Transport of sugars across the plasma membrane of beetroot protoplasts

Protoplasts isolated from beetroot tissue took up glucose preferentially whereas sucrose was transported more slowly. The ¹⁴C-label from [¹⁴C]glucose and [¹⁴C]sucrose taken up by the cells could be detected rapidly in phosphate esters and, after feeding of [¹⁴C]glucose was found also in sucrose. The temperature-dependent uptake process (activation energy E_A about 50 kJ · mol^–1) seems to be carrier mediated as indicated by its substrate saturation and, for glucose, by competition experiments which revealed positions C₁, C₅ and C₆ of the D-glucose molecule as important for effective uptake. The apparent K_m(20° C) for glucose (3-O-methylglucose) was about 1 mM whereas for sucrose a significantly lower apparent affinity was determined (K_m about 10 mM). When higher concentrations of glucose (5 mM) or sucrose (20 mM) were administered, the uptake process followed first-order kinetics. Carrier-mediated transport was inhibited by N,N-dicyclohexylcarbodiimide, Na-orthovanadate, p–chloromercuribenzenesulfonic acid, and by uncouplers and ionophores. The uptake system exhibited a distinct pH optimum at pH 5.0. The results indicate that generation of a proton gradient is a prerequisite for sugar uptake across the plasma membrane. Protoplasts from the bundle regions in the hypocotyl take up glucose at higher rates than those derived from bundle-free regions. The results favour the idea that apoplastic transport of assimilates en route of unloading might be restricted to distinct areas within the storage organ (i.e. the bundle region) whereas distribution in the storage parenchyma is symplastic.Abbreviations CCCP Carbonylcyanide m–chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DOG deoxyglucose - Mes 2-(N-morpholino)ethanesulfonic acid - 3-OMG 3-O-methylglucose - PCMBS p–chloromercuribenzenesulfonic acid - SDS Sodium dodecyl sulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

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.     

Metabolism of maltose and sucrose by microspores isolated from barley (Hordeum vulgare L.)

The aim of this work was to discover why barley (Hordeum vulgare L.) microspores die when cultured on media containing 40 mM sucrose but undergo embryogenesis on 40 mM maltose. Freshly isolated microspores were cultured for 6–24 h on media containing either [U-¹⁴C]maltose or [U-¹⁴C]sucrose at 40 mM, and the detailed distribution of ¹⁴C was determined. The amounts of glycolytic intermediates, ATP, ADP and AMP, in microspores were also measured. Cultures on sucrose differed from those on maltose in that the initial rate of metabolism was faster but declined rapidly, less ¹⁴C was recovered in polymers and more in alanine, there was extensive leakage of assimilated carbon, significant accumulation of ethanol and a lower adenylate energy charge. It is argued that microspores cultured on 40 mM sucrose die because they metabolize the sugar rapidly, become hypoxic and, as a result, accumulate large quantities of ethanol within the cells. Metabolism of maltose is slower and there is sufficient oxygen available to allow cells to survive in culture. Consequently some of the cultured cells undergo embryogenesis.P.S. thanks the Science and Engineering Research Council and Shell Research Ltd., Sittingbourne, for a Cooperative Award in Science and Engineering studentship.     

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.     

Carbohydrate oxidation by leucoplasts from suspension cultures of soybean (Glycine max L.)

The aim of this work was to discover how leucoplasts from suspension cultures of soybean (Glycine max L.) oxidize hexose monophosphates. Leucoplasts were isolated from protoplast lysates on a continuous gradient of Nycodenz with a yield of 28% and an intactness of 80%. Incubation of the leucoplasts with ¹⁴C-labelled substrates led to ¹⁴CO₂ production, that was dependent upon leucoplast intactness, from [U-¹⁴C]glucose 6-phosphate, [U-¹⁴C]glucose 1-phosphate, [U-¹⁴C] fructose 6-phosphate and [U-¹⁴C]glucose+ATP, but not from [U-¹⁴C]fructose-1,6-bisphosphate or [U-¹⁴C]triose phosphate. The yield from [U-¹⁴C]glucose 6-phosphate was at least four times greater than that from any of the other substrates. When [1-¹⁴C]-, [2-¹⁴C]-, [3,4-¹⁴C]-, and [6-¹⁴C]glucose 6-phosphate were supplied to leucoplasts significant ¹⁴CO₂ production that was dependent upon leucoplast intactness was found only for [1-¹⁴C]glucose 6-phosphate. It is argued that soybean cell leucoplasts oxidize glucose 6-phosphate via the oxidative pentose phosphate pathway with very little recycling, and that in these plastids glycolysis to acetyl CoA is negligible.S.A.C. thanks the Science and Engineering Research Council for a research studentship.     

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 [³H]H₂O space of red beet (Beta vulgaris L.) vacuoles has been studied using silicone-layer-filtering centrifugation on both fluorometric and ¹⁴C-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.Abbreviations EDTA ethylenediamine tetraacetic acid - fr. wt. fresh weight - Tris tris-(hydroxymethyl)-aminomethan     

Lipid synthesis in the laticifers of Euphorbia lathyris L. seedlings

Various solutions of labeled precursors were absorbed by the cotyledons of etiolated Euphorbia lathyris L. seedlings. Incorporation of ¹⁴C into triterpenes from [2-¹⁴C]mevalonic acid, [1-¹⁴C]acetate, [3-¹⁴C]pyruvate, [U-¹⁴C]glyoxylate, [U-¹⁴C]glycerol, [U-¹⁴C]serine, [U-¹⁴C]xylose, [U-¹⁴C]glucose, and [U-¹⁴C]sucrose was obtained. The [¹⁴] triterpenes synthesized from [¹⁴C] sugars were mainly of latex origin. [¹⁴C]mevalonic acid was only involved in terpenoid synthesis outside the laticifers. Exogenously supplied glyoxylate, serine, and glycerol were hardly involved in lipid synthesis at all. The ¹⁴C-distribution over the various triterpenols was consistent with the mass distribution of these constituents in gas liquid chromatography when [¹⁴C]sugars, [¹⁴C]acetate, and [¹⁴C]pyruvate were used. These precursors were supplied to the seedlings in the presence of increasing amounts of unlabeled substrates. The amount of substrate directly involved in lipid synthesis as well as the absolute triterpenol yield was calculated from the obtained [¹⁴C]triterpenols. The highest yield was obtained in the sucrose incorporated seedlings, being 25% of the daily increase of latex triterpenes in growing seedlings.     

DCCD induced sodium uptake by Anacystis nidulans

The Na level inside cells of Anacystis nidulans is lower than in the external medium reflecting an effective Na extrusion. Na efflux is an active process and is driven by a Na⁺/H⁺-antiport system. The necessary H⁺-gradient is generated by a proton translocating ATPase in the plasmalemma. This ATPase (electrogenic proton pump) also produces the membrane potential (about -110 mV) responsible for K accumulation. N,N-dicyclohexylcarbodiimide (DCCD) inhibits the ATPase and the H⁺-gradient completely, but the membrane potential is only reduced (<-70 mV), since K efflux initiated by DCCD maintains the potential partly by diffusion potential.With DCCD, active Na efflux is inhibited thus revealing Na uptake and leading by equilibration to the membrane potential to a 5–20 fold accumulation. Na uptake depends on the DCCD concentration with an optimum at (1–2)×10^-4 M DCCD. Pretreatment with DCCD for a few minutes followed by replacement of the medium suffices to induce Na uptake.DCCD induced Na influx is about 5 times faster in light than in darkness, and the steady state is reached much earlier in light; a 5 fold increase by light was also found for Rb uptake with untreated cells. Valinomycin stimulates the influx of Rb to about the same rate in light and dark. Therefore light may unspecifically increase the permeability of the plasma-lemma probably via the ATP level. Similarly to DCCD also 3×10^-3 M N-ethylmaleimide induces Na uptake.Abbreviations Used DCCD N,N-dicyclohexylcarbodiimide - NEM N-ethylmaleimide - CCCP carbonylcyanide m-chlorophenylhydrazone - Pipes piperazine-N,N-bis(2-ethanesulfonic acid) - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

The Effect of Sugars on the Binding of [Hg]-p-Chloromercuribenzenesulfonic Acid to Leaf Tissues

Replacement of mannitol with sucrose decreases the binding of [²⁰³Hg]-p-chloromercuribenzenesulphonic acid (PCMBS) to Vicia faba leaf discs without epidermis. This decrease is optimal for 20 minutes on incubation, is concentration-dependent, and is also found with maltose and raffinose. In parallel experiments, the addition of sucrose, maltose, and raffinose during PCMBS pretreatment was shown to increase subsequent uptake of [U-¹⁴C]sucrose. In contrast, d- or l-glucose, 3-O-methylglucose, galactose, fructose, palatinose, turanose, or melibiose had no effect either on PCMBS binding or on [¹⁴C]sucrose uptake. The sucrose-induced decrease of PCMBS binding is retained after a cold and ionic shock. Measurements of specific activities of membrane fractions prepared from tissues incubated in labeled PCMBS show that the decrease concerns the 120,000 gravity pellet, but that very mild procedures must be chosen to prevent redistribution of label in the supernatant. Altogether, the data provide new support to the hypothesis that the active site of the sucrose carrier contains a group sensitive to PCMBS.     

Light or high (30°C) temperature effects on l-[U-C]leucine incorporation and protein patterns in embryos and endosperm during the early phase of germination of the negatively photoblastic and thermosensitive seeds of Phacelia tanacetifolia

The activities of l-[U-¹⁴C]leucine uptake and incorporation into proteins of embryos and endosperm of seeds of Phacelia tanacetifolia Benth. cv Bleu-Clair were analysed during the first 24 h of incubation under conditions optimal for germination (16°C in darkness) and in two inhibitory conditions: 16°C in the light and 30°C in darkness. Blocking germination induced by light or 30°C was accompanied by the inhibition of l-[U-¹⁴C]leucine uptake and incorporation in embryos. In the endosperm, the activation of l-[U-¹⁴C]leucine uptake was of the same magnitude for the non-inhibited and the light-inhibited seeds and much higher for the 30°C-inhibited seeds; the activation of l-[U-¹⁴C]leucine incorporation was quantitatively similar in all three conditions, with the patterns of newly synthesised proteins qualitatively different in the endosperm from light- or 30°C-inhibited seeds. The results showed that germination of P. tanacetifolia seeds is controlled by light or super-optimal temperature through the inhibition of the activation of transport and protein synthetic activities in embryo without effect on the endosperm. We suggest, on the basis of the translational activity, the possibility that in the inhibitory conditions the blockade of the embryo to operate as a sink affects the transition of the endosperm to operation as a source.     

Palytoxin Acts on Na+,K+-ATPase but not Nongastric H+,K+-ATPase

Palytoxin (PTX) opens a pathway for ions to pass through Na,K-ATPase. We investigate here whether PTX also acts on nongastric H,K-ATPases. The following combinations of cRNA were expressed in Xenopus laevis oocytes: Bufo marinus bladder H,K-ATPase α₂- and Na,K-ATPase β₂-subunits; Bufo Na,K-ATPase α₁- and Na,K-ATPase β₂-subunits; and Bufo Na,K-ATPase β₂-subunit alone. The response to PTX was measured after blocking endogenous Xenopus Na,K-ATPase with 10 μm ouabain. Functional expression was confirmed by measuring ⁸⁶Rb uptake. PTX (5 nm) produced a large increase of membrane conductance in oocytes expressing Bufo Na,K-ATPase, but no significant increase occurred in oocytes expressing Bufo H,K-ATPase or in those injected with Bufo β₂-subunit alone. Expression of the following combinations of cDNA was investigated in HeLa cells: rat colonic H,K-ATPase α₁-subunit and Na,K-ATPase β₁-subunit; rat Na,K-ATPase α₂-subunit and Na,K-ATPase β₂-subunit; and rat Na,K-ATPase β₁- or Na,K-ATPase β₂-subunit alone. Measurement of increases in ⁸⁶Rb uptake confirmed that both rat Na,K and H,K pumps were functional in HeLa cells expressing rat colonic HKα₁/NKβ₁ and NKα₂/NKβ₂. Whole-cell patch-clamp measurements in HeLa cells expressing rat colonic HKα₁/NKβ₁ exposed to 100 nm PTX showed no significant increase of membrane current, and there was no membrane conductance increase in HeLa cells transfected with rat NKβ₁- or rat NKβ₂-subunit alone. However, in HeLa cells expressing rat NKα₂/NKβ₂, outward current was observed after pump activation by 20 mm K⁺ and a large membrane conductance increase occurred after 100 nm PTX. We conclude that nongastric H,K-ATPases are not sensitive to PTX when expressed in these cells, whereas PTX does act on Na,K-ATPase.     

Transport and metabolism of glucose and arabinose in Bifidobacterium breve

Glucose was required for the transport of arabinose into Bifidobacterium breve. The non-metabolisable glucose analogue 2-deoxy-d-glucose (2-DG) did not facilitate assimilation of arabinose. Studies using d-[U-¹⁴C]-labelled arabinose showed that it was fermented to pyruvate, formate, lactate and acetate, whereas the principal metabolic products of d-[U-¹⁴C]-labelled glucose were acetate and formate. In contrast to glucose, arabinose was not incorporated into cellular macromolecules. A variety of metabolic inhibitors and inhibitors of sugar transport (proton ionophores, metal ionophores, compounds associated with electron transport) were used to investigate the mechanisms of sugar uptake. Only NaF, an inhibitor of substrate level phosphorylation, and 2-DG inhibited glucose assimilation. 2-DG had no effect on arabinose uptake, but NaF was stimulatory. High levels of phosphorylation of glucose and 2-DG by PEP and to a lesser degree, ATP were seen in phosphoenolpyruvate: phosphotransferase (PEP:PTS) assays. These data together with strong inhibition of glucose uptake by NaF suggest a role for phosphorylation in the transport process. Arabinose uptake in B. breve was not directly dependent on phosphorylation or any other energy-linked form of transport but may be assimilated by glucose-dependent facilitated diffusion.Abbreviations (2,4-DNP) 2,4-dinitrophenol - (2,4-DNP) carbonylcyanide m-chlorophenylhydrazone - (CCCP) (phosphoenolpyruvate phosphotransferase system) - PEP: PTS trichloroacetic acid - (TCA) 2-deoxy-d-glucose - (2-DG) 2-deoxy-d-glucose     

Selenite uptake and incorporation by Selenomonas ruminatium

Selenite uptake and incorporation in Selenomonas ruminantium was constitutive with an inducible component. It was distinct from sulphate or selenate transport, since sulphate and selenate did not inhbit uptake, nor could sulphate or selenate uptake be demonstrated. Selenite uptake had an apparent K _m of 1.28 mM and a V _max of 148 ng Se min^-1 mg^-1 protein. Uptake was sensitive to inhibition by 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), azide, iodoacetic acid (IAA) and N-ethylmaleimide (NEM), but not chloropromazine (CPZ), N,N-dicyclohexyl-carbodiimide (DCCD), quinine, arsenate, or fluoride. Treatment of cells accumulating ⁷⁵[Se]-Selenite with 2,4,DNP inhibited uptake, but did not cause efflux. Transport of selenite was inhibited by sulphite and nitrite, but not by nitrate, phosphate, sulphate of selenate. ⁷⁵[Se]-Selenite was incorporated into selenocystine, selenoethionine, selenohomocysteine, and selenomethionine and was also reduced to red elemental selenium.     

Close coupling between extrusion of H+ and uptake of K+ by barley roots

Extrusion of H⁺ by intact barley (Hordeum vulgare L.) roots was automatically titrated. Simultaneously, uptake of K⁺ into the roots, transport of K⁺ through the roots, and (as a residual term) accumulation of K⁺ within the root tissue were determined. When no monovalent cation was present in the medium the steady rate of H⁺ release was close to zero. Addition of K⁺ stimulated H⁺ extrusion within less than 1 min. The stimulation of H⁺ release was apparently limited only by the movement of K⁺ through the apoplast of the roots. The steady rate of H⁺ extrusion depended on the availability of external K⁺ and saturated at a K⁺ concentration of about 100 mol· dm^-3. Half-maximum rates of net K⁺ uptake and H⁺ extrusion were reached at a K⁺ concentration of about 10 mol·dm^-3. With (slowly absorbable) sulfate as the only anion present, the stoichoimetry between H⁺ release and net K⁺ uptake was one. In conclusion, the uptake of K⁺ across the plasmalemma of the cells of the root cortex is electrically coupled to H⁺ extrusion.     

Parachloromercuribenzenesulfonic Acid : a potential tool for differential labeling of the sucrose transporter

Vicia faba leaf discs without epidermis were pretreated with parachloromercuribenzenesulfonic acid (PCMBS), rinsed and incubated on [¹⁴C]sucrose (1 or 40 millimolar). Those sucrose concentrations were chosen as representative of the apparent uptake system 1 (1 millimolar) and system 2 (40 millimolar) previously characterized. Pretreatment with 0.5 millimolar PCMBS for 20 minutes inhibited system 1 and system 2 by about 70%.

Addition of unlabeled sucrose during PCMBS-pretreatment protected the carrier(s) from the inhibition, whereas glucose, fructose, and sucrose analogs were unable to afford protection. At 1 millimolar [¹⁴C]sucrose, the protection resulted in a small but consistent reduction of normal inhibition (from 63 to 45%) for sucrose concentrations of 50 millimolar and more during pretreatment. Contrarily, at 40 millimolar [¹⁴C]sucrose, the protection increased linearly with the sucrose concentration in the pretreatment medium, and complete prevention of inhibition was reached for 250 millimolar sucrose.

The protection was not due to exchange diffusion and was located in the veins. Michaelian kinetics indicated that PCMBS and sucrose compete with each other at the active site of the carrier.

Among 14 compounds tested (sugars, amino-acids, hormones, ³²P), sucrose uptake was by far the most sensitive to PCMBS. Sucrose preferentially protected its carrier(s) from inhibition. Treatment with 20 millimolar cysteine or 20 millimolar dithioerythreitol reversed inhibition by PCMBS pretreatment.

     

Delta endotoxin inhibits Rb+ uptake,lowers cytoplasmic pH and inhibits a K+-ATPase inManduca sexta CHE cells

Summary Delta endotoxin, a 68 kilodalton protein isolated fromBacillus thuringiensis spp.Kurstaki, is a potent entomocidal agent that alters a K⁺ current across midgut tissue of many phytophagous insects. This toxin completely inhibited the vanadate-sensitive⁸⁶Rb⁺ uptake and mimicked the vanadate-induced decrease in cytosolic pH in a cell line (CHE) originating fromManduca sexta embryonic tissue. The toxin also inhibited a K⁺-sensitive-ATPase in the plasma membranes isolated from these cells. Using the K⁺-sensitive-ATPase substratp-nitrophenyl phosphate, delta endotoxin was found to have aK _i of 0.4 m. These data suggest that the toxin inhibits a K⁺-ATPase responsible for⁸⁶Pb⁺ uptake in the CHE cells. The relationship between the toxin inhibition of K⁺-ATPase and toxin-altered K⁺ current is discussed.     

Uptake of isopentenyl diphosphate by plastids isolated from Vitis vinifera L. cell suspensions

The uptake of [1-¹⁴C]isopentenyl diphosphate by intact plastids purified from cell suspensions of Vitis vinifera L. cv. Muscat de Frontignan was investigated using vacuum-filtration and silicone-oil-filtering techniques. Transport across the plastid envelope which was stimulated by cations, such as Mg²⁺ and Mn²⁺, was characterized by a K _m of approx. 0.5 mM and a V _max of 25 nmol·(mg protein)^-1·-h^-1. The data showed that isopentenyl diphosphate apparently accumulated in the plastid against a concentration gradient. The involvement of a protein carrier was suggested by the strong inhibition of the uptake by compounds which are known to block SH groups. Thus, the saturation kinetics together with the pH optimum (7.5–8), the temperature dependence (maximum incorporation at 37 °C) and the competitive inhibition by a structural analogue of the substrate (aminophenylethyl diphosphate) provided evidence for a mechanism of uptake by facilitated diffusion. The carrier identified may thus play a major role in supplying the plastid compartment with isopentenyl diphosphate for isoprenoid biosynthesis.Abbreviations APP aminophenylethyl diphosphate - DMAPP dimethylallyl diphosphate - GPP geranyl diphosphate - IPP isopentenyl diphosphate - NEM N-ethylmaleïmide - PCMB p-chloromercuribenzoate     

Light-promoted diffusional amino acid efflux from Commelina leaf disks

The release (=the measured loss) of amino acids was studied in Commelina benghalensis leaf disks. The release is assumed to be the result of influx and efflux, therefore, both movements were investigated.The uptake of ¹⁴C-labeled valine exhibited a biphasic isotherm. The uptake was pH-dependent, especially at low substrate concentrations (pH optimum 4.8). Signals for amino acid/proton co-transport were observed: stimulation of the uptake by fusicoccin (FC), inhibition by diethylstilbestrol (DES) or by high K⁺ concentrations. In the light, the ATP level of the disks was maintained during the uptake period (2 h), in darkness the ATP content decreased from 87 to 24 nmol g^–1 fr. wt. However, light-promoted uptake, which is explained in the proton pump concept by an intensified proton extrusion as the result of high ATP production, was lacking.The release of amino acids was increased by washing with p-chloromercuriphenyl sulphonic acid (PCMBS), nystatin, 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), or KCN. The release (Q₁₀ about 1.5) was independent of the external pH and was linearly related to the intracellular amino acid concentration. Light enhanced the rate of release to the same extent at all intracellular concentrations. The present results suggest that the release is balanced by a, at least partially, proton-driven influx and a diffusional ligh-promoted efflux. A provisional model shows how the diffusional effulx can be indirectly controlled by a counter-flow fueled by the metabolism.Abbreviations PCMBS p-chloromercuriphenylsulphonic acid - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DES diethylstibestrol - DCMU 3 (3,4-dichlorophenyl)-1, 1-dimethyl urea - TRIS 2-amino-2-(hydromethyl)propane-1,3 diol - MES 2-(N¹-morpholino) ethane sulphonic acid monohydrate - FC fusicoccin     

Involvement of polyamines in the inhibiting effect of injury caused by cutting on K+ uptake through the plasma membrane

The inhibition of K⁺ uptake through the plasma membrane resulting from injury caused by cutting, or from application of polyamines (PAs), has been investigated in root segments of maize (Zea mays L.) and pea (Pisum sativum L.). It was found, for both treatments, that K⁺ uptake recovered if the segments were washed for 2 h. The K⁺ uptake inhibited by cutting and that inhibited by spermidine treatment were stimulated to the same extent by fusicoccin. In addition, there was a correlation between the extent of the recovery of K⁺ uptake caused by washing and the distribution, along the root axis, of both PAs and the activities of enzymes responsible for PA degradation. In apical segments of maize, where the PA content and the activity of the degradative enzyme polyamine oxidase (EC 1.5.3.3) were higher than in the more distal segments, the recovery of K⁺ uptake caused by washing was also higher. On the other hand, the opposite trend was observed in root segments of pea, where the PA content and the activity of the degradative enzyme diamine oxidase (EC 1.4.3.6) were higher in distal segments in which K⁺ uptake was greatly stimulated by washing. The effect of the amine-oxidase inhibitor, aminoguanidine, indicates that the degradation products of PAs are involved in the mechanism of inhibition of K⁺ uptake by PAs. The data also seem to indicate that PAs and their degradation products are responsible for the inhibition of K⁺ uptake occurring as a result of injury sustained by cutting roots into segments.Abbreviations DAO diamine oxidase - FC fusicoccin - PA polyamine - PAO polyamine oxidase - PUT putrescine - SPD spermidine     

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.