MICROSATELLITE GENOTYPING OF SINGLE CELLS OF THE DINOFLAGELLATE SPECIES LINGULODINIUM POLYEDRUM (DINOPHYCEAE): A NOVEL APPROACH FOR MARINE MICROBIAL POPULATION GENETIC STUDIES1

In recent years, two new approaches have been introduced in genetic studies of phytoplankton species. One is the application of highly polymorphic microsatellite markers, which allow detailed population genetic studies; the other is the development of methods that enable the direct genetic characterization of single cells as an alternative to clonal cultures. The aim of this study was to combine these two approaches in a method that would allow microsatellite genotyping of single phytoplankton cells, providing a novel tool for high‐resolution population genetic studies. The dinoflagellate species Lingulodinium polyedrum (F. Stein) J. D. Dodge was selected as a model organism to develop this novel approach. The method we describe here is based on several key developments: (i) a simple and efficient DNA extraction method for single cells, (ii) the characterization of microsatellite markers for L. polyedrum, (iii) a protocol for the species identification of single cells through the analysis of partial rRNA gene sequences, and (iv) a two‐step multiplex PCR protocol for the simultaneous amplification of microsatellite markers and partial rRNA gene sequences from single cells. Our protocol allowed the amplification of up to six microsatellite loci together with either the complete ITS1‐5.8S‐ITS2 region or a partial 18S region of the ribosomal gene of L. polyedrum from single motile cells and resting cysts. This article describes and evaluates the developed approach and discusses its significance for population genetic studies of L. polyedrum and other phytoplankton species.     

BACTERIA‐INDUCED MOTILITY REDUCTION IN LINGULODINIUM POLYEDRUM (DINOPHYCEAE)1

Biotic factors that affect phytoplankton physiology and behavior are not well characterized but probably play a crucial role in regulating their population dynamics in nature. We document evidence that some marine bacteria can decrease the swimming speed of motile phytoplankton through the release of putative protease(s). Using the dinoflagellate Lingulodinium polyedrum (F. Stein) J. D. Dodge as a model system, we showed that the motility‐reducing components of bacterial‐algal cocultures were mostly heat labile, were of high molecular weight (>50 kDa), and could be partially neutralized by incubations with protease inhibitors. We further showed that additions of the purified protease pronase E decreased dinoflagellate swimming speed in a concentration‐dependent manner. We propose that motility can be used as a marker for dinoflagellate stress or general unhealthy status due to proteolytic bacteria, among other factors.     

Pharmacological investigation of the bioluminescence signaling pathway of the dinoflagellate Lingulodinium polyedrum: evidence for the role of stretch‐activated ion channels

Dinoflagellate bioluminescence serves as a whole‐cell reporter of mechanical stress, which activates a signaling pathway that appears to involve the opening of voltage‐sensitive ion channels and release of calcium from intracellular stores. However, little else is known about the initial signaling events that facilitate the transduction of mechanical stimuli. In the present study using the red tide dinoflagellate Lingulodinium polyedrum (Stein) Dodge, two forms of dinoflagellate bioluminescence, mechanically stimulated and spontaneous flashes, were used as reporter systems to pharmacological treatments that targeted various predicted signaling events at the plasma membrane level of the signaling pathway. Pretreatment with 200 μM Gadolinium III (Gd³⁺), a nonspecific blocker of stretch‐activated and some voltage‐gated ion channels, resulted in strong inhibition of both forms of bioluminescence. Pretreatment with 50 μM nifedipine, an inhibitor of L‐type voltage‐gated Ca²⁺ channels that inhibits mechanically stimulated bioluminescence, did not inhibit spontaneous bioluminescence. Treatment with 1 mM benzyl alcohol, a membrane fluidizer, was very effective in stimulating bioluminescence. Benzyl alcohol‐stimulated bioluminescence was inhibited by Gd³⁺ but not by nifedipine, suggesting that its role is through stretch activation via a change in plasma membrane fluidity. These results are consistent with the presence of stretch‐activated and voltage‐gated ion channels in the bioluminescence mechanotransduction signaling pathway, with spontaneous flashing associated with a stretch‐activated component at the plasma membrane.     

Use of Quantitative Real-Time PCR to Investigate the Dynamics of the Red Tide Dinoflagellate Lingulodinium polyedrum

A new method based on quantitative real-time polymerase chain reaction (qPCR) was developed and applied to quantify the red tide dinoflagellate Lingulodinium polyedrum in natural seawater samples and in laboratory cultures. The method uses a Molecular Beacon™ approach to target a species-specific region of the small subunit ribosomal RNA gene. The accuracy of the method was verified by microscopical counts using cultures of the dinoflagellate isolated from coastal waters near Los Angeles, CA, and with natural water samples spiked with cultured L. polyedrum. The method was applied to document the pattern and timing of vertical migration by the dinoflagellate in a 2-m water column on an 11:13 h light/dark photoperiod established in the laboratory. Positive phototaxis of L. polyedrum resulted in dense aggregations of the dinoflagellate within the top few centimeters of the water column during the light period. This pattern of distribution was readily established by both methods, although abundances of L. polyedrum determined using qPCR were higher than abundances determined by microscopy in the morning and lower in the afternoon and evening. These differences may have been a consequence of variability in the DNA content per cell because of synchrony of cell division. Counts using both methods to analyze natural samples collected from coastal waters in the Long Beach–Los Angeles area and adjacent San Pedro Channel were in close agreement. However, the qPCR method exhibited greater sensitivity than the microscopical method when L. polyedrum was present at low abundances, and qPCR had a much higher rate of sample throughput than microscopy. The development of this new approach for enumerating L. polyedrum provides a useful tool for studying the ecology of this important red tide species.     

VECTORIAL LABELING OF DINOFLAGELLATE CELL SURFACE PROTEINS1

Our objective was to identify cell surface proteins in the dinoflagellate Lingulodinium polyedrum. Proteins on the surface of living cells that had regions exposed to the external medium were labeled with Na¹²⁵I. After partial purification of membrane proteins and analysis by two‐dimensional gel electrophoresis and autoradiography, a protein of roughly 43 kDa was found to have incorporated the radiolabel. This protein was cloned using a combination of protein microsequencing and PCR amplification. The derived protein sequence in the cDNA has a signal peptide at the N‐terminal end of the protein and thus represents the first plasma membrane protein ever reported for a dinoflagellate. The function of the protein is unknown, but its cloning provides a proof of principle for the general use of vectorial labeling in identifying cell surface proteins of marine algae.     

A dinoflagellate CDK5-like cyclin-dependent kinase

Background information. Mitosis during the dinoflagellate cell cycle is unusual in that the nuclear envelope remains intact and segregation of the permanently condensed chromosomes uses a cytoplasmic mitotic spindle. To examine regulation of the dinoflagellate cell cycle in the context of these unusual nuclear features, it is necessary to isolate and characterize cell cycle regulators such as CDK (cyclin‐dependent kinase). Results. We report the characterization of a CDK from the dinoflagellate Lingulodinium polyedrum. This CDK reacts with an anti‐PSTAIRE antibody and was identified by protein microsequencing after partial purification. The protein microsequence shows homology toward the Pho85/CDK5 clade of CDKs. Neither the amount nor the phosphorylation state changed over the course of the cell cycle, in agreement with results reported for CDK5 family members in other systems. Conclusions. We conclude we have probably isolated a dinoflagellate CDK5‐like protein. The data reported here support the identification of this protein as a CDK5 homologue, and suggest that dinoflagellates may contain several CDK families.     

EFFECTS OF SMALL‐SCALE TURBULENCE ON NET GROWTH RATE AND SIZE OF TEN SPECIES OF MARINE DINOFLAGELLATES1

Field observations and results from previous laboratory studies on the effects of turbulence on dinoflagellates have led to a paradigm in phytoplankton ecology that dinoflagellate growth is negatively affected by turbulence. To test the paradigm, 10 species of autotrophic dinoflagellates were exposed to quantified three‐dimensional turbulence generated by vertically oscillating cylindrical rods in 20‐L rectangular culture tanks. Turbulence was quantified in the tanks (as the turbulent energy dissipation rate, ε ) using an acoustic Doppler velocimeter. Dinoflagellates were exposed to two turbulence treatments: high turbulence ( ε ～ 10 ^{? 4} m²·s ^{? 3}), low turbulence ( ε ～ 10 ^{? 8} m²·s ^{? 3}), and an unstirred control. In accord with the paradigm, Ceratium fusus (Ehrenberg) Dujardin had lower net growth rates in high turbulence, whereas Pyrocystis noctiluca Murray ex Haeckel and Ceratium tripos (O. F. Müller) Nitzsch did not increase their numbers in high turbulence. However, Alexandrium tamarense (Lebour) Balech, Pyrocystis fusiformis Wyville‐Thomson ex Murray, Alexandrium catenella (Whedon and Kofoid) Balech, and a Gyrodinium sp. Kofoid and Swezy were apparently unaffected by turbulence and had the same net growth rates across all turbulence treatments. Contradicting the paradigm, Lingulodinium polyedrum (Stein) Dodge (= Gonyaulax polyedra), Gymnodinium catenatum Graham, and Alexandrium fundyense Balech had increased net growth rates in high turbulence treatments. Cross‐sectional area (CSA) varied little across turbulence treatments for 8 of 10 dinoflagellate species tested, CSA in C. fusus increased when net growth rate decreased in high turbulence, and, conversely, CSA decreased in L. polyedrum when net growth rate increased in high turbulence.     

MECHANISMS OF FLUID SHEAR‐INDUCED INHIBITION OF POPULATION GROWTH IN A RED‐TIDE DINOFLAGELLATE1

Net population growth of some dinoflagellates is inhibited by fluid shear at shear stresses comparable with those generated during oceanic turbulence. Decreased net growth may occur through lowered cell division, increased mortality, or both. The dominant mechanism under various flow conditions was determined for the red‐tide dinoflagellate Lingulodinium polyedrum (Stein) Dodge. Cell division and mortality were determined by direct observation of isolated cells in 0.5‐mL cultures that were shaken to generate unquantified fluid shear. Larger volume cultures were exposed to quantified laminar shear in Couette‐flow chambers (0.004–0.019 N·m ^{? 2} shear stress) and to unquantified flow in shaken flasks. In these larger cultures, cell division frequency was calculated from flow cytometric measurements of DNA·cell^?1. The mechanism by which shear inhibits net growth of L. polyedrum depends on shear stress level and growth conditions. Observations on the isolated cells showed that shaking inhibited growth by lowering cell division without increased mortality. Similar results were found for early exponential‐phase cultures exposed to the lowest experimental shear stress in Couette‐flow chambers. However, mortality occurred when a late exponential‐phase culture was exposed to the same low shear stress and was inferred to occur in cultures exposed to higher shear stresses. Elevated mortality in those treatments was confirmed using behavioral, morphological, and physiological assays. The results predict that cell division in L. polyedrum populations will be inhibited by levels of oceanic turbulence common for near‐surface waters. Shear‐induced mortality is not expected unless shear‐stress levels are unusually high or when cellular condition resembles late exponential/stationary phase cultures.     

An alternative beads‐on‐a‐string chromatin architecture in Thermococcus kodakarensis

We have applied chromatin sequencing technology to the euryarchaeon Thermococcus kodakarensis, which is known to possess histone‐like proteins. We detect positioned chromatin particles of variable sizes associated with lengths of DNA differing as multiples of 30 bp (ranging from 30 bp to >450 bp) consistent with formation from dynamic polymers of the archaeal histone dimer. T. kodakarensis chromatin particles have distinctive underlying DNA sequence suggesting a genomic particle‐positioning code and are excluded from gene‐regulatory DNA suggesting a functional organization. Beads‐on‐a‐string chromatin is therefore conserved between eukaryotes and archaea but can derive from deployment of histone‐fold proteins in a variety of multimeric forms.     

Arabidopsis O‐GlcNAc transferase SEC activates histone methyltransferase ATX1 to regulate flowering

Post‐translational modification of proteins by O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) is catalyzed by O‐GlcNAc transferases (OGTs). O‐GlcNAc modification of proteins regulates multiple important biological processes in metazoans. However, whether protein O‐GlcNAcylation is involved in epigenetic processes during plant development is largely unknown. Here, we show that loss of function of SECRET AGENT (SEC), an OGT in Arabidopsis, leads to an early flowering phenotype. This results from reduced histone H3 lysine 4 trimethylation (H3K4me3) of FLOWERING LOCUS C (FLC) locus, which encodes a key negative regulator of flowering. SEC activates ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1), a histone lysine methyltransferase (HKMT), through O‐GlcNAc modification to augment ATX1‐mediated H3K4me3 histone modification at FLC locus. SEC transfers an O‐GlcNAc group on Ser947 of ATX1, which resides in the SET domain, thereby activating ATX1. Taken together, these results uncover a novel post‐translational O‐GlcNAc modification‐mediated mechanism for regulation of HKMT activity and establish the function of O‐GlcNAc signaling in epigenetic processes in plants.     

GALEIDINIIUM RUGATUM GEN. ET SP. NOV. (DINOPHYCEAE), A NEW COCCOID DINOFLAGELLATE WITH A DIATOM ENDOSYMBIONT1

A new sand‐dwelling dinoflagellate from Palau, Galeidinium rugatum Tamura et Horiguchi gen. et sp. nov., is described. The life cycle of this new alga consists of a dominant nonmotile phase and a brief motile phase. The motile cell transforms itself directly into the nonmotile cell after swimming for a short period, and cell division takes place in the nonmotile phase. The nonmotile cell possesses a dome‐like cell covering, which is wrinkled and equipped with a transverse groove on the surface. The cell has 10–20 chloroplasts and a distinct eyespot. The motile cell is Gymnodinium‐like in shape. The dinoflagellate possesses an endosymbiotic alga to which the chloroplasts belong and which is separated from the host (dinoflagellate) cytoplasm by a unit membrane. The endosymbiont cytoplasm also possesses its own eukaryotic nucleus and mitochondria. The eyespot is surrounded by triple membranes and is located in the host cytoplasm. Photosynthetic pigment analysis, using HPLC, revealed that G. rugatum possesses fucoxanthin as the principal accessory pigment instead of peridinin. The rbcL tree showed that G. rugatum is monophyletic with Durinskia baltica (Levander) Carty et Cox and Kryptoperidinium foliaceum (Stein) Lindemann and that this clade is closely related to the pennate diatom, Cylindrotheca sp. The endosymbiont of G. rugatum is therefore shown to be a diatom. Phylogenetic analysis based on small subunit rDNA sequences demonstrated that G. rugatum, D. baltica, and K. foliaceum, all of which are known to harbor an endosymbiont of diatom origin, are closely related.     

Iron uptake and storage in the HAB dinoflagellate <Emphasis Type="Italic">Lingulodinium polyedrum</Emphasis>

The iron uptake and storage systems of terrestrial/higher plants are now reasonably well understood with two basic strategies being distinguished: Strategy I involves the induction of an Fe(III)-chelate reductase (ferrireductase) along with Fe(II) or Fe(III) transporter proteins while strategy II plants have evolved sophisticated systems based on high-affinity, iron specific, binding compounds called phytosiderophores. In contrast, there is little knowledge about the corresponding systems in marine, plant-like lineages. Herein we report a study of the iron uptake and storage mechanisms in the harmful algal bloom dinoflagellate Lingulodinium polyedrum. L. polyedrum is an armored dinoflagellate with a mixotrophic lifestyle and one of the most common bloom species on Southern California coast widely noted for its bioluminescent properties and as a producer of yessotoxins. Short term radio-iron uptake studies indicate that iron is taken up by L. polyedrum in a time dependent manner consistent with an active transport process. Based on inhibitor and other studies it appears that a reductive–oxidative pathway such as that found in yeast and the green alga Chlamydomonas reinhardtii is likely. Of the various iron sources tested vibrioferrin, a photoactive and relatively weak siderophore produced by potentially mutualistic Marinobacter bacterial species, was the most efficient. Other more stable and non-photoactive siderophores such as ferrioxamine E were ineffective. Several pieces of data including long term exposure to ⁵⁷Fe using Mössbauer spectroscopy suggest that L. polyedrum does not possess an iron storage system but rather presumably relies on an efficient iron uptake system, perhaps mediated by mutualistic interactions with bacteria.     

Devastating farmed abalone mortalities attributed to yessotoxin-producing dinoflagellates

A large dinoflagellate bloom in Walker Bay (South Africa) in January 2017 impacted 3 land-based abalone farms resulting in the death of several million animals. Satellite-derived images of Chl-a from the Ocean and Land Colour Imager (OLCI) on board the European Space Agency Sentinel-3 A showed bloom initiation in late December 2016 and dispersal in mid-February 2017. The bloom was dominated by two dinoflagellate species identified by light microscopy as Gonyaulax spinifera (Claparède & Lachmann) Diesing, 1866 and Lingulodinium polyedrum (Stein) Dodge, 1989. These morphologically based identifications were confirmed by phylogenetic analysis using partial sequences of the large subunit rDNA of both dinoflagellates. The appearance of yessotoxins (YTX) in abalone clearly coincided with increases in dinoflagellate concentrations. Yessotoxins in both the plankton and abalone were dominated by the two analogues homo-YTX and 45-hydroxy-YTX. The absence of toxins in a clonal culture of L. polyedrum implicated G. spinifera as the likely source of YTX. Toxin concentrations were found to be highest in the gills which showed the most significant pathology, including severe, generalized disruption of the gill epithelium characterized by degeneration and necrosis of epithelial cells accompanied by a modest inflammatory response. Some farms undertook pre-emptive or emergency harvesting to reduce financial losses.     

Elg1, an alternative subunit of the RFC clamp loader,preferentially interacts with SUMOylated PCNA

Replication‐factor C (RFC) is a protein complex that loads the processivity clamp PCNA onto DNA. Elg1 is a conserved protein with homology to the largest subunit of RFC, but its function remained enigmatic. Here, we show that yeast Elg1 interacts physically and genetically with PCNA, in a manner that depends on PCNA modification, and exhibits preferential affinity for SUMOylated PCNA. This interaction is mediated by three small ubiquitin‐like modifier (SUMO)‐interacting motifs and a PCNA‐interacting protein box close to the N‐terminus of Elg1. These motifs are important for the ability of Elg1 to maintain genomic stability. SUMOylated PCNA is known to recruit the helicase Srs2, and in the absence of Elg1, Srs2 and SUMOylated PCNA accumulate on chromatin. Strains carrying mutations in both ELG1 and SRS2 exhibit a synthetic fitness defect that depends on PCNA modification. Our results underscore the importance of Elg1, Srs2 and SUMOylated PCNA in the maintenance of genomic stability.     

Changes in antioxidant enzyme activities, malondialdehyde, and glutathione contents in the dinoflagellate Lingulodinium polyedrum (Dinophyceae) grown in batch-cultures

Catalase (CAT; EC 1.11.1.6) and ascorbate peroxidase (APX; EC 1.11.1.11) activities, as well as malondialdehyde (MDA) and reduced glutathione (GSH) and oxidized glutathione (GSSG) contents, were determined during the growth of the unicellular marine alga Lingulodinium polyedrum (Stein) Dodge in batch‐cultures. CAT and APX activity peaks were detected at the beginning of algal exponential growth, although declining trends were subsequently identified in both enzymes, with a slight increase in CAT activity at the end of the experimental period. MDA content attained maximum values from day 0–3 and at the end of the experimental period (day 21), declining halfway from day 10–14. GSH and GSSG contents presented the highest values at the beginning of the growth curve, decreasing from day 3 onwards. Despite the depletion of the GSH pool, an upward trend was observed in the (GSH) (0.5 GSSG + GSH)^?1 ratio, indicating that the L. polyedrum cells were able to maintain an increasing redox potential along exponential and linear growth phases in their efforts to prevent oxidative stress.     

Structural basis for recognition of H3K4 methylation status by the DNA methyltransferase 3A ATRX–DNMT3–DNMT3L domain

DNMT3 proteins are de novo DNA methyltransferases that are responsible for the establishment of DNA methylation patterns in mammalian genomes. Here, we have determined the crystal structures of the ATRX–DNMT3–DNMT3L (ADD) domain of DNMT3A in an unliganded form and in a complex with the amino‐terminal tail of histone H3. Combined with the results of biochemical analysis, the complex structure indicates that DNMT3A recognizes the unmethylated state of lysine 4 in histone H3. This finding indicates that the recruitment of DNMT3A onto chromatin, and thereby de novo DNA methylation, is mediated by recognition of the histone modification state by its ADD domain. Furthermore, our biochemical and nuclear magnetic resonance data show mutually exclusive binding of the ADD domain of DNMT3A and the chromodomain of heterochromatin protein 1α to the H3 tail. These results indicate that de novo DNA methylation by DNMT3A requires the alteration of chromatin structure.