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.
Highlights
Composition of light-harvesting complex (LHC) proteins[15,16,17,18,19]
As the plastoquinone pool becomes highly reduced by unbalanced excitation energy at PSII and PSI, LHCII phosphorylation occurs[29], inducing phosphorylated LHCII to dissociate from PSII in the grana[30,31], which leads to an increase in the rate of energy transfer to PSI in the stroma lamellae[32,33], presumably via migration of phosphorylated LHCII from the grana to stroma lamellae[34,35]
To achieve live-cell visualization of 3D structural dynamics with spatial resolution beyond the diffraction limit at video rate, we developed super-resolution confocal live imaging microscopy (SCLIM)[42]
Summary
Composition of LHC proteins[15,16,17,18,19]. It has been suggested that the degree of PSII ordered array in grana affects the photosynthetic functions, such as energy transfer, electron transport, photoprotection, and protein repair processes[20,21,22,23,24,25]. We observed thylakoid membrane structural dynamics using a live-cell imaging technique.
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