Plastocyanin: Structural and functional analysis

Plastocyanin is one of the best characterized of the photosynthetic electron transfer proteins. Since the determination of the structure of poplar plastocyanin in 1978, the structure of algal (Scenedesmus, Enteromorpha, Chlamydomonas) and plant (French bean) plastocyanins has been determined either by crystallographic or NMR methods, and the poplar structure has been refined to 1.33 Å resolution. Despite the sequence divergence among plastocyanins of algae and vascular plants (e.g., 62% sequence identity between theChlamydomonas and poplar proteins), the three-dimensional structures are remarkably conserved (e.g., 0.76 Å rms deviation in the C positions between theChlamydomonas and poplar proteins). Structural features include a distorted tetrahedral copper binding site at one end of an eight-stranded antiparallel -barrel, a pronounced negative patch, and a flat hydrophobic surface. The copper site is optimized for its electron transfer function, and the negative and hydrophobic patches are proposed to be involved in recognition of physiological reaction partners. Chemical modification, cross-linking, and site-directed mutagenesis experiments have confirmed the importance of the negative and hydrophobic patches in binding interactions with cytochromef and Photosystem I, and validated the model of two functionally significant electron transfer paths in plastocyanin. One putative electron transfer path is relatively short (4 Å) and involves the solvent-exposed copper ligand His-87 in the hydrophobic patch, while the other is more lengthy (12–15 Å) and involves the nearly conserved residue Tyr-83 in the negative patch.     

Heterogeneity of Photosystem I reaction centers in barley leaves as related to the donation from stromal reductants

The light-response curves of P700 oxidation and time-resolved kinetics of P700⁺ dark re-reduction were studied in barley leaves using absorbance changes at 820 nm. Leaves were exposed to 45 °C and treated with either diuron or diuron plus methyl viologen (MV) to prevent linear electron flow from PS II to PSI and ferredoxin-dependent cyclic electron flow around PSI. Under those conditions, P700⁺ could accept electrons solely from soluble stromal reductants. P700 was oxidized under weak far-red light in leaves treated with diuron plus MV, while identical illumination was nearly ineffective in diuron-treated leaves in the absence of MV. When heat-exposed leaves were briefly illuminated with strong far-red light, which completely oxidized P700, the kinetics of P700⁺ dark reduction was fitted by a single exponential term with half-time of about 40 ms. However, two first-order kinetic components of electron flow to P700⁺ (fast and slow) were found after prolonged leaf irradiation. The light-induced modulation of the kinetics of P700⁺ dark reduction was reversed following dark adaptation. The fast component (half time of 80–90 ms) was 1.5 larger than the slow one (half time of about 1 s). No kinetic competition occurred between two pathways of electron donation to P700⁺ from stromal reductants. This suggests the presence of two different populations of PSI. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plastocyanin,an electron-transfer protein

Plastocyanin (Pc) is a copper (Cu)-containing blue protein, that functions as a mobile electron carrier between cytochrome (cyt) f and Photosystem 1 (PS1) in oxygenic organisms. The atomic structure is known and can be described as a -barrel with hydrophobic residues in the interior of the protein. To increase the understanding about structure-function relationships, site-directed mutagenesis of Pc has proven to be very useful. Mainly two spectroscopic techniques, optical and EPR spectroscopy, have been used to investigate how the copper-site is affected by different mutations. The redox properties of the mutants have been investigated and factors that affect the reduction potential are discussed. Absorption and EPR spectra and reduction potentials for the surface mutants are similar to those of the corresponding wild-type. However, mutants around the Cu ion affected the mentioned properties. Comparisons are made with other cupredoxins. Five site-directed mutants of spinach Pc, Pc(Leu12His), Pc(Leu15His), Pc(Thr79His), Pc(Lys81His) and Pc(Tyr83His), have been modified by covalent attachment of a photoactive ruthenium (Ru)-complex at the surface-exposed histidine residues. The rates of the internal electron-transfer reactions exhibit an exponential dependence on the metal-to-metal separation with a decay factor of 1.1 A_-1. A reorganization energy for the Cu-to-Ru electron-transfer reaction of 1.2 eV was determined. Interprotein electron-transfer reactions involving genetically modified Pc are described. Ionic-strength and pH dependencies indicated that electrostatic interactions are involved in the complex formation between Pc and PS 1, which was confirmed by mutations in the acidic patches of Pc. A very specific interaction was further verified by replacements of hydrophobic residues. Position 10, 12, 36, 87 and 90 were found to be very important for the formation of an active complex. A comparison between available structures of Pc and cyt c₆, both effective donors to PS 1, is made. The physiological electron donor to Pc, cyt f, is briefly described.     

Quantification of cyclic electron flow around Photosystem I in spinach leaves during photosynthetic induction

The variation of the rate of cyclic electron transport around Photosystem I (PS I) during photosynthetic induction was investigated by illuminating dark-adapted spinach leaf discs with red + far-red actinic light for a varied duration, followed by abruptly turning off the light. The post-illumination re-reduction kinetics of P700⁺, the oxidized form of the photoactive chlorophyll of the reaction centre of PS I (normalized to the total P700 content), was well described by the sum of three negative exponential terms. The analysis gave a light-induced total electron flux from which the linear electron flux through PS II and PS I could be subtracted, yielding a cyclic electron flux. Our results show that the cyclic electron flux was small in the very early phase of photosynthetic induction, rose to a maximum at about 30 s of illumination, and declined subsequently to <10% of the total electron flux in the steady state. Further, this cyclic electron flow, largely responsible for the fast and intermediate exponential decays, was sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, suggesting an important role of redox poising of the cyclic components for optimal function. Significantly, our results demonstrate that analysis of the post-illumination re-reduction kinetics of P700⁺ allows the quantification of the cyclic electron flux in intact leaves by a relatively straightforward method.     

Reduction of the thylakoid electron transport chain by stromal reductants—evidence for activation of cyclic electron transport upon dark adaptation or under drought

The reduction of P700⁺, the primary electron donor of photosystem I (PSI), following a saturating flash of white light in the presence of the photosystem II (PSII) inhibitor 3-(3.4-dichlorophenyl)-1,1-dimethylurea (DCMU), was examined in barley plants exposed to a variety of conditions. The decay kinetic fitted to a double exponential decay curve, implying the presence of two distinct pools of PSI. A fast component, with a rate constant for decay of around 0.03–0.04 ms^–1 was observed to be sensitive to the duration of illumination. This rate constant was slower than, but comparable to, that observed in non-inhibited samples (i.e. where linear flow was active). It was substantially faster than values typically reported for experiments where PSII activity is inhibited. The magnitude of this component rose in leaves that were dark-adapted or exposed to drought. This component was assigned to PSI centres involved in cyclic electron transport. The remaining slowly decaying P700⁺ population (rate constant of around 0.001–0.002 ms^–1) was assigned to centres normally involved in linear electron transport (but inhibited here because of the presence of DCMU), or inactivated centres involved in the cyclic pathway. Processes that might regulate the relative flux through cyclic electron transport are discussed.     

Electron microscopic structural analysis of Photosystem I,Photosystem II,and the cytochromeb6/f complex from green plants and cyanobacteria

Electron microscopy (EM) in combination with image analysis is a powerful technique to study protein structure at low- and high resolution. Since electron micrographs of biological objects are very noisy, substantial improvement of image quality can be obtained by averaging individual projections. Crystallographic and noncrystallographic averaging methods are available and have been applied to study projections of the large protein complexes embedded in photosynthetic membranes from cyanobacteria and higher plants. Results of EM on monomeric and trimeric Photosystem I complexes, on monomeric and dimeric Photosystem II complexes, and on the monomeric cytochromeb6/f complex are discussed.     

The discovery and function of plastocyanin: A personal account

A brief autobiographical account is presented of the early research that led to the discovery of the copper protein plastocyanin and the identification of its function as an electron carrier in plant photosynthesis. A discussion follows of different approaches employed for the determination of the functional site of plastocyanin in relation to cytochrome f. A summary is provided of a heated controversy about the involvement of two or three light reactions in photosynthesis and an experiment is described that has contributed to resolution of the controversy through the identification of the functional site of plastocyanin. An early history of photosynthesis research in Japan is also discussed.Abbreviation DCIP 2,6-dichlorophenolindophenol Invited and edited by Govindjee.     

Deciphering the 820 nm signal: redox state of donor side and quantum yield of Photosystem I in leaves

By recording leaf transmittance at 820 nm and quantifying the photon flux density of far red light (FRL) absorbed by long-wavelength chlorophylls of Photosystem I (PS I), the oxidation kinetics of electron carriers on the PS I donor side was mathematically analyzed in sunflower (Helianthus annuus L.), tobacco (Nicotiana tabacum L.) and birch (Betula pendula Roth.) leaves. PS I donor side carriers were first oxidized under FRL, electrons were then allowed to accumulate on the PS I donor side during dark intervals of increasing length. After each dark interval the electrons were removed (titrated) by FRL. The kinetics of the 820 nm signal during the oxidation of the PS I donor side was modeled assuming redox equilibrium among the PS I donor pigment (P700), plastocyanin (PC), and cytochrome f plus Rieske FeS (Cyt f + FeS) pools, considering that the 820 nm signal originates from P700⁺ and PC⁺. The analysis yielded the pool sizes of P700, PC and (Cyt f + FeS) and associated redox equilibrium constants. PS I density varied between 0.6 and 1.4 μmol m⁻². PS II density (measured as O₂ evolution from a saturating single-turnover flash) ranged from 0.64 to 2.14 μmol m⁻². The average electron storage capacity was 1.96 (range 1.25 to 2.4) and 1.16 (range 0.6 to 1.7) for PC and (Cyt f + FeS), respectively, per P700. The best-fit electrochemical midpoint potential differences were 80 mV for the P700/PC and 25 mV for the PC/Cyt f equilibria at 22 °C. An algorithm relating the measured 820 nm signal to the redox states of individual PS I donor side electron carriers in leaves is presented. Applying this algorithm to the analysis of steady-state light response curves of net CO₂ fixation rate and 820 nm signal shows that the quantum yield of PS I decreases by about half due to acceptor side reduction at limiting light intensities before the donor side becomes oxidized at saturating intensities. Footnote: This revised version was published online in August 2006 with corrections to the Cover Date.     

Electron transfer from cytochrome f to photosystem I in green algae

The time course of P700⁺ reduction and cytochrome f oxidation following a single-turnover flash excitation of photosystem I was measured under various conditions in different strains of green algae. P700⁺ was reduced with a half-time of 4 s. The rate of cytochrome f oxidation was found to depend widely on physiological factors. Reversible transitions are described from a slow-oxidation state (t _1/2=500 s) to a fast-oxidation state (t _1/2=80 s). The addition of ionophore strongly favours and stabilizes the fast-oxidation state. We suggest that these transitions reflect either reversible association between the cytochrome bf complex and the reaction center of photosystem I or changes in the mobility of oxidized plastocyanin. The transitions might be under the control of the membrane potential or the intracellular ATP content. The relation of these reversible transitions with the light state transitions, and their possible involvement in a switch from linear to cyclic electron transfer, are discussed.Abbreviations cyt cytochrome - DCHC dicyclohexyl-18-crown-6 - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT dinitrophenylether of iodonitrothymol - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - LHC light harvesting complex - PC plastocyanin - PS I photosystem I     

Cytochrome f: Structure,function and biosynthesis

Cytochrome f is an intrinsic membrane component of the cytochrome bf complex, transferring electrons from the Rieske FeS protein to plastocyanin in the thylakoid lumen. The protein is held in the thylakoid membrane by a single transmembrane span located near its C-terminus with a globular hydrophilic domain extending into the lumen. The globular domain of the turnip protein has recently been crystallised, offering the prospect of a detailed three-dimensional structure. Reaction with plastocyanin involves localised positive charges on cytochrome f interacting with the acidic patch on plastocyanin and electron transfer via the surface-exposed tyrosine residue (Tyr83) of plastocyanin. Apocytochrome f is encoded in the chloroplast genome and is synthesised with an N-terminal presequence which targets the protein to the thylakoid membrane. The synthesis of cytochrome f is coordinated with the synthesis of the other subunits of the cytochrome bf complex.     

Electron Fluxes through Photosystem I in Cucumber Leaf Discs Probed by far-red Light

Cucumber leaf discs were illuminated at room-temperature with far-red light to photo-oxidise P700, the chlorophyll dimer in Photosystem (PS) I. The post-illumination kinetics of P700(+) re-reduction were studied in the presence of inhibitors or cofactors of photosynthetic electron transport. The re-reduction kinetics of P700(+) were well fitted as the sum of three exponentials, each with its amplitude and rate coefficient, and an initial flux (at the instant of turning off far-red light) given as the product of the two. Each initial flux is assumed equal to a steady state flux during far-red illumination. The fast phase of re-reduction, with rate coefficient k (1) approximately 10 s(-1), was completely abolished by a saturating concentration of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU); it is attributed to electron flow to P700(+) from PS II, which was stimulated to some extent by far-red light. The intermediate phase, with rate coefficient k (1) approximately 1 s(-1), was only partly diminished by methyl viologen (MV) which diverts electron flow to oxygen. The intermediate phase is attributed to electron donation from reduced ferredoxin to the intersystem pool; reduced ferredoxin could be formed: (1) directly by electron donation on the acceptor of PS I; and/or (2) indirectly by stromal reductants, in line with only a partial inhibition of the intermediate phase by MV. Duroquinol enhanced the intermediate phase in the presence of DCMU, presumably through its interaction with thylakoid membrane components leading to the partial reduction of plastoquinone. The slow phase of P700(+) re-reduction, with rate coefficient k (1) approximately 0.1 s(-1), was unaffected by DCMU and only slightly affected by MV; it could be associated with electron donation to either: (1) the intersystem chain by stromal reductants catalysed by NAD(P)H dehydrogenase slowly; or (2) plastocyanin/P700(+) by ascorbate diffusing across the thylakoid membrane to the lumen. It is concluded that a post-illumination analysis of the fluxes to P700(+) can be used to probe the pathways of electron flow to PS I in steady state illumination.     

Light-dependent,but phytochrome-independent,translational control of the accumulation of the P700 chlorophyll-a protein of photosystem I in barley (Hordeum vulgare L.)

This work reports on the regulation of synthesis of the P700 chlorophyll-a apoprotein of photosystem I in barley. The mRNA for the P700 apoprotein is almost exclusively confined to the plastid membrane-bound polysomes. However, the mRNA for the 32-kDa herbicide-binding protein of photosystem II is found in both the soluble and membrane-bound polysomes.The mRNA for the P700 apoprotein is found in similar amounts in dark-grown and light-grown wild-type as well as mutant xantha-l⁸¹ barley. The latter mutant is deficient in chlorophyll biosynthesis. However, while wild-type leaves accumulate the P700 chlorophyll-a protein only in the light, mutant leaves never accumulate the P700 apoprotein.A more sensitive approach was taken using isolated plastids to study P700 apoprotein synthesis. Etioplasts did not synthesize detectable P700 apoprotein even when the etioplasts were exposed to light. However, only a 1-min exposure of leaves to light was necessary to induce P700 apoprotein synthesis by isolated plastids.Phytochrome involvement in controlling P700 apoprotein synthesis was tested by using red/farred light treatment of leaves. These treatments showed no far-red reversibility of red-induced P700-apoprotein synthesis in isolated plastids even after 3 h of darkness after the light treatments. From these data we conclude that the accumulation of P700 apopootein is not under the control of phytochrome and that the light induction of P700 apoprotein is most likely mediated through the protochlorophyllide/chlorophyllide system. This control, however, may also involve cytoplasmic signals as the synthesis of the P700 apoprotein is not turned on in illuminated etioplasts.     

Cell enlargement and sugar accumulation in the gynaecium of the glasshouse carnation (Dianthus caryophyllus L.) induced by ethylene

Summary Histological examination of the ovary walls from ethylene-treated cut flowering stems of the carnation showed that the cells had enlarged and this appeared to account for the increased growth of the ovary which follows ethylene treatment of this flower. Sugar analyses of the flower parts indicated that growth of the ovary was accompanied by an increase in the ratio of sucrose to reducing sugars in the petals and ovary, and a net increase in sugars in the ovary. A sugar, tentatively identified as xylose, increased in the petals after ethylene treatment. Nitrogen, phosphorus and potassium contents of the ovary also increased after the ethylene treatment. The results, consistent with the hypothesis that sucrose is translocated in response to ethylene, are discussed in relation to previous work relating to the involvement of ethylene in flower senescence.     

Effects of pH on the kinetics of redox reactions in and around the cytochromebf complex in an isolated system

Rate-coefficients describing the electron transfer reactions between P700 and plastocyanin, between cytochromef in cytochromebf complexes and plastocyanin, and between decyl plastoquinol and cytochromebf complexes were determined as a function of pH in the range 4–10 from flash-induced absorbancy changes at four wavelengths. The reactions between P700 and plastocyanin, and between cytochromef and plastocyanin were optimised when there was electrostatic interaction between ionised acidic groups in plastocyanin with a pK_a of 4.3–4.7 and ionised basic constituents in P700 (assumed to be in the PSI-F subunit) and in cytochromef, with a pK_b of 8.9–9.4. The basic groups are thought to be lysine rather than arginine. This mechanism agrees with that inferred from effects of ionic strength changes on rate-coefficients. The relation between the second-order rate-coefficient for decyl plastoquinol oxidation by thebf complex and pH was characterised by a pK_a of 6.1. This is interpreted as showing that the anion radical form of that quinol, which has a pK_a of 6, and which becomes progressively protonated when pH is changed from 7 to 5, is essential to reduce cytochromeb-563 (low potential) during quinol oxidation. Above pH 9, permanent effects were observed on this rate-coefficient, which were absent in the reactions between P700, plastocyanin and cytochromef.     

Inhibitor binding to isolated chloroplast cytochrome bf complex

Effects of three inhibitors of quinol oxidation in the chloroplast cytochrome bf complex (stigmatellin, tridecylstigmatellin and dibromothymoquinone) were studied in an isolated system comprising Photosystem I (PS I) particles, plastocyanin (PC) and cytochrome bf complex, in the absence of quinol or quinone. Addition of these inhibitors increased the extent of cytochrome f oxidation after a laser flash created oxidised PS I reaction centre (P700) and PC, and decreased somewhat the extent of PC oxidation. The re-reduction of oxidised P700 was more complete than when inhibitor was absent. The data were simulated with reactions which included the putative reduction of cytochrome f by the Rieske centre (FeS) and different rate-coefficients according as to whether inhibitor was bound to the bf complex or not. It was concluded that under the conditions studied the Rieske centre donated electrons to oxidised cytochrome f and plastocyanin with an average rate coefficient of 35 s^–1. This electron transfer was prevented by any of the three inhibitors, which also increased the equilibrium coefficient for the cytochrome f/PC reaction by a maximum factor of two. This increase corresponded to a decrease in the back reaction coefficient and an increase in the forward rate. The equilibrium coefficient for the reduction of oxidised P700 by PC was about 2 in the absence of inhibitor but increased to about 20 in their presence, but only if cytochrome bf complex was additionally present. This was attributed to the transient formation of complexes between P700 with bound plastocyanin, and bf complex. The operative mid-point potential of FeS, if that of cytochrome f is 370 mV, was 390 mV. Deviations in midpoint potentials (P700/plastocyanin) from solution values were attributed to the bound state of the reactants. Estimates were made of the binding coefficient of each of the three inhibitors to p-sites in the cytochrome bf complex in the absence of competing quinol. A stoichiometry of two inhibitors per bf dimer was necessary to cause the above changes in reduction potential of cyt f and PC. A result of one inhibitor per dimer was statistically unlikely, particularly in the case of tridecylstigmatellin.Abbreviations Cyt- cytochrome - DBMIB(H₂)- 2,5-dibromo-3--ethyl-6-isopropyl-p-benzoquinone (reduced) - E _m- midpoint reduction potential of a couple relative to the standard hydrogen electrode - e-t- electron transfer - FeS (or R)- Rieske iron-sulphur centre - HEPES- N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - Mega-9- nonoyl-N-methylglucamide - n-site (Q_r-site)- quinone reduction site in cytochrome bf complex - PC- plastocyanin - p-site (Q_o-site)- quinol oxidation site in cytochrome bf complex - PQ- plastoquinone - PSI- Photosystem I - P700- reaction centre in Photosystem I - TDS- tridecyl stigmatellin     

Alternative Photosystem I-Driven Electron Transport Routes: Mechanisms and Functions

In addition to the linear electron transport, several alternative Photosystem I-driven (PS I) electron pathways recycle the electrons to the intersystem electron carriers mediated by either ferredoxin:NADPH reductase, NAD(P)H dehydrogenase, or putative ferredoxin:plastoquinone reductase. The following functions have been proposed for these pathways: adjustment of ATP/NADPH ratio required for CO(2) fixation, generation of the proton gradient for the down-regulation of Photosystem II (PS II), and ATP supply the active transport of inorganic carbon in algal cells. Unlike ferredoxin-dependent cyclic electron transport, the pathways supported by NAD(P)H can function in the dark and are likely involved in chlororespiratory-dependent energization of the thylakoid membrane. This energization may support carotenoid biosynthesis and/or maintain thylakoid ATPase in active state. Active operation of ferredoxin-dependent cyclic electron transport requires moderate reduction of both the intersystem electron carriers and the acceptor side of PS I, whereas the rate of NAD(P)H-dependent pathways under light depends largely on NAD(P)H accumulation in the stroma. Environmental stresses such as photoinhibition, high temperatures, drought, or high salinity stimulated the activity of alternative PS I-driven electron transport pathways. Thus, the energetic and regulatory functions of PS I-driven pathways must be an integral part of photosynthetic organisms and provides additional flexibility to environmental stress.     

Turtle hemoglobin: evidence of a T-state oxyhemoglobin

The 270 MHz ¹H NMR spectra of rabbit skeletal long and short S2 were indistinguishable at 20°C and 30°C and contained only a small proportion of sharp peaks associated with flexible regions. At 60°C both proteins were denatured and had essentially identical spectra. At 40°C and 50°C the long S2 spectrum contained a marginally greater proportion of sharp peaks, representing not more than 25 residues/chain. Our results are consistent with the presence of a small hinge in long S2 but do not support its containing an extensive region which provides contractile force by a helix—coil transition.     

The structure and function of the chloroplast photosynthetic membrane — a model for the domain organization

Recent work on the domain organization of the thylakoid is reviewed and a model for the thylakoid of higher plants is presented. According to this model the thylakoid membrane is divided into three main domains: the stroma lamellae, the grana margins and the grana core (partitions). These have different biochemical compositions and have specialized functions. Linear electron transport occurs in the grana while cyclic electron transport is restricted to the stroma lamellae. This model is based on the following results and considerations. (1) There is no good candidate for a long-range mobile redox carrier between PS II in the grana and PS I in the stroma lamellae. The lateral diffusion of plastoquinone and plastocyanin is severely restricted by macromolecular crowding in the membrane and the lumen respectively. (2) There is an excess of 14±18% chlorophyll associated with PS I over that of PS II. This excess is assumed to be localized in the stroma lamellae where PS I drives cyclic electron transport. (3) For several plant species, the stroma lamellae account for 20±3% of the thylakoid membrane and the grana (including the appressed regions, margins and end membranes) for the remaining 80%. The amount of stroma lamellae (20%) corresponds to the excess (14–18%) of chlorophyll associated with PS I. (4) The model predicts a quantum requirement of about 10 quanta per oxygen molecule evolved, which is in good agreement with experimentally observed values. (5) There are at least two pools of each of the following components: PS I, PS II, cytochrome bf complex, plastocyanin, ATP synthase and plastoquinone. One pool is in the grana and the other in the stroma compartments. So far, it has been demonstrated that the PS I, PS II and cytochrome bf complexes each differ in their respective pools.Abbreviations PS I and PS II Photosystem I and II - P 700 reaction center of PS I - LHC II light-harvesting complex II     

Enhanced rates of P700<Superscript>+</Superscript> dark-reduction in leaves of<Emphasis Type="Italic"> Cucumis sativus</Emphasis> L. photoinhibited at chilling temperature

The changes in electron transport within photosystem I (PSI) were studied in detached leaves of Cucumis sativus L. during the course of irradiation with moderate white light (300 mol photons m^–2 s^–1) at 4°C. When intact leaves were exposed to the combination of moderate light and low temperature, the amplitude of far-red light-induced P700 absorbance changes at 820 nm (A₈₂₀), a relative measure of PSI, progressively decreased as the light treatment time increased. Almost no oxidation of P700 was noticeable after 5 h. Methyl viologen accelerated the oxidation of P700 to a steady-state level and also increased the magnitudes of A₈₂₀ changes in photoinhibited leaves, reflecting the rapid removal of electrons from native carriers. Photoinhibition under moderate light and chilling temperature also accelerated the rate of P700⁺ reduction after far-red light excitation as the half-times of the two exponential components of P700⁺ decay curves decreased relative to the control ones. A detailed analysis of the kinetics of P700⁺ reduction using diuron alone or the combination of diuron and methyl viologen strongly favours an increased rate of electron donation from stromal reductants to PSI through the plastoquinone pool following photoinhibitory treatment. Importantly, the marked acceleration of P700⁺ re-reduction is the consequence of the irradiation of leaf segments at low temperature and not caused by chilling stress alone.Abbreviations A ₀ and A ₁ Primary acceptor chlorophyll and secondary electron acceptor phylloquinone - FR Far-red light - F _X , F _A , and F _B Iron–sulfur centers - MT Multiple-turnover flash - MV Methyl viologen - Ndh NAD(P)H-dehydrogenase - PQ Plastoquinone - PS Photosystem - P700 Reaction-center chlorophyll of PSI - ST Single-turnover flash     

Characterization of the photosynthetic electron transport chain in normal and photobleachedAnabaena cylindrica by flash spectroscopy

Electron transport of normal and photobleachedAnabaena cylindrica was studied using spectral and kinetic analyses of absorbance transients induced by single turnover flashes. Between 500 and 600 nm two positive bands (540 and 566 nm) and two negative bands (515 and 554 nm) were found. Absorbance changes at 515 and 540 nm were partly characterized. None of these absorbance changes represent an electrochromic shift. Absorbance changes at 554 and 566 nm correspond to the oxidation of cytochromef and the reduction of cytochromeb ₅₆₃, respectively. We found a very slight 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) sensitivity of cytochromef in normal cells, while DCMU was completely ineffective for cytochromef reduction in photobleached cells. The absorbance change of cytochromeb ₅₆₃ increased, while the absorbance change of cytochromef was smaller than in normal cells. The increased O₂ evolution in photobleached cells and the negligible electron transport via cytochromef suggest the participation of other electron acceptor(s) in the electron-transport chain of photobleachedAnabaena cylindrica.