Cysteine is playing role in nutrient remobilization during salt stress, extended darkness stress and drought stress. Nitrogen and Sulfur are remobilised from cysteine during stress or senescence to provide energy.

1. Cysteine degradation in nutrient
turnover and energy metabolism
Supervisors:
Prof. Dr. Hans - Peter Braun
Prof. Dr. Traud Winkelmann
Dr. Tatjana Hildebrandt
Dhriti Satya
Leibniz University Hannover, Germany
29 June 2017
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2. Proteins
Amino
acids
CO2
NH4
+
H2S
Protein
biosynthesis
Amino acid
degradation
Amino acid
biosynthesis
Protein
degradation
Introduction: Role of amino acid in nutrient turnover and
energy metabolism
Amino-acids serves the central role for plants to use nutrients efficiently
General overview of amino acid metabolism in cells of germinating plant and Senescent
or stressed cell
Hildebrandt et al. (2015)
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3. Macromolecules are constantly turned-over to adjust cellular changes in
response to stress adaptation and senescence via autophagy process. Thus
controlling Nutrient remobilization.
Introduction: Mechanisms involved in protein degradation
during senescence or stress
Trends in Plant Science 2011 16, modified
3

4. Introduction: Relationship between cysteine degradation
and energy metabolism
• Amino acids (highlighted in purple) are degraded to precursors or intermediates of
the TCA cycle
Hildebrandt et al. (2015)
 Subcellular Localization and Energy
Yield of Amino Acid Catabolic Pathways;
 Most reaction steps of the catabolic
pathways occur either in the
mitochondria or cytosol
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5. Cysteine
Sulfur
assimilation
Nitrogen
assimilation
Carbon
fixation
Reflects the functional role of Cysteine
Introduction: Why I’m interested in Cysteine amino acid?
Hildebrandt et al. (2015)
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6. ethe1-1
Plants grown under short-day conditions for 100 days
ETHE1 deficient plant show an early
senescence phenotype under light-limited
conditions (sd;8 h of light/16 h of dark)
Krüßel et. al. 2014
→ defect in nutrient remobilization?
Introduction: Cysteine degradation pathway and
Introduction to knock-down mutant
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Cysteine degradation pathway. OAS-TL (thiol lyase isoform), GSH Glutathione, GSSH Glutathione persulfide
GSSH Sulfite
O2 GSH
Thiosulfate3-mercaptopyruvate
GSSH GSHGSH pyruvate
L-cysteine
2-oxoglutarate glutamate
ETHE1 Str1Str1Atr1
H2S
OAS-TL
Wt
Mitochondria
ethe1-1
T-DNA
insertion
line

7. Introduction: Significance of ETHE1 during abiotic stress
Abiotic stress datasets
Microarray Datasets for each Stress conditions (Arabidopsis thaliana)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
drought salt darkness cold heat
ETHE1expression
[log2foldchangestress/control]
Expression of the ETHE1 gene under abiotic stress conditions
ETHE1 is strongly induced by Drought, Salt stress and Extended Darkness
(= conditions with increased protein degradation)
→ role in nutrient remobilization?
Gene investigator
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8. Test the physiological role of cysteine degradation in nutrient turnover.
Approach: Comparison of wild type Col0 ecotype with ETHE1 knockdown mutant ethe1-1
a) Under various abiotic stress by analyzing different stress parameters.
b) Under in-vitro conditions of high versus low nitrogen and sulfur concentrations by
analyzing phenotype and leaf composition.
Objectives:
Hypothesis:
CysteineN, S, C
Cysteine degradation
The cysteine degradation pathway is essential for coordinating carbon, nitrogen, and
sulfur turnover.
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9. long-day
conditions (ld;
16 h of light/8
h of dark
3 weeks old plants
55= Wild type
Col-0 ecotype
55= Mutant type
ethe1-1
Wild Type
ethe1-1
* For size match
ethe1-1 were
sown 3 days later
then wild type
7 days
2 weeks old plants
 3 replicates of
each treatment.
55 wild type
55 ethe1-1
X 3
XDarkness
7 days
XDrought
12 days
Experimental set-up:
Long-day
conditions (ld;
16 h of light/8
h of dark
Heat
(37°C)
7 days
Salt Stress
(100mM, 20ml)
5th week4th week
Wild Type
ethe1-1
Wild Type
ethe1-1
Wild Type
ethe1-1
Wild Type
ethe1-1
Arabidopsis thaliana plants
Before stress During stress During recovery
7 days
Control
No stress
9
1hr

10. Parameters were measured:
Phenotype
CorelDraw-X7 was
used for pictures
illustration
Pictures were clicked
using 14MP camera
of Smart phone
Area measurement
(Top-view)
ImageJ Software
Fresh weight
measured
Collected in falcon tube and
kept in liquid nitrogen
Rosette was harvested
Pulverized
Protein analysis
Chlorophyll analysis
Materials and Methods:
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11. Chlorophyll Measurement
Protein Measurement
Bradford protein assay was used
Weighing of pulverized
material
Performed on liquid
nitrogen
(Pre-cooled eppendorf)
~ 20mg for Chlorophyll
test
~ 50mg for Protein test
Materials and Methods:
90%, 80%, 50% ethanol
80°C
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12. In-vitro system
Materials and Methods:
Plating of seeds on pre-prepared and
overnight cooled full nutrient media
Seed grown vertically in phytochamber
until the seedlings are ~8mm long
Seedlings are transferred to the square
plates with different salt concentration
Grown vertically in phytochamber until
‘Control’ has grown upto full length
1.
2.
3. 4.
12Growing conditions: Long-day (ld; 16 h of light/8 h of dark)

13. Weeks after planting
2 3 4 5
Freshweight(mg)
0
500
1000
1500
2000
Wild type
ethe1-1
Stress treatments
Control Salt Darkness Drought
Growthrate(mg/week)
0
50
100
150
Wild type
ethe1-1
Results and Discussion: Growth Rate
13
2 weeks 3 weeks 4 weeks 5 weeks
Variation during recovery
Stress treatments
Control Heat Salt Darkness Drought
Growthrate(mg/week)
0
50
100
150
Wild type
ethe1-1
Variation during stressGrowth rate of control under normal conditions
WT WT WT WTethe1-1 ethe1-1 ethe1-1 ethe1-1
Significant (p<0.05) Stress with respect to control, Vertical bars indicate +S.E.
(0.042)
(0.0017)
(0.0395)
(0.044)
(0.0029)
(0.047)
(0.0005)
(0.008)
(0.000)
(0.0001)

14. Phenotype analysis of 4 weeks old Arabidopsis thaliana plants
Wild type
(Col-0)
Mutant
ethe1-1
Results: Phenotype analysis during stress
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Control
(No stress)
Darkness
Stress
Heat
Stress
Salt
Stress
Drought
Stress
Drought, Salt and darkness treated plants were highly under stress
Heat treated plant appears similar to Control
Nutrients are remobilized in drought, salt and darkness stressed plants for their survival

15. Salt concentration
50mM 100mM
Inhibition%
0
10
20
30
Wild type
ethe1-1
Salt concentration
0mM 50mM 100mM
Rootlength(mm)
0
20
40
60
80
Wild type
ethe1-1
Results: In-vitro analysis of salt stress
Phenotype analysis
15
ethe1-1 ethe1-1 ethe1-1
WT WT WT
Control 50mM NaCl 100mM NaCl
(0.0098)
Significant (p<0.05)
ethe1-1 with respect to wild type
Stress with respect to control
Vertical bars indicate +S.E. and n=3
(0.041317)
(0.018254)
Root length variation
Root toxicity test
(0.0026)
(0.008463)
(0.021663)
No significant difference in wild type and ethe1-1

16. Stress treatments
Control Salt Darkness Drought
Rosettearea(cm2)
0
10
20
30
Wild type
ethe1-1
Stress treatments
Control Salt Darkness Drought
µg/mgFW
0
1
2
3
4
5
Wild type
ethe1-1
Results: Parameters during stress
Area of rosette during stress Protein concentration in rosette
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(0.0035)
(0.0003)
(0.0135)(0.0056)
(0.0149)
Significant (p<0.05) Stress with respect to control, Vertical bars indicate +S.E. and n=3
Significant decrease in area of rosette during various stress
Significant decrease in protein concentration of ethe1-1 during Salt and Darkness stress
with respect to Control (no stress)
No significant difference between wild type and ethe1-1 during various stress in 4
weeks old plants
(0.0412)
(0.028)

17. Stress treatments
Control Salt Darkness Drought
g/mgFW
0
1
2
3
4
Wild type
ethe1-1
Stress treatments
Control Salt Darkness Drought
g/mgFW
0
1
2
3
4
Wild type
ethe1-1
Results: Parameters during stress
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Total chlorophyll (a+b) during stress Chlorophyll a/b ratio during stress
Wild type ethe1-1
Darkness
(Stress)
Control
(No stress)
Significant (p<0.05) Stress with respect to control
Vertical bars indicate +S.E. and n=3
Significant decrease of total chl in darkness
Both Chl a and b changed in same proportion
No significant difference in wild type and ethe1-1
(0.0035)
(0.0029)

18. Phenotype analysis of 5 weeks old Arabidopsis thaliana plants
Wild type
(Col-0)
Mutant
ethe1-1
Results: Phenotype analysis during recovery
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Control
(No stress)
Darkness
Stress
Salt
Stress
Drought
Stress
Drought, Salt and darkness treated plants were recovering
Biosynthesis of proteins and amino acids occurs

19. Results: Parameters During Recovery
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Area of rosette during recovery Protein in leaf rosette during recovery
Stress treatments
Control Salt Darkness Drought
Rosettearea(cm2)
0
20
40
60 Wild type
ethe1-1
Stress treatments
Control Salt Darkness Drought
µg/mgFW
0
1
2
3
4
5
Wild type
ethe1-1
Significant (p<0.05) Stress with respect to control, Vertical bars indicate +S.E. and n=3
Significant decrease in area of rosette during various stress
No significant variation in protein concentration
No significant difference in four weeks old wild type and ethe1-1 during various stress
(0.0035)
(0.0036)
(0.024)
(0.0149)

20. Stress treatments
Control Salt Darkness Drought
g/mgFW
0
1
2
3
4
Wild type
ethe1-1
Results: Parameters During Recovery
20
Stress treatments
Control Salt Darkness Drought
g/mgFW
0
1
2
3
4
Wild type
ethe1-1
Total chlorophyll (a+b) during recovery Chlorophyll a/b ratio during recovery
Significant (p<0.05) Stress with respect to control, Vertical bars indicate +S.E. and n=3
(0.047)
(0.0023)
Significantly less recovery of total chlorophyll during darkness stress
Both Chlorophyll a and b are recovering in same proportion
No significant difference between wild type and ethe1-1

21. Stress Growth Chlorophyll a+b Protein Comments
Salt Protein degradation
Darkness Protein degradation
Drought
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Recovery Growth Chlorophyll a+b Protein Comments
Salt Recovered
Darkness Not much recovery
Drought Recovered
Wild type
ethe1-1
w.r.t. Control
Summary:
 No variation between 4 weeks old wild type and ethe1-1 during various stress treatments
and recovery.
Conclusion:
During stress nutrients are remobilized in same proportion in ethe1-1 and wild type
During these conditions other pathways are degrading cysteine.

22. Thank you for your attention
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