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.     

Efficient Light Harvesting by Photosystem II Requires an Optimized Protein Packing Density in Grana Thylakoids

A recently developed technique for dilution of the naturally high protein packing density in isolated grana membranes was applied to study the dependence of the light harvesting efficiency of photosystem (PS) II on macromolecular crowding. Slight dilution of the protein packing from 80% area fraction to the value found in intact grana thylakoids (70%) leads to an improved functionality of PSII (increased antenna size, enhanced connectivity between reaction centers). Further dilution induces a functional disconnection of light-harvesting complex (LHC) II from PSII. It is concluded that efficient light harvesting by PSII requires an optimal protein packing density in grana membranes that is close to 70%. We hypothesize that the decreased efficiency in overcrowded isolated grana thylakoids is caused by excited state quenching in LHCII, which has previously been correlated with neoxanthin distortion. Resonance Raman spectroscopy confirms this increase in neoxanthin distortion in overcrowded grana as compared with intact thylakoids. Furthermore, analysis of the changes in the antenna size in highly diluted membranes indicates a lipid-induced dissociation of up to two trimeric LHCII from PSII, leaving one trimer connected. This observation supports a hierarchy of LHCII-binding sites on PSII.     

Immunocytochemical and Cytochemical Localization of Photosystems I and II

Cytochemical and immunocytochemical methods were used to localize photosystems I and II in barley (Hordeum vulgare L. cv Himalaya) chloroplasts. PSI activity, monitored by diaminobenzidine oxidation, was associated with the lumen side of the thylakoids of both grana and stroma lamellae. The P₇₀₀ chlorophyll a protein, the reaction center of PSI, was localized on thin sections of barley chloroplasts using monospecific antibodies to this protein and the peroxidase-antiperoxidase procedure. Results obtained by immunocytochemistry were similar to those of the diaminobenzidine oxidation: both grana and stroma lamellae contained immunocytochemically reactive material. Both the grana and stroma lamellae were also labeled when isolated thylakoids were reacted with the P₇₀₀ chlorophyll a protein antiserum and then processed by the peroxidase-antiperoxidase procedure. PSII activity was localized cytochemically by monitoring the photoreduction of thiocarbamyl nitroblue tetrazolium, a reaction sensitive to the PSII inhibitor, DCMU. PSII reactions occurred primarily on the grana lamellae, with weaker reactions on the stroma lamellae.     

Segregation of photosystems in thylakoid membranes as a critical phenomenon

The distribution of the two photosystems, PSI and PSII, in grana and stroma lamellae of the chloroplast membranes is not uniform. PSII are mainly concentrated in grana and PSI in stroma thylakoids. The dynamics and factors controlling the spatial segregation of PSI and PSII are generally not well understood, and here we address the segregation of photosystems in thylakoid membranes by means of a molecular dynamics method. The lateral segregation of photosystems was studied assuming a model comprising a two-dimensional (in-plane), two-component, many-body system with periodic boundary conditions and competing interactions between the photosystems in the thylakoid membrane. PSI and PSII are represented by particles with different values of negative charge. The pair interactions between particles include a screened Coulomb repulsive part and an exponentially decaying attractive part. The modeling results suggest a complicated phase behavior of the system, including quasi-crystalline phase of randomly distributed complexes of PSII and PSI at low ionic screening, well defined clustered state of segregated complexes at high screening, and in addition, an intermediate agglomerate phase where the photosystems tend to aggregate together without segregation. The calculations demonstrated that the ordering of photosystems within the membrane was the result of interplay between electrostatic and lipid-mediated interactions. At some values of the model parameters the segregation can be represented visually as well as by analyzing the correlation functions of the configuration.     

Structural and functional self-organization of Photosystem II in grana thylakoids

The biogenesis of the well-ordered macromolecular protein arrangement of photosystem (PS)II and light harvesting complex (LHC)II in grana thylakoid membranes is poorly understood and elusive. In this study we examine the capability of self organization of this arrangement by comparing the PSII distribution and antenna organization in isolated untreated stacked thylakoids with restacked membranes after unstacking. The PS II distribution was deduced from freeze-fracture electron microscopy. Furthermore, changes in the antenna organization and in the oligomerization state of photosystem II were monitored by chlorophyll a fluorescence parameters and size analysis of exoplasmatic fracture face particles. Low-salt induced unstacking leads to a randomization and intermixing of the protein complexes. In contrast, macromolecular PSII arrangement as well as antenna organization in thylakoids after restacking by restoring the original solvent composition is virtually identical to stacked control membranes. This indicates that the supramolecular protein arrangement in grana thylakoids is a self-organized process.     

Low-light-induced formation of semicrystalline photosystem II arrays in higher plant chloroplasts

Remodeling of photosynthetic machinery induced by growing spinach plants under low light intensities reveals an up-regulation of light-harvesting complexes and down-regulation of photosystem II and cytochrome b6f complexes in intact thylakoids and isolated grana membranes. The antenna size of PSII increased by 40-60% as estimated by fluorescence induction and LHCII/PSII stoichiometry. These low-light-induced changes in the protein composition were accompanied by the formation of ordered particle arrays in the exoplasmic fracture face in grana thylakoids detected by freeze-fracture electron microscopy. Most likely these highly ordered arrays consist of PSII complexes. A statistical analysis of the particles in these structures shows that the distance of neighboring complexes in the same row is 18.0 nm, the separation between two rows is 23.7 nm, and the angle between the particle axis and the row is 26 degrees . On the basis of structural information on the photosystem II supercomplex, a model on the supramolecular arrangement was generated predicting that two neighboring complexes share a trimeric light-harvesting complex. It was suggested that the supramolecular reorganization in ordered arrays in low-light grana thylakoids is a strategy to overcome potential diffusion problems in this crowded membrane. Furthermore, the occurrence of a hexagonal phase of the lipid monogalactosyldiacylglycerol in grana membranes of low-light-adapted plants could trigger the rearrangement by changing the lateral membrane pressure.     

Structural and functional self-organization of Photosystem II in grana thylakoids

The biogenesis of the well-ordered macromolecular protein arrangement of photosystem (PS)II and light harvesting complex (LHC)II in grana thylakoid membranes is poorly understood and elusive. In this study we examine the capability of self organization of this arrangement by comparing the PSII distribution and antenna organization in isolated untreated stacked thylakoids with restacked membranes after unstacking. The PS II distribution was deduced from freeze-fracture electron microscopy. Furthermore, changes in the antenna organization and in the oligomerization state of photosystem II were monitored by chlorophyll a fluorescence parameters and size analysis of exoplasmatic fracture face particles. Low-salt induced unstacking leads to a randomization and intermixing of the protein complexes. In contrast, macromolecular PSII arrangement as well as antenna organization in thylakoids after restacking by restoring the original solvent composition is virtually identical to stacked control membranes. This indicates that the supramolecular protein arrangement in grana thylakoids is a self-organized process.     

Quality control of photosystem II: FtsH hexamers are localized near photosystem II at grana for the swift repair of damage

The reaction center-binding D1 protein of Photosystem II is oxidatively damaged by excessive visible light or moderate heat stress. The metalloprotease FtsH has been suggested as responsible for the degradation of the D1 protein. We have analyzed the distribution and subunit structures of FtsH in spinach thylakoids and various membrane fractions derived from the thylakoids using clear native polyacrylamide gel electrophoresis and Western blot analysis. FtsH was found not only in the stroma thylakoids but also in the Photosystem II-enriched grana membranes. Monomeric, dimeric, and hexameric FtsH proteases were present as major subunit structures in thylakoids, whereas only hexameric FtsH proteases were detected in Triton X-100-solubilized Photosystem II membranes. Importantly, among the membrane fractions examined, hexameric FtsH proteases were most abundant in the Photosystem II membranes. In accordance with this finding, D1 degradation took place in the Photosystem II membranes under light stress. Sucrose density gradient centrifugation analysis of thylakoids and the Photosystem II membranes solubilized with n-dodecyl-β-d-maltoside and a chemical cross-linking study of thylakoids showed localization of FtsH near the Photosystem II light-harvesting chlorophyll-protein supercomplexes in the grana. These results suggest that part of the FtsH hexamers are juxtapositioned to PSII complexes in the grana in darkness, carrying out immediate degradation of the photodamaged D1 protein under light stress.     

Lateral heterogeneity of plant thylakoid protein complexes: early reminiscences

The concept that the two photosystems of photosynthesis cooperate in series, immortalized in Hill and Bendall''s Z scheme, was still a black box that defined neither the structural nor the molecular organization of the thylakoid membrane network into grana and stroma thylakoids. The differentiation of the continuous thylakoid membrane into stacked grana thylakoids interconnected by single stroma thylakoids is a morphological reflection of the non-random distribution of photosystem II/light-harvesting complex of photosystem II, photosystem I and ATP synthase, which became known as lateral heterogeneity.     

TLP18.3, a novel thylakoid lumen protein regulating photosystem II repair cycle

A proteome analysis of Arabidopsis thaliana thylakoid-associated polysome nascent chain complexes was performed to find novel proteins involved in the biogenesis, maintenance and turnover of thylakoid protein complexes, in particular the PSII (photosystem II) complex, which exhibits a high turnover rate. Four unknown proteins were identified, of which TLP18.3 (thylakoid lumen protein of 18.3 kDa) was selected for further analysis. The Arabidopsis mutants (SALK_109618 and GABI-Kat 459D12) lacking the TLP18.3 protein showed higher susceptibility of PSII to photoinhibition. The increased susceptibility of DeltaTLP18.3 plants to high light probably originates from an inefficient reassembly of PSII monomers into dimers in the grana stacks, as well as from an impaired turnover of the D1 protein in stroma exposed thylakoids. Such dual function of the TLP18.3 protein is in accordance with its even distribution between the grana and stroma thylakoids. Notably, the lack of the TLP18.3 protein does not lead to a severe collapse of the PSII complexes, suggesting a redundancy of proteins assisting these particular repair steps to assure functional PSII. The DeltaTLP18.3 plants showed no clear visual phenotype under standard growth conditions, but when challenged by fluctuating light during growth, the retarded growth of DeltaTLP18.3 plants was evident.     

Quality Control of Photosystem II: THYLAKOID UNSTACKING IS NECESSARY TO AVOID FURTHER DAMAGE TO THE D1 PROTEIN AND TO FACILITATE D1 DEGRADATION UNDER LIGHT STRESS IN SPINACH THYLAKOIDS*

Photosystem II is vulnerable to light damage. The reaction center-binding D1 protein is impaired during excessive illumination and is degraded and removed from photosystem II. Using isolated spinach thylakoids, we investigated the relationship between light-induced unstacking of thylakoids and damage to the D1 protein. Under light stress, thylakoids were expected to become unstacked so that the photodamaged photosystem II complexes in the grana and the proteases could move on the thylakoids for repair. Excessive light induced irreversible unstacking of thylakoids. By comparing the effects of light stress on stacked and unstacked thylakoids, photoinhibition of photosystem II was found to be more prominent in stacked thylakoids than in unstacked thylakoids. In accordance with this finding, EPR spin trapping measurements demonstrated higher production of hydroxyl radicals in stacked thylakoids than in unstacked thylakoids. We propose that unstacking of thylakoids has a crucial role in avoiding further damage to the D1 protein and facilitating degradation of the photodamaged D1 protein under light stress.In the chloroplasts of higher plants and green algae, thylakoid membranes are closely associated and stack to form grana. Under electron microscopy, cylindrical grana consisting of 10–20 layers of thylakoids have been observed. They have a diameter of 300–600 nm and are interconnected by lamellae of several hundred nm in length (1, 2). The structure of grana in the chloroplasts of higher plants is well known, but the precise role of grana is incompletely understood. Their possible functions in primary photochemical reactions and subsequent events have been discussed extensively (3–9). Photosystem I (PSI)³ and II (PSII) complexes are segregated from each other in thylakoids, showing lateral heterogeneity in their distribution. The PSII complex is a multisubunit pigment-protein complex responsible for the photochemical oxidation of water and reduction of plastoquinone (8, 10–13). It comprises >25 protein subunits and other low molecular weight cofactors, including chlorophylls, carotenoids, plastoquinones, and manganeses. In the chloroplasts of higher plants, PSII complexes and the associated light-harvesting antenna complex LHCII are not present throughout the thylakoid membranes but are abundant in the grana (2, 14). A densely packed array of PSII complexes in the grana was visualized by electron microscopy (8, 15). Grana formation is more prominent in shade leaves (or shade plants) than in sun leaves (or sun plants), so it has been suggested that enrichment of the PSII·LHCII complex in grana is a strategy of plants to collect excitation energy by PSII under weak light (16). The grana structure probably provides an organized environment for PSII. PSI and ATP synthase are located exclusively in the stroma-exposed thylakoids, including the stroma thylakoids, grana end membranes, and grana margins, because these complexes protrude into the stroma. Cytochrome b₆/f complexes without this protrusion are present uniformly throughout the thylakoids (3). It has been suggested that separation of PSI and PSII complexes on the thylakoids through grana formation is important to prevent “spillover” of excitation energy from PSII to PSI, which lowers photosynthesis efficiency (17).An active PSII complex comprises a homodimer of PSII monomers (13). When thylakoids are exposed to excessive visible light, the PSII dimer dissociates into two monomers (18), but the most significant change takes place inside the monomeric PSII, where the reaction center-binding D1 protein is photodamaged and degraded by specific proteases (19, 20). The photodamage to the D1 protein is a photooxidative process. This is caused by reactive oxygen species (ROS), most probably singlet oxygen (¹O₂) or the hydroxyl radical (HO^•) produced by overreduction of the acceptor side of PSII under excessive illumination or by endogenous cationic radicals, such as the oxidized forms of the primary electron donor P680 and the secondary electron donor Tyr_Z (Tyr¹⁶¹ of D1) to PSII (21). Strong illumination of the grana may readily cause damage to the PSII complexes by ROS and endogenous cationic radicals, because the grana is rich in PSII complexes. Segregation of PSI and PSII in the stacked thylakoids should make the electron transport between PSI and PSII a rate-limiting step in the electron flow, and overexcitation of PSII under these conditions may stimulate ROS production at the acceptor side of PSII. Close association of LHCII with the PSII core complexes should also stimulate ROS generation in the grana. Unstacking of the thylakoids, which is also expected to lead to random distribution of PSI and PSII on the thylakoids and dissociation of the LHCII from the PSII core, may be important to avoid photodamage to PSII.In the proteolysis of the damaged D1 protein in the chloroplasts of higher plants, the N-terminal Thr of the D1 protein is dephosphorylated, and the subsequent degradation produces 23- and 9-kDa fragments as the primary cleavage products (19, 20). The protease(s) and phosphatase(s) involved in these steps are presumably localized in the stroma thylakoids, grana end membranes, and grana margin. Lateral migration of the damaged PSII complexes from the grana to the membrane regions where the damaged PSII complexes are repaired is therefore important for degradation of the D1 protein. Thylakoid unstacking, if it occurs under light stress, should stimulate diffusion of the protein complexes on the thylakoids, thereby stimulating D1 turnover.First, we examined if excessive visible light can induce unstacking of the thylakoids. Second, we studied the effects of strong illumination on stacked and unstacked thylakoids to see if they showed different responses to excessive light. We strongly suggest that unstacking of the thylakoids caused by light stress is necessary to avoid further photodamage to the D1 protein and to facilitate degradation and removal of the photodamaged D1 protein from PSII complexes.     

Transversal and lateral exciton energy transfer in grana thylakoids of spinach

The excitation energy transfer between photosystem (PS) II complexes was studied in isolated grana disks and thylakoids using chlorophyll a fluorescence induction measurements in the presence of DCMU under stacked and destacked conditions. Destacking of grana was achieved using a sonication protocol in a buffer without MgCl(2). The degree of stacking was controlled and quantified by atomic force microscopy and by the concomitant absorption changes. As expected from the literature, intact thylakoids showed a strong dependency of the connectivity of PSII centers, the F(m)/F(o) ratio as well as the fraction of PSIIbeta centers on the MgCl(2) concentration. In contrast, these parameters did not change in isolated grana disks. In particular, the connectivity remained constantly high irrespective of the degree of destacking. These differences were explained by the high protein density in grana disks, which hinders separation and mixing of proteins sufficiently to change energy transfer properties. Due to the occurrence of stroma lamella in intact thylakoids, intermixing of PSII and PSI is possible and allows for changes in F(m)/F(o) ratio as is the separation of LHCII from PSII, thus leading to an increase in the fraction of PSIIbeta. Even if mixing and separation of proteins are impaired in isolated grana disks, destacking should lead to a decrease in connectivity if transversal excitation energy transfer between two opposite membranes is significant. Because the connectivity is constant over all degrees of destacking employed, we conclude that the energy transfer in granas is mainly lateral.     

Quantification of photosystem I and II in different parts of the thylakoid membrane from spinach

Electron paramagnetic resonance (EPR) was used to quantify Photosystem I (PSI) and PSII in vesicles originating from a series of well-defined but different domains of the thylakoid membrane in spinach prepared by non-detergent techniques. Thylakoids from spinach were fragmented by sonication and separated by aqueous polymer two-phase partitioning into vesicles originating from grana and stroma lamellae. The grana vesicles were further sonicated and separated into two vesicle preparations originating from the grana margins and the appressed domains of grana (the grana core), respectively. PSI and PSII were determined in the same samples from the maximal size of the EPR signal from P700(+) and Y(D)( .-), respectively. The following PSI/PSII ratios were found: thylakoids, 1.13; grana vesicles, 0.43; grana core, 0.25; grana margins, 1.28; stroma lamellae 3.10. In a sub-fraction of the stroma lamellae, denoted Y-100, PSI was highly enriched and the PSI/PSII ratio was 13. The antenna size of the respective photosystems was calculated from the experimental data and the assumption that a PSII center in the stroma lamellae (PSIIbeta) has an antenna size of 100 Chl. This gave the following results: PSI in grana margins (PSIalpha) 300, PSI (PSIbeta) in stroma lamellae 214, PSII in grana core (PSIIalpha) 280. The results suggest that PSI in grana margins have two additional light-harvesting complex II (LHCII) trimers per reaction center compared to PSI in stroma lamellae, and that PSII in grana has four LHCII trimers per monomer compared to PSII in stroma lamellae. Calculation of the total chlorophyll associated with PSI and PSII, respectively, suggests that more chlorophyll (about 10%) is associated with PSI than with PSII.     

Arrangement of Photosystem II and ATP Synthase in Chloroplast Membranes of Spinach and Pea

We used cryoelectron tomography to reveal the arrangements of photosystem II (PSII) and ATP synthase in vitreous sections of intact chloroplasts and plunge-frozen suspensions of isolated thylakoid membranes. We found that stroma and grana thylakoids are connected at the grana margins by staggered lamellar membrane protrusions. The stacking repeat of grana membranes in frozen-hydrated chloroplasts is 15.7 nm, with a 4.5-nm lumenal space and a 3.2-nm distance between the flat stromal surfaces. The chloroplast ATP synthase is confined to minimally curved regions at the grana end membranes and stroma lamellae, where it covers 20% of the surface area. In total, 85% of the ATP synthases are monomers and the remainder form random assemblies of two or more copies. Supercomplexes of PSII and light-harvesting complex II (LHCII) occasionally form ordered arrays in appressed grana thylakoids, whereas this order is lost in destacked membranes. In the ordered arrays, each membrane on either side of the stromal gap contains a two-dimensional crystal of supercomplexes, with the two lattices arranged such that PSII cores, LHCII trimers, and minor LHCs each face a complex of the same kind in the opposite membrane. Grana formation is likely to result from electrostatic interactions between these complexes across the stromal gap.     

Lateral heterogeneity in the distribution of chlorophyll-protein complexes of the thylakoid membranes of spinach chloroplasts

The lateral distribution of the main chlorophyll-protein complexes between appressed and non-appressed thylakoid membranes has been studied. The reaction centre complexes of Photosystems I and II and the light-harvesting complex have been resolved by an SDS-polyacrylamide gel electrophoretic method which permits most of the chlorophyll to remain protein-bound.

The analyses were applied to subchloroplast fractions shown to be derived from different thylakoid regions. Stroma thylakoids were separated from grana stacks by centrifugation following chloroplast disruption by press treatment or digitonin. Vesicles derived from the grana partitions were isolated by aqueous polymer two-phase partition. A substantial depletion in the amount of Photosystem I chlorophyll-protein complex and an enrichment in the Photosystem II reaction centre complex and the light-harvesting complex occurred in the appressed grana partition region. The high enrichment in this fraction compared to grana stack fractions derived from press or digitonin treatments, suggests that the grana Photosystem I is restricted mainly to the non-appressed grana end membranes and margins, and that the grana partitions possess mainly Photosystem II reaction centre complex and the light-harvesting complex.

In contrast, stroma thylakoids are highly enriched in the Photosystem I reaction centre complex. They possess also some 10–20% of the total Photosystem II reaction centre complex and the light-harvesting complex.

The ratio of light-harvesting complex to Photosystem II reaction centre complex is rather constant in all subchloroplast fractions suggesting a close association between these complexes. This was not so for the ratio of light-harvesting complex and the Photosystem I reaction centre complex.

The lateral heterogeneity in the distribution of the photosystems between appressed and non-appressed membranes must have a profound impact on current understanding of both the distribution of excitation energy and photosynthetic electron transport between the photosystems.     

Quantification of photosystem I and II in different parts of the thylakoid membrane from spinach

Electron paramagnetic resonance (EPR) was used to quantify Photosystem I (PSI) and PSII in vesicles originating from a series of well-defined but different domains of the thylakoid membrane in spinach prepared by non-detergent techniques. Thylakoids from spinach were fragmented by sonication and separated by aqueous polymer two-phase partitioning into vesicles originating from grana and stroma lamellae. The grana vesicles were further sonicated and separated into two vesicle preparations originating from the grana margins and the appressed domains of grana (the grana core), respectively. PSI and PSII were determined in the same samples from the maximal size of the EPR signal from P700⁺ and Y_D, respectively. The following PSI/PSII ratios were found: thylakoids, 1.13; grana vesicles, 0.43; grana core, 0.25; grana margins, 1.28; stroma lamellae 3.10. In a sub-fraction of the stroma lamellae, denoted Y-100, PSI was highly enriched and the PSI/PSII ratio was 13. The antenna size of the respective photosystems was calculated from the experimental data and the assumption that a PSII center in the stroma lamellae (PSIIβ) has an antenna size of 100 Chl. This gave the following results: PSI in grana margins (PSIα) 300, PSI (PSIβ) in stroma lamellae 214, PSII in grana core (PSIIα) 280. The results suggest that PSI in grana margins have two additional light-harvesting complex II (LHCII) trimers per reaction center compared to PSI in stroma lamellae, and that PSII in grana has four LHCII trimers per monomer compared to PSII in stroma lamellae. Calculation of the total chlorophyll associated with PSI and PSII, respectively, suggests that more chlorophyll (about 10%) is associated with PSI than with PSII.     

Segregation of the photosystems in higher plant thylakoids and short- and long-term regulation by a mesoscopic approach

In this paper we consider the relationship between the lateral segregation of photosystems I and II in the grana and characteristics of the short- and long-term regulation in thylakoids following the mesoscopic approach. Our study is thermodynamic; it is based on the Flory-Huggins theory for binary mixtures and the McMillan-Mayer theory of heterogeneous solutions. We demonstrate that state transitions promote rearrangement of photosystems by either favoring their mixing after LHCII phosphorylation or enhancing their segregation after LHCII dephosphorylation. This regulation influences the entire system properties locally. We also demonstrate that the variations of the photosystem ratio promote rearrangement of the photosystems preserving their segregation. This regulation influences the entire system properties globally. The studies presented are another indication of the importance of the segregation of the photosystems in the grana thylakoids of higher plants. Segregation of PSIs and PSIIs is a signature of the spinodal decomposition, which is a fine regulatory mechanism, related to both the short- and long-term adaptations of the photosynthetic apparatus in higher plant thylakoids.     

A land plant‐specific thylakoid membrane protein contributes to photosystem II maintenance in Arabidopsis thaliana

The structure and function of photosystem II (PSII) are highly susceptible to photo‐oxidative damage induced by high‐fluence or fluctuating light. However, many of the mechanistic details of how PSII homeostasis is maintained under photoinhibitory light remain to be determined. We describe an analysis of the Arabidopsis thaliana gene At5g07020, which encodes an unannotated integral thylakoid membrane protein. Loss of the protein causes altered PSII function under high‐irradiance light, and hence it is named ‘Maintenance of PSII under High light 1’ (MPH1). The MPH1 protein co‐purifies with PSII core complexes and co‐immunoprecipitates core proteins. Consistent with a role in PSII structure, PSII complexes (supercomplexes, dimers and monomers) of the mph1 mutant are less stable in plants subjected to photoinhibitory light. Accumulation of PSII core proteins is compromised under these conditions in the presence of translational inhibitors. This is consistent with the hypothesis that the mutant has enhanced PSII protein damage rather than defective repair. These data are consistent with the distribution of the MPH1 protein in grana and stroma thylakoids, and its interaction with PSII core complexes. Taken together, these results strongly suggest a role for MPH1 in the protection and/or stabilization of PSII under high‐light stress in land plants.     

Investigations of the role of the main light-harvesting chlorophyll- protein complex in thylakoid membranes. Reconstitution of depleted membranes from intermittent-light-grown plants with the isolated complex

The functions of the light-harvesting complex of photosystem II (LHC- II) have been studied using thylakoids from intermittent-light-grown (IML) plants, which are deficient in this complex. These chloroplasts have no grana stacks and only limited lamellar appression in situ. In vitro the thylakoids showed limited but significant Mg2+-induced membrane appression and a clear segregation of membrane particles into such regions. This observation, together with the immunological detection of small quantities of LHC-II apoproteins, suggests that the molecular mechanism of appression may be similar to the more extensive thylakoid stacking seen in normal chloroplasts and involve LHC-II polypeptides directly. To study LHC-II function directly, a sonication- freeze-thaw procedure was developed for controlled insertion of purified LHC-II into IML membranes. Incorporation was demonstrated by density gradient centrifugation, antibody agglutination tests, and freeze-fracture electron microscopy. The reconstituted membranes, unlike the parent IML membranes, exhibited both extensive membrane appression and increased room temperature fluorescence in the presence of cations, and a decreased photosystem I activity at low light intensity. These membranes thus mimic normal chloroplasts in this regard, suggesting that the incorporated LHC-II interacts with photosystem II centers in IML membranes and exerts a direct role in the regulation of excitation energy distribution between the two photosystems.     

Dynamics of photosystem II: a proteomic approach to thylakoid protein complexes

Oxygenic photosynthesis produces various radicals and activeoxygen species with harmful effects on photosystem II (PSII).Such photodamage occurs at all light intensities. Damaged PSIIcentres, however, do not usually accumulate in the thylakoidmembrane due to a rapid and efficient repair mechanism. Theexcellent design of PSII gives protection to most of the proteincomponents and the damage is most often targeted only to thereaction centre D1 protein. Repair of PSII via turnover of thedamaged protein subunits is a complex process involving (i)highly regulated reversible phosphorylation of several PSIIcore subunits, (ii) monomerization and migration of the PSIIcore from the grana to the stroma lamellae, (iii) partial disassemblyof the PSII core monomer, (iv) highly specific proteolysis ofthe damaged proteins, and finally (v) a multi-step replacementof the damaged proteins with de novo synthesized copies followedby (vi) the reassembly, dimerization, and photoactivation ofthe PSII complexes. These processes will shortly be reviewedpaying particular attention to the damage, turnover, and assemblyof the PSII complex in grana and stroma thylakoids during thephotoinhibition–repair cycle of PSII. Moreover, a two-dimensionalBlue-native gel map of thylakoid membrane protein complexes,and their modification in the grana and stroma lamellae duringa high-light treatment, is presented. Key words: Arabidopsis thylakoid membrane proteome, assembly of photosystem II, D1 protein, light stress, photosystem II photoinhibition, repair of photosystem II