Modulation of herbicide-binding by the redox state of Q400, an endogenous component of Photosystem II

Preincubation of isolated chloroplasts with ferricyanide, prior to addition of DCMU, unmasks a high-potential electron acceptor (Q₄₀₀) in Photosystem II that acts as an additional quencher and prolongs the fluorescence induction curve in the presence of DCMU (Ikegami, I. and Katoh, S. (1973) Plant Cell Physiol. 14, 829–836). This study confirms that Q₄₀₀ is endogenous to Photosystem II and is not a bound ferricyanide, and several new characteristics of this high potential acceptor are established. (a) It is accessible to ferricyanide even in the presence of DCMU. The rate of oxidation, however, is very slow, consistent with access only via Q_A. Accessibility may be enhanced by magnesium, reminiscent of the oxidation of Q⁻_A by ferricyanide. (b) Oxidation of Q₄₀₀ drastically suppresses the binding of DCMU at neutral and alkaline pH. Below pH 6, however, DCMU binding is essentially normal. The pH dependence of DCMU binding is consistent with the known pH dependence of the redox midpoint potential of Q₄₀₀. (c) Binding of many other inhibitors of Q_A-to-Q_B electron transfer is much less affected or even completely unaffected. These results have implications for current notions of herbicide binding and may also bear on the origin of slow phases of fluorescence induction in the presence of DCMU.     

Evidence for two types of electron transfer processes through Photosystem II

Inhibition of electron flow from H₂O to methylviologen by 3-(34 dichlorophenyl)-1,1 dimethyl urea (DCMU), yields a biphasic curve — an initial high sensitivity phase and a subsequent low sensitivity phase. The two phases of electron flow have a different pH dependence and differ in the light intensity required for saturation.Preincubation of chloroplasts with ferricyanide causes an inhibition of the high sensitivity phase, but has no effect on the low sensitivity phase. The extent of inhibition increases as the redox potential during preincubation becomes more positive. Tris-treatment, contrary to preincubation with ferricyanide, affects, to a much greater extent, the low sensitivity phase.Trypsin digestion of chloroplasts is known to block electron flow between Q _A and Q _B, allowing electron flow to ferricyanide, in a DCMU insensitive reaction. We have found that in trypsinated chloroplasts, electron flow becomes progressively inhibited by DCMU with increase in pH, and that DCMU acts as a competitive inhibitor with respect to [H⁺]. The sensitivity to DCMU rises when a more negative redox potential is maintained during trypsin treatment. Under these conditions, only the high sensitivity, but not the low sensitivity phase is inhibited by DCMU.The above results indicate the existence of two types of electron transport chains. One type, in which electron flow is more sensitive to DCMU contains, presumably Fe in a Q _A Fe complex and is affected by its oxidation state, i.e., when Fe is reduced, it allows electron flow to Q _B in a DCMU sensitive step; and a second type, in which electron transport is less sensitive to DCMU, where Fe is either absent or, if present in its oxidized state, is inaccessible to reducing agents.Abbreviations DCMU 3-(34 dichlorophenyl)-1, 1 Dimethyl urea - MV methyl viologen - PS II Photosystem II - Tris tris (hydroxymethyl)aminomethane     

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).     

A differential effect of 3-(3′4′ dichlorophenyl)-1,1 dimethyl urea and atrazine on fluorescence kinetics in chloroplasts

It was found that DCMU had a differential effect at two concentration ranges on variable fluorescence kinetics in isolated chloroplasts. The increase in fluorescence rate at low concentrations of DCMU was abolished by preincubation of chloroplasts with ferricyanide or formate, treatments which were shown to convert Fe in the PS II reaction center (i.e., the FeQ_A complex) into a non-oxidizable form, but it was not affected by Tris treatment. Increase in fluorescence kinetics (at the initial linear rate) at high concentrations of DCMU was found to be abolished by Tris treatment but it was only marginally affected by ferricyanide or formate treatments. The effect of Tris could be abolished by addition of hydroquinone-ascorbate, which restored electron flow to the pool of secondary acceptors.Contrary to the effect of DCMU, no such differential concentration dependence of the variable fluorescence kinetics was found for atrazine.The increase in fluorescence kinetics (at the initial linear rate) at a low concentration rate of DCMU is presumably restricted to units which contain an oxidizable Fe in the FeQ_A complex. Increase in fluorescence kinetics (at the initial linear rate) at high DCMU concentration is probably related to the effect of DCMU at the Q_B site.Abbreviations DCMU 3-(34 dichlorophenyl)-1,1 dimethyl urea - PS II Photosystem II - Tris tris (hydroxymethyl) aminomethane     

Phosphorylation in isolated chloroplasts coupled to dichlorophenyldimethylurea-insensitive silicomolybdate reduction

1. The electron transport in isolated chloroplasts with silicomolybdate as electron acceptor has been reinvestigated. The silicomolybdate reduction has been directly measured as ΔA₇₅₀ or indirectly as O₂ evolution (in the presence or absence of ferricyanide).2. Silicomolybdate-dependent O₂ evolution is inhibited to a similar extent by 3-(3,4-dichlorophenyl) 1, 1-dimethylurea (DCMU) or dibromothymoquinone (DBMIB), indicating the existence of two different sites of silicomolybdate reduction: one before the DCMU block (i.e. at Photosystem II) and one after the DBMIB block (i.e. at Photosystem I).3. Silicomolybdate-dependent O₂ evolution is coupled to ATP synthesis with an $\text{ATP}\text{2}\text{e}{}^{\mathrm{?}}$ ratio of 1.0 to 1.1. The presence of ferricyanide inhibits this ATP synthesis ($\text{ATP}\text{2}\text{e}{}^{\mathrm{?}}$ ratio then is about 0.3).4. Silicomolybdate-dependent O₂ evolution is also coupled to ATP-synthesis in the presence of DCMU with an $\text{ATP}\text{2}\text{e}{}^{\mathrm{?}}$ ratio of 0.6–0.8 characteristic of Site II; in this case the electron transport itself is not affected by uncouplers or energy-transfer inbihitors.5. The data are interpreted as a further demonstration that the water-splitting reaction is responsible for the conservation of energy at Photosystem II.     

Dichlorophenylurea-insensitive Reduction of Silicomolybdic Acid by Chloroplast Photosystem II

The photoreduction of silicomolybdate and other heteropoly ions by chloroplasts is insensitive to 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU). Both water and diphenylcarbazide can be used as electron source for the reduction. Three different assays for silicomolybdate reduction are described including oxygen evolution, formation of a reduced heteropoly blue silicomolybdate, or an indirect assay for reduced silicomolybdate by redox indicators, such as ferricyanide or cytochrome c. The effects of detergents and tris washing are consistent with silicomolybdate reduction through photosystem II before the DCMU site. The effects of orthophenanthroline and bathophenanthroline indicate chelator-sensitive sites in photosystem II before the site of DCMU action.     

Exogenous versus endogenous acceptors in photosystem ii in inhibited chloroplasts

The conditions for steady-state Signal IIf formation in response to single turnover flashes in Tris-treated, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-inhibited chloroplasts have been investigated. DCMU inhibits Signal IIf generation as the photoinactive state, Z P680 Q-A, accumulates. Potassium ferricyanide relieves this inhibition so that Signal IIf can be fully developed on each flash in a flash series. The effectiveness of ferricyanide in stimulating Signal IIf formation is dependent on its concentration, the flash repetition rate, and the salt composition of the chloroplast suspension. There are two models in the literature for Q-A oxidation under these inhibitory conditions: direct oxidation of Q-A by exogenous acceptors like ferricyanide or oxidation of Q-A by an endogenous acceptor, AH, which has a midpoint potential of approximately 400 mV. It is found that the direct exogenous acceptor model accounts well for these data, whereas the AH model does not explain several of these results. The apparent rate constant for the direct oxidation of Q-A by ferricyanide at various concentrations of salt has been calculated from our electron paramagnetic resonance (EPR) data and compared with the corresponding rate constant determined by S. Itoh from fluorescence data (Biochim, Biophys. Acta 504, 324-340, 1978); good agreement is found for the two different experimental approaches.     

Studies on chlorophyll fluorescence in chloroplasts II. Effect of ferricyanide on the induction of fluorescence in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea

1. The effect of preincubating spinach chloroplasts with ferricyanideon the time courses of chlorophyll- fluorescence in the presenceof 3-(3,4-dichlorophyl)-1,1-dimethylurea (DCMU) was studied.When DCMU was absent from the preincubation mixture, but wasadded just before the onset of excitation light, preincubationof chloroplasts with ferricyanide markedly affected the fluorescencekinetics. The rise-rate was lowered and consequently the areaabove the induction curve (S/Fv), which is proportional to thepool size of the electron acceptor(s) for photosystem 2, increased.The maximum increase in the S/Fv was attained after 3 min and10 min, respectively, of preincubation with 5?10^–4M and3?10^–5M ferricyanide.
2. When DCMU was present during preincubationwith ferricyanide,the effect of ferricyanide in increasingthe S/Fv, was completelyeliminated.
3. The effect of ferricyanidewas also suppressed by addition offerrocyanide to the preincubationmixture. The redox potentialof the ferri-ferrocyanide mixturewhich produced 50% suppressionof the ferricyanide effect wasabout 360 mV.
4. A similar dependency of the ferricyanide effecton the redoxpotential was observed in Tris-treated chloroplasts.However,the redox potential of cytochrome b-559 was markedlyloweredby Tris-treatment.
5. These results were explained byassuming the occurrence of asecondary electron acceptor, R,between the reaction centerof photosystem 2 and the DCMU-sensitivesite.

(Received February 27, 1973; )     

Characterization of the stimulating effect of low-dose stressors in maize and bean seedlings

The effect of some more or less harmful compounds like Cd, Pb, Ni, Ti salts and DCMU at low concentrations on the development of chloroplasts in maize and bean seedlings was investigated. Chlorophyll content, chlorophyll a/b ratio, photosynthetic activity (14CO2 fixation), chlorophyll-protein composition of thylakoid membranes, fluorescence spectra of chloroplasts, fluorescence induction parameters of leaves and electron microscopic structure of maize and bean chloroplasts as well as growth parameters were studied. Stimulation of chlorophyll synthesis and photosynthetic activity was observed at different intervals during all of the treatments, while chlorophyll a/b ratios and fluorescence properties of leaves or chloroplasts did not change considerably except in DCMU treated plants. Heavy metal treatments increased the amount of photosystem I and light-harvesting complex II, while decreased amount of photosystem I and higher amount of light-harvesting complex II was found in DCMU treated thylakoids. Electron microscopy showed only sligth differences in the morphology of chloroplast lamellar system (mostly in DCMU treated plants), while the status of the plasmalemma and tonoplast seemed to be altered as a result of certain metal treatments. Results showed the expression of a cytokinin-like effect on the development of chloroplasts. It is assumed, that these low-dose stressors generate non-specific alarm reactions in plants, which may involve changes of the hormonal balance.     

Analysis of chlorophyll a fluoresence changes in weak light in heat treated Amaranthus chloroplasts

After preheating of Amaranthus chloroplasts at elevated temperatures (up to 45°C), the chlorophyll a fluorescence level under low excitation light rises as compared to control (unheated) as observed earlier in other chloroplasts (Schreiber U and Armond PA (1978) Biochim Biophys Acta 502: 138–151). This elevation of heat induced fluorescence yield is quenched by addition of 0.1 mM potassium ferricyanide, suggesting that with mild heat stress the primary electron acceptor of photosystem II is more easily reduced than the unheated samples. Furthermore, the level of fluorescence attained after illumination of dithionite-treated samples is independent of preheating (up to 45°C). Thus, these experiments indicate that the heat induced rise of fluorescence level at low light can not be due to changes in the elevation in the true constant F₀ level, that must by definition, be independent of the concentration of Q_A. It is supposed that the increase in the fluorescence level by weak modulated light is either partly associated with dark reduction of Q_A due to exposure of chloroplasts to elevated temperature or due to temperature induced fluorescence rise in the so called inactive photosystem II centre where Q_A are not connected to plastoquinone pool. In the presence of dichlorophenyldimethylurea the fluorescence level triggered by weak modulated light increases at alkaline pH, both in control and heat stressed chloroplasts. This result suggests that the alkaline pH accelerates electron donation from secondary electron donor of photosystem II to Q_A both in control and heat stressed samples. Thus the increase in fluorescence level probed by weak modulated light due to preheating is not solely linked to increase in true F₀ level, but largely associated with the shift in the redox state of Q_A, the primary stable electron acceptor of photosystem II.Abbreviations ADRY Acceleration of Deactivation of Reaction of Enzyme Y - CCCP Carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone - Chl Chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FeCN potassium ferricyanide - HEPES 4-(2-hydroxy ethyl)-1-piperazine ethane sulfonic acid - LHCP Light harvesting chlorophyll protein - MES (4-morpholine ethane sulfonic acid) - PS photosystem - Q_A and Q_B first and second consecutive electron acceptors of photosystem II - TES (2-[tris(hydroxymethyl)-methylamino]-1-ethanesulfonic acid) sulfonic acid - TRICINE N-[tris(hydroxymethyl)methyl] glycine     

Effects of various buffers, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and NaN3 on menadione mediated photophosphorylation in isolated chloroplasts

The effects of DCMU and NaN₃ were studied on menadione-mediated photophosphorylation in broken spinach chloroplasts kept in low oxygen tension in Tricine or HEPES buffers at either high or reduced irradiances. – (A) At high irradiance (131 W. m^?2) and absence of NaN₃ the ATP formation was inhibited by DCMU regardless of the type of buffer used. – (B) At high irradiance and presence of NaN₃ some concentrations of DCMU stimulated, whilst others inhibited the ATP formation in a HEPES buffer. The ATP formation was predominantly inhibited by DCMU in a Tricine buffer. – (C) At reduced irradiance (57 W. m^?2) the chloroplasts in a HEPES buffer were almost insensitive towards DCMU both in the presence and absence of NaN₃. – (D) Chloroplasts in a Tricine buffer were slightly stimulated in their ATP formation by DCMU at reduced irradiance either with or without the presence of NaN₃ in the experimental medium. When menadione acts as a terminal electron acceptor, oxygen is consumed on its reoxidation. The results indicate that this process may occur with oxygen released by the splitting of water as the main oxidant. – The data also demonstrate the importance of caution when selecting buffering substances as well as when choosing light intensities for experiments on photophosphorylation in chloroplasts.     

Inhibition site of the electron transport system in lettuce chloroplasts by fumigation of leaves with SO2

In chloroplasts isolated from SO₂-fumigated leaves at 2.0 ppm,electron flow from water to 2,6-dichloroindophenol (DCIP) wasinhibited, but the electron flow from reduced DCIP to methylviologen was not affected. Neither diphenylcarbazide nor MnCl₂could restore the activity of the DCIP-Hill reaction of SO₂-injuredchloroplasts. Electron flows, from water to ferricyanide orto silicomolybdic acid, were inhibited in a degree similar tothat of the DCIP-Hill reaction. The rate of carotenoid photobleaching in the presence of carbonylcyanide-m-chlorophenylhydrazone was suppressed and paralleledthe inhibition of the DCIP-Hill reaction. In SO₂-injured chloroplasts, the variable part of the fluorescencetransient was diminished, and the fluorescence yield loweredby SO₂ was increased with 3-(3', 4'-dichlorophenyl)-l, l-dimethylurea(DCMU) or more pronouncedly by incubating the sample with sodiumdithionite. However, the yield could not recover to the levelfound in non-fumigated chloroplasts. With SO₂ fumigation, thetime required to reach steady-state level of fluorescence becamelonger in the absence of DCMU, but was not altered in the presenceof DCMU. The pool size of the primary electron acceptors decreasedwith SO₂ fumigation. We concluded that SO₂ inactivated the primaryelectron donor or the reaction center itself. The mode of SO₂action in the electron transport chain is discussed. (Received October 20, 1979; )     

Effects of herbicides on binary oscillations of ultraviolet flash-induced absorption changes in chloroplasts

The competition between various herbicides and plastoquinone, at the Q_B site of Photosystem II, has been studied by measuring absorption changes between 300 and 360 nm in spinach and chenopod chloroplasts, in response to a train of saturating short xenon flashes. A complex pattern was observable without addition of chemicals interfering with electron flow. The effect of potassium ferricyanide, of hydroxylamine and of valinomycine-K⁺ permitted to simplify the pattern and, in particular, to observe binary oscillations with flash number, attributable to the functioning of the two-electron gate Q_B. Herbicides belonging to different classes block electron transfer when added at high concentration. At low concentration, however, inverted binary oscillations become observable. When the chloroplasts have been first oxidized with ferricyanide, this behaviour develops progressively in response to illumination. Varying the herbicide concentration, it appears that the concentration inducing maximum binary oscillations correlates with inhibition of linear electron transfer, within each class of herbicides. Phenolic herbicides induce the largest oscillations and ureas the smallest. The binary oscillations have the spectrum of the plastoquinone anion. The results clearly show that the studied herbicides compete efficiently with Q_B, but not with Q⁻_B, at the Q_B binding site. Atrazine-resistant chenopod chloroplasts still display normal binary oscillations in the absence of herbicide, or in the presence of atrazine alone. They are highly sensitive to DCMU and to i-dinoseb, but no inverted binary oscillations could be observed with these herbicides.     

Stimulation of Photosystem I Electron Transport by High Concentration of 3-(3,4-Dichlorophenyl)-1,1-dimethyl Urea in Uncoupled Chloroplasts

The light saturated rate of photosystem I-dependent electron transport (ascorbate/dichlorophenol-indophenol → methyl vilogen in presence of 1 micromolar 3-[3,4-dichlorophenyl]-1,1-dimethyl urea [DCMU]) was increased by a high concentration of DCMU added to broken and uncoupled chloroplasts isolated from pea (Pisum sativum). At 50 micromolar DCMU, the increase was around 50%. No stimulation was observed under limiting intensity of illumination, indicating that the relative quantum yield of electron transport was not affected by high DCMU. The light-saturated rate in coupled (to proton gradient formation) chloroplasts was unchanged by 50 micromolar DCMU, suggesting that the rate-limitation imposed by energy coupling was not affected. Using N,N,N′,N′-tetramethyl-p-phenylene diamine as electron donor, essentially no DCMU stimulation of the rate was observed, indicating further that the electron donation at a site close to P700 was not affected by high DCMU. It is concluded that DCMU, in the range of 10 to 50 micromolar, affected the thylakoid membranes in such a way that the rate constant of electron donation by dichlorophenol-indophenol at the site prior to the site of energy coupling increased. Further observations that DCMU at 100 micromolar stimulated the rate in coupled chloroplasts indicated an additional DCMU action, presumably by uncoupling the chloroplasts from phosphorylation, as suggested by Izawa (Shibata et al., eds, Comprehensive Biochemistry and Biophysics of Photosynthesis, University Press, State College, Pennsylvania, pp 140-147, 1968). A scheme has been proposed for multiple sites of DCMU action on the electron transport system in chloroplasts.     

Effects of Azide on Photophosphorylation in Isolated Spinach Chloroplasts

The influence of sodium azide on open-chain and flavine mononucleotide mediated cyclic photophosphorylation in isolated spinach chloroplasts was investigated under anaerobic conditions. Open chain phosphorylation was completely inhibited with DCMU both in the presence and absence of sodium azide in the experimental medium. Flavine mononucleotide mediated photophosphorylation was only slightly inhibited by DCMU in the absence of sodium azide but inhibited in two steps by increasing amounts of DCMU when sodium azide was present in the medium. The first step can be explained as being mainly an effect of DCMU on an open chain electron transport, with water and H₂O₂ as electron donors and with flavine mononucleotide — kept in an oxidized state by sodium azide — as the electron acceptor. The second step, as well as the comparatively insensitivity to DCMU in the absence of sodium azide, depends on cyclic photophosphorylation mediated by flavine mononucleotide.     

Guard Cell Chloroplasts Are Essential for Blue Light-Dependent Stomatal Opening in Arabidopsis

Blue light (BL) induces stomatal opening through the activation of H⁺-ATPases with subsequent ion accumulation in guard cells. In most plant species, red light (RL) enhances BL-dependent stomatal opening. This RL effect is attributable to the chloroplasts of guard cell, the only cells in the epidermis possessing this organelle. To clarify the role of chloroplasts in stomatal regulation, we investigated the effects of RL on BL-dependent stomatal opening in isolated epidermis, guard cell protoplasts, and intact leaves of Arabidopsis thaliana. In isolated epidermal tissues and intact leaves, weak BL superimposed on RL enhanced stomatal opening while BL alone was less effective. In guard cell protoplasts, RL enhanced BL-dependent H⁺-pumping and DCMU, a photosynthetic electron transport inhibitor, eliminated this effect. RL enhanced phosphorylation levels of the H⁺-ATPase in response to BL, but this RL effect was not suppressed by DCMU. Furthermore, DCMU inhibited both RL-induced and BL-dependent stomatal opening in intact leaves. The photosynthetic rate in leaves correlated positively with BL-dependent stomatal opening in the presence of DCMU. We conclude that guard cell chloroplasts provide ATP and/or reducing equivalents that fuel BL-dependent stomatal opening, and that they indirectly monitor photosynthetic CO₂ fixation in mesophyll chloroplasts by absorbing PAR in the epidermis.     

Charge accumulation at the reducing side of system 2 of photosynthesis

A study was made of the reactions between the primary and secondary electron acceptors of Photosystem 2 by measurements of the increase of chlorophyll fluorescence induced in darkness by dithionite or by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). The experiments were done either with chloroplasts to which hydroxylamine or carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) was added, or with chloroplasts treated with tris(hydroxymethyl)aminomethane (Tris) to which phenylenediamine and ascorbate were added as donor system. Under these conditions the fluorescence increase induced by dithionite or DCMU added after illumination with short light flashes was dependent on the flash number with a periodicity of two; it was large after an uneven number of flashes, and small after a long darktime or after an even number of flashes. The results are interpreted in terms of a model which involves a hypothetical electron carrier situated between Q and plastoquinone; this electron carrier is thought to equilibrate with plastoquinone in a two-electron transfer reaction; the results obtained with DCMU are explained by assuming that its midpoint potential is lowered by this inhibitor.     

P700 sensitization by low concentration of DCMU in isolated pea chloroplasts

Using steady-state relaxation spectrophotometric technique a P₇₀₀ component ($\text{t}\text{1}\text{2}\text{～20 ms}$) has been detected which was sensitized by low concentration (10^?7M) DCMU in isolated broken chloroplasts of pea. The relative quantum yield of electron flux through DCMU-sensitized P₇₀₀ was similar to that with methyl viologen or NADP as terminal electron acceptor and water as electron donor. Kinetic analysis showed that a small fraction (10%) of the total P₇₀₀ reaction centers was sensitized by low DCMU.     

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.