Antarctic Lichen Research Articles

Anti‐allergic effect of Ramalin from Ramalina terebrata in TNF‐α‐stimulated HaCaT and RBL‐2H3 cells

Allergies are immediate hypersensitive responses to antigens and IL‐4 is involved in the initiation and development of allergic responses. The present study was undertaken to examine whether ramalin, an antioxidant compound from the Antarctic lichen Ramalina terebrata, has an inhibitory effect on allergic response in TNF‐α‐stimulated HaCaT and RBL‐2H3 cells. In this study, the results of Real‐Time PCR analysis showed that mRNA levels of IL‐18, MCP‐1, RANTES and CCL17 were suppressed by ramalin in TNF‐α‐treated human keratinocyte (HaCaT) cells. Western blot analysis also indicated that ramalin inhibited the nuclear translocation of NF‐κB and phosphorylation of MAP kinases induced by TNF‐α. These results suggest that ramalin inhibits TNF‐α‐stimulated chemotactic cytokines through the suppression of NF‐κB signaling pathways and MAP kinase pathways. In addition, Real‐Time PCR analysis and ELISA showed that ramalin suppressed the mRNA levels and secretion of IL‐4. Expression of NFAT2 was also attenuated by ramalin in antigen‐mediated rat basophilic leukemia (RBL‐2H3) cells. Taken together, these results suggest that ramalin has an anti‐allergic effect and is a candidate for the therapeutic agent to treat allergy symptoms.

Microsatellite analyses of the Antarctic endemic lichen Buellia frigida Darb. (Physciaceae) suggest limited dispersal and the presence of glacial refugia in the Ross Sea region

In order to assess the origins of Antarctic lichens (local or long distance), we examined the population genetic structure of the endemic Antarctic lichen Buellia frigida across a latitudinal gradient of roughly 10° along the Transantarctic Mountains, Western Antarctica, using four microsatellite loci. All loci were highly polymorphic. Data were analysed as both biallelic (dikaryotic) and as haploid in order to determine whether different life-cycle phases could influence our interpretation of population structure. For biallelic data, allelic richness (A) ranged from 5.25 to 7.99 and measures of diversity suggested low levels of gene flow among most sites (e.g. F ST = G ST = 0.09–0.31; D EST = 0.03–0.7). For haploid data, allelic richness (A) ranged from 3.5 to 5.46, private allelic richness (A r) ranged from 0.81 to 2.05, Nei’s unbiased genetic distance ranged from 0.15 to 1.42 and Nei’s unbiased genetic identity ranged from 0.24 to 0.86 among locations. Two locations, the McMurdo Dry Valleys and Queen Maud Mountains, stand out as possible glacial refugia, with both having a high number of private alleles. Despite the high potential for wind-dispersed spores, it appears likely that successful colonisation in different areas is restricted. One possible explanation is that the combination of ice-free conditions and water availability occurs only during the short summer period when the prevailing wind patterns may influence dispersal pathways. Dispersal from the southernmost site (Queen Maud Mountains) appears particularly restricted and may be the result of dispersal barriers such as glaciers. We conclude that a combination of prevailing wind patterns and physical barriers restrict spore settlement and therefore dispersal and recruitment among regions.

Production of UV-B screens and changes in photosynthetic efficiency in Antarctic Nostoc commune colonies and a lichen Xanthoria elegans depend on a dose and duration of UV-B stress

The survival of non-vascular autotrophs in the extreme polar conditions and the principles of their high tolerance to extreme physical factors have been intriguing scientists in last decades. Therefore, this study focuses on the capacity of production of UV-B screening pigments in two model Antarctic species, one algal lichen, and colony of a cyanobacterium. Dose-dependent activation of protective mechanisms of Antarctic cyanobacterium (Nostoc commune) and algal lichen (Xanthoria elegans), synthesis of UV-B screening compounds in particular, were studied together with the changes in photosynthetic efficiency induced by a background photosynthetically active radiation (PAR) supplemented with UV-B radiation. The samples were exposed to different doses of UV-B (280–320 nm), low (0.7 W m-2), medium (1.5 W m-2) and high (3.0 W m-2) for 5 days. Untreated samples (control) were shielded from UV-B radiation during experiment. Chlorophyll fluorescence parameters and secondary UV-B protective metabolites were analysed in the intervals of 24 h, 48 h and 120 h. Amount of UV-B screening pigments was measured spectrophotometrically using several specific wavelengths in UV-B absorption range. Results showed that if exposed to a low dose of UV-B radiation or a short-term treatment, both species exhibited an increase in UV-B screening pigments to protect the lichen photobiont against UV-B damage. However, if exposed to a high dose of UV-B radiation or a long-term treatment, a decrease of UV-B screening compounds occured. This implies that Antarctic lichen and cyanobacterium can protect themselves against an increase of stress factors ranging within physiological limits, like e.g. increased synthesis of UV-B screening compounds thanks to a thinning of the ozone layer and consequent increase in UV radiation doses incident on Antarctic terrestrial ecosystems. Nevertheless, the likely increased UV-B radiation due to more intense depletion of stratospheric ozone layer may lead to alterations in UV-B tolerance in Antarctic lichens in future.

PHYTOSOCIOLOGICAL APPROACH OF LICHENS IN THE ICE-FREE AREAS ADJOINING THE ARCTOWSKI REGION, ADMIRALTY BAY, KING GEORGE ISLAND, ANTARCTICA

The Antarctic lichens have been used as bioindicators in biomonitoring studies, having an important contribution to the floristic composition, and its existence is dependent on ice-free regions. The phytosociological data were obtained according to the Braun-Blanquet sampling method. This study compares the lichen formation of Arctowski region, using data collected during the Brazilian Antarctic Program expedition conducted in the austral summer of 2003/2004 and 2012/2013. According to data collected at the moment, increases of the lichens occurrences in the plant formations in the region were found. Studies indicate that abiotic factors, once linked to local factors can to affect the distribution of the communities in the environment. This essay suggests the fragility of lichen formation in the ice-free areas of Arctowski region. However, a long term evaluation as necessary to confirm this hypothesis. The phytosociological studies can contribute to the management of scientific activities involved with the Brazilian Antarctic Program.

Features of chlorophyll fluorescence transients can be used to investigate low temperature induced effects on photosystem II of algal lichens from polar regions ( Short Communication )

Chlorophyll fluorescence is an effective tool for investigating characteristics of any photosynthesizing organisms and its responses due to different stressors. Here, we have studied a short-term temperature response on three Antarctic green algal lichen species: Umbilicaria antarctica, Xanthoria elegans, and Rhizoplaca melanophtalma. We measured slow chlorophyll fluorescence transients in these Antarctic lichen species during slowely cooling of thallus temperature from 20°C to 5, 0 and -5°C with 20 minute acclimation at each temperature. The measurements were supplemented with saturation pulses for the analysis of chlorophyll fluorescence parameters: maximum yield of PS II photochemistry (FV/FM), effective quantum yield of PS II photochemistry (FPSII) and quenching parameters. In response to decreasing thallus temperature, we observed species-specific changes in chlorophyll fluorescence parameters as well as in the shape of the chlorophyll fluorescence transients. We propose that species-specific changes in the slow phase of chlorophyll fluorescence transients can be potentially used as indicators of freezing stress in photosynthetic apparatus of lichen algal photobionts.

English

Recent studies suggest that bacterial communities in lichens contribute structurally and ecologically, but their biological activities are not fully investigated. In this study, we explored biological potential of microorganisms that are isolated from Antarctic lichen Psoroma sp. Using their bacterial cell culture extracts, we evaluated antibacterial and antioxidant properties. Among 20 bacterial species that were isolated from Antarctic lichen Psoroma sp., PAMC 26508 (Streptomyces sp., similarity: 100%) showed antibacterial activities against all target bacteria with inhibition zone diameter of 7 to 9. PAMC 26537 (Burkholderia sordidicola, similarity: 98.828%) exhibited higher antioxidant potential with an inhibition rate of 60.12% in 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 58.69% in 2,2'-azino-bis[3-ethyl benzothiazoline-6-sulphonic acid] (ABTS) assay than control ascorbic acid (29.31%). Our results indicated that they have potential to be used as novel source of antibacterial and antioxidant agents. Key words: Antarctica, antibacterial/antioxidant, lichen associated bacteria, lichen, Psoroma sp.

Changes in photosynthesis, pigment composition and glutathione contents in two Antarctic lichens during a light stress and recovery

Over last decades, several studies have been focused on short-term high light stress in lichens under laboratory conditions. Such studies reported a strong photoinhibition of photosynthesis accompanied by a partial photodestruction of PSII, involvement of photoprotective mechanisms, and resynthetic processes into gradual recovery. In our paper, we applied medium [800 μmol(photon) m-2 s-1] light stress to induce negative changes in PSII funcioning as well as pigment and glutathione (GSH) content in two Antarctic fruticose lichen species. Chlorophyll (Chl) fluorescence parameters, such as potential and effective quantum yield of photosynthetic processes and fast transients (OJIP) recorded during high light exposition and recovery, revealed that Usnea antarctica was less susceptible to photoinhibition than U. aurantiaco-atra. This might be supported by a more pronounced high light-induced reduction in Chl a and b contents in U. aurantiaco-atra compared with U. antarctica. In both experimental species, total GSH showed an initial increase during the first 30-40 min of high light treatment followed by a decrease (60 min) and an increase during dark recovery. Full GSH recovery, however, was not finished in U. aurantiaco-atra even after 5 h indicating lower capacity of photoprotective mechanisms in the species. OJIP curves showed high light-induced decrease in both species, however, the recovery of the OJIPs shape to pre-photoinhibitory values was faster and more apparent in U. antarctica than in U. aurantiaco-atra. The results are discussed in terms of sensitivity of the two species to photoinhibition and their photosynthetic performance in natural environment.

Algal and fungal diversity in Antarctic lichens.

The composition of lichen ecosystems except mycobiont and photobiont has not been evaluated intensively. In addition, recent studies to identify algal genotypes have raised questions about the specific relationship between mycobiont and photobiont. In the current study, we analyzed algal and fungal community structures in lichen species from King George Island, Antarctica, by pyrosequencing of eukaryotic large subunit (LSU) and algal internal transcribed spacer (ITS) domains of the nuclear rRNA gene. The sequencing results of LSU and ITS regions indicated that each lichen thallus contained diverse algal species. The major algal operational taxonomic unit (OTU) defined at a 99% similarity cutoff of LSU sequences accounted for 78.7-100% of the total algal community in each sample. In several cases, the major OTUs defined by LSU sequences were represented by two closely related OTUs defined by 98% sequence similarity of ITS domain. The results of LSU sequences indicated that lichen-associated fungi belonged to the Arthoniomycetes, Eurotiomycetes, Lecanoromycetes, Leotiomycetes, and Sordariomycetes of the Ascomycota, and Tremellomycetes and Cystobasidiomycetes of the Basidiomycota. The composition of major photobiont species and lichen-associated fungal community were mostly related to the mycobiont species. The contribution of growth forms or substrates on composition of photobiont and lichen-associated fungi was not evident.

Ramalin suppresses adipocyte differentiation though the MAPKs pathway in 3T3‐L1 preadipocyte (657.10)

Obesity is a main risk factor for several metabolic disorders such as type 2 diabetes, hypertension, and cardiovascular diseases. Obesity is also recognized as a low‐grade chronic inflammation. In the current study, the inhibitory effect of ramalin, isolated from Antarctic lichen, Ramalina terebrata, on adipocyte differentiation in 3T3‐L1 cells was examined. Ramalin attenuated the accumulation of lipid droplets stained with Oil red O in non‐toxic concentration. Western blot assay showed that ramalin remarkably inhibited the level of peroxisome proliferator‐activated receptor γ (PPAR γ) and CCAAT/enhancer‐binding protein β (c/EBP β) as well as CCAAT/enhancer‐binding protein α (c/EBP alpha) in a concentration dependent manner. Real‐time PCR assay revealed that the expression of MDI‐induced adipogenesis related genes (GLUT4, aP2, leptin) was significantly suppressed by ramalin. Our data also showed that ramalin significantly decreased phosphorylation of ERK1/2, p38 and JNK in a concentration‐dependent manner. Results from ELISA assay demonstrated that ramalin reduced the production of IL‐6 and TNF‐α. Taken together, ramalin can be a novel therapeutic candidate for controlling adipogenesis in preadipocytes.

The apoptotic effect of ramalin on human breast cancer cells (657.11)

Ramalin is the compound isolated from Antarctic lichen, Ramalina terebrata. Previous study showed that ramalin has antioxidant and anti‐inflammatory activity. However, the effect of ramalin on MCF‐7 human cancer cells has not been addressed. We investigated the effect of ramalin on cell proliferation, apoptosis, and its mechanism in MCF‐7 cells. MTT assay and western blotting showed that ramalin decreased concentration‐dependently cell proliferation, induced apoptosis related proteins (Bax, Bid, AIF), and activated caspase‐3 and 8, respectively. In addition, Annexin V/PI analysis revealed that apoptotic cells were remarkably increased in ramalin‐treated MCF‐7. Our data also showed that ramalin increased phosphorylation of ERK1/2, p38 and JNK in a concentration‐ and time‐dependent manner. Taken together, ramalin induces apoptosis through Mitogen‐activated protein kinases (MAPKs) and caspase dependent signaling pathways in MCF‐7 cells and may be a potential anticancer agent for the treatment of human breast cancer.

Anti‐obese effect of ramalin in high‐fat diet‐induced obese mice (824.7)

The recent rise in obesity is tightly associated with an increase in diseases such as type 2 diabetes, cardiovascular disease and hepatic steatosis. Ramalin, isolated from the Antarctic lichen Ramalin terebrata, has been known to have various biological activities including anti‐inflammatory activity. However, other biological activities of ramalin remain unknown. We examined the anti‐obesity effect of ramalin in five week‐old mice that were fed either a control high‐fat diet (HFD), or HFD with oral administration 50 mg/kg or 100 mg/kg of ramalin for 8 weeks. Mice fed with ramalin showed 25% reduced body weight gain and ramalin treatment significantly inhibited epididymal fat‐pad weights compared with mice fed with HFD. Ramalin also reduced the levels of glucose, hepatic triglyceride, serum total, high‐density lipoprotein (HDL) and Low‐density lipoprotein (LDL) cholesterol. ELISA assay revealed that ramalin remarkably inhibited HFD‐induced leptin cytokine level. Furthermore, Real time PCR assay showed that HFD‐induced adipose tissue genes, known as adipogenesis markers, were significantly suppressed by ramalin. Overall, these results suggest that ramalin might be a plausible candidate to control obesity and obesity‐related disorders.

The occurrence and development of peat mounds on King George Island (Maritime Antarctic)

On King George Island, South Shetlands Islands, a type of peat formation has been discovered which has not previously been reported from the Antarctic. These formations are in shape of mounds up to 7x 15 m in area, with a peat layer of about I m thick. About twenty five cm below the surface there is a layer of permanently frozen peat. The mounds are covered by living mosses (<em>Polytrichum alpinum</em> and <em>Drepanocladus uncinatus</em>), Antarctic hair grass (<em>Deschampsia antarctica</em>) and lichens. Erosion fissures occurring on the surface are evidence of contemporary drying and cessation of the mound's growth. The initial phase of the development of the mounds began with a community dominated by <em>Calliergidium austro-stramineum</em> and <em>Deschampsia antarctica</em>, and their further development has been due to peat accumulation formed almost entirely by <em>Calliergidium</em>. The location of the mounds is near a penguin rookery, which clearly conditioned the minerotrophic character of these formations, as compared with the "moss peat banks" formed by <em>Chorisodontium aciphyllum</em> and <em>Polytrichum al-pestre</em>. Moreover, the peat mounds differ from the latter in several ways, e.g. rate of growth and floristic composition. Radiocarbon dating of peat from the base of one mound gave an age of 4090±60 years B.P. This suggests that the age of the tundra on King George Island is about 5000-4000 years.

Photoinhibition of photosynthesis in Antarctic lichen Usnea antarctica. II. Analysis of non-photochemical quenching mechanisms activated by low to medium light doses

The paper focus sensitivity of an Antarctic lichen Usnea antarctica to photoinhibition studied under controlled laboratory conditions. Main emphasis was given to the analysis of quenching mechanisms, i.e. deexcitation pathways of absorbed light energy exploited in non-photochemical processes. Thalli of U. antarctica were collected at the James Ross Island, Antarctica (57°52´57´´ W, 63°48´02´´ S) and transferred in dry state to the Czech Republic. After rewetting in a laboratory, they were exposed to medium light intensities (300, 600 and 1000 mmol m-2 s-1 of photosynthetically active radiation) for 6 h. Before and during photoinhibitory treatments, chlorophyll fluorescence parameters, photoinhibitory (qI), state 1-2 transition (qT), and energy-dependent quenching (qE) in particular were measured to evaluate dose- and time-dependent changes in these parameters. The results showed that among the components forming non-photochemical quenching (qN), qI contributes to the largest extent to qN, while qE and qT contribute less. This finding differs from our earlier studies made in a short term-, and high light-treated U. antarctica that found qE together with qI is the most important part of non-photochemical quenching. Possible explanation is that photoinhibition in PS II in U. ant-arctica, when induced by low to medium light, activates qE to only limited extend and for a relatively short time (tens of minutes). With prolonged high light treatment lasting several hours, qE tends to be reduced to the values close to zero and qI then forms a major part of qN.

Adaptation of an Antarctic lichen to Martian niche conditions can occur within 34 days

Stresses occurring on the Martian surface were simulated in a Mars Simulation Chamber (MSC) and included high UV fluxes (Zarnecki and Catling, 2002), low temperatures, low water activity, high atmospheric CO2 concentrations, and an atmospheric pressure of about 800Pa (Kasting, 1991; Head et al., 2003). The lichen Pleopsidium chlorophanum is an extremophile that lives in very cold, dry, high-altitude habitats, which are Earth's best approximation of the Martian surface. Samples with P. chlorophanum were exposed uninterruptedly to simulated conditions of the unprotected Martian surface (i.e. 6344kJm−2) and protected niche conditions (269kJm−2) for 34 days. Under unprotected Martian surface conditions the fungal symbiont decreases its metabolic activity and it was unclear if the algal symbiont of the lichen was still actively photosynthesizing. However, under “protected site” conditions, the entire lichen not only survived and remained photosynthetically active, it even adapted physiologically by increasing its photosynthetic activity over 34 days.

Understanding lichen diversity on the Antarctic Peninsula using parataxonomic units as a surrogate for species richness

Expert collection of specimens in the field and further determination of species is the best method for determining species richness. However, the relative paucity of botanists working in Antarctica makes this approach impractical for broad‐scale surveys of Antarctic floral biodiversity. Lichens are the dominant macrophytes of terrestrial Antarctica and, as such, play a fundamental part of the ice‐free terrestrial ecosystem. Many distinct ice‐free terrestrial habitats in the Antarctic are not represented in the current network of Antarctic protected areas. However, it is difficult to identify appropriate areas for conservation because comprehensive data on distributional patterns of Antarctic flora are not available, and existing data for most Antarctic lichen species are not compiled. Consequently, cost‐effective survey methods and surrogates for the prediction of species richness are needed to accelerate assessments of local biodiversity and help select areas for conservation. A combination of a photographic “citizen scientist” approach for the collection of data and the use of parataxonomic unit (PU) richness as a surrogate for species richness, might be a possible solution to effectively collect preliminary information and rapidly build databases on species diversity. We have developed a database and gathered photographic information on lichen occurrences for sites that are frequently visited by tourists. We tested the identification capabilities with a reference data set of Antarctic lichen images from the U.S. National Herbarium. We showed that all species used in this test can be detected and that, for 74% of the images, all classifiers were able to identify the genus of the specimen. Twenty‐nine sites were photographically surveyed by researchers and tourists between 2009/2010 and 2011/2012 in the Antarctic Peninsula region. We estimated PU richness as a proxy for species richness for each of the 29 sites surveyed and provide two examples of potential applications. These surveys provide preliminary information for identifying areas for protection and priorities for future research.

A new pseudodepsidone from the Antarctic lichen Stereocaulon alpinum and its antioxidant, antibacterial activity

A new pseudodepsidone from the Antarctic lichen Stereocaulon alpinum and its antioxidant, antibacterial activity

Ramalin inhibits LPS‐induced autophagic response in macrophages through the downregulation of iNOS expression

Activation of macrophages is essential for the host defense system and accumulating evidence suggests that autophagy is an important component in macrophage activity. Prolonged activation of macrophages, however, becomes detrimental and contributes to the progression of chronic inflammatory diseases with abnormal cell death. Therefore, controlling the overactivation of macrophages may be a new therapeutic strategy for various inflammatory diseases. Here we showed that ramalin, an antioxidant compound from the Antarctic lichen Ramalina terebrata, inhibited both LPS‐induced autophagy and iNOS in macrophages. To verify the role of NO in autophagy, we compared autophagy‐related gene expression in LPS‐stimulated and SNP‐stimulated macrophages by western blotting. Interestingly, ramalin did not suppress NO and autophagy in macrophages exposed to NO donor, SNP. In addition, inhibition of NO production by SMT significantly reduced LPS‐induced autophagy. These finding imply that ramalin might suppress LPS‐induced autophagy by inhibiting NO secretion. Ramalin also showed inhibitory effect on LPS‐induced NF‐κB and MAPK activation. Overall, these finding indicate that ramalin suppresses autophagy in LPS‐stimulated macrophages by preventing NO production and downregulating both NF‐κB and MAPK activation.

ChemInform Abstract: New Diterpene Furanoids from the Antarctic lichen Huea sp.

AbstractFour new diterpene furanoids, hueafuranoids A—D, are isolated from the MeOH extract of Antarctic lichen Huea sp.

Photosynthetic symbionts in Antarctic terrestrial ecosystems: the physiological response of lichen photobionts to drought and cold

Lichens form an important part of the biodiversity in terrestrial ecosystems of Antarctica where they represent the dominant vegetation. Previous studies on the genetic diversity of photobionts of lichens have indicated that clade S Trebouxia photobionts are the most widespread in continental Antarctica, predominantly in macrolichens. For the first time, a comparative study of the physiology of a variety of isolated Antarctic lichen photobionts (genus Trebouxia) was performed. Photosynthetic activity was examined by chlorophyll a fluorescence and correlated with freezing and desiccation under laboratory conditions and photosynthetic pigments were quantified in response to desiccation. Data were obtained from photobionts collected from the Antarctic regions of North Victoria Land, Coal Nunatak and Rothera Point, as well as from a European site (Gotland, Sweden). While the isolated algae reacted individually to stress treatments, they were highly susceptible to desiccation stress but could rapidly recover from freezing. Photobiont-specific physiological adaptations are considered to explain the dominance of clade S Trebouxia photobionts.

New diterpene furanoids from the Antarctic lichen Huea sp

In the course of ongoing research on protein tyrosine phosphatase 1B (PTP1B) inhibitory compounds from Antarctic lichens, four new diterpene furanoids, hueafuranoids A–D (1–4) have been isolated from the MeOH extract of Antarctic lichen Huea sp. by various chromatographic methods. The structures of these compounds were elucidated by analysis of NMR and MS data, and comparing their spectral data with those in the literature. Compound 1 showed inhibitory activity against therapeutically targeted protein, PTP1B with an IC50 value of 13.9μM. The kinetic analysis of PTP1B inhibition by hueafuranoid A (1) suggested that the diterpene furanoids encountered in this study inhibited PTP1B activity in a non-competitive manner.