Molecular cloning of heat shock protein 70 and HOP from the freshwater green algae Closterium ehrenbergii and their responses to stress.

Heat shock proteins (HSPs) and HSP70-HSP90 organizing proteins (HOPs) are related, and they function together to maintain cellular homeostasis and respond to stress. In the present study, we reported the first molecular characteristics of HSP70 (designated as CeHSP70) and HOP (designated as CeHOP) genes from the freshwater green algae Closterium ehrenbergii and examined the changes in their expression profiles under heat stress and toxic chemicals treatment. CeHSP70 presented the conserved motif patterns and EEVD domain specific to cytosolic HSP70; CeHOP contained a typical domain of TPR repeats. Real-time PCR analysis showed that thermal stress considerably up-regulated both CeHOP and CeHSP70. In addition, the genes were significantly induced by CuCl2, CuSO4, and NiSO4, but not by K2Cr2O7, herbicide, and endocrine disrupting chemicals. These results suggest that CeHOP and CeHSP70 function together and play a role in responses to specific stressors and indicate their possible use as sensitive specific biomarkers in risk assessments.

The genus Closterium, a new model organism to study sexual reproduction in streptophytes.

Contents Summary 99 I. Introduction 99 II. Life cycle of Closterium 100 III. Sexual reproductive processes in the heterothallic Closterium peracerosum-strigosum-littorale complex 101 IV. Homothallism in the C. psl. complex 102 V. Sexual reproduction and inheritance of mating types in Closterium ehrenbergii 102 VI. mt-determining gene of the C. psl. complex 103 VII. Future perspectives 103 Acknowledgements 103 References 103 SUMMARY: Closterium occupies a key phylogenetic position as an ancestor of land plants and is the best-characterized Charophycean alga in terms of the process of sexual reproduction. Zygospores form as a result of sexual reproduction between genetically determined mating type plus (mt+ ) and mating type minus (mt- ) cells in heterothallic strains, or between clonal cells in homothallic strains. Here we review knowledge on the intercellular communication and mating type determination for successful sexual reproduction in Closterium. Using genomic information and transgenic techniques, the genus could be a model organism to study the mechanisms and evolution of sexual reproduction in streptophytes.

Identification of a new mating group and reproductive isolation in the Closterium peracerosum-strigosum-littorale 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.

Photosynthetic and biochemical responses of the freshwater green algae Closterium ehrenbergii Meneghini (Conjugatophyceae) exposed to the metal coppers and its implication for toxicity testing.

The freshwater green algae Closterium is sensitive to water quality, and hence has been suggested as ideal organisms for toxicity testing. In the present study, we evaluated the photosynthetic and biochemical responses of C. ehrenbergii to the common contaminants, coppers. The 72 h median effective concentrations (EC50) of CuSO4 and CuCl2 on the test organism were calculated to be 0.202 mg/L and 0.245 mg/L, respectively. Exposure to both coppers considerably decreased pigment levels and photosynthetic efficiency, while inducing the generation of reactive oxygen species (ROS) in cells with increased exposure time. Moreover, the coppers significantly increased the levels of lipid peroxidation and superoxide dismutase (SOD) activity, even at relatively lower concentrations. These suggest that copper contaminants may exert deleterious effects on the photosynthesis and cellular oxidative stress of C. ehrenbergii, representing its powerful potential in aquatic toxicity assessments.

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.

Physiological and biochemical responses of the freshwater green algae Closterium ehrenbergii to the common disinfectant chlorine.

Chlorine (Cl2) is widely used as a disinfectant in water treatment plants and for cleaning swimming pools; it is finally discharged into aquatic environments, possibly causing damage to the non-target organisms in the receiving water bodies. Present study evaluated the effects of the biocide Cl2 to the green alga Closterium ehrenbergii (C. ehrenbergii). Growth rate, chlorophyll a levels, carotenoids, chlorophyll autofluorescence, and antioxidant enzymes were monitored up to 72-h after Cl2 exposure. C. ehrenbergii showed dose-dependent decrease in growth rate and cell division after exposure to Cl2. By using cell counts, the median effective concentration (EC50)-72-h was calculated to be 0.071mgL(-1). Cl2 significantly decreased the pigment levels and chlorophyll autofluorescence intensity, indicating that the photosystem was damaged in C. ehrenbergii. In addition, it increased the production of reactive oxygen species (ROS) in the cells. This stressor significantly increased the activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase, and glutathione, and affected the physiology of the cells. These results indicate that Cl2 induces oxidative stress in the cellular metabolic process and leads to physiological and biochemical damages in the green algae. Cl2 discharged in industrial effluents and from water treatment plants may cause harmful effects to the C. ehrenbergii a common freshwater microalgae and other non-target organisms.

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.

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.

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.