Motomichi Matsuzaki

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.

Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells

For more than 140 years, pollen tube guidance in flowering plants has been thought to be mediated by chemoattractants derived from target ovules. However, there has been no convincing evidence of any particular molecule being the true attractant that actually controls the navigation of pollen tubes towards ovules. Emerging data indicate that two synergid cells on the side of the egg cell emit a diffusible, species-specific signal to attract the pollen tube at the last step of pollen tube guidance. Here we report that secreted, cysteine-rich polypeptides (CRPs) in a subgroup of defensin-like proteins are attractants derived from the synergid cells. We isolated synergid cells of Torenia fournieri, a unique plant with a protruding embryo sac, to identify transcripts encoding secreted proteins as candidate molecules for the chemoattractant(s). We found two CRPs, abundantly and predominantly expressed in the synergid cell, which are secreted to the surface of the egg apparatus. Moreover, they showed activity in vitro to attract competent pollen tubes of their own species and were named as LUREs. Injection of morpholino antisense oligomers against the LUREs impaired pollen tube attraction, supporting the finding that LUREs are the attractants derived from the synergid cells of T. fournieri.

A 100%-complete sequence reveals unusually simple genomic features in the hot-spring red alga Cyanidioschyzon merolae

BackgroundAll previously reported eukaryotic nuclear genome sequences have been incomplete, especially in highly repeated units and chromosomal ends. Because repetitive DNA is important for many aspects of biology, complete chromosomal structures are fundamental for understanding eukaryotic cells. Our earlier, nearly complete genome sequence of the hot-spring red alga Cyanidioschyzon merolae revealed several unique features, including just three ribosomal DNA copies, very few introns, and a small total number of genes. However, because the exact structures of certain functionally important repeated elements remained ambiguous, that sequence was not complete. Obviously, those ambiguities needed to be resolved before the unique features of the C. merolae genome could be summarized, and the ambiguities could only be resolved by completing the sequence. Therefore, we aimed to complete all previous gaps and sequence all remaining chromosomal ends, and now report the first nuclear-genome sequence for any eukaryote that is 100% complete.ResultsOur present complete sequence consists of 16546747 nucleotides covering 100% of the 20 linear chromosomes from telomere to telomere, representing the simple and unique chromosomal structures of the eukaryotic cell. We have unambiguously established that the C. merolae genome contains the smallest known histone-gene cluster, a unique telomeric repeat for all chromosomal ends, and an extremely low number of transposons.ConclusionBy virtue of these attributes and others that we had discovered previously, C. merolae appears to have the simplest nuclear genome of the non-symbiotic eukaryotes. These unusually simple genomic features in the 100% complete genome sequence of C. merolae are extremely useful for further studies of eukaryotic cells.

A Plant-Specific Dynamin-Related Protein Forms a Ring at the Chloroplast Division Site

Chloroplasts have retained the bacterial FtsZ for division, whereas mitochondria lack FtsZ except in some lower eukaryotes. Instead, mitochondrial division involves a dynamin-related protein, suggesting that chloroplasts retained the bacterial division system, whereas a dynamin-based system replaced the bacterial system in mitochondria during evolution. In this study, we identified a novel plant-specific group of dynamins from the primitive red alga Cyanidioschyzon merolae. Synchronization of chloroplast division and immunoblot analyses showed that the protein (CmDnm2) associates with the chloroplast only during division. Immunocytochemical analyses showed that CmDnm2 appears in cytoplasmic patches just before chloroplast division and is recruited to the cytosolic side of the chloroplast division site to form a ring in the late stage of division. The ring constricts until division is complete, after which it disappears. These results show that a dynamin-related protein also participates in chloroplast division and that its behavior differs from that of FtsZ and plastid-dividing rings that form before constriction at the site of division. Combined with the results of a recent study of mitochondrial division in Cyanidioschyzon, our findings led us to hypothesize that when first established in lower eukaryotes, mitochondria and chloroplasts divided using a very similar system that included the FtsZ ring, the plastid-dividing/mitochondrion-dividing ring, and the dynamin ring.

The Phylogenetic Position of Red Algae Revealed by Multiple Nuclear Genes from Mitochondria-Containing Eukaryotes and an Alternative Hypothesis on the Origin of Plastids

Red algae are one of the main photosynthetic eukaryotic lineages and are characterized by primitive features, such as a lack of flagella and the presence of phycobiliproteins in the chloroplast. Recent molecular phylogenetic studies using nuclear gene sequences suggest two conflicting hypotheses (monophyly versus non-monophyly) regarding the relationships between red algae and green plants. Although kingdom-level phylogenetic analyses using multiple nuclear genes from a wide-range of eukaryotic lineages were very recently carried out, they used highly divergent gene sequences of the cryptomonad nucleomorph (as the red algal taxon) or incomplete red algal gene sequences. In addition, previous eukaryotic phylogenies based on nuclear genes generally included very distant archaebacterial sequences (designated as the outgroup) and/or amitochondrial organisms, which may carry unusual gene substitutions due to parasitism or the absence of mitochondria. Here, we carried out phylogenetic analyses of various lineages of mitochondria-containing eukaryotic organisms using nuclear multigene sequences, including the complete sequences from the primitive red alga Cyanidioschyzon merolae. Amino acid sequence data for two concatenated paralogous genes (α- and β-tubulin) from mitochondria-containing organisms robustly resolved the basal position of the cellular slime molds, which were designated as the outgroup in our phylogenetic analyses. Phylogenetic analyses of 53 operational taxonomic units (OTUs) based on a 1525-amino-acid sequence of four concatenated nuclear genes (actin, elongation factor-1α, α-tubulin, and β-tubulin) reliably resolved the phylogeny only in the maximum parsimonious (MP) analysis, which indicated the presence of two large robust monophyletic groups (Groups A and B) and the basal eukaryotic lineages (red algae, true slime molds, and amoebae). Group A corresponded to the Opisthokonta (Metazoa and Fungi), whereas Group B included various primary and secondary plastid-containing lineages (green plants, glaucophytes, euglenoids, heterokonts, and apicomplexans), Ciliophora, Kinetoplastida, and Heterolobosea. The red algae represented the sister lineage to Group B. Using 34 OTUs for which essentially the entire amino acid sequences of the four genes are known, MP, distance, quartet puzzling, and two types of maximum likelihood (ML) calculations all robustly resolved the monophyly of Group B, as well as the basal position of red algae within eukaryotic organisms. In addition, phylogenetic analyses of a concatenated 4639-amino-acid sequence for 12 nuclear genes (excluding the EF-2 gene) of 12 mitochondria-containing OTUs (including C. merolae) resolved a robust non-sister relationship between green plants and red algae within a robust monophyletic group composed of red algae and the eukaryotic organisms belonging to Group B. A new scenario for the origin and evolution of plastids is suggested, based on the basal phylogenetic position of the red algae within the large clade (Group B plus red algae). The primary plastid endosymbiosis likely occurred once in the common ancestor of this large clade, and the primary plastids were subsequently lost in the ancestor(s) of the Discicristata (euglenoids, Kinetoplastida, and Heterolobosea), Heterokontophyta, and Alveolata (apicomplexans and Ciliophora). In addition, a new concept of “Plantae” is proposed for phototrophic and nonphototrophic organisms belonging to Group B and red algae, on the basis of the common history of the primary plastid endosymbiosis. The Plantae include primary plastid-containing phototrophs and nonphototrophic eukaryotes that possibly contain genes of cyanobacterial origin acquired in the primary endosymbiosis.

Dynamic recruitment of dynamin for final mitochondrial severance in a primitive red alga

Dynamins are a eukaryote-specific family of GTPases. Some family members are involved in diverse and varied cellular activities. Here, we report that the primitive red alga Cyanidioschyzon merolae retains only one dynamin homolog, CmDnm1, belonging to the mitochondrial division subfamily. Previously, the bacterial cell division protein, FtsZ, was shown to localize at the mitochondrial division site in the alga. We showed that FtsZ and dynamin coexist as mitochondrial division-associated proteins that act during different phases of division. CmDnm1 was recruited from 10–20 cytoplasmic patches (dynamin patches) to the midpoint of the constricted mitochondrion-dividing ring (MD ring), which was observed as an electron-dense structure on the cytoplasmic side. CmDnm1 is probably not required for early constriction; it forms a ring or spiral when the outer mitochondrial membrane is finally severed, whereas the FtsZ and MD rings are formed before constriction. It is thought that the FtsZ, MD, and dynamin rings are involved in scaffolding, constriction, and final separation, respectively. In eukaryotes, mitochondrial severance is probably the most conserved role for the dynamin family.

Cyanidioschyzon merolae Genome. A Tool for Facilitating Comparable Studies on Organelle Biogenesis in Photosynthetic Eukaryotes

The ultrasmall unicellular red alga Cyanidioschyzon merolae lives in the extreme environment of acidic hot springs and is thought to retain primitive features of cellular and genome organization. We determined the 16.5-Mb nuclear genome sequence of C. merolae 10D as the first complete algal genome. BLASTs and annotation results showed that C. merolae has a mixed gene repertoire of plants and animals, also implying a relationship with prokaryotes, although its photosynthetic components were comparable to other phototrophs. The unicellular green alga Chlamydomonas reinhardtii has been used as a model system for molecular biology research on, for example, photosynthesis, motility, and sexual reproduction. Though both algae are unicellular, the genome size, number of organelles, and surface structures are remarkably different. Here, we report the characteristics of double membrane- and single membrane-bound organelles and their related genes in C. merolae and conduct comparative analyses of predicted protein sequences encoded by the genomes of C. merolae and C. reinhardtii. We examine the predicted proteins of both algae by reciprocal BLASTP analysis, KOG assignment, and gene annotation. The results suggest that most core biological functions are carried out by orthologous proteins that occur in comparable numbers. Although the fundamental gene organizations resembled each other, the genes for organization of chromatin, cytoskeletal components, and flagellar movement remarkably increased in C. reinhardtii. Molecular phylogenetic analyses suggested that the tubulin is close to plant tubulin rather than that of animals and fungi. These results reflect the increase in genome size, the acquisition of complicated cellular structures, and kinematic devices in C. reinhardtii.

Two types of FtsZ proteins in mitochondria and red-lineage chloroplasts: the duplication of FtsZ is implicated in endosymbiosis.

The ancestors of plastids and mitochondria were once free-living bacteria that became organelles as a result of endosymbiosis. According to this theory, a key bacterial division protein, FtsZ, plays a role in plastid division in algae and plants as well as in mitochondrial division in lower eukaryotes. Recent studies have shown that organelle division is a process that combines features derived from the bacterial division system with features contributed by host eukaryotic cells. Two nonredundant versions of FtsZ, FtsZ1 and FtsZ2, have been identified in green-lineage plastids, whereas most bacteria have a single ftsZ gene. To examine whether there is also more than one type of FtsZ in red-lineage chloroplasts (red algal chloroplasts and chloroplasts that originated from the secondary endosymbiosis of red algae) and in mitochondria, we obtained FtsZ sequences from the complete sequence of the primitive red alga Cyanidioschyzon merolae and the draft sequence of the stramenopile (heterokont) Thalassiosira pseudonana. Phylogenetic analyses that included known FtsZ proteins identified two types of chloroplast FtsZ in red algae (FtsZA and FtsZB) and stramenopiles (FtsZA and FtsZC). These analyses also showed that FtsZB emerged after the red and green lineages diverged, while FtsZC arose by the duplication of an ftsZA gene that in turn descended from a red alga engulfed by the ancestor of stramenopiles. A comparison of the predicted proteins showed that like bacterial FtsZ and green-lineage FtsZ2, FtsZA has a short conserved C-termmal sequence (the C-terminal core domain), whereas FtsZB and FtsZC, like the green-lineage FtsZ1, lack this sequence. In addition, the Cyanidioschyzon and Dictyostelium genomes encode two types of mitochondrial FtsZ proteins, one of which lacks the C-terminal variable domain. These results suggest that the acquisition of an additional FtsZ protein with a modified C terminus was common to the primary and secondary endosymbioses that produced plastids and that this also occurred during the establishment of mitochondria, presumably to regulate the multiplication of these organelles.

Detection and localization of a chloroplast-encoded HU-like protein that organizes chloroplast nucleoids

Chloroplast DNA (cpDNA) is packed into discrete structures called chloroplast nucleoids (cp-nucleoids). The structure of cpDNA is thought to be important for its maintenance and regulation. In bacteria and mitochondria, histone-like proteins (such as HU and Abf2, respectively) are abundant and play important roles in DNA organization. However, a primary structural protein has yet to be found in cp-nucleoids. Here, we identified an abundant DNA binding protein from isolated cp-nucleoids of the primitive red alga Cyanidioschyzon merolae. The purified protein had sequence homology with the bacterial histone-like protein HU, and it complemented HU-lacking Escherichia coli mutants. The protein, called HC (histone-like protein of chloroplast), was encoded by a single gene (CmhupA) in the C. merolae chloroplast genome. Using immunofluorescence and immunoelectron microscopy, we demonstrated that HC was distributed uniformly throughout the entire cp-nucleoid. The protein was expressed constitutively throughout the cell and the chloroplast division cycle, and it was able to condense DNA. These results indicate that HC, a bacteria-derived histone-like protein, primarily organizes cpDNA into the nucleoid.

Phylogenetic positions of Glaucophyta, green plants (Archaeplastida) and Haptophyta (Chromalveolata) as deduced from slowly evolving nuclear genes

The phylogenetic positions of the primary photosynthetic eukaryotes, or Archaeplastida (green plants, red algae, and glaucophytes) and the secondary photosynthetic chromalveolates, Haptophyta, vary depending on the data matrices used in the previous nuclear multigene phylogenetic studies. Here, we deduced the phylogeny of three groups of Archaeplastida and Haptophyta on the basis of sequences of the multiple slowly evolving nuclear genes and reduced the gaps or missing data, especially in glaucophyte operational taxonomic units (OTUs). The present multigene phylogenetic analyses resolved that Haptophyta and two other groups of Chromalveolata, stramenopiles and Alveolata, form a monophyletic group that is sister to the green plants and that the glaucophytes and red algae are basal to the clade composed of green plants and Chromalveolata. The bootstrap values supporting these phylogenetic relationships increased with the exclusion of long-branched OTUs. The close relationship between green plants and Chromalveolata is further supported by the common replacement in two plastid-targeted genes.