Observation and characterisation of a transient in the yield of chlorophyll fluorescence in intact spinach chloroplasts

A transient in chlorophyll fluorescence, which is associated with a transient in 9-aminoacridine fluorescence and a perturbation in the rate of oxygen evolution, has been observed in intact spinach chloroplasts. The results indicate that changes in the redox state of Q are, at least partially, responsible for the transient in chlorophyll fluorescence. The size of the transient is highly dependent upon the concentration of inorganic phosphate and upon the pH of the medium. The properties of the transient are consistent with the suggestion that it reflects changes in the levels of stromal intermediates during induction.Abbreviations BES NN-Bis(2-hydroxyethyl)2-aminoethanesulphonic acid dihydroxyacetone-P(DHAP): dihydroxyacetone phosphate glycerate-3-P (PGA): glycerate-3-phosphate - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulphonic acid - MES 2-(N-Morpholino)ethanesulphonic acid - Pi inorganic phosphate - qE quenching of chlorophyll fluorescence by the energisation of the thylakoid membrane - qQ quenching of chlorophyll fluorescence by oxidised Q, the electron acceptor of photosystem 2 - ribose-5-P (R5P) ribose-5-phosphate - Rbu-5-P ribulose-5-phosphate     

Light-induced changes of b-type cytochromes in the electron transport chain of Euglena chloroplasts

Light-induced changes of b-type cytochromes in Euglena chloroplastswere studied spectrophotometrically.

1. In the dark and at pH 6.5, most of the cytochrome 558 in chloroplastswas in the reduced state, and most of the cytochrome 563, inthe oxidized state. Illumination of chloroplasts at pH 6.5 induceda rapid, but slight oxidation of cytochrome 552 and cytochrome558. The magnitude of photooxidation of cytochrome 558 was greatlyenhanced by the addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea(DCMU). The rate of photooxidation in the presence of DCMU wasstimulated by the addition of 0.15 µM Euglena cytochrome552, or 100 µM methyl viologen.
2. Euglena chloroplasts,incubated at 55°C for 5 min showedno significant absorbancechanges for about 10 min after theonset of illumination. However,greater photooxidation of cytochrome558 was observed afterprolonged illumination, or in the presenceof DCMU or ethylenediaminetetraaceticacid (EDTA). Similar resultswere obtained with chloroplastspre-treated at pH 9.0–10.0for 5 min.
3. At pH 9.5, andin the dark, both cytochrome 563 and cytochrome558 were inan almost reduced state. On illumination at thispH, both cytochromeswere photooxidized, with a complicatedkinetics, showing aninitial rapid and small absorbance decrease,followed by a stagnantphase of temporary retarded reaction.In the presence of DCMUor EDTA, photooxidation proceeded rapidlywithout a stagnantphase.
4. At pH 6.5 cytochrome 558, on cessation of illumination,wasquickly reduced to the initial level. At pH 9.5, there wasalsoappreciable re-reduction of cytochrome 558 and 563 whenthelight was turned off at an early stage of illumination.Theamounts of re-reduction of the cytochromes in the subsequentdark period, however, decreased as photooxidation of cytochromesproceeded. This decrease was accelerated by the presence ofDCMU.
5. At pH 9.5 ascorbate and manganese served as electrondonorsfor die DCMU-sensitive photooxidation of cytochromes558 and563.
6. Experimental results are discussed with specialreference tothe occurrence of two pools of electron carriers,one at thereducing side and the other at the oxidizing sideof photosystem2. The role of manganese in the latter pool ofelectron carriersis also discussed.

(Received March 11, 1970; )     

Light-induced changes of NADPH fluorescence in isolated chloroplasts: a spectral and fluorescence lifetime study

Isolated chloroplasts show a light-induced reversible increase in blue-green fluorescence (BGF), which is only dependent on NADPH changes. In the present communication, we report a time-resolved and spectral analysis of this BGF in reconstituted chloroplasts and intact isolated chloroplasts, in the dark and under actinic illumination. From these measurements we deduced the contribution of the different forms of NADPH (free and bound to proteins) to the light-induced variation of BGF and conclude that this variation is due only to the redox change of the NADP pool. A simple model estimating the distribution of NADPH between the free and bound form was designed, that explains the differences measured for the BGF of reconstituted chloroplasts and intact chloroplasts. From the decay-associated spectra of the chloroplast BGF, we also deduced the participation of flavins to the green peak of chloroplast fluorescence emission spectrum, and the existence of excitation energy transfer from proteins to bound NADPH in chloroplasts. In addition, we re-examined the use of chloroplast BGF as a quantitative measure of NADPH concentration, and confirmed that chloroplast BGF can be used for non-destructive, continuous and probably quantitative monitoring of light-induced changes in NADP redox state.     

Stoichiometries of electron transport complexes in spinach chloroplasts

The stoichiometric relationship among photosystem II complexes, photosystem I complexes, cytochrome b/f complexes, high-potential cytochrome b-559, and chlorophyll in spinach chloroplasts has been determined. Two features of this data stand out, in contrast to currently proposed stoichiometries in which the ratio of photosystem II to photosystem I is reported to be 2:1 and the chlorophyll to reaction center ratio to be as low as 260:1. Using a variety of techniques it was found that the stoichiometry of photosystem II:photosystem I:cytochrome b/f complex was 1:1:1, within 10%, and that the ratio of total chlorophyll to these components was 600:1, also within 10%. A ratio of two high-potential cytochrome b-559 molecules per 640 chlorophyll, or two molecules per photosystem II reaction center, was found. These ratios were remarkably constant regardless of the time of year or the source of the spinach. The concentration of photosystem II complexes was determined using a pH electrode to measure the flash-induced proton release resulting from water oxidation. The photosystem I reaction center concentration was measured by two different techniques that compared favorably. In the first method a pH electrode was used to measure the amount of flash-induced proton consumption associated with the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive oxidation of N,N,N',N'- tetramethylphenylenediamine , resulting in the production of hydrogen peroxide. In the second method the amount of P700 oxidized by far-red light was determined using dual-wavelength spectroscopy. The concentration of the cytochrome b/f complex was determined assuming 1 mol of cytochrome f per complex. The concentration of cytochrome f was measured spectroscopically by its light-induced turnover and by chemical difference spectra. The concentration of high-potential cytochrome b-559 was determined by chemical difference spectra. In addition to these studies, the light-induced absorbance change exhibiting a peak at 323 nm that has been attributed to the reduction of the primary quinone acceptor of photosystem II has been investigated. This measurement frequently has been used to quantitate the photosystem II to chlorophyll ratio. However, in view of these results it is argued that this technique significantly overestimates the photosystem II concentration.     

Light-induced slow changes in chlorophyll a fluorescence in isolated chloroplasts: Effects of magnesium and phenazine methosulfate

We have investigated the possible relationships between the cation-induced and phenazine methosulfate (PMS)-induced fluorescence changes and their relation to light induced conformational changes of the thylakoid membrane.1. In isolated chloroplasts, PMS markedly lowers the quantum yield of chlorophyll a fluorescence (φ_f) when added either in the presence or the absence of dichloro-phenyldimethylurea (DCMU). In contrast, Mg²⁺ causes an increase in φ_f. However, these effects are absent in isolated chloroplasts fixed with glutaraldehyde that retain (to a large extent) the ability to pump protons, suggesting that structural alteration of the membrane—not the pH changes—is required for the observed changes in φ_f. The PMS triggered decrease in φ_f is not accompanied by any changes in the emission (spectral) characteristics of the two pigment systems, whereas room temperature emission spectra with Mg²⁺ and Ca²⁺ show that there is a relative increase of System II to System I fluorescence.2. Washing isolated chloroplasts with 0.75 mM EDTA eliminates (to a large extent) the PMS-induced quenching and Mg²⁺-induced increase of φ_f, and these effects are not recovered by the further addition of dicyclohexyl carbodiimide. It is known that washing with EDTA removes the coupling factor, and thus, it seems that the coupling factor is (indirectly) involved in conformational change of thylakoid membranes leading to fluorescence yield changes.3. In purified pigment System II particles, neither PMS nor Mg²⁺ causes any change in φ_f. Our data, taken together with those of the others, suggest that a structural modification of the thylakoid membranes (not macroscopic volume changes of the chloroplasts) containing both Photosystems I and II is necessary for the PMS-induced quenching and Mg²⁺-induced increase of φ_f. These two effects can be explained with the assumption that the PMS effect is due to an increase in the rate of internal conversion (k_h), whereas the Mg²⁺ effect is due to a decrease in the rate of energy transfer (k_t), between the two photosystems.4. From the relative ratio of φ_f with DCMU and DCMU plus Mg²⁺, we have calculated k_t (the rate constant of energy transfer between Photosystems II and I to be 4.2·10⁸ s^?1, and φ_t (quantum yield of this transfer) to be 0.12.     

Regulation of electron transport in maize mesophyll chloroplasts: The relationship between chlorophyll a fluorescence quenching and O2 evolution

The relationship between the redox state of the primary electron acceptor of photosystem II (Q_A) and the rate of O₂ evolution in isolated mesophyll chloroplasts from Zea mays L. is examined using pulse-modulated chlorophyll a fluorescence techniques. A linear relationship between photochemical quenching of chlorophyll fluorescence (q_Q) and the rate of O₂ evolution is evident under most conditions with either glycerate 3-phosphate or oxaloacetate as substrates. There appears to be no effect of the transthylakoid pH gradient on the rate of electron transfer from photosystem II into Q_A in these chloroplasts. However, the proportion of electron transport occurring through cyclic-pseudocyclic pathways relative to the non-cyclic pathway appears to be regulated by metabolic demand for ATP. The majority of non-photochemical quenching in these chloroplasts at moderate irradiances appeared to be energy-dependent quenching.Abbreviations and symbols PSII photosystem II - Fm maximum fluorescence obtained on application of a saturating light pulse - Fo basal fluorescence recorded in the absence of actinic light (i.e. all PSII traps are open) - Fv Fm-Fo - q_Q photochemical quenching - q_NP non-photochemical quenching - q_E energy-dependent quenching of chlorophyll fluorescence     

Studies on chlorophyll fluorescence in chloroplasts. I. Effects of dithionite on fluorescence of chlorophyll A in isolated spinach chloroplasts

Effects of dithionite on the time-course of fluorescence emitted from chlorophyll a in isolated spinach chloroplasts were studied. Addition of dithionite markedly shortened the induction period of fluorescence and increased the steady-state level of fluorescence. However, a small but distinct induction, comparable to that observed in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea, was always observed in the presence of dithionite. When the fluorescence change was determined in the presence of DCMU, preincubation of the chloroplasts with dithionite for a prolonged period further shortened, but only slowly, the induction period. However, addition of DCMU during the incubation period abolished most of the effects of dithionite in reducing the induction period. The results obtained were interpreted in terms of the reduction by dithionite of endogenous electron carriers associated with photosystem 2.