Planta 9 Springer-Verlag 1983

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1 Planta (1983)159: Planta 9 Springer-Verlag 1983 Rapid response of the plasma-membrane potential in oat coleoptiles to auxin and other weak acids George W. Bates * and Mary Helen M. Goldsmith Department of Biology, Kline Biology Tower, Yale University, New Haven, CT 06511, USA Abstract. We have compared the effects of the auxin, indole-3-acetic acid (IAA) with that of other weak acids on the plasma-membrane potential of oat (Arena sativa L.) coleoptile cells. Cells treated with ] gm IAA at ph 6 depolarize mv in ]0-12min, but they then repolarize, until by rain their potentials are about 25 mv more negative than the initial value. Similar concentrations of benzoic and butyric acids cause the initial depolarization, but not the subsequent hyperpolarization. The hyperpolarization is therefore specific to IAA. All the weak acids, including IAA, evoke a rapid hyperpolarization when their concentrations are raised to 10 mm. This result indicates that at high concentrations, the uptake of undissociated weak acids activates electrogenic proton pumping, most likely by lowering cytoplasmic ph. In contrast, the hyperpolarization observed with concentrations of IAA four orders of magnitude lower appears to be a specific hormonal effect. This specific, auxin-induced hyperpolarization occurs at the same time as the initiation of net proton secretion and supports the hypothesis that auxin initiates extension growth by increasing proton pumping. Key words: Acid, weak, uptake - Auxin, action and specificity - Arena, IAA and membrane potential - Coleoptile - Cytoplasmic ph - Electrogenic proton transport - Plasma membrane potential. Introduction Proton extrusion by plant cells is widely thought to be the result of an electrogenic pump (for re- * Present address: Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA Abbreviations: FC = fusicoccin; IAA = indole-3-acetic acid views, see Poole 1978; Spanswick ]98]). In higher plants, the observation that the fungal toxin fusicoccin (FC) both stimulates net H + efflux and hyperpolarizes the membrane potential (Marr6 et al. 1974; Cleland et al. 1977; Marr~ ]977, 1979; Felle ]982) provides support for the idea that H + secretion is indeed electrogenic. Although auxin also stimulates H + secretion (Rayle and Cleland 1977) and hyperpolarizes the membrane (Lfittge et al. 1972; Etherton 1970; Marr6 et al. 1974; Mizuno et al. 1980), its effects on membrane potential are different from those of FC. Instead of a monotonic increase in negativity, auxin causes an initial depolarization which is followed, at about the time that H + secretion can be detected, by a more sustained hyperpolarization (Cleland etal. 1977; Nelles 1977; but compare Mizuno et al. 1980). The transient depolarization occurs during the latency for auxin-induced H + secretion and might, along with an increased rate of protoplasmic streaming (Sweeney and Thimann 1937), represent one of the earliest known responses to auxin. Since auxin is a weak acid and is accumulated in the cell to a considerable concentration (Rubery and Sheldrake 1974; reviewed by Goldsmith 1977), we were interested to know whether both the depolarizing and hyperpolarizing phases of the membrane's response were specific to IAA. For this reason, we have investigated, over an extended range of concentration (] M), the time course of the electrical response to IAA and two other weak acids, butyric and benzoic, which, although they lack auxin activity, possess a pk similar to that of IAA. Since for the present study we have selected impalements showing both low resistance and highly negative membrane potential, the responses we report occur primarily at the plasma membrane rather than the tonoplast (Goldsmith et al. 1972; Bates et al. 1982a, b).

2 232 G.W. Bates and M.H.M. Goldsmith: Response of membrane potential of oats to IAA Material and methods All experiments were performed on Arena coleoptile cells using the standard electrophysiological techniques previously described (Bates et al. 1982a). Seedlings of Arena sativa L. cv. Victory (Swedish Seed Assoc., Sval6f, Sweden) were grown for 4 d in the dark. All subsequent manipulations were carried out under normal room light. Coleoptiles were cut, and the primary leaf and 3 mm of the tip were removed. The coleoptiles were then cut into 12-mm sections and were split longitudinally through the sides of the coleoptile not containing the vascular bundles. The split sections were floated for 2 h on a basal medium containing 0.1 mm KCI, 0.1 mm CaC12, 1 mm morpholinoethanesulfonic acid (Mes) adjusted to ph 6 with 2-amino- 2(hydroxymethyl)-l,3-propandiol (Tris-base; Sigma Chemical Co., St. Louis, Mo., USA). The split sections, cut surface up, were secured in a lucite chamber (volume 0.5 ml) (Bates et al. 1982a). The tissue was aerated by a continuous flow of buffer over the section at a rate of about 1 ml rain- 1. The bath was mounted on a microscope stage, and membrane voltage was determined by inserting a micropipette electrode filled with 3 M KC1 into one of the large parenchyma cells in the cell layer just below the cut surface while observing at 160-fold magnification. The reference electrode was in the bath downstream from the tissue. The figures show the typical response of an individual cell, and Table 1 gives the means for the maximum depolarization, and final values of membrane potential in replicate experiments_+ the standard error. All potentials reported here are from low-resistance (< 5 Ms impalements. Although the microelectrode tip is in the vacuole, we have previously presented evidence that when the input resistance is less than about 8 MQ, the contribution of the tonoplast to the measured voltage is greatly diminished by leakage shunts, with the result that such impalements record largely the voltage across the plasma membrane (Goldsmith et al. 1972; Bates et al. 1982a). Solutions of benzoic acid were made with sodium benzoate. Solutions of butyric acid and IAA were adjusted to ph 6 with Tris-base. Changing solutions during electrical recording was accomplished by a manifold. Since FC combines rapidly with a high-affinity receptor (Dohrmann et al. 1977), it was effective when added as a 10-gl drop (10-3 M) to the upstream end of the flow bath. For simultaneous measurements of proton and butyric-acid release, segments of coleoptile were prepared as described above except the epidermal layer was peeled off and a single 10-mm segment cut. Fourteen such segments were preincubated for I h in 1 mm KCl, 0.1 mm CaCIz, 0.1 mm Mes adjusted to ph 6 with NaOH before transferring to 2 ml of a similar solution containing 10 mm KC1 and 10 mm butyric acid labeled with Bq of sodium [1-t4C]butyrate (specific activity 5.10 it Bq mol ~; New England Nuclear, Waltham, Mass., USA), and ph adjusted to 6 with NaOH. After 1 h incubation, the sections were rinsed twice (15 s ) in butyrate-free solution. After each rinse, the solution retained in the central hollow of the cylinder was flushed out with a stream of O2 and the sections blotted dry. The sections were suspended in 2 ml of continuously oxygenated, nnbuffered butyrate-free solution, and the ph of the solution was continuously monitored with a flat-surface combination ph electrode (No ; Beckman Instruments, Fullerton, Calif., USA) connected to a chart recorder. Simultaneously, efflux of radioactivity into the medium was measured by scintillation counting of 20-gl aliquots, taken every minute for the first 6 min, then at 2-min intervals for 6-20 min, and at 4-rain intervals thereafter. At the end of the experiment, the total acidity released by the sections was backtitrated with dilute NaOH to the initial ph. -I 'l I I I I l I I I I I I 1 I l F I t I I I I I I I I I I I I I Fig. 1. Comparison of the response of oat coleoptile plasma membrane to IAA and FC. Arrow indicates addition of IAA by a change of the bath flowing over a split segment from basal salt solution (0.1 mm KCI+0.1 mm CaC12) buffered at ph 6 with 1 mm Mes (see Material and methods) to one with basal salts+10-6 gm IAA. Fusicoccin added (at arrow) as a 10 gl aliquot of 10-3M directly to the upstream end of the bath -I00 I I I I I I I I I I I I I 10-6M IAA /'~ ", ~EE -120 ~ ~. """,. > ~ " i I I I I I I t I _0 24 Fig. 2. Comparison of response of the membrane to 10-4 M and 10-6 M IAA at ph 6.0. Arrows indicate addition of IAA. The composition of the medium flowing by the split segment of oat coleoptile in this and subsequent figures is given in Fig. 1. The difference in the lags before hyperpolarization is significant while the final membrane potentials are not (see Results and Table 1) Results The initial response to 10-s M IAA (Fig. 1) in Mes buffered at ph 6 containing 0.1 mm of both KC1 and CaC12 was a depolarization of mv which lasted about 10 rain. This phase was followed by repolarization, during which the membrane potential ultimately became mV more negative than the original potential. Under these conditions, the potential did not display rhythmic oscillations (compare G6ring et al. 1979; Stahlberg and Polevoy 1979). In contrast to IAA, 10-5 M FC elicited only a rapid hyperpolarization of mv, after a lag of 1 rain or less, with no intervening depolarizing phase (Fig. 1). These results are very similar to those reported by Cleland et al. (1977) except that the initial depolarizations in IAA observed here were substantially

3 G.W. Bates and M.H.M. Goldsmith: Response of membrane potential of oats to IAA 233 Table 1. The initial depolarization and final value of the membrane potential achieved by oat coleoptile cells in indoleacetic acid and other weak acids. The weak acids were administered in a flowing bath that contained 0.1 mm KC1, 0.1 mm CaC12 and 1 mm Mes buffered at ph 6.0 Treatment Concn. No." Depolariza- Final (M) tion b membrane (mv) potential r (my) IAA IAA _2.5 c 14.5_+2.0 c -151_ _+3 Benzoic acid _+3.0 d, e Benzoic acid Butyric acid _+ 3.0 e d - 91 _+ 3-96_+4 a Number of cells impaled b Mean_+ SE c Figure 2 shows the kinetics of the response of one of these cells in I0.6 M IAA and another in 10-4 M a Figure 3 shows the kinetics of the response of one of these cells in 10.6 M benzoic acid and another in 10-6 M butyric acid 9 Figure 4 shows the kinetics of one of these cells in 10.6 M benzoic acid and another in 10-4 M f Stable value of membrane potential achieved after at least 20 min exposure to IAA or other weak acid larger. Although the latency to onset of hyperpolarization is concentration-dependent for both FC (Rubinstein and Cleland 1981) and auxin (see below) the available evidence shows a close correlation in time between hyperpolarization and efflux of H (Cleland 1976a, b; Jacobs and Ray 1976; Cleland et al. 1977; Rubinstein and Cleland 1981). This indicates that both compounds stimulate an electrogenic secretion of protons. At 10-4M IAA, the lag before the beginning of repolarization is significantly reduced (7.5 versus 10 rain, p < 0.01). Consequently cells depolarize less in I0-4M IAA than in 100-fold lower auxin concentrations (Fig. 2). Subsequently the cells repolarize to a final value that is not significantly different in the two concentrations (Table 1). To determine whether the initial depolarization is a specific response to IAA or a more general property of lipophilic weak acids, we investigated the effects of benzoic and butyric acids. Weak acids will accumulate in the cytoplasm because of the ph gradient across the plasma membrane (Collander J 959; Rubery and Sheldrake 1974; Davies and Rubery 1978; Sussman and Goldsmith 1981). Both benzoic and butyric acids at 10-6M produced depolarizations similar to that seen with IAA (compare Figs. 2, 3). Although several experiments lasted for more than 30 min, neither of these weak acids caused any hyperpolarization at such low concentrations (10-6 or 10.4 M; Table 1). Re- -BO i i i i I i i i i -I00 --" > I0-6 M ".EE BENZOATE,,/~~ "" ~-- I0-6 M BUTYRATE -140 J f i I I I I I I Fig. 3. Low concentrations of benzoic and butyric acids cause a sustained depolarization of oat coleoptile cells. Time courses for two typical impalements after addition of the acid at time zero. (See Table 1 for mean data) -80 I ~, ~ i i ~ i -I00 I0-6 M >E B ~ O A..-'''" T E... E > -120 ~--~--'10-4 M BENZOATE -140 f R I J I I I I Fig. 4. The magnitude of the benzoic-acid-induced depolarization of oat coleoptile cells is concentration dependent. Time course for two typical cells after addition of 10-6 or 10-4 benzoic at time zero. (See Table 1 for mean data) moval of the weak acid from the bath resulted in a slow and incomplete recovery of the initial potential over a period of I h (data not shown). The size of the depolarization did not depend strongly on concentration; an increase from 10-6 to IO-~M produced only slightly larger depolarizations (see Fig. 4). Although prior treatment with another weak acid reduced the initial depolarization in response to IAA, the subsequent hyperpolarization was as great in the presence as in the absence of 10-6 M benzoic acid (Fig. 5). Thus the depolarization in weak acid is probably not the result of inactivating the electrogenic proton pump that is stimulated by auxin. At still higher concentrations, the response to weak acids changed dramatically. At 10-2 M, both butyric acid and IAA produced only a rapid, massive hyperpolarization of about 50 mv (Fig. 6). (The transients seen in Fig. 6 on changing from buffer to 10-2 M butyric acid and back to buffer resulted from the different amounts of Tris needed to adjust the solutions to ph 6. Using equal amounts of Tris-base in both the basal and butyric-

4 234 G.W. Bates and M.H.M. Goldsmith: Response of membrane potential of oats to IAA I I I I I I 1 I I I I I I I I I I 10-6 M -I00 - ~ -, I I I I I I I I I l I I I I I I I _L Fig. 5. Pretreatment with 10-6 M benzoic acid starting at time zero reduces the subsequent depolarization of oat coleoptile cells by IAA added after 9 rain without affecting the timing or magnitude of hyperpolarization. At the upward arrow, the solution was changed from the buffered salt solution (ph 6) plus 10 6 M benzoic to one with the same composition plus 10-6 M IAA I I l I I I I I I I I I _80 84 I -ioo[ '~E li-2 M BUTYRATE # Fig. 6, High concentrations of butyric acid administered in the buffered salt solution (ph 6) cause a rapid hyperpolarization that is reversible on rinsing in butyric acid-free buffer (second arrow). Both solutions were adjusted to ph 6 with Tris-base. Similar results were also obtained with 10-z M IAA acid solutions and adjusting the ph of the basal buffer to 6 with HC1, eliminated these voltage transients.) In 10-2 M IAA, the response was also a rapid hyperpolarization similar to that seen with butyric acid in Fig. 6. It is reasonable to postulate that at 10 -z M the accumulation of weak acid acidifies the cytoplasm and leads to an electrogenic efflux of protons. Because solutions of these acids at 10-2M have substantial buffering capacity at ph 6, it was not feasible to verify this postulate experimentally by measuring H + efflux. Unlike the FC response, which is essentially irreversible (Felle 1982), the hyperpolarization produced by 10-2 M butyric acid can be readily reversed by flushing the chamber (Fig. 6). When the 7.0 t I I I ' ph 6.0 I0 ph 5.0 ~ I I I Fig. 7. Efflux of radioactivity and acidity from peeled 10-mm oat coleoptile segments following pretreatment for I h in 10 mm [l~c]butyric acid and rinsing in butyric-acid free solution (Further details given in Material and methods). The numbers on the ~4C axis represent 105 times molarity (M) of [14C]butyric acid in the efflux medium butyric-acid solution was replaced by buffer (Fig. 6, second arrow), the membrane depolarized about 80 mv in 8 min. The potential did not return to its initial value, however, but depolarized further to a value similar to that seen in low concentrations of butyrate (see Fig. 3). This depolarization probably reflects shutting down of electrogenie H + secretion resulting from efflux of butyric acid from the cell (Fig. 7) and the decreased acid load. Not surprisingly, it required a longer time for the efflux of 14C from the entire tissue (Fig. 7) than for the depolarization of a superficial cell (Fig. 6). Discussion We observe that in M IAA, oat coleoptile cells depolarize transiently before hyperpolarizing, while in nonhormonal weak acids (butyric and benzoic) the depolarization occurs but is maintained. When the concentration of IAA or butyric acid is raised to 10-2 M, however, the only response is an immediate hyperpolarization which continues for as long as the weak acid is present in the bath. Similarly, root cells also show an immediate hyperpolarization in I and 2.5 mm butyric acid (Marr6 et al. 1982) which is accompanied by an increased uptake of K + and reduced by erythrosin B, an inhibitor of the plasma membrane's ATPase. In Neurospora, where it has been possible to measure cytoplasmic ph, butyric-acid concentrations in this range are sufficient to overcome the buffering capacity of the cytoplasm, and the fall in cytoplasmic ph triggers increased proton pumping (Sanders et al. 1981). In all probability, 14 c 0

5 G.W. Bates and M.H.M. Goldsmith: Response of membrane potential of oats to IAA 235 the explanation for the hyperpolarization of coleoptile cells at 10-2 M weak acid is similar; however, the sustained hyperpolarization of higherplant cells in 10-2M weak acid differs from the transient one that is obtained with the fungus. In Neurospora, the conductance of the membrane increases and the cell depolarizes (Sanders et al. 1981). Thus, the conductance increase reduces the potential gradient against which the Neurospora cell must work to unload the excess H +. Coleoptile cells, however, do not show a conductance increase under comparable conditions. Lacking this specialization, these cells, which ordinarily do not encounter large quantities of organic acids in their environment, remain hyperpolarized, and therefore eliminate the excess acid less efficiently than Neurospora does. If the hyperpolarization in 10-2 M weak acids is due to acidification of the cell, it is highly unlikely that the IAA-induced hyperpolarization at 10-6 M, a concentration four orders of magnitude lower, can be similarly explained. Other weak acids do not evoke hyperpolarization at such low concentration; thus this response is specific to auxin. The transient depolarization that precedes the hyperpolarization in response to micromolar IAA concentrations has also been observed by others (Cleland et al. 1977; Nelles 1977) and might signal a detectable effect of auxin that precedes the increased proton efflux. Under our conditions, however, other nonhormonal weak acids also cause a similar depolarization which, unlike that in auxin, is maintained for at least half an hour. Therefore, only the hyperpolarization induced by micromolar IAA can be considered to be a specific hormonal response. The coincidence in timing between the repolarization that occurs only in auxin and the measureable decrease in external ph provides strong support for the hypothesis (Cleland 1982) that the auxin-induced ph drop is the result of electrogenic H + secretion. It should be noted that the impalements reported here displayed initial input resistances of 5 Ms or less. With such impalements, the contribution of the tonoplast to the intracellular electrical potential has been largely eliminated (Bates et al. 1982a), and the observed responses to auxin, other weak acids, and FC reside primarily at the plasma membrane. Both aliphatic and aromatic weak acids are known to cause rapid changes in membrane permeability (Jackson and Taylor 1970; Glass and Dunlop 1974) at ph 5. The effect of formic, acetic, or propionic acid on roots is pervasive and rapid; over several hours respiration is severely inhibited, the buffering capacity of the tissue falls, and the roots leak a major fraction of their ionic contents (Jackson and Taylor 1970). Perhaps the most likely explanation for the depolarization seen here with low concentrations of weak acids, as well as for reports of rapid effects of auxin on membrane structure (Helgerson et al. 1976; Morr6 and Bracker 1976; Vian et al. 1976) and permeability (Loros and Taiz 1982), is that weak acids alter membrane permeability by partitioning into the membrane. If changes in membrane permeability are involved, then the ionic composition of the bathing medium as well as the physiological condition of the plant material will likely influence the results. In addition to the present observations on the membrane potential, there are several other reports of an initial, transient effect of IAA that is opposite in sign to that observed after the min lag period. For example, (1) the growth rate of sections of oat and corn coleoptile (Rayle et al. 1970) as well as pea stems (Rayle et al. 1970; Barkley and Evans 1970) may decrease prior to increasing after addition of 10-4 M IAA. (2) Corn coleoptiles with an asymmetric auxin source may execute an initial curvature towards the source before the onset of curvature away (Ullrich 1978) and (3)also may display a small lateral potential opposite in sign (measured with probes on the surface of the coleoptile) to the final electropositive one (Morath and Hertel 1978). Each of these transient responses, including the depolarization by IAA and weak acids reported here, occurs before the growth rate increases, and thus has suggestively similar timing. On the basis of our observation that nonhormonal weak acids cause a similar depolarization, we suggest that these may all be nonspecific effects of IAA unrelated to its hormonal action. Other authors have, however, come to the opposite conclusion, perhaps because they failed to take into account that benzoic acid, phenylacetic acid, 2-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid, being much less readily transported than IAA, would also be less effective than IAA even in a nonspecific action because they would have less access than IAA to many of the cells in segments covered with a cuticle. While recent discussions have focused on a possible action of auxin directly on the plasma membrane, and more specifically on the possibility that auxin stimulates a proton-pumping ATPase, there is as yet no conclusive evidence that the plasma membrane is the primary site of auxin action (e.g. Cleland 1982; Cross et al. 1978). An alternative worthy of further investigation is that auxin alters cellular respiration in such a way as to increase

6 236 G.W. Bates and M.H.M. Goldsmith: Response of membrane potential of oats to IAA the availability of protons to the pump or to change the ATP/ADP ratio. Yamaki (1954) has shown that there is a substantial increment in the respiration of Arena coleoptiles as well as dramatic changes in respiratory quotient occurring within the lag period before auxin stimulates elongation; early enough, therefore, to account for the hyperpolarization caused by physiological concentrations of IAA ( M). Enhanced fixation of CO 2 into malic acid could increase the supply of protons to the pump (Smith and Raven 1976), but in oat coleoptile segments auxin-stimulated CO 2 fixation occurs too late (Haschke and L/ittge 1975) to account for the onset of hyperpolarization. This research is supported by grants from The Whitehall Foundation and the National Science Foundation. We are grateful to T.H. Goldsmith for discussions. Fusicoccin was a gift from Montedison S.P.A., Milano, Italy. References Barkley, G.M., Evans, M.L. (:t970) Timing of the auxin response in etiolated pea stem sections. Plant Physiol. 45, Bates, G.W., Goldsmith, M.H.M., Goldsmith, T.H. (1982a) Separation of tonoplast and plasma membrane potential and resistance in cells of oat coleoptiles. J. Membr. Biol. 66, Bates, G.W., Goldsmith, M.H.M., Goldsmith, T.H. (1982b) Origins and measurement of the membrane potentials in Arena coleoptiles. In: Plasmalemma and tonoplast: their functions in the plant cell. Proc. of Int. Workshop on Plasmalemma and Tonoplast of Plant Cells, pp , Marm~, D., Marr6, E., Hertel, R., eds. Elsevier Biomedical, Amsterdam New York Oxford Collander, R. (1959) Cell membranes: their resistance to penetration and their capacity for transport. In : Plant physiology - A treatise, vol. 2, pp , Steward, F.C., ed. Academic Press, New York London Cleland, R.E. (1976a) Kinetics of hormone-induced H + excretion. Plant Physiol. 58, Cleland, R.E. (1976b) Fusicoccin-induced growth and hydrogen ion excretion of Arena coleoptiles: relation to auxin responses. Planta 128, Cleland, R.E. (1982) The mechanism of auxin-induced proton efflux. In: Plant growth substances 1982, pp , Wareing, P.F., ed. Academic Press, New York London Cleland, R.E., Prins, H.B.A., Harper, J.R., Higinbotham, N. (1977) Rapid hormone-induced hyperpolarization of the oat coleoptile potential. Plant Physiol. 59, Cross, J.W., Briggs, W.R., Dohrmann, U.C., Ray, P.M. (1978) Auxin receptors of maize coleoptiles do not have ATPase activity. Plant Physiol. 61, Davies, P.J., Rubery, P.H. (1978) Components of auxin transport in stem segments of Pisum sativum L. Planta 142, Dohrmann, U., Hertel, R., Pesci, P., Cocucci, S.M., Marr+, E., Randazzo, G., Ballio, A. (1977) Localization of'in vitro' binding of the fungal toxin tusicoccin to plasma membrane rich fractions from corn coleoptiles. Plant Sci. Lett. 9, Etherton, B. (1970) Effect of indole-3-acetic acid on membrane potential of oat coleoptile ceils. Plant Physiol. 45, Felle, H. (1982) Effects of fusicoccin upon membrane potential, resistance and current-voltage characteristics in root hairs in Sinapis alba. Plant Sci. Lett. 25, Glass, A.D.M., Dunlop, J. (1974) Influence of phenolic acids on ion uptake. Plant Physiol. 54, Goldsmith, M.H.M. (1977) The polar transport of auxin. Annu. Rev. Plant Physiol. 28, Goldsmith, M.H.M., Fernandez, H., Goldsmith, T.H. (1972) Electrical properties of parenchymal cell membranes in the oat coleoptile. Planta 102, G6ring, H., Polevoy, V.V., Stahlberg, R., Stumpe, G. (1979) Depolarization of transmembrane potential of corn and wheat coleoptiles under reduced water potential and after IAA application. Plant Cell Physiol. 20, Haschke, H.P., L/ittge, U. (1977) Action of auxin on CO 2 dark fixation in Arena coleoptile segments as related to elongation growth. Plant Sci. Lett. 8, Helgerson, S.L., Cramer, W.A., Morr6, D.J. (1976) Evidence for an increase in microviscosity of plasma membranes from soybean hypocotyls induced by the plant hormone indole-3- acetic acid. Plant Physiol. 58, Jackson, P.C., Taylor, J.M. (1970) Effects of organic acids on ion uptake and retention in barly roots. Plant Physiol. 46, Jacobs, M., Ray, P.M. (1976) Rapid auxin-induced decrease in free space ph and its relationship to auxin-induced growth in maize and pea. Plant Physiol. 58, Loros, J., Taiz, L. (1982) Auxin increases the water permeability of Allium cells. Plant Sci. Lett. 26, Liittge, U., Higinbotham, N., Pallaghy, C.K. (1972) Electrochemical evidence of specific action of indole acetic acid on membranes of Mnium leaves. Z. Naturforsch. Tell B 2"/, Marr+, E. (1977) Effects of fusicoccin and hormones on plant cell membrane activities: observations and hypotheses. In: Regulation of cell membrane activities in plants, pp , Marr6, E., ed. Elsevier/North-Holland, Amsterdam Marr6, E. (1979) Fusicoccin a tool in plant physiology. Annu. Rev. Plant Physiol. 30, MarrY, E., Lado, P., Ferroni, A., Ballarin Denti, A. (1974) Transmembrane potential increase induced by IAA, benzyladenine, and fusicoccin. Correlation with proton extrusion and cell enlargement. Plant Sci. Lett. 2, MarrY, M.T., Romani, G., Cocucci, M., Marr6, E. (1982) Internal ph and transmembrane potential as regulators of the activity of the proton pump of higher plants. International Workshop on Membranes and Transport in Biosystems, pp , Laterza, Bari, Italy Mizuno, A., Katuo, K., Okamoto, H. (1980) Structure and function of the elongation sink in the stems of higher plants. I. Effects of anoxia and IAA on the growth rate and the spatially separate electrogenic ion pumps. Plant Cell Physiol. 21, Morath, M., Hertel, R. (1978) Lateral electric potential following asymmetric auxin application to amize coleoptiles. Planta 140, MorrO, D.J., Bracker, C.E. (1976) Ultrastructural alteration of plant plasma membranes induced by auxin and calcium ions. Plant Physiol. 58, Nelles, A. (1977) Short term effects of plant hormones. Planta 147, Poole, R.J. (1978) Energy coupling for membrane transport. Annu. Rev. Plant Physiol. 29, Rayle, D.L., Evans, Mi., Hertel, R. (1970) Action of auxin on cell elongation. Proc. Natl. Acad. Sci. USA 65,

7 G.W. Bates and M.H.M. Goldsmith: Response of membrane potential of oats to IAA 237 Rayle, D.L., Cleland, R.E. (1977) Control of plant cell enlargement by hydrogen ions. Curr. Top. Dev. Biol. 11, Rubinstein, R., CMand, R.E. (1981) Responses of Arena coleoptiles to suboptimal fusicoccin: kinetics and comparisons with indoleacetic acid. Plant Physiol. 68, Rubery, P.H., Sheldrake, A.R. (1974) Carrier-mediated auxin transport. Planta 118, Sanders, D., Hansen, U.P., Slayman, C.L. (1981) Role of plasma membrane proton pump in ph regulation in non-animal cells. Proc. Natl. Acad. Sci. USA 78, Smith, F.A., Raven, J.A. (1976) H + transport and regulation of cell ph. In: Encyclopedia of plant physiology, N.S., vol. 2: Transport in plants IIA: Cells, pp , Lfittge, U., Pitman, M., eds. Springer, Berlin Heidelberg New York Spanswick, R.M. (1981) Electrogenic ion pumps. Annu. Rev. Plant Physiol. 32, 26%289 Stahlberg, R., Polevoy, V.V. (1979) Nature of the rhythmic oscillations of the membrane potential in corn coleoptile cells [in Russian]. Dokl. Bot. Sci. 247, Sussman, M.R., Goldsmith, M.H.M. (1981) Auxin uptake and action of N-l-naphthylphthalamic acid in corn coleoptiles. Planta 151, Sweeney, B.M., Thimann, K.V. (1937) The effects of auxins on protoplasmic streaming, I1. J. Gen. Physiol. 25, 439~461 Vian, B., Mosiniak, M., Roland, J.-C1. (1976) Alterations ultrastructurales du plasmalemme de Phaeolus aureus iuduites par l'auxine sur cellules enti6res et membranes isol6es. Ann. Sci. Nat. Bot. S~r. 12, 17, Ullrich, C.H. (1978) Continuous measurement of initial curvature of maize coleoptiles induced by lateral auxin application. Ptanta 140, Yamaki, T. (1954) Effect of indoleacetic acid upon oxygen uptake, carbon dioxide fixation and elongation of Arena coleoptile cylinders in darkness. Sci. Pap. Coll. Gen. Educ. Univ. Tokyo 4, ] Received 8 March; accepted 8 June 1983