The cytochrome b6f complex: plastoquinol oxidation and regulation of electron transport in chloroplasts.

In oxygenic photosynthetic systems, the cytochrome b6f (Cytb6f) complex (plastoquinol:plastocyanin oxidoreductase) is a heart of the hub that provides connectivity between photosystems (PS) II and I. In this review, the structure and function of the Cytb6f complex are briefly outlined, being focused on the mechanisms of a bifurcated (two-electron) oxidation of plastoquinol (PQH2). In plant chloroplasts, under a wide range of experimental conditions (pH and temperature), a diffusion of PQH2 from PSII to the Cytb6f does not limit the intersystem electron transport. The overall rate of PQH2 turnover is determined mainly by the first step of the bifurcated oxidation of PQH2 at the catalytic site Qo, i.e., the reaction of electron transfer from PQH2 to the Fe2S2 cluster of the high-potential Rieske iron-sulfur protein (ISP). This point has been supported by the quantum chemical analysis of PQH2 oxidation within the framework of a model system including the Fe2S2 cluster of the ISP and surrounding amino acids, the low-potential heme b6L, Glu78 and 2,3,5-trimethylbenzoquinol (the tail-less analog of PQH2). Other structure-function relationships and mechanisms of electron transport regulation of oxygenic photosynthesis associated with the Cytb6f complex are briefly outlined: pH-dependent control of the intersystem electron transport and the regulatory balance between the operation of linear and cyclic electron transfer chains.

Electron Transport in Chloroplasts: Regulation and Alternative Pathways of Electron Transfer.

This work represents an overview of electron transport regulation in chloroplasts as considered in the context of structure-function organization of photosynthetic apparatus in plants. Main focus of the article is on bifurcated oxidation of plastoquinol by the cytochrome b6f complex, which represents the rate-limiting step of electron transfer between photosystems II and I. Electron transport along the chains of non-cyclic, cyclic, and pseudocyclic electron flow, their relationships to generation of the trans-thylakoid difference in electrochemical potentials of protons in chloroplasts, and pH-dependent mechanisms of regulation of the cytochrome b6f complex are considered. Redox reactions with participation of molecular oxygen and ascorbate, alternative mediators of electron transport in chloroplasts, have also been discussed.

Electrometric and Electron Paramagnetic Resonance Measurements of a Difference in the Transmembrane Electrochemical Potential: Photosynthetic Subcellular Structures and Isolated Pigment-Protein Complexes.

A transmembrane difference in the electrochemical potentials of protons (ΔµH+) serves as a free energy intermediate in energy-transducing organelles of the living cell. The contributions of two components of the ΔµH+ (electrical, Δψ, and concentrational, ΔpH) to the overall ΔµH+ value depend on the nature and lipid composition of the energy-coupling membrane. In this review, we briefly consider several of the most common instrumental (electrometric and EPR) methods for numerical estimations of Δψ and ΔpH. In particular, the kinetics of the flash-induced electrometrical measurements of Δψ in bacterial chromatophores, isolated bacterial reaction centers, and Photosystems I and II of the oxygenic photosynthesis, as well as the use of pH-sensitive molecular indicators and kinetic data regarding pH-dependent electron transport in chloroplasts, have been reviewed. Further perspectives on the application of these methods to solve some fundamental and practical problems of membrane bioenergetics are discussed.

Plastoquinol Oxidation: Rate-Limiting Stage in the Electron Transport Chain of Chloroplasts.

This work is devoted to theoretical study of functioning of the cytochrome (Cyt) b6f complex (plastoquinol:plastocyanin oxidoreductase) of the electron transport chain (ETC) in oxygenic photosynthesis. A composition of the chloroplast ETC and molecular mechanisms of functioning of the Cyt b6f complex, which stands between photosystems II and I (PSII and PSI), are briefly reviewed. The Cyt b6f complex oxidizes plastoquinol (PQH2) molecules formed in PSII, and reduces plastocyanin, which serves as an electron donor to PSI. PQH2 oxidation is the rate-limiting step in the chain of electron transfer processes between PSII and PSI. Using the density functional theory (DFT) method, we have analyzed the two-electron (bifurcated) oxidation of PQH2 in the catalytic center Qo of the Cyt b6f complex. Results of DFT calculations are consistent with the fact that the first step of PQH2 oxidation, electron transfer to the Fe2S2 cluster of the iron-sulfur protein (ISP), is an endergonic (energy-accepting) process (ΔE ≈ 15 kJ·mol-1) that can limit turnover of the Cyt b6f complex. The second stage of bifurcated oxidation of PQH2 - electron transfer from semiquinone (PQH•, formed after the first step of PQH2 oxidation) to heme b6L - is the exergonic (energy-donating) process (ΔE < 0). DFT modeling of this stage revealed that semiquinone oxidation should accelerate after the PQH• radical shift towards the heme b6L (an electron acceptor) and the carboxy group of Glu78 (a proton acceptor). The data obtained are discussed within the framework of the Mitchell Q-cycle model describing PQH2 oxidation at the Qo site of the Cyt b6f complex.

The noninvasive monitoring of the redox status of photosynthetic electron transport chains in Hibiscus rosa-sinensis and Tradescantia leaves.

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.

Structural and Functional Aspects of Electron Transport Thermoregulation and ATP Synthesis in Chloroplasts.

The review is focused on analysis of the mechanisms of temperature-dependent regulation of electron transport and ATP synthesis in chloroplasts of higher plants. Importance of photosynthesis thermoregulation is determined by the fact that plants are ectothermic organisms, whose own temperature depends on the ambient temperature. The review discusses the effects of temperature on the following processes in thylakoid membranes: (i) photosystem 2 activity and plastoquinone reduction; (ii) electron transfer from plastoquinol (via the cytochrome b6f complex and plastocyanin) to photosystem 1; (iii) transmembrane proton transfer; and (iv) ATP synthesis. The data on the relationship between the functional properties of chloroplasts (photosynthetic transfer of electrons and protons, functioning of ATP synthase) and structural characteristics of membrane lipids (fluidity) obtained by electron paramagnetic resonance studies are presented.

Temperature-dependent regulation of electron transport and ATP synthesis in chloroplasts in vitro and in silico.

The significance of temperature-dependent regulation of photosynthetic apparatus (PSA) is determined by the fact that plant temperature changes with environmental temperature. In this work, we present a brief overview of temperature-dependent regulation of photosynthetic processes in class B chloroplasts (thylakoids) and analyze these processes using a computer model that takes into account the key stages of electron and proton transport coupled to ATP synthesis. The rate constants of partial reactions were parametrized on the basis of experimental temperature dependences of partial photosynthetic processes: (1) photosystem II (PSII) turnover and plastoquinone (PQ) reduction, (2) the plastoquinol (PQH2) oxidation by the cytochrome (Cyt) b6f complex, (3) the ATP synthase activity, and (4) the proton leak from the thylakoid lumen. We consider that PQH2 oxidation is the rate-limiting step in the intersystem electron transport. The parametrization of the rate constants of these processes is based on earlier experimental data demonstrating strong correlations between the functional and structural properties of thylakoid membranes that were probed with the lipid-soluble spin labels embedded into the membranes. Within the framework of our model, we could adequately describe a number of experimental temperature dependences of photosynthetic reactions in thylakoids. Computer modeling of electron and proton transport coupled to ATP synthesis supports the notion that PQH2 oxidation by the Cyt b6f complex and proton pumping into the lumen are the basic temperature-dependent processes that determine the overall electron flux from PSII to molecular oxygen and the net ATP synthesis upon variations of temperature. The model describes two branches of the temperature dependence of the post-illumination reduction of [Formula: see text] characterized by different activation energies (about 60 and ≤ 3.5 kJ mol-1). The model predicts the bell-like temperature dependence of ATP formation, which arises from the balance of several factors: (1) the thermo-induced acceleration of electron transport through the Cyt b6f complex, (2) deactivation of PSII photochemistry at sufficiently high temperatures, and (3) acceleration of the passive proton outflow from the thylakoid lumen bypassing the ATP synthase complex. The model describes the temperature dependence of experimentally measured parameter P/2e, determined as the ratio between the rates of ATP synthesis and pseudocyclic electron transport (H2O → PSII → PSI → O2).

Electron transport in Tradescantia leaves acclimated to high and low light: thermoluminescence, PAM-fluorometry, and EPR studies.

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.

Slow induction of chlorophyll a fluorescence excited by blue and red light in Tradescantia leaves acclimated to high and low light.

Tradescantia is a good model for assaying induction events in higher plant leaves. Chlorophyll (Chl) fluorescence serves as a sensitive reporter of the functional state of photosynthetic apparatus in chloroplasts. The fluorescence time-course depends on the leaf growth conditions and actinic light quality. In this work, we investigated slow induction of Chl a fluorescence (SIF) excited by blue light (BL, λmax = 455 nm) or red light (RL, λmax = 630 nm) in dark-adapted leaves of Tradescantia fluminensis acclimated to high light (~ 1000 µmol photons m-2 s-1; HL) or low light (~ 100 µmol photons m-2 s-1; LL). Our special interest was focused on the contribution of the avoidance response to SIF kinetics. Bearing in mind that BL and RL have different impacts on photoreceptors that initiate chloroplast movements within the cell (accumulation/avoidance responses), we have compared the SIF patterns during the action of BL and RL. The time-courses of SIF and kinetics of non-photochemical quenching (NPQ) of Chl a fluorescence revealed a certain difference when leaves were illuminated by BL or RL. In both cases, the yield of fluorescence rose to the maximal level P and then, after the lag-phase P-S-M1, the fluorescence level decreased toward the steady state T (via the intermediate phases M1-M2 and M2-T). In LL-acclimated leaves, the duration of the P-S-M1 phase was almost two times longer that in HL-grown plants. In the case of BL, the fluorescence decay included the transient phase M1-M2. This phase was obscure during the RL illumination. Non-photochemical quenching of Chl a fluorescence has been quantified as [Formula: see text], where [Formula: see text] and [Formula: see text] stand for the fluorescence response to saturating pulses of light applied to dark-adapted and illuminated samples, respectively. The time-courses of such a formally determined NPQ value were markedly different during the action of RL and BL. In LL-grown leaves, BL induced higher NPQ as compared to the action of RL. In HL-grown plants, the difference between the NPQ responses to BL and RL illumination was insignificant. Comparing the peculiarities of Chl a fluorescence induced by BL and RL, we conclude that the avoidance response can provide a marked contribution to SIF and NPQ generation. The dependence of NPQ on the quality of actinic light suggests that chloroplast movements within the cell have a noticeable impact on the formally determined NPQ value. Analyzing kinetics of post-illumination decay of NPQ in the context of solar stress resistance, we have found that LL-acclimated Tradescantia leaves are more vulnerable to strong light than the HL-grown leaves.

Photo-reducible plastoquinone pools in chloroplasts of Tradescentia plants acclimated to high and low light.

In photosynthetic systems of oxygenic type, plastoquinone (PQ) molecules are reduced by photosystem II (PSII). The turnover of PQ determines the rate of PSII operation. PQ molecules are present in surplus with respect to PSII. In this work, using the pulse amplitude modulation-fluorometry technique, we quantified photo-reducible PQ pools in chloroplasts of two contrasting ecotypes of Tradescantia, acclimated either to low light (~ 100 µmol photons·m-2 ·s-1 , LL) or to high light (~ 1000 µmol photons·m-2 ·s-1 , HL). The LL-grown plants are characterized by higher capacity of rapidly reducible PQ pool ([PQ]0 /[PSII] ≈ 8) as compared to HL-grown plants of both species ([PQ]0 /[PSII] ≈ 4). The elevated content of PQ in LL plants favours photosynthetic electron flow at low-solar irradiance.

Three phases of energy-dependent induction of [Formula: see text] and Chl a fluorescence in Tradescantia fluminensis leaves.

In plants, the short-term regulation (STR, seconds to minute time scale) of photosynthetic apparatus is associated with the energy-dependent control in the chloroplast electron transport, the distribution of light energy between photosystems (PS) II and I, activation/deactivation of the Calvin-Benson cycle (CBC) enzymes, and relocation of chloroplasts within the plant cell. In this work, using a dual-PAM technique for measuring the time-courses of P700 photooxidation and Chl a fluorescence, we have investigated the STR events in Tradescantia fluminensis leaves. The comparison of Chl a fluorescence and [Formula: see text] induction allowed us to investigate the contribution of the trans-thylakoid pH difference (ΔpH) to the STR events. Two parameters were used as the indicators of ΔpH generation: pH-dependent component of non-photochemical quenching of Chl a fluorescence, and pHin-dependent rate of electron transfer from plastoquinol (PQH2) to [Formula: see text] (via the Cyt b6f complex and plastocyanin). In dark-adapted leaves, kinetics of [Formula: see text] induction revealed three phases. Initial phase is characterized by rapid electron flow to [Formula: see text] (τ1/2 ~ 5-10 ms), which is likely related to cyclic electron flow around PSI, while the outflow of electrons from PSI is restricted by slow consumption of NADPH in the CBC. The light-induced generation of ΔpH and activation of the CBC promote photooxidation of P700 and concomitant retardation of [Formula: see text] reduction (τ1/2 ~ 20 ms). Prolonged illumination induces additional slowing down of electron transfer to [Formula: see text] (τ1/2 ≥ 30-35 ms). The latter effect is not accompanied by changes in the Chl a fluorescence parameters which are sensitive to ΔpH generation. We suggest the tentative explanation of the latter results by the reversal of Q-cycle, which causes the deceleration of PQH2 oxidation due to the back pressure of stromal reductants.

Light acclimation of shade-tolerant and sun-resistant Tradescantia species: photochemical activity of PSII and its sensitivity to heat treatment.

In this work, we have compared photosynthetic characteristics of photosystem II (PSII) in Tradescantia leaves of two contrasting ecotypes grown under the low light (LL) and high light (HL) regimes during their entire growth period. Plants of the same genus, T. fluminensis (shade-tolerant) and T. sillamontana (sun-resistant), were cultivated at 50-125 µmol photons m-2 s-1 (LL) or at 875-1000 µmol photons m-2 s-1 (HL). Analyses of intrinsic PSII efficiency was based on measurements of fast chlorophyll (Chl) a fluorescence kinetics (the OJIP test). The fluorescence parameters Fv/Fm (variable fluorescence) and F0 (the initial level of fluorescence) in dark-adapted leaves were used to quantify the photochemical properties of PSII. Plants of different ecotypes showed different sustainability with respect to changes in the environmental light intensity and temperature treatment. The sun-resistant species T. sillamontana revealed the tolerance to variations in irradiation intensity, demonstrating constancy of maximum quantum efficiency of PSII upon variations of the growth light. In contrast to T. sillamontana, facultative shade species T. fluminensis demonstrated variability of PSII photochemical activity, depending on the growth light intensity. The susceptibility of T. fluminensis to solar stress was documented by a decrease in Fv/Fm and a rise of F0 during the long-term exposition of T. fluminensis to HL, indicating the loss of photochemical activity of PSII. The short-term (10 min) heat treatment of leaf cuttings caused inactivation of PSII. The temperature-dependent heating effects were different in T. fluminensis and T. sillamontana. Sun-resistant plants T. sillamontana acclimated to LL and HL displayed the same plots of Fv/Fm versus the treatment temperature (t), demonstrating a decrease in Fv/Fm at t ≥ 45 °C. The leaves of shadow-tolerant species T. fluminensis grown under the LL and HL conditions revealed different sensitivities to heat treatment. Plants grown under the solar stress conditions (HL) demonstrated a gradual decline of Fv/Fm at lower heating temperatures (t ≥ 25 °C), indicating the "fragility" of their PSII as compared to T. fluminensis grown at LL. Different responses of sun and shadow species of Tradescantia to growth light and heat treatment are discussed in the context of their biochemical and ecophysiological properties.

Oxygenic photosynthesis: EPR study of photosynthetic electron transport and oxygen-exchange, an overview.

In this review, we consider the applications of electron paramagnetic resonance (EPR) methods to the study of the relationships between the electron transport and oxygen-exchange processes in photosynthetic systems of oxygenic type. One of the purposes of this article is to encourage scientists to use the advantageous EPR oximetry approaches to study oxygen-related electron transport processes in photosynthetic systems. The structural organization of the photosynthetic electron transfer chain and the EPR approaches to the measurements of molecular oxygen (O2) with O2-sensitive species (nitroxide spin labels and solid paramagnetic particles) are briefly reviewed. In solution, the collision of O2 with spin probes causes the broadening of their EPR spectra and the reduction of their spin-lattice relaxation times. Based on these effects, tools for measuring O2 concentration and O2 diffusion in biological systems have been developed. These methods, named "spin-label oximetry," include not only nitroxide spin labels, but also other stable-free radicals with narrow EPR lines, as well as particulate probes with EPR spectra sensitive to molecular oxygen (lithium phthalocyanine, coals, and India ink). Applications of EPR approaches for measuring O2 evolution and consumption are illustrated using examples of photosynthetic systems of oxygenic type, chloroplasts in situ (green leaves), and cyanobacteria.

Cyclic electron transfer around Photosystem I mediated by 2,3-dichloro-1,4-naphtoquinone and ascorbate.

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.

The Cytochrome b 6 f Complex: Biophysical Aspects of Its Functioning in Chloroplasts.

This chapter presents an overview of structural properties of the cytochrome (Cyt) b 6 f complex and its functioning in chloroplasts. The Cyt b 6 f complex stands at the crossroad of photosynthetic electron transport pathways, providing connectivity between Photosystem (PSI) and Photosysten II (PSII) and pumping protons across the membrane into the thylakoid lumen. After a brief review of the chloroplast electron transport chain, the consideration is focused on the structural organization of the Cyt b 6 f complex and its interaction with plastoquinol (PQH2, reduced form of plastoquinone), a mediator of electron transfer from PSII to the Cyt b 6 f complex. The processes of PQH2 oxidation by the Cyt b 6 f complex have been considered within the framework of the Mitchell's Q-cycle. The overall rate of the intersystem electron transport is determined by PQH2 turnover at the quinone-binding site Qo of the Cyt b 6 f complex. The rate of PQH2 oxidation is controlled by the intrathylakoid pHin, which value determines the protonation/deprotonation events in the Qo-center. Two other regulatory mechanisms associated with the Cyt b 6 f complex are briefly overviewed: (i) redistribution of electron fluxes between alternative (linear and cyclic) pathways, and (ii) "state transitions" related to redistribution of solar energy between PSI and PSII.

Photosynthetic Electron and Proton Transport in Chloroplasts: EPR Study of ΔpH Generation, an Overview.

This is a brief overview focused on the electron paramagnetic resonance applications to the study of the proton transport processes in chloroplasts. After brief description of structural and functional organization of the chloroplast electron transport chain, our attention is focused on the measurements of trans-thylakoid pH difference (ΔpH) with pH-sensitive spin-probes. The use of spin-probes is based either (i) on measuring the ΔpH-partitioning of spin-probes between the thylakoid lumen and external volume, or (ii) on monitoring changes in the electron paramagnetic resonance spectra of pH-sensitive nitroxide radicals located in the lumen. Along with the use of spin-probes, the intra-thylakoid pH (pHin) can be determined by the "kinetic" method, which relies on the fact that the rate-limiting step in the chain of photosynthetic electron transfer (plastoquinol oxidation by the cytochrome b 6 f complex) is controlled by pHin. The results of ΔpH determinations in chloroplasts based on the use of pH-sensitive spin-probes and measurements of post-illumination reduction of photoreaction centers of Photosystem I are discussed in the context of the problem of energy coupling in laterally heterogeneous lamellar system of chloroplasts.

Connectivity between electron transport complexes and modulation of photosystem II activity in chloroplasts.

In chloroplasts, photosynthetic electron transport complexes interact with each other via the mobile electron carriers (plastoquinone and plastocyanin) which are in surplus amounts with respect to photosystem I and photosystem II (PSI and PSII), and the cytochrome b 6 f complex. In this work, we analyze experimental data on the light-induced redox transients of photoreaction center P700 in chloroplasts within the framework of our mathematical model. This analysis suggests that during the action of a strong actinic light, even significant attenuation of PSII [for instance, in the result of inhibition of a part of PSII complexes by DCMU or due to non-photochemical quenching (NPQ)] will not cause drastic shortage of electron flow through PSI. This can be explained by "electronic" and/or "excitonic" connectivity between different PSII units. At strong AL, the overall flux of electrons between PSII and PSI will maintain at a high level even with the attenuation of PSII activity, provided the rate-limiting step of electron transfer is beyond the stage of PQH2 formation. Results of our study are briefly discussed in the context of NPQ-dependent mechanism of chloroplast protection against light stress.

Acclimation of shade-tolerant and light-resistant Tradescantia species to growth light: chlorophyll a fluorescence, electron transport, and xanthophyll content.

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.

Lateral heterogeneity of the proton potential along the thylakoid membranes of chloroplasts.

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.