Studi tassonomici, filogenetici e funzionali di microrganismi fotosintetici adattati ad estremi di temperatura.

TAXONOMICAL, PHYLOGENETIC AND FUNCTIONAL STUDIES ON PHOTOSYNTHETIC MICRORGANISMS ADAPTED TO EXTREMES OF TEMPERATURE This research dealt with a morphological, ultrastuctural, physiological and taxonomical study on a cryophilic microalga isolated from a snow field of the Antelao (Dolomiti) glacier and some cyanobacteria from the thermal waters of the Euganean District. The aim of the research on these interesting photosynthetic microrganisms, living in habitats with so severe temperature conditions, was to obtain new information about the ultrastructural features, the taxonomic collocation and the physiological mechanisms enabling them to cope with the severe temperature conditions and also with the changes in this environmental factor. The green cryophilic microalga "Chlorella" saccharophila, was cultured at 4°, 8° and 16°C in order to analyze the effects of the change in growth temperature. The algal cultures responded positively to the temperature rise, showing an increase of the growth rate. The rise of the growth temperature did not affect the ultrastructure of the cells and did not change their content in chlorophylls, while it led to an increase of both carotenoid amount and carotenoid/chlorophyll ratio. The stability and functionality of the photosystems were maintained, as suggested by the constant values of the Fv/Fm ratios, the unchanged quantity of essential polypeptidic components (D1 and LHCII) and by the preservation of the correct ultrastructural organization of the thylakoidal membranes. At the rise of the growth temperature a significant increase of the photosynthetic activity, measured as in vivo oxygen emission, occurred. This was plausibly due to the increase of the rate of enzymatic reactions. The comparison of the in vivo fluorescence emission spectra (at 77K) of the algal cultures growth at the different temperatures evidenced a decrease of the emission peaks of PSII at the higher temperatures, suggesting a state transition of photosystems from state 1 (with LHCII associated to PSII) to state 2 (with LHCII moved to PSI). This change, which modified the excitation energy distribution between the photosystems was thermo-modulated and could be seen as an adaptive response of the algal cells. As regards the respiration rate of the algal cells, it, interestingly, did not show substantial changes in cultures grown at the different temperatures. Respiration analyses, accomplished after treatments with inhibitors of the distinct mitochondrial electron transport pathways, as well as Western analysis carried out with an antibody against the alternative oxidase (AOX) suggested that the lack of decrease in oxygen consumption at the lower temperature could depend on a great activity of the alternative cyanide-resistant pathway. The more suitable temperature for the growth of the cryophilic microalga, however, was that of 4°C. In fact, the higher temperatures, although maintaining and even increasing the growth rate, led in time to an anticipated senescence of the algal cultures. The first cyanobacterium from the Euganean thermal waters which was studied was a coccoid strain that the phylogenetic analysis led to consider as a new species of the Cyanobacterium genus. This cyanobacterium was named Cyanobacterium aponinum. Analyses carried out on cultures maintained at different temperatures showed that C. aponinum grew at temperatures in the range of about 20-40°C. However, in comparison with the ovoidal cells grown at 30°C, those maintained at 40°C, which had become roundish and larger, showed a decrease of photosynthesis (as in vivoO2 emission referred to chlorophyll amount), a reduction of the quantity of D1 protein of the reaction centre of PSII, evidenced by Western analysis, and a lowering of the Fv/Fm value (as chlorophyll fluorescence emission). All these results suggested an inibition and a damage of some PSII units. Interestingly, the analysis of in vivo fluorescence emission at 77K showed that the increase of growth temperature from 30° to 40°C in cells exposed to the same light conditions caused a transition of the photosystems from the state 1, with the phycobilisomes linked to the PSII, to the state 2, with the phycobilisomes linked to PSI. The increase of the temperature at 45°C led to the death of the cyanobacterium in few days. The death occurred surprisingly through an orderly programme of events. More precisely, a gradual and directed demolition of the photosynthetic pigments and thylakoids occurred from the centre towards the periphery of the cells, evidenced by a disappearance of chlorophyll autofluorescence. Moreover, the DAPI staining and the electrophoretic analysis showed that the DNA was degraded and a Western analysis demonstrated the increase in the algasl cells of a protein recognized by an antibody against the human caspase-3. At the end, only ghosts of cells remained, which were apparently integral but lacking in thylakoids and in all the other recognizable cellular components. Another cyanobacterium studied was a filamentous strain, which grew in culture at temperatures higher than 20°C and lower that 55°C. The phylogenetic analysis led to consider the examined strain as a species belonging to the genus Leptolyngbya, so that it was named Leptolyngbya sp. ETS-04. At the transmission electron microscope the strain grown at 30°C was constituted by rectangular cells with peripheral thylakoids arranged parallel to the cell wall. The filaments were surrounded by a well defined sheath. The increase of temperature at 40°C did not change the ultrastructural features of the Leptolyngbya sp. ETS-04 cells. At 50°C the cyanobacterium was characterized by huge mucilaginous sheaths, which could enclode more than one filament. On a dry weight basis not substantial differences in photosynthetic pigments were found between cultures grown at 30 and 40°C. At 50°C, instead, all the pigment contents decreased. The changes of temperature did not affect the phycobiliprotein ratios showing the preminence (about 70%) of phycocyanin. The in vivo fluorescence emission (at 77K) of the cyanobacterium cells, grown at the three temperatures, did not exhibit remarkable differences both with excitation at 560 nm (for phycobilins) and at 440 nm (for chlorophyll a) and showed that the photosystems remained in the transition state 1. The very high peaks of the phycocyanin confirmad the presence of phycobilisomes very rich in this pigment. The photosynthetic activity, measured as in vivo O2 evolution on the basis of chlorophyll amount, decreased in the culture grown at 50°C. Furthermore, also a reduction in the Fv/Fm ratio values (as chlorophyll fluorescence emission) occurred at this temperature. All this suggested some inactivation of PSII. Thus, the temperature of 50°C, although permitting the growth in culture of Leptolyngbya sp. ETS-04, results to be already stressing for this thermal strain. The increase of the temperature at 55°C led to the death of the cyanobacterium in few days. The death occurred through a massive demolition of the cells, with some debrises remaining inside the mucilaginous sheaths. The last cyanobacterium studied was a filamentous strain, which was characterized only from a morphological, ultrastructural and phylogenetic point of view. The morphological features were comparable with those of Conferva duplisecta Pollini, described in 1817 and never more signalized in the Euganean District. Morphological, ultrastructural and molecular analyses allowed a more precise definition of the taxonomy of this cyanobacterium ascribing it to the genus Oscillatoria. On the basis of the obtained results and of the more recent cyanobacterium taxonomy the name suggested for this isolated was Oscillatoria duplisecta.