地衣次生代谢产物及其生物活性研究进展

地衣是一类独特的生物有机体,其体内合成了多种特殊的次生代谢产物,主要为缩酚酸、缩酚酸环醚、缩酚酮、二苯并呋喃等类化合物。这些次生代谢产物具有多样的生物活性,包括抗氧化、抗辐射、抗菌、抗病毒、抗肿瘤、植物生长抑制和昆虫拒食等活性。本文对近40年来有关地衣化学及生物活性方面的研究进行综述,为地衣资源的进一步研究和开发提供参考。     

Cyanolichens can have both cyanobacteria and green algae in a common layer as major contributors to photosynthesis

Background and Aims

Cyanolichens are usually stated to be bipartite (mycobiont plus cyanobacterial photobiont). Analyses revealed green algal carbohydrates in supposedly cyanobacterial lichens (in the genera Pseudocyphellaria, Sticta and Peltigera). Investigations were carried out to determine if both cyanobacteria and green algae were present in these lichens and, if so, what were their roles.

Methods

The types of photobiont present were determined by light and fluorescence microscopy. Small carbohydrates were analysed to detect the presence of green algal metabolites. Thalli were treated with selected strengths of Zn²⁺ solutions that stop cyanobacterial but not green algal photosynthesis. CO₂ exchange was measured before and after treatment to determine the contribution of each photobiont to total thallus photosynthesis. Heterocyst frequencies were determined to clarify whether the cyanobacteria were modified for increased nitrogen fixation (high heterocyst frequencies) or were normal, vegetative cells.

Key Results

Several cyanobacterial lichens had green algae present in the photosynthetic layer of the thallus. The presence of the green algal transfer carbohydrate (ribitol) and the incomplete inhibition of thallus photosynthesis upon treatment with Zn²⁺ solutions showed that both photobionts contributed to the photosynthesis of the lichen thallus. Low heterocyst frequencies showed that, despite the presence of adjacent green algae, the cyanobacteria were not altered to increase nitrogen fixation.

Conclusions

These cyanobacterial lichens are a tripartite lichen symbiont combination in which the mycobiont has two primarily photosynthetic photobionts, ‘co-primary photobionts’, a cyanobacterium (dominant) and a green alga. This demonstrates high flexibility in photobiont choice by the mycobiont in the Peltigerales. Overall thallus appearance does not change whether one or two photobionts are present in the cyanobacterial thallus. This suggests that, if there is a photobiont effect on thallus structure, it is not specific to one or the other photobiont.     

Phytochemical review of the lichen genus <Emphasis Type="Italic">Stereocaulon</Emphasis> (Fam. Stereocaulaceae) and related pharmacological activities highlighted by a focus on nine species

The Stereocaulon genus is one of the fruticose lichen groups distributed worldwide from tropical zones to polar zones. However, the scientific study of this tricky genus is still limited, making it a challenge to study the group further. Detailed morphological studies are essential to discriminate closely shaped species which is illustrated through personal data focused on phyllocladia, apothecia and spores of nine species. Secondary metabolites isolated from Stereocaulon species are mostly some depsides, depsidones, diphenylethers and dibenzofurans which can have a taxonomic value. The use of Stereocaulon lichens as a traditional medicine in several regions of the world and pharmacological studies of extracts and isolated compounds have been compiled. Biological activities as cytotoxic, anti-inflammatory, antibacterial, antifungal or antioxidant are reported.     

Fungal farmers or algal escorts: lichen adaptation from the algal perspective

Domestication of algae by lichen‐forming fungi describes the symbiotic relationship between the photosynthetic (green alga or cyanobacterium; photobiont) and fungal (mycobiont) partnership in lichen associations ( Goward 1992 ). The algal domestication implies that the mycobiont cultivates the alga as a monoculture within its thallus, analogous to a farmer cultivating a food crop. However, the initial photobiont ‘selection’ by the mycobiont may be predetermined by the habitat rather than by the farmer. When the mycobiont selects a photobiont from the available photobionts within a habitat, the mycobiont may influence photobiont growth and reproduction ( Ahmadjian & Jacobs 1981 ) only after the interaction has been initiated. The theory of ecological guilds ( Rikkinen et al. 2002 ) proposes that habitat limits the variety of photobionts available to the fungal partner. While some studies provide evidence to support the theory of ecological guilds in cyanobacterial lichens ( Rikkinen et al. 2002 ), other studies propose models to explain variation in symbiont combinations in green algal lichens ( Ohmura et al. 2006 ; Piercey‐Normore 2006 ; Yahr et al. 2006 ) hypothesizing the existence of such guilds. In this issue of Molecular Ecology, Peksa & ?kaloud (2011) test the theory of ecological guilds and suggest a relationship between algal habitat requirements and lichen adaptation in green algal lichens of the genus Lepraria. The environmental parameters examined in this study, exposure to rainfall, altitude and substratum type, are integral to lichen biology. Lichens have a poikilohydric nature, relying on the availability of atmospheric moisture for metabolic processes. Having no known active mechanism to preserve metabolic thallus moisture in times of drought, one would expect a strong influence of the environment on symbiont adaptation to specific habitats. Adaptation to changes in substrata and its properties would be expected with the intimate contact between crustose lichens in the genus Lepraria. Altitude has been suggested to influence species distributions in a wide range of taxonomic groups. This is one of the first studies to illustrate an ecological guild, mainly for exposure to rainfall (ombrophiles and ombrophobes), with green algal lichens.     

Photobiont selectivity leads to ecological tolerance and evolutionary divergence in a polymorphic complex of lichenized fungi

Background and Aims

The integrity and evolution of lichen symbioses depend on a fine-tuned combination of algal and fungal genotypes. Geographically widespread species complexes of lichenized fungi can occur in habitats with slightly varying ecological conditions, and it remains unclear how this variation correlates with symbiont selectivity patterns in lichens. In an attempt to address this question, >300 samples were taken of the globally distributed and ecologically variable lichen-forming species complex Tephromela atra, together with closely allied species, in order to study genetic diversity and the selectivity patterns of their photobionts.

Methods

Lichen thalli of T. atra and of closely related species T. grumosa, T. nashii and T. atrocaesia were collected from six continents, across 24 countries and 62 localities representing a wide range of habitats. Analyses of genetic diversity and phylogenetic relationships were carried out both for photobionts amplified directly from the lichen thalli and from those isolated in axenic cultures. Morphological and anatomical traits were studied with light and transmission electron microscopy in the isolated algal strains.

Key Results

Tephromela fungal species were found to associate with 12 lineages of Trebouxia. Five new clades demonstrate the still-unrecognized genetic diversity of lichen algae. Culturable, undescribed lineages were also characterized by phenotypic traits. Strong selectivity of the mycobionts for the photobionts was observed in six monophyletic Tephromela clades. Seven Trebouxia lineages were detected in the poorly resolved lineage T. atra sensu lato, where co-occurrence of multiple photobiont lineages in single thalli was repeatedly observed.

Conclusions

Low selectivity apparently allows widespread lichen-forming fungi to establish successful symbioses with locally adapted photobionts in a broader range of habitats. This flexibility might correlate with both lower phylogenetic resolution and evolutionary divergence in species complexes of crustose lichen-forming fungi.     

Pharmaceutically relevant metabolites from lichens

Lichen metabolites exert a wide variety of biological actions including antibiotic, antimycobacterial, antiviral, antiinflammatory, analgesic, antipyretic, antiproliferative and cytotoxic effects. Even though these manifold activities of lichen metabolites have now been recognized, their therapeutic potential has not yet been fully explored and thus remains pharmaceutically unexploited. In this mini-review, particular attention is paid to the most common classes of small-molecule constituents of lichens, from both the chemical viewpoint and with regard to possible therapeutic implications. In particular, aliphatic acids, pulvinic acid derivatives, depsides and depsidones, dibenzofuans, anthraquinones, naphthoquinones as well as epidithiopiperazinediones are described. An improved access to these lichen substances in drug discovery high-throughput screening programs will provide impetus for identifying novel lead-compounds with therapeutic potential and poses new challenges for medicinal chemistry.     

Lead and copper effects on lipid metabolism in cultured lichen photobionts with different phosphorus status

We evaluated the ability of heavy metals (copper, lead) to alter lipid metabolism in four algal lichen photobionts following short term exposure. Metal concentrations (10 microM) were equivalent to environmentally relevant levels that have been reported to have effects on intact algae. The algae were grown under normal or deficient phosphate conditions to assess any interactions with the heavy metal stress. Given the frequent sensitivity of lichens to copper and lead, there were surprisingly small changes on lipid metabolism, as assessed by radiolabelling from [1-14C]acetate. The main effects, which were seen in a number of cases, were an overall inhibition of total lipid labelling and a relative increase in the labelling of triacylglycerols in the non-polar fraction. Both of these changes can be viewed as reflecting general toxicity of heavy metals. The Coccomyxa photobiont species were more sensitive than Trebouxia species, which fits with the general distribution of the latter in lichens inhabiting harsh environments.     

Lichen substance concentrations in the lichen Hypogymnia physodes are correlated with heavy metal concentrations in the substratum

Lichen substances (i.e. lichen-specific carbon-based secondary compounds) are known to be involved in the uptake and immobilization of metal ions, though the biochemical mechanisms of this interaction are largely unexplained. Previous research on potential effects of lichen substances on heavy metal uptake and tolerance mostly focused on lichens in heavily polluted areas with exceptionally high metal concentrations. In the present study, we aimed at gathering information as to whether lichen substances might be involved in the fine-tuning of metal uptake even at not or low-polluted sites. Therefore, we studied lichen substance concentrations in the epiphytic lichen Hypogymnia physodes and metal concentrations in its substratum in a montane spruce forest of Germany. H. physodes produces two depsides and five depsidones, which had been shown to be involved in metal homeostasis, namely in Cu and Mn uptake, in previous laboratory experiments. The amount of lichen substances increased with increasing heavy metal concentration in the substratum, though the latter varied only in the range of a few μmol g⁻¹ between the sample trees. Variability of lichen substance concentrations in H. physodes within the individual trees was low. Among the different lichen substances of H. physodes, the amount of the depsidone physodalic acid relative to the total of lichen substances was most closely correlated to the concentrations of Cu and Mn in the substratum, whereas the amount of the depsidone 3-hydroxyphysodic acid decreased both with increasing concentrations of these two metals and physodalic acid. Thus, our data suggest that lichen substances contribute to metal homeostasis not only in heavy metal-rich habitats, but also at not or low-polluted sites where the lichen substances apparently help to maintain constant intracellular metal concentrations despite of spatially varying availabilities of metal ions.     

Cytotoxic and apoptotic effects on hepatocytes of secondary metabolites obtained from lichens

There are a large number of species of Antarctic lichens, and several studies describing the secondary metabolites present in these lichens, as well as the advances in understanding the chemistry of these metabolites, have been reported. In addition, some derivatives displaying interesting antibacterial effects have been described. The cytotoxic and apoptotic effects of 15 secondary metabolites (depsides, depsidones and usnic acid) obtained from Continental (Chilean) and Antarctic lichens were evaluated in primary cultures of rat hepatocytes. Intracellular lactate dehydrogenase release, caspase 3 activation and DNA fragmentation were measured. In this study, we have evaluated a set of markers associated with pivotal steps in the execution phase of apoptosis, in order to detect compounds with apoptotic effects on hepatocytes before significant necrosis takes place. Flow cytometric analysis of DNA fragmentation revealed an increase in apoptotic nuclei with sub-diploid DNA content after the exposure of hepatocytes to sub-cytotoxic concentrations of the compounds. Among these, salazinic acid, stictic acid and psoromic acid displayed significant apoptotic activities.     

THE RESPONSE OF DIFFERENTCLADONIASPECIES AFTER TREATMENT WITH FERTILIZER OF LIME IN HEATHLAND

The effect of deposition of different fertilizers and of lime on lichens was investigated under field conditions. Three terricolous lichens were tested:Cladonia floerkeanaC. furcataandC. portentosa. The lichens were exposed to three types of powdered fertilizer (NPK-, N-, P-fertilizer) and lime. The applied nutrients were recorded in lichen thalli and soil. the influence on lichen growth rate and vegetation cover was estimated over 2 years. The response was a clear change in vegetation cover with an exceptional stimulating effect of NPK on the mossPolytrichum formosum. A lethal effect was observed onCladonia floerkeanafollowing exposure to lime. NPK and P had the most significant stimulatory effect onC. furcata. Only NPK promoted the growth ofC. portentosaand P, N or Ca had a negative effect.     

Metabolic processes involved with sugar alcohol and secondary metabolite production in the hyperaccumulator lichen Diploschistes muscorum reveal its complex adaptation strategy against heavy-metal stress

The synthesis of various unique secondary metabolites by lichens is the result of mutualistic symbiotic association between the mycobiont and autotrophic photobiont. The function of these compounds and causal factors for their production are not fully understood. This paper examines the effect of heavy-metal bioaccumulation and physiological parameters related to photosynthesis and carbon metabolism on the production of lichen substances in hyperaccumulator Diploschistes muscorum. The obtained model of secondary metabolite concentrations in the thalli demonstrates that the carbon source provided by the photobiont and associated polyols produced by the mycobiont have positive impact on the production; on the contrary, the increased intracellular load of heavy metals and excessive loss of cell membrane integrity adversely affected secondary metabolite contents. Additionally, the production of secondary metabolites appears to be more dependent on intracellular metal concentrations than on soil pollution level. To compensate for metal stress, both efficient functioning of algal component and sufficient production of secondary metabolites are required. The balanced physiological functioning of mycobiont and photobiont constitutes the complex protective mechanism to alleviate the harmful effects of heavy metal stress on primary and secondary metabolism of lichens.     

Extremotolerance and Resistance of Lichens: Comparative Studies on Five Species Used in Astrobiological Research II. Secondary Lichen Compounds

Lichens, which are symbioses of a fungus and one or two photoautotrophs, frequently tolerate extreme environmental conditions. This makes them valuable model systems in astrobiological research to fathom the limits and limitations of eukaryotic symbioses. Various studies demonstrated the high resistance of selected extremotolerant lichens towards extreme, non-terrestrial abiotic factors including space exposure, hypervelocity impact simulations as well as space and Martian parameter simulations. This study focusses on the diverse set of secondary lichen compounds (SLCs) that act as photo- and UVR-protective substances. Five lichen species used in present-day astrobiological research were compared: Buellia frigida, Circinaria gyrosa, Rhizocarpon geographicum, Xanthoria elegans, and Pleopsidium chlorophanum. Detailed investigation of secondary substances including photosynthetic pigments was performed for whole lichen thalli but also for axenically cultivated mycobionts and photobionts by methods of UV/VIS-spectrophotometry and two types of high performance liquid chromatography (HPLC). Additionally, a set of chemical tests is presented to confirm the formation of melanic compounds in lichen and mycobiont samples. All investigated lichens reveal various sets of SLCs, except C. gyrosa where only melanin was putatively identified. Such studies will help to assess the contribution of SLCs on lichen extremotolerance, to understand the adaptation of lichens to prevalent abiotic stressors of the respective habitat, and to form a basis for interpreting recent and future astrobiological experiments. As most of the identified SLCs demonstrated a high capacity in absorbing UVR, they may also explain the high resistance of lichens towards non-terrestrial UVR.     

Epiphytic lichen diversity and its dependence on bark chemistry in the northern Mongolian dark taiga

Epiphytic lichen diversity was studied in a dark taiga forest of Pinus sibirica, Abies sibirica and Picea obovata in the western Khentey Mountains, northern Mongolia. Though most lichen species occurred on all three tree species, lichen diversity was higher on Abies and Picea than on Pinus. On branches, lichen vegetation differed less between tree species than on the trunk. The occurrence of many Parmeliaceae species with a hydrophilic surface and of many species producing the dibenzofuran usnic acid gives evidence of the low deposition of acidic pollutants in the study area. The Mn content of bark, which is known to limit at high values the abundance of epiphytic lichens in coniferous forests of Europe and North America, is apparently not controlling the spatial distribution of epiphytic lichens in the dark taiga of Mongolia. This is attributed to the dry and cold winters in Mongolia, as high Mn is especially leached from the surface of trees under moist conditions at temperatures around the freezing point, when the contact between water droplets and the tree surface is particularly intensive. Such moist and cold weather conditions are frequent in most parts of the northern coniferous forests of Europe and North America, but are rare events in the most continental parts of Asia, i.e. in Mongolia and eastern Siberia.     

Bioaccumulation of copper, lead and zinc by the bivalves Macomona liliana and Austrovenus stutchburyi

Aquatic organisms take up heavy metals from surrounding environments which accumulate in their body tissues. In the region of Auckland, New Zealand, the heavy metals, copper (Cu), lead (Pb) and zinc (Zn) are the primary sediment contaminants of concern. Previous investigations have revealed adverse effects of Cu and Zn, but not of Pb, on estuarine infauna and a higher sensitivity of the deposit-feeding bivalve Macomona liliana than the suspension-feeding bivalve Austrovenus stutchburyi to these metals. In order to further examine the bioavailability of Cu, Pb and/or Zn and their interactive effects, bioaccumulation of Cu, Pb and Zn was measured in M. liliana and A. stutchburyi after 10-day exposure to these metals in the laboratory. Both bivalves accumulated Pb and Zn, while bioaccumulation of Cu only occurred in A. stutchburyi in the presence of Zn. There was some evidence that the presence of Pb could increase bioaccumulation of Zn. Bioaccumulation was generally much higher in M. liliana than in A. stutchburyi, potentially suggesting their higher uptake rates of metals and thus explaining the higher sensitivity of M. liliana to these heavy metals. Bioaccumulation of Pb in the bivalves and its potential influences on the bioavailability of other metals indicated that, despite the lack of any evidence for acute toxicity of Pb in previous studies, it could still pose a potentially important environmental threat. Bioaccumulation of heavy metals found in the present study also highlights the needs for further investigations of potential chronic toxicity of these metals.     

Lichen secondary metabolites affect growth of <Emphasis Type="Italic">Physcomitrella patens</Emphasis> by allelopathy

Lichen secondary metabolites can function as allelochemicals and affect the development and growth of neighboring bryophytes, fungi, vascular plants, microorganisms, and even other lichens. Lichen overgrowth on bryophytes is frequently observed in nature even though mosses grow faster than lichens, but there is still little information on the interactions between lichens and bryophytes.In the present study, we used extracts from six lichen thalli containing secondary metabolites like usnic acid, protocetraric acid, atranorin, lecanoric acid, nortistic acid, and thamnolic acid. To observe the influence of these metabolites on bryophytes, the moss Physcomitrella patens was cultivated for 5 weeks under laboratory conditions and treated with lichen extracts. Toxicity of natural mixtures of secondary metabolites was tested at three selected doses (0.001, 0.01, and 0.1 %). When the mixture contained substantial amounts of usnic acid, we observed growth inhibition of protonemata and reduced development of gametophores. Significant differences in cell lengths and widths were also noticed. Furthermore, usnic acid had a strong effect on cell division in protonemata suggesting a strong impact on the early stages of bryophyte development by allelochemicals contained in the lichen secondary metabolites.Biological activities of lichen secondary metabolites were confirmed in several studies such as antiviral, antibacterial, antitumor, antiherbivore, antioxidant, antipyretic, and analgetic action or photoprotection. This work aimed to expand the knowledge on allelopathic effects on bryophyte growth.     

Further evidence that activation of net photosynthesis by dry cyanobacterial lichens requires liquid water

Abstract: In contrast to green algal lichens, cyanobacterial species of different families, growth forms and habitats proved to be unable to attain positive net CO₂ assimilation when the dry thalli were treated with air of high relative humidity; they needed liquid water for the reactivation of their photosynthetic apparatus. Identical behaviour is shown by all of the 47 lichen species with cyanobacterial photobionts, from six different genera, studied so far. This suggests a widely distributed, if not general, characteristic of cyanobacterial lichens. The difference in performance between both groups of photobionts was maintained when the lichen thallus was macerated. Furthermore, cultures of Chroococcidiopsis were unable to make use of water vapour hydration for positive net photosynthesis, and were similar in this respect to some free-living aerophilic cyanohacteria tested earlier. Possible physiological implications as well as ecological consequences for water-relation-dependent habitat selection of green-algal and cyanobacterial lichens are discussed.     

Insight into the pattern of heavy-metal accumulation in lichen thalli

BackgroundHeavy metals that pass through the plasmalemma are expected to influence on lichen metabolic processes; however, lichens may tolerate high concentrations of metals by sequestrating them extracellularly. Heavy metal accumulation level fundamentally determine the success of lichens in the colonisation of polluted sites; however, the proportions between extra- and intracellular metal concentrations in lichen thalli are still poorly recognized. In this study metal accumulation patterns of selected toxic trace elements, i.e. Pb, Cd, and micronutrients, i.e. Zn, Cu and Ni, in Cladonia cariosa thalli were recognised in relation to extra- and intracellular fractions.MethodsThe intracellular and total concentrations of Zn, Pb, Cd, Cu and Ni in lichen thalli collected from eleven variously polluted sites were determined by means of atomic absorption spectrometry. Additionally, organic carbon and total nitrogen contents as well as pH of soil substrate were measured.ResultsThe accumulation patterns differed between studied metal elements; the major part of Zn, Pb and Cd loads was accumulated extracellularly, whereas Cu and Ni accumulation was mostly intracellular. Like toxic trace elements, Zn was accumulated mainly extracellularly at high polluted sites. The non-linear models most reliably reflect relationships between intracellular and extracellular metal contents in C. cariosa thalli. The intracellular contents of Zn, Pb, Cd and Cu increased slower at higher than at lower extracellular concentrations. Moreover, at higher total concentrations of elements in the thalli, their extracellular proportions were markedly increased.ConclusionThe results suggest that in the face of extreme Zn-enrichment, lichens demonstrate the ability to accumulate the excess of Zn outside the cells. Therefore, it can be concluded that metal accumulation depend not only on the element but also on its abundance in the environment and direct availability for lichens. The studied species showed a defence against excessive intracellular accumulation when a given element is in excess. Such capability may facilitate the colonization of extremely polluted sites by certain pioneer lichens.     

PHOTOBIONT INVENTORY OF A LICHEN COMMUNITY GROWING ON HEAVY-METAL-RICH ROCK

Abstract: The photobiont inventory of a stand of the Acarosporetum sinopicae, a lichen community comprising saxicolous, chalcophilous lichens, has been analysed. Investigated lichen species were Acarospora rugulosa, A. sinopica, Bellemerea diamartha, Lecanora polytropa, L. subaurea, Lecidea silacea, L. lapicida, Rhizocarpon geographicum, and Umbilicaria cylindrica. For all these lichen species this is the first record of the photobionts, except for L. lapicida. The photobionts were cultured axenically and investigated using light microscopical and molecular methods (ITS-sequence analyses). Every lichen species contained only one photobiont species. All photobionts belong toTrebouxia jamesii , but two different subspecies were found with the morphological differences corresponding to molecular differences. The new subspecies T. jamesii subsp. angustilobata is described, differing from the typical T. jamesii by a crenulate chloroplast but identical to the latter taxon in respect to the pyrenoid structure in the light microscope. These results are discussed with respect to the photobiont inventory of the Physcietum adscendentis, analysed in an earlier study.     

Light might regulate divergently depside and depsidone accumulation in the lichen Parmotrema hypotropum by affecting thallus temperature and water potential

Depsides and depsidones are the most common secondary products uniquely produced in lichens by the fungal symbiont, and they accumulate on the outer surface of its hyphae. Their biological roles are subject to debate. Quantitatively the compounds typical of a given lichen can vary dramatically from thallus to thallus. Several studies have addressed whether this variability is correlated with the light reaching different thalli, but the conclusions are contradictory. We addressed the question with the lichen Parmotrema hypotropum growing on unshaded, vertical tree trunks, a controlled natural environment where the light absorbed by each thallus over its lifetime is the only major position-dependent variable. The exact north-east-south-west orientation of each thallus was used to calculate its yearly light exposure based on astronomical and meteorological considerations. The calculated irradiation around the trunk, distributed over a continuous 40-fold intensity range, then was compared with the amount of compound per unit thallus weight, determined by quantitative thin layer chromatography. P. hypotropum accumulates the depside atranorin in the cortex and the depsidone norstictic acid in the medulla and around the algae. A direct correlation was observed between the yearly amount of light reaching the lichen and the amount of atranorin. In contrast, the amount of norstictic acid decreased with increasing light. Although we did not measure thallus temperature and water potential, a unifying interpretation of these and other published data is that depside/depsidone accumulation in lichens is mediated by localized changes in temperature and water potential produced by light absorption within each thallus. This suggests water relations-based functions for depsides and depsidones.