Planta

Planta (1984) 160: 59-65

9 Springer-Verlag t984

Membrane-potential changes in vacuoles isolated from storage roots of red beet (Beta vulgaris L.) Anthony J. Miller, John J. Brimelow and Philip John Department of Agricultural Botany, Plant Science Laboratories, University of Reading, Reading RG6 2AS, U K

Abstract. The membrane potential in vacuoles isolated from storage roots of red beet (Beta vulgaris L.) has been studied by following changes in the fluorescence of the dye 3,3'-diethylthiodicarbocyanine iodide, and by determining the uptake of the lipophilic triphenylmethylphosphonium cation. The vacuoles have a membrane potential, internal negative, which is estimated to be around - 6 0 inV. These potentials become less negative by nearly 10 mV on addition of ATP. This ATP-dependent depolarisation is inhibited by the protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone and by the ATPase inhibitors, N,N'dicyclohexylcarbodiimide and trimethyltin chloride, but it is largely insensitive to sodium orthovanadate. Fusicoccin had no significant effect on the isolated vacuoles, but its addition to excised tissue caused a hyperpolarisation of the cells measured using a microelectrode.

Key words: ATPase - Beta (membrane potential) - Membrane potential - Fusicoccin - Vacuole (membrane potential).

trogenic H + movement (Marin et al. 1981). The proton-motive force so generated is sufficient to account for the 10-fold accumulation of citrate observed with these vacuoles (Matin et al. 1981). Vacuoles isolated from red beet have an ATPase activity which has been well characterised (Leigh and Walker 1980; Walker and Leigh 1981), and has many features in common with the ATPase of the Hevea vacuo-lysosomes (d'Auzac 1977). However, in contrast to the Hevea ATPase, activity of the beet ATPase has been reported to be linked to a hyperpolarisation of the electrically negative vacuole interior (Doll and Hauer 1981). In beet, the translocated ions have not been identified, but it seems likely that H + are involved (Doll and Hauer 1981). Thus in the Hevea and beet vacuoles similar ATPases appear to be driving H + in opposite directions. In view of this apparent important difference between these two types of vacuole we have re-examined the polarity, magnitude and other characteristics of the membrane-potential changes associated with ATP hydroysis by vacuoles isolated from red beet. Material and methods

Introduction

It is generally believed (see Wagner 1982) that the accumulative uptake of solutes into plant vacuoles depends upon the activity of an ATPase located at the tonoplast, although work with isolated vacuoles has provided little evidence of an ATP-dependent energisation of the tonoplast. In those vacuoles which have been examined most thoroughly, the vacuo-lysosomes isolated from the latex of Hevea brasiliensis, it has been shown that ATP hydrolysis drives an inwardly-directed, elecAbbreviations: DCCD=N,N'-dicyclohexylcarbodiimide; DiSCa-(5)=3,Y-diethylthiodicarbocyanine iodide; F C C P = c a r bonylcyanide p-trifluoromethoxyphenylhydrazone; TPMP + = triphenylmethylphosphonium ion

Plant material and chemicals. Storage roots of red beet (Beta vulgaris L.) were purchased locally or were grown in the experimental grounds at the University of Reading, and stored in moist vermiculite at 5 ~ C. The tritiated triphenylmethylphosphonium ion ([3H]TPMP +) as the iodide salt was purchased from Amersham International plc, U K ; metrizamide from Nyegaard UK, Birmingham, U K ; 3,3-diethylthiodicarbocyanine iodide (DiS-C2-(5)) from Eastman Kodak, Rochester, USA; trimethyltin chloride from Aldrich Chemical Co., Dorset, U K ; TPMP + as the bromide salt was obtained from the Chemistry Department, University of Reading; disodium ATP was from Boehringer. Fusicoccin was a generous gift from Professor A. Ballio, University of Rome, Italy. Vacuole isolation. Vacuoles were isolated by two methods, both of which are based on the large-scale, tissue-slicing technique of Leigh and Branton (1976). In the principal method the vacuoles were purified by flotation through a layer of metrizamide. This method was essentially as described by Leigh et al.

60 (1979) for the isolation of vacuoles from washed slices of beet. In this method the collection medium contained 1.0 M sorbitol, 50raM 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris)-C1 (pH 8.0), 5 mM disodium ethylenediaminetetraacetate (Na2EDTA), 20 mM potassium metabisulphite and 0.1% dextran sulphate. The resuspension medium contained 1.2 M sorbitol, 10 mM Tris-2-(N-morpholino)ethanesulfonic acid (Mes); pH 7.6), 1 mM Na z EDTA. After the vacuoles were removed from the metrizamide gradient, they were diluted in approx. 3 vol. of a medium containing 1.2 M sorbitol, 10 mM Tris-MES (pH 7.6), and 5 mM MgC12. Then they were sedimented by centrifugation at 500 g for 5 rain, and resuspended in about 3 ml of the same medium. In the second method, the vacuoles were purified by sedimentation through a dextran density gradient. The method described by Doll et al. (1979) was modified so that the collection and resuspension media were as described above, and the discontinuous density gradient of dextran was composed of 5 ml of 20% (w/v) dextran and 10 ml of 5% (w/v) dextran overlain by vacuoles suspended in the resuspension medium described above. After centrifugation as in Doll et al. (1979), vacuoles were collected from the interface of the 5% (w/v) and 20% (w/v) layers, then washed and resuspended as described above. The betanin present was determined as in Leigh et al. (1979), and expressed as the betanin units defined by Leigh and Branton (1976). Fluorescence measurements. The ATP-dependent membranepotential changes were followed by monitoring the fluorescence changes of the cyanine dye DiS-C2-(5 ). The fluorescence of the DiS-C2-(5) was measured at ambient temperature (20-25 ~ using a Perkin-Elmer LS-3 Fluorescence Spectrometer (PerkinElmer, Beaconsfield, Bucks., UK). Excitation was at 650 nm and emission was measured at 670 nm. Vacuole suspension (0.5 ml) containing 60-100 betanin units was added to a reaction mixture which contained in a final volume of 3 ml: 1.2 M sorbitol, 10 mM Tris-C1 (pH 7.6), 5 mM MgC1z and 0.4 gM DiS-Ca-(5). Uptake of TPMP +. The membrane potential was determined from measurements of the uptake of the lipophilic cation TPMP +, the distribution of which across the tonoplast membrane is assumed to arrive at an electrochemical equilibrium with the membrane potential according to the Nernst equation (see Rottenberg 1979). Vacuoles (20-60 betanin units m1-1) were incubated at 26 ~ C in a medium which contained 10 pM [3H] TPMP + (2.76 M Bq mmol-a), 1.2 M sorbitol, 10 mM Tris-Mes (pH 7.6), and 5 mM MgC12. The mixtures were shaken gently to maintain the vacuoles in suspension. After an appropriate period the vacuoles were separated from the incubation medium using the phthalate-oil-layer separation technique described by Leigh et al. (1981). Aliquots (0.3 ml) of the incubation media were overlain on a 1 : 1 (v/v) mixture of diethyl and dibutyl phthalate and sedimented by centrifugation for 30 s in a Beckman Microfuge (Beckman-RllC, High Wycombe, Bucks., UK). The end of the centrifuge tube containing the pellet was severed, shaken with 1 ml of 35 mM sodium dodecyl sulphate containing 0.8 mM Na 2 EDTA and 40 mM Tris-C1 (pH 7.5) (Leigh et al. 1981), and the mixture was centrifuged for 2 min in a Beckman Microfuge. Betanin in the supernatant was estimated spectrophotometrically as described by Leigh et al. (1979). This procedure ensured that only the betanin contained within vacuoles was used as a basis for the determination of vacuole volumes; it also allowed us to take into account the failure of a proportion (

Membrane-potential changes in vacuoles isolated from storage roots of red beet (Beta vulgaris L.).

The membrane potential in vacuoles isolated from storage roots of red beet (Beta vulgaris L.) has been studied by following changes in the fluorescenc...
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