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Holliday junctions, four-stranded DNA structures formed during homologous recombination, are disentangled by resolvases that have been found in prokaryotes and eukaryotes but not in plant organelles. Here, we identify monokaryotic chloroplast 1 (MOC1) as a Holliday junction resolvase in chloroplasts by analyzing a green alga Chlamydomonas reinhardtii mutant defective in chloroplast nucleoid (DNA-protein complex) segregation. MOC1 is structurally similar to a bacterial Holliday junction resolvase, resistance to ultraviolet (Ruv) C, and genetically conserved among green plants. Reduced or no expression of MOC1 in Arabidopsis thaliana leads to growth defects and aberrant chloroplast nucleoid segregation. In vitro biochemical analysis and high-speed atomic force microscopic analysis revealed that A. thaliana MOC 1 (AtMOC1) binds and cleaves the core of Holliday junctions symmetrically. MOC1 may mediate chloroplast nucleoid segregation in green plants by resolving Holliday junctions.

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.

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.

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 1 , 2 . 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.

In chloroplast division, the plastid-dividing (PD) ring is a main structure of the PD machinery and is a universal structure in the plant kingdom. However, the components and formation of the PD ring have been enigmatic. By proteomic analysis of PD machineries isolated from Cyanidioschyzon merolae, we identified the glycosyltransferase protein plastid-dividing ring 1 (PDR1), which constructs the PD ring and is widely conserved from red alga to land plants. Electron microscopy showed that the PDR1 protein forms a ring with carbohydrates at the chloroplast-division site. Fluorometric saccharide ingredient analysis of purified PD ring filaments showed that only glucose was included, and down-regulation of PDR1 impaired chloroplast division. Thus, the chloroplasts are divided by the PD ring, which is a bundle of PDR1-mediated polyglucan filaments.

Peroxisomes (microbodies) are ubiquitous single-membrane–bounded organelles and fulfill essential roles in the cellular metabolism. They are found in virtually all eukaryotic cells and basically multiply by division. However, the mechanochemical machinery involved in peroxisome division remains elusive. Here, we first identified the peroxisome-dividing (POD) machinery. We isolated the POD machinery from Cyanidioschyzon merolae , a unicellular red alga containing a single peroxisome. Peroxisomal division in C. merolae can be highly synchronized by light/dark cycles and the microtubule-disrupting agent oryzalin. By proteomic analysis based on the complete genome sequence of C. merolae , we identified a dynamin-related protein 3 (DRP3) ortholog, CmDnm1 (Dnm1), that predominantly accumulated with catalase in the dividing-peroxisome fraction. Immunofluorescence microscopy demonstrated that Dnm1 formed a ring at the division site of the peroxisome. The outlines of the isolated dynamin rings were dimly observed by phase-contrast microscopy and clearly stained for Dnm1. Electron microscopy revealed that the POD machinery was formed at the cytoplasmic side of the equator. Immunoelectron microscopy showed that the POD machinery consisted of an outer dynamin-based ring and an inner filamentous ring. Down-regulation of Dnm1 impaired peroxisomal division. Surprisingly, the same Dnm1 serially controlled peroxisomal division after mitochondrial division. Because genetic deficiencies of Dnm1 orthologs in multiperoxisomal organisms inhibited both mitochondrial and peroxisomal proliferation, it is thought that peroxisomal division by contraction of a dynamin-based machinery is universal among eukaryotes. These findings are useful for understanding the fundamental systems in eukaryotic cells.

Ultrasmall algae have attracted the attention of biologists investigating the basic mechanisms underlying living systems. Their potential as effective organisms for producing useful substances is also of interest in bioindustry. Although genomic information is indispensable for elucidating metabolism and promoting molecular breeding, many ultrasmall algae remain genetically uncharacterized. Here, we present the nuclear genome sequence of an ultrasmall green alga of freshwater habitats, Medakamo hakoo . Evolutionary analyses suggest that this species belongs to a new genus within the class Trebouxiophyceae. Sequencing analyses revealed that its genome, comprising 15.8 Mbp and 7629 genes, is among the smallest known genomes in the Viridiplantae. Its genome has relatively few genes associated with genetic information processing, basal transcription factors, and RNA transport. Comparative analyses revealed that 1263 orthogroups were shared among 15 ultrasmall algae from distinct phylogenetic lineages. The shared gene sets will enable identification of genes essential for algal metabolism and cellular functions. Multi-omic and evolutionary analyses of the ultrasmall green algae, Medakomo hakoo , suggest that it might belong to a new genus within the class Trebouxiophyceae and provide further insight into the essential genes for microalgae.

In many eukaryotes, cytokinesis proceeds in two successive steps: first, ingression of the cleavage furrow and second, abscission of the intercellular bridge. In animal cells, the actomyosin contractile ring is involved in the first step, while the endosomal sorting complex required for transport (ESCRT), which participates in various membrane fusion/fission events, mediates the second step. Intriguingly, in archaea, ESCRT is involved in cytokinesis, raising the hypothesis that the function of ESCRT in eukaryotic cytokinesis descended from the archaeal ancestor. In eukaryotes other than in animals, the roles of ESCRT in cytokinesis are poorly understood. To explore the primordial core mechanisms for eukaryotic cytokinesis, we investigated ESCRT functions in the unicellular red alga that diverged early in eukaryotic evolution. provides an excellent experimental system. The cell has a simple organelle composition. The genome (16.5 Mb, 5335 genes) has been completely sequenced, transformation methods are established, and the cell cycle is synchronized by a light and dark cycle. Similar to animal and fungal cells, cells divide by furrowing at the division site followed by abscission of the intercellular bridge. However, they lack an actomyosin contractile ring. The proteins that comprise ESCRT-I-IV, the four subcomplexes of ESCRT, are partially conserved in . Immunofluorescence of native or tagged proteins localized the homologs of the five ESCRT-III components [charged multivesicular body protein (CHMP) 1, 2, and 4-6], apoptosis-linked gene-2-interacting protein X (ALIX), the ESCRT-III adapter, and the main ESCRT-IV player vacuolar protein sorting (VPS) 4, to the intercellular bridge. In addition, ALIX was enriched around the cleavage furrow early in cytokinesis. When the ESCRT function was perturbed by expressing dominant-negative VPS4, cells with an elongated intercellular bridge accumulated-a phenotype resulting from abscission failure. Our results show that ESCRT mediates cytokinetic abscission in . The fact that ESCRT plays a role in cytokinesis in archaea, animals, and early diverged alga supports the hypothesis that the function of ESCRT in cytokinesis descended from archaea to a common ancestor of eukaryotes.

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.