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.     

Sucrose Transport and Phloem Unloading in Stem of Vicia faba: Possible Involvement of a Sucrose Carrier and Osmotic Regulation

Stems of Vicia faba plants were used to study phloem unloading because they are hollow and have a simple anatomical structure that facilitates access to the unloading site. After pulse labeling of a source leaf with ¹⁴CO₂, stem sections were cut and the efflux characteristics of ¹⁴C-labeled sugars into various buffered solutions were determined. Radiolabeled sucrose was shown to remain localized in the phloem and adjacent phloem parenchyma tissues after a 2-hour chase. Therefore, sucrose leakage from stem segments prepared following a 75-minute chase period was assumed to be characteristic of phloem unloading. The efflux of ¹⁴C assimilates from the phloem was enhanced by 1 millimolar p-chloromercuribenzene sulfonic acid (PCMBS) and by 5 micromolar carbonyl cyanide m-chlorophenly hydrazone (CCCP). However, PCMBS inhibited and CCCP enhanced general leakage of nonradioactive sugars from the stem segments. Sucrose at concentrations of 50 millimolar in the free space increased efflux of [¹⁴C]sucrose, presumably through an exchange mechanism. This exchange was inhibited by PCMBS and abolished by 0.2 molar mannitol. Increasing the osmotic concentration of the efflux medium with mannitol reduced [¹⁴C]sucrose efflux. However, this inhibition seems not to be specific to sucrose unloading since leakage of total sugars, nonlabeled sucrose, glucose, and amino acids from the bulk of the tissue was reduced in a similar manner. The data suggest that phloem unloading in cut stem segments is consistent with passive efflux of sucrose from the phloem to the apoplast and that sucrose exchange via a membrane carrier may be involved. This is consistent with the known conductive function of the stem tissues, and contrasts with the apparent nature and function of unloading in developing seeds.     

Phloem Unloading in Developing Leaves of Sugar Beet : I. Evidence for Pathway through the Symplast

Physiological and transport data are presented in support of a symplastic pathway of phloem unloading in importing leaves of Beta vulgaris L. (`Klein E multigerm'). The sulfhydryl reagent p-chloromercuribenzene sulfonic acid (PCMBS) at concentration of 10 millimolar inhibited uptake of exogenous [¹⁴C]sucrose by sink leaf tissue over sucrose concentrations of 0.1 to 5.0 millimolar. Inhibited uptake was 24% of controls. The same PCMBS treatment did not affect import of ¹⁴C-label into sink leaves during steady state labeling of a source leaf with ¹⁴CO₂. Lack of inhibition of import implies that sucrose did not pass through the free space during unloading. A passively transported xenobiotic sugar, l-[¹⁴C]glucose, imported by a sink leaf through the phloem, was evenly distributed throughout the leaf as seen by whole-leaf autoradiography. In contrast, l-[¹⁴C]glucose supplied to the apoplast through the cut petiole or into a vein of a sink leaf collected mainly in the vicinity of the major veins with little entering the mesophyll. These patterns are best explained by transport through the symplast from phloem to mesophyll.     

Phloem loading in Vicia faba leaves: Effect of N-ethylmaleimide and parachloromercuribenzenesulfonic acid on H+ extrusion,K+ and sucrose uptake

The effects of a penetrating (NEM) and a non-penetrating (PCMBS) sulfhydryl-specific reagent on proton extrusion, ⁸⁶Rb and [U-¹⁴C]sucrose uptake by Vicia faba leaves have been studied. Proton extrusion was strongly or completely inhibited by 0.1 mM NEM. ⁸⁶Rb and [U-¹⁴C]sucrose uptake were markedly reduced by NEM concentrations equal to or higher than 0.5 mM. Under our experimental conditions, PCMBS (1 mM) exerted a strong inhibition on [¹⁴C]sucrose uptake but did not inhibit proton extrusion and ⁸⁶Rb uptake. The sensitivity of phloem loading to PCMBS is thought to be a consequence of sugar-carrier blockage and not of inhibition of the proton pump.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - DES diethylstilbestrol - DCCD dicyclohexylcarbodiimide - FC Fusicoccin - NEM N-ethylmaleimide - PCMBS p-chloromercuribenzenesulfonic acid     

Phloem unloading in soybean seed coats: dynamics and stability of efflux into attached ;empty ovules'

The time-course of sucrose efflux from attached seedcoats (having their embryos surgically removed) into aqueous traps placed in the `empty ovules' had three phases. The first phase lasted 10 minutes and probably was a period of apoplastic flushing. The second lasted 2 to 3 hours and is thought to be a phase of equilibration of seed coat symplast with the frequently refreshed liquid. The third phase of relatively steady efflux was postulated to reflect the continued import of sucrose from the plant, and hence to reflect the rate of sieve tube unloading. The average steady state efflux was equal under most conditions to the estimated rate of sucrose import. Efflux and import were unaffected by 150 millimolar osmoticum (mannitol or polyethylene glycol [molecular weight about 400]), by 0.5 millimolar CaCl<sub>2</sub>, or by pretreatments up to 20 minutes with p-chloromercuribenzenesulfonic acid (PCMBS); they were enhanced by 40 micromolar abscisic acid, 40 micromolar indoleacetic acid, 20 micromolar fusicoccin, and 1 millimolar dithiothreitol (DTT) and were inhibited by 100 micromolar KCN, by 0.03% H<sub>2</sub>O<sub>2</sub>, by 20 micromolar and 5 micromolar trifluoromethoxy (carbonyl cyamide) phenylhydrazone, by repeated 5 minutes per hour treatments with 5 millimolar PCMBS, and by 5 millimolar DTT. The `steady state' sucrose efflux was able to account for about half the rate of dry weight growth of the embryo, but stabilization of the system with <1 millimolar DTT taken together with other considerations is likely to give good correspondence between experimental unloading rates and in vivo growth rates.     

Evidence for the presence of a sucrose carrier in immature sugar beet tap roots

The objectives of this work were to determine the path of phloem unloading and if a sucrose carrier was present in young sugar beet (Beta vulgaris L.) taproots. The approach was to exploit the characteristics of the sucrose analog, 1'-fluorosucrose (F-sucrose) which is a poor substrate for acid invertase but is a substrate for sucrose synthase. Ten millimolar each of [³H]sucrose and [¹⁴C]F-sucrose were applied in a 1:1 ratio to an abraded region of an attached leaf for 6 hours. [¹⁴C]F-sucrose was translocated and accumulated in the roots at a higher rate than [³H]sucrose. This was due to [³H]sucrose hydrolysis along the translocation path. Presence of [³H]hexose and [¹⁴C]F-sucrose in the root apoplast suggested apoplastic sucrose unloading with its subsequent hydrolysis. Labeled F-sucrose uptake by root tissue discs exhibited biphasic kinetics and was inhibited by unlabeled sucrose, indicating that immature roots have the ability for carrier-mediated sucrose transport from the apoplast. Collectively, in vivo and in vitro data indicate that despite sucrose hydrolysis by the wall-bound invertase, sucrose hydrolysis is not entirely essential for sugar accumulation in this tissue.     

The cellular pathway of photosynthate transfer in the developing wheat grain. III. A structural analysis and physiological studies of the pathway from the endosperm cavity to the starchy endosperm

The cellular pathway of sucrose transfer from the endosperm cavity to the starchy endosperm of developing grains of wheat (Triticum turgidum) has been elucidated. The modified aleurone and sub-aleurone cells exhibit a dense cytoplasm enriched in mitochondria and endoplasmic relicilium. Significantly, the sub-aleurone cells are characterized by secondary wall ingrowths. Numerous plasmodesmata interconnect all cells between the modified aleurone and starchy endosperm. The pro-tonophore carbonylcyanide-m-chlorophenyl hydrazone (CCCP) slowed [¹⁴C]sucrose uptake by grain tissue slices enriched in modified aleurone and sub-aleurone cells but had no effect on uptake by the starchy endosperm. The fluorescent weak acid sulphorhodamine G (SRG) was preferentially accumulated by the modified aleurone and sub-aleurone cells, and this uptake was sensitive to CCCP. The combined plasma membrane surface areas of the modified aleurone and sub-aleurone cells appeared to be sufficient to support the in vivo rates of sucrose transfer to the starchy endosperm. Plasmolysis of intact excised grain inhibited [¹⁴C]sucrose transfer from the endosperm cavity to the starchy endosperm. The sulphydryl group modifier p-chloromercuribenzenesulphonie acid (PCMBS) decreased [¹⁴C]sucrose uptake by the modified aleurone and sub-aleurone cells but had little effect on uptake by the starchy endosperm. In contrast, when PCMBS and [¹⁴C]sucrose were supplied to the endosperm cavity of intact excised grain, PCMBS slowed accumulation by all tissues equally. Estimates of potential sucrose fluxes through the interconnecting plasmodesmata were found to be within the published range. It is concluded that the bulk of sucrose is accumulated from the endosperm cavity by the modified aleurone and sub-aleurone cells and subsequently transferred through the symplast to the starchy endosperm.     

Fluoride-Induced Inhibition of Starch Biosynthesis in Developing Potato, Solanum tuberosum L., Tubers Is Associated with Pyrophosphate Accumulation

Pretreatment of discs excised from developing tubers of potato (Solanum tuberosum L.) with 10 millimolar sodium fluoride induced a transient increase in 3-phosphoglycerate content. This was followed by increases in triose-phosphate, fructose 1,6-bisphosphate and hexose-phosphate (glucose 6-phosphate + fructose 6-phosphate + glucose 1-phosphate). The effect of fluoride is attributed to an inhibition of glycolysis and a stimulation of triose-phosphate recycling (the latter confirmed by the pattern of ¹³C-labeling [NMR] in sucrose when tissue was supplied with [2-¹³C]glucose). Fluoride inhibited the incorporation of [U-¹⁴C] glucose, [U-¹⁴C]sucrose, [U-¹⁴C]glucose 1-phosphate, and [U-¹⁴C] glycerol into starch. The incorporation of [U-¹⁴C]ADPglucose was unaffected. Inhibition of starch biosynthesis was accompanied by an almost proportional increase in the incorporation of ¹⁴C into sucrose. The inhibition of starch synthesis was accompanied by a 10-fold increase in tissue pyrophosphate (PPi) content. Although the subcellular localization of PPi was not determined, a hypothesis is presented that argues that the PPi accumulates in the amyloplast due to inhibition of alkaline inorganic pyrophosphatase by fluoride ions.     

Energetics of Amino Acid Uptake by Vicia faba Leaf Tissues

The uptake of [U-¹⁴C]threonine and of (α-¹⁴C]aminoisobutyrate (α-AIB) by Vicia faba leaf discs is strongly pH dependent (optimum: pH 4.0) and exhibits biphasic saturation kinetics. Kinetics of α-AIB uptake at different pH values indicate that acidic pH values decrease the K_m of the carriers while the maximal velocity remains nearly unaffected. Similar results were obtained for both system 1 (from 0.5 to 5 millimolar) and system 2 (from 20 to 100 millimolar).

After addition of amino acids to a medium containing leaf fragments, alkalinizations depending both on the amino acid added and on its concentration have been recorded.

The effects of compounds which increase (fusicoccin) or decrease (uncouplers, ATPase inhibitors, high KCl concentrations) the protonmotive force were studied both on the acidification of the medium and on amino acid uptake by the tissues. There is a close relationship between the time required for the effect of these compounds on the acidification and that needed for inhibition of uptake.

Studies with thiol inhibitors show that 0.1 millimolar N-ethylmaleimide preferentially inhibits uptake by the mesophyll whereas 0.1 millimolar parachloromercuribenzenesulfonate affects rather uptake by the veins.

New evidence was found which added to the electrophysiological data already supporting the occurrence of proton amino acid symport in leaf tissues, particularly in the veins.

     

Potassium Transport in Corn Roots : II. The Significance of the Root Periphery

The relative transport capabilities of the cells of the root periphery and cortex were investigated using a variety of experimental techniques. Brief (30 seconds to 1 minute) exposures with the penetrating sulfhydryl reagent, N-ethyl maleimide (NEM), and the impermeant reagent, p-chloromercuribenzene sulfonic acid (PCMBS), dramatically reduced ⁸⁶Rb⁺ (0.2 millimolar RbCl) uptake into 2 centimeter corn (Zea mays [A632 × (C3640 × Oh43)]) root segments. Autoradiographic localization studies with [³H]NEM and [²⁰³Hg]PCMBS demonstrated that, during short term exposures with either reagent, sulfhydryl binding occurred almost exclusively in the cells of the root periphery.

Corn root cortical protoplasts were isolated, and exhibited significant K⁺(⁸⁶Rb⁺) influx. The kinetics for K⁺ uptake were studied; the influx isotherms were smooth, nonsaturating curves that approached linearity at higher K⁺(Rb⁺) concentrations (above 1 millimolar K⁺). These kinetics were identical in shape to the complex kinetics previously observed for K⁺ uptake in corn roots (Kochian, Lucas 1982 Plant Physiol 70: 1723-1731), and could be resolved into a saturable and a first order kinetic component.

The existence of a hypodermal apoplastic barrier was investigated. The apoplastic, cell wall binding dye, Calcofluor White M2R, appeared to be excluded from the cortex by the hypodermis. However, experiments with damaged roots indicated that this result may be an artifact resulting from the binding of dye to the epidermal cell walls. Furthermore, [²⁰³Hg] PCMBS autoradiography demonstrated that the hypodermis was not a barrier to apoplastic movement of PCMBS.

These results suggest that although cortical cells possess the capacity to absorb ions, K⁺ influx at low concentrations is limited to the root periphery. Cortical cell uptake appears to be repressed under these conditions. At higher concentrations, cortical cells may function to absorb K⁺. Such a model may involve regulation of cortical cell ion transport capacity.

     

Potassium transport in corn roots : I. Resolution of kinetics into a saturable and linear component

Influx isotherms were obtained for ⁸⁶Rb⁺ uptake into 2-cm corn (Zea mays [A632 × (C3640 × Oh43)] root segments for both low- (0.2 millimolar CaSO₄) and high-salt (0.2 millimolar CaSO₄ + 5 millimolar KCl) grown roots. Unlike the discontinuous curves usually presented for K⁺ influx, our isotherms were smooth, nonsaturating curves that approached linearity at K⁺ (Rb⁺) concentrations above 1 millimolar. The kinetics for K⁺ transport could be resolved into saturable and linear components. The saturable components yielded K_m values of 16 and 86 micromolar for low- and high-salt roots, respectively, while V_max values were 5.62 and 1.85 moles per gram fresh weight per hour. Results of experiments with the penetrating sulfhydryl reagent, N-ethyl maleimide (NEM), and the impermeant reagent, p-chloromercuribenzene sulfonic acid (PCMBS) indicated that the saturable and linear components were independent mechanisms of K⁺ transport.

Short-term NEM exposures (30 seconds to 5 minutes) selectively inhibited the saturable system, but had little effect on the linear component. Increasing NEM exposures resulted in further inhibition and subsequent abolition of the saturable component; the linear component exhibited limited NEM sensitivity. PCMBS elicited the same general inhibitory trends, although it was less effective as a saturable component inhibitor.

The effects of NEM and PCMBS on K⁺ efflux were also studied. Short NEM exposures had no effect on cytoplasmic efflux, while inhibiting vacuolar efflux significantly. From these data, it is unclear at which site(s) NEM is acting. A more complex response was obtained with PCMBS, where a monophasic efflux curve was observed. Analysis indicated that the vacuolar efflux was stimulated, while the cytoplasmic component was abolished.

The nature of the linear component is discussed, and it is proposed that the mechanism may be more complex than simple facilitated diffusion.

     

Sucrose uptake by sugar beet tap root tissue

Sucrose uptake by discs of mature sugar beet root tissue incubated in [¹⁴C]-sucrose exhibited nonsaturating kinetics over the concentration range of 1 to 500 millimolar. Uptake was inhibited by dinitrophenol, sodium cyanide, low O₂, and penetrating sulfhydryl inhibitors. ATPase inhibitors, sodium fluoride, and oligomycin reduced uptake by 20 and 40%, respectively. Uptake as asymmetrically labeled sucrose ([¹⁴C]glucose) occurred with approximately 80% retention of asymmetry, indicating a nonhydrolytic pathway. Uptake was against a concentration gradient and required metabolic energy.     

Inhibition of Loading of C Assimilates by p-Chloromercuribenzenesulfonic Acid : Localization of the Apoplastic Pathway in Vicia faba

The apoplast of mature leaves excised from broadbean (Vicia faba L.) plants was infiltrated with 2 millimolar p-chloromercuribenzenesulfonic acid (PCMBS) via the transpiration stream, and the ability of the tissues to take up sugars was tested. An infiltration time of 75 minutes was sufficient to obtain a maximal (75%) inhibition of exogenous [¹⁴C]sucrose (1 millimolar) uptake. This infiltration affected neither CO₂ assimilation nor the transmembrane potential difference of leaf cells but strongly inhibited phloem loading of endogenous [¹⁴C] assimilates. The study of the symplastic relations between the different cell types of the mature leaf showed that the density of the plasmodesmata is generally very low in comparison with other species investigated so far, particularly when considering the mesophyll/bundle sheath and the bundle sheath/phloem cells connections, as well as the connections of the transfer cell-sieve tube complex with the surrounding cells. These three successive barriers therefore strongly limit the possibilities of symplastic transit of the assimilates to the conducting cells. The comparison of the densities of plasmodesmata in an importing and an exporting leaf suggests that the maturation of the leaf is characterized by a marked symplastic isolation of the phloem, and, within the phloem itself, by the isolation of the conducting complex. As a consequence, these physiological and cytological data demonstrate the apoplastic nature of loading in the mature leaf of Vicia faba, this species undoubtedly presenting a typical model for apoplastic loading.     

Selective Solubilization of Membrane Proteins Differentially Labeled by p-Chloromercuribenzenesulfonic Acid in the Presence of Sucrose

Broadbean (Vicia faba L.) leaf discs have been incubated with the slowly permeant thiol reagent [²⁰³Hg]-para-chloromercuribenzenesulfonic acid (PCMBS) in the presence or in the absence of sucrose, and the release of PCMBS-labeled proteins has been monitored in media containing various concentrations of urea, ethyleneglycol-bis-(β-aminoethyl ether)-N, N, N′, N′-tetraacetic acid (EGTA), sodium cholate, sodium dodecyl sulfate, Triton X-100, octylglucoside or (3-[3-cholamidopropyl)-dimethylammonio] 1-propane-sulfonate) (CHAPS). The proteins differentially labeled by PCMBS in the presence of sucrose which, on the basis of previous results, are assumed to include the sucrose carrier, were preferentially solubilized by 1% CHAPS, 1% octylglucoside, or 1% Triton X-100. Other PCMBS-labeled proteins (`background' proteins) could be partially removed by EGTA, urea, or 0.1% cholate. Sequential treatment by 10 mm EGTA and 1% CHAPS was found to give a fraction highly enriched in the differentially labeled proteins. Analysis of the specific activity of microsomal pellets suggests that the results obtained with leaf discs give a good account of what is occurring at the plasma membrane level. These data, which suggest that the proteins differentially labeled by PCMBS in the presence of sucrose are intrinsic membrane proteins, can be used to solubilize these proteins from microsomal fractions.     

Pathway of Phloem unloading of sucrose in corn roots

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

Sugar Efflux from Maize (Zea mays L.) Pedicel Tissue

Sugar release from the pedicel tissue of maize (Zea mays L.) kernels was studied by removing the distal portion of the kernel and the lower endosperm, followed by replacement of the endosperm with an agar solute trap. Sugars were unloaded into the apoplast of the pedicel and accumulated in the agar trap while the ear remained attached to the maize plant. The kinetics of ¹⁴C-assimilate movement into treated versus intact kernels were comparable. The rate of unloading declined with time, but sugar efflux from the pedicel continued for at least 6 hours and in most experiments the unloading rates approximated those necessary to support normal kernel growth rates. The unloading process was challenged with a variety of buffers, inhibitors, and solutes in order to characterize sugar unloading from this tissue.

Unloading was not affected by apoplastic pH or a variety of metabolic inhibitors. Although p-chloromercuribenzene sulfonic acid (PCMBS), a nonpenetrating sulfhydryl group reagent, did not affect sugar unloading, it effectively inhibited extracellular acid invertase. When the pedicel cups were pretreated with PCMBS, at least 60% of sugars unloaded from the pedicel could be identified as sucrose. Unloading was inhibited up to 70% by 10 millimolar CaCl₂. Unloading was stimulated by 15 millimolar ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid which partially reversed the inhibitory effects of Ca²⁺. Based on these results, we suggest that passive efflux of sucrose occurs from the maize pedicel symplast followed by extracellular hydrolysis to hexoses.

     

Sulfhydryl Group Involvement in Plasmalemma Transport of HCO(3) and OH in Chara corallina

The effect of the sulfhydryl reagents (—SH) p-chloromercuribenzene-sulfonic acid (PCMBS), N-ethylmaleimide (NEM), and inorganic mercury on H¹⁴CO₃⁻ assimilation in Chara corallina is reported. Commercial grade PCMBS caused severe inhibition of H¹⁴CO₃⁻ assimilation. Results obtained using purified PCMBS (stock solution passed through a chelating resin) indicated that inhibition observed using unpurified PCMBS was due predominantly to the presence of inorganic mercury (as a contaminant). The inhibitory role of inorganic mercury was verified using HgCl₂. This chemical caused a dramatic inhibition of H¹⁴CO₃⁻ assimilation, while it had little effect on cellular ¹⁴CO₂ fixation. Reversal of the Hg²⁺ inhibition of H¹⁴CO₃⁻ assimilation (in presence of 1.0 millimolar dithioerythritol) was extremely slow, requiring 2 to 3 hours for the reestablishment of control rates. This slow recovery may reflect de novo synthesis of transport proteins.     

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     

Effect of Exogenously Supplied Foliar Potassium on Phloem Loading in Beta vulgaris L

The effect of foliar application of K⁺ on processes associated with phloem loading was investigated in source leaves of sugar beet (Beta vulgaris L.). KCI was supplied exogenously at concentrations of up to 100 millimolar in the solution bathing the abraded upper epidermis of source leaves. K⁺ added at concentrations below 30 millimolar generally promoted the rate of export of material derived from ¹⁴CO₂ but not from exogenously applied [¹⁴C]sucrose. Paralleling promotion of export, the level of material derived from photosynthesis, which was released into the bathing solution, also increased in response to addition of K⁺ to the free space. Net photosynthetic rate was not affected. K⁺ at 5 and 15 millimolar concentrations did not stimulate uptake of [¹⁴C]sucrose into source leaf discs.     

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