Insights on desiccation tolerance of the lichen photobiont trebouxia sp. pl. in both thalline and isolated ones

Resumo

The study of desiccation tolerance of lichens, and of their phycobionts in particular, has been mainly focused on the antioxidant system that protects the cell against photo-oxidative stress produced during dehydration and rehydration. However, desiccation tolerance cannot be achieved in lichen phycobionts with antioxidant activity alone. The main objective of the thesis was to expand the knowledge of the poorly understood desiccation tolerant mechanisms. The first chapter presents an introduction to the subject of the thesis, based on the available background information, and explains the rationale of the thesis. The aim of the chapter 2 was to establish the optimal growing conditions for the isolated phycobiont Trebouxia erici Ahmadjian (SAG 32.85 = UTEX 911) and to determine how these working procedures could modify the results of further experiments. We concluded that cellulose-acetate discs for agar cultures should be inoculated with 14-day-old liquid cultures, and growth under irradiance of 30 ?mol m-2 s-1 PAR at 20 ºC. Experiments should be carried out with 21day-old algal discs. The experiments carried out in third chapter were designed on one hand, to determine the grade of desiccation tolerance of the isolated lichen phycobiont T. erici, and on the other hand, we studied the response of some protective mechanisms. The results showed that both long desiccations and rapid drying produced greater membrane damage and lower recovery of metabolic and photosynthetic activity than after brief desiccations and slow drying, respectively. Photosynthesis was never recovered totally neither after rapid nor slow drying. Classical mechanisms involved in the protection of the cell against oxidative stress were present in hydrated algae although were not enhanced during dehydration/rehydration. Likewise, in T. erici, dehydrins were also constitutively expressed. However, a slow drying time was required for activating the conformational change in the photosystem II which protect against photoinhibition in desiccation state. In the fourth chapter we decided to carry out proteomic and genetic expression analyses of the changes associated with desiccation and rehydration in the isolated phycobiont Trebouxia erici, in order to find out other proteins that may be involved on desiccation tolerance mechanisms of phycobionts. Proteomic analysis showed that desiccation caused up-regulation of around 19 proteins and down-regulation of 43 proteins. Among the proteins up-regulated during drying were found: a putative cation transport protein; a protein with an F-box domain; a cell division cycle 48 protein; ?-tubulin protein; a 90 kDa Heat shock protein. We observed that five Hsp90 and two ?-tubulin genes were activated during dehydration and mRNA was accumulated until the cell was completely dried. In the fifth chapter we investigated the role of the NO in the biochemical interaction between lichen symbionts and whether the NO may increase the endurance of lichenized phycobionts under desiccation. Rehydration of the lichen R. farinacea caused the release of NO together with a high production of ROS. The amount of NO detected decreased significantly with the addition of c-PTIO. In the isolated phycobionts, scavenging of the self produced NO caused a decrease in the recovery of photosynthetic activity after dehydration, probably due to the higher levels of photo-oxidative stress. The aim of the work presented in the sixth chapter was to determine the effect of cell ageing in desiccation resistance of T. erici. Phycobiont capacity to recover photosynthesis activity after desiccation was inversely related with cell ageing, suggesting that desiccation tolerance is age-dependent. The results presented in this thesis demonstrate that desiccation tolerance of T. erici is achieved by a complex system of constitutive and induced mechanisms. Moreover, some factors such as drying rate, desiccation duration, lichenization and ageing may alter the recovery capacity of T. erici after desiccation.