It is the study of  “Proteome”. The word "proteome" is a blend of "protein" and "genome”. Large scale study of Proteins. Particularly their structures and functions. Study of full set of proteins in a cell type or tissue, and changes during various conditions

1. 1
SPEAKER
ZINZALA VIVEK N.
Ph.D. (Crop Physiology)
Dept. of GPB, N.M.C.A. ,NAU, Navsari

2. SEMINAR CONTANT
Introduction
Function of Protein
What is proteomics
Why proteomics
Case study
Conclusion
2

3. PROTEOMICS
• It is the study of “Proteome”.
• The word "proteome" is a blend of "protein" and "genome”.
• Large scale study of Proteins.
• Particularly their structures and functions.
• Study of full set of proteins in a cell type or tissue, and changes during various conditions
Involves
 protein-protein interactions
 organelle composition
 protein activity patterns and
 protein profiles.
3

4. Functions of protein
Function Description
Membrane transport
Membrane proteins are used for facilitated diffusion and active transport, and also for
electron transport during cell respiration and photosynthesis.
Hormones Play vital role for growth and development of plant.
Receptors
Binding sites in membranes and cytoplasm for hormones, neurotransmitters, tastes and
smells, and also receptors for light in the eye and in plants.
Packing of DNA
Histones are associated with DNA in eukaryotes and help chromosomes to condense
during mitosis.
4

5. The Virtue of the Proteome
 Proteome = protein compliment of the genome
 Proteomics = study of the proteome
 Protein world = study of less abundant proteins
 Transcriptomics = often insufficient to study functional aspects of genomics
5

6. What is the Proteome?
DNA
RNA
Protein
Modified Proteins/
Functional Protein
Transcription
The collection of proteins in a cell
Translation
Post-translational
modification (PTM)
6

7. Proteomics
Proteomics is an attempt to describe biological state and qualitative
and quantitative changes of protein content of cells and extracellular
biological materials under different conditions to further understand
biological processes.
A large-scale characterization and functional analysis of
the proteins expressed by a genome
The term proteomics was first coined by Marc Wilkins in
1994 to make an analogy with genomics.
7

8. Types of Proteomics
8
3. Functional Proteomics
2. Structural Proteomics
1. Expression Proteomics

9. Why Proteomics?
• Whole Genome Sequence –complete, but it does not show how proteins function or
biological processes occur.
• Post-translational modification –proteins sometimes chemically modified or regulated
after synthesis.
• Protein – protein interactions.
• Proteins fold into specific 3-D structures which determine function.
• Gain insight into alternative splicing
9

10. Why Proteomics?
• Several levels of regulation from gene to function
• Proteins are the ultimate operating molecules producing the physiological effect
10

11. 11

12. Applications of Proteomics
Proteomics
Structural
Proteomics
Proteome
Mining
Post-
translational
Modifications
Protein
Expression
Profiling
Functional
Proteomics
Protein-
protein
Interactions
Glycosylation
Phosphorylation
Proteolysis
Yeast two-hybrid
Co-precipitation
Phage Display
Drug Discovery
Target ID
Differential Display
Yeast Genomics
Affinity Purified
Protein Complexes
Mouse Knockouts
Medical
Microbiology
Signal
Transduction
Disease
Mechanisms
Organelle
Composition
Subproteome
Isolation
Protein
Complexes 12

13. How to do Proteomics
• Two-dimensional electrophoresis
• IEF strip separation
• SDS-PAGE gel separation
• Mass Spectrometry
• Protein sequencing
• Peptide mapping
• Post-translational Modification
• Others
• Dual channel imaging
• Micro array analysis
Bidle et al., 2008 13

14. 14
Protein Mixture
Separation Individual Proteins
Fig. 1. Basic Proteomic Analysis Scheme
MALDI-TOF
Mass Spectroscopy
Protein Identification
Plant

15. Fig. 2. Plant Protein Extraction and Fractionation
15

16. Fig. 3. Two-dimensional Gel Electrophoresis
First dimension: IEF (based on isoelectric point)
SDS-PAGE
(basedonmolecularweight)
+ -
acidic basic
High
MW
Low
MW
Sample
Hahne et al., 2010 16

17. Components of a Mass Spectrometer
1. Source – produces gas-phase ions from the sample
2. Mass analyzer – resolves ions based on their m/z ratio
3. Detector – detects ions resolved by the mass analyzer
Sample
Input Ionization
Source
Mass
Analyzer
Detector
m/z
Intensity
Size of arrows indicates the size of the ions, not the flight order or order of detection 17

19. Fig. 4. Overall Effect of Abiotic stress to Plant
Air pollution
Salinity stress
Drought stress
Temperature
stress
Light stress
Mechanical
damage
Cold stress
19
 Chlorophyll loss,
 Membrane leakage,
 Na+ accumulation and K+
reduction
 ROS (H2O2) accumulation,
 Lipid peroxidation
 Oxidative stress defense
 Metabolisms
Late embryogenesis abundant
(LEA) proteins
Ion transporters
Osmotin
Metabolic processes
Redox regulation
Heat shock protein 1 (HSP1)
Effect of plant Plant response

21. CS-1: Effects of NaCl on protein profiles of
tetraploid and hexaploid wheat species and
their diploid wild progenitors
• Wheat cultivar: Triticum monococcum, Aegilops speltoides, Aegilops tauschii, Triticum durum and Triticum aestivum.
• Wheat seedlings were placed into plastic cups (200 ml)
• Including Hewitt solution without salt and
with salt 100 mmol/l NaCl.
• All solutions were renewed every two days.
• Control and NaCl treated seedlings were grown in a controlled growth during 15 days.
• At the end of this period, the first leaves of control and NaCl-treated seedlings were sampled for electrophoresis analysis.
• Increase in the amount of the proteins may lead to an increase in the tolerance mechanisms towards NaCl salinity of wheat species
Yildiz and Terzi (2008)Afyonkarahisar, Turkey
21

22. Fig. 5. 2D SDS PAGE profiles of the soluble leaf proteins extracted from control (C) and salt (S)-treated seedlings of
Triticum monococcum, Aegilops speltoides, Aegilops tauschii, Triticum durum and Triticum aestivum.
Increased, decreased and completely lost proteins in S
compared to C treatment are indicated by arrow in both
control and salt treatments 22

23. Protein
number
Mr pI
Cultivated wheat species Wild wheat species (progenitors)
Triticum aestivum
(AABBDD)
Triticum durum
(AABB)
Triticum monococcum
(AA)
Aegilops speltoides
(BB)
Aegilops tauschii
(DD)
1 20.3 5.1 -* CL*
2 20.7 5.1 - CL
3 20.7 6.8** - +* - +
4 21.0 5.1 + - CL
5 21.0 6.6 - -
6 21.0 6.8 +
7 21.5 6.6 -
8 21.7 6.7 -
9 21.7 6.9 CL
10 22.0 6.3 + +
11 22.0 6.6 -
12 22.5 6.2 +
13 22.5 6.8 + -
14 22.7 7.1 +
15 22.9 7.0 -
16 23.0 5.4 +
17 23.0 5.5 +
18 23.0 6.7 - +
Table 1. Protein polymorphism in the leaf tissues of cultivated hexaploid and tetraploid wheat species and their diploid wild
progenitors in 100 mmol/l NaCl treatment compared to the control treatment
*CL = completely lost protein; − = protein decreased in amount; + = protein increased in amount **bold numbers in Mr and pI column indicate common proteins between at least two species 23
Cont..

24. Protein
number
Mr pI
Cultivated wheat species Wild wheat species (progenitors)
Triticum aestivum
(AABBDD)
Triticum durum
(AABB)
Triticum monococcum
(AA)
Aegilops speltoides
(BB)
Aegilops tauschii
(DD)
19 23.2 6.5 - - -
20 23.2 7.1 + + - -
21 23.3 6.6 -
22 23.5 6.3 -
23 23.5 6.5 + -
24 23.5 6.7 + -
25 23.5 6.8 + + -
26 23.8 6.3 -
27 23.9 6.2 -
28 24.5 6.3 -
29 29.5 6.2 +
30 30.3 6.2 + -
31 30.4 6.3 +
32 30.6 6.3 +
33 33.4 6.7 +
34 34.8 7.5 + -
35 35.3 7.8 + - +
36 35.4 6.6 +
37 35.4 7.5 + -
*CL = completely lost protein; − = protein decreased in amount; + = protein increased in amount **bold numbers in Mr and pI column indicate common proteins between at least two species
24
Cont..

25. CS-2: Proteomics analysis of salt-induced leaf
proteins in two rice germplasms with different
salt sensitivity
• Seeds of the cultivars Dongjin, and a weedy rice, Dalseongaengmi-44,
were grown hydroponically in a controlled environment growth chamber
• Two week old seedlings were treated with nutrient solution (Sohn et al.
2005) containing 0, 45 and 90 mM NaCl for 4 d.
• The rice leaf samples were then collected and immediately frozen in
liquid nitrogen, after which they were stored at -800C until protein
extraction.
Lee et al. (2011)Jinju, Korea 25

26. Fig. 6. 2-DGE analysis (PEG-fractionated supernatant samples) of leaf proteins in salt-stressed rice
cultivars (Dongjin and Dalseongaengmi-44).
 A total of 100 mg proteins were separated by 2-DGE, and then visualized using silver stain.
 Arrows and indicate proteins that were up-regulated in response to salt stress.
26
control
control
control
control
90 mM NaCl
90 mM NaCl
90 mM NaCl
90 mM NaCl
Protein no.= 2 Class III peroxidase 29 precursor
3 = Beta-1,3-glucanase precursor
4 = Beta-1,3-glucanase precursor
5 = ATP synthase beta subunit
8 = OSJNBa0086A10.7 (putative transcription factor X2)
16 = Rubisco activase small isoform precursor
20 = Drought-induced S-like ribonuclease

27. Fig. 7. Enlarged views of the up-regulated proteins identified from the PEG-
fractionated pellet samples of the rice leaf tissues.
27
Dongjin
DongjinDalseongaengmi-44
Dalseongaengmi-44

28. Table 2. Abundance ratio of the up-regulated proteins in the leaf tissues of a Dongjin and
Dalseongaengmi-44 rice cultivar after 96 h of salt stress treatment
Spot no.
Abundance ratioz
45 mM 90 mM
1 2.57±0.01y 3.27±0.25
3 2.76±0.91 3.77±1.31
4 3.52±0.21 6.22±0.66
6 1.42±0.15 4.44±1.31
7 1.36±0.34 8.29±0.49
8 1.44±0.07 2.98±0.27
10 2.72±0.81 7.41±0.93
11 4.41±2.29 16.80±3.58
12 2.78±0.54 5.05±0.30
14 7.02±2.99 6.38±0.54
15 2.20±0.43 4.48±0.36
16 6.27±0.93 13.11±1.90
17 2.03±0.62 2.66±0.03
21 5.70±1.41 15.63±1.64
22 3.47±0.72 8.42±0.62
23 3.76±1.04 7.87±2.02
28
Spot no.
Abundance ratioz
45 mM 90 mM
2 1.90±0.74y 3.94±0.97
3 1.65±0.33 6.12±1.66
4 6.41±0.33 22.02±3.32
5 1.88±0.48 7.26±1.01
9 3.85±0.43 14.44±2.50
12 1.42±0.86 6.44±2.68
13 1.09±0.51 5.31±4.47
15 1.06±1.02 5.02±5.47
18 4.57±0.94 8.78±0.86
19 3.34±0.48 7.34±0.97
20 5.65±1.01 13.67±0.68
21 16.08±0.78 19.13±0.97
22 2.10±0.49 5.49±1.27
23 7.73±0.92 15.51±1.12
Dalseongaengmi-44Dongjin
 z = The abundance ratios of each spot were measured using a densitometer (Bio-rad) and then
compared with those of controls.
 y = Data represent the means9SE of three biological replicates.

29. Table 3. Protein spots up-regulated by salt stress in the leaves of two rice germplasms
Spot no. Dongjin dalseongaengmi-44
1 +
2 +
3 + +
4 + +
5 +
6 +
7 +
8 +
9 +
10 +
11 +
12 + +
13 +
14 +
15 + +
16 +
17 +
18 +
19 +
20 +
21 + +
22 + +
23 + +
29

30. Table 4. Salt-stress-induced up-regulated proteins identified by PMF with MALDI-TOF MSz
Spot
no.
NCBI
accession
no.
Protein name Organism
Theoretical Observed
MOWSE
score
My SCx
Mr pI Mr pI
1 ABB47307 ATP synthase F1 beta subunit (chloroplast) O. sativa 60.3 5.3 46.0 6.2 2.31+9 13 31
2 CAH69271 Class III peroxidase 29 precursor O. sativa 34.2 5.3 35.3 5.9 8.50+7 14 55
3 ADD10383 Beta-1,3-glucanase precursor O. sativa 34.8 4.7 33.5 5.6 27509 6 18
4 ADD10383 Beta-1,3-glucanase precursor O. sativa 34.8 4.7 33.2 5.7 92493 7 22
5 BAA90397 ATP synthase beta subunit O. sativa 54.0 5.4 33.0 6.0 3.70+10 15 30
6 CAG34174 Rubisco large chain O. sativa 52.8 6.2 32.9 6.6 95575 11 21
7 CAG34174 Rubisco large chain O. sativa 52.8 6.2 32.3 6.5 1.74+8 19 26
8 NP_918125 OSJNBa0086A10.7 (putative transcription factor X2) O. sativa 28.7 6.7 28.0 6.3 1708 4 18
9 AAX95072 Fructose-bisphosphate aldolase class-1 O. sativa 42.0 6.4 26.0 5.8 41360 9 24
10 AAV44199 Dehydroascorbate reductase O. sativa 23.8 5.8 24.0 6.4 12503 5 39
13 NP_917384 Putative ribosomalprotein L12 O. sativa 18.2 5.4 21.0 5.5 2846 4 40
14 CAJ01693 2-Cys peroxiredoxin O. sativa 28.1 5.7 19.2 5.8 23814 5 33
15 BAD36628 Putative chaperon 21 precursor O. sativa 23.1 5.7 18.2 5.8 3632 5 34
16 AAX95286 Rubisco activase small isoform precursor O. sativa 47.9 5.9 41.0 5.8 3.11+13 24 46
17 CAG34174 Rubisco large chain O. sativa 52.8 6.3 33.5 6.1 8.52+8 22 31
18 Q9LSU1 Proteasome subunit alpha type 5 O. sativa 26.0 4.7 29.0 5.5 1.38+7 7 50
30
zPMF = peptide mass fingerprinting;
MALDI-TOF MS = matrix-assisted laser desorption ionization-time of flight mass spectrometry;
Mr = molecular weight;
pI = isoelectric point.
y = Number of matching peptides.
x = Sequence coverage by PMF.

31. Fig. 8. MS analysis of spot 14. Protein spot 14 was excised and then digested with trypsin, after which the resulting
peptides were analyzed using the QSTAR pulsar-i MS system.
31
 (A), MS spectra. The ion (1721.9) marked with an asterisk was analyzed by MS/MS.
 (B), the MS/MS spectra of ion 1721.9.
 The b ions (b1-6), y ions (y1-10) and the corresponding peptide sequences are shown.
 The protein was identified as 2-Cys peroxiredoxin (NCBI accession number AJO1693) by a database
search

32. Table 5. Proteins up-regulated in response to salt-stress-identified by sequence tag analysis with ESI-MS/MSZ
Spot
no.
NCBI
accession no.
Protein name Organism
Theoretical Observed Theoreticaly
(%)
Sequencex
Mr pI Mr pI
11 CAG34174 Rubisco large chain O. sativa 52.8 6.2 23.0 6.0 100 GIQVER
12 AAO65861 Putative actin binding protein O. sativa 16.2 4.9 4.9 5.5 100 SLPADGCR
100 AAGEGGEAPR
100 SPAAADVR
14 CAJ01693 2-Cys peroxiredoxin O. sativa 28.1 5.7 5.7 5.5 100 STINNLAIGR
100 PLISDVTK
100 QGIALR
19 BAD33340 Putative triose phosphate isomerase, Chloroplast precursor O. sativa 32.4 7.0 7.0 5.8 100 AQEVHAAVR
91 VSPEVAGSGIR
100 IGELLEER
100 NSVTSK
20 AAL33776 Drought-induced S-like ribonuclease O. sativa 28.4 5.2 5.2 5.8 100 GVTPGVQ
100 GYPSEDFFVK
100 ENTAVVR
100 YNTAFIK
21 CAG34174 Rubisco large chain O. sativa 52.8 6.2 6.2 5.7 100 GIQVER
22 CAG34174 Rubisco large chain O. sativa 52.8 6.2 6.2 6.8 100 DILAAFR
23 CAG34174 Rubisco large chain O. sativa 52.8 6.2 6.2 5.6 100 EYETK
zESI-MS/MS = electrospray ionization-tandem mass spectrometry;
Mr = molecular weight;
pI = isoelectric point.
y = Percentage of identities for sequence tags.
x = The sequence of the matching peptide.
Increase in concentration of NaCl to increase in conc. of protein,
this upregulated proteins are gives a tolerance mechanism
against a NaCl stress and identify upregulated protein
32

33. CS-3: Differential Proteins Expressed in Rice
Leaves in Response to Salinity and
Exogenous Spermidine Treatments
• protein spots were identified in the leaves of salt-tolerant (Pokkali) and salt sensitive (KDML105) rice cultivars
• Seven-day-old seedlings with three leaves were transplanted to plastic containers holding 20 L full-strength Yoshida nutrient solution (Yoshida et al, 1976)
with pH 5.0 (50 seedlings per container).
• The solution was renewed weekly and the pH was daily adjusted to 5.0.
• Two treatment groups of 30-day old seedlings were pretreated by adding 1.0 mmol/L Spd into nutrient solution.
• After 24 h of Spd pretreatment,
 control,
 Spermidine (Spd), (1.0 mmol/L Spd)
 NaCl (150.0 mmol/L NaCl)
 NaCl+Spd treatments
• After 7 d of salt treatment, the leaves were collected, immediately frozen in liquid nitrogen and stored at -80 °C.
• The experiment was carried out in a greenhouse under natural photoperiod
Saleethong et al. (2016)Surin, Thailand 33

34. Fig. 10. Two-dimensional electrophoretic patterns of soluble proteins in rice leaf.
Protein no. = 8 Oxygen-evolving complex protein 1,
this protein are responsive to salt stress and cause changes in the activity of photosystem II (PSII) in coping
with salt stress
34
Pokkali KDML105
(A) Pokkali
(B) KDML105
obtained from
 control,
 Spermidine (Spd),
 NaCl and
 NaCl+Spd treatments

35. Table 6. Identification of differential proteins and changes in the intensity of each protein spot in the leaves of Pokkali.
Protei
n
No.
Increasing or decreasing component (Fold)
pI
Mw
(kDa)
Accession no.
Homologous
protein
Score Function
C/NaCl C/S S/S+NaCl Pr S/NaCl
1A ↑(4.03) ↓(4.58) ↑(4.67) ↓(3.96) 7.4 32
NP_00105638
9
Os05g0574400 377 Similar to malate dehydrogenase
2A - ↓(8.48) - ↓(2.96) 6.7 32
NP_00104371
7
Os01g0649100 369 Similar to malate dehydrogenase
3A ↓(2.68) ↓(3.92) - ↓(2.20) 5.4 32 ABA91631
Fructose
bisphosphate
aldolase
451 Fructose-bisphosphatealdolase activity
4A ↓(1.05) ↓(3.54) ↑(1.57) ↓(2.15) 4.9 29
NP_00104313
4
Os01g0501800 1633
Similar to photosystem II oxygenevolving
complex protein 1
5A ↑(2.00) ↓(11.78) - ↓(17.03) 5.3 24 AAB63603
Triosephosphate
isomerase
251 Triose-phosphate isomerase activity
6A ↑(2.28) - - ↓(3.34) 6.4 24 EEC78412
Hypothetical
protein OsI_18213
251 Putative uncharacterized protein
7A - ↓(3.71) - ↓(6.71) 4.8 23 BAD35228
Putative
chaperonin 21
precursor
606 Chaperonin ATPase activity
8A ↑(1.04) ↓(1.60) ↑(2.36) ↑(1.12) 5.9 21
NP_00105886
3
Os07g0141400 976
Similar to photosystem II oxygenevolving
enhancer protein 2
9A - ↓(6.06) ↑(2.80) ↓ (2.07) 6.6 18 AAA33917
Superoxide
dismutase
60
Superoxide dismutase copper
chaperone activity
35
C/NaCl = control gel was compared with the NaCl gel
C/S = the control gel was compared with the Spd gel,;
S/S+NaCl = Spd gelwas compared with Spd+NaCl gel
Pr S/NaCl = the plants were pretreated Spd, with the plants treated with NaCl
 pI, Isoelectric point; Mw, Molecular weight.

36. Table 7. Identification of differential proteins and changes in the intensity of each protein spot in the leaves
of KDML105.
Protei
n
No.
Increasing or decreasing component (Fold)
pI
Mw
(kDa)
Accession no. Homologous protein Score Function
C/NaCl C/S S/S+NaCl Pr S/NaCl
1B ↓(2.02) ↓(1.66) ↑(2.39) ↑(2.91) 7.8 34 NP_001056389 Os05g0574400 386 Similar to malate dehydrogenase
2B - ↓(2.33) ↑(1.53) ↓(1.65) 7.0 34 NP_001043717 Os01g0649100 311 Malate dehydrogenase
3B ↓(1.34) ↓(3.43) - ↓(2.73) 6.4 34 ABG66141 Malate dehydrogenase 193 Malate metabolic process
4B ↓(4.11) ↓(2.01) - ↑(1.97) 5.7 35 ABA91631 Fructose-bisphosphate aldolase 806 Fructose-bisphosphatealdolase activity
5B ↑(1.58) ↓(2.65) ↑(2.73) ↓(1.53) 5.6 27 AAB63603 Triosephosphate isomerase 727 Triose-phosphate isomerase activity
6B ↓(3.41) ↓(4.62) ↑(3.81) ↑(2.81) 6.9 27 EEC78412 Hypothetical protein OsI_18213 248 Putative uncharacterized protein
7B ↓(1.18) ↑(1.43) ↓(6.01) ↓(3.58) 4.7 19 NP_001065834 Os11g0165700 89
Jacalin-related lectin domain containing
protein
8B - ↓(1.49) - ↓(1.28) 6.5 20 2002393A
Oxygen-evolving complex
protein 1
97 Oxygen sensor activity
9B ↑(9.85) ↑(7.57) ↓(1.36) ↓(1.77) 6.8 20 AAA33917 Superoxide dismutase 55
Superoxide dismutase copper chaperone
activity
10B ↓(1.59) ↑(1.48) ↓(2.73) - 8.6 20 NP_001067074 Os12g0569500 100
Thaumatin, pathogenesis-related family
protein
11B ↑(11.94) ↑(5.92) - ↓(1.54) 7.2 17 BAC10110 Copper/zinc-superoxide 71 Antioxidant activity
12B ↑(41.74) ↑(31.19) ↓(1.18) ↓(1.58) 5.8 16 BAD09607
dismutasePutative superoxide
dismutase [Cu-Zn] 389 Antioxidant activity
36
C/NaCl = control gel wascompared with the NaCl gel
C/S = the control gel was compared with the Spd gel,;
S/S+NaCl = Spd gelwas compared with Spd+NaCl gel
Pr S/NaCl = the plants were pretreated Spd, with the plants
treated with NaCl
 pI, Isoelectric point; Mw, Molecular weight.
The photosynthetic oxygen-evolving enhancer protein 2 was detected only in Pokkali and
was up-regulated by salt-stress and further enhanced by Spd treatment. that Spd acted directly as
antioxidants and give a tolerant mechanism against NaCl stress.

37. 37

38. CS-4: Proteomic Analysis of Drought Stress-
Responsive Proteins in Rice Endosperm
Affecting Grain Quality
• IR-64 (Indica cultivar)
• Plants were grown to maturity under greenhouse conditions
• were subjected to 4 days of rapid drought stress starting 3 days before heading
(3DBH).
• Properly filled seeds for proteomic analysis were collected at maturity from both
well-watered and drought-stressed rice plants.
• Sample grains were dehusked, embryo was excised from the seed, and the
remaining endosperm was used for protein extraction.
Manila, Philippines Mushtaq et al. (2008) 38

39. Fig. 11. Effect of drought on spikelet fertility per panicle in IR-64
 Two-dimensional gel protein patterns of mature seed endosperm of IR-64 plants drought stressed for 4 days at 3 days
before heading.
 Approximately 120 μg of protein was loaded on each gel.
 Sizes of molecular markers and the pI range of the first dimension (pI 4-7) are indicated. 39
Sky blue color bar represents spikelet fertility in well watered plants and Gray
color Bar represents spikelet fertility in drought-stressed plants.
spikeletfertility

40. Fig. 12. Behavior of (A) Granule-Bound Starch Synthase (GBSS), (B) Nucleoside diphosphate
Kinase and (C) Globulin, on 2-dimensiosnal gels of grain proteome of rice cultivar IR-
64, under well-watered (WW), and drought-stressed (4DS) conditions.
40
(A) Starch is composed of two distinct polymers, amylopectin and
amylose amylose in endosperm of rice grain is synthesized by a
Granule-Bound Starch Synthase (GBSS),
 It is a product of waxy gene, has been improving the quality of rice
grains
 GBSS remained unaltered in its behavior,
 No effect on grain quality.
(B) NDP is involved in nitrogen metabolism.
 In seeds, nitrogen mainly originates from leaves and stems that
mobilize more than 65% of their nitrogen content.
 NDP is down-regulation has affected seed filling under drought stress
conditions and consequently, might have affected seed yield.
(C) Globulins are an important source of major endosperm storage
protein
 Globulin was down-regulated
 Therefore, nutritional quality of rice grains under drought stress
conditions may have been affected

41. Table 8. Abundance ratio of endosperm proteins during drought conditions.
Spot no. pI MW AB ration
1 5.89 41 0.63*
2 6.75 58 1.86*
3 6.73 43 0.31*
4 6.46 43 2.31*
5 5.81 28 0.42*
6 6.84 40 NA
7 5.52 19 4.90*
8 6.55 22 0.55*
9 5.10 36 0.27/0
10 6.40 19 0.73*
11 6.51 65 NA
12 6.80 75 1.05
13 4.17 25 1.56*
NA=data not available 41

42. Table 9. Proteins identified by MS.
Spot
no.
MP/Ca Protein name
Accession
no.
Experimental
Mw/pI
Subcellular
localization/
Probability
5 8/28 19 kDa globulin precursor CAA45400 28/5.84
Mitochondrial outer membrane /
0.850
6 6/25 Glutelin type I precursor XP_463450 40/6.84 outside / 0.820
10 3/38
Nucleoside diphosphate
kinase
XP_478187 19/6.4 Microbody peroxisome /0.640
11 15/28
Granule-bound Starch
Synthase
AAC61675 65/6.51 chloroplast stroma / 0.851
12 2/47 B1160F02.9 XP_470940.1 75/6.7 Cytoplasm/0.650
a the number of matched peptides/the percentage of sequence coverage.
42
Identified proteins such as Granule-Bound Starch Synthase (GBSS, Wx protein), which is
thought to play a very important role in starch biosynthesis and quality and is a very crucial
factor in determining rice grain quality.

43. CS-5: Comparative Analysis of Sorghum
bicolor Proteome in Response to
Drought Stress and following Recovery
• 11434, drought tolerant, and 11431, drought sensitive
• Seeds were germinated in Petri dishes for 48 hours.
• Three seedlings were planted in 11 × 11 × 11 cm3 plastic pots filled with 700g soil
• Plants were kept in a greenhouse
• the soil was left to dry until the level of 10% field capacity and left further for 7 days without any watering.
• The plants were rewatered till water drain for recovery.
• third leaves were collected,
• frozen under liquid N2, and
• stored at −80∘C.
• 24 hours after recovery, samples of the third leaves were collected from the remaining plants,
• frozen under liquid N2, and
• stored at −80∘C.
Jedmowski et al. (2014)Frankfurt, Germany 43

44. Fig. 13. RWC of # 11434, drought
tolerant, and # 11431,
drought sensitive, genotypes.
 Differentially expressed proteins on 10% SDS-PAGE following
separation on 24 cm nonlinear strips pH 3–10, scanning, and
staining with Colloidal Comassie Brilliant Blue.
 Black arrows represent proteins expressed in both genotypes.
 represent expressed proteins in # 11434, drought tolerant
genotype, and
 white arrows with round bottom represent expressed proteins in #
11431, drought sensitive, genotype. 44

45. Table 10. MALDI-TOF-MS results of picked spots from control, drought treatment and recovery of drought tolerant, #
11434, and susceptible, # 11431, genotypes and their expression values in comparison to control
Spot
ID
NCBI Acc. no. Protein name Mr/pI 𝑆∗ %𝐶 𝑀
# 11434 # 11431
D R D R
01: Metabolism
6 gi/18483235 Methionine synthase/Sorghum bicolor 84135/5.93 96 8% 8 6.7 1.6 6.6
7 gi/18483235 Methionine synthase/Sorghum bicolor 84135/5.93 102 12% 12 5.5
16 gi/226502947 S-adenosylmethionine synthase l Z. mays 42986/5.57 193 19% 10 1.7 1.5
29 gi/666089 P-(S)-hydroxymandelonitrile lyase Sorghum bicolor 41796/4.72 154 13% 8 3.6
02: Energy
1 gi/242096062
Hypothetical protein Sorbidraft of S. bicolor homologues to putative C4 phosphoenolpyruvate carboxylase/Saccharum officinarum
CAC08829.1
108988/5.89 306 29% 28 2.1 3.3 -2
2 gi/242096062 Hypothetical protein Sorbidraft of S. bicolor homologues to putative C4 phosphoenolpyruvate carboxylase/S. officinarum CAC08829.1 108988/5.89 504 30% 33 1.7
3 gi/242096062 Hypothetical protein Sorbidraft of S. bicolor homologues to putative C4 phosphoenolpyruvate carboxylase/S. officinarum CAC08829.1 108988/5.89 331 29% 30 2.3
4 gi/242096062 Hypothetical protein Sorbidraft of S. bicolor homologues to putative C4 phosphoenolpyruvate carboxylase/S. officinarum CAC08829.1 108988/5.89 354 26% 30 2.4
5 gi/242080811 Hypothetical protein Sorbidraft of S. bicolor homologues to aconitate hydratase of Oryza sativa Japonica Q6YZX6.1 108402/6.41 101 11% 13 1.8
9 gi/242051769 Hypothetical protein Sorbidraft of S. bicolor homologues to chloroplast NADP-dependent malic enzyme Z. mays NP 001105313.1 69904/6.23 181 28% 21 -2.8 -2.4
10 gi/242051769 Hypothetical protein Sorbidraft of S. bicolor homologues to chloroplast NADP-dependent malic enzyme Z. mays NP 001105313.1 69904/6.23 199 25% 18 -2.8 -2.7
11 gi/242051769 Hypothetical protein Sorbidraft of S. bicolor homologues to chloroplast NADP-dependent malic enzyme Z. mays NP 001105313.1 69904/6.23 193 26% 18 -2.8 -2.3 -2
17 gi/242059597 Hypothetical protein Sorbidraft of S. bicolor homologues to fructose-1,6-bisphosphat aldolase, cytosol Z. mays NP 001105336.1 38990/6.96 278 23% 12 1.6
45
The search carried out against the entire NCBInr database; SID: spot identification number; Mr/pI: calculated molecular weight and isoelectric point of predicted
proteins; 𝑆: Score; ∗protein scores greater than 84 are significant (𝑃 < 0.05); 𝐶%: percentage of coverage;𝑀: number of peptides matched; 𝐷: drought; 𝑅: recovery.
Cont..

46. Spot
ID
NCBI Acc. no. Protein name Mr/pI 𝑆∗ %𝐶 𝑀
# 11434 # 11431
D R D R
24 gi|195634659 Plastid fructose-1,6-bisphosphate aldolase, Z. mays 41924/7.63 395 34% 19 -2.1 -1.9 -1.8
19 gi|108705994 Plastid glyceraldehyde-3-phosphate dehydrogenase/O. sativa Japonica 34024/4.99 121 19% 11 -1.5
20 gi/255540341 Cytosolic glyceraldehyde-3-phosphate dehydrogenase/Ricinus communis 36930/7.10 157 20% 14 1.6 1.7 1.7
30 gi/30385668 Pyruvate phosphate dikinase/Sorghum bicolor 103021/5.68 94 16% 17 1.9 1.6
03: Transcription
21 gi/242085078 Hypothetical protein Sorbidraft of S. bicolor homologues to RNA-binding protein/Arabidopsis thaliana NP 172405.1 42324/8.89 438 40% 25 -2
28 gi|195637410 40S ribosomal protein S3/Z. mays 25605/9.4 117 46% 12 1.8
05: Protein synthesis
15 gi|195620072 Elongation factor alpha/Z. mays 49534/9.15 225 21% 14 8.5 5 7
04: Protein destination/storage
8 gi|162459902 Nucleoredoxin 1/Z. mays 64058/4.8 168 16% 12 4.4 4.9 4.5
12 gi/242094438 Hypothetical protein Sorbidraft of S. bicolor homologues to Heat shock protein 60 61927/5.47 148 31% 16 2.1
13 gi/242094438 Hypothetical protein Sorbidraft of S. bicolor homologues to Heat shock protein 60 61927/5.47 161 29% 14 2.2
14 gi/242094438 Hypothetical protein Sorbidraft of S. bicolor homologues to Heat shock protein 60 61927/5.47 126 19% 12 1.9 2.3
18 gi/242041951 Hypothetical protein Sorbidraft of S. bicolor homologues to pepsin/retropepsin like aspartat protease/Z. mays ACG35399.1 42799/8.96 79 14% 2
31 gi/242056107 Hypothetical protein Sorbidraft of S. bicolor homologues to mitocondrial processing peptidase/Z. mays NP 001150614.1 54054/6.24 198 28% 12 17
32 gi/145666464 Protein disulfide isomerase/Z. mays 56921/5.01 174 19% 18 2.1 2.5
05: Unclear classification
22 gi/242075782
Hypothetical protein Sorbidraft of S. bicolor homologues to ABA stress- and fruit-ripening inducible-like protein Z. mays
CAA72998.1
28466/4.92 258 65% 22 3.6 4.5 7
25 gi/242035869 Osr40Sc1 like protein 39718/6.27 66 9% 3 5.2
26 gi/242035869 Osr40Sc1 like protein 39718/6.27 201 13% 6 3.6 3.3 3
27 gi/242056773 Hypothetical protein Sorbidraft/S. bicolor 31577/6.06 205 23% 9 3.6 2.9 4.6
33 gi/242095250 Hypothetical protein Sorbidraft/S. bicolor 41333/4.91 222 37% 14 1.9 2.3
46
The drought tolerant genotype proteome analysis indicated that the combined activities of several protein groups may enable the plants to
tolerate drought stress and efficiently recover after removing the stress conditions.
Cont..

47. CS-6: Physiological and proteomic analysis of the
response to drought stress in an inbred
Korean maize line
• Inbred maize (Zea mays L.) line KS140 was subjected to drought stress by
withholding water for 10 days at the V5 or V6 leaf stage
• Plants were cultivated in a greenhouse
• Water was withheld for 10 days commencing at the V6 leaf stage.
• protein spots, and these were identified using MALDI-TOF mass spectrometry.
Kim et al. (2015)Suwon, South Korea 47

48. Fig.14. (A) Relative leaf water content, (B) leaf area, dry
matter of aerial tissue(C) and root (D) of well-watered and
drought-stressed plants at 0, 3, and 10 days after
withholding water.
Fig. 15. (A) Stomatal conductance, (B) net CO2
assimilation rate, (C) water use efficiency, and (D) leaf
chlorophyll content of well-watered and drought-stressed
maize plants at 0, 3, and 10 days after withholding water. 48

49.  Representative 2-DE gel of Korean maize inbred line
(KS140).
 (A) Well-watered, (B) Ten days drought stress.
 A total of 500 µg protein was used for each 2-DE
gel.
 Arrows are indicated by differentially expressed
protein spots.
Fig. 16. A close-up view of differentially expressed protein spots is
shown.
49
Protein= 4 = Pathogenesis-related protein 1
5 = TPA: pathogeneis protein 10
7 = Abscisic stress-ripening protein 2-like
29=Heat shock protein 1 (LOC100501536)

50. Table 11. Proteins identified by MALDI-TOF MS.
Spot No. Accession No. Putative Function Score Expect MP SC (%)
Mr(kD)
/pI
Biological
process
Organism
1 B6TA31 Fruit protein PKIWI502 325 1.1e-025 24 54 31.7/6.62 Development Zea mays
2 B6U5I1 Peptidyl-prolyl cis-trans isomerase 146 8.5e-008 22 30 46.8/4.91 Protein folding
Zea mays
3 gi|414883697 TPA: hypothetical protein ZEAMMB73_937583 128 5.4e-006 14 86 14.1/4.83
Zea mays
4 B6SXF5 Pathogenesis-related protein 1 103 0.0017 11 67 17.1/5.39 Plant defense
Zea mays
5 D4HR93 TPA: pathogeneis protein 10 110 0.00034 10 51 17.1/5.36 Plant defense
Zea mays
6 gi|514787580 Cytochrome b6-f complex iron-sulfur subunit 138 5.4e-007 14 40 24.3/8.52 Photosynthesis
Zea mays
7 B6UB73 Abscisic stress-ripening protein 2-like 63 17 7 21 11.5/9.80 Plant stress Setaria italica
8 P12653 APx1 - Cytosolic Ascorbate Peroxidase 254 1.3e-018 19 63 27.5/5.65 Plant stress Zea mays
9 B6TPH0 Glutathione S-transferase 130 3.4e-006 11 27 23.5/5.28 Plant stress Zea mays
10 gi|226533140 Lactoylglutathione lyase 177 6.7e-011 21 53 35.3/6.62 Metabolism Zea mays
11 B6T171 Hypothetical protein 166 8.5e-010 16 40 33.6/5.96 Zea mays
12 B6SSU6 Serine-glyoxylate aminotransferase (LOC100281949) 274 1.3e-020 24 51 44.4/6.72 Metabolism Zea mays
13 K7V067 Fructose-bisphosphate aldolase, cytoplasmic isozyme 1 268 5.4e-020 20 48 38.5/6.26 Metabolism Zea mays
14 B6T9J4 Isocitrate dehydrogenase (ZEAMMB73_038317) 389 4.3e-032 37 59 46.5/6.11 Metabolism Zea mays
15 B6TUD4 Aspartate aminotransferase 356 8.5e-029 28 48 50.5/8.15 Metabolism Zea mays
16 C0PD30 ATP synthase subunit gamma, chloroplastic precursor 288 5.4e-022 19 32 40.1/8.44 Photosynthesis Zea mays
17 gi|226509797 Fructose-1,6-bisphosphate aldolase 187 6.7e-012 18 52 38.4/6.37 Metabolism Zea mays
50SC, sequence coverage.Mr/pI, Theoretical molecular weight/isoelectric point. Cont…

51. Spot No.
Accession No. Putative Function Score Expect MP
SC
(%)
Mr(kD)
/pI
Biological
process
Organism
18 P25462 Uncharacterized protein LOC100274579 586 8.5e-052 31 70 42.5/5.65 Zea mays
19 gi|242083462 Glutamine synthetase 228 5.4e-016 15 28 46.3/6.42 Metabolism Zea mays
20 B6TG70 Hypothetical protein 66 9.5 6 19 72.02/8.44
Sorghum
bicolor
21 B6TG70 Mitochondrial-processing peptidase beta subunit 332 2.1e-026 33 51 58.5/5.87 Proteolysis Zea mays
22 Q6L3A1 ATP synthase subunit alpha 381 2.7e-031 35 50 55.8/5.87 Photosynthesis
Saccharum
hybrid
23 P93804 Phosphoglucomutase 301 2.7e-023 28 41 63.3/5.46
Carbohydrate
metabolism
Zea mays
24 C0P4M0 Pyridine nucleotide-disulphide oxidoreductase 95 0.01 9 29 46.6/5.60 Plant stress Zea mays
25 B6T416 Ribulose bisphosphate carboxylase/oxygenase activase 107 0.00067 9 24 48.1/6.29 Photosynthesis Zea mays
26 K7VII1 Putative actin family protein isoform 1 324 1.3e-025 21 52 41.9/5.24 Structure Zea mays
27 P15719 Malate dehydrogenase (NADP) 207 6.7e-014 23 40 47.3/6.49
Carbohydrate
metabolism
Zea mays
28 B4G072 UDP-glucosyltransferase BX9 376 8.5e-031 29 53 50.6/5.22 Metabolism Zea mays
29 C4J410 Heat shock protein 1 (LOC100501536) 319 4.3e-025 33 46 71.2/5.08 Plant stress Zea mays
51
SC, sequence coverage.Mr/pI, Theoretical molecular weight/isoelectric point.
Cont…
Drought affected the relative leaf water content, leaf area, aerial and root tissue dry matter, stomatal conductance,
net CO2 assimilation rate, and water use efficiency. Up regulated protein like two pathogen related proteins (PR)
(PR-1 and PR-10), abscisic stress-ripening protein 2-like protein and heat shock protein 1 (HSP1) shows drought
tolerant mechanism and for development of selective breeding markers for drought tolerance in maize.

52. CS-7: Physiological, Biochemical and Proteomic
Responses of Rice (Oryza sativa L.)Varieties
Godaheenati and Pokkali for Drought Stress
at the Seedling Stage
• Two traditional rice (Oryza sativa L.) varieties,
 Godaheenati (4049) and
 Pokkali
• were selected to screen for drought stress responses at the vegetative stage.
• A single seed was planted in 8" length soil column with 1" diameter
• Dehydration condition was imposed to the 4-week old seedlings by withdrawing water,
• tissues were harvested at every 24 h after treatment for 5 days.
• The collected leaf tissues were subjected to the following analysis.
• Further screening is recommended for Godaheenati as a drought tolerant rice variety to be used in rice breeding programs.
Jayaweera et al. (2016)Gannoruwa, Sri Lanka 52

53. 53
Fig. 17. Effect of drought on the relative water content of
rice varieties Godaheenati (4049) and Pokkali
Fig. 18. A representative fraction of the 12% acrylamide gel
 Differentially expressed proteins of two-week old rice
leaves of Godaheenati (4049):
 (A) control and
 (B) drought affected
photosystem II oxygen evolving complex protein (N-EGVPPXLTFD) and stated of its role in light harvesting, which could
potentially yield crop plants that are more resistant to Drought stress and prevent inhibitory effects on photosynthesis
Protein no. 2 = photosystem II oxygen evolving complex protein

54. 54

55. CS-8: Metabolomics and proteomics analyses of
grain yield reduction in rice under abrupt
drought-flood alternation
• Wufengyou 286 (Oryza sativa L.) is the dominant double-cropping super hybrid early rice variety
• Rice was planted in plastic buckets of 24.0 cm height and 29.0 cm inner diameter of the upper
portion, and 23.5 cm inner diameter at the bottom
• Each pot contained approximately 10 kg of dry soil
• the rice abrupt drought-flood alteration stage was set at the panicle differentiation stage.
• Drought treatment continued for further 2 d until the soil was white and cracking, and the plants
were wilting and withered (imitating severe drought).
• For submergence treatment, plants in soil-containing pots were completely submerged in a high
water-filled square box (1.35 m height) in a greenhouse
Xiong et al. (2018)Jiangxi, China 55

56. Fig. 19. Analysis of yield and physiological indexes: (a) yield per plant, (b) soluble protein content, (c)
SOD activity, (d) CAT activity, (e) POD activity, (f) MDA content.
56
CK0: Control
CK1: drought
CK2: floods;
T1: abrupt drought-flood alteration
In plants under flooding stress, the electron transport chains of mitochondria and chloroplasts is blocked, and the intracellular energy charge is reduced.
All these factors could promote the production of reactive oxygen species (ROS)
POD, CAT, SOD, and glutathione-S-transferase usually act as ROS scavengers to reduce oxidative damage caused by oxidative stress in plants

57. Fig. 20. Hierarchical cluster analysis of changed metabolite pools.
57
 Hierarchical trees were drawn based on detected changes of metabolites in spikes of rice under different water treatments:
(a) T1 vs CK0 comparison treatment,
(b) T1 vs CK1 comparison treatment, and
(c) T1 vs CK2 comparison treatment.
 Columns represent the repetition between different treatments, while rows represent different metabolites.
 Red and green colors indicate increased and decreased metabolite concentrations, respectively.
CK0: Control
CK1: drought
CK2: floods
T1 : abrupt drought-flood alteration.

58. Fig. 21. Venn diagram the differentially expressed
proteins (DEPs) between alteration.
58
Fig. 22. Summary of up- and down-regulation of
differentially expressed proteins (DEPs)
Results for abrupt drought-flood alternation at the young spike differentiation stage responsive proteins.
CK0: Control
CK1: drought
CK2: floods
T1 : abrupt drought-flood alteration. Activity of SOD activity, CAT activity, POD activity and MDA content
and also up regulated protein protect the rice plant to against drought-
flood effect
T1 vs CK0 T1 vs CK1
T1 vs CK2

59. 59

60. CS-9: Comparative Proteomic Analysis Provides New
Insights into Chilling Stress Responses in Rice
• To gain a better understanding of chilling stress responses in rice (Oryza
sativa L. cv. Nipponbare), we carried out a comparative proteomic analysis.
• Three-week-old rice seedlings were treated at 6 °C for 6 or 24 h and then
recovered for 24 h.
• Chilling treatment resulted in stress phenotypes of rolling leaves, increased
relative electrolyte leakage, and decreased net photosynthetic rate.
Yan et al. (2005)Shanghai, China 60

61. Fig. 23. The physiological responses induced by chilling stress
in rice.
Three-week-old seedlings were treated at 6 °C for 0, 6, and 24 h and then were allowed to recover for 24 h (R24 h).
The relative electrolyte leakage, the Pn = photosynthetic rate, the Gs = stomatat conductance, and the
intercellular CO2 concentration (Ci) are shown in B, C, D, and E, respectively. 61

62. 62
(A) 2-DE gel of the control sample.
(B) 2-DE gel of sample treated at 6 °C for 6 h.
Temporal changes of differentially expressed proteins after
chilling treatment and recovery.
Fig. 24. Representative 2-DE gels of rice leaf proteins.

63. Fig. 25. Venn diagram analysis of the differentially expressed proteins at each
chilling time point.
63
The number of differentially expressed spots up- or down-regulated at a particular
time point(s) are shown in the different segments.
 A, the down-regulated proteins.
 B, the uregulated proteins.
R24 h, recovery for 24 h.

64. 64
 The protein excised from gels was digested with trypsin, and the resulting peptides were analyzed using the 4700 roteomics
Analyzer.
 A, the MS spectra.
 The ion 2047.08 marked with an asterisk was analyzed by MS/MS.
 B, MS/MS spectra of ion 2047.08.
 The y ions (y3– y14) and the corresponding peptide sequence are shown.
 The protein was identified as ascorbate peroxidase (NCBI accession number BAB17666) after database searching.
Fig. 26. Identification of spot 71 by MS

65. 65
 a Sequence coverage of
matched peptides.
 b The sequence of
matched peptides
The identification of novel
cold-responsive proteins
provides not only new
insights into chilling stress
responses but also a good
starting point for further
dissection of their functions
using genetic and other
approaches

66. CS-10: Proteomic analysis of cold acclimation in
winter wheat under field conditions
• wheat (Triticum aestivum L. cv Pishgam) under field conditions
• fully expanded upper leaves of wheat plants in each sampling date were harvested and
then stored at -80 °C.
• proteome analysis was carried out for four sampling dates including
• T1 (4 Nov: before the beginning of cold acclimation),
• T2 (23 Nov: initiation of cold acclimation; LT tolerance=~-6 °C),
• T3 (26 Dec: vernalization fulfillment; LT tolerance=~ -15 °C) and
• T4 (21 Feb: early reproductive growth stage LT tolerance=~-10 °C).
• Changes induced in leaf proteins were studied by two dimensional gel electrophoresis
and quantitatively analysed using image analysis software.
Janmohammadi et al. (2014)Iran 66

67.  2-DE gel analysis of proteins extracted from
leaves of Pishgam winter wheat harvested at
different developmental stages.
 Panel shows the reference map derived from
computerized image analysis performed by
using Progenesis Same Spots software.
 Numbers indicate the variable protein spots.
 A large number of all the selected proteins
were part of the photosynthetic apparatus,
confirming the key role of the chloroplast
machinery during LT acclimation.
 Accordingly, proteome analysis of organelles
such as the chloroplast and plasma membrane
may be applied to widen our information about
LT tolerance.
67
Protein no.=
Fig. 27.

68. Table 12. Differentially expressed proteins during different developmental stages in winter wheat identified by MALDI-
TOF MS
Spot No.a Protein name Accession no. Organism Database Fold of variationb Proteome comparisons
Increased
771 70 kDa heat shock protein gi|290131414 Triticum aestivum NCBI 2.13 T2/T1
1357 Putative fructose-bisphosphate aldolase 35_1820 Oryza sativa HarvestHv 2.08 T2/T1
2432 MADS-box protein 35_27943 Oryza sativa HarvestHv 2.05 T2/T1
1042 RuBisCO large subunit gi|2493650 Triticum aestivum NCBI 1.95 T2/T1
641 Os03g0108400 gi|255674149 Oryza sativa NCBI 1.88 T2/T1
1292 actin gi|281485191 Persea americana NCBI 1.86 T2/T1
1133 NADH dehydrogenase subunit 1 gi|18378414 Cucurbita argyrosperma NCBI 1.76 T3/T2
1169 Thioredoxin-like protein 35_50073 Oryza sativa HarvestHv 1.68 T3/T2
2199 Oxygen-evolving enhancer protein 2 gi|131394 Triticum aestivum NCBI 1.44 T3/T2
2234 Manganese superoxide dismutase gi|125663927 Triticum aestivum NCBI 1.40 T3/T2
2169 NADH dehydrogenase subunit 1 gi|18378406 Cucurbita ecuadorensis NCBI 1.36 T3/T2
2351 metal ion transmembrane transporter gi|240256271 Arabidopsis thaliana NCBI 1.34 T3/T2
2190 Oxygen-evolving enhancer protein 2, 35_1423 Oryza sativa HarvestHv 1.96 T4/T3
1510 ribosomal protein S1 gi|159161283 Cuscuta exaltata NCBI 1.92 T4/T3
1108 UDP-glucose pyrophosphorylase gi|88866516 Oryza sativa NCBI 1.84 T4/T3
1359 Fructose-bisphosphate aldolase Ta_TC235339 Arabidopsis thaliana TIGERPoa 1.79 T4/T3
2648 ribulose 1,5-bisphosphate carboxylase, large subunit Ta_TC263613 Arabidopsis thaliana TIGERPoa 1.78 T4/T3
1007 RuBisCO large subunit-binding protein subunit alpha gi|134102 Triticum aestivum NCBI 1.70 T4/T3
a = Spot number represents the number on the master gel
b = Fold of protein variation is calculated by standardizing the mean of the normalized spot volumes of samples at different harvesting times (T1, T2, T3
and T4) with the mean of the normalized spot.
68

69. Spot No.a Protein name Accession no. Organism Database Fold of variationb Proteome comparisons
Decreased
2199 Oxygen-evolving enhancer protein 2, chloroplastic gi|131394 Triticum aestivum NCBI 0.68 T2/T1
2351 metal ion transmembrane transporter gi|240256271 Arabidopsis thaliana NCBI 0.54 T2/T1
2169 NADH dehydrogenase subunit 1 gi|18378406 Cucurbita ecuadorensis NCBI 0.52 T2/T1
1821 oxygen-evolving complex protein 1 gi|739292 Triticum aestivum NCBI 0.50 T2/T1
2265 undecaprenyl diphosphate synthase, putative gi|255582903 Ricinus communis NCBI 0.50 T2/T1
2191 Oxygen-evolving enhancer protein 2 gi|131394 Triticum aestivum NCBI 0.48 T2/T1
2207 dehydroascorbate reductase gi|259017810 Triticum aestivum NCBI 0.48 T2/T1
1062 Os05g0291700 protein gi|3176645 Oryza sativa HarvestHv 0.58 T3/T2
1237 Transcription factor, putative gi|255574095 Ricinus communis NCBI 0.48 T3/T2
1042 RuBisCO large subunit-binding protein subunit beta, chloroplastic gi|2493650 Triticum aestivum NCBI 0.48 T3/T2
1244 Translational elongation factor Tu 35_976 Oryza sativa HarvestHv 0.46 T3/T2
1069 ATP synthase CF1 beta subunit gi|14017579 Triticum aestivum NCBI 0.46 T3/T2
1366 ribulose 1,5-bisphosphate carboxylase activase isoform 1 gi|167096 Hordeum vulgare NCBI 0.44 T3/T2
1357 Putative fructose-bisphosphate aldolase 35_1820 Oryza sativa HarvestHv 0.43 T3/T2
948 protein P0668H12.12 35_24827 Oryza sativa HarvestHv 0.49 T4/T3
1502 Putative aldehyde oxidase-like protein 35_3416 Oryza sativa HarvestHv 0.44 T4/T3
689 Cytochrome P450-like 35_12050 Oryza sativa NCBI 0.40 T4/T3
525 Electron transporter, putative gi|255576550 Ricinus communis NCBI 0.38 T4/T3
2690 cell-autonomous heat shock cognate protein 70 gi|26985223 Cucurbita ecuadorensis NCBI 0.38 T4/T3
1335 Photosystem II 44 kDa reaction center protein (P6 protein) Og_TC291300 Oryza sativa TIGERPoa 0.36 T4/T3
69
The abundance of some metabolic regulator, ion transporter, redox and photosynthetic proteins increased by achieving maximum LT tolerance in treatment
T3- (26 Dec: vernalization fulfillment; LT tolerance=~ -15 °C). By initiation of the reproductive phase treatment T4- (21 Feb: early reproductive growth stage
LT tolerance=~-10 °C) the abundance of some proteins that mainly participate in photosynthesis and carbon metabolism significantly increased

70. Nandha et al. (2018)Junagadh, India 70
 Identify the characteristic change in two wheat genotypes viz, GW 451 (heat tolerant) and WH 147 (heat
susceptible) from flag leaf.
 Seeds of both genotypes were grown up to boot leaf stage (around 52 days after sowing).
 Flag leaves from both genotypes were collected as control samples and other set of plants were treated at 40ºC for
one hour and treated leaves were collected as treated samples.
 proteins reported in heat tolerant genotype was higher than heat susceptible genotype.

71. Fig. 28. Spots detected on gel photograph of A) control leaf sample of heat susceptible genotype (WH 147);
B) treated leaf sample of heat susceptible genotype (WH 147); C) control leaf sample of heat
tolerant genotype (GW 451); D) treated leaf sample of heat tolerant genotype (GW 451)
A
C
B
D
71
control
control
WH 147 WH 147
GW 451
GW 451
Heat treated
Heat treated

72. Table 13. Comparative analysis of level of expression between treated and control leaf of WH 147
Sr. No. Match ID
Coefficient
of
Variation
(%)
WH 147 (Control leaf) WH 147 (Treated leaf)
pI
MW
(kDa)
% vol pI
MW
(kDa)
% vol
1 152 48.5 8.97 14 0.770 10.11 60 0.267
2 154 51.6 7.78 16 0.125 9.21 44 0.394
3 156 29.9 8.47 18 0.404 9.49 60 0.750
4 157 52.7 8.19 23 0.295 8.97 61 0.954
5 161 50.2 5.98 50 1.013 6.39 38 0.337
6 162 38.0 6.63 58 1.427 6.58 60 0.642
7 163 48.0 8.71 60 1.028 7.69 97 0.362
8 169 18.4 4.34 77 0.379 4.59 22 0.550
9 172 40.6 5.67 95 1.268 5.10 52 0.537
10 176 82.0 4.35 97 0.245 3.85 31 2.480
72

73. Table 14. Comparative analysis of level of expression between treated and control leaf of GW 451
Sr. No. Match ID
Coefficient
of
Variation
(%)
GW 451 (Control leaf) GW 451 (Treated leaf)
pI
MW
(kDa)
% vol pI
MW
(kDa)
% vol
1 48 18.7 4.49 36 0.191 4.83 66 0.130
2 50 31.2 7.74 37 0.309 8.07 63 0.589
3 56 51.5 9.93 47 0.280 10.02 53 0.876
4 68 71.1 4.53 69 0.831 4.86 102 4.913
5 69 39.4 7.67 77 0.207 8.16 97 0.477
6 72 35.3 4.82 83 0.400 5.38 97 0.191
7 73 01.4 8.25 84 0.470 8.61 95 0.484
8 74 38.8 9.56 91 1.079 9.94 97 2.445
9 75 52.6 4.85 94 0.575 5.09 97 0.178
10 76 20.8 4.48 94 0.847 4.93 97 0.555
73

74. Table 15. Numbers of spots identified in 2DE in control and treated leaf sample in both
genotypes
Control Leaves Treated Leaves Total No. of Spots
GW 451 WH 147 GW 451 WH 147 GW 451 WH 147
pI (2-4) 41 17 21 28 62 45
MW (KDa) 14-97 14-97 14-102 14-99
pI (4-6) 35 41 30 45 65 86
MW (KDa) 18-97 12-97 14-107 14-97
pI (6-8) 41 27 27 40 68 67
MW (KDa) 17-97 12-100 18-97 13-97
pI (8-10) 53 33 38 51 91 84
MW (KDa) 12-97 14-97 14-97 14-97
Total Spots 170 118 116 164 286 282
Total 288 280 568
74
Various proteins were reported with highly versatile amount of molecular weight
indicated the tolerance level against heat stress.

75. Plant proteomic analysis allows for the large-scale study of molecular changes occurring at the
protein level.
Proteomics has already been used to evaluate abiotic stress-responsive proteins in important crop
species such as rice, wheat, maize and sorghum.
Proteome studies the complete set of proteins encoded by the genome and thus complement the
transcriptome studies.
The first group includes proteins that function in abiotic stress tolerance such as chaperones, late
embryogenesis abundant (LEA) proteins, osmotin, mRNA-binding proteins, key enzymes for
osmolyte biosynthesis, water channel proteins, metabolites transporters, detoxification enzymes,
and various proteases.
 These proteins can then serve as molecular markers in marker-assisted selection and breeding
programs or in transgenic approaches to improving plant drought tolerance.
75