When injured, the protoplasms come out from the multi‐nucleate giant cell of a green alga Bryopsis plumosa and can generate numerous new cells spontaneously. The cell organelles aggregated rapidly in sea water and became covered with a gelatinous envelope within fifteen minutes. A lipid cell membrane was formed inside the envelope within nine to twelve hours. Cytochemical studies using Nile Red and various enzymes revealed that the primary envelope is initially composed of polysaccharides then becoming a polysaccharide‐lipid complex. Fluorescein diacetate (FDA) staining showed that the primary envelope has some characteristics of cell membranes including semipermeability and selective transport of materials. The aggregation of cell organelles appears to be mediated by two kinds of material; one in vacuolar sap and the other on the surface of cell organelles. About a thousand new cells were generated from a single disintegrated branch and forty percent of them eventually developed into mature plants.
In red algae, fertilization begins with gamete‐gamete contact between the trichogyne cell wall of the female carpogonium and spermatial coverings. During the fertilization in Aglaothamnion oosumiense, reproductive cells interact with each other through sex specific adhesion molecules on the surface of spermatia and trichogyne. The gamete binding is highly selective suggesting the presence of recognition factors along their surfaces. In the previous studies, we have reported that spermatial binding to trichogynes of a red alga, Aglaothamnion oosumiense is mediated by a lectin‐carbohydrate complementary system. Spermatial binding to trichogynes was inhibited by pre‐incubation of trichogynes with N‐acetyl‐D‐galactosamine and D‐glucose and hence lectins specific to these sugars were expected to present on the surfaces of trichogyne cell wall. We have isolated a new lectin from Aglaothamnion oosumiense by the use of agarose bound N‐acetyl‐D‐galactosamine affinity chromatography and named it as rhodobindin. Rhodobindin agglutinated human erythrocytes as well as spermatia of Aglaothmanion oosumiense. The agglutinating activity of this lectin was inhibited by N‐acetyl‐D‐galactosamine and N‐acetyl‐D‐glucosamine. SDS‐PAGE results showed that this lectin may be monomeric. The molecular weight was determined as 21,876 dalton by matrix‐assisted laser desorption ionization (MALDI) mass‐spectrometry. N‐terminal amino acid sequence of the lectin was analyzed and revealed to have no identity with those of known proteins. The complementary male glycoprotein was also isolated and purified by the use of SBA‐agarose affinity chromatography. The subtractive cloning was carried out to characterize the recognition molecules.
Protoplast regeneration from extruded cytoplasm of the multi‐cellular marine green alga Microdictyon umbilicatum (Velley) Zanardini (Cladophorales, Anadyomenaceae) was investigated. The early process of protoplast formation is comprised of two steps: agglutination of cell organelles into protoplasmic masses followed by generation of a temporary enclosing envelope around them. Agglutination of cell organelles was mediated by a lectin‐carbohydrate complementary system. Three sugars, D‐galactosamine, D‐glucosamine, and a‐D‐mannose, inhibited the agglutination process, and three complementary lectins for the above sugars, peanut agglutinin, Ricinus communis agglutinin and concanavalin A, bound to the surfaces of chloroplasts. Agglutination assay using human erythrocytes showed the presence of lectins specific for the above sugars in the algal vacuolar sap. The lectin has been purified by the use of D‐mannose agarose affinity column. Its Molecular weight was shown to be 36,000 dalton by SDS‐PAGE gel electrophoresis. When the basic regeneration process was accomplished, the cells chose one of two developmental strategies; about 70% of one‐celled protoplasts transformed into reproductive cells within two weeks after wounding, while others began cell division and grew into typical Microdictyon plants. Quadriflagellate swarmers were liberated from the reproductive cells, and they germinated into mature plants
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.
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