Identification and characterization of a new conserved motif within the presequence of proteins targeted into complex diatom plastids

Several groups of algae evolved by secondary endocytobiosis, which is defined as the uptake of a eukaryotic alga into a eukaryotic host cell and the subsequent transformation of the endosymbiont into an organelle. Due to this explicit evolutionary history such algae possess plastids that are surrounded by either three or four membranes. Protein targeting into plastids of these organisms depends on N-terminal bipartite presequences consisting of a signal and a transit peptide domain. This suggests that different protein targeting systems may have been combined during establishment of secondary endocytobiosis to enable the transport of proteins into the plastids. Here we demonstrate the presence of an apparently new type of transport into diatom plastids. We analyzed protein targeting into the plastids of diatoms and identified a conserved amino acid sequence motif within plastid preprotein targeting sequences. We expressed several diatom plastid presequence:GFP fusion proteins with or without modifications within that motif in the diatom Phaeodactylum tricornutum and found that a single conserved phenylalanine is crucial for protein transport into the diatom plastids in vivo, thus indicating the presence of a so far unknown new type of targeting signal. We also provide experimental data about the minimal requirements of a diatom plastid targeting presequence and demonstrate that the signal peptides of plastid preproteins and of endoplasmic reticulum-targeted preproteins in diatoms are functionally equivalent. Furthermore we show that treatment of the cells with Brefeldin A arrests protein transport into the diatom plastids suggesting that a vesicular transport step within the plastid membranes may occur.     

Two Types of FtsZ Proteins in Mitochondria and Red-Lineage Chloroplasts: The Duplication of FtsZ Is Implicated in Endosymbiosis

The ancestors of plastids and mitochondria were once free-living bacteria that became organelles as a result of endosymbiosis. According to this theory, a key bacterial division protein, FtsZ, plays a role in plastid division in algae and plants as well as in mitochondrial division in lower eukaryotes. Recent studies have shown that organelle division is a process that combines features derived from the bacterial division system with features contributed by host eukaryotic cells. Two nonredundant versions of FtsZ, FtsZ1 and FtsZ2, have been identified in green-lineage plastids, whereas most bacteria have a single ftsZ gene. To examine whether there is also more than one type of FtsZ in red-lineage chloroplasts (red algal chloroplasts and chloroplasts that originated from the secondary endosymbiosis of red algae) and in mitochondria, we obtained FtsZ sequences from the complete sequence of the primitive red alga Cyanidioschyzon merolae and the draft sequence of the stramenopile (heterokont) Thalassiosira pseudonana. Phylogenetic analyses that included known FtsZ proteins identified two types of chloroplast FtsZ in red algae (FtsZA and FtsZB) and stramenopiles (FtsZA and FtsZC). These analyses also showed that FtsZB emerged after the red and green lineages diverged, while FtsZC arose by the duplication of an ftsZA gene that in turn descended from a red alga engulfed by the ancestor of stramenopiles. A comparison of the predicted proteins showed that like bacterial FtsZ and green-lineage FtsZ2, FtsZA has a short conserved C-termmal sequence (the C-terminal core domain), whereas FtsZB and FtsZC, like the green-lineage FtsZ1, lack this sequence. In addition, the Cyanidioschyzon and Dictyostelium genomes encode two types of mitochondrial FtsZ proteins, one of which lacks the C-terminal variable domain. These results suggest that the acquisition of an additional FtsZ protein with a modified C terminus was common to the primary and secondary endosymbioses that produced plastids and that this also occurred during the establishment of mitochondria, presumably to regulate the multiplication of these organelles.     

EVOLUTIONARY ANALYSES OF THE NUCLEAR‐ENCODED PHOTOSYNTHETIC GENE psbO FROM TERTIARY PLASTID‐CONTAINING ALGAE IN DINOPHYTA1

Although the dinophytes generally possess red‐algal‐derived secondary plastids, tertiary plastids originating from haptophyte and diatom ancestors are recognized in some lineages within the Dinophyta. However, little is known about the nuclear‐encoded genes of plastid‐targeted proteins from the dinophytes with diatom‐derived tertiary plastids. We analyzed the sequences of the nuclear psbO gene encoding oxygen‐evolving enhancer protein from various algae with red‐algal‐derived secondary and tertiary plastids. Based on our sequencing of 10 new genes and phylogenetic analysis of PsbO amino acid sequences from a wide taxon sampling of red algae and organisms with red‐algal‐derived plastids, dinophytes form three separate lineages: one composed of peridinin‐containing species with secondary plastids, and the other two having haptophyte‐ or diatom‐derived tertiary plastids and forming a robust monophyletic group with haptophytes and diatoms, respectively. Comparison of the N‐terminal sequences of PsbO proteins suggests that psbO genes from a dinophyte with diatom‐derived tertiary plastids (Kryptoperidinium) encode proteins that are targeted to the diatom plastid from the endosymbiotic diatom nucleus as in the secondary phototrophs, whereas the fucoxanthin‐containing dinophytes (Karenia and Karlodinium) have evolved an additional system of psbO genes for targeting the PsbO proteins to their haptophyte‐derived tertiary plastids from the host dinophyte nuclei.     

Protein targeting into complex diatom plastids: functional characterisation of a specific targeting motif

Plastids of diatoms and related algae evolved by secondary endocytobiosis, the uptake of a eukaryotic alga into a eukaryotic host cell and its subsequent reduction into an organelle. As a result diatom plastids are surrounded by four membranes. Protein targeting of nucleus encoded plastid proteins across these membranes depends on N-terminal bipartite presequences consisting of a signal and a transit peptide-like domain. Diatoms and cryptophytes share a conserved amino acid motif of unknown function at the cleavage site of the signal peptides (ASAFAP), which is particularly important for successful plastid targeting. Screening genomic databases we found that in rare cases the very conserved phenylalanine within the motif may be replaced by tryptophan, tyrosine or leucine. To test such unusual presequences for functionality and to better understand the role of the motif and putative receptor proteins involved in targeting, we constructed presequence:GFP fusion proteins with or without modifications of the “ASAFAP”-motif and expressed them in the diatom Phaeodactylum tricornutum. In this comprehensive mutational analysis we found that only the aromatic amino acids phenylalanine, tryptophan, tyrosine and the bulky amino acid leucine at the +1 position of the predicted signal peptidase cleavage site allow plastid import, as expected from the sequence comparison of native plastid targeting presequences of P. tricornutum and the cryptophyte Guillardia theta. Deletions within the signal peptide domains also impaired plastid import, showing that the presence of F at the N-terminus of the transit peptide together with a cleavable signal peptide is crucial for plastid import. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. A. Gruber and S. Vugrinec contributed equally to this work.     

IN VIVO CHARACTERIZATION OF THE DIATOM MULTIPARTATE PLASTID TARGETING SIGNAL

Diatoms and related algae have plastids that are surrounded by four membranes. The outer two membranes are continuous with the endoplasmic reticulum and the inner two membranes are analogous to the plastid envelope membranes of higher plants and green algae. Thus the plastids are completely compartmentalized within the ER membranes. The targeting presequences for nuclear‐encoded plastid proteins have two recognizable domains. The first domain is a classic signal sequence, which presumably targets the proteins to the endoplasmic reticulum. The second domain has characteristics of a transit peptide, which targets proteins to the plastids of higher plants. To characterize these targeting domains, the presequence from the nuclear‐encoded plastid protein AtpC was utilized. A series of deletions of this presequence were fused to Green Fluorescent Protein (GFP) and transformed into cells of the diatom, Phaeodactylum tricornutum. The intracelluar localization of GFP was visualized by fluorescence microscopy. This work demonstrates that the first domain of the presequence is responsible for targeting proteins to the ER lumen and is the essential first step in the plastid protein import process. The second domain is responsible to directing proteins from the ER and through the plastid envelope and only a short portion of the transit peptide‐like domain is necessary to complete this second processing step. In vivo data generated from this study in a fully homologous transformation system has confirmed Gibbs' hypothesis regarding a multistep import process for plastid proteins in chromophytic algae.     

The division of pleomorphic plastids with multiple FtsZ rings in tobacco BY-2 cells

Plastids, an essential group of plant cellular organelles, proliferate by division to maintain continuity through cell lineages in plants. In recent years, it was revealed that the bacterial cell division protein FtsZ is encoded in the nuclear genome of plant cells, and plays a major role in the plastid division process forming a ring along the center of plastids. Although the best-characterized type of plastid division so far is the division with a single FtsZ ring at the plastid midpoint, it was recently reported that in some plant organs and tissues, plastids are pleomorphic and form multiple FtsZ rings. However, the pleomorphic plastid division mechanism, such as the formation of multiple FtsZ rings, the constriction of plastids and the behavior of plastid (pt) nucleoids, remains totally unclear. To elucidate these points, we used the cultured cell line, tobacco (Nicotiana tabacum L.) Bright Yellow-2, in which plastids are pleomorphic and show dynamic morphological changes during culture. As a result, it was revealed that as the plastid elongates from an ellipsoid shape to a string shape after medium renewal, FtsZ rings are multiplied almost orderly and perpendicularly to the long axis of plastids. Active DNA synthesis of pt nucleoids is induced by medium transfer, and the division and the distribution of pt nucleoids occur along with plastid elongation. Although it was thought that the plastid divides with simultaneous multiple constrictions at all the FtsZ ring sites, giving rise to many small plastids, we found that the plastids generally divide constricting at only one FtsZ ring site. Moreover, using electron microscopy, we revealed that plastid-dividing (PD) rings are observed only at the constriction site, and not at swollen regions. These results indicate that in the pleomorphic plastid division with multiple FtsZ rings, the formation of PD rings occurs at a limited FtsZ ring site for one division. Multiplied FtsZ rings seem to localize in advance at the expected sites of division, and the formation of a PD ring at each FtsZ ring site occurs in a certain order, not simultaneously. Based on these results, a novel model for the pleomorphic plastid division with multiple FtsZ rings is proposed.     

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.     

A homologue of the bacterial cell division site-determining factor MinD mediates placement of the chloroplast division apparatus

BACKGROUND: Chloroplast division in plant cells occurs by binary fission, yielding two daughter plastids of equal size. Previously, we reported that two Arabidopsis homologues of FtsZ, a bacterial protein that forms a cytokinetic ring during cell division, are essential for plastid division in plants, and may be involved in the formation of plastid-dividing rings on both the stromal and cytosolic surfaces of the chloroplast envelope membranes. In bacteria, positioning of the FtsZ ring at the center of the cell is mediated in part by the protein MinD. Here, we identified AtMinD1, an Arabidopsis homologue of MinD, and investigated whether positioning of the plastid-division apparatus at the plastid midpoint might involve a mechanism similar to that in bacteria. RESULTS: Sequence analysis and in vitro chloroplast import experiments indicated that AtMinD1 contains a transit peptide that targets it to the chloroplast. Transgenic Arabidopsis plants with reduced AtMinD1 expression exhibited variability in chloroplast size and number and asymmetrically constricted chloroplasts, strongly suggesting that the plastid-division machinery is misplaced. Overexpression of AtMinD1 inhibited chloroplast division. These phenotypes resemble those of bacterial mutants with altered minD expression. CONCLUSIONS: Placement of the plastid-division machinery at the organelle midpoint requires a plastid-targeted form of MinD. The results are consistent with a model whereby assembly of the division apparatus is initiated inside the chloroplast by the plastidic form of FtsZ, and suggest that positioning of the cytosolic components of the apparatus is specified by the position of the plastidic components.     

Nucleus-to-nucleus gene transfer and protein retargeting into a remnant cytoplasm of cryptophytes and diatoms

The complex plastid of the cryptophyte Guillardia theta and of the diatom Phaeodactylum tricornutum can both be traced back to an engulfed eukaryotic red alga. The eukaryotic origin of these plastids is most obvious in cryptophytes, where the organelle still possesses a remnant nucleus, the nucleomorph. The nucleomorph itself is embedded in the periplastid compartment (PPC), the remnant of the former red algal cytosol. In the cryptophyte and diatom, the complex plastid is surrounded by 4 membranes, the outer one being continuous with the host rough endoplasmatic reticulum. In a recent report, we have shown that a nuclear encoded PPC protein of G. theta expressed in P. tricornutum leads to a localization, recently described as being a "bloblike structure," which can be obtained by mutation of plastid protein-targeting sequences of the diatom itself. Here we present further nucleus-encoded PPC proteins from G. theta, such as the eukaryotic translation elongation factor-1alpha, evidence for their nucleus-to-nucleus gene transfer, and retargeting of the proteins. We also investigated the first nuclear encoded PPC-targeted protein of P. tricornutum (Hsp70) and analyzed it for in vivo localization together with the identified G. theta PPC proteins. This revealed that all localize to the bloblike structures, which we suggest is the highly reduced PPC of P. tricornutum. Furthermore, the described cryptophyte PPC proteins possibly allow the elucidation of the processes by which proteins are involved in different levels of host control over its eukaryotic organelle.     

Origins of the secondary plastids of Euglenophyta and Chlorarachniophyta as revealed by an analysis of the plastid‐targeting,nuclear‐encoded gene psbO1

Because the secondary plastids of the Euglenophyta and Chlorarachniophyta are very similar to green plant plastids in their pigment composition, it is generally considered that ancestral green algae were engulfed by other eukaryotic host cells to become the plastids of these two algal divisions. Recent molecular phylogenetic studies have attempted to resolve the phylogenetic positions of these plastids; however, almost all of the studies analyzed only plastid‐encoded genes. This limitation may affect the results of comparisons between genes from primary and secondary plastids, because genes in endosymbionts have a higher mutation rate than the genes of their host cells. Thus, the phylogeny of these secondary plastids must be elucidated using other molecular markers. Here, we compared the plastid‐targeting, nuclear‐encoded, oxygen‐evolving enhancer (psbO) genes from various green plants, the Euglenophyta and Chlorarachniophyta. A phylogenetic analysis based on the PsbO amino acid sequences indicated that the chlorarachniophyte plastids are positioned within the Chlorophyta (including Ulvophyceae, Chlorophyceae, and Prasinophyceae, but excluding Mesostigma). In contrast, plastids of the Euglenophyta and Mesostigma are positioned outside the Chlorophyta and Streptophyta. The relationship of these three phylogenetic groups was consistent with the grouping of the primary structures of the thylakoid‐targeting domain and its adjacent amino acids in the PsbO N‐terminal sequences. Furthermore, the serine‐X‐alanine (SXA) motif of PsbO was exactly the same in the Chlorarachniophyta and the prasinophycean Tetraselmis. Therefore, the chlorarachniophyte secondary plastids likely evolved from the ancestral Tetraselmis‐like alga within the Chlorophyta, whereas the Euglenophyte plastids may have originated from the unknown basal lineage of green plants.     

The ultrastructural features and division of secondary plastids

Plastids in heterokonts, cryptophytes, haptophytes, dinoflagellates, chlorarachniophytes, euglenoids, and apicomplexan parasites derive from secondary symbiogenesis. These plastids are surrounded by one or two additional membranes covering the plastid-envelope double membranes. Consequently, nuclear-encoded plastid division proteins have to be targeted into the division site through the additional surrounding membranes. Electron microscopic observations suggest that the additional surrounding membranes are severed by mechanisms distinct from those for the division of the plastid envelope. In heterokonts, cryptophytes and haptophytes, the outermost surrounding membrane (epiplastid rough endoplasmic reticulum, EPrER) is studded with cytoplasmic ribosomes and connected to the rER and the outer nuclear envelope. In monoplastidic species belonging to these three groups, the EPrER and the outer nuclear envelope are directly connected to form a sac enclosing the plastid and the nucleus. This nuclear-plastid connection, referred to as the nucleus-plastid consortium (NPC), may be significant to ensure the transmission of the plastids during cell division. The plastid dividing-ring (PD-ring) is a conserved component of the division machinery for both primary and secondary plastids. Also, homologues of the bacterial cell division protein, FtsZ, may be involved in the division of secondary plastids as well as primary plastids, though in secondary plastids they have not yet been localized to the division site. It remains to be examined whether or not dynamin-like proteins and other protein components known to function in the division of primary plastids are used also in secondary plastids. The nearly completed sequencing of the nuclear genome of the diatom Thalassiosira pseudonana will give impetus to molecular and cell biological studies on the division of secondary plastids.     

How do endosymbionts become organelles? Understanding early events in plastid evolution

What factors drove the transformation of the cyanobacterial progenitor of plastids (e.g. chloroplasts) from endosymbiont to bona fide organelle? This question lies at the heart of organelle genesis because, whereas intracellular endosymbionts are widespread in both unicellular and multicellular eukaryotes (e.g. rhizobial bacteria, Chlorella cells in ciliates, Buchnera in aphids), only two canonical eukaryotic organelles of endosymbiotic origin are recognized, the plastids of algae and plants and the mitochondrion. Emerging data on (1) the discovery of non‐canonical plastid protein targeting, (2) the recent origin of a cyanobacterial‐derived organelle in the filose amoeba Paulinella chromatophora, and (3) the extraordinarily reduced genomes of psyllid bacterial endosymbionts begin to blur the distinction between endosymbiont and organelle. Here we discuss the use of these terms in light of new data in order to highlight the unique aspects of plastids and mitochondria and underscore their central role in eukaryotic evolution. BioEssays 29:1239–1246, 2007. © 2007 Wiley Periodicals, Inc.     

Plastid division is driven by a complex mechanism that involves differential transition of the bacterial and eukaryotic division rings

During plastid division, two structures have been detected at the division site in separate analyses. The plastid-dividing ring can be detected by transmission electron microscopy as two (or three) electron-dense rings: an outer ring on the cytosolic face of the outer envelope, occasionally a middle ring in the intermembrane space, and an inner ring on the stromal face of the inner envelope. The FtsZ ring, which plays a central role in bacterial division, also is involved in plastid division and is believed to have descended to plastids from cyanobacterial endosymbiosis. The relationship between the two structures is not known, although there is discussion regarding whether they are identical. Biochemical and immunocytochemical investigations, using synchronized chloroplasts of the red alga Cyanidioschyzon merolae, showed that the plastid FtsZ ring is distinct and separable from the plastid-dividing ring. The FtsZ ring localizes in stroma and faces the inner plastid-dividing ring at the far side from the inner envelope. The FtsZ ring and the inner and outer plastid-dividing rings form in that order before plastid division. The FtsZ ring disappears at the late stage of constriction before dissociation of the plastid-dividing ring, when the constriction is still in progress. Our results suggest that the FtsZ ring;-based system, which originated from a plastid ancestor, cyanobacteria, and the plastid-dividing ring;-based system, which probably originated from host eukaryotic cells, form a complex and are involved in plastid division by distinct modes.     

FtsZ characterization and immunolocalization in the two phases of plastid reorganization in arbuscular mycorrhizal roots of Medicago truncatula

We have analyzed plastid proliferation in root cortical cells of Medicago truncatula colonized by arbuscular mycorrhizal (AM) fungi by concomitantly labeling fungal structures, root plastids, a protein involved in plastid division (FtsZ1) and a protein involved in the biosynthesis of AM-specific apocarotenoids. Antibodies directed against FtsZ1 have been generated after heterologous expression of the respective gene from M. truncatula and characterization of the gene product. Analysis of enzymatic activity and assembly experiments showed similar properties of this protein when compared with the bacterial proteins. Immunocytological experiments allowed two phases of fungal and plastid development to be clearly differentiated and plastid division to be monitored during these phases. In the early phase of arbuscule development, lens-shaped plastids, intermingled with the arbuscular branches, divide frequently. Arbuscule degradation, in contrast, is characterized by large, tubular plastids, decorated by a considerable number of FtsZ division rings.     

Diversification of ftsZ During Early Land Plant Evolution

The plastid division proteins FtsZ are encoded by a small nuclear gene family in land plants. Although it has been shown for some of the gene products that they are imported into plastids and function in plastid division, the evolution and function of this gene family and their products remain to be unraveled. Here we present two new ftsZ genes from the moss Physcomitrella patens and compare the genomic structure of members of the two plant ftsZ gene families. Comparison of sequence features and phylogenetic analyses confirm the presence of two clusters of paralogues in land plants and demonstrate that these genes were duplicated before the divergence of mosses, ferns and seed plants.     

Three old and one new: protein import into red algal-derived plastids surrounded by four membranes

Engulfment of a red or green alga by another eukaryote and subsequent reduction of the symbiont to an organelle, termed a complex plastid, is a process known as secondary endosymbiosis and is shown in a diverse group of eukaryotic organisms. Important members are heterokontophytes, haptophytes, cryptophytes, and apicomplexan parasites, all of them with complex plastids of red algal origin surrounded by four membranes. Although the evolutionary relationship between these organisms is still debated, they share common mechanisms for plastid protein import. In this review, we describe recent findings and current models on preprotein import into complex plastids with a special focus on the second outermost plastid membrane. Derived from the plasma membrane of the former endosymbiont, the evolution of protein transport across this so-called periplastidal membrane most likely represented the challenge in the transition from an endosymbiont to a host-dependent organelle. Here, remodeling and relocation of the symbiont endoplasmic reticulum-associated degradation (ERAD) machinery gave rise to a translocon complex termed symbiont-specific ERAD-like machinery and provides a fascinating insight into complex cellular evolution.     

Presequence Acquisition During Secondary Endocytobiosis and thePossible Role of Introns

Targeting of nucleus-encoded proteins into chloroplasts is mediated by N-terminal presequences. During evolution of plastids from formerly free-living cyanobacteria by endocytobiosis, genes for most plastid proteins have been transferred from the plastid genome to the nucleus and subsequently had to be equipped with such plastid targeting sequences. So far it is unclear how the gene domains coding for presequences and the respective mature proteins may have been assembled. While land plant plastids are supposed to originate from a primary endocytobiosis event (a prokaryotic cyanobacterium was taken up by a eukaryotic cell), organisms with secondary plastids like diatoms experienced a second endocytobiosis step involving a eukaryotic alga taken up by a eukaryotic host cell. In this group of algae, apparently most genes encoding chloroplast proteins have been transferred a second time (from the nucleus of the endosymbiont to the nucleus of the secondary host) and thus must have been equipped with additional targeting signals. We have analyzed cDNAs and the respective genomic DNA fragments of seven plastid preproteins from the diatom Phaeodactylum tricornutum. In all of these genes we found single spliceosomal introns, generally located within the region coding for the N-terminal plastid targeting sequences or shortly downstream of it. The positions of the introns can be related to the putative phylogenetic histories of the respective genes, indicating that the bipartite targeting sequences in these secondary algae might have evolved by recombination events via introns.The nucleotide sequences have been deposited at Genbank under accession numbers AY191862, AY191863, AY191864, AY191865, AY191866, AY191867, and AY191868.