Lichens are poikilohydrous symbiotic associations between a fungus, photosynthetic partners, and bacteria. They are tolerant to repeated desiccation/rehydration cycles and adapted to anhydrobiosis. Nitric oxide (NO) is a keystone for stress tolerance of lichens; during lichen rehydration, NO limits free radicals and lipid peroxidation but no data on the mechanisms of its synthesis exist. The aim of this work is to characterize the synthesis of NO in the lichen Ramalina farinacea using inhibitors of nitrate reductase (NR) and nitric oxide synthase (NOS), tungstate, and NG-nitro-L-arginine methyl ester (L-NAME), respectively. Tungstate suppressed the NO level in the lichen and caused an increase in malondialdehyde during rehydration in the hyphae of cortex and in phycobionts, suggesting that a plant-like NR is involved in the NO production. Specific activity of NR in R. farinacea was 91 μU/mg protein, a level comparable to those in the bryophyte Physcomitrella patens and Arabidopsis thaliana. L-NAME treatment did not suppress the NO level in the lichens. On the other hand, NADPH-diaphorase activity cytochemistry showed a possible presence of a NOS-like activity in the microalgae where it is associated with cytoplasmatic vesicles. These data provide initial evidence that NO synthesis in R. farinacea involves NR.

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Lichen desiccation/rehydration cycles lead to an increased oxidative stress modulated by the multifaceted mediator nitrogen monoxide (NO). Active cell death, frequently triggered by oxidative damage with NO participation, has been confirmed even in unicellular organisms. This adaptive mechanism has not been studied in lichens and no specific experimental protocols exist. Hoechst 33,342 enters viable cells and DNA binding increases its fluorescence, particularly intense in condensed apoptotic chromatin. YO-PRO-1 can only permeate the altered membrane of apoptotic P2X7-positive cells. Proteolytic caspases are activated upon different types of active cell death. Our objectives are to determine if these markers indicate active cell death in Ramalina farinacea after desiccation/rehydration and to study the effect of NO scavenging. YO-PRO-1, Hoechst 33342, and Caspase 3/7 Green DNA binding were assessed in thalli rehydrated with deionized water and with a cocktail of apoptosis inducers. A 24 h kinetics and a microscopical analysis were performed. YO-PRO-1 fluorescence was not detected, Hoechst 33342 staining abruptly decreases during the first hours, while caspase-like activity associated to phycobionts steadily increases. Whereas the apoptosis inducers cocktail 1x significantly increased caspase-like activity affecting both symbionts, Hoechst staining was only affected at 10x. NO scavenging diminishes caspase-like activation and seems to accelerate Hoechst abrupt decrease during thallus rehydration. In conclusion, the demonstration of caspase-like activity in R. farinacea and its Trebouxia phycobionts point to the presence of active cell death but other methods assessing cell effective death or DNA irreversible fragmentation (i.e. TUNEL assay) are necessary to confirm this feature.

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