptor
potential (7 mV amplitude: 15 s duration). Small-scale PD oscillations are visible during the 4 min recovery period (vertical bars).
Stimulus f1 provoked a receptor potential (15 mV amplitude: 60 s duration) and stimulus f2 induced an AP. (d) Following the
touch-induced AP in isotonic 100 Na/1.0 Ca, the cell briefly recovered the resting PD of -137 mV, and then, 60 s after the touch-induced
AP, began to depolarize. The first panel of the chart excerpt shows this depolarization. At a PD of -70 mV, the cell entered a series of
spontaneous repetitive APs (*). These were of short duration (approximately 6 s) and occurred at similar intervals (170.5, 170.5, 170.5,
162 and 195 s). Following repetitive firing, the cell gradually recovered a stable resting PD of -116 mV (not shown). The cell increased
its sensitivity to mechano-stimulus, the form of the AP changed, and spontaneous repetitive APs followed the touch-induced AP.
(e) Membrane PD of the same cell in isotonic 100 Na/0.1 Ca. The y-axis is situated 35 min after introducing 100 Na/0.1 Ca. This cell
retained a resting PD between -116 and -113 mV, as described previously. However, small-scale PD oscillations (arrow) increased in
amplitude (between 2 and 10 mV) and duration (approximately 2.5 min). Sensitivity to mechanical stimulus increased, and stimulus f0.5
provoked a receptor potential (12 mV amplitude: 30 s duration). Small-scale PD oscillations are visible (vertical bars). Stimulus f1 induced
an AP of extended duration (3 min 30 s). (f) Repetitive APs followed the touch-induced AP. The cell initially recovered the resting PD,
and then gradually depolarized (not shown), initiating repetitive APs at a PD of -26 mV. Compared with repetitive APs in 100 Na/1.0 Ca,
these APs had extended duration (approximately 20 s) and occurred at shorter intervals (26, 30, 37, 36, 33, 33 and 34 s). The APs ceased
when the cell PD hyperpolarized to -75 mV (not shown). Unusually, this cell recovered a stable PD of -107 mV. Following further
repetitive APs, the cell died within 60 min of mechano-stimulation. (g) Cell PD and mechano-responses of another cell, this time
transferred directly from 180sorb into 100 Na/0.1 Ca. The cell PD in 180sorb was -230 mV. The y-axis is situated 10 min after introducing
100 Na/0.1 Ca. After 10 min, the cell had depolarized to a PD of -107 mV. Repetitive APs (*) commenced after 20 min. The time scale of
the chart is in minutes rather than seconds, and the firing is not resolved as described previously. The PD stabilized at -22 mV following
spontaneous firing. The cell was desensitized to mechano-stimulus. Stimulus f1 provoked an extended depolarization, rather than a
receptor potential. The cell subsequently depolarized to 0 mV, and did not recover cytoplasmic streaming after 40 min.
-120 mV, respectively, fired action potentials. Much depolarized cells 1 and 3 (with PDs of -22 and -21 mV, respectively) did not fire. The effect of 100 Na/0.1 Ca on cell 5
was beyond the resolution of the apparatus, because the
lowest energy stimulus elicited action potentials (f0.5). Cell
7 died prior to stimulation. Cell 4 had not experienced
spontaneous firing in 100 Na/0.1 Ca, and this cell alone
increased its sensitivity (Fig. 2e).
Following the touch-induced action potential, all cells
depolarized to zero PD and permanently ceased streaming
within 60 min (Table 2), a markedly different fate from
that of cells in 100 Na/1.0 Ca, which recovered a mean
Table 2. Membrane PDs (mean(SD, n = number of cells) of cells used in mechano-stimulation experiments in APW, 180sorb, 100 Na/0.1
Ca and 100 Na/1.0 Ca
APW
180sorb
100 Na/0.1 Ca
100 Na/1.0 Ca
**100 Na/0.1 Ca
PD (mV)
-228 ⫾ 42 (n = 6)
PD (mV)
(a): -212 ⫾ 23 (n = 6)
(b): -217 ⫾ 16 (n = 7)
PD (mV)
Initial: -106 ⫾ 15 (n = 6)
RAP: -53 ⫾ 41 (n = 6)
noRAP: -121 (one cell)
Post-MS: 0 (n = 6)
PD (mV)
Initial: -123 ⫾ 21 (n = 7)
–
noRAP: -123 ⫾ 21 (n = 7)
Post-MS: -118 ⫾ 10 (n = 7)
PD (mV)
Initial: -82 ⫾ 43 (n = 7)
RAP: -41 ⫾ 34 (n = 3)
noRAP: -122 ⫾ 9 (n = 4)
Post-MS: 0 (n = 6)
The column **100 Na/0.1 Ca shows the PD of cells pre-treated in 100 Na/1.0 Ca. The PD in 180sorb of cells transferred into 100 Na/0.1 Ca
(a) and 100 Na/1.0 Ca (b) was similar. ‘Initial’ signifies the stable PD after 30 min in the solution. RAP signifies repetitive action potentials
(shown in Figures 2d,f,g), and the accompanying value is the mean PD after they ceased. noRAP indicates the absence of repetitive action
potentials. ‘Post-MS’ is the mean PD 60 min after mechano-stimulation in each solution. In column two, the two groups of cells (a) and (b)
were subsequently transferred into 100 Na/0.1 Ca or 100 Na/1.0 Ca.
PDs, potential differences; APW, artificial pond water; MS, mechano-stimulus.
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1575–1591
1582 V. A. Shepherd et al.
Table 3. (a) Mechano-responses of Chara cells with turgor reduced in 180sorb in isotonic 100 Na/1.0 Ca and of the same cells in isotonic
100 Na/0.1 Ca. (b) Mechano-responses of Chara cells with turgor reduced in 180sorb and of the same cells in isotonic 100 Na/0.1 Ca
(a)
180sorb
100 Na/1.0 Ca
Cell
Stable PD
(mV)
1
-222
f0.5: TAP
Stable PD: -220
-126
f0.5: TAP
SRAP
2
-226
f0.5: 32.6
f1: TAP
Stable PD: -230
-122
3
-186
-76
4
-230
5
-203
f0.5: 13
f1: 20.3
f2: TAP
Stable PD: -185
f0.5: 3.7
f1: 8.6
f2: 18
f3: TAP
Stable PD: -230
f0.5: TAP
Stable PD: -200
f0.5: 18
f1: 25
f2: TAP
SRAP
f0.5: 20
f1: 27
f2: TAP
SRAP
f0.5: 7
f1: 13
f2: TAP
SRAP
6
-230
7
-220
Receptor potential
amplitude (mV) at
stimulus
f0.5: 15.5
f1: 19.2
f2: TAP
Stable PD: -230
f0.5: 1
f1: 9.4
f2: 12
f3: TAP
Stable PD: -220
Stable PD
(mV)
-136
100 Na/0.1 Ca
Receptor potential
amplitude (mV) at
stimulus
Stable PD
(mV)
-22
-81
SRAP
f0.5: 4
f1: 15
Depolarization, death (<30 min)
-116
f0.5: 12
f1: TAP
SRAP
Depolarization, death (>60 min)
f0.5: TAP
SRAP
Depolarization, death (<60 min)
f0.5: 13
f1: 22
f2: TAP
Depolarization, death (<60 min)
*Spontaneous action potential
SRAP
Depolarization, death (<60 min)
f0.5: TAP
SRAP
-112
-130
f0.5: 36
f1: TAP
SRAP
-120
-135
f1: TAP
-100
180sorb
Cell
Stable PD
(mV)
Receptor potential
amplitude (mV)
Stable PD
(mV)
1
-170
f0.5: TAP
*Spontaneous single
action potential
-28
*Spontaneous action potential
f0.5: 0.8
f1: no response
Depolarization, death
f0.5: TAP
Depolarization, death (<30 min)
SRAP
-21
-136
(b)
Receptor potential
amplitude (mV) at
stimulus
100 Na/0.1 Ca
Stable PD: -154
2
-220
f0.5: TAP
Stable PD: -225
-121
3
-223
f0.5: TAP
Stable PD: -220
SRAP
-22
4
-230
f0.5: TAP
Stable PD: -230
SRAP
-80
5
-200
f0.5: 10
f1: 18
f2: 31
f3: TAP
Stable PD: -205
SRAP
-21
Receptor potential amplitude (mV)
f0.5: no response
f1: no response
f2: no response
f3: no response
f4: 12.3 mV
X no action potential
Depolarization, death
f0.5: TAP
SRAP
Depolarization, death
f0.5: no response
f1: 2
f3: X no action potential
Depolarization, death
f0.5: TAP
SRAP
Depolarization, death
f0.5: no response
f1: 11
X no action potential
Depolarization, death
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1575–1591
Mechano-perception in Chara cells 1583
Table 3. Continued
(b)
180sorb
100 Na/0.1 Ca
Cell
Stable PD
(mV)
Receptor potential
amplitude (mV)
Stable PD
(mV)
6
-230
f0.5: no response
f1: 8
f2: TAP
Stable PD: -230
SRAP
-112
Receptor potential amplitude (mV)
f0.5: 15
f1: TAP
SRAP
Depolarization, death (<24 h)
TAP, is the level of touch stimulus (f0.5 to f4) that activated an action potential. SRAP, indicates spontaneous repetitive action potentials.
Spontaneous repetitive action potentials appear in Fig. 2d,f,g.
‘Receptor potential amplitude’ is the amplitude in mV of the receptor potential following each level of touch stimulus, from f0.5 to f4. ‘Stable
potential difference (PD)’ (in 180sorb) is the PD recovered following a touch-induced action potential. ‘Depolarization, death’ indicates
depolarization to zero PD coupled with streaming cessation for 30 min or longer (see Fig. 2g). ‘X no action potential’ indicates where touch
stimulus did not initiate an action potential.
PD (-118 ⫾ 10 mV) and continued cytoplasmic streaming
despite having experienced spontaneous firing.
Interestingly, both touch-induced action potentials and
spontaneous repetitive action potentials were of shorter
duration in 100 Na/1.0 Ca than in 100 Na/0.1 Ca (compare
Fig. 2c,d with Fig. 2e,f).
Mechano-responses of cells transferred
directly from 180sorb into 100 Na/0.1 Ca
All cells initially depolarized by approximately 100 mV to a
mean PD of -106 ⫾ 15.1 mV, after transfer from 180sorb to
100 Na/0.1 Ca (Fig. 2g; Tables 2 and 3b). As discussed previously, the amplitudes of small-scale resting PD oscillations
also increased to between 5 and 10 mV with occasional
spikes of approximately 25 mV amplitude. The cells were
significantly depolarized (P0 = 0.00) in comparison with
their PD in APW and 180sorb (Table 2), but only slightly
depolarized in comparison with the PD of the first group
of cells, in 100 Na/1.0 Ca (P0 = 0.19). However, after
approximately 10–20 min in 100 Na/0.1 Ca, cells (except 2)
fired spontaneous repetitive action potentials (Table 3b,
Fig. 2g).
At the conclusion of repetitive firing, cells were further
depolarized to a mean PD of -53 ⫾ 41 mV (Table 2). Half
the cells were much depolarized (with PDs close to
-22 mV), and half had PDs ranging from -80 to -112 mV
(Table 3b). Cells were significantly more depolarized in 100
Na/0.1 Ca than in 100 Na/1.0 Ca (P0 = 0.01) because of
repetitive firing.
Cells responded to mechano-stimulus in 100 Na/0.1 Ca
with a different spectrum of responses compared with their
responses in 180sorb (Table 3b, Fig. 2g).
Cells 1, 3 and 5, which were much depolarized prior to
stimulation (Table 3b), became less sensitive to mechanostimulus. These produced smaller-amplitude receptor
potentials or became entirely unresponsive (Fig. 2g).
Mechano-stimulus was followed by gradual depolarization
to zero PD, cessation of cytoplasmic streaming, and death
within 60 min (Fig. 2g).
In contrast, cells 2 and 4 fired touch-induced action
potentials (Table 3b). The action potentials were followed
in both these cells by spontaneous repetitive firing, after
which cells depolarized to zero PD, ceased streaming, and
died.
Cell 6 alone became more sensitive to mechano-stimulus,
responding to given stimuli with receptor potentials of
larger amplitude and an action potential with a lower
energy stimulus than in 180sorb (Table 3b). This cell also
fired spontaneously and repetitively, depolarized, and died.
Thus, while all cells stimulated in 100 Na/1.0 Ca recovered a relatively hyperpolarized PD (-118.2 ⫾ 9.5 mV) and
survived for longer than 60 min post-stimulus, mechanostimulus in 100 Na/0.1 Ca was quickly lethal.
Current–voltage analysis and
mathematical modelling
Neither the resting PD nor the I/V characteristics
changed significantly when turgor pressure was reduced in
90sorb (Fig. 3a–d, data in APW in black, data in 90sorb in
blue).
The resting PD depolarized from -236 ⫾ 7 to
-184 ⫾ 20 mV, and the conductance increased approximately threefold (Table 4; Fig. 3a–d, data in green) after
60 min in 50 Na/1.0 Ca. Modelling identified classes of
transporters responsible. The background conductance
increased from 0.45 ⫾ 0.15 to 2.0 ⫾ 0.3 S·m-2. The rate constant parameters kio0 and koi of the electrogenic proton
pump both increased from 8000 ⫾ 2000 and 100 ⫾ 20 s-1
in APW to 12 000 ⫾ 3000 and 130 ⫾ 25 s-1, respectively
(parameter values in Table 4). This group of cells did not
survive voltage clamping to potentials more negative than
-280 mV, and so we could not investigate inward rectifiers.
The same cells, when transferred to 50 Na/0.1 Ca, depolarized further to -155 ⫾ 7 mV. The PD range, where
clamping was feasible, also narrowed (Fig. 3a–d, data in
red). Only two of the group of five cells survived voltage
clamping, and only for ~30 min. There was no further
change in the background conductance. Both of the rate
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1575–1591
1584 V. A. Shepherd et al.
Figure 3. Current–voltage (I/V) and conductance–voltage characteristics of cells in different media: artificial pond water (APW) (black;
n = 5); 90sorb (blue; n = 5; average time in medium, 96 min); 50 Na/1.0 Ca (green; n = 5; average time in medium, 65 min); 50 Na/0.1 Ca
(red; statistics from two surviving cells; time in medium, 20–30 min.). A separate group of four cells was transferred directly from 90sorb
to 50 Na/0.1 Ca (orange; average time in medium, 60 min). The data were collected into 15 or 20 mV bins (horizontal error bars).
Standard errors are shown as vertical bars. (a) The data and the fitted I/V characteristics. The model parameters are shown in Table 4. The
extreme fits through the error bars of 50 Na/1.0 Ca and 50 Na/0.1 Ca data are shown as dotted lines, giving the range of the fitted
parameters in Table 4. In the case of the four cells transferred directly from 90sorb to 50 Na/0.1 Ca, the extreme fitted lines were obtained
by the pump contribution to the upper limit and by changing the reversal potential difference (PD) for the background current from -100
to -60 mV for the lower limit. The APW and 90sorb data were processed in a similar fashion, and the resulting parameter ranges are
shown in Table 4, but the overlapping tolerance lines are not shown for clarity. (b) Fitted pump currents; (c) fitted background
conductances; (d) total conductances calculated as differentials of the total currents.
constant parameters kio0 and koi of the electrogenic
proton pump decreased to 8000 ⫾ 2500 and 60 ⫾ 25 s-1,
respectively.
A separate group of four cells was transferred directly
into 50 Na/0.1 Ca. Of these, only one showed a low level of
proton pump activity (Table 4) after 60 min. The averaged
data were modelled by the background conductance only
(Fig. 3a–d in orange). The background conductance
increased significantly to 3.2 ⫾ 0.2 S·m-2.
Viability and rates of cytoplasmic streaming
Figure 4 shows the viability of control cells that were not
mechanically stimulated in each of the media (Table 1c).
Cell viability depended on both the external concentration
of NaCl and Ca2+, but not on the extracellular osmotic
pressure: viability in 180sorb was slightly greater than in
APW. All cells had died within 5 d in 100 Na/0.1 Ca, while
half were still viable after 7 d in 100 Na/1.0 Ca. Viability
depended on extracellular [Ca2+]ext: all cells survived for
7 d in modified APW including 10 mm Ca2+ (not shown),
whereas only 70% survived for 7 d in APW containing
0.1 mm Ca2+. The rate of cytoplasmic streaming
(65 ⫾ 10 mm s-1) did not change significantly after 3 d in
any medium except 100 Na/1.0 Ca and 100 Na/0.1 Ca,
where it slowed to 50–45 mm s-1, respectively. These
unstimulated cells remained viable for significantly longer
in saline media than their mechanically stimulated
counterparts.
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1575–1591
Mechano-perception in Chara cells 1585
Table 4. Parameters used in mathematical modelling
Medium
gbackground (S m-2)
kio0 (s-1)
koi (s-1)
Resting PD (mV)
Pump reversal PD (mV)
APW
90sorb
Average time: 96 min
50 Na/1.0 Ca
Average time: 65 min
50 Na/0.1 Ca after 50 Na/1.0 Ca
Average time: 20–30 min
50 Na/0.1 Ca after 90sorb
Average time: 60 min
0.45 ⫾ 0.15
8 000 ⫾ 2000
100 ⫾ 20
-236 ⫾ 7
-377 ⫾ 13
0.6 ⫾ 0.3
8 800 ⫾ 2800
100 ⫾ 20
-227 ⫾ 7
-379 ⫾ 15
2.0 ⫾ 0.3
12 000 ⫾ 3000
130 ⫾ 25
-184, range: -202, -167
-394 ⫾ 12
2.0 ⫾ 0.3
8 000 ⫾ 2500
60 ⫾ 25
-150, range: -169, -127
-364 ⫾ 23
3.2 ⫾ 0.2
8 000 (max)
60 (max)
-100, range: -132, -60
-364 (max)
The other pump parameters, koi0 and kio, were kept at 0.5 s-1. The inward rectifier was not observed in the data sets. The uncertainties in the
resting PD were estimated from the measured resting PDs in APW and 90sorb. In the 50 Na data sets, the range is defined by an en dash. The
outward rectifier was observed only in the last set of data (bottom row): NKPK = 0.65 ¥ 10-7m s-1, V50+ = -5 mV, zg = 2.0. The curve of the best
fit contained no contribution from the pump, while the upper limit contained the same contribution from the pump as in the previous data
set. The lower limit contained no contribution from the pump, and the reversal PD for the background current changed to -60 mV. Cells were
in the various media for the following average times when sampling was performed: 90sorb, 96 min; 50 Na/1.0 Ca, 65 min; 50 Na/0.1 Ca, after
50 Na/1.0 Ca, 20–30 min; and 50 Na/0.1 Ca after 90sorb, 60 min.
PD, potential difference; APW, artificial pond water.
DISCUSSION
Historically, the use of simpler cell models has facilitated
understanding of complex multicellular systems, and giant
charophyte cells are prototypes of plant cell excitability,
membrane transport (Tazawa & Shimmen 2001) and
mechano-perception (Shimmen 2001). We have investigated the effects of salinity on mechano-perception by
Chara cells, as manifested in receptor and action potentials,
as well as salinity-induced changes in electrophysiology.
Reducing cell turgor pressure increases sensitivity to
mechanical stimulus in Chara cells (Shepherd et al. 2001).
Here, we report that an isotonic increase in salinity has
effects on the receptor potential and excitability, over and
above the effect of reduced turgor pressure. The nature
of these effects depends on the extracellular Ca2+ ion
concentration.
Salinity-induced changes in ion transport:
the points of no return
The PD of plant cells contains contributions from two components: first, the passive (background conductance), and
second, the active and pump-specific. Reducing turgor pressure had negligible effects on these components (Fig. 3,
Table 4; Shepherd et al. 2001). Similarly, mannitol-induced
turgor reduction has little effect on the proton pump
activity in Mung bean (Nakamura et al. 1992). However, I/V
Surviving cells
12
180sorb
10
100Na/10.0Ca
8
APW
6
100Na/1.0Ca
4
2
Figure 4. Viability of cells, assessed by
100Na/0.1Ca
0
1
2
3
4
Number of days
5
6
7
cytoplasmic streaming, in the absence of
mechano-stimulus. Cells were in the
saline and sorbitol-based media shown in
Table 1c. Artificial pond water (APW)
containing 10.0 mm Ca2+ was also tested,
and all cells survived for 7 d.
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Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1575–1591
1586 V. A. Shepherd et al.
analysis reveals four salinity-specific changes in ion transport, the nature of which depends on [Ca2+]ext.
Firstly, a rapid and dramatic increase in the background
conductance partially short-circuits the proton pump
current, and thus depolarizes the membrane PD (Fig. 3,
Table 4). The background conductance increased sevenfold
in 50 Na/0.1 Ca [with low (Ca2+)ext] and fourfold in 50 Na/1.0
Ca [with high (Ca2+)ext]. If the background conductance
in charophyte cells arises from the equivalent of NSCCs
(Beilby & Shepherd 2001), it is also a likely route for Na+
influx, which occurs via NSCCs in embryophyte plant cells
(Demidchik & Maathuis 2007). Calcium ions also inhibit
NSCCs of Arabidopsis protoplasts in a concentrationdependent manner (Demidchik & Tester 2002).
Secondly, currents generated by the electrogenic proton
pump changed in a calcium-dependent manner, increasing
in 50 Na/1.0 Ca and declining to negligible levels in 50
Na/0.1 Ca (Fig. 3b, Table 4). Activation of the proton pump
appears to depend on [Ca2+]ext, or specifically on the ionic
ratios between [Na+]ext and [Ca2+]ext at constant [K+]ext.
Chara cells depolarize to the passive component of the PD
in Ca2+-free APW (Bisson 1984). Stimulation of proton
pumping in 50 Na/1.0 Ca did not counteract salinityinduced depolarization of the resting PD (Fig. 3a), but
established a PD (Table 4) negative to the excitation
threshold (APthreshold) of approximately -100 mV (Beilby
2007). Stimulation of proton pumping in saline media was
described in Mung bean (Nakamura et al.1992) and bean
mesophyll (Shabala 2000). With negligible proton pumping
(Fig. 3b), the background conductance becomes dominant
in 50 Na/0.1 Ca, shifting the resting PD to APthreshold
(Table 4, bottom row).
Thirdly, most cells in 100 Na/0.1 Ca spontaneously initiated trains of repetitive firing (Table 3a,b; Fig. 2g). This
repetitive firing resembles the onset of a critical instability,
of the type described by Hayashi & Hirakawa (1980) in the
Nitella cell, where a time-ordered structure (repetitive
firing) emerged after ramping currents to the excitation
threshold. Most cells in 100 Na/1.0 Ca had PDs negative to
APthreshold (Table 2), and initiated repetitive firing only after
a touch-induced action potential, which shifted the PD transiently to APthreshold (Fig. 2d). Kishimoto (1966) attributed
NaCl-induced repetitive firing in Nitella to calcium depletion at the cell membrane. Our results suggest this is
because proton pump currents decline when [Ca2+]ext is low,
shifting the resting PD to APthreshold.
Finally, the threshold for activation of the outward rectifiers (KORthreshold) is approximately -50 mV (Fig. 3d), and
depolarization to more positive potentials leads to sustained K+ efflux.When [Ca2+]ext is low, increased background
conductance and loss of proton pump activity combine to
gradually depolarize the cell PD. Trains of spontaneous
action potentials, which involve net Cl- and K+ effluxes
(Beilby 2007), accelerate the depolarization because the
deactivation of the proton pump precludes recovery of the
resting PD.
Thus, two critical thresholds, APthreshold and KORthreshold,
serve as ‘points of no return’ when [Ca2+]ext is low. Proton
pump inactivation depolarizes cells to APthreshold, initiating
spontaneous repetitive firing and further depolarization to
KORthreshold. At this point, K+ depletion becomes irrevocable. The background conductance increases to a lesser
extent when [Ca2+]ext is high, and increased proton pump
activity establishes a PD outside the danger zone of the
critical APthreshold. In the absence of mechano-stimulus, this
circumvents both ‘points of no return’.
Interestingly, cells pre-treated in saline media with high
[Ca2+]ext had more negative PDs than those transferred
directly to media with low [Ca2+]ext (Tables 3a and 4). Such
pre-treatment may delay the eventual displacement of Ca2+
by Na+ at negative sites in the cell wall (Shabala & Newman
2000).
We do not yet know whether excitation plays a role in
responses of land plant cells to salinity. In the 1990s, wholecell patch clamp studies of various cereal roots suggested
the possibility that the K+ outward rectifier could maintain
an outward K+ current while less selective cation channels
permit Na+ entry (reviewed by Tyerman & Skerrett 1999).
Salinity induces K+ efflux via the outward rectifier in land
plant cells (Shabala et al. 2006), and this depends on the
magnitude of NaCl-induced depolarization (Chen et al.
2007), or, essentially, on proton pump activity, as in Chara.
Land plants are a sister group to the extant Charales (Karol
et al. 2001), and these cellular responses to salinity are likely
to be fundamental, with ancient antecedents.
The salt-tolerant turgor-regulating charophytes, such as
Chara longifolia (Yao, Bisson & Brzezicki 1992) and Lamprothamnium succinctum (Beilby & Shepherd 2001), avoid
the ‘points of no return’ through activation of the proton
pump, which maintains a negative resting PD (reviewed by
Beilby, Bisson & Shepherd 2006). Decreased turgor pressure activates the proton pump in Lamprothamnium (Al
Khazaaly & Beilby 2007). Similarly, Thellungiella, a salttolerant relative of Arabidopsis, maintains a negative
resting PD and regulates turgor during salt stress (Inan
et al. 2004; Volkov & Amtmann 2006).
Mechano-perception in saline media as a
function of [Ca2+]ext
The receptor potential difference (RPD) involves activation
of mechano-sensitive Ca2+ channels at the plasma membrane, transient elevation of intracellular Ca2+ and efflux of
Cl- via Ca2+-activated Cl- channels, resulting in transient
depolarization (Kaneko et al. 2005). The magnitude of Ca2+
influx increases with increasing stimulus strength, accounting for incremental depolarization (‘RPD amplitude’).
Most cells became hypersensitive to mechanical stimulus
in 100 Na/1.0 Ca. The RPD amplitudes increased, relative to
RPD amplitudes in sorbitol (Fig. 2a–c, Table 3a). This was
an effect of NaCl, not simply a result of turgor reduction.
Cells were thereby more likely to fire a touch-induced
action potential (Table 3a, Fig. 2c), followed by repetitive
firing as the repolarizing PD (-90 ⫾ 23 mV) approached
APthreshold (Fig. 2d).
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Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1575–1591
Mechano-perception in Chara cells 1587
Stimulation of proton pumping enabled restoration of
the resting PD. However, each action potential depletes the
cell of K+ and Cl- (Beilby 2007), transiently inhibits photosynthesis (Bulychev et al. 2004; Krupenina & Bulychev
2007), and presumably has further indirect impacts on
electrogenic pump-driven transport via ATP depletion.
Although cells re-established a PD negative to APthreshold,
and all remained viable for at least 60 min following
mechano-stimulus, even slight mechano-stimulus produces
multiple excitations and K+ efflux. Hypersensitivity to
mechano-stimulus thus increases the propensity for depolarization to APthreshold and KORthreshold.
Cells in 100 Na/0.1 Ca responded to given stimuli more
haphazardly. Following spontaneous repetitive firing, much
depolarized cells (e.g. with a PD of -22 mV) were desensitized to mechano-stimulus, producing smaller-amplitude
RPDs or becoming inexcitable (Table 3a,b; Fig. 2g). In contrast, cells maintaining a more negative PD (e.g. -80 to
-121 mV) retained their sensitivity to mechano-stimulus.
Some cells became hypersensitive, producing RPDs of
larger amplitude and touch-induced action potentials
(Fig. 2e). Following repetitive firing (Fig. 2f), the cells depolarized to a PD more positive than KORthreshold (Table 3a,b).
Unsurprisingly, all cells died within 60 min of mechanostimulus, whereas 50% of non-stimulated cells remained
viable after 2 d in 100 Na/0.1 Ca, and 90% were viable after
2 d in 100 Na/1.0 Ca (Fig. 4). A high [Ca2+]ext to [Na+]ext ratio
in the medium has previously been found to enhance the
survival of C. corallina cells in saline media (Tufariello,
Hoffmann & Bisson 1988).
Furthermore, the ratio between [Ca2+]ext and [Na+]ext has
dramatic effects on action potential. The duration of both
touch-induced and spontaneous repetitive action potentials
was shorter in 100 Na/1.0 Ca than in 100 Na/0.1 Ca
(compare Fig. 2c,d with Fig. 2e,f). Presumably, the
excitation-associated net effluxes of Cl- and K+ were greater
in 100 Na/0.1 Ca. Monovalent cations, including Na+,
prolong the action potential, but divalent ions, including
Ca2+, suppress this effect (Shimmen et al. 1976).
Increased salinity eventually induces fatal depolarization, depending on the [Na+]ext to [Ca2+]ext ratio (Fig. 4),
but mechano-stimulus accelerates the process in a Ca2+dependent manner. A key factor in the fatal synergy
between mechano-stimulus, [Ca2+]ext and [Na+]ext is excitation, both mechanically and spontaneously induced. Postexcitation recovery depends on proton pump activity
re-establishing a PD negative to APthreshold, and the pump is
active only when [Ca2+]ext is high. When [Ca2+]ext is low, cells
depolarize to KORthreshold following excitation.
Cells are either desensitized or hypersensitized
to mechano-stimulus in saline media: the roles
of turgor pressure and Cl- conductance
Superficially, the production of larger-amplitude RPDs
(increased Ca2+ influx and Cl- efflux) in saline media
depends on membrane PD (Table 3a,b), but the generation
of RPD does not depend on the H+ pump (Shimmen
1997b). Two different factors contribute to the increased
RPD amplitudes. The first lacks measurable electrophysiological correlates. As reported previously, cells are sensitized to mechano-stimulus when turgor pressure is reduced
(Shepherd et al. 2001), but neither the cell PD nor the background conductance changes significantly (Fig. 3) (Kiyosawa & Ogata 1987; Shepherd et al. 2001).
We do not know why cells with reduced turgor are sensitized to mechanical stimulation. Decompression stimulus
induces smaller-amplitude RPDs when turgor pressure is
reduced, suggesting that changed membrane tension is a
crucial factor (Iwabuchi et al. 2008). Changed membrane
tension is unlikely to account for increased RPD amplitudes in our experiments, however, because membrane
transfer between plasma membrane and endocytotic
vesicles compensates for osmotic contraction in hypertonic
solution (Wolfe & Steponkus 1983; Wolfe, Dowgert & Steponkus 1985; Dowgert, Wolfe & Steponkus 1987). The
plasma membrane is probably under comparable small
tension in both APW and 180sorb.
Previously, we suggested that a continuum among cell
wall, plasma membrane and cytoskeleton (Baluska et al.
2003; Telewski 2006; Pickard 2007) is critical to mechanoperception in Chara (Shepherd et al. 2001). Future experiments will investigate the effects on generation of receptor
and action potentials of inhibiting either or both actin and
microtubule cytoskeletons.
A second, electrophysiological factor contributes to
increased RPD amplitudes. The hundredfold increase in
[Cl-]ext results in a reversal PD for Cl- (ECl) of approximately -50 mV, assuming a [Cl-]cyt of 10 mm (Coster 1966).
Thus, the driving force for Cl- efflux, on which the RPD
depolarization depends, is negligible in cells with a resting
PD close to ECl. At PDs more positive than ECl, the currents
are diminished by the inwardly rectifying properties of the
Cl- channels (Beilby & Shepherd 2006b). This is borne out
by the data, where cells with PDs close to ECl became unresponsive or showed reduced RPD amplitudes (Table 3a,b).
On the other hand, cells with more negative PDs were
hypersensitized to mechano-stimulus, showing increased
RPD amplitudes with given stimuli (Table 3a,b). Preliminary modelling, based on Beilby & Shepherd (2006b),
shows that an increase in [Cl-]cyt would result in an increase
in Cl- currents, which may contribute to the observed
hypersensitivity.
Potential role of excitation in responses of
land plants to salinity
The action potentials of Chara cells reflect those of land
plant cells (Tazawa & Shimmen 2001; Fisahn et al. 2004),
and mechanically stimulated or spontaneous excitations
may thus be widely relevant to understanding land plant
responses to salinity. Action and/or variation potentials
suppress photosynthesis in Chara (Krupenina & Bulychev
2007) and in land plants (Koziolek et al. 2004; Lautner et al.
2005). Salt (KCl) applied to roots induces action potentials
in leaves (Favre, Greppin & Degli Agosti 2001; Felle &
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1575–1591
1588 V. A. Shepherd et al.
Zimmermann 2007). Salinity reduces photosynthetic efficiency (e.g. Shabala et al. 2005), and mechano-perception
and/or excitation may contribute. Excitation combined with
proton pump inhibition is another possible avenue for the
K+ depletion, which is thought (Maathuis & Amtmann
1999) to underlie NaCl toxicity. Circumvention of the critical APthreshold when [Ca2+]ext/[Na+]ext is high potentially
contributes to well-known ameliorative effects of high
[Ca2+]ext on salt-stressed plants (Cramer 2002).
CONCLUSION
Our results reveal the hitherto unrecognized importance of
mechanically stimulated and spontaneous action potentials
Freshwater
Sorbitol (reduction of
turgor pressure)
Stable:
Membrane potential
Conductance
Salt water
high calcium
Salt water
low calcium
Proton pump stimulated
Proton pump deactivated
Fourfold increase in background
conductance
Sevenfold increase in background
conductance
Membrane PDs negative to
APthreshold
Membrane potential depolarizes to
APthreshold
No spontaneous excitation
Spontaneous repetitive excitations
(of extended duration)
Cell PD is negative to KORthreshold
Cell PD is positive to KORthreshold.
At KORthreshold, K+ efflux sustained
and irrevocable
Cells hypersensitive to mechanostimulus
Cells desensitized to mechanostimulus
Mechano-stimulus leads to
repetitive excitation
No response to mechano-stimulus
PD remains negative to
APthreshold, and KORthreshold
PD continues depolarizing
Cells viable
Cells not viable
Figure 5. Diagrammatic representation of responses to mechano-stimulus in saline media with either high (1.0 mm) or low (0.1 mm) Ca2+
ion concentrations. When [Ca2+]ext is low, deactivation of the proton pump and the increase in background conductance depolarize the
membrane potential to action potential (AP)threshold. Spontaneous repetitive excitations of extended duration are the result, followed by
depolarization to potential differences (PDs) positive to KORthreshold. Although these cells are desensitized to mechano-stimulus, K+ efflux
is then sustained and irrevocable. On the other hand, cells retaining a PD negative to K+ outward rectifier (KOR)threshold (and ECl) become
hypersensitive to mechano-stimulus, and further excitation and K+ efflux then drive the PD to KORthreshold. Cell viability was extended in
the absence of mechano-stimulus. When [Ca2+]ext is high, the smaller background current and stimulation of proton pump currents
establish membrane PDs negative to APthreshold. Cells do not spontaneously fire, but they become hypersensitive to mechano-stimulus.
Even a small mechano-stimulus provokes excitation, followed by repetitive firing as the repolarizing PD crosses APthreshold. Ca2+-dependent
activation of the proton pump re-establishes a PD slightly negative to APthreshold and well negative to KORthreshold, and cells remain viable
for longer when [Ca2+]ext is high. Cell viability was extended in the absence of mechano-stimulus.
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1575–1591
Mechano-perception in Chara cells 1589
in the salt sensitivity of Chara cells. The cells eventually
depolarize and die in saline media, but mechanically stimulated and spontaneous excitation augments the process in a
Ca2+-dependent manner. Two critical membrane potential
thresholds, the excitation threshold (APthreshold) and the
threshold for activation of the K+ outward rectifier
(KORthreshold), act as ‘points of no return’ in determining cell
viability in saline media. This is summarized in Fig. 5.
Molecular approaches have demonstrated the importance of calcium signalling in salt sensitivity (Zhu 2001).
Action potentials function at a higher organizational level
as systemic Ca2+ signals, and their role in the responses of
land plants to increased salinity is an avenue for further
study.
ACKNOWLEDGMENTS
Virginia Shepherd received a grant from the Japan Society
for the Promotion of Science and the Australian Academy
of Science, to visit Professor Shimmen’s lab at the University of Hyogo. We thank Professor N.A. Walker for reading
the manuscript and stimulating discussion.
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Received 2 April 2008; received in revised form 26 June 2008;
accepted for publication 29 July 2008
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