Involvement of plastoquinone and lipids in electron transport reactions mediated by the cytochrome b6-f complex isolated from spinach

The isolation of a cytochrome b₆-f complex from spinach, which is depleted of plastoquinone (and lipid), is reported. The depleted complex no longer functions as a plastoquinol-plastocyanin oxidoreductase but can be reconstituted with plastoquinone and exogenous lipids. The lipid classes digalactosyldiacylglycerol, phosphatidylglycerol and phosphatidylcholine were active in reconstitution while monogalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol were not. Neither plastoquinone nor lipid alone fully reconstitutes electron transport in the depleted complex. Saturation of plastoquinol-plastocyanin oxidoreductase activity in the depleted complex occurs at 1 plastoquinone per cytochrome f.     

The redox state of the plastoquinone pool controls the level of the light-harvesting chlorophyll a/b binding protein complex II (LHC II) during photoacclimation

A cytochrome b ₆ f deficient mutant of Lemna perpusilla maintains a constant and lower level of the light-harvesting chl a/b-binding protein complex II (LHC II) as compared to the wild type plants at low-light intensities. Inhibition of the plastoquinone pool reduction increases the LHC II content of the mutant at both low- and high-light intensities but only at high-light intensity in the wild type plants. Proteolytic activity against LHC II appears during high-light photoacclimation of wild type plants. However, the acclimative protease is present in the mutant at both light intensities. These and additional results suggest that the plastoquinone redox state serves as the major signal-transducing component in the photoacclimation process affecting both, synthesis and degradation of LHC II and appearance of acclimative LHC II proteolysis. The plastoquinol pool cannot be oxidized by linear electron flow in the mutant plants which are locked in a ‘high light’ acclimation state. The cytochrome b ₆ f complex may be involved indirectly in the regulation of photoacclimation via 1) regulation of the plastoquinone redox state; 2) regulation of the redox-controlled thylakoid protein kinase allowing exposure of the dephosphorylated LHC II to acclimative proteolysis. This revised version was published online in June 2006 with corrections to the Cover Date.     

The <Emphasis Type="Italic">pds2</Emphasis> mutation is a lesion in the <Emphasis Type="Italic">Arabidopsis</Emphasis> homogentisate solanesyltransferase gene involved in plastoquinone biosynthesis

Plastoquinone plays critical roles in photosynthesis, chlororespiration and carotenoid biosynthesis. The previously isolated pds2 mutant from Arabidopsis was deficient in tocopherol and plastoquinone accumulation, and the biochemical phenotype of this mutant could not be reversed by externally applied homogentisate, suggesting a later step in tocopherol and/or plastoquinone biosynthesis had been disrupted. Recently, the protein encoded by At3g11950 (AtHST) was shown to condense homogentisate with solanesyl diphosphate (SDP), the substrate for plastoquinone synthesis, but not phytyl diphosphate (PDP), the substrate for tocopherol biosynthesis. We have sequenced the AtHST allele in the pds2 mutant background and identified an in-frame 6 bp (2 aa) deletion in the gene. The pds2 mutation could be functionally complemented by constitutive expression of AtHST, demonstrating that the molecular basis for the pds2 mutation is this 6 bp-lesion in the AtHST gene. Confocal microscopy of EGFP tagged AtHST suggested that AtHST is localized to the chloroplast envelope, supporting the hypothesis that plastoquinone synthesis occurs in the plastid.     

Location,expression and orientation of the putative chlororespiratory enzymes,Ndh and IMMUTANS,in higher-plant plastids

The chloroplasts of many plants contain not only the photosynthetic electron transport chain, but also two enzymes, Ndh and IMMUTANS, which might participate in a chloroplast respiratory chain. IMMUTANS encodes a protein with strong similarities to the mitochondrial alternative oxidase and hence is likely to be a plastoquinol oxidase. The Ndh complex is a homologue of complex I of mitochondria and eubacteria and is considered to be a plastoquinone reductase. As yet these components have not been purified to homogeneity and their expression and orientation within the thylakoid remain ill-defined. Here we show that the IMMUTANS protein, like the Ndh complex, is a minor component of the thylakoid membrane and is localised to the stromal lamellae. Protease digestion of intact and broken thylakoids indicates that both Ndh and IMMUTANS are orientated towards the stromal phase of the membrane in Spinacia oleracea L. Such an orientation is consistent with a role for the Ndh complex in the energisation of the plastid membrane. In expression studies we show that IMMUTANS and the Ndh complex are present throughout the development of both Pisum sativum L. cv Progress No. 9 and Arabidopsis thaliana (L.) Heynh. leaves, from early expansion to early senescence. Interestingly, both the Ndh complex and the IMMUTANS protein accumulate within etiolated leaf tissue, lacking the photosystem II complex, consistent with roles outside photosynthetic electron transport.Abbreviations PQ plastoquinone - PSI, PSII photosystem I, II     

Respiratory control over photosynthetic electron transport in chloroplasts of higher-plant cells: evidence for chlororespiration

Flash-induced primary charge separation, detected as electrochromic absorbance change, the operation of the cytochrome b/f complex and the redox state of the plastoquinone pool were measured in leaves, protoplasts and open-cell preparations of tobacco (Nicotiana tabacum L.), and in isolated intact chloroplasts of peas (Pisum sativum L.). Addition of 0.5–5 mM KCN to these samples resulted in a large increase in the slow electrochromic rise originating from the electrogenic activity of the cytochrome b/f complex. The enhancement was also demonstrated by monitoring the absorbance transients of cytochrome f and b ₆ between 540 and 572 nm. In isolated, intact chloroplasts with an inhibited photosystem (PS) II, low concentrations of dithionite or ascorbate rendered turnover of only 60% of the PSI reaction centers, KCN being required to reactivate the remainder. Silent PSI reaction centers which could be reactivated by KCN were shown to occur in protoplasts both in the absence and presence of a PSII inhibitor. Contrasting spectroscopic data obtained for chloroplasts before and after isolation indicated the existence of a continuous supply of reducing equivalents from the cytosol.Our data indicate that: (i) A respiratory electron-transport pathway involving a cyanide-sensitive component is located in chloroplasts and competes with photosynthetic electron transport for reducing equivalents from the plastoquinone pool. This chlororespiratory pathway appears to be similar to that found in photosynthetic prokaryotes and green algae. (ii) There is an influx of reducing equivalents from the cytosol to the plastoquinone pool. These may be indicative of a complex respiratory control of photosynthetic electron transport in higher-plant cells.Abbreviations and symbols A₅₁₅ flash-induced electrochromic absorbance change at 515 nm - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PS photosystem - SHAM salicylhydroxamic acid     

Spectrophotometric measurement of plastoquinone photoreduction in the blue-green alga,Phormidium uncinatum

The redox state of plastoquinone was measured in vivo in the blue-green alga, Phormidium uncinatum by means of a double beam UV-spectrophotometer. The difference in absorbance of the oxidized and the reduced forms of plastoquinone was amplified, and stored and averaged in a computer. The redox state was changed by two alternating actinic light beams. When one actinic wavelength was kept constant at 700 nm (PSI) variation of the other yielded an action spectrum representing photosystem II. The inhibitors of the photosynthetic electron transport chain, DCMU and DBMIB, reduced the difference in absorbance between the oxidized and reduced forms of plastoquinone.Abbreviations DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea     

State 1-State 2 transitions in the cyanobacterium Synechococcus 6301 are controlled by the redox state of electron carriers between Photosystems I and II

The mechanism by which state 1-state 2 transitions in the cyanobacterium Synechococcus 6301 are controlled was investigated by examining the effects of a variety of chemical and illumination treatments which modify the redox state of the plastoquinone pool. The extent to which these treatments modify excitation energy distribution was determined by 77K fluorescence emission spectroscopy. It was found that treatment which lead to the oxidation of the plastoquinone pool induce a shift towards state 1 whereas treatments which lead to the reduction of the plastoquinone pool induce a shift towards state 2. We therefore propose that state transitions in cyanobacteria are triggered by changes in the redox state of plastoquinone or a closely associated electron carrier. Alternative proposals have included control by the extent of cyclic electron transport around PS I and control by localised electrochemical gradients around PS I and PS II. Neither of these proposals is consistent with the results reported here.Abbreviations DBMIB 2,5-dibromo-3methyl-6-isopropyl-p-benzoquinone - Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DQH₂ duroquinol (tetramethyl-p-hydroquinone) - LHC II light-harvesting chlorophyll a/b-binding protein of PS II - Light 1 light predominantly exciting PS I - Light 2 light predominantly exciting PS II - M.V. methyl viologen - PS photosystem     

Higher plant-like fluorescence induction and thermoluminescence characteristics in cyanobacterium, Spirulina mutant defective in PQH2 oxidation by cytb6/f complex

Characterization of the photosynthetic electron transport in a mutant of Spirulina platensis, generated by chemical mutagenesis, demonstrated that the electron transfer from the plastoquinone (PQ) to cytochrome b6/f was slowed. Thermoluminescence (TL) measurements suggested the presence of reversed energy flow via PQ, which resulted in an emergence of the plant-like after-glow TL band at 45 degrees C that could be enhanced by the transthylakoidal pH gradient and could be eliminated by an uncoupler, FCCP. The localization of the changes in the electron transport of the mutant cells measured by various methods revealed that the re-oxidation of the PQ pool is hampered in the mutant compared to the wild-type cells. The reduction in energy migration was localized between PQ and PS I reaction centers.     

Reconstitution of plastoquinone in the D1/D2/cytochrome b-559 photosystem II reaction centre complex

Reconstitution of plastoquinone in the photosystem II D1/D2/cytochrome b-559 reaction centre complex, in the presence of the detergent Triton X-100, is reported. Illumination of the reconstituted system results in the reduction of cytochrome b-559, the process being partly herbicide-sensitive. In addition, the reconstitution of plastoquinone results in the ability of the isolated reaction centre to catalyse the photoreduction of 2,6-dichlorophenolindophenol in the presence of the exogenous electron donor diphenylcarbazide.     

Molecular analysis of a phytohemagglutinin-defective cultivar of Phaseolus vulgaris L.

Plastoglobuli have been isolated and purified from chloroplasts of beech and spinach leaves and from broom flower chromoplasts by a repeated floating-gradient technique. The main components in plastoglobuli isolated from chloroplasts were triacylglycerols and lipophilic prenyl quinones, mainly plastohydroquinone and -tocopherol. The corresponding oxidized prenyl quinones, plastoquinone (ox), -tocoquinone, and the phylloquinone vitamin K₁, were detected in trace amounts. Plastoglobuli isolated from chromoplasts contained large amounts of carotenoid esters. Triacylglycerols constituted two-thirds of the content of these plastoglobuli. The total prenyl quinone content was low in chromoplast plastoglobuli. Plastoquinone (ox) was the major prenyl quinone constituent. Plastoglobuli contained small amounts of chlorophylls, carotenoids (with the exception of chromoplast plastoglobuli), glycolipids, and proteins due to adsorption phenomena during the isolation process; however, increasing purification of the plastoglobuli fractions resulted in an exponential decline of these components. Adsorption of thylakoid lipids onto the plastoglobuli during the isolation process was demonstrated using an artificial globuli system. Therefore, pigments, glyco- and phospholipids, and proteins were regarded as thylakoid contaminations and not as actual constituents of plastoglobuli.     

Involvement of plastoquinone bound within the isolated cytochrome b6-f complex from chloroplasts in oxidant-induced reduction of cytochrome b6

(1) Oxidant-induced reduction of cytochrome b₆ is completely dependent on a reduced component within the isolated cytochrome b₆-f complex. This component can be reduced by dithionite or by NADH/N-methylphenazonium methosulfate. It is a 2H⁺/2e⁻ carrier with a midpoint potential of 100 mV at pH 7.0, which is very similar to the midpoint potential of the plastoquinone pool in chloroplasts. (2) Oxidant-induced reduction of cytochrome b₆ is stimulated by plastoquinol-1 as well as by plastoquinol-9. The midpoint potential of the transient reduction of cytochrome b₆, however, was not shifted by added plastoquinol. (3) Quinone analysis of the purified cytochrome b₆-f complex revealed about one plastoquinone per cytochrome f. The endogenous quinone is heterogeneous, a form more polar than plastoquinone-A, probably plastoquinone-C, dominating, This is different from the thylakoid membrane where plastoquinone-A is the main quinone. (4) The endogenous quinone can be extracted from the lyophilized cytochrome b₆-f complex by acetone, but not by hydrocarbon solvents. Oxidant-induced reduction of cytochrome b₆ was observed in the lyophilized and hexane-extracted complex, but was lost in the acetone-extracted complex. Reconstitution was achieved either with plastoquinol-1 or plastoquinol-9, suggesting that a plastoquinol molecule is involved in oxidant-induced reduction of cytochrome b₆.     

Participation of plastoquinone,cytochrome c 553 and ferrodoxin-NADP+ oxido-reductase in both photosynthesis and respiration in Spirulina maxima

Dark and light oxidation of NADPH was measured in Spirulina maxima thylakoid membranes. The dark reaction was more cyanide sensitive than the light reaction. In light, 83% of the electrons from NADPH produced H₂O₂ on reducing oxygen, whereas in the dark this number was only 36%. These results are explained by assuming the presence of an electron transport segment common to the photosynthetic and the respiratory chains, so that electrons flowing through the cyanide sensitive oxidase in the dark are diverted to the photosytem (PS) I reaction center (P700). In addition, cytochrome (cyt) c ₅₅₃ was found to be an electron donor for both cyt oxidase and P700. Half maximum reduction rates were obtained with 7 M cyt c ₅₅₃. The intrathylakoidal concentration of cyt c ₅₅₃ was determined to be 83 M. About 60% of the respiratory NADPH oxidation activity was lost by extracting the membranes with pentane and was restored by adding plastoquinone (the main photosythetic quinone). NADPH oxidation activity was also inhibited upon washing the membranes with a low salt buffer. This activity was restored by adding partially purified ferredoxin-NADP⁺ oxido-reductase (FNR). A model for the electron transport in thylakoids, in which cyt c ₅₅₃, plastoquinone and FNR participate in both photosynthesis and respiration is proposed.     

Interplay between photochemical activities and pigment composition in an outdoor culture of Haematococcus pluvialis during the shift from the green to red stage

The transfer of laboratory cultures of H. pluvialis to high irradiance outdoors caused a substantial decline in the maximum quantum yield of photosystem II (PSII), from 0.65 in the morning to 0.45 at midday, as measured by the ratio of variable to maximum fluorescence yields (F_v/F_m), and a steep rise in non-photochemical quenching (NPQ). Chlorophyll fluorescence induction curves of morning samples showed a clear I-step, reflecting a certain PSII heterogeneity. Single turnover flash measurements on samples taken from the outdoor photobioreactor in the middle of day showed an increase in the reoxidation time constant of the reduced plastoquinone Q_A ^–, i.e., the time required for electron transfer from the primary plastoquinone acceptor of PSII Q_A ^– to the secondary plastoquinone acceptor Q_B. Photosynthesis rates were almost constant during the day. Along with the increase in non-photochemical quenching, there was a slight increase in zeaxanthin and antheraxanthin contents and decrease in violaxanthin, showing the presence of an operative xanthophyll cycle in this microalga. A marked increase of secondary carotenoids was found at the end of the first day of exposure to sunlight, mainly astaxanthin monoester, which reached 15.5% of the total carotenoid content. Though cells turned reddish during the second day, the decline in the fluorescence parameter F_v/F_m in the middle of the day was less than during the first day, and there was no further increase in the value for NPQ. Similar behaviour was observed during the third day when the culture was fully red. After four days of exposure to sunlight, the dry weight reached 800 mg L^–1 and the concentration of secondary carotenoids (81% astaxanthin monoester) reached 4.4% dry weight.     

Composition and function of plastoglobuli

The lipid composition of whole leaves and isolated plastoglobul of beech (Fagus sylvatica) has been studied during four natural autumnal senescence stages. Chlorophylls, glycolipids, and phospholipids were extensively degraded in leaves. About 20% of the glycolipids found in leaves during summer, however, remained in the last stage of leaf senescence. Triacylglycerols, also detected in large amounts in summer leaves, were hydrolyzed during senescence. The content of free fatty acids derived from degradation of glycerolipids therefore increased. The total carotenoid and prenyl quinone content was largely unchanged during senescence, except during the last stage investigated, but the reduced forms of prenyl quinones decreased while the oxidized prenyl quinones increased. Plastoglobuli isolated from summer leaves mainly contained triacylglycerols, plastohydroquinone, and -tocopherol. The triacylglycerol content declined in plastoglobuli during senescence. Most of the triacylglycerols must be located outside the plastoglobuli throughout the stages investigated. Carotenoids liberated from thylakoids were esterified and increasingly deposited in plastoglobuli during senescence. In the last senescence stage, carotenoid esters were the main component of plastoglobuli. Prenyl quinones were also transferred into plastoglobuli. Reduced prenyl quinones were sucessively oxidized during senescence and plastoquinone (oxidized) was the predominant prenyl quinone in plastoglobuli isolated from the last senescence stage. The carotenoid and prenyl quinone distribution was identical in leaves and plastoglobuli during late senescence. The main constituents of thylakoids, glycolipids and proteins, were not deposited in plastoglobuli and therefore did not play an important role in plastoglobuli metabolism.Abbreviation PQ plastoquinone     

Characterization of a non-detergent PS II-cytochrome b/f preparation (BS)

A non-detergent photosystem II preparation, named BS, has been characterized by countercurrent distribution, light saturation curves, absorption spectra and fluorescence at room and at low temperature (–196°C). The BS fraction is prepared by a sonication-phase partitioning procedure (Svensson P and Albertsson P-Å, Photosynth Res 20: 249–259, 1989) which removes the stroma lamellae and the margins from the grana and leaves the appressed partition region intact in the form of vesicles. These are closed structures of inside-out conformation. They have a chlorophyll a/b ratio of 1.8–2.0, have a high oxygen evolving capacity (295 mol O₂ per mg chl h), are depleted in P700 and enriched in the cytochrome b/f complex. They have about 2 Photosystem II reaction centers per 1 cytochrome b/f complex.The plastoquinone pool available for PS II in the BS vesicles is 6–7 quinones per reaction center, about the same as for the whole thylakoid. It is concluded, therefore, that the plastoquinone of the stroma lamellae is not available to the PS II in the grana and that plastoquinone does not act as a long range electron transport shuttler between the grana and stroma lamellae.Compared with Photosystem II particles prepared by detergent (Triton X-100) treatment, the BS vesicles retain more cytochrome b/f complex and are more homogenous in their surface properties, as revealed by countercurrent distribution, and they have a more efficient energy transfer from the antenna pigments to the reaction center.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_v variable fluorescence - LHC light-harvesting complex - PpBQ phenyl-p-benzoquinone - PQ plastoquinone pool - P700 reaction center of PS I - PS I, PS II Photosystem I, II - Q_A first bound plastoquinone accepter - RC reaction centre     

Participation of Chloroplasts in Plant Apoptosis

Mitochondria are known to participate in the initiation of programmed cell death (PCD) in animals and in plants. The role of chloroplasts in PCD is still unknown. We describe a new system to study PCD in plants; namely, leaf epidermal peels. The peel represents a monolayer consisting of cells of two types: phototrophic (guard cells) and chemotrophic (epidermal cells). The peels from pea (Pisum sativum L.) leaves were treated by cyanide as an inducer of PCD. We found an apoptosis-enhancing effect of illumination on chloroplast-containing guard cells, but not on chloroplastless epidermal cells. Antioxidants and anaerobiosis prevented the CN^–-induced apoptosis of cells of both types in the dark and in the light. On the other hand, methyl viologen and menadione known as ROS-generating reagents as well as the Hill reaction electron acceptors (BQ, DAD, TMPD, or DPIP) that are not oxidized spontaneously by O₂ were shown to prevent the CN^–-induced nucleus destruction in guard cells. Apoptosis of epidermal cells was potentiated by these reagents, and they had no influence on the CN^– effect. The light-dependent activation of CN^–-induced apoptosis of guard cells was suppressed by DCMU, stigmatellin or DNP-INT, by a protein kinase inhibitor staurosporine as well as by cysteine and serine protease inhibitors. The above data suggest that apoptosis of guard cells is initiated upon a combined action of two factors, i.e., ROS and reduced plastoquinone of the photosynthetic electron transfer chain. As to reduction of ubiquinone in the mitochondrial respiratory chain, it seems to be antiapoptotic for the guard cell.     

Plastoquinol as a singlet oxygen scavenger in photosystem II

It has been found that in Chlamydomonas reinhardtii cells, under high-light stress, the level of reduced plastoquinone considerably increases while in the presence of pyrazolate, an inhibitor of plastoquinone and tocopherol biosynthesis, the content of reduced plastoquinone quickly decreases, similarly to α-tocopherol. In relation to chlorophyll, after 18 h of growth under low light with the inhibitor, the content of α-tocopherol was 22.2 mol/1000 mol chlorophyll and that of total plastoquinone (oxidized and reduced) was 19 mol/1000 mol chlorophyll, while after 2 h of high-light stress the corresponding amounts dropped to 6.4 and 6.2 mol/1000 mol chlorophyll for α-tocopherol and total plastoquinone, respectively. The degradation of both prenyllipids was partially reversed by diphenylamine, a singlet oxygen scavenger. It was concluded that plastoquinol, as well as α-tocopherol is decomposed under high-light stress as a result of a scavenging reaction of singlet oxygen generated in photosystem II. The levels of both α-tocopherol and of the reduced plastoquinone are not affected significantly in the absence of the inhibitor due to a high turnover rate of both prenyllipids, i.e., their degradation is compensated by fast biosynthesis. The calculated turnover rates under high-light conditions were twofold higher for total plastoquinone (0.23 nmol/h/ml of cell culture) than for α-tocopherol (0.11 nmol/h/ml). We have also found that the level of α-tocopherolquinone, an oxidation product of α-tocopherol, increases as the α-tocopherol is consumed. The same correlation was also observed for γ-tocopherol and its quinone form. Moreover, in the presence of pyrazolate under low-light growth conditions, the synthesis of plastoquinone-C, a hydroxylated plastoquinone derivative, was stimulated in contrast to plastoquinone, indicating for the first time a functional role for plastoquinone-C. The presented data also suggest that the two plastoquinones may have different biosynthetic pathways in C. reinhardtii.     

Impaired photosystem I oxidation induces STN7-dependent phosphorylation of the light-harvesting complex I protein Lhca4 in Arabidopsis thaliana

Reduction of the plastoquinone (PQ) pool is known to activate phosphorylation of thylakoid proteins. In the Arabidopsis thaliana mutants psad1-1 and psae1-3, oxidation of photosystem I (PSI) is impaired, and the PQ pool is correspondingly over-reduced. We show here that, under these conditions, the antenna protein Lhca4 of PSI becomes a target for phosphorylation. Phosphorylation of the mature Lhca4 protein at Thr16 is suppressed in stn7 psad1 and stn7 psae1 double mutants. Thus, under extreme redox conditions, hyperactivation of thylakoid protein kinases and/or reorganization of thylakoid protein complex distribution increase the susceptibility of PSI to phosphorylation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Anna Ihnatowicz and Paolo Pesaresi contributed equally to the article.     

The cyclic electron pathways around photosystem I in Chlamydomonas reinhardtii as determined in vivo by photoacoustic measurements of energy storage

The photoacoustic technique was used to measure energy storage by cyclic electron transfer around photosystem I in intact Chlamydomonas reinhardtii cells illuminated with far-red light (>715 nm). The in-vivo cyclic pathway was characterized by investigating the effects of various chemicals on energy storage. Participation of plastoquinone and ferredoxin in the cyclic electron flow was confirmed by the complete suppression of energy storage in the presence of the plastoquinol antagonist 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and the ferredoxin inhibitors/competitors methylviologen, phenylmercuric acetate and p-benzoquinone. Two alternative electron cycles are demonstrated to operate in vivo. One cycle is sensitive to antimycin A, myxothiazol and 2-(n-heptyl)-4-hydroxyquinoline N-oxide (HQNO) and is catalyzed by ferredoxin which reduces plastoquinone through a route involving cytochrome b ₆ and its protonmotive Q-cycle. The other cycle is unaffected by the above-mentioned inhibitors but is sensitive to N-ethylmaleimide (NEM), an inhibitor of the ferredoxin-NADP reductase, and 2-monophosphoadenosine-5-diphosphoribose (PADR), an analogue of NADP, showing that the electron recycling was mediated by NADPH. Possibly, electrons enter the plastoquinone pool through the action of a NAD(P)H dehydrogenase, which is insensitive to classical inhibitors of the mitochondrial NADH dehydrogenase. Loss of energy storage by photosystem-I-driven cyclic electron transfer in farred light was observed only when antimycin A, myxothiazol or HQNO was used in combination with NEM or PADR. Analysis of the light-intensity dependence and the rate of in-vivo cyclic electron transfer in the presence of various inhibitors indicates that the NADPH-dependent electron-cycle is the preferential cyclic pathway in Chlamydomonas cells illuminated with far-red light.Abbreviations A^max maximal photothermal signal - Cyt cytochrome - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - ES photochemical energy storage - FNR ferredoxin NADP⁺ reductase - HQNO 2-(n-heptyl)-4-hydroxyquinoline N-oxide - NEM N-ethylmaleimide - P₇₀₀ reaction-center pigment of PSI - PADR 2-monophosphoadenosine-5-diphosphoribose - pBQ p-benzoquinone - PMA phenylmercuric acetate We are very grateful to Dr. M.-H. Montane (Cadarache, Saint-Paul-lez-Durance, France) for her advice in the electroporation experiments.     

Mechanism of Action of Sulcotrione,a 4-Hydroxyphenylpyruvate Dioxygenase Inhibitor,in Developed Plant Tissues

The herbicides diuron, fluridone, or sulcotrione differently reduced chlorophyll (Chl) and carotenoid (Car) contents. Four days after herbicide treatment, application of sulcotrione resulted in a Chl/Car ratio of 5.88, similar as in untreated controls; diuron resulted in ratio of 5.24, while fluridone induced a greater diminution in Car contents and yielded a final ratio of 7.02. Sulcotrione induced a more rapid decrease than fluridone did in the quantum yield of photosystem 2 (PS2) as monitored by Chl fluorescence. Measurements of DPIP reduction with isolated thylakoids indicated that sulcotrione was a more effective inhibitor of the Hill reaction in cucumber, a herbicide sensitive species, than in maize, a herbicide-insensitive species. These results are consistent with the view that inhibition of electron transport via reduction in plastoquinone contents in plants leads to the major herbicidal effect of sulcotrione in mature green tissues.