Abstract
Freezing of extracellular spaces is the first event when plants are exposed to freezing temperatures, but
mechanisms of ice nucleation and propagation remain poorly understood. Japanese cold hardy dwarf
bamboos grow in one of the northernmost areas of bamboo distribution and are known to employ deep
supercooling in most living tissues as the freeze survival strategy. Studies have been performed on
mechanisms of their deep supercooling capability. However, little is known about the ice nucleating factors
which may initiate freezing at the apoplast of the vascular and epidermal tissues. This study focused on ice
nucleation activity (INA) in aploplast extracts from cold hardy bamboo species, Sasa kurilensis, S. nipponica
and Sasamorpha borealis. We obtained apoplast extracts from leaf blades and culms and determined their
INA following size fractionation using membrane filters. The apoplast extracts from leaf blades and culms
had highly extractable and particulate INA (size: 10 kDa<<0.2 μm). INA of substances larger than 300 kDa
(<<0.2 μm) were similar to the freeze initiation temperatures of the vascular and epidermal tissues. Results
of heat and protease treatments indicated that the major substance(s) responsible for ice nucleation in the
apoplast extracts is proteinaceous, irrespective of the seasons, tissues or species.
