NaCl-stimulated proton efflux and cell expansion in sugar-beet leaf discs

The expansion of illuminated sugar-beet leaf discs floating on aqueous solutions is stimulated by 10 mM NaCl. During expansion, protons are pumped out of the cell and NaCl increases this proton flux by about 40%. The nett flux of K⁺ and Na⁺ into the discs was also evaluated. During the expansion period K⁺ decreases while Na⁺ increases markedly. The results indicate the existence of a sodium-stimulated proton pump which is active during cell enlargement.Abbreviations IAA indole-3-acetic acid - PEG polyethylene glycol     

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     

Development of pH sensitivity of sucrose uptake during ageing of Vicia faba leaf discs

Uptake of [U-¹⁴C]-sucrose (40 m M ) by fresh and aged peeled leaf discs of broad bean ( Vicia faba L. cv. Aguadulce) has been studied. In fresh discs, uptake was nearly insensitive to external pH, whereas the pH response of absorption in discs aged for 12 h was bell-shaped, with an optimum between pH 5 and 6. At this pH, uptake was nearly twice that in fresh tissue. The passive (insensitive to carbonyl cyanide m -chlorophenylhydrazone and to cold treatment) uptake was the same in fresh or aged discs. The development of pH sensitivity of absorption did not appear when ageing was performed in the presence of 10^−H M cycloheximide or 5.7 × 10⁻⁵ M actinomycin D. Similarly, when the tissues were treated with 10⁻³ M spermidine for 2 h after excision and then aged for 10 h, the development of the pH-sensitive uptake system was inhibited. Ca²⁺ (10⁻² M ) supplied together with spermidine prevented the inhibiting effect of spermidine. The appearance of the pH-sensitive system was also markedly reduced if ageing took place in the presence of 10⁻³ M aminoethoxyvinylglycine. Autoradiographs from fresh discs and from discs aged with or without the inhibitors suggest that pH sensitivity developed more intensively in the parenchyma than in the veins.
The results suggest some caution when using excised leaf discs for studies on sucrose uptake and phloem loading. Development of pH sensitivity of uptake may require the synthesis of both DNA-dependent RNA and protein and could be related to ethylene metabolism.     

Dinitrophenol-induced efflux of sucrose from maize scutellum cells

Maize scutellum slices accumulated sucrose during incubation in glucose, fructose or sucrose. Sucrose was accumulated in two compartments, tentatively     

Bicarbonate effects in leaf discs from spinach

In this paper, we show the unique role of bicarbonate ion in stimulating the electron transfer of photosystem II (PS II) in formate-treated leaf discs from spinach. This is referred to as the bicarbonate effect and is independent of the role of CO₂ in CO₂ fixation. It is shown to have two sites of action:

Regulation of cholesterol efflux from macrophages

PURPOSE OF REVIEW: The lipid efflux pathway is important for both HDL formation and the reverse cholesterol transport pathway. This review is focused on recent findings on the mechanism of lipid efflux and its regulation, particularly in macrophages. RECENT FINDINGS: Significant progress has been made on understanding the sequence of events that accompany the interaction of apolipoproteins A-I with cell surface ATP-binding cassette transporter A1 and its subsequent lipidation. Continued research on the regulation of ATP-binding cassette transporter A1 and ATP-binding cassette transporter G1 expression and traffic has also generated new paradigms for the control of lipid efflux from macrophages and its contribution to reverse cholesterol transport. In addition, the mobilization of cholesteryl esters from lipid droplets represents a new step in the control of cholesterol efflux. SUMMARY: The synergy between lipid transporters is a work in progress, but its importance in reverse cholesterol transport is clear. The regulation of efflux implies both the regulation of relevant transporters and the cellular trafficking of cholesterol.     

Regulation of sucrose phosphate synthase by gibberellins in soybean and spinach plants

Exogenous applications of gibberellins (GAs) increased the extractable activity of leaf sucrose phosphate synthase (SPS) in soybean (Glycine max [L.]) and spinach (Spinacia oleracea [L.]). The response to GA applications was detectable within 2 h postapplication and was still observed 6 h, 24 h, and 7 d after treatment. When paclobutrazol, a GA biosynthesis inhibitor, was applied to intact soybean and spinach plants, decreased extractable SPS activity resulted within 24 h following the treatment. Different methods of GA application (spray, injection, capillary wick, and excised leaf systems) produced similar effects on SPS activity of soybean leaves. Protein synthesis in soybean leaves appeared to be necessary for GA-promoted SPS activity because gibberellic acid only partially reversed the inhibitory effect of pretreatment with cycloheximide. Levels of SPS protein from crude extracts of spinach plants were measured by a dot blot technique using monoclonal antibodies against SPS. Application of gibberellic acid to spinach leaves increased levels of SPS protein 2 h, 24 h, and 7 d after treatment. The results suggest that, in both soybean and spinach, GA is one of the endogenous hormonal factors that regulate the steady-state level of SPS protein and, hence, its activity.     

Emission isoprene from leaf discs of hamamelis

lsoprene has been reported and identified as a light dependent natural plant emission. GLC techniques are used to measure isoprene emission from leaf d     

Regulation of senescence in bean leaf discs by light and chemical growth regulators

The senescence of excised discs of primary leaves of Phaseolus vulgaris, L., var. Red Kidney was followed by measuring the net breakdown of protein and chlorophyll. The chemical growth regulators indoleacetic acid, 2,4-dichlorophenoxy-acetic acid, gibberellic acid, kinetin, and 6-benzylaminopurine were relatively ineffective in retarding senescence in this tissue. White light, on the other hand, was very effective in senescence retardation. The response to light did not have the characteristics of a low energy (phytochrome) response and was blocked by concentrations of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea which inhibited photosynthesis in the leaf discs. The light-induced retardation of senescence was concluded to be dependent on photosynthesis.     

Sucrose release from soybean leaf slices

The release of photosynthate from leaf slices of soybean [ Glycine max (L.) Merr. cv. Ransom II], to a bathing medium was studied to ascertain how p -chloromercuribenzenesulfonic acid (PCMBS) can both stimulate and inhibit sucrose release. Soybean leaf slices released photosynthate to a bathing medium at a rate that was approximately linear with time. The photosynthate released was about 20% ionic and 80% non-ionic, and sucrose represented about 75% of the total. Removal of Ca²⁺ from the medium increased the rate of release of all fractions, but amino acid release showed the largest increase. Sucrose was released at a rate estimated to be about 20% of the normal transport rate in intact leaves. The rate of sucrose uptake from 5 m M sucrose into soybean leaf slices was optimum at pH 6.3, and the rate of sucrose release was lowest at the same pH. However, sucrose uptake was found to be insignificant during release experiments. Sucrose release, but not amino acid release, was inhibited 75% by 1 m M PCMBS.
The data support two components of sucrose release in leaves. The first is insensitive to the addition of PCMBS. This component probably represents leakage from phloem tissue. The second component is inhibited by PCMBS and probably represents release from the mesophyll. By comparing sucrose release from leaf slices of 12 different species of plants, 2 groups were found. In the first group, sucrose release was inhibited between 60 and 80% by PCMBS, and in the second group between 0% and 40%. The difference in the two groups can be explained by a relative difference in the size of the two components of sucrose release for each species.     

Characterization of amino Acid efflux from isolated soybean cells

Cells from reproductive soybean (Glycine max [L.] Merr.) plants were isolated using a mechanical-enzymic technique that produced a high yield of uniform, physiologically active cells. Cells were incubated in a pH 6.0 buffered solution and subjected to various treatments in order to determine the nature of net amino acid efflux. Total net amino acid (ninhydrinreactive substances) efflux was not affected by the following conditions: (a) darkness, (b) aeration, (c) K⁺ concentrations of 0.1, 1.0, 10, or 100 millimolar and (d) pH 4, 5, 6, 7, or 8. The Q₁₀ for net amino acid efflux between 10°C and 30°C was 1.6. Thus, it seems that net amino acid efflux requires neither current photosynthetic energy nor a pH/ion concentration gradient. Amino acid analyses of the intra-and extracellular fractions over time showed that each amino acid was exported linearly for at least 210 minutes, but that export rate was not necessarily related to internal amino acid pools. Amino acids that were exported fastest were alanine, lysine, leucine, and glycine. Addition of the inhibitor p-chloromercuriphenyl sulfonic acid, 3(3,4-dichlorophenyl)-1,1-dimethylurea, or carbonylcyanide p-trifluoromethoxyphenylhydrazone increased the rate of total amino acid efflux but had specific effects on the efflux of certain amino acids. For example, p-chloromercuriphenyl sulfonic acid greatly enhanced efflux of γ-aminobutyric acid, which is not normally exported rapidly even though a high concentration normally exists within cells. The data suggest that net amino acid efflux is a selective diffusional process. Because net efflux is the result of simultaneous efflux and influx, we propose that efflux is a facilitated diffusion process whereas influx involves energy-dependent carrier proteins.     

Direct creation of marker-free tobacco plants from agroinfiltrated leaf discs

Agroinfiltration is employed as a fast way to directly create marker-free transgenic tobacco plants. As an example for the efficiency of the method, Agrobacterium cells harboring a marker-free vector coding for β-glucuronidase (GUS) were infiltrated into the leaf discs of Nicotiana tabacum, which were then used as explants for marker-free plant regeneration by tissue culture. Through GUS staining, a large number of small calli were shown to be stably transformed on the treated leaf discs at 17 days after agroinfiltration. Most importantly, after continuous culture of the leaf discs until shoot regeneration, about 15% of the regenerants were proven to be transformants by polymerase chain reaction (PCR) analysis.     

Evidence on the mechanism of enhanced sucrose uptake at low cell turgor in leaf discs of Phaseolus coccinius

Plants often tolerate water deficits by lowering the osmotic potential of their cell sap. This may be achieved by accumulation of solutes which results in the maintenance of a positive turgor potential. In this study, the effect of water deficit on sugar uptake was investigated in leaf discs of Phaseolus coccinius L. (cv. Scarlet). Evidence is presented that cell turgor affects the kinetics of sugar transport at the membrane level. Uptake kinetics of sucrose, glucose and 3-O-methyl glucose by tissues equilibrated in solutions of relatively high (200–400 mOsm) osmotic concentration consisted of a sat-urable and a linear component. Low external osmotic concentration i.e., high cellular turgor inhibited the saturating component of sucrose uptake, resulting in a linear uptake profile. However, high cell turgor had no effect on glucose or 3-O-methyl glucose uptake kinetics. The effect of turgor versus osmotic component of water potential was differentiated by comparing responses to non-penetrating (manmtol) or polyethylene glycol, (3350) and penetrating (ethylene glycal) osmotica. Changes in sucrose uptake rates and kinetics were due to changes in cellular turgor and not osmotic potential. Furthermore, at low cellular turgor, a net increase in sucrose uptake occurred as a consequence of enhanced influx rates and not as a result of reduced efflux rates. The data are consistent with previous findings that sugar uptake rates are enhanced under low turgor. We present first evidence indicating that the mechanism by which higher rates of sucrose uptake are maintained underwater deficit conditions is by the activation of the saturable transport system. This mechanism supports previous suggestions that changes in cell turgor are sensed and manifested at the membrane level.     

Linear sucrose transport in protoplasts from developing soybean cotyledons

Previous studies with isolated soybean cotyledon protoplasts revealed the presence of a saturable, simple diffusion, and nonsaturating carrier-mediated uptake of sucrose into soybean cotyledon cells. A proton/sucrose cotransport may be involved in the saturable sucrose uptake (Lin et al. 1984 Plant Physiol 75: 936-940 and Schmitt et al. 1984 Plant Physiol 75: 941-946). In this study, we investigated the linear sucrose uptake mechanism by treating isolated protoplasts with 15 micromolar p-trifluoromethoxy-carbonylcyanide phenylhydrazone (FCCP) or 100 micromolar p-chloromecuribenzenesulfonic acid to eliminate the saturable uptake. We found: (a) increasing external pH decreases the linear sucrose uptake; (b) fusicoccin at 20 micromolar stimulates and FCCP at 15 micromolar inhibits this linear sucrose uptake; and (c) the ratio of the initial influx of proton to sucrose is close to one in both saturable and nondiffusive linear (difference between the total linear and diffusive components) uptakes. The results suggest that a proton/sucrose cotransport is also involved in the nondiffusive linear sucrose uptake into soybean cotyledon cells.     

Light-promoted diffusional amino acid efflux from Commelina leaf disks

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

Energetics of sucrose transport into protoplasts from developing soybean cotyledons

The accumulation of tetraphenylphosphonium (TPP⁺), 5,5′-dimethyl-oxazolidine-2,4-dione (DMO), and a micro pH electrode were used to measure membrane potential, intracellular and extracellular pH, respectively, upon the addition of exogenous sucrose to soybean cotyledon protoplasts. Addition of sucrose caused a specific and transient (a) depolarization of the membrane potential (measured by TPP⁺ accumulation), (b) acidification of the intracellular pH (measured by DMO accumulation), and (c) alkalization of the external medium (measured by a micro pH electrode). The time course for all these changes was similar (i.e. 5 to 10 minutes). Based on the rate of sucrose uptake and alkalization of the external medium, a stoichiometry of 1.02 to 1.10 for proton to sucrose was estimated. These data strongly support a proton/sucrose cotransporting mechanism in soybean cotyledon cells.     

A sucrose binding protein homologue from soybean affects sucrose uptake in suspension-cultured transgenic tobacco cells

We have obtained Nicotiana tabacum transgenic cell lines expressing a sucrose binding protein (sbp) homologue gene from soybean (Glycine max L.), designated s-64, either in the sense or antisense orientation. Sense cell lines over-accumulated the S-64 protein, whereas the antisense cell lines had reduced levels of the endogenous homologue protein. Sucrose uptake experiments were conducted by incubating suspension-cultured tobacco cells with radiolabeled sucrose at pH 4.5 or 7.0. Raising the extracellular pH to 7.0 caused an inhibition of radiolabeled carbon uptake efficiency, which was attributed to the pH-sensitivity of cell-wall invertase (EC 3.2.1.26), H⁺/hexose transporter and/or H⁺/sucrose symporter activities. Because SBP-mediated sucrose uptake has been shown to be insensitive to extracellular pH in yeast, we performed the sucrose uptake experiments in sense and antisense cultured cells at pH 7.0. Under this condition, the level of SBP homologue correlated with the efficiency of radiolabeled uptake by the transgenic tobacco cells. Furthermore, manipulation of S-64 levels altered sucrose-cleaving activities in a metabolic compensatory manner. Enhanced accumulation of S-64 caused an increase in intracellular sucrose synthase (cleavage, EC 2.4.1.13) activity with a concomitant decline in cell-wall invertase activity. This result may reflect a metabolic adjustment of the sense cell lines caused by its high efficiency of direct sucrose uptake as disaccharide. In contrast, the level of cell-wall invertase activity was remarkably increased in antisense cells, favoring the invertase-dependent sugar uptake system. Collectively, these results may establish a functional link between radiolabeled influx and S-64 accumulation, suggesting that SBP affects sucrose uptake in suspension-cultured cells.     

Development of plants from leaf discs of variegatedColeus and its relation to patterns of leaf chlorosis

Summary Leaf discs approximately 8 mm in diameter taken from green and from chlorotic areas of variegated leaves ofColeus were grown in light under sterile conditions in a mineral salt, sucrose, vitamin medium supplemented with auxin and cytokinin. Green shoots, which later formed roots, grew from both green and chlorotic discs in media containing suitable amounts of auxin and cytokinin. None developed in media supplemented with auxin alone or with cytokinin alone. Discs with young plants were transferred to soil. Plants that grew varied widely from those with no chlorosis to those with more chlorosis than the original variety from which the discs were taken. Plants grown from discs taken from green areas of leaves with chlorosis varied in patterns of chlorosis as much as those that grew from discs from chlorotic areas of leaves. This research was supported, in part, by The Conservation and Research Foundation.     

Regulation of calcium efflux from isolated rat parotid cells

Calcium efflux from isolated rat parotid acinar cells was studied with 45Ca. Carbachol, phenylephrine, substance P, monobutyryl cyclic AMP and isoproterenol stimulated 45Ca efflux. It is suggested that carbachol, phenylephrine and substance P mobilize the same pool of cellular Ca. This suggestion is based on two observations. Firstly, combinations of any two of these three agonists at saturating concentrations result in no more 45Ca efflux than either agonist alone. Secondly, stimulation of 45Ca efflux by any one of the three agonists prevents further stimulation of 45Ca efflux by the same or one of the other two agonists. The pool of calcium mobilized by isoproterenol or monobutyryl cyclic AMP is different from the pool mobilized by carbachol. This conclusion is based on the observation that stimulation of 45Ca efflux by a saturating concentration of carbachol did not inhibit stimulation of 45Ca efflux by isoproterenol. Furthermore the effect of a saturating concentration of isoproterenol on 45Ca efflux is additive with that caused by a saturating concentration of carbachol. The effect of carbachol, phenylephrine and substance P on 45Ca2+ efflux did not require extracellular Ca2+.