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Identification of hyperpolarization-activated calcium channels in apical pollen tubes of Pyrus pyrifolia

Hai-Yong Qu1,3, Zhong-Lin Shang2, Shao-Ling Zhang1, Lian-Mei Liu3 and Ju-You Wu1

1College of Horticulture, NanJing Agricultural University, NanJing, China; 2College of Life Sciences, HeBei Normal University, Shi Jia Zhuang, China; 3HuaiYin Institute of Technology, HuaiAn, China

Summary

• The pollen tube has been widely used to study the mechanisms underlying polarizedtip growth in plants. A steep tip-to-base gradient of free cytosolic calcium ([Ca2+]cyt)is essential for pollen-tube growth. Local Ca2+ influx mediated by Ca2+-permeablechannels plays a key role in maintaining this [Ca2+]cyt gradient.• Here, we developed a protocol for successful isolation of spheroplasts from pollentubes of Pyrus pyrifolia and identified a hyperpolarization-activated cation channelusing the patch-clamp technique.• We showed that the cation channel conductance displayed a strong selectivity fordivalent cations, with a relative permeability sequence of barium (Ba2+) ≈ Ca2+ >magnesium (Mg2+) > strontium (Sr2+) > manganese (Mn2+). This channel conductancewas selective for Ca2+ over chlorine (Cl–) (relative permeability PCa/PCl = 14 in 10 mM

extracellular Ca2+). We also showed that the channel was inhibited by the Ca2+ channelblockers lanthanum (La3+) and gadolinium (Gd3+). Furthermore, channel activitydepended on extracellular pH and pollen viability.• We propose that the Ca2+-permeable channel is likely to play a role in mediatingCa2+ influx into the growing pollen tubes to maintain the [Ca2+]cyt gradient.

Key words: Ca2+-permeable channels, patch clamp, pollen tube spheroplasts, Pyruspyrifolia.

New Phytologist (2007) 174: 524–536

© The Authors (2007). Journal compilation © New Phytologist (2007) doi: 10.1111/j.1469-8137.2007.02069.x

Author for correspondence: Shao-ling Zhang Tel: +86 025 84396580 Fax: +86 025 84395262 Email: [email protected]

Received: 3 December 2006 Accepted: 28 February 2007

Introduction

It has been clearly established that a tip-to-base free cytosoliccalcium ([Ca2+]cyt) gradient is essential for pollen tube growth(Felle & Hepler, 1997). The existence of the [Ca2+]cyt gradientin growing pollen tubes has been repeatedly demonstrated byratiometric Ca2+ imaging techniques in many plant species(Pierson et al., 1994; Franklin-Tong et al., 1997, 2002;Holdaway-Clarke et al., 1997; Messerli & Robinson, 1997;Pierson et al., 1997). A disruption or modification of the[Ca2+]cyt gradient reversibly inhibits pollen tube growth(Pierson et al., 1994; Malhó & Trewavas, 1996), indicatingthat the [Ca2+]cyt gradient plays a critical role in modulatingpolar elongation. Many studies have suggested that a Ca2+

influx through putative Ca2+ channels at the pollen tubeapical plasma membrane is responsible for the formation of

the [Ca2+]cyt gradient (Pierson et al., 1994; Holdaway-Clarkeet al., 1997; Messerli & Robinson, 1997; Messerli et al., 1999).However, the technical difficulties in isolating spheroplastsfrom elongating pollen tubes, together with the problem that thespheroplasts from pollen tube apices often retain hypersecretoryactivity, make it difficult to characterize directly channel activitiesin plasma membranes of pollen tubes (Brownlee et al., 1999).Nevertheless, recent studies with the patch-clamp techniquehave identified stretch-activated cation channels (Dutta &Robinson, 2004) and hyperpolarization-activated potassium(K+) channels (Griessner & Obermeyer, 2003) in plasmamembranes of pollen tubes of Lilium longiflorum.

Two types of voltage-dependent Ca2+-permeable cationchannels have been identified in plasma membranes of higherplant cells: depolarization-activated channels (Thuleau et al.,1994; Thion et al., 1998) and hyperpolarization-activated

© The Authors (2007). Journal compilation © New Phytologist (2007) www.newphytologist.org New Phytologist (2007) 174: 524–536

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channels (Pei et al., 2000; Véry & Davies, 2000; Shang et al.,2005). Depolarization-activated Ca2+ channels have beensuggested to play a role in the transduction of Ca2+-dependentsignals, while hyperpolarization-activated Ca2+ channelsmay be of importance in nutritional acquisition of Ca2+

(Miedema et al., 2001). Véry & Davies (2000) characterizeda hyperoplarization-activated Ca2+-permeable channel in theapical plasma membrane of Arabidopsis root hairs. Theysuggested that Ca2+ conductance may function as a route forlocal Ca2+ influx into the tip of the root hair, thus contributingto its apex-high [Ca2+]cyt gradient. Like pollen tube growth, roothair elongation is a polarized process in which growth is restrictedto the tip. However, whether a similar hyperpolerization-activated Ca2+-permeable channel exists at the apices of elongatingpollen tubes remains unknown. In the present study, we isolatedspheroplasts from apical pollen tubes of Pyrus pyrifolia cv.Housui (a pear variety that has been widely planted in Japanand China) and identified a hyperpolarization-activated Ca2+-permeable cation channel conductance using the whole-cellpatch-clamp configuration.

In addition to calcium, a high proton concentration (lowpH, 4.5–6.5) also facilitates both pollen germination andtube growth (Hepler et al., 2001). Whereas there is considerableconsensus concerning the presence of [Ca2+]cyt gradients andtheir relationship to growth, the role of protons remainsdebatable (Holdaway-Clarke et al., 2003). It has been heldthat protons may be a more fundamental polarizing regulatorthan [Ca2+]cyt, but many issues remain to be clarified. Increasingevidence has suggested that proper control of local pH, bothapoplastically and symplastically, contributes significantly topolarized growth (see e.g. Hepler et al., 2001). In this study,we observed distinct effects of extracellular pH on pollen tubeapical plasma membrane Ca2+ currents.

Materials and Methods

Pyrus pyrifolia Nakai cv. Housui pollen was collected annuallyfrom GaoYou Fruit Experimental Yard ( JiangSu Province, China).Pollen grains were preserved by drying at air temperature for12 h and were stored in silica gel at −20°C.

Isolation of spheroplasts from pollen tubes

Pollen was grown at 24°C for 3 h on modified Brewbaker &Kwack medium comprising (in mM): 0.55 Ca(NO3)2,1.60 H3BO3, 1.60 MgSO4, 1.00 KNO3, 440 sucrose and 52-(N-Morpholino)ethanesulfonic acid hydrate (MES)/Tris(pH 6.0–6.2; pH was adjusted by Tris) (Brewbaker & Kwack,1963). Pollen tubes were washed twice with de-ionized waterand incubated in an enzyme solution for 2.5 h at 32°C torelease spheroplasts. The enzyme solution was composed of1% (weight/volume (w/v)) macerozyme R-10, 2.0% (w/v)Cellulase RS-10 (Onozuka, Tokyo, Japan), 0.7% (w/v) PectolyaseY-23 (Seishin, Tokyo, Japan) and 1% (w/v) bovine serum

albumin (BSA) (Sigma, Mexico City, Mexico). The enzymesolution was then exchanged with the control bathingsolution.

Experimental solutions

The standard bath solution contained (in mM) 0.2 glucose, 10CaCl2 and 5 MES, adjusted to an osmolality of 800 mOsM

and a pH of 5.8 with D-sorbitol and Tris, respectively. Thepipette solution comprised (in mM) 1 MgCl2, 0.1 CaCl2,4 Ca(OH)2, 10 ethyleneglycoltetraacetic acid (EGTA), 2MgATP, 10 HEPES, 100 CsCl and 0.1 GTP, adjusted to a pHof 7.3 and an osmolality of 1100 mOsM by Tris and D-sorbitol,respectively. ATP was incorporated to delay rundown of currents(Forscher & Oxford, 1985) and GTP was incorporated to sustainpossible G-protein-related activity (Edwards et al., 1989; Yawo& Momiyama, 1993). The free calcium concentration in thepipette solution was approx. 10 nM, calculated with the chemicalspeciation program GEOCHEM (Parker et al., 1987). Changesto bath and pipette solutions are given in the figure legends.

Electrophysiology and data analysis

The pipettes were pulled from borosilicate glass blanks andcoated with Sylgard (184 silicone elastomer kit; Dow Corning,Midland, MI, USA). Pipette resistance ranged from 15 to35 Ω in 10 mM CaCl2. Whole-cell plasma membrane currentswere measured using an Axon 200B amplifier (Axon Instrument,Foster City, CA, USA). The whole-cell configuration wasobtained using a short burst of suction applied to the pipetteinterior to rupture the membrane, resulting in a substantialincrease in capacitance. Series resistance and capacitance werecompensated accordingly. The membrane was held at a holdingpotential of 0 mV and then the voltage was either clamped atdiscrete values for 2.5 s or changed rapidly and continuouslyin a ‘ramp’. Voltage protocols for tail current analysis are describedin the figure legends. Data were sampled at 2 kHz and filteredat 0.5 kHz, then analyzed using PCLAMP 9.0 (Axon Instrument).Junction potentials were corrected according to Amtmann &Sanders (1997). All experiments were conducted at roomtemperature (20–22°C). The permeability of the channels toCa2+ relative to chlorine (Cl–) (PCa/PCl) was estimated usingthe equation derived from the Goldman–Hodgkin–Katz (GHK)equation (Goldman, 1943; Hodgkin & Katz, 1949):

(ZCl and Zca, the valency of Cl– and Ca2+ ions, respectively;[Ca]i and [Cl ]i, the activities of intracellular Ca2+ and Cl–,respectively; [Ca]o and [Cl ]o, the activities of extracellular

P

P

Z

Z

Z E F RT

Z E F RT

Cl Z E F RT Cl

Ca Cl Z E F RT

Ca

Cl

Cl

Ca

Ca rev

Cl rev

i Cl rev o

o i Ca rev

exp( / )

exp( / )

[ ] exp( / ) [ ]

[ ] [ ] exp( / )

=−−

−−

2

2

1

1

New Phytologist (2007) 174: 524–536 www.newphytologist.org © The Authors (2007). Journal compilation © New Phytologist (2007)

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Ca2+ and Cl–, respectively; F, R and T have their usual values(R, 8.14J·mol–1·K–1; F, 96500J·V–1·mol–1; T, 298K). Erev, themeasured reversal potential (in volts).)

In addition to the whole-cell recording configuration, theoutside-out excised patch configuration was used to recordsingle-channel currents. The resistance of the pipettes rangedfrom 100 to 120 Ω in 10 mM CaCl2. Data were sampled at1 kHz and the recording time was 51 s. Bessel filtering wasat 2 kHz. Smooth lines were obtained from best fits of data tothe Boltzmann equation:

PO/PO(max) = 1 + exp[(Vmax − V1/2)/k]1

(PO/PO(max), the relative open frequency; Vmax, the membranepotential (the voltage applied); V1/2, the half-maximal voltageof activation; k, the slope factor.) V1/2 and k were determinedfor each current obtained from individual spheroplasts (Salapateket al., 2002; Misonou et al., 2004).

Growth analysis

Photomicrographs (Biological Microscopes Motic BA200;Motic, Amoy, FuJian Province, China) were analyzed usingMOTIC IMAGES ADVANCED 3.2 (Motic) to determine germinationrates and pollen tube lengths.

Statistical analysis

Tests were conducted using Student’s t-test.

Results

Isolation of spheroplasts from pollen tubes

After incubation of the pollen tubes in the enzymaticsolution, spheroplasts were released from the apical regions(Fig. 1). To ensure that spheroplasts were derived from thepollen tube apex, pollen tubes were observed every 10 minduring incubation in the enzyme solution and observedduring solution exchange. The spheroplasts of the pollentubes were distinguished from pollen grain protoplasts bytheir smaller size. The mean diameters of the spheroplastsisolated from the pollen tubes and pollen grain protoplastswere 43.5 ± 1.7 µm (n = 60) and 75.1 ± 2.9 µm (n = 50),respectively. Few pollen grain protoplasts were obtained as itis difficult to remove pollen walls by general enzymolysis,largely because of the many low-molecular-weight com-pounds (carotenes and flavonoids), lipids and proteins in theexine (Stanley & Linskens, 1985; Chay et al., 1992). Thosethat were released often remained securely attached to theircell wall (see Fig. 1, protoplasts 1–3) even if enzymolysis wasprolonged.

Identification and characterization of Ca2+ channels

The patch-clamp whole-cell configuration was used tocharacterize ionic channels in the plasma membrane of apicalspheroplasts derived from the pollen tubes of P. pyrifolia(Fig. 2a). For pollen harvested in 2006, seal resistance was

Fig. 1 Enzymatic release of pollen tube spheroplasts and pollen grain protoplasts of Pyrus pyrifolia cv. Housui. Note the extrusion of a spheroplast from a pollen tube apex. Protoplasts from pollen grains (1–4) were greater in diameter than spheroplasts released from pollen tube apices.


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typically 1 GΩ and such seals were obtained in approx. 40%of attempts. With Ca2+ being the only permeant cation in thebath (10 mM), hyperpolarizing pulses of > −100 mV from theholding potential of 0 mV elicited large, time-dependentinward currents (Fig. 2b,c). Application of 15 successive

hyperpolarizing voltage ramps (with 2-min intervals betweenramps) revealed that the inward Ca2+ currents were stable(Fig. 2d; n = 8). Reversal of the osmotic gradient across themembrane (bath 1200 mOsM, pipette 1100 mOsM) had noeffect on the conductance (data not shown).

Fig. 2 Whole-cell recordings of current obtained from the apical spheroplast plasma membrane of Pyrus pyrifolia cv. Housui. (a) A pipette forming a high-resistance electrical seal with the spheroplast plasma membrane. Seal resistance always exceeded 1 GΩ. (b) Whole-cell currents from an individual spheroplast were elicited by sequential step-wise hyperpolariziation of the membrane to −200 mV from a holding potential of 0 mV. The inset depicts the voltage clamp protocol. The bathing solution contained 10 mM calcium (Ca2+). Polarity convention: a downward current deflection is the entry of positive charge into the spheroplast or the exit of negative charge. (c) Ca2+ currents were recorded in response to a hyperpolarizing voltage ramp from 0 to −200 mV (ramp speed 9.84 mV s−1); 10 mM Ca2+ (n = 8). (d) Ca2+ currents elicited by 15 successive voltage ramps (ramp speed 15.19 mV s−1) recorded in 10 mM Ca2+ from one spheroplast. The interval between each ramp was 2 min. Currents were superimposed. (e) Effect of extracellular CaCl2 on the current–voltage (I/V) relationship. Each data point shows mean ± standard error (closed circles, 1 mM, n = 12; open circles, 5 mM, n = 9; triangles, 10 mM, n = 44). The difference in Ca2+ currents between different Ca2+ concentrations was statistically significant when voltage ranged from −200 to −180 mV (**, P < 0.01), and from −160 to −140 mV (*, P < 0.05).


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To verify the nature of the currents, the concentrationof external CaCl2 was varied. As shown in Fig. 2(e), thehyperpolarization-activated inward currents were markedlyreduced when the CaCl2 concentration was reduced from 10to 1 mM (n = 9–44), indicating that Ca2+ was the charge carrier.The mean (± standard error (SE)) reversal potential (Erev) ofthe inward current (determined from the ‘tail-currents’) was22.5 ± 8.7 mV in 10 mM CaCl2 (Fig. 3a,c; n = 9) and decreasedto 12.6 ± 4.7 mV when the external CaCl2 was reduced to1 mM (Fig. 3b,c; n = 7). As the estimated equilibrium potentialsfor Ca2+ decreased from 174 to 145 mV but those for Cl–

increased from 41 to 99 mV as external CaCl2 was reducedfrom 10 to 1 mM, the variational trend in Erev suggests that the

inward currents were likely to have been carried by Ca2+ influxrather than Cl– efflux. The relative permeability of the channelsto Ca2+ and Cl– (PCa/PCl) was estimated according to themodified GHK equation using tail-current Erev values (Fig. 3c).A permeability ratio of PCa/PCl of 14 was determined, whichsuggests that this conductance is selective for Ca2+ over Cl–.

Selectivity of the hyperpolarization-activated inward conductance

To examine whether the hyperpolarization-activated inwardconductance was selective for divalent cations, the 10 mM

CaCl2 in the bath was substituted with equimolar MgCl2,

Fig. 3 Tail-current analysis reveals the reversal potential of the whole-cell conductance of Pyrus pyrifolia cv. Housui. (a, b) Evidence for a calcium (Ca2+) component in the inward current with 10 mM (a) and 1 mM (b) extracellular CaCl2. After activation of the conductance by a single voltage step from 0 to −200 mV, the voltage was increased in a single step to depolarizing voltages ranging from −10 or −20 mV to 60 mV (see insets). The arrow indicates the reversal current (i.e. no net current is observed); this permits estimation of the reversal potential, the voltage at which no net current passes. (c) Current–voltage (I/V) relationship of the tail currents. The upper and lower arrows indicate the reversal potentials in 1 mM (closed circles, n = 7) and 10 mM (open circles, n = 9) extracellular CaCl2, respectively.


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BaCl2, SrCl2 or MnCl2. By comparing the magnitude of theinward currents from −120 to −200 mV, a permeability sequenceof barium (Ba2+) ≈ Ca2+ > magnesium (Mg2+) > strontium(Sr2+) > manganese (Mn2+) was established (Fig. 4; n = 10–16).Inward currents also were observed when 10 mM KCl replaced10 mM CaCl2 in the bath solution. However, currents wereapprox. 10 times smaller at −200 mV than those recorded withextracellular CaCl2 or BaCl2 (Fig. 5; n = 9). Therefore, it canbe concluded that the hyperpolariszation-activated inwardconductance was strongly selective for Ca2+.

Single-channel Ca2+ currents

Further characterization of the Ca2+ channels was conductedat the single-channel level using outside-out excised membranepatches with high-resistance seals (> 5 GΩ) (Fig. 6a). As thevoltage was hyperpolarized from −100 to −200 mV, the mean(± SE) magnitude of the inward current was significantlyincreased from −0.67 ± 0.95 pA to −8.87 ± 3.10 pA (n = 8).The threshold for channel opening was approx. −100 mV(Fig. 6b). In addition to the change in currents, the mean(± SE) opening frequency (PO) was also increased from0.0413 ± 0.007 to 0.425 ± 0.0360 (Fig. 6c; n = 8). Theseobservations indicate that the activity of the Ca2+ channeldepended on voltage. The PO curve was fitted by the Boltzmannequation (Eqn 6), where V1/2 was −163.79145 ± 3.54645 mV,and K was 17.08056 ± 3.46271 (n = 8).

Effects of gadolinium (Gd3+) and lanthanum (La3+)

Gd3+ and La3+ are two widely used Ca2+ channel blockerseffective in both animal and plant cells. As shown in Fig. 7,100 µM La3+ or Gd3+ markedly inhibited the hyperpolarization-activated inward currents (n = 7). Application of this concentrationof Gd3+ or La3+ abolished germination and markedly inhibitedtube elongation in P. pyrifolia (Table 1). Taken together, theseresults suggest that the Ca2+-permeable cation channel identifiedhere is involved in pollen germination and tube elongation.

Viability of pollen influences Ca2+ influx

There was an extremely significant difference (P < 0.01) ingermination rate and pollen tube length (determined at 3 h ofincubation) between the pollen that was collected in 2004and then stored for 2 yr and that collected in 2006 and thenstored for 6 months. The former exhibited a germination rateof only 4.3% (n = 276) and mean (± SE) pollen tube length

Fig. 4 The effect of different extracellular divalent cations on the whole-cell current–voltage (I/V) relationship of apical spheroplasts of Pyrus pyrifolia cv. Housui. Each curve was based on 10 pollen tube spheroplasts except for calcium (Ca2+) (n = 18) and barium (Ba2+) (n = 16). Each data point represents the mean ± standard error recorded in 10 mM extracellular divalent cation. The inward currents were significantly different between Ca2+ and magnesium (Mg2+); Ca2+ and manganese (Mn2+); and Ca2+ and strontium (Sr2+) when the test voltage was between −180 and −200 mV (**, P < 0.01), and between −160 and −120 mV (*, P < 0.05); but they were not significantly different (P > 0.05) between Ba2+ and Ca2+.

Table 1 Gadolinium (Gd3+) and lanthanum (La3+) affect germination and growth of Pyrus pyrifolia cv. Housui pollen

Control

Treatment

Gd3+ La3+

Germination rate (%) 73 0 0Length of pollen tube at 2 h (µm) 226 ± 21 0 0Length of pollen tube at 4 h (µm) 451 ± 28 0 0Length of pollen tube (µm) 2 h after 451 ± 28** 230 ± 20 228 ± 18addition of inhibitor to growing pollen tubes

GdCl3 or LaCl3 was added to a final concentration of 100 µM before germination and then pollen tube length was determined at 2 and 4 h after germination. Alternatively, pollen grains were germinated under control conditions and inhibitor at a concentration of 100 µM was added 2 h after germination (treatment). At 4 h, the lengths of control pollen tubes were compared with those in the treatments, and the difference was found to be significant (**, P < 0.01). Experiments were repeated three times and data are mean (± standard error) values from at least 300 observations.


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of 151.68 ± 86.41 µm (n = 62), while the latter exhibiteda germination rate of > 70% (n = 369) a pollen tube length of> 400 µm (n = 304). These data suggested that prolongedstorage deleteriously affected pollen viability. Additionally,the Ca2+ current magnitude of apical spheroplasts from the2004 pollen was approx. 12 times smaller than that of apicalspheroplasts from the 2006 pollen (Fig. 8; n = 16). Thus, Ca2+

channel activity at tip of the pollen tube was affected by pollenstorage. The low germination and growth rates of the 2004 pollencoupled with the low success rate in obtaining high-electrical-resistance seals (5%) curtailed further investigation of this pheno-menon. Seal resistances were in the range of 400–700 Ω andwere not improved by coating pipette tips with poly L-lysine.

pH dependence of Ca2+ currents

The pH of the medium was found to influence both pollengermination rate and pollen tube length. The medium pH

was adjusted from 4.5 to 7.0 using MES and/or Tris. Pollengermination differed significantly with medium pH (Fig. 9a).It was highest at pH 5.5 (80.23%; n = 316) and lowest atpH 7.0 (8.95%; n = 410) but there was no significantdifference between pH 5.5 and 6.5. Low germination rateswere observed at pH 4.5 (n = 493) and pollen tube growthwas arrested at this pH (Fig. 9b). Despite showing the lowestgermination rates, pollen tube length after 1 h at pH 7.0 wassignificantly greater than at pH 4.5. During the first 2 h ofgrowth, pollen tube lengths were greatest at pH 5.5 and 6.5,but at 3 h those at pH 6.5 were significantly longer(P < 0.01) than those at pH 5.5 (Fig. 9b). External pH alsoaffected the Ca2+ currents; these decreased gradually as thepH was increased from 4.5 to 6.5 (Fig. 10a,b). For instance,relative to the currents at pH 6.5 (measured at −200 mV)the currents at pH 4.5 and 5.5 were approx. 3 and 2 timeshigher, respectively. These results show that Ca2+ currentswere pH dependent.

Fig. 5 Extracellular potassium (K+) weakly permeates the hyperpolarization-activated conductance. (a) K+ currents were recorded with the whole-cell configuration (10 mM KCl replaced CaCl2 as the charge carrier; the pipette solution contained 100 mM CsCl; n = 21). (b) Comparison of mean (± standard error) current–voltage (I/V) relationships with K+ or calcium (Ca2+) as the charge carrier (closed circles, 10 mM KCl; open circles, 10 mM CaCl2; n = 9). Insets are schematics of the voltage clamping protocol.


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Discussion

Hyperpolarization-activated Ca2+-permeable channels havebeen identified in a number of plant cells (Miedema et al.,2001), such as in the root hairs (Véry & Davies, 2000) andpollen grains (Wang et al., 2004) of Arabidopsis, and in thepollen grains of lily (Lilium davidii Duchartre ; Shang et al.,2005). Previous studies have also isolated spheroplasts from thepollen tubes of lily, identifying K+ inwardly rectifying channels(Griessner & Obermeyer, 2003) and stretch-activated cationchannels (Dutta & Robinson, 2004). The present study revealed

the existence of hyperpolarization-activated Ca2+-permeablechannels in the plasma membrane of pollen tubes ofP. pyrifolia. The hyperpolarization-activated Ca2+ conductanceexhibited similar characteristics to those reported in theliterature (Pei et al., 2000; Véry & Davies, 2000; Wang et al.,2004; Shang et al., 2005) in terms of activation kinetics,selectivity and pharmacology.

It is well established that tip growth, as seen in pollen tubes,algal rhizoids, fungal hyphae and root hairs, is associated withan apex-high [Ca2+]cyt gradient (e.g. Messerli & Robinson, 1997;Felle & Hepler, 1997; Wymer et al., 1997; Messerli et al., 1999).

Fig. 6 Single-channel recordings of calcium (Ca2+) currents. (a) Outside-out patches of the apical pollen tube spheroplast plasma membrane of Pyrus pyrifolia cv. Housui were polarized to different voltages (10 mM extracellular Ca2+). Step-like current events – an indication of the opening (O) and closing (C) of single channels – were seen only when the voltage was between −100 and −200 mV. Different channel open states (O1, O2) occurred at the most hyperpolarized voltage (−200 mV). No activity was observed between −80 and +100 mV. An addition of 100 µM lanthanum (La3+) or gadolinium (Gd3+) to the bath solution inhibited channel activity, as indicated by very brief openings. (b) The current–voltage (I/V) relationship of single-channel inward currents when voltage was varied from −100 to −200 mV. (c) Open probability (PO) as a function of applied voltage. The PO curve was fitted by the Boltzmann equation PO/PO(max) = 1/[1 − exp(V − V1/2)/K], where V1/2 is −163.79145 ± 3.54645 mV and K is 17.08056 ± 3.46271 (n = 8). Closed circles, 10 mM Ca2+.


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Fig. 7 Effects of lanthanum (La3+) and gadolinium (Gd3+) on inward currents of the pollen tube apical spheroplast plasma membrane of Pyrus pyrifolia cv. Housui. (a) Normal calcium (Ca2+) currents recorded in 10 mM extracellular Ca2+. (b) Ca2+ currents with 100 µM La3+ added to the bath solution (representative of seven independent trials). (c) Normal barium (Ba2+) currents recorded in 10 mM extracellular Ba2+. (d) Ba2+ currents with 100 µM Gd3+ added to the bath solution (representative of seven independent trials). (e) Comparison of the mean (± standard error) current–voltage (I/V) relationships from the four conditions shown in (a)–(d) (P < 0.01 for the data points marked **). Note that La3+ and Gd3+ completely blocked the channels (n = 7).


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The [Ca2+]cyt gradient has been suggested to result from alocalized Ca2+ influx through Ca2+-permeable channels at theapical plasma membrane (Wymer et al., 1997; Holdaway-Clarke et al., 2003). However, evidence of the presence offunctional Ca2+-permeable channels in the apices of pollentubes has been limited. In the present study, we successfullyisolated spheroplasts from the apical regions of elongatingpollen tubes in P. pyrifolia and routinely obtained the high-resistance seals (> 1GΩ) that are essential for characterizingthe activity of ionic channels by patch-clamp electrophysiology.A hyperpolarization-activated inwardly rectifying currentwas found to dominate the conductance of the pollen tubespheroplast plasma membrane. The inward current wascarried by a Ca2+ influx (PCa/PCl of 14), but the channel wasalso permeable to other divalent cations with a permeabilitysequence of Ba2+ ≈ Ca2+ > Mg2+ > Sr2+ > Mn2+. This is com-parable to the cation channel identified in the plasma membranesof root hair tips in Arabidopsis (Véry & Davies, 2000). InArabidopsis pollen grains, however, a cation channel with a

different permeability sequence of Mg2+ > Ca2+ > Ba2+ > Sr2+

> Mn2+ was observed (Wang et al., 2004).Similar to the Ca2+-permable channels of Arabidopsis root

hairs and pollen grains (Véry & Davies, 2000; Wang et al.,2004), the channel identified here in P. pyrifolia pollen tubeswas sensitive to La3+ and Gd3+, two broad-spectrum Ca2+-channel blockers effective in both animal and plant systems.These two inhibitors also suppressed pollen germinationand tube elongation. Moreover, the low viability of pollencorresponded to low channel activity at the pollen tube apex.Therefore, we suggest that the Ca2+-permeable channelsidentified in the present study have physiological significancein regulating pollen germination and tube elongation inP. pyrifolia.

Voltage and membrane tension are anticipated to besignificant factors in the regulation of plasma membrane Ca2+

Fig. 8 Pollen viability affects the magnitude of the calcium (Ca2+) currents. (a) Representative whole-cell recording from an apical pollen tube spheroplast from Pyrus pyrifolia cv. Housui pollen collected in 2004 (10 mM extracellular Ca2+). (b) Comparison of mean (± standard error) whole-cell Ca2+ currents in apical pollen tube spheroplasts obtained from pollen collected in 2004 (open circles, n = 16) and 2006 (closed circles, n = 29). Recording conditions were as in (a).

Fig. 9 Effect of extracellular pH on germination and tube growth of Pyrus pyrifolia cv. Housui pollen. (a) Germination rates of pollen at pH 4.5 (n = 493), pH 5.5 (n = 316), pH 6.5 (n = 721), and pH 7.0 (n = 410). (b) Pollen tube lengths were measured at 1, 2, and 3 h after germination (pH 4.5, n = 93; pH 5.5, n = 235; pH 6.5, n = 307; pH 7.0, n = 71). Growth was arrested at pH 4.5 and greatly inhibited at pH 7.0. Growth rate was fastest at pH 6.5, particularly after 2 h.


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channels in polar growth and signalling. There is evidencethat reorienting treatments applied during pollen tube growthinduce a depolarization of the plasma membrane, suggestingthat voltage-gated Ca2+ channels might be activated (Malhóet al., 1995, 2000). Dutta & Robinson (2004) have identifieda stretch-activated Ca2+ channel in pollen-tip spheroplasts ofLilium longiflorum. The selectivity of this channel was notdetermined but it was found to be blocked by spider venom;application of this toxin to growing pollen tubes blocked Ca2+

influx, implicating the stretch-activated channel in growth.A similar stretch-activated Ca2+ channel (which was Gd3+-sensitive and open at negative voltages and showed a verystrong selectivity for Ca2+ over K+) was observed after 1 h ormore of incubation in the germination medium, shortly beforegermination occurred; it was not present in a functional formin newly hydrated pollen grains (Dutta & Robinson, 2004).There is substantial evidence for a role of mechanosensitiveion channels in detecting and eliciting responses to animal

cell volume changes (McCartey & O’Neil, 1992; Chen et al.,1996). In plants, stretch-activated Ca2+-permeable channelshave been found in guard cell plasma membranes (Cosgrove& Hedrich, 1991; Grabov & Blatt, 1998). In tip growingcells, such as pollen tubes, turgor pressure could regulatestretch-activated Ca2+ channels only within narrow limitsdespite variations in cell elongation rate (Benkert et al., 1997).The finding here that the hyperpolarization-activated Ca2+-permeable channel was present in both pollen grains andpollen tubes suggests that the hyperpolarization-activatedCa2+ channels may play an important role in germination,[Ca2+]cyt oscillations and maintenance of the [Ca2+]cyt gradientat the tip of the pollen tube.

Pollen tubes possess an oscillatory pH gradient (6.8 at thetip and 7.5 in the clear zone, detected with BCECF-dextran;Feijó et al., 1999). Tip acidity follows growth by ∼20° (whileionic activities and fluxes show the same period as growth,they usually do not show the same phase and mostly lag behind)whereas the alkaline band anticipates growth by ∼125°(Holdaway-Clarke & Hepler, 2003; Hepler et al., 2005,2006). These results indicate the alkaline band as a centralplayer in growth control. In addition to the internal pH gradient,there are marked currents of protons in the extracellularmedium both in root hairs and in pollen tubes. Apoplastic,apical H+ may promote a more plastic and extensible wall; forexample, acidic pH decreases the activity of pectin methylesterase(PME; Moustacas et al., 1986), thus reducing the number ofcarboxyl residues and the amount of Ca2+ cross-linking. LowpH may also enhance the activity of acidic isoforms of PME(Li et al., 1996, 2002), which, together with pectin hydrolyases,cause the degradation of pectin gels (Bordenave, 1996). However,in lily, the germination rate and the average growth rate ofshort tubes are more sensitive to changes in pH while forlonger tubes the pH has less effect, except at pH 7.0, at whichgrowth is completely stopped (Holdaway-Clarke et al., 2003).In this study, we found that there were no differences ingermination and initial growth between pH 5.5 and 6.5.However, growth rate was significantly increased at pH 6.5after 2 h, suggesting that relatively long pollen tubes could bemore sensitive to changes of pH. High extracellular H+ (low pH)promoted Ca2+ influx through Ca2+ channels in the membraneof apical pollen tubes. The lower the pH of the bath solution,the greater the Ca2+ currents. As extremes of low and high pHinhibited germination and pollen tube growth, we reason thatpH regulation of Ca2+ influx mediated by the hyperpolarization-activated Ca2+ channel must be finely tuned to allow growthto proceed. Too great or too low an influx would be inhibitory.

In summary, apical spheroplasts from elongating pollentubes of P. pyrifolia were isolated and a hyperpolarization-activated Ca2+-permeable inward conductance was identifiedin the plasma membrane. The Ca2+-permeable channel wasalso permeable to other divalent cations including Ba2+, Ca2+,Mg2+, Sr2+, and Mn2+. The calcium-channel blockers La3+

and Gd3+ inhibited the hyperpolarization-activated channel

Fig. 10 The impact of extracellular pH on inward calcium (Ca2+) currents. (a) The inward currents (mean ± standard error) were compared at three bathing solution pH values (closed circles, pH 4.5, n = 15; triangles, pH 5.5, n = 44; open circles, pH 6.5, n = 12). The difference in Ca2+ currents was statistically significant when the voltage was held between −160 and −200 mV (**, P < 0.01), and between −140 and −120 mV (*, P < 0.05). (b) Ca2+ currents at different pH values recorded from a single spheroplast of Pyrus pyrifolia cv. Housui. The dark gray area indicates that the current at pH 5.5 was greater than that at pH 6.5 (n = 3). The light gray area indicates that the current at pH 4.5 was higher than that at pH 5.5. The inset shows the voltage protocol.


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as well as pollen germination and tube elongation. Moreover,the channel was regulated by extracellular pH and activity variedwith pollen viability. We conclude that the Ca2+-permeablechannel characterized in the present study is likely to playa role in pollen germination and tube elongation.

Acknowledgements

We thank all the scientists who helped to improve ourmanuscript. Special thanks to Dr Julia Davies (Department ofPlant Sciences, University of Cambridge) and Dr Wu Li (TheRockefeller University). We are indebted to the two anonymousreviewers, whose insightful comments resulted in a muchimproved final version of the manuscript. This work wassupported by the Science Foundation of Doctoral SubjectPoint of the Chinese Ministry of Education (No: B200523)and the scientific research fund of HYIT (No: HG0606).

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