Dinoflagellate Chloroplast Research Articles

DNA:RNA Hybrids Are Major Dinoflagellate Minicircle Molecular Types.

Peridinin-containing dinoflagellate plastomes are predominantly encoded in nuclear genomes, with less than 20 essential chloroplast proteins carried on "minicircles". Each minicircle generally carries one gene and a short non-coding region (NCR) with a median length of approximately 400-1000 bp. We report here differential nuclease sensitivity and two-dimensional southern blot patterns, suggesting that dsDNA minicircles are in fact the minor forms, with substantial DNA:RNA hybrids (DRHs). Additionally, we observed large molecular weight intermediates, cell-lysate-dependent NCR secondary structures, multiple bidirectional predicted ssDNA structures, and different southern blot patterns when probed with different NCR fragments. In silico analysis suggested the existence of substantial secondary structures with inverted repeats (IR) and palindrome structures within the initial ~650 bp of the NCR sequences, in accordance with conversion event(s) outcomes with PCR. Based on these findings, we propose a new transcription-templating-translation model, which is associated with cross-hopping shift intermediates. Since dinoflagellate chloroplasts are cytosolic and lack nuclear envelope breakdown, the dynamic DRH minicircle transport could have contributed to the spatial-temporal dynamics required for photosystem repair. This represents a paradigm shift from the previous understanding of "minicircle DNAs" to a "working plastome", which will have significant implications for its molecular functionality and evolution.

Genetic transformation of the dinoflagellate chloroplast

Coral reefs are some of the most important and ecologically diverse marine environments. At the base of the reef ecosystem are dinoflagellate algae, which live symbiotically within coral cells. Efforts to understand the relationship between alga and coral have been greatly hampered by the lack of an appropriate dinoflagellate genetic transformation technology. By making use of the plasmid-like fragmented chloroplast genome, we have introduced novel genetic material into the dinoflagellate chloroplast genome. We have shown that the introduced genes are expressed and confer the expected phenotypes. Genetically modified cultures have been grown for 1 year with subculturing, maintaining the introduced genes and phenotypes. This indicates that cells continue to divide after transformation and that the transformation is stable. This is the first report of stable chloroplast transformation in dinoflagellate algae.

Breaking up is hard to do: the complexity of the dinoflagellate chloroplast genome

Breaking up is hard to do: the complexity of the dinoflagellate chloroplast genome

Transformation of the dinoflagellate chloroplast

Dinoflagellate algae are ecologically and environmentally important, as symbionts of corals and many other aquatic organisms, and the causative agents of red tides. However, attempts over the last twenty years to establish genetic manipulation systems for dinoflagellates have met with little success. We have exploited the unusual chloroplast genome of dinoflagellates to establish a system for transformation of this organelle. The chloroplast genome of peridinin-containing (the ancestral state) dinoflagellates is highly reduced and composed of a number of small, plasmid-like molecules, referred to as ‘minicircles’. We have constructed shuttle vectors that are fusions of minicircles and Escherichia coli plasmids and carry selectable markers. We used biolistic transformation to introduce these into the model dinoflagellate Amphidinium carterae. We found that the plasmids confer the expected phenotype on the dinoflagellate cells, and we can detect the plasmid DNA and associated transcripts following selection, indicating successful transformation. This opens up the possibility of studying many aspects of dinoflagellate chloroplast biology, including the maintenance and expression of the minicircles, and the role of the chloroplast in phenomena such as coral bleaching.

The Chloroplast Genome of a Symbiodinium sp. Clade C3 Isolate

Dinoflagellate algae of the genus Symbiodinium form important symbioses within corals and other benthic marine animals. Dinoflagellates possess an extremely reduced plastid genome relative to those examined in plants and other algae. In dinoflagellates the plastid genes are located on small plasmids, commonly referred to as 'minicircles'. However, the chloroplast genomes of dinoflagellates have only been extensively characterised from a handful of species. There is also evidence of considerable variation in the chloroplast genome organisation across those species that have been examined. We therefore characterised the chloroplast genome from an environmental coral isolate, in this case containing a symbiont belonging to the Symbiodinium sp. clade C3. The gene content of the genome is well conserved with respect to previously characterised genomes. However, unlike previously characterised dinoflagellate chloroplast genomes we did not identify any 'empty' minicircles. The sequences of this chloroplast genome show a high rate of evolution relative to other algal species. Particularly notable was a surprisingly high level of sequence divergence within the core polypeptides of photosystem I, the reasons for which are currently unknown. This chloroplast genome also possesses distinctive codon usage and GC content. These features suggest that chloroplast genomes in Symbiodinium are highly plastic.

Functional remodeling of RNA processing in replacement chloroplasts by pathways retained from their predecessors.

Chloroplasts originate through the endosymbiotic integration of a host and a photosynthetic symbiont, with processes established within the host for the biogenesis and maintenance of the nascent chloroplast. It is thought that several photosynthetic eukaryotes have replaced their original chloroplasts with others derived from different source organisms in a process termed "serial endosymbiosis of chloroplasts." However, it is not known whether replacement chloroplasts are affected by the biogenesis and maintenance pathways established to support their predecessors. Here, we investigate whether pathways established during a previous chloroplast symbiosis function in the replacement chloroplasts of the dinoflagellate alga Karenia mikimotoi. We show that chloroplast transcripts in K. mikimotoi are subject to 3' polyuridylylation and extensive sequence editing. We confirm that these processes do not occur in free-living relatives of the replacement chloroplast lineage, but are otherwise found only in the ancestral, red algal-derived chloroplasts of dinoflagellates and their closest relatives. This indicates that these unusual RNA-processing pathways have been retained from the original symbiont lineage and made use of by the replacement chloroplast. Our results constitute an addition to current theories of chloroplast evolution in which chloroplast biogenesis may be radically remodeled by pathways remaining from previous symbioses.

Polyuridylylation and processing of transcripts from multiple gene minicircles in chloroplasts of the dinoflagellate Amphidinium carterae

Although transcription and transcript processing in the chloroplasts of plants have been extensively characterised, the RNA metabolism of other chloroplast lineages across the eukaryotes remains poorly understood. In this paper, we use RT-PCR to study transcription and transcript processing in the chloroplasts of Amphidinium carterae, a model peridinin-containing dinoflagellate. These organisms have a highly unusual chloroplast genome, with genes located on multiple small 'minicircle' elements, and a number of idiosyncratic features of RNA metabolism including transcription via a rolling circle mechanism, and 3' terminal polyuridylylation of transcripts. We demonstrate that transcription occurs in A. carterae via a rolling circle mechanism, as previously shown in the dinoflagellate Heterocapsa, and present evidence for the production of both polycistronic and monocistronic transcripts from A. carterae minicircles, including several regions containing ORFs previously not known to be expressed. We demonstrate the presence of both polyuridylylated and non-polyuridylylated transcripts in A. carterae, and show that polycistronic transcripts can be terminally polyuridylylated. We present a model for RNA metabolism in dinoflagellate chloroplasts where long polycistronic precursors are processed to form mature transcripts. Terminal polyuridylylation may mark transcripts with the correct 3' end.

Re-classification of Pheopolykrikos hartmannii as Polykrikos (Dinophyceae) based partly on the ultrastructure of complex extrusomes

Athecate, pseudocolony-forming dinoflagellates have been classified within two genera of polykrikoids, Polykrikos and Pheopolykrikos, and different views about the boundaries and composition of these genera have been expressed in the literature. The photosynthetic polykrikoid Pheopolykrikos hartmannii, for instance, was originally described within Polykrikos and is now known to branch closely with several Polykrikos species in molecular phylogenetic analyses of ribosomal gene sequences. In this study, we report the first ultrastructural data for this species and demonstrate that Ph. hartmannii has all of the features that characterize the genus Polykrikos, including the synapomorphic “taeniocyst-nematocyst complex”. We also demonstrate that the ultrastructure of the chloroplasts in Ph. hartmannii conforms to the usual peridinin-containing chloroplasts of most photosynthetic dinoflagellates, which improves inferences about the origin(s) and evolution of photosynthesis within the genus. After taking into account all of the ultrastructural data on polykrikoids presented here and in the literature, this species is re-classified to its original status as Polykrikos hartmannii.

Substitutional editing of Heterocapsa triquetra chloroplast transcripts and a folding model for its divergent chloroplast 16S rRNA

Substitutional editing increases genomic plasticity by changing or modifying bases at the RNA level. In this study we sequenced 10 mature chloroplast mRNAs, the chloroplast 16S rRNA and a partial chloroplast 23S rRNA from the dinoflagellate Heterocapsa triquetra, and found multiple types of substitutional editing, with A-to-G editing predominating. A-to-G editing of mRNAs converts two unusual AUA start codons into conventional AUG start codons, but three AUA start codons are not edited, showing that this dinoflagellate chloroplast has three possible start codons: AUG, AUA and UUG. To analyze the editing effects on rRNAs, we computationally predicted the secondary structure of the 16S rRNA based on the E. coli model. There are twenty editing sites in well-conserved regions of the secondary structure and eleven out of them restore conservation with other models. Moreover, A-to-G editing sites are frequently found in loop regions rather than double-stranded regions, suggesting that the A-to-G editing mechanism in dinoflagellate chloroplasts is different from that responsible for animal nuclear A-to-I(G) editing. The model of the edited 16S rRNA derived by the comparative method shares conserved secondary structural elements with other 16S rRNAs in spite of its very divergent primary sequence, supporting its role as a functional component of the chloroplast ribosome.

The remarkable chloroplast genome of dinoflagellates

Dinoflagellates are an economically and ecologically important eukaryotic algal group. The organization of their chloroplast genome appears to be radically different from that in plants and other algae. The gene content has been dramatically reduced in dinoflagellates, with the large-scale transfer of genes to the nucleus. Most of the remaining genes encode subunits of Photosystems I and II, the cytochrome b(6)f complex, and ATP synthase, as well as rRNAs and a few tRNAs. Whereas conventional chloroplast genomes have all their genes physically linked on one molecule, dinoflagellate chloroplast genes are located on small plasmids, termed 'minicircles'. Each minicircle has at most a few genes, and a distinguishable 'core' region. Genes are always in the same orientation with respect to the core region. There are also non-coding minicircles, including aberrant forms of minicircles apparently derived from other minicircles by rearrangement. The evidence that the minicircles are located in the chloroplast and that there is no conventional chloroplast genome in addition to the minicircles is discussed. Transcription of minicircles is probably initiated close to the core, generating transcripts corresponding to an almost entire minicircle. The transcripts are then cleaved to molecules corresponding to individual genes. Post-transcriptional modifications include editing and addition of a polyU tail. It is discussed why these particular genes have been retained in the dinoflagellate chloroplast, together with the possibility that the chloroplast supplies fMet-tRNA to the mitochondrion.

Transcript Analysis of Dinoflagellate Plastid Gene Minicircles

The dinoflagellate chloroplast genome is fragmented into a number of plasmid-like minicircles, mostly containing one or more genes, and with a conserved core. We show here that, in addition to the transcripts of similar sizes to individual genes that have been reported previously, there are larger transcripts beginning and ending close to the core region. These may give rise to the smaller transcripts by processing. We also show that previously reported ORFs (open reading frames) are represented by transcripts that are significantly more abundant than those for non-coding regions, indicating that the ORFs are indeed functional. We show that 'empty' minicircles are also transcribed. We propose a model for linkage of DNA replication and transcription in dinoflagellate chloroplasts.

Identification and transcription of transfer RNA genes in dinoflagellate plastid minicircles

The dinoflagellate chloroplast genome is unique in that the genes are found on small circular DNA molecules carrying from one to three genes. In addition, only 14 of the typical chloroplast-located genes have so far been discovered on minicircles, while a number have been transferred to the nucleus. We have sequenced four new minicircles from the dinoflagellate Heterocapsa triquetra, three of which carry a single protein-coding gene ( psbD, psbE, petD) and one that appears to be an “empty” circle. Using the tRNA prediction programs ARAGORN and tRNAscan-SE, tRNA-Met was found in the petD circle immediately downstream of the end of petD, while tRNA-Trp and tRNA-Pro were detected in the psbE and petD circles as well as in several chimeric circles of H. triquetra and the psbA minicircles of Heterocapsa pygmaea. RT-PCR showed that the tRNAs were co-transcribed with the protein-coding genes that preceded them, and cleaved from the precursor before a poly(U) tail was added to the mRNA.

Baldinia anauniensis gen. et sp. nov.: a ‘new’ dinoflagellate from Lake Tovel, N. Italy

G. Hansen, N. Daugbjerg, and P. Henriksen. 2007. Baldinia anauniensis gen. et sp. nov.: a ‘new’ dinoflagellate from Lake Tovel, N. Italy. Phycologia 46: 86–108. DOI: 10.2216/PH06-23.1The green form of ‘Glenodinium sanguineum Marchesoni’ sensu Baldi was investigated in detail by light microscopy, scanning electron microscopy, transmission electron microscopy and by analysis of the SSU- and LSU rDNA genes. These data demonstrated it to be different from the red form of ‘G. sanguineum’ (now Tovellia sanguinea) and to represent a new species, Baldinia anauniensis gen. et sp. nov. It lacked thecal plates and an apical groove, and was characterised by the presence of a peculiar lamellar body similar to that described from certain dinoflagellates harbouring a diatom endosymbiont. However, B. anauniensis had a typical dinoflagellate chloroplast, and pigment analysis by high-performance liquid chromatography revealed peridinin as the major carotenoid pigment. Phylogenies based on either SSU or LSU rDNA revealed a relationship to suessialoid and woloszynskioid lineages rather than to the diatom-containing species, though morphological support for this relationship could only be argued by the absence of trichocyst and to some extent the intraplastidial eyespot type. A ventral fibre attached to the right side of the longitudinal basal body, an arrangement not observed in any other dinoflagellate before. Numerous food vacuoles with cryptophyte remnants demonstrated that B. anauniensis was mixotrophic.

Preliminary Examination of Short-Term Cellular Toxicological Responses of the Coral Madracis mirabilis to Acute Irgarol 1051 Exposure

Irgarol 1051 is an s-triazine herbicide formulated with Cu2O in antifouling paints. Recent studies have shown that Irgarol 1051 inhibits coral photosynthesis at environmentally relevant concentrations, consistent with its mode of action as a photosystem II inhibitor. Related toxicologic effects of this herbicide on coral cellular physiology have not yet been investigated. We used cellular diagnostics to measure changes in 18 toxicologic cellular parameters in endosymbiotic algal (dinoflagellate) and cnidarian (host) fractions of the common branching coral Madracis mirabilis associated with in vivo 8- and 24-hour exposures to a nominal initial Irgarol 1051 concentration of 10 microg L(-1). Responses measured were (1) xenobiotic response, which includes total and dinoflagellate multixenobiotic resistance (MXR), cnidarian cytochrome (CYP) P450-3 and P450-6 classes, cnidarian, and dinoflagellate glutathione-s-transferase (GST); (2) oxidative damage and response, which includes cnidarian and dinoflagellate Cu/Zn and Mn superoxide dismutase (SOD), cnidarian and dinoflagellate glutathione peroxidase (GPx), cnidarian catalase, and total protein carbonyl); (3) metabolic homeostasis, which includes chloroplast and invertebrate small heat-shock proteins (sHsp), cnidarian protoporphyrinogen oxidase IX (PPO), cnidarian ferrochelatase, and cnidarian heme oxygenase; and (4) protein metabolic condition, which includes cnidarian and dinoflagellate heat shock proteins (hsp70 and hsp60), total ubiquitin, and cnidarian ubiquitin ligase. Acute responses to Irgarol 1051 exposure included significant increases in total and dinoflagellate MXR, dinoflagellate Cu/Zn SOD, dinoflagellate chloroplast sHsp, and cnidarian PPO. Irgarol 1051 exposure resulted in decreases in cnidarian GPx, cnidarian ferrochelatase, cnidarian catalase, and cnidarian CYP 450-3 and -6 classes. Related implications of Irgarol 1051 exposure to coral cellular condition are discussed.

Comparative analysis of dinoflagellate chloroplast genomes reveals rRNA and tRNA genes

BackgroundPeridinin-containing dinoflagellates have a highly reduced chloroplast genome, which is unlike that found in other chloroplast containing organisms. Genome reduction appears to be the result of extensive transfer of genes to the nuclear genome. Unusually the genes believed to be remaining in the chloroplast genome are found on small DNA 'minicircles'. In this study we present a comparison of sets of minicircle sequences from three dinoflagellate species.ResultsPCR was used to amplify several minicircles from Amphidinium carterae so that a homologous set of gene-containing minicircles was available for Amphidinium carterae and Amphidinium operculatum, two apparently closely related peridinin-containing dinoflagellates. We compared the sequences of these minicircles to determine the content and characteristics of their chloroplast genomes. We also made comparisons with minicircles which had been obtained from Heterocapsa triquetra, another peridinin-containing dinoflagellate. These in silico comparisons have revealed several genetic features which were not apparent in single species analyses. The features include further protein coding genes, unusual rRNA genes, which we show are transcribed, and the first examples of tRNA genes from peridinin-containing dinoflagellate chloroplast genomes.ConclusionComparative analysis of minicircle sequences has allowed us to identify previously unrecognised features of dinoflagellate chloroplast genomes, including additional protein and RNA genes. The chloroplast rRNA gene sequences are radically different from those in other organisms, and in many ways resemble the rRNA genes found in some highly reduced mitochondrial genomes. The retention of certain tRNA genes in the dinoflagellate chloroplast genome has important implications for models of chloroplast-mitochondrion interaction.

First ultrastructural investigations of the consortium between a phototrophic eukaryotic endocytobiont and Podolampas bipes (Dinophyceae)

Several species of the genus Podolampas (Dinophyceae), P. antarctica, P. bipes, P. elegans, P. palmipes, P. reliculata, P. spinifera and a new undescribed species, were recurrently found in plankton samples of the Atlantic Ocean, Pacific Ocean and the Mediterranean Sea during several oceanographic expeditions. Only P. bipes and P. reticulata always contained autofluorescent endocytobionts, which were interpreted as dinoflagellate chloroplasts by former investigations based on light microscopy. Our investigations of P. bipes showed a consortium of the dinoflagellate and multiple, endocytic cells, each containing chloroplasts, mitochondria, ribosomes, a dictyosome and a eukaryotic nucleus. In culture, these endocytobionts were persistent in the host cells and were distributed into both daughter cells during mitotic cell division. Although endo-symbionts are known from various dinoflagellates, endocytobiosis of several photosynthetic eukaryotic cells per dinoflagellate is new. Ultrastructural details of the endocytobionts show that they belong to the class Dictyochophyceae (Heterokontophyta), most probably to the order Pedinellales. Additionally, further ultrastructural details of P. bipes, such as the presence of three types of extrusomes, intracellular bacteria, rod-like structures composed of tubules and a presumably motile thecal plate, are described.

Dinoflagellate chloroplasts ? where have all the genes gone?

Dinoflagellate chloroplasts ? where have all the genes gone?

Morphological reinvestigation and characterization of the marine, sand-dwelling dinoflagellate Adenoides eludens (Dinophyceae)

The phototrophic, marine, sand-dwelling dinoflagellate Adenoides eludens (Herdman) Balech was isolated from intertidal sand of the North German Wadden Sea and subsequently cultured. For taxonomic purposes it was examined using light, scanning and transmission electron microscopy. Cells are slightly asymmetrical, oval to round, laterally flattened, 30.0 – 35.0 μm long and 23.0 – 28.0 μm deep. The theca is composed of 26 plates. According to our interpretation the plate formula is Po 4′ 6c 4s 5′′′ 5p 1′′′′ or alternatively it can be interpreted as Po 4′ 6c 5s 5′′′ 3p 2′′′′. All thecal plates are smooth with scattered pores. Dinoflagellate chloroplasts surrounded by three membranes display thylakoids grouped in stacks of three. Two stalked pyrenoids with a starch sheath are present. Spindle trichocysts were present and extruded through trichocyst pores. Additionally, intracellular gram-negative bacteria were found in the cytoplasm. Asexual reproduction is by desmoschisis. The position of Adenoides among the dinoflagellates is discussed, and we tentatively regard it as belonging to the gonyaulacoid lineage.

Evolution of dinoflagellate unigenic minicircles and the partially concerted divergence of their putative replicon origins.

Dinoflagellate chloroplast genes are unique in that each gene is on a separate minicircular chromosome. To understand the origin and evolution of this exceptional genomic organization we completely sequenced chloroplast psbA and 23S rRNA gene minicircles from four dinoflagellates: three closely related Heterocapsa species (H. pygmaea, H. rotundata, and H. niei) and the very distantly related Amphidinium carterae. We also completely sequenced a Protoceratium reticulatum minicircle with a 23S rRNA gene of novel structure. Comparison of these minicircles with those previously sequenced from H. triquetra and A. operculatum shows that in addition to the single gene all have noncoding regions of approximately a kilobase, which are likely to include a replication origin, promoter, and perhaps segregation sequences. The noncoding regions always have a high potential for folding into hairpins and loops. In all six dinoflagellate strains for which multiple minicircles are fully sequenced, parts of the noncoding regions, designated cores, are almost identical between the psbA and 23S rRNA minicircles, but the remainder is very different. There are two, three, or four cores per circle, sometimes highly related in sequence, but no sequence identity is detectable between cores of different species, even within one genus. This contrast between very high core conservation within a species, but none among species, indicates that cores are diverging relatively rapidly in a concerted manner. This is the first well-established case of concerted evolution of noncoding regions on numerous separate chromosomes. It differs from concerted evolution among tandemly repeated spacers between rRNA genes, and that of inverted repeats in plant chloroplast genomes, in involving only the noncoding DNA cores. We present two models for the origin of chloroplast gene minicircles in dinoflagellates from a typical ancestral multigenic chloroplast genome. Both involve substantial genomic reduction and gene transfer to the nucleus. One assumes differential gene deletion within a multicopy population of the resulting oligogenic circles. The other postulates active transposition of putative replicon origins and formation of minicircles by homologous recombination between them.

MOLECULAR PHYLOGENETICS AND THE DINOFLAGELLATE CHLOROPLAST

Delwiche, C. F., and Bachvaroff, T. R.Cell Biology and Molecular Genetics, University of Maryland ‐ College Park MD 20742‐5815 USAThe chloroplasts of dinoflagellates have been among the last chloroplasts to be studied with molecular phylogenetic methods. DNA sequences from peridinin‐containing chloroplasts are now available, and have begun to be examined with molecular phylogenetic methods. The high rate of sequence evolution in these genes increases noise and makes analyses difficult. We have evaluated phylogenetic signal in several dinoflagellate chloroplast genes using parametric bootstrapping and other methods. Although some aspects of the tree topologies found in phylogenetic analyses of dinoflagellate chloroplast genes are clearly artifactual, there are some features that seem to reflect genuine phylogenetic information. We find support for the concept of monophyletic chromophyte chloroplasts, but contradictory information concerning the relationships among the host cells that contain these plastids. The best hope for determining the evolutionary history of chromophytic plastids lies in the analysis of nuclear‐encoded, chloroplast expressed genes.