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Plant Physiol. Feb 2005; 137(2): 567–585.
PMCID: PMC1065357
Focus Issue on Chlamydomonas

Cyanidioschyzon merolae Genome. A Tool for Facilitating Comparable Studies on Organelle Biogenesis in Photosynthetic Eukaryotes1,[w]

Abstract

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.

To date, the genomes of more than 200 prokaryotes and several eukaryotes, including an alga, fungi, plants, animals, and their parasites, are known. However, we have little insight into the genomes of photosynthetic eukaryotes, such as Chlamydomonas reinhardtii, which are evolutionary intermediate organisms between primitive alga (Cyanidioschyzon merolae) and higher plants (Arabidopsis [Arabidopsis thaliana] and Oryza sativa), although such information would prove invaluable for investigations of the fundamental traits, origin, and evolution of eukaryotic and plant cells.

The primitive red alga C. merolae is a small (1.5 μm in diameter) organism that lives in sulfate-rich hot springs (pH 1.5, 45°C; De Luca et al., 1978). It has many characteristics that make it an ideal organism for elucidating the function, biosynthesis, and multiplication of organelles in eukaryotic cells. Figure 1 summarizes the dynamic changes in fine structures during mitosis in C. merolae compared with typical eukaryotic cells. A detailed description of the behavior and genes of each organelle will be shown in “Results and Discussion.” Although a typical eukaryotic cell contains one nucleus, it has many double membrane-bound (nucleus, mitochondria, and plastids) and single membrane-bound organelles (endoplasmic reticulum [ER], Golgi apparatus, microbodies, and lysosomes), division of which occurs at random and cannot be synchronized. In addition, the shape of organelles is very diverse and complicated (Kuroiwa, 1998; Kuroiwa et al., 1998a). On the other hand, the C. merolae cell does not have a cell wall and contains only one nucleus, one mitochondrion, and one plastid, which are simply spherical or disc-like in shape, division of which can be completely synchronized by light treatment (Terui et al., 1995). It is also a eukaryote with one of the smallest genomes, containing minimal ultrastructural constituents of eukaryotic cells: one microbody (peroxisome), one Golgi apparatus with two cisternae and coated vesicles, one ER, a few lysosome-like structures, and a small volume of cytosol (Kuroiwa et al., 1994). Therefore, it is very easy to determine the behavior of organelles during the cell cycle. As for the cellular mechanisms studied using C. merolae, for example, the role of ftsZ or dynamin in organelle division, they are common to both higher animals and plants. The simple characteristics of this alga are sure to provide an understanding of the basic mechanisms of other organelle division. This cell therefore offers unique advantages as a model organism for studies on mitochondrial and plastid divisions (Kuroiwa, 1998; Kuroiwa et al., 1998a; Miyagishima et al., 2003; Nishida et al., 2003).

Figure 1.
The basic courses of mitosis in a typical eukaryotic cell and unicellular red alga (C. merolae) cell. As typical cells contain many double membrane- and single membrane-bound organelles, they are not illustrated. During interphase, the centrosome (CEN) ...

The Cyanidioschyzon Genome Project was launched in 2001 with rough karyotyping (14–17 chromosomes) based on pulsed-field gel electrophoresis (Takahashi et al., 1995), using the 9 nuclear-coded genes known then. Whole-genome shotgun analyses showed the 16,520,305-bp sequence of the 20 chromosomes of C. merolae and allowed identification of the 5,331 genes on them. Now we have complete (100%) sequences of the C. merolae nuclear genome without gaps (H. Nozaki, O. Misumi, M. Matsuzaki, H. Takano, S. Maruyama, K. Tanaka, K. Terasawa, N. Sato, T. Mori, K. Nishida, F. Yagisawa, Y. Yoshida, H. Kuroiwa, and T. Kuroiwa, unpublished data). We performed annotation of the predicted genes and a genome-based evaluation of the relationship between C. merolae and other organisms. Consequently, we obtained complete information on all three genome compartments, nuclear (Matsuzaki et al., 2004), mitochondrial (Ohta et al., 1998), and plastid (Ohta et al., 2003), of this simple photosynthetic eukaryote.

Genome-wide analyses of this alga have provided an understanding of genes related to organelle biogenesis, multiplication, maintenance, and the ways in which progress is modulated as light conditions change. Therefore, C. merolae genome information will allow us to elucidate basic cellular properties common to all eukaryotes. On the other hand, genome information including approximately 25,500 genes in the approximately 115-Mb nuclear genome of the higher plant Arabidopsis is already available for many plant fields. However, C. merolae is markedly different from Arabidopsis taxonomically and with regards to genome size. To understand the fundamental aspects of photosynthetic organisms, before undertaking comparative genome analyses of both organisms, we need comparable genome information of intermediate organisms between C. merolae and Arabidopsis. The unicellular green alga C. reinhardtii contains an approximately 100-Mb nuclear genome, plastid genome, and mitochondria genome (Table I), and has been widely used as a model system for studying the molecular and genetic mechanisms of a number of cellular processes, such as photosynthesis, motility, and sexual reproduction (Harris, 1989).

Table I.
Comparison of cellular components of C. merolae and C. reinhardtii

Since C. reinhardtii is equipped with characteristics of cells, such as flagellar movement, and since it is capable of mating, both of which are not seen in C. merolae, it is very interesting to compare the genomes of both species. Genome information will provide unprecedented opportunities for plant improvements by establishing the detailed structures of and relationships between the genomes of C. merolae and C. reinhardtii. In this report, C. merolae is handled as a model organism of organelle research, and its intracellular structure is classified as follows: double membrane-bound organelles, single membrane-bound organelles, and cytosolic components; they are explained in terms of organelle maintenance.

RESULTS AND DISCUSSION

C. merolae was compared with typical eukaryotes, including C. reinhardtii and Arabidopsis, with regards to behavior of organelles during mitosis in maintaining double membrane-bound organelles, single membrane-bound organelles, and cytosolic components, which are essential for eukaryotic cells (Fig. 1). As the typical eukaryotic cell contains too many cytoplasmic double membrane- and single membrane-bound organelles, it is difficult to illustrate their behavior. By contrast, as the cell nucleus contains a large amount of DNA, the behavior of the chromosomes is well known in mitosis (Fig.1, a–e). During interphase, the centrosome forms outside the nucleus. At prophase, the centrosome divides, and the resulting two asters can be seen to have moved apart. Chromosomes then condense (each chromosome consists of paired chromatids attached by kinetochores), the nuclear envelope breaks down, and the nucleolus dissolves. At metaphase, the bipolar structure of the spindle is clear, and all chromosomes are aligned at the equator of the spindle. At anaphase, sister chromatids separate synchronously, and through microtubules, daughter chromosomes begin to move toward the poles. At telophase, the daughter nuclei and their nucleolei reform using the actin contractile ring; cytokinesis is almost complete.

Figure 1, f to j, shows phase contrast-fluorescent images of the C. merolae interphase and dividing cells exhibiting localization of the cell nucleus, and mitochondrial and plastid nuclei (nucleoids) after 4′,6-diamidino-2-phenylindole staining. Divisions of the plastid, mitochondrion, and nucleus occur in this order and can be highly synchronized by light/dark cycles. During late G2, plastid and mitochondrial divisions start and finish by metaphase. At prophase, condensation of the 20 chromosomes does not occur, thus each chromosome cannot be identified during the metaphase and anaphase. Cytokinesis starts from the plastid side and closes between daughter nuclei.

As the C. merolae cell contains a minimal set of small membrane-bound organelles, it is easy to determine the behavior of organelles during the cell cycle. Figure 1, k to o, shows the behavior of organelles during mitosis, obtained using data from this and previous experiments. The interphase and dividing cells contain single membrane-bound organelles (ER, one Golgi apparatus, lysosomes, and one microbody) and double membrane-bound organelles (one cell nucleus, one mitochondrion, and one plastid). The cell nucleus has a nucleolus, whereas the mitochondrion and plastids contain mitochondrial and plastid nuclei, respectively. During interphase, the centrosome forms the focus for the interphase microtubule array outside the cell nucleus. By early prophase, the centrosome and Golgi apparatus divide, and the resulting two asters and Golgi apparatus can be seen to have moved apart. Chromosomal condensation does not occur during prophase. At prometaphase, the nucleolus is dissolved but the nuclear envelope does not break down. Cell nuclei, centrosomes, ER, and Golgi apparatus behave as if they are linked. Plastid and mitochondrial divisions start in this order in G2 and finish by metaphase. During mitochondrial and plastid divisions, a mitochondrial-dividing ring (MD ring) and plastid-dividing ring (PD ring) appear at the equator of dividing V-shaped mitochondrion and dumbbell-shaped plastids, respectively. The microbody and lysosomes associate with the dividing V-shaped mitochondrion and separate into daughter cells. At metaphase and early anaphase, the bipolar structure of the spindle is clear, and all chromosomes appear to be aligned at the equator of the spindle, but each chromosome cannot be identified. Plastid and mitochondrial divisions finish by metaphase, at which point separation of the microbody starts. The batch-like connection between daughter mitochondrion and the microbody appears to play an important role in microbody division. At anaphase, sister chromatids begin to move toward the poles, but chromosomal condensation doesn't occur during mitosis. At telophase, the daughter nuclei and nucleoli reform, and division of the microbody finishes using the patch. By late telophase, cytokinesis is almost complete. The mitochondrion, lysosome, and microbody behave as if they are linked. It seems that there are connections between daughter mitochondria and the spindle, as if the nuclear family has a relationship with the mitochondrial family. Cytokinesis starts from the plastid side and closes between daughter nuclei. At the final stage of cell division, a tiny contractile-like ring appears at the equator of the cell. When the C. merolae cell was compared with typical eukaryotic cells, there were remarkable differences in condensation of chromosomes and the contractile ring for cytokinesis. Thus, detailed comparison of the structural basis between C. merolae and a typical photosynthetic unicellular microorganism is essential.

Table I summarizes the detailed comparison of the C. merolae and C. reinhardtii cells with regards to the fine structure and genes for maintaining double membrane-bound organelles, single membrane-bound organelles, and cytosolic components, which are essential for eukaryotic cells. There were interesting characteristics common to all as well as differences between, as shown following each organelle in the table.

Figure 2 summarizes the repertoire of C. merolae proteins on the basis of their assignment to eukaryotic clusters of orthologous groups (KOGs). Of the 4,771 predicted proteins, 2,536 were assigned to KOGs by emulating the National Center for Biotechnology Information (NCBI) KOGnitor service (http://www.ncbi.nlm.nih.gov/COG/new/kognitor.html). The prepublication draft sequence (C. reinhardtii version 2.0 gene model) and annotation data of C. reinhardtii used in these analyses are preliminary and might contain errors. The data of C. reinhardtii (gene model version 2.0) were also assigned by the same method. The distribution of the functional classification of C. merolae was compared with that of C. reinhardtii and Arabidopsis, which have similar genome size (100 Mb); in general, the distribution was similar to both species. The lowered proportion of genes for carbohydrate transport and metabolism and for secondary metabolite biosynthesis, transport, and catabolism found in the unicellular algae compared with Arabidopsis might reflect their simple cellular organization.

Figure 2.
Comparison of the functional classification of C. merolae proteins with those of other organisms. Columns represent the proportion of proteins assigned to the KOG classification of each organism; C. merolae, C. reinhardtii, and Arabidopsis in a left-to-right ...

Compared with C. reinhardtii and Arabidopsis, the ratio of genes for information storage and processing is relatively large in C. merolae (Fig. 2). Since the entire C. merolae genome did not undergo gene duplication, it is thought that duplication of gene clusters, other than genes for information storage and processing, occurred in Arabidopsis. In addition, it became clear that C. merolae has relatively few signal transfer and cytoskeleton genes compared with C. reinhardtii and Arabidopsis. The gene lists and general functions of the plastids, such as respiration and photosynthesis, in C. reinhardtii can be found at the U.S. Department of Energy Joint Genome Institute (JGI) Web site (http://genome.jgi-psf.org/cgi-bin/metapathways?db=chlre2). In C. merolae and C. reinhardtii, the cytological features of each organelle and other components, and the related genes were compared; they are explained in the following paragraphs.

Cell Nucleus

Most eukaryotes have nucleoli that contain 100 to 1,000 tandem-repeated arrays of units encoding 18S, 5.8S, and 25S ribosomal RNA (rRNA) genes. The nucleus of C. merolae contains one nucleolus (Fig. 1; Kuroiwa et al., 1994) and intrinsically possesses three separate units of single ribosomal DNA (rDNA; Matsuzaki et al., 2004) distributed between different chromosomal loci (Maruyama et al., 2004). This indicates that the long tandem repeats of rDNA units, which are believed to coalesce in or around the nucleolus in most eukaryotes, are not required for nucleolar structure and innate ribosome functions in C. merolae. Moreover, C. merolae has only three copies of the 5S rRNA gene, the sequences of which are almost identical. The nucleolus structure is organized with rRNAs, small nucleolar RNAs (snoRNAs), and various associated proteins. C. merolae has almost 18 basic protein components, which were assigned by KOG annotation as C/D and H/ACA guide snoRNAs, respectively (Table II). These proteins are essential for growth and snoRNA accumulation in eukaryotes. Three rDNA units, a fibrillar component, and small ribonucleoproten particles colocalize and play an important role in the modification and processing of pre-rRNA. Since nucleolus-associated chromatin, as a condensed region of nucleolar DNA, is absent in C. merolae but develops markedly in C. reinhardtii compared to yeast, the C. reinhardtii nucleolus must have more than 150 repeats of rDNA.

Table II.
Conserved orthologous genes of organelles in C. merolae and C. reinhardtii

The putative telomere repeats in C. merolae are (AATGGGGGG)n, and they are found on the ends of all chromosomes; at most there are only several repeats (H. Takano, O. Misumi, S. Maruyama, M. Matsuzaki, H. Kuroiwa, and T. Kuroiwa, unpublished data). In spite of this telomere structure, the chromosomes are correctly maintained and inherited. The C. reinhardtii telomere repeats (Petracek et al., 1990), (TTTTAGGG)n, are more A+T-rich than the C. merolae sequences (Table I) and are similar in sequence to higher organism such as Arabidopsis (TTTAGGG)n (Richards and Ausubel, 1998) and Homo sapiens (TTAGGG)n (Brown, 1989; Cross et al., 1989). In general, telomeres are separated from coding sequences by repetitive subtelomeric regions measuring several kilobases. C. merolae chromosomes also show multiple subtelomeric duplications; several sequence elements up to 20 kb long were duplicated at 30 of 40 putative subtelomeric regions, that is, at the terminal regions of chromosomes. In C. reinhardtii, the subtelomeric regions are unknown. Subtelomeric duplications have been reported in the vestigial nucleus (nucleomorph) of the cryptomonad Guillardia theta, which has rRNA genes and several open reading frames at both ends of all chromosomes (Douglas et al., 2001). The results support the idea that the origin of the nucleomorph might be a nucleus in a red alga.

While the centromeric region in higher eukaryotes often contains many repetitive species-specific elements and few genes, the chromosomes of C. merolae that were completely sequenced without gaps lack regions filled with repetitive elements (H. Nozaki, O. Misumi, M. Matsuzaki, H. Takano, S. Maruyama, K. Tanaka, K. Terasawa, N. Sato, T. Mori, K. Nishida, F. Yagisawa, Y. Yoshida, H. Kuroiwa, and T. Kuroiwa, unpublished data). Electron microscopic observations of dividing C. merolae cells revealed that the number of kinetochore microtubules is approximately identical to the number of chromosomes (Fig. 1; S. Maruyama, K. Nishida, and T. Kuroiwa, unpublished data). This suggests that C. merolae chromosomes have point or very confined centromeres, which consist of specialized nonrepetitive elements, as in Saccharomyces cerevisiae (Choo, 1997). We are currently in the process of determining these centromeres via immunological experiments using a centromere-specific histone H3 variant, CENP-A, which was identified in the C. merolae genome (S. Maruyama, H. Kuroiwa, S. Miyagishima, K. Tanaka, and T. Kuroiwa, unpublished data). Each chromosome has varying degrees of a single A+T-rich region at the midstream. Each chromosome in C. reinhardtii also has a point centromere (Table I; http://www.botany.duke.edu/chamy/ChlamyGen/maps.html). Since the centromeric regions are generally known to have a biased base composition, the local A+T-rich regions possibly determine the centromeres.

One of the most interesting features of C. merolae is each histone gene. Most eukaryotes possess multiple copies of the gene for each histone because a large amount of new histone proteins is required to make new nucleosomes in each cell cycle. C. merolae has one or a few genes corresponding to the histone encoded in chromosome 14 (Table II; supplemental data), whereas C. reinhardtii has many histone genes and their nuclei contain dense and dispersed chromatin (Tables I and andII).II). The results show that the formation of chromatin does not depend on the number of histone genes. Detailed analysis of the primary structure of chromosome 14 is now under way (K. Terasawa, O. Misumi, H. Kuroiwa, T. Kuroiwa, and N. Sato, unpublished data). In C. reinhardtii, there are many histone genes (Table II).

C. merolae has a nuclear pore (Fig. 1), and nucleocytoplasmic transport is mainly carried out by members of the importin (karyopherin) B family and exportins. C. merolae also has core receptor components, importin a, b1, b3, and exportin1, their regulator of small GTP-binding protein Ran, and three nuclear pore complex components (nucleoporins); however, a few nuclear pore proteins and Ran binding-proteins do not exist. This insufficiency might be related to the simplicity of the nuclear pore structure (Table II). The lamin gene also is absent in C. merolae as well as C. reinhardtii, S. cerevisiae, and Arabidopsis (Table II).

Metaphase chromosomes are segregated into daughter nuclei by kinetochore microtubules in the spindle (Fig. 1). Mitotic spindles of many cells, including C. reinhardtii, are organized by centrosomes, which contain centrioles (basal bodies), and interactions between spindle microtubules and microtubule-based motor proteins play critical roles in spindle formation and function. In C. reinhardtii, the mitotic apparatus consists of many cytoskeletal proteins such as α-, β-, and γ-tubulins, bipolar kinesins, C-terminal kinesins, and so on (Table II). While there are many genes related to cytoskeletal proteins in C. reinhardtii, there are minimal in C. merolae (Table II), and little is known about the mechanism of chromosome separation by mitotic apparatus in C. merolae. Twelve genes encoding the structural maintenance of chromosome (SMC) family (condensin and cohesin), which is involved in metaphase chromosome formation, are included in the C. merolae genome (Table II). However, precise chromosome condensation has not been observed in this organism during mitosis (Fig. 1). These components of the SMC family probably act on segregation of the 20 chromosomes of C. merolae. The chromosome structure might have evolved considerably because metaphase cells showing 16 chromosomes are observed in C. reinhardtii (Loppes and Matagne, 1972).

Although transcribed RNAs are imported through nuclear pores, the genes of the C. merolae genome contain only 27 introns. The infrequent occurrence of introns is likely related to the lack of some known spliceosomal proteins. The splicing process of eukaryotic spliceosomal introns involves some essential small nuclear ribonucleoprotein (snRNP) complexes, the components of which are widely conserved among eukaryotes. C. reinhardtii also has all components of spliceosomes (Table II). However, in the C. merolae genome, conserved protein subunits of U1 snRNP (A, C, and 70 kD) and U4/U6 snRNP were not detected, while those of U2 and U5 snRNP and all the common core components (Sm and Sm-like proteins) were (Table II). The RNA components of these snRNPs need to be identified experimentally because no reliable method is known for finding the genomic sequences of these RNAs. There are two possible explanations for the absence of some protein components related to splicing. First, there are unknown protein components for splicing that functionally replace U1A and other proteins. Second, splicing in C. merolae proceeds without those proteins known to be required in eukaryotic splicing, since the principal functions of snRNPs are generally mediated by the RNA components and support for their interaction is the main role of the protein components.

Mitochondria

Mitochondria contain mitochondrial nucleoids in which their own DNA molecules are organized by basic proteins, including a Grom (Kuroiwa et al., 1976; Kuroiwa, 1982; Sasaki et al., 2003; Sakai et al., 2004), divided by binary fission (Kuroiwa et al., 1977, 1998a), and distributed into daughter cells during each cell cycle. As the C. merolae cell has a single disc-shaped mitochondrion, division of which can be highly synchronized, it is easy to observe the course of mitochondrial division (Fig. 1); mitochondrial fusion does not occur. A mitochondrial dividing apparatus called a MD ring, which is larger than those of Physarum polycephalum (Kuroiwa, 1986), Cyanidium caldarium (Kuroiwa et al., 1998a), and Nannochloropsis oculata (Hashimoto, 2004), is observed at the equatorial region in C. merolae under electron microscopy (Kuroiwa et al., 1993, 1998b). The MD ring consists of double rings: an outer MD ring on the cytoplasmic side and an inner MD ring in the matrix. Previous studies have shown that C. merolae retains mitochondrial FtsZ (Takahara et al., 2000, 2001) and that the FtsZ forms a ring under the inner MD ring. Another primitive eukaryote alga, the chromophyte Mallomonas splendens (Beech et al., 2000), retains the use of FtsZ in mitochondrial division. Nishida et al. (2003) showed that C. merolae uses both FtsZ and dynamin in mitochondrial division. In summary, the FtsZ ring forms early at the site of future division, then the MD rings are formed, contraction of the equatorial region progresses, and, finally, the dynamin ring appears to form later and to function only in final separation, just before the FtsZ rings are dissolved (Fig. 1; Nishida et al., 2003). Therefore, a dynamic trio (FtsZ, MD, and dynamin rings) controls mitochondrial divisions.

However, in the cells of many eukaryotes, there are many mitochondria per cell, which divide at random. Even in unicellular eukaryotes such as yeasts and C. reinhardtii, mitochondrial shape changes dynamically from small spherical structures to a fused giant network during the cell cycle (Ehara et al., 1995). It is therefore difficult to sample a mitochondrial division event, thus C. merolae seems to be a model system for observing mitochondrial division. A large gene family consisting of more than 10 members encoding functionally diverse dynamin-related proteins for membrane pinching has been found in many eukaryotes (Miyagishima et al., 2003). C. reinhardtii also has orthologs of ftsZ and dynamin for mitochondrial division; however, their morphology continually changes with fusion and division throughout the cell cycle, and therefore detailed analyses of the dynamics have not been performed cytologically. It is predicted that C. reinhardtii has at least eight dynamin genes concerned with the severance of various membranes. The translocon of mitochondria is composed of several complexes. Tom40 of the general import pore; Tim23, Tim17, and Tim44 of the presequence translocase; Tim22 of the inner membrane insertion complex; and Tim9, Tim10, Tim8, and Tim13 of the intermembrane space complexes have been found in the C. merolae genome (Table II). But the import receptors Tom20 and Tom70, which mediate the initial step of mitochondrial import, have not been found, and a homolog of the second receptor, Tom22, had only a weak similarity. To show the presence/structure of the mitochondrial translocon in C. reinhardtii, more detailed genome information is required.

Plastids

Plastids contain plastid nucleoids, which show morphologic diversity such as centrally located plastid nucleoids, circular plastid nucleoids, and scattered plastid nucleoids; they are organized by basic protein (Kuroiwa et al., 1981; Sakai et al., 2004). Plastids divide by binary division or pleomorphic division (Kuroiwa et al., 2001; Momoyama et al., 2003). C. merolae contains one centrally located nucleoid surrounded by 5 to 10 concentric thylakoids with semicircular phycobilisomes, and the plastids are divided by binary division accompanied with plastid nucleoidal division (Fig. 1). The plastid nucleoid is organized with a bacterial histone-like protein, which is encoded in the plastid genome (Kobayashi et al., 2002). Plastid division (plastidokinesis) is associated with the formation of a so-called series of distinct PD rings (Mita and Kuroiwa, 1986) and a FtsZ ring (Miyagishima et al., 2003). Two plastid FtsZ genes (FtsZ1-1 and FtsZ1-2), which are clustered into two phylogenetic groups, play a role in plastid division, and it was found that one dynamin gene (Dnm2) is encoded in the C. merolae genome (Miyagishima et al., 2003, 2004). Dynamin forms a third type of ring separated from the FtsZ and PD rings. Time-course experiments of plastid division using electron microscopy and immunofluorescence localization of FtsZ and CmDnm2 showed that the FtsZ ring forms before onset of constriction at the equator of dividing plastids and disassembles during the final stage of plastid constriction. The PD ring forms at a somewhat later stage, at which time contraction at the equatorial region starts (Fig. 1). Dynamin (CmDnm2) begins to form a ring at the final stage of plastid division, and this ring cuts the bridge between daughter plastids formed by the PD ring. Thus, it appears that this dynamic trio of rings (FtsZ, PD, and dynamin) also functions in plastid division as with mitochondrial division.

Concerning plastid division, additional bacterial components, such as MinE and MinD, were not found in C. merolae. Thus, the FtsZ, PD, and dynamin rings were shown to have distinct functions in eukaryotic plastid division. PD ring genes are yet unknown, but finding them should be accelerated by the Cyanidioschyzon Genome Project. In C. reinhardtii, cup-shaped plastids contain their own DNAs and divide by binary divisions. The genes for plastid division, FtsZ, Drp (Dnm), MinD, and MinE, were retained in the nuclear genome of C. reinhardtii as well as in Arabidopsis (Table II).

Among the known translocon proteins in plastids, Toc34, Toc75, Tic110, Tic22, and Tic20 are encoded in the C. merolae genome, but other proteins such as Toc159, Tic40, and Tic55 are not found (Table II). These findings indicate that C. merolae has a prototypical translocon consisting of a minimal set of components, although the presence of additional rhodophyte-specific proteins cannot be excluded. McFadden and van Dooren (2004) discussed the origin and evolution of plastids using possible components of the plastid protein import apparatus based on genome information of C. merolae and other photosynthetic organisms. The C. merolae open reading frame CMM310C, with homology to Tic62 of higher plants, encodes 304 amino acid residues. CMM310C lacks the amino acid residues corresponding to the C-terminal region, which contains a repetitive module; this module interacts with a ferredoxin-NAD(P)(+) oxidoreductase in peas. Since the gene structure resembles the dehydrogenase of cyanobacteria, it is appropriate to think that the gene also functions as a dehydrogenase in C. merolae.

Standard components of photosynthesis genes were observed in C. merolae. Many of them (11 PSI genes and 17 PSII genes) are encoded in the plastid genome, while PsbO, P, U, and Z as well as a distant PsbQ homolog are encoded in the nuclear genome. Genes encoding the energy dissipation system involving the xanthophylls cycle (violaxanthin deepoxidase and zeaxanthin epoxidase) and PsbS as well as ndh genes, except for a gene encoding a homolog of cyanobacterial NADH dehydrogenase type II, are not present in C. merolae. On the other hand, the genes of xanthophylls cycle are all found, except the gene of violaxanthin deepoxidase, in C. reinhardtii.

Enzymes of the Calvin cycle in plants have been shown to be a mosaic of enzymes of cyanobacterial origin and enzymes originating from the eukaryotic host. Red algal Rubisco is known to be a product of horizontal gene transfer. The origin of other Calvin cycle enzymes is essentially identical in C. merolae and Arabidopsis (Matsuzaki et al., 2004). It is highly probable, therefore, that the complex and mosaic origin of Calvin cycle enzymes derived from common ancestors of green plants and red algae, and no essential changes occurred after the separation of the two lineages. If similar analysis is performed after complete data on Calvin cycle genes are determined for C. reinhardtii, this concept will be further supported.

Light signal transduction is critical for photoautotrophic organisms. Since the division of C. merolae cells is synchronized by light, an elaborate mechanism for light signal transduction must operate. For photoreceptors, several putative blue-light receptor (cryptochrome) genes were found in C. merolae, whereas no phytochrome-like genes were identified. Plant phytochromes are receptors of red and far-red light and have similarities with bacterial sensory His kinases. Since cyanobacteria also have ancestral phytochrome genes (Montgomery and Lagarias, 2002), C. merolae might have lost its phytochromes after its divergence from green plants. It should also be noted that the C. merolae nuclear genome encodes only one two-component His kinase candidate and no response regulators. In higher plants, many components of the bacterial two-component system are suggested as being involved in hormonal signaling and circadian oscillation regulation (Urao et al., 2000). Evidence for trimeric G protein and cAMP signaling is also missing; thus, these signal transduction mechanisms in C. merolae appear to be very simple, corroborating the result of KOG analysis. In various biological processes, the light signal is carrying out important roles in C. reinhardtii. Phototaxis is a typical cellular response to light signals in alga. Recently, the function of two rhodopsins, Chlamydomonas sensory rhodopsins A and B, as phototaxis receptors was demonstrated by in vivo analysis of photoreceptor electrical currents and motility responses (Sineshchekov et al., 2002). In addition, blue light controls the sexual life cycle of Chlamydomonas, which is mediated by phototropin, a UV-A/blue-light receptor that plays a prominent role in multiple photoresponses (Huang and Beck, 2003; Huang et al., 2004). It is noteworthy that the mechanism of light signal transduction and the effect on cells differ between C. merolae and C. reinhardtii, as both are photosynthetic alga.

The genome sequences of the plastid (149,987 bp) in C. merolae have been revealed (Ohta et al., 2003), and complete sequences from various plastids have been determined (e.g. Ohyama et al., 1986; Shinozaki et al., 1986). Phylogenetic analyses using multiple plastid genes from a wide range of eukaryotic lineages have also been carried out to resolve the robust phylogenetic relationships among plastids (e.g. Martin et al., 2002; Maul et al., 2002; Yoon et al., 2002; Ohta et al., 2003). Nozaki et al. (2003) reported plastid phylogeny and evolution based on a loss of plastid genes deduced from complete plastid genome sequences from a wide range of eukaryotic phototrophs. They represented a wide range of eukaryotic lineages (including three secondary plastid-containing groups) as two large monophyletic groups with high bootstrap values. Complete genome information of photosynthetic eukaryotes will allow elucidation of phylogenetic relationships according to the transfer of genes between genomes, as well as provide an understanding of the genetic regulation systems of photosynthesis in plastids.

Endoplasmic Reticulum

The cytoplasm of C. merolae contains small, coated vesicles and a rough-surface ER. The double-nuclear membrane is continuously covered with ER, and a Golgi body is usually situated nearby (Fig. 1). The alignment of these membrane systems in the cell of C. reinhardtii is similar, although C. reinhardtii has more single membrane-bound organelles per cell than C. merolae. Signal recognition particles on the ER play a critical role in protein sorting across the membrane. Among the known components of the signal recognition particles, the genes for SRP19, SRP54, SRP68, and SRP72 were found in the C. merolae genome, but the genes for SRP9 and SRP14, which are involved in translational arrest of ribosomes that synthesize signal-containing polypeptides conserved in many organisms, were not detected (Table II).

The C. merolae genome encodes limited subsets of vesicle-coating proteins. We were able to find suites of coatomers for COPI- and COPII-coated vesicles with key GTPases for their formation, namely Arf1 and Sar1p (Kirchhausen, 2000), respectively (Table II). We also found the heavy chain of clathrin but no obvious homolog for the light chain, which regulates the formation of clathrin triskelion and has more sequence diversity than the heavy chain. In yeasts, gene disruption of the light chain causes serious but not complete defects in clathrin-mediated transportation that can be partially rescued by overexpression of the heavy chain. It is therefore possible to assume that the light chain of clathrin might be altered in C. merolae. Furthermore, only one set of adaptor protein (AP) complexes for clathrin exists in C. merolae, whereas at least three of the four sets of subunits exist in all other eukaryotes for which genome information is available.

Golgi Apparatus

One Golgi apparatus was usually located near the centrosome in C. merolae (Fig. 1), whereas several were observed around the cell nucleus in C. reinhardtii. In both organisms, vesicles from the ER to the Golgi apparatus were observed by ultrastructural studies (Kuriyama et al., 1999). Several genes of the Golgi transport system have been annotated (Table II), but details of the vesicle transport system require clarification based on genome information.

Microbodies (Peroxisome)

Microbodies are recognized as electron dense bodies by electron microscopy. The behavior of the microbody was observed and formation of its three-dimensional structure was reconstructed from serial thin sections around one set of cell division cycle in C. merolae (Fig. 1; Miyagishima et al., 1998). The microbody changed shape intricately at the prophase during mitosis and then was divided by binary division. The electron dense patch-like connection between a daughter microbody and daughter mitochondria appeared to be available for separation of daughter microbodies (Fig. 1; Miyagishima et al., 2001). In addition, Pex11p, which is the key regulator of microbody division and proliferation, is present in C. reinhardtii but absent in C. merolae (Table II). Although there are genes coding most of the major proteins involved in protein sorting from the cytosol to organelles, some additional genes are lacking in C. merolae. Two kinds of signals (peroxisome targeting signals), PTS1 and PTS2, are known to be present in precursor proteins of microbodies. C. merolae has a final precursor protein receptor (Pex14p) and initial receptor protein (Pex5p) for PTS1, but lacks an obvious homolog of the PTS2 receptor (Pex7p; Table II). Catalase behaves as a catalyst for the conversion of hydrogen peroxide into water and oxygen in the microbody. C. merolae has a typical catalase gene, and its protein was detected in the microbody by immunoelectron microscopy (Miyagishima et al., 1999). Although such microbody-like structures appear in sections of Chlamydomonas cells, they have received relatively little experimental attention.

Lysosomes

C. merolae cells have a few lysosome-like structures, which contain lysosomal enzymes such as vacuolar ATPase, vacuolar pyrophosphatase, and acid phosphatase (Table II; F. Yagisawa, H. Kuroiwa, T. Nagata, and T. Kuroiwa, unpublished data). In mitosis, lysosomes in C. merolae seem to behave as a mitochondrial family (Fig. 1); the behavior and multiplication of lysosomes during the cell cycle will be reported in detail in the future (F. Yagisawa, unpublished data). None of the genes related to autophagy were found in the C. merolae genome, but several autophagy genes are retained in C. reinhardtii (Apg4, 6), yeast (Hamasaki et al., 2005), and mammalian genomes (Cuervo, 2004). Autophagy is a mechanism for optimizing the abundance of cellular components and for recycling biomolecular resources such as amino acids.

Cytosolic Components and Surface Structure

The tubulin family carries out most fundamental biological functions, such as cytoskeleton, flagella movement and chromosome separation in eukaryotic cells. In C. merolae, a simple spindle consisting of kinetochore microtubules, polar microtubules, and patch-like centrosome is found and was seen to play a role in the separation of the 20 chromosomes (Fig. 1). The formation, behavior, and function of the spindle will be published in detail in the future (K. Nishida, H. Kuroiwa, T. Nagata, and T. Kuroiwa, unpublished data). The genome of C. merolae only includes three genes that code α-, β-, and γ-tubulin proteins, respectively, compared to nine in C. reinhardtii (Table II). In C. merolae, there are no basal bodies or flagella, but there are mitotic spindles. However, C. reinhardtii cells have basal bodies, flagella, and mitotic spindles. Paralogous genes corresponding to α-, β-, and γ-tubulin, respectively, have not been found in the nuclear genome of C. merolae. Comparative analysis of the existence of flagellar structures with the composition of tubulin genes, and their phylogenetic relationship, will be interesting in the future. To confirm the relationships between cytoskeletal or motility machinery in the C. merolae and C. reinhardtii, we examined the phylogeny of tubulin genes. A phylogenetic tree inferred from the amino acid sequences of α-, β-, and γ-tubulin from the KEGG database release 3.0 was constructed for C. reinhardtii (version 2.0 gene model) and C. merolae by the neighbor-joining (NJ) method (Fig. 3). Three major groups were distinguished for the α-, β-, and γ-groups. The gene of C. merolae was positioned basally to the lineage, including Arabidopsis and Plasmodium homologs in each of the three tubulin families, and the C. merolae β-tubulin gene was positioned with the Encephalitozoon cuniculi lineage. The α-, β-, and γ-tubulin genes of C. reinhardtii were shown to be sisters of the Arabidopsis group. It is surprising that each tubulin gene of C. reinhardtii, which is currently widely used as a model system of flagellum equipment, showed a close relationship to a higher plant as a result of molecular phylogenetic analysis. α-, β-, and γ-tubulin genes have not been duplicated in C. merolae after branching with a green lineage, but α- and β-tubulin genes have been observed in C. reinhardtii.

Figure 3.Figure 3.
Schematic representation of the phylogenetic groups of tubulin (a). Phylogenetic relationships of β- and γ-tubulin genes (b) and α-tubulin genes (c). The tree was constructed by the NJ method using Kimura distances. Branch lengths ...

In C. merolae, there is no particular structure outside the plasma membrane. In C. reinhardtii, a specialized region differentiates the narrow membrane zone overlying the plasma membrane at the cell anterior, giving rise to the fertilization tubule of the mating-type plus cells. In cross section, an electron-dense ring appears to be associated with the plasma membrane. During sexual conjugation, the fertilization tubule has been shown to contain an F-actin bundle; an actin gene defect was apparently caused by deficient growth of the fertilization tubule. One actin and several other proteins probably play a crucial role in the formation of the fertilization tubule in C. reinhardtii (KatoMinoura et al., 1997). On the other hand, this structure and phenomenon were not found in C. merolae, which didn't express an actin gene (Takahashi et al., 1995). The absence of myosin is consistent with the fact that actin microfilaments for cytokinesis were not detected by electron microscopy or immunodetection, and that expressed sequence tag clones for the actin gene were not obtained (Matsuzaki et al., 2004). It is proposed that C. merolae cells do not require the actomyosin system, at least under our culture conditions; this is supported by the fact that disruption of the actomyosin system in other organisms does not necessarily cause lethality or complete cell division defectiveness. Probably, cytokinesis might be performed by a primitive contractile ring.

A typical cell wall was not observed in C. merolae by electron microscopy (Kuroiwa et al., 1994). The multilayered cell wall of C. reinhardtii consists of an insoluble Hyp-rich glycoprotein framework and several chaotrope-soluble Hyp-containing glycoproteins. Despite conservation of the genes for cell wall biosynthesis in both algae (Table II), there is no cell wall in C. merolae. There must therefore be a primitive, not rigid, cell surface structure in C. merolae. In addition to the surface structure of C. merolae, the existence of highly expressed transporter genes on the plasma membrane is involved in the mechanism that allows adaptation to strong acid and heavy metal ion-rich environments. The genes of cell adhesion molecules, such as integrin, cadherin, and catenin, which are conserved in animal cells, did not exist in C. merolae.

C. reinhardtii has two mating types that fuse to form diploid zygotes when each gamete is mixed. Uniparental inheritance of plastid DNA occurs during this sexual reproduction process (Kuroiwa et al., 1982; Nishimura et al., 2002). Although some genes involved in uniparental inheritance of C. reinhardtii have been reported (Ferris et al., 2002), the key gene of this phenomenon remains to be elucidated. Whole-genome information of C. reinhardtii will provide us with novel information about organelle inheritance and perhaps help elucidate the sex of C. merolae. Comparative genome analyses between C. merolae and C. reinhardtii with regards to the evolution of sexual reproduction and inheritance of organelles will be an interesting topic of study in the future.

CONCLUSION

The complete genome sequence of C. merolae revealed that this organism possesses unique features in its primary sequence structure and gene composition, making it useful for understanding the basic system and division of organelles and the evolution of photosynthetic eukaryotes. For understanding the maintenance of organelles, the C. merolae and C. reinhardtii genome projects provide complete or sufficient genome sequence data, which allows comparative orthologous analysis of the two algal genomes. Since it is composed of a minimum gene set, C. merolae genome information should accelerate studies on, for example, the establishment of cellular components, and will allow us to elucidate cellular and molecular properties common to other eukaryotes. In addition, the present genome information of C. merolae demonstrates the fundamental attributes of photosynthesis in eukaryotes and the unique photosynthetic features that are distinct from green phototrophs. These unique features of C. merolae should help provide an understanding of the origin, evolution, and fundamental structure and function of eukaryotes.

Recently, the isolation of a mutant (Yagisawa et al., 2004) and nuclear transformation by homologous recombination have been reported in C. merolae (Minoda et al., 2004). C. reinhardtii is also known to be a good model organism for studies of molecular biology with transformation techniques of plastids (Harris, 2001). There is only one report about homologous recombination of the nuclear genome, but the technique used is very complicated (Sodeinde and Kindle, 1993); the procedure is, thus, far from routine for this alga. Homologous recombination of the nuclear genome of plants has so far only been reported in the moss Physcomitrella patens (Schaefer, 2002). Therefore, gene-targeting technology using the unicellular C. merolae system will help solve many problems with regards to plant as well as basic eukaryotic biology.

Despite considerable advances in our understanding of organelle evolution and biogenesis, future proteomic and gene-targeting analyses promise to accelerate our understanding of these vital features of photosynthetic eukaryotes. Now, we have obtained complete sequences of the three genome compartments and are advancing microarray and proteome analyses as post-genome studies of C. merolae.

MATERIALS AND METHODS

Predicted proteins of Cyanidioschyzon merolae and Chlamydomonas reinhardtii were compared by reciprocal WU-BLASTP comparisons; that is, each predicted C. merolae nuclear protein was compared against all the predicted proteins of C. reinhardtii (JGI C. reinhardtii version 2.0 gene model) and vice versa. When a high-scoring pair was detected, we collected all members of the groups from both organisms. Functional classification was performed based on the NCBI eukaryotic cluster of orthologous genes by emulating the KOGnitor service (http://www.ncbi.nlm.nih.gov/COG/new/kognitor.html). Gene lists in the table were basically classified for each organelle by KOG description. The prepublication draft sequence (JGI C. reinhardtii version 2.0 gene model) and annotation data of C. reinhardtii, which were used in the analyses, are preliminary and might contain errors.

The gene lists and metabolic maps of the general functions of mitochondria and plastids, such as respiration and photosynthesis, can be found on the KEGG Web site (http://www.genome.jp/kegg/).

Phylogenetic Analyses of Tubulin Genes

The amino acid sequences of orthologous α-, β-, and γ-tubulin were extracted from the KEGG database release 3.0 and aligned using ClustalX 30 with the default option. After gaps in the alignment were excluded, the three tubulin genes of C. merolae were included and used for phylogenetic analysis. NJ trees based on Kimura distances were calculated using ClustalX. Bootstrap values in the NJ analysis were carried out based on 1,000 replications, also using ClustalX.

Supplementary Material

Supplemental Data:

Acknowledgments

We thank members of C. merolae genome project for helpful discussion.

Notes

1This work was supported by grants-in-aid for Scientific Research on Priority Areas (C) Genome Biology from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (nos. 1320611 and 14204078 to T.K.), and grants-in-aid from the Promotion of Basic Research Activities for Innovative Biosciences (ProBRAIN to T.K.).

[w]The online version of this article contains Web-only data.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.053991.

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