Golgi inheritance in the primitive red alga, Cyanidioschyzon merolae

The Golgi body has important roles in modifying, sorting, and transport of proteins and lipids. Eukaryotic cells have evolved in various ways to inherit the Golgi body from mother to daughter cells, which allows the cells to function properly immediately after mitosis. Here we used Cyanidioschyzon merolae, one of the most suitable systems for studies of organelle dynamics, to investigate the inheritance of the Golgi. Two proteins, Sed5 and Got1, were used as Golgi markers. Using immunofluorescence microscopy, we demonstrated that C. merolae contains one to two Golgi bodies per cell. The Golgi body was localized to the perinuclear region during the G1 and S phases and next to the spindle poles in a microtubule-dependent manner during M phase. It was inherited together with spindle poles upon cytokinesis. These observations suggested that Golgi inheritance is dependent on microtubules in C. merolae.     

Carotenoid biosynthesis in the primitive red alga Cyanidioschyzon merolae

Cyanidioschyzon merolae is considered to be one of the most primitive of eukaryotic photosynthetic organisms. To obtain insights into the origin and evolution of the pathway of carotenoid biosynthesis in eukaryotic plants, the carotenoid content of C. merolae was ascertained, genes encoding enzymes of carotenoid biosynthesis in this unicellular red alga were identified, and the activities of two candidate pathway enzymes of particular interest, lycopene cyclase and beta-carotene hydroxylase, were examined. C. merolae contains perhaps the simplest assortment of chlorophylls and carotenoids found in any eukaryotic photosynthetic organism: chlorophyll a, beta-carotene, and zeaxanthin. Carotenoids with epsilon-rings (e.g., lutein), found in many other red algae and in green algae and land plants, were not detected, and the lycopene cyclase of C. merolae quite specifically produced only beta-ringed carotenoids when provided with lycopene as the substrate in Escherichia coli. Lycopene beta-ring cyclases from several bacteria, cyanobacteria, and land plants also proved to be high-fidelity enzymes, whereas the structurally related epsilon-ring cyclases from several plant species were found to be less specific, yielding products with beta-rings as well as epsilon-rings. C. merolae lacks orthologs of genes that encode the two types of beta-carotene hydroxylase found in land plants, one a nonheme diiron oxygenase and the other a cytochrome P450. A C. merolae chloroplast gene specifies a polypeptide similar to members of a third class of beta-carotene hydroxylases, common in cyanobacteria, but this gene did not produce an active enzyme when expressed in E. coli. The identity of the C. merolae beta-carotene hydroxylase therefore remains uncertain.     

Centromere dynamics in the primitive red alga Cyanidioschyzon merolae

Centromeres are universally conserved functional units in eukaryotic linear chromosomes, but little is known about the structure and dynamics of the centromere in lower photosynthetic eukaryotes. Here we report the identification of a centromere marker protein CENH3 and visualization of centromere dynamics in the ultra-small primitive red alga Cyanidioschyzon merolae. Immunoblotting and immunofluorescence microscopy showed that CENH3 increased rapidly during S phase, followed by a drastic reconstitution into two discrete foci adjacent to the spindle poles at metaphase, suggesting the cell-cycle-regulated expression of CENH3. Immunoelectron microscopy revealed that the CENH3 signals were associated with the nuclear envelope, implying interplay between the kinetochore complex and the nuclear envelope. These results demonstrate dynamic centromere reconstitution during the cell cycle in an organism in which the chromosomes do not condense at metaphase.     

Characterization of phytochelatin synthase produced by the primitive red alga Cyanidioschyzon merolae

Phytochelatins (PCs), non-protein peptides with the general structure [(γ-Glu-Cys)n-Gly (n≥ 2)], are involved in the detoxification of toxic heavy metals mainly in higher plants. The synthesis of the peptides is mediated by phytochelatin synthase (PCS), which is activated by a range of heavy metals. CmPCS, a PCS-like gene found in the genomic DNA of the primitive red alga Cyanidioschyzon merolae, was isolated and a recombinant protein (rCmPCS) fused with a hexahistidine tag at the N-terminus of CmPCS was produced. The finding that this protein mediated PC synthesis from glutathione in a metal-dependent way clearly establishes that rCmPCS is functional. The maximum activity was attained at a reaction temperature of 50 °C, considerably higher than the temperature required for the maximal activity of PCS isolated from the higher plant Silene cucubalus, probably due to the alga being a thermophile. CmPCS showed optimal pH in a slightly higher region than higher plant PCSs, probably due to the less effective charge relay network in the catalytic triad. In addition, the pattern of enzyme activation by metal ions was specific to rCmPCS, with Ag+, Cu2+, and Hg2+ showing only limited activation. In contrast to other eukaryotic PCSs, CmPCS has an extra domain in the N-terminal region from residues 1 to 109, and contains fewer cysteine residues in the C-terminal domain. These differences may be responsible for the metal specificity of the activation of CmPCS. Although the enzyme preparation lost PCS activity progressively when stored at 4 °C, the inclusion of Cd2+ in the preparation effectively prevented the reduction of activity. Furthermore, Cd2+ effectively restored the activity of the inactivated enzyme. These results indicate that Cd2+ ions bind the enzyme to maintain the structural integrity of the peptides.     

The minimal eukaryotic ribosomal DNA units in the primitive red alga Cyanidioschyzon merolae.

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.     

Identification of novel proteins in isolated polyphosphate vacuoles in the primitive red alga Cyanidioschyzon merolae

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.     

A putative mitochondrial ftsZ gene is present in the unicellular primitive red alga Cyanidioschyzon merolae

Two ftsZ homologues were isolated from the unicellular primitive red alga Cyanidioschyzon merolae (CmftsZ1 and CmftsZ2). Phylogenetic analysis revealed that CmftsZ1 is most closely related to the ftsZ genes of alpha-Proteobacteria, suggesting that it is a mitochondrial-type ftsZ gene, whereas CmftsZ2 is most closely related to the ftsZ genes of cyanobacteria, suggesting that it is a plastid-type ftsZ gene. Southern analysis indicates that CmftsZ1 and CmftsZ2 are both single-copy genes located on chromosome XIV in the C. merolae genome. Northern analysis revealed that both CmftsZ1 and CmftsZ2 are transcribed, and accumulate specifically before cell and organelle division. The results of Western analysis suggest that CmFtsZ1 is localized in mitochondria.     

Evolutionary significance of the ring-like plastid nucleus in the primitive red alga Cyanidioschyzon merolae as revealed by drying

Protoplasma - 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...     

Spatiotemporal dynamics of condensins I and II: evolutionary insights from the primitive red alga Cyanidioschyzon merolae

Condensins are multisubunit complexes that play central roles in chromosome organization and segregation in eukaryotes. Many eukaryotic species have two different condensin complexes (condensins I and II), although some species, such as fungi, have condensin I only. Here we use the red alga Cyanidioschyzon merolae as a model organism because it represents the smallest and simplest organism that is predicted to possess both condensins I and II. We demonstrate that, despite the great evolutionary distance, spatiotemporal dynamics of condensins in C. merolae is strikingly similar to that observed in mammalian cells: condensin II is nuclear throughout the cell cycle, whereas condensin I appears on chromosomes only after the nuclear envelope partially dissolves at prometaphase. Unlike in mammalian cells, however, condensin II is confined to centromeres in metaphase, whereas condensin I distributes more broadly along arms. We firmly establish a targeted gene disruption technique in this organism and find, to our surprise, that condensin II is not essential for mitosis under laboratory growth conditions, although it plays a crucial role in facilitating sister centromere resolution in the presence of a microtubule drug. The results provide fundamental insights into the evolution of condensin-based chromosome architecture and dynamics.     

Optimization of cryopreservation conditions for the unicellular red alga Cyanidioschyzon merolae

Cryopreservation is essential for maintaining stable stocks of organisms. We report the development of a method for cryopreservation of the unicellular red alga Cyanidioschyzon merolae, a model organism for the investigation of the basic architecture of photosynthetic eukaryotes. Glycerol, dimethyl sulfoxide and methanol were examined for their ability to protect the cell from cryoinjury and/or cytotoxicity. It was found that methanol was the most effective as a cryoprotectant for C. merolae. After the optimized setting of parameters such as working concentration of cryoprotectant and the period of slow cooling, cultures were supplemented with 5% (v/v) methanol and frozen by slow cooling using a passive-freezing unit, followed by plunging into liquid nitrogen. We found C. merolae cells retained greater than 80% viability for at least 83 days in storage.     

Subcellular distribution of central carbohydrate metabolism pathways in the red alga Cyanidioschyzon merolae

Main conclusion

Comprehensive subcellular localization analysis revealed that the subcellular distribution of carbohydrate metabolic pathways in the red alga Cyanidioschyzon is essentially identical with that in Arabidopsis , except the lack of transaldolase. In plants, the glycolysis and oxidative pentose phosphate pathways (oxPPP) are located in both cytosol and plastids. However, in algae, particularly red algae, the subcellular localization of enzymes involved in carbon metabolism is unclear. Here, we identified and examined the localization of enzymes related to glycolysis, oxPPP, and tricarboxylic acid (TCA) and Calvin–Benson cycles in the red alga Cyanidioschyzon merolae. A gene encoding transaldolase of the oxPPP was not found in the C. merolae genome, and no transaldolase activity was detected in cellular extracts. The subcellular localization of 65 carbon metabolic enzymes tagged with green fluorescent protein or hemagglutinin was examined in C. merolae cells. As expected, TCA and Calvin–Benson cycle enzymes were localized to mitochondria and plastids, respectively. The analyses also revealed that the cytosol contains the entire glycolytic pathway and partial oxPPP, whereas the plastid contains a partial glycolytic pathway and complete oxPPP, with the exception of transaldolase. Together, these results suggest that the subcellular distribution of carbohydrate metabolic pathways in C. merolae is essentially identical with that reported in the photosynthetic tissue of Arabidopsis thaliana; however, it appears that substrates typically utilized by transaldolase are consumed by glycolytic enzymes in the plastidic oxPPP of C. merolae.     

Purification and characterization of organellar DNA polymerases in the red alga Cyanidioschyzon merolae

DNA polymerase gamma, a mitochondrial replication enzyme of yeasts and animals, is not present in photosynthetic eukaryotes. Recently, DNA polymerases with distant homology to bacterial DNA polymerase I were reported in rice, Arabidopsis, and tobacco, and they were localized to both plastids and mitochondria. We call them plant organellar DNA polymerases (POPs). However, POPs have never been purified in the native form from plant tissues. The unicellular thermotrophic red alga Cyanidioschyzon merolae contains two genes encoding proteins related to Escherichia coli DNA polymerase I (PolA and PolB). Phylogenetic analysis revealed that PolB is an ortholog of POPs. Nonphotosynthetic eukaryotes also have POPs, which suggested that POPs have an ancient origin before eukaryotic photosynthesis. PolA is a homolog of bacterial DNA polymerase I and is distinct from POPs. PolB was purified from the C. merolae cells by a series of column chromatography steps. Recombinant protein of PolA was also purified. Sensitivity to inhibitors of DNA synthesis was different in PolA, PolB, and E. coli DNA polymerase I. Immunoblot analysis and targeting studies with green fluorescent protein fusion proteins demonstrated that PolA was localized in the plastids, whereas PolB was present in both plastids and mitochondria. The expression of PolB was regulated by the cell cycle. The available results suggest that PolB is involved in the replication of plastids and mitochondria.     

Transient gene suppression in a red alga, Cyanidioschyzon merolae 10D

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.     

Expression and localization of two SecA homologs in the unicellular red alga Cyanidioschyzon merolae

SecA is an ATP-driven motor for Sec translocase that participates in bacterial protein export and thylakoidal import in plants. We have reported that Cyanidioschyzon merolae, a unicellular red alga, possesses a nuclear-encoded secA(nuc) and a plastid-encoded secA(pt) gene. In this study we found that the amount of SecA(nuc) protein almost quadrupled at high temperature, whereas that of the SecA(pt) protein increased far less. We were also able to determine the localization of both SecAs to the chloroplast by immunofluorescence and immunoelectron microscopy. We suggest that SecA(nuc) has an important role in the chloroplast at high temperatures.     

Characterization of the nuclear- and plastid-encoded secA-homologous genes in the unicellular red alga Cyanidioschyzon merolae

SecA is an ATP-driven motor for protein translocation in bacteria and plants. Mycobacteria and listeria were recently found to possess two functionally distinct secA genes. In this study, we found that Cyanidioschyzon merolae, a unicellular red alga, possessed two distinct secA-homologous genes; one encoded in the cell nucleus and the other in the plastid genome. We found that the plastid-encoded SecA homolog showed significant ATPase activity at low temperature, and that the ATPase activity of the nuclear-encoded SecA homolog showed significant activity at high temperature. We propose that the two SecA homologs play different roles in protein translocation.     

Division of cell nuclei, mitochondria, plastids, and microbodies mediated by mitotic spindle poles in the primitive red alga Cyanidioschyzon merolae

To understand the cell cycle, we must understand not only mitotic division but also organelle division cycles. Plant and animal cells contain many organelles which divide randomly; therefore, it has been difficult to elucidate these organelle division cycles. We used the primitive red alga Cyanidioschyzon merolae, as it contains a single mitochondrion and plastid per cell, and organelle division can be highly synchronized by a light/dark cycle. We demonstrated that mitochondria and plastids multiplied by independent division cycles (organelle G1, S, G2 and M phases) and organelle division occurred before cell–nuclear division. Additionally, organelle division was found to be dependent on microtubules as well as cell–nuclear division. We have observed five stages of microtubule dynamics: (1) the microtubule disappears during the G1 phase; (2) α-tubulin is dispersed within the cytoplasm without forming microtubules during the S phase; (3) α-tubulin is assembled into spindle poles during the G2 phase; (4) polar microtubules are organized along the mitochondrion during prophase; and (5) mitotic spindles in cell nuclei are organized during the M phase. Microfluorometry demonstrated that the intensity peak of localization of α-tubulin changed in the order to spindle poles, mitochondria, spindle poles, and central spindle area, but total fluorescent intensity did not change remarkably throughout mitotic phases suggesting that division and separation of the cell nucleus and mitochondrion is mediated by spindle pole bodies. Inhibition of microtubule organization induced cell–nuclear division, mitochondria separation, and division of a single membrane-bound microbody, suggesting that similar to cell–nuclear division, mitochondrion separation and microbody division are dependent on microtubules.     

Light adaptation of the unicellular red alga,Cyanidioschyzon merolae,probed by time-resolved fluorescence spectroscopy

Photosynthesis Research - Photosynthetic organisms change the quantity and/or quality of their pigment–protein complexes and the interactions among these complexes in response to light...     

Complete sequence and analysis of the plastid genome of the unicellular red alga Cyanidioschyzon merolae.

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.