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Ethylene activates a plasma membrane Ca2+-permeable channel in tobacco suspension cells

Min-Gui Zhao, Qiu-Ying Tian and Wen-Hao Zhang.Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany,

the Chinese Academy of Sciences, Beijing 100093, China

Summary

• Here, the effects of the ethylene-releasing compound, ethephon, and the ethyleneprecursor, 1-aminocyclopropane-1-carboxylic acid (ACC), on ionic currentsacross plasma membranes and on the cytosolic Ca2+ activity ([Ca2+]c) of tobacco(Nicotiana tabacum) suspension cells were characterized using a patch-clamptechnique and confocal laser scanning microscopy.• Exposure of tobacco protoplasts to ethephon and ACC led to activation of aplasma membrane cation channel that was permeable to Ba2+, Mg2+ and Ca2+,and inhibited by La3+, Gd3+ and Al3+.• The ethephon- and ACC-induced Ca2+-permeable channel was abolished by theantagonist of ethylene perception (1-metycyclopropene) and by the inhibitor ofACC synthase (aminovinylglycin), indicating that activation of the Ca2+-permeablechannels results from ethylene. Ethephon elicited an increase in the [Ca2+]c of tobaccosuspension cells, as visualized by the Ca2+-sensitive probe Fluo-3 and confocalmicroscopy. The ethephon-induced elevation of [Ca2+]c was markedly inhibited byGd3+ and BAPTA, suggesting that an influx of Ca2+ underlies the elevation of [Ca2+]c.• These results indicate that an elevation of [Ca2+]c, resulting from activation of theplasma membrane Ca2+-permeable channels by ethylene, is an essential componentin ethylene signaling in plants.

New Phytologist (2007) 174: 507–515

© The Authors (2007). Journal compilation © New Phytologist (2007) doi: 10.1111/j.1469-8137.2007.02037.x

Author for correspondence: Wen-Hao Zhang Tel: +86 10 62836697Fax: +86 10 62592430 Email: [email protected]

Received: 4 January 2007 Accepted: 24 January 2007

Key words: cytosolic Ca2+ activity, ethylene, patch-clamp, plasma membrane Ca2+-permeable channel, tobacco (Nicotiana tabacum) suspension cells.

Introduction

Ethylene is a gaseous plant hormone that is involved in awide spectrum of important physiological processes inplants, ranging from seed germination to nodulation initiation(Bleecker & Kende, 2000; Kwak et al., 2006). Ethylene also

plays an important role in perception and transduction signalsassociated with both biotic (van Loon et al., 2006) andabiotic (Munné-Bosch et al., 2004) stresses. There issubstantial evidence that ethylene is closely related to otherplant hormones, including auxin, cytokinin, abscisic acid(ABA), salicylic acid and jasmonate, in terms of its signaling

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pathways and responses to biotic and abiotic stress in plants(Guo & Ecker, 2004; Etheridge et al., 2006; Kwak et al.,2006). Ethylene is synthesized from methionine throughS-adenosyl-L-Met and 1-aminocyclopropane-1-carboxylic acid(ACC) (Kende, 1993). The rate-limiting step of ethylene biosyn-thesis is believed to lie in the production of ACC by ACC syn-thease, which is followed by the conversion of ACC to ethyleneby ACC oxidase (Kende, 1993; Bleecker & Kende, 2000).

Recent studies have revealed that there is a functionallink between ethylene and reactive oxygen species (ROS) ingeneral, and hydrogen peroxide in particular, in mediatingstomatal closure (Desikan et al., 2005, 2006) and programmedcell death (de Jong et al., 2002). For instance, ethylene-inducedstomatal closure in Arabidopsis is dependent on H2O2production in guard cells, and the ethylene receptor mutantsetr1-1 and etr1-3 are insensitive to ethylene in terms ofstomatal closure and H2O2 production (Desikan et al., 2006).In this context, the function of ethylene is analogous to ABA,which elicits H2O2 production and stomatal closure viaelevating the cytosolic Ca2+ activity ([Ca2+]c) through activat-ing Ca2+-permeable channels in the plasma membranes ofguard cells (Grabov & Blatt, 1998; Hamilton et al., 2000;Pei et al., 2000; Murata et al., 2001). However, it remains tobe characterized whether an elevation of [Ca2+]c, as a resultof activation of the plasma membrane Ca2+-permeablechannels, is involved in the ethylene-induced stomatal closure.In addition to stomatal movement, there is evidence indi-cating that the ethylene-mediated pathogenesis (Raz &Fluhr, 1992) in tobacco and root-hair formation, and growthin pea (Petruzzelli et al., 2003), are dependent on Ca2+. How-ever, there has been no direct experimental evidence to showwhether ethylene affects [Ca2+]c and modulates the plasmamembrane Ca2+-permeable channels. Elevations of [Ca2+]cof plant cells by ABA (Gehring et al., 1990; McAinsh et al.,1990), auxin (Gehring et al., 1990), nitric oxide (Garcia-Mata et al., 2003), jasmonic acid (Sun et al., 2006), H2O2(Pei et al., 2000; Foreman et al., 2003), free oxygen radicals(Demidchik et al., 2003) and calmodulin (Shang et al., 2005)have been reported.

Two types of voltage-gated Ca2+-permeable channels in theplasma membranes of plants have been identified: depolarisation-activated (Thuleau et al., 1994; Thion et al., 1998); and hyper-polarisation-activated (Kiegle et al., 2000; Hamilton et al., 2000;Pei et al., 2000; Very & Davies, 2000; Murata et al., 2001;Klusener et al., 2002; Lecourieux et al., 2002; Perfus-Barbeochet al., 2002; Wang et al., 2004; Shang et al., 2005). Depolarisation-activated channels are likely to play a role in signal transduction(Miedema et al., 2001), whereas hyperpolarisation-activatedCa2+ channels (Miedema et al., 2001), together with voltage-independent Ca2+-permeable nonselective cation channels(Demidchik et al., 2002; White & Davenport, 2002), mayfunction in the acquisition of Ca2+. The depolarisation-activated Ca2+-permeable channels are regulated by microtubules(Thion et al., 1996, 1998). Activity of the hyperpolarisation-

activated plasma membrane Ca2+ channels is modulated byROS (Pei et al., 2000; Murata et al., 2001; Klusener et al., 2002;Lecourieux et al., 2002; Demidchik et al., 2003; Foremannet al., 2003), fungal elicitors (Gelli et al., 1997), cAMP(Lemtiri-Chlieh & Berkowitz, 2004), calmodulin (Shanget al., 2005) and cytochalsin D (Wang et al., 2004). In thepresent study, we demonstrated, using the whole-cell patch-clamp technique, that ethylene activates a Ca2+-permeablechannel in the plasma membranes of tobacco suspensioncells, and found, using the Ca2+-sensitive fluorescent probe,Fluo-3, and confocal laser scanning microscopy, that ethyleneelicits an increase in the cytosolic Ca2+ activities.

Materials and Methods

Tobacco suspension cells (Nicotiana tabacum L. cv. Samsun),initiated from pith parenchyma cells, were grown inGamborg’s B5 liquid medium, supplemented with 1 µM 2,4di-chlorophenoxyacetic acid, under dim lighting with photo-synthetic flux density of approx. 20 µmol m−2 s−1 at 25°C, ona rotary shaker at a speed of 100 r.p.m. and were subculturedevery 14 d. Protoplasts of tobacco cells were isolated byincubating the suspension cells in an enzyme solution of 0.4%(w/v) cellulase (Onozuka RS; Yakult Ltd., Honsha, Japan) and0.04% (w/v) pectolyase (Sigma-Aldrich) polyvinypryrrolidone(PVP) containing 0.5% (w/v) bovine serum albumin, 1 mM

CaCl2, 500 mM sorbitol, 2 mM ascorbic acid and 10 mM Mes/Tris (pH 6.0), for 1 h at 25°C. A sucrose density gradient, asdescribed previously (Zhang et al., 1997), was used to collect cleanprotoplasts. The protoplasts were kept on ice until patch clamped.

Electrophysiology and data analysis

Patch pipettes, pulled from borosilicate glass blanks (ClarkElectromedical, Reading, UK), were coated with SylgardR

(Dow Corning, Midland, MI, USA). The voltage across thepatch was controlled and the current measured using anAxopatch 200 A (Axon Instruments, Foster City, CA, USA).Series resistance and capacitance were compensated. Voltagepulses of approx. 4 s in duration were used to study thevoltage-dependent current. Current–voltage curves fromwhole-cell recordings were constructed from a series of voltagesteps. Current–voltage curves were also obtained by applyingrepetitive voltage ramps of 4 s in duration, ranging from−200 mV to 40 mV. Data were sampled at 2 kHz and filteredat 0.5 kHz by a low-pass 4 pole Bessel filter. Sufficient timebetween voltage pulses was given to allow the current to settleto a steady-state level at each holding potential before a newpulse protocol was initiated. Records were stored and analysedusing pClamp 8.0 (Axon Instruments, Foster City, CA, USA).All experiments were carried out at room temperature (20–22°C). Junction potentials were calculated, and corrected for,by using the program JPCALC (P. H. Barry, University of NewSouth Wales, Sydney, Australia).

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Experimental solutions

The pipette solution was composed of 10 mM BaCl2, 0.1 mM

dithiothreitol, 4 mM EGTA, 10 mM HEPES, pH 7.2, andhad an osmolality of 720 mOsm kg−1, adjusted with Tris andsorbitol, respectively. All bath solutions contained 5 mM Mes,pH 6.0, and had an osmolality of 700 mOsm kg−1, adjustedwith Tris and sorbitol, respectively. Details of bath solutionsare given in the appropriate figure legends.

Measurement of cytosolic Ca2+ activity

Tobacco suspension cells were incubated in a solution containing0.5 mM CaCl2 and 20 µM Ca2+-sensitive fluorescent dye,Fluo-3/AM (Molecular Probe, Eugene, OR, USA), at 4°C for1 h, followed by a 3-h incubation at 25°C in the dark, asdescribed by Zhang et al. (1998). After washing with theincubation solution (0.5 mM CaCl2), the suspension cellswere mounted in a specifically designed chamber flooded withthe standard incubation solution for imaging using a confocalmicroscope. The Fluo-3 fluorescence of the suspension cellswas measured with a laser confocal scanning microscope(LSM 510; Zeiss, Oberkochen, Germany). The wavelength ofexcitation light was 488 nm, and the emission signals of> 515 nm were used to collect the images. Three-dimensionalscanning was performed every 5 min with a 2-µm Z-seriesproject step, and three-dimensional reconstructed imagesof individual suspension cells were used to calculate therelative fluorescence. The fluorescence intensity of the entireindividual suspension cells was determined using the ZEISS LSM

510 software and was expressed in pixel numbers on a scaleranging from 0 to 255.

Results

Ethephon-activated whole-cell current

A small inward and outward current was elicited by membranehyperpolarisation and depolarisation in the bath solutioncontaining 10 BaCl2 in the absence of the ethylene-releasingcompound, ethephon (Fig. 1a). Exposure of tobacco protoplaststo ethephon led to a marked increase in the whole-cell current,and the ethephone-induced whole-cell current was stronglydependent upon the voltages (i.e. the current became moreactivated with membrane hyperpolarizing in the presence ofethephone) (Fig. 1b,c). The ethephon-elicited current wasobserved within 1–2 min of exposure to ethephon, and thecurrent increased with the exposure time and reached amaximum approx. 30 min after the addition of ethephon tothe bath solution (Fig. 1b, inset). The ethephon-activated currentwas found in 74% of the protoplasts examined (n = 32).

When protoplasts were pretreated with 1-metylcyclopropene(1-MCP), which inhibits ethylene binding to the receptor,whole-cell currents across plasma membranes of the tobaccoprotoplasts remained relatively unchanged upon exposure toethephon (Fig. 1d), suggesting that the observed activation ofinward current by ethephon is attributed to the effect of ethylene.

Ethephon-activated current is carried by an influx of divalent cations

To test whether the ethephone-dependent inward currentis caused by cation (Ba2+) influx or anion (Cl–) efflux, theresponse of the inward current to changes in concentrationof the external BaCl2 was investigated. When external BaCl2

Fig. 1 Effect of the ethylene-releasing compound, ethephon, on the whole-cell current (Im) across the plasma membrane of tobacco (Nicotiana tabacum) protoplasts. The whole-cell current was elicited by voltage pulses ranging from −182 to 58 mV from a holding potential of −2 mV before (a) and 30 min after (b) the addition of 100 µM ethephon to the bath solution containing 100 mM BaCl2, 5 mM Mes, pH 6.0. Inset, time-course of changes in current density at −182 mV in response to exposure to 100 µM ethephon. Mean steady current–voltage curves in the absence and presence of ethephon are shown in (c). The data represent the mean ± standard error (SE) of 17 protoplasts. (d) Current–voltage curves for whole-cell currents in 1-metycyclopropene (1-MCP)-pretreated protoplasts before and after the addition of ethephon to the bath (bath solution as in (a)). Data represent the mean ± SE of three protoplasts.

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was reduced from 100 to 10 mM in the bath solution, themagnitude of the ethephon-dependent inward currents wasreduced and the reversal potential of the currents shiftedfollowing the equilibrium potential for Ba2+ (EBa), but itshifted opposite the equilibrium potential for Cl– (ECl)(Fig. 2a). This indicates that the ethephon-activated channelis highly permeable for Ba2+ over Cl–, with the permeabilityratio between Ba2+ and Cl– (PBa/PCl), of 23.4 ± 4.5 (n = 3),calculated using the Goldman equation (Bertil, 1992). Therelative permeability of the ethephon-activated channels toother cations was also evaluated by substitution of Ba2+ inthe bath solution with other equilmolar divalent cation ions(Ca2+, Mg2+, Mn2+) and monovalent cation ions (200 mM

Na+ and TEA+) while maintaining a constant Cl– concentration.The channel responsible for the ethephon-activated inwardcurrent was almost equally permeable to Ba2+, Mg2+ and Ca2+,but less permeable to Mn2+ and displayed little permeabilityto the monvalent cations of Na+ and TEA+ (Fig. 2b,c).

The ethephon-activated current was sensitive to the broad-spectrum calcium-channel blockers, Gd3+ and La3+, and toAl3+ (Fig. 2d). For example, 100 µM Gd3+, La3+ and Al3+

(pH 4.0) inhibited the ethephon-activated current at −182 mVby 68.3 ± 4.6%, 70.5 ± 5.7% and 58.7 ± 6.4% (n = 3),respectively. Taken together, these findings reveal thatethephon activates a cation channel permeable to Ba2+,Mg2+ and Ca2+, and that the ethephon-dependent channel isinhibited by Gd3+, La3+ and Al3+.

Ethylene precursor, ACC, also activated the divalent cation channels

To test, in more detail, whether the ethephon-activated Ca2+-permeable channels results from ethylene, the effect of the

ethylene precursor (ACC) on the whole-cell current of tobaccoprotoplasts was also investigated. As shown in Fig. 3, theexposure of tobacco protoplasts to 500 µM ACC also elicitedan inward current, and the ACC-induced inward currentswere inhibited by La3+ and Gd3+ (Fig. 3d).

To test whether the ACC-induced inward current resultsfrom the conversion of ACC to ethylene by ACC synthase,the effect of the ACC synthase inhibitor, aminovinylglycine(AVG), on the ACC-dependent current was studied. Whenprotoplasts preincubated in 100 µM AVG for 1 h were chal-lenged with ACC, no ACC-induced current was found(n = 5) (Fig. 4a–c), suggesting that the ethylene derived fromACC accounts for the ACC-activated inward current.

The ACC-induced inward current was smaller than theethephon-induced current at the same concentrations. There-fore, ethephon is more potent in activating the divalent cationchannels in tobacco protoplasts than ACC. For example,ethephon, at concentrations as low as 10 µM, resulted in a sig-nificantly enhanced current (Table 1). By contrast, ACC at100 µM only marginally stimulated the current, and the cur-rent magnitude activated by 500 µM ACC was approx. 50%of that activated by 100 µM ethephon (Table 1). Seal resist-ance between the patch-pipette and the protoplasts oftendeteriorated when higher concentrations of ethephon wereused (data not shown), making it impossible to examine theireffect on the whole-cell current.

Effect of ethephon on cytosolic Ca2+ activity in tobacco cells

Activation of the Ca2+-permeable channel in the plasmamembranes of tobacco cells by ethephon will mediate aninflux of Ca2+ into the protoplasts, leading to an elevation of

Fig. 2 Ethephon-induced current, measured in the external solution of 100 mM and 10 mM BaCl2, in the presence of 100 µM ethephon (a). E100Ba, E10Ba, E100Cl and E10Cl are the equilibrium potentials for Ba2+ and Cl– in the 100- and 10-mM BaCl2 solutions, respectively. (b) Current–voltage curves for currents induced by ethephon in bath solutions containing 100 mM BaCl2, 100 mM MgCl2, 100 mM CaCl2, 100 mM MnCl2, 200 mM NaCl and TEACl. (c) Current densities induced by ethephon at −182 mV in varying external cations. Data represent the mean ± standard error (SE) of three to five tobacco (Nicotiana tabacum) protoplasts. (d) Current–voltage curves for ethephon-induced currents treated with channel blockers of 100 µM GdCl3, LaCl3, AlCl3. Data represent the mean ± SE of three to four protoplasts. All bath solutions are identical to those described in Fig. 1, except for the AlCl3 treatment in which the pH of the bath solution was adjusted to 4.0.

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[Ca2+]c. To test whether ethephon induces an increase in[Ca2+]c, the effect of ethephon on the [Ca2+]c of the tobaccocells was investigated using a Ca2+-sensitive fluorescent probe,Fluo-3, and confocal laser scanning microscopy. When thetobacco suspension cells were imaged under confocalmicroscopy, relatively weak fluorescence from the suspensioncells was detected, and a marginal increase in the fluorescenceover time was observed for cells incubated in the controlsolution (Fig. 5a,e). By contrast, a marked increase in theFluo-3-dependent fluorescence was observed upon additionof ethephon to the incubation solution (Fig. 5b,e). Theincrease in Fluo-3-dependent fluorescence indicates thatethephon elicits an increase in the [Ca2+]c. The increase inthe [Ca2+]c could result from either an influx of Ca2+ from theapoplasm and/or Ca2+ release from intracellular stores. Toidentify the source of Ca2+ underlying the ethephon-elicitedincrease in [Ca2+]c, the effects of the Ca2+-channel blocker,Gd3+, and of the Ca2+ chelator, BAPTA, on the ethephon-dependent increase in [Ca2+]c were studied. The ethephon-

elicited increase in [Ca2+]c was abolished when the cells weretreated with Gd3+ and BAPTA (Fig. 5c–e). As shown inFig. 5(e), the Fluo-3 fluorescence intensity (expressed as pixelnumbers) from suspension cells that were not challengedby ethylene remained relatively constant, whereas thefluorescence intensity exhibited a rapid increase in suspensioncells upon exposure to ethephon. The ethephone-elicitedincrease in fluorescence intensity was abolished when thesuspension cells were pretreated with Gd3+ and BAPTA(Fig. 5e). Therefore, these findings suggest that the ethephon-induced increase in [Ca2+]c is likely to result from Ca2+ influxthrough Ca2+-permeable channels in the plasma membranesof tobacco cells.

Discussion

In the present study, we found that, using the whole-cellpatch-clamp technique, the treatment of tobacco suspensioncells with the ethylene-releasing compound, ethephon, andthe ethylene precursor, ACC, activated Ca2+-permeablecation channels in plasma membranes of tobacco suspensioncells (Fig. 1). Inhibitors of ethylene binding to receptors(1-MCP) and ethylene biosynthesis (AVG) diminishedthe ethephon- and ACC-induced plasma membrane Ca2+-permeable channels (Figs 1c and 3), indicating that theactivation of Ca2+-permeable channels by ethephon and ACCcan be attributed to ethylene. Ethephon elicited a rapidelevation of [Ca2+]c of the same tobacco suspension cells, andthe elevation of [Ca2+]c induced by ethephon was diminishedby the Ca2+-channel blocker, Gd3+, and the Ca2+ chealtor,BAPTA (Fig. 5), suggesting that the influx of Ca2+ throughethephon-activated Ca2+-permeable channels underpins theelevation of [Ca2+]c. Free cytosolic Ca2+ ions play a pivotal

Fig. 3 Whole-cell currents (Im) in the absence (a) and presence of 500 µM 1-aminocyclopropane-1-carboxylic acid (ACC) (b). Current–voltage curves for currents measured before and after treatment with 500 µM (c) and currents measured in the absence and presence of 100 µM LaCl3 and GdCl3 (d). Data represent the mean ± standard error of three to five tobacco (Nicotiana tabacum) protoplasts. All bath solutions contained 100 mM BaCl2, 5 mM Mes, pH 6.0, osmolality of 700 mOsM.

Table 1 Effect of ethephon and 1-aminocyclopropane-1-carboxylic acid (ACC) on the whole-cell current density (Im) of tobacco (Nicotiana tabacum) suspension cells measured at −182 mV

Treatment Im (mA m−2)

Control −15.7 ± 5.9 (17)+0.01 mM ethephon −33.2 ± 7.2 (4)+0.1 mM ethephon −58.7 ± 8.6 (17)+0.1 mM ACC −21.8 ± 5.5 (6)+0.5 mM ACC −29.5 ± 7.8 (7)

Data represent the mean ± standard error (SE), with the number of protoplasts given in parenthesis.

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role in the transduction of various hormonal andenvironmental signals to the responsive element of cellularmetabolism in plants (Sanders et al., 2002). It is conceivablethat the ethylene-induced increase in [Ca2+]c acts as a signalto activate Ca2+-dependent proteins and protein kinases, thus

modulating ethylene-dependent physiological processes. Tothe best knowledge of the authors, the results presented in thepresent study show that, for the first time, ethylene modulates[Ca2+]c of higher plant cells through activating Ca2+-permeable channels in the plasma membranes. Therefore, thesefindings highlight that interactions between ethylene and[Ca2+]c could be of critical importance in the perception andsignaling of ethylene in plants.

Hyperpolarisation-activated Ca2+-permeable channels inthe plasma membranes have been characterized in many plantcells, including guard cells (Grabov & Blatt, 1998; Hamiltonet al., 2000; Pei et al., 2000), root elongating cells (Kiegleet al., 2000), root hairs (Very & Davies, 2000), pollens andpollen tubes (Wang et al., 2004; Shang et al., 2005), andtomato suspension cells (Gelli et al., 1997). The ethylene-induced, hyperpolarisation-activated Ca2+-permeable channelsidentified in the present study were comparable to thehyperpolarisation-activated Ca2+-permeable channels in theliterature in terms of permeability, pharmacological profilesand activation kinetics (cf. Kiegle et al., 2000; Pei et al., 2000;Very & Davies, 2000; Shang et al., 2005). Therefore, it islikely that the ethylene-dependent cation channels belong tothe same family of plant divalent cation channels.

The observation that plasma membrane Ca2+-permeablechannels in tobacco suspension cells were activated by ethyl-ene suggests that modulation of Ca2+ influx through theethylene-dependent Ca2+ channels may be a key event in theethylene signaling cascade. In this context, the hyperpolarisation-activated Ca2+-permeable channels have been shown to bemodulated by ROS (Pei et al., 2000; Demidchik et al., 2003;Foremann et al., 2003), ABA (Grabov & Blatt, 1998;Hamilton et al., 2000), fungal elicitor (Gelli et al., 1997),actin cytoskeleton (Wang et al., 2004), cAMP (Lemtiri-Chlieh& Berkowitz, 2004) and calmodulin (Shang et al., 2005).Interestingly, ethylene is closely associated with ABA inmodulating seed germination (Beaudoin et al., 2000) andstomatal closure (Desikan et al., 2005), with ROS in pro-grammed cell death (de Jong et al., 2002; Yakimova et al., 2006),with calmodulin in heat stress (Larkindale & Knight, 2002),and in leaf senescence and death (Yang & Poovaiah, 2000) andfungal and bacterial pathogen attack (van Loon et al., 2006).

The activation of the plasma membrane Ca2+-permeablechannels by ethylene, and the subsequent elevation of [Ca2+]c,may provide an explanation for the findings that ethylene elic-its stomatal closure in Arabidopsis (Desikan et al., 2005), asan increase in [Ca2+]c often precedes stomatal closure inducedby ABA and ROS (McAinsh et al., 1990; Pei et al., 2000). Inaddition to guard cells, there have been reports indicating thepotential involvement of plasma membrane Ca2+-permeablechannels in ethylene signaling and response. For instance,ethylene markedly stimulates cadmium-induced cell death(Yakimova et al., 2006). This can be accounted for by the factthat ethylene stimulates cadmium influx through Ca2+-permeable channels, thus stimulating cadmium-dependent

Fig. 4 Whole-cell currents (Im) in tobacco (Nicotiana tabacum) protoplasts pretreated with 500 µM aminovinylglycin (AVG), an inhibitor of ethylene synthase, before (a) and after challenge with 500 µM 1-aminocyclopropane-1-carboxylic acid (ACC) (b). Current–voltage curves for the currents were determined in the absence and presence of ACC in protoplasts pretreated with AVG. Data represent the mean ± standard error of five protoplasts.

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cell death. There is evidence that cadmium influx into plant cellsoccurs through Ca2+-permeable channels (Perfus-Barbeochet al., 2002). Moreover, our findings also provide some insightsinto the ethylene-dependent enhancement of root hair elon-gation. Elongation of root hair is enhanced by ethylene(Tanimoto et al., 1995; Petruzzelli et al., 2003) and is posi-tively dependent on [Ca2+]c at the tip of root hairs (Wymeret al., 1997). Therefore, the activation of Ca2+-permeableplasma membrane channels by ethylene will lead to a greater[Ca2+]c in the tips of root hairs, which in turn promotes theelongation of root hairs. Furthermore, our findings alsoprovide direct experimental evidence to account for the obser-vations that ethylene-dependent induction of the pathogenesis-related gene is inhibited and stimulated by reducing the Ca2+

influx with EGTA and artificially elevating cytosolic Ca2+

with Ca2+ ionophore (Raz & Fluhr, 1992).

In summary, we demonstrated that an ethylene-releasingcompound (ethephon) and an ethylene precursor (ACC)rapidly activated a Ca2+-permeable channel in the plasmamembrane, eliciting an elevation of the [Ca2+]c of tobaccosuspension cells. These findings unequivocally demonstratethat activation of the plasma membrane Ca2+-permeable channelunderlies the ethylene-elicited elevation of [Ca2+]c. Given thata number of phytohormones (e.g. ABA, auxin and jasmonicacid) and messenger molecules (e.g. nitric oxide, cAMP) acton [Ca2+]c, and that there is a close functional link betweenethylene to these phytohormones and messenger molecules,the findings that ethylene elicited an elevation of [Ca2+]cof plant cells by activating the Ca2+-permeable channelshighlight the fact that [Ca2+]c may serve as a key node forcross-talk between ethylene and other phytohormones andmessenger molecules in plants.

Fig. 5 Representative Fluo-3-dependent fluorescence in tobacco (Nicotiana tabacum) suspension cells to 100 µM ethephon without any pretreatment (b), and with 2 h of pretreatment with 100 µM GdCl3 (c) and 10 mM BAPTA (d), before and after treatment for 5–30 min. The control (a) shows the changes in fluorescence intensity over time, without any treatment. Bar, 100 µm. (e) Time-course of change in Fluo-3-dependent fluorescence intensity (expressed as pixel numbers) relative to the control, in response to ethephon, ethephon + Gd3+, and ethephon + BAPTA, as shown in (a–d). The fluorescence intensity of the suspension cells was measured by delimiting the individual suspension cells and the mean fluorescence of the suspension cells was measured. Data represent the mean ± standard error of 8–10 cells.

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Acknowledgements

This work was supported by the National Science Foundationof China (30570136) and the Chinese Academy of Sciencesthrough its Hundred Talent Program.

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