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     

Mitochondrial division by an electron-dense ring inCyanidioschyzon merolae

Summary The first identification of a mitochondria-dividing ring (MD-ring), which is located in the cytoplasm near the outer envelope membrane at the constricted isthmus of dividing mitochondria in the red algaCyanidioschyzon merolae, is reported. The MD-ring is about 50 nm wide and 10 nm thick at early stage of mitochondrial constriction and is a somewhat electron-dense circular bundle. The MD-ring is believed to be essential for the division of mitochondrion (mitochondriokinesis) since the ring appears at the equatorial region of the mitochondria just before the initiation of mitochondrial division and can be observed throughout mitochondrial division. The MD-ring has features comparable to that of the plastid-dividing (PD) ring.Abbreviations MD mitochondria-dividing - PD plastid-dividing     

The timing and manner of disassembly of the apparatuses for chloroplast and mitochondrial division in the red alga Cyanidioschyzon merolae

The timing and manner of disassembly of the apparatuses for chloroplast division (the plastid-dividing ring; PD ring) and mitochondrial division (the mitochondrion-dividing ring; MD ring) were investigated in the red alga Cyanidioschyzon merolae De Luca, Taddei and Varano. To do this, we synchronized cells both at the final stage of and just after chloroplast and mitochondrial division, and observed the rings in three dimensions by transmission electron microscopy. The inner (beneath the stromal face of the inner envelope) and middle (in the inter-membrane space) PD rings disassembled completely, and disappeared just before completion of chloroplast division. In contrast, the outer PD and MD rings (on the cytoplasmic face of the outer envelope) remained in the cytosol between daughter organelles after chloroplast and mitochondrial division. The outer rings started to disassemble and disappear from their surface just after organelle division, initially clinging to the outer envelopes at both edges before detaching. The results suggest that the two rings inside the chloroplast disappear just before division, and that this does not interfere with completion of division, while the outer PD and MD rings function throughout and complete chloroplast and mitochondrial division. These results, together with previous studies of C. merolae, disclose the entire cycle of change of the PD and MD rings. Received: 19 May 2000 / Accepted: 3 August 2000     

Real-time analyses of chloroplast and mitochondrial division and differences in the behavior of their dividing rings during contraction

The time courses of chloroplast and mitochondrial division and the morphological changes in the plastid-dividing ring (PD ring) and mitochondrion-dividing ring (MD ring) during chloroplast and mitochondrial division were studied in Cyanidioschyzon merolae De Luca, Taddei and Varano. To accomplish this, chloroplast and cell division of living cells were continuously video-recorded under light microscopy, and the morphological changes in the PD and MD rings were analyzed quantitatively and three-dimensionally by transmission electron microscopy (TEM). Under the light microscope, the diameters of the chloroplast and the cell decreased at uniform velocities, the speed depending on the temperature. To study in detail the sequential morphological change of the mitochondrion in M phase and the contractile mechanism in the divisional planes of the chloroplast and the mitochondrion, we observed the PD and MD rings, which are believed to promote contraction, under TEM, using the diameter of the chloroplast as an index of the time. Three PD rings (an outer PD ring on the cytoplasmic face of the outer envelope, a middle PD ring in the intermembrane space, and an inner PD ring on the stromal face of the inner envelope) were clearly observed, but only the outer MD ring could be observed. The PD ring started to contract soon after it formed, while the contraction of the MD ring did not occur immediately after formation, but was delayed until the contraction of the PD ring was almost complete. Once the MD ring began to contract, the rate of decrease of its circumference was 4 times as high as that of the PD ring. As the outer PD and MD rings contracted, they grew thicker and maintained a constant volume, while the thickness of the inner PD ring did not change and its volume decreased at a constant rate with contraction. In the early stage of contraction, the widths of the three PD rings increased in order, from the outer to the inner ring. With contraction, their widths changed at different rates until they came to have much the same width. In cross-section, the MD ring was wider where it was next to the chloroplast than at the opposite side, adjacent to the nucleus in the early stage of contraction. By the late stage, the widths of the two sides became equal. In our observations, the microbody elongated along the outer MD ring and touched the outer PD ring during contraction of the PD and MD rings. These results clearly revealed differences between the mode of contraction of the outer, middle, and inner PD rings, and between the PD and the MD rings. They also revealed the coordinated widening of the three PD rings, and suggested that the microbody plays a role in the contraction of the PD and MD rings. Received: 1 July 1998 / Accepted: 1 September 1998     

Structure, function and evolution of the mitochondrial division apparatus

Mitochondria are derived from free-living alpha-proteobacteria that were engulfed by eukaryotic host cells through the process of endosymbiosis, and therefore have their own DNA which is organized using basic proteins to form organelle nuclei (nucleoids). Mitochondria divide and are split amongst the daughter cells during cell proliferation. Their division can be separated into two main events: division of the mitochondrial nuclei and division of the matrix (the so-called mitochondrial division, or mitochondriokinesis). In this review, we first focus on the cytogenetical relationships between mitochondrial nuclear division and mitochondriokinesis. Mitochondriokinesis occurs after mitochondrial nuclear division, similar to bacterial cytokinesis. We then describe the fine structure and dynamics of the mitochondrial division ring (MD ring) as a basic morphological background for mitochondriokinesis. Electron microscopy studies first identified a small electron-dense MD ring in the cytoplasm at the constriction sites of dividing mitochondria in the slime mold Physarum polycephalum, and then two large MD rings (with outer cytoplasmic and inner matrix sides) in the red alga Cyanidioschyzon merolae. Now MD rings have been found in all eukaryotes. In the third section, we describe the relationships between the MD ring and the FtsZ ring descended from ancestral bacteria. Other than the GTPase, FtsZ, mitochondria have lost most of the proteins required for bacterial cytokinesis as a consequence of endosymbiosis. The FtsZ protein forms an electron transparent ring (FtsZ or Z ring) in the matrix inside the inner MD ring. For the fourth section, we describe the dynamic association between the outer MD ring with a ring composed of the eukaryote-specific GTPase dynamin. Recent studies have revealed that eukaryote-specific GTPase dynamins form an electron transparent ring between the outer membrane and the MD ring. Thus, mitochondriokinesis is thought to be controlled by a mitochondrial division (MD) apparatus including a dynamic trio, namely the FtsZ, MD and dynamin rings, which consist of a chimera of rings from bacteria and eukaryotes in primitive organisms. Since the genes for the MD ring and dynamin rings are not found in the prokaryotic genome, the host genomes may make these rings to actively control mitochondrial division. In the fifth part, we focus on the dynamic changes in the formation and disassembly of the FtsZ, MD and dynamin rings. FtsZ rings are digested during a later period of mitochondrial division and then finally the MD and dynamin ring apparatuses pinched off the daughter mitochondria, supporting the idea that the host genomes are responsible for the ultimate control of mitochondrial division. We discuss the evolution, from the original vesicle division (VD) apparatuses to VD apparatuses including classical dynamin rings and MD apparatuses. It is likely that the MD apparatuses involving the dynamic trio evolved into the plastid division (PD) apparatus in Bikonta, while in Opisthokonta, the MD apparatus was simplified during evolution and may have branched into the mitochondrial fusion apparatus. Finally, we describe the possibility of intact isolation of large MD/PD apparatuses, the identification of all their proteins and their related genes using C. merolae genome information and TOF-MS analyses. These results will assist in elucidating the universal mechanism and evolution of MD, PD and VD apparatuses.     

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.     

Checkpoint control on mitochondrial division inCyanidioschyzon merolae

Summary It is generally accepted that mitochondria proliferate by division. However, since the apparatus for mitochondrial division was discovered only recently, the basic mechanism of mitochondrial division remains poorly understood. The unicellular red algaCyanidioschyzon merolae is the only organism in which the existence of the apparatus for mitochondrial division (mitochondrion-dividing ring) has been proved by electron microscopy. Since mitochondrial division, mitosis, and cytokinesis regularly occurred in that order, we can assume that tight linkage exists between mitochondrial division and the mitotic cycle. To examine this assumption, we performed experiments with aphidicolin, a specific inhibitor of DNA polymerase , using cells that had been synchronized by a 12 h light/12 h dark treatment. The effects of aphidicolin onC. merolae cells were examined by both epifluorescence and electron microscopy. When cells synchronized at the S phase were treated with aphidicolin, neither mitosis nor cytokinesis occurred. Epifluorescence microscopy after staining with 3,3-dihexyloxacarbocyanine iodide (DiOC₆; a mitochondrion-specific fluorochrome) revealed that mitochondrial division was also completely inhibited. Nevertheless, electron-microscopic examination of the aphidicolin-treated cells clearly revealed the presence of a mitochondrion-dividing ring in mitochondria in all cells examined, in spite of the absence of mitochondrial division. Microbodies, which might be related to mitochondrial division inC. merolae, also failed to divide and became attached to the mitochondrion-dividing rings. These results imply the presence of a checkpoint control mechanism that inhibits division of mitochondria and microbodies in the absence of the synthesis of cell-nuclear DNA.Abbreviation DiOC₆ 3,3-dihexyloxacarbocyanine iodide     

Isolation of the cell-nuclear,mitochondrial, and chloroplast DNA from the ultra-small eukaryoteCyanidioschyzon merolae

Summary The amounts of cell-nuclear DNA (cl-DNA), mitochondrial DNA (mt-DNA) and chloroplast DNA (cp-DNA) inCyanidioschyzon merolae were estimated by using a video-intensified microscope (VIM) system.C. merolae had the smallest amount of cell-nuclear DNA among eukaryotes. The results show that a cell-nucleus, a mitochondrion and a chloroplast contain an average 8.0×10³kbp, 1.6×10³kbp, and 5.0×10³kbp, respectively. To confirm these results, cl-DNA, mt-DNA, and cp-DNA were isolated from cells by density centrifugation on Hoechst 33258/CsCl after density centrifugation on ethidium bromide/CsCl. The amounts of cl-DNA, mt-DNA, and cp-DNA obtained from the bands supported the data shown by the VIM-system. The cytochemical and biochemical characteristics were compared with those ofCyanidium caldarium RK-1 andC. caldarium Forma A. The values of cl-DNA and cp-DNA ofC. merolae were about 1.716 and 1.709, respectively. The order in density was different from that ofC. caldarium Forma A but very similar to that ofC. caldarium RK-1. However, the restriction patterns of cp-DNA inC. merolae differed from those ofC. caldarium RK-1.     

Chloroplast division machinery as revealed by immunofluorescence and electron microscopy

The division of chloroplasts (plastids) is critical for the viability of photosynthetic eukaryotes. Previously we reported on the chloroplast division apparatus, which consists of inner and outer double or triple rings (PD rings). Chloroplasts are assumed to arise from bacterial endosymbionts, while bacterial division is instigated by a bacterial cytokinesis Z-ring protein (FtsZ). Here we present immunofluorescence and electron-microscopic evidence of chloroplast division via complex machinery involving the FtsZ and PD rings in the higher plant Pelargonium zonale Ait. Prior to invagination, the FtsZ protein was attached to a ring at the stromal division site. Following formation of the FtsZ ring, the inner stromal and outer cytosolic PD rings appeared, signifying the initiation of invagination. The FtsZ ring and the PD rings were found at the leading edge of chloroplast constriction throughout division. During chloroplast division, neither the FtsZ nor the inner rings changed width, but the volume of the outer ring gradually increased. We suggest that the FtsZ ring determines the division region, after which the inner and outer PD rings are formed as a lining for the FtsZ ring. With the outer ring providing the motivating force, the FtsZ and inner PD rings ultimately decompose to their base components.     

A possible role for actin dots in the formation of the contractile ring in the ultra-micro algaCyanidium caldarium RK-1

Summary In the primitive red algaCyanidium caldarium RK-1, cytokinesis is controlled by a simple contractile ring, as in animal cells. To clarify the mechanism of formation of the contractile ring, we isolated actin genes and performed an immunocytological study.C. caldarium RK-1 has two actin genes encoding proteins with the same sequence of 377 amino acids. The primary structure indicated that the actin molecules ofC. caldarium RK-1 are typical, despite the fact that the organism is considered to be phylogenetically primitive. We prepared antiserum against aC. caldarium RK-1 actin fusion protein and indirect immunofluorescence staining was performed. In interphase cells, many actin dots were observed in the cytoplasm but none at the future cleavage plane. Prior to cytokinesis, some of these dots appeared and became aligned along the equatorial plane. At the same time, a thin immature contractile ring was observed to appear to be formed by connection of the aligned actin dots. This immature contractile ring thickened to nearly its maximum size by the time cytokinesis began. The formation of the contractile ring seemed to be a result of de novo assembly of actin monomers, rather than a result of the accumulation and bundling of pre-existing actin filaments. During the constriction of the contractile ring, no actin dots were observed in the cytoplasm. These observations suggest that actin dots are responsible for the formation of the contractile ring, but are not necessary for its disintegration. Furthermore, immunogold localization specific for actin revealed at electron microscopy level that fine filaments running just beneath the cleavage furrow are, in fact, actin filaments.Abbreviations ORF open reading frame - IPTG isopropyl--D(–)-thiogalactopyranoside - SDS-PAGE sodium dodecyl sulphate-poly-acrylamide gel electrophoresis - DAPI 4,6-diamidino-2-phenylindole     

Isolation of dividing chloroplasts with intact plastid-dividing rings from a synchronous culture of the unicellular red alga Cyanidioschyzon merolae

In order to obtain a three-dimensional view of the plastid-dividing ring (PD ring) and promote the biochemical study of plastid division, we developed a procedure to isolate structurally intact dividing chloroplasts (rhodoplasts) possessing PD rings from a highly synchronized culture of the unicellular red alga Cyanidioschyzon merolae. The procedure consists of five steps. (1) The chloroplast division cycle is synchronized by light/dark cycles and treatment with 5-fluorodeoxyuridine. (2) The synchronized cells are treated with hypotonic solution. (3) The swollen cells are lysed in a French Pressure Cell. (4) The lysate is treated with DNase I. (5) The intact chloroplasts are separated by density-gradient centrifugation. The PD ring was visualized by fluorescence microscopy, after labeling the surface proteins of isolated chloroplasts with N-hydroxy-sulfo-succinimidyl biotin and detecting them with fluorescein isothiocyanate avidin. Scanning electron microscopy (SEM) showed that the outer envelopes and PD rings were conserved on the isolated dividing chloroplasts. These are the first fluorescence microscopic and SEM images of the PD ring and they clearly show PD rings encircling isolated dividing chloroplasts in three dimensions. Received: 15 April 1999 / Accepted: 12 May 1999     

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.     

PLASTID DIVISION IN MALLOMONAS (SYNUROPHYCEAE,HETEROKONTA)1

Laser scanning confocal microscopy and TEM were used to study the morphology of secondary plastids in algae of the genus Mallomonas (Synurophyceae). At interphase, Mallomonas splendens (G. S. West) Playfair, M. rasilis Dürrschm., M. striata Asmund, and M. adamas K. Harris et W. H. Bradley contained a single H‐shaped plastid consisting of two large lobes connected by a narrow isthmus. Labeling of DNA revealed a necklace‐like arrangement of plastid nucleoids at the periphery of the M. splendens plastid and a less‐patterned array in M. rasilis. The TEM of M. splendens and M. rasilis showed an electron‐dense belt surrounding the plastid isthmus in interphase cells; this putative plastid‐dividing ring (PD ring) was adpressed to the inner pair of the four plastid membranes, suggesting that it is homologous to the PD ring of green and red plastids. The PD ring did not contain actin (indicated by lack of staining with phalloidin) and displayed filaments or tubules of 5–10 nm in diameter that may be homologous to the tubules described in red algal PD rings. Confocal microscopy of chl autofluorescence from M. splendens showed that the plastid isthmus was severed as mitosis began, giving rise to two single‐lobed daughter plastids, which, as mitosis and cell division progressed, separated from one another and then each constricted to form the H‐shaped plastids of daughter cells. Similar plastid division cycles were observed in M. rasilis and M. adamas; however, the plastid isthmus of M. striata was retained throughout most of cell division and was eventually severed by the cell cleavage furrow.     

The cellular machineries responsible for the division of endosymbiotic organelles

Chloroplasts (plastids) and mitochondria evolved from endosymbiotic bacteria. These organelles perform vital functions in photosynthetic eukaryotes, such as harvesting and converting energy for use in biological processes. Consistent with their evolutionary origins, plastids and mitochondria proliferate by the binary fission of pre-existing organelles. Here, I review the structures and functions of the supramolecular machineries driving plastid and mitochondrial division, which were discovered and first studied in the primitive red alga Cyanidioschyzon merolae. In the past decade, intact division machineries have been isolated from plastids and mitochondria and examined to investigate their underlying structure and molecular mechanisms. A series of studies has elucidated how these division machineries assemble and transform during the fission of these organelles, and which of the component proteins generate the motive force for their contraction. Plastid- and mitochondrial-division machineries have important similarities in their structures and mechanisms despite sharing no component proteins, implying that these division machineries evolved in parallel. The establishment of these division machineries might have enabled the host eukaryotic ancestor to permanently retain these endosymbiotic organelles by regulating their binary fission and the equal distribution of resources to daughter cells. These findings provide key insights into the establishment of endosymbiotic organelles and have opened new avenues of research into their evolution and mechanisms of proliferation.     

FLORIDOSIDES IN CYANIDIUM CALDARIUM,CYANIDIOSCHYZON MEROLAE AND GALDIERIA SULPHURARIA(RHODOPHYTA,CYANIDIOPHYCEAE)1

Cyanidium caldarium Geitler, Cyanidioschyzon merolae De Luca, Taddei & Varano and Galdieria sulphuraria (Galdieri) Merola are the three thermoacidophilic algae characterized by a chloroplast which is bounded by a single membrane. The presence of this atypical chloroplast made the inclusion of these algae in the Rhodophyta difficult. The discovery in the three algae of floridoside and isofloridoside, typical storage products of red algae, in compatible with their inclusion in the Rhodophyta     

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.     

Analysis of a plastid gene cluster reveals a close relationship betweenCyanidioschyzon andCyanidium

Cyanidioschyzon merolae andCyanidium caldarium are representative species among of the most primitive algae, although the two species are distinctly different in various morphological traits. We determined the nucleotide sequence of therbcL gene and a flanking 8-kb region in the plastid genome of each of these algae. In both algae, 12 genes were identified in this region, in an identical order. This gene order is not conserved in the plastid genomes of other species of the kingdom Plantae that have been sequenced to data. An additional unidentified open reading frame was also found in the two algae that we analyzed, which has not been described in any other species of algae includingPorphyra purpurea. Comparison of the amino acid sequences of selected genes also supported the conclusion thatCyanidioschyzon merolae andCyanidium caldarium are closely related and that they are distinct from other rhodophytes. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession numbers D63675 and D63676.     

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

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

Storage glucan and glucosyltransferase isozymes of cyanidioschyzon merolae: A primitive eukaryote

Cyanidioschyzon merolae, a primitive eukaryotic alga isolated from supposedly pure cultures of the thermoacidophilic alga, Cyanidium caldarium, has many of the characteristics of such prokaryotes as bacteria and the cyanobacteria. Cyanidioschyzon appears to have even more of these prokaryotic features than does Cyanidium. Cyanidioschyzon divides by binary fission as do most bacteria. Its thylakoids are arranged along the periphery of the cell, like the cyanobacteria. Its formation of storage glucan, as well as the type of sugar formed is more like that of the blue-green algae rather than that of the red algae. Cyanidioschyzon merolae may be much more primitive than Cyanidium caldarium, and could be the most primitive eukaryotic cell.     

The dynamic surface of dividing cyanelles and ultrastructure of the region directly below the surface in <Emphasis Type="Italic">Cyanophora paradoxa</Emphasis>

The cyanelles of glaucocystophytes are probably the most primitive of known extant plastids and the closest to cyanobacteria. Their kidney shape and FtsZ arc during the early stage of division define cyanelle division. In order to deepen and expand earlier results (Planta 227:177–187, 2007), cells of Cyanophora paradoxa were fixed with two different chemical and two different freeze-fixation methods. In addition, cyanelles from C. paradoxa were isolated to observe the surface structure of dividing cyanelles using field emission scanning electron microscopy (FE-SEM). A shallow furrow started on one side of the division plane. The furrow subsequently extended, covering the entire division circle, and then invaginated deeply, becoming clearly visible. The typical FtsZ arc was 2.3–3.4 μm long. This length matches that of the cleavage furrow observed using FE-SEM. The cyanelle cleavage furrows are from one-fourth to one-half of the circumference of the division plane. The shallow furrow that appears on the cyanelle outer surface effectively changes the division plane. Using freeze-fixation methods, the electron-dense stroma and peptidoglycan could be distinguished. In addition, an electron-dense belt structure (the cyanelle ring) was observed inside the leading edge at the cyanelle division plane. The FtsZ arc is located at the division plane ahead of the cyanelle ring. Immunogold-TEM localization shows that FtsZ is located interiorly of the cyanelle ring. The lack of an outer PD ring, together with the arch-shaped furrow, suggests that the mechanical force of the initial (arch shaped) septum furrow constriction comes from inside the cyanelle.