Characterization of the Active Sucrose Transport System of Immature Soybean Embryos

Abstract

Immature soybean embryos were isolated from soybean lG4vcine max (L.) Merr Active transport is sensitive to uncoupling agents and the suHhydrylmodifying reagent p-chloromecuribenzene sulfonate, is dependent on more than one energy source, and exhibits well-defined requirements for incubation temperature, pH, and oxygen availability. Under optimal incubation conditions of 35°C, saturating illumination (pH 6), and 21% oxygen, the apparent Km for sucrose is approximately 8 millimolar and V,,. is approximately 0.6 micromoles per hour per 100 milligrams fresh weight. Embryos readily accumulate sucrose from dilute exogenous solutions and, when preloaded with large amounts of sucrose, maintain the internal sucrose pool against steep outward gradients. These and other observations indicate that, although perhaps fully saturated in vivo, active sucrose transport is a significant component of photosynthate uptake in developing soybean embryos, enhancing uptake at physiological sucrose concentrations 2-to 5-fold over diffusion alone.It is tacitly agreed that photosynthate production rather than phloem transport capacity controls photosynthate availability and thus crop productivity. There is growing evidence, however, that processes that may limit the rate of photosynthate uptake and utilization in developing sink tissues may also play an important regulatory role (7,23,24, 27, 28). Among soybeans, for example, genetic differences in seed growth rate and final size appear to be determined more by the number of cells in the embryo than by the photosynthate supply from the leaves (4). This suggests that the rate of sucrose uptake and utilization by embryos on a per cell basis may be similar across soybean cultivars. But not enough is known about these processes to assess their role as determinants of sink strength, carbon partitioning, or economic yield. was provided by a mixture of incandescent and Sylvania cool white fluorescent lamps. Principal growth conditions (24/18°C, 12-h photoperiod) favored rapid seed development. Undamaged embryos of 300 to 400 mg FW' were excised from developing seeds and used for the following experiments. Sucrose Uptake Experiments. Unless otherwise specified, these experiments were performed with complete embryos (embryonic axis attached to both cotyledons), 300 to 400 mg FW, excised 20 to 30 min prior to each experiment. As the seed coats were removed, the embryos were transferred to 5 mm Mes buffer (pH 6.0) at room temperature until sufficient numbers were obtained.All embryos were then subjected to a 20-min pretreatment in aerated Mes buffer before incubation in ["4CJsucrose. Inhibitors, when used, were introduced for this 20-min period. PCMBS was not present during the subsequent '4C uptake period but, instead, any excess was washed from the embryo free space with buffer (three 3-min washes) before '4C incubation. These procedures were used to focus the PCMBS action on the sucrose-translocating protein rather than energy-coupling component (i.e. ATPase) of the active uptake syst...

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).…”

“…Soybean (Glycine max cv Clark) were grown as previously described (14) except that some plants were grown symbiotically by inoculating the seed at sowing with an appropriate strain of Rhizobium Japonicum and deleting nitrogen from the nutrient solution. Plants were utilized for experiments at various stages of seed development.…”

Sugar Uptake by Cotton Tissues

“…The relatively rapid movement of label from sucrose into other chemical forms in both tissues (cf. 21) suggests that the sugar uptake isotherms presented here are likely to involve not only movement of sugars across cell membranes but also the conversion of sucrose into other compounds. Cotton root cells exhibit a significantly faster rate of conversion of sucrose into polymers than leaf cells (Table I) and both tissues convert sucrose to other molecules more rapidly than many other plant systems (21).…”

“…Sucrose accumulation in tissues isolated from several plant species seems to consist of a saturable component, most apparent at low sucrose levels, plus a nonsaturable component ( 17,18,21). Evidence for this interpretation of these kinetics stems from, among other things, the elimination of the saturable, but not the nonsaturable, component of sucrose uptake by inhibitors, particularly the organic mercurials.…”