Carotenoid photobleaching induced by the action, of photosynthetic reaction center II: DGMU-sensitivity

Carotenoid photobleaching in photosynthetic membrane fragmentsof the blue-green alga Anabaena variabilis was studied withspecial reference to DCMU-sensitivity. Carotenoid photobleaching supported by CCCP is strongly enhancedby Ferri, and, at the same time, becomes less sensitive to DCMU(cf. 5). The DCMU-insensitive reaction was found to show characteristicsvery similar to those of DCMU-sensitive reaction in (i) thedependence on the excitation of pigment system II chlorophylla, (ii) the stimulation by CCCP and NaNa and the suppressionby antimycin A, and (iii) the partial dependence on molecularoxygen. In our membrane fragments Ferri was found to act asan electron acceptor for the photosystem II reaction bypassingthe DCMU-sensitive site. We concluded that (i) carotenoid photobleachinginsensitive to DCMU is also driven by reaction center II, and(ii) in the presence of Ferri, Ferri accepts electrons ejectedby reaction center II bypassing the DCMU-sensitive site. (Received January 20, 1977; )     

On the functional site of manganese in photosynthetic electron transport system

Normal Euglena chloroplasts contained 1 atom of Mn per 47±8chlorophyll molecules. The manganese content of chloroplastswas decreased by heat treatment. After complete removal of manganeseby incubation at 45°C for 5 min, Hill activity with DPIPas electron acceptor was abolished, but the activity of DPIPphotoreduction with diphenylcarbazide as electron donor wasunaffected. Hill activity was inactivated by incubating Euglena chloroplastsat alkaline pH. The presence of a high concentration of Trisduring incubation of chloroplasts at an alkaline pH had no additionaleffect on the activity drop. Donor-supported DPIP photoreduction in heated Euglena chloroplasts,as well as the normal Hill reaction in untreated chloroplasts,was inhibited by DCMU, HOQNO and ioxynil which block electrontransport at the reducing side of system II. These reactionswere also inhibited by another group of inhibitors; CCCP, salicylaldoxime,antimycin A and azide, which block electron transport at a sitebetween the electron carriers, Y₁ and Y₂ located on the oxidizingside of system II. Possible sites of inhibition by heat treatment and by inhibitorsand sites for entry of electrons from artificial electron donorsin the photosynthetic electron transport chain, especially inrelation to the functional site of endogenous manganese in chloroplasts,were proposed. (Received October 30, 1971; )     

The Effect of Inhibitors of Photosynthetic and Respiratory Electron Transport on Nitrate Reduction and Nitrite Accumulation in Excised Z. mays L. Leaves

The assimilation of nitrate and nitrite under dark and lightconditions in Zea mays L. leaves was investigated. Nitrate wasassimilated under dark-aerobic conditions. Anaerobiosis stimulatednitrate reduction and nitrite accumulation under dark conditions.Vacuum infiltration of inhibitors of respiratory electron transport,antimycin A and rotenone, stimulated nitrate reduction and nitriteaccumulation under dark-aerobic conditions. Vacuum infiltrationof low concentrations of PCP, DNP and mCCCP depressed nitratereduction and nitrite accumulation under dark-aerobic conditions,whereas, infiltration of higher concentrations stimulated nitratereduction and nitrite accumulation. The greatest level of nitrateand nitrite reduction occurred under light conditions. The inhibitorof photosynthetic electron transport, DCMU, stimulated the accumulationof nitrite in the light, but decreased nitrate reduction. Whenthe inhibitors of respiratory electron transport antimycin Aand rotenone, were supplied together with DCMU in the light,nitrite accumulation was enhanced. Low concentrations of mCCCPdecreased both nitrate reduction and nitrite accumulation underlight conditions when supplied with DCMU. Key words: Nitrate reduction, Nitrite accumulation, Leaves     

Studies on cytochromes in photosynthetic electron transport system I. photoreduction and photo-oxidation of cytochrome b559 by photosystem II in spinach chloroplasts

Light-induced redox-reactions of cytochrome b₅₅₉ in spinachchloroplasts were investigated. Illumination of chloroplastsinduced photoreduction of cytochrorne b₅₅₉ Red light (650 nm)was more effective than far-red light (725 nm), indicating thatthe photoreduction is a photosystem II-mediated reaction. Onaddition of DCMU, the photoreduction was eliminated and a photooxidationof cytochrome b₅₅₉ was observed. The rate of this photooxidationwas faster with photosystem II light than with photo-systemI light. On addition of Mn⁺⁺ the photooxidation was partly suppressed;far-red light became as effective as red light in inducing photooxidationof cytochrome b₅₉₉, in the presence of DCMU and Mn⁺⁺. Ascorbate completely suppressed photooxidation of cytochromeb⁵⁵⁹ In the presence of ascorbate, however, photooxidation wasobserved in the presence of inhibitors or after inhibitory treatmentsof chloroplasts which affected the oxidizing side of systemII. These inhibitors and inhibitory treatments, but not DCMU,decreased the redoxpotential of cytochrome b₅₅₉. Reactivationof Hill reaction in Tris-washed chloroplasts by indophenol-ascorbatetreatment was not accompanied by an abolishment of photooxidationof cytochrome b₅₅₉. A possible mechanism is proposed to account for these reactionsof cytochrome b₅₅₉ in the photosynthetic electron transportin chloroplasts. (Received April 4, 1972; )     

Inhibition site of the electron transport system in lettuce chloroplasts by fumigation of leaves with SO2

In chloroplasts isolated from SO₂-fumigated leaves at 2.0 ppm,electron flow from water to 2,6-dichloroindophenol (DCIP) wasinhibited, but the electron flow from reduced DCIP to methylviologen was not affected. Neither diphenylcarbazide nor MnCl₂could restore the activity of the DCIP-Hill reaction of SO₂-injuredchloroplasts. Electron flows, from water to ferricyanide orto silicomolybdic acid, were inhibited in a degree similar tothat of the DCIP-Hill reaction. The rate of carotenoid photobleaching in the presence of carbonylcyanide-m-chlorophenylhydrazone was suppressed and paralleledthe inhibition of the DCIP-Hill reaction. In SO₂-injured chloroplasts, the variable part of the fluorescencetransient was diminished, and the fluorescence yield loweredby SO₂ was increased with 3-(3', 4'-dichlorophenyl)-l, l-dimethylurea(DCMU) or more pronouncedly by incubating the sample with sodiumdithionite. However, the yield could not recover to the levelfound in non-fumigated chloroplasts. With SO₂ fumigation, thetime required to reach steady-state level of fluorescence becamelonger in the absence of DCMU, but was not altered in the presenceof DCMU. The pool size of the primary electron acceptors decreasedwith SO₂ fumigation. We concluded that SO₂ inactivated the primaryelectron donor or the reaction center itself. The mode of SO₂action in the electron transport chain is discussed. (Received October 20, 1979; )     

Studies on the pathway of cyclic electron flow in mesophyll chloroplasts of a C4 plant

1. Cyclic photophosphorylation driven by white light, as followed by ¹⁴CO₂ fixation by mesophyll chloroplast preparations of the C₄ plant Digitaria sanguinalis, was specifically inhibited by disalicylidenepropanediamine (DSPD), antimycin A, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIb), 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDAC), and KCN suggesting that ferredoxin, cytochrome b₅₆₃, plastoquinone, cytochrome f, and plastocyanin are obligatory intermediates of cyclic electron flow. It was found that 0.2 μM DCMU and 40 μM o-phenanthroline blocked noncyclic electron flow, stimulated cyclic photophosphorylation, and caused a partial reversal (40–100%) of the inhibition by DBMIB and antimycin A, but not DSPD.

2. Cyclic photophosphorylation could also be activated using only far-red illumination. Under this condition, however, cyclic photophosphorylation was much less sensitive to the inhibitors DBMIB, EDAC and antimycin A, but remained completely sensitive to DSPD and KCN. Inhibition in far-red light was not increased by preincubating the chloroplasts with the various inhibitors for several minutes in white light.

3. The striking correspondence between the effects of photosystem II inhibitors, DCMU and o-phenanthroline, on cyclic photophosphorylation under white light and cyclic photophosphorylation under far-red light (in the absence of photosystem II inhibitors) suggests that electrons flowing from photosystem II may regulate the pathway of cyclic electron flow.     

Studies on the Hill reaction of membrane fragments of blue-green algae IV. Garotenoid photobleaching induced by photosystem II action

Carotenoid photobleaching induced by the action of photosystemII was investigated with membrane fragments of Anabaena cylindricaand A. variabilis. Carotenoid photobleaching occurred only when ferricyanide and/orCCCP was present in the reaction mixture. Maximum velocity ofthe reaction was obtained in the presence of ferricyanide andCCCP. Difference spectra (light minus dark) indicated that afast carotenoid photobleaching was accompanied by a slow chlorophyllbleaching. The pattern of the difference spectra was identicalto that reported by Yamashita et al. (1) with spinach chloroplasts.The reaction was DCMU-sensitive, though a portion of the activitywas insensitive to DCMU when ferricyanide was present in thereaction mixture. The effect of CCCP on stimulation of carotenoidphotobleaching showed the same function against CCCP concentrationas that on inhibition of DPIP-photoreduction with DPC. Carotenoidphotobleaching was stimulated by ferricyanide and suppressedby ferrocyanide; thereaction rate was reduced fifty percentwith a ferri- and ferrocyanide mixture giving 510 mV redox potential(pH 7.5). Benzoquinone was stimulatory, but DPIP had no effect. Incubation of membrane fragments in a dilute buffer inactivatedthe Hill reaction but neither the DPC-supported DPIP-photoreductionnor carotenoid photobleaching in A. variabilis. In A. cylindrica,incubation inactivated all three reactions. Inactivation ofthe latter two reactions followed the same kinetics. (Received August 31, 1972; )     

Regulation of the cytosolic adenylate ratio as determined by rapid fractionation of mesophyll protoplasts of oat

Adenylate levels in chloroplasts, mitochondria and the cytosol of oat mesophyll protoplasts were determined under light and dark conditions, in the absence and presence of plasmalemma-permeable inhibitors of electron transfer and uncouplers of phosphorylation. This was achieved using a microgradient technique which allowed an integrated homogenization and fractionation of protoplasts within 60 s (Hampp et al. 1982, Plant Physiol. 69, 448–455), under conditions which quench bulk activities of metabolic interconversion in less than 2 s. In illuminated controls, ATP/ADP ratios were found to be 2.1 in chloroplasts, about unity in mitochondria, and 11 in the cytosol; whereas, in the dark, this ratio only showed a large drop in chloroplasts (0.4). None of the compounds used [carbonylcyanide m-chlorophenylhydrazone (CCCP), carbonylcyanide p-trifluoromethoxy-phenylhydrazone (FCCP), antimycin A, dibromothymoquinone (DBMIB), dichlorophenyldi-methylurea (DCMU), or salicylhydroxamic acid (SHAM)] affected the stroma adenylate ratio in the dark. Under illumination, however, the ATP/ADP ratios were partly reduced in the presence of antimycin (inhibitor of cyclic photophosphorylation) and of DCMU (inhibitor of linear electron flow), while in the presence of DBMIB, DCMU+ antimycin (inhibition of both cyclic and linear electron flow), and CCCP (uncoupling) the ratio obtained was the same as that occurring in the dark. In contrast, mitochondrial adenylate levels did not exhibit large variations under the various treatments. The cytosolic ATP/ADP ratio, however, showed dramatic changes: in darkened protoplasts, cytosolic values dropped to 0.2 and 0.1 in the presence of uncouplers and antimycin, respectively, while SHAM did not induce any significant alteration. In the light, a similar pronounced decrease in ATP levels was observed only after the application of uncouplers or inhibitors of both mitochondrial and photosynthetic electron transport, whereas selective inhibition of the latter was largely ineffective in reducing the cytosolic ATP/ADP ratio. Thus, the results show that the antimycin-sensitive electron transport is, potentially, equally active in light and darkness. In addition, they indicate that antimycin-insensitive electron transport in mitochondria (alternative pathway) does not significantly contribute to the cytosolic energy state.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DBMIB dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropy-p-benzoquinone) - DCMU dichlorophenyldimethylurea - FCCP carbonylcyanide-p-trifluoromethoxy-phenylhydrazone - SHAM sancylhydroxamic acid     

On the possible site of inhibition of photosynthetic electron transport by ultraviolet-B (UV-B) radiation

In Amaranthus chloroplasts that are exposed to ultraviolet-B (UV-B) radiation, the electron flow from water to dichlorophenol indophenol (DCPIP) was inhibited, but the electron flow from reduced DCPIP to methyl viologen remains unaffected. Diphenylcarbazide was ineffective in restoring the activity of DCPIP Hill reaction in UV-B irradiated chloroplasts. Electron flow from water to ferricyanide or dichloro-dimethoxy- p -benzoquinone was inhibited to a degree similar to that of the DCPIP Hill reaction.
The rate of carotenoid photobleaching in the presence of carbonyl cyanide- m -chlorophenylhydrazone, an indicator of the photochemical reaction near the vicinity of reaction centre of photosystem II, was suppressed and paralleled with the inhibition of the DCPIP Hill reaction.
In the UV-B treated chloroplasts, the variable part of the fluorescence transient was diminished. Though the fluorescence yield was lowered by the UV-B radiation, addition of 3-(3,4-dichlorophenyl)-l, l-dimethylurea (DCMU) and/or sodium dithionite increased the emission markedly. With the increase in the dosage of UV-B irradiation, the time required to reach the steady state fluorescence level became longer in the absence of DCMU and shorter in the presence of DCMU. The kinetics of 520 nm absorbance change was markedly unaltered by the UV-B irradiation but its dark decay was prolonged. It is concluded that UV-B irradiation inactivates the photosystem II reaction centre.     

Studies on the Role of Photosynthesis in the Photoperiodic Induction of Flowering in the Short-Day Plants Kalanchoe blossfeldiana Poellniz and Xanthium pensylvanicum Wallr: II. THE EFFECT OF CHEMICAL INHIBITORS OF PHOTOSYNTHESIS

The role of photosynthesis in flower induction in the short-dayplants Kalanchoe blossfeldiana and Xanthium pensylvanicum wasinvestigated by chemical suppression of photosynthesis and preventionof chlorophyll formation in the induced leaf. ‘Bleaching’leaves with streptomycin completely prevented flowering in X.pensylvanicum at concentrations shown to reduce the chlorophylland carotenoid content of the leaf significantly. Such leaveswere unable to induce flowering even when supplied with sugarsand other photosynthetic products. Photosystem II inhibitors,DCMU and cadmium ion, inhibited induction in both species aswell as suppressing photosynthesis (as tested by O₂ evolutionand starch production) whereas the photosystem I inhibitor,metronidazole, had no effect. Antimycin A inhibited floweringin K. blossfeldiana and may have a similar site of action toDCMU. Neither ammonium ion nor DBMIB, which acts upon plastoquinone(i.e. between PS I and PS II in the ‘Z scheme’),had any effect on floral induction and it is argued that theinductive process is independent of photosynthetic phosphorylationbut a step in the electron transport pathway between the sitesof action of DCMU and DBMIB may be crucial. DSPD and its hydrolysisproduct, salicylaldehyde, suppressed flowering in K. blossfeldianabut the uncertainty regarding their chemistry precludes anyfirm conclusions regarding the nature of their action.     

Two molecular forms of pea ferredoxin in the electron transport chain of chloroplasts

The effects of two molecular forms of water-soluble ferredoxin (Fd I and Fd II) on the kinetics of electron transport in bean chloroplasts (class B) were studied. The light-induced redox transitions of the photosystem I reaction center P700 were measured by the intensity of the EPR signal I produced by P700+. Both forms of ferredoxin, Fd I and Fd II, when added to the chloroplasts in catalytic amounts, stimulate the light-induced electron transfer from P700 to NADP+. Nevertheless, Fd I is a better mediator of the back reactions from NADPH to P700+. This electron transfer pathway is sensitive to the cyclic electron transport inhibitor, antimycin A, and to DCMU inhibitor of electron transport between photosystem II and plastoquinone. It may be concluded that the two molecular forms of ferredoxin, Fd I and Fd II, differ in their ability to catalyze cyclic electron transport in photosystem I. The role of Fd I and Fd II in regulation of electron transport at the acceptor site of photosystem I is discussed.     

The site of manganese function in photosynthetic electron transport system

The effects of Mn²⁺ on aerobic photobleaching of carotenoids, on photoreduction of 2,6-dichlorophenolindophenol (DCIP) and on fluorescence above 600 mμ of spinach chloroplasts washed with 0.8 M Tris-HC1 buffer were investigated. Carotenoids (mostly carotenes, lutein and violaxanthin) in the Tris-washed chloroplasts were irreversibly bleached by illumination with red light, while carotenoids in normal chloroplasts prepared with a low concentration of Tris-HC1 underwent no bleaching upon illumination. The photobleaching of carotenoids observed with Tris-washed chloroplasts was inhibited by Mn²⁺ (MnCl₂ or MnSO₄) as well as by some inhibitors of the Hill reaction such as dichlorophenyl-1,1-dimethylurea (DCMU), methylthio-4,6-bis-isopropylamino-s-triazine and o-phenanthroline or by reducing agents such as ascorbate plus tetramethyl-p-phenylene diamine (TMPD). DCIP photoreduction, which was deactivated by Tris, was reactivated to 50–80% of the rate for normal chloroplasts upon addition of Mn²⁺. The restored photoreduction of DCIP was inhibited by DCMU and carbonylcyanide m-chlorophenylhydrazone (CCCP). The steady-state fluorescence yield of normal chloroplasts measured at room temperature was lowered by Tris treatment, and the decreased yield was restored by adding Mn²⁺ as well as ascorbate plus TMPD. CCCP also lowered the yield; the yield was recovered by adding ascorbate plus TMPD. Determination of manganese in normal and Tris-washed chloroplasts showed that 30% of the manganese in chloroplast was removed with Tris. It was postulated that Mn²⁺ functions in the electron transport on the oxidizing side of Photosystem II at a site between water and an electron carrier (Y). CCCP as well as Tris inhibits the reduction of Y⁺ by Mn²⁺, and carotenoids are oxidized by Y⁺ which is reduced by ascorbate plus TMPD.     

Photodestruction of Pigments in Higher Plants by Herbicide Action: I. THE EFFECT OF DCMU (DIURON) ON ISOLATED CHLOROPLASTS

3-(3',4'-Dichlorophenyl)-1',1'-dimethyl urea (DCMU) inducedthe photobleaching of chlorophylls and carotenoids in isolatedchloroplasts of Hordeum vulgare. In chloroplasts illuminatedin both the absence and presence of DCMU (5.0 mmol m^–3),the destruction of carotenoid preceded that of the chlorophylls.The rate of photodestruction was accelerated by the presenceof DCMU. After only 2 h illumination the rates of loss of ß-caroteneand of the epoxyxanthophylls, neoxanthin and violaxanthin, weresimilar (approximately 40–50% loss in the presence of5–0 mmol m^–3 DCMU) but weremuch greater than thatof lutein (25% loss). Analysis of the individual pigment-proteincomplexes, isolated from chloroplasts following such treatment,showed that whilst pigment destruction had occurred in all complexes,the relative content of the LHCP2/CPa complexes (containingthe PSII core) had fallen to the greatest extent. Further illuminationof the chloroplasts, for up to 22 h, resulted in far greaterbleaching but showed a similar pattern of pigment loss, withDCMU again accelerating the rate at which this loss occurred.ß-Carotene-5,6-epoxide was identified as a productof such photo-oxidative conditions. Key words: DCMU, carotenoids, chlorophylls, photobleaching, ß-carotene-5,6-expoxide     

Photoactivation of the Electron Flow from NADPH to Plastoquinone in Spinach Chloroplasts

Intact chloroplasts from spinach showed a transient increasein Chl fluorescence after saturating illumination with actiniclight and its yield depended on the duration of illuminationand the intensity of the actinic light (AL). The increase waspartially suppressed when antimycin A was added immediatelyafter termination of the AL. The inhibited fluorescence increase,therefore, reflected the electron flow from the reductant(s)that had accumulated during the actinic illumination to theplastoquinone (PQ) pool via ferredoxin and the antimycin A-sensitiveCyt b-559 [Miyake et al. (1995) Plant Cell Physiol. 36: 743].Addition of dihydroxyacetone phosphate (DHAP) to chloroplastscaused the enhancement of the increase in fluorescence afterAL, which was inhibited by antimycin A. Decay of the transientlyraised fluorescence was retarded by 2-heptyl-4-hydroxyquinolineN-oxide and stigmatellin, suggesting that re-oxidation of thereduced PQ pool is coupled with the operation of Q-cycle. Althoughthe activity of the stromal enzyme system that supplies NADPHon addition of DHAP was constant irrespective of light or darkness,the capacity of the intact chloroplasts to show a DHAP-dependentfluorescence increase had a limited lifetime after AL was turnedoff. This result suggests that the antimycin A-sensitive Cytb-559 or ferredoxin-NADP reductase is activated by light anddeactivated in the dark. In ruptured chloroplasts, the additionof NADPH increased the dark fluorescence yield only in the presenceof Fd, which also was inhibited by antimycin A. Thus the photoregulatorymechanism of Cyt b-559 (Fd) in intact chloroplasts appearedto be lost when chloroplasts were ruptured. (Received June 21, 1995; Accepted September 25, 1995)     

Regulation of Cyclic Photophosphorylation during Ferredoxin-Mediated Electron Transport : Effect of DCMU and the NADPH/NADP Ratio

Addition of ferredoxin to isolated thylakoid membranes reconstitutes electron transport from water to NADP and to O₂ (the Mehler reaction). This electron flow is coupled to ATP synthesis, and both cyclic and noncyclic electron transport drive photophosphorylation. Under conditions where the NADPH/NADP⁺ ratio is varied, the amount of ATP synthesis due to cyclic activity is also varied, as is the amount of cyclic activity which is sensitive to antimycin A. Partial inhibition of photosystem II activity with DCMU (which affects reduction of electron carriers of the interphotosystem chain) also affects the level of cyclic activity. The results of these experiments indicate that two modes of cyclic electron transfer activity, which differ in their antimycin A sensitivity, can operate in the thylakoid membrane. Regulation of these activities can occur at the level of ferredoxin and is governed by the NADPH/NADP ratio.     

The relationship between state II to state I transitions and cyclic electron flow around photosystem I

The effects of electron acceptors, inhibitors of electron flow and uncouplers and inhibitors of photophosphorylation on a state II to I transition were studied. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) did not inhibit the state II to I transition. By contrast, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), methyl viologen and antimycin A inhibited the transition indicating that the cyclic electron flow around photosystem I, but not the oxidation of electron carriers (such as plastoquinone), induced the state II to I transition. Uncouplers, but not inhibitors of photophosphorylation, inhibited the state transition suggesting that the proton transport through the cyclic electron flow was related to the transition.     

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.     

Effects of dibromothymoquinone and bathophenanthroline on flash-induced cytochrome f oxidation in spinach chloroplasts

The effects of several electron transport inhibitors on themagnitude and kinetics of cytochrome f oxidation induced byflash illumination were studied in the - and -band regions.On the flash excitation only a fraction of cytochrome f presentin the chloroplasts was oxidized with a half time of 0.1 to0.3 msec and then reduced with a half time of 10 to 25 msec. Dibromothymoquinone (DBMIB) at concentrations which severelysuppressed the reduction of cytochrome f approximately doubledthe magnitude of cytochrome f oxidation caused by a flash, mainlyby inducing an additional slow oxidation of cytochrome f witha half time longer than 1 msec. Enhancement of the cytochromef oxidation was also observed in the presence of bathophenanthroline.Such enhanced oxidation in duced by the two inhibitors was largelydiminished with the addition of reduced 2,6-dichlorophenolindophenolwhich accelerated cytochrome f reduction. In contrast, the inhibitionof cytochrome f reduction by 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU) was not associated with an increase in the magnitudeof cytochrome f oxidation. However, addition of DBMIB to theDCMU-poisoned chloroplasts enhanced cytochrome f oxidation,suggesting that this is related to a block of the electron transportbetween plastoquinone and cytochrome f. The results are explainedby assuming the occurrence of an electron carrier between plastoquinoneand cytochrome f. (Received May 10, 1978; )     

Stimulation of Photosystem I Electron Transport by High Concentration of 3-(3,4-Dichlorophenyl)-1,1-dimethyl Urea in Uncoupled Chloroplasts

The light saturated rate of photosystem I-dependent electron transport (ascorbate/dichlorophenol-indophenol → methyl vilogen in presence of 1 micromolar 3-[3,4-dichlorophenyl]-1,1-dimethyl urea [DCMU]) was increased by a high concentration of DCMU added to broken and uncoupled chloroplasts isolated from pea (Pisum sativum). At 50 micromolar DCMU, the increase was around 50%. No stimulation was observed under limiting intensity of illumination, indicating that the relative quantum yield of electron transport was not affected by high DCMU. The light-saturated rate in coupled (to proton gradient formation) chloroplasts was unchanged by 50 micromolar DCMU, suggesting that the rate-limitation imposed by energy coupling was not affected. Using N,N,N′,N′-tetramethyl-p-phenylene diamine as electron donor, essentially no DCMU stimulation of the rate was observed, indicating further that the electron donation at a site close to P700 was not affected by high DCMU. It is concluded that DCMU, in the range of 10 to 50 micromolar, affected the thylakoid membranes in such a way that the rate constant of electron donation by dichlorophenol-indophenol at the site prior to the site of energy coupling increased. Further observations that DCMU at 100 micromolar stimulated the rate in coupled chloroplasts indicated an additional DCMU action, presumably by uncoupling the chloroplasts from phosphorylation, as suggested by Izawa (Shibata et al., eds, Comprehensive Biochemistry and Biophysics of Photosynthesis, University Press, State College, Pennsylvania, pp 140-147, 1968). A scheme has been proposed for multiple sites of DCMU action on the electron transport system in chloroplasts.     

Ageing of Euglena chloroplasts in vitro I. Variations in pigment pattern and in morphology

Isolated chloroplasts of Euglena gracilis Klebs were kept for10 d in complete darkness at 4 C in a maintenance buffer (pH7.5) without shaking. During incubation, the qualitative andquantitative changes in the pattern of photosynthetic pigmentswere evaluated by the combined use of spectrophotometry in thevisible range of whole chloroplasts and their acetone extracts,of in vivo spectrofluorimetry and of reversed-phase HPLC. Microscopicand submicroscopic modifications were also followed by UV andtransmission electron microscopy. The main findings were as follows: (1) a fast decay of all photosyntheticpigments, chiefly chlorophylls, not accompanied by evident signsof alteration of the thylakoid system during the first 5 d;(2) a higher stability of PSII compared to PSI and of antennacomplexes compared to the relative reaction centres during thefirst 24–48 h; (3) a low accumulation of phaeoderivativecompounds in spite of the marked decrease of chlorophyll content;(4) a lack of dephytylated compounds; (5) a quicker decay ofthe intensity of fluorescence emission with respect to the decreasingchlorophyll a content; and (6) a fast degradation of xanthophyllsand ß-carotene with the consequent lack of defencefrom the ageing oxidative stresses. This accounts for the rapidloss of pigments, although the lack of other antioxidant defencemechanisms is not excluded. The characterization of some of the steps involved in plastiddegradation may render this experimental model viable for furtherstudies on plastid senescence, a multifactorial process stillawaiting definite answers. Key words: Euglena gracilis, ageing, isolated chloroplasts, morphological changes, pigment degradation