Download 10. the effect of chloramphenicol on psii photoinhibition and

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