Yoshiki Nishimura

Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D

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. 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.

Active digestion of sperm mitochondrial DNA in single living sperm revealed by optical tweezers

In almost all eukaryotes, mitochondrial (mt) genes are transmitted to progeny mainly from the maternal parent. The most popular explanation for this phenomenon is simple dilution of paternal mtDNA, because the paternal gametes (sperm) are much smaller than maternal gametes (egg) and contribute a limited amount of mitochondria to the progeny. Recently, this simple explanation has been challenged in several reports that describe the active digestion of sperm mtDNA, down-regulation of mtDNA replication in sperm, and proteolysis of mitochondria triggered by ubiquitination. In this investigation, we visualized mt nucleoids in living sperm by using highly sensitive SYBR green I vital staining. The ability to visualize mt nucleoids allowed us to clarify that the elimination of sperm mtDNA upon fertilization is achieved through two steps: (i) gradual decrease of mt nucleoid numbers during spermatogenesis and (ii) rapid digestion of sperm mtDNA just after fertilization. One notable point is that the digestion of mtDNA is achieved before the complete destruction of mitochondrial structures, which may be necessary to avoid the diffusion and transmission of potentially deleterious sperm mtDNA to the progeny.

Composition and physiological function of the chloroplast NADH dehydrogenase‐like complex in Marchantia polymorpha

The chloroplast NADH dehydrogenase-like (NDH) complex mediates cyclic electron transport and chloro-respiration and consists of five sub-omplexes, which in angiosperms further associate with photosystem I (PSI) to form a super-complex. In Marchantia polymorpha, 11 plastid-encoded subunits and all the nuclear-encoded subunits of the A, B, membrane and ferredoxin-binding sub-complexes are conserved. However, it is unlikely that the genome of this liverwort encodes Lhca5 and Lhca6, both of which mediate NDH-PSI super-complex formation. It is also unlikely that the subunits of the lumen sub-complex, PnsL1-L4, are encoded by the genome. Consistent with this in silico prediction, the results of blue-native gel electrophoresis showed that NDH subunits were detected in a protein complex with lower molecular mass in Marchantia than the NDH-PSI super-complex in Arabidopsis. Using the plastid transformation technique, we knocked out the ndhB gene in Marchantia. Although the wild-type genome copies were completely segregated out, the ΔndhB lines grew like the wild-type photoautotrophically. A post-illumination transient increase in chlorophyll fluorescence, which reflects NDH activity in vivo in angiosperms, was absent in the thalli of the ΔndhB lines. In ruptured chloroplasts, antimycin A-insensitive, and ferredoxin-dependent plastoquinone reduction was impaired, suggesting that chloroplast NDH mediates similar electron transport in Marchantia and Arabidopsis, despite its possible difference in structure. As in angiosperms, linear electron transport was not strongly affected in the ΔndhB lines. However, the plastoquinone pool was slightly more reduced at low light intensity, suggesting that chloroplast NDH functions in redox balancing of the inter system, especially under low light conditions.

Antisense Transcript and RNA Processing Alterations Suppress Instability of Polyadenylated mRNA in Chlamydomonas Chloroplasts

In chloroplasts, the control of mRNA stability is of critical importance for proper regulation of gene expression. The Chlamydomonas reinhardtii strain Δ26pAtE is engineered such that the atpB mRNA terminates with an mRNA destabilizing polyadenylate tract, resulting in this strain being unable to conduct photosynthesis. A collection of photosynthetic revertants was obtained from Δ26pAtE, and gel blot hybridizations revealed RNA processing alterations in the majority of these suppressor of polyadenylation (spa) strains, resulting in a failure to expose the atpB mRNA 3′ poly(A) tail. Two exceptions were spa19 and spa23, which maintained unusual heteroplasmic chloroplast genomes. One genome type, termed PS+, conferred photosynthetic competence by contributing to the stability of atpB mRNA; the other, termed PS−, was required for viability but could not produce stable atpB transcripts. Based on strand-specific RT-PCR, S1 nuclease protection, and RNA gel blots, evidence was obtained that the PS+ genome stabilizes atpB mRNA by generating an atpB antisense transcript, which attenuates the degradation of the polyadenylated form. The accumulation of double-stranded RNA was confirmed by insensitivity of atpB mRNA from PS+ genome-containing cells to S1 nuclease digestion. To obtain additional evidence for antisense RNA function in chloroplasts, we used strain Δ26, in which atpB mRNA is unstable because of the lack of a 3′ stem-loop structure. In this context, when a 121-nucleotide segment of atpB antisense RNA was expressed from an ectopic site, an elevated accumulation of atpB mRNA resulted. Finally, when spa19 was placed in a genetic background in which expression of the chloroplast exoribonuclease polynucleotide phosphorylase was diminished, the PS+ genome and the antisense transcript were no longer required for photosynthesis. Taken together, our results suggest that antisense RNA in chloroplasts can protect otherwise unstable transcripts from 3′→5′ exonuclease activity, a phenomenon that may occur naturally in the symmetrically transcribed and densely packed chloroplast genome.

The biparental transmission of the mitochondrial genome in Chlamydomonas reinhardtii visualized in living cells.

In the isogamous green alga Chlamydomonas reinhardtii, the chloroplast genome is transmitted from the mt+ parent, while the mitochondrial genes are believed to be inherited from the mt- parent. Chloroplast nucleoids have been visualized by DAPI (4,6-diamidino-2-phenylindole) staining, and the preferential digestion of the mt- chloroplast nucleoids has been observed in young zygotes. However, the mitochondrial nucleoids have never been visualized, and their behavior is only deduced from genetic and biochemical studies. We discovered that the mitochondrial and chloroplast genomes can be visualized simultaneously in living cells, using the fluorescent dye SYBR Green I. The ability to visualize the mitochondrial and chloroplast genome in vivo permits the direct observation of the number, distribution and behavior of the chloroplast and mitochondrial nucleoids in young zygotes. Using this method, the biparental transmission of the mitochondrial genome was revealed.

Genome analysis and its significance in four unicellular algae, Cyanidioshyzon merolae, Ostreococcus tauri, Chlamydomonas reinhardtii, and Thalassiosira pseudonana

Algae play a more important role than land plants in the maintenance of the global environment and productivity. Progress in genome analyses of these organisms means that we can now obtain information on algal genomes, global annotation and gene expression. The full genome information for several algae has already been analyzed. Whole genomes of the red alga Cyanidioshyzon merolae, the green algae Ostreococcus tauri and Chlamydomonas reinhardtii, and the diatom Thalassiosira pseudonana have been sequenced. Genome composition and the features of cells among the four algae were compared. Each alga maintains basic genes as photosynthetic eukaryotes and possesses additional gene groups to represent their particular characteristics. This review discusses and introduces the latest research that makes the best use of the particular features of each organism and the significance of genome analysis to study biological phenomena. In particular, examples of post-genome studies of organelle multiplication in C. merolae based on analyzed genome information are presented.

Isolation and phenotypic characterization ofChlamydomonas reinhardtii mutants defective in chloroplast DNA segregation

SummaryEach wild-typeChlamydomonas reinhardtii cell has one large chloroplast containing several nuclei (nucleoids). We used DNA insertional mutagenesis to isolate Chlamydomonas mutants which contain a single, large chloroplast (cp) nucleus and which we namedmoc (monokaryotic chloroplast). DAPI-fluorescence microscopy and microphotometry observations revealed thatmoc mutant cells only contain one cp-nucleus throughout the cell division cycle, and that unequal segregation of cpDNA occurred during cell division in themoc mutant. One cell with a large amount of cpDNA and another with a small amount of cpDNA were produced after the first cell division. Unequal segregation also occurred in the second cell division, producing one cell with a large amount (about 70 copies) of cpDNA and three other cells with a small amount (only 2–8 copies) of cpDNA. However, most individualmoc cells contained several dozen cpDNA copies 12 h after the completion of cell division, suggesting that cpDNA synthesis was activated immediately after chloroplast division. In contrast to the cpDNA, the mitochondrial (mt) DNA of themoc mutants was observed as tiny granules scattered throughout the entire cell. These segregated to each daughter cell equally during cell division. Electron-microscopic observation of the ultrastructure ofmoc mutants showed that a low-electron-density area, which was identified as the cp-nucleus by immunoelectron microscopy with anti-DNA antibody, existed near the pyrenoid. However, there were no other structural differences between the chloroplasts of wild-type cells andmoc mutants. The thylakoid membranes and pyrenoid were identical. Therefore, we propose that the novelmoc mutants are only defective in the dispersion and segregation of cpDNA. This strain should be useful to elucidate the mechanism for the segregation of cpDNA.

Gsp1 Triggers the Sexual Developmental Program Including Inheritance of Chloroplast DNA and Mitochondrial DNA in Chlamydomonas reinhardtii

This study examined a mutant of Chlamydomonas reinhardtii, biparental31, defective in the sexual development upon the fusion of male (mating type minus [mt−]) and female (mt+) gametes and demonstrated the importance of a homeoprotein gene (GSP1) and inositol metabolism in orchestrating the sexual program, including uniparental inheritance of chloroplast and mitochondrial DNA. The isogamous green alga Chlamydomonas reinhardtii has emerged as a premier model for studying the genetic regulation of fertilization and sexual development. A key regulator is known to be a homeoprotein gene, GAMETE-SPECIFIC PLUS1 (GSP1), which triggers the zygotic program. In this study, we isolated a mutant, biparental31 (bp31), which lacks GSP1. bp31 mt+ gametes fuse normally to form zygotes, but the sexual development of the resulting diploid cell is arrested and pellicle/zygospore/tetrad formation is abolished. The uniparental inheritance of chloroplast (cp) and mitochondrial (mt) DNA (cytoplasmic inheritance) was also impaired. bp31 has a deletion of ∼60 kb on chromosome 2, including GSP1. The mutant phenotype was not rescued by transformation with GSP1 alone but could be rescued by the cotransformation with GSP1 and another gene, INOSITOL MONOPHOSPHATASE-LIKE1, which is involved in various cellular processes, including the phosphatidylinositol signaling pathway. This study confirms the importance of Gsp1 in mediating the zygotic program, including the uniparental inheritance of cp/mtDNA. Moreover, the results also suggest a role for inositol metabolism in the sexual developmental program.

Comparative analysis of DNA synthesis activity in plastid-nuclei and mitochondrial-nuclei simultaneously isolated from cultured tobacco cells

Abstract Plastid-nuclei (plastid-nucleoids) and mitochondrial-nuclei (mitochondrial-nucleoids) were simultaneously isolated from cultured tobacco cells (Nicotiana tabacum L., line BY-2), and their activity synthesizing DNA in vitro was examined. The isolated plastid- and mitochondrial-nuclei incorporated about 20 and 50 pmol of dCTP, respectively, into DNA per microgram of template DNA during a 60-min incubation. The DNA synthetic activity of the two organelle-nuclei exhibited similar responses to various inhibitors; it was resistant to aphidicolin and sensitive to N-ethylmaleimide and high concentrations of ddCTP, which are all characteristics of γ-like DNA polymerase. The responses of the DNA synthetic activity in the two organelle-nuclei to pH and divalent- and monovalent-metal cation concentrations were also similar. Moreover, an in situ DNA polymerase assay following SDS-polyacrylamide gel electrophoresis revealed that DNA polymerases with an apparent molecular mass of 116 kDa were present in both the isolated plastid- and mitochondrial-nuclei, and that the two 116-kDa DNA polymerases were quite similar in terms of their sensitivity to various inhibitors, optimum assay conditions, and template preferences. These results indicate that DNA replication in plastids and mitochondria may be conducted by DNA polymerases that have quite similar characteristics.

Uniparental inheritance of cpDNA and the genetic control of sexual differentiation in Chlamydomonas reinhardtii.

An intriguing feature of most eukaryotes is that chloroplast (cp) and mitochondrial (mt) genomes are inherited almost exclusively from one parent. Uniparental inheritance of cp/mt genomes was long thought to be a passive outcome, based on the fact that eggs contain multiple numbers of organelles, while male gametes contribute, at best, only a few cp/mtDNA. However, the process is likely to be more dynamic because uniparental inheritance occurs in organisms that produce gametes of identical sizes (isogamous). In Chlamydomonas reinhardtii, the uniparental inheritance of cp/mt genomes is achieved by a series of mating type-controlled events that actively eliminate the mating type minus (mt−) cpDNA. The method by which Chlamydomonas selectively degrades mt− cpDNA has long fascinated researchers, and is the subject of this review.