Search

The acquisition of photosynthesis is a fundamental step in the evolution of eukaryotes. However, few phototrophic organisms are unambiguously recognized in the Precambrian record. The in situ detection of metabolic byproducts in individual microfossils is the key for the direct identification of their metabolisms. Here, we report a new integrative methodology using synchrotron-based X-ray fluorescence and absorption. We evidence bound nickel-geoporphyrins moieties in low-grade metamorphic rocks, preserved in situ within cells of a ~1 Gyr-old multicellular eukaryote, Arctacellularia tetragonala. We identify these moieties as chlorophyll derivatives, indicating that A. tetragonala was a phototrophic eukaryote, one of the first unambiguous algae. This new approach, applicable to overmature rocks, creates a strong new proxy to understand the evolution of phototrophy and diversification of early ecosystems., (© 2022. The Author(s).)

Gaia Pigino studies the molecular mechanisms and principles of self-organization in cilia using 3D cryo-EM., (© 2021 Morgado-Palacin.)

Centrosomes are composed of two orthogonally arranged centrioles surrounded by an electron-dense matrix called the pericentriolar material (PCM). Centrioles are cylinders with diameters of ~250 nm, are several hundred nanometres in length and consist of 9-fold symmetrically arranged microtubules (MT). In dividing animal cells, centrosomes act as the principal MT-organising centres and they also organise actin, which tunes cytoplasmic MT nucleation. In some specialised cells, the centrosome acquires additional critical structures and converts into the base of a cilium with diverse functions including signalling and motility. These structures are found in most eukaryotes and are essential for development and homoeostasis at both cellular and organism levels. The ultrastructure of centrosomes and their derived organelles have been known for more than half a century. However, recent advances in a number of techniques have revealed the high-resolution structures (at Å-to-nm scale resolution) of centrioles and have begun to uncover the molecular principles underlying their properties, including: protein components; structural elements; and biogenesis in various model organisms. This review covers advances in our understanding of the features and processes that are critical for the biogenesis of the evolutionarily conserved structures of the centrosomes. Furthermore, it discusses how variations of these aspects can generate diversity in centrosome structure and function among different species and even between cell types within a multicellular organism., (Copyright © 2021. Published by Elsevier Ltd.)

Background: Global warming directly influencing ozone layer depletion, which eventually is increasing ultraviolet radiation penetration having far-reaching impacts on living biota. This particularly influences the primary producer microalgae which are the basic unit of food webs in the aquatic habitats. Therefore, it is necessary to concentrate the research at this micro-level to understand the harmful impact of increased UV-B radiation ever before. Consequently, the present attempt aimed to focus on the influence of UV-B on growth criteria, photosynthetic pigments, some metabolites, and ultrastructure of the freshwater cyanobacteria, Planktothrix cryptovaginata (Microcoleaceae), Nostoc carneum (Nostocaceae), Microcystis aeruginosa (Microcystaceae), the Chlorophyte Scenedesmus acutus (Scenedesmaceae), and the marine Cyanobacterium Microcystis (Microcystaceae)., Methods: The cultures of investigated algae were subjected directly to different duration periods (1, 3, 5, and 7 h) of artificial UV-B in addition to unirradiated control culture and allowed to grow for 10 days, after which the algal samples were analyzed for growth, photosynthetic activities, primary metabolities and cellular ultrastructure., Results: A remarkable inhibitory influence of UV-B was observed on growth criteria (measured as optical density and dry weight) and photosynthetic pigments of P. cryptovaginata, N. carneum, M. aeruginosa, S. acutus, and marine Microcystis . Where increasing the exposure time of UV-B was accompanied by increased inhibition. The variation in carbohydrate and protein contents under UV stress was based on the exposure periods and the algal species. The variation in algal ultrastructure by UV-B stress was noticed by an Electron Microscope. Cells damage and lysis, cell wall and cell membrane ruptured and release of intracellular substances, loss of cell inclusion, plasmolysis and necrosis, or apoptosis of the algal cells were observed by exposure to 7 h of UV-B., Conclusion: Exposure to UV-B has a marked harmful impact on the growth, pigments, and metabolic activity, as well as the cellular ultrastructure of some cyanobacteria and chlorophytes.

Several species of the genus Tetraselmis (Chlorodendrophyceae, Chlorophyta) were recently discovered to possess unsuspected biomineralization capacities: they produce multiple intracellular inclusions of amorphous calcium carbonate (ACC), called micropearls. Early light-microscopists had spotted rows of refractive granules in some species, although without identifying their mineral nature. Scanning electron microscope (SEM) observations showed that the distribution of the micropearls in the cell forms a pattern, which appears to be characteristic for a given species. The present study shows that this pattern correlates with the shape of the chloroplast, which differs between Tetraselmis species, because micropearls align themselves along the incisions between chloroplast lobes. This was observed both by SEM and in live cells by light microscopy (LM) using Nomarski differential interference contrast. Additionally, molecular phylogenetic analyses, of rbcL and ITS2 gene sequences from diverse strains of Chlorodendrophyceae, corroborated the morphological observations by identifying two groups among nominal Tetraselmis spp. that differ in chloroplast morphology, micropearl arrangement, and ITS2 RNA secondary structure., (Copyright © 2020 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The purpose of this investigation is to evaluate the implementation of ultrasound-assisted liquid biphasic flotation (LBF) system for the recovery of natural astaxanthin from Haematococcus pluvialis microalgae. Various operating conditions of ultrasound-assisted LBF systems such as the position of ultrasound horn, mode of ultrasonication (pulse and continuous), amplitude of ultrasonication, air flowrate, duration of air flotation, and mass of H. pluvialis microalgae were evaluated. The effect of ultrasonication on the cellular morphology of microalgae was also assessed using microscopic analysis. Under the optimized operating conditions of UALBF, the maximum recovery yield, extraction efficiency, and partition coefficient of astaxanthin were 95.08 ± 3.02%, 99.74 ± 0.05%, and 185.09 ± 4.78, respectively. In addition, the successful scale-up operation of ultrasound-assisted LBF system verified the practicability of this integrated approach for an effective extraction of natural astaxanthin., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Lichens provide valuable systems for studying symbiotic interactions. In lichens, these interactions are frequently described in terms of availability, selectivity and specificity of the mycobionts and photobionts towards one another. The lichen-forming, green algal genus Trebouxia Puymaly is among the most widespread photobiont, associating with a broad range of lichen-forming fungi. To date, 29 species have been described, but studies consistently indicate that the vast majority of species-level lineages still lack formal description, and new, previously unrecognized lineages are frequently reported. To reappraise the diversity and the evolutionary relationships of species-level lineages in Trebouxia, we assembled DNA sequence data from over 1600 specimens, compiled from a range of sequences from previously published studies, axenic algal cultures, and lichens collected from poorly sampled regions. From these samples, we selected representatives of the currently known genetic diversity in the lichenized Trebouxia and inferred a phylogeny from multi-locus sequence data (ITS, rbcL, cox2). We demonstrate that the current formally described species woefully underrepresent overall species-level diversity in this important lichen-forming algal genus. We anticipate that an integrative taxonomic approach, incorporating morphological and physiological data from axenic cultures with genetic data, will be required to establish a robust, comprehensive taxonomy for Trebouxia. The data presented here provide an important impetus and reference dataset for more reliably characterizing diversity in lichenized algae and in using lichens to investigate the evolution of symbioses and holobionts., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Members of the Watanabea clade of Trebouxiophyceae are genetically diverse and widely distributed in all kinds of habitats, especially in most terrestrial habitats. Ten new strains of terrestrial algae isolated from the tropical rainforest in China, and four published strains were investigated in this study. Morphological observation and molecular phylogenetic analyses based on the 18S, ITS, rbcL, and tufA genes were used to identify the new strains. Four previously described species were reinvestigated to supplement molecular data and autospores' morphological photographs. The phylogenetic analyses based on 18S only, the concatenated dataset of 18S and ITS, as well as the concatenated dataset of rbcL and tufA, showed the same phylogenetic positions and relationships of these new strains. According to the phylogenetic analysis and morphological comparisons results, we described these 10 strains as four new members within the Watanabea clade, Polulichloris yunnanensis sp. nov., Polulichloris ovale sp. nov., Massjukichlorella orientale sp. nov., and Massjukichlorella minus sp. nov., and two known species, Massjukichlorella epiphytica, and Mysteriochloris nanningensis. Additionally, we provide strong evidence proving that Phyllosiphon, Mysteriochloris, Polulichloris, and Desertella all reproduce through unequal sized autospores., (© 2020 International Society of Protistologists.)

Nucleomorphs are relic endosymbiont nuclei so far found only in two algal groups, cryptophytes and chlorarachniophytes, which have been studied to model the evolutionary process of integrating an endosymbiont alga into a host-governed plastid (organellogenesis). However, past studies suggest that DNA transfer from the endosymbiont to host nuclei had already ceased in both cryptophytes and chlorarachniophytes, implying that the organellogenesis at the genetic level has been completed in the two systems. Moreover, we have yet to pinpoint the closest free-living relative of the endosymbiotic alga engulfed by the ancestral chlorarachniophyte or cryptophyte, making it difficult to infer how organellogenesis altered the endosymbiont genome. To counter the above issues, we need novel nucleomorph-bearing algae, in which endosymbiont-to-host DNA transfer is on-going and for which endosymbiont/plastid origins can be inferred at a fine taxonomic scale. Here, we report two previously undescribed dinoflagellates, strains MGD and TGD, with green algal endosymbionts enclosing plastids as well as relic nuclei (nucleomorphs). We provide evidence for the presence of DNA in the two nucleomorphs and the transfer of endosymbiont genes to the host (dinoflagellate) genomes. Furthermore, DNA transfer between the host and endosymbiont nuclei was found to be in progress in both the MGD and TGD systems. Phylogenetic analyses successfully resolved the origins of the endosymbionts at the genus level. With the combined evidence, we conclude that the host-endosymbiont integration in MGD/TGD is less advanced than that in cryptophytes/chrorarachniophytes, and propose the two dinoflagellates as models for elucidating organellogenesis., Competing Interests: The authors declare no competing interest.

Microalgae are known to respond to salinity stress via mechanisms that include accumulation of compatible solutes and synthesis of antioxidants. Here, we describe a salinity-tolerance mechanism mediated by lipid droplets (LDs). In the alga Parachlorella kessleri grown under salt-stress conditions, we observed significant increases in cell size and LD content. LDs that were closely grouped along the plasma membrane shrank as the plasma membrane expanded, and some LDs were engulfed by vacuoles. Transcriptome analysis showed that genes encoding lysophospholipid acyltransferases (LPLATs) and phospholipase A2 were significantly up-regulated following salt stress. Diacylglycerol kinase and LPLAT were identified in the proteome of salt-induced LDs, alongside vesicle trafficking and plastidial proteins and histone H2B. Analysis of fatty acid composition revealed an enrichment of C18:1 and C18:2 at the expense of C18:3 in response to salt stress. Pulse-chase experiments further suggested that variations of fatty acid composition were associated with LDs. Acetate stimulation research further confirmed a positive role of LDs in cell growth under salt stress. These results suggest that LDs play important roles in salt-stress tolerance, through harboring proteins, participating in cytoplasmic component recycling, and providing materials and enzymes for membrane modification and expansion., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Lobosphaera incisa is a green microalga that accumulates high levels of the valuable omega-6 long-chain polyunsaturated fatty acids (LC-PUFA) arachidonic acid (ARA, 20:4n-6) in triacylglycerols (TAG) under nitrogen (N) starvation. LC-PUFA accumulation is a rare trait in photosynthetic microalgae with insufficiently understood physiological significance. In this study, RNAi was attempted, for the first time in L. incisa, to produce knockdown lines for the Δ5 desaturase gene. Two lines, termed modified lines, which were isolated during screening for transgenic events, demonstrated alterations in their LC-PUFA profile, ARA-biosynthesis gene expression and lipid class distribution. In line M5-78, which appeared to carry a mutation in the Δ6 elongase gene, LC-PUFA were substituted by 18:3n-6 in all glycerolipids. Line M2-35, for which the exact genetic background has not been established, displayed a dramatic reduction in 20:4n-6, concomitant with an augmented proportion of 18:1n-9, in particular in the extraplastidial membrane lipids and TAG. The physiological responses of the modified lines to stressful conditions were compared with the wild type and the Δ5 desaturase mutant. In the N-replete cells of modified lines, the frequency of lipid droplets was reduced, while a number of starch grains increased, suggesting altered partitioning of assimilated carbon into reserve products. Furthermore, both lines exhibited reduced ability to accumulate TAG under N deprivation and recover from N starvation. Both lines demonstrated lower photosynthetic pigment contents, impairments in photosynthesis under a range of stressful conditions, and less efficient functioning of photoprotection under optimal conditions. Possible implications of fatty acids modifications in the stress response of L. incisa are addressed., (� The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The green oleaginous microalga Lobosphaera incisa accumulates storage lipids triacylglycerols (TAG) enriched in the long-chain polyunsaturated fatty acid arachidonic acid under nitrogen (N) deprivation. In contrast, under phosphorous (P) deprivation, the production of the monounsaturated oleic acid prevails. We compared physiological responses, ultrastructural, and metabolic consequences of L. incisa acclimation to N and P deficiency to provide novel insights into the key determinants of ARA accumulation. Differential responses to nutrient deprivation on growth performance, carbon-to-nitrogen stoichiometry, membrane lipid composition and TAG accumulation were demonstrated. Ultrastructural analyses suggested a dynamic role for vacuoles in sustaining cell homeostasis under conditions of different nutrient availability and their involvement in autophagy in L. incisa. Paralleling ARA-rich TAG accumulation in lipid droplets, N deprivation triggered intensive chloroplast dismantling and promoted catabolic processes. Metabolome analysis revealed depletion of amino acids and pyrimidines, and repression of numerous biosynthetic hubs to favour TAG biosynthesis under N deprivation. Under P deprivation, despite the relatively low growth penalties, the presence of the endogenous P reserves and the characteristic lipid remodelling, metabolic signatures of energy deficiency were revealed. Metabolome adjustments to P deprivation included depletion in ATP and phosphorylated nucleotides, increased levels of TCA-cycle intermediates and osmoprotectants. We conclude that characteristic cellular and metabolome adjustments tailor the adaptive responses of L. incisa to N and P deprivation modulating its LC-PUFA production., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Effects on short-term (6 h) and long-term (96 h) exposure to cadmium (Cd) at 0.1, 0.5 and 2.5 mg/L in microalga Dunaliella salina were assessed using both physiological end points and gene expression analysis. Different physiological responses between the short-term and long-term exposures were observed. Upon 6 h after Cd exposure, lipid peroxidation and cell ultrastructure remained unchanged, while contents of chlorophyll a, chlorophyll b, carotenoids were increased at 0.5 and 2.5 mg/L Cd. Contrarily, 96 h after Cd exposure, lipid peroxidation levels were increased, while pigments content was decreased, and damaged cell ultrastructure was apparent at 2.5 mg/L Cd. Activities of antioxidant enzymes (APX, SOD, GST, GPX, and GR) changed differently both at 6 h and 96 h after Cd exposure. Upon 6 h after Cd exposure, SOD and GST activity increased at all three doses, GR and GPX activity increased at 0.5 mg/L Cd while APX activity increased at 0.1 mg/L Cd. Contrarily, 96 h after Cd exposure, activities of all the antioxidant enzymes increased both at 0.1 and 0.5 mg/L Cd; but there was a decrease in SOD and GR activity in D. salina exposed to 2.5 mg/L Cd. RNA-seq and qRT-PCR analyses indicated that genes involved in ROS-scavenge, photosystem, and ribosome functions were differentially expressed. The most significantly enriched function was the ribosome, in which more than 30 ribosome genes were up-regulated at 6 h but down-regulated at 96 h after Cd exposure at 2.5 mg/L. Our study indicated for the first time that genes encoding ribosomal proteins are the primary target for Cd in microalgae, which allowed gaining new insights into temporal dynamics of toxicity and adaptive response pathways in microalgae exposed to metals., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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 72 h of exposure to 5 M 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 3 M 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.

Accurate knowledge of elemental distributions within biological organisms is critical for understanding their cellular roles. The ability to couple this knowledge with overall cellular architecture in three dimensions (3D) deepens our understanding of cellular chemistry. Using a whole, frozen-hydrated Chlamydomonas reinhardtii cell as an example, we report the development of 3D correlative microscopy through a combination of simultaneous cryogenic x-ray ptychography and x-ray fluorescence microscopy. By taking advantage of a recently developed tomographic reconstruction algorithm, termed GENeralized Fourier Iterative REconstruction (GENFIRE), we produce high-quality 3D maps of the unlabeled alga's cellular ultrastructure and elemental distributions within the cell. We demonstrate GENFIRE's ability to outperform conventional tomography algorithms and to further improve the reconstruction quality by refining the experimentally intended tomographic angles. As this method continues to advance with brighter coherent light sources and more efficient data handling, we expect correlative 3D x-ray fluorescence and ptychographic tomography to be a powerful tool for probing a wide range of frozen-hydrated biological specimens, ranging from small prokaryotes such as bacteria, algae, and parasites to large eukaryotes such as mammalian cells, with applications that include understanding cellular responses to environmental stimuli and cell-to-cell interactions.

The chloroplast is the chlorophyll-containing organelle that produces energy through photosynthesis. Within the chloroplast is an intricate network of thylakoid membranes containing photosynthetic membrane proteins that mediate electron transport and generate chemical energy. Historically, electron microscopy (EM) has been a powerful tool for visualizing the macromolecular structure and organization of thylakoid membranes. However, an understanding of thylakoid membrane dynamics remains elusive because EM requires fixation and sectioning. To improve our knowledge of thylakoid membrane dynamics we need to consider at least two issues: (i) the live-cell imaging conditions needed to visualize active processes in vivo; and (ii) the spatial resolution required to differentiate the characteristics of thylakoid membranes. Here, we utilize three-dimensional structured illumination microscopy (3D-SIM) to explore the optimal imaging conditions for investigating the dynamics of thylakoid membranes in living plant and algal cells. We show that 3D-SIM is capable of examining broad characteristics of thylakoid structures in chloroplasts of the vascular plant Arabidopsis thaliana and distinguishing the structural differences between wild-type and mutant strains. Using 3D-SIM, we also visualize thylakoid organization in whole cells of the green alga Chlamydomonas reinhardtii. These data reveal that high light intensity changes thylakoid membrane structure in C. reinhardtii. Moreover, we observed the green alga Chromochloris zofingiensis and the moss Physcomitrella patens to show the applicability of 3D-SIM. This study demonstrates that 3D-SIM is a promising approach for studying the dynamics of thylakoid membranes in photoautotrophic organisms during photoacclimation processes., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Botryococcus braunii is an oleaginous green algae with the distinctive property of accumulating high quantities of hydrocarbons per dry weight in its colonies. Large variation in colony structure exists, yet its implications and influence of oil distribution and diffusion dynamics are not known and could not be answered due to lack of suitable in vivo methods. This publication seeks to further the understanding on oil dynamics, by investigating naturally relevant large (700-1500μm) and extra-large (1500-2500μm) sized colonies of Botryococcus braunii (race B, strain Showa) in vivo, using a comprehensive approach of chemical shift selective imaging, chemical shift imaging and spin echo diffusion measurements at high magnetic field (17.6T). Hydrocarbon distribution in large colonies was found to be localised in two concentric oil layers with different thickness and concentration. Extra-large colonies were highly unstructured and oil was spread throughout colonies, but with large local variations. Interestingly, fluid channels were observed in extra-large colonies. Diffusion-weighted MRI revealed a strong correlation between colony heterogeneity, oil distribution, and diffusion dynamics in different parts of Botryococcus colonies. Differences between large and extra-large colonies were characterised by using T2 weighted MRI along with relaxation measurements. Our result, therefore, provides first non-invasive MRI means to obtain spatial information on oil distribution and diffusion dynamics in Botryococcus braunii colonies., Competing Interests: The authors have declared that no competing interests exist.

The relation between water status and expression profiles of desiccation -related genes has been studied in the desiccation tolerant (DT) aeroterrestrial green microalga Trebouxia gelatinosa, a common lichen photobiont. Algal colonies were desiccated in controlled conditions and during desiccation water content (WC) and water potential (Ψ) were measured to find the turgor loss point (Ψ tlp ). Quantitative real-time PCR was performed to measure the expression of ten genes related to photosynthesis, antioxidant defense, expansins, heat shock proteins (HSPs), and desiccation related proteins in algal colonies collected during desiccation when still at full turgor (WC > 6 g H 2 O g -1 dry weight), immediately before and after Ψ tlp (-4 MPa; WC ∼ 1 g H 2 O g -1 dry weight) and before and after complete desiccation (WC < 0.01 g H 2 O g -1 dry weight), quantifying the HSP70 protein levels by immunodetection. Our analysis showed that the expression of eight out of ten genes changed immediately before and after Ψ tlp . Interestingly, the expression of five out of ten genes changed also before complete desiccation, i.e. between 0.2 and 0.01 g H 2 O g -1 dry weight. However, the HSP70 protein levels were not affected by changes in water status. The study provides new evidences of the link between the loss of turgor and the expression of genes related to the desiccation tolerance of T. gelatinosa, suggesting the former as a signal triggering inducible mechanisms., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

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., (© 2018 Phycological Society of America.)

Microscopic green algae inhabiting desert microbiotic crusts are remarkably diverse phylogenetically, and many desert lineages have independently evolved from aquatic ancestors. Here we worked with five desert and aquatic species within the family Scenedesmaceae to examine mechanisms that underlie desiccation tolerance and release of unicellular versus multicellular progeny. Live cell staining and time-lapse confocal imaging coupled with transmission electron microscopy established that the desert and aquatic species all divide by multiple (rather than binary) fission, although progeny were unicellular in three species and multicellular (joined in a sheet-like coenobium) in two. During division, Golgi complexes were localized near nuclei, and all species exhibited dynamic rotation of the daughter cell mass within the mother cell wall at cytokinesis. Differential desiccation tolerance across the five species, assessed from photosynthetic efficiency during desiccation/rehydration cycles, was accompanied by differential accumulation of intracellular reactive oxygen species (ROS) detected using a dye sensitive to intracellular ROS. Further comparative investigation will aim to understand the genetic, ultrastructural and physiological characteristics supporting unicellular versus multicellular coenobial morphology, and the ability of representatives in the Scenedesmaceae to colonize ecologically diverse, even extreme, habitats., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Main Conclusion: Based on 3D sections through cells of Botryococcus braunii, the structure of three domains of endoplasmic reticulum, and their spatial and functional relationships to other organelles are clarified. Oil production by photosynthetic microalgae has attracted attention since these oils can be converted into renewable, carbon-neutral fuels. The green alga B. braunii accumulates large amounts of hydrocarbons, 30-50% of cell dry weight, in extracellular spaces rather than its cytoplasm. To advance the knowledge of hydrocarbon biosynthesis and transport pathways in this alga, we utilized transmission EM combined with rapid freezing and image reconstruction. We constructed detailed 3D maps distinguishing three ER domains: rdER with ribosomes on both sides, rsER with ribosomes on one side, and sER without ribosomes. The rsER and sER domains were especially prominent during the oil body formation and oil secretion stages. The ER contacted the chloroplasts, oil bodies, or plasma membrane via the rsER domains, oriented with the ribosome-free surface facing the organelles. We discuss the following transport pathway for hydrocarbons and their precursors in the cytoplasm: chloroplast → endoplasmic reticulum (ER) → oil bodies → ER → plasma membrane → secretion. This study represents the first 3D study of the three-domain classification (rdER, rsER and sER) of the ER network among eukaryotic cells. Finally, we propose the novel features of the ERs in plant cells that are distinct from the latest proposed model for the ERs in mammalian cells.

The Oocystaceae family is generally considered to contain common freshwater eukaryotic microalgae, and few are reported living in semi-saline habitats. Our latest ecological survey in Qinghai Lake and Angzicuo Lake, both large, closed, high-altitude, semi-saline lakes located on the Qinghai-Tibet plateau in China, revealed Oocystaceae species as a dominant group among plankton. Since limited knowledge exists about semi-saline species in the Oocystaceae family, a taxonomical study was carried out using morphological and phylogenetic methods. Using this approach, four new strains of Oocystaceae were identified and successfully cultured in the lab. Molecular results correlated with morphological characters and resolved these species into at least three genera. A new genus, Euchlorocystis, with type species Euchlorocystis subsalina, is described here as having the distinctive morphology of multiple pyrenoids per chloroplast among Oocystaceae, and an independent phylogenetic position at the base of the Oocystaceae. Similarly, the genus Densicystis, with type species Densicystis glomerata, is newly proposed here as having a unique colony morphology of dozens or hundreds of little cells tightly embedded in ellipsoid to round mucilage masses. Oocystis marina, originally described from the Baltic Sea, was also identified in Qinghai Lake and Angzicuo Lake and phylogenetically positioned in the semi-saline clade of the Oocystaceae. The result that a marine species was detected in the closed inland lakes implies a further need to reevaluate the origins of these species., (© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists.)

The family Oocystaceae (Chlorophyta) is a group of morphologically and ultrastructurally distinct green algae that constitute a well-supported clade in the class Trebouxiophyceae. Despite the family's clear delimitation, which is based on specific cell wall features, only a few members of the Oocystaceae have been examined using data other than morphological. In previous studies of Trebouxiophyceae, after the establishment of molecular phylogeny, the taxonomic status of the family was called into question. The genus Oocystis proved to be paraphyletic and some species were excluded from Oocystaceae, while a few other species were newly redefined as members of this family. We investigated 54 strains assigned to the Oocystaceae using morphological, ultrastructural and molecular data (SSU rRNA and rbcL genes) to clarify the monophyly of and diversity within Oocystaceae. Oonephris obesa and Nephrocytium agardhianum clustered within the Chlorophyceae and thus are no longer members of the Oocystaceae. On the other hand, we transferred the coenobial Willea vilhelmii to the Oocystaceae. Our findings combined with those of previous studies resulted in the most robust definition of the family to date. The division of the family into three subfamilies and five morphological clades was suggested. Taxonomical adjustments in the genera Neglectella, Oocystidium, Oocystis, and Ooplanctella were established based on congruent molecular and morphological data. We expect further taxonomical changes in the genera Crucigeniella, Eremosphaera, Franceia, Lagerheimia, Oocystis, and Willea in the future., (© 2017 Phycological Society of America.)

Increasing consumer use of engineered nanomaterials has led to significantly increased efforts to understand their potential impact on the environment and living organisms. Currently, no individual technique can provide all the necessary information such as their size, distribution, and chemistry in complex biological systems. Consequently, there is a need to develop complementary instrumental imaging approaches that provide enhanced understanding of these "bio-nano" interactions to overcome the limitations of individual techniques. Here we used a multimodal imaging approach incorporating dark-field light microscopy, high-resolution electron microscopy, and nanoscale secondary ion mass spectrometry (NanoSIMS). The aim was to gain insight into the bio-nano interactions of surface-functionalized silver nanoparticles (Ag-NPs) with the green algae Raphidocelis subcapitata, by combining the fidelity, spatial resolution, and elemental identification offered by the three techniques, respectively. Each technique revealed that Ag-NPs interact with the green algae with a dependence on the size (10 nm vs 60 nm) and surface functionality (tannic acid vs branched polyethylenimine, bPEI) of the NPs. Dark-field light microscopy revealed the presence of strong light scatterers on the algal cell surface, and SEM imaging confirmed their nanoparticulate nature and localization at nanoscale resolution. NanoSIMS imaging confirmed their chemical identity as Ag, with the majority of signal concentrated at the cell surface. Furthermore, SEM and NanoSIMS provided evidence of 10 nm bPEI Ag-NP internalization at higher concentrations (40 μg/L), correlating with the highest toxicity observed from these NPs. This multimodal approach thus demonstrated an effective approach to complement dose-response studies in nano-(eco)-toxicological investigations.

Biological cubic membranes (CM), which are fluid membranes draped onto the 3D periodic parallel surface geometries with cubic symmetry, have been observed within subcellular organelles, including mitochondria, endoplasmic reticulum, and thylakoids. CM transition tends to occur under various stress conditions; however, multilayer CM organizations often appear associated with light stress conditions. This report is about the characterization of a projected gyroid CM in a transmission electron microscopy study of the chloroplast membranes within green alga Zygnema (LB923) whose lamellar form of thylakoid membrane started to fold into multilayer gyroid CM in the culture at the end of log phase of cell growth. Using the techniques of computer simulation of transmission electron microscopy (TEM) and a direct template matching method, we show that these CM are based on the gyroid parallel surfaces. The single, double, and multilayer gyroid CM morphologies are observed in which space is continuously divided into two, three, and more subvolumes by either one, two, or several parallel membranes. The gyroid CM are continuous with varying amount of pseudo-grana with lamellar-like morphology. The relative amount and order of these two membrane morphologies seem to vary with the age of cell culture and are insensitive to ambient light condition. In addition, thylakoid gyroid CM continuously interpenetrates the pyrenoid body through stalk, bundle-like, morphologies. Inside the pyrenoid body, the membranes re-folded into gyroid CM. The appearance of these CM rearrangements due to the consequence of Zygnema cell response to various types of environmental stresses will be discussed. These stresses include nutrient limitation, temperature fluctuation, and ultraviolet (UV) exposure.

The phylogenetic placement of Prasiola calophylla, from an anthropogenic habitat previously shown to contain a novel UV sunscreen compound, was confirmed by analysis of its rbcL gene. This alga has the capacity to tolerate strong water-limiting conditions. The photosynthetic performance and ultrastructural changes under desiccation and osmotic stress were investigated. Freshly harvested thalli showed an effective quantum yield of PSII [Y(II)] of 0.52 ± 0.06 that decreased to ∼60% of the initial value at 3000 mM sorbitol, and 4000 mM sorbitol led to a complete loss of Y(II). The Y(II) of thalli exposed to controlled desiccating conditions at 60% relative humidity (RH) ceased within 240 min, whereas zero values were reached after 120 min at 20% RH. All investigated samples completely recovered Y(II) within ∼100 min after rehydration. Relative electron transport rates (rETR) were temperature dependent, increasing from 5, 10, to 25 °C but strongly declining at 45 °C. Transmission electron microscopy of samples desiccated for 2.5 h showed an electron dense appearance of the entire cytoplasm when compared to control samples. Thylakoid membranes were still visible in desiccated cells, corroborating the ability to recover. Control and desiccated cells contained numerous storage lipids and starch grains, providing reserves. Overall, P. calophylla showed a high capacity to cope with water-limiting conditions on a physiological and structural basis. A lipophilic outer layer of the cell walls might contribute to reduce water evaporation in this poikilohydric organism.

The toxic effects of phenol on four marine microalgae (Dunaliella salina, Platymonas subcordiformis, Phaeodactylum tricornutum Bohlin, and Skeletonema costatum) were evaluated. The 96h EC 50 values were 72.29, 92.97, 27.32, and 27.32mgL -1 , respectively, which were lower than those values of freshwater microalgae reported in the literature. During a 96-h exposure to a sub-lethal concentration of phenol (1/2 96h EC 50 ) with green alga (D. salina) and diatom (S. costatum), reactive oxygen species (ROS) accumulation, and chlorophyll a (Chl a) content decrease were simultaneously observed in diatom cells after 48h treatment. On the contrary, other chlorophylls in both algae were unaffected. Under transmission electron microscopy (TEM), the phenol-induced ultrastructure alterations included disappearance, or shrinkage, of nucleolus and enlargement of vacuoles, which may result in programmed cell death (PCD). The increase in number of lipid droplets may be related to phenol detoxification. These results indicate that the sensitivity of marine microalgae to phenol was dependent on some biotic factors such as cell size, ROS production, and phenol degradation ability in algal cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Chemical fixation is a critical step in the analysis of the ultrastructure of seaweeds because the wrong approach can compromise the ability to distinguish fine-scale cellular composition. Fixation agents, fixation time and type of tissue are important factors to consider for transmission electron microscopy (TEM), and not every protocol is suitable for all cell types. We evaluated a range of fixation agents, post-fixation time and dehydration solutions to determine a TEM protocol for seaweeds in the Family Ulvaceae. We assessed Ulva lactuca using 5 protocols. The level of preservation obtained differed markedly between fixation methods The best result was obtained by fixing the sample with 2.5% glutaraldehyde, 0.05M sodium cacodylate buffer and 2% paraformaldehyde overnight, and 8h post-fixation in 1% in osmium tetroxide 1%. This approach and fixation time ensured that the membranes, especially the thylakoid membranes of chloroplasts, remained intact. Ethanol is recommended for dehydration as the use of acetone for dehydration resulted in the collapse of cellular membranes. This new protocol will ensure the ultrastructure of Ulvacean seaweeds can be clearly ascertained in the future., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Algal endosymbiosis is widely present among eukaryotes including many protists and metazoans. However, the mechanisms involved in their interactions between host and symbiont remain unclear. Here, we used electron microscopy and three-dimensional reconstruction analyses to examine the ultrastructural interactions between the symbiotic zoochlorella and the organelles in the host Paramecium bursaria, which is a model system of endosymbiosis. Although in chemically fixed samples the symbiotic algae show no direct structural interactions with the host organelles and the perialgal vacuole membrane (PVM), in cryofixed P. bursaria samples the intimate connections were identified between the host mitochondria and the symbiotic algae via the PVM. The PVM was closely apposed to the cell wall of the symbiotic algae and in some places it showed direct contacts to the host mitochondrial membrane and the cell wall of the symbiotic algae. Further, the PVM-associated mitochondria formed a mitochondrial network and were also connected to host ER. Our observations propose a new endosymbiotic systems between the host eukaryotes and the symbionts where the benefiting symbiosis is performed through intimate interactions and an active structural modification in the host organelles.

The picoalga Ostreococcus tauri is a minimal photosynthetic eukaryote that has been used as a model system. O. tauri is known to efficiently produce docosahexaenoic acid (DHA). We provide a comprehensive study of the glycerolipidome of O. tauri and validate this species as model for related picoeukaryotes. O. tauri lipids displayed unique features that combined traits from the green and the chromalveolate lineages. The betaine lipid diacylglyceryl-hydroxymethyl-trimethyl-β-alanine and phosphatidyldimethylpropanethiol, both hallmarks of chromalveolates, were identified as presumed extraplastidial lipids. DHA was confined to these lipids, while plastidial lipids of prokaryotic type were characterized by the overwhelming presence of ω-3 C18 polyunsaturated fatty acids (FAs), 18:5 being restricted to galactolipids. C16:4, an FA typical of green microalgae galactolipids, also was a major component of O. tauri extraplastidial lipids, while the 16:4-coenzyme A (CoA) species was not detected. Triacylglycerols (TAGs) displayed the complete panel of FAs, and many species exhibited combinations of FAs diagnostic for plastidial and extraplastidial lipids. Importantly, under nutrient deprivation, 16:4 and ω-3 C18 polyunsaturated FAs accumulated into de novo synthesized TAGs while DHA-TAG species remained rather stable, indicating an increased contribution of FAs of plastidial origin to TAG synthesis. Nutrient deprivation further severely down-regulated the conversion of 18:3 to 18:4, resulting in obvious inversion of the 18:3/18:4 ratio in plastidial lipids, TAGs, as well as acyl-CoAs. The fine-tuned and dynamic regulation of the 18:3/18:4 ratio suggested an important physiological role of these FAs in photosynthetic membranes. Acyl position in structural and storage lipids together with acyl-CoA analysis further help to determine mechanisms possibly involved in glycerolipid synthesis., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

The disintegration of three industry relevant algae (Chlorella vulgaris, Neochloris oleoabundans and Tetraselmis suecica) was studied in a lab scale bead mill at different bead sizes (0.3-1mm). Cell disintegration, proteins and carbohydrates released into the water phase followed a first order kinetics. The process is selective towards proteins over carbohydrates during early stages of milling. In general, smaller beads led to higher kinetic rates, with a minimum specific energy consumption of ⩽0.47kWhkg DW -1 for 0.3mm beads. After analysis of the stress parameters (stress number and stress intensity), it appears that optimal disintegration and energy usage for all strains occurs in the 0.3-0.4mm range. During the course of bead milling, the native structure of the marker protein Rubisco was retained, confirming the mildness of the disruption process., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The unicellular green alga, Penium margaritaceum, represents a novel and valuable model organism for elucidating cell wall dynamics in plants. This organism's cell wall contains several polymers that are highly similar to those found in the primary cell walls of land plants. Penium is easily grown in laboratory culture and is effectively manipulated in various experimental protocols including microplate assays and correlative microscopy. Most importantly, Penium can be live labeled with cell wall-specific antibodies or other probes and returned to culture where specific cell wall developmental events can be monitored. Additionally, live cells can be rapidly cryo-fixed and cell wall surface microarchitecture can be observed with variable pressure scanning electron microscopy. Here, we describe the methodology for maintaining Penium for experimental cell wall enzyme studies.

Background: The multicellular volvocine alga Pleodorina is intermediate in organismal complexity between its unicellular relative, Chlamydomonas, and its multicellular relative, Volvox, which shows complete division of labor between different cell types. The volvocine green microalgae form a group of genera closely related to the genus Volvox within the order Volvocales (Chlorophyta). Embryos of multicellular volvocine algae consist of a cellular monolayer that, depending on the species, is either bowl-shaped or comprises a sphere. During embryogenesis, multicellular volvocine embryos turn their cellular monolayer right-side out to expose their flagella. This process is called 'inversion' and serves as simple model for epithelial folding in metazoa. While the development of spherical Volvox embryos has been the subject of detailed studies, the inversion process of bowl-shaped embryos is less well understood. Therefore, it has been unclear how the inversion of a sphere might have evolved from less complicated processes., Results: In this study we characterized the inversion of initially bowl-shaped embryos of the 64- to 128-celled volvocine species Pleodorina californica. We focused on the movement patterns of the cell sheet, cell shape changes and changes in the localization of cytoplasmic bridges (CBs) connecting the cells. The development of living embryos was recorded using time-lapse light microscopy. Moreover, fixed and sectioned embryos throughout inversion and at successive stages of development were analyzed by light and transmission electron microscopy. We generated three-dimensional models of the identified cell shapes including the localization of CBs., Conclusions: In contrast to descriptions concerning volvocine embryos with lower cell numbers, the embryonic cells of P. californica undergo non-simultaneous and non-uniform cell shape changes. In P. californica, cell wedging in combination with a relocation of the CBs to the basal cell tips explains the curling of the cell sheet during inversion. In volvocine genera with lower organismal complexity, the cell shape changes and relocation of CBs are less pronounced in comparison to P. californica, while they are more pronounced in all members of the genus Volvox. This finding supports an increasing significance of the temporal and spatial regulation of cell shape changes and CB relocations with both increasing cell number and organismal complexity during evolution of differentiated multicellularity.

Life can thrive in extreme environments where inhospitable conditions prevail. Organisms which resist, for example, acidity, pressure, low or high temperature, have been found in harsh environments. Most of them are bacteria and archaea. The bacterium Deinococcus radiodurans is considered to be a champion among all living organisms, surviving extreme ionizing radiation levels. We have discovered a new extremophile eukaryotic organism that possesses a resistance to ionizing radiations similar to that of D. radiodurans. This microorganism, an autotrophic freshwater green microalga, lives in a peculiar environment, namely the cooling pool of a nuclear reactor containing spent nuclear fuels, where it is continuously submitted to nutritive, metallic, and radiative stress. We investigated its morphology and its ultrastructure by light, fluorescence and electron microscopy as well as its biochemical properties. Its resistance to UV and gamma radiation was assessed. When submitted to different dose rates of the order of some tens of mGy · h -1 to several thousands of Gy · h -1 , the microalga revealed to be able to survive intense gamma-rays irradiation, up to 2,000 times the dose lethal to human. The nuclear genome region spanning the genes for small subunit ribosomal RNA-Internal Transcribed Spacer (ITS) 1-5.8S rRNA-ITS2-28S rRNA (beginning) was sequenced (4,065 bp). The phylogenetic position of the microalga was inferred from the 18S rRNA gene. All the revealed characteristics make the alga a new species of the genus Coccomyxa in the class Trebouxiophyceae, which we name Coccomyxa actinabiotis sp. nov., (© 2016 Phycological Society of America.)

The marine, sand-dwelling green alga Kraftionema allantoideum gen. et sp. nov. is described from clonal cultures established from samples collected in coastal, high intertidal pools from south eastern Australia. The species forms microscopic, uniseriate, unbranched, 6-8 μm wide filaments surrounded by a gelatinous capsule of varying thickness. Filaments are twisted, knotted, and variable in length from 4 to 50 cells in field samples but straighter and much longer in culture, up to 1.5 mm in length. Cell division occurs in several planes, resulting in daughter cells of varying shape, from square to rectangular to triangular, giving rise to gnarled filaments. Mature cells become allantoid, elongate with rounded ends, before dividing one time to form bicells comprised of two domed cells. Adjacent bicells separate from one another and mature filaments appeared as a string of loosely arranged sausages. A massive, single, banded chloroplast covered 3/4 of the wall circumference, and contained a single large pyrenoid encased in a starch envelope that measures 1.5-2.5 μm. Filaments were not adhesive nor did they produce specialized adhesive cells or structures. Reproduction was by fragmentation with all cells capable of producing a new filament. No motile or reproductive cells were observed. Filaments in culture grew equally well in freshwater or marine media, as well as at high salinity, and cells quickly recovered from desiccation. Phylogenetic analysis based on the nuclear-encoded small subunit ribosomal RNA (18S) shows the early branching nature of the Kraftionema lineage among Ulotrichales, warranting its recognition as a family (Kraftionemaceae)., (© 2016 Phycological Society of America.)

The biogenesis, assembly, and degradation of ejectisomes of Pyramimonas grossii were investigated by conventional transmission electron microscopy. Premature ejectisomes were mainly found beneath the cell envelope, often in close proximity to the nucleus, and as vesicles with diameters of 100 to 400 nm. Ejectisomes in the early stages of development contained only a few (2-4) turns of the ejectisome tapes. In the course of the ejectisome development, the number of turns and the widths of the coiled tapes increased. It is likely that vesicles, which were up to 650 nm in diameter, with granule- and plate-like structures inside, delivered additional preassembled ejectisome polypeptides to these premature stages. Both types of vesicles, those containing early stages of ejectisomes and those delivering additional ejectisome material, are believed to originate directly from the endoplasmic reticulum. Mature ejectisomes were mainly registered at the apical periphery of the cells. Up to 11 ejectisomes were found within a single cell. Ejectisomes that were most likely being in the process of degradation were registered within the cytoplasm and within vesicles, often together with material which resembled body scales. Mature ejectisomes which were still furled or which were arrested in the process of discharge were also found outside the cells in the medium.

Phosphorus is an essential element for life on earth and is also important for modern agriculture, which is dependent on inorganic fertilizers from phosphate rock. Polyphosphate is a biological polymer of phosphate residues, which is accumulated in organisms during the biological wastewater treatment process to enhance biological phosphorus removal. Here, we investigated the relationship between polyphosphate accumulation and electron-dense bodies in the green alga Parachlorella kessleri. Under sulfur-depleted conditions, in which some symporter genes were upregulated, while others were downregulated, total phosphate accumulation increased in the early stage of culture compared to that under sulfur-replete conditions. The P signal was detected only in dense bodies by energy dispersive X-ray analysis. Transmission electron microscopy revealed marked ultrastructural variations in dense bodies with and without polyphosphate. Our findings suggest that the dense body is a site of polyphosphate accumulation, and P. kessleri has potential as a phosphate-accumulating organism.

Premise of the Study: Discovery and morphological characterization of a novel epiphytic aquatic green alga increases our understanding of Chaetopeltidales, a poorly known order in Chlorophyceae. Chloroplast genomic data from this taxon reveals an unusual architecture previously unknown in green algae., Methods: Using light and electron microscopy, we characterized the morphology and ultrastructure of a novel taxon of green algae. Bayesian phylogenetic analyses of nuclear and plastid genes were used to test the hypothesized membership of this taxon in order Chaetopeltidales. With next-generation sequence data, we assembled the plastid genome of this novel taxon and compared its gene content and architecture to that of related species to further investigate plastid genome traits., Key Results: The morphology and ultrastructure of this alga are consistent with placement in Chaetopeltidales (Chlorophyceae), but a distinct trait combination supports recognition of this alga as a new genus and species-Koshicola spirodelophila gen. et sp. nov. Its placement in the phylogeny as a descendant of a deep division in the Chaetopeltidales is supported by analysis of molecular data sets. The chloroplast genome is among the largest reported in green algae and the genes are distributed on three large (rather than a single) chromosome, in contrast to other studied green algae., Conclusions: The discovery of Koshicola spirodelophila gen. et sp. nov. highlights the importance of investigating even commonplace habitats to explore new microalgal diversity. This work expands our understanding of the morphological and chloroplast genomic features of green algae, and in particular those of the poorly studied Chaetopeltidales., (© 2016 Botanical Society of America.)

Most swarmers (swimming cells) of the stramenopile group, ranging from unicellular protist to giant kelps (brown algae), have two heterogeneous flagella: a long anterior flagellum (AF) and a relatively shorter posterior flagellum (PF). These flagellated cells often exhibit phototaxis upon light stimulation, although the mechanism by which how the phototactic response is regulated remains largely unknown. A flavoprotein concentrating at the paraflagellar body (PFB) on the basal part of the PF, which can emit green autofluorescence under blue light irradiance, has been proposed as a possible blue light photoreceptor for brown algal phototaxis although the nature of the flavoprotein still remains elusive. Recently, we identified helmchrome as a PF-specific flavoprotein protein in a LC-MS/MS-based proteomics study of brown algal flagella (Fu et al. 2014). To verify the conservation of helmchrome, in the present study, the absence or presence and the localization of helmchrome in swarmers of various algal species were investigated. The results showed that helmchrome was only detected in phototactic swarmers but not the non-phototactic ones of the stramenopile group. Electron microscopy further revealed that the helmchrome detectable swarmers bear a conserved PFB-eyespot complex, which may serve as structural basis for light sensing. It is speculated that all three conserved properties: helmchrome, the PFB structure, and the eyespot apparatus, will be essential parts for phototaxis of stramenopile swarmers.

A bacterial endosymbiont was unexpectedly found in the "axenic" culture strain of the streptophyte green alga Mesostigma viride (NIES-995). Phylogenetic analyses based on 16S rRNA gene sequences showed that the symbiont belongs to the order Rickettsiales, specifically to the recently designated clade "Candidatus Megaira," which is closely related to the well-known Rickettsia clade. Rickettsiales bacteria of the "Ca. Megaira" clade are found in a taxonomically diverse array of eukaryotic hosts, including chlorophycean green algae, several ciliate species, and invertebrates such as Hydra. Transmission electron microscopy, fluorescence in situ hybridi-zation, and SYBR Green I staining experiments revealed that the endosymbiont of M. viride NIES-995 is rod shaped, typically occurs in clusters, and is surrounded by a halo-like structure, presumably formed by secretory substances from the bacterium. Two additional M. viride strains (NIES-296 and NIES-475), but not SAG50-1, were found to house the rickettsial endosymbiont. Analyses of strain NIES-995 transcriptome data indicated the presence of at least 91 transcriptionally active genes of symbiont origins. These include genes for surface proteins (e.g., rOmpB) that are known to play key roles in bacterial attachment onto host eukaryotes in related Rickettsia species. The assembled M. viride transcriptome includes transcripts that code for a suite of predicted algal-derived proteins, such as Ku70, WASH, SCAR, and CDC42, which may be important in the formation of the algal-rickettsial association., (© 2016 Phycological Society of America.)

Coccomyxa is a genus of unicellular green algae of the class Trebouxiophyceae, well known for its cosmopolitan distribution and great ecological amplitude. The taxonomy of this genus has long been problematic, due to reliance on badly-defined and environmentally variable morphological characters. In this study, based on the discovery of a new species from an extreme habitat, we reassess species circumscription in Coccomyxa, a unicellular genus of the class Trebouxiophyceae, using a combination of ecological and DNA sequence data (analyzed with three different methods of algorithmic species delineation). Our results are compared with those of a recent integrative study of Darienko and colleagues that reassessed the taxonomy of Coccomyxa, recognizing 7 species in the genus. Expanding the dataset from 43 to 61 sequences (SSU + ITS rDNA) resulted in a different delimitation, supporting the recognition of a higher number of species (24 to 27 depending on the analysis used, with the 27-species scenario receiving the strongest support). Among these, C. melkonianii sp. nov. is described from material isolated from a river highly polluted by heavy metals (Rio Irvi, Sardinia, Italy). Analyses performed on ecological characters detected a significant phylogenetic signal in six different characters. We conclude that the 27-species scenario is presently the most realistic for Coccomyxa and we suggest that well-supported lineages distinguishable by ecological preferences should be recognized as different species in this genus. We also recommend that for microbial lineages in which the overall diversity is unknown and taxon sampling is sparse, as is often the case for green microalgae, the results of analyses for algorithmic DNA-based species delimitation should be interpreted with extreme caution.

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In recent years, the release of chemical pollutants to water bodies has increased due to anthropogenic activities. Ni(2+) is an essential metal that causes damage to aquatic biota at high concentrations. Phytoplankton are photosynthesizing microscopic organisms that constitute a fundamental community in aquatic environments because they are primary producers that sustain the aquatic food web. Nickel toxicity has not been characterized in all of the affected levels of biological organization. For this reason, the present study evaluated the toxic effects of nickel on the growth of a primary producer, the green microalga Ankistrodesmus falcatus, and on its biochemical, enzymatic, and structural levels. The IC50 (96h) was determined for Ni(2+). Based on this result, five concentrations were determined for additional tests, in which cell density was evaluated daily. At the end of the assay, pigments and six biomarkers, including antioxidant enzymes (catalase [CAT], glutathione peroxidase [GPx], superoxide dismutase [SOD]), and macromolecules (proteins, carbohydrates and lipids), were quantified; the integrated biomarker response (IBR) was determined also. The microalgae were observed by SEM and TEM. Population growth was affected starting at 7.5 μg L(-1) (0.028 μM), and at 120 μg L(-1) (0.450 μM), growth was inhibited completely; the determined IC50 was 17 μg L(-1). Exposure to nickel reduced the concentration of pigments, decreased the content of all of the macromolecules, inhibited of SOD activity, and increased CAT and GPx activities. The IBR revealed that Ni(2+) increased the antioxidant response and diminished the macromolecules concentration. A. falcatus was affected by nickel at very low concentrations; negative effects were observed at the macromolecular, enzymatic, cytoplasmic, and morphological levels, as well as in population growth. Ni(2+) toxicity could result in environmental impacts with consequences on the entire aquatic community. Current regulations should be revised to protect primary producers., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Freshwater green algae started to colonize terrestrial habitats about 460 million years ago, giving rise to the evolution of land plants. Today, several streptophyte green algae occur in aero-terrestrial habitats with unpredictable fluctuations in water availability, serving as ideal models for investigating desiccation tolerance. We tested the hypothesis that callose, a β-d-1,3-glucan, is incorporated specifically in strained areas of the cell wall due to cellular water loss, implicating a contribution to desiccation tolerance. In the early diverging genus Klebsormidium, callose was drastically increased already after 30 min of desiccation stress. Localization studies demonstrated an increase in callose in the undulating cross cell walls during cellular water loss, allowing a regulated shrinkage and expansion after rehydration. This correlates with a high desiccation tolerance demonstrated by a full recovery of the photosynthetic yield visualized at the subcellular level by Imaging-PAM. Furthermore, abundant callose in terminal cell walls might facilitate cell detachment to release dispersal units. In contrast, in the late diverging Zygnema, the callose content did not change upon desiccation for up to 3.5 h and was primarily localized in the corners between individual cells and at terminal cells. While these callose deposits still imply reduction of mechanical damage, the photosynthetic yield did not recover fully in the investigated young cultures of Zygnema upon rehydration. The abundance and specific localization of callose correlates with the higher desiccation tolerance in Klebsormidium when compared with Zygnema., (© The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

The green alga Pseudokirchneriella subcapitata has been widely used in ecological risk assessment, usually based on the impact of the toxicants in the alga growth. However, the physiological causes that lead algal growth inhibition are not completely understood. This work aimed to evaluate the biochemical and structural modifications in P. subcapitata after exposure, for 72 h, to three nominal concentrations of Cd(II), Cr(VI), Cu(II) and Zn(II), corresponding approximately to 72 h-EC10 and 72 h-EC50 values and a high concentration (above 72 h-EC90 values). The incubation of algal cells with the highest concentration of Cd(II), Cr(VI) or Cu(II) resulted in a loss of membrane integrity of ~16, 38 and 55%, respectively. For all metals tested, an inhibition of esterase activity, in a dose-dependent manner, was observed. Reduction of chlorophyll a content, decrease of maximum quantum yield of photosystem II and modification of mitochondrial membrane potential was also verified. In conclusion, the exposure of P. subcapitata to metals resulted in a perturbation of the cell physiological status. Principal component analysis revealed that the impairment of esterase activity combined with the reduction of chlorophyll a content were related with the inhibition of growth caused by a prolonged exposure to the heavy metals., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Modern microbialites in Pavilion Lake, BC, provide an analog for ancient non-stromatolitic microbialites that formed from in situ mineralization. Because Pavilion microbialites are mineralizing under the influence of microbial communities, they provide insights into how biological processes influence microbialite microfabrics and mesostructures. Hemispherical nodules and micrite-microbial crusts are two mesostructures within Pavilion microbialites that are directly associated with photosynthetic communities. Both filamentous cyanobacteria in hemispherical nodules and branching filamentous green algae in micrite-microbial crusts were associated with calcite precipitation at microbialite surfaces and with characteristic microfabrics in the lithified microbialite. Hemispherical nodules formed at microbialite surfaces when calcite precipitated around filamentous cyanobacteria with a radial growth habit. The radial filament pattern was preserved within the microbialite to varying degrees. Some subsurface nodules contained well-defined filaments, whereas others contained only dispersed organic inclusions. Variation in filament preservation is interpreted to reflect differences in timing and amount of carbonate precipitation relative to heterotrophic decay, with more defined filaments reflecting greater lithification prior to degradation than more diffuse filaments. Micrite-microbial crusts produce the second suite of microfabrics and form in association with filamentous green algae oriented perpendicular to the microbialite surface. Some crusts include calcified filaments, whereas others contained voids that reflect the filamentous community in shape, size, and distribution. Pavilion microbialites demonstrate that microfabric variation can reflect differences in lithification processes and microbial metabolisms as well as microbial community morphology and organization. Even when the morphology of individual filaments or cells is not well preserved, the microbial growth habit can be captured in mesoscale microbialite structures. These results suggest that when petrographic preservation is extremely good, ancient microbialite growth structures and microfabrics can be interpreted in the context of variation in community organization, community composition, and lithification history. Even in the absence of distinct microbial microfabrics, mesostructures can capture microbial community morphology., (© 2015 John Wiley & Sons Ltd.)

Botryococcus braunii is a colony-forming green alga that accumulates large amounts of liquid hydrocarbons within the colony. The utilization of B. braunii for biofuel production is however hindered by its low biomass productivity. Here we describe a novel bacterial ectosymbiont (BOTRYCO-2) that confers higher biomass productivity to B. braunii. 16S rDNA analysis indicated that the sequence of BOTRYCO-2 shows low similarity (<90%) to cultured bacterial species and located BOTRYCO-2 within a phylogenetic lineage consisting of uncultured alphaproteobacterial clones. Fluorescence in situ hybridization (FISH) studies and transmission electric microscopy indicated that BOTRYCO-2 is closely associated with B. braunii colonies. Interestingly, FISH analysis of a water bloom sample also found BOTRYCO-2 bacteria in close association with cyanobacterium Microcystis aeruginosa colonies, suggesting that BOTRYCO-2 relatives have high affinity to phytoplankton colonies. A PCR survey of algal bloom samples revealed that the BOTRYCO-2 lineage is commonly found in Microcystis associated blooms. Growth experiments indicated that B. braunii Ba10 can grow faster and has a higher biomass (1.8-fold) and hydrocarbon (1.5-fold) yield in the presence of BOTRYCO-2. Additionally, BOTRYCO-2 conferred a higher biomass yield to BOT-22, one of the fastest growing strains of B. braunii. We propose the species name 'Candidatus Phycosocius bacilliformis' for BOTRYCO-2.

The microalgae of the genus Asterochloris are the preferential phycobionts in Cladonia, Lepraria and Stereocaulon lichens. Recent studies have highlighted the hidden diversity of the genus, even though phycobionts hosting species of the genus Cladonia in Mediterranean and Canarian ecosystems have been poorly explored. Phylogenetic analyses were made by concatenation of the sequences obtained with a plastid - LSU rDNA - and two nuclear - internal transcribed spacer (ITS) rDNA and actin - molecular markers of the phycobionts living in several populations of the Cladonia convoluta-Cladonia foliacea complex, Cladonia rangiformis and Cladonia cervicornis s. str. widely distributed in these areas in a great variety of substrata and habitats. A new strongly supported clade was obtained in relation to the previously published Asterochloris phylogenies. Minimum genetic variation was detected between our haplotypes and other sequences available in the GenBank database. The correct identification of the fungal partners was corroborated by the ITS rDNA barcode. In this study we provide a detailed characterization comprising chloroplast morphology, and ultrastructural and phylogenetic analyses of a novel phycobiont species, here described as Asterochloris mediterranea sp. nov. Barreno, Chiva, Moya et Škaloud. A cryopreserved holotype specimen has been deposited in the Culture Collection of Algae of Charles University in Prague, Czech Republic (CAUP) as CAUP H 1015. We suggest the use of a combination of several nuclear and plastid molecular markers, as well as ultrastructural (transmission electron and confocal microscopy) techniques, both in culture and in the symbiotic state, to improve novel species delimitation of phycobionts in lichens.

The extracellular matrix of scaly green flagellates consists of small organic scales consisting of polysaccharides and scale-associated proteins (SAPs). Molecular phylogenies have shown that these organisms represent the ancestral stock of flagellates from which all green plants (Viridiplantae) evolved. The molecular characterization of four different SAPs is presented. Three SAPs are type-2 membrane proteins with an arginine/alanine-rich short cytoplasmic tail and an extracellular domain that is most likely of bacterial origin. The fourth protein is a filamin-like protein. In addition, we report the presence of proteins similar to the integrin-associated proteins α-actinin (in transcriptomes of glaucophytes and some viridiplants), LIM-domain proteins, and integrin-associated kinase in transcriptomes of viridiplants, glaucophytes, and rhodophytes. We propose that the membrane proteins identified are the predicted linkers between scales and the cytoskeleton. These proteins are present in many green algae but are apparently absent from embryophytes. These proteins represent a new protein family we have termed gralins for green algal integrins. Gralins are absent from embryophytes. A model for the evolution of the cell surface proteins in Plantae is discussed., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Microalgal biodiesel is one of the most promising renewable fuels. The wet technique for lipids extraction has advantages over the dry method, such as energy-saving and shorter procedure. The cell disruption is a key factor in wet oil extraction to facilitate the intracellular oil release. Ultrasonication, high-pressure homogenization, enzymatic hydrolysis and the combination of enzymatic hydrolysis with high-pressure homogenization and ultrasonication were employed in this study to disrupt the cells of the microalga Neochloris oleoabundans. The cell disruption degree was investigated. The cell morphology before and after disruption was assessed with scanning and transmission electron microscopy. The energy requirements and the operation cost for wet cell disruption were also estimated. The highest disruption degree, up to 95.41%, assessed by accounting method was achieved by the combination of enzymatic hydrolysis and high-pressure homogenization. A lipid recovery of 92.6% was also obtained by the combined process. The combined process was found to be more efficient and economical compared with the individual process.