Protonation and Light Synergistically Convert Plasmalemma Sugar Carrier System in Mesophyll Protoplasts to its Fully Activated Form

Guy, Micha; Reinhold, Leonora; Rahat, Michaela; Seiden, Aza

doi:10.1104/pp.67.6.1146

Cited by 14 publications

(4 citation statements)

References 17 publications

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“…3). A very powerful effect of light on the uptake of 3-O-methyl-glucose by protoplasts isolated from the mesophyll of Pisum sativum L. has also been reported and it has been suggested that light may greatly stimulate the plasmalemma-sited proton extrusion pump [10,11]. Twenty-four h following isolation, in either dark-or light-kept protoplasts, glucose uptake decreased significantly (Fig.…”

Section: Discussionmentioning

confidence: 96%

Progress in leaf protoplast isolation and culture from virus-free axenic shoot cultures of Vitis vinifera L.

Theodoropoulos¹,

Roubelakis‐Angelakis

1990

Plant Cell Tiss Organ Cult

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Axenic shoot cultures of virus-free Vitis vinifera L. cv. Soultanina were a highly efficient source for isolation of viable protoplasts. Optimum results were obtained with leaves of 50-100mg fresh weight, leaf discs of 0.7 cm in diameter, 100 and 15 U ml-~ Cellulase R-10 and M acerozyme R-10, respectively, and 18 h reaction time in either light or in darkness. Protoplast yield was approx. 25 × 106 viable protoplasts per g fresh weight and their size ranged from 12 to 44 ~tm. During a 20-day culture period, the maximum survival rate obtained was approx. 40%. A plating density of 10 × 105 protoplasts per ml resulted in increased survival rates. Various growth regulators and glutamine did not significantly improve survival rates of protoplasts, whereas extract from coconut added to the culture medium caused an increase in the survival rates of protoplasts. Cell elongation at a significant rate and divisions were observed. [~4C]glucose uptake was studied as an index of cell membrane integrity and functioning. Uptake rate of glucose by protoplasts was linear for up to 60 min, fully inhibited by NaN 3, with an optimum pH of 4.8. Protoplasts 24 h old exhibited significantly lower rates of glucose uptake.

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Section: Discussionmentioning

confidence: 96%

Progress in leaf protoplast isolation and culture from virus-free axenic shoot cultures of Vitis vinifera L.

Theodoropoulos¹,

Roubelakis‐Angelakis

1990

Plant Cell Tiss Organ Cult

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“…2). It is highly likely that the two effects are related, since there is now persuasive evidence (see 1 1) that in many systems, including leaf mesophyll protoplasts (8,9), sugar uptake is driven by H+-symport. From the experiments summarized in Figures 3 to 6, we can conclude that the basis of the effect on sugar flux is unlikely to be promotion of the aging process, suppression of leak (i.e.…”

Section: Discussionmentioning

confidence: 99%

“…We have earlier (8,9) brought evidence which suggests that plasmalemma transport of sugars into pea mesophyll protoplasts in the light (though possibly not in the dark) is driven by pmf2 as is believed to be the case in many other plant systems (11). Apart from its sensitivity to light, the sugar transport system of leaf tissue also shows sensitivity to turgor.…”

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confidence: 88%

Is Modulation of the Rate of Proton Pumping a Key Event in Osmoregulation?

Reinhold¹,

Seiden²,

Volokita³

1984

Plant Physiol.

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ABSTRAC1The net uptake of 3-0-methylglucose into leaf segments obtained from Senecio mikanioides Otto, and net proton efflux from the segments, were both promoted when the osmotic potential of the medium was decreased by addition of mannitol, sorbitol, or polyethylene glycol (optimal osmolarity, 0.3 Osmolar for mannitol and sorbitol). The effect was not due to promotion of 'aging', since the antibiotic cerulenin suppressed aging without reducing the size of the mannitol stimulation; further, mannitol did not accelerate aging. Neither was the effect ascribable to diminished efflux (i.e. reduced 'leak' because: first, visualization of the unidirectional supr fluxes by double labeling indicated that the effect of added osmoticum was to promote influx rather than to reduce effiux; second, compartment analysis did not suggest any effect of mannitol on the rate constants for efflux from either the slowly equilibrating or more rapidly equilibrating compartment. The effect was not specific to poly-ols since it was also obtained with betaine and choline chloride. Since methyl glucose is not taken up into the phloem it could not be ascribed to a turgor effect on phloem loading. We conclude that the effect may reflect osmoregulation. As the sugar flux is probably driven by protonmotive force, it is likely that the effects on proton flux and on supr flux are related. We suggest that the plasmalemma-sited proton pump is sensitive to the hydrostatic pressure gradient across the plasmalemma-cell wall complex, and functions both as detector and as effector in osmoregulation.We have earlier (8, 9) brought evidence which suggests that plasmalemma transport of sugars into pea mesophyll protoplasts in the light (though possibly not in the dark) is driven by pmf2 as is believed to be the case in many other plant systems (11). Apart from its sensitivity to light, the sugar transport system of leaf tissue also shows sensitivity to turgor. Because experiments in which external osmotic pressure is manipulated in the hypotonic range are not practicable with isolated protoplasts, we have carried out these experiments with leaf segments of Senecio mikanioides from which the lower epidermis had been removed. We have observed that a change towards more negative external water potential brings about an increased rate ofproton extrusion (also reported by 5, 7) as well as an increase in sugar influx. The observed effect of turgor on sugar transport may thus well be mediated via the proton extrusion pump and the pmf. In a brief preliminary communication (12), we proposed that these responses to low turgor reflected osmotic regulation, and suggested that the proton pump might play a key role in the mechanism 'Supported by of osmotic adaptation. However, a number of alternative explanations for the observed effects suggested themselves, which we thought necessary to investigate. We have presented the results of our investigation in detail. MATERIALS AND METHODSLeaves which had almost reached their maximum size were collected from plants of Senecio...

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“…When glucose uptake was studied over an extended range of concentrations, multiphasic plots of rate versus sugar concentration were obtained (6)(7)(8)(9). Koshland and associates, who studied enzyme reactions showing the same kinetic behavior, favored a model of mixed cooperativity in their interpretation of these kinetics (1 1).…”

Section: Resultsmentioning

confidence: 99%

The Effect of Dormancy on Glucose Uptake in Gladiolus Cormels

Ginzburg

Ben-Gad²

1986

Plant Physiol.

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The uptake of glucose and of 3-0-methyl-D-glucose by cormel slices of Gladiolus Xgandavensis Van Houtte was studied in relation to cormel dormancy. Uptake was higher in nondormant cormels. Incubation of nondormant cormels with abscisic acid (ABA) reduced their uptake capacity. Treatment of dormant cormels with 6-benzyladenine (BA) did not affect their uptake rate. ABA and BA promoted 02 uptake, indicating that differences in uptake are not related to differences in energy supply.Transport of sugars is a major activity in the starch-storing cells of Gladiolus cormels. The rate of transport is expected to increase when reserves are mobilized during the break of cormel dormancy. Previous communications described the regulation of cormel dormancy and related metabolic changes by 12 were supplied dormant by Avshalom Nurseries, Israel. Cormels were stored at 25C, a temperature at which they remained dormant for at least 15 months, or at 5°C, at which dormancy was broken after 3 months. Germination and viability tests were performed routinely as described previously (1).Uptake Experiments. Cormels were descaled manually and imbibed for 3 d in 10 ml of water or a 0.1 mM solution of BA or ABA in 9 cm Petri dishes. Imbibition was important for uniform uptake. After imbibition, cormels were sliced into 1 mm thick slices, which were then floated on water for about 1 h. One-g lots of slices were incubated with [U-'4C] in 5 ml of 0.1 M citrate-phosphate buffer at pH 4.0 in 50 ml Erlenmeyer flasks for 2 h on a shaking water bath adjusted to 30°C. Incubation was stopped by washing the slices three times with 5 ml of distilled H20 and then grinding them twice in 10 ml of boiling 80% ethanol. The ethanolic extracts were combined, reduced in volume under vacuum, and the radioactivity was measured by scintillation counting. Our washing procedure removed 98% of the radioactivity from the free space.When glucose uptake was measured, a stream ofair was passed through the incubation flasks and collected in 10 ml of a 0. RESULTSThe time course of uptake of glucose and of MG3 by slices prepared from dormant and from nondormant cormels was compared. When 1 g lots of slices were incubated in 5 ml of 0.1 mm solutions of ['4C]glucose (0.2 mCi/mmol) or ['4C]MG (0.25 mCi/mmol) for periods of 10 to 150 min, uptake of both sugars was linear with time in both dormant and nondormant cormel slices (Fig. 1). The difference that was found between dormant and nondormant cormels in the rate of MG uptake also occurred in the slices (data not shown). When nonlabeled glucose was added to a final concentration of 2 mm to a 0.1 mm solution of ['4C]MG, the uptake of MG was suppressed, indicating that glucose and MG compete for the same uptake mechanism.The pH dependence of glucose and MG uptake was followed using dormant cormel slices. One g of slices was incubated for 2 h in 0.1 mm solution of glucose or MG, in 5 ml of citrate phosphate buffer adjusted to pH values between 4.0 and 7.0. Uptake of both sugars decreased with increasing pH (Fig. 2).The...

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Protonation and Light Synergistically Convert Plasmalemma Sugar Carrier System in Mesophyll Protoplasts to its Fully Activated Form

Cited by 14 publications

References 17 publications

Progress in leaf protoplast isolation and culture from virus-free axenic shoot cultures of Vitis vinifera L.

Progress in leaf protoplast isolation and culture from virus-free axenic shoot cultures of Vitis vinifera L.

Is Modulation of the Rate of Proton Pumping a Key Event in Osmoregulation?

The Effect of Dormancy on Glucose Uptake in Gladiolus Cormels

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