Yuki Tsuchikane

Stable Nuclear Transformation of the Closterium peracerosum-strigosum-littorale Complex

Although charophycean algae form a relevant monophyly with embryophytes and hence occupy a fundamental place in the development of Streptophyta, no tools for genetic transformation in these organisms have been developed. Here we present the first stable nuclear transformation system for the unicellular Zygnematales, the Closterium peracerosum-strigosum-littorale complex (C. psl complex), which is one of the most useful organisms for experimental research on charophycean algae. When a vector, pSA106, containing the dominant selectable marker ble (phleomycin-resistant) gene and a reporter cgfp (Chlamydomonas-adapted green fluorescent protein) gene was introduced into cells via particle bombardment, a total of 19 phleomycin-resistant cells were obtained in the presence of a low concentration of phleomycin. Six isogenic strains isolated using conditioned medium showed consecutive cgfp expression and long-term stability for phleomycin resistance. DNA analyses verified single or tandem/redundant integration of ~10 copies of pSA106 into the C. psl complex genome. We also constructed an overexpression vector, pSA1102, and then integrated a CpPI gene encoding minus-specific sex pheromone into pSA1102. Ectopic overexpression of CpPI and the pheromonal function were confirmed when the vector pSA1102_CpPI was introduced into mt(+) cells. The present efficient transformation system for the C. psl complex should provide not only a basis for molecular investigation of Closterium but also an insight into important processes in early development and evolution of Streptophyta.

Gene Expression Profiling Using cDNA Microarray Analysis of the Sexual Reproduction Stage of the Unicellular Charophycean Alga Closterium peracerosum-strigosum-littorale Complex

The desmid Closterium peracerosum-strigosum-littorale complex, which is the closest unicellular sister to land plants, is the best characterized of the charophycean green algae with respect to the process of sexual reproduction. To elucidate the molecular mechanism of intercellular communication during sexual reproduction, we created a normalized cDNA library from mixed cells of the sexual and the vegetative phases and generated a cDNA microarray. In total, 3,236 expressed sequence tags, which were classified into 1,615 nonredundant groups, were generated for cDNA microarray construction. Candidate genes for key factors involved in fertilization, such as those that encode putative receptor-like protein kinase, leucine-rich-repeat receptor-like protein, and sex pheromone homologs, were up-regulated during sexual reproduction and/or by the addition of the purified sex pheromones, and the expression patterns of these genes were confirmed by quantitative real-time polymerase chain reaction analysis. This first transcriptome profile of Closterium will provide critical clues as to the mechanism and evolution of intercellular communication between the egg and sperm cells of land plants.

New insights into the regulation of sexual reproduction in Closterium.

The genus Closterium, which is the closest unicellular relative to land plants, is the best-characterized charophycean green alga with respect to the process of sexual reproduction. In two representative heterothallic species, the steps and methods of intercellular communication were fully described. Glycoproteinaceous sex pheromones involved in the progress of these processes were physiologically and biochemically characterized and the corresponding genes were cloned. These pheromones function in most steps of sexual reproduction. For elucidating the mechanisms of sexual reproduction in detail, molecular tools such as expressed sequence tag, microarray analysis, and genetic transformation systems have been established, and whole genome analyses are ongoing. Finally, sexual reproductive isolation among mating groups was characterized, and the mechanism involved in this isolation was considered with respect to sex pheromones. In homothallic Closterium, the presence of a pheromone orthologous to the heterothallic type and possible sexual differentiation were also described, through the combination of closely related heterothallic cells.

Zygospore formation between homothallic and heterothallic strains of Closterium

Zygospore formation in different strains of the Closterium peracerosum-strigosum-littorale complex was examined in this unicellular isogamous charophycean alga to shed light on gametic mating strains in this taxon, which is believed to share a close phylogenetic relationship with land plants. Zygospores typically form as a result of conjugation between mating-type plus (mt+) and mating-type minus (mt−) cells during sexual reproduction in the heterothallic strain, similar to Chlamydomonas. However, within clonal cells, zygospores are formed within homothallic strains, and the majority of these zygospores originate as a result of conjugation of two recently divided sister gametangial cells derived from one vegetative cell. In this study, we analyzed conjugation of homothallic cells in the presence of phylogenetically closely related heterothallic cells to characterize the reproductive function of homothallic sister gametangial cells. The relative ratio of non-sister zygospores to sister zygospores increased in the presence of heterothallic mt+ cells, compared with that in the homothallic strain alone and in a coculture with mt− cells. Heterothallic cells were surface labeled with calcofluor white, permitting fusions with homothallic cells to be identified and confirming the formation of hybrid zygospores between the homothallic cells and heterothallic mt+ cells. These results show that at least some of the homothallic gametangial cells possess heterothallic mt−-like characters. This finding supports speculation that division of one vegetative cell into two sister gametangial cells is a segregative process capable of producing complementary mating types.

REPRODUCTIVE ISOLATION BY SEX PHEROMONES IN THE CLOSTERIUM PERACEROSUM-STRIGOSUM-LITTORALE COMPLEX (ZYGNEMATALES, CHAROPHYCEAE)(1).

The Closterium peracerosum–strigosum–littorale (C. psl.) complex consists of unicellular algae and is known to be composed of several reproductively isolated mating groups of heterothallic strains. Group I‐E is completely isolated from mating groups II‐A and II‐B, groups II‐A and II‐B are partially isolated from each other, and only mating‐type plus (mt+) cells of group II‐A and mating‐type minus (mt−) cells of group II‐B form zygotes. Based on the alignment of 1506 group I introns, significant phylogenetic relationships were observed among mating groups II‐A and II‐B, while mating group I‐E was distant from groups II‐A and II‐B. Sexual cell division in both mating‐type cells of group II‐A was stimulated in conditioned media in which cells of group II‐B had been cultured. When mt− cells of group II‐B were stimulated in conditioned medium derived from group II‐A, mt+ cells of group II‐B did not respond to the conditioned medium. Conditioned media derived from group I‐E did not exhibit sexual cell division (SCD)–inducing activity against any strain except those within its own group. From the alignment of deduced amino acid sequences from orthologous protoplast‐release‐inducing protein (PR‐IP) Inducer genes, we detected a significant similarity among groups II‐A and II‐B, and mating group I‐E had low similarity to other mating groups. The existing degree of reproductive isolation can be partially explained by differences in molecular structures and physiological activities of sex pheromones of these heterothallic mating groups.

SEXUAL PROCESSES AND PHYLOGENETIC RELATIONSHIPS OF A HOMOTHALLIC STRAIN IN THE CLOSTERIUM PERACEROSUM–STRIGOSUM–LITTORALE COMPLEX (ZYGNEMATALES, CHAROPHYCEAE)1

Members of the Closterium peracerosum–strigosum–littorale (C. psl.) complex are unicellular charophycean algae in which there are two modes of zygospore formation, heterothallic and homothallic. A homothallic strain of Closterium (designation, kodama20) was isolated from a Japanese rice paddy field. Based on alignment of the 1506 group‐I introns, which interrupt nuclear SSU rDNAs, homothallic kodama20 is most closely related to the heterothallic mating group II‐B, which is partially sexually isolated from group II‐A. Time‐lapse photography of the conjugation process in kodama20 revealed that most of the observed zygospores originated from one vegetative cell. The sexual conjugation process consisted of five stages: (1) cell division resulting in the formation of two sister gametangial cells from one vegetative cell, (2) formation of a sexual pair between the two sister gametangial cells (or between gametangial cells of another adjoined individual), (3) formation of conjugation papillae, (4) release of gametic protoplasts from both members of a pair, and (5) formation of the zygospore by protoplast fusion. For conjugation to progress, the cell density and light condition in the culture was critical. We suggested the presence of a conjugation promotion factor.

Characterization and Molecular Cloning of Conjugation- Regulating Sex Pheromones in Homothallic Closterium

Conjugation-regulating pheromones were analyzed in homothallic Closterium for the first time. Members of the Closterium peracerosum-strigosum-littorale complex are unicellular charophycean algae in which there are two modes of zygospore formation: heterothallism and homothallism. A homothallic strain of Closterium (designation, kodama20) forms selfing zygospores via the conjugation of two sister gametangial cells derived from one vegetative cell. Conjugation-promoting and -suppressing activities, against cells at very low (1 x 10(2) cells ml(-1)) and normal (1 x 10(4) cells ml(-1)) cell density, respectively, were detected in the medium in which cells of a normal density had been cultured. Pheromone activities decreased to 20% after incubation at 60 °C for 10  min. The release and action of the pheromones was dependent on light. The culture medium was subjected to gel filtration, and both active substances had an apparent molecular mass of 17  kDa; this was similar to that previously reported for the heterothallic sex-specific pheromone protoplast-release-inducing protein (PR-IP) Inducer. cDNAs encoding the orthologs of PR-IP Inducer were isolated from the homothallic strain. Recombinant PR-IP Inducers produced by yeast cells showed conjugation-promoting activity. These results indicate that conjugation of the homothallic strain is regulated by an ortholog of a heterothallic sex-specific pheromone.

A Receptor-Like Kinase, Related to Cell Wall Sensor of Higher Plants, is Required for Sexual Reproduction in the Unicellular Charophycean Alga, Closterium peracerosum–strigosum–littorale Complex

Here, we cloned the CpRLK1 gene, which encodes a receptor-like protein kinase expressed during sexual reproduction, from the heterothallic Closterium peracerosum-strigosum-littorale complex, one of the closest unicellular alga to land plants. Mating-type plus (mt(+)) cells with knockdown of CpRLK1 showed reduced competence for sexual reproduction and formed an abnormally enlarged conjugation papilla after pairing with mt(-) cells. The knockdown cells were unable to release a naked gamete, which is indispensable for zygote formation. We suggest that the CpRLK1 protein is an ancient cell wall sensor that now functions to regulate osmotic pressure in the cell to allow proper gamete release.

Conjugation processes of Penium margaritaceum (Zygnemophyceae, Charophyta)

Detailed conjugation processes in Penium, a unicellular conjugating green alga, are described for the first time. A homothallic strain of Penium margaritaceum (Ehrenb.) Bréb. (Designation, izu84‐10) was isolated from a rice paddy field in Japan. The species was identified based on its morphology, and a molecular phylogeny confirmed that izu84‐10 was closely related to another identified strain of this species. Using time‐lapse photography, the conjugation processes in P. margaritaceum were observed and then categorized into the following six stages: (1) cell division, resulting in the formation of two sister gametangial cells from one vegetative cell; (2) formation of a sexual pair between the two sister gametangial cells (or between gametangial cells of another nearby individual); (3) formation of conjugation papillae by elongation of the cell wall; (4) release of a gamete from one of the pair members; (5) release of a gamete from the other pair member; and (6) formation of the zygospore by gamete fusion. By alcian blue staining, possible involvement of mucilage to facilitate this cell adhesion and cell–cell communication was suggested.

CRISPR/Cas9-based knockouts reveal that CpRLP1 is a negative regulator of the sex pheromone PR-IP in the Closterium peracerosum-strigosum-littorale complex

Heterothallic strains of the Closterium peracerosum-strigosum-littorale (C. psl.) complex have two sexes, mating-type plus (mt+) and mating-type minus (mt−). Conjugation between these two sexes is regulated by two sex pheromones, protoplast-release-inducing protein (PR-IP) and PR-IP Inducer, which are produced by mt+ and mt− cells, respectively. PR-IP mediates the release of protoplasts from mt− cells during mating. In this study, we examined the mechanism of action of CpRLP1 (receptor-like protein 1), which was previously identified in a cDNA microarray analysis as one of the PR-IP-inducible genes. Using CRISPR/Cas9 technology, we generated CpRLP1 knockout mutants in mt− cells of the C. psl. complex. When the knockout mt− cells were mixed with wild-type mt+ cells, conjugation was severely reduced. Many cells released protoplasts without pairing, suggesting a loss of synchronization between the two mating partners. Furthermore, the knockout mutants were hypersensitive to PR-IP. We conclude that CpRLP1 is a negative regulator of PR-IP that regulates the timing of protoplast release in conjugating C. psl. cells. As the first report of successful gene knockout in the class Charophyceae, this study provides a basis for research aimed at understanding the ancestral roles of genes that are indispensable for the development of land plants.