Linear sucrose transport in protoplasts from developing soybean cotyledons

Previous studies with isolated soybean cotyledon protoplasts revealed the presence of a saturable, simple diffusion, and nonsaturating carrier-mediated uptake of sucrose into soybean cotyledon cells. A proton/sucrose cotransport may be involved in the saturable sucrose uptake (Lin et al. 1984 Plant Physiol 75: 936-940 and Schmitt et al. 1984 Plant Physiol 75: 941-946). In this study, we investigated the linear sucrose uptake mechanism by treating isolated protoplasts with 15 micromolar p-trifluoromethoxy-carbonylcyanide phenylhydrazone (FCCP) or 100 micromolar p-chloromecuribenzenesulfonic acid to eliminate the saturable uptake. We found: (a) increasing external pH decreases the linear sucrose uptake; (b) fusicoccin at 20 micromolar stimulates and FCCP at 15 micromolar inhibits this linear sucrose uptake; and (c) the ratio of the initial influx of proton to sucrose is close to one in both saturable and nondiffusive linear (difference between the total linear and diffusive components) uptakes. The results suggest that a proton/sucrose cotransport is also involved in the nondiffusive linear sucrose uptake into soybean cotyledon cells.     

Energetics of sucrose transport into protoplasts from developing soybean cotyledons

The accumulation of tetraphenylphosphonium (TPP⁺), 5,5′-dimethyl-oxazolidine-2,4-dione (DMO), and a micro pH electrode were used to measure membrane potential, intracellular and extracellular pH, respectively, upon the addition of exogenous sucrose to soybean cotyledon protoplasts. Addition of sucrose caused a specific and transient (a) depolarization of the membrane potential (measured by TPP⁺ accumulation), (b) acidification of the intracellular pH (measured by DMO accumulation), and (c) alkalization of the external medium (measured by a micro pH electrode). The time course for all these changes was similar (i.e. 5 to 10 minutes). Based on the rate of sucrose uptake and alkalization of the external medium, a stoichiometry of 1.02 to 1.10 for proton to sucrose was estimated. These data strongly support a proton/sucrose cotransporting mechanism in soybean cotyledon cells.     

Identification of membrane protein associated with sucrose transport into cells of developing soybean cotyledons

The photolyzable sucrose derivative 6′-deoxy-6′-(4-azido-2-hydroxy)-benzamidosucrose (6′-HABS), competitively inhibited the influx of [¹⁴C] sucrose into protoplasts from developing soybean (Glycine max L. Merr cv Wye) cotyledons. Photolysis of ¹²⁵I-labeled 6′-HABS in the presence of 10 millimolar dithiothreitol and microsomal preparations from developing soybean cotyledons led to label incorporation into a moderately abundant membrane protein with an apparent molecular mass of about 62 kilodalton (kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 62 kD protein was partially protected from labeling by the inclusion of 100 millimolar sucrose in the photolysis medium and also by the inclusion of 10 millimolar phenyl α-d-thioglucopyranoside. Glucose, raffinose, or phenyl α-d-3-deoxy-3-fluoroglucopyranoside did not afford even partial protection from labeling. When the photolyzable moiety of 6′-HABS was attached to 6-deoxy-6-aminoglucose and ¹²⁵I labeled, the resulting photoprobe did not label the 62 kD protein above background. The labeled protein at 62 kD is therefore apparently a specific, sucrose binding protein. Sucrose influx into cotlyedons of less than 25 milligrams fresh weight (approximately 10 days after flowering) occurred by passive processes, but metabolically dependent uptake became dominant over the next 5 to 7 days of development. Both the Coomassie staining protein at 62 kD and label incorporation at that position in analysis of membrane proteins appeared concomitant with the onset of active sucrose influx. Polyclonal antibodies to the purified 62 kD protein bound specifically to a protein in the plasmalemma of thin sections prepared from cotyledons and density stained with colloidal gold-protein A. The results suggest that the 62 kD membrane protein is associated with sucrose transport and may be the plasmalemma sucrose transporter.     

Identification and characterization of a sucrose transporter isolated from the developing cotyledons of soybean

Reduced carbon produced in mature leaves is distributed throughout plants in the form of sucrose. Sucrose transporter proteins (SUT) play a crucial role in transporting sucrose. We isolated a cDNA encoding a sucrose transporter, GmSUT1, which is expressed in the developing cotyledons of soybean (Glycine max). [14C]sucrose uptake assays demonstrate that GmSUT1 has a K(m) of 5.6mM and a V(max) of 5.8 nmol sucrose min(-1)(mg cells)(-1), which are similar to those of the low-affinity-high-capacity sucrose transporter family. GmSUT1 protein accumulates gradually during cotyledon development, correlating with increasing sucrose levels in the maturing cotyledons. Collectively, these data suggest that GmSUT1 plays an active role in the movement of sucrose into the developing seeds.     

Sucrose uptake by developing soybean cotyledons

Sucrose uptake by excised developing soybean cotyledons shows a biphasic dependence on sucrose concentration. At concentrations less than about 50 millimolar external sucrose, uptake can be described as a carrier-mediated process, with a K_m of 8 millimolar. At higher external sucrose concentrations, a linear dependence becomes apparent, which suggests the participation of a nonsaturable component in total uptake. Sucrose absorption is dependent on the presence of an electrochemical potential gradient for protons since agents interfering with the generation or maintenance of this gradient (NaN₃ or carbonylcyanide-m-chlorophenyl hydrazone) decrease sucrose transport to a level at or below that predicted from the operation of the noncarrier-mediated process alone. The saturable component of sucrose uptake is also sensitive to the sulfhydryl-modifying compounds N-ethylmaleimide and p-chloro-mercuribenzenesulfonate. The thiol-reducing agent diethioerythritol reverses fully the p-chloro-mercuri-benzenesulfonate inhibition, but not that of N-ethyl maleim de. Sucrose transport is sensitive to external pH, being decreased at high pH₀. Since sucrose-induced depolarization of the membrane potential and carrier-mediated sucrose influx show similar pH-dependence, inhibitor sensitivity, and values of K_m for sucrose, a sucrose/proton contransport process appears to operate in developing soybean cotyledon cells. Measurement of free space and intracellular sucrose concentrations in vivo suggests that the carrier-mediated process is fully saturated and that sucrose transport may be limiting for sucrose accumulation by the developing seed.     

Lipid molecular species composition in developing soybean cotyledons

The fatty acid composition of triglyceride and phospholipids in developing soybean cotyledons (Glycine max L., var. “Harosoy 63”) was analyzed at several stages of growth between 30 and 70 days after flowering. Changes observed in fatty acid composition within each lipid class were related to the levels of lipid molecular species present in the oil. Thirteen molecular species of triglyceride were identified in developing cotyledons, however three of these groups: trilinolenic, dilinolenic-monolinoleic, and linolenic-linoleic-oleic triglycerides, were not found in the mature seed. In immature cotyledons, trioleic and trilinoleic triglycerides accounted for 50% of the structures found; the level of these molecules decreased to 24.9% in the mature seed. The dilinoleic-monolinolenic triglycerides increased from 0.4 to 23.4% during cotyledon development. Changes in triglyceride composition were compared to the levels of molecular species for each phospholipid class. Dilinoleic and monosaturated monolinoleic phospholipid species were dominant in all phospholipid classes throughout development.     

Role of phloem in sucrose transport by Ricinus cotyledons

Sucrose is taken up and accumulated by cotyledons of Ricinus communis L. Autoradiographic studies reveal a predominant accumulation of sucrose in the phloem of the cotyledons. The export of sucrose from the cotyledons to hypocotyl and roots proceeds in the phloem by mass flow. These results, taken together with previous data, are experimental evidence for proton-sucrose symport as the mechanism of phloem loading.     

In vivo citrate studies with developing soybean cotyledons

The in vivo data presented here are strong evidence for theinvolvement of citrate cleavage enzyme in lipid synthesis indeveloping soybean cotyledons. The incorporation of ¹⁴C fromcitrate into crude lipid fraction in vivo had a pH optimum of4.5; was linear with time; had a temperature optimum of 35?C;and was inhibited by (–)-hydroxycitrate. The point ofcitrate cleavage was between carbons 3 and 4 of the citratemolecule and therefore ¹⁴C was incorporated into crude lipidfraction from citrate-5-¹⁴C but not citrate-1-¹⁴C or citrate-6-¹⁴C. ¹ Cooperative investigations of the Agricultural Research Service,U.S. Department of Agriculture, and the Illinois AgriculturalExperiment Station. ² This research represents partial fulfillment of the Ph.D.requirements of Daniel R. Nelson. Presently at Monsanto AgriculturalProducts Co., St. Louis, MO 63141, U.S.A. (Received September 20, 1976; )     

Involvement of phospholipids in triglyceride biosynthesis by developing soybean cotyledons

The incorporation of phospholipids specifically labeled with glycerol-2³H and acyl-¹⁴C by whole cell tissues of developing soybean cotyledons (Glycine max L.) reveals that phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, N-acylphosphatidylethanolamine, and phosphatidic acid can be metabolized to diglyceride. The diglyceride formed may be recylced into phospholipid or acylated to triglyceride. Diglyceride from phosphatidic acid and phosphatidylethanolamine is used readily in triglyceride biosynthesis compared to the other phospholipids. Incorporation of N-acylphosphatidylethanolamine having [9-10-³H(N)]oleic acid esterified at sn-3 in cotyledons shows rapid acyltransfer of ³H into triglyceride and therefore N-acylphosphatidylethanolamine appears to participate in triglyceride biosynthesis as an acyl donor. These studies emphasize phospholipid metabolism in developing soybean cotyledons is a dynamic process which plays a key role in triglyceride formation.     

Studies on lipid synthesis and degradation in developing soybean cotyledons

The metabolic activity of individual lipid classes found in developing soybean cotyledons (Glycine max.) is estimated by determining the degradation rate of the compound under given conditions. Pulse-labeling and dual substrate labeling are used to evaluate this parameter. These studies indicate first order decay kinetics for phosphatidic acid, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, N-acyl-phosphatidylethanolamine, diglyceride, and zero order kinetics for triglyceride in cotyledons var. "Harosoy 63" at 30 days after flowering. Decay coefficients for acyl groups and lipid-glycerol moieties within specific lipid classes from either method are comparable. Half-life (t((1/2))) calculations from the decay coefficients indicate extremely rapid turn-over rates (0.08 to 3.4 hours at 25 C) and suggest similar turnover rates of acyl groups and lipid-glycerol in diglyceride and all phospholipids except N-acylphosphatidylethanolamine where acyl groups are replaced independent of the glycerol moiety. These experiments reveal not only different metabolic activity between lipid components of soybean cotyledons, but also describe a new method for measuring lipid turnover in plants.     

The role of citrate in lipid synthesis in developing soybean cotyledons

The role of citrate and the citrate cleavage enzyme in lipidsynthesis in developing soybean cotyledons (Glycine max L. Merr.var. Harosoy 63) was investigated. The activity of the enzymewas inhibited by (—) hydroxycitrate, which is a specificinhibitor of citrate cleavage by this enzyme. Incorporationof label from citrate-1-¹⁴C and -5-¹⁴C indicated that the citratemolecule is cleaved between carbons 3 and 4. Acetyl CoA-¹⁴Cand oxaloacetate-¹⁴C phenylhydrazone were isolated as productsof the citrate cleavage reaction. The production of oxaloacetate-¹⁴C-phenylhdrazonefrom citrate-6-¹⁴C was carried out using a nucleotide free enzymepreparation and did not require the addition of ATP or CoA.Therefore it would appear that the citrate cleavage reactionis not CoA dependent in developing soybean seeds. Incorporationof pyruvate-2-¹⁴C into the crude lipid fraction was shown torequire both the particulate and soluble fractions. Apparentlyin soybeans, as in animal systems, pyruvate is oxidized by thepyruvate dehydrogenase complex and the acetyl CoA formed condenseswith oxaloacetate to produce citrate in the mitochondria. Citrateis then transported out of the mitochondria to the cytosol whereit is cleaved to form acetyl CoA for lipid synthesis. ¹ Cooperative investigations of the Agricultural Research Service,U.S. Department of Agriculture, and Illinois Agricultural ExperimentStation. ² This research represents partial fulfillment of the Ph. D.requirements of Daniel R. Nelson. Presently at Monsanto AgriculturalProducts Co., St. Louis, MO 63141, U.S.A. (Received January 12, 1977; )     

Sugar transport into protoplasts isolated from developing soybean cotyledons : I. Protoplast isolation and general characteristics of sugar transport

A procedure is described to isolated functional protoplasts from developing soybean (Glycine max L. Merr. 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 Q₁₀ value and high activation energies).     

Involvement of phospholipids in polyunsaturated Fatty Acid synthesis in developing soybean cotyledons

Developing soybean (cv. Dare) cotyledons harvested at 30 days after flowering were pulse-labeled with [1-(14)C]oleoyl-CoA. The metabolic interrelation of radiolabeled unsaturated fatty acids between the major glycerolipid classes was determined at various time intervals. At chase time zero, [(14)C]oleic acid accounted for 99.2% of the total glycerolipid radioactivity, and phospholipids contained 92% of the total incorporated radioactivity. With time, phospholipids were metabolized in triacylglycerol biosynthesis and radioactivity was detected in polyunsaturated fatty acids. The hypothesis that phospholipids were metabolic intermediates in polyunsaturated fatty acid biosynthesis was tested by comparing the theoretical and the actual amount of radiolabeled oleic acid that was associated with triacylglycerol as a function of time. The radioactive oleic acid found in triacylglycerol at various intervals was derived from phospholipids via a diacylglycerol intermediate. Assuming no phospholipid desaturation, the potential or theoretical amounts of [(14)C]oleic acid that could be transferred to triacylglycerol from phospholipids was defined by a system of differential equations. The results demonstrated that the decline in [(14)C]oleic acid from phospholipid after long chase intervals was equal to the total amount of radioactive unsaturated fatty acids found in neutral lipids. The difference between the theoretical and actual amounts of [(14)C]oleic acid present in triacylglycerol after long time intervals was equal to the amount of radioactivity present in polyunsaturated fatty acids. Based upon those findings in soybeans, the desaturation of oleic acid associated with phospholipids was highly probable.     

Effect of freezing and cold storage on phospholipids in developing soybean cotyledons

Freezing of plant tissue adversely affects lipid composition. Immature soybean cotyledons (Glycine max L. Merr.) var. “Harosoy 63” were frozen with liquid N₂, dry ice, or stored in a freezer (−20 C) before lipid extraction. The effects of freezing temperature, thawing rate, and cold storage on the lipid composition of frozen tissue revealed significantly higher levels of phosphatidic acid, and diminished levels of phosphatidylcholine, phosphatidylethanolamine, and N-acylphosphatidylethanolamine from the control. Regardless of freezing temperature, phosphatidic acid levels increased from 4.7 mole% to nearly 50 mole% of the total phospholipid when frozen tissues were stored 10 days at −20 C. During the same period, N-acylphosphatidylethanolamine decreased from 54.1 mole% to 6.6 mole% phospholipid. At least 8 mole% of the phosphatidic acid increase occurred during slow thawing of the frozen tissues. In autoclaved samples, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, and N-acylphosphatidylethanolamine levels were not different from the control. Labeling of the lipid-glycerol with ³H, and fatty acids with ¹⁴C, demonstrated the degradation product was primarily phosphatidic acid. Apparently enzymic destruction of the phospholipids occurred during freezing, cold storage, and thawing.     

N and C NMR determination of allantoin metabolism in developing soybean cotyledons

The metabolism of allantoin by immature cotyledons of soybean (Glycine max L. cv Elf) grown in culture was investigated using solid state ¹³C and ¹⁵N nuclear magnetic resonance. All of the nitrogens of allantoin were incorporated into protein in a manner similar to that of each other and to the amide nitrogen of glutamine. The C-2 of allantoin was not incorporated into cellular material; presumably it was lost as CO₂. About 50% of the C-5 of allantoin was incorporated into cellular material as a methylene carbon; the other 50% was presumably also lost as CO₂. The ¹³C-¹⁵N bonds of [5-¹³C;1-¹⁵N] and [2-¹³C;1,3-¹⁵N]allantoin were broken prior to the incorporation of the nitrogens into protein. These data are consistent with allantoin's degradation to two molecules of urea and one two-carbon fragment. Cotyledons grown on allantoin as a source of nitrogen accumulated 21% of the nitrogen of cotyledons grown on glutamine. Only 50% of the nitrogen of the degraded allantoin was incorporated into the cotyledon as organic nitrogen; the other 50% was recovered as NH₄⁺ in the media in which the cotyledons had been grown. The latter results suggests that the lower accumulation of nitrogen by cotyledons grown on allantoin was in part due to failure to assimilate NH₄⁺ produced from allantoin. The seed coats had a higher activity of glutamine synthetase and a higher rate of allantoin degradation than cotyledons indicating that seed coats play an important role in the assimilation and degradation of allantoin.     

N and C NMR determination of methionine metabolism in developing soybean cotyledons

The metabolism of d- and l-methionine by immature cotyledons of soybean (Glycine max, L. cv Elf) grown in culture has been investigated using solid-state ¹³C and ¹⁵N nuclear magnetic resonance. d-Methionine is taken up by the cotyledons and converted to an amide, most likely by N-malonylation. About 16% of the l-methionine taken up is incorporated intact into protein, and 25% remains as soluble methionine. Almost two-thirds of the l-methionine that enters the cotyledons is degraded. The largest percentage of this is used in transmethylation of the carboxyl groups of pectin. Methionine is not extensively converted to polyamines. We attribute the stimulation of growth of the cotyledons by exogenous methionine to the bypassing of a rate-limiting methyl-transfer step in the synthesis of methionine itself, and subsequently of pectins and proteins.     

The origin of protein bodies in developing soybean cotyledons: a proposal

Summary The biogenesis of protein bodies is examined in cotyledons of soybean (Glycine max, Merr) at the time when reserve protein is beginning to accumulate in the cotyledons. Reserve protein is deposited in the central vacuoles of parenchyma cells and new protein bodies arise from the central vacuole by pinching-off small masses of reserve protein surrounded by a portion of the tonoplast.Supported by a grant to MJC from the National Science Foundation (Metabolic Biology) and a grant (to BYY) from the National Research Council of Canada.On leave from the Department of Biology, University of New Brunswick, N.B., Canada.     

Seed-specific overexpression of a potato sucrose transporter increases sucrose uptake and growth rates of developing pea cotyledons

During the storage phase, cotyledons of developing pea seeds are nourished by nutrients released to the seed apoplasm by their maternal seed coats. Sucrose is transported into pea cotyledons by sucrose/H+ symport mediated by PsSUT1 and possibly other sucrose symporters. PsSUT1 is principally localised to plasma membranes of cotyledon epidermal and subepidermal transfer cells abutting the seed coat. We tested the hypothesis that endogenous sucrose/H+ symporter(s) regulate sucrose import into developing pea cotyledons. This was done by supplementing their transport activity with a potato sucrose symporter (StSUT1), selectively expressed in cotyledon storage parenchyma cells under control of a vicilin promoter. In segregating transgenic lines, enhanced [(14)C]sucrose influx into cotyledons above wild-type levels was found to be dependent on StSUT1 expression. The transgene significantly increased (approximately 2-fold) transport activity of cotyledon storage parenchyma tissues where it was selectively expressed. In contrast, sucrose influx into whole cotyledons through the endogenous epidermal transfer cell pathway was increased by only 23% in cotyledons expressing the transgene. A similar response was found for rates of biomass gain by intact cotyledons and by excised cotyledons cultured on a sucrose medium. These observations demonstrate that transport activities of sucrose symporters influence cotyledon growth rates. The attenuated effect of StSUT1 overexpression on sucrose and dry matter fluxes by whole cotyledons is consistent with a large proportion of sucrose being taken up at the cotyledonary surface. This indicates that the cellular location of sucrose transporter activity plays a key role in determining rates of sucrose import into cotyledons.     

The cellular pathway of sucrose transport in developing cotyledons ofVicia faba L. andPhaseolus vulgaris L.: a physiological assessment

The cellular pathway of sugar uptake in developing cotyledons of Vicia faba L. and Phaseolus vulgaris L. seed was evaluated using a physiological approach. The cotyledon interface with the seed coat is characterised by a specialised dermal cell complex. In the case of Vicia faba cotyledons, the epidermal component of the dermal cell complex is composed of transfer cells. Sucrose is the major sugar presented to the outer surface of both cotyledons and it is taken up from the apoplasm unaltered. Estimated sucrose concentrations within the apparent free space of Vicia and Phaseolus cotyledons were 105 and 113 mM respectively. Rates of in-vitro uptake of [¹⁴C]sucrose by cotyledon segments or by whole cotyledons following physical removal or porter inactivation of the outer cells demonstrated that, for both Vicia and Phaseolus cotyledons, the dermal cell complexes are the most intense sites of sucrose uptake. Accumulation of [¹⁴C]sucrose in the storage parenchyma of whole cotyledons was directly affected by experimental manipulation of uptake by the outer cell layers and plasmolytic disruption of the interconnecting plasmodesmata. These findings indicated that sucrose accumulated by the dermal cell complexes is transported symplasmically to the storage parenchyma. Overall, it is concluded that the dermal cell complexes of the developing legume embryo, irrespective of the presence or absence of wall ingrowths, are the major sites for the uptake of sucrose released from the maternal tissues to the seed apoplasm. Thereafter, the accumulated sucrose is transported radially inward through the symplast to the storage parenchyma.Abbreviations AFS apparent free space - CF 5-(6)-carboxyfluorescein - CFDA 5-(6)-carboxyfluorescein diacetate - Mes 2-(N-morpholino)ethanesulfonic acid - PCMBS p-chloromercuribenzenesulfonic acid - SRG sulphorhodamine G The investigation was supported by funds from the Research Management Committee, The University of Newcastle and the Australian Research Council. One of us, R. McDonald, gratefully acknowledges the support of an Australian Postgraduate Research Award. We are grateful to Stella Savoury for preparing the photomicrographs.