The structural and functional domains of plant thylakoid membranes

In plants, the stacking of part of the photosynthetic thylakoid membrane generates two main subcompartments: the stacked grana core and unstacked stroma lamellae. However, a third distinct domain, the grana margin, has been postulated but its structural and functional identity remains elusive. Here, an optimized thylakoid fragmentation procedure combined with detailed ultrastructural, biochemical, and functional analyses reveals the distinct composition of grana margins. It is enriched with lipids, cytochrome b₆f complex, and ATPase while depleted in photosystems and light‐harvesting complexes. A quantitative method is introduced that is based on Blue Native Polyacrylamide Gel Electrophoresis (BN‐PAGE) and dot immunoblotting for quantifying various photosystem II (PSII) assembly forms in different thylakoid subcompartments. The results indicate that the grana margin functions as a degradation and disassembly zone for photodamaged PSII. In contrast, the stacked grana core region contains fully assembled and functional PSII holocomplexes. The stroma lamellae, finally, contain monomeric PSII as well as a significant fraction of dimeric holocomplexes that identify this membrane area as the PSII repair zone. This structural organization and the heterogeneous PSII distribution support the idea that the stacking of thylakoid membranes leads to a division of labor that establishes distinct membrane areas with specific functions.     

Towards elucidation of dynamic structural changes of plant thylakoid architecture

Long-term acclimation of shade versus sun plants modulates the composition, function and structural organization of the architecture of the thylakoid membrane network. Significantly, these changes in the macroscopic structural organization of shade and sun plant chloroplasts during long-term acclimation are also mimicked following rapid transitions in irradiance: reversible ultrastructural changes in the entire thylakoid membrane network increase the number of grana per chloroplast, but decrease the number of stacked thylakoids per granum in seconds to minutes in leaves. It is proposed that these dynamic changes depend on reversible macro-reorganization of some light-harvesting complex IIb and photosystem II supracomplexes within the plant thylakoid network owing to differential phosphorylation cycles and other biochemical changes known to ensure flexibility in photosynthetic function in vivo. Some lingering grana enigmas remain: elucidation of the mechanisms involved in the dynamic architecture of the thylakoid membrane network under fluctuating irradiance and its implications for function merit extensive further studies.     

The State of Photosystem I Complexes in the Central Regions of Pea Granal Thylakoids

Grana-core and grana-margin fragments were obtained from pea (Pisum sativum L.) thylakoids, and both fractions contained photosystem I (PSI) complexes. The yield of these fractions exhibited variations for the plants grown during various periods of the summer season. Low-temperature fluorescence spectra, excitation spectra of long-wave fluorescence, and P700 kinetic characteristics were recorded for these fractions. PSI complexes in central granal regions were associated with PSII and the light-harvesting complexes of PSII, which followed from the excitation spectra of long-wave fluorescence and the kinetic characteristics of P700 light oxidation and dark reduction. The characteristics of the margin regions were changed depending on the fraction yield. If the yield was low, marginal fragments contained mainly PSI complexes. When the yield increased, PSI associates with PSII appeared. A spatial distribution and state of PSI complexes in granal thylakoids are discussed as related to the size and composition of the light-harvesting antenna.     

Structural variability of plant photosystem II megacomplexes in thylakoid membranes

Plant photosystem II (PSII) is organized into large supercomplexes with variable levels of membrane‐bound light‐harvesting proteins (LHCIIs). The largest stable form of the PSII supercomplex involves four LHCII trimers, which are specifically connected to the PSII core dimer via monomeric antenna proteins. The PSII supercomplexes can further interact in the thylakoid membrane, forming PSII megacomplexes. So far, only megacomplexes consisting of two PSII supercomplexes associated in parallel have been observed. Here we show that the forms of PSII megacomplexes can be much more variable. We performed single particle electron microscopy (EM) analysis of PSII megacomplexes isolated from Arabidopsis thaliana using clear‐native polyacrylamide gel electrophoresis. Extensive image analysis of a large data set revealed that besides the known PSII megacomplexes, there are distinct groups of megacomplexes with non‐parallel association of supercomplexes. In some of them, we have found additional LHCII trimers, which appear to stabilize the non‐parallel assemblies. We also performed EM analysis of the PSII supercomplexes on the level of whole grana membranes and successfully identified several types of megacomplexes, including those with non‐parallel supercomplexes, which strongly supports their natural origin. Our data demonstrate a remarkable ability of plant PSII to form various larger assemblies, which may control photochemical usage of absorbed light energy in plants in a changing environment.     

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

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

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

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

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

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

A reassessment of the use of herbicide binding to measure photosystem II reaction centres in plant thylakoids

The binding of the herbicide atrazine to thylakoid membranes is often used to quantify Photosystem II reaction centres. Two atrazine binding sites, with high and low affinities, have been observed on the D1 and D2 polypeptides of Photosystem II, respectively (McCarthy S., Jursinic P. and Stemler A. (1988) Plant Physiol. 86S:46). We have observed that the accessibility of the low-affinity binding sites is variable, being limited in freshly isolated thylakoids or in fresh frozen-thawed thylakoids, but increasing during storage of the membranes on ice. In contrast, the accessibility of the high-affinity binding sites, which are titratable at low concentrations (< 500 nM) of herbicide, is much less variable, although the dissociation constant is greatly influenced by ethanol. We conclude that to quantify Photosystem II reaction centres by atrazine binding, it is sufficient and more reliable to assay only the high-affinity binding sites.     

Light‐harvesting superstructures of green plant chloroplasts lacking photosystems

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

The physiological significance of photosystem II heterogeneity in chloroplasts

Photosystem II in green plant chloroplasts displays heterogeneity both in the composition of its light-harvesting antenna and in the ability to reduce the plastoquinone pool. These two features are discussed in terms of chloroplast development and in view of a proposed photosystem II repair cycle.     

Effect of different light qualities on the ultrastructure, thylakoid membrane composition and assimilation metabolism of Chlorella fusca

Chlorella fusca (Shihira et Krauss) strain C-1.1.10 was grown under three different light qualities (red, white or blue light) in homocontinuous cultures. Under electron microscopy, blue light cultures showed enlarged cells, thinner cell walls and lower starch content than red light cells. Under blue light, the degree of stacking of the thylakoid membranes was significantly lower than under white or red light conditions. Changing the light from blue to red the ratio of exposed to appressed membranes was doubled. Compared to red light cells, blue light cells exhibited higher photosynthetic rates per chlorophyll molecule and contained less chlorophyll per dry weight. Blue light stimulated the content of soluble protein as well as that of soluble carbohydrates. The dry weight productivity per unit time was enhanced under blue light conditions. The thylakoid protein complexes which are generally assumed to be localized in the exposed membranes were found in higher concentrations under blue light than under red light. In blue light, both the Photosystem II/Photosystem I ratio and the ratio of light-harvesting chlorophyll protein to P-700 chlorophyll a -protein were lower than in red light. Blue light cells contained twice the concentration of cytochrome f , which correlates well with their higher photosynthetic capacity. When altering the light quality, the degree of change in the reaction center complexes was much lower than expected given the corresponding degree of change in the ratio of exposed to appressed membranes. These results are discussed in light of the question as to whether the variation in the stoichiometry of the laterally distributed complexes can be explained by changes in the degree of stacking alone.     

Quantification of energy-converting protein complexes in plant thylakoid membranes

Knowledge about the exact abundance and ratio of photosynthetic protein complexes in thylakoid membranes is central to understanding structure-function relationships in energy conversion. Recent modeling approaches for studying light harvesting and electron transport reactions rely on quantitative information on the constituent complexes in thylakoid membranes. Over the last decades several quantitative methods have been established and refined, enabling precise stoichiometric information on the five main energy-converting building blocks in the thylakoid membrane: Light-harvesting complex II (LHCII), Photosystem II (PSII), Photosystem I (PSI), cytochrome b₆f complex (cyt b₆f complex), and ATPase. This paper summarizes a few quantitative spectroscopic and biochemical methods that are currently available for quantification of plant thylakoid protein complexes. Two new methods are presented for quantification of LHCII and the cyt b₆f complex, which agree well with established methods. In addition, recent improvements in mass spectrometry (MS) allow deeper compositional information on thylakoid membranes. The comparison between mass spectrometric and more classical protein quantification methods shows similar quantities of complexes, confirming the potential of thylakoid protein complex quantification by MS. The quantitative information on PSII, PSI, and LHCII reveal that about one third of LHCII must be associated with PSI for a balanced light energy absorption by the two photosystems.     

Organization of the Marginal Regions of Pea Granal Thylakoids

Thylakoids of pea chloroplasts isolated from plants grown during various time intervals from June to August were subjected to fragmentation. Using a modified procedure, a fraction of larger particles was separated from those previously considered as fragments of intergranal thylakoids. The particles of the fraction isolated were identified as fragments of marginal regions of granal thylakoids (margins). The relative yield of these fragments depended on the time interval of plant growth. Two types of low-temperature fluorescence spectra corresponding to a high and low yield of the fraction were detected. The characteristics of the first one were a high fluorescence intensity in the short-wave region and the presence of bands with maxima at 687 and 696 nm emitted by photosystem II (PSII). The ratio of PSII to PSI complexes (PSII/PSI) in the fractions characterized by a low and high yield varied from 1 to 5. The analysis of excitation spectra of long-wave fluorescence of PSI showed that PSI complexes in the margin fragments obtained at a low fraction yield were depleted in chlorophyll forms with a 682-nm absorption maximum and enriched in those with a 668-nm maximum. Since an increase in the yield of the margin-fragment fraction is due to an increased unstacking of granal thylakoids, the differences in the characteristics of fragments obtained with a low and a high yield reflect the changes in the composition of granal thylakoids in the direction from the margin to the centrum, that is, a decrease in the relative content of PSI complexes and alterations in the composition and size of its light-harvesting antenna. The consistency between the data obtained and the present view concerning the different functions of PSI located in different thylakoid regions is discussed.     

Counter-current distribution of sonicated inside-out thylakoid vesicles

Summary Inside-out thylakoid vesicles were isolated from spinach chloroplasts, and fragmented by sonication. Different fragments were separated by counter-current distribution and analyzed for chlorophyll and P700. The inside-out vesicles had a chlorophyll a/b ratio of 2.2–2.4 (original chloroplasts 2.8–3.0). After further fragmentation of the inside-out vesicles by sonication and separation by countercurrent distribution three populations of vesicles were obtained having chlorophyll a/b ratios of 1.7, 1.9 and 2.5 respectively. The P-700 was depleted in fractions with lower chlorophyll a/b ratio and was nearly absent in the fraction having a chlorophyll a/b ratio of 1.7 (chlorophyll/P700 > 4500 mol/mol). That PSII membrane vesicles, with such a low chlorophyll a/b ratio and lacking PSI, can be prepared by a non-detergent method provides strong support for the notion that PSI and PSII are segregated along the thylakoid membrane.A plot of P700 per chlorophyll against chlorophyll b/(a+b) fits a straight line connecting the pure PSI membrane (chlorophyll a/b = 6; P700/chlorophyll = 5.6 mmol/mol) with the pure PSII membrane (chlorophyll a/b = 1.7; P700 = 0). These two membranes can be considered as separate phases of a two-dimensional phase system. Models for the thylakoid membrane are discussed.Abbreviations PSI Photosystem I - PSII Photosystem II - PEG Polyethylene Glycol - P700 Reaction Center of PSI     

Role of thylakoid protein kinases in photosynthetic acclimation

Photosynthetic organisms are able to adjust to changes in light quality through state transition, a process which leads to a balancing of the light excitation energy between the antennae systems of photosystem II and photosystem I. A genetic approach has been used in Chlamydomonas with the aim of elucidating the signaling chain involved in state transitions. This has led to the identification of a small family of Ser-Thr protein kinases associated with the thylakoid membrane and conserved in algae and land plants. These kinases appear to be involved both in short and long term adaptations to changes in the light environment.     

Characterization of the photosystem II centers inactive in plastoquinone reduction by fluorescence induction

In order to characterize the photosystem II (PS II) centers which are inactive in plastoquinone reduction, the initial variable fluorescence rise from the non-variable fluorescence level F_o to an intermediate plateau level F_i has been studied. We find that the initial fluorescence rise is a monophasic exponential function of time. Its rate constant is similar to the initial rate of the fastest phase (-phase) of the fluorescence induction curve from DCMU-poisoned chloroplasts. In addition, the initial fluorescence rise and the -phase have the following common properties: their rate constants vary linearly with excitation light intensity and their fluorescence yields are lowered by removal of Mg⁺⁺ from the suspension medium. We suggest that the inactive PS II centers, which give rise to the fluorescence rise from F_o to F_i, belong to the -type PS II centers. However, since these inactive centers do not display sigmoidicity in fluorescence, they thus do not allow energy transfer between PS II units like PS II.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DMQ 2,5-dimethyl-p-benzoquinone - F_o initial non-variable fluorescence yield - F_m maximum fluorescence yield - F_i intermediate fluorescence yield - PS II photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

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.     

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.     

The PsbS protein controls the macro-organisation of photosystem II complexes in the grana membranes of higher plant chloroplasts

The PsbS protein is a critical component in the regulation of non-photochemical quenching (NPQ) in higher plant photosynthesis. Electron microscopy and image analysis of grana membrane fragments from wild type and mutant Arabidopsis plants showed that the semi-crystalline domains of photosystem II supercomplexes were identical in the presence and absence of PsbS. However, the frequency of the domains containing crystalline arrays was increased in the absence of PsbS. Conversely, there was a complete absence of such arrays in the membranes of plants containing elevated amounts of this protein. It is proposed that PsbS controls the macro-organisation of the grana membrane, providing an explanation of its role in NPQ.     

Transverse heterogeneity in lipid fluidity in spinach thylakoids,photosystem II preparations,and thylakoid galactolipid vesicles

We have used three doxyl stearic acid spin labels to study the transverse hetero-geneity in lipid fluidity in thylakoids, photosystem II (PS II) preparations, and thylakoid galactolipid vesicles. This comparative study shows that spin labels incorporated into the membrane of the PS II preparation experience far more immobilization than do the same spin labels incorporated into either thylakoids or vesicles prepared from the polar lipids extracted from thylakoids. The spin label immobilization found in the PS II preparation is manifest even near the center of the bilayer, where lipid mobility is normally at its maximum. Analysis of the lipid content of the PS II preparation, relative to chlorophyll, suggests that the PS II preparation may be lipid depleted. This lipid depletion could explain the results presented. However, electron microscopy [Dunahay et al. (1984) Biochim. Biophys. Acta 764:179–193] has not indicated that major delipidation has occurred, and so it remains possible that the immobilization found in the PS II preparation is due primarily to the normal (but close) juxtaposition of adjacent PS II complexes and the cooperative immobilization of their surrounding lipids. Based on the results presented, we conclude that highly mobile lipids are not required for oxygen evolution, the primary photochemistry or the secondary reduction of exogenously added quinones. Unfortunately, the relationship between the plastoquinone pool and the fluidity of the membrane in the PS II preparation remains ambiguous.Abbreviations PS II photosystem II - SDSA 5-doxylstearic acid - 12DSA 12-doxylstearic acid - 16DSA 16-doxylstearic acid - 7N14 2-heptyl-2-hexyl-5,5-dimethyloxazolidine-N-oxyl - chromium oxalate potassium trioxalatochromiate - EPR electron paramagnetic resonance - Chl chlorophyll - MGDG monogalactosyldiacylglycerol - DGDG digalactosyldiacylglycerol     

Temperature-dependent changes in Photosystem II heterogeneity of attached leaves under high light

Attached leaves of pumpkin ( Cucurbita pepo L. cv. Jattiläismeloni) were exposed to high light intensity at room temperature (ca 23°C) and at 1°C. Fluorescence parameters and electron transport activities measured from isolated thylakoids indicated faster photoinhibition of PSII at low temperature. Separation of the α and β components of the complementary area above the fluorescence induction curve of dichlorophenyl-dimethylurea-poisoned thylakoids revealed that at low temperature only the α-centers declined during exposure to high light intensity while the content of functional β-centers remained constant. Freeze-fracture electron microscopy showed no decrease in the density of particles on the appressed exoplasmic fracture face, indicating that the photoinhibited α-centers remained in the appressed membranes at 1°C. Because of the function of the repair and protective mechanisms of PSII, strong light induced less photoinhibition at room temperature, but more complicated changes occurred in the α/β-heterogeneity of PSII. During the first 30 min at high light intensity the decrease in α-centers was almost as large as at 1°C, but in contrast to the situation at low temperature the decrease in α-centers was compensated for by a significant increase in PSIIβ-centers. Changes in the density and size of freeze-fracture particles suggest that this increase in β-centers was due to migration of phosphorylated light-harvesting complex from appressed to non-appressed thylakoid membranes while the PSII core remained in the appressed membranes. This situation, however, was only transient and was followed by a rapid decrease in the functionalβ-centers.     

Effects of diquat on pigment-protein complexes of thylakoid membranes in soybean and maize plants

Soybean (Glycine max Merrill) and maize (Zea mays L.) plants were exposed for 5 to 48 h to the herbicide diquat under "white light" (WL) or far-red radiation (FR) (photon fluence rate of 30 μmol m^-2 s^-1). The WL enhanced diquat effect on chlorophyll content in soybean plants, while FR had the same effects on maize plants. After 5 h, diquat increased the content of polypeptides bound to light-harvesting proteins in both plants. This revised version was published online in July 2006 with corrections to the Cover Date.