Journal of Experimental Botany

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (58) Request Permissions Disclaimer Google Scholar Articles by Fan, L.-M. Articles by Wu, W.-H. Search for Related Content PubMed PubMed Citation Articles by Fan, L.-M. Articles by Wu, W.-H. Agricola Articles by Fan, L.-M. Articles by Wu, W.-H. Social Bookmarking          What's this?

Journal of Experimental Botany, Vol. 52, No. 361, pp. 1603-1614, August 1, 2001
© 2001 Oxford University Press

Original Papers

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

Top Abstract Introduction Materials and methods Results Discussion References

 
The focus of this study is to investigate the regulatory role of K⁺ 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 µm after a 6 h incubation. A decrease in external K⁺ concentration from 1 mM to 35 µM resulted in 30% inhibition of pollen germination and 40% inhibition of pollen tube growth. An increase in external K⁺ concentration from 1 mM to 30 mM stimulated pollen tube growth but inhibited pollen germination. To study how K⁺ influx is associated with pollen germination and tube growth, regulation of the inward K⁺ channels in the pollen plasma membrane was investigated by conducting patch-clamp whole-cell recording with pollen protoplasts. K⁺ currents were first identified in Arabidopsis pollen protoplasts. The inward K⁺ currents were insensitive to changes in cytoplasmic Ca²⁺ but were inhibited by a high concentration of external Ca²⁺. A decrease of external Ca²⁺ concentration from 10 mM (control) to 1 mM had no significant effect on the inward K⁺ currents, while an increase of external Ca²⁺ concentration from 10 mM to 50 mM inhibited the inward K⁺ currents by 46%. Changes in external pH significantly affected the magnitude, conductance, voltage-independent maximal conductance, and activation kinetics of the inward K⁺ currents. The physiological importance of potassium influx mediated by the inward K⁺-channels during Arabidopsis pollen germination and tube growth is discussed.

Key words: Arabidopsis thaliana, pollen germination, K⁺-channel, patch-clamp, in vitro pollen culture.

	Introduction

Top Abstract Introduction Materials and methods Results Discussion References

 
Pollen germination and pollen tube growth are essential processes that ensure the reproduction of flowering 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 significantly regulated by the transport of inorganic ions, such as Ca²⁺ and K⁺, across the plasma membranes of pollen and/or pollen tubes (Feijó et al., 1995; Taylor and Hepler, 1997). It is also known that K⁺ is required for both pollen germination and tube growth (Brewbaker and Kwack, 1963; Weisenseel and Jaffe, 1976; Feijó et al., 1995). By the application of patch-clamp techniques, the three types of K⁺-channels from Lilium pollen protoplasts were identified and the possible involvement of inward K⁺-channel-mediated K⁺ influx during pollen tube growth was suggested (Obermeyer and Kolb, 1993). Using conventional voltage-clamp techniques, Obermeyer and Blatt observed both an outward K⁺ current and an inward K⁺ current across the plasma membrane of non-germinating Lilium pollen grains. (Obermeyer and Blatt, 1995) They proposed that an inward K⁺ current in a non-germinating pollen grain may play a role in initiating the osmotic water influx required for pollen germination. Fan et al. have used a patch-clamp whole-cell recording technique to identify and characterize K⁺ 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 K⁺-channels. It was also shown that the inward K⁺ channels in Brassica pollen protoplasts are significantly regulated by external Ca²⁺, which is an important regulatory factor for pollen germination and pollen tube growth. These previous studies strongly suggest that the inward K⁺ channels may be essential components involved in the processes of pollen germination and tube growth, and that the regulation of the K⁺-channels may play a regulatory role in pollen germination and tube growth.

To investigate molecular mechanisms of K⁺-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 difficult 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 first 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 first time and the regulatory effects of external K⁺ 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 K⁺ channels in Arabidopsis pollen plasma membranes and the possible regulation of the inward K⁺ channels by Ca²⁺ and external pH was investigated. The physiological importance of potassium influx via the inward K⁺-channels in Arabidopsis pollen germination and tube growth is also discussed.

	Materials and methods

Top Abstract Introduction Materials and methods Results Discussion References

 
Plant growth conditions
Arabidopsis thaliana (ecotype Landsberg erecta) plants were grown in mixed soil in a growth chamber. The light intensity was 120–150 µmol m^-2 s^-1 for a 12 h daily light period and day/night temperatures were 22±2 °C and 18±2 °C, 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 flowers were used for in vitro pollen germination experiments since pollen grains at this developmental stage showed the highest germination percentage. For each experiment, 150 flowers were randomly collected from 30 different plants. The dehisced anthers of five randomly picked flowers 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% (w/v) agar (K⁺-depleted agar, see the text in ‘Results’), 16.6% (w/v) sucrose, 3.65% (w/v) sorbitol, and 10 µg 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 double-distilled water and heated to 100 °C 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 °C with 100% relative humidity (controlled with a humidifier) in the light (30 µmol m^-2 s^-1 supplied with fluorescent 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 flowers of 10 different Arabidopsis plants immediately before the isolation of pollen protoplasts. The pollen grains were first washed in standard solution containing 1 mM KNO₃, 0.2 mM KH₂PO₄, 1 mM MgSO₄, 1 µM KI, 0.1 µM CuSO₄, 5 mM CaCl₂, 5 mM MES (pH 5.8 adjusted with TRIS), 500 mM glucose and sorbitol (osmolality=1.5 Osmol kg^-1) before enzymatic digestion. After filtration through a nylon mesh (diameter=80 µm) and centrifugation at 160 g for 5 min, pollen grains were incubated in 2 ml of enzyme solution at 28 °C for 1 h to release pollen protoplasts. The enzyme solution was prepared with standard solution containing 1% (w/v) cellulase R-10 (Yakult Honsha Co., Japan), 0.5% (w/v) macerozyme R-10 (Yakult Honsha Co., Japan), 0.2% (w/v) PDS (potassium dextran sulphate, CalBiochem, USA), and 0.2% (w/v) BSA. The mixture was centrifuged at 160 g for 5 min and the pellet was resuspended with 2 ml of the standard solution. This centrifugation/resuspension cycle was conducted three times to remove BSA, enzymes, and undigested pollen wall debris completely. The pollen protoplasts were finally 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 Ca²⁺ concentration in this standard pipette solution was 10 nM. The pipette (internal) solution containing 10 µM free Ca²⁺ was made by addition of 4.94 mM CaCl₂ to the standard pipette solution. The calculation of free Ca²⁺ 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 M. The experiments were conducted at room temperature (20±2 °C) in dim light. Cell capacitance was measured for each cell using the capacity compensation device of the amplifier. All data were acquired 5 min after the formation of the whole-cell configuration.

Whole cell currents were measured using an Axopatch-200A amplifier (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 configuration was obtained, membrane potential (V_m) 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 filtered at 1 kHz before storage (1 ms per sample) on a computer disk.

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 first three data points obtained after V_m 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 final 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 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

Top Abstract Introduction Materials and methods Results Discussion References

 
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.

View larger version (24K): [in this window] [in a new window]   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.

 

View larger version (98K): [in this window] [in a new window]   Fig. 2. Effects of external K+ 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 K+’ actually contained 35 µM K+; see text.)

 

Effects of external K⁺, Ca²⁺, Ba²⁺, 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 Ca²⁺, K⁺, boric acid, etc.) was determined based on the results of repeated experiments. As shown in Figs 2 and 3A, 1 mM K⁺ in the medium resulted in the highest germination percentage, and either higher or lower K⁺ resulted in the inhibition of pollen germination. When the medium contained ‘no additional K⁺’, both pollen germination and tube growth was significantly inhibited (Figs 2B, 3A). In fact, the medium with ‘no additional K⁺’ contained approximately 35 µM K⁺ mainly from K⁺-contaminated agar even though ‘K⁺-depleted agar’ was used. K⁺ contents in several different brands of agars were measured and all these agars contain K⁺ that results in K⁺ concentrations between 200 and 300 µM when 1% (w/v) agar was added to medium. To obtain ‘K⁺-depleted agar’, the purchased agar was washed 3–4 times with 2 mM HCl, then rinsed with double-distilled water 5 times, and finally dried in an oven at 40 °C. The addition of 1% (w/v) K⁺-depleted agar to the medium resulted in a final K⁺ concentration of approximately 35 µM. It is possible that pollen germination may be inhibited much more if K⁺ in medium could be completely removed.

View larger version (41K): [in this window] [in a new window]   Fig. 3. Effects of external K+ (A), Ba2+ (B), Ca2+ (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 K+’ in A actually contained 35 µM K+. (# Data point for the control; * Significantly different from the control by t-test, P<0.05; ** Significantly different from the control by t-test, P<0.01.)

 
The optimum external K⁺ concentration for pollen tube growth was 30 mM in the present experiments (Fig. 3A). The average pollen tube length reached 147±7 µm in medium containing 30 mM K⁺ after 6 h incubation, and either higher or lower external K⁺ inhibited pollen tube growth (Fig. 3A). The average pollen tube length under the ‘no additional K⁺’ condition was only 73±5 µm (58% of that under the control conditions) after 6 h incubation. When external K⁺ was higher than 30 mM, pollen tube growth was significantly inhibited. The averaged pollen tube length in the medium containing 50 mM K⁺ was only 82±2 µm after 6 h incubation. In the presence of 1 mM K⁺, addition of 10 mM Ba²⁺ as a K⁺ influx blocker significantly inhibited pollen germination by 53% and tube growth by 46%, respectively (Fig. 3B). These results suggest that external K⁺ does play an important role in the regulation of pollen germination. Although the optimum external K⁺ concentration for pollen germination (1 mM) did not match the optimum K⁺ concentration for pollen tube growth (30 mM), the average pollen tube length reached 126±8 µm (86% of the maximal tube length) in the medium containing 1 mM K⁺. In addition, it was observed that Arabidopsis pollen tubes tended to swell at their tips during incubation when the external K⁺ was equal to or higher than 30 mM (not shown). Therefore, 1 mM K⁺ in the medium was chosen as the ideal K⁺ concentration for in vitro Arabidopsis pollen germination.

Figure 3C clearly shows that external Ca²⁺ is required for in vitro Arabidopsis pollen germination and tube growth. The addition of 1 mM EGTA in the absence of external Ca²⁺ completely blocked pollen germination. The external Ca²⁺ concentration for both pollen germination and tube growth was 10 mM in these experiments. External pH dramatically influenced 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 and the test potentials (V_m) ranged from -180 mV to -40 mV with each increment at 20 mV. The time-activated and voltage-dependent inward currents were observed when V_m was more negative than -80 mV.

View larger version (27K): [in this window] [in a new window]   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).

 
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 first 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) K⁺ concentrations were 100 mM and 10 mM, respectively. The theoretical equilibrium potential for K⁺ (E_K, the Nernst potential) under these conditions is -56.05 mV, while the theoretical equilibrium potentials for Ca²⁺, Cl^- and glutamate ions were approximately +177.42 mV, -62.10 mV, and +56.93 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 K⁺ 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 K⁺ 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 K⁺ under these conditions. To confirm that the inward currents were mainly carried by the influx of K⁺, the dependence of the inward currents on external K⁺ concentrations was investigated. As shown in Fig. 5, the magnitudes of the inward currents were strongly dependent on the external K⁺ concentration. The inward currents increased along with the increase of external K⁺ concentrations (Fig. 5A–E). The current/voltage relations at various external K⁺ concentrations are summarized in Fig. 5F which clearly shows the strong dependence of the inward currents on external K⁺ concentrations. All these data demonstrate that the observed inward currents are mainly carried by channel-mediated K⁺ influx.

View this table: [in this window] [in a new window]   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 K+ concentrations

 

View larger version (35K): [in this window] [in a new window]   Fig. 5. Dependence of the inward whole-cell currents of Arabidopsis pollen protoplasts on the external K+. (A–E) Patch-clamp whole-cell recordings at various external K+ concentrations; (F) current/voltage relations; (G) concentration dependence of inward K+ 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 K+), 14 (1 mM K+), 9 (5 mM K+), 22 (10 mM K+), and 13 (20 mM K+), 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 K+ concentrations, and fitted with the Michaelis–Menten equation.

 
The affinity of potassium ions to the inward K⁺ channels was derived from plotting the normalized chord conductance (pS pF^-1) at -180 mV versus the external K⁺ concentrations (Fig. 5G). The plotted curve shown in Fig. 5G was well fitted to a Michaelis–Menten kinetic equation, and the resulting Michaelis–Menten constant (K_m) for K⁺ influx through the inward channels was approximately 3.945 mM. This K_m value is within the K_m range of the low-affinity K⁺ uptake systems in higher plant cells (Maathuis and Sanders, 1994; Maathuis et al., 1997; Kim et al., 1998).

Effects of external Ba²⁺ and TEA⁺ on the inward K⁺ currents
Figure 6A and B show the inhibitory effects of Ba²⁺and TEA⁺, respectively on the inward whole-cell K⁺ currents in Arabidopsis pollen protoplasts. Addition of 1 mM Ba²⁺ in the external solutions resulted in 51% inhibition of the inward currents at -180 mV, and addition of 10 mM Ba²⁺ completely inhibited the inward currents (Fig. 6A). Addition of 1 mM or 20 mM TEA⁺ in the external medium inhibited the inward whole-cell K⁺ currents by 29% or 74% at -180 mV, respectively (Fig. 6B). The inhibition of the inward currents by Ba²⁺ or TEA⁺ further confirmed that the recorded inward whole-cell currents were mainly carried by K⁺. Considering these results together with the results presented in Fig. 3B that showed the inhibition of pollen germination and tube growth by Ba²⁺, it may be proposed that K⁺-channel-mediated K⁺ influx has a role in the regulation of Arabidopsis pollen germination and tube growth.

View larger version (26K): [in this window] [in a new window]   Fig. 6. Effects of external Ba2+ (A) and TEA+ (B) on the inward whole-cell K+ currents of Arabidopsis pollen protoplasts. Sample sizes for each treatment in (A) were 15 (control), 11 (1 mM Ba2+), and 6 (10 mM Ba2+), respectively. Sample sizes for data shown in (B) were 17 (control), 10 (1 mM TEA+), and 6 (20 mM TEA+), respectively. Each data point represents mean±SE. (** Significantly different from the control by t-test, P<0.01.)

 

Regulation of the inward K⁺ channels by internal and external Ca²⁺
Fluctuations of cytoplasmic Ca²⁺ 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 K⁺ influenced Arabidopsis pollen germination and tube growth in vitro, which suggests that K⁺ influx or inward K⁺ channels may be involved in pollen germination and tube growth. The alteration of cytoplasmic free Ca²⁺ concentrations (from 10 nM to 10 µM) did not affect the inward K⁺ 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 Ca²⁺ concentration from 10 mM to 50 mM inhibited the inward K⁺ currents by 46% although the decrease of external Ca²⁺ concentration from 10 mM (control) to 1 mM had no significant effect on the inward K⁺ currents (Fig. 7B).

View larger version (25K): [in this window] [in a new window]   Fig. 7. Effects of internal (A) and external (B) Ca2+ on the inward whole-cell K+ currents of Arabidopsis pollen protoplasts. Sample sizes for the data shown in (A) were 15 (control, 10 nM Ca2+) and 6 (10 µM Ca2+), respectively. Sample sizes for the data shown in (B) were 11 (1 mM Ca2+), 12 (10 mM Ca2+), and 14 (50 mM Ca2+), respectively. Each data point represents mean±SE. (* Significantly different from the control by t-test, P<0.05; ** Significantly different from the control by t-test, P<0.01.)

 

Regulation of the inward K⁺ channels by external pH
Figure 8 shows the effects of external pH on the inward K⁺ currents. Compared with the inward K⁺ currents recorded at pH 5.8, a more acidic pH (4.5) stimulated the inward K⁺ currents by nearly 22%, while a more alkaline pH (8.5) inhibited the inward K⁺ currents by approximately 70% at -180 mV. The voltage-independent maximal conductance (G_max) and the half-maximal activation voltage (E_1/2) of the inward K⁺ channels at various external pH were obtained by Boltzmann fitting of the steady-state conductance/voltage relations of the inward K⁺ channels. As shown in Table 2, E_1/2 shifted to more positive voltages and the G_max 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 K⁺ currents at -180 mV. The changes in external pH significantly affected the half activation time (t_1/2) of the inward K⁺ currents. Compared with the results at pH 5.8, the acidic external pH (4.5) resulted in the faster activation (shorter t_1/2), while the alkaline external pH (8.5) slowed the activation of the inward K⁺ currents (longer t_1/2). To understand the properties of the inward K⁺ channel activation kinetics further, Chesbyshev fitting methods (included in pCLAMP 6.0.4 software) were applied to fit the inward whole-cell current traces. In most cases, the inward whole-cell current traces were well fitted 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 K⁺ influx 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 K⁺ currents are dominated by the current component with shorter time constant (₁) under acidic pH conditions and with longer time constant (₂) under alkaline conditions.

View larger version (45K): [in this window] [in a new window]   Fig. 8. Effects of external pH on the inward whole-cell K+ currents of Arabidopsis pollen protoplasts. (A–C) The whole-cell recordings at various external pH; (D) current/voltage relations at various external pH; (E) conductance/voltage 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 fitted with the Boltzmann equation. (* Significantly different from the control by t-test, P<0.05; ** Significantly different from the control by t-test, P<0.01.)

 

View this table: [in this window] [in a new window]   Table 2. Effects of external pH on the steady-state properties of the inward whole-cell K+ currents in Arabidopsis pollen protoplasts

 

View this table: [in this window] [in a new window]   Table 3. Effects of external pH on the activation kinetics of the inward whole-cell K+ currents in Arabidopsis pollen protoplasts at -180 mV

 

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

 
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 agar-containing 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 solidified medium with agar seem to be important environmental factors for in vitro pollen germination. It is likely that the solidified surface of the 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 K⁺ influx in pollen germination and tube growth
For in vitro experiments in this study, the optimum external K⁺ concentration for Arabidopsis pollen germination was 1 mM. When the germination medium contained ‘no additional K⁺’, pollen germination was inhibited by 26%. The actual K⁺ concentration under these conditions was approximately 35 µM because of the K⁺ contamination of the agar even though K⁺-depleted agar was used. It is reasonable to expect that a genuine ‘no-K⁺-containing’ medium would result in a greater inhibition of pollen germination. For pollen tube growth, however, the optimum K⁺ concentration in the germination medium was near 30 mM. The results suggest that pollen tube growth may need more K⁺ influx than the pollen germination process. One possible explanation for this phenomenon is that K⁺ 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 K⁺ 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 K⁺ concentration. The increase of cytoplasmic K⁺ 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 K⁺ above 220 mM completely inhibited in vitro protein translation (Weber et al., 1978). With pollen grain rehydration during germination, the K⁺ 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 K⁺ influx 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, K⁺ influx as well as a consequent influx of water is required for the maintenance of the turgor pressure in the pollen tube. The data presented in this paper (Fig. 3A) show that 1 mM external K⁺ was needed for the highest pollen germination percentage, while 30 mM external K⁺ was required for the highest growth rate of the pollen tubes. Interestingly, it was observed in this study that high external K⁺ 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 K⁺ influx was correlated with a larger diameter of lily pollen tube. It is reasonable to propose that K⁺ influx may play an important role in the regulation of pollen turgor pressure. However, excessive K⁺ influx driven by very high (such as 50 mM) external K⁺ significantly inhibited both pollen germination and tube growth (Fig. 3A). Such inhibitory effects of excessive external K⁺ can effectively be removed by the addition of 5 mM TEA⁺ in the medium (data not shown), which further demonstrates that a certain amount of K⁺ influx is required for proper or healthy pollen germination and tube growth.

The addition of Ba²⁺ as a K⁺ influx blocker in the medium significantly inhibited both pollen germination and tube growth in the presence of 1 mM of external K⁺ (Fig. 3B). Consistent with the effects of Ba²⁺ on pollen germination and tube growth, either 10 mM Ba²⁺ or 20 mM TEA⁺ significantly inhibited the inward K⁺ currents in pollen protoplasts in the patch-clamp experiments (Fig. 6A, B). Taking these results together, it is concluded that K⁺ influx does play an important role in pollen germination and tube growth and that the regulation of the inward K⁺ channels in the pollen and pollen tube plasma membranes may be involved in signalling cascades of pollen germination and tube growth.

Regulation of K⁺ channels and pollen germination and tube growth by Ca²⁺ and external pH
When penetrating through the pistil tissues, pollen tubes are thought to encounter relatively high Ca²⁺ environments, particular in the micropylar filiform 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 K⁺ channels by high Ca²⁺ 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 K⁺ channels and pollen germination as well as tube growth, the effects of external and internal Ca²⁺ 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 K⁺ channels are strongly regulated by cytoplasmic Ca²⁺ (Schroeder and Hagiwara, 1989; Blatt et al., 1990; Fairley-Grenot and Assmann, 1992a, b; Lemtri-Chlieh and MacRobbie, 1994; Kelly et al., 1995; Grabov and Blatt, 1997), the inward K⁺ channels in Arabidopsis pollen protoplasts were insensitive to changes in intracellular Ca²⁺ 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 K⁺ channel among the three types of K⁺ channel recorded in the plasma membranes of lily pollen protoplasts was Ca²⁺-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 fluctuations of [Ca²⁺]_c 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 K⁺-channels in the plasma membranes of pollen tube are regulated by changes in [Ca²⁺]_c and it is worthy of further study.

The inward K⁺ 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 acidification that is induced by H⁺-ATPase activity that pumps H⁺ out of the cell. Therefore, the inward K⁺ 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 acidification may increase the activity of the inward K⁺-channels in lily pollen 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 K⁺ 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 K⁺ influx via the inward channels. In fact, as the results presented in Figs 2 and 3 indicate, a higher concentration of external K⁺ (such as 10 or 30 mM compared to 1 mM) that may drive a greater K⁺ influx did not result in a higher pollen germination percentage. Therefore, it is proposed that pollen germination may not require a very high K⁺ influx.

In conclusion, in vitro methods for Arabidopsis pollen germination using agar-containing media have been successfully established for the first time. Combining in vitro pollen germination experiments with patch-clamp whole-cell recording techniques, the present results suggest that K⁺ influx as well as its regulation mediated by the inward K⁺-channels plays an important role in Arabidopsis pollen germination and tube growth.

	Acknowledgments

 
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.

	Notes

 
¹ To whom correspondence should be addressed. Fax: +86 10 6289 3450. wuwh{at}public3.bta.net.cn1.

	Abbreviations

 
E_1/2, half-activation voltage; E_rev, reversal potential; G_max, maximal conductance; K_m, Michaelis–Menten constant; t_1/2, half-activation time; V_m, membrane potential; ₁ and ₂, time constants.

	References

Top Abstract Introduction Materials and methods Results Discussion 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. K⁺ channels of stomatal guard cells: characteristics of the inward rectifier and its control by pH. Journal of General Physiology 99, 615–644.[Abstract/Free Full Text]

Blatt MR, Thiel G, Trentham DR. 1990. Reversible inactivation of K⁺ channels of Vicia stomatal guard cells following the photolysis of caged inositol 1,4,5-trisphosphate. Nature 346, 766–769.[Medline]

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.[Web of Science]

Cheung AY, Wang H, Wu H-M. 1995. A floral transmitting tissue-specific glycoprotein attracts pollen tubes and stimulates their growth. Cell 82, 383–393.[Web of Science][Medline]

Fairley-Grenot KA, Assmann SM. 1992a. Permeation of Ca²⁺ through K⁺ channels in the plasma membrane of Vicia faba guard cells. Journal of Membrane Biology 128, 103–113.[Web of Science][Medline]

Fairley-Grenot KA, Assmann SM. 1992b. Whole-cell K⁺ current across the plasma membrane of guard cells from a grass: Zea mays. Planta 186, 282–293.[Web of Science]

Fan L-M, Wu W-H, Yang H-Y. 1999. Identification and characterization of the inward K⁺ channels in the plasma membranes of Brassica pollen protoplasts. Plant and Cell Physiology 40, 859–865.[Abstract/Free Full Text]

Feijó JA, Malhó R, Obermeyer G. 1995. Ion dynamics and its possible role during in vitro pollen germination and tube growth. Protoplasma 187, 155–167.[Web of Science]

Findlay GP, Tyerman SD, Garill A, Skerrett M. 1994. Pump and K⁺ inward rectifiers in the plasmalemma of wheat root protoplasts. Journal of Membrane Biology 139, 103–116.[Web of Science][Medline]

Franklin-Tong VE. 1999. Signalling and the modulation of pollen tube growth. The Plant Cell 11, 727–738.[Free Full Text]

Grabov A, Blatt MR. 1997. Parallel control of the inward-rectifier K⁺ channel by cytosolic free Ca²⁺ and pH in Vicia guard cells. Planta 201, 84–95.[Web of Science]

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. Pflugers Arch 391, 85–100.[Web of Science][Medline]

Heslop-Harrison J. 1987. Pollen germination and pollen tube growth. International Review of Cytology 107, 1–78.

Holdaway-Clarke TL, Feijó JA, Hackett GR, Kunkel JG, Hepler PK. 1997. Pollen tube growth and the intracellular cytosolic calcium gradient oscillate in phase while extracellular calcium influx is delayed. The Plant Cell 9, 1999–2010.[Abstract]

Ilan N, Schwartz A, Moran N. 1996. External protons enhance the activity of the hyperpolarization-activated K⁺ channels in guard cell protoplasts of Vicia faba. Journal of Membrane Biology 154, 169–181.[Web of Science][Medline]

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-affinity potassium transport activity. The Plant Cell 10, 51–62.[Abstract/Free Full Text]

Lee H-S, Huang S, Kao T-H. 1994. S proteins control rejection of incompatible pollen in Petunia inflata. Nature 367, 560–563.[Medline]

Leeton PRJ, Smyth DR. 1993. An abundant LINE-like element amplified 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.[Web of Science][Medline]

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 influx. The Plant Cell 11, 1731–1742.[Abstract/Free Full Text]

Maathuis FJM, Ichida AM, Sanders D, Schroeder JI. 1997. Roles of higher plant K⁺ channels. Plant Physiology 114, 1141–1149.[Web of Science][Medline]

Maathuis FJM, Sanders D. 1994. Mechanism of high-affinity potassium uptake in roots of Arabidopsis thaliana. Proceedings of the National Academy of Sciences, USA 91, 9272–9276.[Abstract/Free Full Text]

Mascarenhas JP. 1993. Molecular mechanisms of pollen tube growth and differentiation. The Plant Cell 5, 1303–1314.[Free Full Text]

Messerli M, Robinson KR. 1997. Tip localized Ca²⁺ pulses are coincident with peak pulsatile growth rates in pollen tubes of Lilium longiflorum. Journal of Cell Science 110, 1269–1278.[Abstract]

Messerli MA, Danuser G, Robinson KR. 1999. Pulsatile influxes of H⁺, K⁺ and Ca²⁺ lag growth pulses of Lilium longiflorum pollen tubes. Journal of Cell Science 112, 1497–1509.[Abstract]

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 longiflorum: characteristics of the non-germinating pollen grains. Journal of Experimental Botany 46, 803–813.[Abstract/Free Full Text]

Obermeyer G, Kolb H-A. 1993. K⁺ 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 H⁺-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 flux and the intracellular calcium gradient: effect of BAPTA-type buffers and hypertonic media. The Plant Cell 6, 1815–1828.[Abstract/Free Full Text]

Pierson ES, Miller DD, Callaham DA, van Aken J, Hackett G, Hepler PK. 1996. Tip-localized calcium entry fluctuates during pollen tube growth. Developmental Biology 174, 160–173.[Web of Science][Medline]

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.[Abstract/Free Full Text]

Schroeder JI, Hagiwara S. 1989. Cytosolic calcium regulates ion channels in the plasma membrane of Vicia faba guard cells. Nature 338, 427–430.[Web of Science]

Taylor LP, Hepler PK. 1997. Pollen germination and tube growth. Annual Review of Plant Physiology and Plant Molecular Biology 48, 461–491.[Web of Science]

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.[Web of Science][Medline]

Tian HQ, Russell SD. 1997. Calcium distribution in fertilized and unfertilized ovules and embryo sacs of Nicotiana tabacum L. Planta 202, 93–105.[Web of Science]

Weber LA, Hickey ED, Baglioni C. 1978. Influence of potassium salt concentration and temperature on inhibition of mRNA translation by 7-methylguanosine 5'-monophosphate [in the wheat germ and rabbit reticulocyte cell-free systems]. Journal of Biological Chemistry 253, 178–183.[Abstract/Free Full Text]

Weisenseel MH, Jaffe LF. 1976. The major growth current through lily pollen tubes enters as K⁺ and leaves as H⁺. 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.[Abstract/Free Full Text]

Wu WH, Assmann SM. 1994. A membrane-delimited pathway of G-protein regulation of the guard-cell inward K⁺ channels. Proceedings of the National Academy of Sciences, USA 91, 6310–6314.[Abstract/Free Full Text]

Yu C-J, Wu W-H. 1999. Identification and characterization of the inward K⁺-channels in Arabidopsis root cells. Science in China (C) 42, 307–315.

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				R. Kuang, K.-H. Chan, E. Yeung, and B. L. Lim Molecular and Biochemical Characterization of AtPAP15, a Purple Acid Phosphatase with Phytase Activity, in Arabidopsis Plant Physiology, September 1, 2009; 151(1): 199 - 209. [Abstract] [Full Text] [PDF]

				Y. Wang, W.-Z. Zhang, L.-F. Song, J.-J. Zou, Z. Su, and W.-H. Wu Transcriptome Analyses Show Changes in Gene Expression to Accompany Pollen Germination and Tube Growth in Arabidopsis Plant Physiology, November 1, 2008; 148(3): 1201 - 1211. [Abstract] [Full Text] [PDF]

				A. B. Sivitz, A. Reinders, and J. M. Ward Arabidopsis Sucrose Transporter AtSUC1 Is Important for Pollen Germination and Sucrose-Induced Anthocyanin Accumulation Plant Physiology, May 1, 2008; 147(1): 92 - 100. [Abstract] [Full Text] [PDF]

				S. Yamaoka and C. J. Leaver EMB2473/MIRO1, an Arabidopsis Miro GTPase, Is Required for Embryogenesis and Influences Mitochondrial Morphology in Pollen PLANT CELL, March 1, 2008; 20(3): 589 - 601. [Abstract] [Full Text] [PDF]

				S. Masuda, K. Mizusawa, T. Narisawa, Y. Tozawa, H. Ohta, and K.-i. Takamiya The Bacterial Stringent Response, Conserved in Chloroplasts, Controls Plant Fertilization Plant Cell Physiol., February 1, 2008; 49(2): 135 - 141. [Abstract] [Full Text] [PDF]

				T. Shimmen Electrophysiological Characterization of the Node in Chara corallina: Functional Differentiation for Wounding Response Plant Cell Physiol., February 1, 2008; 49(2): 264 - 272. [Abstract] [Full Text] [PDF]

				R. Wen, J. A. Torres-Acosta, L. Pastushok, X. Lai, L. Pelzer, H. Wang, and W. Xiao Arabidopsis UEV1D Promotes Lysine-63-Linked Polyubiquitination and Is Involved in DNA Damage Response PLANT CELL, January 1, 2008; 20(1): 213 - 227. [Abstract] [Full Text] [PDF]

				S. Coimbra, J. Almeida, V. Junqueira, M. L. Costa, and L. G. Pereira Arabinogalactan proteins as molecular markers in Arabidopsis thaliana sexual reproduction J. Exp. Bot., December 1, 2007; 58(15-16): 4027 - 4035. [Abstract] [Full Text] [PDF]

				J. H. Doelling, A. R. Phillips, G. Soyler-Ogretim, J. Wise, J. Chandler, J. Callis, M. S. Otegui, and R. D. Vierstra The Ubiquitin-Specific Protease Subfamily UBP3/UBP4 Is Essential for Pollen Development and Transmission in Arabidopsis Plant Physiology, November 1, 2007; 145(3): 801 - 813. [Abstract] [Full Text] [PDF]

				S. Frietsch, Y.-F. Wang, C. Sladek, L. R. Poulsen, S. M. Romanowsky, J. I. Schroeder, and J. F. Harper A cyclic nucleotide-gated channel is essential for polarized tip growth of pollen PNAS, September 4, 2007; 104(36): 14531 - 14536. [Abstract] [Full Text] [PDF]

				L. L. Ge, H. Q. Tian, and S. D. Russell Calcium function and distribution during fertilization in angiosperms Am. J. Botany, June 1, 2007; 94(6): 1046 - 1060. [Abstract] [Full Text] [PDF]

				S. Dai, T. Chen, K. Chong, Y. Xue, S. Liu, and T. Wang Proteomics Identification of Differentially Expressed Proteins Associated with Pollen Germination and Tube Growth Reveals Characteristics of Germinated Oryza sativa Pollen Mol. Cell. Proteomics, February 1, 2007; 6(2): 207 - 230. [Abstract] [Full Text] [PDF]

				X.-F. Song, C.-Y. Yang, J. Liu, and W.-C. Yang RPA, a Class II ARFGAP Protein, Activates ARF1 and U5 and Plays a Role in Root Hair Development in Arabidopsis Plant Physiology, July 1, 2006; 141(3): 966 - 976. [Abstract] [Full Text] [PDF]

				S. Rehman and S. J. Yun Developmental regulation of K accumulation in pollen, anthers, and papillae: are anther dehiscence, papillae hydration, and pollen swelling leading to pollination and fertilization in barley (Hordeum vulgare L.) regulated by changes in K concentration? J. Exp. Bot., March 1, 2006; 57(6): 1315 - 1321. [Abstract] [Full Text] [PDF]

				L. Jiang, S.-L. Yang, L.-F. Xie, C. S. Puah, X.-Q. Zhang, W.-C. Yang, V. Sundaresan, and D. Ye VANGUARD1 Encodes a Pectin Methylesterase That Enhances Pollen Tube Growth in the Arabidopsis Style and Transmitting Tract PLANT CELL, February 1, 2005; 17(2): 584 - 596. [Abstract] [Full Text] [PDF]

				J. Xu, C. A. Brearley, W.-H. Lin, Y. Wang, R. Ye, B. Mueller-Roeber, Z.-H. Xu, and H.-W. Xue A Role of Arabidopsis Inositol Polyphosphate Kinase, AtIPK2{alpha}, in Pollen Germination and Root Growth Plant Physiology, January 1, 2005; 137(1): 94 - 103. [Abstract] [Full Text] [PDF]

				R. Dutta and K. R. Robinson Identification and Characterization of Stretch-Activated Ion Channels in Pollen Protoplasts Plant Physiology, July 1, 2004; 135(3): 1398 - 1406. [Abstract] [Full Text] [PDF]

				G. R. Hicks, E. Rojo, S. Hong, D. G. Carter, and N. V. Raikhel Geminating Pollen Has Tubular Vacuoles, Displays Highly Dynamic Vacuole Biogenesis, and Requires VACUOLESS1 for Proper Function Plant Physiology, March 1, 2004; 134(3): 1227 - 1239. [Abstract] [Full Text] [PDF]

				K. Kobayashi, K. Awai, K.-i. Takamiya, and H. Ohta Arabidopsis Type B Monogalactosyldiacylglycerol Synthase Genes Are Expressed during Pollen Tube Growth and Induced by Phosphate Starvation Plant Physiology, February 1, 2004; 134(2): 640 - 648. [Abstract] [Full Text] [PDF]

				J. D. Becker, L. C. Boavida, J. Carneiro, M. Haury, and J. A. Feijo Transcriptional Profiling of Arabidopsis Tissues Reveals the Unique Characteristics of the Pollen Transcriptome Plant Physiology, October 1, 2003; 133(2): 713 - 725. [Abstract] [Full Text] [PDF]

				M. Golovkin and A. S. N. Reddy A calmodulin-binding protein from Arabidopsis has an essential role in pollen germination PNAS, September 2, 2003; 100(18): 10558 - 10563. [Abstract] [Full Text] [PDF]

				E. M. Lord Adhesion and guidance in compatible pollination J. Exp. Bot., January 1, 2003; 54(380): 47 - 54. [Abstract] [Full Text] [PDF]

				J. Scholz-Starke, M. Buttner, and N. Sauer AtSTP6, a New Pollen-Specific H+-Monosaccharide Symporter from Arabidopsis Plant Physiology, January 1, 2003; 131(1): 70 - 77. [Abstract] [Full Text] [PDF]

				K. Mouline, A.-A. Very, F. Gaymard, J. Boucherez, G. Pilot, M. Devic, D. Bouchez, J.-B. Thibaud, and H. Sentenac Pollen tube development and competitive ability are impaired by disruption of a Shaker K+ channel in Arabidopsis Genes & Dev., February 1, 2002; 16(3): 339 - 350. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (58) Request Permissions Disclaimer Google Scholar Articles by Fan, L.-M. Articles by Wu, W.-H. Search for Related Content PubMed PubMed Citation Articles by Fan, L.-M. Articles by Wu, W.-H. Agricola Articles by Fan, L.-M. Articles by Wu, W.-H. Social Bookmarking          What's this?

Online ISSN 1460-2431 - Print ISSN 0022-0957

Copyright © 2005 Society for Experimental Biology

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics