Cyt b-559 (Fd) Participating in Cyclic Electron Transport in Spinach Chloroplasts: Evidence for Kinetic Connection with the Cyt b6/f Complex

A small fraction of low potential Cyt b-559, amounting to only13% of total Cyt b-559 in spinach chloroplasts, is analyzedwith the help of a highly selective, computer-controlled spectrophotometer,which simultaneously applies 16 pulse modulated narrow bandmeasuring beams with wavelengths in the cytochrome -band (500–600nm) for recordings of time resolved difference spectra. ThisCyt b-559 fraction remains oxidized upon dark incubation withascorbate and is reduced upon illumination. It can be reducedby cyclic PSI in an antimycin A-sensitive reaction or in thecourse of antimycin A-insensitive linear electron transportvia the Cyt b₆/f complex. Reduction by NADPH in the dark requiresferredoxin. Simultaneous recordings of Cyt b-563 and Cyt f revealclose kinetic connection between this Cyt b-559 fraction andthe low potential chain of the Cyt b₆/f complex. These resultsconfirm and extend previous observations of Miyake et al. 1995(Plant Cell Physiol. 36: 743) in maize mesophyll thylakoids,which led to the hypothesis that Cyt b-559 (Fd) occupies theposition of the postulated ferredoxin-plastoquinone reductase(FQR) in cyclic electron transport. (Received March 9, 1999; Accepted May 21, 1999)     

Characterization of factors affecting the activity of photosystem I cyclic electron transport in chloroplasts

PSI cyclic electron transport is essential for photosynthesis and photoprotection. In higher plants, the antimycin A-sensitive pathway is the main route of electrons in PSI cyclic electron transport. Although a small thylakoid protein, PGR5 (PROTON GRADIENT REGULATION 5), is essential for this pathway, its function is still unclear, and there are numerous debates on the rate of electron transport in vivo and its regulation. To assess how PGR5-dependent PSI cyclic electron transport is regulated in vivo, we characterized its activity in ruptured chloroplasts isolated from Arabidopsis thaliana. The activity of ferredoxin (Fd)-dependent plastoquinone (PQ) reduction in the dark is impaired in the pgr5 mutant. Alkalinization of the reaction medium enhanced the activity of Fd-dependent PQ reduction in the wild type. Even weak actinic light (AL) illumination also markedly activated PGR5-dependent PSI cyclic electron transport in ruptured chloroplasts. Even in the presence of linear electron transport [11 mumol O2 (mg Chl)(-1) h(-1)], PGR5-dependent PSI electron transport was detected as a difference in Chl fluorescence levels in ruptured chloroplasts. In the wild type, PGR5-dependent PSI cyclic electron transport competed with NADP+ photoreduction. These results suggest that the rate of PGR5-dependent PSI cyclic electron transport is high enough to balance the production ratio of ATP and NADPH during steady-state photosynthesis, consistently with the pgr5 mutant phenotype. Our results also suggest that the activity of PGR5-dependent PSI cyclic electron transport is regulated by the redox state of the NADPH pool.     

Light-induced redox changes of cytochrome b-559

Dark incubation of spinach or pea chloroplasts with 10 μm carbonylcyanide m-chlorophenylhydrazone (CCCP) had a negligible effect either on the redox state or the redox potential of the high potential form of cytochrome b-559 (cytochrome b-559_hp). A similar result was obtained with spinach chloroplasts on incubation with 3.3 μm carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), but pea chloroplasts showed a decrease of 10–20% in the amount of reduced cytochrome b-559.Light-induced redox changes of cytochrome b-559 were not observed in untreated spinach chloroplasts. In the presence of CCP or FCCP, cytochrome b-559 was photooxidized both in 655 nm actinic light and in far-red light. Addition of the plastoquinone antagonist, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) to CCCP- or FCCP-treated chloroplasts had only a small effect on the photooxidation of cytochrome b-559 in 655 light, but it completely inhibited the oxidation in far-red light.Electron flow from water to 2,3′,6-trichlorophenolindophenol was partly inhibited by CCCP or FCCP, but the degree of inhibition does not appear to be sufficient to account for the photooxidation of cytochrome b-559.The photooxidation of cytochrome b-559 by 655 nm light at liquid nitrogen temperature was not influenced by prior treatment of the chloroplasts at room temperature with CCCP, DBMIB, or CCCP + DBMIB.The results cannot be explained by the presence of two independent pools of cytochrome b-559 in CCCP-treated chloroplasts, one photooxidized by Photosystem II and the other photooxidized by Photosystem I and photoreduced by Photosystem II.     

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; )     

Developmental Changes in Amounts of Thylakoid Components in Plastids of Barley Leaves

Changes in the amounts of several components of the photosyntheticelectron-transport system during greening of etiolated barleyleaves were studied on a "per plastid" basis. P700 and Q_A, whichwere initially absent from etioplasts, appeared 2 h after thestart of illumination in complete complexes of PS I and PS II,respectively. From 6 h, they increased rapidly in amount witha constant stoichiometric ratio of 1:1. Amounts of Cyt f, Cytb₆, Cyt b-559 and FeS, initially present in etioplasts at levelsthat were one-third to half of those in mature chloroplasts,also increased rapidly after 6 h of illumination. The molarratio of Cyt f, Cyt b₆ and Cyt b-559 was the same in etioplastsand in mature chloroplasts, namely 1:2:2. After 4 h of illumination,levels of FeS increased at nearly the same rate as those ofthe PS I complex. The increase in levels of all components wasmarked after 6 h of illumination, probably due to the energysupplied by developing plastids that had just become photosyntheticallycompetent. The results are discussed in relation to the timeof appearance of chlorophyll-protein complexes and photochemicalactivities. ¹ Present address: Department of Botany, Faculty of Science,Kyoto University, Kyoto, 606-01 Japan.     

Differential inhibition by plastoquinone analogues of photoreduction of cytochrome b-559 in chloroplasts

The high-potential form of cytochrome b-559 (b-559 HP) is closely linked to the oxygenic photosystem (photosystem II) but its relation to other redox components of the photosynthetic apparatus, including plastoquinone, is still obscure. We investigated the photoreduction of cytochrome b-559 HP by isolated chloroplasts in the presence of 3 antagonists of plastoquinone, of which, DBMIB (dibromothymoquinone) and DNP-INT (dinitrophenyl ether of iodonitrothymol) are known to inhibit the oxidation of the plastoquinone pool (PQ) by the FeS-cytochrome ƒ/b₆ complex and one, UHDBT (5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole) is known to inhibit the reduction of PQ by Q_B.Q_B is a protein-bound plastoquinone that serves as a two-electron gate for the reduction of PQ. We found that DBMIB and DNP-INT did not inhibit but low concentrations of UHDBT severely inhibited the photoreduction of cytochrome b-559 HP. These results suggest that the electron donor for the reduction of cytochrome b-559 HP was either Q_B or a portion of the PQ pool that was oxidized by a new pathway free of binding sites for DBMIB and DNP-INT.     

Kinetics of chlorophyll fluorescence at 77 K in Chlorella and chloroplasts. Effects of CCCP,ferricyanide and DCMU

The kinetics of chlorophyll fluorescence at 77 K were studied in Chlorella cells and spinach chloroplasts.During a first illumination, the rise is polyphasic with at least three phases. The slowest one is irreversible and corresponds to the cytochrome oxidation.The dark regeneration of half the variable fluorescence is biphasic, the fast phase being inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) both in Chlorella and chloroplasts.The fluorescence rise during a second illumination is still biphasic.Carbonyl cyanide m-chlorophenylhydrazone (CCCP) slows down the fluorescence rise in Chlorella but has no effect on the dark regeneration. It does not affect the fluorescence of chloroplasts.Ferricyanide which oxidizes cytochrome b-559 at room temperature produces a quenching of the variable fluorescence and an acceleration of the fluorescence rise during the first illumination.Our results fit the idea of the heterogeneity of the Photosystem II centers at low temperature.     

Light-dependent fluorescence variations in intact leaves

Transient variations in the fluorescence from intact Phytolaccaamericana leaves after the onset of illumination were measuredunder various light and dark conditions. Dark-adapted leaveswhen illuminated with strong light underwent an intensity variationwith a peak; the fluorescence intensity reaching its peak severalseconds after the onset of illumination then decreasing to asteady level. The peak height relative to the steady level increasedwith the increasing intensity of actinic light. Pre-illuminationof the dark-adapted leaves with strong light caused a markedlowering of the peak. About 20 min of dark incubation was requiredfor the light-adapted leaves to return to the dark-adapted state.All of the action spectra, for the peak, the steady level andthe effect of light in post-illumination to inhibit recoveryto the dark state, showed high bands due to chlorophyll b andcarotenoid absorption and low bands due to chlorophyll a absorption.We concluded that the light absorbed by photosystem 2 is responsiblefor these phenomena. (Received April 21, 1975; )     

Simultaneous measurement of changes in red and blue fluorescence in illuminated isolated chloroplasts and leaf pieces: The contribution of NADPH to the blue fluorescence signal

A newly developed nitrogen laser fluorimeter insensitive to actinic illumination was used to follow simultaneously the light induced changes in red and blue fluorescence of intact isolated spinach chloroplasts and leaf pieces. The recorded variable blue fluorescence was linked to a water soluble component of intact isolated chloroplasts, depended on Photosystem I, and was related to changes in carbon metabolism. From the comparison of changes in intact and broken chloroplasts and from fluorescence spectra under different conditions, it was concluded that the variation in NADPH was the major cause for the changes in blue fluorescence. This study opens a path towards continuous and non-destructive monitoring of NADPH redox state in chloroplasts and leaves.Abbreviations Chl chlorophyll - DHAP dihydroxyacetone phosphate - DLGA DL-glyceraldehyde - FNR ferredoxin-NADP reductase - FWHM full width at half maximum - LED light emitting diodes - OAA oxaloacetate - q_N non-photochemical quenching - PGA 3-phosphoglycerate - Pi inorganic orthophosphate - q_P photochemical quenching - PPFD photosynthetic photon flux density - Q_A primary quinone acceptor of Photosystem II Preliminary results of this work were presented at the First Conference on the Physiology and Biochemistry of high Mountain Plants, 2–3 July 1992, Villar d'Arene, France.     

Simultaneous Appearance of C-550 and Cytochrome b559HP (High Potential Form) in Barley Leaves Greened under Intermittent Light

Cytochrome b_559HP has been detected by spectrochemical assays in plastids of barley leaves greened under intermittent light (flashed leaves). The amount of cytochrome b_559HP in these plastids was nearly 10-fold lower than in normal chloroplasts when the results were expressed on plastid number. The amount of cytochrome b_559HP phototransformable at -170°C was similar, on a C-550 basis, in the plastids of flashed and normal green leaves. The appearance of the 2 components was simultaneous during the greening process under intermittent light and it is suggested that it was parallel to the increase of appressed regions in thylakoids. The illumination of flashed leaves under continuous light for 5 minutes allowed the appearance of a normal Photosystem-II activity, but had no effect either on the cytochrome b_559HP content of the plastids or on the photoreactivity of this component at low temperature.     

Effects of Light Stress on Redox Potential Forms of Cyt b-559 in Photosystem II Membranes Depleted of Water-Oxidizing Complex

Effects of photoinhibition on the redox properties of Cyt b-559were studied with NH₂OH treated PSII membranes, which are depletedof the water-oxidizing complex. The membranes contained threeredox forms (HP-, IP- and LP-forms) of Cyt b-559, with E_m valuesof +435, +237 and +45 mV, respectively. A novel intermediate-potentialform of Cyt b-559 was generated during photoinhibition on thedonor side of PSII: photoinhibitory illumination (7,000 µEm^–2 s^–1) for 1 min induced a 30% decrease in thelevel of the HP-form, with concomitant generation of the intermediate-potential(IP-) form whose E_m value was about +350mV. Prolonged illumination(10 min) resulted in complete loss of the HP-form and an apparentincrease in the level of the IPform. After further photoinhibitorytreatment (60 min), complete loss of the IP'-form was observedand levels of the IP- and LP-forms each increased to about 50%of the total amount of Cyt b-559. Kinetic analysis of thesedata led to the conclusion that the HP-form is converted tothe LP-form via two intermediate-potential forms (IP' and IP),and that IP'-form appears only at the early phase of photoinhibition. (Received March 30, 1994; Accepted February 27, 1995)     

Plastid development in primary leaves of Phaseolus vulgaris: The light-induced development of the chloroplast cytochromes

Summary Etioplasts obtained from the primary leaves of dark-grown bean plants contained cytochromes f, b-559_LP and b-563 in a molar ratio of approximately 1.0:2.0:1.5. On illumination of the plants there was a lag of between 10 and 15 h before these cytochromes increased in amount, but after 48 h they had increased from 6- to 10-fold on a per plastid basis. The presence of cytochrome b-559_HP in the plastids was first detected after 15 h of illumination, which coincided with the commencement of grana formation and the onset of a number of photosynthetic reactions in the greening leaves. After 48 h of illumination the molar ratio for cytochromes f, b-559_HP, b-559_LP and b-563 was 1.0:1.2:2.8:2.6.Agranal chloroplasts formed by the exposure of dark-grown plants to intense light flashes contained high amounts of cytochromes f, b-559_LP and b-563 but cytochrome b-559_HP could not be detected.As the light-induced formation of cytochromes f, b-559_LP and b-563 was substantially inhibited by D-threo chloramphenicol, but not by the L-threo isomer, it seems likely that their formation was dependent on 70S ribosomes. Both chloramphenicol isomers gave plastids which lacked cytochrome b-559_HP.     

A pathway for the reduction of cytochrome b-559 by Photosystem II in chloroplasts

Cytochrome b-559, which is normally reduced in the dark, was oxidized by preillumination in the presence of N-methyl-phenazonium methosulfate with low intensity far-red light. The average half-time for the photoreduction of oxidized cytochrome b-559 by a long actinic flash ranged from 90 to 110 ms. In the presence of 0.25 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea the half-time for the photoreduction increased to 230 ms although the extent of the absorbance increase was unchanged. Under similar conditions inhibition of electron transport by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and the increase in the chlorophyll fluorescence show that a large fraction of the Photosystem II reaction centers are blocked. These results are consistent with the concept that electrons are shared between different photosynthetic units by a common pool of plastoquinone and imply that the principle pathway for the reduction of cytochrome b-559 by Photosystem II occurs through plastoquinone. In the presence of the uncoupler gramicidin which stimulates non-cyclic electron transport, the rate of photoreduction of cytochrome b-559 is slower ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 180}\text{ms}$), from which it is inferred that cytochrome b-559 competes with cytochrome f for electrons out of this pool. Comparison of cytochrome b-559 photoreduction and electron transport rates using untreated and KCN-treated chloroplasts indicate that, under conditions of basal electron transport from water to ferricyanide, approximately one-fifth of the electrons from Photosystem II go through cytochrome b-559 to ferricyanide. Further support for this pathway is provided by a comparison of the effect of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (dibromothymoquinone) on the rates of reduction of cytochrome b-559 and ferricyanide.     

Photoreactions of cytochrome b-559 and cyclic electron flow in Photosystem II of intact chloroplasts

The high potential cytochrome b-559 of intact spinach chloroplasts was photooxidized by red light with a high quantum efficiency and by far-red light with a very low quantum efficiency, when electron flow from water to Photosystem II was inhibited by a carbonyl cyanide phenylhydrazone (FCCP or CCCP). Dithiothreitol, which reacts with FCCP or CCCP, reversed the photooxidation of cytochrome b-559 and restored the capability of the chloroplasts to photoreduce CO₂ showing that the FCCP/CCCP effects were reversible. The quantum efficiency of cytochrome b-559 photooxidation by red or far-red light in the presence of FCCP was increased by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone which blocks oxidation of reduced plastoquinone by Photosystem I. When the inhibition of water oxidation by FCCP or CCCP was decreased by increased light intensities, previously photooxidized cytochrome b-559 was reduced. Red light was much more effective in photoreducing oxidized high potential cytochrome b-559 than far-red light. The red/far-red antagonism in the redox state of cytochrome b-559 is a consequence of the different sensitivity of the cytochrome to red and far-red light and does not indicate that the cytochrome is in the main path of electrons from water to NADP. Rather, cytochrome b-559 acts as a carrier of electrons in a cyclic path around Photosystem II. The redox state of the cytochrome was shifted to the oxidized side when electron transport from water became rate-limiting, while oxidation of water and reduction of plastoquinone resulted in its shifting to the reduced side.     

Kinetics of NADPH-Induced Non-Photochemical Reduction of the Plastoquinone Pool in Spinach Chloroplasts

Kinetics of non-photochemical reduction of the photosynthetic intersystem electron transport chain by exogenous NADPH was examined in osmotically lysed spinach chloroplasts by chlorophyll (Chl) fluorescence measurements under anaerobic condition. Upon the addition of NADPH, the apparent F₀ increased sigmoidally, and the value of the maximal slope was calculated to give the reduction rate of plastoquinone (PQ) pool. Application of 5 µM antimycin A lowered significantly both the ceiling and the rate of the NADPH-induced Chl fluorescence increase, while the suppressive effect of 10 µM rotenone was slighter. This indicated that dark reduction of the PQ pool by NADPH in spinach chloroplasts under O₂-limitation condition could be attributed mainly to the pathway catalysed sequentially by ferredoxin-NADP⁺ oxidoreductase (FNR) and ferredoxin-plastoquinone reductase (FQR), rather than that mediated by NAD(P)H dehydro- genase (NDH).     

Effects of Far-red Light on the Labelling of Acyl Lipids during the Greening of Barley (Hordeum vulgare)

Etiolated Hordeum vulgare (barley) Plants were greened underwhite light or far-red (> 700 nm) light. Exposure to far-redlight inhibited chlorophyll synthesis (especially chlorophyllb) and the development of photosystem II which were seen whengreening took place under white light. Primary leaves were detachedand the labelling of their acyl lipids from [¹⁴C]acetate wasstudied under white light or far-red light illumination. Greeningwith far-red light caused a reduction in the radiolabellingof polyunsaturated fatty acids and diacylgalactosylglycerol.Total fatty acid labelling rates were unaffected. Phosphatidylethanolamine,which was normally poorly labelled, accounted for up to 15 percent of the total radioactivity in acyl moieties of lipids inleaves greened with far-red light. The results are discussedin connection with the role that acyl lipids may play in normalthylakoid structure and function. Hordeum vulgare, barley, acyl lipids, white light, far-red light, chloroplasts, thylakoids     

Ferredoxin and ferredoxin-NADP-oxidoreductase in leaves ofPhaseolus vulgaris L.

During light-induced greening of 10-dayold etiolated bean seedlings a strong increase is observed of ferredoxin (Fd) and of ferredoxin-NADP-oxidoreductase (FNR; E.C. 1.6.99.4) activity, both known to reside in chloroplasts. The production of Fd starts immediately upon illumination and proceeds almost linearly for at least the next 72 h. It is inhibited by chloramphenicol (CAP) but not by cycloheximide (CHI), beit that in the presence of the latter Fd synthesis was impaired after 48 h of illumination. We conclude that for the elaboration of functional Fd in greening chloroplasts protein synthesis on chloroplast ribosomes is required. The increase of FNR activity shows a lag of about 24 h and is blocked by both CAP and CHI. When CAP is applied at 24 h after the illumination started it has no effect. We suggest that the synthesis of FNR on cytoplasmic ribosomes requires prior synthesis of protein(s) on chloroplast ribosomes.The nature of possible interactions between chloroplasts and cytoplasm is discussed.Abbreviations CAP D-threo-chloramphenicol - CHI cycloheximide - DCIP dichlorophenol-indophenol - DEAE diethylaminoethyl - Fd Ferredoxin - FNR ferredoxin-NADP-oxidoreductase - NAR nitrate reductase - NIR nitrite reductase     

Further investigation of the light-induced cytochrome b-559 reaction in membrane fragments of the blue-green alga Anabaena variabilis

Oxidation-reduction reactions of the low redox potential cytochromeb-559 were studied for membrane fragments of the blue-greenalga Anabaena variabilis. Cytochrome photooxidation was observableat room temperature when the membrane fragments had been preincubatedat room temperature in the dark. A CCCP addition (10^–4M) strongly enhanced the reaction. Oxidation consisted of a DCMU-sensitive and an insensitive reaction.The former depended on actinic illumination of short wavelength.The latter showed a dependency on longer wavelength light. Theformer was assumed to be induced by the action of photosystemII and the latter by that of photosystem I. The photosystem II oxidation was small before preincubation,and was enhanced by added DPIP or Ferro. This was interpretedas photosystem II action inducing oxidation as well as reduction;reduction being inactivated during dark incubation or beingsuppressed by added redox reagents which compete for electronacceptance from photosystem II so that oxidation was apparentlyenhanced. The oxidationreduction reactions of this cytochromewith low redox potential were assumed to be almost identicalwith those of the high redox potential form, at least in themembrane fragments of Anabaena variabilis. (Received June 8, 1975; )     

Cytochrome b560 in chromatophores from Chromatium vinosum

A membrane-bound cytochrome of the B-type in Chromatium chromatophores,cytochrome b₅₆₀, was reduced both by flash light activationand continuous illumination in the presence of antimycin atcontrolled ambient redox potentials. The light-minus-dark differencespectra had peaks at 560 and 430 nm, and troughs at 445 and415 nm. The reduction was observed in the ambient redox potentialfrom 400 to about 200 mV. However, below 200 mV, a re-reductionof photooxidized C-type cytochrome superimposed the reductionof cytochrome b₅₆₀ In the absence of antimycin, the reductionwas not observed, suggesting that the reoxidation of cytochromeb₅₆₀ was faster than the reduction. Dark titrations at various pH values showed that E_m7 of thecytochrome b₅₆₀ was about 40 mV and the E_m value was pH-dependent(–60 mV/pH) from pH 6 to 9. Cytochrome b₅₆₀ had a pK ataround pH 9. The content and some properties of cytochrome b₅₆₀ were similarin chromatophores from either photoautotrophically or photoheterotrophicallygrown cells. The possibility of involvement of cytochrome b₅₆₀ in the photosyntheticelectron transfer is discussed. (Received April 19, 1980; )