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

Abstract

A technique has been developed which permits mechanistic studies of phloem unloading in developing seeds of soybean (Glyrune mar cv Clark) and other legumes. An opening is cut in the pod wall and the embryo surgically removed from the seedcoat without diminishing the capacity of that tissue for assimilate import, phloem unloading, or efflux. The sites of phloem unloading were accessible via the seedcoat apoplast and were challenged with inhibitors, solutes, buffers, etc., to characterize the unloading process.Unloading is stimulated by divalent metal chelators and diethylsdibestrol, and inhibited by metabolic uncouplers and sulfhydryl group modifiers. Solutes released from the seed coat had a carbon/nitrogen ratio of 31 milligrams carbon per milligram nitrogen; sucrose represented 90% of the carbon present and various nitrogenous solutes contributed the remaining 10%. Unloading could be maintained for up to 8 hours at rates of 0.5 to 1.0 micromoles per hour, providing a valid, convenient in viva technique for studies of phloem unloading and seed growth mechanisms.The study of solute exit from the phloem ('unloading') in sink tissue has been severely hampered by the lack of convenient, valid techniques. Access to the phloem for experimental manipulation of the unloading process has been gained in but few systems, most notably sugar cane stalks (6) and Cuscuta-parasitized stems of Viciafaba (16). While much has been learned of the kinetics of photosynthate import, anatomy of the tissues involved, and uptake of assimilates by sink consumer cells (4,5,15), little is known of the mechanisms controlling the exit of solutes from the phloem sieve tubes.The phloem retains its solutes with minimal radial exchange;sites of unloading are notable exceptions. A popular hypothesis is that the sieve tube/companion cell/phloem parenchyma complex counters leakage by continuously reloading assimilates along the entire vascular length, and that sinks permit unloading by locally inhibiting the reloading mechanism (5, 10). This model is consistent with the data suggesting passive efflux from the phloem of developmentally immature sinks incapable of reloading, and for storage sinks where rapid hydrolysis or compartmentation prevents reloading of sucrose (4, 5, 15). However, other studies have provided evidence that, under some conditions, sinks directly effect solute exit from the phloem complex in a controlled, energydependent manner (13, 16). Given the diversity of sink tissues ' Visiting Scientist.which exist, it is probable that more than one unloading mechanism operates. This paper characterizes a novel technique for studying phloem unloading and maternal/embryo transfer of photosynthate in legume fruit. MATERIALS AND METHODSSoybean (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...

Sugar Efflux from Maize (Zea mays L.) Pedicel Tissue

“…MOPS, Mes, and Tes, nonpermeant buffers, gave virtually the same results as citrate phosphate And K-phosphate buffers, which are considered to be permeant buffers (Table II). Several studies have demonstrated that sugar unloading from plant cells may be an ion-mediated process and that external potassium often influences the observed rate of unloading (1,12,22). Wolswinkel and Ammerlaan (27), however, observed that 25 mm K+ had no consistent effect on phloem unloading in Viciafaba.…”

“…Observation (22) observed that 14C-assimilate movement into soybean seeds, altered with agar traps, was identical to that of intact seeds. Other studies on legumes have also suggested that assimilate unloading can continue for 8 h or more after the removal of the embryo and it was concluded that in legumes the embryo is not necessary for continued unloading from the seed coat (17,22,27). Similarly, in maize we found that the embryo and endsoperm are not necessary for the unloading of assimilates from the maternal tissue for at least 4 h.…”

“…Metabolic Inhibitors and PCMBS Studies. Sugar unloading from the seeds of soybean and broad bean, as well as unloading from other plant cells, is an energy requiring process and was shown to be markedly inhibited by a wide range of metabolic inhibitors (1,17,22,26,27). If sugar unloading from maize pedicel cells is also an active process, it should be sensitive to chemical inhibitors.…”

“…Precise determination of the unloading pathways in these species and in many other plant species is hampered by the lack of adequate techniques for studying the unloading process. Recently, Thorne and Rainbird (22) described a technique which they developed for mechanistic studies of assimilate unloading from soybean seed coats. Utilizing this technique, the above authors were able to study the unloading process through its responses to various chemical agents.…”

“…Similar studies have been conducted on the seeds of other legumes ( 17,(25)(26)(27). In general, these studies have indicated that sugar and/ or amino acid unloading from legume seed coats requires metabolic energy, since NaN3, KCN, DNP2, CCCP, NaF, PCMBS, and low temperatures inhibit unloading (17,22,26,27). However, this type ofsystem is probably not ubiquitous in crop plants since there is great diversity in the nature of the primary sink tissues.…”