Differential effects of turgor on sucrose and potassium transport at the tonoplast and plasma membrane of sugar beet storage root tissue

When turgor was increased, by decreasing the concentration of mannitol bathing discs of sugar beet storage root tissue, the rates of sucrose and potassium uptake into the vacuole were decreased. At all external mannitol concentrations the rate of sucrose and potassium uptake across the plasma membrane was an order of magnitude greater than the rate of quasi-steady uptake into the vacuole, implying a very large efflux. Efflux of both sucrose and potassium was increased at high turgor. However, while increasing turgor decreased the rate of K⁺ uptake, the rate of sucrose uptake at the plasma membrane increased with time. Compartmental analysis of tracer exchange kinetics was used to determine unidirectional K⁺ fluxes. From these results, it was estimated that the increase in K⁺ efflux accompanying a 1.5 MPa increase in turgor could lead to a net increase of 140mol^?3h^?1 in the external potassium concentration. It is suggested that the turgor-imposed increase in solute efflux is a means of regulating intracellular osmotic pressure and/or turgor in sugar beet storage roots, but that sucrose is preferentially retrieved from the apoplast, even under conditions of excessively high turgor. However, much of this sucrose is probably lost from the cell, implying a ‘futile’ sucrose transport cycle at the plasma membrane. The turgor-stimulated leak of potassium could play a major role in the regulation of turgor pressure in sugar beet storage root tissue.     

Phloem loading of sucrose: involvement of membrane ATPase and proton transport

p-Chloromercuribenzenesulfonic acid markedly inhibited sucrose accumulation into sugar beet source leaves without inhibiting hexose accumulation. The site of inhibition is proposed to be the plasmalemma ATPase, since the ATPase-mediated H⁺ efflux was completely inhibited by p-chloromercuribenzenesulfonic acid under conditions where intracellular metabolism, as measured by photosynthesis and hexose accumulation, was unaffected. Fusicoccin, a potent activator of active H⁺/K⁺ exchange, stimulated both active sucrose accumulation and proton efflux in the sugar beet leaf tissue. These data provide strong evidence for the phloem loading of sucrose being coupled to a proton transport mechanism driven by a vectorial plasmalemma ATPase.     

Sucrose Efflux from the Maize Scutellum

Sucrose efflux from maize scutellum slices was promoted by high pH and by K+, Na+ or Rb+. Incubation in mannose (which drastically reduces the ATP level) caused high rates of sucrose efflux only when KCl was present at pH 8. The effects of triphenylmethylphosphonium ion (TPMP+, a lipid soluble cation) on sucrose efflux were similar to those of mannose plus KCl. Mannose and TPMP+ caused release of stored sucrose into the cytoplasm, but pH8 and KCl (mannose) or pH 8 (TPMP+) in the bathing solution were necessary for rapid efflux of sucrose. Rb⁺ uptake took place during sucrose efflux. In mannose, rates of Rb⁺ uptake and sucrose efflux were low at pH 5.6 and high at pH 8.0, although the time courses for uptake and efflux were different. It is concluded that sucrose efflux is electrogenic and that it occurs as sucrose-H⁺ symport. A scheme for sucrose transport across plasmalemma and tonoplast is presented.     

Nature of the light-induced h efflux and na uptake in cyanobacteria

We investigated the nature of the light-induced, sodium-dependent acidification of the medium and the uptake of sodium by Synechococcus. The rate of acidification (net H⁺ efflux) was strongly and specifically stimulated by sodium. The rates of acidification and sodium uptake were strongly affected by the pH of the medium; the optimal pH for both processes being in the alkaline pH range. Net proton efflux was severely inhibited by inhibitors of adenosine triphosphatase activity, energy transfer, and photosynthetic electron transport, but was not affected by the presence of inorganic carbon (C_i). Light and C_i stimulated the uptake of sodium, but the stimulation by C_i was observed only when C_i was present at the time sodium was provided. Amiloride, a potent inhibitor of Na⁺/H⁺ antiport and Na⁺ channels, stimulated the rate of acidification but inhibited the rate of sodium uptake. It is suggested that acidification might stem from the activity of a light dependent proton excreting adenosine triphosphatase, while sodium transport seems to be mediated by both Na⁺/H⁺ antiport and Na⁺ uniport.     

The mechanism of sugar uptake by sugarcane suspension cells

Sugarcane cell suspensions took up sugar from the medium at rates comparable to or greater than sugarcane tissue slices or plants in the field. This system offers an opportunity for the study of kinetic and energetic mechanisms of sugar transport in storage parenchyma-like cells in the absence of heterogeneity introduced by tissues. The following results were obtained: (a) The sugar uptake system was specific for hexoses; as previously proposed, sucrose was hydrolyzed by an extracellular invertase before the sugar moieties were taken up; no evidence for multiple sugar uptake systems was obtained. — (b) Uptake of the glucose-analog 3-O-methylglucose (3-OMG) reached a plateau value with an intracellular concentration higher than in the medium (approximately 15-fold). — (c) There was a balance of influx and efflux during steady state; the rate of exchange influx was lower than the rate of net influx; the K_m value was higher (70 M) than for net influx (24 M); the exchange efflux is proposed to be mediated by the same transport system with a K_m value of approximately 2.6 mM for internal 3-OMG; the rate of net efflux of hexoses was less than a third of the rate of exchange efflux. — (d) The uptake of hexoses proceeded as proton-symport with a stoichiometry of 0.87 H⁺ per sugar; during the onset of hexose transport there was a K⁺ exit of 0.94 K⁺ per sugar for charge compensation. (It was assumed that the real stoichiometries are 1 H⁺ and 1 K⁺ per sugar.) The K_m values for sugar transport and sugar-induced proton uptake were identical. Sucrose induced proton uptake only in the presence of cell wall invertase. — (e) There was no net proton uptake with 3-OMG by cells which were preloaded with glucose though there was significant sugar uptake. It is assumed, therefore, that the exit of hexose occurs together with protons. — (f) The protonmotive potential of sugarcane cells corresponded to about 120 mV: pH-gradient 1.1 units, membrane potential of-60 mV (these values increased if vacuolar pH and membrane potential were also considered). It was abolished by uncouplers, and the magnitude of the components depended on the external pH value. We present evidence for the operation of a proton-coupled sugar transport system in cell suspensions that were derived from, and have characteristics of, storage parenchyma. The quantitative rates of sugar transport suggest that the role of this transport system is not limiting for sugar storage.Abbreviations 3-OMG 3-O methylglucose - DMO 5,5-dimethyl-2, 4-oxazolidinedione - TPP tetraphenylphosphonium chloride - CCCP carbonyl cyanide, m-chlorophenylhydrozane     

Accumulation and conversion of sugars by developing wheat grains. 3. Non-diffusional uptake of sucrose, the substrate preferred by endosperm slices

Abstract. Starch synthesis by developing wheat endosperm slices incubated in liquid media was more rapid, at optimum concentration, from sucrose as external substrate than from glucose and/or fructose. Fructose inhibited conversion of sucrose or glucose. The results are consistent with the hypothesis that sucrose is not hydrolysed in the apoplast before uptake.
Besides a diffusional influx and efflux of labelled sucrose there was a non-diffusional influx; it was inhibited by dinitrophenol, potassium arsenate, potassium iodide, and parachloromercuribenzene sulphonate (PCMBS). PCMBS inhibited both uptake and conversion of label from 150 molm⁻³¹⁴C-sucrose by 75%. Uptake and conversion of sucrose were stimulated by lowering pH and by fusicoccin, a promoter of proton extrusion.
Extracellular solutes like raffinosc and polyethylene glycol stimulated net uptake of label from ¹⁴C-sucrose — the larger molecule being more effective — this being due to a non-specific inhibition of diffusional efflux. At too high an osmotic concentration such solutes reduced net uptake; the larger the molecule the lower this transitional concentration.
In conclusion, wheat endosperm is better equipped to convert apoplastic sucrose rather than the hydrolysis products to starch; active loading of sucrose possibly involves proton co-transport; and large molecules in the extracellular solution reduce the diffusional elllux of loaded substrate.     

Sucrose efflux and export from the maize scutellum*

Abstract During incubation of maize scutellum slices in fructose, there was an efflux of sucrose. Efflux was constant for at least 4 h at fructose concentrations of 70 or 100 mol m^?3. Efflux was increased by EDTA, and decreased by Ca²⁺. Efflux was independent of pH after EDTA treatment, but increased from untreated slices when the pH was lowered from 7 to 4. Uranyl ion and PCMBS (p-chloro-mercuribenzenesulfonic acid) abolished sucrose uptake, but were only weak inhibitors of sucrose efflux. These results are consistent with efflux occurring by simple diffusion through aqueous pores, but they do not rule out facilitated diffusion. Rates of sucrose export from the scutellum to the root shoot axis were estimated from measurements of axis respiration and dry weight gain. Sucrose efflux from scutellum slices was only 14-22% of the export rate. Sucrose efflux from the whole scutellum was only 3-4% of the export rate. It is concluded that the observed efflux is from leaky cells and does not represent sucrose on the way to the phloem along a path that includes the apoplast. These results support the idea that the path for sucrose from parenchyma cell to sieve tube in the maize scutellum is entirely symplastic.     

Uptake of sucrose by Saccharomyces cerevisiae

Sucrose transport has been shown to occur in several Suc^? and Suc⁺Saccharomyces cerevisiae strains as an energy-dependent process. Assay conditions have been established to avoid both extra- and intracellular hydrolysis of the disaccharide thus allowing the identification of sucrose as such inside the cell immediately after the uptake; acid pH values (4.0–5.0) were optimal for transport although significant uptake was also detected at neutral pH. Transport of sucrose was not dependent on ATP and seemed to be driven by protonmotive force supplied by the electrochemical gradient of protons across the plasma membrane. The actual symport of protons along with sucrose was directly detected by continuous pH measurement of the reaction mixtures and the initial rate of proton movement in the symport process was determined. KC1 inhibited transport of sucrose suggesting that exit of K⁺ ions might well be involved in maintaining the electroneutrality of the process. On the other hand, NaCl stimulated transport by 50% in our experimental conditions. The specificity of sucrose transport was also tested using different disaccharides.     

ATP-induced sucrose efflux from red-beet tonoplast vesicles

 Sucrose efflux from the vacuole of mobilizing red-beet (Beta vulgaris L.) hypocotyl cells was investigated using purified tonoplast vesicles. Tonoplast vesicle purity was assured by the immunoreactivity to antibodies raised against the vacuolar ATPase and by the strong inhibition exhibited by the H⁺-ATPase to bafilomycin-A and NO₃ ⁻. Inhibition of the H⁺-ATPase by vanadate and azide was negligible. Sucrose was loaded into tonoplast vesicles by using the pH-jump method of energization. Addition of ATP to sucrose-loaded vesicles in the presence of bafilomycin-A resulted in efflux of a significant amount of sucrose. During ATP-induced sucrose efflux, bafilomycin-insensitive ATPase activity increased significantly with no increase in H⁺-translocating activity. The additional bafilomycin-A insensitive ATPase activity observed in sucrose-loaded vesicles was completely inhibited by vanadate as was the efflux of sucrose. Similar to vanadate, thapsigargin was also inhibitory to sucrose efflux and to the bafilomycin-A insensitive ATPase activity. The data indicate that vacuolar sucrose can be actively mobilized by a specific ATP-dependent efflux mechanism. Received: 12 October 1999 / Accepted: 18 November 1999     

Effect of plant hormones on sucrose uptake by sugar beet root tissue discs

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

Dissociation of H + extrusion from K+ uptake by means of lipophilic cations

Abstract Dissociation of active H⁺ extrusion (?ΔH⁺) from K⁺ uptake in pea and maize root segments was attempted by substituting K⁺ in the incubation medium with lipophilic cations assumed to enter the cell by passive, non-specific, permeation through the lipid component of the plasmalemma. Among the compounds tested, tributylbenzylammonium significantly stimulated ?ΔH⁺ in the absence of other monovalent cations in the medium. This effect was much more evident when the experiment was carried out in the presence of fusicoccin, which strongly stimulates proton extrusion and monovalent cation uptake, and hyperpolarizes the trans-membrane electric potential in these materials. Also the lipophilic cations tetraphenylphosphonium, dimethyldibenzylammonium and hexylguanidine markedly stimulated FC-promoted ?ΔH⁺. Octylguanidine at a low concentration induced an early stimulation followed by a strong inhibition of ?ΔH⁺. A complete lack of additivity was observed between the effects of lipophilic cations and that of K⁺ on H⁺ extrusion. Lipophilic cations severely inhibited K⁺ uptake. These data are interpreted as supporting the view of an electric, rather than a chemical, (namely, involving the same carrier system) nature of the coupling of active H⁺ extrusion with K⁺ influx.     

Effect of root environment on proton efflux in wheat roots

Proton net efflux of wheat (Triticum aestivum L.) roots growing in sand culture or hydroponics was determined by measuring the pH values of the solution surrounding the roots by pH microelectrodes, by base titration and by color changes of a pH indicator in solid nutrient media. The proton net efflux was dependent on light, aeration, and source of nitrogen (NH ₄ ⁺ , NO ₃ ^? ). Ammonium ions caused the highest proton efflux, whereas nitrate ions decreased the proton efflux. Iron deficiency had no significant effect on proton efflux. Replacement of ammonium by nitrate inhibited proton efflux, whereas the reverse enhanced proton extrusion. A lag period between changes in plant environment and proton efflux was observed. The proton net efflux occurred at the basal portion of the roots but not in the root tips or at the elongation zone. Under optimal conditions, proton efflux capacity reached a maximum value of 5.7 μmole H⁺ g^?1 fresh weight h^?1 with an average (between different measurements) of 3.4 μmole H⁺ g^?1 fresh wth^?1 whereas the pH value decreased to 3.2–3.7 and reached a minimal value of 2.9. Inhibition of ATPase activity by orthovanadate inhibited proton efflux. The results indicate that proton efflux in wheat roots is ammonium ion and light dependent and probably governed by ATPase activity.     

A model for sucrose transport in the maize scutellum

A model originally developed for transport of neutral substrates in bacterial systems was tested for its suitability for depicting sucrose transport across the plasmalemma of the maize scutellum cell. The model contains a sucrose—proton symporter, a negatively-charged free carrier and a neutral sucrose—proton—carrier complex. Sucrose transport is driven by the sucrose gradient and by a proton electrochemical gradient set up by a proton-translocating ATPase. The results of experiments on sucrose uptake in scutellum slices are in accord with predictions based on the model. Evidence was obtained for an electrogenic proton pump in the plasmalemma, for sucrose—proton symport and for a sucrose transport mechanism driven by both electrical potential and pH gradients. It was found that treatments (dinitrophenol, N-ethylmaleimide or HCl) causing a net proton influx into the slices also caused an efflux of sucrose. Interpretations of these results compatible with the model are given.     

Charge and acidity compensation during proton-sugar symport in Chlorella: The H+-ATPase does not fully compensate for the sugar-coupled proton influx

This study was undertaken in order to demonstrate the extent to which the activity of the plasmalemma H⁺-ATPase compensates for the charge and acidity flow caused by the sugar-proton symport in cells of chlorella vulgaris Beij.. Detailed analysis of H⁺ and K⁺ fluxes from and into the medium together with measurements of respiration, cytoplasmic pH, and cellular ATP-levels indicate three consecutive phases after the onset of H⁺ symport. Phase 1 occurred immediately after addition of sugar, with an uptake of H⁺ by the hexoseproton symport and charge compensation by K⁺ loss from the cells and, to a smaller degree, by loss of another ion, probably a divalent cation. This phase coincided with strong membrane depolarization. Phase 2 started approximately 5 s after addition of sugar, when the acceleration of the H⁺-ATPase caused a slow-down of the K⁺ efflux, a decrease in the cellular ATP level and an increase in respiration. The increased respiration was most probably responsible for a pronounced net acidification of the medium. This phase was inhibited in deuterium oxide. In phase 3, finally, a slow rate of net H⁺ uptake and K⁺ loss was established for several further minutes, together with a slight depolarization of the membrane. There was hardly any pH change in the cytoplasm, because the cytoplasmic buffering capacity was high enough to stabilize the pH for several minutes despite the net H⁺ fluxes. The quantitative participation of the several phases of H⁺ and K⁺ flow depended on the pH of the medium, the ambient Ca²⁺ concentration, and the metabolic fate of the transported sugar. The results indicate that the activity of the H⁺-ATPase never fully compensated for H⁺ uptake by the sugar-symport system, because at least 10% of symport-caused charge inflow was compensated for by K⁺ efflux. The restoration of pH in the cytoplasm and in the medium was probably achieved by metabolic reactions connected to increased glycolysis and respiration.Abbreviations DMO dimethyloxazolidinedione - EDTA ethylcnediaminetetraacetic acid - p.c. packed cell volume     

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.     

A model for proton and potassium co-transport during the uptake of glutamine and sucrose by tomato internode disks

Internode disks of tomato (Lycopersicon esculentum cv. Moneymaker) were shaken in glutamine and sucrose solutions. At low external pH (<±5.5), the uptake of these substances was accompanied with K⁺ efflux, at high pH (>±5.5) with K⁺ influx. Low concentrations of external K⁺ (2 mmol l^-1) stimulated the uptake of glutamine, which was strongly inhibited by the supply of high K⁺ concentrations (20 mmol l^-1). The effect of K⁺ was particularly pronounced at high pH-values. Addition of CCCP in light reduced the uptake of glutamine to the same level as in the dark, and stopped the K⁺ fluxes which coincided with the uptake. A model is presented wherein the movements of K⁺ across the membrane are related to co-transport, depending on the membrane potential and the Nernst potential of K⁺.Abbreviation CCCP carbonylcyanide-m-chlorophenylhydrazone     

Protonation and light synergistically convert plasmalemma sugar carrier system in mesophyll protoplasts to its fully activated form

The course of sugar fluxes into and out of protoplasts isolated from the mesophyll of Pisum sativum L. has been followed over brief time intervals (minutes). Light strongly stimulated net sugar influx at pH 8 as well as at pH 5.5. The proton conductor carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited initial influx in the light, both at pH 8.0 and at pH 5.5. CCCP was without effect in the dark at either pH. All these results applied both to sucrose and to the nonmetabolizable glucose analog 3-O-methyl-d-glucose.When protoplasts at pH 5.5 were transferred from light to darkness, "stored" light driving force maintained uptake in the dark at the full light rate for the first 7 minutes. At pH 8, however, even 4 minutes after transfer to dark, uptake was well below the light rate. Initial uptake rates over a range of external concentrations were derived from progress curves obtained in the light and in the dark, both at pH 5.5 and at 7.7. When initial rate was plotted against concentration, simple Michaelis-Menten kinetics were observed only under the condition pH 5.5, light. In the dark at both pH values, and in the light at pH 7.7, complex curves with intermediate plateaus were obtained, strongly resembling curves reported for systems where mixed negative and positive cooperativity is operating.The same "K(m) for protons" was observed in the dark and in the light (10(-7) molar). Switching protoplasts in the dark from pH 8 to 5.5 failed to drive sugar transport by imposed protonmotive force, as judged by lack of sensitivity to CCCP. Switching protoplasts which had taken up sugar in the dark at pH 5.5 to pH 7 induced net efflux of sugar. Flux analysis showed that this effect was entirely due to the prompt fall in influx.It is concluded from the kinetic experiments that protonation alone is not sufficient to convert the sugar transport system to its fully activated high affinity form. A further light-dependent factor which acts synergistically with protonation is required.     

Sucrose transport into vacuoles isolated from barley mesophyll protoplasts

Sucrose transport has been investigated in vacuoles isolated from barley mesophyll protoplasts. Rates of sucrose transfer across the tonoplast were even higher in vitro than in vivo indicating that the sucrose transport system had not suffered damage during isolation of the vacuoles. Sucrose transport is carrier-mediated as shown by substrate saturation of transport and sensitivity to a metabolic inhibitor and to competitive substrates. A number of sugars, in particular maltose and raffinose, decreased uptake of sucrose. Sorbitol was slowly taken up but had no effect on sucrose transport. The SH-reagent p-chloromercuribenzene sulfonate inhibited sucrose uptake completely. The apparent K_m of the carrier for sucrose uptake was 21 mM. Transport was neither influenced by ATP and pyrophosphate, with or without Mg²⁺ present, nor by protonophores and valinomycin (with K⁺ present). Apparently uptake was not energy dependent. Efflux experiments with preloaded vacuoles indicated that sucrose unloading from the isolated vavuoles is mediated by the same carrier which catalyses uptake. The vacuole of mesophyll cells appears to represent an intermediary storage compartment. Uptake of photosynthetic products into the vacuole during the light apparently minimizes osmotic swelling of the small cytosolic compartment of vacuolated leaf cells when photosynthetic productivity exceeds the capacity of the phloem for translocation of sugars.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazincethane-sulfonic acid - pCMBS p-chloromercuribenzene sulfonate Dedicated to Professor Dr. W. Simonis on the occasion of his 75th birthday     

Characterization of solute transport in plasma membrane vesicles isolated from cotyledons ofRicinus communis L.

Evidence is presented for the proton-coupled transport of sucrose and glutamine in purified plasma membrane vesicles isolated from cotyledons ofRicinus communis. Imposition of a pH gradient (internal alkaline) across the plasma membrane resulted in a rapid uptake of sucrose and glutamine which was inhibited in the presence of carbonyl cyanide-m-chlorophenyl hydrazone. Imposition of a pH gradient plus an internal negative membrane potential stimulated uptake further. Glucose and fructose uptakes were negligible under these conditions. Sucrose uptake into the vesicles demonstrated saturation kinetics with a K_m of 0.87 mol·m^-3, indicating carrier-mediated transport. In support of this, uptake was very sensitive to the protein-modifying reagentp-chloromercuribenzenesulphonic acid. N-Ethylmaleimide, another sulphydryl reagent, was only slightly inhibitory. However, both reagents strongly inhibited sucrose uptake into intact cotyledons; the possible reasons for the difference between the intact and isolated systems are assessed. The value of this system for the study of sucrose and amino acid carriers is discussed.     

H Cotransports in Corn Roots as Related to the Surface pH Shift Induced by Active H Excretion

The surface pH shift induced by active H⁺ excretion in corn (Zea mays L.) roots was estimated using acetic acid influx as a pH probe (H Sentenac, C Grignon 1987 Plant Physiol 84: 1367-1372). At constant bulk pH, buffering the medium strongly reduced the magnitude of the surface pH shift. This was used to study the effect of surface pH shift on H⁺ cotransports. In the absence of buffers, the surface pH shift increased with the bulk pH. Buffers decreased ³²Pi influx and this effect was stronger at pH 7.2 than at pH 5.8, and stronger in the absence than in the presence of an inhibitor of the proton pump (vanadate). Buffers exerted a similar depressive and pH-dependent effect on net NO₃⁻ uptake. They hyperpolarized the cell membrane, and stimulated ⁸⁶Rb⁺ influx, K⁺:H⁺ net exchange, and malate accumulation. These results are consistent with the hypothesis that H⁺ accumulation at the cell surface is effective in driving H⁺ reentry. We concluded that the surface pH shift due to proton pump activity is involved in the energetic coupling of H⁺ cotransports.