When water turns to ice: Control of ice volume and freezing dynamics as important aspects of cold acclimation

In the cold acclimated (CA) state, a reduced tissue water content is considered important to survive subzero temperatures. However, the causal relationship between the reduced water content and increased frost hardiness is unclear. Our aim was to assess whether the seasonally reduced water content affects the freezing dynamics and the amount of ice formed in evergreen leaves: Xeromorph leaves of the woody species Buxus sempervirens and Hedera helix were compared with the herbaceous, soft-leaved Bellis perennis in the non-acclimated (NA) state in summer, during cold acclimation, and in the fully CA state in winter. Freezing dynamics were studied using differential scanning calorimetry in addition to the volume fraction of ice and related to water content, osmotic potential, and frost hardiness. In the CA state, freezing dynamics were slower than in NA state. In xeromorph leaves, displacement from ideal equilibrium freezing was higher than in B. perennis. Freeze dehydration was lower in CA state. In the CA state, water content and osmotic potential were reduced, except for B. sempervirens, where the water content remained unchanged. Active osmoregulation and controlled dehydration (only found in two species), are supporting cellular water retention against the dehydrative force of extracellular ice. B. perennis had the highest water content and the least negative osmotic potential and was the most frost susceptible species (LT₁₀: 8.4 °C CA). The leaves froze at ideal equilibrium. 83 % of the total water froze, occupying more than 60 vol%. H. helix (LT₁₀: 18.4 °C CA) was frost hardier and B. sempervirens (LT₁₀: 28.8 °C CA) the frost hardiest species, but in contrast to the other species tested got frost killed by intracellular freezing. The xeromorph leaves froze at non-ideal equilibrium and had lesser ice masses. Despite an increase in frost hardiness with CA, the volume fraction of ice at LT₁₀ was the same (30–40 vol%). In the CA state, slower freeze dehydration and at the same subzero temperature lesser ice masses appeared to be important for higher frost hardiness. Overall, an important component of cold acclimation in evergreen leaves was the slowing of freezing dynamics, which, depending on the species, involved a specific cell architecture, osmoregulation, and a reduction in water content.