3D Visualization of Thylakoid Membrane Development

Proper biogenesis of the chloroplast is essential for all photosynthetic plant cells. As germinating seedlings are exposed to light, etioplasts in young mesophyll cells become chloroplasts in the developing seedling. The chloroplast thylakoids develop from a paracrystalline tubular structure that is transformed when illuminated into a series of flattened membranes that become the grana thylakoids. Traditional transmission electron microscopy (TEM) shows subcellular fine structure in two-dimensional thin slices, but a comprehensive study of chloroplast biogenesis requires 3D visualization of membrane transformations.

Electron tomography (ET) creates 3D reconstructions of subcellular objects by taking a series of traditional TEMs collected at different angles and using computer algorithms to generate detailed views of the surfaces of structures (Shimoni et al., 2005; Daum and Kühlbrandt, 2011). Kowalewska et al. (2016) have used ET and confocal laser scanning microscopy to follow the development of stacked grana thylakoids as etiolated runner bean (Phaseolus coccineus) seedlings are exposed to light. They characterize membrane structure during the first 3 d of illumination (under a 16-h-light/8-h-dark photoperiod) of dark-adapted seedlings.

After 8 d of etiolation, etioplasts in dark-adapted seedlings showed a regular arrangement of branched membrane tubules. These tubular units are joined as a paracrystalline network in the prolamellar body (PLB), much like the cells in a honeycomb (see figure, A and B). As soon as 1 h after illumination, the symmetry of the PLB was lost as the internal membranes rearranged (figure, C). The tetrahedral network was no longer visible and the margins of the tubules developed flattened stromal slats that would develop into prothylakoids.

Thylakoid development in three dimensions. As dark-adapted runner bean seedlings are illuminated, the paracrystalline prolamellar body of the etioplast ([A] and [B]) becomes disorganized ([C] and [D]), as membranes flatten ([E] and [F]) and are transformed into stacked grana ([G] and [H]). (Reprinted from Kowalewska et al. [2016].)

After 2 h of illumination, only a remnant of the PLB was present and membranes became organized parallel to the long axis of the chloroplast (figure, D). These PLB remnants were continuous, as opposed to the porous membranes seen in prothylakoids (PTs). By 4 h, the PLB was transformed into a parallel arrangement of PTs with local connections, often split dichotomously into two adjacent branches (figure, E). By 8 h, the first stacked membranes had appeared (figure, F). The appressed thylakoids were no longer porous and membranes were seen to be continuous in stacked regions, with each stacked membrane connected to a single split PT.

By the beginning of the second day, small grana stacks were observed, and both stroma thylakoids and grana thylakoids were evident (figure, G). Each stroma thylakoid was connected to two adjacent grana thylakoids at an angle of ∼20°, as seen by Austin and Staehelin (2011). By day 3, stroma thylakoids had split dichotomously, connecting with two grana thylakoids, except at the top and bottom of the grana stack, where the stroma thylakoid connected to only one grana thylakoid (figure, H).

The entire transformation of the paracrystalline tubular membrane network of the PLB to the organized, stacked grana thylakoid membranes is shown in a supplemental movie created by the authors in 2D and 3D (this wonderful video should not be missed!). Every stage of thylakoid development is shown in a traditional TEM view, outlining the membranes to be visualized in 3D. Then, the 3D structures of the outlined areas are presented and rotated to show membrane surfaces from many angles during the membrane transformations.

The authors also use confocal laser scanning microscopy to monitor red chlorophyll fluorescence, as it helps to show the distribution of appressed thylakoids. And, because chlorophyll-protein complexes affect membrane stacking, they use low-temperature fluorescence emission and excitation spectroscopy to characterize these complexes. The authors complement this work with mild denaturing electrophoresis and immunodetection of the chlorophyll-protein complexes. In summary, the authors propose a theoretical model of the membrane changes during grana development and greatly contribute to our understanding of chloroplast and thylakoid biogenesis.