Confocal cryomicroscopic analysis and cryodynamics of endoplasmic reticulum in herbaceous plant cells

Plants that survive subzero temperatures in winter enhance their freezing tolerance upon exposure to non-freezing temperature, a phenomenon termed cold acclimation. Although the mechanism of enhanced freezing tolerance has been widely examined at the molecular level, the understanding of cell physiological aspects remains poor. In our previous study, confocal cryomicroscopic observation of leaf epidermal cells of Allium fistulosum, the cold-hardy Welsh onion, showed that extracellular freezing seems to induce cessation of streaming and development of vesiculation of the endoplasmic reticulum (ER). Further analysis of this unique cryobehavior of ER may lead to a better understanding of how plant cells survive freezing. We confirmed that ER vesiculation occurred in root cells of Arabidopsisthaliana, which suggested that freeze-induced vesiculation of ER might be conserved in herbaceous plants. Because the ER membrane dynamics in Welsh onion cells were more clearly observed than those in Arabidopsis cells, the Welsh onion system was used for more detailed analyses. Average velocities of ER membranes were quantitatively analyzed and resultant Arrhenius plots showed that extracellular freezing rapidly decreased ER streaming, which had ceased completely at −5°C in both non-acclimated and cold-acclimated cells. After thawing, only in cold-acclimated cells, ER networks quickly recovered at 2°C. Furthermore, during supercooling at −5°C, only ER of cold-acclimated cells maintained streaming and networks. These results indicated that ER networks in cold-acclimated cells are more stable at low temperature, and extracellular freezing results in the breakdown of cold-stable ER networks in cold-acclimated cells. One possibility is that the breakdown of ER networks is involved in an enhanced freezing tolerance mechanism.