Abstract
Most of the leaf tissues of cold hardy bamboos employ deep supercooling as the mechanisms of cold hardiness. Their mechanisms of deep supercooling and freezing injury still remain unknown. Here, we analyzed precise freezing behaviors in leaf tissues of Sasa kurilensis. Differential thermal analyses of both leaf blades and leaf sheath revealed that a large low temperature exotherm (LTE) initiated around -20℃. To reveal freezing behaviors during the LTE, we observed their freezing process using cryomicroscopy. Leaf blades have a ladder-like vein network. Mesophyll cells are compartmentalized in a tissue unit surrounded by lateral and longitudinal veins. The large LTE released from the leaf blade is most likely a combination of multiple freezing events: all the mesophyll cells in a tissue unit froze together in two sequential incidents (indicated by darkening of the unit) and tissue units randomly froze one after another in the first half of the LTE. In the latter half of the LTE, cells in the hypodermis and other tissues froze independently one after another (some of which were accompanied by bubble formation). Both types of freezing events were most likely to be intracellular freezing except for bubble formation (cavitation). LTE corresponded well with injuries of the tissues. The large LTE released from the leaf sheath arose from the random (intracellular) freezing of cell clusters. The tissue was not separated by lateral veins but the cluster of cells froze together (cluster size was different in a freezing incident from another). LTE corresponded well with injuries of the leaf sheath. In both leaf blades and leaf sheath, acoustic emission (AE) was observed during LTE. AE in leaf blades was well coincided with the bubble formation (cavitation) in bulliform cells and/or intracellular freezing in hypodermis. While that in the leaf sheath was more likely coincided with intracellular freezing of the component cells.
