Genome Analysis and Its Significance in Four Unicellular Algae

Home , Micromonas, Ostreococcus, Ostreococcus tauri

J Plant Res (2008) 121:3–17 DOI 10.1007/s10265-007-0133-9

INVITED ARTICLE

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

Osami Misumi Æ Yamato Yoshida Æ Keiji Nishida Æ Takayuki Fujiwara Æ Takayuki Sakajiri Æ Syunsuke Hirooka Æ Yoshiki Nishimura Æ Tsuneyoshi Kuroiwa

Received: 5 October 2007 / Accepted: 30 October 2007 / Published online: 12 December 2007 Ó The Botanical Society of Japan and Springer 2007

Abstract Algae play a more important role than land In particular, examples of post-genome studies of organelle plants in the maintenance of the global environment and multiplication in C. merolae based on analyzed genome productivity. Progress in genome analyses of these organ- information are presented. isms means that we can now obtain information on algal genomes, global annotation and gene expression. The full Keywords Algae Á Genome Á Organelle Á Metabolism Á genome information for several algae has already been Photosynthetic organisms analyzed. Whole genomes of the red alga Cyanidioshyzon merolae, the green algae Ostreococcus tauri and Chla- mydomonas reinhardtii, and the diatom Thalassiosira Introduction pseudonana have been sequenced. Genome composition and the features of cells among the four algae were com- Algae are a highly diverse group of organisms that live in a pared. Each alga maintains basic genes as photosynthetic variety of environments, such as in oceans, in fresh water, eukaryotes and possesses additional gene groups to repre- in the soil, on ice, on rock, and so on. This characteristic sent their particular characteristics. This review discusses makes algae very suitable for studies concerning the and introduces the latest research that makes the best use of adaptation of organisms to different environments, and it the particular features of each organism and the signiﬁ- provides the possibility of ﬁnding novel genes related to cance of genome analysis to study biological phenomena. different environmental tolerance characteristics. Cyanid- ioshyzon merolae is an unicellular red alga which lives in extreme environments, such as those at high temperatures or that are strongly acidic (De Luca et al 1978). In addition, O. Misumi Á K. Nishida Á T. Fujiwara Á T. Sakajiri Á algae are highly morphologically diverse. Species vary S. Hirooka Á T. Kuroiwa Department of Life Science, Graduate School of Science, from single-celled to multiple-celled. They vary in size too; Rikkyo University, Tokyo 171-8501, Japan for example, cells of the picoplankton ex. Ostreococcus tauri are *1 lm in diameter (Derelle et al. 2002), whilst Y. Yoshida Ecklonia cava (Laminariaceae) may reach a length of Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, more than 2 m. In addition they vary in terms of their Chiba 277-8562, Japan growth habits (for example, volvocales species form col- onies) or in their physical structure (for example, many Y. Nishimura haptophytes have an exoskeleton of calcareous plates Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan called coccoliths). Thus algae are suitable for studies of cell differentiation. T. Kuroiwa (&) One of the most important topics in algal research is Research Information Center for Extremophile, photosynthesis. The mechanism of photosynthesis in the Rikkyo University, 3-34-1 Nishiikebukuro, Toshima-ku, Tokyo 171-8501, Japan chloroplast of green algae is basically similar to that of e-mail: [email protected] land plants. Therefore, many of the phenomena analyzed 123 4 J Plant Res (2008) 121:3–17

a yt using algae can be applied to higher plants. For example, h a a ta ta xa p se id ts y y a e to o st a n a h h r l n b a zo la zo ia p p o p o o l o P a o a the heterotrophic ability of Chlamydomonas to utilize r o o h m k ol p n i zo b e te d c p o ro r to le n g a e a c o u io ic e te e g e n t o ch a h la il p et e in u re u e m r alternative carbon sources as nutrition allows photosyn- B R G C A H H K E G F M A A thetic mutants to persist (Harris 1989). Algae have Bikonta (Plantae) Oposthokonta Amoebazoa contributed considerably to research in this field. The 4 biomass resources of algae in the ocean are of immense III 3 importance in the global environment. Algae have also 2 contributed to research into a variety of other phenomena 1 in cell biology. Studies of eukaryote sexual reproduction and life cycles have often been investigated using algae. II Cell nucleus In addition, research into flagellar movement (Dutcher Mitochondrion 1995) or organelle multiplication, applicable to eukary- I Chloroplast otic organisms in general, has been developed using unicellular algae with simple morphologies. Analyzing algal genomes provides important information for Fig. 1 Phylogenetic positions of Cyanidioschyzon merolae (1), research into not only the simple acquisition of nucleo- Ostreococcus tauri (2) Chlamydomonas reinhardtii (3) and Thalass- iosira psudonana (4) on a modified version of Nozaki’s phylogenetic tide sequences, but also the physiology, environmental tree (Nozaki et al. 2003). The points of primary endosymbiosis of adaptation mechanisms, morphogenesis and evolution of alphaproteobacteria (I), primary endosymbiosis of cyanobacteria (II) organisms (especially the origin of photosynthetic and secondary endosymbiosis of red algae are represented. C. merolae organisms, and their evolution into land plants). This belongs to the Rhodophyta and O. tauri and C. reinhardtti belong to the green lineage. T. pseudonana belongs to the Heterokontophyta short review covers four algae, C. merolae, O. tauri, C. with secondary endosymbiotic chloroplasts reinhardtii and T. pseudonana, the compositions of their respective algal genomes, and introduces research that relates to this genome information. In particular we suggest that Cyanidioschyzon merolae cells, with heat- stable proteins that are unique among eukaryotes, are fundamental to future studies of proteomic expression analysis using microarrays and those in structural biology.

Outline of sequenced algal genomes

Figure 1 shows the phylogenetic position of each alga on Nozaki’s phylogenetic tree (Nozaki et al. 2003). C. merolae belongs to the Rhodophyta, which diverged earliest among photosynthetic organisms. O. tauri and C. reinhardtti belong to the green lineage. These algae have a plastid acquired by primary endosymbiosis of ancestral cyanobacteria. On the other hand, T. pseudonana has a Fig. 2a–d Fluorescent photomicrographs of C. merolae (a), O. tauri plastid acquired by secondary endosymbiosis and belongs (b), C. reinhardtti (c), and Nitzschia sp. cells (d) after staining with to the Heterokontophyta. Figure 2 shows fluorescent 4,6-diamidino-2-phenylindole (DAPI). The spherical or ovoid-shaped cell nuclei emit blue-white fluorescence (arrows) and chloroplasts photomicrographs of each algal cell stained with DAPI. emit red autofluorescence. The chloroplast nuclei (nucleoids) of C. Blue-white fluorescence in the cells indicates the com- reinhardtii are scattered in the chloroplast while those of sequenced T. partments where genomic DNA exists. The chloroplast psudonana and Nitzschia sp. are located in a ring on the periphery of DNA of C. merolae is located at the center of the chlo- the chloroplast. Scale bar 1 lm roplast, while O. tauri and C. reinhardtii have dispersed DNA similar to the chloroplasts of the higher plants. The chloroplast DNA of T. pseudonana exists along the edge complex forms are found in C. reinhardtii and of the chloroplast envelope. One spherical mitochondrion T. pseudonana. Multiple genome DNA molecules are is contained in the cells in C. merolae and O. tauri. usually contained in the chloroplast and mitochondria; However, in other species mitochondria have altered photosynthesis and respiration of cells functions through forms and sizes through division or fusion, and such replication and distribution of this DNA. 123 J Plant Res (2008) 121:3–17 5

Cyanidioschyzon merolae and sexual differentiation were found in the genome (Mori et al. 2006; Nozaki et al. 2006). Recently, the complete cell- A summary of the cellular components of the four algae nuclear genome sequences were released (Nozaki et al. with complete genome sequences is shown in Table 1, and 2007). Since the completion of the mitochondrial and an outline of the genome, cell and organelle structures for plastid genome sequences, C. meolae has become the ﬁrst these algae is shown in Table 2. C. merolae was the ﬁrst eukaryote in which all three genome compartments—cell alga to provide a complete genome sequence (Matsuzaki nucleus, mitochondrion and plastid—have been completely et al. 2004; http://merolae.biol.s.u-tokyo.ac.jp/). C. merolae sequenced (Nozaki et al. 2007). The complete sequence of has been very useful as a model organism for studies of the C. merolae consists of 16,546,747 nucleotides covering 20 division and multiplication of cell organelles (Kuroiwa linear chromosomes from telomere to telomere (Table 2). 1998; Kuroiwa et al. 1998, 2006). The genome project of C. These 20 linear nuclear chromosomes plus the two circular merolae was performed in order to elucidate these mecha- organelle DNA molecules comprise the entire genome of nisms. C. merolae cells contain a minimal set of organelles the organism, and contain 16,728,945 base pairs. The for eukaryotes, and the cells proliferate by an extremely nuclear genome encodes 4,775 protein-coding genes, and simple binary division process (Kuroiwa et al. 1994, 1998; the mitochondrial and plastid genomes encode 34,205 Misumi et al. 2005). Sexual reproduction has not been protein-coding genes, respectively. The unique feature of recorded, although some genes concerning gamete fusion the genome of C. merolae is that it is simple but that it has

Table 1 General features of four algal genomes Feature C. merolae O. tauri C. reinhardtii T. pseudonana

Nucleus No. of chromosomes 20 20 17 24 Genome size (bp) 16,546,747 12,600,000 121,000,000 31,300,000 G + C content (%) 55 58 64a 47 No. of genes 5,335 7,892 15143a 11,390 Mean gene length (bp) 1,551 1,245 1580a 1745 Gene density (bp per gene) 3,102 1,597 7,851a 3,500 Percent coding 44.8 81.6 16.7a 28.8 Genes with introns (%) 0.5 39 92a ND No. of introns 27 4,730 ND ND Mean length of intron (bp) 248 126 373a ND Mean length of exons (bp) 1,540 750 190a ND No. of tRNA genes 30 174 259a 131 No. of 5S rRNA genes 3 4 3a,b ND No. of 5.8S, 18S and 28S rRNA units 3 4 3a,b *35 Mitochondrion Molecule Circular Circular Linear Circular Genome size (bp) 32,211 44,237 15,758 43,827 G + C content (%) 27.1 38.2 45.2 30.1 No. of protein genes 34 43 8 35 Density (bp per protein genes) 947 1,029 1,970 1,252 Plastid Molecule Circular Circular Circular Circular Genome size (bp) 149,987 71,666 203,828 128,814 G + C content (%) 37.6 39.9 34.5 30.7 No. of protein genes 208 61 69 141 Density (bp per protein genes) 721 1,175 2,954 914 Data for O. tauri, C. reinhardtii, T. pseudonana are from Derelle et al. (2006); Palenik et al. (2007); Armbrust et al. (2004), the JGI website (http://www.jgi.doe.gov/) and the NCBI website (http://www.ncbi.nlm.nih.gov/). ND not determined a These data were obtained from Merchant et al. (2007) after their manuscript was submitted b The number is the outermost copies in tandem arrays on three linkage groups

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Table 2 Comparison of the cellular components of C. merolae, O. tauri, C. reinhardtii and T. pseudonana C. merolae O. tauri C. reinhardtii T. pseudonana

Lineage Rhodophyta, Chlorophyta, Chlorophyta, Heterokontophyta, Cyanidiophyceae Prasinophyceae Chlorophyceae Bacillariophyceae Cell diameter 1–2 lm \1 lm 5–10 lm 5–10 lm Cell division Binary fission Binary fission Endospore division Division with frustule Double-membrane bounded Nucleus Chromosome number 20 20 17 24 Ploidy Haploid Haploid (diploid?) Haploid, diploid Haploid, diploid Genome size 16.5 Mbp 12.6 Mbp 120 Mbp 31.3 Mbp Telomeric repeat GGGGGGAAT TTTAGGG TTTTAGGG TTAGGG Nucleolus + + + + Chromatin, dispersed + - ++ Chromatin, condensed + - ++ Metaphase chromosomes Dispersed ND Condensed Condensed Mitotic spindle with microtubule Internuclear Not observed Internuclear Internuclear Meiosis Unclear Unclear + + Mitochondria Origin Primary endosymbiosis Primary endosymbiosis Primary endosymbiosis Primary endosymbiosis Number per cell 1 1 One-many Many Nucleoids Centrally located Centrally located Centrally located Centrally located Shape Erythrocyte-shaped Ovoid-shaped Oval, elongated, branching Oval, elongated, branching Dynamics Binary division Binary division Division, fusion Division, fusion Plastid Origin Primary endosymbiosis Primary endosymbiosis Primary endosymbiosis Secondary endosymbiosis Number per cell 1 1 1 Many Nucleoids Centrally located Scattered Scattered Ring Shape Spherical Ovoid Cup shape Oval Thylakoid Single layer Multilayer with grana Multilayer with grana Triple layer Pyrenoid No No 1-few 1 Eyespot No No 2–4 layers No Dynamics Binary division Binary division Binary division Binary division Starch granules Cytoplasm Stroma Stroma Cytoplasm Single membrane bounded ER + + + + Golgi apparatus 1 1 10* 10* Microbody 1 1 A few A few Lisosome 2–4 Several vesicles Several Several Contractile vacuole No No 2 No Cell motility No No Two flagella No Cell wall No typical wall Cellulosic wall Cellulosic wall Silicified wall Sexual reproduction Unclear Unclear + (mating type plus, minus) + Mating structure No No + - Data for O. tauri, C. reinhardtii, T. pseudonana are from Derelle et al. (2006); Palenik et al. (2007); Henderson et al. (2007); Armbrust et al. (2004), the JGI website (http://www.jgi.doe.gov/) and our observations

123 J Plant Res (2008) 121:3–17 7 sufficient genes to function as an eukaryote (Misumi et al. also has a small genome size, with 12.6 million base pairs in 2005). A typical example is that of the ribosomal DNA gene the nuclear genome, 20 chromosomes and 7,892 genes units. Most eukaryotes have multiple copies of their rDNA (Derelle et al. 2006; Palenik et al. 2007; Table 2). This alga units in tandem, making a nucleolus, but C. merolae has has 1.5 times the number of genes of C. merolae. The only three copies of its rDNA units although it makes an mitochondria and plastid genomes of this alga have also obvious nucleolus (Maruyama et al. 2004). In many species, been determined, and have 44,237 and 71,666 base pairs, histone genes also tend to make repeating clusters found on respectively (Table 2). On the other hand, the genome size each chromosome. C. merolae histone genes are localized of O. tauri has been estimated, using cytological fluores- on chromosome 14 and form a small cluster. The five major cence quantity analysis of nuclear DNA, to be larger than histone genes encode two copies of the three core histone the sequenced size reported. Although there is a possibility genes (H2A, H2B and H4), three copies of the H3 gene, and that diploid cells exist, the quantitive analysis showed a single copy of the linker-histone H1 gene, respectively 20 million base pairs (Fig. 3; Table 3; Kuroiwa et al. 2004). (Nozaki et al. 2007). This is perhaps the simplest compo- Therefore, the detailed analysis remains to be resolved. The sition of histone genes. Although the encoded genes of genome of the alga was found to be extremely compressed histone are the minimum set, a general chromatin structure and the inter-ORF region between the genes was only is formed. The sequencing of all chromosomal termini 197 base pairs on average. It is by far the smallest organism sequenced has indicated that AATGGGGGG is the telo- among the eukaryotes. A comparatively uniform GC con- mere repeat sequence in all chromosomal ends of C. tent of 58% occurs across almost the entire genome, but merolae (Nozaki et al. 2007). This telomeric nucleotide falls to 54 and 52% for chromosome 19 and for half of sequence is different from that of typical green plants. This chromosome 2, respectively. In addition, about 80% of the may be a characteristic peculiar to red algae, and it would be transposable elements concentrated in these regions also interesting to determine teleomeric sequences in closely show abnormality. A genome sequence for O. lucimarinus, related alga such as Cyanidium cardalium and Galdieria a closely related species of O. tauri, was completed recently suruphuraria. The C. merolae genome contains 26 class I (Palenik et al. 2007). O. lucimarinus has 13.2 million base elements (retrotransposons) and eight class II elements pairs with 21 chromosomes and 7,651 genes. This has which constitute only about 0.7% of the whole nuclear enabled a comparison of genome information for O. tauri genome. The transcripts of three of the 26 retrotransposons and O. lucimarinus. In a comparison to chromosomes 2 and are contained an intact reverse transcriptase open-reading 18 of O. lucimarinus, which correspond to chromosomes 2 frame. A BLASTX search suggested that all 26 retrotrans- and 19 of O. tauri, it was found that the chromosomes of O. posons were closely related to non-long terminal repeat tauri exhibited marked differences in composition. More- retrotransposons. Although LTR retrotransposons are over, chromosome 21 of O. lucimarinus is comprised of widely distributed in eukaryotes, there are none in the parts of chromosomes 9 and 13 of O. tauri. It was found that C. merolae genome. In contrast, there were 253 copies of chromosome 21 arose recently from duplication and fusion a novel interspersed repetitive element similar to the shrimp of the chromosomes. It would be enlightening to cross these spot syndrome virus. These repetitive elements have an two species and observe the meiosis process to examine the average size of 3.2 kbp, and are distributed randomly on all changes in chromosome composition and investigate the chromosomes, and together comprise about 5% of the correlation with the speciation. However, sexual repro- genome (Nozaki et al. 2007). duction has not been recorded for these algae to date.

Ostreococcus tauri Chlamydomonas reinhardtii

Ostreococcus is a member of the so-called picoplankton Chlamydomonas is one of the most popular algae used in family of marine algae. Their cell size of 1 lm is the experimental studies (Harris 2001). This alga has been used smallest among the eukaryotes. Recently, a detailed three- for many years in studies of genetics, biochemistry and cell dimensional structure of O. tauri, obtained using electron biology as a model experimental organism. In addition to a cryotomography, has been described (Henderson et al. long history of classical biological experiments, research 2007). The alga was found to be composed of one nucleus, into the molecular biology of Chlamydomonas has also mitochondrion, chloroplast, peroxisome (microbody), shown marked developments recently. The key advantages Golgi body, and several vesicles and granules. It belongs to of C. reinhardtii for studies of photosynthesis is their family Prasinophyceae, which is believed to be the most ability to grow either photoautotrophically or heterotro- primitive species in the green lineage from which all other phically (using acetate as the sole source of carbon) and to green algae and ancestors of land plants descended. O. tauri assemble its photosynthetic apparatus in the dark. Dark- 123 8 J Plant Res (2008) 121:3–17

Fig. 3a–g Phase contrast/ fluorescent (a, b, f), fluorescent (c, e, g) and phase-contrast (d) photomicrograph images of O. tauri (a, c–g) and C. merolae cells (b–g) with chloroplasts (red autofluorescence) after staining with DAPI. The O. tauri cells (a, c–g) are smaller than the C. merolae cells (b–g). By contrast, the fluorescent intensity of the O. tauri cell nuclei (small arrowheads in c and e) is higher than that of the nuclei in C. merolae (large arrowhead in e) (Kuroiwa et al. 2004). Scale bar 1 lm

Table 3 Fluorescent intensities (number of photons) of O. tauri and consists of circular molecules with an inverted repeat C. merolae cell nuclei similar to the general form for photosynthetic eukaryotes O. tauri C. merolae (Maul et al. 2002). The construction of and phenotype A B A/B analyses for various genes have been examined using genetic recombination techniques. Chlamydomonas is the 3 3 DAPI 1,134 9 10 455 9 10 2.5 most suitable organism for analyzing molecular mecha- 3 3 SYBR Green I 21,983 9 10 6,398 9 10 3.4 nisms of replication, transcription and recombination of the A and B are the mean photon counts over the whole area every second chloroplast genome. The atypical composition of the after staining with DAPI or SYBR Green I (Kuroiwa et al. 2004) nuclear genome was suggested prior to genome analysis. In particular, the high ratio of GC content for the non-coding region is an extremely unique characteristic. It was thought grown cells, grown with acetate medium, maintain normal that this high GC content (about 64%) of nuclear DNA chloroplast structure (Harris 1989; Dent et al. 2001). In interfered with the sequencing analysis. Finally, the outline addition, the characteristic of the algae to move using two of the draft genome sequence was recently completed. The flagellae has greatly contributed to flagellum movement genome size is approximately 120 million base pairs with analysis of eukaryotes. Another advantage of this organism 17 chromosomes (Grossman et al. 2003; Table 2). The size is its susceptability to gene introduction methods, both to of this genome is almost the same as that of the nuclear the plastid (Boynton et al. 1988; Newman et al. 1990) and genome of the Arabidopsis thaliana, which is a model land the established nucleus (Debuchy et al. 1989; Kindle et al. plant. However, 1,557 scaffolds still exist (version 3.0 data 1989; Shimogawara et al. 1998). Because various markers from JGI: http://genome.jgi-psf.org/Chlre3/Chlre3.home. and introduction methods have been established, tagged html). Because contigs are not assigned on each chromo- genes in the nuclear genome can be picked up and cloned some, the sizes of the individual chromosomes cannot be (Tam and Lefebvre 1993). The genomes of mitochondria determined from the results of the genome analysis. and chloroplast have been sequenced. The mitochondria Recently, a karyotype with sufficient resolution has been genome of C. reinhardtti is a linear molecule with a length reported which disputes the number and genome size of the of 15.7 kb, and has only eight protein coding genes (Boer chromosomes in C. reinhardtii (Aoyama et al. 2007). This et al. 1985). Most mitochondrial genomes of photosyn- result should prove useful in the estimation of chromosome thetic organisms are circular molecules; so C. reinhardtti is size. It would be preferable to fill the remaining gaps very unusual. On the other hand, the chloroplast genome between contigs and assign them on the 17 chromosomes.

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These contigs contain over 15,000 predicted genes. The genome size from v3.0 assembled data from JGI was 31.3 known functional proportion of the genes is predicted to be million base pairs (http://genome.jgi-psf.org/Thaps3/ two-thirds, and the function of the remaining genes is still Thaps3.home.html). The genomes of the mitochondria unknown. These genes are annotated by members of the and chloroplast were also sequenced in 2005 and 2006, Chlamydomonas Community. respectively, and information on all three genomes is now available.

Thalassiosira pseudonana Biological analyses based on genome information Diatoms are unicellular eukaryotic algae with unique pig- ments and an ornate silica casing. These algae are found Figure 4 summarizes the repertoire of proteins in each throughout marine and freshwater ecosystems and may be organism based on their assignment to eukaryotic clusters responsible for as much as 20% of global primary pro- of orthologous groups (KOGs). The distribution of the ductivity. Diatoms are the main primary producer in the functional classification of each alga shows the ratio of the oceans. The most characteristic feature of diatoms is the gene compositions according to their characteristics. The form of the silicified cell wall that consists of silicic acid distributions were, on the whole, similar for C. merolae and outside the cell (Table 1). The cell walls display species- O. tauri, which have a similar genome size. The extremely specific nanostructures of extremely fine detail. Genome low proportion of genes for the cytoskeleton found in C. analysis of diatoms has been motivated by considerations merolae, as compared with the other organisms, might of their ecological and evolutionary importance. The dia- reflect its simple cellular organization and systems. As tom on average contains a several-hundred mega base pair compared with the others, the rate of occurrence of genes genome (Veldhuis et al. 1997). T. pseudonana was con- related to transcription, signal transduction mechanisms, sidered to be suitable for complete genome analysis and extracellular structures is very high in C. reinhardtii. because of its comparatively small genome size. The This would reflect its comparatively complicated cell genome of T. pseudonana was sequenced in 2004, pro- structure and life cycle. The ratio of the genes related to viding a clear outline (Armbrust et al. 2004). Twenty-four nuclear structure is markedly higher in T. pseudonana pairs of nuclear chromosomes ranging in size from 0.66 to compared to the other functional categories. Although many 3.32 Mb were characterized based on sequence analyses, genes related to the nuclear structure are assigned and are giving a total of 34.5 million base pairs (Table 2). The nucleolar GTP/ATPase, the reason for this is unknown. The

Fig. 4 Comparison of the functional classifications of proteins among four unicellular algae. Columns represent the proportion of proteins assigned C. merolae O. tauri C. reinhardtii T. pseudonanaTranslation, ribosomal structure and biogenesis to the KOG classification of RNA processing and modification each organism, C. merolae, Transcription O. tauri, C. reinhardtti and Replication, recombination and repair T. pseudonana, in a left to light Chromatin structure and dynamics fashion. The actual numbers of Cell cycle control, cell division, chromosome partitioning proteins assigned to each Nuclear structure classification in O. tauri, Defense mechanisms C. reinhardtti and T. Signal transduction mechanisms pseudonana were obtained Cell wall/membrane/envelope biogenesis from the JGI website Cell motility Cytoskeleton (http://www.jgi.doe.gov/) Extracellular structures Intracellular trafficking, secretion, and vesicular transport Posttranslational modification, protein turnover, chaperones Energy production and conversion Carbohydrate transport and metabolism Amino acid transport and metabolism Nucleotide transport and metabolism Coenzyme transport and metabolism Lipid transport and metabolism Inorganic ion transport and metabolism Secondary metabolites biosynthesis, transport and catabolism General function prediction only Function unknown

123 10 J Plant Res (2008) 121:3–17 global image of gene composition among the four algae is are very important to the formation of the plastid mem- as has been shown above. Considering their unique meta- brane in photosynthetic organisms, and they are bolic systems, some of the biological phenomena analyzed synthesized using prokaryotic and eukaryotic pathways, using this genome information are described below. which exist in the plastid and the endoplasmic reticulum respectively. All of the glycerolipids in photosynthetic algae have been found in most studies. In the case of C. Analyses related to cellular metabolism merolae, the fatty acid composition was very simple, with palmitic, stearic, oleic, and linoleic acids being the major Sulfate metabolism in C. merolae acids found. Unsaturated fatty acids containing two or more double bonds were not detected experimentally and Since C. merolae inhabits extreme (high-temperature, major desaturase genes, such as acyl lipid desaturases of strongly acidic) environments, it makes an interesting cyanobacterial origin and stearoyl acyl carrier protein subject for investigating metabolic systems adapted for desaturase, which is generally conserved in land plants and such extreme conditions. Its sulfur metabolism has been green algae, were not detected in C. merolae from genome particularly well investigated, since C. merolae lives in hot information. These results suggest that polyunsaturated springs typified by high sufide levels. Assimilation is the fatty acids are not indispensable to photosynthetic organ- only way to convert inorganic sulfur to organic sulfur. isms. It has been clearly shown, for the first time, from the Cysteine is the primary sulfur-containing compound and is genome information of C. merolae and experimental used along with other organic compounds. In photosyn- results for lipid biosynthesis, that lipid synthetic pathways thetic eukaryotes, two genes encode the key enzymes in differ in green and red algae. cysteine synthesis: O-acetylserine thiol liase (OASTL) and serine acetyl transferase (SAT). OASTL catalyzes the synthesis of cysteine from sulfide and O-acetylserine, Photosynthesis and selenoprotein genes in O. tauri which is synthesized from serine and acetyl-CoA by SAT. C. merolae is a good model organism for studies of Ostrecoccus tauri displays some unique characteristics adaptation to this environment and the related metabolic with respect to photosynthesis. The alga lacks some genes system. The two genes coding for OASTL and SAT were of importance to the light-harvesting complexes (LHC) of isolated from C. merolae (i.e., four genes in total) (Toda photosystem II, but maintains other LHCI antenna protein et al. 1998, 2001). SAT and OASTL have been demon- genes (Derelle et al. 2006). Furthermore, this unicellular strated to be active in three cellular compartments in higher alga has all of the genes for C4 photosynthesis without the plants—cytoplasm, mitochondria and plastid. All four mechanism for concentrating carbon dioxide. It is very genes of C. merolae were predicted to be of the organellar interesting that these genes exist even though the C4 types. Interestingly, OASTLs of C. merolae were derived photosynthesis system cannot actually function in this from both bacterial and eukaryotic origins, whilst SAT organism. The existence of unique metabolic systems for genes belonged to the bacteria family. Transcription of all carbon dioxide fixation are expected from the variation of of these genes has been confirmed using EST analysis, the genes related to photosynthesis, but these need exper- suggesting that all of these genes are functional and that imental verification. The genes of a transport system for sulfur metabolism systems of C. merolae utilize different nitrogen metabolism have also been completely identified. pathways in different compartments. Complementary However, whether the gene products, transporters, actually experiments using E. coli to investigate different properties function effectively in the uptake of the nitrogen source is of OSATLs also suggest distinct sulfur metabolic pathways not known. While there seems to be a close relationship in different subcellular components, most probably the between the amount of nitrogen supplied and the induction chloroplast and mitochondrion. It has also been suggested, of gametes for many algae, the sexual reproduction process from the KEGG results, that many genes concerned with has not yet been recorded for this alga. As studies regarding the metabolism of cysteine were also encoded in the C. the relationship between nitrogen metabolism and gamete merolae genome. genesis progress, particular characteristics may become clear. Ostreococcus has genes for a large proportion of selenocysteine-containing proteins relative to its genome Lipid metabolism in C. merolae size (Palenik et al. 2007). O. tauri has 26 selenocysteine- containing genes, while C. reinhardtii, with a genome size The entire picture for the lipid metabolism system of C. ten times that of O. tauri, has only 12 genes including merolae has been described recently using biochemistry selenocysteine (Lobanov et al. 2007). One major category and bioinformatics (Sato and Moriyama 2007). Acyl lipids of the selenoproteins in Ostreococcus is the glutathione 123 J Plant Res (2008) 121:3–17 11 peroxidases. There is a possibility that these genes are biosynthesis identified in this way actually took part in the related to stress defense. The detailed function and mean- synthesis of the cell wall, experiments were carried out ing of selenoproteins in eukaryotes remain unknown. O. using an inhibitor. Cells treated with 10 mM 1,3-diami- tauri, which holds many copies of selenoproteins, is a noproprane dihydrochloride, which inhibits polyamine material suited to studies of their function and it is hoped biosynthesis, were inhibited to about 10% of the growth of that detailed cytological analyses of the proteins can be untreated cells and had a rounded morphology. As valves performed. Genes commonly associated with the uptake of from the treated culture were incompletely formed, it was metal ions do not exist in O. tauri; thus a special mecha- suggested that some polyamines are crucial to valve for- nism, different from the general mechanism used by other mation (Frigeri et al. 2006). A unique feature of diatoms is algae, seems to be used. In particular, mechanisms con- their ornately patterned frustule that displays species-spe- cerning Fe uptake are peculiar to O. tauri. With respect to cific nanostructures. Recent attention has focused on the the metabolism of trace elements, the genes for biosyn- biosynthesis of frustules and their nanostructures as a thesizing vitamin 12 do not exist at all. This alga seems to paradigm for future silica nanotechnology. Studies in this depend on extraction of vitamin 12 from the external field will progress based on genomic information. By environment and it uses substrates to synthesize combining transformation methods, it should become methionine. possible to gain a much better understanding of the molecular mechanism of frustule formation in the future.

Frustule biogenesis in T. pseudonana Post-genome analyses in C. reinhardtii The most distinctive aspect of diatoms is the frustule, which consists of hydrated silicon dioxide (silica). The Chlamydomonas reinhardtii is a good experimental model silica structure is formed by expanding and molding the to use to investigate the structure and organization of membrane-bound silica deposition vesicle. Most of the photosystems, chloroplast biogenesis, the construction of biosyntheses and biochemistry relating to the silicon frus- the flagellar apparatus, the sexual reproduction process, the tule are still to be elucidated. As a result of genome uniparental inheritance of the organelle genome, adaptation analysis, three genes encoding transporters for active to various environments, and circadian rhythms via various uptake of silicic acid have been identified (Armbrust et al. molecular biology and genetic techniques. Proteomics or 2004). The results from experiments on transcriptional transcriptomics, based on the available genomic resources, activities and the protein levels for these genes, performed has begun to reveal new details in each field of research. In using a synchronized culture, indicated that a major regu- particular, analyses focusing on proteomes have already latory step for SIT expression occurs at the translational or provided new insights into the dynamics and compositions post-translational level, and that SIT activity is controlled of the photosynthetic apparatus, the chloroplast ribosome, largely by intracellular processes and not by protein levels the respiration machinery of the mitochondria, phospho- (Thamatrakoln and Hildebrand 2007). To study molecular proteome, and the flagellum. DNA microarrays and components of the silica deposition vesicle, and in partic- macroarrays have been used to study transcriptomes of ular membrane-associated proteins involved in structure carbon-concentrating mechanisms (CCMs) in C. rein- formation, isolated cell wall fraction proteomes from the hardtii (Im et al. 2003; Miura et al. 2004; Yoshioka et al. synchronized culture of T. pseudonana have been analyzed 2004). Recent improvements to microarray techniques (Frigeri et al. 2006). Tandem mass spectrometric analysis have also been applied to the analysis of other metabolic based on sequenced genome information identified 31 systems (Zhang et al. 2004; Eberhard et al. 2006). Prote- proteins in the fraction. The patterns of the transcripts ome analyses are generally used to understand the according to the cell cycle have been analyzed with respect construction of the photosynthetic apparatus (Yamaguchi to these protein-encoding genes, and a relationship to et al. 2003; Stauber et al. 2003; Turkina et al. 2006; Gillet frustule formation has been suggested. In particular, glu- et al. 2006; Schmidt et al. 2006), light and circadian pro- tamate acetyltransferase, which is one of the genes related grams (Wagner et al 2004), the composition of to the synthesis of polyamine, was identified in the fraction. mitochondrial protein components (Lis et al. 2003), and so The expression patterns of mRNA for other genes in the on. A large number of different mutant loci have also been polyamine biosynthesis pathway were then analyzed during shown to affect the assembly and function of the flagellar cell wall synthesis. Three genes (ornithine decarboxylase, apparatus. Biochemical analyses of wild-type and mutant- ornithine carbamoyltransferase, and S-adenosylmethionine type flagella have demonstrated that the flagellar axoneme decarboxylase) were correlated to the timing of cell wall alone is composed of more than 200 proteins (Dutcher synthesis. To confirm that the genes of polyamine 1995). Proteomic analyses have been performed on the 123 12 J Plant Res (2008) 121:3–17 polypeptide components of the flagella and basal body (Li et al., 2004; Keller et al. 2005; Pazour et al. 2005; Yang et al. 2006). These post-genome analyses will be exten- sively utilized, in combination with existing genetic, biochemical, and molecular tools, in future algal studies (Stauber and Hippler 2004). Genome information for C. reinhardtii will be useful for studies into the proliferation or inheritance of chloroplasts. Chloroplasts multiply after the replication and distribution of chloroplast DNA; however, the detailed mechanisms of DNA transmission are not well understood. The moc mutants of the altered localization and distribution of chloroplast DNA have been isolated (Misumi et al. 1999); thus, the molecular mechanisms of the transmission of genetic information during vegetative proliferation should be elucidated based on the genome information. On the other hand, the uniparental inheritance of chloroplast and mitochondrial genomes during the sexual reproduction process is a universal trait among the eukaryotes, from algae to vertebrates (Kuroiwa 1991; Gillham 1994; Ni- shimura et al. 2002, 2006). The processes of the degradation of organelle DNA during sexual reproduction have been well studied using C. reinhardtii as a model system (Kuroiwa et al. 1982, 1985; Kuroiwa 1991; Aoy- ama et al. 2006). It is now thought that uniparental inheritance is achieved through active processes, including the rapid digestion, protection, and amplification of organelle DNA. Though the disappearance of organelle DNA in Fig. 5 Model for the molecular mechanism of uniparental inheri- accord with the course of the sexual reproduction process tance in C. reinhardtii based on the active digestion of mt- cpDNA by Mt+-specific DNase (MDN). In vegetative cells, MDN is absent or from gametogenesis to meiosis has been examined cyto- inactivated in both mating types. During gametogenesis, MDN is logically, the detailed molecular mechanism and the genes synthesized or activated only in mt+ cells. At the same time, mt+ related to it are not well understood. Genes encoding these cpDNA becomes resistant to the action of MDN. During gamete factors should be found in the future by mass spectrometry fusion, MDN obtains access to unprotected mt- chloroplasts and digests mt- cpDNA, leading to the uniparental inheritance of analysis based on the genome information presently ana- cpDNA. Several factors may mediate the successful digestion of lyzed (Ogawa and Kuroiwa 1986, 1987, 1985; Uchida et al. mt- cpDNA after zygote formation: (1) entry of MDN into mt- 1992, 1993; Nishimura et al. 1999, 2002). Our research chloroplasts; (2) efficient access of MDN to cpDNA molecules; and 2+ target is to clarify the mechanisms of uniparental inheri- (3) an increase in Ca inside mt- chloroplasts. Zygote-specific gene expression may be crucial to these processes (Nishimura et al. 2002) tance shown in Fig. 5 based on the genome information for C. reinhardtii. are so-called photoreceptors, exist in C. merolae even though the synchronization of the cell cycle is induced Studies of organelle proliferation based on genome using light (Matsuzaki et al. 2004). The existence of an information for C. merolae unknown light receptor mechanism is suggested. More- over, importantly, the condition of isolation of each The use of sequenced genome information to elucidate the organelle has been examined in detail (Miyagishima et al. biological phenomena is important. One example is a study 1999b, 2001a; Yoshida et al. 2006; Nishida et al. 2007; relating to the mechanism of organelle division in C. Yagisawa et al. 2007). The basic processes of mitosis that merolae. Initially, synchronization of cell cycles with light/ have been analyzed in C. merolae cell so far are repre- dark cycles was established in C. merolae (Suzuki et al. sented in Fig. 6. During interphase, the centrosome forms 1994; Terui et al. 1995) and the division process of the cell the focus for the interphase microtubule array outside the was analyzed in detail cytologically prior to studies of nucleus. By early prophase, the centrosome and the Golgi organelle division (Miyagishima et al. 1999a; Misumi et al. apparatus divide, and the resulting two asters and the Golgi 2005). Neither phytochrome nor phototropin, both of which apparatus move apart. Chromosomal condensation does not 123 J Plant Res (2008) 121:3–17 13

a b c d e

N M P

f g h i j ER N NLO CEN LY G MI

Fig. 6a–j The basic course of mitosis in a C. merolae cell. cannot be identified (d). Chromosomes do not condense during Illustrations show the behavior of cellular components during mitosis. mitosis. Cytokinesis starts from the plastid side and closes between Phase contrast–fluorescent images of C. merolae interphase (a) and daughter nuclei (e). Scale bar in e 1 lm. Schematic representation of dividing cells (b–e) showing the localization of nuclear (N), C. merolae interphase (f) and dividing cells (g–j); cell contains basic mitochondrial (M), and plastid DNA (P in blue/white) after DAPI organelles [a nucleus (N), a mitochondrion (M), and a plastid (P), ER, staining are shown. The plastids emit red autofluorescence. Divisions a Golgi apparatus (G), lysosomes (LY), and a microbody (MI)]. The of the plastid, mitochondrion, and nucleus occur in this order (a–e). nucleus has a nucleolus (NLO), and the mitochondrion and plastids During late G2 (a) and prophase (b), the plastid and mitochondrial contain their own nuclei (nucleoids) in the center (blue), respectively divisions advance and finish by the metaphase (c). At anaphase, the (Misumi et al. 2005) chromatids begin to move toward the poles, but each chromatid

occur during prophase. Plastid and mitochondrial divisions division, a tiny contractile-like ring appears at the equator start in this order in late G2 and end by the metaphase. The of the cell. dynamic trio of rings control mitochondrial and plastid The electron-dense plastid dividing ring (PD ring) was divisions. Microbody and lysosomes associate with the first identified outside the plastid envelope at the con- dividing V-shaped mitochondrion. At metaphase and early stricted site of the dividing chloroplast in C. caldarium, anaphase, the bipolar structure of the spindle is clear, and which is a close relation of C. merolae (Mita et al. 1986; all chromosomes appear to be aligned at the equator of the Mita and Kuroiwa 1988). Presently, the favored organism spindle. However, individual chromosomes cannot be for studies of organelle division has changed to C. merolae identified. Plastid and mitochondrial divisions finish by due to its large division ring and simpler structure. In 1993, metaphase, at which time division of the microbody starts. electron-dense deposits in the equatorial region of the The connecting bridge between daughter mitochondrion dividing mitochondria were first identified after an exten- and the microbody appears to play an important role in sive search for a mitochondrial dividing ring (MD ring) microbody division. At anaphase, sister chromatids sepa- throughout the cell and life cycles of C. merolae (Kuroiwa rate synchronously, and daughter chromosomes begin to et al. 1993). Research at the molecular level concerning the move toward the poles. Although this alga has the full organelle division has advanced notably through the use of complement of genes concerned with chromosome con- of synchronized culture techniques and isolation systems densation (Hirano 2005), chromosomal condensation never using C. merolae with its obvious division apparatus occurs during mitosis in C. merolae. At telophase, the (Miyagishima et al 2001a, b; Nishida et al. 2005). Recent daughter nuclei and nucleoli reform, and division of the studies have shown strong similarities between plastid and microbody may occur through the connecting bridge. By mitochondrial division (Takahara et al. 2000; Miyagishima late telophase, cytokinesis is almost complete. Although et al. 2003; Nishida et al. 2003; Kuroiwa et al. 2006; the typical actin gene is encoded in the genome, tran- Miyagishima and Kuroiwa 2006). scription of the gene does not occur and the gene product The division of plastids and mitochondria mainly was not detected (Takahashi et al. 1998). In addition, no involves a dynamic trio: an FtsZ ring of bacterial origin, genes encoding myosin were found. C. merolae cells electron-dense PD/MD rings, and eukaryotic dynamin therefore seem to divide using a system other than that of rings (Miyagishima et al. 2001b, 2003; Nishida et al. 2003; actomyosin. The mitochondrion, lysosome and microbody Kuroiwa et al. 2006; Miyagishima and Kuroiwa 2006). behave as if they are linked. At the final stage of cell Four genes represent mitochondrial FtsZ (FtsZ2-1 and

123 14 J Plant Res (2008) 121:3–17

FtsZ2-2) and plastid FtsZ (FtsZ1-1 and FtsZ1-2), both of (MALDI-TOFMS) method. Mda1 forms a stable homo- which form early at the site of future division, before the oligomer by itself and the protein participates in the formation of PD/MD rings can be confirmed by genome mitochondrial division machinery as a core component analysis (Matsuzaki et al. 2004). Only two dynamin genes (Nishida et al. 2007). Studies on chloroplast division have (Dnm1 and Dnm2) have been found, that play roles in the made similar progress. Isolates of (plastid-dividing, dyn- later stages of mitochondrion and plastid membrane fission, amin, and FtsZ) PDF machineries from dividing respectively. These findings suggest that both mitochondria chloroplasts were obtained from synchronized cells and and plastids in early eukaryotes exhibit similar division treated with detergents. The proteome of the isolated PDF apparatus consisting of complexes of bacterial and machineries was examined using SDS-polyacrylamide gel eukaryotic rings. Components other than this main electrophoresis, MALDI-TOF-MS and immunoblotting dynamic trio are being identified using highly synchronized analysis. A number of novel proteins as well as those cultures and fractionation of division machinery of C. already known, such as dynamin, were identified in the merolae. When the nucleotide sequence of an entire gen- proteome of the isolated PDF machineries (Yoshida et al. ome is sequenced, target genes can be identified from 2006). It was suggested that these components were the peptide mass fingerprints of ultrasmall quantities of pro- motive power for sliding bundles. As a result, the detailed teins. Recently, part of the mitochondrial division molecular mechanisms of chloroplast division by PDF machinery was purified from M phase-arrested C. merolae machineries were proposed (Fig. 7). The functions of cells through biochemical fractionation. The dynamin- several components of these machineries are currently related protein Dnm1 was one of the two major proteins in being elucidated. In addition, experiments to discover the purified fraction and was accompanied by a protein, novel genes related to the multiplication of organelles from Mda1, newly identified by the matrix-assisted laser transcriptome analysis have been performed using highly desorption/ionization time-of-flight mass spectrometry synchronized cultures and oligonucleotide microarrays

Fig. 7 Model of chloroplast division by PDF machinery. In the ﬁrst step, the GTPase dynamin molecules in the PDF machinery probably function as cross-bridges or covers that undergo microscopic movements to drive the sliding of the 5–7 nm ﬁlaments in the PD ring during the early phase of chloroplast division. The dynamin ring generates the dividing force for contraction with the aid of the other rings. In the second step, the dynamin molecules move from the surface of the PDF machinery to the inside of the machinery and play a role in pinching off the narrow bridge between daughter chloroplasts during the late phase of chloroplast division (Yoshida et al. 2006)

123 J Plant Res (2008) 121:3–17 15 containing all of the nuclear, mitochondrion and chloro- sequence analyses of other algae are also now in progress. plast genes (T. Fujiwara et al., unpublished results). Many It is very important to study a variety of species, repre- genetic candidates related to the proliferation of the cell senting algae from different phylogenetic groups, which and organelles have been identiﬁed from this experiment. exhibit both unique and important biological Thus, research into cell or organelle proliferation using C. characteristics. merolae has advanced notably using a combination of the Genetic manipulation methods have been developed and genome information and established experimental reported for various algae. C. reinhardtii is known to be the techniques. best model organism for studies of molecular biology with transformation techniques for both plastids and nucleus. Recent developments allow gene function to be evaluated Conclusion using RNAi suppression of gene activity, and GFP fusion proteins are available to elucidate localization of gene The algal genome sequences revealed that individual products. On the other hand, transformation experiments organisms possess unique features in terms of primary are also reported, such as those for C. merolae (Minoda sequence structures and gene compositions, making each et al. 2004) and several diatoms (Dunahey et al. 1995; Apt useful for understanding basic physiological aspects and et al. 1996, 2002; Falciatore et al. 1999). If functional the evolution of photosynthetic eukaryotes. When investi- analysis using gene disruption becomes possible by gating the maintenance of common cellular components, improving gene manipulation methods for these species, each algal genome project provides enough genome algae will become very useful in many research ﬁelds. A sequence data to allow comparative orthologous analysis of number of notable advances in our understanding of mor- each algal genome. Since such research is based on small phological, physiological and evolutionary aspects have genome repertoires, particularly for unicellular algae, algal been made so far. Needless to say, future proteomic and genome information should accelerate studies on, for gene-targeting analyses promise to accelerate our under- example, the establishment of cellular components, and it standing of these vital features of photosynthetic will allow us to elucidate cellular and molecular properties eukaryotes, including land plants. In particular, studies that are they have in common with land plants or other based on the three genome sequences (nuclear, mitochon- eukaryotes. drion and plastid), which all provide substantial genetic Genome projects are being generated for the green alga information on eukaryotes, are possible with C. merolae. Chlorella vulgaris, Dunaliella salina, Micromonas pusilla, In addition, as revealed by its recently completed and Volvox carteri, the red alga Galdieria sulphuraria, and genome sequence (Nozaki et al. 2007), the smallest the haptophyte Emiliania huxleyi, among others. Genome known histone-gene cluster, a very low density of

Fig. 8 Nuclear genome O. tauri, C. reinhardtii, T. pseudonana comparison of the four algal the number of chromosomes species in a modiﬁed ﬁgure which encode the histone genes t he (Nozaki et al. 2007; ra bsti t y t o http://www.biol.s.u-tokyo.ac.jp/ se 7< ra oc A in ns cu N a po pi rD g s ed users/tayousei/labdetail.html). of en on r om The number of introns, the be e um e n number of rDNA sets, the tsh 4-200 2-10% t h number of chromosomes which e n encode the histone genes, the u m b proportion of the genome occu- e n r o r o pied by transposons, and the t f

in p r number of protein-encoded h o it t e w genes are shown in the diagram. in s e 39 < % 7,892-15,000 e n n The genome size and its orga- e c g o d nization signiﬁcantly vary e d 1 g between all four kinds of uni- e n cellular algae. C. merolae e 3 s appears to have the simplest 4,775 nuclear genome and is extre- 0.7% mely useful for further studies of eukaryotic cells 0.5% Cyanidioschyzon merolae

123 16 J Plant Res (2008) 121:3–17 transposable elements, and other previously described Gillet S, Decottignies P, Chardonnet S, Le Marechal P (2006) simple features of the C. merolae nuclear genome are Photosynth Res 89:201–211 Grossman AR, Harris EE, Hauser C, Lefebvre PA, Martinez D, very distinctive and constitute the simplest set of genomic Rokhsar D, Shrager J, Silflow CD, Stern D, Vallon O, Zhang Z features found in any nonsymbiotic eukaryote yet studied (2003) Eukaryot Cell 2:1137–1150 (Fig. 8). Therefore, C. merolae represents an ideal model Harris EH (1989) The Chlamydomonas sourcebook. Academic, San organism for studying the fundamental relationships Diego, CA Harris EH (2001) Annu Rev Plant Physiol Plant Mol Biol 52:363–406 between information encoded on the three genomes Henderson GP, Gan L, Jensen GJ (2007) PLoS ONE 2:e749 and the functions of organelles. Furthermore, because Hirano T (2005) Curr Biol 15:R265–R275 C. merolae inhabits high-temperature environments (45– Im CS, Zhang Z, Shrager J, Chang CW, Grossman AR (2003) 50 °C), most of its proteins must be unusually heat-stable, Photosynth Res 75:111–125 Keller LC, Romijn EP, Zamora I, Yates JR III, Marshall WF (2005) and so its proteome may well provide important new Curr Biol 15:1090–1098 insights into the structural analysis of proteins and their Kindle KL, Schnell RA, Fernańdez E, Lefebvre PA (1989) J Cell Biol heat stability. 109:2589–2601 Kuroiwa T (1991) Int Rev Cytol 128:1–62 Acknowledgments This work was supported by Grant-in-Aid for Kuroiwa T (1998) Bioessays 20:344–354 Scientific Research A (No. 19207004 to TK), and by grants from Kuroiwa T, Kawano S, Nishibayashi S, Sato C (1982) Nature Frontier Project ‘‘Adaptation and Evolution of Extremophile’’ (to TK) 298:481–483 from the Ministry of Education, Culture, Sports, Science and Tech- Kuroiwa T, Nakamura S, Sato C, Tsubo Y (1985) Protoplasma nology of Japan, and from the Program for the Promotion of Basic 125:43–52 Research Activities for Innovative Biosciences (PROBRAIN to TK). 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