The chlorarachniophyte nucleomorph is supplemented with host cell nucleus-encoded histones.

In chlorarachniophytes, algae originating from secondary endosymbiosis, the complex plastids retained a nucleomorph, the vestigial nucleus of the green algal endosymbiont. The nucleomorph of Bigelowiella natans encodes several plastid-targeted proteins and hundreds of housekeeping proteins. However, many fundamental genes for the maintainance of this subcompartment are missing. In this issue of Molecular Microbiology, Hirakawa et al. (2011) demonstrate nuclear histone genes of dual evolutionary origin in B. natans and convincingly show the targeting of the corresponding proteins to nucleus and nucleomorph respectively. One of the ways through which the nuclear genome exerts control upon its endosymbiotic junior partner is revealed. Insights into the nature of bipartite targeting sequences directing the respective proteins into the periplastidal space (where the nucleomorph resides) are gained. Further, cell cycle-dependent, differential regulation is shown for both nuclear and nucleomorph histone genes.

Metabolic symbiosis and the birth of the plant kingdom.

Eukaryotic cells are composed of a variety of membrane-bound organelles that are thought to derive from symbiotic associations involving bacteria, archaea, or other eukaryotes. In addition to acquiring the plastid, all Archaeplastida and some of their endosymbiotic derivatives can be distinguished from other organisms by the fact that they accumulate starch, a semicrystalline-storage polysaccharide distantly related to glycogen and never found elsewhere. We now provide the first evidence for the existence of starch in a particular species of single-cell diazotrophic cyanobacterium. We provide evidence for the existence in the eukaryotic host cell at the time of primary endosymbiosis of an uridine diphosphoglucose (UDP-glucose)-based pathway similar to that characterized in amoebas. Because of the monophyletic origin of plants, we can define the genetic makeup of the Archaeplastida ancestor with respect to storage polysaccharide metabolism. The most likely enzyme-partitioning scenario between the plastid's ancestor and its eukaryotic host immediately suggests the precise nature of the ancient metabolic symbiotic relationship. The latter consisted in the export of adenosine diphosphoglucose (ADP-glucose) from the cyanobiont in exchange for the import of reduced nitrogen from the host. We further speculate that the monophyletic origin of plastids may lie in an organism with close relatedness to present-day group V cyanobacteria.

Expression of nucleus-encoded genes for chloroplast proteins in the flagellate Euglena gracilis.

Reverse transcription PCRs (RT-PCRs), real-time RT-PCRs and microarrays containing 50-mer oligonucleotides representing nucleus-encoded genes for chloroplast proteins from Euglena gracilis were used to compare light- and dark-grown wild-type mRNA levels to those of light- and dark-grown E. gracilis stable white mutant strains W(gm)ZOflL, W3BUL and W10BSmL. The analyses revealed no light-dependent regulation of mRNA levels. Moreover, the mRNA levels of most genes were unchanged in all white mutants in comparison with wild-type. These results suggest that mRNA levels of nucleus-encoded genes for chloroplast proteins in E. gracilis do not depend on either light or plastid function.

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.

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.

Pathway of cytosolic starch synthesis in the model glaucophyte Cyanophora paradoxa.

The nature of the cytoplasmic pathway of starch biosynthesis was investigated in the model glaucophyte Cyanophora paradoxa. The storage polysaccharide granules are shown to be composed of both amylose and amylopectin fractions, with a chain length distribution and crystalline organization similar to those of green algae and land plant starch. A preliminary characterization of the starch pathway demonstrates that Cyanophora paradoxa contains several UDP-glucose-utilizing soluble starch synthase activities related to those of the Rhodophyceae. In addition, Cyanophora paradoxa synthesizes amylose with a granule-bound starch synthase displaying a preference for UDP-glucose. A debranching enzyme of isoamylase specificity and multiple starch phosphorylases also are evidenced in the model glaucophyte. The picture emerging from our biochemical and molecular characterizations consists of the presence of a UDP-glucose-based pathway similar to that recently proposed for the red algae, the cryptophytes, and the alveolates. The correlative presence of isoamylase and starch among photosynthetic eukaryotes is discussed.

Evolutionary conservation of dual Sec translocases in the cyanelles of Cyanophora paradoxa.

BACKGROUND: Cyanelles, the peptidoglycan-armored plastids of glaucocystophytes, occupy a unique bridge position in between free-living cyanobacteria and chloroplasts. In some respects they side with cyanobacteria whereas other features are clearly shared with chloroplasts. The Sec translocase, an example for "conservative sorting" in the course of evolution, is found in the plasma membrane of all prokaryotes, in the thylakoid membrane of chloroplasts and in both these membrane types of cyanobacteria. RESULTS: In this paper we present evidence for a dual location of the Sec translocon in the thylakoid as well as inner envelope membranes of the cyanelles from Cyanophora paradoxa, i. e. conservative sorting sensu stricto. The prerequisite was the generation of specific antisera directed against cyanelle SecY that allowed immunodetection of the protein on SDS gels from both membrane types separated by sucrose density gradient floatation centrifugation. Immunoblotting of blue-native gels yielded positive but differential results for both the thylakoid and envelope Sec complexes, respectively. In addition, heterologous antisera directed against components of the Toc/Tic translocons and binding of a labeled precursor protein were used to discriminate between inner and outer envelope membranes. CONCLUSION: The envelope translocase can be envisaged as a prokaryotic feature missing in higher plant chloroplasts but retained in cyanelles, likely for protein transport to the periplasm. Candidate passengers are cytochrome c6 and enzymes of peptidoglycan metabolism. The minimal set of subunits of the Toc/Tic translocase of a primitive plastid is proposed.

Monophyly of primary photosynthetic eukaryotes: green plants, red algae, and glaucophytes.

Between 1 and 1.5 billion years ago, eukaryotic organisms acquired the ability to convert light into chemical energy through endosymbiosis with a Cyanobacterium (e.g.,). This event gave rise to "primary" plastids, which are present in green plants, red algae, and glaucophytes ("Plantae" sensu Cavalier-Smith). The widely accepted view that primary plastids arose only once implies two predictions: (1) all plastids form a monophyletic group, as do (2) primary photosynthetic eukaryotes. Nonetheless, unequivocal support for both predictions is lacking (e.g.,). In this report, we present two phylogenomic analyses, with 50 genes from 16 plastid and 15 cyanobacterial genomes and with 143 nuclear genes from 34 eukaryotic species, respectively. The nuclear dataset includes new sequences from glaucophytes, the less-studied group of primary photosynthetic eukaryotes. We find significant support for both predictions. Taken together, our analyses provide the first strong support for a single endosymbiotic event that gave rise to primary photosynthetic eukaryotes, the Plantae. Because our dataset does not cover the entire eukaryotic diversity (but only four of six major groups in), further testing of the monophyly of Plantae should include representatives from eukaryotic lineages for which currently insufficient sequence information is available.

A carboxysomal carbon-concentrating mechanism in the cyanelles of the 'coelacanth' of the algal world, Cyanophora paradoxa?

Cyanelles are the peculiar plastids of glaucocystophyte algae that retained a peptidoglycan wall from the ancestral cyanobacterial endosymbiont. All cyanobacteria and most algae possess an inorganic carbon-concentrating mechanism (CCM) that involves a microcompartment--carboxysomes in prokaryotes and pyrenoids in eukaryotes--harboring the bulk of cellular (plastidic) Rubisco. In the case of the living fossil, Cyanophora paradoxa, the existence of a CCM was a matter of debate. Microarray data revealing 142 CO(2)-responsive genes (induced or repressed through a shift from high to low CO(2) conditions), gas exchange measurements and measurements of photosynthetic affinity provided strong support for a CCM. We favor a recent hypothesis that glaucocystophyte cyanelles as the closest cousins to cyanobacteria among plastids contain 'eukaryotic carboxysomes': bicarbonate enrichment within cyanelles should be considerably higher than in chloroplasts with their pyrenoid-based CCM. Thus, the stress-bearing function of the peptidoglycan layer, the other unique heritage, would be indispensable. An isolation method for cyanelle 'carboxysomes' was developed and the protein components other than Rubisco analyzed by MS. Rubisco activase was identified and corroborated by western blotting. The well-established cyanelle in vitro import system allows to use them as 'honorary cyanobacteria': assembly processes of supramolecular structures as phycobilisomes and carboxysomes thus can be studied after import of nucleus-encoded precursor proteins and subsequent fractionation. Even minor components can easily be tracked and a surprisingly dynamic view is obtained. Labeled pre-activase was imported into isolated cyanelles and 30% of the mature protein was found to be incorporated into the carboxysome fraction. A final decision between carboxysome or pyrenoid must await the identification of cyanelle carbonic anhydrase and, especially, the demonstration of shell proteins.

The invariant phenylalanine of precursor proteins discloses the importance of Omp85 for protein translocation into cyanelles.

BACKGROUND: Today it is widely accepted that plastids are of cyanobacterial origin. During their evolutionary integration into the metabolic and regulatory networks of the host cell the engulfed cyanobacteria lost their independency. This process was paralleled by a massive gene transfer from symbiont to the host nucleus challenging the development of a retrograde protein translocation system to ensure plastid functionality. Such a system includes specific targeting signals of the proteins needed for the function of the plastid and membrane-bound machineries performing the transfer of these proteins across the envelope membranes. At present, most information on protein translocation is obtained by the analysis of land plants. However, the analysis of protein import into the primitive plastids of glaucocystophyte algae, revealed distinct features placing this system as a tool to understand the evolutionary development of translocation systems. Here, bacterial outer membrane proteins of the Omp85 family have recently been discussed as evolutionary seeds for the development of translocation systems. RESULTS: To further explore the initial mode of protein translocation, the observed phenylalanine dependence for protein translocation into glaucophyte plastids was pursued in detail. We document that indeed the phenylalanine has an impact on both, lipid binding and binding to proteoliposomes hosting an Omp85 homologue. Comparison to established import experiments, however, unveiled a major importance of the phenylalanine for recognition by Omp85. This finding is placed into the context of the evolutionary development of the plastid translocon. CONCLUSION: The phenylalanine in the N-terminal domain signs as a prerequisite for protein translocation across the outer membrane assisted by a "primitive" translocon. This amino acid appears to be optimized for specifically targeting the Omp85 protein without enforcing aggregation on the membrane surface. The phenylalanine has subsequently been lost in the transit sequence, but can be found at the C-terminal position of the translocating pore. Thereby, the current hypothesis of Omp85 being the prokaryotic contribution to the ancestral Toc translocon can be supported.

Protein import into cyanelles.

"Cyanelles" are peptidoglycan-armored plastids of glaucocystophyte algae with close morphological and biochemical resemblance to endosymbiotic cyanobacteria. Genome sequencing and phylogenetic analysis have placed cyanelles on the earliest branch of phototrophic eukaryotes after the singular primary endosymbiotic event, the closest relatives to cyanobacteria among extant plastids. This model is supported by similar mechanisms for the targeting of nucleus-encoded cyanelle and chloroplast precursor proteins whose genes were transferred to the nucleus after the endosymbiotic event. As in chloroplasts, a prokaryote-type Sec preprotein translocase is shown to operate in cyanelle thylakoid membranes.

Conservative sorting in a primitive plastid. The cyanelle of Cyanophora paradoxa.

Higher plant chloroplasts possess at least four different pathways for protein translocation across and protein integration into the thylakoid membranes. It is of interest with respect to plastid evolution, which pathways have been retained as a relic from the cyanobacterial ancestor ('conservative sorting'), which ones have been kept but modified, and which ones were developed at the organelle stage, i.e. are eukaryotic achievements as (largely) the Toc and Tic translocons for envelope import of cytosolic precursor proteins. In the absence of data on cyanobacterial protein translocation, the cyanelles of the glaucocystophyte alga Cyanophora paradoxa for which in vitro systems for protein import and intraorganellar sorting were elaborated can serve as a model: the cyanelles are surrounded by a peptidoglycan wall, their thylakoids are covered with phycobilisomes and the composition of their oxygen-evolving complex is another feature shared with cyanobacteria. We demonstrate the operation of the Sec and Tat pathways in cyanelles and show for the first time in vitro protein import across cyanobacteria-like thylakoid membranes and protease protection of the mature protein.

Cyanophora paradoxa genome elucidates origin of photosynthesis in algae and plants.

The primary endosymbiotic origin of the plastid in eukaryotes more than 1 billion years ago led to the evolution of algae and plants. We analyzed draft genome and transcriptome data from the basally diverging alga Cyanophora paradoxa and provide evidence for a single origin of the primary plastid in the eukaryote supergroup Plantae. C. paradoxa retains ancestral features of starch biosynthesis, fermentation, and plastid protein translocation common to plants and algae but lacks typical eukaryotic light-harvesting complex proteins. Traces of an ancient link to parasites such as Chlamydiae were found in the genomes of C. paradoxa and other Plantae. Apparently, Chlamydia-like bacteria donated genes that allow export of photosynthate from the plastid and its polymerization into storage polysaccharide in the cytosol.

A possible role for short introns in the acquisition of stroma-targeting peptides in the flagellate Euglena gracilis.

The chloroplasts of Euglena gracilis bounded by three membranes arose via secondary endosymbiosis of a green alga in a heterotrophic euglenozoan host. Many genes were transferred from symbiont to the host nucleus. A subset of Euglena nuclear genes of predominately symbiont, but also host, or other origin have obtained complex presequences required for chloroplast targeting. This study has revealed the presence of short introns (41-93 bp) either in the second half of presequence-encoding regions or shortly downstream of them in nine nucleus-encoded E. gracilis genes for chloroplast proteins (Eno29, GapA, PetA, PetF, PetJ, PsaF, PsbM, PsbO, and PsbW). In addition, the E. gracilis Pbgd gene contains two introns in the second half of presequence-encoding region and one at the border of presequence-mature peptide-encoding region. Ten of 12 introns present within presequence-encoding regions or shortly downstream of them identified in this study have typical eukaryotic GT/AG borders, are T-rich, 45-50 bp long, and pairwise sequence identities range from 27 to 61%. Thus single recombination events might have been mediated via these cis-spliced introns. A double crossing over between these cis-spliced introns and trans-spliced introns present in 5'-UTRs of Euglena nuclear genes is also likely to have occurred. Thus introns and exon-shuffling could have had an important role in the acquisition of chloroplast targeting signals in E. gracilis. The results are consistent with a late origin of photosynthetic euglenids.

Protein translocation into and within cyanelles (review).

The cyanelles of the glaucocystophyte alga Cyanophora paradoxa resemble endosymbiotic cyanobacteria in morphology, pigmentation and, especially, in the presence of a peptidoglycan wall situated between the inner and outer envelope membranes. However, it is now clear that cyanelles in fact are primitive plastids. Phylogenetic analyses of plastid, nuclear and mitochondrial genes support a single primary endosymbiotic event. In this scenario cyanelles and all other plastid types are derived from an ancestral photosynthetic organelle combining the high plastid gene content of the Porphyra purpurea rhodoplast and the peptidoglycan wall of glaucocystophyte cyanelles. This means that the import apparatus of all primary plastids should be homologous. Indeed, heterologous in vitro import can now be shown in both directions, provided a phenylalanine residue essential for cyanelle import is engineered into the N-terminal part of chloroplast transit peptides. The cyanelle and likely also the rhodoplast import apparatus can be envisaged as prototypes with a single receptor showing this requirement for N-terminal phenylalanine. In chloroplasts, multiple receptors with overlapping and less stringent specificities have evolved explaining the efficient heterologous import of native precursors from C. paradoxa. With respect to conservative sorting in cyanelles, both the Sec and Tat pathways could be demonstrated. Another cyanobacterial feature, the dual location of the Sec translocase in thylakoid and inner envelope membranes, is also unique to cyanelles. For the first time, protease protection of internalized lumenal proteins could be shown for cyanobacteria-like, phycobilisome-bearing thylakoid membranes after import into isolated cyanelles.

Homologous protein import machineries in chloroplasts and cyanelles.

The cyanelles of the glaucocystophyte alga Cyanophora paradoxa resemble endosymbiotic cyanobacteria, especially in the presence of a peptidoglycan wall between the inner and outer envelope membranes. However, it is now clear that cyanelles are in fact primitive plastids. Phylogenetic analyses of plastid, nuclear and mitochondrial genes support a single primary endosymbiotic event. In this scenario, cyanelles and all other plastid types are derived from an ancestral photosynthetic organelle combining the high gene content of rhodoplasts and the peptidoglycan wall of cyanelles. This means that the import apparatuses of all primary plastids, i.e. those from glaucocystophytes, red algae, green algae and higher plants, should be homologous. If this is the case, then transit sequences should be similar and heterologous import experiments feasible. Thus far, heterologous in vitro import has been shown in one direction only: precursors from C. paradoxa were imported into isolated pea or spinach chloroplasts. Cyanelle transit sequences differ from chloroplast stroma targeting peptides in containing in their N-terminal domain an invariant phenylalanine residue which is shown here to be crucial for import. In addition, we now demonstrate that heterologous precursors are readily imported into isolated cyanelles, provided that the essential phenylalanine residue is engineered into the N-terminal part of chloroplast transit peptides. The cyanelle and likely also the rhodoplast import apparatus can be envisaged as prototypes with a single receptor/channel showing this requirement for N-terminal phenylalanine. In chloroplasts, multiple receptors with overlapping and less stringent specificities have evolved, explaining the efficient heterologous import of native precursors from C. paradoxa.

Characterization of apcC, the nuclear gene for the phycobilisome core linker polypeptide L(c)(7.8) from the glaucocystophyte alga Cyanophora paradoxa. Import of the precursor into isolated cyanelles and integration of the mature protein into intact phycobilisomes.

Phycobilisomes are the complex and highly efficient light-harvesting antenna systems of cyanobacteria, glaucocystophyte algae and red algae. In the glaucocystophyte Cyanophora paradoxa, seven genes for (chromophoric) phycobilisome components are known thus far, which all reside on the cyanelle genome. Here, we report the sequence of apcC, specifying the precursor to the colorless polypeptide L(c)(7.8), the first core linker reported for a eukaryote. The precursor was efficiently imported in vitro into isolated cyanelles. Fractionation into thylakoid membranes and stroma and into intact phycobilisomes and soluble proteins, respectively, indicated a low but significant incorporation of the imported linker polypeptide into the phycobilisomes.

Chloroplast SecE: evidence for spontaneous insertion into the thylakoid membrane.

SecE, an essential component of the bacterial SecAYEG translocase, mediates protein translocation across the cytoplasmic membrane. In the thylakoid membranes of chloroplasts an SecE homologue, cpSecE, has recently been identified. In this report we show that insertion of cpSecE does not require stromal extract, indicating that signal recognition particle is not involved. Removal of nucleoside triphosphates has apparently no effect on the integration, again ruling out an involvement of SRP or its partner protein, FtsY. The use of well-known inhibitors of the Sec- and Tat pathways, sodium azide and nigericin, respectively, also had no influence on membrane insertion. The data presented here point towards cpSecE as another passenger of a wholly spontaneous import/insertion pathway in the thylakoids of chloroplasts.