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

以地钱和绿色藻类克雷伯藻和新月藻为材料，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，然后进行冷冻置换或冷冻断裂。对M. polymorpha，特别是K.松弛，细胞结构保存良好。在两种植物的叶绿体分裂的峡部周围清楚地检测到一对内外质体分裂环。在K。在分裂早期的叶绿体中发现了松弛的“不闭合的”质体分裂环双峰。在未闭合的环被环绕的峡部一侧，峡部严重收缩，但环未到达的一侧几乎不收缩。在叶绿体分裂的早期阶段，微体的形状发生变化，形成几个突起，其中一个突起缠绕在分裂的叶绿体的峡部，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峰的形成。虽然有可能制备具有良好分辨率的K.然而，遗憾的是，迄今为止还没有获得质体分裂环的复制品。看起来，需要的是叶绿体高度同步分裂的细胞样品。检查是否含有肌动蛋白的质体分裂环使用免疫电镜后快速冷冻和冷冻置换没有证据表明肌动蛋白的本地化在质体分裂环。

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.