Planta

Planta (1987) 171:235-240

9 Springer-Verlag 1987

Effects of fusicoccin and abscisic acid on, glucose uptake into isolated beetroot protoplasts Hans-Peter Getz, Michael Schulte-Altedorneburg and Johannes Willenbrink Botanisches Institut der Universitfit K61n, Gyrhofstrasse 15, D-5000 K61n 41, Federal Republic of Germany

Abstract. Uptake of glucose, 3-O-methylglucose and sucrose into beetroot protoplasts is considerably stimulated by ] 0 - 6 M fusicoccin. This effect is decreased in the presence of 10 m M Na + or K +, 2 m M Mg 2+ or Ca 2+. Whereas fusicoccin causes no change in the pH-optimum of the sugar uptake (pH 5.0), the apparent Km of this uptake which obeys a biphasic kinetics is decreased by the action of fusicoccin. In the protoplast suspension, fusicoccin induces an acidification which is suppressed by uncoupling agents. Correspondingly, uncouplers as well as vanadate and diethylstilbestrol markedly inhibit the effect of fusicoccin on sugar uptake. The present data support the view that glucose uptake into beetroot protoplasts depend on the proton-pumping activity of the plasmalemma-ATPase, cis-Abscisic acid diminishes significantly the fusicoccin-enhanced glucose uptake. By using a radioimmunoassay, the internal abscisic acid content of the protoplast was estimated to be in the range of 10 -6 M. Protoplasts .isolated from bundle tissue contain twice as much abscisic acid as those derived from storage parenchyma. Because protoplasts from the bundle tissue were shown to take up sugars much faster than those from the storage cells, the observed effect of abscisic acid might reflect an involvement of this hormone in the regulation of carbohydrate partitioning in the beet.

Key words: Abscisic acid (effect on sugar uptake) - ATPase (plasmalemma) - Beta (sugar uptake) - Fusicoccin (effect on sugar uptake) - Protoplast. Abbreviations." ABA=cis-abscisic acid; bundle protoplast= protoplasts isolated from the conducting tissue of beetroots, DES = diethylstilbestrol; FC = fusicoccin; 3-OMG = 3-O-methylglucopyranose; PCMBS =p-chloromercuribenzenesulfonic acid; storage protoplasts=protoplasts isolated from storage parenchyma

Introduction

Fusicoccin (FC) stimulates H+-extrusion at the plasmalemma and causes a rapid increase in the plasma membrane potential (Marr6 1979; Marr6 and Ballarin-Denti 1985). Although not unequivocally accepted (Lin and Giaquinta 1979), a H +/K +exchange mediating ATPase is believed to be involved in this action (Lado et al. 1980; Felle 1982). It is therefore logical to assume that F C stimulates uptake of solute, i.e. sugars (Marr6 1980; Colombo et al. 1978; K o m o r et al. 1981). Recently it could be shown for protoplasts isolated from beetroot that glucose - and to a lesser extent sucrose - is taken up at a rate that is pH-dependent (Getz et al. 1987). The uptake which is highly specific exhibits characteristics typical for systems which are energized by a transport ATPase. Results concerning effects of cis-abscisic acid (ABA) on assimilate transport and partitioning published so far are quite contradictory (Wyse et al. 1980; Saftner and Wyse 1984; Wyse 1985). On the one hand, A B A seems to interfere with substrate uptake, e.g. in stimulating uptake of glucose and glycine in Lemna gibba G1 (Hartung et al. 1980). On the other hand ABA is said to counteract FC-action on sugar uptake (Malek and Baker 1978) and H+/K+-exchange (Jung and Ltittge 1980). Therefore effects of FC on promotion and inhibition of glucose uptake need to be studied. Special attention was paid to interaction between FC and ABA. Since ABA is known as a growth regulator which is supposed to interact with sugar transport, we tried to determine the in vivo ABA-content of the protoplasts in order to avoid application of excessively high concentrations of this substance in the respective experiments.

236

Materials and methods Plant material and chemicals. Uniform seeds of Beta vulgaris L. (red beet, type " R o t e Kugel, new globe or little ball") were obtained from van Waveren Pflanzenzucht G m b H , G6ttingen, and grown as previously described (Willenbrink et al. 1984). Sorbitol, sodium dodecyl sulfate (SDS) and D-glucose were purchased from Merck (Darmstadt, F R G ) , trypsin inhibitor from Serva (Heidelberg, F R G ) and phenyl-methylsilicone oil AP 100 from K a h m a n n and Ellerbrock (Bielefeld, FRG). Radiochemicals were from A m e r s h a m Buchler (Braunschweig, FRG). All other chemicals were provided by Sigma (Miinchen, FRG). Fusicoccin was a kind gift of Dr. Ballio, Roma.

Protoplast isolation and uptake experiments were performed according to Getz et al. (1987). Experiments were run at room temperature (220_+2 ~ C) in 2-(N-morpholino)ethane sulfonic acid (Mes) buffered uptake media. The pH was readjusted after the addition of inhibitors, FC or/and ABA to the protoplast suspension. Protoplasts were preincubated for at least 5 min with these additives prior to starting the uptake experiments by addition of 14C-labelled sugar. If not stated otherwise, the data presented in this paper are mean values of at least four experiments run in triplicate, corrected for controls.

H.-P. Getz et al. : Effect of fusicoccin and ABA on glucose uptake Table 1. Influence of FC-concentration of glucose uptake in beetroot protoplasts. U p t a k e medium: 0.5 m M (14C)D-glucose (9.25 kBq-mmo1-1) in 800 m M sorbitol, 3 m M Mes-Tris, pH 5.0. Uptake rate was linear for 5, 10, and 20 min periods. 106 bundle protoplasts ( = 2 0 3 units betacyanine) per ml were used. For control experiments, the final concentration of ethanol was adjusted to that used in the resp. FC-concentration series (0.1, resp. 0.25 EtOH). u B e t = 100 units betacyanine; so = standard deviation Fusicoccin

nmoles glucose uBet. h

sD

% (control)

0 (0.1% EtOH) 10 .8 10 -v /0 6 5-10 -6

9.8 10.7 14.2 16.9 20.7

3.8 3.4 5.9 4.1 5.2

100 109 144 172 210

0 (0.25% EtOH) 10 .5 5" 10- 5 10 -4

9.4 15.9 9.9 10.5

2.9 3.8 1.7 3.3

100 168 105 lll

pH-Measurement. Changes of H +-concentrations in protoplast suspensions were monitored using a pH-electrode (Ingold, Frankfurt, F R G ) connected with an amplifier to a recorder (Servogor, Metrawatt, N/irnberg, F R G ) according to K o m o r et al. (1977). Unbuffered media (800 m M sorbitol plus 10 m M KCI) were adjusted to pH 6 with diluted solutions of K O H and HCI. Protoplasts were washed twice with this medium until the experiments were started.

s

D+FC

eS ee

15

i

St

st e* s* eS en

F C . , "

Determination of total ABA-content. The preparation of the ABA-protein conjugate as well as the ABA-isolation and the radioimmunoassay were carried out according to Weiler (1979, 1980). Some modifications used here are described elsewhere (Schulte-Altedorneburg et al. 1987).

10

I

I

en

Results

Influence of fusicoccin on glucose uptake. As shown

in Table 1, we found optimal glucose uptake in the presence of 5" 10-6 M FC. The proton pump is reported to be activated by FC (Marr6 and Ballarin-Denti 1985). If the apparent K,, and Vmax values are dependent on external pH, as stated elsewhere (Komor and Tanner 1974a, b; Giaquinta 1977), then FC should affect the kinetic response of the protoplasts to external glucose (Schmitt et al. 1984). We found a decrease of the apparent K~, for glucose transport under the influence of FC (Fig. 1) from about 1.9 mM to about 0.9 mM. The uptake of the nonmetabolizable glucose analogon 3-O-methylglucopyranose is affected in a similar manner ( / m M compared with about 1.8 mM in the absence of FC). At pH 5, glucose uptake by beetroot protoplast is in its optimum. If uptake rate measured at pH 5 is set as 100% in both cases controls and FCtreated samples, the shape of the pH-curve for FCtreated samples appears as a broader peak (Fig. 2),

./o ' " ~ *

X

/

9

"

1.5

Q

x

-6 E c-

1.o

5

0.5 ~s~occin -1~] 1

; 1~3 2

2'0 1Is s(mM)

Fig. i. Influence of glucose concentration on FC-induced glucose uptake. 9 without FC; o 2-10 .6 M FC. Conditions for uptake by 106 protoplasts (derived from the whole beetroot tissue) as described in Table 1. Insert. Lineweaver-Burk plot of the same values after linear regression analysis: apparent Kin: 1 . 9 m M (control); 0 . 9 m M (with 2.10 6 M FC)

whereas the pH optimum for glucose uptake remains virtually unaltered.

Acidification of the uptake medium by FC-action.

The data reported above indicate an involvement of H + in glucose transport across the plasma mem-

H.-P. Getz et al. : Effect of fusicoccin and ABA on glucose uptake

237

I

5 rain

I

,gusicoccin 9

"O,,

t

@

HCI

FC 20 I

I

I

I

I

1

/-,

5

6

7

8

9pH

Fig. 3. Acidification of beetroot protoplast suspensions under the influence of fusicoccin (FC). Original recorder tracings from a series of six different experiments with identical results, a = protoplast suspension as used for uptake experiments but without buffer, b = " d e a d " protoplasts. HCl-addition was 2"10 8 moles (H+). C C C P = Carbonylcyanide-m-chlorophenylhydrazone

Fig. 2. Influence of EC on pH dependent glucose uptake. Uptake conditions and media as described in Table 1, +_2.10 -6 M FC. Samples contained 106 bundle protoplasts per ml. Rates of uptake (in nmoles glucose. (100 units betacyanine)-1, h-1), at pH 5 equalled 100%: 5.4 without FC, 8.7 in the presence of 2-10 -6 M FC

Table 2. Inhibition of FC-enhanced glucose uptake into isolated protoplasts by mono- and divalent cations. Uptake conditions as described in Table 1. K + and Na + were added as chlorideor as nitrate salts ( 1 0 r a M final concentration), Mg 2+ and Ca 2 + as chloride salts only (2 m M final concentration). FC = 2.10 6 M. ( % ) = % c o n t r o l • deviation of ion effect (in % of control)

brane. Therefore we tested whether or not the addition of FC to a protoplast suspension could induce a proton extrusion into the surrounding medium. As shown in Fig. 3, at a concentration of 2"10-6 M FC causes a slight acidification of the unbuffered medium in the presence of about 106 protoplasts per ml. Acidification of the medium is in the range of 3 nmoles H + per rain which equals 2 nmoles H + per 106 protoplasts and rainute. Subsequent addition of 10 -6 M (final concentration) stops the acidifying action of FC on intact protoplasts almost immediately (Fig. 3).

Effect of FC on glucose transport in the presence of mono- and divalent cations and of inhibitors of plasmalemma A TPases. The divalent cations Mg 2 + and Ca 2+ partially counter the stimulating effect of FC on glucose transport. At pH 5, stimulation by FC is abolished completely by Na + and K +, whereas this effect is less pronounced at pH 7 (Table 2). No difference could be detected between chloride- or nitrate salts. Na-ortho-vanadate inhibits specifically the plasma membrane ATPase of higher plants (Gallagher and Leonard 1982; O'Neill and Spanswick 1984a). Its mode of action is compared with its ability to form a trigonal bipyramidal conformation like the phosphate transition state of this enzyme (Briskin et al. 1985). The action of the sterol diethylstilbestrol (DES) as an ATPase inhibitor is not yet fully understood (Balke and Hodges 1979). It was shown by Lado et al. (1980) that vanadate

Glucose (0.5raM) plus

(%) pH 5 nmoles uBet.h

-

5.4

VC FC+K + FC+Na+ FC+Mg 2+ F C + C a 2+ Fc + Ca2+ +K +

8.2 5.5 4.9 4.7 6.5 6.7

(100) (152_+18) (100_+24) (90_+26) (87+_20) (120• (124+_16)

pH 7 nmoles uBet.h

(%)

3.0 5.5 4.2 4.7 4.5 4.3 2.9

(100) (183___42) (140_+40) (157-t-_11) (150_+32) (143_+36) (97_+48)

and DES negate the stimulation of K + / H + - e x change by FC. Therefore, more distinct effects of ATPase inhibitors on glucose transport could be expected in the presence of FC. The data presented in Table 3 show a nearly complete reversal of the FC-stimulation by p-chloromercuribenzenesulfonic acid (PCMBS) and by vanadate whereas DES exerted in these experiments a rather slight effect, if any. More interestingly, 5.10 -5 M ABA negates the FC-stimulation, too.

The effect of externally applied ABA on glucose transport. As far as assimilate partitioning in high-

er plants is concerned, the direction of ABA-action stimulation or inhibition - appears to depend on various factors, such as plant age, the type of tissue investigated, the plant's native ABA-content, and the concentration of externally applied ABA

238

H.-P. Getz et al. : Effect of fusicoccin and ABA on glucose uptake

Table 3. Influence of inhibitors of A B A of FC-stimulated glucose uptake. Mean values from 7 experiments run in triplicate. Uptake conditions as in Table 1. sD ( % ) = s t a n d a r d deviation of inhibitor (ABA)-effect related to 100% control

0.5 mM glucose.+ additive

nmoles uBet. h

0 -I-2-10 -6 M FC ,1,1,2.10 -6 M F C + P C M B S (l '10 -4 M) ,1,2"10 -6 M F C + v a n a d a t e

% (control) SD(%)

6.29 11.5 5.5

100 182 87

_+35 _+37

7.2

114

+18

8.7

139

_+38

6.4

102

_+37

(5-10 -5 M) -t-2" 10 -6 M F C + D E S (5.10 -5 M) + 2 . 1 0 -6 M F C + A B A (5"10 -5 M)

Table 4. Inhibition of glucose uptake by ABA, dependent on final ABA-concentration in the uptake medium. Uptake conditions as described in Table 1, SD= standard deviation

ABA (M)

nmoles uBet-h

sD

% (control)

0 (0.5% EtOH) 10 .8 10 -7 10 -6 10 - s 10 -4

9.9 7.9 7.4 7.4 6.6 5.7

(___2.7) (_+1.5) (_+1.8) (+_1.3) (_+1.2) (___1.4)

100 79 75 71 60 57

(Ackerson 1984; Tietz and Dingkuhn 1981; Trewavas 1982; Marschner et al. 1984). For beetroot storage tissue taken from 120d old beets, an amount of 2.6-10-6 M ABA in bundle protoplasts and 1.2-]0 -6 M ABA in storage protoplasts was determined. Glucose uptake into the protoplasts is reduced in the presence of ABA, but even at 10 -5 M ABA the inhibition does not exceed 40% compared to controls (Table 4).

Discussion As was expected from data contributed by Marr~ (1980) and Marr~ and Ballarin-Denti (1985), fusicoccin (2" 10- 6 M) stimulates H § in beetroot protoplasts as well. While proton extrusion activity appears to be stimulated to a greater extent by FC for Viciafaba leaf fragments (Delrot et al. 1980; Fig. 2b) than in beetroot protoplasts, initial rates of medium acidification compare well with those measured for isolated Asparagus mesophyll cells (Brown and Nicholls 1985) or maize root segments (Colombo et al. 1978). Fusicoccin significantly enhanced the uptake of 0.5 m M glucose (Table 1) and of 3-OMG (data not shown). The

pH-optimum of glucose uptake at pH 5 has not been altered by FC but a broadening of the optim u m curve can be observed which might reflect the known interaction of FC with the plasmalemma ATPase. In addition, FC lowers the apparent Kin-value for glucose (Fig. 1) and 3-OMG uptake (data not shown). To our knowledge a FC-induced change in apparent affinity of a sugar carrier has not been described for other species as yet. Such changes, however, might be due not only to affinity changes of the carrier itself, but also to various factors as ion concentration or membrane potential which both affect just cotransport systems (Wright et al. 1986). Recently, Cleland (1985) reported that FC-treatment of solubilized pea root plasmalemma vesicles prior to reconstitution resulted in vesicles whose Km for MgATP was reduced from 0.6 to about 0.2 raM, without affecting V~ax- O ~Neill and Spanswick (1984 b, c) could not detect any effect of FC on proton pumping activity in beetroot plasma membrane vesicles. In case of vesicles, however, the FC-receptor sites might be located inside as a consequence of reconstitution experiments, as pointed out by Cleland (1985; cf. also Aducci et al. 1984). Stimulation of glucose uptake induced by FC is partially released by Na +, K +, and divalent cations. Inhibition of FC-stimulated glucose uptake by cations could be ascribed to a partial depolarization of the membrane potential as a consequence of ion fluxes. Fairly high concentrations of Ca 2 + and Mg 2+ are known to inhibit plasma membrane ATPase-activity (Bennett et al. 1985). The FC-stimulated glucose uptake is completely abandoned by protonophores (data not shown), PCMBS and vanadate. At a concentration of 2 . 1 0 - S M ABA reduces markedly FC-induced stimulation of glucose-uptake. The data are consistent with the view that glucose uptake in isolated protoplasts depends on the proton-pumping activity in the plasma membrane ATPase. Abscisic acid diminishes FC-stimulation of sugar uptake. This result coincides with those reported elsewhere concerning interaction of fusicoccin and ABA. Abscisic acid could be involved in the regulation of sugar accumulation in storage tissue. Whether the distinct difference in ABA-content between protoplasts from the bundle region and those from the storage parenchyma more than fortuitously parallels the fact that bundle protoplasts take up glucose with rates which are far in excess of those measured for storage parenchyma protoplasts, must be resolved in further studies. It might be promising to investigate the nature of ABA to be found in the different regions of the

H.-P. Getz et al. : Effect of fusicoccin and ABA on glucose uptake

beet plant. Since there is no information available on both conjugation of ABA and its compartmentation in the cell, we can at the moment only speculate whether or not ABA could play a role as a promotor for phloem unloading in storage tissue as proposed by Tanner (1980). Studies are under way to evaluate compartmentation of free and conjugated ABA. Supported by Deutsche Forschungsgemeinschaft (Wi 107/32-5). We thank Mrs. C. Zander for skillful technical assistance, and Dr. A. Maretzki, Aiea, for correcting the English manuscript.

References Ackerson, R.C. (1984) Regulation of soybean embryogenesis by abscisic acid. J. Exp. Bot. 35, 403-413 Aducci, P., Coletta, M., Marra, M. (1984) An improved scatchard analysis of fusicoccin-binding to maize coleoptile membranes. Plant Sci. Lett. 33, 187-193 Balke, N.E., Hodges, T.K. (1979) Inhibition of adenosine triphosphatase activity of the plasma membrane fraction of oat roots by diethylstilbestrol. Plant Physiol. 63, 48-52 Bennett, A., O'Neill, S., Eilmann, M., Spanswick, R. (1985) Modulation of H +-ATPase activity by reaction substrates and products. Plant Physiol. 78, 495-499 Bown, A.W., Nicholls, F. (1985) An investigation into the role of photosynthesis in regulating ATP levels and rates of H § efflux in isolated mesophyll cells. Plant Physiol. 79, 928-934 Briskin, D.P., Thornley, W.R., Poole, R.J. (1985) Vanadatedependent NADH oxidation in microsomal membranes of sugar beet. Arch. Biochem. Biophys. 236, 228537 Cleland, R.E. (1985) Fusicoccin modifies the Km for the MgATP-driven proton pumping into reconstituted pea root vesicles. Plant Physiol. 77, Suppl., 472 Colombo, R., Demichelis, M.I., Lado, P. (1978) 3-O-methylglucose uptake stimulation in plant materials and its relationships with proton extrusion. Planta 138, 249-256 Delrot, S., Despeghel, J.-P., Bonnemain, J.-L. (1980) Phloem loading in Vicia faba leaves: Effect of N-ethylmaleimide and parachloromercuribenzenesulfonic acid on H + extrusion, K + and sucrose uptake. Planta 149, 144-148 Felle, H. (1982) Effects of fusicoccin upon membrane potential, resistance and current-voltage characteristics in root hairs of Sinapis alba. Plant Sci. Lett. 25, 219-225 Gallagher, S.R., Leonard, R.T. (1982) Effect of vanadate, molybdate and azide on membrane-associated ATPase and soluble phosphatase activities of corn roots. Plant Physiol. 70, 1335 1340 Getz, H.P., Knauer, D., Willenbrink, J. (1987) Transport of sugars across the membrane of beetroot protoplasts. Planta, in press Giaquinta, R. (1977) Phloem loading of sucrose, pH dependence and selectivity. Plant Physiol. 59, 750-755 Hartung, W., Ullrich-Eberius, CI., Lfittge, U., B6cher, M., Novacky, A. (1980) Effect of abscisic acid on membrane potential and transport of glucose and glycine in Lemna gibba G1. Planta 148, 256-261 Jung, K.-D., Lfittge, U. (1980) Effects of fusicoccin and abscisic acid on sugar and ion transport from plant glands. Ann. Bot. 45, 339-349 Komor, E., Rotter, M., Tanner, W. (1977) A proton-cotransport system in a higher plant: sucrose transport in Ricinus eommunis. Plant Sci. Lett. 9, 153 162

239 Komor, E., Tanner, W. (1974a) The hexose-proton symport system of Chlorella vulgaris. Specifity, stoichiometry and energetics of sugar-induced proton uptake. Eur. J. Biochem. 44, 219-233 Komor, E., Tanner, W. (1974b) The hexose-cotransport system of Chlorella, pH-dependent change in Km values and translocation constants of the uptake system. J Gen. Physiol. 64, 568-581 Komor, E., Thom, M., Maretzki, A. (1981) The mechanism of sugar uptake by sugarcane suspension ceils. Planta 153, 181-192 Lado, P., Colombo, R., Cerana, R., Bonetti, A. (1980) Effects of plasmalemma ATPase inhibitors on K+-dependent and lipophilic cation-dependent FC-stimulated H + extrusion. In: Plant membrane transport: Current conceptual issues, pp. 535-536, Spanswick, R.M., Lucas, W.J., Dainty, J., eds. Elsevier/North-Holland Biomedical Press, Amsterdam Lin, W., Giaquinta, R.T. (1979) Evidence for a non ATPasemediated plasmalemma H+/K + exchange in corn root tissue. Plant Physiol. 63, Suppl., 13 Malek, F., Baker, D.A. (1978) Effect of fusicoccin on proton cotransport of sugars in the phloem loading of Ricinus communis L. Plant Sci. Lett. 11,233-239 MarrY, E. (1979) Fusicoccin : a tool in plant physiology. Annn. Rev. Plant Physiol. 30, 273-288 Marr6, E. (1980) Mechanism of action of phytotoxins affecting plasmalemma functions. Prog. Phytochem. 6, 253-284 MarrY, E., Ballarin-Denti, A. (1985) The proton pumps of the plasmalemma and the tonoplast of higher plants. J. Bioenerg. Biomembr. 17, 1-21 Marschner, H., Sattelmacher, B., Bangerth, F. (1984) Growth rate of potato tubers and endogenous contents of indolylacetic acid and abscisic acid. Physiol. Plant. 60, 16-20 O'Neill, S.D., Spanswick, R.M. (1984a) Effects of vanadate on the plasma membrane ATPase of red beet and corn. Plant Physiol. 75, 568-591 O'Neill, S.D., Spanswick, R.M. (1984b) Solubilization of a vanadate-sensitive H +-ATPase from the plasma membrane of Beta vulgaris. J. Membr. Biol. 79, 231-245 O'Neill, S.D., Spanswick, R.M. (1984c) Characterization of native and reconstituted plasma membrane H +-ATPase from the plasma membrane of Beta vulgaris. J. Membr. Biol. 79, 245-257 Saftner, R.A., Wyse, R.E. (1984) Effect of plant hormones on sucrose uptake by sugar beet root tissue discs. Plant Physiol. 74, 955-961 Schmitt, R.M., Hitz, W.D., Lin, W., Giaquinta, R.T. (1984) Sugar transport into protoplasts isolated from developing soybean cotyledons. II. Sucrose transport kinetics, selectivity, and modeling systems. Plant Physiol. 75, 941-946 Schulte-Altedorneburg, M., Schneider, Hj.A.W., Willenbrink, J. (1987) Wilt-induced changes of ABA-content in growing sugar beets as determined by a radioimmunoassay. J. Plant Physiol., in press Tanner, W. (1980) On the possible role of abscisic acid on phloem unloading. Ber. Dtsch. Bot. Ges. 93, 349-351 Tietz, A., Dingkuhn, M. (1981) Regulation of assimilate transport in barley (Hordeum vulgare cultivar Sorte Claudia) by the abscisic acid content of young caryopses. Z. Pflanzenphysiol. 104, 475-479 Trewavas, A. (1982) How do plant growth substances work. Plant Cell Environ. 4, 203-228 Weiler, E.W. (1979) Radioimmunoassay for the determination of free and conjugated abscisic acid. Planta 144, 255-263 Weiler, E.W. (1980) Radioimmunoassays for the differential and direct analysis of free and conjugated abscisic acid in plant extracts. Planta 148, 262-272

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Willenbrink, J., Doll, S., Getz, H.-P., Meyer, S. (1984) Zuckeraufnahrne in isolierte Vakuolen und Protoplasten aus dem Speichergewebe von Beta-Riiben. Bet. Dtsch. Bt. Ges. 97, 2~39 Wright, J.K., Seckler, R., Overath, P. (1986) Molecular aspects of sugar-ion cotransport. Annu. Rev. Biochem. 55, 225-248 Wyse, R.E., Daie, J., Saftner, R. (1980) Hormonal control of sink activity in sugar beet. Plant Physiol. 65, Suppl., 662

H.-P. Getz et al. : Effect of fusicoccin and ABA on glucose uptake Wyse, R.E. (1985) Membrane transport of sucrose: Possible site for assimilate allocation. In: Frontiers of membrane research in agriculture, pp 257 271, Beltsville Symposium 9, St. John, J.B., Berlin, E., Jackson, P.C., eds Rowman and Allenheld, Totowa Received 18 September 1986; accepted 4 March 1987