SYNCHRONIZATION OF CHLOROPLAST DIVISION IN THE ULTRAMICROALGA CYANIDIOSCHYZON MEROLAE (RHODOPHYTA) BY TREATMENT WITH LIGHT AND APHIDICOLIN

A system of highly synchronized chloroplast divisions was developed in the unicellular red alga Cyanidioschyzon merolae De Luca, Taddei, & Varano. Chloroplast divisions were examined by epifluorescence microscopy following treatments with light and inhibitors. When the cells during stationary phase were transferred into a new medium under a 12:12 h LD cycle, chloroplasts, mitochondria, and cell nuclei divided synchronously in that order soon after the initiation of dark periods. More than 40% of the cells contained dividing chloroplasts. To obtain a system of highly synchronized cell division and chloroplast division, the cells synchronized by a 12:12 h LD cycle were treated with various inhibitors. Nocodazole and propyzamide did not affect cell and organelle divisions, whereas aphidicolin markedly inhibited cell-nuclear divisions and cytokinesis and induced a delay in chloroplast division. More than 80% of the cells contained dividing chloroplasts when cells synchronized by light were treated with aphidicolin for 12 h. This synchronized system will be useful for studies of the molecular and cellular mechanisms of organelle divisions .     

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.     

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.     

Conserved structure of Snu13 from the highly reduced spliceosome of Cyanidioschyzon merolae

Structural and functional analysis of proteins involved in pre‐mRNA splicing is challenging because of the complexity of the splicing machinery, known as the spliceosome. Bioinformatic, proteomic, and biochemical analyses have identified a minimal spliceosome in the red alga Cyanidioschyzon merolae. This spliceosome consists of only 40 core proteins, compared to ～70 in S. cerevisiae (yeast) and ～150 in humans. We report the X‐ray crystallographic analysis of C. merolae Snu13 (CmSnu13), a key component of the assembling spliceosome, and present evidence for conservation of Snu13 function in this algal splicing pathway. The near identity of CmSnu13's three‐dimensional structure to yeast and human Snu13 suggests that C. merolae should be an excellent model system for investigating the structure and function of the conserved core of the spliceosome.     

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.     

A Reaction Center-dependent Photoprotection Mechanism in a Highly Robust Photosystem II from an Extremophilic Red Alga,Cyanidioschyzon merolae

Members of the rhodophytan order Cyanidiales are unique among phototrophs in their ability to live in extremely low pH levels and moderately high temperatures. The photosynthetic apparatus of the red alga Cyanidioschyzon merolae represents an intermediate type between cyanobacteria and higher plants, suggesting that this alga may provide the evolutionary link between prokaryotic and eukaryotic phototrophs. Although we now have a detailed structural model of photosystem II (PSII) from cyanobacteria at an atomic resolution, no corresponding structure of the eukaryotic PSII complex has been published to date. Here we report the isolation and characterization of a highly active and robust dimeric PSII complex from C. merolae. We show that this complex is highly stable across a range of extreme light, temperature, and pH conditions. By measuring fluorescence quenching properties of the isolated C. merolae PSII complex, we provide the first direct evidence of pH-dependent non-photochemical quenching in the red algal PSII reaction center. This type of quenching, together with high zeaxanthin content, appears to underlie photoprotection mechanisms that are efficiently employed by this robust natural water-splitting complex under excess irradiance. In order to provide structural details of this eukaryotic form of PSII, we have employed electron microscopy and single particle analyses to obtain a 17 Å map of the C. merolae PSII dimer in which we locate the position of the protein mass corresponding to the additional extrinsic protein stabilizing the oxygen-evolving complex, PsbQ′. We conclude that this lumenal subunit is present in the vicinity of the CP43 protein, close to the membrane plane.     

Structures of the Metabolites from Steviol Methyl Ester by Gibberella fujikuroi

Steviol methyl ester (methyl ent-13-hydroxykaur-16-en-19-oate)* was converted into five new metabolites together with a known compound, methyl ent-7α,13-dihydroxykaur-16-en-19-oate, by Gibberella fujikuroi in the presence of a plant growth retardant. The structures of these new metabolites were elucidated to be methyl ent-7β,13-dihydroxykaur-16-en-19-oate, methyl ent-11α,13-dihydroxykaur-16-en-19-oate, methyl ent-7β,11α,13-trihydroxykaur-16-en-19-oate, methyl ent-11α, 13,15β-trihydroxykaur-16-en-19-oate and methyl ent-13,15β-dihydroxy-11-oxokaur-16-en-19-oate mainly by spectroscopic analyses.     

Polyethylene glycol (PEG)-mediated transient gene expression in a red alga, Cyanidioschyzon merolae 10D

DNA introduction into cells is an essential technique for molecular genetic analysis. Here, we show that DNA is easily introduced into cells of the unicellular red alga Cyanidioschyzon merolae by a polyethylene glycol (PEG)-mediated protocol. In this study, the beta-tubulin gene of C. merolae was cloned on a plasmid and a hemagglutinin (HA) tag then added at the C-terminus. This plasmid was then introduced into C. merolae cells by a PEG-mediated transformation protocol. At 24 h after PEG-mediated transformation, intracellular localization of the tagged protein was detected by anti-HA immunocytochemistry, indicating the utility of this transient expression system for molecular genetic analyses.