Plastids house the biology of eukaryotic photosynthesis. While 1000s of plastid genomes have been sequenced, the availability of less than ten proteomes and only two species with full 70S plastid ribosomal structures limit our understanding of plant evolution. We optimized a protocol for the rapid isolation of Marchantia polymorpha plastids that provides a highly enriched and intact organelle fraction from gradient volumes as little as 2 mL. The approach was successfully applied to six other species. Focusing on M. polymorpha, we determined the proteome of the plastid fraction, identifying 1337 nuclear-encoded proteins with a high confidence, where 83% belong to orthologs shared with angiosperms. We further isolated protein complexes by RNA affinity purification using poly-lysine and provide the high-resolution structures of the 50S subunit of the chloroplast ribosome and RuBisCO using cryogenic EM and image reconstruction to 2.23 and 2.12 Å resolution, respectively. For plastids, our data show that the genome reduction event experienced by the bryophyte common ancestor has had little impact on the organelle's complexity and they underscore a high level of structural conservation of key components. Our data provide novel resources to explore the functional evolution of plastid proteomes and major macromolecular complexes of cyanobacterial origin.

Land plants alternate between multicellular haploid and diploid phases, requiring a tight coordination between vegetative growth and sexual reproduction. We investigated SWI3A/B, an ancient core subunit of the SWI/SNF complex, of the non-vascular liverwort Marchantia polymorpha. A mutation in the promoter of MpSWI3A/B affected gametangiophore development and spermiogenesis in males, revealing its male-specific role in reproductive development. The MpSWIA/B mutant line amplifies vegetative propagation in males under conditions that would normally induce reproductive growth. The phenotype was underpinned by transcriptomic changes, showing that MpSWI3A/B modulates key regulators of gametangiophore initiation (e.g. BONOBO, GLID), sperm development and motility (e.g. DUO1, PKAR), and asexual reproduction (e.g. KAI2). We propose that, as a chromatin-level regulator, MpSWI3A/B may contribute to balancing vegetative and reproductive phases and highlight the protein's potential when exploring ancient epigenetic functions that coordinate developmental phase transitions in land plants.

Ribosome stalling caused by polyproline (PPs) motifs is common. Their translation is enhanced by accessory proteins such as YebC in bacteria, whose homolog, TRANSLATIONAL ACTIVATOR OF CYTOCHROME C OXIDASE 1 (TACO1), aids the translation of mitochondria-encoded proteins. The prevalence of PP motifs across plastid-encoded genes and their impact on the translation of photosynthesis-relevant proteins remains unexplored. Equally, a translation-enhancer of PP motifs equivalent to TACO1 for plastid ribosomes has not been reported. Here, we show that plastid genomes encode 24 proteins with a minimum of one PP motif on average, half of which are conserved in their cyanobacterial homologs, and that the vast majority of eukaryotes, including plants, encode a single TACO1 that we demonstrate to be dually targeted to mitochondria and plastids of Marchantia polymorpha. We resolved the MpTACO1 structure at 2.34 Å by X-ray crystallography and the flexibility by small-angle X-ray scattering. Through modelling, we demonstrate that MpTACO1 can fit into the peptidyl transfer centre of plant chlororibosomes in a similar manner as human TACO1 in the mitoribosome. The identification and structure determination of the first plastid-targeted YebC/TACO1 allows us to sketch a unified model for the function and evolution of this ancient family of ribosomal accessory proteins, underscoring their indispensable role in the translation of bioenergetic membrane proteins reaching back almost 4 billion years.

Land plants alternate between multicellular haploid and diploid phases, a life cycle requiring tight coordination between vegetative growth and sexual reproduction. Chromatin regulators such as SWI3, a core subunit of the SWI/SNF complex, are ancestral and highly diversified in angiosperms. As their functions outside of angiosperms remain unclear, we investigated SWI3 of the liverwort Marchantia polymorpha , a non-vascular dioecious land plant. A mutation in the promoter of Mp SWI3 affected gametangiophore development and spermiogenesis in males, revealing its male-specific role in reproductive development. The Mp SWI3 mutant line amplifies vegetative propagation in males under conditions that would normally induce reproductive growth. The phenotype was underpinned by transcriptomic changes, showing that MpSWI3 modulates key regulators of gametangiophore initiation (e.g. BONOBO, GLID), sperm development and motility (e.g. DUO1, PKAR), and asexual reproduction (e.g. KAI2). Together, this positions Mp SWI3 as a chromatin-level regulator balancing vegetative and reproductive phases, highlighting an ancient epigenetic function that coordinates developmental phase transitions in land plants. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, SPP2237–440043394

Plastids and mitochondria are key to plant survival and adaptation. The evolutionary progress of land plants (embryophytes) witnessed gene and genome duplications, and the expansion of organelle-localized proteins. To deal with the increase of nuclear-encoded proteins, targeting to and import by the mitochondrion and plastid are known to have adapted in multiple ways. It included the addition of entirely new import channels and lineage-specific import receptors. Through comparative genomics and experimental biology, we uncover further changes in the organelle import machineries. Their evolution likely served to enhance the rate of protein import and improve its physiological regulation, e.g. via interactions between the import channel and respiratory complex. On the cargo side, nuclear-encoded N-terminal targeting sequences of mitochondrial targeting peptide (TP) and plastidal (pTPs) proteins have diverged in their charge via a preference for phosphorylatable amino acids (AA) (adding negative charges after phosphorylation) and an avoidance of positive charges in the pTPs, which is most evident in eudicots. Using Chlamydomonas and Marchantia, we experimentally underscore that the evolved TP divergence prevents mis-sorting between mitochondria and plastids. In accordance with the increase in phosphorylatable AA in the pTPs, we pinpoint the embryophytic origin of a membrane-anchored phosphatase, PAP2, which is associated with targeting sequence processing. On the whole, we propose a revised model for the evolution of plant organelle protein import from algae to angiosperms, which facilitated the flourishing of this lineage on land.

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.

Mitochondria and plastids import thousands of proteins. Their experimental localisation remains a frequent task, but can be resource-intensive and sometimes impossible. Hence, hundreds of studies make use of algorithms that predict a localisation based on a protein’s sequence. Their reliability across evolutionary diverse species is unknown. Here, we evaluate the performance of common algorithms (TargetP, Localizer and WoLFPSORT) for four photosynthetic eukaryotes (Arabidopsis thaliana, Zea mays, Physcomitrium patens, and Chlamydomonas reinhardtii) for which experimental plastid and mitochondrial proteome data is available, and 171 eukaryotes using orthology inferences. The match between predictions and experimental data ranges from 75% to as low as 2%. Results worsen as the evolutionary distance between training and query species increases, especially for plant mitochondria for which performance borders on random sampling. Specificity, sensitivity and precision analyses highlight cross-organelle errors and uncover the evolutionary divergence of organelles as the main driver of current performance issues. The results encourage to train the next generation of neural networks on an evolutionary more diverse set of organelle proteins for optimizing performance and reliability.

Manganese (Mn) is key to oxygenic photosynthesis as it catalyzes the splitting of water in photosystem II and functions as cofactor of multiple enzymes. A single ABC-type transporter, MntCAB, is so far established for the uptake of the metal under limiting conditions in cyanobacteria. It is unknown, how Mn is imported under replete conditions. We identified two proteins in the model cyanobacterium Synechocystis sp. PCC 6803, which are homologous to the unknown protein family 0016 (UPF0016) member manganese exporter (Mnx). In contrast to Mnx, which consists of six transmembrane domains, the new candidate proteins contain only three transmembrane domains. Hence, we named them hemi manganese exporter (Hmx) 1 and 2. Knock-out mutants in hmx1 and/or hmx2 showed sensitivity toward low Mn supplementation, and reduced intracellular Mn pools. Additional deletion of mntC hindered the cells to thrive unless external Mn was added and enhanced the depletion of their intracellular Mn pool. In accordance with the observed localization of Hmx1 and Hmx2 in the plasma membrane, we postulate a Mn uptake function for a heteromeric Hmx1/2 across the plasma membrane under a wide range of Mn concentrations and a supporting role for the MntCAB system under Mn-limiting conditions. On the basis of their phylogenies, we propose that Hmx1 and Hmx2 are the ancestral progenitors of eukaryote-type UPF0016 proteins with six transmembrane domains. The Mn transport function of Hmx1/2 underscores this as fundamental ancient feature of the UPF0016 family. Likely, Hmx1 and Hmx2 coevolved with the internalization of the oxygen-evolving complex.### Competing Interest StatementThe authors have declared no competing interest.

Mitochondria and plastids import thousands of proteins. Their experimental localisation remains a frequent task, but can be resource-intensive and sometimes impossible. Hence, hundreds of studies make use of algorithms that predict a localisation based on a protein’s sequence. Their reliability across evolutionary diverse species is unknown. Here, we evaluate the performance of common algorithms (TargetP, Localizer and WoLFPSORT) for four photosynthetic eukaryotes for which experimental plastid and mitochondrial proteome data is available, and 171 eukaryotes using orthology inferences. The match between predictions and experimental data ranges from 75% to as low as 2%. Results worsen as the evolutionary distance between training and query species increases, especially for plant mitochondria for which performance borders on random sampling. Specificity, sensitivity and precision analyses highlight cross-organelle errors and uncover the evolutionary divergence of organelles as the main driver of current performance issues. The results encourage to train the next generation of neural networks on an evolutionary more diverse set of organelle proteins for optimizing performance and reliability.### Competing Interest StatementThe authors have declared no competing interest.

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.

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

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. The use of bioinformatics, phylogenetics, and comparative genomics, together with the recent publication of several key bryophyte genome sequences, have greatly advanced the knowledge about bryophytes and their evolution.

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.

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 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.

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.

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.

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.

Significance Statement Ever since the first report of a new archaeal lineage, the asgardarchaea, their metagenome analyses have encouraged continued speculations on a type of cell biology ranging between that of prokaryotes and eukaryotes. While it appears a tempting notion, recent microscopic images of an asgardarchaeon suggest otherwise. We inspected the origin of eukaryotic protein families with respect to their distribution across bacteria and archaea. This reveals that the protein families shared exclusively between asgardarchaea and eukaryotes amounts to only 0.3% of the protein families conserved across all eukaryotes. Asgardarchaeal diversity is likely unrivaled across archaea, but their cell biology remains prokaryotic in nature and lends support for the importance of endosymbiosis in evolving eukaryotic traits. Summary The difference between pro- and eukaryotic biology is evident in their genomes, cell biology, and evolution of complex and macroscopic body plans. The lack of intermediates between the two types of cells places the endosymbiotic acquisition of the mitochondrion through an archaeal host at the event horizon of eukaryote origin. The identification of eukaryote specific proteins in a new archaeal phylum, the asgardarchaea, has fueled speculations about their cellular complexity, suggesting they could be eukaryote-like. Here we analyzed the coding capacity of 150 eukaryotes, 1000 bacteria, and 226 archaea, including the only cultured member of the asgardarchaea, Candidatus Prometheoarchaeon syntrophicum MK-D1. Established clustering methods that recover endosymbiotic contributions to eukaryotic genomes, recover an asgardarchaeal-unique contribution of a mere 0.3% to protein families present in the last eukaryotic common ancestor, while simultaneously suggesting that asgardarchaeal diversity rivals that of all other archaea combined. Furthermore, we show that the number of homologs shared exclusively between asgardarchaea and eukaryotes is only 27 on average. Genomic and in particular cellular complexity remains a eukaryote-specific feature and, we conclude, is best understood as the archaeal host’s solution to housing an endosymbiont and not as a preparation for obtaining one.