Freezing of water bound in lichen thallus as observed by H NMR. II. Freezing protection mechanisms in a cosmopolitan lichen Cladonia mitis and in Antarctic lichen species at different hydration levels

Proton NMR spectra at 300 MHz for dehydrated and hydrated thallus of Cladonia mitis Sandst. =C. arbuscula (Wallr.) Flot ssp. Mitis (Sandst.) Ruoss], Himantormia lugubris (Hue) Lamb and Usnea aurantiaco-atra (Jacq.) Bory were recorded. The temperature was decreased from room temperature down to −45 °C. Pulse length was set to π/2=8.3 μs, which allowed the observation of tightly bound and loosely bound/or free water fraction, whereas the signal from solid matrix of thallus was reduced. In hydrated thallus a narrow Lorentzian line coming from loosely bound/free water fraction was recorded. For the temperature range between −5 and −20 °C a discontinuous increase in line halfwidths, accompanied by a decrease in area under the peak, was observed. This was attributed to the cooperative freezing of bulk water present in lichen thallus. In dehydrated thallus the NMR line consists of two components: a narrow, Lorentzian one (coming from loosely bound/free water fraction) and a broad line (from water tightly bound in lichen thallus). The overall area under peak remains unchanged down to −5 °C, and then between −5 and −20 °C it continuously decreases due to non-cooperative water immobilisation. As the temperature is decreased, for temperatures above −5 °C, the contribution made by the broad line component increases at the expense of the narrow line component. The mechanism of loosely-to-tightly bound water transfer is, at least partially, responsible for the freeze-protection of thallus in the lichen species investigated. No significant differences between the freeze protecting loosely-to-tightly bound water transfer mechanism of Antarctic lichens and that of cosmopolitan lichens was noticed.