Planta (1983)159:329-335

P l ~ J n ~ 9 Springer-Verlag 1983

Energy coupling for membrane hyperpolarization in Lemna: respiration rate, ATP level and membrane potential at low oxygen concentrations H. L6ppert Botanisches Institut der Universit/it fiir Bodenkultur, Gregor Mendel Strasse 33, A-1180 Vienna, Austria

Abstract. Respiration rate, ATP content and membrane potential of L e m n a have been measured as a function of the concentration of dissolved oxygen. Kinetic analysis showed that within the range from I gM to 20 gM O2, the respiration rate of isolated mitochondria and intact plants was a hyperbolic function of the oxygen concentration. The apparent Michaelis constant (Kin) for the oxygen of respiration of intact plants (1.15___0.08 laM) is close to that for isolated mitochondria (1.07 _ 0.06 gM), so that diffusion of oxygen within the tissue was obviously not rate-limiting under the applied experimental conditions. The ATP level decreased in parallel with the respiration rate when the oxygen concentration was reduced. In contrast, the hyperpolarization of the membrane potential above the diffusion potential had already decreased at oxygen concentrations where the respiration rate and ATP level remained practically unchanged and was completely abolished at oxygen concentrations above the K m of respiration. This result is discussed according to the current models for electrogenic pumps. It is concluded that ATP cannot be the fuel for the electrogenic process under investigation. Key words: ATP and hyperpolarization - Electric potential - L e m n a -- Membrane potential (energy coupling) - Respiration (membrane hyperpolarization).

Introduction Electrogenic uniport of protons is commonly regarded to be responsible for a component of the membrane potential (Era) of plant cells which is Abbreviations." E,. = membrane potential, Ea = diffusion poten-

tial

dependent on metabolic activity. Its inhibition by ATPase inhibitors, inhibitors of electron transport and uncouplers of phosphorylation has favoured the view that the proton pump is fuelled by ATP (for a recent review, see Spanswick 1981). However, only in few instances has the dependence of E m on ATP supply been directly demonstrated. The striking correlation of changes of E m with changes of the ATP level in N e u r o s p o r a (Slayman et al. 1970) strongly supports the assumption of an electrogenic ATPase. The affinity of the electrogenic process for MgATP (Slayman et al. 1973) corresponds well with the affinity of an ATPase and a proton pump in membrane vesicles from this organism (Scarborough 1976, 1980). With perfused cells of Chara, it has been possible to demonstrate (without the use of inhibitors) that the membrane potential is strictly dependent upon ATP (Shimmen and Tazawa 1977). Similar unequivocal support for an ATPfuelled electrogenic pump has not been obtained with higher plants. Upon addition of C N - to carrot root tissue, E m depolarized with a half-time of about 10 s (Anderson etal. 1977). Although ATP was not measured, the authors compared their results with the data of Atkinson et al. (1966) and concluded that according to the latter, membrane depolarization is about 20 times too fast to be attributed to ATP decay. A discrepancy between the half-times of C N - - i n d u c e d ATP decay and membrane depolarization has also been observed with cotton cotyledons and L e m n a (UllrichEberius et al. 1983). From experiments with red beet tissue, ATP levels have been compared with membrane potentials at different concentrations of respiratory inhibitors (Mercier and Poole 1980). The dramatic depolarization within a narrow range of the ATP level may be taken as support for an electrogenic ATPase only in the case where proof is obtained for the involvement of regulatory

330

processes. The most striking and still unexplained discord between cellular ATP and E mhas been observed with Lemna (L6ppert 1981). The membrane potential did not respond to a reduction of cellular ATP by aresenate or N,N'-dicyclohexyl carbodiimide (DCCD), and a high ATP level, maintained by cyclic photophosphorylation, could not support the electrogenic process in the absence of oxygen. As these results were found to be incompatible with the commonly accepted concept of an ATPfuelled electrogenic pump in the cell membranes of plants, further experiments seemed indispensable. However, the interpretation of the results from inhibitor experiments is always complicated by the risk that the action of the applied substances may not be confined to ATP generation or consumption, and, therefore, and alternative experimental approach has been used in the present investigation: the ATP supply was controlled by adjusting the respiration rate at different values when the oxygen concentration was reduced to levels below saturation of the terminal oxidases.

H. L6ppert: Energy coupling for membrane hyperpolarization

N2

I

4

inlet suction Fig. 1. Plant chamber for measurement of membrane potential of Lemna at low dissolved-oxygen concentrations. A plant was placed on a glass rod (1) and kept in position with a membrane strip (2); the two parts of the chamber were pressed together with springs (3). Bathing solution from a reservoir (not shown) was forced by gravity through the chamber; constant flow rate is important to obtain stable and reproducible signals from the oxygen electrode (4). A reference electrode, inserted into a bore (5) and a micropipette (6) were used for potential measurement

t

Material and methods Culture conditions. Lemna aequinoctialis Welwitsch ( = L. paucicostata Hegelm.) strain 6746 was cultured under axenic conditions in the medium of Datko et al. (1980). The concentrations of the macronutrients were: 1.0 mK KNO 3 , 0.4 mM KHzPO4, 1.4 mM Ca(NO3) z and 0.4 mM Mg(NO3)z; pH was adjusted to 5.8 with KOH. The plants were illuminated with a highpressure discharge lamp (HQIL 400 W, Osram) for 16 h d 1 with an energy fluence rate of 22 W m -2. For all experiments, plants consisting of a young mother frond with a small daughter in the right meristematic pocket were selected from the cultures. Measurement of membrane potential. The micropipettes for impalement of the tissue were drawn from borosilicate glass capillaries with an inner filament (Clark Electromedical Instruments Pangbourne, UK). They were filled with 3 M KC1 and had tip potentials from - 1 0 to - 1 8 mV and tip resistances from 10 to 15 Mf~. The electric circuit consisted of Ag-AgC1 electrodes, connected to an electrometer amplifier (WPI, New Haven, Conn., USA; Model 707). Current injection through the built-in bridge circuitry was performed with a pulse generator (Gould, Hainault, U K ; Model PG 58 A); the signals from the bridge output and current monitor were displayed on a dualtrace oscilloscope (Gould Model OS 3000 A), the signal from the probe output was registrated with a chart recorder (BBC Goerz, Vienna, Austria; Model 460). The measurements at low oxygen concentrations required a special design of the plant chamber (Fig. 1). A plant was mounted on a glass rod (0.6 mm in diameter) with a 1-mmbroad strip of 10-gin thick polyethylene membrane, so that only a minimum area of the plant surface was not in direct contact with the medium. The oxygen concentration in the bathing solution, measured with a Clark-type electrode (Yellow Springs Instruments, USA, Model 53), could be adjusted to any desired value by mixing Nz-bubbled medium with air-saturated medium. Because of the small volume of the chamber (0.35 ml) and the high flow rate (about 10 ml rain 1), the bathing solution within the chamber was rapidly exchanged and

N2

6

4

o.j inlet

Fig. 2. Plant chamber for respiration measurements. The Lemna plants floated below a nylon net (1) and thus damage by the stirring bars (2) was avoided. Mercury seals (3) at the ground-in joint (4) and the 'O'-ring seal (5) of the oxygen electrode (6) prevented diffusion of atmospheric oxygen into the glass chamber a high streaming velocity was achieved at the plant surface. To exclude diffusion of atmospheric oxygen into the chamber, the 'O'-ring seal and the outer compartment of the chamber were gassed with N z. Positioning of the microelectrode and the plant chamber was performed with micromanipulators (Leitz, Wetzlar, F R G and Emerson, Cambridge, Mass., USA, respectively) during alternate observations through vertically and horizontally mounted stereo microscopes (magnification x 80). Respiration measurements with intact plants. Up to 10 plants were enclosed in an all-glass chamber as shown in Fig. 2. The oxygen concentration of the bathing solution within the chamber was initially brought to the desired level as described above. For measurement of respiration rate, the flow of bathing solution was stopped and the oxygen concentration within the

H. L6ppert: Energy coupling for membrane hyperpolarization

331

ATP determination. Four plants were enclosed between nylon

-I-T-[IIII1!IIIIII21

6

Fig. 3. Plant chamber for freeze-stop removal of Lemna plants from bathing solution at low dissolved-oxygen concentrations. The plants were enclosed between nylon nets (1), covering the bore in an aluminum disc (2). A spinning disc (3) above the oxygen electrode (4) produced a constant streaming velocity at the membrane; temperature was controlled by circulating water (5). Upon pushing the polyethylene plate (6) into the indicated direction, two aluminum cylinders (7) are moved by solenoids towards the plants chamber was maintained constant by automatically performed titration with air-saturated or oxygen-saturated medium. The amplitudes of the oscillations of oxygen concentration, caused by titration, were smaller than 0.13 gM in the range below 5 laM O 2 and 0.25 gM above 5 gM 02. The respiration rate was calculated from the added volume within a certain time interval (usually 20-30 rain).

Respiration measurements with isolated mitoehondria. The method of Day and Wiskich (1974) has been modified for isolation of mitochondria from Lemna. The plants (20 g fresh weight) were kept for 30 rain at 4 ~ C and were then transferred to 60 ml chilled isolation medium, which contained 0.3 M sucrose, 75mM 2-amino-2-(hydroxymethyl)-l,3-propane diol (Tris)HCI of pH 7.2, 15mM ethylene diaminetetraacetic acid (EDTA), 0.1% bovine serum albumin and I mM cysteine. The plants were disrupted with a high-speed cutting rod (Krups, Solingen, FRG), operated for 20 s at full speed. The homogehate was stained through a double layer of "miracloth" (Calbiochem, La Jolla, Calif., USA) and centrifuged at 2500 g for 10 rain. Mitochondria were precipitated from the supernatant by centrifugation at 12,000 g for 15 rain and washed three times by consecutive resuspension and sedimentation, using the reaction medium (0.25 M sucrose, 10 mM Tris-HCl of pH 7.2, t0 mM KHzPO4, 5 mM MgCI2 and 0.5 mM EDTA). All procedures were carried out at 2 ~ C. Oxygen uptake was measured in the chamber used for intact plants (Fig. 2) after addition of K § malate and ADP (final concentrations: 10 mM and 5 raM, respectively). The suspension of mitochondria was gassed with N 2 prior to insertion of the electrode. After closing the chamber, the oxygen concentration was adjusted to the desired value by addition of an air-saturated suspension of mitochondria. The respiration rate was determined in the same way as for whole plants; the values were corrected for dilution of the suspension caused by titration.

nets within the bore in an aluminium disc and were inserted in the oxygen-electrode chamber as shown in Fig. 3. Bathing solution was forced through the chamber by gravity with a flow rate of 10 ml rain -1. After about 30 min, the aluminum disc was pushed out of the chamber and was immediately brought into contact, on both sides, with aluminum cylinders precooIed in liquid nitrogen. The frozen disc of bathing solution, enclosing the plants, was then removed from the bore and subjected to freeze-drying. The plants were extracted as described previously (L6ppert 1981); the ATP content of the samples was analyzed using the luciferin-liciferase assay, using a high-sensitivity test combination (Boehringer, Mannheim, FRG). During all experiments, the temperature was maintained at 25 ~ C. Since for technical reasons, temperature control using circulating water was not possible in certain parts of the plant chambers, the ambient temperature was kept constant and was continuously monitored.

Results and discussion Respiration o f isolated mitochondria and intact plants. T h e r e s p i r a t i o n o f i s o l a t e d m i t o c h o n d r i a s h o w e d the e x p e c t e d h i g h affinity t o w a r d s o x y g e n . W i t h i n the i n v e s t i g a t e d r a n g e o f o x y g e n c o n c e n t r a tions, kinetic analysis yielded a single h y p e r b o l a (Fig. 4a), as s h o w n b y l i n e a r i z a t i o n in a d o u b l e r e c i p r o c a l p l o t (Fig. 4b). A c c o r d i n g to the principles o f e n z y m e kinetics, a n a p p a r e n t Michaelis c o n s t a n t (K,,) w a s c a l c u l a t e d (Fig. 4). It is h i g h e r t h a n the K,, o f the oxidases in m u n g - b e a n m i t o c h o n d r i a (0.1 g M a n d 0.5 ~tM, a c c o r d i n g to I k u m a et al. 1964) a n d l o w e r t h a n the K m o f the oxidases in Chlorella (2.1 g M a n d 6.7 g M , a c c o r d i n g to Sarg e n t a n d T a y l o r 1972). T h e kinetics o f r e s p i r a t i o n o f i n t a c t p l a n t s (Fig. 5a, b) is in g o o d a g r e e m e n t w i t h the results obtained with isolated mitochondria. Therefore, d i f f u s i o n o f o x y g e n w i t h i n the tissue d o e s n o t seem to be rate-limiting u n d e r the a p p l i e d e x p e r i m e n t a l c o n d i t i o n s . This c o n c l u s i o n is a n i m p o r t a n t p r e r e q uisite f o r the i n t e r p r e t a t i o n o f p o t e n t i a l m e a s u r e m e n t s with i n t a c t p l a n t s at l o w o x y g e n c o n c e n t r a tions. T h e d e v i a t i o n f r o m t h e o r y o f the e x p e r i m e n tal values in the r a n g e o f s a t u r a t i o n (Fig. 5) is a p o i n t o f f u r t h e r i n v e s t i g a t i o n a n d will n o t be disc u s s e d in the p r e s e n t c o n t e x t .

A T P content o f intact plants. T h e decrease in respir a t i o n rate w i t h d e c r e a s i n g o x y g e n c o n c e n t r a t i o n is clearly reflected in the A T P c o n t e n t (Fig. 6). Alt h o u g h a h y p e r b o l i c f u n c t i o n c a n n o t be e x p e c t e d b e c a u s e o f r e g u l a t i o n , it a p p e a r s , t h a t the A T P level parallels the r e s p i r a t i o n rate (Fig. 7). T h e r e fore, the A T P s u p p l y in the d a r k c a n be effectively c o n t r o l l e d w h e n the r e s p i r a t i o n rate is limited b y r e d u c i n g the o x y g e n c o n c e n t r a t i o n in the b a t h i n g

332

H. L6ppert: Energy coupling for membrane hyperpolarization

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0.2 014 016 0B 110 1/)JM 0 2 Fig. 4a, b. Respiration rate o f isolated mitochondria o f Lemna as a function of dissolved-oxygen concentration. The values from a are shown in a double reciprocal plot in b. The value of K m, inserted in b, is the mean from four different experiments

0.2 0.4 0.6 0.8 1.0

b

1/pM 02

Fig. 5 a, b. Respiration rate of intact Lemna plants as a function of dissolved-oxygen concentration. The values from a, calculated on a FW basis, are shown in a double reciprocal plot in b. The curve in a corresponds to the linear extrapolation in b. The inserted value of K m is the mean from six different experiments

solution to levels below saturation of the terminal oxidase. Membrane potential in relation to dissolved oxygen concentration. The membrane potentials (Era) reported in the present investigation, represent the electrical potential difference between the vacuole and the external solution and have been derived from stationary-state measurements" After certain changes in the external conditions had taken place, the membrane potential was allowed to attain stable values before readings were made. The hyperpolarization of Em above the diffusion potential (Ed) could be determined after depolarization by C N - or anaerobic conditions in the dark (L6ppert 1981). Mean values of Em and E~, obtained with plants which have been grown in complete culture medium, are summarized in Table 1. The E m- E d values responded in a sensitive way to changes in the dissolved-oxygen concentration of the bathing solution below 20 gM 02

60

~ 50 ,~ -~ 4o E ~ 30 w 20 ~ 10 o_

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8 10 12 14 16 18 20 /uM 02

Fig. 6. The ATP content of intact Lemna plants as a function of dissolved oxygen concentration. The plants were kept at a specific oxygen concentration for about 30 min before freezestop removal from the bathing solution. ATP content was calculated on a FW basis

H. L6ppert: Energy coupling for membrane hyperpolarization

333 100

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w

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Fig, 7. ATP content of intact Lemna plants as a function of respiration rate. For the plot, values of respiration rate and ATP content at various oxygen concentrations have been taken from the curves in Fig. 5 and Fig. 6

Table 1. Steady-state values of the membrane potential and the passive diffusion potential. The values are the m e a n • SE from (N) experiments. To obtain K+-free medium, KNO3 and KHzPO 4 were omitted from the culture medium Vacuolar potential (mY)

Bathing solution

Complete medium K +-free medium

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Fig. 8. The response of the active component of the membrane potential of Lemna to changes in the dissolved-oxygen concentration. A complete curve was obtained from measurements with the same impaled cell. Measurements performed with plants from different harvests yielded practically identical curves. The bathing solution was complete culture medium (e) or culture medium from which K + has been omitted (A)

(Fig. 8). Although presence or absence of K + in the bathing solution affected E m - E d and the kinetics of its changes with decreasing oxygen concentration, Era- Ed was always completely extinguished at oxygen concentrations above the K m

2

40

60 80 t00 ATP content (%)

Fig. 9. The active component of the membrane potential as a function of respiration rate and ATP content of intact Lemna plants. For the plot, graphically interpolated values of respiration rate, ATP content and membrane potential at various oxygen concentrations have been obtained from Figs. 5b, 6 and 8. Curve 1: plants in complete medium, curve 2: plants in K § free medium

of respiration (Fig. 8). When K + was present in the bathing solution, E,, depolarized suddenly to E e when the oxygen concentration reached 4 gM, although the oxygen concentration was reduced in extremely small steps (A[O2]=0.25 gM per step) in this region. An explanation is not possible at present, but is not essential for the argument below. Membrane potential in relation to A T P supply. To elucidate the role of mitochondrial oxidative phosphorylation for the hyperpolarization of E,, above Ed, E m- - E d at different oxygen concentrations may be compared with the corresponding values of respiration rate or ATP content. Such a comparison is justified, as all results have been obtained from stationary-state measurements and plants have been kept under practically identical external conditions in all experiments. From the plots in Fig. 9, it is obvious that E m - E ~ may undergo considerable changes, although the ATP level is constant, and that minor changes in respiration rate and ATP level are paralleled by a depolarization of E,, to E d. The compatibility of this result with the assumption of a pump ATPase may be discussed according to the current concepts for electrogenic pumps (for reviews see: Poole 1978; Bentrup 1979; Spanswick 1981). When the pump is considered to operate near its equilibrium potential Ep and the conductivity of the pump channel exceeds by far the conductivities of passive diffusion channels, Ep and hence E m will be affected as soon as the free-energy change of the non-transported component of the process is changed, according to the equation

334

Ep-

H. L6ppert: Energy coupling for membrane hyperpolarization

AG O' vF

RT ATP vF In A D P . P i

0.059ApH,

where v denotes the H + / A T P ratio, AG o' the standard free energy change in ATP hydrolysis, R the gas constant, T the temperature, F the Faraday constant and Pi orthophosphate. Although it is not possible to calculate reliable values for Ep because the actual situation, seen by the pump, is not known from experimental data, changes of E~ in response to changes in ATP supply may be estimated from the second term of the equation. For this purpose, the following assumptions may be made. Firstly, the cytosolic A T P / A D P ratio may be taken as 8; values of 9.1 and 7.5 have been reported by Goller et al. (1982) and Stitt et al. (1982) for oat and wheat leaf protoplasts in the dark. Secondly, changes in the ATP content, measured with intact plants, may be attributed to changes in cytosolic ATP, as mitochondria give a minor contribution to the cellular ATP content and the ATP level within the chloroplasts undergoes only a small change upon inhibition of energy supply in the dark (Goller et al. 1982). Thirdly, for simplicity, the increase in the cytosolic ADP concentration may be taken as equal to the decrease in ATP concentration, knowing that this will result in an overestimation of the potential change, as the ADP increase will be much smaller than the ATP decrease (Goller et al. 1982). Finally, the cytosolic concentration of inorganic phosphate may be regarded as practically constant at small changes of the ATP level, because in Lemna the concentration of inorganic phosphate in the cytoplasm is about 15 m M in comparison with about 1 m M of ATP (data not shown). Under these presuppositions, a 10% decrease in the ATP content of the plants should depolarize Ep only by about 20 mV when v is assumed to be unity or by 10 mV when v is assumed to be 2. Although these values bear a certain error, they are undoubtedly incompatible with the experimental results (Fig. 9). On the other hand, when the equilibrium potential of the pump is far from E,, and the pump conductivity is negligible by comparison with membrane conductivity, the membrane potential is under kinetic control. The hyperpolarization of E,, above E d should be proportional to'the current through the pump. Since the latter is assumed to depend on ATP, E,, should change as a consequence of a change in the ATP supply. As already mentioned (see Introduction), this situation is encountered in Neurospora. In contrast with Lemna, the results shown in Fig. 9 do not reflect the properties of a pump ATPase, acting as a current source. The steep decrease of E,~- E d might be at-

tributed to regulation of pump activity or changes in membrane resistance. However, a depolarization also occurred without a change of the ATP level, so that coupling of hyperpolarizing pump activity to oxidative phosphorylation is rather unlikely. Although an explanation of the effect of external K + on Era-Ed and on the kinetics of its depolarization is not possible at present, it is important to state that the above conclusions apply, irrespective of the presence or absence of K + in the bathing solution. So it does not appear that in Lemna different mechanisms of energy coupling are operative at different K § concentrations, as has been suggested for corn roots (Cheeseman et al. 1980). The results discussed above seem to be important in two respects. Firstly, the view that ATP is not the fuel for the electrogenic pump in Lemna has gained further support. It seems legitimate, therefore, to continue investigations on the mechanism of coupling of membrane hyperpolarization to cellular metabolism. In this respect, a possible involvement of extramitochondrial electron transport should not be completely neglected. Support for the existence of electron transport in the plasmalemma, which affects the membrane potential, comes from recent investigations on corn root protoplasts (Lin 1982a, b) and cultured carrot cells (Craig and Crane 1981, 1982). Secondly, the finding that the electrogenic pump in Lemna can be inhibited without affecting cellular ATP, may serve to distinguish between ATP-driven primary active transport of solutes and secondary active transport, which is dependent upon the activity of the electrogenic pump. The author wishes to thank Professor Riklef Kandeler for his encouraging interest and support. Thanks are also due to Mr. Ernst Scharfetter for carefully attending to the sterile plant cultures. Financial support from the "Fonds zur F6rderung der wissenschaftlichen Forschung" of the Republic of Austria is gratefully acknowledged.

References Anderson, W.P., Robertson, R.N., Wright, B.J. (1977) Membrane potentials in carrot root cells. Aust. J. Plant Physiol. 4, 241-252 Atkinson, M.R., Eckermann, G., Grant, M., Robertson, R.N. (1966) Salt accumulation and adenosine triphosphate in carrot xylem tissue. Proc. Natl. Acad. Sci. USA 55, 560-564 Bentrup, F.W. (1979) Reception and transduction of electrical and mechanical stimuli. In : Encyclopedia of plant physiology, N.S. vol. 7: Physiology of movements, pp. 42-70, Haupt, W., Feinleib, M.E., eds. Springer, Berlin Heidelberg New York Cheeseman, J.M., La Fayette, P.R., Gronewald, J.W., Hanson, J.B. (1980) Effect of ATPase inhibitors on cell potential and K + influx in corn roots. Plant Physiol. 65, 1139-1145

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Craig, Th.A., Crane, F.L. (1981) A transplasmamembrane electron transport system in plant cells. Plant Physiol. [Suppl.] 67, 99 Craig, Th.A., Crane, F.L. (1982) Transplasmamembrane electron transport shows hormonat control and produces membrane hyperpolarization. Plant Physiol. [Suppl.] 69, 151 Datko, A.H., Mudd, S.H., Giovanelli, J. (1980) Lemna paucicostata Hegelm. 6746 Development of standardized growth conditions suitable for biochemical experimentation. Plant Physiol. 65, 906-912 Day, D.A., Wiskich, J.T. (1974) The oxidation of malate and exogenous reduced nicotinamide adenine dinucleotide by isolated mitochondria. Plant Physiol. 53, 104-109 Goller, M., Hampp, R., Ziegler, H. (1982) Regulation of the cytosolic adenylate ratio as determined by rapid fractionation of mesophyll protoplasts of oat. Effect of electron transfer inhibitors and uncouplers. Planta 156, 255 263 Ikuma, H., Schindler, F.J., Bonner, W.D. (1964) Kinetic analysis of oxidases in tightly coupled plant mitochondria. Plant Physiol. [Suppl.] 39, 60 Lin, W. (1982a) Responses of corn root protoplasts to exogenous NADH: oxygen consumption, ion uptake and membrane potential. Proc. Natl. Acad. Sci. USA. 79, 3773-3776 Lin, W. (1982b) Isolation of NADH oxidation system from the plasmalemma of corn root protoplasts. Plant Physiol. 70, 326-328 L6ppert, H. (1981) Energy coupling for membrane hyperpolarization in Lemna: evidence against an ATP-fueled electrogenic pump as the exclusive mechanism. Planta 151, 293-297 Mercier, A.J., Poote, R.J. (1980) Electrogenic pump activity in red beet: its relation to ATP levels and to cation influx. J. Membr. Biol. 55, 165 174

Poole, R.J. (1978) Energy coupling for membrane transport. Annu. Rev. Plant Physiol. 29, 437460 Sargent, D.F., Taylor, C.P.S. (1972) Terminal oxidases of Chlorella pyrenoidosa. Plant Physiol. 49, 775-778 Scarborough, G.A. (1976) The Neurospora plasma membrane ATPase is an electrogenic pump. Proc. Natl. Acad. Sci. USA 73, 1485-1488 Scarborough, G.A. (1980) Proton translocation catalyzed by the electrogenic ATPase in the plasma membrane of Neurospora. Biochemistry 19, 2925-2931 Shimmen, T., Tazawa, M. (1977) Control of membrane potential and excitability of Chara cells with ATP and Mg 2+ . J. Membr. Biol. 37, 167-192 Slayman, C.L., Lu, Ch.Y.-H., Shane, L. (1970) Correlated changes in membrane potential and ATP concentrations in Neurospora. Nature (London) 226, 274-276 Slayman, C.L., Long, W.S., Lu, C.Y.-H. (1973) The relationship between ATP and an electrogenic pump in the plasma membrane of Neurospora crassa. J. Membr. Biol. 14, 305-338 Spanswick, R.M. (1981) Electrogenic ion pumps. Annu. Rev. Plant Physiol. 32, 267-289 Stitt, M., Lilley, R. McC., Heldt, H.W. (1982) Adenine nucleotide levels in the cytosol, chloroplasts, and mitochondria of wheat leaf protoplasts. Plant Physiol. 70, 971-977 Ullrich-Eberius, C.I., Novacky, A., Ball, E. (1983) Effect of cyanide in dark and light on the membrane potential and the ATP level of young and mature green tissues of higher plants. Plant Physiol. 72, 7-15

Received 7 April; accepted 5 July 1983