Final Report Summary - REAL TUNE (Role of enzymatic activity in lichen tolerance under nitrogen excess) Human activities, mainly combustion of fossil fuels and industrialized agriculture, have detrimental effects on the environment around the world. At a planetary scale, human processes convert more atmospheric nitrogen (N) into reactive forms than all Earth’s natural processes combined, significantly perturbing global cycling of the element. Due to the low use efficiency of the applied N, a large part of this additional reactive N is released to the environment, accumulating in terrestrial and aquatic systems and polluting the atmosphere. It slowly erodes the resilience of important ecological subsystems, and has implications for other systems and processes in ways that we still do not completely understand.Since reactive N production is linked to the daily activities of an increasing global population, a significant reduction of N emissions in the near future is quite unlikely. Consequently, to face the N problem, we can only hope to understand the underlying mechanisms and find alternative solutions.Lichens, symbiotic associations between algae and fungi, are among the most sensitive organisms to NH3 pollution at the ecosystem level, having a differential response depending on their functional response group: oligotrophic species are extremely sensitive to NH3 pollution, while nitrophytic species tolerate such conditions. Several papers have considered N tolerance in lichens, however many questions are still unsolved.The EU funded research project “REAL TUNE - Role of enzymatic activity in lichen tolerance under nitrogen excess” aimed to investigate the mechanisms of N tolerance in lichens in order to provide suitable science-based tools for N management and environmental protection.The project was developed in Portugal, United Kingdom, Italy and Croatia involving three experimental sites (Parque do Alambre na Serra de Arrábida in Portugal; Whim site in Scotland, United Kingdom; Porto Conte experimental site, Italy), 7 institutions (Centro de Biologia Ambiental, Universidade de Lisboa, Portugal; ITQB - Instituto de Tecnologia Quimica e Biologica, Universidade de Lisboa, Portugal; Centre for Ecology and Hydrology, UK; Università di Siena, Università di Viterbo and Università di Sassari, Italy; University of Zagreb, Croatia) and 6 related projects.Physiological tests, proteomic, isotopic and enzymatic analysis, transplant experiments and transmission electronic microscopy were used. Moreover, further results are expected from proteomic analyses (ITQB) on samples still in the field due to technical problems during the implementation of the work.Methods for ergosterol and chitin analyses in lichens have been implemented at the host institution as well as a method for measuring enzymatic activity previously used on mycorrhizae, which was adapted to lichens.Lichens were collected in the field along existing N gradient caused by agricultural activities and/or urban areas, transplanted along gradient of N availability/desertification or treated in experimental sites under controlled conditions.Although data are still being analysed, I obtained important results contributing to the understanding of N tolerance in lichens. Here a brief summary of the main conclusions so far:1) potassium content was well correlated with N tolerance showing a gradient from the least to the most tolerant in samples from different sites, confirming the importance of intracellular cations in N stress response;2) sensitive lichens showed a stronger correlation with the atmospheric ammonia concentration than with the substrate pH, while tolerant species pH seems to be driven by change in substrate pH indicating that tolerance can be linked to the capacity to maintain the pH unaltered;3) the production of secondary compounds (usnic acid and perlatoric acid) in Cladonia portentosa seems to be lower in N-treated samples, but there was no clear relation with N dose or form;4) preliminary results of proteomics analysis from samples subjected to long and short treatments showed that some proteins are correlated with NO3 dose in N treatment, while others were produced in the control but not in treated samples. The former can be involved in storage, detoxification or stress response, while the latter can be related to N transportation and poorly expressed in the presence of high N availability. Selected spots are now being processed for mass spectrometry at the University of Siena in order to determine the amino acid sequence;5) the potential activities of the tested catabolic extracellular enzymes were not correlated with N treatments;6) ultrastructural changes in algae were not specifically related to nitrogen excess, but rather to generic oxidative damage. In this way, the high concentrations of antioxidant substances found in Xanthoria parietina can explain its N tolerance;7) based on N isotopic signature analysis indicating a differential uptake of NH4+ and NO3- in different species, cation exchange capacity was confirmed to be important in determining N tolerance in lichens;8) in conditions of low pollution in a Mediterranean urban area, lichen functional diversity (i.e. the diversity defined from the point of view of ecological characteristics) showed that climatic gradients can be more important than pollution gradients; in fact, species related to water requirements were more significantly correlated with surrounding land-use than any other lichen species, including those associated with N pollution,9) field data combined with data from transplanted experiments showed that X. parietina is affected by N, even if to a lesser extent and at higher concentrations than other species considered sensitive. That suggests that the ecological success of X. parietina at ammonia-rich sites might be more related to indirect effects of increased N availability than to a positive direct effect on its physiological performance.Conclusions: N tolerance in lichens seems to be linked to a modification in protein expression with the increase of a generic antioxidant activity and the reduction of transporters for N (4). Simultaneously, secondary compounds’ production decreases (3), likely because resources are allocated in the previous mentioned activity. This on one hand makes lichens tolerant to N also tolerant to factors that provoke aspecific oxidative stress (6) like drought (8), while on the other implies the existence of a threshold besides which repairing is not possible even in tolerant species (9). On the contrary, those mechanisms do not involve the novo synthesis of catabolic enzymes since their potential activity does not change (5). Cation exchange capacity regulates the entrance of N in the cells (7), more in the sensitive species and less in the resistant, with different effects on thallus pH that is influenced by ammonia atmospheric concentration in the former and by substrate pH in the latter (2). Among intracellular cations, potassium has a protective role since its concentration in higher in resistant species (1). REAL TUNE can provide results relevant to different target groups; which is why my divulgation activities were so varied.In my results, the scientific community can find interesting clues to ecosystems functioning and molecular mechanisms related to N nutrition and N pollution.Policy makers can obtain important information about N management and environment protection. For ex, distinguishing the effects of climatic parameters from the effects of pollution in a monitoring survey (8) can be essential for sustainable urbanization and pollution reduction measures, while to understand that only sensitive species are directly affected by N indicates that only sensitive species must be used for the establishment of critical loads and levels, important instruments of environmental protection.To students from high schools I tried to communicate my passion for my work, explaining how fascinating but how hard it can be and showing them that there are several possibilities for funding at national and European level.Finally, in meetings with general public I aimed at increasing people’s awareness showing that our habits can have great influence on the environment and that we can do a lot for environmental protection both living in a more sustainable way and asking for better policies.