Ultrastructural Studies on plastid-dividing rings using quick freeing and immunocytochemistry

以多形地菜、黄斑克雷伯sormidium flacacium Klebsormidium ehrenberghii绿藻为材料，研究了质体分裂环的超微结构。样品在液氦温度下快速冷冻固定，然后进行冷冻取代或冷冻断裂。对于多形霉萌发的孢子，特别是黄曲霉的细胞，细胞结构保存得很好。在这两种植物的分裂叶绿体峡部周围明显地检测到内外质体分裂环的双重结构。在白莲属植物分裂早期叶绿体中发现了“不闭合”的质体分裂环。未闭合的环被包围的峡部一侧是严重收缩的，但环未到达的一侧几乎没有收缩。在叶绿体分裂的早期阶段，微体的形状发生变化，有几个突起，其中一个环绕在分裂叶绿体的峡部，并与外质体分裂环相连。这些结果表明，未闭合的质体分裂环可能在叶绿体形成闭合环之前施加机械力使叶绿体收缩，微体可能参与了质体分裂环双线体的形成。采用冻裂法和复制法观察质体分裂环。虽然可以制备出高分辨率的冷冻复制体，但遗憾的是，到目前为止还没有获得体细胞分裂环的复制体。看起来，需要的是叶绿体高度同步分裂的细胞样本。在快速冷冻和冷冻取代后使用免疫电镜检查质体分裂环是否含有肌动蛋白，没有发现肌动蛋白在质体分裂环定位的证据。

Ultrastructures of plastid-dividing rings were studied using a liverwort Marchantia polymorpha and green algae of Klebsormidium flaccidum and Closterium ehrenberghii. The samples were fixed by quick freezing at the temperature of liquid helium followed by procedure of freeze-substitution or freeze-fracture. For germinating sporelings of M. polymorpha and, in particular, cells of K. flaccidum, cellular structures were preserved very well. A doublet of inner and outer plastid-dividing rings was clearly detected around the isthmus of the dividing chloroplasts in both plants. In K. flaccidum 'unclosed' plastid dividing-ring doublet was revealed in chloroplasts of an early stage of division. The side of the isthmus us where the unclosed ring is girdled is heavily constricted but the side where the ring has not reached is scarcely constricted. During the early stage of the chloroplast division the microbody changes in shape to have several protuberances, one of which enwinds the isthmus of the dividing chloroplast and associates the outer plastid-dividing ring. These findings suggest, that the unclosed plastid-dividing ring may exert mechanical force to constrict the chloroplast before its forming a closed ring, and that the microbody may be involved in formation of the plastid dividing-ring doublet.Methods of freeze-fracture and replica were conducted to observe plastid-dividing rings. Although it was possible to prepare freeze replicas with good resolution of cells of K. flaccidum, however, unfortunately the replicas of the plastid-dividing rings were not obtained so far. Seemingly, required are cell samples where highly synchronous divisions of the chloroplasts take place. Examination as to whether the plastid-dividing rings contain actin using immunoelectron microscopy following quick freezing and freeze-substitution obtained no evidence for localization of actin at the plastid-dividing rings.