Proton Fluxes Associated with Sugar Uptake in Vicia.faba Leaf. Tissues' .... One or two minutes after the addition of sugar, the apparent proton-extruding activity ...
Plant Physiol. (1981) 68, 706-711 0032-0889/81/68/0706/06/$00.50/0
Proton Fluxes Associated with Sugar Uptake in
Vicia.faba Leaf
Tissues' Received for publication December 8, 1980 and in revised form March 9, 1981
SERGE DELROT Station Biologique de Beau-Site, 25 Faubourg St-Cyprien, 86000 Poitiers, France ABSTRACT
View f.M leaf fragments bring the pH of their incubation medium to about 4.7, whatever the initial pH value. At this pH, addition of 20 mi_imolar sucrose causes a transient (20 to 40 minutes) alkadinization (0.05 to 0.10 pH unit) of the medium. The alaniation is not observed in the presence of p-chloromercurlbenzenesuffonic acid which blocks the sucrose carier involved in phloem loading without affecting the ATPase (DeIrot, Despeghel, Bonemain 1960 Planta 149: 144-148). Addition of 20 milihlar gluose, fructose, or 3-0-methylgucose induces weaker alkainization than sucrose. Sequential additions of sugars show that: (a) sucroseand hexose-induced proton fluxes are nearly saturated at 20 mIHimolar sugar (b) there is no competition between sucrose and hexoses for inducing proton influxes whereas (c) glucose and 3-0-methylglucose are competing for a common system. Autoradlographs performed under the conditions used for the observation of proton fluxes show a slht accumulation of I'4Clsucrose into the veins within 2 minutes of uptake, whereas 114Clglucose and 3-0-methyl [14Clglucose are localized in the mesophyil. These data support the protonsucrose cotransport hypothesis of pblem loading and show that mesophyli cells are able to take up hexoses by symport with protons. The apparent sucrose/proton stoichometry is constant below 5 millimolar sucrose (about 1.9 sucrose per proton taken up) but increases up to lar sucrose. This confirms 6 sucrose per proton, between 5 and 15 our previous study indicating that above 5 miflmolar sucrose, a system which exhibits little pH dependence is involved in the uptake. Simultaneous measurements of H+ and K' fluxes indicate that sucrose uptake is accompani by a reduction of K+ uptake rate, thus suggesting that sucrose and K' uptake can compete in dissipating the protonmotive force.
A recent review (14) pointed out that much of the work sustaining the proton-sugar co-transport hypothesis of phloem loading (12) has been performed with Ricinus cotyledons (15, 16, 18) or petioles (19, 20). Although proton fluxes associated with uptake of sugars have been extensively investigated with this material (15, 18), until now, no work concerning the effect of protonation on the kinetic parameters of sucrose uptake by castor bean cotyledons has been published. On the other hand, concerning mature leaves, where phloem loading normally operates, the effects of protonation on the kinetics of sucrose uptake are well documented (6, 13), whereas no detailed work has appeared about the proton fluxes linked to the operation of the postulated symport. In Beta leaves, the Km of the carrier for sucrose is increased when the pH of the 1 This work was supported by the Centre National de la Recherche Scientifique (ERA 701 and RCP 580).
apoplast is alkaline, whereas the V.2 is not affected (13). In Viciafaba, inasmuch as the sugar concentration of the apoplast is low (5 mM), the uptake exhibits little pH dependence (6). Although these data (6, 13, 14) suggest that conformational changes of the carrier could be induced by the means of protonation/deprotonation processes at the external and internal sides of the transfer cell plasmalemma, we have no direct evidence that protons are really translocated through the membrane during phloem loading in mature leaves. The aim of this work is to show that the uptake of sugars by foliar tissues, and particularly that the uptake of sucrose into the veins, is accompanied by proton fluxes from the medium (the apoplast) into the cells. MATERIALS AND METHODS
Plant Material. Broadbean plants (Viciafaba L. cv Aguadulce) were grown on vermiculite under a controlled environment (7). Our experiments were performed with mature exporting leaves whose lower epidermis had been stripped off, to facilitate the exchanges between the tissues and the incubation medium. Because great amounts of tissue were needed to measure sufficient proton fluxes, in the present work we have generally used leaf fragments which could be more rapidly prepared than the discs previously used (6, 7). pH Measurements. Mature leaf tissues without lower epidermis (0.50 or 1.00 g) were incubated onto 20 ml of a medium containing 250 mm mannitol, 0.5 mm CaCl2, 0.25 mm MgCl2. Unless otherwise specified, the initial pH of the medium was 6.0. The incubation solution was continuously mixed with a rotary stirrer and the pH was monitored with a pH recording unit "RTS" 622 and "RTS" 822 (Radiometer, Copenhagen) provided with K4040 (calomel) and G 2040 C (glass) electrodes (for details, see ref. 7). Measurements of Proton Fluxes during Sucrose Uptake. One g of leaf fragments was incubated as previously described, until the pH of the medium had reached a value of 4.6 to 4.8. At this time, the pH was maintained by an automatic titrator, with a 0.01 pH unit accuracy, at a value chosen between 4.60 and 4.80, by addition of 0.2 mm NaOH. The proton-extruding activity was monitored during 15 to 20 min by continuously recording the amounts of NaOH required for titration, and then 100 pl of a 3Hlabeled sucrose solution (5 to 10 ,uCi) previously adjusted at pH 4.75 was added. Depending on the experiments, final concentration of sucose in the medium was varied between 0.5 and 15 mM. One or two minutes after the addition of sugar, the apparent proton-extruding activity decreased and even completely vanished
2Abbreviations: V.., maximal velocity; 3-0-MeG, 3-0-methylglucopyranoside; PCMBS,p-chloromercuribenzenesulfonic acid; FC, fusicoccin; CCCP, carbonyl cyanide p-chlorophenylhydrazone. 706
Plant Physiol. Vol. 68, 1981
PROTON FLUXES AND SUGAR UPTAKE BY LEAF
when high sucrose concentrations were added. The tissues were usually incubated for 7.5 min or for a shorter time (at least 6 min) if an alkalinization of the medium was evident. Sugar-induced proton influxes were measured by difference between the amounts ofNaOH required for maintaining the pH before and after sucrose addition. After incubation, the leaf fragments were rinsed for 3 x 2 min in three baths of unlabeled solution without sucrose, combusted to 3H20 in an Oxymat "IN" 4101 oxidiser (Kontron), and counted by liquid scintillation spectroscopy. Preliminary experiments showed that the rinsing procedure used ensures a total leakage of effluxable free space sucrose. K+ Uptake. In some experiments, both H+ and K+ potentials were recorded in the same medium with the two recording units ("RTS" 622 and 822) functioning simultaneously. The K+ ion was monitored by a Radiometer K+ selectrode (F 2312 K). Radiochemicals. [U-_4C]Sucrose (477 mCi/mmol, [6,6'(n)3H]sucrose (5 Ci/mmol), D-[U-_4CJglucose (260 mCi/mmol), and 3-O-methyl-D-[U-_4C]glucose (295 mCi/mmol) were from Amersham France (Versailles).
|I
