Presence Of DCMU Research Articles

Photosystem I cyclic electron transport: Measurement of ferredoxin-plastoquinone reductase activity

Absorbance changes of ferredoxin measured at 463 nm in isolated thylakoids were shown to arise from the activity of the enzyme ferredoxin-plastoquinone reductase (FQR) in cyclic electron transport. Under anaerobic conditions in the presence of DCMU and an appropriate concentration of reduced ferredoxin, a light-induced absorbance decrease due to further reduction of Fd was assigned to the oxidation of the other components in the cyclic pathway, primarily plastoquinone. When the light was turned off, Fd was reoxidised and this gave a direct quantitative measurement of the rate of cyclic electron transport due to the activity of FQR. This activity was sensitive to the classical inhibitor of cyclic electron transport, antimycin, and also to J820 and DBMIB. Antimycin had no effect on Fd reduction although this was inhibited by stigmatellin. This provides further evidence that there is a quinone reduction site outside the cytochrome bf complex. The effect of inhibitors of ferredoxin-NADP(+) reductase and experiments involving the modification of ferredoxin suggest that there may be some role for the reductase as a component of FQR. Contrary to expectations, NADPH2 inhibited FQR activity; ATP and ADP had no effect.

Protection of Wheat Chloroplasts from Lipid Peroxidation and Loss of Photosynthetic Pigments by Quercetin under strong Illumination

Summary Exposure of chloroplasts to strong visible light in the presence of DCMU and paraquat resulted in lipid peroxidation and pigment photobleaching, which could be because of increased rates of production of active oxygen species. Quercetin and its glycoside, rutin, protected the chloroplasts from thylakoid lipid peroxidation and pigment photo-oxidation. These flavonoids more effectively protected the chloroplasts when the light treatment was given in the presence of DCMU as compared with that in the presence of paraquat. This differential protection by the flavonoids was investigated by inhibition of crocin photobleaching by quercetin and rutin, which showed that both of the flavonoids effectively quenched singlet oxygen (1O2 On the basis of our results, we suggest that singlet oxygen may be one of the predominant active species produced during strong illumination of chloroplasts, which may further be accelerated in the presence of DCMU. However, strong illumination in the presence of paraquat may lead to production of species such as superoxide and hydroxyl free radicals, which may damage the thylakoid membrane but may not be efficiently quenched by quercetin and rutin.

The dependence of the degrees of sigmoidicities of fluorescence induction curves in spinach chloroplasts on the duration of actinic pulses in pump-probe experiments

Utilizing a pump-probe double flash method, the shapes of the fluorescence induction curves in spinach chloroplasts (0°C), and the induction ratio R = F M/ F 0 ( F 0 and F M are the fluorescence yields when all PS II reaction centers are open, and closed, respectively) were studied as a function of: (1) the duration ( t 1) of the pump flash, (2) the number of pump flashes, (3) absence or presence of DCMU, and (4) the time interval Δt between the pump and the probe flashes. In the pump-probe technique, an actinic pulse (P 1) of different fluences closes a fraction q of the PS II reaction centers. After a variable time interval Δt a second, weak pulse (P 2), is used to measure the variable fluorescence yield F v. The shapes of the fluorescence curves ( F v vs. the fluence of the P 1 pulse) were analyzed in terms of the standard eqution F v = (1 − p) q/(1 − pq), where p is a parameter describing the connectivity between different photosynthetic units (Joliot, A. and Joliot, P. (1964) C. R. Acad. Sci. 13, 4622–4625). The shapes of the F v curves are found to depend only on the width ( t 1) of the pump pulses. For t 1 ⩽ 300 ps, the curves are exponential in shape with p = 0.0 and R < 2.5. However, for pulse durations t 1 in the millisecond range, and employing the same probe flash method for measuring the variable fluorescence, the F v curves assume the same familiar sigmoidal shapes as in the case of conventional steady-state illumination. With t 1 in the microsecond range ( t 1 ≈ 0.7−2 μs), the F v curves are more nearly exponential than sigmoidal in shape with p values generally around ≈0.3 and R < 3. For t 1 >/ 50 μs, the fluorescence induction curves are sigmoidal in shape and, generally, p values of 0.55–0.60 are observed with R > 3. A sequential hit model (Valkunas, L., Geacintov, N.E., France, L. and Breton, J. (1991) Biophys. J. 59, 397–408), in which the PS II reaction centers evolve through different states characterized by differences in fluorescence yields, a process characterized by a dark-time t 1 or ≈2–50 μs between two successive hits, can account for these results. This model is consistent with the concept of interunit transfer of excitations. However, the shapes of the fluorescence induction curves cannot provide any information on the value of p within the context of this model.

Analysis of fluorescence induction in thylakoids with the method of moments reveals two different active Photosystem II centres

There is presently a debate concerning the number of phases in fluorescence induction and on the identification of the several possible heterogeneities in PS II centres. However, the usual methods of analysis present numerical problems, including a lack of 'robustness' (robustness being defined as the ability to give the correct answer in the presence of distortions or artefacts). We present here the adaptation of the method of moments, which was developed for robustness, to the analysis of fluorescence induction. We were thus able to identify three phases in the fluorescence induction in the presence of DCMU. The slowest phase was attributed to the centres inactive in plastoquinone reduction by using duroquinone as electron acceptor. In order to compare fluorescence with and without DCMU, we introduced the 'rate of photochemistry', defined as the product of the area times the rate constant of an exponential. This quantity is invariant for a given centre no matter what the size of the electron acceptor pool is. The two fastest phases in the presence of DCMU were attributed to active centres because their rate of photochemistry was the same as that of the plastoquinone-reducing phases in the absence of DCMU. Because their reduction of plastoquinone showed different kinetics, these two types of active centres were either separated by more than 250 nm or were associated with discrete plastoquinone pools having restricted diffusion domains.

Action of sodium diethyldithiocarbamate (NaDDC) on the HILL activity and chlorophyll fluorescence in Anacystis nidulans IU 625

AbstractEffect of sodium diethyldithiocarbamate (NaDDC) on chlorophyll fluorescence and HILL activity in Anacystis cells exhibited stimulation of both the processes in a concentration‐dependent manner (SINGH et al. 1989). Treatment of cells with sodium dodecyl sulphate (1% w/v) and tris (0.5 M) resulted into further stimulation of NaDDC supported both fluorescence emission and HILL activity. Addition of energy consuming substrates like NaHCO3 (3 mM) and 3‐phosphoglyceric acid (PGA 1 mM) could bring about quenching of NaDDC stimulated fluorescence. However, fluorescence quenching of carbon substrates could be reversed in presence of DCMU (10 mM). Further, the results on cytochrome‐c reduction, fluorescence emission and pigment absorption in the presence of NaDDC exhibited a pH dependence (6–8) of NaDDC on these processes with maximum effect at pH 6. NaDDC‐induced general increase in the absorbance of pigments accompanied by hypsochromic shifts at 680 and 625 nm were indicative of conformational changes in the pigment protein complexes. The results with energy transfer inhibitors showed that uncouplers like FCCP (10 μM), Nigericin (10 μM) and NH4Cl (1 mM) could significantly alter the effect of NaDDC on fluorescence emission and HILL activity, while antimycin (5 μg/ml) and N‐ethylamaleimide (100 mM) had little effect. These results, taken together, suggested that NaDDC‐induced build up of proton gradient might be resulting into cessation of electron transport and stimulation of fluorescence by exerting a back pressure on the charge separation property of membrane.

Physiological sources of reductant for nitrogen fixation activity in Nostoc sp. strain UCD 7801 in symbiotic association with Anthoceros punctatus

Pure cultures of the symbiotic cyanobacterium-bryophyte association with Anthoceros punctatus were reconstituted by using Nostoc sp. strain UCD 7801 or its 3-(3,4-dichlorophenol)-1,1-dimethylurea (DCMU)-resistant mutant strain, UCD 218. The cultures were grown under high light intensity with CO2 as the sole carbon source and then incubated in the dark to deplete endogenous reductant pools before measurements of nitrogenase activities (acetylene reduction). High rates of light-dependent acetylene reduction were obtained both before starvation in the dark and after recovery from starvation, regardless of which of the two Nostoc strains was reconstituted in the association. Rates of acetylene reduction by symbiotic tissue with the wild-type Nostoc strain decreased 99 and 96% after 28 h of incubation in the dark and after reexposure to light in the presence of 5 microM DCMU, respectively. Supplementation of the medium with glucose restored nitrogenase activity in the dark to a rate that was 64% of the illuminated rate. In the light and in the presence of 5 microM DCMU, acetylene reduction could be restored to 91% of the uninhibited rate by the exogenous presence of various carbohydrates. The rate of acetylene reduction in the presence of DCMU was 34% of the uninhibited rate of tissue in association with the DCMU-resistant strain UCD 218. This result implies that photosynthates produced immediately by the cyanobacterium can supply at least one-third of the reductant required for nitrogenase activity on a short-term basis in the symbiotic association. However, high steady-state rates of nitrogenase activity by symbiotic Nostoc strains appear to depend on endogenous carbohydrate reserves, which are presumably supplied as photosynthate from both A. punctatus tissue and the Nostoc strain.

The Role of Secondary Quinone Acceptor Q B of Photosystem II in Photoinhibition of Isolated Wheat Chloroplasts

Summary The role of semiquinone anion radicals in photoinhibition of isolated wheat ( Triticum aestivum ) chloroplasts was investigated by subjecting the chloroplasts to high light stress in the presence or absence of DCMU and DBMIB. The decrease in the efficiency of PS II photochemistry measured as F v /F m ratio and oxygen evolution after photoinhibition of isolated wheat chloroplasts was less in the presence of DCMU. We suggest that the protective effect of DCMU is due to its binding to the 32 kDa Q B -binding protein and reducing the probability of formation of semiquinone anion and other free radical species that have been suggested to be involved in photoinhibition damage. The hypothesis was also tested by using DBMIB during photoinhibition. DBMIB is known to reduce the plastoquinone pool, resulting in an increase in the semiquinone ion population. A greater extent of reduction of F v /F m and oxygen evolution was observed when chloroplasts were photoinhibited in the presence of DBMIB. The results suggest an involvement of reduced semiquinones in the photoinhibition of wheat chloroplasts. A partial recovery of variable chlorophyll fluorescence in the presence of 20 mM hydroxylamine was also observed in chloroplasts subjected to light stress.

The appearance of quenching centres in photosystem II

The variable fluorescence quenching found in the presence of DCMU with isolated chloroplasts which have been exposed previously to a prolonged low light intensity (Sinclair and Spence 1988), is accompanied by a loss of the sigmoidal appearance of the fluorescence induction transient. About 80% of the fluorescence decrease is due to the PS IIα units and 50% of the α centres are inactivated by light exposure. Light incubation slows the PS II partial reaction while the PS I partial reaction is unaffected. We propose that in the light, normal PS IIα centres change into quenching centres which degrade excitation energy to thermal energy. This change can be reversed by 30 min of darkness. A higher flash intensity is needed to saturate the steady state O2 flash yield from light-incubated chloroplasts indicating a light-induced decrease of the average photosynthetic unit size as would happen if PS IIα units were preferentially inactivated. These light-induced changes may relate to an adaptation in leaves to increasing light intensity.

The role of light reactions in the regulation of anthocyanin synthesis in Petunia corollas

The role of light reactions in anthocyanin synthesis was studied in both attached and detached corollas of Petunia hybrida (cv. Hit Parade Rosa), the latter grown in vitro in media containing 150 mM sucrose and 50 μM gibberellic acid (GA). Light was essential for the synthesis of anthocyanin in detached corollas, whereas in intact corollas its effect was only to enhance anthocyanin synthesis. Continuous white light at a fluence rate of at least 20 μmol m−2 s−1 was needed for anthocyanin synthesis in detached corollas. Blue light was more effective than red or green, and far‐red was ineffective. Pigmentation of detached corollas exposed to light was inhibited by the photosynthetic inhibitor 3‐(4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU). The chloroplast uncoupler NH4Cl did not affect anthocyanin synthesis, which was, however, inhibited by the blocking of ATP synthesis in both the chloroplast and the mitochondria by dicyclohexylcarbodiimide (DCCD). Sucrose uptake in vitro was inhibited by DCMU and by darkness, and was promoted equally by blue and red light. The activity of phenylalanine ammonialyase (EC 4.3.1.5) was inhibited in detached corollas grown in the dark or in the light in the presence of DCMU. The activity of chalcone isomerase (EC 5.5.1.6) was not affected by light. These findings suggest that at least two different light reactions are involved in the regulation of anthocyanin synthesis in petunia corollas, namely the high irradiance reaction (HIR) and photosynthesis.

Scavenging of Hydrogen Peroxide in Prokaryotic and Eukaryotic Algae: Acquisition of Ascorbate Peroxidase during the Evolution of Cyanobacteria

Ascorbate (AsA) peroxidase was found in six species of cyanobacteria among ten species tested. Upon the addition of H218O2 to the cells of AsA peroxidase-containing cyanobacteria, 16O2 derived from water and 18O2 derived from H2I8O2 were evolved in the light. The evolution of 16O2 was inhibited by DCMU and did not occur in the dark, but I8O2 was evolved even in the dark or in the presence of DCMU. Similar light-dependent evolution of 16O2 was observed in the cells of AsA peroxidase-containing Euglena and Chlamydomonas. However, the cells of AsA perox-idase-lacking cyanobacteria evolved only 18O2 in either the light or dark. Furthermore, the quenching of chlorophyll fluorescence induced by hydrogen peroxide was observed only in the cells of the AsA peroxidase-containing Synechocystis 6803, and not in the cells of Anacystis nidulans which lacks AsA peroxidase. Thus, cyanobacteria can be divided into two groups, those that has and those that lacks AsA peroxidase. The first group scavenges hydrogen peroxide with the peroxidase using a photoreductant as the electron donor, and the second group only scavenges hydrogen peroxide with catalase.

Electron Transport through photosystem I Stimulates Light Activation of Ribulose Bisphosphate Carboxylase/Oxygenase (Rubisco) by Rubisco Activase

The activation state of ribulose bisphosphate carboxylase/oxygenase (rubisco) in a lysed chloroplast system is increased by light in the presence of a saturating concentration of ATP and a physiological concentration of CO(2) (10 micromolar). Electron transport inhibitors and artificial electron donors and acceptors were used to determine in which region of the photosynthetic electron transport chain this light-dependent reaction occurred. In the presence of DCMU and methyl viologen, the artificial donors durohydroquinone and 2,6-dichlorophenolindophenol (DCPIP) plus ascorbate both supported light activation of rubisco at saturating ATP concentrations. No light activation occurred when DCPIP was used as an acceptor with water as electron donor in the presence of ATP and dibromothymoquinone, even though photosynthetic electron transport was observed. Nigericin completely inhibited the light-dependent activation of rubisco. Based on these results, we conclude that stimulation of light activation of rubisco by rubisco activase requires electron transport through PSI but not PSII, and that this light requirement is not to supply the ATP needed by the rubisco activase reaction. Furthermore, a pH gradient across the thylakoid membrane appears necessary for maximum light activation of rubisco even when ATP is provided exogenously.

Light-dependent degradation of the D1 protein in photosystem II is accelerated after inhibition of the water splitting reaction

Strong illumination of oxygen-evolving organisms inhibits the electron transport through photosystem II (photoinhibition). In addition the illumination leads to a rapid turnover of the D1 protein in the reaction center of photosystem II. In this study the light-dependent degradation of the D1 reaction center protein and the light-dependent inhibition of electron-transport reactions have been studied in thylakoid membranes in which the oxygen evolution has been reversibly inhibited by Cl- depletion. The results show that Cl(-)-depleted thylakoid membranes are very vulnerable to damage induced by illumination. Both the D1 protein and the inhibition of the oxygen evolution are 15-20 times more sensitive to illumination than in control thylakoid membranes. The presence, during the illumination, of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) prevented both the light-dependent degradation of the D1 protein and the inhibition of the electron transport. The protection exerted by DCMU is seen only in Cl(-)-depleted thylakoid membranes. These observations lead to the proposal that continuous illumination of Cl(-)-depleted thylakoid membranes generates anomalously long-lived, highly oxidizing radicals on the oxidizing side of photosystem II, which are responsible for the light-induced protein damage and inhibition. The presence of DCMU during the illumination prevents the formation of these radicals, which explains the protective effects of the herbicide. It is also observed that in Cl(-)-depleted thylakoid membranes, oxygen evolution (measured after the readdition of Cl-) is inhibited before electron transfer from diphenylcarbazide to dichlorophenolindophenol.(ABSTRACT TRUNCATED AT 250 WORDS)

Bicarbonate effects in leaf discs from spinach

In this paper, we show the unique role of bicarbonate ion in stimulating the electron transfer of photosystem II (PS II) in formate-treated leaf discs from spinach. This is referred to as the "bicarbonate effect" and is independent of the role of CO2 in CO2 fixation. It is shown to have two sites of action: (1) the first, described here for the first time, stimulates the electron flow between the hydroxylamine donation site ("Z" or "D") and QA, the first plastoquinone electron acceptor and (2) the other accelerates the electron flow beyond QA, perhaps at the QA QB complex, where QB is the second plastoquinone electron acceptor. The first site of inhibition by formate-treatment is detected by the decrease of the rate of oxygen evolution and the simultaneous quenching of the variable chlorophyll a (Chl a) fluorescence of leaf discs infiltrated with 100 mM formate for about 10 s followed by storage for 10 min in dark. This is referred to as short-term formate treatment. Addition of bicarbonate reverses this short-term formate effect and restores fully both Chl a fluorescence and oxygen evolution rate. Reversible quenching of variable Chl a fluorescence of heated and short-term formate treated leaf discs, in the presence of hydroxylamine as an artificial electron donor to PS II, is also observed. This suggests that the first site of action of the anion effect is indeed between the site of donation of hydroxylamine to PS II (i.e. "Z" or "D") and QA. The second site of the effect, where bicarbonate depletion has its most dramatic effect, as well known in thylakoids, is shown by an increase of Chl a fluorescence of leaf discs infiltrated with 100 mM formate for about 10 min followed by storage for 10 min in dark. This is referred to as the long-term formate treatment. Addition of bicarbonate fully restores the variable Chl a fluorescence of these leaf discs. Chl a fluorescence transient of DCMU-infiltrated (10 min) leaf discs is similar to that of long-term formate-treated one suggesting that the absence of bicarbonate, like the presence of DCMU, inhibits the electron flow beyond QA.

Inhibitory effects of mercury on photosystem II photochemistry in Dunaliella tertiolecta under in vivo conditions

The inhibitory effect of mercury on photosystem II (PSII) photochemistry was investigated in the green alga Dunaliella tertiolecta. In the presence of 80 μM HgCl 2, oxygen evolution is completely inhibited, whereas 50% of the variable fluorescence still remains. The effect of mercury on the fluorescence yield in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) is reversed when the electron donor to PSII is hydroxylamine instead of water. This result confirms that the inhibition site of mercury in the presence of DCMU is associated with the activity of the water-splitting system. Mercury induces a decrease in the complementary area, indicating that the number of functional PSII primary electron acceptors is decreased. Mercury inhibition results in a decrease in the PSII quantum yield in algal cells. However, the reduction rate of the PSII primary electron acceptor (Q A) is not affected. Therefore, this effect on PSII induces a complete inactivation of the PSII reaction centres associated with the mercury-inhibited water-splitting system.

A proportion of photosystem II core complexes are decoupled from the phycobilisome in light-state 2 in the cyanobacterium Synechococcus 6301

Cells of Synechococcus 6301 were briefly exposed to a phycocyanin-absorbed light in the presence of DCMU. PS II trap closure was then estimated from fluorescence induction measurements with excitation light absorbed predominantly either by chlorophyll or by phycocyanin. In cells adapted to light-state 2, the exposure to light absorbed by phycocyanin closed only a proportion of the PS II centres that could be closed by exposure to light absorbed by chlorophyll. This distinction was reduced in cells adapted to light-state 1. We conclude that a proportion of PS II core complexes become decoupled from the phycobilisomes during the transition to light-state 2.

Formation by NO of nitrosyl adducts of redox components of the Photosystem II reaction center. I. NO binds to the acceptor-side non-heme iron

NO is a good electrophile and EPR spin probe, carrying an unpaired electron ( S = 1 2 ). Exposure of Photosystem II reaction centers to NO results in the appearance of an EPR signal at g = 4. Dark titration with NO shows a K d ≈ 30 μM in spinach chloroplasts and 250 μM in BBY preparations. Successive cycles of illumination at 200 K, followed by incubation at 245 K, results in binary oscillations of the amplitude of the g = 4 signal in spinach chloroplasts. This signal is small in states Q − AQ B, Q − AQ − B, but large in states Q AQ B and Q AQ BH 2 of the PS II acceptor side. NO slows electron transfer between Q A and Q B (Diner, B.A and Petrouleas, V. (1990) Biochim. Biophys. Acta 1015, 141–149) and modifies the Mössbauer spectrum of the non-heme Fe(II). These results strongly support the assignment of the g = 4 EPR signal to an acceptor side Fe(II)-NO adduct in an S = 3 2 state. Exchange coupling of this species with the S = 1 2 semiquinones results in an integral spin system, not detectable in X-band EPR. The g = 4 spectrum can be described by a spin hamiltonian. The rhombicity parameter E D is in all cases small (⩽ 0.015), implying a near axial environment. NO can donate electrons to PS II. Charge recombination, following a light flash in the presence of DCMU, is blocked by NO ( Km ≈ 30 μM, 25°C). NO also reacts reversibly in the dark with D + (an oxidized secondary donor), resulting in the disappearance of Signal II dark with a K d of 3 μM. Pumping off of NO in the dark results in full recovery of the signal.

Anaerobic biosynthesis of unsaturated fatty acids in the cyanobacterium, Oscillatoria limnetica.

The mechanism for synthesis of monounsaturated fatty acids under aerobic and anaerobic conditions was studied in the facultative anaerobic cyanobacterium, Oscillatoria limnetica. The hexadecenoic acid (C16:1) of aerobically grown O. limnetica was shown to contain both the delta 7 (79%) and delta 9 (21%) isomers, while the octadecenoic (C18:1) acid was entirely the delta 9 acid. Incorporation of [2-14C] acetate into the fatty acids under aerobic conditions resulted in synthesis of the delta 7 and delta 9 C16:1 and the delta 9 C18:1. Synthesis of unsaturated fatty acids in the presence of DCMU required sulfide. Anaerobic incubations in the presence of DCMU and sulfide (less than 0.003% atmospheric oxygen) resulted in a two-fold increase in monounsaturated fatty acids of both delta 7 and delta 9 C16:1 and delta 9 and delta 11 C18:1. The synthesis of these is characteristic of a bacterial-type, anaerobic pathway.

Regulation of Transplasmalemma Electron Transport in Oat Mesophyll Cells by Sphingoid Bases and Blue Light

Long-chain sphingoid bases inhibit transplasmalemma electron transport in certain animal cells in part by inhibiting protein phosphorylation. As a first step in determining whether similar regulatory processes exist for cell surface redox activity in plants, peeled leaf segments of Avena sativa L. cv Garry were exposed to sphingoid bases and other long chain lipids. Sphingoid bases which are the most active inhibitors of protein kinase C in animal cells inhibit transplasmalemma electron transport by mesophyll cells in the dark as measured by reduction of exogenous ferricyanide. In white light, however, the same compounds markedly stimulate redox activity. The stimulation by sphingoid bases in the light is not eliminated by the inhibitor of photosynthesis, 3-(3,4-dichlorophenyl)-1,1 dimethylurea (DCMU). Redox activity remaining in the presence of DCMU and sphingoid bases can be observed in blue but not red light. A tentative hypothesis considering the involvement of two separate redox systems is presented in an attempt of explain the disparate action of sphingoid bases on electron transport across the plasmalemma.

Mitochondrial Respiration Can Support NO3− and NO2− Reduction during Photosynthesis

Mass spectrometric analysis shows that assimilation of inorganic nitrogen (NH(4) (+), NO(2) (-), NO(3) (-)) by N-limited cells of Selenastrum minutum (Naeg.) Collins results in a stimulation of tricarboxylic acid cycle (TCA cycle) CO(2) release in both the light and dark. In a previous study we have shown that TCA cycle reductant generated during NH(4) (+) assimilation is oxidized via the cytochrome electron transport chain, resulting in an increase in respiratory O(2) consumption during photosynthesis (HG Weger, DG Birch, IR Elrifi, DH Turpin [1988] Plant Physiol 86: 688-692). NO(3) (-) and NO(2) (-) assimilation resulted in a larger stimulation of TCA cycle CO(2) release than did NH(4) (+), but a much smaller stimulation of mitochondrial O(2) consumption. NH(4) (+) assimilation was the same in the light and dark and insensitive to DCMU, but was 82% inhibited by anaerobiosis in both the light and dark. NO(3) (-) and NO(2) (-) assimilation rates were maximal in the light, but assimilation could proceed at substantial rates in the light in the presence of DCMU and in the dark. Unlike NH(4) (+), NO(3) (-) and NO(2) (-) assimilation were relatively insensitive to anaerobiosis. These results indicated that operation of the mitochondrial electron transport chain was not required to maintain TCA cycle activity during NO(3) (-) and NO(2) (-) assimilation, suggesting an alternative sink for TCA cycle generated reductant. Evaluation of changes in gross O(2) consumption during NO(3) (-) and NO(2) (-) assimilation suggest that TCA cycle reductant was exported to the chloroplast during photosynthesis and used to support NO(3) (-) and NO(2) (-) reduction.

Electron transfer through the quinone acceptor complex of Photosystem II after one or two actinic flashes in bicarbonate-depleted spinach thylakoid membranes

We report here the pH dependence of the kinetics of the decay of variable chlorophyll a fluorescence after one or two actinic flashes in the absence or the presence of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethyl-urea) in HCO 2 −-depleted or anion-inhibited spinach thylakoid membranes. All the reported effects of HCO 3 − removal are reversed by the addition of 5 mM HCO 3 −. The initial first-order component for the oxidation of Q A − (the reduced primary plastoquinone acceptor of Photosystem II (PS II) by Q B (the secondary plastoquinone acceptor) was reversibly inhibited in a pH-dependent manner in HCO 3 −-depleted membranes. After a single actinic flash, the half-time of Q A − decay was 630 μs (amplitude, 29%) at pH 6.5 which changed to a value of 320 μs (amplitude, 66%) at pH 7.75. The rate and amplitude at pH 7.75 were approximately the same as found in the restored and control membranes which were pH independent over the same pH range. A similar observation was made after the second actinic flash. Thus, at alkaline pH HCO 3 −-depleted membranes behave as control membranes with respect to electron flow from Q A to Q B or to Q B −. The time ( t 50) at which the [Q A −] is 50% of the maximum [Q A −] during the back reaction between Q A − and the S 2 state of the oxygen-evolving complex, in the presence of 5 μM DCMU, was increased from 1.3 s in control and restored samples to 5.3 s in HCO 3 −-depleted samples below pH 7.0, but was unaffected above pH 7.5 (2.3–2.9 s in all cases). Furthermore, a new pathway of Q A − with a half-time of less than 100 μs was present at pH 8.0 in the presence of DCMU, in approx. one-third of the PS II centers in HCO 3 −-depleted membranes. The apparent equilibrium for the sharing of an electron between Q A and Q B is estimated to decrease by a factor of 4 at pH 6.0 in treated membranes ( K app ≈ 16) as compared to the restored or control membranes ( K app ≈ 62); there was no difference in K app at pH 7.75. Estimates of the operating redox potential for the Q B/Q B − couple from the results presented here indicated that the pH dependence of this parameter is greatly reduced in treated membranes (−60 mV at pH 6.0 to −72 mV at pH 7.75) as compared to restored or control membranes (−25 mV at pH 6.0 to −72 mV at pH 7.75). We iscuss our results in the context of a model that envisages HCO 3 − to act as a proton donor to the protein dissociable group believed to participate in the protonation of Q B −. Finally, the possibility of HCO 3 − being a ligand to Fe 2+ in the Q A-Fe-Q B complex of the PS II reaction center is also discussed.