ABSTRACT
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.
Subject(s)
Regulatory Elements, Transcriptional , Rhodophyta , Transcription, Genetic , Cell Division , Cyclin B/genetics , Rhodophyta/geneticsABSTRACT
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.
Subject(s)
Chloroplasts/physiology , Plant Proteins/metabolism , Rhodophyta/physiology , Cell Cycle , Cell Nucleus/genetics , Cell Nucleus/metabolism , Plant Proteins/genetics , Up-RegulationABSTRACT
Nitrogen starvation is known to induce the accumulation of triacylglycerol (TAG) in many microalgae, and potential use of microalgae as a source of biofuel has been explored. However, nitrogen starvation also stops cellular growth. The expression of cyanobacterial acyl-acyl carrier protein (ACP) reductase in the unicellular red alga Cyanidioschyzon merolae chloroplasts resulted in an accumulation of TAG, which led to an increase in the number and size of lipid droplets while maintaining cellular growth. Transcriptome and metabolome analyses showed that the expression of acyl-ACP reductase altered the activities of several metabolic pathways. The activities of enzymes involved in fatty acid synthesis in chloroplasts, such as acetyl-CoA carboxylase and pyruvate dehydrogenase, were up-regulated, while pyruvate decarboxylation in mitochondria and the subsequent consumption of acetyl-CoA by the tricarboxylic acid (TCA) cycle were down-regulated. Aldehyde dehydrogenase, which oxidizes fatty aldehydes to fatty acids, was also up-regulated in the acyl-ACP reductase expresser. This activation was required for the lipid droplet accumulation and metabolic changes observed in the acyl-ACP reductase expresser. Nitrogen starvation also resulted in lipid droplet accumulation in C. merolae, while cell growth ceased as in the case of other algal species. The metabolic changes that occur upon the expression of acyl-ACP reductase are quite different from those caused by nitrogen starvation. Therefore, there should be a method for further increasing the storage lipid level while still maintaining cell growth that is different from the metabolic response to nitrogen starvation.
Subject(s)
Cyanobacteria/enzymology , Enoyl-(Acyl-Carrier Protein) Reductase (NADPH, B-Specific)/metabolism , Rhodophyta/enzymology , Rhodophyta/metabolism , Triglycerides/metabolism , Enoyl-(Acyl-Carrier Protein) Reductase (NADPH, B-Specific)/genetics , Rhodophyta/geneticsABSTRACT
Septins are a group of GTP-binding proteins that are multi-functional, with a well-known role in cytokinesis in animals and fungi. Although the functions of septins have been thoroughly studied in opisthokonts (fungi and animals), the function and evolution of plant/algal septins are not as well characterized. Here we describe septin localization and expression in the green algae Nannochloris bacillaris and Marvania geminata. The present data suggest that septins localize at the division site when cytokinesis occurs. In addition, we show that septin homologs may be found only in green algae, but not in other major plant lineages, such as land plants, red algae and glaucophytes. We also found other septin homolog-possessing organisms among the diatoms, Rhizaria and cryptomonad/haptophyte lineages. Our study reveals the potential role of algal septins in cytokinesis and/or cell elongation, and confirms that septin genes appear to have been lost in the Plantae lineage, except in some green algae.
Subject(s)
Biological Evolution , Chlorophyta/genetics , Septins/genetics , Algal Proteins/antagonists & inhibitors , Algal Proteins/genetics , Algal Proteins/metabolism , Amino Acid Sequence , Cell Division , Chlorophyta/drug effects , Chlorophyta/metabolism , Cytokinesis , Fluorescent Antibody Technique, Indirect , GTP-Binding Proteins/antagonists & inhibitors , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Microscopy, Electron, Scanning , Microscopy, Fluorescence , Models, Biological , Molecular Sequence Data , Phenylurea Compounds/pharmacology , Phylogeny , Protein Binding , Protein Structure, Tertiary , Pyridines/pharmacology , Septins/antagonists & inhibitors , Septins/metabolismABSTRACT
In unicellular algae with a single chloroplast, two mechanisms coordinate cell and chloroplast division: the S phase-specific expression of chloroplast division genes and the permission of cell cycle progression from prophase to metaphase by the onset of chloroplast division. This study investigated whether a similar mechanism exists in a unicellular alga with multiple chloroplasts using the glaucophyte alga Cyanophora sudae, which contains four chloroplasts (cyanelles). Cells with eight cyanelles appeared after the S phase arrest with a topoisomerase inhibitor camptothecin, suggesting that the mechanism of S phase-specific expression of cyanelle division genes was conserved in this alga. Inhibition of peptidoglycan synthesis by ß-lactam antibiotic ampicillin arrested cells in the S-G2 phase, and inhibition of septum invagination with cephalexin resulted in cells with two nuclei and one cyanelle, despite inhibition of cyanelle division. This indicates that even in the unicellular alga with four chloroplasts, the cell cycle progresses to the M phase following the progression of chloroplast division to a certain division stage. These results suggested that C. sudae has two mechanisms for coordinating cell and cyanelle division, similar to the unicellular algae with a single chloroplast.
Subject(s)
Cyanophora , Cell Cycle , Chloroplasts/metabolism , Cyanophora/genetics , Cyanophora/metabolism , Mitosis , Plastids/metabolismABSTRACT
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 Cyanidioschyzon merolae that diverged early in eukaryotic evolution. C. merolae 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, C. merolae 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 C. merolae. 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 C. merolae. The fact that ESCRT plays a role in cytokinesis in archaea, animals, and early diverged alga C. merolae supports the hypothesis that the function of ESCRT in cytokinesis descended from archaea to a common ancestor of eukaryotes.
ABSTRACT
The transition from G1 to S phase and subsequent nuclear DNA replication in the cells of many species of eukaryotic algae occur predominantly during the evening and night in the absence of photosynthesis; however, little is known about how day/night changes in energy metabolism and cell cycle progression are coordinated and about the advantage conferred by the restriction of S phase to the night. Using a synchronous culture of the unicellular red alga Cyanidioschyzon merolae, we found that the levels of photosynthetic and respiratory activities peak during the morning and then decrease toward the evening and night, whereas the pathways for anaerobic consumption of pyruvate, produced by glycolysis, are upregulated during the evening and night as reported recently in the green alga Chlamydomonas reinhardtii Inhibition of photosynthesis by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) largely reduced respiratory activity and the amplitude of the day/night rhythm of respiration, suggesting that the respiratory rhythm depends largely on photosynthetic activity. Even when the timing of G1/S-phase transition was uncoupled from the day/night rhythm by depletion of retinoblastoma-related (RBR) protein, the same patterns of photosynthesis and respiration were observed, suggesting that cell cycle progression and energy metabolism are regulated independently. Progression of the S phase under conditions of photosynthesis elevated the frequency of nuclear DNA double-strand breaks (DSB). These results suggest that the temporal separation of oxygenic energy metabolism, which causes oxidative stress, from nuclear DNA replication reduces the risk of DSB during cell proliferation in C. merolaeIMPORTANCE Eukaryotes acquired chloroplasts through an endosymbiotic event in which a cyanobacterium or a unicellular eukaryotic alga was integrated into a previously nonphotosynthetic eukaryotic cell. Photosynthesis by chloroplasts enabled algae to expand their habitats and led to further evolution of land plants. However, photosynthesis causes greater oxidative stress than mitochondrion-based respiration. In seed plants, cell division is restricted to nonphotosynthetic meristematic tissues and populations of photosynthetic cells expand without cell division. Thus, seemingly, photosynthesis is spatially sequestrated from cell proliferation. In contrast, eukaryotic algae possess photosynthetic chloroplasts throughout their life cycle. Here we show that oxygenic energy conversion (daytime) and nuclear DNA replication (night time) are temporally sequestrated in C. merolae This sequestration enables "safe" proliferation of cells and allows coexistence of chloroplasts and the eukaryotic host cell, as shown in yeast, where mitochondrial respiration and nuclear DNA replication are temporally sequestrated to reduce the mutation rate.
Subject(s)
Cell Cycle/radiation effects , DNA Replication/radiation effects , Darkness , Energy Metabolism/radiation effects , Light , Rhodophyta/growth & development , Rhodophyta/radiation effects , Aerobiosis , Cell Respiration , Oxygen/metabolism , Photosynthesis , Rhodophyta/genetics , Rhodophyta/metabolismABSTRACT
Chloroplasts have evolved from a cyanobacterial endosymbiont and multiply by dividing. Chloroplast division is performed by constriction of the ring-like protein complex (the PD machinery), which forms at the division site. The PD machinery is composed of cyanobacteria-descended components such as FtsZ and eukaryote-derived proteins such as the dynamin-related protein, DRP5B. In the red alga Cyanidioschyzon merolae, FtsZ ring formation on the stromal side precedes PDR1 and DRP5B ring formation on the cytosolic side. In this study, we impaired FtsZ ring formation in C. merolae by overexpressing FtsZ just before FtsZ ring formation. As a result, PDR1 and DRP5B failed to localize at the chloroplast division site, suggesting that FtsZ ring formation is required for the PDR1 and DRP5B rings. We further found, by expressing a dominant negative form of DRP5B, that DRP5B ring formation begins on the nuclear side of the chloroplast division site. These findings provide insight into how the PD machinery forms in red algae.
ABSTRACT
The unicellular red alga Cyanidioschyzon merolae is a model organism for studying the basic biology of photosynthetic organisms. The C. merolae cell is composed of an extremely simple set of organelles. The genome is completely sequenced. Gene targeting and a heat-shock inducible gene expression system has been recently established. However, a conditional gene knockdown system has not been established, which is required for the examination of function of genes that are essential to cell viability and primary mutant defects. In the current study, we first evaluated the expression of a transgene from two chromosomal neutral loci located in the intergenic region between CMD184C and CMD185C, and a region upstream of the URA5.3 gene. There was no significant difference in expression between them and this result suggests that both may be used as neutral loci. We then designed an inducible and repressible gene expression by using promoters of nitrate-assimilation genes. The expression of nitrate-assimilation genes such as NR (nitrate reductase), NIR (nitrite reductase), and NRT (the nitrate/nitrite transporter) are reversibly regulated by their dependence on nitrogen sources. We constructed stable strains in which a cassette containing the NR, NIR, or NRT promoter and sfGFP gene was inserted in a region upstream of URA5.3 and examined the efficacy of the promoters. The NR, NIR, and NRT promoters were constitutively activated in the nitrate medium, whereas their activities were extremely low in presence of ammonium. The activation of each promoter was immediately inhibited within a period of 1 h by the addition of ammonium. Thus, a conditional knockdown system in C. merolae was successfully established. The activity varies among the promoters, and each is selectable according to the expression level of a target gene estimated by RNA-sequencing. This method is applicable to defects in genes of interest in photosynthetic organism.
ABSTRACT
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.
Subject(s)
Gene Expression , Heat-Shock Response/genetics , Rhodophyta/genetics , Cell Cycle/genetics , Chloroplasts/genetics , Databases, Nucleic Acid , Gene Expression Profiling , Genes, Reporter , Genome , Hot Temperature , Mutation , Promoter Regions, Genetic , TranscriptomeABSTRACT
Circadian rhythms of cell division have been observed in several lineages of eukaryotes, especially photosynthetic unicellular eukaryotes. However, the mechanism underlying the circadian regulation of the cell cycle and the nature of the advantage conferred remain unknown. Here, using the unicellular red alga Cyanidioschyzon merolae, we show that the G1/S regulator RBR-E2F-DP complex links the G1/S transition to circadian rhythms. Time-dependent E2F phosphorylation promotes the G1/S transition during subjective night and this phosphorylation event occurs independently of cell cycle progression, even under continuous dark or when cytosolic translation is inhibited. Constitutive expression of a phospho-mimic of E2F or depletion of RBR unlinks cell cycle progression from circadian rhythms. These transgenic lines are exposed to higher oxidative stress than the wild type. Circadian inhibition of cell cycle progression during the daytime by RBR-E2F-DP pathway likely protects cells from photosynthetic oxidative stress by temporally compartmentalizing photosynthesis and cell cycle progression.
Subject(s)
Cell Division/genetics , Circadian Rhythm/genetics , E2F Transcription Factors/metabolism , Retinoblastoma Protein/genetics , Rhodophyta/genetics , Darkness , E2F Transcription Factors/biosynthesis , G1 Phase , Multiprotein Complexes/metabolism , Oxidative Stress/physiology , Phosphorylation , Photosynthesis/physiology , Protein Biosynthesis , S Phase/physiologyABSTRACT
Chloroplasts of the unicellular green alga Nannochloris bacillaris Naumann cultured under nutrient-enriched conditions have multiple rings of FtsZ, a prokaryote-derived chloroplast division protein. We previously reported that synthesis of excess chloroplast DNA and formation of multiple FtsZ rings occur simultaneously. To clarify the role of multiple FtsZ rings in chloroplast division, we investigated chloroplast DNA synthesis and ring formation in cells cultured under various culture conditions. Cells transferred from a nutrient-enriched medium to an inorganic medium in the light showed a drop in cell division rate, a reduction in chloroplast DNA content, and changes in the shape of chloroplast nucleoids as cells divided. We then examined DNA synthesis by immunodetecting BrdU incorporated into DNA strands using the anti-BrdU antibody. BrdU-labeled nuclei were clearly observed in cells 48 h after transfer into the inorganic medium, while only weak punctate signals were visible in the chloroplasts. In parallel, the number of FtsZ rings decreased from 6 to only 1. When the cells were transferred from an inorganic medium to a nutrient-enriched medium, the number of cells increased only slightly in the first 12 h after transfer; after this time, however, they started to divide more quickly and increased exponentially. Chloroplast nucleoids changed from punctate to rod-like structures, and active chloroplast DNA synthesis and FtsZ ring formation were observed. On the basis of our results, we conclude that multiple FtsZ ring assembly and chloroplast DNA duplication under nutrient-rich conditions facilitate chloroplast division after transfer to oligotrophic conditions without further duplication of chloroplast DNA and formation of new FtsZ rings.
ABSTRACT
We examined the effects of phosphate enrichment on chloroplasts of the unicellular green alga Nannochloris bacillaris Naumann. The doubling time of cells was similar in phosphate-limited (no ß-glycerophosphate) and phosphate-enriched (2 mM ß-glycerophosphate) media. The lengths of cells and chloroplasts were similar, regardless of phosphate concentration. The relationship between the ring formation of the prokaryote-derived chloroplast division protein FtsZ and phosphate concentration was examined using indirect fluorescent antibody staining. The number of FtsZ rings increased as the phosphate concentration of the medium increased. Multiple FtsZ rings were formed in cells in phosphate-enriched medium; up to six FtsZ rings per chloroplast were observed. The number of FtsZ rings increased as the chloroplast grew. The FtsZ ring located near the center of the chloroplast had the strongest fluorescence. The FtsZ ring at the relative center of all FtsZ rings was used for division. Plastid division rings did not multiply in phosphate-enriched culture. The chloroplast DNA content was 2.3 times greater in phosphate-enriched than in phosphate-limited culture and decreased in cells cultured in phosphate-enriched medium containing 5-fluorodeoxyuridine (FdUr). In the presence of FdUr, only one FtsZ ring formed, even under phosphate enrichment. This finding suggests that excessive chloroplast DNA replication induces multiple FtsZ ring formation in phosphate-enriched culture. We propose a multiple FtsZ ring formation model under phosphate enrichment.