Electrogenic Sucrose Transport in Developing Soybean Cotyledons

Abstract

Addition of sucrose to a solution bathing an excised developing soybean cotyledon causes a transient depolarization of the membrane potential, as measured using standard electrophysiological techniques. The magnitude of the depolarization is dependent on the concentration of both sucrose and protons in a manner which suggests carrier mediation; this process has an apparent Km for sucrose of about 10 milimolar. Agents interfering with the generation or maintenance of a proton electrochemical gradient eliminate these depolarizations. Electrogenic sugar transport is sensitive to sulfhydryl-modifying reagents; their effect appears to be through a direct interaction with the carrier protein and/or with the process establishing the proton electrochemical gradient across the plasma membrane. p-Chloromercuribenzene sulfonate appears to be a selective inhibitor of the carrier-mediated process itself.and protons inside and outside the cell, respectively, and A4A is the membrane potential.In physical terms, transport across the cell membrane is facilitated by a carrier which alternately exposes its binding sites for proton(s) and solute on the external and internal side of the membrane (Fig. 1). The movement of protons into the cell upon addition of substrate causes a transient change in the voltage difference across the plasma membrane (8, 10, 22, 26, 29; see 22 for additional references); the value towards which the membrane potential moves is Ecotra. (16), the voltage at which no net current flows through the co-transport system. It can be derived from equation (1) (2) The concept that the energy stored in an existing ion gradient can be used to accomplish the accumulation of organic solutes is based on the early ideas of Crane and Mitchell, now commonly regarded as an outgrowth of the chemiosmotic theory. A history of the development of these ideas has been presented by Crane (4). In this scheme, the movement of an ion down its electrochemical gradient provides energy for the coupled uptake of another solute; the process is termed co-transport or symport. If transport occurs by the system outlined above, several criteria should be fulfilled: first, the uptake velocity should show carriermediated kinetic behavior with respect to substrate and proton concentrations; second, transport should be sensitive to those agents interfering with the generation and maintenance of the proton electrochemical gradient; third, transport should be affected by reagents interfering with the membrane-bound carrier protein.Developing soybean seeds offer an excellent experimental system for investigating transport processes. Since there are no symplasmic connections between parent plant and embryo (37), assim- LICHTNER AND SPANSWICK ilate must negotiate at least one plasma membrane between the site of phloem unloading and that of storage. Photosynthate, 90 to 95% in the form of sucrose (39), reaches the seeds by the ventral phloem bundles traversing the carpel (pod) and anastomosing through the outer integument. Phloem translocate...

Sucrose Uptake by Developing Soybean Cotyledons

“…While the uptake by cells immediately adjacent to the external solution (the epidermal cells of soybean cotyledons) would be influenced by the pH of the bulk solution, those deeper within the tissue may not be. This may explain the large pH effect seen in the sucrose-induced depolarizations measured within the first two or three cell layers (20) versus the smaller pH effect on uptake, which occurs throughout the entire tissue (Fig. 3).…”

“…For measurements of sucrose influx, two to five excised embryos were secured in a cheesecloth bag and placed in a preincubation mixture containing (in mmol/liter): 0.5 K+; 1.6 Cl-; 0.1 Ca2+; 0.1 Mg2+; 0. (20). RESULTS …”

“…cv. Chippewa 64) were grown and harvested at mid pod-fill as described previously (20). Freshly harvested embryos were used in flux measurements and in determinations of free space and intracellular sucrose content.…”

An In Vivo Technique for the Study of Phloem Unloading in Seed Coats of Developing Soybean Seeds

“…10 min, followed by a 10-min water wash) and then added agar with or without 15 mM EGTA. PCMBS, a nonpenetrating sulfhydryl reagent known to bind reversibly to membrane-bound sucrose carriers (2,9,14), markedly inhibited sucrose release to the agar during the 3-h period following PCMBS exposure and 14C02 labeling (Table II). In the absence of EGTA in the agar (Table II, (Table II).…”

“…The similarity of the agar and control-embryo 14C levels suggests that phloem bleeding from the cut ends of the seed coat vascular bundles did not occur. Apparently, the agar simulated the low-sucrose conditions maintained in the free space in vivo by the transport activities of the embryo (9,14).…”