Abstract
AbstractChloroplasts 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.
Highlights
Chloroplasts are essential plant organelles that arose from cyanobacterial ancestors by the process of endosymbiosis
Meristematic cells that harbor smaller plastids or proplastids contained much smaller Z-rings (Fig. 1b) that presumably encircled the plastid, but the shape of these small plastids could not be determined from the micrographs
FtsZ is a key protein of chloroplast division and this report presents the first electron tomographic 3D structure of plant FtsZ assembly in situ
Summary
Chloroplasts are essential plant organelles that arose from cyanobacterial ancestors by the process of endosymbiosis. The size of starch-storing plastids (amyloplasts) affects the properties of starch granules. Controlling the latter in crops is of interest to industries concerned with the applications of starch [1, 2], and to this end it is critical to understand by what mechanism plastid/chloroplast size is controlled. Both bacteria and chloroplasts divide by the process of binary fission. Changes in FtsZ expression levels or assembly lead to dramatic phenotypes such
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