Quercetin interaction with the chloroplast ATPase complex

1. Quercetin, a flavonoid which acts as an energy transfer inhibitor in photophosphorylation is shown to inhibit the P-ATP exchange activity of membrane-bound CF1 and the ATPase activity of isolated CF1. Quercetin, affects also the proton uptake in chloroplasts in a manner similar to that of dicyclohexylcarbodiimide. 2. The light-dependent proton uptake in EDTA-treated chloroplasts is stimulated by quercetin. In untreated chloroplasts quercetin has a dual effect: it enhances at pH above 7.5 while at lower pH values it decreases the extent of H+ uptake. Similar effects were obtained with dicyclohexylcarbodiimide. 3. Like quercetin, dicyclohexylcarbodiimide was also found to inhibit the ATPase activity of isolated CF1. 4. Quercetin inhibits uncoupled electron transport induced by either EDTA-treatment of chloroplasts or by addition of uncouplers. Quercetin restores H+ uptake in both types of uncoupled chloroplasts. 5. The mode of action of quercetin and dicyclohexylcarbodiimide in photophosphorylation is discussed, and interaction with both CF1 and F0 is suggested.     

Binding of 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-diphosphate opens the pathway for protons through the chloroplast ATPase complex

The effect of 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-diphosphate (TNP-ADP) on photophosphorylation and on the proton conductivity of the thylakoid membrane has been investigated. The results show that TNP-ADP is a potent competitive inhibitor of photophosphorylation (Ki = 1-2 microM). Moreover, in the absence of ADP and Pi, TNP-ADP accelerates basal electron transport of chloroplasts. Addition of ADP, which promotes release of the analogue from CF1, completely reverses this effect of TNP-ADP; likewise Pi alone reverses stimulation of electron transport by TNP-ADP. Dicyclohexylcarbodiimide treatment, which is known to close CF0 to H+, completely abolishes the effect of TNP-ADP. The measurements of the alkalization of the medium and the acidification of the thylakoid lumen following single turnover flashes showed that binding of TNP-ADP to CF1 increased membrane permeability for H+. Further results suggest that binding of TNP-ADP to the catalytic site of CF1 opens the CF0-CF1 complex for H+. Since ADP, as well as Pi alone, reverses the effect, it is concluded that TNP-ADP induces a conformation of the CF0-CF1 complex similar to the one triggered by simultaneous binding of ADP plus Pi. This may be achieved by interaction of the TNP residue with the Pi binding site. Thus it seems that the status of the catalytic site(s) in CF1 can be transmitted to the CF0 part to control proton flux through the ATPase complex in an economically reasonable way.     

Inhibition of Thylakoid ATPase by Venturicidin as an Indicator of CF1-CF0 Interaction

Venturicidin inhibits the F0 portion of membrane-located, H+-pumping ATPases. We find it meets the criteria for an energy transfer inhibitor for spinach (Spinacia oleracea) thylakoids: complete inhibition of photophosphorylation and of photophosphorylation-stimulated and basal electron flow rates, but not of electron flow under uncoupled conditions. The extent of H+ uptake in the light is stimulated by venturicidin (vtcd), as expected for a compound blocking H+ efflux through CF0. Vtcd had no effect on the nonproton pumping, methanol-stimulated ATPase of thylakoids or on soluble CF1 ATPase. Under totally uncoupled conditions (saturating NH4Cl + gramicidin), vtcd can still inhibit sulfite-stimulated thylakoid ATPase completely. The concentration of vtcd needed for inhibition of ATPase was proportional to the concentration of thylakoids present in the assay, with an apparent stoichiometry of about 10 vtcd molecules per CF1/CF0 for 50% inhibition. Vtcd raised the Km for ATP somewhat, but had a stronger effect on the Vmax with respect to ATP. Inhibition by saturating vtcd ranged from 50 to 100%, depending on the condition of the thylakoids. Grinding leaves in buffer containing 0.2 M choline chloride (known to provide superior photophosphorylation rates) helped bring on maximum vtcd inhibition; trypsin treatment or aging of thylakoids brought on vtcd-resistant ATPase. We conclude that the extent of inhibition by vtcd can be used as an indicator of the tightness of coupling between CF1 and CF0.     

叶绿体atpE基因表达产物的生化特性

在细菌中表达的叶绿体ａｔｐＥ基因产物ε亚基蛋白对不同方式激活的叶绿体ＡＴ－Ｐａｓｅ均有抑制作用,而其抗血清则促进ＡＴ－Ｐａｓｅ活力。Ｅ．ｃｏｌｉ中表达的ε亚基蛋白在光合磷酸化反应中对循环和非循环光合磷酸化都有促进作用,其抗血清对循环光合磷酸化有抑制作用,而对非循环光合磷酸化则起促进作用。     

Effects of Optically Active 1-(α-Methylbenzyl)-3-(3,4-dichlorophenyl)urea on Reactions of Mitochondria and Chloroplasts

Effects of the R- and S-isomers and racemate of 1-(alpha-methylbenzyl)-3-(3,4-dichlorophenyl)urea (MBPU) were measured on phosphorylation and electron transport in mung bean (Phaseolus aureus L.) mitochondria and spinach (Spinacia oleracea L.) chloroplasts.In chloroplasts, S-MBPU inhibited basal and methylamine-uncoupled electron transport with ferricyanide as the oxidant, both photoreduction and coupled photophosphorylation with water as the electron donor and with ferricyanide and nicotinamide adenine dinucleotide phosphate (NADP) as oxidants, and cyclic photophosphorylation with phenazine methosulfate as the electron mediator under an argon gas phase. With ascorbate 2,6-dichloro-phenolindophenol as the electron donor, phosphorylation coupled to NADP reduction was inhibited, but the reduction of NADP was not inhibited. The R-isomer of MBPU, like the S-isomer, inhibited all of the photophosphorylation reactions studied. However, unlike the S-isomer, the R-isomer either did not inhibit or was a very weak inhibitor of all photoreduction reactions. The effects of the MBPUs on the chloroplast reactions can be explained by action at two different sites: an optically specific site near photosystem II and the oxygen evolution pathway, and a second optically nonspecific site associated with the generation of ATP.In mitochondria, both the R- and S-isomers stimulated state 4 respiration, inhibited state 3 respiration, and released oligomycin-inhibited respiration with malate, succinate, and NADH as substrates. Both enantiomers were equally active in all studies with malate and succinate as substrates. However, with NADH as substrate, R-MBPU was a stronger inhibitor of state 3 respiration and a weaker stimulator of state 4 respiration than S-MBPU.     

Mechanisms by which reactions catalyzed by chloroplast coupling factor 1 are inhibited: ATP synthesis and ATP-H2O oxygen exchange

The ATP-H2O back-exchange reaction catalyzed by membrane-bound chloroplast coupling factor 1 (CF1) in the light is known to be extensive; each reacting ATP molecule nearly equilibrates its gamma-PO3 oxygens with H2O before it dissociates from the enzyme. Pi, ASi, ADP, and GDP, alternate substrates of photophosphorylation, each inhibit the exchange reaction. At all concentrations of these substrate/inhibitor molecules tested, the high extent of exchange per molecule of ATP that reacts remains the same, while the number of ATP molecules experiencing exchange decreases. Thus, these inhibitors appear to act in a competitive-type manner, decreasing ATP turnover, as opposed to modulating the rate constants responsible for the partitioning of E X ATP during the exchange reaction. This is consistent with the identity of CF1 catalytic sites for ATP-H2O back-exchange and ATP synthesis. Carbonyl cyanide m-chlorophenylhydrazone and NH4Cl (uncouplers of photophosphorylation) and phloridzin (an energy-transfer inhibitor) also lower the rate of ATP-H2O back-exchange; they too are found to act by decreasing the turnover of the ATP pool, not the extent of exchange per reacting ATP molecule. The extent of ATP-H2O forward oxygen exchange, which occurs during net ATP synthesis prior to product dissociation, is unaffected by uncouplers, whether catalyzed by native CF1 (ATPase latent) or the dithiothreitol/light-activated ATPase form. The mode of NH4Cl inhibition of the ATP synthesis reaction, therefore, is not through a change in the partitioning of the E X ATP complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

The stoichiometry (ATP/2e− ratio) of non-cyclic photophosphorylation in isolated spinach chloroplasts

1. The stoichiometry of non-cyclic photophosphorylation and electron transport in isolated chloroplasts has been re-investigated. Variations in the isolation and assay techniques were studied in detail in order to obtain optimum conditions necessary for reproducibly higher ADP/O (equivalent to ATP/2e^?) and photosynthetic control ratios.2. Studies which we carried out on the possible contribution of cyclic phosphorylation to non-cyclic phosphorylation suggested that not more than 10% of the total phosphorylation found could be due to cyclic phosphorylation.3. Photosynthetic control, and the uncoupling of electron transport in the presence of NH₄Cl, were demonstrated using oxidised diaminodurene as the electron acceptor. A halving of the ADP/O ratio was found, suggesting that electrons were being accepted between two sites of energy conservation, one of which is associated with Photosystem I and the other associated with Photosystem II.4. ATP was shown to inhibit State 2 and State 3 of electron transport, but not State 4 electron transport or the overall ADP/O ratio, thus confirming its activity as an energy transfer inhibitor. It is suggested that part of the non-phosphorylating electron transport rate (State 2) which is not inhibited by ATP is incapable of being coupled to subsequent phosphorylation triggered by the addition of ADP (State 3). If the ATP-insensitive State 2 electron transport is deducted from the State 3 electron transport when calculating the ADP/O ratio, a value of 2.0 is obtained.5. The experiments reported demonstrate that there are two sites of energy conservation in the non-cyclic electron transfer pathway: one associated with Photosystem II and the other with Photosystem I. Thus, non-cyclic photophosphorylation can probably produce sufficient ATP and NADPH “in vivo” to allow CO₂ fixation to proceed.     

Effects of four benzoxazinoids on gibberellin-induced alpha-amylase activity in barley seeds

Germination of barley seeds was inhibited by 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) and 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA) at concentrations greater than 0.03mmol/L, and 6-methoxy-benzoxazolin-2(3H)-one (MBOA) and benzoxazolin-2(3H)-one (BOA) at concentrations greater than 0.1mmol/L. These benzoxazinoids also inhibited the induction of alpha-amylase activity in the barley seeds, and inhibited gibberellin-induced alpha-amylase activity in de-embryonated barley seeds. Significant inhibition in the germination and alpha-amylase induction were observed as concentrations of DIMBOA, DIBOA, MBOA and BOA increased. These results suggest that DIMBOA, DIBOA, MBOA and BOA may inhibit the germination of barley seeds by inhibiting the gibberellin-induced process, leading to alpha-amylase production. The inhibitory activities of germination and alpha-amylase induction of DIMBOA and DIBOA were greater than those of their degraded substances MBOA and BOA, respectively, and the inhibitory activities of DIMBOA and MBOA were greater than those of their demethoxylated analogues DIBOA and BOA, respectively.     

Kinetics of electron transport, proton transfer and photophosphorylation in chloroplasts and their relation to temperature-induced structural changes in the thylakoid membrane

Effects of various temperatures on the rates of electron transport between two photosystems, the light-induced uptake of protons, kinetics of proton efflux from the chloroplasts in the dark and photophosphorylation were studied in isolated chloroplasts. There are correlations between the physical state of thylakoid membrane and the rates of electron- and proton transport processes. The temperature dependence of "structural" parameter (fluidity of lipids in membrane) as well as the rates of electron- and proton transport processes reveal the breaks under the same temperatures. Stimulation of photophosphorylation by temperature increasing correlates with the heat activation of chloroplasts latent ATPase due to thermoinduced structural changes in the heat activation of chloroplasts latent ATPase due to thermoinduced structural changes in the protein part of CF0-CF1 complex. The rate of photophosphorylation also correlates with the physical state of membrane lipids. Thermoinduced "melting" of the thylakoid membrane inhibits the ATP formation because of a decrease in photosystem 2 photochemical activity and stimulation of membrane conductivity for protons.     

Damage to the chloroplast H+-ATPase complex by low concentrations of glutaraldehyde

The energy-dependent processes coupled to electron transport were studied in isolated pea chloroplasts treated with low concentrations (1-5 mM) of glutaraldehyde (GA) in the dark and in the light sufficient to cause energization of the membrane. After GA treatment the chloroplasts exhibited a strong suppression of cyclic and non-cyclic phosphorylation, coupled (+ADP+Pi) electron transport and diminution of the light-activated Mg2+-ATPase activity. The rate of basal electron transport was unaffected. The GA-treated chloroplasts were found to retain the capacity to form the osmotic component of the transmembrane potential. These data and the results of the effect of florizine and ATP on electron transport suggest that the effect of GA on energy transduction processes associated with photophosphorylation may consist in its action on reversible H+-ATPase. In light-adapted samples treated with GA the data characterizing the formation of a high energy state (rate of photophosphorylation, steady-state level of photo-induced quenching of atebrin fluorescence and its dark recovery; photo-induced absorbance changes at 520 nm; rate of the slow phase of delayed fluorescence increment) appear to be changed to a greater extent as compared to the dark-adapted samples. The observed changes may arise from a greater conductivity of thylakoid membranes due to fixation of the H+-ATPase proton channel in the "open" state.     

Hydroxamic Acid glucosyltransferases from maize seedlings

Hydroxamic acids occur in several forms in maize (Zea mays L.) with 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) being the predominant form and others including 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) being found at lower concentrations. Two enzymes capable of glucosylating hydroxamic acids were identified in maize protein extracts and partially purified and characterized. The total enzyme activity per seedling increased during the first 4 days of germination and was concurrent with the accumulation of DIMBOA. Purification of the enzymes by ammonium sulfate precipitation followed by Sephadex G-200 and Q-Sepharose gel chromatography resulted in a 13-fold increase in specific activity. The enzymes are initially separated into two peaks (peak 1 and peak 2) of activity by Q-Sepharose gel chromatography. The peak 1 glucosyltransferase had 3.6% of the DIMBOA glucosylating activity when DIBOA was used as substrate, whereas this percentage increased to 57% for the peak 2 enzyme. The enzyme in peak 2 has a K_m of 174 micromolar for DIMBOA and a K_m of 638 micromolar for DIBOA; the enzyme in peak 1 has a K_m of 217 micromolar for DIMBOA and its activity on DIBOA was too low to determine a K_m. The identification of two glucosyltransferases capable of glucosylating hydroxamic acids in vitro serves as an initial step in the characterization of the enzymes involved in production of hydroxamic acids in maize.     

Effect of powdery mildew infection on photosynthesis by leaves and chloroplasts of sugar beets

Chloroplasts isolated from powdery mildew-infected (Erysiphe polygoni DC) sugar beet leaves (Beta vulgaris L) showed a reduction in the rate of electron transport and in the accompanying ATP formation in noncyclic photophosphorylation (water as electron donor, NADP as electron acceptor) and little or no change in the rate of ATP formation in cyclic photophosphorylation catalyzed by phenazine methosulfate. The inhibition of noncyclic photophosphorylation appeared to lead in the parent leaves to a decreased rate of photosynthetic CO₂ assimilation and a shift in products resulting in a relative increase of amino acids. These changes were accompanied by alterations in chloroplast ultrastructure and by a reduction in the activity of enzymes necessary for the formation of organic acids (phosphoenolpyruvate carboxylase and malate dehydrogenase). These results are similar to the findings of Montalbini and Buchanan (1974 Physiol. Plant Pathol. 4: 191-196) with chloroplasts from rust-infected Vicia faba leaves.     

Delayed chloroplast luminescence. Activation and suppression

Effect of diethyl ether, detergent triton X-100, glycerine, sucrose and preliminary heating on delayed luminescence (DL) of chloroplasts has been studied. Ether and triton X-100 in concentrations activating electron transport inhibit DL acting similarly to photophosphorylation uncouplers. Preliminary heating to 42-42C, glycerine and sucrose activate both the electron transport and DL of chloroplasts. Activation of electron transport under these agents is suggested to result not from photophosphorylation uncoupling, but from the changes in the conformation of chloroplast memebranes.     

Effect of chemical modification of amino groups by fluorescamine on partial reactions of photosynthesis.

4-Phenylspiro [furan-2(3H),1-phtalan]3,3'-dione (fluorescamine) was used to covalently modify amino groups of thylakoids. Subsequently its effect on parameters of energy transfer and phosphorylating activity was assessed. While electron transport, the extent of proton uptake, 515 nm change and 9-aminoacridine quench were relatively resistant to such treatment, the functions connected to coupling factor 1, namely ATP formation by acid/base transition, ATPase activity and photophosphorylation were affected much earlier. Photophosphorylation appears to be the most sensitive. The data are interpreted as indicating an involvement of free amino groups in energy transfer.     

Effect of chemical modification of amino groups by fluorescamine on partial reactions of photosynthesis

4-Phenylspiro[furan-2(3H),1-phtalan]3,3′-dione (fluorescamine) was used to covalently modify amino groups of thylakoids. Subsequently its effect on parameters of energy transfer and phosphorylating activity was assessed. While electron transport, the extent of proton uptake, 515 nm change and 9-aminoacridine quench were relatively resistant to such treatment, the functions connected to coupling factor 1, namely ATP formation by acid/base transition, ATPase activity and photophosphorylation were affected much earlier. Photophosphorylation appears to be the most sensitive. The data are interpreted as indicating an involvement of free amino groups in energy transfer.     

The potential of hydroxamic acids in tetraploid and hexaploid wheat varieties as resistance factors against the bird‐cherry oat aphid,Rhopalosiphum padi

The potential for exploiting natural wheat resistance to control the cereal aphid Rhopalosiphum padi, the most important aphid pest of small grain cereals in the UK, was investigated as an alternative approach to the use of insecticides. The investigation focussed on a group of secondary metabolites, the hydroxamic acids or benzoxazinones, present naturally as glucosides, but which hydrolyse on tissue damage to give biologically active aglycones, e.g. 2,4‐dihydroxy‐7‐methoxy‐1,4‐benzoxazin‐3‐one (DIMBOA) which are associated with natural plant defence. These can be important for resistance against insects, fungi, bacteria and nematodes for a range of cultivated monocotyledonous plants and could ultimately be combined with other defence mechanisms to provide a general approach to cereal aphid control. Levels of hydroxamic acids, particularly DIMBOA‐glucoside, were determined in hexaploid (Triticum aestivum) and tetraploid (Triticum durum) wheat varieties and differences were found between species and varieties. The effect of feeding by R. padi on the level of hydroxamic acids in the leaf tissue was also investigated. Thus, after 24 h of aphid feeding, as an apparently localised hydrolytic defence reaction in the leaf, levels of DIMBOA‐glucoside decreased noticeably. When aphids were fed on sucrose solution containing low doses of DIMBOA there was a significant mortality compared to the sucrose control. However, the levels of and variation in hydroxamic acids in the wheat varieties investigated were insufficient for significant differences in aphid behaviour and development.     

The role of plastidic cytochrome c in algal electron transport and photophosphorylation.

By an improved isolation procedure chloroplasts could be obtained from the alga Bumilleriopsis filiformis (Xanthophyceae) which exhibited high electron transport rates tightly coupled to ATP formation. Uncouplers both stimulate electron transport and inhibit photophosphorylation. These chloroplasts retain almost all soluble cytochrome c-553 besides a membrane-bound cytochrome c-554.5 (=f-554.5). Sonification or iron deficiency removed the soluble cytochrome only with a concurrent decrease of electron transport from water to methyl viologen or to NADP and decreased non-cyclic and cyclic photophosphorylation. However, photosynthetic control and the P/2e ratios remain unaltered. In Bumilleriopsis, which apparently has no plastocyanin, the soluble cytochrome c-553 seemingly links electron transport between the bound cytochrome c and P-700.     

Chloride ion transport and its inhibition in thylakoid membranes

Cl- translocation across energized and nonenergized thylakoid membranes was found to be inhibited by piretanide, an inhibitor of active Cl- transport in fish intestinal epithelia. Piretanide has no effect on photophosphorylation catalyzed by phenazine methosulfate or on Ca2+-dependent ATPase activity of isolated chloroplast coupling factor (CF1). Light-dependent Cl- uptake, contrary to H+ uptake, is severalfold greater at pH 8.0 than at pH 6.7.     

Bioenergetic Responses of Synechocystis 6803 Fatty Acid Desaturase Mutants at Low Temperatures

Fatty acid composition of the membrane lipids in the mesophilic cyanobacterium Synechocystis sp. PCC 6803 was altered in earlier work by targeted mutagenesis of genes for fatty acid desaturases. In this work, cells of several mutant strains, depleted in the unsaturated fatty acids in membrane lipids, were grown at 34 degrees C. Spheroplasts (permeabilized cells) were prepared by lysozyme digestion of the cell wall followed by gentle osmotic shock. The bioenergetic parameters ATP formation, electron transport, and H+ uptake were measured at various temperatures. All three bioenergetic parameters for spheroplasts from wild-type cells (which had abundant polyunsaturated fatty acids) were active down to the lowest temperatures used (1 degrees - 2 degrees C). In two strains, which lacked the capacity to desaturate fatty acids at the A 12 position and at the A 12 and A6 positions (designated as desA- and desA-/desD-, respectively), the spheroplasts lost the capacity to form ATP (measured as phenazine methosulfate cyclic phosphorylation) at about 5 degrees C but retained electron transport (water oxidation-dependent ferricyanide reduction) and H+ uptake linked to phenazine methosulfate cyclic electron transport. It appears that the absence of the unsaturation of fatty acids in the A 12 and A6 positions blocks the ability of the photosynthetic membranes to couple a bioenergetically competent proton-motive force to the ATP formation mechanism at temperatures below 5 degrees C. It remains to be determined whether the loss of ATP formation in the mutant strains is the failure of available protons to properly flow into the CF0CF1-ATP synthase or a failure in the CF1 part of the complex in coupling the dissipative H+ flow to the enzyme mechanism of the synthase.     

Agents inducing high Mg2+-ATPase activity of isolated coupling factor 1 from spinach chloroplasts

Conditions are reported under which purified coupling factor 1 (CF1) from spinach chloroplasts exhibits Mg2+-dependent ATPase activity of about 120 mumoles/min/mg protein. It is shown that CF1, partially activated by treatment with heat and dithiothreitol (DTT), can be further activated by octyl glucoside. The Mg2+-dependent ATPase activity increases linearly as a function of the concentration of octyl glucoside from about 20 mumoles/min/mg protein in the absence of detergent to 120 mumoles/min/mg protein in the presence 15 mM octyl glucoside. This concentration is below the critical micellar concentration (CMC) of the detergent, indicating that the monomeric form is responsible for the activation. Without treatment with heat and DTT, the Mg2+-dependent ATPase activity of CF1 is virtually zero, but can be stimulated by octyl glucoside. In this case, however, only concentrations around CMC give a substantial increase in activity (about 50 mumoles/min/mg at 28 mM octyl glucoside). Concentrations higher than CMC inhibit both latent and heat-activated CF1.