Diverse eukaryotes have retained mitochondrial homologues of the bacterial division protein FtsZ

Mitochondrial fission requires the division of both the inner and outer mitochondrial membranes. Dynamin-related proteins operate in division of the outer membrane of probably all mitochondria, and also that of chloroplasts--organelles that have a bacterial origin like mitochondria. How the inner mitochondrial membrane divides is less well established. Homologues of the major bacterial division protein, FtsZ, are known to reside inside mitochondria of the chromophyte alga Mallomonas, a red alga, and the slime mould Dictyostelium discoideum, where these proteins are likely to act in division of the organelle. Mitochondrial FtsZ is, however, absent from the genomes of higher eukaryotes (animals, fungi, and plants), even though FtsZs are known to be essential for the division of probably all chloroplasts. To begin to understand why higher eukaryotes have lost mitochondrial FtsZ, we have sampled various diverse protists to determine which groups have retained the gene. Database searches and degenerate PCR uncovered genes for likely mitochondrial FtsZs from the glaucocystophyte Cyanophora paradoxa, the oomycete Phytophthora infestans, two haptophyte algae, and two diatoms--one being Thalassiosira pseudonana, the draft genome of which is now available. From Thalassiosira we also identified two chloroplast FtsZs, one of which appears to be undergoing a C-terminal shortening that may be common to many organellar FtsZs. Our data indicate that many protists still employ the FtsZ-based ancestral mitochondrial division mechanism, and that mitochondrial FtsZ has been lost numerous times in the evolution of eukaryotes.     

Three rings for the evolution of plastid shape: a tale of land plant FtsZ

Nuclear-encoded plant FtsZ genes are derived from endosymbiotic gene transfer of cyanobacteria-like genes. The green lineage (Chloroplastida) and red lineage (Rhodophyta) feature FtsZ1 and FtsZ2 or FtsZB and FtsZA, respectively, which are involved in plastid division. These two proteins show slight differences and seem to heteropolymerize to build the essential inner plastid division ring. A third gene, encoding FtsZ3, is present in glaucophyte and charophyte algae, as well as in land plants except ferns and angiosperms. This gene was probably present in the last common ancestor of the organisms united by having a primary plastid (Archaeplastida) and was lost during vascular plant evolution as well as in the red and green algae. The presence/absence pattern of FtsZ3 mirrors that of a full set of Mur genes and the peptidoglycan wall encoded by them. Based on these findings, we discuss a role for FtsZ3 in the establishment or maintenance of plastid peptidoglycan shells.     

Origin and evolution of the chloroplast division machinery

Chloroplasts were originally established in eukaryotes by the endosymbiosis of a cyanobacterium; they then spread through diversification of the eukaryotic hosts and subsequent engulfment of eukaryotic algae by previously nonphotosynthetic eukaryotes. The continuity of chloroplasts is maintained by division of preexisting chloroplasts. Like their ancestors, chloroplasts use a bacterial division system based on the FtsZ ring and some associated factors, all of which are now encoded in the host nuclear genome. The majority of bacterial division factors are absent from chloroplasts and several new factors have been added by the eukaryotic host. For example, the ftsZ gene has been duplicated and modified, plastid-dividing (PD) rings were most likely added by the eukaryotic host, and a member of the dynamin family of proteins evolved to regulate chloroplast division. The identification of several additional proteins involved in the division process, along with data from diverse lineages of organisms, our current knowledge of mitochondrial division, and the mining of genomic sequence data have enabled us to begin to understand the universality and evolution of the division system. The principal features of the chloroplast division system thus far identified are conserved across several lineages, including those with secondary chloroplasts, and may reflect primeval features of mitochondrial division. Shin-ya Miyagishima is the recipient of the Botanical Society Award for Young Scientists, 2004.     

Visualization of a cytoskeleton-like FtsZ network in chloroplasts

It has been a long-standing dogma in life sciences that only eukaryotic organisms possess a cytoskeleton. Recently, this belief was questioned by the finding that the bacterial cell division protein FtsZ resembles tubulin in sequence and structure and, thus, may be the progenitor of this major eukaryotic cytoskeletal element. Here, we report two nuclear-encoded plant ftsZ genes which are highly conserved in coding sequence and intron structure. Both their encoded proteins are imported into plastids and there, like in bacteria, they act on the division process in a dose-dependent manner. Whereas in bacteria FtsZ only transiently polymerizes to a ring-like structure, in chloroplasts we identified persistent, highly organized filamentous scaffolds that are most likely involved in the maintenance of plastid integrity and in plastid division. As these networks resemble the eukaryotic cytoskeleton in form and function, we suggest the term "plastoskeleton" for this newly described subcellular structure.     

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.     

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.     

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.     

Isolation of mitochondrial and plastid ftsZ genes and analysis of the organelle targeting sequence in the diatom Chaetoceros neogracile (Diatoms,Bacillariophyceae)

Mitochondria and plastids multiply by division in eukaryotic cells. Recently, the eukaryotic homolog of the bacterial cell division protein FtsZ was identified and shown to play an important role in the organelle division process inside the inner membrane. To explore the evolution of FtsZ proteins, and to accumulate data on the protein import system in mitochondria and plastids of the red algal lineage, one mitochondrial and three plastid ftsZ genes were isolated from the diatom Chaetoceros neogracile, whose plastids were acquired by secondary endosymbiotic uptake of a red alga. Protein import into organelles depends on the N‐terminal organelle targeting sequences. N‐terminal bipartite presequences consisting of an endoplasmic reticulum signal peptide and a plastid transit peptide are required for protein import into diatom plastids. To characterize the organelle targeting peptides of C. neogracile, we observed the localization of each green fluorescent protein‐tagged predicted organelle targeting peptide in cultured tobacco cells and diatom cells. Our data suggested that each targeting sequences functioned both in tobacco cultured cells and diatom cells.     

ARC3 is a stromal Z-ring accessory protein essential for plastid division

In plants, chloroplast division is an integral part of development, and these vital organelles arise by binary fission from pre-existing cytosolic plastids. Chloroplasts arose by endosymbiosis and although they have retained elements of the bacterial cell division machinery to execute plastid division, they have evolved to require two functionally distinct forms of the FtsZ protein and have lost elements of the Min machinery required for Z-ring placement. Here, we analyse the plastid division component accumulation and replication of chloroplasts 3 (ARC3) and show that ARC3 forms part of the stromal plastid division machinery. ARC3 interacts specifically with AtFtsZ1, acting as a Z-ring accessory protein and defining a unique function for this family of FtsZ proteins. ARC3 is involved in division site placement, suggesting that it might functionally replace MinC, representing an important advance in our understanding of the mechanism of chloroplast division and the evolution of the chloroplast division machinery.     

MULTIPLE FtsZ RING FORMATION AND REDUPLICATED CHLOROPLAST DNA IN NANNOCHLORIS BACILLARIS (CHLOROPHYTA,TREBOUXIOPHYCEAE) UNDER PHOSPHATE‐ENRICHED CULTURE1

We examined the effects of phosphate enrichment on chloroplasts of the unicellular green alga Nannochloris bacillaris Naumann. The doubling time of cells was similar in phosphate‐limited (no β‐glycerophosphate) and phosphate‐enriched (2 mM β‐glycerophosphate) media. The lengths of cells and chloroplasts were similar, regardless of phosphate concentration. The relationship between the ring formation of the prokaryote‐derived chloroplast division protein FtsZ and phosphate concentration was examined using indirect fluorescent antibody staining. The number of FtsZ rings increased as the phosphate concentration of the medium increased. Multiple FtsZ rings were formed in cells in phosphate‐enriched medium; up to six FtsZ rings per chloroplast were observed. The number of FtsZ rings increased as the chloroplast grew. The FtsZ ring located near the center of the chloroplast had the strongest fluorescence. The FtsZ ring at the relative center of all FtsZ rings was used for division. Plastid division rings did not multiply in phosphate‐enriched culture. The chloroplast DNA content was 2.3 times greater in phosphate‐enriched than in phosphate‐limited culture and decreased in cells cultured in phosphate‐enriched medium containing 5‐fluorodeoxyuridine (FdUr). In the presence of FdUr, only one FtsZ ring formed, even under phosphate enrichment. This finding suggests that excessive chloroplast DNA replication induces multiple FtsZ ring formation in phosphate‐enriched culture. We propose a multiple FtsZ ring formation model under phosphate enrichment.     

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.     

A plant-specific dynamin-related protein forms a ring at the chloroplast division site

Chloroplasts have retained the bacterial FtsZ for division, whereas mitochondria lack FtsZ except in some lower eukaryotes. Instead, mitochondrial division involves a dynamin-related protein, suggesting that chloroplasts retained the bacterial division system, whereas a dynamin-based system replaced the bacterial system in mitochondria during evolution. In this study, we identified a novel plant-specific group of dynamins from the primitive red alga Cyanidioschyzon merolae. Synchronization of chloroplast division and immunoblot analyses showed that the protein (CmDnm2) associates with the chloroplast only during division. Immunocytochemical analyses showed that CmDnm2 appears in cytoplasmic patches just before chloroplast division and is recruited to the cytosolic side of the chloroplast division site to form a ring in the late stage of division. The ring constricts until division is complete, after which it disappears. These results show that a dynamin-related protein also participates in chloroplast division and that its behavior differs from that of FtsZ and plastid-dividing rings that form before constriction at the site of division. Combined with the results of a recent study of mitochondrial division in Cyanidioschyzon, our findings led us to hypothesize that when first established in lower eukaryotes, mitochondria and chloroplasts divided using a very similar system that included the FtsZ ring, the plastid-dividing/mitochondrion-dividing ring, and the dynamin ring.     

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.     

Short FtsZ filaments can drive asymmetric cell envelope constriction at the onset of bacterial cytokinesis

FtsZ, the bacterial homologue of eukaryotic tubulin, plays a central role in cell division in nearly all bacteria and many archaea. It forms filaments under the cytoplasmic membrane at the division site where, together with other proteins it recruits, it drives peptidoglycan synthesis and constricts the cell. Despite extensive study, the arrangement of FtsZ filaments and their role in division continue to be debated. Here, we apply electron cryotomography to image the native structure of intact dividing cells and show that constriction in a variety of Gram‐negative bacterial cells, including Proteus mirabilis and Caulobacter crescentus, initiates asymmetrically, accompanied by asymmetric peptidoglycan incorporation and short FtsZ‐like filament formation. These results show that a complete ring of FtsZ is not required for constriction and lead us to propose a model for FtsZ‐driven division in which short dynamic FtsZ filaments can drive initial peptidoglycan synthesis and envelope constriction at the onset of cytokinesis, later increasing in length and number to encircle the division plane and complete constriction.     

Two Dictyostelium orthologs of the prokaryotic cell division protein FtsZ localize to mitochondria and are required for the maintenance of normal mitochondrial morphology

In bacteria, the protein FtsZ is the principal component of a ring that constricts the cell at division. Though all mitochondria probably arose through a single, ancient bacterial endosymbiosis, the mitochondria of only certain protists appear to have retained FtsZ, and the protein is absent from the mitochondria of fungi, animals, and higher plants. We have investigated the role that FtsZ plays in mitochondrial division in the genetically tractable protist Dictyostelium discoideum, which has two nuclearly encoded FtsZs, FszA and FszB, that are targeted to the inside of mitochondria. In most wild-type amoebae, the mitochondria are spherical or rod-shaped, but in fsz-null mutants they become elongated into tubules, indicating that a decrease in mitochondrial division has occurred. In support of this role in organelle division, antibodies to FszA and FszA-green fluorescent protein (GFP) show belts and puncta at multiple places along the mitochondria, which may define future or recent sites of division. FszB-GFP, in contrast, locates to an electron-dense, submitochondrial body usually located at one end of the organelle, but how it functions during division is unclear. This is the first demonstration of two differentially localized FtsZs within the one organelle, and it points to a divergence in the roles of these two proteins.     

Rings and networks: the amazing complexity of FtsZ in chloroplasts

Bacteria have proteins that can form filaments and rings, and these are thought to be the evolutionary progenitors of actin and tubulin. Plant homologues of the most intensively studied bacterial FtsZ protein are nuclear-encoded by a small gene family, are plastid-bound and participate in the plastid division process. The hypothesis is put forward that FtsZ and other proteins form a filamentous network in plastids, a plastoskeleton, which keeps these organelles in shape and helps them to divide.     

Orderly formation of the double ring structures for plastid and mitochondrial division in the unicellular red alga Cyanidioschyzon merolae

The formation of the plastid-dividing ring (PD ring) and mitochondrion-dividing ring (MD ring) was studied in a highly synchronous culture of the unicellular red alga Cyanidioschyzon merolae. The timing and the order of formation of the MD and PD rings were determined by observing organelles around the onset of their division, using transmission electron microscopy. In  C. merolae, there is one chloroplast and one mitochondrion per cell, and the shape of the chloroplast changes sequentially from acorn-like, to round, to trapezoidal, to peanut-shaped, in that order, during the early stage of chloroplast division. None of the cells with acorn-shaped or round chloroplasts contained organelles with PD rings or MD rings, while all of the cells with peanut-shaped chloroplasts contained organelles with both PD rings and MD rings. In cells with peanut-shaped chloroplasts, the PD and MD rings were double ring structures, with an outer ring located on the cytoplasmic face of the outer membrane of the organelle, and an inner ring located in the matrix beneath the inner membrane. These results suggested that the double ring structures of the PD ring and the MD ring form when chloroplasts are trapezoidal in shape. Detailed three-dimensional observation of cells with trapezoidal chloroplasts revealed the following steps in the formation of the double ring structures of the PD and MD rings: (i) the inner ring of the PD ring forms first, followed by the outer ring; (ii) then the MD ring forms and becomes visible; (iii) when the double ring structures of the two rings have formed, the microbody then moves from its remote location to the plane of division of the mitochondrion and contraction of the PD and MD rings commences. These steps were also confirmed by computer-aided three-dimensional reconstruction of the images from serial thin sections. This study reveals the order of formation of the double ring structures of the PD and MD rings, and the behavior of the microbody around the onset of division of plastids and mitochondria. The results also provide the first evidence that the inner PD ring is not a tension element formed by the contractile pressure but a definite structure, independent of the outer ring. Received: 31 March 1998 / Accepted: 14 May 1998     

Endosymbiosis,cell evolution,and speciation

In 1905, the Russian biologist C. Mereschkowsky postulated that plastids (e.g., chloroplasts) are the evolutionary descendants of endosymbiotic cyanobacteria-like organisms. In 1927, I. Wallin explicitly postulated that mitochondria likewise evolved from once free-living bacteria. Here, we summarize the history of these endosymbiotic concepts to their modern-day derivative, the “serial endosymbiosis theory”, which collectively expound on the origin of eukaryotic cell organelles (plastids, mitochondria) and subsequent endosymbiotic events. Additionally, we review recent hypotheses about the origin of the nucleus. Model systems for the study of “endosymbiosis in action” are also described, and the hypothesis that symbiogenesis may contribute to the generation of new species is critically assessed with special reference to the secondary and tertiary endosymbiosis (macroevolution) of unicellular eukaryotic algae.     

Developmentally regulated association of plastid division protein FtsZ1 with thylakoid membranes in Arabidopsis thaliana

FtsZ is a key protein involved in bacterial and organellar division. Bacteria have only one ftsZ gene, while chlorophytes (higher plants and green alga) have two distinct FtsZ gene families, named FtsZ1 and FtsZ2. This raises the question of why chloroplasts in these organisms need distinct FtsZ proteins to divide. In order to unravel new functions associated with FtsZ proteins, we have identified and characterized an Arabidopsis thaliana FtsZ1 loss-of-function mutant. ftsZ1-knockout mutants are impeded in chloroplast division, and division is restored when FtsZ1 is expressed at a low level. FtsZ1-overexpressing plants show a drastic inhibition of chloroplast division. Chloroplast morphology is altered in ftsZ1, with chloroplasts having abnormalities in the thylakoid membrane network. Overexpression of FtsZ1 also induced defects in thylakoid organization with an increased network of twisting thylakoids and larger grana. We show that FtsZ1, in addition to being present in the stroma, is tightly associated with the thylakoid fraction. This association is developmentally regulated since FtsZ1 is found in the thylakoid fraction of young developing plant leaves but not in mature and old plant leaves. Our results suggest that plastid division protein FtsZ1 may have a function during leaf development in thylakoid organization, thus highlighting new functions for green plastid FtsZ.