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Abstract: Chloroplast thylakoid membranes accommodate densely packed protein complexes in ordered, often semi-crystalline arrays and are assembled into highly organized multilamellar systems, an organization warranting a substantial degree of stability. At the same time, they exhibit remarkable structural flexibility, which appears to play important – yet not fully understood – roles in different short-term adaptation mechanisms in response to rapidly changing environmental conditions. In this review I will focus on dynamic features of the hierarchically organized photosynthetic machineries at different levels of structural complexity: (i) isolated light harvesting complexes, (ii) molecular macroassemblies and supercomplexes, (iii) thylakoid membranes and (iv) their multilamellar membrane systems. Special attention will be paid to the most abundant systems, the major light harvesting antenna complex, LHCII, and to grana. Two physical mechanisms, which are less frequently treated in the literature, will receive special attention: (i) thermo-optic mechanism —elementary structural changes elicited by ultrafast local heat transients due to the dissipation of photon energy, which operates both in isolated antenna assemblies and the native thylakoid membranes, regulates important enzymatic functions and appears to play role in light adaptation and photoprotection mechanisms; and (ii) the mechanism by which non-bilayer lipids and lipid phases play key role in the functioning of xanthophyll cycle de-epoxidases and are proposed to regulate the protein-to-lipid ratio in thylakoid membranes and contribute to membrane dynamics. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: Electron transport processes were investigated in barley leaves in which the oxygen-evolution was fully inhibited by a heat pulse (48 °C, 40 s). Under these circumstances, the K peak (∼ F 400 μs) appears in the chl a fluorescence (OJIP) transient reflecting partial QA reduction, which is due to a stable charge separation resulting from the donation of one electron by tyrozine Z. Following the K peak additional fluorescence increase (indicating QA − accumulation) occurs in the 0.2–2 s time range. Using simultaneous chl a fluorescence and 820 nm transmission measurements it is demonstrated that this QA − accumulation is due to naturally occurring alternative electron sources that donate electrons to the donor side of photosystem II. Chl a fluorescence data obtained with 5-ms light pulses (double flashes spaced 2.3–500 ms apart, and trains of several hundred flashes spaced by 100 or 200 ms) show that the electron donation occurs from a large pool with t 1/2 ∼30 ms. This alternative electron donor is most probably ascorbate. [Copyright &y& Elsevier]

Energization of thylakoid membranes brings about the acidification of the lumenal aqueous phase, which activates important regulatory mechanisms. Earlier Jajoo and coworkers (2014 FEBS Lett. 588:970) have shown that low pH in isolated plant thylakoid membranes induces changes in the excitation energy distribution between the two photosystems. In order to elucidate the structural background of these changes, we used small-angle neutron scattering on thylakoid membranes exposed to low p 2 H (pD) and show that gradually lowering the p 2 H from 8.0 to 5.0 causes small but well discernible reversible diminishment of the periodic order and the lamellar repeat distance and an increased mosaicity — similar to the effects elicited by light-induced acidification of the lumen. Our data strongly suggest that thylakoids dynamically respond to the membrane energization and actively participate in different regulatory mechanisms. [ABSTRACT FROM AUTHOR]

Macro-organisation of the protein complexes in plant thylakoid membranes plays important roles in the regulation and fine-tuning of photosynthetic activity. These delicate structures might, however, undergo substantial changes during isolating the thylakoid membranes or during sample preparations, e.g., for electron microscopy. Circular-dichroism (CD) spectroscopy is a non-invasive technique which can thus be used on intact samples. Via excitonic and psi-type CD bands, respectively, it carries information on short-range excitonic pigment–pigment interactions and the macro-organisation (chiral macrodomains) of pigment–protein complexes (psi, polymer or salt-induced). In order to obtain more specific information on the origin of the major psi-type CD bands, at around (+)506, (−)674 and (+)690 nm, we fingerprinted detached leaves and isolated thylakoid membranes of wild-type and mutant plants and also tested the effects of different environmental conditions in vivo. We show that (i) the chiral macrodomains disassemble upon mild detergent treatments, but not after crosslinking the protein complexes; (ii) in different wild-type leaves of dicotyledonous and monocotyledonous angiosperms the CD features are quite robust, displaying very similar excitonic and psi-type bands, suggesting similar protein composition and (macro-) organisation of photosystem II (PSII) supercomplexes in the grana; (iii) the main positive psi-type bands depend on light-harvesting protein II contents of the membranes; (iv) the (+)506 nm band appears only in the presence of PSII–LHCII supercomplexes and does not depend on the xanthophyll composition of the membranes. Hence, CD spectroscopy can be used to detect different macro-domains in the thylakoid membranes with different outer antenna compositions in vivo. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In photosynthetic organisms, carotenoids (carotenes and xanthophylls) are important for light harvesting, photoprotection and structural stability of a variety of pigment–protein complexes. Here, we investigated the consequences of altered carotenoid composition for the functional organization of photosynthetic complexes in wild-type and various mutant strains of the cyanobacterium Synechocystis sp. PCC 6803. Although it is generally accepted that xanthophylls do not play a role in cyanobacterial photosynthesis in low-light conditions, we have found that the absence of xanthophylls leads to reduced oligomerization of photosystems I and II. This is remarkable because these complexes do not bind xanthophylls. Oligomerization is even more disturbed in crtH mutant cells, which show limited carotenoid synthesis; in these cells also the phycobilisomes are distorted despite the fact that these extramembranous light-harvesting complexes do not contain carotenoids. The number of phycocyanin rods connected to the phycobilisome core is strongly reduced leading to high amounts of unattached phycocyanin units. In the absence of carotenoids the overall organization of the thylakoid membranes is disturbed: Photosystem II is not formed, photosystem I hardly oligomerizes and the assembly of phycobilisomes remains incomplete. These data underline the importance of carotenoids in the structural and functional organization of the cyanobacterial photosynthetic machinery. [ABSTRACT FROM AUTHOR]

Abstract: By using low temperature fluorescence spectroscopy, it has been shown that exposing chloroplast thylakoid membranes to acidic pH reversibly decreases the fluorescence of photosystem II while the fluorescence of photosystem I increases [P. Singh-Rawal et al. (2010) Evidence that pH can drive state transitions in isolated thylakoid membranes from spinach, Photochem Photobiol Sci, 9 830–837]. In order to shed light on the origin of these changes, we performed circular dichroism (CD) spectroscopy on freshly isolated pea thylakoid membranes. We show that the magnitude of the psi-type CD, which is associated with the presence of chirally ordered macroarrays of the chromophores in intact thylakoid membranes, decreases gradually and reversibly upon gradually lowering the pH of the medium from 7.5 to 4.5 (psi, polymer or salt induced). The same treatment, as shown on thylakoid membranes washed in hypotonic low salt medium possessing no psi-type bands, induces no discernible change in the excitonic CD. These data show that while no change in the pigment–pigment interactions and thus in the molecular organization of the bulk protein complexes can be held responsible for the observed changes in the fluorescence, acidification of the medium significantly alters the macro-organization of the complexes, hence providing an explanation for the pH-induced redistribution of the excitation energy between the two photosystems. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial. [Copyright &y& Elsevier]

Abstract: The multilamellar organization of freshly isolated spinach and pea chloroplast thylakoid membranes was studied using small-angle neutron scattering. A broad peak at ~0.02Å−1 is ascribed to diffraction from domains of ordered, unappressed stroma lamellae, revealing a repeat distance of 294ű7Å in spinach and 345ű11Å in pea. The peak position and hence the repeat distance of stroma lamellae is strongly dependent on the osmolarity and the ionic strength of the suspension medium, as demonstrated by varying the sorbitol and the Mg++-concentration in the sample. For pea thylakoid membranes, we show that the repeat distance decreases when illuminating the sample with white light, in accordance with our earlier results on spinach, also regarding the observation that addition of an uncoupler prohibits the light-induced structural changes, a strong indication that these changes are driven by the transmembrane proton gradient. We show that the magnitude of the shrinkage is strongly dependent on light intensity and that the repeat distance characteristic of the dark state after illumination is different from the initial dark state. Prolonged strong illumination leads to irreversible changes and swelling as reflected in increased repeat distances. The observed reorganizations are discussed within the frames of the current structural models of the granum-stroma thylakoid membrane assembly and the regulatory mechanisms in response to variations in the environmental conditions in vivo. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial. [Copyright &y& Elsevier]

Abstract: Experiments were carried out to identify a process co-determining with QA the fluorescence rise between F0 and FM. With 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU), the fluorescence rise is sigmoidal, in its absence it is not. Lowering the temperature to −10°C the sigmoidicity is lost. It is shown that the sigmoidicity is due to the kinetic overlap between the reduction kinetics of QA and a second process; an overlap that disappears at low temperature because the temperature dependences of the two processes differ. This second process can still relax at −60°C where recombination between QA − and the donor side of photosystem (PS) II is blocked. This suggests that it is not a redox reaction but a conformational change can explain the data. Without DCMU, a reduced photosynthetic electron transport chain (ETC) is a pre-condition for reaching the FM. About 40% of the variable fluorescence relaxes in 100ms. Re-induction while the ETC is still reduced takes a few ms and this is a photochemical process. The fact that the process can relax and be re-induced in the absence of changes in the redox state of the plastoquinone (PQ) pool implies that it is unrelated to the QB-occupancy state and PQ-pool quenching. In both +/−DCMU the process studied represents ~30% of the fluorescence rise. The presented observations are best described within a conformational protein relaxation concept. In untreated leaves we assume that conformational changes are only induced when QA is reduced and relax rapidly on re-oxidation. This would explain the relationship between the fluorescence rise and the ETC-reduction. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: By using absorption and fluorescence spectroscopy, we compared the effects of heat and light treatments on the phycobilisome (PBS) antenna of Synechocystis sp. PCC 6803 cells. Fluorescence emission spectra obtained upon exciting predominantly PBS, recorded at 25 °C and 77 K, revealed characteristic changes upon heat treatment of the cells. A 5-min incubation at 50 °C, which completely inactivated the activity of photosystem II, led to a small but statistically significant decrease in the F 680/F 655 fluorescence intensity ratio. In contrast, heat treatment at 60 °C resulted in a much larger decrease in the same ratio and was accompanied by a blue-shift of the main PBS emission band at around 655 nm (F 655), indicating an energetic decoupling of PBS from chlorophylls and reorganizations in its internal structure. (Upon exciting PBS, F 680 originates from photosystem II and from the terminal emitter of PBS.). Very similar changes were obtained upon exposing the cells to high light (600–7500 μmol photons m−2 s−1) for different time periods (10 min to 3 h). In cells with heat-inactivated photosystem II, the variations caused by light treatment could clearly be assigned to a similar energetic decoupling of the PBS from the membrane and internal reorganizations as induced at around 60 °C. These data can be explained within the frameworks of thermo-optic mechanism [Cseh et al. 2000, Biochemistry 39, 15250]: in high light the heat packages originating from dissipation might lead to elementary structural changes in the close vicinity of dissipation in heat-sensitive structural elements, e.g. around the site where PBS is anchored to the membrane. This, in turn, brings about a diminishment in the energy supply from PBS to the photosystems and reorganization in the molecular architecture of PBS. [Copyright &y& Elsevier]

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. • Absorption, fluorescence, linear and circular dichroism spectra were recorded. • Additional chlorophyll b spectral forms were identified compared with plant LHCII. • Energy transfer takes place in hundreds of femtoseconds to tens of picoseconds. • Weakly coupled chlorophylls slow down the excitation migration. • LHCII is a spectrally tunable antenna. [ABSTRACT FROM AUTHOR]