Active Uptake Of Sucrose Research Articles

Downregulating the sucrose transporter VpSUT1 in Verbascum phoeniceum does not inhibit phloem loading

Sucrose is loaded into the phloem in the minor veins of leaves before export. Two active, species-specific loading mechanisms have been proposed. One involves transporter-mediated sucrose transfer from the apoplast into the sieve element-companion cell complex, so-called apoplastic loading. In the putative second mechanism, sucrose follows an entirely symplastic pathway, and the solute concentration is elevated by the synthesis of raffinose and stachyose in the phloem, not by transporter activity. Several sucrose-transporting plants have been shown to be apoplastic loaders by downregulating sucrose transporter 1 (SUT1), leading to accumulation of sugars and leaf chlorosis. In this study we compared the effect of downregulating SUT1 in Nicotiana tabacum, a sucrose transporter, and Verbascum phoeniceum, a species that transports raffinose and stachyose. To test the effectiveness of RNAi downregulation, we measured SUT1 mRNA levels and sucrose-H(+) symport in leaf discs. Mild NtSUT1 downregulation in N. tabacum resulted in the pronounced phenotype associated with loading inhibition. In contrast, no such phenotype developed when VpSUT1 was downregulated in V. phoeniceum, leaving minimal sucrose transport activity. Only those plants with the most severe VpSUT1 downregulation accumulated more carbohydrate than usual and these plants were normal by other criteria: growth rate, photosynthesis, and ability to clear starch during the night. The results provide direct evidence that the mechanism of phloem loading in V. phoeniceum does not require active sucrose uptake from the apoplast and strongly supports the conclusion that the loading pathway is symplastic in this species.

Sucrose (JrSUT1) and hexose (JrHT1 and JrHT2) transporters in walnut xylem parenchyma cells: their potential role in early events of growth resumption

In temperate woody species, the vegetative growth period is characterized by active physiological events (e.g., bud break), which require an adequate supply of soluble sugars imported in the xylem sap stream. One-year-old shoots of walnut (Juglans regia L. cv. 'Franquette') trees, which have an acrotonic branching pattern (only apical and distal vegetative buds burst), were used to study the regulation of xylem sugar transporters in relation to bud break. At the end of April (beginning of bud break), a higher xylem sap sucrose concentration and a higher active sucrose uptake by xylem parenchyma cells were found in the apical portion (bearing buds able to burst) than in the basal portion (bearing buds unable to burst) of the sample shoots. At the same time, xylem parenchyma cells of the apical portion of the shoots exhibited greater amounts of both transcripts and proteins of JrSUT1 (Juglans regia putative sucrose transporter 1) than those of the basal stem segment. Conversely, no pronounced difference was found for putative hexose transporters JrHT1 and JrHT2 (Juglans regia hexose transporters 1 and 2). These findings demonstrate the high capacity of bursting vegetative buds to import sucrose. Immunological analysis revealed that sucrose transporters were localized in all parenchyma cells of the xylem, including vessel-associated cells, which are highly specialized in nutrient exchange. Taken together, our results indicate that xylem parenchyma sucrose transporters make a greater contribution than hexose transporters to the imported carbon supply of bursting vegetative buds.

Transport of amino acids (L-valine, L-lysine, L-glutamic acid) and sucrose into plasma membrane vesicles isolated from cotyledons of developing pea seeds.

Transport of the amino acids L-valine, L-lysine, and L-glutamic acid and of sucrose was studied in plasma membrane vesicles isolated from developing cotyledons of pea (Pisum sativum L. cv. Marzia). The vesicles were obtained by aqueous polymer two-phase partitioning of a microsomal fraction and the uptake was determined after the imposition of a H(+)-gradient (DeltapH, inside alkaline) and/or an electrical gradient (Deltapsi, inside negative) across the vesicle membrane. In the absence of gradients, a distinct, time-dependent uptake of L-valine was measured, which could be enhanced about 2-fold by the imposition of DeltapH. The imposition of Deltapsi stimulated the influx of valine by 20%, both in the absence and in the presence of DeltapH. Uptake of L-lysine was more strongly stimulated by Deltapsi than by DeltapH, and its DeltapH-dependent uptake was enhanced about 6-fold by the simultaneous imposition of Deltapsi. In the absence of gradients the uptake of L-glutamic acid was about 2-fold higher than that of L-valine, but it was not detectably affected by DeltapH or Deltapsi. Although the transport of sucrose was very low, a stimulating effect of DeltapH could be clearly demonstrated. The results lend further support to the contention that during seed development cotyledonary cells employ H(+)-symporters for the active uptake of sucrose and amino acids.

Functional characterization of sucrose binding protein-mediated sucrose uptake in yeast

A sucrose binding protein was identified whose temporal and spatial patterns of mRNA expression and localization patterns of protein were tightly correlated with the active uptake of sucrose in several cell types. Heterologous expression of the sucrose binding protein in yeast strains have allowed a functional characterization of sucrose uptake as mediated by this protein. Ectopic expression of the sucrose binding protein in the susy7/ura3 yeast strain restored the ability of this strain to grow on medium containing sucrose as a sole carbon source. Further characterization of the kinetics of [(14)C]-sucrose uptake indicate that the sucrose binding protein mediates the uptake of sucrose in a non-saturable, linear manner and that this uptake is relatively insensitive to pH or treatment with protonophores. These biochemical attributes of sucrose binding protein-mediated sucrose uptake mimic the well characterized linear, non-saturable uptake of sucrose described in higher plants. The sucrose binding protein is a unique plasma membrane protein and shares sequence similarity to the seed storage proteins. The sucrose binding protein may mediate sucrose uptake across the plasma membrane in a plant-specific manner.

Sulfur dioxide inhibits the sucrose carrier of the plant plasma membrane

Plasma membrane vesicles were prepared by phase partition from a microsomal fraction of broad bean ( Vicia faba L.) leaf. In order to study the effects of sodium sulfite on active uptake of sucrose, the vesicles were artificially energized by a transmembrane pH gradient (ΔpH) and/or a transmembrane electrical gradient (Δψ). At 1 mM, sulfite strongly inhibited sucrose uptake but did not affect the two components of the proton motive force, ΔpH (measured by dimethyloxazolidine dione) and Δψ (measured by tetraphenylphosphonium). Moreover, sulfic did not inhibit the proton-pumping ATPase of the plasma membrane vesicles. These data demonstrate that sulfite may inhibit transport of photoassimilates in plant by a direct inhibition of the sucrose carrier of the plasma membrane.

Selective Inhibition of Active Uptake of Sucrose into Plasma Membrane Vesicles by Polyclonal Sera Directed against a 42 Kilodalton Plasma Membrane Polypeptide

Several polyclonal sera were raised in rabbits and in mice against putative sucrose carrier proteins, i.e. a 42 kilodalton (O Gallet, R Lemoine, C Larsson, S Delrot [1989] Biochim Biophys Acta 978: 56-64) and a 62 kD (KG Ripp, PV Viitanen, WD Hitz, VR Fransceschi [1988] Plant Physiol 88: 1435-1445) polypeptide of the plasma membrane. The effects of these sera on the active uptake of sucrose and of valine into purified plasma membrane vesicles from sugar beet (Beta vulgaris L.) leaves and roots were studied. At a dilution of 1/50, the anti-42 kilodalton sera consistently inhibited sucrose uptake in plasma membranes from leaves or from roots. They had no effect on valine uptake. Under the same experimental conditions, the anti-62 kilodalton sera had no effect on active uptake of sucrose. The data further support the view that a 42 kilodalton polypeptide is a component of the transport system mediating sucrose uptake across the plasma membrane of plant cells.

Active uptake of sucrose by plant plasma membrane vesicles: determination of some important physical and energetical parameters

The present paper investigates some characteristics of plant plasma membrane vesicles in relation to sucrose uptake. The plasma membrane vesicles purified by aqueous two‐phase partitioning from sugar beet (Beta vulgaris L.) leaf microsomes had an average diameter of 291 ± 13 nm and their internal volume was 2.2 μl (mg protein)−1. Although the vesicles were subjected to one freeze‐thaw cycle for the purpose of storage, they retained mainly (78 %) their original right‐side‐out orientation as estimated either by labelling with peroxidase‐labelled Concanavalin A or by measurements of ATPase latency. The use of higher concentrations of protecting agents [5 mM ethylenediaminetetraacetic acid (EDTA) and 2 mM dithiothreitol (DTT)] in the grinding buffer led to an increase of sucrose uptake by the vesicles. Measurements of the two components (pH and electrical gradients) of the proton motive force (PMF) showed that a high PMF (‐200 mV) was created under the experimental conditions used. The accumulation of sucrose which theoretically could be obtained by this PMF is much larger than the accumulation actually measured. Although the data support the conclusion that an active uptake of sucrose is driven by the PMF in plasma membrane vesicles from sugar beet leaves, they also stress some limits of this experimental system.

Active uptake of sucrose by plant plasma membrane vesicles: determination of some important physical and energetical parameters

Active uptake of sucrose by plant plasma membrane vesicles: determination of some important physical and energetical parameters

Bioregulator enhancement of sink activity in sugar beet

The sink mobilizing abillity is partially determined by sugar uptake rates of storage cells. Two synthetic growth regulators (Pix and BAS 106W) were tested for their effect on sucrose uptake in root tissue discs or glucose uptake in cell cultures of sugar beet. In tissue discs, uptake at the plasmalemma was determined by incubating the discs for 1 h in the presence of 5 mM sucrose and at the tonoplast for 4 h in the presence of 40 mM sucrose. Cell cultures were incubated for 1 h in the presence of 1 mM glucose. Pix (10 mg l−1) caused a 20% stimulation of active sucrose uptake at the plasmalemma. Active sucrose uptake at the tonoplast was increased 67% by 100 mg l−1 Pix. No effect of BAS 106W was observed on sucrose uptake in tissue discs. In cell cultures, a 65% enhancement of active glucose uptake was observed with both Pix and BAs 106W. When the bioregulators were applied to the root medium of seedlings, Pix but not BAS 106W resulted in increased root/shoot ratio, translocation of 14C-assimilates, and allocation of more biomass to the root sink. The data suggested that sugar transport and translocation may be used as biochemical criteria for rapid screening of effective yield enhancing bioregulators.

Effect of Plant Hormones on Sucrose Uptake by Sugar Beet Root Tissue Discs

Abscisic acid (ABA), auxins, cytokinins, gibberellic acid, alone or in combination were tested for their effects on short-term sucrose uptake in sugar beet (Beta vulgaris cv USH-20) roots. The effect of ABA on active sucrose uptake varied from no effect to the more generally observed 1.4-to 3.0-fold stimulation. A racemic mixture of ABA and its trans isomer were more stimulatory than ABA alone. Pretreating and/or simultaneously treating the tissue with K(+) or IAA prevented the ABA response while cytokinins and gibberellic acid did not. While the variable sensitivities of beet root to ABA may somehow be related to the auxin and alkali cation status of the tissue, tissue sensitivity to ABA was not correlated with ABA uptake, accumulation, or metabolic patterns. In contrast to ABA, indoleacetic acid (IAA) and other auxins strongly inhibited active sucrose uptake in beet roots. Cytokinins enhanced the auxin-induced inhibition of sucrose uptake but ABA and gibberellic acid did not modify or counteract the auxin effect. Trans-zeatin, benzyladenine, kinetin, and gibberellins had no effect on active sucrose uptake. None of the hormones or hormone mixtures tested had any significant effect on passive sucrose uptake. The effects of IAA and ABA on sucrose uptake were detectable within 1 h suggesting a rather close relationship between the physiological activities of IAA and ABA and the operation of the active transport system.

Sucrose Uptake and Compartmentation in Sugar Beet Taproot Tissue

Active sucrose uptake by discs of mature sugar beet (Beta vulgaris L. cv GW-D2 and USH-20) root tissue shows a biphasic dependence on external sucrose. At concentrations up to 20 millimolar sucrose, the active uptake mechanism appears to approach saturation, with an apparent K(m) of 3.6 millimolar. At higher external sucrose concentrations, a linear dependence becomes obvious indicating the probable presence of a nonsaturable, metabolically dependent uptake component. Active transport was not observed at external sucrose concentrations that caused tissue plasmolysis. Passive sucrose uptake in unplasmolyzed tissue showed a linear dependence on external sucrose concentration. The mitochondrial and/or suspected vacuolar ATPase inhibitors oligomycin, diethylstilbestrol, and N,N-dicyclohexylcarbodiimide strongly inhibited active sucrose uptake, whereas the putative plasmalemma-specific ATPase inhibitor orthovanadate was without effect.Sucrose efflux patterns from root discs indicated three distinct sucrose compartments having efflux kinetics consistent with those for cell wall, cytoplasm, and vacuole with the vacuole being the slowest releasing compartment. The sucrose contents and volumes of the compartments indicated that sucrose uptake into the vacuole was against a concentration gradient. Combined sucrose uptake/efflux analyses indicated that sucrose uptake into the vacuole is primarily an active transport process while transport into the cytoplasm is apparently passive, at least at external sucrose concentrations above 20 millimolar. We discuss the possibility that active sucrose uptake into the vacuoles of sugar beet storage cells is rate limited by passive sucrose transport to the active uptake site.

Alkali Cation/Sucrose Co-transport in the Root Sink of Sugar Beet

The mechanism of sucrose transport into the vacuole of root parenchyma cells of sugar beet was investigated using discs of intact tissue. Active sucrose uptake was evident only at the tonoplast. Sucrose caused a transient 8.3 millivolts depolarization of the membrane potential, suggesting an ion co-transport mechanism. Sucrose also stimulated net proton efflux. Active (net) uptake of sucrose was strongly affected by factors that influence the alkali cation and proton gradients across biological membranes. Alkali cations (Na(+) and K(+)) at 95 millimolar activity stimulated active uptake of sucrose 2.1- to 4-fold, whereas membrane-permeating anions inhibited active sucrose uptake. The pH optima for uptake was between 6.5 and 7.0, pH values slightly higher than those of the vacuole. The ionophores valinomycin, gramicidin D, and carbonyl cyanide m-chlorophenylhydrazone at 10 micromolar concentrations strongly inhibited active sucrose uptake. These data are consistent with the hypothesis that an alkali cation influx/proton efflux reaction is coupled to the active uptake of sucrose into the vacuole of parenchyma cells in the root sink of sugar beets.

Characteristics of the sucrose uptake system of vacuoles isolated from red beet tissue. Kinetics and specifity of the sucrose uptake system

The uptake of sucrose against a concentration gradient into the dextran-impermeable [(3)H]H2O space of red beet (Beta vulgaris L.) vacuoles has been studied using silicone-layer-filtering centrifugation on both fluorometric and (14)C-measurement of sucrose. Sucrose transport into vacuoles proceeds partly by an active transport system and partly by passive permeation. The K M(20°C) for active sucrose uptake was found to be about 22 mM and the V Max(20°C) was about 174 nmol sucrose x (unit betacyanin)(-1) x h(-1). The temperature dependency of sucrose transport appears to have an activation energy of 35,0 KJ×mol(-1). Among various mono-, di-, and trisaccharides tested, raffinose acts as a competitive inhibitor of sucrose uptake.

A proton-cotransport system in a higher plant: Sucrose transport in Ricinus communis

Sucrose uptake into cotyledons of castor beans ( Ricinus communis) proceeds partly by an active transport system and partly by passive permeation; α-methylglucoside (α-MG) is almost exclusively transported by the passive route. The Km for active sucrose uptake is 18 mM and the Vmax 4–5 μmoles/100 mg fresh weight per 1 h. The tissue is able to accumulate sucrose more than 100-fold. Sucrose but not α-MG induces a respiratory increase, which shows a similar sucrose concentration dependence as active sucrose transport. The addition of saturating amounts of sucrose to the incubation medium leads to a transient alkalinization of the medium. A second addition does not show this effect, neither does α-MG at any time. In the presence of 100 mM K +, which depolarizes the membrane potential, the uptake of sucrose is strongly inhibited. The results suggest that active sucrose transport in this tissue is mediated by an electrogenic proton cotransport system.

Sugar Selectivity and Other Characteristics of Phloem Loading in Beta vulgaris L.

The rate of phloem loading, its selectivity, and the disposition of labeled carbon were studied following application of (14)C-labeled sugars to the free space of source leaves of sugar beet (Beta vulgaris L.). Buffered 10 mm solutions of (14)C-labeled sucrose, fructose, stachyose, mannitol, 3-0-methyl glucose or l-glucose were applied to the abraded epidermis of source leaves held in the dark. Distribution of the labeled carbon from sugar taken up from the free space was studied by micro-densitometry of autoradiographs. Uptake of labeled sugar from the free space, partition between mesophyll and minor veins, metabolic conversions, export and respiration were followed during the 3-hr time course studies. Rates of sugar uptake into the minor veins, flux rates through the sieve element-companion cell complex membrane and concentration ratios between free space and the interior of the minor vein phloem cells were compared for the six sugars studied for evidence of active uptake. The composition of the free space solution in leaves photosynthesizing in (14)CO(2) was studied by vacuum infiltration of the source leaf air spaces and removal of the solution by centrifugation. Labeled compounds in this solution were compared to those in an aqueous ethanol extract of the same leaf pieces.The results in sugar beet source leaves support the concept of direct, active uptake of sucrose from free space into minor veins. This is not the case for fructose, 3-0-methyl glucose, mannitol, or stachyose. The latter two sugars, which are translocated in some plants, are not loaded into the minor veins at a rate sufficient to make them a significant component of the material translocated. The rate of phloem loading is controlled in part by mesophyll metabolism, especially as it affects the availability of sucrose to the free space. Both the rate and selectivity of export are controlled by uptake from the free space into the sieve element-companion cell complex of the minor veins.

The effects of sucrose and various salts on the growth and lysosomal enzyme activity of L5178Y cells.

ABSTRACT L5178 Y murine leukemic cells do not seem to be affected by sucrose in the same manner as other mammalian cells. Although there is active uptake of sucrose with no concomitant metabolism, there is no increase in vacuolation or cell size and there is only a slight increase in acid phosphatase, acid protease and β-glucuronidase activities. However, the glycosidases are significantly elevated in the presence of 0·08 M sucrose. This may be due to a specific induction of these enzymes by the sucrose not related to a general increase in lysosomes. As the sucrose concentration is raised, a depression of the growth rate and an elevation of glycosidase activities occurs, reaching a maximum at 0·08 M sucrose. This depression of growth with a concomitant increase in generation time is primarily a result of the increased osmolarity of the medium; the same effect is observed with increased concentrations of NaCl. Although the growth effects are similar with either sucrose or NaCl, the elevation of glycosidase activity occurs only with increased concentrations of sucrose. Sucrose-induced vacuolation with an increase in cell size is not evident in L5178Y cells; therefore this effect does not seem to be common to all mammalian cells, even though it has been reported for LS cells, Chinese hamster fibroblasts, and chick bone rudiments and in vivo for liver cells.

Effect of Mercury Vapour on the Active Uptake of Sucrose by Leaf Tissues

ALTHOUGH the poisonous action of mercury vapour on plants was noticed as far back as 1797 1, little attention has been paid to this compared with the action of mercury compounds. In 1934, Zimmerman and Crocker2, investigating the damage done to roses by mercuric chloride applied to the soil, found that the effect was due to the formation of free mercury in the soil, the vapour of which attacked the shoot. In the course of studying the uptake of sucrose by floating leaf disks of Atropa belladonna (deadly nightshade) and Lactuca sativa var. capitata (lettuce), we have found that the active uptake is greatly reduced by the presence of free mercury.