Ok-Kyong Chah’s research while affiliated with Kongju National University and other places

A cytogenetic investigation on male and female reproductive cells of Aglaothamnion oosumiense Itono indicates that the sexuality of this species might be determined by a sex chromosome. Chromosome counts in female and male gametophytes gave 37 and 36, respectively. Sex ratio of gametophytes was 1:1. Both male-derived and female-derived bisexual plants were observed. Bisexual plants were different in gross morphology and position of carpogonial branches from normal unisexual gametophytes. The chromosome number of female-derived bisexual plants was N=37 and male-derived bisexual plants was N=36. Some male plants developed parasporangia in addition. The paraspore germlings showed the same chromosome number as the male plants. The fertilized carpogonium and gonimoblast cells had 2N = ca. 70 chromosomes.

Regeneration of protoplasts from extruded cytoplasm and successive development of aplanospores within regenerated cells are described in the marine green alga Chaetomorpha aerea (Dillwyn) Kützing (Cladophorales, Cladophoraceae). Agglutination of cell organelles in seawater seemed to be mediated by a complementary lectin-carbohydrate system. Three carbohydrates – D-galactosamine, D-glucosamine and α-D-mannose – inhibited agglutination of cell organelles. The presence of these sugar moieties on the surface of cell organelles was verified with their complementary fluorescein isothiocyanate lectins. Agglutination assays using human erythrocytes showed the presence of lectins specific for the above sugars in the protoplasm. The fluorescent probe l-(4-trimethylammoniumphenyl)-6-phenyl-a,3,5-hexatriene revealed that the envelope initially surrounding protoplasts was not a lipid-based cell membrane. Fluorescein diacetate staining showed esterase activity in the protoplasts from the beginning of the regeneration process, suggesting that their envelopes were intact. Enzyme digestion revealed that the enclosing envelope includes polysaccharides as essential structural components and then transforms into a polysaccharide–lipid complex, before a complete plasma membrane develops within 6 h after wounding. Electron microscopic observation suggested that new plasma membrane formed by incorporation of original cell membrane into the primary envelope. An extensive agglutination of cytoplasmic vesicles was observed in the protoplasts. New cell walls developed within 24 h after wounding. Thereafter, the cells developed aplanospores or swarmers 4–10 days after wounding. The released aplanospore divided into 8 cells, which became motile biflagellate swarmers capable of germination.

Sex determination and development of reproductive structures in Aglaothamnion oosumiense were examined by cytogenetic and molecular studies. Chromosome studies of male and female reproductive cells indicated that the sexuality of this species might be determined by a sex chromosome. Chromosome counts in female and male gametophytes were 37 and 36, respectively. The sex ratio of the gametophytes was 1:1. Also male-derived bisexual plants were observed. They were different in morphology, position of carpogonial branches and chromosome number. Some male plants also developed parasporangia. Chromosome number of the paraspore germlings was the same as in the male plants. A novel method combining element of suppression subtractive hybridization (SSH) with high throughput differential screening permitted an efficient and rapid cloning of rarely transcribed differentially expressed genes. Potential of the method is demonstrated by the isolation of 212 subtractive clones that were differentially expressed in male and female plants. About 18 subtractive clones were confirmed by reverse northern blotting. Two genes, AOMS-1 and AOMS-2, which showed male specific expression were analyzed. These genes seem to be involved in differentiation of male reproductive structures.

Spermatial development and fertilization processes were investigated by electron microscopy in Aglaothamnion oosumiense Itono. The spermatium is composed of two parts, an ovoidal head and two appendages projecting from each distal end. The appendages originate from spermatangial vesicles (SVs) and follow a developmental sequence beginning as amorphous material and ending as a fully-formed fibrous structure compressed within the SVs. SV formation is due to contributions initially from endoplasmic reticulum and later from dictyosome-derived vesicles. Chemical differentiation of the spermatial wall occurs early in its development. Calcofluor white does not label spermatial walls, indicating an absence of cellulose polysaccharides, which are abundant in vegetative cell walls. Liberated spermatia had a prophase arrested nucleus with a pair of polar rings. The cytoplasm of the trichogyne was connected with that of spermatia at the fertilization canal. The cytoplasm of the trichogyne was empty when the nuclear fusion between spermatium and carpogonium occurred.

A cDNA library from a mixed population of female and male gametophytes of Griffithsia japonica Okamura was constructed, and a cDNA clone, designated GjFP-1 (G. japonica female predominant-1), was isolated by differential screening of the cDNA library. The transcript corresponding to GjFP-1 was abundant in female gametophytes, but only basal levels of the transcript were detected in male and tetrasporangial thalli. Determination of the nucleotide sequence of GjFP-1 identified a putative open reading frame encoding 313 amino acids and a 3′ untranslated region of 116 nucleotides. The deduced amino acid sequence of GjFP-1 for the putative open reading frame shared high homology with the previously reported amino acid sequences of heat shock protein 90 (hsp90). RNA blot hybridization analysis for the transcripts of heat-shocked G. japonica and DNA hybridization analysis for the genomic DNA of G. japonica with GjFP-1 as a probe suggested that GjFP-1 is a cDNA clone for the hsp90 gene in G. japonica.In situ hybridization for the GjFP-1 transcript again showed a differential specificity of the transcript in female gametophytes. The data reported here suggest a possibility of hsp90 function linked to the development of the female gametophytes in G. japonica.

Life history and post-fertilization process of Aglaothamnion oosumiense from Korea were studied in culture. This species showed a Polysiphonia-type life history with dioecious gametophyte as well as asexual reproduction via parasporangium on male plants. The occurrence of parasporangiate plants was not affected by environmental factors nor inherited strictly to the next generation. Most (>95%) of the paraspores developed into male plants while less than 5% of paraspores became parasporangiate male plants again. Bisexual plants were also observed. Each carpogonial branch had two auxiliary cells, which divided distally to produce the primary gonimoblast initials, respectively, before the foot cells were cut off proximately from it by intercalary divisions, Two carposporophytes resulted from a successful fertilization. Morphologically, Aglaothamnion oosumiense resembled Aglaothamnion callophyllidicola (Yamada) Boo, Lee, Rueness et Yoshida, and the taxonomic characters separating them were found to be unstable. The taxonomic relationship between the two species, therefore, should be critically reassessed with a thorough reexamination including their type specimens.

A new species of the genus Antithamnion Naegeli, A. aglandum sp. nov., is described from Korea. The species is similar to A. nipponicum Yamada et Inagaki in branching pattern and reproductive development, but is distinguished by having no gland cells and producing hair cells on the female frond. Isolates of A. aglandum were investigated in culture, comparing their growth and reproduction with A. nipponicum from Korea. A, aglandum showed a Polysiphonia type life history and exhibited neither mixed phases nor apomixis. In culture, under various environmental conditions, it developed hair cells consistently and did not produce any gland cells. Procarps occurred successively in pairs on upper segments of main and lateral axes. After fertilization, a connecting cell was formed between carpogonium and auxiliary cell. Hybridization attempts between A. aglandum and A. nipponicum were negative.