Regulation of Amino Acid Uptake into Oat Mesophyll Cells: A Comparison between Protoplasts and Leaf Segments

Rubinstein, Benjamin I. P.; Tattar, Terry A.

doi:10.1093/jxb/31.1.269

Cited by 38 publications

(32 citation statements)

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“…3B) In all systems, many structural and biochemical changes occur during the transition from cell to protoplast (reviewed in Galun [6] Pulvinar protoplasts from Samanea extrude HI in response to FC slightly more rapidly than corn root protoplasts (12), but only about one-third as fast as leaf protoplasts from oat (19), tobacco (18), or spinach (18). In the experiments presented here, however, acidification was allowed to continue for 100 mn before the final aliquot was measured in order to obtain a large pH change.…”

Section: Resultsmentioning

confidence: 99%

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Extensor and Flexor Protoplasts from Samanea Pulvini

Gorton

Satter²

1984

Plant Physiol.

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Section: Resultsmentioning

confidence: 99%

“…Although protoplasts undergo severe stresses during isolation, in many systems they retain physiological responses observed in intact cells (9,12,15,19). Protoplasts from stomatal guard cells, for example, swell in response to light (24) and shrink in response to ABA (23) Solutions.…”

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confidence: 99%

Extensor and Flexor Protoplasts from Samanea Pulvini

Gorton

Satter²

1984

Plant Physiol.

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“…The exposed mesophyll cells appear intact and uninjured (Fig. ID) (22) and internal pH (10), the excretion of H+ by control and FC-treated segments ( Fig. 2A) Attempts were made to determine the effects of osmotic shock on uptake of H+ by floating peeled leaf segments on a pH below their equilibrium value and monitoring the resulting increase in pH (Fig.…”

Section: Resultsmentioning

confidence: 99%

Regulation of H⁺ Excretion

Rubinstein¹

1982

Plant Physiol.

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Osmotic shock, a 15-minute plasmolysis followed by a 15-minute rehydration in the cold, is a nondestructive technique which inhibits fusicoccinstimulated H+ excretion from oat mesophyll cells (Avena sativa L.). Osmotic shock also causes a loss of intracellular solutes and stimulates H+ uptake, but osmoregulation can still occur, and enhanced H+ uptake is observed only at low external pH. It Cl-CCP, carbonylcyanide, m-chlorophenyl-hydrazone.(Econowatt, Westinghouse, Corp.) and incandescent light at 145 M-2 One-quarter strength Hoagland solution was applied at time of sowing and on a daily basis after 4 d. The primary leaves were harvested 7 d after sowing, the lower (abaxial) epidermis was peeled away, and one or two 1-cm segments were cut approximately 1 cm below the tip.Segments to be osmotically shocked were first plasmolyzed on 0.6 M sorbitol, 29 mm sucrose, 5 mm Hepes (pH 7.0) for 15 min. This solution was then aspirated, the tissue moved to an ice bucket, and cold 5 mm Hepes (pH 7.0) was added. After rehydration for 15 min, the sections were transferred to an appropriate medium for estimates of H+ flux. Unshocked sections were treated identically except that the sorbitol plus sucrose was omitted from the Hepes buffer during the first 15-min treatment. Sucrose alone is without effect, but was included for future comparisons with protoplasts which are suspended in media containing sucrose.The presumptive net efflux of H+ was measured by floating five 1-cm peeled leaf segments on 2.5 ml of 1 mm Hepes (pH 6.5) plus 1 mm KCI in 20-ml beakers. To determine the alkalinization of the medium, segments were floated on Hepes-KCl adjusted to pH 4.8 with HCI. For both types of measurements, the beakers were shaken continuously at ambient light and temperature, and the pH of the medium was determined at intervals with a Model 26 Radiometer pH meter equipped with a Radiometer combination electrode. Data were converted to H+ equivalents with the aid of a titration curve for the solution used. All treatments were run in duplicate and experiments repeated on at least 2 separate d. Tissue osmolarity was determined by the methods of Van Volkenburgh and Cleland (personal communication). Ten peeled leaf segments were wrapped in aluminum foil after treatment and frozen on dry ice for I to 2 h. After thawing for 2 to 5 min, 5 to 10 pl of cytoplasm was expressed and placed on a filter paper disc in a Wescor Model 5100B vapor pressure osmometer. Osmolarities of incubation media were determined by freezing point depression with an Advanced Model 3L osmometer.For scanning electron microscopy, peeled or unpeeled leaf segments were fixed overnight in osmium vapor and then dehydrated in acetone and critical point exchanged. The tissue was mounted on copper pegs, coated with gold/palladium in a sputter coater, and examined with a JEOL Model JS35 SEM at an accelerating voltage of 25 kev. RESULTSPreliminary experiments indicated that leaf tissue must be peeled in order to measure H' flux or to perturb the membrane by osmotic shoc...

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“…In studies using leaf discs, PCMBS has been shown to increase the release of valine (24), but to decrease sucrose release (1). There is considerable evidence in the literature that amino acid uptake occurs via a proton-mediated carrier system (8,16,22,24); thus it seems that the increased net effiux observed with inhibitors may have resulted from decreased uptake of amino acids.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Amino Acid Efflux from Isolated Soybean Cells

Secor

Schrader²

1984

Plant Physiol.

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Cells from reproductive soybean (Glycine max [L.] Menf.) 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 Qio 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 -y-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.Mature soybean seeds contain about 6% N, primarily as storage protein. Approximately half of this N is remobilized (translocated) from vegetative tissue when the plant has a reduced capacity for assimilating atmospheric and soil N during the time of rapid seed growth (9). This syndrome of satisfying the N demand by degradation of leaf protein and the subsequent remobilization of N to the seeds has been referred to as the "self destruction" hypothesis by Sinclair and de Wit (21). Attempts to overcome this "self destruction" and improve soybean yields by using soil or foliar N fertilizer applications have been inconclusive (3,4,7,25). This suggests an inability of the leaf to absorb and/or assimilate the applied N, or a limitation on N transport within the plant.

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Regulation of Amino Acid Uptake into Oat Mesophyll Cells: A Comparison between Protoplasts and Leaf Segments

Cited by 38 publications

References 1 publication

Extensor and Flexor Protoplasts from Samanea Pulvini

Extensor and Flexor Protoplasts from Samanea Pulvini

Regulation of H⁺ Excretion

Characterization of Amino Acid Efflux from Isolated Soybean Cells

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Regulation of Amino Acid Uptake into Oat Mesophyll Cells: A Comparison between Protoplasts and Leaf Segments

Cited by 38 publications

References 1 publication

Extensor and Flexor Protoplasts from Samanea Pulvini

Extensor and Flexor Protoplasts from Samanea Pulvini

Regulation of H+ Excretion

Characterization of Amino Acid Efflux from Isolated Soybean Cells

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Product

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Regulation of H⁺ Excretion