Xylem parenchyma cells (XPCs) of boreal hardwood species adapt to sub-freezing temperatures by deep supercooling to maintain a liquid state of intracellular water near -40°C. Our previous study found that crude xylem extracts from such tree species exhibited anti-ice nucleation activity to promote supercooling of water. In the present study, thus, we attempted to identify the causative substances of supercooling. Crude xylem extracts from katsura tree (Cercidiphyllum japonicum), of which XPCs exhibited deep supercooling to -40°C, were prepared by methanol extraction. The crude extracts were purified by liquid-liquid extraction and then by silica gel column chromatography. Although all the fractions obtained after each purification step exhibited some levels of anti-ice nucleation activity, only the most active fraction was retained to proceed to the subsequent level of purification. High-performance liquid chromatography (HPLC) analysis of a fraction with the highest level of activity revealed four peaks with high levels of anti-ice nucleation activity in the range of 2.8-9.0°C. Ultraviolet (UV), mass and nuclear magnetic resonance (NMR) spectra revealed that these four peaks corresponded to quercetin-

The supercooling capability of xylem parenchyma cells (XPCs) in boreal hardwood species differs depending not only on species, but also season. In this study, the roles of cell walls and intracellular contents in supercooling capability of XPCs were examined in three boreal hardwood species, Japanese beech, katsura tree and mulberry, whose supercooling capability differs largely depending on species and season. XPCs in these species harvested in winter and summer were treated by rapid freezing and thawing (RFT samples) or by RFT with further washing (RFTW samples) to remove intracellular contents from XPCs in order to examine the roles of cell walls in supercooling. RFT samples were also treated with glucose solution (RFTG samples) to examine roles of intracellular contents in supercooling. The supercooling capabilities of these samples were examined by differential thermal analysis after ultrastructural observation of XPCs by a cryo-scanning electron microscope to confirm effects of the above treatments. XPCs in RFTW samples showed a large reduction in supercooling capability to similar temperatures regardless of species or season. On the other hand, XPCs in RFTG samples showed a large increase in supercooling capability to similar temperatures regardless of species or season. These results indicate that although cell walls have an important role in maintenance of supercooling, change in supercooling capability of XPCs is induced by change in intracellular contents, but not by change in cell wall properties.