Light-harvesting Complex II Trimers Research Articles

Regulatory dynamics of the higher-plant PSI–LHCI supercomplex during state transitions

State transition is a fundamental light acclimation mechanism of photosynthetic organisms in response to the environmental light conditions. This process rebalances the excitation energy between photosystem I (PSI) and photosystem II through regulated reversible binding of the light-harvesting complex II (LHCII) to PSI. However, the structural reorganization of PSI–LHCI, the dynamic binding of LHCII, and the regulatory mechanisms underlying state transitions are less understood in higher plants. In this study, using cryoelectron microscopy we resolved the structures of PSI–LHCI in both state 1 (PSI–LHCI-ST1) and state 2 (PSI–LHCI–LHCII-ST2) from Arabidopsis thaliana. Combined genetic and functional analyses revealed novel contacts between Lhcb1 and PsaK that further enhanced the binding of the LHCII trimer to the PSI core with the known interactions between phosphorylated Lhcb2 and the PsaL/PsaH/PsaO subunits. Specifically, PsaO was absent in the PSI–LHCI-ST1 supercomplex but present in the PSI–LHCI–LHCII-ST2 supercomplex, in which the PsaL/PsaK/PsaA subunits undergo several conformational changes to strengthen the binding of PsaO in ST2. Furthermore, the PSI–LHCI module adopts a more compact configuration with shorter Mg-to-Mg distances between the chlorophylls, which may enhance the energy transfer efficiency from the peripheral antenna to the PSI core in ST2. Collectively, our work provides novel structural and functional insights into the mechanisms of light acclimation during state transitions in higher plants.

Regulation of light-harvesting systems in plants: Flexible conformation of light-harvesting complex II trimer depending on its binding site.

Photosynthesis is a fundamental energy conversion process that sustains the Earth's ecosystem. As the environment has changed, photosynthetic systems of photosynthetic organisms have been diversified and optimized. Recent studies indicate that photosynthesis has been designed not only for ‘efficiency’ in energy conversion but also for ‘flexibility' in sustaining itself in a changing environment. In particular, light-harvesting systems were found to have the greatest diversity and flexibility to cope with the changing light conditions. In this presentation, I would like to introduce my recent studies on the regulation of light-harvesting systems by protein-protein interactions in plants and green algae. Our results revealed that the conformation of a light-harvesting complex II (LHCII) trimer in the green alga Chlamydomonas reinhardtii was dependent on the binding site. We propose that this conformational flexibility of the LHCII trimer possibly modulates its light-harvesting properties by altering excitation-energy flow over the photosynthetic supercomplex.

Conformation of Light-Harvesting Complex II Trimer Depends upon Its Binding Site.

The light-harvesting complex II (LHCII) trimer in plants functions as a major antenna complex and a quencher to protect it from photooxidative damage. Theoretical studies on the structure of an LHCII trimer have demonstrated that excitation energy transfer between chlorophylls (Chls) in LHCII can be modulated by its exquisite conformational fluctuation. However, conformational changes depending on its binding location have not yet been investigated, even though reorganization of protein complexes occurs by physiological regulations. In this study, we investigated conformational differences in LHCII by comparing published structures of an identical LHCII trimer in the three different photosystem supercomplexes from the green alga Chlamydomonas reinhardtii. Our results revealed distinct differences in Chl configurations as well as polypeptide conformations of the LHCII trimers depending on its binding location. We propose that these configurational differences readily modulate the function of LHCII and possibly lead to a change in excitation-energy flow over the photosynthetic supercomplex.

Protein dynamics and lipid affinity of monomeric, zeaxanthin-binding LHCII in thylakoid membranes

The xanthophyll cycle in the antenna of photosynthetic organisms under light stress is one of the most well-known processes in photosynthesis, but its role is not well understood. In the xanthophyll cycle, violaxanthin (Vio) is reversibly transformed to zeaxanthin (Zea) that occupies Vio binding sites of light-harvesting antenna proteins. Higher monomer/trimer ratios of the most abundant light-harvesting protein, the light-harvesting complex II (LHCII), usually occur in Zea accumulating membranes and have been observed in plants after prolonged illumination and during high-light acclimation. We present a combined NMR and coarse-grained simulation study on monomeric LHCII from the npq2 mutant that constitutively binds Zea in the Vio binding pocket. LHCII was isolated from 13C-enriched npq2 Chlamydomonas reinhardtii (Cr) cells and reconstituted in thylakoid lipid membranes. NMR results reveal selective changes in the fold and dynamics of npq2 LHCII compared with the trimeric, wild-type and show that npq2 LHCII contains multiple mono- or digalactosyl diacylglycerol lipids (MGDG and DGDG) that are strongly protein bound. Coarse-grained simulations on npq2 LHCII embedded in a thylakoid lipid membrane agree with these observations. The simulations show that LHCII monomers have more extensive lipid contacts than LHCII trimers and that protein-lipid contacts are influenced by Zea. We propose that both monomerization and Zea binding could have a functional role in modulating membrane fluidity and influence the aggregation and conformational dynamics of LHCII with a likely impact on photoprotection ability.

Accumulation of geranylgeranylated chlorophylls in the pigment-protein complexes of Arabidopsis thaliana acclimated to green light: effects on the organization of light-harvesting complex II and photosystem II functions

Light quality significantly influences plant metabolism, growth and development. Recently, we have demonstrated that leaves of barley and other plant species grown under monochromatic green light (500–590 nm) accumulated a large pool of chlorophyll a (Chl a) intermediates with incomplete hydrogenation of their phytyl chains. In this work, we studied accumulation of these geranylgeranylated Chls a and b in pigment-protein complexes (PPCs) of Arabidopsis plants acclimated to green light and their structural–functional consequences on the photosynthetic apparatus. We found that geranylgeranylated Chls are present in all major PPCs, although their presence was more pronounced in light-harvesting complex II (LHCII) and less prominent in supercomplexes of photosystem II (PSII). Accumulation of geranylgeranylated Chls hampered the formation of PSII and PSI super- and megacomplexes in the thylakoid membranes as well as their assembly into chiral macrodomains; it also lowered the temperature stability of the PPCs, especially that of LHCII trimers, which led to their monomerization and an anomaly in the photoprotective mechanism of non-photochemical quenching. Role of geranylgeranylated Chls in adverse effects on photosynthetic apparatus of plants acclimated to green light is discussed.

Pigment structure in the light-harvesting protein of the siphonous green alga Codium fragile

The siphonaxanthin-siphonein-chlorophyll-a/b-binding protein (SCP), a trimeric light-harvesting complex isolated from photosystem II of the siphonous green alga Codium fragile, binds the carotenoid siphonaxanthin (Sx) and/or its ester siphonein in place of lutein, in addition to chlorophylls a/b and neoxanthin. SCP exhibits a higher content of chlorophyll b (Chl-b) than its counterpart in green plants, light-harvesting complex II (LHCII), increasing the relative absorption of blue-green light for photosynthesis. Using low temperature absorption and resonance Raman spectroscopies, we reveal the presence of two non-equivalent Sx molecules in SCP, and assign their absorption peaks at 501 and 535 nm. The red-absorbing Sx population exhibits a significant distortion that is reminiscent of lutein 2 in trimeric LHCII. Unexpected enhancement of the Raman modes of Chls-b in SCP allows an unequivocal description of seven to nine non-equivalent Chls-b, and six distinct Chl-a populations in this protein.

Characterization of fluorescent chlorophyll charge-transfer states as intermediates in the excited state quenching of light-harvesting complex II.

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 4K, 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.

How paired PSII\u2013LHCII supercomplexes mediate the stacking of plant thylakoid membranes unveiled by structural mass-spectrometry

Grana are a characteristic feature of higher plants’ thylakoid membranes, consisting of stacks of appressed membranes enriched in Photosystem II (PSII) and associated light-harvesting complex II (LHCII) proteins, together forming the PSII-LHCII supercomplex. Grana stacks undergo light-dependent structural changes, mainly by reorganizing the supramolecular structure of PSII-LHCII supercomplexes. LHCII is vital for grana formation, in which also PSII-LHCII supercomplexes are involved. By combining top-down and crosslinking mass spectrometry we uncover the spatial organization of paired PSII-LHCII supercomplexes within thylakoid membranes. The resulting model highlights a basic molecular mechanism whereby plants maintain grana stacking at changing light conditions. This mechanism relies on interactions between stroma-exposed N-terminal loops of LHCII trimers and Lhcb4 subunits facing each other in adjacent membranes. The combination of light-dependent LHCII N-terminal trimming and extensive N-terminal α-acetylation likely affects interactions between pairs of PSII-LHCII supercomplexes across the stromal gap, ultimately mediating membrane folding in grana stacks.

Structural insight into light harvesting for photosystem II in green algae.

Green algae and plants rely on light-harvesting complex II (LHCII) to collect photon energy for oxygenic photosynthesis. In Chlamydomonas reinhardtii, LHCII molecules associate with photosystem II (PSII) to form various supercomplexes, including the C2S2M2L2 type, which is the largest PSII-LHCII supercomplex in algae and plants that is presently known. Here, we report high-resolution cryo-electron microscopy (cryo-EM) maps and structural models of the C2S2M2L2 and C2S2 supercomplexes from C. reinhardtii. The C2S2 supercomplex contains an LhcbM1-LhcbM2/7-LhcbM3 heterotrimer in the strongly associated LHCII, and the LhcbM1 subunit assembles with CP43 through two interfacial galactolipid molecules. The loosely and moderately associated LHCII trimers interact closely with the minor antenna complex CP29 to form an intricate subcomplex bound to CP47 in the C2S2M2L2 supercomplex. A notable direct pathway is established for energy transfer from the loosely associated LHCII to the PSII reaction centre, as well as several indirect routes. Structure-based computational analysis on the excitation energy transfer within the two supercomplexes provides detailed mechanistic insights into the light-harvesting process in green algae.

The Kinase STATE TRANSITION 8 Phosphorylates Light Harvesting Complex II and Contributes to Light Acclimation in Arabidopsis thaliana.

Phosphorylation of the light-harvesting complex II (LHCII) is a central trigger for the reorganization of the photosynthetic complexes in the thylakoid membrane during short-term light acclimation. The major kinase involved in LHCII phosphorylation is STATE TRANSITION 7 (STN7), and its activity is mostly counteracted by a thylakoid-associated phosphatase, PROTEIN PHOSPHATASE 1/THYLAKOID ASSOCIATED PHOSPHATASE 38 (PPH1/TAP38). This kinase/phosphatase pair responds to the redox status of the photosynthetic electron transport chain. In Arabidopsis thaliana, Lhcb1 and Lhcb2 subunits of the LHCII trimers are the major targets of phosphorylation and have different roles in the acclimation of the photosynthetic machinery. Another antagonistic kinase and phosphatase pair, STATE TRANSITION 8 (STN8) and PHOTOSYSTEM II PHOSPHATASE (PBCP) target a different set of thylakoid proteins. Here, we analyzed double, triple, and quadruple knockout mutants of these kinases and phosphatases. In multiple mutants, lacking STN7, in combination with one or both phosphatases, but not STN8, the phosphorylation of LHCII was partially restored. The recovered phosphorylation favors Lhcb1 over Lhcb2 and results in a better adaptation of the photosynthetic apparatus and increased plant growth under fluctuating light. This set of mutants allowed to unveil a contribution of STN8-dependent phosphorylation in the acclimation to rapid light variations.

Four distinct trimeric forms of light-harvesting complex II isolated from the green alga Chlamydomonas reinhardtii.

Light-harvesting complex II (LHCII) absorbs light energy and transfers it primarily to photosystem II in green algae and land plants. Although the trimeric structure of LHCII is conserved between the two lineages, its subunit composition and function are believed to differ significantly. In this study, we purified four LHCII trimers from the green alga Chlamydomonas reinhardtii and analyzed their biochemical properties. We used several preparation methods to obtain four distinct fractions (fractions 1-4), each of which contained an LHCII trimer with different contents of Type I, III, and IV proteins. The pigment compositions of the LHCIIs in the four fractions were similar. The absorption and fluorescence spectra were also similar, although the peak positions differed slightly. These results indicate that this green alga contains four types of LHCII trimer with different biochemical and spectroscopic features. Based on these findings, we discuss the function and structural organization of green algal LHCII antennae.

Lipid Fingerprints and Cofactor Dynamics of Light-Harvesting Complex II in Different Membranes

Plant light-harvesting complex II (LHCII) is the key antenna complex for plant photosynthesis. We present coarse-grained molecular dynamics simulations of monomeric and trimeric LHCII in a realistic thylakoid membrane environment based on the Martini force field. The coarse-grained protein model has been optimized with respect to atomistic reference simulations. Our simulations provide detailed insights in the thylakoid lipid fingerprint of LHCII which compares well with experimental data from membrane protein purification. Comparing the monomer and trimeric LHCII reveals a stabilizing effect of trimerization on the chromophores as well as the protein. Moreover, the average chromophore distance shortens in the trimer leading to stronger excitonic couplings. When changing the native thylakoid environment to a model membrane the protein flexibility remains constant, whereas the chromophore flexibility is reduced. Overall, the presented LHCII model lays the foundation to investigate the μs dynamics of this key antenna protein of plants.

The positive regulation of putrescine on light-harvesting complex II and excitation energy dissipation in salt-stressed cucumber seedlings

Non-photochemical quenching (NPQ) is an important mechanism employed by higher plants to protect themselves against the adverse effects of excess excitation energy. The regulatory mechanism of exogenous putrescine (Put) on the status of light-harvesting complex II (LHCII) and its dissipation of excess excitation energy in cucumber seedlings (Cucumis sativus L.) were investigated under salt stress in this study. Put significantly increased the maximum quantum photochemical efficiency in photosystem II (Fv/Fm), NPQ, and the actual yield of PSII (ΦPSII) under salt stress. Quantum yield of regulated energy dissipation in PSII (ΦNPQ) significantly increased and non-regulated energy dissipation (ΦNO) decreased when was sprayed on stressed plants. Put increased the expression levels of D1, D2, Lhcb4 and PsbS, thereby minimizing degradation to the monomeric and trimeric states of LHCII in the thylakoid membranes of salt-stressed cucumber leaves. In addition, Put up-regulated the activity of transglutaminase (TGase) and significantly increased the levels of total endogenous polyamines in monomeric and trimeric LHCII under salt stress. These results suggest that Put could play a crucial role in dissipating excess excitation energy by regulating endogenously increased polyamines to alter the adaptation of LHCII, which are most probably involved in an improvement of photochemical efficiency in salt stressed-cucumber seedlings.

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Non-photochemical quenching (NPQ) is an important mechanism employed by higher plants to protect themselves against the adverse effects of excess excitation energy. The regulatory mechanism of exogenous putrescine (Put) on the status of light-harvesting complex II (LHCII) and its dissipation of excess excitation energy in cucumber seedlings (Cucumis sativus L.) were investigated under salt stress in this study. Put significantly increased the maximum quantum photochemical efficiency in photosystem II (Fv/Fm), NPQ, and the actual yield of PSII (ΦPSII) under salt stress. Quantum yield of regulated energy dissipation in PSII (ΦNPQ) significantly increased and non-regulated energy dissipation (ΦNO) decreased when was sprayed on stressed plants. Put increased the expression levels of D1, D2, Lhcb4 and PsbS, thereby minimizing degradation to the monomeric and trimeric states of LHCII in the thylakoid membranes of salt-stressed cucumber leaves. In addition, Put up-regulated the activity of transglutaminase (TGase) and significantly increased the levels of total endogenous polyamines in monomeric and trimeric LHCII under salt stress. These results suggest that Put could play a crucial role in dissipating excess excitation energy by regulating endogenously increased polyamines to alter the adaptation of LHCII, which are most probably involved in an improvement of photochemical efficiency in salt stressed-cucumber seedlings.

Thylakoid membrane dynamics and state transitions in Chlamydomonas reinhardtii under elevated temperature.

Moderately elevated temperatures can induce state transitions in higher plants by phosphorylation of light-harvesting complex II (LHCII). In this study, we exposed unicellular algae Chlamydomonas reinhardtii to moderately elevated temperatures (38°C) for short period of time in the dark to understand the thylakoid membrane dynamics and state transition mechanism. Here we report that under elevated temperatures (1) LHCII gets phosphorylated similar to higher plants and (2) there is decreased absorption cross section of photosystem II (PSII), whereas (3) there is no change in absorption cross section of photosystem I (PSI) indicating that LHCII trimers are largely disconnected with both photosystems under moderately elevated temperatures and (4) on return to room temperature after elevated temperature treatment there is a formation of state transition complex comprising of PSII-LHCII-PSI. The temperature-induced state transition mechanism also depends on stt7 kinase-like in light-induced state transition. The protein content was stable at the moderately elevated temperature treatment of 40°C; however, at 45°C severe downregulation in photosynthetic performance and protein content was observed. In addition to the known changes to photosynthetic apparatus, elevated temperatures can destabilize the PSII-LHCII complex that can result in decreased photosynthetic efficiency in C. reinhardtii. We concluded that the membrane dynamics of light-induced state transitions differs considerably from temperature-induced state transition mechanisms in C. reinhardtii.

Correlated fluorescence quenching and topographic mapping of Light-Harvesting Complex II within surface-assembled aggregates and lipid bilayers

Light-Harvesting Complex II (LHCII) is a chlorophyll-protein antenna complex that efficiently absorbs solar energy and transfers electronic excited states to photosystems I and II. Under excess light intensity LHCII can adopt a photoprotective state in which excitation energy is safely dissipated as heat, a process known as Non-Photochemical Quenching (NPQ). In vivo NPQ is triggered by combinatorial factors including transmembrane ΔpH, PsbS protein and LHCII-bound zeaxanthin, leading to dramatically shortened LHCII fluorescence lifetimes. In vitro, LHCII in detergent solution or in proteoliposomes can reversibly adopt an NPQ-like state, via manipulation of detergent/protein ratio, lipid/protein ratio, pH or pressure. Previous spectroscopic investigations revealed changes in exciton dynamics and protein conformation that accompany quenching, however, LHCII-LHCII interactions have not been extensively studied. Here, we correlated fluorescence lifetime imaging microscopy (FLIM) and atomic force microscopy (AFM) of trimeric LHCII adsorbed to mica substrates and manipulated the environment to cause varying degrees of quenching. AFM showed that LHCII self-assembled onto mica forming 2D-aggregates (25–150 nm width). FLIM determined that LHCII in these aggregates were in a quenched state, with much lower fluorescence lifetimes (~0.25 ns) compared to free LHCII in solution (2.2–3.9 ns). LHCII-LHCII interactions were disrupted by thylakoid lipids or phospholipids, leading to intermediate fluorescent lifetimes (0.6–0.9 ns). To our knowledge, this is the first in vitro correlation of nanoscale membrane imaging with LHCII quenching. Our findings suggest that lipids could play a key role in modulating the extent of LHCII-LHCII interactions within the thylakoid membrane and so the propensity for NPQ activation.

The causes of altered chlorophyll fluorescence quenching induction in the Arabidopsis mutant lacking all minor antenna complexes

Non-photochemical quenching (NPQ) of chlorophyll fluorescence is the process by which excess light energy is harmlessly dissipated within the photosynthetic membrane. The fastest component of NPQ, known as energy-dependent quenching (qE), occurs within minutes, but the site and mechanism of qE remain of great debate. Here, the chlorophyll fluorescence of Arabidopsis thaliana wild type (WT) plants was compared to mutants lacking all minor antenna complexes (NoM). Upon illumination, NoM exhibits altered chlorophyll fluorescence quenching induction (i.e. from the dark-adapted state) characterised by three different stages: (i) a fast quenching component, (ii) transient fluorescence recovery and (iii) a second quenching component. The initial fast quenching component originates in light harvesting complex II (LHCII) trimers and is dependent upon PsbS and the formation of a proton gradient across the thylakoid membrane (ΔpH). Transient fluorescence recovery is likely to occur in both WT and NoM plants, but it cannot be overcome in NoM due to impaired ΔpH formation and a reduced zeaxanthin synthesis rate. Moreover, an enhanced fluorescence emission peak at ~679 nm in NoM plants indicates detachment of LHCII trimers from the bulk antenna system, which could also contribute to the transient fluorescence recovery. Finally, the second quenching component is triggered by both ΔpH and PsbS and enhanced by zeaxanthin synthesis. This study indicates that minor antenna complexes are not essential for qE, but reveals their importance in electron stransport, ΔpH formation and zeaxanthin synthesis.

Investigation of Thermodynamic Dissociation Kinetics to Determine the Binding Strengths within a Membrane Protein Complex: Photosystem II Supercomplex

Protein-protein interactions as well as the formations of protein complexes have been intensively studied by various methods in both in vivo and in vitro. However, although membrane proteins have important roles in cells, interactions within membrane protein complexes have been hardly studied than that within soluble proteins due to the lack of suitable methods, though one approach using mass spectrometry has been recently developed to elucidate membrane protein complexes. Here, we present an approach to determine binding strengths within a membrane protein complex by investigating the thermodynamic dissociation kinetics of an isolated membrane protein complex using time-resolved fluorescence spectroscopy. We demonstrated different bindings of light-harvesting complex II (LHCII) trimers in a photosystem II (PSII) supercomplex isolated from a green alga Chlamydomonas reinhardtii, where the different binding types of the LHCII trimers have been indirectly predicted by the presence or absence of LHCII trimers in negatively stained PSII supercomplex particles observed by electron microscopy but have not been determined. As a result of analysis on thermodynamic dissociation kinetics, we identified three different activation energies for the dissociations of LHCII trimers, which imply the presence of multiple hydrogen bonds and/or ion-dipole bonds between LHCII trimers and core complexes.

Comparison of phosphorylation and assembly of photosystem complexes and redox homeostasis in two wheat cultivars with different drought resistance

Reversible phosphorylation of proteins and the assembly of thylakoid complexes are the important protective mechanism against environmental stresses in plants. This research was aimed to investigate the different responses of the antioxidant defense system and photosystem II (PSII) to osmotic stress between drought-resistant and drought-susceptible wheat cultivars. Results showed that the decrease in PSII photochemistry and six enzyme activities was observed in drought-susceptible wheat compared with drought-resistant wheat under osmotic stress. In addition, a lower accumulation of reactive oxygen species (ROS) and cell death were found in the resistant wheat compared with the susceptible wheat under osmotic stress. Western blot analysis revealed that osmotic stress led to a remarkable decline in the steady state level of D1 protein in drought-susceptible wheat. However, the CP29 protein was strongly phosphorylated in drought-resistant wheat compared with the susceptible wheat under osmotic stress. Our results also showed that drought-resistant wheat presented higher phosphorylated levels of the light-harvesting complex II (LHCII), D1, and D2 proteins and a more rapid dephosphorylated rate than drought-susceptible wheat under osmotic stress. Furthermore, the PSII-LHCII supercomplexes and LHCII trimers were more rapidly disassembled in drought-susceptible wheat than the drought-resistant wheat under osmotic stress. These findings provide that reversible phosphorylation of thylakoid membrane proteins and assembly of thylakoid membrane complexes play important roles in plant adaptation to environmental stresses.

Two mechanisms for dissipation of excess light in monomeric and trimeric light-harvesting complexes.

Oxygenic photoautotrophs require mechanisms for rapidly matching the level of chlorophyll excited states from light harvesting with the rate of electron transport from water to carbon dioxide. These photoprotective reactions prevent formation of reactive excited states and photoinhibition. The fastest response to excess illumination is the so-called non-photochemical quenching which, in higher plants, requires the luminal pH sensor PsbS and other yet unidentified components of the photosystem II antenna. Both trimeric light-harvesting complex II (LHCII) and monomeric LHC proteins have been indicated as site(s) of the heat-dissipative reactions. Different mechanisms have been proposed: energy transfer to a lutein quencher in trimers, formation of a zeaxanthin radical cation in monomers. Here, we report on the construction of a mutant lacking all monomeric LHC proteins but retaining LHCII trimers. Its non-photochemical quenching induction rate was substantially slower with respect to the wild type. A carotenoid radical cation signal was detected in the wild type, although it was lost in the mutant. We conclude that non-photochemical quenching is catalysed by two independent mechanisms, with the fastest activated response catalysed within monomeric LHC proteins depending on both zeaxanthin and lutein and on the formation of a radical cation. Trimeric LHCII was responsible for the slowly activated quenching component whereas inclusion in supercomplexes was not required. This latter activity does not depend on lutein nor on charge transfer events, whereas zeaxanthin was essential.