Planta

Planta 149, 144 148 (1980)

9 by Springer-Verlag 1980

Phloem Loading in Viciafaba Leaves: Effect of N-Ethylmaleimide and Parachloromercuribenzenesulfonic Acid on H + Extrusion, K + and Sucrose Uptake Serge Delrot, Jean-Pierre Despeghel, and Jean-Louis Bonnemain Laboratoire de Biologic et Physiologic V6g~tales, Universit~ de Poitiers, 25 Faubourg Saint Cyprien, F-86000 Poitiers, France

Abstract. The effects of a penetrating (NEM) and a non-penetrating (PCMBS) sulfhydryl-specific reagent on proton extrusion, S6Rb and [U-14C]sucrose uptake by Viciafaba leaves have been studied. Proton extrusion was strongly or completely inhibited by 0.1 mM NEM. S6Rb and [U-l~C]sucrose uptake were markedly reduced by NEM concentrations equal to or higher than 0.5 mM. Under our experimental conditions, PCMBS (1 mM) exerted a strong inhibition on [14C]sucrose uptake but did not inhibit proton extrusion and 86Rb uptake. The sensitivity of phloem loading to PCMBS is thought to be a consequence of sugar-carrier blockage and not of inhibition of the proton pump.

Key words: Phloem loading - Potassium uptake Proton extrusion - Sucrose uptake - Sulfhydrylspecific reagents - Uptake (H + , K + , sucrose) - Vicia.

Introduction

Since Giaquinta (1977) first suggested that phloem loading of sucrose in higher plants would occur by a proton sugar cotransport process, several other researchers (Malek and Baker 1977, 1978; Komor 1977; Hutchings 1978; Delrot and Bonnemain 1978, 1979a, 1979b) have also proposed and substantiated such a mechanism. With this hypothesis, active absorption of solute is coupled to the electrogenic transport of protons through the membrane and is energized by the proton gradient. This coupling has been demonstrated in bacteria (West and Mitchell 1972), in fungi (Seaston et al. 1973), in algae (Komor 1973), and in animal cells where Na + ions replace the protons Abbreviations : CCCP = carbonylcyanide-m-chlorophenylhydrazone; DES = diethylstilbestrol; DCCD = dicyclohexylcarbodiimide; FC=Fusicoccin; NEM=N-ethylmaleimide; PCMBS=pchloromercuribenzenesulfonic acid

0032-0935/80/0149/0144/$01.00

(Crane 1962). Sugar accumulation by foliar minor veins is an important prerequisite for long-distance transport of assimilates and may be involved in the onset of translocation in leaves (Geiger et al. 1973). Unlike an ATPase carrier model, a cotransport model depends on the activity of two proteins: the energizing pump, and the carrier protein. Any substance which will inhibit the activity of one of these proteins will also reduce solute uptake. Giaquinta (1976) demonstrated that PCMBS markedly inhibits sucrose uptake into Beta vulgaris leaf tissues. Since this sulfhydrylspecific reagent does not enter the cell, the site o f inhibition is located at the plasmalemma level and PCMBS was thought to react with the sulfhydryl groups of a carrier protein. However, because of the dual dependency of the cotransport phenomena, it is difficult to assume that sucrose uptake inhibition by PCMBS is solely due to the blockage of the carrier but not to that of the proton pump. Such an ambiguity has been ruled out in the case of Chlorella cells by Komor etal. (1978), who have shown that the facilitated sugar diffusion system induced by nystatin is NEM-sensitive. Recent work in this laboratory (Delrot and Bonnemain 1979 a) has given evidence for a K +-activated, Na +-insensitive H +/K + exchange system in Viciafaba leaves. Autoradiographs with S6Rb showed that this system is more active in the veins than in the surrounding mesophyll tissues. Since proton extrusion and the uptake of S6Rb (or K +) were stimulated by FC in this material, the H+/K + exchange can be ascribed to the activity of the FC-binding ATPase (Dohrmann et al., 1977). Moreover, addition of sucrose (30 mM), but not of glucose (30 mM), causes a weak (0.07 pH units) and transient (15-20 rain) alkalinization of the incubation medium of Viciafaba leaf fragments (Delrot and Bonnemain 1979b). Recently, Giaquinta (1979) confirmed the existence of a proton pump in foliar tissues (Beffagna

S. Delrot et al.: Phloem Loading in Vicia Leaves

et al. 1977; Delrot and Bonnemain 1979 a). However, studying the effect of PCMBS, the author reached conclusions quite different from those in his first paper on this subject (Giaquinta 1976), since the inhibition of sucrose uptake by PCMBS was ascribed to the action of this substance on the ATPase. We have carried out some experiments to test the sensitivity of Viciafaba leaves to sulflaydryl-specific chemical modifiers PCMBS and NEM and to identify the inhibition site. The results that we present in this paper indicate that PCMBS can inhibit phloem loading without affecting proton extrusion and 86Rb (or K +) uptake.

145

Autoradiography. After-lyophilization, the discs labeled with 14C were exposed to "biemulsion" Kodirex films.

Radioactivity Measurements. The tissues labeled with 14C were combusted to ~4CO2 by an Intertechnique IN 4101 oxidizer. The radioactivity recovered in a phenethylamine/toluene/water coktail was counted by liquid scintillation spectroscopy (Intertechnique SL 33). The discs labeled with 86Rb were counted by using a Philips PW 4237 counter. The results were corrected for decay and counting efficiency.

Results

Proton Extrusion Activity. V a r i o u s N E M t i o n s (0.01, 0.1, 0.5, 1,5 m M )

concentrahave been tested for

t h e i r e f f e c t o n p r o t o n e x t r u s i o n in t h e Viciafaba leaf' Material

and Methods

Plant Material. Broad bean (Viciafaba cv. Aguadulce) plants were grown on vermiculite in a controlled environment under the following conditions: 16h of light (14 W m 2, Sylvania tubes F 65 W gro-lux) at 22_+ I ~ and 8 h of dark at 20+ I ~ with a relative humidity of 65+ 5% throughout. The plants were watered daily with Hoagland's solution.

fragments. A low NEM c o n c e n t r a t i o n (0.01 m M ) markedly inhibited the acidification of the incubation m e d i u m (Fig. 1 b); an acidification w h i c h usually oc-

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Proton Fluxes Measurements. Measurements of proton extrusion activity were made with a Radiometer RTS 622 titration chain including a pH meter PHM 62 provided with the K 4040 and G 2040 C electrodes. The pH meter was connected to an ABU 12 autoburette and a TTT 60 titrator. Using this apparatus as a pH star, the amounts of NaOH (1 mM) required for maintenance of the pH at a selected value (with a precision of 0.01 pH unit) were determined by the titrator, added by the autoburette, and recorded on a REC 61 servograph recorder provided with the REA 160 titrigraph module. As a standard condition, 480 mg (fresh weight) of tissue excised from a leaf of which the lower epidermis had been removed were incubated in 20 ml of a solution containing 250 mM mannitol, 0.5 mM CaClz, and 0.25 mM MgCI> The pH value for titration measurements was always chosen between 5.90 and 6.00. Measurements were made under light (14 W m 2) or dark conditions at 20_+ 1~ C. Sensitivity of proton extrusion to NEM and PCMBS was found to be the same under both light and dark conditions.

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Labelling with [U-14C]Sucrose. 12 mm diameter discs were excised from mature leaves of 3 week-old plants after the lower epidermis had been stripped off. 15 discs were first preincubated in the dark for 2 h in a solution containing 250 mM mannitol (as an osmoticure) and 20 mM sodium phosphate buffer pH 5.80. The discs were then transferred to 10 mI of a solution containing 250 mM mannitol, 20 mM sodium phosphate buffer pH 5.80, and 20 mM sucrose with 1.13-10 s Bq [U-14C]sucrose. Different concentrations of PCMBS or NEM were added to the preincubation and incubation solutions. After one hour of incubation in the dark (20 _+1~ C), the tissues were rinsed in three changes of unlabeled medium for 3 min each, frozen in powdered solid CO2 and lyophilized. [U14C]sucrose (specific activity: 1.52-101~ Bq mmol-1) was supplied by the Radiochemical Centre, Amersham, U.K.

Labeling with 86Rb CI. The experiments were made in the same way as described above except that the incubation solution (250 mM mannitol, 20 mM sodium phosphate buffer pH 5.80) contained 10 mM KC1 with 7.54- 104 Bq S6Rb C1. S6Rb (specific activity: 2. l0 s Bq mg -~) was supplied by CEA, Gif sur Yvette, France.

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Time aF[er beginning oF [rea[menl(min) Fig. 1 a-c. pH change of the incubation medium induced by Vicia faba leaf fragments (without the lower epidermis) in light conditions, a control, b the effect of 0.01 mM NEM (arrow) on the acidification of the medium by leaf fragments, e the effect of 0.1 mM PCMBS (arrow) on the acidification of the medium by leaf fragments

S. Delrot et al. : Phloem Loading in Vicia Leaves

146 Table 1. The proton extruding activity during the 1~%2~cl and 3~a hour of treatment with 1 mM PCMBS or 1 mM NEM compared with control values. With 1 mM NEM, the measurements were started 10 min after the addition of the inhibitor. The data (neq/g fr.wt.) are the mean of 11 experiments _+2 s.e Time (hour)

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610_+ 176 911 _+192 1,149 _+242

1,024_+306 1,038 _+223 1,460 _+428

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curred u n d e r o u r e x p e r i m e n t a l c o n d i t i o n s (Fig. 1 a). A t 0.1 m M , N E M i n h i b i t i o n o f p r o t o n e x t r u d i n g activity always s t a r t e d within 2 min (Fig. 2 a), and t o t a l i n h i b i t i o n was reached within 7-15 m i n in the h a l f of the experiments. This m a y suggest t h a t N E M can directly inhibit the p r o t o n - p u m p i n g system before red u c i n g the a m o u n t of A T P in the cell. N E M concen, t r a t i o n s higher t h a n 0.5 m M i n d u c e d an a l k a l i n i z a tion o f the i n c u b a t i o n m e d i u m a n d n o p r o t o n extrusion could be d e t e c t e d (Table 1). U n l i k e N E M (0.1 m M ) , P C M B S (0.01 and 0.1 m M ) d i d n o t affect the a c i d i f i c a t i o n o f the i n c u b a tion m e d i u m b y the leaf f r a g m e n t s (Fig. 1 c). T h e add i t i o n of 1 m M P C M B S d u r i n g the i n c u b a t i o n of leaf f r a g m e n t s p r o v e d to be an u n s u i t a b l e m e t h o d for the m e a s u r e m e n t of p r o t o n extrusion b e c a u s e it i n d u c e d a t r a n s i e n t acidification of the m e d i u m , even

when the p H o f the a d d e d s o l u t i o n h a d been carefully adjusted. U n d e r these c o n d i t i o n s , it was h a r d to visualize the s h o r t - t e r m effect o f this s u b s t a n c e on the tissues b y titrating the e x t r u d e d p r o t o n s . Therefore, the i n h i b i t o r was a d d e d first to the m e d i u m in o r d e r to reach a s t e a d y p H b e f o r e the leaf f r a g m e n t s were i n c u b a t e d . P r o t o n extrusion activity m e a s u r e d in this w a y can be c o m p a r e d with the c o n t r o l values. T a b l e 1 clearly shows t h a t 1 m M P C M B S does n o t inhibit the p r o t o n e x t r u s i o n in o u r m a t e r i a l b u t tends to s t i m u l a t e it d u r i n g the first h o u r o f t r e a t m e n t . M o r e over, the acidification o f the m e d i u m b y foliar tissue in the presence o f 1 m M P C M B S can be ascribed to the activity o f a p r o t o n p u m p , since this p h e n o m e n o n is highly sensitive to fusicoccin (Fig. 2b) a n d i n h i b i t e d b y C C C P after a lag time o f 5 - 1 5 m i n (Fig. 2c). Thus, o u r results c a n n o t be explained by s o m e effect o f P C M B S on the p e r m e a b i l i t y o f p l a s m a l e m m a , i n d u c i n g a l e a k a g e o f i n t r a c e l l u l a r acidic substances.

86Rb Uptake. T h e effect o f v a r i o u s i n h i b i t o r concent r a t i o n s on the S6Rb u p t a k e by f o l i a r discs is s h o w n in Fig. 3. C o n c e n t r a t i o n o f N E M equal to or higher t h a n 0.5 m M s t r o n g l y inhibited the u p t a k e o f 86Rb. T h e i n h i b i t i o n was less effective with 0.1 m M N E M , a l t h o u g h p r o t o n extrusion was s t r o n g l y o r c o m p l e t e l y b l o c k e d b y this c o n c e n t r a t i o n (Fig. 2a). This indicates

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Time after" beginnin9 or Ir'ealmenl(min) Fig. 2 a-e. Proton extrusion activity of the leaf fragments as shown by automatic titration with 1 mM NaOH (light' conditions), a the effect of 0.1 mM NEM (arrow) on proton extrusion; b incubation medium + 1 mM PCMBS + 10 jaM FC (arrow) ; e incubation medium + 1 mM PCMBS + 5 jaM CCCP (arrow)

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concentration (mM) Fig. 3. The effect of various concentrations of NEM ( o - - o ) and PCMBS (u---m) on the 86Rb (or K +) uptake by Viciafaba leaf discs (absorption time: 1 h). Each value is the mean of 15 1 cm2 foliar discs _+2 s.e. This experiment was repeated three times with similar results

S. Delrot et al.: Phloem Loading in Vicia Leaves

147

to the inhibitor effect on the permeability of plasmalemma to water. Depending on the experiments, 3040% of total 86Rb uptake could not be inhibited by NEM. The same percentage has been found with various concentrations of other inhibitors (0.5 mM DES, 0.1 mM DCCD, 0.02mM CCCP, data not shown) and thus can be accounted for by a passive entry of this label in the tissue.

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Fig. 4. The effect of various concentrations of N E M ( 9 and PCMBS (m - - - i ) on the [14C]sucrose uptake by foliar discs (time of uptake: 1 h). Each value is the mean of 5 triplicates + 2 s.e.

that proton extrusion can be stopped without strongly affecting the K § uptake. PCMBS which does not inhibit the proton pump at 1 mM exerted no clear-cut effect on S6Rb uptake in the concentration range of between 0.01 mM and 1 mM, and a stimulation of the 86 Rb uptake with 5 mM PCMBS was found (Fig. 3). The fresh weight of the discs treated with PCMBS (1 mM or 5 mM) and the control discs were equal under the conditions of 86Rb uptake experiments. Thus, the stimulating effect of 5 mM PCMBS on 86Rb uptake cannot be ascribed to the passive entry of 86Rb in the cell due

[U-14CJSucrose Uptake. The data in Fig. 4 show that both NEM and PCMBS inhibit sucrose uptake. However, the optimal effect of NEM is obtained at 0.5 mM, whereas the inhibiting action of PCMBS steadily increases from 0.1 mM up to 5 mM. The autoradiographs (Fig. 5) suggest that the two substances are acting in a somewhat different way since the minor vein network contains much less label with PCMBS than with NEM. As for 86Rb, some component of the sucrose uptake seems to be passive.

Discussion Obviously, phloem loading in Viciafaba leaves is sensitive to the sulfhydryl-specific reagents NEM and PCMBS, in accordance with previous data from Giaquinta (1976) on Beta vulgaris leaves. However, our experiments give additional information on the site of inhibition by these substances. During preliminary work, respiration measurements performed on our material (Auger, unpublished results) indicated that NEM does enter the cell whereas PCMBS does not, since only NEM inhibits respiration. The acidification of the incubation medium by our material cannot be accounted for by chloroplasts or mitochondria, since FC has no effect on these organelles (Beffagna et al. 1977). Furthermore, the rapid action (< 2 min) of substances such as FC concurs with the idea that

Fig. 5a-c. Autoradiographs of the distribution of [lr in non-treated (a), 1 mM PCMBS (b) or 1 mM NEM-treated (c) leaf discs. The discs were first used for autoradiography and then combusted to 1r for counting (see Fig. 4)

148 the p r o t o n extruding system is located at the plasmal e m m a (De!rot a n d B o n n e m a i n 1979 a). Since P C M B S exerts n o i n h i b i t o r y effect on the p r o t o n extrusion n o r on the 86Rb uptake, one m u s t conclude that n o sulfhydryl group essential for the A T P a s e activity is exposed to the outer side of the p l a s m a l e m m a . P l a n t ATPases often require the integrity of the SH g r o u p for their activity (Poole 1978). Such a group, if needed for p r o t o n extrusion in o u r material, has to be t u r n e d inside the m e m b r a n e and this could therefore explain the rapid effect ( < 2 min) of the p e n e t r a t i n g chemical modifier N E M . O u r results are n o t in agreement with those from recent w o r k which suggests that the p r o t o n p u m p is P C M B S - s e n s i t i v e in B e t a v u l g a r i s leaves ( G i a q u i n t a 1979) or in P h a s e o l u s a u r e u s hypocotyls ( G o l d b e r g a n d Prat 1979). Except for the difference in p l a n t material, we have f o u n d n o e x p l a n a t i o n for this app a r e n t discrepancy. C o m p a r i s o n of Figs. 2 a a n d 3 shows that N E M 0.1 m M can completely abolish p r o t o n extrusion w i t h o u t drastically affecting 86Rb uptake. This indicates that the coupling between H § a n d K § movem e n t is n o t direct, as in the N a § + A T P a s e of a n i m a l cells, b u t rather that K § entry in the cell is i n d u c e d by H § electrogenic extrusion. Since P C M B S exerts n o effect on the H + / K § exchange system b u t strongly reduces sugar a c c u m u l a tion in the veins, it m u s t be c o n c l u d e d that this inhibitor is acting on the carrier p r o t e i n involved in p h l o e m loading, thus c o n f i r m i n g the first idea proposed by G i a q u i n t a (1976). The fusicoccin was kindly provided by Prof. A. Ballio. This research was supported by the "Centre National de la Recherche Scientifique" (ERA 701 and RCP 580). The authors wish to thank R. Lacotte for his technical assistance.

References Beffagna, N., Cocucci, S., Marr6, E. (1977) Stimulating effect of fusicoccin on K-activated ATPase in plasmalemma preparations from higher plant tissues. Plant Sci. Lett. 8, 91 98 Crane, R.K. (1962) Hypothesis of mechanism of intestinal active transport of sugar. Fed. Proc. 21, 891 895

s. Delrot et al. : Phloem Loading in Vicia Leaves Delrot, S., Bonnemain, J.L. (1978) Etude du m6canisme de l'accumulation des produits de la photosynth~se dans les nervures. C.R. Acad. Sci. (Paris) 278D, 125-130 Delrot, S., Bonnemain, J.L. (1979a) Echanges H§ + et cotransport H§ dans les tissus foliaires de Vicia faba L. C.R. Acad. Sci. (Paris) 288D, 71-76 Delrot, S., Bonnemain, J.L. (1979b) Protonsugar cotransport in leaf tissues of Viciafaba L. Plant Physiol. 63, S-44 Dohrman, U., Hertel, R., Pesci, P., Cocucci, S.M., MarrY, E., Randazzo, G., Ballio, A. (1977) Localization of "in vitro" binding of the fungal toxin fusicoccin to plasma-membrane-rich fractions from corn coleoptiles. Plant Sci. Lett. 10, 291~99 Geiger, D.R., Giaquinta, R.T., Sovonick, S.A., Fellows, R.J. (1973) Solute distribution in sugar beet leaves in relation to phloem loading and translocation. Plant Physiol. 52, 585-589 Giaquinta, R.T. (1976) Evidence for phloem loading from the apoplast. Chemical modification of membrane sulfhydryl groups. Plant Physiol. 57, 872-875 Giaquinta, R.T. (1977) Possible role of pH gradient and membrane ATPase in the loading of sucrose into the sieve tubes. Nature (London) 267, 369-370 Giaquinta, R.T. (1979) Phloem loading of sucrose. Involvement of membrane ATPase and proton transport. Plant Physiol. 63, 744-748 Goldberg, R., Prat, R. (i979) Comparaison des effets de la fusicoccine et de l'auxine sur la croissance de segments excis6s d'hypocotyles de Phaseolus aureus. Physiol. V6g. 17, 83 94 Hutchings, V.M. (1978) Sucrose and proton cotransport in Ricinus cotyledons. I. H + influx associated with sucrose uptake. Planta 138, 229 235 Komor, E. (1973) Proton-coupled hexose transport in Chlorella vulgaris. FEBS Lett. 38, 16-18 Komor, E. (1977) Sucrose uptake by cotyledons of Ricinus cornrnunis L.: characteristics, mechanism, and regulation. Planta 137, 119 131 Komor, E., Weber, H., Tanner, W. (1978) Essential sulfhydryl group in the transport catalyzing protein of the hexose-proton cotransport system of Chlorella. Plant Physiol. 61, 785-786 Malek, F., Baker, D.A. (1977) Proton cotransport of sugars in phloem loading. Planta 135, 297 299 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 Poole, R.J. (1978) Energy coupling for membrane transport. Ann. Rev. Plant Physiol. 29, 437-460 Seaston, A., Inkson, C., Eddy, A.A. (1973) The absorption of protons with specific aminoacids and carbohydrates by yeast. Biochem. J. 134, i031 1043 West, I., Mitchell, P. (1972) Proton-coupled galactoside translocation in non-metabolizing Escherichia coll. J. Bioenerg. 3, 445462 Received 17 October 1979; accepted 23 January 1980