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.

Evolution: The great photosynthesis heist.

Many eukaryotes acquired chloroplasts by endosymbiotic acquisition of photosynthetic bacteria or already-domesticated chloroplasts from other eukaryotes. However, the ciliate Mesodinium rubrum acquires the nucleus of a photosynthetic eukaryote, as well as its chloroplast, resulting in dramatic metabolic remodelling in the ciliate.

In silico identification of Theileria parva surface proteins.

East Coast Fever is a devastating African cattle disease caused by the apicomplexan parasite, Theileria parva. Little is known about the cell surface, and few proteins have been identified. Here, we take an in silico approach to identify novel cell surface proteins, and predict the structure of four key proteins.

The Evolution of the Cytochrome c6 Family of Photosynthetic Electron Transfer Proteins.

During photosynthesis, electrons are transferred between the cytochrome b6f complex and photosystem I. This is carried out by the protein plastocyanin in plant chloroplasts, or by either plastocyanin or cytochrome c6 in many cyanobacteria and eukaryotic algal species. There are three further cytochrome c6 homologs: cytochrome c6A in plants and green algae, and cytochromes c6B and c6C in cyanobacteria. The function of these proteins is unknown. Here, we present a comprehensive analysis of the evolutionary relationship between the members of the cytochrome c6 family in photosynthetic organisms. Our phylogenetic analyses show that cytochromes c6B and c6C are likely to be orthologs that arose from a duplication of cytochrome c6, but that there is no evidence for separate origins for cytochromes c6B and c6C. We therefore propose renaming cytochrome c6C as cytochrome c6B. We show that cytochrome c6A is likely to have arisen from cytochrome c6B rather than by an independent duplication of cytochrome c6, and present evidence for an independent origin of a protein with some of the features of cytochrome c6A in peridinin dinoflagellates. We conclude with a new comprehensive model of the evolution of the cytochrome c6 family which is an integral part of understanding the function of the enigmatic cytochrome c6 homologs.

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.

An essential pentatricopeptide repeat protein in the apicomplexan remnant chloroplast.

The malaria parasite Plasmodium and other apicomplexans such as Toxoplasma evolved from photosynthetic organisms and contain an essential, remnant plastid termed the apicoplast. Transcription of the apicoplast genome is polycistronic with extensive RNA processing. Yet little is known about the mechanism of apicoplast RNA processing. In plants, chloroplast RNA processing is controlled by multiple pentatricopeptide repeat (PPR) proteins. Here, we identify the single apicoplast PPR protein, PPR1. We show that the protein is essential and that it binds to RNA motifs corresponding with previously characterized processing sites. Additionally, PPR1 shields RNA transcripts from ribonuclease degradation. This is the first characterization of a PPR protein from a nonphotosynthetic plastid.

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.

Transcripts in the Plasmodium Apicoplast Undergo Cleavage at tRNAs and Editing, and Include Antisense Sequences.

The apicoplast, an organelle found in Plasmodium and many other parasitic apicomplexan species, is a remnant chloroplast that is no longer able to carry out photosynthesis. Very little is known about primary transcripts and RNA processing in the Plasmodium apicoplast, although processing in chloroplasts of some related organisms (chromerids and dinoflagellate algae) shows a number of unusual features, including RNA editing and the addition of 3' poly(U) tails. Here, we show that many apicoplast transcripts are polycistronic and that there is extensive RNA processing, often involving the excision of tRNA molecules. We have identified major RNA processing sites, and have shown that these are associated with a conserved sequence motif. We provide the first evidence for the presence of RNA editing in the Plasmodium apicoplast, which has evolved independently from editing in dinoflagellates. We also present evidence for long, polycistronic antisense transcripts, and show that in some cases these are processed at the same sites as sense transcripts. Together, this research has significantly enhanced our understanding of the evolution of chloroplast RNA processing in the Apicomplexa and dinoflagellate algae.

Identification of Sequences Encoding Symbiodinium minutum Mitochondrial Proteins.

The dinoflagellates are an extremely diverse group of algae closely related to the Apicomplexa and the ciliates. Much work has previously been undertaken to determine the presence of various biochemical pathways within dinoflagellate mitochondria. However, these studies were unable to identify several key transcripts including those encoding proteins involved in the pyruvate dehydrogenase complex, iron-sulfur cluster biosynthesis, and protein import. Here, we analyze the draft nuclear genome of the dinoflagellate Symbiodinium minutum, as well as RNAseq data to identify nuclear genes encoding mitochondrial proteins. The results confirm the presence of a complete tricarboxylic acid cycle in the dinoflagellates. Results also demonstrate the difficulties in using the genome sequence for the identification of genes due to the large number of introns, but show that it is highly useful for the determination of gene duplication events.

Evolution of chloroplast transcript processing in Plasmodium and its chromerid algal relatives.

It is well understood that apicomplexan parasites, such as the malaria pathogen Plasmodium, are descended from free-living algae, and maintain a vestigial chloroplast that has secondarily lost all genes of photosynthetic function. Recently, two fully photosynthetic relatives of parasitic apicomplexans have been identified, the 'chromerid' algae Chromera velia and Vitrella brassicaformis, which retain photosynthesis genes within their chloroplasts. Elucidating the processes governing gene expression in chromerid chloroplasts might provide valuable insights into the origins of parasitism in the apicomplexans. We have characterised chloroplast transcript processing pathways in C. velia, V. brassicaformis and P. falciparum with a focus on the addition of an unusual, 3' poly(U) tail. We demonstrate that poly(U) tails in chromerids are preferentially added to transcripts that encode proteins that are directly involved in photosynthetic electron transfer, over transcripts for proteins that are not involved in photosynthesis. To our knowledge, this represents the first chloroplast transcript processing pathway to be associated with a particular functional category of genes. In contrast, Plasmodium chloroplast transcripts are not polyuridylylated. We additionally present evidence that poly(U) tail addition in chromerids is involved in the alternative processing of polycistronic precursors covering multiple photosynthesis genes, and appears to be associated with high levels of transcript abundance. We propose that changes to the chloroplast transcript processing machinery were an important step in the loss of photosynthesis in ancestors of parasitic apicomplexans.

Evolution: unveiling early alveolates.

The isolation and characterisation of a novel protist lineage enables the reconstruction of early evolutionary events that gave rise to ciliates, malaria parasites, and coral symbionts. These events include dramatic changes in mitochondrial genome content and organisation.

Cryptic organelle homology in apicomplexan parasites: insights from evolutionary cell biology.

The economic and clinical significance of apicomplexan parasites drives interest in their many evolutionary novelties. Distinctive intracellular organelles play key roles in parasite motility, invasion, metabolism, and replication, and understanding their relationship with the organelles of better-studied eukaryotic systems suggests potential targets for therapeutic intervention. Recent work has demonstrated divergent aspects of canonical eukaryotic components in the Apicomplexa, including Golgi bodies and mitochondria. The apicoplast is a relict plastid of secondary endosymbiotic origin, harboring metabolic pathways distinct from those of host species. The inner membrane complex (IMC) is derived from the cortical alveoli defining the superphylum Alveolata, but in apicomplexans functions in parasite motility and replication. Micronemes and rhoptries are associated with establishment of the intracellular niche, and define the apical complex for which the phylum is named. Morphological, cell biological and molecular evidence strongly suggest that these organelles are derived from the endocytic pathway.

An analysis of dinoflagellate metabolism using EST data.

The dinoflagellates are an important group of eukaryotic, single celled algae. They are the sister group of the Apicomplexa, a group of intracellular parasites and photosynthetic algae including the malaria parasite Plasmodium. Many apicomplexan mitochondria have a number of unusual features, including the lack of a pyruvate dehydrogenase and the existence of a branched TCA cycle. Here, we analyse dinoflagellate EST (expressed sequence tag) data to determine whether these features are apicomplexan-specific, or if they are more widespread. We show that dinoflagellates have replaced a key subunit (E1) of pyruvate dehydrogenase with a subunit of bacterial origin and that transcripts encoding many of the proteins that are essential in a conventional ATP synthase/Complex V are absent, as is the case in Apicomplexa. There is a pathway for synthesis of starch or glycogen as a storage carbohydrate. Transcripts encoding isocitrate lyase and malate synthase are present, consistent with ultrastructural reports of a glyoxysome. Finally, evidence for a conventional haem biosynthesis pathway is found, in contrast to the Apicomplexa, Chromera and early branching dinoflagellates (Perkinsus, Oxyrrhis).

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.

Transcript level responses of Plasmodium falciparum to antimycin A.

The mitochondrial electron transport chain is essential to Plasmodium and is the target of the antimalarial drug atovaquone. The mitochondrial genomes of Plasmodium sp. are the most reduced known, and the majority of mitochondrial proteins are encoded in the nucleus and imported into the mitochondrion post-translationally. Many organisms have signalling pathways between the mitochondria and the nucleus to regulate the expression of nuclear-encoded mitochondrially-targeted proteins, for example in response to mitochondrial dysfunction. We have studied the transcript profiles of synchronous Plasmodium falciparum treated with an LD(50) concentration of the complex III inhibitor antimycin A, to investigate whether such pathways exist in the parasite. There was a broad perturbation of gene expression. The differentially expressed genes were enriched for transcripts encoding proteins involved in invasion, stress response, nucleotide biosynthesis and respiration. Some effects were attributable to a delay in the gene expression phase of drug-treated parasites. However, our data indicated regulation of mitochondrial stress response genes and genes involved in pyrimidine biosynthesis, implying the existence of a signalling pathway from the mitochondrion to the nucleus.

Transcript-level responses of Plasmodium falciparum to thiostrepton.

The antimalarial activity of the antibiotic thiostrepton has long been attributed to inhibition of apicoplast protein synthesis through binding of apicoplast ribosomal RNA. However, the kinetics of parasite death upon thiostrepton treatment differ from those seen for other inhibitors of apicoplast housekeeping functions. We have analysed global changes in gene expression of the malaria parasite, Plasmodium falciparum, in an attempt to shed light on the responses of the parasite to this drug. Our results indicate a delay in gene expression profiles of thiostrepton-treated parasites. A small number of genes appear to be regulated outside of this trend; our data suggest a response from genes encoding components of the mitochondrial translational machinery, while little response is seen from genes encoding apicoplast-targeted proteins. Our findings are consistent with an effect of thiostrepton on mitochondrial protein synthesis, and thus warrant a re-evaluation of the target of thiostrepton in Plasmodium. They also provide some suggestion of mitochondrion-nucleus signalling in the parasite.

Conservation of structural and functional elements of TSC1 and TSC2: a bioinformatic comparison across animal models.

The tuberous sclerosis complex 1/2-mammalian target of rapamycin (TSC1/2-mTOR) proteins act as integrators of a range of intracellular signalling pathways. Various genetic disorders associated with learning and behavioural deficits, including TSC, Fragile X, Neurofibromatosis Type 1, Noonan and Leopard syndromes, are associated with abnormalities in TSC-mTOR signalling. Based on the assumption that signalling proteins and their structural and functional components are widely conserved, a number of animal models are used to study aspects of the physical and behavioural phenotypes of these human disorders. Model organisms include rat (Rattus norvegicus), mouse (Mus musculus), zebrafish (Danio rerio), fruitfly (Drosophila melanogaster) and fission yeast (Schizosaccharomyces pombe). Here we used a bioinformatic approach to examine the presence of structural and functional elements of TSC1 and TSC2 across these organisms, together with Strongylocentrotus purpuratus and Dictyostelium discoideum. Results suggest that while Rattus norvegicus and Mus musculus TSC1 and TSC2 showed very high similarity to the human sequences, this was not the case for Danio rerio, Drosophila melanogaster, Strongylocentrotus purpuratus, Schizosaccharomyces pombe or Disctyostelium discoideum. Findings indicate that caution should be exercised in detailed interpretation of results from some model organisms.