Zinc-inhibited Electron Transport of Photosynthesis in Isolated Barley Chloroplasts

In isolated barley chloroplasts, the presence of 2 millimolar ZnSO₄ inhibits the electron transport activity of photosystem II, as measured by photoreduction of dichlorophenolindophenol, O₂ evolution, and chlorophyll a fluorescence. The inhibition of photosystem II activity can be restored by the addition of the electron donor hydroxylamine or diphenylcarbazide, but not by benzidine and MnCl₂. These observations suggest that Zn inhibits electron flow at the oxidizing side of photosystem II at a site prior to the electron donating site(s) of hydroxylamine and diphenylcarbazide. No inhibition of photosystem I-dependent electron transport by 3 millimolar ZnSO₄ is observed. However, with concentrations of ZnSO₄ above 5 millimolar, photosystem I activity is partially inactivated. Washing Zn²⁺-treated chloroplasts partially restores the O₂-evolving activity.     

Effect of metal chelators on thermoluminescence peaks of spinach chloroplasts and photosystem II particles: probing the water oxidation cycle with 8-hydroxyquinoline

Inhibition of photosystem II (PS II) activity by 8-hydroxyquinoline (8-HQ) has been investigated in case of spinach chloroplasts and isolated photosystem II particles using the thermoluminescence technique. In presence of 8-HQ, water to methylviologen (MV) photoreduction in isolated chloroplasts is inhibited while the reduction of dichlorophenol indophenol is inhibited in both chloroplasts as well as in photosystem II particles. The activity can be restored fully by addition of diphenylcarbazide (DPC), suggesting that the donor side of water oxidation complex is affected. The changes in the thermoluminescence peaks indicate that the charge recombination processes involving S2 or S3 states of the Kok's cycle are probably affected by 8-HQ treatment.     

Comparative study of the action of ultraviolet-C and ultraviolet- B radiation on photosynthetic electron transport

The effect of ultraviolet-C (UV-C, mainly 254 nm radiation) and ultraviolet-B (UV-B, 290-320 nm) radiation on the photosynthetic electron transport reactions has been investigated. The rates of Hill activity mediated by ferricyanide and dichlorodimethoxy-p-benzoquinone (DCDMQ) were differently sensitive to UV-C but equally inhibited by UV-B. Replacement of water with diphenylcarbazide was ineffective in restoring the activity of dichlorophenol indophenol (DCPIP) Hill reaction in UV-B treated chloroplasts, but had significant effect in UV-C treated chloroplasts.
Photobleaching of carotenoids in the presence of carbonyl cyanide-m-chlorophenyl-hydrazone, an indicator of the photochemical reaction associated with the reaction centre of photosystem II, was suppressed and is paralleled by the changes in Hill activity only in UV-B-treated chloroplasts. Carotenoid photobleaching occurred even in UV-C treated chloroplasts showing no measurable Hill activity. UV-C and UV-B irradiation diminished variable fluorescence. With UV-B treated, but not with UV-C treated chloroplasts, an increase in the fluorescence yield was observed upon the addition of 3-(3,4-dichIorophenyl)-l,l-dimethylurea (DCMU) and/or Na dithionite.
Photosystem I activity was found to be unaffected by both UV-C and UV-B radiation at the fluences tested. Kinetics of P700 photooxidation and dark reversal in UV treated chloroplasts indicate that only the electron flow from photosystem II to photosystem I is impaired. It is concluded that while UV-B radiation inactivates specifically the photosystem II reaction centre, UV-C radiation acts at plastoquinone, the quencher Q, and the water oxidizing enzyme system.     

Photooxidation reactions of diphenylcarbazide and their DCMU-sensitivity in thylakoids of the blue-green alga Oscillatoria chalybea

Thylakoids of Oscillatoria chalybea are able to split water. The Hill reaction of these thylakoids is sensitive to DCMU. Diphenylcarbazide can substitute for water as the electron donor to photosystem II with these fully functioning thylakoids. However, the diphenylcarbazide photooxidation is completely insensitive to 3-(3,4-dichlorophenyl)-N-N-dimethyl urea (DCMU) at high diphenylcarbazide concentrations. In with Tris-treated Oscillatoria thylakoids the water splitting capacity is lost and diphenylcarbazide restores electron transport through photosystem II as occurs with higher plant chloroplasts. However, also these photoreactions are insensitive to DCMU. If diphenylcarbazide acts in Oscillatoria as an electron donor to photosystem II the result suggests that diphenylcarbazide feeds in its electrons behind the DCMU inhibition site. This in turn indicates that in Oscillatoria the site of inhibition of DCMU is on the donor side of photosystem II.Abbreviations Used DCMU 3-(3,4-dichlorophenyl)-N-N-dimethyl urea - DPC diphenylcarbazide - DCPiP 2,6-dichlorophenol indophenol - TMB tetramethyl benzidine - A-2-sulf anthraquinone-2-sulfonate     

Chemical modification studies of chloroplast membranes. Water oxidation inhibition by diazonium-benzenesulfonic acid

The water-soluble chemical modifier, diazonium benzene-sulfonic acid, significantly inhibited photosystem II-dependent water oxidation (oxygen evolution) when the compound was reacted with chloroplast membranes in the light but not in the dark. The photochemistry of photosystem II was not affected by the diazonium treatment, shown by complete restoration of photosystem II-dependent electron flow from the alternate electron donor diphenylcarbazide.Paralleling the inhibition of oxygen evolution the illuminated chloroplasts bound significantly more diazonium reagent than did chloroplasts treated in the dark. Both the inhibition of oxygen evolution and the increased binding of the diazonium to the membranes were dependent on photosystem II electron flux, which could not be replaced by photosystem I cyclic electron flow. A dark base to acid or acid to base transition resulted in a similar inhibition of water oxidation and increased diazonium binding.The results suggest a membrane conformational change associated with photosystem II electron flow that exposes otherwise buried diazo reactive groups at the external grana membrane surface.     

Dichlorophenylurea-insensitive Reduction of Silicomolybdic Acid by Chloroplast Photosystem II

The photoreduction of silicomolybdate and other heteropoly ions by chloroplasts is insensitive to 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU). Both water and diphenylcarbazide can be used as electron source for the reduction. Three different assays for silicomolybdate reduction are described including oxygen evolution, formation of a reduced heteropoly blue silicomolybdate, or an indirect assay for reduced silicomolybdate by redox indicators, such as ferricyanide or cytochrome c. The effects of detergents and tris washing are consistent with silicomolybdate reduction through photosystem II before the DCMU site. The effects of orthophenanthroline and bathophenanthroline indicate chelator-sensitive sites in photosystem II before the site of DCMU action.     

Effects of DDT on photosynthetic electron flow in Secale species

Following a survey of a range of varieties of rye, mainly Secale cereale, for reaction to DDT, the mode of action of the pesticide in a susceptible variety was studied. Two sites of interaction of DDT with the photosynthetic electron transport chain were demonstrated. The first site of inhibition was on the oxidizing side of photosystem 2, between the sites of electron donation from diphenylcarbazide at pH 6.0 and pH 8.0 in Tris-washed chloroplasts. The second site of DDT inhibition was in the intermediate electron transport chain, and was demonstrated by using dichlorophenol-indophenol and phenyldiamines as electron donors in chloroplasts where electron flow from photosystem 2 was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The sites are distinct from those characteristic of herbicides which affect photosynthetic electron flow.     

Donor function of phosphate ions in photosystem 2

Evidence was obtained for the interaction between the photosystem 2 (PS2) reaction centre (RC) chlorophyll (Chl) P680 and inorganic phosphate, Pi. The light-induced endogenous basal electron transport to ferricyanide in PS2 depended on endogenous Pi. The electron transport in phosphate deficient chloroplasts was absent, and could be resumed upon the addition of exogenous Pi or of the exogenous electron donor, diphenylcarbazide. Some chloroplast Chl molecules were apparently bound with Pi to a complex via the magnesium atom that was detected by the increase in absorbance in the Chl a absorption maximum at 435 nm observed after the consumption of endogenous Pi in the photophosphorylation reactions. The electron paramagnetic resonance (EPR) Signal I, found in the spectra at 77 K after irradiation of frozen samples in chloroplasts poor in endogenous Pi, was the sum of P700+ and P680+ signals. The P680+ signal disappeared after addition of Pi, diphenylcarbazide or diuron to the chloroplasts before freezing. In addition, the EPR doublet signal of the phosphate anion radicals was recorded at 77 K after irradiation in the ethanol solutions of Chl a containing potassium phosphate. The same doublet signal was discovered in the difference EPR spectrum "chloroplasts minus chloroplasts with diuron" at 77 K after irradation. The results are a possible evidence of the participation of phosphate ions in the primary light reactions of PS2. This revised version was published online in June 2006 with corrections to the Cover Date.     

Donor function of phosphate ions in photosystem 2

Evidence was obtained for the interaction between the photosystem 2 (PS2) reaction centre (RC) chlorophyll (Chl) P680 and inorganic phosphate, Pi. The light-induced endogenous basal electron transport to ferricyanide in PS2 depended on endogenous Pi. The electron transport in phosphate deficient chloroplasts was absent, and could be resumed upon the addition of exogenous Pi or of the exogenous electron donor, diphenylcarbazide. Some chloroplast Chl molecules were apparently bound with Pi to a complex via the magnesium atom that was detected by the increase in absorbance in the Chl a absorption maximum at 435 nm observed after the consumption of endogenous Pi in the photophosphorylation reactions. The electron paramagnetic resonance (EPR) Signal I, found in the spectra at 77 K after irradiation of frozen samples in chloroplasts poor in endogenous Pi, was the sum of P700+ and P680+ signals. The P680+ signal disappeared after addition of Pi, diphenylcarbazide or diuron to the chloroplasts before freezing. In addition, the EPR doublet signal of the phosphate anion radicals was recorded at 77 K after irradiation in the ethanol solutions of Chl a containing potassium phosphate. The same doublet signal was discovered in the difference EPR spectrum "chloroplasts minus chloroplasts with diuron" at 77 K after irradation. The results are a possible evidence of the participation of phosphate ions in the primary light reactions of PS2.     

Stabilization of Thylakoid Membranes by Spermine during Stress-induced Senescence of Barley Leaf Discs

The effect of spermine on photochemical activity and polypeptide composition of chloroplasts from barley leaf discs during senescence in the dark was studied. Chloroplast membranes did not show photosystem II activity after spermine treatment when water was the electron donor, but in the presence of diphenylcarbazide, this activity was observed. The diphenylcarbazide-stimulated photoreduction of dichloroindophenol was 3-fold greater in leaf discs incubated for 72 hours in spermine than in water. Photosystem I activity was reduced by about 90% within the first 24 hours in the spermine-treated samples. This reduction, however, was not due to a decrease in the photosynthetic unit size. A preferential loss of polypeptides other than those associated with photosystem II was observed during senescence of the leaf discs in water, but this loss was reduced by spermine. Spermine treatment also prevented the appearance of several additional chlorophyll proteins found in the controls during senescence. The results have been interpreted on the basis of the interaction of spermine with thylakoid membranes resulting in stabilization of membrane function during senescence.     

Sedimentation equilibrium in reacting systems. VII. The temperature-dependent self-association of beta-lactoglobulin A at pH 2.46

The polyene antibiotic filipin inhibits the activities of both photosystem I and photosystem II in maize mesophyll chloroplasts and pea chloroplasts. Maximum inhibition of photosystem II activity was observed at a filipin concentration of about 0.4 mm in maize mesophyll chloroplasts and 1.0 mm in pea chloroplasts. Inhibition of photosystem II activity was temperature dependent, being much less if the antibiotic and chloroplasts were incubated at 0 °C compared to 25 °C. The inhibition of photosystem I activity of both maize mesophyll and pea chloroplasts caused by filipin, could be overcome by the addition of the soluble electron transfer protein, plastocyanin. It is concluded that the inhibition of photochemical activity caused by filipin is a secondary effect resulting from a change in membrane conformation induced by the antibiotic.     

Dependence of fluorescence spectra of chloroplasts from the activity of photosystem 2]

By means of high sensitive spectrofluorometer the fluorescence spectra have been measured of normal chloroplasts and those with blocked photosystem 2 activity due to photoinhibition or treatment with 0.6 M tris-buffer. At room temperature fluorescence spectra of inactivated chloroplasts are similar to the spectrum of normal chloroplasts measured at low light intensity. Under excitation by intense light a decrease of intensity at 685 nm is appeared (about 3-4 times) in the fluorescence spectra of inactivated chloroplasts as compared to the spectrum of normal chloroplasts. The sharp intensity decrease of maxima at 685 and 695 nm (3-4 times) and small decrease at 680 and 730 nm (by 30-50%) are observed in low temperature fluorescence spectra of inactivated chloroplasts. Thus, the damage of photosystem 2 reaction centres is not accompanied by the preferential decrease of the only fluorescence band. The similarity of fluorescence difference spectra of chloroplasts distinguished by the state of photosystem 2 reaction centre, and the complex structure of difference spectra indicate that the variable fluorescence of chloroplasts during the induction is due to the emission of bulk chlorophyll alpha of the photosystem 2.     

Electron donor pool of photosystem II in Tris-washed chloroplasts in a study of low temperature delayed fluorescence

Transient time courses ("induction") and the intensity of thedelayed fluorescence of chlorophyll a (measured between 0.1and 3.9 msec after a 0.9 msec excitation period) were studiedwith a phosphoroscope at temperatures between 40 and –170°Cin Tris-washed chloroplasts. Tris-washing of chloroplasts changed the temperature dependenciesof the induction and the intensity of the delayed fluorescence.From the analysis of the induction each photosystem II reactioncenter appears to be linked to a donor pool which can supplyone electron to the acceptor pool in Tris-washed chloroplasts. An artificial electron donor, diphenylcarbazide affected thedelayed fluorescence above –100°C evidence that electronsare donated to photosystem II in at least two different ways. An electron transport inhibitor, 3-(3',4'-dichlorophenyl)-l,l-dimethylurea,changed the induction of the delayed fluorescence at temperaturesabove –60°C. The temperature dependence of the electron transport in thevicinity of photosystem II was characterized from these results. (Received May 27, 1980; )     

Photosynthesis and growth of Erythrina variegata as affected by water stress and triacontanol

Erythrina variegata Lam. seedlings were grown under water stress (Ψ = -3.2 MPa) and subsequently sprayed with triacontanol (Tria). Water stress significantly reduced shoot growth rate, while roots continued to grow. Content of chlorophyll (Chl) a decreased more than that of Chl b. Water stress also reduced photosynthetic activity of chloroplasts as measured by Chl fluorescence induction. Stress effect was identified at the oxidation site of photosystem (PS) 2 prior to the hydroxylamine donating site and perhaps close to or after the diphenylcarbazide donating site. The loss of O₂ evolving thylakoid polypeptides (33, 23, 17 kDa) and the large (55 kDa) and small (15 kDa) subunits of ribulose-1,5-bisphosphate carboxylase (RuBPC) were found in water stressed seedlings. The reduction in RuBPC activity was accompanied by reduction of CO₂ fixation and stomatal conductance. All photosynthetic parameters were improved by Tria. This revised version was published online in August 2006 with corrections to the Cover Date.     

Photophosphorylation Associated with Photosystem II: I. Photosystem II Cyclic Photophosphorylation Catalyzed by p-Phenylenediamine

Incubation of spinach chloroplast membranes for 90 minutes in the presence of 50 mm KCN and 100 mum HgCl(2) produces an inhibition of photosystem I activity which is stable to washing and to storage of the chloroplasts at -70 C. Subsequent exposure of these preparations to NH(2)OH and ethylenediaminetetraacetic acid destroys O(2) evolution and flow of electrons from water to oxidized p-phenylenediamine, but two types of phosphorylating cyclic electron flow can still be observed. In the presence of 3-(3,4-dichlorophenyl)-1,1'-dimethylurea, phenazinemethosulfate catalyzes ATP synthesis at a rate 60% that observed in uninhibited chloroplasts. C-Substituted p-phenylenediamines will also support low rates of photosystem I-catalyzed cyclic photophosphorylation, but p-phenylenediamine is completely inactive. When photosystem II is not inhibited, p-phenylenediamine will catalyze ATP synthesis at rates up to 90 mumol/hr.mg chlorophyll. This reaction is unaffected by anaerobiosis, and an action spectrum for ATP synthesis shows a peak at 640 nm. These results are interpreted as evidence for the existence of photosystem II-dependent cyclic photophosphorylation in these chloroplast preparations.     

Development of photosynthetic electron transport in Pinus silvestris

Photosynthetic electron transport activity has been measured in chloroplasts isolated from dark-grown seedlings of Pinus silvestris L. and in chloroplasts isolated from seedlings subjected to illumination for periods of up to 48 h. Activities of photosystem 2, photosystem 1 and photosystem 2 plus 1 have been measured. Chloroplasts isolated from dark-grown seedlings showed significant electron transport activity through both photosystems and through the entire electron transport chain from water to NADP. Illumination of the seedlings for only 5 min markedly promoted photosystem 2 activity. The artificial electron donor, diphenylcarbazide. promoted activity in chloroplasts from dark-grown seedlings and in chloroplasts from seedlings illuminated for up to 30 min. In comparison to photosystem 2 and overall electron transport from water to NADP, photosystem 1 activity increased only slightly during illumination. Measurements of electron transport and fluorescence kinetics have confirmed that photosynthetic electron transport capacity is limited on the water splitting side of photosystem 2 in dark-grown seedlings, whereas the primary and secondary electron acceptors of photosystem 2 are fully synthesized and functioning in darkness. Polyethylene glycol must be used as a protective agent when isolating photoactive chloroplasts from secondary needles of conifers. However, the presence of polyethylene glycol, when isolating chloroplasts from dark-grown pine cotyledons, caused a total inhibition of the activity of photosystem 2. The failure of others to show a substantial electron transport activity in chloroplasts from dark-grown Pinus silvestris might depend on their use of polyethylene glycol in the preparation medium and/or on their use of suboptimal reaction conditions for the electron transport measurements.     

Analysis of Rice Mutants by Low Temperature-Derivative Spectrophotometry in Relation to Pigment Compositions and Photochemical Activities

The chloroplasts of five rice (Oryza sativa) mutants examined in the present study possessed the following pigment and activity characteristics as compared with those of normal strain (Nohrin No. 8); a) less chlorophylls (especially, chlorophyll b, the molar ratio of a to b= 6 ～ 41) and less carotenoids but higher ratios of β-carotene to chlorophylls; b) only photosystem 1 particles were obtained by density gradient centrifugation of digitonin-treated mutant chloroplasts while both photosystem 1 and 2 particles were obtained from normal strain chloroplasts; c) the photosystem 2 activities of mutant chloroplasts were lower (48 ～ 81 %) than that of normal chloroplasts while their photosystem 1 activities were 1.9 ～ 2.4 times higher. The derivative absorption spectrum of the normal chloroplasts (a/b= 4) measured at liquid nitrogen temperature showed many small but distinct maxima and minima in the red region in addition to those observable for chlorophylls in solution. These band structures including the French inflection were lost partially in the derivative spectra of three mutants with a/b= 6, 8 and 11 and almost completely in those of the remaining two mutants with a/b= 18 and 41. The lack of such band structures reflecting specific states of chlorophylls in vivo was attributed to the absence of some conformational structures such as those expected in photosystem-2 particles, which are formed by the presence of chlorophyll b and are resistant to the digitonin treatment. Chlorophylls in specific states in such structures were thought to exhibit a higher photosystem 2 activity.     

Tetracaine inhibition of electron transport in pea chloroplasts is coupled to calcium displacement by the local anesthetic

Chloroplasts isolated from pea seadlings grown on water containing 45Ca2+ were treated with local anesthetic tetracaine. Addition of tetracaine inactivated the electron transport activity of donor side photosystem II. This inhibition was accompanied by 45Ca2+ release from the chloroplast membranes as the whole and destroyed by osmotic shock. No such effect was observed when Tris or hydroxylamine were used to inhibit the donor side photosystem II. Upon thermal inactivation of chloroplasts 45Ca2+ release occurred but at temperatures above 80 degrees. The functional role of Ca2+ in photosystem II is discussed.     

A Lipid Requirement for Photosystem I Activity in Heptane-extracted Spinach Chloroplasts

A lipid requirement for photosystem I activity in Spinacia oleracea chloroplasts has been characterized. The transfer of electrons from tetramethyl-p-phenylenediamine through the chloroplast photosystem to viologen dye was used as an assay of photosystem I activity. Activity is diminished by prolonged heptane extraction and is partially restored by readdition of the extracted lipid. Extracted chloroplasts require plastocyanin for maximal restoration of activity. The effect of lipid extract in restoration is partially replaced by triglycerides containing unsaturated, C₁₈ fatty acids. Various potential redox carriers which occur naturally in chloroplasts do not substitute for extracted lipid. Galacto-lipids, sulfolipids, and phospholipids are not involved in the restoration of activity.     

Stabilization of Photosystem II (O(2) Evolution) of Spinach Chloroplasts by Radiation-induced Immobilization

Spinach chloroplasts were immobilized with vinyl monomers by radiation-induced polymerization at low temperature and stored in buffer containing bovine serum albumin. The lifetime of O₂ evolution activity in photosystem II was prolonged remarkably in immobilized chloroplasts. Thermostability of immobilized chloroplasts stored in buffer containing bovine serum albumin was far better than that of immobilized chloroplasts in pure buffer and that of intact chloroplasts. When immobilized chloroplasts were stored in buffer including polyethylene glycol, the lifetime of O₂ evolution activity was longer than for those stored in buffer containing bovine serum albumin.