A Novel Method of Natural Cryoprotection: Intracellular Glass Formation in Deeply Frozen Populus

Correlating measurements from differential scanning calorimetry, freeze-fracture freeze-etch electron microscopy, and survival of twigs after two-step cooling experiments, we provide strong evidence that winter-hardened Populus balsamifera v. virginiana (Sarg.) resists the stresses of freezing below -28°C by amorphous solidification (glass formation) of most of its intracellular contents during slow cooling (≤5°C per hour). It is shown that other components of the intracellular medium go through glass transitions during slow cooling at about -45°C and below -70°C. This 'three glass' model was then used to predict the results of differential scanning calorimetry, freeze-fracture freeze-etch electron microscopy, and biological experiments. This model is the first definitive explanation for the resistance of a woody plant to liquid N2 temperatures even if quench cooling (1200°C per minute) begins at temperatures as high as -20°C and warming is very slow (≤5°C per hour). It is also the first time high temperature natural intracellular glass formation has been demonstrated.