Dinoflagellates are a diverse group of ecologically significant micro-eukaryotes that can serve as a model system for plastid symbiogenesis due to their susceptibility to plastid loss and replacement via serial endosymbiosis. Kareniaceae harbor fucoxanthin-pigmented plastids instead of the ancestral peridinin-pigmented ones and support them with a diverse range of nucleus-encoded plastid-targeted proteins originating from the haptophyte endosymbiont, dinoflagellate host, and/or lateral gene transfers (LGT). Here, we present predicted plastid proteomes from seven distantly related kareniaceans in three genera (Karenia, Karlodinium, and Takayama) and analyze their evolutionary patterns using automated tree building and sorting. We project a relatively limited ( ~ 10%) haptophyte signal pointing towards a shared origin in the family Chrysochromulinaceae. Our data establish significant variations in the functional distributions of these signals, emphasizing the importance of micro-evolutionary processes in shaping the chimeric proteomes. Analysis of plastid genome sequences recontextualizes these results by a striking finding the extant kareniacean plastids are in fact not all of the same origin, as two of the studied species (Karlodinium armiger, Takayama helix) possess plastids from different haptophyte orders than the rest.

• Klíčová slova
• Automated Tree Sorting, Myzozoa, Post-Endosymbiotic Organelle Evolution, Protists, Shopping Bag Model,
• MeSH
• Dinoflagellata * genetika   metabolismus   MeSH
• fylogeneze MeSH
• plastidy genetika   MeSH
• proteom genetika   metabolismus   MeSH
• symbióza genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteom MeSH

Ochrophyta is an algal group belonging to the Stramenopiles and comprises diverse lineages of algae which contribute significantly to the oceanic ecosystems as primary producers. However, early evolution of the plastid organelle in Ochrophyta is not fully understood. In this study, we provide a well-supported tree of the Stramenopiles inferred by the large-scale phylogenomic analysis that unveils the eukaryvorous (nonphotosynthetic) protist Actinophrys sol (Actinophryidae) is closely related to Ochrophyta. We used genomic and transcriptomic data generated from A. sol to detect molecular traits of its plastid and we found no evidence of plastid genome and plastid-mediated biosynthesis, consistent with previous ultrastructural studies that did not identify any plastids in Actinophryidae. Moreover, our phylogenetic analyses of particular biosynthetic pathways provide no evidence of a current and past plastid in A. sol. However, we found more than a dozen organellar aminoacyl-tRNA synthases (aaRSs) that are of algal origin. Close relationships between aaRS from A. sol and their ochrophyte homologs document gene transfer of algal genes that happened before the divergence of Actinophryidae and Ochrophyta lineages. We further showed experimentally that organellar aaRSs of A. sol are targeted exclusively to mitochondria, although organellar aaRSs in Ochrophyta are dually targeted to mitochondria and plastids. Together, our findings suggested that the last common ancestor of Actinophryidae and Ochrophyta had not yet completed the establishment of host-plastid partnership as seen in the current Ochrophyta species, but acquired at least certain nuclear-encoded genes for the plastid functions.

• Klíčová slova
• Actinophryidae, aminoacyl-tRNA synthase, gene transfer, organellar DNA, phylogenomics, plastid evolution,
• MeSH
• ekosystém MeSH
• fylogeneze MeSH
• genom plastidový * MeSH
• Heterokontophyta * genetika   MeSH
• molekulární evoluce MeSH
• plastidy genetika   MeSH
• rostliny genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Plastids, organelles that evolved from cyanobacteria via endosymbiosis in eukaryotes, provide carbohydrates for the formation of biomass and for mitochondrial energy production to the cell. They generate their own energy in the form of the nucleotide adenosine triphosphate (ATP). However, plastids of non-photosynthetic tissues, or during the dark, depend on external supply of ATP. A dedicated antiporter that exchanges ATP against adenosine diphosphate (ADP) plus inorganic phosphate (Pi) takes over this function in most photosynthetic eukaryotes. Additional forms of such nucleotide transporters (NTTs), with deviating activities, are found in intracellular bacteria, and, surprisingly, also in diatoms, a group of algae that acquired their plastids from other eukaryotes via one (or even several) additional endosymbioses compared to algae with primary plastids and higher plants. In this review, we summarize what is known about the nucleotide synthesis and transport pathways in diatom cells, and discuss the evolutionary implications of the presence of the additional NTTs in diatoms, as well as their applications in biotechnology.

• Klíčová slova
• adenosine triphosphate (ATP), endosymbiosis, evolution, photosynthesis, plastid, synthetic biology, transport,
• MeSH
• biologická evoluce MeSH
• biologický transport MeSH
• biotechnologie MeSH
• membránové transportní proteiny chemie   metabolismus   MeSH
• nukleotidy biosyntéza   metabolismus   MeSH
• rozsivky metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• membránové transportní proteiny MeSH
• nukleotidy MeSH

Diatoms are unicellular algae and evolved by secondary endosymbiosis, a process in which a red alga-like eukaryote was engulfed by a heterotrophic eukaryotic cell. This gave rise to plastids of remarkable complex architecture and ultrastructure that require elaborate protein importing, trafficking, signaling and intracellular cross-talk pathways. Studying both plastids and mitochondria and their distinctive physiological pathways in organello may greatly contribute to our understanding of photosynthesis, mitochondrial respiration and diatom evolution. The isolation of such complex organelles, however, is still demanding, and existing protocols are either limited to a few species (for plastids) or have not been reported for diatoms so far (for mitochondria). In this work, we present the first isolation protocol for mitochondria from the model diatom Thalassiosira pseudonana. Apart from that, we extended the protocol so that it is also applicable for the purification of a high-quality plastids fraction, and provide detailed structural and physiological characterizations of the resulting organelles. Isolated mitochondria were structurally intact, showed clear evidence of mitochondrial respiration, but the fractions still contained residual cell fragments. In contrast, plastid isolates were virtually free of cellular contaminants, featured structurally preserved thylakoids performing electron transport, but lost most of their stromal components as concluded from Western blots and mass spectrometry. Liquid chromatography electrospray-ionization mass spectrometry studies on mitochondria and thylakoids, moreover, allowed detailed proteome analyses which resulted in extensive proteome maps for both plastids and mitochondria thus helping us to broaden our understanding of organelle metabolism and functionality in diatoms.

• Klíčová slova
• Chloroplast, Organelle isolation, Photosynthesis, Proteomics, Respiration, Thylakoids,
• MeSH
• mitochondrie metabolismus   MeSH
• plastidy metabolismus   MeSH
• proteom metabolismus   MeSH
• rozsivky metabolismus   MeSH
• tylakoidy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteom MeSH

The establishment of the mitochondrion is seen as a transformational step in the origin of eukaryotes. With the mitochondrion came bioenergetic freedom to explore novel evolutionary space leading to the eukaryotic radiation known today. The tight integration of the bacterial endosymbiont with its archaeal host was accompanied by a massive endosymbiotic gene transfer resulting in a small mitochondrial genome which is just a ghost of the original incoming bacterial genome. This endosymbiotic gene transfer resulted in the loss of many genes, both from the bacterial symbiont as well the archaeal host. Loss of genes encoding redundant functions resulted in a replacement of the bulk of the host's metabolism for those originating from the endosymbiont. Glycolysis is one such metabolic pathway in which the original archaeal enzymes have been replaced by bacterial enzymes from the endosymbiont. Glycolysis is a major catabolic pathway that provides cellular energy from the breakdown of glucose. The glycolytic pathway of eukaryotes appears to be bacterial in origin, and in well-studied model eukaryotes it takes place in the cytosol. In contrast, here we demonstrate that the latter stages of glycolysis take place in the mitochondria of stramenopiles, a diverse and ecologically important lineage of eukaryotes. Although our work is based on a limited sample of stramenopiles, it leaves open the possibility that the mitochondrial targeting of glycolytic enzymes in stramenopiles might represent the ancestral state for eukaryotes.

• MeSH
• biologická evoluce MeSH
• Blastocystis cytologie   enzymologie   genetika   metabolismus   MeSH
• energetický metabolismus MeSH
• genom mitochondriální MeSH
• glykolýza * MeSH
• mitochondrie genetika   metabolismus   MeSH
• rozsivky cytologie   enzymologie   genetika   metabolismus   MeSH
• symbióza MeSH
• transformace genetická MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Diatoms are unicellular algae and important primary producers. The process of carbon fixation in diatoms is very efficient even though the availability of dissolved CO2 in sea water is very low. The operation of a carbon concentrating mechanism (CCM) also makes the more abundant bicarbonate accessible for photosynthetic carbon fixation. Diatoms possess carbonic anhydrases as well as metabolic enzymes potentially involved in C4 pathways; however, the question as to whether a C4 pathway plays a general role in diatoms is not yet solved. While genome analyses indicate that the diatom Phaeodactylum tricornutum possesses all the enzymes required to operate a C4 pathway, silencing of the pyruvate orthophosphate dikinase (PPDK) in a genetically transformed cell line does not lead to reduced photosynthetic carbon fixation. In this study, we have determined the intracellular location of all enzymes potentially involved in C4-like carbon fixing pathways in P. tricornutum by expression of the respective proteins fused to green fluorescent protein (GFP), followed by fluorescence microscopy. Furthermore, we compared the results to known pathways and locations of enzymes in higher plants performing C3 or C4 photosynthesis. This approach revealed that the intracellular distribution of the investigated enzymes is quite different from the one observed in higher plants. In particular, the apparent lack of a plastidic decarboxylase in P. tricornutum indicates that this diatom does not perform a C4-like CCM.

• Klíčová slova
• C4 photosynthesis, Carboxylation, Chloroplast, Decarboxylation, Green fluorescent protein (GFP),
• MeSH
• Arabidopsis fyziologie   MeSH
• fosfoenolpyruvátkarboxylasa klasifikace   metabolismus   MeSH
• fotosyntéza fyziologie   MeSH
• koloběh uhlíku MeSH
• kukuřice setá fyziologie   MeSH
• mitochondrie enzymologie   MeSH
• pyruvátkarboxylasa genetika   metabolismus   MeSH
• regulace genové exprese enzymů fyziologie   MeSH
• regulace genové exprese u rostlin fyziologie   MeSH
• rozsivky enzymologie   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fosfoenolpyruvátkarboxylasa MeSH
• pyruvátkarboxylasa MeSH

The acquisition and assimilation of inorganic C have been investigated in several of the 15 clades of the Ochrophyta other than diatoms, with biochemical, physiological and genomic data indicating significant mechanistic variation. Form ID Rubiscos in the Ochrophyta are characterized by a broad range of kinetics values. In spite of relatively high K0.5CO2 and low CO2 : O2 selectivity, diffusive entry of CO2 occurs in the Chrysophyceae and Synurophyceae. Eustigmatophyceae and Phaeophyceae, on the contrary, have CO2 concentrating mechanisms, usually involving the direct or indirect use of [Formula: see text] This variability is possibly due to the ecological contexts of the organism. In brown algae, C fixation generally takes place through a classical C3 metabolism, but there are some hints of the occurrence of C4 metabolism and low amplitude CAM in a few members of the Fucales. Genomic data show the presence of a number of potential C4 and CAM genes in Ochrophyta other than diatoms, but the other core functions of many of these genes give a very limited diagnostic value to their presence and are insufficient to conclude that C4 photosynthesis is present in these algae.This article is part of the themed issue 'The peculiar carbon metabolism in diatoms'.

• Klíčová slova
• CO2 concentrating mechanism, Rubisco, brown algae, diffusive CO2 entry, inorganic carbon, photosynthesis,
• MeSH
• anorganické látky metabolismus   MeSH
• fylogeneze MeSH
• Heterokontophyta klasifikace   metabolismus   MeSH
• uhlík metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• anorganické látky MeSH
• uhlík MeSH

Mitochondrial evolution entailed the origin of protein import machinery that allows nuclear-encoded proteins to be targeted to the organelle, as well as the origin of cleavable N-terminal targeting sequences (NTS) that allow efficient sorting and import of matrix proteins. In hydrogenosomes and mitosomes, reduced forms of mitochondria with reduced proteomes, NTS-independent targeting of matrix proteins is known. Here, we studied the cellular localization of two glycolytic enzymes in the anaerobic pathogen Trichomonas vaginalis: PPi-dependent phosphofructokinase (TvPPi-PFK), which is the main glycolytic PFK activity of the protist, and ATP-dependent PFK (TvATP-PFK), the function of which is less clear. TvPPi-PFK was detected predominantly in the cytosol, as expected, while all four TvATP-PFK paralogues were imported into T. vaginalis hydrogenosomes, although none of them possesses an NTS. The heterologous expression of TvATP-PFK in Saccharomyces cerevisiae revealed an intrinsic capability of the protein to be recognized and imported into yeast mitochondria, whereas yeast ATP-PFK resides in the cytosol. TvATP-PFK consists of only a catalytic domain, similarly to "short" bacterial enzymes, while ScATP-PFK includes an N-terminal extension, a catalytic domain, and a C-terminal regulatory domain. Expression of the catalytic domain of ScATP-PFK and short Escherichia coli ATP-PFK in T. vaginalis resulted in their partial delivery to hydrogenosomes. These results indicate that TvATP-PFK and the homologous ATP-PFKs possess internal structural targeting information that is recognized by the hydrogenosomal import machinery. From an evolutionary perspective, the predisposition of ancient ATP-PFK to be recognized and imported into hydrogenosomes might be a relict from the early phases of organelle evolution.

• MeSH
• adenosintrifosfát farmakologie   MeSH
• difosfáty metabolismus   MeSH
• ferredoxiny metabolismus   MeSH
• fosfofruktokinasy chemie   metabolismus   MeSH
• fylogeneze MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• organely účinky léků   metabolismus   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• Saccharomyces cerevisiae účinky léků   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• transport proteinů účinky léků   MeSH
• Trichomonas vaginalis účinky léků   enzymologie   MeSH
• vodík metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• difosfáty MeSH
• ferredoxiny MeSH
• fosfofruktokinasy MeSH
• vodík MeSH