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.     

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.     

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.     

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.     

Molecular crowding and order in photosynthetic membranes

The integrity and maintenance of the photosynthetic apparatus in thylakoid membranes of higher plants requires lateral mobility of their components between stacked grana thylakoids and unstacked stroma lamellae. Computer simulations based on realistic protein densities suggest serious problems for lateral protein and plastoquinone diffusion especially in grana membranes, owing to strong retardation by protein complexes. It has been suggested that three structural features of grana thylakoids ensure efficient lateral transport: the organization of protein complexes into supercomplexes; the arrangement of supercomplexes into structured assemblies, which facilitates diffusion process in crowded membranes; the limitation of the diameter of grana discs to less than approximately 500 nm, which keeps diffusion times short enough to support regulation of light harvesting and repair of photodamaged photosystem II.     

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.     

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.     

From chloroplasts to photosystems: in situ scanning force microscopy on intact thylakoid membranes

Envelope-free chloroplasts were imaged in situ by contact and tapping mode scanning force microscopy at a lateral resolution of 3-5 nm and vertical resolution of approximately 0.3 nm. The images of the intact thylakoids revealed detailed structural features of their surface, including individual protein complexes over stroma, grana margin and grana-end membrane domains. Structural and immunogold-assisted assignment of two of these complexes, photosystem I (PS I) and ATP synthase, allowed direct determination of their surface density, which, for both, was found to be highest in grana margins. Surface rearrangements and pigment- protein complex redistribution associated with salt-induced membrane unstacking were followed on native, hydrated specimens. Unstacking was accompanied by a substantial increase in grana diameter and, eventually, led to their merging with the stroma lamellae. Concomitantly, PS IIalpha effective antenna size decreased by 21% and the mean size of membrane particles increased substantially, consistent with attachment of mobile light-harvesting complex II to PS I. The ability to image intact photosynthetic membranes at molecular resolution, as demonstrated here, opens up new vistas to investigate thylakoid structure and function.     

Probing the organization of photosystem II in photosynthetic membranes by atomic force microscopy

Efficient photosynthetic energy transduction and its regulation depend on a precise supramolecular arrangement of the plant photosystem II (PSII) complex in grana membranes of chloroplasts. The topography of isolated photosystem II supercomplexes and the supramolecular organization of this complex in grana membrane preparations are visualized by high-resolution atomic force microscopy (AFM) in air in tapping mode with an active feedback control to minimize tip-sample interactions. Systematic comparison between topographic characteristics of the protrusions in atomic force microscopic images and well-established high-resolution and freeze-fracture electron microscopic data shows that the photosystem II organization can be properly imaged by AFM in air. Taking the protruding water-splitting apparatus as a topographic marker for PSII, its distribution and orientation in isolated grana membrane were analyzed. For the latter a new mathematical procedure was established, which revealed a preference for a parallel alignment of PSII that resembles the organization in highly ordered semicrystalline arrays. Furthermore, by analyzing the height of grana membrane stacks, we conclude that lumenal protrusions of adjacent photosystem II complexes in opposing membranes are displaced relative to each other. The functional consequences for lateral migration processes are discussed.     

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.     

Reversible particle movements associated with unstacking and restacking of chloroplast membranes in vitro

Freeze-fracture and freeze-etch techniques have been employed to study the supramolecular structure of isolated spinach chloroplast membranes and to monitor structural changes associated with in vitro unstacking and restacking of these membranes. High-resolution particle size histograms prepared from the four fracture faces of normal chloroplast membranes reveal the presence of four distinct categories of intramembranous particles that are nonrandomly distributed between grana and stroma membranes. The large surface particles show a one to one relationship with the EF-face particles. Since the distribution of these particles between grana and stroma membranes coincides with the distribution of photosystem II (PS II) activity, it is argued that they could be structural equivalents of PS II complexes. An interpretative model depicting the structural relationship between all categories of particles is presented. Experimental unstacking of chloroplast membranes in low-salt medium for at least 45 min leads to a reorganization of the lamellae and to a concomitant intermixing of the different categories of membrane particles by means of translational movements in the plane of the membrane. In vitro restacking of such experimentally unstacked chloroplast membranes can be achieved by adding 2-20 mM MgCl2 or 100-200 mM NaCl to the membrane suspension. Membranes allowed to restack for at least 1 h at room temperature demonstrate a resegregation of the EF-face particles into the newly formed stacked membrane regions to yield a pattern and a size distribution nearly indistinguishable from the normally stacked controls. Restacking occurs in two steps: a rapid adhesion of adjoining stromal membrane surfaces with little particle movement, and a slower diffusion of additional large intramembranous particles into the stacked regions where they become trapped. Chlorophyll a:chlorophyll b ratios of membrane fraction obtained from normal, unstacked, and restacked membranes show that the particle movements are paralleled by movements of pigment molecules. The directed and reversible movements of membrane particles in isolated chloroplasts are compared with those reported for particles of plasma membranes.     

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.     

Photosystem II solubilizes as a monomer by mild detergent treatment of unstacked thylakoid membranes*

We studied the aggregation state of Photosystem II in stacked and unstacked thylakoid membranes from spinach after a quick and mild solubilization with the non-ionic detergent n-dodecyl-α,D-maltoside, followed by analysis by diode-array-assisted gel filtration chromatography and electron microscopy. The results suggest that Photosystem II (PS II) isolates either as a paired, appressed membrane fragment or as a dimeric PS II-LHC II supercomplex upon mild solubilization of stacked thylakoid membranes or PS II grana membranes, but predominantly as a core monomer upon mild solubilization of unstacked thylakoid membranes. Analysis of paired grana membrane fragments reveals that the number of PS II dimers is strongly reduced in single membranes at the margins of the grana membrane fragments. We suggest that unstacking of thylakoid membranes results in a spontaneous disintegration of the PS II-LHC II supercomplexes into separated PS II core monomers and peripheral light-harvesting complexes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes

ABSTRACT: BACKGROUND: The thylakoid system in plant chloroplasts is organized into two distinct domains: granaarranged in stacks of appressed membranes and non-appressed membranes consisting ofstroma thylakoids and margins of granal stacks. It is argued that the reason for thedevelopment of appressed membranes in plants is that their photosynthetic apparatus need tocope with and survive ever-changing environmental conditions. It is not known however,why different plant species have different arrangements of grana within their chloroplasts. Itis important to elucidate whether a different arrangement and distribution of appressed andnon-appressed thylakoids in chloroplasts are linked with different qualitative and/orquantitative organization of chlorophyll-protein (CP) complexes in the thylakoid membranesand whether this arrangement influences the photosynthetic efficiency. RESULTS: Our results from TEM and in situ CLSM strongly indicate the existence of differentarrangements of pea and bean thylakoid membranes. In pea, larger appressed thylakoids areregularly arranged within chloroplasts as uniformly distributed red fluorescent bodies, whileirregular appressed thylakoid membranes within bean chloroplasts correspond to smaller andless distinguished fluorescent areas in CLSM images. 3D models of pea chloroplasts show adistinct spatial separation of stacked thylakoids from stromal spaces whereas spatial divisionof stroma and thylakoid areas in bean chloroplasts are more complex. Structural differencesinfluenced the PSII photochemistry, however without significant changes in photosyntheticefficiency. Qualitative and quantitative analysis of chlorophyll-protein complexes as well asspectroscopic investigations indicated a similar proportion between PSI and PSII corecomplexes in pea and bean thylakoids, but higher abundance of LHCII antenna in pea ones.Furthermore, distinct differences in size and arrangements of LHCII-PSII and LHCI-PSIsupercomplexes between species are suggested. CONCLUSIONS: Based on proteomic and spectroscopic investigations we postulate that the differences in thechloroplast structure between the analyzed species are a consequence of quantitativeproportions between the individual CP complexes and its arrangement inside membranes.Such a structure of membranes induced the formation of large stacked domains in pea, orsmaller heterogeneous regions in bean thylakoids. Presented 3D models of chloroplasts showed that stacked areas are noticeably irregular with variable thickness, merging with eachother and not always parallel to each other.     

Structural changes of the thylakoid membrane network induced by high light stress in plant chloroplasts

Land plants live in a challenging environment dominated by unpredictable changes. A particular problem is fluctuation in sunlight intensity that can cause irreversible damage of components of the photosynthetic apparatus in thylakoid membranes under high light conditions. Although a battery of photoprotective mechanisms minimize damage, photoinhibition of the photosystem II (PSII) complex occurs. Plants have evolved a multi-step PSII repair cycle that allows efficient recovery from photooxidative PSII damage. An important feature of the repair cycle is its subcompartmentalization to stacked grana thylakoids and unstacked thylakoid regions. Thus, understanding the crosstalk between stacked and unstacked thylakoid membranes is essential to understand the PSII repair cycle. This review summarizes recent progress in our understanding of high-light-induced structural changes of the thylakoid membrane system and correlates these changes to the efficiency of the PSII repair cycle. The role of reversible protein phosphorylation for structural alterations is discussed. It turns out that dynamic changes in thylakoid membrane architecture triggered by high light exposure are central for efficient repair of PSII.     

Immunogold localization of light-harvesting and photosystem I complexes in the thylakoids ofFucus serratus (Phaeophyceae)

Summary The repartition of light-harvesting complex (LHC) and photosystem I (PS I) complex has been examined in isolated plastids ofFucus serratus by immunocytochemical labelling. LHC is distributed equally all along the length of thylakoid membranes, without any special repartition in the appressed membranes of the three associated thylakoids ofFucus. PS I is present on all the thylakoid membranes, but the external membranes of the three associated thylakoids are largely enriched relatively to the inner ones. This specific repartition of PSI on non-appressed membranes can be compared to the localization of PSI on stroma thylakoid membranes of higher plants and green algae. Consequently, although they share some common features with those of higher plants and green algae, the appressions of thylakoids in brown algae has neither the same structure nor the same functional role as typical grana stacked membranes in the repartition of the harvested energy.Abbreviations BSA bovine serum albumin - GAR goat anti-rabbit immunoglobulin G - LHC light-harvesting complex - PBS phosphatebuffered saline - PS I photosystem I - PS II photosystem II     

Excitation energy transfer in native and unstacked thylakoid membranes studied by low temperature and ultrafast fluorescence spectroscopy

In this work, the transfer of excitation energy was studied in native and cation-depletion induced, unstacked thylakoid membranes of spinach by steady-state and time-resolved fluorescence spectroscopy. Fluorescence emission spectra at 5 K show an increase in photosystem I (PSI) emission upon unstacking, which suggests an increase of its antenna size. Fluorescence excitation measurements at 77 K indicate that the increase of PSI emission upon unstacking is caused both by a direct spillover from the photosystem II (PSII) core antenna and by a functional association of light-harvesting complex II (LHCII) to PSI, which is most likely caused by the formation of LHCII-LHCI-PSI supercomplexes. Time-resolved fluorescence measurements, both at room temperature and at 77 K, reveal differences in the fluorescence decay kinetics of stacked and unstacked membranes. Energy transfer between LHCII and PSI is observed to take place within 25 ps at room temperature and within 38 ps at 77 K, consistent with the formation of LHCII-LHCI-PSI supercomplexes. At the 150–160 ps timescale, both energy transfer from LHCII to PSI as well as spillover from the core antenna of PSII to PSI is shown to occur at 77 K. At room temperature the spillover and energy transfer to PSI is less clear at the 150 ps timescale, because these processes compete with charge separation in the PSII reaction center, which also takes place at a timescale of about 150 ps.     

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.     

Distribution of the early light-inducible protein in the thylakoids of developing pea chloroplasts

The distribution of the early light-inducible protein (ELIP) of pea (Pisum sativum) between grana and stroma thylakoids was studied. An antibody raised against a bacterial-expressed fusion protein containing ELIP sequences was used. Illumination of dark-grown pea seedlings causes an accumulation of the ELIP in the thylakoid membranes with a maximum level at 16 h. During continuous illumination exceeding 16 h the level decreases again. The fractionation of thylakoid membranes of 48-h-illuminated pea seedlings in grana and stroma thylakoids reveals that there is no uniform distribution of ELIP in the thylakoids. Rather 60-70% of ELIP was found in the stroma thylakoids and 30-40% in the grana thylakoids. This distribution is in accordance with that of photosystem I but not with that of photosystem II. After Triton-X-100 solubilization almost all ELIP is found in the photosystem-I-containing fraction. This also supports an association of ELIP with photosystem I.     

The importance of grana stacking for xanthophyll cycle-dependent NPQ in the thylakoid membranes of higher plants

In the present study we have examined the effects of grana stacking on the rate of violaxanthin (Vx) de-epoxidation and the extent of non-photochemical quenching of chlorophyll a fluorescence (NPQ) in isolated thylakoid membranes of spinach. Our results show that partial and complete unstacking of thylakoids in reaction media devoid of sorbitol and MgCl₂ did not significantly affect the efficiency of Vx de-epoxidation. Under high light (HL) illumination we found slightly higher values of Vx conversion in stacked membranes, whereas in thylakoids incubated at pH 5.2 in the dark, representing the pH-optimum of Vx de-epoxidase, de-epoxidation was slightly increased in the unstacked membranes. Partial and complete unstacking of grana membranes, however, had a dramatic effect on the HL-induced NPQ. High NPQ values could only be achieved in stacked thylakoid membranes in the presence of MgCl₂ and sorbitol. In unstacked membranes NPQ was drastically decreased. The effects of grana stacking on the xanthophyll cycle-dependent component of NPQ were even more pronounced, and complete unstacking of thylakoid membranes led to a total loss of this quenching component. Our data imply that grana stacking in the thylakoid membranes of higher plants is of high importance for the process of overall NPQ. For the xanthophyll cycle-dependent component of NPQ it may even be essential. Possible effects of grana stacking on the mechanism of zeaxanthin-dependent quenching are discussed.