THE SYMBIOTIC BIRTH AND SPREAD OF PLASTIDS: HOW MANY TIMES AND WHODUNIT?

I discuss the evidence for a single origin of primary plastids in the context of a paper in this issue challenging this view, and I review recent evidence concerning the number of secondary plastid endosymbioses and the controversy over whether the relic plastid of apicomplexans is of red or green algal origin. A broad consensus has developed that the plastids of green algae, red algae, and glaucophytes arose from the same primary, cyanobacterial endosymbiosis. Although the analyses in this issue by Stiller and colleagues firmly undermine one of many sources of data, gene content similarities among plastid genomes used to argue for a monophyletic origin of primary plastids, the overall evidence still clearly favors monophyly. Nonetheless, this issue should not be considered settled and new data should be sought from better sampling of cyanobacteria and glaucophytes, from sequenced nuclear genomes, and from careful analysis of such key features as the plastid import apparatus. With respect to the number of secondary plastid symbioses, it is completely unclear as to whether the secondary plastids of euglenophytes and chlorarachniophytes arose by the same or two different algal endosymbioses. Recent analyses of certain plastid and nuclear genes support the chromalveolate hypothesis of Cavalier-Smith, namely, that the plastids of heterokonts, haptophytes, cryptophytes, dinoflagellates, and apicomplexans all arose from a common endosymbiosis involving a red alga. However, another recent paper presents intriguing conflicting data on this score for one of these groups—apicomplexans—arguing instead that they acquired their plastids from green algae.     

Is Protein Import into Plastids with Four‐Membrane Envelopes Dependent on Two Toc Systems Operating in Tandem?

Abstract: Plastids with four‐membrane envelopes have evolved by several independent endosymbioses involving a eukaryotic alga as the endosymbiont and a protozoan predator as the host. It is assumed that their outermost membrane is derived from the phagosomal membrane of the host and that protein targeting to and across this membrane proceeds co‐translationally, including ER and the Golgi apparatus (e.g., chlorarachniophytes) or only ER (e.g., heterokonts). Since the two inner membranes (or the plastid envelope) of such a complex plastid are derived from the endosymbiont plastid, they are probably provided with Toc and Tic systems, enabling post‐translational passage of the imported proteins into the stroma. The third envelope membrane, or the periplastid one, originates from the endosymbiont plasmalemma, but what import apparatus operates in it remains enigmatic. Recently, Cavalier‐Smith (1999^[5]) has proposed that the Toc system, pre‐existing in the endosymbiont plastid, has been relocated to the periplastid membrane, and that plastids having four envelope membranes contain two Toc systems operating in tandem and requiring the same targeting sequence, i.e., the transit peptide. Although this model is parsimonious, it encounters several serious obstacles, the most serious one resulting from the complex biogenesis of Toc75 which forms a translocation pore. In contrast to most proteins targeted to the outer membrane of the plastid envelope, this protein carries a complex transit peptide, indicating that a successful integration of the Toc system into the periplastid membrane would have to be accompanied by relocation of the stromal processing peptidase to the endosymbiont cytosol. However, such a relocation would be catastrophic because this enzyme would cleave the transit peptide off all plastid‐destined proteins, thus disabling biogenesis of the plastid compartment. Considering these difficulties, I suggest that in periplastid membranes two Toc‐independent translocation apparatuses have evolved: a porin‐like channel in chlorarachniophytes and cryptophytes, and a vesicular pathway in heterokonts and haptophytes. Since simultaneous evolution of a new transport system in the periplastid membrane and in the phagosomal one would be complicated, it is argued that plastids with four‐membrane envelopes have evolved by replacement of plastids with three‐membrane envelopes. I suggest that during the first round of secondary endosymbioses (resulting in plastids surrounded by three membranes), myzocytotically‐engulfed eukaryotic alga developed a Golgi‐mediated targeting pathway which was added to the Toc/Tic‐based apparatus of the endosymbiont plastid. During the second round of secondary endosymbioses (resulting in plastids surrounded by four membranes), phagocytotically‐engulfed eukaryotic alga exploited the Golgi pathway of the original plastid, and a new translocation system had to originate only in the periplastid membrane, although its emergence probably resulted in modification of the import machinery pre‐existing in the endosymbiont plastid.     

Plastid genome sequence of the cryptophyte alga Rhodomonas salina CCMP1319: lateral transfer of putative DNA replication machinery and a test of chromist plastid phylogeny

Cryptophytes are a group of unicellular algae with chlorophyll c-containing plastids derived from the uptake of a secondary (i.e., eukaryotic) endosymbiont. Biochemical and molecular data indicate that cryptophyte plastids are derived from red algae, yet the question of whether or not cryptophytes acquired their red algal plastids independent of those in heterokont, haptophyte, and dinoflagellate algae is of long-standing debate. To better understand the origin and evolution of the cryptophyte plastid, we have sequenced the plastid genome of Rhodomonas salina CCMP1319: at 135,854 bp, it is the largest secondary plastid genome characterized thus far. It also possesses interesting features not seen in the distantly related cryptophyte Guillardia theta or in other red secondary plastids, including pseudogenes, introns, and a bacterial-derived gene for the tau/gamma subunit of DNA polymerase III (dnaX), the first time putative DNA replication machinery has been found encoded in any plastid genome. Phylogenetic analyses indicate that dnaX was acquired by lateral gene transfer (LGT) in an ancestor of Rhodomonas, most likely from a firmicute bacterium. A phylogenomic survey revealed no additional cases of LGT, beyond a noncyanobacterial type rpl36 gene similar to that recently characterized in other cryptophytes and haptophytes. Rigorous concatenated analysis of 45 proteins encoded in 15 complete plastid genomes produced trees in which the heterokont, haptophyte, and cryptophyte (i.e., chromist) plastids were monophyletic, and heterokonts and haptophytes were each other's closest relatives. However, statistical support for chromist monophyly disappears when amino acids are recoded according to their chemical properties in order to minimize the impact of composition bias, and a significant fraction of the concatenate appears consistent with a sister-group relationship between cryptophyte and haptophyte plastids.     

Symbiotic origin of a novel actin gene in the cryptophyte Pyrenomonas helgolandii

Cryptophytes are photosynthetic protists that have acquired their plastids through the secondary symbiotic uptake of a red alga. A remarkable feature of cryptophytes is that they maintain a reduced form of the red algal nucleus, the nucleomorph, between the second and third plastid membranes (periplastidial compartment; PC). The nucleomorph is thought to be a transition state in the evolution of secondary plastids, with this genome ultimately being lost when photosynthesis comes under full control of the "host" nucleus (e.g., as in heterokonts, haptophytes, and euglenophytes). Genes presently found in the nucleomorph seem to be restricted to those involved in its own maintenance and to that of the plastid; other genes were lost as the endosymbiont was progressively reduced to its present state. Surprisingly, we found that the cryptophyte Pyrenomonas helgolandii possesses a novel type of actin gene that originated from the nucleomorph genome of the symbiont. Our results demonstrate for the first time that secondary symbionts can contribute genes to the host lineage which are unrelated to plastid function. These genes are akin to the products of gene duplication or lateral transfer and provide a source of evolutionary novelty that can significantly increase the genetic diversity of the host lineage. We postulate that this may be a common phenomenon in algae containing secondary plastids that has yet to be fully appreciated due to a dearth of evolutionary studies of nuclear genes in these taxa.     

SYMBIOTIC ORIGIN OF A NOVEL ACTIN GENE IN THE CRYPTOPHYTE,PYRENOMONAS HELGOLANDII

Cryptophytes are photosynthetic protists that have acquired their plastids through the secondary symbiotic uptake of a red alga. A remarkable feature of cryptophytes is that they maintain a reduced form of the red algal nucleus, the nucleomorph, between the second and third plastid membranes (periplastidial compartment, PC). The nucleomorph is thought to be a transition state in the evolution of secondary plastids with this genome ultimately being lost (e.g., as in heterokonts, haptophytes, euglenophytes) when photosynthesis comes under full control of the “host” nucleus. For this to happen, all genes for plastid function must be transferred from the nucleomorph to the nucleus. In this regard, it is generally assumed that nucleomorph genes with functions unrelated to plastid or PC maintenance are lost. Surprisingly, we show here the existence of a novel type of actin gene in the host nucleus of the cryptophyte, Pyrenomonas helgolandii, that has originated from the nucleomorph genome of the symbiont. Our results demonstrate for the first time that secondary symbionts can contribute genes to the host lineage that are unrelated to plastid function. These genes are akin to the products of gene duplication and provide a source of evolutionary novelty that could significantly increase the genetic diversity of the host lineage. We postulate that this may be a common phenomenon in algae containing secondary plastids that has yet to be fully appreciated due to a dearth of evolutionary studies of nuclear genes in these taxa.     

Nucleus-encoded,plastid-targeted glyceraldehyde-3-phosphate dehydrogenase (GAPDH) indicates a single origin for chromalveolate plastids

Plastids (the photosynthetic organelles of plants and algae) originated through endosymbiosis between a cyanobacterium and a eukaryote and subsequently spread to other eukaryotes by secondary endosymbioses between two eukaryotes. Mounting evidence favors a single origin for plastids of apicomplexans, cryptophytes, dinoflagellates, haptophytes, and heterokonts (together with their nonphotosynthetic relatives, termed chromalveolates), but so far, no single molecular marker has been described that supports this common origin. One piece of evidence comes from plastid-targeted glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which originated by a gene duplication of the cytosolic form. However, no plastid GAPDH has been characterized from haptophytes, leaving an important piece of the puzzle missing. We have sequenced genes encoding cytosolic, mitochondrion-targeted, and plastid-targeted GAPDH proteins from a number of haptophytes and heterokonts and found haptophyte homologs that branch within a strongly supported clade of chromalveolate plastid-targeted genes, being more closely related to an apicomplexan homolog than was expected. The evolution of plastid-targeted GAPDH supports red algal ancestry of apicomplexan plastids and raises a number of questions about the importance of plastid loss and the possibility of cryptic plastids in nonphotosynthetic lineages such as ciliates.     

THE EVOLUTION OF RNA POLYMERASE II IN RED ALGAE

Cryptophytes are photosynthetic protists that have acquired their plastids through the secondary symbiotic uptake of a red alga. A remarkable feature of cryptophytes is that they maintain a reduced form of the red algal nucleus, the nucleomorph, between the second and third plastid membranes (periplastidial compartment, PC). The nucleomorph is thought to be a transition state in the evolution of secondary plastids with this genome ultimately being lost (e.g., as in heterokonts, haptophytes, euglenophytes) when photosynthesis comes under full control of the "host" nucleus. For this to happen, all genes for plastid function must be transferred from the nucleomorph to the nucleus. In this regard, it is generally assumed that nucleomorph genes with functions unrelated to plastid or PC maintenance are lost. Surprisingly, we show here the existence of a novel type of actin gene in the host nucleus of the cryptophyte, Pyrenomonas helgolandii , that has originated from the nucleomorph genome of the symbiont. Our results demonstrate for the first time that secondary symbionts can contribute genes to the host lineage that are unrelated to plastid function. These genes are akin to the products of gene duplication and provide a source of evolutionary novelty that could significantly increase the genetic diversity of the host lineage. We postulate that this may be a common phenomenon in algae containing secondary plastids that has yet to be fully appreciated due to a dearth of evolutionary studies of nuclear genes in these taxa.     

64 Nucleus-encoded, plastid-targeted glyceraldehyde-3-phosphate dehydrogenase (GAPDH) indicates a single origin for chromalveolate plastids

Plastids (the photosynthetic organelles of plants and algae) ultimately originated through an endosymbiosis between a cyanobacterium and a eukaryote. Subsequently, plastids spread to other eukaryotes by secondary endosymbioses that took place between a eukaryotic alga and a second eukaryote. Recently, evidence has mounted in favour of a single origin for plastids of apicomplexans, cryptophytes, dinoflagellates, haptophytes, and heterokonts (together with their non-photosynthetic relatives, collectively termed chromalveolates). As of yet, however, no single molecular marker has been described which supports a common origin for all of these plastids. One piece of the evidence for a single origin of chromalveolate plastids came from plastid-targeted glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which originated by a gene duplication of the cytosolic form. However, no plastid GAPDH has been characterized from haptophytes, leaving an important piece of the puzzle missing. We have sequenced genes encoding cytosolic, mitochondrial-targeted, and plastid-targeted GAPDH proteins from a number of haptophytes and heterokonts, and found the haptophyte homologues to branch within the strongly supported clade of chromalveolate plastid-targeted GAPDH genes. Interestingly, plastid-targeted GAPDH genes from the haptophytes were more closely related to apicomplexan genes than was expected. Overall, the evolution of plastid-targeted GAPDH reinforces other data for a red algal ancestry of apicomplexan plastids, and raises a number of questions about the importance of plastid loss and the possibility of cryptic plastids in non-photosynthetic lineages such as ciliates.     

PHYLOGENOMICS AND SECONDARY PLASTIDS: A LOOK BACK AND A LOOK AHEAD1

Despite their importance to evolution, ecology, and cell biology, eukaryotes that acquired plastids through secondary endosymbiosis remain poorly studied from a genomic standpoint. Chromalveolata, a eukaryotic supergroup proposed to have descended from a heterotrophic eukaryote that acquired a red algal plastid by secondary endosymbiosis, includes four major lineages (alveolates, cryptophytes, haptophytes, and heterokonts). The chromalveolates exhibit remarkable diversity of cellular organization, and the available data suggest that they exhibit equal diversity in their genome organization. One of the most obvious differences in cellular organization is the retention of a highly reduced red algal nucleus in cryptophytes (also known as cryptomonads), but there are other major differences among chromalveolate lineages, including the loss of photosynthesis in multiple lineages. Although the hypothesis of chromalveolate monophyly is appealing, there is limited support for the hypothesis from nuclear genes, and questions have even been raised about the monophyly of chromalveolate plastids. Evidence for the chromalveolate hypothesis from large‐scale nuclear data sets is reviewed, and alternative hypotheses are described. The potential for integrating information from chromalveolate genomics into functional genomics is described, emphasizing both the methodological challenges and the opportunities for future phylogenomic analyses of these groups.     

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.     

Chlorophyll c-containing plastid relationships based on analyses of a multigene data set with all four chromalveolate lineages

The chlorophyll c-containing algae comprise four major lineages: dinoflagellates, haptophytes, heterokonts, and cryptophytes. These four lineages have sometimes been grouped together based on their pigmentation, but cytological and rRNA data had suggested that they were not a monophyletic lineage. Some molecular data support monophyly of the plastids, while other plastid and host data suggest different relationships. It is uncontroversial that these groups have all acquired plastids from another eukaryote, probably from the red algal lineage, in a secondary endosymbiotic event, but the number and sequence of such event(s) remain controversial. Understanding chlorophyll c-containing plastid relationships is a first step towards determining the number of endosymbiotic events within the chromalveolates. We report here phylogenetic analyses using 10 plastid genes with representatives of all four chromalveolate lineages. This is the first organellar genome-scale analysis to include both haptophytes and dinoflagellates. Concatenated analyses support the monophyly of the chlorophyll c-containing plastids and suggest that cryptophyte plastids are the basal member of the chlorophyll c-containing plastid lineage. The gene psbA, which has at times been used for phylogenetic purposes, was found to differ from the other genes in its placement of the dinoflagellates and the haptophytes, and in its lack of support for monophyly of the green and red plastid lineages. Overall, the concatenated data are consistent with a single origin of chlorophyll c-containing plastids from red algae. However, these data cannot test several key hypothesis concerning chromalveolate host monophyly, and do not preclude the possibility of serial transfer of chlorophyll c-containing plastids among distantly related hosts.     

Review

Most photosynthetic dinoflagellates harbour the peridinin plastid. This plastid is surrounded by three membranes and its characteristic pigments are chlorophyll c and the carotenoid peridinin. The evolutionary origin of this peculiar plastid remains controversial and is hotly debated. On the recently published tree of concatenated plastid-encoded proteins, dinoflagellates emerge from within the Chromista (clade containing cryptophytes, heterokonts, and haptophytes) and cluster specifically with Heterokonta. These data inspired a new version of the ‘chromalveolate’ model, according to which the peridinin plastid evolved by ‘descent with modification’ from a heterokont-like plastid that had been acquired from a rhodophyte by an ancestral chromalveolate. However, this model of plastid evolution encounters serious obstacles. Firstly, the heterokont plastid is surrounded by four membranes, which means that the ancestral peridinin plastid must have lost one of these primary membranes. However, such a loss could be traumatic, because it could potentially disturb protein import into and/or within the plastid. Secondly, on the phylogenetic tree of Dinoflagellata and Heterokonta, the first to diverge are not plastid, but heterotrophic, aplastidal taxa. Thus, when accepting the single origin of the heterokont and peridinin plastids, we would have to postulate multiple plastid losses, but such a scenario is highly doubtful when the numerous non-photosynthetic functions of plastids and their existence in heterotrophic protists, including parasitic lineages, are considered. Taking these obstacles into account, we suggest an alternative interpretation of the concatenated tree of plastid-encoded proteins. According to our hypothesis, the peridinin plastid evolved from a heterokont alga through tertiary endosymbiosis.     

A HYPOTHESIS FOR PLASTID EVOLUTION IN CHROMALVEOLATES1

Four eukaryotic lineages, namely, haptophytes, alveolates, cryptophytes, and heterokonts, contain in most cases photosynthetic and nonphotosynthetic members—the photosynthetic ones with secondary plastids with chl c as the main photosynthetic pigment. These four photosynthetic lineages were grouped together on the basis of their pigmentation and called chromalveolates, which is usually understood to imply loss of plastids in the nonphotosynthetic members. Despite the ecological and economic importance of this group of organisms, the phylogenetic relationships among these algae are only partially understood, and the so‐called chromalveolate hypothesis is very controversial. This review evaluates the evidence for and against this grouping and summarizes the present understanding of chromalveolate evolution. We also describe a testable hypothesis that is intended to accommodate current knowledge based on plastid and nuclear genomic data, discuss the implications of this model, and comment on areas that require further examination.     

Phylogeny of Dinoflagellate Plastid Genes Recently Transferred to the Nucleus Supports a Common Ancestry with Red Algal Plastid Genes

It is generally accepted that peridinin-containing dinoflagellate plastids are derived from red alga, but whether they are secondary plastids equivalent to plastids of stramenopiles, haptophytes, or cryptophytes, or are tertiary plastids derived from one of the other secondary plastids, has not yet been completely resolved. As secondary plastids, plastid gene phylogeny should mirror that of nuclear genes, while incongruence in the two phylogenies should be anticipated if their origin was as tertiary plastids. We have analyzed the phylogeny of plastid-encoded genes from Lingulodinium as well as that of nuclear-encoded dinoflagellate homologues of plastid-encoded genes conserved in all other plastid genome sequences. Our analyses place the dinoflagellate, stramenopile, haptophyte, and cryptophyte plastids firmly in the red algal lineage, and in particular, the close relationship between stramenopile plastid genes and their dinoflagellate nuclear-encoded homologues is consistent with the hypothesis that red algal-type plastids have arisen only once in evolution.     

Heterotachy processes in rhodophyte-derived secondhand plastid genes: Implications for addressing the origin and evolution of dinoflagellate plastids

Serial transfer of plastids from one eukaryotic host to another is the key process involved in evolution of secondhand plastids. Such transfers drastically change the environment of the plastids and hence the selection regimes, presumably leading to changes over time in the characteristics of plastid gene evolution and to misleading phylogenetic inferences. About half of the dinoflagellate protists species are photosynthetic and unique in harboring a diversity of plastids acquired from a wide range of eukaryotic algae. They are therefore ideal for studying evolutionary processes of plastids gained through secondary and tertiary endosymbioses. In the light of these processes, we have evaluated the origin of 2 types of dinoflagellate plastids, containing the peridinin or 19'-hexanoyloxyfucoxanthin (19'-HNOF) pigments, by inferring the phylogeny using "covarion" evolutionary models allowing the pattern of among-site rate variation to change over time. Our investigations of genes from secondary and tertiary plastids derived from the rhodophyte plastid lineage clearly reveal "heterotachy" processes characterized as stationary covarion substitution patterns and changes in proportion of variable sites across sequences. Failure to accommodate covarion-like substitution patterns can have strong effects on the plastid tree topology. Importantly, multigene analyses performed with probabilistic methods using among-site rate and covarion models of evolution conflict with proposed single origin of the peridinin- and 19'-HNOF-containing plastids, suggesting that analysis of secondhand plastids can be hampered by convergence in the evolutionary signature of the plastid DNA sequences. Another type of sequence convergence was detected at protein level involving the psaA gene. Excluding the psaA sequence from a concatenated protein alignment grouped the peridinin plastid with haptophytes, congruent with all DNA trees. Altogether, taking account of complex processes involved in the evolution of dinoflagellate plastid sequences (both at the DNA and amino acid level), we demonstrate the difficulty of excluding independent, tertiary origin for both the peridinin and 19'-HNOF plastids involving engulfment of haptophyte-like algae. In addition, the refined topologies suggest the red algal order, Porphyridales, as the endosymbiont ancestor of the secondary plastids in cryptophytes, haptophytes, and heterokonts.     

Evolution of Protein Targeting into “Complex” Plastids: The “Secretory Transport Hypothesis”

Abstract: In algae different types of plastids are known, which vary in pigment content and ultrastructure, providing an opportunity to study their evolutionary origin. One interesting feature is the number of envelope membranes surrounding the plastids. Red algae, green algae and glaucophytes have plastids with two membranes. They are thought to originate from a primary endocytobiosis event, a process in which a prokaryotic cyanobacterium was engulfed by a eukaryotic host cell and transformed into a plastid. Several other algal groups, like euglenophytes and heterokont algae (diatoms, brown algae, etc.), have plastids with three or four surrounding membranes, respectively, probably reflecting the evolution of these organisms by so‐called secondary endocytobiosis, which is the uptake of a eukaryotic alga by a eukaryotic host cell. A prerequisite for the successful establishment of primary or secondary endocytobiosis must be the development of suitable protein targeting machineries to allow the transport of nucleus‐encoded plastid proteins across the various plastid envelope membranes. Here, we discuss the possible evolution of such protein transport systems. We propose that the secretory system of the respective host cell might have been the essential tool to establish protein transport into primary as well as into secondary plastids.     

After the primary endosymbiosis: an update on the chromalveolate hypothesis and the origins of algae with Chl <Emphasis Type="Italic">c</Emphasis>

The chromalveolate hypothesis proposed by Cavalier-Smith (J Euk Microbiol 46:347–366, 1999) suggested that all the algae with chlorophyll c (heterokonts, haptophytes, cryptophytes, and dinoflagellates), as well as the ciliates, apicomplexans, oomycetes, and other non-photosynthetic relatives, shared a common ancestor that acquired a chloroplast by secondary endosymbiosis of a red alga. Much of the evidence from plastid and nuclear genomes supports a red algal origin for plastids of the photosynthetic lineages, but the number of secondary endosymbioses and the number of plastid losses have not been resolved. The issue is complicated by the fact that nuclear genomes are mosaics of genes acquired over a very long time period, not only by vertical descent but also by endosymbiotic and horizontal gene transfer. Phylogenomic analysis of the available whole-genome data has suggested major alterations to our view of eukaryotic evolution, and given rise to alternative models. The next few years may see even more changes once a more representative collection of sequenced genomes becomes available.     

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.     

INTERMEDIATE FEATURES OF CYANELLE DIVISION OF CYANOPHORA PARADOXA (GLAUCOCYSTOPHYTA) BETWEEN CYANOBACTERIAL AND PLASTID DIVISION1

Cyanelles of glaucocystophytes may be the most primitive of the known plastids based on their peptidoglycan content and the sequence phylogeny of cyanelle DNA. In this study, EM observations have been made to characterize the cyanelle division of Cyanophora paradoxa Korshikov and to gain insights into the evolution of plastid division. Constriction of cyanelles involves ingrowth of the septum at the cleavage site with the inner envelope membrane invaginating at the leading edge and the outer envelope membrane invaginating behind the septum. This means the inner and outer envelope membranes do not constrict simultaneously as they do in plastid division in other plants. The septum and the cyanelle envelope became stained after a silver‐methenamine staining was applied for in situ detection of polysaccharides. Septum formation was inhibited by β‐lactams and vancomycin, which are potent inhibitors of bacterial peptidoglycan biosynthesis. These results suggest the presence of peptidoglycan at the septum and the cyanelle envelope. In dividing cyanelles, a single electron‐dense ring (cyanelle ring) was observed on the stromal face of the inner envelope membrane at the isthmus, but no ring‐like structures were detected on the outer envelope membrane. Thus a single, stromal cyanelle ring such as this is quite unique and also distinct from FtsZ rings, which are not detectable by TEM. These features suggest that the cyanelle division of glaucocystophytes represents an intermediate stage between cyanobacterial and plastid division. If monophyly of all plastids is true, the cyanelle ring and the homologous inner plastid dividing ring might have evolved earlier than the outer plastid dividing ring.