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Lichen: from genome to ecosystem in a changing world 3I-P (3I – P8) Submission ID: IAL0242-00001 DIFFERENT STRATEGIES TO ACHIEVE Pb-TOLERANCE IN THE TWO TREBOUXIA PHYCOBIONTS OF THE LICHEN RAMALINA FARINACEA Casano L.M. 1 , Guera A. 1 , Del Campo E. M. 1 , Barreno E. 2 , Garcia-Breijo F. J. 3 , Alvarez R. 1 , Del Hoyo A. 1 , Reig-Armiñana J. 4 1 Plant Biology, University of Alcala, Madrid, Alcala de Henares, Spain 2 Botany, Icbibe, University of Valencia, Valencia, Burjassot, Spain 3 Ecosistemas Agroforestales, Universidad Politecnica de Valencia, Valencia, Valencia, Spain 4 Botany, Icbibe-jardi Botanic , University of Valencia, Valencia, burjassot, Suriname Ramalina farinacea L. (Ach.) has a relatively high tolerance to Pb (Branquinho et al., 1999). On the other hand, our group demonstrated that this lichen contains two Trebouxia phycobionts (provisionally named TR1 and TR9). TR1 and TR9 showed distinct physiological responses to oxidative stress caused by a strong ROS propagator, which produced a more severe decay in photosynthesis, photosynthetic pigments and proteins in TR1. Antioxidant enzymes were decreased by oxidative stress in TR1, but increased in TR9. Since Pb toxicity is caused by enhanced ROS formation, we hypothesized that TR9 phycobiont would be relatively more Pbtolerant than TR1. Therefore, in the present study we searched for physiological differences between isolated TR1 and TR9 under Pb exposure by analyzing the extracellular and intracellular Pb accumulation, photosynthetic pigments and chlorophyll fluorescence parameters. Additionally, the detoxification response to Pb was estimated by several important antioxidant enzymes: glutathione reductase (GR), superoxide dismutase (SOD), ascorbate peroxidase (APx) and catalase. As expected, extracellular Pb increased with the augment of Pb in both TR1 and TR9. However, TR9 immobilized significantly more metal than TR1 at extracellular level. The intracellular Pb uptake followed an opposite trend to that observed for the extracellular Pb, being approximately three times higher in TR1 than in TR9. However, photosynthetic pigments were not strongly affected by the presence of Pb within cells in either TR1 or TR9, since it was not observed any pigment decay higher than 10% (respect to controls). Accordingly, photosynthetic electron transport was not affected by the presence of Pb, as evidence by absence of PSII photoinhibition and the lack of effect on the ΦPSII in both phycobionts. Control levels of GR, SOD and APx were significantly higher in TR1 that in TR9. However, Pb induced the three enzymes in TR9 while it had no effect on TR1, so that antioxidant activities were quantitatively similar in both phycobionts under Pb treatments. In conclusion, each algal species seem to have acquired similar levels of Pb tolerance through the integration of distinct morphological, biochemical and physiological features. [MCINN (CGL2009-13429-C02- 01/02), AECID (PCI_A_l024755/09) and Generalitat Valenciana (PROMETEO 174/2008 GVA)] 136
The 7 th International Association for Lichenology Symposium 2012 (3I – P9) Submission ID: IAL0249-00001 AMYLOLYTIC ACTIVITIES OF SOME THERMOPHILIC MYCOBIONTS ISOLATED IN NIGERIA Ogunleye A. O. 1 1 Department of Biochemistry and Microbiology, Lead City University, Ibadan, Oyo State, Nigeria A total of 16 species of fungi were isolated from 3 locations viz:-two refuse-dump sites and palm kernel stack in Ibadan, Nigeria. Out of the 16 isolates obtained at 45 ° C incubation temperature, 9 were found to be thermotolerant while the other 7 were found to be thermophilic. The thermophilic and thermotolerant fungi were separated from the others by incubating at a low temperature of 12 °C at which the thermotolerant fungi grew, while the thermophilic did not grow at this temperature. All the fungi (thermophilic and thermotolerant) were screened for possible antimicrobial activities using different media sources. However, none of the screened organisms showed sign of antimicrobial activity by having no clear zone of inhibition against the test organisms. The test organisms used were Bacillus subtilis and Staphylococcus aureus. The conditions necessary for the production of extracellular amylase enzyme of thermophilic fungi isolated were determined using a stationary liquid medium of starch-yeast extract. Amylases were produced by all the thermophilic fungi. The amylase activities of all the fungi used were determined at pH 6.9. The peak activities for the enzyme (amylase) were shown to be at 45 ° C. The possible use of extracellular enzymes from these fungi for various purposes is discussed. (3I – P10) Submission ID: IAL0266-00001 PHYCOBIONTS IN SUSPENSION: METHODS OF QUANTIFICATION Catala M. 1 , Dominguez N. 1 , Moreno H. 1 , Barreno E. 2 1 Biology and Geology, Rey Juan Carlos University, Mostoles, Spain 2 Inst. Cavanilles of Biodiversity and Evolutionary Biology, Botany Dept., Faculty of Biology, University of Valencia, Burjassot, Spain The quantification of phycobiont concentration in suspensions is usually performed under the microscope by means of hemocytometric chambers. This method is time consuming and the presence of phycobiont clusters increases experimental error. Future advances in phycobiont biology require rapid and precise surrogate methods. Optical density (OD) has long been used in microbiology since it is simple and rapid. Chlorophyll autofluorescence can be used as a measure of cell number, a widespread method in green algae research. Finally, the use of specific stoichiometric fluorescent probes, such as Hoechst, allows precise quantification of DNA. Fluorometric methods are extremely sensitive and very low concentrations can be detected. Furthermore, the use of microplates and plate-readers can greatly assist in this task, since tens of low volume samples can be measured simultaneously. The aim of this work is the study of different surrogate methods for the quantification of phycobiont suspensions. Two axenic strains, Asterochloris erici Ahmadjian (SAG 32.85 = UTEX 911), and Trebouxia sp.(TR9) isolated from Ramalina farinacea, were studied. Suspensions were grown in 3NBBM (16:8 h light:dark) at 20ºC and 15ºC, respectively. A Fuchs-Rosenthal hemocytometric chamber was used for cell counts. OD (650 nm) was measured in fresh 100 µl-aliquots with an Anthos 2010 plate-reader. Chlorophyll autofluorescence (λexc 485 nm, λem 635 nm) and DNA-bound Hoechst fluorescence (λexc 360, λem 465 nm) were measured in a TECAN SPECTRAFluor Plus plate-reader. Frozen 200 µl aliquots were used for fluorometric methods. Chlorophyll samples were thawed and directly measured, DNA samples were previously lysed in a DNA buffer. The results show that OD gives reproducible, rapid results, using low volumes. However, its sensitivity is the lowest. Chlorophyll autofluorescence is rapid and sensitive but requires the availability of a fluorometer, as DNA quantification, that also needs a short sample processing. However, both OD and chlorophyll autofluorescence may be affected by changes during phycobiont cell cycle (i.e. debris, cluster/cell size, metabolism). We conclude that the measurements studied may be used as surrogates of cell number but the limitations of each method must always be considered. [MCINN (CGL2009-13429-C02-01/02), AECID (PCI_A_l024755/09) and Generalitat Valenciana (PROMETEO 174/2008 GVA)] 137 3I-P
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The 7 th IAL Symposium 2012 Lichens
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The 7 th International Association
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Report The 7 th International Assoc
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IAL COUNCIL (2008-2012): The 7 th I
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