Steady State of Photosynthetic Electron Transport in Cells of the Cyanophyti Synechocystis PCC 6714 Having Different Stoichiometry between PS I and PS II: Analysis of Flash-Induced Oxidation-Reduction of Cytochrome f and P700 under Steady State of Photosynthesis

The steady state of photosynthetic electron transport drivenby two photosystems was studied with cells of the cyanophyteSynechocystis PCC 6714 by analyzing the flash-induced oxidation-reductionof Cyt f and P700 under continuous background illumination.We first analyzed the spectra and the kinetics of flash-inducedabsorption changes in the 400 to 440 nm wavelength region anddefined the absorption changes due to oxidation-reduction ofCyt f and P700. Results indicated that the flash-induced absorptionchanges at 420 and 435 nm are due to the oxidation-reductionof Cyt f and P700, respectively. Determination of the steadystate of Cyt f (420 nm) and P700 (435 nm) was made for the cellsgrown under a weak orange light exciting mainly PS II (PS IIlight) and having a high ratio of PS I to PS II (PS I/PS II),and those grown under a weak red light exciting preferentiallyPS I (PS I light) and having a low PS I/PS II. The steady stateof electron transport in cells of the two types were comparedunder PS I and PS II lights. The results indicated that: (1)under the light conditions used for growth (both red and orangelight), the intermediate electron pool between the two photosystemsremained in a redox state so as to keep both photosystems inthe open state. (2) When shifted to PS I light, the intermediatepool and PS I in cells of high PS I/PS II became extremely electron-poor,and so most of the PS I reaction centers were closed. (3) Theintermediate pool in cells of low PS I/PS II became extremelyelectron-rich when shifted to PS II light, and most of the PSII reaction centers were closed. The electron transport stateis released from such biased states by regulation of PS I/PSII. Results supported our previously proposed hypothesis thatthe stoichiometry between PS I and PS II is regulated so asto keep the two photosystems in the open state. The relationshipbetween the steady state of electron transport and the regulationof PS I/PS II is discussed. (Received August 2, 1990; Accepted December 10, 1990)     

Regulation of Electron Transport Composition in Cyanobacterial Photosynthetic System: Stoichiometry among Photosystem I and II Complexes and Their Light-Harvesting Antennae and Cytochrome b6/fComplex

This study was done to confirm our previous observation withthe pattern of changes in electron transport composition inducedby an imbalance of the electron transport state. Contents ofphotosystem (PS) I and II complexes and their antennae and Cytb₆/f complex were determined for systems of cyanobacterium SynechocystisPCC 6714 of different PS I/PS II ratios. The results indicatedthat (1) the observed changes in the PS I/PS II ratio are not-dueto regulation of the activities of the respective PS's but tochanges in their contents, (2) the molar ratio between PS IIand Cyt b₆/f complexes was fairly constant when marked changesoccurred in the PS I content, and (3) the PS II and Cyt b₆/fcontents per cell remained fairly constant while the PS I contentchanged markedly. These findings agree with our previous observationwith autotrophic cells of Anacystis nidulans Tx 20 and supportour argument that in cyanobacterial and red algal electron transportsystems, the content of the terminalcomponent(s), such as PSI complex, is regulated in order to maintain a balance betweenthe electron influx by PS II action to the system and the effluxby PS I action from it. (Received June 3, 1987; Accepted September 20, 1987)     

Studies on thylakoid phosphorylation and noncyclic electron transport

The effect of thylakoid phosphorylation on noncyclic electron transport in spinach chloroplasts was investigated by measuring both the reduction of nicotinamide adenine dinucleotide phosphate (NADP) and the steady-state redox level of the primary electron acceptor quinone of photosystem II (Q) during electron flow to NADP. These data are compared with the theoretical predictions for an electron transport model which relates both the redox levels of Q and the photosystem II optical cross section to the overall velocity of noncyclic electron flow. It is demonstrated that transfer of 15-20% of the photosystem II antenna to photosystem I may stimulate electron flow to NADP only if Q is less than 60-70% oxidized (this condition exists with our thylakoids, even at extremely low absorption fluxes, when the illumination is not specifically enriched in photosystem I absorbed wavelengths); in phosphorylated thylakoids the steady-state redox level Q is substantially shifted to a more oxidized one (measurements of this parameter using light of different wavelengths quantitatively support the idea that thylakoid phosphorylation leads to increased photosystem I and decreased photosystem II cross sections); thylakoid phosphorylation leads to stimulated noncyclic electron flow to NADP only when the increased photosystem I antenna is able to bring about large increases in the steady-state level of oxidized Q.     

Photosynthetic Electron Transport During Senescence of the Primary Leaves of Phaseolus vulgaris L.: II. THE ACTIVITY OF PHOTOSYSTEMS ONE AND TWO, AND A NOTE ON THE SITE OF REDUCTION OF FERRICYANIDE

The activity of photosystems one and two (PS I and PS II) wasmeasured in chloroplasts isolated from the primary leaves ofPhaseolus vulgaris. During foliar senescence, the rates of electrontransport through PS I and PS II declined by approximately 25%and 33% respectively. These losses of activity could not accountfor the decrease of 80% in the rate of coupled, non-cyclic electrontransport during senescence. It is therefore suggested thatan impairment of electron flow between the photosystems limitednon-cyclic electron transport in chloroplasts from older leaves.In this study the activity of PS II was measured using oxidizedp-phenylenediamine as the electron acceptor, and trifluralinas an inhibitor of electron transport between PS II and PS I.In chloroplasts from young leaves the reduction of ferricyanidewas a measure of non-cyclic electron transport, but in preparationsfrom older leaves ferricyanide received a large proportion ofelectrons from PS II.     

Regulation of Photosystem Stoichiometry in the Photosynthetic System of the Cyanophyte Synechocystis PCC 6714 in Response to Light-Intensity

Light-induced changes in stoichiometry among three thylakoidcomponents, PS I, PS II and Cyt b₆-f complexes, were studiedwith the cyanophyte Synechocystis PCC 6714. Special attentionwas paid to two aspects of the stoichiometric change; first,a comparison of the patterns of regulation in response to differencesin light-intensity with those induced by differences in light-quality,and second, the relationship between regulation of the stoichiometryand the steady state of the electron transport system. Resultsfor the former indicated that (1) the abundance of PS I on aper cell basis was reduced under white light at the intensityas high as that for light-saturation of photosynthesis, butPS I per cell was increased under low light-intensity, (2) PSII and Cyt b₆-f complexes remained fairly constant, and (3)changes in the abundance of PS I depended strictly on proteinsynthesis. The pattern was identical with that of chromaticregulation. For the second problem, the redox steady-statesof Cyt f and P700 under white light of various intensities weredetermined by flash-spectroscopy. Results indicated that (1)Cyt f and P700 in cells grown under low light-intensity [highratio of PS I to PS II (PS I/PS II)] were markedly oxidizedwhen the cells were exposed to high light-intensity, while theyremained in the reduced state under low light-intensity. (2)After a decrease in the abundance of PS I, most of P700 remainedin the reduced state even under high light-intensity, whilethe level of reduced Cyt f remained low. (3) Both Cyt f andP700 in cells of low PS I/PS II were fully reduced under lowlight-intensity, and Cyt f reduction following the flash wasrapid, which indicates that the turnover of PS I limits theoverall rate of electron flow. After an increase in the abundanceof PS I, the electron transport recovered from the biased state.(4) The redox steady-state of the Cyt b₆-f complex correlatedwell with the regulation of PS I/PS II while the state of thePQ pool did not. Based on these results, a working model ofthe regulation of assembly of the PS I complex, in which theredox steady-state of the Cyt b₆-f complex is closely relatedto the primary signal, is proposed. (Received August 2, 1990; Accepted December 10, 1990)     

Interaction of Amaranthin with the Electron Transport Chain of Chloroplasts

The electron paramagnetic resonance method was used to study the interactions of amaranthin with isolated class B chloroplasts from broad bean (Vicia faba L.) and amaranth (Amaranthus tricolor L.) during the light-driven electron and proton transport. Amaranthin was shown to interact with electron transport chain of chloroplasts at the PS II level; it also affects the electron transport near PS I. At the same time, amaranthin had no significant inhibitory effect on the light-dependent formation of the transmembrane pH gradient.     

Bioenergetic consequences from xenotopic expression of a tunicate AOX in mouse mitochondria: Switch from RET and ROS to FET

Electron transfer from all respiratory chain dehydrogenases of the electron transport chain (ETC) converges at the level of the quinone (Q) pool. The Q redox state is thus a function of electron input (reduction) and output (oxidation) and closely reflects the mitochondrial respiratory state. Disruption of electron flux at the level of the cytochrome bc₁ complex (cIII) or cytochrome c oxidase (cIV) shifts the Q redox poise to a more reduced state which is generally sensed as respiratory stress. To cope with respiratory stress, many species, but not insects and vertebrates, express alternative oxidase (AOX) which acts as an electron sink for reduced Q and by-passes cIII and cIV. Here, we used Ciona intestinalis AOX xenotopically expressed in mouse mitochondria to study how respiratory states impact the Q poise and how AOX may be used to restore respiration. Particularly interesting is our finding that electron input through succinate dehydrogenase (cII), but not NADH:ubiquinone oxidoreductase (cI), reduces the Q pool almost entirely (>90%) irrespective of the respiratory state. AOX enhances the forward electron transport (FET) from cII thereby decreasing reverse electron transport (RET) and ROS specifically when non-phosphorylating. AOX is not engaged with cI substrates, however, unless a respiratory inhibitor is added. This sheds new light on Q poise signaling, the biological role of cII which enigmatically is the only ETC complex absent from respiratory supercomplexes but yet participates in the tricarboxylic acid (TCA) cycle. Finally, we delineate potential risks and benefits arising from therapeutic AOX transfer.     

Evidence for two sites of superoxide production by mitochondrial NADH-ubiquinone oxidoreductase (complex I)

Complex I (NADH-ubiquinone oxidoreductase) can form superoxide during forward electron flow (NADH-oxidizing) or, at sufficiently high protonmotive force, during reverse electron transport from the ubiquinone (Q) pool (NAD(+)-reducing). We designed an assay system to allow titration of the redox state of the superoxide-generating site during reverse electron transport in rat skeletal muscle mitochondria: a protonmotive force generated by ATP hydrolysis, succinate:malonate to alter electron supply and modulate the redox state of the Q pool, and inhibition of complex III to prevent QH(2) oxidation via the Q cycle. Stepwise oxidation of the QH(2)/Q pool by increasing malonate concentration slowed the rates of both reverse electron transport and rotenone-sensitive superoxide production by complex I. However, the superoxide production rate was not uniquely related to the resultant potential of the NADH/NAD(+) redox couple. Thus, there is a superoxide producer during reverse electron transport at complex I that responds to Q pool redox state and is not in equilibrium with the NAD reduction state. In contrast, superoxide production during forward electron transport in the presence of rotenone was uniquely related to NAD redox state. These results support a two-site model of complex I superoxide production; one site in equilibrium with the NAD pool, presumably the flavin of the FMN moiety (site I(F)) and the other dependent not only on NAD redox state, but also on protonmotive force and the reduction state of the Q pool, presumably a semiquinone in the Q-binding site (site I(Q)).     

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; )     

Light-induced electron transport pathways in membrane preparations from Rhodopseudomonas capsulata

The photosynthetic electron transport chain in Rhodopseudomonas capsulata cells was investigated by studying light-induced noncyclic electron transport from external donors to O₂. Two membrane preparations with opposite membrane polarity, heavy chromatophores and regular chromatophores, were used to characterize this electron transport. It was shown that with lipophylic electron donors such as dichloroindophenol, diaminobenzidine, and phenazine methosulfate the electron transport activities were similar in both types of chromatophores, whereas horse heart cytochrome c, K₄Fe(CN)₆, 3-sulfonic acid phenazine methosulfate, and ascorbate, which cannot penetrate the membrane, were more active in the heavy chromatophores than in the regular chromatophores. Partial depletion of cytochrome c₂ from the heavy chromatophores caused a decrease in the light-induced O₂ uptake from reduced dichloroindophenol or ascorbate. The activity could be restored with higher concentrations of dichloroindophenol or with purified cytochrome c₂ from Rps. capsulata. It is assumed that in the heavy chromatophores the artificial electron donors are oxidized on the cytochrome c₂ level which faces the outside medium. However, cytochrome c₂ is not exposed to the outside medium in the regular chromatophores. Therefore, only lipophylic donors would interact with cytochrome c₂ in this system, while hydrophylic donors would be oxidized by another component of the electron transport chain which is exposed to the external medium. Studies with inhibitors of photophosphorylation show that antimycin A enhances the light-dependent electron transport to O₂ whereas 1:10 phenanthroline inhibited the reaction, but dibromothymoquinone did not affect it. It is assumed that a nonheme iron protein is taking part in this electron transport but not a dibromothymoquinone-sensitive quinone. The terminal oxidase of the light-dependent pathway is different from the two oxidases of the respiratory chain. The ratio between electrons entering the system and molecules of O₂ consumed is 4, which means that the end product of O₂ reduction is H₂O.     

Rates of vectorial proton transport supported by cyclic electron flow during oxygen reduction by illuminated intact chloroplasts

The light-dependent quenching of 9-aminoacridine fluorescence was used to monitor the state of the transthylakoid proton gradient in illuminated intact chloroplasts in the presence or absence of external electron acceptors. The absence of appreciable light-dependent fluorescence quenching under anaerobic conditions indicated inhibition of coupled electron transport in the absence of external electron acceptors. Oxygen relieved this inhibition. However, when DCMU inhibited excessive reduction of the plastoquinone pool in the absence of oxygen, coupled cyclic electron transport supported the formation of a transthylakoid proton gradient even under anaerobiosis. This proton gradient collapsed in the presence of oxygen. Under aerobic conditions, and when KCN inhibited ribulose bisphosphate carboxylase and ascorbate peroxidase, fluorescence quenching indicated the formation of a transthylakoid proton gradient which was larger with oxygen in the Mehler reaction as electron acceptor than with methylviologen at similar rates of linear electron transport. Apparently, cyclic electron transport occured simultaneously with linear electron transport, when oxygen was available as electron acceptor, but not when methylviologen accepted electrons from Photosystem I. The ratio of cyclic to linear electron transport could be increased by low concentrations of DCMU. This shows that even under aerobic conditions cyclic electron transport is limited in isolated intact chloroplasts by excessive reduction of electron carriers. In fact, P₇₀₀ in the reaction center of Photosystem I remained reduced in illuminated isolated chloroplasts under conditions which resulted in extensive oxidation of P₇₀₀ in leaves. This shows that regulation of Photosystem II activity is less effective in isolated chloroplasts than in leaves. Assuming that a Q-cycle supports a H⁺/e ratio of 3 during slow linear electron transport, vectorial proton transport coupled to Photosystem I-dependent cyclic electron flow could be calculated. The highest calculated rate of Photosystem I-dependent proton transport, which was not yet light-saturated, was 330 mol protons (mg chlorophyll h)^–1 in intact chloroplasts. If H⁺/e is not three but two proton transfer is not 330 but 220 mol (mg Chl H)^–1. Differences in the regulation of cyclic electron transport in isolated chloroplasts and in leaves are discussed.     

On the functional site of manganese in photosynthetic electron transport system

Normal Euglena chloroplasts contained 1 atom of Mn per 47±8chlorophyll molecules. The manganese content of chloroplastswas decreased by heat treatment. After complete removal of manganeseby incubation at 45°C for 5 min, Hill activity with DPIPas electron acceptor was abolished, but the activity of DPIPphotoreduction with diphenylcarbazide as electron donor wasunaffected. Hill activity was inactivated by incubating Euglena chloroplastsat alkaline pH. The presence of a high concentration of Trisduring incubation of chloroplasts at an alkaline pH had no additionaleffect on the activity drop. Donor-supported DPIP photoreduction in heated Euglena chloroplasts,as well as the normal Hill reaction in untreated chloroplasts,was inhibited by DCMU, HOQNO and ioxynil which block electrontransport at the reducing side of system II. These reactionswere also inhibited by another group of inhibitors; CCCP, salicylaldoxime,antimycin A and azide, which block electron transport at a sitebetween the electron carriers, Y₁ and Y₂ located on the oxidizingside of system II. Possible sites of inhibition by heat treatment and by inhibitorsand sites for entry of electrons from artificial electron donorsin the photosynthetic electron transport chain, especially inrelation to the functional site of endogenous manganese in chloroplasts,were proposed. (Received October 30, 1971; )     

Polyphenol Oxidation by Vicia faba Chloroplast Membranes: STUDIES ON THE LATENT MEMBRANE-BOUND POLYPHENOL OXIDASE AND ON THE MECHANISM OF PHOTOCHEMICAL POLYPHENOL OXIDATION

The mechanism whereby light effects polyphenol oxidation was examined with Vicia faba chloroplast membranes known to contain a bound latent polyphenol oxidase. Results obtained with the inhibitors 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-idopropyl-p-benzoquinone (DBMIB) indicated an involvement of the non-cyclic electron transport pathway in the light-dependent oxidation of polyphenols, such as dihydroxyphenylalanine (DOPA). Further evidence was provided by experiments in which (a) DOPA replaced H₂O as electron donor for the photoreduction of NADP, (b) NADP replaced O₂ as electron acceptor in the photochemical oxidation of DOPA, and (c) the variable fluorescence associated with photosystem II was increased by DOPA. The photochemical oxidation of DOPA by V. faba chloroplast membranes was insensitive to KCN and to antibodies against purified latent polyphenol oxidase. The results are consistent with the conclusion that the light-dependent oxidation of polyphenols by V. faba chloroplast membranes is achieved independently of the latent membrane-bound polyphenol oxidase. Electrons derived from polyphenols seem to enter the noncyclic electron transport chain on the oxidizing side of photosystem II and to react with O₂ at an unidentified site on the photosystem I side of the DCMU/DBMIB blocks.     

Kinetics of cyclosporine A-induced inhibition of succinate-coenzyme Q dehydrogenase in rat renal cortical mitochondria

The kinetic mechanism of succinate-coenzyme Q dehydrogenase (Complex II) inhibition by cyclosporine A (CS) on rat renal cortical mitochondria was investigated. CS showed two modes of inhibition of Complex II of the mitochondrial electron transport system: (a) a mixed linear noncompetitive inhibition of resting succinate-limited and ADP-stimulated respirations suggesting that CS binds to Complex II at a different site than the substrate, affecting the dissociation constant for the enzyme-substrate complex and (b) a competitive inhibition of the DNP-stimulated electron transport system suggesting competition with the oxidized form of a component of Complex II. CS action to renal mitochondrial Complex II limits its function, an effect which may be related to CS nephrotoxicity.     

Some properties of the chlorophyll fluorescence of the diatom Phaeodactylum tricornutum

Fluorescence transients were investigated with the diatom Phaeodactylumtricornutum. Supplementary experiments were done with Chaetocerossp. Under weak excitation ({small tilde}10³ erg/cm²sec), fluorescencetransients were induced simply by die oxidation-reduction reactionof Q, the primary reductant of photosystem II. The action spectraindicated that the electron transfer components between thetwo photosystems were in the most reduced state when fucoxanthinwas excited. The transients were observed with the 681 run emissionand with the 707 nm emission at room temperature. At –196°C,induction due to the reduction of Q. appeared both at the 681and 707 nm emissions. Similar results were also obtained withChaetoceros sp. Under strong excitation (10⁴–10⁵ erg/cm²-sec), the fluorescencetransients due to the interconversion between States 1 and 2of die pigment system (cf. ref. 27, 29) were observed. The transientswere induced by die alternate excitation of chlorophyll a andfucoxanthin or chlorophyll c. Conversion from State 2 to State1 was inhibited by DNP and CCCP, indicating that die processwas energy-dependent. Fluorescence spectra at –196°Cwere not altered by die state-conversion of die pigment system. These results suggest diat all die fluorescence bands whichappeared at room temperature and at –196°C were dueto die chlorophyll a of pigment system II in Phaeodactylum andChaetoceros. (Received September 7, 1972; )     

Photo-inhibition and the Effects of Protein Phosphorylation on Electron Transport in Wheat Thylakoids

The kinetics of changes in photosystem I (PSI), photosystemII (PSII), and whole chain (PSII and PSI) electron transport,chlorophyll fluorescence parameters, the capacity to bind atrazineand the polypeptide profiles of thylakoids isolated from wheatleaves on exposure to a photon flux density of 2000 µmolm^–2 s^–1 were determined. Severe and similar levelsof photo-inhibitory damage to both PSII and whole chain electrontransport occurred and were correlated with decreases in theratio of variable to maximal fluorescence, the proportionalcontribution of the rapid a phase of the fluorescence kineticsand the capacity to bind atrazine. Severe photo-inhibition ofelectron transport was not associated with a major loss of chlorophyllor total thylakoid protein. However, a small decrease in a 70kDa polypeptide together with increases in a number of low molecularmass polypeptides (8–24 kDa) occurred. Phosphorylation of thylakoid polypeptides alleviated photo-inhibitionof PSII electron transport but stimulated photoinhibitory damageto whole chain electron transport. The consequences of suchphosphorylation-induced effects on photoinhibition in vivo areconsidered. Key words: Chlorophyll fluorescence, electron transport, photo-inhibition, protein phosphorylation, thylakoid membranes, wheat (Triticum aestivum)     

Demonstration of Two Sites of Inhibition of Electron Transport by Protein Phosphorylation in Wheat Thylakoids

The effect of protein phosphorylation on electron transportactivities of thylakoids isolated from wheat leaves was investigated.Protein phosphorylation resulted in a reduction in the apparentquantum yield of whole chain and photosystem II (PSII) electrontransport but had no effect on photosystem I (PSI) activity.The affinity of the D1 reaction centre polypeptide of PSII tobind atrazine was diminished upon phosphorylation, however,this did not reduce the light-saturated rate of PSII electrontransport. Phosphorylation also produced an inhibition of thelight-saturated rate of electron transport from water or durohydroquinoneto methyl viologen with no similar effect being observed onthe light-saturated rate of either PSII or PSI alone. This suggeststhat phosphorylation produces an inhibition of electron transportat a site, possibly the cytochrome b₆/f complex, between PSIIand PSI. This inhibition of whole-chain electron transport wasalso observed for thylakoids isolated from leaves grown underintermittent light which were deficient in polypeptides belongingto the light-harvesting chlorophyll-protein complex associatedwith photosystem II (LHCII). Consequently, this phenomenon isnot associated with phosphorylation of LCHII polypeptides. Apossible role for cytochrome b₆/f complexes in the phosphorylation-inducedinhibition of whole chain electron transport is discussed. Key words: Electron transport, light harvesting, photosystem 2, protein phosphorylation, thylakoid membranes, wheat (Triticum aestivum)     

Acifluorfen and Oxidized Ubiquinone in Soybean Mitochondria

Intact mitochondria from soybean [Glycine max (L.) Merrill cvBragg] seedlings were incubated with acifluorfen {sodium-5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoate}. Respiration, measured as O₂ consumption, wasinhibited by acifluorfen with a 50% inhibition occurring with10 µM of the herbicide. The inhibition was unaffectedby illumination; moreover, prior illumination by either UV orvisible light did not alter the inhibition capacity of acifluorfen.Further examination of the locus of acifluorfen inhibition atthe electron transport chain level showed no inhibition in Cytc oxidation capacity as well as no effect on NADH dehydrogenase,succinate dehydrogenase, malate dehydrogenase and isocitratedehydrogenase. It was observed, however, that in mitochondriaincubated for 10 min with 0.2 µM acifluorfen, ubiquinoneexisted predominantly in the oxidized state. We conclude thatat the mitochondrial level, a possible major site of actionof acifluorfen is either a direct or indirect modulation ofthe redox potential of ubiquinone. (Received July 27, 1985; Accepted October 7, 1985)     

Simultaneous oxidation of glycine and malate by pea leaf mitochondria

Mitochondria isolated from pea leaves (Pisum sativum L.) readily oxidized malate and glycine as substrates. The addition of glycine to mitochondria oxidizing malate in state 3 diminished the rate of malate oxidation. When glycine was added to mitochondria oxidizing malate in state 4, however, the rate of malate oxidation was either unaffected or stimulated. The reason both glycine and malate can be metabolized in state 4 appears to be that malate only used part of the electron transport capacity available in these mitochondria in this state. The remaining electron transport capacity was used by glycine, thus allowing both substrates to be oxidized simultaneously. This can be explained by differential use of two NADH dehydrogenases by glycine and malate and an increase in alternate oxidase activity upon glycine addition. These results help explain why photorespiratory glycine oxidation and its associated demand for NAD do not inhibit citric acid cycle function in leaves.     

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 CO₂ (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.