Electrogenic sucrose transport in developing soybean cotyledons

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 K_m for sucrose of about 10 millimolar. 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.     

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.     

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; )     

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.     

Kinetics of C-photosynthate uptake by developing soybean fruit

By pulse-labeling field-grown soybean leaves for 60 seconds at midday with ¹⁴CO₂ and then sequentially harvesting, dissecting, and extracting the radioactive fruit tissues (of pod and seeds), the route, uptake kinetics, and metabolic fate of ¹⁴C-photosynthate as it was imported by 35- to 40-day-old pods were determined. As the [¹⁴C]sucrose pulse entered the pods, the seeds became radioactive immediately but a lag of nearly 30 minutes occurred before label could be detected in the pod wall pericarp.     

Biosynthesis of Fatty acids by a soluble extract from developing soybean cotyledons

Fractionation of developing soybean cotyledons into cellular components demonstrates that most of the activity necessary to incorporate acetate-1-¹⁴C into lipid remains in the supernatant from a 198,000g spin for 1 hr. The system studied is dependent upon ATP, CoA, and CO₂. Concentrations of ATP greater than 4 × 10⁻³m are inhibitory, while 1 × 10⁻⁴m CoA is needed for optimal activity. Avidin inhibition of acetate incorporation into lipid could be reversed by biotin. Studies indicated that NADPH is a better source of reducing power than NADH. The system studied is inhibited by p-chloromercuribenzoic acid and this inhibition can be reversed by an excess of GSH. The system studied shows maximum activity in tris buffer at pH 8.6 or in glycine buffer, pH 9.4.     

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.     

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.     

D-Mannose uptake by fenugreek cotyledons

The uptake of D-mannose was studied in detached cotyledons of germinated fenugreek (Trigonella foenum-graecum L.) seeds. Uptake kinetics indicate the involvement of two components, a saturable component operating at low concentrations and a diffusion-like one at high concentrations. Treatment of cotyledons with carbonyl-cyanide-m-chlorophenylhydrazone and p-chloromercuribenzenesulfonic acid reduced D-mannose-uptake rates by about 35% and 35–65%, respectively. No difference in the uptake rates was observed in the presence of D-galactose or 3-O-methylglucose. D-Mannose uptake was not very much affected by pH. The optimum pH for its uptake was 6.5 while at pH 8.5 its uptake was reduced by 22%. D-Mannose addition to fenugreek cotyledons did not induce alkalinization of the medium. Furthermore, low turgor, which enhances proton/sugar cotransport, decreased D-mannose uptake while the uptake of 3-O-methylglucose was increased. The rate of D-mannose uptake by fenugreek cotyledons depended on the hours of imbibition. These changes of uptake were not followed by analogous changes in the turgor pressure (_p) of fenugreek cotyledons, which remained fairly constant. Results indicate that D-mannose is partially taken up by a carrier which has high specificity for D-mannose, but not by a H⁺-sugar cotransport system. It is further concluded that the carrier plays an important role in switching on and off the uptake capacity of fenugreek cotyledons during seedling development.Abbreviations and symbols CCCP carbonylcyanide-m-chlorophenylhydrazone - DTT dithiothreitol - 3-OMG 3-O-methylglucose - PCMBS p-chloromercuribenzensulfonic acid - water potential - _s osmotic potential - _p turgor pressure     

Sucrose uptake by cotyledons of Ricinus communis L.: Characteristics,mechanism, and regulation

Cotyledons of Ricinus communis take up externally supplied sucrose at a rate of up to 150 mol/h/g fresh weight, which is very high when compared with other sugar transport systems of higher plants. The uptake of sucrose is catalysed with a K _m of 25 mmol l^–1; at high sucrose concentrations a linear (diffusion) component becomes obvious. Other mono-, di-, or trisaccharides do not compete for sucrose uptake. Sucrose is accumulated by the cotyledons up to 100-fold, whereby most of the transported, externally supplied sucrose mixes with sucrose present in the tissue. At low sucrose concentrations, however; a small unexchangeable internal pool of sucrose becomes evident. Poisons of energy metabolism such as FCCP inhibit uptake and accumulation of sucrose. The transport of sucrose induces an increase of respiration, from which an energy requirement of 1.4 ATP/sucrose taken up can be calculated. Sucrose is taken up together with protons at an apparent stoichiometry of 0.3 protons/sucrose. Other sugars do not cause proton uptake. The K _m for sucrose induced proton uptake is 5 mmol l^–1; the discrepancy to the K _m for sucrose uptake as well as the low proton: sucrose stoichiometry might possibly be caused by a large contribution of diffusion barriers. The estimated proton-motive potential difference would by sufficient to explain an electrogenic sucrose accumulation. The rate of uptake of sucrose is subject to feedback inhibition by internal sucrose. It is also regulated during growth of the seedlings since it develops rapidly during the first days of germination and declines again after the 4th day of germination, though no substantial increase of passive permeability resistance was observed.Abbreviations DMO dimethyloxazolidinedione - FCCP trifluoromethoxy (carbonyl-cyanide) phenylhydrazon - fr. wt. fresh weight     

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; )     

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.     

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.     

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.     

Sugar uptake by the dermal transfer cells of developing cotyledons of Vicia faba L.

Two experimental systems were developed to study the uptake of sucrose by the dermal transfer cells of developing cotyledons of Vicia faba L. First, the in-vivo state was approximated by short-term (10 min) incubation of whole cotyledons in [¹⁴C]sucrose solutions. Under these conditions, a minimum of 67% of the ¹⁴C label entered the dermal transfer cell complex. Of this, at least 40% crossed the plasma membranes of the epidermal transfer cells. Second, a protocol was developed to enzymatically isolate and purify dermal transfer cell protoplasts. The yields of the transfer cell protoplasts were relatively low and their preparation incurred a significant loss of plasma membrane. However, the protoplasts remained viable up to 24 h following purification and proved to be a suitable system to verify transport properties observed with whole cotyledons. Using these two experimental systems, it was established that [¹⁴C]sucrose uptake by the dermal transfer cells exhibited features consistent with mediated energy-dependent transport. This included saturation kinetics, competition for uptake between structurally similar molecules, and inhibition of uptake by p-chloromercuribenzenesulfonic acid and several other metabolic inhibitors. For comparative purposes, sugar uptake by the storage parenchyma of the Vicia cotyledons was also examined. In contrast to the dermal transfer cell complex, sucrose uptake by the storage parenchyma displayed characteristics consistent with simple diffusion.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DNP 2,4-dinitrophenol - NEM N-ethylmaleimide - PCMBS p-chloromercuribenzenesulfonic acid 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 indebted to Stella Savory for preparing the ultrathin sections for electron microscopy.     

Sugar uptake by the dermal transfer cells of developing cotyledons of Vicia faba L.

The mechanism of carrier-mediated sucrose uptake by the dermal transfer cells of developing Vicia faba L. cotyledons was studied using excised cotyledons and isolated transfer cell protoplasts. Addition of sucrose resulted in a transitory alkalinization of the bathing solution whereas additions of glucose, fructose or raffinose had no effect. Dissipating the proton motive force by exposing cotyledons and isolated transfer cell protoplasts to an alkaline pH, carbonylcyanide m-chlorophenylhydrazone, weak acids (propionic acid and 5,5-dimethyl-oxazolidine-2,4-dione) or tetraphenylphos-phonium ion resulted in a significant reduction of sucrose uptake. The ATPase inhibitors, erythrosin B (EB), diethylstilbestrol (DES) and N,N-dicyclohexylcarbodiimide (DCCD) were found to abolish the sucrose-induced medium alkanization as well as reduce sucrose uptake. Cytochemical localization of the ATPase, based on lead precipitation, demonstrated that the highest activity was present in the plasma membranes located in wall ingrowth regions of the dermal transfer cells. The presence of a transplasma-membrane redox system was detected by the extracellular reduction of the electron acceptor, hexacyanoferrate III. The reduction of the ferric ion was coupled to a release of protons. The redox-induced proton extrusion was abolished by the ATPase inhibitors EB, DES and DCCD suggesting that proton extrusion was solely through the H⁺-ATPase. Based on these findings, it is postulated that cotyledonary dermal transfer cells take up sucrose by a proton symport mechanism with the proton motive force being generated by a H ⁺ -ATPase. Sucrose uptake by the storage parenchyma and inner epidermal cells of the cotyledons did not exhibit characteristics consistent with sucrose-proton symport.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - EB erythrosin B - Em membrane potential - FC fusicoccin - HCF II hexacyanoferrate II - HCF III hexacyanoferrate III - Mes 2-(N-morpholino)ethanesulfonic acid - pmf proton motive force - TPP⁺ tetraphenylphosphonium ion 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 indebted to Stella Savory for preparing the ultrathin sections for electron microscopy.     

Sucrose and proton cotransport in Ricinus cotyledons

In Ricinus cotyledons, evidence for proton extrusion came from observation of direct acidification of the medium in the presence of potassium salts. Increasing K⁺ influx with increasing pH suggested a link between K⁺ influx and H⁺ efflux by an H⁺ pump. The kinetics of K⁺ influx and H⁺ efflux were consistent with a 1:1 stoichiometry K⁺:H⁺, which may indicate either electrical coupling or carrier mediated exchange. The results were consistent with an H⁺ pump setting up an electrochemical potential gradient which provides the driving force for an H⁺-sucrose cotransport and the movement of K⁺. With reference to this, a model for phloem loading is suggested.