Planta (1988)174:340-348

P l a n t a 9 Springer-Verlag 1988

Stimulation of sugar exit from leaf tissues of Vicia faba L. Bertrand M ' B a t c h i and Serge D e l r o t * Station Biologique de Beau-Site, ERA 701, CNRS, Universit6 de Poitiers, 25 rue du Faubourg St-Cyprien, F-86000 Poitiers, France

Abstract. After removal of the lower epidermis,

leaf discs of Vicia faba L. were loaded with either [14C]sucrose or [3H]3-O-methylglucose (3-0MEG). The exit of preloaded sucrose was strongly stimulated when sucrose was present in the bathing medium, and the exit of 3-O-MeG was also markedly increased in the presence of 3-O-MeG. This specific stimulation exhibited single saturation dependence on the external concentration of sugar (Kin= 9 mM for sucrose, 5 mM for 3-O-MeG), and was sensitive to low temperature, uncouplers and thiol reagents. Sucrose exit was never affected by 3-O-MeG in the bathing medium. Sucrose did not affect the exit of 3-O-MeG in fresh discs, but promoted this exit in discs previously aged for 12 h, indicating partial external hydrolysis of sucrose in the latter tissues. Ageing also dramatically increased the exit of 3-O-MeG induced by 3-O-MeG but had no effect on the exit of sucrose induced by sucrose. The ability of 53 compounds (pentoses, hexoses, hexose-phosphates, polyols, di- and trisaccharides, phenyl- and nitrophenyl-derivatives, sweeteners) to interact with the sucrose carrier and with the hexose carrier was tested. Sucrose, maltose, e-phenylglucoside and p-nitrophenyl-~-glucoside interacted with the sucrose carrier. D-glucose, D-xylose, D-fucose, D-galactose, D-mannose, 3-O-MeG and 2-deoxyglucose interacted with the hexose carrier. Key words: Carrier specificity Hexose transport - Leaf (sugar transport) - Membrane (sugar transport) - Sucrose transport- Vieia (sugar transport). * To whom correspondence should be addressed CCCP =carbonyleyanide-m-chlorophenylhydrazone; EGTA = ethylene glycol-bis(fl-aminoethyl ether)N,N,N',N'-tetraacetic acid; 3-O-MeG = 3-O-methylglucose; PCMBS =p-chloromercuribenzenesulphonic acid

Abbreviations:

Introduction

At the level of the plant cell plasmalemma, sucrose may be absorbed either without hydrolysis or hydrolysed to hexoses which are subsequently taken up. The cells of the conducting complex in phloem absorb sucrose but are unable to take up hexoses (Fondy and Geiger 1977; Giaquinta 1977; Delrot 1981 ; Daie 1985) while the reverse situation occurs for cells of sugarcane stalk (Bowen and Hunter 1972; Maretzki and Thorn 1972) and parenchyma cells of castor-bean cotyledons (Cho and Komor 1985). The specificity of the sucrose carrier and of the hexose carrier is important for basic understanding of the molecular mechanisms of sugar transport in plants. Additionally, the sucrose carrier may be envisaged as a " d o o r " to the phloem for xenobiotics which would include in their structure molecular arrangements recognized by the carrier, and these xenobiotics would then benefit from good systemic properties. Alternatively, a compound hitting the sucrose carrier might be a ' potent herbicide without the need to enter the plant cell. Relatively little information is still available on the specificity of sugar carriers in plants. Use of various sucrose derivatives has indicated that, in soybean cotyledon cells, a large portion of substrate recognition by the sucrose carrier arises from the interaction of a relatively hydrophobic portion (mainly fructose) of the sucrose molecule and a hydrophobic region of the carrier-protein binding site (Card et al. 1986; Hitz et al. 1986). In sugarbeet leaf tissues (Maynard and Lucas 1982), in barley mesophyll vacuoles (Kaiser and Heber 1984) and in broad-bean leaf tissues (M'Batchi et al. 1985), the sucrose carrier recognizes, besides sucrose, maltose and raffinose to some extent. In sugarcane, the hexose carrier recognizes glu-

B. M'Batchi and S. Delrot: M e m b r a n e transport of sugars

cose, galactose and 3-O-methylglucose, but not ketohexoses or pentoses (Maretzki and Thorn 1972; K o m o r et al. 1981). In Chlorella vulgaris (Komor et al. 1985) and in sugar-beet cells (Zamski and Wyse 1985), the steric positions of hydroxyl groups at carbon 1, 2 or 3 of glucose are particularly important for efficient transport. In Riccia fluitans, phlorizin binds to the hexose carrier of the plasmalemma (Felle et al. 1983). Most studies on carrier specificity rely on competition in uptake experiments. A strong reduction of uptake of one sugar by another may be evidence for a common carrier but does not rule out the possibility of non-specific cytoplasmic or membrane interactions that feed back on uptake. To exclude this possibility, kinetic experiments must be carried out to demonstrate competitive patterns of inhibition. Besides, a compound only recognized at the external side of the membrane, but not transported by the carrier studied, may inhibit the uptake of the natural substrate. Another way to gain evidence for interaction with a common carrier is to study the effect of one substrate on the exit of another one. In this paper, we characterize the stimulation of sugar exit from leaf tissues and we study its possible use as a screening test for interaction of various molecules with the sugar carriers.

341 by the external standard method. All experiments were run at least two times independently. In some cases, leaf discs were aged for 12 h in the dark on " N " medium before beginning the experiments. In some experiments, the amounts of sugars released were assayed enzymatically as described previously (Delrot et al. 1983).

Results

Evidence for stimulation of sugar exit. Detailed time-course studies showed that the release of label from broad-bean leaf tissues preloaded with [14C]sucrose or [3H] 3-O-MeG was linear with time for about 50 min (Fig. 1). Paper chromatography showed that the label released from discs preloaded with [~4C]sucrose was exclusively associated with sucrose. Enzymatic measurements of the rate of sucrose release in " N " medium (no sugar added) yielded values of 58 pmol- cm 2. m i n - ~. Combination of these data with those presented in Fig. 1 allowed a specific activity of 2.6 M B q - m m o l - ~ for the sucrose released to be calculated. Under these conditions where exit sensu stricto is measured, lOO

I

I

I

I

I

I

G

90

'~ 80

9O 8O

70 Material and methods Plant material. Broad bean (Viciafaba L. cv. Aguadulce) plants

70

v

60 x 50

60 .'50 (9

were grown as described elsewhere (Delrot et al. 1980). Discs (12 m m diameter) were punched with a cork-borer from mature leaves after stripping of the lower epidermis. After excision, the discs were floated for :15 to 30 min on a solution containing 0.5 m M CaC12, 0.25 m M MgC12, 250 m M mannitol, buffered at pH 6.0 with 20 m M Na/diNa-phosphate ( " N " medium). Unless stated otherwise, the experiments were run in the light at room temperature.

40 (5 a0

Efflux experiments. After preincubation, randomized sets of 15 discs were loaded for 30 min with either 1 m M [U-14C]sucrose (44.4 kBq. ml - ~; 44.4 MBq. mmol - 1) or 1 m M 3-O-methyl-D[1-3H]glucose (3-O-MeG, 74 k B q . m l - ~; 74 M B q - m m o l - ~). The discs were then rinsed 3 x 3 min on " N " medium. This rinse is sufficient to remove all apoplastic label since, in this material, the half-life time of the apoplastic compartment is about 1-2 min (Delrot et al. 1983; El Ibaoui et al. 1986). Afterwards, the discs were transferred to 8 ml of bathing medium containing various concentrations of various sugars (see Results). The initial content of the fresh discs before the start of efflux was about 182.5_+31.7 B q . c m 2 [~4C]sucrose, and 3.06_+0.41 nmol [3H]3-O-MeG cm -2 (190.2_+24.3 B q . c m -2) (means of nine determinations + standard errors). The sucrose and the hexose contents of the tissues were, respectively, 95 and 33 n m o l . c m -2 of leaf tissue (Delrot et al. 1983). The osmolarity of all media used was adjusted at 270 moslnol. Aliquots (100 g[) of bathing medium were sampled at selected times and their radioactivity was counted by liquid scintillation spectroscopy after addition of 5 mI scintillation cocktail (PSII; Amersham, Les Ulis, France). D a t a were corrected for quenching

; 701

40 30

& 20 10 0 100~

20 10 0 100, 90 80 70

~

601

8

lOO

60 ~o 50, 40

40]1 3~ 0

CO

30c0 20 10 20

40 60 80 100 Duration of exit (min)

120

Fig. 1A, B. Time-course study of the exit of [14~]sucrose or of [3H]3-O-MeG from leaf discs of Viciafaba in ~he presence of various sugars. A Exit of 3-O-MeG; o, 9 in the presence of mannitol (control); n, 9 in the presence of 50 m M 3-O-MeG. B Exit of sucrose; o, 9 in the presence of mannitbl (control); [], 9 in the presence of 50 m M sucrose. Data are expressed in absolute values (closed symbols) or as a percentage of the initial radioactivity of the discs (open symbols'). Thd experiment was repeated another time with similar results

342

B. M'Batchi and S. Delrot: Membrane transport of sugars [

I

I

I

0,9

40 i

E

? 30. Or'C,

~ 20.

(9 ~

Q 10" 03

,

0

0

.

.

3

2

~

~

0.1

0

0.0

e4 4 0 'E

0

50

100

150

200

250

[Sugar] in the exit medium (raM)

o

30 164 O3 e3 O

20

14t

=~10

09

0

i

0

10

i

!

!

20

30

40

Duration of exit (min) Fig. 2 A, B. Measurements of the initial rate of exit of 3-O-MeG and sucrose from broad-bean leaf discs in the presence of various sugars. A Exit of 3-O-MeG; 9 control; o in the presence of 50 mM sucrose; [] in the presence of 50 mM 3-O-MeG. B Exit of sucrose; zx control; ~ in the presence of 50 mM 3-0MeG; o in the presence of 50 mM sucrose

the external specific activity of sucrose is the same as in the internal compartment, presumably the cytoplasm. The amount of hexose released was too low to be measured enzymatically, thus preventing an estimation of its specific activity. Exit of [14C]sucrose was strongly promoted by the presence of unlabeled sucrose in the bathing medium (Fig. 1 B), and the exit of [3H]3-O-MeG also increased dramatically in the presence of 3-0MeG (Fig. 1 A). In contrast, the exit of label from discs preloaded with [x4C]sucrose was insensitive to 3-O-MeG (Fig. 2B), and exit of [3H]3-O-MeG was insensitive to sucrose (Fig. 2A). Routinely, the rate of exit was monitored by sampling five aliquots over a 42-min time, as shown in Fig. 2. This strong and specific stimulation of release of label in the presence of peculiar substrates can be explained in terms of cis-inhibition of influx or of trans-stimulation of effiux (see Discussion). Because the exit rates may vary somewhat among different experiments, all experiments described below included a control treatment (exit on mannitol, without addition of another sugar) and all data were expressed by the difference "rate of exit in

._.E iElO! E -~ ~I:Y"

m6-

>4 o ,

0.O 0,1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1/[Sugar] (raM- 1 ) Fig. 3A, B. Concentration dependence of the promotion of sucrose exit from broad-bean leaf discs by sucrose (o) and of the promotion of 3-O-MeG exit by 3-O-MeG (u). A MichaelisMenten plot; B Lineweaver-Burk plot. Tissues were preloaded with either 1 mM [14C]sucrose or 1 mM [3H]3-O-MeG, and the rate of exit was measured (as in Fig. 2), in the presence or in the absence of various concentrations of the corresponding unlabeled sugar (either sucrose or 3-O-MeG). V was measured as the difference between the rate of exit in the presence of a given concentration of tested sugar in the bathing medium minus the rate of exit in the presence of mannitol only (control). Data are means o f two experiments 4- SD

the presence of tested sugar minus rate of exit in the control treatment.". Expressing the data by the ratio "exit in the presence of tested sugar/exit in the control treatment" (data not shown) also led to conclusions similar to the ones presented below.

Dependence of stimulation of exit on external sugar concentration. The stimulation of [x4C]sucrose release as a function of external sucrose concentration displayed a single-phase saturation curve, and the same was true for the release of [3H]3-O-MeG as a function of external 3-O-MeG concentration (Fig. 3A). Double-reciprocal plots yielded appar-

B. M'Batchi and S. Delrot: M e m b r a n e transport of sugars

343

Table 1. Effect of ageing on the stimulation of sucrose exit and 3-O-MeG exit from leaf discs of Vicia faba. Discs aged for 12 h (in the dark) were loaded either with [14C]sucrose or [aH]3-O-MeG, and then transferred to a medium containing mannitol(control), or 50 m M sucrose or 50 m M 3-O-MeG. The rates of exit were monitored as described in Fig. 2. Data are the means of two experiments __ SD Rate of exit Stimulation of exit (Bq-min - l ' c m -z leaf) x 102 3-O-MeG exit control + 5 0 m M sucrose + 5 0 m M 3-O-MeG

33 _+ 3 103-+ 5 548+_21

70+ 2 515+_18

Sucrose exit control + 50 m M sucrose +50mM3-O-MeG

12_+ 0.1 62-+ 1 10+ 2

50• -2+

1 2

ent K,, values of about 9 mM for sucrose and 5 mM for 3-O-MeG (Fig. 3B). Unless otherwise stated, further experiments were conducted with test sugars added at 50 mM in the bathing medium. Increasing the volume of the bathing medium from 8 to 24 ml had no effect on the rates of exit of sucrose or of 3-O-MeG (data not shown).

Effects of ageing on stimulation of exit. Ageing of leaf tissues is accompanied by dramatic changes in the properties of hexose uptake (Ltger et al. 1982) and sucrose uptake (Lemoine et al. 1984). Therefore, we also investigated the release of sugars with leaf discs aged for 12 h. Ageing strongly increased the stimulation of exit of labeled 3-0MeG induced by 3-O-MeG, while the stimulation of exit of labeled sucrose induced by sucrose was

not markedly affected. Although sucrose was unable to promote the exit of 3-O-MeG in fresh discs, the disaccharide significantly increased the exit of 3-O-MeG in aged discs (Table 1).

Effects of inhibitors on stimulation of exit. In fresh discs, low temperature (4~ C) inhibited the stimulation of sucrose exit induced by sucrose by about 74% and that of 3-O-MeG exit by about 86%. In aged discs, the stimulation of 3-O-MeG exit induced by 3-O-MeG was also drastically inhibited (82%) by low temperature. Yet, the stimulation of sucrose release in the presence of sucrose was much less sensitive to temperature in aged discs (20% inhibition) than in fresh discs (data not shown). The uncoupler carbonylcyanide-m-chlorophenylhydrazone (CCCP, 10 gM) decreased the promotion of 3-O-MeG exit induced by 3-O-MeG by 36% in fresh discs and by 52% in aged discs. The inhibition induced by CCCP on the stimulation of sucrose exit induced by sucrose was much less, about 26% in fresh discs, and no inhibition appeared in aged tissues (data not shown). Electrophysiological data, pH and uptake measurements have shown that a treatment with 0.5 mM p-chloromercuribenzenesulphonic acid (PCMBS) for 20 min blocks the sucrose carrier of broad-bean leaf without affecting the general permeability of the membrane nor the proton--pumping ATPase (Delrot et al. 1980; M'Batchi and Delrot 1984; M'Batchi et al. 1986). This non-(or slowly)permeant thiol reagent did not inhibit the stimulation of 3-O-MeG exit induced by 3-O-MeG, either in fresh or in aged discs (Table 2A). In contrast, the stimulation of sucrose exit induced by sucrose

Table 2. Effect of PCMBS on the stimulation of sucrose exit and 3-O-MeG exit from leaf discs of Vicia faba. Fresh discs or discs aged for 12 h were loaded in the absence of PCMBS, and transferred to a medium containing no PCMBS or 500 gM PCMBS. Data are the means of four experiments • SD for fresh tissues and of two experiments • SD for aged tissues Rate of exit [(Bq. m i n - 1 , c m - 2 leaf) x 10 2] Fresh discs

Aged discs

-- PCMBS

+ PCMBS

- PCMBS

+ PCMBS

14-t- 1 87_+ 7 72 _+ 6

13___4 83___7 70 _+4

2 2 + 0.8 559-+ 110 537-+ 109

34-t- 5.6 608-+ 107 574_+ 102

18+ 5 68-I-14 50+10

6+1 15_+3 9-+2

3-O-MeG exit control + 5 0 m M 3-O-MeG Stimulation of exit Sucrose exit control +50mMsucrose Stimulation of exit

14-+ 59• 45+

0.4 6 6

1 6 + 0.6 3 5 + 10 19_+ 9

344

B. M'Batchi and S. Delrot: Membrane transport of sugars

Table 3. Exit from leaf discs of Vicia faba of [14C]sucrose and of [3H]-3-O-methylglucose in the presence of various substrates. The data reported represent the increase in the rate of release induced by the presence of the compound tested in the bathing medium, as shown in Fig. 2. All compounds tested were present at 50 mM, unless otherwise stated. Data are means-+ SD (number of experiments) Compound tested

Promoted exit [(Bq- m i n - 1. c m - 2 leaf) x 102] Sucrose

D-Arabinose L-Arabinose D-Ribose D-Xylose D-Xylitol L-Ascorbic acid D-Fructose Fructose-6-phosphate D-Fucose L-Fucose D-Galactose o-Glucose o-Glucose (25 mM) + p-Fructose (25 raM) Glucose-l-phosphate Glucose-6-phosphate 2-deoxyglucose L-Glucose o-Glucosamine N-Methylglucamine D-Mannose

3 9 2 4 3 21

+0.5 • _+0.5 +1 _+0.2 _+2

(2) (2) (2) (2) (2) (2)

7 64 --2 102 2 15

-+ 0.1 _+10 _+ 0.3 + 0.9 _+ 0.1 _+ 1

(2) (3) (2) (3) (2) (2)

_

(2)

1

_+0.3

(2)

22

___2 +0.5 _1 -+0.2 _+0.2 _+0.2

(2) (2) (2) (2) (2) (2)

27 116 21 92 74 70

4-1 _+ 5 + 8 -+ 5 • _+ 0.8

(2) (2) (2) (2) (2) (2)

27 20 6 --1 S 4

+1 _+0.5 _+0.2 • _+0.6 _+0.4 _+0.2 -+0.4

(2) (2) (2) (2) (2) (2) (2) (2)

45 ___21 143 +32 137 _+15 34 + 1 57 _+ 2 16 + 1 94 _ 8 0.5_+ 0.1

(2) (2) (2) (2) (2) (2)

1 +0.3 3-O-MeG - 1 _+0.2 L-Rhamnose -1 ___0.2 D-Sorbitol 4 _+1 L-Sorbose 4 • a-Methylglucose 0.6+0.1 fl-Methylglucose p-Nitrophenyl-ct-galactoside 20 _+4 (30 mM)

(3)

87

(2)

-5

(2)

4

(2) (2) (2) (2)

5 21 23 35

--1

--1

1

_+14

•

1

(2)

(2)

(11) (2)

_-4- 1

(2)

-+ -+ + _+

(2) (2) (2) (2)

1 0.4 1 4

Promoted exit [(Bq. m i n - 1. cm 2 leaf) x 10 2] Sucrose

3-O-MeG

26 2 6 5 3 2

Myo-inositol

Compound tested

was sensitive to PCMBS, and the inhibition was stronger in fresh (82%) than in aged (58%) tissues (Table 2).

Specificity of stimulation of sugar exit. Fifty three compounds have been tested for their ability to interact with the sucrose carrier and with the hexose carrier. These compounds included pentoses, hexoses, hexose-phosphates, polyols, natural diand tri-saccharides, phenyl- and nitrophenyl-glucosides and -galactosides, sweeteners, and some miscellaneous molecules (Table 3). Because of the high number of compounds tested, the experiments were repeated only twice in most cases. Although this may not allow definitive conclusions to be drawn for some of the compounds tested, the data clearly show the validity of the test to assess interactions with the sugar carriers. The exit of labeled sugars induced by the compounds should be corn-

p-Nitrophenyl-fl-galactoside (30 mM) o-Nitrophenyl-fl-galactoside (30 raM) o-Nitrophenyl-fl-glucoside (30 raM) p-Nitrophenyl-a-glucoside (30 raM) ~-Phenylglucoside fl-Phenylglucoside Lactitol a-D-Lactose r-D-Lactose Lactulose Maltose Melibiose Palatinit Palatinose Sucrose Turanose Raffinose Stachyose ATP EGTA Acesulfame Aspartame Cyclamate Saccharin Phlorizin

3-O-MeG

30 _+ 4

(2) 105_+ 5

11 _ 4

(4)

6___ 0.4 (4)

7 + 1

(4)

7 + 0.4 (4)

44 + 6

(2)

12_+ 4

39 + 3 (2) 12_+ I (2) 5+ 3 10 _+ 4 (2) 7+ 0.9 - 5 _+ 1 - 2 • 0.3 (2) 9_+ 0.2 - 3 + 0.1 (2) 17-1- 2 - 3 • 0.1 (2) 4+ 0.1 33 _ 0.2 (2) 22_+ 3 - 3 _+ 0.1 (2)--2+ 0.1 - 5 _+ 0.3 (2) 2+ 0.~ (2) 2_+ 0.5 0.1 53 + 17 (10) 0 4 + 0.4 (2) 3+ 0.4 (2) 6___ 0.1 10 _ 1 1

+ 0.1 (2)

103 -+ 17 88 _+5

(2) (2) (2) -8 _+1 (2) 6_+3 10 + 0.7 (2) 37 _+ 2 (6) 3 + 1

(4)

(2)

(2)

(2) (2) (2)

(2) (2) (2) (2) (2) (2) (2) (3) (2)

(2) 4_+ 0.1 (2)

90+ 2 79 + 15 25-t- 2 11 7+ 0 42+13

(2) (2) (2) (1) (2) (6)

34__ 1

(4)

pared with the increase induced by sucrose on [ a 4 C ] s u c r o s e exit (53 B q ' m i n -1 c m - 2 leaf), and with the increase induced by 3-O-MeG on [3H]3-OM e G exit (87 Bq" rain-1 c m - 2 leaf). For the purpose of discussion, only the compounds inducing a promotion of efflux equal to at least 50% of the corresponding controls (sucrose and 3-O-MeG) were considered as interacting with the carriers. Neither pentoses (arabinose, ribose, xylose) nor neutral hexoses (1)- or L-fucose, o-galactose, o- or L-glucose, L-rhamnose, L-sorbose, 3-O-MeG, ~- or fl-methylglucose were able to stimulate the exit of sucrose. Among disaccharides, melibiose, lactose, lactulose and the sucrose analogues palatinose and turanose were also inefficient. N o promotion of sucrose exit was apparent in the presence of a trisaccharide (raffinose) or of a tetrasaccharide (stachyose). By contrast, various molecules significantly increased the exit of preloaded [14C]sucrose.

B. M ' B a t c h i and S, Delrot : M e m b r a n e t r a n s p o r t o f sugars

These compounds included natural disaccharides (sucrose, maltose), e-phenylglucoside, p-nitrophenyl-~-glucoside, p-nitrophenyPfl-galactoside, hexose-phosphates (glucose-l-phosphate, fructose6-phosphate) and ATP. The most efficient compounds were sucrose and p-nitrophenyl-/?-galactoside. Adenosine triphosphate also induced a strong exit of preloaded sucrose, but this effect seems rather non-specific, and due to the chelating properties of ATP. Indeed, EGTA exerted a similar increase in sucrose exit, and both ATP and EGTA also promoted the exit of 3-O-MeG. Among various sweeteners (acesulfame, aspartame, cyclamate, saccharin), only the latter compound proved efficient in stimulating the exit of sucrose. Again, this effect was not specific since saccharin also stimulated the exit of 3-O-MeG. Phlorizin, which is a competitive inhibitor of sucrose and hexose uptake in broad-bean leaf (Lemoine and Delrot 1987) did not promote sucrose exit, while it stimulated 3-O-MeG efflux to some extent. None of the natural di- and tri-saccharides tested was able to increase the exit of 3-O-MeG. Various neutral hexoses (D-fucose, D-galactose, oglucose,/~-methylglucose mannose, 3-O-MeG) and the pentose D-xylose induced a strong release of 3-O-MeG. Glucose-6-phosphate was the most efficient compound tested, while glucose-l-phosphate was three times less active, and fructose-6-phosphate was only poorly active. Except L-arabinose which induced a strong exit of 3-O-MeG, other L-sugars (glucose, fucose, rhamnose, sorbose) were poorly active. Fructose and fructose-6-phosphate were also poorly efficient. Surprisingly, p-nitrophenyl-/?-galactoside induced a strong exit of 3-0MeG, while other phenyl- or nitrophenyl-derivafives exhibited little, if any, effect on the exit of 3-O-MeG.

Discussion

This paper shows that the release of preloaded sugars from leaf discs can be specifically promoted by the presence of adequate sugars in the bathing medium, and the data presented characterize this phenomenon. Although stimulation of sucrose release after addition of sucrose in the bathing medium has been mentioned in several plant materials (M'Batchi and Delrot 1984; Turgeon 1984; Aloni et al. 1986), it has not yet been fully characterized. Besides, it may not be a general phenomenon, since the presence of sucrose in the bathing medium inhibits the release of ~4C-assimilates from soybean

345

leaf slices (Anderson 1986) but has no effect on the release of preloaded [14C]sucrose from leaf discs of Phaseolus coccineus (Daie 1985). Exchange diffusion of hexoses has been described with algae (Komor et al. 1981), with sugarcane suspension cells (Komor et al. 1981), and in Riccia fluitans (Gogarten and Bentrup 1983).

Characterization of sugar-induced increase of exit. Two possible explanations, namely cis-inhibition of influx or trans-stimulation of effiux, might account for the specific increase of sugar release induced by the presence of sugars in the bathing medium. In the cis-inhibition mechanism, the presence of a compound recognized (but not necessarily transported) by the sugar carrier at the external side of the plasmalemma would decrease the retrieval of labeled sugar leaked out from the tissues. In the trans-stimulation mechanism, it is assumed that the efflux of labeled sugar depends on the number of carriers reaching the internal side of the membrane. If the "movement" of the ternaryloaded complex proton-sucrose carrier is faster than that of the unloaded carrier, the presence of a transported sugar in the external medium will increase the rate of exit by increasing the number of carriers reaching the internal side of the membrane (counter-exchange mechanism). Although both explanations are not mutually exclusive and cannot be distinguished easily, several observations lead us to favor trans-sfimulation as playing the most important role in the experiments where exit of preloaded sugar is stimulated by the same sugar (cold) added in the bathing medium (Figs. 1, 2, Tables 1, 2). First, the bathing medium, which was constantly shaken, had a volume 30 times larger than the tissues, and any label released would be rapidly diluted, thus mostly escaping retrieval, even in a medium without added sugar. Rapid dilution is favoured by the lacunous nature of this tissue and the fast exchange of the apoplastic compartment (Delrot et al. 1983; El Ibaoui et al. 1986). The rates of release measured did not depend on the volumes of bathing medium, also indicating that apparent release did not result from dilution of label by external unlabeled sugar. Second, the number of carriers reaching the internal side of the membrane in the presence of 50 mM sucrose (at least 1.3 nmol-cm-2.min -1, Detrot and Bonnemain 1981) is largely sufficient to account for the increase of label release (about 0.6 Bq.cm -2 rain- 1). Indeed, even if one takes into acciount that the initial specific activity of internal sucrose (2.6MBq'mmo1-1) will be diluted about two times by unlabeled sucrose taken up (computed

346 from the initial sucrose content of the discs and from the rate of sucrose uptake from a 50 mM sucrose solution), the radioactivity released would represent only 0.8 nmol-cm-2-min -1. Third, sucrose uptake into broad-bean leaf tissues is mediated by two saturable systems superimposed with a diffusion-like component (Delrot 1981; Delrot and Bonnemain 1981). If competition alone at the external site of the membrane could explain the apparent stimulation of sucrose efflux, then dual kinetics (two/(ms) would be expected for the promotion of sucrose release as a function of external sucrose concentration. Figure 3 shows that simple kinetics are obtained, with a Km for sucrose of 9 mM, which does not differ significantly from the Km determined for the influx by the high-affinity system in parallel experiments (7 to 11 mM, data not shown). Fourth, the addition of phlorizin (alone) in the bathing medium is unable to increase sucrose exit (Table 3). Since this compound is a competitive inhibitor of sucrose uptake (Lemoine and Delrot 1987), this result indicates that recognition is not sufficient to promote the release of label and that transport is required. The same explanation holds for the inhibition of sucrose efflux by phlorizin when this compound is added together with sucrose in the bathing medium (Lemoine and Delrot 1987). At saturating concentrations, release of sucrose is promoted about five times above the basal rate of exit, while 3-O-MeG effiux is promoted about 15 times (Table 1). This is mainly the consequence of a basal rate of exit smaller for 3-O-MeG than for sucrose. That retrieval is less efficient for sucrose than for hexose might be due to the high sucrose concentrations in the internal compartment from which sucrose is escaping. Effect o f ageing on sugar release. The most striking feature induced by ageing is the dramatic increase in the stimulation of 3-O-MeG exit (Table 1), which is quite specific, since the stimulation of sucrose exit is not affected. This increase cannot be ascribed to the higher initial radioactivity content of aged discs compared to fresh discs. Indeed, aged discs contained about twice as much 3-O-MeG (or sucrose) as fresh discs, while the exit of labeled 3-O-MeG induced by 3-O-MeG was stimulated sevenfold by ageing. This phenomenon must be related to a change in the compartmentation of intracellular 3-O-MeG (increase in exchangeable pool) or to a change in the characteristics of the hexose carrier. The sensitivity of the stimulation of 3-0MeG exit to temperature, to CCCP and to PCMBS was not markedly changed upon ageing, which

B. M'Batchi and S. Delrot: Membranetransport of sugars agrees more with the former hypothesis (compartmentation) than with the latter (transport). While there is no stimulation of 3-O-MeG exit by sucrose in fresh discs, sucrose does promote the exit of 3-O-MeG in aged discs, which indicates that sucrose is partially and externally hydrolysed during ageing of leaf discs. The uptake of hexoses derived from sucrose can only occur in the mesophyll (Fondy and Geiger 1977; Delrot 1981), and this may explain the increase in label observed in the mesophyll of aged tissues incubated with p4C]sucrose (Lemoine et al. 1984). The decrease in PCMBS sensitivity after ageing (Table 2) is at least partly the result of extracellular hydrolysis of sucrose, since the hexose carrier is not sensitive to PCMBS (M'Batchi and Delrot 1984). Interaction with the sucrose carrier. The effects of the compounds listed in Table 3 were studied in the absence of cold sucrose (or 3-O-MeG) in the bathing medium. Although several strong arguments have been given above to sustain our interpretation of the data in terms of transport and counter-exchange, this experimental situation may allow apparent promotion of label release by cisinhibition, and deals with the exit of labeled sugar rather than with its effiux. Since this precludes well-defined mechanistic conclusions, the general term interaction (be it by cis- or trans-inhibition) of the compounds with the carriers will be used in the following discussion. Obviously, the sucrose carrier and the hexose carrier possess very different specificities, and the sucrose carrier is unable to interact with pentoses and hexoses. Differential affinity labeling methods with intact leaf discs (M'Batchi and Delrot 1984), with microsomes (Pichelin-Poitevin etal. 1987) and with purified plasmalemma (Pichelin-Poitevin and Delrot 1987) have given some insight on the recognition of sugars by the sucrose carrier of broad-bean leaf. The data showed that the sucrose analogues palatinose and turanose are not recognized, while sucrose, maltose, raffinose and ~phenylglucoside are recognized by the sucrose carrier. As expected, sucrose analogues not recognized (palatinose, turanose) are inefficient in promoting the release of sucrose in the exit test (Table 3). Sucrose, maltose and ~-phenylglucoside interact with the sucrose carrier (Table 3), confirming previous data (M'Batchi etal. 1985; Hitz 1986; PichelinPoitevin and Delrot 1987). Raffinose is able to protect the sucrose carrier from PCMBS inhibition and binding (M'Batchi and Delrot 1984; M'Batchi et al. 1985), indicating recognition by the carrier at the external side of the plasmalemma, yet raffin-

B. M'Batchi and S. Delrot: Membrane transport of sugars

ose showed no interaction with the sucrose carrier in the exit test (Table 3). Again, these data may be interpreted if the interaction measured in the exit test involves not only recognition, but also transport of the tested compounds. Thus, raffinose seems to be recognized, but not transported by the sucrose carrier. Although various fructosyl-substituted sucrose derivatives have been synthesized and tested for transport (Hitz et al. 1986), nitrophenyl glucosides have not yet been tested. Table 3 shows that pnitrophenyl-0~-glucoside interacted efficiently with the sucrose carrier, but not with the hexose carrier. In relative terms, the promotion of sucrose release by the other nitrophenyl derivatives tested is similar to or less than the promotion they exert on 3-O-MeG release, thus indicating that these effects are unspecific. By contrast, ~-phenylglucoside interacted almost as efficiently as sucrose on sucrose exit, but had no effect on 3-O-MeG exit. This specific interaction agrees with the idea that the fructosyl moiety of sucrose interacts mainly in a hydrophobic way with the sucrose carrier (Hitz et al. 1986). Addition of the polar nitro group in the para position is not detrimental to transport (compare c~-phenylglucoside and p-nitrophenyl-~-glucoside). Bock and Lemieux (1982) have underlined the near isomorphous relationship of saccharin with the hydrophobic region of sucrose. Yet, the apparent increase induced by this compound on sucrose exit is not specific (see 3-O-MeG) and saccharin (50 mM) was unable to inhibit sucrose uptake (1 to 5 mM) in competition experiments (data not shown). Therefore, the promotion of sucrose exit by saccharin is a side-effect. Similarly, the apparent effect of various hexose phosphates on sucrose exit seems mainly due to a general effect on membrane leakiness, because the discs incubated in these conditions did not look normal and took the apparency of plasmolyzed tissues. Besides, fructose-6phosphate and glucose-6-phosphate promoted the exit of 3-O-MeG to the same extent as the exit of sucrose. Interaction with the hexose carrier. Table 3 shows that D-glucose, 3-O-MeG and glucosamine interacted specifically with the hexose carrier, while sorbose and myo-inositol did not. These data confirm a recent study on sugar-cane suspension cells (Zamski and Wyse 1986). However, the latter authors concluded that galactose was not transported by the hexose carrier, while the present data clearly show a specific stimulation of 3-O-MeG exit by galactose. Our data are in agreement with those

347

of Maretzki and Thorn (1972), who found that, in sugarcane suspension cells, galactose and 3-0MeG competed with glucose uptake, while ketohexoses and pentoses did not. However, mannose interacted with the hexose carrier in broad bean leaf (Table 3), while it did not in sugarcane (Maretzki and Thom 1972). The specific interaction of 2-deoxyglucose and mannose with the hexose carrier in broad bean does not support the conclusion previously drawn for sugar beet (Zamski and Wyse 1986), that the OH group on carbon 2 is important for recognition by this carrier. In this regard, our data agree with the conclusion that this OH group per se is not absolutely necessary for transport (Komor et al. 1985). In broad bean, the most important OH are at carbons 3 and 4 since galactose, 3-O-MeG and glucose interact with the same carrier. The CHaOH group at carbon 6 is not essential, since fucose (6-deoxyglucose) also interacts with the carrier. Overall, the present data fit in well with the idea stemming from studies with animal cells, which showed that the preferred form of transport among the chair forms is CI (Lefevre and Marshall 1958). However, this preference is not exclusive since L-glucose, stable as the IC form, interacted to some extent. The data also show that the most efficient sugars are those which have the smallest number of axial substituents (OH) in the C1 conformation (D-glucose, D-mannose, D-xylose, o-mannose, D-fucose, 2-deoxyglucose). The strong interaction of L-arabinose with the hexose carrier, compared to that of D-arabinose, is not surprising, since in its C1 form, L-arabinose closely matches the well-transported D-galactose (C1), except it lacks the CH2OH group at carbon 6. Exit and counter-exchange experiments may provide a convenient screening test to estimate rapidly the ability of various compounds to interact and to be transported by the sugar carriers. Indeed, any compound transported by the carriers is able to increase the efflux of the corresponding preloaded label. Yet, an increase in efflux does not necessarily imply that the compound is transported, since potential side-effects may mimick counter-exchange. Therefore, normal competition experiments should also be done for those compounds which have passed successfully the screening tests based on release of preloaded sugars. We thank Mr. B. Thiriet (Beghin-Say, Paris, France) for kindly supplying the sweeteners.

References Aloni, B., Wyse, R.Z., Griffith, S. (1986) Sucrose transport and phloem unloading in stem of Vicia faba: possible in-

348 volvement of a sucrose carrier and osmotic regulation. Plant Physiol. 81,482-486 Anderson, J.M. (1986) Sucrose release from soybean leaf slices. Physiol. Plant. 66, 319-327 Bock, K., Lemieux, R.U. (1982) The conformational properties of sucrose in aqueous solution: intramolecular hydrogen bonding. Carbohydr. Res. 100, 63-74 Bowen, J.E., Hunter, J.E. (1972) Sugar transport in immature internodal tissue of sugarcane, lI. Mechanism of sucrose transport. Plant Physiol. 49, 789-793 Card, P.J., Hitz, W.D., Ripp, K.G. (1986) Chemoenzymatic syntheses of fructose-modified sucroses via multienzyme systems. Some topographical aspects of the binding of sucrose to a sucrose carrier protein. J. Am. Chem. Soc. 108, 158-161 Cho, B.H., Komor, E. (1985) Comparison of suspension cells and cotyledons of Ricinus with respect to sugar uptake. J. Plant Physiol. 118, 381-390 Daie, J. (1985) Sugar transport in leaf discs of Phaseolus coecineus. Physiol. Plant. 64, 553-558 Delrot, S. (1981) Proton fluxes associated with sugar uptake in Viciafaba leaf tissues. Plant Physiol. 68, 706-711 Delrot, S., Bonnemain, J.L. (1981) Involvement of protons as a substrate for the sucrose carrier during phloem loading in Viciafaba leaves. Plant Physiol. 67, 560-564 Delrot, S., Despeghel, J.P., Bonnemain, J.L. (1980) Phloem loading in Viciafaba leaves: effect of N-ethylmaleimide and parachloromercuribenzensulphonic acid on H + extrusion, K + and sucrose uptake. Planta 149, 141-148 Delrot, S., Faucher, M., Bonnemain, J.L., Bonmort, J. (1983) Nycthemeral changes in intracellular and apoplastic sugars in Viciafaba leaves. Physiol. V6g. 21, 459-467 E1 Ibaoui H., Delrot, S., Besson, J., Bonnemain, J.L. (/986) Uptake and release of a phloem-mobile (glyphosate) and of a non-phloem-mobile (iprodione) xenobiotic by broadbean leaf tissues. Physiol. V6g. 24, 431-442 Felle, H., Gogarten, J.P., Bentrup, F.W. (1983)Phlorizin inhibits the hexose transport across the plasmalemma of Riccia fluitans. Planta 157, 267 270 Fondy, B.R., Geiger, D.R. (1977) Sugar selectivity and other characteristics of phloem loading in Beta vulgaris L. Plant Physiol. 59, 953-960 Giaquinta, R.T. (1977) Phloem loading of sucrose, pH dependence and selectivity. Plant Physiol. 59, 750-755 Gogarten, J.P., Bentrup, F.W. (1983) Fluxes and compartmenration of 3-O-methyl-D-glucose in Riccia fluitans. Planta 159, 423-431 Hitz, W.D. (1986) Molecular determinants of sugar carrier specificity. In: Phloem transport, pp. 27-39, Cronshaw, J., Lucas, W.J., Giaquinta, R.T., eds. Alan R. Liss, New York Hitz, W.D., Card, P.J., Ripp, K.G. (1986) Substrate recognition by a sucrose transporting protein. J. Biol. Chem. 261, 11986-11991

B. M'Batchi and S. Delrot: Membrane transport of sugars Kaiser, G., Heber, U. (1984) Sucrose transport into vacuoles isolated from barley mesophyll protoplasts. Planta 161, 562-568 Komor, E., Haass, D., Tanner, W. (1971) Unusual features of the active hexose uptake system of Chlorella vulgaris. Biochim. Biophys. Acta 266, 649-660 Komor, E., Schobert, C., Cho, B.H. (1985) Sugar specificity and sugar proton interaction for the hexose-proton-symport system of Chlorella. Eur. J. Biochem. 145, 649-656 Komor, E., Thorn, M., Maretzki, A. (1981) The mechanism of sugar uptake by sugarcane suspension ceils. Planta 153, 181-192 Lefevre, P.G., Marshall, J.K. (1958) Conformational specificity in a biological sugar transport system. Am. J. Physiol. 194, 333 337 L6ger, A., Delrot, S., Bonnemain, J.L. (1982) Properties of sugar uptake by wheat leaf segments: effects of ageing and pH dependence. Physiol. V6g. 20, 651 659 Lemoine, R., Delrot, S. (/987) Recognition of phlorizin by the carriers of sucrose and hexose in broad bean leaves. Physiol. Plant. 69, 639-644 Lemoine, R., Delrot, S., Auger, E. (1984) pH sensitization of sucrose uptake during ageing of broad bean leaf tissues. Physiol. Plant. 61, 571-576 Maretzki, A., Thorn, M. (1972) Membrane transport of sugars in cell suspensions of sugarcane. I. Evidence for sites and specificity. Plant Physiol. 49, 177-182 Maynard, J.W., Lucas, W.J. (1982) A reanalysis of the two component phloem loading system in Beta vulgaris. Plant Physiol. 69, 734-739 M'Batchi, B., Delrot, S. (1984) Parachloromercuribenzesutfonic acid: a potential tool for differential labeling of the sucrose transporter. Plant Physiol. 75, 154-160 M'Batchi, B., E1 Ayadi, R., Delrot, S. (1986) Direct versus indirect effects ofp-chloromercuribenzenesulphonic acid on sucrose uptake by plant tissues: the electrophysiological evidence. Physiol. Plant. 68, 391-395 M'Batchi, B., Pichelin, D., Delrot, S. (1985) The effects of sugars on the binding of [Z~ sulfonic acid to leaf tissues. Plant Physiol. 79, 537-542 Pichelin-Poitevin, D., Delrot, S. (1987) Differential labeling of a 42 kD membrane polypeptide in the presence of sucrose. C. R. Acad. Sci. Ser. D 304, 371-375 Pichelin-Poitevin, D., Delrot, S., M'Batchi, B., Everat-Bourbouloux, A. (1987) Differential labeling of membrane proteins by N-ethylmaleimide in the presence of sucrose. Plant Physiol. Biochem. 25, 597-607 Turgeon, R. (1984) Efflux of sucrose from minor veins of tobacco leaves. Planta 161, 120-/28 Zamski, E., Wyse (1985) Stereospecificity of the glucose carrier in sugar beet suspension cells. Plant Physiol. 78, 291-295 Received 12 August; accepted 9 November 1987