Dinoflagellate chloroplasts as a model for extreme genome reduction and fragmentation in organelles - The COCOA principle for gene retention.

The genomes of peridinin-containing dinoflagellate chloroplasts have a very unusual organisation. These genomes are highly fragmented and greatly reduced, with most of the usual complement of chloroplast genes relocated to the nucleus. Dinoflagellate chloroplasts highlight evolutionary changes that are found to varying extents in a number of other organelle genomes. These include the chloroplast genome of the green alga Boodlea and other Cladophorales, and the mitochondrial genomes of blood-sucking and chewing lice, the parasitic plant Rhopalocnemis phalloides, the red alga Rhodosorus marinus and other members of the Stylonematophyceae, diplonemid flagellates, and some Cnidaria. Consideration of the coding content of the remnant chloroplast genomes indicates that organelles may preferentially retain genes for proteins important in initiating assembly of complexes, and the same is largely true for mitochondria. We propose a new principle, of CO-location for COntrol of Assembly (COCOA), indicating the importance of retaining these genes in the organelle. This adds to, but does not invalidate, the existing hypotheses of the multisubunit completion principle, CO-location for Redox Regulation (CORR) and Control by Epistasy of Synthesis (CES).

Evolution: The plasticity of plastids.

Many chloroplast-bearing plants and algae lost their photosynthetic activity during evolution but retained their chloroplasts for other functions. A group of dinoflagellate algae apparently lost one half of their photosynthetic machinery but retained the other, providing a novel mechanism for light perception.

Publisher Correction: Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

Integrated Genomic and Transcriptomic Analysis of the Peridinin Dinoflagellate Amphidinium carterae Plastid.

The plastid genomes of peridinin-containing dinoflagellates are highly unusual, possessing very few genes, which are located on small chromosomal elements termed "minicircles". These minicircles may contain genes, or no recognisable coding information. Transcripts produced from minicircles may undergo unusual processing events, such as the addition of a 3' poly(U) tail. To date, little is known about the genetic or transcriptional diversity of non-coding sequences in peridinin dinoflagellate plastids. These sequences include empty minicircles, and regions of non-coding DNA in coding minicircles. Here, we present an integrated plastid genome and transcriptome for the model peridinin dinoflagellate Amphidinium carterae, identifying a previously undescribed minicircle. We also profile transcripts covering non-coding regions of the psbA and petB/atpA minicircles. We present evidence that antisense transcripts are produced within the A. carterae plastid, but show that these transcripts undergo different end cleavage events from sense transcripts, and do not receive 3' poly(U) tails. The difference in processing events between sense and antisense transcripts may enable the removal of non-coding transcripts from peridinin dinoflagellate plastid transcript pools.

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.

The genetic intractability of Symbiodinium microadriaticum to standard algal transformation methods.

Modern transformation and genome editing techniques have shown great success across a broad variety of organisms. However, no study of successfully applied genome editing has been reported in a dinoflagellate despite the first genetic transformation of Symbiodinium being published about 20 years ago. Using an array of different available transformation techniques, we attempted to transform Symbiodinium microadriaticum (CCMP2467), a dinoflagellate symbiont of reef-building corals, with the view to performing subsequent CRISPR-Cas9 mediated genome editing. Plasmid vectors designed for nuclear transformation containing the chloramphenicol resistance gene under the control of the CaMV p35S promoter as well as several putative endogenous promoters were used to test a variety of transformation techniques including biolistics, electroporation and agitation with silicon carbide whiskers. Chloroplast-targeted transformation was attempted using an engineered Symbiodinium chloroplast minicircle encoding a modified PsbA protein expected to confer atrazine resistance. We report that we have been unable to confer chloramphenicol or atrazine resistance on Symbiodinium microadriaticum strain CCMP2467.

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.

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.

Organization and expression of organellar genomes.

Protist mitochondrial genomes show a very wide range of gene content, ranging from three genes for respiratory chain components in Apicomplexa and dinoflagellates to nearly 100 genes in Reclinomonas americana. In many organisms the rRNA genes are fragmented, although still functional. Some protist mitochondria encode a full set of tRNAs, while others rely on imported molecules. There is similarly a wide variation in mitochondrial genome organization, even among closely related groups. Mitochondrial gene expression and control are generally poorly characterized. Transcription probably relies on a 'viral-type' RNA polymerase, although a 'bacterial-type' enzyme may be involved in some cases. Transcripts are heavily edited in many lineages. The chloroplast genome generally shows less variation in gene content and organization, although greatly reduced genomes are found in dinoflagellate algae and non-photosynthetic organisms. Genes in the former are located on small plasmids in contrast to the larger molecules found elsewhere. Control of gene expression in chloroplasts involves transcriptional and post-transcriptional regulation. Redox poise and the ATP/ADP ratio are likely to be important determinants. Some protists have an additional extranuclear genome, the nucleomorph, which is a remnant nucleus. Nucleomorphs of two separate lineages have a number of features in common.

Dinoflagellates: a mitochondrial genome all at sea.

Dinoflagellate algae are notorious for their highly unusual organization of nuclear and chloroplast genomes. Early studies on the dinoflagellate mitochondrial genome indicated that it encodes the same three protein-coding genes found in Plasmodium spp., but with a complex organization and transcript editing. Recent work has extended this view, showing that the dinoflagellate mitochondrial genome contains a wide array of gene fragments and genes interspersed with noncoding inverted repeats. The genome seems to require noncanonical start and stop codons, as well as high levels of editing, trans-splicing and the addition of oligonucleotide caps at the 5' and 3' ends of transcripts. Despite its small coding content, the dinoflagellate mitochondrial genome is one of the most complex known.

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.

Organization of the mitochondrial genome in the dinoflagellate Amphidinium carterae.

We have characterized the mitochondrial genome of the dinoflagellate Amphidinium carterae. It contains just 3 identifiable protein-coding genes: cox1, cox3, and cob. No evidence for rRNA or tRNA genes was found. Expressed sequence tags (EST) sequences for the 3 genes suggest that RNA editing occurs in 2 cases removing an in-frame stop codon. Two of the transcripts (cob and cox1) lack a stop codon at the end of the gene. The genome contains a large amount of noncoding DNA including many fragmented copies of all the 3 genes and large numbers of inverted repeats. The genome, which contains about 70% AT, has undergone extensive recombination, possibly due to the inverted repeats. The highly reduced mitochondrial gene content supports the relationship of the dinoflagellates and apicomplexa as sister groups.

Comparative analysis of dinoflagellate chloroplast genomes reveals rRNA and tRNA genes.

BACKGROUND: Peridinin-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. RESULTS: PCR 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. CONCLUSION: Comparative 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.

Molecular diversity of dinoflagellate symbionts of Cnidaria: the psbA minicircle of Symbiodinium.

Dinoflagellate algae of the genus Symbiodinium are important symbionts within corals and other benthic marine animals. The molecular diversity of Symbiodinium has been described mainly by use of ribosomal DNA sequence data. We tested whether minicircle sequences, which appear to form the chloroplast genome in many dinoflagellates, could be used as a marker for molecular diversity among symbionts found in corals and sea anemones. Partial and full-length sequences for psbA were obtained from environmental samples of coral and sea anemones of wide-ranging geographical distribution. Phylogenetic trees constructed with partial psbA sequences were consistent with the known phylotypes of the isolates. Further sequencing suggested that the psbA gene is present on a minicircle in all Symbiodinium phylotypes. The length and DNA sequence of the non-coding portion of the minicircles varied considerably among Symbiodinium phylotypes. In two Symbiodinium isolates from different phylotypes an elaborate pattern of repeat sequences of unknown function was found in the non-coding region. Phylogenetic analysis of the non-coding region of the psbA minicircle indicates that minicircle sequences could be a useful chloroplast-derived marker for differentiating both closely related and distantly related Symbiodinium isolates.

Why are plastid genomes retained in non-photosynthetic organisms?

The evolution of the plastid from a photosynthetic bacterial endosymbiont involved a dramatic reduction in the complexity of the plastid genome, with many genes either discarded or transferred to the nucleus of the eukaryotic host. However, this evolutionary process has not gone to completion and a subset of genes remains in all plastids examined to date. The various hypotheses put forward to explain the retention of the plastid genome have tended to focus on the need for photosynthetic organisms to retain a genetic system in the chloroplast, and they fail to explain why heterotrophic plants and algae, and the apicomplexan parasites all retain a genome in their non-photosynthetic plastids. Here we consider two additional explanations: the 'essential tRNAs' hypothesis and the 'transfer-window' hypothesis.

Novel plastid gene minicircles in the dinoflagellate Amphidinium operculatum.

Seven new minicircles, forming part of the fragmented plastid genome of the dinoflagellate Amphidinium operculatum, have been identified by PCR. Three minicircles are full-length, one of 2.6 kb encoding the 23S rRNA gene, one of 2.4 kb containing the psaB gene, and a third of 2.5 kb containing the psbD, psbE and psbI genes. This is the first report of a three-gene minicircle. All three genes are conventional in length, with the same codon bias found in other minicircle genes. The psbI gene sequence is very divergent. One empty minicircle, of 1.7 kb, and three 'microcircles', between 400 and 600 bp, have also been identified by PCR. They appear to be formed from full-length minicircles by homologous recombination and internal deletion. All three microcircles contain the core region common to minicircles, but are missing a coding region, providing further evidence that the core region is necessary for minicircle replication and maintenance.

Phylogenetics of artificial manuscripts.

Biological evolution has parallels with the development of natural languages, man-made artifacts, and manuscript texts. As a result, phylogenetic methods developed for evolutionary biology are increasingly being used in linguistics, anthropology, archaeology, and textual criticism. Despite this popularity, there have been few critical tests of their suitability. Here, we apply phylogenetic methods to artificial manuscripts with a known true phylogeny, produced by modern 'scribes'. Although the survival of ancestral forms and multiple descendants from a single ancestor are probably much more common in manuscript evolution than biological evolution, we were able to reconstruct most of the true phylogeny. This is important because phylogenetic methods are influencing the production of critical editions of major written works. We also show that the variation in rates of change at different locations in the text follows a gamma distribution, as is often the case in DNA sequences.