Imaging of Photochemical Properties of Thylakoid

Shigeichi Kumazakia, Eunchul Kima,b, Shuho Nozuea, Masahide Terazimaa, Tae Kyu Ahnb
aDepartment of Chemistry, Graduate School of Science, Kyoto University, Kyoto, 606-8502, Japan; bDepartment of Energy Science, Sungkyunkwan University, Suwon, 440-746 Korea

Constituents and/or morphology of thylakoid membranes (TM) have been reported to be dependent not only on the type of photosynthetic organisms but also on many factors like their environmental light, nutrition, their growth phase, cellular differentiation, tissue types etc. How the properties of the TM are biologically regulated still remains to be directly visualized at subchloroplast and individual-cell levels, which requires highly informative imaging methods. Stacked and non-stacked regions of TM in plant chloroplasts, grana and stroma-lamellae, are rich in photosystem II and I (PSII and PSI), respectively. Unbalanced excitation between PSI and PSII is known to be compensated by state-transition mechanims, in which antenna sizes of PSI and/or PSII are regulated. We examined if changes in the antenna sizes of PSI and PSII in the grana and stroma-lamellae can be simultaneously estimated by our fluorescence spectral microscopy [1,2]. Deformation of fluorescence spectra may mask spectral changes attributable to state-transitions, but it was minimized by global spectral decomposition at multiple focal planes of chloroplasts in which average transmission spectrum inside chloroplasts was incooperated. Given miminal assumptions for the relationship between fluorescence quantum yield and antenna sizes of PSI and PSII, our results supports a model where a single trimer of light-harvesting complex II (LHCII) is dissociated from PSII and associated with PSI by the transition from state 1 to state 2 [2]. More detailed interpratation, depending on the TM regions, will be described in the meeting.

In contrast to the fluorescence spectra, time constants of fluorescence decay are robust parameters against attenuation. We are thus recently exploring effective applications of fluorescence lifetime-imaging microscopy (FLIM). Our FLIM system enables one to obtain multiple images with systematically varied excitation laser powers [3], by which PSI-rich cells are differentiated from PSII rich ones due to their different behaviors. We have selected a filamentous diazotroph containing differentiated cell, heterocyst. PSII content in matured heterocyst is known to be far lower than that of vegetative cells. The laser-intensity-varied FLIM in principle enables one to estimate residual PSII in heterocys, for example. In addition, we have composed a numerical simulation that can largely reproduce experimental sensitivity of the average fluorescence lifetime to the absolute laser power by taking all relevant parameters into account [3]. The successful simulation suggests that no substantial photoprotective quenching is activated under the experimental conditions employed. The system is thus ready for studies on a wide range of quenching mechanisms as well as imaging of photochemical reactions in individual cells.


[1] S. Kumazaki et al., J. Microsc., 228 (2007) 240 - 254.
[2] E. Kim, et al., Plant Cell Physiol. 56(4) (2015) 759 - 768.
[3] S. Nozue et al., Biochim. Biophys. Acta 1857 (2016) 46–59.