Structural changes in acclimated and unacclimated leaves during freezing and thawing

Freeze-induced damage to leaf tissues was studied at different states of acclimation to low temperatures in snow gum, Eucalyptus pauciflora Sieber ex Sprengel. Intact, attached leaves of plants grown under glasshouse or field conditions were frozen at natural rates (frost-freezing) and thawed under laboratory conditions. Leaves were cryo-fixed unfrozen, during frost-freezing or after thawing for observation in a cryo-scanning electron microscope. Frost-freezing in unacclimated tissues caused irreversible tissue damage consistent with tissue death. Intracellular ice formed in the cambium and phloem, killing the cells and leaving persistent gaps between xylem and phloem. Many other cells were damaged by frost-freeze-induced dehydration and failed to resorb water from thawed extracellular ice, leaving substantial amounts of liquid water in intercellular spaces. In contrast, acclimated leaves showed reversible tissue displacements consistent with leaf survival. In these leaves during freezing, massive extracellular ice formed in specific expansion zones within the midvein. On thawing, water was resorbed by living cells, restoring the original tissue shapes. Possible evolutionary significance of these expansion zones is discussed. Acclimated leaves showed no evidence of intracellular freezing, nor tissue lesions caused by extracellular ice. While the observations accord with current views of freeze-sensitivity and tolerance, cryo-microscopy revealed diverse responses in different tissue types.