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The membrane potential of growing lily pollen
Institut fiir Botanik und Pharmazeutische Biologie der Universitat Erlangen-Nurnberg, D-8520 Erlangen, Federal Republic of Germany
The Membrane Potential of Growing Lily Pollen M. H.
WElSENSEEL
and H. H.
WENISCH
With 4 figures Received March 15, 1980 . Accepted May 9, 1980
Summary Pollen grains of Lilium longiflorum growing in a liquid medium at between 20 and 25°C in continuous white light were penetrated with single microelectrodes. Two different electric potentials could be recorded from germinated grains: (i) In unvacuolated, recently germinated grains potentials of -90 to -130 mV were measured. These probably reflect the average potential difference between the cytoplasm and the medium. (ii) In pollen grains that had grown a tube of several hundred ,Hm and had become largely vacuolated an average potential of -60 mV was recorded. This might represent the potential of the vacuole. Changing the ionic composition of the bathing medium during impalement, the potentials of the cytoplasm responded to a 10-fold decrease in the medium's K+-concentration with a hyperpolarization of ca. 50 mV, and to a 10-fold increase with a depolarization of ca. 70 mV. Ten-fold changes in the K+ -concentration of the medium caused the potentials of the vacuole to respond with only 15 mV hyperpolarization, and 30 mV depolarization, respectively. Decreasing the H+ -concentration of the medium by 100-fold caused a hyperpolarization in the cytoplasm and in the vacuole of ca. 15 mV. Decreasing or increasing the Ca2 +-concentration or the CI--concentration of the medium had no significant effects on the electric potentials of the cytoplasm and of the vacuole. Lowering the temperature of the standard medium (pH 4) by about 10 or 20°C resulted in a depolarization of the plasma membrane by about 35 mV and 43 mV, respectively. No temperature-related depolarizations of the plasma membrane were observed in a growth medium of pH 6. The data obtained during this study indicate that the plasma membrane of growing lily pollen is mainly permeable to K+ -ions and that an electrogenic ion transport mechanism (probably an H+ -efflux) can contribute to the membrane potential. Key words: pollen grains, membrane potential, relative ion permeabilities, temperature effects, Lilium longiflorum. Abbreviations: CO = carbon monoxide; Ery( = electric potential of the cytoplasm; Evac = electric potential of the vacuole; EGTA = Ethyleneglycol-bis (fJ-aminoethylether) N,N'-tetraacetic acid; FCCP = Carbonyl cyanide, p-trifluoro-methoxyphenyl hydrazone, MES = 2 (N-morpholino) ethane sulfonic acid; Tris = Tris (hydroxymethyl) -aminomethane; S.E.M. = standard error of the mean.
Z. Pflanzenphysiol. Ed. 99. S. 313-323. 1980.
314
M. H.
WEISENSEEL
and H. H.
WENISCH
Introduction
Pollen grains have several attractive properties for electrophysiological investigations: (i) Dry pollen grains are in a state of arrested development that very quickly turns into active development after contact with a suitable medium, resulting in the growth of a tube within a few minutes to hours. This feature offers the opportunity to study changes in membrane properties related to development and localized growth in a higher plant cell. (ii) Ungerminated pollen grains have nO large vacuole but are rather densely filled with cytoplasm. During growth of the tube they slowly fill with a vacuole. It therefore should be possible to measure the electrical properties of the plasmalemma and of the tonoplast in the same type of a higher plant cell separately. (iii) Pollen grains contain no chlorophyll but probably do contain other pigments, as for instance carotenoids, flavins and phytochrome (JOHRI and VASIL, 1961; CHHABRA and MALIK, 1978) which may be bound to the plasma membrane as in other green or potentially green plant cells (cf. MARME, 1977). The effects of such pigments on membrane properties might therefore be studied without the interference of photosynthesis-related events. The few electrophysiological studies performed with pollen grains and tubes so far have shown that the membrane of Lilium pollen grains hyperpolarizes by about 40-50 m V before visihle growth begins (MATSCHKAL, see WEISENSEEL, 1977), and that the membrane potential and membrane resistance of Oenothera pollen tubes respond to temperature changes in a hysteretic way (MELAMED-HAREL and REINHOLD, 1979). It has also been found that growing lily pollen drive an electric current through themselves. This current enters the future site of growth and the growing tube, and leaves the non-growing part of the grain (WEISENSEEL et aI., 1975). Since the inward current consists mainly of K+ (and some Ca 2+ entering at the tip), a high permeability of the plasmalemma for K+ is suggested (WEISENSEEL and JAFFE,1976; JAFFE et aI., 1975). The outward current that is carried mainly by H+ indicates an active extrusion mechanism for H+ (WEISENSEEL and JAFFE, 1976). The aim of the present investigation was to continue our electrophysiological studies of pollen by tackling the following questions: Can the electric potentials of the cytoplasm and of the vacuole of lily pollen grains be measured separately? What are the major ions to which the plasmalemma is mainly permeable? Is the postulated H+ -transport mechanism of the growing lily pollen electrogenic? Materials and Methods Preparation and early development of pollen grains
Pollen grains were collected with a brush from the anthers of fully developed flowers of Lilium longiflorum and sputtered sparsely onto nylon nets (Scrynel HD, 85 [lm mesh width, Ziirich Bolting Cloth Mfg, Switzerland) that were stretched over plastic rings cemented to the bottom of 5 cm wide plastic petri dishes. The dishes were filled with 8 ml of medium covering the pollen grains about 0.5 mm high. The time when the dishes were filled with medium was taken as time zero for the experiment, and from this time on the Z. Pflanzenphysiol. Bd. 99. S. 313-323. 1980.
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dishes were kept in a room at 21 ± 1 cC or 24 ± 1 °C under white fluorescent light. The standard medium in which the lily pollen was grown contained the following components: 1.65 mM CaCl., 1.0 mM KN0 3 , 0.13 mM H 3 BO a, 5 mM MES-buffer and 300 mM mannitOl; pH 3.9 ± 0.1. Before use, the medium was saturated with air. In all batches ca. 50-70 °/0 of the pollen grains germinated within 60-100 min and grew a tube that elongated with a rate of up to 8.lIm min-I. Pollen grains with tubes of 100-200,Hm length appeared completely filled with cytOplasm. When the tubes elongated further a vacuole began to form at the site opposite to the location of germination. This vacuole enlarged rapidly with further elongation of the tube and filled the whole grain when the tube had reached a length of several hundred ,11m. Measurement of the electric potentials
Single microelectrodes were pushed from above into the center of growing pollen grains, under observation with a stereo microscope (Leitz, Germany). The micropipettes were fabricated from 1.5 mm wide glass capillaries containing an inner glass filament (Clark Electrochemical lnstr., England, Type GC 150 F). They were pulled to a tip of ca. 1 ,11m diameter with the aid of a vertical pipette puller (David Kopf lnstr., USA, Model 700 C). The micropipettes were then filled with cold 1 M KCl-solution by first submersing the ends into the KCI-solution and then filling the shaft with a syringe. Most of these micropipettes had excellent electrical properties, i.e. low resistances and low tip potentials. In a batch of 100 micropipettes, the average tip resistance was 5.0 ± 0.4 megohms before puncturing. The tip potentials of these pipettes, when dipped in the standard growth medium, were -3 ± 0.3 m V in 51 micropipettcs, 0 m V in 16 micropipettes and + 2.5 ± 0.3 m V in 33 micropipettes. The puncturing of pollen grains normally caused an increase in the resistance and in the tip potential of a micropipette. For instance, in 50 micropipettes checked after retraction from the grains the resistance had increased to 50 ± 4 megohms and the tip potentials had increased to -12 ± 1.2 or to + 6 ± 1.3 m V. The micropipettes were connected via a short piece of Ag-AgCI wire to a DC-preamplifier with an input impedance of larger than 1013 ohms and a leakage current of less than 4· 10-13 amps (Bioelectric Instr., USA, Model NF 1). A small fiber junction electrode with an Ag-AgCI internal (Beckman Instr., USA), grounded via a calibrator (Bioelectric lnstr., USA, Model CA 5), served as a reference electrode in the bathing medium. The electric potentials were recorded simultaneously on the screen of a storage oscilloscope (Tektronix Instr., USA, Model 5031) and on a chart recorder (Heathkit, USA, Model lR 18 M). Both the microelectrode and the reference electrode were mounted on micromanipulators (Prior, England) and the dish with the pollen grains was placed on a micropositioner table for fine horizontal adjustments (Line Tool Comp., USA). To monitOr the temperature of the bathing medium a small, electrically insulated, telethermometer probe (Yellow Springs lnstr., USA, Model 520) was dipped into the medium. In addition to the room light the dishes were illuminated from the side with a light guide (Schott a. Gen., Germany, Model KL 150 W). Medium changes around impaled pollen grains
To measure the relative permeabilities of various ions in the plasma membrane the effect of changes in the ion concentration of the medium on the electric potentials was monitored when 30 ml of new medium were introduced at one end of the dish and 30 ml were withdrawn at the opposite end. The medium change took about 1-2 min to complete and caused no change in the height of the medium above the grains. Since with this method new medium mixed with old medium, we always measured the actual concentration of the ions with ion selective electrodes (Orion Research Corp., USA) before and after the medium change. To test the effect of carbon monoxide (CO) on the electric potentials another type of medium change was employed: The dish with the pollen was placed in a larger dish and flushed Z. Pflanzenphysiol. Bd. 99. S. 313-323. 1980.
316
M. H.
WEISENSEEL
and H. H.
WENISCH
with ca. 300 ml of CO-saturated medium. The overflow was collected in the large dish a level of about 35 mm above the grains. This increase of medium height was intended in order to bring the pollen grains further away from the surface and therewith to keep the CO-concentration around the pollen more or less constant. Temperature changes in the bathing medium were accomplished by surrounding the dishes on the outside with cold water of ca. 14 DC and ca. 4 DC, respectively. to
Results
The results presented below are from recordings of 5-30 min duration in germinated pollen grains that continued to grow during electrode penetration. The values given for electric potentials are the potential differences between an «inside» compartment e.g. the cytoplasm, and the potential of the micropipette in the bathing medium immediately after withdrawal. The latter potential, although in most cases not more than a few m V different from the potential of the micropipette before puncturing, was chosen as a reference potential in order to account for changes in the tip potential of the micropipette during puncturing. The electric potentials of growing pollen grains
When the tip of the microelectrode touched a pollen grain, an unstable and transient potential of -18 to -59 mV was recorded in many cases. This potential which averaged about -40 m V in 50 such cases, most likely represents the Donnanpotential of the thick cell wall of the pollen grains. This conclusion is supported by the fact that this transient potential could be recorded only when the electrode was advanced very slowly into the grain, and by the fact that further advancement of the electrode always resulted in a jump to a new and more negative potential (Fig. 1). time (mini
r---r-~1~-----------T9--~\~ reterence potential
Fig. 1: Examples of the three types of electric potentials recorded from individual pollen grains of Lilium longiflorum growing in standard growth medium. Type 1 was recorded from a grain shortly after its germination, type 2 from a grain that had grown a tube of a few hundred 11m, and type 3 from a grain that had grown a tube of several hundred JIm and become filled with a vacuole. The electrodes entered the grains at 0 min and were withdrawn at about 15 min. Z. Pflanzenphysiol. Bd. 99. S. 313-323. 1980.
Membrane potential of growing pollen
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After puncturing the protoplast with the microelectrode three distinct types of potentials could be recorded: In pollen grains that had germinated a short time ago (i.e. ca. 10-30 min) the microelectrode measured potentials of about -90 to -130 mY. These potentials remained very stable during the entire time of measurement (Type 1 in Fig. 1). In pollen grains that had grown a tube of a few hundred flm and that had begun to form a vacuole, the electrode normally recorded a potential of about -90 to -130 mV immediately after puncturing. This potential was stable for a few minutes only, then it depolarized spontaneously by 40-50 mV and thereafter remained stable at the new level (Type 2 in Fig. 1). When the pollen grains had grown a tube of several hundred 11m or more and had become largely vacuolated, the puncturing electrode sensed a potential that was always less negative than -90 mV. These potentials depolarized for only a few mV at the beginning of the measurements and then remained stable (Type 3 in Fig. 1). The majority of the latter potentials measured about -50 to -70 m V. A quantitative comparison of the magnitudes of the potentials of type 1 and of type 2 before the spontaneous depolarizations shows that both values are of the same magnitude. The same is true for the potentials of type 3 and of type 2 after the spontaneous depolarizations. When the final, stable magnitudes of all 174 successful measurements are graphed into 10m V wide categories, two significant peaks appear in the histogram (Fig. 2). One peak appears at -100 to -110 mV and a second peak at around -60 m V. It seems not unlikely that the more negative potentials are the potentials of the cytoplasm (E,'yt) and that the less negative ones are the potentials of the vacuole (E,a,.). The responses of Ecut and of E ,w to changes of the medium
In a second series of experiments we tried to estimate the relative permeability of the plasma membrane of growing pollen grains to the major ions of the medium. )0
.
-
c
E ~
n
,----
20
f
E
,-----
,---
r-
r-r-
r-
II
40
60
80
100
- 120
l!;l
potential difference (mV)
Fig. 2: Histogram of the final, stable values of the electric potentials of 5-15 min long measurements in 174 pollen grains of Lilium longiflorum. Every potential was assigned to one of the 10mV wide categories.
Z. Pjlanzenphysiol. Bd. 99. S. 313-323. 1980.
318
M. H.
WEISENSEEL
and H. H.
WENISCH
When the ionic composition of the medium around impaled grains was rapidly changed, monitoring the effect of the changes on the electric potentials of the cytoplasm and of the vacuole showed the following results. a) Changes in the K+-concentration of the medium Decreasing the K+ -concentration of the medium caused hyperpolarizations of the membrane, increasing the K+ -concentration induced depolarizations. Quite surprisingly, the magnitude of the potential changes was always much less in recordings from the vacuole than in those from the cytoplasm. In more detail, these results were obtained (Fig. 3): o ,'
f
K+-concentratlon of the medium (mo[·[-t)
10- 3
-50
potentials of the cytoptasm
-150
'10
Fig. 3: The response of the electric potentials of the vacuole and of the cytoplasm of growing lily pollen to rapid changes in the medium's K+-concentration. Dots (e) indicate the initial K+-concentration and arrowheads (.... ) the final K+-concentration of the medium. Solid lines connect changes in K+ -concentrations that caused hyperpolarizations, dashed lines connect those that caused depolarizations. Bars indicate S.E.M. The numbers next to each line show the number of measurements compiled. The initial and final K+ -concentrations of the medium were measured with K+ -selective electrodes. A decrease of the K+ -concentration from 1.1 . 10- 3 M to 2.5 . 10-4 M induced an average hyperpolarization of the Ecyt of 36 mV (12 measurements). In another 10 measurements a decrease of the K+-concentration from 1.1 . 10-3 M to 9 . 10-5 M resulted in a hyperpolarization of the plasma membrane of 49 m V. The same two changes in the medium's K+-concentration caused much smaller hyperpolarizations of E vac , namely 12 m V in 16 measurements, and 14 m V in 11 measurements, respectively. The pH of all the media was kept at 3.9 ± 0.1. An increase in the medium's K+-concentration from 1.1 . 10-3 M to 9· 10-3 M induced an average depolarization of the Ecyt's of 66 m V (10 measurements). After several minutes the potentials began to repolarize slowly by an average of 30 m V. Upon the same increase in the K+ -concentration the Evac's depolarized by only 30 mV (28 measurements). Z. Pjlanzenphysiol. Bd. 99. S. 313-323. 1980.
Membrane potential of growing pollen
319
b) Changes in the H+ -concentration of the medium A rapid decrease of the H+ -concentration of the medium resulted in a hyperpolarization of the membrane, a rapid increase of the H+ -concentration caused a depolarization. Within the pH-range tested, i.e. between pH 4.0 and pH 6.5, the magnitude of the induced potential changes was about equal for the cytoplasm and for the vacuole. These results were obtained (Fig. 4): H· - concentration of the medium (mol·t-') O.5x
:; -50 .§
potentials of
..
the vacuole
~-100
the cytoplasm
iJ
-6
O,5x
10-~
16~ ___ ~
9, --FI---;.-~='-='-----~~~
~ potentials of
13,
~
Fig. 4: The responses of the electric potentials of the vacuole and of the cytoplasm of growing lily pollen to rapid changes in the medium's pH. The initial and final H+-concentrations of the media were measured with pH-electrodes. Otherwise as Fig. 3. Decreasing the H+ -concentration from 0.6 . 10- 4 M to 0.3 . 10- 6 M induced an average hyperpolarization of 19 m V in the E,·yt's (8 measurements). Similar hyperpolarizations of the Ent's, i.e. 5-10 mV per 10-fold decrease in H+-concentration, were obtained in 24 other pollen grains with smaller steps in changes of the pH. Also the E,'ae's responded to a 10-fold decrease in the medium's H+ -concentration with a 5-10 m V hyperpolarization, e.g. when the H+ -concentration was decreased from 0.6 . 10-4 M to 0.4 . 10- 6 M an average hyperpolarization of 22 m V was induced (12 measurements). When the H+-concentration of the medium was decreased to 1O- 6-10-'M, spontaneous and transient spikes of 50-100mV depolarization and of a duration of about half a min occurred quite frequently. We also observed that about 40 % of all pollen grains ejected small amounts of cytoplasm at their tips some time after the elevation of the pH to about 7. Increasing the H+concentration of the medium from 0.6 . 10- 5 M to 0.8 . 10- 4 M induced an average depolarization of 11 m V of the E"u.'s in 16 experiments. Experiments with larger increases in H+-concentration were not successful for two reasons: (i) Only a very small number of pollen grains germinated at pH 6 or higher, and (ii) most pollen tubes stopped growth below ca. pH 3.5. The media with the different H+ -concentrations, used in the above experiments, were obtained by adding increasing amounts of Tris-buffer to a growth medium Z, Pjlanzenphysiol. Bd. 99. S. 313-323.1980.
320
7v1. H.
WEISENSEEL
and H. H.
WENISCH
that contained 2 mM MES (instead of the usual 5 mM): Addition of 1.0 mM Tris yielded a H+-concentration of ca. 1.3 . 10- 6 M, addition of 2.0 mM, a H+-concentration of ca. 1.3 . 10- 7 M and addition of 3.0 mM Tris, a H+ -concentration of a 1.6 . 10-8 M. (The H+ -concentrations given in Fig. 4 are those in the dishes after medium exchange and deviate from the added medium's H+-concentration due to mixing.) In 18 experiments we added to the medium the H+-ionophore FCCP, to a final concentration of 2 . 10- 5 M (and 0.75 Ofo ethanol). Within 2 min after addition of FCCP the Eryt's depolarized by an average of 49 m V (6 experiments) and the Evac's depolarized by 45 mV (12 experiments). We consider these results as a hint for an electrogenic component but as still preliminary because the solvent ethanol alone could cause depolarizations, although with a much slower rate. c) Changes of the medium's Ca 2+- and Cl--concentrations Neither a decrease nor an increase in the Ca 2+-concentration of the medium induced a significant change in the electric potentials of the cytoplasm and of the vacuole. For instance, before and after decreasing the Ca 2+-concentration of the medium from 1.5 . 10- 3 M to 2.1 . 10-4 M, the Ecyt's were -106 ± 2.8 (S.E.M.) m V and -102 ± 2.0 m V, respectively (13 experiments). Under the same conditions the Evac's of 19 pollen grains were -60 ± 3.0 mV and -65 ± 3.9 m V. Lowering the Ca2+-concentration further by adding the Ca 2+-chelating agent EGTA at a concentration of 1 mM caused an almost immediate rupturing of nearly all pollen tubes at their tips. In those few pollen tubes that did not burst the clear caps filled with granular contents and all growth stopped. Before and after increasing the Ca 2+_ concentration of the medium from 1.5 . 10- 3 M to 1.2 . 10-2M the Ecyt's of pollen grains were -101 ± 4.8mV and -108 ± 4.7mV, respectively. The Evac's of 15 pollen grains were -56 ± 2.3 m V and -58 ± 2.6 m V. Decreasing the CI--concentration of the medium also had no significant effect on the electric potentials of the cytoplasm and of the vacuole. Decreasing the CI-concentration from 3.3 . 10-3 M to 3.7 . 10-4 M resulted in no change of the Ecyt of 5 pollen grains (average Ecyt before the medium exchange -113 ± 2.9 mY, after the medium exchange -113 ± 2.7 m V). In another 13 pollen grains the Evac's before and after the same change in Cl--concentration were -63 ± 3.1 mV and -61 ± 3.3 mY.
The effects
0/ CO and 0/ low temperature on Ecyt
Our previous measurements of the endogenous electric currents of growing lily pollen have suggested that the grains extrude H+, and we speculated that this H+efflux might be electrogenic and hyperpolarize the plasmalemma (WEISENSEEL and JAFFE, 1976). To test this idea, we applied CO-medium or low-temperature-medium to electrode-impaled pollen grains with the intention of halting or slowing the putative electrogenic pump. Z. PJlanzenphysiol. Bd. 99. S. 313-323. 1980.
Membrane potential of growing pollen
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When the dishes were flushed with medium that had been degassed under low pressure and then saturated over night with 99 Ofo pure CO, the pollen grains did not show any significant changes of the Eeyt's in all 6 experiments carried out (average E"yt before the medium exchange -107 ± 4.7 mY, after the medium exchange -110 ± 3.7 m V). But decreasing the temperature of the bathing medium caused substantial depolarizations of the plasma membrane: When the temperature was decreased within 5 min from 24 ± 1 c'C to 13 ± 1 °c the E"yt's depolarized by 35 mY, i.e. from an average of -110 ± 1.6 mV to -75 ± 3.3 mV (7 measurements). Applying an even larger temperature change to impaled pollen grains, namely from 24 ± 1 DC to 6 ± l~C, caused a decrease in the membrane potentials from -106 ± 2.4 mV to -63 ± 3.5 mV (14 measurements). These temperature-dependent depolarizations were found only in medium of pH 4. When we did the same experiments with pollen grains growing in medium of pH 6 (containing 2 mM MES and 1 mM Tris) the El'yj's did not respond to temperature changes and, unexpectedly, the average membrane potential was lower than in medium of pH 4. Before and after a decrease in temperature from 24 ± 1 DC to 14 ± 1°C the average Eeyt was -88 ± 2.7 mV and -85 ± 2.9 mY, respectively (6 measurements). When we decreased the temperature from 24 ± 1 DC to 5 ± 1 DC the El'yt's before and after the change were -92 ± 2.4 mV and -92 ± 2.2 mV (15 measurements). Discussion
Three types of electric potentials have been measured in the protoplasts of growing lily pollen: Potentials of ca. -90 to -130 mV, potentials of ca. -40 to -80 mV, and potentials that dropped from high values to lower ones during individual measurements. The more negative potentials were always recorded from pollen grains that had germinated quite recently and that were still densely filled with cytoplasm. The less negative potentials were recorded from highly vacuolated pollen grains that had grown fairly long tubes. In pollen grains that had started to form a vacuole the potentials of many grains showed typically large negative values at the beginning and a few minutes later the potentials dropped spontaneously to less negative values, but never the other way around. This close correlation between a large negative potential and a plasma-filled grain, and a less negative potential and a vacuolized grain suggests the interpretation that the more negative potentials are those of the cytoplasm, and that the less negative potentials are those of the vacuole. The cases with a transition of the potential during the course of a measurement then probably mean that the electrode first stuck in the cytoplasm and then penetrated into the vacuole. Although this interpretation of the measurements seems straight forward we nevertheless cannot exclude that the membrane potentials of growing lily pollen have undergone a depolarization during elongation of the tubes, resulting in less negative values in pollen grains with longer tubes. Z. Pjlanzenphysiol. Ed. 99. S. 313-323. 1980.
322
M. H.
WEISENSEEL
and H. H.
WENISCH
When the concentration of each major ion was changed during a measurement, only a change in the concentrations of K+ and H+ induced a significant response whereas changes in CI- and Ca 2+ did not cause a change in potentials. For the potentials of the cytoplasm this response was a ca. 50 m V hyperpolarization and a ca. 70 m V depolarization for a 10-fold decrease and increase in the K+ -concentration of the medium, respectively. These large responses of the Eeyt's to K+ -changes suggest a high relative permeability of the plasmalemma of growing lily pollen to K+. We were therefore quite surprised about the weak responses of the apparent Evae's to changes in the K+-concentration of the medium, i.e. a 10-fold decrease causing a hyperpolarization of ca. 15 mY, a 10-fold increase causing a depolarization of ca. 30 m V. This unexpected result could mean (i) that the tonoplast responds separately and oppositely to the plasmalemma, i.e. when the plasmalemma hyperpolarizes the tonoplast depolarizes, and vice versa. The question of how the necessary movement of charge across the tonoplast is affected when the potential of the plasmalemma is changed must remain open. It could mean (ii) that the plasma membrane of pollen grains with longer tubes has become less K+ -selectively permeable. Decreasing the H+ -concentration of the medium by 10-fold resulted in a ca. 8 m V hyperpolarization of the Eeyt's and of the Evac's, increasing it by 10-fold induced a depolarization of 10 m V of the Evac's. These results suggest that the permeability of the plasmalemma to H+ may be rather high. If we assume that the potential changes caused by changes of the pH of the medium reflect real changes of the plasmalemma, we can estimate that the H+-permeability is 8-times the K+-permeability. But, of course, a pH-change can have other effects that cause a measurable change of the Ecyt. e.g. changes in the potential of the cell wall and/or effects on the permeabilities of other ions. Our present measurements also indicate that the Ecyt of pollen grains might contain an electrogenic component: Decreasing the temperature of the medium by about 10 or 20°C resulted in large depolarizations as one might expect from decreases in the rate of temperature-sensitive pumps. On the other hand, surrounding pollen grains with CO-saturated medium did not affect their membrane potentials, in contrast to e.g. pea stem cells (ANDERSON et ai., 1974). This result is not, however, conclusive, since lily pollen grains might use other sources of energy like glycolysis or even CO- independent respiration, as do some strains of yeast and some species of Arum (TROLL and HOHN, 1973). Not so easy to interpret are the results from the experiments with temperature changes around pollen grains growing in a medium of pH 6. We had expected that this pH would cause a hyperpolarization of the plasma membrane because of the less steep gradient for an active H+ -efflux. But just the opposite happened, the membrane potentials were smaller at pH 6 than at pH 4. In order to explain this phenomenon we can only speculate that a low external pH might somehow stimulate the H+-pumps in the membrane, perhaps via an acidification of the cytoplasm. Z. Pflanzenphysiol. Bd. 99. S. 313-323. 1980.
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Acknowledgements We gratefully acknowledge reading of the manuscript and gtvmg several really helpful comments by Dr. LAURINDA JAFFE and Mr. M. BLATT. We also acknowledge the financial suppOrt of the Deutsche Forschungsgemeinschaft.
References ANDERSON, W. P., D. L. HENDRIX, and N. HIGINBOTHAM: The effect of cyanide and carbon monoxide on the electrical potential and resistance of cell membranes. Plant Physio!. 54, 712-716 (1974). CHHABRA, N. and C. P. MALIK: Influence of spectral quality of light on pollen tube elongation in Arachis hypogaea. Ann. Bot. 42, 1109-1117 (1978). JAFFE, L. A., M. H. WEISENSEEL, and L. F. JAFFE: Calcium accumulations within the growing tips of pollen tubes. J. Cell Bio!. 67, 488-492 (1975). JOHRI, B. M. and 1. K. VASIL: Physiology of pollen. The Botanical Rev. 27, 325-381 (1961). MARME, D.: Phytochrome: membranes as possible sites of primary action. Ann. Rev. Plant Physio!. 28, 173-198 (1977). MELAMED-HAREL, H. and L. REINHOLD: Hysteresis in the responses of membrane potential, membrane resistance, and growth rate to cyclic temperature change. Plant Physio!. 63, 1089-1094 (1979). TROLL, W. and K. HOHN: Allgemeine Botanik. Ferdinand Enke Verlag, Stuttgart, 572 (1973). WEISENSEEL, M. H., R. NUCCITELLI, and L. F. JAFFE: Large electrical currents traverse growing pollen tubes. J. Cell Bio!. 66, 556-567 (1975). WEISENSEEL, M. H. and L. F. JAFFE: The major growth current through lily pollen tubes enters as K+ and leaves as H+. Planta 133, 1-7 (1976). WEISENSEEL, M. H.: Changes in membrane properties and transcellular ion movements in developing plant cells. In: MARRE, E. and O. CIFERRI (Eds.): Regulation of Cell Membrane Activities in Plants, 267-274. Elsevier/North Holland Biomedical Press, Amsterdam, 1977.
M. H. WElSENSEEL, Institut fur Botanik der Universitat, Schloilgarten 4, D-8520 Erlangen.
Z. PJlanzenphysiol. Bd. 99. S. 313-323. 1980.
The Membrane Potential of Growing Lily Pollen M. H.
WElSENSEEL
and H. H.
WENISCH
With 4 figures Received March 15, 1980 . Accepted May 9, 1980
Summary Pollen grains of Lilium longiflorum growing in a liquid medium at between 20 and 25°C in continuous white light were penetrated with single microelectrodes. Two different electric potentials could be recorded from germinated grains: (i) In unvacuolated, recently germinated grains potentials of -90 to -130 mV were measured. These probably reflect the average potential difference between the cytoplasm and the medium. (ii) In pollen grains that had grown a tube of several hundred ,Hm and had become largely vacuolated an average potential of -60 mV was recorded. This might represent the potential of the vacuole. Changing the ionic composition of the bathing medium during impalement, the potentials of the cytoplasm responded to a 10-fold decrease in the medium's K+-concentration with a hyperpolarization of ca. 50 mV, and to a 10-fold increase with a depolarization of ca. 70 mV. Ten-fold changes in the K+ -concentration of the medium caused the potentials of the vacuole to respond with only 15 mV hyperpolarization, and 30 mV depolarization, respectively. Decreasing the H+ -concentration of the medium by 100-fold caused a hyperpolarization in the cytoplasm and in the vacuole of ca. 15 mV. Decreasing or increasing the Ca2 +-concentration or the CI--concentration of the medium had no significant effects on the electric potentials of the cytoplasm and of the vacuole. Lowering the temperature of the standard medium (pH 4) by about 10 or 20°C resulted in a depolarization of the plasma membrane by about 35 mV and 43 mV, respectively. No temperature-related depolarizations of the plasma membrane were observed in a growth medium of pH 6. The data obtained during this study indicate that the plasma membrane of growing lily pollen is mainly permeable to K+ -ions and that an electrogenic ion transport mechanism (probably an H+ -efflux) can contribute to the membrane potential. Key words: pollen grains, membrane potential, relative ion permeabilities, temperature effects, Lilium longiflorum. Abbreviations: CO = carbon monoxide; Ery( = electric potential of the cytoplasm; Evac = electric potential of the vacuole; EGTA = Ethyleneglycol-bis (fJ-aminoethylether) N,N'-tetraacetic acid; FCCP = Carbonyl cyanide, p-trifluoro-methoxyphenyl hydrazone, MES = 2 (N-morpholino) ethane sulfonic acid; Tris = Tris (hydroxymethyl) -aminomethane; S.E.M. = standard error of the mean.
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Introduction
Pollen grains have several attractive properties for electrophysiological investigations: (i) Dry pollen grains are in a state of arrested development that very quickly turns into active development after contact with a suitable medium, resulting in the growth of a tube within a few minutes to hours. This feature offers the opportunity to study changes in membrane properties related to development and localized growth in a higher plant cell. (ii) Ungerminated pollen grains have nO large vacuole but are rather densely filled with cytoplasm. During growth of the tube they slowly fill with a vacuole. It therefore should be possible to measure the electrical properties of the plasmalemma and of the tonoplast in the same type of a higher plant cell separately. (iii) Pollen grains contain no chlorophyll but probably do contain other pigments, as for instance carotenoids, flavins and phytochrome (JOHRI and VASIL, 1961; CHHABRA and MALIK, 1978) which may be bound to the plasma membrane as in other green or potentially green plant cells (cf. MARME, 1977). The effects of such pigments on membrane properties might therefore be studied without the interference of photosynthesis-related events. The few electrophysiological studies performed with pollen grains and tubes so far have shown that the membrane of Lilium pollen grains hyperpolarizes by about 40-50 m V before visihle growth begins (MATSCHKAL, see WEISENSEEL, 1977), and that the membrane potential and membrane resistance of Oenothera pollen tubes respond to temperature changes in a hysteretic way (MELAMED-HAREL and REINHOLD, 1979). It has also been found that growing lily pollen drive an electric current through themselves. This current enters the future site of growth and the growing tube, and leaves the non-growing part of the grain (WEISENSEEL et aI., 1975). Since the inward current consists mainly of K+ (and some Ca 2+ entering at the tip), a high permeability of the plasmalemma for K+ is suggested (WEISENSEEL and JAFFE,1976; JAFFE et aI., 1975). The outward current that is carried mainly by H+ indicates an active extrusion mechanism for H+ (WEISENSEEL and JAFFE, 1976). The aim of the present investigation was to continue our electrophysiological studies of pollen by tackling the following questions: Can the electric potentials of the cytoplasm and of the vacuole of lily pollen grains be measured separately? What are the major ions to which the plasmalemma is mainly permeable? Is the postulated H+ -transport mechanism of the growing lily pollen electrogenic? Materials and Methods Preparation and early development of pollen grains
Pollen grains were collected with a brush from the anthers of fully developed flowers of Lilium longiflorum and sputtered sparsely onto nylon nets (Scrynel HD, 85 [lm mesh width, Ziirich Bolting Cloth Mfg, Switzerland) that were stretched over plastic rings cemented to the bottom of 5 cm wide plastic petri dishes. The dishes were filled with 8 ml of medium covering the pollen grains about 0.5 mm high. The time when the dishes were filled with medium was taken as time zero for the experiment, and from this time on the Z. Pflanzenphysiol. Bd. 99. S. 313-323. 1980.
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dishes were kept in a room at 21 ± 1 cC or 24 ± 1 °C under white fluorescent light. The standard medium in which the lily pollen was grown contained the following components: 1.65 mM CaCl., 1.0 mM KN0 3 , 0.13 mM H 3 BO a, 5 mM MES-buffer and 300 mM mannitOl; pH 3.9 ± 0.1. Before use, the medium was saturated with air. In all batches ca. 50-70 °/0 of the pollen grains germinated within 60-100 min and grew a tube that elongated with a rate of up to 8.lIm min-I. Pollen grains with tubes of 100-200,Hm length appeared completely filled with cytOplasm. When the tubes elongated further a vacuole began to form at the site opposite to the location of germination. This vacuole enlarged rapidly with further elongation of the tube and filled the whole grain when the tube had reached a length of several hundred ,11m. Measurement of the electric potentials
Single microelectrodes were pushed from above into the center of growing pollen grains, under observation with a stereo microscope (Leitz, Germany). The micropipettes were fabricated from 1.5 mm wide glass capillaries containing an inner glass filament (Clark Electrochemical lnstr., England, Type GC 150 F). They were pulled to a tip of ca. 1 ,11m diameter with the aid of a vertical pipette puller (David Kopf lnstr., USA, Model 700 C). The micropipettes were then filled with cold 1 M KCl-solution by first submersing the ends into the KCI-solution and then filling the shaft with a syringe. Most of these micropipettes had excellent electrical properties, i.e. low resistances and low tip potentials. In a batch of 100 micropipettes, the average tip resistance was 5.0 ± 0.4 megohms before puncturing. The tip potentials of these pipettes, when dipped in the standard growth medium, were -3 ± 0.3 m V in 51 micropipettcs, 0 m V in 16 micropipettes and + 2.5 ± 0.3 m V in 33 micropipettes. The puncturing of pollen grains normally caused an increase in the resistance and in the tip potential of a micropipette. For instance, in 50 micropipettes checked after retraction from the grains the resistance had increased to 50 ± 4 megohms and the tip potentials had increased to -12 ± 1.2 or to + 6 ± 1.3 m V. The micropipettes were connected via a short piece of Ag-AgCI wire to a DC-preamplifier with an input impedance of larger than 1013 ohms and a leakage current of less than 4· 10-13 amps (Bioelectric Instr., USA, Model NF 1). A small fiber junction electrode with an Ag-AgCI internal (Beckman Instr., USA), grounded via a calibrator (Bioelectric lnstr., USA, Model CA 5), served as a reference electrode in the bathing medium. The electric potentials were recorded simultaneously on the screen of a storage oscilloscope (Tektronix Instr., USA, Model 5031) and on a chart recorder (Heathkit, USA, Model lR 18 M). Both the microelectrode and the reference electrode were mounted on micromanipulators (Prior, England) and the dish with the pollen grains was placed on a micropositioner table for fine horizontal adjustments (Line Tool Comp., USA). To monitOr the temperature of the bathing medium a small, electrically insulated, telethermometer probe (Yellow Springs lnstr., USA, Model 520) was dipped into the medium. In addition to the room light the dishes were illuminated from the side with a light guide (Schott a. Gen., Germany, Model KL 150 W). Medium changes around impaled pollen grains
To measure the relative permeabilities of various ions in the plasma membrane the effect of changes in the ion concentration of the medium on the electric potentials was monitored when 30 ml of new medium were introduced at one end of the dish and 30 ml were withdrawn at the opposite end. The medium change took about 1-2 min to complete and caused no change in the height of the medium above the grains. Since with this method new medium mixed with old medium, we always measured the actual concentration of the ions with ion selective electrodes (Orion Research Corp., USA) before and after the medium change. To test the effect of carbon monoxide (CO) on the electric potentials another type of medium change was employed: The dish with the pollen was placed in a larger dish and flushed Z. Pflanzenphysiol. Bd. 99. S. 313-323. 1980.
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with ca. 300 ml of CO-saturated medium. The overflow was collected in the large dish a level of about 35 mm above the grains. This increase of medium height was intended in order to bring the pollen grains further away from the surface and therewith to keep the CO-concentration around the pollen more or less constant. Temperature changes in the bathing medium were accomplished by surrounding the dishes on the outside with cold water of ca. 14 DC and ca. 4 DC, respectively. to
Results
The results presented below are from recordings of 5-30 min duration in germinated pollen grains that continued to grow during electrode penetration. The values given for electric potentials are the potential differences between an «inside» compartment e.g. the cytoplasm, and the potential of the micropipette in the bathing medium immediately after withdrawal. The latter potential, although in most cases not more than a few m V different from the potential of the micropipette before puncturing, was chosen as a reference potential in order to account for changes in the tip potential of the micropipette during puncturing. The electric potentials of growing pollen grains
When the tip of the microelectrode touched a pollen grain, an unstable and transient potential of -18 to -59 mV was recorded in many cases. This potential which averaged about -40 m V in 50 such cases, most likely represents the Donnanpotential of the thick cell wall of the pollen grains. This conclusion is supported by the fact that this transient potential could be recorded only when the electrode was advanced very slowly into the grain, and by the fact that further advancement of the electrode always resulted in a jump to a new and more negative potential (Fig. 1). time (mini
r---r-~1~-----------T9--~\~ reterence potential
Fig. 1: Examples of the three types of electric potentials recorded from individual pollen grains of Lilium longiflorum growing in standard growth medium. Type 1 was recorded from a grain shortly after its germination, type 2 from a grain that had grown a tube of a few hundred 11m, and type 3 from a grain that had grown a tube of several hundred JIm and become filled with a vacuole. The electrodes entered the grains at 0 min and were withdrawn at about 15 min. Z. Pflanzenphysiol. Bd. 99. S. 313-323. 1980.
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After puncturing the protoplast with the microelectrode three distinct types of potentials could be recorded: In pollen grains that had germinated a short time ago (i.e. ca. 10-30 min) the microelectrode measured potentials of about -90 to -130 mY. These potentials remained very stable during the entire time of measurement (Type 1 in Fig. 1). In pollen grains that had grown a tube of a few hundred flm and that had begun to form a vacuole, the electrode normally recorded a potential of about -90 to -130 mV immediately after puncturing. This potential was stable for a few minutes only, then it depolarized spontaneously by 40-50 mV and thereafter remained stable at the new level (Type 2 in Fig. 1). When the pollen grains had grown a tube of several hundred 11m or more and had become largely vacuolated, the puncturing electrode sensed a potential that was always less negative than -90 mV. These potentials depolarized for only a few mV at the beginning of the measurements and then remained stable (Type 3 in Fig. 1). The majority of the latter potentials measured about -50 to -70 m V. A quantitative comparison of the magnitudes of the potentials of type 1 and of type 2 before the spontaneous depolarizations shows that both values are of the same magnitude. The same is true for the potentials of type 3 and of type 2 after the spontaneous depolarizations. When the final, stable magnitudes of all 174 successful measurements are graphed into 10m V wide categories, two significant peaks appear in the histogram (Fig. 2). One peak appears at -100 to -110 mV and a second peak at around -60 m V. It seems not unlikely that the more negative potentials are the potentials of the cytoplasm (E,'yt) and that the less negative ones are the potentials of the vacuole (E,a,.). The responses of Ecut and of E ,w to changes of the medium
In a second series of experiments we tried to estimate the relative permeability of the plasma membrane of growing pollen grains to the major ions of the medium. )0
.
-
c
E ~
n
,----
20
f
E
,-----
,---
r-
r-r-
r-
II
40
60
80
100
- 120
l!;l
potential difference (mV)
Fig. 2: Histogram of the final, stable values of the electric potentials of 5-15 min long measurements in 174 pollen grains of Lilium longiflorum. Every potential was assigned to one of the 10mV wide categories.
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When the ionic composition of the medium around impaled grains was rapidly changed, monitoring the effect of the changes on the electric potentials of the cytoplasm and of the vacuole showed the following results. a) Changes in the K+-concentration of the medium Decreasing the K+ -concentration of the medium caused hyperpolarizations of the membrane, increasing the K+ -concentration induced depolarizations. Quite surprisingly, the magnitude of the potential changes was always much less in recordings from the vacuole than in those from the cytoplasm. In more detail, these results were obtained (Fig. 3): o ,'
f
K+-concentratlon of the medium (mo[·[-t)
10- 3
-50
potentials of the cytoptasm
-150
'10
Fig. 3: The response of the electric potentials of the vacuole and of the cytoplasm of growing lily pollen to rapid changes in the medium's K+-concentration. Dots (e) indicate the initial K+-concentration and arrowheads (.... ) the final K+-concentration of the medium. Solid lines connect changes in K+ -concentrations that caused hyperpolarizations, dashed lines connect those that caused depolarizations. Bars indicate S.E.M. The numbers next to each line show the number of measurements compiled. The initial and final K+ -concentrations of the medium were measured with K+ -selective electrodes. A decrease of the K+ -concentration from 1.1 . 10- 3 M to 2.5 . 10-4 M induced an average hyperpolarization of the Ecyt of 36 mV (12 measurements). In another 10 measurements a decrease of the K+-concentration from 1.1 . 10-3 M to 9 . 10-5 M resulted in a hyperpolarization of the plasma membrane of 49 m V. The same two changes in the medium's K+-concentration caused much smaller hyperpolarizations of E vac , namely 12 m V in 16 measurements, and 14 m V in 11 measurements, respectively. The pH of all the media was kept at 3.9 ± 0.1. An increase in the medium's K+-concentration from 1.1 . 10-3 M to 9· 10-3 M induced an average depolarization of the Ecyt's of 66 m V (10 measurements). After several minutes the potentials began to repolarize slowly by an average of 30 m V. Upon the same increase in the K+ -concentration the Evac's depolarized by only 30 mV (28 measurements). Z. Pjlanzenphysiol. Bd. 99. S. 313-323. 1980.
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b) Changes in the H+ -concentration of the medium A rapid decrease of the H+ -concentration of the medium resulted in a hyperpolarization of the membrane, a rapid increase of the H+ -concentration caused a depolarization. Within the pH-range tested, i.e. between pH 4.0 and pH 6.5, the magnitude of the induced potential changes was about equal for the cytoplasm and for the vacuole. These results were obtained (Fig. 4): H· - concentration of the medium (mol·t-') O.5x
:; -50 .§
potentials of
..
the vacuole
~-100
the cytoplasm
iJ
-6
O,5x
10-~
16~ ___ ~
9, --FI---;.-~='-='-----~~~
~ potentials of
13,
~
Fig. 4: The responses of the electric potentials of the vacuole and of the cytoplasm of growing lily pollen to rapid changes in the medium's pH. The initial and final H+-concentrations of the media were measured with pH-electrodes. Otherwise as Fig. 3. Decreasing the H+ -concentration from 0.6 . 10- 4 M to 0.3 . 10- 6 M induced an average hyperpolarization of 19 m V in the E,·yt's (8 measurements). Similar hyperpolarizations of the Ent's, i.e. 5-10 mV per 10-fold decrease in H+-concentration, were obtained in 24 other pollen grains with smaller steps in changes of the pH. Also the E,'ae's responded to a 10-fold decrease in the medium's H+ -concentration with a 5-10 m V hyperpolarization, e.g. when the H+ -concentration was decreased from 0.6 . 10-4 M to 0.4 . 10- 6 M an average hyperpolarization of 22 m V was induced (12 measurements). When the H+-concentration of the medium was decreased to 1O- 6-10-'M, spontaneous and transient spikes of 50-100mV depolarization and of a duration of about half a min occurred quite frequently. We also observed that about 40 % of all pollen grains ejected small amounts of cytoplasm at their tips some time after the elevation of the pH to about 7. Increasing the H+concentration of the medium from 0.6 . 10- 5 M to 0.8 . 10- 4 M induced an average depolarization of 11 m V of the E"u.'s in 16 experiments. Experiments with larger increases in H+-concentration were not successful for two reasons: (i) Only a very small number of pollen grains germinated at pH 6 or higher, and (ii) most pollen tubes stopped growth below ca. pH 3.5. The media with the different H+ -concentrations, used in the above experiments, were obtained by adding increasing amounts of Tris-buffer to a growth medium Z, Pjlanzenphysiol. Bd. 99. S. 313-323.1980.
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WEISENSEEL
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WENISCH
that contained 2 mM MES (instead of the usual 5 mM): Addition of 1.0 mM Tris yielded a H+-concentration of ca. 1.3 . 10- 6 M, addition of 2.0 mM, a H+-concentration of ca. 1.3 . 10- 7 M and addition of 3.0 mM Tris, a H+ -concentration of a 1.6 . 10-8 M. (The H+ -concentrations given in Fig. 4 are those in the dishes after medium exchange and deviate from the added medium's H+-concentration due to mixing.) In 18 experiments we added to the medium the H+-ionophore FCCP, to a final concentration of 2 . 10- 5 M (and 0.75 Ofo ethanol). Within 2 min after addition of FCCP the Eryt's depolarized by an average of 49 m V (6 experiments) and the Evac's depolarized by 45 mV (12 experiments). We consider these results as a hint for an electrogenic component but as still preliminary because the solvent ethanol alone could cause depolarizations, although with a much slower rate. c) Changes of the medium's Ca 2+- and Cl--concentrations Neither a decrease nor an increase in the Ca 2+-concentration of the medium induced a significant change in the electric potentials of the cytoplasm and of the vacuole. For instance, before and after decreasing the Ca 2+-concentration of the medium from 1.5 . 10- 3 M to 2.1 . 10-4 M, the Ecyt's were -106 ± 2.8 (S.E.M.) m V and -102 ± 2.0 m V, respectively (13 experiments). Under the same conditions the Evac's of 19 pollen grains were -60 ± 3.0 mV and -65 ± 3.9 m V. Lowering the Ca2+-concentration further by adding the Ca 2+-chelating agent EGTA at a concentration of 1 mM caused an almost immediate rupturing of nearly all pollen tubes at their tips. In those few pollen tubes that did not burst the clear caps filled with granular contents and all growth stopped. Before and after increasing the Ca 2+_ concentration of the medium from 1.5 . 10- 3 M to 1.2 . 10-2M the Ecyt's of pollen grains were -101 ± 4.8mV and -108 ± 4.7mV, respectively. The Evac's of 15 pollen grains were -56 ± 2.3 m V and -58 ± 2.6 m V. Decreasing the CI--concentration of the medium also had no significant effect on the electric potentials of the cytoplasm and of the vacuole. Decreasing the CI-concentration from 3.3 . 10-3 M to 3.7 . 10-4 M resulted in no change of the Ecyt of 5 pollen grains (average Ecyt before the medium exchange -113 ± 2.9 mY, after the medium exchange -113 ± 2.7 m V). In another 13 pollen grains the Evac's before and after the same change in Cl--concentration were -63 ± 3.1 mV and -61 ± 3.3 mY.
The effects
0/ CO and 0/ low temperature on Ecyt
Our previous measurements of the endogenous electric currents of growing lily pollen have suggested that the grains extrude H+, and we speculated that this H+efflux might be electrogenic and hyperpolarize the plasmalemma (WEISENSEEL and JAFFE, 1976). To test this idea, we applied CO-medium or low-temperature-medium to electrode-impaled pollen grains with the intention of halting or slowing the putative electrogenic pump. Z. PJlanzenphysiol. Bd. 99. S. 313-323. 1980.
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When the dishes were flushed with medium that had been degassed under low pressure and then saturated over night with 99 Ofo pure CO, the pollen grains did not show any significant changes of the Eeyt's in all 6 experiments carried out (average E"yt before the medium exchange -107 ± 4.7 mY, after the medium exchange -110 ± 3.7 m V). But decreasing the temperature of the bathing medium caused substantial depolarizations of the plasma membrane: When the temperature was decreased within 5 min from 24 ± 1 c'C to 13 ± 1 °c the E"yt's depolarized by 35 mY, i.e. from an average of -110 ± 1.6 mV to -75 ± 3.3 mV (7 measurements). Applying an even larger temperature change to impaled pollen grains, namely from 24 ± 1 DC to 6 ± l~C, caused a decrease in the membrane potentials from -106 ± 2.4 mV to -63 ± 3.5 mV (14 measurements). These temperature-dependent depolarizations were found only in medium of pH 4. When we did the same experiments with pollen grains growing in medium of pH 6 (containing 2 mM MES and 1 mM Tris) the El'yj's did not respond to temperature changes and, unexpectedly, the average membrane potential was lower than in medium of pH 4. Before and after a decrease in temperature from 24 ± 1 DC to 14 ± 1°C the average Eeyt was -88 ± 2.7 mV and -85 ± 2.9 mY, respectively (6 measurements). When we decreased the temperature from 24 ± 1 DC to 5 ± 1 DC the El'yt's before and after the change were -92 ± 2.4 mV and -92 ± 2.2 mV (15 measurements). Discussion
Three types of electric potentials have been measured in the protoplasts of growing lily pollen: Potentials of ca. -90 to -130 mV, potentials of ca. -40 to -80 mV, and potentials that dropped from high values to lower ones during individual measurements. The more negative potentials were always recorded from pollen grains that had germinated quite recently and that were still densely filled with cytoplasm. The less negative potentials were recorded from highly vacuolated pollen grains that had grown fairly long tubes. In pollen grains that had started to form a vacuole the potentials of many grains showed typically large negative values at the beginning and a few minutes later the potentials dropped spontaneously to less negative values, but never the other way around. This close correlation between a large negative potential and a plasma-filled grain, and a less negative potential and a vacuolized grain suggests the interpretation that the more negative potentials are those of the cytoplasm, and that the less negative potentials are those of the vacuole. The cases with a transition of the potential during the course of a measurement then probably mean that the electrode first stuck in the cytoplasm and then penetrated into the vacuole. Although this interpretation of the measurements seems straight forward we nevertheless cannot exclude that the membrane potentials of growing lily pollen have undergone a depolarization during elongation of the tubes, resulting in less negative values in pollen grains with longer tubes. Z. Pjlanzenphysiol. Ed. 99. S. 313-323. 1980.
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When the concentration of each major ion was changed during a measurement, only a change in the concentrations of K+ and H+ induced a significant response whereas changes in CI- and Ca 2+ did not cause a change in potentials. For the potentials of the cytoplasm this response was a ca. 50 m V hyperpolarization and a ca. 70 m V depolarization for a 10-fold decrease and increase in the K+ -concentration of the medium, respectively. These large responses of the Eeyt's to K+ -changes suggest a high relative permeability of the plasmalemma of growing lily pollen to K+. We were therefore quite surprised about the weak responses of the apparent Evae's to changes in the K+-concentration of the medium, i.e. a 10-fold decrease causing a hyperpolarization of ca. 15 mY, a 10-fold increase causing a depolarization of ca. 30 m V. This unexpected result could mean (i) that the tonoplast responds separately and oppositely to the plasmalemma, i.e. when the plasmalemma hyperpolarizes the tonoplast depolarizes, and vice versa. The question of how the necessary movement of charge across the tonoplast is affected when the potential of the plasmalemma is changed must remain open. It could mean (ii) that the plasma membrane of pollen grains with longer tubes has become less K+ -selectively permeable. Decreasing the H+ -concentration of the medium by 10-fold resulted in a ca. 8 m V hyperpolarization of the Eeyt's and of the Evac's, increasing it by 10-fold induced a depolarization of 10 m V of the Evac's. These results suggest that the permeability of the plasmalemma to H+ may be rather high. If we assume that the potential changes caused by changes of the pH of the medium reflect real changes of the plasmalemma, we can estimate that the H+-permeability is 8-times the K+-permeability. But, of course, a pH-change can have other effects that cause a measurable change of the Ecyt. e.g. changes in the potential of the cell wall and/or effects on the permeabilities of other ions. Our present measurements also indicate that the Ecyt of pollen grains might contain an electrogenic component: Decreasing the temperature of the medium by about 10 or 20°C resulted in large depolarizations as one might expect from decreases in the rate of temperature-sensitive pumps. On the other hand, surrounding pollen grains with CO-saturated medium did not affect their membrane potentials, in contrast to e.g. pea stem cells (ANDERSON et ai., 1974). This result is not, however, conclusive, since lily pollen grains might use other sources of energy like glycolysis or even CO- independent respiration, as do some strains of yeast and some species of Arum (TROLL and HOHN, 1973). Not so easy to interpret are the results from the experiments with temperature changes around pollen grains growing in a medium of pH 6. We had expected that this pH would cause a hyperpolarization of the plasma membrane because of the less steep gradient for an active H+ -efflux. But just the opposite happened, the membrane potentials were smaller at pH 6 than at pH 4. In order to explain this phenomenon we can only speculate that a low external pH might somehow stimulate the H+-pumps in the membrane, perhaps via an acidification of the cytoplasm. Z. Pflanzenphysiol. Bd. 99. S. 313-323. 1980.
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Acknowledgements We gratefully acknowledge reading of the manuscript and gtvmg several really helpful comments by Dr. LAURINDA JAFFE and Mr. M. BLATT. We also acknowledge the financial suppOrt of the Deutsche Forschungsgemeinschaft.
References ANDERSON, W. P., D. L. HENDRIX, and N. HIGINBOTHAM: The effect of cyanide and carbon monoxide on the electrical potential and resistance of cell membranes. Plant Physio!. 54, 712-716 (1974). CHHABRA, N. and C. P. MALIK: Influence of spectral quality of light on pollen tube elongation in Arachis hypogaea. Ann. Bot. 42, 1109-1117 (1978). JAFFE, L. A., M. H. WEISENSEEL, and L. F. JAFFE: Calcium accumulations within the growing tips of pollen tubes. J. Cell Bio!. 67, 488-492 (1975). JOHRI, B. M. and 1. K. VASIL: Physiology of pollen. The Botanical Rev. 27, 325-381 (1961). MARME, D.: Phytochrome: membranes as possible sites of primary action. Ann. Rev. Plant Physio!. 28, 173-198 (1977). MELAMED-HAREL, H. and L. REINHOLD: Hysteresis in the responses of membrane potential, membrane resistance, and growth rate to cyclic temperature change. Plant Physio!. 63, 1089-1094 (1979). TROLL, W. and K. HOHN: Allgemeine Botanik. Ferdinand Enke Verlag, Stuttgart, 572 (1973). WEISENSEEL, M. H., R. NUCCITELLI, and L. F. JAFFE: Large electrical currents traverse growing pollen tubes. J. Cell Bio!. 66, 556-567 (1975). WEISENSEEL, M. H. and L. F. JAFFE: The major growth current through lily pollen tubes enters as K+ and leaves as H+. Planta 133, 1-7 (1976). WEISENSEEL, M. H.: Changes in membrane properties and transcellular ion movements in developing plant cells. In: MARRE, E. and O. CIFERRI (Eds.): Regulation of Cell Membrane Activities in Plants, 267-274. Elsevier/North Holland Biomedical Press, Amsterdam, 1977.
M. H. WElSENSEEL, Institut fur Botanik der Universitat, Schloilgarten 4, D-8520 Erlangen.
Z. PJlanzenphysiol. Bd. 99. S. 313-323. 1980.