A Freeze-Fracture Study Designed to Clarify the Mechanisms of Freezing Injury Due to the Freezing-Induced Close Apposition of Membranes in Cortical Parenchyma Cells of Mulberry

Abstract

Freezing injury in cortical parenchyma cells of mulberry (Morus bombycis Koidz. cv. Goroji) collected in autumn and spring was closely associated with the occurence of ultrastructural changes in the plasma membrane, namely, formation of aparticulate domains (APD) with accompanying "fracture-jump lesions" and fine irregularities, that were produced by the close apposition of membranes caused by freezing. In order to identify the cause of freezing injury due to the formation of these ultrastructural changes, frozen samples were warmed to near subzero temperatures and structural changes in plasma membranes were examined by freeze-fracture electron microscopy. The results showed that, upon warming to -5°C, stuctures in the APD in the plasma membranes were changed in a variety of ways, which include formation of small pores, shallow slits, deep slits, and wide slits with visible contents, in addition to the absence of fracture jumps. Furthermore, fusion between plasma membranes and closely apposed endomembranes was produced at the APD upon warming to -5°C. It is suggested that structures in the APD seen upon warming to -5°C might represent the variety of structures that form during the process of membrane fusion by interlamellar attachment. Further warming to -3°C resulted in more distinct structural changes in the APD, perhaps as a result of membrane fusion, and led to further structural changes in entire plasma membranes. It is suggested that the occurrence of nonphysiological fusion of membranes at the APD including fracture-jump lesions and fine irregularities might result in freezing injury.