Cytochemical and ultrastructural studies on protoplast formation from disintegrated cells of the marine alga Chaetomorpha aerea (Chlorophyta)

Abstract

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.