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Expression of the Nucleus-Encoded Chloroplast Division Genes and Proteins Regulated by the Algal Cell Cycle Shin-ya Miyagishima,*,1 Kenji Suzuki,2 Kumiko Okazaki,à,2 and Yukihiro Kabeya1 1 Center for Frontier Research, National Institute of Genetics, Shizuoka, Japan Advanced Science Institute, RIKEN, Saitama, Japan àPresent address: Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan *Corresponding author: E-mail: [email protected]. Associate editor: Charles Delwiche 2 Abstract Key words: cell cycle, chloroplast division, endosymbiosis, endosymbiotic gene transfer. Introduction Mitochondria and chloroplasts function as energy-converting organelles in eukaryotic cells. Both organelles arose from endosymbiotic events in which bacterial cells became integrated into primitive eukaryotic cells (Archibald 2009; Keeling 2011). In addition to these particular organelles, there are many other endosymbiotic events, which have integrated new functions into eukaryotic host cells. Specifically, all chloroplasts (and nonphotosynthetic plastids) trace their origin to a primary endosymbiotic event that took place more than a billion years ago in which an ancestral cyanobacterium was integrated into a previously nonphotosynthetic eukaryote possessing mitochondria. The ancient alga, which resulted from this primary endosymbiotic event, evolved into the Glaucophyta (glaucophyte algae), Rhodophyta (red algae), and Viridiplantae (green algae, streptophyte algae, and land plants), which together are grouped as the Plantae or Archaeplastida. After these primitive green and red algae had been established, chloroplasts then spread into other lineages of eukaryotes through secondary endosymbiotic events in which a red or a green alga became integrated into a previously nonphotosynthetic eukaryote (Archibald 2009; Keeling 2011). In a manner reminiscent of their free-living ancestor, chloroplasts have retained the bulk of their bacterial biochemistry and replicate by the division of the preexisting organelle. However, chloroplasts have also been substantially remodeled by the host cell so as to function as organelles within the eukaryotic host cell (Rodriguez-Ezpeleta and Philippe 2006; Archibald 2009; Keeling 2011). In particular, most of the genes once present in the cyanobacterial endosymbiont have either been lost or relocated to the nucleus (a process called endosymbiotic gene transfer). A protein import system was developed that translocates proteins encoded by the nucleus from the cytosol to the chloroplast. Several transporters that exchange metabolites between the cytoplasm and the chloroplast also evolved. Chloroplast division ultimately came to be a process tightly regulated by the host cell, which ensured © The Author 2012. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: [email protected] Mol. Biol. Evol. 29(10):2957–2970. 2012 doi:10.1093/molbev/mss102 Advance Access publication April 3, 2012 Downloaded from https://academic.oup.com/mbe/article-abstract/29/10/2957/1025493/Expression-of-the-Nucleus-Encoded-Chloroplast by guest on 03 August 2017 2957 Research article Chloroplasts have evolved from a cyanobacterial endosymbiont and their continuity has been maintained by chloroplast division, which is performed by the constriction of a ring-like division complex at the division site. It is believed that the synchronization of the endosymbiotic and host cell division events was a critical step in establishing a permanent endosymbiotic relationship, such as is commonly seen in existing algae. In the majority of algal species, chloroplasts divide once per specific period of the host cell division cycle. In order to understand both the regulation of the timing of chloroplast division in algal cells and how the system evolved, we examined the expression of chloroplast division genes and proteins in the cell cycle of algae containing chloroplasts of cyanobacterial primary endosymbiotic origin (glaucophyte, red, green, and streptophyte algae). The results show that the nucleus-encoded chloroplast division genes and proteins of both cyanobacterial and eukaryotic host origin are expressed specifically during the S phase, except for FtsZ in one graucophyte alga. In this glaucophyte alga, FtsZ is persistently expressed throughout the cell cycle, whereas the expression of the nucleus-encoded MinD and MinE as well as FtsZ ring formation are regulated by the phases of the cell cycle. In contrast to the nucleus-encoded division genes, it has been shown that the expression of chloroplast-encoded division genes is not regulated by the host cell cycle. The endosymbiotic gene transfer of minE and minD from the chloroplast to the nuclear genome occurred independently on multiple occasions in distinct lineages, whereas the expression of nucleusencoded MIND and MINE is regulated by the cell cycle in all lineages examined in this study. These results suggest that the timing of chloroplast division in algal cell cycle is restricted by the cell cycle-regulated expression of some but not all of the chloroplast division genes. In addition, it is suggested that the regulation of each division-related gene was established shortly after the endosymbiotic gene transfer, and this event occurred multiple times independently in distinct genes and in distinct lineages. MBE Miyagishima et al. · doi:10.1093/molbev/mss102 permanent inheritance of the chloroplasts during the course of cell division and from generation to generation. The majority of algae (both unicellular and multicellular) have just one or at most only a few chloroplasts per cell, and it is obvious that a direct and precise relationship must exist between the host cell and chloroplast division. That is, chloroplasts must divide once per cell cycle before the host cell completes cytokinesis (Possingham and Lawrence 1983). In contrast, land plants and certain algal species contain dozens of chloroplasts per cell that divide nonsynchronously, even within the same cell. Because land plants evolved from algae (streptophyte algae/charophytes) (Becker and Marin 2009; Delwiche and Timme 2011), there should have been a linkage between the cell cycle and chloroplast division in their algal ancestor that was subsequently lost during land plant evolution. Thus, it is probable that the continuity of chloroplasts in host cells was originally established by the synchronization of endosymbiotic cell division with host cell division in an ancient alga, as is seen in existing algae. However, the mechanism underlying the regulation of chloroplast division is poorly understood. Nevertheless, recent progress on the understanding of the chloroplast division machinery has allowed an examination of how the host cell regulates the timing of chloroplast division (Yang et al. 2008; Maple and Moller 2010; Pyke 2010; Yoshida et al. 2010; Miyagishima et al. 2011). Recent studies have shown that chloroplast division is performed by the constriction of a protein complex that forms at the division site, spanning both the inside and the outside of the two chloroplast envelope membranes (Yang et al. 2008; Maple and Moller 2010; Pyke 2010; Yoshida et al. 2010; Miyagishima et al. 2011). Consistent with the endosymbiotic origin of chloroplasts, the complex has retained certain components of the cyanobacterial division machinery, such as FtsZ and ARC6, inside the chloroplast (i.e., on the inner envelope membrane and the stromal side). In addition, proteins and structures of eukaryotic host origin, such as DRP5B and the PD rings, are involved in the division complex both inside and outside of the chloroplasts. The formation of the FtsZ ring is the first known event that occurs at the chloroplast division site. As in bacteria, positioning of the FtsZ ring formation is, at least in part, regulated by Min proteins of cyanobacterial origin. Normally, all the chloroplast division genes of cyanobacterial origin are encoded in the nuclear genome, indicating that these genes were translocated from the endosymbiont to the host genome. However, there are exceptions in which a few of the division genes, such as FtsI, FtsW, SepF, and MinD, are still retained by the chloroplast genome in certain algal lineages. The identification of the chloroplast division proteins provides a strategy for elucidating how the timing of chloroplast division is coupled with the host cell cycle. This requires a determination of when the chloroplast division genes and proteins are expressed and when the chloroplast division complex is formed during the algal cell cycle. Previous studies examined the expression patterns of chloroplast division genes using the red alga Cyanidioschyzon merolae (Takahara et al. 2000; Fujiwara et al. 2009; Yoshida 2958 et al. 2010), the green alga Chlamydomonas reinhardtii (Adams et al. 2008), and the diatom Seminavis robusta (which possesses chloroplasts of a red algal secondary endosymbiotic origin) (Gillard et al. 2008), the cell cycles of which are synchronized by a 24-h light–dark cycle. It was shown that transcripts of certain nucleus-encoded chloroplast division genes accumulate during a specific period of the synchronous cultures when chloroplasts divide. These studies raised the possibility that the cell cycle–based expression of the chloroplast division genes allows the chloroplast to divide once in a specific phase of the host cell cycle. Because cells were synchronized by a 24-h light and dark cycle and chloroplast division occurred early in the dark period, it is still unclear whether this expression of chloroplast division genes is directly controlled by the cell cycle or instead is regulated by light and/or circadian rhythms. In this study, we examined the relationship between the cell cycle and expression of the chloroplast division genes and proteins in various algal lineages that possess primary chloroplasts (glaucophytes and red, green, and streptophyte algae). In addition, the expression patterns of the division-related genes and proteins that are encoded in some of these algal chloroplast genomes were analyzed. The results show that most but not all the nucleus-encoded chloroplast division genes and proteins, regardless of origin, are expressed during the S phase and that the expression of the chloroplast-encoded division genes is not regulated by the host cell cycle. It is also shown that endosymbiotic gene transfer of minD and minE occurred on multiple independent occasions in distinct lineages, even though the expression of nucleus-encoded MIND and MINE and the corresponding proteins are restricted to S phase in these distinct lineages (Note that the genes in the chloroplast genome are indicated in lowercase, whereas those in the nuclear genome are in uppercase.). Our results suggest that the division synchronization of the host cell and chloroplasts relies on the cell cycle–based expression of a certain set of chloroplast division genes. The results also suggest that the cell cycle–based regulation of a division gene expression was established shortly after the gene was transferred into the host nuclear genome and that this event has occurred on multiple occasions. Materials and Methods Algal Culture Cyanophora paradoxa UTEX555 (NIES-547), Chlorella vulgaris C-27 (NIES-2170), Nephroselmis olivacea NIES-484, and Mesostigma viride NIES-296 were obtained from the Microbial Culture Collection at the National Institute for Environmental Studies in Japan (NIES). C. paradoxa, C. vulgaris, and M. viride cells were cultured in C medium. C. reinhardtii 137c cells were cultured in Sueoka’s high salt medium (HSM). N. olivacea cells were cultured in AF-6 medium. C. merolae 10D cells were cultured in Allen’s medium. All media were prepared according to the description provided by the NIES (http://mcc.nies.go.jp/02medium-e.html). Downloaded from https://academic.oup.com/mbe/article-abstract/29/10/2957/1025493/Expression-of-the-Nucleus-Encoded-Chloroplast by guest on 03 August 2017 Algal Cell Cycle and Chloroplast Division · doi:10.1093/molbev/mss102 For the synchronization of C. merolae, the cells in stationary culture were subcultured to ,1 107 cells/ml and subjected to a 12-h light/12-h dark cycle (100 lmol photons m2s1) at 42°C under aeration with ordinary air (Suzuki et al. 1994). The cells in the second cycle were used for further analyses. To arrest the cells in the S phase, a 1/50,000 volume of 10 mg/ml camptothecin solution in dimethyl sulfoxide (DMSO) was added 8 h after the onset of the second light period. To arrest the cells in the M phase, 1/500 volume of 50 mM MG132 solution in DMSO was added at the onset of the second dark period. For the synchronization of C. reinhardtii, the cells in log phase were subcultured to ,2 105 cells/ml and subjected to a 12-h light/12-h dark cycle (100 lmol photons m2s1) at 24°C under aeration with 1% CO2 in air (Surzycki 1971). The cells in the second cycle were used for further analyses. To arrest the cells in the S phase, 2deoxyadenosine was added to the culture at a concentration of 5 mM 8 h after the onset of the second light period. For the synchronization of C. vulgaris, the cells in log phase were subcultured to ,5 105 cells/ml and subjected to a 16-h light/8-h dark cycle (100 lmol photons m2s1) at 24°C under aeration with 2% CO2 in air (Tamiya et al. 1953). The cells in the third cycle were used for further analyses. For the synchronization of C. paradoxa, the cells were cultured to a cell density of 1 106 cells/ml at 24°C under continuous light (40 lmol photons m2s1) under aeration with ordinary air. To arrest the cells in the S phase, a1/1,000 volume of 5 mg/ml aphidicolin solution in DMSO was added to culture and cells were cultured for 24 h. To remove aphidicolin, cells were washed twice with fresh medium by centrifugation at 200 g for 10 min and then cultured under the same conditions as above. M. viride cells were cultured to a cell density of 2 106 cells/ml at 24°C under continuous light (40 lmol photons m2s1) on a rotary shaker. One microliter of the culture was subjected to a l Cell Sorter (Advance, Japan) to fractionate the smaller and larger cells. Gene Cloning In this study, we newly cloned C. paradoxa MIND and MINE, C. vulgaris FTSZ1, MINE, and DRP5B, M. viride PCNA (AB673435), FTSZ2, DRP5B, and N. olivacea FTSZ2 genes (The GenBank accession numbers other than that of M. viride PCNA are listed in supplementary table 1, Supplementary Material online). All the genes were cloned by reverse transcription polymerase chain reaction (RT–PCR) using cDNA as the template. cDNA was synthesized from total RNA using a random hexamer with ThermoScript RT (Invitrogen). For 3#-rapid amplification of cDNA ends (RACE), cDNA was synthesized using the primer 3race1 (poly-T and an adapter sequence). RNA was extracted as described below. A partial cDNA sequence of C. paradoxa MIND (EH035858) and a full-length cDNA sequences of MINE (EC659992) were found in the expressed sequence tag (EST) data (Reyes-Prieto et al. 2006). Expression of MINE MBE was confirmed by RT–PCR using the primers Cp-MINEf1 and Cp-MINE-r1. The upstream flanking sequence of the MIND fragment was amplified by degenerate RT– PCR using Cp-MINE-f1 and Cp-MINE-r1 for the first PCR and Cp-MINE-f1 and Cp-MINE-r2 for the second nested PCR. Partial cDNA sequences of C. vulgaris FTSZ1, MINE, and DRP5B were obtained by degenerate PCR using the primers Cv-FTSZ-f1 and Cv-FTSZ-r1 for FTSZ1, Cv-MINE-f1, and CvMINE-r1 for MINE and Cv-DRP5B-f1 and Cv-DRP5B-r1 for DRP5B, respectively. The downstream flanking sequences of ftsZ1 were obtained by 3#-RACE using the primers Cv-FTSZ-race-f1 and 3race2 for the first PCR and CvFTSZ-race-f2 and 3race2 for the second nested PCR. The downstream flanking sequences of MINE were obtained by 3#-RACE using Cv-MINE-race-f1 and 3race2 for the first PCR and Cv-MINE-race-f2 and 3race2 for the second nested PCR. Partial cDNA sequences of M. viride PCNA, FTSZ2, and DRP5B were obtained by degenerate PCR using the primers Mv-PCNA-f1 and Mv-PCNA-r1 for PCNA, Mv-FTSZ-f1 and Mv-FTSZ-r1 for FTSZ2, and Mv-DRP5B-f1 and Mv-DRP5Br1 for DRP5B, respectively. The downstream flanking sequences of DRP5B were obtained by 3#-RACE using Mv-DRP5B-race-f1 and 3race2. A partial cDNA sequence of N. olivacea FTSZ2 was obtained by degenerate PCR using the primers No-FTSZ-f1 and No-FTSZ-r1. Semiquantitative RT–PCR Cells were harvested from 1 to 10 ml culture by centrifugation and stored at 80°C until RNA extraction. Cells were disrupted with TRIzol reagent (Invitrogen). For the disruption of C. reinhardtii, C. vulgaris, and C. paradoxa, cells were homogenized in TRIzol with acid-washed glass beads (425–600 lm) by vigorous shaking. After the addition of chloroform and phase separation, total RNA was extracted from the upper aqueous phase by using an RNeasy Plant Mini Kit (QIAGEN). After DNaseI treatment, cDNA was synthesized from the RNA using random hexamer with ThermoScript RT (Invitrogen). The cDNA synthesized from 5 ng of total RNA was used for the PCRs. The PCRs were performed using the primers listed in supplementary table 2 (Supplementary Material online). Preparation of Antibodies The antibodies against C. paradoxa FtsZ, MinD, MinE, and SepF, C. reinhardtii FtsZ and DRP5B, and C. vulgaris MinD and MinE were raised in rabbits using the respective recombinant proteins as described previously (Miyagishima et al. 2003). The cDNA sequence encoding the full-length or a partial fragment of the respective protein was amplified by PCR using the primers listed in supplementary table 2 (Supplementary Material online). These PCR products were cloned into a pET100 expression vector (Invitrogen) and 6xHis fusion polypeptides were expressed in Rosetta Downloaded from https://academic.oup.com/mbe/article-abstract/29/10/2957/1025493/Expression-of-the-Nucleus-Encoded-Chloroplast by guest on 03 August 2017 2959 MBE Miyagishima et al. · doi:10.1093/molbev/mss102 (DE3) Escherichia coli cells, purified, and used as antigens. Antibodies were affinity-purified from the respective antisera by using respective recombinant proteins coupled to a HiTrap NHS-activated HP column (GE Healthcare). To prepare antibodies against C. vulgaris and C. reinhardtii Drp5B, both recombinant proteins were mixed and injected into the same rabbits. Immunoblot Analyses Cells were harvested from 5 to 50 ml of culture by centrifugation and stored at 80°C until immunoblot analysis. Cells were suspended in the protein extraction buffer (50 mM Tris, pH 7.5, 8 M Urea, and 0.1% Triton X-100). To disrupt C. reinhardtii, C. vulgaris, and C. paradoxa, cells were homogenized in the protein extraction buffer with acidwashed glass beads (425–600 lm) by vigorous shaking for 10 min. Protein content was determined with Bradford assay and equal amounts of the total protein were subjected to a immunoblot analysis. Immunoblotting assays were performed as previously described (Nakanishi et al. 2009). Antibodies against C. merolae Drp5B (Miyagishima et al. 2003) (1:1000 to detect C. merolae Drp5B), C. reinhardtii FtsZ1 (1:2000 to detect C. reinhardtii and C. vulgaris FtsZ1) and DRP5B (1:2000 to detect C. reinhardtii and C. vulgaris Drp5B), C. vulgaris MinD (1:5000 to detect C. reinhardtii and C. vulgaris MinD) and MinE (1:5000 to detect C. reinhardtii and C. vulgaris MinE), C. paradoxa FtsZ (1:2000 to detect C. merolae FtsZ2-1 and C. paradoxa FtsZ), MinD (1:1000 to detect C. paradoxa MinD), MinE (1:1000 to detect C. paradoxa MinE), and SepF (1:2000 to detect C. paradoxa SepF) were diluted as indicated. The primary antibodies were detected by horseradish peroxidase– conjugated goat anti-mouse or anti-rabbit antibody diluted to 1:20,000. The signal was detected by SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and the VersaDoc 5000 imaging system (Bio-Rad). Immunofluorescence Microscopy For the immunofluorescent detection of FtsZ1 in C. reinhardtii, cells were fixed in methanol at 20°C for 10 min and washed twice with phosphate-buffered saline, or PBS-T (0.1% Tween 20 in PBS). The cells were permeabilized for 30 min at room temperature with 10% DMSO in PBS-T. After blocking with 2% bovine serum albumin in PBS-T (blocking buffer) for 30 min, cells were labeled at room temperature for 2 h with anti-C. paradoxa FtsZ antibodies diluted to 1:2000 in blocking buffer (anti-C. reinhardtii FtsZ1 antibodies were not used because of the high background level on immunofluorescence microscopy). Cells were then washed twice with blocking buffer and incubated with Alexa Fluor 488 goat anti-rabbit IgG (H þ L) (Invitrogen) diluted in the blocking buffer at a concentration of 1:1000 at room temperature for 1 h. After washing twice with PBS-T, cells were observed under fluorescence microscopy (BX-51; Olympus). Immunofluorescent detection of FtsZ in C. paradoxa and M. viride was performed using a method modified from that used for C. reinhardtii. Cells were fixed in the fixation 2960 buffer (3% paraformaldehyde, 50 mM Pipes, pH 6.8, 10 mM ethylene glycol tetraacetic acid, and 5 mM MgSO4) for 1 h at room temperature and washed twice with PBS. Instead of using DMSO, the fixed cells were permeabilized for 30 min at room temperature with 0.1% Triton X-100 in PBS. For C. patadoxa, cells were further treated with 0.2 mg ml1 lysozyme dissolved in Tris–HCl, pH 7.5, 10 mM ethylenediaminetetraacetic acid for 10 min at 37°C. Anti-C. paradoxa FtsZ antibodies were diluted to a concentration of 1:500. Immunofluorescent detection of FtsZ2-1 in C. merolae was performed as described previously (Miyagishima et al. 2003). Results Distribution of the Chloroplast Division Genes of Cyanobacterial Origin and the Deduced Timing of Endosymbiotic Gene Transfer In order to examine and compare the relationship between the eukaryotic cell cycle and the timing of chloroplast division in the eukaryotic algae that possess primary chloroplasts, in this study, we used the red alga C. merolae, the green algae C. reinhardtii, and Chlorella vulgaris, the glaucophyte alga Cyanophora paradoxa and the streptophyte alga Mesostgma viride. C. merolae and C. reinhardtii were chosen because the whole nuclear and chloroplast genome sequence data are available and the methods for cell cycle synchronization by means of a light and dark cycle have been established (Surzycki 1971; Suzuki et al. 1994). In both organisms, all the known chloroplast division proteins are encoded in the nuclear genome (fig. 1B). The other algal species were chosen because chloroplast genomes have been fully sequenced and the cyanobacteria-descended MinD, FtsI, FtsW, and SepF are still encoded in the chloroplast genome (fig. 1B) (it was previously reported that MinE is encoded in the chloroplast genome of C. vulgaris, but see below), although the whole nuclear genome sequence data are not available at present. MinD was shown to regulate the positioning of the chloroplast FtsZ ring, as does its cyanobacterial counterpart (Yang et al. 2008; Maple and Moller 2010; Yoshida et al. 2010; Miyagishima et al. 2011). Although the functions of FtsI, FtsW, and SepF have not been determined in eukaryotic algae, these proteins localize at the bacterial division site and have been shown to be involved in cyanobacterial cell division (Marbouty et al. 2009a, 2009b). One of the aims of this study is to examine the relationship between the regulation of chloroplast division gene expression and the endosymbiotic gene transfer from the chloroplast to the nuclear genome. Thus, before characterizing each organism separately, we compared the repertoire of the known chloroplast division genes of cyanobacterial origin and the location of the genes (i.e., nuclear or chloroplast genome) to deduce the timing of endosymbiotic gene transfer (fig. 1B). Among the chloroplast division genes, FTSZ, ARC6, and MINC (MinC is involved in the positioning of FtsZ ring in bacteria, but its function in eukaryotic algae have not been determined) have been Downloaded from https://academic.oup.com/mbe/article-abstract/29/10/2957/1025493/Expression-of-the-Nucleus-Encoded-Chloroplast by guest on 03 August 2017 Algal Cell Cycle and Chloroplast Division · doi:10.1093/molbev/mss102 MBE FIG. 1. Schematic view of the chloroplast division complex and distribution of the chloroplast division proteins of cyanobacterial origin. (A) Localization of the components of the division complex (modified from Miyagishima et al. 2011). Only the known division site-localized components and proteins that determine the division site are shown (Yang et al. 2008; Maple and Moller 2010; Yoshida et al. 2010; Miyagishima et al. 2011). Some lineages possess two types of FtsZ that are involved in chloroplast division. In Viridiplantae, these are called FtsZ1 and FtsZ2. The division proteins unique to land plants such as PDV1, PDV2, MCD1, and PARC6 are not shown. Although omitted from the diagram, ARC3 is also involved in the positioning of the FtsZ ring and is encoded in nuclear genomes of land plants and certain lineages of green algae. Because ARC3 was not found in Cyanidioschyzon merolae and Chlamydomonas reinhardtii, in which complete nuclear genome sequences are available, we could not examine the expression in this study. MinC, FtsW, FtsI, and SepF were shown to be involved in cyanobacterial division (Marbouty et al. 2009a, 2009b), but the function and localization of these proteins in chloroplasts have not been determined. (B) Distribution of the chloroplast division genes of cyanobacterial origin and the deduced timing of endosymbitotic gene transfer (updated from Miyagishima et al. 2011). The tree topology is based on Becker and Marin (2009) and Archibald (2009). Branch lengths do not represent phylogenetic distances. Proteins were searched by Blast against the whole-genome database of C. merolae, Ostreococcus lucimarinus, C. reinhardtii, Physcomitrella patens, and Arabidopsis thaliana. In other cases, searches were performed in the chloroplast genome databases and EST databases of Cyanophora paradoxa, Guillardia theta, Nephroselmis olivacea, Chlorella vulgaris, and Mesostigma viride. Because the complete genomic information on the latter five organisms is not available, there are blank columns. The asterisks indicate the genes that were identified in this study. In the FtsZ column, ‘‘nm’’ indicates that FtsZ is encoded in the nucleomorph, a relic nucleus that originated from a red algal secondary endosymbiont. ‘‘D,’’ ‘‘E,’’ ‘‘Z,’’ ‘‘6,’’ and ‘‘C’’ indicate the deduced timing of endosymbiotic gene transfer of minD, minE, ftsZ, arc6 (ftn2 in cyanobacteria), and minC, respectively. C. paradoxa, Cyanophora paradoxa UTEX555; C. merolae, Cyanidioschyzon merolae 10D; G. theta, Guillardia theta; N. olivacea, Nephroselmis olivacea NIES-484; O. lucimarinus, Ostreococcus lucimarinus CCE9901; C. vulgaris, Chlorella vulgaris C-27; C. reinhardtii, Chlamydomonas reinhardtii; M. virde, Mesostigma viride NIES-296; P. patens, Physcomitrella patens; A. thaliana, Arabidopsis thaliana. The GenBank accession numbers of the amino acid sequences are summarized in supplementary table 1 (Supplementary Material online). Downloaded from https://academic.oup.com/mbe/article-abstract/29/10/2957/1025493/Expression-of-the-Nucleus-Encoded-Chloroplast by guest on 03 August 2017 2961 MBE Miyagishima et al. · doi:10.1093/molbev/mss102 found only in the nuclear genome, suggesting that these genes were transferred into the nuclear genome before the emergence of the extant algal lineages (fig. 1B). In contrast, ftsI, ftsW, and sepF have been found only in the chloroplast genome, suggesting that these genes were lost instead of being transferred into the host nuclear genome. The distribution patterns of ftsI and ftsW suggest that these genes were lost multiple times independently in distinct lineages (fig. 1B). The distribution patterns of the MIND (minD) and MINE (minE) genes suggest that these genes were transferred into the nuclear genome multiple times independently in distinct lineages, as described below. Using Blast searches of the EST database of C. paradoxa (Reyes-Prieto et al. 2006), we found the nucleus-encoded MINE (including full length of the orf; GenBank accession number EC659992) and MIND (containing 210 bp of the orf; EH035858) cDNA sequences. Their expression was confirmed by RT–PCR, and we further cloned a longer MIND cDNA (756 bp of the orf). Although it was previously reported that MinE together with MinD is encoded in the chloroplast genome of C. vulgaris (Wakasugi et al. 1997) (C-27, the same strain that was used in this study), the nucleotide sequence and the deduced amino acid sequence (NP_045873) showed no significant similarity to genes or proteins of other organisms, including minE genes and MinE proteins in our Blast search. In addition, we found MINE cDNA (CAB42593) in the previously characterized nuclear cDNA clones of Auxenochlorella protothecoides (Hortensteiner et al. 2000), which is closely related to C. vulgaris. Therefore, we expected that MinE would be encoded in the nuclear genome of C. vulgaris and, in fact, nuclear MINE cDNA was found by RT– PCR. MinE and MinD were not found to be encoded in the C. merolae genome, suggesting that these genes were lost in the ancestor. However, both proteins are encoded in the chloroplast genome of the cryptomonad Guillardia theta, which possesses chloroplasts of a red algal secondary endosymbiotic origin, suggesting that MinD and MinE were originally encoded in the chloroplast genome of a red algal ancestor. Given these distribution patterns of the MIND (minD) and MINE (minE) genes, endosymbiotic gene transfer of minD probably occurred on at least four independent occasions in the ancestors of glaucophytes, chlorophyceae (a group in green algae), Mamiellophyceae (a group in green algae), and land plants (fig. 1B). Endosymbiotic gene transfer of minE probably occurred independently at least twice in the ancestors of glaucophytes and Viridiplantae (green algae and streptophytes) (fig. 1B). Because the times of endosymbiotic gene transfer are deduced based on parsimonious considerations, it is likely that there may be more occasions of independent transfer for each gene. The Cell Cycle–Regulated Expression of Chloroplast Division Genes and Proteins in the Red Alga C. merolae The unicellular red alga C. merolae contains a single chloroplast per cell, which divides once before cytokinesis 2962 (fig. 2). Previous studies using C. merolae synchronized by a light/dark cycle showed that chloroplast division gene transcripts and proteins accumulate specifically around the onset of the dark period when chloroplast division commences (Takahara et al. 2000; Fujiwara et al. 2009; Yoshida et al. 2010). Because of the use of a light/dark cycle, it was still unclear whether the expression of the chloroplast division genes and proteins is regulated by the cell cycle rather than light or circadian rhythms. In addition, the timing of chloroplast division was assigned to the G2 and M phases (Fujiwara et al. 2009; Yoshida et al. 2010), essentially based on cell morphology observations, but the exact timing of chloroplast division gene expression and chloroplast division in relation to the cell cycle has not been reported. In order to examine whether the timing of chloroplast division is directly controlled by the cell cycle and, if so, to define the cell cycle stage in which the chloroplast divides, we arrested the cells in the synchronous culture in the S or M phase using inhibitors of cell cycle progression. It has been shown that cell cycle is regulated by circadian rhythms, but the circadian clock mechanism oscillates independently of cell cycle progression (Goto and Johnson 1995; Mori and Johnson 2001). To arrest the cells in the S phase, camptothecin, a specific inhibitor of DNA topoisomerase I, was added to the culture (Nishida et al. 2005). RT–PCR analyses showed that the level of PCNA (an Sphase marker gene; Pcna is involved in leading strand synthesis during DNA replication, Morgan 2006) remains constant and CDC20 (an M-phase marker gene; Cdc20 functions in the metaphase-to-anaphase transition, Morgan 2006) is not transcribed, which is in contrast to the culture without the inhibitor, in which the PCNA transcript disappears after the S phase and then the CDC20 transcript accumulates (fig. 2B). These results indicate that cells were efficiently arrested in the S -phase by camptothecin. To arrest the cells in the M phase, MG-132, a proteasome inhibitor, was added to the culture (Nishida et al. 2005). RT–PCR analyses showed that the level of CDC20 remains constant, in contrast to the culture lacking the inhibitor, in which the CDC20 transcript disappears after the M phase (fig. 2B). These results indicate that cells were efficiently arrested in the M phase by MG-132. When the cells were arrested in the S phase, although the cells did not divide the chloroplasts continued to divide, producing abnormal cells containing four chloroplasts per cell (fig. 2A), as described previously (Itoh et al. 1996; Nishida et al. 2005). RT–PCR analyses showed that the transcript levels of all the known chloroplast division genes remained constant after the emergence of the transcripts at approximately the 12th h in the synchronous culture (fig. 2B). Immunoblot analyses showed that the levels of FtsZ2-1 and Drp5B proteins also remained constant after the first appearance at approximately 12th h in the synchronous culture (fig. 2C). In contrast, when the cells were arrested in the M phase, chloroplast division stopped after one round of division during the S phase (fig. 2A). RT–PCR and immunoblot analyses showed that the transcripts of the chloroplast division genes and the FtsZ2-1 and Drp5B Downloaded from https://academic.oup.com/mbe/article-abstract/29/10/2957/1025493/Expression-of-the-Nucleus-Encoded-Chloroplast by guest on 03 August 2017 Algal Cell Cycle and Chloroplast Division · doi:10.1093/molbev/mss102 MBE FIG. 2. Expression of the chloroplast division genes and proteins during the cell cycle of the red alga Cyanidioschyzon merolae. C. merolae 10D was used in this study. (A) Progression of the cell cycle in the synchronous culture of C. merolae. Cells were entrained by a 12-h light and 12-h dark cycle. The cells in the second cycle are shown. To arrest the cells in the S or M phase, camptothecin (an inhibitor of DNA topoisomerase-I) or MG-132 (an inhibitor of proteasomes) was added to the culture at the indicated time point. (B) Semiquantitative RT–PCR analyses showing the change in the mRNA levels of the chloroplast division genes during synchronous culture. The chloroplast division genes are boxed. 18S rRNA (CMQ305R) was used as the quantitative control. PCNA (CMS101C) and CDC20 (CMA138C) were used as the S- and M-phase markers, respectively. (C) Immunoblot analyses showing the change in the levels of the chloroplast division proteins during synchronous culture. (D) Immunofluorescence microscopy showing the timing of FtsZ ring formation. The localization of FtsZ is shown by the green fluorescence and red is the autofluorescence of chlorophyll. Scale bar 5 5 lm (A), 2 lm (D). proteins disappeared after the S phase, as was the case in the culture without the inhibitor (fig. 2B and C). These results in C. merolae suggest that 1) the chloroplast division occurs only during S phase, 2) this is because the chloroplast division complex is formed specifically during the S phase (fig. 2D), and 3) this regulation is at least in part Downloaded from https://academic.oup.com/mbe/article-abstract/29/10/2957/1025493/Expression-of-the-Nucleus-Encoded-Chloroplast by guest on 03 August 2017 2963 MBE Miyagishima et al. · doi:10.1093/molbev/mss102 based on the expression and degradation of the components of the chloroplast division complex during the S phase and M phase, respectively. Cell Cycle–Regulated Expression of the Chloroplast Division Genes and Proteins in the Green Alga C. reinhardtii In order to explore the common features and differences in the regulation of chloroplast division among the eukaryotic algae, we examined the relationship between the cell cycle and the expression of the chloroplast division genes and proteins in the green alga C. reinhardtii. C. reinhardtii cell contains a single chloroplast, and this chloroplast divides during the S/M phase. Cells grow in size by many fold during the G1 phase and then enter into two to four rounds of S/M phases to produce 4–16 daughter cells (fig. 3A) (Surzycki 1971). Previous studies using C. reinhardtii synchronized by a light and dark cycle showed that the levels of the FTSZ1, FTSZ2, MIND, and MINE transcripts oscillate in the early dark period (Adams et al. 2008; Hu et al. 2008). Because this oscillation persisted under either a continuous light or a dark condition (peaking in the subjective-dark period; under the dark condition, cells still divided in a heterotrophic medium) after previous entrainment by a light and dark cycle, it was suggested that the expression correlates with the circadian rhythm (Hu et al. 2008). However, the cell cycle of C. reinhardtii is itself also regulated by the circadian rhythm (Goto and Johnson 1995), and therefore, it is still unclear whether the expression of the chloroplast division genes are regulated by the circadian rhythm or the cell cycle. The RT–PCR analyses performed using the synchronous culture showed that the transcripts of all the known chloroplast division genes accumulated specifically during the S/M phases (in addition to FTSZ1, there were also FTSZ2, MIND, and MINE, ARC6, MINC, and DRP5B) (fig. 3B). This pattern was also observed under the continuous light condition after entrainment by a light and dark cycle. When the cells were arrested in the S/M phase by deoxyadenosine, which has been shown to inhibit DNA synthesis in C. reinhardtii (Harper and John 1986), the transcript levels of the chloroplast division genes and an S/M-phase marker in C. reinhardtii, CYCA (Bisova et al. 2005) remained constant after its appearance at around 12th h (fig. 3B). This result suggests that the expression of chloroplast division genes is regulated by the cell cycle. In addition, immunoblot analyses showed that the FtsZ1, MinD, and Drp5B proteins specifically accumulate during the S/M phases and then are degraded (fig. 3C). The protein levels remained constant when the cells were arrested during the S phase (fig. 3C). Immunofluorescence microscopy showed that the FtsZ ring forms only during the S/M phases (fig. 3D). These results suggest that the expression of the chloroplast division machinery components during the S/M phase and degradation of these proteins after chloroplast division restrict the timing of chloroplast division complex formation in the green alga C. reinhardtii in a similar manner as in the red alga C. merolae. In contrast to C. merolae (fig. 2A), chloroplast division appeared 2964 to stop during constriction of the division site in C reinhardtii when the cell was arrested in the S/M phases (fig. 3A), but the reason is not clear at present. Relationship between the Cell Cycle and Expression of the Chloroplast Division Genes and Proteins in the Green Alga C. vulgaris, in Which the MinD Protein Is Encoded in the Chloroplast Genome In both C. merolae and C. reinhardtii, all the chloroplast division genes are encoded in the nuclear genome. In contrast, a few chloroplast division genes of cyanobacterial origin are still encoded in the chloroplast genome in certain algal lineages (fig. 1B). In order to examine the relationship between endosymbiotic gene transfer of chloroplast division genes from the chloroplast to the nuclear genome and also the regulation of the gene expression by the host cell cycle, we examined the expression of chloroplast division genes and proteins in the green alga C. vulgaris, in which the MinD protein is encoded in the chloroplast genome, as described above. C. vulgaris cells contain a single chloroplast and the cell cycle can be synchronized by a light and dark cycle (Tamiya et al. 1953). Similar to C. reinhardtii, the cells grow during the G1 phase and then enter into two to four rounds of the S/M phases so as to produce 4–16 daughter cells (fig. 4A) (Tamiya et al. 1953). RT–PCR analyses of C. vulgaris synchronized by a light and dark cycle showed that the nucleus-encoded FTSZ1, DRP5B, and MINE transcripts accumulated around the time of chloroplast division (fig. 4B). The transcript level of the chloroplast-encoded minD also oscillated, peaking around the light/dark transition, which corresponds to the time of chloroplast division. However, under continuous light after entrainment by a light and dark cycle, the minD transcript level continued to increase, in contrast to FTSZ1, DRP5B, and MINE, which decreased after chloroplast division (fig. 4B). These results suggest that the transcript level of the chloroplast-encoded minD changes in accordance with the light condition rather than the cell cycle and timing of chloroplast division. Immunoblot analyses showed that the nucleus-encoded FtsZ1, Drp5B, and MinE proteins accumulated prior to (MinE) or around (FtsZ1 and Drp5B) the time of chloroplast division, whereas the level of the chloroplast-encoded MinD remained constant throughout the cell cycle. This pattern of chloroplast-encoded MinD protein contrasts with the nucleus-encoded MinD protein in C. reinhardtii, suggesting that the regulatory system of MinD expression by the host cell cycle was established after the endosymbiotic gene transfer of minD from the chloroplast to the nuclear genome, at least in this lineage of green algae. We could not observe the timing of the chloroplast division complex formation by immunofluorescence microscopy because of the very thick cell walls of C. vulgaris. However, the Drp5B protein is expressed exclusively at the time of chloroplast division, suggesting that the mature division complex forms only during a specific period of the cell cycle in C. vulgaris. Downloaded from https://academic.oup.com/mbe/article-abstract/29/10/2957/1025493/Expression-of-the-Nucleus-Encoded-Chloroplast by guest on 03 August 2017 Algal Cell Cycle and Chloroplast Division · doi:10.1093/molbev/mss102 MBE FIG. 3. Expression of the chloroplast division genes and proteins during the cell cycle of the green alga Chlamydomonas reinhardtii. C. reinhardtii 137c (Chlorophyceae) was used in this study. (A) Progression of the cell cycle in the synchronous culture of C. reinhardtii. Cells were entrained by a 12-h light and 12-h dark cycle. The cells in the second cycle are shown. To arrest the cells in the S phase, deoxyadenosine, which blocks DNA synthesis, was added to the culture at the indicated time point. (B) Semiquantitative RT–PCR analyses showing the change in the mRNA levels of the chloroplast division genes during synchronous culture. The chloroplast division genes are boxed. EF-1a (XM_001696516) was used as the quantitative control. CYCA (XM_001693115) was used as the S-phase marker. (C) Immunoblot analyses showing the change in the levels of the chloroplast division proteins during synchronous culture. (D) Immunofluorescence microscopy showing the timing of FtsZ ring formation. The localization of FtsZ is shown by the green fluorescence and red is the autofluorescence of chlorophyll. Scale bar 5 10 lm (A), 2 lm (D). Relationship between the Cell Cycle and Expression of the Chloroplast Division Genes and Proteins in the Glaucophyte Alga C. paradoxa In order to determine whether chloroplast division is regulated by the host cell cycle also in glaucophytes, we examined the expression of the chloroplast division genes and proteins in the glaucophyte C. paradoxa. Glaucophytes have retained a peptidoglycan layer between the two chloroplast envelope membranes, unlike the chloroplasts in other lineages, and are suggested to have branched off earliest in the evolution of the Plantae (Reyes-Prieto and Bhattacharya 2007). The number of chloroplasts (called cyanelles) per C. paradoxa cell varies from one to six, but the majority of the cells in our culture contained two or four Downloaded from https://academic.oup.com/mbe/article-abstract/29/10/2957/1025493/Expression-of-the-Nucleus-Encoded-Chloroplast by guest on 03 August 2017 2965 MBE Miyagishima et al. · doi:10.1093/molbev/mss102 FIG. 4. Expression of the chloroplast division genes and proteins during the cell cycle of the green alga Chlorella vulgaris. C. vulgaris C-27 (Trebouxiophyceae) was used in this study. (A) Progression of cell cycle in the synchronous culture of Chlamydomonas reinhardtii. Cells were entrained by a 16-h light and 8-h dark cycle. The cells in the third cycle are shown. (B) Semiquantitative RT–PCR analyses showing the change in the mRNA levels of the chloroplast division genes during synchronous culture. The chloroplast division genes are boxed. EF-1a and chloroplast-encoded rpoA (NC_001865) were used as the quantitative controls. ‘‘N’’ and ‘‘CP’’ indicate genes that are encoded in the nuclear and chloroplast genome, respectively. (C) Immunoblot analyses showing the change in the levels of the chloroplast division proteins during synchronous culture. N and CP indicate the proteins that are encoded in the nuclear and chloroplast genome, respectively. Note that the cells were cultured for 4 more hours in the dark in (B) and (C). Scale bar 5 5 lm. chloroplasts per cell, as reported previously (Pickett-Heaps 1972; Sato et al. 2007). We tested several conditions in an effort to synchronize the cell cycle with a light and dark cycle, but we did not achieve synchronization. Therefore, we synchronized the cells by arresting the cells in the S phase with aphidicolin and restarting the cell cycle by removing aphidicolin under a continuous light condition. Cells during cytokinesis were predominantly observed around 6 h after aphidicolin removal (fig. 5A), suggesting that the cells had entered into M phase synchronously. RT– PCR analyses of an S-phase marker, PCNA, and an M-phase marker, CYCB (encoding ann M-phase cyclin), also showed that the culture was synchronized (fig. 5B). RT–PCR showed that throughout the cell cycle, the transcript levels of the nucleus-encoded MIND and MINE changed, peaking during the S phase (fig. 5B), whereas 2966 the transcript levels of the nucleus-encoded FTSZ and the chloroplast-encoded ftsW and sepF stayed constant (fig. 5B). Immunoblot analyses showed that levels of the MinD and MinE proteins change, peaking during S phase, whereas the FtsZ level is constant throughout the cell cycle (fig. 5C), as are levels of the corresponding transcripts (fig. 5B). In contrast to the constant expression of sepF transcript (fig. 5B), the SepF protein level changed, peaking during the S phase (fig. 5C). Although FtsZ level remained constant during the cell cycle, the FtsZ rings were predominantly observed in cells during the S phase (fig. 5D). These results indicate that the transcription of chloroplastencoded division genes is not regulated by the host cell cycle as in the case of C. vulgaris minD. The results also indicate that the host cell is able to determine the timing of chloroplast division by the cell cycle–based regulation of Downloaded from https://academic.oup.com/mbe/article-abstract/29/10/2957/1025493/Expression-of-the-Nucleus-Encoded-Chloroplast by guest on 03 August 2017 Algal Cell Cycle and Chloroplast Division · doi:10.1093/molbev/mss102 MBE FIG. 5. Expression of the chloroplast division genes and proteins during the cell cycle of the glaucophyte alga C. paradoxa. C. paradoxa UTEX555 was used in this study. (A) Progression of the cell cycle in the synchronous culture of Cyanophora paradoxa. Cells were arrested in the S phase with aphidicolin (an inhibitor of DNA polymerase) for 24 h under continuous light and then the cell cycle was restarted under continuous light along with the removal of aphidicolin. (B) Semiquantitative RT–PCR analyses showing the change in the mRNA levels of the chloroplast division genes during synchronous culture. The chloroplast division genes are boxed. EF-1a was used as the quantitative control. Pcna and cycB were used as the S- and M-phase markers. ‘‘N’’ and ‘‘CP’’ indicate the genes that are encoded in the nuclear and chloroplast genome, respectively. (C) Immunoblot analyses showing the change in the levels of the chloroplast division proteins during synchronous culture. N and CP indicate the proteins that are encoded in the nuclear and chloroplast genome, respectively. (D) Immunofluorescence microscopy showing the timing of FtsZ ring formation. The localization of FtsZ is shown by the green fluorescence and red is the autofluorescence of chlorophyll. Scale bars 5 10 lm (A and D). not all but only some of the nucleus-encoded chloroplast division genes/proteins. Relationship between the Cell Cycle and Expression of the Chloroplast Division Genes and Proteins in the Streptophyte Alga M. viride We have examined the regulation of the timing of chloroplast division in three major groups of eukaryotic algae, which possess primary chloroplasts (glaucophytes, as well as red and green algae). Finally, we examined the relationship in the streptophyte alga M. viride, one of the earliest diverging members of streptophytes, which include certain algal lineages and land plants (Becker and Marin 2009; Delwiche and Timme 2011). M. viride contains a single cup-shaped chloroplast per cell. Because we could not synchronize M. viride by a variety of light and dark cycles or drug treatments, we fractionated the nonsynchronous proliferating cells under continuous light using a microfluidic device into two fractions containing smaller and larger cells, respectively (fig. 6A). The fractionation yielded a sufficient number of cells for RT–PCR analysis but not immunoblotting. RT–PCR confirmed that cells in the S phase (expressing PCNA) and M phase (expressing CYCB) are enriched in the fraction of larger cells (fig. 6B). The analyses showed that the transcripts of the nucleus-encoded FTSZ and DRP5B are also enriched in the fraction of larger cells, whereas the transcripts of the chloroplast-encoded minD, ftsI, and ftsW are evenly distributed in the smaller and larger cell fractions (fig. 6B). Immunofluorescence microscopy revealed that the FtsZ ring exists only in the larger cells (fig. 6C). These results indicate that the formation of the chloroplast division complex and transcription of the nucleusencoded but not chloroplast-encoded chloroplast division Downloaded from https://academic.oup.com/mbe/article-abstract/29/10/2957/1025493/Expression-of-the-Nucleus-Encoded-Chloroplast by guest on 03 August 2017 2967 MBE Miyagishima et al. · doi:10.1093/molbev/mss102 FIG. 6. Expression of the chloroplast division genes during the cell cycle of Mesostigma viride. M. viride NIES-296 (Streptophyta, Mesostigmatophyceae) was used in this study. (A) Smaller cells and larger dividing cells were separated by a microfluidic device from log-phase culture under continuous light. (B) Semiquantitative RT–PCR analyses comparing the mRNA levels of the chloroplast division genes between the smaller cells (S) and the larger dividing cells (L). Chloroplast division genes are boxed. EF-1a (DQ394295) was used as the quantitative control. PCNA (DN262911) and CYCB (EC728922) were used as the S- and M-phase markers. ‘‘N’’ and ‘‘CP’’ indicate the genes that are encoded in the nuclear and chloroplast genome, respectively. (C) Immunofluorescence microscopy showing the timing of FtsZ ring formation. The localization of FtsZ is shown by green fluorescence. The differential interference contrast images and fluorescent images are merged. Scale bar 5 20 lm (A) and 10 lm (C). genes are regulated by the host cell cycle in M. viride, as had been observed in the green alga C. vulgaris and the glaucophyte alga C. paradoxa. Discussion Regulation of the Chloroplast Division Genes by the Algal Cell Cycle Because cells in many algal lineages contain just one or only a few chloroplasts per cell, chloroplasts have to be duplicated during a specific phase of the cell cycle before cytokinesis. Thus, there is a correlation between the timing of chloroplast division and the cell cycle. Previous studies showed that the transcripts (and proteins in the red alga C. merolae) of certain chloroplast division genes accumulate during a period of chloroplast division in 24-h light/ 2968 dark synchronous culture of a few species of algae (Takahara et al. 2000; Adams et al. 2008; Gillard et al. 2008; Hu et al. 2008; Fujiwara et al. 2009; Yoshida et al. 2010). However, it had remained unclear whether this gene expression is directly controlled by the cell cycle or is regulated by light and/or circadian rhythms. In this study, we showed that the expression of the chloroplast division genes and proteins is controlled by the cell cycle by examining cells arrested in the S phase or M phase. This regulation was observed in glaucophytes as well as red, green, and streptophyte algae, which include all the lineages of algae possessing primary chloroplasts. In addition, the results showed that the expression of all the known nucleus-encoded chloroplast division genes and proteins (i.e., components of the chloroplast division complex and its direct regulators) of both cyanobacterial and eukaryotic host origin, except for FtsZ in C. paradoxa, are regulated by the host cell cycle. Based on these results, there is probably a mechanism that controls the transcription of chloroplast division genes in the nuclear genome specifically during the S phase, which results in the expression of these proteins. In addition, nucleus-encoded chloroplast division proteins (except for C. paradoxa FtsZ and chloroplast-encoded SepF proteins) disappeared in the late M phase, suggesting that degradation of the chloroplast division proteins also likely plays a role in the regulation of chloroplast division. At present, it is not clear whether this degradation is just because of the instability of the proteins or involves proteases or other systems for proteolysis specific to the chloroplast division proteins. If the latter is the case, there should be at least two kinds of proteases or mechanisms, which are responsible for the degradation of the stromal (FtsZ, MinD, MinE, and SepF) and cytosolic (Drp5B) chloroplast division proteins, respectively. Because Drp5B disappeared during the M phase in C. merolae even when cells were arrested by the proteasome inhibitor MG-132, it is unlikely that proteasomes are responsible for the degradation of Drp5B. In contrast to the nucleus-encoded division genes and proteins, the expression of the chloroplast-encoded division genes and proteins was found to not be regulated by the host cell cycle. In addition, nucleus-encoded FtsZ in C. paradoxa was expressed throughout the cell cycle, whereas FtsZ ring formation was restricted to the S phase. Thus, the regulation of the expression of some but not all the chloroplast division genes and proteins are apparently sufficient to restrict the timing of chloroplast division to a period between the S phase and late M phase. It is possible that posttranslational modification of the chloroplast division proteins is also involved in the regulation of the timing of chloroplast division, such as phosphorylation or processing that is observed in mitochondrial division proteins (Nishida et al. 2007; Taguchi et al. 2007; Yoshida et al. 2009). Nevertheless, the regulation of both expression and the degradation of certain chloroplast division proteins is apparently sufficient to inhibit chloroplast division in cells during the G1 phase and late M phase because the cells do not express the set of components that is required to form a functional chloroplast division complex. Downloaded from https://academic.oup.com/mbe/article-abstract/29/10/2957/1025493/Expression-of-the-Nucleus-Encoded-Chloroplast by guest on 03 August 2017 Algal Cell Cycle and Chloroplast Division · doi:10.1093/molbev/mss102 The Relationship between the Expression of the Chloroplast Division Genes and Endosymbiotic Gene Transfer Unlike the nucleus-encoded division genes and proteins, it was shown that the chloroplast-encoded division genes and proteins do not correlate with the host cell cycle. One exception was the SepF protein in C. paradoxa, which predominantly accumulates during S phase, but the level of the sepF transcript remained constant through the cell cycle. Thus, it is suggested that the system for the transcriptional regulation of each chloroplast division gene by the host cell cycle was established after endosymbiotic gene transfer into the host nuclear genome. In addition, despite the fact that MIND was independently transferred into the nuclear genome at least four times and MINE was transferred at least twice in distinct lineages, the expression of these genes came to be regulated by the host cell cycle in each case. These observations indicate that there is an evolutionary trend, such that a division gene comes under the regulatory control of the cell cycle after endosymbiotic gene transfer, even though there are exceptions, such as FTSZ in C. paradoxa. It should be noted that the parallel independent endosymbiotic gene transfers predicted for minD and minE are not a rare occurrence. A previous study indicated that both independent gene loss and transfer have occurred frequently in the chloroplast genome (Martin et al. 1998). In general, a gene transferred from an organelle to the nuclear genome must acquire a eukaryotic promoter in order to be transcribed by the eukaryotic transcription machinery. In addition, the gene also needs to acquire a transit peptide-coding sequence in order to be imported into the organelle (Timmis et al. 2004). In the case of chloroplast division genes, each gene most likely acquired a promoter that is regulated by the cell cycle. In this regard, the immediately upstream genomic region of FTSZ and DRP5B in C. merolae possesses sequences that are similar to an E2F-biding site, TTTCGCGC, which is known to be recognized by the S-phase transcription factor E2F (Morgan 2006). Characterization of the promoter region and transcription factors of the chloroplast division genes are expected to yield important insights into this matter in the near future. This study suggests that endosymbiotic gene transfer and subsequent acquisition of transcriptional regulation by the host cell cycle have contributed to the synchronization of the host cell and primary chloroplast division. An additional question is whether this scheme is applicable to other endosymbiotic systems, such as mitochondria, secondary chloroplasts, and the chromatophore of Paulinella chromatophore (Marin et al. 2005). In C. merolae, the transcripts of the nucleus-encoded mitochondrial division genes DRP3, MDA1, ZED, and a-proteobacteria-descended FTSZ along with the corresponding proteins accumulate in a specific period of the 24-h light–dark cycle when the mitochondria divide (Nishida et al. 2007; Fujiwara et al. 2009; Yoshida et al. 2009). In the diatom (a stramenopile) Seminavis robusta, a single cell of which contains two MBE chloroplasts of a red algal secondary endosymbiotic origin, the nucleus-encoded FTSZ transcript accumulates in a specific period of the 24-h light–dark cycle in which the chloroplasts divide (Gillard et al. 2008). Thus, the scheme presented in this study for primary chloroplasts appears to be applicable to secondary chloroplasts and mitochondria. In addition, a similar scheme might also be applicable to the regulation of eukaryotic cell cycle of an endosymbiont by a eukaryotic host cell. Chlorarachniophyte algae possess a relic nucleus (nucleomorph) derived from a green algal endosymbiont. In Bigelowiella natans, nuceomorph division occurs during a specific period before mitosis (Moestrup and Sengco 2001). A recent study showed that endosymbiont-derived histone proteins are encoded in the host nuclear genome and are targeted to the nucleomorph. Transcripts of these histone genes were shown to accumulate during the time corresponding to S phase in a culture, which was partially synchronized by a 24-h light–dark cycle (Hirakawa et al. 2011). Further study of the mechanism of cell cycle–based expression of chloroplast division genes and other endosymbiotic systems will yield important insights into the common features that underlie the establishment and evolution of permanent endosymbiotic relationships. Supplementary Material Supplementary tables 1 and 2 are available at Molecular Biology and Evolution online (http://www.mbe. oxfordjournals.org/). Acknowledgments We thank Y. Ono and A. Yamashita for technical support and BSI’s Research Resources Center of RIKEN for production of antibodies. We are also grateful to National Institute for Environmental Studies (NIES) for providing some algal strains. This study was supported by grant-in-aid for Scientific Research from Japan Society for the Promotion of Science (no. 19870033 to S.M.) and by Core Research for Evolutional Science and Technology (CREST) Program of Japan Science and Technology Agency (JST) (to S.M.). References Adams S, Maple J, Moller SG. 2008. Functional conservation of the MIN plastid division homologues of Chlamydomonas reinhardtii. Planta 227:1199–1211. Archibald JM. 2009. The puzzle of plastid evolution. Curr Biol. 19:R81–R88. Becker B, Marin B. 2009. Streptophyte algae and the origin of embryophytes. 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