Molecular architecture of the thylakoid membrane: lipid diffusion space for plastoquinone

We have determined the stoichiometric composition of membrane components (lipids and proteins) in spinach thylakoids and have derived the molecular area occupied by these components. From this analysis, the lipid phase diffusion space, the fraction of lipids located in the first protein solvation shell (boundary lipids), and the plastoquinone (PQ) concentration are derived. On the basis of these stoichiometric data, we have analyzed the motion of PQ between photosystem (PS) II and cytochrome (cyt.) bf complexes in this highly protein obstructed membrane (protein area about 70%) using percolation theory. This analysis reveals an inefficient diffusion process. We propose that distinct structural features of the thylakoid membrane (grana formation, microdomains) could help to minimize these inefficiencies and ensure a non-rate limiting PQ diffusion process. A large amount of published evidence supports the idea that higher protein associations exist, especially in grana thylakoids. From the quantification of the boundary lipid fraction (about 60%), we conclude that protein complexes involved in these associations should be spaced by lipids. Lipid-spaced protein aggregations in thylakoids are qualitatively different to previously characterized associations (multisubunit complexes, supercomplexes). We derive a hierarchy of protein and lipid interactions in the thylakoid membrane.     

Voltammetric measurement of the plastoquinone redox state in isolated thylakoids

The oxidation of plastoquinol by the cytochrome bf complex is commonly believed to be the rate limiting step in photosynthetic electron transport. When input of electrons from PS II exceeds electron flow through the cytochrome bf complex the plastoquinone pool becomes reduced. A voltammetric technique previously used to measure the redox state of the ubiquinone pool in plant mitochondria, was modified to measure the redox state of the plastoquinone pool in thylakoids. The presence or absence of a proton gradient strongly influenced the relationship between the redox state of the plastoquinone pool and other photosynthetic parameters. A linear relationship between the rate of electron transport and the reduction of plastoquinone was found. The slope of this relationship was greater in coupled than in uncoupled thylakoids, indicating that under coupled conditions the plastoquinone pool is more reduced at any given rate of electron flow. A complex relationship was found between QA reduction, calculated as 1 – q_p, and the redox state of the plastoquinone pool. The extent of Q_A reduction was similar in coupled and uncoupled thylakoids, but at any given level of Q_A reduction, PQ was always more reduced in coupled thylakoids. These results suggest that the presence of a proton gradient changes the equilibrium constant between Q_A and PQ.     

Plastocyanin redox kinetics in spinach chloroplasts: evidence for disequilibrium in the high potential chain

Reduction kinetics of cytochrome f, plastocyanin (PC) and P(700) ('high-potential chain') in thylakoids from spinach were followed after pre-oxidation by a saturating light pulse. We describe a novel approach to follow PC redox kinetics from deconvolution of 810-860 nm absorption changes. The equilibration between the redox-components was analyzed by plotting the redox state of cytochrome f and PC against that of P(700). In thylakoids with (1) diminished electron transport rate (adjusted with a cytochrome bf inhibitor) or (2) de-stacked grana, cytochrome f and PC relaxed close to their thermodynamic equilibriums with P(700). In stacked thylakoids with non-inhibited electron transport, the equilibration plots were complex and non-hyperbolic, suggesting that during fast electron flux, the 'high-potential chain' does not homogeneously equilibrate throughout the membrane. Apparent equilibrium constants <5 were calculated, which are below the thermodynamic equilibrium known for the 'high potential chain'. The disequilibrium found in stacked thylakoids with high electron fluxes is explained by restricted long-range PC diffusion. We develop a model assuming that about 30% of Photosystem I mainly located in grana end-membranes and margins rapidly equilibrate with cytochrome f via short-distance transluminal PC diffusion, while long-range lateral PC migration between grana cores and distant stroma lamellae is restricted. Implications for the electron flux control are discussed.     

Plastocyanin redox kinetics in spinach chloroplasts: evidence for disequilibrium in the high potential chain

Reduction kinetics of cytochrome f, plastocyanin (PC) and P₇₀₀ (‘high-potential chain’) in thylakoids from spinach were followed after pre-oxidation by a saturating light pulse. We describe a novel approach to follow PC redox kinetics from deconvolution of 810-860 nm absorption changes. The equilibration between the redox-components was analyzed by plotting the redox state of cytochrome f and PC against that of P₇₀₀. In thylakoids with (1) diminished electron transport rate (adjusted with a cytochrome bf inhibitor) or (2) de-stacked grana, cytochrome f and PC relaxed close to their thermodynamic equilibriums with P₇₀₀. In stacked thylakoids with non-inhibited electron transport, the equilibration plots were complex and non-hyperbolic, suggesting that during fast electron flux, the ‘high-potential chain’ does not homogeneously equilibrate throughout the membrane. Apparent equilibrium constants <5 were calculated, which are below the thermodynamic equilibrium known for the ‘high potential chain’. The disequilibrium found in stacked thylakoids with high electron fluxes is explained by restricted long-range PC diffusion. We develop a model assuming that about 30% of Photosystem I mainly located in grana end-membranes and margins rapidly equilibrate with cytochrome f via short-distance transluminal PC diffusion, while long-range lateral PC migration between grana cores and distant stroma lamellae is restricted. Implications for the electron flux control are discussed.     

Non-photochemical reduction of thylakoid photosynthetic redox carriers in vitro: Relevance to cyclic electron flow around photosystem I?

Non-photochemical (dark) increases in chlorophyll a fluorescence yield associated with non-photochemical reduction of redox carriers (F_npr) have been attributed to the reduction of plastoquinone (PQ) related to cyclic electron flow (CEF) around photosystem I. In vivo, this rise in fluorescence is associated with activity of the chloroplast plastoquinone reductase (plastid NAD(P)H:plastoquinone oxidoreductase) complex. In contrast, this signal measured in isolated thylakoids has been attributed to the activity of the protein gradient regulation-5 (PGR5)/PGR5-like (PGRL1)-associated CEF pathway. Here, we report a systematic experimentation on the origin of F_npr in isolated thylakoids. Addition of NADPH and ferredoxin to isolated spinach thylakoids resulted in the reduction of the PQ pool, but neither its kinetics nor its inhibitor sensitivities matched those of F_npr. Notably, F_npr was more rapid than PQ reduction, and completely insensitive to inhibitors of the PSII Q_B site and oxygen evolving complex as well as inhibitors of the cytochrome b₆f complex. We thus conclude that F_npr in isolated thylakoids is not a result of redox equilibrium with bulk PQ. Redox titrations and fluorescence emission spectra imply that F_npr is dependent on the reduction of a low potential redox component (E_m about − 340 mV) within photosystem II (PSII), and is likely related to earlier observations of low potential variants of Q_A within a subpopulation of PSII that is directly reducible by ferredoxin. The implications of these results for our understanding of CEF and other photosynthetic processes are discussed.     

Macromolecular crowding and its influence on possible reaction mechanisms in photosynthetic electron flow

The diffusion of plastoquinol and its binding to the Qo site of the cyt bf complex in the course of photosynthetic electron transport was studied by following the sigmoidal flash-induced re-reduction kinetics of P700 after previous oxidation of the intersystem electron carriers. The data resulting from these experiments were matched with a simulation of electron transport using Monte Carlo techniques. The simulation was able to account for the experimental observations. Two different extreme cases of reaction mechanism at the Qo site were compared: a diffusion limited collisional mechanism and a non-diffusion limited tight binding mechanism. Assuming a tight binding mechanism led to best matches due to the high protein density in thylakoids. The varied parameters resulted in values well within the range of published data. The results emphasise the importance of structural characteristics of thylakoids in models of electron transport.     

Model for the fluorescence induction curve of photoinhibited thylakoids.

The fluorescence induction curve of photoinhibited thylakoids measured in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea was modeled using an extension of the model of Lavergne and Trissl (Biophys. J. 68:2474-2492), which takes into account the reversible exciton trapping by photosystem II (PSII) reaction centers and exciton exchange between PSII units. The model of Trissl and Lavergne was modified by assuming that PSII consists of photosynthetically active and photoinhibited (inactive in oxygen evolution) units and that the inactive PSII units can efficiently dissipate energy even if they still retain the capacity for the charge separation reaction. Comparison of theoretical and experimental fluorescence induction curves of thylakoids, which had been subjected to strong light in the presence of the uncoupler nigericin, suggests connectivity between the photoinhibited and active PSII units. The model predicts that photoinhibition lowers the yield of radical pair formation in the remaining active PSII centers. However, the kinetics of PSII inactivation in nigericin-treated thylakoids upon exposure to photoinhibitory light ranging from 185 to 2650 micromol photons m-2 s-1 was strictly exponential. This may suggest that photoinhibition occurs independently of the primary electron transfer reactions of PSII or that increased production of harmful substances by photoinhibited PSII units compensates for the protection afforded by the quenching of excitation energy in photoinhibited centers.     

Structural and functional self-organization of Photosystem II in grana thylakoids

The biogenesis of the well-ordered macromolecular protein arrangement of photosystem (PS)II and light harvesting complex (LHC)II in grana thylakoid membranes is poorly understood and elusive. In this study we examine the capability of self organization of this arrangement by comparing the PSII distribution and antenna organization in isolated untreated stacked thylakoids with restacked membranes after unstacking. The PS II distribution was deduced from freeze-fracture electron microscopy. Furthermore, changes in the antenna organization and in the oligomerization state of photosystem II were monitored by chlorophyll a fluorescence parameters and size analysis of exoplasmatic fracture face particles. Low-salt induced unstacking leads to a randomization and intermixing of the protein complexes. In contrast, macromolecular PSII arrangement as well as antenna organization in thylakoids after restacking by restoring the original solvent composition is virtually identical to stacked control membranes. This indicates that the supramolecular protein arrangement in grana thylakoids is a self-organized process.     

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

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

Structural and functional self-organization of Photosystem II in grana thylakoids

The biogenesis of the well-ordered macromolecular protein arrangement of photosystem (PS)II and light harvesting complex (LHC)II in grana thylakoid membranes is poorly understood and elusive. In this study we examine the capability of self organization of this arrangement by comparing the PSII distribution and antenna organization in isolated untreated stacked thylakoids with restacked membranes after unstacking. The PS II distribution was deduced from freeze-fracture electron microscopy. Furthermore, changes in the antenna organization and in the oligomerization state of photosystem II were monitored by chlorophyll a fluorescence parameters and size analysis of exoplasmatic fracture face particles. Low-salt induced unstacking leads to a randomization and intermixing of the protein complexes. In contrast, macromolecular PSII arrangement as well as antenna organization in thylakoids after restacking by restoring the original solvent composition is virtually identical to stacked control membranes. This indicates that the supramolecular protein arrangement in grana thylakoids is a self-organized process.     

Functional effects of chemical modification of unstacked and stacked chloroplasts with glutaraldehyde.

Although glutaraldehyde alkylates protein NH₂ groups to the same extent in unstacked and stacked thylakoids, the photosynthetic electron transport of the stacked membranes is always more inhibited. Inhibition of photosystem II electron transport, measured in the presence of lipophilic Hill oxidants, is 20–30% in unstacked and 60–70% in stacked thylakoids. Photosystem I electron transport is nearly completely inhibited in both preparations, but in the case of stacked thylakoids maximal inhibition occurs at a lower glutaraldehyde level than in unstacked thylakoids. In contrast, the photooxidation of the reaction center chromophore of photosystem I (P700) is unaffected by the glutaraldehyde treatment of either stacked or unstacked chloroplasts. The results are discussed with regard to the accessibility of membrane sites to exogenous electron transport cofactors, in view of the observation that N-methylphenazonium methosulfate, a quencher of electronically excited chlorophyll a, partitions more easily into the pigment domains of the glutaraldehyde-fixed unstacked thylakoids.     

A theoretical study on effect of the initial redox state of cytochrome b559 on maximal chlorophyll fluorescence level (F(M)): implications for photoinhibition of photosystem II

In this work, we extended the reversible radical pair model which describes energy utilization and electron transfer up to the first quinone electron acceptor (Q(A)) in photosystem II (PSII), by redox reactions involving cytochrome (cyt) b559. In the model, cyt b559 accepts electrons from the reduced primary electron acceptor in PSII, pheophytin, and donates electrons to the oxidized primary electron donor in PSII (P680+). Theoretical simulations of chlorophyll fluorescence rise based on the model show that the maximal fluorescence, F(M), increases with an increasing amount of initially reduced cyt b559. In this work we applied, the first to our knowledge, metabolic control analysis (MCA) to a model of reactions in PSII. The MCA was used to determine to what extent the reactions occurring in the model control the F(M) level and how this control depends on the initial redox state of cyt b559. The simulations also revealed that increasing the amount of initially reduced cyt b559 could protect PSII against photoinhibition. Also experimental data, which might be used to validate our theory, are presented and discussed.     

Photooxidation of cytochrome b559 in oxygen-evolving photosystem II.

Cytochrome b559 (cyt b559) is an intrinsic and essential component of the photosystem II (PSII) protein complex, but its function, stoichiometry, and electron-transfer kinetics in the physiological system are not well-defined. In this study, we have used flash-detection optical spectroscopy to measure the kinetics and yields of photooxidation and dark reduction of cyt b559 in untreated, O2-evolving PSII-enriched membranes at room temperature. The dark redox states of cyt b559 and the primary electron acceptor, QA, were determined over the pH range 5.0-8.5. Both the fraction of dark-oxidized cyt b559 and dark-reduced QA increased with increasing acidity. Consistent with these results, an acid-induced drop in pH from 8.5 to 4.9 in a dark-adapted sample caused the oxidation of cyt b559, indicating a shift in the redox state during the dark reequilibration. As expected from the dark redox state of cyt b559, the rate and extent of photooxidation of cyt b559 during continuous illumination decreased toward more acidic pH values. After a single, saturating flash, the rate of photooxidation of cyt b559 was of the same order of magnitude as the rate of S2QA- charge recombination. In untreated PSII samples at pH 8.0 with 42% of cyt b559 oxidized and 15% of QA reduced in the dark, 4.7% of one copy of cyt b559 was photooxidized after one flash with a t1/2 of 540 +/- 90 ms. On the basis of our previous work [Buser, C. A., Thompson, L. K., Diner, B. A., & Brudvig, G. W (1990) Biochemistry 29, 8977] and the data presented here, we conclude that Sn+1, YZ., and P680+ are in redox equilibrium and cyt b559 (and YD) are oxidized via P680+. After a period of illumination sufficient to fully reduce the plastoquinone pool, we also observed the pH-dependent dark reduction of photooxidized cyt b559, where the rate of reduction decreased with decreasing pH and was not observed at pH < 6.4. To determine the direct source of reductant to oxidized cyt b559, we studied the dark reduction of cyt b559 and the reduction of the PQ pool as a function of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) concentration. We find that DCMU inhibits the reduction of cyt b559 under conditions where the plastoquinone pool and QA are reduced. We conclude that QB-. (H+) or QBH2 is the most likely source of the electron required for the reduction of oxidized cyt b559.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of α-tocopherol quinone, α-tocopherol and other prenyllipids with photosystem II

We have found that plastoquinone-A (PQ-A) and α-tocopherol (α-Toc) increased the reduction level of the high-potential form of cytochrome b-559 (cyt. b-559 HP) and α-tocopherol quinone (α-TQ) decreased the level of this cytochrome form in Scenedesmus obliquus wild-type, while the investigated prenyllipids were not active in the restoration of the cyt. b-559 HP form in Scenedesmus PS28 mutant and Synechococcus 6301 (Anacystis nidulans) where the cyt. b-559 HP form is naturally not present. Among the tested prenyllipids, α-TQ quenched fluorescence in thylakoids of S. obliquus wild-type, the PS28 mutant and tobacco to the highest extent, while PQ-A was less effective in this respect. α-Tocopherol showed the opposite effect to α-TQ and it was rather small. The fluorescence quenching measurements of thylakoids in the presence of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) showed that the α-Toc and FCCP (carbonylcyanide-p-trifluoromethoxy-phenyl-hydrazone) did not quench non-photochemically chlorophyll fluorescence while PQ-9 and α-TQ were effective fluorescence quenchers at higher concentrations (> 15 μM). However, at the lower α-TQ concentrations where its effective fluorescence quenching was found in DCMU-free samples, there was nearly no quenching effect by α-TQ observed in DCMU-treated thylakoids. This suggested a specific, not non-photochemical, DCMU sensitive, fluorescence quenching of photosystem II (PSII) at low α-TQ concentrations which is probably connected with the cyclic electron transport around PSII and might have a function of excess light energy dissipation. The effects of α-TQ on PSII resembled those of FCCP under many respects which might suggest similar mechanism of action of these compounds on PSII, i.e. the catalytic deprotonation and/or redox changes of some components of PSII such as the water splitting system, tyrosine D, Chl_z or cytochrome b-559.     

Synthetic quinones influencing herbicide binding and Photosystem II electron transport. The effects of triazine-resistance on quinone binding properties in thylakoid membranes

We have analyzed the binding of synthetic quinones and herbicides which inhibit electron transport at the acceptor side of Photosystem II (PS II) of the photosynthetic electron-transport chain in thylakoid membranes. These data show that quinones and PS II-directed herbicides compete for binding to a common binding environment within a PS II region which functions as the Q⁻ / PQ oxidoreductase. We observed that (1) synthetic quinones cause a parallel inhibition of electron transport and [¹⁴C]herbicide displacement, and (2) herbicide binding is affected both by the fully oxidized and fully reduced form of a quinone. Quinone function and inhibitor binding were also investigated in thylakoids isolated from triazine-resistant weed biotypes. We conclude the following. (1) The affinity of the secondary accepting quinone, B, is decreased in resistant thylakoids. (2) The observation that the equilibrium concentration of reduced Q after transferring one electron to the acceptor side of PS II is increased in resistant as compared to susceptible chloroplasts may be explained both by a decrease in the affinity of PQ for the herbicide / quinone binding environment, and by a decrease of the midpont redox potential of the B / B⁻ couple. (3) The binding environment regulating quinone and herbicide affinity may be divided roughly into two domains; we suggest that the domain regulating quinone head-group binding is little changed in resistant membranes, whereas the domain-regulating quinone side-group binding (and atrazine) is altered. This results in increased inhibitory activity of tetrachloro-p-benzoquinone and phenolic herbicides, which are hypothesized to utilize the quinone head-group domain. The two domains appear to be spatially overlapping because efficient atrazine displacement by tetrachloro-p-benzoquinone is observed.     

Quality Control of Photosystem II: THYLAKOID UNSTACKING IS NECESSARY TO AVOID FURTHER DAMAGE TO THE D1 PROTEIN AND TO FACILITATE D1 DEGRADATION UNDER LIGHT STRESS IN SPINACH THYLAKOIDS*

Photosystem II is vulnerable to light damage. The reaction center-binding D1 protein is impaired during excessive illumination and is degraded and removed from photosystem II. Using isolated spinach thylakoids, we investigated the relationship between light-induced unstacking of thylakoids and damage to the D1 protein. Under light stress, thylakoids were expected to become unstacked so that the photodamaged photosystem II complexes in the grana and the proteases could move on the thylakoids for repair. Excessive light induced irreversible unstacking of thylakoids. By comparing the effects of light stress on stacked and unstacked thylakoids, photoinhibition of photosystem II was found to be more prominent in stacked thylakoids than in unstacked thylakoids. In accordance with this finding, EPR spin trapping measurements demonstrated higher production of hydroxyl radicals in stacked thylakoids than in unstacked thylakoids. We propose that unstacking of thylakoids has a crucial role in avoiding further damage to the D1 protein and facilitating degradation of the photodamaged D1 protein under light stress.In the chloroplasts of higher plants and green algae, thylakoid membranes are closely associated and stack to form grana. Under electron microscopy, cylindrical grana consisting of 10–20 layers of thylakoids have been observed. They have a diameter of 300–600 nm and are interconnected by lamellae of several hundred nm in length (1, 2). The structure of grana in the chloroplasts of higher plants is well known, but the precise role of grana is incompletely understood. Their possible functions in primary photochemical reactions and subsequent events have been discussed extensively (3–9). Photosystem I (PSI)³ and II (PSII) complexes are segregated from each other in thylakoids, showing lateral heterogeneity in their distribution. The PSII complex is a multisubunit pigment-protein complex responsible for the photochemical oxidation of water and reduction of plastoquinone (8, 10–13). It comprises >25 protein subunits and other low molecular weight cofactors, including chlorophylls, carotenoids, plastoquinones, and manganeses. In the chloroplasts of higher plants, PSII complexes and the associated light-harvesting antenna complex LHCII are not present throughout the thylakoid membranes but are abundant in the grana (2, 14). A densely packed array of PSII complexes in the grana was visualized by electron microscopy (8, 15). Grana formation is more prominent in shade leaves (or shade plants) than in sun leaves (or sun plants), so it has been suggested that enrichment of the PSII·LHCII complex in grana is a strategy of plants to collect excitation energy by PSII under weak light (16). The grana structure probably provides an organized environment for PSII. PSI and ATP synthase are located exclusively in the stroma-exposed thylakoids, including the stroma thylakoids, grana end membranes, and grana margins, because these complexes protrude into the stroma. Cytochrome b₆/f complexes without this protrusion are present uniformly throughout the thylakoids (3). It has been suggested that separation of PSI and PSII complexes on the thylakoids through grana formation is important to prevent “spillover” of excitation energy from PSII to PSI, which lowers photosynthesis efficiency (17).An active PSII complex comprises a homodimer of PSII monomers (13). When thylakoids are exposed to excessive visible light, the PSII dimer dissociates into two monomers (18), but the most significant change takes place inside the monomeric PSII, where the reaction center-binding D1 protein is photodamaged and degraded by specific proteases (19, 20). The photodamage to the D1 protein is a photooxidative process. This is caused by reactive oxygen species (ROS), most probably singlet oxygen (¹O₂) or the hydroxyl radical (HO^•) produced by overreduction of the acceptor side of PSII under excessive illumination or by endogenous cationic radicals, such as the oxidized forms of the primary electron donor P680 and the secondary electron donor Tyr_Z (Tyr¹⁶¹ of D1) to PSII (21). Strong illumination of the grana may readily cause damage to the PSII complexes by ROS and endogenous cationic radicals, because the grana is rich in PSII complexes. Segregation of PSI and PSII in the stacked thylakoids should make the electron transport between PSI and PSII a rate-limiting step in the electron flow, and overexcitation of PSII under these conditions may stimulate ROS production at the acceptor side of PSII. Close association of LHCII with the PSII core complexes should also stimulate ROS generation in the grana. Unstacking of the thylakoids, which is also expected to lead to random distribution of PSI and PSII on the thylakoids and dissociation of the LHCII from the PSII core, may be important to avoid photodamage to PSII.In the proteolysis of the damaged D1 protein in the chloroplasts of higher plants, the N-terminal Thr of the D1 protein is dephosphorylated, and the subsequent degradation produces 23- and 9-kDa fragments as the primary cleavage products (19, 20). The protease(s) and phosphatase(s) involved in these steps are presumably localized in the stroma thylakoids, grana end membranes, and grana margin. Lateral migration of the damaged PSII complexes from the grana to the membrane regions where the damaged PSII complexes are repaired is therefore important for degradation of the D1 protein. Thylakoid unstacking, if it occurs under light stress, should stimulate diffusion of the protein complexes on the thylakoids, thereby stimulating D1 turnover.First, we examined if excessive visible light can induce unstacking of the thylakoids. Second, we studied the effects of strong illumination on stacked and unstacked thylakoids to see if they showed different responses to excessive light. We strongly suggest that unstacking of the thylakoids caused by light stress is necessary to avoid further photodamage to the D1 protein and to facilitate degradation and removal of the photodamaged D1 protein from PSII complexes.     

Modulation of photosynthetic electron transport in the absence of terminal electron acceptors: characterization of the rbcL deletion mutant of tobacco

Tobacco rbcL deletion mutant, which lacks the key enzyme Rubisco for photosynthetic carbon assimilation, was characterized with respect to thylakoid functional properties and protein composition. The Delta rbcL plants showed an enhanced capacity for dissipation of light energy by non-photochemical quenching which was accompanied by low photochemical quenching and low overall photosynthetic electron transport rate. Flash-induced fluorescence relaxation and thermoluminescence measurements revealed a slow electron transfer and decreased redox gap between Q(A) and Q(B), whereas the donor side function of the Photosystem II (PSII) complex was not affected. The 77 K fluorescence emission spectrum of Delta rbcL plant thylakoids implied a presence of free light harvesting complexes. Mutant plants also had a low amount of photooxidisible P700 and an increased ratio of PSII to Photosystem I (PSI). On the other hand, an elevated level of plastid terminal oxidase and the lack of F0 'dark rise' in fluorescence measurements suggest an enhanced plastid terminal oxidase-mediated electron flow to O2 in Delta rbcL thylakoids. Modified electron transfer routes together with flexible dissipation of excitation energy through PSII probably have a crucial role in protection of PSI from irreversible protein damage in the Delta rbcL mutant under growth conditions. This protective capacity was rapidly exceeded in Delta rbcL mutant when the light level was elevated resulting in severe degradation of PSI complexes.     

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

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

Targeted mutagenesis of the psbE and psbF genes blocks photosynthetic electron transport: evidence for a functional role of cytochrome b559 in photosystem II.

The genes encoding the two subunits (alpha and beta) of the cytochrome b559 (cyt b559) protein, psbE and psbF, were cloned from the unicellular, transformable cyanobacterium, Synechocystis 6803. Cyt b559, an intrinsic membrane protein, is a component of photosystem II, a membrane-protein complex that catalyzes photosynthetic oxygen evolution. However, the role of cyt b559 in photosynthetic electron transport is yet to be determined. A high degree of homology was found between the cyanobacterial and green plant chloroplastidic psbE and psbE genes and in the amino acid sequences of their corresponding protein products. Cartridge mutagenesis techniques were used to generate a deletion mutant of Synechocystis 6803 in which the psbE and psbF genes were replaced by a kanamycin-resistance gene cartridge. Physiological analyses indicated that the PSII complexes of the mutant were inactivated. We conclude that cyt b559 is an essential component of PSII.     

Further identification of the exoplasmic face particles on the freeze-fractured thylakoid membranes: a study using double and triple mutants from Chlamydomonas reinhardtii lacking various photosystem II subunits and the cytochrome b6/f complex.

About 20% of the exoplasmic face (EF) particles present in the freeze-fractured thylakoid membranes of the wild type strain of Chlamydomonas reinhardtii remain in mutants lacking photosystem II (PSII) because of the absence of either one of the two PSII subcomplexes CP43 or D1/D2/CP47. We show that about half of these residual EF particles can be accounted for by PSII subcomplexes still present in such mutants, and by cytochrome (cyt) b6/f complexes. Analysis of double mutants lacking both types of protein complexes points to an association of cyt b6/f complexes with PSII subcomplexes in some of these EF particles and to a requirement in cyt b6/f complexes for the translocation of each of the two PSII subcomplexes (the CP43 subunit and the D1/D2/CP47 subcomplex) from the unstacked to the stacked regions of the thylakoid membranes.