PsbS enhances nonphotochemical fluorescence quenching in the absence of zeaxanthin

Leaves and chloroplasts from Arabidopsis plants with increased amounts of PsbS protein showed the same percentage increase in nonphotochemical quenching in comparison to the wild type both in the presence and absence of zeaxanthin. The absorption change at 525-535 nm was also more pronounced in both cases. It is suggested that PsbS alone can cause the quenching, supporting the model in which zeaxanthin acts as an allosteric activator and is not the primary cause of the process. It is proposed that PsbS acts as a trigger of the conformational change that leads to the establishment of nonphotochemical quenching.     

Arabidopsis plants lacking PsbS protein possess photoprotective energy dissipation

It is commonly accepted that the photosystem II subunit S protein, PsbS, is required for the dissipation of excess light energy in a process termed ‘non‐photochemical quenching’ (NPQ). This process prevents photo‐oxidative damage of photosystem II (PSII) thus avoiding photoinhibition which can decrease plant fitness and productivity. In this study Arabidopsis plants lacking PsbS (the npq4 mutant) were found to possess a competent mechanism of excess energy dissipation that protects against photoinhibitory damage. The process works on a slower timescale, taking about 1 h to reach the same level of NPQ achieved in the wild type in just a few minutes. The NPQ in npq4 was found to display very similar characteristics to the fast NPQ in the wild type. Firstly, it prevented the irreversible light‐induced closure of PSII reaction centres. Secondly, it was uncoupler‐sensitive, and thus triggered by the ΔpH across the thylakoid membrane. Thirdly, it was accompanied by significant quenching of the fluorescence under conditions when all PSII reaction centres were open (F_o state)_. Fourthly, it was accompanied by NPQ‐related absorption changes (ΔA535). Finally, it was modulated by the presence of the xanthophyll cycle carotenoid zeaxanthin. The existence of a mechanism of photoprotective energy dissipation in plants lacking PsbS suggests that this protein plays the role of a kinetic modulator of the energy dissipation process in the PSII light‐harvesting antenna, allowing plants to rapidly track fluctuations of light intensity in the environment, and is not the primary cause of NPQ or a direct carrier of the pigment acting as the non‐photochemical quencher.     

The xanthophyll cycle pool size controls the kinetics of non-photochemical quenching in Arabidopsis thaliana

Arabidopsis plants overexpressing beta-carotene hydroxylase 1 accumulate over double the amount of zeaxanthin present in wild-type plants. The final amplitude of non-photochemical quenching (NPQ) was found to be the same in these plants, but the kinetics were different. The formation and relaxation of NPQ consistently correlated with the de-epoxidation state of the xanthophyll cycle pool and not the amount of zeaxanthin. These data indicate that zeaxanthin and violaxanthin antagonistically regulate the switch between the light harvesting and photoprotective modes of the light harvesting system and show that control of the xanthophyll cycle pool size is necessary to optimize the kinetics of NPQ.     

The Lhcb protein and xanthophyll composition of the light harvesting antenna controls the DeltapH-dependency of non-photochemical quenching in Arabidopsis thaliana

Nonphotochemical quenching (NPQ) is the photoprotective dissipation of energy in photosynthetic membranes. The hypothesis that the DeltapH-dependent component of NPQ (qE) component of non-photochemical quenching is controlled allosterically by the xanthophyll cycle has been tested using Arabidopsis mutants with different xanthophyll content and composition of Lhcb proteins. The titration curves of qE against DeltapH were different in chloroplasts containing zeaxanthin or violaxanthin, proving their roles as allosteric activator and inhibitor, respectively. The curves differed in mutants deficient in lutein and specific Lhcb proteins. The results show that qE is determined by xanthophyll occupancy and the structural interactions within the antenna that govern allostericity.     

Action potential in a plant cell lowers the light requirement for non-photochemical energy-dependent quenching of chlorophyll fluorescence

This study deals with effects of membrane excitation on photosynthesis and cell protection against excessive light, manifested in non-photochemical quenching (NPQ). In Chara corallina cells, NPQ and pericellular pH displayed coordinated spatial patterns along the length of the cell. The NPQ values were lower in H⁺-extruding cell regions (external pH ∼ 6.5) than in high pH regions (pH ∼ 9.5). Generation of an action potential by applying a pulse of electric current caused NPQ to increase within 30-60 s. This effect, manifested as a long-lived drop of maximum chlorophyll fluorescence (F_m′), occurred at lower photosynthetic flux densities (PFD) in the alkaline as compared to acidic cell regions. The light response curve of NPQ shifted, after generation of an action potential, towards lower PFD. The release of NPQ by nigericin and the rapid reversal of action potential-triggered NPQ in darkness indicate its relation to thylakoid ΔpH. Generation of an action potential shortly after darkening converted the chloroplasts into a latent state with the F_m identical to that of unexcited cells. This state transformed to the quenched state after turning on weak light that was insufficient for NPQ prior to membrane excitation of the cells. The ionophore, A23187, shifted NPQ plots similarly to the action potential effect, consistent with a likely role of a rise in the cytosolic Ca²⁺ level in the action potential-induced quenching. The results suggest that a rapid electric signal, across the plasma membrane, might exert long-lived effects on photosynthesis and chlorophyll fluorescence through ion flux-mediated pathways.     

The PsbS protein controls the organization of the photosystem II antenna in higher plant thylakoid membranes

The PsbS subunit of photosystem II (PSII) plays a key role in nonphotochemical quenching (NPQ), the major photoprotective regulatory mechanism in higher plant thylakoid membranes, but its mechanism of action is unknown. Here we describe direct evidence that PsbS controls the organization of PSII and its light harvesting system (LHCII). The changes in chlorophyll fluorescence amplitude associated with the Mg(2+)-dependent restacking of thylakoid membranes were measured in thylakoids prepared from wild-type plants, a PsbS-deficient mutant and a PsbS overexpresser. The Mg(2+) requirement and sigmoidicity of the titration curves for the fluorescence rise were negatively correlated with the level of PsbS. Using a range of PsbS mutants, this effect of PsbS was shown not to depend upon its efficacy in controlling NPQ, but to be related only to protein concentration. Electron microscopy and fluorescence spectroscopy showed that this effect was because of enhancement of the Mg(2+)-dependent re-association of PSII and LHCII by PsbS, rather than an effect on stacking per se. In the presence of PsbS the LHCII.PSII complex was also more readily removed from thylakoid membranes by detergent, and the level of PsbS protein correlated with the amplitude of the psi-type CD signal originating from features of LHCII.PSII organization. It is proposed that PsbS regulates the interaction between LHCII and PSII in the grana membranes, explaining how it acts as a pH-dependent trigger of the conformational changes within the PSII light harvesting system that result in NPQ.     

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.     

Acclimation- and mutation-induced enhancement of PsbS levels affects the kinetics of non-photochemical quenching in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The efficiency of photosystem II antenna complexes (LHCs) in higher plants must be regulated to avoid potentially damaging overexcitation of the reaction centre in excess light. Regulation is achieved via a feedback mechanism known as non-photochemical quenching (NPQ), triggered the proton gradient (ΔpH) causing heat dissipation within the LHC antenna. ΔpH causes protonation of the LHCs, the PsbS protein and triggers the enzymatic de-epoxidation of the xanthophyll, violaxanthin, to zeaxanthin. A key step in understanding the mechanism is to decipher whether PsbS and zeaxanthin cooperate to promote NPQ. To obtain clues about their respective functions we studied the effects of PsbS and zeaxanthin on the rates of NPQ formation and relaxation in wild-type Arabidopsis leaves and those overexpressing PsbS (L17) or lacking zeaxanthin (npq1). Overexpression of PsbS was found to increase the rate of NPQ formation, as previously reported for zeaxanthin. However, PsbS overexpression also increased the rate of NPQ relaxation, unlike zeaxanthin, which is known decrease the rate. The enhancement of PsbS levels in plants lacking zeaxanthin (npq1) by either acclimation to high light or crossing with L17 plants showed that the effect of PsbS was independent of zeaxanthin. PsbS levels also affected the kinetics of the 535 nm absorption change (ΔA535), which monitors the formation of the conformational state of the LHC antenna associated with NPQ, in an identical way. The antagonistic action of PsbS and zeaxanthin with respect to NPQ and ΔA535 relaxation kinetics suggests that the two molecules have distinct regulatory functions.     

Adjustment of photosynthetic activity to drought and fluctuating light in wheat

Drought is a major cause of losses in crop yield. Under field conditions, plants exposed to drought are usually also experiencing rapid changes in light intensity. Accordingly, plants need to acclimate to both, drought and light stress. Two crucial mechanisms in plant acclimation to changes in light conditions comprise thylakoid protein phosphorylation and dissipation of light energy as heat by non-photochemical quenching (NPQ). Here, we analyzed the acclimation efficacy of two different wheat varieties, by applying fluctuating light for analysis of plants, which had been subjected to a slowly developing drought stress as it usually occurs in the field. This novel approach allowed us to distinguish four drought phases, which are critical for grain yield, and to discover acclimatory responses which are independent of photodamage. In short-term, under fluctuating light, the slowdown of NPQ relaxation adjusts the photosynthetic activity to the reduced metabolic capacity. In long-term, the photosynthetic machinery acquires a drought-specific configuration by changing the PSII-LHCII phosphorylation pattern together with protein stoichiometry. Therefore, the fine-tuning of NPQ relaxation and PSII-LHCII phosphorylation pattern represent promising traits for future crop breeding strategies.     

The relationship between maximum tolerated light intensity and photoprotective energy dissipation in the photosynthetic antenna: chloroplast gains and losses

The principle of quantifying the efficiency of protection of photosystem II (PSII) reaction centres against photoinhibition by non-photochemical energy dissipation (NPQ) has been recently introduced by Ruban & Murchie (2012 Biochim. Biophys. Acta 1817, 977–982 (doi:10.1016/j.bbabio.2012.03.026)). This is based upon the assessment of two key parameters: (i) the relationship between the PSII yield and NPQ, and (ii) the fraction of intact PSII reaction centres in the dark after illumination. In this paper, we have quantified the relationship between the amplitude of NPQ and the light intensity at which all PSII reaction centres remain intact for plants with different levels of PsbS protein, known to play a key role in the process. It was found that the same, nearly linear, relationship exists between the levels of the protective NPQ component (pNPQ) and the tolerated light intensity in all types of studied plants. This approach allowed for the quantification of the maximum tolerated light intensity, the light intensity at which all plant leaves become photoinhibited, the fraction of (most likely) unnecessary or ‘wasteful’ NPQ, and the fraction of photoinhibited PSII reaction centres under conditions of prolonged illumination by full sunlight. It was concluded that the governing factors in the photoprotection of PSII are the level and rate of protective pNPQ formation, which are often in discord with the amplitude of the conventional measure of photoprotection, the quickly reversible NPQ component, qE. Hence, we recommend pNPQ as a more informative and less ambiguous parameter than qE, as it reflects the effectiveness and limitations of the major photoprotective process of the photosynthetic membrane.     

毛竹PsbS基因的克隆及其原核表达特征研究

采用RT-PCR技术从毛竹(Phyllostachys edulis)叶片中克隆到1个PsbS基因,命名为PePsbS (GenBank No. FJ600727),其编码区为810 bp,编码269个氨基酸。序列分析表明,PePsbS编码的蛋白与其它单子叶植物的PsbS蛋白有很高的相似性。蛋白结构分析表明,PePsbS基因编码蛋白包含导肽部分和成熟蛋白,其中成熟蛋白包含4个跨膜结构域。将PePsbS基因编码成熟蛋白的序列构建到原核表达载体pET23a中,并转入大肠杆菌,用IPTG进行诱导表达。结果表明, 41℃下诱导4 h的表达效果最好,目的蛋白含量约占总蛋白的21.5%,分子量约为22.0 kD。这说明温度和诱导时间明显影响PePsbS基因的表达。     

Dark-adapted spinach thylakoid protein heterogeneity offers insights into the photosystem II repair cycle

In higher plants, thylakoid membrane protein complexes show lateral heterogeneity in their distribution: photosystem (PS) II complexes are mostly located in grana stacks, whereas PSI and adenosine triphosphate (ATP) synthase are mostly found in the stroma-exposed thylakoids. However, recent research has revealed strong dynamics in distribution of photosystems and their light harvesting antenna along the thylakoid membrane. Here, the dark-adapted spinach (Spinacia oleracea L.) thylakoid network was mechanically fragmented and the composition of distinct PSII-related proteins in various thylakoid subdomains was analyzed in order to get more insights into the composition and localization of various PSII subcomplexes and auxiliary proteins during the PSII repair cycle. Most of the PSII subunits followed rather equal distribution with roughly 70% of the proteins located collectively in the grana thylakoids and grana margins; however, the low molecular mass subunits PsbW and PsbX as well as the PsbS proteins were found to be more exclusively located in grana thylakoids. The auxiliary proteins assisting in repair cycle of PSII were mostly located in stroma-exposed thylakoids, with the exception of THYLAKOID LUMEN PROTEIN OF 18.3 (TLP18.3), which was more evenly distributed between the grana and stroma thylakoids. The TL29 protein was present exclusively in grana thylakoids. Intriguingly, PROTON GRADIENT REGULATION5 (PGR5) was found to be distributed quite evenly between grana and stroma thylakoids, whereas PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE1 (PGRL1) was highly enriched in the stroma thylakoids and practically missing from the grana cores. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

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.     

Restoration of rapidly reversible photoprotective energy dissipation in the absence of PsbS protein by enhanced DeltapH

Variations in the light environment require higher plants to regulate the light harvesting process. Under high light a mechanism known as non-photochemical quenching (NPQ) is triggered to dissipate excess absorbed light energy within the photosystem II (PSII) antenna as heat, preventing photodamage to the reaction center. The major component of NPQ, known as qE, is rapidly reversible in the dark and dependent upon the transmembrane proton gradient (ΔpH), formed as a result of photosynthetic electron transport. Using diaminodurene and phenazine metasulfate, mediators of cyclic electron flow around photosystem I, to enhance ΔpH, it is demonstrated that rapidly reversible qE-type quenching can be observed in intact chloroplasts from Arabidopsis plants lacking the PsbS protein, previously believed to be indispensible for the process. The qE in chloroplasts lacking PsbS significantly quenched the level of fluorescence when all PSII reaction centers were in the open state (F(o) state), protected PSII reaction centers from photoinhibition, was modulated by zeaxanthin and was accompanied by the qE-typical absorption spectral changes, known as ΔA(535). Titrations of the ΔpH dependence of qE in the absence of PsbS reveal that this protein affects the cooperativity and sensitivity of the photoprotective process to protons. The roles of PsbS and zeaxanthin are discussed in light of their involvement in the control of the proton-antenna association constant, pK, via regulation of the interconnected phenomena of PSII antenna reorganization/aggregation and hydrophobicity.     

Photoprotective energy quenching in the red alga Porphyridium purpureum occurs at the core antenna of the photosystem II but not at its reaction center

Photosynthetic organisms have evolved light-harvesting antennae over time. In cyanobacteria, external phycobilisomes (PBSs) are the dominant antennae, whereas in green algae and higher plants, PBSs have been replaced by proteins of the Lhc family that are integrated in the membrane. Red algae represent an evolutionary intermediate between these two systems, as they employ both PBSs and membrane LHCR proteins as light-harvesting units. Understanding how red algae cope with light is not only interesting for biotechnological applications, but is also of evolutionary interest. For example, energy-dependent quenching (qE) is an essential photoprotective mechanism widely used by species from cyanobacteria to higher plants to avoid light damage; however, the quenching mechanism in red algae remains largely unexplored. Here, we used both pulse amplitude-modulated (PAM) and time-resolved chlorophyll fluorescence to characterize qE kinetics in the red alga Porphyridium purpureum. PAM traces confirmed that qE in P. purpureum is activated by a decrease in the thylakoid lumen pH, whereas time-resolved fluorescence results further revealed the quenching site and ultrafast quenching kinetics. We found that quenching exclusively takes place in the photosystem II (PSII) complexes and preferentially occurs at PSII’s core antenna rather than at its reaction center, with an overall quenching rate of 17.6 ± 3.0 ns⁻¹. In conclusion, we propose that qE in red algae is not a reaction center type of quenching, and that there might be a membrane-bound protein that resembles PsbS of higher plants or LHCSR of green algae that senses low luminal pH and triggers qE in red algae.     

Significance of the lipid phase in the dynamics and functions of the xanthophyll cycle as revealed by PsbS overexpression in tobacco and in-vitro de-epoxidation in monogalactosyldiacylglycerol micelles

The dynamics of the xanthophyll cycle relative to non-photochemical quenching (NPQ) were examined in tobacco plants overexpressing violaxanthin de-epoxidase (VDE), PsbS and PsbS+VDE for effects on NPQ and violaxanthin (V) de-epoxidation over a range of light intensities. Induction of de-epoxidation and NPQ increased in overexpressed VDE and PsbS plants, respectively. Surprisingly, under low light, overexpressing PsbS enhanced de-epoxidation in addition to NPQ. The effect was hypothesized as due to PsbS binding zeaxanthin (Z) or inducing the binding of Z within the quenching complex, thus shifting the equilibrium toward higher de-epoxidation states. Studies in model systems show that Z can stereospecifically inhibit VDE activity against violaxanthin. This effect, observed under conditions of limiting lipid concentration, was interpreted as product feedback inhibition. These results support the hypothesis that the capacity of the thylakoid lipid phase for xanthophylls is limited and modulates xanthophyll-cycle activity, in conjunction with the release of V and binding of Z by pigment-binding proteins. These modulating factors are incorporated into a lipid-matrix model that has elements of a signal transduction system wherein the light-generated protons are the signal, VDE the signal receptor, Z the secondary messenger, the lipid phase the transduction network, and Z-binding proteins the targets.     

PsbS contributes to photoprotection in Chlamydomonas reinhardtii independently of energy dissipation

Photosynthetic organisms are frequently exposed to excess light conditions and hence to photo-oxidative stress. To counteract photo-oxidative damage, land plants and most algae make use of non- photochemical quenching (NPQ) of excess light energy, in particular the rapidly inducible and relaxing qE-mechanism. In vascular plants, the constitutively active PsbS protein is the key regulator of qE. In the green algae C. reinhardtii, however, qE activation is only possible after initial high-light (HL) acclimation for several hours and requires the synthesis of LHCSR proteins which act as qE regulators. The precise function of PsbS, which is transiently expressed during HL acclimation in C. reinhardtii, is still unclear. Here, we investigated the impact of different PsbS amounts on HL acclimation characteristics of C. reinhardtii cells. We demonstrate that lower PsbS amounts negatively affect HL acclimation at different levels, including NPQ capacity, electron transport characteristics, antenna organization and morphological changes, resulting in an overall increased HL sensitivity and lower vitality of cells. Contrarily, higher PsbS amounts do not result in a higher NPQ capacity, but nevertheless provide higher fitness and tolerance towards HL stress. Strikingly, constitutively expressed PsbS protein was found to be degraded during HL acclimation. We propose that PsbS is transiently required during HL acclimation for the reorganization of thylakoid membranes and/or antenna proteins along with the activation of NPQ and adjustment of electron transfer characteristics, and that degradation of PsbS is essential in the fully HL acclimated state.     

The photoprotective protein PsbS exerts control over CO(2) assimilation rate in fluctuating light in rice

A direct impact of chloroplastic protective energy dissipation (qE) on photosynthetic CO(2) assimilation has not been shown directly in plants in the absence of photoinhibition. To test this empirically we transformed rice to possess higher (overexpressors, OE) and lower (RNA interference, RNAi) levels of expression of the regulatory psbS gene and analysed CO(2) assimilation in transformants in a fluctuating measurement light regime. Western blots showed a several-fold difference in levels of PsbS protein between RNAi and OE plants with the wild type (WT) being intermediate. At a growth light intensity of 600 μmol m(-2) sec(-1) , the carboxylation capacity, electron transport capacity and dark adapted F(v)/F(m) (ratio of variable to maximum fluorescence) were inhibited in RNAi plants compared with WT and OE. The PsbS content had a significant impact on qE (measured here as non-photochemical quenching, NPQ) but the strongest effect was observed transiently, immediately following the application of light. This capacity for qE was several-fold lower in RNAi plants and significantly higher in OE plants during the first 10 min of illumination. At steady state the differences were reduced: notably at 500 μmol m(-2) sec(-1) all plants had the same NPQ values regardless of PsbS content. During a series of light-dark transitions the induction of CO(2) assimilation was inhibited in OE plants, reducing integrated photosynthesis during the light period. We conclude that the accumulation of PsbS and the resultant qE exerts control over photosynthesis in fluctuating light, showing that optimization of photoprotective processes is necessary for maximum photosynthetic productivity even in the absence of photoinhibitory stress.     

A novel method produces native light-harvesting complex II aggregates from the photosynthetic membrane revealing their role in nonphotochemical quenching

Nonphotochemical quenching (NPQ) is a mechanism of regulating light harvesting that protects the photosynthetic apparatus from photodamage by dissipating excess absorbed excitation energy as heat. In higher plants, the major light-harvesting antenna complex (LHCII) of photosystem (PS) II is directly involved in NPQ. The aggregation of LHCII is proposed to be involved in quenching. However, the lack of success in isolating native LHCII aggregates has limited the direct interrogation of this process. The isolation of LHCII in its native state from thylakoid membranes has been problematic because of the use of detergent, which tends to dissociate loosely bound proteins, and the abundance of pigment–protein complexes (e.g. PSI and PSII) embedded in the photosynthetic membrane, which hinders the preparation of aggregated LHCII. Here, we used a novel purification method employing detergent and amphipols to entrap LHCII in its natural states. To enrich the photosynthetic membrane with the major LHCII, we used Arabidopsis thaliana plants lacking the PSII minor antenna complexes (NoM), treated with lincomycin to inhibit the synthesis of PSI and PSII core proteins. Using sucrose density gradients, we succeeded in isolating the trimeric and aggregated forms of LHCII antenna. Violaxanthin- and zeaxanthin-enriched complexes were investigated in dark-adapted, NPQ, and dark recovery states. Zeaxanthin-enriched antenna complexes showed the greatest amount of aggregated LHCII. Notably, the amount of aggregated LHCII decreased upon relaxation of NPQ. Employing this novel preparative method, we obtained a direct evidence for the role of in vivo LHCII aggregation in NPQ.