Intercellular Communication During Sexual Reproduction of Closterium (Conjugatophyceae)

Closterium were reviewed. In the case of Closterium peracerosum-strigosum-littorale complex, two sex-specific pheromones and their receptors were involved in sexual reproduction. These pheromones were glycoproteins and the expression of corresponding genes was critically regulated by the sex and environmental conditions. In the case of Closterium ehrenbergii, chemotactic and sexual cell division-inducing activities for mating-type plus cells were detected and characterized. Although many processes remain to be elucidated, the present results will be helpful for understanding not only the mode of sexual reproduction in Closterium but also the variety of intercellular communication in the plant kingdom especially during sexual reproduction. Received 10 June 2000/ Accepted in revised form 6 July 2000     

Isolation of myosin XI genes from the Closterium peracerosum-strigosum-littorale complex and analysis of their expression during sexual reproduction

Myosins comprise a large superfamily of molecular motors that generate mechanical force in ATP-dependent interactions with actin filaments. On the basis of their conserved motor domain sequences, myosins can be divided into at least 17 classes, 3 of which (VIII, XI, XIII) are found in plants. Although full sequences of myosins are available from several species of green plants, little is known about the functions of these proteins. Additionally, sequence information for algal myosin is incomplete, and little attention has been given to the molecular evolution of myosin from green plants. In the present study, the Closterium peracerosum-strigosum-littorale complex was used as a model system for investigating a unicellular basal charophycean alga. This organism has been well studied with respect to sexual reproduction between its two mating types. Three types of partial sequences belonging to class XI myosins were obtained using degenerate primers designed to amplify motor domain sequences. Real-time polymerase chain reaction analysis of the respective myosin genes during various stages of the algal life cycle showed that one of the genes was more highly expressed during sexual reproduction, and that expression was cell-cycle-dependent in vegetatively grown cells.     

Protein and RNA Synthesizing Activities during Sexual Process of Heterothallic Closterium

Protein and RNA synthesizing activities increased markedly during the mating process and decreased during the maturation stage of zygotes in heterothallic strains of Closterium peracerosum-strigosum-littoale, KAS-4-29 (mating-type minus) and KAS -4-30 (mating-type plus) and a homothallic Closterium acerosum. Different proteins were synthesized at the different stages of the mating process, suggesting that a sequential expression and repression of mating genes occur for the mating-specific protein synthesis during the sexual reproduction.     

Molecular characterization of the cytoplasmic interacting protein of the receptor kinase IRK expressed in the inflorescence and root apices of Arabidopsis

Meristem maintenance and differentiation is regulated by intercellular communication through receptor-like kinases (RLKs) in plants, but the underlying molecular mechanisms of RLK signaling remain largely unknown. A cytoplasmic interactor for inflorescence and root apices receptor-like kinase (IRK), which is a typical meristematic RLK with leucine-rich repeats in Arabidopsis, was identified using a yeast two-hybrid assay and named IRK-interacting protein (IRKI). IRKI is a novel but highly conserved protein found in higher plants. The interaction between IRK and IRKI was confirmed by an in vitro pull-down assay and supported by their simultaneous expression in actively dividing cells in meristems. In the root tip, IRKI expression and localization visualized by green fluorescence protein (GFP) were observed in the quiescent center, initial cells, and immature stele cells. IRKI expression was expanded by exogenous auxin treatment and repressed by inhibitor treatment of polar auxin transport.     

A sex pheromone, protoplast release-inducing protein (PR-IP) inducer, induces sexual cell division and production of PR-IP in Closterium

The sex pheromone protoplast release-inducing protein (PR-IP) inducer and a sexual cell division-inducing pheromone-minus (SCD-IP-minus) that mediates the sexual reproduction of the heterothallic Closterium peracerosum-strigosum-littorale (C. psl) complex were investigated in this study. Recombinant PR-IP inducer produced by yeast cells was prepared and assayed for production of PR-IP and induction of SCD. Both biological activities were observed after treating mating-type plus (mt+) cells with the recombinant pheromone. SCD was induced by exposure to a lower concentration of the same pheromone and by a shorter treatment period with the pheromone than was production of PR-IP. This indicates that the previously characterized PR-IP inducer has both PR-IP-inducing and SCD-inducing activities with mt+ cells, although the inducing mechanisms of the two pheromones differ.     

Identification of a new mating group and reproductive isolation in the <Emphasis Type="Italic">Closterium peracerosum–strigosum–littorale</Emphasis> complex

Reproductive isolation is essential for the process of speciation. In order to understand speciation, it is necessary to compare one mating group with other phylogenetically related but reproductively isolated groups. The Closterium peracerosum–strigosum–littorale (C. psl.) complex is a unicellular isogamous zygnematophycean alga, which is believed to share a close phylogenetic relationship with the land plants. In this study, we identified a new mating group, named group G, of C. psl. complex and compared its physiological and biochemical characteristics with the mating group I-E, which was closely related to the mating group G. Zygospores are typically formed as a result of conjugation between mating-type plus (mt⁺) and mating-type minus (mt^?) cells in the same mating group during sexual reproduction. Crossing experiments revealed mating groups G and I-E were reproductively isolated from each other, but the release of lone protoplasts from mt^? cells of mating group G was induced in the presence of mt⁺ cells of mating group I-E. In fact, the sex pheromone, protoplast-release-inducing protein of mating group I-E induced the release of protoplasts from mt^? cells of mating group G. When mt⁺ and mt^? cells of both mating groups I-E and G were co-cultured (multiple-choice matings), the zygospore formation of mating group G, but not that of mating group I-E, was inhibited. Based on these results, we propose a possible mechanism of reproductive isolation between the two mating groups and suggest the presence of sexual interference between mating group G and mating group I-E.     

Treatment with antibiotics that interfere with peptidoglycan biosynthesis inhibits chloroplast division in the desmid Closterium

Charophytes is a green algal group closely related to land plants. We investigated the effects of antibiotics that interfere with peptidoglycan biosynthesis on chloroplast division in the desmid Closterium peracerosum-strigosum-littorale complex. To detect cells just after division, we used colchicine, which inhibits Closterium cell elongation after division. Although normal Closterium cells had two chloroplasts before and after cell division, cells treated with ampicillin, D-cycloserine, or fosfomycin had only one chloroplast after cell division, suggesting that the cells divided without chloroplast division. The antibiotics bacitracin and vancomycin showed no obvious effect. Electron microscopic observation showed that irregular-shaped chloroplasts existed in ampicillin-treated Closterium cells. Because antibiotic treatments resulted in the appearance of long cells with irregular chloroplasts and cell death, we counted cell types in the culture. The results suggested that cells with one chloroplast appeared first and then a huge chloroplast was generated that inhibited cell division, causing elongation followed by cell death.     

Molecular cloning and sexually dimorphic expression of DM-domain genes in Daphnia magna

Daphnia magna is known to switch between sexual and asexual reproduction depending on the environment. It reproduces asexually when in an optimal environment for food, photoperiod, and population density. Once the environment declines, it changes reproductive strategy from asexual to sexual reproduction. However, the molecular bases of environmental sex determination are largely unknown. To understand the molecular mechanisms of environmental sex determination in Daphnia, it is essential to isolate the genes related to sex determination. As DM-domain genes are well known as sex-related genes, we aimed to identify DM-domain genes from Daphnia. Based on degenerate PCR of conserved DM domains using Daphnia cDNA, we identified three DM-domain genes that corresponded to DMRT11E, DMRT93B, and DMRT99B of Drosophila melanogaster. Quantitative gene expression analysis in gonads revealed that DMRT93B was expressed only in the testis. This finding contributes to an improved understanding of the switching mechanism from an asexual to a sexual life cycle depending on the environment.     

Sexual reproduction and sex determination in green algae

The sexual reproductive processes of some representative freshwater green algae are reviewed. Chlamydomonas reinhardtii is a unicellular volvocine alga having two mating types: mating type plus (mt⁺) and mating type minus (mt^?), which are controlled by a single, complex mating-type locus. Sexual adhesion between the gametes is mediated by sex-specific agglutinin molecules on their flagellar membranes. Cell fusion is initiated by an adhesive interaction between the mt⁺ and mt^? mating structures, followed by localized membrane fusion. The loci of sex-limited genes and the conformation of sex-determining regions have been rearranged during the evolution of volvocine algae; however, the essential function of the sex-determining genes of the isogamous unicellular Chlamydomonas reinhardtii is conserved in the multicellular oogamous Volvox carteri. The sexual reproduction of the unicellular charophycean alga, Closterium peracerosum-strigosum-littorale complex, is also focused on here. The sexual reproductive processes of heterothallic strains are controlled by two multifunctional sex pheromones, PR-IP and PR-IP Inducer, which independently promote multiple steps in conjugation at the appropriate times through different induction mechanisms. The molecules involved in sexual reproduction and sex determination have also been characterized.     

Properties of cell surface carbohydrates in sexual reproduction of the Closterium peracerosum–strigosum–littorale complex (Zygnematophyceae,Charophyta)

The Closterium peracerosum–strigosum–littorale complex is a best characterized zygnematophycean green alga with respect to the process of sexual reproduction. Intercellular communication mediated by two sex pheromones has been well documented in this organism, but information concerning direct cell–cell recognition and fusion of cells involved in conjugation processes has not yet been clarified. In this study, we examined the properties of cell surface carbohydrates in vegetative and reproductive cells using a variety of fluorescein isothiocyanate labeled lectins as probes. Among 20 lectins tested, 10 bound to the Closterium cell surface, and eight of these were specific for the cells involved in sexual reproduction. In addition, some of the lectins inhibited the progress of zygote formation. In particular, Lycopersicon esculentum lectin (LEL) and ConcanavalinA (ConA) considerably inhibited zygote formation (23.6% and 0% of zygotes formed, respectively, compared with the control). LEL mainly accumulated on conjugation papillae and on the surface and lumens of empty cell walls remaining after zygote formation. ConA bound to both vegetative and sexually reproductive cells and strongly accumulated on the conjugation papillae of the latter, indicating ConA binding material(s) are non‐specifically present in Closterium cells but some of the material(s) would be essential for zygote formation. These results suggest that different carbohydrates specifically recognized by these lectins are involved in cell recognition and/or fusion during conjugation processes in the C. psl. complex.     

Uncovering gene regulatory networks during mouse fetal germ cell development

The commitment of germ cells to either oogenesis or spermatogenesis occurs during fetal gonad development: germ cells enter meiosis or mitotic arrest, depending on whether they reside within an ovary or a testis, respectively. Despite the critical importance of this step for sexual reproduction, gene networks underlying germ cell development have remained only partially understood. Taking advantage of the W(v) mouse model, in which gonads lack germ cells, we conducted a microarray study to identify genes expressed in fetal germ cells. In addition to distinguishing genes expressed by germ cells from those expressed by somatic cells within the developing gonads, we were able to highlight specific groups of genes expressed only in female or male germ cells. Our results provide an important resource for deciphering the molecular pathways driving proper germ cell development and sex determination and will improve our understanding of the etiology of human germ cell tumors that arise from dysregulation of germ cell differentiation.