Directional Cytotoxic Reactions between Incompatible Plasmodia of DIDYMIUM IRIDIS

In vitro studies with chloroplasts isolated from maize populations differing in juvenile productivity by the sixth leaf stage of growth demonstrated differences in rates of ferricyanide reduction and of non-cyclic photophosphorylation. Similar results were obtained for two independent sets of replicate selections for high and for low productivity. Chloroplasts from low productive selections had about 115 percent of the reduction rate and 129 percent of the phosphorylation rate found for the high productive selections. Differences in ferricyanide reduction held for a fourfold range of experimentally induced rates. The contrasting rates of electron flow did not result from differential extraction environments following cell disruption nor from the failure to protect chloroplasts during extraction. Light-dependent phosphate uptake gave evidence, though inconclusive, of being so modified. No evidence was found for reciprocal effects between low-high and high-low hybrids. No difference in activities was found between chloroplasts isolated from two populations selected for narrow and for wide leaf widths. These two selections differed markedly in leaf areas. While in vitro measures of chloroplast efficiency reflected differences which were under genetic control, the results did not follow those expected from photosynthetic studies. The differences in rates appeared to reflect modification of processes other than those in the photosynthetic pathway.     

Effects of Diffusion and Topological Factors on the Efficiency of Energy Coupling in Chloroplasts with Heterogeneous Partitioning of Protein Complexes in Thylakoids of Grana and Stroma. A Mathematical Model

In this work, we studied theoretically the effects of diffusion restrictions and topological factors that could influence the efficiency of energy coupling in the heterogeneous lamellar system of higher plant chloroplasts. Our computations are based on a mathematical model for electron and proton transport in chloroplasts coupled to ATP synthesis in chloroplasts that takes into account the nonuniform distribution of electron transport and ATP synthase complexes in the thylakoids of grana and stroma. Numerical experiments allowed the lateral profiles of pH in the thylakoid lumen and in the narrow gap between grana thylakoids to be simulated under different metabolic conditions (in the state of photosynthetic control and under conditions of photophosphorylation). This model also provided an opportunity to simulate the effects of steric constraints (the extent of appression of thylakoids in grana) on the rates of non-cyclic electron transport and ATP synthesis. This model demonstrated that there might be two mechanisms of regulation of electron and proton transport in chloroplasts: 1) slowing down of non-cyclic electron transport due to a decrease in the intra-thylakoid pH, and 2) retardation of plastoquinone reduction due to slow diffusion of protons inside the narrow gap between the thylakoids of grana. Numerical experiments for model systems that differ with respect to the arrangement of thylakoids in grana allowed the effects of osmolarity on the photophosphorylation rate in chloroplasts to be explained.     

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

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

The kinetics of photoinhibition of the photosynthetic apparatus in pea chloroplasts

Abstract The kinetics of a range of chlorophyll fluorescence parameters, non-cyclic electron transport and the capacity of the thylakoids to bind Atrazine were examined during photoinhibition treatment of intact pea chloroplasts. Parameters of fluorescence induction of chloroplasts in the presence and absence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea at 20 °C and at 77 K were determined. The contributions of photochemical and non-photochemical quenching processes to the loss of fluorescence during photoinhibitory treatment were assessed. Two distinct phases of photoinhibitory damage were observed. During the initial 5 min period of exposure to light the minimal fluorescence level (F_o) increased, whilst the maximal fluorescence level (F_P) decreased, both coupled and uncoupled non-cyclic electron transport to methyl viologen decreased and the ability to bind Atrazine to the thylakoids decreased. Fluorescence analyses demonstrated that during this period thylakoids were becoming increasingly less efficient at generating and maintaining a transmembrane proton electrochemical gradient. Photoinhibitory damage that occurred at later times between 5 and 20 min was of a very different nature. Both F_o and F_P declined, a loss of coupled and uncoupled non-cyclic electron transport was observed together with a loss of the capacity to photo-oxidize water. However, no further loss of Atrazine-binding was associated with such changes. A consistent decrease in the quantum yield of non-cyclic electron transport was also observed throughout photoinhibition treatment. The possibility of two distinct mechanisms of photoinhibitory damage to the photosynthetic apparatus is discussed.     

Inhibition of electron-transport in chloroplasts by a quinone analogue: evidence for two sites of DPIPH2 oxidation

A new inhibitor of photoreactions in chloroplasts, 2,3-dimethyl 5-dybroxy 6-phytol benzoquinone is shown to be an electron transfer inhibitor which blocks both cyclic and non-cyclic electron flow. Basal levels of electron transport from reduced dichlorophenol-indophenol to methyl viologen are not affected by the inhibitor, but uncoupler stimulated electron transport in the same system is inhibited. It is concluded that reduced dichlorophenol-indophenol can be oxidized by the photosynthetic electron transport chain in isolated chloroplasts at two sites: site I proximal to P₇₀₀ and site II distal to P₇₀₀. Site I has a low affinity for the electron donor. Electron flow from this site to methyl viologen does not suppert ATP formation and it is resistant to inhibition by the quinone analogue. Electron donation at site II, located on the linear portion of the electron transport chain between the two photosystems, has a higher affinity for reduced dichlorophenol-indophenol and precedes a phosphorylation site. The electron flow from this site is stimulated by uncouplers and inhibited by the quinone analogue.Abbreviations DPIP 2,6-dichlorophenol indophenol - MeV methyl viologen - DCMU s-(s, t-dichlocophenyl-1,1-dimethylurca - CCP m-chlorocyanocarbonyl phenylthydrazone - DTE dithioerythritol - PMS phenaxine methosulfate - DMHPB 2,3-dimethyl 5-hydroxy 6-phytol benzoquinone Contribution No. 422 from the Charles F. Kettering Research Laboratory. This research supported in part by the National Science Foundation Grant No. G88432.Supported by an NSF Post-doctoral Fellowship No. 49032.     

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

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

Chloroplastic activity in response to gibberellic acid treatment of dwarf and normal cultivars of pea (Pisum sativum L.)

Levels of ferricyanide reduction, cyclic and non-cyclic photophosphorylation were measured in chloroplasts of two cultivars of pea and a comparison of their P/2e⁺ ratios were made. No differences were observed in cyclic photophosphorylation or ferricyanide reduction but non-cyclic photophosphorylation was lower in chloroplasts from the dwarf than the normal cultivar. Thus the P/2e⁺ ratio of the dwarf was lower than the normal. Dwarf seedlings treated with gibberellic acid (GA₃) had similar rates of cyclic photophosphorylation as the untreated dwarf but non-cyclic photophosphorylation was lower as was ferricyanide reduction. This resulted in P/2e⁺ ratios that were higher in chloroplasts from the GA₃ treated dwarf seedlings than the untreated, and were the same as the untreated normal. Addition of GA₃ directly to the chloroplasts did not alter the activity in any way. Hence gibberellins do not directly affect changes in chloroplastic activity but may conceivably be involved in a feed-back control system.     

Photosynthetic Electron Transport,Photophosphorylation, and Antioxidants in Two Ecotypes of Reed (Phragmites Communis Trin.) from Different Habitats

We compared chloroplast photochemical properties and activities of some chloroplast-localised enzymes in two ecotypes of Phragmites communis, swamp reed (SR, C₃-like) and dune reed (DR, C₄-like) plants growing in the desert region of north-west China. Electron transport rates of whole electron transport chain and photosystem (PS) 2 were remarkably lower in DR chloroplasts. However, the electron transport rate for PS1 in DR chloroplasts was more than 90 % of the activity similar in the SR chloroplasts. Activities of Mg²⁺-ATPase and cyclic and non-cyclic photophosphorylations were higher in DR chloroplasts than in the SR ones. The activities of chloroplast superoxide dismutase (SOD) and ascorbate peroxidase (APX), both localised at or near the PS1 complex and serving to scavenge active oxygen around PS1, and the content of ascorbic acid, a special substrate of APX in chloroplast, were all higher in DR chloroplasts. Hence reed, a hydrophytic plant, when subjected to intense selection pressure in dune habitat, elevates its cyclic electron flow around PS1. In consequence, it provides extra ATP required by C₄ photosynthesis. Combined high activities of active oxygen scavenging components in DR chloroplasts might improve protection of photosynthetic apparatus, especially PS1, from the damage of reactive oxygen species. This offers new explanation of photosynthetic performance of plant adaptation to long-term natural drought habitat, which is different from those, subjected to the short-term stress treatment or even to the artificial field drought.     

Inhibition of Hill Reaction and Photophosphorylation by l,l,l-Trichloro-2,2-bis(p-chlorophenyl)ethane

The effect of DDT on DCIP and Fe(CN)₆^3– photoreductions,and cyclic and non-cyclic photo-phosphorylations, in some 30varieties of barley from widely different parts of the worldis reported. Whereas resistant barleys were not affected byDDT treatment, chloroplasts from treated susceptible barleysshowed parallel inhibitions of all the investigated aspectsof photosynthesis. However, in a few susceptible varieties inhibitionsof Fe(CN)₆^3– photoreduction or non-cyclic photophosphorylationwere not so pronounced. Possible reasons for these anomaliesare discussed; in particular earlier reports that DDT had noeffect on these latter photosynthetic activities may have beendue to the use of hypotonic media during chloroplast isolation.     

Electron transport pathways in spinach chloroplasts. Reduction of the primary acceptor of Photosystem II by reduced nicotinamide adenine dinucleotide phosphate in the dark

Addition of NADPH to osmotically lysed spinach chloroplasts results in a reduction of the primary acceptor (Q) of Photosystem II. This reduction of Q reaches a maximum of 50% in chloroplasts maintained under weak illumination and requires added ferredoxin and Mg²⁺. The reaction is inhibited by (i) an antibody to ferredoxin-NADP⁺ reductase (EC 1.6.7.1), (ii) treatment of chloroplasts with N-ethylmaleimide in the presence of NADPH, (iii) disulfodisalicylidenepropanediamine, (iv) antimycin, and (v) acceptors of non-cyclic electron transport. Uncouplers of phosphorylation do not affect NADPH-driven reduction of Q.It is proposed that electron flow from NADPH to Q may occur in the dark by a pathway utilising portions of the normal cyclic and non-cyclic electron carrier sequences. The possible in vivo role for such a pathway in redox poising of cyclic electron transport and hence in controlling the ATP/NADPH supply ratio is discussed.     

Photosynthetic Electron Transport During Senescence of the Primary Leaves of Phaseolus vulgaris L.: II. THE ACTIVITY OF PHOTOSYSTEMS ONE AND TWO, AND A NOTE ON THE SITE OF REDUCTION OF FERRICYANIDE

The activity of photosystems one and two (PS I and PS II) wasmeasured in chloroplasts isolated from the primary leaves ofPhaseolus vulgaris. During foliar senescence, the rates of electrontransport through PS I and PS II declined by approximately 25%and 33% respectively. These losses of activity could not accountfor the decrease of 80% in the rate of coupled, non-cyclic electrontransport during senescence. It is therefore suggested thatan impairment of electron flow between the photosystems limitednon-cyclic electron transport in chloroplasts from older leaves.In this study the activity of PS II was measured using oxidizedp-phenylenediamine as the electron acceptor, and trifluralinas an inhibitor of electron transport between PS II and PS I.In chloroplasts from young leaves the reduction of ferricyanidewas a measure of non-cyclic electron transport, but in preparationsfrom older leaves ferricyanide received a large proportion ofelectrons from PS II.     

Responses of Photosynthetic Electron Transport in Stomatal Guard Cells and Mesophyll Cells in Intact Leaves to Light, CO2, and Humidity

High-resolution images of the chlorophyll fluorescence parameter Fq'/Fm' from attached leaves of commelina (Commelina communis) and tradescantia (Tradescantia albiflora) were used to compare the responses of photosynthetic electron transport in stomatal guard cell chloroplasts and underlying mesophyll cells to key environmental variables. Fq'/Fm' estimates the quantum efficiency of photosystem II photochemistry and provides a relative measure of the quantum efficiency of non-cyclic photosynthetic electron transport. Over a range of light intensities, values of Fq'/Fm' were 20% to 30% lower in guard cell chloroplasts than in mesophyll cells, and there was a close linear relationship between the values for the two cell types. The responses of Fq'/Fm' of guard and mesophyll cells to changes of CO2 and O2 concentration were very similar. There were similar reductions of Fq'/Fm' of guard and mesophyll cells over a wide range of CO2 concentrations when the ambient oxygen concentration was decreased from 21% to 2%, suggesting that both cell types have similar proportions of photosynthetic electron transport used by Rubisco activity. When stomata closed after a pulse of dry air, Fq'/Fm' of both guard cell and mesophyll showed the same response; with a marked decline when ambient CO2 was low, but no change when ambient CO2 was high. This indicates that photosynthetic electron transport in guard cell chloroplasts responds to internal, not ambient, CO2 concentration.     

Studies on the Mechanism of Photoinhibition in Higher Plants: I. EFFECTS OF HIGH LIGHT INTENSITY ON CHLOROPLAST ACTIVITIES IN CUCUMBER ADAPTED TO LOW LIGHT

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: O₂ 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.     

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

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

Electron transport pathways in spinach chloroplasts. Reduction of the primary acceptor of photosystem II by reduced nicotinamide adenine dinucleotide phosphate in the dark.

Addition of NADPH to osmotically lysed spinach chloroplasts results in a reduction of the primary acceptor (Q) of photosystem II. This reduction of Q reaches a maximum of 50% in chloroplasts maintained under weak illumination and requires added ferredoxin and Mg2+. The reaction is inhibited by (I) an antibody to ferredoxin-NADP+ reductases (EC 1.6.7.1), (ii) treatment of chloroplasts with N-ethylmaleimide in the presence of NADPH, (iii) disulfodisalicylidenepropanediamine, (iv) antimycin, and (v) acceptors of non-cyclic electron transport. Uncouplers of phosphorylation do not affect NADPH-driven reduction of Q. It is proposed that electron flow from NADPH to Q may occur in the dark by a pathway utilising portions of the normal cyclic and non-cyclic electron carrier sequences. The possible in vivo role for such a pathway in redox poising of cyclic electron transport and hence in controlling the ATP/NADPH supply ratio is discussed.     

Alkaloids as inhibitors of photophosphorylation in spinach chloroplasts

A group of 12 alkaloids were tested as inhibitors of photophosphorylation in spinach chloroplasts. Ajmaline, a dihydroindole alkaloid, was found to be the strongest inhibitor of both cyclic and non-cyclic photophosphorylation. Low concentrations of ajmaline also inhibited the dark and light ATPases, and the coupled electron flow from water to ferricyanide, measured either as ferrocyanide formed or as oxygen evolved, but not the uncoupled electron transport or the pH rise of illuminated unbuffered suspensions of chloroplasts. Higher concentrations of ajmaline stimulated, instead of inhibiting, photosynthetic electron transport or oxygen evolution and decreased the pH rise, thus behaving as an uncoupler, such as ammonia.Photophosphorylation was partially inhibited by 100 μM dihydrosanguinarine, 100 μM dihydrochelerythrine (benzophenanthridine alkaloids); 500 μM O,O'-dimethylmagnoflorine, 500 μM N-methylcorydine (aporphine alkaloids) and 1 mM julocrotine. They also inhibited coupled oxygen evolution and only partially (dihydrosanguinarine and dihydrochelerythrine) or not at all (the other alkaloids) uncoupled oxygen evolution.Spegazzinine (dihydroindole alkaloid), magnoflorine, N-methylisocorydine, coryneine (aporphine alkaloids), candicine and ribalinium chloride were without effect on photophosphorylation at 500 μM.     

Inhibition of photosynthetic reactions by light

Illumination of isolated intact chloroplasts of Spinacia oleracea L. for 10 min with 850 W m^-2 red light in the absence of substrate levels of bicarbonate caused severe inhibition of subsequently measured photosynthetic activities. The capacity of CO₂-dependent O₂ evolution and of non-cyclic electron transport were impaired to similar degrees. This photoinactivation was prevented by addition of bicarbonate which allowed normal carbon metabolism to proceed during preillumination. Photoinhibition of electron transport was observed likewise upon illumination of intact or broken chloroplasts when efficient electron acceptors were absent. Addition of uncouplers did not influence the extent of inhibition. Studies of partial electron-transport reactions indicated that the activity of both photosystems was affected by light. In addition, the water-oxidation system or its connection to photosystem II seemed to be impaired. Preillumination did not cause uncoupling of photophosphorylation. Chlorophyll-fluorescence data obtained at room temperature and at 77 K are consistent with the view that photosystem-II reaction centers were altered. Addition of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) or 1,4-diazabicyclo(2,2,2)octane to isolated thylakoids prior to preillumination substantially diminished photoinhibition. This result shows that reactive oxygen species were involved in the damage. It is concluded that bright light, which normally does not damage the photosynthetic apparatus, may exert the described destructive effects under conditions that restrict metabolic turnover of photosynthetic energy.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PSI photosystem I - PSII photosystem II     

Effects of uncouplers on Mg2+-dependent fluorescence quenching in isolated chloroplasts

Uncoupling concentrations (about 1 mol l^-1) of desaspidin or carbonyl cyanide-4-trifluoromethoxyphenyl hydrazone reverse the slow light-induced, Mg²⁺-dependent quenching of fluorescence of chlorophyll a in isolated (intact and broken) spinach chloroplasts. Likewise, uncoupling inhibits the light-induced increase of the Mg²⁺ concentration in the stroma of intact chloroplasts, as determined with Eriochrome Blue SE. Addition of higher amounts of the uncouplers to the chloroplasts leads to a slow, light-dependent fluorescence lowering which appears to be promoted by high light intensities and is not reversed in the dark. The reversal of the fluorescence quenching by uncoupling is interpreted to reflect exchange of protons for Mg²⁺ ions at negative sites of the inner thylakoid face, caused by the collapse of the proton gradient across the membrane. The secondary fluorescence lowering caused by high levels of the uncouplers and high light intensities is suggested to be related to an inhibition of non-cyclic photosynthetic electron transport.Abbreviation FCCP carbonyl cyanide-4-trifluoromethoxyphenyl hydrazone     

The site of phosphorylation associated with Photosystem I

1. 1. Small particles prepared from spinach chloroplasts after treatment with digitonin, exhibited Photosystem I reactions, including phosphorylation, at rates as high as those in chloroplasts, whereas electron flow from water to NADP⁺ or ferricyanide through Photosystem II was completely lost. Mediators of cyclic electron flow, such as pyocyanine, or N-methylphenazonium methosulfate in red light, had to be reduced to support photophosphorylation.Diaminodurene at high concentrations catalyzed cyclic phosphorylation under anaerobic conditions without addition of a reductant. In fact, addition of ascorbate gave rise to a marked inhibition which was released by addition of a suitable electron acceptor such as methylviologen.

2. 2. Under aerobic conditions a low O₂ uptake, observed in the presence of diaminodurene, was stimulated several-fold upon addition of methylviologen and was stimulated again several-fold on further addition of ascorbate. The rate of phosphorylation, however, remained the same. The low P/2e ratio obtained under these conditions was not decreased at lower light intensities.

3. 3. These findings suggest a phosphorylation site associated with cyclic electron flow through Photosystem I without participation of the electron carriers of Photosystem II. A non-cyclic electron flow to O₂ can be induced in this system by addition of methylviologen which effectively competes with the electron acceptors of cyclic flow. This non-cyclic electron flow still involves the same phosphorylation site. A scheme for electron transport and for the location of phosphorylation sites in chloroplasts is proposed.