Kinetics of delayed chlorophyll a fluorescence registered in milliseconds time range

Delayed fluorescence dark decays in the time interval from 0.35 to 5.5ms are measured during dark to light adaptation in whole barley leaves and isolated thylakoid membranes, using a disc phosphoroscope. The changes in delayed fluorescence features are compared with variable chlorophyll fluorescence simultaneously registered with the same apparatus as well as in parallel by Handy PEA (Hansatech Instruments Ltd.), and absorbance changes at 820 nm. The registered delayed fluorescence signal is a sum of three components – submillisecond with lifetime of about 0.6 ms, millisecond decayed 2–4 ms and slow component with lifetime > >5.5 ms. The submillisecond delayed fluorescence component is proposed to be a result of radiative charge recombination in Photosystem II reaction centers in the state Z⁺PQ_A⁻Q_B⁻, and its lifetime is determined by the rate of electron transfer from Q_A⁻ to Q_B⁻. The millisecond delayed fluorescence component is associated with recombination in Z⁺PQ_A⁻Q_B⁼ centers with a lifetime determined by the sum of the rate constants of electron transfer from the oxygen-evolving complex to Z⁺ and of the exchange between the reduced and oxidized plastoquinone pool in the Q_B-site. On the basis of these assumptions and of the different share of the three components in the integral delayed fluorescence during induction, an attempt has been made to interpret the changes in the delayed fluorescence intensity during the transition of the photosynthetic apparatus from dark to light adapted state.     

Evidence for the contribution of the S-state transitions of oxygen evolution to the initial phase of fluorescence induction

Fluorescence induction of isolated spinach chloroplasts was measured by using weak continuous light. It is found that the kinetics of the initial phase of fluorescence induction as well as the initial fluorescence level F_j are influenced by the number of preilluminating flashes, and shows damped period 4 oscillation. Evidence is given to show that it is correlated with the S-state transitions of oxygen evolution. Based on the previous observations that the S states can modulate the fluorescence yield of Photosystem II, a simulating calculation suggests that, in addition to the Photosystem II centers inactive in the plastoquinone reduction, the S-state transitions can also make a contribution to the intial phase of fluorescence induction.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - F₀ non-variable fluorescence level emitted when all PS II centers are open - F_i initial fluorescence level immediately after shutter open - F_pt intermediate plateau fluorescence level - F_m maximum fluorescence level emitted when all PS II centers are closed - PS II Photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

The effects of low temperature acclimation and photoinhibitory treatments on Photosystem 2 studied by thermoluminescence and fluorescence decay kinetics

The effects of low temperature acclimation and photoinhibitory treatment on Photosystem 2 (PS 2) have been studied by thermoluminescence and chlorophyll fluorescence decay kinetics after a single turnover saturating flash. A comparison of unhardened and hardened leaves showed that, in the hardened case, a decrease in overall and B-band thermoluminescence emissions occurred, indicating the presence of fewer active PS 2 reaction centers. A modification in the form of the B-band emission was also observed and is attributed to a decrease in the apparent activation energy of recombination in the hardened leaves. The acclimated leaves also produced slower Q_A ^– reoxidation kinetics as judged from the chlorophyll fluorescence decay kinetics. This change was mainly seen in an increased lifetime of the slow reoxidation component with only a small increase in its amplitude. Similar changes in both thermoluminescence and fluorescence decay kinetics were observed when unhardened leaves were given a high light photoinhibitory treatment at 4°C, whereas the hardened leaves were affected to a much lesser extent by a similar treatment. These results suggest that the acclimated plants undergo photoinhibition at 4°C even at low light intensities and that a subsequent high light treatment produces only a small additive photoinhibitory effect. Furthermore, it can be seen that photoinhibition eventually gives rise to PS 2 reaction centers which are no longer functional and which do not produce thermoluminescence or variable chlorophyll fluorescence.Abbreviations D1 The 32 kDa protein of Photosystem 2 reaction center - F_m maximum chlorophyll fluorescence yield - F₀ minimal chlorophyll fluorescence yield obtained when all PS 2 centers are open - F_i intermediate fluorescence level corresponding to PS 2 centers which are loosely or not connected to plastoquinone (non-B centers) - F_v maximum variable chlorophyll fluorescence yield (F_v=F_m–F₀) - PS 2 Photosystem 2 - Q_A and Q_B respectively, primary and secondary quinonic acceptors of PS 2 - S1, S2 and S3 respectively, the one, two and three positively charged states of the oxygen evolving system - Z secondary donor of PS 2     

Recovery of photosystem I and II activities during re-hydration of lichen<Emphasis Type="Italic"> Hypogymnia physodes</Emphasis> thalli

Photochemical efficiencies of photosystem I (PSI) and photosystem II (PSII) were studied in dry thalli of the lichen Hypogymnia physodes and during their re-hydration. In dry thalli, PSII reaction centers are photochemically inactive, as evidenced by the absence of variable chlorophyll (Chl) fluorescence, whereas the primary electron donor of PSI, P700, exhibits irreversible oxidation under continuous light. Upon application of multiple- and, particularly, single-turnover pulses in dry lichen, P700 oxidation partially reversed, which indicated recombination between P700⁺ and the reduced acceptor F_X of PSI. Re-wetting of air-dried H. physodes initiated the gradual restoration of reversible light-induced redox reactions in both PSII and PSI, but the recovery was faster in PSI. Two slow components of P700⁺ reduction occurred after irradiation of partially and completely hydrated thalli with strong white light. In contrast, no slow component was found in the kinetics of re-oxidation of Q_A^–, the reduced primary acceptor of PSII, after exposure of such thalli to white light. This finding indicated the inability of PSII in H. physodes to provide the reduction of the plastoquinone pool to significant levels. It is concluded that slow alternative electron transport routes may contribute to the energetics of photosynthesis to a larger extent in H. physodes than in higher plants.Abbreviations A₀ and A₁ Primary acceptor chlorophyll and secondary electron acceptor phylloquinone - Chl a Chlorophyll a - F_m Maximal level of chlorophyll fluorescence when all PSII centers are closed - F_o Minimal level of fluorescence when all PSII centers are open after dark adaptation - FR Far-red - F_v Variable fluorescence (=F_m–F_o) - F_X, F_A, and F_B Iron–sulfur centers - MT pulse Multiple-turnover pulse - PS Photosystem - P700 Reaction center chlorophyll of PSI - Q_A Primary quinone acceptor of PSII - Q_B Secondary quinone acceptor of PSII - ST pulse Single-turnover pulse     

Characterization of a non-detergent PS II-cytochrome b/f preparation (BS)

A non-detergent photosystem II preparation, named BS, has been characterized by countercurrent distribution, light saturation curves, absorption spectra and fluorescence at room and at low temperature (–196°C). The BS fraction is prepared by a sonication-phase partitioning procedure (Svensson P and Albertsson P-Å, Photosynth Res 20: 249–259, 1989) which removes the stroma lamellae and the margins from the grana and leaves the appressed partition region intact in the form of vesicles. These are closed structures of inside-out conformation. They have a chlorophyll a/b ratio of 1.8–2.0, have a high oxygen evolving capacity (295 mol O₂ per mg chl h), are depleted in P700 and enriched in the cytochrome b/f complex. They have about 2 Photosystem II reaction centers per 1 cytochrome b/f complex.The plastoquinone pool available for PS II in the BS vesicles is 6–7 quinones per reaction center, about the same as for the whole thylakoid. It is concluded, therefore, that the plastoquinone of the stroma lamellae is not available to the PS II in the grana and that plastoquinone does not act as a long range electron transport shuttler between the grana and stroma lamellae.Compared with Photosystem II particles prepared by detergent (Triton X-100) treatment, the BS vesicles retain more cytochrome b/f complex and are more homogenous in their surface properties, as revealed by countercurrent distribution, and they have a more efficient energy transfer from the antenna pigments to the reaction center.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_v variable fluorescence - LHC light-harvesting complex - PpBQ phenyl-p-benzoquinone - PQ plastoquinone pool - P700 reaction center of PS I - PS I, PS II Photosystem I, II - Q_A first bound plastoquinone accepter - RC reaction centre     

Light saturation curves show competence of the water splitting complex in inactive Photosystem II reaction centers

Photosystem II complexes of higher plants are structurally and functionally heterogeneous. While the only clearly defined structural difference is that Photosystem II reaction centers are served by two distinct antenna sizes, several types of functional heterogeneity have been demonstrated. Among these is the observation that in dark-adapted leaves of spinach and pea, over 30% of the Photosystem II reaction centers are unable to reduce plastoquinone to plastoquinol at physiologically meaningful rates. Several lines of evidence show that the impaired reaction centers are effectively inactive, because the rate of oxidation of the primary quinone acceptor, Q_A, is 1000 times slower than in normally active reaction centers. However, there are conflicting opinions and data over whether inactive Photosystem II complexes are capable of oxidizing water in the presence of certain artificial electron acceptors. In the present study we investigated whether inactive Photosystem II complexes have a functional water oxidizing system in spinach thylakoid membranes by measuring the flash yield of water oxidation products as a function of flash intensity. At low flash energies (less that 10% saturation), selected to minimize double turnovers of reaction centers, we found that in the presence of the artificial quinone acceptor, dichlorobenzoquinone (DCBQ), the yield of proton release was enhanced 20±2% over that observed in the presence of dimethylbenzoquinone (DMBQ). We argue that the extra proton release is from the normally inactive Photosystem II reaction centers that have been activated in the presence of DCBQ, demonstrating their capacity to oxidize water in repetitive flashes, as concluded by Graan and Ort (Biochim Biophys Acta (1986) 852: 320–330). The light saturation curves indicate that the effective antenna size of inactive reaction centers is 55±12% the size of active Photosystem II centers. Comparison of the light saturation dependence of steady state oxygen evolution in the presence of DCBQ or DMBQ support the conclusion that inactive Photosystem II complexes have a functional water oxidation system.Abbreviations DCBQ 2,6-dichloro-p-benzoquinone - DMBQ 2,5-dimethyl-p-benzoquinone - F_o initial fluorescence level using dark-adapted thylakoids - Inactive reaction centers reaction centers inactive in plastoquinone reduction - PS II Photosystem II - Q_A primary quinone acceptor of Photosystem II - Q_B secondary quinone acceptor of Photosystem II Department of Plant Biology, University of IllinoisDepartment of Physiology & Biophysics, University of Illinois     

The non-photochemical reduction of plastoquinone in leaves

Although it is generally assumed that the plastoquinone pool of thylakoid membranes in leaves of higher plants is rapidly oxidized upon darkening, this is often not the case. A multiflash kinetic fluorimeter was used to monitor the redox state of the plastoquinone pool in leaves. It was found that in many species of plants, particularly those using the NAD-malic enzyme C₄ system of photosynthesis, the pool actually became more reduced following a light to dark transition. In some Amaranthus species, plastoquinone remained reduced in the dark for several hours. Far red light, which preferentially drives Photosystem I turnover, could effectively oxidize the plastoquinone pool. Plastoquinone was re-reduced in the dark within a few seconds when far red illumination was removed. The underlying mechanism of the dark reduction of the plastoquinone pool is still uncertain but may involve chlororespiratory activity.Abbreviations apparent F_o observed fluorescence yield after dark adaptation - F_m maximum fluorescence when all Q_A is fully reduced - F_o minimum fluorescence yield when Q_A is fully oxidized and non-photochemical quenching is fully relaxed - F_s steady state fluorescence yield - PPFD photosynthetic photon flux density - PQ plastoquinone - Q_A primary quinone acceptor of the Photosystem II reaction center - Q_B secondary quinone acceptor to the Photosystem II reaction center - F F_m minus F_s     

Photoelectric study on the kinetics of trapping and charge stabilization in oriented PS II membranes

Excitation energy trapping and charge separation in Photosystem II were studied by kinetic analysis of the fast photovoltage detected in membrane fragments from peas with picosecond excitation. With the primary quinone acceptor oxidized the photovoltage displayed a biphasic rise with apparent time constants of 100–300 ps and 550±50 ps. The first phase was dependent on the excitation energy whereas the second phase was not. We attribute these two phases to trapping (formation of P-680⁺ Phe^-) and charge stabilization (formation of P-680⁺ Q_A ^-), respectively. A reversibility of the trapping process was demonstrated by the effect of the fluorescence quencher DNB and of artificial quinone acceptors on the apparent rate constants and amplitudes. With the primary quinone acceptor reduced a transient photoelectric signal was observed and attributed to the formation and decay of the primary radical pair. The maximum concentration of the radical pair formed with reduced Q_A was about 30% of that measured with oxidized Q_A. The recombination time was 0.8–1.2 ns.The competition between trapping and annihilation was estimated by comparison of the photovoltage induced by short (30 ps) and long (12 ns) flashes. These data and the energy dependence of the kinetics were analyzed by a reversible reaction scheme which takes into account singlet-singlet annihilation and progressive closure of reaction centers by bimolecular interaction between excitons and the trap. To put on firmer grounds the evaluation of the molecular rate constants and the relative electrogenicity of the primary reactions in PS II, fluorescence decay data of our preparation were also included in the analysis. Evidence is given that the rates of radical pair formation and charge stabilization are influenced by the membrane potential. The implications of the results for the quantum yield are discussed.Abbreviations DCBQ 2,6-dichloro-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNB m-dinitrobenzene - PPBQ phenyl-p-benzoquinone - PS I photosystem I of green plants - PS II photosystem II of green plants - PSU photosynthetic unit - P-680 primary donor of PS II - Phe intermediary pheophytin acceptor of PS II - Q_A primary quinone acceptor of PS II - RC reaction center     

Relaxation of variable chlorophyll fluorescence after illumination of dark-adapted barley leaves as influenced by the redox states of electron carriers

Kinetics of the dark relaxation of variable chlorophyll fluorescence, Fv, were studied after brief illumination of dark-adapted barley leaves in order to understand the rapid reversibility of pulse-induced fluorescence increases, which is observed even when fast linear electron transport to an external electron acceptor is not possible. Four kinetically distinct components were observed which reveal complexity in the oxidation of the reduced primary quinone acceptor of Photosystem II, Q_A ⁻: the slowest component accounted for 4–5% of maximal Fv and had a life-time of several seconds. It is suggested to represent a minor population of inactive Photosystem II centers. The other three components displayed first-order kinetics with half-time of 6–8 ms (`fast' component), 60–80 ms (`middle' component) and 650–680 ms (`slow' component). The fast component dominated Fv when methyl viologen or far-red light accelerated oxidation of plastohydroquinone. It shows rapid oxidation of Q_A ⁻ during electron flow to plastoquinone commensurate with maximum linear electron flow through the electron transport chain. The other two components were observed under conditions of restricted electron flow and excessive reduction of electron carriers. Unexpectedly, the slow component, which is interpreted to reflect the recombination between Q_A ⁻ and an intermediate on the oxidizing side of Photosystem II, saturated already at low irradiances of actinic light when plastoquinone was not yet strongly reduced suggesting that dark-adaptation of leaves results not only in the loss of activity of light-regulated enzymes of the carbon cycle but affects also electron flow from Q_A ⁻ to plastoquinone. KCN poisoning or high temperature treatment of leaves produced a nonexponential pattern of slow Fv relaxation. This effect was largely (heat treatment) or even completely (KCN) abolished by far-red light. This revised version was published online in August 2006 with corrections to the Cover Date.     

Chimaeric CP47 mutants of the cyanobacterium Synechocystis sp. PCC 6803 carrying spinach sequences: Construction and function

Chimaeric mutants of the cyanobacterium Synechocystis sp. PCC 6803 have been generated carrying part or all of the spinach psbB gene, encoding CP47 (one of the chlorophyll-binding core antenna proteins in Photosystem II). The mutant in which the entire psbB gene had been replaced by the homologous gene from spinach was an obligate photoheterotroph and lacked Photosystem II complexes in its thylakoid membranes. However, this strain could be transformed with plasmids carrying selected regions of Synechocystis psbB to give rise to photoautotrophs with a chimaeric spinach/cyanobacterial CP47 protein. This process involved heterologous recombination in the cyanobacterium between psbB sequences from spinach and Synechocystis 6803; which was found to be reasonably effective in Synechocystis. Also other approaches were used that can produce a broad spectrum of chimaeric mutants in a single experiment. Functional characterization of the chimaeric photoautotrophic mutants indicated that if a decrease in the photoautotrophic growth rates was observed, this was correlated with a decrease in the number of Photosystem II reaction centers (on a chlorophyll basis) in the thylakoid membrane and with a decrease in oxygen evolution rates. Remaining Photosystem II reaction centers in these chimaeric mutants appeared to function rather normally, but thermoluminescence and chlorophyll a fluorescence measurements provided evidence for a destabilization of Q_B ^–. This illustrates the sensitivity of the functional properties of the PS II reaction center to mild perturbations in a neighboring protein.Abbreviations diuron 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_v variable chlorophyll a fluorescence - HEPES N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) - (k)bp (kilo)base pairs - PS II Photosystem II - Q_A primary electron-accepting plastoquinone in Photosystem II - Q_B secondary electron-accepting plastoquinone in Photosystem II - SDS sodium dodecyl sulfate     

Fluorescence induction curves registered from individual microalgae cenobiums in the process of population growth

Registration of chlorophyll fluorescence induction curves (IC) from individual microalgae cenobiums was performed during Scenedesmus quadricauda culture growth. Emphasis was placed on the analysis of patterns of the slow phase of IC, since these slow fluorescence transitions reflect complex interactions between primary and secondary photosynthetic processes. A classification was performed of the ICs obtained according to the patterns of their slow phase. Four different types of such patterns were distinguished. The microalgae population structure with respect to IC patterns was investigated at different stages of culture growth. The distribution of microalgae cenobiums over the patterns of IC was found to change in accordance with the stage of population development. At the stage of the population growth enhancement, nonmonotonous IC dominated with a high steady-state level of fluorescence. The stage of linear growth was characterized by IC with monotonous decay kinetics and low steady-state level of fluorescence. At the third stage including the phases of growth inhibition, stationary state and the beginning of cell death the population structure was the most heterogeneous, with all IC patterns observed.Abbreviations CO₂ carbon dioxide - ETC electron transfer chain - Fl fluorescence - FNR ferredoxin-NADPH reductase - IC induction curve of chlorophyll fluorescence - PQ plastoquinone - PS I Photosystem I - PS II Photosystem II - Q_A primary quinone acceptor of PS II     

Photosystem II heterogeneity: the acceptor side

It is well known that two photosystems, I and II, are needed to transfer electrons from H₂O to NADP⁺ in oxygenic photosynthesis. Each photosystem consists of several components: (a) the light-harvesting antenna (L-HA) system, (b) the reaction center (RC) complex, and (c) the polypeptides and other co-factors involved in electron and proton transport. First, we present a mini review on the heterogeneity which has been identified with the electron acceptor side of Photosystem II (PS II) including (a) L-HA system: the PS II and PS II units, (b) RC complex containing electron acceptor Q₁ or Q₂; and (c) electron acceptor complex: Q_A (having two different redox potentials Q_L and Q_H) and Q_B (Q_B-type; Q'_B type; and non-Q_B type); additional components such as iron (Q-400), U (E_m,7=–450 mV) and Q-318 (or Aq) are also mentioned. Furthermore, we summarize the current ideas on the so-called inactive (those that transfer electrons to the plastoquinone pool rather slowly) and active reaction centers. Second, we discuss the bearing of the first section on the ratio of the PS II reaction center (RC-II) and the PS I reaction center (RC-I). Third, we review recent results that relate the inactive and active RC-II, obtained by the use of quinones DMQ and DCBQ, with the fluorescence transient at room temperature and in heated spinach and soybean thylakoids. These data show that inactive RC-II can be easily monitored by the OID phase of fluorescence transient and that heating converts active into inactive centers.Abbreviations DCBQ 2,5 or 2,6 dichloro-p-benzoquinone - DMQ dimethylquinone - Q_A primary plastoquinone electron acceptor of photosystem II - Q_B secondary plastoquinone electron acceptor of photosystem II - IODP successive fluorescence levels during time course of chlorophyll a fluorescence: O for origin, I for inflection, D for dip or plateau, and P for peak     

Photoinhibition and light-dependent turnover of the D1 reaction-centre polypeptide of photosystem II are enhanced by mineral-stress conditions

The function of photosystem II (PSII) and the turnover of its D1 reaction-center protein were studied in spinach (Spinacia oleracea L.) plants set under mineral stress. The mineral deficiencies were induced either by supplying the plants with an acidic nutrient solution or by strongly reducing the supply of magnesium alone or together with sulfur. After exposure for 8–10 weeks to the different media, the plants were characterized by a loss of chlorophyll and an increase in starch content, indicating a disturbance in the allocation of assimilates. Depending on the severity of the mineral deficiencies the plants lost their ability to adapt even to moderate iradiances of 400 mol photons·m^–2·s^–1 and became photoinhibited, as indicated by the decrease in F_v/F_m (the ratio of yield of variable fluorescence to yield of maximal fluorescence when all reaction centers are closed). The loss of PSII function was induced by changes on the acceptor side of PSII. Fast fluorescence decay showed a loss of PSII centers with bound Q_B, the secondary quinone acceptor of PSII, and a fast reoxidation kinetic of q _a ^- , the primary quinone acceptor of PSII, in the photoinactivated plants. No appreciable change could be observed in the amount of PSII centers with unbound Q_B and in Q_B-nonreducing PSII centers. Immunological studies showed that the contents of the D1 and D2 proteins of the PSII reaction center and of the 33-kDa protein of the water-splitting complex were diminished in the photoinhibited plants, and the occurrance of a new polypetide of 14 kDa that reacted with an antibody against the C-termius of the D1 protein. As shown by pulse-labelling experiments with [¹⁴C]leucine both degradation and synthesis of the D1 protein were enhanced in the mineral-deficient plants when compared to non-deficient plants. A stimulation of D1-protein turnover was also observed in pH 3-grown plants, which were not inhibited at growth-light conditions. Obviously, stimulation of D1-protein turnover prevented photoinhibition in these plants. However, in the Mg- and Mg/S-deficient plants even a further stimulation of D1-protein turnover could not counteract the increased rate of photoinactivation.Abbreviations amp_(f,m,s) amplitude of the fast, (medium and slow) exponential component of fluorescence decay - F_m yield of maximum fluorescenc when all reaction centers are closed - F_o yield of intrinsic fluorescence at open PSII reaction centers in the dark - F_v yield of variable fluorescence, (difference between F_m and F_o) - LHC light-harvesting complex - PFD photon flux density - Q_A primary quinone acceptor of PSII - Q_B secondary quinone acceptor of PSII Dedicated to Professor Dr. Dres. hc. Achim Trebst on the occasion of his 65th birthdayThis work was supported by grants from the BMFT and the Ministerium für Umwelt, Raumordnung and Landwirtschaft, Nordrhein-Westfalen. The authors thank H. Wietoska and M. Bronzel for skilful technical assistance.     

Light-dependent modification of Photosystem II in spinach leaves

In dark-adapted spinach leaves approximately one third of the Photosystem II (PS II) reaction centers are impaired in their ability to transfer electrons to Photosystem I. Although these inactive PS II centers are capable of reducing the primary quinone acceptor, Q_A, oxidation of Q_A ^– occurs approximately 1000 times more slowly than at active centers. Previous studies based on dark-adapted leaves show that minimal energy transfer occurs from inactive centers to active centers, indicating that the quantum yield of photosynthesis could be significantly impaired by the presence of inactive centers. The objective of the work described here was to determine the performance of inactive PS II centers in light-adapted leaves. Measurements of PS II activity within leaves did not indicate any increase in the concentration of active PS II centers during light treatments between 10 s and 5 min, showing that inactive centers are not converted to active centers during light treatment. Light-induced modification of inactive PS II centers did occur, however, such that 75% of these centers were unable to sustain stable charge separation. In addition, the maximum yield of chlorophyll fluorescence associated with inactive PS II centers decreased substantially, despite the lack of any overall quenching of the maximum fluorescence yield. The effect of light treatment on inactive centers was reversed in the dark within 10–20 mins. These results indicate that illumination changes inactive PS II centers into a form that quenches fluorescence, but does not allow stable charge separation across the photosynthetic membrane. One possibility is that inactive centers are converted into centers that quench fluorescence by formation of a radical, such as reduced pheophytin or oxidized P680. Alternatively, it is possible that inactive PS II centers are modified such that absorbed excitation energy is dissipated thermally, through electron cycling at the reaction center.Abbreviations A518 absorbance change at 518 nm, reflecting the formation of an electric field across the thylakoid membrane - A_FL1 amplitude of the fast (<100 ms) phase of A518 induced by the first of two saturating, single-turnover flashes spaced 30 ms apart - A_FL2 amplitude of the fast (<100 ms) phase of A518 induced by the second of two saturating, single-turnover flashes spaced 50 ms apart - DCBQ 2,6-dichloro-p-benzoquinone - Fo yield of chlorophyll fluorescence when Q_A is fully oxidized - Fm yield of chlorophyll fluorescence when Q_A is fully reduced - Fx yield of chlorophyll fluorescence when Q_A is fully reduced at inactive PS II centers, but fully oxidized at active PS II centers - Pheo pheophytin - P680 the primary donor of Photosystem II - PPFD photosynthetic photon flux density - Q_A Primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

Low-Intensity picosecond fluorescence kinetics and excitation dynamics in barley chloroplasts

The fluorescence decays of barley chloroplasts have been measured by single-photon counting with tunable picosecond dye laser excitation. The fluorescence decays of dark-adapted chloroplasts are best fitted to a sum of three exponential lifetime components with lifetimes of 112, 380 and 2214 ps. The relative magnitude of each component is shown to be dependent on the excitation wavelength and collected emission wavelength. The excitation wavelength dependence is correlated with the Photosystem (PS) I and PS II action study of Ried [36] and with the measured pigment distributions in the photosynthetic unit [37,41]. Experiments varying the single excitation pulse intensity from 10⁸ to 10¹² photons/cm² pulse show that our results are not distorted by singlet-singlet annihilation. Unflowed samples where the cloroplasts are under constant illumination show 2-fold increases in quantum yield of fluorescence primarily in the two longer lifetime components. Theoretical calculations of Shipman [31] on an isolated reaction center with a homogeneous antenna are discussed and the principles extended to discussion of the measured barley chloroplast fluorescence decay components in terms of photosynthetic unit light-harvesting array models and earlier experimental work. Our data support a photosynthetic unit model in which 70–90% of the photons absorbed are quenched by either PS I or efficiently quenching PS II in a process where the fluorescence lifetime is 100 ps. The origin of the intermediate 380 ps. component is probably due to excitation transfer to a PS II reaction center in a redox state which quenches less efficiently.     

Mechanism of photoinhibition: photochemical reaction center inactivation in system II of chloroplasts

Photoinhibition of photosynthesis is manifested at the level of the leaf as a loss of CO₂ fixation and at the level of the chloroplast thylakoid membrane as a loss of photosystem II electron-transport capacity. At the photosystem II level, photoinhibition is manifested by a lowered chlorophyll a variable fluorescence yield, by a lowered amplitude of the light-induced absorbance change at 320 nm (A₃₂₀) and 540-minus-550 nm (A_540–550), attributed to inhibition of the photoreduction of the primary plastoquinone Q_A molecule. A correlation of the kinetics of variable fluorescence yield loss with the inhibition of Q_A photoreduction suggested that photoinhibited reaction centers are incapable of generating a stable charge separation but are highly efficient in the trapping and non-photochemical dissipation of absorbed light. The direct effect of photoinhibition on primary photochemical parameters of photosystem II suggested a permanent reaction center modification the nature of which remains to be determined.Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement     

Fluorescence induction of Photosystem II membranes shows the steps till reduction and protonation of the quinone pool

Chlorophyll fluorescence induction (Chl-F) was investigated in Photosystem II (PSII)-enriched membranes, which predominantly include active (Q_B reducing) PSII reaction centres (RCs) and lack Photosystem I (PSI). The Chl-F curve of these preparations show a polyphasic rise from F₀, the minimal fluorescence, to F_P, the maximal fluorescence, with several intermediate transitions. Analyses of these transitions revealed three exponential rise components with lifetimes of 18 ms, 400 ms and 800 ms. The 18 ms component was assigned to the photoaccumulation of reduced Q_A. The two slowest components, of 400 ms and 800 ms, were assigned to Q_B reduction (Q_B⁻ and Q_B⁼) and further Q_B⁼ protonation (till Q_BH₂), respectively. These assignments were based on the observation of specific quenching of the phases by DCMU or by different oxidized, reduced and protonated quinones. The work is done in low light conditions which are saturating to avoid photoinhibition or PSII inactivation effects. The results suggest that the Chl-F curve observed in PSII-enriched membranes can be attributed to the sequential steps till the photoaccumulation (reduction and protonation) of plastoquinone (PQ) by PSII. These results are in good agreement with the molecular models that show a correspondence between Chl-F and PQ reduction steps, like the models that propose and explain the O-J-I-P transients.     

Cyclic electron flow around Photosystem II in vivo

The oxygen flash yield (Y_O2) and photochemical yield of PS II (_{PS II}) were simultaneously detected in intact Chlorella cells on a bare platinum oxygen rate electrode. The two yields were measured as a function of background irradiance in the steady-state and following a transition from light to darkness. During steady-state illumination at moderate irradiance levels, Y_O2 and _{PS II} followed each other, suggesting a close coupling between the oxidation of water and Q_A reduction (Falkowski et al. (1988) Biochim. Biophys. Acta 933: 432–443). Following a light-to-dark transition, however, the relationship between Q_A reduction and the fraction of PS II reaction centers capable of evolving O₂ became temporarily uncoupled. _{PS II} recovered to the preillumination levels within 5–10 s, while the Y_O2 required up to 60 s to recover under aerobic conditions. The recovery of Y_O2 was independent of the redox state of Q_A, but was accompanied by a 30% increase in the functional absorption cross-section of PS II (_{PS II}). The hysteresis between Y_O2 and the reduction of Q_A during the light-to-dark transition was dependent upon the reduction level of the plastoquinone pool and does not appear to be due to a direct radiative charge back-reaction, but rather is a consequence of a transient cyclic electron flow around PS II. The cycle is engaged in vivo only when the plastoquinone pool is reduced. Hence, the plastoquinone pool can act as a clutch that disconnects the oxygen evolution from photochemical charge separation in PS II.Abbreviations ADRY acceleration of the deactivation reactions of the water-splitting enzyme (agents) - Chl chlorophyll - cyt cytochrome - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_O minimum fluorescence yield in the dark-adapted state - F_I minimum fluorescence yield under ambient irradiance or during transition from the light-adapted state - F_M maximum fluorescence yield in the dark-adapted state - F_M maximum fluorescence yield under ambient irradiance or during transition from light-adapted state - F_V, F_V variable fluorescence (F_V=F_M–F_O ; F_V=F_M–F_I) - FRR fast repetition rate (fluorometer) - _{PS II} quantum yield of Q_A reduction (_{PS II}=(F_M – F_O)/F_M or _{PS II})=(F_M= – F_I=)/F_M=) - LHCII Chl a/b light harvesting complexes of Photosystem II - OEC oxygen evolving complex of PS II - P680 reaction center chlorophyll of PS II - PQ plastoquinone - POH₂ plastoquinol - PS I Photosystem I - PS II Photosystem II - RC II reaction centers of Photosystem II - PS II the effective absorption cross-section of PHotosystem II - TL thermoluminescence - Y_O2 oxygen flash yield The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

Acceptor side photoinhibition in PS II: On the possible effects of the functional integrity of the PS II donor side on photoinhibition of stable charge separation

Photoinhibition under aerobic and anaerobic conditions was analyzed in O₂-evolving and in Tris-treated PS II-membrane fragments from spinach by measuring laser-flash-induced absorption changes at 826 nm reflecting the transient P680⁺ formation and the chlorophyll fluorescence lifetime. It was found that anaerobic photoinhibitory treatment leads in both types of samples to the appearence of two long-lived fluorescence components with lifetimes of 7 ns and 16 ns, respectively. The extent of these fluorescence kinetics depends on the state of the reaction center (open/closed) during the fluorescence measurements: it is drastically higher in the closed state. It is concluded that this long-lived fluorescence is mainly emitted from modified reaction centers with singly reduced Q_A(Q_A ^-). This suggests that the observation of long-lived fluorescence components cannot necessarily be taken as an indicator for reaction centers with missing or doubly reduced and protonated Q_A (Q_AH₂). Time-resolved measurements of 826 nm absorption changes show that the rate of photoinhibition of the stable charge separation (P680^*Q_A P680⁺Q_A ^-), is nearly the same in O₂-evolving and in Tris-treated PS II-membrane fragments. This finding is difficult to understand within the framework of the Q_AH₂-mechanism for photoinhibition of stable charge separation because in that case the rate of photoinhibition should strongly depend on the functional integrity of the donor side of PS II. Based on the results of this study it is inferred, that several processes contribute to photoinhibition within the PS II reaction center and that a mechanism which comprises double reduction and protonation of Q_A leading to Q_AH₂ formation is only of marginal – if any – relevance for photoinhibition of PS II under both, aerobic and anaerobic, conditions.