Search

The term phycobiome was recently introduced to designate all the microalgae (primary or non-primary) associated with lichen symbioses. Abundant non-primary symbiotic microalgae are usually obtained from lichen isolations, confirming that thalli are a source of biodiversity and new species. In this study, microalgae were isolated from thalli of Buellia zoharyi, Ramalina farinacea and Parmotrema pseudotinctorum collected in the Iberian Peninsula and the Canary Islands. Excluding Trebouxia phycobionts, 17 strains similar to Stichococcus (Prasiola clade) were obtained. Molecular identification was carried out by nuclear ITS sequencing, and a phylogenetic tree was generated from these sequences, and grouping them into 4 clades: Diplosphaera chodatti, Diplosphaera sp.1. Deuterostichocuccus sp.1. and Tritostichococcus coniocybes. It is also noteworthy that Diplosphaera sp.1 was detected and isolated from three phylogenetically distant lichenized fungi (B. zoharyi, R. farinacea and P. pseudotinctorum), which were sampled in ecologically different localities, namely Tenerife, La Gomera and Castellón. These results reinforce the idea of the constant presence of certain microalgae associated with the lichen thalli which, despite not being the main primary photobiont, probably form part of the lichen’s phycobiomes.

The decline observed in many populations of highly sensitive lichens has led to the inclusion of some species on regional Red Lists, but this has seldom been accompanied by an examination of the genetic variation of the fungal and algal symbiotic partners. Here, the red-listed epiphytic lichen Seirophora villosa was surveyed in 14 Mediterranean coastal populations to examine the genetic diversity of both partners. The geographical distribution of four-locus haplotypes followed the same trend in mycobiont and photobiont data sets, some being widespread and others restricted to single localities. The fungus showed a moderate selectivity in its photobiont choice, associating with two undescribed Trebouxia microalgae with a biased distribution across the western Mediterranean. One alga species occurred exclusively in the Italian Peninsula. This region also hosted the highest number of haplotypes for both symbionts. The use of 454-pyrosequencing revealed no clear signs of algal coexistence within selected thalli. The phylogenetic reconstruction for the mycobiont showed uncertain limits among Seirophora spp., stressing the need for further taxonomic evaluation. Additionally, we found that Ramalina lacera, a co-occurring epiphytic lichen in the studied area, shared photobiont genotypes with S. villosa. Our results thus suggest that the protection not only of the red-listed species S. villosa, but also of the whole epiphytic lichen community investigated here might be advisable.

Trebouxiophyceae is a wide class of green algae comprising coccoid and elliptic unicells, filaments, blades and colony-forming species that occur in diverse terrestrial and aquatic environments. Within this class, the genus Trebouxia Puymaly is among the most widespread lichen phycobionts worldwide. However, the 29 formally described species based on the combination of morphological traits and genetic diversity woefully underrepresented the overall species-level diversity recognized in the genus. In Trebouxia, reliable differentiation and characterization of the species-level lineages can be achieved by studying the diversity of key diagnostic features of pyrenoid ultrastructure and chloroplast morphology of axenically grown algal cultures. Here, we used transmission electron microscopy (TEM) coupled with confocal laser scanning microscopy (CLSM) to analyze the pyrenoid and the chloroplast of 20 Trebouxia species-level lineages grown directly on solid agar medium and on cellulose-acetate discs laid over the agar medium. With the new, detailed morphoanatomical characterization of these species-level lineages, we reappraise Trebouxia taxonomy in light of the most recent phylogenetic delimitation provided by Muggia et al. (2020).

Lichen phycobiomes have recently emerged as a source of biodiversity and new species of microalgae. Although in the genus Diplosphaera free-living microalgae are common, numerous strains belonging to this genus have frequently been recognized or isolated from lichen thalli. In this study, a comprehensive analysis of the strain Diplosphaera sp. ASUV135, isolated from a lichen thallus, has been carried out using an integrative taxonomic approach. The SSU and nuclear-encoding ITS rDNA, as well as the chloroplast rbcL gene, were sequenced and analyzed to ascertain its taxonomic position and phylogenetic relationships within the genus Diplosphaera. This strain was also analyzed by light, confocal and transmission microscopy for morphological and ultrastructural characterization. The phenotypic plasticity in this strain was also confirmed by changes in its morphology under different growth conditions, as well as those of modulated Chlorophyll a fluorescence emissions, to understand its photosynthetic functioning. Our results pointed out that this strain represents a new taxon within the genus Diplosphaera (Prasiola group), described here as Diplosphaera elongate sp. nova. This study also provides tools for future research on organisms with high phenotypic plasticity by using molecular, morphological, ultrastructural and physiological approaches.

Fungal–algal relationships—both across evolutionary and ecological scales—are finely modulated by the presence of the symbionts in the environments and by the degree of selectivity and specificity that either symbiont develop reciprocally. In lichens, the green algal genus Trebouxia Puymaly is one of the most frequently recovered chlorobionts. Trebouxia species-level lineages have been recognized on the basis of their morphological and phylogenetic diversity, while their ecological preferences and distribution are still only partially unknown. We selected two cosmopolitan species complexes of lichen-forming fungi as reference models, i.e., Rhizoplaca melanophthalma and Tephromela atra, to investigate the diversity of their associated Trebouxia spp. in montane habitats across their distributional range worldwide. The greatest diversity of Trebouxia species-level lineages was recovered in the altitudinal range 1,000–2,500 m a.s.l. A total of 10 distinct Trebouxia species-level lineages were found to associate with either mycobiont, for which new photobionts are reported. One previously unrecognized Trebouxia species-level lineage was identified and is here provisionally named Trebouxia “A52.” Analyses of cell morphology and ultrastructure were performed on axenically isolated strains to fully characterize the new Trebouxia “A52” and three other previously recognized lineages, i.e., Trebouxia “A02,” T. vagua “A04,” and T. vagua “A10,” which were successfully isolated in culture during this study. The species-level diversity of Trebouxia associating with the two lichen-forming fungi in extreme habitats helps elucidate the evolutionary pathways that this lichen photobiont genus traversed to occupy varied climatic and vegetative regimes.

New resources of food, pharmaceuticals or biotechnological products are needed. The huge biodiversity of aero-terrestrial lichen-symbiont microalgae belonging to the Chlorophyta group remains unexplored despite they present interesting features such as extreme stress tolerance and growth in water shortage. Appropriateness for human consumption demands the demonstration of the absence of toxic effects. In vitro biocompatibility of crude homogenates of axenic microalga Asterochloris erici, isolated from the lichen Cladonia cristatella, was analyzed after treatment of cultured L929 fibroblasts with different concentrations of microalgal homogenates. The microalgal protein content (37%) was similar to spirulina or soybean. Antioxidant capacity (10.6 ± 0.6 µmol TE/g WW) or phenolic content (7.5 ± 0.5 mg GAE/g DW) were high compared to Chlorella. The results show that crude homogenates of A. erici do not induce cytotoxicity but seem to have some cytostatic effect inducing slight cell cycle alterations and intracellular reactive oxygen species (ROS) increase at the highest concentration. Carotenoid analysis demonstrates high contents of lutein (1211 µg/g microalga DW), a xanthophyll with antioxidant and cytostatic properties in vivo and high commercial added value. These findings confirm that Asterochloris erici can be suitable for the development of alimentary or pharmaceutical applications and further in vivo animal testing. The cytostatic effects should be further investigated for antitumor agents.

The Trebouxiophyceae is the class of Chlorophyta algae from which the highest number of chloroplast genome (cpDNA) sequences has been obtained. Several species in this class participate in symbioses with fungi to form lichens. However, no cpDNA has been obtained from any Trebouxia lichen-symbiont microalgae, which are present in approximately half of all lichens. Here, we report the sequence of the completely assembled cpDNA from Trebouxia sp. TR9 and a comparative study with other Trebouxio-phyceae. The organization of the chloroplast genome of Trebouxia sp. TR9 has certain features that are unusual in the Trebouxiophyceae and other green algae. The most remarkable characteristics are the presence of long intergenic spacers, a quadripartite structure with short inverted repeated sequences (IRs), and the loss of the rps4 gene. The presence of long intergenic spacers accounts for a larger cpDNA size in comparison to other closely related Trebouxiophyceae. The IRs, which were thought to be lost in the Trebouxiales, are distinct from most of cpDNAs since they lack the rRNA operon and uniquely includes the rbcL gene. The functional transfer of the rps4 gene to the nuclear genome has been confirmed by sequencing and examination of the gene architecture, which includes three spliceosomal introns as well as the verification of the presence of the corresponding transcript. This is the first documented transfer of the rps4 gene from the chloroplast to the nucleus among Viridiplantae. Additionally, a fairly well-resolved phylogenetic reconstruction, including Trebouxia sp. TR9 along with other Trebouxiophyceae, was obtained based on a set of conserved chloroplast genes.

New resources of food, pharmaceuticals or biotechnological products are needed. The huge biodiversity of aero-terrestrial lichen-symbiont microalgae belonging to the Chlorophyta group remains unexplored despite they present interesting features such as extreme stress tolerance and growth in water shortage. Appropriateness for human consumption demands the demonstration of the absence of toxic effects. In vitro biocompatibility of crude homogenates of axenic microalga Asterochloris erici, isolated from the lichen Cladonia cristatella, was analyzed after treatment of cultured L929 fibroblasts with different concentrations of microalgal homogenates. The microalgal protein content (37%) was similar to spirulina or soybean. Antioxidant capacity (10.6 ± 0.6 µmol TE/g WW) or phenolic content (7.5 ± 0.5 mg GAE/g DW) were high compared to Chlorella. The results show that crude homogenates of A. erici do not induce cytotoxicity but seem to have some cytostatic effect inducing slight cell cycle alterations and intracellular reactive oxygen species (ROS) increase at the highest concentration. Carotenoid analysis demonstrates high contents of lutein (1211 µg/g microalga DW), a xanthophyll with antioxidant and cytostatic properties in vivo and high commercial added value. These findings confirm that Asterochloris erici can be suitable for the development of alimentary or pharmaceutical applications and further in vivo animal testing. The cytostatic effects should be further investigated for antitumor agents.

This study analyses the interactions among crustose and lichenicolous lichens growing on gypsum biocrusts. The selected community was composed of Acarospora nodulosa, Acarospora placodiiformis, Diploschistes diacapsis, Rhizocarpon malenconianum and Diplotomma rivas-martinezii. These species represent an optimal system for investigating the strategies used to share phycobionts because Acarospora spp. are parasites of D. diacapsis during their first growth stages, while in mature stages, they can develop independently. R. malenconianum is an obligate lichenicolous lichen on D. diacapsis, and D. rivas-martinezii occurs physically close to D. diacapsis. Microalgal diversity was studied by Sanger sequencing and 454-pyrosequencing of the nrITS region, and the microalgae were characterized ultrastructurally. Mycobionts were studied by performing phylogenetic analyses. Mineralogical and macro- and micro-element patterns were analysed to evaluate their influence on the microalgal pool available in the substrate. The intrathalline coexistence of various microalgal lineages was confirmed in all mycobionts. D. diacapsis was confirmed as an algal donor, and the associated lichenicolous lichens acquired their phycobionts in two ways: maintenance of the hosts’ microalgae and algal switching. Fe and Sr were the most abundant microelements in the substrates but no significant relationship was found with the microalgal diversity. The range of associated phycobionts are influenced by thallus morphology.

Lichens undergo desiccation/rehydration cycles and are permeable to heavy metals, which induce free radicals. Nitrogen monoxide (NO) regulates important cellular functions, but the research on lichen NO is still very scarce. In Ramalina farinacea thalli, NO seems to be involved in the peroxidative damage caused by air pollution, antioxidant defence and regulation of lipid peroxidation and photosynthesis. Our hypothesis is that NO also has a critical role during the rehydration and in the responses to lead of its isolated phycobionts (Trebouxia sp. TR9 and Trebouxia jamesii). Therefore, we studied the intracellular reactive oxygen species (ROS) production, lipid peroxidation and chlorophyll autofluorescence during rehydration of thalli and isolated microalgae in the presence of a NO scavenger and Pb(NO3)2. During rehydration, NO scavenging modulates free radical release and chlorophyll autofluorescence but not lipid peroxidation in both thalli and phycobionts. Pb(NO3)2 reduced free radical release (hormetic effect) both in the whole thallus and in microalgae. However, only in TR9, the ROS production, chlorophyll autofluorescence and lipid peroxidation were dependent on NO. In conclusion, Pb hormetic effect seems to depend on NO solely in TR9, while is doubtful for T. jamesii and the whole thalli.

Three vagrant (Circinaria hispida, Circinaria gyrosa, and Circinaria sp. 'paramerae') and one crustose (semi-vagrant, Circinaria sp. 'oromediterranea') lichens growing in very continental areas in the Iberian Peninsula were selected to study the phycobiont diversity. Mycobiont identification was checked using nrITS DNA barcoding: Circinaria sp. 'oromediterranea' and Circinaria sp. 'paramerae' formed a new clade. Phycobiont diversity was analyzed in 50 thalli of Circinaria spp. using nrITS DNA and LSU rDNA, with microalgae coexistence being found in all the species analyzed by Sanger sequencing. The survey of phycobiont diversity showed up to four different Trebouxia spp. as the primary phycobiont in 20 thalli of C. hispida, in comparison with the remaining Circinaria spp., where only one Trebouxia was the primary microalga. In lichen species showing coexistence, some complementary approaches are needed (454 pyrosequencing and/or ultrastructural analyses). Five specimens were selected for high-throughput screening (HTS) analyses: 22 Trebouxia OTUs were detected, 10 of them not previously known. TEM analyses showed three different cell morphotypes (Trebouxia sp. OTU A12, OTU S51, and T. cretacea) whose ultrastructure is described here in detail for the first time. HTS revealed a different microalgae pool in each species studied, and we cannot assume a specific pattern between these pools and the ecological and/or morphological characteristics. The mechanisms involved in the selection of the primary phycobiont and the other microalgae by the mycobiont are unknown, and require complex experimental designs. The systematics of the genus Circinaria is not yet well resolved, and more analyses are needed to establish a precise delimitation of the species.

The identification of new organisms for environmental toxicology bioassays is currently a priority, since these tools are strongly limited by the ecological relevance of taxa used to study global change. Lichens are sensitive bioindicators of air quality and their microalgae are an untapped source for new low-cost miniaturized bioassays with ecological importance. In order to increase the availability of a wider range of taxa for bioassays, the sensitivity of two symbiotic lichen microalgae, Asterochloris erici and Trebouxia sp. TR9, to atrazine was evaluated. To achieve this goal, axenic cultures of these phycobionts in suspension were exposed to a range of environmental concentrations of the herbicide atrazine, a common water pollutant. Optical density and chlorophyll autofluorescence were used as endpoints of ecotoxicity and ecophysiology on cell suspensions. Results show that lichen microalgae show high sensitivity to very low doses of atrazine, being higher in Asterochloris erici than in Trebouxia sp. TR9. We conclude that environmental concentrations of atrazine could modify population dynamics probably through a shift in reproduction strategies of these organisms. This seminal work is a breakthrough in the use of lichen microalgae in the assessment of micropollution effects on biodiversity.

Lichens are symbiotic organisms, originated by mutualistic associations of heterotrophic fungi (mycobiont), photosynthetic partners (photobionts) which can be either cyanobacteria (cyanobionts) or green microalgae (phycobionts), and bacterial consortia. They are poikilohydric organisms without cuticles or nutrient absorption organs adapted to anhydrobiosis. They present a large range of tolerance to abiotic stress (UV radiation, extreme temperatures, high salinity, mineral excess, etc.) and prosper all around the Earth, especially in harsh habitats, including Antarctica and warm deserts. Their biodiversity is widely used as a bioindicator of environmental quality due to this diversity of tolerance in different species, and they are included in air Pb monitoring programmes worldwide. Their ability to bioaccumulate environmental substances, including some air pollutants and heavy metals, makes them excellent passive biomonitors of Pb. Heavy metal tolerance is related to diverse mechanisms: cell walls and exclusion systems (such as extracellular polymeric substances), intracellular chelators and an extraordinary antioxidant and repair capacity. But recent data show that the most powerful mechanism is related with the upregulation of mutual systems by symbiosis.

Lichens are poikilohydrous symbiotic associations between a fungus, photosynthetic partners, and bacteria. They are tolerant to repeated desiccation/rehydration cycles and adapted to anhydrobiosis. Nitric oxide (NO) is a keystone for stress tolerance of lichens, during lichen rehydration, NO limits free radicals and lipid peroxidation but no data on the mechanisms of its synthesis exist. The aim of this work is to characterize the synthesis of NO in the lichen Ramalina farinacea using inhibitors of nitrate reductase (NR) and nitric oxide synthase (NOS), tungstate, and NG-nitro-L-arginine methyl ester (L-NAME), respectively. Tungstate suppressed the NO level in the lichen and caused an increase in malondialdehyde during rehydration in the hyphae of cortex and in phycobionts, suggesting that a plant-like NR is involved in the NO production. Specific activity of NR in R. farinacea was 91 &mu, U/mg protein, a level comparable to those in the bryophyte Physcomitrella patens and Arabidopsis thaliana. L-NAME treatment did not suppress the NO level in the lichens. On the other hand, NADPH-diaphorase activity cytochemistry showed a possible presence of a NOS-like activity in the microalgae where it is associated with cytoplasmatic vesicles. These data provide initial evidence that NO synthesis in R. farinacea involves NR.

Trebouxiophyceae (Chlorophyta) is a species-rich class of green algae with a remarkable morphological and ecological diversity. Currently, there are a few completely sequenced mitochondrial genomes (mtDNA) from diverse Trebouxiophyceae but none from lichen symbionts. Here, we report the mitochondrial genome sequence of Trebouxia sp. TR9 as the first complete mtDNA sequence available for a lichen-symbiont microalga. A comparative study of the mitochondrial genome of Trebouxia sp. TR9 with other chlorophytes showed important organizational changes, even between closely related taxa. The most remarkable change is the enlargement of the genome in certain Trebouxiophyceae, which is principally due to larger intergenic spacers and seems to be related to a high number of large tandem repeats. Another noticeable change is the presence of a relatively large number of group II introns interrupting a variety of tRNA genes in a single group of Trebouxiophyceae, which includes Trebouxiales and Prasiolales. In addition, a fairly well-resolved phylogeny of Trebouxiophyceae, along with other Chlorophyta lineages, was obtained based on a set of seven well-conserved mitochondrial genes.

Aim: The historical causes responsible for the wide distribution of terricolous, crustose lichenized fungi across the Mediterranean Basin and the Canary Islands have never been explored. Here, we used the terricolous, circum-Mediterranean/Macaronesian species Buellia zoharyi (Caliciaceae, Ascomycota) to infer the time frame, and the climatic, geological and ecological factors influencing the origin and current spatial distribution of this species. Location: Mediterranean Basin and Canary Islands. Methods: Data from two nuclear markers (nrITS and tef1) obtained from 226 specimens of 23 populations covering the entire distribution range of B. zoharyi were used to calculate genetic diversity indices and haplotype networks and to investigate population size changes and structure. Three secondary calibrations were used to estimate the timing of the divergence of B. zoharyi from its hypothesized sister species, B. elegans, and the diversification of B. zoharyi. Results: We found low nucleotide diversity and two geographically differentiated haplogroups, with a contact zone in the Iberian Peninsula. The three dating approaches established wide temporal windows for the divergence of B. zoharyi from B. elegans (Eocene-Pliocene) and its diversification (Miocene-Pleistocene). These intervals overlap with the origin and diversification ages found in other lichen-forming fungi and vascular plants inhabiting the Mediterranean region. Main conclusions: In the context of lichen biogeography, our results support ecological specialization as well as geological and climatic events as drivers of the evolutionary history of B. zoharyi in the Mediterranean. In particular, the combined effects of the Messinian salinity crisis and the subsequent Zanclean Flood on the availability of gypsum soils in the Mediterranean Basin, as well as the Quaternary climatic oscillations, seem to have collectively shaped the amount and distribution of B. zoharyi population genetic diversity.

Buellia zoharyi is a crustose placodioid lichen, usually occurring on biocrusts of semiarid ecosystems in circum-Mediterranean/Macaronesian areas. In previous work, we found that this lichenized fungus was flexible in its phycobiont choice in the Canary Islands. Here we test whether geography and habitat influence phycobiont diversity in populations of this lichen from the Iberian Peninsula and Balearic Islands using Sanger and high throughput sequencing (HTS). Additionally, three thallus section categories (central, middle and periphery) were analyzed to explore diversity of microalgal communities in each part. We found that B. zoharyi populations hosted at least three different Trebouxia spp., and this lichen can associate with distinct phycobiont strains in different habitats and geographic regions. This study also revealed that the Trebouxia composition of this lichen showed significant differences when comparing the Iberian Peninsula with the Balearics thalli. No support for differences in microalgal communities was found among thallus sections, however, several thalli showed different predominant Trebouxia spp. at each section. This result corroborate that thallus parts selected for DNA extraction in metabarcoding analyses are key to not bias the total phycobiont diversity detected. This study highlights that inclusion of HTS analysis is crucial to understand lichen symbiotic microalgal diversity.

[EN] Main conclusion. For the first time we provide a study on the physiological, ultrastructural and molecular effects of salt stress on a terrestrial symbiotic green microalga, Trebouxia sp. TR9. Although tolerance to saline conditions has been thoroughly studied in plants and, to an extent, free-living microalgae, scientific data regarding salt stress on symbiotic lichen microalgae is scarce to non-existent. Since lichen phycobionts are capable of enduring harsh, restrictive and rapidly changing environments, it is interesting to study the metabolic machinery operating under these extreme conditions. We aim to determine the effects of prolonged exposure to high salt concentrations on the symbiotic phycobiont Trebouxia sp. TR9, isolated from the lichen Ramalina farinacea. Our results suggest that, when this alga is confronted with extreme saline conditions, the cellular structures are affected to an extent, with limited chlorophyll content loss and photosynthetic activity remaining after 72h of exposure to 5M NaCl. Furthermore, this organism displays a rather different molecular response compared to land plants and free-living halophile microalgae, with no noticeable increase in ABA levels and ABA-related gene expression until the external NaCl concentration is raised to 3M NaCl. Despite this, the ABA transduction pathway seems functional, since the ABA-related genes tested are responsive to exogenous ABA. These observations could suggest that this symbiotic green alga may have developed alternative molecular pathways to cope with highly saline environments., Supported by the Ministerio de Economía y Competitividad (MINECO, Spain) and FEDER (CGL2016-79158-P), and the PROMETEO Excellence in Research Program (Generalitat Valenciana, Spain) (PROMETEO/2017/039). Funding for Ernesto Hinojosa-Vidal was also provided by MINECO (BES-2013-065511).

The class Trebouxiophyceae is comprised of coccoid to ellipsoid unicells, filaments, blades and colony-forming species of green algae occurring in diverse terrestrial and aquatic environments. Some representatives have evolved parasitic heterotrophic lifestyles, others have been investigated for their biotechnological potential and others have evolved as integral components of lichen symbioses. In this review, we provide an overview of the current understanding of diversity, taxonomy and evolutionary context for the important lichen-forming algal class Trebouxiophyceae (Chlorophyta). In particular, we focus on members of the family Trebouxiaceae (Trebouxiales), the best-known, most widespread and most species-rich group of terrestrial, lichenised green algae. Recent investigations on the diversity of lichen phycobionts demonstrate the importance of implementing integrative taxonomic approaches. Therefore, combining analyses of morphological and anatomical traits with genetic data has improved our perspective of diversity in lichenised algae. More accurate recognition of diversity in Trebouxiophyceae will enhance our understanding of phylogenetic relationships and trait evolution, specimen identification in genomic and meta–bar-coding studies and patterns of specificity and selectivity among the lichen symbionts. We conclude with a discussion of the roles and transformative potential of high-throughput sequencing in research related to lichen-associated algae.

Lichens are poikilohydric symbiotic organisms that can survive in the absence of water. Photosynthesis must be highly regulated in these organisms, which live under continuous desiccation-rehydration cycles, to avoid photooxidative damage. Analysis of chlorophyll a fluorescence induction curves in the lichen microalgae of the Trebouxiophyceae Asterochloris erici and in Trebouxia jamesii (TR1) and Trebouxia sp. (TR9) phycobionts, isolated from the lichen Ramalina farinacea, shows differences with higher plants. In the presence of the photosynthetic electron transport inhibitor DCMU, the kinetics of Q(A) reduction is related to variable fluorescence by a sigmoidal function that approaches a horizontal asymptote. An excellent fit to these curves was obtained by applying a model based on the following assumptions: (1) after closure, the reaction centers (RCs) can be converted into "energy sink" centers (sRCs); (2) the probability of energy leaving the sRCs is very low or zero and (3) energy is not transferred from the antenna of PSII units with sRCs to other PSII units. The formation of sRCs units is also induced by repetitive light saturating pulses or at the transition from dark to light and probably requires the accumulation of reduced Q(A), as well as structural changes in the reaction centers of PSII. This type of energy sink would provide a very efficient way to protect symbiotic microalgae against abrupt changes in light intensity.

Trebouxia TR1 and T. TR9 are the two microalgae that coexist within Ramalina farinacea thalli. In the presence of Pb, TR9 formed extracellular aggregates, while TR1 showed a lower wall lead retention capability. Herein, we studied the cell walls and extracellular polymers (EPS) of TR1 and TR9, and their possible implication in the different Pb retention capacity of these microalgae. The proportion of cell walls on the overall cell biomass was 2.6 times higher in TR9 than in TR1. Glycosyl linkage analysis indicated the presence of hot-water soluble β-galactofuranan(s) in both cell walls, distinct from that previously described in Asterochloris, with increased rhamnose content in TR9 and a higher substitution by side chains in TR1. In the EPS of both microalgae, monosaccharide composition resembled their cell walls; however, TR9 produced two times more EPS than TR1. EPS also contained proteins, displaying distinct polypeptide patterns, modulated by Pb in TR9. Uronic acid content in EPS was higher in TR9, and increased in this phycobiont after the addition of Pb. Our findings suggest that the differential extracellular Pb retention capabilities of TR1 and TR9 microalgae can be related to differences in the main features of their cell walls and EPS.

The study of desiccation tolerance of lichens, and of their chlorobionts in particular, has frequently focused on the anti- oxidant system that protects the cell against photo-oxidative stress during dehydration/rehydration cycles. In this study, we used proteomic and transcript analyses to assess the changes associated with desiccation in the isolated phycobiont Aste- rochloris erici. Algae were dried either slowly (5-6 h) or rapidly (

Classically, lichen phycobionts are described as poikilohydric organisms able to undergo desiccation due to the constitutive presence of molecular protection mechanisms. However, little is known about the induction of cellular responses in lichen phycobionts during drying. The analysis of the lipid composition of the desiccated lichen microalga Asterochloris erici revealed the unusual accumulation of highly polar lipids (oligogalactolipids and phosphatidylinositol), which prevents the fusion of membranes during stress, but also the active degradation of cone-shaped lipids (monogalactosyldiacylglycerol and phosphatidylethanolamine) to stabilize membranes in desiccated cells. The level of phosphatidic acid increased 7-fold during desiccation, implicating a possible role for phospholipase D (PLD) in the response to osmotic stress. Inhibition of PLD with 1-butanol markedly impaired the recovery of photosynthesis activity in A. erici upon desiccation and salt stress (2 M NaCl). These two hyperosmotic stresses caused the phosphorylation of c-Jun N-terminal kinase (JNK) and p38-like mitogen-activated protein kinase (MAPK) and the dephosphorylation of extracellular signal-regulated kinase (ERK). The incubation with 1-butanol reduced the phosphorylation of JNK-like proteins and increased the dephosphorylation of ERK-like proteins, which indicates an upstream control of MAPK cascades by PLD. The phosphoproteome showed that desiccation caused the phosphorylation of several proteins in A. erici, most of them involved in protein turnover. The results demonstrate that lichen phycobionts possess both constitutive and inducible protective mechanisms to acquire desiccation tolerance. Among others, these responses are controlled by the PLD pathway through the activation of MAPK cascades.

Desiccation tolerant (DT) organisms are able to withstand an extended loss of body water and rapidly resume metabolism upon rehydration. This ability, however, is strongly dependent on a slow dehydration rate. Fast dehydration affects membrane integrity leading to intracellular solute leakage upon rehydration and thereby impairs metabolism recovery. We test the hypothesis that the increased cell membrane damage and membrane permeability observed under fast dehydration, compared with slow dehydration, is related to an increase in lipid peroxidation. Our results reject this hypothesis because following rehydration lipid peroxidation remains unaltered, a fact that could be due to the high increase of NO upon rehydration. However, in fast-dried samples we found a strong signal of red autofluorescence upon rehydration, which correlates with an increase in ROS production and with membrane leakage, particularly the case of phenolics. This could be used as a bioindicator of oxidative stress and membrane damage.