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...
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 3-0-methyl-Dglucose.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,. 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.
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