JXB Advance Access originally published online on March 26, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Journal of Experimental Botany, Vol. 55, No. 399, pp. 993-1001, May 1, 2004
© 2004 Oxford University Press
Cell and Molecular Biology, Biochemistry and Molecular Physiology |
Pulsing Cl– channels in coat cells of developing bean seeds linked to hypo-osmotic turgor regulation
Received 1 August 2003; Accepted 2 February 2004
1 Department of Horticulture, Viticulture and Oenology, The University of Adelaide, Waite Campus PMB 1, Glen Osmond, SA 5064, Australia
2 Biophysics Department, School of Physics, The University of NSW, Kensington, NSW, 2052, Australia
3 School of Life and Environmental Sciences, The University of Newcastle, Newcastle, NSW 2308, Australia
* To whom correspondence should be addressed. Fax: +61 8 8303 7316. E-mail: wen-hao.zhang{at}adelaide.edu.au
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Seed coat cells in the developing seeds of grain legumes release nutrients to the developing embryo. This occurs into an apoplastic space that separates the maternal (seed coat) and filial (embryo) generations. Protoplasts of seed coat cells from coats of Phaseolus vulgaris L. seeds were isolated and whole-cell current across their plasma membranes was characterized using the patch-clamp technique. A pulsing inward current that displayed a spontaneous activation and voltage-dependent inactivation was observed. The frequency and magnitude of the current pulses were positively dependent on cytoplasmic Cl– concentrations and independent of external cations. The pulse current was inhibited by DIDS and La3+, but not by Gd3+. Single channel events (conductance=18 pS) could be identified with the inactivating phase of the pulses. Together, these findings are consistent with the current being carried by a burst of Cl– efflux through Cl–-permeable channels that activate almost simultaneously. Neomycin caused a reversible inhibition of the pulsed current, suggesting that its activation is likely to be modulated by an IP3-dependent intracellular Ca2+ release. The pharmacological profiles of Cl– efflux from excised seed coats were comparable with those of the Cl– channels in the whole cell configuration, suggesting that the Cl– channels may underpin Cl– efflux from the seed coats. Efflux of Cl– from the seed coats was also stimulated by hypo-osmotic treatment as was the frequency and magnitude of Cl– channel in whole-cell patch clamp experiments. This implies that the Cl– channels responsible for the pulsed Cl– currents are likely to be a component of the turgor-regulatory mechanism in developing bean seeds.
Key words: Hypo-osmotic treatment, neomycin, patch-clamp, Phaseolus vulgaris L., pulsed Cl– channels, seed coat.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In developing seeds of grain legumes, the maternal coat and enclosed filial embryo (consisting of two large cotyledons) is symplasmically isolated (Patrick and Offler, 2001). Therefore, all nutrients required by developing cotyledons must be released from seed coats to the seed apoplasm (Patrick and Offler, 2001). In developing seeds of French bean (Phaseolus vulgaris L.), phloem-imported nutrients move through the seed coat symplasm to specialized ground parenchyma cells where they are released across their plasma membranes to the seed apoplasm (Wang et al., 1995). Sucrose, K+, Cl– and assimilated nitrogen are the principal nutrients transported by this route (Patrick, 1984; Walker et al., 1995). Sucrose efflux is characterized by both passive and energy-coupled components, the latter mediated by proton–sucrose antiport (Walker et al., 1995, 2000).
The protoplasts derived from ground parenchyma cells of bean seed coats displayed distinctive morphology, i.e. a granulated cytoplasm, relative large nucleus, off-centre vacuoles, and lack of chloroplasts (cf. Zhang et al., 2002). This allows specific investigation of ion transport across their plasma membranes by the patch-clamp technique (Zhang et al., 2002). Two types of non-selective channels (currents) have been characterized in the plasma membranes of ground parenchyma protoplasts that differ in their activation kinetics and selectivity between K+ and Ca2+ (Zhang et al., 2000, 2002). A slowly-activating outward current, found in about 25% of patched protoplasts, is more selective for K+ than Cl– (PK:PCl=4.00), weakly selective between K+ and Ca2+ (PK:PCa=0.75) and non-selective between univalent cations (Zhang et al., 2002). A ubiquitous fast-activating current is weakly selective between K+ and Cl– (PK:PCl =1.8–2.5), highly selective for K+ over Ca2+ (PK:PCa >10) and non-selective between univalent cations (Zhang et al., 2000, 2002). These non-selective channels may provide a low-resistance pathway for the release of phloem-transported ions into the seed apoplasm. In the present paper, the discovery of pulsed Cl– inward current in the plasma membranes of ground parenchyma cells of bean seed coats is reported. The pulsed Cl– current was stimulated by hypo-osmotic treatments. Therefore, the channels that underlie this Cl– current may account for Cl– efflux from seed coats under hypo-osmotic conditions as part of a turgor-regulatory mechanism (Zhang et al., 1996).
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant materials and protoplast isolation
Beans (Phaseolus vulgaris L. cv. Redland pioneer) were raised under glasshouse conditions and seeds harvested as described previously (Wang et al., 1995). Seed coats were surgically removed from the underlying embryos, cut longitudinally into small pieces and digested in an enzyme solution for 2 h at 20 °C. The solution contained 0.8% cellulase (Onozuka RS; Yakult Honsha, Tokyo) and 0.08% pectolyase (Sigma), 0.5% PVP, 0.5% bovine serum albumin (BSA), 1 mM CaCl2, and 5 mM MES/TRIS (pH 6.0), and osmolality of 500 mOsm kg–1 adjusted with sorbitol. A sucrose density gradient, as described previously (Zhang et al., 1997), was used to collect clean protoplasts. The protoplasts were kept on ice until patch-clamped. Protoplasts of ground parenchyma cells, that function to release solutes (Wang et al., 1995), were identified on the basis of their distinctive morphology (Zhang et al., 2002). The mean diameter of these protoplasts was 29.2±4.3 µm (SD, n=86).
Electrophysiology and data analysis
Patch pipettes, pulled from borosilicate glass blanks (Clark Electromedical, Readings, UK), were coated with SylgardR (Dow Corning, Midland, MI). Voltage across the patch was controlled and current measured using an Axopatch 200B (Axon Instruments, Foster City, CA, USA). Whole-cell preparations were obtained by forming gigaseals in the cell-attached mode and then an extra suction was applied to rupture the plasma membrane. Successful whole-cell configurations were indicated by a substantial increase in capacitance. Series resistance was compensated to about 50% and capacitance was compensated. Voltage pulses, between 4 s and 20 s in duration, were used to elicit inward currents. Data were sampled at 2 kHz and filtered at 0.5 kHz by a low pass 4 pole Bessel filter. Records were stored and analysed using pClamp 8.0 (Axon Instruments, Foster City, CA, USA). All experiments were carried out at room temperature (20–22 °C). Junction potentials were calculated, and corrected for, using the program JPCalc (PH Barry, University of New South Wales, Sydney, Australia).
Experimental solutions
Two types of pipette solution were commonly used in the present study. They were composed of (mM): High Cl–: 100 KCl, 2.3 CaCl2, 2 MgCl2, 2 Na2ATP, 10 EGTA, 10 HEPES; Low Cl–: 10 KCl, 90 K-glutamate, 2.3 CaCl2, 2 MgCl2, 2 Na2ATP, 10 EGTA, 10 HEPES. Free calcium concentrations in both types of pipette solution were approximately 50 nM calculated using the chemical speciation program GEOCHEM (Parker et al., 1987). Both solutions were adjusted to an osmolality of 720 mOsM with sorbitol and pH 7.2 with TRIS. All bath solutions contained, in addition to other solutes, 5 mM 2-(N-morpholino)ethanesulphonic acid (MES), pH 6.0, and 700 mOsm kg–1 or 600 mOsm kg–1 adjusted with TRIS and sorbitol, respectively. The details of the bath and pipette solutions are given in appropriate figure legends.
Measurement of Cl– efflux
Six uniform seed coats were cut longitudinally around the integumentary fusion line and embryos carefully removed. The coat halves were arranged in their respective pairs with their longitudinal axes vertical and their cut surfaces uppermost. One coat half of each pair served as the control and the other as the treatment. Wash solutions, comprised of 0.5 mM CaCl2, 5 mM MES, 300 mM or 80 mM sorbitol, pH 6.0 adjusted with TRIS, were dispensed into the space vacated by the embryo. The apoplasmic content of the seed coat was removed during the first 10 min of wash-out. These solutions were discarded. Subsequent solution changes were collected at 10 min intervals and the collected solutions bulked for Cl– analysis. The wash protocol was terminated after 30–40 min and fresh weights of the seedcoat halves determined. Cl– concentrations in the wash solutions were measured by capillary zone electrophoresis using a Quanta 4000 instrument (Waters, Milford, USA) as described by Frachisse et al. (1999).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Spontaneous pulses of inward current in whole cell configuration caused by the simultaneous opening of channels
A spontaneously activating, ‘pulse-like’ inward current was observed in 39% of the protoplasts derived from ground-parenchyma cells (n=121) with pipette solution containing 108 mM Cl– (Fig. 1A). Following the spontaneous activation, the inward current decayed over time at constant voltage (inactivation) (Fig. 1A). Often when the current was almost completely inactivated single channel events could be observed (lower graph in Fig. 1A) and from these records it was possible to obtain amplitudes as a function of voltage from which a single channel conductance could be obtained. The single channel conductance was 18±1 pS (95% confidence interval, 4 protoplasts) in a bath solution composed of 10 mM KCl, 10 mM CaCl2, and high chloride pipette solution.
|
The time-course of this inactivation was best fitted by a single exponential (Fig. 1B). Time constants of inactivation were reduced from 252±42 ms at –40 mV to 80± 8 ms (n=16) at –180 mV (Fig. 1C). Note that there were large variations in current magnitude among current pulses at a given voltage level (cf. Fig. 2). An inward current pulse was defined when the current magnitude was greater than –5 pA relative to the background level, and when it displayed an apparent time-dependent inactivation (cf. Fig. 1).
|
The pulsed inward current was carried by Cl– efflux
The pulsed inward current was hardly affected when external K+ was substituted with less permeant organic cation tetraethylammonium (TEA+) (Fig. 2A, B). The current occurred when K+ was omitted from the bath (Fig. 2C, D). For instance, the mean frequency of the pulsed current at membrane voltages between –60 mV and –180 mV was 0.54±0.17 Hz and 0.52±0.14 Hz for three protoplasts measured in 10 mM KCl and 10 mM TEACl solutions, respectively. Both the frequency and peak current magnitude of the pulses were independent of external Ca2+ concentrations between 10 mM and 0.2 mM Ca2+ (Fig. 2E, F). The mean frequency of the pulsed current, was 0.61±0.22 Hz and 0.53±0.24 Hz for three protoplasts bathed in 10 mM and 0.2 mM CaCl2, respectively. The current magnitude, measured as peak current of all the current pulses between –60 mV and –180 mV, of the three protoplasts at high and low Ca2+ bath solutions were –71.4±38.4 pA (n=18) and –73.6± 38.8 pA (n=16), respectively.
The pulsed inward current was observed in a smaller proportion of protoplasts when low Cl– concentration was present in the pipette solution ([Cl–]=18 mM) (11% of patched protoplasts n=54 as opposed to 39% in high [Cl–]). Furthermore, the peak current magnitude was positively dependent upon the Cl– concentrations in both pipette and external solutions (Fig. 3A, B). With 10 mM KCl and 10 mM CaCl2 in the bath, the current reversed at a potential close to the equilibrium potential for Cl– (ECl) (ECl= +25 mV) and more positive than EK (EK= –58 mV) (Fig. 3B). Moreover, the reversal potential shifted from +19 mV to –20 mV when the external KCl concentration was changed from 10 to 100 mM (Fig. 3B). A relative permeability for Cl– over K+ (PCl/PK) of 9.0 was calculated using the Goldman equation (cf. Wegner and De Boer, 1997). Furthermore, the single channel current–voltage curve obtained from some whole cell recordings (Fig. 1A) intersected the voltage axis at a voltage not significantly different from the equilibrium potential for Cl–. Taken together, these results suggest that the pulsed current is likely to be carried by a burst of Cl– efflux through many single channels that activate almost simultaneously.
|
In contrast to the peak current (Fig. 3A, B), the mean Cl– current as a function of voltage was non-linear (Fig. 3C), with the slope conductance increasing at more negative voltages. This indicates that the probability of pulses occurring increased with more negative voltages.
Pharmacology of the Cl– current pulses
The pulsed Cl– current was sensitive to 4,4'-di-isothiocyanatostillbene-2,2'-disulphonic acid (DIDS) (Fig. 4A, B), a Cl– channel antagonist that inhibits R-type anion channels in guard cells (Marten et al., 1993). The inhibition of the current by DIDS was not readily reversible upon wash-out of the inhibitor (data not shown). The pulsed Cl– current was reversibly, and completely, inhibited by 100 µM LaCl3 (Fig. 4C, D). By contrast, GdCl3, up to a concentration of 1 mM, had no effect on the pulsed current (Fig. 4E, F).
|
In Chara cells, a pulsed Cl– current associated with the action potential is activated by an inositol 1,4,5-triphosphate (IP3)-dependent internal Ca2+ release (Biskup et al., 1999). It was examined whether a similar phenomenon occurred in ground parenchyma protoplasts by treating them with neomycin. Neomycin inhibits phospholipase C (PLC), which mobilizes IP3 from its membrane-bound precursor phosphotidylinositol 4,5-biphosphate (PIP2) (Epstein et al., 1985). As shown in Fig. 5, the pulsed Cl– current was completely abolished upon the addition of neomycin to the bath, and the inhibitory effect was fully reversed upon removal of neomycin (Fig. 5C).
|
The Cl– pulsed current was stimulated by hypo-osmotic shock
The release of phloem-imported nutrients from bean seed coats is distinguished by its marked stimulation when the turgor of the seed coat unloading cells is elevated (Patrick, 1994; Zhang et al., 1996). The response of pulsed Cl– current in ground parenchyma protoplasts to a hypo-osmotic treatment was investigated. Figure 6 shows that both the frequency and magnitude of the pulsed current markedly increased when the protoplast was exposed to a reduction in external osmolality from 700 to 600 mOsm kg–1. The mean frequency of the pulsed current, at membrane voltages between –100 mV and –180 mV, was increased from 0.16±0.07 Hz (n=6) to 0.63±0.22 Hz (n=6) when external osmolality was reduced from 700 to 600 mOsm kg–1 with 1 mM KCl and 1 mM CaCl2 in the bath. The current magnitude, measured as peak current of all the current pulses at membrane voltages between –100 mV and –180 mV, was increased from –39±33 pA (n=9) to –107±64 pA (n=36) in response to the decrease in the external osmolality from 700 to 600 mOsm kg–1. Moreover, the hypo-osmotically induced activity was largely reversed upon; Returning the protoplast to iso-osmotic conditions (Fig. 6C).
|
Effect of neomycin, La3+, Gd3+ and hypo-osmotic treatment on Cl– efflux from seed coats
To examine whether the characterized Cl– current in patch-clamp experiments accounted for Cl– efflux from seed coats, the effects were tested of treatments on Cl– efflux from seed coat halves that either inhibited or stimulated the Cl– current in patch clamp experiments. Chloride efflux was inhibited by approximately 40% and 30% by 1 mM neomycin and La3+, respectively (Table 1). By contrast, Cl– efflux from seed coat halves was insensitive to Gd3+ (Table 1). Determination of Cl– concentrations by capillary zone electrophoresis was found to be unreliable in the presence of DIDS (data not shown), making it impossible to examine the effect of DIDS on Cl– efflux from seed coats. Efflux of Cl– from seed coat halves was enhanced when incubated in external solution of 80 mOsm kg–1 compared with that of 320-mOsm kg–1 (Table 1). These findings are consistent with the effects of these treatments on the Cl– currents, suggesting that the Cl– channels are involved in mediation of Cl– efflux from seed coat.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
One of the important functions of the ground parenchyma cells in coats of developing bean seeds is to release phloem-imported nutrients from seed coats to the seed apoplasm (Wang et al., 1995; Patrick and Offler, 2001). Two types of channels (currents) that are non-selective among univalent cations and weakly selective for K+ over Cl– in the plasma membranes of the ground parenchyma protoplasts have previously been characterized (Zhang et al., 2000; 2002). In the present paper, an inward current was identified that was activated in a pulse-like manner at membrane potentials negative of ECl in protoplasts derived from ground parenchyma cells of Phaseolus seed coats (Fig. 1). The frequency, current magnitude, and activation potential were positively dependent on the Cl– concentration in the cytoplasm (Fig. 3). The current was independent of external cation concentrations (Fig. 2) and sensitive to the Cl– channel blocker DIDS (Fig. 4A, B). Single channel records obtained from decaying parts of the inactivation of the pulsed current showed a conductance of 18 pS and from extrapolation of the current voltage curve reversed close to ECl. These findings are consistent with the current being carried by a burst of Cl– efflux through almost simultaneously activating channels, of the order of 50–100 channels per pulse. The Cl– current was inhibited by La3+ and neomycin (Figs 4, 5), but not by Gd3+ (Fig. 4). A similar effect of these agents was observed on Cl– efflux from seed coats (Table 1), suggesting that the Cl– channels characterized in the present study are involved in Cl– efflux from seed coats. Neomycin and La3+ abolished the Cl– currents (Figs 4, 5), while these agents inhibited the Cl– efflux from seed coats by approximately 40% and 30%, respectively (Table 1). This suggests that the Cl– channels described here could only account for about 40% of the total Cl– released from seed coats and that there are other transport systems involved in Cl– release.
The involvement of cytoplasmic Ca2+ activity ([Ca2+]c) causing activation of Cl– channels in plasma membranes of plant cells has been documented (White and Broadley, 2001). Therefore, it is likely that an increase in [Ca2+]c due to Ca2+ influx through Ca2+-permeable channels may act as a primary trigger to elicit the Cl– current. The inhibition of the current by La3+ (Fig. 4), a known Ca2+ channel blocker (Very and Davies, 2000; White, 2000), is in line with this suggestion. However, La3+ has been shown to block Cl– currents in plasma membranes of Chara cells (Tyerman et al., 1986; Biskup et al., 1999) and blue-light induced anion channels in Arabidopsis cells (Lewis and Spadling, 1998). The finding that Gd3+, another widely known Ca2+ channel blocker of plant cells (Very and Davies, 2000; White, 2000), did not inhibit the Cl– current (Fig. 4), also argues against the contribution of Ca2+ to activation of the Cl– current. Alternatively, Ca2+ channels that are sensitive to La3+, but not to Gd3+, could be involved in modulation of the Cl– current.
One important finding in the present study is that the Cl– inward current was sensitive to neomycin (Fig. 5), an antagonist of PLC-mediated cleavage of PIP2 to IP3 and diacylglycerol (Berridge, 1993). A pulsed current carried by Cl– efflux that is transiently activated by IP3-induced intracellular Ca2+ release has been characterized in Chara cells (Biskup et al., 1999; Wacke and Thiel, 2001; Wacke et al., 2003). In Chara cells, IP3-dependent intracellular Ca2+ liberation occurs as an all-or-none event (Wacke and Thiel, 2001; Wacke et al., 2003). Whether a similar all-or-none increase in cytoplasmic Ca2+ activity operates in ground parenchyma cells of bean seed coats remains to be evaluated. The random activation of the Cl– current could be accounted for by an uneven distribution of Ca2+ hot spots, resulting from spontaneous activation of IP3-dependent intracellular Ca2+ release, and the proximity of Ca2+ hot spots to Cl– channels in the plasma membranes. Alternatively, the inhibition of Cl– current by neomycin may result from its direct blockade of Ca2+-permeable cation channels or the Cl– channel directly. Neomycin has been shown to block capacitative Ca2+ entry in cultured rat spinal cord neurons (Zhou et al., 2000). To the best of the authors’ knowledge, there have been no reports of blockade of plant or animal Cl– channels by neomycin.
Both the pulsed Cl– current and the Cl– efflux from seed coat halves were stimulated by hypo-osmotic treatments (Fig. 6; Table 1), implying that the Cl– conductance characterized in the present study functions as a route for Cl– release from the seed coats under hypo-osmotic conditions. A biphasic increase in the cytoplasmic Ca2+ activity, resulting from Ca2+ influx and then intracellular Ca2+ release, appears to be a common phenomenon in plant cells in response to a hypo-osmotic treatment (Cessna and Low, 2001). It is envisaged that the hypo-osmotically-induced Ca2+ influx through a mechano-sensitive Ca2+-permeable channel stimulates PLC activity, leading to an IP3-mediated intracellular Ca2+ release. However, the lack of effect of Gd3+ on the Cl– inward current (Fig. 4) seems to discount this possibility as Gd3+ is a known antagonist of Ca2+-permeable channels of plant cells (White, 2000). Nevertheless, the possibility cannot be ruled out that a Ca2+-permeable and relatively Gd3+-insensitive channel mediates Ca2+ influx to activate PLC. In this context, two types of membrane-depolarization activated Ca2+-permable Ca2+ channels, differing in sensitivity to La3+ and Gd3+, in the plasma membranes of maize root cells have been identified (Marshall et al., 1994). It has been shown that hypo-osmotic treatment depolarized membrane potential of the ground parenchyma cells (Walker et al., 2000). Therefore, an increase in [Ca2+]c resulting from activation of a Gd3+-insensitive Ca2+ channel by membrane depolarization under hypo-osmotic treatment directly, or indirectly through Ca2+-dependent protein kinases, activate Cl– conductance in the plasma membrane to mediate Cl– efflux, leading to downward turgor regulation (Okazaki, 1996).
The spontaneous activation of Cl– current, in particular under hypo-osmotic conditions, is reminiscent of turgor-regulated pulsed burst of Cl– efflux in marine algal Acetabularia mediterranea (Wendler et al., 1983). Like the seed coat unloading cells, the burst of Cl– release from the algal cells is believed to involve downward turgor regulation (Wendler et al., 1983). Nutrient efflux from coats of developing bean seeds is closely regulated by turgor of the seed coat cells (Patrick, 1994; Walker et al., 2000). An increase in turgor, caused by an enhanced uptake of nutrients by the embryos from the seed apoplasmic pool, elicits a rapid increase in efflux of solutes (Patrick et al., 1986; Walker et al., 2000). This downward regulated turgor is used to maintain a constant hydrostatic pressure difference between source and sink, thus allowing for a sustained rate of phloem import into seed coats to match rates of nutrient uptake by cotyledons (Zhang et al., 1996). The finding that hypo-osmotic treatment stimulated the pulsed Cl– inward current (Fig. 6) provides a mechanistic explanation for the hypo-osmotically induced Cl– efflux from seed coats (Table 1). A spontaneous burst of Cl– efflux through the Cl– channel will occur once phloem-imported Cl– is built up to a certain level in the ground parenchyma cells. Furthermore, activation of the Cl– current at a wide range of membrane potentials would ensure that the Cl– transported into the seed coat unloading cells will be released rapidly to the seed apoplasm. The PLC activity may act as an important regulator to modulate the Cl– channel activity through IP3-mediated intracellular Ca2+ release in the seed coat unloading cells.
|
Acknowledgements |
|---|
This study was supported by the Australian Research Council. We thank Louise Hetherington and Wendy Sullivan for their expert technical assistance and Kevin Stokes for supplying experimental plant materials.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Berridge MJ. 1993. Inositol triphosphate and calcium signalling. Nature 361, 315–325.[CrossRef][Medline]
Biskup B, Gradmann D, Thiel G. 1999. Calcium release from InsP3-sensitive internal stores initiates action potential in Chara. FEBS Letters 453, 72–76.[CrossRef][Web of Science][Medline]
Cessna SG, Low PS. 2001. Activation of the oxidative burst in aequorin-transformed Nicotiana tabacum cells is mediated by protein kinase- and anion-channel-dependent release of Ca2+ from internal stores. Planta 214, 126–134.[Web of Science][Medline]
Epstein PA, Prentki M, Attie MF. 1985. Modulation of intracellular Ca2+ in the parathyroid cell: release of Ca2+ from non-mitochondrial pools by inositol triphosphate. FEBS Letters 188, 141–144.[CrossRef][Web of Science][Medline]
Frachisse JM, Thomine S, Colcombet J, Guern J, Barbier-Brygoo H. 1999. Sulphate is both a substrate and an activator of the voltage-dependent anion channel of Arabidopsis hypocotyl cells. Plant Physiology 121, 253–261.
Lewis BD, Spadling EP. 1998. Non-selective block by La3+ of Arabidopsis ion channels in signal transduction. Journal of Membrane Biology 162, 81–90.[CrossRef][Web of Science][Medline]
Marshall J, Corzo A, Leigh RA, Sanders D. 1994. Membrane potential-dependent calcium transport in right-side-out plasma membrane vesicles. The Plant Journal 5, 683–694.[CrossRef][Web of Science]
Marten I, Busch H, Raschke K, Hedrich R. 1993. Modulation and block of the plasma membrane anion channel of guard cells by stilbene derivatives. European Biophysics Journal 21, 403–408.
Okazaki Y. 1996. Turgor regulation in a brackish water charophyte, Lamprothamnium succinctum. Journal of Plant Research 109, 107–112.[CrossRef]
Parker DR, Zelazny LW, Kinraide TB. 1987. Improvements to the program GEOCHEM. Soil Science Society of American 51, 488–491.
Patrick JW. 1984. Photosynthate unloading from seed coats of Phaseolus vulgaris L.: control by water relations. Journal of Plant Physiology 115, 297–310.
Patrick JW. 1994. Turgor-dependent unloading of assimilates from coats of developing legume seed: assessment of the significance of the phenomenon in the whole plant. Physiologia Plantarum 90, 645–654.[CrossRef]
Patrick JW, Jacobs E, Offler CE, Cram WJ. 1986. Photosynthate unloading from seed coats of Phaseolus vulgaris L.: nature and cellular location of turgor-sensitive unloading. Journal of Experimental Botany 37, 1006–1019.
Patrick JW, Offler CE. 2001. Compartmentation of transport and transfer events in developing seeds. Journal of Experimental Botany 52, 551–564.
Tyerman SD, Findlay GP, Paterson GJ. 1986. Inward membrane current in Chara inflata. II. Effects of pH, Cl– channel blockers and NH4+, and significance for the hyperpolarized state. Journal of Membrane Biology 89, 153–161.[CrossRef]
Very AA, Davies JM. 2000. Hyperpolarization-activated calcium channels at the tip of Arabidopsis root hairs. Proceedings of the National Academy of Sciences, USA 97, 9801–9806.
Wacke M, Thiel G. 2001. Electrically triggered all-or-none Ca2+-liberation during action potential in the giant alga Chara. Journal of General Physiology 118, 11–21.
Wacke M, Thiel G, Hütt M-T. 2003. Ca2+ dynamics during membrane excitation of the green alga Chara: model simulations and experimental data. Journal of Membrane Biology 191, 179–192.[CrossRef][Web of Science][Medline]
Walker NA, Patrick JW, Zhang WH, Fieuw S. 1995. Efflux of photosynthate and acid from developing seed coats of Phaseolus vulgaris L.: a chemiosmotic analysis of pump-driven efflux. Journal of Experimental Botany 46, 539–549.
Walker NA, Zhang WH, Harrington G, Holdaway N, Patrick JW. 2000. Effluxes of solutes from developing seed coats of Phaseolus vulgaris L. and Vicia faba L.: locating the effects of turgor in a coupled chemisosmotic system. Journal of Experimental Botany 51, 1047–1055.
Wang X-D, Harrington G, Patrick JW, Offler CE, Fieuw S. 1995. Cellular pathways of photosynthate transport in coats of developing seed of Vicia faba L. and Phaseolus vulgaris L.: principal cellular sites of efflux. Journal of Experimental Botany 45, 49–63.
Wegner LH, De Boer AH. 1997. Properties of two outward-rectifying channels in root xylem parenchyma cells suggest a role in K+ homeostasis and long distance signalling. Plant Physiology 115, 1707–1719.[Abstract]
Wendler S, Zimmermann U, Bentrup FW. 1983. Relationship between cell turgor pressure, electrical membrane potential and chloride efflux in Acetabularia mediterranea. Journal of Membrane Biology 72, 75–84.[CrossRef][Web of Science]
White PJ. 2000. Calcium channels in higher plants. Biochimica et Biophysica Acta 1465, 171–189.[Medline]
White PJ, Broadley MR. 2001. Chloride in soils and its uptake and movement within the plant. Annals of Botany 88, 967–988.
Zhang WH, Atwell BJ, Patrick JW, Walker NA. 1996. Turgor-dependent efflux of assimilate from coats of developing seeds of Phaseolus vulgaris L.: water relations of the cells involved in efflux. Planta 199, 25–33.[Web of Science]
Zhang WH, Skerrett M, Walker NA, Patrick JW, Tyerman SD. 2002. Non- selective currents and channels in plasma membranes of protoplasts from coats of developing seeds of bean seeds. Plant Physiology 128, 388–399.
Zhang WH, Walker NA, Patrick JW, Tyerman SD. 1997. Mechanism of solute efflux from seed coat: whole-cell K+ currents in the plasma membrane of protoplasts derived from Vicia faba L. seed coats. Journal of Experimental Botany 48, 1565–1572.
Zhang WH, Walker NA, Tyerman SD, Patrick JW. 2000. Fast activation of a time- dependent outward current in protoplasts derived from coats of developing Phaseolus vulgaris seeds. Planta 211, 894–898.[CrossRef][Web of Science][Medline]
Zhou Z-S, Shu Y-S, Zhao Z-Q. 2000. Neomycin blocks substance P-induced calcium entry in cultured rat spinal cord neurons. Neuroscience Letters 287, 57–60.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||