Role of novel dimeric Photosystem II (PSII)-Psb27 protein complex in PSII repair

The multisubunit membrane protein complex Photosystem II (PSII) catalyzes one of the key reactions in photosynthesis: the light-driven oxidation of water. Here, we focus on the role of the Psb27 assembly factor, which is involved in biogenesis and repair after light-induced damage of the complex. We show that Psb27 is essential for the survival of cyanobacterial cells grown under stress conditions. The combination of cold stress (30 °C) and high light stress (1000 μmol of photons × m(-2) × s(-1)) led to complete inhibition of growth in a Δpsb27 mutant strain of the thermophilic cyanobacterium Thermosynechococcus elongatus, whereas wild-type cells continued to grow. Moreover, Psb27-containing PSII complexes became the predominant PSII species in preparations from wild-type cells grown under cold stress. Two different PSII-Psb27 complexes were isolated and characterized in this study. The first complex represents the known monomeric PSII-Psb27 species, which is involved in the assembly of PSII. Additionally, a novel dimeric PSII-Psb27 complex could be allocated in the repair cycle, i.e. in processes after inactivation of PSII, by (15)N pulse-label experiments followed by mass spectrometry analysis. Comparison with the corresponding PSII species from Δpsb27 mutant cells showed that Psb27 prevented the release of manganese from the previously inactivated complex. These results indicate a more complex role of the Psb27 protein within the life cycle of PSII, especially under stress conditions.     

Cyclic electron flow within PSII protects PSII from its photoinhibition in thylakoid membranes from spinach chloroplasts

Cyclic electron flow within PSII (CEF-PSII) was proven to alleviate the photoinhibition of PSII. We set the conditions where CEF-PSII functioned or did not, by adding nigericin to the reaction mixture for the dissipation of DeltapH across thylakoid membranes, and then the thylakoids were illuminated. When CEF-PSII did not function and the activity of linear electron flow (LEF) was low, light-treated thylakoid membranes largely lost the activity of LEF. The inactivation of LEF was due to the loss of the activity of PSII, but not that of PSI. The inactivation of PSII was suppressed, when CEF-PSII functioned or LEF was enhanced. These results imply that CEF-PSII contributes to the protection of PSII from its photoinhibition with LEF, as an electron sink.     

Damage of PSII by Sulfite

The photoreduction of DCIP by PSⅡ pasticles isolated from spinach leaves was inhibited by sulfite and the degree of inhibition was increased with the increase of sulfite concentration. The site of sulfite damage was on the oxidation side. In dark, electron flow from H2O to DCIP and from DPC to DCIP was not affected by sulfite. With certain concentrations of sulfite, the damage to PSⅡ particle varied with time of sulfite treatment and the mechanism of the damage might be related to the discretion of 33 kD polypeptide from thylakoid membrane and the leakage of Mn. Sulfite did not specifically damage the newly prepared thyla- koid, but this was the case with aged thylakoid. The rate of DCIP photoreduction decreased as the aging process was prolonged. Decrease in Mn content correlated with the decrease of DCIP photoreduction. Especially in the presence of EDTA, with the decrease of Mn, the rate of electron transport was severely reduced.     

亚硫酸对PSII的伤害

从菠菜叶中提取 PSII 颗粒和叶绿体、经亚硫酸处理后发现:由 PSII 颗粒催化的 DCIP光还原速率依 SO_3~(2-)浓度增高而降低、伤害部位发生于 PSII 的氧化侧,接近水的部位。在黑暗条件下 H_2O→DCIP 和 DPC→DCIP 的电子传递均不受影响。在特定 SO_3~(2-)浓度下,PSII 颗粒的伤害随处理时间的延长而加重,其伤害机理与33kD 多肽的解离和 Mn 的流失有关。SO_3~(2-)对新鲜叶绿体并不伤害;对老化的叶绿体则伤害明显,DCIP 光还原速率依老化时间的延长而降低。Mn 含量的减少与 DCIP 光还原速率的降低呈正相关,试样中添加 EGTA后电子传递速率受害更为严重。     

The PSII calcium site revisited

Oxidation of H₂O by photosystem II is a unique redox reaction in that it requires Ca²⁺ as well as Cl⁻ as obligatory activators/cofactors of the reaction, which is catalyzed by Mn atoms. The properties of the binding site for Ca²⁺ in this reaction resemble those of other Ca²⁺ binding proteins, and recent X-ray structural data confirm that the metal is in fact ligated at least in part by amino acid side chain oxo anions. Removal of Ca²⁺ blocks water oxidation chemistry at an early stage in the cycle of redox reactions that result in O-O bond formation, and the intimate involvement of Ca²⁺ in this reaction that is implied by this result is confirmed by an ever-improving set of crystal structures of the cyanobacterial enzyme. Here, we revisit the photosystem II Ca²⁺ site, in part to discuss the additional information that has appeared since our earlier review of this subject (van Gorkom HJ, Yocum CF In: Wydrzynski TJ, Satoh K (eds) Photosystem II: the light-driven water:plastoquinone oxidoreductase), and also to reexamine earlier data, which lead us to conclude that all S-state transitions require Ca²⁺.     

The mechanism for dioxygen formation in PSII studied by quantum chemical methods.

The availability of an X-ray structure for PSII including the water-oxidizing cluster, where the metal atoms and the amino acids are assigned, has opened up new possibilities to study the mechanism for dioxygen formation. In the present paper the main results of an ongoing hybrid DFT study are presented. The model used follows the structure suggested by the X-ray analysis as closely as possible. After nearly one thousand optimizations of different structures, each one with about 70 atoms, the main features of a water oxidizing mechanism start to emerge. The key intermediate is an oxyl radical state in S(3), stabilized by a weak trans effect to a bridging oxo in the cube. To reach this radical state a structural rearrangement appears necessary, in which one additional bridging oxo is formed between the dangling manganese and a manganese in the cube. The calculated energetics is reasonable but still not fully consistent with a correct mechanism. It is suggested that some part of the structure is not correct, probably the presence of the bicarbonate.     

Cyclic flow of electrons within PSII in thylakoid membranes

In photosynthesis, the electrons released from PSII are considered to be shared mainly by carbon metabolism and the water-water cycle. We demonstrated previously that some electrons are utilized in a CO2- and O2-independent manner in leaves of wild watermelon [Miyake and Yokota (2000) Plant Cell Physiol: 41: 335]. In the present study, we examined the mechanism of this alternative flow of electrons in thylakoid membranes, isolated from fresh spinach leaves, by simultaneously measuring the quantum yield of PSII and the flux of the linear flow of electrons. In the presence of the protonophore nigericin, which eliminates the pH gradient across thylakoid membranes, the quantum yield and the flux of the linear flow of electrons were directly proportional to one another. The quantum yield at a given linear flux of electrons was much higher in the absence of nigericin than in its presence, indicating that an additional or alternative flow of electrons can occur independently of the linear flow in the absence of nigericin. In the presence of nigericin, the alternative flux of electrons increased with decreasing pH and with increasing reduction of the plastoquinone pool. Cyclic flow of electrons in PSII appears to be the most plausible candidate for the alternative flow of electrons. The flux reached 280 micromol x e(-) (mg Chl)(-1) x h(-1) and was similar to that of the CO2- and O2-independent alternative flow of electrons that we found in leaves of wild watermelon. The cyclic, alternative flow of electrons in PSII provides a possible explanation for the alternative flow of electrons observed in vivo.     

Thylakoid Membrane Maturation and PSII Activation Are Linked in Greening Synechocystis sp. PCC 6803 Cells

Thylakoid membranes are typical and essential features of both chloroplasts and cyanobacteria. While they are crucial for phototrophic growth of cyanobacterial cells, biogenesis of thylakoid membranes is not well understood yet. Dark-grown Synechocystis sp. PCC 6803 cells contain only rudimentary thylakoid membranes but still a relatively high amount of phycobilisomes, inactive photosystem II and active photosystem I centers. After shifting dark-grown Synechocystis sp. PCC 6803 cells into the light, “greening” of Synechocystis sp. PCC 6803 cells, i.e. thylakoid membrane formation and recovery of photosynthetic electron transport reactions, was monitored. Complete restoration of a typical thylakoid membrane system was observed within 24 hours after an initial lag phase of 6 to 8 hours. Furthermore, activation of photosystem II complexes and restoration of a functional photosynthetic electron transport chain appears to be linked to the biogenesis of organized thylakoid membrane pairs.Thylakoid membranes are typical and essential features of both chloroplasts and cyanobacteria. The intracellular thylakoid membranes of cyanobacteria harbor the protein complexes of the photosynthetic electron transport chain (Nowaczyk et al., 2010; Bernat and Rögner, 2011). The photosynthetic electron transport chain is composed of three large membrane protein complexes, i.e. PSII, the cytochrome b₆f complex, and PSI. Excitation energy trapping by PSII results in water splitting at the PSII donor side within the thylakoid lumen and transport of electrons to the primary and secondary electron accepting quinone molecules Q_A and Q_B, respectively. Following double reduction and protonation, Q_B is released from PSII into the plastoquinone (PQ) pool and delivers electrons to the cytochrome b₆f complex. The cytochrome b₆f complex transfers the electrons to the soluble electron carrier plastocyanin or cytochrome c₆, which subsequently reduces PSI. For efficient light harvesting, cyanobacteria contain soluble light-harvesting antenna proteins, the phycobilisomes (PBSs), which transfer light energy to the photosynthetic reaction centers. In cyanobacteria, the PSI-to-PSII ratio is controlled by light and by the redox state of the PQ/PQH₂-pool (Fujita et al., 1987), and under high-light growth conditions, typically less PSI is present in cyanobacterial thylakoid membranes compared with low-light conditions (Fujita et al., 1994).Thylakoid membranes and photosynthetic electron transport are essential for phototrophic growth of cyanobacterial cells. Despite their importance for survival of cyanobacteria, biogenesis of thylakoid membranes is yet not well understood. It still is an ongoing debate whether the internal membrane systems (cytoplasmic and thylakoid membranes) are connected in cyanobacteria or not, and thus whether thylakoids represent a completely separated membrane entity (Liberton et al., 2006; van de Meene et al., 2006, 2012; Schneider et al., 2007). Up to now, only few proteins have been described to be involved in thylakoid membrane biogenesis. Among them the Vipp1 protein (vesicle inducing protein in plastids1) seems to play an important role in thylakoid membrane biogenesis, as in chloroplasts of Arabidopsis (Arabidopsis thaliana) and in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis), depletion of Vipp1 results in a reduced thylakoid membrane system (Kroll et al., 2001; Westphal et al., 2001). While the exact physiological function of the protein is not yet known (Vothknecht et al., 2012), depletion of Vipp1 in Synechocystis not only results in reduced thylakoid membrane formation, but also affects the activity and structure of components of the photosynthetic electron transport chain (Fuhrmann et al., 2009; Gao and Xu, 2009).As complexes of the respiratory electron transport chain are also localized in cyanobacterial thylakoids, the photosynthetic and respiratory electron transport pathways are highly interconnected and both contribute to formation of an electrochemical gradient across the thylakoid membrane and energy production. Due to this, Synechocystis is able to grow completely heterotrophically under light-activated photoheterotrophic growth (LAHG) conditions in the presence of high Glc concentrations (Anderson and McIntosh, 1991; Smart et al., 1991).In this study, we have used dark-grown Synechocystis cells to investigate “greening” of Synechocystis cells, i.e. thylakoid membrane formation and recovery of photosynthetic electron transport reactions. Following transfer of Synechocystis cells into the light, complete restoration of a typical thylakoid membrane system was observed within 24 h. While dark-grown Synechocystis cells contained only rudimentary thylakoid membranes, they still contained a high concentration of PBSs, active PSI as well as inactive PSII complexes. Activation of PSII complexes appears to be linked to the biogenesis of organized thylakoid membrane pairs.     

Characterization of PSII photochemistry and thermostability in salt-treated Rumex leaves

A study was conducted, using chlorophyll fluorescence, rapid fluorescence induction kinetics, and polyphasic fluorescence transients, to determine the effect of salt treatment and heat stress on PSII photochemistry in Rumex leaves. Salt treatment was accomplished by adding NaCl solutions of different concentrations ranging from 50 to 200 mmol/L. Heat stress was induced by exposing the plant leaves to temperatures ranging from 29 to 47 degrees C. The control plants were grown without NaCl treatment. The data acquired in this study showed that NaCl treatment alone had no effect on the maximal photochemistry of PSH or the polyphasic rise of chlorophyll fluorescence. However, the NaCl treatment modified heat stress on PSII photochemistry in Rumex leaves, which was manifested by a lesser heat-induced decrease in photochemical quenching (qP), efficiency of excitation energy capture by open PSII reaction centers (Fv'/Fm'), and quantum yield of PSII electron transport (phiPSII). The data also showed that NaCl treatment compromised the impact of heat stress on the capacity of transferring electrons from Q(A)- to Q(B). Furthermore, the NaCl treatment promoted heat resistance of O2-evolving complex (OEC). In summary, NaCl treatment enhanced the thermostability of PSII.     

The chloroplast gene ycf9 encodes a photosystem II (PSII) core subunit, PsbZ, that participates in PSII supramolecular architecture

We have characterized the biochemical nature and the function of PsbZ, the protein product of a ubiquitous open reading frame, which is known as ycf9 in Chlamydomonas and ORF 62 in tobacco, that is present in chloroplast and cyanobacterial genomes. After raising specific antibodies to PsbZ from Chlamydomonas and tobacco, we demonstrated that it is a bona fide photosystem II (PSII) subunit. PsbZ copurifies with PSII cores in Chlamydomonas as well as in tobacco. Accordingly, PSII mutants from Chlamydomonas and tobacco are deficient in PsbZ. Using psbZ-targeted gene inactivation in tobacco and Chlamydomonas, we show that this protein controls the interaction of PSII cores with the light-harvesting antenna; in particular, PSII-LHCII supercomplexes no longer could be isolated from PsbZ-deficient tobacco plants. The content of the minor chlorophyll binding protein CP26, and to a lesser extent that of CP29, also was altered substantially under most growth conditions in the tobacco mutant and in Chlamydomonas mutant cells grown under photoautotrophic conditions. These PsbZ-dependent changes in the supramolecular organization of the PSII cores with their peripheral antennas cause two distinct phenotypes in tobacco and are accompanied by considerable modifications in (1) the pattern of protein phosphorylation within PSII units, (2) the deepoxidation of xanthophylls, and (3) the kinetics and amplitude of nonphotochemical quenching. The role of PsbZ in excitation energy dissipation within PSII is discussed in light of its proximity to CP43, in agreement with the most recent structural data on PSII.     

Reaction System for Violaxanthin De-Epoxidase with PSII Membranes

PSII membranes were used as a substrate for violaxanthin de-epoxidase(VDE) that had been solubilized from spinach thylakoids by sonication.Inclusion of Tween 20 in the assay mixture was essential, althoughthe detergent apparently inhibited the activity in the conventionalassay with purified violaxanthin and lipid as substrate. Themaximum enhancing effect of the detergent was observed nearits critical micellar concentration. It is likely that the monomerof the detergent helped VDE react with the substrate in themembranes. Dependence of the activity on the substrate concentrationsuggested that VDE functions at least at two sites in the membranes,probably on both their lumenal and stromal surfaces. The abilityof the enzyme to function on the stromal surface in in vitroassays was demonstrated by using intact thylakoids as the substrate.Under such conditions where the endogenous VDE was functioningin the lumen, the exogenously added VDE converted an-theraxanthinto zeaxanthin in the absence of Tween 20. This result suggeststhat, in the reaction with PSII membranes, the detergent wasrequired for VDE to react with violaxanthin but not with antheraxanthin.Otherwise, the detergent was necessary for the reaction on thelumenal surface. (Received September 5, 1997; Accepted October 19, 1997)     

S.stapfianus and E. curvula cv. Consol in vivo photosynthesis, PSII activity and ABA content during dehydration

Changes in photosynthetic activity, CO₂ assimilation rate, _PSII by fluorescence andABA content, were monitored in the grasses Eragrostis curvula cv. Consol and Sporobolusstapfianus Gandoger in response to dehydration. Thefirst being a warm season grass well adapted todrought and the second a desiccation-tolerant orresurrection plant. The trial was performed on intactleaves during a whole plant drying course. After acycle of dehydration (down to 5% RWC) andrehydration to full turgor the resurrection plantshowed recovery of photosynthetic capability. E.curvula is drought-resistant but notdrought-tolerant being not capable of recovering whendried to 20% RWC. The sensitivity of photosynthesisto the drying treatment was different in E.curvula and S. stapfianus. During dryingtreatment, up to a leaf water loss of 40%, E.curvula photosynthesis seemed to be inhibited bycarbon metabolism, because PSII activity was not yetaffected. In S. stapfianus at the same point ofdehydration photosynthesis still worked though adown-regulation of PSII activity (Fv/Fm) occurred ata higher RWC. Non-photochemical chlorophyllfluorescence quenching (qN) was analysed. Duringdrying qN increased in both plants, but more in theresurrection plant though its assimilation rate wasless affected. The importance of ABA in regulating CO₂ assimilation rate is discussed.     

PSII photochemistry and carboxylation efficiency in Liriodendron tulipifera under ozone exposure

Liriodendron tulipifera is an important forest plant which is commonly used in urban environments as a shade tree. Young plants have been exposed (under controlled conditions) to 120 ppb of O₃ for 45 consecutive days (5 h d⁻¹). The aim of this investigation was to clarify if O₃ limits the physiological performance of L. tulipifera. In treated plants, dynamics related to membrane injury, gas exchange and chlorophyll a fluorescence leads to: (i) increase in lipid peroxidation (maximum value of +78% 15 days after the fumigation, compared to controls); (ii) reduction of photosynthetic activity (up to 66% 28 days after the exposure), twinned with a partial stomatal closure and a store of CO₂ in substomatal chambers; (iii) reduction in carboxylation efficiency (−11% at the end of exposure); (iv) damage to PSII, as demonstrated by the increase in the PSII excitation pressure (−57% 28 days after the treatment). On this basis, O₃ should be considered very harmful to L. tulipifera, although the reduction of total chlorophylls content and the activation of xanthophyll cycle take place in order to attempt to regulate light absorbed energy limiting oxidative damage.     

Acclimation to temperature and irradiance modulates PSII charge recombination

Acclimation of wild type and the chlorina F2 mutant of barley to either high light or low temperature results in a 2- to 3-fold increase in non-photochemical quenching which occurred independently of either energy-dependent quenching (qE), xanthophyll cycle-mediated antenna quenching or state transitions. Results of in vivo thermoluminescence measurements used to address this conundrum indicated that excitation pressure regulates the temperature gap for S(2)Q(B)(-) and S(2)Q(A)(-) charge recombinations within photosystem II reaction centers. This is discussed in terms of photoprotection through non-radiative charge recombination.     

Time-resolved oxygen production by PSII: chasing chemical intermediates

Photosystem II (PSII) produces dioxygen from water in a four-stepped process, which is driven by four quanta of light and catalysed by a Mn-cluster and tyrosine Z. Oxygen is liberated during one step, coined S(3)=>S(0). Chemical intermediates on the way from reversibly bound water to dioxygen have not yet been tracked, however, a break in the Arrhenius plot of the oxygen-evolving step has been taken as evidence for its existence. We scrutinised the temperature dependence of (i) UV-absorption transients attributable to the reduction of the Mn-cluster and tyrosine Z by water, and (ii) polarographic transients attributable to the release of dioxygen. Using a centrifugatable and kinetically competent Pt-electrode, we observed no deviation from a linear Arrhenius plot of oxygen release in the temperature range from -2 to 32 degrees C, and hence no evidence, by this approach, for a sufficiently long-lived chemical intermediate. The half-rise times of oxygen release differed between Synechocystis WT* (at 20 degrees C: 1.35 ms) and a point mutant (D1-D61N: 13.1 ms), and the activation energies differed between species (Spinacia oleracea, 30 kJ/mol versus Synechocystis, 41 kJ/mol) and preparations (PSII membranes, 41 kJ/mol versus core complexes, 33 kJ/mol, Synechocystis). Correction for polarographic artefacts revealed, for the first time, a temperature-dependent lag-phase of the polarographic transient (duration at 20 degrees C: 0.45 ms, activation energy: 31 kJ/mol), which was indicative of a short-lived intermediate. It was, however, not apparent in the UV-transients. Thus the "intermediate" was probably newly formed and transiently bound oxygen.     

毛尖紫萼藓(Grimmia pilifera P.Beauv) PSII光化学效率对脱水和复水的响应

利用快速叶绿素荧光动力学技术研究了毛尖紫萼藓脱水和复水过程中叶绿素荧光变化,结果显示在脱水过程中毛尖紫萼藓PSⅡ的最大光化学效率（Fv/Fm）、光合机构电子传递的量子产额（ETo／ABS）、捕获的激子将电子传递到电子传递链中超过QA的其它电子受体的概率（ETo／TRo）、单位叶面积的反应中心的数量（RC／CSo）以及PSⅡ受体库（Area）对叶片含水量的响应等均存在相对含水量阈值。在阈值范围内脱水,对以上荧光参数影响不大,低于阈值后,各荧光参数值迅速下降,直至PSⅡ反应中心完全关闭以及光化学过程结束。再复水后,毛尖紫萼藓光合机构的最大捕光效率、实际光化学效率、PSⅡ反应中心受体侧的电子传递链以及反应中心均能得到快速而有效的恢复。表明一定时间内脱水不会对毛尖紫萼藓的光合器官造成严重伤害,光合系统仍维持在可恢复状态。     

Production of reactive oxygen species in decoupled, Ca2+-depleted PSII and their use in assigning a function to chloride on both sides of PSII

Extraction of Ca²⁺ from the oxygen-evolving complex of photosystem II (PSII) in the absence of a chelator inhibits O₂ evolution without significant inhibition of the light-dependent reduction of the exogenous electron acceptor, 2,6-dichlorophenolindophenol (DCPIP) on the reducing side of PSII. The phenomenon is known as “the decoupling effect” (Semin et al. Photosynth Res 98:235–249, 2008). Extraction of Cl^? from Ca²⁺-depleted membranes (PSII[–Ca]) suppresses the reduction of DCPIP. In the current study we investigated the nature of the oxidized substrate and the nature of the product(s) of the substrate oxidation. After elimination of all other possible donors, water was identified as the substrate. Generation of reactive oxygen species HO, H₂O₂, and O ₂ ^·? , as possible products of water oxidation in PSII(–Ca) membranes was examined. During the investigation of O ₂ ^·? production in PSII(–Ca) samples, we found that (i) O ₂ ^·? is formed on the acceptor side of PSII due to the reduction of O₂; (ii) depletion of Cl^? does not inhibit water oxidation, but (iii) Cl^? depletion does decrease the efficiency of the reduction of exogenous electron acceptors. In the absence of Cl^? under aerobic conditions, electron transport is diverted from reducing exogenous acceptors to reducing O₂, thereby increasing the rate of O ₂ ^·? generation. From these observations we conclude that the product of water oxidation is H₂O₂ and that Cl^? anions are not involved in the oxidation of water to H₂O₂ in decoupled PSII(–Ca) membranes. These results also indicate that Cl^? anions are not directly involved in water oxidation by the Mn cluster in the native PSII membranes, but possibly provide access for H₂O molecules to the Mn₄CaO₅ cluster and/or facilitate the release of H⁺ ions into the lumenal space.