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Photosystem I is the most efficient photosynthetic enzyme with structure and composition highly conserved among all oxygenic phototrophs. Cyanobacterial Photosystem I is typically associated into trimers for reasons that are still debated. Almost universally, Photosystem I contains a number of long-wavelength-absorbing ‘red’ chlorophylls (Chls), that have a sizeable effect on the excitation energy transfer and trapping. Here we present spectroscopic comparison of trimeric Photosystem I from Synechocystis PCC 6803 with a monomeric complex from the ΔpsaL mutant and a ‘minimal’ monomeric complex ΔFIJL, containing only subunits A, B, C, D, E, K and M. The quantum yield of photochemistry at room temperature was the same in all complexes, demonstrating the functional robustness of this photosystem. The monomeric complexes had a reduced far-red absorption and emission equivalent to the loss of 1.5–2 red Chls emitting at 710–715 nm, whereas the longest-wavelength emission at 722 nm was not affected. The picosecond fluorescence kinetics at 77 K showed spectrally and kinetically distinct red Chls in all complexes and equilibration times of up to 50 ps. We found that the red Chls are not irreversible traps at 77 K but can still transfer excitations to the reaction centre, especially in the trimeric complexes. Structure-based Förster energy transfer calculations support the assignment of the lowest-energy state to the Chl pair B37/B38 and the trimer-specific red Chl emission to Chls A32/B7 located at the monomer–monomer interface. These intermediate-energy red Chls facilitate energy migration from the lowest-energy states to the reaction centre.

Photosystem II (PSII) uses solar energy to oxidize water and delivers electrons for life on Earth. The photochemical reaction center of PSII is known to possess two stationary states. In the open state (PSIIO), the absorption of a single photon triggers electron-transfer steps, which convert PSII into the charge-separated closed state (PSIIC). Here, by using steady-state and time-resolved spectroscopic techniques on Spinacia oleracea and Thermosynechococcus vulcanus preparations, we show that additional illumination gradually transforms PSIIC into a light-adapted charge-separated state (PSIIL). The PSIIC-to-PSIIL transition, observed at all temperatures between 80 and 308 K, is responsible for a large part of the variable chlorophyll-a fluorescence (Fv) and is associated with subtle, dark-reversible reorganizations in the core complexes, protein conformational changes at noncryogenic temperatures, and marked variations in the rates of photochemical and photophysical reactions. The build-up of PSIIL requires a series of light-induced events generating rapidly recombining primary radical pairs, spaced by sufficient waiting times between these events--pointing to the roles of local electric-field transients and dielectric relaxation processes. We show that the maximum fluorescence level, Fm, is associated with PSIIL rather than with PSIIC, and thus the Fv/Fm parameter cannot be equated with the quantum efficiency of PSII photochemistry. Our findings resolve the controversies and explain the peculiar features of chlorophyll-a fluorescence kinetics, a tool to monitor the functional activity and the structural-functional plasticity of PSII in different wild-types and mutant organisms and under stress conditions. VC American Society of Plant Biologists 2021. All rights reserved

Light-harvesting complex II (LHCII) is the major antenna complex in higher plants and green algae. It has been suggested that a major part of the excited state energy dissipation in the so-called "non-photochemical quenching" (NPQ) is located in this antenna complex. We have performed an ultrafast kinetics study of the low-energy fluorescent states related to quenching in LHCII in both aggregated and the crystalline form. In both sample types the chlorophyll (Chl) excited states of LHCII are strongly quenched in a similar fashion. Quenching is accompanied by the appearance of new far-red (FR) fluorescence bands from energetically low-lying Chl excited states. The kinetics of quenching, its temperature dependence down to 4 K, and the properties of the FR-emitting states are very similar both in LHCII aggregates and in the crystal. No such FR-emitting states are found in unquenched trimeric LHCII. We conclude that these states represent weakly emitting Chl-Chl charge-transfer (CT) states, whose formation is part of the quenching process. Quantum chemical calculations of the lowest energy exciton and CT states, explicitly including the coupling to the specific protein environment, provide detailed insight into the chemical nature of the CT states and the mechanism of CT quenching. The experimental data combined with the results of the calculations strongly suggest that the quenching mechanism consists of a sequence of two proton-coupled electron transfer steps involving the three quenching center Chls 610/611/612. The FR-emitting CT states are reaction intermediates in this sequence. The polarity-controlled internal reprotonation of the E175/K179 aa pair is suggested as the switch controlling quenching. A unified model is proposed that is able to explain all known conditions of quenching or non-quenching of LHCII, depending on the environment without invoking any major conformational changes of the protein.

The phycobilisome (PBS) serves as the major light-harvesting system, funnelling excitation energy to both photosystems (PS) in cyanobacteria and red algae. The picosecond kinetics involving the excitation energy transfer has been studied within the isolated systems and intact filaments of the cyanobacterium Anabaena variabilis PCC 7120. A target model is proposed which resolves the dynamics of the different chromophore groups. The energy transfer rate of 8.5 ± 1.0/ns from the rod to the core is the rate-limiting step, both in vivo and in vitro. The PBS-PSI-PSII supercomplex reveals efficient excitation energy migration from the low-energy allophycocyanin, which is the terminal emitter, in the PBS core to the chlorophyll a in the photosystems. The terminal emitter of the phycobilisome transfers energy to both PSI and PSII with a rate of 50 ± 10/ns, equally distributing the solar energy to both photosystems. Finally, the excitation energy is trapped by charge separation in the photosystems with trapping rates estimated to be 56 ± 6/ns in PSI and 14 ± 2/ns in PSII. Electronic supplementary material The online version of this article (10.1007/s11120-020-00723-0) contains supplementary material, which is available to authorized users.

Light-harvesting complex II (LHCII) from the marine green macroalga Bryopsis corticulans is spectroscopically characterized to understand the structural and functional changes resulting from adaptation to intertidal environment. LHCII is homologous to its counterpart in land plants but has a different carotenoid and chlorophyll (Chl) composition. This is reflected in the steady-state absorption, fluorescence, linear dichroism, circular dichroism and anisotropic circular dichroism spectra. Time-resolved fluorescence and two-dimensional electronic spectroscopy were used to investigate the consequences of this adaptive change in the pigment composition on the excited-state dynamics. The complex contains additional Chl b spectral forms - absorbing at around 650 nm and 658 nm - and lacks the red-most Chl a forms compared with higher-plant LHCII. Similar to plant LHCII, energy transfer between Chls occurs on timescales from under hundred fs (mainly from Chl b to Chl a) to several picoseconds (mainly between Chl a pools). However, the presence of long-lived, weakly coupled Chl b and Chl a states leads to slower exciton equilibration in LHCII from B. corticulans. The finding demonstrates a trade-off between the enhanced absorption of blue-green light and the excitation migration time. However, the adaptive change does not result in a significant drop in the overall photochemical efficiency of Photosystem II. These results show that LHCII is a robust adaptable system whose spectral properties can be tuned to the environment for optimal light harvesting. Ministry of Education (MOE) Published version The work is supported by grants from the Hungarian Ministry of Finance (GINOP-2.3.2-15-2016-00001), the National Research, Development and Innovation Fund (NKFI NN 124904; 2018-1.2.1-NKP-2018-00009), the Singapore Ministry of Education Academic Research Fund (MOE2015-T2-1-039), the National Key Research and Development Program of China (2017YFA0503700), a CAS Key Research Program for Frontier Science (QYZDY-SSW-SMC003), and the National Natural Science Foundation of China (31600191). CD measurements at the B23 beamline of the Diamond Light Source Ltd. were supported by the project CALIPSOplus under Grant Agreement 730872 from the EU Framework Programme for Research and Innovation HORIZON 2020.

Excitation energy transfer (EET) and trapping in Anabaena variabilis (PCC 7120) intact cells, isolated phycobilisomes (PBS) and photosystem I (PSI) complexes have been studied by picosecond time-resolved fluorescence spectroscopy at room temperature. Global analysis of the time-resolved fluorescence kinetics revealed two lifetimes of spectral equilibration in the isolated PBS, 30–35 ps and 110–130 ps, assigned primarily to energy transfer within the rods and between the rods and the allophycocyanin core, respectively. An additional intrinsic kinetic component with a lifetime of 500–700 ps was found, representing non-radiative decay or energy transfer in the core. Isolated tetrameric PSI complexes exhibited biexponential fluorescence decay kinetics with lifetimes of about 10 ps and 40 ps, representing equilibration between the bulk antenna chlorophylls with low-energy “red” states and trapping of the equilibrated excitations, respectively. The cascade of EET in the PBS and in PSI could be resolved in intact filaments as well. Virtually all energy absorbed by the PBS was transferred to the photosystems on a timescale of 180–190 ps. Electronic supplementary material The online version of this article (10.1007/s11120-020-00719-w) contains supplementary material, which is available to authorized users.

Photosystem I is a robust and highly efficient biological solar engine. Its capacity to utilize virtually every absorbed photon’s energy in a photochemical reaction generates great interest in the kinetics and mechanisms of excitation energy transfer and charge separation. In this work, we have employed room-temperature coherent two-dimensional electronic spectroscopy and time-resolved fluorescence spectroscopy to follow exciton equilibration and excitation trapping in intact Photosystem I complexes as well as core complexes isolated from Pisum sativum. We performed two-dimensional electronic spectroscopy measurements with low excitation pulse energies to record excited-state kinetics free from singlet–singlet annihilation. Global lifetime analysis resolved energy transfer and trapping lifetimes closely matches the time-correlated single-photon counting data. Exciton energy equilibration in the core antenna occurred on a timescale of 0.5 ps. We further observed spectral equilibration component in the core complex with a 3–4 ps lifetime between the bulk Chl states and a state absorbing at 700 nm. Trapping in the core complex occurred with a 20 ps lifetime, which in the supercomplex split into two lifetimes, 16 ps and 67–75 ps. The experimental data could be modelled with two alternative models resulting in equally good fits—a transfer-to-trap-limited model and a trap-limited model. However, the former model is only possible if the 3–4 ps component is ascribed to equilibration with a “red” core antenna pool absorbing at 700 nm. Conversely, if these low-energy states are identified with the P700 reaction centre, the transfer-to-trap-model is ruled out in favour of a trap-limited model. MOE (Min. of Education, S’pore) Accepted version

The light reactions of photosynthesis are hosted and regulated by the chloroplast thylakoid membrane (TM) — the central structural component of the photosynthetic apparatus of plants and algae. The two-dimensional and three-dimensional arrangement of the lipid–protein assemblies, aka macroorganisation, and its dynamic responses to the fluctuating physiological environment, aka flexibility, are the subject of this review. An emphasis is given on the information obtainable by spectroscopic approaches, especially circular dichroism (CD). We briefly summarise the current knowledge of the composition and three-dimensional architecture of the granal TMs in plants and the supramolecular organisation of Photosystem II and light-harvesting complex II therein. We next acquaint the non-specialist reader with the fundamentals of CD spectroscopy, recent advances such as anisotropic CD, and applications for studying the structure and macroorganisation of photosynthetic complexes and membranes. Special attention is given to the structural and functional flexibility of light-harvesting complex II in vitro as revealed by CD and fluorescence spectroscopy. We give an account of the dynamic changes in membrane macroorganisation associated with the light-adaptation of the photosynthetic apparatus and the regulation of the excitation energy flow by state transitions and non-photochemical quenching.

During the past two decades, two-dimensional electronic spectroscopy (2DES) and related techniques have emerged as a potent experimental toolset to study the ultrafast elementary steps of photosynthesis. Apart from the highly engaging albeit controversial analysis of the role of quantum coherences in the photosynthetic processes, 2DES has been applied to resolve the dynamics and pathways of energy and electron transport in various light-harvesting antenna systems and reaction centres, providing unsurpassed level of detail. In this paper we discuss the main technical approaches and their applicability for solving specific problems in photosynthesis. We then recount applications of 2DES to study the exciton dynamics in plant and photosynthetic light-harvesting complexes, especially light-harvesting complex II (LHCII) and the fucoxanthin-chlorophyll proteins of diatoms, with emphasis on the types of unique information about such systems that 2DES is capable to deliver. This article is part of a Special Issue entitled Light harvesting, edited by Dr. Roberta Croce.

International audience; 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 (Magyar et al. 2018). 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 2 ns (~60%) and 10 ns (~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. This article is protected by copyright. All rights reserved.

In higher-plant Photosystem I (PSI), the majority of “red” chlorophylls (absorbing at longer wavelengths than the reaction centre P700) are located in the peripheral antenna, but contradicting reports are given about red forms in the core complex. Here we attempt to clarify the spectroscopic characteristics and quantify the red forms in the PSI core complex, which have profound implication on understanding the energy transfer and charge separation dynamics. To this end we compare the steady-state absorption and fluorescence spectra and picosecond time-resolved fluorescence kinetics of isolated PSI core complex and PSI–LHCI supercomplex from Pisum sativum recorded at 77 K. Gaussian decomposition of the absorption spectra revealed a broad band at 705 nm in the core complex with an oscillator strength of three chlorophylls. Additional absorption at 703 nm and 711 nm in PSI–LHCI indicated up to five red chlorophylls in the peripheral antenna. Analysis of fluorescence emission spectra resolved states emitting at 705, 715 and 722 nm in the core and additional states around 705–710 nm and 733 nm in PSI–LHCI. The red states compete with P700 in trapping excitations in the bulk antenna, which occurs on a timescale of ~20 ps. The three red forms in the core have distinct decay kinetics, probably in part determined by the rate of quenching by the oxidized P700. These results affirm that the red chlorophylls in the core complex must not be neglected when interpreting kinetic experimental results of PSI.

Diatoms possess special light-harvesting proteins involved in the photoprotection mechanism called non-photochemical quenching (NPQ). These Lhcx proteins were shown to be subunits of trimeric fucoxanthin-chlorophyll complexes (FCPa) in centric diatoms, but their mode of action is still unclear. Here we investigated the influence of Fcp6, an orthologue to Lhcx1 of Thalassiosira pseudonana in the diatom Cyclotella meneghiniana, by reducing its amount using an antisense approach. Whereas the pigment interactions inside FCPa were not influenced by the presence or absence of Fcp6, as demonstrated by unaltered spectra of circular dichroism, changes could be observed on the level of thylakoids and cells in the mutants compared to WT. This fits to recent models of NPQ in diatoms, where FCP aggregation or supramolecular reorganisation is thought to be a major feature. Thus, Fcp6 (Lhcx1) appears to alter pigment-pigment interactions inside the aggregates, but not inside (un-aggregated) FCPa itself.

Extraction of plant light-harvesting complex II (LHCII) from the native thylakoid membrane or from aggregates by the use of surfactants brings about significant changes in the excitonic circular dichroism (CD) spectrum and fluorescence quantum yield. To elucidate the cause of these changes, e.g. trimer-trimer contacts or surfactant-induced structural perturbations, we compared the CD spectra and fluorescence kinetics of LHCII aggregates, artificial and native LHCII-lipid membranes, and LHCII solubilized in different detergents or trapped in polymer gel. By this means we were able to identify CD spectral changes specific to LHCII-LHCII interactions, at (-)-437 and (+)-484 nm, and changes specific to the interaction with the detergent n-dodecyl-β-maltoside (β-DM) or membrane lipids, at (+)-447 and (-)-494 nm. The latter change is attributed to the conformational change of the LHCII-bound carotenoid neoxanthin, by analyzing the CD spectra of neoxanthin-deficient plant thylakoid membranes. The neoxanthin-specific band at (-)-494 nm was not pronounced in LHCII in detergent-free gels or solubilized in the α isomer of DM but was present when LHCII was reconstituted in membranes composed of phosphatidylcholine or plant thylakoid lipids, indicating that the conformation of neoxanthin is sensitive to the molecular environment. Neither the aggregation-specific CD bands, nor the surfactant-specific bands were positively associated with the onset of fluorescence quenching, which could be triggered without invoking such spectral changes. Significant quenching was not active in reconstituted LHCII proteoliposomes, whereas a high degree of energetic connectivity, depending on the lipid:protein ratio, in these membranes allows for efficient light harvesting.

Light-harvesting complex II (LHCII), the major peripheral antenna of Photosystem II in plants, participates in several concerted mechanisms for regulation of the excitation energy and electron fluxes in thylakoid membranes. In part, these include interaction of LHCII with Photosystem I (PSI) enhancing the latter's absorption cross-section – for example in the well-known state 1 – state 2 transitions or as a long-term acclimation to high light. In this work we examined the capability of LHCII to deliver excitations to PSI in reconstituted membranes in vitro. Proteoliposomes with native plant thylakoid membrane lipids and different stoichiometric ratios of LHCII:PSI were reconstituted and studied by steady-state and time-resolved fluorescence spectroscopy. Fluorescence emission from LHCII was strongly decreased in PSI–LHCII membranes due to trapping of excitations by PSI. Kinetic modelling of the time-resolved fluorescence data revealed the existence of separate pools of LHCII distinguished by the time scale of energy transfer. A strongly coupled pool, equivalent to one LHCII trimer per PSI, transferred excitations to PSI with near-unity efficiency on a time scale of less than 10 ps but extra LHCIIs also contributed significantly to the effective antenna size of PSI, which could be increased by up to 47% in membranes containing 3 LHCII trimers per PSI. The results demonstrate a remarkable competence of LHCII to increase the absorption cross-section of PSI, given the opportunity that the two types of complexes interact in the membrane.

The functional domain size for efficient excited singlet state quenching was studied in artificial aggregates of the main light-harvesting complex II (LHCIIb) from spinach and in native thylakoid membranes by picosecond time-resolved fluorescence spectroscopy and quantum yield measurements. The domain size was estimated from the efficiency of added exogenous singlet excitation quenchers—phenyl- p -benzoquinone (PPQ) and dinitrobenzene (DNB). The mean fluorescence lifetimes τ av were quantified for a range of quencher concentrations. Applying the Stern–Volmer formalism, apparent quenching rate constants k q were determined from the dependencies on quencher concentration of the ratio τ 0 av / τ av , where τ 0 av is the average fluorescence lifetime of the sample without addition of an exogenous quencher. The functional domain size was gathered from the ratio k q ′/ k q , i.e., the apparent quenching rate constants determined in aggregates (or membranes), k q ′, and in detergent-solubilised LHCII trimers, k q , respectively. In LHCII macroaggregates, the resulting values for the domain size were 15–30 LHCII trimers. In native thylakoid membranes the domain size was equivalent to 12–24 LHCII trimers, corresponding to 500–1000 chlorophylls. Virtually the same results were obtained when membranes were suspended in buffers promoting either membrane stacking or destacking. These domain sizes are orders of magnitude smaller than the number of physically connected pigment–protein complexes. Therefore our results imply that the physical size of an antenna system beyond the numbers of a functional domain size has little or no effect on improving the light-harvesting efficiency.

In photosynthesis research, circular dichroism (CD) spectroscopy is an indispensable tool to probe molecular architecture at virtually all levels of structural complexity. At the molecular level, the chirality of the molecule results in intrinsic CD; pigment-pigment interactions in protein complexes and small aggregates can give rise to excitonic CD bands, while "psi-type" CD signals originate from large, densely packed chiral aggregates. It has been well established that anisotropic CD (ACD), measured on samples with defined non-random orientation relative to the propagation of the measuring beam, carries specific information on the architecture of molecules or molecular macroassemblies. However, ACD is usually combined with linear dichroism and can be distorted by instrumental imperfections, which given the strong anisotropic nature of photosynthetic membranes and complexes, might be the reason why ACD is rarely studied in photosynthesis research. In this study, we present ACD spectra, corrected for linear dichroism, of isolated intact thylakoid membranes of granal chloroplasts, washed unstacked thylakoid membranes, photosystem II (PSII) membranes (BBY particles), grana patches, and tightly stacked lamellar macroaggregates of the main light-harvesting complex of PSII (LHCII). We show that the ACD spectra of face- and edge-aligned stacked thylakoid membranes and LHCII lamellae exhibit profound differences in their psi-type CD bands. Marked differences are also seen in the excitonic CD of BBY and washed thylakoid membranes. Magnetic CD (MCD) spectra on random and aligned samples, and the largely invariable nature of the MCD spectra, despite dramatic variations in the measured isotropic and anisotropic CD, testify that ACD can be measured without substantial distortions and thus employed to extract detailed information on the (supra)molecular organization of photosynthetic complexes. An example is provided showing the ability of CD data to indicate such an organization, leading to the discovery of a novel crystalline structure in macroaggregates of LHCII.

The induction and relaxation of non-photochemical quenching (NPQ) under steady-state conditions, i.e. during up to 90min of illumination at saturating light intensities, was studied in Arabidopsis thaliana. Besides the well-characterized fast qE and the very slow qI component of NPQ, the analysis of the NPQ dynamics identified a zeaxanthin (Zx) dependent component which we term qZ. The formation (rise time 10–15min) and relaxation (lifetime 10–15min) of qZ correlated with the synthesis and epoxidation of Zx, respectively. Comparative analysis of different NPQ mutants from Arabidopsis showed that qZ was clearly not related to qE, qT or qI and thus represents a separate, Zx-dependent NPQ component.

The diatom algae, responsible for at least a quarter of the global photosynthetic carbon assimilation in the oceans, are capable of switching on rapid and efficient photoprotection, which helps them cope with the large fluctuations of light intensity in the moving waters. The enhanced dissipation of excess excitation energy becomes visible as non-photochemical quenching (NPQ) of chlorophyll a fluorescence. Intact cells of the diatoms Cyclotella meneghiniana and Phaeodactylum tricornutum, which show different NPQ induction kinetics under high light illumination, were investigated by picosecond time-resolved fluorescence under dark and NPQ-inducing high light conditions. The fluorescence kinetics revealed that there are two independent sites responsible for NPQ. The first quenching site is located in an FCP antenna system that is functionally detached from both photosystems, while the second quenching site is located in the PSII-attached antenna. Notwithstanding their different npq induction and reversal kinetics, both diatoms showed identical NPQ via both mechanisms in the steady-state. Their fluorescence decays in the dark-adapted states were different, however. A detailed quenching model is proposed for NPQ in diatoms.

Higher plant thylakoid membranes contain a protein kinase that phosphorylates certain threonine residues of light-harvesting complex II (LHCII), the main light-harvesting antenna complexes of photosystem II (PSII) and some other phosphoproteins (Allen, Biochim Biophys Acta 1098:275, 1992). While it has been established that phosphorylation induces a conformational change of LHCII and also brings about changes in the lateral organization of the thylakoid membrane, it is not clear how phosphorylation affects the dynamic architecture of the thylakoid membranes. In order to contribute to the elucidation of this complex question, we have investigated the effect of duroquinol-induced phosphorylation on the membrane ultrastructure and the thermal and light stability of the chiral macrodomains and of the trimeric organization of LHCII. As shown by small angle neutron scattering on thylakoid membranes, duroquinol treatment induced a moderate (~10%) increase in the repeat distance of stroma membranes, and phosphorylation caused an additional loss of the scattering intensity, which is probably associated with the partial unstacking of the granum membranes. Circular dichroism (CD) measurements also revealed only minor changes in the chiral macro-organization of the complexes and in the oligomerization state of LHCII. However, temperature dependences of characteristic CD bands showed that phosphorylation significantly decreased the thermal stability of the chiral macrodomains in phosphorylated compared to the non-phosphorylated samples (in leaves and isolated thylakoid membranes, from 48.3 degrees C to 42.6 degrees C and from 47.5 degrees C to 44.3 degrees C, respectively). As shown by non-denaturing PAGE of thylakoid membranes and CD spectroscopy on EDTA washed membranes, phosphorylation decreased by about 5 degrees C, the trimer-to-monomer transition temperature of LHCII. It also enhanced the light-induced disassembly of the chiral macrodomains and the monomerization of the LHCII trimers at 25 degrees C. These data strongly suggest that phosphorylation of the membranes considerably facilitates the heat- and light-inducible reorganizations in the thylakoid membranes and thus enhances the structural flexibility of the membrane architecture.

The major chlorophyll (Chl) a/b complexes of photosystem II (LHCIIb), in addition to their primary light-harvesting function, play key roles in the organization of the granal ultrastructure of the thylakoid membranes and in various regulatory processes. These functions depend on the structural stability and flexibility of the complexes. The lumenal side of LHCIIb is exposed to broadly variable pH environments, due to the build-up and decay of the pH gradient during photosynthesis. Therefore, the negatively charged amino acids in the lumenal loop might be of paramount importance for adjusting the structure and functions of LHCIIb. In order to clarify the structural roles of these residues, we investigated the pigment stoichiometries, absorption, linear and circular dichroism spectra of the reconstituted LHCIIb complexes, in which the negatively charged amino acids in the lumenal loop were exchanged to neutral ones (E94G, E107V and D111V). The mutations influenced the pigment binding and the molecular architecture of the complexes. Exchanging E94 to G destabilized the 310 helix in the lumenal loop structure and led to an acquired pH sensitivity of the LHCIIb structure. We conclude that these amino acids are important not only for pigment binding in the complexes, but also in stabilizing the conformation of LHCIIb at different pHs.

Using the mass-measuring capability of scanning transmission electron microscopy, we demonstrate that membrane crystals of the main light-harvesting complex of plants possess the ability to undergo light-induced dark-reversible disassociations, independently of the photochemical apparatus. This is the first direct visualization of light-driven reversible reorganizations in an isolated photosynthetic antenna. These reorganizations, identified earlier by circular dichroism (CD), can be accounted for by a biological thermo-optic transition: structural changes are induced by fast heat transients and thermal instabilities near the dissipation, and self-association of the complexes in the lipid matrix. A comparable process in native membranes is indicated by earlier findings of essentially identical kinetics, and intensity and temperature dependences of the ΔCD in granal thylakoids.

Diatoms possess effective photoprotection mechanisms, which may involve reorganizations in the photosynthetic machinery. We have shown earlier, by using circular dichroism (CD) spectroscopy, that in Phaeodactylum tricornutum the pigment–protein complexes are arranged into chiral macrodomains, which have been proposed to be associated with the multilamellar organization of the thylakoid membranes and shown to be capable of undergoing light-induced reversible reorganizations (Szabo et al. Photosynth Res 95:237, 2008). Recently, by using small-angle neutron scattering (SANS) on the same algal cells we have determined the repeat distances and revealed reversible light-induced reorganizations in the lamellar order of thylakoids (Nagy et al. Biochem J 436:225, 2011). In this study, we show that in moderately heat-treated samples, the weakening of the lamellar order is accompanied by the diminishment of the psi-type CD signal associated with the long-range chiral order of the chromophores (psi, polymer or salt-induced). Further, we show that the light-induced reversible increase in the psi-type CD is associated with swelling in the membrane system, with magnitudes larger in high light than in low light. In contrast, shrinkage of the membrane system, induced by sorbitol, brings about a decrease in the psi-type CD signal; this shrinkage also diminishes the non-photochemical quenching capability of the cells. These data shed light on the origin of the psi-type CD signal, and confirm that both CD spectroscopy and SANS provide valuable information on the macro-organization of the thylakoid membranes and their dynamic properties; these parameters are evidently of interest with regard to the photoprotection in whole algal cells.

The kinetics of non-photochemical quenching (NPQ) of chlorophyll fluorescence was studied in pea leaves at different temperatures between 5 and 25 degrees C and during rapid jumps of the leaf temperature. At 5 degrees C, NPQ relaxed very slowly in the dark and was sustained for up to 30 min. This was independent of the temperature at which quenching was induced. Upon raising the temperature to 25 degrees C, the quenched state relaxed within 1 min, characteristic for qE, the energy-dependent component of NPQ. Measurements of the membrane permeability (delta A515) in dark-adapted and preilluminated leaves and NPQ in the presence of dithiothreitol strongly suggest that the effect of low temperature on NPQ was not because of limitation by the lumenal pH or the de-epoxidation state of the xanthophylls. These data are consistent with the notion that the transition from the quenched to the unquenched state and vice versa involves a structural reorganization in the photosynthetic apparatus. An eight-state reaction scheme for NPQ is proposed, extending the model of Horton and co-workers (FEBS Lett 579:4201-4206, 2005), and a hypothesis is put forward concerning the nature of conformational changes associated with qE.

Aggregates and solubilized trimers of LHCII were characterized by circular dichroism (CD), linear dichroism and time-resolved fluorescence spectroscopy and compared with thylakoid membranes in order to evaluate the native state of LHCII in vivo. It was found that the CD spectra of lamellar aggregates closely resemble those of unstacked thylakoid membranes whereas the spectra of trimers solubilized in n-dodecyl-β,d-maltoside, n-octyl-β,d-glucopyranoside, or Triton X-100 were drastically different in the Soret region. Thylakoid membranes or LHCII aggregates solubilized with detergent exhibited CD spectra similar to the isolated trimers. Solubilization of LHCII was accompanied by profound changes in the linear dichroism and increase in fluorescence lifetime. These data support the notion that lamellar aggregates of LHCII retain the native organization of LHCII in the thylakoid membranes. The results indicate that the supramolecular organization of LHCII, most likely due to specific trimer–trimer contacts, has significant impact on the pigment interactions in the complexes.

Delayed fluorescence dark decays in the time interval from 0.35 to 5.5ms are measured during dark to light adaptation in whole barley leaves and isolated thylakoid membranes, using a disc phosphoroscope. The changes in delayed fluorescence features are compared with variable chlorophyll fluorescence simultaneously registered with the same apparatus as well as in parallel by Handy PEA (Hansatech Instruments Ltd.), and absorbance changes at 820 nm. The registered delayed fluorescence signal is a sum of three components – submillisecond with lifetime of about 0.6 ms, millisecond decayed 2–4 ms and slow component with lifetime > >5.5 ms. The submillisecond delayed fluorescence component is proposed to be a result of radiative charge recombination in Photosystem II reaction centers in the state Z+PQ − Q − , and its lifetime is determined by the rate of electron transfer from Q − to Q − . The millisecond delayed fluorescence component is associated with recombination in Z+PQ − Q = centers with a lifetime determined by the sum of the rate constants of electron transfer from the oxygen-evolving complex to Z+ and of the exchange between the reduced and oxidized plastoquinone pool in the Q B -site. On the basis of these assumptions and of the different share of the three components in the integral delayed fluorescence during induction, an attempt has been made to interpret the changes in the delayed fluorescence intensity during the transition of the photosynthetic apparatus from dark to light adapted state.

We studied the periodicity of the multilamellar membrane system of granal chloroplasts in different isolated plant thylakoid membranes, using different suspension media, as well as on different detached leaves and isolated protoplasts—using small-angle neutron scattering. Freshly isolated thylakoid membranes suspended in isotonic or hypertonic media, containing sorbitol supplemented with cations, displayed Bragg peaks typically between 0.019 and 0.023 A − 1 , corresponding to spatially and statistically averaged repeat distance values of about 275–330 A. Similar data obtained earlier led us in previous work to propose an origin from the periodicity of stroma thylakoid membranes. However, detached leaves, of eleven different species, infiltrated with or soaked in D 2 O in dim laboratory light or transpired with D 2 O prior to measurements, exhibited considerably smaller repeat distances, typically between 210 and 230 A, ruling out a stromal membrane origin. Similar values were obtained on isolated tobacco and spinach protoplasts. When NaCl was used as osmoticum, the Bragg peaks of isolated thylakoid membranes almost coincided with those in the same batch of leaves and the repeat distances were very close to the electron microscopically determined values in the grana. Although neutron scattering and electron microscopy yield somewhat different values, which is not fully understood, we can conclude that small-angle neutron scattering is a suitable technique to study the periodic organization of granal thylakoid membranes in intact leaves under physiological conditions and with a time resolution of minutes or shorter. We also show here, for the first time on leaves, that the periodicity of thylakoid membranes in situ responds dynamically to moderately strong illumination. This article is part of a Special Issue entitled: Photosynthesis research for sustainability: Keys to produce clean energy.