Rieske FeS overexpression in tobacco provides increased abundance and activity of Cytochrome b₆f

Photosynthesis is fundamental for plant growth and yield. The Cytochrome b₆ f complex catalyses a rate-limiting step in thylakoid electron transport and therefore represents an important point of regulation of photosynthesis. Here we show that overexpression of a single core subunit of Cytochrome bA ) and plastoquinone pool and faster electron transport from the plastoquinone pool to Photosystem I upon changes in irradiance, compared to control plants. A faster establishment of qE , the energy-dependent component of non-photochemical quenching, in transgenic plants suggests a more rapid build-up of the transmembrane proton gradient, also supporting the increased in vivo Cytochrome b₆ f activity. However, there was no consistent increase in steady-state rates of electron transport or CO₂ assimilation in plants overexpressing Rieske FeS grown in either laboratory conditions or field trials, suggesting that the in vivo activity of the complex was only transiently increased upon changes in irradiance. Our results show that overexpression of Rieske FeS in tobacco enhances the abundance of functional Cytochrome b₆ f and may have the potential to increase plant productivity if combined with other traits

Enzymes impliqués dans la production des formes réactives de l'oxygène dans les membranes plasmiques, les mitochrondries et les chloroplastes

Production of reactive oxygen species (ROS) was studied in different subcellular compartments using spectroscopic methods (UV/VIS, fluorescence, infrared and electron paramagnetic resonance). The identities and catalytic mechanisms of ROS-producing enzymes in the plasma membrane (PM) were studied as well as mitochondrial ROS accumulation in response to cadmium (Cd2+) and the protective role of the plastid terminal oxidase (PTOX). Cd2+ was shown to be an inhibitor of the PM superoxide (O2•-)-producing NADPH oxidase in vitro. In vivo Cd2+ inhibited the extracellular production of O2•- but stimulated the accumulation of hydrogen peroxide (H2O2). Cd2+ induced ROS production in isolated mitochondria. Mitochondrial ROS-inducing inhibitors increased extracellular H2O2 production confirming these organelles the main source of Cd2+-induced extracellular H2O2 generation in vivo. A ROS-producing quinone reductase (QR) was isolated from PMs. ROS production occured via the pH-dependent deprotonation of the end product menadiol leading to its intermediate forms that react with O2 forming O2•- and H2O2. A potential naphthoquinone species in PMs was identified that could serve as a QR substrate in vivo. The protection of the photosynthetic electron transfer chain by the PTOX, a plastoquinol: O2 oxidoreductase, was studied in PTOX-overexpressing plants (PTOX+). Based on the altered response to low and high light conditions in PTOX+ it was proposed that PTOX is coupled to an SOD in order to function as a protective enzyme. The absence of additional chloroplastic SOD in PTOX+ lines might have limited the plant's ability to detoxify O2•- produced by elevated levels of PTOX+.Les formes réactives de l'oxygène (FRO) ont été analysées dans différents compartiments cellulaires en utilisant des méthodes spectroscopiques (UV/VIS, fluorescence, infrarouge, résonance paramagnétique électronique). L'identité et les mécanismes catalytiques des enzymes qui produisent les FRO dans les membranes plasmiques (MP) et les mitochondries ont été étudiés, ainsi que le rôle protectif de l'oxydase terminale plastidiale (PTOX) des chloroplastes. Cd2+ s'est révélé être un inhibiteur de la NADPH oxydase des MP. In vivo Cd2+ inhibait la production extracellulaire de O2•- mais stimulait l'accumulation de H2O2. Dans des mitochondries isolées, Cd2+ a augmenté la production de FRO. Antimycin A a entraîné une élévation du H2O2 extracellulaire, confirmant que la mitochondrie est le site principal de production de l'H2O2 extracellulaire induite par Cd2+ in vivo. Une quinone réductase (QR) génératrice de FRO a été isolée des MP. La déprotonation pH-dépendante du quinole a produit des formes intermédiaires instables qui génèrent des FRO par réaction avec O2. Des espèces quinoniques ont été détectées dans la MP et pourraient servir de substrat aux QR in vivo. La protection de la chaine photosynthétique de transfert d'électron par la plastoquinol:O2 oxydoréductase a été étudiée chez des plantes PTOX+ surexprimant PTOX. En raison de leur réponse altérée en conditions de faible et forte intensité lumineuse, il a été proposé que pour fonctionner comme enzyme protectrice, PTOX est couplée à une SOD. Chez les lignées PTOX+, le niveau de SOD chloroplastique n'était pas plus élevé, limitant probablement leur capacité à détoxifier les taux élevés de O2•- généré

Enzymes impliqués dans la production des formes réactives de l'oxygène dans les membranes plasmiques, les mitochondries et les chloroplastes

Les formes réactives de l oxygène (FRO) ont été analysées dans différents compartiments cellulaires en utilisant des méthodes spectroscopiques (UV/VIS, fluorescence, infrarouge, résonance paramagnétique électronique). L identité et les mécanismes catalytiques des enzymes qui produisent les FRO dans les membranes plasmiques (MP) et les mitochondries ont été étudiés, ainsi que le rôle protectif de l oxydase terminale plastidiale (PTOX) des chloroplastes. Cd2+ s est révélé être un inhibiteur de la NADPH oxydase des MP. In vivo Cd2+ inhibait la production extracellulaire de O2.- mais stimulait l accumulation de H2O2. Dans des mitochondries isolées, Cd2+ a augmenté la production de FRO . Antimycin A a entrainé une élévation du H2O2 extracellulaire, confirmant que la mitochondrie est le site principal de production de l H2O2 extracellulaire induite par Cd2+ in vivo. Une quinone réductase (QR) génératrice de FRO a été isolée des MP. La déprotonation PH dépendante du quinole a produit des formes intermédiaires instables qui génèrent des FRO par réaction avec O2. Des espèces quinoniques ont été détectées dans la MP et pourraient servir de substrat aux QR in vivo. La protection de la chaine photosynthétique de transfert d électron par la plastoquinol ; O2 oxydoréductase a été étudiée chez des plantes PTOX+ surexprimant PTOX. En raison de leur réponse altérée en conditions de faible et de forte intensité lumineuse, il a été proposé que pour fonctionner comme enzyme protectrice, PTOX est couplée à une SOD. Chez les lignées PTOX+, le niveau de SOD chloroplastique n était pas plus élevé, limitant probablement leur capacité à détoxifier les taux élevés de O2.- généré.Production of reactive oxygen species (ROS) was studied in different subcellular compartments using spectroscopic methods (UV/VIS, fluorescence, infrared and electron paramagnetic resonance). The identities and catalytic mechanisms of ROS-producing enzymes in the plasma membrane (PM) were studied as well as mitochondrial ROS accumulation in response to cadmium (Cd2+) and the protectrice role of the plastid terminal oxidase (PTOX). Cd2+ was shown to be an inhibitor of the PM superoxide (O2.-) producing NADPH oxidase in vitro. In vivo Cd2+ inhibited the extracellular production of O2.- but stimulated the accumulation of hydrogen peroxide (H2O2 ). Cd2+ induced ROS production in isolated mitochondria. Mitochondrial ROS- inducing inhibitors increased extracellular H2O2 production confirming these organelles the main source of Cd2+ induced extracellular H2O2 generation in vivo. A ROS-producing quinone reductase(QR) was isolated from PMs. ROS production occurred via the pH dependent deprotonation of the end product menadiol leading to its intermediate forms that react with O2 forming O2.- and H2O2. A potential naphthoquinose species in PMs was identified that could serve as a QR substrate in vivo. The protection of the photosynthetic electron transfer chain by the PTOX, a plastoquinol : O2 oxidoreductase, was studied in PTOX-overexpressing plants (PTOX+ ). Based on the altered response to low and high light conditions in PTOX+ it was proposed that PTOX is coupled to an SOD in order to function as a protective enzyme. The absence of additional chloroplastic SOD in PTOX+ lines might have limited the plant s ability to detoxify O2.- produced by elevated levels of PTOX+ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Naphthoquinone-Dependent Generation of Superoxide Radicals by Quinone Reductase Isolated from the Plasma Membrane of Soybean[W]

Using a tetrazolium-based assay, a NAD(P)H oxidoreductase was purified from plasma membranes prepared from soybean (Glycine max) hypocotyls. The enzyme, a tetramer of 85 kD, produces O2·− by a reaction that depended on menadione or several other 1,4-naphthoquinones, in apparent agreement with a classification as a one-electron-transferring flavoenzyme producing semiquinone radicals. However, the enzyme displayed catalytic and molecular properties of obligatory two-electron-transferring quinone reductases of the DT-diaphorase type, including insensitivity to inhibition by diphenyleneiodonium. This apparent discrepancy was clarified by investigating the pH-dependent reactivity of menadionehydroquinone toward O2 and identifying the protein by mass spectrometry and immunological techniques. The enzyme turned out to be a classical NAD(P)H:quinone-acceptor oxidoreductase (EC 1.6.5.2, formerly 1.6.99.2) that reduces menadione to menadionehydroquinone and subsequently undergoes autoxidation at pH ≥ 6.5. Autoxidation involves the production of the semiquinone as an intermediate, creating the conditions for one-electron reduction of O2. The possible function of this enzyme in the generation of O2·− and H2O2 at the plasma membrane of plants in vivo is discussed

Role of the NAD(P)H quinone oxidoreductase NQR and the cytochrome b AIR12 in controlling superoxide generation at the plasma membrane.

International audienceThe quinone reductase NQR and the b-type cytochrome AIR12 of the plasma membrane are important for the control of reactive oxygen species in the apoplast. AIR12 and NQR are two proteins attached to the plant plasma membrane which may be important for generating and controlling levels of reactive oxygen species in the apoplast. AIR12 (Auxin Induced in Root culture) is a single gene of Arabidopsis that codes for a mono-heme cytochrome b. The NADPH quinone oxidoreductase NQR is a two-electron-transferring flavoenzyme that contributes to the generation of O 2(•-) in isolated plasma membranes. A. thaliana double knockout plants of both NQR and AIR12 generated more O 2(•-) and germinated faster than the single mutant affected in AIR12. To test whether NQR and AIR12 are able to interact functionally, recombinant purified proteins were added to plasma membranes isolated from soybean hypocotyls. In vitro NADH-dependent O 2(•-) production at the plasma membrane in the presence of NQR was reduced upon addition of AIR12. Electron donation from semi-reduced menadione to AIR12 was shown to take place. Biochemical analysis showed that purified plasma membrane from soybean hypocotyls or roots contained phylloquinone and menaquinone-4 as redox carriers. This is the first report on the occurrence of menaquinone-4 in eukaryotic photosynthetic organisms. We propose that NQR and AIR12 interact via the quinone, allowing an electron transfer from cytosolic NAD(P)H to apoplastic monodehydroascorbate and control thereby the level of reactive oxygen production and the redox state of the apoplast

Plastid alternative oxidase (PTOX) promotes oxidative stress when overexpressed in tobacco

International audiencePhotoinhibition and production of reactive oxygen species were studied in tobacco plants overexpressing the plastid terminal oxidase (PTOX). In high light, these plants was more susceptible to photoinhibition than wild-type plants. Also oxygen-evolving activity of isolated thylakoid membranes from the PTOX-overexpressing plants was more strongly inhibited in high light than in thylakoids from wild-type plants. In contrast in low light, in the PTOX overexpressor, the thylakoids were protected against photoinhibition while in wild type they were significantly damaged. The production of superoxide and hydroxyl radicals was shown by EPR spin-trapping techniques in the different samples. Superoxide and hydroxyl radical production was stimulated in the overexpressor. Two-thirds of the superoxide production was maintained in the presence of DNP-INT, an inhibitor of the cytochrome b(6)f complex. No increase of the SOD content was observed in the overexpressor compared with the wild type. We propose that superoxide is produced by PTOX in a side reaction and that PTOX can only act as a safety valve under stress conditions when the generated superoxide is detoxified by an efficient antioxidant syste

Assignment of the slowly exchanging substrate water of nature's water-splitting cofactor

Identifying the two substrate water sites of nature's water-splitting cofactor (Mn4CaO5 cluster) provides important information toward resolving the mechanism of O-O bond formation in Photosystem II (PSII). To this end, we have performed parallel substrate water exchange experiments in the S1 state of native Ca-PSII and biosynthetically substituted Sr-PSII employing Time-Resolved Membrane Inlet Mass Spectrometry (TR-MIMS) and a Time-Resolved 17O-Electron-electron Double resonance detected NMR (TR-17O-EDNMR) approach. TR-MIMS resolves the kinetics for incorporation of the oxygen-isotope label into the substrate sites after addition of H218O to the medium, while the magnetic resonance technique allows, in principle, the characterization of all exchangeable oxygen ligands of the Mn4CaO5 cofactor after mixing with H217O. This unique combination shows i) that the central oxygen bridge (O5) of Ca-PSII core complexes isolated from Thermosynechococcus vestitus has, within experimental conditions, the same rate of exchange as the slowly exchanging substrate water (WS) in the TR-MIMS experiments and ii) that the exchange rates of O5 and WS are both enhanced by Ca2+→Sr2+ substitution in a similar manner. In the context of previous TR-MIMS results, this shows that only O5 fulfills all criteria for being WS. This strongly restricts options for the mechanism of water oxidation

Thylakoid Membrane Architecture in Synechocystis Depends on CurT, a Homolog of the Granal CURVATURE THYLAKOID1 Proteins

International audienc