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     

Evidence for a uridine-5′-diphosphate-glucose-protectedp-chloromercuribenzene sulfonic acid-binding site in sugarcane vacuoles

The uptake of uridine-5-diphosphate (UDP) glucose into vacuoles isolated fromSaccharum sp. cells was fully inhibited by pretreatment with 50 Mp-chloromercuribenzenesulfonic acid (PCMBS) and was not affected by N-ethylmaleimide up to a concentration of 5 mM. The addition of 10 mM UDP-glucose during the pretreatment partially protected the uptake mechanism from PCMBS inhibition, while the presence of adenosine-5-diphosphate (ADP) glucose or of various hexose-phosphates had no protective effect. Parallel experiments on the binding of [²⁰³Hg]PCMBS to the vacuoles showed that UDP-glucose and UDP added at 10 mM concentrations caused a 40% decrease in the binding of PCMBS while ADP-glucose did not inhibit the binding. The results indicate the presence in a previously proposed group translocator of at least one site that can bind UDP-glucose. This site, which is blocked by PCMBS, interacts with the nucleotide moiety of UDP-glucose.Abbreviations ADP-glucose adenosine-5-diphosphate glucose - PCMB p-chloromercuribenzoic acid - PCMBS p-chloromercuribenzenesulfonic acid - UDP uridine-5-diphosphate - UDP-glucose uridine-5-diphosphate glucose     

Changes of cytoplasmic free Ca2+ in the green alga Mougeotia scalaris as monitored with indo-1, and their effect on the velocity of chloroplast movements

The fluorescent calcium-sensitive dye 1-[2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,N,N-tetraacetic acid (indo-1) was loaded by a transplasmalemma pH gradient into filamentous cells and protoplasts of Mougeotia scalaris, such that most of the indo-1 fluorescence originated from the cytoplasm. Incubation of M. scalaris filaments in ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA)-buffered media (-log [Ca²⁺] (=pCa) 8 versus pCa 3) caused a consistent and significant decrease in the cytoplasmic free [Ca²⁺]. Pulses of the fluorescence excitation light (UV-A 365 nm, 0.7 s) caused an increase in cytoplasmic free [Ca²⁺] in M. scalaris that was nearly independent of the external [Ca²⁺] and of chloroplast dislocation by centrifugation. This calcium flux, highest in UV-A light, compared with blue or red light, probably resulted from a release of Ca²⁺ from intracellular stores. Increased cytoplasmic [Ca²⁺] may affect the velocity of chloroplast rotation since UV-A-light-mediated chloroplast movement was faster than in blue or red light. Consistently, the calcium ionophore A23187 and the calcium-channel agonist Bay-K8644 both increased the velocity of the red-light-mediated chloroplast rotation. Based on these and other observations, a Ca²⁺-induced decrease in cytoplasmic viscosity in Mougeotia is presumed to occur.Abbreviations EGTA ethylene glycol-bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid - indo-1 1-[2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,N,Ntetraacetic acid - pCa log [Ca²⁺] - P_fr far-red-absorbing form of phytochrome - P_r red-absorbing form of phytochrome - x_G geometric mean Dedicated to Professor Wolfgang Haupt on the occasion of his 70th birthdayThis paper is part of the Ph.D. thesis of U. Russ at the Justus-Liebig-Universitat Giessen (FRG). Part of this work has been presented at a meeting on Calcium and intracellular signalling in plants in Plymouth, UK, Dec. 1990We are indebted to Dr. G. Seibold and Dipl. Phys. H. Weintraut for their advice on the technique of microspectrofluorometry and for allowing access to the microspectrophotometric facilities in the Strahlenzentrum der Justus-Liebig-Universität, Giessen, FRG. We thank Mrs. A. Quanz for reliable culture of the algae and evaluation of the videotapes. Bay-K8644 was a generous gift of Bayer AG, Wuppertal, FRG. U. russ was supported by a scholarship according to the Hessisches Graduierten Förderungsgesetz. This work was supported by the Deutsche Forschungsgemeinschaft.     

Abscisic acid metabolism —vacuolar/extravacuolar distribution of metabolites

The biotransformation of abscisic acid (ABA) was studied in cell suspension cultures of Lycopersicon esculentum. The ABA was converted by the cells to phaseic acid, nigellic acid, dihydrophaseic acid, abscisic acid--D-glucopyranosyl ester (ABA-Glc) and other ABA and phaseic acid conjugates. Investigation of their cellular distribution showed that the conjugated forms were located only in the vacuoles whereas ABA and its acidic metabolites were found mainly in the extravacuolar fractions. Our results, together with a number of studies on the increase of ABA-Glc as a response to stress, allow us to propose that ABA-Glc is irreversibly compartmented in the vacuoles of plant cells.Abbreviations ABA abscisic acid - ABA-Glc -D-glucopyranosyl ester of ABA - DPA 4-dihydrophaseic acid; nigellic acid=3-methyl-5-(1-hydroxy-2-hydroxymethyl-6-dimethyl-4-oxo-cyclohex-2-enyl)-penta-2Z, 4E-dienoic acid - PA phaseic acid     

Membrane-potential changes in vacuoles isolated from storage roots of red beet (Beta vulgaris L.)

The membrane potential in vacuoles isolated from storage roots of red beet (Beta vulgaris L.) has been studied by following changes in the fluorescence of the dye 3,3-diethylthiodicarbocyanine iodide, and by determining the uptake of the lipophilic triphenylmethylphosphonium cation. The vacuoles have a membrane potential, internal negative, which is estimated to be around-60 mV. These potentials become less negative by nearly 10 mV on addition of ATP. This ATP-dependent depolarisation is inhibited by the protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone and by the ATPase inhibitors, N,N-dicyclohexylcarbodiimide and trimethyltin chloride, but it is largely insensitive to sodium orthovanadate. Fusicoccin had no significant effect on the isolated vacuoles, but its addition to excised tissue caused a hyperpolarisation of the cells measured using a microelectrode.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - DiS-C₂-(5) 3,3-diethylthiodicarbocyanine iodide - FCCP carbonylcyanide p-trifluoromethoxyphenylhydrazone - TPMP⁺ triphenylmethylphosphonium ion     

Detoxification of xenobiotics by plant cells: characterisation of vacuolar amphiphilic organic anion transporters

The transport activities of two primary ATP-dependent organic-anion transporters in the tonoplast of isolated barley (Hordeum vulgare L. cv. Klaxon) vacuoles have been characterised with N-ethylmaleimide glutathione (NEM-SG) and taurocholate as substrates. The transporters showed different sensitivities to organic anions and a variety of transport inhibitors and drugs. The vacuolar uptake of NEM-SG was inhibited by carbonylcyanide 4-trifluoromethoxyphenylhydrazone, 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), S-(2,4-dinitrophenyl)glutathione, alkyl-S-glutathione derivatives and taurocholate but stimulated by probenecid. The uptake of taurocholate was inhibited by vinblastine, DIDS and probenecid. Both transporters were unaffected by verapamil. The kinetic properties of the transporters indicate a general preference for amphiphilic anions with some substrate overlap. These characteristics of the transporters are similar to those displayed by the multidrug resistance protein of mammalian drug-resistant cells. We suggest that these vacuolar transporters be described as plant multispecific organic anion transporters (pMOATs).Abbreviations Bm-S bimane S-glutathione - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - DNP-SG S-(2,4-dinitrophenyl)glutathione - FCCP carbonylcyanide 4-trifluoromethoxyphenylhydrazone - LTC₄ cysteinyl leukotriene - MDR multidrug transporter - MRP multidrug resistance protein - NEM-SG N-ethylmaleimide glutathione We thank Prof E. Martinoia for technical advice on the uptake experiments and Prof J. Palmer for helpful discussions and suggestions. M.B.-K. was partially sponsored by a grant from Stichting VSB Fonds, The Netherlands. IACR receives grant-aided support from the Biotechnology and Biological Science Research Council of the United Kingdom     

Alkaloid N-oxides as transport and vacuolar storage compounds of pyrrolizidine alkaloids in Senecio vulgaris L

Cell-suspension cultures of pyrrolizidinealkaloid-producing species selectively take up and accumulate senecionine (sen) and its N-oxide (sen-Nox). Cultures established from non-alkaloid-producing species are unable to accumulate the alkaloids. The uptake and accumulation of ¹⁴C-labelled alkaloids was studied using a Senecio vulgaris cell-suspension culture as well as protoplasts and vacuoles derived from it. The alkaloid uptake exhibits all characteristics of a carrier-mediated transport. The uptake of sen-Nox follows a multiphasic saturation kinetics. The K_m-values for sen Nox of 53 M and 310 M are evaluated. Senecionine competitively inhibits sen-Nox uptake, indicating that the tertiary alkaloid and its N-oxide share the same membrane carrier. The N-oxide of sen shows a pH optimum below 5.5, whereas sen is taken up over a range from pH 4 to 8. Activation energies of 90 and 53 kJ·mol^-1 are calculated for sen-Nox and sen transport, respectively. At concentrations of 10 to 100 M, sen-Nox is rapidly taken up by cells and protoplasts; within 2 h >90% of total N-oxide is within the cells. By contrast the uptake of sen is less efficient. Vacuoles isolated from protoplasts preloaded with sen-Nox totally retained the alkaloid N-oxide, whereas sen is rapidly lost during the procedure of vacuole preparation. N-oxidation converts the weak lipophilic tertiary base into a charged polar molecule which is excellently adapted to serve as the cellular transport and storage form of pyrrolizidine alkaloids.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - DNP 2,4-dinitrophenol - sen senecionine - sen-Nox senecionine N-oxide     

PEG-enhanced,electric field-induced fusion of tonoplast and plasmalemma of grape protoplasts

Cultured grape cells accumulate anthocyanins in vacuoles rather than secreting them into the nutrient medium. Therefore, grape cells that contain tonoplast segments in their plasmalemma should be capable of excreting anthocyanins rather than sequestering them in their vacuoles. In initial attempts to construct such novel cells, small vacuoles were fused with the plasmalemma of cultured plant cells. Protoplasts were isolated from grape calluses that produce and accumulate anthocyanins. Small vacuoles were formed by gently rupturing vacuoles isolated from grape protoplasts. Although small vacuoles and protoplasts became aligned in an AC field, the tonoplast and plasmalemma did not readily fuse when subjected to 3 DC pulses of 1200 V cm^–1 for 50 s each. Changes in the intensity, number and/or duration of the DC pulses had no effect on the fusion process. When 1.0% polyethylene glycol was added to the electrofusion buffer, however, small vacuoles and protoplasts fused within a few minutes after the DC pulses were applied. These novel grape cells remained viable for several hours.Abbreviations BSA bovine serum albumin - 2,4-D 2,4-dichloro-phenoxyacetic acid - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-amino-ethyl ether)-N,N,N,N - MES 2-[N-Morpholino]ethanesulfonic acid - MOPS 3-[N-Morpholino]propanesulfonic acid - PVP polyvinylpyrrolidone     

Interaction between photosynthetic and respiratory electron-transfer chains in the membranes of Anabaena variabilis

The rate of CO₂- and p-benzoquione-dependent photosynthetic O₂ evolution by Anabaena variabilis cells remained unaltered and the rate of O₂ uptake observed after switching off the light (endogenous respiration) was enhanced by a factor of 6–8 when the O₂ concentration was increased from 200 to 400 M. Photosystem-I-linked O₂ uptake and respiration of the cells incubated with ascorbate and N,N,NN-tetramethyl-p-phenylenediamine was not appreciable influenced by the O₂ concentration. 2-Iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether, blocking electron transfer at the plastoquinone level, suppressed O₂ evolution and had no influence on endogenous respiration. 2-n-Heptyl-4-hydroxyquinoline-N-oxide, an inhibitor of electron transfer between photosystems II and I, as well as the cytochrome-oxidase inhibitors N ₃ ^- , CN^- and NH₂OH, caused a 35–50% retardation of endogenous respiration and blocked photosynthetic O₂ evolution. The molar ratio of cytochromes b₆, f, c-553, aa₃ and photosystem-I reaction centers in the isolated membranes equalled approx. 2:1:2:0.7:2. It is inferred that endogenous respiration of A. variabilis cells is inhibited by the light-induced electron flow through both photosystems at the level of the plastoquinone-plastocyanin-oxidoreductase complex.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT 2-iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether - Hepes 4-(2-hydroxyethyl)-1-piperazine ethansulfonic acid - TMPD N,N,NN-tetramethyl-p-phenylenediamine     

Uptake and accumulation of ajmalicine into isolated vacuoles of cultured cells of Catharanthus roseus (L.) G. Don. and its conversion into serpentine

Isolated vacuoles from ajmalicine-producing cell suspensions of Catharanthus roseus accumulated the alkaloid ajmalicine. Dissipation of the transtonoplast pH gradient with nigericin abolished ajmalicine accumulation, whereas dissipation of the transtonoplast potential with valinomycin had no effect. Addition of Mg-ATP resulted in a higher ajmalicine accumulation. Serpentine produced by the cells was largely recovered in isolated vacuoles; in contrast, ajmalicine was lost. Ajmalicine was converted in vitro into serpentine by horseradish basic peroxidases (EC 1.11.1.7). In cultured cells there was a striking conformity between the time course of serpentine content and that of the activity of basic peroxidases. Ajmalicine was converted efficiently into serpentine by basic peroxidases extracted from vacuoles and by intact isolated vacuoles. The results are consistent with the hypothesis that ajmalicine accumulates by an ion-trap mechanism and that the accumulated ajmalicine is converted into serpentine inside the vacuoles. By the transformation of ajmalicine into the charged serpentine a trap is created to retain the alkaloids more efficiently in the vacuole.Abbreviations and Symbols DCCD N,N-dicyclohexylcar-bodiimide - TPP⁺ tetraphenylphosphonium - pH trans-tonoplast pH gradient - transmembrane potential difference We thank Dr W. Kreis, Universität Tübingen, FRG for fruitful discussions and for his suggestions in isolation of vacuoles.     

Implication of tyramine in the biosynthesis of morphinan alkaloids in Papaver

Doubly-labeled [³H, ¹⁴C]tyrosines, [1-¹³C-]tyramine or [2-¹⁴C]tyramine, administered to the stems of intact Papaver somniferum L. plants, were found to be incorporated into the morphinan alkaloids of the plant with comparable efficiency. ³H/¹⁴C ratios of alkaloids from plants fed the tyrosines were consistent with an almost equal conversion of this amino acid into the tetrahydroisoquinoline (TIQ) and benzyl-derived segments. Nuclear magnetic resonance (NMR) analyses of morphine isolated after administration of [1-¹³C]tyramine demonstrated selective labeling of C-16 of the alkaloid, indicating the conversion of this amine primarily into the TIQ-derived moiety. Morphine and thebaine labeled by [2-¹⁴C]tyramine were degraded to phenanthridines and N,N-dimethyl ethylamines. Of the total radioactivity in the alkaloids 97% was found to be associated with the ethylamines, a distribution consistent with the NMR data. This preferential utilization of tyramine in the biosynthesis of morphinan alkaloids can be explained by the compartmentalization of intermediates and enzymes of the pathway.Abbreviations L-dopa L-3,4-dihydroxyphenylalanine - HPLC high-pressure liquid chromatography - NMR nuelear magnetic resonance - TIQ tetrahydroisoquinoline     

The relationship of arginine groups to photosynthetic HCO 3 - uptake inUlva sp. mediated by a putative anion exchanger

The marine macroalgaUlva sp. can take up HCO ₃ ^- via a process which chemically resembles that of anion exchange in red blood cells (Drechsler et al. 1993, Planta191, 34–40). In this work we explore the possibility that high-pK amino-acid residues could be functionally involved in the binding/transport of HCO ₃ ^- . It was found that the specific arginyl-reacting agents phenylglyoxal and 2,3-butanedione inhibited photosynthesis ofUlva competitively with inorganic carbon at pH 8.2–8.4 (which is close to the pH of normal seawater), where HCO ₃ ^- was the predominant inorganic carbon form taken up. The inhibition by phenylglyoxal was irreversible at 32°C and high pH values, while that of butanedione became irreversible in the presence of borate. These interactions, as well as the protection of the irreversible phenylglyoxal-inhibition by inorganic carbon and by the membrane-impermeant agents 4,4-diisothiocyanostilbene 2,2-disulfonate and 4,4-dinitrostilbene-2,2-disulfonate indicate that arginine (and possibly also lysine) are involved in the HCO ₃ ^- uptake process, probably at the plasmalemma level. The photosynthetic affinity ofUlva to external inorganic carbon gradually decreased with increasing pH from 8.2 to 10.5, and this decrease parallels the decline in protonation of amino acids with a pK of around 10. Based on this information, as well as the inhibition studies, it is suggested that arginine and lysine residues are essential proteinaceous constituents involved in anionic inorganic carbon (HCO ₃ ^- and possibly also CO ₃ ^2- ) uptake into theUlva cells.Abbreviations AE1 anion exchanger 1 (of red blood cells) - BD 2,3-butanedione - CA carbonic anhydrase - CI inorganic carbon - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonate - DNDS 4,4-dinitrostilbene-2,2-disulfonate - PG phenylglyoxal This paper is in partial fulfillment of a Ph.D. study by R. Sharkia. Supported by the Israel Academy of Sciences, grant 441/93 (to S.B.), and by the Fund for Encouragement of Research, Histadrut, Israel (to R.S.).     

Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Light dependence of protoplasmic streaming in Nitella flexilis L. as measured by means of laser-velocimetry

Laser-velocimetry was applied in order to study the effect of light on the velocity of protoplasmic streaming (pps) in Characean cells. A change from dark to light (= 6 W · m^–2) leads to an acceleration of streaming by about 15–30% with a time-constant of approx. 300 s. The transition from light to dark causes a transient decrease of velocity below the original dark level. This response occurs with a time constant of about 500 s. It returns to its initial value with a time-constant of about 2000 s. This may indicate that a control loop of cytosolic homeostasis takes a decrease in pCa more seriously than an increase. A possible involvement of temperature effects caused by illumination was excluded by measuring the influence of temperature. Steady-state velocity of streaming changed by 5% per 1° C. Irradiation with infra-red light ( > 780 nm) did not cause a change in velocity. The absence of a light effect on streaming velocity in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) shows that photosynthesis and not phytochrome is involved. The role of light-induced changes of pCa is discussed, especially with respect to the hypothesis of Vanselow and Hansen (1989, J. Membr. Biol. 110, 175–187) that photosynthesis acts on the plasmalemma K⁺-channel via light-induced uptake of Ca²⁺ into the chloroplasts.Abbreviations and Symbols ASF auto structure function - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - pps protoplasmic streaming - _L, _D, _C time-constants of the light and dark responses, and of a putative Ca-control system Financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged. The first author was granted a scholarship by the state of Schleswig-Holstein. We are indebted to Prof. Dr. G. Pfister for technical advice and helpful discussions and to Mrs. E. Götting for drawing the figures.     

Regulation of electrical coupling between Arabidopsis root hairs

Voltage clamp was used to measure the voltage dependence of cell-to-cell coupling via plasmodesmata between higher-plant cells (root hairs of Arabidopsis thaliana (L.) Heynh.). In addition, ionophoresis was used to introduce a variety of ions [Ca²⁺, inositol-trisphosphate, Li⁺, K⁺, Mg²⁺, ethylene glycol-bis(-aminoethyl ether)-N,N,N, N-tetraacetic acid (EGTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA), H⁺, and OH^–] to examine whether they regulate cell-to-cell coupling. Electrical coupling showed high variability in this single cell type at the same developmental stage; the coupling ratio ranged from near 0% to about 90% with a mean value of 32%. It was voltage independent for intracellular voltage gradients (transplasmodesmatal) of -163 to 212 mV. While Ca²⁺ closes the plasmodesmatal connections (at concentrations higher than those causing cessation of cytoplasmic streaming), inositol-trisphosphate and lithium are without effect. Apparently, inositol-trisphosphate may not cause increased cytosolic Ca²⁺ in root hairs. Alkalinization by OH ionophoresis caused a modest decline in cell-to-cell coupling, as did acidification by H⁺ ionophoresis (to an extent causing the cell to become flacid). Increases in cytosolic K⁺, Mg²⁺, and the calcium chelator BAPTA by ionophoresis had no effect on cell-to-cell coupling. The regulation (and lack thereof) reported here for plant plasmodesmata is quite similar to that of gap junctions.Abbreviations BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N, N-tetraacetic acid     

Determination of the membrane potential of vacuoles isolated from red-beet storage tissue

The membrane potential of isolated vacuoles of red beet (Beta vulgaris L.) was estimated using several methods. The quenching of the fluorescence of the cyanine dyes 3,3-diethylthiodicarbocyanine iodide (DiS-C₂–(5)) and 3,3-dipropylthiodicarbocyanine iodide (DiS-C₃–(5)) in vacuoles indicated a transmembrane potential difference, negative inside at low external potassium concentrations. The was found to be-55 mV with two other methods, the distribution of ²⁰⁴T1⁺ in the presence of valinomycin and the distribution of the lipophilic cation triphenylmethylphosphonium. Uncouplers reduced this value to-35 mV. High external potassium concentrations, comparable to cytosolic values, abolished the membrane potential almost completely. The addition of 1 mM Tris-Mg²⁺-ATP markedly hyperpolarized the membrane to-75 mV. This effect was prevented by inhibitors of the ATPase activity located in isolated vacuole membranes.Abbreviations ANS aminonaphthalene sulfonate - DiS-C₂–(5) 3,3-diethylthiodicarbocyanine iodide - DiS-C₃–(5) 3,3-dipropylthiodicarbocyanine iodide - EDAC 1-ethyl-3-C-3dimethylaminopropylcarbodiimide - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - MES morpholinoethylsulfonic acid - TPP⁺ tetraphenylphoshonium - TPMP triphenylmethylphosphonium - Tris tris(hydroxymethyl)aminomethane     

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     

Redistribution of potassium,chloride and calcium during the gravitropically induced movement of Mimosa pudica pulvinus

When the leaves of Mimosa pudica are changed from their normal position in the gravitational field, they perform reversible compensatory movements by means of pulvini. These movements are not the result of growth processes but involve reversible turgor variations. These variation are concomitant with ion migrations within pulvini: during the gravitropic movement, K⁺ and Cl^- shift towards the adaxial half of the motor organ whereas Ca²⁺ shifts towards the abaxial half. Compounds known to affect K⁺ transport, tetraethylammonium chloride and valinomycin, do not hinder the gravitropic movement but inhibit strongly the seismonastic reaction. The same general result is obtained with compounds affecting anion transport, disulfonic stilbenes and 9-anthracene carboxylic acid. Calcium chelators inhibit the gravitropic movement more efficiently than the seismonastic reaction and the calcium ionophore A 23 187 increases both movements. The data obtained with these various compounds indicate that ions do not have the same functional importance in the regulation of the two different pulvinar movements.Abbreviations abx abaxial half of the pulvinus - adx adaxial half of the pulvinus - 9-AC 9-anthracene carboxylic acid - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - SITS 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid - TEA tetraethylammonium chloride     

Sodium efflux from perfused giant algal cells

Internodal cells of the giant alga Chara corallina were perfused internally to replace the native cytoplasm, tonoplast and vacuole with artificial cytoplasm. Sodium efflux from perfused cells, measured by including ²²Na in the perfusion media, was increased by increasing the internal sodium concentration and by decreasing the external pH, and was inhibited by external application of the renal diuretic amiloride. The sodium efflux was markedly ATP-dependent, with a 50-fold decrease in efflux observed after perfusion with media lacking ATP. Efflux in the presence of ATP was reduced by 33% by inclusion of 10 M N,N-dicyclohexylcarbodiimide in the perfusion medium. The membrane potential of the perfused cells approximated that of intact cells from the same culture. It is suggested that sodium efflux in perfused Chara cells proceeds via a secondary antiporter with protons, regulated by ATP in a catalytic role and with the proton motive force acting as the energy source.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Mes 2-(N-morpholino)ethanesulphonic acid - Mops 3(N-morpholino)propanesulphonic acid - Taps tris(hydroxymethyl)methylaminopropanesulphonic acid     

Properties of a high-affinity cytokinin-binding protein from wheat germ

A soluble protein that interacts with a range of cytokinins was extensively purified from wheat (Triticum aestivum L.) germ. This protein has a K _d for kinetin of 2×10^-7 M. The binding of kinetin to the protein is inhibited by low concentrations of synthetic and naturally-occurring cytokinins including N⁶-benzyladenine, N⁶-benzyladenosine, kinetin riboside, N⁶-dimethylallyladenine, N⁶-dimethylallyladenosine, zeatin, zeatin riboside, N⁶-dimethyladenine and N⁶-dimethyladenosine. Adenine, adenosine and several non-N⁶-substituted adenine derivatives were ineffective as inhibitors of kinetin binding. While N⁶-butyryl-3,5-cyclic AMP, N⁶,2-O-dibutyryl-3,5-cyclic AMP and 2,3-cyclic AMP inhibited binding of kinetin to the protein, 3,5-cyclic AMP was ineffective. The kinetin-binding protein is heat-labile and pronase-sensitive. Kinetin-binding activity exactly co-chromatographs with a single peak of carbohydrate and protein on gel-filtration and is displaced from concanavalin A-Sepharose 4B by -methylglucoside. On gel filtration, the kinetin-binding protein behaves as a soluble protein with an apparent molecular weight of 180,000 daltons.