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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. [ABSTRACT FROM AUTHOR]

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 × 102 cells ml−1) and normal (1 × 104 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. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In the unicellular charophycean alga Closterium peracerosum-strigosum-littorale complex, the protoplast-release-inducing protein (PR-IP), a sex pheromone responsible for gametic protoplast release from mating-type minus (mt–) cells, was found to stimulate secretion of mucilage from the cells. Induction of sexual cell division by PR-IP was also confirmed. Bioassays were used to determine the minimum doses required to induce these functions, revealing that 5 · 10−16 M of PR-IP stimulated mucilage secretion, and that 5 · 10−10 M of PR-IP were required for protoplast release. Exposure of the cells to 5 · 10−11 M of PR-IP resulted in the induction of sexual cell division as well as mucilage secretion. These results strongly suggest that PR-IP is a multifunctional pheromone that independently promotes multiple steps in conjugation at the appropriate times through different induction mechanisms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]