Live-cell visualization of excitation energy dynamics in chloroplast thylakoid structures

Abstract

The intricate molecular processes underlying photosynthesis have long been studied using various analytic approaches. However, the three-dimensional (3D) dynamics of such photosynthetic processes remain unexplored due to technological limitations related to investigating intraorganellar mechanisms in vivo. By developing a system for high-speed 3D laser scanning confocal microscopy combined with high-sensitivity multiple-channel detection, we visualized excitation energy dynamics in thylakoid structures within chloroplasts of live Physcomitrella patens cells. Two distinct thylakoid structures in the chloroplast, namely the grana and stroma lamellae, were visualized three-dimensionally in live cells. The simultaneous detection of the shorter (than ~670 nm) and longer (than ~680 nm) wavelength regions of chlorophyll (Chl) fluorescence reveals different spatial characteristics—irregular and vertical structures, respectively. Spectroscopic analyses showed that the shorter and longer wavelength regions of Chl fluorescence are affected more by free light-harvesting antenna proteins and photosystem II supercomplexes, respectively. The high-speed 3D time-lapse imaging of the shorter and longer wavelength regions also reveals different structural dynamics—rapid and slow movements within 1.5 seconds, respectively. Such structural dynamics of the two wavelength regions of Chl fluorescence would indicate excitation energy dynamics between light-harvesting antenna proteins and photosystems, reflecting the energetically active nature of photosynthetic proteins in thylakoid membranes.

Cytological analysis and structural quantification of FtsZ1-2 and FtsZ2-1 network characteristics in Physcomitrella patens

“…Recent studies have found that moss chloroplasts are surrounded by a peptidoglycan wall, emphasising the evolutionary position of P. patens chloroplasts as intermediate between free-living bacteria and fully domesticated plastids of higher plants 25 – 27 . Because of this intermediate position and its experimental advantages, P. patens chloroplasts are suitable objects for live-cell visualization and subsequent modelling 28 , 29 .…”

Subdiffraction‐resolution live‐cell imaging for visualizing thylakoid membranes

“…Thylakoid membranes form intricate networks of grana and stroma lamellae structures that are about 5–10 µm in size. It is possible to visualize thylakoid membranes in the moss Physcomitrella patens with increased spatial resolution using 3D image deconvolution processing and determining photon-emitting positions through reverse calculation of the convolved point spread function (Iwai et al 2014, Iwai et al 2016). In this study, we applied 3D structured illumination microscopy (3D-SIM) to characterize thylakoid membranes in chloroplasts of A. thaliana and C. reinhardtii at subdiffraction resolution.…”

Small-Angle X-Ray and Neutron Scattering on Photosynthetic Membranes

“…1-200 nm in near in vivo conditions and is widely applied in structural biology and soft matter sciences. Small-angle scattering complements various microscopic techniques, such as TEM (Menke, 1960;Paolillo and Paolillo, 1970;Staehelin, 1986;Austin and Staehelin, 2011;Armbruster et al, 2013;Heinz et al, 2016;Kowalewska et al, 2016;Wood et al, 2018;Kowalewska et al, 2019;Wood et al, 2019;Li et al, 2020), scanning electron microscopy (Mustárdy and Jánossy, 1979;Armbruster et al, 2013), cryo-EM (Ford et al, 2002;Kirchhoff et al, 2011;Engel et al, 2015), cryoelectron tomography (Shimoni et al, 2005;Austin and Staehelin, 2011;Daum and Kühlbrandt, 2011;Kouřil et al, 2011;Ford and Holzenburg, 2014;Bussi et al, 2019;Rast et al, 2019), atomic force microscopy (Kaftan et al, 2002;Sturgis et al, 2009;Sznee et al, 2011;Grzyb et al, 2013;Onoa et al, 2014), confocal laser scanning microscopy (Kowalewska et al, 2016;Mazur et al, 2019), and live cell imaging (Iwai et al, 2014;Iwai et al, 2016). The first scattering studies on photosynthetic membranes were performed in 1953 by Finean et al (1953) and has continued ever since.…”