Evidence for a fluorescence yield change driven by a light-induced conformational change within photosystem II during the fast chlorophyll a fluorescence rise

Experiments were carried out to identify a process co-determining with Q(A) the fluorescence rise between F(0) and F(M). With 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU), the fluorescence rise is sigmoidal, in its absence it is not. Lowering the temperature to -10°C the sigmoidicity is lost. It is shown that the sigmoidicity is due to the kinetic overlap between the reduction kinetics of Q(A) and a second process; an overlap that disappears at low temperature because the temperature dependences of the two processes differ. This second process can still relax at -60°C where recombination between Q(A)(-) and the donor side of photosystem (PS) II is blocked. This suggests that it is not a redox reaction but a conformational change can explain the data. Without DCMU, a reduced photosynthetic electron transport chain (ETC) is a pre-condition for reaching the F(M). About 40% of the variable fluorescence relaxes in 100ms. Re-induction while the ETC is still reduced takes a few ms and this is a photochemical process. The fact that the process can relax and be re-induced in the absence of changes in the redox state of the plastoquinone (PQ) pool implies that it is unrelated to the Q(B)-occupancy state and PQ-pool quenching. In both +/-DCMU the process studied represents ~30% of the fluorescence rise. The presented observations are best described within a conformational protein relaxation concept. In untreated leaves we assume that conformational changes are only induced when Q(A) is reduced and relax rapidly on re-oxidation. This would explain the relationship between the fluorescence rise and the ETC-reduction.     

Parameterization of photosystem II photoinactivation and repair

The photoinactivation (also termed photoinhibition or photodamage) of Photosystem II (PSII) and the counteracting repair reactions are fundamental elements of the metabolism and ecophysiology of oxygenic photoautotrophs. Differences in the quantification, parameterization and terminology of Photosystem II photoinactivation and repair can erect barriers to understanding, and particular parameterizations are sometimes incorrectly associated with particular mechanistic models. These issues lead to problems for ecophysiologists seeking robust methods to include photoinhibition in ecological models. We present a comparative analysis of terms and parameterizations applied to photoinactivation and repair of Photosystem II. In particular, we show that the target size and quantum yield approaches are interconvertible generalizations of the rate constant of photoinactivation across a range of incident light levels. Our particular emphasis is on phytoplankton, although we draw upon the literature from vascular plants. This article is part of a Special Issue entitled: Photosystem II.     

A simple chlorophyll fluorescence parameter that correlates with the rate coefficient of photoinactivation of Photosystem II

A method of partitioning the energy in a mixed population of active and photoinactivated Photosystem II (PS II) complexes based on chlorophyll fluorescence measurements is presented. There are four energy fluxes, each with its quantum efficiency: a flux associated with photochemical electron flow in active PS II reaction centres (J_{PS II}), thermal dissipation in photoinactivated, non-functional PS IIs (J_NF), light-regulated thermal dissipation in active PS IIs (J_NPQ) and a combined flux of fluorescence and constitutive, light-independent thermal dissipation (J_f,D). The four quantum efficiencies add up to 1.0, without the need to introduce an ‘excess’ term E, which in other studies has been claimed to be linearly correlated with the rate coefficient of photoinactivation of PS II (k_pi). We examined the correlation of k_pi with various fluxes, and found that the combined flux (J_NPQ + J_f,D= J_pi) is as well correlated with k_pi as is E. This combined flux arises from F_s/F_m^′, the ratio of steady-state to maximum fluorescence during illumination, which represents the quantum efficiency of combined non-photochemical dissipation pathways in active PS IIs. Since F_s/F_m^′ or its equivalent, J_pi, is a likely source of events leading to photoinactivation of PS II, we conclude that F_s/F_m^′ is a simple predictor of k_pi.     

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.     

Analysis of elevated temperature-induced inhibition of photosystem II using chlorophyll a fluorescence induction kinetics in wheat leaves (Triticum aestivum)

Wheat is the major crop plant in many parts of the world. Elevated temperature-induced changes in photosynthetic efficiency were studied in wheat (T. aestivum) leaves by measuring Chl a fluorescence induction kinetics. Detached leaves were subjected to elevated temperature stress of 35 °C, 40 °C or 45 °C. Parameters such as Fv/Fm, performance index (PI), and reaction centre to absorbance ratio (RC/ABS) were deduced using radial plots from fluorescence induction curves obtained with a plant efficiency analyser (PEA). To derive precise information on fluorescence induction kinetics, energy pipeline leaf models were plotted using biolyzer hp3 software. At 35 °C, there was no effect on photosynthetic efficiency, including the oxygen-evolving complex, and the donor side of PSII remained active. At 40 °C, activity was reduced by 14%, while at 45 °C, a K intermediate step was observed, indicating irreversible damage to the oxygen-evolving complex. This analysis can be used to rapidly screen for vitality and stress tolerance characteristics of wheat growing in the field under high temperature stress.     

Thermodynamic investigation into the mechanism of the chlorophyll fluorescence quenching in isolated photosystem II light-harvesting complexes

Chlorophyll fluorescence quenching can be stimulated in vitro in purified photosystem II antenna complexes. It has been shown to resemble nonphotochemical quenching observed in isolated chloroplasts and leaves in several important respects, providing a model system for study of the mechanism of photoprotective energy dissipation. The effect of temperature on the rate of quenching in trimeric and monomeric antenna complexes revealed the presence of two temperature-dependent processes with different activation energies, one between approximately 15 and 35 degrees C and another between approximately 40 and 60 degrees C. The temperature of the transition between the two phases was higher for trimers than for monomers. Throughout this temperature range, the quenching was almost completely reversible, the protein CD was unchanged, and pigment binding was maintained. The activation energy for the low temperature phase was consistent with local rearrangements of pigments within some of the protein domains, whereas the higher temperature phase seemed to arise from large scale conformational transitions. For both phases, there was a strong linear correlation between the quenching rate and the appearance of an absorption band at 685 nm. In addition, quenching was correlated with a loss of CD at approximately 495 nm from Lutein 1 and at 680 nm from chlorophylls a1 and a2, the terminal emitters. The results obtained indicate that quenching of chlorophyll fluorescence in antenna complexes is brought about by perturbation of the lutein 1/chlorophyll a1/chlorophyll a2 locus, forming a poorly fluorescing chlorophyll associate, either a dimer or an excimer.     

Chlorophyll a fluorescence rise induced by high light illumination of dark-adapted plant tissue studied by means of a model of photosystem II and considering photosystem II heterogeneity

Chlorophyll a fluorescence rise (FLR) measured in vivo in dark-adapted plant tissue immediately after the onset of high light continuous illumination shows complex O-K-J-I-P transient. The steps typically appear at about 400 micros (K), 2 ms (J), 30 ms (I), and 200 - 500 ms (P) and a transient decrease of fluorescence to local minima (dips D) can be observed after the K, J, and I steps. As the FLR reflects a function of photosystem II (PSII) and to more understand the FLR, a PSII reactions model was formulated comprising equilibrium of excited states among all light harvesting and reaction centre pigments and P680, reversible radical pair formation and the donor and acceptor side functions. Such a formulated model is the most detailed and complex model of PSII reactions used so far for simulations of the FLR. By varying of selected model parameters (rate constants and initial conditions) several conclusions can be made as for the origin of and changes in shape of the theoretical FLR and compare them with in-literature-reported results. For homogeneous population of PSII and using standard in-literature-reported values of the model parameters, the simulated FLR is characterized by reaching the minimal fluorescence F(0) at about 3 ns after the illumination is switched on lasting to about 1 micros, followed by fluorescence rise to a plateau located at about 2 ms and subsequent fluorescence rise to a global maximum that is reached at about 60 ms. Varying of the values of rate constants of fast processes that can compete for utilization of the excited states with fluorescence emission does not change qualitatively the shape of the FLR. However, primary photochemistry of PSII (the charge separation, recombination and stabilization), non-radiative loss of excited states in light harvesting antennae and excited states quenching by oxidized plastoquisnone (PQ) molecules from the PQ pool seem to be the main factors controlling the maximum quantum yield of PSII photochemistry as expressed by the F(V)/F(M) ratio. The appearance of the plateau at about 2 ms in the FLR is affected by several factors: the height of the plateau in the FLR increases when the fluorescence quenching by oxidized P680(+) is not considered in the simulations or when the electron transfer from Q(A)(-) to Q(B)((-)) is slowed down whereas the height of the plateau decreases and its position is shifted to shorter times when OEC is initially in higher S state. The plateau at about 2 ms is changed into the local fluorescence maximum followed by a dip when the fluorescence quenching by oxidized PQ molecules or the charge recombination between P680(+) and Q(A)(-) is not considered in the simulations or when all OEC is initially in the S(0) state or when the S -state transitions of OEC are slowed down. Slowing down of the S -state transitions of OEC as well as of the electron transfer from Q(A)(-) to Q(B)((-)) also causes a decrease of maximal fluorescence level. In the case of full inhibition of the S -state transitions of OEC as well as in the case of full inhibition of the electron donation to P680(+) by Y(Z), the local fluorescence maximum becomes the global fluorescence maximum. Assuming homogeneous PSII population, theoretical FLR curve that only far resembles experimentally measured O-J-I-P transient at room temperature can be simulated when slowly reducing PQ pool is considered. Assuming heterogeneous PSII population (i.e. the alpha/beta and the Q(B) -reducing/Q(B)-non-reducing heterogeneity and heterogeneity in size of the PQ pool and rate of its reduction) enables to simulate the FLR with two steps between minimal and maximal fluorescence whose relative heights are in agreement with the experiments but not their time positions. A cause of this discrepancy is discussed as well as different approaches to the definition of fluorescence signal during the FLR.     

Thermostability and photostability of photosystem II of the resurrection plant Haberlea rhodopensis studied by chlorophyll fluorescence

The stability of PSII in leaves of the resurrection plant Haberlea rhodopensis to high temperature and high light intensities was studied by means of chlorophyll fluorescence measurements. The photochemical efficiency of PSII in well-hydrated Haberlea leaves was not significantly influenced by temperatures up to 40 degrees C. Fo reached a maximum at 50 degrees C, which is connected with blocking of electron transport in reaction center II. The intrinsic efficiency of PSII photochemistry, monitored as Fv/Fm was less vulnerable to heat stress than the quantum yield of PSII electron transport under illumination (phiPSII). The reduction of phiPSII values was mainly due to a decrease in the proportion of open PSII centers (qP). Haberlea rhodopensis was very sensitive to photoinhibition. The light intensity of 120 micromol m(-2) s(-1) sharply decreased the quantum yield of PSII photochemistry and it was almost fully inhibited at 350 micromol m(-2) s(-1). As could be expected decreased photochemical efficiency of PSII was accompanied by increased proportion of thermal energy dissipation, which is considered as a protective effect regulating the light energy distribution in PSII. When differentiating between the three components of qN it was evident that the energy-dependent quenching, qE, was prevailing over photoinhibitory quenching, qI, and the quenching related to state 1-state 2 transitions, qT, at all light intensities at 25 degrees C. However, the qE values declined with increasing temperature and light intensities. The qI was higher than qE at 40 degrees C and it was the major part of qN at 45 degrees C, indicating a progressing photoinhibition of the photosynthetic apparatus.     

The time course of photoinactivation of photosystem II in leaves revisited

Since photosystem II (PS II) performs the demanding function of water oxidation using light energy, it is susceptible to photoinactivation during photosynthesis. The time course of photoinactivation of PS II yields useful information about the process. Depending on how PS II function is assayed, however, the time course seems to differ. Here, we revisit this problem by using two additional assays: (1) the quantum yield of oxygen evolution in limiting, continuous light and (2) the flash-induced cumulative delivery of PS II electrons to the oxidized primary donor (P700(+)) in PS I measured as a 'P700 kinetics area'. The P700 kinetics area is based on the fact that the two photosystems function in series: when P700 is completely photo-oxidized by a flash added to continuous far-red light, electrons delivered from PS II to PS I by the flash tend to re-reduce P700(+) transiently to an extent depending on the PS II functionality, while the far-red light photo-oxidizes P700 back to the steady-state concentration. The quantum yield of oxygen evolution in limiting, continuous light indeed decreased in a way that deviated from a single-negative exponential. However, measurement of the quantum yield of oxygen in limiting light may be complicated by changes in mitochondrial respiration between darkness and limiting light. Similarly, an assay based on chlorophyll fluorescence may be complicated by the varying depth in leaf tissue from which the signal is detected after progressive photoinactivation of PS II. On the other hand, the P700 kinetics area appears to be a reasonable assay, which is a measure of functional PS II in the whole leaf tissue and independent of changes in mitochondrial respiration. The P700 kinetics area decreased in a single-negative exponential fashion during progressive photoinactivation of PS II in a number of plant species, at least at functional PS II contents ≥6?% of the initial value, in agreement with the conclusion of Sarvikas et al. (Photosynth Res 103:7-17, 2010). That is, the single-negative-exponential time course does not provide evidence for photoprotection of functional PS II complexes by photoinactivated, connected neighbours.     

Global changes of chlorophyll excitonic interactions in photosystem II during thermal denaturation

Photosystem II (PSII) is a multisubunit chlorophyll-binding enzyme that absorbs light to catalyze water oxidation and plastoquinone reduction. Chlorophyll excitonic interaction changes in PSII were studied by absorption and circular dichroism spectra from 25 degrees C to 80 degrees C, and protein subunit denaturation was monitored by differential scanning calorimetry. A four-stage process of chlorophyll excitonic interaction change was observed being correlated with the denaturation of protein subunits.     

Thermally-induced delayed fluorescence of photosystem I and II chlorophyll in thermophilic cyanobacterium Synechococcus elongatus.

Stationary delayed fluorescence (DF) of chlorophyll in isolated membrane preparations from thermophilic cyanobacterium Synechococcus elongatus was investigated as a function of temperature. Two peaks at different temperatures were observed. The low-temperature peak (54-60 degrees C) coincided with the main maximum of the thermally-induced delayed fluorescence of chlorophyll in intact cells and PSII-particles with active oxygen-evolving system. The high-temperature peak (78 degrees C) coincided with the minor band of delayed light emitted by intact cells. It was also observed in the delayed fluorescence emission from a PSI-enriched fraction preparation. The intensities of the DF peaks were dependent on the presence of inhibitors, donors and acceptors that cause specific effects on electron transport of the two photosystems. The low-temperature and high-temperature peaks were related to PSII and PSI, respectively. The manifestation of delayed fluorescence from PSI and PSII at different temperatures seems to be a specific property of thermophilic cyanobacteria. The reason for this may be a high thermal stability of the photosystems and the lack of the PSII antenna complex in isolated membranes. Consequently, the relative yield of delayed fluorescence from PSI markedly increases. Thermally-induced fluorescence seen in membranes of cyanobacteria showed a high sensitivity to structural and functional membrane alterations induced by pH changes, different electron transport stabilizing agents or different concentrations of MgCl2.     

Photoinhibition of photosystems I and II using chlorophyll fluorescence measurements

In this study the photoinhibition of photosystems (PS) I and II caused by exposure to high intensity light in oat (Avena sativa, var Prevision) is measured by the emission of chlorophyll fluorescence in intact leaves adapted to darkness. The maximal quantum yield of PS II was lower in plants grown under high light intensity than in plants grown under low intensity, which indicates that PS II is photoinhibited by such conditions. PS I was more stable than PS II in plants exposed to strong light for a moderate time (five photoperiods) since the oxidised plastoquinone pool size under far-red (FR) light was similar in plants grown under high light intensity to plants grown under low intensity, probably as a result of the cyclic electron flow around PS I being stimulated in response to high light intensity. However, over longer times (10 photoperiods) the PS I was photoinhibited, since the oxidised plastoquinone pool size under FR light increased as a consequence of the decrease in PS I activity caused by high light intensity. This practical is intended for advanced students of plant biochemistry and plant physiology.     

The fluorescence decay kinetics of in vivo chlorophyll measured using low intensity excitation

We report fluorescence lifetimes for in vivo chlorophyll a using a time-correlated single-photon counting technique with tunable dye laser excitation. The fluorescence decay of dark-adapted chlorella is almost exponential with a lifetime of 490 ps, which is independent of excitation from 570 nm to 640 nm.Chloroplasts show a two-component decay of 410 ps and approximately 1.4 ns, the proportion of long component depending upon the fluorescence state of the chloroplasts. The fluorescence lifetime of Photosystem I was determined to be 110 ps from measurements on fragments enriched in Photosystem I prepared from chloroplasts with digitonin.     

Evidence for variable chlorophyll fluorescence of photosystem I in vivo

Photosynthesis Research - Room temperature fluorescence in vivo and its light-induced changes are dominated by chlorophyll a fluorescence excited in photosystem II, F(II), peaking around...     

Determination of the quantum efficiency of photosystem II and of non-photochemical quenching of chlorophyll fluorescence in the field

A newly developed portable chlorophyll fluorometer in combination with a special leaf clip holder was used for assessing photosynthetic activity of attached sun leaves of Fagus sylvatica and Cucurbita pepo under field conditions. During diurnal time courses, fluorescence yield, photosynthetic photon flux density (PPFD) incident on the leaf plane, and leaf temperature were measured and quantum efficiency of photosystem II (PS II), apparent relative electron transport rates, and non-photochemical fluorescence quenching (NPQ) calculated. In both species, quantum efficiency followed closely the incident PPFD and no hysteresis could be observed during the day. Apparent electron transport rate showed light saturation above a PPFD of 700 mol m^–2 s^–1 in F. sylvatica, while in C. pepo no saturation was visible up to 1400 mol m^–2 s^–1. NPQ was closely correlated to excessive PPFD calculated from the PS II quantum yield. Maximal NPQ observed was 3.3 Although the beech leaf was exposed for a considerable time to PPFD values of 1400–1500 mol m^–2 s^–1 and leaf temperatures between 30 and 35°C, no obvious signs for sustained photodamage could be observed. The data demonstrate the potential of chlorophyll fluorescence measurements to analyse photosynthetic performance under field conditions with minimal disturbance of the plant. Potential error sources due to the geometry of the leaf clip holder used are discussed.Dedicated to Prof. Dr. F.-C. Czygan on the occasion of his 60th birthday     

Statistical properties of chlorophyll fluorescence induction parameters

Parameters of the fast chlorophyll (Chl) fluorescence induction (the O-J-I-P curve) of plants of winter wheat grown in the field canopy were statistically tested for Gaussian distribution. Five different statistical methods showed that the obtained values did not obey the Gaussian distribution law. The presentation of the parameters with the help of the mean and standard deviation masks the information about statistical properties of the values. Thus, we recommend to present the parameters by means of median, quartiles, and minimum and maximum values rather than by means of the mean and standard deviation.     

A model of chlorophyll a fluorescence induction kinetics with explicit description of structural constraints of individual photosystem II units

Chlorophyll a fluorescence induction (FI) kinetics, in the microseconds to the second range, reflects the overall performance of the photosynthetic apparatus. In this paper, we have developed a novel FI model, using a rule-based kinetic Monte Carlo method, which incorporates not only structural and kinetic information on PSII, but also a simplified photosystem I. This model has allowed us to successfully simulate the FI under normal or different treatment conditions, i.e., with different levels of measuring light, under 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea treatment, under 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone treatment, and under methyl viologen treatment. Further, using this model, we have systematically studied the mechanistic basis and factors influencing the FI kinetics. The results of our simulations suggest that (1) the J step is caused by the two-electron gate at the Q _B site; (2) the I step is caused by the rate limitation of the plastoquinol re-oxidation in the plastoquinone pool. This new model provides a framework for exploring impacts of modifying not only kinetic but also structural parameters on the FI kinetics.     

A three-state model for energy trapping and chlorophyll fluorescence in photosystem II incorporating radical pair recombination

The multiphasic fluorescence induction kinetics upon a high intensity light pulse have been measured and analyzed at a time resolution of 10 micros in intact leaves of Peperomia metallica and Chenopodium album and in chloroplasts isolated from the latter. Current theories and models on the relation between chlorophyll fluorescence yield and primary photochemistry in photosystem II (PSII) are inadequate to describe changes in the initial phase of fluorescence induction and in the dark fluorescence level F(0) caused by pre-energization of the system with single turnover excitation(s). A novel model is presented, which gives a quantitative relation between the efficiencies of primary photochemistry, energy trapping, and radical pair recombination in PSII. The model takes into account that at least two turnovers are required for stationary closure of a reaction center. An open reaction center is transferred with high efficiency into its semiclosed (-open) state. This state is characterized by Q(A) and P680 in the fully reduced state and a lifetime equal to the inverse of the rate constant of Q(A)(-) oxidation (approx. 250 micros). The fluorescence yield of the system with 100% of the centers in the semiclosed state is 50% of the maximal yield with all centers in the closed state at fluorescence level F(m). A situation with approximately 100% of the centers in the semiclosed state is reached after a single turnover excitation in the presence of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU). The lifetime of this state under these conditions is approximately 10 s. Closure of a semiclosed (-open) center occurs with low efficiency in a second turnover. The low(er) efficiency is caused by the rate of P(+) reduction by the secondary donor Y(Z) being competitive with the rate of radical pair recombination in second and following turnovers. The single-turnover-induced alterations in the initial kinetics of the fluorescence concomitantly with a 15-25% increase in F(o) can be simulated with the present so called three-state model of energy trapping. The experimental data suggest evidence for an electrostatic effect of local charges in the vicinity of the reaction center affecting the rate of radical pair recombination in the reaction center.