Segregation of the photosystems in thylakoids depends on their size

Lateral segregation of two types of photosystems in thylakoid membranes of green plants is one of the key factors that provide the stability and fine-tuning of the light quanta supply by pigment proteins and non-cyclic electron transport. Due to this specific feature of the membrane structural organization, the photosynthetic units function in the green plants with optimal performance. In this report a mesoscopic theory is outlined to address the physical aspects of segregation phenomenon. Results of theoretical studies and computer simulations suggest that charge mismatch and the size difference between two photosystems in grana are most responsible for their lateral segregation, which is driven by the screened electrostatic and lipid-induced interactions. Comparative simulations of photosystems of different sizes show the crucial dependence of their ordering on a geometrical parameter. It seems that the size effect alone may prevent photosystems from segregated arrangement in cyanobacterial thylakoids.     

Phosphorylation-dependent regulation of excitation energy distribution between the two photosystems in higher plants

Phosphorylation-dependent movement of the light-harvesting complex II (LHCII) between photosystem II (PSII) and photosystem I (PSI) takes place in order to balance the function of the two photosystems. Traditionally, the phosphorylatable fraction of LHCII has been considered as the functional unit of this dynamic regulation. Here, a mechanical fractionation of the thylakoid membrane of Spinacia oleracea was performed from leaves both in the phosphorylated state (low light, LL) and in the dephosphorylated state (dark, D) in order to compare the phosphorylation-dependent protein movements with the excitation changes occurring in the two photosystems upon LHCII phosphorylation. Despite the fact that several LHCII proteins migrate to stroma lamellae when LHCII is phosphorylated, no increase occurs in the 77 K fluorescence emitted from PSI in this membrane fraction. On the contrary, such an increase in fluorescence occurs in the grana margin fraction, and the functionally important mobile unit is the PSI-LHCI complex. A new model for LHCII phosphorylation driven regulation of relative PSII/PSI excitation thus emphasises an increase in PSI absorption cross-section occurring in grana margins upon LHCII phosphorylation and resulting from the movement of PSI-LHCI complexes from stroma lamellae and subsequent co-operation with the P-LHCII antenna from the grana. The grana margins probably give a flexibility for regulation of linear and cyclic electron flow in plant chloroplasts.     

Phosphorylation-dependent regulation of excitation energy distribution between the two photosystems in higher plants

Phosphorylation-dependent movement of the light-harvesting complex II (LHCII) between photosystem II (PSII) and photosystem I (PSI) takes place in order to balance the function of the two photosystems. Traditionally, the phosphorylatable fraction of LHCII has been considered as the functional unit of this dynamic regulation. Here, a mechanical fractionation of the thylakoid membrane of Spinacia oleracea was performed from leaves both in the phosphorylated state (low light, LL) and in the dephosphorylated state (dark, D) in order to compare the phosphorylation-dependent protein movements with the excitation changes occurring in the two photosystems upon LHCII phosphorylation. Despite the fact that several LHCII proteins migrate to stroma lamellae when LHCII is phosphorylated, no increase occurs in the 77 K fluorescence emitted from PSI in this membrane fraction. On the contrary, such an increase in fluorescence occurs in the grana margin fraction, and the functionally important mobile unit is the PSI-LHCI complex. A new model for LHCII phosphorylation driven regulation of relative PSII/PSI excitation thus emphasises an increase in PSI absorption cross-section occurring in grana margins upon LHCII phosphorylation and resulting from the movement of PSI-LHCI complexes from stroma lamellae and subsequent co-operation with the P-LHCII antenna from the grana. The grana margins probably give a flexibility for regulation of linear and cyclic electron flow in plant chloroplasts.     

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.     

A supramolecular light-harvesting complex from chloroplast photosystem-II membranes.

In this study, we report on the composition of a photosystem-II antenna preparation which contains three chlorophyll-a/b proteins (CP), CP29, CP24 and light-harvesting complex (LHC) II obtained from Zea mays grana membranes as previously described [Dainese, P. & Bassi, R. (1991) J. Biol. Chem. 266, 8136-8142]. We demonstrate that the three chlorophyll proteins are present in the preparation with a 3:3:9 molar ratio and that they form a supramolecular antenna complex which represents one third of the photosystem-II antenna system. Phosphorylation experiments show that this complex is involved in the mechanism of regulation of excitation-energy distribution between photosystems: phosphorylation of the membranes induces dissociation of the LHCII moiety from the CP29-CP24 moiety and changes in the aggregation state of LHCII components of the CP29-CP24-LHCII complex. The LHCII subpopulations of the complex are shown to be distinct from the total LHCII population by isoelectrofocusing analysis. On the basis of these data and in the light of the stoichiometry of photosystem-II chlorophyll-binding proteins, we propose a model for the organization of photosystem-II antenna system.     

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.     

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.     

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.     

Light regulation of photosynthetic membrane structure,organization, and function

The light environment during plant growth determines the structural and functional properties of higher plant chloroplasts, thus revealing a dynamically regulated developmental system. Pisum sativum plants growing under intermittent illumination showed chloroplasts with fully functional photosystem (PS) II and PSI reaction centers that lacked the peripheral chlorophyll (Chi) a/b and Chl a light-harvesting complexes (LHC), respectively. The results suggest a light flux differential threshold regulation in the biosynthesis of the photosystem core and peripheral antenna complexes. Sun-adapted species and plants growing under far-red-depleted illumination showed grana stacks composed of few (3–5) thylakoids connected with long intergrana (stroma) thylakoids. They had a PSII/PSI reaction center ratio in the range 1.3–1.9. Shade-adapted species and plants growing under far-red-enrichcd illumination showed large grana stacks composed of several thylakoids, often extending across the entire chloroplast body, and short intergrana stroma thylakoids. They had a higher PSII/PSI reaction center ratio, in the range of 2.2–4.0. Thus, the relative extent of grana and stroma thylakoid formation corresponds with the relative amounts of PSII and PSI in the chloroplast, respectively. The structural and functional adaptation of the photosynthetic membrane system in response to the quality of illumination involves mainly a control on the rate of PSII and PSI complex biosynthesis.     

Proteomic characterization of hierarchical megacomplex formation in Arabidopsis thylakoid membrane

Conversion of solar energy into chemical energy in plant chloroplasts concomitantly modifies the thylakoid architecture and hierarchical interactions between pigment–protein complexes. Here, the thylakoids were isolated from light‐acclimated Arabidopsis leaves and investigated with respect to the composition of the thylakoid protein complexes and their association into higher molecular mass complexes, the largest one comprising both photosystems (PSII and PSI) and light‐harvesting chlorophyll a/b‐binding complexes (LHCII). Because the majority of plant light‐harvesting capacity is accommodated in LHCII complexes, their structural interaction with photosystem core complexes is extremely important for efficient light harvesting. Specific differences in the strength of LHCII binding to PSII core complexes and the formation of PSII supercomplexes are well characterized. Yet, the role of loosely bound L‐LHCII that disconnects to a large extent during the isolation of thylakoid protein complexes remains elusive. Because L‐LHCII apparently has a flexible role in light harvesting and energy dissipation, depending on environmental conditions, its close interaction with photosystems is a prerequisite for successful light harvesting in vivo. Here, to reveal the labile and fragile light‐dependent protein interactions in the thylakoid network, isolated membranes were subjected to sequential solubilization using detergents with differential solubilization capacity and applying strict quality control. Optimized 3D‐lpBN‐lpBN‐sodium dodecyl sulfate–polyacrylamide gel electrophoresis system demonstrated that PSII–LHCII supercomplexes, together with PSI complexes, hierarchically form larger megacomplexes via interactions with L‐LHCII trimers. The polypeptide composition of LHCII trimers and the phosphorylation of Lhcb1 and Lhcb2 were examined to determine the light‐dependent supramolecular organization of the photosystems into megacomplexes.     

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.     

Appearance of type 1, 2, and 3 light-harvesting complex II and light-harvesting complex I proteins during light-induced greening of barley (Hordeum vulgare) etioplasts.

Monospecific antibodies directed against typical domains of type 1, 2, and 3 light-harvesting complex (LHC) II apoproteins have been used (a) to identify these apoproteins on denaturing sodium dodecyl sulfate gels of barley (Hordeum vulgare) thylakoids, (b) to determine their distribution between grana and stroma membranes, and (c) to follow their accumulation during light-induced greening of etioplasts. In addition, we have studied the light-induced assembly of chlorophyll-protein complexes with a native green gel system (K.D. Allen, L.A. Staehelin [1991] Anal Biochem 194: 214-222). Western blot analysis of the three major LHCII apoprotein bands has identified the highest molecular mass band at 27.5 kD as containing the type 2 LHCII apoproteins, the middle band at 26.9 kD as containing the type 1 LHCII apoproteins, and the lowest band at 26.0 kD as containing the type 3 LHCII apoproteins. During light-induced greening of 6-d-old etiolated barley seedlings, the type 1, 2, and 3 LHCII apoproteins accumulate simultaneously and at similar rates but appear somewhat sooner (< 4 h) in thylakoids from apical than from basal (4-8 h) leaf segments. LHCI polypeptides accrue with similar kinetics, whereas the 33-kD oxygen-evolving complex polypeptides can be detected already in the 0-h light samples. During the most rapid phase of thylakoid development (8-24 h), two slightly larger (28.3 and 28.7 kD) type 2 LHCII apoproteins (precursor intermediates?) also accumulate in the thylakoids. No corresponding higher molecular mass forms of type 1 and 3 LHCII apoproteins could be detected. It is interesting that differences are still apparent in the composition of chlorophyll-protein complexes of light-control plants and those of etiolated plants greened for 8 d.     

Resolution of 16 to 20 chlorophyll-protein complexes using a low ionic strength native green gel system

Conventional native "green gel" systems resolve at most 10 chlorophyll-protein complexes from thylakoid membranes of higher plants and green algae. Such analyses suggest a simplicity of the thylakoid membrane that is not supported by a growing body of evidence on the heterogeneity of photosystems I and II (PSI and PSII) and their associated antennae (LHCI and LHCII). We report here the development and characterization of a low ionic strength native "green gel" system that resolves from 16 to 20, mostly large chlorophyll-protein complexes from a variety of higher plant and green algal species with very little release of free pigment. In Chlamydomonas, this system resolves multiple PSI-LHCI complexes, multiple PSII-LHCII complexes, four oligomeric LHCII complexes, as well as several low electrophoretic mobility reaction center complexes, and a number of small complexes. We have obtained similar resolution with a large number of higher plant and green algal species. We also demonstrate how this system can be used as a sort of "fingerprinting" technique to distinguish thylakoids of different species, and for the analysis of photosynthetic mutants, using the chlorophyll b-less chlorina f2 mutant of barley as an example.     

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.     

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.     

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.     

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.     

Light affects the accessibility of the thylakoid light harvesting complex II (LHCII) phosphorylation site to the membrane protein kinase(s)

Redox-controlled, reversible phosphorylation of the thylakoid light harvesting complex II (LHCII) regulates its association with photosystems (PS) I or II and thus, energy distribution between the two photosystems (state transition). Illumination of solubilized LHCII enhances exposure of the phosphorylation site at its N-terminal domain to protein kinase(s) and tryptic cleavage in vitro [Zer et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8277-8282]. Here we report that short illumination (5-10 min, 15-30 micromol m(-2) s(-1)) enhances the accessibility of LHCII phosphorylation site to kinase(s) activity also in isolated thylakoids. However, prolonged illumination or higher light intensities (30 min, 80-800 micromol m(-2) s(-1)) prevent phosphorylation of LHCII in the isolated membranes as well as in vivo, although redox-dependent protein kinase activity persists in the illuminated thylakoids toward exogenous solubilized LHCII. This phenomenon, ascribed to light-induced inaccessibility of the phosphorylation site to the protein kinase(s), affects in a similar way the accessibility of thylakoid LHCII N-terminal domain to tryptic cleavage. The illumination effect is not redox related, decreases linearly with temperature from 25 to 5 degrees C and may be ascribed to light-induced conformational changes in the complex causing lateral aggregation of dephosphorylated LHCII bound to and/or dissociated from PSII. The later state occurs under conditions allowing turnover of the phospho-LHCII phosphate. The light-induced inaccessibility of LHCII to the membrane-bound protein kinase reverses readily in darkness only if induced under LHCII-phosphate turnover conditions. Thus, phosphorylation prevents irreversible light-induced conformational changes in LHCII allowing lateral migration of the complex and the related state transition process.     

Light‐harvesting superstructures of green plant chloroplasts lacking photosystems

The light‐harvesting antenna of higher plant photosystem II (LHCII) is the major photosynthetic membrane component encoded by an entire family of homologous nuclear genes. On the contrary, the great majority of proteins of photosystems and electron transport components are encoded by the chloroplast genome. In this work, we succeeded in gradually inhibiting the expression of the chloroplast genes that led to the disappearance of the photosystem complexes, mimicking almost total photoinhibition. The treated plants, despite displaying only some early signs of senescence, sustained their metabolism and growth for several weeks. The only major remaining membrane component was LHCII antenna that formed superstructures – stacks of dozens of thylakoids or supergrana. Freeze‐fracture electron microscopy revealed specific organization, directly displaying frequently bifurcated membranes with reduced or totally absent photosystem II (PSII) reaction centre complexes. Our findings show that it is possible to accumulate large amounts of light‐harvesting membranes, organized into three‐dimensional structures, in the absence of reaction centre complexes. This points to the reciprocal role of LHCII and PSII in self‐assembly of the three‐dimensional matrix of the photosynthetic membrane, dictating its size and flexible adaptation to the light environment.     

Supramolecular photosystem II organization in grana thylakoid membranes: evidence for a structured arrangement

The distribution of photosystem (PS) II complexes in stacked grana thylakoids derived from electron microscopic images of freeze-fractured chloroplasts are examined for the first time using mathematical methods. These characterize the particle distribution in terms of a nearest neighbor distribution function and a pair correlation function. The data were compared with purely random distributions calculated by a Monte Carlo simulation. The analysis reveals that the PSII distribution in grana thylakoids does not correspond to a random protein mixture but that ordering forces lead to a structured arrangement on a supramolecular level. Neighboring photosystems are significantly more separated than would be the case in a purely random distribution. These results are explained by structural models, in which boundary lipids and light-harvesting complex (LHC) II trimers are arranged between neighboring PSII. Furthermore, the diffusion of PSII was analyzed by a Monte Carlo simulation with a protein density of 80% area occupation (determined for grana membranes). The mobility of the photosystems is severely reduced by the high protein density. From an estimate of the mean migration time of PSII from grana thylakoids to stroma lamellae, it becomes evident that this diffusion contributes significantly to the velocity of the repair cycle of photoinhibited PSII.