Transition to the glassy state upon dehydration as viewed through the activity of the xanthophyll cycle in the lichen Flavoparmelia caperata

Lichens can dehydrate to water contents (WC) below 10% of dry mass and survive in this condition for weeks or even months. As water is lost, the cytoplasm undergoes a transition from a liquid to a glassy state, a process termed vitrification. In the glassy state molecular mobility is strongly reduced, slowing down chemical reactions, including those that cause cellular deterioration. Recently, we showed that vitrification in the lichen Flavoparmelia caperata occurs at WC in-between 17 and 3% by measurements of molecular mobility and activity of the xanthophyll cycle. Here, we assessed at which WC enzymatic activity ceases as a consequence of vitrification. Hydrated thalli of the lichen F. caperata were subjected to fast dehydration (1.5 h) to 16±1% WC. Then, separate sets of thalli were equilibrated for 15, 30 and 45 days at standard air relative humidity (RH) of 94, 75, 55, 35 and 5% obtained with saturated solutions of KNO3, NaCl, Mg(NO3)2, MgCl2 and silica gel, respectively. Photosynthetic pigments were quantified and the enzymedependent de-epoxidation of violaxanthin was used to assess at which WC enzymatic activity ceases. Chlorophyll a fluorescence was measured before the experiments and after an equilibration period of 45 days. Photosynthetic pigments did not change during dehydration. Violaxanthin de-epoxidation and the resulting zeaxanthin accumulation was observed during equilibration at 94, 75 and 55%, but not at 35 and 5% RH. Chlorophyll contents did not change in thalli kept at 5 to 55% RH, but decreased after 30 and 15 days in thalli kept at 75 and 94% RH, respectively. Permanent damage to PS II was observed only in thalli kept at 94% RH. Our results suggest that upon dehydration the vitrification process occurs in-between 12 and 8% WC, whereas prolonged periods in partially hydrated conditions (WC > 12%) are deleterious for this lichen.