Methylviologen and dibromothymoquinone treatments of pea leaves reveal the role of photosystem I in the Chl a fluorescence rise OJIP

The effects of dibromothymoquinone (DBMIB) and methylviologen (MV) on the Chl a fluorescence induction transient (OJIP) were studied in vivo. Simultaneously measured 820-nm transmission kinetics were used to monitor electron flow through photosystem I (PSI). DBMIB inhibits the reoxidation of plastoquinol by binding to the cytochrome b₆/f complex. MV accepts electrons from the FeS clusters of PSI and it allows electrons to bypass the block that is transiently imposed by ferredoxin-NADP⁺-reductase (FNR) (inactive in dark-adapted leaves). We show that the IP phase of the OJIP transient disappears in the presence of DBMIB without affecting F_m. MV suppresses the IP phase by lowering the P level compared to untreated leaves. These observations indicate that PSI activity plays an important role in the kinetics of the OJIP transient. Two requirements for the IP phase are electron transfer beyond the cytochrome b₆/f complex (blocked by DBMIB) and a transient block at the acceptor side of PSI (bypassed by MV). It is also observed that in leaves, just like in thylakoid membranes, DBMIB can bypass its own block at the cytochrome b₆/f complex and donate electrons directly to PC⁺ and P700⁺ with a donation time τ of 4.3 s. Further, alternative explanations of the IP phase that have been proposed in the literature are discussed.     

In vivo analysis of chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence induction

Quantitative characteristics of photosynthetic electron transport were evaluated in vivo on the basis of the multi-exponential analysis of OJIP fluorescence transients induced by saturating actinic light. The OJIP fluorescence curve F(t), measured in Chlamydomonas reinhardtii cells, was transformed into the (1 − F _O/F(t)) × (F _V /F _M)⁻¹ transient, which is shown to relate to PS 2 closure. We assumed that kinetics of PS 2 closure during OJIP rise reflects time-separated processes related to the establishment of redox equilibrium at the PS 2 acceptor side (OJ), PQ pool (JI), and beyond Cyt b/f (IP). Three-exponential fitting was applied to (1 − F _O/F(t)) × (F _V /F _M)⁻¹ transient to obtain lifetimes and amplitudes of the OJ, JI, and IP components of PS 2 closure, which were used to calculate overall rates of reduction and re-oxidation of the PS 2 acceptor side, PQ pool, and intermediates beyond Cyt b/f complex. The results, obtained in the presence of inhibitors, oxidative reagents, and under different stress conditions prove the suggested model and characterize the introduced parameters as useful indicators of photosynthetic function.     

Polyphasic rise of chlorophyll a fluorescence in herbicide-resistant D1 mutants of Chlamydomonas reinardtii

Chlorophyll (Chl) a fluorescence transient, a sensitive and non-invasive probe of the kinetics and heterogeneity of the filling up of the electron acceptor pool of Photosystem II (PS II), was used to characterize D1-mutants of Chlamydomonas reinhardtii. Using a shutter-less system (Plant Efficiency Analyzer, Hansatech, UK), which provides the first measured data point at 10 s and allows data accumulation over several orders of magnitude of time, we have characterized, for the first time, complete Chl a fluorescence transients of wild type (WT), cell wall less (CW-15) C. reinhardtii and several herbicide-resistant mutants of the D1 proteins: D1-V219I A251V, F255Y, S264A G256D and L275F. In all cases, the Chl a fluorescence induction transients follow a pattern of O-J-I-P where J and I appear as two steps between the minimum Fo (O) and the maximum Fmax (Fm, P) levels. The differences among the mutants are in the kinetics of the filling up of the electron acceptor pool of PS II (this paper) in addition to those in the re-oxidation kinetics of Q_A ^– to Q_A, published elsewhere (Govindjee et al. (1992) Biochim Biophys. Acta: 1101: 353–358; Strasser et al. (1992) Archs. Sci. Genève 42: 207–224) and not in the ratio of the maximal fluorescence Fm to the initial fluorescence Fo. The value of this experimental ratio is Fm/Fo = 4.4±0.21 independent of the mutation. At 600 W m^–2 of 650 nm excitation, distinct hierarchy in the fraction of variable Chl a fluorescence at the J level is observed: S264A > A251V G256A > L275F V219I > F255Y CW-15 WT. At 300 and 60 W m^–2 excitation, a somewhat similar hierarchy among the mutants was observed for the intermediate levels J and I. Addition of bicarbonate-reversible inhibitor formate did not change the O to J phases, slowed the I to P rise, and in many cases, slowed the decay of fluorescence beyond the P level. These observations are interpreted in terms of formate effect being on the acceptor rather than on the donor side (S-states) of PS II. The formate effect was different in different mutants, with L275F being the most insensitive mutant followed by others (V219I, F255Y, WT, A251V and S264A). Further, in the presence of high concentrations of DCMU, identical transients were observed for all the mutants and the WT.The quantum yield of photochemistry of PS II, calculated from 1-(Fo/Fm), is in the range of 0.73 to 0.82 for the WT as well as for the mutants examined. Thus, in contrast to differences in the kinetics of the electron acceptor side of PS II, there were no significant differences in the maximum quantum yield of PS II, among the mutants tested. We suggest that earlier photochemistry yield values were much lower (0.4–0.6) than those reported here due to either higher measured values of Fo by instruments using camera shutters, or due to the use of cells grown in less than-optimal conditions.

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Methylviologen and dibromothymoquinone treatments of pea leaves reveal the role of photosystem I in the Chl a fluorescence rise OJIP

The effects of dibromothymoquinone (DBMIB) and methylviologen (MV) on the Chl a fluorescence induction transient (OJIP) were studied in vivo. Simultaneously measured 820-nm transmission kinetics were used to monitor electron flow through photosystem I (PSI). DBMIB inhibits the reoxidation of plastoquinol by binding to the cytochrome b(6)/f complex. MV accepts electrons from the FeS clusters of PSI and it allows electrons to bypass the block that is transiently imposed by ferredoxin-NADP(+)-reductase (FNR) (inactive in dark-adapted leaves). We show that the IP phase of the OJIP transient disappears in the presence of DBMIB without affecting F(m). MV suppresses the IP phase by lowering the P level compared to untreated leaves. These observations indicate that PSI activity plays an important role in the kinetics of the OJIP transient. Two requirements for the IP phase are electron transfer beyond the cytochrome b(6)/f complex (blocked by DBMIB) and a transient block at the acceptor side of PSI (bypassed by MV). It is also observed that in leaves, just like in thylakoid membranes, DBMIB can bypass its own block at the cytochrome b(6)/f complex and donate electrons directly to PC(+) and P700(+) with a donation time tau of 4.3 s. Further, alternative explanations of the IP phase that have been proposed in the literature are discussed.     

Photoinhibition in Chlamydomonas reinhardtii: Effect on state transition,intersystem energy distribution and Photosystem I cyclic electron flow

The energy distribution, state transitions and photosynthetic electron flow during photoinhibition of Chlamydomonas reinhardtii cells have been studied in vivo using photoacoustics and modulated fluorescence techniques. In cells exposed to 2500 W/m² light at 21 °C for 90 min, 90% of the oxygen evolution activity was lost while photochemical energy storage as expressed by the parameter photochemical loss (P.L.) at 710–720 nm was not impaired. The energy storage vs. modulation frequency profile indicated an endothermic step with a rate constant of 2.1 ms. The extent of the P.L. was not affected by DCMU but was greatly reduced by DBMIB. The regulatory mechanism of the state 1 to state 2 transition process was inactivated and the apparent light absorption cross section of photosystem II increased during the first 20 min of photoinhibition followed by a significant decrease relative to that of photosystem I. These results are consistent with the inactivation of the LHC II kinase and the presence of an active cyclic electron flow around photosystem I in photoinhibited cells.Abbreviations PS I, PS II Photosystem I and Photosystem II respectively - P.L. photochemical loss - DCMU 3-(3,4-dichlorophenyl-1,1-dimethyl urea - LHC II light harvesting chlorophyll a,b-protein complex of PS II - DBMIB 2,5 dibromo-3-methyl-6-isopropyl-p-benzoquinone     

Quantification of non-Q B -reducing centers in leaves using a far-red pre-illumination

An alternative approach to quantification of the contribution of non-Q_B-reducing centers to Chl a fluorescence induction curve is proposed. The experimental protocol consists of a far-red pre-illumination followed by a strong red pulse to determine the fluorescence rise kinetics. The far-red pre-illumination induces an increase in the initial fluorescence level (F_{25 μs}) that saturates at low light intensities indicating that no light intensity-dependent accumulation of Q_A⁻ occurs. Far-red light-dose response curves for the F_{25 μs}-increase give no indication of superimposed period-4 oscillations. F_{25 μs}-dark-adaptation kinetics following a far-red pre-pulse, reveal two components: a faster one with a half-time of a few seconds and a slower component with a half-time of around 100 s. The faster phase is due to the non-Q_B-reducing centers that re-open by recombination between Q_A⁻ and the S-states on the donor side. The slower phase is due to the recombination between Q_B⁻ and the donor side in active PS II reaction centers. The pre-illumination-induced increase of the F_{25 μs}-level represents about 4–5% of the variable fluorescence for pea leaves (∼2.5% equilibrium effect and 1.8–3.0% non-Q_B-reducing centers). For the other plant species tested these values were very similar. The implications of these values will be discussed.     

Probing of photosynthetic reactions in four phytoplanktonic algae with a PEA fluorometer

High-resolution light-induced kinetics of chlorophyll fluorescence (OJIP transients) were recorded and analyzed in cultures of diatoms (Thalassiosira weissflogii, Chaetoceros mulleri) and dinoflagellates (Amphidinium carterae, Prorocentrum minimum). Fluorescence transients showed the rapid exponential initial rise from the point O indicating low connectivity between PS II units and high absorption cross-section of PS II antenna. Dark-adapted dinoflagellates revealed capability to maintain the PS I-mediated re-oxidation of the PQ pool at the exposure to strong actinic light that may lead to the underestimation of F _M value. In OJIP transients recorded in phytoplanktonic algae the fluorescence yield at the point O exceeded F _O level because Q_A has been already partly reduced at 50 μs after the illumination onset. PEA was also employed to study the recovery of photosynthetic reactions in T. weissflogii during incubation of nitrogen starved cells in N-replete medium. N limitation caused the impairment of electron transport between Q_A and PQs, accumulation of closed PS II centers, and the reduced ability to generate transmembrane ΔpH upon illumination, almost fully restored during the recovery period. The recovered cells showed much higher values of NPQ than control ones suggesting maximization of photoprotection mechanisms in the population with a ‘stress history.’     

Novel effects of methyl viologen on photosystem II function in spinach leaves

Methyl viologen (MV) is a well-known electron mediator that works on the acceptor side of photosystem I. We investigated the little-known, MV-induced inhibition of linear electron flow through photosystem II (PS II) in spinach-leaf discs. Even a low [MV] decreased the (1) average, light-adapted photochemical efficiency of PS II traps, (2) oxidation state of the primary quinone acceptor Q_A in PS II during illumination, (3) photochemical efficiency of light-adapted open PS II traps, (4) fraction of absorbed light energy dissipated constitutively in a light-independent manner or as chlorophyll (Chl) a fluorescence emission, (5) Chl a fluorescence yield corresponding to dark-adapted open reaction-center traps (F _o) and closed reaction-center traps (F _m), and (6) half-time for re-oxidation of Q_A⁻ in PS II after a single-turnover flash. These effects suggest that the presence of MV accelerates various “downhill” electron-transfer steps in PS II. Therefore, when using the MV to quantify cyclic electron flow, the inhibitory effect of MV on PS II should be taken into account.     

The Cytochrome cbo from the Obligate Methylotroph Methylobacillus flagellatus KT Is a Cytochrome c Oxidase

The cbo-type oxidase of Methylobacillus flagellatus KT was purified to homogeneity by preparative native gel electrophoresis, and the kinetic properties and substrate specificity of the enzyme were studied. Ascorbate and ascorbate/N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) were oxidized by cytochrome cbo with a pH optimum of 8.3. With TMPD as an electron donor for the cbo-type oxidase, the optimal pH (7.0 to 7.6) was determined from the difference between respiration rates in the presence of ascorbate/TMPD and only ascorbate. The kinetic constants determined at pH 7.0 were as follows: oxidation by the enzyme of reduced TMPD was characterized by K _M = 0.86 mM and V _max = 1.1 mol O₂/(min mg protein), and oxidation of reduced horse heart cytochrome c was characterized by K _M = 0.09 mM and V _max = 0.9 mol O₂/(min mg protein). Cyanide inhibited ascorbate/TMPD–oxidase activity (K _i = 4.5–5.0 M). The soluble cytochrome c _H (12 kDa), partially purified from M. flagellatus KT, was found to serve as a natural electron donor for the cbo-type oxidase.     

Inhibition of photosystem II activity by Cu++ ion. Choice of buffer and reagent is critical

Cupric ion (Cu⁺⁺) inhibits the rate of photosystem II electron transport and the intensity of the variable part of chl a fluorescence in isolated chloroplast thylakoids. The inhibition is markedly dependent on the nature of the buffer used in the assay medium. In MES and HEPES buffers, complete inhibition of photosystem II occurs at 30 M of Cu⁺⁺, while in Tricine no inhibition occurred even at 200 M Cu⁺⁺. In other buffers used (TES, Phosphate, Tris), the efficacy of Cu⁺⁺ inhibition is intermediate. The calculated binding constants are found to correspond to the observed I₅₀ values for the six buffers used. It is concluded that the previous reports on copper inhibition, where buffers have been used indiscriminately should be reconsidered. Certain reagents such as hydroxylamine, ascorbate and diphenyl carbazide, which react with Cu⁺⁺, should be avoided.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenol indophenol - DCMU 3-(3,4 dichlorophenyl)-1,1-dimethyl urea - DAD diaminodurene - DPC diphenyl carbazide - Fv variable chl fluorescence - HEPES N-2-hydroxyethyl piperazine sulfonic acid - I ₃₀ inhibitor concentration causing 30% inhibition of Fv - MES 2-(N-morpholino) ethane sulfonic acid - MV Methyl viologen - PS II Photosystem II - PS I Photosystem I - TES N-tris(hydroxymethyl)-methyl-2-amino sulfonic acid - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - Tricine N-tris(hydroxymethyl) ethylglycine - Tris N-tris(hydroxymethyl)amino ethane     

Purification of a Photosystem II reaction center from a thermophilic cyanobacterium using immobilized metal affinity chromatography

Oxygen-evolving PS II particles from the thermophilic cyanobacterium Synechococcus elongatus are partially purified by centrifugation on a sucrose gradient and are bound to a Chelating Sepharose column loaded with Cu²⁺ ions. Bound particles are then transformed into PS II RC complexes by two washing steps. First, washing with a phosphate buffer (pH=6.5) containing 0.02% of SB 12 removes the rest of phycobilins and leaves pure PS II core particles on the column. Second, washing with a phosphate buffer (pH=6.2) containing 0.2 M LiClO₄ and 0.05% of DM removes CP 47 and CP 43 and leaves bare PS II RC complexes on the column. These are then eluted with a phosphate buffer containing 1% of dodecylmaltoside (DM). The molar ratio of pigments in the eluate changes with the progress of elution but around the middle of the elution period a nearly stable ratio is maintained of Chl a: Pheo a: Car: Cyt b 559 equal to 2.9: 1: 0.9: 0.8. In these fractions the photochemical separation of charges could be demonstrated by accumulation of reduced pheophytin (A of 430–440 nm) and by the flash induced formation of P680⁺ (A at 820 nm). The relatively slow relaxation kinetics of the latter signal (t_1/2 1 ms) may suggest that in a substantial fraction of the RCs Q_A remains bound to the complex.Abbreviations Car -carotene - Chl a chlorophyll a - CP43, CP47 chlorophyll-proteins, with R_m 43 and 47 kDa - DBMIB dibromothymoquinone,2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone - DM -dodecyl-d-maltoside - HPLC high-performance liquid chromatography - OG n-octyl--d-glucopyranoside - IMAC immobilied metal affinity chromatography - Pheo a pheophytin a - PQ-9 plastoquinone-9 - P680 primary electron donor in PS II - PS II RC Photosystem II reaction centre - Q_A primary electron acceptor in PS II - SB-12 N-dodecyl-N,N-dimethyl-3-amino-1-propanesulphonate, (sulphobetain 12)     

Photosynthetic electron transport in thylakoids from cotton cultivars (Gossypium sp.) differing in tolerance to prometryn

A method to determine photosynthetic electron transport in thylakoid membranes is described for Gossypium barbadense (cv. Pima S-7) and G. hirsutum (cv. DP 5415). These cultivars differed markedly in tolerance to prometryn, a PS II inhibitor. The rates of photosynthetic electron transport obtained were 245 mole oxygen mg^–1 chl h¹. Plant age and leaf size influenced the activity of the thylakoid preparations. Thylakoids from leaves of plants 24 to 37 d and 50–70 mm in diameter had the highest activities; thylakoids from cotyledons, fully expanded leaves and young leaves had low activity. Thylakoids from both species had similar photosynthetic activities and I₅₀'s for prometryn, atrazine and diuron. Thus, tolerance to prometryn was not due to differential binding at D1 protein.Abbreviations PSII photosystem II - DAP day after planting - DQ duroquinone - DBMIB dibromothymoquinone - DMBQ 2,5-dimethyl-p-benzoquinone - I₅₀ concentration to inhibit reaction by 50% - Q_A quinone A - Q_B quinone B     

Analysis of dark-relaxation kinetics of variable fluorescence in intact leaves

The dark-relaxation kinetics of variable fluorescence, F_v, in intact green leaves of Pisum stativum L. and Dolichos lablab L. were analyzed using modulated fluorometers. Fast (t_1/2 = 1 s) and slow (t_1/2 = 7–8 s) phases in f_v dark-decay kinetics were observed; the rate and the relative contribution of each phase in total relaxation depended upon the fluence rate of the actinic light and the point in the induction curve at which the actinic light was switched off. The rate of the slow phase was accelerated markedly by illumination with far-red light; the slow phase was abolished by methyl viologen. The halftime of the fast phase of F_v dark decay decreased from 250 ms in dark-adapted leaves to 12–15 ms upon adaptation to red light which is absorbed by PSII. The analysis of the effect of far-red light, which is absorbed mainly by PSI, on F_v dark decay indicates that the slow phase develops when a fraction of Q_A ^– (the primary stable electron acceptor of PSII) cannot transfer electrons to PSI because of limitation on the availability of P700⁺ (the primary electron donor of PSI). After prolonged illumination of dark-adapted leaves in red (PSII-absorbed) light, a transient. F_v rise appears which is prevented by far-red (PSI-absorbed) light. This transient f_v rise reflects the accumulation of Q_A ^– in the dark. The observation of this transient F_v rise even in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydrazone (CCCP) indicates that a mechanism other than ATP-driven back-transfer of electrons to QA may be responsible for the phenomenon. It is suggested that the fast phase in F_v dark-decay kinetics represents the reoxidation of Q_A ^– by the electron-transport chain to PSI, whereas the slow phase is likely to be related to the interaction of Q_A ^– with the donor side of PSII.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - F_O initial fluorescence level - F_v variable fluorescence - P700 primary electron donor of PSI - PSI, II photosystem I, II - Q_A (Q_A ^–) Q_B (Q_B ^–) primary and secondary stable electron acceptor of PSII in oxidized (reduced) state Supported by grant B6.1/88 DST, Govt. of India.     

Photosystem II chlorophyll a fluorescence lifetimes and intensity are independent of the antenna size differences between barley wild-type and chlorina mutants: Photochemical quenching and xanthophyll cycle-dependent nonphotochemical quenching of fluorescence

Photosystem II (PS II) chlorophyll (Chl) a fluorescence lifetimes were measured in thylakoids and leaves of barley wild-type and chlorina f104 and f2 mutants to determine the effects of the PS II Chl a+b antenna size on the deexcitation of absorbed light energy. These barley chlorina mutants have drastically reduced levels of PS II light-harvesting Chls and pigment-proteins when compared to wild-type plants. However, the mutant and wild-type PS II Chl a fluorescence lifetimes and intensity parameters were remarkably similar and thus independent of the PS II light-harvesting antenna size for both maximal (at minimum Chl fluorescence level, F_o) and minimal rates of PS II photochemistry (at maximum Chl fluorescence level, F_m). Further, the fluorescence lifetimes and intensity parameters, as affected by the trans-thylakoid membrane pH gradient (pH) and the carotenoid pigments of the xanthophyll cycle, were also similar and independent of the antenna size differences. In the presence of a pH, the xanthophyll cycle-dependent processes increased the fractional intensity of a Chl a fluorescence lifetime distribution centered around 0.4–0.5 ns, at the expense of a 1.6 ns lifetime distribution (see Gilmore et al. (1995) Proc Natl Acad Sci USA 92: 2273–2277). When the zeaxanthin and antheraxanthin concentrations were measured relative to the number of PS II reaction center units, the ratios of fluorescence quenching to [xanthophyll] were similar between the wild-type and chlorina f104. However, the chlorina f104, compared to the wild-type, required around 2.5 times higher concentrations of these xanthophylls relative to Chl a+b to obtain the same levels of xanthophyll cycle-dependent fluorescence quenching. We thus suggest that, at a constant pH, the fraction of the short lifetime distribution is determined by the concentration and thus binding frequency of the xanthophylls in the PS II inner antenna. The pH also affected both the widths and centers of the lifetime distributions independent of the xanthophyll cycle. We suggest that the combined effects of the xanthophyll cycle and pH cause major conformational changes in the pigment-protein complexes of the PS II inner or core antennae that switch a normal PS II unit to an increased rate constant of heat dissipation. We discuss a model of the PS II photochemical apparatus where PS II photochemistry and xanthophyll cycle-dependent energy dissipation are independent of the Peripheral antenna size.Abbreviations Ax antheraxanthin - BSA bovine serum albumin - c_x lifetime center of fluorescence decay component x - CP chlorophyll binding protein of PS II inner antenna - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DTT dithiothreitol - f_x fractional intensity of fluorescence lifetime component x - F_m, F_m maximal PS II Chl a fluorescence intensity with all Q_A reduced in the absence, presence of thylakoid membrane energization - F_o minimal PS II Chl a fluorescence intensity with all Q_A oxidized - F_v=F_m–F_o variable level of PS II Chl a fluorescence - HPLC high performance liquid chromatography - k_A rate constant of all combined energy dissipation pathways in PS II except photochemistry and fluorescence - k_F rate constant of PS II Chl a fluorescence - LHCIIb main light harvesting pigment-protein complex (of PS II) - N_pig mols Chl a+b per PS II - NPQ=(F_m/F_m–1) nonphotochemical quenching of PS II Chl a fluorescence - PAM pulse-amplitude modulation fluorometer - PFD photon-flux density, mols photons m^–2 s^–1 - PS II Photosystem II - P680 special-pair Chls of PS II reaction center - Q_A primary quinone electron acceptor of PS II - V_x violaxanthin - w_x width at half maximum of Lorentzian fluorescence lifetime distribution x - Zx zeaxanthin - pH trans-thylakoid proton gradient - % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqef0uAJj3BZ9Mz0bYu% H52CGmvzYLMzaerbd9wDYLwzYbItLDharqqr1ngBPrgifHhDYfgasa% acOqpw0xe9v8qqaqFD0xXdHaVhbbf9v8qqaqFr0xc9pk0xbba9q8Wq% Ffea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qqQ8frFve9Fve9Ff0dme% GabaqaaiGacaGaamqadaabaeaafiaakeaacqGH8aapcqaHepaDcqGH% +aGpdaWgaaWcbaGaamOraiaad2gaaeqaaaaa!4989!\[< \tau > _{Fm}\],% MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqef0uAJj3BZ9Mz0bYu% H52CGmvzYLMzaerbd9wDYLwzYbItLDharqqr1ngBPrgifHhDYfgasa% acOqpw0xe9v8qqaqFD0xXdHaVhbbf9v8qqaqFr0xc9pk0xbba9q8Wq% Ffea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qqQ8frFve9Fve9Ff0dme% GabaqaaiGacaGaamqadaabaeaafiaakeaacqGH8aapcqaHepaDcqGH% +aGpdaWgaaWcbaGaamOraiaad+gaaeqaaOGaeyypa0Zaaabqaeaaca% WGMbWaaSbaaSqaaiaadIhaaeqaaOGaam4yamaaBaaaleaacaWG4baa% beaaaeqabeqdcqGHris5aaaa!50D3!\[< \tau > _{Fo} = \sum {f_x c_x }\] average lifetime of Chl a fluorescence calculated from a multi-exponential model under F_m, F_o conditions     

An improved method of measuring photosynthetic electron transport in Euglena under in vivo conditions

A method is described to measure photochemical activity in intact cells of Euglena under in vivo conditions. The method employs a cell wall digesting enzyme (cellulysin) to induce enough permeability in the cell walls and membranes in order to allow dyes, commonly used to investigate light-dependent electron transport reactions to enter, but without inducing a concomittant efflux of metabolites. Between 1 and 2 h of incubation in 5% (w/v) cellulysin provided conditions which allowed measurement of photosystem I-, II- and I+II-dependent electron transport with rates up to 600% higher than in control cells; whereas other cell wall degrading enzymes (cellulase and pectinase) still did not increase the entry of the dyes. Cellulysin up to 2 h of incubation had little or no effect on whole cell respiration, photosynthetic O₂ evolution, or the export of potassium and (¹⁴C) labeled compounds out of cells; therefore cellulysin obviously did not change the normal habit or physiology of Euglena. Cellulysin (4 h digestion), cellulase and pectinase (2–4 h of incubation) on the other hand led to a lowering of respiration and light-dependent O₂ evolution, and increased the efflux of K⁺, but apparently decreased that of (¹⁴C)labeled fixation products.Abbreviations DBMIB dibromothymoquinone - DCPIP 2,6-dichlorophenol-indophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMMIB 2,3-dimethyl-5,6-methylenedioxy-p-benzoquinone - MV methylviologen - PSI photosystem I - PS II photosystem II     

Similarity between electron donor side reactions in the solubilized Photosystem II–LHC II supercomplex and Photosystem-II-containing membranes

The PS II–LHC II supercomplex is a novel type of oxygen evolving Photosystem II (PS II) core particle that contains the light harvesting complex proteins Lhcb1/2/4/5 in addition to the PS II reaction centre, oxygen evolving complex (OEC) and inner antennae [Hankamer et al. (1997) Eur J Biochem 243: 422–429]. The 33 and 23 kDa extrinsic proteins in these particles have been localised by image analysis of electron micrographs and averaging techniques [Boekema et al. (1998) Eur J Biochem 252: 268–276]. To assay the functionality of the water splitting complex, we compared the single flash P680⁺ reduction kinetics in these supercomplexes with those of PS II-rich granal stack membranes (BBYs). We found that the P680⁺ reduction kinetics in PS II–LHC II supercomplexes were indistinguishable from those in BBYs. We also examined a number of PS II core particles lacking the Lhcb components. All of these had different P680⁺ reduction kinetics, which we attributed to partial loss of OEC function before and during the measurements.     

Simultaneous photoreduction and photooxidation of cytochrome b-559 in Photosystem II treated with carbonylcyanide-m-chlorophenylhydrazone

The possibility of a Photosystem II (PS II) cyclic electron flow via Cyt b-559 catalyzed by carbonylcyanide m-chlorophenylhydrazone (CCCP) was further examined by studying the effects of the PS II electron acceptor 2,6-dichloro-p-benzoquinone (DCBQ) on the light-induced changes of the redox states of Cyt b-559. Addition to barley thylakoids of micromolar concentrations of DCBQ completely inhibited the changes of the absorbance difference corresponding to the photoreduction of Cyt b-559 observed either in the presence of 10 M ferricyanide or after Cyt b-559 photooxidation in the presence of 2 M CCCP. In CCCP-treated thylakoids, the concentration of photooxidized Cyt b-559 decreased as the irradiance of actinic light increased from 2 to 80 W m^-2 but remained close to the maximal concentration (0.53 photooxidized Cyt b-559 per photoactive Photosystem II) in the presence of 50 M DCBQ. The stimulation of Cyt b-559 photooxidation in parallel with the inhibition of its photoreduction caused by DCBQ demonstrate that the extent of the light-induced changes of the redox state of Cyt b-559 in the presence of CCCP is determined by the difference between the rates of photooxidation and photoreduction of Cyt b-559 occuring simultaneously in a cyclic electron flow around PS II.We also observed that the Photosystem I electron acceptor methyl viologen (MV) at a concentration of 1 mM barely affected the rate and extent of the light-induced redox changes of Cyt b-559 in the presence of either FeCN or CCCP. Under similar experimental conditions, MV strongly quenched Chl-a fluorescence, suggesting that Cyt b-559 is reduced directly on the reducing side of Photosystem II.Abbreviations ADRY acceleration of the deactivation reactions of the water-splitting system Y - ANT-2p 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene - CCCP carbonylcyanide-m-chlorophenylhydrazone - DCBQ 2,6-dichloro-p-benzoquinone - FeCN ferricyanide - MV methyl viologen - P₆₈₀ Photosystem II reaction center Chl-a dimer CIW-DPB publication No. 1118.     

In situ photosynthetic differentiation of the green algal and the cyanobacterial photobiont in the crustose lichen Placopsis contortuplicata

In situ photosynthetic activity in the green algal and the cyanobacterial photobionts of Placopsis contortuplicata was monitored within the same thallus using chlorophyll a fluorescence methods. It proved possible to show that the response to hydration of the green algal and the cyanobacterial photobionts is different within the same thallus. Measurements of the photochemical efficiency of PS II, Fv/Fm, reveal that in the dry lichen thallus photosynthetic activity could be induced in the green algal photobiont by water vapour uptake, in the cyanobacterial photobiont only if it was hydrated with liquid water. However, rates of apparent electron flow through PS II as well as rates of CO₂ gas exchange were suboptimal after hydration with water vapour alone and maximum rates could only be observed when the thallus was saturated with liquid water. The differences in the waterrelated photosynthetic performance and different light response curves of apparent electron transport rate through PS II indicate that the two photobionts act highly independently of each other. It was shown that the cyanobacteria from the cephalodia in P. contortuplicata act as photobiont. The rate of electron flow through PS II was found to be saturated at 1500 mol photon m^–2 s^–1, despite a considerable increase of non-photochemical quenching in the green algal photobiont which is lacking in the cyanobacterial photobiont. No evidence of photoinhibition could be found in either photobiont. Pronounced competition between the green algal and the cyanobacterial thallus can be observed in the natural habitat, indicating that the symbiosis in P. contortuplicata should be regarded as a very variable adaptation to the extreme environmental conditions in the maritime Antarctic.Abbreviations DR dark respiration - ETR apparent rate of electron flow of PS II (=F/Fm×PFD) - F difference in yield of fluorescence and maximal Fm and steady state Fs under ambient light - Fo minimum level of fluorescence yield in dark-adapted state - Fo minimum level of fluorescence yield after transient darkening and far-red illumination - Fm maximum level of dark-adapted fluorescence yield - Fm maximum yield of fluorescence under ambient light - Fs yield of fluorescence at steady state - Fv difference in minimum fluorescence and maximum fluorescence in dark-adapted state - NP net photosynthesis - NPQ coefficient for non-photochemical quenching - PAR photosynthetically active radiation (400–700 nm) - PFD photon flux density in PAR - PS II photosystem II - qN coefficient for non-photochemical quenching - qP coefficient for photochemical quenching     

Characterization of synchronized cultures of Bumilleriopsis filiformis

Activity of the photosynthetic apparatus of synchronized cultures was studied with the xanthophycean alga Bumilleriopsis filiformis, following the kinetics of fluorescence induction and photooxidation of cytochrome f (= cytochrome c-553) of intact cells. During the beginning of the cell-division phase, minimum cellular photosynthetic activity is observed and a maximum after its completion, which is accompanied by corresponding changes in Hill reaction activity and re-reduction of cytochrome f by photosystem II light. At minimum activity, the level of steady state fluorescence was higher than at the maximum. This is due, at least in part, to the diminished electron flow between the two photosystems seemingly caused by decreased photosystem I activity. This explanation was suported by the kinetics of cytochrome-f photooxidation.Thus, electron transport activity of both photosystems appears to vary during the cell cycle.Abbreviations pBQ p-benzoquinone - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCIP dichlorophenolindophenol - MV methylviologen (paraquat) - Q fluorescence quencher (in photosystem II)     

Efficiency of hydrogen photoproduction by photosystem I-enriched subchloroplast vesicles combined with Proteus mirabilis cells. Effects of some exogenous electron donors

High rates of hydrogen photoproduction are obtained when glutaraldehyde-fixed Photosystem I-enriched vesicles (Photosystem II-depleted) are added to hydrogenase-containing cells of Proteus mirabilis in the presence of the mediator methylviologen and a suitable electron donating system. This donor system includes ascorbate, dithioerythritol (DTE) and the mediator tetramethylphenylene-diamine (TMPD) and reduces the photosynthetic electron transfer chain at the level of plastocyanin. Both DTE and ascorbate are required for hydrogen photoproduction, DTE being the ultimate electron donor and ascorbate only having a catalytic function. Whereas the aerobic photoreduction of methylviologen is similar in the presence of DTE, ascorbate or both, under anaerobic conditions only combination of both compounds results in a high and stable amount of reduced methylviologen that can be utilized by the hydrogenase. It is concluded that oxidation reactions of reduced methylviologen, competing with the hydrogenase, rather than methylviologen photoreduction, limit hydrogen photoproduction in the presence of either DTE or ascorbate. These oxidation reactions are suggested to involve back reactions to the oxidized form(s) of ascorbate and DTE but backflow to the photosynthetic electron transfer chain (i.e. cyclic electron transfer) can not be excluded.Abbreviations Tes N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid - DTE 1,4-dithioerythritol - TMPD, N,N,N N-tetramethyl-p-phenylenediamine - DCMU 3-(3, 4-dichlorophenyl)-1, 1,-dimethylureum - EDAC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide - DNP-INT 2-iodo-6-isopropyl-3-methyl-2, 4, 4-trinitrodiphenyl ether - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone - PS photosystem - Chl chlorophyll