Cryotolerance in the Antarctic lichen Umbilicaria antarctica is driven by supercooling and structural confinement

The Antarctic lichen Umbilicaria antarctica exhibits exceptional resilience to extreme cold, enabling survival in harsh polar environments. This study investigates the molecular and structural adaptations underlying its tolerance to subzero temperatures. Nuclear Magnetic Resonance analysis showed that at intermediate hydration level, water undergoes a transition from loosely bound to more strongly bound states between - 17 °C and - 24 °C indicating a non-cooperative water immobilization process rather than conventional freezing. Activation energy and average distances between relaxing proton pairs were determined using the Bloembergen-Purcell-Pound (BPP) model. Differential Scanning Calorimetry showed that freezing strongly depends on hydration, with all thermal peaks occurring below - 10 °C, confirming the presence of supercooled water. Heating protocols incorporating isothermal holding steps revealed an additional peak, consistent with diffusion of supercooled water toward pre-existing ice crystallites. Scanning Electron Microscopy detected sub-micrometer compartments within the thallus that physically confine water and solutes. This confinement-synergy effect stabilizes supercooled water, suppresses ice nucleation, and modulates molecular mobility, playing an important role in freezing avoidance. This integrative approach advances our understanding of cold tolerance mechanisms and highlights how coupled structural confinement and physicochemical interactions govern water behavior in extremophiles, offering broader insight into adaptation strategies in polar environments.