Secondary Plastids Research Articles

Internal plastid-targeting signal found in a RubisCO small subunit protein of a chlorarachniophyte alga

In all plants and algae, most plastid proteins are encoded by the nuclear genome and, consequently, need to be transported into plastids across multiple membranes. In organisms with secondary plastids, which evolved by secondary endosymbioses, and are surrounded by three or four envelope membranes, precursors of nuclear-encoded plastid proteins generally have an N-terminal bipartite targeting sequence that consists of an endoplasmic reticulum (ER)-targeting signal peptide (SP) and a transit peptide-like (TPL) sequence. The bipartite targeting sequences have been demonstrated to be necessary and sufficient for targeting proteins into the plastids of many algal groups, including chlorarachniophytes. Here, we report a new type of targeting signal that is required for delivering a RubisCO small subunit (RbcS) protein into the secondary plastids of chlorarachniophyte algae. In this study, we analyzed the plastid-targeting ability of an RbcS pre-protein, using green fluorescent protein (GFP) as a reporter molecule in chlorarachniophyte cells. We demonstrate that the N-terminal bipartite targeting sequence of the RbcS pre-protein is not sufficient, and that a part of the mature protein is also necessary for plastid targeting. By deletion analyses of amino acids, we determined the approximate location of an internal plastid-targeting signal within the mature protein, which is involved in targeting the protein from the ER into the chlorarachniophyte plastids.

Eukaryotic algae: where lies the diversity of oxygenic photosynthesis

Due to their fast growth, homogeneity as cell populations and easy handling, microalgae attracted plant biologists as laboratory organisms for the study of the metabolism and physiology of photosynthetic cells. This led, for example, to the extensive use of the green alga Chlamydomonas reinhardtii for studying photosynthesis, to such a degree that this alga was nicknamed the green yeast (e.g. Goodenough 1992). Reinforcing the dominant position of Chlamydomonas, the availability of its nuclear genome sequence (Merchant et al. 2007) made also possible the identification of a minimal set of proteins (designated the GreenCut) that were likely involved specifically in chloroplast function within the green lineage. Recent advances in approaching the functions of these proteins are highlighted in this special issue (Grossman et al. 2010). Many pioneering works and more recent findings on the regulation of photosynthetic electron transport arose from studies on eukaryotic microalgae, such as chlororespiration (Bennoun 1982) and cyclic electron flow (Iwai et al. 2010) in Chlamydomonas, or state transitions in the green alga Chlorella pyrenoidosa (Bonaventura and Meyers 1969). Recent developments concerned with state transitions and auxiliary electron transfer pathways are reviewed in this issue (Alric 2010; Lemeille and Rochaix 2010; Peltier et al. 2010). Oxygenic photosynthesis in eukaryotes is not restricted to terrestrial plants and plant-model algal systems (mainly green algae). Indeed photosynthesis in eukaryotic cell was acquired laterally through a primary endosymbiotic event with a cyanobacteria and this gave rise to plants, green algae, red algae and glaucophytes (e.g. Rodriguez-Ezpeleta et al. 2005). As examples, two contributions to this issue highlight the unique architecture of the photosynthetic apparatus in red algae (Neilson and Durnford 2010; Su et al. 2010). Photosynthesis then spread throughout different eukaryotic kingdoms laterally via secondary endosymbiosis, most commonly through the engulfment by a nonphotosynthetic host of a red alga or green alga, giving rise for example to diatoms and euglena, respectively (e.g. Archibald 2009). Among eukaryotic algae, diatoms play a considerable role in the primary productivity of oceans and thus in biogeochemical carbon cycle, comparable to that of cyanobacteria. The acquisition of these so-called secondary plastids also accounts for much of the photosynthetic diversity on the planet, i.e. it was associated with a variety of adaptation strategies involving the photosynthetic process. Some of these peculiarities are dealt with here in reviews on carotenoid biosynthesis in diatoms (Bertrand 2010), light-harvesting processes (Neilson and Durnford 2010), photoprotective mechanisms (Goss and Jakob 2010), and inorganic carbon acquisition (Raven 2010). At a time when human societies are facing major challenges in terms of climate control, renewable energy production, and nutrition of populations across the planet, the understanding of photosynthetic processes and their features in different groups of algae forms a basis for the development of algal biotechnology. The availability of suitable algal strains and the optimization of the mass P. Cardol (&) Laboratory of Genetics of Microorganisms, Department of Life Sciences, Institute of Plant Biology, B22, University of Liege, 27 Boulevard du Rectorat, 4000 Liege, Belgium e-mail: pierre.cardol@ulg.ac.be

Morphology and Ultrastructure of Multiple Life Cycle Stages of the Photosynthetic Relative of Apicomplexa, Chromera velia

Chromera veliais a photosynthetic alga with a secondary plastid that represents the closest known photosynthetic relative of the apicomplexan parasites. The original description of this organism was based on brownish, immotile coccoid cells, which is the predominating stage ofC. veliain the culture. Here we provide a detailed light and electron microscopy description of coccoid cells ofC. veliaand a previously undocumented bi-flagellated stage that is highly motile and moves in a characteristic zig-zag pattern. Transformation from a coccoid into a flagellate stage occurs in exponentially growing cultures, and is accelerated by exposure to light. TheC. veliacells contain a pseudoconoid, which is likely homologous to the corresponding structure in the apical complex of Apicomplexa, cortical alveoli subtended by subpellicular microtubules, mitochondrion with tubular cristae, a micropyle, and a distinctive chromerosome, an apparently novel type of extrusion organelle. Ultrastructural analysis of the flagellate supports its close association with colpodellids and apicomplexans and provides important insight into their evolution.

A phylogenetic mosaic plastid proteome and unusual plastid-targeting signals in the green-colored dinoflagellate Lepidodinium chlorophorum

BackgroundPlastid replacements through secondary endosymbioses include massive transfer of genes from the endosymbiont to the host nucleus and require a new targeting system to enable transport of the plastid-targeted proteins across 3-4 plastid membranes. The dinoflagellates are the only eukaryotic lineage that has been shown to have undergone several plastid replacement events, and this group is thus highly relevant for studying the processes involved in plastid evolution. In this study, we analyzed the phylogenetic origin and N-terminal extensions of plastid-targeted proteins from Lepidodinium chlorophorum, a member of the only dinoflagellate genus that harbors a green secondary plastid rather than the red algal-derived, peridinin-containing plastid usually found in photosynthetic dinoflagellates.ResultsWe sequenced 4,746 randomly picked clones from a L. chlorophorum cDNA library. 22 of the assembled genes were identified as genes encoding proteins functioning in plastids. Some of these were of green algal origin. This confirms that genes have been transferred from the plastid to the host nucleus of L. chlorophorum and indicates that the plastid is fully integrated as an organelle in the host. Other nuclear-encoded plastid-targeted protein genes, however, are clearly not of green algal origin, but have been derived from a number of different algal groups, including dinoflagellates, streptophytes, heterokonts, and red algae. The characteristics of N-terminal plastid-targeting peptides of all of these genes are substantially different from those found in peridinin-containing dinoflagellates and green algae.ConclusionsL. chlorophorum expresses plastid-targeted proteins with a range of different origins, which probably arose through endosymbiotic gene transfer (EGT) and horizontal gene transfer (HGT). The N-terminal extension of the genes is different from the extensions found in green alga and other dinoflagellates (peridinin- and haptophyte plastids). These modifications have likely enabled the mosaic proteome of L. chlorophorum.

The acquisition of phototrophy: adaptive strategies of hosting endosymbionts and organelles

Many non-photosynthetic species of protists and metazoans are capable of hosting viable algal endosymbionts or their organelles through adaptations of phagocytic pathways. A form of mixotrophy combining phototrophy and heterotrophy, acquired phototrophy (AcPh) encompasses a suite of endosymbiotic and organelle retention interactions, that range from facultative to obligate. AcPh is a common phenomenon in aquatic ecosystems, with endosymbiotic associations generally more prevalent in nutrient poor environments, and organelle retention typically associated with more productive ones. All AcPhs benefit from enhanced growth due to access to photosynthetic products; however, the degree of metabolic integration and dependency in the host varies widely. AcPh is found in at least four of the major eukaryotic supergroups, and is the driving force in the evolution of secondary and tertiary plastid acquisitions. Mutualistic resource partitioning characterizes most algal endosymbiotic interactions, while organelle retention is a form of predation, characterized by nutrient flow (i.e., growth) in one direction. AcPh involves adaptations to recognize specific prey or endosymbionts and to house organelles or endosymbionts within the endomembrane system but free from digestion. In many cases, hosts depend upon AcPh for the production of essential nutrients, many of which remain obscure. The practice of AcPh has led to multiple independent secondary and tertiary plastid acquisition events among several eukaryote lineages, giving rise to the diverse array of algae found in modern aquatic ecosystems. This article highlights those AcPhs that are model research organisms for both metazoans and protists. Much of the basic biology of AcPhs remains enigmatic, particularly (1) which essential nutrients or factors make certain forms of AcPh obligatory, (2) how hosts regulate and manipulate endosymbionts or sequestered organelles, and (3) what genomic imprint, if any, AcPh leaves on non-photosynthetic host species.

Did Trypanosomatid Parasites Contain a Eukaryotic Alga–Derived Plastid in Their Evolutionary Past?

The Trypanosomatidae is closely related to euglenids that harbor plastids acquired from a green alga via secondary endosymbiosis. This discovery led to the idea that trypanosomatid parasites contained a green alga-derived plastid in their evolutionary past, an evolutionary scenario that was criticized based on the rarity of plant/plastid/cyanobacterium-like genes in the completely sequenced genomes of Trypanosoma and Leishmania species. Because it is difficult to identify such genes, however, their apparent rarity does not preclude a previous plastid endosymbiosis in the Trypanosomatidae. The genome of the plastid-less apicomplexan Cryptosporidium parvum preserves only a handful of plant/plastid/cyanobacterium-like genes, suggesting massive loss of plastid genes after elimination of its plastid. Additional support for such wholesale gene loss comes from fucoxanthin-containing dinoflagellates. Trypanosomatid nuclear genomes contain cyanobacterium-, green plant-, and haptophyte alga-derived genes, suggesting that they could have possessed a plastid in their evolutionary past; however, these genes also could represent examples of more typical horizontal gene transfer that did not accompany a plastid endosymbiosis. Thus, the presence of host cell genes that were adapted for use in the plastid would be much stronger evidence for a past plastid endosymbiosis in the Trypanosomatidae. Good examples of such genes are those encoding superoxide dismutases (SODs). Trypanosomatid parasites possess 4 iron-containing SODs, with 2 of them, SODA and SODC, targeted to the mitochondrion. In contrast with SODAs with classical single-domain mitochondrial targeting signals, SODCs carry bipartite pre-sequences composed of a signal peptide, followed by a transit peptide. Interestingly, these N-terminal extensions show striking similarities in length, hydropathy profiles, amino acid composition, and targeting properties to pre-sequences of proteins targeted to eukaryotic alga-derived plastids of euglenids and dinoflagellates. In turn, phylogenetic analyses indicate that SODCs originated from a mitochondrion-targeted SOD via gene duplication and were inherited vertically in the trypanosomatid lineage. These data represent a new kind of evidence for a past plastid endosymbiosis in the Trypanosomatidae, but the nature of this plastid remains unclear. It is usually assumed that the trypanosomatid plastid shared a common origin with that of euglenids, but Delta 4 desaturase phylogenies suggest that it could have originated via an independent, tertiary endosymbiosis involving a haptophyte alga. It is also possible that ancestors of the Trypanosomatidae initially possessed a primary plastid that later was replaced by a secondary or tertiary plastid.

Characterization of Periplastidal Compartment–Targeting Signals in Chlorarachniophytes

Secondary plastids are acquired by the engulfment and retention of eukaryotic algae, which results in an additional surrounding membrane or pair of membranes relative to the more familiar primary plastids of land plants. In most cases, the endocytosed alga loses its eukaryotic genome as it becomes integrated, but in two algal groups, the cryptophytes and chlorarachniophytes, the secondary plastids retain a vestigial nucleus in the periplastidal compartment (PPC), the remnant eukaryotic cytoplasm between the inner and the outer membrane pairs. Many essential housekeeping genes are missing from these reduced genomes, suggesting that they are now encoded in the host nucleus and their products are targeted to the PPC. One such nucleus-encoded, PPC-targeted protein, the translation elongation factor like (EFL) was recently identified in chlorarachniophytes. It bears an N-terminal-targeting sequence comprising a signal peptide and a transit peptide-like sequence (TPL) similar to the plastid-targeted proteins of chlorarachniophytes as well as a hydrophilic C-terminal extension rich in lysine and aspartic acid. Here, we characterize the function of the N- and C-terminal extensions of PPC-targeted EFL in transformed chlorarachniophyte cells. Using green fluorescent protein as a reporter molecule, we demonstrate that several negatively charged amino acids within the TPL are essential for accurate targeting to the PPC. Our findings further reveal that the C-terminal extension functions as a PPC retention signal in combination with an N-terminal plastid-targeting peptide, which suggests that plastid and PPC proteins may be sorted in the PPC.

Filling the Gap, Evolutionarily Conserved Omp85 in Plastids of Chromalveolates

Chromalveolates are a diverse group of protists that include many ecologically and medically relevant organisms such as diatoms and apicomplexan parasites. They possess plastids generally surrounded by four membranes, which evolved by engulfment of a red alga. Today, most plastid proteins must be imported, but many aspects of protein import into complex plastids are still cryptic. In particular, how proteins cross the third outermost membrane has remained unexplained. We identified a protein in the third outermost membrane of the diatom Phaeodactylum tricornutum with properties comparable to those of the Omp85 family. We demonstrate that the targeting route of P. tricornutum Omp85 parallels that of the translocation channel of the outer envelope membrane of chloroplasts, Toc75. In addition, the electrophysiological properties are similar to those of the Omp85 proteins involved in protein translocation. This supports the hypothesis that P. tricornutum Omp85 is involved in precursor protein translocation, which would close a gap in the fundamental understanding of the evolutionary origin and function of protein import in secondary plastids.

Genetic Evidence that an Endosymbiont-derived Endoplasmic Reticulum-associated Protein Degradation (ERAD) System Functions in Import of Apicoplast Proteins

Most apicomplexan parasites harbor a relict chloroplast, the apicoplast, that is critical for their survival. Whereas the apicoplast maintains a small genome, the bulk of its proteins are nuclear encoded and imported into the organelle. Several models have been proposed to explain how proteins might cross the four membranes that surround the apicoplast; however, experimental data discriminating these models are largely missing. Here we present genetic evidence that apicoplast protein import depends on elements derived from the ER-associated protein degradation (ERAD) system of the endosymbiont. We identified two sets of ERAD components in Toxoplasma gondii, one associated with the ER and cytoplasm and one localized to the membranes of the apicoplast. We engineered a conditional null mutant in apicoplast Der1, the putative pore of the apicoplast ERAD complex, and found that loss of Der1(Ap) results in loss of apicoplast protein import and subsequent death of the parasite.

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.

The luggage hypothesis: Comparisons of two phototrophic hosts with nitrogen-fixing cyanobacteria and implications for analogous life strategies for kleptoplastids/secondary symbiosis in dinoflagellates

Nostoc and Richelia belong to a group of heterocystous cyanobacteria and are unique within this group in forming intracellular symbioses with phototrophic hosts, the angiosperm Gunnera and the diatoms (algae) Rhizosolenia and Hemiaulus, respectively. The function of the cyanobiont is similar in the symbioses, namely providing fixed atmospheric nitrogen to their hosts; also the cyanobionts are contained in a host compartment, the symbiosome. The evolutionary timescale for the cyanobiont-endosymbiosis formation is in both instances about ≈90 Ma. However, the potentials for further co-evolution of host and microsymbiont, are different. Nostoc is regarded as preyed upon by its host, while in the Richelia-Rhizosolenia symbiosis example the evolution towards a new type of permanent organelle is possible. It is proposed that symbiosis is ruled by divergent host strategies. In the case of Richelia-Rhizosolenia the evolution of a permanent symbiosis is linked to diatom hosts needing to carry the cyanobiont permanently, as it is not available free-living in the oceans. However, in the case of Nostoc/Gunnera, the host exploits an abundant cyanobacterial species. A model where the relative abundance of microsymbionts determines the nature of the symbiosis comes into view: If environmental ratios of host/microsymbiont are so that hosts are the dominating party, then the host has to carry the microsymbiont as luggage (vertical transmission). Likewise, if the ratio of microsymbiont is higher than host, than the host will prey on the microsymbiont (horizontal transmission). The article also discusses the retention of secondary plastids in dinoflagellates. We show that dinoflagellates are organisms that exemplify both types of strategies that is either preying or harbouring a permanent organelle. The difference from the cyanobacterial example is that only parts of the eukaryotic microsymbionts are kept, usually only the plastid. We emphasize that the dinoflagellates can obtain their plastids from various different organisms. The luggage theory offers an explanation to why some dinoflagellate species contain kleptoplastids, while others have permanent, secondary plastids and some have tertiary plastids.

Molecular Phylogeny and Description of the Novel Katablepharid Roombia truncata gen. et sp. nov., and Establishment of the Hacrobia Taxon nov

BackgroundPhotosynthetic eukaryotes with a secondary plastid of red algal origin (cryptophytes, haptophytes, stramenopiles, dinoflagellates, and apicomplexans) are hypothesized to share a single origin of plastid acquisition according to Chromalveolate hypothesis. Recent phylogenomic analyses suggest that photosynthetic “chromalveolates” form a large clade with inclusion of several non-photosynthetic protist lineages. Katablepharids are one such non-photosynthetic lineage closely related to cryptophytes. Despite their evolutionary and ecological importance, katablepharids are poorly investigated.Methodology/Principal FindingsHere, we report a newly discovered flagellate, Roombia truncata gen. et sp. nov., that is related to katablepharids, but is morphologically distinct from othermembers of the group in the following ways: (1) two flagella emerge from a papilla-like subapical protrusion, (2) conspicuous ejectisomes are aligned in multiple (5–11) rows, (3) each ejectisome increases in size towards the posterior end of the rows, and (4) upon feeding, a part of cytoplasm elastically stretch to engulf whole prey cell. Molecular phylogenies inferred from Hsp90, SSU rDNA, and LSU rDNA sequences consistently and strongly show R. truncata as the sister lineage to all other katablepharids, including lineages known only from environmental sequence surveys. A close association between katablepharids and cryptophytes was also recovered in most analyses. Katablepharids and cryptophytes are together part of a larger, more inclusive, group that also contains haptophytes, telonemids, centrohelids and perhaps biliphytes. The monophyly of this group is supported by several different molecular phylogenetic datasets and one shared lateral gene transfer; therefore, we formally establish this diverse clade as the “Hacrobia.”Conclusions/SignificanceOur discovery of R. truncata not only expands our knowledge in the less studied flagellate group, but provide a better understanding of phylogenetic relationship and evolutionary view of plastid acquisition/losses of Hacrobia. Being an ancestral to all katablepharids, and readily cultivable, R. truncata is a good candidate for multiple gene analyses that will contribute to future phylogenetic studies of Hacrobia.

Protein targeting into secondary plastids of chlorarachniophytes.

Most plastid proteins are encoded by the nuclear genome, and consequently, need to be transported into plastids across multiple envelope membranes. In diverse organisms possessing secondary plastids, nuclear-encoded plastid precursor proteins (preproteins) commonly have an N-terminal extension that consists of an endoplasmic reticulum (ER)-targeting signal peptide and a transit peptide-like sequence (TPL). This bipartite targeting peptide is believed to be necessary for targeting the preproteins into the secondary plastids. Here, we newly demonstrate the function of the bipartite targeting peptides of an algal group, chlorarachniophytes, and characterize the functional domains of the TPL in the precursor of a plastid protein, ATP synthase delta subunit (AtpD), using a GFP as a reporter molecule. We show that the C-terminal portion of the TPL is important for targeting the AtpD preprotein from the ER into the chlorarachniophyte plastids, and several positively charged amino acids in the TPL are also necessary for transporting the preprotein across the 2 innermost plastid membranes. Compared with other groups with secondary plastids, the TPL functional domains of the chlorarachniophytes are unique, which might be caused by independent acquisition of their plastids.

On the origin of chloroplasts, import mechanisms of chloroplast-targeted proteins, and loss of photosynthetic ability — review

Primary plastids of green algae (including land plants), red algae and glaucophytes are bounded by two membranes and are thought to be derived from a single primary endosymbiosis of a cyanobacterium in a eukaryotic host. Complex plastids of euglenids and chlorarachneans bounded by three and four membranes, respectively, most likely arose via two separate secondary endosymbioses of a green alga in a eukaryotic host. Secondary plastids of cryptophyta, haptophyta, heterokontophyta and apicomplexan parasites bounded by four membranes, and plastids of dinoflagellates bounded by three membranes could have arisen via a single secondary endosymbiosis of a red alga in a eukaryotic host (chromalveolate hypothesis). However, the scenario of separate tertiary origins (symbioses of an alga possessing secondary plastids in a eukaryotic host) of some (or even most) chromalveolate plastids can be also consistent with the current data. The protein import into complex plastids differs from the import into primary plastids, as complex plastids contain one or two extra membrane(s). In organisms with primary plastids, plastid-targeted proteins contain N-terminal transit peptide which ferries proteins through the protein import machineries (multiprotein complexes) of the two (originally cyanobacterial) membranes. In organisms with complex plastids, the secretory signal sequence directing proteins to endomembrane system and afterwards through extra outermost membrane(s) is generally present upstream of the classical transit peptide. Several free-living as well as parasitic eukaryotes possess non-photosynthetic plastids. These plastids have generally retained the plastid genome, functional plastid transcriptional and translational apparatus, and various metabolic pathways, suggesting that though these plastids lost their photosynthetic ability, they are essential for the mentioned organisms. Nevertheless, some eukaryotes could have lost chloroplast compartment completely.

An Unusual ERAD-Like Complex Is Targeted to the Apicoplast ofPlasmodium falciparum

Many apicomplexan parasites, including Plasmodium falciparum, harbor a so-called apicoplast, a complex plastid of red algal origin which was gained by a secondary endosymbiotic event. The exact molecular mechanisms directing the transport of nuclear-encoded proteins to the apicoplast of P. falciparum are not well understood. Recently, in silico analyses revealed a second copy of proteins homologous to components of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) system in organisms with secondary plastids, including the malaria parasite P. falciparum. These proteins are predicted to be endowed with an apicoplast targeting signal and are suggested to play a role in the transport of nuclear-encoded proteins to the apicoplast. Here, we have studied components of this ERAD-derived putative preprotein translocon complex in malaria parasites. Using transfection technology coupled with fluorescence imaging techniques we can demonstrate that the N terminus of several ERAD-derived components targets green fluorescent protein to the apicoplast. Furthermore, we confirm that full-length PfsDer1-1 and PfsUba1 (homologues of yeast ERAD components) localize to the apicoplast, where PfsDer1-1 tightly associates with membranes. Conversely, PfhDer1-1 (a host-specific copy of the Der1-1 protein) localizes to the ER. Our data suggest that ERAD components have been "rewired" to provide a conduit for protein transport to the apicoplast. Our results are discussed in relation to the nature of the apicoplast protein transport machinery.

Protein Targeting into Secondary Plastids1

Most of the coding capacity of primary plastids is reserved for expressing some central components of the photosynthesis machinery and the translation apparatus. Thus, for the bulk of biochemical and cell biological reactions performed within the primary plastids, many nucleus-encoded components have to be transported posttranslationally into the organelle. The same is true for plastids surrounded by more than two membranes, where additional cellular compartments have to be supplied with nucleus-encoded proteins, leading to a corresponding increase in complexity of topogenic signals, transport and sorting machineries. In this review, we summarize recent progress in elucidating protein transport across up to five plastid membranes in plastids evolved in secondary endosymbiosis. Current data indicate that the mechanisms for protein transport across multiple membranes have evolved by altering pre-existing ones to new requirements in secondary plastids.

Unusual Nuclear Division in Nannochloropsis oculata (Eustigmatophyceae, Heterokonta) which May Ensure Faithful Transmission of Secondary Plastids

Nuclear and plastid division in the monoplastidic, unicellular eustigmatophyte alga Nannochloropsis oculata (Heterokonta) was investigated by electron microscopy. The outermost of four membranes of the secondary plastid is continuous with the outer nuclear envelope membrane to form a nucleus-plastid continuum (NPC). Such physical continuity between the nucleus and the plastid is maintained throughout the cell cycle. Mitosis takes place in a closed spindle. In prophase, a barrel-shaped nuclear pole body (BR-NPB), emanating microtubules towards the cytoplasm, was detected in the vicinity of the nuclear poles. In metaphase, instead of the BR-NPB, a boomerang-shaped nuclear pole body (BM-NPB) occupied the spindle poles, projecting microtubules towards the opposite pole. The BR- and BM-NPB may function as a microtubule organizing centre (MTOC) but are distinct in morphology from any known MTOCs. During anaphase/telophase, the nucleus undergoes constriction with the microtubules penetrating the nucleus along the pole-to-pole spindle axis. The final stage of the nuclear division takes place in an unusual fashion such that the compartment of the inner nuclear envelope divides in advance of that of the outer nuclear envelope. Such unusual nuclear division is discussed in relevance to transmission of the secondary plastid. The present study provides the first report for nuclear and plastid division in Eustigmatophyceae.

A HYPOTHESIS FOR PLASTID EVOLUTION IN CHROMALVEOLATES(1).

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.

Toxoplasma gondii Tic20 is essential for apicoplast protein import

Apicomplexan parasites harbor a secondary plastid that has lost the ability to photosynthesize yet is essential for the parasite to multiply and cause disease. Bioinformatic analyses predict that 5-10% of all proteins encoded in the parasite genome function within this organelle. However, the mechanisms and molecules that mediate import of such large numbers of cargo proteins across the four membranes surrounding the plastid remain elusive. In this work, we identify a highly diverged member of the Tic20 protein family in Apicomplexa. We demonstrate that Tic20 of Toxoplasma gondii is an integral protein of the innermost plastid membrane. We engineer a conditional null-mutant and show that TgTic20 is essential for parasite growth. To characterize this mutant functionally, we develop several independent biochemical import assays to reveal that loss of TgTic20 leads to severe impairment of apicoplast protein import followed by organelle loss and parasite death. TgTic20 is the first experimentally validated protein import factor identified in apicoplasts. Our studies provide experimental evidence for a common evolutionary origin of import mechanisms across the innermost membranes of primary and secondary plastids.

Evolutionary Origins and Functions of the Carotenoid Biosynthetic Pathway in Marine Diatoms

Carotenoids are produced by all photosynthetic organisms, where they play essential roles in light harvesting and photoprotection. The carotenoid biosynthetic pathway of diatoms is largely unstudied, but is of particular interest because these organisms have a very different evolutionary history with respect to the Plantae and are thought to be derived from an ancient secondary endosymbiosis between heterotrophic and autotrophic eukaryotes. Furthermore, diatoms have an additional xanthophyll-based cycle for dissipating excess light energy with respect to green algae and higher plants. To explore the origins and functions of the carotenoid pathway in diatoms we searched for genes encoding pathway components in the recently completed genome sequences of two marine diatoms. Consistent with the supplemental xanthophyll cycle in diatoms, we found more copies of the genes encoding violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZEP) enzymes compared with other photosynthetic eukaryotes. However, the similarity of these enzymes with those of higher plants indicates that they had very probably diversified before the secondary endosymbiosis had occurred, implying that VDE and ZEP represent early eukaryotic innovations in the Plantae. Consequently, the diatom chromist lineage likely obtained all paralogues of ZEP and VDE genes during the process of secondary endosymbiosis by gene transfer from the nucleus of the algal endosymbiont to the host nucleus. Furthermore, the presence of a ZEP gene in Tetrahymena thermophila provides the first evidence for a secondary plastid gene encoded in a heterotrophic ciliate, providing support for the chromalveolate hypothesis. Protein domain structures and expression analyses in the pennate diatom Phaeodactylum tricornutum indicate diverse roles for the different ZEP and VDE isoforms and demonstrate that they are differentially regulated by light. These studies therefore reveal the ancient origins of several components of the carotenoid biosynthesis pathway in photosynthetic eukaryotes and provide information about how they have diversified and acquired new functions in the diatoms.