Phenotypic screening of drought stress tolerance of starch related mutants in Arabidopsis
thaliana by using Chlorophyll Fluorescence Imaging in
Plantscreen system
Sharmila Madhavan1, Klara Simkova1, Daniel Horrer2, Diana Santelia2 and Martin Trtilek1
1 Photon Systems Instruments spol s.r.o. Drasov, Czech Republic
2 Universität Zurich, Molecular Plant Physiology, Zurich, Switzerland
Aim:
The aim of the study is to screen the starch related mutants for progressive drought
stress tolerance by means of high throughput kinetic Chlorophyll Fluorescence
Imaging and RGB imaging.
Background:
Starch is a major carbohydrate storage molecule and a major product of photosynthesis in the leaves of plants.
Starch synthesis occurs during the day in chloroplast and collapse in the night. The ß-amylase (BAM) is a major
enzyme involved in starch degradation producing maltose from Glucans. Besides the normal pathway of starch
degradation, an alternative stress-induced pathway has been proposed. Such pathway includes enzymes
BAM1(ß-amylase 1), AMY3(α-amylase 3) and PGM1(phosphoglucomutase 1) and might be activated in response
to drought stress resulting in starch degradation. Such a pathway would enable the degradation of starch in the
light, providing metabolites and osmo-protectans leading to an increased drought stress tolerance. In this study,
we have used the high-throughput phenotyping platform, the PlantScreen System developed in Photon System
Instruments, s.r.o, for drought stress tolerance screening in various starch related mutants.
Methods:
Timeline showing treatment of progressive
drought stress :
Last
watering
(5mins)
Measurement
starting (t= 1)
Rewatering
(t= 13)
Quenching kinetics curve displaying various analyzed
photosynthetic parameters in dark and light phase:
• Quenching Kinetics measures and calculates set of
photosynthetic parameters which is used to study the
photosynthetic apparatus
Count survival
rate (t= 14)
• Analysed parameters are:
Fo, Fm, Fv, Fo', Fm', Ft, Ft1, Fp1, Fp2
Conveyor and Robotic PlantScreen system:
• Calculated parameters are:
i)
Fv/Fm(maximum Quantum yield) = (FmF0)/Fm
ii) Dryf_4 (Drying factor_4) = Fp1/F0
Results:
pgm-1
0.9
7
0.9
6
stress
0.8
control
stress
0.8
control
Dryf_4
Fv/Fm
0.75
4
3
control
2
1
0.75
0
0.7
0.7
1 2 4 5 6 7 8 9 10 11
7
bam-1
0.9
Fv/Fm
0.9
0.85
0.85
0.8
control
0.75
Days
6
5
5
4
stress
3
control
6
7
8
9 10 11
Days
• From Fv/Fm, no difference was observed between wild type
and starch mutants and also between watered and non-watered
plants
70
60
50
40
30
10
1 2 4 5 6 7 8 9 10 11
Days
1 2 4 5 6 7 8 9 10 11
80
20
2
0
5
amy-3
control
0
4
control
stress
control
2
stress
3
1
1
90
4
1
0.7
1 2 4 5 6 7 8 9 10 11
6
stress
0.75
0.7
7
bam-1
2
stress
0.8
Dryf_4
amy-3
100
1 2 4 5 6 7 8 9 10 11
1 2 4 5 6 7 8 9 10 11
1 2 4 5 6 7 8 9 10 11
Fv/Fm
stress
Dryf_4
Fv/Fm
0.85
Dryf_4
5
0.85
7
6
5
4
3
2
1
0
survival rate (%)
col-0
pgm-1
col-0
Days
•It was observed significant difference between col-0 and pgm1, which
suggests pgm1 might be resistant to drought stress, but not bam1 and
amy3
0
col-0
bam1
amy3
• pgm1 showed higher survival rate compared with
col-0 and other mutants
•Also, the early stress response was observed on day 5 after drought
stress initiation
•Dryf_4 detect early stress response and also allows to discriminate
between drought stress resistant and drought stress sensitive
bam1 amy3
ii) Images of col-0 and pgm1
plants on day 14 after
i) Images of drought stress col-0 rewatering; pgm1 has higher
and pgm1 plants on day 13:
survival rate than col-0
Visual Images of watered and non-watered col-0, bam1 and amy3
plants during progressive drought stress treatment:
non-watered
watered
col-0
Day 1
Day 6
Day 9
Day 11
Day 12
col-0
pgm-1
col-0
pgm-1
Conclusion:
• Under progressive drought stress condition, pgm1 mutant showed higher survival rate than bam1 and amy3 and this
seems to correlate with fluorescence kinetics data.
•This results suggests pgm1 mutant has enhanced capacity to withstand water deprivation than other starch mutants.
Reference:
Zeeman S.C, Smith S.M, Smith A.M (2007): The diurnal metabolism of leaf
starch. The Biochemical Journal 401,13–28
pgm1
Contact : sharmilamadhavan712@gmail.com
Acknowledgement: I would like to acknowledge HARVEST(research programme of
European union) for the financial support for our research