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Colorless plastids incapable of photosynthesis evolved in many plant and algal groups, but what functions they perform is still unknown in many cases. Here, we study the elusive plastid of Euglena longa , a nonphotosynthetic cousin of the familiar green flagellate Euglena gracilis . We document an unprecedented combination of metabolic functions that the E. longa plastid exhibits in comparison with previously characterized nonphotosynthetic plastids. For example, and truly surprisingly, it has retained the synthesis of tocopherols (vitamin E) and a phylloquinone (vitamin K) derivative. In addition, we offer a possible solution of the long-standing conundrum of the presence of the CO 2 -fixing enzyme RuBisCO in E. longa . Our work provides a detailed account on a unique variant of relic plastids, the first among nonphotosynthetic plastids that evolved by secondary endosymbiosis from a green algal ancestor, and suggests that it has persisted for reasons not previously considered in relation to nonphotosynthetic plastids. Most secondary nonphotosynthetic eukaryotes have retained residual plastids whose physiological role is often still unknown. One such example is Euglena longa , a close nonphotosynthetic relative of Euglena gracilis harboring a plastid organelle of enigmatic function. By mining transcriptome data from E. longa , we finally provide an overview of metabolic processes localized to its elusive plastid. The organelle plays no role in the biosynthesis of isoprenoid precursors and fatty acids and has a very limited repertoire of pathways concerning nitrogen-containing metabolites. In contrast, the synthesis of phospholipids and glycolipids has been preserved, curiously with the last step of sulfoquinovosyldiacylglycerol synthesis being catalyzed by the SqdX form of an enzyme so far known only from bacteria. Notably, we show that the E. longa plastid synthesizes tocopherols and a phylloquinone derivative, the first such report for nonphotosynthetic plastids studied so far. The most striking attribute of the organelle could be the presence of a linearized Calvin-Benson (CB) pathway, including RuBisCO yet lacking the gluconeogenetic part of the standard cycle, together with ferredoxin-NADP + reductase (FNR) and the ferredoxin/thioredoxin system. We hypothesize that the ferredoxin/thioredoxin system activates the linear CB pathway in response to the redox status of the E. longa cell and speculate on the role of the pathway in keeping the redox balance of the cell. Altogether, the E. longa plastid defines a new class of relic plastids that is drastically different from the best-studied organelle of this category, the apicoplast. IMPORTANCE Colorless plastids incapable of photosynthesis evolved in many plant and algal groups, but what functions they perform is still unknown in many cases. Here, we study the elusive plastid of Euglena longa , a nonphotosynthetic cousin of the familiar green flagellate Euglena gracilis . We document an unprecedented combination of metabolic functions that the E. longa plastid exhibits in comparison with previously characterized nonphotosynthetic plastids. For example, and truly surprisingly, it has retained the synthesis of tocopherols (vitamin E) and a phylloquinone (vitamin K) derivative. In addition, we offer a possible solution of the long-standing conundrum of the presence of the CO 2 -fixing enzyme RuBisCO in E. longa . Our work provides a detailed account on a unique variant of relic plastids, the first among nonphotosynthetic plastids that evolved by secondary endosymbiosis from a green algal ancestor, and suggests that it has persisted for reasons not previously considered in relation to nonphotosynthetic plastids.

Chloroplasts are generally known as eukaryotic organelles whose main function is photosynthesis. They perform other functions, however, such as synthesizing isoprenoids, fatty acids, heme, iron sulphur clusters and other essential compounds. In non-photosynthetic lineages that possess plastids, the chloroplast genomes have been reduced and most (or all) photosynthetic genes have been lost. Consequently, non-photosynthetic plastids have also been reduced structurally. Some of these non-photosynthetic or “cryptic” plastids were overlooked or unrecognized for decades. The number of complete plastid genome sequences and/or transcriptomes from non-photosynthetic taxa possessing plastids is rapidly increasing, thus allowing prediction of the functions of non-photosynthetic plastids in various eukaryotic lineages. In some non-photosynthetic eukaryotes with photosynthetic ancestors, no traces of plastid genomes or of plastids have been found, suggesting that they have lost the genomes or plastids completely. This review summarizes current knowledge of non-photosynthetic plastids, their genomes, structures and potential functions in free-living and parasitic plants, algae and protists. We introduce a model for the order of plastid gene losses which combines models proposed earlier for land plants with the patterns of gene retention and loss observed in protists. The rare cases of plastid genome loss and complete plastid loss are also discussed.

Euglena gracilis is a flagellate photosynthetic microalga that, thanks to its metabolic adaptability, can grow under both autotrophic and heterotrophic conditions. This adaptability makes euglena an interesting species for applied biotechnology. We focused on the proteome of E. gracilis cultivated in Cramer-Myers medium (supplemented with ethanol) in dark and light conditions. Cultures grown in the light showed a characteristic green coloration, while cultures incubated in the dark were bright yellow. When cultured in the dark, microalga showed reduced concentration of chlorophylls ( a , b , and total) and carotenoids compared to cells cultured in the light. Conversely, there was an increase in proline content in the dark compared to light cultivation. Using proteomic approach, we revealed 162 differentially accumulated proteins in light- and dark-grown cells classified into 12 functional groups. Notably, alterations in the metabolism of fatty acids and amino acids, secondary metabolism, and accumulation of stress- and detoxification-related proteins in microalgal cells cultivated in darkness with ethanol as a carbon source may help euglena adapt to these conditions. Based on our results and literature, we hypothesize that vitamin B12 potentially plays an important role in light/dark metabolic switch, similarly as in bacteria.

Calpains are cysteine proteases involved in many cellular processes. They are an ancient and large superfamily of enzymes responsible for the cleavage and irreversible modification of a large variety of substrates. They have been intensively studied in humans and other mammals, but information about calpains in bacteria is scarce. Calpains have not been found among Archaea to date. In this study, we have investigated the presence of calpains in selected cyanobacterial species using in silico analyses. We show that calpains defined by possessing CysPC core domain are present in cyanobacterial genera Anabaena , Aphanizomenon , Calothrix , Chamaesiphon , Fischerella , Microcystis , Scytonema and Trichormus . Based on in silico protein interaction analysis, we have predicted putative interaction partners for identified cyanobacterial calpains. The phylogenetic analysis including cyanobacterial, other bacterial and eukaryotic calpains divided bacterial and eukaryotic calpains into two separate monophyletic clusters. We propose two possible evolutionary scenarios to explain this tree topology: (1) the eukaryotic ancestor or an archaeal ancestor of eukaryotes obtained calpain gene from an unknown bacterial donor, or alternatively (2) calpain gene had been already present in the last common universal ancestor and subsequently lost by the ancestor of Archaea, but retained by the ancestor of Bacteria and by the ancestor of Eukarya. Both scenarios would require multiple independent losses of calpain genes in various bacteria and eukaryotes.

Trans -splicing is a process by which 5′- and 3′-ends of two pre-RNA molecules transcribed from different sites of the genome can be joined together to form a single RNA molecule. The spliced leader (SL) trans -splicing is mediated by the spliceosome and it allows the replacement of 5′-end of pre-mRNA by 5′(SL)-end of SL-RNA. This form of splicing has been observed in many phylogenetically unrelated eukaryotes. Either the SL trans -splicing (SLTS) originated in the last eukaryotic common ancestor (LECA) (or even earlier) and it was lost in most eukaryotic lineages, or this mechanism of RNA processing evolved several times independently in various unrelated eukaryotic taxa. The bioinformatic comparisons of SL-RNAs from various eukaryotic taxonomic groups have revealed the similarities of secondary structures of most SL-RNAs and a relative conservation of their splice sites (SSs) and Sm-binding sites (SmBSs). We propose that such structural and functional similarities of SL-RNAs are unlikely to have evolved repeatedly many times. Hence, we favor the scenario of an early evolutionary origin for the SLTS and multiple losses of SL-RNAs in various eukaryotic lineages.

Euglena gracilis is a freshwater protist possessing secondary chloroplasts of green algal origin. Various physical factors (e.g. UV) and chemical compounds (e.g. antibiotics) cause the bleaching of E. gracilis cells—the loss of plastid genes leading to the permanent inability to photosynthesize. Bleaching can be prevented by antimutagens (i.e. lignin, vitamin C and selenium). Besides screening the mutagenic and antimutagenic activity of chemicals, E. gracilis is also a suitable model for studying the biological effects of many organic pollutants. Due to its capability of heavy metal sequestration, it can be used for bioremediation. E. gracilis has been successfully transformed, offering the possibility of genetic modifications for synthesizing compounds of biotechnological interest. The novel design of the “next generation” transgenic expression cassettes with respect to the specificities of euglenid gene expression is proposed. Moreover, E. gracilis is a natural source of commercially relevant bioproducts such as (pro)vitamins, wax esters, polyunsaturated fatty acids and paramylon (β-1,3-glucan). One of the highest limitations of large-scale cultivation of E. gracilis is its disability to synthesize essential vitamins B1 and B12. This disadvantage can be overcome by co-cultivation of E. gracilis with other microorganisms, which can synthesize sufficient amounts of these vitamins. Such co-cultures can be used for the effective accumulation and harvesting of Euglena biomass by bioflocculation.

Phenol, a monocyclic aromatic hydrocarbon with various commercial uses, is a major pollutant in industrial wastewater. Euglena gracilis is a unicellular freshwater flagellate possessing secondary chloroplasts of green algal origin. This protist has been widely used for monitoring the biological effect of various inorganic and organic environmental pollutants, including aromatic hydrocarbons. In this study, we evaluate the influence of different phenol concentrations (3.39 mM, 3.81 mM, 4.23 mM, 4.65 mM, 5.07 mM, 5.49 mM and 5.91 mM) on the growth, morphology and cell division of E. gracilis. The cell count continually decreases (p < 0.05–0.001) over time with increasing phenol concentration. While phenol treatment does not induce bleaching (permanent loss of photosynthesis), the morphological changes caused by phenol include the formation of spherical (p < 0.01–0.001), hypertrophied (p < 0.05) and monster cells (p < 0.01) and lipofuscin bodies. Phenol also induces an atypical form of cell division of E. gracilis, simultaneously producing more than 2 (3–12) viable cells from a single cell. Such atypically dividing cells have a symmetric “star”-like shape. The percentage of atypically dividing cells increases (p < 0.05) with increasing phenol concentration. Our findings suggest that E. gracilis can be used as bioindicator of phenol contamination in freshwater habitats and wastewater.

Euglenophytes are a familiar algal group with green alga-derived secondary plastids, but the knowledge of euglenophyte plastid function and evolution is still highly incomplete. With this in mind we sequenced and analysed the transcriptome of the non-photosynthetic species Euglena longa . The transcriptomic data confirmed the absence of genes for the photosynthetic machinery, but provided candidate plastid-localised proteins bearing N-terminal bipartite topogenic signals (BTSs) of the characteristic euglenophyte type. Further comparative analyses including transcriptome assemblies available for photosynthetic euglenophytes enabled us to unveil salient aspects of the basic euglenophyte plastid infrastructure, such as plastidial targeting of several proteins as C-terminal translational fusions with other BTS-bearing proteins or replacement of the conventional eubacteria-derived plastidial ribosomal protein L24 by homologs of archaeo-eukaryotic origin. Strikingly, no homologs of any key component of the TOC/TIC system and the plastid division apparatus are discernible in euglenophytes, and the machinery for intraplastidial protein targeting has been simplified by the loss of the cpSRP/cpFtsY system and the SEC2 translocon. Lastly, euglenophytes proved to encode a plastid-targeted homolog of the termination factor Rho horizontally acquired from a Lambdaproteobacteria-related donor. Our study thus further documents a substantial remodelling of the euglenophyte plastid compared to its green algal progenitor.

One group of hypotheses suggests archaeal and/or bacterial ancestry of eukaryotes, while the second group suggests that the ancestor of eukaryotes was different. Especially, the followers of the first group of hypotheses should ask the following: is the replacement of archaeal lipids by bacterial (or vice versa) possible? Do the phylogenies support the origin of one domain from another (or the others)? Can we consider the nutritional mode to resolve the problems of cell origin(s)? Is there any evidence that the ancestor of eukaryotes was intron-free? Would the symbiosis of α-proteobacterial ancestors of mitochondria be successful in an asexual host? Is there evidence that the last universal common ancestor (LUCA) or the last eukaryotic common ancestor was bounded by one membrane only? With respect to the current knowledge about cells and their molecular components, the answer to most of these questions is: No! A model for the origins of domains is briefly presented which cannot be assigned as false through the current scientific data, and is rather consistent with the assumption that eukaryotes are direct descendants of neither archaea nor bacteria. It is proposed that the domain Bacteria arose the first, and that the last common ancestor of Archaea and Eukarya was a pre-cell or a progenote similar to LUCA. The pre-karyote (the host entity for α-proteobacterial ancestors of mitochondria) was probably bounded by two membranes, possessed spliceosomal introns and spliceosomes, was sexual, and α-proteobacterial ancestors of mitochondria were most likely parasites of the pre-karyote periplasm (intermembrane space).

Euglena gracilis growth with antibacterial agents leads to bleaching, permanent plastid gene loss. Colorless Euglena ( Astasia ) longa resembles a bleached E. gracilis . To evaluate the role of bleaching in E. longa evolution, the effect of streptomycin, a plastid protein synthesis inhibitor, and ofloxacin, a plastid DNA gyrase inhibitor, on E. gracilis and E. longa growth and plastid DNA content were compared. E. gracilis growth was unaffected by streptomycin and ofloxacin. Quantitative PCR analyses revealed a time dependent loss of plastid genes in E. gracilis demonstrating that bleaching agents produce plastid gene deletions without affecting cell growth. Streptomycin and ofloxacin inhibited E. longa growth indicating that it requires plastid genes to survive. This suggests that evolutionary divergence of E. longa from E. gracilis was triggered by the loss of a cytoplasmic metabolic activity also occurring in the plastid. Plastid metabolism has become obligatory for E. longa cell growth. A process termed “ intermittent bleaching ”, short term exposure to subsaturating concentrations of reversible bleaching agents followed by growth in the absence of a bleaching agent, is proposed as the molecular mechanism for E. longa plastid genome reduction. Various non-photosynthetic lineages could have independently arisen from their photosynthetic ancestors via a similar process.