Light-induced Redox Changes Research Articles

Differences in Ionic, Enzymatic, and Photosynthetic Features Characterize Distinct Salt Tolerance in Eucalyptus Species.

In the face of rising salinity along coastal regions and in irrigated areas, molecular breeding of tolerant crops and reforestation of exposed areas using tolerant woody species is a two-way strategy. Thus, identification of tolerant plants and of existing tolerance mechanisms are of immense value. In the present study, three Eucalyptus ecotypes with potentially differential salt sensitivity were compared. Soil-grown Eucalyptus plants were exposed to 80 and 170 mM NaCl for 30 days. Besides analysing salt effects on ionic/osmotic balance, and hydrolytic enzymes, plants were compared for dynamics of light-induced redox changes in photosynthetic electron transport chain (pETC) components, namely plastocyanin (PC), photosystem I (PSI) and ferredoxin (Fd), parallel to traditional chlorophyll a fluorescence-based PSII-related parameters. Deconvoluted signals for PC and Fd from PSI allowed identification of PC and PSI as the prime salinity-sensitive components of pETC in tested Eucalyptus species. Eucalyptus loxophleba portrayed efficient K+-Na+ balance (60–90% increased K+) along with a more dynamic range of redox changes for pETC components in old leaves. Young leaves in Eucalyptus loxophleba showed robust endomembrane homeostasis, as underlined by an increased response of hydrolytic enzymes at lower salt concentration (~1.7–2.6-fold increase). Findings are discussed in context of salinity dose dependence among different Eucalyptus species.

Water-splitting manganese complex controls light-induced redox changes of cytochrome b 559 in Photosystem II

The effect of water-splitting Mn complex on light-induced redox changes of cytochrome b(559) (cyt b(559)) was studied in spinach photosystem II (PSII) membranes. Photoreduction of the heme iron in the intact PSII membranes was completely suppressed by DCMU, whereas photoreduction and photooxidation of the heme iron in the Mn-depleted PSII membranes were unaffected by DCMU. Interestingly, photoreduction and photooxidation of the heme iron in the Mn-depleted PSII membranes were completely diminished by exogenous superoxide dismutase (SOD), whereas no effect of SOD on photoreduction of the heme iron was observed in the intact PSII membranes. The current work shows that the light-induced redox changes of cyt b(559) proceed via a different mechanism in the both types of PSII membranes. In the intact PSII membranes, photoreduction of the heme iron is mediated by plastoquinol. However, in the Mn-depleted PSII membranes, photoreduction and photooxidation of the heme iron are mediated by superoxide anion radical formed in PSII.

Evaluation of the participation of ferredoxin in oxygen reduction in the photosynthetic electron transport chain of isolated pea thylakoids.

The contribution to reduction of oxygen by ferredoxin (Fd) to the overall reduction of oxygen in isolated pea thylakoids was studied in the presence of Fd versus Fd + NADP(+). The overall rate of electron transport was measured using a determination of Photosystem II quantum yield from chlorophyll fluorescence parameters, and the rate of oxidation of Fd was measured from the light-induced redox changes of Fd. At low light intensity, increasing Fd concentration from 5 to 30 microM in the absence of NADP(+) increased the proportion of oxygen reduction by Fd from 25-35 to 40-60% in different experiments. This proportion decreased with increasing light intensity. When NADP(+) was added in the presence of 15 microM Fd, which was optimal for the NADP(+) reduction rate, the participation of Fd in the reduction of oxygen was low, no more than 10%, and it also decreased with increasing light intensity. At high light intensity, the overall oxygen reduction rates in the presence of Fd + NADP(+) and in the presence of Fd alone were comparable. The significance of reduction of dioxygen either by water-soluble Fd or by the membrane-bound carriers of the photosynthetic electron transport chain for redox signaling under different light intensities is discussed.

A redox-regulated chloroplast protein phosphatase binds to starch diurnally and functions in its accumulation.

Starch is the ultimate storage molecule formed in the photosynthetic fixation of carbon dioxide by chloroplasts. Starch accumulates during the day and is degraded at night to intermediates that are exported to heterotrophic organs. The mechanism by which diurnal cycles control the transitory biosynthesis and degradation of chloroplast starch has long remained a mystery. We now report evidence that a dual-specificity protein phosphatase, DSP4, binds to starch granules during the day and dissociates at night. Disruption of the DSP4 gene resulted in a dramatic increase in the level of starch in mutant Arabidopsis plants. Moreover, although composition was apparently unchanged, the morphology of the starch granule was significantly altered compared to the wild type counterpart. Two regulatory factors linked to light (i.e., pH and redox status) changed both the activity and the starch-binding capacity of DSP4. The results further revealed that DSP4 represents a major fraction of granule-bound phosphatase activity during the day but not at night. Our study suggests that DSP4 acts as a bridge between light-induced redox changes and protein phosphorylation in the regulation of starch accumulation.

EPR study of light-induced regulation of photosynthetic electron transport in Synechocystis sp. strain PCC 6803

The kinetics of the light-induced redox changes of the photosystem 1 (PS 1) primary donor P 700 in whole cells of the cyanobacteria Synechocystis sp. PCC 6803 were studied by the electron paramagnetic resonance method. It was shown that the linear photosynthetic electron transport in cyanobacteria was controlled by two main mechanisms: (i) oxygen-dependent acceleration of electron transfer from PS 1 to NADP + due to activation of the Calvin cycle reactions and (ii) retardation of electron flow between two photosystems governed by a transmembrane proton gradient. In addition to the linear photosynthetic electron transport, cyanobacteria were capable of maintaining alternative pathways involving cyclic electron transfer around PS 1 and respiratory chains.

LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): Binding sites for the chromophore flavin mononucleotide

Phototropism, the bending response of plant organs to or away from a directional light source, is one of the best studied blue light responses in plants. Although phototropism has been studied for more than a century, recent advances have improved our understanding of the underlying signaling mechanisms involved. The NPH1 gene of Arabidopsis thaliana encodes a blue light-dependent autophosphorylating protein kinase with the properties of a photoreceptor for phototropism. NPH1 apoprotein noncovalently binds FMN to form the holoprotein nph1. The N-terminal region of the protein contains two LOV (light, oxygen, or voltage) domains that share homology with sensor proteins from a diverse group of organisms. These include the bacterial proteins NIFL and AER, both of which bind FAD, and the phy3 photoreceptor from Adiantium capillus-veneris. The LOV domain has therefore been proposed to reflect a flavin-binding site, regulating nph1 kinase activity in response to blue light-induced redox changes. Herein we demonstrate that the LOV domains of two nph1 proteins and phy3 bind stoichiometric amounts of FMN when expressed in Escherichia coli. The spectral properties of the chromopeptides are similar to the action spectrum for phototropism, implying that the LOV domain binds FMN to function as a light sensor. Thus, our findings support the earlier model that nph1 is a dual-chromophoric flavoprotein photoreceptor regulating phototropic responses in higher plants. We therefore propose the name phototropin to designate the nph1 holoprotein.

Arabidopsis NPH1: a protein kinase with a putative redox-sensing domain.

The NPH1 (nonphototropic hypocotyl 1) gene encodes an essential component acting very early in the signal-transduction chain for phototropism. Arabidopsis NPH1 contains a serine-threonine kinase domain and LOV1 and LOV2 repeats that share similarity (36 to 56 percent) with Halobacterium salinarium Bat, Azotobacter vinelandii NIFL, Neurospora crassa White Collar-1, Escherichia coli Aer, and the Eag family of potassium-channel proteins from Drosophila and mammals. Sequence similarity with a known (NIFL) and a suspected (Aer) flavoprotein suggests that NPH1 LOV1 and LOV2 may be flavin-binding domains that regulate kinase activity in response to blue light-induced redox changes.

Antenna Sizes of Photosystem I and Photosystem II in Chlorophyll b-Deficient Mutants of Rice. Evidence for an Antenna Function of Photosystem II Centers That are Inactive in Electron Transport

Light-harvesting capacities of photosystem I (PSI) and photosystem II (PSII) in a wild-type and three chlorophyll 6-deficient mutant strains of rice were determined by measuring the initial slope of light-respons e curve of PSI and PSII electron transport and kinetics of light-induced redox changes of P-700 and QA, respectively. The light-harvesting capacity of PSI determined by the two methods was only moderately reduced by chlorophyll 6-deficiency. Analysis of the fluorescence induction that monitors time course of QA photoreduction showed that both relative abundance and antenna size of PSIIa decrease with increasing deficiency of chlorophyll b and there is only PSII,, in chlorina 2 which totally lacks chlorophyll b. The numbers of antenna chlorophyll molecules associated with the mutant PSII centers were, therefore, three to five times smaller than that of PSII a in the wild type rice. Rates of PSII electron transport determined on the basis of PSII centers in the three mutants were 60-70% of that in the normal plant at all photon flux densities examined, indicating that substantial portions of the mutant PSII centers are inactive in electron transport. The initial slopes of light-respons e curves of PSII electron transport revealed that the functional antenna sizes of the active populations of PSII centers in the mutants correspond to about half that of PSII a in the wild type rice. Thus, the numbers of chlorophyll molecules that serve as antenna of the oxygen-evolving PSII centers in the mutants are significantly larger than those that are actually associated with each PSII center. It is proposed that the inactive PSII serves as an antenna of the active PSII in the three chlorophyll ^-deficient mutants of rice. In spite of the reduced antenna size of PSII, therefore, the total light-harvesting capacity of PSII approximately matches that of PSI in the mutants.

Simultaneous photoreduction and photooxidation of cytochrome b-559 in Photosystem II treated with carbonylcyanide-m-chlorophenylhydrazone

The possibility of a Photosystem II (PS II) cyclic electron flow via Cyt b-559 catalyzed by carbonylcyanide m-chlorophenylhydrazone (CCCP) was further examined by studying the effects of the PS II electron acceptor 2,6-dichloro-p-benzoquinone (DCBQ) on the light-induced changes of the redox states of Cyt b-559. Addition to barley thylakoids of micromolar concentrations of DCBQ completely inhibited the changes of the absorbance difference corresponding to the photoreduction of Cyt b-559 observed either in the presence of 10 μM ferricyanide or after Cyt b-559 photooxidation in the presence of 2 μM CCCP. In CCCP-treated thylakoids, the concentration of photooxidized Cyt b-559 decreased as the irradiance of actinic light increased from 2 to 80 W m(-2) but remained close to the maximal concentration (0.53 photooxidized Cyt b-559 per photoactive Photosystem II) in the presence of 50 μM DCBQ. The stimulation of Cyt b-559 photooxidation in parallel with the inhibition of its photoreduction caused by DCBQ demonstrate that the extent of the light-induced changes of the redox state of Cyt b-559 in the presence of CCCP is determined by the difference between the rates of photooxidation and photoreduction of Cyt b-559 occuring simultaneously in a cyclic electron flow around PS II.We also observed that the Photosystem I electron acceptor methyl viologen (MV) at a concentration of 1 mM barely affected the rate and extent of the light-induced redox changes of Cyt b-559 in the presence of either FeCN or CCCP. Under similar experimental conditions, MV strongly quenched Chl-a fluorescence, suggesting that Cyt b-559 is reduced directly on the reducing side of Photosystem II.

Location of cytochrome b-559 between photosystem II and Photosystem I in noncyclic electron transport

Selective light-induced redox changes of cytochrome b-559 in relation to cytochrome f have been investigated at 20°C in fresh spinach thylakoids under physiological conditions and in the presence of specific inhibitors and uncouplers. Light absorbed by PS II (650 nm) promotes the sequential reduction of cytochrome b-559 and cytochrome f, whereas light absorbed by PS I (720 nm) brings about the oxidation of cytochrome f and, by way of this, the oxidation of cytochrome b-559. Either DCMU or DBMIB completely blocks the reduction of the two cytochromes by PS II light, but not the oxidation of cytochrome f by PS I light. On the other hand, cyanide as well as polylysine inhibits almost totally the oxidation of both cytochromes f by PSI light. From these and previous results, it is concluded that photoreduction of cytochrome b-559 by PS II occurs through the plastoquinone pool and that its photooxidation by PS I involves both plastocyanin and cytochrome f. It thus appears that cytochrome b-559 is located between plastoquinone and cytochrome f in noncyclic electron transport in chloroplasts.

Reconstitution of cyclic electron transport and photophosphorylation by incorporation of the reaction center, cytochrome bc1 complex and ATP synthase from Rhodobacter capsulatus into ubiquinone-10/phospholipid vesicles

The photosynthetic reaction center, the ubiquinol-cytochrome c reductase (EC 1.10.2.2) and the ATP synthase (EC 3.6.1.4) were selectively solubilized and isolated from the intracytoplasmic membranes of photoheterotrophically grown Rhodobacter capsulatus with n-octyl β- d -glucopyranoside. The purified enzymes were co-reconstituted in predetermined stoichiometries in ubiquinol-10 phospholipid vesicles by a two-step procedure. In the first step, reaction centers and ubiquinol-cytochrome c reductase were incorporated randomly by detergent dialysis. Cyclic electron transport between these two membrane complexes was selected by including cytochrome c in the interior of the proteoliposomes. In the second step, the ATP synthase molecules were incorporated by detergent dilution into the preformed proteoliposomes from the outer face of the vesicles. Analysis of the light-induced redox changes of the cytochromes and of the bacteriochlorophyll dimer, in combination with the effect of specific inhibitors demonstrated that an electron transport pathway closely resembling that of the physiological system was reconstructed in the proteoliposomes. Light-driven ATP synthesis was detected at a maximal rate of 36 nmol ATP/min per mg ATP synthase. Variation in the stoichiometry of the protein components indicated that the rate-limiting reaction was ATP synthesis.

The adaptation of the electron transfer chain of Rhodopseudomonas capsulata to different light intensities

(1) Cells of Rhodopseudomonas capsulata (wild-type) were grown photoheterotrophically in a turbidostat under very high and very low light intensity. Membranes were isolated from cells adapted to the respective light conditions and fractionated by sucrose density centrifugation. The molar ratios of ubiquinone and cytochromes c 2, c 1, b-561 and b-566 per reaction center were 3-fold to 5-fold higher in high-light than in low-light membranes. (2) Most of the Cyt( c 1 + c 2) and Cyt b-561 detected in dark redox titrations undergoes light-induced redox changes, both in high- and in low-light membranes. (3) The fractions of the total photooxidizable reaction center and Cyt( c 1 + c 2) oxidized under continuous light in the absence of antimycin are higher in membranes from low-light- than from high-light-grown cells. (4) From these data and results of kinetic studies it is proposed that cyclic electron flow under saturating light intensities is faster in high-light-grown cells.

Light-induced redox changes in chloroplast cytochrome f after phosphorylation of membrane proteins

Light-induced redox changes in chloroplast cytochrome f after phosphorylation of membrane proteins

A theory of charge separation, ion, electron and proton transport in photosynthetic membranes based on asymmetry of surface charges

We present a model for the light-induced charge separation, proton and ion transport across photosynthetic membranes based on an assumption of the transmembrane surface charge asymmetry. In dark equilibrium, this asymmetry gives rise to an internal membrane electric field whose direction is perpendicular to the membrane surfaces. The role of the field in the light-induced charge separation is similar to the function of the built-in electric field across a solid-state p-n junction. Light-generated free charge carriers in the membrane flow according to its direction and upon recombination on the surface give rise to an electrochemical potential difference for electrons across the membrane. The associated coupled electron-proton transport, and ion diffusion can be viewed as a response of the system to the light-induced redox and electric potential changes.

The relationship of the cyclic and non-cyclic electron transport pathways in chloroplasts

Light-induced redox changes of plastocyanin, the Rieske iron-sulfur center, and P-700 have been studied in situ in spinach chloroplasts. Plastocyanin and the Rieske center behaved in an analogous manner in that their steady states were fully oxidized in the light in the presence or absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea when an electron acceptor is present. After illumination under conditions of non-cyclic electron transfer from water to an electron acceptor, followed by a short dark period, the steady state of both shifted to a more reduced level. A 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive photoreduction of the Rieske center was observed in ferricyanide-washed chloroplast fragments. With reduced ferredoxin as electron donor, it was possible to demonstrate a reduction in the dark of these electron carriers and of P-700; this reduction was insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea but was inhibited by antimycin A. These findings are discussed in relation to a function for these electron carriers in the cyclic electron transport pathway in chloroplasts and to their function in the non-cyclic electron transport pathway.

Photooxidation of the high-potential iron—sulfur center in chloroplasts

The high-potential iron-sulfur center associated with the g = 1.89-1.90 electron paramagnetic resonance signal in spinach chloroplasts is fully reduced in the dark. It is photooxidized in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and reduced after illumination with red actinic light in the absence of inhibitors. The light-induced redox changes of the high-potential iron-sulfur center are shown to correlate with those of plastocyanin detected at g = 2.05. Demonstration of light-induced redox changes in the high-potential iron-sulfur center provides support for a functional role of this component in the chloroplast electron transport chain.

Cytochrome b-563 redox changes in intact CO 2-fixing spinach chloroplasts and in developing pea chloroplasts

Intact spinach chloroplasts, capable of high rates of photochemical oxygen evolution with CO 2 as electron acceptor (120–350 μmol 0 2 mg chlorophyll −1 · h −1) were examined for cytochrome redox changes. The response of the cytochromes in intact chloroplasts to oxidants and reductants appears to be governed by the permeability of the chloroplast envelope. The low potential cytochromes ( b-559 LP and b-563) were more slowly reduced at 25 °C by dithionite than is the case with broken chloroplasts. At 0 °C, the reduction of the low potential cytochromes in intact chloroplasts was extremely slow. The chloroplast envelope is impermeable to ferricyanide, slowly permeable to ascorbate and rapidly permeable to reduced dichlorophenolindophenol. Light-induced redox changes of cytochrome b-563 in intact chloroplasts were examined both at 0° and 25 °C. A red/far-red antagonism on the redox changes of cytochrome b-563 was observed at 0° C under anaerobic conditions. 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) inhibited the photoreduction of cytochrome b-563 in red light following far-red illumination. The photooxidation of cytochrome b-563 under anaerobic conditions was not influenced by DCMU or 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone (DBMIB). The photoreduction of cytochrome b-563 under aerobic conditions was much less efficient than its photooxidation under anaerobic conditions. Developing pea chloroplasts showed much greater light-induced redox changes of cytochrome b-563 than did intact spinach chloroplasts. Our data are consistent with the view that cytochrome b-563 functions on a cyclic pathway around Photosystem I, but it appears that cyclic flow is sensitive to the relative poising of the redox levels of cytochrome b-563 and the components of the non-cyclic pathway.

Effect of NADP + on light-induced cytochrome changes in membrane fragments from a blue-green alga

The effect of NADP + on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragments prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP + and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH 2) to NADP +. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH 2 as the electron donor, NADP + had no effect on the light-induced redox changes of cytochromes: with or without NADP +, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an α peak at 549 nm. With 664 nm illumination and water as the electron donor, NADP + had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b 559. Cytochrome b 559 appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP +) for the electron flow from water.

Light-induced redox changes of cytochrome b-559

Dark incubation of spinach or pea chloroplasts with 10 μ m 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 μ m 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.