Chloroplasts are essential plant organelles that divide by binary fission through a coordinated ring-shaped division machinery located both on the outside and inside of the chloroplast. The first step in chloroplast division is the assembly of an internal division ring (Z-ring) that is composed of the key filamentous chloroplast division proteins FtsZ1 and FtsZ2. How the individual FtsZ filaments assemble into higher-order structures to form the dividing Z-ring is not well understood and the most detailed insights have so far been gleaned from prokaryotic FtsZ. Here, we present in situ data of chloroplast FtsZ making use of a smaller ring-like FtsZ assembly termed mini-rings that form under well-defined conditions. Structured illumination microscopy (SIM) permitted their mean diameter to be determined as 208 nm and also showed that 68 % of these rings are terminally attached to linear FtsZ filaments. A correlative microscopy-compatible specimen preparation based on freeze substitution after high-pressure freezing is presented addressing the challenges such as autofluorescence and specific fluorescence attenuation. Transmission electron microscopy (TEM) and scanning TEM (STEM) imaging of thin sections exhibited ring-like densities that matched in size with the SIM data, and TEM tomography revealed insights into the molecular architecture of mini-rings demonstrating the following key features: (1) overall, a roughly bipartite split into a more ordered/curved and less ordered/curved half is readily discernible; (2) the density distribution in individual strands matches with the X-ray data, suggesting they constitute FtsZ protofilaments; (3) in the less ordered half of the ring, the protofilaments are able to assemble into higher-order structures such as double helices and supercoiled structures. Taken together, the data suggest that the state of existence of mini-rings could be described as metastable and their possible involvement in filament storage and Z-ring assembly is discussed.

中文翻译：

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拟南芥叶绿体中FtsZ微型环的原位结构

叶绿体是必需的植物细胞器，它们通过位于叶绿体外部和内部的协调的环状分裂机构通过二元裂变分裂。叶绿体分裂的第一步是组装内部分裂环（Z环），该环由关键的丝状叶绿体分裂蛋白FtsZ1和FtsZ2组成。各个FtsZ细丝如何组装成更高阶的结构以形成Z形分割环的方法尚未广为人知，迄今为止，最详细的见解还是来自原核FtsZ。在这里，我们介绍了叶绿体FtsZ的原位数据，该数据使用了在定义明确的条件下形成的称为微型环的较小的环状FtsZ组件。结构照明显微镜（SIM）允许将其平均直径确定为208 nm，并且还显示这些环中有68％最终连接到线性FtsZ灯丝。提出了一种基于显微镜的相关显微镜兼容标本，该标本基于高压冷冻后的冷冻替代，解决了诸如自发荧光和特定荧光衰减等难题。薄层的透射电子显微镜（TEM）和扫描TEM（STEM）成像显示出与SIM数据大小相匹配的环状密度，而TEM断层扫描揭示了对微型环分子结构的深刻见解，证实了以下关键特征： 1）总的来说，可以很容易地辨别出一个大致两部分，分为一个有序/弯曲的部分和一个有序/弯曲较少的部分；（2）各个链中的密度分布与X射线数据相匹配，表明它们构成了FtsZ原型丝。（3）在环的顺序不规则的一半中，原丝能够组装成更高阶的结构，例如双螺旋和超螺旋结构。综上所述，数据表明迷你环的存在状态可以描述为亚稳态，并讨论了它们可能参与灯丝存储和Z环组装。