Restoration of the high potential form of cytochrome b-559 through the photoreactivation of Tris-inactivated oxygen-evolving center

Restoration of a high potential (HP) form of cytochrome b-559 (Cyt b-559) from a low potential (LP) form was the primary process in the reconstitution of O₂-evolving center during the photoreactivation of Tris-inactivated chloroplasts. In normal chloroplasts, about 0.5 to 0.7 mol of Cyt b-559 was present in the HP form per 400 chlorophyll molecules. However, the HP form was converted to the LP form when the O₂-evolving center was inactivated by 0.8 M alkaline Tris-washing (pH 9.1). The inactivation was reversible and both the Cyt b-559 HP form and the O₂-evolving activity were restored by incubating the inactivated chloroplasts with weak light, Mn²⁺, Ca²⁺ and an electron donor (photoreactivation). The recovery of the HP form preceded the recovery of O₂-evolving activity. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) did not inhibit the recovery of the HP form. Thus, the recovery of Cyt b-559 HP form was the primary reaction in the photoreactivation, which was stimulated by the light-induced redox reaction of the PS-II core center.Abbreviations ASC ascorbate - BSA bovine serum albumin - Chl chlorophyll - Cyt b-559 HP form high potential form of cytochrome b-559 - Cyt b-559 LP form low potential form of cytochrome b-559 - Cyt b-559 VLP form very low potential form of cytochrome b-559 - Cyt f cytochrome f - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenol indophenol - Hepes N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid - HQ hydroquinone - SHN chloroplast-preparation medium containing 0.4 M sucrose, 50 mM Hepes-Na (pH 7.8) and 20 mM NaCl - PS-II Photosystem II     

Effects of Nanoanatase TiO2 on Photosynthesis of Spinach Chloroplasts Under Different Light Illumination

With a photocatalyzed characteristic, nanoanatase TiO₂ under light could cause an oxidation–reduction reaction. Our studies had proved that nano-TiO₂ could promote photosynthesis and greatly improve spinach growth. However, the mechanism of nano-TiO₂ on promoting conversion from light energy to electron energy and from electron energy to active chemistry energy remains largely unclear. In this study, we report that the electron transfer, oxygen evolution, and photophosphorylation of chloroplast (Chl) from nanoanatase-TiO₂-treated spinach were greatly increased under visible light and ultraviolet light illumination. It was demonstrated that nanoanatase TiO₂ could greatly improve whole chain electron transport, photoreduction activity of photosystem II, O₂-evolving and photophosphorylation activity of spinach Chl not only under visible light, but also energy-enriched electron from nanoanatase TiO₂, which entered Chl under ultraviolet light and was transferred in photosynthetic electron transport chain and made NADP⁺ be reduced into NADPH, and coupled to photophosphorylation and made electron energy be transformed to ATP. Moreover, nanoanatase h⁺, which photogenerated electron holes, captured an electron from water, which accelerated water photolysis and O₂ evolution.     

Modification of oxygen evolving center by Tris-washing

Tris-washing inhibits the O₂-evolving center of chloroplasts and their particles specifically and reversibly, and it was applied to many investigations on O₂-evolving center and PS II reaction center. In this review are introduced the various photosynthetic investigations in which Tris-washing was applied and are also discussed briefly on the site and the mechanism of Tris-inactivation, properties of P680 and Z, characteristic change in fluorescence and delayed light emission, and reactivation of O₂-evolving center by DCPIP.H₂-treatment and photo-reactivation of Tris-washed chloroplasts and their particles.     

Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations

Chloroplasts with high rates of photosynthetic O₂ evolution (up to 120 mol O₂· (mg Chl)^-1·h^-1 compared with 130 mol O₂· (mg Chl)^-1·h^-1 of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO₂ concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O₂ uptake could be observed in the dark by high- and low-CO₂ adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 mol photons·m^-2·s^-1 for high- and low-CO₂ adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200–300 M for low-CO₂ adapted chloroplasts, whereas high-CO₂ adapted chloroplasts were not saturated even at 700 M DIC. The concentrations of DIC required to reach half-saturated rates of net O₂ evolution (K_m(DIC)) was 31.1 and 156 M DIC for low- and high-CO₂ adapted chloroplasts, respectively. These results demonstrate that the CO₂ concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon - K_m(DIC) coneentration of dissolved inorganic carbon required for the rate of half maximal net O₂ evolution - PFR photon fluence rate - SPGM silicasol-PVP-gradient medium     

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.Abbreviations Asc ascorbate - Chl chlorophyll a+b - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(dichlorophenyl)-1,1-dimethylurea - DCPIP dichlorophenolindophenol - HL high light - LL low light - MV methylviologen - PhAR photosynthetically active radiation - PS photosystem     

Characterization of photosynthetic electron transport in bundle sheath cells of maize. I. Ascorbate effectively stimulates cyclic electron flow around PSI

Redox changes of the reaction-center chlorophyll of photosystem I (P700) and chlorophyll fluorescence yield were measured in bundle sheath strands (BSS) isolated from maize (Zea mays L.) leaves. Oxidation of P700 in BSS by actinic light was suppressed by nigericin, indicating the generation of a proton gradient across the thylakoid membranes of BSS chloroplasts. Methyl viologen, which transfers electrons from photosystem I (PSI) to O₂, caused a considerable decrease in the reduction rate of P700⁺ in BSS after turning off actinic light, showing that electron flow from the acceptor side of PSI to stromal components is critical for this reduction. Ascorbate (Asc), and to a lesser extent malate (Mal), caused a lower level of P700⁺ in BSS under aerobic conditions in far-red light, implying electron donation from these substances to the intersystem carriers. When Asc or Mal was added to BSS during pre-illumination under anaerobic conditions in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), the far-red-induced level of P700⁺ was lowered. The results suggest Asc and Mal can cause reduction of stromal donors, which in turn establishes conditions for rapid PSI-driven P700⁺ reduction. Addition of these metabolites also strongly stimulated the development of a proton gradient in thylakoids under aerobic conditions in the absence of DCMU, i.e. under conditions analogous to those in vivo. Ascorbate was a much more effective electron donor than Mal, suggesting it has a physiological role in activation of cyclic electron flow around PSI.     

Interactions between magnesium ions,pH, glucose-6-phosphate,and NADPH/NADP+ ratios in the modulation of chloroplast glucose-6-phosphate dehydrogenase in vitro

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from spinach chloroplasts is strongly affected by interactions between Mg²⁺, proton, and substrate concentrations. Mg²⁺ activates the enzyme to different degrees; however, it is not essential for enzyme activity. The Mg²⁺-dependent activation follows a maximum curve, magnitude and position of the maximum being dependent on pH and NADPH/NADP⁺ ratios. At a ratio of zero and pH 7.2, maximum activity is observed at 10 mM Mg²⁺. Increasing the NADPH/NADP⁺ ratio up to 1.7 (a ratio measured in the stroma during a light period), maximum activity is shifted to much lower Mg²⁺ concentrations. At pH 8.2 (corresponding to the pH of the stroma in the light) and at a high NADPH/NADP⁺ ratio, enzyme activity is not affected by the Mg²⁺ ion. The results are discussed in relation to dark-light-dark regulation of the oxidative pentose phosphate cycle in spinach chloroplasts.Abbreviations DTT dithiothreitol - G-6-P glucose-6-phosphate - G-6-PDH glucose-6-phosphate dehydrogenase (EC 1.1.1.49) - PPC pentose phosphate cycle     

5-O-β-d-galactopyranosyl-7-methoxy-3′,4′-dihydroxy-4-phenylcoumarin,an inhibitor of photophosphorylation in spinach chloroplasts

5-O--d-galactopyranosyl-7-methoxy-3,4-dihydroxy-4-phenylcoumarin isolated from Exostema caribaeum (Rubiaceae) has been found to act as an energy-transfer inhibitor in spinach chloroplasts. ATP synthesis and phosphorylating (coupled) electron flow were inhibited by 89 and 72%, respectively, at a concentration of 400 M. H⁺-uptake, basal and uncoupled electron transport were not affected by the coumarin. The light-activated Mg⁺²-ATPase activity from bound membrane thylakoid chloroplasts was slightly inhibited by the coumarin. Also, the heat-activated Ca⁺²-ATPase activity of the isolated coupling factor protein was insensitive to this compound. In chloroplasts partially stripped of coupling factor 1 by an EDTA treatment, the coumarin showed a restoration of the proton uptake process. These results suggest that the 4-phenylcoumarin under investigation inhibited phosphorylation in chloroplasts by specifically blocking the transport of protons through a membrane-bound component or a carrier channel (CF_O) located in a hydrophobic region at or near the functional binding site for the coupling factor 1.Abbreviations CF₁ chloroplast coupling factor 1 - CF_O coupling factor zero - DCCD dicyclohexylcarbodiimide - DTT dithiothreitol - EDTA ethylene-diaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - MES 2-(N-morpholino) ethanesulphonic acid - TCA trichloroacetic acid Taken in part from PhD thesis of M.R. Calera.     

Weak Light-Induced Oxygen Consumption Observed during Photoreactivation Is Coupled to the Recovery of Oxygen Evolving Activity

During photoreactivation of the O₂-evolving center in Tris-inactivated/Mn-depletedthylakoids, a slow O₂-consumption occurred. This O₂-consumptionbecame detectable when the O₂-evolving activity of thylakoidswas inactivated by Tris-treatment and decreased as photoreactivationproceeded. The O₂-consumption and photoreactivation similarlyrequired Mn²⁺ at µM levels in addition to PSII electrondonors and shared severa common characteristics. Stimulationof O₂-consumption and photoreactivation by these cofactors werealways accompanied by enhancement in chlorophyll fluorescenceinduction, suggesting the involvement of a Mehler-type reactionin photoreactivation. Although the electron transport due tothis O₂-consumption was rapid enough to oxidize 4 Mn²⁺ ionsto reconstitute the tetranuclear Mn-cluster in each O₂-evolvingcenter in a few seconds, actual recovery of O₂-evolving activityoccurred more slowly in a few minutes. It was inferred thatphotoreactivation in Tris-inactivated thylakoids is not a simplephotooxidation of Mn2²⁺ but involves more complicated processeswhich are coupled to the Mehlertype electron transport fromPSII to oxygen via PSI. (Received July 11, 1994; Accepted August 23, 1996)     

Increases in peroxide formation by the Photosystem II oxygen evolving reactions upon removal of the extrinsic 16, 22 and 33 kDa proteins are reversed by CaCl2 addition

This communication introduces a new spectrophotometric assay for the detection of peroxide generated by Photosystem II (PS II) under steady state illumination in the presence of an electron acceptor. The assay is based on the formation of an indamine dye in a horseradish peroxidase coupled reaction between 3-(dimethylamino)benzoic acid and 3-methyl-2-benzothiazolinone hydrazone. Using this assay, we found that as the O₂ evolution activity of PS II-enriched membrane fragments is decreased by treatments which cause the dissociation of the 33 and/or 23 and 16 kDa extrinsic proteins (i.e., CaCl₂-washing, NaCl-washing, lauroylcholine-treatment and ethylene glycol-treatment), light-induced peroxide formation increases. Both the losses of O₂ evolution and increases in peroxide formation seen under these conditions are reversed by CaCl₂ addition, indicating that the two activities originate from the water-splitting site. However, the increased rates of peroxide formation do not quantitatively match the losses in O₂ evolution activity. We suggest that a rapid consumption of the peroxide takes place via a catalase/peroxidase activity at the water-splitting site which competes with both the O₂ evolution and peroxide formation reactions. The observed peroxide formation is interpreted as arising from enhanced water accessibility to the catalytic site upon perturbation of the extrinsic proteins which then leads to alternate water oxidation side reactions.Abbreviations Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU 3-(3,4-dichloro)-1,1-dimethylurea - DCPIP 1,6-dichlorophenolindophenol - DMAB 3-(dimethylamino)benzoic acid - DMBQ 2,6-dimethyl-p-benzoquinone - DPC diphenylcarbazide - HEPES 4-(2-hydroxyethyl)-1-piperazinesulfonic acid - HMD HRP, MBTH, DMAB - HRP horseradish peroxidase - LCC lauroylcholine chloride - MBTH 3-methyl-2-benzothiazolinone hydrazone - MES 4-morpholinoethanesulfonic acid     

Artificial inhibitory effects of sulfite on photosystem II activity measured by oxygen evolution in chloroplasts

In this report we demonstrate sulfite interaction with oxygen and PSII electron acceptors (ferricyanide and para-benzoquinone) during measurement of oxygen evolution in chloroplasts. Redox potentials of oxygen, ferricyanide and para-benzoquinone allow them to compete for sulfite. Without taking this into account, sulfite inhibition of oxygen evolution can be overestimated, since sulfite consumes oxygen and reduces ferricyanide or para-benzoquinone during the measurement. In order to correctly measure the rate of oxygen evolution in chloroplasts, it is necessary to avoid presence of sulfite during the measurement. After overcoming the artifact, mentioned above, we confirm the sulfite inhibition of oxygen evolution in chloroplasts but at a lesser extent than earlier reported. This, however, is a pretreatment effect.Abbreviations Chl Chlorophyll - EDTA Ethylenediamine Tetraacetic Acid - FeCN Potassium Ferricyanide - Hepes N-2-Hydroxyethylpiperazine-N¹-2-ethanesulfonic acid - pBQ Para-benzoquinone - PSII photosystem II     

Photoconsumption of molecular oxygen on both donor and acceptor sides of photosystem II in Mn-depleted subchloroplast membrane fragments

Oxygen consumption in Mn-depleted photosystem II (PSII) preparations under continuous and pulsed illumination is investigated. It is shown that removal of manganese from the water-oxidizing complex (WOC) by high pH treatment leads to a 6-fold increase in the rate of O₂ photoconsumption. The use of exogenous electron acceptors and donors to PSII shows that in Mn-depleted PSII preparations along with the well-known effect of O₂ photoreduction on the acceptor side of PSII, there is light-induced O₂ consumption on the donor side of PSII (nearly 30% and 70%, respectively). It is suggested that the light-induced O₂ uptake on the donor side of PSII is related to interaction of O₂ with radicals produced by photooxidation of organic molecules. The study of flash-induced O₂ uptake finds that removal of Mn from the WOC leads to O₂ photoconsumption with maximum in the first flash, and its yield is comparable with the yield of O₂ evolution on the third flash measured in the PSII samples before Mn removal. The flash-induced O₂ uptake is drastically (by a factor of 1.8) activated by catalytic concentration (5-10 μM, corresponding to 2-4 Mn per RC) of Mn²⁺, while at higher concentrations (> 100 μM) Mn²⁺ inhibits the O₂ photoconsumption (like other electron donors: ferrocyanide and diphenylcarbazide). Inhibitory pre-illumination of the Mn-depleted PSII preparations (resulting in the loss of electron donation from Mn²⁺) leads to both suppression of flash-induced O₂ uptake and disappearance of the Mn-induced activation of the O₂ photoconsumption. We assume that the light-induced O₂ uptake in Mn-depleted PSII preparations may reflect not only the negative processes leading to photoinhibition but also possible participation of O₂ or its reactive forms in the formation of the inorganic core of the WOC.     

Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography

Glutathione reductase (EC 1.6.4.2) was purified from spinach (Spinacia oleracea L.) leaves by affinity chromatography on ADP-Sepharose. The purified enzyme has a specific activity of 246 enzyme units/mg protein and is homogeneous by the criterion of polyacrylamide gel electrophoresis on native and SDS-gels. The enzyme has a molecular weight of 145,000 and consists of two subunits of similar size. The pH optimum of spinach glutathione reductase is 8.5–9.0, which is related to the function it performs in the chloroplast stroma. It is specific for oxidised glutathione (GSSG) but shows a low activity with NADH as electron donor. The pH optimum for NADH-dependent GSSG reduction is lower than that for NADPH-dependent reduction. The enzyme has a low affinity for reduced glutathione (GSH) and for NADP⁺, but GSH-dependent NADP⁺ reduction is stimulated by addition of dithiothreitol. Spinach glutathione reductase is inhibited on incubation with reagents that react with thiol groups, or with heavymetal ions such as Zn²⁺. GSSG protects the enzyme against inhibition but NADPH does not. Pre-incubation of the enzyme with NADPH decreases its activity, so kinetic studies were performed in which the reaction was initiated by adding NADPH or enzyme. The Km for GSSG was approximately 200 M and that for NADPH was about 3 M. NADP⁺ inhibited the enzyme, assayed in the direction of GSSG reduction, competitively with respect to NADPH and non-competitively with respect to GSSG. In contrast, GSH inhibited non-competitively with respect to both NADPH and GSSG. Illuminated chloroplasts, or chloroplasts kept in the dark, contain equal activities of glutathione reductase. The kinetic properties of the enzyme (listed above) suggest that GSH/GSSG ratios in chloroplasts will be very high under both light and dark conditions. This prediction was confirmed experimentally. GSH or GSSG play no part in the light-induced activation of chloroplast fructose diphosphatase or NADP⁺-glyceraldehyde-3-phosphate dehydrogenase. We suggest that GSH helps to stabilise chloroplast enzymes and may also play a role in removing H₂O₂. Glucose-6-phosphate dehydrogenase activity may be required in chloroplasts in the dark in order to provide NADPH for glutathione reductase.Abbreviations GSH reduced form of the tripeptide glutathione - GSSG oxidised form of glutathione     

Jasmonate: A decision maker between cell death and acclimation in the response of plants to singlet oxygen

Under stress conditions that bring about excessive absorption of light energy in the chloroplasts, the formation of singlet oxygen (¹O₂) can be strongly enhanced, triggering programmed cell death. However, the ¹O₂ signaling pathway can also lead to acclimation to photooxidative stress, when ¹O₂ is produced in relatively low amounts. This acclimatory response is associated with a strong downregulation of the jasmonate biosynthesis pathway and the maintenance of low jasmonate levels, even under high light stress conditions that normally induce jasmonate synthesis. These findings suggest a central role for this phytohormone in the orientation of the ¹O₂ signaling pathway toward cell death or acclimation. This conclusion is confirmed here in an Arabidopsis double mutant obtained by crossing the ¹O₂-overproducing mutant ch1 and the jasmonate-deficient mutant dde2. This double mutant was found to be constitutively resistant to ¹O₂ stress and to display a strongly stimulated growth rate compared with the single ch1 mutant. However, the involvement of other phytohormones, such as ethylene, cannot be excluded.     

Inactivation of photosynthetic oxygen evolution by o-phenanthroline and LiClO4 in Photosystem 2 of the pea

We examined the effects of o-phenanthroline and LiClO₄ on oxygen evolution and electron transport in the Photosystem 2 complex of the pea. Treatment of Photosystem 2 particles with a combination of 3.0 mM o-phenanthroline and 1.0 M LiClO₄ for 30–40 min at 0°C decreased the oxygen-evolving activity with the electron acceptor (either phenyl-p-benzoquinone or 2,6-dichlorophenol indophenol) to less than 5% of the original level. However with the same treatment, the electron-transport activity from an artificial electron donor, 1,5-diphenylcarbohydrazide, to 2,6-dichlorophenol indophenol remained at 60% of the original activity. The amount of manganese in the Photosystem 2 complex decreased in parallel with the loss of oxygen evolution following treatment. These observations suggest that the treatment of the Photosystem 2 complex with o-phenanthroline and LiClO₄ inhibits electron transport on the oxygen-evolving side much more significantly than on the electron-acceptor side.Abbreviations Chl chlorophyll - DCPIP 2,6-dichlorophenol indophenol - DPC 1,5-diphenylcarbo hydrazide - EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes 4-morpholineethanesulfonic acid - PBQ phenyl-p-benzoquinone - PS 2 Photosystem 2     

Effect of phosphon-D on photosynthetic light reactions and on reactions of the oxidative and reductive pentose phosphate cycle in a reconstituted spinach (Spinacia oleracea L.) chloroplast system

Phosphon-D (tributyl-2, 4-dichlorobenzylphosphonium chloride), known as an inhibitor of gibberellin biosynthesis, enhances photosynthetic electron transport by up to 200%, with Fe(CN) ₆ ^3- and NADP⁺ being the electron acceptors. Maximum stimulation is reached at phosphon-D concentrations around 2–5 M. At the same time photosynthetic ATP formation is gradually inhibited. Phosphon-D concentrations over 0.1 mM inhibit electron transport. The uncoupling activity of phosphon-D is manifested by inhibition of noncyclic ATP synthesis and by stimulation of light-induced electron flow. The inhibition of ATP synthesis drastically decreases photosynthetic carbon assimilation in a reconstituted spinach chloroplast system. The two ATP-dependent kinase reactions of the reductive pentose phosphate cycle become the rate-limiting steps. On the other hand a stimulated photoelectron transport increases the NADPH/NADP⁺ ratio, resulting in a drastic inhibition of chloroplast glucose-6-phosphate dehydrogenase (EC 1.1.1.49), the key enzyme of the oxidative pentose phosphate cycle. When light-induced electron flow is inhibited by high phosphon-D concentrations and the NADPH/NADP⁺ ratio is low, the light-dependent inhibition of glucose-6-phosphate dehydrogenase is gradually abolished.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride - B-Nine N-dimethylaminosuccinamic acid - CCC (2-chloroethyl)-trimethylammonium chloride - DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea - DCPIP dichlorophenolindophenol - G-6-PDH glucose-6-phosphate dehydrogenase - FBP fructose bisphosphate - F-6-P fructose-6-phosphate - 3-PGA 3-phosphoglyceric acid - Posphon-D tributyl-2,4-dichlorobenzylphosphonium chloride - PMP pentose monophosphates - PPC pentose phosphate cycle - RuBP ribulose bisphosphate - Ru-5-P ribulose-5-phosphate Dedicated to Prof. Dr. Drs.h.c. Adolf Butenandt on the occasion of his 75. birthday     

On the decarboxylation of α-keto acids by isolated chloroplasts

The mechanism of the decarboxylation of α-keto acids by isolated chloroplasts has been studied with the aid of superoxide dismutase and catalase. Using photosynthetic and enzymatic systems, which are known to catalyze peroxidic oxidations, we have been able to demonstrate that both the superoxide free radical ion and H₂O₂ are necessary for maximal rates of decarboxylation. In isolated chloroplasts, an auto-oxidizable electron acceptor as well as an electron donor for Photosystem I are absolute requirements for the decarboxylation. H₂O₂ seems to be the primary oxidant in the decarboxylation of pyruvate or glyoxylate by isolated chloroplasts. A secondary rate of decarboxylation is superimposed on the primary one, mediated by superoxide free radical ion. Mn²⁺ stimulates the decarboxylation probably via intermediarily-formed Mn³⁺ in a reaction, which is neither inhibited by catalase nor by superoxide dismutase. A decarboxylation of pyruvate or glyoxylate by isolated chloroplasts in the presence of NADP⁺ is initiated, as soon as the available NADP⁺ is fully reduced. In this case, the open-chain electron transport seems to switch from NADP⁺ to oxygen as the terminal electron acceptor.     

Analysis of oxygen evolution during photosynthetic induction and in multiple-turnover flashes in sunflower leaves

Exchange of CO₂ and O₂ and chlorophyll fluorescence were measured in the presence of 360 1 · 1^–1 CO₂ in nitrogen in Helianthus annuss L. leaves which had been preconditioned in the dark or at a photon flux density (PFD) of 24 mol · m^–2 · s^–1 either in 21 or 0% O₂. An initial light-dependent O₂ outburst of 6 mol · m^–2 was measured after aerobic dark incubation. It was attributed to the reduction of electron carriers, predominantly plastoquinone. The maximum initial rate of O₂ evolution at PFD 8000 mol · m^–2 · s^–1 was 170 mol · m^–2 · s^–2 or about four times the steady CO₂-and light-saturated rate of photosynthesis. Fluorescence measurements showed that the rate was still acceptor-limited. Fast O₂ evolution ceased after electron carriers were reduced in the dark-adapted leaf, but continued for a short time at the lower rate of 62 mol · m^–2 · s^–1 in the light-adapted leaf. The data are interpreted to show that enzymes involved in 3-phosphoglycerate reduction are dark-inhibited, but were fully active in low light. In a dark-adapted leaf, respiratory CO₂ evolution continued under nitrogen; it was partially inhibited by illumination. Prolonged exposure of a leaf to anaerobic conditions caused reducing equivalents to accumulate. This was shown by a slowly increasing chlorophyll fluorescence yield which indicated the reduction of the PSII acceptor Q_A in the dark. When the leaf was illuminated, no O₂ evolution was detected from short light pulses, although transient O₂ production was appreciable during longer light pulses. This indicates that an electron donor (pool size about 2–3 e/PSII reaction center) became reduced in the dark and the first photons were used to oxidise this donor instead of water.Abbreviations Chl chlorophyll - CRC carbon reduction cycle - GAPDH NADP-glyceraldehyde-phosphate dehydrogenase - PFD photon flux density - PGA 3-phosphoglycerate - RuBP ribulose bisphosphate - TCA tricarboxylic acid cycle To whom correspondence should be addressedThis work received support by the Estonian Academy of Sciences, the Gottfried-Wilhelm-Leibniz Program of the Deutsche For-schungsgemeinschaft and the Sonderforschungsbereich 251 of the University of Würzburg.     

Carbon dioxide and the reduction of indophenol and ferricyanide by chloroplasts

Pea chloroplasts isolated in salt media show decreased rates of 2:6 dichlorophenolindophenol (DCPIP) and ferricyanide reduction when depleted of CO₂ at pH values below 7.5. The greatest effect of CO₂ was on uncoupled systems. The incorporation of 10⁻², 2 × 10⁻² and 4 × 10⁻² m sodium acetate into the reaction mixtures progressively increased the bicarbonate concentration required for half maximal rates of reduction of DCPIP. The reaction was saturated by bicarbonate concentrations of 1 to 4 × 10⁻² m. With both DCPIP and ferricyanide, the addition of bicarbonate to illuminated chloroplast systems depleted of CO₂ gave very rapid increases in the rates of reduction. Bicarbonate also stimulated oxygen uptake by the illuminated chloroplasts when added hydrogen acceptors had been reduced. There was no effect of bicarbonate on ferricyanide reduction at low light intensities, but with DCPIP reduction, the apparent magnitude of the effect was independent of light intensity. This suggests that DCPIP reacts with the chloroplast electron transport chain at a site nearer to a photochemical stage than does ferricyanide. It also suggests that CO₂ has at least 2 sites of action.     

Two sites of photoinhibition of the electron transfer in oxygen evolving and Tris-treated PS II membrane fragments from spinach

Photoinhibition was analyzed in O₂-evolving and in Tris-treated PS II membrane fragments by measuring flash-induced absorption changes at 830 nm reflecting the transient P680⁺ formation and oxygen evolution. Irradiation by visible light affects the PS II electron transfer at two different sites: a) photoinhibition of site I eliminates the capability to perform a stable charge separation between P680⁺ and Q_A ^- within the reaction center (RC) and b) photoinhibition of site II blocks the electron transfer from Y_Z to P680⁺. The quantum yield of site I photoinhibition (2–3×10^-7 inhibited RC/quantum) is independent of the functional integrity of the water oxidizing system. In contrast, the quantum yield of photoinhibition at site II depends strongly on the oxygen evolution capacity. In O₂-evolving samples, the quantum yield of site II photoinhibition is about 10^-7 inhibited RC/quantum. After selective elimination of the O₂-evolving capacity by Tris-treatment, the quantum yield of photoinhibition at site II depends on the light intensity. At low intensity (<3 W/m²), the quantum yield is 10^-4 inhibited RC/quantum (about 1000 times higher than in oxygen evolving samples). Based on these results it is inferred that the dominating deleterious effect of photoinhibition cannot be ascribed to an unique target site or a single mechanism because it depends on different experimental conditions (e.g., light intensity) and the functional status of the PS II complex.Abbreviations A₈₃₀ absorption change at 830 nm - P680 primary electron donor of PS II - PS II photosystem II - Mes 2(N-morpholino)ethansulfonic acid - Q_A, Q_B primary and secondary acceptors of PS II - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenylcarbohydrazide - FWHM fullwidth at half maximum - Ph-p-BQ phenyl-p-benzoquinone - PFR photon fluence rate - Pheo pheophytin - RC reaction center