Water Relations of Pachysandra Leaves during Freezing and Thawing : Evidence for a Negative Pressure Potential Alleviating Freeze-Dehydration Stress

The evergreen herb Pachysandra terminalis becomes moderately frost-hardy in winter. The water relations of its frost-hardy leaves were studied during a freeze-thaw cycle. Leaf water potentials, measured by psychrometry at subfreezing temperatures, were identical with those of ice, indicating equilibrium freezing. Microscopic observations showed extracellular freezing of tissue water. As evidenced by thermal analysis, the freezing process starts with the crystallization of a minor volume which was identified as apoplasmic water. The following long-lasting exotherm indicated slow export of water from the protoplasts driven by extracellular crystallization. In partially frozen leaves, the fractions of liquid water were measured at several subfreezing temperatures by nuclear magnetic resonance spectroscopy. They were consistently greater than those calculated from the osmotic potentials of cellular fluid, and the differences increased with decreasing temperature. About 50% of the differences could be abolished by freeze-killing of the leaf and was thus ascribed to the effect of a (negative) pressure reinforcing the osmotic potential. The persistent part of the differences may have reflected a matric component. At −7°C, the absolute values of both potentials were −1.7 megapascals each. The water relations of Pachysandra leaves clearly indicate nonideal equilibrium freezing where negative pressures and matric potentials contribute to the leaf water potential and thus alleviate freeze-dehydration of the tissue.