21-permeable channels in the plasma membrane of ... · ca21-permeable channels in the plasma...

Ca21-Permeable Channels in the PlasmaMembrane of Arabidopsis Pollen Are Regulated byActin Microfilaments1

Yong-Fei Wang, Liu-Min Fan, Wen-Zheng Zhang, Wei Zhang, and Wei-Hua Wu*

State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences,China Agricultural University, Beijing 100094, China

Cytosolic free Ca21 and actin microfilaments play crucial roles in regulation of pollen germination and tube growth. The focus ofthis study is to test the hypothesis that Ca21 channels, as well as channel-mediated Ca21 influxes across the plasma membrane(PM) of pollen and pollen tubes, are regulated by actin microfilaments and that cytoplasmic Ca21 in pollen and pollen tubes isconsequently regulated. In vitro Arabidopsis (Arabidopsis thaliana) pollen germination and tube growth were significantlyinhibited by Ca21 channel blockers La31 or Gd31 and F-actin depolymerization regents. The inhibitory effect of cytochalasin D(CD) or cytochalasin B (CB) on pollen germination and tube growth was enhanced by increasing external Ca21. Ca21 fluo-rescence imaging showed that addition of actin depolymerization reagents significantly increased cytoplasmic Ca21 levels inpollen protoplasts and pollen tubes, and that cytoplasmic Ca21 increase induced by CD or CBwas abolished by addition of Ca21

channel blockers. By using patch-clamp techniques, we identified the hyperpolarization-activated inward Ca21 currents acrossthe PM of Arabidopsis pollen protoplasts. The activity of Ca21-permeable channels was stimulated by CB or CD, but not byphalloidin. However, preincubation of the pollen protoplasts with phalloidin abolished the effects of CD or CB on the channelactivity. The presented results demonstrate that the Ca21-permeable channels exist in Arabidopsis pollen and pollen tube PMs,and that dynamic actin microfilaments regulate Ca21 channel activity and may consequently regulate cytoplasmic Ca21.

The primary function of pollen and pollen tubes is todeliver sperms to egg apparatus for double fertiliza-tion that is required for sexual reproduction of flower-ing plants. Pollen germination and pollen tube growthare a continuous and highly polarized process char-acteristic of tip growth; thus pollen and pollen tubesprovide an ideal model system for the study of cellpolarity control and tip growth. It is well known thatextracellular Ca21 is required for pollen germinationand tube growth (for review, see Steer and Steer, 1989;Taylor and Hepler, 1997; Malho, 1998; Franklin-Tong,1999), which indicates a possible involvement of Ca21

influx in pollen germination and tube growth. Uponpollen hydration and germination, cytoplasmic Ca21

concentration ([Ca21]i) at the germinal aperture wherethe pollen tube emerges increases to a higher levelthan other regions, and a tip-focused Ca21 gradient isthen established and sustained while a pollen tubegrows forward (Rathore et al., 1991; Pierson et al.,1994; Feijo et al., 1995). Disruption or modification ofthe Ca21 gradient inhibits pollen tube growth (Milleret al., 1992) or changes its growth direction (Malho and

Trewavas, 1996). The tip-focused Ca21 gradient oscil-lates in the pollen tubes of lily (Lilium longiflorum) andother species (Holdaway-Clarke et al., 1997; Messerliand Robinson, 1997; Messerli et al., 2000). Ca21 mobi-lization from intracellular Ca21 sink (such as vacuoleor endoplasmic reticulum) and extracellular Ca21 in-flux at pollen tube tips are believed to be two possibleCa21 sources for establishing the Ca21 gradient andoscillation. It is well established that the phospholi-pase C (PLC)-inositol triphosphate (IP3) system mobi-lizes the intracellular Ca21 in animal cells (for review,see Berridge et al., 2000). In lily pollen tubes, PLCactivity has also been demonstrated (Helsper et al.,1987). This result was confirmed later in Papaver rhoeaspollen tubes, and the PLC-IP3 system was furtherdemonstrated to be involved in pollen tube growth bypropagating slow-moving calcium waves (Franklin-Tong et al., 1996). On the other hand, several lines ofevidence indicate that the tip-focused Ca21 gradient,as well as its oscillation, requires tip-localized extra-cellular Ca21 influxes (Pierson et al., 1994, 1996; forreview, see Rudd and Franklin-Tong, 1999). Further-more, studies using Mn21-quenching techniques im-ply the presence of plasma membrane (PM)-localizedCa21 channels in pollen tubes (Malho et al., 1995).How-ever, definitive identification and characterization ofCa21-permeable channels in pollen and pollen tubesrequires application of patch-clamp techniques to di-rectly detect activity of Ca21-permeable channels. Un-fortunately, all attempts so far to apply the patch-clamptechniques to pollen tubes have been unsuccessful

1 This work was supported by the National Science Foundation ofChina (key research grant no. 39930010 and competitive grant no.30070050 to L.M.F.), and by the Chinese National Key Basic ResearchProject (no. G1999011701 to W.H.W.).

* Corresponding author; e-mail [email protected]; fax8610–6289–3491.

Article, publication date, and citation information can be found atwww.plantphysiol.org/cgi/doi/10.1104/pp.104.042754.

3892 Plant Physiology, December 2004, Vol. 136, pp. 3892–3904, www.plantphysiol.org � 2004 American Society of Plant Biologists

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because protoplasts from the pollen tube apex are hy-persecretory, thereby preventing giga-ohm seal forma-tion between the membrane and the patch-clampelectrodes (Sanders et al., 1999). However, applicationof the patch-clamp techniques to pollen protoplasts hasbeen successful (Obermeyer and Kolb, 1993; Fan et al.,1999, 2001; Fan andWu, 2000; Griessner and Obermey-er, 2003). Considering that pollen germination and tubegrowth are a continuous process, it is reasonable topostulate that there may exist the same types of Ca21-permeable channels in both pollen and pollen tubePMs.A number of previous studies have demonstrated

that the actin cytoskeleton plays a critical role inregulation of pollen germination and pollen tubegrowth (Fu et al., 2001; for additional refs., see reviewsin Staiger, 2000; Vidali and Hepler, 2000). Pollenactivation and germination are accompanied by thegradual replacement of large F-actin aggregates pres-ent in the vegetative cytoplasm by a filamentousnetwork that converges on the germinal aperture andeventually ramifies the pollen tube. This arrangementof actin microfilaments may be required for particlemovement and contributes to tip growth by directingvesicle traffic to the tip. However, the experimentalresults for existence of actin microfilaments at thevery tip of pollen tubes had been controversial.Early studies with chemical fixation and fluorescentphalloidin-labeling techniques have suggested thepresence of a dense actin network at the extreme tip(Derksen et al., 1995). In disagreement with this result,rapid freezing, freeze substitution, and electron andlight microscopy techniques revealed only thin actinbundles and short individual actin microfilaments inrandom orientation (Lancelle et al., 1987; Doris andSteer, 1996). Also, no actin networkwas observed in liv-ing lily pollen tubes stained by injected fluorescence-labeled phalloidin (Miller et al., 1996). These studieshave led to a notion of the tube tip actin-free clear zoneproposition that is free of F-actin (for review, see Vidaliand Hepler, 2000). However, by using improvedfixation methods (Staiger et al., 1994) or using expres-sion of green fluorescent protein-mTalin techniques(Kost et al., 1998; Fu et al., 2001), an actin ring in thesubapical region of pollen tubes has been revealed. Acollar structure of F-actin was also described in chem-ically fixed maize and Papaver pollen tubes (Gibbonet al., 1999; Geitmann et al., 2000). The collar structureseems to be composed of fine actin microfilamentsinstead of thick F-actin bundles like those in a pollentube shank. Gibbon et al. (1999) showed that tipelongation of either pollen tubes or root hairs wasinhibited by treatment with a low concentration ofcytochalasin D (CD) or latrunculin, which disruptedthe subapical F-actin ring but did not markedly affectthe thick axis actin bundles and cytoplasm streaming.By expressing green fluorescent protein-mTalin, Fuet al. (2001) have clearly demonstrated the presence ofa dynamic change of tip-localized F-actin in livingtobacco pollen tubes, and the dynamic tip actin ap-

pears as short actin bundles. Time-course analysis alsosuggests that the actin collar structure appears toalternate with the F-actin dynamics (Fu et al., 2001).They have also shown that the amount of tip F-actinoscillates in the opposite phase with pollen tubeelongation rates and the oscillation of tip [Ca21]i, andthat the peak of tip F-actin precedes that of tubegrowth. These observations led to a hypothesis that thedynamic F-actin may cross-talk with the tip [Ca21]idynamics, playing in concert a crucial role in theregulation of pollen tube growth. Nevertheless, mech-anisms underlying the interaction between dynamicF-actin and oscillating cytoplasmic Ca21 at the pollentube tip are not yet established.

The involvement of actin microfilaments in ionchannel regulation has been well established in animalcells (Negulyaev et al., 2000), and the regulation of PMK1 channels by actin microfilaments has also beenreported in plant cells (Hwang et al., 1997; Liu andLuan, 1998). We hypothesize that actin microfilamentsmay also play an important role in regulating PMCa21-permeable channels during pollen germination andtube growth processes. In this article, using the patch-clamp techniques, we have identified a Ca21-perme-able channel in the PM of Arabidopsis (Arabidopsisthaliana) pollen protoplasts. In combination with invitro pollen germination assay and [Ca21]i measure-ment with the laser-scanning confocal microscopy(LSCM) technique, we show that the actin cytoskeletonregulates the activity of Ca21-permeable channels inthe pollen PM. Our results support a tight and finelycontrolled connection between actin dynamics and[Ca21]i oscillation at the tip of a pollen tube.

RESULTS

Actin Depolymerization Reagent-Caused Inhibition of

Arabidopsis Pollen Germination and Tube Growth IsEnhanced by Increasing [Ca21]o and Ca21 Influx

Inorganic Ca21 channel blockers Gd31 and La31 andorganic blocker verapamil, which are commonly usedto identify PM Ca21 channels (Pineros and Tester,1997), were applied to test their effects on Arabidopsispollen germination and tube growth. The previousstudies have shown that Ca21 channel blocker-induced inhibition of pollen germination and tubegrowth is possibly correlated with the inhibition ofCa21 influx (Feijo et al., 1995, and refs. therein). Asshown in Figure 1A, both in vitro pollen germinationand tube growth were significantly inhibited in a dose-dependent manner by Gd31 at the concentrationshigher than 50 mM. The addition of 200 mM Gd31 inthe medium almost completely inhibited pollen ger-mination and tube growth (Fig. 1A). The addition ofLa31 to the medium resulted in very similar inhibitoryeffects on pollen germination and tube growth (datanot shown) as the addition of Gd31 did, while additionof 600 mM verapamil inhibited pollen germination by88% and tube growth by 46% (data not shown). These

Ca21-Permeable Channels in Arabidopsis Pollen

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results suggest that Ca21 influx mediated by Ca21-permeable channels in the PM of Arabidopsis pollenor pollen tubes is involved in the regulation of pollengermination and tube growth.

To test whether the actin cytoskeleton is involved inregulation of pollen germination and tube growth,actin depolymerization reagents were added to thegermination medium. The pollen germination andtube growth were significantly inhibited by those actindepolymerization reagents in a dose-dependent man-ner. Addition of 5 mM CD (Fig. 1B), 2 mM cytochalasin B(CB; data not shown), or 1 nM latrunculin A (Lat A;data not shown) completely inhibited pollen germina-tion and tube growth. It should also be noted that thetube tips showed swollen shapes subject to addition ofany of these reagents (data not shown). However,addition of either oryzalin, a microtubule depolymer-ization reagent, or taxol, a microtubule stabilizer, in thegerminationmedium at concentrations from 1 to 50 mM

did not affect Arabidopsis pollen germination andtube growth (data not shown), indicating that micro-tubules do not play an important role in the regulationof pollen germination and tube growth.

Interestingly, the inhibitory effects of low concen-trations of actin depolymerization reagents on pollengermination and tube growth were significantly en-hanced by increasing the external Ca21 concentration([Ca21]o) from 1 mM to 5 or 30 mM (Fig. 1, C and D).Along with the increase of [Ca21]o from 1 mM to 5 or30 mM, the inhibition of pollen germination by 0.3 mM

CD increased from 16.34% to 34.05% or to 56.14% (Fig.1C), while the inhibition of tube growth increasedfrom 30.03% to 39.30% or to 69.78% (Fig. 1D), respec-tively. Similarly, increasing [Ca21]o also enhanced in-hibition of pollen germination and tube growthinduced by 0.2 mM CB (data not shown).

These findings suggest that the inhibition of pollengermination and tube growth by depolymerization ofactin microfilaments may be, at least in part, due to itsenhancement of Ca21 influx into pollen or pollentubes, and that activity of the Ca21-permeable PMchannels may be regulated by actin microfilaments.

Actin Depolymerization Reagent-Induced Elevationsof [Ca21]i in Pollen Protoplasts and Pollen Tubes

Are [Ca21]o Dependent

If actin microfilaments affect extracellular Ca21 in-flux, we would expect that the cytosolic Ca21 levels

Figure 1. Effects of Ca21 channel blocker Gd31 (A) and actin depoly-merizing reagent CD (B–D) on Arabidopsis pollen germination and

tube growth. The ingredients of the in vitro germination media and thedetailed experimental protocols were described in ‘‘Materials andMethods.’’ The external Ca21 concentration for A and B was 5 mM. Allexperiments were repeated three times and each treatment in oneexperiment had four replicates. For each replicate, 400 pollen grainswere counted for calculation of pollen germination rate and 80 pollentubes were measured for their growth length. Data were presented asmean 6 SE from three independent experiments. #, Data point for thecontrol; **, significantly different from the control at P , 0.01 byStudent’s t test.

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will be elevated by the depolymerization of actinmicrofilaments. To test this hypothesis, Fluo-3/AM,a Ca21-specific fluorescence indicator, was preloadedinto pollen protoplasts and pollen tubes, and changesin the relative cytosolic Ca21 levels were monitoredwith LSCM upon application of various reagents. Asshown in Figure 2, the intensity of Fluo-3 fluorescencein the pollen protoplasts was increased significantlyafter incubating the protoplasts with 10 mM CB (Fig. 2Bversus 2A), and 18 out of 21 tested protoplasts showedthis positive response. This CB-induced [Ca21]i in-crease was eliminated by the addition of 200 mM La31

(Fig. 2C) or 200 mM Gd31 (Fig. 2D) to the incubationbuffer. Addition of 5 mM 1,2-bis(2-aminophenoxy)-ethane-N,N,N#,N#-tetraacetic acid (BAPTA) abolishedCB-induced [Ca21]i increase even in the presence of5 mM external Ca21 (Fig. 2E). Similar results wereobserved for the addition of 20 mM CD to the in-cubation buffer (Fig. 2, F–H), and 18 out of 21 testedprotoplasts showed CD-induced [Ca21]i increase. Thetime kinetics of quantitative changes in cytosoliccalcium concentrations induced by CD is shown inFigure 2K. Compared to the treatments with CB or CD,Lat A was more efficient in elevating [Ca21]i in pollenprotoplasts at as low as 10 nM (Fig. 2I). Similarly, thefluorescence in a pollen tube was also enhanced by20 mM CD (Fig. 2J), 10 mM CB, or 10 nM Lat A (data notshown). Among 12 tested pollen tubes, there were 10pollen tubes showing CB-induced [Ca21]i increase. Fortreatment with CD, eight out of nine tested pollentubes showed the positive response. However, neither50 mM oryzalin nor 50 mM taxol had a detectable effecton the intensity of Fluo-3 fluorescence in either pollenprotoplasts or pollen tubes (data not shown), whichsuggests that the microtubule cytoskeleton may notplay role in the regulation of Ca21 influx. These resultsdemonstrate that the F-actin depolymerization-in-duced [Ca21]i elevation was due to extracellular Ca21

influx, likely through the Ca21-permeable channels inthe PM of pollen or pollen tubes.

Identification and Characterization ofHyperpolarization-Activated Ca21-PermeableChannels in Arabidopsis Pollen Protoplasts

The results presented above suggest that Ca21-permeable channelsmay exist in the PMofArabidopsispollen protoplasts and the Ca21-permeable channelsmay play a role in the regulation of pollen germinationand tube growth. To further test this notion, patch-clamp techniques were applied to directly identify andcharacterize the PM Ca21 channels in Arabidopsispollen protoplasts in this study.Figure 3 shows patch-clamp recordings of the in-

ward currents from Arabidopsis pollen protoplastsunder the control conditions as described in ‘‘Materialsand Methods.’’ The whole-cell inward currents re-corded at 2160 mV occurred in a periodic pattern asshown in Figure 3A. This periodic current burstingmay reflect a natural periodic oscillation of the channel

activity across the PM. Figure 3B shows a typicalrecording of the inward single-channel currents froman outside-out membrane patch, and Figure 3C showsthe current/voltage (I/V) relationship derived fromthe recording data as shown in Figure 3B. The reversalpotential of the recorded channel was approximately110 mV obtained by extrapolation of the I/V curveas shown in Figure 3C. A conductance of 6.5 6 0.5picoSiemens (pS; n5 6) for the recorded channel underthe described conditions was derived according to theequation G 5 I/(E 2 Erev), where G, I, E, and Erevrepresent the single-channel conductance, the currentamplitude, the membrane potential applied, and thereversal membrane potential, respectively. In theory,both influx of cations and efflux of anions may result inobservation of inward current signals. To ensure thatthe recorded inward currents only represent the sig-nals of extracellularCa21 influx, the external (extracellu-lar) and internal (intracellular) solutions for patch-clamprecording were carefully designed. For the solutionsused for the control conditions, the major ions areCa21 and Cl2. According to the Nernst equation, theErev for Ca21 and Cl2 was 1194 mV and 2174 mV,respectively, under the control conditions. BecauseCl2 efflux can only occur at a more negative voltagethan 2174 mV, it is impossible for Cl2 efflux throughthe PM when an applied voltage was more positivethan 2174 mV. Considering that the voltages ap-plied in our experiments were always more positivethan 2160 mV, the possibility that Cl2 efflux contrib-utes to the recorded inward current signals can beexcluded. The theoretical Nernst potential of1194 mVfor Ca21 ions was the closest to the experimentalreversal potential of 145 mV, which led us to deter-mine the nature of the recorded currents as Ca21

currents. In addition, Glu is another anion includedin the solutions. However, Glu is believed to be animpermeable anion in ion channel studies (Pei et al.,1998), and also there was no change of either single-channel conductance or reversal potential observed inour experiment when the intracellular Glu concentra-tion was changed (data not shown). Thus, contributionof Glu efflux to the recorded inward currents can alsobe excluded. Therefore, considering Ca21 as the onlycation in the external solutions, Ca21 influx throughCa21 channels could be the only source that accountedfor the inward currents under the given conditions inour experiments.

The extracellular Ca21 dependence of the single-channel conductance of the inward currents supportsthe above notion. If the inward current mainly resultedfrom Ca21 influx, its single-channel conductanceshould increase along with increasing Ca21 gradientin an outside-to-inside direction across the PM. It wasobserved that the single-channel conductance of theinward currents increased from 3.8 pS to 6.5 or 8.64 pSwhen the extracellular Ca21 concentration waschanged from 10 mM to 50 or 100 mM, respectively,when the intracellular Ca21 concentration remainedunchanged (Fig. 4, A and B). The relationship of





Figure 2. Actin depolymerization reagent-induced elevations of [Ca21]i in pollen protoplasts or pollen tubes resulted from Ca21

influx. Fluo-3 fluorescent images were acquired by LSCM and the time (min) before or after the specific treatment is indicated atthe bottom left of each image. The relative fluorescence intensities were presented as pseudocolors. A, Control, showing that 1%DMSO (the final concentration of DMSO used for making the stock solutions of CB, CD, and Lat A) had no effect on Fluo-3fluorescence intensity in protoplasts. B, A total of 10 mM CB markedly increased Fluo-3 fluorescence intensity. C and D, A total of200 mM La31 or Gd31 impaired CB-induced increase in Fluo-3 fluorescence intensity, respectively. E, A total of 5 mM BAPTAinhibited CB-induced increase in Fluo-3 fluorescence intensity even after addition of 5 mM Ca21 to the incubation buffer (thearrows indicate the time when CB or Ca21 was added). F, A total of 20 mM CD induced increase of Fluo-3 fluorescence intensity.G and H, A total of 200 mM La31 or Gd31 abolished CD-induced increase in Fluo-3 fluorescence intensity, respectively. I, A totalof 10 nM Lat A dramatically induced increase of Fluo-3 fluorescence intensity. J, The left four images show Fluo-3 fluorescenceintensity in pollen tubes increased with time after treatment with 20 mM CD, and the far right image shows the pollen tube inbright field. K, Time kinetics of quantitative changes in cytosolic calcium concentrations induced by CD. The vertical colored barat the right illustrates the relative cytosolic Ca21 levels.

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single-channel conductance versus extracellular Ca21

concentration was well fitted to the Michaelis-Mentenequation as shown in Figure 4B, which gives a half-conductance Ca21 concentration of approximately18 mM. In addition, with the increases in external Ca21,the reversal potential (Erev) of the Ca

21 currents shift tomore positive voltages (Fig. 4B, inset), which is con-sistent with the shifting direction of Ca21 equilibriumpotential (ECa). The result that the inward current wasno longer observed in the absence of external Ca21

further confirms that the recorded inward currentswere due to Ca21 influx.It has been reported that Ca21 channels may also be

permeable to other divalent cations, such as Ba21,

Mg21, Sr21, and Mn21 (Pineros and Tester, 1997). Theresults presented in Figure 4, C and D show that therecorded channels have different permeability to var-ious divalent cations in a sequence of relative per-meability as Mg21 . Ca21 . Ba21 . Sr21 . Mn21. Tofurther identify whether the recorded inward currentsweremediated byCa21 channels, effects ofwidely usedCa21 channel blockers on the channel activity weretested. It was observed that the inward currents weredramatically inhibited by La31 (Fig. 5, A and B) or Gd31

(Fig. 5, C and D) in a dose-dependent manner. Totalopen probability (Po) of the channels at 2160 mVdecreased by 95% or 98% in the presence of 150 mM

La31 or 150 mM Gd31, respectively. Notably, the in-hibition of the inward currents by La31 or Gd31 wasremoved reversibly by washing out the drugs from theextracellular solution (Fig. 5, A–D). However, the in-ward currents were not affected by verapamil (data notshown). These results clearly demonstrate that therecorded inward currents in this study were mediatedby hyperpolarization-activated and La31- and Gd31-sensitive Ca21-permeable channels in the PM of Arabi-dopsis pollen protoplasts.

Ca21-Permeable Channels in Arabidopsis PollenProtoplasts Are Regulated by Dynamic Actins

Following the experiments to identify Ca21-perme-able channels in Arabidopsis pollen protoplasts, aspresented in Figures 3 to 5, further patch-clamp experi-ments were conducted to test the hypothesis that Ca21

channel-mediated Ca21 influx was regulated byF-actin. The previous study (Horber et al., 1995) haddemonstrated that cytoskeleton structures are stillpresent in the excised membrane patches. CD or CBwas applied to the excised membrane patches to ob-serve their possible effect on the calcium channelactivity. Figure 6 illustrates the effects of CD and CBon the Ca21 channel activity at 2160 mV. After theprotoplasts were incubated with 20 mM CD for 20 to30min, the Po of the Ca

21 channels increasedmarkedly,on the average by 194.7% for the inside-out membranepatches (Fig. 6, A and B) and by 273.6% for the outside-out membrane patches (Fig. 6, C and D). The similarresults were observed for the treatment with 10 mM CBas shown in Figure 6, E to H. A pretreatment with 100mM phalloidin abolished the inhibitory effect of CD onthe channel activity, although 100 mM phalloidin alonedid not have any detectable effect (Fig. 7, A and B). Theresults presented in Figure 8 show that Lat A (a morespecific actin-depolymerizing drug) significantly acti-vated Ca21 currents at the whole-cell level, and thewhole-cell Ca21 currents were similarly inhibited bythe Ca21 channel blocker La31. In addition, the treat-ments with 50 mM oryzalin or 50 mM taxol did not resultin any detectable effect on the Ca21 currents (data notshown), indicating that the Ca21-permeable channelsare not regulated by themicrotubule cytoskeleton. Thisis consistent with our observations that these drugs did

Figure 3. Patch-clamp whole-cell (A) and single-channel recordings (B)of the inward currents from Arabidopsis pollen protoplasts. A, A whole-cell recording from an Arabidopsis pollen protoplast at 2160 mVshows the periodic inward currents. B, Single-channel recordings froman outside-out membrane patch of an Arabidopsis pollen protoplast atvarious voltages. C, I/V relationship derived from six membranepatches. The data points in C represent mean 6 SD (n 5 6). The dashedlines in A and B represent the closed state of channels.





not affect pollen germination and tube growth orcytoplasmic Ca21 levels. These results suggest that thedisassembly of F-actin induces activation of the Ca21-permeable channels in the PM of Arabidopsis pollenprotoplasts.

Taking all presented results together, it is concludedthat the actin cytoskeleton plays a role in regulatingthe activity of the inward Ca21-permeable channelspresent in the Arabidopsis pollen PM and consequentcytoplasmic free Ca21 levels. The results support thehypothesis that dynamic change between F-actin andG-actin at the tip of a pollen tube provides an impor-tant mechanism for the Ca21 oscillation at the tip ofa pollen tube.

DISCUSSION

A cytosolic Ca21 gradient as well as its oscillation isconsidered to play a central role in the regulation ofpollen germination and tube growth (Rathore et al.,1991; Miller et al., 1992; Pierson et al., 1994; Malho andTrewavas, 1996; Holdaway-Clarke et al., 1997; forreview, see Feijo et al., 1995; Holdaway-Clarke andHepler, 2003). Several lines of evidence suggest thatPM Ca21 channels may be prominently involved in theregulation of this Ca21 gradient (Pierson et al., 1994,1996; Malho et al., 1995). Ca21 influx in pollen or pollen

tubes has been investigated by using 45Ca21 (Jaffe et al.,1975), Ca21-selective vibrating probes (Kuhtreiber andJaffe, 1990; Pierson et al., 1994; Holdaway-Clarke et al.,1997; Messerli et al., 1999; Franklin-Tong et al., 2002),and Mn21-quenching techniques (Malho et al., 1995).However, direct detection of Ca21 channels in thePM of pollen or pollen tubes by using patch-clamptechniques has not been reported. In this study, wehave identified Ca21-permeable channels in pollenprotoplasts using patch-clamp whole-cell and single-channel recording techniques. We also show that Ca21

channels in pollen protoplasts are regulated by actindynamics, supporting the hypothesis that the actincytoskeleton may regulate Ca21 channel activity andsubsequently Ca21 influx as well as cytosolic Ca21

oscillation at the tip of pollen tubes.

Identification of Ca21 Channels in Arabidopsis

Pollen Protoplasts

The most significant and important aspect of thisstudy is the identification and characterization of theCa21-permeable channels in the PM of pollen proto-plasts. First, our careful design of the solutions usedfor patch-clamp recordings ensured that the natureof the recorded currents was Ca21 currents. Underthe control conditions, the Erev for Ca21 or Cl2 was1194 mV or 2174 mV, respectively, according to the

Figure 4. Dependence of the inwardcurrent conductance on external Ca21

concentrations (A and B) and the per-meability of various divalent cations tothe PM of Arabidopsis pollen proto-plasts (C). A, Single-channel record-ings from the same outside-outmembrane patch at 2160 mV at differ-ent external Ca21 concentrations. B,The relationship of single-channel con-ductance versus external Ca21 concen-tration derived from the recordingsas shown in A, which was well fit bythe Michaelis-Menten equation. Thechanges in Erev at different externalCa21 are also shown in inset of B. C,Single-channel recordings from thesame outside-out membrane patch at2160 mV with different divalent cat-ions in external (bath) solutions. D,Histograms of single-channel conduc-tance derived from the recordings asshown in C. Dashed line in A and Cindicates the closed state of the chan-nels. Time and current scale bars aredisplayed in A and C. Data in B and Dare presented as mean6 SE (n55 for B;n 5 3 for D).

Wang et al.




Nernst equation. This made Cl2 efflux impossible tooccur since all the applied clamp voltages were morepositive than 2174 mV. Second, a possible Glu effluxcan also be excluded given that this anion is generallybelieved to be impermeable to the PM (Pei et al., 1998)and that there was no change of either single-channelconductance or reversal potential observed when theintracellular Glu concentration was changed in thisstudy. Third, the single-channel conductance of therecorded inward currents was dependent upon extra-cellular Ca21. Therefore, as the only cation in theextracellular solutions, Ca21 influx through Ca21 chan-nels could be the only source that accounted for therecorded inward currents under the given conditionsin the presented experiments.Further support for the existence of PM Ca21 chan-

nels in pollen comes from the experiments using Ca21

channel blockers. We found that the inward currentswere dramatically inhibited by Gd31 or La31 in a dose-dependent manner (Fig. 5, A–D). Gd31 and La31 havebeen successfully used to inhibit Mn21 entry intoAgapanthus pollen tubes (Malho et al., 1995), whichreflects Gd31 and La31 inhibition on Ca21 influx andextracellular Ca21 fluxes into P. rhoeas pollen tubes(Franklin-Tong et al., 2002). We showed that the in-ward currents were not affected by low concentrationsof verapamil, consistent with the previous report thatthere was no significant inhibition of Mn21 uptakeobserved for Agapanthus pollen tubes in the presence

of the organic channel blocker verapamil or nifedipine(Malho et al., 1995). These two organic Ca21 channelblockers are commonly used to block animal Ca21

channels (Hess, 1990). The findings of this study andof Malho et al. (1995) suggest that different types ofCa21 channels may exist in pollen and pollen tubesfrom those in animal cells. In addition, we observedanother type of Ca21 channel, which was stretch-activated and Gd31- or La31-sensitive, in Arabidopsisand Brassica pollen protoplasts (Y.F. Wang and W.H.Wu, unpublished data). The stretch-activated PMCa21

channels have been proposed to be present in pollenand pollen tubes and may function in pollen germi-nation and tube growth (Pierson et al., 1994; Malhoet al., 1995; Malho and Trewavas, 1996). The features ofthe stretch-activated Ca21 channels differ from those ofthe hyperpolarization-activated Ca21 channels in pol-len and pollen tubes reported in this study. These twotypes of Ca21 channels may act in concert to regulatepollen germination and tube growth.

Consistent with the results of patch-clamp experi-ments, we found that both F-actin-disrupting drugsand Ca21 channel blockers affected in vitro pollengermination and pollen tube growth as well as[Ca21]i in pollen. The only discrepancy is the effectof verapamil (50 mM), which markedly inhibitedpollen germination but did not affect Ca21 channelactivity. A possible explanation could be that verapa-mil may inhibit pollen germination and tube growth

Figure 5. Calcium channel blockerLa31 (A and B) or Gd31 (C and D)induced decrease of Po of the channelsin a dose-dependent manner. A, Aseries of single-channel recordingsfrom the same outside-out membranepatch at 2160 mV without or withLa31 at different concentrations inexternal solutions. B, Histograms ofsingle-channel conductance versusLa31 concentration, derived from therecordings as shown in A. C, Single-channel recordings from the same out-side-out membrane patch at 2160 mVwithout or with Gd31 at different con-centrations in external solutions. D,Histograms of single-channel conduc-tance versus Gd31 concentration, de-rived from the recordings as shown inC. Dashed lines in A and C indicate theclosed state of the channels. Time andcurrent scale bars are displayed in Aand C. Data in B and D are presentedas mean 6 SE (n 5 5). #, Data pointfor the control; *, significantly differ-ent from the control at P , 0.05 byStudent’s t test; **, significantly differ-ent from the control at P , 0.01 byStudent’s t test.





by nonspecifically affecting some other factors insteadof Ca21 channels involved in these processes.

Do Pollen Tubes Possess the Same Type of Ca21

Channels at Their Tips as Do Pollens?

As discussed by Sanders et al. (1999), pollen tube tipsare hypersecretory,making them too difficult for patch-

clamp recordings on pollen tube protoplasts. Althoughthere is no direct evidence to demonstrate what type ofCa21 channel exists in pollen tubes, particularly atpollen tube tips, the results presented here implythat pollen tubes may have the same type of Ca21

channels as those in pollen. Our results showedthat Ca21 channel inhibitors and actin depolymeriza-tion reagents similarly affect the three physiological

Figure 6. Actin depolymerization re-agent CD (A–D) or CB (E–H) inducedincreases of the Po of the Ca21 chan-nels. A and C, Single-channel record-ings at 2137 mV from an inside-outmembrane patch or an outside-outmembrane patch, respectively, in theabsence (control; upper trace) or pres-ence (lower trace) of 20 mM CD. B andD, Po of the channels derived fromthe recordings as shown in A andC), respectively. E and G, Single-channel recordings at 2137 mV froman inside-out membrane patch or anoutside-out membrane patch, respec-tively, in the absence (control; uppertrace) or presence (lower trace) of 10mM CB. F and H, Po of the chan-nels derived from the recordings asshown in E and G, respectively. Thedashed lines in A, C, E, and G indicatethe closed state of the channels. Timeand current scale bars are shown in A,C, E, and G. Data in B, D, F, and Hare presented as mean 6 SE (n 5 5). #,Data point for the control; *, signifi-cantly different from the control at P,

0.05 by Student’s t test; **, signifi-cantly different from the control at P,

0.01 by Student’s t test.

Wang et al.




processes: both pollen germination and tube growth(Fig. 1), cytoplasmic Ca21 elevation in pollen proto-plasts and pollen tubes (Fig. 2), and Ca21-permeablechannel activity (Figs. 6–8). Since pollen germinationand tube growth are a continuous and integral process,it is plausible to propose that pollen and pollen tubesuse the same type of Ca21 channels in the wholepolarized process. However, direct demonstration ofthis notion requires patch-clamp recording of Ca21

channel activity in the PM of pollen tubes as well asmolecular cloning and functional characterization ofCa21 channels in pollen and pollen tubes.

Cross-Talk between Dynamic F-Actin and Oscillating

[Ca21]i in Pollen Germination and Tube Growth

The tip-focused Ca21 gradients and the actin cyto-skeleton are two key factors controlling pollen germi-nation and tube growth. It has been known for yearsthat [Ca21]i is highly dynamic or oscillatory at the tipof pollen tubes (Holdaway-Clarke et al., 1997; Messerliand Robinson, 1997; Messerli et al., 2000). Similarly,dynamic or oscillatory F-actin has also been shown toexist at the tip of pollen tubes (Fu et al., 2001). In-terestingly, the amount of F-actin at the tip oscillates inthe opposite phase with pollen tube growth and[Ca21]i. Rop GTPase signaling has been shown to

coordinate these two processes (Lin and Yang, 1997;Li et al., 1999; Fu et al., 2001; Gu et al., 2003), but thedetailed regulatory mechanisms underlying these twodynamic processes, especially the regulation of Ca21

oscillation, are not yet fully understood.As discussed in the introduction, the involvement of

actinmicrofilaments in ion channel regulation has beenwell established in animal cells (Negulyaev et al., 2000),and F-actin regulation of K1 channels has also beenreported in plant cells (Hwang et al., 1997; Liu andLuan, 1998). A critical role for the actin cytoskeleton inpollen germination and tube growth (Gibbon et al.,1999;Vidali et al., 2001; this study) ledus to hypothesizethat actin microfilaments may function in regulatingthe PM Ca21 channels during pollen germination andtube growth processes. This ideawas also prompted bythe observation of Fu et al. (2001) that actin dynamics atthe tip of pollen tubes are in the opposite phase withpollen tube growth and Ca21 oscillation, where [Ca21]ireaches the highest level when F-actin reaches mini-mum and vice versa. Our results provide direct evi-dence that F-actin negatively affects Ca21-permeablechannels in pollen, representing the first example ofactin regulation of Ca21-permeable channels in plants.We found that actin depolymerization reagentsCD,CB,or Lat A elevated [Ca21]i in either pollen protoplasts

Figure 8. Hyperpolarization-activated whole-cell Ca21 currents inArabidopsis pollen protoplasts. Whole-cell currents were measuredduring a 4-s voltage ramp between 2160 and 120 mV (the voltageprotocols are shown in A). A, Whole-cell Ca21 currents were in-hibited by 100 mM La31. B, Whole-cell Ca21 currents were stimulatedby 1 nM Lat A.

Figure 7. Actin stabilizer phalloidin eliminates the increase in Po of theCa21 channels by 20 mM CD. A, Single-channel recordings from thesame inside-out membrane patch at 2137 mV without (upper trace) orwith (middle trace) treatment by 100 mM phalloidin or by 100 mM

phalloidin plus 20 mM CD (lower trace). B, Po of the channels derivedfrom the recordings as shown in A. The dashed lines in A indicate theclosed state of the channels. Time and current scale bars are displayedin A. The data in B are presented as mean 6 SE (n 5 5).





or pollen tubes (Fig. 2) and activated the PM Ca21-permeable channels (Figs. 6 and 8). On the other hand,phalloidin alone did not affect the activity of the Ca21

channels, but pretreatment with phalloidin abolishedsubsequent potential CD or CB activation of the Ca21

channels (Fig. 7). Thus, our findings can explain whyF-actin oscillates in the opposite phase with [Ca21]i (Fuet al., 2001).Moreover, actin depolymerization reagent-induced inhibition of Arabidopsis pollen germinationand tube growth is dependent on [Ca21]o. This may beexplained by actin depolymerization-exerted inhibi-tory effects, at least in part by enhancing Ca21 influxthrough PM Ca21-permeable channels in pollen andpollen tubes.HighCa21has long been shown to disruptthe actin cytoskeleton in lily pollen tubes (Kohno andShimmen, 1987). Actin-binding protein profilins se-quester pollen G-actin in a Ca21-dependent mannerand are believed to contribute to actin dynamics inpollen tubes (Kovar et al., 2000; Fu et al., 2001). In-terestingly, the peak of tip Ca21 is estimated to be ashigh as 3 to 10 mM, falling in the range of Ca21

concentration of 5 mM optimal for profilin-mediatedG-actin sequestration (Kovar et al., 2000). Thus, aspreviously proposed by Fu et al. (2001), Ca21-regulatedpotential oscillation of profilin-dependent G-actin se-questration could well contribute to actin dynamics atthe pollen tube tip.

As discussed above and also proposed by Fu et al.(2001), there may exist an interaction or a cross-talkbetween actin dynamics and Ca21 dynamics at the tipof pollen tubes. Based on this potential cross-talk,a positive loop may occur; namely, Ca21-activatedactin depolymerization may lead to further elevationof [Ca21]i, and conversely, actin depolymerization-elevated [Ca21]i may in turn accelerate actin depoly-merization as well. However, this positive interactingloop must be limited by a negative regulatory mech-anism; i.e. high [Ca21]i feedback may inhibit Ca21

channel-mediated extracellular Ca21 influxes. SinceRop or Rac activates both F-actin assembly and [Ca21]i,it is possible that this potential negative feedbackmechanismmay involve Ca21 inhibition of Rop activa-tion (Zheng and Yang, 2000). This counter-regulationof F-actin and Ca21 at the tip may be coordinated withtheir potential roles in the respective regulation ofsecretory vesicle transport to and fusion with the tubeapex. Such a spatial and temporal coordinationmay becritical for the rapid and directional membrane exten-sion and wall material deposit characteristic of tipgrowth in pollen tubes. However, further studies areneeded to understand the spatial and temporal re-lationship and functional interaction between thedynamic Ca21 and F-actin at the tip of pollen tubes.

Hyperpolarization-activated Ca21-permeable chan-nels have been reported in other cell types in severalplant species, such as tomato suspension cells (Gelliet al., 1997), Arabidopsis root hairs (Very and Davies,2000; Foreman et al., 2003), Vicia guard cells (Hamiltonet al., 2000), Arabidopsis guard cells (Pei et al., 2000),etc. Although it is difficult to quantitatively compare

our data to previously reported results because ofdifferent experimental conditions as well as differenttypes of cells, it might be plausible to postulate thatthose channels in different types of cells may sharesimilar regulatory mechanisms by actins. For example,a growing root hair, similar to a growing pollen tube, isanother tip-growing cell model for studying the reg-ulationmechanism of cell growth. The tip of a growingroot hair also has an oscillating calcium gradient.Interestingly, our recent results show that both inwardK1 channels and Ca21-permeable channels in roothairs of Medicago sativa are regulated by actins (L.W.Fan and W.H. Wu, unpublished data), which suggeststhat similar mechanisms may exist for calcium channelregulation by actin cytoskeleton in both root hairs andpollen tubes.

MATERIALS AND METHODS

Plant Materials

Arabidopsis (Arabidposis thaliana) ecotype Landsberg erecta plants were

grown in mixed soil in a growth chamber under a 12-h light/12-h dark cycle

(100 mmol m22 s21) and temperatures of 22�C 6 1�C and 15�C 6 4�C for

daylight and night, respectively. Plants were watered twice a week with tap

water, and the relative humidity was kept at approximately 70%.

In Vitro Pollen Germination Assay

In vitro Arabidopsis pollen germination experiments were conducted as

described previously (Fan et al., 2001), except that the medium was slightly

modified. The medium was composed of 1 mM KCl, 5 mM CaCl2, 0.8 mM

MgSO4, 1.5 mM boric acid, 1% (w/v) agar, 16.6% (w/v) Suc, 3.65% (w/v)

sorbitol, 10mg L21 myoinositol, and the pHwas adjusted to 5.8 withMES-Tris.

Other specific conditions were indicated in the text and the figure legends.

Double distilled water was used to prepare all media. Each 1.5 mL of the

heated solution (100�C, 2 min) was poured into a small petri dish (diameter5

35 mm) and cooled down to form a thin layer. The dehisced anthers were

carefully dipped onto the surface of the media to make pollen grains stick to it.

The dishes were incubated for 6 h in a climatic chamber (continuous light,

25�C 6 0.2�C, 100% relative humidity), frozen at 220�C for 3 min to quickly

terminate the pollen germination and tube growth, and kept on ice for

counting the pollen germination rate and measuring pollen tube length. All

the experiments were repeated three times and at least four replicates were

carried out for each treatment. For each replicate, there were no less than 400

pollen grains counted for calculation of the pollen germination rate, and

approximately 80 pollen tubes were measured for their growth length. The

pollen grains with emerging tubes longer than their diameter were considered

as germinated.

Cytosolic Ca21 Measurement

Arabidopsis pollen protoplasts were isolated as described previously (Fan

et al., 2001). The isolated pollen protoplasts were incubatedwith 10mM Fluo-3/

AM in the standard washing solutions at 20�C for 30 min before Ca21 imaging

measurement. The standard washing solutions contained 1 mM KNO, 0.2 mM

KH2PO4, 5 mM CaCl2, 1 mMMgSO4, 1 mM KI, 0.1 mM CuSO4, 0.5 M sorbitol, 0.8 M

Glc, 10 mg L21 myoinositol, 5 mM MES, and the pH was adjusted to 5.5 with

Tris. The osmolality of the standard washing solutions was 1.81 mol kg21. The

mixture was centrifuged at 160g for 5 min, and the protoplasts were re-

suspended in the standardwashing solution. The centrifugation/resuspension

cycle was conducted two times to ensure the removal of any remaining ex-

ternal Fluo-3/AM in the washing solutions. The protoplasts were finally

resuspended in the standard washing solutions and kept on ice before use. For

the cytoplasmic Ca21 imaging of pollen tubes, Arabidopsis pollens were

germinated first as described above and the germinated pollens were in-

cubated with 10 mM Fluo-3/AM in the standard washing solutions at 20�C for

Wang et al.




1 h before Ca21 imaging measurement. The Fluo-3 fluorescence of the

protoplasts and pollen tubes was measured with LSCM (MRC 1024, equipped

with krypton/argon laser light; Bio-Rad Spectroscopy Group, Cambridge,

MA). The wavelength of excitation light was 488 nm and the emission signals

at 515 nm were collected. Three-dimensional scanning was conducted every

5minwith a 1.5-mmZ-series project step, and three-dimensional reconstructed

images were used to show the relative [Ca21]i.

Patch-Clamp Single-Channel Recordings

Standard single-channel recording techniques (Hamill et al., 1981) were

applied in this study. For the control conditions, the external (bath) solutions

contained 50mMCaCl2, 5 mMMES (pH 5.8 with Tris), and osmolality at 1.5 mol

kg21 adjustedwith sorbitol, and the internal (pipette) solutions contained 2mM

BAPTA, 0.05 mmol/L CaCl2, 0.09 mM calcium gluconate, 92 mM potassium

glutamate, 5 mM HEPES (pH 7.2 with Tris), and osmolality at 1.55 mol kg21

adjusted with sorbitol. The final free Ca21 concentration for the internal

solutions was 100 nM. The recordings were performed at room temperature

(20�C 6 2�C) under dim light. Pipette capacitance was compensated for each

membrane patch using the capacity compensation device of the amplifier. All

data were acquired 5 min after formation of the single-channel configuration.

Single-channel currents were measured using an Axopatch-200A amplifier

(Axon Instruments, Foster City, CA) that was connected to a microcomputer

via an interface (TL-1 DMA Interface; Axon Instruments). pCLAMP (version

6.0.4; Axon Instruments) software was used to acquire and analyze the single-

channel events. After the single-channel configuration was obtained, mem-

brane potential (Vm) was clamped to 0 mV (holding potential). Liquid junction

potentials were considered and corrected for all the data presented. Single-

channel current data were filtered at 1 kHz before storage (125 ms/sample)

onto a computer hard disc.

All data were presented as mean 6 SE. SigmaPlot software was used to

analyze and plot the data.

Preparation of Stock Solutions

All chemicals were obtained from Sigma (St. Louis) unless otherwise

indicated. CD, CB, or paclitaxel (taxol) was dissolved in dimethyl sulfoxide

(DMSO) at a concentration of 20 mM, respectively, and Lat Awas dissolved in

DMSO at a concentration of 200 mM. Phalloidin was dissolved in methanol at

a concentration of 20 mM. Fluo-3/AM was dissolved in DMSO at a concentra-

tion of 1 mM. Oryzalin was dissolved in distilled water at a concentration of

100 mg mL21. All the stock solutions were kept at 220�C before use.

ReceivedMarch 15, 2004; returned for revision June 28, 2004; accepted June 28,

2004.

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