LS-8-P-3283 Sun and shade chloroplast ultrastructure of Norway spruce needles 3D-visualized using dual-axis TEM tomography and serial sectioning

The most common method used for visualization of chloroplast ultrastructure is transmission electron microscopy (TEM). However, due to the size of chloroplasts (diameter of about 3 to 8 µm), there is only a very limited extent of three-dimensional (3D) structural information, when standard ultrathin sections (70-80 nm) are used. Both serial sectioning and electron tomography offer a powerfull tool for 3D reconstruction and visualization of chloroplast ultrastructure described in detail in the abstract of Radochová et al. (IMC2014).

In the present study, dual-axis TEM tomography and serial sectioning were used for visualization of the spatial arrangement of chloroplast ultrastructure. Norway spruce (Picea abies L. Karst.) needles were sampled at the experimental site Bílý Kříž (Beskids Mts., Czech Republic). Samples were chemically fixed, processed using microwave tissue processor and embedded into Spurr´s epoxy resin. Electron tomography projections of two orthogonal axes were acquired from 200 nm thick sections using JEOL JEM-2100F microscope equipped with Gatan camera Orius SC 1000 and Serial EM automated acquisition software. Tomograms were processed by specialized computer program (IMOD package, Boulder Laboratory, Colorado). Serial sections (at least five consecutive sections) were viewed in JEOL JEM-1010 microscope and acquired image data were aligned using special software (Link MRC). Chloroplast components (e.g. thylakoids, starch grains and plastoglobuli) were traced in reconstructed series of images (from both electron tomography and serial sectioning) in a process called interactive segmentation and then visualized using 3D plug-in in Ellipse software.

Based on a previous quantitative study on chloroplast ultrastructure using ultrathin sections and TEM, we observed differentiation into two types of chloroplasts within one sun-exposed needle. The chloroplasts from the light exposed side of the sun needle appeared to have less and lower grana (GT) and more plastoglobuli, which can indicate high light stress (Fig. 1). The chloroplasts from the light unexposed side of the sun needle showed more grana, less plastoglobuli and higher starch accumulation. The 3D-visualization using dual-axis electron tomography and serial sectioning (Fig. 2) enabled comparison of spatial arrangement of thylakoid membranes of the two types of chloroplasts from light exposed and unexposed sides of the sun needle.