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Primary plastids originated from a free-living cyanobacterial ancestor and possess their own genomes-probably a few DNA copies. These genomes, which are organized in centrally located plastid nuclei (CN-type pt-nuclei), are produced from preexisting plastids by binary division. Ancestral algae with a CN-type pt-nucleus diverged and evolved into two basal eukaryotic lineages: red algae with circular (CL-type) pt-nuclei and green algae with scattered small (SN-type) pt-nuclei. Although the molecular dynamics of pt-nuclei in green algae and plants are now being analyzed, the process of the conversion of the original algae with a CN-type pt-nucleus to red algae with a CL-type one has not been studied. Here, we show that the CN-type pt-nucleus in the primitive red alga Cyanidioschyzon merolae can be changed to the CL-type by application of drying to produce slight cell swelling. This result implies that CN-type pt-nuclei are produced by compact packing of CL-type ones, which suggests that a C. merolae-like alga was the original progenitor of the red algal lineage. We also observed that the CL-type pt-nucleus has a chain-linked bead-like structure. Each bead is most likely a small unit of DNA, similar to CL-type pt-nuclei in brown algae. Our results thus suggest a C. merolae-like alga as the candidate for the secondary endosymbiont of brown algae.

Mitochondria, which evolved from a free-living bacterial ancestor, contain their own genomes and genetic systems and are produced from preexisting mitochondria by binary division. The mitochondrion-dividing (MD) ring is the main skeletal structure of the mitochondrial division machinery. However, the assembly mechanism and molecular identity of the MD ring are unknown. Multi-omics analysis of isolated mitochondrial division machinery from the unicellular alga Cyanidioschyzon merolae revealed an uncharacterized glycosyltransferase, MITOCHONDRION-DIVIDING RING1 (MDR1), which is specifically expressed during mitochondrial division and forms a single ring at the mitochondrial division site. Nanoscale imaging using immunoelectron microscopy and componential analysis demonstrated that MDR1 is involved in MD ring formation and that the MD ring filaments are composed of glycosylated MDR1 and polymeric glucose nanofilaments. Down-regulation of MDR1 strongly interrupted mitochondrial division and obstructed MD ring assembly. Taken together, our results suggest that MDR1 mediates the synthesis of polyglucan nanofilaments that assemble to form the MD ring. Given that a homolog of MDR1 performs similar functions in chloroplast division, the establishment of MDR1 family proteins appears to have been a singular, crucial event for the emergence of endosymbiotic organelles.

Most microalgae accumulate neutral lipids, including triacylglycerol (TAG), into spherical structures called lipid bodies (LBs) under environmental stress conditions such as nutrient depletion. In green algae, starch accumulation precedes TAG accumulation, and the starch is thought to be a substrate for TAG synthesis. However, the relationship between TAG synthesis and the starch content in red algae, as well as how TAG accumulation is regulated, is unclear. In this study, we cultured the primitive red alga Cyanidioschyzon merolae under nitrogen-depleted conditions, and monitored the formation of starch granules (SGs) and LBs using microscopy. SGs stained with potassium iodide were observed at 24 h; however, LBs stained specifically with BODIPY 493/503 were observed after 48 h. Quantitative analysis of neutral sugar and cytomorphological semi-quantitative analysis of TAG accumulation also supported these results. Thus, the accumulation of starch occurred and preceded the accumulation of TAG in cells of C. merolae. However, TAG accumulation was not accompanied by a decrease in the starch content, suggesting that the starch is a major carbon storage sink, at least under nitrogen-depleted conditions. Quantitative real-time PCR revealed that the mRNA levels of genes involved in starch and TAG synthesis rarely changed during the culture period, suggesting that starch and TAG synthesis in C. merolae are not controlled through gene transcription but at other stages, such as translation and/or enzymatic activity.

Triacylglycerol (TAG) produced by microalgae is a potential source of biofuel. Although various metabolic pathways in TAG synthesis have been identified in land plants, the pathway of TAG synthesis in microalgae remains to be clarified. The unicellular rhodophyte Cyanidioschyzon merolae has unique properties as a producer of biofuel because of easy culture and feasibility of genetic engineering. Additionally, it is useful in the investigation of the pathway of TAG synthesis, because all of the nuclear, mitochondrial and plastid genomes have been completely sequenced. We found that this alga accumulated TAG under nitrogen deprivation. Curiously, the amount and composition of plastid membrane lipids did not change significantly, whereas the amount of endoplasmic reticulum (ER) lipids increased with considerable changes in fatty acid composition. The nitrogen deprivation did not decrease photosynthetic oxygen evolution per chlorophyll significantly, while phycobilisomes were degraded preferentially. These results suggest that the synthesis of fatty acids is maintained in the plastid, which is used for the synthesis of TAG in the ER. The accumulated TAG contained mainly 18 : 2(9,12) at the C-2 position, which could be derived from phosphatidylcholine, which also contains this acid at the C-2 position.

Chloroplast Division Machinery The machinery for photosynthesis, which captures the Sun's energy to generate carbohydrates, generally resides in subcellular chloroplasts of plant cells. Chloroplasts must divide as the plant cell divides, but to do so requires their own plastid dividing machinery. Yoshida et al. (p. 949 : see the cover) have now analyzed the plastid dividing machinery of the single-celled alga Cyanidioschyzon merolae , whose cells each contain a single chloroplast. The plastid dividing machinery is made up of polysaccharide chains and the proteins that make them, which together generate a ring that constricts to physically divide the chloroplast.

Vacuoles/lysosomes function in endocytosis and in storage and digestion of metabolites. These organelles are inherited by the daughter cells in eukaryotes. However, the mechanisms of this inheritance are poorly understood because the cells contain multiple vacuoles that behave randomly. The primitive red alga Cyanidioschyzon merolae has a minimum set of organelles. Here, we show that C. merolae contains about four vacuoles that are distributed equally between the daughter cells by binding to dividing mitochondria. Binding is mediated by VIG1, a 30-kD coiled-coil protein identified by microarray analyses and immunological assays. VIG1 appears on the surface of free vacuoles in the cytosol and then tethers the vacuoles to the mitochondria. The vacuoles are released from the mitochondrion in the daughter cells following VIG1 digestion. Suppression of VIG1 by antisense RNA disrupted the migration of vacuoles. Thus, VIG1 is essential for tethering vacuoles to mitochondria during vacuole inheritance in C. merolae.

Summary Bacterial cell division systems that include FtsZ are found throughout prokaryotes [1]. Mitochondria arose from an endosymbiotic α-proteobacterial ancestor and proliferate by division [2–4]. However, how the mitochondrial division system was established from bacterial division is not clear. Here, we have isolated intact mitochondrial division (MD) machineries from the primitive red alga Cyanidioschyzon merolae and identified a bacterial ZapA-like protein, ZED, that constricts the basal structure of MD machinery with FtsZ. ZED contains a predicted mitochondrial transit signal and two coiled-coil regions and has partial homology with the bacterial division protein ZapA [5]. Cytological studies revealed that ZED accumulates to form a ring structure that colocalizes with FtsZ beneath the inner membrane. ZED proteins are expressed just before mitochondrial division. The short-form ZED (S-ZED) then appears at the mitochondrial constriction phase. Protein-protein interaction analysis and transient expression of antisense against ZED showed that S-ZED interacts with FtsZ1 to constitute the basal structure of the MD machinery and is required for mitochondrial division. We also demonstrate compelling functional similarity between bacterial ZapA and mitochondrial ZED, suggesting that the bacterial cell division system was incorporated into the MD machinery with remodeling of bacterial division proteins during evolution.

Previous cell cycle studies have been based on cell-nuclear proliferation only. Eukaryotic cells, however, have double membranes-bound organelles, such as the cell nucleus, mitochondrion, plastids and single-membrane-bound organelles such as ER, the Golgi body, vacuoles (lysosomes) and microbodies. Organelle proliferations, which are very important for cell functions, are poorly understood. To clarify this, we performed a microarray analysis during the cell cycle of Cyanidioschyzon merolae. C. merolae cells contain a minimum set of organelles that divide synchronously. The nuclear, mitochondrial and plastid genomes were completely sequenced. The results showed that, of 158 genes induced during the S or G2-M phase, 93 were known and contained genes related to mitochondrial division, ftsZ1-1, ftsz1-2 and mda1, and plastid division, ftsZ2-1, ftsZ2-2 and cmdnm2. Moreover, three genes, involved in vesicle trafficking between the single-membrane organelles such as vps29 and the Rab family protein, were identified and might be related to partitioning of single-membrane-bound organelles. In other genes, 46 were hypothetical and 19 were hypothetical conserved. The possibility of finding novel organelle division genes from hypothetical and hypothetical conserved genes in the S and G2-M expression groups is discussed.

Antisense suppression is a powerful tool to analyze gene function. In this study, we show that antisense RNA suppressed the expression of a target gene in the unicellular red alga, Cyanidioschyzon merolae. In this study, the antisense strand of the catalase gene was cloned and inserted into an expression vector upstream of the GFP gene. This plasmid was introduced into C. merolae cells using a polyethylene glycol-mediated transformation protocol. Using the expression of GFP as a marker of transformed cells, the expression of catalase was examined by immunocytochemistry. Decreased expression of catalase was observed in cells that were transformed with the antisense strand of the catalase gene. These results indicate the utility of this antisense suppression system.

Chloroplast division involves plastid-dividing, dynamin, and FtsZ (PDF) rings. We isolated intact supertwisted (or spiral) and circular PDF machineries from chloroplasts of the red alga Cyanidioschyzon merolae . After individual intact PDF machineries were stretched to four times their original lengths with optical tweezers, they spontaneously returned to their original sizes. Dynamin-released PDF machineries did not retain the spiral structure and could not be stretched. Thus, dynamin may generate the motive force for contraction by filament sliding in dividing chloroplasts, in addition to pinching-off the membranes.

The ultrasmall unicellular red alga Cyanidioschyzon merolae lives in the extreme environment of acidic hot springs and is thought to retain primitive features of cellular and genome organization. We determined the 16.5-Mb nuclear genome sequence of C. merolae 10D as the first complete algal genome. BLASTs and annotation results showed that C. merolae has a mixed gene repertoire of plants and animals, also implying a relationship with prokaryotes, although its photosynthetic components were comparable to other phototrophs. The unicellular green alga Chlamydomonas reinhardtii has been used as a model system for molecular biology research on, for example, photosynthesis, motility, and sexual reproduction. Though both algae are unicellular, the genome size, number of organelles, and surface structures are remarkably different. Here, we report the characteristics of double membrane- and single membrane-bound organelles and their related genes in C. merolae and conduct comparative analyses of predicted protein sequences encoded by the genomes of C. merolae and C. reinhardtii. We examine the predicted proteins of both algae by reciprocal BLASTP analysis, KOG assignment, and gene annotation. The results suggest that most core biological functions are carried out by orthologous proteins that occur in comparable numbers. Although the fundamental gene organizations resembled each other, the genes for organization of chromatin, cytoskeletal components, and flagellar movement remarkably increased in C. reinhardtii. Molecular phylogenetic analyses suggested that the tubulin is close to plant tubulin rather than that of animals and fungi. These results reflect the increase in genome size, the acquisition of complicated cellular structures, and kinematic devices in C. reinhardtii.

In the mitochondria of primitive eukaryotes, FtsZ and dynamin are part of the machinery involved in division of the inner and outer membranes, respectively. These genes also commonly function in the same manner during chloroplast division. In this study, a relationship between the localization of the inner and outer division machinery was directly shown for the first time. Triple immunofluorescent labeling was performed in the red alga Cyanidioschyzon merolae by a device using narrow bandpass filter sets and bright photostable dyes. FtsZ (CmFtsZ1) and dynamin (CmDnm1) localizations were examined simultaneously throughout the mitochondrial division cycle with an alternative mitochondrial marker protein, the mitochondrial translation elongation factor EF-Tu, whose localization was also shown to be identical to the mitochondrial matrix. FtsZ and dynamin did not necessarily co-localize when both were recruited to the mitochondrial constriction site, indicating that inner and outer dividing machineries are not in tight association during the late stage of division. (J Histochem Cytochem 52:843–849, 2004)

Small, compact genomes of ultrasmall unicellular algae provide information on the basic and essential genes that support the lives of photosynthetic eukaryotes, including higher plants. Here we report the 16,520,305-base-pair sequence of the 20 chromosomes of the unicellular red alga Cyanidioschyzon merolae 10D as the first complete algal genome. We identified 5,331 genes in total, of which at least 86.3% were expressed. Unique characteristics of this genomic structure include: a lack of introns in all but 26 genes; only three copies of ribosomal DNA units that maintain the nucleolus; and two dynamin genes that are involved only in the division of mitochondria and plastids. The conserved mosaic origin of Calvin cycle enzymes in this red alga and in green plants supports the hypothesis of the existence of single primary plastid endosymbiosis. The lack of a myosin gene, in addition to the unexpressed actin gene, suggests a simpler system of cytokinesis. These results indicate that the C. merolae genome provides a model system with a simple gene composition for studying the origin, evolution and fundamental mechanisms of eukaryotic cells.

Cyanidioschyzon merolae is a small unicellular red alga that is considered to belong to one of the most deeply branched taxa in the plant kingdom. Its genome size is estimated to be 16.5 Mbp, one of the smallest among free-living eukaryotes. In the nucleus containing this small genome, one nucleolus is clearly observed, but the molecular basis for the intranuclear structure including ribosomal DNA organization is still unclear. We constructed a bacterial artificial chromosome library for C. merolae 10D composed of two subsets with different insert size distributions. The two subsets have average insert sizes of 97 and 48 kb, representing 10.0- and 6.9-fold genome-equivalent coverage of the haploid genome, respectively. For application to whole-genome shotgun sequencing, the termini of each clone were sequenced as sequence-tagged connectors and mapped on the contigs assigned to chromosomes. Screening for rRNA genes by conventional colony hybridization with high-density filter blots and subsequent sequencing revealed that the C. merolae genome contained the smallest number of ribosomal DNA units among all the eukaryotes examined to date. They consist of only 3 single units of rRNA genes distributed on separate chromosomal loci, representing an implication for concerted evolution. Based on these results, the origin and evolution of the nucleolus are discussed.

The cell of the unicellular red alga Cyanidioschyzon merolae contains a single chloroplast and mitochondrion, the division of which is tightly synchronized by a light/dark cycle. The genome content is extremely simple, with a low level of genetic redundancy, in photosynthetic eukaryotes. In addition, transient transformation and stable transformation by homologous recombination have been reported. However, for molecular genetic analyses of phenomena that are essential for cellular growth and survival, inducible gene expression/suppression systems are needed. Here, we report the development of a heat-shock inducible gene expression system in C. merolae. CMJ101C, encoding a small heat shock protein, is transcribed only when cells are exposed to an elevated temperature. Using a superfolder GFP as a reporter protein, the 200-bp upstream region of CMJ101C orf was determined to be the optimal promoter for heat-shock induction. The optimal temperature to induce expression is 50°C, at which C. merolae cells are able to proliferate. At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression. After the heat shock, the mRNA level decreases rapidly. As an example of the system, the expression of a dominant negative form of chloroplast division DRP5B protein, which has a mutation in the GTPase domain, was induced. Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure. This result suggests that the dominant negative DRP5B inhibited the final scission of the chloroplast division site, but not the earlier stages of division site constriction. It is also suggested that cell cycle progression is not arrested by the impairment of chloroplast division at the final stage.

Summary The cell cycle usually refers to the mitotic cycle, but the cell-division cycle in the plant kingdom consists of not only nuclear but also mitochondrial and chloroplast division cycle. However, an integrated control system that initiates division of the three organelles has not been found. We report that a novel C-terminal kinesin-like protein, three-organelle division-inducing protein (TOP), controls nuclear, mitochondrial and chloroplast divisions in the red alga Cyanidioschyzon merolae . A proteomics study revealed that TOP is a member of a complex of mitochondrial-dividing (MD) and plastid-dividing (PD) machineries (MD/PD machinery complex) just prior to constriction. After TOP localizes at the MD/PD machinery complex, mitochondrial and chloroplast divisions occur and the components of the MD/PD machinery complexes are phosphorylated. Furthermore, we found that TOP downregulation impaired both mitochondrial and chloroplast divisions. MD/PD machinery complexes were formed normally at each division site but they were neither phosphorylated nor constricted in these cells. Immunofluorescence signals of Aurora kinase (AUR) were localized around the MD machinery before constriction, whereas AUR was dispersed in the cytosol by TOP downregulation, suggesting that AUR is required for the constriction. Taken together our results suggest that TOP induces phosphorylation of MD/PD machinery components to accomplish mitochondrial and chloroplast divisions prior to nuclear division, by relocalization of AUR. In addition, given the presence of TOP homologs throughout the eukaryotes, and the involvement of TOP in mitochondrial and chloroplast division may illuminate the original function of C-terminal kinesin-like proteins.

The ability of the primitive red alga Cyanidioschyzon merolae to adapt to high temperatures was utilized to produce thermotolerant transgenic plants. C. merolae inhabits an extreme environment (42 degrees C, pH 2.5) and the nuclear, mitochondrial, and plastid genomes have been sequenced. We analyzed expressed sequence tag (EST) data to reveal mechanisms of tolerance to high temperatures. The stromal ascorbate peroxidase (CmstAPX) that scavenges reactive oxygen species (ROS) was expressed at high levels (4th of 4,479 entries), thus, it offers clues to understanding high-temperature tolerance. CmstAPX has a chloroplast transit peptide (cTP) and a peroxidase domain. The peroxidase domain of CmstAPX has deletions and insertions when compared with that of Arabidopsis thaliana stromal APX (AtstAPX). To clarify aspects of tolerance to oxidative and high-temperature stress, we produced transgenic A. thaliana plants overexpressing CmstAPX and AtstAPX. CmstAPX plants showed higher activities of soluble APX than those of wild-type and AtstAPX plants. Fluorescence signals of a GFP fusion protein, immuno-fluorescence, and immunogold electron microscopy showed that CmstAPX was localized in the stroma of chloroplasts. Compared with wild-type plants and AtstAPX plants, CmstAPX plants were more tolerant to oxidative stress induced by methylviologen (MV, 0.4 muM) and high-temperature stress (33 degrees C). CmstAPX plants retained the highest chlorophyll content when treated with MV and high temperature, and their stroma and chloroplasts remained intact in their chloroplasts, whereas they disintegrated in wild-type plants. Our results suggest that the increased activity of APX in the chloroplasts of CmstAPX plants increased thermotolerance by increasing ROS-scavenging capacity at high temperatures.

Plant vacuoles are organelles bound by a single membrane, and involved in various functions such as intracellular digestion, metabolite storage, and secretion. To understand their evolution and fundamental mechanisms, characterization of vacuoles in primitive plants would be invaluable. Algal cells often contain polyphosphate-rich compartments, which are thought to be the counterparts of seed plant vacuoles. Here, we developed a method for isolating these vacuoles from Cyanidioschyzon merolae, and identified their proteins by MALDI TOF-MS. The vacuoles were of unexpectedly high density, and were highly enriched at the boundary between 62 and 80% w/v iodixanol by density-gradient ultracentrifugation. The vacuole-containing fraction was subjected to SDS-PAGE, and a total of 46 proteins were identified, including six lytic enzymes, 13 transporters, six proteins for membrane fusion or vesicle trafficking, five non-lytic enzymes, 13 proteins of unknown function, and three miscellaneous proteins. Fourteen proteins were homologous to known vacuolar or lysosomal proteins from seed plants, yeasts or mammals, suggesting functional and evolutionary relationships between C. merolae vacuoles and these compartments. The vacuolar localization of four novel proteins, namely CMP249C (metallopeptidase), CMJ260C (prenylated Rab receptor), CMS401C (ABC transporter) and CMT369C (o-methyltransferase), was confirmed by labeling with specific antibodies or transient expression of hemagglutinin-tagged proteins. The results presented here provide insights into the proteome of C. merolae vacuoles and shed light on their functions, as well as indicating new features.

Background All previously reported eukaryotic nuclear genome sequences have been incomplete, especially in highly repeated units and chromosomal ends. Because repetitive DNA is important for many aspects of biology, complete chromosomal structures are fundamental for understanding eukaryotic cells. Our earlier, nearly complete genome sequence of the hot-spring red alga Cyanidioschyzon merolae revealed several unique features, including just three ribosomal DNA copies, very few introns, and a small total number of genes. However, because the exact structures of certain functionally important repeated elements remained ambiguous, that sequence was not complete. Obviously, those ambiguities needed to be resolved before the unique features of the C. merolae genome could be summarized, and the ambiguities could only be resolved by completing the sequence. Therefore, we aimed to complete all previous gaps and sequence all remaining chromosomal ends, and now report the first nuclear-genome sequence for any eukaryote that is 100% complete. Results Our present complete sequence consists of 16546747 nucleotides covering 100% of the 20 linear chromosomes from telomere to telomere, representing the simple and unique chromosomal structures of the eukaryotic cell. We have unambiguously established that the C. merolae genome contains the smallest known histone-gene cluster, a unique telomeric repeat for all chromosomal ends, and an extremely low number of transposons. Conclusion By virtue of these attributes and others that we had discovered previously, C. merolae appears to have the simplest nuclear genome of the non-symbiotic eukaryotes. These unusually simple genomic features in the 100% complete genome sequence of C. merolae are extremely useful for further studies of eukaryotic cells.

The complete nucleotide sequence of the plastid genome of the unicellular primitive red alga Cyanidioschyzon merolae 10D (Cyanidiophyceae) was determined. The genome is a circular DNA composed of 149,987 bp with no inverted repeats. The G + C content of this plastid genome is 37.6%. The C. merolae plastid genome contains 243 genes, which are distributed on both strands and consist of 36 RNA genes (3 rRNAs, 31 tRNAs, tmRNA, and a ribonuclease P RNA component) and 207 protein genes, including unidentified open reading frames. The striking feature of this genome is the high degree of gene compaction; it has very short intergenic distances (approximately 40% of the protein genes were overlapped) and no genes have introns. This genome encodes several genes that are rarely found in other plastid genomes. A gene encoding a subunit of sulfate transporter (cysW) is the first to be identified in a plastid genome. The cysT and cysW genes are located in the C. merolae plastid genome in series, and they probably function together with other nuclear-encoded components of the sulfate transport system. Our phylogenetic results suggest that the Cyanidiophyceae, including C. merolae, are a basal clade within the red lineage plastids.