Ultrastructural Changes Arising from Freezing of Leaf Blade Cells of Cold Acclimated Rye (Secale cereale)

Abstract

The survival at sub-zero temperatures of leaf blade cells of rye ( Secale cereale cv. Voima) that had been cold-acclimated at 5 °C for 4 weeks was determined by measuring the efflux of ninhydrin-positive substances: 50 % of the cells were dead at - 8 °C (LT 50 ) and none survived at -16 °C or below. Specimens for transmission electron microscopy were prepared from leaf blade segments by fast freezing followed by freeze-substitution in acetone with O S O 4 fixation. In specimens from plants maintained at 5 °C, chloroplasts and mitochondria were well-structured and the endoplasmic reticulum and dictyosomes easily discernible. Many membrane bound vesicles, ranging in cross sectional diameter from ~ 60 nm upwards were seen in the cytoplasm. Sometimes stacks of membrane strands running parallel to the plasmalemma for up to 2 µm were also observed. Upon freezing to -16 °C, the cells were severely dehydrated and distorted, the vacuoles severely shrunken, and the cytoplasm and mitochondria disorganized; cytoplasmic vesicles and stacks of membrane strands seen in the controls were absent. Upon freezing to - 8 °C, some cells were as disorganized as those at -16 °C, although the cell wall did not always adhere to the collapsed protoplast; others were relatively intact and some showed evidence of intracellular ice crystal formation.