Ultrastructural alterations in cells of hardened and non-hardened winter rye during hyperosmotic and extracellular freezing stresses

Abstract

Epicotyl tissues of cold-hardened and non-hardened winter rye (Secale cereale L. cv. Puma) were subjected to extracellular freezing (−10 °C) and hyperosmotic stresses (1–4 M salt solutions), and were fixed such that the cellular plasmolytic and freezing patterns were retained to allow for examination of ultrastructural alterations during stress. Loss of integrity of the plasmalemma and the appearance of osmiophilic regions on the protoplasmic surface were observed and could be correlated with lethal cellular injury due to both hyperosmotic and extracellular freezing stresses. Complete loss of ultrastructure of organelles such as etioplasts and mitochondria also occurred, but only at levels of hyperosmotic stress higher than that required to kill the cells. The formation of osmiophilic granules during hyperosmotic stress was irreversible since on deplasmolysis they were observed to outline the protoplasm at the point where it burst. Occasionally membraneous connections between such osmiophilic granules could be observed after deplasmolysis. Similar osmiophilic granules were also observed on the outer membrane of some mitochondria. In the frozen-fixed cells, ultrastructural examination showed that the cell wall collapsed with the protoplast, indicating that the freezing pattern was retained by fixation. Osmiophilic regions on the protoplasmic surface and disruptions of the plasmalemma were also observed. These results support the idea that primary freezing injury to the cell occurs during the freezing process. Although the stresses between plasmolysis and freezing may be different in some respects, some of the ultrastructural manifestation of injury in both cases are similar.