A molecular atlas of plastid and mitochondrial proteins reveals organellar remodeling during plant evolutionary transitions from algae to angiosperms.

Algae and plants carry 2 organelles of endosymbiotic origin that have been co-evolving in their host cells for more than a billion years. The biology of plastids and mitochondria can differ significantly across major lineages and organelle changes likely accompanied the adaptation to new ecological niches such as the terrestrial habitat. Based on organelle proteome data and the genomes of 168 phototrophic (Archaeplastida) versus a broad range of 518 non-phototrophic eukaryotes, we screened for changes in plastid and mitochondrial biology across 1 billion years of evolution. Taking into account 331,571 protein families (or orthogroups), we identify 31,625 protein families that are unique to primary plastid-bearing eukaryotes. The 1,906 and 825 protein families are predicted to operate in plastids and mitochondria, respectively. Tracing the evolutionary history of these protein families through evolutionary time uncovers the significant remodeling the organelles experienced from algae to land plants. The analyses of gained orthogroups identifies molecular changes of organelle biology that connect to the diversification of major lineages and facilitated major transitions from chlorophytes en route to the global greening and origin of angiosperms.

A mysterious cloak: the peptidoglycan layer of algal and plant plastids.

The plastids of algae and plants originated on a single occasion from an endosymbiotic cyanobacterium at least a billion years ago. Despite the divergent evolution that characterizes the plastids of different lineages, many traits such as membrane organization and means of fission are universal-they pay tribute to the cyanobacterial origin of the organelle. For one such trait, the peptidoglycan (PG) layer, the situation is more complicated. Our view on its distribution keeps on changing and little is known regarding its molecular relevance, especially for land plants. Here, we investigate the extent of PG presence across the Chloroplastida using a phylogenomic approach. Our data support the view of a PG layer being present in the last common ancestor of land plants and its remarkable conservation across bryophytes that are otherwise characterized by gene loss. In embryophytes, the occurrence of the PG layer biosynthetic toolkit becomes patchier and the availability of novel genome data questions previous predictions regarding a functional coevolution of the PG layer and the plastid division machinery-associated gene FtsZ3. Furthermore, our data confirm the presence of penicillin-binding protein (PBP) orthologs in seed plants, which were previously thought to be absent from this clade. The 5-7 nm thick, and seemingly unchanged, PG layer armoring the plastids of glaucophyte algae might still provide the original function of structural support, but the same can likely not be said about the only recently identified PG layer of bryophyte and tracheophyte plastids. There are several issues to be explored regarding the composition, exact function, and biosynthesis of the PG layer in land plants. These issues arise from the fact that land plants seemingly lack certain genes that are believed to be crucial for PG layer production, even though they probably synthesize a PG layer.

Mitochondrial evolution: Gene shuffling, endosymbiosis, and signaling.

Genes for cardiolipin and ceramide synthesis occur in some alphaproteobacterial genomes. They shed light on mitochondrial origin and signaling in the first eukaryotic cells.

Endosymbiotic selective pressure at the origin of eukaryotic cell biology.

The dichotomy that separates prokaryotic from eukaryotic cells runs deep. The transition from pro- to eukaryote evolution is poorly understood due to a lack of reliable intermediate forms and definitions regarding the nature of the first host that could no longer be considered a prokaryote, the first eukaryotic common ancestor, FECA. The last eukaryotic common ancestor, LECA, was a complex cell that united all traits characterising eukaryotic biology including a mitochondrion. The role of the endosymbiotic organelle in this radical transition towards complex life forms is, however, sometimes questioned. In particular the discovery of the asgard archaea has stimulated discussions regarding the pre-endosymbiotic complexity of FECA. Here we review differences and similarities among models that view eukaryotic traits as isolated coincidental events in asgard archaeal evolution or, on the contrary, as a result of and in response to endosymbiosis. Inspecting eukaryotic traits from the perspective of the endosymbiont uncovers that eukaryotic cell biology can be explained as having evolved as a solution to housing a semi-autonomous organelle and why the addition of another endosymbiont, the plastid, added no extra compartments. Mitochondria provided the selective pressures for the origin (and continued maintenance) of eukaryotic cell complexity. Moreover, they also provided the energetic benefit throughout eukaryogenesis for evolving thousands of gene families unique to eukaryotes. Hence, a synthesis of the current data lets us conclude that traits such as the Golgi apparatus, the nucleus, autophagosomes, and meiosis and sex evolved as a response to the selective pressures an endosymbiont imposes.

The greening ashore.

More than half a billion years ago a streptophyte algal lineage began terraforming the terrestrial habitat and the Earth's atmosphere. This pioneering step enabled the subsequent evolution of all complex life on land, and the past decade has uncovered that many traits, both morphological and genetic, once thought to be unique to land plants, are conserved across some streptophyte algae. They provided the common ancestor of land plants with a repertoire of genes, of which many were adapted to overcome the new biotic and abiotic challenges. Exploring these molecular adaptations in non-tracheophyte species may help us to better prepare all green life, including our crops, for the challenges precipitated by the climate change of the Anthropocene because the challenges mostly differ by the speed with which they are now being met.

Loss of Plastid Developmental Genes Coincides With a Reversion to Monoplastidy in Hornworts.

The first plastid evolved from an endosymbiotic cyanobacterium in the common ancestor of the Archaeplastida. The transformative steps from cyanobacterium to organelle included the transfer of control over developmental processes, a necessity for the host to orchestrate, for example, the fission of the organelle. The plastids of almost all embryophytes divide independently from nuclear division, leading to cells housing multiple plastids. Hornworts, however, are monoplastidic (or near-monoplastidic), and their photosynthetic organelles are a curious exception among embryophytes for reasons such as the occasional presence of pyrenoids. In this study, we screened genomic and transcriptomic data of eleven hornworts for components of plastid developmental pathways. We found intriguing differences among hornworts and specifically highlight that pathway components involved in regulating plastid development and biogenesis were differentially lost in this group of bryophytes. Our results also confirmed that hornworts underwent significant instances of gene loss, underpinning that the gene content of this group is significantly lower than other bryophytes and tracheophytes. In combination with ancestral state reconstruction, our data suggest that hornworts have reverted back to a monoplastidic phenotype due to the combined loss of two plastid division-associated genes, namely, ARC3 and FtsZ2.

An overview of bioinformatics, genomics, and transcriptomics resources for bryophytes.

Bryophytes are useful models for the study of plant evolution, development, plant-fungal symbiosis, stress responses, and gametogenesis. Additionally, their dominant haploid gametophytic phase makes them great models for functional genomics research, allowing straightforward genome editing and gene knockout via CRISPR or homologous recombination. Until 2016, however, the only bryophyte genome sequence published was that of Physcomitrium patens. Throughout recent years, several other bryophyte genomes and transcriptome datasets became available, enabling better comparative genomics in evolutionary studies. The increase in the number of bryophyte genome and transcriptome resources available has yielded a plethora of annotations, databases, and bioinformatics tools to access the new data, which covers the large diversity of this clade and whose biology comprises features such as association with arbuscular mycorrhiza fungi, sex chromosomes, low gene redundancy, or loss of RNA editing genes for organellar transcripts. Here we provide a guide to resources available for bryophytes with regards to genome and transcriptome databases and bioinformatics tools.

Signatures of Transcription Factor Evolution and the Secondary Gain of Red Algae Complexity.

Red algae (Rhodophyta) belong to the superphylum Archaeplastida, and are a species-rich group exhibiting diverse morphologies. Theory has it that the unicellular red algal ancestor went through a phase of genome contraction caused by adaptation to extreme environments. More recently, the classes Porphyridiophyceae, Bangiophyceae, and Florideophyceae experienced genome expansions, coinciding with an increase in morphological complexity. Transcription-associated proteins (TAPs) regulate transcription, show lineage-specific patterns, and are related to organismal complexity. To better understand red algal TAP complexity and evolution, we investigated the TAP family complement of uni- and multi-cellular red algae. We found that the TAP family complement correlates with gain of morphological complexity in the multicellular Bangiophyceae and Florideophyceae, and that abundance of the C2H2 zinc finger transcription factor family may be associated with the acquisition of morphological complexity. An expansion of heat shock transcription factors (HSF) occurred within the unicellular Cyanidiales, potentially as an adaption to extreme environmental conditions.

Genetic autonomy and low singlet oxygen yield support kleptoplast functionality in photosynthetic sea slugs.

The kleptoplastic sea slug Elysia chlorotica consumes Vaucheria litorea, stealing its plastids, which then photosynthesize inside the animal cells for months. We investigated the properties of V. litorea plastids to understand how they withstand the rigors of photosynthesis in isolation. Transcription of specific genes in laboratory-isolated V. litorea plastids was monitored for 7 days. The involvement of plastid-encoded FtsH, a key plastid maintenance protease, in recovery from photoinhibition in V. litorea was estimated in cycloheximide-treated cells. In vitro comparison of V. litorea and spinach thylakoids was applied to investigate reactive oxygen species formation in V. litorea. In comparison to other tested genes, the transcripts of ftsH and translation elongation factor EF-Tu (tufA) decreased slowly in isolated V. litorea plastids. Higher levels of FtsH were also evident in cycloheximide-treated cells during recovery from photoinhibition. Charge recombination in PSII of V. litorea was found to be fine-tuned to produce only small quantities of singlet oxygen, and the plastids also contained reactive oxygen species-protective compounds. Our results support the view that the genetic characteristics of the plastids are crucial in creating a photosynthetic sea slug. The plastid's autonomous repair machinery is likely enhanced by low singlet oxygen production and elevated expression of FtsH.

Evidence for a Syncytial Origin of Eukaryotes from Ancestral State Reconstruction.

Modern accounts of eukaryogenesis entail an endosymbiotic encounter between an archaeal host and a proteobacterial endosymbiont, with subsequent evolution giving rise to a unicell possessing a single nucleus and mitochondria. The mononucleate state of the last eukaryotic common ancestor (LECA) is seldom, if ever, questioned, even though cells harboring multiple (syncytia, coenocytes, and polykaryons) are surprisingly common across eukaryotic supergroups. Here, we present a survey of multinucleated forms. Ancestral character state reconstruction for representatives of 106 eukaryotic taxa using 16 different possible roots and supergroup sister relationships, indicate that LECA, in addition to being mitochondriate, sexual, and meiotic, was multinucleate. LECA exhibited closed mitosis, which is the rule for modern syncytial forms, shedding light on the mechanics of its chromosome segregation. A simple mathematical model shows that within LECA's multinucleate cytosol, relationships among mitochondria and nuclei were neither one-to-one, nor one-to-many, but many-to-many, placing mitonuclear interactions and cytonuclear compatibility at the evolutionary base of eukaryotic cell origin. Within a syncytium, individual nuclei and individual mitochondria function as the initial lower-level evolutionary units of selection, as opposed to individual cells, during eukaryogenesis. Nuclei within a syncytium rescue each other's lethal mutations, thereby postponing selection for viable nuclei and cytonuclear compatibility to the generation of spores, buffering transitional bottlenecks at eukaryogenesis. The prokaryote-to-eukaryote transition is traditionally thought to have left no intermediates, yet if eukaryogenesis proceeded via a syncytial common ancestor, intermediate forms have persisted to the present throughout the eukaryotic tree as syncytia but have so far gone unrecognized.

The Asgard Archaeal-Unique Contribution to Protein Families of the Eukaryotic Common Ancestor Was 0.3.

The identification of the asgard archaea has fueled speculations regarding the nature of the archaeal host in eukaryogenesis and its level of complexity prior to endosymbiosis. Here, we analyzed the coding capacity of 150 eukaryotes, 1,000 bacteria, and 226 archaea, including the only cultured member of the asgard archaea. Clustering methods that consistently recover endosymbiotic contributions to eukaryotic genomes recover an asgard archaeal-unique contribution of a mere 0.3% to protein families present in the last eukaryotic common ancestor, while simultaneously suggesting that this group's diversity rivals that of all other archaea combined. The number of homologs shared exclusively between asgard archaea and eukaryotes is only 27 on average. This tiny asgard archaeal-unique contribution to the root of eukaryotic protein families questions claims that archaea evolved complexity prior to eukaryogenesis. Genomic and cellular complexity remains a eukaryote-specific feature and is best understood as the archaeal host's solution to housing an endosymbiont.

Gene Duplications Trace Mitochondria to the Onset of Eukaryote Complexity.

The last eukaryote common ancestor (LECA) possessed mitochondria and all key traits that make eukaryotic cells more complex than their prokaryotic ancestors, yet the timing of mitochondrial acquisition and the role of mitochondria in the origin of eukaryote complexity remain debated. Here, we report evidence from gene duplications in LECA indicating an early origin of mitochondria. Among 163,545 duplications in 24,571 gene trees spanning 150 sequenced eukaryotic genomes, we identify 713 gene duplication events that occurred in LECA. LECA's bacterial-derived genes include numerous mitochondrial functions and were duplicated significantly more often than archaeal-derived and eukaryote-specific genes. The surplus of bacterial-derived duplications in LECA most likely reflects the serial copying of genes from the mitochondrial endosymbiont to the archaeal host's chromosomes. Clustering, phylogenies and likelihood ratio tests for 22.4 million genes from 5,655 prokaryotic and 150 eukaryotic genomes reveal no evidence for lineage-specific gene acquisitions in eukaryotes, except from the plastid in the plant lineage. That finding, and the functions of bacterial genes duplicated in LECA, suggests that the bacterial genes in eukaryotes are acquisitions from the mitochondrion, followed by vertical gene evolution and differential loss across eukaryotic lineages, flanked by concomitant lateral gene transfer among prokaryotes. Overall, the data indicate that recurrent gene transfer via the copying of genes from a resident mitochondrial endosymbiont to archaeal host chromosomes preceded the onset of eukaryotic cellular complexity, favoring mitochondria-early over mitochondria-late hypotheses for eukaryote origin.

Anomalous Phylogenetic Behavior of Ribosomal Proteins in Metagenome-Assembled Asgard Archaea.

Metagenomic studies permit the exploration of microbial diversity in a defined habitat, and binning procedures enable phylogenomic analyses, taxon description, and even phenotypic characterizations in the absence of morphological evidence. Such lineages include asgard archaea, which were initially reported to represent archaea with eukaryotic cell complexity, although the first images of such an archaeon show simple cells with prokaryotic characteristics. However, these metagenome-assembled genomes (MAGs) might suffer from data quality problems not encountered in sequences from cultured organisms due to two common analytical procedures of bioinformatics: assembly of metagenomic sequences and binning of assembled sequences on the basis of innate sequence properties and abundance across samples. Consequently, genomic sequences of distantly related taxa, or domains, can in principle be assigned to the same MAG and result in chimeric sequences. The impacts of low-quality or chimeric MAGs on phylogenomic and metabolic prediction remain unknown. Debates that asgard archaeal data are contaminated with eukaryotic sequences are overshadowed by the lack of evidence indicating that individual asgard MAGs stem from the same chromosome. Here, we show that universal proteins including ribosomal proteins of asgard archaeal MAGs fail to meet the basic phylogenetic criterion fulfilled by genome sequences of cultured archaea investigated to date: These proteins do not share common evolutionary histories to the same extent as pure culture genomes do, pointing to a chimeric nature of asgard archaeal MAGs. Our analysis suggests that some asgard archaeal MAGs represent unnatural constructs, genome-like patchworks of genes resulting from assembly and/or the binning process.

More than just a ticket canceller: the mitochondrial processing peptidase tailors complex precursor proteins at internal cleavage sites.

Most mitochondrial proteins are synthesized as precursors that carry N-terminal presequences. After they are imported into mitochondria, these targeting signals are cleaved off by the mitochondrial processing peptidase (MPP). Using the mitochondrial tandem protein Arg5,6 as a model substrate, we demonstrate that MPP has an additional role in preprotein maturation, beyond the removal of presequences. Arg5,6 is synthesized as a polyprotein precursor that is imported into mitochondria and subsequently separated into two distinct enzymes. This internal processing is performed by MPP, which cleaves the Arg5,6 precursor at its N-terminus and at an internal site. The peculiar organization of Arg5,6 is conserved across fungi and reflects the polycistronic arginine operon in prokaryotes. MPP cleavage sites are also present in other mitochondrial fusion proteins from fungi, plants, and animals. Hence, besides its role as a "ticket canceller" for removal of presequences, MPP exhibits a second conserved activity as an internal processing peptidase for complex mitochondrial precursor proteins.

Major Changes in Plastid Protein Import and the Origin of the Chloroplastida.

Core components of plastid protein import and the principle of using N-terminal targeting sequences are conserved across the Archaeplastida, but lineage-specific differences exist. Here we compare, in light of plastid protein import, the response to high-light stress from representatives of the three archaeplastidal groups. Similar to land plants, Chlamydomonas reinhardtii displays a broad response to high-light stress, not observed to the same degree in the glaucophyte Cyanophora paradoxa or the rhodophyte Porphyridium purpureum. We find that only the Chloroplastida encode both Toc75 and Oep80 in parallel and suggest that elaborate high-light stress response is supported by changes in plastid protein import. We propose the origin of a phenylalanine-independent import pathway via Toc75 allowed higher import rates to rapidly service high-light stress, but with the cost of reduced specificity. Changes in plastid protein import define the origin of the green lineage, whose greatest evolutionary success was arguably the colonization of land.

Characterization of the BspA and Pmp protein family of trichomonads.

BACKGROUND: Trichomonas vaginalis is a human-infecting trichomonad and as such the best studied and the only for which the full genome sequence is available considering its parasitic lifestyle, T. vaginalis encodes an unusually high number of proteins. Many gene families are massively expanded and some genes are speculated to have been acquired from prokaryotic sources. Among the latter are two gene families that harbour domains which share similarity with proteins of Bacteroidales/Spirochaetales and Chlamydiales: the BspA and the Pmp proteins, respectively. RESULTS: We sequenced the transcriptomes of five trichomonad species and screened for the presence of BspA and Pmp domain-containing proteins and characterized individual candidate proteins from both families in T. vaginalis. Here, we demonstrate that (i) BspA and Pmp domain-containing proteins are universal to trichomonads, but specifically expanded in T. vaginalis; (ii) in line with a concurrent expansion of the endocytic machinery, there is a high number of BspA and Pmp proteins which carry C-terminal endocytic motifs; and (iii) both families traffic through the ER and have the ability to increase adhesion performance in a non-virulent T. vaginalis strain and Tetratrichomonas gallinarum by a so far unknown mechanism. CONCLUSIONS: Our results initiate the functional characterization of these two broadly distributed protein families and help to better understand the origin and evolution of BspA and Pmp domains in trichomonads.

Development of the Mitochondrial Intermembrane Space Disulfide Relay Represents a Critical Step in Eukaryotic Evolution.

The mitochondrial intermembrane space evolved from the bacterial periplasm. Presumably as a consequence of their common origin, most proteins of these compartments are stabilized by structural disulfide bonds. The molecular machineries that mediate oxidative protein folding in bacteria and mitochondria, however, appear to share no common ancestry. Here we tested whether the enzymes Erv1 and Mia40 of the yeast mitochondrial disulfide relay could be functionally replaced by corresponding components of other compartments. We found that the sulfhydryl oxidase Erv1 could be replaced by the Ero1 oxidase or the protein disulfide isomerase from the endoplasmic reticulum, however at the cost of respiration deficiency. In contrast to Erv1, the mitochondrial oxidoreductase Mia40 proved to be indispensable and could not be replaced by thioredoxin-like enzymes, including the cytoplasmic reductase thioredoxin, the periplasmic dithiol oxidase DsbA, and Pdi1. From our studies we conclude that the profound inertness against glutathione, its slow oxidation kinetics and its high affinity to substrates renders Mia40 a unique and essential component of mitochondrial biogenesis. Evidently, the development of a specific mitochondrial disulfide relay system represented a crucial step in the evolution of the eukaryotic cell.

Nutrient exchange in arbuscular mycorrhizal symbiosis from a thermodynamic point of view.

To obtain insights into the dynamics of nutrient exchange in arbuscular mycorrhizal (AM) symbiosis, we modelled mathematically the two-membrane system at the plant-fungus interface and simulated its dynamics. In computational cell biology experiments, the full range of nutrient transport pathways was tested for their ability to exchange phosphorus (P)/carbon (C)/nitrogen (N) sources. As a result, we obtained a thermodynamically justified, independent and comprehensive model of the dynamics of the nutrient exchange at the plant-fungus contact zone. The predicted optimal transporter network coincides with the transporter set independently confirmed in wet-laboratory experiments previously, indicating that all essential transporter types have been discovered. The thermodynamic analyses suggest that phosphate is released from the fungus via proton-coupled phosphate transporters rather than anion channels. Optimal transport pathways, such as cation channels or proton-coupled symporters, shuttle nutrients together with a positive charge across the membranes. Only in exceptional cases does electroneutral transport via diffusion facilitators appear to be plausible. The thermodynamic models presented here can be generalized and adapted to other forms of mycorrhiza and open the door for future studies combining wet-laboratory experiments with computational simulations to obtain a deeper understanding of the investigated phenomena.

On plant defense signaling networks and early land plant evolution.

All land plants must cope with phytopathogens. Algae face pathogens, too, and it is reasonable to assume that some of the strategies for dealing with pathogens evolved prior to the origin of embryophytes - plant terrestrialization simply changed the nature of the plant-pathogen interactions. Here we highlight that many potential components of the angiosperm defense toolkit are i) found in streptophyte algae and non-flowering embryophytes and ii) might be used in non-flowering plant defense as inferred from published experimental data. Nonetheless, the common signaling networks governing these defense responses appear to have become more intricate during embryophyte evolution. This includes the evolution of the antagonistic signaling pathways of jasmonic and salicylic acid, multiple independent expansions of resistance genes, and the evolution of resistance gene-regulating microRNAs. Future comparative studies will illuminate which modules of the streptophyte defense signaling network constitute the core and which constitute lineage- and/or environment-specific (peripheral) signaling circuits.

Membranes and evolution.

Biological membranes are thin amphiphilic sheaths, only a few nanometres thick, that define both the boundaries of all cells as well as the diversity of the internal compartments in eukaryotes. The plasma membrane of a typical prokaryote houses about 20-30% of the cell's expressed proteins, and its lipids account for approximately 10% of the cell's dry mass. The numbers for eukaryotic cells are comparable - the difference in surface area to volume ratio is overall compensated by the eukaryotic endomembrane system. Roughly a fourth of the protein encoded by the human genome carries at least one stretch of sequence predicted to serve as a transmembrane domain. Membranes host substrate exchange, sensing and communication, and life-giving energy conservation via chemiosmotic ATP synthesis.