Sexual Reproduction in the Closterium peracerosum–Strigosum–littorale Complex

Overview of sexual reproduction in Closterium

The desmid Closterium belongs to the Zygnematophyceae and is the most successfully characterized unicellular charophycean in terms of the maintenance of strains and sexual reproduction (Ichimura 1971). Recently, studies have suggested that either the Zygnematophyceae or a clade consisting of Zygnematophyceae and Coleochaetophyceae might be a likely sister group of land plants (Turmel et al. 2006; Wodniok et al. 2011).

Additional data are required to confirm this and biological studies of the Closterium are likely to generate great interest in the near future. The sexual reproduction of species in the genus Closterium has been of interest to many investigators for more than 100 years, and the morphological details and modes of sexual reproduction are well documented (Cook 1963; Lippert 1967; Pickett-Heaps and Fowke 1971; Ichimura 1973; Noguchi and Ueda 1985; Noguchi 1988). Closterium has no flagellum-like machinery for active movement and has been considered to use diffusible substances for the intercellular communication essential for sexual reproduction. Ichimura (1971) reported a technique for promoting the sexual reproduction of Closterium in an axenic culture using a synthetic culture medium and many studies using this system have subsequently been published (Hamada et al. 1982; Watanabe and Ichimura 1982; Ichimura 1983; Kato et al. 1983; Ichimura and Kasai 1987; Kasai and Ichimura 1987, 1990; Ichimura and Kasai 1995).

In Closterium, two types of conjugation produce zygotes (Tsuchikane et al. 2010b; Sekimoto et al. 2012). One is a conjugation between two complementary mating-type cells (mt+ and mt–) and the other is a conjugation between clonal cells. The former is referred to as heterothallism and the latter as homothallism (Graham and Wilcox 2000). The conjugation process can be divided into several steps: sexual cell division (SCD), which produces sexually competent gametangial cells, pairing, formation of conjugation papillae, condensing of their cytoplasm, release and fusion of gametic protoplasts (gametes), and the formation of zygotes.

After the formation of zygotes, they become dormant and acquire resistance against dryness. Once they are exposed to dry conditions followed by a water supply, they start meiosis. Two non-sister nuclei of the second meiotic division survive and the other two degenerate. As a result, the two surviving nuclei carry opposite mating type genes in the absence of crossing over, and a pair of mt+ and mt– cells would arise from one zygote in the case of heterothallic strains (Brandham and Godward 1965; Lippert 1967; Hamada et al. 1982; Watanabe and Ichimura 1982).

Sex Pheromones in the Heterothallic Closterium peracerosum–strigosum–littorale Complex

When mt+ and mt– cells of the heterothallic Cl. psl. Complex are mixed together in a nitrogen-depleted mating medium under light conditions, cells of both types differentiate to gametangial cells as a result of SCD and become paired. These paired cells then release their protoplasts to form zygotes (Fig. 3).

A pheromone, named protoplast-release-inducing protein (PR-IP), was isolated from the Cl. psl. Complex (Sekimoto et al. 1990). This pheromone is a glycoprotein that consists of subunits of 42- and 19-kDa. It is released by mt+ cells (NIES-67, obtained from the National Institute for Environmental Studies, Ibaraki, Japan) and is responsible for inducing the release of protoplasts from mt– cells (NIES-68). The latter process proceeds only after appropriate pre-culture under continuous light conditions, during which the mt– cells differentiate from vegetative cells into sexually competent cells (Sekimoto and Fujii 1992) and PR-IP receptors appear on the plasma membranes of mt– cells. Specific binding of the biotinylated 19-kDa subunit of PR-IP to the cells has been clearly demonstrated (Sekimoto et al. 1993b).

Another pheromone, which induces the synthesis and release of PR-IP, has been detected in a medium in which only mt– cells had been cultured (Sekimoto et al. 1993a). The pheromone, named PR-IP Inducer, was subsequently purified and found to be a glycoprotein with a molecular mass of 18.7 kDa (Nojiri et al. 1995). PR-IP Inducer is released constitutively from mt– cells in the presence of light and directly induces the production and release of PR-IP from mt+ cells. Furthermore, cDNAs encoding the subunits of PR-IP (Sekimoto et al. 1994a, b) and PR-IP Inducer (Sekimoto et al. 1998) have been isolated. A computer search using the nucleotide sequences and the deduced amino-acid sequences failed to reveal any homologies to known proteins. Genes for these pheromones can be detected in cells of both mating types by genomic Southern hybridization analysis, but are only expressed in cells of the respective mating types, suggesting the sex-specific regulation of gene expression (Sekimoto et al. 1994c, 1998). The sequences of 500 bp immediately upstream of the transcriptional initiation sites from mt– and mt+ cells are almost identical, indicating the existence of the putative mt+ cells specific trans-acting factor(s) (Endo et al. 1997). From the recent whole genome analysis, both 19-kDa and 42-kDa subunits are encoded by a single gene locus each, but PR-IP Inducer is encoded by a multigene family (unpublished data). Also, many paralogous genes may be encoding PR-IP Inducer-like proteins.

In the sexual reproductive processes of Closterium species, gametangial cells are produced from haploid vegetative cells. Ichimura (1971) reported that vegetative cells of the Cl. psl. Complex divided before the formation of sexual pairs when both mating type cells were mixed (Ichimura 1971). This SCD of each mating-type cell could be induced in a medium in which both mating type cells had been co-cultured (Tsuchikane et al. 2003). The mt– cells release an SCD-inducing pheromone specific for the mt+ cells and are designated SCD-IP-minus (sexual-cell division-inducing pheromone-minus), whereas a pheromone specific to mt– cells released from mt+ cells is designated SCD-IP-plus. Time-lapse video analyses have revealed that SCD was not always required for successful pairing because some of the non-divided vegetative cells can form pairs (unpublished data).

Closterium exhibits a gliding locomotory behavior, mediated by the forceful extrusion of mucilage from one pole of the cell that causes the cell to glide in the opposite direction (Domozych et al. 1993). Substances with the ability to stimulate the secretion of uronic-acid-containing mucilage rom mt+ and mt– cells were detected in media in which mt– and mt+ cells had been cultured separately, and were designated as mucilage-secretion-stimulating pheromone (MS SP)-minus and MS-SP-plus, respectively (Akatsuka et al. 2003).

Both MS-SP-minus and SCD-IP-minus displayed similar characteristics to the PR-IP Inducer, whereas both MS-SP-plus and SCD-IP-plus displayed similar characteristics to the PR-IP, with respect to molecular mass, heat stability, and their dependency on light for secretion and function, indicating the presence of close relationships among these pheromones. Recombinant PR-IP Inducer produced in yeast cells generated the induction of both PR-IP and SCD by mt+ cells, although SCD could be induced by exposure to lower concentrations of recombinant PR-IP Inducer (Sekimoto 2002; Tsuchikane et al. 2005). Moreover, the SCD could be induced by a shorter period of treatment with the pheromone than the production of PR-IP (Tsuchikane et al. 2005). In addition, PR-IP Inducer also displayed mucilage-secretion-stimulating activity against mt+ cells (Akatsuka et al. 2003).

However, purified PR-IP also exhibited mucilage-secretion-stimulating, SCD-inducing, and protoplast-releasing activities against mt– cells, although the effective concentrations were different (Akatsuka et al. 2006). These results strongly suggest that both PR-IP and PR-IP Inducer are multifunctional pheromones that independently promote multiple steps in conjugation at the appropriate times through different induction mechanisms.

Mode of Sexual Reproduction in the Closterium peracerosum– strigosum–littorale Complex

Based on the results described here, postulated sexual reproductive events can be summarized. The PR-IP Inducer is released from mt– cells when cells are exposed to nitrogen-depleted conditions in a light environment. The mt+ cells then receive a signal and begin to release PR-IP into the medium. During this communication, mucilage is secreted into the surrounding medium. Concentrations of these pheromones are gradually elevated and SCD is then induced with respective gametangial cells being formed as a result. The mt+ and mt– cells then move together and become paired due to the effects of unknown chemotactic pheromones. After the final communication by PR-IP and PR-IP Inducer, the mt– cells begin to release their protoplasts. The release of protoplasts from mt+ cells is eventually induced by the direct adhesion of cells, and these protoplasts fuse to form a zygote (Fig. 3).

EST and Microarray Analyses to Elucidate Sexual Reproduction

To elucidate the molecular mechanism of intercellular communication during sexual reproduction, a normalized cDNA library was established from a mixture of cDNA libraries prepared from cells at various stages of sexual reproduction and from a mixture of vegetative mt+ and mt– cells. The aim was to reduce redundancy, and 3236 ESTs were generated, which were classified into 1615 non-redundant groups (Sekimoto et al. 2003, 2006).

The EST sequences were compared with non-redundant protein sequence databases in the public domain using the BLASTX program, and 1045 non-redundant sequences displaying similarity to previously registered genes in the public databases were confirmed. The source group with the highest similarity was land plants, including Arabidopsis thaliana.

A cDNA microarray was then constructed and expression profiles were obtained using mRNA isolated from cells in various stages of the life cycle. Finally, 88 pheromone-inducible, conjugation-related, and/or sex-specific genes were identified (Sekimoto et al. 2006), although their functions during sexual reproduction have not been characterized.

Of the 88 genes identified, a gene encoding receptor-like protein kinase (RLK) was the most notable and named CpRLK1. The gene is expressed during sexual reproduction and treatment of mt+ cells with the PR-IP Inducer also induces the expression, indicating that the CpRLK1 protein probably functions during sexual reproduction (Sekimoto et al. 2006). The full-length cDNA has been isolated and an amino acid sequence containing an extracellular domain (ECD) was obtained (unpublished data). In A. thaliana, the RLK family is the largest gene family with more than 600 family members (Shiu and Bleecker 2001, 2003; Shiu et al. 2004), although the functions of most of these genes are still unknown. Only two RLK genes have been found in the genome of Ch. Reinhardtii; however, the predicted proteins do not have recognizable ECDs. No RLK gene was found in the genome of Ostreococcus tauri (Lehti-Shiu et al. 2009). In contrast, RLKs having transmembrane domains and/or ECDs have been isolated from two charophyceans (Nitella axillaris and Closterium ehrenbergii) (Sasaki et al. 2007), indicating that the receptor configuration was likely established before the divergence of land plants from charophyceans but after the divergence of charophyceans from chlorophytes (Graham and Wilcox 2000; Karol et al. 2001). The receptor configuration is likely to function for intercellular communication, especially during sexual reproduction; however, the confirmation of genomic information from early diversified nonsexual charophyceans such as Klebsormidiophyceae and Chlorokybophyceae is necessary to confirm this assumption.

Recently, a nuclear transformation system for Cl. psl. Complex was developed (Abe et al. 2008a, 2008b, 2011). It should provide not only a basis for molecular investigation of Closterium but also an insight into important processes regarding the mechanism and evolution of intercellular communication between the egg and sperm cells of land plants.

Conjugation Processes of the Homothallic Closterium Peracerosum–strigosum–littorale Complex

In isogamous organisms, if gametes from the same individual are able to conjugate to each other and produce viable progeny, the organism is termed homothallic (self-fertile). If gametes from two individuals of different genetic makeup are required for successful mating, the organism is termed heterothallic (self-sterile; Graham and Wilcox 2000). These two types of zygote formation exist in natural populations of Closterium.

The detailed conjugation processes of the homothallic strain in the Cl. psl. Complex (kodama20; NIES-2666) were revealed by a time-lapse analysis (Tsuchikane et al. 2010b). The first step in the conjugation process is cell division resulting in the formation of two sister gametangial cells from one vegetative cell. Two gametangial cells form a pair and then form a zygote. In contrast to the heterothallic cells, the formation of gametangial cells by cell division is absolutely indispensable for the next pairing step. Approximately 90% of homothallic zygotes originate as a result of conjugation of two sister gametangial cells derived from one vegetative cell (sister conjugation; Fig. 4B left). Hence, sister gametangial cells of the homothallic strain can recognize each other. The resultant zygotes are referred to as sister zygotes. The remaining 10% of zygotes originate from the gametangial cells of separately adjoined individuals (non-sister conjugation; Fig. 4B right) and are referred to as non-sister zygotes.

Conjugation-regulating Sex Pheromones in Homothallic Strains

For conjugation to occur in the homothallic cells, cell density in the culture is critical. Cells likely discern and regulate their density to achieve conjugation through a mechanism similar to the quorum sensing observed in some types of bacteria (Camilli and Bassler 2006). Two conjugation-related activities were successfully detected in a cell-free cultured medium (Tsuchikane et al. 2010a). One of the activities stimulated the formation of gametangial cells by cell division and promoted the formation of zygotes (conjugation-promoting activity). The other suppressed the progress of the steps in conjugation (conjugation-suppressing activity). Both active substances displayed similar characteristics to those of the heterothallic sex-pheromone, PR-IP Inducer. The cDNAs encoding orthologous PR-IP Inducer were cloned from homothallic cells using a combination of degenerate and rapid amplification of cDNA ends (RACE)-polymerase chain reaction (PCR).

Three representative recombinant PR-IP Inducers produced by yeast cells were shown to display conjugation-promoting activity, but not –suppressing activity (Tsuchikane et al. 2010a).

As explained previously, PR-IP Inducer from the heterothallic strain is released from mt– cells in a nitrogen-depleted medium under light conditions. In homothallic cells, conjugation is also regulated by a pheromone, which is an ortholog of heterothallic PR-IP Inducer; however, both the homothallic cells and the resultant gametangial cells are theoretically clones and do not appear to be differentiated in either mating type. In addition, most homothallic zygotes originated by sister conjugation, apparently recognizing each other (Fig. 4B). To confirm this, the relationship between homothallic cells and heterothallic cells has been further characterized as discussed below.

Relationships between Heterothallism and Homothallism

Heterothallic mating group II-B and homothallic strains (kodama20) are phylogenetically closely related (Tsuchikane et al. 2010b; Tsuchikane et al. 2012). One can assume that the type of conjugation (heterothallic vs. homothallic) has been shifted by the mutation of a few important genes.

Because approximately 90% of the homothallic zygotes are sister zygotes, originating as a result of the conjugation of two sister gametangial cells, one can hypothesize that these sister gametangial cells are sexually differentiated to their respective mating-type cells, as with hetorothallic strains. In laboratory studies, homothallic cells have been mixed with heterothallic group II-B cells, which had been surface labeled with calcofluor white, permitting fusions with homothallic cells to be identified. The formation of hybrid zygotes between the homothallic cells and heterothallic mt+ cells was confirmed (Tsuchikane et al. 2012). These results suggest that at least some of the homothallic gametangial cells possess the same characteristics as heterothallic mt– cells. In heterothallic strains, mt+ and mt– cells recognize each other through the mating-type-specific sex pheromones PR-IP Inducer and PR-IP. Thus, homothallic cells and heterothallic mt+ cells may recognize each other through sex pheromones. These findings support the idea that the division of one vegetative cell into two sister gametangial cells is a segregative process capable of producing complementary mating types.

The sister conjugation has also been observed in other unicellular isogamous charophycean alga (Penium margaritaceum; Tsuchikane et al. 2011), as well as the Cl. psl. Complex. Whether homothallism or heterothallism represents the ancestral reproductive strategy has not yet been determined. To clarify the evolution of sex within algal species in detail, the phylogenetic relationship of homothallic and heterothallic strains in various taxonomic groups must be studied in the near future.