Spin-Probe Studies during Freezing of Cells Isolated from Cold-Hardened and Nonhardened Winter Rye

Mesophyll cells isolated from cold-hardened and nonhardened winter rye (Secale cereale L. cv. Puma) were spin-labeled with the fatty-acid spin probe N-oxyl-4,4-dimethyloxazolidine 5-ketostearic acid. The probe was intercalated within the cellular membranes and changes in probe motion were followed during extracellular freezing of the cells. A correlation was observed between the lethal freezing temperatures (LT(50)) of the cells and the maximum hyperfine splitting value achieved by the incorporated probe. Rigid limit spectra indicated that a more ordered average packing was attained by membranes of hardened cells which survived freezing to lower temperatures.Nonhardened cells fixed with osmic acid at lethal freezing temperatures, in the frozen state, showed both conversion of normal bilayered cellular membrane ultrastructure to an amorphous state and condensation of cellular membranes to form densely packed multibilayered vesicles. Taken together with the spin-labeling data, these results suggest that at least one molecular mechanism of extracellular freezing injury involves the irreversible conversion of planar membrane bilayers to structures having less ordered packing and increased surface curvatures.