Induction patterns of delayed luminescence from isolated chloroplasts. I. Response of delayed luminescence to changes in the prompt fluorescence yield

1. Using a phosphoroscope, delayed luminescence and prompt chlorophyll fluorescence from isolated chloroplasts have been compared during the induction period.2. Two distinct decay components of delayed luminescence were measured a “fast” component (from ≈1 ms to ≈6 ms) and a “slow” component (at ≈6 ms).3. The fast luminescence component often did not correlate with the fluorescence changes while the slow component significantly changed its intensity during the induction period in a manner which could usually be linearly correlated with variable portion of the fluorescence yield change.4. This correlation was evident after preillumination with far-red light or after allowing a considerable time for dark relaxation.5. The close relationship between the slow luminescence component and variable fluorescence yield was observed with a large range of light intensities and also in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea which considerably changes the fluorescence induction kinetics.6. Valinomycin and other antibiotics reduced the amplitude of the 6 ms (slow) luminescence without affecting its relation with the fluorescence induction suggesting possibly that a constant electrical gradient exist in the dark or formed very rapidly in the light, which effects the emission intensity.7. Changes in salt levels of suspending media equally affected the amplitude of both delayed luminescence and variable fluorescence under conditions when the reduction of Q is maximal and constant.8. The results are discussed in terms of several models. It is concluded that the model of independent Photosystem II units together with photosynthetic back reaction concept is incompatible with the data. Other alternative models (the “lake” model and photosynthetic back reaction; recombination of charges in the antenna chlorophyll; the “W” hypothesis) were in closer agreement with the results.     

Heterogeneity of system II secondary electron acceptors in Tris-washed chloroplasts

Tris-washed chloroplasts were submitted to saturating short flashes, and then rapidly mixed with dichlorophenyldimethylurea (DCMU). The amount of singly reduced secondary acceptor was estimated from the DCMU-induced increase in fluorescence, caused by the reverse electron flow from secondary to primary acceptor. The back-transfer from the singly reduced secondary acceptor to the primary acceptor Q induced by DCMU addition affects only a part (60%) of the variable fluorescence (ΔF_max). As previously shown, the quenchers involved in this phenomenon, ‘B-type’ quenchers, are different from those controlling the complementary part of the fluorescence, the non-B-type. In this report, we show that at pH 8.5 in the B-type systems, there exist two kinds of secondary electron acceptors: B, a two-electron acceptor, the corresponding Q accounting for 40% of the variable fluorescence; B′, a one-electron acceptor, the corresponding Q accounting for 20% of the variable fluorescence. The lifetimes of B^? and B′^? in the absence of DCMU are 40 and 1 s, respectively. The primary acceptors of the B and B′ systems can be considered as corresponding to the Q₁s defined previously (Joliot, P. and Joliot, A. (1981) in Proceedings of the 5th International Congress on Photosynthesis (Akoynoglou, G., ed.), pp. 885–899, Balaban International Science Services, Philadelphia). The B′ centers seems to be equivalent to the Q_β centers as defined by other workers (Van Gorkom, H.J., Thielen, A.P.G.M. and Gorren, A.C.F. (1982) in The Function of Quinones in Energy Conserving Systems (Trumpower, B.L., ed.), Academic Press, New York, in the press).     

The back reaction in the primary electron transfer couple of photosystem II of photosynthesis

Changes of C-550, cytochrome b₅₅₉ and fluorescence yield induced in chloroplasts by single saturating flashes were studied at low temperature. A single saturating flash at −196°C was quite ineffective in reducing C-550, oxidizing cytochrome b₅₅₉ or increasing the fluorescence yield, presumably because most of the charge separation induced by the flash was dissipated by a direct back reaction in the primary electron transfer couple. The back reaction, which competes with the dark reduction of the oxidized primary electron donor by a secondary electron donor, becomes increasingly important as the temperature is lowered because of the temperature coefficient of the reaction with the secondary donor. The effect of the back reaction is to lower the quantum yield for the production of stable photochemical products by steady irradiation. Assuming a quantum yield of unity for the photoreduction of C-550 at room temperature, the quantum yield for the reaction is about 0.40 at −100°C and 0.27 at −196°C.     

Stimulation by manganese and other divalent cations of the electron donation reactions of Photosystem II

Using inside-out thylakoid membranes, it has been shown that the oxidation of water and associated reduction of dichlorophenol indophenol is partially inhibited by low concentrations of cation chelators. This inhibition correlates with a removal of two manganese ions per Photosystem II reaction centre. The chelator-induced inhibition was completely reversed by the addition of low levels of Mn²⁺ ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 20 μ}\text{M}$) and higher levels of Mg²⁺ and Ca²⁺ ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 1}\text{mM}$). Other cations were not effective, indicating that the ability to overcome the inhibition did not involve a general electrostatic screening process. The degree of inhibition by chelators was greater at lower light intensities and after treatment with glutaraldehyde. In the presence of glutaraldehyde the stimulatory effect of Mn²⁺ was lost, while pretreatment with Mn²⁺ prevented the glutaraldehyde effect. These results are discussed in terms of conformational changes of the electron donation chains involving cation- (preferentially Mn-) dependent coupling between the oxygen evolving and reaction-centre complexes of Photosystem II.     

Inhibition of the reoxidation of the secondary electron acceptor of Photosystem II by bicarbonate depletion

In bicarbonate-depleted chloroplasts, the chlorophyll a fluorescence decayed with a halftime of about 150 ms after the third flash, and appreciably faster after the first and second flash of a series of flashes given after a dark period. After the fourth to twentieth flashes, the decay was also slow. After addition of bicarbonate, the decay was fast after all the flashes of the sequence. This indicates that the bicarbonate depletion inhibits the reoxidation of the secondary acceptor R²⁻ by the plastoquinone pool; R is the secondary electron acceptor of pigment system II, as it accepts electrons from the reduced form of the primary electron acceptor (Q⁻). This conclusion is consistent with the measurements of the DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea)-induced chlorophyll a fluorescence after a series of flashes in the presence and the absence of bicarbonate, if it is assumed that DCMU not only causes reduction of Q if added in the state QR⁻, but also if added in the state QR²⁻.     

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.     

Kinetics of reduction of the oxidized primary electron donor of Photosystem II in spinach chloroplasts and in Chlorella cells in the microsecond and nanosecond time ranges following flash excitation

Absorption changes (ΔA) at 820 nm, following laser flash excitation of spinach chloroplasts and Chlorella cells, were studied in order to obtain information on the reduction time of the photooxidized primary donor of Photosystem II at physiological temperatures.In the microsecond time range the difference spectrum of ΔA between 750 and 900 nm represents a peak at 820 nm, attributable to a radical-cation of chlorophyll a. In untreated dark-adapted material the signal can be attributed solely to P⁺?700; it decays in a polyphasic manner with half-times of 17 μs, 210 μs and over 1 ms. The oxidized primary donor of Photosystem II (P⁺_II) is not detected with a time resolution of 3 μs. After treatment with 3–10 mM hydroxylamine, which inhibits the donor side of Photosystem II, P⁺_II is observed and decays biphasically (a major phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 20–40 μ}\text{s}$, and a minor phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 200 μ}\text{s}$), probably by reduction by an accessory electron donor.In the nanosecond range, which was made accessible by a new fast-response flash photometer operating at 820 nm, it was found the P⁺_II is reduced with a half-time of 25–45 ns in untreated dark-adapted chloroplasts. It is assumed that the normal secondary electron donor is responsible for this fast reduction.     

Charge accumulation and recombination in Photosystem II studied by thermoluminescence. I. Participation of the primary acceptor Q and secondary acceptor B in the generation of thermoluminescence of chloroplasts

In the glow curves of chloroplasts excited by a series of flashes at +1°C the intensity of the main thermoluminescence band appearing at +30°C (B band; B, secondary acceptor of Photosystem II) exhibits a period-4 oscillation with maxima on the 2nd and 6th flashes indicating the participation of the S₃ state of the water-splitting system in the radiative charge recombination reaction. After long-term dark adaptation of chloroplasts (6 h), when the major part of the secondary acceptor pool (B pool) is oxidized, a period-2 contribution with maxima occurring at uneven flash numbers appears in the oscillation pattern. The B band can even be excited at ?160°C as well as by a single flash in which case the water-splitting system undergoes only one transition (S₁ → S₂). The experimental observations and computer simulation of the oscillatory patterns suggest that the B band originates from charge recombination of the S₂B^? and S₃B^? redox states. The half-time of charge recombination responsible for the B band is 48 s. When a major part of the plastoquinone pool is reduced due to prolonged excitation of the chloroplasts by continuous light, a second band (Q band; Q, primary acceptor of Photosystem II) appears in the glow curve at +10°C which overlaps with the B band. In chloroplasts excited by flashes prior to DCMU addition only the Q band can be observed showing maxima in the oscillation pattern at flash numbers 2, 6 and 10. The Q band can also be induced by flashes after DCMU addition which allows only one transition of the water-splitting system (S₁ → S₂). In the presence of DCMU, electrons accumulate on the primary acceptor Q, thus the Q band can be ascribed to the charge recombination of either the S₂Q^? or S₃Q^? states depending on whether the water-splitting system is in the S₂ or the S₃ state. The half-time of the back reaction of Q^? with the donor side of PS II (S₂ or S₃ states) is 3 s. It was also observed that in a sequence of flashes the peak positions of the Q and B bands do not depend on the advancement of the water-splitting system from the S₂ state to the S₃ state. This result implies that the midpoint potential of the water-splitting system remains unmodified during the S₂ → S₃ transition.     

Primary and secondary electron donors in Photosystem II of chloroplasts. Rates of electron transfer and location in the membrane

Absorption changes at 820 or 515 nm after a short laser flash were studied comparatively in untreated chloroplasts and in chloroplasts in which oxygen evolution is inhibited.In chloroplasts pre-treated with Tris, the primary donor of Photosystem II (P-680) is oxidized by the flash, as observed by an absorption increase at 820 nm. After the first flash it is re-reduced in a biphasic manner with half-times of 6 μs (major phase) and 22 μs. After the second flash, the 6 μs phase is nearly absent and P-680⁺ decays with half-times of 130 μs (major phase) and 22 μs. Exogenous electron donors (MnCl₂ or reduced phenylenediamine) have no direct influence on the kinetics of P-680⁺.In untreated chloroplasts the 6 and 22 μs phases are of very small amplitude, either at the 1st, 2nd or 3rd flash given after dark-adaptation. They are observed, however, after incubation with 10 mM hydroxylamine.These results are interpreted in terms of multiple pathways for the reduction of P-680⁺: a rapid reduction (<1 μs) by the physiological donor D₁; a slower reduction (6 and 22 μs) by donor D′₁, operative when O₂ evolution is inhibited; a back-reaction (130 μs) when D′₁ is oxidized by the pre-illumination in inhibited chloroplasts. In Tris-treated chloroplasts the donor system to P-680⁺ has the capacity to deliver only one electron.The absorption change at 515 nm (electrochromic absorption shift) has been measured in parallel. It is shown that the change linked to Photosystem II activity has nearly the same magnitude in untreated chloroplasts or in chloroplasts treated with hydroxylamine or with Tris (first and subsequent flashes). Thus we conclude that all the donors (P-680, D₁, D′₁) are located at the internal side of the thylakoid membrane.     

Photosystem II energy coupling in chloroplasts with H2O2 as electron donor

NH₂OH-treated, non-water-splitting chloroplasts can oxidize H₂O₂ to O₂ through Photosystem II at substantial rates (100–250 μequiv · h^?1 · mg^?1 chlorophyll with 5 mM H₂O₂) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H₂O₂ → Photosystem II → dimethylquinone reaction supports phosphorylation with a $\text{P}\text{e}{}_2$ ratio of 0.25–0.35 and proton uptake with $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.67 (pH 8)–0.85 (pH 6). These are close to the $\text{P}\text{e}{}_2$ value of 0.3–0.38 and the $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.7–0.93 found in parallel experiments for the H₂O → Photosystem II → dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor → Photosystem II → dibromothymoquinone (→O₂) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I^?, ferrocyanide).     

The effect of redox potential on the kinetics of fluorescence induction in pea chloroplasts. II. Sigmoidicity

The shape of the fluorescence induction curve in chloroplasts inhibited by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea has been determined at different redox potentials. At ?10 mV a monophasic and sigmoidal curve is seen which is transformed into an exponential curve when the potential is poised at ?150 mV. At this potential, the quencher with high midpoint, Q_H, is reduced but that with low midpoint, Q_L, is oxidized. Thus, a sigmoidal induction is observed during photoreduction of Q_L and Q_H but photoreduction of Q_L proceeds with exponential kinetics. A correlation between the relative proportions of Q_L and Q_H observed in redox titration and the sigmoidicity of induction is also seen upon depletion of Mg²⁺ and after alkalinization to pH 9.5. Several models are discussed to explain the relationship between Photosystem II interactions and Q heterogeneity.     

Charge accumulation and recombination in Photosystem II studied by thermoluminescence. II. Oscillation of the C band induced by flash excitation

The characteristics of the thermoluminescence band appearing at +50°C in the glow curve (C band) was investigated in maize chloroplasts. The C band, which had a half-time of 10 min, could be charged in the presence of DCMU, and its amplitude significantly increased if preilluminated chloroplasts were reexcited after DCMU addition. Inactivation of the water-splitting system by hydroxylamine- or Tris-treatment did not abolish the C band. In chloroplasts subjected to various numbers of flashes before DCMU addition, the amplitude of the C band exhibited oscillation patterns which were markedly dependent upon dark adaptation of chloroplasts. Flash excitation of chloroplasts preilluminated by continuous light for 30 s prior to 5 min dark adaptation resulted in a period-4 oscillation with maxima occurring at flash numbers 0, 4, 8, 12. After a 6-h dark-adaptation of chloroplasts the period-4 oscillation was superimposed with a period-2 oscillation. The oscillatory patterns were simulated by model calculations and the possible origin of the C band is discussed.     

Studies on the water-oxidizing system by the effects of different treatments in chloroplasts

Treatments such as trypsinization (50 μg/ml per mg Chl for 1 h), osmotic shock of the chloroplasts or mild heating altered the oxygen evolution in such a way that the properties of the Photosystem II were simplified. After these treatments, the damping of the oscillation pattern of O₂ yields induced by a flash series remained the same, irrespective of the level of inhibition induced by the treatment. This damping did not decrease with increasing flash energy, as observed in untreated chloroplasts. The light saturation curve of the S₂ → S₃ transition of the O₂ evolving system no more exhibited the slow-increasing phase at high flash energy observed under normal conditions. The kinetic properties of the O₂-evolving system were also simplified. After the treatments cited above, deactivation of S₂ and S₃ were identical and accelerated with respect to untreated chloroplasts. Turnover kinetics of the transitions S₁ → S₂ and S₂ → S₃ were also similar and simpler without a lag for S₂ → S₃. These results indicate that the treatments mentioned above disconnect one donor from the O₂-evolving complex. This donor, under normal conditions, contributes to the increase of the quantum yield of the transition S₂ → S₃ at high flash energy. This donor is here denoted by D. Our results are in agreement with the following working hypothesis: the large miss, observed on the S₂ → S₃ transition without any contribution of the donor D, may be due to the fact that the system needs a conformation change of the O₂-evolving complex in the S₂ state, so that the main donor Y can oxidize the second H₂O molecule in the water-splitting complex. In the inactive state corresponding to the absence of a conformation change, the donor D, being different in configuration, is likely to oxidize the S₂ state into an S₃ state at high light intensity.     

Measurement of degree of chlorophyll fluorescence polarization in relation to the regulation of excitation energy transfer between Photosystems I and II in pea chloroplasts

The degree of chlorophyll fluorescence polarization (p) at 740 nm was measured at room temperature for pea chloroplasts subjected to various conditions. (1) In agreement with previous published observations, p decreased when the Photosystem (PS) II traps were closed by illumination in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. (2) Under these conditions, the magnitude of p was also sensitive to the presence of salts. Under conditions when ‘spillover’ of the excitation energy from PS II to PS I was low, p was also low, being consistent with increased migration of energy between the PS II light-harvesting chlorophylls. In contrast, when spillover was at a maximum p increased. (3) The change in p in the presence of salts was dependent on the concentration and valency of the cations in such a way as to suggest the changes were mediated through electrostatic forces. The dependency of p on ionic composition of the experimental medium was closely related to the associated changes in fluorescence yield. (4) Membrane stacking, caused by lowering pH of the chloroplast suspension, did not induce a significant change in p, suggesting that this pH-induced process is different from the membrane stacking brought about by manipulating the salt levels. (5) Incubation of thylakoids with ATP induces light-dependent phosphorylation of the light-harvesting chlorophyll-protein complexes, and regulates excitation energy transfer between PS I and PS II (Bennett, J., Steinback, K.R. and Arntzen, C.J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5253–5257). Under conditions which bring about this phosphorylation it was found that p increased to a value indicative of spillover.     

Analysis of chlorophyll fluorescence quenching by DBMIB as a means of investigating the consequences of thylakoid membrane phosphorylation

The effect of Mg²⁺ concentration and phosphorylation of the light harvesting chlorophyll $\text{a}\text{b}$ protein on the ability of DBMIB to quench chlorophyll fluorescence of isolated pea thylakoids has been studied. Over a wide range of Mg²⁺ concentrations (5?0.33 mM), the observed changes in fluorescence yield are mirrored by similar changes in the quenching ability of DBMIB, indicating that the cation-induced phenomenon involves alterations in radiative lifetimes. In contrast, phosphorylation at 10 mM Mg²⁺ brings about a lowering of the chlorophyll fluorescence yield, while having no effect on the quenching capacity of DBMIB. This result can be interpreted as a phosphorylation-induced decrease in PS II absorption cross-section. At Mg²⁺ levels between 5 and 1 mM, phosphorylation leads to a change in the quenching of fluorescence by DBMIB, when compared with non-phosphorylated thylakoids. At these cation levels, the degree of DBMIB-induced quenching cannot wholly account for the observed changes in chlorophyll fluorescence due to phosphorylation. It is concluded that the phosphorylation- and Mg²⁺-induced changes in fluorescence yield are independent but inter-related processes which involve surface charge screening as emphasised by the change in cation sensitivity of the DBMIB quenching before and after phosphorylation.     

The rapid component of electron paramagnetic resonance signal II: A candidate for the physiological donor to Photosystem II in spinach chloroplasts

Rapid light-induced transients in EPR Signal IIf (F^?+) are observed in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated, Tris-washed chloroplasts until the state F P680 Q^? is reached. In the absence of exogenous redox mediators several flashes are required to saturate this photoinactive state. However, the Signal IIf transient is observed on only the first flash following DCMU addition if an efficient donor to Signal IIf, phenylenediamine or hydroquinone, is present. Complementary polarographic measurements show that under these conditions oxidized phenylenediamine is produced only on the first flash of a series. The DCMU inhibition of Signal IIf can be completely relieved by oxidative titration of a one-electron reductant with E′_08.0 = +480 mV. At high reduction potentials the decay time of Signal IIf is constant at about 300 ms, whereas in the absence of DCMU the decay time is longer and increases with increasing reduction potential.A model is proposed in which Q^?, the reduced Photosystem II primary acceptor, and D, a one-electron 480 mV donor endogenous to the chloroplast suspension, compete in the reduction of Signal IIf (F^?+). At high potentials D is oxidized in the dark, and the (Q^? + F^?+) back reaction regenerates the photoactive F P680 Q state. The electrochemical and kinetic evidence is consistent with the hypothesis that the Signal IIf species, F, is identical with Z, the physiological donor to P680.     

The role of pH and membrane potential in the reactions of Photosystem II as measured by effects on delayed fluorescence

(1) If DCMU is added to chloroplasts which have been preilluminated (0–8 flashes) the turnover of the water-splitting enzyme is limited to one further transition upon continuous illumination. (2) The intensity of millisecond delayed fluorescence measured in the presence of mediators of cyclic electron transport around Photosystem I and of DCMU added after pre-flashing is stimulated above the level in the presence of DCMU alone and varies according to the number of pre-flashes (Bowes, J.M. and Crofts, A.R. (1978) Z. Naturforsch 33c, 271–275). (3) Separate contributions of the following energetic terms to the induction kinetics and extent of millisecond delayed fluorescence under these conditions have been examined with a view to assessing their involvement in and the mechanism of the stimulation of the emission above the level in dark-adapted chloroplasts in the presence of DCMU: (a) the initial pH of the phase in equilibrium with the water-splitting enzyme; (b) the change in internal pH which occurred when Photosystem I acted as a proton pump; (c) the electrical potential difference across the membrane resulting from rapid charging of the membrane capacitance. (4) It was confirmed that delayed light was stimulated as a result of the interaction of the intrathylakoid pH (3a and b) with the equilibria of the S-states involving proton release according to the model in which this occurs on all except the transition S₁ → S₂; the stimulation was qualitatively proportional to the number of protons released. (5) There was no marked variation of the membrane potential as a function of the number of pre-flashes.