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In vitro Arabidopsis pollen germination and characterization of the inward potassium currents in Arabidopsis pollen grain protoplasts. Liu-Min Fan, Yong-Fei ...
Journal of Experimental Botany, Vol. 52, No. 361, pp. 1603±1614, August 2001

In vitro Arabidopsis pollen germination and characterization of the inward potassium currents in Arabidopsis pollen grain protoplasts Liu-Min Fan, Yong-Fei Wang, Hong Wang and Wei-Hua Wu1 Department of Plant Sciences, College of Biological Sciences, Key Research Laboratory in Plant Physiology and Biochemistry, China Agricultural University, Beijing 100094, China Received 11 December 2000; Accepted 5 April 2001

Abstract The focus of this study is to investigate the regulatory role of Kq influx in Arabidopsis pollen germination and pollen tube growth. Using agar-containing media, in vitro methods for Arabidopsis pollen germination have been successfully established for the first time. The pollen germination percentage was nearly 75% and the average pollen tube length reached 135 mm after a 6 h incubation. A decrease in external Kq concentration from 1 mM to 35 mM resulted in 30% inhibition of pollen germination and 40% inhibition of pollen tube growth. An increase in external Kq concentration from 1 mM to 30 mM stimulated pollen tube growth but inhibited pollen germination. To study how Kq influx is associated with pollen germination and tube growth, regulation of the inward Kq channels in the pollen plasma membrane was investigated by conducting patchclamp whole-cell recording with pollen protoplasts. Kq currents were first identified in Arabidopsis pollen protoplasts. The inward Kq currents were insensitive to changes in cytoplasmic Ca2q but were inhibited by a high concentration of external Ca2q. A decrease of external Ca2q concentration from 10 mM (control) to 1 mM had no significant effect on the inward Kq currents, while an increase of external Ca2q concentration from 10 mM to 50 mM inhibited the inward Kq currents by 46%. Changes in external pH significantly affected the magnitude, conductance, voltage-independent maximal conductance, and activation kinetics of the inward Kq currents. The physiological importance of potassium 1

influx mediated by the inward Kq-channels during Arabidopsis pollen germination and tube growth is discussed. Key words: Arabidopsis thaliana, pollen germination, Kq-channel, patch-clamp, in vitro pollen culture.

Introduction Pollen germination and pollen tube growth are essential processes that ensure the reproduction of ¯owering plants. These complex processes involve a number of signalling events, including cell±environment interaction, intercellular communication and intracellular signalling (Taylor and Hepler, 1997; Franklin-Tong, 1999). Physiological and molecular mechanisms of regulation of pollen germination and pollen tube growth have been extensively studied in the past (Heslop-Harrison, 1987; Mascarenhas, 1993; Taylor and Hepler, 1997; Franklin-Tong, 1999). It is known that pollen germination and tube growth are signi®cantly regulated by the transport of inorganic ions, such as Ca2q and Kq, across the plasma membranes of pollen anduor pollen tubes (Feijo et al., 1995; Taylor and Hepler, 1997). It is also known that Kq is required for both pollen germination and tube growth (Brewbaker and Kwack, 1963; Weisenseel and Jaffe, 1976; Feijo et al., 1995). By the application of patch-clamp techniques, the three types of Kq-channels from Lilium pollen protoplasts were identi®ed and the possible involvement of inward Kq-channel-mediated Kq in¯ux during pollen tube

To whom correspondence should be addressed. Fax: q86 10 6289 3450. E-mail: [email protected] Abbreviations: E1u2, half-activation voltage; Erev, reversal potential; Gmax, maximal conductance; Km, Michaelis±Menten constant; t1u2, half-activation time; Vm, membrane potential; t1 and t2, time constants. Published by Oxford University Press

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growth was suggested (Obermeyer and Kolb, 1993). Using conventional voltage-clamp techniques, Obermeyer and Blatt observed both an outward Kq current and an inward Kq current across the plasma membrane of nongerminating Lilium pollen grains. (Obermeyer and Blatt, 1995) They proposed that an inward Kq current in a nongerminating pollen grain may play a role in initiating the osmotic water in¯ux required for pollen germination. Fan et al. have used a patch-clamp whole-cell recording technique to identify and characterize Kq currents in Brassica pollen protoplasts (Fan et al., 1999) and the results showed that the inward whole-cell currents in Brassica pollen protoplasts are mainly carried by the inward Kq-channels. It was also shown that the inward Kq channels in Brassica pollen protoplasts are signi®cantly regulated by external Ca2q, which is an important regulatory factor for pollen germination and pollen tube growth. These previous studies strongly suggest that the inward Kq channels may be essential components involved in the processes of pollen germination and tube growth, and that the regulation of the Kq-channels may play a regulatory role in pollen germination and tube growth. To investigate molecular mechanisms of Kq-channel regulation as well as its association with signal transduction cascades in pollen germination and tube growth, Arabidopsis was considered as a model system for this study. The plant species used for most of the previous studies of pollen germination and tube growth regulation were Lilium (Pierson et al., 1994, 1996; Holdaway-Clarke et al., 1997; Messerli and Robinson, 1997; Messerli et al., 1999), Nicotiana (Cheung et al., 1995) and Petunia (Lee et al., 1994), probably due to the large size of the pollen grains and also the easy collection of large amounts of pollen. However, due to their large genome size (Leeton and Smyth, 1993) and limited molecular genetic information compared with Arabidopsis, it may be too dif®cult to use those species to conduct further molecular and genetic studies. It is obvious that there are a number of advantages if Arabidopsis could be used as a model system to study regulatory mechanisms in pollen germination and tube growth due to its relatively small genome and short life cycle (Meyerowitz and Somerville, 1994). In order to use Arabidopsis as a model system to study its pollen germination and tube growth, there must ®rst be a method for the in vitro germination of Arabidopsis pollen grains. Arabidopsis pollen belongs to the trinucleate type of pollen that usually does not germinate well in vitro and it has been reported that the in vitro germination percentage of Arabidopsis pollen grains was less than 20% (Preuss et al., 1993). Thus, it has usually been considered that Arabidopsis pollen grains barely germinate in vitro (Taylor and Hepler, 1997). In the present study, a method for in vitro Arabidopsis pollen germination has been established for the ®rst time

and the regulatory effects of external Kq and other factors on Arabidopsis pollen germination and tube growth have been investigated. In addition, patch-clamp whole-cell recording techniques were applied to identify and characterize the inward Kq channels in Arabidopsis pollen plasma membranes and the possible regulation of the inward Kq channels by Ca2q and external pH was investigated. The physiological importance of potassium in¯ux via the inward Kq-channels in Arabidopsis pollen germination and tube growth is also discussed.

Materials and methods Plant growth conditions

Arabidopsis thaliana (ecotype Landsberg erecta) plants were grown in mixed soil in a growth chamber. The light intensity was 120 ±150 mmol m 2 s 1 for a 12 h daily light period and dayunight temperatures were 22"2 8C and 18"2 8C, respectively. Plants were watered once every 5 d with tap water and the relative humidity in the growth chamber was kept near 70%. In vitro pollen germination

Only freshly anther-dehisced ¯owers were used for in vitro pollen germination experiments since pollen grains at this developmental stage showed the highest germination percentage. For each experiment, 150 ¯owers were randomly collected from 30 different plants. The dehisced anthers of ®ve randomly picked ¯owers were carefully dipped onto the surface of agar plates to transfer the pollen grains. Each treatment had four replicates (four agar plates). The Basic Medium for in vitro pollen germination contained 5 mM MES ( pH 5.8 adjusted with TRIS), 1 mM KCl, 10 mM CaCl2, 0.8 mM MgSO4, 1.5 mM boric acid, 1% (wuv) agar (Kq-depleted agar, see the text in `Results'), 16.6% (wuv) sucrose, 3.65% (wuv) sorbitol, and 10 mg ml 1 myo-inositol. Changes in the concentrations of KCl, CaCl2, and boric acid in the medium as well as medium pH are indicated in the text. The medium was prepared with doubledistilled water and heated to 100 8C for 2 min. Each agar plate (35 mm diameter Petri dish) contained 1.5 ml medium forming a thin layer. Following pollen application, the dishes were immediately transferred to a chamber at 25 8C with 100% relative humidity (controlled with a humidi®er) in the light (30 mmol m 2 s 1 supplied with ¯uorescent tubes). The total and germinated pollen grains were counted and pollen tube length was measured under a microscope after incubation for 6 h. All experiments were repeated three times and each treatment in one experiment included four agar plates (replicates). For each agar plate 500 pollen grains were counted for calculation of germination percentage and 100 pollen tubes were measured. Isolation of pollen protoplasts Matured pollen grains were collected from 30 ¯owers of 10 different Arabidopsis plants immediately before the isolation of pollen protoplasts. The pollen grains were ®rst washed in standard solution containing 1 mM KNO3, 0.2 mM KH2PO4, 1 mM MgSO4, 1 mM KI, 0.1 mM CuSO4, 5 mM CaCl2, 5 mM MES (pH 5.8 adjusted with TRIS), 500 mM glucose and sorbitol (osmolality ˆ 1.5 Osmol kg 1) before enzymatic digestion. After ®ltration through a nylon mesh (diameter ˆ 80 mm) and centrifugation at 160 g for 5 min, pollen grains were

Kq-channels in Arabidopsis pollen protoplasts 1605 incubated in 2 ml of enzyme solution at 28 8C for 1 h to release pollen protoplasts. The enzyme solution was prepared with standard solution containing 1% (wuv) cellulase R-10 (Yakult Honsha Co., Japan), 0.5% (wuv) macerozyme R-10 (Yakult Honsha Co., Japan), 0.2% (wuv) PDS (potassium dextran sulphate, CalBiochem, USA), and 0.2% (wuv) BSA. The mixture was centrifuged at 160 g for 5 min and the pellet was resuspended with 2 ml of the standard solution. This centrifugationuresuspension cycle was conducted three times to remove BSA, enzymes, and undigested pollen wall debris completely. The pollen protoplasts were ®nally resuspended in the standard solution and kept on ice before use in patch-clamp experiments.

Patch-clamp whole-cell recording experiments Standard whole-cell recording techniques (Hamill et al., 1981) were applied in this study. The bath solution contained 5 mM MES (pH 5.8 with TRIS), 10 mM CaCl2, 1 mM MgCl2, 10 mM potassium glutamate, and osmolality at 1.5 Osmol kg 1 adjusted with sorbitol. The standard pipette solution contained 5 mM HEPES ( pH 7.2 with TRIS), 5 mM EGTA, 0.3 mM CaCl2, 100 mM potassium glutamate, 1 mM MgCl2, and osmolality at 1.5 Osmol kg 1 adjusted with sorbitol. The free Ca2q concentration in this standard pipette solution was 10 nM. The pipette (internal) solution containing 10 mM free Ca2q was made by addition of 4.94 mM CaCl2 to the standard pipette solution. The calculation of free Ca2q concentration in the pipette solutions was conducted with MAX Chelator software (Version 6, by Dr Chris Patton at Stanford University, CA, USA). The whole-cell recordings conducted under these conditions were taken as the control. The resistance of the electrodes under these conditions was between 15 and 20 MO. The experiments were conducted at room temperature (20"2 8C) in dim light. Cell capacitance was measured for each cell using the capacity compensation device of the ampli®er. All data were acquired 5 min after the formation of the whole-cell con®guration. Whole cell currents were measured using an Axopatch-200A ampli®er (Axon Instruments, Foster City, CA, USA) which was connected to a microcomputer via an interface (TL-1 DMA Interface, Axon Instruments, USA). pCLAMP (Version 6.0.4, Axon Instruments) software was used to acquire and analyse the whole-cell currents. After the whole cell con®guration was obtained, membrane potential (Vm) was clamped to 58 mV. Voltage pulse protocols as shown in the text were generated using pCLAMP software and applied to the clamped cell for data acquisition. Whole-cell current data were ®ltered at 1 kHz before storage (1 ms per sample) on a computer disk.

was applied to test the differences between the control and the treatments. Chemicals All chemicals were obtained from Sigma (St Louis, USA) unless otherwise indicated.

Results In vitro Arabidopsis pollen germination and tube growth

The `Basic Medium' described in `Materials and methods' was used in the control experiments for in vitro Arabidopsis pollen germination. Figure 1 shows the time-dependence of pollen germination and tube growth under the control conditions. Nearly 75% of pollen grains germinated after incubation for 6 h (Figs 1, 2A). The maximal germination percentage (;80%) was reached after 12 h incubation. The pollen tubes continued to grow after 12 h incubation (Fig. 1) and active cytoplasmic streaming was clearly observed even after 24 h incubation. Effects of external Kq, Ca2q, Ba2q, pH, and boron on pollen germination and tube growth in vitro

The concentration suitable for the pollen germination of each component in the medium (such as Ca2q, Kq, boric acid, etc.) was determined based on the results of repeated experiments. As shown in Figs 2 and 3A, 1 mM Kq in the medium resulted in the highest germination percentage, and either higher or lower Kq resulted in the inhibition of pollen germination. When the medium contained `no additional Kq', both pollen germination and tube growth was signi®cantly inhibited (Figs 2B, 3A). In fact, the medium with `no additional Kq' contained

Data analysis of the whole-cell recordings

Leak currents of each whole-cell recording were subtracted before generating whole-cell current-voltage relations. Leak current for each cell was determined from the ®rst three data points obtained after Vm was stepped from the holding voltage to the test voltages (Wu and Assmann, 1994). The mean values of whole-cell currents were determined as the average of data points obtained between 1.6 and 1.9 s (300 data points total) after imposition of the test voltage (when current amplitude had reached its plateau). After subtraction of leak currents, the ®nal whole-cell currents were expressed as the currents per unit capacitance ( pA pF 1) to account for variations in cell surface area. All data are given as means"SE. SigmaPlot software was used to draw I±V plots and the function of t-test in this software

Fig. 1. Time-courses of Arabidopsis pollen germination and pollen tube growth in vitro. All experiments were repeated three times and each treatment in one experiment had four replicates (four dishes for in vitro germination). Five hundred pollen grains were counted for germination and 100 pollen tubes were measured for pollen tube length for each replicate. Each data point is presented as mean"SE.

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Fig. 2. Effects of external Kq on Arabidopsis pollen germination and tube growth in vitro. The scale bar shown in (A) is for all treatments (A±C). (Note: the treatment indicated with `No additional Kq' actually contained 35 mM Kq; see text.)

approximately 35 mM Kq mainly from Kq-contaminated agar even though `Kq-depleted agar' was used. Kq contents in several different brands of agars were measured and all these agars contain Kq that results in Kq concentrations between 200 and 300 mM when 1% (wuv) agar was added to medium. To obtain `Kq-depleted agar', the purchased agar was washed 3±4 times with 2 mM HCl, then rinsed with double-distilled water 5 times, and ®nally dried in an oven at 40 8C. The addition of 1% (wuv) Kq-depleted agar to the medium resulted in a ®nal Kq concentration of approximately 35 mM. It is possible

that pollen germination may be inhibited much more if Kq in medium could be completely removed. The optimum external Kq concentration for pollen tube growth was 30 mM in the present experiments (Fig. 3A). The average pollen tube length reached 147"7 mm in medium containing 30 mM Kq after 6 h incubation, and either higher or lower external Kq inhibited pollen tube growth (Fig. 3A). The average pollen tube length under the `no additional Kq' condition was only 73"5 mm (58% of that under the control conditions) after 6 h incubation. When external Kq was higher than 30 mM, pollen tube growth was signi®cantly inhibited. The averaged pollen tube length in the medium containing 50 mM Kq was only 82"2 mm after 6 h incubation. In the presence of 1 mM Kq, addition of 10 mM Ba2q as a Kq in¯ux blocker signi®cantly inhibited pollen germination by 53% and tube growth by 46%, respectively (Fig. 3B). These results suggest that external Kq does play an important role in the regulation of pollen germination. Although the optimum external Kq concentration for pollen germination (1 mM) did not match the optimum Kq concentration for pollen tube growth (30 mM), the average pollen tube length reached 126"8 mm (86% of the maximal tube length) in the medium containing 1 mM Kq. In addition, it was observed that Arabidopsis pollen tubes tended to swell at their tips during incubation when the external Kq was equal to or higher than 30 mM (not shown). Therefore, 1 mM Kq in the medium was chosen as the ideal Kq concentration for in vitro Arabidopsis pollen germination. Figure 3C clearly shows that external Ca2q is required for in vitro Arabidopsis pollen germination and tube growth. The addition of 1 mM EGTA in the absence of external Ca2q completely blocked pollen germination. The external Ca2q concentration for both pollen germination and tube growth was 10 mM in these experiments. External pH dramatically in¯uenced both pollen germination and tube growth (Fig. 3D). Both pollen germination and tube growth were completely inhibited at or below pH 4.5 and at or above pH 8.0. Among the pH conditions tested, pH 5.8 was the most suitable for both pollen germination and tube growth. For external boron that is known as an essential factor for pollen germination (Benkert et al., 1997), the optimal concentration was 1.5 mM (added as boric acid) for both Arabidopsis pollen germination and tube growth (data not shown). Identification of the inward whole-cell currents of Arabidopsis pollen protoplasts

Figure 4A shows a typical whole-cell patch-clamp recording from an Arabidopsis thaliana pollen protoplast under the control conditions as described in `Materials and methods'. The holding potential was clamped at 58 mV

Kq-channels in Arabidopsis pollen protoplasts 1607

Fig. 3. Effects of external Kq (A), Ba2q (B), Ca2q (C), and pH (D) on Arabidopsis pollen germination and pollen tube growth in vitro. All experiments were repeated three times and each treatment in one experiment had four replicates (four dishes for in vitro germination). Five hundred pollen grains were counted for germination and 100 pollen tubes were measured for pollen tube length for each replicate. Each data point is expressed as mean"SE. The treatment indicated with `0 mM Kq' in A actually contained 35 mM Kq. (# Data point for the control; * Signi®cantly different from the control by t-test, P-0.05; ** Signi®cantly different from the control by t-test, P-0.01.)

and the test potentials (Vm) ranged from 180 mV to 40 mV with each increment at 20 mV. The timeactivated and voltage-dependent inward currents were observed when Vm was more negative than 80 mV. The whole-cell tail-currents were recorded and analysed (Fig. 4B, C) in order to identify the ions that are mainly responsible for the observed currents across the plasma membrane of a pollen protoplast. The tail-current recordings were conducted following the voltage protocols shown in Fig. 4B. The membrane potential was ®rst clamped from 58 mV to 180 mV to activate the inward current for 1 s, and was subsequently stepped to a more sitive voltage. The membrane potential that resulted in zero tail-current as shown in Fig. 4C was determined as the reversal potential of the observed currents. As shown in Fig. 4C, the measured reversal potential was near 40 mV when the internal (cytoplasm or pipette solution) and external (bath solution) Kq concentrations were 100 mM and 10 mM, respectively. The theoretical equilibrium potential for Kq (EK, the Nernst potential) under these conditions is 56.05 mV,

while the theoretical equilibrium potentials for Ca2q, Cl and glutamate ions were approximately q177.42 mV, 62.10 mV, and q56.93 mV, respectively. Therefore, the reversal potential of the observed currents across the plasma membrane of an Arabidopsis pollen protoplast shown in Fig. 4 was close to the Kq equilibrium potential. Furthermore, the reversal potentials for the inward whole-cell currents at various external Glu-K (potassium glutamate) concentrations were measured and compared to the theoretical equilibrium potentials for Kq and Cl under these conditions (Table 1). As shown in Table 1, the measured reversal potential shifted to more positive potentials with the increase of external Glu-K concentrations, which was similar to the changes of theoretical equilibrium potentials for Kq under these conditions. To con®rm that the inward currents were mainly carried by the in¯ux of Kq, the dependence of the inward currents on external Kq concentrations was investigated. As shown in Fig. 5, the magnitudes of the inward currents were strongly dependent on the external Kq concentration. The inward currents

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of the inward currents at 180 mV, and addition of 10 mM Ba2q completely inhibited the inward currents (Fig. 6A). Addition of 1 mM or 20 mM TEAq in the external medium inhibited the inward whole-cell Kq currents by 29% or 74% at 180 mV, respectively (Fig. 6B). The inhibition of the inward currents by Ba2q or TEAq further con®rmed that the recorded inward whole-cell currents were mainly carried by Kq. Considering these results together with the results presented in Fig. 3B that showed the inhibition of pollen germination and tube growth by Ba2q, it may be proposed that Kq-channel-mediated Kq in¯ux has a role in the regulation of Arabidopsis pollen germination and tube growth. Regulation of the inward Kq channels by internal and external Ca2q

Fig. 4. A typical whole-cell patch-clamp recording (A) and analysis of tail-currents (B, C) of an Arabidopsis pollen protoplast. A portion of the tail-currents in (B) is enlarged and presented in (C). The arrow in (C) indicates the reversal potential of the whole-cell recording. The voltage protocols for the whole-cell recording and the tail-current recording are shown in (A) and (B), respectively. The current and time scale bars for (A) and (B) are shown in (A).

increased along with the increase of external Kq concentrations (Fig. 5A±E). The currentuvoltage relations at various external Kq concentrations are summarized in Fig. 5F which clearly shows the strong dependence of the inward currents on external Kq concentrations. All these data demonstrate that the observed inward currents are mainly carried by channel-mediated Kq in¯ux. The af®nity of potassium ions to the inward Kq channels was derived from plotting the normalized chord conductance ( pS pF 1) at 180 mV versus the external Kq concentrations (Fig. 5G). The plotted curve shown in Fig. 5G was well ®tted to a Michaelis±Menten kinetic equation, and the resulting Michaelis±Menten constant (Km) for Kq in¯ux through the inward channels was approximately 3.945 mM. This Km value is within the Km range of the low-af®nity Kq uptake systems in higher plant cells (Maathuis and Sanders, 1994; Maathuis et al., 1997; Kim et al., 1998). Effects of external Ba2q and TEAq on the inward Kq currents

Figure 6A and B show the inhibitory effects of Ba2qand TEAq, respectively on the inward whole-cell Kq currents in Arabidopsis pollen protoplasts. Addition of 1 mM Ba2q in the external solutions resulted in 51% inhibition

Fluctuations of cytoplasmic Ca2q in pollen as well as in pollen tube cytoplasm have been demonstrated to regulate pollen germination and tube growth (Pierson et al., 1996; Holdaway-Clarke et al., 1997; Messerli and Robinson, 1997). The results presented in Figs 2 and 3A show that external Kq in¯uenced Arabidopsis pollen germination and tube growth in vitro, which suggests that Kq in¯ux or inward Kq channels may be involved in pollen germination and tube growth. The alteration of cytoplasmic free Ca2q concentrations (from 10 nM to 10 mM) did not affect the inward Kq currents (Fig. 7A), which is consistent with the previous report with Brassica pollen protoplasts (Fan et al., 1999) and with Arabidopsis root cell protoplasts (Yu and Wu, 1999). However, an increase of external Ca2q concentration from 10 mM to 50 mM inhibited the inward Kq currents by ;46% although the decrease of external Ca2q concentration from 10 mM (control) to 1 mM had no signi®cant effect on the inward Kq currents (Fig. 7B). Regulation of the inward Kq channels by external pH

Figure 8 shows the effects of external pH on the inward Kq currents. Compared with the inward Kq currents recorded at pH 5.8, a more acidic pH (4.5) stimulated the inward Kq currents by nearly 22%, while a more alkaline pH (8.5) inhibited the inward Kq currents by approximately 70% at 180 mV. The voltage-independent maximal conductance (Gmax) and the half-maximal activation voltage (E1u2) of the inward Kq channels at various external pH were obtained by Boltzmann ®tting of the steady-state conductanceuvoltage relations of the inward Kq channels. As shown in Table 2, E1u2 shifted to more positive voltages and the Gmax value increased in relation to a decrease in external pH. Table 3 shows the effects of external pH on the activation kinetics of the inward Kq currents at 180 mV. The changes in external pH signi®cantly affected the half

Kq-channels in Arabidopsis pollen protoplasts 1609 Table 1. Comparison of the measured reversal potentials (Erev) for the whole-cell currents and the theoretical equilibrium potentials for the various ions at various external Kq concentrations External Kqa (mM)

0

1

5

10

20

b Erev

95.00"4.23 (n ˆ 7) ` q178.13 q3.04 62.57 ndc

76.80"2.64 (n ˆ 10) 113.75 q178.13 q3.04 62.57 q114.72

45.75"2.19 (n ˆ 8) 73.31 q177.80 q2.77 62.43 q74.24

34.69"1.70 (n ˆ 13) 56.05 q177.42 q2.35 62.10 q56.93

23.30"1.87 (n ˆ 10) 39.00 q176.77 q1.91 61.64 q39.79

EK ECa EMg ECl EGlu a

Kq was supplied as a glutamate salt. Each data point for Erev represents mean"SE. c nd, not determined. b

Fig. 5. Dependence of the inward whole-cell currents of Arabidopsis pollen protoplasts on the external Kq. (A±E) Patch-clamp whole-cell recordings at various external Kq concentrations; (F) currentuvoltage relations; (G) concentration dependence of inward Kq current conductance at 180 mV. Voltage protocols, and current and time scale bars are shown in (A) and (D), respectively. Sample sizes for each treatment in (F) and (G) were 11 (0 mM Kq), 14 (1 mM Kq), 9 (5 mM Kq), 22 (10 mM Kq), and 13 (20 mM Kq), respectively. Each data point in (F) and (G) is expressed as mean"SE. The kinetic curve in (G) was plotted with the normalized chord conductances at 180 mV versus the external Kq concentrations, and ®tted with the Michaelis±Menten equation.

activation time (t1u2) of the inward Kq currents. Compared with the results at pH 5.8, the acidic external pH (4.5) resulted in the faster activation (shorter t1u2), while the alkaline external pH (8.5) slowed the activation of the inward Kq currents (longer t1u2). To understand the properties of the inward Kq channel activation kinetics further, Chesbyshev ®tting methods (included in pCLAMP 6.0.4 software) were applied to ®t the inward whole-cell current traces. In most cases, the inward

whole-cell current traces were well ®tted by the sum of two exponential functions. Two-component channel activation kinetics may be explained by considering that the recorded whole-cell currents may be attributed to the Kq in¯ux across one type of channel with two closed states, or two types of channel with one closed state (Findlay et al., 1994; White and Lemtiri-Chlieh, 1995). The results presented in Table 3 also show that the inward Kq currents are dominated by the current

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Fig. 6. Effects of external Ba2q (A) and TEAq (B) on the inward whole-cell Kq currents of Arabidopsis pollen protoplasts. Sample sizes for each treatment in (A) were 15 (control), 11 (1 mM Ba2q), and 6 (10 mM Ba2q), respectively. Sample sizes for data shown in (B) were 17 (control), 10 (1 mM TEAq), and 6 (20 mM TEAq), respectively. Each data point represents mean"SE. (** Signi®cantly different from the control by t-test, P-0.01.)

component with shorter time constant (t1) under acidic pH conditions and with longer time constant (t2) under alkaline conditions.

Discussion Methods for Arabidopsis pollen germination in vitro

As trinucleate type pollen, Arabidopsis pollen grains have been thought to germinate poorly in vitro. Li et al. conducted in vitro germination of Arabidopsis pollen in their experiments although they did not report pollen germination percentages (Li et al., 1999). Using agarcontaining media, in vitro methods for Arabidopsis pollen germination have been successfully established in this study and the maximal pollen germination percentage reached 80% or even greater. The constant saturated humidity and solidi®ed medium with agar seem to be important environmental factors for in vitro pollen germination. It is likely that the solidi®ed surface of the

Fig. 7. Effects of internal (A) and external (B) Ca2q on the inward whole-cell Kq currents of Arabidopsis pollen protoplasts. Sample sizes for the data shown in (A) were 15 (control, 10 nM Ca2q) and 6 (10 mM Ca2q), respectively. Sample sizes for the data shown in (B) were 11 (1 mM Ca2q), 12 (10 mM Ca2q), and 14 (50 mM Ca2q), respectively. Each data point represents mean"SE. (* Signi®cantly different from the control by t-test, P-0.05; ** Signi®cantly different from the control by t-test, P-0.01.)

germination medium could mimic the micro-milieu conditions for pollen germination in vivo (as on the Arabidopsis dry-type stigma). The protocols for in vitro Arabidopsis pollen germination presented in this paper may provide a convenient method for using Arabidopsis pollen as a model system to study the regulation of pollen germination and tube growth. Possible physiological roles of Kq influx in pollen germination and tube growth

For in vitro experiments in this study, the optimum external Kq concentration for Arabidopsis pollen germination was 1 mM. When the germination medium contained `no additional Kq', pollen germination was inhibited by 26%. The actual Kq concentration under these conditions was approximately 35 mM because of the Kq contamination of the agar even though Kq-depleted agar was used. It is reasonable to expect that a genuine

Kq-channels in Arabidopsis pollen protoplasts 1611

Fig. 8. Effects of external pH on the inward whole-cell Kq currents of Arabidopsis pollen protoplasts. (A±C) The whole-cell recordings at various external pH; (D) currentuvoltage relations at various external pH; (E) conductanceuvoltage relations at various external pH. Voltage protocols for the whole-cell recordings and the time and current scale bars are shown in (C). Each data point in (D) and (E) represents mean"SE. The curve in (E) was plotted with the normalized chord conductances at 180 mV versus the membrane potentials, and ®tted with the Boltzmann equation. (* Signi®cantly different from the control by t-test, P-0.05; ** Signi®cantly different from the control by t-test, P-0.01.)

Table 2. Effects of external pH on the steady-state properties of the inward whole-cell Kq currents in Arabidopsis pollen protoplasts External pH 4.5 5.8 8.5

E1u2 (mV) 133.41"2.73 (n ˆ 10)** 146.59"1.74 (n ˆ 16) 153.58"6.53 (n ˆ 5)

Gmax (pS pF 1) 481.83"38.59 (n ˆ 10)** 370.68"19.04 (n ˆ 16) 217.46"49.46 (n ˆ 5)**

Each data point represents mean"SE. ** Signi®cantly different from the control (pH 5.8) by t-test, P-0.01.

`no-Kq-containing' medium would result in a greater inhibition of pollen germination. For pollen tube growth, however, the optimum Kq concentration in the germination medium was near 30 mM. The results suggest that pollen tube growth may need more Kq in¯ux than the pollen germination process. One possible explanation for this phenomenon is that Kq plays a more important role as a major osmotica in pollen tube growth than in pollen germination. Pollen tube growth is accompanied by a rapid increase in cytoplasmic volume and, therefore, more

Kq ions may be needed to maintain proper cytoplasmic solute concentration. Pollen grains experience a dehydration process before anther dehiscence (Heslop-Harrison, 1987), which results in an increase in the intracellular Kq concentration. The increase of cytoplasmic Kq may reach a very high level, such as 280 mM in Tradescantia pollen grains (Bashe and Mascarenhas, 1984). It was reported that high concentrations of Kq above 220 mM completely inhibited in vitro protein translation (Weber et al., 1978). With pollen grain rehydration during germination, the Kq concentration reaches a level suitable for the initiation of protein synthesis required for pollen germination and pollen tube growth in most species studied (reviewed by Mascarenhas, 1993). These previous studies suggest that an excessive Kq in¯ux seems unnecessary and even harmful to the initiation of pollen germination, particularly during the early stages of pollen germination. However, once the tube appears from a germination pore and begins to grow rapidly, Kq in¯ux as well as a consequent in¯ux of water is required for the maintenance of the

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Table 3. Effects of external pH on the activation kinetics of the inward whole-cell Kq currents in Arabidopsis pollen protoplasts at 180 mV External pH

4.5 5.8 8.5

t1u2 (ms)

29.22"3.32 (n ˆ 9)** 63.00"5.76 (n ˆ 18) 192.13"36.48 (n ˆ 8)**

Time constant (ms)

t1

t2

39.29"3.31 (n ˆ 11)** 68.38"7.75 (n ˆ 9) 133.71"16.19 (n ˆ 6)**

368.24"45.44 (n ˆ 11) 419.85"55.98 (n ˆ 9) 513.98"56.74 (n ˆ 6)**

Each data point represents mean"SE. ** Signi®cantly different from the control (pH 5.8) by t-test, P-0.01.

turgor pressure in the pollen tube. The data presented in this paper (Fig. 3A) show that 1 mM external Kq was needed for the highest pollen germination percentage, while 30 mM external Kq was required for the highest growth rate of the pollen tubes. Interestingly, it was observed in this study that high external Kq conditions (higher than 30 mM) resulted in swelling at the tips of pollen tubes. This result is consistent with the previous report (Messerli et al., 1999) that larger Kq in¯ux was correlated with a larger diameter of lily pollen tube. It is reasonable to propose that Kq in¯ux may play an important role in the regulation of pollen turgor pressure. However, excessive Kq in¯ux driven by very high (such as 50 mM) external Kq signi®cantly inhibited both pollen germination and tube growth (Fig. 3A). Such inhibitory effects of excessive external Kq can effectively be removed by the addition of 5 mM TEAq in the medium (data not shown), which further demonstrates that a certain amount of Kq in¯ux is required for proper or healthy pollen germination and tube growth. The addition of Ba2q as a Kq in¯ux blocker in the medium signi®cantly inhibited both pollen germination and tube growth in the presence of 1 mM of external Kq (Fig. 3B). Consistent with the effects of Ba2q on pollen germination and tube growth, either 10 mM Ba2q or 20 mM TEAq signi®cantly inhibited the inward Kq currents in pollen protoplasts in the patch-clamp experiments (Fig. 6A, B). Taking these results together, it is concluded that Kq in¯ux does play an important role in pollen germination and tube growth and that the regulation of the inward Kq channels in the pollen and pollen tube plasma membranes may be involved in signalling cascades of pollen germination and tube growth. Regulation of Kq channels and pollen germination and tube growth by Ca2q and external pH

When penetrating through the pistil tissues, pollen tubes are thought to encounter relatively high Ca2q environments, particular in the micropylar ®liform apparatus and the receptive synergid (Tian and Russell, 1997). Considering that the pollen tube stops extension and releases sperms in the receptive synergid, the inhibition

of pollen inward Kq channels by high Ca2q externally may be an important regulatory mechanism for pollen tube growth in vivo. In order to understand the association between the regulation of the inward Kq channels and pollen germination as well as tube growth, the effects of external and internal Ca2q and external pH that are all well-known important regulatory factors for pollen germination and tube growth were investigated in this study. Unlike the case in stomatal guard cells in which the inward Kq channels are strongly regulated by cytoplasmic Ca2q (Schroeder and Hagiwara, 1989; Blatt et al., 1990; Fairley-Grenot and Assmann, 1992a, b; LemtriChlieh and MacRobbie, 1994; Kelly et al., 1995; Grabov and Blatt, 1997), the inward Kq channels in Arabidopsis pollen protoplasts were insensitive to changes in intracellular Ca2q in the present study. This result is consistent with the previous report with Brassica pollen protoplasts (Fan et al., 1999). However, Obermeyer and Kolb reported that an inward Kq channel among the three types of Kq channel recorded in the plasma membranes of lily pollen protoplasts was Ca2q-regulated (Obermeyer and Kolb, 1993). In addition to the different plant species used in two different lines of experiments, differing experimental conditions may also cause contrasting results. Since the ¯uctuations of wCa2qxc in the tip of the pollen tube in Lilium regulates pollen tube growth (Pierson et al., 1994, 1996; Messerli and Robinson, 1997; Holdaway-Clarke et al., 1997), it is of interest whether the inward Kq-channels in the plasma membranes of pollen tube are regulated by changes in wCa2qxc and it is worthy of further study. The inward Kq channels in plant cells are generally activated by hyperpolarized membrane potentials across the plasma membranes (Thiel et al., 1992). The hyperpolarization of the plasma membrane is associated with extracellular acidi®cation that is induced by Hq-ATPase activity that pumps Hq out of the cell. Therefore, the inward Kq channels in plant cells are usually activated by an acidic external environment, as is the case in guard cells (Blatt, 1992; Ilan et al., 1996). Obermeyer et al. have suggested that an extracellular acidi®cation may increase the activity of the inward Kq-channels in lily pollen

Kq-channels in Arabidopsis pollen protoplasts 1613

plasma membrane (Obermeyer et al., 1996). It was observed in the present study that an acidic external pH (such as pH 4.5) did enhance the inward Kq currents in Arabidopsis pollen protoplasts. However, the suitable external pH for in vitro Arabidopsis pollen germination and subsequent tube growth in this study was more alkaline (pH 5.8) than the external pH (pH 4.5) that resulted in the greater Kq in¯ux via the inward channels. In fact, as the results presented in Figs 2 and 3 indicate, a higher concentration of external Kq (such as 10 or 30 mM compared to 1 mM) that may drive a greater Kq in¯ux did not result in a higher pollen germination percentage. Therefore, it is proposed that pollen germination may not require a very high Kq in¯ux. In conclusion, in vitro methods for Arabidopsis pollen germination using agar-containing media have been successfully established for the ®rst time. Combining in vitro pollen germination experiments with patch-clamp wholecell recording techniques, the present results suggest that Kq in¯ux as well as its regulation mediated by the inward Kq-channels plays an important role in Arabidopsis pollen germination and tube growth. Acknowledgements We would like to thank Dr Sarah Assmann for her critical reading of the manuscript and helpful comments. This work was supported by the Chinese National Key Basic Research Project (No G1999011701) and a NSFC (National Natural Science Foundation of China) key research grant (No 39930010) to W-HW, and partially supported by a NSFC competitive research grant (No 39870397) to L-MF.

References Bashe D, Mascarenhas JP. 1984. Changes in potassium ion concentrations during pollen dehydration and germination in relation to protein synthesis. Plant Science Letters 35, 55±60. Benkert R, Obermeyer R, Bentrup F-W. 1997. The turgor pressure of growing lily pollen tubes. Protoplasma 198, 1±8. Blatt M. 1992. Kq channels of stomatal guard cells: characteristics of the inward recti®er and its control by pH. Journal of General Physiology 99, 615±644. Blatt MR, Thiel G, Trentham DR. 1990. Reversible inactivation of Kq channels of Vicia stomatal guard cells following the photolysis of caged inositol 1,4,5-trisphosphate. Nature 346, 766±769. Brewbaker JL, Kwack BH. 1963. The essential role of calcium ion in pollen germination and pollen tube growth. American Journal of Botany 50, 859±865. Cheung AY, Wang H, Wu H-M. 1995. A ¯oral transmitting tissue-speci®c glycoprotein attracts pollen tubes and stimulates their growth. Cell 82, 383±393. Fairley-Grenot KA, Assmann SM. 1992a. Permeation of Ca2q through Kq channels in the plasma membrane of Vicia faba guard cells. Journal of Membrane Biology 128, 103±113. Fairley-Grenot KA, Assmann SM. 1992b. Whole-cell Kq current across the plasma membrane of guard cells from a grass: Zea mays. Planta 186, 282±293.

Fan L-M, Wu W-H, Yang H-Y. 1999. Identi®cation and characterization of the inward Kq channels in the plasma membranes of Brassica pollen protoplasts. Plant and Cell Physiology 40, 859±865. Feijo JA, Malho R, Obermeyer G. 1995. Ion dynamics and its possible role during in vitro pollen germination and tube growth. Protoplasma 187, 155±167. Findlay GP, Tyerman SD, Garill A, Skerrett M. 1994. Pump and Kq inward recti®ers in the plasmalemma of wheat root protoplasts. Journal of Membrane Biology 139, 103±116. Franklin-Tong VE. 1999. Signalling and the modulation of pollen tube growth. The Plant Cell 11, 727±738. Grabov A, Blatt MR. 1997. Parallel control of the inwardrecti®er Kq channel by cytosolic free Ca2q and pH in Vicia guard cells. Planta 201, 84±95. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. P¯ugers Arch 391, 85±100. Heslop-Harrison J. 1987. Pollen germination and pollen tube growth. International Review of Cytology 107, 1±78. Holdaway-Clarke TL, Feijo JA, Hackett GR, Kunkel JG, Hepler PK. 1997. Pollen tube growth and the intracellular cytosolic calcium gradient oscillate in phase while extracellular calcium in¯ux is delayed. The Plant Cell 9, 1999±2010. Ilan N, Schwartz A, Moran N. 1996. External protons enhance the activity of the hyperpolarization-activated Kq channels in guard cell protoplasts of Vicia faba. Journal of Membrane Biology 154, 169±181. Kelly WB, Esser JE, Schroeder JI. 1995. Effects of cytosolic calcium and limited, possible dual, effects of G protein modulators on guard cell inward potassium channels. The Plant Journal 8, 479±489. Kim EJ, Kwak JM, Uozumi N, Schroeder JI. 1998. AtKUP1: an Arabidopsis gene encoding high-af®nity potassium transport activity. The Plant Cell 10, 51±62. Lee H-S, Huang S, Kao T-H. 1994. S proteins control rejection of incompatible pollen in Petunia in¯ata. Nature 367, 560±563. Leeton PRJ, Smyth DR. 1993. An abundant LINE-like element ampli®ed in the genome of Lilium speciosum. Molecular and General Genetics 237, 97±104. Lemtri-Chlieh F, MacRobbie EAC. 1994. Role of calcium in the modulation of Vicia guard cell potassium channels by abscisic acid: a patch-clamp study. Journal of Membrane Biology 137, 99±107. Li H, Lin Y, Heath RM, Zhu MX, Yang Z. 1999. Control of pollen tube tip growth by a Rop GTPase-dependent pathway that leads to tip-localized calcium in¯ux. The Plant Cell 11, 1731±1742. Maathuis FJM, Ichida AM, Sanders D, Schroeder JI. 1997. Roles of higher plant Kq channels. Plant Physiology 114, 1141±1149. Maathuis FJM, Sanders D. 1994. Mechanism of highaf®nity potassium uptake in roots of Arabidopsis thaliana. Proceedings of the National Academy of Sciences, USA 91, 9272±9276. Mascarenhas JP. 1993. Molecular mechanisms of pollen tube growth and differentiation. The Plant Cell 5, 1303±1314. Messerli M, Robinson KR. 1997. Tip localized Ca2q pulses are coincident with peak pulsatile growth rates in pollen tubes of Lilium longi¯orum. Journal of Cell Science 110, 1269±1278. Messerli MA, Danuser G, Robinson KR. 1999. Pulsatile in¯uxes of Hq, Kq and Ca2q lag growth pulses of Lilium longi¯orum pollen tubes. Journal of Cell Science 112, 1497±1509.

1614

Fan et al.

Meyerowitz EM, Somerville CR. (eds) 1994. Arabidopsis. New York: Cold Spring Harbor Laboratory Press. Obermeyer G, Blatt MR. 1995. Electrical properties of intact pollen grains of Lilium longi¯orum: characteristics of the non-germinating pollen grains. Journal of Experimental Botany 46, 803±813. Obermeyer G, Kolb H-A. 1993. Kq channels in the plasma membrane of lily pollen protoplasts. Botanica Acta 106, 26±31. Obermeyer G, Kriechbaumer R, Strasser D, Maschessnig A, Bentrup FW. 1996. Boric acid stimulates the plasma membrane Hq-ATPase of ungerminated lily pollen grains. Physiologia Plantarum 98, 281±290. Pierson ES, Miller DD, Callaham DA, Shipley AM, Rivers BA, Cresti M, Hepler PK. 1994. Pollen tube growth is coupled to the extracellular calcium ion ¯ux and the intracellular calcium gradient: effect of BAPTA-type buffers and hypertonic media. The Plant Cell 6, 1815±1828. Pierson ES, Miller DD, Callaham DA, van Aken J, Hackett G, Hepler PK. 1996. Tip-localized calcium entry ¯uctuates during pollen tube growth. Developmental Biology 174, 160±173. Preuss D, Lemieux B, Yen G, Davis RW. 1993. A conditional sterile mutation eliminates surface components from Arabidopsis pollen and disrupts cell signalling during fertilization. Genes Development 7, 974±985. Schroeder JI, Hagiwara S. 1989. Cytosolic calcium regulates ion channels in the plasma membrane of Vicia faba guard cells. Nature 338, 427±430.

Taylor LP, Hepler PK. 1997. Pollen germination and tube growth. Annual Review of Plant Physiology and Plant Molecular Biology 48, 461±491. Thiel G, MacRobbie EAC, Blatt MR. 1992. Membrane transport in stomatal guard cells: the importance of voltage control. Journal of Membrane Biology 126, 1±18. Tian HQ, Russell SD. 1997. Calcium distribution in fertilized and unfertilized ovules and embryo sacs of Nicotiana tabacum L. Planta 202, 93±105. Weber LA, Hickey ED, Baglioni C. 1978. In¯uence of potassium salt concentration and temperature on inhibition of mRNA translation by 7-methylguanosine 59-monophosphate win the wheat germ and rabbit reticulocyte cell-free systemsx. Journal of Biological Chemistry 253, 178±183. Weisenseel MH, Jaffe LF. 1976. The major growth current through lily pollen tubes enters as Kq and leaves as Hq. Planta 133, 1±7. White PJ, Lemtiri-Chlieh F. 1995. Potassium currents across the plasma membrane of protoplasts derived from rye roots: a patch-clamp study. Journal of Experimental Botany 46, 497±511. Wu WH, Assmann SM. 1994. A membrane-delimited pathway of G-protein regulation of the guard-cell inward Kq channels. Proceedings of the National Academy of Sciences, USA 91, 6310±6314. Yu C-J, Wu W-H. 1999. Identi®cation and characterization of the inward Kq-channels in Arabidopsis root cells. Science in China (C) 42, 307±315.