This study compared the biochemical and physiological responses of six maize genotypes under waterlogging stress conditions. The genotypes differed in their canopy cover, chlorophyll content, membrane damage, and antioxidant enzyme activity when exposed to waterlogging over six days. CML 54 x CML 487, BIL 219 and CML 487 showed the best performance under stress, with higher antioxidant enzyme activities and less membrane damage and chlorophyll loss. CML 54 and CML 486 were the most susceptible. The tolerant genotypes will be targets for future breeding programs to develop waterlogging tolerance in maize.

1. Comparative Analysis of Biochemical
& Physiological Responses of Maize
Genotypes under Waterlogging Stress
Tahmina Akter
Khulna University
Bangladesh

2. Outline
 Introduction
 Objectives
 Materials and Methods
 Result and Discussion
 Conclusion
 Prospective
 Acknowledgement
2

3. 3
Introduction
Figure: Total maize production and area in Bangladesh
Maize is the third most important cereal crop in
Bangladesh as well as most of the countries of Asia
BBS, 2018; PP: 91-96
0.8 0.8 1.0
2.3 2.4
3.0
2014-2015 2015-2016 2016-2017
Total Maize area and production in Bangladesh
Area (acres)(Million) Production (M. Ton)(Million)

4. 4
Why waterlogging stress?
 Climate change, high sea level rise, off-season
rainfall
 In Bangladesh, >10% of the total maize
growing area is affected
 18% of the total maize production area in
South and Southeast Asia causing production
losses of 25–30% annually
Introduction
Uddin MS, 2014; Zaidi et al. 2010

5. Background
5
Abiotic
stresses
Oxidation and
degradation of cellular
components
 Membrane damage
 Loss of organelle
function
 Reduction of
metabolic efficiency
 Reduced carbon
fixation
 Reduced growth
 Yield loss
Functional
and
structural
damage
 Lipids and
fatty acids
 Nucleic
acids
 Proteins
 Pigments
 DNA
CELL DEATH
ROS and toxins in cells
 O2•- (Super oxide)
 H2O2 (Hydrogen peroxide)
 OH• (Hydroxyl Radicle)
 LPO (Lipid per oxidation)
 MG (Methylglyoxal)

6. Objective
i. To demonstrate the role of anti-oxidative
enzymes in scavenging ROS elements
i. To compare the physiological and biochemical
changes of maize genotypes
ii. To screen the waterlogging tolerant and sensitive
maize genotypes in terms of physiological and
biochemical changes
6

7. 7

8. Methodology
8
Seed sowing
Seed
germination
Transplant in
hydroponic
condition
Hardening
Waterlogging
stress induction
Physiological
tests
Biochemical
tests
Plasma
membrane
integrity
Canopy
cover
Chlorophyll
content
Activity of anti-
oxidative enzyme
(CAT, POD, APX, GPX)
Lipid
Peroxidation
Leaf extract and
Spectrophotometer
SPAD
meter
Crop sensor
Evan’s
blue
Schiff’s
reagent

9. 9
 Plant Materials (Maize Genotypes)
 CML 54 × CML 487
 BIL 219
 CML 54
 CML486 × CML 487
 CML 486
 CML 487
Materials and Methods

10. 10
Materials and Methods
 Seedlings Treatments
 Control (Hoagland solution + Oxygen
supply)
 Waterlogging stress (Hoagland solution
only)

11. Materials and Methods
11
 Determination of Chlorophyll Content using leaf extract in
spectrophotometer (Lichtenthaler, 1987)
 Determination of chlorophyll using SPAD meter (Soil Plant
Analysis Development) (Rambo et al., 2010)
 Determination of canopy cover using Green Seeker Hand
Held Optical Sensor (Tremblay et al., 2009)
 Histochemical Detection of Membrane Damage (Chen et al.,
2010)
 Measurement of Lipid Peroxidation (Heath and Packer, 1968)

12. Materials and Methods
12
 Determination of protein (Bradford, 1976)
 Native PAGE and activity staining (Rohman et al., 2016)
 Statistical analysis (STATISTIX 10)
Enzymatic Antioxidants:
 Peroxide Dismutase (POD): Hemeda and Klein, 1990
 Catalase (CAT): Csiszar et al., 2007
 Ascorbate peroxidase (APX): Nakano and Asada,1981
 Glutathione peroxidase (GPX): Elia et al., 2003

13. 13

14. Effect of Waterlogging Stress on Canopy Cover
Canopy Cover
Genotypes Day 0 Day 2 Day 4 Day 6
CML 54×CML487 0.53±0.00a 0.49±0.01ab 0.44±0.00a-d 0.39±0.03b-e
BIL 219 0.40±0.06b-e 0.37±0.04c-f 0.33±0.02e-h 0.29±0.01gh
CML54 0.37±0.02c-f 0.31±0.02e-h 0.27±0.01e-h 0.19±0.01h
CML486×CML487 0.39±0.01b-e 0.37±0.02c-f
0.33±0.01d-h 0.27±0.01f-h
CML 486 0.46±0.02a-c 0.34±0.02d-g 0.31±0.02
e-h 0.24±0.01gh
CML 487 0.39±0.01ab 0.36±0.03c-f 0.34±0.03e-h 0.33±0.00gh
14

15. Effect of Waterlogging Stress on Chlorophyll Content
(SPAD)
SPAD (Soil Plant Analysis Development)
Day 0 Day 2 Day 4 Day 6
CML 54×CML487 36.55±0.58b-e 34.15±0.31d-g 29.98±0.20f-i 26.08±0.98hi
BIL 219 34.18±0.71a 33.53±0.59ab 31.33±0.37bc 27.53±0.25e-h
CML54 32.60±0.46ab 26.69± 0.20b-d 23.85±0.25c-g 18.30±0.11f-i
CML486×CML487 36.47±0.07ab 27.45±0.78e-h 25.30±0.20g-i 21.88±0.91i
CML 486 32.10±0.12b-e 27.75±0.43e-h 22.43±0.36e-h 18.03±0.95hi
CML 487 31.10±0.84b-f 29.43±0.51e-h 26.98±0.13g-i 24.83±0.72j
15

16. Effect of Waterlogging Stress on Total Chlorophyll and
Carotenoid Content (Leaf Extract)
Total Chlorophyll
Day 0 Day 2 Day 4 Day 6
CML 54 ×CML487 0.49±0.07a-c 0.43±0.06bc 0.39±0.04b-d 0.36±0.04c
BIL 219 0.67±0.08a-d 0.62±0.02a-e 0.59±0.07cd 0.57±0.009b-d
CML 54 0.46±0.06a-c 0.32±0.04a-d 0.28±0.07c 0.16±0.02d
CML486×CML487 0.85±0.08ab 0.70±0.07a-c 0.56±0.02bc 0.48±0.03a-d
CML 486 0.63±0.04a-d 0.51±0.01ab 0.44±0.05a-c 0.32±0.06cd
CML 487 0.76±0.07a 0.67±0.06d 0.60±0.06b-d 0.52±0.01a-d
16
Carotenoid
Day 0 Day 2 Day 4 Day 6
CML 54 ×CML487 1.37±0.05d-f 1.30±0.04bc 1.18±0.00a-e 1.03±0.01bc
BIL 219 1.56±0.03a-c 1.35±0.06b-d 1.27±0.03de 1.13±0.08ab
CML 54 1.70±0.03c-e 1.33±0.05a-d 1.15±0.01ab 0.98±0.01c-f
CML486×CML487 1.58±0.00bd 1.25±0.08c-f 0.95±0.03a-c 0.81±0.05ab
CML 486 1.27±0.01a-e 1.09±0.03de 0.86±0.01ef 0.67±0.04d
CML 487 1.20±0.01cd 1.09±0.05b 0.96±0.03a-c 0.88±0.06f

17. Histochemical Staining of Roots
17
Figure: Lipid peroxidation test using Schiff’s reagent
Figure: Plasma membrane integrity test using Evan’s blue

18. Waterlogging Stress Effect on POD Enzyme Activity
18
Bars with the same letters are not significantly different at P≤0.05
Day 0 Day 2 Day4 Day 6 Day 0 Day 2 Day4 Day 6
CML 54 × CML 487 BIL 219
POD 1
POD 2
POD 3
Day 0 Day 2 Day4 Day 6 Day 0 Day 2 Day4 Day 6
CML 54 CML 486×CML 487
POD 1
POD 2
POD 3
Day0 Day2 Day4 Day6 Day0 Day2 Day4 Day6
CML 486 CML 487
POD 1
POD 2
POD 3
ef c-f
a-c
f
ef
d-f
a-c
a-d
a
a-c a-d
b-f
a-c a-c
a-e a-d
b-f
b-f
a-c
0.0
0.2
0.4
0.6
0.8
1.0
1.2
CML 54 × CML487 BIL 219 CML54 CML486 × CML487 CML 486 CML 487
POD(nmmin-1mg-1protein)
Day 0 Day 2 Day 4 Day 6
a-c
ab
c-f
a-d
b-f

19. Effect of Waterlogging Stress on CAT Enzyme Activity
19
Day 0 Day2 Day4 Day 6 Day 0 Day 2 Day4 Day 6
CML 54 × CML 487 BIL 219
CAT 1
CAT 2
Day0 Day2 Day4 Day6 Day0 Day2 Day4 Day6
CML 54 CML 486×CML 487
CAT 1
CAT 2
Bars with the same letters are not significantly different at P≤0.05
Day0 Day2 Day4 Day 6 Day0 Day2 Day4 Day6
CML 486 CML 487
CAT 1
CAT 2
a-h a-h
c-h
d-h gh
b-h
a-e
a-c
c-h
a-h f-h e-h
a-g
ab
a-f
h
a-h
a
a-d
a
a-e
a-e a
a-c
0
10
20
30
40
50
60
CML 54 × CML487 BIL 219 CML54 CML486 × CML487 CML 486 CML 487
CAT(nmm-1mg-1protein)
Day 0 Day 2 Day 4 Day 6

20. Effect of Waterlogging Stress on APX Enzyme Activity
20
Bars with the same letters are not significantly different at P≤0.05
c
a-c
bc
bc
a-c
a-c
bc
a-c
a-c
a-c
a-c ab
bc
a-c
a-c
a
a-c
a-c
bc
a-c
c
a-c
a-c
a-c
0.0
0.5
1.0
1.5
2.0
CML 54 × CML487 BIL 219 CML54 CML486 × CML487 CML 486 CML 487
APX(nmmin-1mg-1Protein)
Day 0 Day 2 Day 4 Day 6
Day0 Day2 Day4 Day6 Day0 Day2 Day4 Day6
CML54 CML 486×CML 487
APX 1
APX 2
APX 3
APX 4
Day0 Day2 Day4 Day6 Day0 Day2 Day4 Day6
CML 54 × CML 487 BIL 219
APX 1
APX 2
APX 3
APX 4
Day 0 Day 2 Day4 Day 6 Day0 Day 2 Day4 Day6
CML 486 CML 487
APX 1
APX 4
APX 2
APX 3

21. Effect of Waterlogging Stress on GPX Enzyme Activity
21Bars with the same letters are not significantly different at P≤0.05
Day 0 Day 2 Day4 Day 6 Day 0 Day 2 Day4 Day 6
GPX 1
GPX 2
BIL 219CML 54× CML 487
GPX 3
Day 0 Day 2 Day4 Day6 Day0 Day2 Day4 Day6
CML 54
GPX 1
GPX 2
CML 486×CML 487
GPX 3
Day0 Day2 Day4 Day6 Day0 Day2 Day4 Day6
GPX 1
GPX 2
GPX 3
CML 486 CML 487
a-d
ab
de
c-e
b-e
a-e
a
ab
e
b-e
a-e
a-e
a
a
b-e b-e b-e
a
a a
a
b-e a-c
a
0
20
40
60
80
100
120
140
160
180
CML 54 × CML487 BIL 219 CML54 CML486 × CML487 CML 486 CML 487
GPX(nmmin-1mg-1protein)
Day 0 Day 2 Day 4 Day 6

22. 22
More intensive pink red and blue color indicates
more lipid peroxidation and cell membrane damage
 SPAD value and leaf extract measurement of
chlorophyll content showed the similar results
 Reduced canopy cover indicates N2 deficiency
suggesting reduced chlorophyll content

23. 23
 Increased anti-oxidative enzyme activities
suggests the better ROS scavenging activity
CML 54 × CML 487, BIL 219 and CML 487
showed the best performance under waterlogging
stress condition
 CML 54 and CML 486 were found to be
susceptible genotypes

24. 24
CML 54 × CML 487, BIL 219 and CML 487 are
going to be the target of researchers for future
breeding programs
There was irregular up and down-regulation of
some anti-oxidative enzyme activities. Advanced
study is needed to identify the cause of this
unusual activity of the anti-oxidative enzymes

25. 25
 Biotechnology & Genetic Engineering Discipline,
Khulna University, Bangladesh.
 Molecular Breeding Laboratory, Bangladesh
Agricultural Research Institute (BARI).
 Organizing Committee of 13th Asian Maize
Conference.

26. 26

27. 27
Figure: Maize seedlings in controlled condition
(Hoagland solution + O2 supply)

28. 28
Figure: Maize seedlings under waterlogging stress
condition (Deprived of O2 supply)