Lichen photobiology in relation to climate change : protection in peltigeralean lichens against excess ultraviolet (UV) radiation using induced melanins and the effects of UV on melanin synthesizing enzymes.

Master of Science in Biological sciences.

Depletion of the stratospheric ozone has allowed more solar ultraviolet (UV) radiation (100-400 nm) to reach the lower atmosphere. Despite the success of the continued implementation of Montreal protocol of 1987 and its amendments aimed at the protection of the ozone layer, it is possible that UV will remain elevated in some regions due to decreasing removal by clouds and aerosols. Strong interactions between ozone depletion and changes in climate induced by increasing greenhouse gases could result in increased UV radiation levels reaching the surface. Increased exposure to UV-B can cause significant damage to organisms including protein damage, inhibition of photosynthesis, formation of high energy reactive oxygen species (ROS), disruption of nitrogen fixation and induction of cell mutation and inhibition of growth. Lichens are composite organisms that arise from a symbiotic association between fungi and photoautotrophic algal partners, which may be microalgae and/or cyanobacteria. They have developed several protective strategies to protect themselves from excessive radiation. Lichens such as Cladonia rangiferina, Lobaria pulmonaria and Umbilicaria rossica are known to synthesize melanins in response to elevated levels of UV-B radiation. Synthesis of melanins is generally catalysed by enzymes that are involved in phenol coupling including P450 monooxygenases, ascorbate oxidases, peroxidases, laccases and tyrosinases (Nezbedová et al. 2001). The work presented in this thesis investigated the enzymes involved in melanin biosynthesis in lichens from suborder Peltigerineae. We present a detailed characterization of redox enzymes from Lobaria pulmonaria and Pseudocyphellaria aurata with a special focus on tyrosinases. Furthermore, an evaluation of the response of L. pulmonaria to UV radiation was done. The role of the redox enzymes tyrosinases, laccases and peroxidases in melanin synthesis is elucidated by exposing L. pulmonaria thalli to various combinations of UV and PAR. Laboratory experiments clearly indicated that L. pulmonaria and P. aurata have redox enzymes that are of similar nature to those of their free-living symbionts and other lichens. Tyrosinase from L. pulmonaria oxidized monophenolic compounds and was able to metabolize L-DOPA, thus showing characteristics of a typical fungal tyrosinase. L-DOPA can also be metabolized by peroxidases and laccase suggesting that peroxidases and laccases may participate in melanisation reactions. A field experiment showed that exposing shade-adapted L. pulmonaria to solar radiation in the field induces DOPA melanin synthesis. The synthesis of the brown cortical compounds occurred when lichen thalli were exposed to direct sunlight or placed under a wavelength neutral filter that marginally reduced overall light. Melanin synthesis was accompanied by an increase in laccase activity in lichens that were exposed to natural sunlight. By contrast, no changes in enzyme activity occurred in lichen thalli placed under the wavelength neutral filter. Placing lichen thalli under filters that removed both UV-A and UV-B prevent melanisation and was accompanied by an increase in tyrosinase activity. Filtering out UV-B had no effect on enzyme activity but slight browning occurred. Results showed that laccases could be involved in melanin biosynthesis under some conditions, but provided no evidence for a role of tyrosinases in melanisation. However, it could be that melanisation requires tyrosinase, but rates of melanisation are controlled by the levels of melanin precursors such as tyrosine. No differences in maximal PSII efficiency and chlorophyll contents occurred between the radiation treatments indicating that no deterioration of photosynthetically apparatus occurred and the photobionts remained relatively healthy even when exposed to UV. Taken together these results suggest that further research in needed into determining the levels of melanin precursors, the roles of lichen redox enzymes in melanin synthesis and whether a transcriptional activation of these redox enzymes by UV radiation in lichens exists or not. Furthermore, additional roles for tyrosinase need to be investigated, for example defence against pathogens.