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We describe the types of polymerase chain reaction (PCR) markers that we have isolated using amplified fragment length polymorphisms (AFLP) in closely related taxa from diverse plant genera. With these markers, both inter- and intraspecific differences have been identified. The characterization of the nucleotide sequences and fragment length polymorphisms of such AFLP-derived PCR markers is promising for investigating the ecology and evolution of closely related plant taxa. [ABSTRACT FROM AUTHOR]

The “ k0-method” of neutron activation analysis has been tested for applicability to the determination of trace elements in samples of thyroids of inhabitants from regions in Belarus affected to different degree by thyroid cancer among children after the accident of the Chemobyl power plant. It is shown that the k0-method produces results identical to the “relative method” in neutron activation analysis. Significant differences in the elemental composition of thyroids from the regions of Gomel and Minsk are observed and may be related to the different levels of occurrence of thyroid cancer among children. [ABSTRACT FROM AUTHOR]

Abstract Background Poly-3-hydroxybutyrate (PHB) is a polyester with thermoplastic properties that is naturally occurring and produced by such bacteria as Ralstonia eutropha H16 and Bacillus megaterium. In contrast to currently utilized plastics and most synthetic polymers, PHB is biodegradable, and its production is not dependent on fossil resources making this bioplastic interesting for various industrial applications. Results In this study, we report on introducing the bacterial PHB pathway of R. eutropha H16 into the diatom Phaeodactylum tricornutum, thereby demonstrating for the first time that PHB production is feasible in a microalgal system. Expression of the bacterial enzymes was sufficient to result in PHB levels of up to 10.6% of algal dry weight. The bioplastic accumulated in granule-like structures in the cytosol of the cells, as shown by light and electron microscopy. Conclusions Our studies demonstrate the great potential of microalgae like the diatom P. tricornutum to serve as solar-powered expression factories and reveal great advantages compared to plant based production systems.

Abstract Background The gene expression system of chloroplasts is far more complex than that of their cyanobacterial progenitor. This gain in complexity affects in particular RNA metabolism, specifically the transcription and maturation of RNA. Mature chloroplast RNA is generated by a plethora of nuclear-encoded proteins acquired or recruited during plant evolution, comprising additional RNA polymerases and sigma factors, and sequence-specific RNA maturation factors promoting RNA splicing, editing, end formation and translatability. Despite years of intensive research, we still lack a comprehensive explanation for this complexity. Results We inspected the available literature and genome databases for information on components of RNA metabolism in land plant chloroplasts. In particular, new inventions of chloroplast-specific mechanisms and the expansion of some gene/protein families detected in land plants lead us to suggest that the primary function of the additional nuclear-encoded components found in chloroplasts is the transgenomic suppression of point mutations, fixation of which occurred due to an enhanced genetic drift exhibited by chloroplast genomes. We further speculate that a fast evolution of transgenomic suppressors occurred after the water-to-land transition of plants. Conclusion Our inspections indicate that several chloroplast-specific mechanisms evolved in land plants to remedy point mutations that occurred after the water-to-land transition. Thus, the complexity of chloroplast gene expression evolved to guarantee the functionality of chloroplast genetic information and may not, with some exceptions, be involved in regulatory functions.

Abstract Background Most genes introduced into phototrophic eukaryotes during the process of endosymbiosis are either lost or relocated into the host nuclear genome. In contrast, groEL homologues are found in different genome compartments among phototrophic eukaryotes. Comparative sequence analyses of recently available genome data, have allowed us to reconstruct the evolutionary history of these genes and propose a hypothesis that explains the unusual genome distribution of groEL homologues. Results Our analyses indicate that while two distinct groEL genes were introduced into eukaryotes by a progenitor of plastids, these particular homologues have not been maintained in all evolutionary lineages. This is of significant interest, because two chaperone proteins always co-occur in oxygenic photosynthetic organisms. We infer strikingly different lineage specific processes of evolution involving deletion, duplication and targeting of groEL proteins. Conclusion The requirement of two groEL homologues for chaperon function in phototrophs has provided a constraint that has shaped convergent evolutionary scenarios in divergent evolutionary lineages. GroEL provides a general evolutionary model for studying gene transfers and convergent evolutionary processes among eukaryotic lineages.

Background: Heart failure (HF) remains a challenging healthcare issue necessitating innovative therapies like cardiac resynchronization-defibrillation therapy (CRT-D). However, the definition of a CRT-D response lacks uniformity, impeding effective clinical evaluation. This study explores diverse CRT-D responder definitions encompassing functional, echocardiographic and laboratory criteria. Materials & Methods: A single-center study involving 132 CRT-D patients scrutinized responder criteria including NYHA stage, LVEF increase and proBNP decrease. Statistical analyses such as Kaplan-Meier curves and Cox hazard regression were employed to evaluate responder characteristics and survival outcomes. Results: Responder rates varied across criteria, revealing nuanced patient profiles. CRT-D responders defined by NYHA decrease, LVEF increase or proBNP decrease exhibit improved survival rates after 2 and 3 years ( p < 0.050). Young age, absence of recent myocardial infarction and normal right ventricular echocardiographic parameters emerge as predictors for positive response. In part, drug-based HF therapy correlates with increased responder rates. Cox regression identified LVEF ≥ 5% and proBNP decrease ≥ 25% as independent predictors of extended survival. Conclusions: CRT-D responder definitions exhibit considerable variability, emphasizing the need for a nuanced patient-centered approach. Factors like right ventricular function, drug therapy, atrial fibrillation and renal function influence responses. This study enriches our understanding of CRT-D response and contributes to the foundation for personalized HF management.

Bivalves from the family Lucinidae host sulfur-oxidizing bacterial symbionts, which are housed inside specialized gill epithelial cells and are assumed to be acquired from the environment. However, little is known about the Lucinidae life cycle and symbiont acquisition in the wild. Some lucinid species broadcast their gametes into the surrounding water column, however, a few have been found to externally brood their offspring by the forming gelatinous egg masses. So far, symbiont transmission has only been investigated in one species that reproduces via broadcast spawning. Here, we show that the lucinid Loripes orbiculatus from the West African coast forms egg masses and these are dominated by diverse members of the Alphaproteobacteria, Clostridia, and Gammaproteobacteria. The microbial communities of the egg masses were distinct from those in the environments surrounding lucinids, indicating that larvae may shape their associated microbiomes. The gill symbiont of the adults was undetectable in the developmental stages, supporting horizontal transmission of the symbiont with environmental symbiont acquisition after hatching from the egg masses. These results demonstrate that L. orbiculatus acquires symbionts from the environment independent of the host's reproductive strategy (brooding or broadcast spawning) and reveal previously unknown associations with microbes during lucinid early development., (© 2022. The Author(s).)

In the ocean, most hosts acquire their symbionts from the environment. Due to the immense spatial scales involved, our understanding of the biogeography of hosts and symbionts in marine systems is patchy, although this knowledge is essential for understanding fundamental aspects of symbiosis such as host-symbiont specificity and evolution. Lucinidae is the most species-rich and widely distributed family of marine bivalves hosting autotrophic bacterial endosymbionts. Previous molecular surveys identified location-specific symbiont types that "promiscuously" form associations with multiple divergent cooccurring host species. This flexibility of host-microbe pairings is thought to underpin their global success, as it allows hosts to form associations with locally adapted symbionts. We used metagenomics to investigate the biodiversity, functional variability, and genetic exchange among the endosymbionts of 12 lucinid host species from across the globe. We report a cosmopolitan symbiont species, Candidatus Thiodiazotropha taylori, associated with multiple lucinid host species. Ca. T. taylori has achieved more success at dispersal and establishing symbioses with lucinids than any other symbiont described thus far. This discovery challenges our understanding of symbiont dispersal and location-specific colonization and suggests both symbiont and host flexibility underpin the ecological and evolutionary success of the lucinid symbiosis., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Unicellular organisms that live in marine environments must cope with considerable fluctuations in the availability of inorganic phosphate (P i ). Here, we investigated the extracellular P i concentration-dependent expression, as well as the intracellular or extracellular localization, of phosphatases and phosphate transporters of the diatom Phaeodactylum tricornutum . We identified P i -regulated plasma membrane-localized, ER-localized, and secreted phosphatases, in addition to plasma membrane-localized, vacuolar membrane-localized, and plastid-surrounding membrane-localized phosphate transporters that were also regulated in a P i concentration-dependent manner. These studies not only add further knowledge to already existing transcriptomic data, but also highlight the capacity of the diatom to distribute P i intracellularly and to mobilize P i from extracellular and intracellular resources., (Copyright © 2020 Dell’Aquila, Zauner, Heimerl, Kahnt, Samel-Gondesen, Runge, Hempel and Maier.)

One of the most abundant sources of organic carbon in the ocean is glycolate, the secretion of which by marine phytoplankton results in an estimated annual flux of one petagram of glycolate in marine environments 1 . Although it is generally accepted that glycolate is oxidized to glyoxylate by marine bacteria 2-4 , the further fate of this C 2 metabolite is not well understood. Here we show that ubiquitous marine Proteobacteria are able to assimilate glyoxylate via the β-hydroxyaspartate cycle (BHAC) that was originally proposed 56 years ago 5 . We elucidate the biochemistry of the BHAC and describe the structure of its key enzymes, including a previously unknown primary imine reductase. Overall, the BHAC enables the direct production of oxaloacetate from glyoxylate through only four enzymatic steps, representing-to our knowledge-the most efficient glyoxylate assimilation route described to date. Analysis of marine metagenomes shows that the BHAC is globally distributed and on average 20-fold more abundant than the glycerate pathway, the only other known pathway for net glyoxylate assimilation. In a field study of a phytoplankton bloom, we show that glycolate is present in high nanomolar concentrations and taken up by prokaryotes at rates that allow a full turnover of the glycolate pool within one week. During the bloom, genes that encode BHAC key enzymes are present in up to 1.5% of the bacterial community and actively transcribed, supporting the role of the BHAC in glycolate assimilation and suggesting a previously undescribed trophic interaction between autotrophic phytoplankton and heterotrophic bacterioplankton.

Nucleomorphs are small nuclei that evolved from the nucleus of former eukaryotic endosymbionts of cryptophytes and chlorarachniophytes. These enigmatic organelles reside in their complex plastids and harbor the smallest and most compacted eukaryotic genomes investigated so far. Although the coding capacity of the nucleomorph genomes is small, a significant percentage of the encoded proteins (predicted nucleomorph-encoded proteins, pNMPs) is still not functionally annotated. We have analyzed pNMPs with unknown functions via Phyre2, a bioinformatic tool for prediction and modeling of protein structure, resulting in a functional annotation of 215 pNMPs out of 826 uncharacterized open reading frames of cryptophytes. The newly annotated proteins are predicted to participate in nucleomorph-specific functions such as chromosome organization and expression, as well as in modification and degradation of nucleomorph-encoded proteins. Additionally, we have functionally assigned nucleomorph-encoded, putatively plastid-targeted proteins among the reinvestigated pNMPs. Hints for a putative function in the periplastid compartment, the cytoplasm surrounding the nucleomorphs, emerge from the identification of pNMPs that might be homologs of endomembrane system-related proteins. These proteins are discussed in respect to their putative functions., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

CRISPR/Cas9 is a powerful tool for genome editing. We constructed an easy-to-handle expression vector for application in the model organism Phaeodactylum tricornutum and tested its capabilities in order to apply CRISPR/Cas9 technology for our purpose. In our experiments, we targeted two different genes, screened for mutations and analyzed mutated diatoms in a three-step process. In the end, we identified cells, showing either monoallelic or homo-biallelic targeted mutations. Thus, we confirm that application of the CRISPR/Cas9 system for P. tricornutum is very promising, although, as discussed, overlooked pitfalls have to be considered.

Nonphotosynthetic plastids retain important biological functions and are indispensable for cell viability. However, the detailed processes underlying the loss of plastidal functions other than photosynthesis remain to be fully understood. In this study, we used transcriptomics, subcellular localization, and phylogenetic analyses to characterize the biochemical complexity of the nonphotosynthetic plastids of the apochlorotic diatom Nitzschia sp. NIES-3581. We found that these plastids have lost isopentenyl pyrophosphate biosynthesis and ribulose-1,5-bisphosphate carboxylase/oxygenase-based carbon fixation but have retained various proteins for other metabolic pathways, including amino acid biosynthesis, and a portion of the Calvin-Benson cycle comprised only of glycolysis/gluconeogenesis and the reductive pentose phosphate pathway (rPPP). While most genes for plastid proteins involved in these reactions appear to be phylogenetically related to plastid-targeted proteins found in photosynthetic relatives, we also identified a gene that most likely originated from a cytosolic protein gene. Based on organellar metabolic reconstructions of Nitzschia sp. NIES-3581 and the presence/absence of plastid sugar phosphate transporters, we propose that plastid proteins for glycolysis, gluconeogenesis, and rPPP are retained even after the loss of photosynthesis because they feed indispensable substrates to the amino acid biosynthesis pathways of the plastid. Given the correlated retention of the enzymes for plastid glycolysis, gluconeogenesis, and rPPP and those for plastid amino acid biosynthesis pathways in distantly related nonphotosynthetic plastids and cyanobacteria, we suggest that this substrate-level link with plastid amino acid biosynthesis is a key constraint against loss of the plastid glycolysis/gluconeogenesis and rPPP proteins in multiple independent lineages of nonphotosynthetic algae/plants., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Diatoms are unicellular organisms evolved by secondary endosymbiosis. Although studied in many aspects, the functions of vacuolar-like structures of these organisms are rarely investigated. One of these structures is a dominant central vacuole-like compartment with a marbled phenotype, which is supposed to represent a chrysolaminarin-storing and carbohydrate mobilization compartment. However, other functions as well as targeting of proteins to this compartment are not shown experimentally. In order to study trafficking of membrane proteins to the vacuolar membrane, we scanned the genome for intrinsic vacuolar membrane proteins and used one representative for targeting studies. Our work led to the identification of several proteins located in the vacuolar membrane as well as the sub-compartmentalized localization of one protein. In addition, we show that a di-leucine-based motif is an important signal for correct targeting to the central vacuole of diatoms, like it is in plants., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Many protists with high ecological and medical relevance harbor plastids surrounded by four membranes. Thus, nucleus-encoded proteins of these complex plastids have to traverse these barriers. Here we report on the identification of the protein translocators located in two of the plastid surrounding membranes and present recent findings on the mechanisms of protein import into the plastids of diatoms., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Many important algae groups like diatoms, dinoflagellates and ‘kelp’ but also apicomplexan parasites evolved in secondary endosymbiosis. Here, a eukaryote-eukaryote endosymbiosis created chimeric cells, in which a eukaryotic symbiont was reduced to a complex plastid. Although having lost nearly all of the eukaryotic compartments of the symbiont, a tiny lumen representing the remnant of the cytoplasm of the symbiont is still present in most of these organisms. This compartment, the periplastidal compartment, shows different degrees of reductions as in two algal groups the former nucleus is still present in a minimized form, called nucleomorph, whereas most others have lost the genetic system completely. Thus, the natural reduction of eukaryotic cytoplasms can be studied in terms of evolution and functionality, giving additionally advices for the design of synthetic minimized compartments.

Predicting the sub-cellular localization of proteins is an important task in bioinformatics, for which many standard prediction tools are available. While these tools are powerful in general and capable of predicting protein localization for the most common compartments, their performance strongly depends on the organism of interest. More importantly, there are special compartments, such as the apicoplast of apicomplexan parasites, for which these tools cannot provide a prediction at all. In the absence of a highly conserved targeting signal, even motif searches may not be able to provide a lead for the accurate prediction of protein localization for a compartment of interest. In order to approach difficult cases of that kind, we propose an alternative method that complements existing approaches by using a more targeted protein sequence model. Moreover, our method makes use of (weighted) measures for time series comparison. To demonstrate its performance, we use this method for predicting localization in special compartments of three different species, for which existing methods yield only sub-optimal results. As shown experimentally, our method is indeed capable of producing reliable predictions of sub-cellular localization for difficult cases, i.e. if training data is scarce and a potential protein targeting signal may not be well conserved.

Plastid and mitochondrial genomes have undergone parallel evolution to encode the same functional set of genes. These encode conserved protein components of the electron transport chain in their respective bioenergetic membranes and genes for the ribosomes that express them. This highly convergent aspect of organelle genome evolution is partly explained by the redox regulation hypothesis, which predicts a separate plastid or mitochondrial location for genes encoding bioenergetic membrane proteins of either photosynthesis or respiration. Here we show that convergence in organelle genome evolution is far stronger than previously recognized, because the same set of genes for ribosomal proteins is independently retained by both plastid and mitochondrial genomes. A hitherto unrecognized selective pressure retains genes for the same ribosomal proteins in both organelles. On the Escherichia coli ribosome assembly map, the retained proteins are implicated in 30S and 50S ribosomal subunit assembly and initial rRNA binding. We suggest that ribosomal assembly imposes functional constraints that govern the retention of ribosomal protein coding genes in organelles. These constraints are subordinate to redox regulation for electron transport chain components, which anchor the ribosome to the organelle genome in the first place. As organelle genomes undergo reduction, the rRNAs also become smaller. Below size thresholds of approximately 1,300 nucleotides (16S rRNA) and 2,100 nucleotides (26S rRNA), all ribosomal protein coding genes are lost from organelles, while electron transport chain components remain organelle encoded as long as the organelles use redox chemistry to generate a proton motive force.

Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote-eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have >21,000 protein genes and are intron rich, and B. natans exhibits unprecedented alternative splicing for a single-celled organism. Phylogenomic analyses and subcellular targeting predictions reveal extensive genetic and biochemical mosaicism, with both host- and endosymbiont-derived genes servicing the mitochondrion, the host cell cytosol, the plastid and the remnant endosymbiont cytosol of both algae. Mitochondrion-to-nucleus gene transfer still occurs in both organisms but plastid-to-nucleus and nucleomorph-to-nucleus transfers do not, which explains why a small residue of essential genes remains locked in each nucleomorph.

Protein import into complex plastids of red algal origin is a multistep process including translocons of different evolutionary origins. The symbiont-derived ERAD-like machinery (SELMA), shown to be of red algal origin, is proposed to be the transport system for preprotein import across the periplastidal membrane of heterokontophytes, haptophytes, cryptophytes, and apicomplexans. In contrast to the canonical endoplasmic reticulum-associated degradation (ERAD) system, SELMA translocation is suggested to be uncoupled from proteasomal degradation. We investigated the distribution of known and newly identified SELMA components in organisms with complex plastids of red algal origin by intensive data mining, thereby defining a set of core components present in all examined organisms. These include putative pore-forming components, a ubiquitylation machinery, as well as a Cdc48 complex. Furthermore, the set of known 20S proteasomal components in the periplastidal compartment (PPC) of diatoms was expanded. These newly identified putative SELMA components, as well as proteasomal subunits, were in vivo localized as PPC proteins in the diatom Phaeodactylum tricornutum. The presented data allow us to speculate about the specific features of SELMA translocation in contrast to the canonical ERAD system, especially the uncoupling of translocation from degradation.

RNA editing is a post-transcriptional process that can act upon transcripts from mitochondrial, nuclear, and chloroplast genomes. In chloroplasts, single-nucleotide conversions in mRNAs via RNA editing occur at different frequencies across the plant kingdom. These range from several hundred edited sites in some mosses and ferns to lower frequencies in seed plants and the complete lack of RNA editing in the liverwort Marchantia polymorpha. Here, we report the sequence and edited sites of the chloroplast genome from the liverwort Pellia endiviifolia. The type and frequency of chloroplast RNA editing display a pattern highly similar to that in seed plants. Analyses of the C to U conversions and the genomic context in which the editing sites are embedded provide evidence in favor of the hypothesis that chloroplast RNA editing evolved to compensate mutations in the first land plants.

Background: Poly-3-hydroxybutyrate (PHB) is a polyester with thermoplastic properties that is naturally occurring and produced by such bacteria as Ralstonia eutropha H16 and Bacillus megaterium. In contrast to currently utilized plastics and most synthetic polymers, PHB is biodegradable, and its production is not dependent on fossil resources making this bioplastic interesting for various industrial applications., Results: In this study, we report on introducing the bacterial PHB pathway of R. eutropha H16 into the diatom Phaeodactylum tricornutum, thereby demonstrating for the first time that PHB production is feasible in a microalgal system. Expression of the bacterial enzymes was sufficient to result in PHB levels of up to 10.6% of algal dry weight. The bioplastic accumulated in granule-like structures in the cytosol of the cells, as shown by light and electron microscopy., Conclusions: Our studies demonstrate the great potential of microalgae like the diatom P. tricornutum to serve as solar-powered expression factories and reveal great advantages compared to plant based production systems.