Modification of the intracellular sugar content alters the incidence of freeze-induced membrane lesions of protoplasts isolated from Arabidopsis thaliana leaves

Abstract

Sugar content and freezing tolerance of protoplasts of Arabidopsis thaliana leaves were manipulated by incubating seedlings in a sucrose solution before protoplast isolation. Incubation in a 400 mM sucrose solution at 2°C in the dark increased their freezing tolerance equivalent to that achieved after a conventional cold acclimation at 2°C. The increased freezing tolerance was due to a decrease in the incidence of freeze-induced lesions: expansion-induced lysis (EIL) between -2 and -4°C and loss of osmotic responsiveness (LOR) between -5 and -12°C. The concentration of sucrose in the incubation medium required to minimize the incidence of the lesions was substantially different: 10-35 mM for EIL and 30-400 mM for LOR. Incubation in the sucrose solution at 23°C decreased LOR only at -5 and -6°C but less than that incubated at 2°C, and there was no effect on EIL. Incubation in sorbitol solutions at 2°C also decreased LOR at -5 and -6°C but much less than in the sucrose solution. These results suggest that low concentrations of sucrose act as a metabolic substrate for the low-temperature-induced alterations required for the amelioration of EIL and, at higher concentrations, sucrose has a direct cryoprotective effect to minimize LOR.