Membrane potential changes during pollen germination and tube growth

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We have found the polar distribution of the membrane potential along the protoplast surface and the longitudinal potential gradient along the pollen tube.
ISSN 1990-519X, Cell and Tissue Biology, 2009, Vol. 3, No. 6, pp. 573–582. © Pleiades Publishing, Ltd., 2009. Original Russian Text © M.A. Breygina, A.V. Smirnova, N.P. Matveeva, I.P. Yermakov, 2009, published in Tsitologiya, Vol. 51, No. 10, 2009, pp. 815–823.

Membrane Potential Changes during Pollen Germination and Tube Growth M. A. Breygina*, A. V. Smirnova, N. P. Matveeva, and I. P. Yermakov Biological Faculty, Moscow State University, Moscow, Russia *e-mail: [email protected] Received March 17, 2009

Abstract—Using methods of quantitative fluorescent microscopy, we studied membrane potential changes during pollen germination and in growing pollen tubes. Two voltage-sensitive dyes were used, i.e., DiBAC4(3), to determine the mean membrane potential values in pollen grains and isolated protoplasts, and Di-4-ANEPPS, to map the membrane potential distribution on the surfaces of the pollen protoplast and pollen tube. We have shown that the activation of the tobacco pollen grain is accompanied by the hyperpolarization of the vegetative cell plasma membrane by about 8 mV. Lily pollen protoplasts were significantly hyperpolarized (–108 mV) with respect to the pollen grains (–23 mV) from which they were isolated. We have found the polar distribution of the membrane potential along the protoplast surface and the longitudinal potential gradient along the pollen tube. In the presence of plasma membrane H+-ATPase inhibitor sodium orthovanadate (1 mM) or its activator fusicoccin (1 μM), the longitudinal voltage gradient was modified, but did not disappear. Anion channel blocker NPPB (40 μM) fully discarded the gradient in pollen tubes. The obtained results indicate the hyperpolarization of the plasma membrane during pollen germination and uneven potential distribution on the pollen grain and tube surfaces. An inhibitory analysis of the distribution of the potential in the tube has revealed the involvement of the plasma membrane H+-ATPase and anion channels in the regulation of its value. Key words: membrane potential, pollen grain germination, polar growth. DOI: 10.1134/S1990519X0906011X

Abbreviations: DiBAC4(3), Bis(1,3-dibutylbarbituric acid(5)) trimethine oxonol; Di-4-ANEPPS-3-(4-(2(6-(dibutylamino)-2-naphthyl)-trans-ethenyl)pyridinium)propane sulfonate; NPPB, (5-nitro-2-(3-phenylpropylamino) benzoic acid

along the tube, while the outward currents were shifted towards the pollen grain. According to these authors (Weisenseel and Wenisch, 1980), the membrane potential values of a vegetative cell of lily pollen grain varied from –90 to –130 mV. In later studies with the use of improved microelectrode methods, similar results of – 110 to –150 mV were obtained for lily pollen (Obermeyer and Blatt, 1995). For pollen grains of other plant species, more positive membrane potential values have been reported, including –30 mV in Petunia hybrida and –37 mV in Narcissus (Feijo et al., 1995), while the potential of isolated pollen protoplasts of Brassica chinensis was –79 mV (Fan et al., 2003). The pollen tube membrane potential also differed in different objects: thus, in Agapanthus umbellatus, it amounted to –55 mV (Malho et al., 1995), whereas, in Arabidopsis, it was around –100 mV (Mouline et al., 2002).

INTRODUCTION The pollen grains of angiospermous plants germinate on pistil stigmas and form pollen tubes that deliver spermia to the places of fertilization. The formation of the tube is preceded by the hydration of the pollen grain, the activation of its metabolic processes, and the structural rearrangement of the cytoplasm connected with the formation of the polarity axis. The pollen tube is one of the best-studied objects for analyzing the mechanisms of polar growth (Taylor and Helper, 1997). Studies of the ion regulation of pollen germination and tube growth started more than 30 years ago, when it was shown using nonselective microelectrodes that the activation of lily pollen grain leads to the appearance of transmembrane ion currents (Weisenseel et al., 1975). The inward current reached maximal values in the place of would-be germination, while the outward current reached maximal values on the opposite pole. After germination, the inward currents were distributed

The works in the last few years on the inhibitory analysis and/or study of mutants for plasmalemma transport proteins have revealed the important role of H+, K+, Ca2+, and Cl– transmembrane transfer in the regulation of pollen germination and pollen tube growth (Matveeva et al., 2003; Holdaway-Clarke and Hepler, 2003; Hepler et al., 2006; Breygina et al., 2009). Furthermore, a connection between growth processes and the formation of ion gradients in the tube 573

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cytoplasm has been established: the calcium concentration decreases with an increase in the distance from the tip, while the intracellular pH value is minimal at the apex and changes gradually along the tube length (Holdaway-Clarke and Hepler, 2003; Hepler et al., 2006) At the same time, previously obtained data (Weisenseel et al., 1975) on the spatial distribution of ion currents in the pollen tube was confirmed (Michard et al., 2009). It has been established that H+, K+, and Ca2+ enter the tube in its apical part. Outward proton currents have been revealed in more distal zones and their intensity changed along the tube length (Certal et al., 2008; Michard et al., 2009). The current distribution of ion currents, at least in the case of protons, greatly depends on the polar distribution of ion-transporting proteins on the plasmalemma. This is indicated by the experiments on the simultaneous study of proton currents and the distribution of fluorescent labeled H+-ATPase in the plasmalemma of the tobacco pollen tube (Certal et al., 2008). The fluorescence was absent in the tube apex, but it was present in the subapical area; with an increase in the distance from the apex, the amount of H+-ATPase in the plasmalemma rose simultaneously with increasing intensity in the outward proton current. Moreover, orthovanadate, an inhibitor of H+-ATPase activity, inhibited the outward proton current. The problem of the Cl– current distribution has not been solved completely. According to some data (Zonia et al., 2002), Cl– exits the pollen tube in its apical part, while enters in more distal regions; with increasing distance from the apex, the current first increases, then flattens to plateau. Other authors, by using similar experiments, have concluded that the inward H+ currents can be erroneously interpreted as the outward Cl– currents (Messerli et al., 2004). Data on the localization of ion-transporting proteins in the pollen grain plasmalemma are scarce. In isolated lily pollen protoplasts, a polar distribution of mechanosensitive calcium channels has been revealed (Dutta and Robinson, 2004); it is suggested that they are located in the place of the possible tube appearance. In correspondence with this, it has been established for Arabidopsis pollen that the local increase in the calcium concentration in the area of the functional pore is a necessary condition for germination in vivo (Iwano et al., 2004). The above-considered microelectrode studies of ion regulation of pollen germination have revealed a complex network of interconnected ion currents that, in combination, determine the difference of potentials on both sides of the plasma membrane. In turn, the membrane potential controls the processes of ion and metabolite transport and the interaction of the cell with the surrounding medium. The idea of the spatial and/or temporal changes of the membrane potential during pollen germination and tube growth has been discussed (Holdaway-Clarke and Hepler, 2003; Robinson and

Messerli, 2003); however, it has not been solved in microelectrode studies. The goal of the present work was to reveal changes of membrane potential during pollen germination. To solve this task, we used methods of fluorescent microscopy with vital staining of cells with potential-dependent dyes that had previously been applied successfully in studies of animal and fungal cells. MATERIALS AND METHODS Plant material and sample preparation. Objects were cut plants of lily Lilum longiflorum Thunb., variety White Europe, and plants of the tobacco Nicotiana tabacum L., variety Petit Havana SR1, grown from seeds in a climatic chamber (25°C, 16-h light day). Anthers were removed from flowers on the eve of their opening and placed into a thermostat (25°C) for 3 days. Pollen from the opened anthers was collected in test tubes and stored at –20°C. After thawing, the pollen grains were cleansed of tryphine with hexane and air dried. Dry pollen samples were incubated in a moist chamber at 25°C for 1–2 h before use to obtain the pollen grain cultures or protoplasts. Pollen was incubated in standard medium in 2.5-cm Petri dishes or in 35-μl plastic cultural chambers (CoverWell, Schleicher and Schuell, Germany) covered inside with 0.01% poly-L-lysine. In the latter case, the pollen tubes attached to the upper chamber wall were analyzed. For plasmolysis experiments pollen tubes were incubated for 30 min in the standard medium supplemented with 0.9 M mannite. To isolate protoplasts, lily pollen was incubated in the medium with enzymes (see below) for 2 h at 30°C, then washed out with the same medium without enzymes and used immediately for staining or fixation. Protoplasts isolation was controlled by the commonly used method (Tanaka et al., 1987) by staining samples with Calcofluor White M2R fluorescent dye (Fluorescent Brightener 28, Sigma), which reveals the presence of the cell wall. Reagents. DiBAC4(3), i.e., Bis(1,3-dibutylbarbituric acid(5)) trimethine oxonol, and Di-4-ANEPPS, i.e., 3-(4-(2-(6-(dibutylamino)-2-naphthyl)-trans-ethenyl)pyridinium)propane sulfonate (Molecular Probes, the Netherlands); fusicoccin (Serva, Germany); sodium orthovanadate (ISN, United States); Fluorescent Brightener; NPPB, i.e., (5-nitro-2-(3-phenylpropylamino)benzoic acid; and poly-L-lysine (Sigma, United States); and cellulose and pectinase (ICN, United States). Composition of growth media and added reagents. The standard medium included 0.3 M sucrose, 1.6 mM H3BO3, 3 mM Ca(NO3)2, 0.8 mM MgSO4, and 1 mM KNO3 in 25 mM MES-Tris buffer, pH 5.9. The medium for protoplast isolation (Tanaka et al., 1987) differed from the standard one by the pH value (5.8), sucrose concentration (0.5 M), and the CELL AND TISSUE BIOLOGY

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presence of cellulase and pectinase (1% each). Fusicoccin, sodium orthovanadate, and NPPB were added to the pollen grain suspensions after 75 min of incubation in standard medium. The final fusicoccin concentration amounted to 1 μM, the final concentration of orthovanadate was 1 mM, and the final concentration of NPPB was 40 μM. The time of action of each of these reagents on the pollen tubes was 10 min. Changes in the membrane potential value were revealed using two dyes, i.e., DiBAC4(3) and Di-4ANEPPS. DiBAC4(3) belongs to the group of slow dyes; charged molecules of these dyes are distributed between the cell cytoplasm and surrounding medium in correspondence with the Nernst’s equation (Placˇ ek and Sigler, 1996). By measuring the fluorescence intensity of living and fixed (completely depolarized) cells, the membrane potential values (mV) can be calculated (Emri et al., 1998). The membrane potential value of the vegetative cell of the pollen grain was determined by the previously described procedure (Breygina et al., 2009). For this, the cells were stained with DiBAC4(3) solution at a concentration of 5 μM for 10 min. Fixed cells were used as completely depolarized control (Emri et al., 1998; Breygina et al., 2009). Di-4-ANEPPS belongs to the group of fast dyes that are inserted into the membrane. With the change in membrane potential, the charge in the dye molecule migrates, which leads to a shift in the excitation and emission spectra (Loew, 1996). Usually, the intensity of the fluorescence excited in two spectral regions (the blue and green ones) is measured and their ratio (Fb/Fg) is a measure of the membrane potential at a certain membrane area. Thus, the use of this dye allows one to detect local changes in the membrane potential within a single cell. Protoplasts were suspended in Di-4ANEPPS solution (10 μM) and microscoped immediately after sedimentation by centrifugation (250 g). The pollen tubes were stained in a drop on the microscope slide by mixing the suspension of grown pollen grains with the dye solution (10 μM) in a 1 : 1 ratio and were microscoped immediately. In experiments with double staining, protoplasts were first stained with DiBAC4(3), then Di-4-ANEPPS solution (20 μM) was added to the suspension in a 1 : 1 ratio. Microscopy and computer image analysis. An Axioplan 2 imaging MOT microscope supplied with corresponding filter sets, a mercury lamp, and an AxioCam HRc digital camera (Carl Zeiss, Germany) was used in the study. Fluorescence was excited in the wavelength range of 359–371 nm and recorded at the wavelengths higher than 397 nm (in the case of Calcofluor White M2R) or excited in the range of 475–495 nm and recorded at 515–565 nm (DiBAC4(3)) or excited in the blue (475–495 nm) or green (540–552 nm) spectral range, while recorded at wavelengths higher than 590 nm (Di-4-ANEPPS). Objects were photographed using a high-rate automatic shutter that allowed one to illuminate the preparation only at the moment of shootCELL AND TISSUE BIOLOGY

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Membrane potential values of protoplasts and pollen grains (PG) determined using potential-dependent dye DiBAC4(3) Object

Potential, mV

The protoplast isolated from lily PG The lily PG, hydrated (10 min) The tobacco PG, hydrated (10 min) The tobacco PG, activated (30 min)

–108 ± 3.0 –23 ± 1.0 –37 ± 1.5 – 45 ± 1.9

ing and allowed one to take series of photographs at a certain exposure. The images were obtained and analyzed with AxioVision 4.7 software (Carl Zeiss, Germany). Statistics. All experiments were performed in no less than five biological repeats. The statistical significance of differences was determined by Student’s criterion at the level of 0.05 or 0.01. The figure and table present the mean values and their standard errors. RESULTS Two fluorescent dyes were used to reveal changes in the membrane potential: Di-4-ANEPPS and DiBAC4(3) (Haugland, 2005). DiBAC4(3) was used in earlier studies of the tobacco pollen grain and pollen tube (Matveeva et al., 2004; Breygina et al., 2009); Di4-ANEPPS had not been used previously to study pollen. Therefore, it was necessary to determine the possible limitations of the method due to peculiarities of the object in a preliminary study. The staining of pollen grains with Di-4-ANEPPS has revealed an intensive, nonspecific binding of the dye with exine, i.e., the hydrophobic external layer of the pollen wall (data not shown). This prevents the detection of the plasma membrane fluorescence and makes Di-4-ANEPPS unavailable for studies of the ungerminated pollen grain. Therefore, freshly isolated lily pollen protoplasts were used for staining. After one day, these protoplasts already intensively regenerated the cell wall, which, in some cases, was clearly seen as patches (Figs. 1a, 1b). In protoplasts stained with Di-4ANEPPS, the cell contour was distinctly seen (Fig. 2a). The undamaged protoplast did not include the dye into the cytoplasm for at least 15 min after the addition of the dye. In the pollen tube, the clear contour was also obvious (Fig. 3a); however, in the apical area (throughout the apical 3 μm of the tube), the dye appeared in the cytoplasm relatively quickly. Therefore, this part of the tube was excluded from analysis. Control experiments with the plasmolysis of the pollen tube showed (Figs. 3b, 3c) that the plasmolyzed area contains a brightly stained plasma membrane, while the pollen tube lateral walls are unstained; furthermore, in the periplasmic space, which separates the wall and the plasma membrane, dye-binding components appear, which are presumably membranes of vesicles (Kroh and Knuiman, 1985). These data demonstrate the binding of the dye

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(a)

(a)

(b) Fig. 1. Protoplast isolated from lily pollen grain after 1 day of incubation without enzymes. (a) In bright field; (b) fluorescence, patches of the wall material stained with Calcofluor White M2R are seen. Scale bar: 20 μm.

with the plasma membrane and indicate that the pollen tube wall is not an obstacle for analyzing changes in the membrane potential. To establish the degree of agreement between the results obtained by using the two dyes, isolated protoplasts were stained simultaneously with DiBAC4(3) and Di-4-ANEPPS. For each protoplast, the intensity of the fluorescence of DiBAC4(3) in the cytoplasm and the mean fluorescence of Di-4-ANEPPS bound to the membrane were determined. The fluorescence intensity of Di-4-ANEPPS was recorded at excitation in the blue (Fb) and green (Fg) spectral ranges and their ratio Fb/Fg was calculated (Montana et al., 1989). The diameter of protoplasts chosen for analysis varied from 111 to 153 μm; the intensity of the measured fluorescence also differed. We have failed to reveal the correlation of

(b) Fig. 2. Protoplast isolated from lily pollen grain and stained with Di-4-ANEPPS: (a) excitation of fluorescence in the blue spectral range; (b) ratio image obtained by dividing this image by the other one obtained at excitation of fluorescence in the green range. Scale bar: 20 μm.

fluorescence with the protoplasm diameter in the indicated limits; the correlation coefficient r for DiBAC4(3) amounted to 0.09, whereas, for Fb/Fg Di-4-ANEPPS, it amounted to –0.23. At the same time, a high correlation (r = 0.93) has been found between the DiBAC4(3) fluorescence intensity and the Fb/Fg ratio for Di-4ANEPPS (Fig. 4). This agrees well with the known data that, upon cell depolarization, the DiBAC4(3) content CELL AND TISSUE BIOLOGY

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Fb/Fg 2.7

2.3

(a) 1.9

1.5 500 1000 1500 2000 DiBAC4(3) fluorescence intensity, rel. units

(b)

(c)

Fig. 3. Tobacco pollen tubes stained with Di-4-ANEPPS: (a) pollen tube in the standard isotonic medium; (b, c) pollen tube under conditions of plasmolysis (0.9 M mannitol); (a, c) in fluorescent light; (b) in bright field. Scale bar: 10 μm.

and the Fb/Fg value increase, whereas, at hyperpolarization, both values decrease (Brauner et al., 1984; Zhang et al., 1998). Further study of the Di-4-ANEPPS-stained protoplasts included the visualization and analysis of the distribution of the membrane potential (the Fb/Fg value) on their surface. For this purpose, a ratio image was obtained using AxioVision by dividing one image by the other. The first image was obtained under the excitation of fluorescence in the blue, while the other in the green spectral range. The results of the division of images are presented in Fig. 2b and the results of measurements in the Fb/Fg value at various points in the stained membrane are shown in Fig. 5. The maximal value was reached at the point of the transaction of axis 1 with the protoplast surface (Figs. 2b, 5), while the minimal value was achieved at axis 6. A similar calculation of various protoplasts has shown that the maximal and minimal values differ on average by 1.5 times and both differ from the Fb/Fg value averaged at the circumference (p < 0.01). Thus, membrane zones that differ by the Fb/Fg value have been revealed, which indicates nonuniform distribution of membrane potential on the protoplast surface. The membrane potential values of protoplast and pollen grains in millivolts were determined with aid of DiBAC4(3). In the living lily pollen grains (Fig. 6a, the CELL AND TISSUE BIOLOGY

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Fig. 4. Correlation of parameters characterizing the membrane potential value of individual protoplasts determined with two methods. Under conditions of double staining for each protoplast, the intensity of DiBAC4(3) fluorescence in the cytoplasm and the mean Fb/Fg ratio for Di-4-ANEPPS bound to the plasma membrane were measured; Fb is the intensity of fluorescence measured in the red spectral range at excitation in the blue range and Fg is the same at excitation in the green range.

upper cell), the stained cytoplasm is seen. The dead pollen grain (Fig. 6a, the lower cell) is depolarized and intensively stained; fixed cells look similarly (not shown). The isolated protoplast (Fig. 6b) binds the dye very weakly. Figure 6c shows the same protoplast photographed in transmitted light. The fixed protoplast presented in Figs. 6d and 6c is photographed at the same exposure as the living one (Fig. 6b), but brightly stained. The performed measurements and calculations have revealed a significant hyperpolarization of protoplasts with respect to the pollen grains from which they were isolated (table). To study the membrane potential during pollen grain activation at the initial stage of germination, we used tobacco pollen, which germinates faster than lily pollen. The first pollen tubes appeared 35 min after the beginning of hydration in liquid medium in vitro, while the lily pollen tubes were detected 1 h after the beginning of incubation. During tobacco pollen activation (30 min), the hyperpolarization of the plasmalemma occurred (table), which, however, did not reach the level of protoplasts. It should be noted that the membrane potential values for tobacco and lily pollen incubated in vitro for 10 min differed significantly; for tobacco, they were more negative (table). To obtain a picture of the membrane potential distribution in the pollen tube, we measured the fluorescence

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2

1.8 1.2 0.6 0

7

3

6

4 5

Fig. 5. Distribution of Fb/Fg ratio value at circumference of protoplast presented in Fig. 2. Intensity of fluorescence exited in blue (Fb) and green (Fg) spectral ranges was measured at eight sites distributed uniformly at the protoplast circumference. The Fb/Fg ratio is placed on the corresponding radial axes.

intensity of its plasma membrane 3–35 μm from the tip of the tube after staining with Di-4-ANEPPS. The Fb/Fg ratio was calculated based on these data. At the site of 3–20 μm, we observed a decrease in this value with an increase in the distance from the tip with sub(a)

(b)

(d)

sequent flattening to a plateau (Fig. 7), which indicates the existence of a gradient of membrane potential along the tube. It was possible to suggest the participation of the plasmalemma H+-ATPase in its creation. To check this suggestion, we studied the effects of fusicoccin (1 μM), which stimulates H+-ATPase, and its inhibitor orthovanadate (1 mM) on the membrane potential distribution. Both treatments changed the shape of the curve (Fig. 7). Fusicoccin produced hyperpolarization in areas close to the apex (p < 0.05), while vanadate caused depolarization in the subapical tube part (p < 0.05). However, in both cases, the potential gradient was present. These data confirm the participation of the proton pump in the regulation of the membrane potential in the pollen tube; at the same time, they also indicate the existence of alternative regulatory mechanisms that are the result of the functioning of other ion-transporting systems. One could suggest the participation of transmembrane anion transfer in these processes. The inhibition of anion channels with specific NPPB blocker (40 μM) completely eliminated the potential gradient along the tube (Fig. 7). DISCUSSION Changes in the membrane potential during pollen grain germination and in the growing tube were studied in the present work by optical methods using two dyes widely applied for estimating the membrane potentials of animal cells, i.e., DiBAC4(3) and Di-4-ANEPPS (Gamalei et al., 1998; Johnson, 1998). In previously published works, data on the use of Di-4-ANEPPS for (c)

(e)

Fig. 6. Lily pollen grains (a) and isolated protoplasts (b–e) stained with DiBAC4(3) vitally (a, b) and after fixation (d); (c, e) the same protoplasts in the bright field. Scale bar: 20 μm. CELL AND TISSUE BIOLOGY

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membrane potential studies in plant cells are very scanty. A visual analysis of the pollen grains, of the protoplasts isolated from them, and of the pollen tubes stained with this dye (Figs. 2 and 3) has shown that the cell wall of the pollen tube is not an obstacle for staining the plasmalemma. Since the tube wall has similar properties to the primary walls of the somatic cell, it can be suggested that this dye could be applied in studies on plant cells, except for cases when the cell surface contains large amounts of lipophilic compounds capable of binding the dye nonspecifically, such as the pollen exine sporopollenin. The second possible limitation for the use of Di-4-ANEPPS is its internalization into the cytoplasm, since this occurs, e.g., in the tip of the pollen tube. It should be noted that the tube is one of the fastest growing plant objects and its tip is characterized by extremely high endocytosis intensity (Cheung and Wu, 2008). By using the slow dye DiBAC4(3), we have managed to reveal the changes in the membrane potential during pollen grain activation (table). The hyperpolarization of the plasma membrane agrees well with the data on the intensification of respiration and the shift in the intracellular pH (Matveeva et al., 2002), as well as with the concept of the intensification of biosynthetic processes and the transport of metabolites across the plasma membrane at this period (Heslop-Harrison, 1987). The lower absolute value of the pollen grain potential in lily as compared with tobacco can be explained by its longer activation period. The membrane potential values that we have obtained for the lily protoplast (table) agree well with data of microelectrode studies of this object (Dutta and Robinson, 2004). The hyperpolarization of the isolated protoplast plasmalemma with respect to the pollen grain (Fig. 6; table) is quite an expected fact, since protoplast intensively synthesizes the cell wall, which can initially be laid in the form of patches (Fig. 1). In the absence of enzymes, these patches are revealed in lily pollen protoplasts after as little as 1–2 h of incubation (Zhao et al., 2004). At the same time, the membrane potential values of lily pollen grain, which were measured by optical methods (–23 mV, table) or using microelectrodes (–90 to −150 mV) (Obermeyer and Blatt, 1995; Weisenseel and Wenisch, 1980), differ significantly. One can suggest that, in experiments using microelectrodes, the pollen grain responded to mechanical actions and/or long incubation in growth medium. It is possible that the elimination of borate from this medium, which was used to stop the pollen germination (Obermeyer and Blatt, 1995), did not prevent pollen grain activation. As was expected, the quantitative analysis of the stained protoplasts has revealed a correlation between their ability to incorporate DiBAC4(3) into the cytoplasm and the value of the Fb/Fg ratio (Fig. 4). The use of Di-4-ANEPPS has allowed us to reveal the nonuniformity of the membrane potential distribution on the surface of the pollen protoplast and the pollen tube (Figs. 2, 3, 5, and 7). A polar potential distribuCELL AND TISSUE BIOLOGY

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Fb/Fg 2.2 1 2 3 1.7 4

1.2 0

10 20 30 Distance from the tube tip, μm

Fig. 7. Longitudinal membrane potential gradient in tobacco pollen tube, revealed by a change of the Fb/Fg ratio in control (1) and under effect of 1 mM sodium orthovanadate (2), 1 μM fusicoccin (3) or 40 μM NPPB (4). See captions in Fig. 4 for explanations of Fb and Fg.

tion is observed in the protoplast (Figs. 2 and 5). This may be connected with the localization of the would-be wall patch (Fig. 1), but this phenomenon needs further study. Previously, using the same dye, the polar potential distribution has been observed on the surface of the neuroblastoma cell placed in the electrical field (Gross et al., 1986). In the later work, Bedlack et al. (1992) established the spatial and temporal correlation of the field-induced morphogenesis with the local incorporation of calcium into the neuroblastoma cells in the place of membrane depolarization. The uneven distribution of the membrane potential on the surface of neuroblastoma cells has also been shown in the study using both microelectrodes and optical methods with application of the Di-8-ANEPPS dye, similar to Di-4-ANEPPS (Zhang et al., 1998). In the tobacco pollen tube, in a distance diapason of 3–20 μm from the tip, a gradual rise in the absolute value of the membrane potential with an increase in the distance from the tip has been found (Fig. 7), which confirms the earlier suggestion put forward by Robinson and Messerli (2003) about the existence of a gradient of potential in the pollen tube. This suggestion failed to be checked experimentally due to the limitations of microelectrode methods. The zone of expansion of this potential gradient corresponds approximately to the area in which, according to Certal et al. (2008), the intensity of proton release from the tube increases. Therefore, it could have been expected that the gradient of the potential was mainly formed due to the nonuniform H+-ATPase activity. The activation or inhibition of this enzyme with fusicoccin or vanadate

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affected the potential distribution by producing local hyper- or depolarization, respectively (Fig. 7); however, these actions did not eliminate the gradient. This means that an important role in the maintenance of the gradient is played by other plasmalemma ion-transporting systems. It seemed useful to check the participation of anion channels in this process, as we had previously revealed that a block of the transmembranous Cl– efflux had a significant effect on the membrane potential value in the pollen grain and tube as well as the pollen grain germination and the tube growth (Matveeva et al., 2003; Breygina et al., 2009). Experiments with use of NPPB, a specific blocker of anion channels (Roberts, 2006), have shown that the inhibition of these channels completely eliminated the potential gradient (Fig. 7). Thus, anion channels act as key factors in the maintenance of the membrane potential gradient in the pollen tube. It should be noted that the disturbance of the potential gradient under the effect of NPPB (Fig. 7) is combined with the disturbance of the compartmentalization of the cytoplasm (Breygina et al., 2009) that underlies the polar growth (Cheung and Wu, 2007). However, additional studies are needed to establish the cause-and-effect connections between these phenomena and the elucidation of the sequence of events that results in the arrest of growth. The use of optical methods of analyzing the membrane potential changes in the present work allowed us to establish that the activation of the pollen grain (at preparation for germination or after removal of the cell wall) is accompanied by the hyperpolarization of the plasmalemma. Furthermore, the polar distribution of the membrane potential value has been detected on the surface of the pollen protoplast and tube. Thus, we have supplemented the results of microelectrode studies on the nonuniform distribution of transmembrane ion currents and confirmed the ideas on the bioelectrical regulation of the pollen grain germination and tube growth (Holdaway-Clarke and Hepler, 2003; Hepler et al., 2006; Michard et al., 2009). At the same time, we have shown that anion channels play an important role in maintaining the membrane potential gradient in the growing tube. This agrees well with data about the existence of the local inward and outward Cl– currents in the tube (Zonia et al., 2002). However, according to our data, functions of these currents are not restricted by the regulation of the cell volume (Zonia et al., 2002) and the key role played by Cl– seems to be the regulation of the membrane potential. A comparison of the germinated pollen grain with other cells allows us to reveal some general regularities. Examples of the hyperpolarization of the plasma membrane during cell differentiation and morphogenesis can be found in studies of both plant (Kropf, 1992) and animal cells (Zhang et al., 1998). Recently, using the example of stem cells, it has been established that the hyperpolarization of the plasmalemma precedes (and triggers) cell differentiation (Sundelacruz et al., 2008). The uneven distribution of transmembrane ion currents,

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