The organic air pollutant cumene hydroperoxide interferes with NOantioxidant role in rehydrating lichen

Organic pollutants effects on lichens have not been addressed. Rehydration is critical for lichens, a burst of free radicals involving NO occurs. Repeated dehydrations with organic pollutants could increase oxidative damage. Our aim is to learn the effects of cumene hydroperoxide (CP) during lichen rehydration using Ramalina farinacea (L.) Ach., its photobiont Trebouxia spp. and Asterochloris erici. Confocal imaging shows intracellular ROS and NO production within myco and phycobionts, being the chloroplast the main source of free radicals. CP increases ROS, NO and lipid peroxidation and reduces chlorophyll autofluorescence, although photosynthesis remains unaffected. Concomitant NO inhibition provokes a generalized increase of ROS and a decrease in photosynthesis. Our results suggest that CP induces a ompensatory hormetic response in Ramalina farinacea that could reduce the lichen s antioxidant resources after repeated desiccation-rehydration cycles. NO is important in the protection from CP.This project was funded by the Spanish Ministry of Education and Science [project numbers CGL2012-40058-C02-01 and CGL2009-13429-C02-01], project Prometeo 2008/174 of the Generalitat Valenciana and the project AECID PCI/A/024755/09 of the Spanish Ministry of Foreign Affaires.Catalá, M.; Gasulla Vidal, F.; Pradas Del Real, A.; García Breijo, FJ.; Reig Armiñana, J.; Barreno Rodriguez, E. (2013). The organic air pollutant cumene hydroperoxide interferes with NOantioxidant role in rehydrating lichen. Environmental Pollution. 179:277-284. https://doi.org/10.1016/j.envpol.2013.04.015S27728417

Coexistence of different intrathalline symbiotic algae and bacterial biofilms in the foliose Canarian lichen Parmotrema pseudotinctorum

[EN] Parmotrema pseudotinctorum (des Abb.) Hale is a foliose lichen able to colonize large areas on rock surfaces in semiarid and warm localities in the Canary Islands. In this contribution, we investigate the phycobionts of this successful lichen under these extreme environmental conditions using ultrastructural and genetic methodologies. Two populations from La Gomera and La Palma islands were analyzed. After TEM analyses, three algal types were clearly distinguished in intrathalline symbiosis, provisionally named Ph1, Ph2, and Ph3. Two of them (Ph1 and Ph2) were Trebouxia showing a well visible pyrenoid corticolatype the chloroplast thylakoids being very different in both. The type Ph3 could be a taxon included in the genus Asterochloris. Our molecular approach consisted in sequencing two different DNA loci: a portion of the chloroplast psbA gene and nuclear ITS. Sequences of the psbA gene resulted in electrophoretograms showing double peaks when DNA extracted from the whole lichen thallus was used as template. Such double peaks were interpreted as single nucleotide polymorphisms (SNPs). This interpretation was confirmed by cloning. However, no intrathalline polymorphisms were detected among the nrITS sequences. Phylogenetic analyses on the basis of the psbA gene revealed three distinct clades. It is likely that these clades corresponded to the the three different morphotypes revealed by TEM. One of these clades, was closely related to T. corticola, other was related to Asterochloris glomerata and the third did not grouped with any specific taxa. These results are the first piece of evidence that algal coexistence may even be established between species of different genera of the Trebouxiophyceae (Asterochloris and Trebouxia at least). Moreover, the coexistence of several microalgal taxa evidenced in this study appears as a consistent character among the populations of this foliose lichen. Further isolation and cultivation of the three different algal types and physiological studies should shed light on the ecological plasticity of the entire holobiont. Along with such variety of intrathalline coexisting algae, another unexpected result was the observation of an almost continuous layer of bacterial-communities coating the lower cortex in all the studied samples of P. pseudotinctorum. The function of these biofilms in the lichen symbiosis remains to be elucidated. The existence of such particular symbiosis involving different algal species and bacteria could be explained by an increased fitness in particular habitats or under specific environmental conditions[ES] Parmotrema pseudotinctorum (des Abb.) Hale es un liquen foliáceo que coloniza roquedos volcánicos y es frecuente en localidades semiáridas y cálidas de las islas Canarias. En este trabajo, se investigan los ficobiontes de este liquen utilizando métodos ultraestructurales y genéticos. Se analizaron dos poblaciones de La Gomera y La Palma. Mediante análisis TEM, se han distinguido claramente tres tipos de algas en simbiosis intratalinas, llamados provisionalmente Ph1, Ph2 y Ph3. Dos de ellas (Ph1 y Ph2), pertenecientes al género Trebouxia, muestran un pirenoide bien visible del tipo corticola, pero los tilacoides del cloroplasto son muy diferentes en ambos. El tipo Ph3 podría ser un taxón incluido en el género Asterochloris. El enfoque molecular consistió en la secuenciación de dos loco diferentes de ADN: una porción del gen psbA del cloroplasto e ITS nuclear. Las secuencias del gen psbA proporcionaron electroforetogramas que mostraban dobles picos cuando se usó como patrón el ADN extraído de los talos completos. Estos dobles picos se interpretaron como polimorfismos de nucleótido simple (SNP). Esta interpretación se confirmó mediante técnicas de clonación. Sin embargo, no se detectaron polimorfismos intratalinos con las secuencias nrITS. Los análisis filogenéticos basados en las secuencias del gen psbA constataron la presencia de tres clados diferenciados. Es probable que éstos correspondan a los tres diferentes morfotipos puestos en evidencia por TEM. Uno de ellos está estrechamente relacionado con T. corticola, otro con Asterochloris glomerata, y el tercero no se agrupaba a ningún taxón especifico. Estos resultados son la primera evidencia de que en los líquenes la coexistencia de algas intratalinas se puede establecer incluso entre especies de difierentes géneros de Trebouxiophyceae (Asterochloris y Trebouxia al menos). Por otra parte, la coexistencia de varios taxones de microalgas, constatada en este estudio, puede considerarse como una sólida característica entre las poblaciones de este liquen foliáceo. Posteriores aislamientos y el cultivo de los tres tipos de algas, además de estudios fisiológicos, podrán explicar la plasticidad ecológica que muestra el holobionte. Otro resultado inesperado fue la observación de una capa casi continua de comunidades bacterianas que recubren el córtex inferior en todas las muestras estudiadas de P. pseudotinctorum. Está por determinar la función que estos biofilms puedan tener en las simbiosis liquénicas. La existencia de estos tipos de simbiosis tan particulares, que involucran tanto a diferentes especies de algas como a bacterias, podría explicarse por un incremento en la capacidad de colonizar hábitats peculiares o con condiciones ambientales específicasThis study was funded by the Spanish Ministry of Economy and Innovation (MINECO CGL2012-40058-C02-01/02), FEDER and the Generalitat Valenciana (PROMETEO 021/2013 GVA).Molins, A.; García-Breijo, F.; Reig Armiñana, J.; Del Campo, EM.; Casano, L.; Barreno Rodriguez, E. (2013). Coexistence of different intrathalline symbiotic algae and bacterial biofilms in the foliose Canarian lichen Parmotrema pseudotinctorum. Vieraea. Folia Scientarum Biologicarum Canariensium. 41:349-370. http://hdl.handle.net/10251/77348S3493704

Fungal-associated NO is involved in the regulation of oxidative stress during rehydration in lichen symbiosis

[EN] Background Reactive oxygen species (ROS) are normally produced in respiratory and photosynthetic electron chains and their production is enhanced during desiccation/rehydration. Nitric oxide (NO) is a ubiquitous and multifaceted molecule involved in cell signaling and abiotic stress. Lichens are poikilohydrous organisms that can survive continuous cycles of desiccation and rehydration. Although the production of ROS and NO was recently demonstrated during lichen rehydration, the functions of these compounds are unknown. The aim of this study was to analyze the role of NO during rehydration of the lichen Ramalina farinacea (L.) Ach., its isolated photobiont partner Trebouxia sp. and Asterochloris erici (Ahmadjian) Skaloud et Peksa (SAG 32.85 = UTEX 911). Results Rehydration of R. farinacea caused the release of ROS and NO evidenced by the fluorescent probes DCFH2-DA and DAN respectively. However, a minimum in lipid peroxidation (MDA) was observed 2 h post-rehydration. The inhibition of NO in lichen thalli with c-PTIO resulted in increases in both ROS production and lipid peroxidation, which now peaked at 3 h, together with decreases in chlorophyll autofluorescence and algal photobleaching upon confocal laser incidence. Trebouxia sp. photobionts generate peaks of NO-endproducts in suspension and show high rates of photobleaching and ROS production under NO inhibition which also caused a significant decrease in photosynthetic activity of A. erici axenic cultures, probably due to the higher levels of photo-oxidative stress. Conclusions Mycobiont derived NO has an important role in the regulation of oxidative stress and in the photo-oxidative protection of photobionts in lichen thalli. The results point to the importance of NO in the early stages of lichen rehydration.This project was funded by the Spanish Ministry of Education and Science [project numbers CGL2006 12917 C02 0 and CGL2009 13429 C02 01], project Prometeo 2008/1/4 of the Generalitat Valenciana and the project AECID PCI/A/024755/09 of the Spanish Ministry of Foreign Affaires. We are grateful to F. Gasulla, J. Gimeno-Romeu, E. Barreno, (ICBIBE, University of Valencia) and A. Guera (Plant Biology, University of Alcala) for communicating unpublished data, to Dr. R. Catala (CIB, Madrid), Dr. P. D'Ocon (UVEG, Valencia) and Dr. J. Medina (INIA, Madrid) for critical revision of the manuscript, and J. L. Rodriguez Gil for MDA protocol optimization. English revision was done by Wendy Ran.Catalá, M.; Gasulla Vidal, F.; Pradas Del Real, AE.; García-Breijo, F.; Reig Armiñana, J.; Barreno Rodriguez, E. (2010). Fungal-associated NO is involved in the regulation of oxidative stress during rehydration in lichen symbiosis. BMC Microbiology. 10. https://doi.org/10.1186/1471-2180-10-297S1

Nitric oxid is involved in oxidative stress during rehydration of Ramalina farinacea (L.) Ach. in the presence of the oxidative air pollutant cumene hydroperoxide

[EN] Air pollution has dramatically decreased lichen diversity in polluted areas. However, the causes underlying this sensitivity are poorly known, hindering the prevention of biodiversity loss. Nitric oxide (NO) is a bioactive gas involved in stress signalling and free radical (ROS) defence, in addition to playing a role in complex atmospheric pollution chemistry. NO production in lichens during their rehydration was recently demonstrated. The aim of this study was to analyze the role of NO in the lichen Ramalina farinacea during rehydration in the presence of the oxidative air pollutant cumene hydroperoxide. To this end, ROS were visualized in vivo using the fluorescent probe DCFH2-DA, NO action was specifically inhibited with c-PTIO, and lipid peroxidation during rehydration in the absence or presence of cumene hydroperoxide was quantified. The results indicated that cumene hydroperoxide causes an increase in the release of intracellular free radicals within the fungal hyphae, together with lipid peroxidation of the whole lichen. Lipid peroxidation, but not oxidative stress reverted to physiological values after NO inhibition. We conclude that, in lichens, NO may be involved in the regulation of the oxidative stress caused by air pollution.This project was funded by the Spanish Ministry of Education and Science [CGL2006-12917-C02-01, CGL2009-13429_C02-00], and the Generalitat Valenciana [PROMETEO 174/2008 GVA].Catalá, M.; Gasulla Vidal, F.; García-Breijo, F.; Reig Armiñana, J.; Barreno Rodriguez, E. (2010). Nitric oxid is involved in oxidative stress during rehydration of Ramalina farinacea (L.) Ach. in the presence of the oxidative air pollutant cumene hydroperoxide. Bibliotheca Lichenologica. 105:87-92. http://hdl.handle.net/10251/85604S879210

South European populations of Ramalina farinacea (L.) Ach. share different Trebouxia algae

[EN] Little is currently known about the population structure of lichen photobionts. Accordingly, in this study we analyzed the nature and distribution of phycobionts in different Spanish populations of Ramalina farinacea. Two different Trebouxia algae were isolated and identified on the basis of anatomical and chloroplast LSU rDNA sequence studies. Interestingly, most of the thalli contained the same two algae. To search for possible genetic variability among these algae, the internal transcribed sequences (ITS) of nuclear rDNA were compared. Surprisingly, only one ITS sequence was found in each thallus, even in those exhibiting multiple morphologically differentiated algae observed in thallus TEM sections. Therefore, ITS sequence analyses are probably only indicative of the predominant photobiont while the other photobiont remains undetected. One photobiont seemed to predominate in the Canary Islands populations and the other in peninsular ones. Additionally, here we describe a new potential DNA marker, based on plastid LSU rDNA, which may allow rapid identification of coexisting algae within the same thallus.This project was funded by the Spanish Ministry of Education and Science [CGL2006-12917-C00, CGL2009-13429-C02-00], and Generalitat Valenciana [PROMETEO 174/2008 GVA]. Dr. A. BECK (Germany) and reviewers are thanked for helpful comments. Wendy Ran revised the manuscript in English.Del Campo, EM.; Gimeno, J.; De Nova, J.; Casano, L.; Gasulla Vidal, F.; García-Breijo, F.; Reig Armiñana, J.... (2010). South European populations of Ramalina farinacea (L.) Ach. share different Trebouxia algae. Bibliotheca Lichenologica. 105:247-256. http://hdl.handle.net/10251/85603S24725610