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PROJECT PROPOSAL for applicants for ITC fellowships (2017/18) supervisor: institution: contact: co-supervisor: Ateeq Ur REHMAN Institute of Plant Biology [email protected] Imre VASS, Ph.D., D.Sc. project title: THE EFFECT OF CHLORAMPHENICOL ON PSII PHOTOINHIBITION AND THE PRODUCTION OF SUPEROXIDE IN ISOLATED SPINACH THYLAKOID MEMBRANES PROJECT SUMMARY Photoinhibition is the light induced damage of photosynthetic activity, which affects primarily the Photosystem II (PSII) complex. In intact organisms photodamage can be restored via a repair process of PSII, which includes de novo synthesis of the D1 reaction center protein. The photodamage and repair processes can be separated from each other by the application protein synthesis inhibitors, such as the frequently used chloramphenicol. However, chloramphenicol might induce unwanted side effects, such as the production of superoxide, which can interfere with the process of photoinhibition. The aim of the project is to characterize the effect of chloramphenicol on photoinhibition studied in isolated thylakoid membranes. BACKGROUND Photosynthetic organisms are able to use sunlight to synthesize carbohydrates from inorganic carbon (CO2). The photosynthetic apparatus contains four major pigment protein complexes in the thylakoid membrane, namely photosystem I (PSI), photosystem II (PSII), cytochrome b6/f complex (Cyt) and ATP synthase. PSII is the first major complex in the electron transport chain, which performs light induced splitting of water into electrons, protons and molecular oxygen. Light is not only the driving force of photosynthesis, but also a major stress factor, which targets primarily the PSII complex. This process is called photoinhibition, which leads to the loss of PSII activity and the degradation of its reaction centre D1-subunit. The photodamaged PSII complex can be repaired in intact photosynthetic organisms, via de novo synthesis the D1-subunit. Reactive oxygen species (ROS), which are formed during the process of photosynthesis, have an important role in photoinhibition. One of the ROS involved in photodamage is superoxide, which is produced as a result of electron transfer from photosynthetic components of PSI and PSII to molecular oxygen. In order to separate the photodamage and repair processes protein synthesis inhibitors are applied, which prevent the re-synthesis of damaged D1 subunit of PSII. One of the frequently applied inhibitors is chloramphenicol, which is used both in cyanobacteria and higher plants. In spite of its frequent use in photoinhibitory studies some reports indicate that chloramphenicol might have unwanted side effects, such as promotion of superoxide production that may enhance light induced damage of PSII. In the present study we aim to clarify the effect of chloramphenicol on PSII, and its involvement in the production of superoxide with special emphasis on its possible interaction with photodamage in thylakoid membranes. CURRENT RESEARCH Our research group is involved in the study of photodamage and photo-protection mechanism of the PSII complex in photosynthetic organism from the last 25 years. Previously we have developed a model for photodamage and photoprotection mechanism of the PSII complex. Our group is also involved in developing a method for the detection of various ROS in intact Synechosystis, algal and plant cells. Most recently, we established a Hismediated chemical trapping method for the detection of singlet oxygen in intact Synechosystis cells, which has already been successfully applied in different Synechosystis strains. Our preliminary results also show that chloramphenicol is not only involved in D1 protein synthesis inhibition, but also takes electrons from PSI to produce superoxide and accelerates photodamage. Rate of O2 uptake (µmolO2/mgChl/h) 0 SOD -50 No add. -100 Chloram +SOD -150 Spinach thylakoid Chloram Effect of SOD and chloramphenicol on the rate of oxygen uptake in spinach thylakoid membranes SPECIFIC AIMS The general aim of this research project is to investigate the effect of chloramphenicol on PSII photoinhibition in plant thylakoid membranes, and also to gain knowledge on the production of superoxide. Specific aims of the project include: To understand the mechanisms of photodamage of the PSII complex by chloramphenicol in isolated thylakoid membranes. To detect and quantify O2- production in the presence and absence of chloramphenicol in isolated thylakoids. METHODS TO BE LEARNED / APPLIED Supported by the TÁMOP 4.1.1.C -13/1/KONV.2014-0001 project To address the above aims, a series of experiments will be performed. We will employ a combination of biochemical and biophysical approaches to study the effect of chloramphenicol on the photosynthetic efficiency and superoxide production in isolated thylakoid membrane. Thylakoid membranes will be isolated from spinach by standard method. The membranes will be stored in -80 °C and will be used for further experiments. The sample concentration for chlorophyll fluorescence measurement will be 10µg Chl/ml and for oxygen measurements 25 µg Chl/ml will be used. Light treatment of resuspended thylakoid membranes in the presence or absence of chloramphenicol. Application of oxygen polarography by using a DW2 oxygen electrode (Hansatech) to follow PSII activity. Application steady-state and flash-induced chlorophyll fluorescence to monitor light induced changes of PSII electron transport characteristics. SUGGESTED READINGS Okada K, et al.: Chloramphenicol is an inhibitor of photosynthesis. FEBS Lett., 295:155-158(1991) Vass I and Cser K: Janus-faced charge recombinations in photosystem II photoinhibition. Trends Plant Sci, 14:20-205(2009) Vass I: Role of charge recombination process in photodamage and photoprotection of the photosystem II complex. Physiologia Plantarum, 142:6-16(2011) Vass I: Molecular mechanisms of photodamage in the Photosystem II complex. Biochim Biophys Acta, 1817:209-217(2012) Tsermentseli S, et al.: The anatomy of cognitive impairment in amyotrophic lateral sclerosis: more than frontal lobe dysfunction. Cortex, 48:166182(2012) Valori CF, et al.: The multifaceted role of glial cells in amyotrophic lateral sclerosis. Cell Mol Life Sci, 71:287-297(2014) SNAPSHOTS FROM THE HOST LABORATORY Significant publications Zhang P, et al.: Operon flv4-flv2 provides cyanobacterial Photosystem II with flexibility of electron transfer. Plant Cell, 24:19521971(2012) Rehman AU, et al.: Characterization of singlet oxygen production and its involvement in photodamage of Photosystem II in the cyanobacterium Synechocystis PCC 6803 by histidine mediated chemical trapping. Biochim Biophys Acta, 1827:689698(2013) Sedoud A, et al.: Cyanobacterial photoactive Orange Carotenoid Protein is an excellent singlet oxygen quencher. Plant Cell, 26:1781-1791(2014) Bersanini L, et al.: Flv2/Flv4 related photoprotective mechanism dissipates excitation pressure of photosystem II in cooperation with phycobilisomes in cyanobacteria. Plant Physiology, 164:805-818(2014) Hill R, et al.: Inhibition of photosynthetic CO2 fixation in the coral Pocillopora damicornis and its relationship to thermal bleaching. Journal of Experimental Biology, 217:2150-2162(2014) Hakkila K, et al.: Oxidative stress and photoinhibition can be separated in the cyanobacterium Synechocystis sp. PCC 6803. Biochim Biophys Acta, 1837:217-225(2014) Shunmugam S, et al.: Secondary metabolite from Nostoc XPORK14A inhibits photosynthesis and growth of Synechocystis PCC 6803. Plant Cell and Environment, 37:1371-1381(2013) Representative recent research grants “Exploration of alternative electron transport patways in cyanobacteria by computer modelling and experimental approaches: From regulation of light reactions to biosolar fuel production” (OTKA, NN-110960) Some of the latest students in the laboratory Rehman A, Ph.D., 2011-2014; “Detection of singlet oxygen in cyanobacterial systems” Vass I-Z, ITC + Ph.D, 2010-14; “Interaction of DNA damage and repair with protein synthesis dependent repair of photodamaged PSII” Paul K, ITC + Ph.D., 2010-2014; “Application of plant phenotyping to study the effects of drought stress in wheat and barley” Kodru S, ITC, 2014-recent; “Detection of superoxide in isolated thylakoid membranes” Kovács S, B.Sc., 2013-recent; ”Effect of silver nanoparticles on the photosynthetic efficiency of Synechocystis PCC 6803 cells” Supported by the TÁMOP 4.1.1.C -13/1/KONV.2014-0001 project