Phylogenomic analysis identifies red algal genes of endosymbiotic origin in the chromalveolates

Endosymbiosis has spread photosynthesis to many branches of the eukaryotic tree; however, the history of photosynthetic organelle (plastid) gain and loss remains controversial. Fortuitously, endosymbiosis may leave a genomic footprint through the transfer of endosymbiont genes to the "host" nucleus (endosymbiotic gene transfer, EGT). EGT can be detected through comparison of host genomes to uncover the history of past plastid acquisitions. Here we focus on a lineage of chlorophyll c-containing algae and protists ("chromalveolates") that are postulated to share a common red algal secondary endosymbiont. This plastid is originally of cyanobacterial origin through primary endosymbiosis and is closely related among the Plantae (i.e., red, green, and glaucophyte algae). To test these ideas, an automated phylogenomics pipeline was used with a novel unigene data set of 5,081 expressed sequence tags (ESTs) from the haptophyte alga Emiliania huxleyi and genome or EST data from other chromalveolates, red algae, plants, animals, fungi, and bacteria. We focused on nuclear-encoded proteins that are targeted to the plastid to express their function because this group of genes is expected to have phylogenies that are relatively easy to interpret. A total of 708 genes were identified in E. huxleyi that had a significant Blast hit to at least one other taxon in our data set. Forty-six of the alignments that were derived from the 708 genes contained at least one other chromalveolate (i.e., besides E. huxleyi), red and/or green algae (or land plants), and one or more cyanobacteria, whereas 15 alignments contained E. huxleyi, one or more other chromalveolates, and only cyanobacteria. Detailed phylogenetic analyses of these data sets turned up 19 cases of EGT that did not contain significant paralogy and had strong bootstrap support at the internal nodes, allowing us to confidently identify the source of the plastid-targeted gene in E. huxleyi. A total of 17 genes originated from the red algal lineage, whereas 2 genes were of green algal origin. Our data demonstrate the existence of multiple red algal genes that are shared among different chromalveolates, suggesting that at least a subset of this group may share a common origin.     

Evolution of the glucose-6-phosphate isomerase: the plasticity of primary metabolism in photosynthetic eukaryotes

Glucose-6-phosphate isomerase (GPI) has an essential function in both catabolic glycolysis and anabolic gluconeogenesis and is universally distributed among Eukaryotes, Bacteria, and some Archaea. In addition to the cytosolic GPI, land plant chloroplasts harbor a nuclear encoded isoenzyme of cyanobacterial origin that is indispensable for the oxidative pentose phosphate pathway (OPPP) and plastid starch accumulation. We established 12 new GPI sequences from rhodophytes, the glaucophyte Cyanophora paradoxa, a ciliate, and all orders of complex algae with red plastids (haptophytes, diatoms, cryptophytes, and dinoflagellates). Our comprehensive phylogenies do not support previous GPI-based speculations about a eukaryote-to-prokaryote horizontal gene transfer from metazoa to gamma-proteobacteria. The evolution of cytosolic GPI is largely in agreement with small subunit analyses, which indicates that it is a specific marker of the host cell. A distinct subtree comprising alveolates (ciliates, apicomplexa, Perkinsus, and dinoflagellates), stramenopiles (diatoms and Phytophthora [oomycete]), and Plantae (green plants, rhodophytes, and Cyanophora) might suggest a common origin of these superensembles. Finally, in contrast to land plants where the plastid GPI is of cyanobacterial origin, chlorophytes and rhodophytes independently recruited a duplicate of the cytosolic GPI that subsequently acquired a transit peptide for plastid import. A secondary loss of the cytosolic isoenzyme and the plastid localization of the single GPI in chlorophycean green algae is compatible with physiological studies. Our findings reveal the fundamental importance of the plastid OPPP for Plantae and document the plasticity of primary metabolism.     

An assessment of vertical inheritance versus endosymbiont transfer of nucleus-encoded genes for mitochondrial proteins following tertiary endosymbiosis in Karlodinium micrum

Most photosynthetic dinoflagellates harbour a red alga-derived secondary plastid. In the dinoflagellate Karlodinium micrum, this plastid was replaced by a subsequent endosymbiosis, resulting in a tertiary plastid derived from a haptophyte. Evolution of endosymbionts entails substantial relocation of endosymbiont genes to the host nucleus: a process called endosymbiotic gene transfer (EGT). In K. micrum, numerous plastid genes from the haptophyte nucleus are found in the host nucleus, providing evidence for EGT in this system. In other cases of endosymbiosis, notably ancient primary endosymbiotic events, EGT has been inferred to contribute to remodeling of other cell functions by expression of proteins in compartments other than the endosymbiont from which they derived. K. micrum provides a more recently derived endosymbiotic system to test for evidence of EGT and gain of function in non-plastid compartments. In this study, we test for gain of haptophyte-derived proteins for mitochondrial function in K. micrum. Using molecular phylogenies we have analysed whether nucleus-encoded mitochondrial proteins were inherited by EGT from the haptophyte endosymbiont, or vertically inherited from the dinoflagellate host lineage. From this dataset we found no evidence of haptophyte-derived mitochondrial genes, and the only cases of non-vertical inheritance were genes derived from lateral gene transfer events.     

Chimeric plastid proteome in the Florida "red tide" dinoflagellate Karenia brevis

Current understanding of the plastid proteome comes almost exclusively from studies of plants and red algae. The proteome in these taxa has a relatively simple origin via integration of proteins from a single cyanobacterial primary endosymbiont and the host. However, the most successful algae in marine environments are the chlorophyll c-containing chromalveolates such as diatoms and dinoflagellates that contain a plastid of red algal origin derived via secondary or tertiary endosymbiosis. Virtually nothing is known about the plastid proteome in these taxa. We analyzed expressed sequence tag data from the toxic "Florida red tide" dinoflagellate Karenia brevis that has undergone a tertiary plastid endosymbiosis. Comparative analyses identified 30 nuclear-encoded plastid-targeted proteins in this chromalveolate that originated via endosymbiotic or horizontal gene transfer (HGT) from multiple different sources. We identify a fundamental divide between plant/red algal and chromalveolate plastid proteomes that reflects a history of mixotrophy in the latter group resulting in a highly chimeric proteome. Loss of phagocytosis in the "red" and "green" clades effectively froze their proteomes, whereas chromalveolate lineages retain the ability to engulf prey allowing them to continually recruit new, potentially adaptive genes through subsequent endosymbioses and HGT. One of these genes is an electron transfer protein (plastocyanin) of green algal origin in K. brevis that likely allows this species to thrive under conditions of iron depletion.     

A first glimpse into the pattern and scale of gene transfer in Apicomplexa

Reports of plant-like and bacterial-like genes for a number of parasitic organisms, most notably those within the Apicomplexa and Kinetoplastida, have appeared in the literature over the last few years. Among the apicomplexan organisms, following discovery of the apicomplexan plastid (apicoplast), the discovery of plant-like genes was less surprising although the extent of transfer and the relationship of transferred genes to the apicoplast remained unclear. We used new genome sequence data to begin a systematic examination of the extent and origin of transferred genes in the Apicomplexa combined with a phylogenomic approach to detect potential gene transfers in four apicomplexan genomes. We have detected genes of algal nuclear, chloroplast (cyanobacterial) and proteobacterial origin. Plant-like genes were detected in species not currently harbouring a plastid (e.g. Cryptosporidium parvum) and putatively transferred genes were detected that appear to be unrelated to the function of the apicoplast. While the mechanism of acquisition for many of the identified genes is not certain, it appears that some were most likely acquired via intracellular gene transfer from an algal endosymbiont while others may have been acquired via horizontal gene transfer.     

Phylogeny of nuclear-encoded plastid-targeted proteins supports an early divergence of glaucophytes within Plantae

The phylogenetic position of the glaucophyte algae within the eukaryotic supergroup Plantae remains to be unambiguously established. Here, we assembled a multigene data set of conserved nuclear-encoded plastid-targeted proteins of cyanobacterial origin (i.e., through primary endosymbiotic gene transfer) from glaucophyte, red, and green (including land plants) algae to infer the branching order within this supergroup. We find strong support for the early divergence of glaucophytes within the Plantae, corroborating 2 important putatively ancestral characters shared by glaucophyte plastids and the cyanobacterial endosymbiont that gave rise to this organelle: the presence of a peptidoglycan deposition between the 2 organelle membranes and carboxysomes. Both these traits were apparently lost in the common ancestor of red and green algae after the divergence of glaucophytes.     

Expression of the nucleus-encoded chloroplast division genes and proteins regulated by the algal cell cycle

Chloroplasts have evolved from a cyanobacterial endosymbiont and their continuity has been maintained by chloroplast division, which is performed by the constriction of a ring-like division complex at the division site. It is believed that the synchronization of the endosymbiotic and host cell division events was a critical step in establishing a permanent endosymbiotic relationship, such as is commonly seen in existing algae. In the majority of algal species, chloroplasts divide once per specific period of the host cell division cycle. In order to understand both the regulation of the timing of chloroplast division in algal cells and how the system evolved, we examined the expression of chloroplast division genes and proteins in the cell cycle of algae containing chloroplasts of cyanobacterial primary endosymbiotic origin (glaucophyte, red, green, and streptophyte algae). The results show that the nucleus-encoded chloroplast division genes and proteins of both cyanobacterial and eukaryotic host origin are expressed specifically during the S phase, except for FtsZ in one graucophyte alga. In this glaucophyte alga, FtsZ is persistently expressed throughout the cell cycle, whereas the expression of the nucleus-encoded MinD and MinE as well as FtsZ ring formation are regulated by the phases of the cell cycle. In contrast to the nucleus-encoded division genes, it has been shown that the expression of chloroplast-encoded division genes is not regulated by the host cell cycle. The endosymbiotic gene transfer of minE and minD from the chloroplast to the nuclear genome occurred independently on multiple occasions in distinct lineages, whereas the expression of nucleus-encoded MIND and MINE is regulated by the cell cycle in all lineages examined in this study. These results suggest that the timing of chloroplast division in algal cell cycle is restricted by the cell cycle-regulated expression of some but not all of the chloroplast division genes. In addition, it is suggested that the regulation of each division-related gene was established shortly after the endosymbiotic gene transfer, and this event occurred multiple times independently in distinct genes and in distinct lineages.     

Host origin of plastid solute transporters in the first photosynthetic eukaryotes

Background  

It is generally accepted that a single primary endosymbiosis in the Plantae (red, green (including land plants), and glaucophyte algae) common ancestor gave rise to the ancestral photosynthetic organelle (plastid). Plastid establishment necessitated many steps, including the transfer and activation of endosymbiont genes that were relocated to the nuclear genome of the 'host' followed by import of the encoded proteins into the organelle. These innovations are, however, highly complex and could not have driven the initial formation of the endosymbiosis. We postulate that the re-targeting of existing host solute transporters to the plastid fore-runner was critical for the early success of the primary endosymbiosis, allowing the host to harvest endosymbiont primary production.     

Mosaic origin of the heme biosynthesis pathway in photosynthetic eukaryotes

Heme biosynthesis represents one of the most essential metabolic pathways in living organisms, providing the precursors for cytochrome prosthetic groups, photosynthetic pigments, and vitamin B(12). Using genomic data, we have compared the heme pathway in the diatom Thalassiosira pseudonana and the red alga Cyanidioschyzon merolae to those of green algae and higher plants, as well as to those of heterotrophic eukaryotes (fungi, apicomplexans, and animals). Phylogenetic analyses showed the mosaic character of this pathway in photosynthetic eukaryotes. Although most of the algal and plant enzymes showed the expected plastid (cyanobacterial) origin, at least one of them (porphobilinogen deaminase) appears to have a mitochondrial (alpha-proteobacterial) origin. Another enzyme, glutamyl-tRNA synthase, obviously originated in the eukaryotic nucleus. Because all the plastid-targeted sequences consistently form a well-supported cluster, this suggests that genes were either transferred from the primary endosymbiont (cyanobacteria) to the primary host nucleus shortly after the primary endosymbiotic event or replaced with genes from other sources at an equally early time, i.e., before the formation of three primary plastid lineages. The one striking exception to this pattern is ferrochelatase, the enzyme catalyzing the first committed step to heme and bilin pigments. In this case, two red algal sequences do not cluster either with the other plastid sequences or with cyanobacterial sequences and appear to have a proteobacterial origin like that of the apicomplexan parasites Plasmodium and Toxoplasma. Although the heterokonts also acquired their plastid via secondary endosymbiosis from a red alga, the diatom has a typical plastid-cyanobacterial ferrochelatase. We have not found any remnants of the plastidlike heme pathway in the nonphotosynthetic heterokonts Phytophthora ramorum and Phytophthora sojae.     

Genes of cyanobacterial origin in plant nuclear genomes point to a heterocyst-forming plastid ancestor

Plastids are descended from a cyanobacterial symbiosis which occurred over 1.2 billion years ago. During the course of endosymbiosis, most genes were lost from the cyanobacterium's genome and many were relocated to the host nucleus through endosymbiotic gene transfer (EGT). The issue of how many genes were acquired through EGT in different plant lineages is unresolved. Here, we report the genome-wide frequency of gene acquisitions from cyanobacteria in 4 photosynthetic eukaryotes--Arabidopsis, rice, Chlamydomonas, and the red alga Cyanidioschyzon--by comparison of the 83,138 proteins encoded in their genomes with 851,607 proteins encoded in 9 sequenced cyanobacterial genomes, 215 other reference prokaryotic genomes, and 13 reference eukaryotic genomes. The analyses entail 11,569 phylogenies inferred with both maximum likelihood and Neighbor-Joining approaches. Because each phylogenetic result is dependent not only upon the reconstruction method but also upon the site patterns in the underlying alignment, we investigated how the reliability of site pattern generation via alignment affects our results: if the site patterns in an alignment differ depending upon the order in which amino acids are introduced into multiple sequence alignment--N- to C-terminal versus C- to N-terminal--then the phylogenetic result is likely to be artifactual. Excluding unreliable alignments by this means, we obtain a conservative estimate, wherein about 14% of the proteins examined in each plant genome indicate a cyanobacterial origin for the corresponding nuclear gene, with higher proportions (17-25%) observed among the more reliable alignments. The identification of cyanobacterial genes in plant genomes affords access to an important question: from which type of cyanobacterium did the ancestor of plastids arise? Among the 9 cyanobacterial genomes sampled, Nostoc sp. PCC7120 and Anabaena variabilis ATCC29143 were found to harbor collections of genes which are-in terms of presence/absence and sequence similarity-more like those possessed by the plastid ancestor than those of the other 7 cyanobacterial genomes sampled here. This suggests that the ancestor of plastids might have been an organism more similar to filamentous, heterocyst-forming (nitrogen-fixing) representatives of section IV recognized in Stanier's cyanobacterial classification. Members of section IV are very common partners in contemporary symbiotic associations involving endosymbiotic cyanobacteria, which generally provide nitrogen to their host, consistent with suggestions that fixed nitrogen supplied by the endosymbiont might have played an important role during the origin of plastids.     

Translocation of proteins across the multiple membranes of complex plastids.

Secondary endosymbiosis describes the origin of plastids in several major algal groups such as dinoflagellates, euglenoids, heterokonts, haptophytes, cryptomonads, chlorarachniophytes and parasites such as apicomplexa. An integral part of secondary endosymbiosis has been the transfer of genes for plastid proteins from the endosymbiont to the host nucleus. Targeting of the encoded proteins back to the plastid from their new site of synthesis in the host involves targeting across the multiple membranes surrounding these complex plastids. Although this process shows many overall similarities in the different algal groups, it is emerging that differences exist in the mechanisms adopted.     

Hypothesis: Gene‐rich plastid genomes in red algae may be an outcome of nuclear genome reduction

Red algae (Rhodophyta) putatively diverged from the eukaryote tree of life >1.2 billion years ago and are the source of plastids in the ecologically important diatoms, haptophytes, and dinoflagellates. In general, red algae contain the largest plastid gene inventory among all such organelles derived from primary, secondary, or additional rounds of endosymbiosis. In contrast, their nuclear gene inventory is reduced when compared to their putative sister lineage, the Viridiplantae, and other photosynthetic lineages. The latter is thought to have resulted from a phase of genome reduction that occurred in the stem lineage of Rhodophyta. A recent comparative analysis of a taxonomically broad collection of red algal and Viridiplantae plastid genomes demonstrates that the red algal ancestor encoded ~1.5× more plastid genes than Viridiplantae. This difference is primarily explained by more extensive endosymbiotic gene transfer (EGT) in the stem lineage of Viridiplantae, when compared to red algae. We postulate that limited EGT in Rhodophytes resulted from the countervailing force of ancient, and likely recurrent, nuclear genome reduction. In other words, the propensity for nuclear gene loss led to the retention of red algal plastid genes that would otherwise have undergone intracellular gene transfer to the nucleus. This hypothesis recognizes the primacy of nuclear genome evolution over that of plastids, which have no inherent control of their gene inventory and can change dramatically (e.g., secondarily non‐photosynthetic eukaryotes, dinoflagellates) in response to selection acting on the host lineage.     

Chlamydiae has contributed at least 55 genes to Plantae with predominantly plastid functions

Background

The photosynthetic organelle (plastid) originated via primary endosymbiosis in which a phagotrophic protist captured and harnessed a cyanobacterium. The plastid was inherited by the common ancestor of the red, green (including land plants), and glaucophyte algae (together, the Plantae). Despite the critical importance of primary plastid endosymbiosis, its ancient derivation has left behind very few “footprints” of early key events in organelle genesis.

Methodology/Principal Findings

To gain insights into this process, we conducted an in-depth phylogenomic analysis of genomic data (nuclear proteins) from 17 Plantae species to identify genes of a surprising provenance in these taxa, Chlamydiae bacteria. Previous studies show that Chlamydiae contributed many genes (at least 21 in one study) to Plantae that primarily have plastid functions and were postulated to have played a fundamental role in organelle evolution. Using our comprehensive approach, we identify at least 55 Chlamydiae-derived genes in algae and plants, of which 67% (37/55) are putatively plastid targeted and at least 3 have mitochondrial functions. The remainder of the proteins does not contain a bioinformatically predicted organelle import signal although one has an N-terminal extension in comparison to the Chlamydiae homolog. Our data suggest that environmental Chlamydiae were significant contributors to early Plantae genomes that extend beyond plastid metabolism. The chlamydial gene distribution and protein tree topologies provide evidence for both endosymbiotic gene transfer and a horizontal gene transfer ratchet driven by recurrent endoparasitism as explanations for gene origin.

Conclusions/Significance

Our findings paint a more complex picture of gene origin than can easily be explained by endosymbiotic gene transfer from an organelle-like point source. These data significantly extend the genomic impact of Chlamydiae on Plantae and show that about one-half (30/55) of the transferred genes are most closely related to sequences emanating from the genome of the only environmental isolate that is currently available. This strain, Candidatus Protochlamydia amoebophila UWE25 is an endosymbiont of Acanthamoeba and likely represents the type of endoparasite that contributed the genes to Plantae.     

Evidence of a chimeric genome in the cyanobacterial ancestor of plastids

Background

Horizontal gene transfer (HGT) is a vexing fact of life for microbial phylogeneticists. Given the substantial rates of HGT observed in modern-day bacterial chromosomes, it is envisaged that ancient prokaryotic genomes must have been similarly chimeric. But where can one find an ancient prokaryotic genome that has maintained its ancestral condition to address this issue? An excellent candidate is the cyanobacterial endosymbiont that was harnessed over a billion years ago by a heterotrophic protist, giving rise to the plastid. Genetic remnants of the endosymbiont are still preserved in plastids as a highly reduced chromosome encoding 54 – 264 genes. These data provide an ideal target to assess genome chimericism in an ancient cyanobacterial lineage.

Results

Here we demonstrate that the origin of the plastid-encoded gene cluster for menaquinone/phylloquinone biosynthesis in the extremophilic red algae Cyanidiales contradicts a cyanobacterial genealogy. These genes are relics of an ancestral cluster related to homologs in Chlorobi/Gammaproteobacteria that we hypothesize was established by HGT in the progenitor of plastids, thus providing a 'footprint' of genome chimericism in ancient cyanobacteria. In addition to menB, four components of the original gene cluster (menF, menD, menC, and menH) are now encoded in the nuclear genome of the majority of non-Cyanidiales algae and plants as the unique tetra-gene fusion named PHYLLO. These genes are monophyletic in Plantae and chromalveolates, indicating that loci introduced by HGT into the ancestral cyanobacterium were moved over time into the host nucleus.

Conclusion

Our study provides unambiguous evidence for the existence of genome chimericism in ancient cyanobacteria. In addition we show genes that originated via HGT in the cyanobacterial ancestor of the plastid made their way to the host nucleus via endosymbiotic gene transfer (EGT).

     

Conservative sorting in the muroplasts of Cyanophora paradoxa: a reevaluation based on the completed genome sequence

After primary endosymbiosis, massive gene transfer occurred from the genome of the cyanobacterial endosymbiont to the nucleus of the protist host cell. In parallel, a specific protein import apparatus arose for reimport of many, but not all products of the genes moved to the nuclear genome. Presequences evolved to allow recognition of plastid proteins at the envelope and their translocation to the stroma. However, plastids (and cyanobacteria) also comprise five other subcompartments. Protein sorting to the cyanobacterial thylakoid membrane, the thylakoid lumen, the inner envelope membrane, the periplasmic space, and the outer envelope membrane is achieved by prokaryotic protein translocases recognizing, e.g., signal sequences. The “conservative sorting” hypothesis postulates that these translocases remained functional in endosymbiotic organelles and obtained their passengers not only from imported proteins but also from proteins synthesized in organello. For proteins synthesized in the cytosol, a collaboration of the general import apparatus and the former prokaryotic translocase is necessary which is often reflected by the use of bipartite presequences, e.g., stroma targeting peptide and signal peptide. For plants, this concept has been experimentally proven and verified. The muroplasts from Cyanophora paradoxa, that have several features more in common with cyanobacteria than with plastids, were analyzed with the availability of the recently completed nuclear genome sequence. Interesting findings include the absence of the post-translational signal recognition particle pathway, dual Sec translocases in thylakoid and inner envelope membranes that are produced from a single set of genes, and a co-translational signal recognition pathway operating without a 4.5S RNA component.     

Transketolase from Cyanophora paradoxa: In Vitro Import into Cyanelles and Pea Chloroplasts and a Complex History of a Gene Often, But Not Always, Transferred in the Context of Secondary Endosymbiosis

ABSTRACT. The glaucocystophyte Cyanophora paradoxa is an obligatorily photoautotrophic biflagellated protist containing cyanelles, peculiar plastids surrounded by a peptidoglycan layer between their inner and outer envelope membranes. Although the 136-kb cyanelle genome surpasses higher plant chloroplast genomes in coding capacity by about 50 protein genes, these primitive plastids still have to import >2,000 polypeptides across their unique organelle wall. One such protein is transketolase, an essential enzyme of the Calvin cycle. We report the sequence of the pre-transketolase cDNA from C. paradoxa and in vitro import experiments of precursor polypeptides into cyanelles and into pea chloroplasts. The transit sequence clearly indicates the localization of the gene product to cyanelles and is more similar to the transit sequences of the plant homologues than to transit sequences of other cyanelle precursor polypeptides with the exception of a cyanelle consensus sequence at the N-terminus. The mature sequence reveals conservation of the thiamine pyrophosphate binding site. A neighbor-net planar graph suggests that Cyanophora , higher plants, and the photosynthetic protist Euglena gracilis acquired their nuclear-encoded transketolase genes via endosymbiotic gene transfer from the cyanobacterial ancestor of plastids; in the case of Euglena probably entailing two transfers, once from the plastid in the green algal lineage and once again in the secondary endosymbiosis underlying the origin of Euglena's plastids. By contrast, transketolase genes in some eukaryotes with secondary plastids of red algal origin, such as Thalassiosira pseudonana , have retained the pre-existing transketolase gene germane to their secondary host.     

Fates of Evolutionarily Distinct,Plastid‐type Glyceraldehyde 3‐phosphate Dehydrogenase Genes in Kareniacean Dinoflagellates

The ancestral kareniacean dinoflagellate has undergone tertiary endosymbiosis, in which the original plastid is replaced by a haptophyte endosymbiont. During this plastid replacement, the endosymbiont genes were most likely flowed into the host dinoflagellate genome (endosymbiotic gene transfer or EGT). Such EGT may have generated the redundancy of functionally homologous genes in the host genome—one has resided in the host genome prior to the haptophyte endosymbiosis, while the other transferred from the endosymbiont genome. However, it remains to be well understood how evolutionarily distinct but functionally homologous genes were dealt in the dinoflagellate genomes bearing haptophyte‐derived plastids. To model the gene evolution after EGT in plastid replacement, we here compared the characteristics of the two evolutionally distinct genes encoding plastid‐type glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) in Karenia brevis and K. mikimotoi bearing haptophyte‐derived tertiary plastids: “gapC1h” acquired from the haptophyte endosymbiont and “gapC1p” inherited from the ancestral dinoflagellate. Our experiments consistently and clearly demonstrated that, in the two species examined, the principal plastid‐type GAPDH is encoded by gapC1h rather than gapC1p. We here propose an evolutionary scheme resolving the EGT‐derived redundancy of genes involved in plastid function and maintenance in the nuclear genomes of dinoflagellates that have undergone plastid replacements. Although K. brevis and K. mikimotoi are closely related to each other, the statuses of the two evolutionarily distinct gapC1 genes in the two Karenia species correspond to different steps in the proposed scheme.     

Genome-based reconstruction of the protein import machinery in the secondary plastid of a chlorarachniophyte alga

Most plastid proteins are encoded by their nuclear genomes and need to be targeted across multiple envelope membranes. In vascular plants, the translocons at the outer and inner envelope membranes of chloroplasts (TOC and TIC, respectively) facilitate transport across the two plastid membranes. In contrast, several algal groups harbor more complex plastids, the so-called secondary plastids, which are surrounded by three or four membranes, but the plastid protein import machinery (in particular, how proteins cross the membrane corresponding to the secondary endosymbiont plasma membrane) remains unexplored in many of these algae. To reconstruct the putative protein import machinery of a secondary plastid, we used the chlorarachniophyte alga Bigelowiella natans, whose plastid is bounded by four membranes and still possesses a relict nucleus of a green algal endosymbiont (the nucleomorph) in the intermembrane space. We identified nine homologs of plant-like TOC/TIC components in the recently sequenced B. natans nuclear genome, adding to the two that remain in the nucleomorph genome (B. natans TOC75 [BnTOC75] and BnTIC20). All of these proteins were predicted to be localized to the plastid and might function in the inner two membranes. We also show that the homologs of a protein, Der1, that is known to mediate transport across the second membrane in the several lineages with secondary plastids of red algal origin is not associated with plastid protein targeting in B. natans. How plastid proteins cross this membrane remains a mystery, but it is clear that the protein transport machinery of chlorarachniophyte plastids differs from that of red algal secondary plastids.     

Proteomic analysis of the Cyanophora paradoxa muroplast provides clues on early events in plastid endosymbiosis

Glaucophytes represent the first lineage of photosynthetic eukaryotes of primary endosymbiotic origin that diverged after plastid establishment. The muroplast of Cyanophora paradoxa represents a primitive plastid that resembles its cyanobacterial ancestor in pigment composition and the presence of a peptidoglycan wall. To attain insights into the evolutionary history of cyanobiont integration and plastid development, it would thus be highly desirable to obtain knowledge on the composition of the glaucophyte plastid proteome. Here, we provide the first proteomic analysis of the muroplast of C. paradoxa. Mass spectrometric analysis of the muroplast proteome identified 510 proteins with high confidence. The protein repertoire of the muroplast revealed novel paths for reduced carbon flow and export to the cytosol through a sugar phosphate transporter of chlamydial origin. We propose that C. paradoxa possesses a primordial plastid mirroring the situation in the early protoalga.