Plastoquinone Research Articles

A Novel Nucleus-Encoded Chloroplast Protein, PIFI, Is Involved in NAD(P)H Dehydrogenase Complex-Mediated Chlororespiratory Electron Transport in Arabidopsis

A transient rise in chlorophyll fluorescence after turning off actinic light reflects nonphotochemical reduction of the plastoquinone (PQ) pool. This process is dependent on the activity of the chloroplast NAD(P)H dehydrogenase (NDH) complex, which mediates electron flow from stromal reductants to the PQ pool. In this study, we characterized an Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutant pifi (for postillumination chlorophyll fluorescence increase), which possesses an intact NDH complex, but lacks the NDH-dependent chlorophyll fluorescence increase after turning off actinic light. The nuclear gene PIFI (At3g15840) containing the T-DNA insertion encodes a chloroplast-targeted protein localized in the stroma and is annotated as a protein of unknown function. The pifi mutant exhibited a lower capacity for nonphotochemical quenching, but similar CO(2) assimilation rates, photosystem II (PSII) quantum efficiencies (PhiPSII), and reduction levels of the primary electron acceptor of PSII (1 - qL) as compared with the wild type. The pifi mutant grows normally under optimal conditions, but exhibits greater sensitivity to photoinhibition and long-term mild heat stress than wild-type plants, which is consistent with lower capacity of nonphotochemical quenching. We conclude that PIFI is a novel component essential for NDH-mediated nonphotochemical reduction of the PQ pool in chlororespiratory electron transport.

Chlororespiration and cyclic electron flow around PSI during photosynthesis and plant stress response

Besides major photosynthetic complexes of oxygenic photosynthesis, new electron carriers have been identified in thylakoid membranes of higher plant chloroplasts. These minor components, located in the stroma lamellae, include a plastidial NAD(P)H dehydrogenase (NDH) complex and a plastid terminal plastoquinone oxidase (PTOX). The NDH complex, by reducing plastoquinones (PQs), participates in one of the two electron transfer pathways operating around photosystem I (PSI), the other likely involving a still uncharacterized ferredoxin-plastoquinone reductase (FQR) and the newly discovered PGR5. The existence of a complex network of mechanisms regulating expression and activity of the NDH complex, and the presence of higher amounts of NDH complex and PTOX in response to environmental stress conditions the phenotype of mutants, indicate that these components likely play a role in the acclimation of photosynthesis to changing environmental conditions. Based on recently published data, we propose that the NDH-dependent cyclic pathway around PSI participates to the ATP supply in conditions of high ATP demand (such as high temperature or water limitation) and together with PTOX regulates cyclic electron transfer activity by tuning the redox state of intersystem electron carriers. In response to severe stress conditions, PTOX associated to the NDH and/or the PGR5 pathway may also limit electron pressure on PSI acceptor and prevent PSI photoinhibition.

A non-invasive assay of the plastoquinone pool redox state based on the OJIP-transient.

The plastoquinone (PQ) pool of the photosynthetic electron transport chain becomes reduced under anaerobic conditions. Here, anaerobiosis was used as a tool to manipulate the PQ-pool redox state in darkness and to study the effects of the PQ-redox state on the Chl-a fluorescence (OJIP) kinetics in pea leaves (Pisum sativum L.). It is shown that the F(J) (fluorescence intensity at 3 ms) is linearly related to the area above the OJ-phase (first 3 ms) representing the reduction of the acceptor side of photosystem II (PSII) and F(J) is also linearly related to the area above the JI-phase (3-30 ms) that parallels the reduction of the PQ-pool. This means that F(J) depends on the availability of oxidized PQ-molecules bound to the Q(B)-site. The linear relationships between F(J) and the two areas indicate that F(J) is not sensitive to energy transfer between PSII-antennae (connectivity). It is further shown that a approximately 94% reduced PQ-pool is in equilibrium with a approximately 19% reduction of Q(A) (primary quinone acceptor of PSII). The non-linear relationship between the initial fluorescence value (F(20 micros)) and the area above the OJ-phase supports the idea that F(20 mus )is sensitive to connectivity. This is reinforced by the observation that this non-linearity can be overcome by transforming the F(20 micros)-values into [Q(A) (-)]-values. Based on the F(J)-value of the OJIP-transient, a simple method for the quantification of the redox state of the PQ-pool is proposed.

Expression of psbA Genes is Regulated at Multiple Levels in the Cyanobacterium Synechococcus sp. PCC 7942

In cyanobacterium Synechococcus sp. PCC 7942 the photosystem II reaction-center protein D1 is encoded by three psbA genes. The psbAI gene encodes D1:1 protein, the form used for acclimated growth, and psbAII and psbAIII genes encode the stress-induced form, D1:2 protein. Strong light and low temperature have been shown to induce the expression of psbAII/III genes and down-regulate the expression of psbAI gene. Recently, we reported the involvement of reduced thiols in the up-regulation of psbAII/III genes. In this study, we have analyzed the regulation of psbA gene expression in Synechococcus further, at both the transcriptional and post-transcriptional levels. We show that the inhibitors of the photosynthetic electron-transfer chain, which have different effects on the redox state of the plastoquinone (PQ) pool, have similar effect on the transcription of psbA genes. The inhibitors 3-(3,4 dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) do not cause any changes in psbA gene expression when added under low-light conditions, but dramatically reduce the high-light induction of psbAII/III genes when added upon a high-light shift. Moreover, when the thiol reductant, dithiothreitol, is added to Synechococcus cells together with DCMU concomitant with the high-light shift, no inhibition of psbAII/III gene up-regulation takes place, indicating that the thiol redox state rather than the redox state of the PQ pool regulates psbA gene transcription. We also provide evidence for post-transcriptional regulation of psbA gene expression, particularly, inhibition of translation of psbAI transcripts at high light, and demonstrate that the D1 protein synthesis and degradation processes are coregulated in Synechococcus.

Regulation of Energy Dissipation in Photosystem I by the Redox State of the Plastoquinone Pool

The effect of exogenous plastoquinone (PQ) on the different deexcitation pathways of photosystem I (PSI) was investigated. Addition of oxidized decyl-plastoquinone (dPQ) and PQ-2 strongly quenched the chlorophyll (Chl) emission spectra of PSI submembrane fractions over all wavelengths. This quenching increased with the concentration of exogenous PQ added and followed the modified Stern-Volmer law. The Stern-Volmer constants found for dPQ and PQ-2 were 1.25 x 10(6) M-1 and 0.55 x 10(6) M-1, respectively, and the fraction of fluorescence accessible to the quencher was 0.7 for both exogenous PQ. dPQ and PQ-2 also retarded the P700 photooxidation measured under limiting actinic light irradiances. Photoacoustic measurements showed that addition of dPQ increased the heat dissipation and decreased the photochemical capacity of PSI. From these results, exogenous oxidized PQ were shown to efficiently quench the Chl excited state in the PSI antenna and change the balance between Chl deexcitation pathways. Moreover, reduction of the endogenous PQ pool in whole thylakoid membranes by NADPH increased PSI fluorescence by 65%, indicating the importance of the redox state of the PQ pool on PSI energy dissipation.

Structural characteristics of channels and pathways in photosystem II including the identification of an oxygen channel

Here we use crystal structures to investigate and review channels and pathways for the transfer of substrates (water, plastoquinone (PQ)) and products (electrons, protons, oxygen, reduced PQ (PQH 2)) to, and from, the redox active catalytic sites of photosystem II (PSII). A putative oxygen channel has been identified which is about 21 Å in length, leading from the water splitting site to the lumen. This channel follows a path along the lumenal surface of CP43, passing across the interface of the large extrinsic loop which joins the fifth and sixth transmembrane helices of this chlorophyll binding protein. In so doing it seems to minimise interactions with the excited states of chlorophylls bound within the PSII complex, especially those that constitute the primary electron donor, P680. Two additional channels leading from the water splitting site, and also exiting at the lumen, were also identified. Their hydrophilic nature suggests that they probably facilitate the delivery of water to, and protons from, the catalytic site. Also discussed are unique features in the electron transfer pathway of PSII, as compared with those of purple photosynthetic bacteria, and structural implications of the PSII Q B-site in terms of PQ protonation and PQ/PQH 2 diffusion.

Cloning and expression of the gene encoding solanesyl diphosphate synthase from Hevea brasiliensis

Summary Plastoquinones play important roles as electron carriers in the light-dependent reactions of photosynthesis and also as a cofactor of phytoene desaturation in the synthesis of carotenoid. A plastoquinone-9 (PQ-9) was identified in Frey-Wyssling organelles of fresh rubber latex from Hevea brasiliensis. This indicates that a Hevea solanesyl diphosphate synthase (HbSDS) must be present for the synthesis of the C45 prenyl side chain of the PQ-9 found in the rubber latex. Based on the sequence information of other trans-prenyl diphosphate synthases, the cDNA encoding HbSDS was cloned from aHevearubberlatexcDNAlibrary.Sequence analysis revealed the presence of an ORFconsisting of 1254 bpcapable of encoding a 46,095 Da polypeptide. The deduced amino acid sequence of the clone contained all conserved regions found in typical trans-prenyl chain elongating enzymes. This cDNA, expressed in Escherichia coli cells as an insoluble but Trx-His-tagged fusion protein, showed a distinct solanesyl diphosphate synthase activity in the presence of Triton X-100. The expression level of HbSDS mRNA in latex was found to be higher than those in leaves, stems and roots. The expressed HbSDS is suggested to be a specific enzyme in the Frey-Wyssling particles of rubber latex. # 2007 Elsevier Ireland Ltd. All rights reserved.

Evidence for a Novel Quinone-Binding Site in the Photosystem II (PS II) Complex That Regulates the Redox Potential of Cytochrome b559

The present study provides a thorough analysis of effects on the redox properties of cytochrome (Cyt) b559 induced by two photosystem II (PS II) herbicides [3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,4-dinitro-6-sec-butylphenol (dinoseb)], an acceleration of the deactivation reactions of system Y (ADRY) agent carbonylcyanide-m-chlorophenylhydrazone (CCCP), and the lipophilic PS II electron-donor tetraphenylboron (TPB) in PS II membrane fragments from higher plants. The obtained results revealed that (1) all four compounds selectively affected the midpoint potential (E(m)) of the high potential (HP) form of Cyt b559 without any measurable changes of the E(m) values of the intermediate potential (IP) and low potential (LP) forms; (2) the control values from +390 to +400 mV for HP Cyt b559 gradually decreased with increasing concentrations of DCMU, dinoseb, CCCP, and TPB; (3) in the presence of high TPB concentrations, a saturation of the E(m) decrease was obtained at a level of about +240 mV, whereas no saturation was observed for the other compounds at the highest concentrations used in this study; (4) the effect of the phenolic herbicide dinoseb on the E(m) is independent of the occupancy of the Q(B)-binding site by DCMU; (5) at high concentrations of TPB or dinoseb, an additional slow and irreversible transformation of HP Cyt b559 into IP Cyt b559 or a mixture of the IP and LP Cyt b559 is observed; and (6) the compounds stimulate autoxidation of HP Cyt b559 under aerobic conditions. These findings lead to the conclusion that a binding site Q(C) exists for the studied substances that is close to Cyt b559 and different from the Q(B) site. On the basis of the results of the present study and former experiments on the effect of PQ extraction and reconstitution on HP Cyt b559 [Cox, R. P., and Bendall, D. S. (1974) The functions of plastoquinone and beta-carotene in photosystem II of chloroplasts, Biochim. Biophys. Acta 347, 49-59], it is postulated that the binding of a plastoquinone (PQ) molecule to Q(C) is crucial for establishing the HP form of Cyt b559. On the other hand, the binding of plastoquinol (PQH2) to Q(C) is assumed to cause a marked decrease of E(m), thus, giving rise to a PQH2 oxidase function of Cyt b559. The possible physiological role of the Q(C) site as a regulator of the reactivity of Cyt b559 is discussed.

フェレドキシン（Fd）葉緑体過剰発現によるプラストキノン（PQ）還元とその酸化還元（レドックス）レベルの植物代謝生理への影響 －葉緑体レドックス制御ネットワーク解明のために－

フェレドキシン（Fd）葉緑体過剰発現によるプラストキノン（PQ）還元とその酸化還元（レドックス）レベルの植物代謝生理への影響 －葉緑体レドックス制御ネットワーク解明のために－

An HPLC-based method of estimation of the total redox state of plastoquinone in chloroplasts, the size of the photochemically active plastoquinone-pool and its redox state in thylakoids of Arabidopsis

We have described a direct, high-performance liquid chromatography-based method of estimation of the total level of plastoquinone (PQ) in leaves, the redox state of total (photoactive and non-photoactive) PQ, as well as the redox state of the PQ-pool that is applicable to any illumination conditions. This method was applied to Arabidopsis thaliana leaves but it can be applied to any other plant species. The obtained results show that the level of total PQ was 25 ± 3 molecules/1000 chlorophyll (Chl) molecules in relation to foliar total Chl content. The level of the photoactive PQ, i.e., the PQ-pool, was about 31% of the total PQ present in Arabidopsis leaves that corresponds to about 8 PQ molecules/1000 Chl molecules. The reduction level of the non-photoactive PQ fraction, present outside thylakoids in chloroplasts, was estimated to account for about 49%. The measurements of the redox state of the PQ-pool showed that the pool was reduced during the dark period in about 24%, and during the light period (150 μmol/m 2·s) the reduction of the PQ-pool increased to nearly 100%. The obtained results were discussed in terms of the activity of chlororespiration pathways in Arabidopsis and the regulatory role of the redox state of PQ-pool in various physiological and molecular processes in plants.

IMMUTANS does not act as a stress-induced safety valve in the protection of the photosynthetic apparatus of Arabidopsis during steady-state photosynthesis.

IMMUTANS (IM) encodes a thylakoid membrane protein that has been hypothesized to act as a terminal oxidase that couples the reduction of O(2) to the oxidation of the plastoquinone (PQ) pool of the photosynthetic electron transport chain. Because IM shares sequence similarity to the stress-induced mitochondrial alternative oxidase (AOX), it has been suggested that the protein encoded by IM acts as a safety valve during the generation of excess photosynthetically generated electrons. We combined in vivo chlorophyll fluorescence quenching analyses with measurements of the redox state of P(700) to assess the capacity of IM to compete with photosystem I for intersystem electrons during steady-state photosynthesis in Arabidopsis (Arabidopsis thaliana). Comparisons were made between wild-type plants, im mutant plants, as well as transgenics in which IM protein levels had been overexpressed six (OE-6 x) and 16 (OE-16 x) times. Immunoblots indicated that IM abundance was the only major variant that we could detect between these genotypes. Overexpression of IM did not result in increased capacity to keep the PQ pool oxidized compared to either the wild type or im grown under control conditions (25 degrees C and photosynthetic photon flux density of 150 micromol photons m(-2) s(-1)). Similar results were observed either after 3-d cold stress at 5 degrees C or after full-leaf expansion at 5 degrees C and photosynthetic photon flux density of 150 micromol photons m(-2) s(-1). Furthermore, IM abundance did not enhance protection of either photosystem II or photosystem I from photoinhibition at either 25 degrees C or 5 degrees C. Our in vivo data indicate that modulation of IM expression and polypeptide accumulation does not alter the flux of intersystem electrons to P(700)(+) during steady-state photosynthesis and does not provide any significant photoprotection. In contrast to AOX1a, meta-analyses of published Arabidopsis microarray data indicated that IM expression exhibited minimal modulation in response to myriad abiotic stresses, which is consistent with our functional data. However, IM exhibited significant modulation in response to development in concert with changes in AOX1a expression. Thus, neither our functional analyses of the IM knockout and overexpression lines nor meta-analyses of gene expression support the model that IM acts as a safety valve to regulate the redox state of the PQ pool during stress and acclimation. Rather, IM appears to be strongly regulated by developmental stage of Arabidopsis.

Digalactosyl-Diacylglycerol Deficiency Impairs the Capacity for Photosynthetic Intersystem Electron Transport and State Transitions in Arabidopsis thaliana Due to Photosystem I Acceptor-Side Limitations

Compared with wild type, the dgd1 mutant of Arabidopsis thaliana exhibited a lower amount of PSI-related Chl-protein complexes and lower abundance of the PSI-associated polypeptides, PsaA, PsaB, PsaC, PsaL and PsaH, with no changes in the levels of Lhca1-4. Functionally, the dgd1 mutant exhibited a significantly lower light-dependent, steady-state oxidation level of P700 (P700(+)) in vivo, a higher intersystem electron pool size, restricted linear electron transport and a higher rate of reduction of P700(+) in the dark, indicating an increased capacity for PSI cyclic electron transfer compared with the wild type. Concomitantly, the dgd1 mutant exhibited a higher sensitivity to and incomplete recovery of photoinhibition of PSI. Furthermore, dgd1 exhibited a lower capacity to undergo state transitions compared with the wild type, which was associated with a higher reduction state of the plastoquinone (PQ) pool. We conclude that digalactosyl-diacylglycerol (DGDG) deficiency results in PSI acceptor-side limitations that alter the flux of electrons through the photosynthetic electron chain and impair the regulation of distribution of excitation energy between the photosystems. These results are discussed in terms of thylakoid membrane domain reorganization in response to DGDG deficiency in A. thaliana.

Evidence that cytochrome b559 is involved in superoxide production in photosystem II: effect of synthetic short-chain plastoquinones in a cytochrome b559 tobacco mutant

Light-induced production of superoxide (O2*-) in spinach PSII (photosystem II) membrane particles was studied using EPR spin-trapping spectroscopy. The presence of exogenous PQs (plastoquinones) with a different side-chain length (PQ-n, n isoprenoid units in the side-chain) enhanced O2*- production in the following order: PQ-1>PQ-2>>PQ-9. In PSII membrane particles isolated from the tobacco cyt (cytochrome) b559 mutant which carries a single-point mutation in the beta-subunit and also has a decreased amount of the alpha-subunit, the effect of PQ-1 was less than in the wild-type. The increase in LP (low-potential) cyt b559 content, induced by the incubation of spinach PSII membrane particles at low pH, resulted in a significant increase in O2*- formation in the presence of PQ-1, whereas it had little effect on O2*- production in the absence of PQ-1. The enhancement of O2*- formation induced by PQ-1 was not abolished by DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea]. Under anaerobic conditions, dark oxidation of LP cyt b559 increased, as pH was decreased. The presence of molecular oxygen significantly enhanced dark oxidation of LP cyt b559. Based on these findings it is suggested that short-chain PQs stimulate O2*- production via a mechanism that involves electron transfer from Pheo- (pheophytin) to LP cyt b559 and subsequent auto-oxidation of LP cyt b559.

A dip in the chlorophyll fluorescence induction at 0.2–2 s in Trebouxia-possessing lichens reflects a fast reoxidation of photosystem I. A comparison with higher plants

An unusual dip (compared to higher plant behaviour under comparable light conditions) in chlorophyll fluorescence induction (FI) at about 0.2–2 s was observed for thalli of several lichen species having Trebouxia species (the most common symbiotic green algae) as their native photobionts and for Trebouxia species cultured separately in nutrient solution. This dip appears after the usual O(J)IP transient at a wide range of excitation light intensities (100–1800 μmol photons m −2 s −1). Simultaneous measurements of FI and 820-nm transmission kinetics (I 820) with lichen thalli showed that the decreasing part of the fluorescence dip (0.2–0.4 s) is accompanied by a decrease of I 820, i.e., by a reoxidation of electron carriers at photosystem I (PSI), while the subsequent increasing part (0.4–2 s) of the dip is not paralleled by the change in I 820. These results were compared with that measured with pea leaves—representatives of higher plants. In pea, PSI started to reoxidize after 2-s excitation. The simultaneous measurements performed with thalli treated with methylviologen (MV), an efficient electron acceptor from PSI, revealed that the narrow P peak in FI of Trebouxia-possessing lichens (i.e., the I–P-dip phase) gradually disappeared with prolonged MV treatment. Thus, the P peak behaves in a similar way as in higher plants where it reflects a traffic jam of electrons induced by a transient block at the acceptor side of PSI. The increasing part of the dip in FI remained unaffected by the addition of MV. We have found that the fluorescence dip is insensitive to antimycin A, rotenone (inhibitors of cyclic electron flow around PSI), and propyl gallate (an inhibitor of plastid terminal oxidase). The 2-h treatment with 5 μM nigericin, an ionophore effectively dissipating the pH-gradient across the thylakoid membrane, did not lead to significant changes either in FI nor I 820 kinetics. On the basis of the presented results, we suggest that the decreasing part of the fluorescence dip in FI of Trebouxia-lichens reflects the activation of ferredoxin–NADP +–oxidoreductase or Mehler–peroxidase reaction leading to the fast reoxidation of electron carriers in thylakoid membranes. The increasing part of the dip probably reflects a transient reduction of plastoquinone (PQ) pool that is not associated with cyclic electron flow around PSI. Possible causes of this MV-insensitive PQ reduction are discussed.

Force field development on pigments of photosystem 2 reaction centre

We developed new parameters for molecular dynamics (MD) simulations, namely partial atomic charges, equilibrium bond-lengths, angles, dihedrals, atom types, and force constants of chlorophyll a (Chl) and plastoquinone (PQ), and both reduced and neutral form of primary acceptor (PHO) molecule. These parameters are essential for MD simulations that can interpret various structure functional relationships during primary processes of charge separation and stabilization in photosystem 2 reaction centres.

The Effect of Sulfur Re-Addition on H2 Photoproduction by Sulfur-Deprived Green Algae

Sulfur deprivation of algal cultures selectively and partially inactivates photosystem II (PSII)-catalyzed O(2) evolution, induces anaerobiosis and hydrogenase expression, and results in sustained H(2) photoproduction for several days. We show that re-addition of limiting amounts of sulfate (1-10 microM final concentration) to the cultures during the H(2)-production phase temporarily reactivates PSII photochemical and O(2)-evolution activity and re-establishes higher rates of electron transport through the photosynthetic electron transport chain. The reactivation of PSII occurs by de novo D1 protein synthesis, but does not result in the re-establishment of aerobic conditions in the reactor, detectable by dissolved-O(2) sensors. However, concomitant H(2) photoproduction is inhibited, possibly due to excessive intra-cellular levels of photosynthetically-evolved O(2). The partial recovery of electron transport rates correlates with the re-oxidation of the plastoquinone (PQ) pool, as observed by pulse-amplitude modulated (PAM) and fluorescence-induction measurements. These results show that the presence of a more oxidized PQ pool releases some of the down-regulation of electron transport caused by the anaerobic conditions.

The pgr1 Mutation in the Rieske Subunit of the Cytochrome b6f Complex Does Not Affect PGR5-dependent Cyclic Electron Transport around Photosystem I

Although photosystem I (PSI) cyclic electron transport is essential for plants, our knowledge of the route taken by electrons is very limited. To assess whether ferredoxin (Fd) donates electrons directly to plastoquinone (PQ) or via a Q-cycle in the cytochrome (cyt) b(6)f complex in PSI cyclic electron transport, we characterized the activity of PSI cyclic electron transport in an Arabidopsis mutant, pgr1 (proton gradient regulation). In pgr1, Q-cycle activity was hypersensitive to acidification of the thylakoid lumen because of an amino acid alteration in the Rieske subunit of the cyt b(6)f complex, resulting in a conditional defect in Q-cycle activity. In vitro assays using ruptured chloroplasts did not show any difference in the activity of PGR5-dependent PQ reduction by Fd, which functions in PSI cyclic electron transport in vivo. In contrast to the pgr5 defect, the pgr1 defect did not show any synergistic effect on the quantum yield of photosystem II in crr2-2, a mutant in which NDH (NAD(P)H dehydrogenase) activity was impaired. Furthermore, the simultaneous determination of the quantum yields of both photosystems indicated that the ratio of linear and PSI cyclic electron transport was not significantly affected in pgr1. All the results indicated that the pgr1 mutation did not affect PGR5-dependent PQ reduction by Fd. The phenotypic differences between pgr1 and pgr5 indicate that maintenance of the proper balance of linear and PSI cyclic electron transport is essential for preventing over-reduction of the stroma.

Inhibitor studies on non-photochemical plastoquinone reduction and H 2 photoproduction in Chlamydomonas reinhardtii

In the absence of PSII, non-photochemical reduction of plastoquinones (PQs) occurs following NADH or NADPH addition in thylakoid membranes of the green alga Chlamydomonas reinhardtii. The nature of the enzyme involved in this reaction has been investigated in vitro by measuring chlorophyll fluorescence increase in anoxia and light-dependent O 2 uptake in the presence of methyl viologen. Based on the insensitivity of these reactions to rotenone, a type-I NADH dehydrogenase (NDH-1) inhibitor, and their sensitivity to flavoenzyme inhibitors and thiol blocking agents, we conclude to the involvement of a type-II NADH dehydrogenase (NDH-2) in PQ reduction. Intact Chlamydomonas cells placed in anoxia have the property to produce H 2 in the light by a Fe-hydrogenase which uses reduced ferredoxin as an electron donor. H 2 production also occurs in the absence of PSII thanks to the existence of a non-photochemical pathway of PQ reduction. From inhibitors effects, we suggest the involvement of a plastidial NDH-2 in PSII-independent H 2 production in Chlamydomonas. These results are discussed in relation to the absence of ndh genes in Chlamydomonas plastid genome and to the existence of 7 ORFs homologous to type-II NDHs in its nuclear genome.

Recruitment of a Foreign Quinone into the A1 Site of Photosystem I: CHARACTERIZATION OF A menB rubA DOUBLE DELETION MUTANT IN SYNECHOCOCCUS SP. PCC 7002 DEVOID OF FX, FA, AND FB AND CONTAINING PLASTOQUINONE OR EXCHANGED 9,10-ANTHRAQUINONE

A photosystem I (PS I) complex containing plastoquinone-9 (PQ-9) but devoid of F(X), F(B), and F(A) was isolated and characterized from a mutant strain of Synechococcus sp. PCC 7002 in which the menB and rubA genes were insertionally inactivated. In isolated PS I trimers, the decay of P700+ measured in the near-IR and the decay of A1- measured in the near-UV were found to be biphasic, with (averaged) room temperature lifetimes of 12 and 350 micros. The decay-associated spectra of both kinetic phases are characteristic of the oxidized minus reduced difference spectrum of a semiquinone, consistent with charge recombination between P700+ and PQ-9-. The amplitude of the flash-induced absorbance changes in both the near-IR and the near-UV show that approximately one-half of the A1 binding sites are either empty or nonfunctional. A spin-polarized chlorophyll triplet is observed by time-resolved EPR, and it is attributed to the 3P700 product of P700+A0- charge recombination via the T0 spin level in those PS I complexes that do not contain a functional quinone. In those A1 sites that are occupied, the P700+Q- polarization pattern indicates that PQ-9 is oriented in a similar manner to that in the menB mutant. When excess 9,10-anthraquinone is added in vitro, it displaces PQ-9 and occupies the A1 binding site more readily than in the menB mutant. This can be explained by a greater accessibility to the A1 site in the menB rubA mutant due to the absence of F(X) and the stromal ridge polypeptides. The relatively low binding affinity of 9,10-anthraquinone allows it to be readily removed from the A1 site by washing. However, all A1 sites are shown to bind napthoquinones with high affinity and thus are proven to be functionally competent in quinone binding. The ability to readily displace PQ-9 from the A1 site makes the menB rubA mutant ideal for introducing novel quinones, particularly anthraquinones, into PS I.

Higher plant-like fluorescence induction and thermoluminescence characteristics in cyanobacterium, Spirulina mutant defective in PQH 2 oxidation by cyt b6/f complex

Characterization of the photosynthetic electron transport in a mutant of Spirulina platensis, generated by chemical mutagenesis, demonstrated that the electron transfer from the plastoquinone (PQ) to cytochrome b6/f was slowed. Thermoluminescence (TL) measurements suggested the presence of reversed energy flow via PQ, which resulted in an emergence of the plant-like after-glow TL band at 45 degrees C that could be enhanced by the transthylakoidal pH gradient and could be eliminated by an uncoupler, FCCP. The localization of the changes in the electron transport of the mutant cells measured by various methods revealed that the re-oxidation of the PQ pool is hampered in the mutant compared to the wild-type cells. The reduction in energy migration was localized between PQ and PS I reaction centers.