Light-induced electron transport and coupled processes in digitonin-fractionated pea subchloroplast particles enriched in photosystem I I. Experimental study

This paper is concerned with the light-induced oxidation-reductions of chlorophyll P-700 in ‘ligh’ digitonin-fractionated pea subchloroplast particles enriched in PS I. In the absence of exogenous cofactors of cyclic and non-cyclic electron transfer, the dark recovery of P-700 after its oxidation in the light occurs slowly. Upon addition of Cl₂ Ind (2,6-dichlorophenol indophenol) it proceeds considerably faster, the steady-state level of P-700 oxidation being reduced and the saturation level being shifted towards higher intensities of actinic light. MV (methylviologen) has an opposite effect. Similar behaviour of the dependence of P-700 oxidation on light was detected after the addition of both uncouplers and substrates of phosphorylation. The subchloroplast fragments, in the presence of Cl₂Ind-H₂, are capable of carrying out the electron transport-coupled processes of energy transduction. The site of coupling is supposed to be located at the level of the acceptors of the photosynthetic electron transport chain. This site can arise artificially via a lipophylic cofactor shuttle as a consequence of a vectorial orientation of functional components of the electron transport chain in photosynthetic membranes.     

On the functional organization of electron transport from plastoquinone to Photosystem I

Signal I, the EPR signal of P-700, induced by long flashes as well as the rate of linear electron transport are investigated at partial inhibition of electron transport in chloroplasts. Inhibition of plastoquinol oxidation by dibromothymoquinone and bathophenanthroline, inhibition of plastocyanin by KCN and HgCl₂, and inhibition by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are used to study a possible electron exchange between electron-transport chains after plastoquinone. (1) At partial inhibition of plastocyanin the reduction kinetics of P-700⁺ show a fast component comparable to that in control chloroplasts and a new slow component. The slow component indicates P-700⁺ which is not accessible to residual active plastocyanin under these conditions. We conclude that P-700 is reduced via complexed plastocyanin. (2) The rate of linear electron transport at continuous illumination decreases immediately when increasing amounts of plastocyanin are inhibited by KCN incubation. This is not consistent with an oxidation of cytochrome f by a mobile pool of plastocyanin with respect to the reaction rates of plastocyanin being more than an order of magnitude faster than the rate-limiting step of linear electron transport. It is evidence for a complex between the cytochrome b₆ - f complex and plastocyanin. The number of these complexes with active plastocyanin is concluded to control the rate-limiting plastoquinol oxidation. (3) Partial inhibition of the electron transfer between plastoquinone and cytochrome f by dibromothymoquinone and bathophenanthroline causes decelerated monophasic reduction of total P-700⁺. The P-700 kinetics indicate an electron transfer from the cytochrome b₆ - f complex to more than ten Photosystem I reaction center complexes. This cooperation is concluded to occur by lateral diffusion of both complexes in the membrane. (4) The proposed functional organization of electron transport from plastoquinone to P-700 in situ is supported by further kinetic details and is discussed in terms of the spatial distribution of the electron carriers in the thylakoid membrane.     

Measurements of cytochrome f and P-700 in intact leaves of Sinapis alba grown under high-light and low-light conditions

The oxidation and reduction of cytochrome f and P-700 is measured spectrophotometrically in leaves of low-light and high-light plants. After illumination with red light, an induction phenomenon for cytochrome f oxidation is observed which indicates a regulation of photosystem I activity through energy distribution between the pigment systems by the energy state of the membrane. After far-red excitation the reduction of cytochrome f in the dark is much slower in low-light leaves. This shows that cyclic electron transport is not improved in low-light plants under these conditions. P-700 is oxidized on excitation with far-red light. However, with high intensities of far-red light, P-700 is partially reduced again which is due to a low extent of photosystem II excitation with the far-red used in the experiments. The low-light leaves show greater sensitivity of photosystem II to this excitation. The initial rate of the cytochrome f oxidation-rate is the same in low-light and high-light leaves. This shows that several P-700 are connected with only one electron transport chain. The consequences of these results concerning the tripartite concept and the photosynthetic unit are discussed. In the high-light plants the experimental data can be well explained by the tripartite organization of the photosynthetic unit. In low-light plants, however, a multipartite organization has to be postulated. In the partition regions of the grana, several antennae systems I, antennae systems II, and light-harvesting complexes can communicate with one electron transport chain.Abbreviations CP I P-700-chlorophyll a-protein - Cyt f cytochrome f - DCMU 3-(3,4 dichlorophenyl)-1,1-dimethylurea - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - LA leaf-area - PhAR photosynthetically active radiation - PS photosystem     

Transient kinetics of the electron transfer between P-700, plastocyanin and cytochrome f in chloroplasts suspended in fluid media at sub-zero temperatures

The kinetics of redox changes of P-700, plastocyanin and cytochrome f in chloroplasts suspended in a fluid medium at sub-zero temperatures have been studied following excitation of the chloroplasts with either a single-turnover flash, a series of flashes or continuous light. The results show that: (1) The kinetics of reduction of P-700⁺ and those of oxidation of plastocyanin are consistent with a bimolecular reaction between these two components as previously suggested (Olsen, L.F., Cox, R.P. and Barber, J. (1980) FEBS Lett. 122, 13–16). (2) Cytochrome f shows heterogeneity with respect to its kinetics of oxidation by Photosystem I. (3) In contrast to the situation when plastoquinol is the electron donor, reduction of cytochrome f by electrons derived from diaminodurene occurs with sigmoidal kinetics that shows a good fit to an apparent equilibrium constant of 12 between the cytochrome and P-700. (4) The rate of electron transfer from plastoquinol to Photosystem I depends on the redox state of the plastoquinone pool. (5) In relation to current ideas about the lateral heterogeneity of Photosystem I and Photosystem II in the thylakoid membrane, the results are consistent with the function of plastocyanin as a mobile carrier of electrons in the intrathylakoid space.     

Variable chlorophyll a fluorescence from P-700 enriched photosystem I particles dependent on the redox state of the reaction centre.

1. Photosystem I particles enriched in P-700 prepared by Triton X-100 treatment of chloroplasts show a light-induced increase in fluorescence yield of more than 100% in the presence of dithionite but not in its absence. 2. Steady state light maintains the P-700, of these particles, in the oxidised state when ascorbate is present but in the presence of dithionite only a transient oxidation occurs. 3 EPR data show that, in these particles, the primary electron acceptor (X) is maintained in the reduced state by light at room temperature only when the dithionite is also present. In contrast, the secondary electron acceptors are reduced in the dark by dithionite. 4. Fluorescence emission and excitation spectra and fluorescence lifetime measurements for the constant and variable fluorescence indicate a heterogeneity of the chlorophyll in these particles. 5. It is concluded that the variable fluorescence comes from those chlorophylls which can transfer their energy to the reaction centre and that the states PX and P+X are more effective quenchers of chlorophyll fluorescence than PX-, where P is P-700.     

The reduction kinetics of chlorophyll aI as an indicator for proton uptake between the light reactions in chloroplasts.

The flash-induced oxidation kinetics of the primary acceptor of light Reaction II (X-320) and the reduction kinetics of chlorophyll aI (P-700) after far-red preillumination have been studied with high time resolution in spinach chloroplasts. 1. The kinetics of chlorophyll aI exhibits a pronounced lag phase of 2--3 ms at the onset of reduction as would be expected for the final product of consecutive reactions. Because the oxidation of the plastoquinone pool is the rate-limiting step for the electron transport between the two light reactions, the lag indicates the maximal electron transfer time over all preceding reactions after light Reaction II. 2. The observation that the lag phase decreases with decreasing pH is evidence of an electron transfer step coupled to a proton uptake reaction. 3. Protonation of X-320 after reduction in the flash is excluded because a slight increase of the decay time is found at decreasing pH values. 4. The time course of plastohydroquinone formation is deduced from the first derivative of the reduction kinetics of chlorophyll aI. This approach covers those plastohydroquinone molecules being available to the electron carriers of System I via the rate-limiting step. Direct measurements of absorbance changes would not allow to discriminate between these and functionally different plastohydroquinone molecules. 5. The derived time course of plastohydroquinone at different pH gives evidence for an additional electron transfer step with a half time of about 1 ms following the proton uptake and preceding the rate-limiting step. It is tentatively attributed to the diffusion of neutral plastohydroquinone across the hydrophobic core of the thylkaloid membrane. 6. The lower limit of the rate constant for proton uptake by an electron carrier, consistent with the lag of chlorophyll aI reduction, is estimated as greater than 10(11) M-1s-1. The value is higher than that of the fastest diffusion controlled protonations of organic molecules in solution. Possible mechanisms of linear electron transport between light Reaction II and the rate-limiting oxidation of neutral plastohydroquinone are thoroughly discussed.     

Adaptation of photosynthesis under iron deficiency in maize

This paper explores the effects of high light stress on Fe-deficient plants. Maize (Zea mays) plants were grown under conditions of Fe deficiency and complete nutrition. Attached, intact leaves of Fe-deficient and control plants were used for gas exchange experiments under suboptimal, optimal and photoinhibitory illumination. Isolated chloroplasts were used to study photosynthetic electron transport system, compromised by the induction of Fe deficiency. The reaction centers of PS II (measured as reduction of Q, the primary electron acceptor of P 680) and PS I (measured as oxidation of P 700) were estimated from the amplitude of light induced absorbance change at 320 and 700 nm, respectively. Plants were subjected to photoinhibitory treatment for different time periods and isolated chloroplasts from these plants were used for electron transport studies. Carbon dioxide fixation in control as well as in Fe-deficient plants decreased in response to high light intensities. Total chlorophyll, P 700 and Q content in Fe-deficient chloroplasts decreased, while Chl a/b ratio and Q/P 700 ratio increased. However, electron transport through PS II suffered more after photoinhibitory treatment as compared to electron transport through PS I or whole chain. Electron transfer through PS I+PS II, excluding the water oxidation complex showed a decrease in Fe-deficient plants. However, electron transport through this part of the chain did not suffer much as a result of photoinhibition, suggesting a defect in the oxidising side of PS II.     

Changes in P-700 Oxidation during the Early Stages of the Induction of Photosynthesis

Following dark adaptation, the response to irradiance of chlorophyll (Chl) fluorescence, the light-induced absorbance change around 820 nm (to measure reaction center Chl of photosystem I [PSI] P-700 oxidation), and CO2 fixation were examined in pea (Pisum sativum L.) leaves under a range of conditions. Initially, P-700 oxidation is restricted by a lack of regeneration of PSI electron acceptors, and the increase of oxidized P-700 (P-700+) that occurs during approximately the first 60 s of irradiation is largely independent of the resistance to electron flow between the two photosystems. Under these conditions, the quantum efficiency for linear electron flow is directly positively related to P-700+ accumulation, which is in contrast to the direct negative correlation that is the most frequently reported relationship between P-700+ accumulation and the quantum efficiency for linear electron flow.     

The site of KCN inhibition in the photosynthetic electron transport pathway

Treatment of chloroplasts with high concentrations of KCN inhibits reactions which involve Photosystem I (e.g. electron transport from water or diaminodurene to methylviologen), but not those assumed to by-pass Photosystem I (e.g. electron transport from water to quinonediimides). The spectrophotometric experiments described in this paper showed that KCN inhibits the oxidation of cytochrome f by far-red light without blocking its reduction by red light. Both optical and EPR experiments indicated that KCN does not inhibit the photooxidation of P700 but markedly slows down the subsequent dark decay (reduction). Reduction of P700 by Photosystem II is prevented by KCN. It is concluded that KCN blocks electron transfer between cytochrome f and P700, i.e. the reaction step which is believed to be mediated by plastocyanin. In KCN-poisoned chloroplasts the slow dark reduction of P700 following photooxidation is greatly accelerated by reduced 2,6-dichlorophenolindophenol or by reduced N-methylphenazonium methosulfate (PMS), but not by diaminodurene. It appears that the reduced indophenol dye and reduced PMS are capable of donating electrons directly to P700, at least partially by-passing the KCN block.     

Flash photolysis-electron spin resonance studies of photosystem I. A fast reduction of component of P-700+.

A 300 mus decay component of ESR Signal I (P-700+) in chloroplasts is observed following a 10 mus actinic xenon flash. This transient is inhibited by treatments which block electron transfer from Photosystem II to Photosystem I (e.g. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), KCN and HgCl2). The fast transient reduction of P-700+ can be restored in the case of DCMU or DBMIB inhibition by addition of an electron donor couple (2,6-dichlorophenol indophenol (Cl2Ind)/ascorbate) which supplies electrons to cytochrome f. However, this donor couple is inefficient in restoring electron transport in chloroplasts which have been inhibited with the plastocyanin inactivators, KCN and HgCl2. Oxidation-reduction measurements reveal that the fast P-700+ reduction component reflects electron transfer from a component with Em = 375 +/- 10 mV (pH = 7.5). These data suggest the assignment of the 300-mus decay kinetics to electron transfer from cytochrome f (Fe2+) to P-700+, thus confirming the recent observations of Haehnel et al. (Z. Naturforsch. 26b, 1171-1174 (1971)).     

Correlation of redox levels of component electron carriers with total electron flux in an electron-transport system. P-700 and the photoreduction of NADP+ in chloroplast fragments.

A mathematical analysis is described which measures the effects of actinic light intensity and concentration of an artificial electron donor on the steady-state light-induced redox level of a reaction-center pigment (e.g. P-700) and on the overall light-induced electron flux (e.g. reduction of NADP+). The analysis led to a formulation (somewhat similar to the Michaelis-Menten equation for enzyme kinetics) in which a parameter, I1/2, is defined as the actinic light intensity that, at a given concentration of electron donro, renders the reaction-center pigment half oxidized and half reduced. To determine the role of a presumed reaction-center pigment, I1/2 is compared with another parameter, equivalent to I1/2, that is obtained independently of the reaciton-center pigment by measuring the effect of actinic light intensity and concentration of electron donor on the overall electron flow. The theory was tested and validated in a model system with spinach Photosystem I chloroplast fragments by measurements of photooxidation of P-700 and light-induced reduction of NADP+ by reduced 2,6-dichlorophenolindophenol. A possible extension of this mathematical analysis to more general electron-transport systems is discussed.     

Evidence for two types of P-700 in membrane fragments from a blue-green alga.

The mathematical analysis described in the preceding paper (Biochim. Biophys. Acta (1977) 460, 65-75), in which the steady-state photooxidation of P-700 was compared with overall electron flux in Photosystem I chloroplast fragments, was applied to membrane fragments from the blue-gree alga Nostoc muscorum (Strain 7119) noted for their high activity of both Photosystem I and Photosystem II. The same analysis, which gave good agreement between the photooxidation of P-700 and the overall light-induced electron flux (measured as NADP+ reduction) in Photosystem I chloroplast fragments, revealed in the algal membrane fragments two P-700 components: one responding to high light intensity (P-700 HI), the photooxidation of which was in good agreement with the overall electron flux (measured as NADP+ reduction by reduced 2,6-dichlorophenolindophenol), and the other component responding to low light intensity (P-700 LI), the photooxidation of which was not correlated with the reduction of NADP+ by reduced 2,6-dichlorophenolindophenol.     

Nonmonotonic Redox Conversions of P700 in Leaves Irradiated with Far-Red Light Originate from Variable Contributions of Several Alternative Electron Transport Pathways during the Induction Period

The origin of nonmonotonic changes in the redox state of P700, the primary electron donor of PSI, was investigated on predarkened barley (Hordeum vulgare L.) leaves exposed to far-red light. To accomplish this, the relaxation kinetics of absorbance changes at 830 nm, reflecting the dark reduction of P700⁺, were measured at different stages of the induction curve. The onset of far-red light resulted in rapid oxidation of P700, which was followed by its partial reduction and subsequent slow oxidation of P700 to a steady-state level. This steady-state level was usually attained within 10 s under far-red light. The relative contribution of the slow kinetic component of P700⁺ reduction decreased in parallel with the transient photoreduction of P700⁺ and increased upon a subsequent stage of P700 photooxidation. The contribution of the middle component to the dark reduction of P700⁺ increased monotonically with the length of far-red light irradiation. The relative amplitude of the fast component of P700⁺ reduction increased sharply during the first 3 s of irradiation and decreased upon longer light exposures. The rates of fast and slow components of dark reduction of P700⁺ remained constant upon illumination of dark-adapted leaves with far-red light for 1 s and longer periods. Thus, nonmonotonic changes in the redox state of P700 in barley leaves exposed to far-red light reflect variable contributions of few alternative electron transport pathways characterized by different rates of electron donation to PSI. The results show the principle possibility of switching-over between alternative pathways of PSI-related electron transfer within one complex of this photosystem. Such switching may occur irrespective of active operation or inhibition of ferredoxin-dependent electron transport.     

Light-dependent development of photosynthetic competence in Scenedesmus mutant No. 8.

The heterotrophically grown, P-700-free mutant No. 8 of Scenedesmus obliquus is unable to carry out photosynthesis. Yet, chloroplast particles isolated from the alga reduced ferricyanide. They also reduced methyl viologen in the presence of the artificial donor reduced 2,6-dichlorophenol indophenol with a low yield but an appreciable saturation rate. NADP reduction or P-700 turn-over could not be detected. When grown mixotrophically, the mutant showed increasing P-700 activity with a concomitant increase in the rate of photosynthesis. Both activities were lost again when the algae were returned to darkness. Isolated chloroplast particles showed a good P-700 turn-over and reasonable rates of NADP reduction. The data suggest that the mutation occurred at a site preceding the formation of the pigment. The results on the photochemical activities are discussed in the light of reports concerning the involvement of P-700 in linear electron transport.     

The use of light-induced absorbance changes at 820 nm to monitor the oxidation state of P-700 in leaves

Abstract The use of the light-induced absorbance change at 820 nm (ΔA 820) to monitor the oxidation and reduction of P-700 in irradiated leaves is examined. Results obtained from leaves irradiated with a range of wavelengths of light, poisoned with DCMU, or lacking PS I, are consistent with the proposition that the light-induced ΔA 820 can be used to monitor P-700 oxidation in leaves.     

Two molecular forms of pea ferredoxin in the electron transport chain of chloroplasts

The effects of two molecular forms of water-soluble ferredoxin (Fd I and Fd II) on the kinetics of electron transport in bean chloroplasts (class B) were studied. The light-induced redox transitions of the photosystem I reaction center P700 were measured by the intensity of the EPR signal I produced by P700+. Both forms of ferredoxin, Fd I and Fd II, when added to the chloroplasts in catalytic amounts, stimulate the light-induced electron transfer from P700 to NADP+. Nevertheless, Fd I is a better mediator of the back reactions from NADPH to P700+. This electron transfer pathway is sensitive to the cyclic electron transport inhibitor, antimycin A, and to DCMU inhibitor of electron transport between photosystem II and plastoquinone. It may be concluded that the two molecular forms of ferredoxin, Fd I and Fd II, differ in their ability to catalyze cyclic electron transport in photosystem I. The role of Fd I and Fd II in regulation of electron transport at the acceptor site of photosystem I is discussed.     

The role of PGR5 in the redox poising of photosynthetic electron transport

The pgr5 mutant of Arabidopsis thaliana has been described as being deficient in cyclic electron flow around photosystem I, however, the precise role of the PGR5 protein remains unknown. To address this issue, photosynthetic electron transport was examined in intact leaves of pgr5 and wild type A. thaliana. Based on measurements of the kinetics of P700 oxidation in far red light and re-reduction following oxidation in the presence of DCMU, we conclude that this mutant is able to perform cyclic electron flow at a rate similar to the wild type. The PGR5 protein is therefore not essential for cyclic flow. However, cyclic flow is affected by the pgr5 mutation under conditions where this process is normally enhanced in wild type leaves, i.e. high light or low CO(2) concentrations resulted in enhancement of cyclic electron flow. This suggests a different capacity to regulate cyclic flow in response to environmental stimuli in the mutant. We also show that the pgr5 mutant is affected in the redox poising of the chloroplast, with the electron transport chain being substantially reduced under most conditions. This may result in defective feedback regulation of photosynthetic electron transport under some conditions, thus providing a rationale for the reduced efficiency of cyclic electron flow.     

Energy transfer from photosystem II to photosystem I in Porphyridium cruentum

Rates of photooxidation of P-700 by green (560 nm) or blue (438 nm) light were measured in whole cells of porphyridium cruentum which had been frozen to -196 degrees C under conditions in which the Photosystem II reaction centers were either all open (dark adapted cells) or all closed (preilluminated cells). The rate of photooxidation of P-700 at -196 degrees C by green actinic light was approx. 80% faster in the preilluminated cells than in the dark-adapted cells. With blue actinic light, the rates of P-700 photooxidation in the dark-adapted and preilluminated cells were not significantly different. These results are in excellent agreement with predictions based on our previous estimates of energy distribution in the photosynthetic apparatus of Porphyridium cruentum including the yield of energy transfer from Photosystem II to Photosystem I determined from low temperature fluorescence measurements.     

Donation of Electrons from Cytosolic Components to the Intersystem Chain in the Cyanobacterium Synechococcus sp. PCC 7002 as Determined by the Reduction of P700+

Cells, of Synechococcus sp. PCC 7002 showed a low oxidationlevel of P700 under a far-red light at 6 W m^–2 which inducednearly complete oxidation of P700 in spinach leaves, and a strongerfar-red light was required to observe the oxidation of P700.DCMU did not affect the level of P700⁺² but 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinoneinduced the oxidation of P700 under far-red light, indicatingthat the low oxidation level of P700 was due to the donationof electrons to P700⁺² from the cytosolic respiratory donorsthrough the intersystem chain at the plastoquinone pool. Theelectron transfer from the cytosolic donors to the intersystemchain was inhibited by HgCl₂ but not by antimycin A. The reductionof P700⁺ in Synechococcus cells, after illumination by strongfar-red light was mostly accounted for by the electron flowto the inter system chain from the respiratory donors (t     

The difficulty of estimating the electron transport rate at photosystem I

It is still a controversial issue how the electron transport reaction is carried out around photosystem I (PSI) in the photosynthetic electron transport chain. The measurable component in PSI is the oxidized P700, the reaction center chlorophyll in PSI, as the absorbance changes at 820–830 nm. Previously, the quantum yield at PSI [Y(I)] has been estimated as the existence probability of the photo-oxidizable P700 by applying the saturated-pulse illumination (SP; 10,000–20,000 µmol photons m^?2 s^?1). The electron transport rate (ETR) at PSI has been estimated from the Y(I) value, which was larger than the reaction rate at PSII, evaluated as the quantum yield of PSII, especially under stress-conditions such as CO₂-limited and high light intensity conditions. Therefore, it has been considered that the extra electron flow at PSI was enhanced at the stress condition and played an important role in dealing with the excessive light energy. However, some pieces of evidence were reported that the excessive electron flow at PSI would be ignorable from other aspects. In the present research, we confirmed that the Y(I) value estimated by the SP method could be easily misestimated by the limitation of the electron donation to PSI. Moreover, we estimated the quantitative turnover rate of P700⁺ by the light-to-dark transition. However, the turnover rate of P700 was much slower than the ETR at PSII. It is still hard to quantitatively estimate the ETR at PSI by the current techniques.