Kinetics of photosystem II electron transport: a mathematical analysis based on chlorophyll fluorescence induction

The OJDIP rise in chlorophyll fluorescence during induction at different light intensities was mathematically modeled using 24 master equations describing electron transport through photosystem II (PSII) plus ordinary differential equations for electron budgets in plastoquinone, cytochrome f, plastocyanin, photosystem I, and ferredoxin. A novel feature of the model is consideration of electron in- and outflow budgets resulting in changes in redox states of Tyrosine Z, P680, and Q_A as sole bases for changes in fluorescence yield during the transient. Ad hoc contributions by transmembrane electric fields, protein conformational changes, or other putative quenching species were unnecessary to account for primary features of the phenomenon, except a peculiar slowdown of intra-PSII electron transport during induction at low light intensities. The lower than F _m post-flash fluorescence yield F _f was related to oxidized tyrosine Z. The transient J peak was associated with equal rates of electron arrival to and departure from Q_A and requires that electron transfer from Q_A ^? to Q_B be slower than that from Q_A ^? to Q_B ^?. Strong quenching by oxidized P680 caused the dip D. Reduced plastoquinone, a competitive product inhibitor of PSII, blocked electron transport proportionally with its concentration. Electron transport rate indicated by fluorescence quenching was faster than the rate indicated by O₂ evolution, because oxidized donor side carriers quench fluorescence but do not transport electrons. The thermal phase of the fluorescence rise beyond the J phase was caused by a progressive increase in the fraction of PSII with reduced Q_A and reduced donor side.     

pH-dependent photoreactions of the high- and low-potential forms of cytochrome b559 in spinach PS II-enriched membranes

Cytochrome b559 (Cyt b559) is a well-known intrinsic component of Photosystem II (PS II) reaction center in all photosynthetic oxygen-evolving organisms, but its physiological role remains unclear. This work reports the response of the two redox forms of Cyt b559 (i.e. the high- (HP) and low-potential (LP) forms) to inhibition of the donor or acceptor side of PS II. The photooxidation of HP Cyt b559 induced by red light at room temperature was pH-dependent under conditions in which electron flow from water was diminished. This photooxidation was observed only at pH values higher than 7.5. However, in the presence of 1 M CCCP, a limited oxidation of HP Cyt b559 was observed at acidic pH, At pH 8.5 and in the presence of the protonophore, this photooxidation of the HP form was accompanied by its partial transformation into the LP form. On the other hand, a partial photoreduction of LP Cyt b559 was induced by red light under aerobic conditions when electron transfer through the primary quinone acceptor Q_A was impaired by strong irradiation in the presence of DCMU. This photoreduction was enhanced at acidic pH values. To the best of our knowledge, this is the first time that both photoreduction and photooxidation of Cyt b559 is described under inhibitory conditions using the same kind of membrane preparations. A model accommodating these findings is proposed.Abbreviations CCCP carbonylcyanide 3-chlorophenylhydrazone - Cyt cytochrome - DCBQ 2,5-dichloro-p-benzoquinone - DCMU dichlorophenyldimethylurea - E _m midpoint redox potential - HP and LP high- and low-potential forms of Cyt b559 - P680 primary donor - I_A acceptor side inhibition - I_D donor side inhibition - Pheo pheophytin - PS II photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

Alteration in chloroplast structure and thylakoid membrane composition due to in vivo heat treatment of rice seedlings: correlation with the functional changes

Exposure of 25 °C-grown, seven-day-old rice seedlings to mild heat stress of 40 °C for 24 h in dark did not cause any change in protein or pigment content of the thylakoids, but produced major disorganization of chloroplast ultrastructure. This heat induced disorganization of thylakoid structure/organization caused significant (65 percnt;) loss in PSII activity, slight loss in PSI activity, and brought about a decrease in relative quantum efficiency of PSII. The herbicide ¹⁴C atrazine binding assay revealed a decreased number of binding sites of the herbicide and altered the herbicide dissociation constant, suggesting that the heat induced disorganization of the thylakoids affects the acceptor side of PSII. Cation induced Chla fluorescence analyses at room temperature and low temperature indicated thatin vivo heat exposure of rice seedlings altered the extent of energy transfer in favor of PSI. Immunoblotting analysis of several PSII polypeptides such as D1/D2 reaction dimer and Cyt b₅₅₉ showed no major changes due to mild heat exposure except for the PSII core antenna polypeptide (CP43), which could reflect the reduction in PSII activity observed in light saturation studies. Similarly, haeme staining did not indicate any change in other cytochrome related polypeptides. Our results therefore clearly suggest thatin vivo exposure of rice seedlings to elevated (40 °C) temperature caused thylakoid structural disorganization, and this disorganization of some of the thylakoid complexes resulted in a loss in thylakoid photochemical function.     

‘Birth defects’ of photosystem II make it highly susceptible to photodamage during chloroplast biogenesis

High solar flux is known to diminish photosynthetic growth rates, reducing biomass productivity and lowering disease tolerance. Photosystem II (PSII) of plants is susceptible to photodamage (also known as photoinactivation) in strong light, resulting in severe loss of water oxidation capacity and destruction of the water‐oxidizing complex (WOC). The repair of damaged PSIIs comes at a high energy cost and requires de novo biosynthesis of damaged PSII subunits, reassembly of the WOC inorganic cofactors and membrane remodeling. Employing membrane‐inlet mass spectrometry and O₂‐polarography under flashing light conditions, we demonstrate that newly synthesized PSII complexes are far more susceptible to photodamage than are mature PSII complexes. We examined these ‘PSII birth defects’ in barley seedlings and plastids (etiochloroplasts and chloroplasts) isolated at various times during de‐etiolation as chloroplast development begins and matures in synchronization with thylakoid membrane biogenesis and grana membrane formation. We show that the degree of PSII photodamage decreases simultaneously with biogenesis of the PSII turnover efficiency measured by O₂‐polarography, and with grana membrane stacking, as determined by electron microscopy. Our data from fluorescence, Q_B‐inhibitor binding, and thermoluminescence studies indicate that the decline of the high‐light susceptibility of PSII to photodamage is coincident with appearance of electron transfer capability Q_A^? → Q_B during de‐etiolation. This rate depends in turn on the downstream clearing of electrons upon buildup of the complete linear electron transfer chain and the formation of stacked grana membranes capable of longer‐range energy transfer.     

Heterogeneity in chloroplast photosystem II

Photosystem-two (PSII) in the chloroplasts of higher plants and green algae is not homogeneous. A review of PSII heterogeneity is presented and a model is proposed which is consistent with much of the data presented in the literature. It is proposed that the non-quinone electron acceptor of PSII is preferentially associated with the sub-population of PSII known as PSIIß.Abbreviations and symbols ATP Adenosine triphosphate - Chl Chlorophyll - C₅₅₀ Absorbance bandshift at 550 nm; proportional to [Q_A ^-] - D, D Components involved ir electron donation to P680 - pH Transthylakoid proton gradient - Transthylakoid electrical gradient - DCMU 3-(3,4-Dichlorophenyl)-1,1-dimethylurea referred to as diuron - E _h Oxidation-reduction potential - E _m Cxidation-reduction midpoint potential - EPR Electron paramagnetic resonance - Fm Fluorescence yield when all traps are closed - Fo Fluorescence yield when all traps are open - Fv Variable fluorescence equal to Fm-Fo - Fi Initial fluorescence yield, (usually equivalent to Fo in dark-adapted thylakoids) - Hepes 2-hydroxyethylpiperazine-N-2-ethane sulphonic acid - LHCP Light-harvesting chlorophyll a/b binding protein - PQ Plastoquinone - PSII Photosystem II - P680 Reaction centre chlorophyll of PSII - P₅₁₈ Absorbance bandshift at 518 nm, reflects asymmetric charge distribution across the thylakoid membrane - Q_A, _QH , Q₁ Primary stable plastoquinone electron acceptor of PSII; a quencher of fluorescence - Q _B , B, R Plastoquinone associated with the Q _B -protein, the two-electron gate - Q _D , Q₂, X _a Non-quinone electron acceptor of PSII - X₃₂₀ Absorbance bandshift at 320 nm; semiquinone anion indicator     

Endogenous Control of Photosynthetic Activity during Progressive Drought: Influence of Final Products of Photosynthesis

Photosynthetic activities and the redox states of photosystem I (PSI) and photosystem II (PSII) in intact leaves of cucumber plants (Cucumis sativus L.), as well as the sucrose and starch contents were examined under conditions of ongoing soil water deficit imposed by the cessation of watering. As the soil drought progressed, the maximum rate of photosynthetic CO₂ fixation was shown to decrease. These changes in the maximum photosynthetic rate occurred synchronously with changes in the maximum quantum yield of photosynthesis. Under soil water deficit, the reduced form of PSII primary acceptor Q _A was accumulated only at photon flux densities of about 100 mol/(m² s). At such photon flux densities, the changes in nonphotochemical quenching (qN) induced by soil water deficit were opposite to changes in photochemical quenching parameter (1 – qP). Irrespective of the duration of soil drought, the relationship between steady-state concentrations of photochemically inactive reaction centers of PSI and PSII (the fractions of P700 and Q _A in the oxidized and reduced state, respectively) was almost linear, which provides evidence for the concerted operation of both photosystems. The conditions of soil water deficit promoted sucrose accumulation in the source leaf, which was paralleled by a substantial decrease in the amount of starch in the same leaf. The highest content of sucrose in the leaf after a 7-day drought was correlated with the largest decrease in photosynthetic activity. It is concluded that the progressive drought triggers an endogenous mechanism that regulates photosynthesis through feedback relations, namely, the inhibition of photosynthesis by its end products.     

Antimycin A inhibits cytochrome b559-mediated cyclic electron flow within photosystem II

The light reactions of photosynthesis are known to comprise both linear and cyclic electron flow in order to convert light energy into chemical energy in the form of NADPH and ATP. Antimycin A (AA) has been proposed as an inhibitor of ferredoxin-dependent cyclic electron flow around photosystem I (CEF-PSI) in photosynthesis research. However, its precise inhibitory mechanism and target site had not been elucidated yet. Here we show that AA inhibits the cyclic (alternative) electron flow via cytochrome b₅₅₉ (Cyt b₅₅₉) within photosystem II (CEF-PSII). When AA was applied to thylakoid membranes isolated from spinach leaves, the high potential form of Cyt b₅₅₉, which was reduced in the dark, was transformed into the lower potential forms and readily oxidized by molecular oxygen. In the absence of AA, the reduced Cyt b₅₅₉ was oxidized by P680⁺ upon light illumination and re-reduced in the dark, mainly by the electron from the Q_B site on the acceptor side of PSII. In contrast, AA suppressed the oxidation of Cyt b₅₅₉ and induced its reduction under the illumination. This inhibition of Cyt b₅₅₉ oxidation by AA enhanced photoinhibition of PSII. Based on the above results, we propose caution regarding the use of AA for evaluating CEF-PSI per se and concurrently propose that AA provides for new insights into, and interpretations of, the physiological importance of Cyt b₅₅₉, rather than that of CEF-PSI in photosynthetic organisms.

     

Acclimation of barley to changes in light intensity: photosynthetic electron transport activity and components

Barley seedlings (Hordeum vulgare L. Boone) were grown at 20°C with 16 h/8 h light/dark cycle of either high (H) intensity (500 mole m^-2 s^-1) or low (L) intensity (55 mole m^-2 s^-1) white light. Plants were transferred from high to low (H L) and low to high (L H) light intensity at various times from 4 to 8 d after leaf emergence from the soil. Primary leaves were harvested at the beginning of the photoperiod. Thylakoid membranes were isolated from 3 cm apical segments and assayed for photosynthetic electron transport, Photosystem II (PS II) atrazine-binding sites (Q_B), cytochrome f(Cytf) and the P-700 reaction center of Photosystem I (PS I). Whole chain, PS I and PS II electron transport activities were higher in H than in L controls. Q_B and Cytf were elevated in H plants compared with L plants. The acclimation of H L plants to low light occurred slowly over a period of 7 days and resulted in decreased whole chain and PS II electron transport with variable effects on PS I activity. The decrease in electron transport of H L plants was associated with a decrease in both Q_B and Cytf. In L H plants, acclimation to high light occurred slowly over a period of 7 days with increased whole chain, PS I and PS II activities. The increase in L H electron transport was associated with increased levels of Q_B and Cytf. In contrast to the light intensity effects on Q_B levels, the P-700 content was similar in both control and transferred plants. Therefore, PS II/PS I ratios were dependent on light environment.Abbreviations Asc ascorbate - BQ 2,5-dimethyl-p-benzoquinone - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCIP 2,6-dichlorophenolindophenol - H control plants grown under high light intensity - H L plants transferred from high to low light intensity - L low control plants grown under low light intensity - L H plants transferred from low to high light intensity - MV methyl viologen - P-700 photoreaction center of Photosystem I - Q_B atrazine binding site - TMPD N,N,N,N-tetramethyl-p-phenylenediamine Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC. Paper No. 11990 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643, USA.     

Excitonic connectivity between photosystem II units: what is it, and how to measure it?

In photosynthetic organisms, light energy is absorbed by a complex network of chromophores embedded in light-harvesting antenna complexes. In photosystem II (PSII), the excitation energy from the antenna is transferred very efficiently to an active reaction center (RC) (i.e., with oxidized primary quinone acceptor Q _A), where the photochemistry begins, leading to O₂ evolution, and reduction of plastoquinones. A very small part of the excitation energy is dissipated as fluorescence and heat. Measurements on chlorophyll (Chl) fluorescence and oxygen have shown that a nonlinear (hyperbolic) relationship exists between the fluorescence yield (Φ _F ) (or the oxygen emission yield, $ \Phi _{{{\text{O}}_{2} }} $ ) and the fraction of closed PSII RCs (i.e., with reduced Q _A). This nonlinearity is assumed to be related to the transfer of the excitation energy from a closed PSII RC to an open (active) PSII RC, a process called PSII excitonic connectivity by Joliot and Joliot (CR Acad Sci Paris 258: 4622–4625, 1964). Different theoretical approaches of the PSII excitonic connectivity, and experimental methods used to measure it, are discussed in this review. In addition, we present alternative explanations of the observed sigmoidicity of the fluorescence induction and oxygen evolution curves.     

DAC Is Involved in the Accumulation of the Cytochrome b6/f Complex in Arabidopsis

The biogenesis and assembly of photosynthetic multisubunit protein complexes is assisted by a series of nucleus-encoded auxiliary protein factors. In this study, we characterize the dac mutant of Arabidopsis (Arabidopsis thaliana), which shows a severe defect in the accumulation of the cytochrome b₆/f complex, and provide evidence suggesting that the efficiency of cytochrome b₆/f complex assembly is affected in the mutant. DAC is a thylakoid membrane protein with two predicted transmembrane domains that is conserved from cyanobacteria to vascular plants. Yeast (Saccharomyces cerevisiae) two-hybrid and coimmunoprecipitation analyses revealed a specific interaction between DAC and PetD, a subunit of the cytochrome b₆/f complex. However, DAC was found not to be an intrinsic component of the cytochrome b₆/f complex. In vivo chloroplast protein labeling experiments showed that the labeling rates of the PetD and cytochrome f proteins were greatly reduced, whereas that of the cytochrome b₆ protein remained normal in the dac mutant. DAC appears to be a novel factor involved in the assembly/stabilization of the cytochrome b₆/f complex, possibly through interaction with the PetD protein.The cytochrome b₆/f (Cyt b6/f) complex is a multisubunit complex that resides in the thylakoid membrane and functions in linear and cyclic electron transport. In the linear process, the complex receives electrons from PSII and transfers them to PSI, a process that is accompanied by the generation of a proton gradient, which is essential for ATP synthesis (Mitchell, 1961; Saraste, 1999). The native form of this complex is present as a dimer with a mass of 310 kD that can be converted into a 140-kD monomer with increasing detergent concentrations (Huang et al., 1994; Breyton et al., 1997; Mosser et al., 1997; Baniulis et al., 2009). In higher plants, the Cyt b6/f monomer contains at least eight subunits: Cyt f, Cyt b₆, PetC, PetD, PetM, PetL, PetG, and PetN (Wollman, 2004). PetC and PetM are encoded by nuclear genes, whereas the others are encoded by plastid genes. It has been shown that PetG and PetN are necessary for complex stability in tobacco (Nicotiana tabacum; Schwenkert et al., 2007). By contrast, PetL is not required for the accumulation of other subunits of the Cyt b6/f complex, even though it is involved in the stability and formation of the functional dimer (Bendall et al., 1986; Schwenkert et al., 2007). Inactivation of PetC in Arabidopsis (Arabidopsis thaliana) resulted in significantly reduced amounts of Cyt b6/f subunits and completely blocked linear electron transport, indicating that PetC participates in the formation of the functionally assembled Cyt b6/f complex (Maiwald et al., 2003). In Synechocystis sp. PCC 6803, the PetM subunit has no essential role in Cyt b6/f complex electron transfer or accumulation; however, the absence of this subunit apparently affects the levels of other protein complexes involved in energy transduction (Schneider et al., 2001). In addition to the other proteins, FNR was identified as a subunit of the Cyt b6/f complex isolated from spinach (Spinacia oleracea) thylakoid membranes (Zhang et al., 2001).Previous research has revealed how the Cyt b6/f complex assembles into a functional dimer (Bendall et al., 1986; Lemaire et al., 1986; Kuras and Wollman, 1994). In the Cyt b6/f complex, Cyt b₆ and PetD form a mildly protease-resistant subcomplex that serves as a template for the assembly of Cyt f and PetG, producing a protease-resistant cytochrome moiety (Wollman, 2004). The PetC and PetL proteins then participate in the assembly of the functional dimer (Schwenkert et al., 2007). PetD becomes more unstable in the absence of Cyt b₆, and the synthesis of Cyt f is greatly reduced when either Cyt b₆ or PetD is inactivated, indicating that both Cyt b₆ and PetD are prerequisite for the synthesis of Cyt f (Kuras and Wollman, 1994). The reduced synthesis of Cyt f can be explained by the so-called CES (for controlled by epistasy of synthesis) mechanism. It is suggested that, in this mechanism, the synthesis rate of some chloroplast-encoded subunits of photosynthetic protein complexes is regulated by the availability of their assembly partners from the same complexes (Choquet et al., 2001). The mechanism of CES for Cyt f has been studied in detail in Chlamydomonas reinhardtii (Choquet et al., 1998; Choquet and Vallon, 2000). In it, the unassembled Cyt f inhibits its own translation through a negative feedback mechanism, and MCA1 and TCA1 have been demonstrated to be involved in the regulation of Cyt f synthesis (Boulouis et al., 2011).Many studies have focused on understanding the conversion of apocytochrome to holocytochrome via the covalent binding of heme in Cyt f and Cyt b₆ during the assembly of Cyt b6/f through the CCS and CCB pathways (Nakamoto et al., 2000; Wollman, 2004; de Vitry, 2011). The CCS pathway was originally discovered in the green alga C. reinhardtii through genetic studies of ccs mutants (for cytochrome c synthesis) that display a specific defect in membrane-bound Cyt f and soluble Cyt c₆, two thylakoid lumen-resident c-type cytochromes functioning in photosynthesis (Xie and Merchant, 1998). In the CCS pathway, six loci that include plastid ccsA and nuclear CCS1 to CCS5 have been found in C. reinhardtii (Xie and Merchant, 1998). In these mutants, the apocytochrome is normally synthesized, targeted, and processed, but heme attachment is perturbed. The CCB pathway is involved in the covalent attachment of heme c(i) to Cyt b₆ on the stromal side of the thylakoid membranes (Kuras et al., 2007). The ccb mutants show defects in the accumulation of subunits of the Cyt b6/f complex and covalent binding of heme to Cyt b₆ (Lyska et al., 2007; Lezhneva et al., 2008). However, heme binding is not a prerequisite for the assembly of Cyt b₆ into the Cyt b6/f complex, although the fully formed Cyt b6/f showed an increased sensitivity to protease (Saint-Marcoux et al., 2009).The assembly of the Cyt b6/f complex is a multistep process, and current studies have shown that the covalent binding of heme to Cyt f and Cyt b₆ is highly regulated. Thus, it is reasonable to speculate that, similar to the other photosynthetic protein complexes (Mulo et al., 2008; Nixon et al., 2010; Rochaix, 2011), the assembly of the Cyt b6/f complex is also assisted by many nucleus-encoded factors. In this study, we characterized an Arabidopsis protein, DAC (for defective accumulation of Cyt b6/f complex), that seems to be involved in the assembly of the Cyt b6/f complex. In addition, we provide evidence that DAC interacts directly with PetD before it assembles within the Cyt b6/f complex.     

PHOTOPHYSIOLOGICAL RESPONSES OF FRAGILARIOPSIS CYLINDRUS (BACILLARIOPHYCEAE) TO NITROGEN DEPLETION AT TWO TEMPERATURES1

The photosynthetic efficiency and photoprotective capacity of the sea‐ice diatom, Fragilariopsis cylindrus (Grunow) W. Krieg., grown in a matrix of nitrogen repletion and depletion at two different temperatures (?1°C and +6°C) was investigated. Temperature showed no significant effect on photosynthetic efficiency or photoprotection in F. cylindrus. Cultures under nitrogen depletion showed enhanced photoprotective capacity with an increase in nonphotochemical quenching (NPQ) when compared with nitrogen‐replete cultures. This phenomenon was achieved at no apparent cost to the photosynthetic efficiency of PSII (F_V/F_M). Nitrogen depletion yielded a partially reduced electron transport chain in which maximum fluorescence (F_M) could only be obtained by adding 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU). reoxidation curves showed the presence of Q_B nonreducing PSII centers under nitrogen depletion. Fast induction curves (FICs) and electron transport rates (ETRs) revealed slowing of the electrons transferred from the primary (Q_A) to the secondary (Q_B) quinone electron acceptors of PSII. The data presented show that nitrogen depletion in F. cylindrus leads to the formation of Q_B nonreducing PSII centers within the photosystem. On a physiological level, the formation of Q_B nonreducing PSII centers in F. cylindrus provides the cell with protection against photoinhibition by facilitating the rapid induction of NPQ. This strategy provides an important ecological advantage, especially during the Antarctic spring, maintaining photosynthetic efficiency under high light and nutrient‐limiting conditions.     

Functional size of Photosystem II determined by radiation inactivation

The functional size of Photosystem II (PS II) was investigated by radiation inactivation. The technique provides an estimate of the functional mass required for a specific reaction and depends on irradiating samples with high energy -rays and assaying the remaining activity. The analysis is based on target theory that has been modified to take into account the temperature dependence of radiation inactivation of proteins. Using PS II enriched membranes isolated from spinach we determined the functional size of primary charge separation coupled to water oxidation and quinone reduction at the Q_B site: H₂O (Mn)₄ Y_z P680 Pheophytin Q phenyl-p-benzoquinone. Radiation inactivation analysis indicates a functional mass of 88 ± 12 kDa for electron transfer from water to phenyl-p-benzoquinone. It is likely that the reaction center heterodimer polypeptides, D1 and D2, contribute approximately 70 kDa to the functional mass, in which case polypeptides adding up to approximately 20 kDa remain to be identified. Likely candidates are the and subunits of cytochrome b ₅₅₉and the 4.5 kDa psbI gene product.Abbreviations Cyt cytochrome - PS Photosystem - P680 primary electron donor of Photosystem II - Q_A primary quinone acceptor of Photosystem II - Q_B secondary quinone acceptor of Photosystem II - Y_z tyrosine donor to P680     

pH sensitivity of the redox state of cytochrome b559 may regulate its function as a protectant against donor and acceptor side photoinhibition

A series of experiments have been conducted with isolated reaction centers of photosystem two (PS II) with the aim to elucidate the functional role of cytochrome (Cyt b ₅₅₉). At pH 6.5 it was found that Cyt b ₅₅₉ was reversibly photoreduced by red actinic light when Mn²⁺ was present as an electron donor while at pH 8.5 a photo-oxidation was observed under the same lighting conditions, which was dark reversible in the presence of hydroquinone. These pH dependent light induced changes were measured under anaerobic conditions and correlated with changes in the relative levels of high (HP) and low (LP) potential forms of the cytochrome. At pH 6.5 the cytochrome was mainly in its LP form while at pH 8.5 a significant proportion was converted to the HP form as detected by dark titrations with hydroquinone. This pH dependent difference in the levels of HP and LP Cyt b ₅₅₉ was also detected when bright white light was used to monitor the level of the LP form using a novel reaction involving direct electron donation from the flavin of glucose oxidase (present in the medium and used together with glucose and catalase as an oxygen trap). The results suggest that PS II directly oxidises and reduces the HP and LP forms, respectively and that the extent of these photo-reactions is dependent on the relative levels of the two forms, which are in turn governed by the pH. This conclusion is interpreted in terms of the model presented previously (Barber J and De Las Rivas J (1993) Proc Natl Acad Sci USA 90: 10942–10946) whereby the pH induced effect is considered as a possible mechanism by which interconversion of LP and HP forms of Cyt b ₅₅₉ is achieved. In agreement with this was the finding that as the extent of photo-oxidisable HPCyt b ₅₅₉ increases, with increasing pH, the rate of irreversible photo-oxidation of -carotene decreases, a result expected if the HP form protects against donor side photoinhibition.Abbreviations -car -carotene - CCCP carbonylcyanide m-chloro-phenylhydrazone - Chl chlorophyll - Cyt b ₅₅₉ cytochrome b ₅₅₉ - HPCyt b ₅₅₉ high potential form of cytochrome b ₅₅₉ which is reducible by hydroquinone - LPCyt b ₅₅₉ low potential form of cytochrome b ₅₅₉ which is non-reducible by hydroquinone - D1 and D2 products of the psbA and psbD genes, respectively - LHC II light-harvesting chlorophyll protein complex associated with PS II - Mes 2-(N-morpholino) ethanesulphonic acid - P680 primary electron donor of PS II - Pheo pheophytin - PQ plastoquinone - PS II Photosystem II - Q_A first stable quinone electron acceptor of PS II - Q_B second stable quinone electron acceptor of PS II - RC reaction center - SDS sodium dodecyl sulphate - SiMo silicomolybdate - Tris tris(hydroxymethyl) amino methane - Y_Z and Y_D tyrosine residues 161 in D1 and D2 proteins of the PS II RC which act as secondary electron donors to P680     

Restoration of the high potential form of cytochrome b-559 through the photoreactivation of Tris-inactivated oxygen-evolving center

Restoration of a high potential (HP) form of cytochrome b-559 (Cyt b-559) from a low potential (LP) form was the primary process in the reconstitution of O₂-evolving center during the photoreactivation of Tris-inactivated chloroplasts. In normal chloroplasts, about 0.5 to 0.7 mol of Cyt b-559 was present in the HP form per 400 chlorophyll molecules. However, the HP form was converted to the LP form when the O₂-evolving center was inactivated by 0.8 M alkaline Tris-washing (pH 9.1). The inactivation was reversible and both the Cyt b-559 HP form and the O₂-evolving activity were restored by incubating the inactivated chloroplasts with weak light, Mn²⁺, Ca²⁺ and an electron donor (photoreactivation). The recovery of the HP form preceded the recovery of O₂-evolving activity. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) did not inhibit the recovery of the HP form. Thus, the recovery of Cyt b-559 HP form was the primary reaction in the photoreactivation, which was stimulated by the light-induced redox reaction of the PS-II core center.Abbreviations ASC ascorbate - BSA bovine serum albumin - Chl chlorophyll - Cyt b-559 HP form high potential form of cytochrome b-559 - Cyt b-559 LP form low potential form of cytochrome b-559 - Cyt b-559 VLP form very low potential form of cytochrome b-559 - Cyt f cytochrome f - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenol indophenol - Hepes N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid - HQ hydroquinone - SHN chloroplast-preparation medium containing 0.4 M sucrose, 50 mM Hepes-Na (pH 7.8) and 20 mM NaCl - PS-II Photosystem II     

Fractional control of photosynthesis by the QB protein,the cytochrome f/b 6 complex and other components of the photosynthesic apparatus

In order to obtain information on fractional control of photosynthesis by individual catalysts, catalytic activities in photosynthetic electron transport and carbon metabolism were modified by the addition of inhibitors, and the effect on photosynthetic flux was measured using chloroplasts of Spinacia oleracea L. In thylakoids with coupled electron transport, light-limited electron flow to ferricyanide was largely controlled by the Q_B protein of the electron-transport chain. Fractional control by the cytochrome f/b ₆ complex was insignificant under these conditions. Control by the cytochrome f/b ₆ complex dominated at high energy fluence rates where the contribution to control of the Q_B protein was very small. Uncoupling shifted control from the cytochrome f/b ₆ complex to the Q_B protein. Control of electron flow was more complex in assimilating chloroplasts than in thylakoids. The contributions of the cytochrome f/b ₆ complex and of the Q_B protein to control were smaller in intact chloroplasts than in thylakoids. Thus, even though the transit time for an electron through the electron-transport chain may be below 5 ms in leaves, oxidation of plastohydroquinone was only partially responsible for limiting photosynthesis under conditions of light and CO₂ saturation. The energy fluence rate influenced control coefficients. Fractional control of photosynthesis by the ATP synthetase, the cytochrome f/b ₆ complex and by ribulose-1,5-bisphosphate carboxylase increased with increasing fluence rates, whereas the contributions of the Q_B protein and of enzymes sensitive to SH-blocking agents decreased. The results show that the burdens of control are borne by several components of the photosynthetic apparatus, and that burdens are shifted as conditions for photosynthesis change.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT 2,4-dinitro phenylether of 2-iodo-4-nitrothymol - pCMBS p-chloromercuribenzosulfonate     

Enhanced susceptibility of the oxidized and unprotonated forms of high potential cytochrome b-559 toward DCMU

The nature of interaction of cytochrome b-559 high potential (HP) with electron transport on the reducing side of photosystem II was investigated by measuring the susceptibility of cytochrome b-559HP to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) under different conditions. Submicromolar DCMU concentrations decreased the rate of absorbance change corresponding to cytochrome b-559HP photoreduction while the amplitude was lowered at higher concentrations (up to 10 M). Appreciable extents of cytochrome b-559HP photoreduction were observed at DCMU concentrations which completely abolished the electron transport from water to methyl viologen under the same experimental conditions. However, the susceptibility of cytochrome b-559HP to DCMU increased with the degree of cytochrome b-559HP oxidation, induced either by ferricyanide or by illumination of low intensity (2 W/m²) of red light in the presence of 2 M carbonyl cyanide-m-chlorophenylhydrazone. Also, the DCMU inhibition was more severe when the pH increased from 6.5 to 8.5, indicating that the unprotonated form of cytochrome b-559HP is more susceptible to DCMU. These results demonstrate that cytochrome b-559HP can accept electrons prior to the Q_B site, probably via Q_A although both Q_A and Q_B can be involved to various extents in this reaction. We suggest that the redox state and the degree of protonation of cytochrome b-559HP alter its interaction with the reducing side of photosystem II.Abbreviations ADRY acceleration of the deactivation reactions of the water-splitting system Y - CCCP carbonylcyanide m-chlorophenylhydrazone - FeCN ferricyanide - HP high potential - MV methylviologen CIW-DPB Publication No.1096.     

Chlorophyll a fluorescence of typical desert plant Alhagi sparsifolia Shap. at two light levels

Alhagi sparsifolia Shap. is exposed to a high-irradiance environment as the main vegetation found in the forelands of the Taklamakan Desert. We investigated chlorophyll a fluorescence emission of A. sparsifolia seedlings grown under ambient (HL) and shade (LL) conditions. Our results indicated that the fluorescence intensity in the leaves was significantly higher for LL-grown plants than that under HL. High values of the maximum quantum yield of PSII for primary photochemistry (φP_o) and the quantum yield that an electron moves further than Q_A - (φE_o) in the plants under LL conditions suggested that the electron flow from Q_A - (primary quinone electron acceptors of PSII) to Q_B (secondary quinone acceptor of PSII) or Q_B - was enhanced at LL compared to natural HL conditions. The efficiency/probability with which an electron from the intersystem electron carriers was transferred to reduce end electron acceptors at the PSI acceptor side and the quantum yield for the reduction of end electron acceptors at the PSI acceptor side were opposite to φP_o, and φE_o. Thus, we concluded that the electron transport on the donor side of PSII was blocked under LL conditions, while acceptor side was inhibited at the HL conditions. The PSII activity of electron transport in the plants grown in shade was enhanced, while the energy transport from PSII to PSI was blocked compared to the plants grown at HL conditions. Furthermore, PSII activity under HL was seriously affected in midday, while the plants grown in shade enhanced their energy transport.     

Cyclic electron flow around PSI monitored by afterglow luminescence in leaves of maize inbred lines (<Emphasis Type="Italic">Zea mays</Emphasis> L.): correlation with chilling tolerance

Maize (Zea mays L.) inbred lines of contrasting chilling sensitivity (three tolerant, three sensitive lines) were acclimated to 280 mol photons m^–2 s^–1 white light at a 17°C sub-optimal temperature. They showed no symptoms of photoinhibition, despite slight changes in photosystem II (PSII) fluorescence and thermoluminescence properties in two tolerant lines. A luminescence afterglow emission [Bertsch and Azzi (1965) Biochim Biophys Acta 94:15–26], inducible by a far-red (FR) illumination of unfrozen leaf discs, was detected either as a bounce in decay kinetics at constant temperatures or as a sharp thermoluminescence afterglow band at about 45°C, in dark-adapted leaves. This band reflects the induction by warming of an electron pathway from stromal reductants to plastoquinones and to the Q_B secondary acceptor of PSII, resulting in a luminescence-emitting charge recombination in the fraction of centres that were initially in the S_2/3Q_B non-luminescent state. A 5-h exposure of plants to growth chamber light shifted this luminescence emission towards shorter times and lower temperatures for several hours in the three chilling-tolerant lines. This downshift was not observed, or only transiently, in the three sensitive lines. In darkness, the downshifted afterglow band relaxed within hours to resume its dark-adapted location, similar for all maize lines. A faster dark re-reduction of P700⁺ oxidized by FR light (monitored by 820-nm absorbance) and an increase of photochemical energy storage under FR excitation (determined by photoacoustic spectroscopy) confirmed that a cyclic pathway induced by white actinic light remained activated for several hours in the tolerant maize lines.     

EFFECT OF CHROMIUM(VI) ON PHOTOSYSTEM II ACTIVITY AND HETEROGENEITY OF SYNECHOCYSTIS SP. (CYANOPHYTA): STUDIED WITH IN VIVO CHLOROPHYLL FLUORESCENCE TESTS1

The inhibitory effect of Cr(VI) on the PSII of Synechocystis sp. was studied. Cr(VI) reduced O₂ evolution and inhibited the water‐splitting system in PSII. S‐states test and flash induction test showed that Cr(VI) exposure increased the proportion of inactivated PSII (PSII_X) and PSII_β reaction centers, which increased the fluxes of dissipated energy. JIP test and Q_A^? reoxidation test demonstrated that Cr(VI) treatment induces inhibition of electron transport from Q_A^? to Q_B/Q_B^? and accumulation of P₆₈₀⁺. More Q_A^? had to be oxidized through S₂(Q_AQ_B)^? charge recombination and oxidation by PQ9 molecules in PSII under Cr(VI) stress. These changes finally decreased the index of photosynthesis performance.     

Spectroscopic studies of bound cytochrome c and an iron-sulfur center in a purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Flash-induced optical kinetics at room temperature of cytochrome (Cyt) c ₅₅₁ and an Fe-S center (CF_A/CF_B) bound to a purified reaction center (RC) complex from the green sulfur photosynthetic bacterium Chlorobium tepidum were studied. At 551 nm, the flash-induced absorbance change decayed with a t _1/2 of several hundred ms, and the decay was accelerated by 1-methoxy-5-methylphenazinium methyl sulfate (mPMS). In the blue region, the absorbance change was composed of mPMS-dependent (Cyt) and mPMS-independent component (CF_A/CF_B) which decayed with a t _1/2 of 400–650 ms. Decay of the latter was effectively accelerated by benzyl viologen (Em –360 mV) and methyl viologen (–440 mV), and less effectively by triquat (–540 mV). The difference spectrum of Cyt c had negative peaks at 551, 520 and 420 nm, with a positive rise at 440 to 500 nm. The difference spectrum of CF_A/CF_B resembled P430 of PSI, and had a broad negative peak at 430435 nm.Abbreviations (B)Chl (bacterio)chlorophyll - Cyt cytochrome - F_A, F_B and F_X iron-sulfur center A, B and X of Photosystem I - CF_A, CF_B and CF_X F_A-,F_B- and F_X-like Fe-S center of Chlorobium - mPMS 1-methoxy-5-methylphenazinium methyl sulfate - PSI Photosystem I - RC reaction center