ROS i antyoksydanty w infekcji roślin, Publikacje naukowe

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//-->1Ph.D. School Title:Crop SciencesCrop Sciences and HorticultureSciences (Plant Pathology andPathophysiology)Prof. Dr. Ferenc Virányi, DScDepartment of Plant ProtectionSzent István University,Gödöllő, HungaryProf. Dr. Zoltán Király,AcademicianPlant Protection Institute,Hungarian Academy ofSciences, Budapest, HungarySZENT ISTVÁN UNIVERSITYField of Science:BIOCHEMICAL AND MOLECULARSTUDIES ON THE ROLE OF REACTIVEOXYGEN SPECIES AND ANTIOXIDANTS INPLANT DISEASE RESISTANCEPh.D. School Leader:PhD THESISSupervisor:YASSER MOHAMED HAFEZGÖDÖLLŐ2005……….………………Approval of Ph.D. School Leader.………………………Approval of Supervisor2NTRODUCTIONReactive oxygen species (free radicals)Recently, it turned out that there is a certain balance between the actionof reactive oxygen species (ROS) and antioxidants in microorganisms, plant aswell as in animal cells. As a result of stress or infection, this balance is abnormalor does not exist. Thus, the role of ROS in different forms of disease resistance orin symptom expression of susceptible plants seems to be pivotal.Reactive oxygen species (ROS) are involved in many important processesin plants (Elstner et al., 1994). ROS are believed to have important roles in plantsin general and in plant-pathogen interactions in particular. They are involved insignal transduction, cell wall reinforcement, hypersensitive response (HR) andphytoalexin production, and have direct antimicrobial effects (Abdu et al., 1993;Galal et al., 1993; Király et al., 1993). The main toxic ROS are the superoxideanion radical (O2·-), hydrogen peroxide (H2O2), hydroxyl radical (OH·) and singletoxygen (1O2) in the biological systems (Elstner, 1982, 1987; Tzeng and DeVay1993). As is seen, some ROS are free radicals and some are reactive molecules.As a result of senescence, superoxide is produced in a high level in themembranes and, at the same time, some antioxidants such as SOD are reduced inthe activity (Droillard et al., 1987, 1989).Role of antioxidantsAntioxidants are substances that delay or inhibit oxidative damage totarget molecules such as lipids, proteins, nucleic acids and carbohydrates.Antioxidants might protect a target by scavenging oxygen-derived species orminimizing the formation of oxygen-derived species.Induced resistanceA common response to necrogenic pathogen infection is the developmentof systemic acquired resistance (SAR) to a subsequent pathogen attack. Thisinduced resistance or SAR results in broad-spectrum, non-specific immunity innon-infected parts of the plant and provides protection against several subsequentpathogens and non-pathogens (Ryals et al., 1994, 1995).The accumulation of salicylic acid (SA) is an important component in thesignal transduction pathway leading to SAR (Métraux et al., 1990; Malamy et al.,1990). It was suggested that the role of SA in SAR signal transduction can inhibitcatalase activity, leading to elevated levels of H2O2which could in turn function asa second messanger of SA in SAR signal transduction (Chen and Klessig, 1991;Chen et al., 1993).The use of chemicals to activate SAR-type reaction provides novelalternatives for disease control in agronomic systems. 2,6-dichloroisonicotinic acidOxidative stressOxidative stress is caused by herbicides, infections (biotic stress) andabiotic stresses, such as air pollution, high light intensity, heat shock etc.Oxidative stress in the cells results in damaging the membranes, the lipids, aminoacids, nucleic acids, pigments and proteins. The end result could be senescence orcell death.3and its methyl ester (both referred to as INA) were the first synthetic chemicalcompounds shown to activate SAR-type reaction, thus providing broad spectrumdisease resistance. Thus, INA seems to be a proper compound for practicalagronomic use.PathogensTobacco mosaic virus (TMV) U1 strain andPseudomonas savastanoi(syringae)pv.phaseolicolaGSPB 1205 were used in this study. The followingfungal pathogens were involved in this study: barley powdery mildew (Blumeriagraminisf. sp.hordeirace A6) andBotrytis cinereastrain Bc-1.The aim of my research••••I tried to have a deeper insight into the role of ROS (H2O2) in inducingsymptom expression in powdery mildew infected barley.Induction of HR type necroses in virus-infected local lesion tobacco even at30°C with the application of ROS.Chemically induced ROS and resistance caused by 2,6-dichloroisonicotinicacid (INA) treatment in susceptible barley plants.Immunization of tobacco plants with very low concentrations of H2O2.Artificial production of necroses at 30°CThe riboflavin/methionine photochemical systemInoculated tobacco leaves were detached three days after infection withTMV and put on the riboflavin/methionine solutions in Petri dishes. Six ml ofmixture containing 266 or 532 µM riboflavin as well as 10 or 20 mM L-methionine, respectively was poured on filter paper in each Petri dish. The Petridishes were illuminated (100 µE m-2s-1) in an incubator at 30°C for three days.The same treatments were conducted with inoculated intact leaves by infiltrationwith a syringe. TMV-infected leaves (without riboflavin treatment) and riboflavintreated healthy leaves (without infection) were used as controls.MATERIALS AND METHODSPlant materialsTobacco (Nicotianatabacum)cultivar Xanthi-nc, which is a local lesion(HR) host of TMV, was used in this study. Susceptible barley (Hordeumvulgare)expressing the geneMloand near-isogenic lines of cultivar Ingrid carrying thegenesmlo5, Mla12andMlgfor resistance against the powdery mildew were alsoused.ROS-producing glucose-glucose oxidase system and directapplication of H2O2Six ml of solutions containing 50, 100, 150, 200, 250 and 300 units ofglucose oxidase/ml and 2 mM glucose were poured on filter paper in each Petridish or injected into intact leaves. TMV inoculated leaves were detached threedays after inoculation and put into these Petri dishes for three days. TMV-infected4leaves (without glucose-glucose oxidase) and glucose–glucose oxidase treatedhealthy leaves (without virus infection) were used as controls.Direct application of 10, 25, 50, 100, 150 and 200 mM H2O2was carriedout by injecting the intact leaves or treating the detached leaves in Petri dishes asdescribed above.held at 20°C served as controls. Four days after inoculation necroses (HR)appeared. ELISA test performed for determining concentration of TMV accordingto Clark and Adams (1977) and Tobiás et al. (1982).Xanthi-nc tobacco plants pre-treated with low concentration of H2O2were inoculated with TMV one day after the treatment. Plants infected with TMVwere kept in the greenhouse for three days. TMV-infected untreated plants servedas controls.ELISA test performed for determining concentration of TMV as mentioned above.The reversible action of antioxidant enzymesIn tobacco infected with TMV, the action of ROS at 30°C was reversedby treatment with 4000 U/ml of superoxide dismutase (SOD) and 5000 U/ml ofcatalase (CAT). SOD and CAT were applied to leaves treated withriboflavin/methionine three days after inoculation with TMV. CAT was applied onleaves treated with glucose-glucose oxidase or directly treated with H2O2threedays after inoculation with TMV.Barley leaves were injected with a water solution contained 2500 unitssuperoxide dismutase (SOD) and 5000 units catalase (CAT)/ ml and leaves weresprayed with H2O2after water evaporation. Leaves were infected with thepowdery mildew fungus after H2O2treatment.Tobacco leaves inoculated with TMV,P. syringaepv.phaseolicolaandBotrytis cinereawere injected immediately after inoculation with a solution whichcontained 5000 units CAT and 2500 units SOD/ ml.Determination of concentration of bacteriaInfection withP. syringaepv.phaseolicolawas carried out one day aftertreatment with low concentration of H2O2and kept in the greenhouse for one day.Plants infected with the bacterium served as controls. To determine the bacterialconcentration, we used the plate-count technique 24, 48 and 72 hours afterinoculation.Application of H2O2to barley leavesLeaves of 8-10-day-old barley seedlings were inoculated withBlumeriagraminisf. sp.hordei.Some intact inoculated leaves were detached at differenttime periods after inoculation. Either intact or detached leaves were sprayed with25-50 mM H2O2one, two and three days after inoculation. The water solution ofH2O2contained 0.5% Tween. Leaves, which were treated with H2O2only orinfected only with the pathogen, served as controls.Determination of concentration of TMVXanthi-nc tobacco plants infected with TMV and treated with riboflavin,glucose-glucose oxidase or H2O2were kept at 30°C. TMV-infected tobacco pants5Application of low concentration of H2O2in tobaccoThe fourth and fifth true leaves of 8-10-week-old Xanthi-nc plants weretreated by spraying the plant leaves with an aqueous solution of H2O2. Plants weresprayed with 5, 7, 10, and 12.5 mM H2O2solution. The control plants were treatedwith water alone. After one day of H2O2treatments, the leaves infected with TMV,Pseudomonas syringaepv.phaseolicolaor withBotrytis cinerea.To detect H2O2spectrophotometrically with a peroxidase independentreaction, a xylenol orange based method was used according to Gay et al. (1999).To detect H2O2by the spectrofluorometer, 2’, 7’-dichlorofluoresceindiacetate (DCFH-DA) dye was used. This dye reacts with H2O2in the presence ofperoxidase yielding the fluorescent dichlorofluorescein (DCF). We used thismethod described by Lu and Higgins (1998).Chemical induction of resistance by INASusceptible barley seedlings (Hordeumvulgarecultivar Ingrid) weretreated with 2,6-dichloroisonicotinic acid (INA) applied as a soil-drench (6-mg/litre soil) 3-4 days after sowing. Inoculation with the powdery mildew fungus(Blumeriagraminisf. sp.hordei)was carried out 4 days after INA treatment (7-8days after sowing). Leaf samples were harvested 12, 18, 24, 36, 48, 60, 72, 84 and96 hours after inoculation with the powdery mildew fungus.Biochemical and gene expression assays of the antioxidantenzymesActivities of antioxidant enzymes, such as ascorbate peroxidase (APX),catalase (CAT), superoxide dismutase (SOD), dehydroascorbate reductase(DHAR), glutathione reductase (GR), glutathione S-transferase (GST), guaiacolperoxidase (POX) as well as activity of NADPH oxidase were determined bybiochemical (spectrophotometric) assays. The gene expression of antioxidantenzymes, alternative oxidase and BAX inhibitor gene were determined using theRT-PCR technique.Histochemical analysis of ROSHistochemical staining for superoxide production in leaf tissue was basedon the ability of O2·-3. RESULTSRole of hydrogen peroxide in symptom expression of barleysusceptible and resistant to powdery mildewUnder natural conditions barley leaves, carrying the geneMlo,exhibitedsusceptible response to infection, themloandMlgbarley leaves were resistant butdid not develop HR necrotic symptoms. TheMla12barley was resistant anddeveloped HR-type symptoms. Under the influence of treatment with H2O2(25-50to reduce nitro blue tetrazolium (NBT). Superoxide wasvisualised as a purple discoloration of NBT. Discoloration of leaf discs wasquantified using a ChemiImager 4000 digital imaging system .H2O2was visualised as a reddish-brown discoloration of 3,3-diaminobenzidine (DAB). Detection of H2O2was performed using 0.1% DAB, asdescribed by Thordal-Christensen et al. (1997) and Hückelhoven et al. (1999). [ Pobierz całość w formacie PDF ]

ROS i antyoksydanty w infekcji roślin, Publikacje naukowe

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