The unique features of starch metabolism in red algae.

Red algae (Rhodophyceae) are photosynthetic eukaryotes that accumulate starch granules outside of their plastids. The starch granules from red algae (floridean starch) show structural similarities with higher plant starch granules but lack amylose. Recent studies have indicated that the extra-plastidic starch synthesis in red algae proceeds via a UDP glucose-selective alpha-glucan synthase, in analogy with the cytosolic pathway of glycogen synthesis in other eukaryotes. On the other hand, plastidic starch synthesis in green cells occurs selectively via ADP glucose in analogy with the pathway of glycogen synthesis in prokaryotes from which plastids have evolved. Given the emerging consensus of a monophyletic origin of plastids, it would appear that the capacity for starch synthesis selectively evolved from the alpha-glucan synthesizing machinery of the host ancestor and its endosymbiont in red algae and green algae, respectively. This implies the evolution of fundamentally different functional relationships between the different subcellular compartments with regard to photosynthetic carbon metabolism in these organisms. It is suggested that the biochemical and molecular elucidation of floridean starch synthesis may offer new insights into the metabolic strategies of photosynthetic eukaryotes.     

Did some red alga‐derived plastids evolve via kleptoplastidy? A hypothesis

The evolution of plastids has a complex and still unresolved history. These organelles originated from a cyanobacterium via primary endosymbiosis, resulting in three eukaryotic lineages: glaucophytes, red algae, and green plants. The red and green algal plastids then spread via eukaryote–eukaryote endosymbioses, known as secondary and tertiary symbioses, to numerous heterotrophic protist lineages. The number of these horizontal plastid transfers, especially in the case of red alga‐derived plastids, remains controversial. Some authors argue that the number of plastid origins should be minimal due to perceived difficulties in the transformation of a eukaryotic algal endosymbiont into a multimembrane plastid, but increasingly the available data contradict this argument. I suggest that obstacles in solving this dilemma result from the acceptance of a single evolutionary scenario for the endosymbiont‐to‐plastid transformation formulated by Cavalier‐Smith & Lee (1985). Herein I discuss data that challenge this evolutionary scenario. Moreover, I propose a new model for the origin of multimembrane plastids belonging to the red lineage and apply it to the dinoflagellate peridinin plastid. The new model has several general and practical implications, such as the requirement for a new definition of cell organelles and in the construction of chimeric organisms.     

Chromalveolates and the Evolution of Plastids by Secondary Endosymbiosis1

ABSTRACT. The establishment of a new plastid organelle by secondary endosymbiosis represents a series of events of massive complexity, and yet we know it has taken place multiple times because both green and red algae have been taken up by other eukaryotic lineages. Exactly how many times these events have succeeded, however, has been a matter of debate that significantly impacts how we view plastid evolution, protein targeting, and eukaryotic relationships. On the green side it is now largely accepted that two independent events led to plastids of euglenids and chlorarachniophytes. How many times red algae have been taken up is less clear, because there are many more lineages with red alga‐derived plastids (cryptomonads, haptophytes, heterokonts, dinoflagellates and apicomplexa) and the relationships between these lineages are less clear. Ten years ago, Cavalier‐Smith proposed that these plastids were all derived from a single endosymbiosis, an idea that was dubbed the chromalveolate hypothesis. No one observation has yet supported the chromalveolate hypothesis as a whole, but molecular data from plastid‐encoded and plastid‐targeted proteins have provided strong support for several components of the overall hypothesis, and evidence for cryptic plastids and new photosynthetic lineages (e.g. Chromera) have transformed our view of plastid distribution within the group. Collectively, these data are most easily reconciled with a single origin of the chromalveolate plastids, although the phylogeny of chromalveolate host lineages (and potentially Rhizaria) remain to be reconciled with this plastid data.     

PRIMARY AND SECONDARY ENDOSYMBIOSIS AND THE ORIGIN OF PLASTIDS

The theory of endosymbiosis describes the origin of plastids from cyanobacterial-like prokaryotes living within eukaryotic host cells. The endosymbionts are much reduced, but morphological, biochemical, and molecular studies provide clear evidence of a prokaryotic ancestry for plastids. There appears to have been a single (primary) endosymbiosis that produced plastids with two bounding membranes, such as those in green algae, plants, red algae, and glaucophytes. A subsequent round of endosymbioses, in which red or green algae were engulfed and retained by eukaryotic hosts, transferred photosynthesis into other eukaryotic lineages. These endosymbiotic plastid acquisitions from eukaryotic algae are referred to as secondary endosymbioses, and the resulting plastids classically have three or four bounding membranes. Secondary endosymbioses have been a potent factor in eukaryotic evolution, producing much of the modern diversity of life.     

The evolution of glycogen and starch metabolism in eukaryotes gives molecular clues to understand the establishment of plastid endosymbiosis

Solid semi-crystalline starch and hydrosoluble glycogen define two distinct physical states of the same type of storage polysaccharide. Appearance of semi-crystalline storage polysaccharides appears linked to the requirement of unicellular diazotrophic cyanobacteria to fuel nitrogenase and protect it from oxygen through respiration of vast amounts of stored carbon. Starch metabolism itself resulted from the merging of the bacterial and eukaryote pathways of storage polysaccharide metabolism after endosymbiosis of the plastid. This generated the three Archaeplastida lineages: the green algae and land plants (Chloroplastida), the red algae (Rhodophyceae), and the glaucophytes (Glaucophyta). Reconstruction of starch metabolism in the common ancestor of Archaeplastida suggests that polysaccharide synthesis was ancestrally cytosolic. In addition, the synthesis of cytosolic starch from the ADP-glucose exported from the cyanobacterial symbiont possibly defined the original metabolic flux by which the cyanobiont provided photosynthate to its host. Additional evidence supporting this scenario include the monophyletic origin of the major carbon translocators of the inner membrane of eukaryote plastids which are sisters to nucleotide-sugar transporters of the eukaryote endomembrane system. It also includes the extent of enzyme subfunctionalization that came as a consequence of the rewiring of this pathway to the chloroplasts in the green algae. Recent evidence suggests that, at the time of endosymbiosis, obligate intracellular energy parasites related to extant Chlamydia have donated important genes to the ancestral starch metabolism network.     

THE MESOZOIC RADIATION OF EUKARYOTIC ALGAE: THE PORTABLE PLASTID HYPOTHESIS1

Although all chloroplasts appear to have been derived from a common ancestor, a major schism occurred early in the evolution of eukaryotic algae that gave rise to red and green photoautotrophic lineages. In Paleozoic and earlier times, the fossil record suggests that oceanic eukaryotic phytoplankton were dominated by the green (chl b‐containing) algal line. However, following the end‐Permian extinction, a diverse group of eukaryotic phytoplankton evolved from secondary symbiotic associations in the red (chl c‐containing) line and subsequently rose to ecological prominence. In the contemporary oceans, red eukaryotic phytoplankton taxa continue to dominate marine pelagic food webs, whereas the green line is relegated to comparatively minor ecological and biogeochemical roles. To help elucidate why the oceans are not dominated by green taxa, we analyzed and compared whole plastid genomes in both the red and green lineages. Our results suggest that whereas all algal plastids retain a core set of genes, red plastids retain a complementary set of genes that potentially confer more capacity to autonomously express proteins regulating oxygenic photosynthetic and energy transduction pathways. We hypothesize that specific gene losses in the primary endosymbiotic green plastid reduced its portability for subsequent symbiotic associations. This corollary of the plastid “enslavement” hypothesis may have limited subsequent evolutionary advances in the green lineage while simultaneously providing a competitive advantage to the red lineage.     

The endosymbiotic origin,diversification and fate of plastids

Plastids and mitochondria each arose from a single endosymbiotic event and share many similarities in how they were reduced and integrated with their host. However, the subsequent evolution of the two organelles could hardly be more different: mitochondria are a stable fixture of eukaryotic cells that are neither lost nor shuffled between lineages, whereas plastid evolution has been a complex mix of movement, loss and replacement. Molecular data from the past decade have substantially untangled this complex history, and we now know that plastids are derived from a single endosymbiotic event in the ancestor of glaucophytes, red algae and green algae (including plants). The plastids of both red algae and green algae were subsequently transferred to other lineages by secondary endosymbiosis. Green algal plastids were taken up by euglenids and chlorarachniophytes, as well as one small group of dinoflagellates. Red algae appear to have been taken up only once, giving rise to a diverse group called chromalveolates. Additional layers of complexity come from plastid loss, which has happened at least once and probably many times, and replacement. Plastid loss is difficult to prove, and cryptic, non-photosynthetic plastids are being found in many non-photosynthetic lineages. In other cases, photosynthetic lineages are now understood to have evolved from ancestors with a plastid of different origin, so an ancestral plastid has been replaced with a new one. Such replacement has taken place in several dinoflagellates (by tertiary endosymbiosis with other chromalveolates or serial secondary endosymbiosis with a green alga), and apparently also in two rhizarian lineages: chlorarachniophytes and Paulinella (which appear to have evolved from chromalveolate ancestors). The many twists and turns of plastid evolution each represent major evolutionary transitions, and each offers a glimpse into how genomes evolve and how cells integrate through gene transfers and protein trafficking.     

Multiple gene phylogenies support the monophyly of cryptomonad and haptophyte host lineages

Cryptomonad algae acquired their plastids by the secondary endosymbiotic uptake of a eukaryotic red alga. Several other algal lineages acquired plastids through such an event [1], but cryptomonads are distinguished by the retention of a relic red algal nucleus, the nucleomorph [2]. The nucleomorph (and its absence in other lineages) can reveal a great deal about the process and history of endosymbiosis, but only if we know the relationship between cryptomonads and other algae, and this has been controversial. Several recent analyses have suggested a relationship between plastids of cryptomonads and some or all other red alga-containing lineages [3-6], but we must also know whether host nuclear genes mirror this relationship to determine the number of endosymbiotic events, and this has not been demonstrated. We have carried out an expressed sequence tag (EST) survey of the cryptomonad Guillardia theta. Phylogenetic analyses of 102 orthologous nucleus-encoded proteins (18,425 amino acid alignment positions) show a robust sister-group relationship between cryptomonads and the haptophyte algae, which also have a red secondary plastid. This relationship demonstrates that loss of nucleomorphs must have taken place in haptophytes independently of any other red alga-containing lineages and that the ancestor of both already contained a red algal endosymbiont.     

Diatom plastids: Secondary endocytobiosis, plastid genome and protein import

Plastids of diatoms and other chromophytic algae have four surrounding membranes. In contrast to plastids of green algae, higher plants and red algae chromophytic cells are thought to have evolved by secondary endocytobiosis, i.e. by uptake of a eukaryotic photosynthetic organism by a eukaryotic host cell. This review gives a brief summary of the current views about the origin of diatom plastids and discusses possible mechanisms the cells might employ to transport nucleus-encoded plastid proteins into these organelles.     

Plastids and protein targeting.

Plastids with two bounding membranes--as exemplified by red algae, green algae, plants, and glaucophytes--derive from primary endosymbiosis; a process involving engulfment and retention of a cyanobacterium by a phagotrophic eukaryote. Plastids with more than two bounding membranes (such as those of euglenoids, dinoflagellates, heterokonts, haptopytes, apicomplexa, cryptomonads, and chlorarachniophytes) probably arose by secondary endosymbiosis, in which a eukaryotic alga (itself the product of primary endosymbiosis) was engulfed and retained by a phagotroph. Secondary endosymbiosis transfers photosynthetic capacity into heterotrophic lineages, has apparently occurred numerous times, and has created several major eukaryotic lineages comprising upwards of 42,600 species. Plastids acquired by secondary endosymbiosis are sometimes referred to as "second-hand." Establishment of secondary endosymbioses has involved transfer of genes from the endosymbiont nucleus to the secondary host nucleus. Limited gene transfer could initially have served to stabilise the endosymbioses, but it is clear that the transfer process has been extensive, leading in many cases to the complete disappearance of the endosymbiont nucleus. One consequence of these gene transfers is that gene products required in the plastid must be targeted into the organelle across multiple membranes: at least three for stromal proteins in euglenoids and dinoflagellates, and across five membranes in the case of thylakoid lumen proteins in plastids with four bounding membranes. Evolution of such targeting mechanisms was obviously a key step in the successful establishment of each different secondary endosymbiosis. Analysis of targeted proteins in the various organisms now suggests that a similar system is used by each group. However, rather than interpreting this similarity as evidence of an homologous origin, I believe that targeting has evolved convergently by combining and recycling existing protein trafficking mechanisms already existing in the endosymbiont and host. Indeed, by analyzing the multiple motifs in targeting sequences of some genes it is possible to infer that they originated in the plastid genome, transferred from there into the primary host nucleus, and subsequently moved into the secondary host nucleus. Thus, each step of the targeting process in "second-hand" plastids recapitulates the gene's previous intracellular transfers.     

Tightly Constrained Genome Reduction and Relaxation of Purifying Selection during Secondary Plastid Endosymbiosis

Endosymbiosis, the establishment of a former free-living prokaryotic or eukaryotic cell as an organelle inside a host cell, can dramatically alter the genomic architecture of the endosymbiont. Plastids or chloroplasts, the light-harvesting organelle of photosynthetic eukaryotes, are excellent models to study this phenomenon because plastid origin has occurred multiple times in evolution. Here, we investigate the genomic signature of molecular processes acting through secondary plastid endosymbiosis—the origination of a new plastid from a free-living eukaryotic alga. We used phylogenetic comparative methods to study gene loss and changes in selective regimes on plastid genomes, focusing on green algae that have given rise to three independent lineages with secondary plastids (euglenophytes, chlorarachniophytes, and Lepidodinium). Our results show an overall increase in gene loss associated with secondary endosymbiosis, but this loss is tightly constrained by the retention of genes essential for plastid function. The data show that secondary plastids have experienced temporary relaxation of purifying selection during secondary endosymbiosis. However, this process is tightly constrained, with selection relaxed only relative to the background in primary plastids. Purifying selection remains strong in absolute terms even during the endosymbiosis events. Selection intensity rebounds to pre-endosymbiosis levels following endosymbiosis events, demonstrating the changes in selection efficiency during different origin phases of secondary plastids. Independent endosymbiosis events in the euglenophytes, chlorarachniophytes, and Lepidodinium differ in their degree of relaxation of selection, highlighting the different evolutionary contexts of these events. This study reveals the selection–drift interplay during secondary endosymbiosis and evolutionary parallels during organellogenesis.     

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.     

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.     

Cyanobacterial Genes Transmitted to the Nucleus Before Divergence of Red Algae in the Chromista

The plastids of red algae, green plants, and glaucophytes may have originated directly from a cyanobacterium-like prokaryote via primary endosymbiosis. In contrast, the plastids of other lineages of eukaryotic phototrophs appear to be the result of secondary or tertiary endosymbiotic events involving a phototrophic eukaryote and a eukaryotic host cell. Although phylogenetic analyses of multiple plastid genes from a wide range of eukaryotic lineages have been carried out, the phylogenetic positions of the secondary plastids of the Chromista (Heterokontophyta, Haptophyta and Cryptophyta) are ambiguous in a range of different analyses. This ambiguity may be the result of unusual substitutions or bias in the plastid genes established by the secondary endosymbiosis. In this study, we carried out phylogenetic analyses of five nuclear genes of cyanobacterial origin (6-phosphogluconate dehydrogenase [gnd], oxygen-evolving-enhancer [psbO], phosphoglycerate kinase [pgk], delta-aminolevulinic acid dehydratase [aladh], and ATP synthase gamma [atpC] genes), using the genome sequence data from the primitive red alga Cyanidioschyzon merolae 10D. The sequence data robustly resolved the origin of the cyanobacterial genes in the nuclei of the Chromista (Heterokontophyta and Haptophyta) and Dinophyta, before the divergence of the extant red algae (including Porphyra [Rhodophyceae] and Cyanidioschyzon [Cyadidiophyceae]). Although it is likely that gnd genes in the Chromista were transmitted from the cyanobacterium-like ancestor of plastids in the primary endosymbiosis, other genes might have been transferred from nuclei of a red algal ancestor in the secondary endosymbiosis. Therefore, the results indicate that the Chromista might have originated from the ancient secondary endosymbiosis before the divergence of extant red algae.     

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.     

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.     

Gene replacement of fructose-1,6-bisphosphate aldolase supports the hypothesis of a single photosynthetic ancestor of chromalveolates

Plastids (photosynthetic organelles of plants and algae) are known to have spread between eukaryotic lineages by secondary endosymbiosis, that is, by the uptake of a eukaryotic alga by another eukaryote. But the number of times this has taken place is controversial. This is particularly so in the case of eukaryotes with plastids derived from red algae, which are numerous and diverse. Despite their diversity, it has been suggested that all these eukaryotes share a recent common ancestor and that their plastids originated in a single endosymbiosis, the so-called "chromalveolate hypothesis." Here we describe a novel molecular character that supports the chromalveolate hypothesis. Fructose-1,6-bisphosphate aldolase (FBA) is a glycolytic and Calvin cycle enzyme that exists as two nonhomologous types, class I and class II. Red algal plastid-targeted FBA is a class I enzyme related to homologues from plants and green algae, and it would be predicted that the plastid-targeted FBA from algae with red algal secondary endosymbionts should be related to this class I enzyme. However, we show that plastid-targeted FBA of heterokonts, cryptomonads, haptophytes, and dinoflagellates (all photosynthetic chromalveolates) are class II plastid-targeted enzymes, completely unlike those of red algal plastids. The chromalveolate enzymes form a strongly supported group in FBA phylogeny, and their common possession of this unexpected plastid characteristic provides new evidence for their close relationship and a common origin for their plastids.     

Phylogeny of Plastids Based on Cladistic Analysis of Gene Loss Inferred from Complete Plastid Genome Sequences

Based on the recent hypothesis on the origin of eukaryotic phototrophs, red algae, green plants, and glaucophytes constitute the primary photosynthetic eukaryotes (whose plastids may have originated directly from a cyanobacterium-like prokaryote via primary endosymbiosis), whereas the plastids of other lineages of eukaryotic phototrophs appear to be the result of secondary or tertiary endosymbiotic events (involving a phototrophic eukaryote and a host cell). Although phylogenetic analyses using multiple plastid genes from a wide range of eukaryotic lineages have been carried out, some of the major phylogenetic relationships of plastids remain ambiguous or conflict between different phylogenetic methods used for nucleotide or amino acid substitutions. Therefore, an alternative methodology to infer the plastid phylogeny is needed. Here, we carried out a cladistic analysis of the loss of plastid genes after primary endosymbiosis using complete plastid genome sequences from a wide range of eukaryotic phototrophs. Since it is extremely unlikely that plastid genes are regained during plastid evolution, we used the irreversible Camin-Sokal model for our cladistic analysis of the loss of plastid genes. The cladistic analysis of the 274 plastid protein-coding genes resolved the 20 operational taxonomic units representing a wide range of eukaryotic lineages (including three secondary plastid-containing groups) into two large monophyletic groups with high bootstrap values: one corresponded to the red lineage and the other consisted of a large clade composed of the green lineage (green plants and Euglena) and the basal glaucophyte plastid. Although the sister relationship between the green lineage and the Glaucophyta was not resolved in recent phylogenetic studies using amino acid substitutions from multiple plastid genes, it is consistent with the rbcL gene phylogeny and with a recent phylogenetic study using multiple nuclear genes. In addition, our analysis robustly resolved the conflicting/ambiguous phylogenetic positions of secondary plastids in previous phylogenetic studies: the Euglena plastid was sister to the chlorophycean (Chlamydomonas) lineage, and the secondary plastids from the diatom (Odontiella) and cryptophyte (Guillardia) were monophyletic within the red lineage.     

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.