Changes in protein metabolism during the acquisition of tolerance to cryopreservation of carrot somatic embryos

High concentrations of sucrose are often used to cryopreserve regenerable plant cell cultures in liquid nitrogen. A 21-h pretreatment of carrot somatic embryos in medium containing 0.4 M sucrose allows 80 % of them to germinate after freezing. Substitution of sucrose by polyethylene glycol 6000 led to lower germination rates. However, a high level of freezing tolerance was restored by addition of 1 μM abscisic acid in the pretreatment medium. Using these different media, both total water soluble protein, using SDS-PAGE, and boiling-stable protein, using 2-D electrophoresis, were studied in relation to acquisition of cryopreservation tolerance. Only boiling-stable protein patterns showed some changes: five polypeptides accumulated in 0.4 M sucrose-pretreated embryos or in embryos pretreated by media containing abscisic acid. This accumulation was not detected with polyethylene glycol 6000 used as sole cryoprotectant. Although over-accumulation of polypeptides was highest with media containing ABA, the best germination rates were linked to pretreatment with 0.4 M sucrose. The addition of okadaic acid in 0.4 M sucrose medium led to embryo death after freezing, confirming the existence of a message leading to metabolic changes and acquisition of cryotolerance. Water-soluble proteins obtained from 0.4 M sucrose-pretreated embryos appeared more active than those extracted from control embryos in protecting in vitro a freeze-labile enzyme. Boiling-stable proteins, corresponding to a part of total proteins, were more active than total proteins. These results suggest that these polypeptides may be involved in a mechanism of protection needed for cell survival during freezing stress.