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1.
Plant Physiol Biochem ; 185: 233-243, 2022 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-35716433

RESUMO

We present an approach to the noninvasive determination of the electron capacity of the intersystem pool of electron carriers in chloroplasts in situ. As apt experimental models, we used the leaves of Hibiscus rosa-sinensis and Tradescantia species. Electron paramagnetic resonance and optical response of P700 (the primary electron donor in Photosystem I) were applied to measuring electron transport in chloroplasts. Electron capacities of the intersystem electron transport chain (ETC) were determined from redox transients of P700 upon chromatic transitions (white light → far-red light). During the induction period, we observed the nonmonotonic changes in the number of electron equivalents in the intersystem ETC per P700 (parameter Q). In Hibiscus rosa-sinensis, the light-induced rise of Q from ≈2.5 (in the dark) to Q ≈ 12 was followed by its decrease to Q ≈ 6. The data obtained are discussed in the context of pH-dependent regulation of electron transport in chloroplasts, which provides the well-balanced operation of the intersystem ETC. The decay of Q is explained by the attenuation of Photosystem II activity due to the lumen acidification and the acceleration of plastoquinol re-oxidation as a result of the Calvin-Benson cycle activation. Our computer model of electron and proton transport coupled to ATP synthesis in chloroplasts was used to analyze the up and down feedbacks responsible for pH-dependent regulation of electron transport in chloroplasts. The procedures introduced here may be important for subsequent works aimed at defining the plastoquinone participation in regulation of photosynthetic processes in chloroplasts in situ.


Assuntos
Cloroplastos/metabolismo , Hibiscus/metabolismo , Fotossíntese/fisiologia , Tradescantia/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Luz , Oxirredução , Complexo de Proteína do Fotossistema I/metabolismo , Folhas de Planta/metabolismo
2.
Biochemistry (Mosc) ; 86(12): 1590-1598, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34937538

RESUMO

Photosynthetic organisms have developed a set of mechanisms aimed at preventing photo-oxidative reactions in the photosynthetic apparatus (PSA) initiated by excessively absorbed light energy. Along with high irradiance, other stressors, e.g., chilling temperatures, can lead to the absorption of the excess of light energy and hence to photo-oxidative stress. Here, we studied induction of photoprotective mechanisms in response to chilling (0°C) at a low irradiance (50 µmol PAR photons m-2·s-1) in the cells of microalga Lobosphaera incisa IPPAS C-2047. After 4 days of incubation at a low temperature, L. incisa IPPAS C-2047 cells showed a notable decrease in the photochemical activity of photosystem II (PSII) and in the efficiency of photosynthetic electron transport, as well as a significant increase in the thermal dissipation of the absorbed light energy in the light-harvesting antenna. In contrast, most conventional markers of PSA acclimation to excess light energy [total chlorophyll and carotenoid content; violaxanthin cycle pigment content and de-epoxidation state; photosynthetic antenna, PSII, and photosystem I (PSI) ratio] remained virtually unchanged. The content of major unsaturated fatty acids also remained almost unaffected, except for arachidonic acid (increased by 40%) recently assumed to activate violaxanthin de-epoxidase by adjusting its lipid microenvironment. Significant changes (4-7-fold increase) were observed in the expression of the gene encoding protective protein LhcSR. Pre-conditioning at 5°C prior to the acclimation to 0°C augmented the PSA photochemical activity. Our data show that the mid-term (4-d) acclimation of L. incisa IPPAS C-2047 to a chilling temperature at a low irradiance triggers the PSA response resembling, in part, the response to high light but relying mostly on the LhcSR protein-dependent quenching of excitation in the photosynthetic antenna.


Assuntos
Clorófitas/enzimologia , Temperatura Baixa , Microalgas/metabolismo , Fotossíntese , Complexo de Proteína do Fotossistema II/metabolismo , Clorófitas/química , Microalgas/química , Complexo de Proteína do Fotossistema II/química
3.
Photosynth Res ; 146(1-3): 123-141, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32594291

RESUMO

Using thermoluminescence, PAM-fluorometry, and electron paramagnetic resonance (EPR) for assaying electron transport processes in chloroplasts in situ, we have compared photosynthetic characteristics in Tradescantia fluminensis leaves grown under low light (LL, 50-125 µmol photons m-2 s-1) or high light (HL, 875-1000 µmol photons m-2 s-1) condition. We found differences in the thermoluminescence (TL) spectra of LL- and HL-acclimated leaves. The LL and HL leaves show different proportions of the Q (~ 0 °C) and B (~ 25-30 °C) bands in their TL spectra; the ratios of the "light sums" of the Q and B bands being SQ/SB ≈ 1/1 (LL) and SQ/SB ≈ 1/3 (HL). This suggests the existence of different redox states of electron carriers on the acceptor side of PSII in LL and HL leaves, which may be affected, in particular, by different capacities of their photo-reducible PQ pools. Enhanced content of PQ in chloroplasts of LL leaves may be the reason for an efficient performance of photosynthesis at low irradiance. Kinetic studies of slow induction of Chl a fluorescence and measurements of P700 photooxidation by EPR demonstrate that HL leaves have faster (about 2 times) response to switching on actinic light as compared to LL leaves grown at moderate irradiation. HL leaves also show higher non-photochemical quenching (NPQ) of Chl a fluorescence. These properties of HL leaves (faster response to light and generation of enhanced NPQ) reflect the flexibility of their photosynthetic apparatus, providing sustainability and rapid response to fluctuations of environmental light intensity and solar stress resistance. Analysis of time-courses of the EPR signals of [Formula: see text] induced by far-red (λmax = 707 nm), exciting predominantly PSI, and white light, exciting both PSI and PSII, suggests that there is a contribution of cyclic electron flow around PSI to electron flow through PSI in HL leaves. The data obtained are discussed in terms of photosynthetic apparatus sustainability of HL and LL leaves under variable irradiation conditions.


Assuntos
Transporte de Elétrons/efeitos da radiação , Tradescantia/metabolismo , Clorofila/metabolismo , Cloroplastos/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , Luz , Fotossíntese , Folhas de Planta/metabolismo , Tradescantia/efeitos da radiação
4.
FEBS Lett ; 592(13): 2220-2226, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29885280

RESUMO

In this work, we investigated electron transport around the photosynthetic pigment-protein complex of Photosystem I (PS I) mediated by external high-potential electron carrier 2,3-dichloro-1,4-naphtoquinone (Cl2 NQ) and ascorbate. It has been demonstrated that the oxidized species of Cl2 NQ and ascorbate serve as intermediates capable of accepting electrons from the iron-sulfur cluster FX of PS I. Reduced species of Cl2 NQ and ascorbate are oxidized by photooxidized PS I primary donor P700+ and/or by molecular oxygen. We have found the synergistic effect of Cl2 NQ and ascorbate on the rate of P700+ reduction. Accelerated electron flow to P700+, observed in the presence of both Cl2 NQ and ascorbate, is explained by an increase in the reduced species of Cl2 NQ due to electron transfer from ascorbate.


Assuntos
Ácido Ascórbico/farmacologia , Transporte de Elétrons/efeitos dos fármacos , Naftoquinonas/farmacologia , Complexo de Proteína do Fotossistema I/efeitos dos fármacos , Complexo de Proteína do Fotossistema I/metabolismo , Elétrons , Cinética , Luz , Oxirredução/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos , Synechocystis
5.
Photosynth Res ; 133(1-3): 87-102, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28176042

RESUMO

In this study, we have compared the photosynthetic characteristics of two contrasting species of Tradescantia plants, T. fluminensis (shade-tolerant species), and T. sillamontana (light-resistant species), grown under the low light (LL, 50-125 µmol photons m-2 s-1) or high light (HL, 875-1000 µmol photons m-2 s-1) conditions during their entire growth period. For monitoring the functional state of photosynthetic apparatus (PSA), we measured chlorophyll (Chl) a emission fluorescence spectra and kinetics of light-induced changes in the heights of fluorescence peaks at 685 and 740 nm (F 685 and F 740). We also compared the light-induced oxidation of P700 and assayed the composition of carotenoids in Tradescantia leaves grown under the LL and HL conditions. The analyses of slow induction of Chl a fluorescence (SIF) uncovered different traits in the LL- and HL-grown plants of ecologically contrasting Tradescantia species, which may have potential ecophysiological significance with respect to their tolerance to HL stress. The fluorometry and EPR studies of induction events in chloroplasts in situ demonstrated that acclimation of both Tradescantia species to HL conditions promoted faster responses of their PSA as compared to LL-grown plants. Acclimation of both species to HL also caused marked changes in the leaf anatomy and carotenoid composition (an increase in Violaxanthin + Antheraxantin + Zeaxanthin and Lutein pools), suggesting enhanced photoprotective capacity of the carotenoids in the plants grown in nature under high irradiance. Collectively, the results of the present work suggest that the mechanisms of long-term PSA photoprotection in Tradescantia are based predominantly on the light-induced remodeling of pigment-protein complexes in chloroplasts.


Assuntos
Aclimatação/efeitos da radiação , Clorofila/metabolismo , Luz , Tradescantia/crescimento & desenvolvimento , Tradescantia/efeitos da radiação , Xantofilas/metabolismo , Aclimatação/fisiologia , Clorofila A , Escuridão , Transporte de Elétrons/efeitos da radiação , Cinética , Oxirredução , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Espectrometria de Fluorescência , Fatores de Tempo , Tradescantia/fisiologia
6.
Biochim Biophys Acta Biomembr ; 1859(3): 388-401, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27916634

RESUMO

This work is devoted to critical analysis and reexamination of the problem of lateral heterogeneity of the trans-thylakoid pH difference (ΔpH=pHout-pHin) in thylakoid membranes of chloroplasts. Correct measurements of ΔpH may be complicated by nonuniform partitioning of the protons pumped into the lumen of granal (stacked) and stroma-exposed thylakoids. We have compared results of ΔpH estimations in isolated bean chloroplasts by two different methods. One of the methods is based on the use of pH-sensitive spin-probes. Another approach relies on the analysis of pH-dependent post-illumination reduction of P700+ - oxidized reaction center of photosystem I (PSI). Both methods lead to virtually the same values of ΔpH, as measured in the state of photosynthetic control when the ATP synthase complexes are inactive (ΔpH~2.3-2.5 at pHout~8). Under the photophosphorylation conditions (metabolic state 3), ΔpH decreases due to the proton drain from the lumen to stroma via active ATP synthases (ΔpH~1-2 at pHout~8). In this state, ΔpH values derived from kinetic data are smaller than ΔpH measured with the pH-probing amines. Such a discrepancy can be explained by the coexistence of thylakoids with different pHin established in granal and stromal thylakoids. The kinetic method is equivalent to the use of a "local pH-meter", which is sensitive to less significant decrease in pHin inside the stroma-exposed thylakoids. Otherwise, pH-indicating probes give information on pHin values averaged out at both granal and stromal thylakoids. Experimental results have been analyzed within the framework of our mathematical model developed for simulation of electron and proton transport processes in laterally heterogeneous thylakoids. The model provides a reasonable description of experimental data, supporting the notion that the long-range diffusion of protons within the lumen and obstructed diffusion of mobile electron carriers (PQH2 and Pc) influence the lateral profiles of pH along the thylakoid membranes. The model predicts significant alkalization of the inter-thylakoid gap and the establishment of nonuniform lateral profiles of ΔpH under the photophosphorylation conditions. These results are discussed in the context of the problem of energy coupling in laterally heterogeneous lamellar system of chloroplasts.


Assuntos
Cloroplastos/metabolismo , Tilacoides/metabolismo , ATPases de Cloroplastos Translocadoras de Prótons/química , ATPases de Cloroplastos Translocadoras de Prótons/metabolismo , Transporte de Elétrons , Concentração de Íons de Hidrogênio , Cinética , Luz , Modelos Biológicos , Fotossíntese , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/metabolismo , Prótons , Marcadores de Spin , Tilacoides/química , Vicia faba/crescimento & desenvolvimento , Vicia faba/metabolismo
7.
Photosynth Res ; 130(1-3): 275-291, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27037825

RESUMO

In this work, we have compared photosynthetic performance and expression of the PsbS and Lhcb1 proteins in two contrast ecotypes of Tradescantia species, T. fluminensis (shade-tolerant) and T. sillamontana (light-resistant), grown at two intensities of light: 50-125 µmol photons m-2 s-1 (low light, LL) and 875-1000 µmol photons m-2 s-1 (high light, HL). Using the EPR method for measuring the P700 content, we have found that LL-grown plants of both species have higher (by a factor of ≈1.7-1.8) contents of PSI per fresh weight unit as compared to HL-grown plants. Acclimation of plants to LL or HL irradiation also influences the Chl(a + b) level and expression of the PsbS and Lhcb1 proteins. Immunoblotting analysis showed that acclimation to HL stimulates (by a factor of ≈1.7-1.8) the level of PsbS related to the total number of P700 centers. In light-resistant species T. sillamontana, the ratio PsbS/P700 is about 2-times higher than in shade-tolerant species T. fluminensis grown under the same conditions. This should enhance the capacity of their leaves for protection against the light stress. In agreement with these observations, the capacity of leaves for NPQ induction was enhanced during plant acclimation to HL. Kinetic studies of P700 photooxidation and light-induced changes in the yield of Chl a fluorescence also revealed that the short-term regulation of electron transport processes in chloroplasts, which manifested themselves in the kinetics of [Formula: see text] induction and the rate of Chl a fluorescence quenching, occurred more rapidly in HL-grown plants than in LL-grown plants. Thus, both factors, enhanced expression of PsbS and more rapid response of the photosynthetic electron transport chain to dark-to-light transitions should increase the capacity of HL-grown plants for their resistance to rapid fluctuations of solar light.


Assuntos
Aclimatação , Clorofila/metabolismo , Tradescantia/metabolismo , Aclimatação/fisiologia , Clorofila/fisiologia , Clorofila A , Fluorescência , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Complexos de Proteínas Captadores de Luz/metabolismo , Complexos de Proteínas Captadores de Luz/fisiologia , Oxirredução , Proteínas de Plantas/metabolismo , Tradescantia/fisiologia
8.
J Photochem Photobiol B ; 152(Pt B): 400-15, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26300376

RESUMO

In this work, using the EPR and PAM-fluorometry methods, we have studied induction events of photosynthetic electron transport in Hibiscus rosa-sinensis leaves. The methods used are complementary, providing efficient tools for in situ monitoring of P700 redox transients and photochemical activity of photosystem II (PSII). The induction of P700(+) in dark-adapted leaves is characterized by the multiphase kinetics with a lag-phase, which duration elongates with the dark-adaptation time. Analyzing effects of the uncoupler monensin and artificial electron carrier methylviologen (MV) on photooxidation of P700 and slow induction of chlorophyll a fluorescence (SIF), we could ascribe different phases of transient kinetics of electron transport processes in dark-adapted leaves to the following regulatory mechanisms: (i) acceleration of electron transfer on the acceptor side of PSI, (ii) pH-dependent modulation of the intersystem electron flow, and (iii) re-distribution of electron fluxes between alternative (linear, cyclic, and pseudocyclic) pathways. Monensin significantly decreases a level of P700(+) and inhibits SIF. MV, which mediates electron flow from PSI to O2 with consequent formation of H2O2, promotes a rapid photooxidation of P700 without any lag-phase peculiar to untreated leaves. MV-mediated water-water cycle (H2O→PSII→PSI→MV→O2→H2O2→H2O) is accompanied by generation of ascorbate free radicals. This suggests that the ascorbate peroxidase system of defense against reactive oxygen species is active in chloroplasts of H. rosa-sinensis leaves. In DCMU-treated chloroplasts with inhibited PSII, the contribution of cyclic electron flow is insignificant as compared to linear electron flow. For analysis of induction events, we have simulated electron transport processes within the framework of our generalized mathematical model of oxygenic photosynthesis, which takes into account pH-dependent mechanisms of electron transport control and re-distribution of electron fluxes between alternative pathways. The model adequately describes the main peculiarities of P700(+) induction and dynamics of the intersystem electron transport.


Assuntos
Hibiscus/metabolismo , Fotossíntese , Clorofila/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Hibiscus/efeitos dos fármacos , Cinética , Modelos Biológicos , Monensin/farmacologia , Paraquat/farmacologia , Fotossíntese/efeitos dos fármacos , Complexo de Proteína do Fotossistema I/metabolismo
9.
Photosynth Res ; 122(2): 215-31, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24965848

RESUMO

Ascorbate is one of the key participants of the antioxidant defense in plants. In this work, we have investigated the interaction of ascorbate with the chloroplast electron transport chain and isolated photosystem I (PSI), using the EPR method for monitoring the oxidized centers [Formula: see text] and ascorbate free radicals. Inhibitor analysis of the light-induced redox transients of P700 in spinach thylakoids has demonstrated that ascorbate efficiently donates electrons to [Formula: see text] via plastocyanin. Inhibitors (DCMU and stigmatellin), which block electron transport between photosystem II and Pc, did not disturb the ascorbate capacity for electron donation to [Formula: see text]. Otherwise, inactivation of Pc with CN(-) ions inhibited electron flow from ascorbate to [Formula: see text]. This proves that the main route of electron flow from ascorbate to [Formula: see text] runs through Pc, bypassing the plastoquinone (PQ) pool and the cytochrome b 6 f complex. In contrast to Pc-mediated pathway, direct donation of electrons from ascorbate to [Formula: see text] is a rather slow process. Oxidized ascorbate species act as alternative oxidants for PSI, which intercept electrons directly from the terminal electron acceptors of PSI, thereby stimulating photooxidation of P700. We investigated the interaction of ascorbate with PSI complexes isolated from the wild type cells and the MenB deletion strain of cyanobacterium Synechocystis sp. PCC 6803. In the MenB mutant, PSI contains PQ in the quinone-binding A1-site, which can be substituted by high-potential electron carrier 2,3-dichloro-1,4-naphthoquinone (Cl2NQ). In PSI from the MenB mutant with Cl2NQ in the A1-site, the outflow of electrons from PSI is impeded due to the uphill electron transfer from A1 to the iron-sulfur cluster FX and further to the terminal clusters FA/FB, which manifests itself as a decrease in a steady-state level of [Formula: see text]. The addition of ascorbate promoted photooxidation of P700 due to stimulation of electron outflow from PSI to oxidized ascorbate species. Thus, accepting electrons from PSI and donating them to [Formula: see text], ascorbate can mediate cyclic electron transport around PSI. The physiological significance of ascorbate-mediated electron transport is discussed.


Assuntos
Ácido Ascórbico/química , Complexo de Proteína do Fotossistema I/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Flavoproteínas Transferidoras de Elétrons , Regulação Bacteriana da Expressão Gênica/fisiologia , Luz , Mutação , Spinacia oleracea , Synechocystis/genética , Synechocystis/metabolismo , Tilacoides
10.
Biochim Biophys Acta ; 1777(3): 285-94, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18226594

RESUMO

Nitroxide radicals are widely used as molecular probes in different fields of chemistry and biology. In this work, we describe pH-sensitive imidazoline- and imidazolidine-based nitroxides with pK values in the range 4.7-7.6 (2,2,3,4,5,5-hexamethylperhydroimidazol-1-oxyl, 4-amino-2,2,5,5-tetramethyl-2,5-dihydro-1H-imidazol-1-oxyl, 4-dimethylamino-2,2-diethyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl, and 2,2-diethyl-5,5-dimethyl-4-pyrrolidyline-1-yl-2,5-dihydro-1H-imidazol-1-oxyl), which allow the pH-monitoring inside chloroplasts. We have demonstrated that EPR spectra of these spin-probes localized in the thylakoid lumen markedly change with the light-induced acidification of the thylakoid lumen in chloroplasts. Comparing EPR spectrum parameters of intrathylakoid spin-probes with relevant calibrating curves, we could estimate steady-state values of lumen pHin established during illumination of chloroplasts with continuous light. For isolated bean (Vicia faba) chloroplasts suspended in a medium with pHout=7.8, we found that pHin approximately 5.4-5.7 in the state of photosynthetic control, and pHin approximately 5.7-6.0 under photophosphorylation conditions. Thus, ATP synthesis occurs at a moderate acidification of the thylakoid lumen, corresponding to transthylakoid pH difference DeltapH approximately 1.8-2.1. These values of DeltapH are consistent with a point of view that under steady-state conditions the proton gradient DeltapH is the main contributor to the proton motive force driving the operation of ATP synthesis, provided that stoichiometric ratio H+/ATP is n> or =4-4.7.


Assuntos
Cloroplastos/metabolismo , Óxidos N-Cíclicos/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Imidazolidinas/química , Imidazolinas/química , Marcadores de Spin , Tilacoides/metabolismo , Vicia faba/metabolismo , Trifosfato de Adenosina/metabolismo , Calibragem , Cloroplastos/efeitos dos fármacos , Cloroplastos/efeitos da radiação , Espectroscopia de Ressonância de Spin Eletrônica/normas , Concentração de Íons de Hidrogênio , Modelos Biológicos , Modelos Químicos , Oxalatos/farmacologia , Fotossíntese , Bombas de Próton/metabolismo , Força Próton-Motriz , Tilacoides/efeitos dos fármacos , Tilacoides/efeitos da radiação , Vicia faba/efeitos dos fármacos , Vicia faba/efeitos da radiação
11.
Biochim Biophys Acta ; 1708(2): 238-49, 2005 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-15953480

RESUMO

In this work, we investigated electron transport processes in the cyanobacterium Synechocystis sp. PCC 6803, with a special emphasis focused on oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains. Redox transients of the photosystem I primary donor P700 and oxygen exchange processes were measured by the EPR method under the same experimental conditions. To discriminate between the factors controlling electron flow through photosynthetic and respiratory electron transport chains, we compared the P700 redox transients and oxygen exchange processes in wild type cells and mutants with impaired photosystem II and terminal oxidases (CtaI, CydAB, CtaDEII). It was shown that the rates of electron flow through both photosynthetic and respiratory electron transport chains strongly depended on the transmembrane proton gradient and oxygen concentration in cell suspension. Electron transport through photosystem I was controlled by two main mechanisms: (i) oxygen-dependent acceleration of electron transfer from photosystem I to NADP(+), and (ii) slowing down of electron flow between photosystem II and photosystem I governed by the intrathylakoid pH. Inhibitor analysis of P700 redox transients led us to the conclusion that electron fluxes from dehydrogenases and from cyclic electron transport pathway comprise 20-30% of the total electron flux from the intersystem electron transport chain to P700(+).


Assuntos
Oxigênio/metabolismo , Fotossíntese , Synechocystis/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Cinética , Luz , Oxirredução , Marcadores de Spin , Synechocystis/fisiologia
12.
J Magn Reson ; 163(2): 257-69, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12914841

RESUMO

We present a method for measuring the transmembrane pH difference (deltapH=pHin-pHout) in chloroplasts with a spin label TEMPAMINE (4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl) accumulating inside the thylakoids in response to generation of deltapH. Experiments with chloroplasts suspended in the media of different osmolarity demonstrated that most of TEMPAMINE (TA) molecules taken up by chloroplasts were localized in the bulk of the thylakoid lumen. The DeltapH value was determined from the relationship deltapH=lg([H+]in/[H+]out) approximately equal to lg(Cin/Cout), where Cin and Cout are the concentrations of TA inside and outside the thylakoids, respectively. To quantify the internal concentration Cin, we used the threshold nature of the concentration-dependent broadening of the EPR signal from TA. It was demonstrated that spin-exchange interactions between TA molecules caused an observable broadening of the signal only when the concentration of TA exceeded the threshold level, [TA]theta approximately 2.0-2.2mM. The concentration dependencies of the signal parameters (the peak-to-peak amplitude, App, and the linewidth, deltaHpp) were described within a model of the non-homogeneous broadening of an unresolved hyperfine multiplet from the protons of TA molecule. If the concentration of TA inside the thylakoids went beyond the threshold level, the spin-exchange broadening of the EPR signal was accompanied by a reversible decrease in the signal height (parameter deltaA). By measuring the signal behavior at different levels of microwave power, we were able to discriminate between the line broadening effects caused by concentrating TA molecules inside the thylakoids or the light-induced changes in the concentration of oxygen. We developed a general algorithm for determination of the deltapH value and the internal volume of thylakoids, Vin, from the non-linear dependence of parameter deltaA on the concentration C0 of TA in a chloroplast suspension. Advantages of this method are: (i) it avoids the use of a broadening agent; (ii) it allows the internal volume of thylakoids to be evaluated; and (iii) the concentrations of TA used to measure the deltapH are below the range of concentrations that could cause the uncoupling electron transport to ATP synthesis in chloroplasts. Results of our measurements are consistent with the literature data on deltapH determinations by other methods.


Assuntos
Cloroplastos/química , Cloroplastos/metabolismo , Óxidos N-Cíclicos/química , Óxidos N-Cíclicos/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Tilacoides/química , Tilacoides/metabolismo , Algoritmos , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Simulação por Computador , Óxidos N-Cíclicos/farmacocinética , Concentração de Íons de Hidrogênio , Luz , Potenciais da Membrana , Modelos Biológicos , Folhas de Planta/química , Folhas de Planta/metabolismo , Marcadores de Spin , Tilacoides/efeitos da radiação , Vicia faba/química , Vicia faba/metabolismo
13.
FEBS Lett ; 544(1-3): 15-20, 2003 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-12782283

RESUMO

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.


Assuntos
Cianobactérias/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Transporte de Elétrons , Antibacterianos/farmacologia , Cianatos/farmacologia , Diurona/farmacologia , Herbicidas/farmacologia , Cinética , Luz , NADP/metabolismo , Nigericina/farmacologia , Nitrogênio/metabolismo , Oxirredução , Oxigênio/metabolismo , Prótons , Espectrofotometria , Fatores de Tempo , Raios Ultravioleta , Valinomicina/farmacologia
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