Spliceosome assembly and regulation: insights from analysis of highly reduced spliceosomes.

Premessenger RNA splicing is catalyzed by the spliceosome, a multimegadalton RNA-protein complex that assembles in a highly regulated process on each intronic substrate. Most studies of splicing and spliceosomes have been carried out in human or S. cerevisiae model systems. There exists, however, a large diversity of spliceosomes, particularly in organisms with reduced genomes, that suggests a means of analyzing the essential elements of spliceosome assembly and regulation. In this review, we characterize changes in spliceosome composition across phyla, describing those that are most frequently observed and highlighting an analysis of the reduced spliceosome of the red alga Cyanidioschyzon merolae We used homology modeling to predict what effect splicing protein loss would have on the spliceosome, based on currently available cryo-EM structures. We observe strongly correlated loss of proteins that function in the same process, for example, in interacting with the U1 snRNP (which is absent in C. merolae), regulation of Brr2, or coupling transcription and splicing. Based on our observations, we predict splicing in C. merolae to be inefficient, inaccurate, and post-transcriptional, consistent with the apparent trend toward its elimination in this lineage. This work highlights the striking flexibility of the splicing pathway and the spliceosome when viewed in the context of eukaryotic diversity.

Development of a rapamycin-inducible protein-knockdown system in the unicellular red alga Cyanidioschyzon merolae.

An inducible protein-knockdown system is highly effective for investigating the functions of proteins and mechanisms essential for the survival and growth of organisms. However, this technique is not available in photosynthetic eukaryotes. The unicellular red alga Cyanidioschyzon merolae possesses a very simple cellular and genomic architecture and is genetically tractable but lacks RNA interference machinery. In this study, we developed a protein-knockdown system in this alga. The constitutive system utilizes the destabilizing activity of the FRB domain of human target of rapamycin (TOR) kinase or its derivatives to knock down target proteins. In the inducible system, rapamycin treatment induces the heterodimerization of the human FKBP12-rapamycin binding (FRB) domain fused to the target proteins with the human FK506-binding protein 12 (FKBP) fused to S-phase kinase associated protein 1 (SKP1) or Cullin 1 (CUL1), subunits of the SCF E3 ubiquitin ligase. This results in the rapid degradation of the target proteins through the ubiquitin-proteasome pathway. With this system, we successfully degraded endogenous essential proteins such as the chloroplast division protein Dynamin related protein 5B (DRP5B) and E2 transcription factor (E2F), a regulator of the G1/S transition, within 2-3 hours after rapamycin administration, enabling the assessment of resulting phenotypes. This rapamycin-inducible protein-knockdown system contributes to the functional analysis of genes whose disruption leads to lethality.

Investigation of bionano additives in red algae Cyanidioschyzon merolae ultrasonified MgO/MWCNT catalyzed biodiesel in optimized engine performance.

Renewable energy research is burgeoning with the anticipation of finding neat liquid fuel. Ultra sonification assisted biodiesel was derived from red algae Cyanidioschyzon merolae, with biodiesel yield of 98.9%. The results of GC MS of the prepared biodiesel showed higher concentration of methyl palmitate, methyl oleate, and stearate. This composition is appreciable, as this plays significance in desirable pour & cloud point properties. NMR spectrum revealed the ester linkages, presence of olefins, and α methyl position in olefins. Mixture of 30 wt% of biodiesel in diesel exhibited work efficiency, and also exhibited low pour point and, lower viscosity values. CeO2 and Fe2O3 nano particles were bio reduced, and were added as nano additive in biodiesel. 1:1 ratio of CeO2 and Fe2O3 added to biodiesel maximised the combustion ability of fuel owing to the oxygen storage capacity of CeO2. Further, this combination produced a satisfactory calorific value. Imbalanced ratios disrupted the catalytic and oxygen storage effects, reduced the overall energy release and calorific value of the biodiesel blend. Pour point and cetane number value of biodiesel blend ultrasonifacted with 1:1 mass ratio of Fe2O3 and CeO2 was observed to be around -7 °C and 53 °C respectively, and was better than other compositions. 1:1 mass ratio of NPS blended with 30 wt% BD in diesel showed tremendous increase in brake thermal efficiency, torque, and power. HC, NOX, and SOX emissions were reduced by 42.8%, 19.3%, and 57% respectively with 1:1 Fe2O3 and CeO2 mixed biodiesel blend. CeO2 favourably improved the oxygen storage capacity of the fuel, whereas Fe2O3 showed decrease in formation of gums and sediments in biodiesel.

Study of Dispersed Repeats in the Cyanidioschyzon merolae Genome.

In this study, we applied the iterative procedure (IP) method to search for families of highly diverged dispersed repeats in the genome of Cyanidioschyzon merolae, which contains over 16 million bases. The algorithm included the construction of position weight matrices (PWMs) for repeat families and the identification of more dispersed repeats based on the PWMs using dynamic programming. The results showed that the C. merolae genome contained 20 repeat families comprising a total of 33,938 dispersed repeats, which is significantly more than has been previously found using other methods. The repeats varied in length from 108 to 600 bp (522.54 bp in average) and occupied more than 72% of the C. merolae genome, whereas previously identified repeats, including tandem repeats, have been shown to constitute only about 28%. The high genomic content of dispersed repeats and their location in the coding regions suggest a significant role in the regulation of the functional activity of the genome.

Citrate synthase from Cyanidioschyzon merolae exhibits high oxaloacetate and acetyl-CoA catalytic efficiency.

Citrate synthase (CS) catalyzes the reaction that produces citrate and CoA from oxaloacetate and acetyl-CoA in the tricarboxylic acid (TCA) cycle. All TCA cycle enzymes are localized to the mitochondria in the model organism, the red alga Cyanidioschyzon merolae. The biochemical properties of CS have been studied in some eukaryotes, but the biochemical properties of CS in algae, including C. merolae, have not been studied. We then performed the biochemical analysis of CS from C. merolae mitochondria (CmCS4). The results showed that the kcat/Km of CmCS4 for oxaloacetate and acetyl-CoA were higher than those of the cyanobacteria, such as Synechocystis sp. PCC 6803, Microcystis aeruginosa PCC 7806 and Anabaena sp. PCC 7120. Monovalent and divalent cations inhibited CmCS4, and in the presence of KCl, the Km of CmCS4 for oxaloacetate and acetyl-CoA was higher in the presence of MgCl2, the Km of CmCS4 for oxaloacetate and acetyl-CoA was higher and kcat lower. However, in the presence of KCl and MgCl2, the kcat/Km of CmCS4 was higher than those of the three cyanobacteria species. The high catalytic efficiency of CmCS4 for oxaloacetate and acetyl-CoA may be a factor in the increased carbon flow into the TCA cycle in C. merolae.

CZON-cutter - a CRISPR-Cas9 system for multiplexed organelle imaging in a simple unicellular alga.

The unicellular alga Cyanidioschyzon merolae has a simple cellular structure; each cell has one nucleus, one mitochondrion, one chloroplast and one peroxisome. This simplicity offers unique advantages for investigating organellar proliferation and the cell cycle. Here, we describe CZON-cutter, an engineered clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system for simultaneous genome editing and organellar visualization. We engineered a C. merolae strain expressing a nuclear-localized Cas9-Venus nuclease for targeted editing of any locus defined by a single-guide RNA (sgRNA). We then successfully edited the algal genome and visualized the mitochondrion and peroxisome in transformants using fluorescent protein reporters with different excitation wavelengths. Fluorescent protein labeling of organelles in living transformants allows us to validate phenotypes associated with organellar proliferation and the cell cycle, even when the edited gene is essential. Combined with the exceptional biological features of C. merolae, CZON-cutter will be instrumental for investigating cellular and organellar division in a high-throughput manner. This article has an associated First Person interview with the first author of the paper.

The carbon-concentrating mechanism of the extremophilic red microalga Cyanidioschyzon merolae.

Cyanidioschyzon merolae is an extremophilic red microalga which grows in low-pH, high-temperature environments. The basis of C. merolae's environmental resilience is not fully characterized, including whether this alga uses a carbon-concentrating mechanism (CCM). To determine if C. merolae uses a CCM, we measured CO2 uptake parameters using an open-path infra-red gas analyzer and compared them to values expected in the absence of a CCM. These measurements and analysis indicated that C. merolae had the gas-exchange characteristics of a CCM-operating organism: low CO2 compensation point, high affinity for external CO2, and minimized rubisco oxygenation. The biomass δ13C of C. merolae was also consistent with a CCM. The apparent presence of a CCM in C. merolae suggests the use of an unusual mechanism for carbon concentration, as C. merolae is thought to lack a pyrenoid and gas-exchange measurements indicated that C. merolae primarily takes up inorganic carbon as carbon dioxide, rather than bicarbonate. We use homology to known CCM components to propose a model of a pH-gradient-based CCM, and we discuss how this CCM can be further investigated.

Cyanidiales as Polyextreme Eukaryotes.

Cyanidiales were named enigmatic microalgae due to their unique polyextreme properties, considered for a very long time unattainable for eukaryotes. Cyanidiales mainly inhabit hot sulfuric springs with high acidity (pH 0-4), temperatures up to 56°C, and ability to survive in the presence of dissolved heavy metals. Owing to the minimal for eukaryotes genome size, Cyanidiales have become one of the most important research objects in plant cell physiology, biochemistry, molecular biology, phylogenomics, and evolutionary biology. They play an important role in studying many aspects of oxygenic photosynthesis and chloroplasts origin. The ability to survive in stressful habitats and the corresponding metabolic pathways were acquired by Cyanidiales from archaea and bacteria via horizontal gene transfer (HGT). Thus, the possibility of gene transfer from prokaryotes to eukaryotes was discovered, which was a new step in understanding of the origin of eukaryotic cell.

The Unicellular Red Alga Cyanidioschyzon merolae-The Simplest Model of a Photosynthetic Eukaryote.

Several species of unicellular eukaryotic algae exhibit relatively simple genomic and cellular architecture. Laboratory cultures of these algae grow faster than plants and often provide homogeneous cellular populations exposed to an almost equal environment. These characteristics are ideal for conducting experiments at the cellular and subcellular levels. Many microalgal lineages have recently become genetically tractable, which have started to evoke new streams of studies. Among such algae, the unicellular red alga Cyanidioschyzon merolae is the simplest organism; it possesses the minimum number of membranous organelles, only 4,775 protein-coding genes in the nucleus, and its cell cycle progression can be highly synchronized with the diel cycle. These properties facilitate diverse omics analyses of cellular proliferation and structural analyses of the intracellular relationship among organelles. C. merolae cells lack a rigid cell wall and are thus relatively easily disrupted, facilitating biochemical analyses. Multiple chromosomal loci can be edited by highly efficient homologous recombination. The procedures for the inducible/repressive expression of a transgene or an endogenous gene in the nucleus and for chloroplast genome modification have also been developed. Here, we summarize the features and experimental techniques of C. merolae and provide examples of studies using this alga. From these studies, it is clear that C. merolae-either alone or in comparative and combinatory studies with other photosynthetic organisms-can provide significant insights into the biology of photosynthetic eukaryotes.

A cotransformation system of the unicellular red alga Cyanidioschyzon merolae with blasticidin S deaminase and chloramphenicol acetyltransferase selectable markers.

BACKGROUND: The unicellular red alga Cyanidioschyzon merolae exhibits a very simple cellular and genomic architecture. In addition, procedures for genetic modifications, such as gene targeting by homologous recombination and inducible/repressible gene expression, have been developed. However, only two markers for selecting transformants, uracil synthase (URA) and chloramphenicol acetyltransferase (CAT), are available in this alga. Therefore, manipulation of two or more different chromosomal loci in the same strain in C. merolae is limited. RESULTS: This study developed a nuclear targeting and transformant selection system using an antibiotics blasticidin S (BS) and the BS deaminase (BSD) selectable marker by homologous recombination in C. merolae. In addition, this study has succeeded in simultaneously modifying two different chromosomal loci by a single-step cotransformation based on the combination of BSD and CAT selectable markers. A C. merolae strain that expresses mitochondrion-targeted mSCARLET (with the BSD marker) and mVENUS (with the CAT marker) from different chromosomal loci was generated with this procedure. CONCLUSIONS: The newly developed BSD selectable marker enables an additional genetic modification to the already generated C. merolae transformants based on the URA or CAT system. Furthermore, the cotransformation system facilitates multiple genetic modifications. These methods and the simple nature of the C. merolae cellular and genomic architecture will facilitate studies on several phenomena common to photosynthetic eukaryotes.

Photosynthesis of the Cyanidioschyzon merolae cells in blue, red, and white light.

Photosynthesis and respiration rates, pigment contents, CO2 compensation point, and carbonic anhydrase activity in Cyanidioschizon merolae cultivated in blue, red, and white light were measured. At the same light quality as during the growth, the photosynthesis of cells in blue light was significantly lowered, while under red light only slightly decreased as compared with white control. In white light, the quality of light during growth had no effect on the rate of photosynthesis at low O2 and high CO2 concentration, whereas their atmospheric level caused only slight decrease. Blue light reduced markedly photosynthesis rate of cells grown in white and red light, whereas the effect of red light was not so great. Only cells grown in the blue light showed increased respiration rate following the period of both the darkness and illumination. Cells grown in red light had the greatest amount of chlorophyll a, zeaxanthin, and ß-carotene, while those in blue light had more phycocyanin. The dependence on O2 concentration of the CO2 compensation point and the rate of photosynthesis indicate that this alga possessed photorespiration. Differences in the rate of photosynthesis at different light qualities are discussed in relation to the content of pigments and transferred light energy together with the possible influence of related processes. Our data showed that blue and red light regulate photosynthesis in C. merolae for adjusting its metabolism to unfavorable for photosynthesis light conditions.

Cis-acting elements involved in the G2/M-phase-specific transcription of the cyclin B gene in the unicellular alga Cyanidioschyzon merolae.

M-specific activator (MSA) cis-acting elements have been determined to be involved in the regulation of G2/M-phase-specific transcription in spermatophytes. In this study, the involvement of MSA-core elements in G2/M-phase-specific transcription was examined in the unicellular red alga Cyanidioschyzon merolae. In the C. merolae genome, MSA-core elements do not accumulate specifically in the upstream of mitosis-specific transcriptional genes. Mutations of the four MSA-core elements of the cyclin B gene, which encodes a central factor of the G2-to-M-phase transition, have resulted in the abolishment of transcription or permission of transcription even in the G1 phase. These results suggest that all four MSA-core elements located in the upstream region of cyclin B are involved in G2/M-phase-specific transcription in C. merolae; however, the nature of the involvement of MSA-core elements in G2/M-phase-specific transcription differed among the four elements. Thus, MSA-core-element-mediated G2/M-phase-specific transcription in C. merolae seems to be regulated by a complex mechanism.

Development of a Novel Nanoarchitecture of the Robust Photosystem I from a Volcanic Microalga Cyanidioschyzon merolae on Single Layer Graphene for Improved Photocurrent Generation.

Here, we report the development of a novel photoactive biomolecular nanoarchitecture based on the genetically engineered extremophilic photosystem I (PSI) biophotocatalyst interfaced with a single layer graphene via pyrene-nitrilotriacetic acid self-assembled monolayer (SAM). For the oriented and stable immobilization of the PSI biophotocatalyst, an His6-tag was genetically engineered at the N-terminus of the stromal PsaD subunit of PSI, allowing for the preferential binding of this photoactive complex with its reducing side towards the graphene monolayer. This approach yielded a novel robust and ordered nanoarchitecture designed to generate an efficient direct electron transfer pathway between graphene, the metal redox center in the organic SAM and the photo-oxidized PSI biocatalyst. The nanosystem yielded an overall current output of 16.5 µA·cm-2 for the nickel- and 17.3 µA·cm-2 for the cobalt-based nanoassemblies, and was stable for at least 1 h of continuous standard illumination. The novel green nanosystem described in this work carries the high potential for future applications due to its robustness, highly ordered and simple architecture characterized by the high biophotocatalyst loading as well as simplicity of manufacturing.

Target of rapamycin-signaling modulates starch accumulation via glycogenin phosphorylation status in the unicellular red alga Cyanidioschyzon merolae.

The target of rapamycin (TOR) signaling pathway is involved in starch accumulation in various eukaryotic organisms; however, the molecular mechanism behind this phenomenon in eukaryotes has not been elucidated. We report a regulatory mechanism of starch accumulation by TOR in the unicellular red alga, Cyanidioschyzon merolae. The starch content in C. merolae after TOR-inactivation by rapamycin, a TOR-specific inhibitor, was increased by approximately 10-fold in comparison with its drug vehicle, dimethyl sulfoxide. However, our previous transcriptome analysis showed that the expression level of genes related to carbohydrate metabolism was unaffected by rapamycin, indicating that starch accumulation is regulated at post-transcriptional levels. In this study, we performed a phosphoproteome analysis using liquid chromatography-tandem mass spectrometry to investigate potential post-transcriptional modifications, and identified 52 proteins as candidate TOR substrates. Among the possible substrates, we focused on the function of CmGLG1, because its phosphorylation at the Ser613 residue was decreased after rapamycin treatment, and overexpression of CmGLG1 resulted in a 4.7-fold higher starch content. CmGLG1 is similar to the priming protein, glycogenin, which is required for the initiation of starch/glycogen synthesis, and a budding yeast complementation assay demonstrated that CmGLG1 can functionally substitute for glycogenin. We found an approximately 60% reduction in the starch content in a phospho-mimicking CmGLG1 overexpression strain, in which Ser613 was substituted with aspartic acid, in comparison with the wild-type CmGLG1 overexpression cells. Our results indicate that TOR modulates starch accumulation by changing the phosphorylation status of the CmGLG1 Ser613 residue in C. merolae.

Regulation of photosystem I-light-harvesting complex I from a red alga Cyanidioschyzon merolae in response to light intensities.

Photosynthetic organisms use different means to regulate their photosynthetic activity in respond to different light conditions under which they grow. In this study, we analyzed changes in the photosystem I (PSI) light-harvesting complex I (LHCI) supercomplex from a red alga Cyanidioschyzon merolae, upon growing under three different light intensities, low light (LL), medium light (ML), and high light (HL). The results showed that the red algal PSI-LHCI is separated into two bands on blue-native PAGE, which are designated PSI-LHCI-A and PSI-LHCI-B, respectively, from cells grown under LL and ML. The former has a higher molecular weight and binds more Lhcr subunits than the latter. They are considered to correspond to the two types of PSI-LHCI identified by cryo-electron microscopic analysis recently, namely, the former with five Lhcrs and the latter with three Lhcrs. The amount of PSI-LHCI-A is higher in the LL-grown cells than that in the ML-grown cells. In the HL-grown cells, PSI-LHCI-A completely disappeared and only PSI-LHCI-B was observed. Furthermore, PSI core complexes without Lhcr attached also appeared in the HL cells. Fluorescence decay kinetics measurement showed that Lhcrs are functionally connected with the PSI core in both PSI-LHCI-A and PSI-LHCI-B obtained from LL and ML cells; however, Lhcrs in the PSI-LHCI-B fraction from the HL cells are not coupled with the PSI core. These results indicate that the red algal PSI not only regulates its antenna size but also adjusts the functional connection of Lhcrs with the PSI core in response to different light intensities.

Glycosyltransferase MDR1 assembles a dividing ring for mitochondrial proliferation comprising polyglucan nanofilaments.

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.

Phototaxis of the Unicellular Red Alga Cyanidioschyzon merolae Is Mediated by Novel Actin-Driven Tentacles.

Phototaxis, which is the ability to move towards or away from a light source autonomously, is a common mechanism of unicellular algae. It evolved multiple times independently in different plant lineages. As of yet, algal phototaxis has been linked mainly to the presence of cilia, the only known locomotive organelle in unicellular algae. Red algae (Rhodophyta), however, lack cilia in all stages of their life cycle. Remarkably, multiple unicellular red algae like the extremophile Cyanidioschyzon merolae (C. merolae) can move towards light. Remarkably, it has remained unclear how C. merolae achieves movement, and the presence of a completely new mechanism has been suggested. Here we show that the basis of this movement are novel retractable projections, termed tentacles due to their distinct morphology. These tentacles could be reproducibly induced within 20 min by increasing the salt concentration of the culture medium. Electron microscopy revealed filamentous structures inside the tentacles that we identified to be actin filaments. This is surprising as C. merolae's single actin gene was previously published to not be expressed. Based on our findings, we propose a model for C. merolae's actin-driven but myosin-independent motility. To our knowledge, the described tentacles represent a novel motility mechanism.

The sole LSm complex in Cyanidioschyzon merolae associates with pre-mRNA splicing and mRNA degradation factors.

Proteins of the Sm and Sm-like (LSm) families, referred to collectively as (L)Sm proteins, are found in all three domains of life and are known to promote a variety of RNA processes such as base-pair formation, unwinding, RNA degradation, and RNA stabilization. In eukaryotes, (L)Sm proteins have been studied, inter alia, for their role in pre-mRNA splicing. In many organisms, the LSm proteins form two distinct complexes, one consisting of LSm1-7 that is involved in mRNA degradation in the cytoplasm, and the other consisting of LSm2-8 that binds spliceosomal U6 snRNA in the nucleus. We recently characterized the splicing proteins from the red alga Cyanidioschyzon merolae and found that it has only seven LSm proteins. The identities of CmLSm2-CmLSm7 were unambiguous, but the seventh protein was similar to LSm1 and LSm8. Here, we use in vitro binding measurements, microscopy, and affinity purification-mass spectrometry to demonstrate a canonical splicing function for the C. merolae LSm complex and experimentally validate our bioinformatic predictions of a reduced spliceosome in this organism. Copurification of Pat1 and its associated mRNA degradation proteins with the LSm proteins, along with evidence of a cytoplasmic fraction of CmLSm complexes, argues that this complex is involved in both splicing and cytoplasmic mRNA degradation. Intriguingly, the Pat1 complex also copurifies with all four snRNAs, suggesting the possibility of a spliceosome-associated pre-mRNA degradation complex in the nucleus.

Unequal misses during the flash-induced advancement of photosystem II: effects of the S state and acceptor side cycles.

Photosynthetic water oxidation is catalyzed by the oxygen-evolving complex (OEC) in photosystem II (PSII). This process is energetically driven by light-induced charge separation in the reaction center of PSII, which leads to a stepwise accumulation of oxidizing equivalents in the OEC (Si states, i = 0-4) resulting in O2 evolution after each fourth flash, and to the reduction of plastoquinone to plastoquinol on the acceptor side of PSII. However, the Si-state advancement is not perfect, which according to the Kok model is described by miss-hits (misses). These may be caused by redox equilibria or kinetic limitations on the donor (OEC) or the acceptor side. In this study, we investigate the effects of individual S state transitions and of the quinone acceptor side on the miss parameter by analyzing the flash-induced oxygen evolution patterns and the S2, S3 and S0 state lifetimes in thylakoid samples of the extremophilic red alga Cyanidioschyzon merolae. The data are analyzed employing a global fit analysis and the results are compared to the data obtained previously for spinach thylakoids. These two organisms were selected, because the redox potential of QA/QA- in PSII is significantly less negative in C. merolae (Em = - 104 mV) than in spinach (Em = - 163 mV). This significant difference in redox potential was expected to allow the disentanglement of acceptor and donor side effects on the miss parameter. Our data indicate that, at slightly acidic and neutral pH values, the Em of QA-/QA plays only a minor role for the miss parameter. By contrast, the increased energy gap for the backward electron transfer from QA- to Pheo slows down the charge recombination reaction with the S3 and S2 states considerably. In addition, our data support the concept that the S2 → S3 transition is the least efficient step during the oxidation of water to molecular oxygen in the Kok cycle of PSII.

Chloroplast division checkpoint in eukaryotic algae.

Chloroplasts evolved from a cyanobacterial endosymbiont. It is believed that the synchronization of endosymbiotic and host cell division, as is commonly seen in existing algae, was a critical step in establishing the permanent organelle. Algal cells typically contain one or only a small number of chloroplasts that divide once per host cell cycle. This division is based partly on the S-phase-specific expression of nucleus-encoded proteins that constitute the chloroplast-division machinery. In this study, using the red alga Cyanidioschyzon merolae, we show that cell-cycle progression is arrested at the prophase when chloroplast division is blocked before the formation of the chloroplast-division machinery by the overexpression of Filamenting temperature-sensitive (Fts) Z2-1 (Fts72-1), but the cell cycle progresses when chloroplast division is blocked during division-site constriction by the overexpression of either FtsZ2-1 or a dominant-negative form of dynamin-related protein 5B (DRP5B). In the cells arrested in the prophase, the increase in the cyclin B level and the migration of cyclin-dependent kinase B (CDKB) were blocked. These results suggest that chloroplast division restricts host cell-cycle progression so that the cell cycle progresses to the metaphase only when chloroplast division has commenced. Thus, chloroplast division and host cell-cycle progression are synchronized by an interactive restriction that takes place between the nucleus and the chloroplast. In addition, we observed a similar pattern of cell-cycle arrest upon the blockage of chloroplast division in the glaucophyte alga Cyanophora paradoxa, raising the possibility that the chloroplast division checkpoint contributed to the establishment of the permanent organelle.