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.     

Sucrose partitioning between vascular bundles and storage parenchyma in the sugarcane stem: a potential role for the ShSUT1 sucrose transporter

A transporter with homology to the SUT/SUC family of plant sucrose transporters was isolated from a sugarcane (Saccharum hybrid) stem cDNA library. The gene, designated ShSUT1, encodes a protein of 517 amino acids, including 12 predicted membrane-spanning domains and a large central cytoplasmic loop. ShSUT1 was demonstrated to be a functional sucrose transporter by expression in yeast. The estimated K_m for sucrose of the ShSUT1 transporter was 2 mM at pH 5.5. ShSUT1 was expressed predominantly in mature leaves of sugarcane that were exporting sucrose and in stem internodes that were actively accumulating sucrose. Immunolocalization with a ShSUT1-specific antiserum identified the protein in cells at the periphery of the vascular bundles in the stem. These cells became lignified and suberized as stem development proceeded, forming a barrier to apoplasmic solute movement. However, the movement of the tracer dye, carboxyfluorescein from phloem to storage parenchyma cells suggested that symplasmic connections are present. ShSUT1 may have a role in partitioning of sucrose between the vascular tissue and sites of storage in the parenchyma cells of sugarcane stem internodes.     

Effect of magnesium and ATP on ATPase of sugarcane vacuoles

Kinetic analysis of the Mg²⁺-dependence of tonoplast ATPase from suspension-cultured cells of sugarcane showed that the enzyme activity increased with increasing magnesium concentrations till 1–3 mM and then decreased consideably for higher concentrations. This kinetic could be explained by the assumption that MgATP^2- is the substrate of ATPase: MgATP^2- concentration increases with increasing concentration of magnesium till, at high concentrations of magnesium, Mg₂ATP is formed. No evidence for a direct role of Mg²⁺ as activator or inhibitor was found. These data corroborate previous findings that MgATP^2- is the sole substrate of the vacuolar ATPase of sugarcane (Thom and Komor 1984). High concentrations of ATP seemed to inhibit the ATPase. This result, however, could be traced back to interference of ATP with the Fiske-Subbarow method of phosphate determination. After adjustment of the test conditions, inhibition by ATP was no longer found. Reported data for ATPases of other plant materials, showing inhibition of enzyme activity with high magnesium or ATP concentrations, might be explicable in a similar way.Abbreviation Mes 2-(N-morpholino)ethane+Sulfonic acid     

Permeabilization of the plasmalemma and wall of soybean root cells to macromolecules

A technique has been developed that results in the reversible permeabilization of the cell wall and plasmalemma of soybean (Glycine max (L.) Merr.) root cells grown in suspension and callus culture. Cells in culture are treated with saponin (0.1 mg/ml) for 15 min at room temperature. They are then coincubated in separate experiments with fluorescent-derivatized dextrans (20–70 kDa) or fluorescein-conjugated goat anti-rabbit immunoglobulin G to ascertain the exclusion size of macromolecules capable of diffusing across the cell wall and plasmalemma into the cytoplasm. Following an incubation period of 30 min, it was observed by conventional and confocal fluorescence microscopy that all derivatized macromolecules tested (20–140 kDa) could be incorporated into the cytoplasm, but not into the vacuole. This procedure did not appear to affect cell viability adversely. A normal doubling time was observed for these cells following the permeabilization procedure.Abbreviations FDA fluorescein diacetate - FITC-20 kDa, FITC-40 kDa, FITC-70 kDa dextrans fluorescein-derivatized 20-kDa, 40-kDa, and 70-kDa dextrans - IgG immunoglobulin G - kDa kilodalton Paramjit K. Gharyal wishes to thank the Nitrogen Availability Program at Michigan State University for financial support. We also thank Edwin de Feijter of Meridian Instruments for technical assistance in performing the confocal measurements. This work was supported by a grant from the U.S. — Israel Binational Agricultural Research and Development Fund (BARD project No. US-1384-87).     

Nitrate reductase is not accumulated in chloroplast-ribosome-deficient mutants of higher plants

The activities of nitrite reductase (EC 1.7.7.1) are 60–70% of wild-type activity in pigment-deficient leaves of the chloroplast-ribosomedeficient mutants albostrians (Hordeum vulgare) and iojap (Zea mays). The activity and apoprotein of nitrate reductase (EC 1.6.6.1.) are lacking in the barley mutant. Only very low activities of nitrate reductase can be extracted from leaves of the maize mutant. The molybdenum cofactor of nitrate reductase and xanthine dehydrogenase (EC 1.2.3.2) is present in maize and barley mutant plants. However, it is not inducible by nitrate in pigment-deficient leaves of albostrians. From these results we conclude: (i) Nitrite reductase (a chloroplast enzyme) is synthesized in the cytoplasm and does not need the presence of nitrate reductase for the induction and maintenance if its activity. (ii) The loss or low activity of nitrate reductase is a consequence of the inability of the mutants to accumulate the apoprotein of this enzyme. (iii) The chloroplasts influence the accumulation (i.e. most probably the synthesis) of the nonchloroplast enzyme, nitrate reductase. The accumulation of nitrate reductase needs a chloroplast factor which is not provided by mutant plastids blocked at an early stage of their development.Abbreviations CRM cross-reacting material - Mo-co molybdenum cofactor - NiR nitrite reductase - NR nitrate reductase     

The oxidation of extracellular NADH by sugarcane cells: Coupling to ferricyanide reduction,oxygen uptake and pH change

Suspension-cultured cells of sugarcane (Saccharum sp. hybrids) did not oxidize exogenously supplied NADH in the absence of ferricyanide (potassium hexacyanoferrate [III]), whereas they did at a low rate in the presence of ferricyanide. Concomitantly, ferricyanide was reduced at a slow rate. Neither a pH change nor a change in respiration was caused by the addition of NADH and-or ferricyanide, but ferricyanide was a strong inhibitor of sugar transport. In contrast to cells, protoplasts rapidly oxidized exogenous NADH. This oxidation was accompanied by an increase in oxygen consumption and a net proton disappearance from the medium. Exogenous ferricyanide was reduced only slowly by protoplasts. Simultaneous presence of NADH and ferricyanide produced two effects: 1) a very rapid stoichiometric oxidation of NADH and reduction of ferricyanide until one of the reaction compounds was exhausted, and 2) a nearly instantaneous inhibition of the slower phase of NADH oxidation, which was observed in the presence of NADH but absence of ferricyanide. The extra oxygen consumption and the alkalinization of the medium, as observed with NADH, were also immediately stopped by ferric ions and ferrous ions. The presence of NADH and ferricyanide caused a fast stoichiometric acidification of the medium. These results were taken as evidence that the oxidation of NADH in the absence of ferricyanide is not related to the NADH-ferricyanide-coupled redox reaction. Furthermore, addition of NADH caused some uncoupling of the protoplasts, an effect which would explain the strong acidification of the cell cytoplasm and the inhibition of various transport systems. The NADH-oxidizing systems oxidized both the -configurated pyridine nucleotide and the -configurated form. Since NADH-linked dehydrogenases usually do not work with -NADH (with the exception of the endoplasmic-reticulum-bound electron-transport system), the observed activities could have been derived from contaminating membranes and dying protoplasts in the suspension. All reported reactions partly or predominantly occurred in the supernatant of the protoplast suspension and increased considerably during incubation of the protoplasts. The rates and quantities of oxygen consumption, pH change, and ferricyanide reduction fitted with NADH oxidation in a stoichiometric ratio, which implied that all these reactions occurred in the extracellular space, without involving transmembrane steps. No evidence for a physiological role in energization of the plasmalemma was found.Abbreviation NADH -nicotinamide adenine dinucleotide reduced form     

Sucrose transport in tonoplast vesicles of red beet roots is linked to ATP hydrolysis

Sucrose uptake into tonoplast vesicles, which were prepared from red beet (Beta vulgaris L.) vacuoles isolated by two different methods, was stimulated by MgATP. Using the same medium as for osmotic disruption of vacuoles, membrane vesicles were prepared from tissue homogenates of dormant red beet roots and separated by high-speed centrifugation through a discontinuous dextran gradient. A low-density microsomal fraction highly enriched in tonoplast vesicles could be further purified from contaminating ER vesicles by inclusion of 5 mM MgCl₂ in the homogenization medium. These vesicles were able to transport sucrose in an ATP-dependent manner against a concentration gradient, whereas vesicles from regions of other densities lacked this feature, indicating that ATP stimulation of sucrose uptake took place only at the tonoplast membrane. Sucrose uptake was optimal at pH 7 in the presence of MgATP and could be stimulated by superimposed pH gradients (vesicle interior acidic) in the absence of MgATP, which is consistent with the operation of a sucrose/H⁺-antiporter at the tonoplast. Tonoplast vesicles, obtained in high yield from tissue homogenates of red beet roots, exhibited sugar-uptake characteristics comparable to those of intact vacuoles; these characteristics included similarities in K _m (1.7 mM), sensitivity to inhibitors and specificity for sucrose.Many experiments were carried out at the Experiment Station of the HSPA, Aiea, Hawaii and financed by an NSF grant to Dr. Maretzki and Mrs. M. Thom.     

Accumulation of alkaloids in plant vacuoles does not involve an ion-trap mechanism

Alkaloid uptake into vacuoles isolated from a Fumaria capreolata L. cell suspension culture was investigated. The uptake is carrier-mediated as shown by its substrate saturation, its sensitivity to metabolic inhibitors and especially by its exclusive preference for the (S)-forms of reticuline and scoulerine while the (R)-enantiomers which do not occur in this plant species were strictly discriminated. The carrier has a high affinity for (S)-reticuline with a K _m=0.3 M. The rate of alkaloid uptake was 6 pmol·h^-1·l^-1 vacuole, and 0.03 mg alkaloid·mg^-1 vacuolar protein were taken up. Transport was stimulated five-to seven-fold by ATP and was inhibited by the ATPase inhibitors N,N-dicyclohexylcarbodiimide and 4-4-diisothiocyanatostilbene-2,2 disulfonic acid, as well as by the protonophore carbonyl cyanide m-chlorophenylhydrazone. A number of alkaloids did not compete with labelled (S)-reticuline for uptake into vacuoles. The uptake system is absolutely specific for alkaloids indigenous to the plant from which the vacuoles were isolated. Slight modifications of the topography of an alkaloid molecule even with full retention of its electrical charge results in its exclusion. Alkaloid efflux was also shown to be mediated by a highly specific energy-dependent carrier. These results contradict the previously proposed ion-trap mechanism for alkaloid accumulation in vacuoles. A highly specific carrier-mediated and energy-dependent proton antiport system for alkaloid uptake and release is postulated.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DIDS 4-4-diisothiocyanatostilbene-2,2 disulfonic acid Dedicated to Professor Harry Beevers, Santa Cruz, on the occasion of his 60^th birthday     

Secretion and membrane recycling in plant cells: novel intermediary structures visualized in ultrarapidly frozen sycamore and carrot suspension-culture cells

Freeze-fracture electron microscopy of propane-jet-frozen samples has been employed to investigate vesicle-mediated secretion and membrane recycling events in carrot (Daucus carota L.) and sycamore maple (Acer pseudoplatanus L.) suspension-culture cells. Stabilization of the cells by means of ultrarapid freezing has enabled us to preserve the cells in a turgid state and to visualize new intermediate membrane configurations related to these events. Indeed, many of the observed membrane configurations, such as flattened membrane vesicles with slit-shaped membrane fusion sites and horseshoe-shaped membrane infoldings, appear to result from the action of turgor forces on the plasma membrane. Individual cells exhibited great variations in numbers and types of membrane configurations postulated to be related to secretion and membrane-recycling events. In the majority of cells, the different membrane profiles displayed a patchy distribution, and within each patch the membrane configurations tended to be of the same stage. This result indicates that secretory events are triggered in domains measuring from 0.1 to about 10 μm in diameter. Based on an extensive analysis of the different membrane configurations seen in our samples, we have formulated the following model of vesicle-mediated secretion in plant cells: Fusion of a secretory vesicle with the plasma membrane leads to the formation of a single, narrow-necked pore that increases in diameter up to about 60 nm. During discharge, the vesicle is flattened, forming a disc-shaped structure perpendicular to the plane of the plasma membrane. As the vesicle is flattened, the pore is converted to a slit, the maximum length of which coincides with the diameter of the flattened vesicle. The flattened vesicle then tips over and concomitantly the plasma-membrane slit becomes curved into a horseshoe-shaped configuration as it extends along the outer margins of the tipped-over vesicle. Some coated pits are present interspersed between the above-mentioned structures, but their numbers appear insufficient to account for an exclusively endocytotic mechanism of membrane recycling. Instead, our micrographs are more consistent with a mixed mode of recycling of membrane components to the cortical endoplamic reticulum and to Golgi cisternae that involves both internalization of membrane by endocytosis and of individual lippid molecules by unknown mechanisms (lipid exchange proteins?). To this end, overall flattening out of the horseshoe-shaped membrane infoldings is accompanied by a retraction and reduction in size of their central, tongue-like structure.     

X-ray microanalysis of ion distribution within root cortical cells of the halophyte Suaeda maritima (L.) Dum.

The ion content of compartments within cortical cells of mature roots of the halophyte Suaeda maritima grown at 200 mol·m^-3 NaCl has been studied by X-ray microanalysis of freeze-substituted thin sections. Sodium and Cl were found in the vacuoles at about four-times the concentration in the cytoplasm or cell walls, whereas K was more concentrated in the cell walls and cytoplasm than in vacuoles. The vacuolar Na concentration was 12- to 13-times higher than that of K. The Na concentration of cell walls of cortical cells was about 95 mol·m^-3 of analysed volume. The cytoplasmic K concentration within the mature cortical cells was estimated to be 55 mol·m^-3 of analysed volume.     

Ultrastructural changes associated with the accumulation and secretion of sanguinarine in Papaver bracteatum suspension cultures treated with fungal elicitor

Suspension cultures of Papaver bracteatum Arya II Lindl., grown without hormone in the presence of conidial extracts of Verticillium dahliae Kleb., accumulate millimolar quantities of the benzophenanthridine alkaloid, sanguinarine. Under the fluorescence microscope, the elicitor-treated cells display an orange-yellow fluorescence characteristic of sanguinarine, primarily near the periphery of the cells. Electron-microscopic inspection showed the presence of slightly dilated endoplasmic reticulum and of electron-dense protuberances on the tonoplast of large central vacuoles. These osmiophilic aggregates lining the tonoplast bud into spherical bodies, appear to become detached from the membrane and are released into the vacuole. Upon subcellular fractionation of elicited cells on Renografin step gradients, sanguinarine was found to be distributed in all bands but with 86% concentrated in the gradient pellet. Analysis of the pellet by electron microscopy showed that it contained electron-dense fragments similar to the osmiophilic bodies observed on the tonoplast of intact elicited cells. In elicited cell cultures, most of the sanguinarine was recovered from medium in a 100·g sedimenting, cell-free, particulate fraction accounting for as much as 85% of the media sanguinarine and 62% of the total sanguinarine. The sanguinarine-rich 100·g media pellet was determined to be two-thirds protein, one-third RNA and was essentially devoid of phenolics, phospholipid and DNA. The pellet consisted of electrondense material and cytoplasmic remnants resembling those found in the Renografin pellet and tonoplast aggregates of intact cells. When placed under hypotonic conditions or extracted with aqueous buffer, pH 3–11, the pellet did not release sanguinarine. These observations provide evidence for storage of sanguinarine at electron-dense deposits which occur on the tonoplast and as freely floating bodies in vacuoles.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - EM electron microscopy - ER endoplasmic reticulum - HPLC high-pressure liquid chromatography - MRST Murashige and Skoog's revised tobacco medium     

Factors affecting the binding between fusicoccin and plasma membranes from maize roots

Plasma membranes have been purified from roots of maize (Zea mays L.) using a two-phase aqueous polymer system, dextran-polyethylene glycol. The plant material was homogenized in the presence of a mixture of natural protease inhibitors from potato (Solanum tuberosum L.); these inhibitors have been shown to be more effective than phenylmethylsulfonyl fluoride in suppressing the endogenous proteases in maize roots. Inhibition of proteolysis in the homogenization medium markedly increased (about tenfold) the number of lowaffinity binding sites for fusicoccin (FC). In addition, storage of plasma membranes at −20° C decreased both the number of the low-affinity sites and their dissociation constant (K_D); this effect was in all probability caused by lipid peroxidation. The presence of EDTA throughout isolation and storage of the plasma membranes stabilized the parameters of FC binding to the membranes. The kinetics of binding of [³H]dihydroFC and the competition between [³H]dihydroFC and FCs A, C, J, and H were determined for the low-affinity sites. It was found that (i) the rate constant of association between FC and the low-affinity binding sites is about two orders of magnitude lower than that for the high-affinity sites; (ii) different FCs can be arranged in the order of decreasing avidity for the low-affinity FCbinding site: FC A>FC C>FC J>FC H. The authors are indebted to Dr. L.M. Krasnopolskaya (Institute of Agricultural Biotechnology, Moscow, Russia) for fusicoccins A, C, J, and H, and to Dr. A.V. Galkin (Institute of Agricultural Biotechnology, Moscow, Russia) for valuable comments and ren dering the paper into English.     

Microtubule organization differs between acid and alkaline bands in internodal cells ofChara but bands can develop in the absence of microtubules

We have studied the relationship between pH banding and the organization of cortical microtubules in the alga Chara corallina Klein ex Willd. Microtubules were visualized by immunofluorescence and also by imunogold-silver enhancement to allow immediate comparison of microtubule arrangement with visible structural cell features. In cells that are nearing growth completion, microtubule number and alignment change between acidic and alkaline bands over a distance of a few micrometres. Thus, it appears that the still unknown mechanisms for microtubule organization respond to the localized differences in membrane properties. Band formation was not prevented when microtubules were depolymerized with the herbicide oryzalin, demonstrating that microtubules are not necessary for pH bands to develop in these cells.Abbreviations DMSO dimethylsulfoxide - MT microtubule We thank Frank Gubler for helpful advice on immunogold-silver enhancement procedures, Brian Gunning for tuition in confocal microscopy, Ann Cork for assistance with photography and Dean Price for helpful discussions. G.O.W. gratefully acknowledges the receipt of a National Research Fellowship and a Queen Elizabeth II Fellowship from the Australian Research Council.     

Intracellular compartmentation of two enzymes of berberine biosynthesis in plant cell cultures

Out of the eight enzymes involved in the biosynthesis of the isoquinoline alkaloid berberine, at least, two enzymes, berberine bridge enzyme and (S)-tetrahydroprotoberberine oxidase, are exclusively located in a vesicle with a specific gravity of =1.14 g·cm^–3 as shown by direct enzymatic assay as well as immunoelectrophoresis. Electronmicroscopic examination of the enzyme-containing particulate preparation from Berberis wilsoniae var. subcaulialata cultured cells demonstrated that it is composed mainly of membranous vesicles. The protein composition of this preparation reveals the presence of only about 20 separable proteins, of which two major ones are berberine bridge enzyme and (S)-tetrahydroprotoberberine oxidase. Incubation of these vesicles with the substrate (S)-reticuline in the presence and absence of S-adenosyl-l-methionine leads to the formation of a red product which was identified as dehydroscoulerine. If the cytoplasmic enzyme S-adenosyl-l-methionine:(S)-scoulerine-9-O-methyltransferase is added to the vesicle preparation in the presence of (S)-reticuline and S-adenosyl-l-methionine, not dehydroscoulerine but columbamine, the immediate precursor of berberine is formed. Some of the quaternary alkaloids are located inside the vesicles; fusion of these vesicles leads to vacuoles containing the quaternary alkaloids. These vesicles are the first highly specific and unique compartment serving only alkaloid biosynthesis; they are found in members of four different plant families and in cell cultures as well as in differentiated tissue.Abbreviations BBE berberine bridge enzyme - STOX (S)-tetrahydroprotoberberine oxidase Dedicated to Professor Karl Decker, Freiburg, on the occasion of his 60^th birthday     

Physical properties of the cell wall of photoautotrophic suspension cells fromChenopodium rubrum L.

Photoautotrophic suspension cells ofChenopodium rubrum were used to determine Donnan potential, charge density and pore-radius distribution in the cell wall. Experiments were done either with turgescent cells or with isolated cell walls. Titration of a cell-wall-generated 9-aminoacridine fluorescence quench with salts of mono- and divalent cations was used to determine Donnan potential and charge density. The experiments and theory were adapted from measurements of membrane surface charges. A tenfold increase in ionic strength, which decreases the repellant forces between charges of the same sign, led to an approximately threefold increase in the measured charge density, thus resulting in a much smaller decrease of the Donnan potential than would be expected if the charge density remained fixed. This decreased influence of ionic strength on the Donnan potential, resulting from the elasticity of the cell wall, was also measurable but less pronounced when the wall of intact cells was stretched by turgor. The porosity of the cell wall was determined by longterm uptake of polyethylene glycols of different molecular weights, and by gel filtration of polyethylene glycols and dextrans as well as mono- and disaccharides using intact suspension cells as matrix. Both methods gave a mean pore diameter of about 4.5 nm and a maximum pore size of 5.5 nm. The resulting pores-size distribution was slightly broader with the latter method.Abbreviations 9-AA 9-aminoacridine - DMBr₂ decamethoniumbromide=N,N,N,N,N,N hexamethyldecane-1,10-diaminebromide - DW dry weight after lyophilization - EDTA ethylene diaminetetra acetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FW fresh weight - Mops 3-(N-morpholino)propanesulfonic acid - MW molecular weight - PEG polyethylene glycol     

The degrees of polymerization and N-acetylation of chitosan determine its ability to elicit callose formation in suspension cells and protoplasts of Catharanthus roseus

Partially and fully deacetylated chitosan fragments and oligomers were compared for their potency to elicit formation of the 1.3--glucan callose in suspension-cultured cells and protoplasts of Catharanthus roseus (line 385). Chitosan oligomers induced little callose formation, while callose synthesis increased with the degree of polymerization of chitosan up to several thousand corresponding to a molecular mass near 10⁶ Da. At a comparable degree of polymerization, partially N-acetylated chitosan fragments were less effective. Colloidal chitin and chitin oligomers induced only trace callose synthesis in protoplasts. These results indicate that the primary interaction involved the amino groups of chitosan and numerous negative charges at the surface of the plasma membrane with spacing in the nanometer range and occurring regularly over micrometer stretches. Charged phospholipid head-groups may fulfill these requirements. The resulting alteration of membrane fluidity may lead to the changes in ion transport known to be associated with the induction of callose formation.Abbreviations DP degree of polymerization - FDA fluorescein diacetate - PE pachyman equivalents     

Transport of glucose,fructose and sucrose by Streptanthus tortuosus suspension cells

Streptanthus tortuosus Kell. suspension cells will grow in a medium with sucrose as carbohydrate source. It was investigated whether the cells are able to take up sucrose or whether sucrose has to be hydrolyzed to glucose and fructose which eventually are taken up. The detailed quantitative analysis of sugar-uptake rates in the low concentration range up to 1 mM showed the following features: (i) There is definitely no sucrose-uptake system working in the low concentration range; any uptake of radioactivity from labelled sucrose proceeds via hydrolysis of sucrose by cell-wallbound invertase. (ii) Hexoses are taken up by two systems, a glucose-specific system with a K _m of 45 M and a high V _max for glucose and a K _m of 6 mM and a low V _max for fructose, and a fructosespecific system with a K _m of 500 M and high a V _max for fructose and a K _m of 650 M and a low V _max for glucose. (iii) There is a more than tenfold preference for uptake of the fructose derived from sucrose versus uptake of free fructose, with the result that the kinetic disadvantage of the fructoseuptake system compared to the glucose-uptake system is diminished if sucrose is supplied as the carbon source. It is speculated that invertase might work as an enzyme aiding in fructose transport.Abbreviations FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - FW fresh weight     

Calmodulin regulates the Ca2+-dependent slow-vacuolar ion channel in the tonoplast of Chenopodium rubrum suspension cells

The patch-clamp technique was applied to vacuoles isolated from a photoautotrophic suspension cell culture of Chenopodium rubrum L. and vacuolar clamp currents, which are predominantly carried by the previously identified Ca²⁺-dependent slow vacuolar (SV) ion channels, were recorded. These currents, which were activated by 1-s voltage pulses of -100 mV (vacuolar interior negative) in the presence of 100 M Ca²⁺ (cytosolic side), could be blocked completely and reversibly by the calmodulin antagonist W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] and its chlorine-deficient analogue W-5; half-maximum inhibition was found at approx. 6 M for W-7 and 70 M for W-5. Inhibition was reversed by addition of 1 g · ml^–1 calmodulin purified from Chenopodium cell suspensions; reversal by bovine brain calmodulin was scarcely appreciable. We conclude that cytosolic calmodulin mediates the Ca²⁺ dependence of the SV-channel in the Chenopodium tonoplast.Abbreviations SV-channel slowly activated, vacuolar ion channel - W-5 N-(6-aminohexyl)-1-naphthalenesulfonamide - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide We acknowledge support by the Deutsche Forschungsgemeinschaft and the Bundesminister für Forschung und Technologie, Bonn, and by the Justus-Liebig-Universität Giessen (to W.B.)