Differential Expression of Stress Adaptation Genes in a Diatom Ulnaria acus under Different Culture Conditions.

Diatoms are a group of unicellular eukaryotes that are essential primary producers in aquatic ecosystems. The dynamic nature of their habitat necessitates a quick and specific response to various stresses. However, the molecular mechanisms of their physiological adaptations are still underexplored. In this work, we study the response of the cosmopolitan freshwater diatom Ulnaria acus (Bacillariophyceae, Fragilariophycidae, Licmophorales, Ulnariaceae, Ulnaria) in relation to a range of stress factors, namely silica deficiency, prolonged cultivation, and interaction with an algicidal bacterium. Fluorescent staining and light microscopy were used to determine the physiological state of cells under these stresses. To explore molecular reactions, we studied the genes involved in the stress response-type III metacaspase (MC), metacaspase-like proteases (MCP), death-specific protein (DSP), delta-1-pyrroline-5-carboxylate dehydrogenase (ALDH12), and glutathione synthetase (GSHS). We have described the structure of these genes, analyzed the predicted amino acid sequences, and measured their expression dynamics in vitro using qRT-PCR. We demonstrated that the expression of UaMC1, UaMC3, and UaDSP increased during the first five days of silicon starvation. On the seventh day, it was replaced with the expression of UaMC2, UaGSHS, and UaALDH. After 45 days of culture, cells stopped growing, and the expression of UaMC1, UaMC2, UaGSHS, and UaDSP increased. Exposure to an algicidal bacterial filtrate induced a higher expression of UaMC1 and UaGSHS. Thus, we can conclude that these proteins are involved in diatoms' adaptions to environmental changes. Further, these data show that the molecular adaptation mechanisms in diatoms depend on the nature and exposure duration of a stress factor.

Analysis of Predicted Amino Acid Sequences of Diatom Microtubule Center Components.

Diatoms synthesize species-specific exoskeletons inside cells under the control of the cytoskeleton and microtubule center. Previous studies have been conducted with the visualization of the microtubule center; however, its composition has not been studied and reliably established. In the present study, several components of MTOC in diatoms, GCP (gamma complex proteins), Aurora A, and centrins have been identified. Analysis of the predicted amino acid sequences of these proteins revealed structural features typical for diatoms. We analyzed the conserved amino acids and the motives necessary for the functioning of proteins. Phylogenetic analysis of GCP showed that all major groups of diatoms are distributed over phylogenetic trees according to their systematic position. This work is a theoretical study; however, it allows drawing some conclusions about the functioning of the studied components and possible ways to regulate them.

Molecular Evolution of Tubulins in Diatoms.

Microtubules are formed by α- and ß-tubulin heterodimers nucleated with γ-tubulin. Tubulins are conserved eukaryotic proteins. Previously, it was shown that microtubules are involved in diatom silica frustule morphogenesis. Diatom frustules are varied, and their morphology is species-specific. Despite the attractiveness of the problem of elucidating the molecular mechanisms of genetically programmed morphogenesis, the structure and evolution of diatom tubulins have not been studied previously. Based on available genomic and transcriptome data, we analyzed the phylogeny of the predicted amino acid sequences of diatom α-, ß- and γ-tubulins and identified five groups for α-tubulins, six for ß-tubulins and four for γ-tubulins. We identified characteristic amino acids of each of these groups and also analyzed possible posttranslational modification sites of diatom tubulins. According to our results, we assumed what changes occurred in the diatom tubulin structures during their evolution. We also identified which tubulin groups are inherent in large diatom taxa. The similarity between the evolution of diatom tubulins and the evolution of diatoms suggests that molecular changes in α-, ß- and γ-tubulins could be one of the factors in the formation of a high morphological diversity of diatoms.

Early structural and functional changes in Baikal Sculpin gills exposed to suspended soot microparticles in experiment.

The toxic influence of soot microparticles on terrestrial organisms has been well studied, although there is scarce data on how microparticles could affect hydrobionts. We performed a first-ever study of the short-term (5 days) impact of furnace soot (0.005 g/L) on the structural and functional features of gill cells in the Baikal Sculpin species Paracottus knerii, Dybowski, 1874. The soot samples used in the experiment were composed of small (10-100 nm) particles and larger (up to 20 µm) aggregates. The dominant fractions of the polycyclic aromatic hydrocarbons of these microparticles were phenanthrene, fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzofluoranthenes, benzopyrenes, indeno[1,2,3-c,d]pyrenes, and benzo[ghi]perylene. Trace element analysis of the soot detected the presence of C, S, Si, Al, Ca, K, Mg, P, and Fe. The gill condition was assessed with electron scanning, transmission, and laser confocal microscopy. Soot induces degenerative changes in the macrostructure and surface of secondary lamellae and increases mucus production in fish gills. A decrease in mitochondrial activity, an increase in reactive oxygen species production, and an increase in the frequency of programmed cell death in gill epithelium were observed under the influence of soot. In chloride cells, an induction of macroautophagy was detected. In general, the changes in fish gills after the short-term influence of soot microparticles indicate the stress of respiratory and osmotic regulation systems in fish. The data obtained are important for forming a coherent picture of the impact of soot on hydrobionts and for developing bioindication methods for evaluating the risks of their influence on aquatic ecosystems.

Phylogeny and structural peculiarities of the EB proteins of diatoms.

The end-binding proteins are a family of microtubule-associated proteins; this family belongs to plus-end-tracking proteins (+TIPs) that regulate microtubule growth and stabilisation. Although the genes encoding EB proteins are found in all eukaryotic genomes, most studies of them have centred on one or another taxonomic group, without a broad comparative analysis. Here, we present a first phylogenetic analysis and a comparative analysis of domain structures of diatom EB proteins in comparison with other phyla of Chromista, red and green algae, as well as model organisms A. thaliana and H. sapiens. Phylogenetically, diatom EB proteins are separated into six clades, generally corresponding to the phylogeny of their respective organisms. The domain structure of this family is highly variable, but the CH and EBH domains responsible for binding tubulin and other MAPs are mostly conserved. Homologous modelling of the F. cylindrus EB protein shows that conserved motifs of the CH domain are positioned on the protein surface, which is necessary for their functioning. We hypothesise that high variance of the diatom C-terminal domain is caused by previously unknown interactions with a CAP-GLY motif of dynactin subunit p150. Our findings contribute to wider possibilities for further investigations of the cytoskeleton in diatoms.

Anomalies in the valve morphogenesis of the centric diatom alga Aulacoseira islandica caused by microtubule inhibitors.

Of all unicellular organisms possessing a cell wall, diatoms are the most adept at micro- and nanoscale embellishment of their frustules. Elements of their cell walls are formed inside the cell under cytoskeletal control. In this work, we used laser scanning microscopy and electron microscopy to describe the major stages of cell wall formation in the centric diatom algae Aulacoseira islandica and to study the effect of various microtubule inhibitors on the morphogenesis of frustule elements. Our results show that colchicine inhibits karyokinesis and cytokinesis in A. islandica colonies. In contrast, valve morphogenesis is changed, rather than inhibited altogether. In normal cells, this process starts simultaneously in both daughter cells, beginning with the formation of two adjacent discs that later become valve faces and connecting spines. Under colchicine treatment, however, the cleavage furrow is blocked and a single lateral valve forms on the side of the cylindrical frustule. As a result, a single hollow pipe forms instead of two separate drinking glass-shaped frustules; such pipes can form up to 35% of all forming frustules. Colchicine inhibits the formation of connecting spines, whereas paclitaxel causes spines to form a complex, branching shape. At the same time, inhibitors do not affect the formation of areolae (openings) in the frustule. We discuss the possibility that various processes of the diatom frustule morphogenesis are controlled by two different mechanisms: membrane-related micromorphogenesis and cytoskeleton-mediated macromorphogenesis.

Frustule morphogenesis of raphid pennate diatom Encyonema ventricosum (Agardh) Grunow.

Diatoms stand out among other microalgae due to the high diversity of species-specific silica frustules whose components (valves and girdle bands) are formed within the cell in special organelles called silica deposition vesicles (SDVs). Research on cell structure and morphogenesis of frustule elements in diatoms of different taxonomic groups has been carried out since the 1950s but is still relevant today. Here, cytological features and valve morphogenesis in the freshwater raphid pennate diatom Encyonema ventricosum (Agardh) Grunow have been studied using light and transmission electron microscopy of cleaned frustules and ultrathin sections of cells, and scanning electron and atomic force microscopy of the frustule surface. Data have been obtained on chloroplast structure: the pyrenoid is spherical, penetrated by a lamella (a stack of two thylakoids); the girdle lamella consists of several short lamellae. The basic stages of frustule morphogenesis characteristic of raphid pennate diatoms have been traced, with the presence of cytoskeletal elements near SDVs being observed throughout this process. Degradation of the plasmalemma and silicalemma is shown to take place when the newly formed valve is released into the space between sister cells. The role of vesicular transport and exocytosis in the gliding of pennate diatoms is discussed.

Changes in the micro- and nanostructure of siliceous valves in the diatom Synedra acus under the effect of colchicine treatment at different stages of the cell cycle.

The important role of the cytoskeleton in the morphogenesis of siliceous frustule components, which are synthesized within the diatom cells, has been revealed due to experiments with microtubule inhibitors. It has been shown that colchicine entering the diatom cell inhibits polymerization of tubulin, the main protein of microtubules, thereby disrupting the normal processes of biogenic silica deposition and daughter valve morphogenesis. In this study, experiments with a synchronized culture of the pennate diatom Synedra acus have been performed to determine the timing and duration of the formation of various valve components and analyze the effect of colchicine at a subtoxic concentration on the structure of daughter valves at different stages of their morphogenesis. Electron microscopic analysis has revealed several types of micro- and nanoscale anomalies in daughter valve morphology, with their frequency varying depending on the time of colchicine treatment. Laser scanning microscopy of preparations vitally stained with Tubulin Tracker Green has shown that polymerized tubulin at early stages of valve morphogenesis is localized along the periphery of the developing valve. This is evidence for an important role of microtubules in the horizontal growth of the valve at the stage when its general structural pattern is established, including its shape and arrangement of basic micro- and nanostructures. Treatment with a microtubule inhibitor at a certain stage of valve morphogenesis makes it possible to obtain new forms with a specific structure of siliceous components that hold promise for use in nanotechnologies.

Effect of mixotrophic growth on the ultrastructure and fatty acid composition of the diatom Synedra acus from Lake Baikal.

BACKGROUND: Interest in studies concerning the effect of organic carbon sources on the growth of diatoms is largely aimed at subsequent physiological changes occurring in their cells. There are no data on structural changes in the cytoplasm and their relationship with changes in the composition of fatty acids in the course of mixotrophic culturing of freshwater diatoms. To elucidate the role of lipids in the growth of diatom cells in autotrophic and mixotrophic cultures, it is necessary to obtain information on the distribution of fatty acids among intracellular compartments and on possible ultrastructural changes in the cells. RESULTS: In this study, the results demonstrated that Synedra acus cells cultured in the presence of 80 mM glycerol contained lipid bodies of increased size, while cells from cultures supplemented with 40 mM glucose accumulated polysaccharide (chrysolaminarin) granules. An increase in the relative amounts of palmitic and stearic acids was revealed at the exponential growth phase of S. acus in the medium with 80 mM glycerol, which was indicative of the accumulation of fatty acids contained in triacylglycerols. CONCLUSIONS: Synedra acus subsp. radians have an ability to proliferate in the presence of high concentrations of organic substances and their transport into cells is evidence for its high adaptation potential, which, along with other factors, accounts for the dominance of this diatom in the spring-summer plankton of the oligotrophic Lake Baikal.