Anja Krieger-Liszkay

Herbicide-induced oxidative stress in photosystem II

Some herbicides act by binding to the exchangeable quinone site in the photosystem II (PSII) reaction centre, thus blocking electron transfer. In this article, it is hypothesized that the plant is killed by light-induced oxidative stress initiated by damage caused by formation of singlet oxygen in the reaction centre itself. This occurs when light-induced charge pairs in herbicide-inhibited PSII decay by a charge recombination route involving the formation of a chlorophyll triplet state that is able to activate oxygen. The binding of phenolic herbicides favours this pathway, thus increasing the efficiency of photodamage in this class of herbicides.

Singlet oxygen production in herbicide‐treated photosystem II

Photo‐generated reactive oxygen species in herbicide‐treated photosystem II were investigated by spin‐trapping. While the production of OH and O2 − was herbicide‐independent, 1O2 with a phenolic was twice that with a urea herbicide. This correlates with the reported influence of these herbicides on the redox properties of the semiquinone QA − and fits with the hypothesis that 1O2 is produced by charge recombination reactions that are stimulated by herbicide binding and modulated by the nature of the herbicide. When phenolic herbicides are bound, charge recombination at the level of P+ Pheo− is thermodynamically favoured forming a chlorophyll triplet and hence 1O2. With urea herbicides this pathway is less favourable.

Efficient Assembly of Photosystem II in Chlamydomonas reinhardtii Requires Alb3.1p, a Homolog of Arabidopsis ALBINO3

Alb3 homologs Oxa1 and YidC have been shown to be required for the integration of newly synthesized proteins into membranes. Here, we show that although Alb3.1p is not required for integration of the plastid-encoded photosystem II core subunit D1 into the thylakoid membrane of Chlamydomonas reinhardtii, the insertion of D1 into functional photosystem II complexes is retarded in the Alb3.1 deletion mutant ac29. Alb3.1p is associated with D1 upon its insertion into the membrane, indicating that Alb3.1p is essential for the efficient assembly of photosystem II. Furthermore, levels of nucleus-encoded light-harvesting proteins are vastly reduced in ac29; however, the remaining antenna systems are still connected to photosystem II reaction centers. Thus, Alb3.1p has a dual function and is required for the accumulation of both nucleus- and plastid-encoded protein subunits in photosynthetic complexes of C. reinhardtii.

Towards structural determination of the water-splitting enzyme

A photosystem II preparation from the thermophilic cyanobacterium Synechococcus elongatus, which is especially suitable for three-dimensional crystallization in a fully active form was developed. The efficient purification method applied here yielded 10 mg of protein of a homogenous dimeric complex of about 500 kDa within 2 days. Detailed characterization of the preparation demonstrated a fully active electron transport chain from the manganese cluster to plastoquinone in the QB binding site. The oxygen-evolving activity, 5000–6000 μmol of O2/(h·mg of chlorophyll), was the highest so far reported and is maintained even at temperatures as high as 50u2009°C. The crystals obtained by the vapor diffusion method diffracted to a resolution of 4.3 Å. The space group was determined to be P212121 with four photosystem II dimers per unit cell. Analysis of the redissolved crystals revealed that activity, supramolecular organization, and subunit composition were maintained during crystallization.

Production, Detection, and Signaling of Singlet Oxygen in Photosynthetic Organisms

SIGNIFICANCEnIn photosynthetic organisms, excited chlorophylls (Chl) can stimulate the formation of singlet oxygen ((1)O(2)), a highly toxic molecule that acts in addition to its damaging nature as an important signaling molecule. Thus, due to this dual role of (1)O(2), its production and detoxification have to be strictly controlled.nnnRECENT ADVANCESnRegulation of pigment synthesis is essential to control (1)O(2) production, and several components of the Chl synthesis and pigment insertion machineries to assemble and disassemble protein/pigment complexes have recently been identified. Once produced, (1)O(2) activates a signaling cascade from the chloroplast to the nucleus that can involve multiple mechanisms and stimulate a specific gene expression response. Further, (1)O(2) signaling was shown to interact with signal cascades of other reactive oxygen species, oxidized carotenoids, and lipid hydroperoxide-derived reactive electrophile species.nnnCRITICAL ISSUESnDespite recent progresses, hardly anything is known about how and where the (1)O(2) signal is sensed and transmitted to the cytoplasm. One reason for that is the limitation of available detection methods challenging the reliable quantification and localization of (1)O(2) in plant cells. In addition, the process of Chl insertion into the reaction centers and antenna complexes is still unclear.nnnFUTURE DIRECTIONSnUnraveling the mechanisms controlling (1)O(2) production and signaling would help clarifying the specific role of (1)O(2) in cellular stress responses. It would further enable to investigate the interaction and sensitivity to other abiotic and biotic stress signals and thus allow to better understand why some stressors activate an acclimation, while others provoke a programmed cell death response.

Influence of the Redox Potential of the Primary Quinone Electron Acceptor on Photoinhibition in Photosystem II

We report the characterization of the effects of the A249S mutation located within the binding pocket of the primary quinone electron acceptor, QA, in the D2 subunit of photosystem II in Thermosynechococcus elongatus. This mutation shifts the redox potential of QA by ∼–60 mV. This mutant provides an opportunity to test the hypothesis, proposed earlier from herbicide-induced redox effects, that photoinhibition (light-induced damage of the photosynthetic apparatus) is modulated by the potential of QA. Thus the influence of the redox potential of QA on photoinhibition was investigated in vivo and in vitro. Compared with the wild-type, the A249S mutant showed an accelerated photoinhibition and an increase in singlet oxygen production. Measurements of thermoluminescence and of the fluorescence yield decay kinetics indicated that the charge-separated state involving QA was destabilized in the A249S mutant. These findings support the hypothesis that a decrease in the redox potential of QA causes an increase in singlet oxygen-mediated photoinhibition by favoring the back-reaction route that involves formation of the reaction center chlorophyll triplet. The kinetics of charge recombination are interpreted in terms of a dynamic structural heterogeneity in photosystem II that results in high and low potential forms of QA. The effect of the A249S mutation seems to reflect a shift in the structural equilibrium favoring the low potential form.

Plastid Alternative Oxidase (PTOX) Promotes Oxidative Stress When Overexpressed in Tobacco

Photoinhibition 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 b6f 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 system.

The glutathione peroxidase homologous gene Gpxh in Chlamydomonas reinhardtii is upregulated by singlet oxygen produced in photosystem II

The expression of the glutathione peroxidase homologous gene Gpxh, known to be specifically induced by the formation of singlet oxygen (1O2), was analyzed in cells of Chlamydomonas reinhardtii exposed to environmental conditions causing photoinhibition. Illumination with high light intensities, leading to an increased formation of 1O2 in photosystem II, continuously induced the expression of Gpxh in cell for at least 2xa0h. Phenolic herbicides like dinoterb, raise the rate of 1O2 formation by increasing the probability of charge recombination in photosystem II via the formation of the primary radical pair and thereby 3P680 formation (Fufezan C etxa0al. 2002, FEBS Letters 532, 407–410). In the presence of dinoterb the light-induced loss of the D1 protein in C. reinhardtii was increased and the high light-induced Gpxh expression was further stimulated. DCMU, a urea-type herbicide, causing reduced 1O2 generation in photosystem II, protected the D1 protein slightly against degradation and downregulated the expression of the Gpxh gene compared to untreated cells exposed to high light intensities. This indicates that the Gpxh expression is induced by 1O2 under environment conditions causing photoinhibition.

Photosynthetic electron flow affects H2O2 signaling by inactivation of catalase in Chlamydomonas reinhardtii

A specific signaling role for H2O2 in Chlamydomonas reinhardtii was demonstrated by the definition of a promoter that specifically responded to this ROS. Expression of a nuclear-encoded reporter gene driven by this promoter was shown to depend not only on the level of exogenously added H2O2 but also on light. In the dark, the induction of the reporter gene by H2O2 was much lower than in the light. This lower induction was correlated with an accelerated disappearance of H2O2 from the culture medium in the dark. Due to a light-induced reduction in catalase activity, H2O2 levels in the light remained higher. Photosynthetic electron transport mediated the light-controlled down-regulation of the catalase activity since it was prevented by 3-(3′4′-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II. In the presence of light and DCMU, expression of the reporter gene was low while the addition of aminotriazole, a catalase inhibitor, led to a higher induction of the reporter gene by H2O2 in the dark. The role of photosynthetic electron transport and thioredoxin in this regulation was investigated by using mutants deficient in photosynthetic electron flow and by studying the correlation between NADP-malate dehydrogenase and catalase activities. It is proposed that, contrary to expectations, a controlled down-regulation of catalase activity occurs upon a shift of cells from dark to light. This down-regulation apparently is necessary to maintain a certain level of H2O2 required to activate H2O2-dependent signaling pathways.

The Cyanobacterial Photoactive Orange Carotenoid Protein Is an Excellent Singlet Oxygen Quencher

This work shows that the cyanobacterial photoactive orange carotenoid protein (OCP) protects Synechocystis cells from photoinhibition even under conditions in which it is unable to quench excess energy absorbed by phycobilisomes. OCP plays a dual role under light stress conditions, protecting cells against photooxidative stress by quenching excess energy and singlet oxygen. Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the photosynthetic reaction centers under high-light conditions. The photoactive orange carotenoid protein (OCP) is essential in this mechanism as a light sensor and energy quencher. When OCP is photoactivated by strong blue-green light, it is able to dissipate excess energy as heat by interacting with phycobilisomes. As a consequence, charge separation and recombination leading to the formation of singlet oxygen diminishes. Here, we demonstrate that OCP has another essential role. We observed that OCP also protects Synechocystis cells from strong orange-red light, a condition in which OCP is not photoactivated. We first showed that this photoprotection is related to a decrease of singlet oxygen concentration due to OCP action. Then, we demonstrated that, in vitro, OCP is a very good singlet oxygen quencher. By contrast, another carotenoid protein having a high similarity with the N-terminal domain of OCP is not more efficient as a singlet oxygen quencher than a protein without carotenoid. Although OCP is a soluble protein, it is able to quench the singlet oxygen generated in the thylakoid membranes. Thus, OCP has dual and complementary photoprotective functions as an energy quencher and a singlet oxygen quencher.