The C-type Arabidopsis thioredoxin reductase ANTR-C acts as an electron donor to 2-Cys peroxiredoxins in chloroplasts

2-Cys peroxiredoxins (Prxs) play important roles in the antioxidative defense systems of plant chloroplasts. In order to determine the interaction partner for these proteins in Arabidopsis, we used a yeast two-hybrid screening procedure with a C175S-mutant of Arabidopsis 2-Cys Prx-A as bait. A cDNA encoding an NADPH-dependent thioredoxin reductase (NTR) isotype C was identified and designated ANTR-C. We demonstrated that this protein effected efficient transfer of electrons from NADPH to the 2-Cys Prxs of chloroplasts. Interaction between 2-Cys Prx-A and ANTR-C was confirmed by a pull-down experiment. ANTR-C contained N-terminal TR and C-terminal Trx domains. It exhibited both TR and Trx activities and co-localized with 2-Cys Prx-A in chloroplasts. These results suggest that ANTR-C functions as an electron donor for plastidial 2-Cys Prxs and represents the NADPH-dependent TR/Trx system in chloroplasts.     

Photosystem II energy coupling in chloroplasts with H2O2 as electron donor.

NH2OH-treated, non-water-splitting chloroplasts can oxidize H2O2 to O2 through Photosystem II at substantial rates (100--250 muequiv . h-1 . mg-1 chlorophyll with 5 mM H2O2) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H2O2 leads to Photosystem II leads to dimethylquinone reaction supports phosphorylation with a P/e2 ratio of 0.25--0.35 and proton uptake with H+/e values of 0.67 (pH 8)--0.85 (pH 6). These are close to the P/e2 value of 0.3--0.38 and the H+/e values of 0.7--0.93 found in parallel experiments for the H2O leads to Photosystem II leads to dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor leads to Photosystem II leads to dibromothymoquinone (leads to O2) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I-, ferrocyanide).     

The rapid component of electron paramagnetic resonance signal II: a candidate for the physiological donor to photosystem II in spinach chloroplasts.

Rapid light-induced transients in EPR Signal IIf (F-+) are observed in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated, Tris-washed chloroplasts until the state F P680 Q minus is reached. In the absence of exogenous redox mediators several flashes are required to saturate this photoinactive state. However, the Signal IIf transient is observed on only the first flash following DCMU addition if an efficient donor to Signal IIf, phenylenediamine or hydroquinone, is present. Complementary polarographic measurements show that under these conditions oxidized phenylenediamine is produced only on the first flash of a series. The DCMU inhibition of Signal IIf can be completely relieved by oxidative titration of a one-electron reductant with E'Os.o equals to + 480 mV. At high reduction potentials the decay time of Signal IIf is constant at about 300 ms, whereas in the absence of DCMU the decay time is longer and increases with increasing reduction potential. A model is proposed in which Q minus, the reduced Photosystem II primary acceptor, and D, a one-electron 480 mV donor endogenous to the chloroplast suspension, compete in the reduction of Signal IIf (F-+). At high potentials D is oxidized in the dark, and the (Q-+F-+) back reaction regenerates the photoactive F P680 Q state. The electrochemical and kinetic evidence is consistent with the hypothesis that the Signal IIf species, F, is identical with Z, the physiological donor to P680.     

Inhibitors of electron transport associated with photosystem II in chloroplasts

The modes of actions of six inhibitors on the electron transportsystem in the vicinity of system II in chloroplasts were studied. The first group, including piericidin A, ioxynil and broxynil,showed relatively simple modes of action on the Hill reaction,fluorescence of chlorophyll and the photobleaching of photosyntheticpigments, which are similar to the action of DCMU. As compared with inhibitors of the first group, the inhibitoryactions of salicylaldoxime, antimycin A and azide on the Hillreaction were more complicated in that they were influencedmore strongly by reaction conditions, i.e. duration of incubation,pH of the reaction mixture and illumination of chloroplasts.Inhibitors of the second group suppressed the rise in fluorescencein the induction period. However, this effect was not observedin the presence of DCMU or dithionite. Salicylaldoxime and azidewere effective in inducing photobleaching of photosyntheticpigments, whereas antimycin A inhibited the photobleaching inducedby ferricyanide or CCCP. Inhibition sites of the inhibitors in the first group are assumedto be similar to that of DCMU, whereas the inhibitors in thesecond group are effective in blocking electron transport onthe oxidizing side of system II between the primary electrondonor of system II and an intermediary electron carrier whichreceives electrons from artificial electron donors for systemII. (Received October 30, 1971; )     

P700: the primary electron donor of photosystem I.

The primary electron donor of photosystem I, P700, is a chlorophyll species that in its excited state has a potential of approximately -1.2 V. The precise chemical composition and electronic structure of P700 is still unknown. Recent evidence indicates that P700 is a dimer of one chlorophyll (Chl) a and one Chl a'. The Chl a' and Chl a are axially coordinated by His residues provided by protein subunits PsaA and PsaB, respectively. The Chl a', but not the Chl a, is also H-bonded to the protein. The H-bonding is likely responsible for selective insertion of Chl a' into the reaction center. EPR studies of P700(+*) in frozen solution and single crystals indicate a large asymmetry in the electron spin and charge distribution towards one Chl of the dimer. Molecular orbital calculations indicate that H-bonding will specifically stabilize the Chl a'-side of the dimer, suggesting that the unpaired electron would predominantly reside on the Chl a. This is supported by results of specific mutagenesis of the PsaA and PsaB axial His residues, which show that only mutations of the PsaB subunit significantly alter the hyperfine coupling constants associated with a single Chl molecule. The PsaB mutants also alter the microwave induced triplet-minus-singlet spectrum indicating that the triplet state is localized on the same Chl. Excitonic coupling between the two Chl a of P700 is weak due to the distance and overlap of the porphyrin planes. Evidence of excitonic coupling is found in PsaB mutants which show a new bleaching band at 665 nm that likely represents an increased intensity of the upper exciton band of P700. Additional properties of P700 that may give rise to its unusually low potential are discussed.     

Energy transfer between photosystem II and photosystem I in chloroplasts.

A model for the photochemical apparatus of photosynthesis is presented which accounts for the fluorescence properties of Photosystem II and Photosystem I as well as energy transfer between the two photosystems. The model was tested by measuring at - 196 degrees C fluorescence induction curves at 690 and 730 nm in the absence and presence of 5mMMgCl2 which presumably changes the distrubution of excitation energy between the two photosystems. The equations describing the fluorescence properties involve terms for the distribution of absorbed quanta, alpha, being the fraction distributed to Photosystem I, and beta, the fraction to Photosystem II to Photosystem I, KT(II yields I). The data, analyzed within the context of the model, permit a direct comparison of alpha and kt(II yields I) in the absence (minus) and presence (+) of Mg-2+ :alpha minus/alpha-+ equals 1.2 and k-minus t)II yields I)/K-+T(II yields I) equal to 1.9. If the criterion that alpha + beta equal to 1 is applied absolute values can be calculated: in the presence of Mg-2+, alpha-+ equal to 0.27 and the yield of energy transfer, phi-+ t(II yields I) varied the presence of Mg-2+, alpha-+ equal to 0.27 and the yield of energy transfer, phi-+ t(II yields I) varied from 0.065 when the Photosystem II reaction centers were all open to 0.23 when they were closed. In the absence of Mg-2+, alpha-minus equal to 0.32 and phi t(II yields I) varied from 0.12 to 0.28. The data were also analyzed assuming that two types of energy transfer could be distinguished; a transfer from the light-harvesting chlorophyll of Photosystem II to Photosystem I, kt(II yields I), and a transfer from the reaction centers of Photosystem II to Photosystem I, kt(II yields I). In that case alpha-minus/alpha+ equal to 1.3, k-minus t(II yields I)/k+ t(II yields I)equal to 1.3 and k-minus t(II yields I) equal to 3.0. It was concluded, however, that both of these types of energy transfer are different manifestations of a single energy transfer process.     

N,N-diethylhydroxylamine: a new electron donor to photosystem II

Diethylhydroxylamine, when added to beet spinach thylakoid membranes in the reaction mixture enhanced both photosystem II mediated dichlorophenolindophenol photoreduction and whole chain electron transport supported by methyl viologen. Diethylhydroxylamine supports dichlorophenolindophenol photoreduction when oxygen evolving complex is inactivated by hydroxylamine washings. All the electron transport assays were found to be highly sensitive to diuron, indicating that diethylhydroxylamine donates electrons to the photosystem II before the herbicide binding site. The stimulation of the photochemical activity by diethylhydroxylamine is not solely due to its action as an uncoupler. It was also observed that the action of diethylhydroxylamine was not altered by preincubations of thylakoids in light in the presence of diethylhydroxylamine. Also, thylakoid membranes did not lose their benzoquinone Hill activity by the pre-incubations with diethylhydroxylamine either in light or in dark. Thus, unlike the photosystem II electron donor, hydroxylamine, diethylhydroxylamine was found to donate electrons without the inactivations of oxygen evolving complex. It is suggested that diethylhydroxylamine is a useful electron donor to the photosystem II.     

Primary and secondary electron donors in photosystem II of chloroplasts. Rates of electron transfer and location in the membrane.

Absorption changes at 820 or 515 nm after a short laser flash were studied comparatively in untreated chloroplasts and in chloroplasts in which oxygen evolution is inhibited. In chloroplasts pre-treated with Tris, the primary donor of Photosystem II (P-680) is oxidized by the flash it is re-reduced in a biphasic manner with half-times of 6 microseconds (major phase) and 22 microseconds. After the second flash, the 6 microseconds phase is nearly absent and P-680+ decays with half-times of 130 microseconds (major phase) and 22 microseconds. Exogenous electron donors (MnCl2 or reduced phenylenediamine) have no direct influence on the kinetics of P-680+. In untreated chloroplasts the 6 and 22 microseconds phases are of very small amplitude, either at the 1st, 2nd or 3rd flash given after dark-adaptation. They are observed, however, after incubation with 10 mM hydroxylamine. These results are interpreted in terms of multiple pathways for the reduction of P-680+: a rapid reduction (less than 1 microseconds) by the physiological donor D1; a slower reduction (6 and 22 microseconds) by donor D'1, operative when O2 evolution is inhibited; a back-reaction (130 microseconds) when D'1 is oxidized by the pre-illumination in inhibited chloroplasts. In Tris-treated chloroplasts the donor system to P-680+ has the capacity to deliver only one electron. The absorption change at 515 nm (electrochromic absorption shift) has been measured in parallel. It is shown that the change linked to Photosystem II activity has nearly the same magnitude in untreated chloroplasts or in chloroplasts treated with hydroxylamine or with Tris (first and subsequent flashes). Thus we conclude that all the donors (P-680, D1, D'1) are located at the internal side of the thylakoid membrane.     

Mechanisms and functions of photosystem I-related alternative electron transport pathways in chloroplasts

This review analyzes various alternative pathways of chloroplast electron transport mediated by photoreactions of photosystem I (PSI) and unrelated to activity of photosystem II (PSII). The mechanisms and functional significance of the alternative pathways are considered. These pathways are complexly organized and comprise ferredoxin-dependent electron recycling around PSI, as well as electron donation to noncyclic chain in the region between PSII and PSI from reduced substances localized in the chloroplast stroma. For each of the alternative pathways, the origin of corresponding enzymes and their compartmentalization in the complex membrane system of the chloroplast are discussed. It is shown that operation of alternative electron transport pathways contributes to energy transduction and cell defense function, facilitates the absorption of inorganic carbon, and is significant for chloroplast respiration. Multiple mechanisms for regulation of alternative pathways have been revealed. It is concluded that PSI-related alternative electron transport pathways constitute an integral part of entire system of photosynthetic electron transport, this system being principally responsible for energy supply of phototrophic cells and whole plants.     

Calmodulin antagonists inhibit electron transport in photosystem II of spinach chloroplasts

Chlorpromazine, phenothiazine and trifluoperazine, known as calmodulin antagonists, inhibit electron transport in Photosystem II of spinach chloroplasts in concentrations from 20–500 μM. The inhibition site is located on the diphenyl carbazide to indophenol pathway in Tris-treated chloroplasts, indicating that water oxidation is not affected by these drugs. Ca²⁺ ions, bound to chloroplast membranes before the addition of calmodulin antagonists, can protect against inhibition up to 25% of the electron transport rate. In presence of A23187, the Ca²⁺-specific ionophore, Ca²⁺ ions provide less protection against inhibition by the 3 calmodulin antagonists used. A possible role of a calmodulin-like protein in spinach chloroplasts is postulated.     

Heterogeneous photosystem 2 activity in isolated spinach chloroplasts

A study was made of the fluorescence induction curves from gently-broken spinach chloroplasts inhibited with DCMU. It was found that there were four kinetically different phases associated with such curves of which only the fastest did not appear to follow exponential kinetics. A comparison of the effects of various concentrations of DCMU on the rate of oxygen evolution and on the fluorescence induction curve did not support the hypothesis that any of the kinetic phases was simply an artefact caused by incomplete inhibition of electron transport. It was also found that 5 min of dark incubation did not maximally oxidize the electron acceptors to photosystem 2 since some acceptors were only oxidized following far-red illumination, suggesting a heterogeneity among these acceptors with respect to their re-oxidation properties. Investigation of the effect of the Q₄₀₀ oxidation state on the fluorescence induction curve revealed that it only influenced the slowest kinetic phase and that Q₄₀₀ did not seem to be associated with the other phases.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1 - 1 dimethylurea - PS 1 photosystem 1 - PS2 photosystem 2 - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - EDTA ethylene-diaminetetraacetic acid - F_max maximum yield of fluorescence emission - F₀ initial yield of fluorescence emission - F_v variable yield of fluorescence emission - N.E. non-exponential kinetics     

Photoreactions of Cytochrome b-559 and cyclic electron flow in photosystem II of intact chloroplasts.

The high potential cytochrome b-559 of intact spinach chloroplasts was photooxidized by red light with a high quantum efficiency and by far-red light with a very low quantum efficiency, when electron flow from water to Photosystem II was inhibited by a carbonyl cyanide phenylhydrazone (FCCP or CCP). Dithiothreitol, which reacts with FCCP or CCCP, reversed the photooxidation of cytochrome b-559 and restored the capability of the chloroplasts to photoreduce CO2 showing that the FCCP/CCCP effects were reversible. The quantum efficiency of cytochrome b-559 photooxidation by red or far-red light in the presence of FCCP was increased by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone which blocks oxidation of reduced plastoquinone by Photosystem I. When the inhibition of water oxidation by FCCP or CCP was decreased by increased light intensities, previously photooxidized cytochrome b-559 was reduced. Red light was much more effective in photoreducing oxidized high potential cytochrome b-559 than far-red light. The red/far-red antagonism in the redox state of cytochrome b-559 is a consequence of the different sensitivity of the cytochrome to red and far-red light and does not indicate that the cytochrome is in the main path of electrons from water to NADP. Rather, cytochrome b-559 acts as a carrier of electrons in a cyclic path around Photosystem II. The redox state of the cytochrome was shifted to the oxidized side when electron transport from water became rate-limiting, while oxidation of water and reduction of plastoquinone resulted in its shifting to the reduced side.     

Anomalous oxygen uptake from isolated chloroplasts inhibited in photosystem II and without external electron donors.

Light induced modulated signal of oxygen uptake by isolated chloroplasts in the presence of methyl viologen, when Photosystem II activity was inhibited and in the absence of any electron donors, was detected by a modulated oxygen Pt electrode, polarized negatively. Evidence is brought to show that an electrochemical process which takes place on the surface of the negatively polarized Pt-cathode produces an intermediate which serves as an electron donor to Photosystem I. Atempts to identify this intermediate show that it may be very probably the superoxide radical generated by the electrochemical reduction of oxygen which continuously diffuses from the external circulating medium to the electrode.     

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.     

On the inhibitory action of cadmium on the donor side of photosystem II in isolated chloroplasts.

The inhibitory effect of the Cd2+ in the electron transport of the isolated chloroplasts has been observed by measuring the oxygen uptake from the solution and the fluorescence induction. Cd2+ is found to be an inhibitor on the donor side of Photosystem II and its action site, as determined by experiments using hydroxylamine and exogenous Mn, is supposed to be on the water-splitting enzyme itself. Moreover, physicochemical and physiological studies indicate that only the ionic form of Cd is acting at the level of the manganoprotein. It is not possible, from this work, to define precisely in which form Cd is taken up through the thylakoid membranes.