Sugar Transport into Protoplasts Isolated from Developing ... - NCBI

Plant Physiol. (1984) 75, 936-940 0032-0889/84/75/0936/05/$0 1.00/0

Sugar Transport into Protoplasts Isolated from Developing Soybean Cotyledons1 I. PROTOPLAST ISOLATION AND GENERAL CHARACTERISTICS OF SUGAR TRANSPORT Received for publication January 23, 1984 and in revised form April 18, 1984

WILLY LIN*, MARK R. SCHMITT2, WILLIAM D. HITZ, AND ROBERT T. GIAQUINTA Central Research and Development Department, Experimental Station, E. I. du Pont de Nemours and

Company, Wilmington, Delaware 19898 ABSTRACT A procedure is described to isolated functional protoplasts from developing soybean (Glycine max L. Meff. cv Wye) cotyledons. Studies of sucrose and hexose uptake into these protoplasts show that the plasmalemma of cotyledons during the stage of rapid seed growth contains a sucrose-specific carrier which is energetically and kinetically distinct from the system(s) involved in hexose transport. For example, sucrose, but not hexose uptake: (a) is inhibited by alkaline pH and the nonpermeant SH modifier, p-chloromercuribenzene sulfonic acid; (b) is stimulated by fusicoccin; (c) shows both a saturable and a linear component of uptake in response to substrate concentration; and (d) displays a sharp temperature response (high Qio value and high activation energies).

The partitioning ofassimilates between source and sink regions is a major determinant of crop yield (6). Although there is a growing body of evidence that sucrose loading into the phloem in source leaves occurs from the apoplast via an energy-dependent sucrose/proton cotransport mechanism within the phloem membranes (4), relatively little is known about the mechanisms of movement of assimilates from the phloem into various sinks. In fact, recent studies suggest, perhaps not too surprisingly, that there are different pathways for assimilate movement into different types of sinks or even during various stages of sink development (2, 3, 5). In developing soybean seeds, the subject of this study, recent evidence demonstrates that sucrose is unloaded from the seedcoat phloem into the apoplast prior to its accumulation into the developing seeds ( 17, 18). Recent studies have characterized the kinetics and energy dependence of the sucrose accumulation into excised whole soybean cotyledons (9, 10, 19). These studies showed that sucrose uptake into excised cotyledons occurred by both a substrate-saturable component observable at low exogenous sucrose concentrations and a linear component apparent at high sucrose concentrations. The use of isolated protoplasts to study the mechanisms of

sugar uptake into developing soybean embryos offers a number of distinct advantages over the intact cotyledon system. First, bulk diffusion and tissue penetration barriers are absent, thereby

aiding kinetic studies. Second, the cell membrane is readily accessible for challenging with either sugar analogs or with membrane-modifying reagents or binding probes to facilitate carrier identification and isolation. Third, lacking cell walls and their associated hydrolyases, protoplasts are amenable to study of the uptake of mono- versus di-, tri-, tetra-, and pentasaccharide transport sugars (e.g., glucose, sucrose, raffinose, stachyose, verbascose) without complications due to oligosaccharide hydrolysis. Fourth, sugar/proton cotransport stoichiometry can be studied without the complications caused by proton buffering by negatively charged cell wall groups. Fifth, protoplasts permit the use of lipophilic indicators of transmembrane electrochemical gradients for studies on transport energetics. Finally, protoplasts are useful as a starting material for vacuole and organelle separation for transport, metabolism, and compartmentation studies. Although protoplasts have been isolated from many plant tissues, we are aware of no reports on the isolation of protoplasts from developing seeds and hence no detailed mechanistic studies on sugar uptake into this tissue. This study and the accompanying paper (15): (a) describe a procedure for isolating functional protoplasts from developing soybean seeds; (b) characterize the substrate concentration, pH, temperature, and metabolic dependence of both sucrose and glucose transport into these protoplasts; (c) demonstrate that the mechanism and energetics of sucrose and glucose transport are different; and (d) report on the sugar specificity of the sucrose transport system.

MATERIALS AND METHODS Plant Materials and Protoplast Isolation. Soybean (Glycine max L. Merr. cv Wye) plants were grown under the conditions as previously described (12). Cotyledons (100 mg fresh weight each) from 50-d-old plants were used for protoplast isolation. After removal of the pod walls, seedcoats, and embryonic axes, soybean cotyledons were sliced with a razor blade into