Photooxidation of ferrocyanide and iodide ions and associated phosphorylation in NH2OH-treated chloroplasts

NH₂OH-treated, non-water oxidizing chloroplasts are shown to be capable of oxidizing ferrocyanide and I^? via Photosystem II at appreciable rates (? 200 μequiv/h per mg chlorophyll). Using methylviologen as electron acceptor, ferrocyanide oxidation can be measured as O₂ uptake, as ferricyanide formation, or as H⁺ consumption (2 Fe²⁺ + 2H⁺ + O₂ → 2 Fe³⁺ + H₂O₂). I^? oxidation can be measured as methylviologen-mediated O₂ uptake, or spectrophotometrically, using ferricyanide as electron acceptor. The oxidation product I₂ is re-reduced, as it is formed, by unknown reducing substances in the reaction system.The rate-saturating concentrations of these donors are very high: 30 mM with ferricyanide and 15 mM with I^?. Relatively lipophilic Photosystem II donors such as catechol, benzidine and p-aminophenol saturate the photooxidation rate at much lower concentrations (< 0.5 mM). It thus seems that the oxidation of hydrophilic reductants such as ferricyanide and I^? is limited by permeability barriers. Very likely the site of Photosystem II oxidation is embedded in the thylakoid membrane or is situated on the inner surface of the membrane.The efficiency of phosphorylation (P/e₂) is 0.5 to 0.6 with ferrocyanide and about 0.5 with I^?. In contrast the P/e₂ ratio is 1.0 to 1.2 when water, catechol, p-aminophenol or benzidine serves as electron donor. These differences imply that only one of two phosphorylation sites operate when ferrocyanide and I^? are oxidized. Ferrocyanide and I^? are also chemically distinct from other Photosystem II donors in that their oxidation does not involve proton release. It is suggested that the mechanism of energy conservation associated with Photosystem II may be only operative when the removal of electrons from the donor results in release of protons (i.e. with water, hydroquinones, phenylamines, etc.).     

Adaptation to quantum flux by the emerson photosynthetic unit

The size of the Emerson photosynthetic unit was measured in Chlorella pyrenoidosa strain no. 252 grown at light intensities between 50 and 1000 foot candles. The Emerson photosynthetic unit changed from a minimum size of 1970 molecules chlorophyll a + b/O₂ per flash in cells grown at 1000 foot candles to a maximum size of 3150 molecules chlorophyll a + b/O₂ per flash for cells grown at 50 foot candles. The size changes were interpreted as a partial adaptation where the trapping center antenna responded to changes in incident light intensity. Light-induced changes in chlorophyll content and size of the Emerson photosynthetic unit were directly related.     

Photosynthetic Unit Size during the Synchronous Life Cycle of Scenedesmus

Apparent size of the photosynthetic unit (chlorophyll/O₂ per flash) was estimated by O₂ yield of repetitive short flashes on cell samples taken at various times from a synchronized culture (14-hour light, 10-hour dark) of Scenedesmus obliquus. Unit size was essentially invariant (< 10% variation) with a mean value of 1750 chlorophyll/O₂ per flash. In contrast, the light-saturated photosynthetic rate per unit chlorophyll, or turnover rate of the photosynthetic unit, varied with the life cycle, rising 40% in the first three hours of the light period and decaying slowly thereafter. The results are taken as evidence that the metabolic machinery is subject to far greater control and adjustment than is the photochemical machinery.     

Respiration in energy-transducing membranes of the thermophilic cyanobacterium Mastigocladus laminosus: I. Relation of the respiratory and photosynthetic electron transport

The thermophilic cyanobacterium Mastigocladus laminosus was grown at different CO₂ concentrations and temperatures. Respiratory and photosynthetic electron transport in isolated membranes were measured and their activities were compared. Cells grown at low CO₂ concentration showed respiratory electron transport, whereas Photosystem-II-dependent transport was optimal in cells grown at high CO₂ concentrations. The respiratory electron transport from NADH and succinate were KCN-sensitive, whereas NADPH-dependent O₂ uptake was not. It could be shown that NADH and succinate donate electrons in the photosynthetic electron pathway via Photosystem I. In cytochrome-c-553-depleted membranes added cytochrome c-553 could stimulate photosynthetic and respiratory electron transport. A common electron transport pathway between the quinone and cytochrome c is postulated.     

Rates and properties of endogenous cyclic photophosphorylation of isolated intact chloroplasts measured by CO2 fixation in the presence of dihydroxyacetone phosphate

1. Dihydroxyacetone phosphate in concentrations ? 2.5 mM completely inhibits CO₂-dependent O₂ evolution in isolated intact spinach chloroplasts. This inhibition is reversed by the addition of equimolar concentrations of P_i, but not by addition of 3-phosphoglycerate. In the absence of P_i, 3-phosphoglycerate and dihydroxyacetone phosphate, only about 20% of the ¹⁴C-labelled intermediates are found in the supernatant, whereas in the presence of each of these substances the percentage of labelled intermediates in the supernatant is increased up to 70–95%. Based on these results the mechanism of the inhibition of O₂ evolution by dihydroxyacetone phosphate is discussed with respect to the function of the known phosphate translocator in the envelope of intact chloroplasts.2. Although O₂ evolution is completely suppressed by dihydroxyacetone phosphate, CO₂ fixation takes place in air with rates of up to 65μ mol · mg^?1 chlorophyll · h^?1. As non-cyclic electron transport apparently does not occur under these conditions, these rates must be due to endogenous pseudocyclic and/or cyclic photophosphorylation.3. Under anaerobic conditions, the rates of CO₂ fixation in presence of dihydroxyacetone phosphate are low (2.5–7 μmol · mg^?1 chlorophyll · h^?1), but they are strongly stimulated by addition of dichlorophenyl-dimethylurea (e.g. 2 · 10^?7 M) reaching values of up to 60 μmol · mg^?1 chlorophyll · h^?1. As under these conditions the ATP necessary for CO₂ fixation can be formed by an endogenous cyclic photophosphorylation, the capacity of this process seems to be relatively high, so it might contribute significantly to the energy supply of the chloroplast. As dichlorophenyl-dimethylurea stimulates CO₂ fixation in presence of dihydroxyacetone phosphate under anaerobic but not under aerobic conditions, it is concluded that only under anaerobic conditions an “overreduction” of the cyclic electron transport system takes place, which is removed by dichlorophenyl-dimethylurea in suitable concentrations. At concentrations above 5 · 10^?7 M dichlorophenyl-dimethylurea inhibits dihydroxyacetone phosphate-dependent CO₂ fixation under anaerobic as well as under aerobic conditions in a similar way as normal CO₂ fixation. Therefore, we assume that a properly poised redox state of the electron transport chain is necessary for an optimal occurrence of endogenous cyclic photophosphorylation.4. The inhibition of dichlorophenyl-dimethylurea-stimulated CO₂ fixation in presence of dihydroxyacetone phosphate by dibromothymoquinone under anaerobic conditions indicates that plastoquinone is an indispensible component of the endogenous cyclic electron pathway.     

The photochemical and fluorescence properties of whole cells,spheroplasts and spheroplast particles from the blue-green alga Phormidium luridum

The photochemical activities and fluorescence properties of cells, spheroplasts and spheroplast particles from the blue-green alga Phormidium luridum were compared. The photochemical activities were measured in a whole range of wavelengths and expressed as quantum yield spectra (quantum yield vs. wavelength). The following reactions were measured: Photosynthesis (O₂ evolution) in whole cells; Hill reaction (O₂ evolution) with Fe(CN)₆^3? and NADP as electron acceptors (Photosystem II and Photosystem II+Photosystem I reactions); electron transfer from reduced 2,6-dichlorophenolindophenol to diquat (Photosystem I reaction). The fluorescence properties were emission spectra, quantum yield spectra and the induction pattern.On the basis of comparison between the quantum yield spectra and the pigments compositions the relative contribution of each pigment to each photosystem was estimated. In normal cells and spheroplasts it was found that Photosystem I (Photosystem II) contains about 90 % (10 %) of the chlorophyll a, 90 % (10 %) of the carotenoids and 15 % (85 %) of the phycocyanin. In spheroplast particles there is a reorganization of the pigments: they loose a certain fraction (about half) of the phycocyanin but the remaining phycocyanin attaches itself exclusively to Photosystem I (!). This is reflected by the loss of Photosystem II activity, a flat quantum yield vs. wavelength dependence and a loss of the fluorescence induction.The fluorescence quantum yield spectra conform qualitatively to the above conclusion. More quantitative estimation shows that only a fraction (20–40 %) of the chlorophyll of Photosystem II is fluorescent. Total emission spectrum and the ratio of variable to constant fluorescence are in agreement with this conclusion.The fluorescence emission spectrum shows characteristic differences between the constant and variable components. The variable fluorescence comes exclusively from chlorophyll a; the constant fluorescence is contributed, in addition to chlorophyll a, by phycocyanine and an unidentified long wavelength component.The variable fluorescence does not change in the transition from whole cells to spheroplasts. However, the constant fluorescence increases considerably. This indicates the release of a small fraction of pigments from the photosynthetic photochemical apparatus which then become fluorescent.     

Mode of Action Studies on Nitrodiphenyl Ether Herbicides : II. The Role of Photosynthetic Electron Transport in Scenedesmus obliquus

The nitrodiphenyl ether herbicide 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitroacetophenone oxime-o-(acetic acid, methyl ester) (DPEI) induces light- and O₂-dependent lipid peroxidation and chlorophyll (Chl) bleaching in the green alga Scenedesmus obliquus. Under conditions of O₂-limitation, these effects are diminished by prometyne and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), both inhibitors of photosynthetic electron transport. Mutants in which photosynthetic electron transport is blocked are also resistant to DPEI under conditions of O₂-limitation. Light- and O₂-dependent lipid peroxidation and Chl bleaching are also induced by 5-[2-chloro-4-(trifluoromethyl)phenoxy]-3-methoxyphthalide (DPEII), a diphenyl ether whose redox properties preclude reduction by photosystem I. However, these effects of DPEII are also inhibited by DCMU. Under conditions of high aeration, DCMU does not protect Scenedesmus cells from Chl bleaching induced by DPEI, but does protect against paraquat. DPEI, but not paraquat, induces tetrapyrrole formation in treated cells in the dark. This is also observed in a mutant lacking photosystem I but is suppressed under conditions likely to lead to O₂ limitation. Our results indicate that, in contrast to paraquat, the role of photosynthetic electron transport in diphenyl ether toxicity in Scenedesmus is not to reduce the herbicide to a radical species which initiates lipid peroxidation. Its role is probably to maintain a sufficiently high O₂ concentration, through water-splitting, in the algal suspension.     

Preparation of O2-evolving Photosystem-II subchloroplasts from spinach

An O₂-evolving Photosystem II subchloroplast preparation was obtained from spinach chloroplasts, using low concentrations of digitonin and Triton X-100. The preparation showed an O₂ evolution activity equivalent to 20% of the uncoupled rate of fresh broken chloroplasts, but had no significant Photosystem-I-dependent O₂ uptake activity. The preparation showed a chlorophyll $\text{a}\text{b}$ ratio of 1.9 and a $\text{P-700}\text{chlorophyll}$ ratio of $\text{1}\text{2400}$. Absorption spectra at room temperature and fluorescence emission spectra of chlorophyll at 77 K suggested a significant decrease in Photosystem I antenna chlorophylls in the O₂-evolving Photosystem II preparation.     

Biochemical characterization of a highly active O2-evolving Photosystem II preparation from maize

An O₂-evolving Photosystem (PS) II preparation was isolated from maize by a Triton X-100 procedure (Kuwabara, T. and Murata, N. (1982) Plant Cell Physiol. 23, 533–539). A highly active O₂-evolving preparation was obtained which evolved O₂ at 76% the rate of fresh chloroplasts (H₂O → 2,6-dichloro-p-benzoquinone) and was very sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. There was no detectable PS I activity in the preparation (2,3,5,6-tetramethyl-p-phenylenediamine → methyl viologen). When analyzed by lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis the O₂-evolving preparation was shown to be highly depleted in CP I, CF₁, and devoid of cytochromes f and b-563 (the absence of which was confirmed by difference spectroscopy). The preparation was enriched in the PS II reaction center polypeptides I and II, the 34 kDa polypeptide (Metz, J., Wong, J. and Bishop, N.I. (1980) FEBS Lett. 114, 61–66), the Coomassie blue-stainable 32 kDa polypeptide (Kuwabara, T. and Murata, N. (1979) Biochim. Biophys. Acta 581, 228–236), LHCP-associated polypeptides and cytochrome b-559. Polypeptides of unknown function at 40.5, 25, 24, 22, 16.6 and 14 kDa were also present in the O₂-evolving preparation. Triton X-114 phase partitioning (Bricker, T.M. and Sherman, L.A. (1982) FEBS Lett. 149, 197–202) indicated that the majority of these polypeptides were intrinsic. Only the polypeptides at 32, 25, 24 and 14 kDa were extrinsic. When examined by the octylglucoside procedure of Camm and Green (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432) the PS II O₂-evolving preparation was shown to contain the chlorophyll-proteins CP 27, CP 29, CP II1, D, and CP a-1 and CP a-2. Chlorophyll-proteins associated with PS I were highly depleted. The visible absorption spectra indicated an enrichment of chlorophyll b and carotenoids in the preparation. The 77 K fluorescence emission spectrum (excitation wavelength = 435 nm) exhibits a strong F-686 with little F-695 shoulder and a broad, low-intensity F-735 emission.     

Picosecond time-resolved energy transfer in Porphyridium cruentum. Part I. In the intact alga

The wavelength-resolved fluorescence emission kinetics of the accessory pigments and chlorophyll a in Porphyridium cruentum have been studied by picosecond laser spectroscopy. Direct excitation of the pigment B-phycoerythrin with a 530 nm, 6 ps pulse produced fluorescence emission from all of the pigments as a result of energy transfer between the pigments to the reaction centre of Photosystem II. The emission from B-phycoerythrin at 576 nm follows a nonexponential decay law with a mean fluorescence lifetime of 70 ps, whereas the fluorescence from R-phycocyanin (640 nm), allophycocyanin (660 nm) and chlorophyll a (685 nm) all appeared to follow an exponential decay law with lifetimes of 90 ps, 118 ps and 175 ps respectively. Upon closure of the Photosystem II reaction centres with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and preillumination the chlorophyll a decay became non-exponential, having a long component with an apparent lifetime of 840 ps. The fluorescence from the latter three pigments all showed finite risetimes to the maximum emission intensity of 12 ps for R-phycocyanin, 24 ps for allophycocyanin and 50 ps for chlorophyll a.A kinetic analysis of these results indicates that energy transfer between the pigments is at least 99% efficient and is governed by an exp $\text{?At}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ transfer function. The apparent exponential behaviour of the fluorescence decay functions of the latter three pigments is shown to be a direct result of the energy transfer kinetics, as are the observed risetimes in the fluorescence emissions.     

Mode of action studies on nitrodiphenyl ether herbicides: I. Use of barley mutants to probe the role of photosynthetic electron transport

5-[2-Chloro-4-(trifluoromethyl)phenoxy]-2-nitroacetophenone oxime-o-(acetic acid, methyl ester) (DPEI), is a potent nitrodiphenyl ether herbicide which causes rapid leaf wilting, membrane lipid peroxidation, and chlorophyll destruction in a process which is both light- and O₂-dependent. These effects resemble those of other nitrodiphenyl ether herbicides. Unlike paraquat, the herbicidal effects of DPEI are only slightly reduced by pretreatment with the photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. DPEI is a weak inhibitor of photosynthetic electron transport (I₅₀ 15 micromolar for water to paraquat) in vitro, with at least one site of action at the cytochrome b₆f complex. Ultrastructural studies and measurements of ethane formation resulting from lipid peroxidation indicate that mutants of barley lacking photosystem I (PSI) (viridis-zb⁶³) or photosystem II (viridis-zd⁶⁹) are resistant to paraquat but susceptible to DPEI. The results indicate that electron transfer through both photosystems is not essential for the toxic effects of nitrodiphenyl ether herbicides. Furthermore, the results show that neither cyclic electron transport around PSI, nor the diversion of electrons from PSI to O₂ when NADPH consumption is blocked are essential for the phytotoxicity of nitrodiphenyl ether herbicides.     

Development of Photosystem I and Photosystem II Activities in Leaves of Light-grown Maize (Zea mays)

To compare chloroplast development in a normally grown plant with etiochloroplast development, green maize plants (Zea mays), grown under a diurnal light regime (16-hour day) were harvested 7 days after sowing and chloroplast biogenesis within the leaf tissue was examined. Determination of total chlorophyll content, ratio of chlorophyll a to chlorophyll b, and O₂-evolving capacity were made for intact leaf tissue. Plastids at different stages of development were isolated and the electron-transporting capacities of photosystem I and photosystem II measured. Light saturation curves were produced for O₂-evolving capacity of intact leaf tissue and for photosystem I and photosystem II activities of isolated plastids. Structural studies were also made on the developing plastids. The results indicate that the light-harvesting apparatus becomes increasingly efficient during plastid development due to an increase in the photosynthetic unit size. Photosystem I development is completed before that of photosystem II. Increases in O₂-evolving capacity during plastid development can be correlated with increased thylakoid fusion. The pattern of photosynthetic membrane development in the light-grown maize plastids is similar to that found in greening etiochloroplasts.     

Promotive effect of 5-aminolevulinic acid on the growth and photosynthesis of Spirulina platensis

Photosynthetic activity in terms of O₂ evolution and the growth of Spirulina platensis was stimulated by adding 5-aminolevulinic acid (ALA, 500 mg/l) to photoautotrophically growing cells. After ALA was added to the medium, intracellular accumulations of phycocyanin and chlorophyll were stimulated simultaneously, followed by enhancement of the photosynthetic activities of photosystems I and II, and lastly, growth was promoted. ALA did not directly activate the photosynthetic electron transport system. However, during a 3-h incubation of intact cells with ALA, photosynthetic activity was enhanced.     

Hydrogen peroxide in artificial photosynthesizing systems

From the point of view of the concepts of hydrogen peroxide as a source of photosynthetic oxygen (hydrogen) coordination and photochemical properties of chlorophyll and its aggregates towards hydrogen peroxide were considered. The binding energy of H₂O and H₂O₂ with chlorophyll and chlorophyllide depending on their form (monomers, dimers and trimers) was estimated by quantum chemical calculations. It is shown that at an increase of the degree of the pigment aggregation binding energy of H₂O₂ was more than the energy of H₂O. Analysis of experimental results of the photochemical decomposition of hydrogen peroxide using chlorophyll was carried out. Estimates of the thermodynamic parameters (ΔG° and ΔH°) of the formation of organic compounds from CO₂ with water and hydrogen peroxide were compared. The interaction of CO₂ with H₂O₂ requires much less energy consumption than with water for all considered cases. The formation of organic products (formaldehyde, alcohols, carboxylic and carbonylic compounds) and simultaneous production of O₂ under the influence of visible light in the systems of inorganic carbon-hydrogen peroxide - chlorophyll (phthalocyanine) is detected by GC/MS method, FTIR spectroscopy, and chemical analysis.     

地表臭氧浓度增加和UV-B辐射增强及其复合处理对大豆光合特性的影响

基于开顶式气室(OTC),系统开展了地表O3增加和UV-B增强及其复合处理下(自然空气,CK;10%UV-B增强,T1;100nmol/mol O3,T2;100 nmol/mol O3+10%UV-B增强,T3)大豆光合气体交换、光响应、光合色素和类黄酮含量等参数的观测与分析研究。结果表明,与对照相比,T1和T2单因子处理组的如下指标有相似变化:气孔导度、气孔限制值下降,胞间二氧化碳浓度上升,净光合速率、最大净光合速率、半饱和光强显著降低,表观量子效率和暗呼吸速率先升后降。T1的叶绿素含量降低不显著,类胡萝卜素含量先降后升,类黄酮含量上升,而T2的叶绿素和类胡萝卜素含量显著降低,类黄酮含量先降后升。复合处理下,与CK相比各指标的变化和单因子相似,影响程度均强于两单因子组。因此,100 nmol/mol O3浓度增加和10%UV-B辐射增强复合处理对大豆叶绿素含量的影响存在协同作用,且O3胁迫起了主导作用。光合作用下降的主要原因均是非气孔因素,复合处理对大豆光合作用的影响比两因子单独胁迫有所加深,是O3和UV-B共同作用的结果。     

The effect of magnesium ions on action spectra for reactions mediated by Photosystems I and II in spinach chloroplasts

Action spectra were measured for positive changes in variable fluorescence (emission > 665 nm) excited by a beam of 485 nm chopped at 75 Hz. The action of two further beams was compared, one being variable, the other (reference) constant with respect to wavelength and intensity. Comparison was achieved by alternating the reference and the variable wavelength beams at 0.3 Hz and adjusting the intensity of the latter such as to cancel out any 0.3 Hz component in the 75 Hz fluorescence signal. The relative action then was obtained as the reciprocal of the intensity of the variable wavelength beam. Similarly, action spectra were measured for O₂ evolution with ferricyanide/p-phenylenediamine as electron acceptor, and for O₂ uptake mediated by methyl viologen with ascorbate 3-(p-chlorophenyl)-1,1-dimethylurea as electron donor in the presence of 2,6-dichlorophenolindophenol.Addition of 5 mM MgCl₂ increases the relative action around 480 nm for the change in variable fluorescence and p-phenylenediamine-dependent O₂ evolution, and decreases it for methyl viologen-mediated O₂ uptake with 2,6-dichlorophenolindophenol/ascorbate as electron donor in the presence of 3-(p-chlorophenyl-1,1-dimethylurea. The change in variable fluorescence and O₂ evolution are stimulated by MgCl₂, whereas O₂ uptake is inhibited by it.The results are discussed in terms of a model assuming a tripartite organization. of the photosynthetic pigments (Thornber, J. P. and Highkin, H. R. (1974) Eur. J. Biochem. 41, 109–116; Butler, W. L. and Kitajima, M. (1975) Biochim. Biophys. Acta 396, 72–85). MgCl₂ is thought to promote energy transfer to Photosystem II from a light-harvesting pigment complex serving both photosystems.     

Metabolic activities in Cyanophora paradoxa and its cyanelles

Isolated cyanelles of Cyanophora paradoxa perform photosystem I and II dependent Hill reactions. The photosynthetic electron transport of the cyanelles does not show special features uncommon in cyanobacteria or chloroplasts of red algae. A preparation of cyanelles performs photosynthetic O₂-evolution with approximately 1/3 of the rate of intact Cyanophora, in only, however, the first three minutes of the experiment. All attempts to stabilize the CO₂-fixation activity of isolated cyanelles failed. Isolated cyanelles do not perform KCN-sensitive O₂-uptake, indicating that respiratory cytochrome oxidase is lacking in cyanelles. O₂-consumption by crude extracts from Cyanophora is inhibited by KCN when N-tetramethyl-p-phenylenediamine/ascorbate or NADH but not NADPH are supplied as the electron donors in contrast to the situation in cyanobacteria. These findings suggest that cyanelles do not respire. It is concluded that cyanelles are not so much related to cyanobacteria as formerly believed, but share many properties with chloroplasts of eukaryotic cells.Abbreviations Chl chlorophyll - DCPIP dichlorophenol-indophenol - TMPD N-tetramethyl-p-phenylenediamine To whom correspondence should be addressed