Masakazu Iwai

Isolation of the elusive supercomplex that drives cyclic electron flow in photosynthesis

Photosynthetic light reactions establish electron flow in the chloroplast’s thylakoid membranes, leading to the production of the ATP and NADPH that participate in carbon fixation. Two modes of electron flow exist—linear electron flow (LEF) from water to NADP+ via photosystem (PS) II and PSI in series and cyclic electron flow (CEF) around PSI (ref. 2). Although CEF is essential for satisfying the varying demand for ATP, the exact molecule(s) and operational site are as yet unclear. In the green alga Chlamydomonas reinhardtii, the electron flow shifts from LEF to CEF on preferential excitation of PSII (ref. 3), which is brought about by an energy balancing mechanism between PSII and PSI (state transitions). Here, we isolated a protein supercomplex composed of PSI with its own light-harvesting complex (LHCI), the PSII light-harvesting complex (LHCII), the cytochrome b6f complex (Cyt bf), ferredoxin (Fd)-NADPH oxidoreductase (FNR), and the integral membrane protein PGRL1 (ref. 5) from C. reinhardtii cells under PSII-favouring conditions. Spectroscopic analyses indicated that on illumination, reducing equivalents from downstream of PSI were transferred to Cyt bf, whereas oxidised PSI was re-reduced by reducing equivalents from Cyt bf, indicating that this supercomplex is engaged in CEF (Supplementary Fig. 1). Thus, formation and dissociation of the PSI–LHCI–LHCII–FNR–Cyt bf–PGRL1 supercomplex not only controlled the energy balance of the two photosystems, but also switched the mode of photosynthetic electron flow.

Molecular Remodeling of Photosystem II during State Transitions in Chlamydomonas reinhardtii

State transitions, or the redistribution of light-harvesting complex II (LHCII) proteins between photosystem I (PSI) and photosystem II (PSII), balance the light-harvesting capacity of the two photosystems to optimize the efficiency of photosynthesis. Studies on the migration of LHCII proteins have focused primarily on their reassociation with PSI, but the molecular details on their dissociation from PSII have not been clear. Here, we compare the polypeptide composition, supramolecular organization, and phosphorylation of PSII complexes under PSI- and PSII-favoring conditions (State 1 and State 2, respectively). Three PSII fractions, a PSII core complex, a PSII supercomplex, and a multimer of PSII supercomplex or PSII megacomplex, were obtained from a transformant of the green alga Chlamydomonas reinhardtii carrying a His-tagged CP47. Gel filtration and single particles on electron micrographs showed that the megacomplex was predominant in State 1, whereas the core complex was predominant in State 2, indicating that LHCIIs are dissociated from PSII upon state transition. Moreover, in State 2, strongly phosphorylated LHCII type I was found in the supercomplex but not in the megacomplex. Phosphorylated minor LHCIIs (CP26 and CP29) were found only in the unbound form. The PSII subunits were most phosphorylated in the core complex. Based on these observations, we propose a model for PSII remodeling during state transitions, which involves division of the megacomplex into supercomplexes, triggered by phosphorylation of LHCII type I, followed by LHCII undocking from the supercomplex, triggered by phosphorylation of minor LHCIIs and PSII core subunits.

CP29, a monomeric light-harvesting complex II protein, is essential for state transitions in Chlamydomonas reinhardtii.

In oxygen-evolving photosynthesis, the two photosystems, photosystem I (PSI) and photosystem II (PSII), function in parallel, and their excitation levels must be balanced to maintain an optimal photosynthetic rate under various light conditions. State transitions balance excitation energy between the two photosystems by redistributing light-harvesting complex II (LHCII) proteins. Here we describe two RNA interference (RNAi) mutants of the green alga Chlamydomonas reinhardtii with one of the minor monomeric LHCII proteins, CP29 or CP26, knocked down. These two proteins have been identified in PSI-LHCI supercomplexes that harbor mobile LHCII proteins from PSII under a state where PSII is preferentially excited (State 2). We show that both the CP29 and CP26 RNAi mutants undergo reductions in the PSII antenna size during a transition from State 1 (a state where PSI is preferentially excited) to State 2, as reflected by nonphotochemical quenching of fluorescence, low temperature fluorescence spectra, and functional absorption cross-section. However, the undocked LHCIIs from PSII do not re-associate with PSI in the CP29-RNAi (b4i) mutant because the antenna size of PSI was not complementary increased. The mobile LHCIIs in the CP26-RNAi (b5i) mutant, however, re-associate with PSI, whose PSI-LHCI/II supercomplex is visualized on a sucrose density gradient. This study clarifies that CP29, not CP26, is an essential component in state transitions and demonstrates that CP29 is crucial when mobile LHCIIs re-associate with PSI under State 2 conditions.

Visualizing structural dynamics of thylakoid membranes

To optimize photosynthesis, light-harvesting antenna proteins regulate light energy dissipation and redistribution in chloroplast thylakoid membranes, which involve dynamic protein reorganization of 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 fine thylakoid membrane structure in live cells. The time-lapse imaging shows that the entire thylakoid membrane network is structurally stable, but the individual thylakoid membrane structure is flexible in 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 essential for dynamic protein reorganization under fluctuating light environments.

Characterization of photosystem I antenna proteins in the prasinophyte Ostreococcus tauri

Prasinophyceae are a broad class of early-branching eukaryotic green algae. These picophytoplankton are found ubiquitously throughout the ocean and contribute considerably to global carbon-fixation. Ostreococcus tauri, as the first sequenced prasinophyte, is a model species for studying the functional evolution of light-harvesting systems in photosynthetic eukaryotes. In this study we isolated and characterized O. tauri pigment-protein complexes. Two photosystem I (PSI) fractions were obtained by sucrose density gradient centrifugation in addition to free light-harvesting complex (LHC) fraction and photosystem II (PSII) core fractions. The smaller PSI fraction contains the PSI core proteins, LHCI, which are conserved in all green plants, Lhcp1, a prasinophyte-specific LHC protein, and the minor, monomeric LHCII proteins CP26 and CP29. The larger PSI fraction contained the same antenna proteins as the smaller, with the addition of Lhca6 and Lhcp2, and a 30% larger absorption cross-section. When O. tauri was grown under high-light conditions, only the smaller PSI fraction was present. The two PSI preparations were also found to be devoid of the far-red chlorophyll fluorescence (715-730 nm), a signature of PSI in oxygenic phototrophs. These unique features of O. tauri PSI may reflect primitive light-harvesting systems in green plants and their adaptation to marine ecosystems. Possible implications for the evolution of the LHC-superfamily in photosynthetic eukaryotes are discussed.

Photosystem II antenna phosphorylation-dependent protein diffusion determined by fluorescence correlation spectroscopy

Flexibility of chloroplast thylakoid membrane proteins is essential for plant fitness and survival under fluctuating light environments. Phosphorylation of light-harvesting antenna complex II (LHCII) is known to induce dynamic protein reorganization that fine-tunes the rate of energy conversion in each photosystem. However, molecular details of how LHCII phosphorylation causes light energy redistribution throughout thylakoid membranes still remain unclear. By using fluorescence correlation spectroscopy, we here determined the LHCII phosphorylation-dependent protein diffusion in thylakoid membranes isolated from the green alga Chlamydomonas reinhardtii. As compared to the LHCII dephosphorylation-induced condition, the diffusion coefficient of LHCII increased nearly twofold under the LHCII phosphorylation-induced condition. We also verified the results by using the LHCII phosphorylation-deficient mutant. Our observation suggests that LHCII phosphorylation-dependent protein reorganization occurs along with the changes in the rate of protein diffusion, which would have an important role in mediating light energy redistribution throughout thylakoid membranes.

Light-harvesting complex Lhcb9 confers a green alga-type photosystem I supercomplex to the moss Physcomitrella patens

Light-harvesting complex (LHC) proteins in chloroplast thylakoid membranes not only transfer absorbed light energy to the two photosystems but also regulate the rate of energy transfer to avoid photodamage. Here we demonstrate that Lhcb9, a recently discovered LHC protein in the moss Physcomitrella patens, functions to connect LHC proteins with photosystem I (PSI), resulting in the formation of two different types of PSI supercomplexes in thylakoid membranes. We observed that the Lhcb9-containing PSI supercomplex is disassembled in response to excess light conditions. On the basis of our phylogenetic analysis, it appears that P. patens acquired Lhcb9 by horizontal gene transfer from the earlier green algal lineage, leading to the presence of both green alga-type and vascular plant-type PSI supercomplexes, which would have been crucial for conquering the dynamic environmental interface between aquatic and terrestrial conditions it faced during evolution.

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

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.

Toward understanding the multiple spatiotemporal dynamics of chlorophyll fluorescence

Dynamic reorganization of photosystems I and II is suggested to occur in chloroplast thylakoid membranes to maintain the efficiency of photosynthesis under fluctuating light conditions. To directly observe the process in action, live-cell imaging techniques are necessary. Using live-cell imaging, we have shown that the fine thylakoid structures in the moss Physcomitrella patens are flexible in time. However, the spatiotemporal resolution of a conventional confocal microscopy limits more precise visualization of entire thylakoid structures and understanding of the structural dynamics. Here, we discuss the issues related to observing chlorophyll fluorescence at multiple spatiotemporal scales in vivo and in vitro.

Simulation of Excitation Energy Transfer within the PSI-LHCI/II Supercomplex from Chlamydomonas reinhardtii

State transition in photosynthesis is a short-term balancing mechanism of energy distribution between photosystem I (PSI) and II (PSII). When PSII is preferentially excited (state 2), a pool of mobile light-harvesting complex II antenna proteins migrates from PSII to PSI. We previously identified three of those mobile proteins (CP26, CP29, and LhcbM5) in the PSI-LHCI/ II supercomplex isolated from the green alga Chlamydomonas reinhardtii placed in state 2. Here, we demonstrate the functional interaction between the chlorophylls in the mobile light-harvesting complex (LHC) II proteins and those in the PSILHCI supercomplex by examining time-resolved fluorescence spectra of (1) the “state 2-type” PSI-LHCI/II supercomplex, (2) the LHCII fraction removed from the supercomplex, and (3) the remained “state 1-type” PSI-LHCI supercomplex. The lifetime of most of the kinetic components were longer in the PSI-LHCI/II supercomplex, suggesting that the functional size of the antenna was increased by the attached LHCII. Furthermore, we propose possible energy transfer processes based on a simulation of energy migration within the PSILHCI/ II supercomplex.