Cyclic Photophosphorylation Research Articles

The interaction of phenylglyoxal with soluble and membrane-bound chloroplast coupling factor 1

The rate of inhibition of cyclic photophosphorylation in chloroplast thylakoids by the arginine reagent phenylglyoxal was enhanced in the light, i.e., under conditions where membrane energization occurred. Uncouplers, but not energy-transfer inhibitors, prevented the effect of light. Chemical modification of chloroplast thylakoids by phenylglyoxal under dark or in light conditions affected differently the light-induced exchange of tightly bound ADP. In both cases the exchange was less inhibited than photophosphorylation. Complete inhibition of ATPase activity of soluble CF 1 was correlated with the incorporation of 8 mol [ 14C]phenylglyoxal per mol enzyme. About 50% of the incorporated radioactivity was lost at different rates depending on the buffer present and suggesting a change in the stoichiometry of the adduct from 2:1 to 1:1. Inhibition of ATPase and photophosphorylating activities of chloroplasts by modification with [ 14C]phenylglyoxal in the dark was associated with the incorporation of 1 and 2 mol reagent per mol membrane-bound CF 1, respectively. In the light the rate of incorporation was enhanced and both reactions were inactivated when 2 mol [ 14 C]phenylglyoxal CF 1 were bound. In all the labelling experiments the radioactivity was mainly recovered from the α- and β-subunits.

Effect of Osmotic Stress on Photosynthesis Studied with the Isolated Spinach Chloroplast

The effect of increasing assay medium sorbitol concentration from 0.33 to 1.0 molar on the photosynthetic reactions of intact and broken spinach (Spinacia oleracea L. var. Long Standing Bloomsdale) chloroplasts was investigated by monitoring O(2) evolution supported by the addition of glyceric acid 3-phosphate (PGA), oxaloacetic acid (OAA), 2,5-dimethyl-p-benzoquinone, and 2,6-dichlorophenolindophenol or as O(2) uptake with methyl viologen as acceptor.Uncoupled 2,6-dichlorophenolindophenol-supported whole chain electron transport (photosystems I and II) was inhibited from the 0.33 molar rate by 14% and 48.6% at 0.67 and 1.0 molar sorbitol in the intact chloroplast and by only 0.4% and 25.0% in the broken chloroplast preparation. Whole chain electron flow from water to other oxidants (OAA, methyl viologen) was also inhibited at increased osmoticum in intact preparations while electron flow from water to methyl viologen, ferricyanide, and NADP in broken preparations did not demonstrate the osmotic response. Electron transport to 2,5-dimethyl-p-benzoquinone (photosystem II) from H(2)O and to methyl viologen (photosystem I) from 3,3'-diaminobenzidine were found to be unaffected by osmolarity in both intact and broken preparations.The stress response was more pronounced (26-38%) with PGA as substrate in the presence of 0.67 molar sorbitol than the inhibition found with uncoupled and coupled linear electron flow. In addition, substrate availability and ATP generated by cyclic photophosphorylation evaluated by addition of Antimycin A were found not to be mediating the full osmotic inhibition of PGA-supported O(2) evolution. In a reconstituted (thylakoids plus stromal protein) chloroplast system to which a substrate level of PGA was added, O(2) evolution was only slightly (7.8%) inhibited by increased osmolarity (0.33-0.67 molar sorbitol) indicating that the level of osmotic inhibition above that contributed by adverse effects on electron flow can be attributed to the functioning of the photosynthetic carbon reduction cycle within the intact chloroplasts.

Photosynthetic Electron Transfer in Preparations of the Cyanobacterium Spirulina platensis

Electron transfer activity in intact trichomes of Spirulina platensis (Nordst.) Geitl. can be observed with either CO(2) or methylviologen as the Hill acceptor. Ferricyanide cannot penetrate the intact trichomes, but photoreduction of this oxidant can be observed when mediated by lipophilic oxidants such as p-phenylenediamine or 2,5-dimethyl-p-benzoquinone. The insensitivity of these reactions to dibromothymoquinone indicates that they are due largely to the activity of photosystem II. Direct photoreduction of ferricyanide can be observed in spheroplasts of Spirulina, indicating that such preparations have altered permeability properties when compared with intact trichomes. Preparation of these spheroplasts, which are osmotically fragile, requires that intact trichomes be washed with KCl and EDTA to induce lysozyme sensitivity and thereby allow digestion of the cell wall. The KCl/EDTA washing procedure used for spheroplast preparation alters the permeability of Spirulina trichomes, as evidenced by the ability of these preparations to photoreduce ferricyanide. This photoreduction reaction is insensitive to dibromothymoquinone, and is stimulated by high concentrations of divalent cations. During assays, the reaction is inhibited by the inclusion of polyethyleneglycol as an osmotic protectant. Photoreduction of methylviologen and NADP(+) is also observed in the washed trichomes, along with an endogenously catalyzed photoreduction of O(2) to H(2)O(2). Photophosphorylation cannot be observed in the washed preparations, but cyclic photophosphorylation with phenazinemethosulfate is observed after mild sonication. These results indicate that KCl/EDTA-washed trichomes of S. platensis retain the full range of energy transducing capacities associated with thylakoid membranes of the intact trichomes; the washing procedure facilitates spheroplast formation and alters, but does not abolish, permeability barriers in these preparations.

Temperature Dependence of Photosynthesis in Agropyron smithii Rydb.

As part of an analysis of the factors regulating photosynthesis in Agropyron smithii Rydb., a C(3) grass, the response of electron transport and photophosphorylation to temperature in isolated chloroplast thylakoids has been examined. The response of the light reactions to temperature was found to depend strongly on the preincubation time especially at temperatures above 35 degrees C. Using methyl viologen as a noncyclic electron acceptor, coupled electron transport was found to be stable to 38 degrees C; however, uncoupled electron transport was inhibited above 38 degrees C. Photophosphorylation became unstable at lower temperatures, becoming progressively inhibited from 35 to 42 degrees C. The coupling ratio, ATP/2e(-), decreased continuously with temperature above 35 degrees C. Likewise, photosystem I electron transport was stable up to 48 degrees C, while cyclic photophosphorylation became inhibited above 35 degrees C. Net proton uptake was found to decrease with temperatures above 35 degrees C supporting the hypothesis that high temperature produces thermal uncoupling in these chloroplast thylakoids. Previously determined limitations of net photosynthesis in whole leaves in the temperature region from 35 to 40 degrees C may be due to thermal uncoupling that limits ATP and/or changes the stromal environment required for photosynthetic carbon reduction. Previously determined limitations to photosynthesis in whole leaves above 40 degrees C correlate with inhibition of photosynthetic electron transport at photosystem II along with the cessation of photophosphorylation.

Cryoprotection of spinach chloroplast membranes by dextrans.

The cryoprotective properties of dextrans have been investigated in freezing experiments with isolated spinach thylakoids (Spinacia oleracea L.). The activity of cyclic photophosphorylation was used as an assay for membrane integrity. Dextrans of average molecular weights between 10,000 and 70,000 daltons proved to be fairly nontoxic to chloroplast membranes. On a molar basis, cryoprotective action increased with increasing molecular weight; on a unit weight basis, the cryoprotective effectiveness of different dextrans was comparable. In the presence of low dextran concentrations which are not sufficient for complete membrane preservation, the effectiveness of the polymers could be considerably increased by the addition of electrolytes. This is in contrast to cryoprotection exerted by sugars. At a given dextran concentration, membrane activity is a function of the electrolyte concentration and follows an optimum curve. If membrane-toxic action of the electrolytes and salt crystallization during freezing which complicate the situation, are not taken into consideration, the increase in membrane protection during freezing by salts was dependent on the concentration of the salts and was not much influenced by the nature of the cations and anions. At 0 °C, dextrans delayed the inactivation of thylakoids suspended in NaCl solutions. From the results it is concluded that cryoprotection produced by dextrans is caused in part by specific membrane stabilization.

Regulation of nitrate reduction in wheat leaves

Wheat leaves exposed to 710 nm monochromatic light, when only photosystem 1 operates, reduced small but significant amount of nitrate to nitrite. This could be due to partial inhibition of mitochondrial oxidation of NADH, brought about by cyclic photo-phosphorylation. Under dark aerobic conditions, citric acid cycle intermediates only slightly stimulated nitrate reduction. Under dark anaerobic conditions, when maximum reduction of nitrate occurred, the time course showed a 1:1 stoichiometry between nitrite and CO2. It is suggested that for maximum reduction of nitrate under physiological conditions, CO2 fixation and export of ATP via triose phosphate shuttle is essential.

Photophosphorylation in isolated heterocysts from the blue-green alga Nostoc muscorum

Isolated heterocysts of the blue-green alga Nostoc muscorum (Anabaena 7119) exhibit high rates of photophosphorylation in systems with cyclic and non-cyclic electron transport. Cyclic photophosphorylation mediated by N-methylphenazonium methosulfate is found to be sensitive to antimycin A, but not to 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinon (DBMIB). Non-cyclic electron transport (diaminodurol → methylviologen) coupled to phosphorylation is affected by DBMIB, but not by antimycin A. Studies with uncouplers indicate that ΔpH is the main component of the protonmotive force under continuous illumination. A different effect of NH 4Cl on dark- and photophosphorylation is observed and discussed with respect to localization of respiration in blue-green algae.

Photosynthetic regeneration of ATP using a strain of thermophilic blue–green algae

Photosynthetic ATP accumulation was shown in the presence of exogenous ADP plus orthophosphate on illumination to the intact cells of a strain of thermophilic blue-green algae isolated from Matsue hot springs, Mastigocladus sp. Kinetic studies of various effectors on the ATP accumulation proved that the ATP synthesis depends mainly on the cyclic photophosphorylation system around photosystem I (PS-I) in the algal cells. The temperature and pH optima for the accumulation were found at 45 degrees C and pH 7.5. Maximum yield was obtained with light intensity higher than 15 mW/cm(2). Borate ion exerted pronounced enhancement on the ATP synthesis. With a continuous reactor at a flow rate of 1 ml/hr at 45 degrees C and pH 7.5, efficient photoconversion of ADP (2mM, at substrate reservoir) to ATP (1mM, at product outlet) has been maintained for a period of 2.5 days, though the efficiency has decreased in a further 2-day period to the level of 0.5mM ATP/9.5 h of residence time.

Effects of simetryne on the photochemical reactions of isolated chloroplasts of rice and barnyardgrass plants.

The effects of simetryne on the photosynthetic electron transport and photophosphorylation activities in chloroplasts isolated from rice (tolerant) and barnyardgrass (susceptible) plants were investigated. Simetryne severely inhibited the electron transport of photosystem II in both the chloroplasts as measured by O2 evolution (pI50 value was 7.3). No inhibition of photosystem I dependent electron transport was observed up to 10-4M simetryne. Non-cyclic photoposphorylation was inhibited by simetryne at the same concentration as in inhibition of photosystem II. Cyclic photophosphorylation was also inhibited, but not as severely as in non-cyclic reaction. The primary herbicidal action of simetryne seems to be inhibition of photosystem II and photophosphorylation. No differences were observed in the degree of inhibition between chloroplasts isolated from rice and barnayrdgrass among all photochemical reactions tested. It cannot therefore be considered that the selectivity of simetryne is caused by the difference in sensitivity of the chloroplasts.

Cyclic and noncyclic photophosphorylation during the ontogenesis of high-light and low-light leaves of Sinapis alba

Noncyclic electron transport to ferricyanide and photophosphorylation as well as the methylviologen mediated aerobic and anaerobic photophosphorylation with dichlorophenolindophenol-ascorbate as the electron donor of photosystem I were measured during the development of high-light and low-light adapted leaves of Sinapis alba. Anaerobic methylviologen-catalyzed phosphorylation is more than twice as high as aerobic phosphorylation. The difference between the rates of aerobic and anaerobic phosphorylation is sensitive to dibromothymoquinone. Thus, under anaerobic conditions, methylviologen mediates a cyclic phosphorylation including plastoquinone. All photochemical activities of high-light chloroplasts are about twice as high as that of low-light chloroplasts and show a permanent decline with increasing plant age. The lower activities of low-light chloroplasts correlate with a decrease of electron transport components, such as cytochrome f. This indicates that the number of electron transport chains is decreased under low-light conditions and more chlorophyll molecules interact with one electrontransport chain.

Effects of Hydroxamic Acids Isolated from Gramineae on Adenosine 5′-triphosphate Synthesis in Chloroplasts

Two hydroxamic acids isolated from maize extracts, 2,4-dihydroxy-7-methoxy-1,4-(2H)-benzoxazin-3(4H)-one (DIMBOA) and the 2-O-beta-d-glucopyranoside of DIMBOA, inhibit photophosphorylation by spinach chloroplasts. Both cyclic and noncyclic photophosphorylations were inhibited to the same extent. The concentrations producing 50% inhibition for DIMBOA and its glucoside were about 1 and 4 millimolar, respectively. These compounds inhibit coupled electron transport but do not affect basal or uncoupled electron transport. Both acids inhibit the ATPase activities of membrane-bound coupling factor 1 (CF(1)) and of purified CF(1). On the basis of these results, it is concluded that DIMBOA and its glucoside act as energy transfer inhibitors of photophosphorylation.

The effect of electron transport inhibitors on nitrogenase activity in the photosynthetic bacterium,Rhodopseudomonas capsulata

It is generally believed that ferredoxin is the in vivo reductant for nitrogenase in photosynthetic bacteria, as in other nitrogen-fixing organisms [1]. PhotosYnthetic bacteria are capable of diazotrophic growth under at least five different growth modes, but the mechanism for the reduction of ferredoxin, or other in vivo nitrogenase reductants, is unknown at present. Although it has been suggested that a ferredoxin may be involved in cyclic photophosphorylation [2], and photoinduced e.p.r, signal changes corresponding to reduction of ferredoxin II have been observed in R. rubrum [3], it is not known if the primary acceptor has a redox potential low enough to reduce ferredoxin [4,5]. In photosynthetic bacteria, nitrogenase activity is ultimately dependent upon an added organic substrate, or hydrogen, as source of electrons. The requirement for ATP for N2ase functioning is presumably met by photophosphorylation or, aerobically in the dark, by oxidative phosphoryla-

Studies of the function of the membrane-bound iron-sulfur centers of the photosynthetic bacterium Chromatium vinosum

Chromatophores from the photosynthetic bacterium, Chromatium vinosum, have been prepared which photoreduce NAD + with either succinate or reduced dichlorophenolindophenol as electron donors. NAD + reduction is inhibited by uncouplers as well as inhibitors of cyclic photophosphorylation. These chromatophores contain several bound iron-sulfur centers which have been detected by low-temperature EPR spectroscopy. One center, having a g 2.01 EPR signal in the oxidized state, has E m7.5 = +50 mV and is partially reduced by succinate in the dark. Three iron-sulfur centers having g 1.93 EPR signals have been resolved by redox titration, and the E m7.5 values of these centers are −50, −175 and −250 mV, respectively. Studies of the involvement of these centers in electron transfer from donors to NAD + have indicated that the center with E m = −50 mV is succinate reducible in the dark and appears to be analogous to center S-1 of succinic dehydrogenase in other systems. An additional g 1.93 iron-sulfur center can be photoreduced in the presence of electron donors and this reduction is inhibited by uncouplers. The possible role of the two low-potential iron-sulfur centers in relation to the dehydrogenases functioning in NAD + reduction is considered.

Studies on the Mechanism of Photoinhibition in Higher Plants

Cucumber plants (Cucumis sativus L.), grown at low quantum flux density (120-150 microeinsteins per square meter per second) were photoinhibited by a three-hour exposure in air to ten times the light intensity experienced during growth. Chloroplasts were isolated from photoinhibited and control leaves and the following activities determined: O(2) evolution in the presence of ferricyanide, photosystem I activity, noncyclic and cyclic photophosphorylation, and light-induced proton uptake. Chlorophyll and chloroplast absorbance spectra, and chloroplast fluorescence were also measured. It was found that photosystem II electron transport and non-cyclic photophosphorylation were inhibited by about 50%, while cyclic photophosphorylation was less inhibited and photosystem I electron transport and light-induced proton uptake were unaffected. Electron transport to methylviologen could not be fully restored by electron donation to photosystem II. Chloroplast fluorescence induction at room temperature was strongly reduced following photoinhibition. There was no difference in the absorption spectra of the extracted chlorophylls from control and photoinhibited chloroplasts, but an increase of the absorption in the blue wavelength region was observed in the photoinhibited chloroplasts. It is suggested that high light stress does not result in alteration of the membrane properties, as is the case in low-temperature stress for example, but affects directly the photosynthetic reaction centers, primarily of photosystem II.

Proton transport in photooxidation of water: A new perspective on photosynthesis

The currently prevalent concept of the generation of photosynthetic reducing power in oxygen-evolving cells envisions a linear (noncyclic) electron flow from water to ferredoxin (and thence to NADP(+)) that requires the collaboration of photosystems I and II (PSI and PSII) joined by plastoquinone and other electron carriers (the Z scheme). The essence of the Z scheme is that only PSI can reduce ferredoxin-i.e., that, after being energized to an intermediate reducing potential by PSII, electrons from water are transported via plastoquinone to PSI which energizes the electrons to their ultimate reducing potential adequate for the reduction of ferredoxin. Basic to the Z scheme is the function of plastoquinone as the obligatory link in electron transport from PSII to PSI. However, we have found that, when plastoquinone function was inhibited, ferredoxin was photoreduced by water without the collaboration of PSI. We now report evidence for an important function of plastoquinone in the translocation of protons liberated inside the thylakoid membrane by photooxidation of water. When the oxygenic photoreduction (i.e., by water) of ferredoxin was blocked by plastoquinone inhibitors, dibromothymoquinone or dinitrophenol ether of iodonitrothymol, the photoreduction of ferredoxin was restored by each of four chemically diverse uncouplers, similar only in their ability to facilitate proton movement across membranes. Similar results were obtained for the oxygenic reduction of NADP(+). Our results suggest that the light-induced electron flow from water cannot be maintained unless the simultaneously liberated protons are removed from inside the membrane via plastoquinone. The new evidence is embodied in a concept of an oxygenic photosystem for photosynthetic electron and proton transport, which we propose as an alternative to the Z scheme, to account for photoreduction of ferredoxin-NADP(+) by water and the coupled oxygenic (formerly noncyclic) ATP formation without involving PSI. The role of the anoxygenic photosystem (formerly called PSI) is ATP formation by cyclic photophosphorylation.

Photophosphorylation Associated with Photosystem II

Photosystem II-dependent cyclic photophosphorylation activity produced by addition of p-phenylenediamines to KCN-Hg-NH(2)OH-inhibited chloroplasts is the product of two separate reactions when a proton/electron donor is the catalyst. The activity observed with an electron donor as catalyst consists of a single reaction. One of the cyclic reactions, evoked by low (</=40 micromolar) concentrations of a proton/electron donor is sensitive to dibromothymoquinone and to perturbation of membrane organization by sonication. The second reaction, requiring higher catalyst concentrations, is less sensitive to either dibromothymoquinone or membrane perturbation. These results indicate that at low concentrations, proton/electron or electron donor catalysts act to produce a photosystem II cyclic reaction which is dependent on membrane-bound electron carriers. High concentrations of proton/electron donors, on the other hand, can produce a phosphorylation reaction in which the catalyst itself is largely responsible for cyclic activity.

Photosynthetic electron transport in the bundle sheath of maize

Initial investigations into the mechanism of C4 photosynthesis showed that in species such as maize, CO* fmed in the mesophyll cells is transported in the form of malate to the bundle sheath cells, where its decarboxylation is catalyzed in the chloroplasts by NADP’-dependent malic enzyme [ 11. The presence of agranal chloroplasts in the bundle sheath of these species [2] and the functional deficiency of photosystem II activity [ 1,3,4] led to the view that the NADPH formed during the decarboxylation of malate is essential for reduction of part of the glycerate-1,3-bisphosphate formed in the reductive pentose phosphate pathway. The marked stimulation of photosynthesis which malate causes in isolated bundle sheath tissue is consistent with this view [3,5]. Although the extent of the deficiency in photosystem II has since been questioned [6,7], recent work with bundle sheath strands having photosynthesis rates equal to those of the parent tissue implied that photosynthetic Oa evolution (and uptake) with COz as the acceptor is limited and that the high ATP demand for COa fucation is largely met by cyclic photophosphorylation mediated by photosystem I rather than via noncyclic or pseudocyclic photophosphorylation [5]. Cyclic photophosphorylation requires activation by electron flow from a suitable reductanr, which establishes appropriate redox poise in the electron carriers. In thisletter, flash spectrophotometric studies are described which indicate that photosystem II activity is inadequate to poise cyclic electron flow in bundle sheaths of maize, and that malate decarboxylation supplies the necessary electrons.

Adenylate Energy Charge and Phosphorylation Potential in the Blue-Green Bacterium Anacystis nidulans

In the blue-green bacterium Anacystis nidulans total pool sizes of AMP, ADP, ATP, and Pi have been measured under steady-state conditions in dark and light both under air + CO2 and under N2 By extraction of freeze-dried cells with dilute Tris buffer attempts have been made to determine the free concentration of Pi Mg2+ was released from the cells by the ionophore A 23187. Cell volume measurements in combination with extmction flata permitted calculation of intracellular concentrations. Energy charge, mass action ratios of the adenylate kinase reaction, ATPjADP ratios and phosphorylation potentials were estimated. Desaspidin which inhibits glycolysis and cyclic photophosphorylation and DCMU which inhibits non-cyclic photophosphorylation were used to study the contribution of different reactions to cellular ATP levels. In light, under air + CO2 and under N2 , the energy charge remained at a constant level of 0.83; the ATP/ADP ratios behave similarly and stay at a value of about 3. The phosphorylation potential in light is about constant nnder air + CO2 whereas under N2 it decreases in presence of inhibitors because of a rise in Pi. The mass action ratios of adenylate kinase ranged between 0.5 and 1 under all conditions used.

Studies on photosynthetic electron transport, photophosphorylation and CO2fixation in zinc deficient leaf cells ofZea mavs. L.

Abstract The effect of Zn deficiency on rate of photosynthesis of leaf discs, isolated mesophyll and bundle sheath cells and chloroplasts of maize (Zea mays. L) was studied. The yield of mesophyll and bundle sheath cells obtained by enzymic digestion of the leaf tissues from Zn deficient plants is lower than the identical tissues from normal plants which suggests that Zn deficiency brings about some structural changes in the leaf cell. Photosynthetic oxygen evolution measured in the leaf discs is low due to Zn deficiency. Photosystem‐II dependent Hill reaction and non cyclic photophosphorylation of chloroplasts were also affected by Zn deficiency. Rate of photosynthetic carbon dioxide fixation by both bundle sheath and mesophyll cells obtained from Zn deficient leaf‐tissue waslower than the cells free from Zn deficiency. Addition of various metabolites like NADPH, ATP and PEP to Zn deficient mesophyll cells whowed marked enhancement in 14‐CO2 fixation. However, addition of NADPH, ATP and RuBP to Zn defici...

Influence of External pH on Noncyclic Photophosphorylation in Chloroplasts Isolated from Higher Plants

Two optimal pH values for non cyclic photophosphorylation activity in chloroplasts isolated from cucumber primary leaves and leaves were found. Two optimal pH values were also found for chloroplasts isolated from pea leaves. Experiments were performed with chloroplasts isolated from pea cultured under low and high light intensity. Photophosphorylation was also investigated in chloroplasts in which either pigment system PSi or PS2 was blocked. These experiments enable us to ascribe one optimal pH value to the action of pigment system PSi and plastoquinone pool, while the second optimal pH value to the fucntion of PS2 and water splitting enzyme system.