Doctoral Thesis · Deepak Kumar Yadav and Pavel Pospíšil, 2011 (Poster) Hydroxyl radical...
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Doctoral Thesis MOLECULAR MECHANISMS OF PRODUCTION AND SCAVENGING OF REACTIVE OXYGEN SPECIES IN PHOTOSYSTEM II OF HIGHER PLANTS Deepak Kumar Yadav Department of Biophysics Centre of the Region Haná for Biotechnological and Agricultural Research Faculty of Science, Palacký University Olomouc, Czech Republic Olomouc, 2013
Transcript of Doctoral Thesis · Deepak Kumar Yadav and Pavel Pospíšil, 2011 (Poster) Hydroxyl radical...
Doctoral Thesis
MOLECULAR MECHANISMS OF PRODUCTION AND SCAVENGING
OF REACTIVE OXYGEN SPECIES IN PHOTOSYSTEM II
OF HIGHER PLANTS
Deepak Kumar Yadav
Department of Biophysics
Centre of the Region Haná for Biotechnological and Agricultural Research
Faculty of Science, Palacký University
Olomouc, Czech Republic
Olomouc, 2013
Bibliographical identification
Name and family name of the author: Deepak Kumar YADAV
Title of doctoral thesis: Molecular mechanisms of production and scavenging of reactive oxygen
species in photosystem II of higher plants
Degree program field (specialization): Biophysics
Duration of Ph.D. study: 2009-2013
Year of defense: 2013
Supervisor: Doc. RNDr. Pavel Pospíšil, Ph.D
Keywords: reactive oxygen species; photosystem II; oxidative stress; plastoquinol; tocopherol;
plastochromanol; electron paramagnetic resonance (EPR) spectroscopy; high-pressure liquid
chromatography (HPLC); confocal laser scanning microscope (CLSM).
© Deepak Kumar YADAV, Palacký University, Olomouc, Czech Republic
Contents
Declaration…………………………………………………………………………………...IV
List of publications...………………………………………………………………………….V
Curriculum Vitae….………………………………………………….……………………....VI
Acknowledgement…………………………………………………………………………...IX
Abbreviations………………………………………………………………………………..XI
Abstract…………………………………………………………………………………….XIII
Chapter 1, Introduction (Overview on structure and function of photosystem II)
1. Photosynthesis………………...…………………………………………………………….2
1.1. Photosynthetic apparatus.…………………………………………………………………2
1.2. Energy and electron transfer in photosynthesis…………………………………………...3
1.3. Photosystem II….……..…...……………………………………………………………...3
1.3.1. Structure of PSII…………………………………………………………………......4
1.3.2. Reaction center chlorophyll and pheophytin………………………………………...7
1.3.3. Plastoquinones and non-heme iron………………………………………………….8
1.3.4. Water-splitting manganese complex………………………………………………...9
1.3.5. Inorganic cofactors: calcium and chlorides………………………………………...10
1.3.6. Extrinsic proteins…………………………………………………………………...11
1.3.7. Cytochrome b559……………………………………………………………………12
Chapter 2, Reactive Oxygen Species (Production and scavenging of reactive oxygen species
in PSII)
2. Reactive oxygen species (ROS)…………………………………………………………...15
2.1. Types of ROS…...……………………………………………………………………….15
2.1.1. Singlet oxygen……………………………………………………………………...15
2.1.2. Superoxide anion radical…………………………………………………………...16
2.1.3. Hydrogen peroxide…………………………………………………………………16
2.1.4. Hydroxyl radical……………………………………………………………………17
2.2. Reactive oxygen species production in PSII.……………………………….....………...17
2.2.1. Singlet oxygen production in PSII…………………….…………………………...17
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2.2.1.1. Singlet oxygen production in antenna complex…………………….………...17
2.2.1.2. Singlet oxygen production in acceptor side photoinhibition of PSII………....18
2.2.1.3. Singlet oxygen production in donor side photoinhibition of PSII…………....19
2.2.2. Superoxide anion radical production in PSII………………………………….…...20
2.2.3. Hydrogen peroxide production in PSII…………………….……………………....21
2.2.4. Hydroxyl radical production in PSII……………………………..………………...21
2.3. Scavenging of ROS……………………………………………………………………...22
2.3.1. Synthesis of tocopherol, plastoquinol and plastochromanol……………………….22
2.3.2. Singlet oxygen scavenging by tocopherol………………………………………….24
2.3.3. Singlet oxygen scavenging by plastoquinol………………………………………..25
2.3.4. Singlet oxygen scavenging by plastochromanol…………………………………...26
Chapter 3, Materials and Methodology
3. Materials and Methods…………………………………………………………………….28
3.1. Chemicals………………………………………………………………………………..28
3.2. Preparation of PSII membranes from Spanacea oleracea……...............……………….28
3.3. Preparation of water-splitting manganese complex depleted PSII membranes…………29
3.4. Preparation of PQ-depleted PSII membranes……………………………………………29
3.5. Growing of Arabidopsis plants………………………………………………………….29
3.6. Chloroplasts isolation from Arabidopsis plants leaves………………………………….29
3.7. High-light treatment for photoinhibition………………………………………………...29
3.8. Heat treatment…………………………………………………………………………...30
3.9. Electron paramagnetic resonance spin-trapping spectroscopy..………...........………….30
3.10. Spectroflourimeterical detection of hydrogen peroxide…………………………..……30
3.11. Confocal laser scanning microscopy (CLSM) ………………………………………...31
3.12. High pressure liquid chromatography (HPLC) ………………………………………..31
3.13. Two-dimensional imaging of ultra-weak photon emission…………………………….31
3.14. Measurement of redox form of cyt b559………………………………………………...31
Chapter 4, Results and Discussion
4. Results and Discussion..…………………………………………………………………...33
4.1. Singlet oxygen scavenging activity of plastoquinol in PSII (Paper I) ………………….33
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4.1.1. Singlet oxygen scavenging by plastoquinol in chemical system…………………..33
4.1.2. Singlet oxygen scavenging by plastoquinol in PQ-depleted PSII membranes...…..34
4.2. Role of chloride ion in hydroxyl radical production in PSII under heat stress (Paper II).37
4.2.1. Hydroxyl radical production in PSII membranes under heat stress………………..37
4.2.2. Effect of halides on hydroxyl radical production in PSII membranes under heat
Stress..................................................................................................................................37
4.2.3. Effect of halides on hydrogen peroxide production in PSII membranes under heat
stress…………...................................................................................................................38
4.3. Evidence on singlet oxygen production in PSII in donor side of photoinhibition of PSII
(Paper III).................................................................................................................................40
4.3.1. Singlet oxygen production in Tris-treated PSII membranes……………………….40
4.3.2. Carbon-centered radical production in Tris-treated PSII membranes…………...…40
4.3.3. Singlet oxygen production in donor side of photoinhibition of PSII………..……..41
4.4. Singlet oxygen scavenging by tocopherol and plastochromanol in Arabidopsis thaliana
(Paper IV) ……………………………………………………………………………………42
4.4.1. HPLC analysis of the content of tocopherol and plastochromanol in WT and vte1
Arabidopsis leaves……………………………………………………………………….42
4.4.2. Singlet oxygen imaging by singlet oxygen sensor green in WT and vte1 Arabidopsis
leaves…..............................................................................................................................43
4.4.3. Singlet oxygen production in chloroplasts isolated from WT and vte1 Arabidopsis
leaves……………………………………………………………………………………..44
4.4.4. Malondialdehyde detection in WT and vte1 Arabidopsis leaves…….............….....46
4.5. Involvement of plastosemiquinone in superoxide anion radical production in PSII……47
4.5.1. Light-induced superoxide anion radical production in PSII membranes…….…….47
4.5.2. Effect of DCMU and dinoseb on light-induced superoxide anion radical production
in PSII membranes……………………………………………………………………….48
4.5.3. Effect of DCMU and dinoseb on photoreduction of cyt b559 in PSII membranes.…48
Chapter 5, Conclusion......…………………………………………………………………..52
Chapter 6, References………………………………………………………………………55
Chapter 7, Publications……………………………………………………………………..76
Chapter 8, Appendix
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Declaration
Hereby I declare that the Ph.D. thesis is my original work and effort that I have written it by
myself using the literature listed in the section “References”.
In Olomouc, ……… -----------------------------
Deepak Kumar Yadav
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List of Publications
This thesis is based on the following research papers. These research papers are referred in
the text by the corresponding roman numbers and are enclosed at the end of the thesis.
I. Yadav D.K., Kruk J., Sinha R.K., Pospíšil P. (2010) Singlet oxygen scavenging
activity of plastoquinol in photosystem II of higher plants: electron paramagnetic
resonance spin-trapping study, Biochimica et Biophysica Acta 1797, 1807-1811.
II. Yadav D.K., Pospíšil P. (2012) Role of chloride ion in hydroxyl radical production in
photosystem II under heat stress: Electron paramagnetic resonance spin-trapping
study, Journal of Bioenergetics and Biomembranes 44, 365-372.
III. Yadav D.K., Pospíšil P. (2012) Evidence on the formation of singlet oxygen in the
donor side photoinhibition of photosystem II: EPR spin-trapping study. PLoS ONE
7(9): e45883.
IV. Rastogi* A., Yadav* D.K., Szymańska R., Kruk J., Sedlářová M., Pospíšil P. (2013)
Singlet oxygen scavenging activity of tocopherol and plastochromanol in Arabidopsis
thaliana: Relevance to photooxidative stress condition (manuscript under revision,
Plant, Cell & Environment).
* These authors contributed equally to this work.
V. Yadav D.K., Kruk J., Pospíšil P. (2013) Evidence on the involvement of
plastosemiquinone in superoxide anion radical production in photosystem II
membranes of higher plant (submitted manuscript).
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Curriculum Vitae
Personal profile:
Name : Deepak Kumar Yadav
Email: [email protected]
Date of Birth : 10.01.1985
Languages known : English & Hindi
Citizenship: India
Current adress: N II, Třída Míru 113, 77111 Olomouc, Czech Republic
Permanent Address: Village Pasiyahi Kala (Ghamhapur) , P.O. Dharaon
Jalalpur, Jaunpur, Uttar Pradesh, India. Pin code- 222136
Educational Qualifications:
2003-2006 Bachelor of Science (B. Sc.)
Ewing Christian College, Allahabad University
Allahabad, Uttar Pradesh, India
Subjects: Botany, Zoology and Chemistry
2007-2009 Master of Science (M. Sc.)
School of Life Sciences, Devi Ahilya University, Indore
Indore, Madhya Pradesh, India
Subject: Life Science
2009-present Doctorate of Philosophy (Ph.D) pursuing
Department of Biophysics, Palacky University, Olomouc
Olomouc, Czech Republic
Study Field: Biophysics
Research topic: Molecular mechanisms of production and scavenging of
reactive oxygen species in photosystem II of higher plants.
Awards and national level competitive examination qualified:
Awarded “Director’s award for excellence in scientific publication” for 2012 by Centre of the
Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 11, Olomouc,
Czech Republic.
Awarded “Dean award” for scientific publication by Faculty of Science, Palacký University,
Olomouc. Czech Republic.
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2009 Graduate Aptitude Test in Engineering (GATE) conducted by IIT
Roorkee, Roorkee, Uttarakhand, India
2009 1st rank in Master of Science (M. Sc.)
2006 Meritorious student Certificate in Bachelor of Science (B. Sc.)
Research experiences at other institutions:
01.08.2012 to 30.10.2012 Department of Physics, Freie University Berlin
Berlin, Germany
Supervisor: Prof. Holger Dau
Project: Low pH induced inhibition of water-oxidation by
photosystem II
02.01.2009 to 31.06.2009 Department of Biochemistry, Maharaj Sayajirao
University, Vadodara, Gujarat India
Supervisor: Prof. G. Naresh Kumar
Project: Improving the residence time ability of
probiotic Escherichia Coli containing vitreoscilla
hemoglobin (vhb) gene.
21.05.2008 to 31.06.2008 Hamadard Laboratories, Ghaziabad, India
Project: Summer training in QA and QC
Conference presentations:
Deepak Kumar Yadav and Pavel Pospíšil, 2011 (Oral) Evidence on the formation of singlet
oxygen in donor side photoinhibition of photosystem II: EPR spin trapping study, summer
school “EBSA biophysics course on solar energybiological and biomimetic solutions” at
Biological research centre of the Hungarian academy of sciences, Szeged, Hungary on
August 27-31, 2011
Deepak Kumar Yadav and Pavel Pospíšil, 2011 (Poster) Hydroxyl radical production in
photosystem II under heat heat stress: EPR spin trapping study, conference “Photosynthesis
research for sustainability” at Baku, Azerbaijan on July 24-30, 2011.
Workshop and Seminar attended:
Attended two days seminar titled “Plant response to UV radiation” organized by department
of physics, faculty of science, Ostrava university, Ostrava, Czech Republic on 21.10 and
22.10.2010.
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Attended three days International conference titled “Photosynthesis in Global Perspective”
held in honor of Govindjee, organized by School of Life Sciences D.A.V.V. Indore, India on
November 27-29, 2008.
Attended a two days seminar titled “Bioinformatics research and application” jointly
organized by College Development Council and Institute of Engineering and Technology
D.A.V.V. Indore, India.
Publications:
Yadav D.K., Kruk J., Pospíšil P. (2013) Evidence on the involvement of plastosemiquinone
in superoxide anion radical production in photosystem II membranes of higher plant
(submitted manuscript).
Rastogi* A., Yadav* D.K., Szymańska R., Kruk J., Sedlářová M., Pospíšil P. (2013) Singlet
oxygen scavenging activity of tocopherol and plastochromanol in Arabidopsis thaliana:
Relevance to photooxidative stress condition (manuscript under revision).
* These authors contributed equally to this work.
Kumar P., Ranawade A.V., Yadav D.K., Kumar G.N., (2013) Potential probiotic Escherichia
coli 16 harboring the Vitreoscilla haemoglobin gene improves colonization and ameliorates
CCl4 induced oxidative stress in rats (manuscript under preparation).
Yadav D.K., Pospíšil P. (2012) Evidence on the Formation of Singlet Oxygen in the Donor
Side Photoinhibition of Photosystem II: EPR Spin-Trapping Study. PLoS ONE 7(9): e45883.
Yadav D.K., Pospíšil P. (2012) Role of chloride ion in hydroxyl radicalproduction in
photosystem II under heat stress: Electron paraagnetic resonance spin-trapping study.
Journal of Bioenergetics and Biomembranes 44, 365-372.
Yadav D.K., Kruk J., Sinha R.K., Pospíšil P. (2010) Singlet oxygen scavenging activity of
plastoquinol in photosystem II of higher plants: electron paramagnetic resonance spin-
trapping study. Biochimica et Biophysica Acta 1797, 1807-1811.
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Acknowledgements
As I write these lines, the names and face of several people comes including my friends,
colleagues and family, who have contributed intellectually and emotionally to this work, over
and above to my overall strengthening in science world.
I would like to convey my profound gratitude to Doc. RNDr. Pavel Pospíšil, Ph.D., for giving
me valuable guidance about my work and presentations, an opportunity to learn, think and
work in a healthy environment.
I am extremely grateful to Prof. Holger Dau for allowing me to pursue my three month
foreign research stay in his laboratory at Department of Physics, Freie University, Berlin
Germany and also giving generous guidance and support during research stay.
Sincere thanks to Prof. Jerzy Kruk for his collaboration and support for my work as well as
his efforts to do work in his lab related to my study.
I would like to extend my thanks to Dr. Michaela Sedlářová and Dr. Jan Hrbáč for support
with respect to confocal scanning laser microscopy and EPR spin trapping measurements
respectively.
I am grateful to faculty members Prof. RNDr. Petr Ilík, Ph.D., Prof. RNDr. Jan Nauš, CSc.,
Doc. RNDr. Dušan Lazár, Ph.D., RNDr. Martina Špundová Ph.D., RNDr. Jan
G. Švec, Ph.D., and other staff members of the Department of Biophysics for their help and
support during my research work and stay in Olomouc.
Special thanks to Dr. Ivelina Zaharieva and Dr. Petko Chernev to teach and help me with
the measurements during my research stay at Freie University Berlin, Dr. Arjun Tiwari to
discuss and answer the all queries and some stupid question about the experiments and
related theories, Dr. Anshu Rastogi and Dr. Rakesh Kumar Sinha and for his support with
experiments. Thank you all of you for the long discussions which helped me understand the
work better, all the times you’ve patiently taught me the techniques and laboratory skills
which have been truly invaluable!
It is a pleasure to thank Ankush Prasad whose encouragement, unstinted support and critical
evaluation have been a great source of inspiration for me during my Ph.D. Thanks to Pedro
and Navdip for their gentleness, correcting and pointing out my english mistakes in
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manuscripts writing. Thanks to Abhishek, Rajbardhan, Zora for endless supports and also
help me to improve self confidence. I will never forget the moments which we spent together
during my stay in Czech Republic.
It is also a pleasure to thanks my colleagues Marek, Jan, Dr. Marika, Miroslav,
Eliška, Hana, Vit, Lukáš, Pavla, Tea, Irma, Parwez, Ravi and others for their help,
encouragement and support.
My deepest gratitude to my grandparents, parents, sisters Manisha, Nisha, Sunita and whole
family member for always being their for me and providing me with their unconditional love,
support and constant encouragement. Ultimately, I bow down to God!
This work was supported by the grant no. MSM 6198959215 (Ministry of Education, Youth
and Sports of the Czech Republic), grant nos. CZ.1.07/2.3.00/20.0057 (Operational
Programme Education for competitiveness from Ministry of Education Youths and Sports,
Czech Republic), ED0007/01/01 (Centre of the Region Haná for Biotechnological and
Agricultural Research), student project PrF_2010_050 and Prf_2011_024 of the Palacky
University.
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Abbreviations
ATP adenosine triphosphate
CCD charge coupled device
Chl chlorophyll
CLSM confocal laser scanning microscopy
Cyt b559 cytochrome b559
Cyt b559 LP, IP, HP low, intermediate and high potential form of cytochrome b559
D1, D2 D1 and D2 proteins of photosystem II
DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea
DMBQ 2,3-dimethyl-6-phytyl-benzoquinol
EMPO 2-ethoxycarbonyl-2-methyl-3,4-dihydro 2H-pyrrole-1oxide
EMPO-OH hydroxyl radical adduct of EMPO
EMPO-OOH superoxide anion radical adduct of EMPO
EMPO-R carbon centered radical adduct of EMPO
EPR electron paramagnetic resonance
HGA homogentisic acid
His histidine
HL high light
H2O2 hydrogen peroxide
HO
hydroxyl radical
HPLC high pressure liquid chromatography
kDa kilo dalton
LL low light
MDA malondialdehyde
MES 2-(N-morpholino)-ethanesulfonic acid
MPBQ 2-methyl-6-phytyl-benzoquinol
MSBQ 2-methyl-6-solanesyl-benzoquinol
NADP nicotinamide adenine dinucleotide phosphate
NADPH reduced nicotinamide adenine dinucleotide phosphate
P680 chlorophyll in the reaction centre of PSII
PC plastochromanol
PC-OH hydroxy-plastochromanol
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PQH2 plastoquinol
Pheo pheophytin
POBN α-phenyl N-tert-butylnitrone
POBN-R carbon centered radical adduct of POBN
PQ plastoquinone
PQH2 plastoquinol
PSI photosystem I
PSII photosystem II
QA, QB primary and secondary quinone in PSII
QC 3rd
plastoquinone
R
carbon centered radical
ROO peroxyl radical
ROS reactive oxygen species
1O2 singlet oxygen
SAM s-adenosyl methionine
SOSG singlet oxygen sensor green
O2-
superoxide anion radical
TMPD 2,2,6,6-tetramethylpiperidone
TEMPONE 2,2,6,6-tetramethylpiperidone-1-oxyl
TyrZ redox active tyrosine-161 of the D1 protein in PSII
UV ultra-violet
vte1 tocoperol cyclase mutant
vte2 homogentisic acid phytyl transferase
vte3 methyl transferase
vte4 γ-tocopherol methyl transferase
WT wild type
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Abstract
Photosynthesis is the biological process where solar energy is converted to chemical energy
by the photosynthetic organisms (higher plants, algae and cyanobacteria). In the
photosynthetic process, water is split into molecular oxygen and protons are released into
lumen of thylakoid. The atmospheric molecular oxygen in our planet is produced by
photosynthetic water oxidation. The molecular oxygen is utilized by oxygen dependent
organism for respiration, thus serving as vital resource for oxygen dependent life on the earth.
Photosystem II is a proteins-pigments complex which is associated with various redox active
cofactors, which is embedded in the lipid bilayer of thylakoid membranes of photosynthetic
organisms. Recently, crystal structure of PSII isolated from cyanobacteria
Thermosynechococcus elongatus and Thermosynechococcus vulcanus has been reported to
comprise of 20 protein subunits, 35 chlorophylls, 12 carotenoids and 25 integral lipids per
monomer (Ferreira et al. 2004, Guskov et al. 2009, Umena et al. 2011). In photosynthesis, the
oxidative stress and related protection mechanism is one of the most intensively studied
topics (Chow and Aro 2005, Asada 2006, Vass and Aro 2007, Murata et al. 2007, Krieger-
Liszkay et al. 2008, Tyystjärvi 2008, 2013, Vass 2012, Pospíšil 2012). Understanding
oxidative stress and related protection mechanisms is very important because it could pave
the way towards development of modified plants that have stress resistance abilities, which
can therefore produce food in adverse atmospheric conditions such as high light, heat stress
etc.
In photosynthesis, it is believed that ROS play a crucial role in PSII under stressed
conditions (Aro et al. 1993, Krieger-Liszkay 2005, Pospíšil 2009, Triantaphylidès and
Havaux 2009, Vass 2012). This current work examines the molecular mechanism of ROS
production and scavenging in PSII under stress condition. Light induced formation of singlet
oxygen (1O2) in Tris-treated PSII membranes was studied by using EPR spin trapping
technique. We measured 1O2 and carbon centered radicals (R
) as monitored by TEMPONE
and POBN-R adduct EPR signal in Tris-treated PSII membranes, respectively. It is proposed
here that the 1O2 formation occurred in Tris-treated PSII membranes via the Russell
mechanism. In this mechanism the recombination of two peroxyl radicals (ROO) formed by
the interaction of R with molecular oxygen leads to
1O2 formation in the donor side
photoinhibition of PSII. On other hand, we have measured hydroxyl radical (HO) formation
in PSII membranes under heat stress as monitored EPMO-OH adduct EPR signals. The
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exogenous addition of chloride and its competitor or blocker for its binding site reduced the
EPMO-OH adduct EPR signal in PSII. It is concluded here that the chloride ion plays a very
important role to the formation of HO in PSII. Chloride ion protects the HO
formation in
PSII membranes under heat stress. We also measured superoxide anion radical (O2-
)
formation in PSII membranes under high-light stress as monitored EPMO-OOH adduct EPR
signals. The exogenous herbicide DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] and
dinoseb reduced the adduct EPR signal in presence of exogenous plastoquinone to PSII
membranes. Similarly, DCMU and dinoseb inhibited the photoreduction of cytochrome b559
in absence and presence of exogenous plastoquinone to PSII membranes. It is concluded that
under high-light stress, plastosemiquinone is involved in the formation of O2-
in PSII.
Oxidative stress is associated with the damage of the systems; therefore plants have
developed strategies to protect themselves against oxidative damage (Mittler 2002, Apel and
Hirt 2004, Foyer and Noctor 2009, Foyer and Shigeoka 2011). In response to oxidative stress,
plants produce a low molecular weight antioxidant within chloroplasts (Munné-Bosch and
Alegre 2002, Apel and Hirt 2004). We demonstrated the 1O2 scavenging activity of
plastoquinol and tocochromanol (tocopherol and plastochromanol). Singlet oxygen
scavenging activity of plastoquinol was studied in PQ-depleted PSII membranes. The
addition of exogenous plastoquinol suppressed the TEMPONE EPR signal in chemical (rose
bengal) as well as biological systems (PSII membranes). It showed direct evidence on the 1O2
scavenging activity of plastoquinol in PSII. On the other hand, to study the 1O2 scavenging
activity of tocochromanol, we have used wild type (WT) and tocopherol cyclase mutant
(vte1) lacking plastochromanol and tocopherol in Arabidopsis thaliana leaves. Our results
showed the light induced 1O2 formation in leaves and chloroplast of vte1 mutant is higher
compared to WT plants. Whereas the exposure of vte1 to high-light resulted in the
pronounced enhancement of MDA formation and ultra-weak photon emission compared to
WT Arabidopsis leaves. These observations revealed that tocopherol and plastochromanol
function as 1O2 scavengers in Arabidopsis and protect against photooxidative stress.
-----------------------------------------------
Introduction
(Overview on structure and function of photosystem II)
-----------------------------------------------
Chapter 1
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1. Photosynthesis
Photosynthesis is a biological process, which leads to the conversion of solar energy into
chemical energy. Photosynthetic organisms oxidize water into molecular oxygen and reduce
carbon dioxide (CO2) to sugar. Oxygenic photosynthesis occurs in photosystem II (PSII) of
cyanobacteria, algae and higher plants according to the following equation and figure 1.1.
6CO2 + 6H2O + light energy C6H12O6 + 6O2
Figure 1.1. Illustration of photosynthetic reaction.
(figure adapted from http://hyperphysics.phy-astr.gsu.edu/hbase/biology/psetran.html )
1.1. Photosynthetic apparatus
Photosynthesis takes place in sub-cellular organelles known as chloroplast, found in
photosynthetic organisms. Chloroplast is covered by an envelope containing outer and inner
bilayer membranes. An inter-membrane space is present between these two layers. The
thylakoid membranes are present inside the chloroplast region covered by the inner
membrane (i.e. stroma of chloroplast). The stacks of thylakoids are called grana (singular-
granum). The stroma of chloroplast consists of the enzymes to catalyze the CO2 fixation and
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other biosynthetic pathways. Thylakoid membranes serve as the place for light reaction in
photosynthesis. It contains protein pigment complexes such as photosystem I (PSI),
Photosystem (PSII), light harvesting complex II (LHCII), cytochrome b6f (cyt b6 f) etc. along
with enzymes ATP synthase. The inner space within thylakoid is called lumen.
1.2. Energy and electron transfer in photosynthesis
Thylakoid membranes consist of a large number of chlorophyll and accessory light absorbing
pigments. The light reaction is the part of photosynthesis which absorbs and converts the
light energy into chemical energy by utilizing the activity of protein pigment complexes (PSI,
PSII, LHCII and cyt b6 f) (Figure 1.2.). The absorption and transfer of light energy from the
pigments of antenna complex to the reaction center facilitates electron transfer to electron
acceptor cofactors in the electron transport chain. Finally, electron transfer through the
electron transport chain reduces NADP to produce NADPH (Figure 1.2.).
Figure 1.2. Schematic view shows light reaction pathway. The protein complexes such as
photosystem I (PSI), photosystem II (PSII), light harvesting complex II (LHCII) and cytochrome b6f
(cyt b6 f) are embedded in the thylakoid membrane (figure adapted from Kern and Guskov 2011).
1.3. Photosystem II
Photosystem II is a pigment-protein complex present in the thylakoid membranes of
photosynthetic organisms. Photosystem II is a homodimeric multisubunit complex with a
molecular weight of 350 kDa per monomer (Ferreira et al. 2004, Loll et al. 2005, Guskov et
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al. 2009, Umena et al. 2011). Photosystem II monomer consists of about 100 cofactors and 20
protein subunits (Guskov et al. 2009, 2010, Gabdulkhakov et al. 2009). In addition to this,
structure of PSII at 1.9 Å showed more than 1300 water molecules per monomer (Umena et
al. 2011). It is involved in electron transfer from water to plastoquinone known as water-
plastoquinone oxido-reductase activity of PSII (Diner and Rappaport 2002, Renger and
Holzwarth 2005, Kern and Renger 2007). Light-driven water oxidation proceeds by water-
splitting manganese complex via abstraction of electrons and release of molecular oxygen
and protons (Dau and Haumann 2008, Brudvig 2008, Cady et al. 2008). The structure of PSII
dimer from cytoplasmic side shows in figure 1.3.
Figure 1.3. View of structure of PSII dimer from the cytoplasmic side (figure adapted from Kern and
Guskov 2011).
1.3.1. Structure of PSII
The structure of PSII has been given by different research groups at different resolutions.
Photosystem II is composed of abundant organic component with various cofactors.
Photosystem II is situated across the lipid membranes from lumen to stroma. The lumenal
and stromal sides of the PSII are called donor and acceptor sides, respectively. The reaction
center of PSII consists of two homologous D1 (encoded by psbA gene) and D2 (encoded by
5
psbD gene) proteins (Karabin et al. 1984, Hollingsworth et al. 1984, Christopher et al. 1999,
Thum et al. 2001, Rutherford and Foller 2003). These proteins are attached to four
chlorophylls a (PD1, PD2, ChlD1, ChlD2), two pheophytins (PheoD1, PheoD2), two
plastoquinones (QA and QB) and a non-heme iron. Furthermore, it is also attached to two
peripheral chlorophylls a (ChlZD1, ChlZD2) and two carotenes (CarD1 and CarD2) (Guskov et al.
2009, Kern and Guskov 2011). The arrangement of cofactors in PSII is shown in figure 1.4.
Figure 1.4. Schematic view shows the arrangement of PSII in higher plants and green algae. Figure
shows only core proteins, acceptor (stromal site) and donor (lumenal site) site of PSII (figure adapted
from Shevela et al. 2012).
In addition to D1/D2 proteins, PSII is composed of many other protein subunits
responsible for the proper organization and function of PSII. The two large protein subunits
CP43 and CP47 (encoded by genes psbC and psbB) are located on the two sides of the D1/D2
with molecular weights of 43 and 47 kDa, respectively (Vermaas et al. 1987, Chisholm and
Williams 1988, Rochaix et al. 1989). Additionally, 11 small protein subunits (encoded by
psbH to psbM, ycf12, psbT and psbX to psbZ gene) are the components of PS II (Guskov et
al. 2009, Kern et al. 2009, Umena et al. 2011). The lumenal region of PS II consists of at least
three membrane-extrinsic protein subunits. These protein subunits are 33 kDa manganese
stabilizing protein (encoded by psbO gene), 12 kDa protein (encoded by psbU gene) and 15
6
kDa, cyt c550 (encoded psbV gene) (Guskov et al. 2009, Kern et al. 2009). For general
information about proteins subunits a complete list is given below in table 1.1. Figure 1.5
shows the pathway of electron flow in PSII along with time scale and distances between the
cofactors in Å.
---------------------------------------------------------------------------------
Subunit name Size (amino acids)
---------------------------------------------------------------------------------
D1, RC subunit 344
CP47, antenna subunit 510
CP43, antenna subunit 461
D2, reaction center subunit 352
Cytochrome b559 α-subunit 84
Cytochrome b559 β-subunit 45
PsbH 66
PsbI 38
PsbJ 40
PsbK 37
PsbL 37
PsbM 36
PsbO 246
PsbT 32
12 kDa extrinsic protein 46
ycf12 46
PsbX 41
PsbY 41
PsbZ 62
---------------------------------------------------------------------------------
Table 1.1. Protein subunit of PSII (Guskov et al. 2009).
7
Figure 1.5. (A) Localization of various cofactors in PSII. This figure arrangement is based on PSII
structure on 2.9 Å resolution (Guskov et al. 2009). (B) Schematic representation of the cofactor
arrangement in PSII (edge-to-edge distances are given in Å) (figure adapted from Müh et al. 2012).
1.3.2. Reaction center chlorophyll and pheophytin
Chlorophyll is a pigment present in chloroplasts of photosynthetic organisms. Structurally, it
is composed of a chlorin ring with magnesium central metal and attached with the long
phytol chain. Photosynthetic organisms possess different types of chlorophyll such as Chl a,
Chl b, Chl d etc. The primary photochemical and subsequent electron transfer reaction occurs
in PSII (Amesz and Gorkom 1978, Nanba and Satoh 1987, Hillier and Babcock 2001,
8
Rappaport and Diner 2008). The charge separation process leads to the formation of primary
radical pair in PSII during electron transport chain of light reaction (Dekker and van
Grondelle 2000, Diner et al. 2001, Diner and Rappaport 2002, Holzwarth et al. 2006, Saito et
al. 2011, Cardona et al. 2012).
Pheophytin is a chlorophyll molecule without central magnesium metal and the
shortest distance (edge to edge distance) between PheoD1 and ChlD1 is 5 Å (Guskov 2009,
Müh and Zouni 2011). The photoreduction of pheophytin has been reported in the past by the
Klimov and workers (Klimov et al. 1977). Historically, the discovery and function of
pheophytin is reviewed by Klimov (2003) in detail. It is concluded that pheophytin works as
an electron acceptor during light induced charge separation in PSII (Klimov and Krasnovsky
1981, Rappaport et al. 2005, Holzwarth et al. 2006, Kato et al. 2009, Palencar et al. 2009,
Allakhverdiev et al. 2010). Reduction of pheophytin forms radical pair at acceptor site of
PSII and transfer the electron to quinone molecules (QA) to further progress the
photochemical process. Ultimately, light-induced charge separation results in the oxidation of
oxidation and the reduction of plastoquinone which consequently leads to the production of
sugars by the Calvin cycle (Kern and Renger 2007, Renger and Renger 2008).
1.3.3. Plastoquinones and non-heme iron
Photosystem II contains two plastoquinone binding sites, designated as primary
plastoquinone binding site QA and secondary plastoquinone binding site QB. Recent crystal
structure of cyanobacteria Thermosynechococcus elongatus reported the presence of a 3rd
plastoquinone binding site (QC) (Guskov et al. 2009), whereas later structure from
Thermosynechococcus vulcanus did not observe the Qc site (Umena et al. 2011). It is
suggested that this could be due to the difference in preparation or crystallization condition of
PSII in both the studies. The two electron reduction of plastoquinone and subsequent
protonation at QB site leads to the formation of plastoquinol during electron transport in PSII.
Plastoquinol is a mobile molecule known to transfer the electron to cyt b6f complex and
release the proton in the lumen of thylakoid (Allen 2003, Barr and Crane 2005, Kern and
Renger 2007, Loll et al. 2007, Müh and Zouni 2011, Müh et al. 2012). The release of protons
in the lumen creates a proton gradient across the thylakoid membranes and ultimately drives
the adenosine triphosphate (ATP) synthesis by enzyme ATP synthase (Allen 2003, Baker et
al. 2007, Joliot and Johnson 2011). To sustain the light-induced electron transfer in PSII, the
9
numbers of plastoquinone molecules required to maintain the fast exchange between PQ
binding site and PQ pool (Guskov et al. 2009, Gabdulkhakov et al. 2009).
In addition to this, the non-heme iron is situated in close proximity of plastoquinone
binding sites QA and QB and ligated by the histidine residue in PSII (Guskov et al. 2009,
Umena et al. 2011, Müh et al. 2012). In general, the exact role of non-heme iron is still
unclear, although the literatures suggest that the non-heme iron is involved in regulation of
quinone reduction in PSII in association with bicarbonate (Ishikita and Knapp 2005, Müh and
Zouni 2011, Müh et al. 2012, Shevela et al. 2012). Recent crystal structures show the
presence of bicarbonate bound to the non-heme iron at the acceptor side of PSII (Umena et al.
2011). In the past, the role of bicarbonate on both acceptor and donor sides of PSII have been
discussed (Govindjee et al. 1992, Xiong et al. 1996, Allakhverdiev et al. 1997, van Rensen et
al. 1999, Klimov and Baranov 2001, van Rensen 2002, Komenda et al. 2002, Baranov et al.
2004, Dasgupta et al. 2008, Müh et al. 2012, Shevela et al. 2012).
1.3.4. Water-splitting manganese complex
Crystal structure of PSII from Thermosynechococcus vulcanus at a resolution of 1.9 Å
showed that the water-splitting manganese complex is composed of 4 Mn, 1 Ca, and 5
oxygen atoms (Mn4CaO5 cluster). Water-splitting manganese complex is attached to the
lumenal side of the PSII. Structures of PSII showed that water-splitting manganese complex
is directly associated with the amino acid residues of D1 and CP43 subunits of PSII and
indirectly with other polypeptides (Ferreira et al. 2004, Guskov et al. 2009, Umena et al.
2011, Kawakami et al. 2011). The process of water oxidation is energetically driven by the
light-induced formation of chlorophyll cation radical P680+
which is formed by the charge
separation in PSII. Subsequently P680+
oxidizes a redox-active tyrosine which leads to
formation of TyrZ at the PSII donor side (Renger and Holzwarth 2005, Rappaport and Diner
2008, Brudvig 2008, Cardona et al. 2012, Grundmeier and Dau 2012). The highly oxidizing
redox potential of TyrZ• (TyrZ
•/TyrZ = 1.2 to 1.4 V) facilitates the stepwise electron transfer
from water-splitting manganese complex to P680+
(Rappaport et al. 2002, Rutherford and
Boussac 2004, McEvoy and Brudvig 2006, Dau and Haumann 2008). Electron transfer is
coupled with the abstraction and release of protons i.e. so-called proton coupled electron
transfer (PCET) in PSII (Jenson and Barry 2009, Barry 2011, Gagliardi et al. 2012).
10
Figure 1.6. Basic reaction cycle of water oxidation by photosystem II (figure adapted from Zaharieva
et al. 2011).
Light-induced sequential abstraction of electrons by P680+
accumulates oxidizing
equivalents at the water-splitting manganese complex. This reaction is shown in figure 1.6 as
the modified Kok cycle involving four S-state transitions of the manganese-calcium complex
(S1, S2, S3 and S4) (Kok et al. 1970, Zaharieva et al. 2011). By the absorption of 4 photons, the
water-splitting manganese complex advances from S0 to S4 states and water oxidizes into
molecular oxygen in the transition between S4 to S0 states (Figure 1.6.) (Haumann et al. 2005,
Dau and Haumann 2007, Barber 2008, Brudvig 2008, Dau et al. 2012, Bondar and Dau
2012). However, the exact molecular mechanism of water oxidation and O=O bond formation
in PSII is unclear and remains to be elucidated.
1.3.5. Inorganic cofactors: calcium and chloride
Calcium and chloride are the essential inorganic cofactors associated with the water-splitting
manganese complex (Homann 2002, Popelková and Yocum 2007, Miqyass et al. 2008,
Yocum 2008). Both the PSII structures showed that one calcium atom is present in the core
of water-splitting manganese complex. The calcium atom of the water-splitting manganese
complex is associated with two water molecules (W3 and W4) (Umena et al. 2011,
Kawakami et al. 2011). It regulates the redox chemistry and provides structural integrity to
water-splitting manganese complex (Gorkom and Yocum 2005, Yocum 2008). Calcium is
11
also important for the oxygen evolution activity of PSII (Rutherford 1989, Ono et al. 2001,
Vrettos et al. 2001, Yocum 2008, Yachandra and Yano 2011).
On the other hand, the structure from the Thermosynechococcus elongatus and
Thermosynechococcus vulcanus showed one and two chloride binding sites in PSII,
respectively (Guskov et al. 2009, Umena et al. 2011). The PSII crystal structures showed the
presence of water molecules situated between chloride ion and water-splitting manganese
complex. The position of chloride is stabilized by its interaction with the amino acid residues
of D1 and D2 proteins (D1-Glu333 and D2-Lys317). Chloride ion is associated with the
amino group of D2-Lys317, backbone nitrogen of D1-Glu333 and provides structural
stability to water-splitting manganese complex of PSII. In literatures, it has also been
reported that chloride provides protection against heat inactivation of PSII activity and loss of
polypeptides (Nash et al. 1985, Thompson et al. 1989). It protects PSII by preventing the
formation of HO under heat stress condition (Paper II). Chloride is also required for the
water oxidation (required for S2→S3 and S3→S0 transitions, but not for S0→S1 and S1→S2
transitions) and proton transfer pathway (Ono et al. 1986, Rutherford 1989, van Vliet and
Rutherford 1996, Wincencjusz et al. 1997, Ishikita et al. 2006, Yocum 2008, Gabdulkhakov
et al. 2009, Kawakami et al. 2009, Rivalta et al. 2011, Pokhrel et al. 2011).
1.3.6. Extrinsic proteins
Photosystem II of higher plants and eukaryotic algae consists of extrinsic proteins (PsbO,
PsbP and PsbQ) and a possibly suggested fourth extrinsic protein PsbR. Extrinsic proteins
PsbO, CyanoP and CyanoQ along with PsbU and PsbV are present in cyanobacteria (Roose
et al. 2007, Enami et al. 2008, Bricker et al. 2012). The PsbO protein (molecular weight 33
kDa or manganese stabilizing protein) is present in all the oxygenic photosynthetic
organisms. The presence of other extrinsic proteins, PsbP (23 or 24 kDa protein), PsbQ (16–
18 kDa protein in plants, 20 kDa protein in some algae), PsbR (10 kDa protein), PsbU (12
kDa protein), PsbV (cytochrome c550) varied in photosynthetic organisms (Ljungberg et al.
1986, Webber et al. 1989, Shen et al. 1992, Kashino et al. 2002, De Las Rivas and Barber
2004, De Las Rivas et al. 2004, Ifuku et al. 2004, Balsera et al. 2005, Roose et al. 2007,
Enami et al. 2008, Guskov et al. 2009, Michoux et al. 2010, Jackson et al. 2010, Umena et al.
2011, Bricker et al. 2012). It is believed that these extrinsic proteins are required to stabilize
the water-splitting manganese complex either directly or indirectly. These proteins provide
the optimal condition for maximal oxygen evolution activity of PSII under physiological
12
conditions (De Las Rivas et al. 2007, Nagao et al. 2010, Ifuku et al. 2011, Bricker and
Frankel 2011, Roncel et al. 2012, Bricker et al. 2013). In addition to that, these proteins can
act as protectors of the water-splitting manganese complex against oxidative damage or
reductants (Enami et al. 2008, Popelková et al. 2011).
1.3.7. Cytochrome b559
Cytochrome b559 (cyt b559) is an intrinsic component of PSII in cyanobacteria, algae and
higher plants. It is tightly associated with the D1 and D2 proteins. Cytochrome b559 is a
heterodimeric, heme-bridged protein consisting of two subunits (α and β) encoded by psbE
and psbF genes, respectively (Herrmann et al. 1984, Pakrasi et al. 1988, Alizadeh et al. 1994,
Mor et al. 1995, Stewart and Brudvig 1998, Morais et al. 1998). Recent crystal structure of
PSII from different thermophilic cyanobacteria Thermosynechococcus elongatus and
Theromosynechococcus vulcanus showed that cyt b559 is located in close proximity to the D2
protein of PSII (Guskov et al. 2009, Umena et al. 2011). The histidine residues (His23 and
His24) of α- and β-subunits of cyt b559 are coordinated to the heme iron, forming a cross
linked structure (Figure 1.7.) (Shinopoulos and Brudvig 2012).
Figure 1.7. Schematic view cyt b559 in PSII (figure adapted from Shinopoulos and Brudvig 2012).
Cytochrome b559 is involved in secondary electron transfer pathway to protect PSII
against photodamage under conditions when the primary electron transfer pathway is
inhibited (Tracewell and Brudvig 2008a, 2008b, Shinopoulos and Brudvig 2012).
Cytochrome b559 is oxidized by P680+
via electron abstraction from β-carotene (Car) and
chlorophyll (Chl) cofactors at the donor side of PSII and it may also be reduced by gain of
electrons from the acceptor site of PSII. Therefore, it forms a cyclic electron transfer pathway
that connects the donor and acceptor site of PSII to eliminate the highly harmful oxidizing
13
equivalents (Thompson and Brudvig 1988, Shuvalov 1994, Poulson et al. 1995, Pospíšil
2011). On the other hand, several experimental evidences have been provided on the
enzymatic function of cyt b559 in PSII. It is suggested that cyt b559 may function as
plastoquinol oxidase, superoxide reductase, superoxide oxidase and oxygen reductase (Buser
et al. 1992, Barber and De Las Rivas 1993, Ananyev et al. 1994, Kruk and Strzałka 1999,
2001, Pospíšil et al. 2006, Tiwari and Pospíšil 2009, Bondarava et al. 2010, Sinha et al. 2010,
Pospíšil 2011).
14
-----------------------------------------------------
Reactive oxygen species
(Production and scavenging of reactive oxygen species in PSII)
-----------------------------------------------------
Chapter 2
15
2. Reactive oxygen species (ROS)
Reactive oxygen species (oxygen containing reactive molecules) are known to damage the
organic components of the cell due to a high oxidizing capability. Reactive oxygen species
posses either paired or unpaired numbers of electrons in the molecular orbital of the
molecules. The ROS with unpaired number of electron are known as radical ROS, whereas
the paired ones are known as non-radical ROS. Half-life times of ROS are very small. In this
chapter, production and scavenging of ROS in PSII are discussed.
2.1. Types of ROS
On the basis of the mechanism of formation of ROS, it can be divided into two groups; 1)
ROS formed by the energy transfer pathway such as singlet oxygen (1O2) (Type II
mechanism). 2) Reactive oxygen species formed by the electron transfer pathway such as
superoxide anion radical (O2-
), hydrogen peroxide (H2O2), hydroxyl radical (HO) etc. (Type
I mechanism). The positioning of electron in molecular orbital for different ROS is shown in
figure 2.1.
Figure 2.1. Molecular orbital diagram of molecular oxygen and ROS.
2.1.1. Singlet oxygen
Singlet oxygen is the highly energized state of molecular oxygen. It is produced by the
excited molecules possessing higher energy than the triplet energy level of molecular oxygen
16
(Halliwell and Gutteridge 2007). When the photosensitizer molecules absorb the radiation,
molecules excite from their ground state (lower energy level) to excited state (high energy
level). The singlet excited state molecules convert into triplet excited state via intersystem
crossing. Triplet excited state molecule transfers the excited energy to molecular oxygen to
form 1O2. Two types of
1O2 are known;
1O2 which possess low energy (
1∆g) of 95 KJ mol
-1
(22.5 kcal mol-1
) or high energy (1∑g
+) of 158 KJ mol
-1 (31.5 kcal mol
-1) (DeRosa and
Crutchley 2002, Schweitzer and Schmidt 2003). Both types of 1O2 differ with molecular
oxygen in only electronic arrangement in π–antibonding orbital. Electronic arrangement of
1∆g
O2 shows the vacant π–antibonding orbital, whereas electronic arrangement of
1∑g
+ O2 is
similar to molecular oxygen, except the last two electrons with antiparallel spin (Figure 2.1.).
2.1.2. Superoxide anion radical
Superoxide anion radical is formed by the one electron reduction of molecular oxygen in a
biological system (Halliwell and Gutteridge 2007). For the formation of O2-
, a highly
reducing compound is required to reduce the molecular oxygen. Superoxide anion radical is
radical ROS because it contains an unpaired number of electron in molecular orbital (Figure
2.1). The average half life time of O2-
is microsecond (µs) in biological system (Møller et al.
2007) with negative standard redox potential of O2/ O2-
redox couple (E0´ = - 160 mV, pH 7)
(Wood 1987).
2.1.3. Hydrogen peroxide
Hydrogen peroxide is formed by the dismutation of O2-
, which is catalyzed by either non-
enzymatic (spontaneous dismutation reaction) or by enzymatic reactions {such as in the
presence of superoxide dismutase (SOD) enzyme} (Halliwell and Gutteridge 2007). It is non-
radical ROS because it contains a paired number of electrons in molecular orbital (figure 2.1).
It is one of the more stable and least reactive ROS. The average half-life time of H2O2 is
several milliseconds (ms) in a biological system (Møller et al. 2007) with positive standard
redox potential of O2-
/H2O2 redox couple (E0´ = + 890 mV, pH 7) (Wood 1987). It can be
also formed directly by a two electron reduction of molecular oxygen. This reaction happens
in the presence of enzymes such as urate oxidase, oxalate oxidase, monoamine oxidase etc. In
some cases, H2O2 is formed by the two electron oxidation of water in presence of enzyme
such as water-oxidase.
17
2.1.4. Hydroxyl radical
Hydroxyl radical is a well known ROS which is produced by the one electron reduction of
H2O2 in the presence of transition metals which is known as the Fenton reaction or homolytic
fission of the O-O bond of H2O2 (Halliwell and Gutteridge 2007). It is a radical ROS because
it contains a unpaired number of electron in molecular orbital. It is the most reactive among
the other ROS and average half life time of HO is nanoseconds (ns) in a biological system
(Møller et al. 2007) with positive standard redox potential of H2O2/HO redox couple (E0´ =
+ 460 mV, pH 7) (Pierre and Fontecave 1999).
2.2. Reactive oxygen species production in PSII
In photosynthesis, ROS is formed when the absorption of solar energy exceeds its utilization
during photochemical processes. In particular, PSII leads the formation of ROS, when the
transfer of energy from antenna complex to reaction center is inhibited. Furtheremore, ROS
are also formed by the leakage of electron to molecular oxygen during electron transport
processes in PSII. Since, PSII is composed of pigments (suitable for the photosensitization
process) and cofactors (posses a broad range of redox potential can reduce the molecular
oxygen), it could be a suitable complex for the formation of ROS under oxidative stress
condition.
2.2.1. Singlet oxygen production in PSII
2.2.1.1. Singlet oxygen production in antenna complex
The major and minor chlorophyll binding proteins and core antenna complex are involved in
absorption and transfer of the excited energy to PSII reaction center. Singlet oxygen
formation occurs in the PSII antenna complex by energy transfer from triplet chlorophyll to
molecular oxygen (Figure 2.2.). Under the environmental stress conditions, such a highly
organized energy transfer from one chlorophyll to other chlorophyll is disturbed, the life-time
of singlet excited chlorophyll molecules is enhanced and converted into triplet chlorophyll
via intersystem crossing. Intersystem crossing is involved in the spin conversion, which leads
to the conversion of singlet chlorophyll to triplet chlorophyll. Singlet oxygen was measured
in the isolated light harvesting complex II (LHCII) by using EPR spin-trapping technique
(Zolla and Rinalducci 2002, Rinalducci et al. 2004). The formation of triplet chlorophyll in
PSII antenna complex has been demonstrated by means of fluorescence and action
spectroscopy (Santabarbara et al. 2001, 2002). It has been proposed that 1O2 can be generated
18
from either weakly coupled or energetically uncoupled triplet chlorophylls in PSII
(Santabarbara et al. 2001, 2002). On other hand, several studies reported that proper assembly
and repair of protein subunits of PSII is important for photosynthetic organisms (Melis 1999,
Nixon et al. 2010, Komenda et al. 2007, 2008, 2012). Due to the improper assembly and
repair of damage proteins subunits in PSII, some chlorophyll temporarily remains unbound.
Under such conditions, the probability of the formation of triplet chlorophyll is increased by
unbound chlorophyll. The authors concluded that 1O2 production in antenna complex occurs
via Type II photosensitization reaction.
2.2.1.2. Singlet oxygen production in acceptor side photoinhibition of PSII
When the photosynthetic organisms are exposed to high-light stress, inactivation of PSII
activity occurs by the process known as photoinhibition i.e. acceptor or donor side
photoinhibition of PSII (Eckert et al. 1991, Prášil et al. 1992, Aro et al. 1993, Adir et al.
2003). The light-induced charge separation and subsequent charge stabilization process leads
to the formation of radical pair [P680+
QA-
] during the electron transport process in PSII.
Under environmental conditions such as high light stress, overreduction of PSII acceptor side
causes the formation of singlet radical pair 1[P680
+Pheo
-] due to back electron transfer from
QA-
to Pheo-
. The singlet radical pair 1[P680
+Pheo
-] undergoes the two way; either
recombines to form P680 or alters into the triplet chlorophyll by charge recombination
pathway (Aro et al. 1993, Rappaport et al. 2002, Krieger-Liszkay et al. 2008, Pospíšil 2012,
Vass 2012). Ultimately, 1O2 is produced by the energy transfer from triplet chlorophyll
(formed by charge recombination pathway) to molecular oxygen (Figure 2.2.) in acceptor
side photoinhibition of PSII (Krieger-Liszkay 2005, Pospíšil 2009, Vass and Cser 2009, Vass
2011). Singlet oxygen were detected in vitro by using different techniques such as chemical
trapping (Telfer et al. 1994), luminescence at 1270 nm (Macpherson et al.1993) and EPR
spin-trapping in thylakoids and PSII membranes (Hideg et al. 1994, Fischer et al. 2007, Paper
I, Paper III). In addition to that, 1O2 also was measured in vivo by using fluorescent
1O2
sensor (SOSG, DanPy etc.) in plants leaves and cyanobacteria (Kálai et al. 1998, Hideg et al.
1998, Flors et al. 2006, Driever et al. 2009, Fischer et al. 2010, Dall’Osto et al. 2010,
Alboresi et al. 2011, Sinha et al. 2012).
19
Figure 2.2. Singlet oxygen generation in PSII (figure adapted from Pospíšil 2012).
2.2.1.3. Singlet oxygen production in donor side photoinhibition of PSII
In the donor side photoinhibition of PSII, the formation of highly long-lived oxidizing
molecules P680+
/TyrZ causes oxidation of organic molecules such as proteins and lipids. It
has been reported that light-induced oxidation of organic molecules forms carbon-centered
radical (R) in PSII (Hideg et al. 1994, Krieger et al. 1998). Recent studies showed that
photoconsumption of molecular oxygen in PSII membranes deprived water-splitting
manganese complex was increased in comparison to PSII membranes. The increase of
photoconsumption of molecular oxygen occurs due to the formation of R, which interacts
with molecular oxygen and forms peroxyl radicals (ROO) (Khorobrykh et al. 2002, Ivanov
and Khorobrykh 2003, Yanykin et al. 2010, Khorobrykh et al. 2011). On other hand, several
studies have been reported the formation 1O2 in chemical system via the Russell mechanism
(Russell 1957, Howard and Ingold 1968). This mechanism explains that the formation of
ROO leading to
1O2 production via decomposition of linear tetraoxide intermediate
(ROOOOR) (Russell 1957, Howard and Ingold 1968, Dean et al. 1997, Miyamoto et al. 2003,
2007, Sun et al. 2007). Similarly, we proposed that the formation of 1O2 occurs via the
Russell mechanism in donor side photoinhibition of PSII (Paper III).
20
2.2.2. Superoxide anion radical production in PSII
Superoxide anion radical is formed by the one electron reduction of molecular oxygen.
Several studies have reported that the formation of O2-
in PSII occurs due to the leakage of
electron to molecular oxygen during electron transport chain in PSII. Superoxide anion
radical formation could be mediated by the pheophytin (Ananyev et al. 1994, Pospíšil et al.
2004, Arató et al. 2004), quinones (QA and QB) (Cleland and Grace 1999, Zhang et al. 2003),
plastoquinone (PQ) in PQ pool (Mubarakshina et al. 2006, Mubarakshina and Ivanov 2010)
and cyt b559 (Pospíšil et al. 2006). The mechanism for the formation of O2-
in PSII is shown
in schematic representation (Figure 2.3). Negative midpoint redox potential of pheophytin
{Em (Pheo/Pheo-
) = - 610 mV, pH 7} (Klimov et al. 1979), quinones {Em (QA/QA-
) = - 80
mV, (QB/QB-
) = - 40 mV, pH 7} and LP form of cyt b559 {Em (Fe3+
/Fe2+
) = - 40 to + 80 mV,
pH 7} (Hauska et al. 1983, Krieger et al. 1995, Pospíšil 2012) are the favourable cofactor for
the formation of O2-
in PSII. In addition to that, the free plastosemiquinone (formed by
interaction of plastoquinol to plastoquinone) also posses a low midpoint redox potential {Em
(PQ/PQ-
) = -170 mV, pH 7} (Hauska et al. 1983) and could be an efficient precursor for the
transfer of electron to molecular oxygen. Similarly, our study showed the involvement of
plastosemiquinone in the formation of O2-
in PSII membranes (Paper V).
Figure 2.3. Hydrogen peroxide, hydroxyl radical and superoxide anion radical production in PSII
(figure adapted from Pospíšil 2012).
21
2.2.3. Hydrogen peroxide production in PSII
Several lines of study have reported the formation of H2O2 in PSII. Hydrogen peroxide is
produced by different reaction pathways such as non-enzymatic or enzymatic reaction. In the
spontaneous dismutation pathway, two molecules of O2-
interact to form H2O2 in PSII
(Klimov et al. 1993, Pospíšil et al. 2004). Since huge number of O2-
is formed by leakage of
electron to molecular oxygen via electron transport reaction in PSII, spontaneous dismutation
of O2-
could be a favourable way to form H2O2 in PSII (Klimov et al. 1993). In addition to
that, the interaction of O2-
with the non heme iron leads the formation of bound peroxide in
acceptor side of PSII (Petrouleas and Diner 1987, Pospíšil et al. 2004). The one electron
reduction of O2-
by plastoquinol is also known to form H2O2 (Kruk et al. 2003). It is
suggested that the plastoquinol reduced O2-
to H2O2 in PQ-pool (Mubarakshina et al. 2006,
Mubarakshina and Ivanov 2010). Recently, it has been suggested that cyt b559 also reduced
O2-
to H2O2 by superoxide reductase activity. Reduction of O2-
to H2O2 is mediated by the
HP form of cyt b559, by converting itself into the intermediate-potential (IP) form of cyt b559
(Tiwari and Pospíšil 2009).
Apart from the above mechanism, it has been proposed that the formation of H2O2
occurs due to the incomplete water oxidation by water-splitting manganese complex in PSII.
According to the literature, H2O2 can be formed either by S2 to S0 state transition (Fine and
Frasch 1992, Taoka et al. 1993) or S1 to S-1 state transition (Thompson et al. 1989) of the Kok
cycle of water-splitting manganese complex. It has been proposed that the heat-induced
release of extrinsic proteins and improper water accessibility to the water-splitting manganese
complex forms H2O2 (Thompson et al. 1989, Wydrzynski et al. 1996). In agreement with this,
we have shown the formation of H2O2 in PSII under heat stress (Paper II) and suggested that
the controlled water environment around water-splitting manganese complex is required for
the proper water oxidation.
2.2.4. Hydroxyl radical production in PSII
Hydroxyl radical is formed by the one electron reduction of free or bound H2O2 (Branchaud
1999, Liochev 1999). The reduction of free H2O2 forming HO together with hydroxyl ion
catalyzed by metal ions via the Fenton reaction. Similarly, metal-catalyzed reduction of
bound peroxide into HO has been reported on acceptor side of PSII (Pospíšil et al. 2004).
Recently, it has been suggested that formation of HO is linked to heat-induced disturbance of
the structural organization of water-splitting manganese complex on the electron donor side
22
of PSII (Pospíšil et al. 2007, Yamashita et al. 2008). In paper II, we proposed that the
chloride ion is required to avoid the formation of HO and maintain the binding of proteins to
water-splitting manganese complex for proper water oxidation. Heat stress destruction of
PSII results in HO formation by Fenton reaction due to improper water oxidation.
2.3. Scavenging of ROS
For the survival of organisms, the formation and removal of ROS should be balanced. Plants
have developed many types of protective mechanism against the oxidative stress condition.
The activation of low molecular weight antioxidant synthesis pathway is among one of them.
The antioxidant such as tocopherols, plastoquinol and plastochromanol are lipid-soluble
molecules constituting of head with side chain. As similar with lipid molecules, the head of
the antioxidants is lipophilic and side chain is hydrophilic in nature. The length of side chain
is variable from one molecule to others. These molecules serve different biological roles in
the cell. Predominantly, these compounds work as antioxidants and protect the cell against
oxidative damage.
2.3.1. Synthesis of tocopherol, plastoquinol and plastochromanol
Tocopherol, plastochromanol and plastoquinol are the lipid soluble essential molecules
synthesized by plants, cyanobacteria and algae for protection against oxidative damage
(Munné-Bosch and Alegre 2002, Sattler et al. 2003, Kruk et al. 2005, Dörmann 2007).
Synthesis of tocopherols occurs in chloroplasts by utilizing homogentisate and phytyl
diphosphate. In the first reaction step of tocopherol synthesis pathway, 2-methyl-6-phytyl-
benzoquinol (MPBQ) is formed by the catalysis of homogentisate and phytyl diphosphate in
the presence of homogentisate phytyltransferase enzyme (vte2) (Soll et al. 1985, Collakova
and DellaPenna 2001, Zbierzak et al. 2010). In the next step, conversion of MPBQ occurs
into 2,3-dimethyl-5-phytyl-1,4-benzoquinol (DMBQ) due to methylation of MPBQ by the
enzymatic activity of vte3 (Cheng et al. 2003). The reaction product DMBQ leads to the
formation of γ-tocopherol in the presence of tocopherol cyclase (vte1) enzyme (Porfirova et
al. 2002, Sattler et al. 2003, 2004, Motohashi et al. 2003, Cheng et al. 2003, van Eenennaam
et al. 2003). At the final step of the synthetic pathway, methylation of γ-tocopherol results in
the formation of α-tocopherol by the activity of methyl transferase enzyme (vte4) by
transferring the methyl group from S-adenosyl methionine (SAM) to γ-tocopherol (Shintani
et al. 1998, Grusak and DellaPenna 1999, DellaPenna 2005, Dörmann 2007).
23
Synthesis of plastoquinol and plastochromanol also occurs in chloroplasts by utilizing
homogentisate and solanesyl-diphosphate (Zbierzak et al. 2010, Mène-Saffrané et al. 2010,
Piller et al. 2012). In the first reaction step of synthetic pathway, 2-methyl-6-solanesyl-
benzoquinol (MSBQ) is formed by the catalysis of homogentisate and solanesyl-diphosphate
in the presence of homogentisate solanesyltransferase (pds2) enzyme (Sadre et al. 2006,
Venkatesh et al. 2006). In further reaction steps, conversion of MSBQ occurs into
plastoquinol (PQH2-9) due to methylation of MSBQ by the enzymatic activity of vte3, a
common methyltransferase enzyme involved in both tocopherol and plastoquinol synthesis
pathways (Motohashi et al. 2003, Cheng et al. 2003, van Eenennaam et al. 2003). The
reaction product, plastoquinol is converted into plastochromanol (PC) in the presence of
tocopherol cyclase (vte1); a common enzyme involved in both tocopherol and
plastochromanol synthesis pathways (Kumar et al. 2005, Raclaru et al. 2006). The schematic
reaction of biosynthetic pathway of tocopherols, plastoquinol and plastochromanol is shown
in figure 2.4.
Figure 2.4. Biosynthetic pathway of tocopherols, plastoquinol and plastochromanol (figure adapted
from Szymańska and Kruk 2010a).
24
2.3.2. Singlet oxygen scavenging by tocopherol
The structure of tocopherol consists of a chromanol ring and a short side chain shown in
figure 2.5. Tocopherol is a lipid soluble antioxidant (commonly known as vitamin E) that
protects animal and plant cells from oxidative damage (DellaPenna and Mène-Saffrané
2011). Tocopherols are synthesized by the photosynthetic organism as described in section
2.3.1. At the physiological condition, tocopherol is predominately present in seeds and
chloroplast of plants. Furthermore, recent studies also support the presence of tocopherols in
plastoglobuli (Austin et al. 2006, Ytterberg et al. 2006, Bréhélin et al. 2007, Piller et al. 2011,
2012).
Figure 2.5. Chemical structure of tocopherol (figure adapted from Gruszka et al. 2008).
Singlet oxygen is one of the most harmful ROS which is known to damage proteins
and lipids in PSII either directly or in an indirect way. Several studies show that the damage
of PSII occurs due to direct degradation of D1 and D2 proteins by the action of 1O2 (Aro et al.
1993, Yamamoto 2001, Lupínková and Komenda 2004, Yamamoto et al. 2008, Vass 2012).
On the other hand, it is also reported that 1O2 affects only the repair cycle of PSII under high-
light stress (Nishiyama et al. 2001, 2006, 2011, Murata et al. 2007, Tyystjärvi 2008, 2013).
Thus, the scavenging of 1O2 is a much needed process to protect the PSII against
photooxidative stress in photosynthetic organisms. Low molecular weight antioxidant
scavenges 1O2 by two ways. First,
1O2 scavenging occurs by the removal or dissipation of
energy from 1O2 into heat, which is known as physical scavenging of
1O2 (Truscott 1990,
Kaiser et al. 1990, Stahl and Sies 2003, Telfer 2002, 2005, Dall’Osto et al. 2007). Second, the
direct interaction of 1O2 with the antioxidant results in the oxidative product which is known
as chemical scavenging of 1O2 (Trebst et al. 2002, Kruk et al. 2005, Penuelas and Munné-
Bosch 2005, Krieger-Liszkay and Trebst 2006, Kruk and Trebst 2008, Gruzska et al. 2008).
Tocopherols are known to scavenge 1O2 by both ways i.e. physical as well as
chemical scavenging. In physical scavenging, tocopherols dissipate the energy from 1O2 into
heat and convert 1O2 back to molecular oxygen, whereas in chemical scavenging, tocopherol
25
interacts with 1O2 forming tocopherol quinone via 8-hydroperoxy-tocopherone. Subsequently,
tocopherol quinone could convert into tocopherol quinol by enzymatic reaction (Kruk and
strzałka 1995, Siegel et al. 1997, Lass and Sohal 1998, Kruk et al. 2000, Munné-Bosch and
Alegre 2002, Munné-Bosch et al. 2005). In literatures, the numerous studies show that
tocopherol protects plants, algae and cyanobacteria against oxidative damage (Sattler et al.
2003, 2004, Havaux et al. 2005, Kanwischer et al. 2005, Liu et al. 2008, Mène-saffrané et al.
2010, Mène-saffrané and DennaPenna 2010, Hakala-Yatkin et al. 2011, Inoue et al. 2011). In
agreement with these, we have shown the 1O2 scavenging activity of tocopherol by EPR-spin
trapping technique and singlet oxygen sensor green (SOSG) fluorescence imaging through
confocal laser scanning microscopy (CLSM) (Paper IV).
2.3.3. Singlet oxygen scavenging by plastoquinol
Plastoquinol is prenyllipid containing quinol head with long side chain as shown in figure
2.6. Plastoquinol is known as electron carrier molecules during electron transport in PSII. It is
synthesized and stored in plastoglobuli (a globular lipid bilayer structure) attached to the
thylakoid membranes. Plastoquinol is formed by the enzymatic conversation of MSBQ in
photosynthetic organisms. The mechanistic pathway of plastoquinol synthesis is described in
section 2.4.1.
Figure 2.6. Chemical structure of plastoquinol (figure adapted from Gruszka et al. 2008).
In addition to electron transfer, the antioxidant activity of plastoquinol has been
suggested in literature (Hundal et al. 1995, Kruk and Trebst 2008, Gruszka et al. 2008,
Szymańska and Kruk 2010a). Recently, The degradation of D1 and D2 proteins in
Chlamydomonas reinhardtii grown in the presence of an inhibitor of plastoquinol
biosynthesis under high-light stress has also been measured. Furthermore, the use of
exogenous plastoquinol homologue containing short side chain to Chlamydomonas
reinhardtii protect against the photodamage of D1 and D2 proteins (Kruk et al. 2005). Later,
consumption of plastoquinol in the presence of inhibitor of plastoquinol biosynthesis is
reported in Chlamydomonas reinhardtii exposed to high-light (Kruk and Trebst 2008). High-
26
light induced synthesis and storage of plastoquinol has been shown in Arabidopsis thaliana
(Szymańska and Kruk 2010a). Plastoquinol oxidation product analysis by HPLC suggests the
scavenging of 1O2 by plastoquinol occurs via chemical scavenging. In this process, interaction
of 1O2 with plastoquinol leads to the formation of oxidized plastoquinol (Gruszka et al. 2008).
In agreement with these studies, our study shows the direct evidence on the 1O2 scavenging
by plastoquinol in PSII (Paper I).
2.3.4. Singlet oxygen scavenging by plastochromanol
Plastochromanol is another prenyllipid, which is getting more attention for its role against the
environmental stress condition. It is structurally homologous to γ -tocotrienol having long
side chain as shown in figure 2.7. It is naturally synthesized by photosynthetic organisms.
Conversion of plastoquinol to plastochromanol is catalyzed by the enzyme tocopherol cyclase
(Kumar et al. 2005, Szymańska and Kruk 2010a) as described in section 2.3.1.
Figure 2.7. Chemical structure of plastochromanol (figure adapted from Gruszka et al. 2008).
In plants, plastochromanol is considerably stored in seeds and leaves which is known
to posses potential antioxidant properties similar to other prenyllipids. Recent studies showed
the accumulation of plastochromanol in leaves under high-light stress in vivo (Zbierzak et al.
2010, Szymańska and Kruk 2010a, 2010b). Singlet oxygen scavenging by plastochromanol is
also measured in vitro (Gruszka et al. 2008). HPLC analysis of leaves and seeds form
Arabidopsis plants showed the presence of hydroxyl-plastochromanol in leaves but not in
seeds. Based on these results, authors suggest that hydroxy-plastochromanol could be the
oxidative product of plastochromanol by the action of 1O2 (Szymańska and Kruk 2010b). To
confirm this proposal, we measured the 1O2 formation in leaves and isolated chloroplast in
vte1 Arabidopsis plants lacking plastochromanol and tocopherol. By comparing the results
from WT and vte1 Arabidopsis plants, we concluded that the plastochromanol acts as a potent
1O2 scavenger in plants (Paper IV).
27
-----------------------------------------
Materials and Methodology
-----------------------------------------
Chapter 3
28
3. Materials and Methods
This chapter briefly describes the different methods used during my research study.
3.1. Chemicals
Major important chemicals used during the work are listed in this short paragraph. Different
spin traps 2, 2, 6, 6-tetramethyl-4-piperidone (TEMPD), POBN (4-pyridyl-1-oxide-N-tert-
butylnitrone) were purchased from Sigma, Aldrich (Germany). 5-(ethoxycorbonyl)-5-methyl-
1-pyrroline N-oxide (EMPO) spin trap was obtained from Alexis Biochemicals
(Switzerland). Capillary tube used for EPR measurements was purchased from Blaubrand
intraMARK, Brand, Germany. Singlet oxygen sensor green (SOSG) reagent was obtained by
Molecular Probes Inc. (U.S.A.).
A brief list of other used chemicals are as follows; sucrose (C12H22O11), sodium
chloride (NaCl), magnesium chloride (MgCl2), calcium chloride (CaCl2), sodium bicarbonate
(NaHCO3), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ), C8H18N2O4S),
sodium-ascorbate (C6H7NaO6), BSA (bovine serum albumin), MES (2-(N-morpholino)-
ethanesulfonic acid) (C6H13NO4S), Triton X-100 {(C14H22O(C2H4O)n}, amplex red (10-
acetyl-3,-7-dihydroxyphenoxazine), HRP (Horseradish peroxidase), MDA (malondialdehyde,
C3H4O2), DCMU [{3-(3,4-dichlorophenyl)-1,1-dimethylurea}, C9H10Cl2N2O], PQ
(plastoquinone), PQH2 (Plastoquinol), heptane (C7H16), hexane (C6H14), acetone (C3H6O),
Isobutanol (C4H10O), ethanol (C2H5OH), TBA (thaiobarbituric acid, C4H4N2O2S), TCA
(trichloroacetic acid, C2HCl3O2), BHT (butylated hydroxytoluene, C15H24O) etc.
3.2. Preparation of PSII membranes from Spanacea oleracea
Photosystem II enriched membranes were prepared from fresh spinach leaves using the
method of Berthold et al. (1981) with the modifications described in Ford and Evans (1983).
Spinach leaves were purchased from the local market and isolation has been done at 4°C in
green light condition using different buffers (buffer A and B). The composition of buffer A
(pH 7.5) was 400 mM sucrose, 15 mM NaCl, 5 mM MgCl2, 5 mM CaCl2, 40 mM HEPES
(pH 7.5), 5 mM Na-ascorbate and 2 gm/l bovine serum albumin, whereas buffer B prepared
by using 400 mM sucrose, 15 mM NaCl and 5 mM MgCl2, 40 mM MES (pH 6.5). Bovine
serum albumin and Na-ascorbate were added just before crushing the spinach leaves. Spinach
leaves were washed twice with deionised water and kept in dark for further use. Dark adapted
leaves (400 gm) were homogenized with 500 ml of buffer A, after that homogenized
mixture was filtered through 2 layers of nylon bolting cloth. Filtrate was transferred into ice
29
chilled centrifugation tubes and centrifuged at 9950 x g for 10 min at 4 ºC. The supernatant
was thrown out and pellet was mixed properly with paint brush and resuspended in 600 ml of
buffer B. Suspension was again centrifuged at 9950 x g for 10 min at 4 ºC. After
centrifugation supernatant was discarded and resuspended pellet in buffer B, at this step
concentration of chlorophyll was measured. The suspension was treated with 5 % Triton X-
100 on ice bath with continuous stirring for 17 min, and then it was centrifuged at 7000 x g
for 7 min. The pellet was discarded and supernatant centrifuged again at 48000 x g for 20 min
at 4 ºC. Pellet was washed (1-3 times) with buffer B at the final step, and chlorophyll
concentration was measured. Photosystem II membranes were diluted to final Chl
concentration (3-6 mg Chl ml-1
) and stored at -80 ºC.
3.3. Preparation of water-splitting manganese complex depleted PSII membranes
Removal of water-splitting manganese complex from PSII membranes were done by Tris
treatment according to method described in Tiwari and Pospíšil (2009). For details, see the
method and material section described in Paper II.
3.4. Preparation of PQ-depleted PSII membranes
The depletion of plastoquinone from PSII membranes were performed in two-steps phase
preparation and phase separation by using the method of Wydrzynski and Inoue (1987). For
details, see the method and material section described in Paper I.
3.5. Growing of Arabidopsis plants
The Arabidopsis thaliana plants (wild type WT Col-0 and tocopherol cyclase vte1 mutant)
were grown under low light (100 mol photons m-2
s-1
) condition. The plants were grown at
a photoperiod of 16 h at a temperature of 25° C in phytotron (Weiss Gallenkamp, United
Kingdom). The Arabidopsis leaves (7-8 weeks) were collected for further experiments.
3.6. Chloroplasts isolation from Arabidopsis plants leaves
Chloroplasts were prepared from Arabidopsis leaves using the method of Aronsson and Jarvis
(2002) with the modifications described in Seigneurin-Berny et al. (2008). For details, see the
method and material section described in Paper IV.
3.7. High-light treatment for photoinhibition
Chloroplasts or PSII membranes were exposed to continuous white light (1000 µmol photons
m-2
s-1
) for time period as required for the study. The illumination was performed using a
30
halogen lamp with a light guide (Schott KL 1500, Germany) under slow continuous stirring
with a tiny bar magnet. The light intensity was measured by quantum radiometer LI-189 (LI-
COR Inc., Lincoln, U.S.A.).
3.8. Heat treatment
Photosystem II membranes in tightly sealed Eppendorf tubes were immersed in water bath
with water circulation maintained by a digitally controlled heater (Cole Parmer, U.S.A.) in
darkness at 40 ºC. After the heat treatment, the sample was immediately transferred and
proceeded for further measurement in dark.
3.9. Electron paramagnetic resonance spin-trapping spectroscopy
Singlet oxygen was measured in biological (PSII membranes, Tris-treated PSII membranes,
chloroplasts) and chemical (rose bengal) system. Singlet oxygen was detected in presence of
2, 2, 6, 6-tetramethyl-4-piperidone (TEMPD) spin trap (Moan and Wold 1979) (Sigma,
U.S.A.) by EPR spin trapping technique (see the method and material section of Paper I, III,
IV). To measure HO• production in PSII membranes under heat stress, we have used EMPO
{5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide} (Alexis Biochemicals, Switzerland) as
spin trap (Olive et al. 2000) (see the method and material section of Paper II). Light-induced
O2-
formation in PSII membranes was measured by EMPO spin trap (Zhang et al. 2000) (see
the method and material section of Paper V). To measure carbon-centered radicals, POBN
and EMPO have been used in this study (North et al. 1992, Stolze et al. 2005) (see the
method and material section of Paper III). The radical spin trap adducts EPR spectra were
recorded using an EPR spectrometer MiniScope MS200 and MS400 (Magnettech GmbH,
Berlin, Germany). EPR conditions were as follows: microwave power, 10 mW; modulation
amplitude, 1 G; modulation frequency, 100 kHz; sweep width, 100 G; scan rate, 1.62 G s-1
.
3.10. Spectroflourimeterical detection of hydrogen peroxide
The formation of H2O2 was measured in PSII membranes using amplex red fluorescent assay.
In this assay, fluorescent probe amplex red (10-acetyl-3, 7-dihydroxyphenoxazine) reacts
with H2O2 in the presence of horseradish peroxidase enzyme to form the fluorescence
compound resorufin (Zhou et al. 1997). Fluorescence emission spectra were measured by
using spectroflourimeter F-4500 (Hitachi, Japan). For details, see the method and material
section described in Paper II.
31
3.11. Confocal laser scanning microscopy (CLSM)
Singlet oxygen imaging in Arabidopsis leaves was performed by CSLM (Olympus Fluorview
1000 confocal unit) with inverted microscope IX 80. Singlet oxygen was measured in
Arabidopsis leaves and cyanobacteria in presence of singlet oxygen sensor green (SOSG;
excitation by a 488 nm line of argon laser and detection by 505-525 nm emission filter set)
(Flors et al. 2006, Ragas et al. 2009). The proper intensity of laser was set according to
unstained samples at the beginning of each experiment (Sedlářová et al. 2011). The intensity
of signal and percentage of pixel in image were evaluated using software FV10-ASW 3.0
Viewer (Olympus). For details, see the method and material section described in Paper IV.
3.12. High pressure liquid chromatography (HPLC)
For quantitative analysis of tocopherol, plastochromanol, hydroxy-plastochromanol and
MDA content in Arabidopsis leaves, HPLC technique was used. Content of prenyllipids was
determined by HPLC according to method described in Szymańska and Kruk (2010a and
2010b). MDA content was measured in Arabidopsis leaves according to Havaux et al. (2005).
For details, see the method and material section described in Paper IV. Loosely bound
plastoquinone was extracted from PSII membranes by method described in Wydrzynski and
Inoue (1987) and content determined by HPLC according method described in Kruk and
Karpinski (2006). For details, see the method and material section described in Paper V.
3.13. Two-dimensional imaging of ultra-weak photon emission
Highly sensitive charge coupled device (CCD) camera VersArray 1300B (Princeton
instruments, U.S.A.) was used for two-dimensional photon imaging. Imaging of ultra-weak
photon emission in Arabidopsis leaves was done according to Prasad and Pospíšil (2011). For
details, see the method and material section described in Paper IV.
3.14. Measurement of redox form of cyt b559
To study the different redox states and redox properties of cyt b559 optical absorption
spectroscopy was used (Olis RSM 1000 spectrometer, Olis Inc., U.S.A.). The different redox
state and content of cyt b559 were determined from the absorbance changes measured at 559
nm according to method describe in Tiwari and Pospíšil (2009). For details, see the method
and material section described in Paper V.
32
-----------------------------------
Results and Discussion
-----------------------------------
Chapter 4
33
4. Results and discussion
During the phase of research work, I focused on the production and scavenging of ROS in
PSII. The production of HO under heat stress (Paper II),
1O2 production in donor side of
photoinhibition (Paper III) and O2-
production under high-light illumination (Paper V) in
PSII are studied. Furthermore, 1O2 scavenging by plastoquinol (Paper I), tocopherol and
plastochromanol (Paper IV) were also measured in isolated PSII, chloroplasts and
Arabidopsis thaliana leaves. In this section, the results related to these papers are briefly
summarized and to see details, please refer to the attached papers, which are enclosed at the
end of thesis.
4.1. Singlet oxygen scavenging activity of plastoquinol in PSII (Paper I)
4.1.1. Singlet oxygen scavenging by plastoquinol in chemical system
To measure the 1O2 scavenging by plastoquinol in chemical system, we used rose bengal, a
known photosensitizer. Illumination of rose bengal with white light results in the formation of
1O2 by energy transfer. Light-induced triplet rose bengal transfers excited energy to
molecular oxygen to form 1O2. The production of
1O2 was measured using EPR spin-trapping
technique. The paramagnetic 2, 2, 6, 6-tetramethyl-4-piperidone-1-oxyl (TEMPONE) EPR
signal is accomplished by the interaction of diamagnetic 2, 2, 6, 6-tetramethyl-4-piperidone
(TMPD) with 1O2. The exposure of rose bengal to white light resulted the production of
TEMPONE EPR signal (Figure 4.1.). Addition of exogenous short-chain plastoquinol (PQH2-
1) completely suppressed the light-induced TEMPONE EPR signal from the rose bengal
(Figure 4.1.). Suppression of TEMPONE EPR signal by plastoquinol concludes the 1O2
scavenging activity of plastoquinol in chemical system.
34
Figure 4.1. Singlet oxygen scavenging by plastoquinol measured in a chemical system. Rose Bengal
is illuminated with white light 1000 mol photons m-2
s-1
intensity in absence and presence of 100 M
plastoquinol (light + PQH2-1) with 50 mM TMPD and 25 mM phosphate buffer (pH 7.0).
4.1.2. Singlet oxygen scavenging by plastoquinol in PQ-depleted PSII membranes
To confirm the scavenging of 1O2 by plastoquinol in biological system, we measured
1O2
production in PQ-depleted PSII membranes in absence and presence of exogenous PQH2-1.
Similarly as in chemical system, we measured light-induced TEMPONE EPR signal in PQ-
depleted PSII membrane (Figure 4.2.). TEMPONE EPR signal increased with the time of
illumination. Due to the impurity of spin trap, a small negligible TEMPONE EPR signal was
observed in PQ-depleted PSII membrane without illumination. Time profile of TEMPONE
EPR signal shows that the illumination of PQ-depleted PSII membranes in the presence of
exogenous PQH2-1 suppressed the 1O2 production (Figure 4.3.). The scavenging of
1O2 in the
presence of PQH2-1 is gradually increased with the increase of concentration of exogenous
PQH2-1 (see figure 4, Paper I). Similarly, the plastoquinol containing different side chains
(PQH2-2, PQH2-4 and PQH2-9) also showed the 1O2 scavenging activity in PQ-depleted PSII
membranes (see figure 5, Paper I).
330 332 334 336 338 340 342
Light+PQH2-1
Dark
Light
B (mT)
35
Figure 4.2. Singlet oxygen production measured in PQ-depleted PSII membranes in absence (A) and
presence of 100 M exogenous PQH2-1 (B). TEMPONE EPR spectra were measured after
illumination of PQ-depleted PSII membranes (150 g Chl ml-1
) with white light 1000 mol photons
m-2
s-1
intensity in the presence of 50 mM TMPD and 40 mM Mes (pH 6.5).
330 332 334 336 338 340 342
90 min
60 min
30 min
15 min
10 min
5 min
0 min
B (mT)
330 332 334 336 338 340 342
90 min
60 min
30 min
15 min
10 min
5 min
0 min
B (mT)
36
Figure 4.3. Time dependence of TEMPONE EPR signal intensity measured in PQ-depleted PSII
membranes in absence and presence of exogenous PQH2-1 by evaluating the relative height of central
peak of 1st derivative of EPR signal. Each data represent the means value of three set of experiments.
Photodamage of PSII is an extensively studied during the last two decades. It is
considered that 1O2 formed in PSII triggers the degradation of D1 protein in PSII under
photoinhibitory conditions. However, many studies also reported that β-carotene, tocopherol
and recently proposed plastoquinol protect PSII against photooxidative stress (Trebst et al.
2002, Kruk et al. 2005, Krieger-Liszkay and Trebst 2006, Kruk and Trebst 2008, Durchan et
al. 2010, Arellano et al. 2011). In agreement with previous proposals, this study shows
evidence on the 1O2 scavenging by plastoquinol in both chemical and biological systems. The
scavenging of 1O2 in the interior of the thylakoid membrane is crucial to prevent the
interaction of 1O2 with proteins and lipids. Due to the fact that plastoquinol is a mobile
molecule, which is able to diffuse in the thylakoid membrane for long distances, plastoquinol
is one of the most efficient scavengers of 1O2 in the thylakoid membranes. In agreement with
this, it has been recently shown that synthesis of plastoquinol is increased in Arabidopsis
plants under high-light stress (Szymańska and Kruk 2010a, 2010b), it also supports that
0 20 40 60 80 1000
500
1000
1500
2000
Control
PQH2-1
TE
MP
ON
E E
PR
sig
nal
(r.u
.)
Time (min)
37
plants should produce more plastoquinol under stress conditions, which provides protection
against oxidative stress by scavenging 1O2.
4.2. Role of chloride ion in hydroxyl radical production in PSII under heat stress (Paper
II)
4.2.1. Hydroxyl radical production in PSII membranes under heat stress
In this study, the detection of HO in PSII under heat stress was performed by using EMPO
spin trap compound. Spin-trap EMPO reacts with HO and results in the formation of
EMPO-OH adduct EPR signal, detected by EPR spin trapping technique. Our result shows
that heating of PSII membranes at 40 °C results in HO· production (Figure 4.4.). Previously,
we have reported that complete suppression of heat-induced EMPO-OH EPR signal occurred
in the presence of catalase or by removal of water-splitting manganese complex from PSII
(Pospíšil et al. 2007, Yamashita et al. 2008).
Figure 4.4. Hydroxyl radical formation measured in PSII membranes (500 g Chl ml-1
) under heat
stress (at 40 °C) in the presence of 75 mM EMPO in 40 mM MES-NaOH buffer (pH 6.5).
4.2.2. Effect of halides on hydroxyl radical production in PSII membranes under heat
stress
Recent studies suggest that HO production is related to the damage of PSII donor side
(Pospíšil et al. 2007, Yamashita et al. 2008). To find out the correlation between chloride ion
and heat-induced HO production in PSII, we measured the EMPO-OH EPR signal in PSII
exposed to 40 °C in the presence of exogenous halides NaCl, NaBr and NaI. Results show the
330 332 334 336 338 340 342
5 min
2.5 min
1 min
0 min
B (mT)
38
significant suppression of heat-induced EMPO-OH EPR signal in presence of exogenous
halides (Figure 4.5). It reveals the involvement of chloride ion in the suppression of HO
production in PSII membranes under heat stress. In addition to this results, chloride channel
blocker DIDS and chloride binding site competitor acetate also suppress the EMPO-OH EPR
signal (see figure 6, Paper II).
Figure 4.5. Hydroxyl radical formation measured in PSII membranes in presence of 100 mM
different exogenous halides (NaCl, NaBr, NaI) under heat stress (at 40 °C) for 5 minutes. All other
experimental settings are identical as described in figure 1.
4.2.3. Effect of halides on hydrogen peroxide production in PSII membranes under heat
stress
It is well known that HO is formed via the Fenton reaction (conversion of H2O2 to HO
in
presence of metal ion) in both chemical and biological systems. According to this, we have
also suggested that the same reaction is responsible for the formation of HO• in PSII under
heat stress. To confirm this, we measured the formation of heat-induced H2O2 in PSII by
amplex red fluorescent assay. Results show production of heat-induced resorufin signal in
PSII membrane and significant suppression of fluorescence signal in presence of halides
(Figure 4.6). Similarly, chlorides channel blocker DIDS and competitive inhibitor for the
chloride binding site acetate also suppress the resorufin fluorescence signal (see figure 3,
Paper II). These observations confirm the involvement of chloride ion in suppression of HO
formation in PSII under heat stress.
Contr
ol
NaC
l
NaB
rNaI
0
1000
2000
3000
4000
5000
EM
PO
-OH
ad
du
ct
EP
R
sig
na
l (r
.u.)
39
Figure 4.6. Hydrogen peroxide (resorufin fluorescence) formation measured in PSII membranes in
presence of 100 mM of different halides (NaCl, NaBr, NaI) under heat stress (at 40 °C) for 5 min,
using amplex red fluorescent assay.
X-ray crystallographic reports from different cyanobacteria show that the chloride ion
is positioned near the water-splitting manganese complex (Guskov et al. 2009, Umena et al.
2011). To make proper water oxidation to molecular oxygen, PSII have different channel for
the movement of water to water-splitting manganese complex and release of molecular
oxygen to the medium (Ishikita et al. 2006, Murray and Barber 2007, Ho 2008, Ho and
Styring 2008, Gabdulkhakov et al. 2009, Guskov et al. 2010). Chloride ion is located at the
water channel and regulates the proper water availability to water-splitting manganese
complex (Guskov et al. 2009, Gabdulkhakov et al. 2009). Previously, it has been proposed
that H2O2 is formed due to the disturbance of protein matrix and water accessibility around
water-splitting manganese complex under stress conditions (Thompson et al. 1989,
Wydrzynski et al. 1996). In agreement with these reports, we proposed that heat-induced
release of chloride ion from its binding site causes the production of H2O2 in PSII. The
production of H2O2 occurs by the unrestrained movement of water molecules around water-
splitting manganese complex, where H2O2 subsequently converts into HO via Fenton
reaction.
Contr
ol
NaC
l
NaB
rNaI
0
5
10
15
20
25
30
35
Flu
ore
sc
en
ce
(r.
u.)
40
4.3. Evidence on singlet oxygen production in donor side of photoinhibition of PSII
(Paper III)
4.3.1. Singlet oxygen production in Tris-treated PSII membranes
To study the 1O2 production in donor side photoinhibition, we utilized Tris-treated PSII
membranes. The hydrophilic spin trap TMPD is used for the detection of 1O2 by EPR spin-
trapping technique. The illumination of Tris-treated PSII membranes in the presence of
TMPD spin trap compound results the TEMPONE EPR signal (Figure 4.7.). Furthermore, the
illumination of Tris-treated PSII at high pH enhanced the TEMPONE EPR signal (see figure
3, Paper III). These results show the formation of 1O2 in donor side photoinhibition.
Figure 4.7. Singlet oxygen formation under donor side photoinhibition. Tris-treated PSII membranes
(200 g Chl ml-1
) were exposed to white light (1000 mol photons m-2
s-1
intensity) in the presence 50
mM TMPD spin trap and 40 mM Mes buffer (pH 6.5).
4.3.2. Carbon-centered radical production in Tris-treated PSII membranes
To decipher the involvement of lipid and protein oxidation in the 1O2 production in donor
side photoinhibition, the carbon centered radical was measured in Tris-treated PSII
membranes. The POBN is a commonly used spin trap for the detection of carbon centered
radical by EPR spin-trapping technique. The illumination of Tris-treated PSII membranes in
presence of POBN spin trap results in the POBN-R adduct EPR signal (Figure 4.8.).
Furthermore, the illumination of Tris-treated PSII at high pH enhanced the POBN-R adduct
330 332 334 336 338 340 342
A30 min
25 min
20 min
15 min
10 min
5 min
0 min
B (mT)
41
EPR signal (see figure 4, Paper III). These results indicate indirectly the involvement of
oxidation of lipids and proteins in 1O2 formation in donor side photoinhibition.
Figure 4.8. Carbon-centered radical formation under donor side photoinhibition. Tris-treated PSII
membranes (200 µg Chl ml-1
) was illuminated to white light (1000 mol photons m-2
s-1
intensity) in
the presence 50 mM POBN spin trap and 40 mM Mes buffer (pH 6.5).
4.3.3. Singlet oxygen production in donor side of photoinhibition of PSII
Based on these observations, we suggested that the formation of 1O2 occurs in donor side of
photoinhibition of PSII via Russell mechanism. The Russell mechanism is well known in
chemical system (Russell 1957, Howard and Ingold 1968, Kanofsky and Axelrod 1986), and
it is believed that 1O2 is formed via ROOOOR intermediate (Dean et al. 1997, Miyamoto et
al. 2003, 2007, Sun et al. 2007). Similarly, we proposed that the light-induced formation of
R in Tris-treated PSII membranes leads to the formation of ROO
by interacting with
molecular oxygen. Combination of two ROO generates
1O2 oxygen in donor side
photoinhibition of PSII via linear ROOOOR intermediate (Scheme 4.1.).
330 332 334 336 338 340 342
A30 min
25 min
20 min
15 min
10 min
5 min
0 min
B (mT)
42
R•
O2
ROO•
ROO•
ROOOOR
1O2 + ROH + RO
P680•+
Light
TyrZ•
Mn4CaO5
Pheo•-
Scheme 4.1. Scheme shows the proposed reaction mechanism at molecular basis about the 1O2
formation in PSII in donor side photoinhibition.
4.4. Singlet oxygen scavenging by tocopherol and plastochromanol in Arabidopsis
thaliana (Paper IV)
4.4.1. HPLC analysis of the content of tocopherol and plastochromanol in WT and vte1
Arabidopsis leaves
In this study, we used the tocopherol cyclase (vte1) mutant (lacking tocopherol and
plastochromanol) of Arabidopsis in comparison with WT. The absence of tocopherol and
plastochromanol in vte1 Arabidopsis is confirmed by the HPLC analysis. Our result shows the
presence of these compounds in WT, whereas vte1 Arabidopsis lacks both tocopherol and
plastochromanol in leaves (Figure 4.9.).
43
0 2 4 6 8 10 12
F
luo
resce
nce
in
ten
sity (
a.u
.)
Retention time (min)
WT
vte1
PC-8
PQH2-9
PC-OH
-Toc
Figure 4.9. HPLC chromatogram shows the content of different prenyllipids in WT and vte1
Arabidopsis leaves.
4.4.2. Singlet oxygen imaging by singlet oxygen sensor green in WT and vte1 Arabidopsis
leaves
To monitor the effect of tocopherol and plastochromanol in vivo, we measured 1O2 formation
in WT and vte1 Arabidopsis leaves by CSLM. Singlet oxygen sensor green (SOSG) was used
for the imaging of 1O2 in leaves. Interaction of
1O2 with SOSG provides fluorescence image.
SOSG fluorescence images show no fluorescence in leaves exposed to low light (left panel)
whereas, the illumination of leaves with high-light results in SOSG fluorescence (right
panel). Figure 4.10 shows that the SOSG fluorescence is more prominent in vte1 Arabidopsis
leaves compared to WT. The result shows that more 1O2 in vte1 Arabidopsis leaves is due to
lack of tocopherol and plastochromanol. It indicates the scavenging of 1O2 occurs by
tocopherol and plastochromanol.
44
Figure 4.10. Singlet oxygen formation measured in leaves of vte1 and WT Arabidopsis by SOSG
fluorescence imaging. SOSG fluorescence images were measured in leaves section of both type
Arabidopsis treated under low light (100 μmol photons m−2
s−1
) and high-light (1000 μmol photons
m−2
s−1
) for 6 h, afterward immersed in SOSG for 30 min in the dark for SOSG infiltration. SOSG
fluorescence images (excitation with a 488 nm line of argon laser and detection with 505-525
nm emission filter set) were measured by CSLM.
4.4.3. Singlet oxygen production in chloroplasts isolated from WT and vte1 Arabidopsis
leaves
To confirm the effect of tocopherol and plastochromanol on 1O2 scavenging in Arabidopsis
plants, light-induced 1O2 formation was measured in chloroplasts isolated from WT and vte1
Arabidopsis leaves. The illumination of chloroplast in the presence of TMPD spin trap
resulted in TEMPONE EPR signal. Figure 4.11 shows the higher amount of light-induced 1O2
45
formed in chloroplasts isolated form vte1 Arabidopsis leaves compared to WT. The data
observed using EPR spin-trapping were in agreement with the SOSG fluorescence images.
These observations clearly indicate the 1O2 scavenging by tocopherol and plastochromanol in
Arabidopsis plants.
Figure 4.11. Light-induced 1O2 production in chloroplasts isolated from WT and vte1 Arabidopsis
leaves detected by EPR-spin trapping technique. Chloroplasts were exposed to white light 1000 μmol
photons m−2
s−1
intensity in the presence of 50 mM TMPD spin trap compound and 40 mM HEPES
(pH 7.6). Time profile of 1O2 formation in chloroplasts isolated from WT and vte1 Arabidopsis leaves
evaluated by relative height of central peak of 1st derivative of TEMPONE EPR signal.
0 5 10 15 20 25 300
1000
2000
3000
4000
5000
6000
C
WT
vte1
TE
MP
ON
E E
PR
Sig
na
l
(r.u
.)
Time (min)
330 332 334 336 338 340 342
B
30 min
25 min
20 min
15 min
10 min
5 min
0 min
B (mT)
330 332 334 336 338 340 342
A
30 min
25 min
20 min
15 min
10 min
5 min
0 min
B (mT)
46
4.4.4. Malondialdehyde detection in WT and vte1 Arabidopsis leaves
To monitor the effect of tocopherol and plastochromanol against the photodamage of lipids,
malondialdehyde (MDA; a secondary product of lipid peroxidation) was quantified by HPLC.
Figure 4.12 shows significantly higher MDA content in vte1 Arabidopsis leaves exposed to
high-light in comparison to WT. Furthermore, similar effect was observed by ultra-weak
photon emission measurement by a charge coupled device (CCD) (see figure 8, Paper IV). Our
results suggest that the tocopherol and plastochromanol protect the Arabidopsis plants against
photooxidative stress.
Figure 4.12. Malondialdehyde (MDA) content in WT and vte1 Arabidopsis leaves grown under low
light (100 μmol photons m−2
s−1
) (LL) and exposed to white light (1000 μmol photons m−2
s−1
) for 30
min (HL) was quantified by high-performance liquid chromatography.
Recent studies on vte1 mutant in Synechocystis sp. PCC 6803 and Arabidopsis
thaliana show the role of tocopherol and plastochromanol in protection of photoinactivation
and repair cycle of PSII (Inou et al. 2011, Hakala-Yatkin et al. 2011). In literature, it has been
suggested that plastochromanol in plants bears 1O2 scavenging activity (Gruszka et al. 2008,
Szymańska and Kruk 2010a, Mène-Saffrané et al. 2010, Zbierzak et al. 2010) with limited
experimental data. In agreement with that, we showed here the evidence on 1O2 scavenging
by tocopherol and plastochromanol in Arabidopsis plant in vivo by SOSG fluorescence
imaging and in vitro by EPR spin-trapping technique. At physiological level, it is reported
that synthesis of tocopherol and plastochromanol is enhanced under high-light stress and
predominantly stored in plastoglobuli attached to thylakoid membranes (Austin 2006,
Lichtenthaler 2007, Piller et al. 2012). Similarly, we proposed that the light-induced storage
of excess tocopherol and plastochromanol in plastoglobuli is involved in protection of plants
against photooxidative stress.
0.0
0.2
0.4
0.6
0.8
1.0
LL HLWT vte1 WT vte1
MD
A (
g/g
FW
)
47
5.5. Involvement of plastosemiquinone in superoxide anion radical production in PSII
(Paper V)
5.5.1. Light-induced superoxide anion radical production in PSII membranes
Light-induced O2-
production in PSII membrane was measured by spin trap compound
EMPO using EPR spin-trapping spectroscopy. There is no EMPO-OOH EPR signal in the
dark, whereas exposure of PSII membranes to light results in EMPO-OOH adduct EPR signal
(Figure 4.13. A). In addition to this, the exposure of PSII membranes to high-light in the
presence of exogenous plastoquinone (PQ-1) enhances the EMPO-OOH adduct EPR signal
(Figure 4.13 B). EMPO-OOH adduct EPR signal increased with the longer time illumination.
These results indicate the involvement of plastosemiquinone/plastoquinol in the O2-
in PSII
membranes.
Figure 4.13. Light-induced production of O2-
in PSII membranes. The EMPO-OOH adduct EPR
signals were measured after the exposure of the PSII membranes (150 μg Chl ml−1
) absence [A] and
in presence of exogenous plastoquinone (PQ-1) (100 μM) [B] with white light 1000 μmol m−2
s−1
intensity in the presence of 25 mM EMPO spin trap, 100 μM desferal and 40 mM MES (pH 6.5). Bar
diagram show the relative intensity of O2-
production in absence (PSII) and presence (PSII+PQ-1) of
exogenous PQ-1.
330 332 334 336 338 340 342
A
90S
180S
120S
60S
30S
00S
B (mT)
330 332 334 336 338 340 342
B
180S
120S
90S
60S
30S
00S
B (mT)
0
2000
4000
6000
8000
10000PSII + PQ-1PSII
1801209060300
EM
PO
-OO
H a
dd
uct
EP
R
sig
nal (r
.u.)
Time (s)
48
4.5.2. Effect of DCMU and dinoseb on light-induced superoxide anion radical
production in PSII membranes
To find out the evidence on the involvement of plastosemiquinone in O2-
production in PSII,
the herbicide DCMU (binds the QB site) and dinoseb (binds the QD site) were used. Light-
induced EMPO-OOH adduct EPR signal was significantly suppressed in the presence of
DCMU, whereas dinoseb did not affect EPR signal in PSII membranes (Figure 4.14).
Furthermore, DCMU and dinoseb both suppressed the EMPO-OOH adduct EPR signal in
PQ-supplemented PSII membranes (Figure 4.14). Results reveal that the plastoquinol/
plastosemiquinone are involved in O2-
formation in PSII membranes.
Figure 4.14. Superoxide anion radical production was measured in spinach PSII membranes and PQ-
1 supplemented PSII membranes in the presence of DCMU and dinoseb. DCMU and dinoseb were
added to PSII membranes before the experiments. Other experimental conditions were same as
described in figure 4.13.
4.5.3. Effect of DCMU and dinoseb on photoreduction of cyt b559 in PSII membranes
To see the effect of light on cyt b559 redox property, the photoreduction of cyt b559 were
observed in PSII membranes in the absence and presence of exogenous PQ-1 (see figure 4,
Paper V). To find out the involvement of plastoquinol in photoreduction of cyt b559, we
measured photoreduction of cyt b559 in the presence of DCMU or dinoseb in PSII and PQ-
supplemented PSII membranes. Our results show that photoreduction of cyt b559 is inhibited
in the presence of DCMU (Figure 4.15 A and B) or dinoseb (Figure 4.16 A and B) in PSII
and PQ-suplemented PSII membranes. These results suggest that the photoreduction of cyt
b559 is mediated by plastoquinol in PSII membranes.
0
2000
4000
6000
8000
10000
EM
PO
-OO
H a
dd
uc
t E
PR
sig
na
l (r
.u.)
Control DCMU Dinoseb
PSII + PQ-1PSII
49
Figure 4.15. PSII membranes [A] and PQ-supplemented PSII membranes [B] were exposed to 1000
μmol photons m−2
s−1
white light intensity for 180s in the presence of DCMU. The spectra represent
the difference of light minus ferricyanide-oxidized [photoreduced HP form of cyt b559, (PH)],
hydroquinone-reduced minus ferricyanide-oxidized spectra [HP form of cyt b559, (HP)], ascorbate-
reduced minus hydroquinone-reduced spectra [IP form of cyt b559, (IP)] and dithionite-reduced minus
ascorbate-reduced spectra [LP form of cyt b559, (LP)]. In this study, PSII membrane with exogenous
PQ-1 is termed as PQ-supplemented PSII membranes.
Figure 4.16. PSII membranes [A] and PQ-supplemented PSII membranes [B] were exposed to 1000
μmol photons m−2
s−1
white light intensity for 180s in the presence of dinoseb. Other experimental
conditions are same as in figure 4.15.
540 560 580
A
LP
IP
HP
PH
Wavelength (nM)
540 560 580
B
LP
IP
HP
PH
Wavelength (nM)
540 560 580
ALP
IP
HP
PH
Wavelength (nM)
540 560 580
BLP
IP
HP
PH
Wavelength (nM)
50
QB site
O2 O2·-
PQ
PQH2 PQ 2PQ·̄
O2·-O2
QDH·
PQH2Cyt b559ox
Cyt b559red
+
QB QB·̄
QD site
QB site
O2 O2·-
PQ
PQH2 PQ 2PQ·̄
O2·-O2
QDH·
PQH2Cyt b559ox
Cyt b559red
+
QB QB·̄
QD site
Scheme 4.2 Scheme shows the proposed reaction mechanism on the involvement of
plastosemiquinone in O2-
formation in PSII.
Based on the results and considering the fact that the herbicide DCMU binds at QB
site and according to recent reports, dinoseb has been shown to bind at QD site (Kaminskaya
et al. 2007a, 2007b, Kaminskaya and Shuvalov 2013), we proposed the possible reactions
mechanism on the involvement of plastosemiquinone in O2-
formation in PSII (Scheme 4.2).
We proposed two places for O2-
formation in PSII. 1) It could be formed at the QB site, by
the electron transfer from the QB bound plastosemiquinone (QB-
) to molecular oxygen. This
reaction is thermodynamically favourable, as to the low midpoint midpoint redox potentials
of redox potential of QB bound plastosemiquinone QB/QB-
(Em = – 45 mV, pH 7) (Hauska et
al. 1983). 2) In agreement with previous reports (Mubarakshina and Ivanov 2010), O2-
could
also be formed by the interaction of free plastosemiquinone (PQ-
) and molecular oxygen.
Formation of free PQ-
occurs due to the interaction of plastoquinol (PQH2) to plastoquinone
(PQ).
51
On the other hand, several studies have also reported the binding of dinoseb at the QB-
binding site and act as ADRY (reagent accelerating deactivation reactions of the water-
splitting manganese complex) compound (Oettmeier and Masson 1980, Rutherford et al.
1984, Mathis and Rutherford 1984, Oettmeier 1999, Klimov et al. 2000, Lambrev and
Goltsev 2001). In addition to this, it has also been suggested that the charge separation in
presence of DCMU is reduced due to electrostatic effect of QA-
which resulted in decrease of
O2-
production in PSII (Pospíšil et al. 2006). Keeping these in mind, we could not
completely rule out the other possibilities of the effect of herbicides on the formation of O2-
in PSII. Therefore, apart from above proposed mechanism, the formation of O2-
predominately by other mechanism like leakage of electron from other cofactors of PSII
(Pheo-
, QA-
and cyt b559) cannot be completely excluded.
52
-----------------
Conclusion
-----------------
Chapter 5
53
Conclusion:
On the basis of the results reported in this thesis, we conclude that:
Exposure of PQ-depleted PSII membranes to the high-light results in 1O2 formation.
Singlet oxygen is scavenged by the addition of exogenous plastoquinol in both
chemical (rose bengal) and biological (PQ-depleted PSII membranes) systems.
Singlet oxygen scavenging by plastoquinol in PSII leads to the protection of PSII
against oxidative stress.
Exposure of PSII membranes to heat stress (40 ºC) results in the formation of HO
and H2O2.
Hydroxyl radical and H2O2 production is suppressed by the addition of exogenous
halides, chloride channel blocker and acetates in PSII membranes.
Heat-induced destabilization of water-splitting manganese complex causes the
formation of HO via the Fenton reaction in PSII due to the release of chloride ion
from its binding site.
We proposed that chloride ions control the HO production in PSII under heat stress
by regulating the proper water accessibility to water splitting manganese complex.
Illumination of Tris-treated PSII membranes with high-light results in the formation
of 1O2 and carbon-centered radical.
Our results provide the evidence on the formation of 1O2 in the donor side
photoinhibition of PSII.
We proposed a molecular mechanism for the production of 1O2 in donor side
photoinhibition. Oxidation of organic molecules (lipids and protein) by highly
oxidizing species P680+
/TyrZ leads to the production of
1O2 in donor side
photoinhibition of PSII via the Russell mechanism.
HPLC analysis of vte1 Arabidopsis leaves confirmed the absence of tocopherol and
plastochromanol in vte1 mutant.
54
In vivo imaging of 1O2 by SOSG in Arabidopsis leaves showed a higher amount of
1O2 production in vte1 Arabidopsis compared to WT after high-light treatment.
Similarly, we observed more light-induced 1O2 production in chloroplasts isolated
from vte1 Arabidopsis leaves in comparison to WT in vitro.
HPLC measurement showed more lipid peroxidation in vte1 Arabidopsis leaves
exposed to high-light in comparison to WT. It is proposed that the tocopherol and
plastochromanol are involved in the protection against photodamage of organic
molecules.
Tocopherol and plastochromanol serve as efficient 1O2 scavenger and protect the
plants against photooxidtive stress.
Light-induced O2-
formation in PSII membranes was enhanced in the presence of
exogenous plastoquinone.
Production of O2-
was suppressed by herbicides DCMU in PSII and PQ-1
supplemented PSII membranes, dinoseb did not affect EMPO-OOH EPR signal in
PSII membranes, whereas EMPO-OOH EPR signal was suppressed in PQ-1
supplemented PSII membranes.
Photoreduction of cyt b559 was inhibited by DCMU and dinoseb in the absence and
presence of PQ-1 to PSII membranes.
Plastosemiquinone is involved in the formation of O2-
in PSII membranes under
high-light stress.
55
-----------------
References
-----------------
Chapter 6
56
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Chapter 8
