PSII Core Research Articles

PH-Sensing Machinery of Excitation Energy Transfer in Diatom PSI–FCPI Complexes

Excitation energy-transfer processes in photosynthetic light-harvesting complexes are strongly affected by the surrounding environments of pigments. Here we report on the effects of pH changes on excitation energy dynamics in both diatom photosystem I-fucoxanthin chlorophyll a/ c-binding protein (PSI-FCPI) and PSI core complexes by means of fluorescence spectroscopies. The steady-state fluorescence spectra of the PSI-FCPI showed similar features among three samples at pH 5.0, 6.5, and 8.0. However, fluorescence decay-associated spectra of the pH 5.0- and 8.0-adapted PSI-FCPI within 100 ps exhibit peak shifts to longer and shorter wavelengths, respectively, than the peaks in the pH 6.5 spectra. Because such spectral changes hardly occur in the PSI complexes, the peak shifts at pH 5.0 and 8.0 in the PSI-FCPI can be ascribed to alterations of pigment-pigment and/or pigment-protein interactions around/within FCPI caused by the pH changes. These findings provide novel physical insights into the pH-sensing light-harvesting strategy in diatom PSI-FCPI.

Thylakoid Protein Phosphorylation Dynamics in a Moss Mutant Lacking SERINE/THREONINE PROTEIN KINASE STN8.

In all eukaryotes, protein phosphorylation is a key regulatory mechanism in several cellular processes, including the acclimation of photosynthesis to environmental cues. Despite being a well-conserved regulatory mechanism in the chloroplasts of land plants, distinct differences in thylakoid protein phosphorylation patterns have emerged from studies on species of different phylogenetic groups. Here, we analyzed thylakoid protein phosphorylation in the moss Physcomitrella patens, assessing the thylakoid phospho-protein profile and dynamics in response to changes in white light intensity. Compared with Arabidopsis (Arabidopsis thaliana), parallel characterization of wild-type P patens and the knockout mutant stn8 (depleted in SER/THR PROTEIN KINASE8 [STN8]) disclosed a moss-specific pattern of thylakoid protein phosphorylation, both with respect to specific targets and to their dynamic phosphorylation in response to environmental cues. Unlike vascular plants, (1) phosphorylation of the PSII protein D1 in P patens was negligible under all light conditions, (2) phosphorylation of the PSII core subunits CP43 and D2 showed only minor changes upon fluctuations in light intensity, and (3) absence of STN8 completely abolished all PSII core protein phosphorylation. Further, we detected light-induced phosphorylation in the minor light harvesting complex (LHC) antenna protein LHCB6, which was dependent on STN8 kinase activity, and found specific phosphorylations on LHCB3. Data presented here provide further insights into the appearance and physiological role of thylakoid protein phosphorylation during evolution of oxygenic photosynthetic organisms and their colonization of land.

Redox transients of P680 associated with the incremental chlorophyll-a fluorescence yield rises elicited by a series of saturating flashes in diuron-treated photosystem II core complex of Thermosynechococcus vulcanus.

Recent chlorophyll-a fluorescence yield measurements, using single-turnover saturating flashes (STSFs), have revealed the involvement of a rate-limiting step in the reactions following the charge separation induced by the first flash. As also shown here, in diuron-inhibited PSII core complexes isolated from Thermosynechococcus vulcanus the fluorescence maximum could only be reached by a train of STSFs. In order to elucidate the origin of the fluorescence yield increments in STSF series, we performed transient absorption measurements at 819 nm, reflecting the photooxidation and re-reduction kinetics of the primary electron donor P680. Upon single flash excitation of the dark-adapted sample, the decay kinetics could be described with lifetimes of 17 ns (∼50%) and 167 ns (∼30%), and a longer-lived component (∼20%). This kinetics are attributed to re-reduction of P680•+ by the donor side of PSII. In contrast, upon second-flash (with Δt between 5 μs and 100 ms) or repetitive excitation, the 819 nm absorption changes decayed with lifetimes of about 2ns (∼60%) and 10ns (∼30%), attributed to recombination of the primary radical pair P680•+ Pheo•- , and a small longer-lived component (∼10%). These data confirm that only the first STSF is capable of generating stable charge separation-leading to the reduction of QA ; and thus, the fluorescence yield increments elicited by the consecutive flashes must have a different physical origin. Our double-flash experiments indicate that the rate-limiting steps, detected by chlorophyll-a fluorescence, are not correlated with the turnover of P680.

Transcriptome Profile of the Variegated Ficus microcarpa c.v. Milky Stripe Fig Leaf.

Photosynthetic properties and transcriptomic profiles of green and white sectors of Ficus microcarpa (c.v. milky stripe fig) leaves were examined in naturally variegated plants. An anatomic analysis indicated that chloroplasts of the white sectors contained a higher abundance of starch granules and lacked stacked thylakoids. Moreover, no photosynthetic rate was detected in the white sectors. Transcriptome profile and differential expressed gene (DEG) analysis showed that genes encoding PSII core proteins were down-regulated in the white sectors. In genes related to chlorophyll metabolism, no DEGs were identified in the biosynthesis pathway of chlorophyll. However, genes encoding the first step of chlorophyll breakdown were up-regulated. The repression of genes involved in N-assimilation suggests that the white sectors were deprived of N. The mutation in the transcription factor mitochondrial transcription termination factor (mTERF) suggests that it induces colorlessness in leaves of the milky stripe fig.

Antenna arrangement and energy transfer pathways of a green algal photosystem-I-LHCI supercomplex.

During oxygenic photosynthesis, photosystems I and II (PSI and PSII) are essential for light-driven electron transport. Excitation energy transfer in PSI occurs extremely quickly, making it an efficient energy converter. In the alga Chlamydomonas reinhardtii (Cr), multiple units of light-harvesting complex I (LHCI) bind to the PSI core and function as peripheral antennae, forming a PSI-LHCI supercomplex. CrPSI-LHCI shows significantly larger antennae compared with plant PSI-LHCI while maintaining highly efficient energy transfer from LHCI to PSI. Here, we report structures of CrPSI-LHCI, solved by cryo-electron microscopy, revealing that up to ten LHCIs are associated with the PSI core. The structures provide detailed information about antenna organization and pigment arrangement within the supercomplexes. Highly populated and closely associated chlorophylls in the antennae explain the high efficiency of light harvesting and excitation energy transfer in CrPSI-LHCI.

A theoretical study on the dynamics of light harvesting in the dimeric photosystem II core complex: regulation and robustness of energy transfer pathways.

Here we present our theoretical investigations into the light reaction in the dimeric photosystem II (PSII) core complex. An effective model for excitation energy transfer (EET) and primary charge separation (CS) in the PSII core complex was developed, with model parameters constructed based on molecular dynamics (MD) simulation data. Compared to experimental results, we demonstrated that this model faithfully reproduces the absorption spectra of the RC and core light-harvesting complexes (CP43 and CP47) as well as the full EET dynamics among the chromophores in the PSII core complex. We then applied master equation simulations and network analysis to investigate detailed EET plus CS dynamics in the system, allowing us to identify key EET pathways and produce a coarse-grained cluster model for the light reaction in the dimeric PSII core complex. We show that non-equilibrium energy transfer channels play important roles in the efficient light harvesting process and that multiple EET pathways exist between subunits of PSII to ensure the robustness of light harvesting in the system. Furthermore, we revealed that inter-monomer energy transfer dominated by the coupling between the two CLA625 molecules enables efficient energy exchange between two CP47s in the dimeric PSII core complex, which leads to significant energy pooling in the CP47 domain during the light reaction. Our study provides a blueprint for the design of light harvesting in the PSII core and show that a structure-based approach using molecular dynamics simulations and quantum chemistry calculations can be effectively utilized to elucidate the dynamics of light harvesting in complex photosynthetic systems.

Silicon improves photosynthetic performance by optimizing thylakoid membrane protein components in rice under drought stress

For revealing the internal mitigation mechanism of silicon to drought stress, a super high-yield hybrid rice LYP9 was employed to study the change of photosynthetic fluorescence, structure and composition of thylakoid membrane as well as its physiological property by polyethylene glycol (PEG) and silicon (Si) treatment. Results showed that K step appeared in OJIP transient and ET0/RC, ET0/CS0, φE0 of rice seedlings were decreased under drought stress, while these parameters were increased by applying silicon. We found that silicon application could relieve the degradation of membrane protein complexes under drought condition, such as supercomplexes, PSI core binding LHCI, PSI core, F1-ATPase binding Cytb6/f complex, PSII core, trimeric LHCII and monomeric LHCII. Thylakoid membranes proteins were further detected by BN-SDS-PAGE, 20 differential protein spots between PEG treatment and PEG treatment applying with silicon were identified. These alterations were involved in light harvesting, stability of PS I, function of PS II reaction centers, electron transport, PS II core antenna content, and consequent synthesis of ATP. Our results indicated that Si treatment might play roles in absorption, transformation and transfer of light energy by optimizing the thylakoid membrane protein components in rice seedlings under drought stress observed by BN-PAGE and BN-SDS-PAGE.

Combined effects of NaCl and Cd2+ stress on the photosynthetic apparatus of Thellungiella salsuginea

Plants show complex responses to abiotic stress while, the effect of the stress combinations can be different to those seen when each stress is applied individually. Here, we report on the effects of salt and/or cadmium on photosynthetic apparatus of Thellungiella salsuginea. Our results showed a considerable reduction of plant growth with some symptoms of toxicity, especially with cadmium treatment. The structural integrity of both photosystems (PSI and PSII) was mostly maintained under salt stress. Cadmium induced a considerable decrease of both PSI and PSII quantum yields and the electron transport rate ETR(I) and ETR(II) paralleled by an increase of non-photochemical quenching (NPQ). In addition, cadmium alone affects the rate of primary photochemistry by an increase of fluorescence at O-J phase and also the photo-electrochemical quenching at J-I phase. A positive L-band appeared with (Cd) treatment as an indicator of lower PSII connectivity, and a positive K-band reflecting the imbalance in number of electrons at donor and acceptor side. In continuity to our previous studies which showed that NaCl supply reduced Cd2+ uptake and limited its accumulation in shoot of divers halophyte species, here as a consequence, we demonstrated the NaCl-induced enhancement effect of Cd2+ toxicity on the PSII activity by maintaining the photosynthetic electron transport chain as evidenced by the differences in ψO, φEo, ABS/RC and TR0/RC and by improvement of performance index PI(ABS), especially after short time of treatment. A significant decrease of LHCII, D1 and CP47 amounts was detected under (Cd) treatment. However, NaCl supply alleviates the Cd2+ effect on protein abundance including LHCII and PSII core complex (D1 and CP47).

Non-photochemical quenching-dependent acclimation and thylakoid organization of Chlamydomonas reinhardtii to high light stress.

Light is essential for all photosynthetic organisms while an excess of it can lead to damage mainly the photosystems of the thylakoid membrane. In this study, we have grown Chlamydomonas reinhardtii cells in different intensities of high light to understand the photosynthetic process with reference to thylakoid membrane organization during its acclimation process. We observed, the cells acclimatized to long-term response to high light intensities of 500 and 1000µmolm-2s-1 with faster growth and more biomass production when compared to cells at 50µmolm-2s-1 light intensity. The ratio of Chl a/b was marginally decreased from the mid-log phase of growth at the high light intensity. Increased level of zeaxanthin and LHCSR3 expression was also found which is known to play a key role in non-photochemical quenching (NPQ) mechanism for photoprotection. Changes in photosynthetic parameters were observed such as increased levels of NPQ, marginal change in electron transport rate, and many other changes which demonstrate that cells were acclimatized to high light which isan adaptive mechanism. Surprisingly, PSII core protein contents have marginally reduced when compared to peripherally arranged LHCII in high light-grown cells. Further, we also observed alterations in stromal subunits of PSI and low levels of PsaG, probably due to disruption of PSI assembly and also its association with LHCI. During the process of acclimation, changes in thylakoid organization occurred in high light intensities with reduction of PSII supercomplex formation. This change may be attributed to alteration of protein-pigment complexes which are in agreement with circular dichoismspectra of high light-acclimatized cells, where decrease in the magnitude of psi-type bands indicates changes in ordered arrays of PSII-LHCII supercomplexes. These results specify that acclimation to high light stress throughNPQ mechanism by expression of LHCSR3 and also observedchanges in thylakoid protein profile/supercomplex formation lead to low photochemical yield and more biomass production in high light condition.

ONE-HELIX PROTEIN2 (OHP2) Is Required for the Stability of OHP1 and Assembly Factor HCF244 and Is Functionally Linked to PSII Biogenesis.

The members of the light-harvesting complex protein family, which include the one-helix proteins (OHPs), are characterized by one to four membrane-spanning helices. These proteins function in light absorption and energy dissipation, sensing light intensity, and triggering photomorphogenesis or the binding of chlorophyll and intermediates of chlorophyll biosynthesis. Arabidopsis (Arabidopsis thaliana) contains two OHPs, while four homologs (named high-light-induced proteins) exist in Synechocystis PCC6803. Various functions have been assigned to high-light-induced proteins, ranging from photoprotection and the assembly of photosystem I (PSI) and PSII to regulation of the early steps of chlorophyll biosynthesis, but little is known about the function of the two plant OHPs. Here, we show that the two Arabidopsis OHPs form heterodimers and that the stromal part of OHP2 interacts with the plastid-localized PSII assembly factor HIGH CHLOROPHYLL FLUORESCENCE244 (HCF244). Moreover, concurrent accumulation of the two OHPs and HCF244 is critical for the stability of all three proteins. In particular, the absence of OHP2 leads to the complete loss of OHP1 and HCF244. We used a virus-induced gene silencing approach to minimize the expression of OHP1 or OHP2 in adult Arabidopsis plants and revealed that OHP2 is essential for the accumulation of the PSII core subunits, while the other photosynthetic complexes and the major light-harvesting complex proteins remained unaffected. We examined the potential functions of the OHP1-OHP2-HCF244 complex in the assembly and/or repair of PSII and propose a role for this heterotrimeric complex in thylakoid membrane biogenesis.

Structure of the maize photosystem I supercomplex with light-harvesting complexes I and II.

Plants regulate photosynthetic light harvesting to maintain balanced energy flux into photosystems I and II (PSI and PSII). Under light conditions favoring PSII excitation, the PSII antenna, light-harvesting complex II (LHCII), is phosphorylated and forms a supercomplex with PSI core and the PSI antenna, light-harvesting complex I (LHCI). Both LHCI and LHCII then transfer excitation energy to the PSI core. We report the structure of maize PSI-LHCI-LHCII solved by cryo-electron microscopy, revealing the recognition site between LHCII and PSI. The PSI subunits PsaN and PsaO are observed at the PSI-LHCI interface and the PSI-LHCII interface, respectively. Each subunit relays excitation to PSI core through a pair of chlorophyll molecules, thus revealing previously unseen paths for energy transfer between the antennas and the PSI core.

Antenna switches partners in the shade

Photosynthesis A cloudy day or an overshadowing tree causes fluctuations in light that can throw off the balance of energy flow in plant photosystems I and II (PSI and PSII). Pan et al. solved structures of PSI bound to two light-harvesting complexes (LHCs). One LHC is permanently associated with PSI. The other LHC delivers light energy to PSII under optimal conditions but can switch to a PSI-associated state after phosphorylation by a kinase that senses the redox environment of the chloroplast. The movement of LHCs between the photosystems helps maintain even energy flux. Two chlorophyll-containing subunits are visible in the structure that connect the PSI core to each LHC. Science , this issue p. [1109][1] [1]: /lookup/doi/10.1126/science.aat1156

Rice tolerance to suboptimal low temperatures relies on the maintenance of the photosynthetic capacity

The purpose of this research was to identify differences between two contrasting rice cultivars in their response to suboptimal low temperatures stress. A transcriptomic analysis of the seedlings was performed and results were complemented with biochemical and physiological analyses. The microarray analysis showed downregulation of many genes related with PSII and particularly with the oxygen evolving complex in the sensitive cultivar IR50. Complementary studies indicated that the PSII performance, the degree of oxygen evolving complex coupling with the PSII core and net photosynthetic rate diminished in this cultivar in response to the stress. However, the tolerant cultivar Koshihikari was able to maintain its energy equilibrium by sustaining the photosynthetic capacity. The increase of oleic acid in Koshihikari could be related with membrane remodelling of the chloroplasts and hence contribute to tolerance. Overall, these results work as a ground for future analyses that look forward to characterize possible mechanisms to tolerate this stress.

Effects of PSII Manganese-Stabilizing Protein Succinylation on Photosynthesis in the Model Cyanobacterium Synechococcus sp. PCC 7002

Lysine succinylation is a newly identified protein post-translational modification and plays important roles in various biological pathways in both prokaryotes and eukaryotes, but its extent and function in photosynthetic organisms remain largely unknown. Here, we performed the first systematic studies of lysine succinylation in cyanobacteria, which are the only prokaryotes capable of oxygenic photosynthesis and the established model organisms for studying photosynthetic mechanisms. By using mass spectrometry analysis in combination with the enrichment of succinylated peptides from digested cell lysates, we identified 1,704 lysine succinylation sites on 691 proteins in a model cyanobacterium Synechococcus sp. PCC 7002. Bioinformatic analysis revealed that a large proportion of the succinylation sites were present on proteins in photosynthesis and metabolism. Among all identified succinylated proteins involved in photosynthesis, the PSII manganese-stabilizing protein (PsbO) was found to be succinylated on Lys99 and Lys234. Functional studies of PsbO were performed by site-directed mutagenesis, and mutants mimicking either constitutively succinylated (K99E and K234E) or non-succinylated states (K99R and K234R) were constructed. The succinylation-mimicking K234E mutant exhibited a decreased oxygen evolution rate of the PSII center and the efficiency of energy transfer during the photosynthetic reaction. Molecular dynamics simulations suggested a mechanism that may allow succinylation to influence the efficiency of photosynthesis by altering the conformation of PsbO, thereby hindering the interaction between PsbO and the PSII core. Our findings suggest that reversible succinylation may be an important regulatory mechanism during photosynthesis in Synechococcus, as well as in other photosynthetic organisms.

Structure and function of photosystem I in Cyanidioschyzon merolae.

The evolution of photosynthesis from primitive photosynthetic bacteria to higher plants has been driven by the need to adapt to a wide range of environmental conditions. The red alga Cyanidioschyzon merolae is a primitive organism, which is capable of performing photosynthesis in extreme acidic and hot environments. The study of its photosynthetic machinery may provide new insight on the evolutionary path of photosynthesis and on light harvesting and its regulation in eukaryotes. With that aim, the structural and functional properties of the PSI complex were investigated by biochemical characterization, mass spectrometry, and X-ray crystallography. PSI was purified from cells grown at 25 and 42°C, crystallized and its crystal structure was solved at 4Å resolution. The structure of C. merolae reveals a core complex with a crescent-shaped structure, formed by antenna proteins. In addition, the structural model shows the position of PsaO and PsaM. PsaG and PsaH are present in plant complex and are missing from the C. merolae model as expected. This paper sheds new light onto the evolution of photosynthesis, which gives a strong indication for the chimerical properties of red algae PSI. The subunit composition of the PSI core from C. merolae and its associated light-harvesting antennae suggests that it is an evolutionary and functional intermediate between cyanobacteria and plants.

Lack of tocopherols influences the PSII antenna and the functioning of photosystems under low light

As tocopherols are expected to protect PSII against toxic singlet oxygen it is surprising that the null tocopherol mutant vte1 has been reported to show only a weak enhancement of photosystem II photoinhibition under high irradiance. Based on the view that singlet oxygen is formed also in unstressed conditions, such as low light (LL), we hypothesized that some defense strategies are activated in vte1 in these light conditions. In support for that we noted several symptoms of stress at PSII in the mutant under LL, by means of parameters of fast and slow kinetics of chlorophyll fluorescence and of changes in the relative contribution of PSII antenna in comparison to those of PSI. This was associated with a lower extent of phosphorylation of PSII core proteins (D1 and CP43). PSII RCs do not totally recover from stress in vte1 even after the nocturnal phase. As a clear compensation for the impeded performance of PSII in the vte1 we noted an increased quantum efficiency of PSI. A pronounced changes between WT and the vte1 mutant were also related to conformation of LHCII at the beginning of photoperiod, suggesting the absence of LHCII trimers in the mutant. The thylakoids thickness was similar in WT and vte1 under LL, but a pronounced unstacking of thylakoids was evoked by HL only in vte1. In conclusion, we postulate that action of 1O2 on PSII in vte1 leads to some permanent damage at PSII core and at LHCII already under LL.

Isolation of the cyanobacterial YFP-tagged photosystem I using GFP-Trap®

A strain of Synechocystis sp. PCC 6803 expressing the yellow fluorescent protein (YFP) fused to the C-terminus of the PsaF subunit of PSI has been constructed and used to isolate native PSI complexes employing the GFP-Trap®, an efficient immunoprecipitation system which recognizes the green fluorescent protein (GFP) and its variants. The protein analysis and spectroscopic characterization of the preparation revealed an isolate of trimeric and monomeric PSI complexes, which showed minimal unspecific contamination as demonstrated by comparison with the wild type control. Interestingly, we detected CP43 subunits of PSII and small amounts of PSII core complexes specifically pulled-down with the YFP-PSI, supporting the association of PSII assembly modules and intermediate assembly complexes with PSI, as observed in our previous studies. The results demonstrate that the GFP-Trap® system represents an excellent tool for studies of PSI biogenesis and interconnection of PSI and PSII assembly processes.

Deletion of CGLD1 Impairs PSII and Increases Singlet Oxygen Tolerance of Green Alga Chlamydomonas reinhardtii.

The green alga Chlamydomonas reinhardtii is a key model organism for studying photosynthesis and oxidative stress in unicellular eukaryotes. Using a forward genetics approach, we have identified and characterized a mutant x32, which lacks a predicted protein named CGLD1 (Conserved in Green Lineage and Diatom 1) in GreenCut2, under normal and stress conditions. We show that loss of CGLD1 resulted in minimal photoautotrophic growth and PSII activity in the organism. We observed reduced amount of PSII complex and core subunits in the x32 mutant based on blue-native (BN)/PAGE and immunoblot analysis. Moreover, x32 exhibited increased sensitivity to high-light stress and altered tolerance to different reactive oxygenic species (ROS) stress treatments, i.e., decreased resistance to H2O2/or tert-Butyl hydroperoxide (t-BOOH) and increased tolerance to neutral red (NR) and rose bengal (RB) that induce the formation of singlet oxygen, respectively. Further analysis via quantitative real-time PCR (qRT-PCR) indicated that the increased singlet-oxygen tolerance of x32 was largely correlated with up-regulated gene expression of glutathione-S-transferases (GST). The phenotypical and physiological implications revealed from our experiments highlight the important roles of CGLD1 in maintaining structure and function of PSII as well as in protection of Chlamydomonas under photo-oxidative stress conditions.

In the lycophyte Selaginella martensii is the “extra-qT” related to energy spillover? Insights into photoprotection in ancestral vascular plants

Lycophytes are early diverging vascular plants, representing a minor group as compared to the dominating euphyllophytes, mostly angiosperms. Having maximally developed in a CO2-rich atmosphere, extant lycophytes are characterized by a low carbon fixing capacity, which is compensated by a marked ability to induce the non-photochemical quenching of chlorophyll fluorescence (NPQ). Different kinetic components contribute to NPQ, in particular the fast relaxing high-energy quenching qE, the middle relaxing qT, and the slowly relaxing qI. Unlike angiosperms, lycophytes enhance the qT component under high light, originating from an “extra-qT”. In this research, we analyze whether in Selaginella martensii the extra-qT can reflect a photosystem (PS) I-based quenching mechanism activated upon saturation of qE capacity. From comparative analyses of fluorescence quenching parameters, carbon fixation, in vivo low- and room-temperature fluorescence spectroscopy, and thylakoid protein phosphorylation, it is proposed that the extra-qT is not mechanistically separate from the ordinary qT. The results suggest a relationship between qT and photoprotective energy spillover to PSI, which is activated upon sensing the excitation energy pressure inside PSII and is possibly facilitated by phosphorylation of Lhcb6, a minor antenna protein of PSII. Energy spillover emphasizes 77K fluorescence emission from PSI core (F714) and becomes more relevant at irradiance levels corresponding to the CO2-limited, potentially photoinhibiting phase of photosynthesis. At the highest irradiances, when Lhcb6 phosphorylation potential has been saturated, the major LHCII increases in turn its phosphorylation level, probably leading to the full exploitation of PSI as a safe excitation sink. It is suggested that the low photosynthetic capacity of lycophytes could allow an easier experimental access to the use of PSI as a safe excitation quencher for PSII, a debated, emerging issue about thylakoid photoprotection in angiosperms.

Proteomic characterization of hierarchical megacomplex formation in Arabidopsis thylakoid membrane.

Conversion of solar energy into chemical energy in plant chloroplasts concomitantly modifies the thylakoid architecture and hierarchical interactions between pigment-protein complexes. Here, the thylakoids were isolated from light-acclimated Arabidopsis leaves and investigated with respect to the composition of the thylakoid protein complexes and their association into higher molecular mass complexes, the largest one comprising both photosystems (PSII and PSI) and light-harvesting chlorophyll a/b-binding complexes (LHCII). Because the majority of plant light-harvesting capacity is accommodated in LHCII complexes, their structural interaction with photosystem core complexes is extremely important for efficient light harvesting. Specific differences in the strength of LHCII binding to PSII core complexes and the formation of PSII supercomplexes are well characterized. Yet, the role of loosely bound L-LHCII that disconnects to a large extent during the isolation of thylakoid protein complexes remains elusive. Because L-LHCII apparently has a flexible role in light harvesting and energy dissipation, depending on environmental conditions, its close interaction with photosystems is a prerequisite for successful light harvesting invivo. Here, to reveal the labile and fragile light-dependent protein interactions in the thylakoid network, isolated membranes were subjected to sequential solubilization using detergents with differential solubilization capacity and applying strict quality control. Optimized 3D-lpBN-lpBN-sodium dodecyl sulfate-polyacrylamide gel electrophoresis system demonstrated that PSII-LHCII supercomplexes, together with PSI complexes, hierarchically form larger megacomplexes via interactions with L-LHCII trimers. The polypeptide composition of LHCII trimers and the phosphorylation of Lhcb1 and Lhcb2 were examined to determine the light-dependent supramolecular organization of the photosystems into megacomplexes.