Yoshihisa Hirakawa

Protein targeting into secondary plastids of chlorarachniophytes.

Most plastid proteins are encoded by the nuclear genome, and consequently, need to be transported into plastids across multiple envelope membranes. In diverse organisms possessing secondary plastids, nuclear-encoded plastid precursor proteins (preproteins) commonly have an N-terminal extension that consists of an endoplasmic reticulum (ER)-targeting signal peptide and a transit peptide-like sequence (TPL). This bipartite targeting peptide is believed to be necessary for targeting the preproteins into the secondary plastids. Here, we newly demonstrate the function of the bipartite targeting peptides of an algal group, chlorarachniophytes, and characterize the functional domains of the TPL in the precursor of a plastid protein, ATP synthase delta subunit (AtpD), using a GFP as a reporter molecule. We show that the C-terminal portion of the TPL is important for targeting the AtpD preprotein from the ER into the chlorarachniophyte plastids, and several positively charged amino acids in the TPL are also necessary for transporting the preprotein across the 2 innermost plastid membranes. Compared with other groups with secondary plastids, the TPL functional domains of the chlorarachniophytes are unique, which might be caused by independent acquisition of their plastids.

TRANSIENT TRANSFORMATION OF A CHLORARACHNIOPHYTE ALGA, LOTHARELLA AMOEBIFORMIS (CHLORARACHNIOPHYCEAE), WITH uidA AND egfp REPORTER GENES1

A transient genetic transformation system was established for a chlorarachniophyte alga, Lotharella amoebiformis K. Ishida et Y. Hara. We first isolated sequences that contain a putative promoter for a RUBISCO SSU (rbcS) gene and a terminator for another copy of rbcS gene from L. amoebiformis. With those promoter and terminator sequences, we developed two expression vectors, pLaRGus and pLaRGfp, which code uidA and egfp genes, respectively. The cells were then transformed with each vector using a microparticle bombardment system. When the cells were transformed with the pLaRGus, β‐glucuronidase (GUS) staining dyed several cells blue. Green fluorescent protein (GFP) fluorescence was observed in the cells transformed with pLaRGfp. The highest transient transformation efficiency, 35 per 2 × 107 cells, was detected from the GUS staining. This study demonstrates that two reporter genes are expressed in L. amoebiformis cells when rbcS promoter and terminator are used. The conditions of transformation were also optimized. This is the first report of successful genetic transformation in chlorarachniophyte algae.

Characterization of Periplastidal Compartment–Targeting Signals in Chlorarachniophytes

Secondary plastids are acquired by the engulfment and retention of eukaryotic algae, which results in an additional surrounding membrane or pair of membranes relative to the more familiar primary plastids of land plants. In most cases, the endocytosed alga loses its eukaryotic genome as it becomes integrated, but in two algal groups, the cryptophytes and chlorarachniophytes, the secondary plastids retain a vestigial nucleus in the periplastidal compartment (PPC), the remnant eukaryotic cytoplasm between the inner and the outer membrane pairs. Many essential housekeeping genes are missing from these reduced genomes, suggesting that they are now encoded in the host nucleus and their products are targeted to the PPC. One such nucleus-encoded, PPC-targeted protein, the translation elongation factor like (EFL) was recently identified in chlorarachniophytes. It bears an N-terminal-targeting sequence comprising a signal peptide and a transit peptide-like sequence (TPL) similar to the plastid-targeted proteins of chlorarachniophytes as well as a hydrophilic C-terminal extension rich in lysine and aspartic acid. Here, we characterize the function of the N- and C-terminal extensions of PPC-targeted EFL in transformed chlorarachniophyte cells. Using green fluorescent protein as a reporter molecule, we demonstrate that several negatively charged amino acids within the TPL are essential for accurate targeting to the PPC. Our findings further reveal that the C-terminal extension functions as a PPC retention signal in combination with an N-terminal plastid-targeting peptide, which suggests that plastid and PPC proteins may be sorted in the PPC.

Nucleomorph Genome Sequences of Two Chlorarachniophytes, Amorphochlora amoebiformis and Lotharella vacuolata.

Many algal groups acquired complex plastids by the uptake of green and red algae through multiple secondary endosymbioses. As a result of gene loss and transfer during the endosymbiotic processes, algal endosymbiont nuclei disappeared in most cases. However, chlorarachniophytes and cryptophytes still possess a relict nucleus, so-called the nucleomorph, of the green and red algal endosymbiont, respectively. Nucleomorph genomes are an interesting and suitable model to study the reductive evolution of endosymbiotically derived genomes. To date, nucleomorph genomes have been sequenced in four cryptophyte species and two chlorarachniophyte species, including Bigelowiella natans (373 kb) and Lotharella oceanica (610 kb). In this study, we report complete nucleomorph genome sequences of two chlorarachniophytes, Amorphochlora amoebiformis and Lotharella vacuolata, to gain insight into the reductive evolution of nucleomorph genomes in the chlorarachniophytes. The nucleomorph genomes consist of three chromosomes totaling 374 and 432 kb in size in A. amoebiformis and L. vacuolata, respectively. Comparative analyses among four chlorarachniophyte nucleomorph genomes revealed that these sequences share 171 function-predicted genes (86% of total 198 function-predicted nucleomorph genes), including the same set of genes encoding 17 plastid-associated proteins, and no evidence of a recent nucleomorph-to-nucleus gene transfer was found. This suggests that chlorarachniophyte nucleomorph genomes underwent most of their reductive evolution prior to the radiation of extent members of the group. However, there are slight variations in genome size, GC content, duplicated gene number, and subtelomeric regions among the four nucleomorph genomes, suggesting that the genomes might be undergoing changes that do not affect the core functions in each species.

Plastid genome sequences of Gymnochlora stellata, Lotharella vacuolata, and Partenskyella glossopodia reveal remarkable structural conservation among chlorarachniophyte species

Chlorarachniophyte algae have complex plastids acquired by the uptake of a green algal endosymbiont, and this event is called secondary endosymbiosis. Interestingly, the plastids possess a relict endosymbiont nucleus, referred to as the nucleomorph, in the intermembrane space, and the nucleomorphs contain an extremely reduced and compacted genome in comparison with green algal nuclear genomes. Therefore, chlorarachniophyte plastids consist of two endosymbiotically derived genomes, i.e., the plastid and nucleomorph genomes. To date, complete nucleomorph genomes have been sequenced in four different species, whereas plastid genomes have been reported in only two species in chlorarachniophytes. To gain further insight into the evolution of endosymbiotic genomes in chlorarachniophytes, we newly sequenced the plastid genomes of three species, Gymnochlora stellata, Lotharella vacuolata, and Partenskyella glossopodia. Our findings reveal that chlorarachniophyte plastid genomes are highly conserved in size, gene content, and gene order among species, but their nucleomorph genomes are divergent in such features. Accordingly, the current architecture of the plastid genomes of chlorarachniophytes evolved in a common ancestor, and changed very little during their subsequent diversification. Furthermore, our phylogenetic analyses using multiple plastid genes suggest that chlorarachniophyte plastids are derived from a green algal lineage that is closely related to Bryopsidales in the Ulvophyceae group.

Polyploidy of Endosymbiotically Derived Genomes in Complex Algae

Chlorarachniophyte and cryptophyte algae have complex plastids that were acquired by the uptake of a green or red algal endosymbiont via secondary endosymbiosis. The plastid is surrounded by four membranes, and a relict nucleus, called the nucleomorph, remains in the periplastidal compartment that is the remnant cytoplasm of the endosymbiont. Thus, these two algae possess four different genomes in a cell: Nuclear, nucleomorph, plastid, and mitochondrial. Recently, sequencing of the nuclear genomes of the chlorarachniophyte Bigelowiella natans and the cryptophyte Guillardia theta has been completed, and all four genomes have been made available. However, the copy number of each genome has never been investigated. It is important to know the actual DNA content of each genome, especially the highly reduced nucleomorph genome, for studies on genome evolution. In this study, we calculated genomic copy numbers in B. natans and G. theta using a real-time quantitative polymerase chain reaction approach. The nuclear genomes were haploid in both species, whereas the nucleomorph genomes were estimated to be diploid and tetraploid, respectively. Mitochondria and plastids contained a large copy number of genomic DNA in each cell. In the secondary endosymbioses of chlorarachniophytes and cryptophytes, the endosymbiont nuclear genomes were highly reduced in size and in the number of coding genes, whereas the chromosomal copy number was increased, as in bacterial endosymbiont genomes. This suggests that polyploidization is a general characteristic of highly reduced genomes in broad prokaryotic and eukaryotic endosymbionts.

Diurnal Transcriptional Regulation of Endosymbiotically Derived Genes in the Chlorarachniophyte Bigelowiella natans

Abstract Chlorarachniophyte algae possess complex plastids acquired by the secondary endosymbiosis of a green alga, and the plastids harbor a relict nucleus of the endosymbiont, the so-called nucleomorph. Due to massive gene transfer from the endosymbiont to the host, many proteins involved in plastid and nucleomorph are encoded by the nuclear genome. Genome sequences have provided a blueprint for the fate of endosymbiotically derived genes; however, transcriptional regulation of these genes remains poorly understood. To gain insight into the evolution of endosymbiotic genes, we performed genome-wide transcript profiling along the cell cycle of the chlorarachniophyte Bigelowiella natans, synchronized by light and dark cycles. Our comparative analyses demonstrated that transcript levels of 7,751 nuclear genes (35.7% of 21,706 genes) significantly oscillated along the diurnal/cell cycles, and those included 780 and 147 genes for putative plastid and nucleomorph-targeted proteins, respectively. Clustering analysis of those genes revealed the existence of transcriptional networks related to specific biological processes such as photosynthesis, carbon metabolism, translation, and DNA replication. Interestingly, transcripts of many plastid-targeted proteins in B. natans were induced before dawn, unlike other photosynthetic organisms. In contrast to nuclear genes, 99% nucleomorph genes were found to be constitutively expressed during the cycles. We also found that the nucleomorph DNA replication would be controlled by a nucleus-encoded viral-like DNA polymerase. The results of this study suggest that nucleomorph genes have lost transcriptional regulation along the diurnal cycles, and nuclear genes exert control over the complex plastid including the nucleomorph.

Overexpression of Molecular Chaperone Genes in Nucleomorph Genomes

Chlorarachniophytes and cryptophytes possess complex plastids that were acquired by the ingestion of a green and red algal endosymbiont, respectively. The plastids are surrounded by four membranes, and a relict nucleus, called the nucleomorph, remains in the periplastidal compartment, which corresponds to the remnant cytoplasm of the endosymbiont. Nucleomorphs contain a greatly reduced genome that possesses only several hundred genes with high evolutionary rates. We examined the relative transcription levels of the genes of all proteins encoded by the nucleomorph genomes of two chlorarachniophytes and three cryptophytes using an RNA-seq transcriptomic approach. The genes of two heat shock proteins, Hsp70 and Hsp90, were highly expressed under normal conditions. It has been shown that molecular chaperone overexpression allows an accumulation of genetic mutations in bacteria. Our results suggest that overexpression of heat shock proteins in nucleomorph genomes may play a role in buffering the mutational destabilization of proteins, which might allow the high evolutionary rates of nucleomorph-encoded proteins.

Prospective function of FtsZ proteins in the secondary plastid of chlorarachniophyte algae

BackgroundDivision of double-membraned plastids (primary plastids) is performed by constriction of a ring-like division complex consisting of multiple plastid division proteins. Consistent with the endosymbiotic origin of primary plastids, some of the plastid division proteins are descended from cyanobacterial cell division machinery, and the others are of host origin. In several algal lineages, complex plastids, the “secondary plastids”, have been acquired by the endosymbiotic uptake of primary plastid-bearing algae, and are surrounded by three or four membranes. Although homologous genes for primary plastid division proteins have been found in genome sequences of secondary plastid-bearing organisms, little is known about the function of these proteins or the mechanism of secondary plastid division.ResultsTo gain insight into the mechanism of secondary plastid division, we characterized two plastid division proteins, FtsZD-1 and FtsZD-2, in chlorarachniophyte algae. FtsZ homologs were encoded by the nuclear genomes and carried an N-terminal plastid targeting signal. Immunoelectron microscopy revealed that both FtsZD-1 and FtsZD-2 formed a ring-like structure at the midpoint of bilobate plastids with a projecting pyrenoid in Bigelowiella natans. The ring was always associated with a shallow plate-like invagination of the two innermost plastid membranes. Furthermore, gene expression analysis confirmed that transcripts of ftsZD genes were periodically increased soon after cell division during the B. natans cell cycle, which is not consistent with the timing of plastid division.ConclusionsOur findings suggest that chlorarachniophyte FtsZD proteins are involved in partial constriction of the inner pair of plastid membranes, but not in the whole process of plastid division. It is uncertain how the outer pair of plastid membranes is constricted, and as-yet-unknown mechanism is required for the secondary plastid division in chlorarachniophytes.

Multiple losses of photosynthesis and convergent reductive genome evolution in the colourless green algae Prototheca.

Autotrophic eukaryotes have evolved by the endosymbiotic uptake of photosynthetic organisms. Interestingly, many algae and plants have secondarily lost the photosynthetic activity despite its great advantages. Prototheca and Helicosporidium are non-photosynthetic green algae possessing colourless plastids. The plastid genomes of Prototheca wickerhamii and Helicosporidium sp. are highly reduced owing to the elimination of genes related to photosynthesis. To gain further insight into the reductive genome evolution during the shift from a photosynthetic to a heterotrophic lifestyle, we sequenced the plastid and nuclear genomes of two Prototheca species, P. cutis JCM 15793 and P. stagnora JCM 9641, and performed comparative genome analyses among trebouxiophytes. Our phylogenetic analyses using plastid- and nucleus-encoded proteins strongly suggest that independent losses of photosynthesis have occurred at least three times in the clade of Prototheca and Helicosporidium. Conserved gene content among these non-photosynthetic lineages suggests that the plastid and nuclear genomes have convergently eliminated a similar set of photosynthesis-related genes. Other than the photosynthetic genes, significant gene loss and gain were not observed in Prototheca compared to its closest photosynthetic relative Auxenochlorella. Although it remains unclear why loss of photosynthesis occurred in Prototheca, the mixotrophic capability of trebouxiophytes likely made it possible to eliminate photosynthesis.