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⁻.     

Light-induced changes of fluorescence and absorbance in spinach chloroplasts at −40 °C

1. 1. Spinach chloroplasts were stored in the dark for at least 1 h, rapidly cooled to −40 °C, and illuminated with continuous light or short saturating flashes. In agreement with the measurements of Joliot and Joliot, chloroplasts that had been preilluminated with one or two flashes just before cooling showed a less efficient increase in the yield of chlorophyll a fluorescence upon illumination at −40 °C than dark-adapted chloroplasts. The effect disappeared below −150 °C, but reappeared again upon warming to −40 °C. Little effect was seen at room temperature in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), added after the preillumination.

2. 2. Light-induced absorbance difference spectra at −40 °C in the region 500–560 nm indicated the participation of two components, the socalled 518-nm change (P518) and C-550. After preillumination with two flashes the absorbance change at 518 nm was smaller, and almost no C-550 was observed. After four flashes, the bands of C-550 were clearly visible again.

3. 3. The fluorescence increase and the absorbance change at 518 nm showed the same type of flash pattern with a minimum after the second and a maximum at the fourth flash. In the presence of 100 μM hydroxylamine, the fluorescence response was low after the fourth and high again after the sixth flash, which confirmed the hypothesis that the flash effect was related to the so-called S-state of the electron transport pathway from water to Photosystem 2.

4. 4. The kinetics of the light-induced absorbance changes were the same at each wavelength, and, apart from the size of the deflection, they were independent of preillumination. Flash experiments indicated that the absorbance changes were a one-quantum reaction. This was also true for the fluorescence increase in dark-adapted chloroplasts, but with preilluminated chloroplasts several flashes were needed to approximately saturate the fluorescence yield.

5. 5. The results are discussed in terms of a mechanism involving two electron donors and two electron acceptors for System 2 of photosynthesis.

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.     

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.     

Fluorescence induction in intact spinach chloroplasts

Under conditions in which the Photosystem II quencher is rapidly reduced upon illumination, either after a preillumination or following treatment with dithionite, the fluorescence-induction curve of intact spinach chloroplasts (class I type) displays a pronounced dip. This dip is probably identical with that observed after prolonged anaerobic incubation of whole algal cells (“I-D dip”). It is inhibited by 3(3,4-dichlorophenyl)-1,1-dimethylurea and occurs in the presence of dithionite, sufficient to reduce the plastoquinone pool. It is influenced by far red light, methylviologen, anaerobiosis and uncouplers in a manner consistent with the interpretation that it represents a photochemical quenching of fluorescence by an electron transport component situated between the Photosystem II quencher and plastoquinone. Glutaraldehyde inhibition may indicate that protein structural changes are involved.     

Effects of carbonyl cyanide m-chlorophenylhydrazone and hydroxylamine on the Photosystem II electron exchange mechanism in 3-(3,4-dichlorophenyl)-1,1-dimethylurea treated algae and chloroplasts

The effects of NH₂OH and carbonyl cyanide m-chlorophenylhydrazone (CCCP) on 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated algae and chloroplasts were studied. In the presence of DCMU, the photochemically separated charges can only disappear through a recombination back reaction; both substances induce an irreversible reduction of the donor side and after sufficient illumination their action in the presence of DCMU leads to the formation of a permanent fluorescent state.

In the DCMU + CCCP system, a fast fluorescence induction curve is observed. The fluorescence yield is brought to its maximum by two flashes. The luminescence emission is strongly inhibited and most centers reach their permanent fluorescent state after one flash.

In the DCMU + NH₂OH system, a slow fluorescence rise is observed and several saturating flashes are needed for the fluorescence yield to reach its maximum. The exhaustion of the NH₂OH oxidizing capacity and the complete transformation to a permanent fluorescent state also require a large number of flashes.

The reduction pathway catalyzed by CCCP appears to be a good competitor to the back reaction, while NH₂OH seems to be a relatively inefficient donor.

In addition the action of NH₂OH and CCCP on fluorescence suggests that the donor side influences the quenching properties of Photosystem II centers. A possible mechanism is proposed.     

Inhibition of O2 evolution by NADP in isolated potato tuber chloroplast and its identity with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea inhibition site.

Chloroplast from greening potato tuber showed good photosynthetic capacity. The evolution of O₂ was dependent upon the intensity of light. A light intensity of 30 lux gave maximum O₂ evolution. At higher intensities inhibition was observed. The presence of bicarbonate in the reaction mixture was essential for O₂ evolution. NADP was found to be a potent inhibitor of O₂ evolution in this system. NADP and 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) inhibited the O₂ evolution completely at a 3 μm concentration level, which was reversed by oxidized 2,6-dichlorophenol-indophenol (DCIP). Cyanide (CN)-treated chloroplasts showed full O₂ evolution capacity, when a lipophilic electron acceptor like N-tetramethyl-p-phenylenediamine (TMPD) or DCIP was used along with ferricyanide. Ferricyanide alone showed only 20% reduction. NADP or DCMU could inhibit O₂ evolution only when TMPD was the acceptor but not with DCIP. Photosystem II (PS II) isolated from these chloroplasts also showed inhibition by NADP or DCMU and its reversal by DCIP. Here also the evolution of O₂ with only TMPD as acceptor was sensitive to NADP or DCMU. In the presence of added silicotungstate in PS II NADP or DCMU did not affect ferricyanide reduction or oxygen evolution. The chloroplasts were able to bind exogenously added NADP to the extent of 120 nmol/mg chlorophyll. It is concluded that the site of inhibition of NADP is the same as in DCMU, and it is between the DCIP and TMPD acceptor site in the electron transport from the quencher (Q) to plastoquinone (PQ).     

Effects of Anaerobiosis on Chlorophyll Fluorescence Yield in Spinach (Spinacia oleracea) Leaf Discs

When spinach (Spinacia oleracea) leaf discs were incubated in a dark anaerobic environment, the chlorophyll fluorescence yield was much increased relative to the aerobic control. Occasionally, the fluorescence yield of the darkened anaerobic samples approached 80% of the maximum fluorescence. The anaerobic incubation period also induced in a leaf disc the capacity to exhibit a low light-mediated chlorophyll fluorescence induction phenomenon. This involved a rapid and slow increase in fluorescence yield, followed by a slow quenching. This could be induced by light levels as low as 400 [mu]W m-2. The anaerobic-dependent increase in chlorophyll fluorescence yield could be relaxed by either far-red light, O2, or a saturating pulse of white light. It was concluded that the anaerobic-dependent increase in chlorophyll fluorescence yield was due to a dark reduction of the plastoquinone pool and its relaxation by reoxidation. Darkened isolated chloroplasts did not exhibit a fluorescence yield increase under anaerobic conditions. Fluorescence slowly increased only when dithiothreitol or dithionite was added.     

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).     

Comparative study of the action of ultraviolet-C and ultraviolet- B radiation on photosynthetic electron transport

The effect of ultraviolet-C (UV-C, mainly 254 nm radiation) and ultraviolet-B (UV-B, 290-320 nm) radiation on the photosynthetic electron transport reactions has been investigated. The rates of Hill activity mediated by ferricyanide and dichlorodimethoxy-p-benzoquinone (DCDMQ) were differently sensitive to UV-C but equally inhibited by UV-B. Replacement of water with diphenylcarbazide was ineffective in restoring the activity of dichlorophenol indophenol (DCPIP) Hill reaction in UV-B treated chloroplasts, but had significant effect in UV-C treated chloroplasts.
Photobleaching of carotenoids in the presence of carbonyl cyanide-m-chlorophenyl-hydrazone, an indicator of the photochemical reaction associated with the reaction centre of photosystem II, was suppressed and is paralleled by the changes in Hill activity only in UV-B-treated chloroplasts. Carotenoid photobleaching occurred even in UV-C treated chloroplasts showing no measurable Hill activity. UV-C and UV-B irradiation diminished variable fluorescence. With UV-B treated, but not with UV-C treated chloroplasts, an increase in the fluorescence yield was observed upon the addition of 3-(3,4-dichIorophenyl)-l,l-dimethylurea (DCMU) and/or Na dithionite.
Photosystem I activity was found to be unaffected by both UV-C and UV-B radiation at the fluences tested. Kinetics of P700 photooxidation and dark reversal in UV treated chloroplasts indicate that only the electron flow from photosystem II to photosystem I is impaired. It is concluded that while UV-B radiation inactivates specifically the photosystem II reaction centre, UV-C radiation acts at plastoquinone, the quencher Q, and the water oxidizing enzyme system.     

On the possible site of inhibition of photosynthetic electron transport by ultraviolet-B (UV-B) radiation

In Amaranthus chloroplasts that are exposed to ultraviolet-B (UV-B) radiation, the electron flow from water to dichlorophenol indophenol (DCPIP) was inhibited, but the electron flow from reduced DCPIP to methyl viologen remains unaffected. Diphenylcarbazide was ineffective in restoring the activity of DCPIP Hill reaction in UV-B irradiated chloroplasts. Electron flow from water to ferricyanide or dichloro-dimethoxy- p -benzoquinone was inhibited to a degree similar to that of the DCPIP Hill reaction.
The rate of carotenoid photobleaching in the presence of carbonyl cyanide- m -chlorophenylhydrazone, an indicator of the photochemical reaction near the vicinity of reaction centre of photosystem II, was suppressed and paralleled with the inhibition of the DCPIP Hill reaction.
In the UV-B treated chloroplasts, the variable part of the fluorescence transient was diminished. Though the fluorescence yield was lowered by the UV-B radiation, addition of 3-(3,4-dichlorophenyl)-l, l-dimethylurea (DCMU) and/or sodium dithionite increased the emission markedly. With the increase in the dosage of UV-B irradiation, the time required to reach the steady state fluorescence level became longer in the absence of DCMU and shorter in the presence of DCMU. The kinetics of 520 nm absorbance change was markedly unaltered by the UV-B irradiation but its dark decay was prolonged. It is concluded that UV-B irradiation inactivates the photosystem II reaction centre.     

Light-induced de-epoxidation of violaxanthin in lettuce chloroplasts IV. The effects of electron-transport conditions on violaxanthin availability

1. In isolated chloroplasts of Lactuca sativa var. Manoa, the size of the violaxanthin fraction which is available for de-epoxidation is not directly dependent on electron transport but rather related to the reduced level of some electron carrier between the photosystems. This is concluded from the effects of various electrontransport conditions on violaxanthin availability: Under conditions of electron transport through both photosystems, availability was saturated at a lower electron-transport rate with actinic light at 670 than at 700 nm. Under conditions of electron transport through Photosystem I, availability was smaller for linear electron flow from reduced N-methylphenazonium methosulfate via methylviologen to oxygen than for cyclic electron flow mediated by either N-methylphenazonium methosulfate or 2,6-dichlorophenolindophenol; in addition for linear r flow from reduced N-methylphenazonium methosulfate via methylviologen to oxygen, availability increased with decreasing light intensity.2. The postulated carrier whose reduced level is related to availability seems to be some carrier between plastoquinone and the primary acceptor of Photosystem II or plastoquinone itself. This conclusion follows from the fact that availability increased with increasing light intensity under conditions of electron flow through both photosystems and that 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (≤ μM) had no effect on availability, whereas low levels of 3,3-(3′,4′-dichlorophenyl)-1,1-dimethylurea resulted in decreased availability (50% decrease at 1 μM). Furthermore, availability in 3,3-(3′,4′-dichlorophenyl)-1,1-dimethylurea-poisoned chloroplasts was fully restored by 2-methyl-1,4-naphtoquinone (menadione) which mediates cyclic electron flow through plastoquinone.3. Violaxanthin availability was zero in the dark and increased in the light to a maximum of 67% of the total violaxanthin in chloroplasts. It is proposed that this variable violaxanthin availability reflects conformational changes on the internal surface of the thylakoid membrane which result in variable exposure of violaxanthin to the de-epoxidase. The fact that not all of the violaxanthin was available for de-epoxidation may indicate a heterogenous distribution of violaxanthin in the membrane.     

Studies on chlorophyll fluorescence in chloroplasts III. Effect of artificial electron donors for photosystexn 2 on reoxidation of the fluorescence quencher, Q, in spinach chloroplasts

1. Effects of various reducing reagents including dithionite,whichserve as artificial electron donors for photosystem 2,on therecovery of fluorescence induction in the presence of3-(3,4-dichlorophenyl)-l,l-dimethylurea(DCMU) during the darkincubation of preilluminated chloroplastswere investigated.
2. The dark recovery of fluorescence induction was not affectedby the addition of the p-phenylenediamine-ascorbate couple,the hydroquinone-ascorbate couple or manganese. Incubation ofchloroplasts with dithionite caused gradual suppression of thedark recovery.
3. Preillumination of chloroplasts caused partialinhibition ofthe recovery of fluorescence induction.
4. At lowintensities of excitation light, the fluorescence yieldincreasedvery slowly and continuously, and never reached asteady state.This continuous increase in fluorescence yieldunder weak lightwas due to photoinhibition of the dark recovery.A techniquewas devised to determine the steady state yieldof fluorescence,without the intervention of photoinhibition,at weak light intensities.The steady state yield of fluorescencein the presence of DCMUwas suppressed at lower excitation intensities.This drop inthe fluorescence yield was not altered by the presenceof addedreducing reagents but was suppressed after long preincubationof chloroplasts with dithionite.
5. The delayed fluorescencewith a decay time of seconds was affectedby dithionite butnot by other reductants.
6. Results are discussed in terms ofreoxidation of the reducedprimary electron acceptor, Q, bythe oxidized primary electrondonor for photosystem 2. A modelof the electron transport associatedwith photoreaction 2 isproposed to account for the experimentalresults obtained.

(Received February 27, 1973; )     

Localization of electron transport inhibition in plastoquinone reactions

Reduction kinetics of P700 following a short flash are measured in spinach chloroplasts after oxidation of the electron carriers between the two photoreactions by far-red light. Three features of the kinetics allow us to localize simultaneously inhibition at different sites between photoreaction II and the reducing site of plastoquinol. These are the initial lag, the halftime, and the area under the transient of the P700 absorbance change, which indicate the electron transfer time from photoreaction II to the reducing site of plastoquinol, the rate of plastoquinol oxidation, and the number of electrons transferred to the special plastoquinone B functioning as secondary electron acceptor of photosystem II, respectively. As an additional diagnostic parameter for inhibition before and after the plastoquinone pool, the area under the transient of the P700 absorbance change is used after long flashes. This area is proportional to the amount of reduced plastoquinone as shown by the absorbance change at 265 nm. The effects of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) are compared with those of 2-bromo-4-nitrothymol, 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol, and Illoxan as representatives for new classes of inhibitors. While 2-halogeno-4-nitrothymols inhibit the reduction of plastoquinone similarly to DCMU, their diphenyl ether derivatives inhibit selectively the oxidation of plastoquinol.     

On the functional organization of electron transport from plastoquinone to Photosystem I

Signal I, the EPR signal of P-700, induced by long flashes as well as the rate of linear electron transport are investigated at partial inhibition of electron transport in chloroplasts. Inhibition of plastoquinol oxidation by dibromothymoquinone and bathophenanthroline, inhibition of plastocyanin by KCN and HgCl₂, and inhibition by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are used to study a possible electron exchange between electron-transport chains after plastoquinone. (1) At partial inhibition of plastocyanin the reduction kinetics of P-700⁺ show a fast component comparable to that in control chloroplasts and a new slow component. The slow component indicates P-700⁺ which is not accessible to residual active plastocyanin under these conditions. We conclude that P-700 is reduced via complexed plastocyanin. (2) The rate of linear electron transport at continuous illumination decreases immediately when increasing amounts of plastocyanin are inhibited by KCN incubation. This is not consistent with an oxidation of cytochrome f by a mobile pool of plastocyanin with respect to the reaction rates of plastocyanin being more than an order of magnitude faster than the rate-limiting step of linear electron transport. It is evidence for a complex between the cytochrome b₆ - f complex and plastocyanin. The number of these complexes with active plastocyanin is concluded to control the rate-limiting plastoquinol oxidation. (3) Partial inhibition of the electron transfer between plastoquinone and cytochrome f by dibromothymoquinone and bathophenanthroline causes decelerated monophasic reduction of total P-700⁺. The P-700 kinetics indicate an electron transfer from the cytochrome b₆ - f complex to more than ten Photosystem I reaction center complexes. This cooperation is concluded to occur by lateral diffusion of both complexes in the membrane. (4) The proposed functional organization of electron transport from plastoquinone to P-700 in situ is supported by further kinetic details and is discussed in terms of the spatial distribution of the electron carriers in the thylakoid membrane.     

Further characterization of a photosystem ii particle isolated from spinach chloroplasts by triton treatment delayed light emission

Delayed light emission from the Triton-fractionated Photosystem II subchloroplast fragments (TSF-IIa) was measured between 0.5 and 10 ms after the termination of illumination. The delayed light emission was diminished by Photosystem II inhibitors, DCMU and o-phenanthroline, which act between the reduced primary acceptor and the plastoquinone pool.Secondary electron donors to Photosystem II, diphenylcarbazide, phenylenediamine, Mn²⁺, and ascorbate inhibited delayed light emission. Secondary electron acceptors such as ferricyanide, dichlorophenol indophenol, and dimethyl benzoquinone enhanced delayed light emission. The addition of secondary electron acceptors to TSF-IIa particles containing Mn²⁺ restored delayed light emission to almost the control level. The plastoquinone antagonist, 2,5-dibromo-3-methyl-6-isopropyl p-benzoquinone, increased delayed light emission at low concentrations but decreased it at higher concentrations. Silicomolybdate enhanced the delayed light emission of TSF-IIa particles markedly, and reversed the inhibition by DCMU. Silicomolybdate showed a similar stimulatory effect on the delayed-light intensity in broken spinach chloroplasts at shorter times after the termination of illumination. Carbonyl cyanide m-chloro (or p-trifluoromethoxy) phenylhydrazones inhibited the delayed light emission, but NH₄Cl had no effect.     

Light-dependent quenching of chlorophyll fluorescence in pea chloroplasts induced by adenosine 5′-triphosphate

Addition of ATP to chloroplasts causes a reversible 25–30% decrease in chlorophyll fluorescence. This quenching is light-dependent, uncoupler insensitive but inhibited by DCMU and electron acceptors and has a half-time of 3 minutes. Electron donors to Photosystem I can not overcome the inhibitory effect of DCMU, suggesting that light activation depends on the reduced state of plastoquinone. Fluorescence emission spectra recorded at ?196°C indicate that ATP treatment increases the amount of excitation energy transferred to Photosystem I. Examination of fluorescence induction curves indicate that ATP treatment decreases both the initial (F_o) and variable (F_v) fluorescence such that the ratio of F_v to the maximum (F_m) yield is unchanged. The initial sigmoidal phase of induction is slowed down by ATP treatment and is quenched 3-fold more than the exponential slow phase, the rate of which is unchanged. A plot of F_v against area above the induction curve was identical plus or minus ATP. Thus ATP treatment can alter quantal distribution between Photosystems II and I without altering Photosystem II-Photosystem II interaction. The effect of ATP strongly resembles in its properties the phosphorylation of the light-harvesting complex by a light activated, ATP-dependent protein kinase found in chloroplast membranes and could be the basis of physiological mechanisms which contribute to slow fluorescence quenching in vivo and regulate excitation energy distribution between Photosystem I and II. It is suggested that the sensor for this regulation is the redox state of plastoquinone.     

Determination and modification of the redox state of the secondary acceptor of Photosystem II in the dark

The redox state of the secondary electron acceptor B of Photosystem II was studied using fluorescence measurements. Preillumination of algae or chloroplasts with a variable number of short saturating flashes followed rapidly by the addition of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea induces oscillations of the initial level of fluorescence. The phase of these oscillations is characteristic of a given $\text{B}\text{B}{}^{\mathrm{?}}$ ratio in the dark-adapted samples.We conclude from our results that about 50% of the secondary electron acceptors are singly reduced in the dark in Chlorella cells, but that more than 70% are fully oxidized in the dark adapted chloroplasts.Benzoquinone treatment modifies this distribution in Chlorella leading to the same situation as in chloroplasts, i.e. more than 70% of the secondary acceptors are oxidized in the dark.The same ratio is observed if these algae are illuminated and then dark-adapted, unless an artificial donor (hydroxylamine) is added before this illumination. In that case about 50% B^? is generated and stabilized in the dark.