Analysis of the reaction products from incubation of sugarcane vacuoles with uridine-diphosphate-glucose: no evidence for the group translocator

Isolated sugarcane (Saccharum spp. hybrid H50-7209) vacuoles incorporate radioactivity during incubation with labeled UDP-glucose by a mechanism which was postulated to be responsible for sucrose storage in the vacuoles (UDP-glucose group translocator). Analysis of the reaction products in the medium revealed that several enzymic processes are going on during incubation with UDP-glucose such as production of hexose phosphates, UMP, and sugars, all of which seem unrelated to the incorporation of radioactivity into vacuoles. The incorporated radioactivity was identified mainly as (1→3)-β-glucan (callose) of polymerization grades up to more than 20. Callose occurs as a contaminant at the surface of isolated vacuoles coming from the plasmalemma. The properties of UDP-glucose incorporation into the vacuolar preparation compared favorably with known properties of callose synthase. The low mol wt glucans that are found are probably degradation products of labeled callose due to hydrolases, which are liberated by centrifugation of vacuoles. The labeled disaccharide, which chromatographically had been formerly identified as sucrose, is laminaribiose. No sucrose (or sucrose phosphate) could be identified in the vacuole preparation after incubation with UDP-glucose. Thus, the mechanism of sucrose storage in sugarcane vacuoles is still open.     

Pathway of Phloem unloading of sucrose in corn roots

The pathway of phloem unloading and the metabolism of translocated sucrose were determined in corn (Zea mays) seedling roots. Several lines of evidence show that exogenous sucrose, unlike translocated sucrose, is hydrolyzed in the apoplast prior to uptake into the root cortical cells. These include (a) presence of cell wall invertase activity which represents 20% of the total tissue activity; (b) similarity in uptake and metabolism of [¹⁴C]sucrose and [¹⁴C]hexoses; and (c) randomization of ¹⁴C within the hexose moieties of intracellular sucrose following accumulation of [¹⁴C] (fructosyl)sucrose. Conversely, translocated sucrose does not undergo apoplastic hydrolysis during unloading. Asymmetrically labeled sucrose ([¹⁴C](fructose)sucrose), translocated from the germinating kernels to the root, remained intact indicating a symplastic pathway for unloading. In addition, isolated root protoplasts and vacuoles were used to demonstrate that soluble invertase activity (V_max = 29 micromoles per milligram protein per hour, K_m = 4 millimolar) was located mainly in the vacuole, suggesting that translocated sucrose entered via the symplasm and was hydrolyzed at the vacuole prior to metabolism.     

Characterization of proton and sugar transport at the tonoplast of sweet lime (Citrus limmetioides) juice cells

Citrus fruits accumulate high levels of sucrose and hexoses, although most photoas-similates arrive in the form of sucrose. In sweet limes, faster rates of sugar accumulation take place early in development when sucrose catabolic enzymes are most active. The present investigation was aimed at providing information on the mechanisms of sucrose (and hexose) uptake into the vacuole of cells containing high levels of sucrose hydrolytic activity. Tonoplast vesicles of high purity were isolated in a discontinuous sucrose gradient. The vesicles were capable of forming a pH gradient in the presence of ATP. Both bafilomycin and NO₃⁻ (but not vanadate) inhibited ATP hydrolysis and prevented the formation of the pH gradient, confirming the tonoplast origin. Energized vesicles (either by addition of ATP or by artificial pH gradient) did not accumulate sucrose or hexoses against a concentration gradient. In the presence of either sucrose or hexoses, the established ΔpH; was not disrupted as was the case with tonoplast vesicles from red beet hypocotyl. Therefore, a sucrose/H⁺ (hexose) antiport may not be the mechanism of sucrose and hexose transport into the vacuoles of sweet lime juice cells. The data indicated that sucrose uptake into vacuoles of sweet lime occurs by facilitated diffusion. Hexoses originate from the hydrolytic action of acid invertase on sucrose within the vacuole, and by the action of cytosolic sucrose synthase.     

Effects in adipocytes of diamide on GSH levels,glucose uptake and cell integrity

Concentrations of insulin and chemical agents (H₂O₂, vitamin K-5) which stimulate hexose transport in fat cells do not alter the cellular levels of glutathione (reduced form; GSH). Diamide, another agent used in studies of insulin action, markedly reduces GSH levels and increases the movement of sugar into the cell. However, unlike insulin, H₂O₂ or vitamin K-5, diamide causes a change in the permeability of fat cells that allows entry of compounds (inulin, sucrose, l-glucose) which are normally excluded by the plasma membrane. Moreover, the accelerated rate of methylglucose uptake produced by diamide treatment is not inhibited by cytochalasin B, an agent that blocks basal and insulin-stimulated methylglucose transport. These results indicate that diamide does not cause a stimulation of the glucose transport system and should not be used (or used with caution) in transport studies. Furthermore, oxidation of GSH does not appear to be necessary for the stimulation of hexose transport in adipocytes by insulin, H₂O₂ or vitamin K-5.     

Assessment of glycinebetaine and proline compartmentation by analysis of isolated beet vacuoles

Vacuoles isolated from storage root tissue of red beet (Beta vulgaris L.) do not leak significant quantities of betanin, sucrose, Na⁺ or K⁺ during isolation. This indicates that analysis of vacuoles in vitro gives meanigful information about the compartmentation of solutes in vivo. Preparations of vacouoles were used to determine the distribution of glycinebetaine and proline between vacuole and cytoplasm in beet cells. Both compounds were detected in preparations of isolated beet vacuoles. In the case of glycinebetaine it was shown that this solute was associated with the vacuoles, not with the small number of other organelles which contaminated the preparations. The vacuolar pool accounted for 26 to 84% of the total tissue glycinebetaine and 17 to 57% of the proline. Concentrations of these compounds in vacuole and cytoplasm were calculated and were always higher in the cytoplasm than in the vacuole. The concentration gradient across the tonoplast varied considerably. The significance of these results is discussed in relation to the hypothesis that glycinebetaine and proline function as benign cytoplasmic osmotica.Abbreviations A₅₃₇ absorbance at 537 nm - MES 2-(N-morpholino)-ethanesulphonic acid - Na₂EDTA ethylenediaminetetraacetic acid, disodium salt - SDS sodium dodecyl sulphate - Tris tris(hydroxymethyl)methylamine     

Evidence for the involvement of a UDP-glucose-dependent group translocator in sucrose uptake into vacuoles of storage roots of red beet

Vacuoles isolated from the storage roots of red beet (Beta vulgaris L.) accumulate sucrose via two different mechanisms. One mechanism transports sucrose directly, and its rate is increased by the addition of MgATP. The other mechanism utilizes uridine diphosphate glucose (UDP-glucose) to synthesize and simultaneously transport sucrose phosphate and sucrose into the vacuole. This group translocation mechanism has also been found in sugarcane vacuoles. As in sugarcane, the beet group translocator does not require fructose 6-phosphate, nor is the latter substance transported into the vacuole. The uptake of UDP[¹⁴C]glucose in inhibited by high concentrations of osmoticum.Abbreviations EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - UDP uridine 5-diphosphate     

Vacuoles from Sugarcane Suspension Cultures : I. ISOLATION AND PARTIAL CHARACTERIZATION

Vacuoles were isolated from suspension cultures of sugarcane (Saccharum sp.) cells by centrifugation of protoplasts at high g force against a 12% (w/v) Ficoll solution. Distribution of marker enzymes and Concanavalin A binding showed an 11% contamination of the vacuole preparation by cytoplasmic components, mitochondria, and endoplasmic reticulum, and 18% contamination by plasma membrane. Acid phosphatase, carboxypeptidase, protease, peroxidase, and ribonuclease activities were enriched in isolated vacuoles. Carboxypeptidase was tonoplast-bound, whereas the other enzymes were soluble. Sucrose, reducing sugars, and free amino acids were measured in protoplasts and vacuoles during growth of cells in suspension culture. Sucrose and reducing sugar content of vacuoles increased as the culture aged, while free amino acids decreased sharply.     

Glucose transport and metabolism in the perfused hindquarters of lean and obese-hyperglecemic (db/db) mice effects of insulin and electrical stimulation

Changes in glucose transport and metabolism in skeletal muscles of the obese-diabetic mice (db/db) was characterized using the perfused mouse hindquarter preparation. Metabolism of [5-³H]glucose, uptake of 3-O-[methyl-³H]glucose (methylglucose) and [2-¹⁴C]deoxyglucose (deoxyglucose) was studied under resting, electrically stimulated contracting, and insulin-stimulated conditions. Basal rate of methylglucose uptake was 255 ± 18 and 180 ± 9 μl/15 min per ml intracellular fluid space for lean and db/db mice, respectively. The V_? of methylglucose transport was decreased with no change in K_m in the db/db mice. Both electrical stimulation and insulin (1/mU/ml) increased methylglucose uptake rate 2-fold in both lean and obese mice. We observed no significant change in insulin sensitivity in the db/db mice in stimulating methylglucose uptake which was subnormal under all conditions. Similar results were obtained using deoxyglucose. Likewise, uptake of glucose and ³H₂O production from [5-³H]glucose were significantly reduced, both at rest and during electrically stimulated contraction in the db/db mouse. However, lactate production in the electrically stimulated db/db mouse preparations was not significantly different from that in the lean mice. These data suggest a major contribution from an impaired glucose transport activity to the reduction in glucose metabolism in the db/db mouse skeletal muscle.     

Isolation and Characterization of Vacuoles from the Ergot Fungus Claviceps purpurea

Protoplasts of Claviceps purpurea were prepared by treatment of mycelium with a lytic mixture of snail gut enzyme and cellulase from Trichoderma viride. Such protoplasts could be efficiently lysed by Triton X-100 treatment at high osmotic pressure without Ca²⁺ or Mg²⁺, allowing the release of intact vacuoles in high yields. Vacuoles obtained from cells grown in modified Vogel medium (vegetative-type cells not producing alkaloids) were isolated and purified by centrifugation from a 5% Ficoll 400 (wt/vol) phase into the interphase between two layers, one containing 0.25 M each of mannitol and sucrose, and one containing 0.5 M mannitol. Vacuoles derived from cells grown in a medium favoring ergot alkaloid synthesis (sclerotia-like cells) were isolated by gentle centrifugation of filtered protoplast lysates without addition of Ficoll 400. Biochemical analyses of the vacuole fraction isolated from either kind of cell revealed their function as compartments harboring several hydrolytic enzymes. However, the enrichment of free amino acids in vacuoles of sclerotia-like cells was less pronounced than that in vacuoles of vegetative-type cells, indicating a difference in metabolic compartmentation in the two types of cells.     

The regulation of sugar uptake and accumulation in bean pod tissue

The identity, localization and physiological significance of enzymes involved in sugar uptake and accumulation were determined for endocarp tissue of pods of Kentucky Wonder pole beans (Phaseolus vulgaris). An intracellular, alkaline invertase (pH optimum, 8) was assayed in extracted protein, as well as enzymes involved in sucrose synthesis, namely, uridinediphosphate (UDP-glucose pyrophosphorylase and UDP-glucose-fructose transglucosylase). Indirect evidence indicated the presence also of hexokinase, phosphohexoseisomerase and phosphoglucomutase. The data suggested that sucrose synthesis occurred in the cytoplasm, and that both sugar storage and an alkaline invertase occurred in the vacuole. The latter functions to hydrolyze accumulated sucrose. An outer space invertase (pH optimum, 4.0) was detected, but was variable in occurrence. Although its activity at the cell surface enhanced sucrose uptake, sucrose may be taken up unaltered.

Over a wide range of concentrations of exogenous glucose the sucrose/reducing sugar ratio of accumulated sugars remained unchanged at about 20. Synthesis of sucrose appears to be requisite to initial accumulation from glucose or fructose, as free hexoses do not increase at the apparent saturating concentration for uptake. Sucrose accumulation from exogenous hexose represents a steady-state value, in which sucrose is transported across the tonoplast into the vacuole at a rate equivalent to its rate of synthesis. Evidence indicates that this component of the accumulation process involves active transport of sucrose against a concentration gradient. The ratio of sucrose/reducing sugars in the accumulated sugars immediately after a period of uptake was inversely related to the level of inner space invertase. Within 16 hours after a period of accumulation, practically all of the sugar occurs as glucose and fructose.

The absence of competition among hexoses and sucrose indicated that a common carrier was not involved in their uptake. From a series of studies on the kinetics of uptake of glucose and fructose, including competition studies, the effects of inhibitors, radioactive assay of accumulated sugars and the distribution of label in accumulated sucrose it appeared that rate limitation for glucose or fructose uptake resides in the sequence of reactions leading to sucrose synthesis, rather than in a process mediated by a carrier protein.

     

Sucrose Compartmentation in the Palisade Parenchyma of Vicia faba L

Intracellular sucrose compartmentation in the palisade parenchyma of Vicia faba L. leaflets was investigated by comparing the specific radioactivity of photosynthetically labeled [¹⁴C]sucrose in samples enriched in vacuole to that in samples enriched in cytoplasm. Brief centrifugation of leaflet punches was used to sediment most of the palisade parenchyma cytoplasm in the adaxial ends of the cells. The punches were quick-frozen, freeze-substituted, and embedded in methacrylate. Samples enriched in cytoplasm or in vacuoles were obtained from paradermal sections. After pulse-labeling, the sucrose specific radioactivity in vacuole-enriched samples was fairly constant. Sucrose specific radioactivity in cytoplasm-enriched samples was about 2.5 times that in vacuole-enriched samples initially and declined thereafter. Earlier interpretation of intracellular sucrose compartmentation (Plant Physiol 1975 55: 704-711) had predicted larger specific activity differences (up to 20 times) between the cytoplasm and vacuole. The difference between the actual and predicted behavior is ascribed to the observed extent of cross-contamination in samples and, more importantly, to the confinement of sucrose to extrachloroplastic regions of the cytoplasm.     

Intracellular Localization of Peptide Hydrolases in Wheat (Triticum aestivum L.) Leaves

Protoplasts from 8- to 9-day-old wheat (Triticum aestivum L.) leaves were used to isolate organelles which were examined for their contents of peptide hydrolase enzymes and, in the case of vacuoles, other acid hydrolases. High yields of intact chloroplasts were obtained using both equilibrium density gradient centrifugation and velocity sedimentation centrifugation on sucrose-sorbitol gradients. Aminopeptidase activity was found to be distributed, in approximately equal proportions, between the chloroplasts and cytoplasm. Leucyltyrosine dipeptidase was mainly found in the cytoplasm, although about 27% was associated with the chloroplasts. Vacuoles shown to be free from Cellulysin contamination contained all of the protoplast carboxypeptidase and hemoglobin-degrading activities. The acid hydrolases, phosphodiesterase, acid phosphatase, α-mannosidase, and β-N-acetylglucosamidase were found in the vacuole to varying degrees, but no β-glucosidase was localized in the vacuole.     

In Vitro Sugar Transport in Zea mays L. Kernels : II. Characteristics of Sugar Absorption and Metabolism by Isolated Developing Embryos

In vitro sugar transport into developing isolated maize embryos was studied. Embryo fresh and dry weight increased concomitantly with endogenous sucrose concentration and glucose uptake throughout development. However, endogenous glucose and fructose concentration and sucrose uptake remained constant. The uptake kinetics of radiolabeled sucrose, glucose, and fructose showed a biphasic dependence on exogenous substrate concentration. Hexose uptake was four to six times greater than sucrose uptake throughout development. Carbonylcyanide-m-chlorophenylhydrazone and dinitrophenol inhibited sucrose and glucose uptake significantly, but 3-O-methyl glucose uptake was less affected. The uptake of 1 millimolar sucrose was strongly pH dependent while glucose was not. Glucose and fructose were readily converted to sucrose and insoluble products soon after absorption into the embryo. Thus, sucrose accumulated, while glucose pools remained low. Based on the findings of this and other studies a model for sugar transport in the developing maize kernel is presented.     

Isolation of Vacuoles from Immature Apple Fruit Flesh and Compartmentation of Sugars, Organic Acids, Phenolic Compounds and Amino Acids

Vacuoles of immature apple fruit (Malus pumila Mill. var. domesticaSchneid.) were obtained by purification using Ficoll densitygradient centrifugation after lysis of the protoplasts by bothmild osmotic shock and the addition of EDTA and BSA. The recoverywas about 35% of the protoplasts. The isolated vacuoles hada mean diameter of about 100 µm. The distribution of sugars, organic acids, phenolic compoundsand amino acids in the vacuole, the cytoplasm and the free spacewas determined. Almost all of the fructose and glucose, themajor sugars of the tissue, were found in the vacuole. Sorbitolwas mainly located in the free space and the vacuole, and sucrosein the free space and the cytoplasm. More than 90% of the malicacid, the main organic acid, was located in the vacuole. Almostall of the phenolic compounds were also deposited in the vacuole. The volumes of the vacuole, the cytoplasm and the free spacein the whole tissue were calculated from the cell numbers ofthe whole tissue, the volume of the isolated protoplasts, andthe volume of the vacuoles present in the protoplast. The soluteconcentration in each compartment was estimated: vacuoles, 888mM; cytoplasm, 37 mM; free space, 57 mM. How these compartmentationsof solutes affected the translocation of sugars into the fruitand the cell expansion is discussed. ¹This paper is contribution A-159 of the Fruit Tree ResearchStation. (Received July 7, 1983; Accepted November 14, 1983)     

The role of the vacuole in the accumulation and mobilization of sucrose

The contribution that isolated vacuoles have made to understanding sucrose storage and mobilization is reviewed briefly, with particular reference to the storage root of red beet (Beta vulgaris L.). Work with isolated vacuoles has shown that in this tissue sucrose is confined to the vacuole and some progress has been made in elucidating the possible mechanism of sucrose transport into the vacuole. The evidence that this is a H⁺: sucrose antiport, dependent on the activity of a proton-translocating ATPase is examined. It is concluded that while there is some evidence for the presence of a proton pump, a link between this and sucrose uptake has still to be established. Using isolated vacuoles it has been demonstrated that during mobilization of sucrose, hydrolysis occurs within the vacuole but the mechanism of unloading of hexoses from the vacuole remains to be elucidated.     

Changes in biochemical composition of vacuoles isolated from Acer pseudoplatanus L. during cell culture

Vacuoles were isolated from Acer pseudoplatanus cell suspension culture using a one-step procedure involving the lysis of the protoplast plasmalemma through a gradient of Ficoll containing DEAE-Dextran. The vacuole suspensions were slightly contaminated by other organelles (less than 5%) and the isolated vacuoles readily accumulated neutral red. Since α-mannosidase was located exclusively in the vacuoles it was used as a convenient marker. It was shown that the number of vacuoles per protoplast decreased as the cell aged. Studies on the biochemical composition of the isolated vacuoles indicated that amino acids, organic acids and protein contents varied with the cell culture cycle, emphasizing the dynamic status of the vacuolar system in cell suspension cultures of Acer pseudoplatanus.     

In Vitro Sugar Transport in Zea mays L. Kernels : I. Characteristics of Sugar Absorption and Metabolism by Developing Maize Endosperm

Short-term transport studies were conducted using excised whole Zea mays kernels incubated in buffered solutions containing radiolabeled sugars. Following incubation, endosperms were removed and rates of net ¹⁴C-sugar uptake were determined. Endogenous sugar gradients of the kernel were estimated by measuring sugar concentrations in cell sap collected from the pedicel and endosperm. A sugar concentration gradient from the pedicel to the endosperm was found. Uptake rates of ¹⁴C-labeled glucose, fructose, and sucrose were linear over the concentration range of 2 to 200 millimolar. At sugar concentrations greater than 50 millimolar, hexose uptake exceeded sucrose uptake. Metabolic inhibitor studies using carbonylcyanide-m-chlorophenylhydrazone, sodium cyanide, and dinitrophenol and estimates of Q₁₀ suggest that the transport of sugars into the developing maize endosperm is a passive process. Sucrose was hydrolyzed to glucose and fructose during uptake and in the endosperm was either reconverted to sucrose or incorporated into insoluble matter. These data suggest that the conversion of sucrose to glucose and fructose may play a role in sugar absorption by endosperm. Our data do not indicate that sugars are absorbed actively. Sugar uptake by the endosperm may be regulated by the capacity for sugar utilization (i.e. starch synthesis).     

Sucrose uptake into vacuoles of sugarcane suspension cells

Uptake of sucrose into vacuoles of suspension cells of Saccharum sp. (sugarcane) was investigated using a vacuole-isolation method based on osmotic- and pH-dependent lysis of protoplasts. Vacuoles took up sucrose at high rates without the influence of tonoplast energization on sucrose transport. Neither addition of ATP or pyrophosphate nor dissipation of the membrane potential or the pH gradient by ionophores changed uptake rates appreciably. Generation of an ATP-dependent pH gradient across the tonoplast was measured in vacuoles and tonoplast vesicles by fluorescence quenching of quinacrine. No H⁺ efflux could be measured by addition of sucrose to energized vacuoles or vesicles so that there was no evidence for a sucrose/H⁺ antiport system. Uptake rates of glucose and other sugars were similar to those of sucrose indicating a relatively non-specific sugar uptake into the vacuoles. Sucrose uptake was concentration-dependent, but no clear saturation kinetics were found. Strict dependence on medium pH and inhibition of sucrose transport by p-chloromercuriphenylsulfonic acid (PCMBS) indicate that sucrose uptake into sugarcane vacuoles is a passive, carrier-mediated process.Abbreviations FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - Mes 2-(N-morpholino)ethanesulfonic acid - Mops 3-(N-morpholino)propanesulfonic acid - PCMBS p-chloromercuriphenylsulfonic acid - PPi pyrophosphate This research was supported by the Deutsche Forschungsgemeinschaft. The technical assistance of H. Schroer is gratefully acknowledged.     

Vacuolar localization of 1-sinapolglucose: l-malate sinapoyltransferase in protoplasts from cotyledons of Raphanus sativus

The distribution of l-malate, sinapic acid esters and 1-sinapoylglucose: l-malate sinapoyltransferase (SMT) which catalyzes the synthesis of sinapoyl-l-malate were examined in preparations of protoplasts obtained from cotyledons of red radish (Raphanus sativus L. var. sativus). Vacuoles isolated from the protoplasts contained all of the SMT activity, all of the accumulated sinapic acid esters and about 50% of free l-malate present initially in the protoplasts. An esterase activity, acting on 1-sinapoyglucose, was found to be exclusively localized in the cytoplasm and a large proportion was found to be recoverable in a 100 000-g pellet obtained from protoplast lysates. The vacuoles were obtained after lysis of the protoplasts by osmotic shock and purification on a Ficoll gradient. The cytoplasmic contamination of vacuole preparations was found to be about 10%, as judged by enzymatic markers and microscopic inspection. No SMT activity was found in a 100 000-g pellet obtained from vacuole lysates. The results indicate that biosynthesis of sinapoyl-l-malate takes place within the central vacuoles of redradish cotyledons.Abbreviation SMT 1-sinapoylglucose: l-malate sinapol-transferase     

Localization and Substrate Specificity of Glycosidases in Vacuoles of Nicotiana rustica

Vacuoles isolated from Nicotiana rustica var brasilia have been shown to contain significant levels of glycosidase activity when assayed using p-nitrophenyl-glycosides as substrates. The substrate specificity for the glycosidases in the vacuolar fraction closely paralleled that found in the protoplasts, and the leaf tissue from which the vacuoles were isolated. The substrate specificity of the vacuolar enzyme(s) was different from glycosidic activity found in the commercial digestive enzyme preparations used to isolate the protoplasts from leaf tissue. It was demonstrated that 70 to 90% of the glycosidases that were found in the protoplasts appeared to be localized within the vacuole, when the p-nitrophenyl substrates α- and β-;d-galactose, β-d-glucose, and α-d-mannose were used. Neither the vacuolar nor the protoplast enzymes were active towards the naturally occurring phenolic glycoside, rutin. α-Mannosidase appears to be a valuable marker enzyme for vacuoles isolated from mesophyll leaf cells of tobacco.