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Ca2+ channels control the rapid expansions in pulsating growth of Petunia hybrida pollen tubes
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© 1998 by Gustav Fischer Verlag, Jena
- Ca2 + Channels Control the Rapid Expansions it; Pulsating Growth of Petunia hybrida Pollen Tubes ANJA GEITMANN*
and MAURO
CRESTI
Department of Environmental Biology, University of Siena, Via P.A. Mattioli 4, 1-53100 Siena, Italy Received March 3, 1997 . Accepted July 7, 1997
Summary Pollen tubes exhibit a tip-focused gradient of the cytosolic Ca2 + concentration, which is .rresumably governed by the activity of Ca2 + channels in the plasma membrane. The putative role of Ca4 as a signalling factor in pulsating growth of Petunia ,?brida pollen tubes was examined. The application of inorganic Ca2 + channel inhibitors La3 + and Gd3 caused the pulsating pollen tubes to abandon their rhythm and continue growth with rather steady rates. This effect was reversible upon removal of these ions. The organic inhibitors of ci+ channels nifedipine and verapamil caused the arrest of overalt pollen tube growth at higher concentrations, whereas intermediate concentrations caused a slowdown of pulse frequency, but not the inhibition of pulses. This leads to the hypothesis, that the activity of a certain group of Ca2+ channels, which is sensitive to La3+ and Gd3 + would be responsible for the control of pulsating growth. Another group of Ca2 + channels, which is sensitive to organic inhibitors, would be required for pollen tube growth in general and provide the basic Ca2 + influx, but would not be directly involved in the growth oscillations. Based on these findings a hypothetical model for the mechanism that cO,ntrols pulsating . : growth in pollen tubes is proposed.
Key words: Petunia hybrida, calcium channel gadolinium, lanthanum, nifedipine,: pollen tube, pulsating growth, verapamiL " Introduction
Pollen tubes are tip-growing cells, their elongation occurs in one direction and the growth process is basically confined to the apical tip of the cylindrical cell. Video observation of in vitro growing pollen tubes and fungal hyphae evidenced that tip growth is not a steady process, but that the growth rates are subject to constant fluctuations. Two types of fluctuations can be distinguished (Geitmann et al., 1996). Type I can be described as sinusoidal oscillations of the growth rate with periods in the range of seconds and amplitudes not higher than two to four times the basic growth rate. This mode of growth was observed in many fungal hyphae (Kaminskyj et al., 1992; L6pez-Franco et al., 1994; Bracker et al., 1995) and the pollen tubes of Aloe zebrina (Tang et al., 1992) and Lilium longiflorum (Pierson et al., 1996). In con-
* Correspondence. RSVS, Pavilion Marchand, Universite Laval, Sainte-Foy, Quebec GIK7P4, Canada. J Plant PhysioL W,L 152. pp. 439-447 (1998)
trast, Type II shows spike-shaped oscillations and was described as pulsatory growth: This mode of growth was observed in pollen tubes of Pitur!a hybrida and Nicotiana tabacum (Pierson et al., 1995;' Gdtmann et al., 1996) and Gasteria verrucosa (Plyushch ecal., 1995), where' periods of slow growth lasting several minutes are interrupted by pulselike elongations lasting for ab6,u'r 10 to 20 s and involving an increase of growth rate up to 50 fold: Type I oscillations. in fungal hyphae were proposed to be caused by fluctuations in the liberation of secretory vesicles (L6pez-Franco et al., 1994; Bracker et al., 1995) or to be controlled by the cytoskeleton accompanied by changes in turgor fressure (Kaminsky; et al., 1992). In Lilium pollen tubes caZ measurements indicated that fluctuations in Ca2 + influx at the apical tip might be involved in the alterations of growth rate causing type I oscillations (Pierson et al., 1996). Whether or not similar models can explain the more extreme pulse-like elongations in Nicotiana tabacum and Petunia hybrida has so far only been subject to speculation (Geitmann et al., 1996; Derksen, 1996).
440
ANJA GEITMANN and MAURO CREST!
An essential aspect in polar growth is represented by ion dynamics. The spatial cytoplasmic differentiation aIong the 10ngitudinaI axis of polarly growing cells, like pollen tubes or fungal hyphae, is believed to be regulated in part by cytosolic ion gradients, particularly Ca2 + and/or H+ gradients Gaffe et aI., 1974; Reiss and Herth, 1978; Herth et aI., 1990; Pierson and Cresti, 1992; Miller et aI., 1992; Jackson and Heath, 1993; Pierson et aI., 1994; Hepler et aI., 1994; Feij6 et aI., 1995; Derksen et aI., 1995). The formation of these gradients is possible because of low rates of Ca2+ diffusion in the cytosol (Speksnijder et aI.,1989), the fragmented nature of the plant cytoplasm, the nonhomogeneous distribution of Ca2+ stores and Ca2 + channels (Reiss and Traxel, 1987; Bednarska, 1989; Feij6 et aI., 1995; Pierson et aI., 1996), and Ca2+ flux throughout the cell (Kilfureiber and Jaffe, 1990). In pollen tubes hiff concentrations of membrane-bound and cytosolic free Ca were shown in the apical part of the cytoplasm of growing pollen tubes Gaffe et aI., 1974; Reiss et aI., 1985 a, 1965 b; Rathore et aI., 1991; Obermeyer and Weisenseel, 1991; Miller et aI., 1992; Pierson et aI., 1994). The presence of this tip-focused Ca2+ gradient is associated with growing cells; its dissipation caused pollen tubes to arrest growth (Miller et aI., 1992; Pierson et aI., 1994; Franklin-Tong et aI., 1996). It is believed that the Ca2+ gradient is generated by Ca2+ ion influx at the,: apical tip of the polarly growing cells. It was f9rthet demoll6tra~ed that high local cytosolic (Weisenseel ef aI." 1975; MaIM et aI." 1994; Pierson et aI., 1996) or membraqe-bound (Reiss et aI., 1985 b; TIrlapur and Cresti, 1992) Ca2 + concentrations precede and localize the germination of pollen grains, and are involved in reorientation of pollen tubes. This suggs:sts that the presence of high Ca2 + concentrations denotes the site of growth or development in these cells. It was therefore temptiag to assume fluctuations of intracellular Ca2 +, conCentrations to be involved in the growth rate oscillations occurring in pulsating pollen tubes. However, pollen tubes of the species exhibiting this phenomenon were extremely sensitive to dye injection, which made Ca2 + imaging in these cells impossible for the time being (Li, personaI communication). The present paper therefore describes a different approach to the question whether or not Ca2 + fluxes playa role in pulsating growth. Ca2 + influx occurs mainly via membragDAS ci+ channels, which in pollen tubes are predominantly tip-located (Reiss and Traxel, 1987; Bednarska, 1989; Obermeyer ,and Weisenseel, 1991; MaIM et aI., 1994; Feij6 et aL~ .~995). Their activity controls the Ca2+ influx, which in turn,&eems to ,be positively correlated to the pollen tube growth ra.te (Pierson et aI., 1994; Feij6 et aI., 1994). In the presertt W:Qrk the role of Ca2+ channels in pulsating growth was -investigated with the help of drugs which inhibit their funetion. ",' ['J .,' I
Materials 8nd
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LaCI3, GdCI3, verapamir -~d nifedipine were obrained from Sigma. Stock solutions were prepared in distilled water and stored at 4·C. The stock solution pf nifedipine,was prepared in ethanol and stored at -20·C in the dark. WQ~king·solutions were prepared by
dilution with culture medium (the working solution of nifedipine was kept in the dark until use).
Video observation ofin vitro growing pollen tubts P. hybrida pollen was dried and stored at - 20·C until use. For in vitro culture pollen was thawed and rehydrated in humid atmos-
phere at room temperature for 90 min. Hot agarose (grade VII, Sigma; 1.5 % in Brewbaker and Kwack (1963) medium with 120/0 sucrose) was spread on a microscope slide, and mer solidification hydrated pollen was brushed on and covered with a drop of medium. Pollen tubes were observed in an inverted microscope (Nikon) equipped with video camera and monitor. The image of the pollen tube tip was traced at intervals of 60, 30 or 15 s on transparent sheets attached to the surface of the monitor. The increase in length over each time interval was measured with a calibrated ruler (Pierson et al., 1995). The image was adjusted for movements of the pollen and pollen tube before each measurement. Registrations of tubes exhibiting typical behavior were chosen for the figures showing pollen tube elongation and growth rate plotted vtrSus time. For each of the different experimental approaches at least ten pollen tubes were monitored. Pollen tubes showing clear and regular pulsing patterns were chosen for the experiments. The growth rate was observed for the duration of about four pulse periods in order to determine pulse frequency and growth rates. Drops of medium conraining the respective inhibitor were added and the behavior of the pollen tube was observed for about another 20 min. It has to be considered that the concentration of the drug is diluted by the medium already present and by the agarose layer. In order to test reversibility of the drug, the overlying medium was sucked off several times and replaced by fresh medium.
Results
Inorganic Gl+ channel blockers In order to inhibit Ca2+ channel activity at the plasma membrane La3 + and Gd3 + ions were added in form of solutions of LaCl3 and GdCI3, respectively, to in vitro growing pollen tubes. Growth was inhibited by 100 IlmollL LaCl3 in the working solution. Similar concentrations were needed for growth inhibition in the case of GdCI3• It has to be considered that the finaI solution which is effective on the cells is diluted approximately between 1: 2 to 1: 5 by the agarose layer. Concentrations around 10 to 50 JlmollL of either ion in the working solution affected growth, usually without arresting it. The pollen tubes abandoned pulsating growth upon the addition of the ions, and continued to grow at rather constant rates, which were usuaIly higher than the respective growth rates between two pulses before addition of the ions. This effect was reversible, removaI of the ions by washing with originaI medium allowed the tubes to resume ~ulsating growth within minutes (Figs. 1, 2). In the case of La +,. pulse frequency after washing was usually similar to the one before addition of the ion (Fig. 1), whereas after treatment with Gd3+ the frequency was lower than before (Fig. 2). '
Organic ctl+ channel blockers The organic Ca2 + channel inhibitors verapamil and nifedipine were tested for their effects on pulsating pollen tubes growing in vitro. In the case of verapamil the behavior of the pollen tubes was rather heterogeneous. Some tubes arrested
441
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Figs. 1-7: Typical examples of time course of pollen tube growth of Paunia hybrida. Pollen tube length (0) was measured every 30 or 60s during slow growth and every 15s during pulses. Growth rate (.) was determined from these data. Fig. I: Example for treatment with LaH . Addition of 10 /lmollL LaCl3 impaired the growth rhythm considerably. The effect was reversible, tubes resumed pulsating growth 8 to 12 min after washing with fresh medium.
Fag. 2: Treatment with GdH . Addition of 10 /lmollL GdCl3 caused pollen tubes to abandon pulsating growth. The effect was reversible after thorough washing, even though the previous pulse frequency was not achieved.
growth at concentrations as low as 250 ~oUL, whereas others continued growing even after addition of 2 mmollL verapamil. In 70 % of the tubes a decrease in tube diameter was observed and in 60 % the growth direction changed upon addition of the drug. Pulsating growth was affected inasmuch as slow growth rate slowed down and pulse periods became longer (Fig. 3). In most tubes pulsating growth was maintained, however, many tubes showed a reduced amplitude of the pulses (Fig. 4). Only two out of 13 tubes abandoned the pulses and continued to grow with constant growth rates (Fig. 5). The effect of verapamil was reversible in some cases (Fig. 4), but not in others (Fig. 5). Nifedipine did not inhibit pollen tube growth neither in concentrations usually used on plant material (10 to 100 ~ollL,) nor in extremely high concentrations (up to 1 mmollL). Pulsating growth was not inhibited, the only visi-
ble effect was a slowdown of the initial phase of the pulses in some cases (Fig. 6) or a decrease in slow growth rate in others (Fig. 7). However these results have to be interpreted cautiously, since it cannot completely be excluded, that nifedipine was photodeactivated rather rapidly during the experiments. Even though the solutions were prepared at dim light and kept in the dark until use, they were inevitably exposed to the light 'in the microscope upon addition to the sample. It was impossible to assess, how fast nifedipine would actually have been deactivated by this exposition to light. Discussion
Oscillations of the growth rate with periods in the range of minutes have been monitored in entire plants (Bose, 1927;
442
ANJA GElTMANN
and MAURO CREST! ____________
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F1po3-S: Behavior of pollen tubes treated with verapamil. The drug caused a decrease of slow growth rate and of pulse frequency (Figs. 3, 4). In some cases growth rate during pulses and the amplitude of the expansions were impaired (Fig. 4), which was in some cases reversible. In most cases pulses were not completely inhibited, only two out of 13 tubes showed steady growth rates after addition of the drug (Fig. 5).
Morgan et aI., 1980; Cosgrove, 1981; Kristie and Jolliffe, 1986) as weU as in tip-growing single cells, such as poUen tubes of cenain plant species (Plyushch, 1995; Pierson et aI.,
1995; Geitmann et aI., 1996). To our knowledge the control mechanism for these oscillations has not been satisfactorily clarified. The present work was undertaken based on the ob-
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Fip. 6, 7: Behavior of pollen tubes treated with nifedipine. The drug did not inhibit pulsating growth. The only visible effects were a slowdown in the initial phase of the pulse (Fig. 6) or a decrease in slow growth rate (Fig. 7).
servation that fluctuations in Ca2+ influx seem to be correlated with pollen tube growth rate (Pierson et al., 1996). The authors however did not provide any evidence for their postulation that these Ca2+ fluctuations which they observed in pollen tubes showing small sinusoidal growth oscillations of higher frequency («type h oscillations) are the cause of growth rate fluctuations and not their consequence. Using specific inhibitors for Ca2 + channels it was shown here that the regulated activity of certain Ca2+ channels governing Ca2 + influx through the plasma membrane is indeed likely to represent a key factor in the control of pollen tube growth rate, at least as far as pulsating growth («type II» oscillations) is concerned.
Tht tjfict oftht inhibition ofCtl+ channtls by inorganic inhibitors on pulsating pollm tubt growth Inorganic Ca2+ channel blockers such as La3+ and Gd3 + inhibit a great range of Ca2+ channels by competing with the Ca2+ ions. The concentrations of La3 + with inhibiting effect are in the high micro- to millimolar range (Tester and Mac-
Robbie, 1990; Herth et al., 1990; Ding and Pickard, 1993; Herrmann and Felle, 1995), whereas Gd3+ was found to be active in concentrations as low as 10 ~moUL (Alexandre and Lassalles, 1991). Regarding pollen germination 100 ~oUL to 1 mmollL La3+ were observed to have an inhibiting effect on pollen of Hannanthus a/biflos and Omothn"a bimnis (Bednarska, 1989), whereas already 10~moUL La3 + were found to reduce the high Ca2+ concentration in the tips of lily pollen tubes, which indicates, that Ca2+ channel activity is indeed responsible for the tip focused Ca2+ gradient in growing pollen tubes (Obermeyer and Weisenseel, 1991; Malh6 et al., 1994). The results fresented here are consistent with these reports. La3+ or Gd + concentrations between 10 ~oUL and 100 ~ollL inhibited Pttunia pollen tube elongation. It can therefore be confirmed that the functioning of the Ca2+ channels is essential for pollen tube growth in general. In the case of ~ulsating tubes, intermediate concentrations of La3+ and Gd + which did not cause the total arrest of growth, caused the tubes to abandon their pulsating rh~, thus clearly indicating the important function of the Ca2+ channels in the control of the pulses.
444
ANJA GEITMANN and MAURO CRESTI
As always when using inhibitors, it has to be considered, however, that their specificity is not absolute. For Gd3 + it was shown by Jackson and Heath (1993), that in fungal hyphae this ion blocks stretch-activated Ca2 + channels, but not K+ channels. Channel inhibition in patch clamp experiments on these cells occurred within minutes or seconds, depending on the concentration of Gd3 + (Garrill et al., 1993). On rl),e other hand it cannot be ignored, however, that La3 + apart from inhibiting Ca2+ channels has been shown to block both inward-rectifying (Tester and MacRobbie, 1990) and outwardrectifying (Ketchum and Poole, 1991) K+ channels. The results obtained in the present work have to be interpreted considering this restriction in specificity.
Polevoy and Stahlberg, 1978; Goring et al., 1979; Fisahn et al .• 1986; Felle et al., 1986; Felle, 1988; Souda et al .• 1990; Toko et al .• 1986, 1990; McAinsh et al .• 1995; Ehrhardt et al .• 1996). For oscillations of the cytosolic Ca2+ concentration many different models for negative or positive feedback mechanisms have been proposed, many of which involve either stable or oscillating levels of inositol-l,4,5-trisphosphate as a signal for Ca2+ release from internal stores (Tsien and Tsien, 1990; Fewtrell, 1993; Berridge, 1990; 1993 and references therein). These models were established for animal cells. but it was shown, that an inositol-l.4.5-trisphosphatemediated signal pathway exists in pollen tubes (FranklinTong et al .• 1996) which provides further support for the proposed feedback mechanism. In general, elevated cytosolic free Ca2+ concentrations are Inhibition ofCtl+ channels with organic inhibitors associated with stimulated exocytosis in various animal (PlattThe organic channel blockers nifedipine and verapamil ner, 1989; Wollheim and Lang. 1994) and plant cell types showed an effect on the overall pollen tube growth rate and/- (Blackbourn et al., 1991; Zorec and Tester, 1992) which or on pulse amplitude, but in general did not inhibit pulsa- opens intriguing possibilities. Interpreting the pollen tube as tory growth. The reduction of the slow growth rate and the a cell with repeated triggered exocytosis, one might regard the arrest of growth upon application of higher concentrations presumable increase in cytosolic Ca2+ as a signal for the rapid evidences that verapamil- and perhaps nifedipine-sensitive release of secretory vesicles. This hypothesis is surr.rted by channels are present in the plasma membrane of pollen tubes Picton and Steer (1982; 1985) who stated that Ca is necesof Petunia hybrida, as already shown for pollen tubes of other sary for vesicle fusion in pollen tube elongation. It is very plant species (Reiss and Herth, 1985; Bednarska, 1989; Feij6 tempting to assume, that the Ca2+ level regulates the rate of et al., 1995) and for the tips of fungal hyphae (Dicker and vesicle fusion at the tip of the pollen tube, possibly involving Turian, 1990; Robson et al., 1991). However, because of the the activity of a functioning cytoskeleton (Geitmann et al .• lack of an effect as dramatic as with inorganic inhibitors, in 1996), of annexins (Battey and Blackbourn, 1993) or of other the case of pulsating pollen tubes one might presume that Ca2+ -dependent proteins. Related to this issue is the question how the Ca2+ channels these Channels only provide the constant basic level of Ca2+ influx at the pollen tube apex. This influx presumably main- located in the apical pollen tube tip are gated. In the case of tains the internal Ca2 + gradient along the longitudinal axis of pulsating pollen tubes it seems likely, that during a complete the cell wich is necessary for general pollen tube elongation. cycle of slow growth and pulse, the membrane tension at the One might assume that these channels are necessary for tip apical tip undergoes changes. Even if most of the internal growth in pollen tubes, but that they probably open and pressure is probably borne by the cell wall (Cosgrove and close independently of the pulse rhythm.. . Hedrich, 1991; Ramahaleo et al., 1996). changes in either cell Again, the results obtained with nifedipine and verapamil wall resistance or internal turgor should cause alterations in have to be interpreted with caution, since they also seem to tension in the plasma membrane. It is therefore tempting to have an effect on K+ -selective channels (Terry et al., 1992). assume, that the Ca2+ channels responsible for the control of Furthermore it cannot be exguded that nifedipine was pho- pulsating growth in pollen tubes are gated by mechanical todeactivated during the experiments, especially since con- stress the gating parameter being either the elastic tension in centrations as low as 10~, which were reported to have an the membrane (Ramahaleo et al., 1996) or the submembrane effect (swelling of the tip, formation of protuberances) on lily cytoskeleton (Sokabe et al., 1991). It is reasonable to assume pollen tubes (Reiss and Herth, 1985), seemingly had no dra- the presence of stretch-sensitive ci+ in the apical ends of matic effect in our experiments, neither did higher concen- pollen tubes since their activity has been demonstrated for trations. other tip growing cells, i.e. ~ngal hyphae (Zhou et al.) 1991; Garrill et al., 1992, 1993; Levina et al., 1995). Furthermore the involvement of stretch-activated ion channels in rhythmic The significance ofthe ctl+ influx in pulsating pollen tubes plant movements has p~eviously been claimed by Schwenke The results d~cribed above tempt to establish the hypo- and Wagner (1992), who observed that Gd3+ inhibits xylem thesis that the pQlse-like elongations in pollen tubes are regu- exudation. which occurs with pulses in the period range of . lated by transient increases of ~2+ . influx into the apical pol- 8 min. len tube tip, which w~uld be likely to have signal function. This suggestion is reasona~le since it has been .repqrted reHypothetical model for the mechanism governing pulsating peatedly that not only in animal but also in plant cells Ca2+ growth influx can undergo periodic fluc~tions causing alteratiop,s in the membrane potential either in. sinusoidal or in spikeThe inhibition of the pulsating rhyth}n in pollen tubes of like form. with period$ in the range of seconds or minutes Petunia by inorganic inhibitors of Ca2+ channels suggests (Scott. 1957; Jenkinson and Scott, 1961; Jenkinson, 1962; that an oscillating cytosolic Ca2+ level .caused by fluctuations Nishizaki. 1968; Lefebvre et al.• 1970; Pickard, 1972. 1973; in the Ca2+ influx through the plasma membrane might be
Ca2 + channels in pulsating pollen tubes
the basic mechanism governing the periodic alterations in growth rate. One can only speculate about a putative feedback mechanism which regulates the Ca2 + influx, but it seems plausible that a temporarily elevated level of cytosolic Ca2+ has a signal function. The signal might induce a sudden liberation of secretory vesicles resulting in a local cell wall relaxation, which in turn might allow a sudden turgor-driven expansion of the cell wall at the apical tip of the pollen tube. A pulse would thus consist of two consecutive steps - the sudden liberation of vesicles followed by the expansion of the newly released cell wall material. The distinction in two phases is favored by the observation of Plyushch et al. (1995) who describe the occurrence of a wall thickening at the pollen tube tip (which might correspond to the vesicle release proposed here) prior to each pulse-like expansion in Gasteria pollen tubes. Whether or not this two-step-model describes the real situation remains to be investigated. In any case however, pulsating pollen tubes seem to provide a suitable model for the investigation of the intracellular signaling pathway based on Ca2 + influx in growing plant cells. References ALExANDRE, J. and J. WSALLES: Hydrostatic and osmotic pressure activated channel in plant vacuole. Biophys. J. 60, 1326-1336 (1991). ' BATIEY, N. H. and H. D. BLACKBouRN: The control of exocytosis in plant cells. New Phytol. 125,307-338 (1993). BEDNARSKA, E.: The effect of exogenous Ca2 + ions on pollen grain germination and pollen tube growth. Sex. Plant Reprod. 2, 5358 (1989). BERRIDGE, M. J.: Calcium oscillations. J. BioI. Chern. 265, 95839586 (1990). - Inositol trisphosphate and calcium signalling. Nature 361, 315325 (1993) . BLACKBOURN, H. D., J. H. WALKER, and N. H . BAlTEY: Calciumdependent phospholipid-binding proteins in plants. Planta 184, 67-73 (1991) . BOSE, J. Plant autographs and their revelations. Longmans, Green, and Co., London (1927). BRACKER, C. E., R. L6PEz-FRANCO, S. BARTNICKI-GARCIA, D. M. MuRPHY, and R. J. HOWARD: Satellite Spitzenkorper, pulsed growth, and the determination of cell shape in growing hyphal tips of fungi. Proc. Royal Micr. Soc. 30, 17 (1995). BREWBAKER, J. L. and B. H. KWACK: The essential role of calcium ion in pollen germination and pollen tube growth. Amer. J. Bot. 50, 859-865 (1963). CoSGROVE, D . J.: Rapid suppression of growth by blue light. Plant Physiology 67, 584-590 (1981). COSGROVE, D. J. and R. HEDRICH: Stretch-activated chloride, potassium, and calcium channels coexisting in plasma membranes of guard cells of Vida foba L. Planta 186, 143-153 (1991). DERKSEN, J. : Pollen tubes: a model system for plant cell growth. Bot. Acta 109, 341-345 (1996). DERKSEN, J., T. RUTIEN, I. K LICHTSCHEIDL, A. H . N. DE WIN, E. S. PIERSON, and G. RONGEN: Quantitative analysis of the distribution of organelles in tobacco pollen tubes: implications for exocytosis and endocytosis. Protoplasma 188,267-276 (1995). DICKER, J. W, ' and G. TURIAN: Calcium deficiencies and apical hyperbranching in wild-type and the .frost> and .spray» morphological mutants of Neurospora crassa. J. Gen. Microbiol. 136, 1413-1420 (1990). '
c.:
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DING, J. P. and B. G. PICKARD: Mechanosensory calcium-selective cation channels in epidermal cells. PlantJ. 3, 83-110 (1993). EHRHARDT, D. w., R. WAIS, and S. R. loNG: Calcium spiking in plant root hairs responding to Rhizobium nodulation signals. Cell 85,673-681 (1996). FEIJ6, J. A., R. MALH6, and G. OBERMEYER: Ion dynamics and its possible role during in vitro pollen germination and tube growth. Protoplasma 187, 155-167 (1995). FEIJ6, J. A., A. SHIPLEY, and L. JAFFE: Spatial and temporal patterns of electric and ionic currents around in vitro germinating pollen of Lilium longiflorum: a vibrating probe study. In: HEBERLEBORS, E., M. HESSE, and O. VINCENTE (eds.): Frontiers in Sexual Plant Reproduction Research. 13th International Congress on Sexual Plant Reproduction, University of Vienna, p. 40 (1994). FELLE, H.: Auxin causes oscillations of cytosolic free calcium and pH in Zea mayscoleoptiles. Planta 174,495-499 (1988). FELLE, H., B. BRUMMER, A. BERTL, and R. PARISH: Indole-3-acetic acid and fusicoccin cause cytosolic acidification of corn coleoptile cells. Proc. Nat!. Acad. Sci. USA 83, 8992-8995 (1986). FEWTRELL, Ca2 + oscillations in non-excitable cells. Annu. Rev. Physiol. 55,427-454 (1993). FISAHN, J., E. MIKSCHL, and U. HANSEN: Separate oscillations of the electrogenic pump and of a K+ -channel in Nitella as revealed by simultaneous measurement of membrane potential and of resistance. J. Exp. Bot. 37, 34-47 (1986). FRANKLIN-TONG, E., B. K DROEBAK, A. C. ALLAN, P. A. C. WATKINS, and A.. J. TREWAVAS: Growth of pollen tubes of Papaver rhoeas is regulated by a slow-moving calcium ware propagated by inositoll,4,5-trisphosphate. Plant Cell 8, 1305-1321 (1996). GARRILL, A., S. L. JACKSON, R. R. LEw, and I. B. HEATH: Ion channel activity and tip growth: tip-localized stretch-activated channels generate an essential Ca2 + gradient in the oomycete Saprokgnia flrax. Europ. J. Cell BioI. 60,358-365 (1993). GARRILL, A., R. R. LEW, and I. B. HEATH: Stretch-activated Ca2 + and Ca2+ -activated K+ channels in the hyphal tip plasma membrane of the oomycete Saprokgnia flrax. J. Cell Sci. 101,721-730 (1992) . GEITMANN, A., Y. Q. LI, and M. CRESTI: The role of the cytoskeleton and dictyosome activity in the pulsatory growth of Nicotiana tabacum and Petunia hybrida. Bot. Acta 109, 102-109 (1996) . GORING, H., V. V. POLEVOY, R. STAHLBERG, and G. STUMPF: Depolarization of transmembrane potential of corn and wheat colooptiles under reduced water potential and afrer IAA application. Plant Cell Physiol. 20. 649-656 (1979) . HEPLER, P. K, D. D . MILLER, E. S. PIERSON, and D . A. CALLAHAM: Calcium and pollen tube growth. In: STEPHENSON, A. G. and Y.H. KAo (eds.): Pollen-pistil interactions and pollen tube growth, Amer. Soc. Plant Physiol., Rockville, Maryland, USA, pp. 111123 (1994). HERRMANN, A. and H. H. FELLE: Tip growth in root hair cells of Sinapis alba L.: significance of internal and external Ca2+ and pH. New Phytol. 129,532-533 (1995). HERTH, w., H. D. REISS, and E. HARTMANN: Role of calcium ions in tip growth of pollen tubes and moss protonema cells. In: HEATH, I. B. (ed.): Tip growth in plant and fungal cells. Academic Press, San Diego, pp. 91-118 (1990). JACKSON, S. L. and I. B. HEATH: Roles of calcium ions in hyphal tip growth. Microbiol. Rev. 57, 367-382 (1993). JAFFE, L. E, K R. ROBINSON, and R. NUCITELLI: Local cation entry and self-electrophoresis as an intracellular localization mechanism. Annu. NY Acad. Sci. 238, 372-389 (1974). JENKINSON, I. S.: Bioelectric oscillations of bean roots: further evidence for a feedback oscillator II. Intracellular plant root potentials. Austral. J. BioI. Sci. 15, 101-114 (1962).
c.:
v.
446
ANJA GBlTMANN and MAURO CRESTI
JBNKINSON, I. S. and B. I. H. SCOTr: Bioelectric oscillations of bean roots: further evidence for a feedback oscillator I. Extracellular response to oscillations in osmotic pressure and auxin. Austral. J. BioI. Sci. 14, 231-247 (1961). KAMINSKY], S. G. w., A. GARRILL, and I. B. HEATH: The relation between turgor and tip growth in Saprolegnia frrax: Turgor is necessary, but not sufficient to explain apical extension rates. Exp. Mycol. 16, 64-75 (1992). KBTCHUM, K A. and R. J. POOLE: Cytosolic calcium regulates a potassium current in corn (ha mays) protoplasts. J. Membrane BioI. 119,277-288 (1991). KIuSTIB, D. N. and P. A. JOLLIFFB: High-resolution studies of growth oscillations during stem elongation. Can. J. Bot. 64, 2399-2405 (1986). KOHTRBIBBR, w. M. and L. F. JAFFB: Detection of extracellular calcium gradients with a calcium-specific vibrating electrode. J. Cell BioI. 110, 1565-1573 (1990). LEFBBVRE, J., R. LBFEVER, and C. GILLET: Oscillations auto-entretenues des potentiels de membrane de Nitella. Bull. Soc. R. Bot. Belg. 103, 157-165 (1970). LEvINA, N. N., R. R. LEw, G. J. HYDB, I. B. HEATH: The roles of Ca2+ and plasma membrane ion channels in hyphal tip growth of Neurospora crassa. J. Cell Sci. 106, 3405-3417 (1995). L6PBz-FRANco, R., S. BARTNICKI-GARCIA, and C. E. BRACKER: Pulsed growth of fungal hyphal tips. Proc. Natl. Acad. Sci. USA 91, 12228-12232 (1994). MALH6, R., N. D. READ, M. SALOME PAIS, and A. J. TREWAVAS: Role of cytosolic free calcium in the reorientation of pollen tube growth. PlantJ. 5, 331-341 (1994). McAINSH, M. R., A. A. R. WEBB, J. E. TAYWR, and A. M. HBTHERINGTON: Stimulus-induced oscillations in guard cell cytosolic free calcium. Plant Cell 7, 1207-1219 (1995). MIl.LEa, D. D., D. A. CAu..uIAM, D. J. GROSS, and P. K HEPLER: Free Ca2+ gradient in growing pollen tubes of Li/ium. J. Cell Sci. 101, 7-12 (1992). MORGAN, D. c., T. O'BRIAN, and H. SMITH: Rapid photomodulation of stem extension in light-grown Sinapis alba L. Planta 150, 95-101 (1980). NISHIZAKI, Y.: Rhythmic changes in the resting potential of a single cell. Plant Cell Physiol. 9, 613-616 (1968). OBERMEYER, G. and M. H. W'BISENSBEL: Calcium channel blocker and calmodulin antagonists affect the gradient of free calcium ions in lily pollen tubes. Europ. J. Cell BioI. 56, 319-327 (1991). PICKARD, B. G.: Spontaneous electrical activity in shoots of Ipomoea, Pisum, and Xanthium. Planta 102,91-14 (1972). - Action potentials in higher plants. Bot. Rev. 39, 172-201 (1973). PICTON, J. M. and M. W. STEER: A model for the mechanism of tip extension in pollen tubes. J. theor. BioI. 98, 15-20 (1982). - - The effects of ruthenium red, lanthanum, fluorescein isothiocyanate and trifluoperazine on vesicle transport, vesicle fusion and tip extension in pollen tubes. Planta 163,20-26 (1985). PIBRSON, E. S. and M. CRBSTI: Cytoskeleton and cytoplasm'ic organization of pollen and pollen tubes. Int. Rev. Cytol. 140, 73125 (1992). PIBRSON, E. S., Y. Q. U, H. Q. ZHANG, M. T. M. WILLBMSB, H. F. UNSKBNS, and M. CRBSTI: Pulsatory growth of pollen tubes: investigation of a possible relationship with the periodic distribution of cell wall components. Acta Bot. Neerl. 44, 121-128 (1995). PIERSON, E. S., D. D. MILLER, D. A. CAu..uIAM, A. M. SHIPLEY, B. A. RIvERS, M. CRBSTI, and P. K. HEPLER: Pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient: effect of BAPTA-type buffers and hypertonic media. Plant Cell 6, 1815-1828 (1994). PIBRSON, E. S., D. D. MILLER, D. A. CAu..uIAM, J. VAN AKBN, G. HACKETr, and P. K HEPLER: Tip-localized calcium entry fluc-
tuates during pollen tube growth. Develop. BioI. 174, 160-173 (1996). PLATrNBR, H.: Regulation of membrane fusion during exocytosis. Int. Rev. Cytol. 119, 197-286 (1989). PLYUSHCH, T. A., M. T. M. WILLEMSE, M. A. W. FRANSSBN-VERHEIJBN, and M. C. REINDBRS: Structural aspects of in vitro pollen tube growth and micropylar penetration in Gasteria verrucosa (Mill.) H. Duval and Lilium longiflorum Thunb. Protoplasma 187, 13-21 (1995). POLEVOY, v. V. and R. STAHLBBRG: Auxin-induzierte langsarne Schwingungen des Membranpotentials bei Maiskoleoptilen. BioI. Rundsch. 16, 38-40 (1978). RAMAHALBo, T., J. ALExANDRE, and J. LAsSALLBS: Stretch activated channels in plant cells. A new model for osmoelastic coupling. Plant Physiol. Biochem. 34, 327-334 (1996). RATHORE, K S., R. J. CORK, and K. R. ROBINSON: A cytoplasmic gradient of Ca2 + is correlated with the growth of lily pollen tubes. Developm. BioI. 148, 612-619 (1991). REISS, H. and W. HBRTH: Visualization of the Ca2+ -gradient in growing pollen tubes of Lilium longiflorum with chlorotetracycline fluorescence. Protoplasma 97, 373-377 (1978). - - Nifedipine-sensitive calcium channels are involved in polar growth oflily pollen tubes. J. Cell Sci. 76, 247-254 (1985). REISS, H. and K TRAXBL: Hint of polar distribution in calcium channels under PIXE analysis. BioI. Trace Element Res. 13, 135142 (1987). REISS, H., G. W. GRIMB, M. Q. u, J. TAKACS, and F. WATr: Distribution of elements in the lily pollen tube tip, determined with the Oxford scanning proton microprobe. Protoplasma 126, 147152 (1985 a). REISS, H., W. HBRTH, and R. NOBILING: Development of membrane- and calcium-gradients during pollen germination of Lilium longiflorum. Planta 163, 84-90 (1985 b). ROBSON, G. D., M. G. WIEBB, and A. P. J. TRINCI: Involvement of Ca2+ in the regulation of hyphal extension and branching in FusariumgraminearumA3/5. Exp. Mycol. 15,263-272 (1991). SCHWENKE, H. and E. WAGNBR: A new concept of root exudation. Plant Cell Environ. 15,289-299 (1992). SCOTr, B. I. H.: Electric oscillations generated by plant roots and a possible feedback mechanism responsible for them. Austral. J. BioI. Sci. 10, 164-179 (1957). SOKABB, M., F. SACHS, and Z. JING: Quantitative video microsCopy of patch clamped membranes: stress, strain, capacitance, and stretch channel activation. Biophys. J. 59,722-728 (1991). SOUDA, M., K TOKO, K. HAYASHI, T. FUJIYOSHI, S. EZAKI, and K. YAMAFuJI: Relationship between growth and electric oscillations in bean roots. Plant Physiol. 93, 532-536 (1990). SPBKSNIJDER, J. E., A. L. MILLER, M. H. WBISENSBBL, T. CHBN, and L. E. JAFFB: Calcium buffer injections block fucoid egg development by facilitating calcium diffusion. Proc. Natl. Acad. Sci. USA 86, 6607-6611 (1989). TANG, x. w., G. Q. LIU, Y. YANG, W. L. ZHBNG, B. C. Wu, and D. T. NIB: Quantitative measurement of pollen tube growth and particle movement. Acta Bot. Sinica 34, 893-898 (1992). TBRRY, B. R., G. P. FINDLAY, and S. D. TYERMAN: Direct effects of Ca2 + -channel blockers on plasma membrane cation channels of Amaranthus tricolor protoplasts. J. Exp. Bot. 43, 14757-11473 (1992). TESTER, M. and E. A. C. MAcRoBBIB: Cytosplasmic calcium affects the gating of potassium channels in the plasma membrane of Chara corallina: a whole-cell study using calcium-channel effectors. Planta 180, 569-581 (1990). TIRLAPUR, U. and M. CRBSTI: Computer-assisted video image analysis of spatial variations in membrane-associated Ca2 + and calmo-
Ca2 + channels in pulsating pollen tubes dulin during pollen hydration, germination and tip growth in Nicotiana tabacum L. Ann. Bot. 69,503-508 (1992). TOKO, K., K. HAYASHI, and K. "YAMAFUJI: Spatio-temporal organization of electricity in biological growth. Trans. IECE of Japan 69, 485-487 (1986). TOKO, K., M. SOUDA, T. MATSUNO, and K. "YAMAFUJI: Oscillations of electrical potential along a root of a higher plant. Biophys. J. 57, 269-272 (1990). TSIEN, R. W. and R. Y. TSIEN: Calcium channels, stores, and oscillations. Annu. Rev. Cell BioI. 6, 715-760 (1990).
447
WEISENSEEL, M. H., R. NUCCITELLI, and L. F. JAFFE: Large electrical currents traverse growing pollen tubes. J. Cell BioI. 66, 556-567 (1975). WOLLHEIM, c. B. and J. lANG: A game plan for exocytosis. Trends Cell BioI. 4,339-341 (1994). ZHOU, X., M. A. STUMPF, H. C. HocH, and C. KUNG: A mechanosensitive channel in whole cells and in membrane patches of the fungus Uromycts. Science 253, 1415-1417 (1991). ZOREC, R. and M. TESTER: Cytoplasmic calcium stimulates exocytosis in a plant secretory cell. Biophys. J. 63,864-867 (1992).
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© 1998 by Gustav Fischer Verlag, Jena
- Ca2 + Channels Control the Rapid Expansions it; Pulsating Growth of Petunia hybrida Pollen Tubes ANJA GEITMANN*
and MAURO
CRESTI
Department of Environmental Biology, University of Siena, Via P.A. Mattioli 4, 1-53100 Siena, Italy Received March 3, 1997 . Accepted July 7, 1997
Summary Pollen tubes exhibit a tip-focused gradient of the cytosolic Ca2 + concentration, which is .rresumably governed by the activity of Ca2 + channels in the plasma membrane. The putative role of Ca4 as a signalling factor in pulsating growth of Petunia ,?brida pollen tubes was examined. The application of inorganic Ca2 + channel inhibitors La3 + and Gd3 caused the pulsating pollen tubes to abandon their rhythm and continue growth with rather steady rates. This effect was reversible upon removal of these ions. The organic inhibitors of ci+ channels nifedipine and verapamil caused the arrest of overalt pollen tube growth at higher concentrations, whereas intermediate concentrations caused a slowdown of pulse frequency, but not the inhibition of pulses. This leads to the hypothesis, that the activity of a certain group of Ca2+ channels, which is sensitive to La3+ and Gd3 + would be responsible for the control of pulsating growth. Another group of Ca2 + channels, which is sensitive to organic inhibitors, would be required for pollen tube growth in general and provide the basic Ca2 + influx, but would not be directly involved in the growth oscillations. Based on these findings a hypothetical model for the mechanism that cO,ntrols pulsating . : growth in pollen tubes is proposed.
Key words: Petunia hybrida, calcium channel gadolinium, lanthanum, nifedipine,: pollen tube, pulsating growth, verapamiL " Introduction
Pollen tubes are tip-growing cells, their elongation occurs in one direction and the growth process is basically confined to the apical tip of the cylindrical cell. Video observation of in vitro growing pollen tubes and fungal hyphae evidenced that tip growth is not a steady process, but that the growth rates are subject to constant fluctuations. Two types of fluctuations can be distinguished (Geitmann et al., 1996). Type I can be described as sinusoidal oscillations of the growth rate with periods in the range of seconds and amplitudes not higher than two to four times the basic growth rate. This mode of growth was observed in many fungal hyphae (Kaminskyj et al., 1992; L6pez-Franco et al., 1994; Bracker et al., 1995) and the pollen tubes of Aloe zebrina (Tang et al., 1992) and Lilium longiflorum (Pierson et al., 1996). In con-
* Correspondence. RSVS, Pavilion Marchand, Universite Laval, Sainte-Foy, Quebec GIK7P4, Canada. J Plant PhysioL W,L 152. pp. 439-447 (1998)
trast, Type II shows spike-shaped oscillations and was described as pulsatory growth: This mode of growth was observed in pollen tubes of Pitur!a hybrida and Nicotiana tabacum (Pierson et al., 1995;' Gdtmann et al., 1996) and Gasteria verrucosa (Plyushch ecal., 1995), where' periods of slow growth lasting several minutes are interrupted by pulselike elongations lasting for ab6,u'r 10 to 20 s and involving an increase of growth rate up to 50 fold: Type I oscillations. in fungal hyphae were proposed to be caused by fluctuations in the liberation of secretory vesicles (L6pez-Franco et al., 1994; Bracker et al., 1995) or to be controlled by the cytoskeleton accompanied by changes in turgor fressure (Kaminsky; et al., 1992). In Lilium pollen tubes caZ measurements indicated that fluctuations in Ca2 + influx at the apical tip might be involved in the alterations of growth rate causing type I oscillations (Pierson et al., 1996). Whether or not similar models can explain the more extreme pulse-like elongations in Nicotiana tabacum and Petunia hybrida has so far only been subject to speculation (Geitmann et al., 1996; Derksen, 1996).
440
ANJA GEITMANN and MAURO CREST!
An essential aspect in polar growth is represented by ion dynamics. The spatial cytoplasmic differentiation aIong the 10ngitudinaI axis of polarly growing cells, like pollen tubes or fungal hyphae, is believed to be regulated in part by cytosolic ion gradients, particularly Ca2 + and/or H+ gradients Gaffe et aI., 1974; Reiss and Herth, 1978; Herth et aI., 1990; Pierson and Cresti, 1992; Miller et aI., 1992; Jackson and Heath, 1993; Pierson et aI., 1994; Hepler et aI., 1994; Feij6 et aI., 1995; Derksen et aI., 1995). The formation of these gradients is possible because of low rates of Ca2+ diffusion in the cytosol (Speksnijder et aI.,1989), the fragmented nature of the plant cytoplasm, the nonhomogeneous distribution of Ca2+ stores and Ca2 + channels (Reiss and Traxel, 1987; Bednarska, 1989; Feij6 et aI., 1995; Pierson et aI., 1996), and Ca2+ flux throughout the cell (Kilfureiber and Jaffe, 1990). In pollen tubes hiff concentrations of membrane-bound and cytosolic free Ca were shown in the apical part of the cytoplasm of growing pollen tubes Gaffe et aI., 1974; Reiss et aI., 1985 a, 1965 b; Rathore et aI., 1991; Obermeyer and Weisenseel, 1991; Miller et aI., 1992; Pierson et aI., 1994). The presence of this tip-focused Ca2+ gradient is associated with growing cells; its dissipation caused pollen tubes to arrest growth (Miller et aI., 1992; Pierson et aI., 1994; Franklin-Tong et aI., 1996). It is believed that the Ca2+ gradient is generated by Ca2+ ion influx at the,: apical tip of the polarly growing cells. It was f9rthet demoll6tra~ed that high local cytosolic (Weisenseel ef aI." 1975; MaIM et aI." 1994; Pierson et aI., 1996) or membraqe-bound (Reiss et aI., 1985 b; TIrlapur and Cresti, 1992) Ca2 + concentrations precede and localize the germination of pollen grains, and are involved in reorientation of pollen tubes. This suggs:sts that the presence of high Ca2 + concentrations denotes the site of growth or development in these cells. It was therefore temptiag to assume fluctuations of intracellular Ca2 +, conCentrations to be involved in the growth rate oscillations occurring in pulsating pollen tubes. However, pollen tubes of the species exhibiting this phenomenon were extremely sensitive to dye injection, which made Ca2 + imaging in these cells impossible for the time being (Li, personaI communication). The present paper therefore describes a different approach to the question whether or not Ca2 + fluxes playa role in pulsating growth. Ca2 + influx occurs mainly via membragDAS ci+ channels, which in pollen tubes are predominantly tip-located (Reiss and Traxel, 1987; Bednarska, 1989; Obermeyer ,and Weisenseel, 1991; MaIM et aI., 1994; Feij6 et aL~ .~995). Their activity controls the Ca2+ influx, which in turn,&eems to ,be positively correlated to the pollen tube growth ra.te (Pierson et aI., 1994; Feij6 et aI., 1994). In the presertt W:Qrk the role of Ca2+ channels in pulsating growth was -investigated with the help of drugs which inhibit their funetion. ",' ['J .,' I
Materials 8nd
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LaCI3, GdCI3, verapamir -~d nifedipine were obrained from Sigma. Stock solutions were prepared in distilled water and stored at 4·C. The stock solution pf nifedipine,was prepared in ethanol and stored at -20·C in the dark. WQ~king·solutions were prepared by
dilution with culture medium (the working solution of nifedipine was kept in the dark until use).
Video observation ofin vitro growing pollen tubts P. hybrida pollen was dried and stored at - 20·C until use. For in vitro culture pollen was thawed and rehydrated in humid atmos-
phere at room temperature for 90 min. Hot agarose (grade VII, Sigma; 1.5 % in Brewbaker and Kwack (1963) medium with 120/0 sucrose) was spread on a microscope slide, and mer solidification hydrated pollen was brushed on and covered with a drop of medium. Pollen tubes were observed in an inverted microscope (Nikon) equipped with video camera and monitor. The image of the pollen tube tip was traced at intervals of 60, 30 or 15 s on transparent sheets attached to the surface of the monitor. The increase in length over each time interval was measured with a calibrated ruler (Pierson et al., 1995). The image was adjusted for movements of the pollen and pollen tube before each measurement. Registrations of tubes exhibiting typical behavior were chosen for the figures showing pollen tube elongation and growth rate plotted vtrSus time. For each of the different experimental approaches at least ten pollen tubes were monitored. Pollen tubes showing clear and regular pulsing patterns were chosen for the experiments. The growth rate was observed for the duration of about four pulse periods in order to determine pulse frequency and growth rates. Drops of medium conraining the respective inhibitor were added and the behavior of the pollen tube was observed for about another 20 min. It has to be considered that the concentration of the drug is diluted by the medium already present and by the agarose layer. In order to test reversibility of the drug, the overlying medium was sucked off several times and replaced by fresh medium.
Results
Inorganic Gl+ channel blockers In order to inhibit Ca2+ channel activity at the plasma membrane La3 + and Gd3 + ions were added in form of solutions of LaCl3 and GdCI3, respectively, to in vitro growing pollen tubes. Growth was inhibited by 100 IlmollL LaCl3 in the working solution. Similar concentrations were needed for growth inhibition in the case of GdCI3• It has to be considered that the finaI solution which is effective on the cells is diluted approximately between 1: 2 to 1: 5 by the agarose layer. Concentrations around 10 to 50 JlmollL of either ion in the working solution affected growth, usually without arresting it. The pollen tubes abandoned pulsating growth upon the addition of the ions, and continued to grow at rather constant rates, which were usuaIly higher than the respective growth rates between two pulses before addition of the ions. This effect was reversible, removaI of the ions by washing with originaI medium allowed the tubes to resume ~ulsating growth within minutes (Figs. 1, 2). In the case of La +,. pulse frequency after washing was usually similar to the one before addition of the ion (Fig. 1), whereas after treatment with Gd3+ the frequency was lower than before (Fig. 2). '
Organic ctl+ channel blockers The organic Ca2 + channel inhibitors verapamil and nifedipine were tested for their effects on pulsating pollen tubes growing in vitro. In the case of verapamil the behavior of the pollen tubes was rather heterogeneous. Some tubes arrested
441
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Figs. 1-7: Typical examples of time course of pollen tube growth of Paunia hybrida. Pollen tube length (0) was measured every 30 or 60s during slow growth and every 15s during pulses. Growth rate (.) was determined from these data. Fig. I: Example for treatment with LaH . Addition of 10 /lmollL LaCl3 impaired the growth rhythm considerably. The effect was reversible, tubes resumed pulsating growth 8 to 12 min after washing with fresh medium.
Fag. 2: Treatment with GdH . Addition of 10 /lmollL GdCl3 caused pollen tubes to abandon pulsating growth. The effect was reversible after thorough washing, even though the previous pulse frequency was not achieved.
growth at concentrations as low as 250 ~oUL, whereas others continued growing even after addition of 2 mmollL verapamil. In 70 % of the tubes a decrease in tube diameter was observed and in 60 % the growth direction changed upon addition of the drug. Pulsating growth was affected inasmuch as slow growth rate slowed down and pulse periods became longer (Fig. 3). In most tubes pulsating growth was maintained, however, many tubes showed a reduced amplitude of the pulses (Fig. 4). Only two out of 13 tubes abandoned the pulses and continued to grow with constant growth rates (Fig. 5). The effect of verapamil was reversible in some cases (Fig. 4), but not in others (Fig. 5). Nifedipine did not inhibit pollen tube growth neither in concentrations usually used on plant material (10 to 100 ~ollL,) nor in extremely high concentrations (up to 1 mmollL). Pulsating growth was not inhibited, the only visi-
ble effect was a slowdown of the initial phase of the pulses in some cases (Fig. 6) or a decrease in slow growth rate in others (Fig. 7). However these results have to be interpreted cautiously, since it cannot completely be excluded, that nifedipine was photodeactivated rather rapidly during the experiments. Even though the solutions were prepared at dim light and kept in the dark until use, they were inevitably exposed to the light 'in the microscope upon addition to the sample. It was impossible to assess, how fast nifedipine would actually have been deactivated by this exposition to light. Discussion
Oscillations of the growth rate with periods in the range of minutes have been monitored in entire plants (Bose, 1927;
442
ANJA GElTMANN
and MAURO CREST! ____________
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F1po3-S: Behavior of pollen tubes treated with verapamil. The drug caused a decrease of slow growth rate and of pulse frequency (Figs. 3, 4). In some cases growth rate during pulses and the amplitude of the expansions were impaired (Fig. 4), which was in some cases reversible. In most cases pulses were not completely inhibited, only two out of 13 tubes showed steady growth rates after addition of the drug (Fig. 5).
Morgan et aI., 1980; Cosgrove, 1981; Kristie and Jolliffe, 1986) as weU as in tip-growing single cells, such as poUen tubes of cenain plant species (Plyushch, 1995; Pierson et aI.,
1995; Geitmann et aI., 1996). To our knowledge the control mechanism for these oscillations has not been satisfactorily clarified. The present work was undertaken based on the ob-
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timelminutes)
Fip. 6, 7: Behavior of pollen tubes treated with nifedipine. The drug did not inhibit pulsating growth. The only visible effects were a slowdown in the initial phase of the pulse (Fig. 6) or a decrease in slow growth rate (Fig. 7).
servation that fluctuations in Ca2+ influx seem to be correlated with pollen tube growth rate (Pierson et al., 1996). The authors however did not provide any evidence for their postulation that these Ca2+ fluctuations which they observed in pollen tubes showing small sinusoidal growth oscillations of higher frequency («type h oscillations) are the cause of growth rate fluctuations and not their consequence. Using specific inhibitors for Ca2 + channels it was shown here that the regulated activity of certain Ca2+ channels governing Ca2 + influx through the plasma membrane is indeed likely to represent a key factor in the control of pollen tube growth rate, at least as far as pulsating growth («type II» oscillations) is concerned.
Tht tjfict oftht inhibition ofCtl+ channtls by inorganic inhibitors on pulsating pollm tubt growth Inorganic Ca2+ channel blockers such as La3+ and Gd3 + inhibit a great range of Ca2+ channels by competing with the Ca2+ ions. The concentrations of La3 + with inhibiting effect are in the high micro- to millimolar range (Tester and Mac-
Robbie, 1990; Herth et al., 1990; Ding and Pickard, 1993; Herrmann and Felle, 1995), whereas Gd3+ was found to be active in concentrations as low as 10 ~moUL (Alexandre and Lassalles, 1991). Regarding pollen germination 100 ~oUL to 1 mmollL La3+ were observed to have an inhibiting effect on pollen of Hannanthus a/biflos and Omothn"a bimnis (Bednarska, 1989), whereas already 10~moUL La3 + were found to reduce the high Ca2+ concentration in the tips of lily pollen tubes, which indicates, that Ca2+ channel activity is indeed responsible for the tip focused Ca2+ gradient in growing pollen tubes (Obermeyer and Weisenseel, 1991; Malh6 et al., 1994). The results fresented here are consistent with these reports. La3+ or Gd + concentrations between 10 ~oUL and 100 ~ollL inhibited Pttunia pollen tube elongation. It can therefore be confirmed that the functioning of the Ca2+ channels is essential for pollen tube growth in general. In the case of ~ulsating tubes, intermediate concentrations of La3+ and Gd + which did not cause the total arrest of growth, caused the tubes to abandon their pulsating rh~, thus clearly indicating the important function of the Ca2+ channels in the control of the pulses.
444
ANJA GEITMANN and MAURO CRESTI
As always when using inhibitors, it has to be considered, however, that their specificity is not absolute. For Gd3 + it was shown by Jackson and Heath (1993), that in fungal hyphae this ion blocks stretch-activated Ca2 + channels, but not K+ channels. Channel inhibition in patch clamp experiments on these cells occurred within minutes or seconds, depending on the concentration of Gd3 + (Garrill et al., 1993). On rl),e other hand it cannot be ignored, however, that La3 + apart from inhibiting Ca2+ channels has been shown to block both inward-rectifying (Tester and MacRobbie, 1990) and outwardrectifying (Ketchum and Poole, 1991) K+ channels. The results obtained in the present work have to be interpreted considering this restriction in specificity.
Polevoy and Stahlberg, 1978; Goring et al., 1979; Fisahn et al .• 1986; Felle et al., 1986; Felle, 1988; Souda et al .• 1990; Toko et al .• 1986, 1990; McAinsh et al .• 1995; Ehrhardt et al .• 1996). For oscillations of the cytosolic Ca2+ concentration many different models for negative or positive feedback mechanisms have been proposed, many of which involve either stable or oscillating levels of inositol-l,4,5-trisphosphate as a signal for Ca2+ release from internal stores (Tsien and Tsien, 1990; Fewtrell, 1993; Berridge, 1990; 1993 and references therein). These models were established for animal cells. but it was shown, that an inositol-l.4.5-trisphosphatemediated signal pathway exists in pollen tubes (FranklinTong et al .• 1996) which provides further support for the proposed feedback mechanism. In general, elevated cytosolic free Ca2+ concentrations are Inhibition ofCtl+ channels with organic inhibitors associated with stimulated exocytosis in various animal (PlattThe organic channel blockers nifedipine and verapamil ner, 1989; Wollheim and Lang. 1994) and plant cell types showed an effect on the overall pollen tube growth rate and/- (Blackbourn et al., 1991; Zorec and Tester, 1992) which or on pulse amplitude, but in general did not inhibit pulsa- opens intriguing possibilities. Interpreting the pollen tube as tory growth. The reduction of the slow growth rate and the a cell with repeated triggered exocytosis, one might regard the arrest of growth upon application of higher concentrations presumable increase in cytosolic Ca2+ as a signal for the rapid evidences that verapamil- and perhaps nifedipine-sensitive release of secretory vesicles. This hypothesis is surr.rted by channels are present in the plasma membrane of pollen tubes Picton and Steer (1982; 1985) who stated that Ca is necesof Petunia hybrida, as already shown for pollen tubes of other sary for vesicle fusion in pollen tube elongation. It is very plant species (Reiss and Herth, 1985; Bednarska, 1989; Feij6 tempting to assume, that the Ca2+ level regulates the rate of et al., 1995) and for the tips of fungal hyphae (Dicker and vesicle fusion at the tip of the pollen tube, possibly involving Turian, 1990; Robson et al., 1991). However, because of the the activity of a functioning cytoskeleton (Geitmann et al .• lack of an effect as dramatic as with inorganic inhibitors, in 1996), of annexins (Battey and Blackbourn, 1993) or of other the case of pulsating pollen tubes one might presume that Ca2+ -dependent proteins. Related to this issue is the question how the Ca2+ channels these Channels only provide the constant basic level of Ca2+ influx at the pollen tube apex. This influx presumably main- located in the apical pollen tube tip are gated. In the case of tains the internal Ca2 + gradient along the longitudinal axis of pulsating pollen tubes it seems likely, that during a complete the cell wich is necessary for general pollen tube elongation. cycle of slow growth and pulse, the membrane tension at the One might assume that these channels are necessary for tip apical tip undergoes changes. Even if most of the internal growth in pollen tubes, but that they probably open and pressure is probably borne by the cell wall (Cosgrove and close independently of the pulse rhythm.. . Hedrich, 1991; Ramahaleo et al., 1996). changes in either cell Again, the results obtained with nifedipine and verapamil wall resistance or internal turgor should cause alterations in have to be interpreted with caution, since they also seem to tension in the plasma membrane. It is therefore tempting to have an effect on K+ -selective channels (Terry et al., 1992). assume, that the Ca2+ channels responsible for the control of Furthermore it cannot be exguded that nifedipine was pho- pulsating growth in pollen tubes are gated by mechanical todeactivated during the experiments, especially since con- stress the gating parameter being either the elastic tension in centrations as low as 10~, which were reported to have an the membrane (Ramahaleo et al., 1996) or the submembrane effect (swelling of the tip, formation of protuberances) on lily cytoskeleton (Sokabe et al., 1991). It is reasonable to assume pollen tubes (Reiss and Herth, 1985), seemingly had no dra- the presence of stretch-sensitive ci+ in the apical ends of matic effect in our experiments, neither did higher concen- pollen tubes since their activity has been demonstrated for trations. other tip growing cells, i.e. ~ngal hyphae (Zhou et al.) 1991; Garrill et al., 1992, 1993; Levina et al., 1995). Furthermore the involvement of stretch-activated ion channels in rhythmic The significance ofthe ctl+ influx in pulsating pollen tubes plant movements has p~eviously been claimed by Schwenke The results d~cribed above tempt to establish the hypo- and Wagner (1992), who observed that Gd3+ inhibits xylem thesis that the pQlse-like elongations in pollen tubes are regu- exudation. which occurs with pulses in the period range of . lated by transient increases of ~2+ . influx into the apical pol- 8 min. len tube tip, which w~uld be likely to have signal function. This suggestion is reasona~le since it has been .repqrted reHypothetical model for the mechanism governing pulsating peatedly that not only in animal but also in plant cells Ca2+ growth influx can undergo periodic fluc~tions causing alteratiop,s in the membrane potential either in. sinusoidal or in spikeThe inhibition of the pulsating rhyth}n in pollen tubes of like form. with period$ in the range of seconds or minutes Petunia by inorganic inhibitors of Ca2+ channels suggests (Scott. 1957; Jenkinson and Scott, 1961; Jenkinson, 1962; that an oscillating cytosolic Ca2+ level .caused by fluctuations Nishizaki. 1968; Lefebvre et al.• 1970; Pickard, 1972. 1973; in the Ca2+ influx through the plasma membrane might be
Ca2 + channels in pulsating pollen tubes
the basic mechanism governing the periodic alterations in growth rate. One can only speculate about a putative feedback mechanism which regulates the Ca2 + influx, but it seems plausible that a temporarily elevated level of cytosolic Ca2+ has a signal function. The signal might induce a sudden liberation of secretory vesicles resulting in a local cell wall relaxation, which in turn might allow a sudden turgor-driven expansion of the cell wall at the apical tip of the pollen tube. A pulse would thus consist of two consecutive steps - the sudden liberation of vesicles followed by the expansion of the newly released cell wall material. The distinction in two phases is favored by the observation of Plyushch et al. (1995) who describe the occurrence of a wall thickening at the pollen tube tip (which might correspond to the vesicle release proposed here) prior to each pulse-like expansion in Gasteria pollen tubes. Whether or not this two-step-model describes the real situation remains to be investigated. In any case however, pulsating pollen tubes seem to provide a suitable model for the investigation of the intracellular signaling pathway based on Ca2 + influx in growing plant cells. References ALExANDRE, J. and J. WSALLES: Hydrostatic and osmotic pressure activated channel in plant vacuole. Biophys. J. 60, 1326-1336 (1991). ' BATIEY, N. H. and H. D. BLACKBouRN: The control of exocytosis in plant cells. New Phytol. 125,307-338 (1993). BEDNARSKA, E.: The effect of exogenous Ca2 + ions on pollen grain germination and pollen tube growth. Sex. Plant Reprod. 2, 5358 (1989). BERRIDGE, M. J.: Calcium oscillations. J. BioI. Chern. 265, 95839586 (1990). - Inositol trisphosphate and calcium signalling. Nature 361, 315325 (1993) . BLACKBOURN, H. D., J. H. WALKER, and N. H . BAlTEY: Calciumdependent phospholipid-binding proteins in plants. Planta 184, 67-73 (1991) . BOSE, J. Plant autographs and their revelations. Longmans, Green, and Co., London (1927). BRACKER, C. E., R. L6PEz-FRANCO, S. BARTNICKI-GARCIA, D. M. MuRPHY, and R. J. HOWARD: Satellite Spitzenkorper, pulsed growth, and the determination of cell shape in growing hyphal tips of fungi. Proc. Royal Micr. Soc. 30, 17 (1995). BREWBAKER, J. L. and B. H. KWACK: The essential role of calcium ion in pollen germination and pollen tube growth. Amer. J. Bot. 50, 859-865 (1963). CoSGROVE, D . J.: Rapid suppression of growth by blue light. Plant Physiology 67, 584-590 (1981). COSGROVE, D. J. and R. HEDRICH: Stretch-activated chloride, potassium, and calcium channels coexisting in plasma membranes of guard cells of Vida foba L. Planta 186, 143-153 (1991). DERKSEN, J. : Pollen tubes: a model system for plant cell growth. Bot. Acta 109, 341-345 (1996). DERKSEN, J., T. RUTIEN, I. K LICHTSCHEIDL, A. H . N. DE WIN, E. S. PIERSON, and G. RONGEN: Quantitative analysis of the distribution of organelles in tobacco pollen tubes: implications for exocytosis and endocytosis. Protoplasma 188,267-276 (1995). DICKER, J. W, ' and G. TURIAN: Calcium deficiencies and apical hyperbranching in wild-type and the .frost> and .spray» morphological mutants of Neurospora crassa. J. Gen. Microbiol. 136, 1413-1420 (1990). '
c.:
445
DING, J. P. and B. G. PICKARD: Mechanosensory calcium-selective cation channels in epidermal cells. PlantJ. 3, 83-110 (1993). EHRHARDT, D. w., R. WAIS, and S. R. loNG: Calcium spiking in plant root hairs responding to Rhizobium nodulation signals. Cell 85,673-681 (1996). FEIJ6, J. A., R. MALH6, and G. OBERMEYER: Ion dynamics and its possible role during in vitro pollen germination and tube growth. Protoplasma 187, 155-167 (1995). FEIJ6, J. A., A. SHIPLEY, and L. JAFFE: Spatial and temporal patterns of electric and ionic currents around in vitro germinating pollen of Lilium longiflorum: a vibrating probe study. In: HEBERLEBORS, E., M. HESSE, and O. VINCENTE (eds.): Frontiers in Sexual Plant Reproduction Research. 13th International Congress on Sexual Plant Reproduction, University of Vienna, p. 40 (1994). FELLE, H.: Auxin causes oscillations of cytosolic free calcium and pH in Zea mayscoleoptiles. Planta 174,495-499 (1988). FELLE, H., B. BRUMMER, A. BERTL, and R. PARISH: Indole-3-acetic acid and fusicoccin cause cytosolic acidification of corn coleoptile cells. Proc. Nat!. Acad. Sci. USA 83, 8992-8995 (1986). FEWTRELL, Ca2 + oscillations in non-excitable cells. Annu. Rev. Physiol. 55,427-454 (1993). FISAHN, J., E. MIKSCHL, and U. HANSEN: Separate oscillations of the electrogenic pump and of a K+ -channel in Nitella as revealed by simultaneous measurement of membrane potential and of resistance. J. Exp. Bot. 37, 34-47 (1986). FRANKLIN-TONG, E., B. K DROEBAK, A. C. ALLAN, P. A. C. WATKINS, and A.. J. TREWAVAS: Growth of pollen tubes of Papaver rhoeas is regulated by a slow-moving calcium ware propagated by inositoll,4,5-trisphosphate. Plant Cell 8, 1305-1321 (1996). GARRILL, A., S. L. JACKSON, R. R. LEw, and I. B. HEATH: Ion channel activity and tip growth: tip-localized stretch-activated channels generate an essential Ca2 + gradient in the oomycete Saprokgnia flrax. Europ. J. Cell BioI. 60,358-365 (1993). GARRILL, A., R. R. LEW, and I. B. HEATH: Stretch-activated Ca2 + and Ca2+ -activated K+ channels in the hyphal tip plasma membrane of the oomycete Saprokgnia flrax. J. Cell Sci. 101,721-730 (1992) . GEITMANN, A., Y. Q. LI, and M. CRESTI: The role of the cytoskeleton and dictyosome activity in the pulsatory growth of Nicotiana tabacum and Petunia hybrida. Bot. Acta 109, 102-109 (1996) . GORING, H., V. V. POLEVOY, R. STAHLBERG, and G. STUMPF: Depolarization of transmembrane potential of corn and wheat colooptiles under reduced water potential and afrer IAA application. Plant Cell Physiol. 20. 649-656 (1979) . HEPLER, P. K, D. D . MILLER, E. S. PIERSON, and D . A. CALLAHAM: Calcium and pollen tube growth. In: STEPHENSON, A. G. and Y.H. KAo (eds.): Pollen-pistil interactions and pollen tube growth, Amer. Soc. Plant Physiol., Rockville, Maryland, USA, pp. 111123 (1994). HERRMANN, A. and H. H. FELLE: Tip growth in root hair cells of Sinapis alba L.: significance of internal and external Ca2+ and pH. New Phytol. 129,532-533 (1995). HERTH, w., H. D. REISS, and E. HARTMANN: Role of calcium ions in tip growth of pollen tubes and moss protonema cells. In: HEATH, I. B. (ed.): Tip growth in plant and fungal cells. Academic Press, San Diego, pp. 91-118 (1990). JACKSON, S. L. and I. B. HEATH: Roles of calcium ions in hyphal tip growth. Microbiol. Rev. 57, 367-382 (1993). JAFFE, L. E, K R. ROBINSON, and R. NUCITELLI: Local cation entry and self-electrophoresis as an intracellular localization mechanism. Annu. NY Acad. Sci. 238, 372-389 (1974). JENKINSON, I. S.: Bioelectric oscillations of bean roots: further evidence for a feedback oscillator II. Intracellular plant root potentials. Austral. J. BioI. Sci. 15, 101-114 (1962).
c.:
v.
446
ANJA GBlTMANN and MAURO CRESTI
JBNKINSON, I. S. and B. I. H. SCOTr: Bioelectric oscillations of bean roots: further evidence for a feedback oscillator I. Extracellular response to oscillations in osmotic pressure and auxin. Austral. J. BioI. Sci. 14, 231-247 (1961). KAMINSKY], S. G. w., A. GARRILL, and I. B. HEATH: The relation between turgor and tip growth in Saprolegnia frrax: Turgor is necessary, but not sufficient to explain apical extension rates. Exp. Mycol. 16, 64-75 (1992). KBTCHUM, K A. and R. J. POOLE: Cytosolic calcium regulates a potassium current in corn (ha mays) protoplasts. J. Membrane BioI. 119,277-288 (1991). KIuSTIB, D. N. and P. A. JOLLIFFB: High-resolution studies of growth oscillations during stem elongation. Can. J. Bot. 64, 2399-2405 (1986). KOHTRBIBBR, w. M. and L. F. JAFFB: Detection of extracellular calcium gradients with a calcium-specific vibrating electrode. J. Cell BioI. 110, 1565-1573 (1990). LEFBBVRE, J., R. LBFEVER, and C. GILLET: Oscillations auto-entretenues des potentiels de membrane de Nitella. Bull. Soc. R. Bot. Belg. 103, 157-165 (1970). LEvINA, N. N., R. R. LEw, G. J. HYDB, I. B. HEATH: The roles of Ca2+ and plasma membrane ion channels in hyphal tip growth of Neurospora crassa. J. Cell Sci. 106, 3405-3417 (1995). L6PBz-FRANco, R., S. BARTNICKI-GARCIA, and C. E. BRACKER: Pulsed growth of fungal hyphal tips. Proc. Natl. Acad. Sci. USA 91, 12228-12232 (1994). MALH6, R., N. D. READ, M. SALOME PAIS, and A. J. TREWAVAS: Role of cytosolic free calcium in the reorientation of pollen tube growth. PlantJ. 5, 331-341 (1994). McAINSH, M. R., A. A. R. WEBB, J. E. TAYWR, and A. M. HBTHERINGTON: Stimulus-induced oscillations in guard cell cytosolic free calcium. Plant Cell 7, 1207-1219 (1995). MIl.LEa, D. D., D. A. CAu..uIAM, D. J. GROSS, and P. K HEPLER: Free Ca2+ gradient in growing pollen tubes of Li/ium. J. Cell Sci. 101, 7-12 (1992). MORGAN, D. c., T. O'BRIAN, and H. SMITH: Rapid photomodulation of stem extension in light-grown Sinapis alba L. Planta 150, 95-101 (1980). NISHIZAKI, Y.: Rhythmic changes in the resting potential of a single cell. Plant Cell Physiol. 9, 613-616 (1968). OBERMEYER, G. and M. H. W'BISENSBEL: Calcium channel blocker and calmodulin antagonists affect the gradient of free calcium ions in lily pollen tubes. Europ. J. Cell BioI. 56, 319-327 (1991). PICKARD, B. G.: Spontaneous electrical activity in shoots of Ipomoea, Pisum, and Xanthium. Planta 102,91-14 (1972). - Action potentials in higher plants. Bot. Rev. 39, 172-201 (1973). PICTON, J. M. and M. W. STEER: A model for the mechanism of tip extension in pollen tubes. J. theor. BioI. 98, 15-20 (1982). - - The effects of ruthenium red, lanthanum, fluorescein isothiocyanate and trifluoperazine on vesicle transport, vesicle fusion and tip extension in pollen tubes. Planta 163,20-26 (1985). PIBRSON, E. S. and M. CRBSTI: Cytoskeleton and cytoplasm'ic organization of pollen and pollen tubes. Int. Rev. Cytol. 140, 73125 (1992). PIBRSON, E. S., Y. Q. U, H. Q. ZHANG, M. T. M. WILLBMSB, H. F. UNSKBNS, and M. CRBSTI: Pulsatory growth of pollen tubes: investigation of a possible relationship with the periodic distribution of cell wall components. Acta Bot. Neerl. 44, 121-128 (1995). PIERSON, E. S., D. D. MILLER, D. A. CAu..uIAM, A. M. SHIPLEY, B. A. RIvERS, M. CRBSTI, and P. K. HEPLER: Pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient: effect of BAPTA-type buffers and hypertonic media. Plant Cell 6, 1815-1828 (1994). PIBRSON, E. S., D. D. MILLER, D. A. CAu..uIAM, J. VAN AKBN, G. HACKETr, and P. K HEPLER: Tip-localized calcium entry fluc-
tuates during pollen tube growth. Develop. BioI. 174, 160-173 (1996). PLATrNBR, H.: Regulation of membrane fusion during exocytosis. Int. Rev. Cytol. 119, 197-286 (1989). PLYUSHCH, T. A., M. T. M. WILLEMSE, M. A. W. FRANSSBN-VERHEIJBN, and M. C. REINDBRS: Structural aspects of in vitro pollen tube growth and micropylar penetration in Gasteria verrucosa (Mill.) H. Duval and Lilium longiflorum Thunb. Protoplasma 187, 13-21 (1995). POLEVOY, v. V. and R. STAHLBBRG: Auxin-induzierte langsarne Schwingungen des Membranpotentials bei Maiskoleoptilen. BioI. Rundsch. 16, 38-40 (1978). RAMAHALBo, T., J. ALExANDRE, and J. LAsSALLBS: Stretch activated channels in plant cells. A new model for osmoelastic coupling. Plant Physiol. Biochem. 34, 327-334 (1996). RATHORE, K S., R. J. CORK, and K. R. ROBINSON: A cytoplasmic gradient of Ca2 + is correlated with the growth of lily pollen tubes. Developm. BioI. 148, 612-619 (1991). REISS, H. and W. HBRTH: Visualization of the Ca2+ -gradient in growing pollen tubes of Lilium longiflorum with chlorotetracycline fluorescence. Protoplasma 97, 373-377 (1978). - - Nifedipine-sensitive calcium channels are involved in polar growth oflily pollen tubes. J. Cell Sci. 76, 247-254 (1985). REISS, H. and K TRAXBL: Hint of polar distribution in calcium channels under PIXE analysis. BioI. Trace Element Res. 13, 135142 (1987). REISS, H., G. W. GRIMB, M. Q. u, J. TAKACS, and F. WATr: Distribution of elements in the lily pollen tube tip, determined with the Oxford scanning proton microprobe. Protoplasma 126, 147152 (1985 a). REISS, H., W. HBRTH, and R. NOBILING: Development of membrane- and calcium-gradients during pollen germination of Lilium longiflorum. Planta 163, 84-90 (1985 b). ROBSON, G. D., M. G. WIEBB, and A. P. J. TRINCI: Involvement of Ca2+ in the regulation of hyphal extension and branching in FusariumgraminearumA3/5. Exp. Mycol. 15,263-272 (1991). SCHWENKE, H. and E. WAGNBR: A new concept of root exudation. Plant Cell Environ. 15,289-299 (1992). SCOTr, B. I. H.: Electric oscillations generated by plant roots and a possible feedback mechanism responsible for them. Austral. J. BioI. Sci. 10, 164-179 (1957). SOKABB, M., F. SACHS, and Z. JING: Quantitative video microsCopy of patch clamped membranes: stress, strain, capacitance, and stretch channel activation. Biophys. J. 59,722-728 (1991). SOUDA, M., K TOKO, K. HAYASHI, T. FUJIYOSHI, S. EZAKI, and K. YAMAFuJI: Relationship between growth and electric oscillations in bean roots. Plant Physiol. 93, 532-536 (1990). SPBKSNIJDER, J. E., A. L. MILLER, M. H. WBISENSBBL, T. CHBN, and L. E. JAFFB: Calcium buffer injections block fucoid egg development by facilitating calcium diffusion. Proc. Natl. Acad. Sci. USA 86, 6607-6611 (1989). TANG, x. w., G. Q. LIU, Y. YANG, W. L. ZHBNG, B. C. Wu, and D. T. NIB: Quantitative measurement of pollen tube growth and particle movement. Acta Bot. Sinica 34, 893-898 (1992). TBRRY, B. R., G. P. FINDLAY, and S. D. TYERMAN: Direct effects of Ca2 + -channel blockers on plasma membrane cation channels of Amaranthus tricolor protoplasts. J. Exp. Bot. 43, 14757-11473 (1992). TESTER, M. and E. A. C. MAcRoBBIB: Cytosplasmic calcium affects the gating of potassium channels in the plasma membrane of Chara corallina: a whole-cell study using calcium-channel effectors. Planta 180, 569-581 (1990). TIRLAPUR, U. and M. CRBSTI: Computer-assisted video image analysis of spatial variations in membrane-associated Ca2 + and calmo-
Ca2 + channels in pulsating pollen tubes dulin during pollen hydration, germination and tip growth in Nicotiana tabacum L. Ann. Bot. 69,503-508 (1992). TOKO, K., K. HAYASHI, and K. "YAMAFUJI: Spatio-temporal organization of electricity in biological growth. Trans. IECE of Japan 69, 485-487 (1986). TOKO, K., M. SOUDA, T. MATSUNO, and K. "YAMAFUJI: Oscillations of electrical potential along a root of a higher plant. Biophys. J. 57, 269-272 (1990). TSIEN, R. W. and R. Y. TSIEN: Calcium channels, stores, and oscillations. Annu. Rev. Cell BioI. 6, 715-760 (1990).
447
WEISENSEEL, M. H., R. NUCCITELLI, and L. F. JAFFE: Large electrical currents traverse growing pollen tubes. J. Cell BioI. 66, 556-567 (1975). WOLLHEIM, c. B. and J. lANG: A game plan for exocytosis. Trends Cell BioI. 4,339-341 (1994). ZHOU, X., M. A. STUMPF, H. C. HocH, and C. KUNG: A mechanosensitive channel in whole cells and in membrane patches of the fungus Uromycts. Science 253, 1415-1417 (1991). ZOREC, R. and M. TESTER: Cytoplasmic calcium stimulates exocytosis in a plant secretory cell. Biophys. J. 63,864-867 (1992).