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The cytochrome b 6  f (cytb 6  f ) complex has a central role in oxygenic photosynthesis, linking electron transfer between photosystems I and II and converting solar energy into a transmembrane proton gradient for ATP synthesis 1-3 . Electron transfer within cytb 6  f occurs via the quinol (Q) cycle, which catalyses the oxidation of plastoquinol (PQH 2 ) and the reduction of both plastocyanin (PC) and plastoquinone (PQ) at two separate sites via electron bifurcation 2 . In higher plants, cytb 6  f also acts as a redox-sensing hub, pivotal to the regulation of light harvesting and cyclic electron transfer that protect against metabolic and environmental stresses 3 . Here we present a 3.6 Å resolution cryo-electron microscopy (cryo-EM) structure of the dimeric cytb 6  f complex from spinach, which reveals the structural basis for operation of the Q cycle and its redox-sensing function. The complex contains up to three natively bound PQ molecules. The first, PQ1, is located in one cytb 6  f monomer near the PQ oxidation site (Q p ) adjacent to haem b p and chlorophyll a. Two conformations of the chlorophyll a phytyl tail were resolved, one that prevents access to the Q p site and another that permits it, supporting a gating function for the chlorophyll a involved in redox sensing. PQ2 straddles the intermonomer cavity, partially obstructing the PQ reduction site (Q n ) on the PQ1 side and committing the electron transfer network to turnover at the occupied Q n site in the neighbouring monomer. A conformational switch involving the haem c n propionate promotes two-electron, two-proton reduction at the Q n site and avoids formation of the reactive intermediate semiquinone. The location of a tentatively assigned third PQ molecule is consistent with a transition between the Q p and Q n sites in opposite monomers during the Q cycle. The spinach cytb 6  f structure therefore provides new insights into how the complex fulfils its catalytic and regulatory roles in photosynthesis.

Understanding the effects of many essential non-steroidal anti-inflammatory drugs (NSAIDs) on plants is still limited, especially at environmentally realistic concentrations. This paper presents the influence of three of the most frequently used NSAIDs (diclofenac, ibuprofen, and naproxen) at environmentally realistic concentrations on the autochthonous green leafy vegetables: orache (Atriplex patula L.), spinach (Spinacia oleracea L.) and lettuce (Lactuca sativa L.). Our research was focused on the determination of the photosynthetic parameters, the emission rate of volatile organic compounds, and the evaluation of the ultrastructure of leaves of studied vegetables after exposure to abiotic stress induced by environmental pollutants, namely NSAIDs. The data obtained indicate a moderate reduction of foliage physiological activity as a response to the stress induced by NSAIDs to the selected green leafy vegetables. The increase of the 3-hexenal and monoterpene emission rates with increasing NSAIDs concentration could be used as a sensitive and a rapid indicator to assess the toxicity of the NSAIDs. Microscopic analysis showed that the green leafy vegetables were affected by the selected NSAIDs. In comparison to the controls, the green leafy vegetables treated with NSAIDs presented irregular growth of glandular trichomes on the surface of the adaxial side of the leaves, less stomata, cells with less cytoplasm, irregular cell walls and randomly distributed chloroplasts. Of the three NSAIDs investigated in this study, ibuprofen presented the highest influence. The results obtained in this study can be used to better estimate the impact of drugs on the environment and to improve awareness on the importance of the responsible use of drugs.

Upon transition of plants from darkness to light the initiation of photosynthetic linear electron transfer (LET) from H 2 O to NADP + precedes the activation of CO 2 fixation, creating a lag period where cyclic electron transfer (CET) around photosystem I (PSI) has an important protective role. CET generates ΔpH without net reduced NADPH formation, preventing overreduction of PSI via regulation of the cytochrome b 6 f (cytb 6 f) complex and protecting PSII from overexcitation by inducing non-photochemical quenching. The dark-to-light transition also provokes increased phosphorylation of light-harvesting complex II (LHCII). However, the relationship between LHCII phosphorylation and regulation of the LET/CET balance is not understood. Here, we show that the dark-to-light changes in LHCII phosphorylation profoundly alter thylakoid membrane architecture and the macromolecular organization of the photosynthetic complexes, without significantly affecting the antenna size of either photosystem. The grana diameter and number of membrane layers per grana are decreased in the light while the number of grana per chloroplast is increased, creating a larger contact area between grana and stromal lamellae. We show that these changes in thylakoid stacking regulate the balance between LET and CET pathways. Smaller grana promote more efficient LET by reducing the diffusion distance for the mobile electron carriers plastoquinone and plastocyanin, whereas larger grana enhance the partition of the granal and stromal lamellae plastoquinone pools, enhancing the efficiency of CET and thus photoprotection by non-photochemical quenching.

Protein synthesis in the chloroplast is mediated by the chloroplast ribosome (chloro-ribosome). Overall architecture of the chloro-ribosome is considerably similar to the Escherichia coli (E. coli) ribosome but certain differences are evident. The chloro-ribosome proteins are generally larger because of the presence of chloroplast-specific extensions in their N- and C-termini. The chloro-ribosome harbours six plastid-specific ribosomal proteins (PSRPs); four in the small subunit and two in the large subunit. Deletions and insertions occur throughout the rRNA sequence of the chloro-ribosome (except for the conserved peptidyl transferase center region) but the overall length of the rRNAs do not change significantly, compared to the E. coli. Although, recent advancements in cryo-electron microscopy (cryo-EM) have provided detailed high-resolution structures of ribosomes from many different sources, a high-resolution structure of the chloro-ribosome is still lacking. Here, we present a cryo-EM structure of the large subunit of the chloro-ribosome from spinach (Spinacia oleracea) at an average resolution of 3.5 Å. High-resolution map enabled us to localize and model chloro-ribosome proteins, chloroplast-specific protein extensions, two PSRPs (PSRP5 and 6) and three rRNA molecules present in the chloro-ribosome. Although comparable to E. coli, the polypeptide tunnel and the tunnel exit site show chloroplast-specific features.

Uranyl acetate (UAc) has been generally used not only as a superb staining reagent for ultrathin sections of plastic-embedded biological materials, but also as high-contrast negative stains for biological macromolecules such as particles of protein or virus. However, the use and purchase of radioactive UAc have been restricted. In this study, we determine the performance of ytterbium triacetate, lutetium triacetate, samarium triacetate and gadolinium triacetate as new staining reagents for biological electron microscopy. We observed chemically fixed spinach (Spinacia oleracea) leaves stained with these reagents. Ultrathin sections were stained with these reagents. Some of them were counterstained with lead citrate. The transmission electron microscopy contrast of spinach organelles was evaluated in sections exposed to the conventional stain and new stains. We show acetate salts of samarium, gadolinium, ytterbium and lutetium could be excellent substitutes for UAc for thin section staining and for negative staining. In addition, each reagent showed appreciable negative-staining effects., (© The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Under light stress, the reaction center-binding protein D1 of PSII is photo-oxidatively damaged and removed from PSII complexes by proteases located in the chloroplast. A protease considered to be responsible for degradation of the damaged D1 protein is the metalloprotease FtsH. We showed previously that the active hexameric FtsH protease is abundant at the grana margin and the grana end membranes, and this homo-complex removes the photodamaged D1 protein in the grana. Here, we showed a change in the distribution of FtsH in spinach thylakoids during excessive illumination by transmission electron microscopy (TEM) and immunogold labeling of FtsH. The change in distribution of the protease was accompanied by structural changes to the thylakoids, which we detected using spinach leaves by TEM after chemical fixation of the samples. Quantitative analyses showed several characteristic changes in the structure of the thylakoids, including shrinkage of the grana, outward bending of the marginal portions of the thylakoids and an increase in the height of the grana stacks under excessive illumination. The increase in the height of the grana stacks may include swelling of the thylakoids and an increase in the partition gaps between the thylakoids. These data strongly suggest that excessive illumination induces partial unstacking of the thylakoids, which enables FtsH to access easily the photodamaged D1 protein. Finally three-dimensional tomography of the grana was recorded to observe the effect of light stress on the overall structure of the thylakoids., (© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The luminol bioluminescence system containing luminol, hydrogen peroxide and HRP was used as a potential substitute light source of sunlight for the photosynthesis of plants, in which the electron flow of the photosynthesis process was proven using chloroplasts isolated from spinach leaves.

The multilamellar organization of freshly isolated spinach and pea chloroplast thylakoid membranes was studied using small-angle neutron scattering. A broad peak at ~0.02Å(-1) is ascribed to diffraction from domains of ordered, unappressed stroma lamellae, revealing a repeat distance of 294ű7Å in spinach and 345ű11Å in pea. The peak position and hence the repeat distance of stroma lamellae is strongly dependent on the osmolarity and the ionic strength of the suspension medium, as demonstrated by varying the sorbitol and the Mg(++)-concentration in the sample. For pea thylakoid membranes, we show that the repeat distance decreases when illuminating the sample with white light, in accordance with our earlier results on spinach, also regarding the observation that addition of an uncoupler prohibits the light-induced structural changes, a strong indication that these changes are driven by the transmembrane proton gradient. We show that the magnitude of the shrinkage is strongly dependent on light intensity and that the repeat distance characteristic of the dark state after illumination is different from the initial dark state. Prolonged strong illumination leads to irreversible changes and swelling as reflected in increased repeat distances. The observed reorganizations are discussed within the frames of the current structural models of the granum-stroma thylakoid membrane assembly and the regulatory mechanisms in response to variations in the environmental conditions in vivo. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The thylakoid membrane system is a complex membrane system that organizes and reorganizes itself to provide plants optimal chemical energy from sunlight under different and varying environmental conditions. Grana membranes are part of this system and contain the light-driven water-splitting enzyme Photosystem II (PSII) and light-harvesting antenna complexes. Here, we present a direct visualization of PSII complexes within grana membranes from spinach. By means of jumping mode atomic force microscopy in liquid, minimal forces were applied between the scanning tip and membrane or protein, allowing complexes to be imaged with high detail. We observed four different packing arrangements of PSII complexes, which occur primarily as dimers: co-linear crystalline rows, nanometric domains of straight or skewed rows, and disordered domains. Upon storing surface-adhered membranes at low temperature prior to imaging, large-scale reorganizations of supercomplexes between PSII and light-harvesting complex II could be induced. The highest resolution images show the existence of membrane domains without obvious topography extending beyond supercomplexes. These observations illustrate the possibility for diffusion of proteins and smaller molecules within these densely packed membranes.

Background and Aims: Within Chenopodioideae, Atripliceae have been distinguished by two bracteoles enveloping the female flowers/fruits, whereas in other tribes flowers are described as ebracteolate with persistent perianth. Molecular phylogenetic hypotheses suggest 'bracteoles' to be homoplastic. The origin of the bracteoles was explained by successive inflorescence reductions. Flower reduction was used to explain sex determination. Therefore, floral ontogeny was studied to evaluate the nature of the bracteoles and sex determination in Atripliceae., Methods: Inflorescences of species of Atriplex, Chenopodium, Dysphania and Spinacia oleracea were investigated using light microscopy and scanning electron microscopy., Key Results: The main axis of the inflorescence is indeterminate with elementary dichasia as lateral units. Flowers develop centripetally, with first the formation of a perianth primordium either from a ring primordium or from five individual tepal primordia fusing post-genitally. Subsequently, five stamen primordia originate, followed by the formation of an annular ovary primordium surrounding a central single ovule. Flowers are either initially hermaphroditic remaining bisexual and/or becoming functionally unisexual at later stages, or initially unisexual. In the studied species of Atriplex, female flowers are strictly female, except in A. hortensis. In Spinacia, female and male flowers are unisexual at all developmental stages. Female flowers of Atriplex and Spinacia are protected by two accrescent fused tepal lobes, whereas the other perianth members are absent., Conclusions: In Atriplex and Spinacia modified structures around female flowers are not bracteoles, but two opposite accrescent tepal lobes, parts of a perianth persistent on the fruit. Flowers can achieve sexuality through many different combinations; they are initially hermaphroditic, subsequently developing into bisexual or functionally unisexual flowers, with the exception of Spinacia and strictly female flowers in Atriplex, which are unisexual from the earliest developmental stages. There may be a relationship between the formation of an annular perianth primordium and flexibility in floral sex determination.

Plants must regulate their use of absorbed light energy on a minute-by-minute basis to maximize the efficiency of photosynthesis and to protect photosystem II (PSII) reaction centers from photooxidative damage. The regulation of light harvesting involves the photoprotective dissipation of excess absorbed light energy in the light-harvesting antenna complexes (LHCs) as heat. Here, we report an investigation into the structural basis of light-harvesting regulation in intact spinach (Spinacia oleracea) chloroplasts using freeze-fracture electron microscopy, combined with laser confocal microscopy employing the fluorescence recovery after photobleaching technique. The results demonstrate that formation of the photoprotective state requires a structural reorganization of the photosynthetic membrane involving dissociation of LHCII from PSII and its aggregation. The structural changes are manifested by a reduced mobility of LHC antenna chlorophyll proteins. It is demonstrated that these changes occur rapidly and reversibly within 5 min of illumination and dark relaxation, are dependent on ΔpH, and are enhanced by the deepoxidation of violaxanthin to zeaxanthin.

We have investigated the three-dimensional (3D) architecture of the thylakoid membranes of Arabidopsis (Arabidopsis thaliana), tobacco (Nicotiana tabacum), and spinach (Spinacia oleracea) with a resolution of approximately 7 nm by electron tomography of high-pressure-frozen/freeze-substituted intact chloroplasts. Higher-plant thylakoids are differentiated into two interconnected and functionally distinct domains, the photosystem II/light-harvesting complex II-enriched stacked grana thylakoids and the photosystem I/ATP synthase-enriched, nonstacked stroma thylakoids. The grana thylakoids are organized in the form of cylindrical stacks and are connected to the stroma thylakoids via tubular junctions. Our data confirm that the stroma thylakoids are wound around the grana stacks in the form of multiple, right-handed helices at an angle of 20° to 25° as postulated by a helical thylakoid model. The junctional connections between the grana and stroma thylakoids all have a slit-like architecture, but their size varies tremendously from approximately 15 × 30 nm to approximately 15 × 435 nm, which is approximately 5 times larger than seen in chemically fixed thylakoids. The variable slit length results in less periodicity in grana/stroma thylakoid organization than proposed in the original helical model. The stroma thylakoids also exhibit considerable architectural variability, which is dependent, in part, on the number and the orientation of adjacent grana stacks to which they are connected. Whereas some stroma thylakoids form solid, sheet-like bridges between adjacent grana, others exhibit a branching geometry with small, more tubular sheet domains also connecting adjacent, parallel stroma thylakoids. We postulate that the tremendous variability in size of the junctional slits may reflect a novel, active role of junctional slits in the regulation of photosynthetic function. In particular, by controlling the size of junctional slits, plants could regulate the flow of ions and membrane molecules between grana and stroma thylakoid membrane domains.

Electrospun fibers consisting of poly(3,4-ethylenedioxythiophene)/poly(styrene sulfonate) (PEDOT/PSS) and poly(ethylene oxide) (PEO) have been used to successfully encapsulate and stabilize thylakoid membrane vesicles isolated from spinach. Light-driven electronic properties were measured. Fibers with immobilized thylakoids show higher electrical conductivity compared with fibers without thylakoids under white light conditions. This is attributed to the electron-generating photosynthetic reactions from the thylakoids. Electron and optical microscopy show the presence of thylakoid vesicles within the fibers using lipid-specific stains. After electrospinning into fibers, the thylakoid vesicles still exhibit an ability to produce a light-driven electron gradient, indicating that activity is preserved during the electrospinning process. These electrospun fibers provide an excellent example of incorporating photosynthetic function into an artificial system.

Background: Thylakoid-stabilised emulsions have been reported to possess satiety-promoting effects and inhibit pancreatic lipase-colipase activity in vitro, which prompted the investigation of their interfacial properties., Results: Thylakoid membranes isolated from spinach were used as an emulsifier/stabiliser in oil (triglyceride)-in-water emulsions. Emulsions were characterised with respect to droplet size, interfacial tension, creaming, surface load and electron microscopy. The effects of pH and thylakoid concentration were also considered. Droplet size decreased with increasing thylakoid concentration, reaching a plateau around 15 microm beyond concentrations of 2 mg protein mL(-1) oil. The resulting emulsions were stable against coalescence but were subject to creaming. The surface pressure (air/water interface) of the thylakoid isolate was 44 mN m(-1) and the surface load 13 mg m(-2) at 10 mg protein mL(-1) oil. Electron micrographs showed thylakoids adsorbed as bunched vesicles on the drop surfaces. The stabilisation mechanism can be described as a combined effect of surface-active molecules, mainly membrane proteins but also membrane lipids, exposed on surfaces of thylakoid membrane vesicles adsorbed as particles., Conclusion: Thylakoid membranes effectively stabilise oil-in-water emulsions, which should facilitate their incorporation in food with satiety-promoting effects. To the authors' knowledge, this is the first study on the emulsifying properties of an isolated biological membrane as a functional ingredient., (2010 Society of Chemical Industry.)

We demonstrate far-field optical imaging at the nanoscale with unlabeled samples. Subdiffraction resolution images of autofluorescent samples are obtained by depleting the ground state of natural fluorophores by transferring them to a metastable dark state and simultaneously localizing those fluorophores that are transiently returning. Our approach is based on the insight that nanoscopy methods relying on stochastic single-molecule switching require only a single fluorescence on-off cycle to yield an image, a condition fulfilled by various biomolecules. The method is exemplified by recording label-free nanoscopy images of thylakoid membranes of spinach chloroplasts.

Escherichia coli O157:H7 contamination of leafy green vegetables is an ongoing concern for consumers. Biofilm-associated pathogens are relatively resistant to chemical treatments, but little is known about their response to irradiation. Leaves of Romaine lettuce and baby spinach were dip inoculated with E. coli O157:H7 and stored at 4 degrees C for various times (0, 24, 48, 72 h) to allow biofilms to form. After each time, leaves were treated with either a 3-min wash with a sodium hypochlorite solution (0, 300, or 600 ppm) or increasing doses of irradiation (0, 0.25, 0.5, 0.75, or 1 kGy). Viable bacteria were recovered and enumerated. Chlorine washes were generally only moderately effective, and resulted in maximal reductions of 1.3 log CFU/g for baby spinach and 1.8 log CFU/g for Romaine. Increasing time in storage prior to chemical treatment had no effect on spinach, and had an inconsistent effect on 600 ppm applied to Romaine. Allowing time for formation of biofilm-like aggregations reduced the efficacy of irradiation. D(10) values (the dose required for a 1 log reduction) significantly increased with increasing storage time, up to 48 h postinoculation. From 0 h of storage, D(10) increased from 0.19 kGy to a maximum of 0.40 to 0.43 kGy for Romaine and 0.52 to 0.54 kGy for spinach. SEM showed developing biofilms on both types of leaves during storage. Bacterial colonization of the stomata was extensive on spinach, but not on Romaine. These results indicate that the protection of bacteria on the leaf surface by biofilm formation and stomatal colonization can reduce the antimicrobial efficacy of irradiation on leafy green vegetables.

Millions of tons of pesticides are applied worldwide annually in agriculture. Among them, herbicides such as triazines and ureas, originating from agricultural runoff, can contaminate soils and surface and ground waters with severe toxic effects on humans. Nowadays, different analytical techniques are available for the detection of these chemicals; however, most of them are expensive and time-consuming, especially in the case of routine analyses. For this reason, on the basis of results collected through many years of experience in the field of photosynthetic organisms, we designed a biosensor platform intended for the easy, low-cost, and fast prescreening of photosynthetic herbicides. The platform combines the possibilities of amperometric and optical transduction systems, which have proven to be highly sensitive (limits of detection = 10(-10)-10(-8) M). The use of genetically modified algae strengthens the power of the platform, allowing different subclasses of herbicides to be recognized. The system has been validated for the analysis of environmental water and is proposed to support laboratories involved in the control of water pollution.

Thylakoid membranes in higher plant chloroplasts are composed by two distinct domains: stacked grana and stroma lamellae. We developed a procedure for biochemical isolation of grana membranes using mild detergent to maintain membrane structure. Pigment and polypeptide analyses of membrane preparation showed the preparations were indeed enriched in grana membranes. The method was shown to be effective in four different plant species, although with small changes in detergent concentration. Electron microscopy analyses also showed that the preparation consisted of large membrane patches with roughly round shape and diameter comparable with grana membranes in vivo. Furthermore, protein complexes distribution was shown to be maintained with respect to freeze fracture studies, demonstrating that the protocol was successful in isolating membranes close to their in vivo state.

Thylakoids are membranes isolated from plant chloroplasts which have previously been shown to inhibit pancreatic lipase/colipase catalysed hydrolysis of fat in vitro and induce short-term satiety in vivo. The purpose of the present study was to examine if dietary supplementation of thylakoids could affect food intake and body weight during long-term feeding in mice. Female apolipoprotein E-deficient mice were fed a high-fat diet containing 41% of fat by energy with and without thylakoids for 100 days. Mice fed the thylakoid-enriched diet had suppressed food intake, body weight gain and body fat compared with the high-fat fed control mice. Reduced serum glucose, serum triglyceride and serum free fatty acid levels were found in the thylakoid-treated animals. The satiety hormone cholecystokinin was elevated, suggesting this hormone mediates satiety. Leptin levels were reduced, reflecting a decreased fat mass. There was no sign of desensitization in the animals treated with thylakoids. The results suggest that thylakoids are useful to suppress appetite and body weight gain when supplemented to a high-fat food during long-term feeding., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

Membrane vesicles, originating from grana, grana core (appressed grana regions), grana margins and stroma lamellae/end membranes, were analysed by counter current distribution (CCD) using aqueous dextran-polyethylene glycol two-phase systems. Each vesicle population gave rise to distinct peaks in the CCD diagram representing different vesicle subpopulations. The grana vesicles and grana core vesicles each separated into 3 different subpopulations having different chlorophyll a/b ratios and PSI/PSII ratios. Two of the grana core subpopulations had a chlorophyll a/b ratio of 2.0 and PSI/PSII ratio of 0.10 and are among the most PSII enriched thylakoid vesicle preparation obtained so far by a non detergent method. The margin vesicles separated into 3 different populations, with about the same chlorophyll a/b ratios, but different fluorescence emission spectra. The stroma lamellae/end membrane vesicles separated into 4 subpopulations. Plastoglobules, connected to membrane vesicles, were highly enriched in 2 of these subpopulations and it is proposed that these 2 subpopulations originate from stroma lamellae while the 2 others originate from end membranes. Fragmentation and separation analysis shows that the margins of grana constitute a distinct domain of the thylakoid and also allows the estimation of the chlorophyll antenna sizes of PSI and PSII in different thylakoid domains.

Linear and cyclic electron fluxes through Photosystem I in 1% CO(2) were quantified in spinach leaf tissue under severe water stress. Using actinic light with a peak at 697 nm for preferential light absorption by Photosystem I while also stimulating Photosystem II to improve redox poising, the cyclic electron flux after 60 s of illumination was a substantial proportion (33-44%) of the total electron flux through PSI at irradiances up to ~1,070 micromol photons m(-2) s(-1). At the maximum irradiance, the cyclic electron flux changed little with the progressive water loss from leaf tissue up to ~60%; by contrast, the linear electron flux was approximately halved. A reason for this differential effect of water stress on the capacity for cyclic and linear electron flow could be the increased crowding of soluble proteins in the stroma due to chloroplast shrinkage. Indeed the confinement of soluble proteins to a smaller chloroplast volume was indicated by cryo-scanning electron microscopy. It is known that the diffusion coefficient of large proteins is decreased when the background concentration of small proteins is raised; by contrast, the diffusion coefficient of small proteins is not affected by increasing the concentration of a large protein (Muramatsu and Minton in Proc Natl Acad Sci USA 85:2984-2988, 1988). Therefore, we suggest that linear electron flow, being coupled to the Calvin-Benson cycle, is limited by the diffusion of large macromolecules, especially the ribulose 1, 5-bisphosphate carboxylase/oxygenase complex. By contrast, cyclic electron flow, involving relatively small macromolecules such as ferredoxin, is less susceptible to inhibition by crowding in the stroma.

Through bioactivity-guided chemical analysis of Selaginella lepidophylla, biflavonoids robustaflavone (1), 2,3-dihydrorobustaflavone (2), and 2,3-dihydrorobustaflavone-5-methyl ether (3) were isolated and their structures confirmed by spectroscopic and spectrometric analyses. Their NMR resonances were unambiguously assigned from HMBC, NOESY, and NOESY-1D experiments, and absolute configurations of 2 and 3 were established. Compound 3 has not been reported, and although structure of 2 was described before, the (13)C NMR assignment does not correlate with the structure reported. Therefore, this is the first report of 2. All compounds inhibited ATP production. Compounds 1 and 2 behaved as Hill reaction inhibitors. 1 interacted with photosystem II, transforming the reaction centers to silent centers at 300 and 600 microM. The interaction and inhibition target of 2 was located on Cyt b6f to PC. The three compounds also behaved as energy transfer inhibitors, 3 being the most active.

We report electron paramagnetic resonance (EPR) studies on photosystem II (PSII) from higher plants in five different domains of the thylakoid membrane prepared by sonication and two-phase partitioning. The domains studied were the grana core, the entire grana stack, the grana margins, the stroma lamellae and the purified stromal fraction, Y100. The electron transport properties of both donor and acceptor sides of PSII such as oxygen evolution, cofactors Y D, Q A, the CaMn 4-cluster, and Cytb 559 were investigated. The PSII content was estimated on the basis of oxidized Y D and Q A (-) Fe (2+) signal from the acceptor side vs Chl content (100% in the grana core fraction). It was found to be about 82% in the grana, 59% in the margins, 35% in the stroma and 15% in the Y100 fraction. The most active PSII centers were found in the granal fractions as was estimated from the rates of electron transfer and the S 2 state multiline EPR signal. In the margin and stroma fractions the multiline signal was smaller (40 and 33%, respectively). The S 2 state multiline could not be induced in the Y100 fraction. In addition, the oxidized LP Cytb 559 prevailed in the stromal fractions while the HP form dominated in the grana core. The margins and entire grana fractions have Cytb 559 in both potential forms. These data together with previous analyses indicate that the sequence of activation of the PSII properties can be represented as: PSII content > oxygen evolution > reduced Cytb 559 > dimerization of PSII centers in all fractions of the thylakoid membrane with the gradual increase from stromal fractions via margin to the grana core fraction. The results further support the existence of a PSII activity gradient which reflects lateral movement and photoactivation of PSII centers in the thylakoid membrane. The possible role of the PSII redox components in this process is discussed.

Pathogenic bacteria that become internalized in leaf tissues are protected from the antimicrobial effects of surface treatments. Ionizing radiation is known to penetrate food tissues, but the efficacy of the process against internalized bacteria is unknown. Leaves of Romaine lettuce and baby spinach were cut into pieces, submerged in a cocktail mixture of three isolates of Escherichia coli O157:H7, and subjected to a vacuum perfusion process to force the bacterial cells into the intercellular spaces in the leaves. Scanning electron microscopy was used to evaluate the efficacy of the perfusion process. The inoculated leaves were then treated with a 3-min water wash, a 3-min wash with a sodium hypochlorite sanitizing solution (300 or 600 ppm), or various doses of ionizing radiation (0.25 to 1.5 kGy). Leaves were stomached to recover the internalized pathogen cells, which were enumerated. The vacuum perfusion effectively forced bacteria into the leaf vasculature and apoplast, as confirmed by scanning electron microscopy. For spinach leaf pieces, neither the water nor the sodium hypochlorite washes resulted in significant reductions of E. coli O157:H7 cells relative to the untreated control. For Romaine lettuce leaf pieces, 300 and 600 ppm sodium hypochlorite each resulted in less than 1-log reduction; water wash was ineffective. Ionizing radiation, in contrast, significantly reduced the pathogen population, with 4-log (Romaine lettuce) or 3-log (spinach) reductions at the highest dose tested. In Romaine leaves, the reduction was dose dependent across the range of doses tested, with a D10-value (the amount of irradiation necessary to reduce the population by 1 log unit) of 0.39 kGy. In spinach leaves, the pathogen had a biphasic response, with a D10-value of 0.27 kGy in the range of 0 to 0.75 kGy but only slight additional reductions from 0.75 to 1.5 kGy. In this study, ionizing radiation but not chemical sanitizers effectively reduced viable E. coli O157:H7 cells internalized in leafy green vegetables, but the response of the pathogen to irradiation was more complex in spinach leaves than in Romaine lettuce leaves.

Four natural products were isolated from the fungus Botryosphaeria rhodina, and their effects on photosynthesis were tested. Only lasiodiplodin (1) inhibited ATP synthesis and electron flow from water to methylviologen; therefore, it acts as a Hill reaction inhibitor in freshly lysed spinach thylakoids. Photosystem I and II and partial reactions as well as ATPase were measured in the presence of 1. Three new different sites of 1 interaction and inhibition were found: one at CF1, the second in the water-splitting enzyme, and the third at the electron-transfer path between P680 and QA; these targets are different from that of the synthetic herbicides present. Electron transport chain inhibition by 1 was corroborated by fluorescence induction kinetics studies.

This manuscript reviews the principles and recent advances of scanning near-field optical microscopy based on tip-induced field enhancement. These scanning microscopes utilize minute probes to locally enhance an electromagnetic field through a complex interplay between surface plasmon excitation and localization of electric charges by geometrical singularities. The necessary conditions leading to an electromagnetic enhancement will be reviewed, as well as the means to characterize it. A brief account of the theoretical framework will be given, together with applications of the technique ranging from chemical imaging to nanolithography., (Copyright (c) 2006 Wiley-Liss, Inc.)

The expression of the glutathione peroxidase homologous gene Gpxh, known to be specifically induced by the formation of singlet oxygen (1O2), was analyzed in cells of Chlamydomonas reinhardtii exposed to environmental conditions causing photoinhibition. Illumination with high light intensities, leading to an increased formation of 1O2 in photosystem II, continuously induced the expression of Gpxh in cell for at least 2 h. Phenolic herbicides like dinoterb, raise the rate of 1O2 formation by increasing the probability of charge recombination in photosystem II via the formation of the primary radical pair and thereby 3P680 formation (Fufezan C et al. 2002, FEBS Letters 532, 407-410). In the presence of dinoterb the light-induced loss of the D1 protein in C. reinhardtii was increased and the high light-induced Gpxh expression was further stimulated. DCMU, a urea-type herbicide, causing reduced 1O2 generation in photosystem II, protected the D1 protein slightly against degradation and downregulated the expression of the Gpxh gene compared to untreated cells exposed to high light intensities. This indicates that the Gpxh expression is induced by 1O2 under environment conditions causing photoinhibition.

The influence of thermal treatment and light exposure on degradation and isomerization of the predominant carotenoids (lutein and beta-carotene) occurring in green leafy vegetables was assessed. The effect of lipid addition on carotenoid stability was also evaluated. For the first time, the stabilities of pure carotenoids and chloroplast-bound carotenoids were compared. Besides degradation, heating caused carotenoid isomerization in all samples. Whereas pure carotenoids favor 13-cis isomers, in native chloroplasts and heated chloroplasts 9-cis isomers were predominant. Illumination of freshly prepared chloroplast isolates caused an initial increase in the level of lutein (9.6%) and beta-carotene (29.8%), while pure carotenoids exhibited time-dependent degradation. The addition of lipids to chloroplast preparations had the reverse effects on the retention of both carotenoids after heating; isomerization was not significantly affected. It was demonstrated that carotenoid stability has to be evaluated for every individual pigment in its genuine environment. Stability data based on model systems (e.g., pure carotenoids) may not be transferred to complex food matrices without intensive investigation.

Confocal laser scanning microscopy was used to study the distribution of the smallest detectable autofluorescing, chlorophyll-bearing structures in fresh, 40 microm thick longitudinal sections of the shoot apex of four dicotyledonous plants (Arabidopsis thaliana, Nicotiana glauca, Lupinus alba, and Spinacia oleracea). In all species, the smallest chlorophyll-bearing particles were found in the outermost cell layers (L1 and L2) of the shoot apex. Their distribution between these layers differed in each species. The smallest such particles were about 0.5-1.0 microm in maximum dimension, approximating the size of a single granum in the developing leaf. Their size and abundance increased with increasing cell age and distance from the peak of the apex. Immediately beneath the L1 and L2 layers was a zone largely devoid of these particles. Below this nonfluorescing zone, in the region where the derivatives of the meristematic zone differentiate into cells of the central pith region, the size and abundance of the chlorophyll-bearing particles increased progressively with increasing distance from the nonfluorescing zone. The presence of these small autofluorescing particles in the L1 and L2 cell layers of the shoot apex places the development of photosystem II fluorescence at an earlier stage of leaf development than previously observed. The use of confocal laser scanning microscopy to study unfixed sections provides another useful metabolic marker for mapping patterns of differentiation and development in the cells of the shoot apex.

The conserved residue Gly47 of the chloroplast ATP synthase beta subunit was substituted with Leu, Arg, Ala and Glu by site-directed mutagenesis. This process generated the mutants epsilon G47L, epsilon G47R, epsilon G47A and epsilon G47E, respectively. All the beta variants showed lower inhibitory effects on the soluble CF1(-epsilon) Ca2+-ATPase compared with wild-type epsilon. In reduced conditions, epsilon G47E and epsilon G47R had a lower inhibitory effect on the oxidized CF1(-epsilon) Ca2+-ATPase compared with wild-type epsilon. In contrast, epsilon G47L and epsilon G47A increased the Ca2+-ATPase activity of soluble oxidized CF1(-epsilon). The replacement of Gly47 significantly impaired the interaction between the subunit epsilon and gamma in an in vitro binding assay? Further study showed that all epsilon variants were more effective in blocking proton leakage from the thylakoid membranes. This enhanced ATP synthesis of the chloroplast and restored ATP synthesis activity of the reconstituted membranes to a level that was more efficient than that achieved by wild-type epsilon. These results indicate that the conserved Gly47 residue of the epsilon subunit is very important for maintaining the structure and function of the epsilon subunit and may affect the interaction between the epsilon subunit, beta subunit of CF1 and subunit III of CFo, thereby regulating the ATP hydrolysis and synthesis, as well as the proton translocation role of the subunit III of CFo.

We investigated a new EPR signal that gives a broad line shape around g=2 in Ca(2+)-depleted Photosystem (PS) II. The signal was trapped by illumination at 243 K in parallel with the formation of Y(Z)*. The ratio of the intensities between the g=2 broad signal and the Y(Z)* signal was 1:3, assuming a Gaussian line shape for the former. The g=2 broad signal and the Y(Z)* signal decayed together in parallel with the appearance of the S(2) state multiline at 243 K. The g=2 broad signal was assigned to be an intermediate S(1)X* state in the transition from the S(1) to the S(2) state, where X* represents an amino acid radical nearby manganese cluster, such as D1-His337. The signal is in thermal equilibrium with Y(Z)*. Possible reactions in the S state transitions in Ca(2+)-depleted PS II were discussed.

The Y(Z)* decay kinetics in a formal S(-1) state, regarded as a reduced state of the oxygen evolving complex, was determined using time-resolved EPR spectroscopy. This S(-1) state was generated by biochemical treatment of thylakoid membranes with hydrazine. The steady-state oxygen evolution of the sample was used to optimize the biochemical procedure for performing EPR experiments. A high yield of the S(-1) state was generated as judged by the two-flash delay in the first maximum of oxygen evolution in Joliot flash-type experiments. We have shown that the Y(Z)* re-reduction rate by the S(-1) state is much slower than that of any other S-state transition in hydrazine-treated samples. This slow reduction rate in the S(-1) to S(0) transition, which is in the order of the S(3) to S(0) transition rate, suggests that this transition is accompanied by some structural rearrangements. Possible explanations of this unique, slow reduction rate in the S(-1) to S(0) transition are considered, in light of earlier observations by others on hydrazine/hydroxylamine reduced PS II samples.

The function of the extrinsic 23 kDa protein of Photosystem II (PSII) was studied with respect to Mn binding and its ability to supply Mn to PSII during photoactivation, i.e. the light-dependent assembly of the tetramanganese cluster. The extrinsic proteins and the Mn cluster were removed by TRIS treatment from PSII-enriched membrane fragments and purified by anion exchange chromatography. Room temperature EPR spectra of the purified 23 kDa protein demonstrated the presence of Mn. Photoactivation was successful with low Mn concentrations when the 23 kDa protein was present, while in its absence a higher Mn concentration was needed to reach the same level of oxygen evolution activity. In addition, the rate of photoactivation was significantly accelerated in the presence of the 23 kDa protein. It is proposed that the 23 kDa protein plays an important role in providing Mn during the process of PSII assembly and that it acquires Mn during the light-induced turnover of D1 in the PSII damage-repair cycle and delivers Mn to repaired PSII.

The common precursor for isoprenoid biosynthesis in plants, isopentenyl diphosphate (IPP), is synthesized by two pathways, the cytosolic mevalonate pathway and the plastidic 1-deoxy-D-xylulose 5-phosphate/methylerythritol phosphate (DOXP/MEP) pathway. The DOXP/MEP pathway leads to the formation of various phosphorylated intermediates, including DOXP, 4-hydroxy-3-methylbutenyl diphosphate (HMBPP), and finally IPP. There is ample evidence for metabolic cross-talk between the two biosynthetic pathways. The present study addresses the question whether isoprenoid intermediates could be exchanged between both compartments by members of the plastidic phosphate translocator (PT) family that all mediate a counter-exchange between inorganic phosphate and various phosphorylated compounds. Transport experiments using intact chloroplasts, liposomes containing reconstituted envelope membrane proteins or recombinant PT proteins showed that HMBPP is not exchanged between the cytosol and the chloroplasts and that the transport of DOXP is preferentially mediated by the recently discovered plastidic transporter for pentose phosphates, the xylulose 5-phosphate translocator. Evidence is presented that transport of IPP does not proceed via the plastidic PTs although IPP transport is strictly dependent on various phosphorylated compounds on the opposite side of the membrane. These phosphorylated trans compounds are, in part, also used as counter-substrates by the plastidic PTs but appear to only trans activate IPP transport without being transported.

Selected cell organelles were investigated at a high level of resolution with the transmission electron microscope, using ultrathin serial sections to create three-dimensional reconstructions. On the basis of these reconstructions, morphological data of chloroplast fine structures, mitochondria, and peroxisomes from control and drought-stressed spinach leaves were evaluated and compared. Mesophyll cell chloroplasts of control plants contained 60% stroma, 23% thylakoids, and 16% starch. In drought-stressed plants, the volume of both the stroma and the thylakoids increased to 68% and 32%, respectively. The amount of plastoglobuli was about 0.3% in both samples. Chloroplasts of stressed plants differed from control plants not only in the thylakoid and stroma values but also in the lack of starch grains. Mitochondria occurred in variable forms in control and stressed samples. In stressed plants, mitochondria had only 65% of the volume compared with control plants. Peroxisomes were inconspicuous.

Our understanding of the factors determining the bioavailability of carotenoids from fruits and vegetables is poor. The apolar nature of carotenoids precludes their simple diffusion from the food structure to the absorption site at the enterocyte. Therefore, there is interest in the potential pathways for solubilization in the gut before absorption. We have studied the transfer of carotenoids from carrot juice and homogenized spinach into lipid phases that mimic the intestinal lumen at the start of digestion. In this paper we report on their transfer into olive oil under conditions pertaining to the gastric environment. A comparison between preparations of raw spinach and of carrot, in which the intact cells have been largely broken, suggests that the membrane-bound carotenoids of spinach are more resistant to transfer than the crystalline carotenoids of carrot. Lowering the pH and pepsin treatment enhance the transfer from raw vegetables. The process of blanching and freezing spinach destroys the chloroplast ultrastructure and leads to (i) a substantial increase in transfer of the carotenoids to oil and (ii) an attenuation or reversal of the enhancement of transfer seen with reduced pH or with pepsin treatment. Similar effects are seen after blanching carrot juice. Our results show that removal of soluble protein and denaturation of membrane proteins enhances the partition of carotenoids into oil. For both vegetables there is no evidence of preference in the extent of transfer of one carotenoid over another. This suggests that partitioning into oil under gastric conditions is not the stage of digestion that could lead to differences in carotenoid bioavailability.

We report a structural characterization by electron microscopy and image analysis of a supramolecular complex consisting of photosystem I and light-harvesting complex I from the unicellular green alga Chlamydomonas reinhardtii. The complex is a monomer, has longest dimensions of 21.3 and 18.2 nm in projection, and is significantly larger than the corresponding complex in spinach. Comparison with photosystem I complexes from other organisms suggests that the complex contains about 14 light-harvesting proteins, two or three of which bind at the side of the PSI-H subunit. We suggest that special light-harvesting I proteins play a role in the binding of phosphorylated light-harvesting complex II in state 2.

A structure of photosystem II recently determined by X-ray crystallography at 3.8 A resolution complements structural studies using high-resolution electron microscopy and represents a major step towards understanding how photosynthetic organisms use light energy to oxidise water.

We obtained topographic images of etioplasts and chloroplasts and measured their elasticity in a physiological buffer using an atomic force microscope (AFM) and found a possible correlation between the morphological and mechanical properties during the light conversion of etioplasts to chloroplasts. Alcian blue 8GX dye was found to be effective for immobilizing the plant organelles stably on a glass surface for AFM experiments. We employed the tapping-mode AFM with a cantilever soft enough to measure the elasticity of the organelles in a liquid solution. The best images of soft, spherical organelles were obtained using the tapping-mode AFM with oscillation at the thermal vibration frequency of the cantilever of around 3 kHz. Whereas etioplasts were found to be smooth-surfaced and stiff against compression by the AFM tip, before light conversion to chloroplasts, they became rough-surfaced and mechanically soft after exposure to light. The elasticity of etioplasts was 20 times higher than that of chloroplasts, probably reflecting changes in their inner structures.

The determination of the structure of PSII at high resolution is required in order to fully understand its reaction mechanisms. Two-dimensional crystals of purified highly active Synechococcus elongatus PSII dimers were obtained by in vitro reconstitution. Images of these crystals were recorded by electron cryo-microscopy, and their analysis revealed they belong to the two-sided plane group p22(1)2(1), with unit cell parameters a = 121 A, b = 333 A, and alpha = 90 degrees. From these crystals, a projection map was calculated to a resolution of approximately 16 A. The reliability of this projection map is confirmed by its close agreement with the recently presented three-dimensional model of the same complex obtained by X-ray crystallography. Comparison of the projection map of the Synechococcus elongatus PSII complex with data obtained by electron crystallography of the spinach PSII core dimer reveals a similar organization of the main transmembrane subunits. However, some differences in density distribution between the cyanobacterial and higher plant PSII complexes exist, especially in the outer region of the complex between CP43 and cytochrome b(559) and adjacent to the B-helix of the D1 protein. These differences are discussed in terms of the number and organization of some of the PSII low molecular weight subunits.

The interaction between water and biological macromolecules in living organisms is of fundamental importance in a range of processes. We have studied water-DNA and water-proteolipid membrane systems over a range of hydration states using inelastic incoherent neutron scattering. We find a relatively sharp transition for both systems at a water concentration above which bulk solvent can be detected. Below this concentration, bulk water is essentially absent, i.e., all the water in the system is interacting with the biological macromolecules. This water is strongly perturbed as judged by its energy transfer spectrum, with a broader and lower energy transition than bulk water in the 50-75 meV (approximately 400-600 cm(-1)) range. Taking into account the differing geometry of (cylindrical) DNA and (planar) membranes, the number of water shells perturbed by each system was estimated. A conclusion is that in living organisms a large proportion of the cellular water will be in a state quite distinct from bulk water. The data add to the growing evidence that water structure in the vicinity of biological macromolecules is unusual and that the proximal water behaves differently compared to the bulk solvent.

This paper describes experiments using optical tweezers to probe chloroplast arrangement, shape and consistency in cells of living leaf tissue and in suspension. Dual optical tweezers provided two-point contact on a single chloroplast or two-point contact on two adhered chloroplasts for manipulation in suspension. Alternatively, a microstirrer consisting of a birefringent particle trapped in an elliptically polarized laser trap was used to induce motion and tumbling of a selected chloroplast suspended in a solution. We demonstrate that displacement of chloroplasts inside the cell is extremely difficult, presumably due to chloroplast adhesion to the cytoskeleton and connections between organelles. The study also confirms that the chloroplasts are very thin and extremely cup-shaped with a concave inner surface and a convex outer surface.

In this study, we present the location of the ferredoxin-binding site in photosystem I from spinach. Image analysis of negatively stained two-dimensional crystals indicates that the addition of ferredoxin and chemical cross-linkers do not significantly alter the unit cell parameters (for untreated photosystem I, a = 26.4 nm, b = 27.6 nm, and gamma = 90 degrees, space group p22(1)2(1) and for ferredoxin cross-linked photosystem I, a = 26.2 nm, b = 27.2 nm, and gamma = 90 degrees, space group p22(1)2(1)). Fourier difference analysis reveals that ferredoxin is bound on top of the stromal ridge principally interacting with the extrinsic subunits PsaC and PsaE. This location would be accessible to the stroma, thereby promoting efficient electron transfer away from photosystem I. This observation is significantly different from that of the ferredoxin binding site proposed for cyanobacteria. A model for the binding of ferredoxin in vascular plants is proposed and is discussed relative to observations in cyanobacteria.

The chloroplast thylakoid membrane of green plants is organized in stacked grana membranes and unstacked stroma membranes. We investigated the structural organization of Photosystem II (PSII) in paired grana membrane fragments by transmission electron microscopy. The membrane fragments were obtained by a short treatment of thylakoid membranes with the mild detergent n-dodecyl-alpha, d-maltoside and are thought to reflect the grana membranes in a native state. The membranes frequently show crystalline macrodomains in which PSII is organized in rows spaced by either 26.3 nm (large-spaced crystals) or 23 nm (small-spaced crystals). The small-spaced crystals are less common but better ordered. Image analysis of the crystals by an aperiodic approach revealed the precise positions of the core parts of PSII in the lattices, as well as features of the peripheral light-harvesting antenna. Together, they indicate that the so-called C(2)S(2) and C(2)S(2)M supercomplexes form the basic motifs of the small-spaced and large-spaced crystals, respectively. An analysis of a pair of membranes with a well-ordered large-spaced crystal reveals that many PSII complexes in one layer face only light-harvesting complexes (LHCII) in the other layer. The implications of this type of organization for the efficient transfer of excitation energy from LHCII to PSII and for the stacking of grana membranes are discussed., (Copyright 2000 Academic Press.)

In spinach thylakoids prepared from intact chloroplasts by shocking in the presence of ascorbate to preserve the operation of ascorbate peroxidase, the rate of oxygen uptake with methyl viologen as acceptor decreased in response to the addition of H2O2. Such a decrease was not observed in the presence of KCN or when the thylakoids lost ascorbate peroxidase activity. Illumination of intact chloroplasts in the presence of H2O2 and methyl viologen showed an initial rate of oxygen exchange, which is intermediate between the initial rate of oxygen evolution in the presence of H2O2 alone and steady-state oxygen uptake in the presence of methyl viologen. The data showed that monodehydroascorbate radical generated in ascorbate peroxidase reaction could compete with methyl viologen for electrons supplied by the electron transport chain in both thylakoids and intact chloroplasts. During the illumination of intact chloroplasts the rate of oxygen uptake increased. The presence of nigericin swiftly led to steady-state oxygen uptake, and to a clear-cut 1:1 relationship between the electron transport rate estimated from fluorescence assay and the electron transport rate determined from oxygen uptake, taking the stoichiometry 1 O2:4 e. The increase in oxygen uptake was attributed to the cessation of monodehydroascorbate radical generation brought about by consumption of intrachloroplast ascorbate in the peroxidase reactions, and the effects of nigericin were explained by acceleration of such consumption. The competition between methyl viologen and monodehydroascorbate radical in the intact chloroplasts was estimated under various conditions.

Protoporphyrinogen oxidase (Protox) is the last common enzyme in the biosynthesis of chlorophylls and heme. In plants, there are two isoenzymes of Protox, one located in plastids and other in the mitochondria. We cloned the cDNA of spinach (Spinacia oleracea) plastidal Protox and purified plastidal Protox protein from spinach chloroplasts. Sequence analysis of the cDNA indicated that the plastid Protox of spinach is composed of 562 amino acids containing the glycine-rich motif GxGxxG previously proposed to be a dinucleotide binding site of many flavin-containing proteins. The cDNA of plastidal Protox complemented a Protox mutation in Escherichia coli. N-terminal sequence analysis of the purified enzyme revealed that the plastidal Protox precursor is processed at the N-terminal site of serine-49. The predicted transit peptide (methionine-1 to cysteine-48) was sufficient for the transport of precursors into the plastid because green fluorescent protein fused with the predicted transit peptide was transported to the chloroplast. Immunocytochemical analysis using electron microscopy showed that plastidal Protox is preferentially associated with the stromal side of the thylakoid membrane, and a small portion of the enzyme is located on the stromal side of the chloroplast inner envelope membrane.

Here we describe the first 3D structure of the photosystem II (PSII) supercomplex of higher plants, constructed by single particle analysis of images obtained by cryoelectron microscopy. This large multisubunit membrane protein complex functions to absorb light energy and catalyze the oxidation of water and reduction of plastoquinone. The resolution of the 3D structure is 24 A and emphasizes the dimeric nature of the supercomplex. The extrinsic proteins of the oxygen-evolving complex (OEC) are readily observed as a tetrameric cluster bound to the lumenal surface. By considering higher resolution data, obtained from electron crystallography, it has been possible to relate the binding sites of the OEC proteins with the underlying intrinsic membrane subunits of the photochemical reaction center core. The model suggests that the 33 kDa OEC protein is located towards the CP47/D2 side of the reaction center but is also positioned over the C-terminal helices of the D1 protein including its CD lumenal loop. In contrast, the model predicts that the 23/17 kDa OEC proteins are positioned at the N-terminus of the D1 protein incorporating the AB lumenal loop of this protein and two other unidentified transmembrane helices. Overall the 3D model represents a significant step forward in revealing the structure of the photosynthetic OEC whose activity is required to sustain the aerobic atmosphere on our planet.

Arbutin (hydroquinone-beta-D-glucopyranoside) is an abundant solute in the leaves of many freezing- or desiccation-tolerant plants. Its physiological role in plants, however, is not known. Here we show that arbutin protects isolated spinach (Spinacia oleracea L.) thylakoid membranes from freeze-thaw damage. During freezing of liposomes, the presence of only 20 mM arbutin led to complete leakage of a soluble marker from egg PC (EPC) liposomes. When the nonbilayer-forming chloroplast lipid monogalactosyldiacylglycerol (MGDG) was included in the membranes, this leakage was prevented. Inclusion of more than 15% MGDG into the membranes led to a strong destabilization of liposomes during freezing. Under these conditions arbutin became a cryoprotectant, as only 5 mM arbutin reduced leakage from 75% to 20%. The nonbilayer lipid egg phosphatidylethanolamine (EPE) had an effect similar to that of MGDG, but was much less effective, even at concentrations up to 80% in EPC membranes. Arbutin-induced leakage during freezing was accompanied by massive bilayer fusion in EPC and EPC/EPE membranes. Twenty percent MGDG in EPC bilayers completely inhibited the fusogenic effect of arbutin. The membrane surface probes merocyanine 540 and 2-(6-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosph ocholi ne (NBD-C(6)-HPC) revealed that arbutin reduced the ability of both probes to partition into the membranes. Steady-state anisotropy measurements with probes that localize at different positions in the membranes showed that headgroup mobility was increased in the presence of arbutin, whereas the mobility of the fatty acyl chains close to the glycerol backbone was reduced. This reduction, however, was not seen in membranes containing 20% MGDG. The effect of arbutin on lipid order was limited to the interfacial region of the membranes and was not evident in the hydrophobic core region. From these data we were able to derive a physical model of the perturbing or nonperturbing interactions of arbutin with lipid bilayers.

Scanning tunneling microscopy (STM) and spectroscopy (STS) were used to obtain the first direct high resolution ( approximately 0.3 nm) images of single isolated Photosystem II (PS II) molecules, and to determine the supramolecular organization of oxygen-evolving PS II core complexes and PS II membrane fragments including the identification, assignment, location and dimensions of the polypeptide units. Our results predict a unique structural model which we then compare with alternative models. We show that the combination of quasi-constant-height mode STM operation, STS and suitable choice of sample-substrate preparations can be used to enable investigation of the structure and function of single PS II particles under normal thermodynamic and hydration conditions without the requirement and complications of ordered PS II arrays or crystals. STS was also used to characterize single-molecule electron conduction and tunneling mechanisms in PS II including the semiconduction and photoconduction behavior of the reaction center and photoexcitation effects in the light-harvesting complex LHC II.

A three-dimensional image of the spinach photosystem II core complex composed of CP47, D1, D2, cytochrome b-559, and psbI gene product was reconstructed at 20-A resolution from the two-dimensional crystals negatively stained with phosphotungstate. Confirming the previous proposal, the crystal had a p22121 symmetry. One PSII core complex was measured to be 80 x 80 A in the membrane plane and 88 A normal to it. The mass distribution was asymmetric about the lipid bilayer, consistent with predictions from the amino acid sequences. The lumenal mass consisted of three domains forming a characteristic triangular platform with another domain on top of it. Three stromal domains were smaller and linearly arranged. Due to strong stain exclusion in the hydrophobic core part of the lipid bilayer, the transmembrane region appeared to be imaged with a reversed contrast. Inverting the contrast resulted in a reasonable density distribution for that part. Thus, though the information on the transmembrane region is limited, the domain structure of the PSII core complex was revealed and allowed us to propose a model for the arrangement of subunits in the PSII core complex., (Copyright 1998 Academic Press.)