Quantifying liquid water in frozen plant tissues by isothermal calorimetry

Abstract

An equation to calculate the percentage of water remaining unfrozen at any temperature due to colligative properties of solutions was derived from the freezing point depression equation. The accuracy of the equation was demonstrated with a 0.1 M sucrose solution frozen at temperatures from −0.5 to −6 °C in an isothermal calorimeter. Empirical measurements using latent heat as a measure of the amount of water frozen were within 1% of the expected values calculated from the equation. The extent to which percentages of water freezing in oat crown tissue at varying temperatures follows the expected freezing curve indicates how closely the system follows colligative freezing processes. The freezing curve for non-acclimated crowns followed a colligative freezing pattern more closely than did the curve for crowns from cold-acclimated plants. This suggests that water in crowns from non-acclimated plants may remain unfrozen primarily by colligative means while other mechanisms of keeping water unfrozen are important in cold-acclimated crowns. This may help explain contradictory results of studies that attempt to correlate carbohydrate concentrations with freezing tolerance.

Introduction

Calorimetry has been an important tool in the study of different forms of freezing stress because water freezing, that results in tissue damage to biological systems, can be detected thermally (see review by Mazur [1]). Greathouse [2] used calorimetry to measure water transitions in potato and in roots of clover and found considerably more water freezing in non-acclimated tissues than in cold-acclimated tissues; he also provided a review of earlier calorimetry studies in which amounts of free and bound water were measured. Levitt (cited by [3]) used calorimetry to show that more than three times the amount of water remained unfrozen in non-acclimated cabbage as compared to acclimated plants. Tumanov et al. [4] measured unfrozen water in wheat and found that the water retaining power of cells in a plant have a major effect on their frost tolerance. They stated that cells in different organs of the same plant do not retain water to the same extent. Johansson [5] used calorimetry to determine the amount of water freezing in wheat and rye plants, and reported conflicting results between unfrozen water and freezing tolerance. Olien [6], [7] found that a shift in latent heat occurred while plants were frozen and attributed this shift to a release of sugar into the apoplast which could have relieved adhesions. Calorimetry was used to demonstrate that pressure caused by an increase in respiratory CO₂ in a closed system induces CO₂ dissolution in water which acts in a colligative manner to reduce the amount of water freezing in oat crowns [8].

Calorimetric experiments with partially frozen systems do not generally provide information as to how unfrozen water is kept in the liquid state. Knowing this could help researchers determine how plants resist various forms of freezing stress. An equation that would determine the percentage of water remaining unfrozen due to colligative properties could allow researchers to help understand stress resistance mechanisms by measuring whether the amount of water freezing in a biological system is following or deviating from a freezing curve based on colligative properties.