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Mechano-perception in Chara cells: the influence ofsalinity and calcium on touch-activated receptorpotentials, action potentials and ion transport

VIRGINIA A. SHEPHERD1, MARY J. BEILBY1, SABAH A.S. AL KHAZAALY1 & TERUO SHIMMEN2

1Department of Biophysics, School of Physics, The University of New South Wales, Sydney, New South Wales 2052, Australiaand 2Department of Life Science, Graduate School of Life Science, University of Hyogo, Harima Science City, Hyogo678-1297, Japan

ABSTRACT

This paper investigates the impact of increased salinity ontouch-induced receptor and action potentials of Charainternodal cells. We resolved underlying changes in iontransport by current/voltage analysis. In a saline mediumwith a low Ca2+ ion concentration [(Ca2+)ext], the cell back-ground conductance significantly increased and protonpump currents declined to negligible levels, depolarizingthe membrane potential difference (PD) to the excitationthreshold [action potential (AP)threshold]. The onset of spon-taneous repetitive action potentials further depolarized thePD, activating K+ outward rectifying (KOR) channels. K+

efflux was then sustained and irrevocable, and cells weredesensitized to touch. However, when [Ca2+]ext was high, thebackground conductance increased to a lesser extent andproton pump currents were stimulated, establishing a PDnarrowly negative to APthreshold. Cells did not spontaneouslyfire, but became hypersensitive to touch. Even slight touchstimulus induced an action potential and further repetitivefiring. The duration of each excitation was extended when[Ca2+]ext was low. Cell viability was prolonged in the absenceof touch stimulus. Chara cells eventually depolarize and diein the saline media, but touch-stimulated and spontaneousexcitation accelerates the process in a Ca2+-dependentmanner. Our results have broad implications for under-standing the interactions between mechano-perception andsalinity stress in plants.

Key-words: excitation; plant mechano-perception; protonpump; salt stress.

INTRODUCTION

The sometimes-dramatic responses of plants to touch haveintrigued biologists for over a century. A voluminous litera-ture is devoted to the well-known thigmonastic behavioursof Mimosa, the Venus’s Flytrap Dionaea, the bladderwortAldrovanda (Sibaoka 1991; Shimmen 2001) and the trigger-plant Stylidium (Hill & Findlay 1981; Findlay 1984). In

recent years, it has become clear that the capacity for per-ceiving and responding to mechanical stimuli is also funda-mental to subtle plant behaviours including responses togravity, temperature, environmental osmolarity, and turgor-controlled growth and development (reviewed by Fasano,Massa & Gilroy 2002; Jaffe, Leopold & Staples 2002;Baluska et al. 2003; Telewski 2006; Haswell 2007; Pickard2007).

It is thought that mechano-perception in plant cells com-mences with membrane deformation, whether this occursdirectly, or indirectly, via transmembrane protein tetherscoupled to the cytoskeleton and the cell wall (reviewed byFasano et al. 2002). Specifically, force perception translatesinto the activity of mechano-sensory Ca2+ ion channels,modulated in a multitude of ways accounting for multipletypes of response (Pickard 2007). Mechano-perceptionrests upon Ca2+-dependent second messenger systems, andcytoplasmic Ca2+ is elevated following different kinds ofmechanical stimulus (Haley et al. 1995). In Trewavas’ model(Trewavas 1999a,b), each kind of stimulus elicits a uniqueCa2+ signal with distinct topology.

The electrophysiology of mechano-perception is difficultto study in small cells of complex tissues. Giant internodalcells of the Charales have been widely used to researchmechano-perception at the cellular level.

Embryophytes (land plants) are often viewed as beingsynonymous with the entire plant kingdom. Phylogeneti-cally, the Charales are the extant sister group to all embryo-phytes, with which they share some fundamentalbiochemical and physiological processes (McCourt, Del-wiche & Karol 2004). These processes include aspects ofexcitability, membrane transport (Tazawa & Shimmen2001) and mechano-perception (Shimmen 2001).

In giant cells of the Charales, mechanical stimuli produc-ing compressive or decompressive mechanical stress on thecell wall deform (Iwabuchi, Kaneko & Kikuyama 2005) oralter the tension (Iwabuchi, Kaneko & Kikuyama 2008) ofthe cell membrane, activating mechano-sensory ion chan-nels. These channels transduce the touch stimulus into anelectrical signal or receptor potential (Shimmen 1996,1997a,b,c). The receptor potential is a small depolariza-tion brought about by mechano-sensory Ca2+ channels at

Correspondence: V. A. Shepherd. Fax: +61 2 9385 6060; e-mail:[email protected]

Plant, Cell and Environment (2008) 31, 1575–1591 doi: 10.1111/j.1365-3040.2008.01866.x

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd 1575

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the plasma membrane. The Ca2+ released stimulates Ca2+-activated Cl- channels (Kaneko et al. 2005), producing a Cl-

efflux (Shimmen 1996, 1997a,b,c). The receptor potentialincreases in amplitude and/or duration more or less incre-mentally with increasing magnitude of stimulus (Kishimoto1968; Shimmen 1996; Kaneko et al. 2005) until a criticalthreshold voltage initiates an action potential (reviewed byBeilby 2007). In contrast to the action potential, the recep-tor potential does not propagate intercellularly.

An action potential involves a significant elevation ofcytoplasmic Ca2+ from 200 to 700 nm (Plieth & Hansen1996). The elevated Ca2+ activates Cl- efflux via Ca2+-activated Cl- channels, further depolarizing the cell andactivating outwardly directed voltage-dependent K+ chan-nels (reviewed, Beilby 2007). Under normal conditions, theaction potential has a characteristic threshold and form, andpropagates from cell to cell (Beilby 2007). The result is anefflux of Cl- and K+ (Oda 1976; Beilby 1984, 2007; Wayne1994), water efflux and transient turgor reduction (Zimmer-mann & Beckers 1978), and cell contraction (Oda & Lin-stead 1975). The motif of elevated cytoplasmic Ca2+, effluxof Cl- and K+, and turgor loss and contractility, is elaboratedinto turgor-based osmotic machinery that enables a widerepertoire of mechano-responses in land plants (Hill &Findlay 1981).

We established that cell turgor pressure modulatesmechano-perception in Chara internodal cells (Shepherd,Shimmen & Beilby 2001; Shepherd, Beilby & Shimmen2002). The critical receptor potential, which triggers anaction potential, occurs with weaker stimulus when turgorpressure is decreased (Shepherd et al. 2001, 2002). Onlythe receptor potential is affected. The voltage threshold forinitiating an action potential is unchanged (Staves & Wayne1993; Shepherd et al. 2001). As discussed earlier, the recep-tor potential is the electrophysiological manifestation ofmechano-perception. Furthermore, it is a change in turgorpressure, rather than its absolute magnitude, that elicitssensitization to mechanical stimulus (Shepherd et al. 2001).

The ability to regulate cell volume and/or turgor pressureis fundamental to the lives of cells. Action potentials prob-ably evolved from osmo-regulatory processes of ancientbacteria inhabiting environments of variable salinity (Grad-mann & Mummert 1980).The responses of charophyte cellsto changes in environmental osmolarity and salinity involvemechano-perception (Shepherd et al. 2002). This mayextend to other plant cells. For example, Arabidopsis leafcells respond to hypotonic or hypertonic media with Ca2+

transients, mediated by stretch-activated (or mechano-sensory) channels (Hayashi et al. 2006), and increasedsalinity also elevates cytoplasmic Ca2+ in Arabidopsis cells(Knight, Trewavas & Knight 1997).

Considering that Chara cells become increasingly sensi-tive to mechanical stimulation as turgor decreases (Shep-herd et al. 2001), it is probable that mechano-perceptionplays a role in their responses to increased environmentalsalinity. Broadly speaking, an increase in environmentalsalinity can impact on plant cells in two ways: firstly, throughosmotic reduction of cell turgor pressure, essentially a

mechanical stimulus, and secondly, through the increasedinflux of Na+, which may then compete with and displace K+

from carboxylate groups of cell proteins such as actin.Theseimpacts are coupled in salt-sensitive Chara cells. Whenturgor pressure is reduced in the presence of NaCl, themagnitude of Na+ influx increases (Davenport, Reid &Smith 1996).

In this paper, we set out to compare and contrast theeffects of turgor pressure reduction and elevated environ-mental salinity on mechano-perception in Chara cells.Because elevated extracellular Ca2+ ion concentrationsreduce the negative impact of high salinity in a wide rangeof plant cells (reviewed by Cramer 2002), including Chara(Hoffmann, Tufariello & Bisson 1989; Whittington & Smith1992), we conducted our experiments in saline mediaincluding either high or low Ca2+ concentrations. Elevatedextracellular Ca2+ diminishes both Na+ influx via non-selective cation channels (NSCCs), and Na+-induced K+

efflux through outward rectifier channels in Arabidopsis(Shabala et al. 2006).We employed the powerful techniquesof current–voltage (I/V) analysis and mathematical model-ling (Beilby & Walker 1996) to identify changes in ion trans-porters induced both by turgor pressure reduction and bysaline media.

Plant physiological experiments frequently focus on asingle environmental factor, such as salinity, or mechanicalstimulus, when both factors are present in the natural world.Giant charophyte cells are unique experimental materialsthat enable investigation of the electrophysiologicalphenomena associated with mechano-perception underconditions of high salinity. Cells of embryophytes andcharophytes respond to mechanical stimulus with a similarelectrophysiological modus of receptor and action poten-tials (Shimmen 2001). Our results may have broad im-plications for understanding the interactions betweenmechano-perception and salinity stress in plant cells.

MATERIALS AND METHODS

Cell preparation

Chara corallina was cultured in Japan as described previ-ously (Mimura & Shimmen 1994). Chara australis Brown(Garcia & Chivas 2006) was collected from Little Bay,Sydney, New South Wales, Australia, and was planted inaquaria containing a handful of autoclaved garden soil, ahandful of rotting leaves and rainwater. C. australis wascultured under equal numbers of Sylvania Gro-Lux fluores-cent tubes (Sylvania Australasia Pty. Ltd., Lisarow,New South Wales, Australia) and cool white fluorescenttubes, providing a photosynthetically active radiation of80 mmol m-2 s-1, on a cycle of 10 h light and 14 h darkness.

The C. corallina internodal cells used in mechano-stimulus experiments were between 45 and 53 mm long.Cells were cut from vigorous plants and were kept in arti-ficial pond water (APW; Table 1a) in a growth cabinet for atleast a week before experiments. Experiments were con-ducted at room temperature (28 °C).

1576 V. A. Shepherd et al.

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Plant, Cell and Environment, 31, 1575–1591

The C. australis internodal cells used in voltage-clampexperiments were about 10–15 mm in length. Sub-apicalinternodal cells were cut from healthy plants and were leftto recover in APW for at least 3 d. Experiments were con-ducted at room temperature (26 °C). C. australis internodalcells were also used in cell viability tests.

Experimental protocols

We used three experimental protocols: (1) mechano-stimulation, (2) I/V measurements and (3) cell viability tests.

Mechano-stimulation experiments

We measured and compared responses with mechano-stimulus in the media listed in Table 1a.

Figure 1 shows the experimental protocols. Firstly,internodal cells were placed into a Perspex chamber (in-house), with two compartments (A and B) connected by agroove in the Perspex. The compartments A and B wereelectrically isolated using petroleum jelly. Both compart-ments initially contained APW, in which cells remained forat least 30 min.

Mechano-stimulus dislodges impaled microelectrodesand damages the cells, and we used extracellular electrodesto record difference potentials (Shimmen 1996). A differ-ence potential is the difference in electrical potentialbetween the two electrically isolated ends A and B of thecell. The membrane potential difference (PD) at each endof the cell is similar in APW, and hence EA - EB is initiallyclose 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 elec-trodes were amplified using a differential microelectrodeamplifier (MEZ7101; Nihon Kohden,Tokyo, Japan), and thesignal EA - EB (the difference potential) was recorded usinga chart recorder (VP-6521A; National, Tokyo, Japan) withvariable speed and sensitivity.

We used the K+ anesthesia technique to measure cell PDs(Shimmen, Kikuyama & Tazawa 1976). The 180sorb solu-tion was introduced into both compartments to reduceturgor pressure (Fig. 1a). The 180sorb in compartment Bwas 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 incompartment A.

Cells were mechanically stimulated using the deviceconstructed by Shimmen (1996). Briefly, a small polyacry-late stimulator (dimensions, 5 ¥ 8 ¥ 1 mm) was suspendedadjacent to the cell flank in compartment A. The stimula-tor was impacted by a thin glass rod (weight, 1.28 g)released from increasing heights (0.5–6 cm). The devicedelivers 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 (Kish-imoto 1968) by leaving 4–5 min between stimulations.Cells were allowed to recover for at least an hour follow-ing an AP.

In separate experiments, we mechanically stimulatedcells in 100 Na/1.0 Ca and in 100 Na/0.1 Ca (Fig. 1c). The180sorb in compartment A was exchanged for either 100Na/1.0 Ca or 100 Na/0.1 Ca, and the cell was left for 60 minto stabilize Ca2+/Na+ exchange in the cell wall.

In one group of experiments, we compared mechano-responses of the same cells in 100 Na/1.0 Ca and in 100

Table 1. Chemical composition andacronyms for media used in (a)mechano-perception experiments,(b) voltage-clamp experiments and (c)cell viability tests

Solution acronym

Chemical constituents

KCl (mm) NaCl (mm) CaCl2 (mm) Sorbitol (mm)

(a)APW 0.1 1.0 0.1 –180sorb 0.1 1.0 0.1 180.0100 KCl 100.0 – – –100 Na/1.0 Ca 0.1 100.0 1.0 –100 Na/0.1 Ca 0.1 100.0 0.1 –

(b)APW 0.1 1.0 0.1 –90sorb 0.1 1.0 0.1 90.050 Na/1.0 Ca 0.1 50.0 1.0 –50 Na/0.1 Ca 0.1 50.0 0.1 –

(c)APW 0.1 1.0 0.1 –180sorb 0.1 1.0 0.1 180.0100 Na/10 Ca 0.1 100.0 10.0 –100 Na/1.0 Ca 0.1 100.0 1.0 –100 Na/0.1 Ca 0.1 100.0 0.1 –

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.

Mechano-perception in Chara cells 1577


Na/0.1 Ca. Cells stimulated in 100 Na/1.0 Ca were left torecover for 60 min, and the 100 Na/1.0 Ca was exchangedfor 100 Na/0.1 Ca. Cells were mechanically stimulated30–45 min later (Fig. 1d).

We recorded the cell resting membrane PDs, the peakamplitudes of receptor potentials, the occurrence of touch-induced action potentials, and PD oscillations.

In a control experiment, we subjected a group of cells tothe protocol of repetitive solution changes and mechano-stimulus, using only APW. As found previously (Shepherdet al. 2001), these procedures had only slight effect on themechano-responses.

The I/V measurements

Cells did not survive voltage-clamping protocols in 100 mmNaCl solutions, regardless of the Ca2+ concentration. Wereduced the NaCl concentration to 50 mm, containing0.1 mm Ca2+ or 1.0 mm Ca2+. This increased the activity ofCa2+ from 0.58 (in 100 mm NaCl) to 0.65 in 50 mm NaCl(Butler 1968). We reduced cell turgor pressure using iso-tonic 90sorb. Table 1b lists the chemical composition of themedia.

The voltage-clamping procedure was as previouslydescribed (Beilby & Shepherd 1996). Cells were

Figure 1. Diagrammatic representationof the protocols for mechano-sensingexperiments. (a) Cell placed intwo-compartment chamber. Bothchambers filled with 180sorb solutionto reduce cell turgor pressure. (b) The100 KCl solution introduced intocompartment B eliminates the membranePD in this region, enabling measurementof cell PD. (c) Either 100 Na/1.0 Ca or100 Na/0.1 Ca is introduced into chamberA. After 45 min. cells are mechanicallystimulated. (d) Cells that survivedmechano-stimulus in 100 Na/1.0 Ca nowexposed to 100 Na/0.1 Ca, so thatresponses can be com pared. PD,potential difference.

EA – EBA B

EA – EBA B

EA – EBA B

EA – EBA B

180sorb

(a)

(b)

(c)

(d)

180sorb

180sorb 100KCl

(PD in A)

100KCl 100Na/0.1Ca OR100Na/1.0Ca

100KCl 100Na/0.1Ca

Reduction of cell turgor pressure

K+ anesthesia. 100KCl introduced to chamber B for measuring cell PD in A

100Na/0.1Ca OR 100Na/1.0Ca introduced to chamber A for ~45 minmechano-stimulus

Cells surviving mechano-stimulus in 100Na/1.0Ca transferred into100Na/0.1Ca: further mechano-stimulus

Difference PD

Stimulator



compartment-clamped, with a microelectrode located in thecytoplasm or vacuole.The plasma membrane I/V character-istics dominate because of the high tonoplast conductance(Beilby 1990). We measured I/V characteristics using abipolar staircase voltage command, with pulses of widthbetween 60 and 100 ms, separated by 120–250 ms, at theresting PD.

The order and time course of solution changes mimickedthose in mechano-stimulus experiments. We first obtainedI/V characteristics of cells in APW and in 90sorb. We thenmeasured I/V characteristics of cells transferred into either50 Na/1.0 Ca or 50 Na/0.1 Ca. We also compared I/V char-acteristics of the same cells in 50 Na/1.0 Ca and 50 Na/0.1Ca.

Modelling and analysis of I/V data

We resolved the total current into contributions made byparallel populations of ion transporters. We modelled theelectrogenic proton pump at the plasma membrane usingthe two-state Hansen, Gradmann, Sanders and Slayman(HGSS) model (Hansen et al. 1981). We fitted the inwardand outward rectifiers using the Goldmann, Hodgkin andKatz (GHK) model, supplemented by the Boltzmann dis-tribution (Amtmann & Sanders 1999). We used an empiri-cal model of the ‘background current’, with a reversal PDof -100 mV and a PD-independent conductance (Beilby &Shepherd 2006a; Al Khazaaly & Beilby 2007). Channelspassing the ‘background current’ are the probable equiva-lent 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 cytoplasmicstreaming in control cells, where mechano-stimulus wasavoided. Cells cut from healthy plants were left in APW for1 week. Ten cells were transferred into each of the media(Table 1c). The media were replaced every second day, andcare was taken to avoid mechano-stimulus. We estimatedthe rate of cytoplasmic streaming as the time taken forcytoplasmic particles to traverse a 32¥ field of view. Thenumber of viable cells was counted daily over 7 d. Deadcells were removed.

RESULTS

Figure 2 shows excerpts from chart records of cell PDs,and cell mechano-responses, in 180sorb, 100 Na/1.0 Ca and100 Na/0.1 Ca. Table 1 shows the composition of the mediaand explains the acronyms used for each. Table 2 showsthe mean resting membrane PDs of cells in these media.Table 3a,b summarizes mechano-responses of cells in allexperiments.

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

PDs in these media were not significantly different. Asreported previously (Shepherd et al. 2001), cells were sen-sitized to mechano-stimulus when turgor pressure wasreduced. In 180sorb, cells responded to given stimuli withlarger amplitude receptor potentials than in APW. Stimulibetween f1 and f4 (modal value f4) induced action poten-tials 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 ofreceptor potentials increased incrementally with increasedstimulus energy, until an action potential was induced(Fig. 2a,b; Table 3a,b).

Mechano-responses in 100 Na/1.0 Ca

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 differentto that in APW (P0 = 0.00). Small magnitude PD oscillationsobserved in APW and 180sorb increased in amplitude andperiod in all cells (Fig. 2c). These oscillations are not clearlyvisible at the scale of reproduction of the figure. We willdescribe such oscillations in detail in a forthcoming paper.

Most cells (3, 4, 6 and 7 in Table 3a) became moresensitive to mechano-stimulus in 100 Na/1.0 Ca (Fig. 2c,Table 3a). Cells 1 and 5 did not decrease their sensitivity tostimulus. However, the limited resolution of the apparatusdid not enable us to determine if the sensitivity of thesecells had increased. Because 100 Na/1.0 Ca and 180sorbwere isotonic media, the increased sensitivity of most cellsresults from increased NaCl concentration. Cell 2 alonedecreased its sensitivity to mechano-stimulus.

Initially, all cells recovered the resting PD following thetouch-induced action potential. However, all cells (with theexception 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 cellsentered a series of spontaneous repetitive action potentials(Fig. 2d). The duration and form of these action potentialswas unique to each cell and repeated over a period of up to60 min. At their conclusion, cells gradually hyperpolarizedto a mean PD of -118.2 � 9.5 mV (Table 2). This PDremained stable for a further 60 min. Cell 7 alone did notdepolarize further from its post-action potential PD of-135 mV, neither did it begin repetitive firing.

Mechano-responses of the same cells in100 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 ofsmall-scale resting PD oscillations increased to between 5and 10 mV (Fig. 2e) with occasional spikes of approxi-mately 25 mV (not shown). We distinguished three types ofelectrophysiological behaviour prior to mechano-stimulus(Table 3a): (1) cells 1 and 7 fired a single spontaneous actionpotential. Cell 1 subsequently remained much depolarized.



Cell 7 recovered its PD for a few seconds, then depolarizedto zero PD and ceased streaming, and died; (2) cells 2 and 3spontaneously entered repetitive action potentials, and thendepolarized significantly; and (3) cells 4, 5 and 6 retained asimilar PD to that in 100 Na/1.0 Ca (Fig. 2e).

Initiation of spontaneous action potentials was a mark-edly different behaviour. Spontaneous action potentials

had only followed touch-induced action potentials in100 Na/1.0 Ca.

Responses to mechano-stimulus differed from those in100 Na/1.0 Ca. Most cells (1, 2, 3 and 6 in Table 3a)decreased their sensitivity to mechano-stimulus andresponded to given stimuli with smaller amplitude receptorpotentials. Of these, only cells 2 and 6, with PDs of -81 and



-120 mV, respectively, fired action potentials. Much depo-larized cells 1 and 3 (with PDs of -22 and -21 mV, respec-tively) did not fire. The effect of 100 Na/0.1 Ca on cell 5was beyond the resolution of the apparatus, because thelowest energy stimulus elicited action potentials (f0.5). Cell7 died prior to stimulation. Cell 4 had not experienced

spontaneous firing in 100 Na/0.1 Ca, and this cell aloneincreased its sensitivity (Fig. 2e).

Following the touch-induced action potential, all cellsdepolarized to zero PD and permanently ceased streamingwithin 60 min (Table 2), a markedly different fate fromthat of cells in 100 Na/1.0 Ca, which recovered a mean

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 themedia is shown in Table 1. Results (a–f) are from the same cell throughout. Vertical bars indicate where the chart recorder speed wasslowed, and the time scale is in minutes. (a) Cell difference potentials (see Fig. 1a), with turgor pressure reduced in 180sorb. The cellresponds to increasing mechanical stimuli f1 and f2 with receptor potentials; small, transient depolarizations. Stimulus f3 induces an actionpotential (AP). The asterisk (*) symbol marks the threshold receptor potential that initiates the AP. The AP appears biphasic because atouch-induced AP occurred in both ends of the cell. Note that the difference potential does not necessarily show the true amplitude ofthe response. (b) Cell membrane potential differences (PDs), measured by K+ anesthesia, with turgor pressure reduced in 180sorb (seeFig. 1b). The resting membrane PD was -225 mV. As described previously, the cell responds to mechanical stimuli f1 and f2 with receptorpotentials 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 thetouch-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 cellbegan depolarizing immediately (not shown), reaching a stable PD of -137 mV after 45 min. Small-scale PD oscillations (within verticalbars) were measured with the chart speed set to a time scale of minutes. These oscillations are not clearly visible at the scale ofreproduction of the figure. We will describe them in detail in a forthcoming paper. Oscillation amplitudes were between 2 and 5 mV, andtheir duration, approximately 1 min. These amplitudes and periods were larger than small-scale oscillations in 180sorb (not shown). Thecell increased its sensitivity to mechanical stimulus, in comparison with its responses in 180sorb. Stimulus f0.5 provoked a receptorpotential (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 thetouch-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-inducedAP, began to depolarize. The first panel of the chart excerpt shows this depolarization. At a PD of -70 mV, the cell entered a series ofspontaneous 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 increasedits 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 cellretained a resting PD between -116 and -113 mV, as described previously. However, small-scale PD oscillations (arrow) increased inamplitude (between 2 and 10 mV) and duration (approximately 2.5 min). Sensitivity to mechanical stimulus increased, and stimulus f0.5provoked a receptor potential (12 mV amplitude: 30 s duration). Small-scale PD oscillations are visible (vertical bars). Stimulus f1 inducedan 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 ceasedwhen the cell PD hyperpolarized to -75 mV (not shown). Unusually, this cell recovered a stable PD of -107 mV. Following furtherrepetitive APs, the cell died within 60 min of mechano-stimulation. (g) Cell PD and mechano-responses of another cell, this timetransferred 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 introducing100 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 ofthe chart is in minutes rather than seconds, and the firing is not resolved as described previously. The PD stabilized at -22 mV followingspontaneous firing. The cell was desensitized to mechano-stimulus. Stimulus f1 provoked an extended depolarization, rather than areceptor potential. The cell subsequently depolarized to 0 mV, and did not recover cytoplasmic streaming after 40 min.

Table 2. Membrane PDs (mean(SD, n = number of cells) of cells used in mechano-stimulation experiments in APW, 180sorb, 100 Na/0.1Ca 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) PD (mV) PD (mV) PD (mV) PD (mV)-228 � 42 (n = 6) (a): -212 � 23 (n = 6) Initial: -106 � 15 (n = 6) Initial: -123 � 21 (n = 7) Initial: -82 � 43 (n = 7)

(b): -217 � 16 (n = 7) RAP: -53 � 41 (n = 6) – RAP: -41 � 34 (n = 3)noRAP: -121 (one cell) noRAP: -123 � 21 (n = 7) noRAP: -122 � 9 (n = 4)Post-MS: 0 (n = 6) Post-MS: -118 � 10 (n = 7) 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 actionpotentials. ‘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.



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 isotonic100 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 100 Na/0.1 Ca

CellStable PD(mV)

Receptor potentialamplitude (mV) atstimulus

Stable PD(mV)


Stable PD(mV)


1 -222 f0.5: TAP -126 f0.5: TAP -22 *Spontaneous action potentialStable PD: -220 SRAP f0.5: 0.8

f1: no responseDepolarization, death

2 -226 f0.5: 32.6 -122 f0.5: 18 -81 f0.5: TAPf1: TAP f1: 25 SRAP Depolarization, death (<30 min)Stable PD: -230 f2: TAP

SRAP3 -186 f0.5: 13 -76 f0.5: 20 SRAP f0.5: 4

f1: 20.3 f1: 27 -21 f1: 15f2: TAP f2: TAP Depolarization, death (<30 min)Stable PD: -185 SRAP

4 -230 f0.5: 3.7 -136 f0.5: 7 -116 f0.5: 12f1: 8.6 f1: 13 f1: TAPf2: 18 f2: TAP SRAPf3: TAP SRAP Depolarization, death (>60 min)Stable PD: -230

5 -203 f0.5: TAP -136 f0.5: TAP -112 f0.5: TAPStable PD: -200 SRAP SRAP

Depolarization, death (<60 min)6 -230 f0.5: 15.5 -130 f0.5: 36 -120 f0.5: 13

f1: 19.2 f1: TAP f1: 22f2: TAP SRAP f2: TAPStable PD: -230 Depolarization, death (<60 min)

7 -220 f0.5: 1 -135 f1: TAP -100 *Spontaneous action potentialf1: 9.4 SRAPf2: 12 Depolarization, death (<60 min)f3: TAPStable PD: -220

(b) 180sorb 100 Na/0.1 Ca

CellStable PD(mV)

Receptor potentialamplitude (mV)

Stable PD(mV) Receptor potential amplitude (mV)

1 -170 f0.5: TAP *Spontaneous singleaction potential

f0.5: no response

Stable PD: -154 -28 f1: no responsef2: no responsef3: no responsef4: 12.3 mVX no action potentialDepolarization, death

2 -220 f0.5: TAP -121 f0.5: TAPStable PD: -225 SRAP

Depolarization, death3 -223 f0.5: TAP SRAP f0.5: no response

Stable PD: -220 -22 f1: 2f3: X no action potentialDepolarization, death

4 -230 f0.5: TAP SRAP f0.5: TAPStable PD: -230 -80 SRAP

Depolarization, death5 -200 f0.5: 10 SRAP f0.5: no response

f1: 18 -21 f1: 11f2: 31 X no action potentialf3: TAP Depolarization, deathStable PD: -205



PD (-118 � 10 mV) and continued cytoplasmic streamingdespite having experienced spontaneous firing.

Interestingly, both touch-induced action potentials andspontaneous repetitive action potentials were of shorterduration in 100 Na/1.0 Ca than in 100 Na/0.1 Ca (compareFig. 2c,d with Fig. 2e,f).

Mechano-responses of cells transferreddirectly from 180sorb into 100 Na/0.1 Ca

All cells initially depolarized by approximately 100 mV to amean PD of -106 � 15.1 mV, after transfer from 180sorb to100 Na/0.1 Ca (Fig. 2g; Tables 2 and 3b). As discussed pre-viously, the amplitudes of small-scale resting PD oscillationsalso increased to between 5 and 10 mV with occasionalspikes of approximately 25 mV amplitude. The cells weresignificantly depolarized (P0 = 0.00) in comparison withtheir PD in APW and 180sorb (Table 2), but only slightlydepolarized in comparison with the PD of the first groupof cells, in 100 Na/1.0 Ca (P0 = 0.19). However, afterapproximately 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 furtherdepolarized to a mean PD of -53 � 41 mV (Table 2). Halfthe 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 100Na/0.1 Ca than in 100 Na/1.0 Ca (P0 = 0.01) because ofrepetitive firing.

Cells responded to mechano-stimulus in 100 Na/0.1 Cawith a different spectrum of responses compared with theirresponses in 180sorb (Table 3b, Fig. 2g).

Cells 1, 3 and 5, which were much depolarized prior tostimulation (Table 3b), became less sensitive to mechano-stimulus. These produced smaller-amplitude receptorpotentials or became entirely unresponsive (Fig. 2g).Mechano-stimulus was followed by gradual depolarizationto zero PD, cessation of cytoplasmic streaming, and deathwithin 60 min (Fig. 2g).

In contrast, cells 2 and 4 fired touch-induced actionpotentials (Table 3b). The action potentials were followedin both these cells by spontaneous repetitive firing, afterwhich cells depolarized to zero PD, ceased streaming, anddied.

Cell 6 alone became more sensitive to mechano-stimulus,responding to given stimuli with receptor potentials oflarger amplitude and an action potential with a lowerenergy stimulus than in 180sorb (Table 3b). This cell alsofired spontaneously and repetitively, depolarized, and died.

Thus, while all cells stimulated in 100 Na/1.0 Ca recov-ered a relatively hyperpolarized PD (-118.2 � 9.5 mV) andsurvived for longer than 60 min post-stimulus, mechano-stimulus in 100 Na/0.1 Ca was quickly lethal.

Current–voltage analysis andmathematical modelling

Neither the resting PD nor the I/V characteristicschanged significantly when turgor pressure was reduced in90sorb (Fig. 3a–d, data in APW in black, data in 90sorb inblue).

The resting PD depolarized from -236 � 7 to-184 � 20 mV, and the conductance increased approxi-mately threefold (Table 4; Fig. 3a–d, data in green) after60 min in 50 Na/1.0 Ca. Modelling identified classes oftransporters responsible. The background conductanceincreased from 0.45 � 0.15 to 2.0 � 0.3 S·m-2. The rate con-stant parameters kio

0 and koi of the electrogenic protonpump 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 notsurvive 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, depo-larized further to -155 � 7 mV. The PD range, whereclamping was feasible, also narrowed (Fig. 3a–d, data inred). Only two of the group of five cells survived voltageclamping, and only for ~30 min. There was no furtherchange in the background conductance. Both of the rate

Table 3. Continued

(b) 180sorb 100 Na/0.1 Ca

CellStable PD(mV)

Receptor potentialamplitude (mV)

Stable PD(mV) Receptor potential amplitude (mV)

6 -230 f0.5: no response SRAP f0.5: 15f1: 8 -112 f1: TAPf2: TAP SRAPStable PD: -230 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. ‘Stablepotential difference (PD)’ (in 180sorb) is the PD recovered following a touch-induced action potential. ‘Depolarization, death’ indicatesdepolarization to zero PD coupled with streaming cessation for 30 min or longer (see Fig. 2g). ‘X no action potential’ indicates where touchstimulus did not initiate an action potential.



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 directlyinto 50 Na/0.1 Ca. Of these, only one showed a low level ofproton pump activity (Table 4) after 60 min. The averageddata were modelled by the background conductance only(Fig. 3a–d in orange). The background conductanceincreased 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 notmechanically stimulated in each of the media (Table 1c).

Cell viability depended on both the external concentrationof NaCl and Ca2+, but not on the extracellular osmoticpressure: viability in 180sorb was slightly greater than inAPW. All cells had died within 5 d in 100 Na/0.1 Ca, whilehalf were still viable after 7 d in 100 Na/1.0 Ca. Viabilitydepended on extracellular [Ca2+]ext: all cells survived for7 d in modified APW including 10 mm Ca2+ (not shown),whereas only 70% survived for 7 d in APW containing0.1 mm Ca2+. The rate of cytoplasmic streaming(65 � 10 mm s-1) did not change significantly after 3 d inany medium except 100 Na/1.0 Ca and 100 Na/0.1 Ca,where it slowed to 50–45 mm s-1, respectively. Theseunstimulated cells remained viable for significantly longerin saline media than their mechanically stimulatedcounterparts.

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 90sorbto 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. Theextreme 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 fittedparameters 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 obtainedby the pump contribution to the upper limit and by changing the reversal potential difference (PD) for the background current from -100to -60 mV for the lower limit. The APW and 90sorb data were processed in a similar fashion, and the resulting parameter ranges areshown in Table 4, but the overlapping tolerance lines are not shown for clarity. (b) Fitted pump currents; (c) fitted backgroundconductances; (d) total conductances calculated as differentials of the total currents.



DISCUSSION

Historically, the use of simpler cell models has facilitatedunderstanding of complex multicellular systems, and giantcharophyte cells are prototypes of plant cell excitability,membrane transport (Tazawa & Shimmen 2001) andmechano-perception (Shimmen 2001). We have investi-gated the effects of salinity on mechano-perception byChara cells, as manifested in receptor and action potentials,as well as salinity-induced changes in electrophysiology.Reducing cell turgor pressure increases sensitivity tomechanical stimulus in Chara cells (Shepherd et al. 2001).Here, we report that an isotonic increase in salinity haseffects on the receptor potential and excitability, over and

above the effect of reduced turgor pressure. The natureof these effects depends on the extracellular Ca2+ ionconcentration.

Salinity-induced changes in ion transport:the points of no return

The PD of plant cells contains contributions from two com-ponents: first, the passive (background conductance), andsecond, the active and pump-specific. Reducing turgor pres-sure had negligible effects on these components (Fig. 3,Table 4; Shepherd et al. 2001). Similarly, mannitol-inducedturgor reduction has little effect on the proton pumpactivity in Mung bean (Nakamura et al. 1992). However, I/V

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 0.45 � 0.15 8 000 � 2000 100 � 20 -236 � 7 -377 � 1390sorb

Average time: 96 min 0.6 � 0.3 8 800 � 2800 100 � 20 -227 � 7 -379 � 1550 Na/1.0 Ca

Average time: 65 min 2.0 � 0.3 12 000 � 3000 130 � 25 -184, range: -202, -167 -394 � 1250 Na/0.1 Ca after 50 Na/1.0 Ca

Average time: 20–30 min 2.0 � 0.3 8 000 � 2500 60 � 25 -150, range: -169, -127 -364 � 2350 Na/0.1 Ca after 90sorb

Average time: 60 min 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. Theoutward 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 bestfit contained no contribution from the pump, while the upper limit contained the same contribution from the pump as in the previous dataset.The lower limit contained no contribution from the pump, and the reversal PD for the background current changed to -60 mV. Cells werein 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, after50 Na/1.0 Ca, 20–30 min; and 50 Na/0.1 Ca after 90sorb, 60 min.PD, potential difference; APW, artificial pond water.

Figure 4. Viability of cells, assessed bycytoplasmic streaming, in the absence ofmechano-stimulus. Cells were in thesaline and sorbitol-based media shown inTable 1c. Artificial pond water (APW)containing 10.0 mm Ca2+ was also tested,and all cells survived for 7 d.

0

2

4

6

8

10

12

1 2 3 4 5 6 7

Surviving cells

100Na/0.1Ca

100Na/1.0Ca

APW

100Na/10.0Ca

180sorb

Number of days



analysis reveals four salinity-specific changes in ion trans-port, the nature of which depends on [Ca2+]ext.

Firstly, a rapid and dramatic increase in the backgroundconductance partially short-circuits the proton pumpcurrent, and thus depolarizes the membrane PD (Fig. 3,Table 4). The background conductance increased sevenfoldin 50 Na/0.1 Ca [with low (Ca2+)ext] and fourfold in 50 Na/1.0Ca [with high (Ca2+)ext]. If the background conductancein 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 inhibitNSCCs of Arabidopsis protoplasts in a concentration-dependent manner (Demidchik & Tester 2002).

Secondly, currents generated by the electrogenic protonpump changed in a calcium-dependent manner, increasingin 50 Na/1.0 Ca and declining to negligible levels in 50Na/0.1 Ca (Fig. 3b, Table 4). Activation of the proton pumpappears to depend on [Ca2+]ext, or specifically on the ionicratios between [Na+]ext and [Ca2+]ext at constant [K+]ext.Chara cells depolarize to the passive component of the PDin Ca2+-free APW (Bisson 1984). Stimulation of protonpumping in 50 Na/1.0 Ca did not counteract salinity-induced depolarization of the resting PD (Fig. 3a), butestablished a PD (Table 4) negative to the excitationthreshold (APthreshold) of approximately -100 mV (Beilby2007). Stimulation of proton pumping in saline media wasdescribed in Mung bean (Nakamura et al.1992) and beanmesophyll (Shabala 2000). With negligible proton pumping(Fig. 3b), the background conductance becomes dominantin 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 initi-

ated trains of repetitive firing (Table 3a,b; Fig. 2g). Thisrepetitive firing resembles the onset of a critical instability,of the type described by Hayashi & Hirakawa (1980) in theNitella cell, where a time-ordered structure (repetitivefiring) emerged after ramping currents to the excitationthreshold. Most cells in 100 Na/1.0 Ca had PDs negative toAPthreshold (Table 2), and initiated repetitive firing only aftera touch-induced action potential, which shifted the PD tran-siently to APthreshold (Fig. 2d). Kishimoto (1966) attributedNaCl-induced repetitive firing in Nitella to calcium deple-tion at the cell membrane. Our results suggest this isbecause proton pump currents decline when [Ca2+]ext is low,shifting the resting PD to APthreshold.

Finally, the threshold for activation of the outward recti-fiers (KORthreshold) is approximately -50 mV (Fig. 3d), anddepolarization to more positive potentials leads to sus-tained K+ efflux.When [Ca2+]ext is low, increased backgroundconductance and loss of proton pump activity combine togradually depolarize the cell PD. Trains of spontaneousaction potentials, which involve net Cl- and K+ effluxes(Beilby 2007), accelerate the depolarization because thedeactivation of the proton pump precludes recovery of theresting 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, initiatingspontaneous repetitive firing and further depolarization toKORthreshold. At this point, K+ depletion becomes irrevo-cable. The background conductance increases to a lesserextent when [Ca2+]ext is high, and increased proton pumpactivity establishes a PD outside the danger zone of thecritical APthreshold. In the absence of mechano-stimulus, thiscircumvents both ‘points of no return’.

Interestingly, cells pre-treated in saline media with high[Ca2+]ext had more negative PDs than those transferreddirectly to media with low [Ca2+]ext (Tables 3a and 4). Suchpre-treatment may delay the eventual displacement of Ca2+

by Na+ at negative sites in the cell wall (Shabala & Newman2000).

We do not yet know whether excitation plays a role inresponses of land plant cells to salinity. In the 1990s, whole-cell patch clamp studies of various cereal roots suggestedthe possibility that the K+ outward rectifier could maintainan outward K+ current while less selective cation channelspermit Na+ entry (reviewed by Tyerman & Skerrett 1999).Salinity induces K+ efflux via the outward rectifier in landplant cells (Shabala et al. 2006), and this depends on themagnitude 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 (Karolet al. 2001), and these cellular responses to salinity are likelyto be fundamental, with ancient antecedents.

The salt-tolerant turgor-regulating charophytes, such asChara longifolia (Yao, Bisson & Brzezicki 1992) and Lam-prothamnium succinctum (Beilby & Shepherd 2001), avoidthe ‘points of no return’ through activation of the protonpump, which maintains a negative resting PD (reviewed byBeilby, Bisson & Shepherd 2006). Decreased turgor pres-sure activates the proton pump in Lamprothamnium (AlKhazaaly & Beilby 2007). Similarly, Thellungiella, a salt-tolerant relative of Arabidopsis, maintains a negativeresting PD and regulates turgor during salt stress (Inanet al. 2004; Volkov & Amtmann 2006).

Mechano-perception in saline media as afunction of [Ca2+]ext

The receptor potential difference (RPD) involves activationof mechano-sensitive Ca2+ channels at the plasma mem-brane, transient elevation of intracellular Ca2+ and efflux ofCl- via Ca2+-activated Cl- channels, resulting in transientdepolarization (Kaneko et al. 2005). The magnitude of Ca2+

influx increases with increasing stimulus strength, account-ing for incremental depolarization (‘RPD amplitude’).

Most cells became hypersensitive to mechanical stimulusin 100 Na/1.0 Ca.The RPD amplitudes increased, relative toRPD amplitudes in sorbitol (Fig. 2a–c, Table 3a). This wasan effect of NaCl, not simply a result of turgor reduction.Cells were thereby more likely to fire a touch-inducedaction potential (Table 3a, Fig. 2c), followed by repetitivefiring as the repolarizing PD (-90 � 23 mV) approachedAPthreshold (Fig. 2d).



Stimulation of proton pumping enabled restoration ofthe resting PD. However, each action potential depletes thecell of K+ and Cl- (Beilby 2007), transiently inhibits photo-synthesis (Bulychev et al. 2004; Krupenina & Bulychev2007), and presumably has further indirect impacts onelectrogenic pump-driven transport via ATP depletion.Although cells re-established a PD negative to APthreshold,and all remained viable for at least 60 min followingmechano-stimulus, even slight mechano-stimulus producesmultiple excitations and K+ efflux. Hypersensitivity tomechano-stimulus thus increases the propensity for depo-larization to APthreshold and KORthreshold.

Cells in 100 Na/0.1 Ca responded to given stimuli morehaphazardly. Following spontaneous repetitive firing, muchdepolarized cells (e.g. with a PD of -22 mV) were desensi-tized to mechano-stimulus, producing smaller-amplitudeRPDs or becoming inexcitable (Table 3a,b; Fig. 2g). In con-trast, cells maintaining a more negative PD (e.g. -80 to-121 mV) retained their sensitivity to mechano-stimulus.Some cells became hypersensitive, producing RPDs oflarger amplitude and touch-induced action potentials(Fig. 2e). Following repetitive firing (Fig. 2f), the cells depo-larized to a PD more positive than KORthreshold (Table 3a,b).Unsurprisingly, all cells died within 60 min of mechano-stimulus, whereas 50% of non-stimulated cells remainedviable after 2 d in 100 Na/0.1 Ca, and 90% were viable after2 d in 100 Na/1.0 Ca (Fig. 4).A high [Ca2+]ext to [Na+]ext ratioin the medium has previously been found to enhance thesurvival of C. corallina cells in saline media (Tufariello,Hoffmann & Bisson 1988).

Furthermore, the ratio between [Ca2+]ext and [Na+]ext hasdramatic effects on action potential. The duration of bothtouch-induced and spontaneous repetitive action potentialswas shorter in 100 Na/1.0 Ca than in 100 Na/0.1 Ca(compare Fig. 2c,d with Fig. 2e,f). Presumably, theexcitation-associated net effluxes of Cl- and K+ were greaterin 100 Na/0.1 Ca. Monovalent cations, including Na+,prolong the action potential, but divalent ions, includingCa2+, suppress this effect (Shimmen et al. 1976).

Increased salinity eventually induces fatal depolariza-tion, 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 synergybetween mechano-stimulus, [Ca2+]ext and [Na+]ext is excita-tion, both mechanically and spontaneously induced. Post-excitation recovery depends on proton pump activityre-establishing a PD negative to APthreshold, and the pump isactive only when [Ca2+]ext is high. When [Ca2+]ext is low, cellsdepolarize to KORthreshold following excitation.

Cells are either desensitized or hypersensitizedto mechano-stimulus in saline media: the rolesof turgor pressure and Cl- conductance

Superficially, the production of larger-amplitude RPDs(increased Ca2+ influx and Cl- efflux) in saline mediadepends on membrane PD (Table 3a,b), but the generationof RPD does not depend on the H+ pump (Shimmen

1997b). Two different factors contribute to the increasedRPD amplitudes. The first lacks measurable electrophysi-ological correlates. As reported previously, cells are sensi-tized to mechano-stimulus when turgor pressure is reduced(Shepherd et al. 2001), but neither the cell PD nor the back-ground conductance changes significantly (Fig. 3) (Kiy-osawa & Ogata 1987; Shepherd et al. 2001).

We do not know why cells with reduced turgor are sen-sitized to mechanical stimulation. Decompression stimulusinduces smaller-amplitude RPDs when turgor pressure isreduced, suggesting that changed membrane tension is acrucial factor (Iwabuchi et al. 2008). Changed membranetension is unlikely to account for increased RPD ampli-tudes in our experiments, however, because membranetransfer between plasma membrane and endocytoticvesicles compensates for osmotic contraction in hypertonicsolution (Wolfe & Steponkus 1983; Wolfe, Dowgert & Ste-ponkus 1985; Dowgert, Wolfe & Steponkus 1987). Theplasma membrane is probably under comparable smalltension in both APW and 180sorb.

Previously, we suggested that a continuum among cellwall, plasma membrane and cytoskeleton (Baluska et al.2003; Telewski 2006; Pickard 2007) is critical to mechano-perception in Chara (Shepherd et al. 2001). Future experi-ments will investigate the effects on generation of receptorand action potentials of inhibiting either or both actin andmicrotubule cytoskeletons.

A second, electrophysiological factor contributes toincreased RPD amplitudes. The hundredfold increase in[Cl-]ext results in a reversal PD for Cl- (ECl) of approxi-mately -50 mV, assuming a [Cl-]cyt of 10 mm (Coster 1966).Thus, the driving force for Cl- efflux, on which the RPDdepolarization depends, is negligible in cells with a restingPD close to ECl.At PDs more positive than ECl, the currentsare diminished by the inwardly rectifying properties of theCl- channels (Beilby & Shepherd 2006b). This is borne outby the data, where cells with PDs close to ECl became unre-sponsive or showed reduced RPD amplitudes (Table 3a,b).

On the other hand, cells with more negative PDs werehypersensitized to mechano-stimulus, showing increasedRPD amplitudes with given stimuli (Table 3a,b). Prelimi-nary modelling, based on Beilby & Shepherd (2006b),shows that an increase in [Cl-]cyt would result in an increasein Cl- currents, which may contribute to the observedhypersensitivity.

Potential role of excitation in responses ofland plants to salinity

The action potentials of Chara cells reflect those of landplant cells (Tazawa & Shimmen 2001; Fisahn et al. 2004),and mechanically stimulated or spontaneous excitationsmay thus be widely relevant to understanding land plantresponses to salinity. Action and/or variation potentialssuppress photosynthesis in Chara (Krupenina & Bulychev2007) and in land plants (Koziolek et al. 2004; Lautner et al.2005). Salt (KCl) applied to roots induces action potentialsin leaves (Favre, Greppin & Degli Agosti 2001; Felle &



Zimmermann 2007). Salinity reduces photosynthetic effi-ciency (e.g. Shabala et al. 2005), and mechano-perceptionand/or excitation may contribute. Excitation combined withproton pump inhibition is another possible avenue for theK+ depletion, which is thought (Maathuis & Amtmann1999) to underlie NaCl toxicity. Circumvention of the criti-cal 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 ofmechanically stimulated and spontaneous action potentials

Proton pump stimulated

Fourfold increase in background conductance

Membrane PDs negative to APthreshold

No spontaneous excitation

Cell PD is negative to KORthreshold

Cells hypersensitive to mechano-stimulus

Mechano-stimulus leads to repetitive excitation

PD remains negative to APthreshold, and KORthreshold

Cells viable

Proton pump deactivated

Sevenfold increase in background conductance

Membrane potential depolarizes to APthreshold

Spontaneous repetitive excitations (of extended duration)

Cell PD is positive to KORthreshold.

At KORthreshold, K+ efflux sustained

and irrevocable

Cells desensitized to mechano-stimulus

No response to mechano-stimulus

PD continues depolarizing

Cells not viable

Stable: Membrane potential Conductance

Sorbitol (reduction of turgor pressure)

Salt water high calcium

Salt water low calcium

Freshwater

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 themembrane potential to action potential (AP)threshold. Spontaneous repetitive excitations of extended duration are the result, followed bydepolarization to potential differences (PDs) positive to KORthreshold. Although these cells are desensitized to mechano-stimulus, K+ effluxis then sustained and irrevocable. On the other hand, cells retaining a PD negative to K+ outward rectifier (KOR)threshold (and ECl) becomehypersensitive to mechano-stimulus, and further excitation and K+ efflux then drive the PD to KORthreshold. Cell viability was extended inthe absence of mechano-stimulus. When [Ca2+]ext is high, the smaller background current and stimulation of proton pump currentsestablish 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+-dependentactivation of the proton pump re-establishes a PD slightly negative to APthreshold and well negative to KORthreshold, and cells remain viablefor longer when [Ca2+]ext is high. Cell viability was extended in the absence of mechano-stimulus.



in the salt sensitivity of Chara cells. The cells eventuallydepolarize and die in saline media, but mechanically stimu-lated and spontaneous excitation augments the process in aCa2+-dependent manner. Two critical membrane potentialthresholds, the excitation threshold (APthreshold) and thethreshold for activation of the K+ outward rectifier(KORthreshold), act as ‘points of no return’ in determining cellviability in saline media. This is summarized in Fig. 5.

Molecular approaches have demonstrated the impor-tance of calcium signalling in salt sensitivity (Zhu 2001).Action potentials function at a higher organizational levelas systemic Ca2+ signals, and their role in the responses ofland plants to increased salinity is an avenue for furtherstudy.

ACKNOWLEDGMENTS

Virginia Shepherd received a grant from the Japan Societyfor the Promotion of Science and the Australian Academyof Science, to visit Professor Shimmen’s lab at the Univer-sity of Hyogo. We thank Professor N.A. Walker for readingthe 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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