Ministry of
Education
and Science
Prenylquinones and carotenoids potential mediators of tolerance of
higher plants to combined light
and temperature stress
IZEBZO - 143169/1
BSRP, May 2014, Sofia
Ministry of
Education
and Science
Partner organizations:
Institute of Biology, University of Neuchâtel,
Neuchâtel, Switzerland
and
Institute of Biophysics and Biomedical Engineering,
Bulgarian Academy of Sciences, Sofia,
Bulgaria
BSRP, May 2014, Sofia
Ministry of
Education
and Science
Research teams:
Switzerland
Felix Kessler – PI
Gaétan Glauser
Celine Besagni
Livia Spicher- PhD student
Bulgaria
Maya Velitchkova - PI
Antoaneta Popova
Milena Gerganova - PhD student
Auyguyn Faik - PhD student
Daniela Stanoeva
Dimitar Vasilev
BSRP, May 2014, Sofia
Ministry of
Education
and Science
OBJECTIVES
Agriculture and ecosystems are under increasing climate pressure
in the changing environment. In most places on the planet
temperatures are increasing concomitantly with increasing dryness
and light intensities. Therefore in the future crops more resistant to
this type of multi-stress will be required. Plant productivity largely
depends on photosynthetic activity.
This project aims :
- to discover how the photosynthetic system performs
under combinations of various temperatures and light intensities;
- to investigate protective mechanisms at the
photosystem, membrane and lipid levels
- tо address in a collaborative effort both the physical
parameters of photosystems as well as the lipid composition of the
surrounding thylakoid membranes.
BSRP, May 2014, Sofia
Ministry of
Education
and Science
METHODOLOGY
CH Group
- Non-targeted lipidomics
- Lipid profiling to analyze carotenoids, prenylquinones
and membrane lipids under different types of stress
- Electron microscopy
BG Group
- Pulse Amplitude Modulated fluorescence
- Oxygen evolution by leaves and thylakoid membranes
- 77K fluorescence
- Flash oxygen yields
- Electrophoresis and Western blot
- UV-VIS spectroscopy
BSRP, May 2014, Sofia
Ministry of
Education
and Science
Research plan
Work package 1
Prenylquinone, carotenoid and lipid remodeling under temperature and
high light stress
Responsible: Prof. F. Kessler
Work package 2
Temperature dependent effects on chloroplast ultrastructure and changes
in number and size of plastoglobules
Responsible: Prof. F. Kessler
Work package 3
Changes in the main pigment-protein complex stoichiometry during
growth at different combinations of light-temperature
Responsible: Prof. M. Velitchkova
Work package 4
Test the possibility of improving the tolerance of photosynthesis to
photoinhibition by combination of the growth temperature and growth light
regimes to which the organism is exposed.
Responsible: Assoc. Prof. Antoaneta Popova
BSRP, May 2014, Sofia
Ministry of
Education
and Science
Collaboration aspects
Exchange of expertise and knowledge:
Experiment’s design
Methods that complement each other
Exchange of samples for measurements
Discussing and solving methodological problems
Share and discuss data and results
Exchange visits:
Progress meetings of research teams or WP-RSs in Swiss
and Bulgaria
Work visits of PhD students for learning of partner's
methods and techniques
Participation in scientific events - conferences, congress
and workshops
BSRP, May 2014, Sofia
Ministry of
Education
and Science
Milestones of joint project
9Recruitment of young scientists – two PhD students in BG
and one in CH.
9Acquisition and upgrade of research equipment (BG only)
9High quality collaborative research over 3 years in
Arabidopsis and tomato
9Implementation of an efficient scientific network between
BG and Swiss teams
9Publication of data in general interest and plant science
oriented journals
BSRP, May 2014, Sofia
Ministry of
Education
and Science
Effect of low growth temperature
0th day
6th day
BSRP, May 2014, Sofia
5 days of recovery
Ministry of
Education
and Science
In vivo studies
WP 4
Investigations of whole leaves for determination of:
- physiological characteristics – PS1 (P700) and PS2
(O2 evolution, fluorescence)
- pigments content - chlorophylls and total carotenoids
- membrane integrity - MDA
- synthesis of protective compounds
BSRP, May 2014, Sofia
Ministry of
Education
and Science
Redox state of PSI, control tomato plants (K) and grown at low temperature (Low).
160
140
120
PSI [%]
100
80
60
40
20
K
Low
0
0
2
4
6
Time [days]
BSRP, May 2014, Sofia
8
10
12
WP 4
Ministry of
Education
and Science
Lipid peroxidation and anthocyanins content in control and LT grown plants
500
350
450
300
350
MDA [nmol/g FW] [%]
Anthocyanins [%]
400
250
300
200
250
150
200
100
150
100
50
l…
K
0
0
2
4
6
8
10
Time [days]
K
50
12
BSRP, May 2014, Sofia
0
0
2
4
6
8
10
Time [days]
12
WP 4
Ministry of
Education
and Science
Maximum quantum yield of PSII of tomato plants
110
100
Fv/Fm [%]
90
80
70
K
60
Low
50
0
2
4
6
Time [days]
BSRP, May 2014, Sofia
8
10
12
WP 4
Ministry of
Education
and Science
In vitro studies
WP 3
Investigations of isolated chloroplast and thylakoid membranes
for determination of:
- photochemical activity of PS1 and PS2
- flash oxygen yields
- excitation energy distribution and interaction
- protein degradation of core complexes and antennas
BSRP, May 2014, Sofia
Ministry of
Education
and Science
Photochemical activity of PS1 and PS2
PS2, % PS1, % 120
120
100
80
60
40
20
0
100
80
NT‐NL
60
NT‐NL
LT‐NL
40
LT‐NL
20
0
0
2
4
6
R5
0
2
Days
4
6
R5
Days
PS2, %
PS1, %
160
160
140
140
120
120
100
100
80
PS1: NT‐NL
60
PS1: NT‐HL
40
80
60
PS2: NT‐NL
PS2: NT‐HL
40
20
20
0
0
0
2
4
6
R5
0
2
4
6
R5
Days
Days
BSRP, May 2014, Sofia
WP 3
Ministry of
Education
and Science
77K fluorescence - excitation energy distribution
Effect of exc. 436 nm and 472 nm
Effect of LT on emission spectrum
500
th
Fluorescence (a.u.)
exc. 436 nm
NT-NL
Fluorescence (a.u.)
0 day
th
6 day
5 days recov
LT-NL
0
640
660
680
700
720
740
760
26%
(14.10 k11 i k12)
780
660
800
680
700
720
740
760
780
800
Wavelenghts (nm)
NT-NL
Fluorescence (a.u.)
500
0
LT-NL
th
0 day
th
6 day
5 days recov
exc. 436 nm
Fluorescence (a.u.)
500
exc. 436 nm
exc. 472 nm
exc. 436 nm
exc. 472 nm
500
22%
0
0
640
660
680
700
720
740
760
780
800
Wavelenghts (nm)
BSRP, May 2014, Sofia
20.10-s11 i s12
660
680
700
720
740
760
780
800
Wavelenghts (nm)
WP 3
Ministry of
Education
and Science
Analysis of fluorescence excitation spectra of PS1 and PS2
400
0 d NT-NL
6 d NT-NL
6 d LT-NL
5 d 5 rec
5 d recov-LT
NT-NL
LT-NL
300
250
200
150
100
50
Em. 735 nm
0 d NT-NL
6 d NT-NL
6 d LT-NL
5 d rec.
5 d recov. LT
NT-NL
LT-NL
600
500
Fluorescence (a.u)
350
Fluorescence (a.u.)
700
Em. 685 nm
400
300
200
100
0
0
400
420
440
460
480
500
Wavelenghts (nm)
BSRP, May 2014, Sofia
600
620
640
660
680
700
Wavelenghts (nm)
WP 3
Ministry of
Education
and Science
Protein degradation
Photosystem 2
Photosystem 1
BSRP, May 2014, Sofia
WP 3
Ministry of
Education
and Science
Preliminary conclusions
Tomato plants respond to high light illumination treatment by:
- An increase of α and γ tocopherols and anthocyanins;
- Higher degree of lipid peroxidation – damages of membrane integrity;
- A decrease of antenna size of both photosystems and/or detachment of
LHC2 from PS1;
- An increase of NPQ as protective mechanism against high light intensity.
Low temperature affects tomato plants as follow:
- Strong increase of anthocyanins content;
- An increase of MDA content up to 4th day of treatment;
- Smaller antenna of PS1 and/or less connection between LHC2 and PS1 higher extent of thylakoid stacking and lateral dissociation of both photosystems;
- Photochemical activity of PSI and PSII, PSI activity being more affected.
BSRP, May 2014, Sofia
Ministry of
Education
and Science
Research output
At the end of first year of project Bulgarian PhD students took part in
International conference
"Bioscience - development and new opportunities" - KLIMENT'S
DAYS (Sofia, November 20-22, 2013), presenting their preliminary
results in three posters:
1. M. Gerganova, D. Vasilev, A. Faik, M. Velitchkova. Effect of low
growth temperature on oxygen evolution reactions in tomato and
Arabidopsis thaliana plants
2. A. Faik, M. Gerganova, M. Velitchkova, Changes in energy
distribution and photochemical activity of Photosystem I and
Photosystem II of tomato plants grown under different light
intensities.
3. K. Dobrev, D. Stanoeva, A.V.Popova. Effect of low growth
temperature on photosynthetic parameters of tomato plants in vivo.
BSRP, May 2014, Sofia
Ministry of
Education
and Science
Cooperative and organizational aspects
Strengths:
Easy and freely communications between all members of both
research teams
Competent and timely responses and answers from the SD of
MES and from SNSF
Timely transfers of funds from MES and Swiss partner
Weaknesses:
The inability of BG institutes to open separate bank accounts in
Swiss francs, which would greatly facilitate the management
and accounting of project
BSRP, May 2014, Sofia
Ministry of
Education
and Science
Acknowledgements:
We greatly appreciate:
Swiss National Science Foundation
Ministry of Education and Science of Bulgaria
Swiss Agency for Development and Cooperation
Swiss Embassy in Bulgaria
Science Directorate of MES
BSRP, May 2014, Sofia