Comparative Transcriptomic and Proteomic Analyses Provide New Insights into the Tolerance to Cyclic Dehydration in a Lichen Phycobiont.

Desiccation tolerance (DT) is relatively frequent in non-vascular plants and green algae. However, it is poorly understood how successive dehydration/rehydration (D/R) cycles shape their transcriptomes and proteomes. Here, we report a comprehensive analysis of adjustments on both transcript and protein profiles in response to successive D/R cycles in Coccomyxa simplex (Csol), isolated from the lichen Solorina saccata. A total of 1833 transcripts and 2332 proteins were differentially abundant as a consequence of D/R; however, only 315 of these transcripts/proteins showed similar trends. Variations in both transcriptomes and proteomes along D/R cycles together with functional analyses revealed an extensive decrease in transcript and protein levels during dehydration, most of them involved in gene expression, metabolism, substance transport, signalling and folding catalysis, among other cellular functions. At the same time, a series of protective transcripts/proteins, such as those related to antioxidant defence, polyol metabolism and autophagy, was upregulated during dehydration. Overall, our results show a transient decrease in most cellular functions as a result of drying and a gradual reactivation of specific cell processes to accommodate the hydration status along successive D/R cycles. This study provides new insights into key mechanisms involved in the DT of Csol and probably other dehydration-tolerant microalgae. In addition, functionally characterising the high number of genes/proteins of unknown functions found in this study may lead to the discovery of new DT mechanisms.

Now Is the Time to Build a National Data Ecosystem for Materials Science and Chemistry Research Data.

Research organizations are critically in need of directed growth toward future interoperability and federation. The purpose of this Viewpoint is to alert the government, academia, professional societies, foundations, and industries of a further need for consideration of data in chemistry and materials as a long-term and sustained development in the US. This paper is a call for coordinated action from the government, academia, and industry to establish a national strategy and concomitant infrastructure focused on research data.

Advances in Understanding of Desiccation Tolerance of Lichens and Lichen-Forming Algae.

Lichens are symbiotic associations (holobionts) established between fungi (mycobionts) and certain groups of cyanobacteria or unicellular green algae (photobionts). This symbiotic association has been essential in the colonization of terrestrial dry habitats. Lichens possess key mechanisms involved in desiccation tolerance (DT) that are constitutively present such as high amounts of polyols, LEA proteins, HSPs, a powerful antioxidant system, thylakoidal oligogalactolipids, etc. This strategy allows them to be always ready to survive drastic changes in their water content. However, several studies indicate that at least some protective mechanisms require a minimal time to be induced, such as the induction of the antioxidant system, the activation of non-photochemical quenching including the de-epoxidation of violaxanthin to zeaxanthin, lipid membrane remodeling, changes in the proportions of polyols, ultrastructural changes, marked polysaccharide remodeling of the cell wall, etc. Although DT in lichens is achieved mainly through constitutive mechanisms, the induction of protection mechanisms might allow them to face desiccation stress in a better condition. The proportion and relevance of constitutive and inducible DT mechanisms seem to be related to the ecology at which lichens are adapted to.

The Under-explored Extracellular Proteome of Aero-Terrestrial Microalgae Provides Clues on Different Mechanisms of Desiccation Tolerance in Non-Model Organisms.

Trebouxia sp. (TR9) and Coccomyxa simplex (Csol) are desiccation-tolerant lichen microalgae with different adaptive strategies in accordance with the prevailing conditions of their habitats. The remodelling of cell wall and extracellular polysaccharides depending on water availability are key elements in the tolerance to desiccation of both microalgae. Currently, there is no information about the extracellular proteins of these algae and other aero-terrestrial microalgae in response to limited water availability. To our knowledge, this is the first report on the proteins associated with the extracellular polymeric substances (EPS) of aero-terrestrial microalgae subjected to cyclic desiccation/rehydration. LC-MS/MS and bioinformatic analyses of the EPS-associated proteins in the two lichen microalgae submitted to four desiccation/rehydration cycles allowed the compilation of 111 and 121 identified proteins for TR9 and Csol, respectively. Both sets of EPS-associated proteins shared a variety of predicted biological functions but showed a constitutive expression in Csol and partially inducible in TR9. In both algae, the EPS-associated proteins included a number of proteins of unknown functions, some of which could be considered as small intrinsically disordered proteins related with desiccation-tolerant organisms. Differences in the composition and the expression pattern between the studied EPS-associated proteins would be oriented to preserve the biochemical and biophysical properties of the extracellular structures under the different conditions of water availability in which each alga thrives.

Disentangling the role of extracellular polysaccharides in desiccation tolerance in lichen-forming microalgae. First evidence of sulfated polysaccharides and ancient sulfotransferase genes.

Trebouxia sp. TR9 and Coccomyxa simplex are desiccation-tolerant microalgae with flexible cell walls, which undergo species-specific remodelling during dehydration-rehydration (D/R) due to their distinct ultrastructure and biochemical composition. Here, we tested the hypothesis that extracellular polysaccharides excreted by each microalga could be quantitatively and/or qualitatively modified by D/R. Extracellular polysaccharides were analysed by size exclusion and anion exchange chromatography, specific stains after gel electrophoresis and gas chromatography/mass spectrometry of trimethylsilyl derivatives (to determine their monosaccharide composition). The structure of a TR9-sulfated polymer was deduced from nuclear magnetic resonance (NMR) analyses. In addition, sugar-sulfotransferase encoding genes were identified in both microalgae, and their expression was measured by RT-qPCR. D/R did not alter the polydispersed profile of extracellular polysaccharides in either microalga but did induce quantitative changes in several peaks. Furthermore, medium-low-sized uronic acid-containing polysaccharides were almost completely substituted by higher molecular mass carbohydrates after D/R. Sulfated polysaccharide(s) were detected, for the first time, in the extracellular polymeric substances of both microalgae, but only increased significantly in TR9 after cyclic D/R, which induced a sugar-sulfotransferase gene and accumulated sulfated ß-D-galactofuranan(s). Biochemical remodelling of extracellular polysaccharides in aeroterrestrial desiccation-tolerant microalgae is species-specific and seems to play a role in the response to changes in environmental water availability.

The chloroplast genome of the lichen-symbiont microalga Trebouxia sp. Tr9 (Trebouxiophyceae, Chlorophyta) shows short inverted repeats with a single gene and loss of the rps4 gene, which is encoded by the nucleus.

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.

Ultrastructural and biochemical analyses reveal cell wall remodelling in lichen-forming microalgae submitted to cyclic desiccation-rehydration.

BACKGROUND AND AIMS: One of the most distinctive features of desiccation-tolerant plants is their high cell wall (CW) flexibility. Most lichen microalgae can tolerate drastic dehydration-rehydration (D/R) conditions; however, their mechanisms of D/R tolerance are scarcely understood. We tested the hypothesis that D/R-tolerant microalgae would have flexible CWs due to species-specific CW ultrastructure and biochemical composition, which could be remodelled by exposure to cyclic D/R. METHODS: Two lichen microalgae, Trebouxia sp. TR9 (TR9, adapted to rapid D/R cycles) and Coccomyxa simplex (Csol, adapted to seasonal dry periods) were exposed to no or four cycles of desiccation [25-30 % RH (TR9) or 55-60 % RH (Csol)] and 16 h of rehydration (100 % RH). Low-temperature SEM, environmental SEM and freeze-substitution TEM were employed to visualize structural alterations induced by D/R. In addition, CWs were extracted and sequentially fractionated with hot water and KOH, and the gel permeation profile of polysaccharides was analysed in each fraction. The glycosyl composition and linkage of the main polysaccharides of each CW fraction were analysed by GC-MS. KEY RESULTS: All ultrastructural analyses consistently showed that desiccation caused progressive cell shrinkage and deformation in both microalgae, which could be rapidly reversed when water availability increased. Notably, the plasma membrane of TR9 and Csol remained in close contact with the deformed CW. Exposure to D/R strongly altered the size distribution of TR9 hot-water-soluble polysaccharides, composed mainly of a ß-3-linked rhamnogalactofuranan and Csol KOH-soluble ß-glucans. CONCLUSIONS: Cyclic D/R induces biochemical remodelling of the CW that could increase CW flexibility, allowing regulated shrinkage and expansion of D/R-tolerant microalgae.

Dynamic evolution of mitochondrial genomes in Trebouxiophyceae, including the first completely assembled mtDNA from a lichen-symbiont microalga (Trebouxia sp. TR9).

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.

Tolerance to Cyclic Desiccation in Lichen Microalgae is Related to Habitat Preference and Involves Specific Priming of the Antioxidant System.

Oxidative stress is a crucial challenge for lichens exposed to cyclic desiccation and rehydration (D/R). However, strategies to overcome this potential stress are still being unraveled. Therefore, the physiological performance and antioxidant mechanisms of two lichen microalgae, Trebouxia sp. (TR9) and Coccomyxa simplex (Csol), were analyzed. TR9 was isolated from Ramalina farinacea, a Mediterranean fruticose epiphytic lichen adapted to xeric habitats, while Csol is the phycobiont of Solorina saccata, a foliaceous lichen that grows on humid rock crevices. The tolerance to desiccation of both species was tested by subjecting them to different drying conditions and to four consecutive daily cycles of D/R. Our results show that a relative humidity close to that of their habitats was crucial to maintain the photosynthetic rates. Concerning antioxidant enzymes, in general, manganese superoxide dismutases (MnSODs) were induced after desiccation and decreased after rehydration. In TR9, catalase (CAT)-A increased, and its activity was maintained after four cycles of D/R. Ascorbate peroxidase activity was detected only in Csol, while glutathione reductase increased only in TR9. Transcript levels of antioxidant enzymes indicate that most isoforms of MnSOD and FeSOD were induced by desiccation and repressed after rehydration. CAT2 gene expression was also upregulated and maintained at higher levels even after four cycles of D/R in accordance with enzymatic activities. To our knowledge, this is the first study to include the complete set of the main antioxidant enzymes in desiccation-tolerant microalgae. The results highlight the species-specific induction of the antioxidant system during cyclic D/R, suggesting a priming of oxidative defence metabolism.

Contrasting strategies used by lichen microalgae to cope with desiccation-rehydration stress revealed by metabolite profiling and cell wall analysis.

Most lichens in general, and their phycobionts in particular, are desiccation tolerant, but their mechanisms of desiccation tolerance (DT) remain obscure. The physiological responses and cell wall features of two putatively contrasting lichen-forming microalgae, Trebouxia sp. TR9 (TR9), isolated from Ramalina farinacea (adapted to frequent desiccation-rehydration cycles), and Coccomyxa solorina-saccatae (Csol), obtained from Solorina saccata (growing in usually humid limestone crevices, subjected to seasonal dry periods) was characterized. Microalgal cultures were desiccated under 25%-30% RH and then rehydrated. Under these conditions, RWC and ψw decreased faster and simultaneously during dehydration in Csol, whereas TR9 maintained its ψw until 70% RWC. The metabolic profile indicated that polyols played a key role in DT of both microalgae. However, TR9 constitutively accumulated higher amounts of polyols, whereas Csol induced the polyol synthesis under desiccation-rehydration. Csol also accumulated ascorbic acid, while TR9 synthesized protective raffinose-family oligosaccharides (RFOs) and increased its content of phenolics. Additionally, TR9 exhibited thicker and qualitatively different cell wall and extracellular polymeric layer compared with Csol, indicating higher water retention capability. The findings were consistent with the notion that lichen microalgae would have evolved distinct strategies to cope with desiccation-rehydration stress in correspondence with the water regime of their respective habitats.

Coordinated ultrastructural and phylogenomic analyses shed light on the hidden phycobiont diversity of Trebouxia microalgae in Ramalina fraxinea.

The precise boundary delineations between taxa in symbiotic associations are very important for evolutionary and ecophysiological studies. Growing evidence indicates that in many cases, the use of either morphological characters or molecular markers results in diversity underestimation. In lichen symbioses, Trebouxia is the most common genus of lichen phycobionts, however, the diversity within this genus has been poorly studied and as such there is no clear species concept. This study constitutes a multifaceted approach incorporating aspects of ultrastructural characterization by TEM and phylogenomics to evaluate the morphological and genetic diversity of phycobionts within the sexually reproducing lichen Ramalina fraxinea in the context of Mediterranean and temperate populations. Results reveal an association with at least seven different Trebouxia lineages belonging to at least two species, T. decolorans and T. jamesii, and diverse combinations of such lineages coexisting within the same thallus depending on the analyzed sample. Some of these lineages are shared by several other non-related lichen taxa. Our findings indicate the existence of a highly diverse assemblage of Trebouxia algae associating with R. fraxinea and suggest a possible incipient speciation within T. decolorans rendering a number of lineages or even actual species. This study stresses the importance of coordinated ultrastructural and molecular analyses to improve estimates of diversity and reveal the coexistence of more than one Trebouxia species within the same thallus. It is also necessary to have clearer species delimitation criteria within the genus Trebouxia and microalgae in general.

Differences in the cell walls and extracellular polymers of the two Trebouxia microalgae coexisting in the lichen Ramalina farinacea are consistent with their distinct capacity to immobilize extracellular Pb.

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 ß-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.

Nanotube liquid crystal elastomers: photomechanical response and flexible energy conversion of layered polymer composites.

Elastomeric composites based on nanotube liquid crystals (LCs) that preserve the internal orientation of nanotubes could lead to anisotropic physical properties and flexible energy conversion. Using a simple vacuum filtration technique of fabricating nanotube LC films and utilizing a transfer process to poly (dimethyl) siloxane wherein the LC arrangement is preserved, here we demonstrate unique and reversible photomechanical response of this layered composite to excitation by near infra-red (NIR) light at ultra-low nanotube mass fractions. On excitation by NIR photons, with application of small or large pre-strains, significant expansion or contraction of the sample occurs, respectively, that is continuously reversible and three orders of magnitude larger than in pristine polymer. Schlieren textures were noted in these LC composites confirming long range macroscopic nematic order of nanotubes within the composites. Order parameters of LC films ranged from S(optical) = 0.51-0.58 from dichroic measurements. Film concentrations, elastic modulus and photomechanical stress were all seen to be related to the nematic order parameter. For the same nanotube concentration, the photomechanical stress was almost three times larger for the self-assembled LC nanotube actuator compared to actuator based on randomly oriented carbon nanotubes. Investigation into the kinetics of photomechanical actuation showed variation in stretching exponent ß with pre-strains, concentration and orientation of nanotubes. Maximum photomechanical stress of ∼ 0.5 MPa W(-1) and energy conversion of ∼ 0.0045% was achieved for these layered composites. The combination of properties, namely, optical anisotropy, reversible mechanical response to NIR excitation and flexible energy conversion all in one system accompanied with low cost makes nanotube LC elastomers important for soft photochromic actuation, energy conversion and photo-origami applications.

Thermo-Active Behavior of Ethylene-Vinyl Acetate | Multiwall Carbon Nanotube Composites Examined by in Situ near-Edge X-ray Absorption Fine-Structure Spectroscopy.

NEXAFS spectroscopy was used to investigate the temperature dependence of thermally active ethylene-vinyl acetate | multiwall carbon nanotube (EVA|MWCNT) films. The data shows systematic variations of intensities with increasing temperature. Molecular orbital assignment of interplaying intensities identified the 1s → π*C=C and 1s → π*C=O transitions as the main actors during temperature variation. Furthermore, enhanced near-edge interplay was observed in prestrained composites. Because macroscopic observations confirmed enhanced thermal-mechanical actuation in prestrained composites, our findings suggest that the interplay of C=C and C=O π orbitals may be instrumental to actuation.

Drosophila embryos as model to assess cellular and developmental toxicity of multi-walled carbon nanotubes (MWCNT) in living organisms.

Different toxicity tests for carbon nanotubes (CNT) have been developed to assess their impact on human health and on aquatic and terrestrial animal and plant life. We present a new model, the fruit fly Drosophila embryo offering the opportunity for rapid, inexpensive and detailed analysis of CNTs toxicity during embryonic development. We show that injected DiI labelled multi-walled carbon nanotubes (MWCNTs) become incorporated into cells in early Drosophila embryos, allowing the study of the consequences of cellular uptake of CNTs on cell communication, tissue and organ formation in living embryos. Fluorescently labelled subcellular structures showed that MWCNTs remained cytoplasmic and were excluded from the nucleus. Analysis of developing ectodermal and neural stem cells in MWCNTs injected embryos revealed normal division patterns and differentiation capacity. However, an increase in cell death of ectodermal but not of neural stem cells was observed, indicating stem cell-specific vulnerability to MWCNT exposure. The ease of CNT embryo injections, the possibility of detailed morphological and genomic analysis and the low costs make Drosophila embryos a system of choice to assess potential developmental and cellular effects of CNTs and test their use in future CNT based new therapies including drug delivery.

Near-edge X-ray absorption fine structure studies of electrospun poly(dimethylsiloxane)/poly(methyl methacrylate)/multiwall carbon nanotube composites.

This work describes the near conduction band edge structure of electrospun mats of multiwalled carbon nanotube (MWCNT)-polydimethylsiloxane-poly(methyl methacrylate) by near edge X-ray absorption fine structure (NEXAFS) spectroscopy. Effects of adding nanofillers of different sizes were addressed. Despite observed morphological variations and inhomogeneous carbon nanotube distribution, spun mats appeared homogeneous under NEXAFS analysis. Spectra revealed differences in emissions from glancing and normal spectra, which may evidence phase separation within the bulk of the micrometer-size fibers. Further, dichroic ratios show polymer chains did not align, even in the presence of nanofillers. Addition of nanofillers affected emissions in the C-H, C═O, and C-C regimes, suggesting their involvement in interfacial matrix-carbon nanotube bonding. Spectral differences at glancing angles between pristine and composite mats suggest that geometric conformational configurations are taking place between polymeric chains and carbon nanotubes. These differences appear to be carbon nanotube-dimension dependent and are promoted upon room temperature mixing and shear flow during electrospinning. CH-π bonding between polymer chains and graphitic walls, as well as H-bonds between impurities in the as-grown MWCNTs and polymer pendant groups are proposed bonding mechanisms promoting matrix conformation.

Balanced gene losses, duplications and intensive rearrangements led to an unusual regularly sized genome in Arbutus unedo chloroplasts.

Completely sequenced plastomes provide a valuable source of information about the duplication, loss, and transfer events of chloroplast genes and phylogenetic data for resolving relationships among major groups of plants. Moreover, they can also be useful for exploiting chloroplast genetic engineering technology. Ericales account for approximately six per cent of eudicot diversity with 11,545 species from which only three complete plastome sequences are currently available. With the aim of increasing the number of ericalean complete plastome sequences, and to open new perspectives in understanding Mediterranean plant adaptations, a genomic study on the basis of the complete chloroplast genome sequencing of Arbutus unedo and an updated phylogenomic analysis of Asteridae was implemented. The chloroplast genome of A. unedo shows extensive rearrangements but a medium size (150,897 nt) in comparison to most of angiosperms. A number of remarkable distinct features characterize the plastome of A. unedo: five-fold dismissing of the SSC region in relation to most angiosperms; complete loss or pseudogenization of a number of essential genes; duplication of the ndhH-D operon and its location within the two IRs; presence of large tandem repeats located near highly re-arranged regions and pseudogenes. All these features outline the primary evolutionary split between Ericaceae and other ericalean families. The newly sequenced plastome of A. unedo with the available asterid sequences allowed the resolution of some uncertainties in previous phylogenies of Asteridae.

The genetic structure of the cosmopolitan three-partner lichen Ramalina farinacea evidences the concerted diversification of symbionts.

The epiphytic lichen Ramalina farinacea is distributed throughout the northern hemisphere in which the same two algal Trebouxia species (provisionally named TR1 and TR9) coexist in every thallus. Ramalina farinacea symbionts were characterized based on the two fungal nuclear loci (nrITS and rpb2) along with the primary and secondary structures of nrITS from each Trebouxia species in the Iberian Peninsula and Canary Islands. The results indicated a noticeable genetic differentiation between mycobionts from these two geographic areas and also suggested concerted changes in the three partners of a lichen symbiosis toward two clearly distinguishable 'holobiont' lineages. Modeling of ITS2 RNA secondary structures suggested their temperature sensitivity in TR1 but not in TR9, which was consistent with the observed superior physiological performance of TR9 phycobionts under relatively high temperatures. Both TR1 and TR9 phycobionts have been also found in a variety of taxonomically distinct lichens with a preferably Mediterranean distribution, being TR1 much more widespread than TR9. Our observations support a model in which ecological diversification and speciation of lichen symbionts in different habitats could include a transient phase consisting of associations with more than one photobiont in individual thalli. Such diversification is likely to be promoted by different physiological backgrounds.

Evolutionary implications of intron-exon distribution and the properties and sequences of the RPL10A gene in eukaryotes.

The RPL10A gene encodes the RPL10 protein, required for joining 40S and 60S subunits into a functional 80S ribosome. This highly conserved gene, ubiquitous across all eukaryotic super-groups, is characterized by a variable number of spliceosomal introns, present in most organisms. These properties facilitate the recognition of orthologs among distant taxa and thus comparative studies of sequences as well as the distribution and properties of introns in taxonomically distant groups of eukaryotes. The present study examined the multiple ways in which RPL10A conservation vs. sequence changes in the gene over the course of evolution, including in exons, introns, and the encoded proteins, can be exploited for evolutionary analysis at different taxonomic levels. At least 25 different positions harboring introns within the RPL10A gene were determined in different taxa, including animals, plants, fungi, and alveolates. Generally, intron positions were found to be well conserved even across different kingdoms. However, certain introns seemed to be restricted to specific groups of organisms. Analyses of several properties of introns, including insertion site, phase, and length, along with exon and intron GC content and exon-intron boundaries, suggested biases within different groups of organisms. The use of a standard primer pair to analyze a portion of the intron-containing RPL10A gene in 12 genera of green algae within Chlorophyta is presented as a case study for evolutionary analyses of introns at intermediate and low taxonomic levels. Our study shows that phylogenetic reconstructions at different depths can be achieved using RPL10A nucleotide sequences from both exons and introns as well as the amino acid sequences of the encoded protein.

Different strategies to achieve Pb-tolerance by the two Trebouxia algae coexisting in the lichen Ramalina farinacea.

Lichen thalli are permeable to airborne substances, including heavy metals, which are harmful to cell metabolism. Ramalina farinacea shows a moderate tolerance to Pb. This lichen comprises two Trebouxia phycobionts, provisionally referred to as TR1 and TR9, with distinct physiological responses to acute oxidative stress. Thus, there is a more severe decay in photosynthesis and photosynthetic pigments in TR1 than in TR9. Similarly, under oxidative stress, antioxidant enzymes and HSP70 protein decrease in TR1 but increase in TR9. Since Pb toxicity is associated with increased ROS formation, we hypothesized greater Pb tolerance in this phycobiont. Accordingly, the aim of the present study was to characterize the physiological differences in the responses of TR1 and TR9 to Pb exposure. Liquid cultures of isolated phycobionts were incubated for 7 days in the presence of Pb(NO3)2. Thereafter, extracellular and intracellular Pb accumulation, photosynthetic pigments, and photosynthesis (as modulated chlorophyll fluorescence) were analyzed along with the antioxidant enzymes glutathione reductase (GR), superoxide dismutase (SOD), ascorbate peroxidase (APx), and catalase (CAT), and the stress-related protein HSP70. Pb uptake increased with the amount of supplied Pb in both algae. However, while significantly more metal was immobilized extracellularly by TR9, the amount of intracellular Pb accumulation was three times higher in TR1. In neither of the phycobionts were significant effects on photosynthetic pigments or photosynthetic electron transport observed. While under control conditions GR, SOD, and APx levels were significantly higher in TR1 than in TR9, only in the latter were these enzymes induced by Pb. This resulted in quantitatively similar antioxidant activities in the two algae when exposed to Pb. In conclusion, the phycobionts of R. farinacea make use of two different strategies against stress, in which the integration of distinct anatomical and physiological features affords similar levels of Pb tolerance.