Chlorophyll fluorescence as a probe for the determination of the photo-induced proton gradient in isolated chloroplasts.

Gramicidin D-treated chloroplasts show an acid-induced quenching of the chlorophyll fluorescence, which is composed of a reversible and irreversible part. The reversible quenching is analogous to the photo-induced quenching in coupled chloroplasts and can be taken to determine the light induced delta pH.

Identification of S2 as the sensitive state to alkaline photoinactivation of photosystem II in chloroplasts.

1. Chloroplasts have been preilluminated by a sequence of n short saturating flashes immediately before alkalinization to pH 9.3, and brought back 2 min later to pH 7.8. The assay of Photosystem II activity through dichlorophenolindophenol photoreduction, oxygen evolution, fluorescence induction, shows that part of the centers is inactivated and that this part depends on the number of preilluminating flashes (maximum inhibition after one flash) in a way which suggests identification of state S2 as the target for alkaline inactivation. 2. As shown by Reimer and Trebst ((1975) Biochem. Physiol. Pflanz. 168, 225-232) the inactivation necessitates the presence of gramicidin, which shows that the sensitive site is on the internal side of the thylakoid membrane. 3. The electron flow through inactivated Photosystem II is restored by artificial donor addition (diphenylcarbazide or hydroxylamine); this suggests that the water-splitting enzyme itself is blocked. The inactivation is accompanied by a solubilization of bound Mn2+ and by the occurence of EPR Signal II "fast". 4. Glutaraldehyde fixation before the treatment does not prevent the inactivation which thus does not seem to involve a protein structural change.

Kinetics of chlorophyll fluorescence at 77K in Chlorella and chloroplasts. Effects of CCCP, ferricyanide and DCMU.

The kinetics of chlorophyll fluorescence at 77K were studied in Chlorella cells and spinach chloroplasts. During a first illumination, the rise is polyphasic with at least three phases. The slowest one is irreversible and corresponds to the cytochrome oxidation. The dark regeneration of half the variable fluorescence is biphasic, the fast phase being inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) both in Chlorella and chloroplasts. The fluorescence rise during a second illumination is still biphasic. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) slows down the fluorescence rise in Chlorella but has no effect on the dark regeneration. It does not affect the fluorescence of chloroplasts. Ferricyanide which oxidizes cytochrome beta-559 at room temperature produces a quenching of the variable fluorescence and an acceleration of the fluorescence rise during the first illumination. Our results fit the idea of the heterogeneity of the Photosystem II centers at low temperature.

A quantitative study of the slow decline of chlorophyll a fluorescence in isolated chloroplasts.

A detailed study of the photo-induced decline in chlorophyll a fluorescence intensity (Kautsky phenomenon) in coupled isolated chloroplasts from a high level (P) to a low stationary level (S) is presented. 1. A linear relationship between P leads to S quenching and intrathylakoid H+ concentration was found. When the light-induced proton gradient was abolished by uncoupling, the fluorescence emission at room temperature was lowered proportionally to increased H+ concentration in the medium. 2. Fluorescence spectra at -196 degrees C of samples frozen at the P and S states showed no significant differences in the Photosystem I/Photosystem II ratio of fluorescence emission. Furthermore, freezing to -196 degrees C reversed the P leads to S quenching. This indicates that the P leads to S quenching is not related to an increase of spillover of excitation energy from Photosystem II to Photosystem I. 3. When Mg2+ was added to thylakoids suspended in a medium free of divalent cations, the inhibition of spillover required lower Mg2+ concentrations (half saturation at 0.6 mM). Increased proton concentration in the medium also inhibited spillover. 4. The results are interpreted in terms of two sites of Mg2+ and H+ effects on excitation deactivation in Photosystem II. One site is located on the outer face of the thylakoid membrane; action of both Mg2+ and H+ at this side diminishes spillover. The second site is located on the inner face of the membrane; as Mg2+ is displaced there by protons, a non-photochemical quenching of Photosystem II fluorescence is induced, which is manifested by the P leads to S decline.

Decreased Photosystem II Core Phosphorylation in a Yellow-Green Mutant of Wheat Showing Monophasic Fluorescence Induction Curve.

In the present work we study the regulation of the distribution of the phosphorylated photosystem II (PSII) core populations present in grana regions of the thylakoids from several plant species. The heterogeneous nature of PSII core phosphorylation has previously been reported (M.T. Giardi, F. Rigoni, R. Barbato [1992] Plant Physiol 100: 1948-1954; M.T. Giardi [1993] Planta 190: 107-113). The pattern of four phosphorylated PSII core populations in the grana regions appears to be ubiquitous in higher plants. In the dark, at least two phosphorylated PSII core populations are always detected. A mutant of wheat (Triticum durum) that shows monophasic room-temperature photoreduction of the primary quinone electron acceptor of PSII as measured by chlorophyll fluorescence increase in the presence and absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and by fluorescence upon flash illumination in intact leaves also lacks the usual distribution of phosphorylated PSII core populations. In this mutant, the whole PSII core population pattern is changed, probably due to altered threonine kinase activity, which leads to the absence of light-induced phosphorylation of CP43 and D2 proteins. The results, correlated to previous experiments in vivo, support the idea that the functional heterogeneity observed by fluorescence is correlated in part to the PSII protein phosphorylation in the grana.

Photoinactivation of the photosynthetic electron transport chain by accumulation of over-saturating light pulses given to dark adapted pea leaves.

The effect of cumulative over-saturating pulses (OSP) of white light (1 s, >10 000 mumol photons m(-2) s(-1)), applied every 20 min on pea leaves, was investigated during a complete diurnal cycle of 24 h. In dark-adapted leaves, this treatment leads to a progressive decline of the optimum Photosystem II (PS II) quantum yield. Continuous low background light (except far-red light) had a protective effect against this OSP-induced photoinactivation. The lack of far-red effect could be due to its absorption mainly in PS I and not in PS II, but could be also due to the general low absorption in this wavelength region. The photoinactivation was enhanced in leaves that had been previously infiltrated with chloramphenicol. The quantum yield of CO(2) assimilation, but not its maximal capacity, was inhibited by the OSP treatment. The most spectacular effects observed, in addition to an irreversible quenching of Fm, was a strong inhibition of Q(A) (-) reoxidation revealed by a large increase in the Fs level and consequently by a decrease of DeltaF/Fm'. Under such conditions, we observed that the electron flow deduced from DeltaF/Fm' underestimated the real electron flow to CO(2). Time-resolved Chlorophyll a fluorescence measurements showed that the reduced capacity of Q(A) (-) reoxidation in OSP treated leaves was accompanied by the appearance of a 4.7 ns component attributed to PS II charge recombination. We suggest that a modification at the Q(B) site may influence the redox potential of Q(A)/Q(A) (-), facilitating the reversion of the primary charge separation. In addition, a 1.2 ns fluorescence component accumulated, which appeared to be responsible for the underestimation of PS II electron flow. The observed photoinactivation seemed to be different from the photoinhibition often described in the literature, which occurs under continuous light.

Light-harvesting chlorophyll a-b complex requirement for regulation of Photosystem II photochemistry by non-photochemical quenching.

Recently, it has been suggested (Horton et al. 1992) that aggregation of the light-harvesting a-b complex (LHC II) in vitro reflects the processes which occur in vivo during fluorescence induction and related to the major non-photochemical quenching (qE). Therefore the requirement of this chlorophyll a-b containing protein complex to produce qN was investigated by comparison of two barley mutants either lacking (chlorina f2) or depressed (chlorina(104)) in LHC II to the wild-type and pea leaves submitted to intermittent light (IL) and during their greening in continuous light.It was observed that qN was photoinduced in the absence of LHC II, i.e. in IL grown pea leaves and the barley mutants. Nevertheless, in these leaves qN had no (IL, peas) or little (barley mutants) inhibitory effect on the photochemical efficiency of QA reduction measured by flash dosage response curves of the chlorophyll fluorescence yield increase induced by a single turn-over flashDuring greening in continuous light of IL pea leaves, an inhibitory effect on QA photoreduction associated to qN developed as Photosystem II antenna size increased with LHC II synthesis. Utilizing data from the literature on connectivity between PS II units versus antenna size, the following hypothesis is put forward to explain the results summarized above. qN can occur in the core antenna or Reaction Center of a fraction of PS II units and these units will not exhibit variable fluorescence. Other PS II units are quenched indirectly through PS II-PS II exciton transfer which develops as the proportion of connected PS II units increases through LHC II synthesis.

Partitioning of photosynthetic electron flow between CO2 and O 2 reduction in a C 3 leaf (Phaseolus vulgaris L.) at different CO 2 concentrations and during drought stress.

Photosystem II chlorophyll fluorescence and leaf net gas exchanges (CO2 and H2O) were measured simultaneously on bean leaves (Phaseolus vulgaris L.) submitted either to different ambient CO2 concentrations or to a drought stress. When leaves are under photorespiratory conditions, a simple fluorescence parameter ΔF/ Fm (B. Genty et al. 1989, Biochem. Biophys. Acta 990, 87-92; ΔF = difference between maximum, Fm, and steady-state fluorescence emissions) allows the calculation of the total rate of photosynthetic electron-transport and the rate of electron transport to O2. These rates are in agreement with the measurements of leaf O2 absorption using (18)O2 and the kinetic properties of ribulose-1,5bisphosphate carboxylase/oxygenase. The fluorescence parameter, ΔF/Fm, showed that the allocation of photosynthetic electrons to O2 was increased during the desiccation of a leaf. Decreasing leaf net CO2 uptake, either by decreasing the ambient CO2 concentration or by dehydrating a leaf, had the same effect on the partitioning of photosynthetic electrons between CO2 and O2 reduction. It is concluded that the decline of net CO2 uptake of a leaf under drought stress is only due, at least for a mild reversible stress (causing at most a leaf water deficit of 35%), to stomatal closure which leads to a decrease in leaf internal CO2 concentration. Since, during the dehydration of a leaf, the calculated internal CO2 concentration remained constant or even increased we conclude that this calculation is misleading under such conditions.

Chlorophyll fluorescence and photoinhibition in a tropical rainforest understory plant.

The data presented here deal with the effects of high-light exposure on the 77 K fluorescence characteristics of Elatostema repens. It is shown that the decrease of the variable fluorescence during the treatment is biphasic. The reactions responsible for the first phase of fluorescence quenching are saturated under 700 µmol photon m(-2) s(-1) and insensitive to streptomycin, whereas those responsible for the second phase are not yet saturated under 700 µ mol photon m(-2) s(-1) and sensitive to streptomycin. It is concluded that only the second phase of fluorescence quenching is associated with photoinhibitory processes. Rate and amplitude of recovery from photoinhibition are maximum under very low light (3.5 µ mol photon m(-2) s(-1)), and very small at a moderate light (160 µ mol photon m(-2) s(-1)) which does not cause photoinhibition. It is concluded that recovery processes are inhibited during photoinhibition. It is suggested that they could be associated with damage occuring on the oxidizing side of PSII.

Effects of drought on primary photosynthetic processes of cotton leaves.

The effects of drought on Photosystem II (PSII) fluorescence and photosynthetic electron transport activities were analyzed in cotton. Water stress did not modify the amplitude of leaf variable fluorescence at room temperature in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) nor at 77 K. It is therefore concluded that photon collection, their distribution between the two photosystems, and PSII photochemistry are unaffected by the stress. In droughted leaves at room temperature under low exciting light, the transitory maximum (F(p)) and steady state (F(t)) fluorescence levels are increased; under high exciting light, F(p) level and the rise time from the initial level (F(o)) to F(p) are unchanged, whereas F(p) to F(t) decay time is increased. These results infer that the drought slows the rate of plastoquinone reoxidation. This conclusion agrees with a larger proportion of reduced primary PSII electron acceptor Q(A) measured at the steady state under low light. In thylakoids isolated from droughted leaves, PSII mediated electron flow was the same as in thylakoids from control leaves, whereas PSI mediated electron transport was inhibited. It is shown that water stress does not induce sensitization to photoinhibition in cotton.

Evidence that the variable chlorophyll fluorescence in Chlamydomonas reinhardtii is not recombination luminescence.

Room temperature single photon timing measurements on intact, Chlamydomonas reinhardtii cells at low excitation energies have been analysed using a four exponential kinetic model. Closing the PSII reaction centres produced two major variable lifetime and two minor constant lifetime components. The yield of each component mirrored the changes in lifetime. Such observations indicate the presence of well-connected PSII centres favoring excitation energy transfer. A Chlamydomonas mutant lacking PSII reaction centre proteins exhibited decay components equivalent to those seen at FM in the wild-type. A titration of in vivo fluorescence, in both the mutant and wild-type algae, using DNB, produced decay components similar to those seen on opening PSII reaction centres. Such observations indicate that the luminescence hypothesis for the origin of the long-lived lifetime component is not the case.

The quenching characteristics of potassium iridic chloride and their meaning for the origin of chlorophyll fluorescence components.

To understand the origins of the different lifetime components of photosystem 2 (PS2) chlorophyll (Chl) fluorescence we have studied their susceptibility to potassium iridic chloride (K2IrCl6) which has been shown to bleach antenna pigments of photosynthetic bacteria (Loach et al. 1963). The addition of K2IrCl6 to PS2 particles gives rise to a preferential quenching of the variable Chl fluorescence (Fv). At concentrations lower than 20 µM, this is brought about mainly by a decrease in the yield, but not in the lifetime, of the slowest component when all the PS2 reaction centres are closed (FM). The yield of the middle and fast decays are not significantly altered. This type of quenching is not seen with DNB. The iridate-induced quenching of the initial fluorescence level (F0) is due to a proportional decrease in the yield and lifetime of the three components and correlates with the observed modification in the relative quantum yield of oxygen evolution. In this concentration range a bleaching of Chl a is seen. At higher iridate levels, greater than 20 µM, a proportional decrease in the lifetimes and yields of the three kinetic components is seen at FM. These changes are associated with a carotenoid bleaching. In isolated light harvesting Chl a/b complexes of PS2 (LHC2), iridate addition converts a 4 ns decay into a 200 ps emission and both types of bleaching are observed. By also measuring the rate of PS2 trap closure versus iridate concentration, we have discussed the results in terms of excitation energy transfer.

Changes in the flash-induced oxygen yield pattern by thylakoid membrane phosphorylation.

Phosphorylation of thylakoid membrane proteins results in a partial inhibition (approximately 15-20%) of the light-saturated rate of oxygen evolution. The site of inhibition is thought to be located on the acceptor side of photosystem 2 (PS2) between the primary, QA, and secondary, QB, plastoquinone acceptors (Hodges et al. 1985, 1987). In this paper we report that thylakoid membrane phosphorylation increases the damping of the quaternary oscillation in the flash oxygen yield and increases the extent of the fast component in the deactivation of the S2 oxidation state. These results support the proposal that thylakoid membrane protein phosphorylation decreases the equilibrium constant for the exchange of an electron between QA and QB. An analysis of the oxygen release patterns using the recurrence matrix model of Lavorel (1976) indicates that thylakoid membrane phosphorylation increases the probability that PS2 miss a S-state transition by 20%. This is equivalent, however, to an insignificant inhibition (approximately 2.4%) of the light-saturated oxygen evolution rate. If a double miss in the S-state transitions is included when the PS2 centres are in S2 the fit between the experimental and theoretical oxygen yield sequences is better, and sufficient to account for the 15-20% inhibition in the steady-state oxygen yield. A double miss in the S-state transition is a consequence of an increased population of PS2 centres retaining QA (-): not only will these PS2 centres fail to catalyse photochemical charge transfer until QA (-) is reoxidized, but the re-oxidation reaction will also result in the deactivation of S2 to S1.