. Author manuscript; available in PMC: 2020 Dec 14.
Published in final edited form as:J Phycol. 2020 May 5;56(4):837–843. doi:
10.1111/jpy.13003 AbstractThe uptake and conversion of a free-living cyanobacterium into a photosynthetic organelle by the single-celled Archaeplastida ancestor helped transform the biosphere from low to high oxygen. There are two documented, independent cases of plastid primary endosymbiosis. The first is the well-studied instance in Archaeplastida that occurred ca. 1.6 billion years ago, whereas the second occurred 90–140 million years ago, establishing a permanent photosynthetic compartment (the chromatophore) in amoebae in the genus Paulinella. Here, we briefly summarize knowledge about plastid origin in the Archaeplastida and then focus on Paulinella. In particular, we describe features of the Paulinella chromatophore that make it a model for examining earlier events in the evolution of photosynthetic organelles. Our review stresses recently gained insights into the evolution of chromatophore and nuclear encoded DNA sequences in Paulinella, metabolic connectivity between the endosymbiont and cytoplasm, and systems that target proteins into the chromatophore. We also describe future work with Paulinella, and the potential rewards and challenges associated with developing further this model system.
Key index words: cyanobacteria, gene expression, Paulinella, photosynthesis, phylogeny, symbiosis
Plastid primary endosymbiosis involved the uptake and retention of a free-living cyanobacterium by a eukaryotic heterotroph. These intracellular organelles typically perform photosynthesis and a variety of other metabolic activities such as protein, fatty acid, and isoprenoid and chlorophyll biosynthesis. The canonical plastid primary endosymbiosis occurred about 1.6 billion years ago, giving rise to the ancestor of the Archaeplastida (formerly Plantae) that diversified into the three “primary” photosynthetic lineages, the Viridiplantae (green algae and land plants), Rhodophyta, and Glaucophyta (Bhattacharya et al. 2012, Shih and Matzke 2013, Rockwell et al. 2014, Zhang et al. 2017, Nowack and Weber 2018). Recent studies have identified the nonphotosynthetic, phagotrophic Rhodelphis species, which are sister organisms to the Rhodophyta. These findings alter the way in which we think about the origins of the Rhodophyta and Archaeplastida and substantiate that both phototrophy and predation were integral to their evolutionary histories (Gawryluk et al. 2019). The Archaeplastida not only provided a novel lineage of primary producers in many aquatic habitats, but the plastid in these cells spread throughout the eukaryotic tree of life through secondary and higher order eukaryote-eukaryote endosymbioses. These endosymbiotic events led to the establishment of widespread marine algal lineages such as diatoms, dinoflagellates, haptophytes, and kelps, all of which play key roles in global carbon cycling.
Despite the importance and impact of Archaeplastida to the evolution of complex life, many aspects of primary endosymbiosis, such as details of endosymbiont uptake, retention, and integration into the metabolic and developmental biology of the “host” are not well understood. The ancient origin of the Archaeplastida plastid, the loss of evolutionary intermediates and the highly derived nature of extant photosynthetic eukaryotes make it challenging to reconstruct many steps in photosynthetic organelle evolution. A significant leap forward in this area came with the discovery (Lauterborn 1895) and subsequent genomic work performed with the photosynthetic amoeba, Paulinella. This lineage has received great attention because it is the only case of primary endosymbiosis that led to the establishment of a photosynthetic organelle (the “chromatophore”) other than the event that established the Archaeplastida. The photosynthetic compartment in Paulinella originated more recently in a rhizarian amoeba host. This makes Paulinella a unique model from which we can gain fundamental insights into the “evolutionary maturation” of a plastid (Marin et al. 2005, Reyes-Prieto et al. 2007, Keeling 2010, Delaye et al. 2016, Nowack and Weber 2018). In this review, we will first briefly discuss the endosymbiotic event that established the Archaeplastida and then describe the characteristics of Paulinella species that support the idea that these species contain the only known case of a bona fide photosynthetic organelle, albeit in the earlier stages (relative to the Archaeplastida) of host-plastid integration. We note that the definition of organelle is in flux due to the finding of other prokaryote-derived compartments (e.g., in mealybugs; Bublitz et al. 2019) that mirror the complex genetic interactions typical of plastids and mitochondria. We do not strive here to use Paulinella as a general model for defining all organelles. Rather, we show, using compelling molecular data the massive evolutionary innovations that define the Paulinella example, including endosymbiont genome erosion, origin of protein import pathways, and host-chromatophore metabolic connectivity that unambiguously identify the chromatophore as a photosynthetic organelle, directly comparable to the more highly derived compartments in Archaeplastida. We also describe the goals of our work with Paulinella, the challenges we are encountering and potential and future solutions to those challenges, which will make this amoeba a stronger model system.
LANDMARKS OF THE ARCHAEPLASTIDA PLASTID ENDOSYMBIOSISFeatures of endosymbiosis that lay the foundation for host-endosymbiont metabolic integration include genome reduction of the endosymbiont, transfer of many endosymbiont genes to the host nuclear genome via a process referred to as endosymbiotic gene transfer (EGT), and the origin of a protein translocation system to move nuclear-encoded, cytosol-translated, and organelle-localized proteins into the plastid, where they perform their functions (Martin et al. 2002, Timmis et al. 2004, Schleiff and Becker 2011). The necessity to achieve these complex innovations may explain why plastid primary endosymbiosis events are exceptionally rare and may ultimately not be sustained. Added to these developments, is the role of external players such as prokaryotic symbionts and phagocytosed prey that may contribute DNA to the host via horizontal gene transfer (HGT); this introduced DNA may play key roles in organelle development and integration of organelle and host metabolisms. The Archaeplastida endosymbiosis appears to have been facilitated by a major input (30–100 genes) from chlamydial pathogens that provided pathway components for carbohydrate, tryptophan, menaquinone, and isoprenoid biosynthesis (Huang and Gogarten 2007, Deschamps 2014, Rockwell et al. 2014, Ball et al. 2015).
Genome reduction has been noted for plastids and all long-term symbionts, and is believed to be driven by Mullers ratchet (Martin and Herrmann 1998, Pettersson and Berg 2007) which predicts a stepwise accumulation of DNA lesions in non-recombining genomes. This process would have occurred in the endosymbiotic cyanobacterium and resulted in the massive loss of coding material and EGT of hundreds of critical genes to the recombining “host” nuclear genome. The genome size of free-living cyanobacteria is anywhere between 1.6 and 12 Mbp, encoding ca. 1,800–12,000 proteins (Dufresne et al. 2003, Dagan et al. 2013). In contrast, Archaeplastida plastid genomes range in size from ca. 80–200 kbp and encode between 80 and 230 proteins (Ponce-Toledo et al. 2019). This size reduction impacts key metabolic processes including amino acid and lipid biosynthesis as well as photosynthesis. Consequently, plastids rely on their host cells to compensate for essential, lost functions, often of host or HGT origin, whose encoded proteins are delivered via the conserved translocons embedded in the outer and inner chloroplast (plastid) envelope membranes (TOC/TIC; Chan et al. 2011, Shi and Theg 2013, Paila et al. 2016). Protein import primarily relies on an N-terminal chloroplast transit peptide (cTPs) that serves as a tag for the import of proteins into the organelle (Bruce 2000). The estimated number of EGTs differs widely between Archaeplastida species Price et al. (2012). Dagan et al. (2013) estimated that 8.7%–11.5% of nuclear genes in photosynthetic eukaryotes sampled branched with cyanobacterial homologs with >80% bootstrap support, although the identification of these sequences may be challenging due to the existence of genomic rearrangements, mutations, and chimeric genes (e.g., symbiogenetic [S]-genes) that encode a protein domain of endosymbiont origin fused to another of non-endosymbiont provenance (Meheust et al. 2016). Moustafa and Bhattacharya (2008) estimated the number of cyanobacterium-derived EGTs to be 3.5–6% of the Chlamydomonas reinhardtii nuclear gene inventory. For plastids and their hosts to be metabolically integrated, a variety of metabolite transporters (e.g., carbohydrates, ions, ATP/ADP) evolved de novo, originating from EGT/HGT sequences, or were recruited from existing host cell membrane transporters that were retargeted to the organelle, allowing for the establishment of efficient crosstalk (Pfeil et al. 2014). Therefore, optimization of metabolic exchange between the endosymbiont and the Archaeplastida host was crucial to ensure survival and maximize fitness of the now chimeric cell.
THE PAULINELLA MODELPhotosynthetic Paulinella species are thecate filose amoebae that belong to the Cercozoa (Rhizaria). This monophyletic lineage transitioned from a heterotrophic to a phototrophic lifestyle by capturing and retaining a cyanobacterium (Marin et al. 2005, Yoon et al. 2009, Bhattacharya et al. 2012). First described in 1895 by Robert Lauterborn, P. chromatophora contains two sausage-shaped chromatophores inside an oval-shaped shell (theca) covered in silica scales (Fig. 1, A and B). These cells contain a “mouth” opening at the anterior end and have filopodia that are used for movement. Initially isolated from an old riverbed in Germany, P. chromatophora has been reported worldwide in shaded sediments of freshwater habitats with low pH (Nowack 2014). Moreover, recent studies identified other species of photosynthetic Paulinella, indicating that this monophyletic lineage split into (at least) two clades after chromatophore origin. The described species not only differ morphologically but also show genetic divergence (Lhee et al. 2017, 2019a). Currently, three photosynthetic Paulinella species (and several closely related strains) have been described: P. chromatophora, P. micropora (both of which are freshwater), and the marine P. longichromatophora. A phylogenetic tree inferred from analysis of 18S rDNA sequences (Bhattacharya et al. 2013) shows that species in this clade that lack a chromatophore, and live a heterotrophic lifestyle, are sisters to photosynthetic Paulinella.
Fig. 1.The photosynthetic amoeba Paulinella. (A) Light microscopy image of P. micropora J129 showing the blue-green sausage-shaped chromatophores inside an oval-shaped shell (theca) covered in silica scales. (B) Cell division stage showing newly formed test with a daughter cell (small cell) containing one parental chromatophore. The scale bar is 10 μm. (C) Nuclear small-subunit rRNA phylogeny of the Rhizaria (based on (A) in Bhattacharya et al. 2013) showing the interrelationships of heterotrophic and photosynthetic lineages. GenBank accession numbers are shown for some taxa. Paulinella micropora FK01 is a close relative of P. micropora KR01 and the position of P. longichromatophora is based on Lhee et al. (2017). Chromatophore genome sizes are shown for photosynthetic Paulinella species.
The data discussed above strongly argue for a single endosymbiosis event giving rise to the chromatophore and that photosynthetic Paulinella had a heterotrophic (phagotrophic) ancestry (Bhattacharya et al. 2012; Fig. 1C). These observations also suggest a plausible mechanism (i.e., cell engulfment as a food source) that led to the capture of the chromatophore ancestor in photosynthetic Paulinella lineages. Bayesian relaxed molecular clock analysis of chromatophore genome data suggests that photosynthetic Paulinella diverged (i.e., the likely time of chromatophore origin) from its sister species 90–140 million years ago (Nowack 2014, Delaye et al. 2016). The independent nature (from the primary endosymbiosis associated with the Archaeplastida) of the Paulinella plastid primary endosymbiosis is supported by various types of direct evidence, in addition to the amoeba phylogenetic data. Analysis of various chromatophore markers, including 16S rDNA, show the grouping of chromatophores with α-cyanobacteria (type 1A RuBisCO [α-carboxysomes]; Prochlorococcus-Synechococcus lineage) and a distant relationship to Archaeplastida plastids that were derived from a β-cyanobacterium which contain type 1B RuBisCO with β-carboxysomes (Marin et al. 2005, 2007, Yoon et al. 2009, Reyes-Prieto et al. 2010, Rae et al. 2013).
CHROMATOPHORE EVOLUTION AND EGTAlthough the Paulinella chromatophore retains key cyanobacterial features such as a bacterial peptidoglycan cell wall, carboxysomes (cellular compartments that house ribulose-1,5-bisphosphate carboxylase/oxygenase), and phycobilisomes (light harvesting antenna complexes), it also has morphological traits that are consistent with a photosynthetic organelle status such as its inability to survive outside of the host cell, its residence in the host cytoplasm, not being encapsulated within a vacuole, and the coordination of chromatophore number and division with host cell division (i.e., two chromatophores per mature host cell; Fig. 1). More convincing are the findings that the chromatophore genome is greatly reduced in size (~1 Mbp) and gene content (ca. 850 protein-coding genes) relative to free-living α-cyanobacteria (generally 2–5 Mbp, ca. ~3,000 protein-coding genes), genes encoding key enzymes in several biosynthetic pathways are absent on the chromatophore genome and therefore must be present on the host genome, and an import system has evolved to move proteins from the cytoplasm into the chromatophore (Nowack et al. 2008, 2016, Nowack and Grossman 2012, Nowack 2014, Singer et al. 2017, Lhee et al. 2019a). To date, the five complete chromatophore genome sequences that have been generated are from P. chromatophora (CCAC 0815), P. micropora (NZ27, KR01, and FK01), and P. longichromatophora (Lhee et al. 2017). All of these strains encode 42 tRNAs and 2 rRNA clusters. However, the P. chromatophora genome encodes the largest number of proteins at 878, compared to 830–860 for P. micropora strains and 867 for P. longichromatophora. In accordance, CCAC 0815 has the largest chromatophore genome size of 1.02 Mbp, whereas the genomes of chromatophores from the other Paulinella spp. range in size from 0.976 to 0.979 Mbp (Nowack et al. 2008, Lhee et al. 2017). Comparison of chromatophore genome structure among these three species shows the presence of six inversions that distinguish P. longichromatophora and P. chromatophora. Three inversions are found when comparing the genomes of P. longichromatophora and P. micropora. The chromatophore genome sequences of the P. micropora species KR01 and FK01 are 99.9% identical (Lhee et al. 2019a).
The genome of the free-living Synechococcus sp. WH5701 (genome size of ~3.0 Mbp, with 3,346 protein-coding genes) is approximately three times the size of the chromatophore genome of Paulinella chromatophore; the latter has retained only ~26% of the putative donor-encoded protein-coding capacity. All genes relating to the TCA cycle are missing from the chromatophore genome. Unlike Archaeplastida plastids, the chromatophore retains most photosynthetic genes with the exception of psaE and psaK. Moreover, most genes involved in chromatophore division are retained on the chromatophore genome. Current data indicate that ~ 40 gene families of α-cyanobacteria origin were transferred to the nuclear genome via EGT (Nowack et al. 2011, 2016, Nowack 2014). In the case of chromatophore encoded psaI, a subunit of photosystem I (PSI), one gene copy, which has an intron, also exists in the host genome. This finding shows that “gene duplications” associated with EGT allows time for the nuclear copy to evolve activity, leading ultimately to the loss of one copy, usually the organelle version. These results provide an interesting comparison to the Archaeplastida, which has also experienced massive plastid gene loss, but in this case, the plastid only retains ~80–200 genes, with ~600–1,000 genes relocated to the host nuclear genome (Moustafa and Bhattacharya 2008, Nowack and Weber 2018). This comparison highlights the putative “incomplete” state of chromatophore transition to a photosynthetic organelle and provides an indication (not proof) of the potential evolutionary trajectory of this organelle.
HOST-CHROMATOPHORE METABOLIC INTEGRATIONAs described above, chromatophore genome reduction resulted in the loss of genes involved in biosynthetic pathways, many of which are well conserved and essential to the survival of free-living cyanobacteria. These gene functions include the biosynthesis of amino acids (e.g., Glu, Arg, His, Try, and Met) and cofactors (e.g., NAD+, riboflavin, thiamine, biotin, cobalamine; Nowack et al. 2008). Genes for other metabolic pathways such as those required for peptidoglycan biosynthesis are partially retained in the chromatophore. Metabolic reconstruction using transcriptome data from Paulinella chromatophora shows that many of the genes that function in the same biosynthetic pathway are distributed between the host and chromatophore genomes. An example is provided by the biosynthetic pathway for arginine, which is primarily nuclear encoded, except for the argD and argE genes, which are on the chromatophore genome (Nowack et al. 2016). Moreover, pathways for metabolites that are missing from the chromatophore are most likely provided by the host, for example, the complete pathway for the biosynthesis of methionine is expressed from the host genome. These data not only suggest intricate host-endosymbiont metabolic integration but also demonstrate the need/utility of a protein import system to facilitate metabolite translocation.
CHROMATOPHORE PROTEIN IMPORTEvidence of transport of nuclear-encoded proteins into the chromatophore was initially established by studying the PsaE and PsaK polypeptides. These proteins are small PSI subunits whose encoding genes were relocated from the chromatophore genome to the host genome. Now synthesized in the cytosol, these proteins were shown to be localized in the chromatophore based on immunogold microscopy, to be present in isolated PSI particles. Lacking an N-terminal localization signal, these small proteins were also found to be enriched in the Golgi apparatus, suggesting a role for the secretory pathway in the import of small proteins into the photosynthetic organelle (Nowack and Grossman 2012).
Recently, Singer et al. (2017) have shown, based on chromatophore proteome analysis, that Paulinella chromatophora has evolved a protein targeting system for two classes of chromatophore-destined proteins. One class includes proteins that are >268 amino acids (aa) in length and contain a putative conserved N-terminal targeting signal of ~200 aa. Many of these proteins, as hypothesized previously, provide missing steps in chromatophore-encoded metabolic pathways or processes. There was found to be a chromatophore amino terminal targeting sequence on these proteins that was able to confer chloroplast localization in Nicotiana benthamiana, suggesting the presence of common features in these two independently derived organelle import signals. Interestingly, it has been proposed that Archaeplastida primary plastid and mitochondrial targeting signals were derived from eukaryotic antibacterial peptides (Wollman 2016). Singer et al. (2017) also found that the great majority of imported proteins are host (i.e., nuclear gene)-derived (of eukaryotic origin) or of unknown origin, suggesting that a major redeployment of Paulinella genetic resources was the primary means of organelle integration, in addition to HGT and EGT.
CHALLENGES AND FUTURE DIRECTIONAt present, photosynthetic Paulinella is the only known (and recent) case of primary plastid endosymbiosis. This makes the amoeba a valuable model to study the transformation of free-living cyanobacteria into a well-integrated photosynthetic compartment, and the transition of a “host” from a heterotrophic to a phototrophic lifestyle (summarized in Fig. 2). Although described 125 years ago, examining the complex evolutionary history and biology of this organism happened more recently with the establishment of clonal and axenic cultures. Moreover, the rapid development and progress made thus far with this model has been explained by advancements in genomics and computational biology. In fact, preliminary analysis of the first two draft genome assemblies of Paulinella (P. micropora strains KR01, MynA) have recently been made available and demonstrate the dynamic nature of genome evolution in this phototrophic lineage (Lhee et al. 2019b, Matsuo et al. 2019). However, these data only scratch the surface of the complex biology of these organisms. Many questions remain unanswered about this system, including: (i) What role does HGT play in plastid acquisition and integration and which host-derived genes are crucial to the maintenance of the organelle, particularly during the earliest phases of organellogenesis? (ii) Are there pre-adaptive genes that existed in the genomes of heterotrophic Paulinella that may have arisen through HGT and that facilitated survival and maintenance of the endocytosed α-cyanobacterium? (iii) Which host-derived genes are not chromatophore targeted but play a role in regulating plastid functions, such as division? (iv) What are the biophysical features of the chromatophore targeting signal that allow it to be recognized by the independently-derived Archaeplastida system? (v) What are key functionalities critical for establishing integration of host-endosymbiont metabolisms? It is clear that the rich toolkit of multi-omics methods should be applied to this model to more fully determine the tailoring of functions that accompany the establishment of a successful plastid. For example, metabolomic studies can provide insights into metabolic exchange, partitioning, and rewiring during the earlier phases of photosynthetic organelle establishment. DNA methylation, histone modification, and small RNA studies can shed light on the role of epigenetic regulation in endosymbiosis. Finally, functional studies using CRISPR/Cas9 editing would be ideal for validating the functions of key symbiosis related-genes, testing the impact of overexpressing stress response related-genes on cell growth and health, and understanding the function of novel genes expressed in response to highlight stress that might play a role in the evolution of light adaptation.
Fig 2.Key landmarks of plastid endosymbiosis in photosynthetic Paulinella. Bacterial internalization in phagotrophic Paulinella (step 1). This event is followed by endosymbiont genome reduction, whereby the chromatophore genome loses genes out-right or genes are transferred to the host nuclear genome through endosymbiotic gene transfer (EGT; steps 1–2). Translation occurs of chromatophore-destined gene products on cytosolic ribosomes and these are translocated into the organelle where they express their function (steps 2–3). Metabolic rewiring takes place as the crosstalk between the host and chromatophore is established, leading to endosymbiont integration (step 4).
Although omics methods are readily applied to most model organisms, they may prove challenging in the photosynthetic Paulinella. One major road-block that impedes progress with this amoeba is its extremely slow growth rate (doubling time of ca. 6–7 d), light sensitivity and its very large and repetitive genome. Many of the experiments described above rely on adequate biomass to obtain reliable data for downstream analysis. Whereas this was not a major issue for genomic and transcriptomics studies, it could prove challenging for proteomic studies and may be an obstacle for metabolomics. Advancements in single-cell methods (e.g., single-cell transcriptomics [scRNA-seq], metabolomics) will ultimately overcome these obstacles. However, these cutting-edge approaches will first need to be optimized for non-mammalian (or other model) systems such as Paulinella. Ultimately, the confluence of a remarkable biology, as exemplified by photosynthetic Paulinella species, and novel omics tools will allow us to reconstruct the history of plastid origin and integration with the prospect that these data will enable the development of stable, artificial symbioses in the near future.
Abbreviations:chloroplast transit peptide
endosymbiotic gene transfer
horizontal gene transfer
translocons embedded in the inner chloroplast envelope membranes
translocons embedded in the outer chloroplast envelope membranes
Arwa Gabr, School of Graduate Studies, Graduate Program in Molecular Bioscience and Program in Microbiology and Molecular Genetics, Rutgers University, New Brunswick, New Jersey 08901, USA.
Arthur R. Grossman, Department of Plant Biology, The Carnegie Institution for Science, Stanford, California 94305, USA
Debashish Bhattacharya, Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, New Jersey 08901, USA.
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