Light-harvesting Complex Of Photosystem II Research Articles

Structure, Dynamics, and Function in the Major Light-Harvesting Complex of Photosystem II

In natural light-harvesting systems, pigment-protein complexes (PPC) convert sunlight to chemical energy with near unity quantum efficiency. PPCs exhibit emergent properties that cannot be simply extrapolated from knowledge of their component parts. In this Perspective, we examine the design principles of PPCs, focussing on the major light-harvesting complex of Photosystem II (LHCII), the most abundant PPC in green plants. Studies using two-dimensional electronic spectroscopy (2DES) provide an incisive tool to probe the electronic, energetic, and spatial landscapes that enable the efficiency observed in photosynthetic light-harvesting. Using the information about energy transfer pathways, quantum effects, and excited state geometry contained within 2D spectra, the excited state properties can be linked back to the molecular structure. This understanding of the structure-function relationships of natural systems constitutes a step towards a blueprint for the construction of artificial light-harvesting devices that can reproduce the efficacy of natural systems.

Violaxanthin and diadinoxanthin cycles as an important photoprotective mechanism in photosynthesis

The molecular mechanism and regulation of violaxanthin (Vx) and diadinoxanthin (Ddx) cycle are discussed. The influence of lipids on the de-epoxidation of the xanthophyll cycle pigments was investigated. Experiments on the significance of physical properties of the aggregates formed by inverted lipid micelles, which are necessary for de-epoxidation, on the conversion of Vx into antheraxanthin (Ax) and zeaxanthin (Zx) and diadinoxanthin (Ddx) to diatoxanthin (Dtx) were performed. Thickness of the hydrophobic fraction of the aggregates, size of the inverted micelles, suggested by mathematical description of the structures, and solubility of Vx and Ddx in various kinds of lipids were the next tested parameters. Obtained results show that the rate of de-epoxidation was strongly dependent on physicochemical properties of lipids. The key role for enzyme activation play non-bilayer lipids and the parameters of inverted micelles created by them, such as thickness, molecular dynamics of hydrophobic core, and their diameter. Additionally the influence of thylakoid lipids on the de-epoxidation of Vx, associated with the light-harvesting complex of PSII (LHCII) were carried out. Almost complete Vx de-epoxidation in the LHCII fractions containing high amounts of endogenous monogalactosyldiacylglycerol (MGDG) was observed in in vitro assays. LHCII preparations with low concentrations of MGDG exhibited a strongly reduced Vx de-epoxidation, which could be increased by addition of exogenous, pure MGDG. The presented results showed that MGDG and other nonlamellar lipids are necessary for the solubilization of Vx and Ddx and they provide also the three-dimensional structures, which are needed for the binding of de-epoxidases and for the accessibility of Vx and Ddx to these enzymes.

Acclimation to intense light implies changes at the level of trimeric subunits involved in the structural organization of the main light-harvesting complex of photosystem II (LHCII) and their isoforms

When plants are grown under stable light conditions their photosynthetic apparatus undergoes a long-term acclimation process. Acclimation to different light intensities involves changes in the organization and/or abundance of protein complexes in the thylakoid membranes. In this study, spinach plants were exposed to differing light intensities, and the structural organization of the major light-harvesting chlorophyll a/b-protein complex of photosystem II (LHCII) was investigated by analysing their trimeric subunits. Plants were exposed to three different light intensities, 100 μmol quanta m⁻² s⁻¹, 200 μmol quanta m⁻² s⁻¹ and an elevated light intensity, 400 μmol quanta m⁻² s⁻¹, sufficient to provoke a moderate stress response in the form of down regulation of PSII. "MicroRotofor" analysis showed the presence of LHCII with different pIs and revealed a clear decline in their abundance as light intensity increased from 100 to 400 μmol quanta m⁻² s⁻¹. The three subunits (Lhcb1, Lhcb2, Lhcb3) behaved differently from each other as: Lhcb1 decreased more significantly than Lhcb2, whereas Lhcb3 was reduced only at a light window at which Lhcb1 and Lhcb2 abundance has already been depleted under intense irradiation. Interestingly, we also found that isoforms of Lhcb1 subunit (Lhcb1.1; 1.2; 1.3) behaved differently in response to elevated light intensity, suggesting an essential role of these isoforms to light adaption and consequently explaining the presence of this multigenic family, often identified among higher plants.

Elevated ΔpH restores rapidly reversible photoprotective energy dissipation in Arabidopsis chloroplasts deficient in lutein and xanthophyll cycle activity

The xanthophylls of the light-harvesting complexes of photosystem II (LHCII), zeaxanthin, and lutein are thought to be essential for non-photochemical quenching (NPQ). NPQ is a process of photoprotective energy dissipation in photosystem II (PSII). The major rapidly reversible component of NPQ, qE, is activated by the transmembrane proton gradient, and involves the quenching of antenna chlorophyll excited states by the xanthophylls lutein and zeaxanthin. Using diaminodurene (DAD), a mediator of cyclic electron flow around photosystem I, to enhance ΔpH we demonstrate that qE can still be formed in the absence of lutein and light-induced formation of zeaxanthin in chloroplasts derived from the normally qE-deficient lut2npq1 mutant of Arabidopsis. The qE induced by high ΔpH in lut2npq1 chloroplasts 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 sensitive to the uncoupler nigericin, and was accompanied by absorption changes in the 410-565nm region. Titrations show the ΔpH threshold for activation of qE in lut2npq1 chloroplasts lies outside the normal physiological range and is highly cooperative. Comparison of quenching in isolated trimeric (LHCII) and monomeric (CP26) light-harvesting complexes from lut2npq1 plants revealed a similarly shifted pH dependency compared with wild-type LHCII. The implications for the roles of lutein and zeaxanthin as direct quenchers of excitation energy are discussed. Furthermore, we argue that the control over the proton-antenna association constant, pK, occurs via influence of xanthophyll structure on the interconnected phenomena of light-harvesting antenna reorganization/aggregation and hydrophobicity.

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).

Anisotropic circular dichroism signatures of oriented thylakoid membranes and lamellar aggregates of LHCII

In photosynthesis research, circular dichroism (CD) spectroscopy is an indispensable tool to probe molecular architecture at virtually all levels of structural complexity. At the molecular level, the chirality of the molecule results in intrinsic CD; pigment-pigment interactions in protein complexes and small aggregates can give rise to excitonic CD bands, while "psi-type" CD signals originate from large, densely packed chiral aggregates. It has been well established that anisotropic CD (ACD), measured on samples with defined non-random orientation relative to the propagation of the measuring beam, carries specific information on the architecture of molecules or molecular macroassemblies. However, ACD is usually combined with linear dichroism and can be distorted by instrumental imperfections, which given the strong anisotropic nature of photosynthetic membranes and complexes, might be the reason why ACD is rarely studied in photosynthesis research. In this study, we present ACD spectra, corrected for linear dichroism, of isolated intact thylakoid membranes of granal chloroplasts, washed unstacked thylakoid membranes, photosystem II (PSII) membranes (BBY particles), grana patches, and tightly stacked lamellar macroaggregates of the main light-harvesting complex of PSII (LHCII). We show that the ACD spectra of face- and edge-aligned stacked thylakoid membranes and LHCII lamellae exhibit profound differences in their psi-type CD bands. Marked differences are also seen in the excitonic CD of BBY and washed thylakoid membranes. Magnetic CD (MCD) spectra on random and aligned samples, and the largely invariable nature of the MCD spectra, despite dramatic variations in the measured isotropic and anisotropic CD, testify that ACD can be measured without substantial distortions and thus employed to extract detailed information on the (supra)molecular organization of photosynthetic complexes. An example is provided showing the ability of CD data to indicate such an organization, leading to the discovery of a novel crystalline structure in macroaggregates of LHCII.

A Small Zinc Finger Thylakoid Protein Plays a Role in Maintenance of Photosystem II in Arabidopsis thaliana

This work identifies LOW QUANTUM YIELD OF PHOTOSYSTEM II1 (LQY1), a Zn finger protein that shows disulfide isomerase activity, interacts with the photosystem II (PSII) core complex, and may act in repair of photodamaged PSII complexes. Two mutants of an unannotated small Zn finger containing a thylakoid membrane protein of Arabidopsis thaliana (At1g75690; LQY1) were found to have a lower quantum yield of PSII photochemistry and reduced PSII electron transport rate following high-light treatment. The mutants dissipate more excess excitation energy via nonphotochemical pathways than wild type, and they also display elevated accumulation of reactive oxygen species under high light. After high-light treatment, the mutants have less PSII-light-harvesting complex II supercomplex than wild-type plants. Analysis of thylakoid membrane protein complexes showed that wild-type LQY1 protein comigrates with the PSII core monomer and the CP43-less PSII monomer (a marker for ongoing PSII repair and reassembly). PSII repair and reassembly involve the breakage and formation of disulfide bonds among PSII proteins. Interestingly, the recombinant LQY1 protein demonstrates a protein disulfide isomerase activity. LQY1 is more abundant in stroma-exposed thylakoids, where key steps of PSII repair and reassembly take place. The absence of the LQY1 protein accelerates turnover and synthesis of PSII reaction center protein D1. These results suggest that the LQY1 protein may be involved in maintaining PSII activity under high light by regulating repair and reassembly of PSII complexes.

LHCII organization and thylakoid lipids affect the sensitivity of the photosynthetic apparatus to high-light treatment.

Pulse-amplitude-modulated (PAM) chlorophyll fluorescence and photosynthetic oxygen evolution were used to investigate the role of the different amount and organization of light-harvesting complexes of photosystem II (LHCII) in four pea species on the susceptibility of the photosynthetic apparatus to high-light treatment. In this work we analyzed the thylakoid membrane lipid composition of the studied pea plants. A relationship between the structural organization of LHCII proteins, the amount of the main lipid classes and the sensitivity of the photosynthetic apparatus to high-light treatment was found. The results reveal that the photosynthetic apparatus, enriched in oligomeric forms of LHCII concomitant with decreased amount of anionic lipids and increased content of the monogalactosyldiacylglycerol (MGDG), is less sensitive to high light. Our data also suggest that the degree of LHCII oligomerization, as well as the lipid composition do not influence the degree of recovery of the PSII photochemistry after excess light exposure.

Regulation of LHCII aggregation by different thylakoid membrane lipids

In the present study the influence of the lipid environment on the organization of the main light-harvesting complex of photosystem II (LHCII) was investigated by 77K fluorescence spectroscopy. Measurements were carried out with a lipid-depleted and highly aggregated LHCII which was supplemented with the different thylakoid membrane lipids. The results show that the thylakoid lipids are able to modulate the spectroscopic properties of the LHCII aggregates and that the extent of the lipid effect depends on both the lipid species and the lipid concentration. Addition of the neutral galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) seems to induce a modification of the disorganized structures of the lipid-depleted LHCII and to support the aggregated state of the complex. In contrast, we found that the anionic lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) exert a strong disaggregating effect on the isolated LHCII. LHCII disaggregation was partly suppressed under a high proton concentration and in the presence of cations. The strongest suppression was visible at the lowest pH value (pH 5) and the highest Mg 2+ concentration (40 mM) used in the present study. This suggests that the negative charge of the anionic lipids in conjunction with negatively charged domains of the LHCII proteins is responsible for the disaggregation. Additional measurements by photon correlation spectroscopy and sucrose gradient centrifugation, which were used to gain information about the size and molecular mass of the LHCII aggregates, confirmed the results of the fluorescence spectroscopy. LHCII treated with MGDG and DGDG formed an increased number of aggregates with large particle sizes in the μm-range, whereas the incubation with anionic lipids led to much smaller LHCII particles (around 40 nm in the case of PG) with a homogeneous distribution.

Protein–protein interactions by molecular modeling and biochemical characterization of PSI-LHCI supercomplexes from Chlamydomonas reinhardtii

The physiological function of Photosystem I (PSI) is a sunlight energy converter, catalyzing one of the initial steps in driving oxygenic photosynthesis in cyanobacteria, algae and higher plants. The Chlamydomonas reinhardtii PSI structure was not known since it contains a unique structure having additional light harvesting complex I (LHCI) subunits, which play a major role in the transfer of sunlight energy to the reaction center. Here, individual subunits of LHC and core subunits are built based on the PDB taken from RCSB Protein Data Bank. The model gives information about the geometrical existence of subunits following a flanking order of Lhca5, Lhca1, Lhca6, Lhca4, Lhca2, Lhca8, Lhca9, Lhca7, and Lhca3. The new subunit PsaO is located close to the PsaH, PsaI and PsaL subunits, thus it may be involved in the state transition mechanism and stabilization of PSI-LHCI supercomplexes. The modeled PSI-LHCI structure of C. reinhardtii shows a unique arrangement of PsaN, PsaO of PSI core subunits and Lhca5 to Lhca9 of LHCI subunits. There are many non-covalent interactions among the PSI and LHCI subunits, which suggest that C. reinhardtii PSI-LHCI supercomplexes are more complex than higher plants. These results strongly support the experimental data that, even with harsh treatment of the PSI-LHCI supercomplexes with detergent, the complexes do not dissociate due to strong interactions between the PSI core and LHCI. Thus, our 3D model may give valid structural information of the PSI-LHCI arrangement and its physiological role in C. reinhardtii.

Efficient Light Harvesting in a Dark, Hot, Acidic Environment: The Structure and Function of PSI-LHCI from Galdieria sulphuraria

Photosystem I-light harvesting complex I (PSI-LHCI) was isolated from the thermoacidophilic red alga Galdieria sulphuraria, and its structure, composition, and light-harvesting function were characterized by electron microscopy, mass spectrometry, and ultrafast optical spectroscopy. The results show that Galdieria PSI is a monomer with core features similar to those of PSI from green algae, but with significant differences in shape and size. A comparison with the crystal structure of higher plant (pea) PSI-LHCI indicates that Galdieria PSI binds seven to nine light-harvesting proteins. Results from ultrafast optical spectroscopy show that the functional coupling of the LHCI proteins to the PSI core is tighter than in other eukaryotic PSI-LHCI systems reported thus far. This tight coupling helps Galdieria perform efficient light harvesting under the low-light conditions present in its natural endolithic habitat.

Period‐four Modulation of Photosystem II Primary Quinone Acceptor (QA) Reduction/Oxidation Kinetics in Thylakoid Membranes

Photosystem II (PSII), a multiprotein complex mainly coded by the chloroplast genome in higher plants and algae, contains the oxygen-evolving complex with four manganese atoms responsible for the oxidation of water. After each absorption of a light quantum by pigment molecules in the light harvesting complexes of PSII, the Mn cluster advances in its oxidation states denoted from S(0) to S(4) . The S(4) state decays to S(0) in the dark with the concurrent release of molecular oxygen. Therefore, the oxygen production in PSII exposed to successive single turnover excitations follows a period-four oscillation pattern. The intensity of chlorophyll a fluorescence of PSII is also known to be influenced by the oxidation state of the Mn cluster. In the present work, fluorescence induction kinetics was measured in isolated thylakoids with various initial S-state populations settled by preflashes. The shape of the fluorescence induction traces was strongly affected by preflashes. O-J and J-I phases of the induction followed a period-four oscillation pattern. The results indicate that these changes reflect the influence of the oxidation rate of the Mn cluster on the reduction/oxidation kinetics of the primary quinone acceptor (Q(A) ) of PSII.

Spectroscopic elucidation of uncoupled transition energies in the major photosynthetic light-harvesting complex, LHCII

Electrostatic couplings between chromophores in photosynthetic pigment-protein complexes, and interactions of pigments with the surrounding protein environment, produce a complicated energy landscape of delocalized excited states. The resultant electronic structure absorbs light and gives rise to energy transfer steps that direct the excitation toward a site of charge separation with near unity quantum efficiency. Knowledge of the transition energies of the uncoupled chromophores is required to describe how the wave functions of the individual pigments combine to form this manifold of delocalized excited states that effectively harvests light energy. In an investigation of the major light-harvesting complex of photosystem II (LHCII), we develop a method based on polarized 2D electronic spectroscopy to experimentally access the energies of the S(0)-S(1) transitions in the chromophore site basis. Rotating the linear polarization of the incident laser pulses reveals previously hidden off-diagonal features. We exploit the polarization dependence of energy transfer peaks to find the angles between the excited state transition dipole moments. We show that these angles provide a spectroscopic method to directly inform on the relationship between the delocalized excitons and the individual chlorophylls through the site energies of the uncoupled chromophores.

Identification and Characterization of an Assembly Intermediate Subcomplex of Photosystem I in the Green Alga Chlamydomonas reinhardtii

Photosystem I (PSI) is a multiprotein complex consisting of the PSI core and peripheral light-harvesting complex I (LHCI) that together form the PSI-LHCI supercomplex in algae and higher plants. The supercomplex is synthesized in steps during which 12-15 core and 4-9 LHCI subunits are assembled. Here we report the isolation of a PSI subcomplex that separated on a sucrose density gradient from the thylakoid membranes isolated from logarithmic growth phase cells of the green alga Chlamydomonas reinhardtii. Pulse-chase labeling of total cellular proteins revealed that the subcomplex was synthesized de novo within 1 min and was converted to the mature PSI-LHCI during the 2-h chase period, indicating that the subcomplex was an assembly intermediate. The subcomplex was functional; it photo-oxidized P700 and demonstrated electron transfer activity. The subcomplex lacked PsaK and PsaG, however, and it bound PsaF and PsaJ weakly and was not associated with LHCI. It seemed likely that LHCI had been integrated into the subcomplex unstably and was dissociated during solubilization and/or fractionation. We, thus, infer that PsaK and PsaG stabilize the association between PSI core and LHCI complexes and that PsaK and PsaG bind to the PSI core complex after the integration of LHCI in one of the last steps of PSI complex assembly.

Isolation of the elusive supercomplex that drives cyclic electron flow in photosynthesis

Photosynthetic light reactions establish electron flow in the chloroplast's thylakoid membranes, leading to the production of the ATP and NADPH that participate in carbon fixation. Two modes of electron flow exist-linear electron flow (LEF) from water to NADP(+) via photosystem (PS) II and PSI in series and cyclic electron flow (CEF) around PSI (ref. 2). Although CEF is essential for satisfying the varying demand for ATP, the exact molecule(s) and operational site are as yet unclear. In the green alga Chlamydomonas reinhardtii, the electron flow shifts from LEF to CEF on preferential excitation of PSII (ref. 3), which is brought about by an energy balancing mechanism between PSII and PSI (state transitions). Here, we isolated a protein supercomplex composed of PSI with its own light-harvesting complex (LHCI), the PSII light-harvesting complex (LHCII), the cytochrome b(6)f complex (Cyt bf), ferredoxin (Fd)-NADPH oxidoreductase (FNR), and the integral membrane protein PGRL1 (ref. 5) from C. reinhardtii cells under PSII-favouring conditions. Spectroscopic analyses indicated that on illumination, reducing equivalents from downstream of PSI were transferred to Cyt bf, whereas oxidised PSI was re-reduced by reducing equivalents from Cyt bf, indicating that this supercomplex is engaged in CEF (Supplementary Fig. 1). Thus, formation and dissociation of the PSI-LHCI-LHCII-FNR-Cyt bf-PGRL1 supercomplex not only controlled the energy balance of the two photosystems, but also switched the mode of photosynthetic electron flow.

The main thylakoid membrane lipid monogalactosyldiacylglycerol (MGDG) promotes the de-epoxidation of violaxanthin associated with the light-harvesting complex of photosystem II (LHCII)

In higher plants, the major part of the xanthophyll cycle pigment violaxanthin (Vx) is non-covalently bound to the main light-harvesting complex of PSII (LHCII). Under saturating light conditions Vx has to be released from its binding site into the surrounding lipid phase, where it is converted to zeaxanthin (Zx) by the enzyme Vx de-epoxidase (VDE). In the present study we investigated the influence of thylakoid lipids on the de-epoxidation of Vx, which was still associated with the LHCII. We isolated LHCII with different concentrations of native, endogenous lipids and Vx by sucrose gradient centrifugation or successive cation precipitation. Analysis of the different LHCII preparations showed that the concentration of LHCII-associated Vx was correlated with the concentration of the main thylakoid lipid monogalactosyldiacylglycerol (MGDG) associated with the complexes. Decreases in the MGDG content of the LHCII led to a diminished Vx concentration, indicating that a part of the total Vx pool was located in an MGDG phase surrounding the LHCII, whereas another part was bound to the LHCII apoproteins. We further studied the convertibility of LHCII-associated Vx in in-vitro enzyme assays by addition of isolated VDE. We observed an efficient and almost complete Vx conversion in the LHCII fractions containing high amounts of endogenous MGDG. LHCII preparations with low concentrations of MGDG exhibited a strongly reduced Vx de-epoxidation, which could be increased by addition of exogenous, pure MGDG. The de-epoxidation of LHCII-associated Vx was saturated at a much lower concentration of native, endogenous MGDG compared with the concentration of isolated, exogenous MGDG, which is needed for optimal VDE activity in in-vitro assays employing pure isolated Vx.

Distribution and functional role of carbonic anhydrase Cah3 associated with thylakoid membranes in the chloroplast and pyrenoid of Chlamydomonas reinhardtii

Localization of lumenal carbonic anhydrase Cah3 in thylakoid membranes of Chlamydomonas reinhardtii was studied using wild-type algae and photosynthetic mutants with different composition of chlorophyll-protein complexes in the photosystems. In addition, the photosynthetic characteristics of wild-type C. reinhardtii and cia3 mutants lacking the activity of carbonic anhydrase Cah3 were examined. Western blot analysis revealed the lack of cross reaction with antibodies to Cah3 in the mutant lacking the photosystem II (PSII) reaction center, in contrast to the mutant deficient in light-harvesting complex of PSII. These data show that the lumenal Cah3 is associated with polypeptides on the donor side of PSII reaction center. Using immunoelectron microscopy and antibodies to Cah3 from C. reinhardtii, we showed for the first time that the major part of thylakoid Cah3 is localized in the pyrenoid where the bulk of Rubisco is located. The rate of photosynthetic oxygen evolution and PSII photochemical efficiency were lower in C. reinhardtii cia3 mutant than in the wild type, especially in the cells grown at limiting CO2 concentrations. These observations show that Cah3 takes part in CO2-concentrating mechanism of the chloroplast. The results support our hypothesis [1, 2] that the carboxylation reaction in microalgae proceeds in the pyrenoid, a specific Rubisco-containing part of the chloroplast, which acquires CO2 from the lumen of intrapyrenoid thylakoids. We discuss significance of the pyrenoid as an autonomous metabolic microcompartment, in which Cah3 plays a key role in the production and concentration of CO2 for Rubisco. These functions may promote the photosynthetic efficiency owing to the effective CO2 supply for the Calvin cycle.

Heat-induced disassembly and degradation of chlorophyll-containing protein complexes in vivo

Gradual heating of green leaves up to non-physiological temperatures is often used to estimate thermal stability of photosynthetic apparatus. However, a complete sequence of heat-induced disassembly and denaturation of chlorophyll-containing protein complexes (CPCs) has not been reported yet. In this work, we heated (1 °C·min − 1 ) barley leaves to temperatures selected according to the changes in the chlorophyll fluorescence temperature curve (FTC) and we analyzed CPC stability by two-dimensional native Deriphat/SDS–PAGE . The first distinct change in both structure and function of photosystem II (PSII) appeared at 40–50 °C. PSII core (CCII) dimers began to dissociate monomers, which was accompanied by a decrease in PSII photochemistry and reflected in FTC as the first fluorescence increase. Further changes in CPCs appeared at 57–60 °C, when FTC increases to its second maximum. Photosystem I (PSI) cores (CCI) partially dissociated from light-harvesting complexes of PSI (LHCI) and formed aggregates. The rest of CCI–LHCI complexes, as well as the CCI aggregates, degraded to the PSI-A/B heterodimer in leaves heated to 70 °C. Heating to these temperatures led to a complete degradation of CCII components and corresponding loss of PSII photochemistry. Trimeric light-harvesting complexes of PSII (LHCII) markedly dissociated to monomers and denatured, as evidenced by a release of large amount of free chlorophylls. Between 70 and 80 °C, a complete degradation of LHCII occurred, leaving the PSI-A/B heterodimer as the only detectable CPC in the membrane. This most thermostable CPC disappeared after heating to 90 °C, which corresponded to a loss of PSI photochemistry.

Cysteine modification of a specific repressor protein controls the translational status of nucleus-encoded LHCII mRNAs in Chlamydomonas

The cytosolic RNA-binding protein NAB1 represses translation of LHCII (light-harvesting complex of photosystem II) encoding mRNAs by sequestration into translationally silent mRNP complexes in the green alga Chlamydomonas reinhardtii. NAB1 contains 2 cysteine residues, Cys-181 and Cys-226, within its C-terminal RRM motif. Modification of these cysteines either by oxidation or by alkylation in vitro was accompanied by a decrease in RNA-binding affinity for the target mRNA sequence. To confirm the relevance of reversible NAB1 cysteine oxidation for the regulation of its activity in vivo, we replaced both cysteines with serines. All examined cysteine single and double mutants exhibited a reduced antenna at PSII caused by a perturbed NAB1 deactivation mechanism, with double mutations and Cys-226 single mutations causing a stronger and more distinctive phenotype compared with the Cys-181 mutation. Our data indicated that the responsible redox control mechanism is mediated by modification of single cysteines. Polysome analyses and RNA co-immunoprecipitation experiments demonstrated the interconnection of the NAB1 thiol state and its activity as a translation repressor in vivo. NAB1 is fully active in its dithiol state and is reversibly deactivated by modification of its cysteines. In summary, this work is an example that cytosolic translation of nucleus encoded photosynthetic genes is regulated via a reversible cysteine-based redox switch in a RNA-binding translation repressor protein.

On the regulation of photosynthesis by excitonic interactions between carotenoids and chlorophylls

Selective 2-photon excitation (TPE) of carotenoid dark states, Car S(1), shows that in the major light-harvesting complex of photosystem II (LHCII), the extent of electronic interactions between carotenoid dark states (Car S(1)) and chlorophyll (Chl) states, phi(Coupling)(Car S(1)-Chl), correlates linearly with chlorophyll fluorescence quenching under different experimental conditions. Simultaneously, a linear correlation between both Chl fluorescence quenching and phi(Coupling)(Car S(1)-Chl) with the intensity of red-shifted bands in the Chl Q(y) and carotenoid absorption was also observed. These results suggest quenching excitonic Car S(1)-Chl states as origin for the observed effects. Furthermore, real time measurements of the light-dependent down- and up-regulation of the photosynthetic activity and phi(Coupling)(Car S(1)-Chl) in wild-type and mutant (npq1, npq2, npq4, lut2 and WT+PsbS) Arabidopsis thaliana plants reveal that also in vivo the quenching parameter NPQ correlates always linearly with the extent of electronic Car S(1)-Chl interactions in any adaptation status. Our in vivo measurements with Arabidopsis variants show that during high light illumination, phi(Coupling)(Car S(1)-Chl) depends on the presence of PsbS and zeaxanthin (Zea) in an almost identical way as NPQ. In summary, these results provide clear evidence for a very close link between electronic Car S(1)-Chl interactions and the regulation of photosynthesis. These findings support a photophysical mechanism in which short-living, low excitonic carotenoid-chlorophyll states serve as traps and dissipation valves for excess excitation energy.