Subdiffraction‐resolution live‐cell imaging for visualizing thylakoid membranes

Abstract

The chloroplast is the chlorophyll-containing organelle that produces energy through photosynthesis. Within the chloroplast is an intricate network of thylakoid membranes containing photosynthetic membrane proteins that mediate electron transport and generate chemical energy. Historically, electron microscopy (EM) has been a powerful tool for visualizing the macromolecular structure and organization of thylakoid membranes. However, an understanding of thylakoid membrane dynamics remains elusive because EM requires fixation and sectioning. To improve our knowledge of thylakoid membrane dynamics we need to consider at least two issues: (i) the live-cell imaging conditions needed to visualize active processes in vivo; and (ii) the spatial resolution required to differentiate the characteristics of thylakoid membranes. Here, we utilize three-dimensional structured illumination microscopy (3D-SIM) to explore the optimal imaging conditions for investigating the dynamics of thylakoid membranes in living plant and algal cells. We show that 3D-SIM is capable of examining broad characteristics of thylakoid structures in chloroplasts of the vascular plant Arabidopsis thaliana and distinguishing the structural differences between wild-type and mutant strains. Using 3D-SIM, we also visualize thylakoid organization in whole cells of the green alga Chlamydomonas reinhardtii. These data reveal that high light intensity changes thylakoid membrane structure in C. reinhardtii. Moreover, we observed the green alga Chromochloris zofingiensis and the moss Physcomitrella patens to show the applicability of 3D-SIM. This study demonstrates that 3D-SIM is a promising approach for studying the dynamics of thylakoid membranes in photoautotrophic organisms during photoacclimation processes.

A key role for phosphorylation of PsbH in the biogenesis and repair of photosystem II in Chlamydomonas

“…The mutants stn8 and pbcp show an altered organization of thylakoid membranes. In stn8 , and particularly in the double mutant stn7stn8 , the diameter of the thylakoid grana is larger compared to the wild type (Fristedt et al 2009, Iwai et al 2018, Nath, et al 2013). In contrast in the pbcp phosphatase mutant, the diameter of the grana is similar to the wild type, but the number of membrane layers in grana stacks is reduced (Samol, et al 2012).…”

The Role of Phosphorylation Dynamics of CURVATURE THYLAKOID 1B in Plant Thylakoid Membranes

“…Both in vivo and in vitro studies have indicated a decrease in grana diameter and the total area of appressed membranes under red or blue light, i.e. when both the PSII core and LHCII become phosphorylated (Chuartzman et al, 2008;Iwai et al, 2018). However, a similar decrease in appressed thylakoid area was observed in dark-acclimated plants exposed directly to high light (HL; Khatoon et al, 2009;Herbstová et al, 2012) when there is high PSII core phosphorylation but complete dephosphorylation of LHCII (Tikkanen et al, 2008a).…”

Chloroplast ultrastructure in plants

“…individual chloroplasts and higher). In addition to FLIM, 3D structural illumination microscopy (3D‐SIM) offers interesting opportunities for studying chloroplast ultrastructures in intact cells (Iwai et al ., ). Although the resolution of this fluorescence‐based technique is lower than EM, it is highly complementary to EM‐based techniques (that monitor only snapshots in time of fixed samples) because it allows in vivo studies on chloroplast dynamics in real time.…”