Cytologia Focus
Nucleomorph: A Fascinating Remnant of Endosymbiosis
2022 Volume 87 Issue 3 Pages 203-208
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2022 Volume 87 Issue 3 Pages 203-208
Nucleomorphs are the reduced nuclei derived from endosymbionts found in some secondary symbiotic algae. Research into the nucleomorph has provided many insights into its role as a product of secondary endosymbiosis, as well as the resulting genome reduction and gene transfer. Recently developed biological techniques and bioinformatic tools have enabled us to examine the characteristics of nucleomorphs more comprehensively than was previously possible. Moreover, new approaches in synthetic biology have begun to emerge that may provide a valuable platform to study artificial endosymbiosis. Together, these technologies could allow greater insights into the mechanisms and regulation of genomic dynamics within the nucleomorph.
Algae play an important biological role as producers of organic matter through photosynthesis and have attracted much attention as the organisms responsible for the most efficient production of biomass on earth (Hosokawa and Kawano 2020, Kuroiwa et al. 2020, 2021, Miyagishima and Fujiwara 2020, Yoshida et al. 2020, Takeshita et al. 2021, Kinoshita et al. 2022, Ota et al. 2022). Algae originally emerged through evolutionary endosymbiosis; furthermore, some algae exist that emerged through a secondary endosymbiotic event. These organisms are interesting from both evolutionary and cell biological perspectives (Gentil et al. 2017, Matsunaga et al. 2018, Okabe and Matsunaga 2022). In this review, we discuss recent findings concerning organelles that provide direct evidence of secondary endosymbiosis, known as nucleomorphs.
Eukaryote photosynthetic organisms are believed to have been established through the engulfment of a cyanobacterium into a host cell, a phenomenon known as primary endosymbiosis. According to recent phylogenetic analyses, however, this process may have been considerably more complicated than a single biological event (Irisarri et al. 2021). This conclusion is further supported by studies on the differences between primary plastid membranes across the phylogenetic spectrum (Sato 2021). There are three major lineages of photosynthetic eukaryotes: Glaucophyta, Rhodophyta, and Viridiplantae. The major difference between these is the identity of the photosynthetic pigment within the plastid membrane. Throughout evolution, some heterotrophic organisms became phototrophic through the acquisition of plastids from the primary endosymbiont. This process is known as secondary endosymbiosis. It is believed that secondary endosymbiosis likely occurred independently multiple times, promoting the evolution of eukaryotes (Curtis et al. 2012, Keeling 2013). Furthermore, recent research suggests that the majority of nuclear genes of plants containing secondary endosymbionts consist of a mosaic of genes from both green and red algae, indicating that their origin is even more complicated than previously thought (Ponce-Toledo et al. 2019, Sibbald and Archibald 2020). The mechanism that underlies secondary endosymbiosis is still unclear; however, a model was proposed in which an alga, containing a primary plastid, is engulfed by a protist host without digestion and is retained inside the host cell. The algal genes are then gradually transferred to the host genome and the alga ultimately becomes a secondary plastid (Fig. 1A) (Yamaguchi et al. 2014). In some species, a remnant of gene transfer remains, known as a relic nucleus or nucleomorph (Curtis et al. 2012). Gaining a better understanding of the mechanisms and consequences of secondary endosymbiosis could provide important insights into the evolutionary process that underlies species diversity today.
The development of new biological techniques has enabled more research into the characteristics of nucleomorphs. Nucleomorphs were first identified through the microscopic study of cryptophytes (Greenwood 1974). The plastids within cryptophytes are surrounded by four membranes, and the nucleomorph exists in the space between the outer and inner pair of the four membranes, called the periplastidal compartment (PPC). The PPC is thought to derive from the cytoplasm of the secondary endosymbiont (Maier et al. 2000). The nucleomorph is therefore surrounded by two nuclear-like membranes and contains DNA (Ludwig and Gibbs 1985). A similar structure was also observed in chlorarachniophytes (Hibberd and Norris 1984). Studies have shown that these two organisms have independent origins; however, convergent evolution has resulted in certain similarities. Cryptophytes originally derived from the engulfment of a red alga by a colorless biflagellated host (Douglas and Penny 1999), while chlorarachniophytes are a group of rhizarian amoeboflagellate algae derived from the engulfment of a green alga by a colorless cercozoan protist (McFadden et al. 1994, 1995, Ishida et al. 2011). The genomes of chlorarachniophytic nucleomorphs are typically smaller than those of cryptophytes (Gilson and McFadden 1999). The nucleomorph genomes of both cryptophytes and chlorarachniophytes are only a few hundred kilobase size, and consist of three chromosomes of approximately equal size (Eschbach et al. 1991, Lane and Archibald 2006, Silver et al. 2007, Phipps et al. 2008, Tanifuji et al. 2010, Ishida et al. 2011). Since the first two nucleomorph genomes were sequenced in the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans (Douglas et al. 2001, Gilson et al. 2006), the nucleomorph genomes of three further cryptophytes and chlorarachniophytes have been sequenced (Lane et al. 2007, Suzuki et al. 2015, Tanifuji et al. 2011, 2014a, Moore et al. 2012). The majority of housekeeping genes present in the chlorarachniophytic nucleomorph are also present in the nucleomorphic genome of cryptophytes (Suzuki et al. 2015, Tanifuji et al. 2011, 2014a). However, several additional genes are also encoded in the nucleomorphs of cryptophytes, but not chlorarachniophytes (Suzuki et al. 2015, Tanifuji et al. 2011, 2014a). This suggests that the genome of the chlorarachniophytic nucleomorph has been more reduced than the cryptophytic nucleomorph genome. In recent years, improvements in sequencing technology and the development of new bioinformatic tools for analysis, such as transcriptomic analysis, have enabled new insights into the characteristics of the nucleomorph.
To better understand transcription within the nucleomorph, Wong et al. (2018) examined pre-mRNA splicing in the cryptophyte G. theta and the chlorarachniophyte B. natans. G. theta contains a nucleomorph with sparse introns, which typically remain unspliced in the mature mRNA transcripts. Conversely, the nucleomorph of B. natans contains many short introns that are highly spliced in the mature transcriptome, at similar levels to eukaryotes (Wong et al. 2018). These differences in splicing may result from the evolutionary difference of the endosymbiotic origin, or alternatively could be due to the differing intron ratios (Slamovits and Keeling 2009). Wong et al. (2018) also observed a high level of both antisense transcription and gene expression in the nucleomorphs in comparison with the host nuclei. The mechanisms underlying this observation are unclear: one possibility is that increased gene expression in the nucleomorph could be a compensatory mechanism against the antisense transcription (Tanifuji et al. 2014b); alternatively, high expression of antisense transcripts could be required to downregulate abnormally high gene expression. Other studies have suggested that overexpression of nucleomorphic genes could be compensating for aberrant splicing (Grisdale et al. 2013, Tanifuji et al. 2014b). These results suggest new ideas about the relationship between RNA splicing, antisense transcription, and regulation of the nucleomorphic genome.
DNA replication within the nucleomorph is thought to be tightly regulated by the host cell cycle. This is supported by the observation that Bigelowiella natans generally contains a single plastid and nucleomorph per cell (Moestrup and Sengco 2001). A recent RNA-seq study in B. natans (Suzuki et al. 2016b) revealed that almost all genes encoded in the nucleomorph are constantly expressed, while gene expression from the host nucleus changes over time. Certain DNA polymerases (POLA and POLD) and replication factors (RFC and RPA) are encoded in the host nucleus and are predicted to be targeted to the nucleomorph. Consistently, fluorescence microscopy in the chlorarachniophyte Amorphochlora amoebiformis has shown these DNA replication proteins to be localized to the nucleomorph (Suzuki et al. 2016b). Interestingly, it has been suggested that the POLD gene is phylogenetically related to giant viruses found in green algae (Blanc et al. 2015). Furthermore, a gene homologous to the nucleomorph-targeted POLD was identified in another chlorarachniophyte, Lotharella globose. This finding implies that the original endosymbiont of chlorarachniophytes may have been infected with a giant virus during the secondary endosymbiosis event, resulting in the host DNA polymerase being replaced with the viral DNA polymerase. Similar cases have been reported for both mitochondria and plastids (Filée and Forterre 2005), suggesting that their endosymbiotic evolution may have occurred under similar selective pressures. Analysis of the cryptophyte G. theta has shown that genes encoded in the nucleomorph genome that are related to the cell cycle, such as cyclin B and histones, are expressed constantly throughout the cell cycle. However, the histone H2A is expressed from the host genome and then is imported into the nucleomorph during a specific phase of cell cycle (Onuma et al. 2017), in a similar manner to that observed in other eukaryotes (Osley 1991). Together, these data indicate that the nucleomorph has lost facets of its own regulatory mechanisms, and the host nucleus and host cell cycle are able to regulate both plastids and nucleomorphs at the level of transcription.
Recent advances in bioinformatics have made it possible to ascertain the functions of uncharacterized open reading frames (ORFs). Using the wealth of information on protein structure that has emerged in recent years, Zauner et al. (2019) were able to predict the function of unannotated genes within the nucleomorph of cryptophytes using the bioinformatic tool Phyre2 (Kelley et al. 2015). This resulted in the assignment of 215 of 826 uncharacterized nucleomorph-encoded genes to a range of cellular functions, including regulation of the cell cycle. Further biochemical studies will be required to confirm these conclusions; however, these predictions are a valuable tool when considering future studies of the nucleomorph.
The three-dimensional organization of the nucleomorph genome has recently been unveiled using modern functional genomic tools. Data from KAS-seq and ATAC-seq, which respectively detect ssDNA and open chromatin region, have shown that most genes encoded in the nucleomorph of the chlorarachniophyte B. natans are highly accessible (Marinov et al. 2022). This corresponds with previous reports using RNA-seq data (Tanifuji et al. 2014b, Suzuki et al. 2016b, Rangsrikitphoti and Durnford 2019). Moreover, Hi-C analysis suggests that while nucleomorphic chromosomes form telomere-to-telomere contacts, the majority of the genes do not interact. The data also suggest that the centromeres do not interact with each other (Marinov et al. 2022), unlike many other eukaryotes (Hoencamp et al. 2021, Sakamoto et al. 2022). Surprisingly, it was also observed that the genomes of the host-derived mitochondrion and the nucleomorph interact (Marinov et al. 2022). This observation is supported by previous studies that reported that these organelles exist in close proximity (Moestrup and Sengco 2001, Hopkins et al. 2012). Together, these data provide novel insights into the physical and genetic characteristics of the nucleomorph.
Studying just two types of nucleomorphs makes it difficult to determine the precise genus of the ancestral endosymbiont of cryptophytes and chlorarachniophytes. Recent studies have unveiled information concerning the phylum and class (Tanifuji et al. 2014a, Archibald 2015, Suzuki et al. 2016a, Kim et al. 2017); however, a more comprehensive history would enable a greater understanding of how endosymbiosis altered the algal endosymbiont genome. A recent study has reported the discovery of two novel dinoflagellate strains, MGD and TGD, which contain new types of nucleomorphs (Sarai et al. 2020). According to phylogenetic analyses, both endosymbionts are predicted to be derived from close relatives of a green algal genus, Pedinomonas (Sarai et al. 2020). Transcriptomic analysis has suggested that the nucleomorphic genomes of the two novel strains are likely to retain their introns. They also show high global expression levels and AT enrichment, as is commonly observed in the conventional nucleomorphic genomes of chlorarachniophytes and cryptophytes (Sarai et al. 2020, Nakayama et al. 2020). This suggests that convergent evolution has occurred during the process of eukaryotic genome reduction. However, many genes are retained in the novel nucleomorphs, including housekeeping genes as well as genes related to photosynthesis (Sarai et al. 2020). These data suggest that genome reduction and endosymbiotic gene transfer in MGD and TGD may be at an earlier stage compared with the conventional cryptophytes and chlorarachniophytes (Fig. 1B) (Nakayama et al. 2020, Sarai et al. 2020). Further examination of the novel nucleomorphs will provide more information on the characteristics and genetics of nucleomorphs in general.
Nucleomorphs have been a subject of much interest over the last 50 years; however, many questions remain unanswered. For instance, the identification of the nuclear genes that directly regulate the nucleomorphic cell cycle would be extremely valuable to increase our understanding of the mechanisms that underlie nucleomorphic division. Furthermore, the fate of nucleomorphs is still unknown and it is unclear whether they will undergo further degradation, or eventually disappear altogether.
Recently, a collection of studies has emerged that have succeeded in experimentally reproducing endosymbiotic genome reduction. Mehta et al. (2018) succeeded in creating artificial endosymbiosis using an engineered strain of Escherichia coli as an endosymbiont and a mutant version of Saccharomyces cerevisiae as a host. In addition, Mehta et al. (2018, 2019) were able to mimic genome reduction using their artificial endosymbiotic system. Another study examined plastid evolution using an artificial, metabolically obligated, endosymbiotic system between a genetically modified cyanobacterium, Synechococcus elongatus, and an S. cerevisiae mutant (Cournoyer et al. 2022). These experiments provide an important platform to investigate the evolution of endosymbiotic organelles and their ongoing genome reduction. It is important to note, however, that these experiments mimic primary endosymbiosis and not secondary endosymbiosis. The development of a system that precisely replicates secondary endosymbiosis between a eukaryotic host and a eukaryotic alga endosymbiont would therefore be extremely valuable for further research into the genome reduction of nucleomorphs (Matsunaga 2018).
This research was supported by grants from MXT/JSPS KAKENHI (20H05911), JST-CREST Grant Number JPMJCR20S6, JST-OPERA program Grant Number JPMJOP1832 to SM, and Basic Science Research Projects from the Sumitomo Foundation Grand Number 200251 to NS. We thank Alison Inglis, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.
