Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Visualizing structural dynamics of thylakoid membranes

Visualizing structural dynamics of thylakoid membranes OPEN Visualizing structural dynamics of thylakoid membranes SUBJECT AREAS: PHOTOSYNTHESIS 1,2 3 1,4 Masakazu Iwai , Makio Yokono & Akihiko Nakano 3-D RECONSTRUCTION Live Cell Molecular Imaging Research Team, Extreme Photonics Research Group, RIKEN Center for Advanced Photonics, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan, PRESTO, Japan Science and Technology Agency (JST), Honcho, Kawaguchi, Received Saitama 332-0012 Japan, Institute of Low Temperature Science, Hokkaido University, Sapporo, Hokkaido 060-0819 Japan, 2 August 2013 Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan. Accepted 27 December 2013 To optimize photosynthesis, light-harvesting antenna proteins regulate light energy dissipation and Published redistribution in chloroplast thylakoid membranes, which involve dynamic protein reorganization of 20 January 2014 photosystems I and II. However, direct evidence for such protein reorganization has not been visualized in live cells. Here we demonstrate structural dynamics of thylakoid membranes by live cell imaging in combination with deconvolution. We observed chlorophyll fluorescence in the antibiotics-induced macrochloroplast in the moss Physcomitrella patens. The three-dimensional reconstruction uncovered the Correspondence and fine thylakoid membrane structure in live cells. The time-lapse imaging shows that the entire thylakoid requests for materials membrane network is structurally stable, but the individual thylakoid membrane structure is flexible in should be addressed to vivo. Our observation indicates that grana serve as a framework to maintain structural integrity of the entire thylakoid membrane network. Both the structural stability and flexibility of thylakoid membranes would be M.I. (miwai@riken.jp) essential for dynamic protein reorganization under fluctuating light environments. 1,2 hotosynthetic organisms have developed flexible machinery for effective light energy use . To increase the light energy absorption in photosystem II (PSII), the association of light-harvesting antenna complex II P (LHCII) with PSII is induced in chloroplast thylakoid membranes, forming the PSII-LHCII supercomplex . The rate of light energy absorption in photosystem I (PSI) is reversibly raised by the dissociation of LHCII from 4,5 6–8 PSII and also the association of LHCII with PSI . In green algae, the association of LHCII with PSI not only 9,10 11 increases PSI excitation but also causes the interaction of cytochrome b f complex with PSI , leading to the 12,13 switch from linear electron transport to cyclic electron transport around PSI . In higher plants, cyclic electron transport is stimulated by the association of chloroplast NADH dehydrogenase-like complex with PSI . Moreover, the reorganization of PSII-LHCII supercomplex is essential for energy dissipation mechanism under 15–17 intense light conditions to protect PSII from excess energy , in which the protein interactions with LHCSR3 18,19 and PSBS are involved in green algae and higher plants, respectively . When PSII is damaged, disassembly of PSII occurs after its migration from the stacked, appressed membranes, or grana, to the single-layer, stroma- 20,21 exposed membranes, or stroma lamellae, where PSII subunits are replaced . Based on these facts, the structure and arrangement of thylakoid membranes have to be flexible for such protein reorganization to be taken place in response to changing light environments. The structure and arrangement of thylakoid membranes have long been studied since the first observation using light microscopy by Hugo von Mohl in 1837. The grana inside chloroplasts are already identified by light microscopy as dense, dot-like structures . Introducing electron microscopy has deepened our understanding of the structural complexity of thylakoid membranes, showing the remarkable architecture in which stroma lamellae 23–26 connect to grana in the helical configuration . Recently, electron tomography has determined the three- 27,28 dimensional (3D) structure of thylakoid membranes in higher plants , revealing the distinctive image of the junctional connections between grana and stroma lamellae. Intriguingly, the junctional slits where stroma 27,28 lamellae connect grana show significant structural variations , reflecting the variability of the membrane structure. Although electron tomography provides a comprehensive picture of thylakoid membrane structure with ,1 nm resolution, it cannot determine the spatiotemporal dynamics. Therefore, to demonstrate the dynamic aspect of thylakoid membrane structure in vivo, the visualization by live cell imaging is essential. In this work, we used conventional confocal microscopy to observe chlorophyll (Chl) fluorescence structures inside chloroplasts. Previous studies have already shown Chl fluorescence images of chloroplasts in various green 29,30 algae and higher plants . Although confocal microscopy exhibits Chl fluorescence structure in live cells, the fine membrane structure is hardly visible by conventional confocal microscopy due to the diffraction limited SCIENTIFIC REPORTS | 4 : 3768 | DOI: 10.1038/srep03768 1 www.nature.com/scientificreports 31 32,33 resolution . Also, autofluorescence from numerous Chl pigments in microscopy . We then applied 3D deconvolution to the observed thylakoid membrane proteins causes too much signal to resolve the serial sections of Chl fluorescence confocal images. The recon- structed 3D image demonstrated the more detailed thylakoid mem- fine membrane structure. In addition, chloroplasts are normally 5 , 10 mm in size, which makes difficult to discern the inside membrane brane network inside the macrochloroplast (Figs. 1g, h). Within the membrane network, there were two distinct structures—the dot-like structure. To overcome these problems, we used the moss Physcomitrella patens protonemata to observe the membrane struc- and thread-like structures (Fig. 1i, arrows and arrow heads, respec- tively). Comparing the images before and after 3D deconvolution ture in the macrochloroplast, which is more than 10 times larger than 32,33 normal chloroplasts , and applied 3D deconvolution to the serial indicated that the reconstructed 3D image does not generate any 34,35 unnatural structures but effectively diminishes the out-of-focus optical sections of confocal images . We also performed 3D time- blur of Chl fluorescence (Fig. S1). The average diameter of the dot- lapse imaging to determine the spatiotemporal dynamics of thyla- like structures was 425 6 70 nm (n 5 25; Fig. S2), which is equivalent koid membrane structure. Our observation suggests that thylakoid to the size of grana as suggested previously . Thus, the dot-like membranes contain significantly flexible structures in vivo. The structures most likely represent grana. dynamic aspect of thylakoid membrane structure in relation to the photoacclimation mechanisms will be discussed. Structural stability and flexibility of thylakoid membranes. We next performed 3D time-lapse imaging of P. patens macrochloro- Results plasts. The time resolution of our confocal microscopy setup was The reconstructed 3D image of thylakoid membrane structure in ,1.3 s per slice, taking ,7 s to obtain each 3D image, so we could the P. patens macrochloroplast. To visualize thylakoid membrane not resolve the fast movement occurring within ,7 s intervals. The structure inside chloroplasts, we used the moss P. patens protone- reconstructed 3D time-lapse images demonstrated the dynamic mata, which usually contain ,50 chloroplasts in each cell (Fig. 1a). movements of thylakoid membrane structure (Fig. 2a and Although confocal microscopy techniques have improved image Supplementary Movie 1). The entire thylakoid membrane network quality, the optical aberrations and the out-of-focus blur will easily was not stable but rather showing random oscillations. Interestingly, lower the actual resolution under the theoretical limit, especially the location of the dot-like structures was almost stationary during using complicated biological samples. In case of chloroplasts, numer- the observation (Figs. 2b, c). This also suggests that the dot-like ous Chls exist in photosynthetic proteins in thylakoid membranes, so structures represent grana. The sum image of the 28 observed Chl the internal membrane structure is merely visible because of too fluorescence images during the observation also showed that the much Chl fluorescence signal (Fig. 1b). To increase the image con- structural patterns were mostly comparable to that of the initial trast and to decrease the effect of out-of-focus signals, we performed image (Fig. 2d). This implies that the entire thylakoid membrane 3D deconvolution . The reconstructed 3D image showed that the network is not largely rearranged during the observation. blurred Chl signals were significantly reduced, revealing the Chl As compared to the dot-like structures, the thread-like structures fluorescence structures inside the chloroplasts (Figs. 1c, d). Since showed more dynamic movement. The spatial arrangement of the Chl pigments present in thylakoid membrane proteins, the struc- thread-like structures changed within the 7 s intervals during the 3D ture shown by Chl fluorescence indicated solely thylakoid mem- time-lapse imaging (Fig. 3a). It was evident that the thread-like branes. But, it was still difficult to analyze thylakoid membrane structures stretched out from a dot-like structure to the neighboring structure because of the ,10 mm size of chloroplasts and their structures. Although the location of the dot-like structures was random movement during the observation. Interestingly, P. patens mostly unchanged as described in Fig. 2, the dot shape looked flex- chloroplast division is involved with peptidoglycan synthesis .We ible, being circular, ovate, and irregularly oblong (Fig. 3a, b). thus treated the protonemata with ampicillin to inhibit peptidogly- Moreover, 3D time-lapse imaging also demonstrated structural flex- can synthesis, leading to the macrochloroplast formation in each cell ibility of the interconnection between the dot-like and thread-like (Figs. 1e, f). Previous studies have confirmed no difference in the structures (Fig. 3b). The thread-like structures were extended from at shape and size of thylakoid membrane structure between nor- least four different locations around the dot-like structure. Since we mal chloroplasts and macrochloroplasts as examined by electron observed Chl fluorescence, we could not determine whether such an Figure 1 | Thylakoid membrane network revealed by confocal microscopy in combination with 3D deconvolution. (a–d) The normal chloroplasts in P. patens protonema grown on the agar media in a glass-bottom dish were directly observed. (e–i) The formation of macrochloroplasts was induced by growing on the agar media containing 1 mM ampicillin in a glass-bottom dish. (a), (e) The differential interference contrast images. (b), (f) The chlorophyll fluorescence images. (c), (g) The reconstructed 3D images of chlorophyll fluorescence. (d), (h) The enlarged images of the squares in (c) and (g), respectively. (i) Surface plot after linear contrast adjustments of the enlarged image of the square in (h). The arrows and arrowheads indicate the dot- like and thread-like structures, respectively. Scale bars, 5 mm. SCIENTIFIC REPORTS | 4 : 3768 | DOI: 10.1038/srep03768 2 www.nature.com/scientificreports Figure 2 | The spatiotemporal dynamics of thylakoid membrane structure in the macrochloroplast under the control conditions. (a) The 3D time-lapse images of the P. patens macrochloroplast showed the Figure 3 | Structural dynamics of the thread-like structures. (a) Chl random oscillation of thylakoid membrane structure (see also fluorescence signal indicating the dot-like (black) and thread-like Supplementary Movie 1). Numbers indicate the elapsed time in seconds structures (transparent green) was extracted from Supplementary Movie 1. during the observation. (b) The kymograph of the square in (a) suggested The structures observed in the previous panel are faintly outlined in the that the location of the dot-like structures were mostly unchanged during next panel to compare the structural changes between the sequential the observation. (c) Surface plot of kymograph in (b). Arrows indicate the images. Numbers indicate the elapsed time in seconds during the directions for fluorescence intensity (i), the distance (d), and the time (t). observation. Scale bar, 2 mm. (b) The enlarged images of the square in (a) (d) The entire thylakoid membrane network of the initial image was almost with the transparent green indicating the thread-like structures. M, the the same as the sum image of the 28 recorded images during the merged image, focusing on the structural variations of the dot-like observation for 196 s. Scale bars, 2 mm. structures (transparent grey) during the observation. Numbers indicate the elapsed time in seconds during the observation. Scale bar, 1 mm. observed dynamic movement implies the movement of the mem- brane proteins or the membrane itself. Also, it is almost unavoidable moving randomly in most of the chloroplast stromal space (Fig. 4d). that each 3D image contains different time axes between Z-planes, These results suggest that the reduced number of PSII by lincomycin leading to the possible artifactual structures introduced by the 3D treatment makes the dot-like structures unstable so that the entire deconvolution in case of fast moving objects in live cell (see also thylakoid membrane network becomes more randomly oscillated Supplementary Note for the evaluation of the effect in our obtained (Fig. 4e). Previous study shows that lincomycin treatment increases results). Nevertheless, our 3D time-lapse imaging could distinguish the amount of LHCII relative to that of PSII in thylakoid mem- the two structurally different objects inside the chloroplasts—the branes . Therefore, the enhanced structural dynamics of the dot-like structures that are structurally stable at least 7 second and thread-like structures was caused by the increased number of the thread-like structures that are not structurally stable as compared LHCII and/or the decreased number of PSII in the total thylakoid to the dot-like structures. membranes. Enhanced structural flexibility of thylakoid membranes caused by Discussion the decreased number of PSII. Earlier studies have suggested that The extensive studies by using a variety of microscopy have deter- the subcompartmentalization of thylakoid membranes into grana and stroma lamellae largely depends on the localization of PSI and mined the structural aspect of thylakoid membranes . Also, the 37–40 reversible structural modification that affects the gross multilamellar PSII . To examine whether the number of PSII affects the organization of thylakoid membranes has been demonstrated in observed dot-like structures, we next treated the protonemata with 43,44 lincomycin, which prevents chloroplast-encoded protein synthesis, vivo . However, the correlation between thylakoid membrane structure and its dynamic aspect has not been directly visualized in thereby decreasing the number of PSII (Fig. S4). The reconstructed 3D image of the lincomycin-treated macrochloroplast still showed live cell. In this study, we visualized for the first time the spatiotem- the thylakoid membrane network, but the dot-like structures poral dynamics of thylakoid membrane structure by using live cell imaging technique. Our observation revealed the complex network appeared to be smaller (Fig. 4a). The average diameter of the dot- like structures was 310 6 50 nm (n 5 25; Fig. S2), about 27% smaller of thylakoid membranes in the P. patens macrochloroplast, which consists of the dot-like structures interconnected by the thread-like than the ones observed under the control conditions. The result suggested that the decreased number of PSII reduces the size of the structures (Fig. 1). The formation of macrochloroplasts in the P. dot-like structures, indicating again that the dot-like structures patens protonemata is crucial for our live cell imaging. Because the normal chloroplasts continuously move around in the cytosol, it is represent grana. The 3D time-lapse imaging of the lincomycin-treated macrochlor- almost impossible to differentiate whether the observed structural oplast indicated that most of the thylakoid membrane network dynamics is caused by the actual thylakoid membrane dynamics or the chloroplast movement. Forming the macrochloroplast in the showed dynamic movement (Fig. 4a and Supplementary Movie 2). It was apparent that the entire thylakoid membrane network moved protonemal cell effectively prohibits the chloroplast movement since more dynamically than the one observed under the control condi- it becomes large enough to fill in the cytoplasm (Fig. 1e). Also, it has tions. Because of the more dynamic movement and also the smaller been verified that thylakoid membrane structure in the P. patens 32,33 size of the dot-like structures, the distinction between the two struc- macrochloroplast is the same as in the normal chloroplast . Therefore, the macrochloroplast formation is essential for not only tures became less obvious. Moreover, the location of the dot-like structures became unstable, along with the dynamic movement of visualizing the detailed thylakoid membrane network but also con- the thread-like structures (Figs. 4b, c). The movement of the thread- firming that our observation demonstrates the actual structural like structures was also not restricted to one particular location but dynamics of thylakoid membranes in vivo. SCIENTIFIC REPORTS | 4 : 3768 | DOI: 10.1038/srep03768 3 www.nature.com/scientificreports determinable. Since our observation suggests that the dot-like struc- tures most likely indicate grana, the thread-like structures, which interconnect the dot-like structures, reflect most likely stroma lamel- lae, where PSI is known to be abundant. However, PSI fluorescence is hardly visible at room temperature , so the observed Chl fluor- escence of the thread-like structures is mostly originated from PSII and/or LHCII. There are two possibilities to describe the thread-like structures. First, the significant structural flexibility of the thread-like structures reflects the dynamic protein interactions involved with PSII and LHCII, which are known to be important processes to 3–5 regulate photoacclimation mechanisms such as state transitions 15–19 and energy dissipation mechanism . Thus, the apparent structural dynamics of the thread-like structures could be caused by the changes in Chl fluorescence lifetime due to the changes in the func- tional antenna size of PSII . Second, the observed thread-like struc- tures represent free LHCII existing as a pool, which is suggested to be critical to readily redistribute the excitation energy to PSI in state 50,51 transitions . In that case, the thread-like structures could indicate the grana margins, where PSI is known to be involved in state transi- tions . As we observed the thread-like structures extending from the dot-like structures (Fig. 3a), it is possible that such LHCII pools are visible in the stroma lamellae particularly when there is no functional Figure 4 | The enhanced structural dynamics of thylakoid membranes in interaction with PSI. Therefore, the observed structural dynamics the lincomycin-treated macrochloroplast. (a) The 3D time-lapse imaging could be caused by the dynamic interactions of LHCII with PSI of the P. patens macrochloroplast treated with 1 mM lincomycin showed occurring in grana margins or stroma lamellae. more dynamic movement of thylakoid membrane structure as compared According to the lamellar phase characteristics of the membrane to the control conditions (see also Supplementary Movie 2). Numbers as described above, we cannot exclude the possibility that such struc- indicate the elapsed time in seconds during the observation. (b) The tural dynamics reflects the fusion and fission of the membrane inter- kymograph of the square in (a) shows that the location of the dot-like acting with grana. Previously, the similar structural rearrangement structures was randomly changed during the observation. (c) Surface plot has been suggested by the observation using de-enveloped chloro- of kymograph in (b). Arrows indicate the directions for fluorescence plasts . It is possible that such fusion and fission of the membranes intensity (i), the distance (d), and the time (t). (d) The entire thylakoid may cause the transport of a large amount of proteins between neigh- membrane network of the initial image became mostly vague in the sum image of the 28 recorded images during the observation for 196 s. (e) Chl boring grana. However, even if it is the case, we cannot visualize the process by our microscopy setup used in this study because such a fluorescence signal indicating the dot-like (black) and thread-like structures (transparent green) was extracted from Supplementary Movie 2. membrane fusion occurs within a millisecond . The structures observed in the previous panel were faintly outlined in the We observed that the thread-like structures stretched out from next panel to compare the structural changes between the sequential various locations of margins of the dot-like structures (Fig. 3b). images as in Fig. 3a. Numbers indicate the elapsed time in seconds during Such structural variability could reflect the inter-grana membrane the observation. Scale bars, 2 mm. regions, which have been observed by earlier electron microscopy . Recent electron tomography has also confirmed the variable size of 27,28 junctional connections between grana and stroma lamellae . Also, Our live cell imaging shows that the entire thylakoid membrane the reorganization of thylakoid membrane ultrastructure has been network is stable (Fig. 2). In particular, the location of the dot-like 55,56 demonstrated in vivo . From these results, our observation implies structure is mostly unchanged during the observation. The result is that the structural variability shown by electron microscopy actually quite reasonable if we assume that the dot-like structures are grana, reflects structural flexibility of grana margins in vivo (Fig. 3b). Such which are cylindrical stacks of tightly appressed membrane layers as 27,28 structural flexibility could be essential because grana margins are shown by electron tomography . The similar observation has also suggested to be important regions for various regulatory pro- been shown previously using fluorescence recovery after photo- 53,57 cesses . This observation can also be explained by the changes in bleaching technique . Interestingly, although the entire thylakoid Chl fluorescence lifetime and/or the possible fusion and fission pro- membrane network is stable, the individual dot-like structure is cess involving the thread-like structures as described above. not completely fixed as showing the random oscillations Moreover, our observation indicates the correlation between the (Supplementary Movie 1). Besides, our observation indicates that the shape of the dot-like structure is variable in time (Fig. 3b). This dot-like structures and structural dynamics of the entire thylakoid membrane network. Previously, it has been shown that the number implies that the dot-like structures are structurally stable but also flexible in vivo. These observations could indicate the unique feature of LHCII affects the grana formation . Our result shows that the decrease in the number of PSII (Fig. S3) also influences the size of the of smectic mesophases of the biological membrane. Previously, the in vitro observation using atomic force microscopy has suggested such dot-like structures, which most likely indicates the decrease in grana size (Fig. 4a and Fig. S2). These suggest that the formation of PSII- dynamic aspects of grana . There are certain mass-free spaces within PSII arrays in grana, indicating the presence of membrane domains LHCII supercomplex is essential for the structural stability of grana with optimal size . As a matter of fact, our observation reveals that where protein interaction and reorganization can occur. LHCII is also suggested to regulate the reorganization of the multibilayer the decrease in the size of the dot-like structures enhances the 4,47 architecture of grana upon its phosphorylation . Therefore, the dynamic movement of the entire thylakoid membrane network, membrane conditions of the dot-like structures, or grana, could be especially the thread-like structures, moving much more randomly much more flexible than previously thought. as compared to the control conditions (Fig. 4 and Supplementary In addition, our observation revealed that the thread-like struc- Movie 2). One of the possiblities to explain such situations is that the tures showed considerable structural flexibility in vivo (Fig. 3a). observed structural dynamics indicates mostly PSII fluorescence, but What the thread-like structures actually reflect is not yet certainly because of the increased number of LHCII relative to that of PSII SCIENTIFIC REPORTS | 4 : 3768 | DOI: 10.1038/srep03768 4 www.nature.com/scientificreports 8. Takahashi, H., Iwai, M., Takahashi, Y. & Minagawa, J. Identification of the mobile (Fig. S3), there should be some portions of LHCII, which are not light-harvesting complex II polypeptides for state transitions in Chlamydomonas connected with PSII. Such unconnected LHCIIs most probably form reinhardtii. Proc. Natl. Acad. Sci. USA 103, 477–482 (2006). the aggregation, which would not be visualized in our measurement 9. Telfer, A., Bottin, H., Barber, J. & Mathis, P. The effect of magnesium and condition because of the energy-dissipative state of LHCII aggrega- phosphorylation of light-harvesting chlorophyll a/b-protein on the yield of P- 5,60,61 700-photooxidation in pea chloroplasts. Biochim. Biophys. Acta 764, 324–330 tion . Thus, the observed structural dynamics might reflect the (1984). changes in Chl fluorescence lifetime due to the interaction between 10. Delosme, R., Olive, J. & Wollman, F. A. Changes in light energy distribution upon PSII-LHCII supercomplex and LHCII aggregation. It is also possible state transitions: An in vivo photoacoustic study of the wild type and 50,51 that the free LHCII existing as a pool might be visualized, as it is photosynthesis mutants from Chlamydomonas reinhardtii. Biochim. Biophys. migrating between the two photosystems during state transitions . Acta 1273, 150–158 (1996). 11. Vallon, O. et al. Lateral redistribution of cytochrome b6/f complexes along The other explanation is that the distortion of thylakoid membrane thylakoid membranes upon state transitions. Proc. Natl. Acad. Sci. USA 88, structure due to the presence of lincomycin would simply be 8262–8266 (1991). visualized. 12. Finazzi, G. et al. Involvement of state transitions in the switch between linear and In conclusion, our live cell imaging has visualized the structural cyclic electron flow in Chlamydomonas reinhardtii. EMBO Rep. 3, 280–285 flexibility of thylakoid membranes in vivo. Our observation provides (2002). 13. Iwai, M. et al. Isolation of the elusive supercomplex that drives cyclic electron flow evidence for the importance of structural stability of the entire thy- in photosynthesis. Nature 464, 1210–1213 (2010). lakoid membrane network, while the membrane structure itself is 14. Peng, L., Fukao, Y., Fujiwara, M. & Shikanai, T. Multistep Assembly of Chloroplast structurally flexible. The significance of such structural flexibility to NADH Dehydrogenase-Like Subcomplex A Requires Several Nucleus-Encoded protein interaction and reorganization will be an issue to be answered Proteins, Including CRR41 and CRR42, in Arabidopsis. Plant Cell 24, 202–214 (2012). next. The dynamic aspects of photoacclimation mechanisms in green 15. de Bianchi, S., Dall’Osto, L., Tognon, G., Morosinotto, T. & Bassi, R. Minor algae and higher plants have recently been shown by biochemical antenna proteins CP24 and CP26 affect the interactions between photosystem II analysis. This study will lead to the future live cell imaging to uncover subunits and the electron transport rate in grana membranes of Arabidopsis. Plant more detailed information of the spatiotemporal dynamics of thyla- Cell 20, 1012–1028 (2008). 16. Johnson, M. P. et al. Photoprotective energy dissipation involves the koid membrane proteins regulating the photoacclimation mechan- reorganization of photosystem II light-harvesting complexes in the grana isms in vivo. membranes of spinach chloroplasts. Plant Cell 23, 1468–1479 (2011). 17. de Bianchi, S. et al. Arabidopsis mutants deleted in the light-harvesting protein Methods Lhcb4 have a disrupted photosystem II macrostructure and are defective in Strain and growth conditions. Wild-type P. patens (Gransden 2004) protonemata photoprotection. Plant Cell 23, 2659–2679 (2011). 62 22 21 were grown on BCDATG agar media at 25uC under 20 mmol photons m s .For 18. Tokutsu, R. & Minagawa, J. Energy-dissipative supercomplex of photosystem II confocal microscopy, the protonemata were grown on a glass-bottom dish with the associated with LHCSR3 in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. agar media in similar conditions. To generate macrochloroplasts, the protonemata USA 110, 10016–10021 (2013). were grown on the agar contained 1 mM ampicillin for 5 to 7 days . To inhibit 19. Goral, T. K. et al. Light-harvesting antenna composition controls the chloroplast-encoded protein synthesis, the protonema were grown on the agar macrostructure and dynamics of thylakoid membranes in Arabidopsis. Plant J. contained 1 mM lincomycin for 5 to 7 days in similar conditions . 69, 289–301 (2012). 20. Aro, E. M. et al. Dynamics of photosystem II: a proteomic approach to thylakoid Confocal microscopy and 3D deconvolution. We used a Zeiss LSM510 confocal protein complexes. J. Exp. Bot. 56, 347–356 (2005). laser scanning microscope with a 363 Plan Apochromat 1.4 NA oil objective. An 21. Kato, Y., Sun, X., Zhang, L. & Sakamoto, W. Cooperative D1 degradation in the argon laser (488 nm, 10% laser power) and a 650 nm long-pass filter were used for photosystem II repair mediated by chloroplastic proteases in Arabidopsis. Plant observation with the image size of 512 3 512 pixel mode. The scan speed was 1.61 ms Physiol. 159, 1428–1439 (2012). per pixel. The pinhole size was adjusted to 1.35 airy units for the better image 22. Gunning, B., Koenig, F. & Govindjee. A dedication to pioneers of research on acquisition and data processing. The gain was adjusted to obtain the optimal Chl chloroplast structure. The Structure and Function of Plastids, xxiii–xxxi (2007). fluorescence image. The optical serial sections were taken 5 slices with 0.2 mm 23. Paolillo Jr, D. J. The three-dimensional arrangement of intergranal lamellae in intervals. For the time-lapse imaging, the 28 series of 3D image were acquired chloroplasts. J. Cell Sci. 6, 243–255 (1970). sequentially without the additional interval time, taking,196 s in total, roughly,7s 24. Wehrmeyer, W. Zur Kla¨rung der strukturellen Variabilita¨t der per each 3D image. The point spread function of the confocal microscope was Chloroplastengrana des Spinats in Profil und Aufsicht. Planta 62, 272–293 measured by using the 0.1-mm TetraSpeck beads (Invitrogen). The measured point (1964). spread function was used for 3D deconvolution. Also, because each 3D image 25. Brangeon, J. & Mustardy, L. Ontogenetic assembly of intra-chloroplastic lamellae contains different time axes between Z-planes, we adjusted the fluorescence intensity viewed in 3-dimension. Biol. Cell. 36, 71–80 (1979). of all pixels according to the correction of the difference in time axis between Z-planes 26. Staehelin, L. A. Chloroplast structure and supramolecular organization of (see Supplementary Note for details). With the data corrected the difference in time photosynthetic membranes. Photosynthesis III: Photosynthetic Membranes and axis, the 3D reconstruction of deconvolution images was done by using the Volocity Light-Harvesting Systems 19, 1–84 (1986). software (Improvision). The kymographs, the 3D surface plots, the sum images, and 27. Mustardy, L., Buttle, K., Steinbach, G. & Garab, G. The three-dimensional the extraction of Chl fluorescence signals for the dot-like and thread-like structures network of the thylakoid membranes in plants: quasihelical model of the granum- were performed by using ImageJ software (US National Institutes of Health). The stroma assembly. Plant Cell 20, 2552–2557 (2008). average diameter of the dot-like structure was measured as full width at half 28. Austin 2nd, J. R. & Staehelin, L. A. Three-dimensional architecture of grana and maximum of the fluorescence intensity profile obtained by using ImageJ software stroma thylakoids of higher plants as determined by electron tomography. Plant (Fig. S2). Physiol. 155, 1601–1611 (2011). 29. Gunning, B. E. S. & Schwartz, O. M. Confocal microscopy of thylakoid autofluorescence in relation to origin of grana and phylogeny in the green algae. 1. Eberhard, S., Finazzi, G. & Wollman, F. A. The dynamics of photosynthesis. Annu. Aust. J. Plant Physiol. 26, 695–708 (1999). Rev. Genet. 42, 463–515 (2008). 30. Mehta, M., Sarafis, V. & Critchley, C. Thylakoid membrane architecture. Aust. J. 2. Li, Z., Wakao, S., Fischer, B. B. & Niyogi, K. K. Sensing and responding to excess Plant Physiol. 26, 709–716 (1999). light. Annu. Rev. Plant Biol. 60, 239–260 (2009). 31. Abbe, E. Beitra¨ge zur Theorie des Mikroskops und der mikroskopischen 3. Allen, J. F. & Forsberg, J. Molecular recognition in thylakoid structure and Wahrnehmung. Archiv. Mikrosk. Anat. 9, 413–418 (1873). function. Trends Plant Sci. 6, 317–326 (2001). 32. Kasten, B. & Reski, R. beta-lactam antibiotics inhibit chloroplast division in a moss 4. Iwai, M., Takahashi, Y. & Minagawa, J. Molecular remodeling of photosystem II (Physcomitrella patens) but not in tomato (Lycopersicon esculentum). J. Plant during state transitions in Chlamydomonas reinhardtii. Plant Cell 20, 2177–2189 Physiol. 150, 137–140 (1997). (2008). 33. Machida, M. et al. Genes for the peptidoglycan synthesis pathway are essential for 5. Iwai, M., Yokono, M., Inada, N. & Minagawa, J. Live-cell imaging of photosystem chloroplast division in moss. Proc. Natl. Acad. Sci. USA 103, 6753–6758 (2006). II antenna dissociation during state transitions. Proc. Natl. Acad. Sci. USA 107, 2337–2342 (2010). 34. Agard, D. A. & Sedat, J. W. Three-dimensional architecture of a polytene nucleus. Nature 302, 676–681 (1983). 6. Kourˇ il, R. et al. Structural characterization of a complex of photosystem I and light-harvesting complex II of Arabidopsis thaliana. Biochemistry 44, 35. Agard, D. A., Hiraoka, Y., Shaw, P. & Sedat, J. W. Fluorescence microscopy in 10935–10940 (2005). three dimensions. Method. Cell Biol. 30, 353–377 (1989). 7. Kargul, J. et al. Light-harvesting complex II protein CP29 binds to photosystem I 36. Herbstova, M., Tietz, S., Kinzel, C., Turkina, M. V. & Kirchhoff, H. Architectural of Chlamydomonas reinhardtii under State 2 conditions. FEBS J. 272, 4797–4806 switch in plant photosynthetic membranes induced by light stress. Proc. Natl. (2005). Acad. Sci. USA 109, 20130–20135 (2012). SCIENTIFIC REPORTS | 4 : 3768 | DOI: 10.1038/srep03768 5 www.nature.com/scientificreports 37. Barber, J. An explanation for the relationship between salt-induced thylakoid 54. Cevc, G. & Richardsen, H. Lipid vesicles and membrane fusion. Advd. Drug stacking and the chlorophyll fluorescence changes associated with changes in Deliver. Rev. 38, 207–232 (1999). spillover of energy from photosystem II to photosystem I. FEBS Lett. 118, 1–10 55. Nagy, G. et al. Reversible membrane reorganizations during photosynthesis in (1980). vivo: revealed by small-angle neutron scattering. Biochem. J. 436, 225–230 (2011). 38. Anderson, J. M. Consequences of spatial separation of photosystem-1 and 56. Posselt, D. et al. Small-angle neutron scattering study of the ultrastructure of photosystem-2 in thylakoid membranes of higher-plant chloroplasts. FEBS Lett. chloroplast thylakoid membranes - periodicity and structural flexibility of the 124, 1–10 (1981). stroma lamellae. Biochim. Biophys. Acta 1817, 1220–1228 (2012). 39. Trissl, H. W. & Wilhelm, C. Why do thylakoid membranes from higher plants 57. Albertsson, P. A. A quantitative model of the domain structure of the form grana stacks? Trends Biochem. Sci. 18, 415–419 (1993). photosynthetic membrane. Trends Plant Sci. 6, 349–354 (2001). 40. Chow, W. S., Kim, E. H., Horton, P. & Anderson, J. M. Granal stacking of 58. Kim, E. H. et al. The multiple roles of light-harvesting chlorophyll a/b-protein thylakoid membranes in higher plant chloroplasts: the physicochemical forces at complexes define structure and optimize function of Arabidopsis chloroplasts: a work and the functional consequences that ensue. Photochem. Photobiol. Sci. 4, study using two chlorophyll b-less mutants. Biochim. Biophys. Acta 1787, 1081–1090 (2005). 973–984 (2009). 41. Belgio, E., Johnson, M. P., Juric, S. & Ruban, A. V. Higher plant photosystem II 59. Kirchhoff, H. Molecular crowding and order in photosynthetic membranes. light-harvesting antenna, not the reaction center, determines the excited-state Trends Plant Sci. 13, 201–207 (2008). lifetime-both the maximum and the nonphotochemically quenched. Biophys. J. 60. Miloslavina, Y. et al. Far-red fluorescence: a direct spectroscopic marker for 102, 2761–2771 (2012). LHCII oligomer formation in non-photochemical quenching. FEBS Lett. 582, 42. Nevo, R., Charuvi, D., Tsabari, O. & Reich, Z. Composition, architecture and 3625–3631 (2008). dynamics of the photosynthetic apparatus in higher plants. Plant J. 70, 157–176 61. Holzwarth, A. R., Miloslavina, Y., Nilkens, M. & Jahns, P. Identification of two (2012). quenching sites active in the regulation of photosynthetic light-harvesting studied 43. Szabo, M. et al. Structurally flexible macro-organization of the pigment-protein by time-resolved fluorescence. Chem. Phys. Lett. 483, 262–267 (2009). complexes of the diatom Phaeodactylum tricornutum. Photosynth. Res. 95, 62. Nishiyama, T., Hiwatashi, Y., Sakakibara, I., Kato, M. & Hasebe, M. Tagged 237–245 (2008). mutagenesis and gene-trap in the moss, Physcomitrella patens by shuttle 44. Nagy, G. et al. Kinetics of structural reorganizations in multilamellar mutagenesis. DNA Res. 7, 9–17 (2000). photosynthetic membranes monitored by small-angle neutron scattering. Eur. Phys. J. E 36, 69 (2013). 45. Goral, T. K. et al. Visualizing the mobility and distribution of chlorophyll proteins in higher plant thylakoid membranes: effects of photoinhibition and protein phosphorylation. Plant J. 62, 948–959 (2010). Acknowledgments 46. Sznee, K. et al. Jumping mode atomic force microscopy on grana membranes from We thank Drs. Mitsuyasu Hasebe and Yuji Hiwatashi for providing the P. patens ecotype spinach. J. Biol. Chem. 286, 39164–39171 (2011). strain and the extensive technical advice for using the moss; Kaoru Kotoshiba and Hiroe 47. Janik, E. et al. Molecular architecture of plant thylakoids under physiological and Watanabe for support with sample preparation. This work was supported by JST PRESTO, light stress conditions: a study of lipid-light-harvesting complex II model JSPS KAKENHI Grant Numbers 21870047 and 23687008, and grants from RIKEN Center membranes. Plant Cell 25, 2155–2170 (2013). for Advanced Photonics, Extreme Photonics Research Project. 48. Owens, T. G., Webb, S. P., Mets, L., Alberte, R. S. & Fleming, G. R. Antenna size dependence of fluorescence decay in the core antenna of photosystem I: estimates Author contributions of charge separation and energy transfer rates. Proc. Natl. Acad. Sci. USA 84, M.I. designed and performed research. M.I. and M.Y. analyzed data. A.N. contributed new 1532–1536 (1987). 49. Veerman, J. et al. Functional heterogeneity of photosystem II in domain specific analytical tool. M.I. prepared figures and wrote the paper. All authors reviewed and regions of the thylakoid membrane of spinach (Spinacia oleracea L.). Biochemistry discussed the manuscript. 46, 3443–3453 (2007). 50. Wientjes, E., van Amerongen, H. & Croce, R. Quantum yield of charge separation Additional information in photosystem II: functional effect of changes in the antenna size upon light Supplementary information accompanies this paper at http://www.nature.com/ acclimation. J. Phys. Chem. B 117, 11200–11208 (2013). scientificreports 51. Galka, P. et al. Functional analyses of the plant photosystem I-light-harvesting complex II supercomplex reveal that light-harvesting complex II loosely bound to Competing financial interests: The authors declare no competing financial interests. photosystem II is a very efficient antenna for photosystem I in state II. Plant Cell How to cite this article: Iwai, M., Yokono, M. & Nakano, A. Visualizing structural dynamics 24, 2963–2978 (2012). of thylakoid membranes. Sci. Rep. 4, 3768; DOI:10.1038/srep03768 (2014). 52. Tikkanen, M. et al. Phosphorylation-dependent regulation of excitation energy distribution between the two photosystems in higher plants. Biochim. Biophys. This work is licensed under a Creative Commons Attribution- Acta 1777, 425–432 (2008). NonCommercial-NoDerivs 3.0 Unported license. To view a copy of this license, 53. Chuartzman, S. G. et al. Thylakoid membrane remodeling during state transitions visit http://creativecommons.org/licenses/by-nc-nd/3.0 in Arabidopsis. Plant Cell 20, 1029–1039 (2008). SCIENTIFIC REPORTS | 4 : 3768 | DOI: 10.1038/srep03768 6 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Scientific Reports Springer Journals

Visualizing structural dynamics of thylakoid membranes

Loading next page...
 
/lp/springer-journals/visualizing-structural-dynamics-of-thylakoid-membranes-VVJPIclqUV

References (78)

Publisher
Springer Journals
Copyright
Copyright © 2014 by The Author(s)
Subject
Science, Humanities and Social Sciences, multidisciplinary; Science, Humanities and Social Sciences, multidisciplinary; Science, multidisciplinary
eISSN
2045-2322
DOI
10.1038/srep03768
Publisher site
See Article on Publisher Site

Abstract

OPEN Visualizing structural dynamics of thylakoid membranes SUBJECT AREAS: PHOTOSYNTHESIS 1,2 3 1,4 Masakazu Iwai , Makio Yokono & Akihiko Nakano 3-D RECONSTRUCTION Live Cell Molecular Imaging Research Team, Extreme Photonics Research Group, RIKEN Center for Advanced Photonics, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan, PRESTO, Japan Science and Technology Agency (JST), Honcho, Kawaguchi, Received Saitama 332-0012 Japan, Institute of Low Temperature Science, Hokkaido University, Sapporo, Hokkaido 060-0819 Japan, 2 August 2013 Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan. Accepted 27 December 2013 To optimize photosynthesis, light-harvesting antenna proteins regulate light energy dissipation and Published redistribution in chloroplast thylakoid membranes, which involve dynamic protein reorganization of 20 January 2014 photosystems I and II. However, direct evidence for such protein reorganization has not been visualized in live cells. Here we demonstrate structural dynamics of thylakoid membranes by live cell imaging in combination with deconvolution. We observed chlorophyll fluorescence in the antibiotics-induced macrochloroplast in the moss Physcomitrella patens. The three-dimensional reconstruction uncovered the Correspondence and fine thylakoid membrane structure in live cells. The time-lapse imaging shows that the entire thylakoid requests for materials membrane network is structurally stable, but the individual thylakoid membrane structure is flexible in should be addressed to vivo. Our observation indicates that grana serve as a framework to maintain structural integrity of the entire thylakoid membrane network. Both the structural stability and flexibility of thylakoid membranes would be M.I. (miwai@riken.jp) essential for dynamic protein reorganization under fluctuating light environments. 1,2 hotosynthetic organisms have developed flexible machinery for effective light energy use . To increase the light energy absorption in photosystem II (PSII), the association of light-harvesting antenna complex II P (LHCII) with PSII is induced in chloroplast thylakoid membranes, forming the PSII-LHCII supercomplex . The rate of light energy absorption in photosystem I (PSI) is reversibly raised by the dissociation of LHCII from 4,5 6–8 PSII and also the association of LHCII with PSI . In green algae, the association of LHCII with PSI not only 9,10 11 increases PSI excitation but also causes the interaction of cytochrome b f complex with PSI , leading to the 12,13 switch from linear electron transport to cyclic electron transport around PSI . In higher plants, cyclic electron transport is stimulated by the association of chloroplast NADH dehydrogenase-like complex with PSI . Moreover, the reorganization of PSII-LHCII supercomplex is essential for energy dissipation mechanism under 15–17 intense light conditions to protect PSII from excess energy , in which the protein interactions with LHCSR3 18,19 and PSBS are involved in green algae and higher plants, respectively . When PSII is damaged, disassembly of PSII occurs after its migration from the stacked, appressed membranes, or grana, to the single-layer, stroma- 20,21 exposed membranes, or stroma lamellae, where PSII subunits are replaced . Based on these facts, the structure and arrangement of thylakoid membranes have to be flexible for such protein reorganization to be taken place in response to changing light environments. The structure and arrangement of thylakoid membranes have long been studied since the first observation using light microscopy by Hugo von Mohl in 1837. The grana inside chloroplasts are already identified by light microscopy as dense, dot-like structures . Introducing electron microscopy has deepened our understanding of the structural complexity of thylakoid membranes, showing the remarkable architecture in which stroma lamellae 23–26 connect to grana in the helical configuration . Recently, electron tomography has determined the three- 27,28 dimensional (3D) structure of thylakoid membranes in higher plants , revealing the distinctive image of the junctional connections between grana and stroma lamellae. Intriguingly, the junctional slits where stroma 27,28 lamellae connect grana show significant structural variations , reflecting the variability of the membrane structure. Although electron tomography provides a comprehensive picture of thylakoid membrane structure with ,1 nm resolution, it cannot determine the spatiotemporal dynamics. Therefore, to demonstrate the dynamic aspect of thylakoid membrane structure in vivo, the visualization by live cell imaging is essential. In this work, we used conventional confocal microscopy to observe chlorophyll (Chl) fluorescence structures inside chloroplasts. Previous studies have already shown Chl fluorescence images of chloroplasts in various green 29,30 algae and higher plants . Although confocal microscopy exhibits Chl fluorescence structure in live cells, the fine membrane structure is hardly visible by conventional confocal microscopy due to the diffraction limited SCIENTIFIC REPORTS | 4 : 3768 | DOI: 10.1038/srep03768 1 www.nature.com/scientificreports 31 32,33 resolution . Also, autofluorescence from numerous Chl pigments in microscopy . We then applied 3D deconvolution to the observed thylakoid membrane proteins causes too much signal to resolve the serial sections of Chl fluorescence confocal images. The recon- structed 3D image demonstrated the more detailed thylakoid mem- fine membrane structure. In addition, chloroplasts are normally 5 , 10 mm in size, which makes difficult to discern the inside membrane brane network inside the macrochloroplast (Figs. 1g, h). Within the membrane network, there were two distinct structures—the dot-like structure. To overcome these problems, we used the moss Physcomitrella patens protonemata to observe the membrane struc- and thread-like structures (Fig. 1i, arrows and arrow heads, respec- tively). Comparing the images before and after 3D deconvolution ture in the macrochloroplast, which is more than 10 times larger than 32,33 normal chloroplasts , and applied 3D deconvolution to the serial indicated that the reconstructed 3D image does not generate any 34,35 unnatural structures but effectively diminishes the out-of-focus optical sections of confocal images . We also performed 3D time- blur of Chl fluorescence (Fig. S1). The average diameter of the dot- lapse imaging to determine the spatiotemporal dynamics of thyla- like structures was 425 6 70 nm (n 5 25; Fig. S2), which is equivalent koid membrane structure. Our observation suggests that thylakoid to the size of grana as suggested previously . Thus, the dot-like membranes contain significantly flexible structures in vivo. The structures most likely represent grana. dynamic aspect of thylakoid membrane structure in relation to the photoacclimation mechanisms will be discussed. Structural stability and flexibility of thylakoid membranes. We next performed 3D time-lapse imaging of P. patens macrochloro- Results plasts. The time resolution of our confocal microscopy setup was The reconstructed 3D image of thylakoid membrane structure in ,1.3 s per slice, taking ,7 s to obtain each 3D image, so we could the P. patens macrochloroplast. To visualize thylakoid membrane not resolve the fast movement occurring within ,7 s intervals. The structure inside chloroplasts, we used the moss P. patens protone- reconstructed 3D time-lapse images demonstrated the dynamic mata, which usually contain ,50 chloroplasts in each cell (Fig. 1a). movements of thylakoid membrane structure (Fig. 2a and Although confocal microscopy techniques have improved image Supplementary Movie 1). The entire thylakoid membrane network quality, the optical aberrations and the out-of-focus blur will easily was not stable but rather showing random oscillations. Interestingly, lower the actual resolution under the theoretical limit, especially the location of the dot-like structures was almost stationary during using complicated biological samples. In case of chloroplasts, numer- the observation (Figs. 2b, c). This also suggests that the dot-like ous Chls exist in photosynthetic proteins in thylakoid membranes, so structures represent grana. The sum image of the 28 observed Chl the internal membrane structure is merely visible because of too fluorescence images during the observation also showed that the much Chl fluorescence signal (Fig. 1b). To increase the image con- structural patterns were mostly comparable to that of the initial trast and to decrease the effect of out-of-focus signals, we performed image (Fig. 2d). This implies that the entire thylakoid membrane 3D deconvolution . The reconstructed 3D image showed that the network is not largely rearranged during the observation. blurred Chl signals were significantly reduced, revealing the Chl As compared to the dot-like structures, the thread-like structures fluorescence structures inside the chloroplasts (Figs. 1c, d). Since showed more dynamic movement. The spatial arrangement of the Chl pigments present in thylakoid membrane proteins, the struc- thread-like structures changed within the 7 s intervals during the 3D ture shown by Chl fluorescence indicated solely thylakoid mem- time-lapse imaging (Fig. 3a). It was evident that the thread-like branes. But, it was still difficult to analyze thylakoid membrane structures stretched out from a dot-like structure to the neighboring structure because of the ,10 mm size of chloroplasts and their structures. Although the location of the dot-like structures was random movement during the observation. Interestingly, P. patens mostly unchanged as described in Fig. 2, the dot shape looked flex- chloroplast division is involved with peptidoglycan synthesis .We ible, being circular, ovate, and irregularly oblong (Fig. 3a, b). thus treated the protonemata with ampicillin to inhibit peptidogly- Moreover, 3D time-lapse imaging also demonstrated structural flex- can synthesis, leading to the macrochloroplast formation in each cell ibility of the interconnection between the dot-like and thread-like (Figs. 1e, f). Previous studies have confirmed no difference in the structures (Fig. 3b). The thread-like structures were extended from at shape and size of thylakoid membrane structure between nor- least four different locations around the dot-like structure. Since we mal chloroplasts and macrochloroplasts as examined by electron observed Chl fluorescence, we could not determine whether such an Figure 1 | Thylakoid membrane network revealed by confocal microscopy in combination with 3D deconvolution. (a–d) The normal chloroplasts in P. patens protonema grown on the agar media in a glass-bottom dish were directly observed. (e–i) The formation of macrochloroplasts was induced by growing on the agar media containing 1 mM ampicillin in a glass-bottom dish. (a), (e) The differential interference contrast images. (b), (f) The chlorophyll fluorescence images. (c), (g) The reconstructed 3D images of chlorophyll fluorescence. (d), (h) The enlarged images of the squares in (c) and (g), respectively. (i) Surface plot after linear contrast adjustments of the enlarged image of the square in (h). The arrows and arrowheads indicate the dot- like and thread-like structures, respectively. Scale bars, 5 mm. SCIENTIFIC REPORTS | 4 : 3768 | DOI: 10.1038/srep03768 2 www.nature.com/scientificreports Figure 2 | The spatiotemporal dynamics of thylakoid membrane structure in the macrochloroplast under the control conditions. (a) The 3D time-lapse images of the P. patens macrochloroplast showed the Figure 3 | Structural dynamics of the thread-like structures. (a) Chl random oscillation of thylakoid membrane structure (see also fluorescence signal indicating the dot-like (black) and thread-like Supplementary Movie 1). Numbers indicate the elapsed time in seconds structures (transparent green) was extracted from Supplementary Movie 1. during the observation. (b) The kymograph of the square in (a) suggested The structures observed in the previous panel are faintly outlined in the that the location of the dot-like structures were mostly unchanged during next panel to compare the structural changes between the sequential the observation. (c) Surface plot of kymograph in (b). Arrows indicate the images. Numbers indicate the elapsed time in seconds during the directions for fluorescence intensity (i), the distance (d), and the time (t). observation. Scale bar, 2 mm. (b) The enlarged images of the square in (a) (d) The entire thylakoid membrane network of the initial image was almost with the transparent green indicating the thread-like structures. M, the the same as the sum image of the 28 recorded images during the merged image, focusing on the structural variations of the dot-like observation for 196 s. Scale bars, 2 mm. structures (transparent grey) during the observation. Numbers indicate the elapsed time in seconds during the observation. Scale bar, 1 mm. observed dynamic movement implies the movement of the mem- brane proteins or the membrane itself. Also, it is almost unavoidable moving randomly in most of the chloroplast stromal space (Fig. 4d). that each 3D image contains different time axes between Z-planes, These results suggest that the reduced number of PSII by lincomycin leading to the possible artifactual structures introduced by the 3D treatment makes the dot-like structures unstable so that the entire deconvolution in case of fast moving objects in live cell (see also thylakoid membrane network becomes more randomly oscillated Supplementary Note for the evaluation of the effect in our obtained (Fig. 4e). Previous study shows that lincomycin treatment increases results). Nevertheless, our 3D time-lapse imaging could distinguish the amount of LHCII relative to that of PSII in thylakoid mem- the two structurally different objects inside the chloroplasts—the branes . Therefore, the enhanced structural dynamics of the dot-like structures that are structurally stable at least 7 second and thread-like structures was caused by the increased number of the thread-like structures that are not structurally stable as compared LHCII and/or the decreased number of PSII in the total thylakoid to the dot-like structures. membranes. Enhanced structural flexibility of thylakoid membranes caused by Discussion the decreased number of PSII. Earlier studies have suggested that The extensive studies by using a variety of microscopy have deter- the subcompartmentalization of thylakoid membranes into grana and stroma lamellae largely depends on the localization of PSI and mined the structural aspect of thylakoid membranes . Also, the 37–40 reversible structural modification that affects the gross multilamellar PSII . To examine whether the number of PSII affects the organization of thylakoid membranes has been demonstrated in observed dot-like structures, we next treated the protonemata with 43,44 lincomycin, which prevents chloroplast-encoded protein synthesis, vivo . However, the correlation between thylakoid membrane structure and its dynamic aspect has not been directly visualized in thereby decreasing the number of PSII (Fig. S4). The reconstructed 3D image of the lincomycin-treated macrochloroplast still showed live cell. In this study, we visualized for the first time the spatiotem- the thylakoid membrane network, but the dot-like structures poral dynamics of thylakoid membrane structure by using live cell imaging technique. Our observation revealed the complex network appeared to be smaller (Fig. 4a). The average diameter of the dot- like structures was 310 6 50 nm (n 5 25; Fig. S2), about 27% smaller of thylakoid membranes in the P. patens macrochloroplast, which consists of the dot-like structures interconnected by the thread-like than the ones observed under the control conditions. The result suggested that the decreased number of PSII reduces the size of the structures (Fig. 1). The formation of macrochloroplasts in the P. dot-like structures, indicating again that the dot-like structures patens protonemata is crucial for our live cell imaging. Because the normal chloroplasts continuously move around in the cytosol, it is represent grana. The 3D time-lapse imaging of the lincomycin-treated macrochlor- almost impossible to differentiate whether the observed structural oplast indicated that most of the thylakoid membrane network dynamics is caused by the actual thylakoid membrane dynamics or the chloroplast movement. Forming the macrochloroplast in the showed dynamic movement (Fig. 4a and Supplementary Movie 2). It was apparent that the entire thylakoid membrane network moved protonemal cell effectively prohibits the chloroplast movement since more dynamically than the one observed under the control condi- it becomes large enough to fill in the cytoplasm (Fig. 1e). Also, it has tions. Because of the more dynamic movement and also the smaller been verified that thylakoid membrane structure in the P. patens 32,33 size of the dot-like structures, the distinction between the two struc- macrochloroplast is the same as in the normal chloroplast . Therefore, the macrochloroplast formation is essential for not only tures became less obvious. Moreover, the location of the dot-like structures became unstable, along with the dynamic movement of visualizing the detailed thylakoid membrane network but also con- the thread-like structures (Figs. 4b, c). The movement of the thread- firming that our observation demonstrates the actual structural like structures was also not restricted to one particular location but dynamics of thylakoid membranes in vivo. SCIENTIFIC REPORTS | 4 : 3768 | DOI: 10.1038/srep03768 3 www.nature.com/scientificreports determinable. Since our observation suggests that the dot-like struc- tures most likely indicate grana, the thread-like structures, which interconnect the dot-like structures, reflect most likely stroma lamel- lae, where PSI is known to be abundant. However, PSI fluorescence is hardly visible at room temperature , so the observed Chl fluor- escence of the thread-like structures is mostly originated from PSII and/or LHCII. There are two possibilities to describe the thread-like structures. First, the significant structural flexibility of the thread-like structures reflects the dynamic protein interactions involved with PSII and LHCII, which are known to be important processes to 3–5 regulate photoacclimation mechanisms such as state transitions 15–19 and energy dissipation mechanism . Thus, the apparent structural dynamics of the thread-like structures could be caused by the changes in Chl fluorescence lifetime due to the changes in the func- tional antenna size of PSII . Second, the observed thread-like struc- tures represent free LHCII existing as a pool, which is suggested to be critical to readily redistribute the excitation energy to PSI in state 50,51 transitions . In that case, the thread-like structures could indicate the grana margins, where PSI is known to be involved in state transi- tions . As we observed the thread-like structures extending from the dot-like structures (Fig. 3a), it is possible that such LHCII pools are visible in the stroma lamellae particularly when there is no functional Figure 4 | The enhanced structural dynamics of thylakoid membranes in interaction with PSI. Therefore, the observed structural dynamics the lincomycin-treated macrochloroplast. (a) The 3D time-lapse imaging could be caused by the dynamic interactions of LHCII with PSI of the P. patens macrochloroplast treated with 1 mM lincomycin showed occurring in grana margins or stroma lamellae. more dynamic movement of thylakoid membrane structure as compared According to the lamellar phase characteristics of the membrane to the control conditions (see also Supplementary Movie 2). Numbers as described above, we cannot exclude the possibility that such struc- indicate the elapsed time in seconds during the observation. (b) The tural dynamics reflects the fusion and fission of the membrane inter- kymograph of the square in (a) shows that the location of the dot-like acting with grana. Previously, the similar structural rearrangement structures was randomly changed during the observation. (c) Surface plot has been suggested by the observation using de-enveloped chloro- of kymograph in (b). Arrows indicate the directions for fluorescence plasts . It is possible that such fusion and fission of the membranes intensity (i), the distance (d), and the time (t). (d) The entire thylakoid may cause the transport of a large amount of proteins between neigh- membrane network of the initial image became mostly vague in the sum image of the 28 recorded images during the observation for 196 s. (e) Chl boring grana. However, even if it is the case, we cannot visualize the process by our microscopy setup used in this study because such a fluorescence signal indicating the dot-like (black) and thread-like structures (transparent green) was extracted from Supplementary Movie 2. membrane fusion occurs within a millisecond . The structures observed in the previous panel were faintly outlined in the We observed that the thread-like structures stretched out from next panel to compare the structural changes between the sequential various locations of margins of the dot-like structures (Fig. 3b). images as in Fig. 3a. Numbers indicate the elapsed time in seconds during Such structural variability could reflect the inter-grana membrane the observation. Scale bars, 2 mm. regions, which have been observed by earlier electron microscopy . Recent electron tomography has also confirmed the variable size of 27,28 junctional connections between grana and stroma lamellae . Also, Our live cell imaging shows that the entire thylakoid membrane the reorganization of thylakoid membrane ultrastructure has been network is stable (Fig. 2). In particular, the location of the dot-like 55,56 demonstrated in vivo . From these results, our observation implies structure is mostly unchanged during the observation. The result is that the structural variability shown by electron microscopy actually quite reasonable if we assume that the dot-like structures are grana, reflects structural flexibility of grana margins in vivo (Fig. 3b). Such which are cylindrical stacks of tightly appressed membrane layers as 27,28 structural flexibility could be essential because grana margins are shown by electron tomography . The similar observation has also suggested to be important regions for various regulatory pro- been shown previously using fluorescence recovery after photo- 53,57 cesses . This observation can also be explained by the changes in bleaching technique . Interestingly, although the entire thylakoid Chl fluorescence lifetime and/or the possible fusion and fission pro- membrane network is stable, the individual dot-like structure is cess involving the thread-like structures as described above. not completely fixed as showing the random oscillations Moreover, our observation indicates the correlation between the (Supplementary Movie 1). Besides, our observation indicates that the shape of the dot-like structure is variable in time (Fig. 3b). This dot-like structures and structural dynamics of the entire thylakoid membrane network. Previously, it has been shown that the number implies that the dot-like structures are structurally stable but also flexible in vivo. These observations could indicate the unique feature of LHCII affects the grana formation . Our result shows that the decrease in the number of PSII (Fig. S3) also influences the size of the of smectic mesophases of the biological membrane. Previously, the in vitro observation using atomic force microscopy has suggested such dot-like structures, which most likely indicates the decrease in grana size (Fig. 4a and Fig. S2). These suggest that the formation of PSII- dynamic aspects of grana . There are certain mass-free spaces within PSII arrays in grana, indicating the presence of membrane domains LHCII supercomplex is essential for the structural stability of grana with optimal size . As a matter of fact, our observation reveals that where protein interaction and reorganization can occur. LHCII is also suggested to regulate the reorganization of the multibilayer the decrease in the size of the dot-like structures enhances the 4,47 architecture of grana upon its phosphorylation . Therefore, the dynamic movement of the entire thylakoid membrane network, membrane conditions of the dot-like structures, or grana, could be especially the thread-like structures, moving much more randomly much more flexible than previously thought. as compared to the control conditions (Fig. 4 and Supplementary In addition, our observation revealed that the thread-like struc- Movie 2). One of the possiblities to explain such situations is that the tures showed considerable structural flexibility in vivo (Fig. 3a). observed structural dynamics indicates mostly PSII fluorescence, but What the thread-like structures actually reflect is not yet certainly because of the increased number of LHCII relative to that of PSII SCIENTIFIC REPORTS | 4 : 3768 | DOI: 10.1038/srep03768 4 www.nature.com/scientificreports 8. Takahashi, H., Iwai, M., Takahashi, Y. & Minagawa, J. Identification of the mobile (Fig. S3), there should be some portions of LHCII, which are not light-harvesting complex II polypeptides for state transitions in Chlamydomonas connected with PSII. Such unconnected LHCIIs most probably form reinhardtii. Proc. Natl. Acad. Sci. USA 103, 477–482 (2006). the aggregation, which would not be visualized in our measurement 9. Telfer, A., Bottin, H., Barber, J. & Mathis, P. The effect of magnesium and condition because of the energy-dissipative state of LHCII aggrega- phosphorylation of light-harvesting chlorophyll a/b-protein on the yield of P- 5,60,61 700-photooxidation in pea chloroplasts. Biochim. Biophys. Acta 764, 324–330 tion . Thus, the observed structural dynamics might reflect the (1984). changes in Chl fluorescence lifetime due to the interaction between 10. Delosme, R., Olive, J. & Wollman, F. A. Changes in light energy distribution upon PSII-LHCII supercomplex and LHCII aggregation. It is also possible state transitions: An in vivo photoacoustic study of the wild type and 50,51 that the free LHCII existing as a pool might be visualized, as it is photosynthesis mutants from Chlamydomonas reinhardtii. Biochim. Biophys. migrating between the two photosystems during state transitions . Acta 1273, 150–158 (1996). 11. Vallon, O. et al. Lateral redistribution of cytochrome b6/f complexes along The other explanation is that the distortion of thylakoid membrane thylakoid membranes upon state transitions. Proc. Natl. Acad. Sci. USA 88, structure due to the presence of lincomycin would simply be 8262–8266 (1991). visualized. 12. Finazzi, G. et al. Involvement of state transitions in the switch between linear and In conclusion, our live cell imaging has visualized the structural cyclic electron flow in Chlamydomonas reinhardtii. EMBO Rep. 3, 280–285 flexibility of thylakoid membranes in vivo. Our observation provides (2002). 13. Iwai, M. et al. Isolation of the elusive supercomplex that drives cyclic electron flow evidence for the importance of structural stability of the entire thy- in photosynthesis. Nature 464, 1210–1213 (2010). lakoid membrane network, while the membrane structure itself is 14. Peng, L., Fukao, Y., Fujiwara, M. & Shikanai, T. Multistep Assembly of Chloroplast structurally flexible. The significance of such structural flexibility to NADH Dehydrogenase-Like Subcomplex A Requires Several Nucleus-Encoded protein interaction and reorganization will be an issue to be answered Proteins, Including CRR41 and CRR42, in Arabidopsis. Plant Cell 24, 202–214 (2012). next. The dynamic aspects of photoacclimation mechanisms in green 15. de Bianchi, S., Dall’Osto, L., Tognon, G., Morosinotto, T. & Bassi, R. Minor algae and higher plants have recently been shown by biochemical antenna proteins CP24 and CP26 affect the interactions between photosystem II analysis. This study will lead to the future live cell imaging to uncover subunits and the electron transport rate in grana membranes of Arabidopsis. Plant more detailed information of the spatiotemporal dynamics of thyla- Cell 20, 1012–1028 (2008). 16. Johnson, M. P. et al. Photoprotective energy dissipation involves the koid membrane proteins regulating the photoacclimation mechan- reorganization of photosystem II light-harvesting complexes in the grana isms in vivo. membranes of spinach chloroplasts. Plant Cell 23, 1468–1479 (2011). 17. de Bianchi, S. et al. Arabidopsis mutants deleted in the light-harvesting protein Methods Lhcb4 have a disrupted photosystem II macrostructure and are defective in Strain and growth conditions. Wild-type P. patens (Gransden 2004) protonemata photoprotection. Plant Cell 23, 2659–2679 (2011). 62 22 21 were grown on BCDATG agar media at 25uC under 20 mmol photons m s .For 18. Tokutsu, R. & Minagawa, J. Energy-dissipative supercomplex of photosystem II confocal microscopy, the protonemata were grown on a glass-bottom dish with the associated with LHCSR3 in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. agar media in similar conditions. To generate macrochloroplasts, the protonemata USA 110, 10016–10021 (2013). were grown on the agar contained 1 mM ampicillin for 5 to 7 days . To inhibit 19. Goral, T. K. et al. Light-harvesting antenna composition controls the chloroplast-encoded protein synthesis, the protonema were grown on the agar macrostructure and dynamics of thylakoid membranes in Arabidopsis. Plant J. contained 1 mM lincomycin for 5 to 7 days in similar conditions . 69, 289–301 (2012). 20. Aro, E. M. et al. Dynamics of photosystem II: a proteomic approach to thylakoid Confocal microscopy and 3D deconvolution. We used a Zeiss LSM510 confocal protein complexes. J. Exp. Bot. 56, 347–356 (2005). laser scanning microscope with a 363 Plan Apochromat 1.4 NA oil objective. An 21. Kato, Y., Sun, X., Zhang, L. & Sakamoto, W. Cooperative D1 degradation in the argon laser (488 nm, 10% laser power) and a 650 nm long-pass filter were used for photosystem II repair mediated by chloroplastic proteases in Arabidopsis. Plant observation with the image size of 512 3 512 pixel mode. The scan speed was 1.61 ms Physiol. 159, 1428–1439 (2012). per pixel. The pinhole size was adjusted to 1.35 airy units for the better image 22. Gunning, B., Koenig, F. & Govindjee. A dedication to pioneers of research on acquisition and data processing. The gain was adjusted to obtain the optimal Chl chloroplast structure. The Structure and Function of Plastids, xxiii–xxxi (2007). fluorescence image. The optical serial sections were taken 5 slices with 0.2 mm 23. Paolillo Jr, D. J. The three-dimensional arrangement of intergranal lamellae in intervals. For the time-lapse imaging, the 28 series of 3D image were acquired chloroplasts. J. Cell Sci. 6, 243–255 (1970). sequentially without the additional interval time, taking,196 s in total, roughly,7s 24. Wehrmeyer, W. Zur Kla¨rung der strukturellen Variabilita¨t der per each 3D image. The point spread function of the confocal microscope was Chloroplastengrana des Spinats in Profil und Aufsicht. Planta 62, 272–293 measured by using the 0.1-mm TetraSpeck beads (Invitrogen). The measured point (1964). spread function was used for 3D deconvolution. Also, because each 3D image 25. Brangeon, J. & Mustardy, L. Ontogenetic assembly of intra-chloroplastic lamellae contains different time axes between Z-planes, we adjusted the fluorescence intensity viewed in 3-dimension. Biol. Cell. 36, 71–80 (1979). of all pixels according to the correction of the difference in time axis between Z-planes 26. Staehelin, L. A. Chloroplast structure and supramolecular organization of (see Supplementary Note for details). With the data corrected the difference in time photosynthetic membranes. Photosynthesis III: Photosynthetic Membranes and axis, the 3D reconstruction of deconvolution images was done by using the Volocity Light-Harvesting Systems 19, 1–84 (1986). software (Improvision). The kymographs, the 3D surface plots, the sum images, and 27. Mustardy, L., Buttle, K., Steinbach, G. & Garab, G. The three-dimensional the extraction of Chl fluorescence signals for the dot-like and thread-like structures network of the thylakoid membranes in plants: quasihelical model of the granum- were performed by using ImageJ software (US National Institutes of Health). The stroma assembly. Plant Cell 20, 2552–2557 (2008). average diameter of the dot-like structure was measured as full width at half 28. Austin 2nd, J. R. & Staehelin, L. A. Three-dimensional architecture of grana and maximum of the fluorescence intensity profile obtained by using ImageJ software stroma thylakoids of higher plants as determined by electron tomography. Plant (Fig. S2). Physiol. 155, 1601–1611 (2011). 29. Gunning, B. E. S. & Schwartz, O. M. Confocal microscopy of thylakoid autofluorescence in relation to origin of grana and phylogeny in the green algae. 1. Eberhard, S., Finazzi, G. & Wollman, F. A. The dynamics of photosynthesis. Annu. Aust. J. Plant Physiol. 26, 695–708 (1999). Rev. Genet. 42, 463–515 (2008). 30. Mehta, M., Sarafis, V. & Critchley, C. Thylakoid membrane architecture. Aust. J. 2. Li, Z., Wakao, S., Fischer, B. B. & Niyogi, K. K. Sensing and responding to excess Plant Physiol. 26, 709–716 (1999). light. Annu. Rev. Plant Biol. 60, 239–260 (2009). 31. Abbe, E. Beitra¨ge zur Theorie des Mikroskops und der mikroskopischen 3. Allen, J. F. & Forsberg, J. Molecular recognition in thylakoid structure and Wahrnehmung. Archiv. Mikrosk. Anat. 9, 413–418 (1873). function. Trends Plant Sci. 6, 317–326 (2001). 32. Kasten, B. & Reski, R. beta-lactam antibiotics inhibit chloroplast division in a moss 4. Iwai, M., Takahashi, Y. & Minagawa, J. Molecular remodeling of photosystem II (Physcomitrella patens) but not in tomato (Lycopersicon esculentum). J. Plant during state transitions in Chlamydomonas reinhardtii. Plant Cell 20, 2177–2189 Physiol. 150, 137–140 (1997). (2008). 33. Machida, M. et al. Genes for the peptidoglycan synthesis pathway are essential for 5. Iwai, M., Yokono, M., Inada, N. & Minagawa, J. Live-cell imaging of photosystem chloroplast division in moss. Proc. Natl. Acad. Sci. USA 103, 6753–6758 (2006). II antenna dissociation during state transitions. Proc. Natl. Acad. Sci. USA 107, 2337–2342 (2010). 34. Agard, D. A. & Sedat, J. W. Three-dimensional architecture of a polytene nucleus. Nature 302, 676–681 (1983). 6. Kourˇ il, R. et al. Structural characterization of a complex of photosystem I and light-harvesting complex II of Arabidopsis thaliana. Biochemistry 44, 35. Agard, D. A., Hiraoka, Y., Shaw, P. & Sedat, J. W. Fluorescence microscopy in 10935–10940 (2005). three dimensions. Method. Cell Biol. 30, 353–377 (1989). 7. Kargul, J. et al. Light-harvesting complex II protein CP29 binds to photosystem I 36. Herbstova, M., Tietz, S., Kinzel, C., Turkina, M. V. & Kirchhoff, H. Architectural of Chlamydomonas reinhardtii under State 2 conditions. FEBS J. 272, 4797–4806 switch in plant photosynthetic membranes induced by light stress. Proc. Natl. (2005). Acad. Sci. USA 109, 20130–20135 (2012). SCIENTIFIC REPORTS | 4 : 3768 | DOI: 10.1038/srep03768 5 www.nature.com/scientificreports 37. Barber, J. An explanation for the relationship between salt-induced thylakoid 54. Cevc, G. & Richardsen, H. Lipid vesicles and membrane fusion. Advd. Drug stacking and the chlorophyll fluorescence changes associated with changes in Deliver. Rev. 38, 207–232 (1999). spillover of energy from photosystem II to photosystem I. FEBS Lett. 118, 1–10 55. Nagy, G. et al. Reversible membrane reorganizations during photosynthesis in (1980). vivo: revealed by small-angle neutron scattering. Biochem. J. 436, 225–230 (2011). 38. Anderson, J. M. Consequences of spatial separation of photosystem-1 and 56. Posselt, D. et al. Small-angle neutron scattering study of the ultrastructure of photosystem-2 in thylakoid membranes of higher-plant chloroplasts. FEBS Lett. chloroplast thylakoid membranes - periodicity and structural flexibility of the 124, 1–10 (1981). stroma lamellae. Biochim. Biophys. Acta 1817, 1220–1228 (2012). 39. Trissl, H. W. & Wilhelm, C. Why do thylakoid membranes from higher plants 57. Albertsson, P. A. A quantitative model of the domain structure of the form grana stacks? Trends Biochem. Sci. 18, 415–419 (1993). photosynthetic membrane. Trends Plant Sci. 6, 349–354 (2001). 40. Chow, W. S., Kim, E. H., Horton, P. & Anderson, J. M. Granal stacking of 58. Kim, E. H. et al. The multiple roles of light-harvesting chlorophyll a/b-protein thylakoid membranes in higher plant chloroplasts: the physicochemical forces at complexes define structure and optimize function of Arabidopsis chloroplasts: a work and the functional consequences that ensue. Photochem. Photobiol. Sci. 4, study using two chlorophyll b-less mutants. Biochim. Biophys. Acta 1787, 1081–1090 (2005). 973–984 (2009). 41. Belgio, E., Johnson, M. P., Juric, S. & Ruban, A. V. Higher plant photosystem II 59. Kirchhoff, H. Molecular crowding and order in photosynthetic membranes. light-harvesting antenna, not the reaction center, determines the excited-state Trends Plant Sci. 13, 201–207 (2008). lifetime-both the maximum and the nonphotochemically quenched. Biophys. J. 60. Miloslavina, Y. et al. Far-red fluorescence: a direct spectroscopic marker for 102, 2761–2771 (2012). LHCII oligomer formation in non-photochemical quenching. FEBS Lett. 582, 42. Nevo, R., Charuvi, D., Tsabari, O. & Reich, Z. Composition, architecture and 3625–3631 (2008). dynamics of the photosynthetic apparatus in higher plants. Plant J. 70, 157–176 61. Holzwarth, A. R., Miloslavina, Y., Nilkens, M. & Jahns, P. Identification of two (2012). quenching sites active in the regulation of photosynthetic light-harvesting studied 43. Szabo, M. et al. Structurally flexible macro-organization of the pigment-protein by time-resolved fluorescence. Chem. Phys. Lett. 483, 262–267 (2009). complexes of the diatom Phaeodactylum tricornutum. Photosynth. Res. 95, 62. Nishiyama, T., Hiwatashi, Y., Sakakibara, I., Kato, M. & Hasebe, M. Tagged 237–245 (2008). mutagenesis and gene-trap in the moss, Physcomitrella patens by shuttle 44. Nagy, G. et al. Kinetics of structural reorganizations in multilamellar mutagenesis. DNA Res. 7, 9–17 (2000). photosynthetic membranes monitored by small-angle neutron scattering. Eur. Phys. J. E 36, 69 (2013). 45. Goral, T. K. et al. Visualizing the mobility and distribution of chlorophyll proteins in higher plant thylakoid membranes: effects of photoinhibition and protein phosphorylation. Plant J. 62, 948–959 (2010). Acknowledgments 46. Sznee, K. et al. Jumping mode atomic force microscopy on grana membranes from We thank Drs. Mitsuyasu Hasebe and Yuji Hiwatashi for providing the P. patens ecotype spinach. J. Biol. Chem. 286, 39164–39171 (2011). strain and the extensive technical advice for using the moss; Kaoru Kotoshiba and Hiroe 47. Janik, E. et al. Molecular architecture of plant thylakoids under physiological and Watanabe for support with sample preparation. This work was supported by JST PRESTO, light stress conditions: a study of lipid-light-harvesting complex II model JSPS KAKENHI Grant Numbers 21870047 and 23687008, and grants from RIKEN Center membranes. Plant Cell 25, 2155–2170 (2013). for Advanced Photonics, Extreme Photonics Research Project. 48. Owens, T. G., Webb, S. P., Mets, L., Alberte, R. S. & Fleming, G. R. Antenna size dependence of fluorescence decay in the core antenna of photosystem I: estimates Author contributions of charge separation and energy transfer rates. Proc. Natl. Acad. Sci. USA 84, M.I. designed and performed research. M.I. and M.Y. analyzed data. A.N. contributed new 1532–1536 (1987). 49. Veerman, J. et al. Functional heterogeneity of photosystem II in domain specific analytical tool. M.I. prepared figures and wrote the paper. All authors reviewed and regions of the thylakoid membrane of spinach (Spinacia oleracea L.). Biochemistry discussed the manuscript. 46, 3443–3453 (2007). 50. Wientjes, E., van Amerongen, H. & Croce, R. Quantum yield of charge separation Additional information in photosystem II: functional effect of changes in the antenna size upon light Supplementary information accompanies this paper at http://www.nature.com/ acclimation. J. Phys. Chem. B 117, 11200–11208 (2013). scientificreports 51. Galka, P. et al. Functional analyses of the plant photosystem I-light-harvesting complex II supercomplex reveal that light-harvesting complex II loosely bound to Competing financial interests: The authors declare no competing financial interests. photosystem II is a very efficient antenna for photosystem I in state II. Plant Cell How to cite this article: Iwai, M., Yokono, M. & Nakano, A. Visualizing structural dynamics 24, 2963–2978 (2012). of thylakoid membranes. Sci. Rep. 4, 3768; DOI:10.1038/srep03768 (2014). 52. Tikkanen, M. et al. Phosphorylation-dependent regulation of excitation energy distribution between the two photosystems in higher plants. Biochim. Biophys. This work is licensed under a Creative Commons Attribution- Acta 1777, 425–432 (2008). NonCommercial-NoDerivs 3.0 Unported license. To view a copy of this license, 53. Chuartzman, S. G. et al. Thylakoid membrane remodeling during state transitions visit http://creativecommons.org/licenses/by-nc-nd/3.0 in Arabidopsis. Plant Cell 20, 1029–1039 (2008). SCIENTIFIC REPORTS | 4 : 3768 | DOI: 10.1038/srep03768 6

Journal

Scientific ReportsSpringer Journals

Published: Jan 20, 2014

There are no references for this article.