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.     

A possible molecular basis for photoprotection in the minor antenna proteins of plants

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.     

Thylakoid protein phosphorylation in dynamic regulation of photosystem II in higher plants

In higher plants, the photosystem (PS) II core and its several light harvesting antenna (LHCII) proteins undergo reversible phosphorylation cycles according to the light intensity. High light intensity induces strong phosphorylation of the PSII core proteins and suppresses the phosphorylation level of the LHCII proteins. Decrease in light intensity, in turn, suppresses the phosphorylation of PSII core, but strongly induces the phosphorylation of LHCII. Reversible and differential phosphorylation of the PSII-LHCII proteins is dependent on the interplay between the STN7 and STN8 kinases, and the respective phosphatases. The STN7 kinase phosphorylates the LHCII proteins and to a lesser extent also the PSII core proteins D1, D2 and CP43. The STN8 kinase, on the contrary, is rather specific for the PSII core proteins. Mechanistically, the PSII-LHCII protein phosphorylation is required for optimal mobility of the PSII-LHCII protein complexes along the thylakoid membrane. Physiologically, the phosphorylation of LHCII is a prerequisite for sufficient excitation of PSI, enabling the excitation and redox balance between PSII and PSI under low irradiance, when excitation energy transfer from the LHCII antenna to the two photosystems is efficient and thermal dissipation of excitation energy (NPQ) is minimised. The importance of PSII core protein phosphorylation is manifested under highlight when the photodamage of PSII is rapid and phosphorylation is required to facilitate the migration of damaged PSII from grana stacks to stroma lamellae for repair. The importance of thylakoid protein phosphorylation is highlighted under fluctuating intensity of light where the STN7 kinase dependent balancing of electron transfer is a prerequisite for optimal growth and development of the plant. This article is part of a Special Issue entitled: Photosystem II.     

Photoprotection in plants involves a change in lutein 1 binding domain in the major light-harvesting complex of photosystem II

Nonphotochemical quenching (NPQ) is the fundamental process by which plants exposed to high light intensities dissipate the potentially harmful excess energy as heat. Recently, it has been shown that efficient energy dissipation can be induced in the major light-harvesting complexes of photosystem II (LHCII) in the absence of protein-protein interactions. Spectroscopic measurements on these samples (LHCII gels) in the quenched state revealed specific alterations in the absorption and circular dichroism bands assigned to neoxanthin and lutein 1 molecules. In this work, we investigate the changes in conformation of the pigments involved in NPQ using resonance Raman spectroscopy. By selective excitation we show that, as well as the twisting of neoxanthin that has been reported previously, the lutein 1 pigment also undergoes a significant change in conformation when LHCII switches to the energy dissipative state. Selective two-photon excitation of carotenoid (Car) dark states (Car S(1)) performed on LHCII gels shows that the extent of electronic interactions between Car S(1) and chlorophyll states correlates linearly with chlorophyll fluorescence quenching, as observed previously for isolated LHCII (aggregated versus trimeric) and whole plants (with versus without NPQ).     

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.     

Psychrophily is associated with differential energy partitioning, photosystem stoichiometry and polypeptide phosphorylation in Chlamydomonas raudensis

Chlamydomonas raudensis UWO 241 and SAG 49.72 represent the psychrophilic and mesophilic strains of this green algal species. This novel discovery was exploited to assess the role of psychrophily in photoacclimation to growth temperature and growth irradiance. At their optimal growth temperatures of 8 degrees C and 28 degrees C respectively, UWO 241 and SAG 49.72 maintained comparable photostasis, that is energy balance, as measured by PSII excitation pressure. Although UWO 241 exhibited higher excitation pressure, measured as 1-qL, at all growth light intensities, the relative changes in 1-qL were similar to that of SAG 49.72 in response to growth light. In response to suboptimal temperatures and increased growth irradiance, SAG 49.72 favoured energy partitioning of excess excitation energy through inducible, down regulatory processes (Phi(NPQ)) associated with the xanthophyll cycle and antenna quenching, while UWO 241 favoured xanthophyll cycle-independent energy partitioning through constitutive processes involved in energy dissipation (Phi(NO)). In contrast to SAG 49.72, an elevation in growth temperature induced an increase in PSI/PSII stoichiometry in UWO 241. Furthermore, SAG 49.72 showed typical threonine-phosphorylation of LHCII, whereas UWO 241 exhibited phosphorylation of polypeptides of comparable molecular mass to PSI reaction centres but the absence of LHCII phosphorylation. Thus, although both strains maintain an energy balance irrespective of their differences in optimal growth temperatures, the mechanisms used to maintain photostasis were distinct. We conclude that psychrophily in C. raudensis is complex and appears to involve differential energy partitioning, photosystem stoichiometry and polypeptide phosphorylation.     

Control of the light harvesting function of chloroplast membranes: the LHCII-aggregation model for non-photochemical quenching

Dissipation of excess excitation energy within the photosystem II light-harvesting antenna (LHCII) by non-photochemical quenching (NPQ) is an important photoprotective process in plants. An update to a hypothesis for the mechanism of NPQ [FEBS Letters 292, 1991] is presented. The impact of recent advances in understanding the structure, organisation and photophysics of LHCII is assessed. We show possible locations of the predicted regulatory and quenching pigment-binding sites in the structural model of the major LHCII. We suggest that NPQ is a highly regulated concerted response of the organised thylakoid macrostructure, which can include different mechanisms and sites at different times.     

Thylakoid Protein Phosphorylation in Higher Plant Chloroplasts Optimizes Electron Transfer under Fluctuating Light

Several proteins of photosystem II (PSII) and its light-harvesting antenna (LHCII) are reversibly phosphorylated according to light quantity and quality. Nevertheless, the interdependence of protein phosphorylation, nonphotochemical quenching, and efficiency of electron transfer in the thylakoid membrane has remained elusive. These questions were addressed by investigating in parallel the wild type and the stn7, stn8, and stn7 stn8 kinase mutants of Arabidopsis (Arabidopsis thaliana), using the stn7 npq4, npq4, npq1, and pgr5 mutants as controls. Phosphorylation of PSII-LHCII proteins is strongly and dynamically regulated according to white light intensity. Yet, the changes in phosphorylation do not notably modify the relative excitation energy distribution between PSII and PSI, as typically occurs when phosphorylation is induced by “state 2” light that selectively excites PSII and induces the phosphorylation of both the PSII core and LHCII proteins. On the contrary, under low-light conditions, when excitation energy transfer from LHCII to reaction centers is efficient, the STN7-dependent LHCII protein phosphorylation guarantees a balanced distribution of excitation energy to both photosystems. The importance of this regulation diminishes at high light upon induction of thermal dissipation of excitation energy. Lack of the STN7 kinase, and thus the capacity for equal distribution of excitation energy to PSII and PSI, causes relative overexcitation of PSII under low light but not under high light, leading to disturbed maintenance of fluent electron flow under fluctuating light intensities. The physiological relevance of the STN7-dependent regulation is evidenced by severely stunted phenotypes of the stn7 and stn7 stn8 mutants under strongly fluctuating light conditions.Several proteins of PSII and its light-harvesting antenna (LHCII) are reversibly phosphorylated by the STN7 and STN8 kinase-dependent pathways according to the intensity and quality of light (Bellafiore et al., 2005; Bonardi et al., 2005). The best-known phosphorylation-dependent phenomenon in the thylakoid membrane is the state transition: a regulatory mechanism that modulates the light-harvesting capacity between PSII and PSI. According to the traditional view, “state 1” prevails when plants are exposed to far-red light (state 1 light), which selectively excites PSI. Alternatively, thylakoids are in “state 2” when plants are exposed to blue or red light (state 2 light), favoring PSII excitation. In state 1, the yield of fluorescence from PSII is higher in comparison with state 2 (for review, see Allen and Forsberg, 2001). State transitions are dependent on the phosphorylation of LHCII proteins (Bellafiore et al., 2005) and their association with PSI proteins, particularly PSI-H (Lunde et al., 2000). Under state 2 light, both the PSII core and LHCII proteins are strongly phosphorylated, whereas the state 1 light induces dephosphorylation of both the PSII core and LHCII phosphoproteins (Piippo et al., 2006; Tikkanen et al., 2006). In nature, however, such extreme changes in light quality rarely occur. The intensity of light, on the contrary, fluctuates frequently in all natural habitats occupied by photosynthetic organisms, thus constantly modulating the extent of thylakoid protein phosphorylation in a highly dynamic manner (Tikkanen et al., 2008a).The regulation of PSII-LHCII protein phosphorylation by the quantity of light is much more complex than the regulatory circuits induced by the state 1 and state 2 lights. Whereas changes in light quality induce a concurrent increase or decrease in the phosphorylation levels of both the PSII core (D1, D2, and CP43) and LHCII (Lhcb1 and Lhcb2) proteins, the changes in white light intensity may influence the kinetics of PSII core and LHCII protein phosphorylation in higher plant chloroplasts even in opposite directions (Tikkanen et al., 2008a). Indeed, it is well documented that low light (LL; i.e. lower than that generally experienced during growth) induces strong phosphorylation of LHCII but relatively weak phosphorylation of the PSII core proteins. Exposure of plants to high light (HL) intensities, on the contrary, promotes the phosphorylation of PSII core proteins but inhibits the activity of the LHCII kinase, leading to dephosphorylation of LHCII proteins (Rintamäki et al., 2000; Hou et al., 2003).Thylakoid protein phosphorylation induces dynamic migrations of PSII-LHCII proteins along the thylakoid membrane (Bassi et al., 1988; Iwai et al., 2008) and modulation of thylakoid ultrastructure (Chuartzman et al., 2008). According to the traditional state transition theory, the phosphorylation of LHCII proteins decreases the antenna size of PSII and increases that of PSI, which is reflected as a quenched fluorescence emission from PSII. Alternatively, subsequent dephosphorylation of LHCII increases the antenna size of PSII and decreases that of PSI, which in turn is seen as increased PSII fluorescence (Bennett et al., 1980; Allen et al., 1981; Allen and Forsberg, 2001). This view was recently challenged based on studies with thylakoid membrane fractions, revealing that modulations in the relative distribution of excitation energy between PSII and PSI by LHCII phosphorylation specifically occur in the areas of grana margins, where both PSII and PSI function under the same antenna system, and the energy distribution between the photosystems is regulated via a more subtle mechanism than just the robust migration of phosphorylated LHCII (Tikkanen et al., 2008b). It has also been reported that most of the PSI reaction centers are located in the grana margins in a close vicinity to PSII-LHCII-rich grana thylakoids (Kaftan et al., 2002), providing a perfect framework for the regulation of excitation energy distribution from LHCII to both PSII and PSI.When considering the natural light conditions, the HL intensities are the only known light conditions that in higher plant chloroplasts specifically dephosphorylate only the LHCII proteins but not the PSII core proteins. However, such light conditions do not lead to enhanced function of PSII. Instead, the HL conditions strongly down-regulate the function of PSII via nonphotochemical quenching of excitation energy (NPQ) and PSII photoinhibition (for review, see Niyogi, 1999). On the other hand, after dark acclimation of leaves and relaxation of NPQ, PSII functions much more efficiently when plants/leaves are transferred to LL despite strong phosphorylation of LHCII, as compared with the low phosphorylation state of LHCII upon transfer to HL conditions.The delicate regulation of thylakoid protein phosphorylation in higher plant chloroplasts according to prevailing light intensity is difficult to integrate with the traditional theory of state transitions (i.e. the regulation of the absorption cross-section of PSII and PSI by reversible phosphorylation of LHCII). Moreover, besides LHCII proteins, reversible phosphorylation of the PSII core proteins may also play a role in dynamic light acclimation of plants. Recently, we demonstrated that the PSII core protein phosphorylation is a prerequisite for controlled turnover of the PSII reaction center protein D1 upon photodamage (Tikkanen et al., 2008a). This, however, does not exclude the possibility that the strict regulation of PSII core protein phosphorylation is also connected to the regulation of light harvesting and photosynthetic electron transfer. Moreover, the interactions between PSII and LHCII protein phosphorylation, nonphotochemical quenching, and cyclic electron flow around PSI in the regulation of photosynthetic electron transfer reactions remain poorly understood. To gain a deeper insight into such regulatory networks, we explored the effect of strongly fluctuating white light on chlorophyll (chl) fluorescence in Arabidopsis (Arabidopsis thaliana) mutants differentially deficient in PSII-LHCII protein phosphorylation and/or the regulatory systems of NPQ.     

Two lutein molecules in LHCII have different conformations and functions: Insights into the molecular mechanism of thermal dissipation in plants

When LHCII forms aggregates, the internal conformational changes will result in chlorophyll fluorescence quenching. Uncovering the molecular mechanism of this phenomenon will help us to understand how plants dissipate the excess excitation energy through non-photochemical quenching (NPQ) process. The crystal structure of spinach and pea LHCII have been published, and recently, we solved another crystal structure of LHCII from cucumber at 2.66A resolution. Here we present the first direct structural evidence indicating that the two lutein(Lut) molecules bound in each LHCII monomer have different conformations, Lut621 has a more twisted conformation than that of Lut620. The intimate interaction between the Lut620 and Chla612/Chla611 dimer leads to form a hetero-trimer, which is considered to be a potential quenching site. We also discovered that the dehydration of the LHCII crystals resulted in a notable shrinkage of the crystal unit cell dimensions which was accompanied by a red-shift of the fluorescence emission spectra of the crystals. These phenomena suggest the changes in the crystal packing during dehydration might be the cause of internal conformational changes within LHCII. We proposed a conformational change related NPQ model based on the structure analysis.     

Aggregation of light-harvesting complex II leads to formation of efficient excitation energy traps in monomeric and trimeric complexes

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.     

Assembly of the major light-harvesting complex II in lipid nanodiscs

Self-aggregation of isolated plant light-harvesting complexes (LHCs) upon detergent extraction is associated with fluorescence quenching and is used as an in vitro model to study the photophysical processes of nonphotochemical quenching (NPQ). In the NPQ state, in vivo induced under excess solar light conditions, harmful excitation energy is safely dissipated as heat. To prevent self-aggregation and probe the conformations of LHCs in a lipid environment devoid from detergent interactions, we assembled LHCII trimer complexes into lipid nanodiscs consisting of a bilayer lipid matrix surrounded by a membrane scaffold protein (MSP). The LHCII nanodiscs were characterized by fluorescence spectroscopy and found to be in an unquenched, fluorescent state. Remarkably, the absorbance spectra of LHCII in lipid nanodiscs show fine structure in the carotenoid and Q_y region that is different from unquenched, detergent-solubilized LHCII but similar to that of self-aggregated, quenched LHCII in low-detergent buffer without magnesium ions. The nanodisc data presented here suggest that 1), LHCII pigment-protein complexes undergo conformational changes upon assembly in nanodiscs that are not correlated with downregulation of its light-harvesting function; and 2), these effects can be separated from quenching and aggregation-related phenomena. This will expand our present view of the conformational flexibility of LHCII in different microenvironments.     

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.     

Origin of absorption changes associated with photoprotective energy dissipation in the absence of zeaxanthin

To prevent photo-oxidative damage to the photosynthetic membrane in strong light, plants dissipate excess absorbed light energy as heat in a mechanism known as non-photochemical quenching (NPQ). NPQ is triggered by the trans-membrane proton gradient (ΔpH), which causes the protonation of the photosystem II light-harvesting antenna (LHCII) and the PsbS protein, as well as the de-epoxidation of the xanthophyll violaxanthin to zeaxanthin. The combination of these factors brings about formation of dissipative pigment interactions that quench the excess energy. The formation of NPQ is associated with certain absorption changes that have been suggested to reflect a conformational change in LHCII brought about by its protonation. The light-minus-dark recovery absorption difference spectrum is characterized by a series of positive and negative bands, the best known of which is ΔA(535). Light-minus-dark recovery resonance Raman difference spectra performed at the wavelength of the absorption change of interest allows identification of the pigment responsible from its unique vibrational signature. Using this technique, the origin of ΔA(535) was previously shown to be a subpopulation of red-shifted zeaxanthin molecules. In the absence of zeaxanthin (and antheraxanthin), a proportion of NPQ remains, and the ΔA(535) change is blue-shifted to 525 nm (ΔA(525)). Using resonance Raman spectroscopy, it is shown that the ΔA(525) absorption change in Arabidopsis leaves lacking zeaxanthin belongs to a red-shifted subpopulation of violaxanthin molecules formed during NPQ. The presence of the same ΔA(535) and ΔA(525) Raman signatures in vitro in aggregated LHCII, containing zeaxanthin and violaxanthin, respectively, leads to a new proposal for the origin of the xanthophyll red shifts associated with NPQ.     

Role of Plastid Protein Phosphatase TAP38 in LHCII Dephosphorylation and Thylakoid Electron Flow

Short-term changes in illumination elicit alterations in thylakoid protein phosphorylation and reorganization of the photosynthetic machinery. Phosphorylation of LHCII, the light-harvesting complex of photosystem II, facilitates its relocation to photosystem I and permits excitation energy redistribution between the photosystems (state transitions). The protein kinase STN7 is required for LHCII phosphorylation and state transitions in the flowering plant Arabidopsis thaliana. LHCII phosphorylation is reversible, but extensive efforts to identify the protein phosphatase(s) that dephosphorylate LHCII have been unsuccessful. Here, we show that the thylakoid-associated phosphatase TAP38 is required for LHCII dephosphorylation and for the transition from state 2 to state 1 in A. thaliana. In tap38 mutants, thylakoid electron flow is enhanced, resulting in more rapid growth under constant low-light regimes. TAP38 gene overexpression markedly decreases LHCII phosphorylation and inhibits state 1→2 transition, thus mimicking the stn7 phenotype. Furthermore, the recombinant TAP38 protein is able, in an in vitro assay, to directly dephosphorylate LHCII. The dependence of LHCII dephosphorylation upon TAP38 dosage, together with the in vitro TAP38-mediated dephosphorylation of LHCII, suggests that TAP38 directly acts on LHCII. Although reversible phosphorylation of LHCII and state transitions are crucial for plant fitness under natural light conditions, LHCII hyperphosphorylation associated with an arrest of photosynthesis in state 2 due to inactivation of TAP38 improves photosynthetic performance and plant growth under state 2-favoring light conditions.     

Enhanced function of non-photoinhibited photosystem II complexes upon PSII photoinhibition

Light induced photosystem (PS)II photoinhibition inactivates and irreversibly damages the reaction center protein(s) but the light harvesting complexes continue the collection of light energy. Here we addressed the consequences of such a situation on thylakoid light harvesting and electron transfer reactions. For this purpose, Arabidopsis thaliana leaves were subjected to investigation of the function and regulation of the photosynthetic machinery after a distinct portion of PSII centers had experienced photoinhibition in the presence and absence of Lincomycin (Lin), a commonly used agent to block the repair of damaged PSII centers. In the absence of Lin, photoinhibition increased the relative excitation of PSII and decreased NPQ, together enhancing the electron transfer from still functional PSII centers to PSI. In contrast, in the presence of Lin, PSII photoinhibition increased the relative excitation of PSI and led to strong oxidation of the electron transfer chain. We hypothesize that plants are able to minimize the detrimental effects of high-light illumination on PSII by modulating the energy and electron transfer, but lose such a capability if the repair cycle is arrested. It is further hypothesized that dynamic regulation of the LHCII system has a pivotal role in the control of excitation energy transfer upon PSII damage and repair cycle to maintain the photosynthesis safe and efficient.     

植物光合机构的状态转换

植物光合机构的状态转换是一种通过光系统Ⅱ的捕光天线色素蛋白复合体（LHCⅡ）的可逆磷酸化调节激发能在两个光系统间的分配来适应环境中光质等短期变化的机制．一般植物光合机构的LHCⅡ磷酸化主要受电子递体质醌和细胞色素b6f复合体氧化还原状态的调节,从而影响其在两种光系统间的移动。植物光合机构的状态转换也可以通过两种光系统相互接近导致激发能满溢来平衡两个光系统的激发能分配。外界离子浓度骤变可以引起盐藻LHCⅡ磷酸化,其调节过程与电子递体的氧化还原状态无关。绿藻的状态转换可以调节细胞内的ATP供求关系。     

Redox signalling and the structural basis of regulation of photosynthesis by protein phosphorylation

In photosynthesis in chloroplasts and cyanobacteria, redox control of thylakoid protein phosphorylation regulates distribution of absorbed excitation energy between the two photosystems. When electron transfer through chloroplast photosystem II (PSII) proceeds at a rate higher than that through photosystem I (PSI), chemical reduction of a redox sensor activates a thylakoid protein kinase that catalyses phosphorylation of light-harvesting complex II (LHCII). Phosphorylation of LHCII increases its affinity for PSI and thus redistributes light-harvesting chlorophyll to PSI at the expense of PSII. This short-term redox signalling pathway acts by means of reversible, post-translational modification of pre-existing proteins. A long-term equalisation of the rates of light utilisation by PSI and PSII also occurs: by means of adjustment of the stoichiometry of PSI and PSII. It is likely that the same redox sensor controls both state transitions and photosystem stoichiometry. A specific mechanism for integration of these short- and long-term adaptations is proposed. Recent evidence shows that phosphorylation of LHCII causes a change in its 3-D structure, which implies that the mechanism of state transitions in chloroplasts involves control of recognition of PSI and PSII by LHCII. The distribution of LHCII between PSII and PSI is therefore determined by the higher relative affinity of phospho-LHCII for PSI, with lateral movement of the two forms of the LHCII being simply a result of their diffusion within the membrane plane. Phosphorylation-induced dissociation of LHCII trimers may induce lateral movement of monomeric phospho-LHCII, which binds preferentially to PSI. After dephosphorylation, monomeric, unphosphorylated LHCII may trimerize at the periphery of PSII.     

First solid-state NMR analysis of uniformly ¹³C-enriched major light-harvesting complexes from Chlamydomonas reinhardtii and identification of protein and cofactor spin clusters

The light-harvesting complex II (LHCII) is the main component of the antenna system of plants and green algae and plays a major role in the capture of sun light for photosynthesis. The LHCII complexes have also been proposed to play a key role in the optimization of photosynthetic efficiency through the process of state 1-state 2 transitions and are involved in down-regulation of photosynthesis under excess light by energy dissipation through non-photochemical quenching (NPQ). We present here the first solid-state magic-angle spinning (MAS) NMR data of the major light-harvesting complex (LHCII) of Chlamydomonas reinhardtii, a eukaryotic green alga. We are able to identify nuclear spin clusters of the protein and of its associated chlorophyll pigments in 13C-13C dipolar homonuclear correlation spectra on a uniformly 13C-labeled sample. In particular, we were able to resolve several chlorophyll 131 carbon resonances that are sensitive to hydrogen bonding to the 131-keto carbonyl group. The data show that 13C NMR signals of the pigments and protein sites are well resolved, thus paving the way to study possible structural reorganization processes involved in light-harvesting regulation through MAS solid-state NMR.     

Slow zeaxanthin accumulation and the enhancement of CP26 collectively contribute to an atypical non-photochemical quenching in macroalga Ulva prolifera under high light

Non-photochemical quenching (NPQ) is an important photoprotective mechanism in plants, which dissipates excess energy and further protects the photosynthetic apparatus under high light stress. NPQ can be dissected into a number of components: qE, qZ, and qI. In general, NPQ is catalyzed by two independent mechanisms, with the faster-activated quenching catalyzed by the monomeric light-harvesting complex (LHCII) proteins and the slowly activated quenching catalyzed by LHCII trimers, both processes depending on zeaxanthin but to different extent. Here, we studied the NPQ of the intertidal green macroalga, Ulva prolifera, and found that the NPQ of U. prolifera lack the faster-activated quenching, and showed much greater sensitivity to dithiothreitol (DTT) than to dicyclohexylcarbodiimide (DCCD). Further results suggested that the monomeric LHC proteins in U. prolifera included only CP29 and CP26, but lacked CP24, unlike Arabidopsis thaliana and the moss Physcomitrella patens. Moreover, the expression levels of CP26 increased significantly following exposure to high light, but the concentrations of the two important photoprotective proteins (PsbS and light-harvesting complex stress-related [LhcSR]) did not change upon the same conditions. Analysis of the xanthophyll cycle pigments showed that, upon exposure to high light, zeaxanthin synthesis in U. prolifera was gradual and much slower than that in P. patens, and could effectively be inhibited by DTT. Based on these results, we speculate the enhancement of CP26 and slow zeaxanthin accumulation provide an atypical NPQ, making this green macroalga well adapted to the intertidal environments.     

The photoprotective molecular switch in the photosystem II antenna

We have reviewed the current state of multidisciplinary knowledge of the photoprotective mechanism in the photosystem II antenna underlying non-photochemical chlorophyll fluorescence quenching (NPQ). The physiological need for photoprotection of photosystem II and the concept of feed-back control of excess light energy are described. The outline of the major component of nonphotochemical quenching, qE, is suggested to comprise four key elements: trigger (ΔpH), site (antenna), mechanics (antenna dynamics) and quencher(s). The current understanding of the identity and role of these qE components is presented. Existing opinions on the involvement of protons, different LHCII antenna complexes, the PsbS protein and different xanthophylls are reviewed. The evidence for LHCII aggregation and macrostructural reorganization of photosystem II and their role in qE are also discussed. The models describing the qE locus in LHCII complexes, the pigments involved and the evidence for structural dynamics within single monomeric antenna complexes are reviewed. We suggest how PsbS and xanthophylls may exert control over qE by controlling the affinity of LHCII complexes for protons with reference to the concepts of hydrophobicity, allostery and hysteresis. Finally, the physics of the proposed chlorophyll-chlorophyll and chlorophyll-xanthophyll mechanisms of energy quenching is explained and discussed. This article is part of a Special Issue entitled: Photosystem II.