Involvement of Protons as a Substrate for the Sucrose Carrier during Phloem Loading in Vicia faba Leaves

Abstract

The effect of pH on uptake of exogenous sucrose by broadbean (Vicia faba L.) leaf discs without the lower epidermis has been investigated at various sucrose concentrations. The concentration dependence of sucrose uptake showed a biphasic saturation response. At high sucrose concentrations (>20 mHHmolar), sucrose uptake showed no pH dependence. At low sugar concentrations (<5 millmolar), plots of 1/V against 1/H give straight lines which all intercept at the same point at the left of the ordinal axis. Calculations show that these data agree well with two-substrate kinetics for the carrier, the substrates being the protons and the sucrose molecules. Our results provide further evidence that protonation/deproton-ation processes of the carrier are involved in phloem loading, especially for low sucrose concentrations of the apoplast. In the past few years, the mechanism of sugar uptake has been extensively investigated with various plant materials: Ricinus cot-yledons (15-17) or petioles (20), sugar beet leaf (9-14), and broadbean leaf (5, 6, 8). Komor (17) has provided evidence that a proton-sugar co-transport process is involved for sucrose uptake in castor bean cotyledons, although the tissue responsible for the observed phenomena cannot be precisely defined. Concerning the leaf tissues, where phloem loading normally has to operate in the plants, we have some indications that proton-sugar symport is involved. Giaquinta (13) showed that exogenous AC]sucrose uptake by sugar beet leaf tissue was stronger at an apoplast pH of 5.0 than at a pH of 8.0. Alkaline pH values increased the Km of the carrier for sucrose, without affecting the Vm.2 A proton-extruding activity has been described in leaf tissues and, on the basis of its sensitivity to effectors such as fusicoccin, K+, Na+, it has been attributed to the existence of a plasmalemma proton-pump (1, 6, 14). Autoradiographs of leaf discs incubated on "6RbCl suggested this proton-pump to be more concentrated in the veins than in the surrounding mesophyll cells (6). However, although a proton-pumping ATPase is a necessary prerequisite for proton-substrate symport, its existence is not sufficient to prove such coupled transport. The carrier kinetic data reported here show that at low external sugar concentrations (I to 5 mm), the carrier responsible for exogenous sucrose accumulation in Viciafaba leaf follows two-substrate kinetics, with the proton as one of the substrates. For high sucrose concentrations, another carrier is involved, which exhibits no pH dependence. Our material was chosen because the lower epidermis can be 'This work was supported by Grants ERA 701 and RCP 580 from the Centre National de la Recherche Scientifique. 2Abbreviations: Vm, maximal velocity; CCCP, carbonyl cyanide m-chlorophenylhydrazone. easily stripped off. The floating of discs prepared in this way ensures a good contact between the tissue and the incubation solution. Moreover, this allows easy manipulations of the apoplast pH and suppresses the effects which would be due to a differential stomatal aperture. Geiger et al. (11, 23) and Giaquinta (13) have shown that leaf discs can be a valuable tool for studies on sucrose phloem loading when incubation times are kept short. MATERIALS AND METHODS Plant Material. Growth of seedlings and leaf disc preparation were performed as described (8). Determination of Kinetic Parameters. After removal of the lower epidermis, the discs (12 mm diameter) were floated for 30 min on a buffered solution of 20 mm Mes (pH 5.0) containing 250 mM mannitol. To determine the kinetics of uptake, the discs then were transferred to incubation media containing sucrose concentrations from 1 to 150 mm. To avoid a loss in counting accuracy through too great a decrease of specific radioactivity, increasing amounts of [U-'4C]sucrose (3 to 12 ,uCi/10 ml) were added to the media. Mannitol (as the osmoticum) was added in varying amounts so that the total sugar (mannitol + sucrose) molarity was 250 mm. After 30-min incubation, the discs were rinsed in three changes of unlabeled solution for 2 min each, dry-ice frozen, lyophilized, and combusted to "CO2 with an Oxymat Intertech-nique IN 4101 oxidiser. A 100-p1 aliquot ofthe incubation medium was combusted under the same conditions in order to convert the value for the recovered radioactivity to 1 representing theamount of sucrose absorbed by the tissues. Similar rates of uptake were measured using either [U-14C1-or [6,6'-(n)-3H]sucrose. In the latter case, the label was recovered as 3H20 after combustion in the Oxymat apparatus. The [U-'4C]sucrose (403 mCi/mmol) and [6,6'-(n)-3H]sucrose (2.1 Ci/mmol) were supplied by Amersham France (Versailles). Effect of pH on Sucrose Uptake. Leaf discs without lower epidermis were floated for 30 min on a 250 mm mannitol solution containing various buffers: 10 mm citrate-20 mm Na-phosphate (pH 3.0, 3.5, 4.0, and 4.5), 20 mm Mes (pH 5.0, 5.5, 6.0, and 6.5), or 20 mm Tricine (pH 7.0, 7.5, 8.0, and 8.5). After preincubation, the discs were incubated for 30 min in the same buffered solution with 1 (set I) or 20 mM (set II) [14CJsucrose. For set II, the mannitol concentration was 230 mm. After incubation, the tissues were rinsed and collected as described above. Effect of pH on Kinetic Parameters of Sucrose Uptake. The results of the experiments described above led us to select five pH values for studying the effect of proton concentration on the kinetics of sucrose uptake at low and high sucrose concentrations. Thus, exogenous sugar uptake was studied at pH 4.0, 5.5, 7.0, 7.5, and 8.0 with 1, 1.5, 2, 3, and 5 mm sucrose (set I), respectively, or 20, 40, 60, 80, 100 mm sucrose (set II), respectively. In experiment I, besides the buffer, all media contained 250 mm mannitol. In experiment II, mannitol was added to keep the overall sugar concentration at 250 mm. These experiments were performed in 560 https://plantphysiol.org Downloaded on December 5, 2020.-Published by