Influence of albumen on aggregation and O-2 evolution of protoplasm from Bryopsis hypnoides Lamourou

The extruded protoplasm from the coenocytic green alga, Bryopsis hypnoides Lamouroux, was able to reform a cell wall and develop further into a mature alga in seawater. In this paper, the influence of albumen on the ability of aggregation and on the photosynthesis of protoplasm was examined. Results show that the protoplasm of B. hypnoides could aggregate in either albumen or chicken egg, which is similar to that in seawater. However unlike in seawater, the aggregation from B. hypnoides in albumen and chicken egg failed to develop into a mature individual. Interestingly, the protoplasm of B. hypnoides could maintain its photosynthetic O-2 evolution in albumen and chicken egg, while the time in chicken egg was longer than that in albumen.

Assembly of the protoplasm of Codium fragile (Bryopsidales, Chlorophyta) into new protoplasts

The cell organelles of the coenocytic alga Codium fragile (Sur.) Hariot aggregated rapidly and protoplasts were formed when its protoplasm was extruded out in seawater. Continuous observation showed that there were long and gelatinous threads connecting the cell organelles. The threads contracted, and thus the cell organelles aggregated into protoplasmic masses. The enzyme digestion experiments and Coomassie Brilliant Blue and Anthrone stainings showed that the long and gelatinous threads involved in the formation of the protoplasts might include protein and saccharides as structure components. Nile Red staining indicated that the protoplast primary envelope was non-lipid at first, and then lipid materials integrated into its surface gradually. The fluorescent brightener staining indicated that the cell wall did not regenerate in the newly formed protoplasts and they all disintegrated within 72 h after formation. Transmission electron microscopy of the cell wall of wild C. fragile showed electron-dense material embedded in the whole cell wall at regular intervals. The experiments indicated that C. fragile would be a suitable model alga for studying the formation of protoplasts.

Assembly of the subcellular parts of Bryopsis hypnoides Lamouroux into new protoplasts

The chloroplasts, mitochondria, and protoplasm devoid of mature chloroplasts (PMC) of Bryopsis hypnoides Lamouroux were isolated by low-speed and sucrose density centrifugation. The PMC aggregated in artificial seawater, and then protoplasts without mature chloroplasts (PtMCs) were formed. Transmission electron microscopy and cytochemical studies indicated that there were mitochondria, nuclei, vesicles, and other small cell organelles in the PtMCs. Scanning electron microscopy showed that there were holes on the surface of 1-h PtMCs and then fewer holes on the surface of 24-h PtMCs, suggesting that a healing process occurred. The plasma membrane was formed over the surface of the PtMCs. However, the cell wall was not regenerated, and the newly formed PtMCs were ruptured and died in 3 days. Light intensity during alga maintenance before use influenced significantly (one-way ANOVA, P < 0.0001) on the number of PtMCs formed; the highest number of PtMCs was formed at 20A mu mol/(m(2) s). When isolated chloroplasts were transferred into seawater, there were only two or three chloroplasts aggregated together. However, isolated mitochondria and the mixed six layers of cell organelles (separated by sucrose density centrifugation) could not aggregate in the artificial seawater. This indicates that the conjunction of cell organelles is important for their aggregation.

Characterization of a new lectin involved in the protoplast regeneration of Bryopsis hypnoides

A group of coenocytic marine algae differs from higher plants, whose totipotency depends on an intact cell (or protoplast). Instead, this alga is able to aggregate its extruded protoplasm in sea water and generate new mature individuals. It is thought that lectins play a key role in the aggregation process. We purified a lectin associated with the aggregation of cell organelles in Bryopsis hypnoides. The lectin was ca. 27 kDa with a pI between pH 5 and pH 6. The absence of carbohydrate suggested that the lectin was not a glycoprotein. The hemagglutinating activity (HA) of the lectin was not dependent on the. presence of divalent cations and was inhibited by N-Acetylgalactosamine, N-Acetylglucosamine, and the glycoprotein bovine submaxillary mucin. The lectin preferentially agglutinated Gram-negative bacterium. The HA of this lectin was stable between pH 4 to pH 10. Cell organelles outside the cytoplasm were agglutinated by the addition of lectin solution (0.5 mg ml(-1)). Our results suggest that the regeneration of B. hypnoides is mediated by this lectin. We also demonstrated that the formation of cell organelle aggregates was inhibited by nigericin in natural seawater (pH 8.0). Given that nigericin dissipates proton gradients across the membrane, we hypothesize that the aggregation of cell organelles was proton-gradient dependent.

单细胞多核管状绿藻原生质团聚及发育研究

在实验室条件下研究了三种单细胞多核管状绿藻（藓羽藻、刺松藻和齿形蕨藻）原生质的团聚和发育过程。结果表明： 藓羽藻原生质团聚过程中，Na+或Ca2+浓度分别为0.5M和0.2 M时形成的藓羽藻亚原生质体数量最多，在原生质体团聚过程中细胞核发生了形变；藓羽藻无成熟叶绿体原生质能够在海水中团聚，并形成无成熟叶绿体的亚原生质体，该亚原生质体有细胞膜形成，但是无细胞壁形成，72小时后会死亡，分离纯化的线粒体在海水中不能团聚，分离纯化的叶绿体在海水中的团聚不是很明显，只是有2-3个叶绿体形成团聚体，而且数量也很少；在藓羽藻团聚过程中外源壳孢子和藓羽藻原生质会很快分开，这样壳孢子就不会被包围到藓羽藻原生质团聚体中，即使有的壳孢子被团聚进藓羽藻亚原生质体中，最后也是以藓羽藻亚原生质体的破裂而壳孢子发育而告终，同时加入壳孢子后还导致藓羽藻亚原生质体的发育方式发生了一定的变化，从以前的单极萌发变成二极萌发；在实验室条件下继代培养了藓羽藻原生质体三代，而且在团聚数量、大小和发育能力方面能够稳定遗传；野生藓羽藻原生质在6-12月份能够团聚，但是只能在8-12月份间发育； 刺松藻原生质体在碱性人工海水中会团聚形成亚原生质体，但是亚原生质体在72h内全部破裂，酶学和染色实验显示刺松藻原生质团聚过程中的絮凝胶状物质含有蛋白质和多糖类物质，松藻是一种非常合适的研究亚原生质体形成的实验模式材料；刺松藻的营养芽、游动细胞、丝状体和果胞都能够再生，刺松藻片断生长的最佳碳和氮浓度分别为：HCO3—C 3mM和NO3—N 70μM，刺松藻对所研究的不同氮源吸收能力基本相同； 齿形蕨藻的光饱和点为200 μmol/m2 s，最佳生长温度为25-30℃；齿形蕨藻片断的再生与光照强度成反比，而且只能在20-35℃间再生。齿形蕨藻原生质在碱性海水中能够团聚，但是与藓羽藻相比较数量非常的少。