TIME-DEPENDENT AND TEMPERATURE-DEPENDENT SOLUTE LOADING OF ISOLATED THYLAKOIDS DURING FREEZING

The influx of sucrose into thylakoids during freezing was investigated by measuring the changes in vesicle volume after a freeze-thaw cycle by hematocrit centrifugation. The time course of solute loading was biphasic. The initial phase of 15-30 min showed a linear time dependence of volume increase. During this phase, solute loading was a direct function of the solute gradient across the membranes when this gradient was corrected for the influence of temperature on solute diffusion. In the second phase, thylakoid volume increase was slowed down. This apparent reduction of solute loading was the result of increased vesicle rupture, measured as the release of the lumenal protein plastocyanin after thawing. This led to a reduced average vesicle volume as measured by hematocrit centrifugation. The observed solute loading of thylakoids during freezing in the presence of the cryoprotectant sucrose could be completely ascribed to passive diffusion of the sugar across the membranes. Solute loading led to osmotic swelling of the vesicles during thawing and to rupture when swelling was excessive. Solute loading, however, was not the only reason for rupture. It was a complex function of temperature, incubation time, and the solute concentration of the suspending solution. © 1992.