Christian Neubauer

O2-dependent electron flow, membrane energization and the mechanism of non-photochemical quenching of chlorophyll fluorescence.

Recent progress in chlorophyll fluorescence research is reviewed, with emphasis on separation of photochemical and non-photochemical quenching coefficients (qP and qN) by the ‘saturation pulse method’. This is part of an introductory talk at the Wageningen Meeting on ‘The use of chlorophyll fluorescence and other non-invasive techniques in plant stress physiology’. The sequence of events is investigated which leads to down-regulation of PS II quantum yield in vivo, expressed in formation of qN. The role of O2-dependent electron flow for ΔpH- and qN-formation is emphasized. Previous conclusions on the rate of ‘pseudocyclic’ transport are re-evaluated in view of high ascorbate peroxidase activity observed in intact chloroplasts. It is proposed that the combined Mehler-Peroxidase reaction is responsible for most of the qN developed when CO2-assimilation is limited. Dithiothreitol is shown to inhibit part of qN-formation as well as peroxidase-induced electron flow. As to the actual mechanism of non-photochemical quenching, it is demonstrated that quenching is favored by treatments which slow down reactions at the PS II donor side. The same treatments are shown to stimulate charge recombination, as measured via 50 μs luminescence. It is suggested that also in vivo internal thylakoid acidification leads to stimulation of charge recombination, although on a more rapid time scale. A unifying model is proposed, incorporating reaction center and antenna quenching, with primary control of ΔpH at the PS II reaction center, involving radical pair spin transition and charge recombination to the triplet state in a first quenching step. In a second step, triplet excitation is trapped by zeaxanthin (if present) which in its triplet excited state causes additional quenching of singlet excited chlorophyll.

Measuring P700 Absorbance Changes around 830 nm with a New Type of Pulse Modulation System

Abstract A new measuring system for monitoring absorbance changes around 830 n m is described, which was developed by modification of a commercially available pulse modulation fluorometer. All modifications concern the emitter-detector unit of the fluorometer, such that only this unit needs to be exchanged when changing from fluorescence to absorbance measurements and vice versa. The new system is shown to be well-suited for measuring redox changes of P700, the reaction center of photosystem I, in intact leaves and isolated chloroplasts. The observed kinetic changes at 830 nm in response to single turnover or multiple turnover saturating flashes are practically identical to those previously measured around 700 nm . The signal/noise ratio is sufficiently high to give well-resolved kinetics without signal averaging. W h e n P700 is oxidized by far-red background light, valuable information on the state of the intersystem electron transport chain is given by the re-reduction kinetics induced by single or multiple turnover saturating flashes. Such measurements are facilitated by the use of poly-furcated fiberoptics. With intact leaves, almost identical responses are found when measuring through the leaf (transmission mode) or from the leaf surface (remission mode). Modulated chlorophyll fluorescence can be measured in parallel; application of saturation pulses for fluorescence quenching analysis produces transient P700 oxidation without oversaturating the measuring system. The information on the P700 redox state complements that obtained from fluorescence measurements, yielding a new practical tool in plant physiological research.

PAM fluorometer based on medium-frequency pulsed Xe-flash measuring light: A highly sensitive new tool in basic and applied photosynthesis research.

A newly developed modulation fluorometer is described which employs repetitive 1 μs Xe-flashes for excitation light. Similar to the standard PAM Chlorophyll Fluorometer, which uses 1 μs LED pulses for measuring light, the integrated measuring light intensity is sufficiently low to monitor the dark-fluorescence level, Fo. The maximal fluorescence yield, Fm, can be determined with high selectivity upon application of a saturating light pulse. The Xe-PAM displays exceptionally high sensitivity, enabling quenching analysis at chlorophyll concentrations as low as 1 μg/l, thus allowing to assess photosynthesis of phytoplankton in natural waters like lakes, rivers and oceans. Due to high flexibility in the choice of excitation and emission wavelengths, this system also provides the experimental basis for a thorough study of fluorescence and photosynthesis properties of various algae classes with differing antenna organisation. By appropriate modifications, the instrument may as well be used to measure with great sensitivity and selectivity other types of fluorescence (e.g. NADPH-fluorescence), as well as light-scattering and absorbance changes.

Membrane barriers and Mehler-peroxidase reaction limit the ascorbate available for violaxanthin de-epoxidase activity in intact chloroplasts

The presence of an acidic lumen and the xanthophylls, zeaxanthin and antheraxanthin, are minimal requirements for induction of non-radiative dissipation of energy in the pigment bed of Photosystem II. We recently reported that ascorbate, which is required for formation for these xanthophylls, also can mediate the needed lumen acidity through the Mehler-peroxidase reaction [Neubauer and Yamamoto (1992) Plant Physiol 99: 1354–1361]. It is demonstrated that in non-CO2-fixing intact chloroplasts and thylakoids of Lactuca sativa, L. c.v. Romaine, the ascorbate available to support de-epoxidase activity is influenced by membrane barriers and the ascorbate-consuming Mehler-peroxidase reaction. In intact chloroplasts, this results in biphasic kinetic behavior for light-induced de-epoxidation. The initial relatively high activity is due to ascorbate preloaded into the thylakoid before light-induction and the terminal low activity due to limiting ascorbate from the effects of chloroplast membranes barriers and a light-dependent process. A five-fold difference between the initial and final activities was observed for light-induced de-epoxidation in chloroplasts pre-incubated with 120 mM ascorbate for 40 min. The light-dependent activity is ascribed to the competitive use of ascorbic acid by ascorbate peroxidase in the Mehler-peroxidase reaction. Thus, stimulating ascorbic peroxidase with H2O2 transiently inhibited de-epoxidase activity and concomitantly increased photochemical quenching. Also, the effects inhibiting ascorbate peroxidase with KCN, and the KM values for ascorbate peroxidase and violaxanthin de-epoxidase of 0.36 and 3.1 mM, respectively, support this conclusion. These results indicate that regulation of xanthophyll-dependent non-radiative energy dissipation in the pigment bed of Photosystem II is modulated not only by lumen acidification but also by ascorbate availability.

Intact chloroplasts display pH 5 optimum of O2-reduction in the absence of methyl viologen: Indirect evidence for a regulatory role of superoxide protonation.

The pH-dependence of light-driven O2-reduction in intact spinach chloroplasts is studied by means of chlorophyll fluorescence quenching analysis and polarographic O2-uptake measurements. Most experiments are carried out in presence of KCN, which blocks activities of Calvin cycle, ascorbate peroxidase and superoxide dismutase. pH is varied by equilibration with external buffers in presence of nigericin. Vastly different pH-optima for O2-dependent electron flow are observed in the presence and absence of the redox catalyst methyl viologen. Both fluorescence quenching analysis and O2-uptake reveal a distinct pH 5 optimum of O2-reduction in the absence of methyl viologen. In the presence of this catalyst, O2-reduction is favoured in the alkaline region, with an optimum around pH 8, similar to other types of Hill reaction. It is suggested that in the absence of methyl viologen the extent of irreversibility of O2-reduction is determined by the rate of superoxide protonation. This implies that O2-reduction takes place within the aprotic phase of the thylakoid membrane and that superoxide-reoxidation via oxidized PS I donors competes with protonation. Superoxide protonation is proposed to occur at the internal surface of the thylakoid membrane. There is no competition between superoxide reoxidation and protonation when in the presence of methyl viologen the site of O2-reduction is shifted into the protic stroma phase. In confirmation of this interpretation, fluorescence measurements in the absence of KCN reveal, that non-catalysed O2-dependent electron flow is unique in beingstimulated by the transthylakoidal pH-gradient. On the basis of these findings a major regulatory role of O2-dependent electron flow under excess light conditions is postulated.

Photochemical and Non-Photochemical Quenching of Chlorophyll Fluorescence Induced by Hydrogen Peroxide

Abstract Chlorophyll Fluorescence. Fluorescence Quenching, Hydrogen Peroxide. Active Oxygen. Ascorbate Peroxidase Chlorophyll fluorescence quenching induced by H 20 2 in intact spinach chloroplasts was investigated with a modulation fluorometer which allows to distinguish between photochemical and non photochemical quenching components by the so-called saturation pulse method. Residual catalase activity was removed by washing and percoll gradient centrifugation. H2O2 was found to induce pronounced photochem ical and non-photochemical quenching, characteristic for the action of a Hill reagent, with a half-maximal rate already observed at 5 × 10-6 m . The saturation characteristics and maximal rate of H2O2-reduction were very similar to those of methylviologen reduction. H2O2-dependent quenching was stimulated by ascorbate and inhibited by cyanide and azide in agreement with previous findings by other researchers that H2O2-reduction involves the ascorbate peroxidase scavenging system and that the actual “Hill acceptor” is an oxidation product of ascorbate, i.e. monodehydroascorbate or dehydroascorbate. With well-coupled intact chloroplasts reducing CO2 at 150 (μmol (mg Chl)-1h-1, iodoacetamide stopped CO2-dependent O2-evolution and consequent addition of 10″3 m H2O2 produced an O2-Solution rate of 240 (μmol (mg Chl)-1h-1 .It is concluded that light-dependent H 20 2 reduction is a very efficient reaction in intact chloroplasts. As H2O2 formation and consequent reduction also occur in vivo, the corresponding quenching should be considered when assimilatory electron flow is estimated from quenching coefficients. It is suggested that proton flux associated with H2O2-formation and reduction may be important for the adjustment of appropriate ATP /NADPH ratios required for CO2-fixation in vivo. Furthermore, H2O2-reduction may serve as a valve reaction whenever Calvin cycle activity is limited by factors different from NADPH supply, thus protecting against photo-inhibitory damage.

On the relationship between chlorophyll fluorescence quenching and the quantum yield of electron transport in isolated thylakoids

The relationship between the empirical fluorescence index ΔF/Fm′ and the quantum yield of linear electron flow, Φs, was investigated in isolated spinach thylakoids. Conditions were optimised for reliable determination of ΔF/Fm′ and Φs with methyl viologen or ferricyanide as electron acceptors under coupled and uncoupled conditions. Ascorbate in combination with methyl viologen was found to stimulate light-induced O2-uptake which is not reflected in ΔF/Fm′ and interpreted to reflect superoxide reduction by ascorbate. In the absence of ascorbate, the plot of ΔF/Fm′ vs. Φs was mostly linear, except for the range of high quantum yields, i.e. at rather low photon flux densities. With ferricyanide as acceptor, use of relatively low concentrations (0.1–0.3 mM) was essential for correct Fm′-determinations, particularly under uncoupled conditions. Under coupled and uncoupled conditions the same basic relationship between ΔF/Fm′ and Φs was observed, irrespective of Φs being decreased by increasing light intensity or by DCMU-addition. The plots obtained with methyl viologen and ferricyanide as acceptors were almost identical and similar to corresponding plots reported previously by other researchers for intact leaves. It is concluded that the index ΔF/Fm′ can be used with isolated chloroplasts for characterisation of such types of electron flow which are difficult to assess otherwise, as e.g. O2 dependent flux. The origin of the ‘non-linear’ part of the relationship is discussed. An involvement of ‘inactive’ PS II centers with separate units and inefficient QA-QB electron transfer is considered likely.

Comparison of effects of air pollutants (SO2, O3, NO2) on intact leaves by measurements of chlorophyll fluorescence and P700 absorbance changes

The immediate effects of short exposures to high concentrations of different air pollutants (20 min SO2, 2 h O3, and 4 h NO2, 5 ppm each) on chlorophyll fluorescence and P700 absorbance changes at 830 nm of intact spinach leaves were investigated. Three different types of fluorescence measurements were used: Fluorescence rise kinetics in saturating light, fast fluorescence induction kinetics (Kautsky-effect), and slow induction kinetics with repetitive application of saturation pulses (saturation pulse method).The results show that the various air pollutants caused rather different damage in the photosynthetic apparatus of the leaves:1.SO2: The main effect is due to the acidifying action, weakening the PS II donor side (suppression of I1-I2-P phase in fluorescence) and inhibiting Calvin cycle activation (no relaxation of membrane energization).2.O3: Ozone has apparently no specific point of attack due to its high reactivity. It obviously reacts with all cell membranes, but primarily with the plasma membrane which it first passes on the way into the leaf.3.NO2: NO2 produces HNO3 and HNO2, when dissolved in the leaf water. The nitrite reductase, however, is highly effective, so that (in the light) nearly all nitrite is reduced. By the reduction of nitrite to ammonia, OH- is produced preventing net acidification. Obviously, the electron transport rates, which are possible with nitrite as acceptor are very high, being comparable to those observed with the well-known Hill reagent methylviologen, as revealed by P700 measurements in saturating light. Such high reactivities with NO2- must prevent assimilatory electron flow.

An active Mehler-peroxidase reaction sequence can prevent cyclic PS I electron transport in the presence of dioxygen in intact spinach chloroplasts

Simultaneous measurements of 9-aminoacridine (9-AA) fluorescence quenching, O2-uptake and chlorophyll fluorescence of intact spinach chloroplasts were carried out to assess the relationship between the transthylakoidal ΔpH and linear electron flux passing through Photosystem II. Three different types of O2-dependent electron flow were investigated: (1) Catalysed by methyl viologen; (2) in the absence of a catalyst and presence of an active ascorbate peroxidase (Mehler-peroxidase reaction); (3) in the absence of a catalyst and with the ascorbate peroxidase being inhibited by KCN (Mehler reaction). The aim of this study was to assess the relative contribution of ΔpH-formation which is not associated with electron flow through Photosystem II and, which should reflect Photosystem I cyclic flow under the different conditions. The relationship between the extent of 9-AA fluorescence quenching and O2-uptake rate was found to be almost linear when methyl viologen was present. In the absence of methyl viologen (Mehler reaction) an increase of 9-AA fluorescence quenching to a value of 20% at low light intensities was associated with considerably less O2-uptake than in the presence of methyl viologen, indicating the involvement of cyclic flow. These findings are in agreement with a preceding study of Kobayashi and Heber (1994). However, when no KCN was added, such that the complete Mehler-peroxidase reaction sequence was operative, the relationship between 9-AA fluorescence quenching and the flux through PS II, as measured via the chlorophyll fluorescence parameter ΔF/Fm′ × PAR, was identical to that observed in the presence of methyl viologen. Under the assumption that methyl viologen prevents cyclic flow, it is concluded that there is no significant contribution of cyclic electron flow to ΔpH-generation in intact spinach chloroplasts.

Contrasting pH-Optima of Light-Driven O2-and H2O2-Reduction in Spinach Chloroplasts as Measured via Chlorophyll Fluorescence Quenching

Abstract Quenching analysis of chlorophyll fluorescence by the saturation pulse method is used to investigate the pH-dependency of O2-dependent electron flow in intact spinach chloroplasts with high ascorbate peroxidase activity. When carboxylase/oxygenase activity is blocked, photochemical and non-photochemical quenching are initially low and increase with illumination time. Quenching shows a pH-optimum around pH 6.5, but only when ΔpH-formation is al lowed. It is suggested that overall O2-dependent electron flow involves two major components, namely O2-reduction (Mehlerreaction) and reduction of the H2O2 formed in the Mehlerreaction, involving enzymic activity of ascorbate peroxidase and monodehydroascorbate reductase. The separated pH-dependencies of light driven O2-reduction (presence of KCN) and of H2O2-reduction (anaerobic conditions) reveal contrasting pH-optima around pH 5 and 8.5, respectively. Energy-dependent, dark relaxable non-photochemical quenching is not observed with O2-reduction but with H2O2-reduction, and only at pH-values above 6.5. The relevance of these findings with respect to regulation of photosynthetic electron flow is discussed. It is suggested that upon limitation of assimilatory electron flow O2-dependent non-assimilatory flow is responsible for ΔpH-formation, by which it is autocatalytically stimulated. It is proposed that this autocatalytical reaction sequence is the basis of the so-called “Kautsky effect” of chlorophyll fluorescence induction.