Cyclic Photophosphorylation Research Articles

Inhibition of photosystem II in Chlorogloeopsis fritschii with shikonin acetate

Shikonin acetate extracted from the roots of the desert plant, Arnebia decumbens, was tested for its effect on the photosynthetic electron transport system of C. fritschii. Photosystem (PS) II-Hill reaction with water and diphenylcarbazide (DPC) as electron donors and ferricyanide as electron acceptor was inhibited completely. Shikonin acetate has little effect on 2,6-dichlorophenolindophenol (DCPIP)-Hill reaction and no effect on PSI with durohydroquinone as electron donor. Inhibition of Mehler reaction from water to methylviologen with whole cells was studied using 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea (DCMU) and shikonin acetate. About 70% inhibition of cyclic and non-cyclic photophosphorylation was found with 10 −7 M shikonin acetate. These results suggest that shikonin acetate inhibits photosynthetic electron flow at the plastoquinone pool.

Photosynthetic Decline from High Temperature Stress during Maturation of Wheat

High temperature stress reduces grain growth in wheat (Triticum aestivum L.) by altering source activity and sink capacity. The impact of stress on source and sink interactions in two wheat cultivars of differing source thermotolerance was monitored by analysis of chlorophyll fluorescence transients, Fv (variable fluorescence) and PSM (peak, stationary, maximum), of attached flag leaves on intact and decapitated tillers grown at optimum (20 degrees C) and stress (35 degrees C) temperatures after anthesis. The thermotolerant cultivar Waverly had reduced Fv and PS quenching and a large increase of SM during heat stress. The less thermotolerant cultivar, Len, exhibited increased Fv and PS quenching and a small increase of SM. Fluorescence induction was similar in intact and decapitated tillers of Len, indicating diminished sinksource interaction during heat stress. The present results and previous observations of photosynthetic activities indicate that cyclic electron transport and photophosphorylation in flag leaves of the thermotolerant cultivar were stimulated by sink demand (increased SM in intact plants). Reduced grain development in the thermolabile cultivar resulted from limited capacity to support cyclic electron transport and photophosphorylation (slight increase in SM of intact plants and large reduction of Cytochrome f/b(6)-mediated electron transport capacity). It was concluded that heat stress injures the photosynthetic apparatus during reproductive growth of wheat and that diminished source activity and sink capacity may be equally important in reducing productivity.

On an Inhibitor of Ferredoxin Catalyzed Cyclic Photophosphorylation in Thylakoid Membranes*

AbstractDibromo‐ and diiodo‐naphthoquinones are shown to be inhibitors of the cytochrome b6/f complex in isolated thylakoid membranes from spinach chloroplasts. Dibromo‐naphthoquinone inhibits ferredoxin catalyzed cyclic photophosphorylation at 0.1 μM concentrations, but non cyclic e‐flow only at 10 μM. It does not inhibit cyclic systems with artifical cofactors, nor non‐cyclic electron flow from duroquinol through photosystem I via the cytochrome b6/f complex. Dibromo‐naphthoquinone does however, lower the stoichiometry for ATP formation in the duroquinol donor system. This inhibitory pattern is quite different from that of DBMIB, but very similar to that of antimycin. This antimycin‐like behaviour of these inhibitors is interpreted to indicate a) the existence of a Qc site in the cytochrome b6/f complex and its obligate function in ferredoxin catalyzed cyclic electron flow and b) a non‐essential role of the Qc site in non‐cyclic electron flow, but which — when operative — pumps an extra proton across the thylakoid membrane increasing the ATP yield.

Localization and Orientation of Subunit Delta of Spinach Chloroplast ATP-Synthase within the CF0 CF1 Complex 2. Identification of C-Terminal Residues of Delta, Exposed on the Thylakoid Membrane

The amino acid residues of spinach CF1 subunit delta are identified which are accessible and thus exposed within the quaternary structure of the ATP-synthase complex on the thylakoid membrane. Two types of antibodies in the monospecific polyclonal antiserum 306 against CF1 delta, described in the previous publication [Z. Naturforsch. 44c, 153-160 (1989)], were separated by virtue of their different affinity to thylakoid membranes and used for specific analysis of the products of proteolytic digestion of delta in situ. Polypeptide delta in situ, i.e. within the CF0 CF1 complex on the membrane, is not susceptible to digestion by aminopeptidase M and trypsin, but is shortened by about 1 kDa by carboxypeptidase Y and digested at residues Glu173 and Glu179 by the Staphylococcus aureus protease V8. The epitope on delta reacting with the agglutinating antibodies from serum 306 is lost after these proteolytical treatments and therefore situated on residues Met180-Val187. Since trypsin destroys this epitope only after prolonged incubation and with at least 50 micrograms trypsin/mg Chl, residue Lys169 of delta probably is inaccessible in situ. We conclude that the C-terminal amphipathic alpha-helix of spinach CF1 subunit delta is exposed on the thylakoid membrane, with the hydrophilic face directed to the outside, and that CF1 delta starts to be shielded within the quaternary structure of the CF0 CF1 complex between Glu173 and Lys169. The hydrophobic face of the c-terminal helix may be part of the binding surface towards CF0. Antibodies from serum 306 inhibit the PMS mediated cyclic photophosphorylation by reacting with C-terminal residues of delta.

Reconstitution of cyclic electron transport and photophosphorylation by incorporation of the reaction center, cytochrome bc1 complex and ATP synthase from Rhodobacter capsulatus into ubiquinone-10/phospholipid vesicles

The photosynthetic reaction center, the ubiquinol-cytochrome c reductase (EC 1.10.2.2) and the ATP synthase (EC 3.6.1.4) were selectively solubilized and isolated from the intracytoplasmic membranes of photoheterotrophically grown Rhodobacter capsulatus with n-octyl β- d -glucopyranoside. The purified enzymes were co-reconstituted in predetermined stoichiometries in ubiquinol-10 phospholipid vesicles by a two-step procedure. In the first step, reaction centers and ubiquinol-cytochrome c reductase were incorporated randomly by detergent dialysis. Cyclic electron transport between these two membrane complexes was selected by including cytochrome c in the interior of the proteoliposomes. In the second step, the ATP synthase molecules were incorporated by detergent dilution into the preformed proteoliposomes from the outer face of the vesicles. Analysis of the light-induced redox changes of the cytochromes and of the bacteriochlorophyll dimer, in combination with the effect of specific inhibitors demonstrated that an electron transport pathway closely resembling that of the physiological system was reconstructed in the proteoliposomes. Light-driven ATP synthesis was detected at a maximal rate of 36 nmol ATP/min per mg ATP synthase. Variation in the stoichiometry of the protein components indicated that the rate-limiting reaction was ATP synthesis.

Cross‐reconstitution of the FOF1‐ATP synthases of chloroplasts and Escherichia coli with special emphasis on subunit δ

F0F1-ATP synthases catalyse ATP formation from ADP and Pi by using the free energy supplied by the transmembrane electrochemical potential of the proton. The delta subunit of F1 plays an important role at the interface between the channel portion F0 and the catalytic portion F1. In chloroplasts it can plug the protonic conductance of CF0 and in Escherichia coli it is required for binding of EF1 to EF0. We wanted to know whether or not delta of one species was effective between F0 and F1 of the other species and vice versa. To this end the respective coupling membrane (thylakoids, everted vesicles from E. coli) was (partially) depleted of F1 and purified F1, F1(-delta), and delta were added in various combinations to the F1-depleted membranes. The efficiency or reconstitution was measured in thylakoids via the rate of phenazinemethosulfate-mediated cyclic photophosphorylation and in E. coli everted vesicles via the degree of 9-amino-6-chloro-2-methoxyacridine fluorescence quenching. Addition of CF1 to partially CF1-depleted thylakoid vesicles restored photophosphorylation to the highest extent. CF1(-delta)+chloroplast delta, EF1, EF1(-delta)+E. coli delta were also effective but to lesser extent. CF1(-delta)+E. coli delta and EF1(-delta)+chloroplast delta restored photophosphorylation to a small but still significant extent. With F1-depleted everted vesicles prepared by repeated EDTA treatment of E. coli membranes, addition of CF1, CF1 (-delta)+chloroplast delta and CF1(-delta)+E. coli delta gave approximately half the extent of 9-amino-6-chloro-2-methoxyacridine fluorescence quenching as compared to EF1 or EF1(-delta)+E. coli delta by energization of the vesicles with NADH, while Ef1(-delta)+chloroplast delta was ineffective. All 'mixed' combinations were probably reconstitutively active only by plugging the protonic leak through the exposed F0 (structural reconstitution) rather than by catalytic activity. Nevertheless, the cross-reconstitution is stunning in view of the weak sequence similarity between chloroplast delta and E. coli delta. It favors a role of delta as a conformational transducer rather than as a proton conductor between F0 and F1.

Changes in photoelectron transport activity in cyanophage N-1-infected cells ofNostoc muscorum

Cyanophage N-1-infectedNostoc muscorum cells were unable to carry out oxygen evolution and photosystem-II-dependent electron transport (H2O→DCPIP). This was associated with preferential degradation of phycobiliproteins. Such cells also exhibited decreased rate of ferredoxin:NADP+ oxidoreductase activity. However, Ca2+-dependent ATPase activity was maintained at a higher level (80%). The results suggested that virus development proceeds in the absence of photosystem-II activity, and the energy is provided by cyclic photophosphorylation aside from that possibly obtained via degradation of carbohydrate reserves.

Localization and Orientation of Subunit Delta of Spinach Chloroplast ATP-Synthase within the CF0CF1 Complex. 1. Distinction of Shielded and Exposed Surfaces of Delta on the Thylakoid Membrane

A new polyclonal antiserum against spinach CF1 subunit delta was produced in rabbits. It decorates only one band at 21 kDa in Western immunoblots of thylakoid proteins and does not react in ELISA with delta-free four subunit CF1(-delta); therefore it is regarded monospecific. The polypeptide used as immunogen had been purified by HPLC. Earlier antisera against CF1 delta cross-react with CF1 subunit beta. The new antiserum 306 contains different antibodies; some can be absorbed with thylakoids, i.e. by delta within the assembled CF0CF1 complex on the membrane, others still react in ELISA with isolated CF1. The former antibodies agglutinate thylakoids and inhibit PMS cyclic photophosphorylation. Therefore we conclude that part of the surface of CF1 subunit delta is exposed within the quaternary structure of the ATP-synthase complex of photosynthetically active thylakoids, but part of the surface of delta is shielded. Trypsination of isolated delta occurs at several sites, but this protease does not attack delta in situ, nor does aminopeptidase. Staphylococcus aureus protease V8 digests CF1 delta after isolation at residues Asp53, Glu61, Glu95 and Glu106, but has no access to these residues of delta in situ. Thus CF1 subunit delta seems to be shielded within the CF0CF1 complex to a large degree. Direct agglutination of thylakoids by anti delta serum 306 was weak and could be improved tenfold by a Coombs serum (goat anti rabbit gammaglobulin), whereas an anti beta serum agglutinated directly. From this we conclude that delta is not accessible at the top of the enzyme; the exposed part is extending below the large subunits alpha and beta and oriented towards the membrane.

Effects of poly (amino acid)s on photophosphorylation in a cyanobacterial thylakoid.

National Chemical Laboratory for Industry, 1-1 Higashi, Tsukuba-shi 305, Japan Poly-L-glutamic acid accelerated by about 25% the cyclic photophosphorylation in the thylakoid a thermophilic cyanobacterium. A mixture of poly-L-glutamic acid and poly-L ornithine of a ratio 2/1 (w/w) also enhanced by 20-30% the photophosphorylation. The acceleration behaviors by poly-L-glutamic acid alone are different from those by the mixture of acidic and basic poly (amino acid) s.

Effect of photoperiod on photosynthetic characteristics of two marine diatoms

Photoadaptation to changes in photoperiod was studied in the marine diatoms Skeletonema costatum (Grev.) Cleve and Phaeodactylum tricomutum Bohlin. The algae were grown under cycling light of 2 h : 2 h and 12 h : 12 h light-dark periods. The daily C assimilation · cell −1 was enhanced by a factor 1.5 in 2 : 2 light regime. In cells grown under 12 : 12 h light regime, the pigment content, the number of PSI, PSUO 2 size, and to a lesser extent, the photosystem II-antenna size were modulated by the light-dark alternations, whereas these characteristics were almost constant over the circadian cycle in 2 : 2 cells. The modifications in the cellular content of photosynthetic pigments were correlated with no change in the relative pigment composition of the photosynthetic units. In S. costatum, the changes in pigment content corresponded to an increase of the ratio PSI : PSII which is emphasized at least by a factor 2.12 and to modifications of the photosystem-antenna sizes (increase of 28% for PSII and decrease of 40% for PSI) when the cells are transferred from 2 : 2 to 12 : 12 light regime (light period). The responses of diatoms to a change of the photoperiod is different according to the species. Indeed, P. tricomutum did not show the same variations in cellular contents of pigments and PSI; moreover, the photosystem-antenna sizes were weakly affected by a change in the photoperiod. The modifications observed in S. costatum, inducing variations of PSI : PSII ratio, were related to the existence of an active cyclic photophosphorylation around PSI in diatoms which modulate the ATP : NADPH 2 ratio in response to the requirements of the metabolic processes.

QSAR of Nitro-diphenylethers as Inhibitors of Cyclic Photophosphorylation

Abstract A number of 25 nitro-diphenylethers have been synthesized and assayed for their inhibitory activity in N-methylphenazonium-methosulfate mediated cyclic photophosphorylation. Their pI50-values were in the range from 3.5 to 5.3. A QSAR-analysis revealed that the lipophilicity of the nitro-diphenylethers as expressed by ∑π played a major role. In addition, the Taft steric parameter Es in positions 6′, 4′ and 2′ (in decreasing order) contributed to the inhibitory activity. For maximal biological activity, positions 4′ and 6′ should be left unsubstituted whereas a bulky substituent is required for position 2′.

Shikonin isovalerate: An inhibitor of photosystem II in Chlorogloeopsis fritschi

Shikonin isovalerate, extracted from the roots of the desert plant Arnebia decumbens, was tested for its effect on photosynthetic electron transport system of Chlorogloeopsis fritschii. The ferricyanide-Hill reaction with water and DPC as electron donors was inhibited completely with 10-5 M shikonin isovalerate. The photoreduction of DCPIP through photosystem II was only slightly inhibited. Photosystem I from durohydroquinone to methyl viologen was not affected using 10-6 M shikonin isovalerate. The same concentration caused 49% inhibition of cyclic photophosphorylation. These results suggest that shikonin isovalerate inhibits photosynthetic electron flow at the plastoquinone pool.

The antimycin sensitivity of flash-induced ATP synthesis in photosystem I-enriched subchloroplast vesicles

Photosystem I-enriched vesicles, derived from spinach thylakoids by mild digitonin treatment, reconstituted with native ferredoxin and redox-poised with NADPH and oxygen, display antimycin-sensitive cyclic photophosphorylation. The effects of 1–30 μM antimycin point to electron transfer inhibition: (i) they are cancelled after addition of 10 μM pyocyanin and (ii) they resemble the effects of 1–5 μM DBMIB, but differ from those of FCCP, nigericin or valinomycin. Some vesicle preparations fails to show antimycin sensitivity in the absence of pyocyanin [(1985) FEBS Lett. 192, 271–274] and have apparently lost the antimycin-sensitive component.

Active Transport of CO2 by the Cyanobacterium Synechococcus UTEX 625

Mass spectrometry has been used to confirm the presence of an active transport system for CO(2) in Synechococcus UTEX 625. Cells were incubated at pH 8.0 in 100 micromolar KHCO(3) in the absence of Na(+) (to prevent HCO(3) (-) transport). Upon illumination the cells rapidly removed almost all the free CO(2) from the medium. Addition of carbonic anhydrase revealed that the CO(2) depletion resulted from a selective uptake of CO(2), rather than a total uptake of all inorganic carbon species. CO(2) transport stopped rapidly (<3 seconds) when the light was turned off. Iodoacetamide (3.3 millimolar) completely inhibited CO(2) fixation but had little effect on CO(2) transport. In iodoacetamide poisoned cells, transport of CO(2) occurred against a concentration gradient of about 18,000 to 1. Transport of CO(2) was completely inhibited by 10 micromolar diethylstilbestrol, a membrane-bound ATPase inhibitor. Studies with DCMU and PSI light indicated that CO(2) transport was driven by ATP produced by cyclic or pseudocyclic photophosphorylation. Low concentrations of Na(+) (<100 microequivalents per liter), but not of K(+), stimulated CO(2) transport as much as 2.4-fold. Unlike Na(+)-dependent HCO(3) (-) transport, the transport of CO(2) was not inhibited by high concentrations (30 milliequivalents per liter) of Li(+). During illumination, the CO(2) concentration in the medium remained far below its equilibrium value for periods up to 15 minutes. This could only happen if CO(2) transport was continuously occurring at a rapid rate, since the continuing dehydration of HCO(3) (-) to CO(2) would rapidly raise the CO(2) concentration to its equilibrium value if transport ceased. Measurement of the rate of dissolved inorganic carbon accumulation under these conditions indicated that at least part of the continuing CO(2) transport was balanced by HCO(3) (-) efflux.

Some characteristics of cyclic photophosphorylation in maize bundle sheath chloroplasts

PMS-dependent photophosphorylation in bundle sheath chloroplasts isolated from Zea mays was monitored by using a continuous method. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and venturicidin were shown to inhibit the ATP-synthesis. Venturicidin has been shown to inhibit ATP-formation in both mesophyll and bundle sheath chloroplasts. In contrast to the case in mesophyll chloroplasts, FMN was not able to promote photophosphorylation in bundle sheath chloroplasts. The effects of other cofactors and inhibitors on the ATP-synthesis in bundle sheath chloroplasts are shown. No photoinduced synthesis of inorganic pyrophosphate was seen, neither in bundle sheath chloroplasts, nor in mesophyll chloroplasts.

塩基性ポリアミノ酸のラン藻光合成膜系への作用

Effects of basic poly (amino acid) s on the photosynthetic activities of the thylakoid membranes of thermophilic cyanobacterium Synechococcus sp. were examined. Poly-L-ornithine, poly-L-arginine, poly-L-lysine and poly-D-lysine uncouple the thylakoid membranes, dissipating ΔpH generated across the membranes and inhibiting cyclic photophosphorylation mediated by phenazine methosulfate. Contrary to the previous reports with some algae, poly-L-ornithine, poly-L-lysine and poly-D-lysine inhibit photosystem 1-dependent electron flow from tetramethyl-p-hydroquinone to methylviologen, presumably by affecting the function of soluble acidic protein cytochrome c-553. Poly-L-histidine gives no effect on photochemical activities of the cyanobacterial thylakoid membranes.

Regulation of photosynthesis in isolated barley protoplasts: the contribution of cyclic photophosphorylation

The response of photosynthetic oxygen evolution and chlorophyll a fluorescence in isolated barley protoplasts to a change in light intensity (from approx. 1 10 saturating to saturating light levels) under saturating CO 2 concentrations was examined. Marked oscillations in the rate of O 2 evolution and chlorophyll fluorescence were observed, resembling those seen in leaves under similar conditions. The effect of antimycin A addition on O 2 evolution at various light intensities and at different times during photosynthetic induction was examined to determine the role of cyclic photophosphorylation during steady-state photosynthesis and during oscillatory behaviour. Antimycin (1 μM) addition reduced the photosynthetic rate by 50–75% but only at light intensities over 80–100 μE · m −2 · s −1. Below this threshold light level, antimycin had no inhibitory effect. Thus, antimycin had a progressively greater effect at higher light intensity. When antimycin was added at various points along the time course of photosynthetic induction, the rate of O 2 evolution was reduced to the same level regardless of the time of addition. Antimycin addition also had a marked effect on the oscillations in O 2 evolution observed after a transition in light intensity from 100 to 1000 μE · m −2 · s −1, increasing the frequency and reducing the damping of the oscillations. Measurements of adenylate levels indicated that antimycin prevented a rapid rise in ATP ADP ratio observed in the chloroplast compartment after the light transition. These results are discussed with reference to regulation of photosynthesis during a transition in light intensity and the limitation of photosynthesis by photophosphorylation under various environmental conditions.

Na+-Stimulation of Photosynthesis in the Cyanobacterium Synechococcus UTEX 625 Grown on High Levels of Inorganic Carbon

Photosynthesis of washed cells of Synechococcus UTEX 625 grown on 5% CO(2) was markedly stimulated (647 +/- 50%) at pH 8.0 by the addition of low concentrations of NaCl (concentration required for half-maximal response, K((1/2),) = 18 micromolar). Studies with KCl and Na(2)SO(4) showed that the stimulation was due to Na(+). Photosynthesis at pH 6.1 was only slightly stimulated by Na(+). The response of photosynthesis at pH 8.0 to [Na(+)] was strongly sigmoidal for dissolved inorganic carbon ([DIC] </= 500 micromolar). Cells grown with high total [DIC], but air-levels of CO(2), at pH 9.6 showed the same response to low [Na(+)]. The absence of Na(+) could be partially, but not completely overcome, by higher [DIC]. Various methods for examining CO(2) or HCO(3) (-) use (K((1/2)) (CO(2) ) determination; isotopic disequilibrium; and consideration of HCO(3) (-) dehydration rate) were consistent with CO(2) use by the cells, but HCO(3) (-) use could not be ruled out. Isotopic disequilibrium studies showed that CO(2) use was stimulated by Na(+). Cells grown on 5% CO(2) accumulated DIC against a concentration gradient by a process (or processes) dependent on Na(+). No evidence for uptake of Na(+) concomitant with DIC uptake could be found. The lack of O(2) evolution during the initial and most rapid period of DIC accumulation suggested that the required energy was obtained from cyclic photophosphorylation.

Regulation of Cyclic Photophosphorylation during Ferredoxin-Mediated Electron Transport

Addition of ferredoxin to isolated thylakoid membranes reconstitutes electron transport from water to NADP and to O(2) (the Mehler reaction). This electron flow is coupled to ATP synthesis, and both cyclic and noncyclic electron transport drive photophosphorylation. Under conditions where the NADPH/NADP(+) ratio is varied, the amount of ATP synthesis due to cyclic activity is also varied, as is the amount of cyclic activity which is sensitive to antimycin A. Partial inhibition of photosystem II activity with DCMU (which affects reduction of electron carriers of the interphotosystem chain) also affects the level of cyclic activity. The results of these experiments indicate that two modes of cyclic electron transfer activity, which differ in their antimycin A sensitivity, can operate in the thylakoid membrane. Regulation of these activities can occur at the level of ferredoxin and is governed by the NADPH/NADP ratio.

Evolution of Reaction Centers in Photosynthetic Prokaryotes

Publisher Summary This chapter focuses on reaction centers (RCs) of photosynthetic prokaryotes and those phylogenetic characteristics that are most significant to reconstruct the ancestry of these bacteria. Photosynthesis is defined as the biological conversion of light energy to chemical energy in the form of electrochemical gradients and/or ATP. The diverse photosynthetic prokaryotes discussed are grouped into six categories: (1) filamentous photosynthetic bacteria, (2) green sulfur bacteria, (3) heliobacterium chlorum —the gram-positive line, (4) purple bacteria (including bacteriochlorophyll (BChl) a -containing nonphototrophic bacteria), (5) cyanobacteria (including Prochlorophytes), and (6) halobacteria. The cyanobacteria are the most diverse and ubiquitous of all the photosynthetic prokaryotes. In several species of cyanobacteria, the source of electrons for reduction of the oxidized RC-1 primary donor can be sulfide, as in the green sulfur bacteria. When photochemically generated electrons from RC-1 are not consumed for pyridine nucleotide reduction, they maybe cycled back to the primary donor and the energy recovered in the synthesis of ATP by cyclic photophosphorylation. In the absence of sulfide, all cyanobacteria provide electrons to reduce the primary donor to RC-1 from RC-2. RC-2 is similar to the RC in the purple bacteria.