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Oxygenic photosynthesis is driven by light absorption in photosystem I (PSI) and photosystem II (PSII). A balanced excitation pressure between PSI and PSII is required for optimal photosynthetic efficiency. State transitions serve to keep this balance. If PSII is overexcited in plants and green algae, a mobile pool of light-harvesting complex II (LHCII) associates with PSI, increasing its absorption cross-section and restoring the excitation balance. This is called state 2. Upon PSI overexcitation, this LHCII pool moves to PSII, leading to state 1. Whether the association/dissociation of LHCII with the photosystems occurs between thylakoid grana and thylakoid stroma lamellae during state transitions or within the same thylakoid region remains unclear. Furthermore, although state transitions are thought to be accompanied by changes in thylakoid macro-organization, this has never been observed directly in functional leaves. In this work, we used confocal fluorescence lifetime imaging to quantify state transitions in single Arabidopsis (Arabidopsis thaliana) chloroplasts in folio with sub-micrometer spatial resolution. The change in excitation-energy distribution between PSI and PSII was investigated at a range of excitation wavelengths between 475 and 665 nm. For all excitation wavelengths, the PSI/(PSI + PSII) excitation ratio was higher in state 2 than in state 1. We next imaged the local PSI/(PSI + PSII) excitation ratio for single chloroplasts in both states. The data indicated that LHCII indeed migrates between the grana and stroma lamellae during state transitions. Finally, fluorescence intensity images revealed that thylakoid macro-organization is largely unaffected by state transitions. This single chloroplast in folio imaging method will help in understanding how plants adjust their photosynthetic machinery to ever-changing light conditions., Competing Interests: Conflict of interest statement. The authors declare no conflict of interest., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Photosynthetic light-harvesting antennae are pigment-binding proteins that perform one of the most fundamental tasks on Earth, capturing light and transferring energy that enables life in our biosphere. Adaptation to different light environments led to the evolution of an astonishing diversity of light-harvesting systems. At the same time, several strategies have been developed to optimize the light energy input into photosynthetic membranes in response to fluctuating conditions. The basic feature of these prompt responses is the dynamic nature of antenna complexes, whose function readily adapts to the light available. High-resolution microscopy and spectroscopic studies on membrane dynamics demonstrate the crosstalk between antennae and other thylakoid membrane components. With the increased understanding of light-harvesting mechanisms and their regulation, efforts are focusing on the development of sustainable processes for effective conversion of sunlight into functional bio-products. The major challenge in this approach lies in the application of fundamental discoveries in light-harvesting systems for the improvement of plant or algal photosynthesis. Here, we underline some of the latest fundamental discoveries on the molecular mechanisms and regulation of light harvesting that can potentially be exploited for the optimization of photosynthesis., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Light drives photosynthesis. In plants it is absorbed by light-harvesting antenna complexes associated with Photosystem I (PSI) and photosystem II (PSII). As PSI and PSII work in series, it is important that the excitation pressure on the two photosystems is balanced. When plants are exposed to illumination that overexcites PSII, a special pool of the major light-harvesting complex LHCII is phosphorylated and moves from PSII to PSI (state 2). If instead PSI is over-excited the LHCII complex is dephosphorylated and moves back to PSII (state 1). Recent findings have suggested that LHCII might also transfer energy to PSI in state 1. In this work we used a combination of biochemistry and (time-resolved) fluorescence spectroscopy to investigate the PSI antenna size in state 1 and state 2 for Arabidopsis thaliana. Our data shows that 0.7 ± 0.1 unphosphorylated LHCII trimers per PSI are present in the stroma lamellae of state-1 plants. Upon transition to state 2 the antenna size of PSI in the stroma membrane increases with phosphorylated LHCIIs to a total of 1.2 ± 0.1 LHCII trimers per PSI. Both phosphorylated and unphosphorylated LHCII function as highly efficient PSI antenna., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The bioenergetics of light-harvesting by photosynthetic antenna proteins in higher plants is well understood. However, investigation into the regulatory non-photochemical quenching (NPQ) mechanism, which dissipates excess energy in high light, has led to several conflicting models. It is generally accepted that the major photosystem II antenna protein, LHCII, is the site of NPQ, although the minor antenna complexes (CP24/26/29) are also proposed as alternative/additional NPQ sites. LHCII crystals were shown to exhibit the short excitation lifetime and several spectral signatures of the quenched state. Subsequent structure-based models showed that this quenching could be explained by slow energy trapping by the carotenoids, in line with one of the proposed models. Using Fluorescence Lifetime Imaging Microscopy (FLIM) we show that the crystal structure of CP29 corresponds to a strongly quenched conformation. Using a structure-based theoretical model we show that this quenching may be explained by the same slow, carotenoid-mediated quenching mechanism present in LHCII crystals., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Marine diatoms are prominent phytoplankton organisms that perform photosynthesis in extremely variable environments. Diatoms possess a strong ability to dissipate excess absorbed energy as heat via nonphotochemical quenching (NPQ). This process relies on changes in carotenoid pigment composition (xanthophyll cycle) and on specific members of the light-harvesting complex family specialized in photoprotection (LHCXs), which potentially act as NPQ effectors. However, the link between light stress, NPQ, and the existence of different LHCX isoforms is not understood in these organisms. Using picosecond fluorescence analysis, we observed two types of NPQ in the pennate diatom Phaeodactylum tricornutum that were dependent on light conditions. Short exposure of low-light-acclimated cells to high light triggers the onset of energy quenching close to the core of photosystem II, while prolonged light stress activates NPQ in the antenna. Biochemical analysis indicated a link between the changes in the NPQ site/mechanism and the induction of different LHCX isoforms, which accumulate either in the antenna complexes or in the core complex. By comparing the responses of wild-type cells and transgenic lines with a reduced expression of the major LHCX isoform, LHCX1, we conclude that core complex-associated NPQ is more effective in photoprotection than is the antenna complex. Overall, our data clarify the complex molecular scenario of light responses in diatoms and provide a rationale for the existence of a degenerate family of LHCX proteins in these algae., (© 2018 American Society of Plant Biologists. All rights reserved.)

Photosystems I and II (PSI and PSII) work in series to drive oxygenic photosynthesis. The two photosystems have different absorption spectra, therefore changes in light quality can lead to imbalanced excitation of the photosystems and a loss in photosynthetic efficiency. In a short-term adaptation response termed state transitions, excitation energy is directed to the light-limited photosystem. In higher plants a special pool of LHCII antennae, which can be associated with either PSI or PSII, participates in these state transitions. It is known that one LHCII antenna can associate with the PsaH site of PSI. However, membrane fractions were recently isolated in which multiple LHCII antennae appear to transfer energy to PSI. We have used time-resolved fluorescence-streak camera measurements to investigate the energy transfer rates and efficiency in these membrane fractions. Our data show that energy transfer from LHCII to PSI is relatively slow. Nevertheless, the trapping efficiency in supercomplexes of PSI with ~2.4 LHCIIs attached is 94%. The absorption cross section of PSI can thus be increased with ~65% without having significant loss in quantum efficiency. Comparison of the fluorescence dynamics of PSI-LHCII complexes, isolated in a detergent or located in their native membrane environment, indicates that the environment influences the excitation energy transfer rates in these complexes. This demonstrates the importance of studying membrane protein complexes in their natural environment., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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 (+)690nm, 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 (+)506nm 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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In response to changes in the reduction state of the plastoquinone pool in its thylakoid membrane, the green alga Chlamydomonas reinhardtti is performing state transitions: remodelling of its thylakoid membrane leads to a redistribution of excitations over photosystems I and II (PSI and PSII). These transitions are accompanied by marked changes in the 77 K fluorescence spectrum, which form the accepted signature of state transitions. The changes are generally thought to reflect a redistribution of light-harvesting complexes (LHCs) over PSII (fluorescing below 700 nm) and PSI (fluorescing above 700 nm). Here we studied the picosecond fluorescence properties of C. reinhardtti over a broad range of wavelengths with very low excitation intensities (0.2 nJ per laser pulse). Cells were directly used for time-resolved fluorescence measurements at 77 K without further treatment, such as medium exchange with glycerol. It is observed that upon going from state 1 (relatively more fluorescence below 700 nm) to state 2 (relatively more fluorescence above 700 nm), a large part of the fluorescence of LHC/PSII becomes substantially quenched in concurrence with LHC detachment from PSII, whereas the absolute amount of PSI fluorescence hardly changes. These results are in agreement with the recent proposal that the amount of LHC moving from PSII to PSI upon going from state 1 to state 2 is rather limited (Unlu et al. Proc Natl Acad Sci USA 111 (9):3460-3465, 2014).

Complex multi-exponential fluorescence decay kinetics observed in various photosynthetic systems like photosystem II (PSII) have often been explained by the reversible quenching mechanism of the charge separation taking place in the reaction center (RC) of PSII. However, this description does not account for the intrinsic dynamic disorder of the light-harvesting proteins as well as their fluctuating dislocations within the antenna, which also facilitate the repair of RCs, state transitions, and the process of non-photochemical quenching. Since dynamic fluctuations result in varying connectivity between pigment-protein complexes, they can also lead to non-exponential excitation decay kinetics. Based on this presumption, we have recently proposed a simple conceptual model describing excitation diffusion in a continuous medium and accounting for possible variations of the excitation transfer pathways. In the current work, this model is further developed and then applied to describe fluorescence kinetics originating from very diverse antenna systems, ranging from PSII of various sizes to LHCII aggregates and even the entire thylakoid membrane. In all cases, complex multi-exponential fluorescence kinetics are perfectly reproduced on the entire relevant time scale without assuming any radical pair equilibration at the side of the excitation quencher, but using just a few parameters reflecting the mean excitation energy transfer rate as well as the overall average organization of the photosynthetic antenna.

Photosynthetic organisms are crucial for life on Earth as they provide food and oxygen and are at the basis of most energy resources. They have a large variety of light-harvesting strategies that allow them to live nearly everywhere where sunlight can penetrate. They have adapted their pigmentation to the spectral composition of light in their habitat, they acclimate to slowly varying light intensities and they rapidly respond to fast changes in light quality and quantity. This is particularly important for oxygen-producing organisms because an overdose of light in combination with oxygen can be lethal. Rapid progress is being made in understanding how different organisms maximize light harvesting and minimize deleterious effects. Here we summarize the latest findings and explain the main design principles used in nature. The available knowledge can be used for optimizing light harvesting in both natural and artificial photosynthesis to improve light-driven production processes.

Plants and green algae optimize photosynthesis in changing light conditions by balancing the amount of light absorbed by photosystems I and II. These photosystems work in series to extract electrons from water and reduce NADP(+) to NADPH. Light-harvesting complexes (LHCs) are held responsible for maintaining the balance by moving from one photosystem to the other in a process called state transitions. In the green alga Chlamydomonas reinhardtii, a photosynthetic model organism, state transitions are thought to involve 80% of the LHCs. Here, we demonstrate with picosecond-fluorescence spectroscopy on C. reinhardtii cells that, although LHCs indeed detach from photosystem II in state 2 conditions, only a fraction attaches to photosystem I. The detached antenna complexes become protected against photodamage via shortening of the excited-state lifetime. It is discussed how the transition from state 1 to state 2 can protect C. reinhardtii in high-light conditions and how this differs from the situation in plants.

Water oxidation in photosynthesis takes place in photosystem II (PSII). This photosystem is built around a reaction center (RC) where sunlight-induced charge separation occurs. This RC consists of various polypeptides that bind only a few chromophores or pigments, next to several other cofactors. It can handle far more photons than the ones absorbed by its own pigments and therefore, additional excitations are provided by the surrounding light-harvesting complexes or antennae. The RC is located in the PSII core that also contains the inner light-harvesting complexes CP43 and CP47, harboring 13 and 16 chlorophyll pigments, respectively. The core is surrounded by outer light-harvesting complexes (Lhcs), together forming the so-called supercomplexes, at least in plants. These PSII supercomplexes are complemented by some "extra" Lhcs, but their exact location in the thylakoid membrane is unknown. The whole system consists of many subunits and appears to be modular, i.e., both its composition and organization depend on environmental conditions, especially on the quality and intensity of the light. In this review, we will provide a short overview of the relation between the structure and organization of pigment-protein complexes in PSII, ranging from individual complexes to entire membranes and experimental and theoretical results on excitation energy transfer and charge separation. It will become clear that time-resolved fluorescence data can provide invaluable information about the organization and functioning of thylakoid membranes. At the end, an overview will be given of unanswered questions that should be addressed in the near future.

This review focuses on the light-harvesting properties of photosystem I (PSI) and its LHCI outer antenna. LHCI consists of different chlorophyll a/b binding proteins called Lhca's, surrounding the core of PSI. In total, the PSI-LHCI complex of higher plants contains 173 chlorophyll molecules, most of which are there to harvest sunlight energy and to transfer the created excitation energy to the reaction center (RC) where it is used for charge separation. The efficiency of the complex is based on the capacity to deliver this energy to the RC as fast as possible, to minimize energy losses. The performance of PSI in this respect is remarkable: on average it takes around 50 ps for the excitation to reach the RC in plants, without being quenched in the meantime. This means that the internal quantum efficiency is close to 100% which makes PSI the most efficient energy converter in nature. In this review, we describe the light-harvesting properties of the complex in relation to protein and pigment organization/composition, and we discuss the important parameters that assure its very high quantum efficiency. Excitation energy transfer and trapping in the core and/or Lhcas, as well as in the supercomplexes PSI-LHCI and PSI-LHCI-LHCII are described in detail with the aim of giving an overview of the functional behavior of these complexes.

LHCII, the most abundant membrane protein on earth, is the major light-harvesting complex of plants. It is generally accepted that LHCII is associated with Photosystem II and only as a short-term response to overexcitation of PSII a subset moves to Photosystem I, triggered by its phosphorylation (state1 to state2 transition). However, here we show that in most natural light conditions LHCII serves as an antenna of both Photosystem I and Photosystem II and it is quantitatively demonstrated that this is required to achieve excitation balance between the two photosystems. This allows for acclimation to different light intensities simply by regulating the expression of LHCII genes only. It is demonstrated that indeed the amount of LHCII that is bound to both photosystems decreases when growth light intensity increases and vice versa. Finally, time-resolved fluorescence measurements on the photosynthetic thylakoid membranes show that LHCII is even a more efficient light harvester when associated with Photosystem I than with Photosystem II., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In this work, we have investigated the role of the individual antenna complexes and of the low-energy forms in excitation energy transfer and trapping in Photosystem I of higher plants. To this aim, a series of Photosystem I (sub)complexes with different antenna size/composition/absorption have been studied by picosecond fluorescence spectroscopy. The data show that Lhca3 and Lhca4, which harbor the most red forms, have similar emission spectra (λ(max) = 715-720 nm) and transfer excitation energy to the core with a relative slow rate of ∼25/ns. Differently, the energy transfer from Lhca1 and Lhca2, the "blue" antenna complexes, occurs about four times faster. In contrast to what is often assumed, it is shown that energy transfer from the Lhca1/4 and the Lhca2/3 dimer to the core occurs on a faster timescale than energy equilibration within these dimers. Furthermore, it is shown that all four monomers contribute almost equally to the transfer to the core and that the red forms slow down the overall trapping rate by about two times. Combining all the data allows the construction of a comprehensive picture of the excitation-energy transfer routes and rates in Photosystem I., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

In 2005, it was found that the fluorescence of crystals of the major light-harvesting complex LHCII of green plants is significantly quenched when compared to the fluorescence of isolated LHCII (A. A. Pascal et al., Nature, 2005, 436, 134-137). The Raman spectrum of crystallized LHCII was also found to be different from that of isolated LHCII but very similar to that of aggregated LHCII, which has often been considered a good model system for studying nonphotochemical quenching (NPQ), the major protection mechanism of plants against photodamage in high light. It was proposed that in the crystal LHCII adopts a similar (quenching) conformation as during NPQ and indeed similar changes in the Raman spectrum were observed during NPQ in vivo (A. V. Ruban et al., Nature, 2007, 450, 575-579). We now compared the fluorescence of various types of crystals, differing in morphology and age. Each type gave rise to its own characteristic mono-exponential fluorescence lifetime, which was 5 to 10 times shorter than that of isolated LHCII. This indicates that fluorescence is not quenched by random impurities and packing defects (as proposed recently by T. Barros et al., EMBO Journal, 2009, 28, 298-306), but that LHCII adopts a particular structure in each crystal type, that leads to fluorescence quenching. Most interestingly, the extent of quenching appears to depend on the crystal morphology, indicating that also the crystal structure depends on this crystal morphology but at the moment no data are available to correlate the crystals' structural changes to changes in fluorescence lifetime., (This journal is © the Owner Societies 2011)

We performed picosecond fluorescence measurements on well-defined Photosystem II (PSII) supercomplexes from Arabidopsis with largely varying antenna sizes. The average excited-state lifetime ranged from 109 ps for PSII core to 158 ps for the largest C(2)S(2)M(2) complex in 0.01% α-DM. Excitation energy transfer and trapping were investigated by coarse-grained modeling of the fluorescence kinetics. The results reveal a large drop in free energy upon charge separation (>700 cm(-1)) and a slow relaxation of the radical pair to an irreversible state (∼150 ps). Somewhat unexpectedly, we had to reduce the energy-transfer and charge-separation rates in complexes with decreasing size to obtain optimal fits. This strongly suggests that the antenna system is important for plant PSII integrity and functionality, which is supported by biochemical results. Furthermore, we used the coarse-grained model to investigate several aspects of PSII functioning. The excitation trapping time appears to be independent of the presence/absence of most of the individual contacts between light-harvesting complexes in PSII supercomplexes, demonstrating the robustness of the light-harvesting process. We conclude that the efficiency of the nonphotochemical quenching process is hardly dependent on the exact location of a quencher within the supercomplexes., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Three phyla of bacteria include phototrophs that contain unique antenna systems, chlorosomes, as the principal light-harvesting apparatus. Chlorosomes are the largest known supramolecular antenna systems and contain hundreds of thousands of BChl c/d/e molecules enclosed by a single membrane leaflet and a baseplate. The BChl pigments are organized via self-assembly and do not require proteins to provide a scaffold for efficient light harvesting. Their excitation energy flows via a small protein, CsmA embedded in the baseplate to the photosynthetic reaction centres. Chlorosomes allow for photosynthesis at very low light intensities by ultra-rapid transfer of excitations to reaction centres and enable organisms with chlorosomes to live at extraordinarily low light intensities under which no other phototrophic organisms can grow. This article reviews several aspects of chlorosomes: the supramolecular and molecular organizations and the light-harvesting and spectroscopic properties. In addition, it provides some novel information about the organization of the baseplate.

In this work we have investigated the origin of the multi-exponential fluorescence decay and of the short excited-state lifetime of Lhca4. Lhca4 is the antenna complex of Photosystem I which accommodates the red-most chlorophyll forms and it has been proposed that these chlorophylls can play a role in fluorescence quenching. Here we have compared the fluorescence decay of Lhca4 with that of several Lhca4 mutants that are affected in their red form content. The results show that neither the multi-exponentiality of the decay nor the fluorescence quenching is due to the red forms. The data indicate that Lhca4 exists in multiple conformations. The presence of the red forms, which are very sensitive to changes in the environment, allows to spectrally resolve the different conformations: a "blue" conformation with a short lifetime and a "red" one with a long lifetime. This finding strongly supports the idea that the members of the Lhc family are able to adopt different conformations associated with their light-harvesting and photoprotective roles. The ratio between the conformations is modified by the substitution of lutein by violaxanthin. Finally, it is demonstrated that the red forms cannot be present in the quenched conformation.

A high-throughput Förster resonance energy transfer (FRET) study was performed on the approximately 100 amino acids long N-terminal domain of the photosynthetic complex CP29 of higher plants. For this purpose, CP29 was singly mutated along its N-terminal domain, replacing one-by-one native amino acids by a cysteine, which was labeled with a BODIPY fluorescent probe, and reconstituted with the natural pigments of CP9, chlorophylls and xanthophylls. Picosecond fluorescence experiments revealed rapid energy transfer (approximately 20-70 ps) from BODIPY at amino-acid positions 4, 22, 33, 40, 56, 65, 74, 90, and 97 to Chl a molecules in the hydrophobic part of the protein. From the energy transfer times, distances were estimated between label and chlorophyll molecules, using the Förster equation. When the label was attached to amino acids 4, 56, and 97, it was found to be located very close to the protein core (approximately 15 A), whereas labels at positions 15, 22, 33, 40, 65, 74, and 90 were found at somewhat larger distances. It is concluded that the entire N-terminal domain is in close contact with the hydrophobic core and that there is no loop sticking out into the stroma. Most of the results support a recently proposed topological model for the N-terminus of CP29, which was based on electron-spin-resonance measurements on spin-labeled CP29 with and without its natural pigment content. The present results lead to a slight refinement of that model.

The topology of the long N-terminal domain (approximately 100 amino-acid residues) of the photosynthetic Lhc CP29 was studied using electron spin resonance. Wild-type protein containing a single cysteine at position 108 and nine single-cysteine mutants were produced, allowing to label different parts of the domain with a nitroxide spin label. In all cases, the apoproteins were either solubilized in detergent or they were reconstituted with their native pigments (holoproteins) in vitro. The spin-label electron spin resonance spectra were analyzed in terms of a multicomponent spectral simulation approach, based on hybrid evolutionary optimization and solution condensation. These results permit to trace the structural organization of the long N-terminal domain of CP29. Amino-acid residues 97 and 108 are located in the transmembrane pigment-containing protein body of the protein. Positions 65, 81, and 90 are located in a flexible loop that is proposed to extend out of the protein from the stromal surface. This loop also contains a phosphorylation site at Thr81, suggesting that the flexibility of this loop might play a role in the regulatory mechanisms of the light-harvesting process. Positions 4, 33, 40, and 56 are found to be located in a relatively rigid environment, close to the transmembrane protein body. On the other hand, position 15 is located in a flexible region, relatively far away from the transmembrane domain.

Over the past several years, many crystal structures of photosynthetic pigment-protein complexes have been determined, and these have been used extensively to model spectroscopic results obtained on the same proteins in solution. However, the crystal structure is not necessarily identical to the structure of the protein in solution. Here, we studied picosecond fluorescence of photosystem I light-harvesting complex I (PSI-LHCI), a multisubunit pigment-protein complex that catalyzes the first steps of photosynthesis. The ultrafast fluorescence of PSI-LHCI crystals is identical to that of dissolved crystals, but differs considerably from most kinetics presented in the literature. In contrast to most studies, the data presented here can be modeled quantitatively with only two compartments: PSI core and LHCI. This yields the rate of charge separation from an equilibrated core (22.5 +/- 2.5 ps) and rates of excitation energy transfer from LHCI to core (k(LC)) and vice versa (k(CL)). The ratio between these rates, R = k(CL)/k(LC), appears to be wavelength-dependent and scales with the ratio of the absorption spectra of LHCI and core, indicating the validity of a detailed balance relation between both compartments. k(LC) depends slightly but nonsystematically on detection wavelength, averaging (9.4 +/- 4.9 ps)(-1). R ranges from 0.5 (<690 nm) to approximately 1.3 above 720 nm.

In bright sunlight, the amount of energy harvested by plants exceeds the electron transport capacity of Photosystem II in the chloroplasts. The excess energy can lead to severe damage of the photosynthetic apparatus and to avoid this, part of the energy is thermally dissipated via a mechanism called non-photochemical quenching (NPQ). It has been found that LHCII, the major antenna complex of Photosystem II, is involved in this mechanism and it was proposed that its quenching site is formed by the cluster of strongly interacting pigments: chlorophylls 611 and 612 and lutein 620 [A.V. Ruban, R. Berera, C. Ilioaia, I.H.M. van Stokkum, J.T.M. Kennis, A.A. Pascal, H. van Amerongen, B. Robert, P. Horton and R. van Grondelle, Identification of a mechanism of photoprotective energy dissipation in higher plants, Nature 450 (2007) 575-578.]. In the present work we have investigated the interactions between the pigments in this cluster not only for LHCII, but also for the homologous minor antenna complexes CP24, CP26 and CP29. Use was made of wild-type and mutated reconstituted complexes that were analyzed with (low-temperature) absorption and circular-dichroism spectroscopy as well as by biochemical methods. The pigments show strong interactions that lead to highly specific spectroscopic properties that appear to be identical for LHCII, CP26 and CP29. The interactions are similar but not identical for CP24. It is concluded that if the 611/612/620 domain is responsible for the quenching in LHCII, then all these antenna complexes are prepared to act as a quencher. This can explain the finding that none of the Lhcb complexes seems to be strictly required for NPQ while, in the absence of all of them, NPQ is abolished.

Under conditions of excess sunlight the efficient light-harvesting antenna found in the chloroplast membranes of plants is rapidly and reversibly switched into a photoprotected quenched state in which potentially harmful absorbed energy is dissipated as heat, a process measured as the non-photochemical quenching of chlorophyll fluorescence or qE. Although the biological significance of qE is established, the molecular mechanisms involved are not. LHCII, the main light-harvesting complex, has an inbuilt capability to undergo transformation into a dissipative state by conformational change and it was suggested that this provides a molecular basis for qE, but it is not known if such events occur in vivo or how energy is dissipated in this state. The transition into the dissipative state is associated with a twist in the configuration of the LHCII-bound carotenoid neoxanthin, identified using resonance Raman spectroscopy. Applying this technique to study isolated chloroplasts and whole leaves, we show here that the same change in neoxanthin configuration occurs in vivo, to an extent consistent with the magnitude of energy dissipation. Femtosecond transient absorption spectroscopy, performed on purified LHCII in the dissipative state, shows that energy is transferred from chlorophyll a to a low-lying carotenoid excited state, identified as one of the two luteins (lutein 1) in LHCII. Hence, it is experimentally demonstrated that a change in conformation of LHCII occurs in vivo, which opens a channel for energy dissipation by transfer to a bound carotenoid. We suggest that this is the principal mechanism of photoprotection.

Non-photochemical quenching (NPQ) protects plants against photodamage by converting excess excitation energy into harmless heat. In vitro aggregation of the major light-harvesting complex (LHCII) induces similar quenching, the molecular mechanism of which is frequently considered to be the same. However, a very basic question regarding the aggregation-induced quenching has not been answered yet. Are excitation traps created upon aggregation, or do existing traps start quenching excitations more efficiently in aggregated LHCII where trimers are energetically coupled? Time-resolved fluorescence experiments presented here demonstrate that aggregation creates traps in a significant number of LHCII trimers, which subsequently also quench excitations in connected LHCIIs.

In this work we modeled the circular dichroism (CD) spectrum of LHCII, the main light harvesting antenna of photosystem II of higher plants. Excitonic calculations are performed for a monomeric subunit, taken from the crystal structure of trimeric LHCII from spinach [Liu, Z. F., Yan, H. C., Wang, K. B., Kuang, T. Y., Zhang, J. P., Gui, L. L., An, X. M., and Chang, W. R. (2004) Nature 428, 287-292]. All of the major features of the CD spectrum above 450 nm are satisfactorily reproduced, and possible orientations of the Chl and carotenoid transition dipole moments are identified. The obtained modeling parameters are used to simulate the CD spectra of two complexes with altered pigment composition: a mutant lacking Chls a 611-612 and a complex lacking the carotenoid neoxanthin. By removing the relevant pigment(s) from the structure, we are able to reproduce their spectra, which implies that the alteration does not disturb the overall structure. The CD spectrum of trimeric LHCII shows a reversed relative intensity of the two negative bands around 470 and 490 nm as compared to monomeric LHCII. The simulations reproduce this reversal, indicating that it is mainly due to interactions between chromophores in different monomeric subunits, and the trimerization does not induce observable changes in the monomeric structure. Our simulated spectrum resembles one of two different trimeric CD spectra reported in literature. We argue that the differences in the experimental trimeric CD spectra are caused by changes in the strength of the monomer-monomer interactions due to the differences in detergents used for the purification of the complexes.

In order to maximize their use of light energy in photosynthesis, plants have molecules that act as light-harvesting antennae, which collect light quanta and deliver them to the reaction centres, where energy conversion into a chemical form takes place. The functioning of the antenna responds to the extreme changes in the intensity of sunlight encountered in nature. In shade, light is efficiently harvested in photosynthesis. However, in full sunlight, much of the energy absorbed is not needed and there are vitally important switches to specific antenna states, which safely dissipate the excess energy as heat. This is essential for plant survival, because it provides protection against the potential photo-damage of the photosynthetic membrane. But whereas the features that establish high photosynthetic efficiency have been highlighted, almost nothing is known about the molecular nature of the dissipative states. Recently, the atomic structure of the major plant light-harvesting antenna protein, LHCII, has been determined by X-ray crystallography. Here we demonstrate that this is the structure of a dissipative state of LHCII. We present a spectroscopic analysis of this crystal form, and identify the specific changes in configuration of its pigment population that give LHCII the intrinsic capability to regulate energy flow. This provides a molecular basis for understanding the control of photosynthetic light-harvesting.

We have modeled steady-state spectra and energy-transfer dynamics in the peripheral plant light-harvesting complex LHCII using new structural data. The dynamics of the chlorophyll (Chl) b-->Chl a transfer and decay of selectively excited "bottleneck" Chl a and b states have been studied by femtosecond pump-probe spectroscopy. We propose an exciton model of the LHCII trimer (with specific site energies) which allows a simultaneous quantitative fit of the absorption, linear-dichroism, steady-state fluorescence spectra, and transient absorption kinetics upon excitation at different wavelengths. In the modeling we use the experimental exciton-phonon spectral density and modified Redfield theory. We have found that fast b-->a transfer is determined by a good connection of the Chls b to strongly coupled Chl a clusters, i.e., a610-a611-a612 trimer and a602-a603 and a613-a614 dimers. Long-lived components of the energy-transfer kinetics are determined by a quick population of red-shifted Chl b605 and blue-shifted Chl a604 followed by a very slow (3 ps for b605 and 12 ps for a604) flow of energy from these monomeric bottleneck sites to the Chl a clusters. The dynamics within the Chl a region is determined by fast (with time constants down to sub-100 fs) exciton relaxation within the a610-a611-a612 trimer, slower 200-300 fs relaxation within the a602-a603 and a613-a614 dimers, even slower 300-800 fs migration between these clusters, and very slow transfer from a604 to the quasi-equilibrated a sites. The final equilibrium is characterized by predominant population of the a610-a611-a612 cluster (mostly the a610 site). The location of this cluster on the outer side of the LHCII trimer probably provides a good connection with the other subunits of PSII.

Three pulse echo peak shift and transient grating (TG) measurements on the plant light-harvesting complexes LHCII and CP29 are reported. The LHCII complex is by far the most abundant light-harvesting complex in higher plants and fulfills several important physiological functions such as light-harvesting and photoprotection. Our study is focused on the light-harvesting function of LHCII and the very similar CP29 complex and reveals hitherto unresolved excitation energy transfer processes. All measurements were performed at room temperature using detergent isolated complexes from spinach leaves. Both complexes were excited in their Chl b band at 650 nm and in the blue shoulder of the Chl a band at 670 nm. Exponential fits to the TG and three pulse echo peak shift decay curves were used to estimate the timescales of the observed energy transfer processes. At 650 nm, the TG decay can be described with time constants of 130 fs and 2.2 ps for CP29, and 300 fs and 2.8 ps for LHCII. At 670 nm, the TG shows decay components of 230 fs and 6 ps for LHCII, and 300 fs and 5 ps for CP29. These time constants correspond to well-known energy transfer processes, from Chl b to Chl a for the 650 nm TG and from blue (670 nm) Chl a to red (680 nm) Chl a for the 670 nm TG. The peak shift decay times are entirely different. At 650 nm we find times of 150 fs and 0.5-1 ps for LHCII, and 360 fs and 3 ps for CP29, which we can associate mainly with Chl b <--> Chl b energy transfer. At 670 nm we find times of 140 fs and 3 ps for LHCII, and 3 ps for CP29, which we can associate with fast (only in LHCII) and slow transfer between relatively blue Chls a or Chl a states. From the occurrence of both fast Chl b <--> Chl b and fast Chl b --> Chl a transfer in CP29, we conclude that at least two mixed binding sites are present in this complex. A detailed comparison of our observed rates with exciton calculations on both CP29 and LHCII provides us with more insight in the location of these mixed sites. Most importantly, for CP29, we find that a Chl b pair must be present in some, but not all, complexes, on sites A(3) and B(3). For LHCII, the observed rates can best be understood if the same pair, A(3) and B(3), is involved in both fast Chl b <--> Chl b and fast Chl a <--> Chl a transfer. Hence, it is likely that mixed sites also occur in the native LHCII complex. Such flexibility in chlorophyll binding would agree with the general flexibility in aggregation form and xanthophyll binding of the LHCII complex and could be of use for optimizing the role of LHCII under specific circumstances, for example under high-light conditions. Our study is the first to provide spectroscopic evidence for mixed binding sites, as well as the first to show their existence in native complexes.

The pigment-protein complexes CP43 and CP47 transfer excitation energy from the peripheral antenna of photosystem II toward the photochemical reaction center. We measured the excitation dynamics of the chlorophylls in isolated CP43 and CP47 complexes at 77 K by time-resolved absorbance-difference and fluorescence spectroscopy. The spectral relaxation appeared to occur with rates of 0.2-0.4 ps and 2-3 ps in both complexes, whereas an additional relaxation of 17 ps was observed only in CP47. Using the 3.8-A crystal structure of the photosystem II core complex from Synechococcus elongatus (A. Zouni, H.-T. Witt, J. Kern, P. Fromme, N. Krauss, W. Saenger, and P. Orth, 2001, Nature, 409:739-743), excitation energy transfer kinetics were calculated and a Monte Carlo simulation of the absorption spectra was performed. In both complexes, the rate of 0.2-0.4 ps can be ascribed to excitation energy transfer within a layer of chlorophylls near the stromal side of the membrane, and the slower 2-3-ps process to excitation energy transfer to the calculated lowest excitonic state. We conclude that excitation energy transfer within CP43 and CP47 is fast and does not contribute significantly to the well-known slow trapping of excitation energy in photosystem II.