Evolution of metabolic capabilities and molecular features of diplonemids, kinetoplastids, and euglenids.

BACKGROUND: The Euglenozoa are a protist group with an especially rich history of evolutionary diversity. They include diplonemids, representing arguably the most species-rich clade of marine planktonic eukaryotes; trypanosomatids, which are notorious parasites of medical and veterinary importance; and free-living euglenids. These different lifestyles, and particularly the transition from free-living to parasitic, likely require different metabolic capabilities. We carried out a comparative genomic analysis across euglenozoan diversity to see how changing repertoires of enzymes and structural features correspond to major changes in lifestyles. RESULTS: We find a gradual loss of genes encoding enzymes in the evolution of kinetoplastids, rather than a sudden decrease in metabolic capabilities corresponding to the origin of parasitism, while diplonemids and euglenids maintain more metabolic versatility. Distinctive characteristics of molecular machines such as kinetochores and the pre-replication complex that were previously considered specific to parasitic kinetoplastids were also identified in their free-living relatives. Therefore, we argue that they represent an ancestral rather than a derived state, as thought until the present. We also found evidence of ancient redundancy in systems such as NADPH-dependent thiol-redox. Only the genus Euglena possesses the combination of trypanothione-, glutathione-, and thioredoxin-based systems supposedly present in the euglenozoan common ancestor, while other representatives of the phylum have lost one or two of these systems. Lastly, we identified convergent losses of specific metabolic capabilities between free-living kinetoplastids and ciliates. Although this observation requires further examination, it suggests that certain eukaryotic lineages are predisposed to such convergent losses of key enzymes or whole pathways. CONCLUSIONS: The loss of metabolic capabilities might not be associated with the switch to parasitic lifestyle in kinetoplastids, and the presence of a highly divergent (or unconventional) kinetochore machinery might not be restricted to this protist group. The data derived from the transcriptomes of free-living early branching prokinetoplastids suggests that the pre-replication complex of Trypanosomatidae is a highly divergent version of the conventional machinery. Our findings shed light on trends in the evolution of metabolism in protists in general and open multiple avenues for future research.

Respiratory chain Complex I of unparalleled divergence in diplonemids.

Mitochondrial genes of Euglenozoa (Kinetoplastida, Diplonemea, and Euglenida) are notorious for being barely recognizable, raising the question of whether such divergent genes actually code for functional proteins. Here we demonstrate the translation and identify the function of five previously unassigned y genes encoded by mitochondrial DNA (mtDNA) of diplonemids. As is the rule in diplonemid mitochondria, y genes are fragmented, with gene pieces transcribed separately and then trans-spliced to form contiguous mRNAs. Further, y transcripts undergo massive RNA editing, including uridine insertions that generate up to 16-residue-long phenylalanine tracts, a feature otherwise absent from conserved mitochondrial proteins. By protein sequence analyses, MS, and enzymatic assays in Diplonema papillatum, we show that these y genes encode the subunits Nad2, -3, -4L, -6, and -9 of the respiratory chain Complex I (CI; NADH:ubiquinone oxidoreductase). The few conserved residues of these proteins are essentially those involved in proton pumping across the inner mitochondrial membrane and in coupling ubiquinone reduction to proton pumping (Nad2, -3, -4L, and -6) and in interactions with subunits containing electron-transporting Fe-S clusters (Nad9). Thus, in diplonemids, 10 CI subunits are mtDNA-encoded. Further, MS of D. papillatum CI allowed identification of 26 conventional and 15 putative diplonemid-specific nucleus-encoded components. Most conventional accessory subunits are well-conserved but unusually long, possibly compensating for the streamlined mtDNA-encoded components and for missing, otherwise widely distributed, conventional subunits. Finally, D. papillatum CI predominantly exists as a supercomplex I:III:IV that is exceptionally stable, making this protist an organism of choice for structural studies.

Extensive molecular tinkering in the evolution of the membrane attachment mode of the Rheb GTPase.

Rheb is a conserved and widespread Ras-like GTPase involved in cell growth regulation mediated by the (m)TORC1 kinase complex and implicated in tumourigenesis in humans. Rheb function depends on its association with membranes via prenylated C-terminus, a mechanism shared with many other eukaryotic GTPases. Strikingly, our analysis of a phylogenetically rich sample of Rheb sequences revealed that in multiple lineages this canonical and ancestral membrane attachment mode has been variously altered. The modifications include: (1) accretion to the N-terminus of two different phosphatidylinositol 3-phosphate-binding domains, PX in Cryptista (the fusion being the first proposed synapomorphy of this clade), and FYVE in Euglenozoa and the related undescribed flagellate SRT308; (2) acquisition of lipidic modifications of the N-terminal region, namely myristoylation and/or S-palmitoylation in seven different protist lineages; (3) acquisition of S-palmitoylation in the hypervariable C-terminal region of Rheb in apusomonads, convergently to some other Ras family proteins; (4) replacement of the C-terminal prenylation motif with four transmembrane segments in a novel Rheb paralog in the SAR clade; (5) loss of an evident C-terminal membrane attachment mechanism in Tremellomycetes and some Rheb paralogs of Euglenozoa. Rheb evolution is thus surprisingly dynamic and presents a spectacular example of molecular tinkering.

The tetrapyrrole synthesis pathway as a model of horizontal gene transfer in euglenoids.

The history of euglenoids may have begun as early as ~2 bya. These early phagotrophs ate cyanobacteria, archaea, and eubacteria, and the subsequent appearance of red algae and chromalveolates provided euglenoids with additional food sources. Following the appearance of green algae, euglenoids acquired a chloroplast via a secondary endosymbiotic event with a green algal ancestor. This endosymbiosis also involved a massive transfer of nuclear-encoded genes from the symbiont nucleus to the host. Expecting these genes to have a green algal origin, this research has shown, through the use of DNA-sequences and the analysis of phylogenetic relationships, that many housekeeping genes have a red algal/chromalveolate ancestry. This suggested that many other endosymbiotic/horizontal gene transfers, which brought genes from chromalveolates to euglenoids, may have been taking place long before the acquisition of the chloroplast. The investigation of the origin of the enzymes involved in the tetrapyrrole synthesis pathway provided insights into horizontal gene transfer in euglenoids and demonstrated that the euglenoid nuclear genome is a mosaic comprised of genes from the ancestral lineage plus genes transferred endosymbiotically/horizontally from green, red, and chromalveolates lineages.

Differential remodelling of peroxisome function underpins the environmental and metabolic adaptability of diplonemids and kinetoplastids.

The remodelling of organelle function is increasingly appreciated as a central driver of eukaryotic biodiversity and evolution. Kinetoplastids including Trypanosoma and Leishmania have evolved specialized peroxisomes, called glycosomes. Glycosomes uniquely contain a glycolytic pathway as well as other enzymes, which underpin the physiological flexibility of these major human pathogens. The sister group of kinetoplastids are the diplonemids, which are among the most abundant eukaryotes in marine plankton. Here we demonstrate the compartmentalization of gluconeogenesis, or glycolysis in reverse, in the peroxisomes of the free-living marine diplonemid, Diplonema papillatum Our results suggest that peroxisome modification was already under way in the common ancestor of kinetoplastids and diplonemids, and raise the possibility that the central importance of gluconeogenesis to carbon metabolism in the heterotrophic free-living ancestor may have been an important selective driver. Our data indicate that peroxisome modification is not confined to the kinetoplastid lineage, but has also been a factor in the success of their free-living euglenozoan relatives.

Novel modes of RNA editing in mitochondria.

Gene structure and expression in diplonemid mitochondria are unparalleled. Genes are fragmented in pieces (modules) that are separately transcribed, followed by the joining of module transcripts to contiguous RNAs. Some instances of unique uridine insertion RNA editing at module boundaries were noted, but the extent and potential occurrence of other editing types remained unknown. Comparative analysis of deep transcriptome and genome data from Diplonema papillatum mitochondria reveals ∼220 post-transcriptional insertions of uridines, but no insertions of other nucleotides nor deletions. In addition, we detect in total 114 substitutions of cytosine by uridine and adenosine by inosine, amassed into unusually compact clusters. Inosines in transcripts were confirmed experimentally. This is the first report of adenosine-to-inosine editing of mRNAs and ribosomal RNAs in mitochondria. In mRNAs, editing causes mostly amino-acid additions and non-synonymous substitutions; in ribosomal RNAs, it permits formation of canonical secondary structures. Two extensively edited transcripts were compared across four diplonemids. The pattern of uridine-insertion editing is strictly conserved, whereas substitution editing has diverged dramatically, but still rendering diplonemid proteins more similar to other eukaryotic orthologs. We posit that RNA editing not only compensates but also sustains, or even accelerates, ultra-rapid evolution of genome structure and sequence in diplonemid mitochondria.

Identification of divergent WH2 motifs by HMM-HMM alignments.

BACKGROUND: The actin cytoskeleton is a hallmark of eukaryotic cells. Its regulation as well as its interaction with other proteins is carefully orchestrated by actin interaction domains. One of the key players is the WH2 motif, which enables binding to actin monomers and filaments and is involved in the regulation of actin nucleation. Contrasting conserved domains, the identification of this motif in protein sequences is challenging, as it is short and poorly conserved. FINDINGS: To identify divergent members, we combined Hidden-Markov-Model (HMM) to HMM alignments with orthology predictions. Thereby, we identified nearly 500 proteins containing so far not annotated WH2 motifs. This included shootin-1, an actin binding protein involved in neuron polarization. Among others, WH2 motifs of 'proximal to raf' (ptr)-orthologs, which are described in the literature, but not annotated in genome databases, were identified. CONCLUSION: In summary, we increased the number of WH2 motif containing proteins substantially. This identification of candidate regions for actin interaction could steer their experimental characterization. Furthermore, the approach outlined here can easily be adapted to the identification of divergent members of further domain families.

Melatonin in plants and other phototrophs: advances and gaps concerning the diversity of functions.

Melatonin is synthesized in Alphaproteobacteria, Cyanobacteria, Dinoflagellata, Euglenoidea, Rhodophyta, Phae ophyta, and Viridiplantae. The biosynthetic pathways have been identified in dinoflagellates and plants. Other than in dinoflagellates and animals, tryptophan is not 5-hydroxylated in plants but is first decarboxylated. Serotonin is formed by 5-hydroxylation of tryptamine. Serotonin N-acetyltransferase is localized in plastids and lacks homology to the vertebrate aralkylamine N-acetyltransferase. Melatonin content varies considerably among species, from a few picograms to several micrograms per gram, a strong hint for different actions of this indoleamine. At elevated levels, the common and presumably ancient property as an antioxidant may prevail. Although melatonin exhibits nocturnal maxima in some phototrophs, it is not generally a mediator of the signal 'darkness'. In various plants, its formation is upregulated by visible and/or UV light. Increases are often induced by high or low temperature and several other stressors including drought, salinity, and chemical toxins. In Arabidopsis, melatonin induces cold- and stress-responsive genes. It has been shown to support cold resistance and to delay experimental leaf senescence. Transcriptome data from Arabidopsis indicate upregulation of genes related to ethylene, abscisic acid, jasmonic acid, and salicylic acid. Auxin-like actions have been reported concerning root growth and inhibition, and hypocotyl or coleoptile lengthening, but effects caused by melatonin and auxins can be dissected. Assumptions on roles in flower morphogenesis and fruit ripening are based mainly on concentration changes. Whether or not melatonin will find a place in the phytohormone network depends especially on the identification of molecular signals regulating its synthesis, high-affinity binding sites, and signal transduction pathways.

When, how and why glycolysis became compartmentalised in the Kinetoplastea. A new look at an ancient organelle.

A characteristic, well-studied feature of the pathogenic protists belonging to the family Trypanosomatidae is the compartmentalisation of the major part of the glycolytic pathway in peroxisome-like organelles, hence designated glycosomes. Such organelles containing glycolytic enzymes appear to be present in all members of the Kinetoplastea studied, and have recently also been detected in a representative of the Diplonemida, but they are absent from the Euglenida. Glycosomes therefore probably originated in a free-living, common ancestor of the Kinetoplastea and Diplonemida. The initial sequestering of glycolytic enzymes inside peroxisomes may have been the result of a minor mistargeting of proteins, as generally observed in eukaryotic cells, followed by preservation and its further expansion due to the selective advantage of this specific form of metabolic compartmentalisation. This selective advantage may have been a largely increased metabolic flexibility, allowing the organisms to adapt more readily and efficiently to different environmental conditions. Further evolution of glycosomes involved, in different taxonomic lineages, the acquisition of additional enzymes and pathways - often participating in core metabolic processes - as well as the loss of others. The acquisitions may have been promoted by the sharing of cofactors and crucial metabolites between different pathways, thus coupling different redox processes and catabolic and anabolic pathways within the organelle. A notable loss from the Trypanosomatidae concerned a major part of the typical peroxisomal H(2)O(2)-linked metabolism. We propose that the compartmentalisation of major parts of the enzyme repertoire involved in energy, carbohydrate and lipid metabolism has contributed to the multiple development of parasitism, and its elaboration to complicated life cycles involving consecutive different hosts, in the protists of the Kinetoplastea clade.

3' processing in protists.

Molecular biologists have traditionally focused on the very small corner of eukaryotic evolution that includes yeast and animals; even plants have been neglected. In this article, we describe the scant information that is available concerning RNA processing in the other four major eukaryotic groups, especially pathogenic protists. We focus mainly on polyadenylation and nuclear processing of stable RNAs. These processes have--where examined--been shown to be conserved, but there are many novel details. We also briefly mention other processing reactions such as splicing.

[Diversity of parasitic protozoan mitochondria and adaptive evolution].

Eukaryotic mitochondrion generally possess a definite and canonical structure and function. However, in the unicellular parasitic protozoa, various atypical mitochondria with respect to the number, structure, and function, have been discovered consecutively, revealing the variability, plasticity and rich diversity of mitochondrion. Here, we review the mitochondrial diversity in diverse parasitic protozoa, and the underlying reason for such diversity--the adaptive evolution of mitochondrion to the micro-oxygen or anaero parasitic environment of these parasites is also analyzed and discussed.