Lateral mobility of the light-harvesting complex in chloroplast membranes controls excitation energy distribution in higher plants

Chloroplast thylakoid protein phosphorylation produces changes in light-harvesting properties and in membrane structure as revealed by freeze-fracture electron microscopy. Protein phosphorylation resulted in an increase in the 77 °K fluorescence signal at 735 nm relative to that at 685 nm. In addition, a decrease in connectivity between Photosystem II centers (PS II) and a dynamic quenching of the room temperature variable fluorescence was observed upon phosphorylation. Accompanying these fluorescence changes was a 23% decrease in the amount of stacked membranes. Microscopic analyses indicated that 8.0-nm particles fracturing on the P-face moved from the stacked into the unstacked regions upon phosphorylation. The movement of the 8.0-nm particles was accompanied by the appearance of chlorophyll b and 25 to 29 kD polypeptides in isolated stroma lamellae fractions. We conclude that phosphorylation of a population of the light-harvesting chlorophyll $\text{a}\text{b}$ protein complexes (LHC) in grana partitions causes the migration of these pigment proteins from the PS II-rich appressed membranes into the Photosystem I (PS I) enriched unstacked regions. This increases the absorptive cross section of PS I. In addition, we suggest that the mobile population of LHC functions to interconnect PS II centers in grana partitions; removal of this population of LHC upon phosphorylation limits PS II → PS II energy transfer and thereby favors spillover of energy from PS II to PS I.     

Arrangement of photosystem II supercomplexes in crystalline macrodomains within the thylakoid membrane of green plant chloroplasts

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.     

Solubilization of green plant thylakoid membranes with n-dodecyl-α,D-maltoside. Implications for the structural organization of the Photosystem II,Photosystem I,ATP synthase and cytochrome b6 f complexes

A biochemical and structural analysis is presented of fractions that were obtained by a quick and mild solubilization of thylakoid membranes from spinach with the non-ionic detergent n-dodecyl-α,D-maltoside, followed by a partial purification using gel filtration chromatography. The largest fractions consisted of paired, appressed membrane fragments with an average diameter of about 360 nm and contain Photosystem II (PS II) and its associated light-harvesting antenna (LHC II), but virtually no Photosystem I, ATP synthase and cytochrome b ₆ f complex. Some of the membranes show a semi-regular ordering of PS II in rows at an average distance of about 26.3 nm, and from a partially disrupted grana membrane fragment we show that the supercomplexes of PS II and LHC II represent the basic structural unit of PS II in the grana membranes. The numbers of free LHC II and PS II core complexes were very high and very low, respectively. The other macromolecular complexes of the thylakoid membrane occurred almost exclusively in dispersed forms. Photosystem I was observed in monomeric or multimeric PS I-200 complexes and there are no indications for free LHC I complexes. An extensive analysis by electron microscopy and image analysis of the CF₀F₁ ATP synthase complex suggests locations of the δ (on top of the F₁ headpiece) and ∈ subunits (in the central stalk) and reveals that in a substantial part of the complexes the F₁ headpiece is bended considerably from the central stalk. This kinking is very likely not an artefact of the isolation procedure and may represent the complex in its inactive, oxidized form. This revised version was published online in June 2006 with corrections to the Cover Date.     

An alternative model for photosystem II/light harvesting complex II in grana membranes based on cryo-electron microscopy studies.

The photosynthetic protein complexes in plants are located in the chloroplast thylakoid membranes. These membranes have an ultrastructure that consists of tightly stacked 'grana' regions interconnected by unstacked membrane regions. The structure of isolated grana membranes has been studied here by cryo-electron microscopy. The data reveals an unusual arrangement of the photosynthetic protein complexes, staggered over two tightly stacked planes. Chaotrope treatment of the paired grana membranes has allowed the separation and isolation of two biochemically distinct membrane fractions. These data have led us to an alternative model of the ultrastructure of the grana where segregation exists within the grana itself. This arrangement would change the existing view of plant photosynthesis, and suggests potential links between cyanobacterial and plant photosystem II light harvesting systems.     

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.     

Further investigations on structural and catalytic properties of O2 evolving preparations from tobacco and two chlorophyll deficient tobacco mutants

Previous investigations (Specht, S., Pistorius, E.K. and Schmid, G.H.: Photosynthesis Res. 13, 47–56, 1987) of Photosystem II membranes from tobacco (Nicotiana tabacum L. cv. John William's Broadleaf) which contain normally stacked thylakoid membranes and from two chlorophyll deficient tobacco mutants (Su/su and Su/su var. Aurea) which have low stacked or essentially unstacked thylakoids with occasional membrane doublings, have been extended by using monospecific antisera raised against the three extrinsic polypeptides of 33,21 and 16 kDa. The results show that all three peptides are synthesized as well in wild type tobacco as in the two mutants to about the same level and that they are present in thylakoid membranes of all three plants. However, in the mutants the 16 and 21 kDa peptides (but not the 33 kDa peptide) are easily lost during solubilization of Photosystem II membranes. In the absence of the 16 and 21 kDa peptide Photosystem II membranes from the mutants have a higher O₂ evolving activity without addition of CaCl₂ than the wild type Photosystem II membranes. On the other hand, after removal of the 33 kDa peptide no significant differences in the binding of Mn could be detected among the three plants. The results also show that reaction center complexes from wild type tobacco and the mutant Su/su are almost identical to the Triton-solubilized Photosystem II membranes from the mutant Su/su var. Aurea.Abbreviations PS photosystem - chl chlorophyll - LHCP light harvesting chlorophyll a/b protein complex - WT wild type - OEE1, OEE2 and OEE3 oxygen evolution enhancing complex of 29–36 kDa, 21–24 kDa and 16–18 kDa, respectively     

A chloroplast membrane lacking Photosystem I. Changes in unstacked membrane regions

The structure and polypeptide composition of the photosynthetic membrane of a mutant of maize has been investigated. The thylakoid membranes of the mutant plants are deficient in Photosystem I activity, although Photosystem II is at near normal levels. SDS polyacrylamide gel electrophoresis of thylakoid membranes from the mutant shows them to be deficient in two polypeptide bands which have been associated with Photosystem I. Freeze-fracture studies of the membrane show that the absence of these polypeptides is associated with a measurable reduction in particle diameter on the unstacked protoplasmic fracture face. This fracture face is derived from the splitting of membranes in unstacked regions of the thylakoid membrane system. It is suggested that in membranes stacked by salts in vitro, Photosystem I activity may be confined to this region.     

A study on the lateral distribution of the plastoquinone pool with respect to Photosystem II in stacked and unstacked spinach chloroplasts

The quenching of Photosystem II (PS II) chlorophyll fluorescence by oxidised plastoquinone has been used in an attempt to determine their relative distribution in the partition zone and stroma-exposed thylakoid membranes. Thus, the PS II-plastoquinone interaction was determined in stacked (2.5 mM MgCl₂) and largely unstacked (0.25 mM MgCl₂) membranes. A method to correct for spillover or other quenching changes at the different MgCl₂ concentrations, which would compete with the plastoquinone-induced quenching, was devised utilising the quinone dibromothymoquinone. This compound is demonstrated to behave as an ideal (theoretically) PS II quencher at both high and low MgCl₂ concentrations, which indicates that it distributes itself homogeneously between partition zone and stroma-exposed membrane regions. In passing from the stacked to the unstacked configuration, the PS II-plastoquinone interaction decreases less than the PS II-dibromothymoquinone interaction. This is interpreted to mean that plastoquinone is present in both the partition zone and stroma-exposed membranes, with somewhat higher concentrations in the stroma-exposed membranes. Thus, plastoquinone is well placed to transport reducing equivalents from the partition zones to the stroma-exposed membranes.     

Fine structure of the chloroplast membranes ofEuglena gracilis as revealed by freeze-cleaving and deep-etching techniques

Summary The chloroplasts ofEuglena gracilis have been examined by freeze-cleaving and deep-etching techniques.The two chloroplast envelope membranes exhibit distinct fracture faces which do not resemble any of the thylakoid fracture faces.Freeze-cleaved thylakoid membranes reveal four split inner faces. Two of these faces correspond to stacked membrane regions, and two to unstacked regions. Analysis of particle sizes on the exposed faces has revealed certain differences from other chloroplast systems, which are discussed. Thylakoid membranes inEuglena are shown to reveal a constant number of particles per unit area (based on the total particle number for both complementary faces) whether they are stacked or unstacked.Deep-etchedEuglena thylakoid membranes show two additional faces, which correspond to true inner and outer thylakoid surfaces. Both of these surfaces carry very uniform populations of particles. Those on the external surface (the A surface) are round and possess a diameter of approximately 9.5 nm. Those on the inner surface (the D surface) appear rectangular (as paired subunits) and measure approximately 10 nm in width and 18 nm in length. Distribution counts of particles show that the number of particles per unit area revealed by freeze-cleaving within the thylakoid membrane approximates closely the number of particles exposed on the external thylakoid surface (the A surface) by deep-etching. The possible significance of this correlation is discussed. The distribution of rectangular particles on the inner surface of the thylakoid sac (D surface) seems to be the same in both stacked and unstacked membrane regions. We have found no correlation between the D surface particles and any clearly defined population of particles on internal, freeze-cleaved membrane faces. These and other observations suggest that stacked and unstacked membranes are similar, if not identical in internal structure.     

Structural,biochemical and biophysical characterization of four oxygen-evolving Photosystem II preparations from spinach

Four procedures utilizing different detergent and salt conditions were used to isolate oxygen-evolving Photosystem II (PS II) preparations from spinach thylakoid membranes. These PS II preparations have been characterized by freeze-fracture electron microscopy, SDS-polyacrylamide gel electrophoresis, steady-state and pulsed oxygen evolution, 77 K fluorescence, and room-temperature electron paramagnetic resonance. All of the O₂-evolving PS II samples were found to be highly purified grana membrane fractions composed of paired, appressed membrane fragments. The lumenal surfaces of the membranes and thus the O₂-evolving enzyme complex, are directly exposed to the external environment. Biochemical and biophysical analyses indicated that all four preparations are enriched in the chlorophyll $\text{a}\text{b-}\text{light-harvesting}$ complex and Photosystem II, and depleted to varying degrees in the stroma-associated components, Photosystem I and the CF₁-ATPase. The four PS II samples also varied in their cytochrome f content. All preparations showed enhanced stability of oxygen production and oxygen-rate electrode activity compared to control thylakoids, apparently promoted by low concentrations of residual detergent in the PS II preparations. A model is presented which summarizes the effects of the salt and detergent treatments on thylakoid structure and, consequently, on the configuration and composition of the oxygen-evolving PS II samples.     

Supramolecular organization of thylakoid membrane proteins in green plants

The light reactions of photosynthesis in green plants are mediated by four large protein complexes, embedded in the thylakoid membrane of the chloroplast. Photosystem I (PSI) and Photosystem II (PSII) are both organized into large supercomplexes with variable amounts of membrane-bound peripheral antenna complexes. PSI consists of a monomeric core complex with single copies of four different LHCI proteins and has binding sites for additional LHCI and/or LHCII complexes. PSII supercomplexes are dimeric and contain usually two to four copies of trimeric LHCII complexes. These supercomplexes have a further tendency to associate into megacomplexes or into crystalline domains, of which several types have been characterized. Together with the specific lipid composition, the structural features of the main protein complexes of the thylakoid membranes form the main trigger for the segregation of PSII and LHCII from PSI and ATPase into stacked grana membranes. We suggest that the margins, the strongly folded regions of the membranes that connect the grana, are essentially protein-free, and that protein-protein interactions in the lumen also determine the shape of the grana. We also discuss which mechanisms determine the stacking of the thylakoid membranes and how the supramolecular organization of the pigment-protein complexes in the thylakoid membrane and their flexibility may play roles in various regulatory mechanisms of green plant photosynthesis.     

The structure and function of the chloroplast photosynthetic membrane — a model for the domain organization

Recent work on the domain organization of the thylakoid is reviewed and a model for the thylakoid of higher plants is presented. According to this model the thylakoid membrane is divided into three main domains: the stroma lamellae, the grana margins and the grana core (partitions). These have different biochemical compositions and have specialized functions. Linear electron transport occurs in the grana while cyclic electron transport is restricted to the stroma lamellae. This model is based on the following results and considerations. (1) There is no good candidate for a long-range mobile redox carrier between PS II in the grana and PS I in the stroma lamellae. The lateral diffusion of plastoquinone and plastocyanin is severely restricted by macromolecular crowding in the membrane and the lumen respectively. (2) There is an excess of 14±18% chlorophyll associated with PS I over that of PS II. This excess is assumed to be localized in the stroma lamellae where PS I drives cyclic electron transport. (3) For several plant species, the stroma lamellae account for 20±3% of the thylakoid membrane and the grana (including the appressed regions, margins and end membranes) for the remaining 80%. The amount of stroma lamellae (20%) corresponds to the excess (14–18%) of chlorophyll associated with PS I. (4) The model predicts a quantum requirement of about 10 quanta per oxygen molecule evolved, which is in good agreement with experimentally observed values. (5) There are at least two pools of each of the following components: PS I, PS II, cytochrome bf complex, plastocyanin, ATP synthase and plastoquinone. One pool is in the grana and the other in the stroma compartments. So far, it has been demonstrated that the PS I, PS II and cytochrome bf complexes each differ in their respective pools.Abbreviations PS I and PS II Photosystem I and II - P 700 reaction center of PS I - LHC II light-harvesting complex 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.     

Prolonged high light treatment of plant cells results in changes of the amount,the localization and the electrophoretic behavior of several thylakoid membrane proteins

The effect of a 30 h high light treatment on the amount and the localization of thylakoid proteins was analysed in low light grown photoautotrophic cells of Marchantia polymorpha and Chenopodium rubrum. High light treatment resulted in a net loss of D1 protein which was accompanied by comparable losses of other proteins of the PS II core (reaction center with inner antenna). LHC II proteins were not reduced correspondingly, indicating that these complexes are less affected by prolonged high light. High light influenced the distribution of PS II components between the grana and the stroma region of the thylakoid membrane, probably by translocation of the respective PS II proteins. Additionally, modifications of several thylakoid proteins were detected in high light treated cells of C. rubrum. These effects are discussed in relation to photoinhibitory damage and repair processes.Abbreviations BCA bioinchonic acid - chl chlorophyll - CF₁ coupling factor - CYC cycloheximide - GT grana thylakoids - HL high light - LL low light - PAGE polyacrylamide gel electrophoresis - PFD photon flux density - PS I Photosystem I - PS II Photosystem II - RC reaction center - SDS sodium dodecylsulfate - ST stroma thylakoids - Thyl unfractionated thylakoids     

The role of chlorophyll-protein complexes in the function and structure of chloroplast thylakoids

Summary The photosynthetic pigments of chloroplast thylakoid membranes are complexed with specific intrinsic polypeptides which are included in three supramolecular complexes, photosystem I complex, photosystem II complex and the light-harvesting complex. There is a marked lateral heterogeneity in the distribution of these complexes along the membrane with photosystem II complex and its associated light-harvesting complex being located mainly in the stacked membranes of the grana partitions, while photosystem I complex is found mainly in unstacked thylakoids together with ATP synthetase. In contrast, the intermediate electron transport complex, the cylochrome b-f complex, is rather uniformly distributed in these two membrane regions. The consequences of this lateral heterogeneity in the location of the thylakoid complexes are considered in relation to the function and structure of chloroplasts of higher plants.     

Senescence induced structural reorganization of thylakoid membranes in Cucumis sativus cotyledons; LHC II involvement in reorganization of thylakoid membranes

We report the formation and appearance of loosely stacked extended grana like structures along with plastoglobuli in the chloroplasts isolated from 27-day old senescing cucumber cotyledons. The origin and the nature of these extended grana structures have not been elucidated earlier. We isolated Photosystem I complexes from 6-day-old control and 27-day-old senescing cotyledons. The chlorophyll a/b ratio of the isolated Photosystem I complex obtained from 6-day cotyledons was 5–5.5 as against a ratio of 2.9 was found in Photosystem I complexes obtained from 27-day-old senescing cotyledons. We also found that the presence of LHC II in the Photosystem I complexes isolated from 27-day cotyledonary chloroplasts. The presence of LHC II in Photosystem I complexes in senescing and not in control samples, clearly suggest the detachment and diffusion of LHC II complexes from stacked grana region to Photosystem I enriched stroma lamellar region thereby, forming loose disorganized extended grana structures seen in the transmission electron microscope. Furthermore, we show that under in vitro condition the senescing cotyledon chloroplasts exhibited lower extent of light induced phosphorylation of LHC II than the control samples suggesting a possible irreversible phosphorylation and diffusion of LHC II in vivo during the progress of senescence in Cucumis cotyledons. From these findings, we suggest that the senescence induced phosphorylation of LHC II and its migration towards Photosystem I may be a programmed one some how causing the destruction of the thylakoid membrane. The released membrane components may be stored in the plastoglobuli prior to their mobilization to the younger plant parts. This revised version was published online in June 2006 with corrections to the Cover Date.     

The polypeptides of stacked and unstacked Chlamydomonas reinhardi chloroplast membranes and their relation to photosystem II activity

When cells of the ac-5 mutant strain of Chlamydomonas reinhardi are cultured mixotrophically, their chloroplast membranes are unstacked and they lack a group of membrane polypeptides that have been reported to be associated with a membrane fraction enriched for Photosystem II activity. On the other hand, the chloroplast membranes of cells grown phototrophically are stacked and they possess the membrane polypeptides. Since the unstacked membranes possess Photosystem II activity, we suggest that the polypeptides must be present in the chloroplast membrane if stacking is to occur.     

Properties of thylakoids and thylakoid particles derived from structurally different chloroplasts

Structurally and functionally different tobacco chloroplasts were subjected to digitonin treatment and subsequent fractional centrifugation. The light-harvesting $\text{chlorophyll}\text{a}\text{chlorophyll}\text{b-}\text{protein}$ complex was found to be enriched in the most dense fraction regardless of the presence of grana in the original preparation. It is suggested that isolated thylakoid membranes and fragments thereof which contain sufficient light-harvesting protein may, under appropriate ionic conditions, form aggregates even when they originate from unstacked thylakoid systems. Comparative studies of fluorescence properties and polypeptide composition of the thylakoids suggest that the light-harvesting protein does not contribute significantly to the fluorescence spectrum of isolated chloroplasts as long as this protein is intimately associated with the Photosystem II (PS II) pigment-protein complex responsible for the 685 nm emission. While the PS II-deficient mutant chloroplasts of the variegated tobacco variety NC 95 lacked both the 685 nm fluorescence component and two or three PS II proteins, one of these proteins was found to be very prominent in our chlorophyll b-deficient mutant thylakoids which also displayed an intense 685 nm fluorescence peak. This correlation supports the contention that a 45 kdalton polypeptide is an apoprotein of pigments associated with the PS II reaction center.     

Molecular crowding and order in photosynthetic membranes

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

Polyamines in the photosynthetic apparatus

The three main polyamines putrescine (Put), spermidine (Spd) and spermine (Spm) were characterized by HPLC in intact spinach leaf cells, intact chloroplasts, thylakoid membranes, Photosystem II membranes, the light-harvesting complex and the PS II complex. All contain the three polyamines in various ratios; the HPLC polyamine profiles of highly resolved PS II species (a Photosystem II core and the rection center) suggest an enrichment in the polyamine Spm.Abbreviations Chl chlorophyll - HPLC high performance liquid chromatography - LHC light-harvesting complex - PS II Photosystem II - PS II-RC Photosystem II reaction center - Put putrescine - Spd spermidine - Spm spermine - 10%S-core D₁-D₂-Cyt b559-47 kD-43 kD complex