International Gluten Workshop, 11th; Beijing (China); 12-15 Aug 2012

1. 11th Int. Wheat Gluten Workshop, Beijing August 2012
Monitoring of storage proteins : accumulation and impact of
high temperature in wheat kernel using proteomics
A TASLEEM-TAHIR, E. BANCEL, P. MARTRE and G. BRANLARD
INRA
63039 Clermont Ferrand
France
branlard@clermont.inra.fr

2. Content
Wheat proteomics
Introduction
What we’ve learned from
wheat proteomics
Proteome : all the proteins expressed in a cell (an organite or a tissue) at a
Proteomics of given time
developping kernel
Wheat kernel proteomics on:
-The endosperm soluble
proteins Storage proteins (since several decades)
- The storage proteins Specific proteins
Puroindolines, Thioredoxins, Glutenins
Proteomics of heat
stressed kernel Amphiphilic proteins, Albumins and Globulins
- Heat effect Proteins associated to starch granules and plastides
Concluding remarks Aleurone layer proteins
- Regulations of WSP Aneuploid and isogenic lines
2x, 4x, 6x wheats
Kernel development
Kernel response to Heat stress

3. Content Some studies of kernel responses to Heat stress
Introduction Physiology and biochemistry
What we’ve learned from Bullar and Jenner 1985
wheat proteomics Blumenthal et al. 1990
Composition and quality variations High T.: Starch synthesis
Proteomics of Peterson et al. 1992 Grain Protein content
developping kernel Blumenthal et al. 1993, 1998
Stone et al. 1994
Glutenins, Gliadins
-The endosperm soluble Wardlaw and Wrigley 1994 Grain filling time
proteins Craft et al. 1996
Gibson et al. 1998
Dough strength
- The storage proteins Uhlen et al. 1998
QTLs analysis
Jordan et al 2007
Proteomics of heat Proteomics
stressed kernel Skylas et al. 2000, 2002
Majoul et al. 2003, 2004
- Heat effect Vensel et al. 2005
Dupont et al. 2006 High T.: Enzymes associated to
Concluding remarks Hurkman et al. 2009
Laino et al. 2010 - Energy metabolism
- Regulations of WSP Transcriptomics - Starch synthesis
Dupont and Altenbach 2003
Altenbach and Kothari 2004 - Stress responses (HSPs)
Altenbach et al. 2009 - Protein synthesis
Kawaura et al. 2005
Laudencia-Chingcuanco et al. 2007 - Signal transduction
McIntosh et al. 2007
Wan et al. 2008

4. Content
Proteomic of endosperm soluble proteins : material
Introduction
What we’ve learned
from wheat proteomics Triticum aestivum L. cv Récital
Proteomics of
developping kernel
°C days
-The endosperm
soluble proteins 0 57 93 152 192 247 309 343 401 457 497 547 597 655 706 764 804 857 903 957 1006
- The storage proteins
Proteomics of heat
stressed kernel Binocular Wheat grain Outer Pericarp & Embryo removed PL ESM
- Heat effect
Concluding remarks
- Regulations of WSP
4

5. Content
Proteomic of endosperm soluble proteins : some results
Introduction
What we’ve learned
from wheat proteomics
152°Cd 1006°Cd
Proteomics of
developping kernel
-The endosperm
soluble proteins
- The storage proteins
Proteomics of heat
stressed kernel
- Heat effect
Concluding remarks
- Regulations of WSP
950 significantly varying spots 487 identified
17 biochemical functions 9 expression profiles
Tasleem Tahir A et al., J. Proteome Res. 2012, 11, 2754−2773
5

6. Content Proteomic of endosperm soluble proteins : protein classification and
expression profiles
Introduction
What we’ve learned
from wheat proteomics Carbohydrate M.
Stress / defense
Proteomics of cytoskeleton
developping kernel
Cell division
-The endosperm Protein turn-over
soluble proteins Protein synthesis
Protein transport
- The storage proteins
Transcription / translation
Amino acid M.
Proteomics of heat Lipid M.
stressed kernel
Protein folding
- Heat effect ATP Interconversion
Signal transduction
Concluding remarks
Cell wall
- Regulations of WSP Folic acid M.
Storage proteins
Unknown proteins
6
Tasleem Tahir A et al., J. Proteome Res. 2012, 11, 2754−2773

7. Content Proteomic of endosperm soluble proteins : proteome map of stress defence
Introduction
related proteins
What we’ve learned kDa
from wheat proteomics
82
Proteomics of
63
developping kernel
-The endosperm P1
soluble proteins P2
P3
- The storage proteins 39
P4
P5
Proteomics of heat P6
stressed kernel 21
P7
- Heat effect P8
P9
Concluding remarks 15
- Regulations of WSP In early stages proteins, Catalase, ascorbate reductase,
were abundant on acidic side
pI 4.8 6.1 7.8 9.5
At later stages proteins (Serpins, alpha amylase and xylanase inhibitor)
7
on basic side

8. Content Proteomic of endosperm soluble proteins : predicted subcellular localization
Introduction of stress defence proteins
What we’ve learned
from wheat proteomics Predicted Sub-cellular Localization of SD Proteins
Proteomics of Number of identified proteins
developping kernel 45 Peroxisome
40
-The endosperm Plastid
35
soluble proteins
30 Nucleus
- The storage proteins 25
Mitochondria
20
Proteomics of heat 15 Extracellular
stressed kernel
10
Endoplasmic Reticulum
- Heat effect 5
0 cytosol
Concluding remarks 1 2 3 4 5 6 7 8 9
Expression profiles
- Regulations of WSP
Presence of SD proteins in these localizations (Extr, P, ER) at
grain maturity help for grain protection
Tasleem Tahir A et al., J. Proteome Res. 2012, 11, 2754−2773

9. Content Proteomic of storage proteins : Main protein spots and profiles
Introduction
What we’ve learned
from wheat proteomics
Proteomics of 0 57 93 152 192 247 309 343 401 457 497 547 597 655 706 764 804 857 903 957 1006
developping kernel
3 IPGE 11 Type Spots Profiles
-The endosperm HMW 22 3
soluble proteins
LMW 47 2
- The storage proteins
S a- b-Gli 15 1
Proteomics of heat d
stressed kernel s g- Gli 11 2
P
- Heat effect
a w- Gli 9 2
Concluding remarks g
e
- Regulations of WSP

10. Content Proteomic of storage proteins : accumulation of major protein types
Introduction
What we’ve learned mg protein/grain
0.7
from wheat proteomics
0.6 alpha-beta
Proteomics of 0.5
gamma
developping kernel omega
0.4
-The endosperm 0.3
soluble proteins
0.2
ratio
- The storage proteins 0.1 0.8
0.7
0
192 247 343 457 547 655 764 1006 0.6
Proteomics of heat stages of development °Cd 0.5
stressed kernel
0.4
mg protein/grain
1.6 0.3
- Heat effect
1.4 0.2 HMW/LMW
HMW
gli/glu
Concluding remarks 1.2 LMW 0.1
1 0
- Regulations of WSP 0.8
192 247 343 457 547 655 764 1006
stages of development °Cd
0.6
0.4
0.2
0
192 247 343 457 547 655 764 1006
stages of development °Cd

11. Content
Proteomics of medium thermal stressed kernel: cv Arche 28/15 vs Arche
Introduction control 23/11 (°C Day/ Night)
What we’ve learned
from wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble
proteins
- The storage proteins
Proteomics of heat S1 S2 S3 S4 S5 S6 S7 Harvest Day / Night
stressed kernel 28 °C / 15 °C
- Heat effect 23 °C / 11°C
Concluding remarks Anthesis 10 20 30 40 50 DAP
- Regulations of WSP Stressed 218 288 382 476 546 640 781 Cumulative average
Control 163 233 376 443 545 646 778 day temperature
Total protein extraction from whole developing seed
4-6 2DEs (IPG pH 3-10 x SDS PAGE) for each sampling

12. Content Proteomics of medium thermal stressed kernel:2DE comparison for total
Introduction proteins cv Arche stressed ( 28/15) compared to control (23/11), Stage S7
What we’ve learned
from wheat proteomics 3 IPGE NL 11
97
Proteomics of 57 up-regulated
developping kernel spots
-The endosperm soluble 66
proteins
- The storage proteins
40
Proteomics of heat
stressed kernel
- Heat effect 26
Concluding remarks
- Regulations of WSP 22
14

13. Content Proteomics of medium thermal stressed kernel:2DE comparison for total
Introduction proteins cv Arche stressed ( 28/15) compared to control (23/11), Stage S7
What we’ve learned IPGE NL
from wheat proteomics 3 11
97
Proteomics of 25 down-
developping kernel regulated spots
-The endosperm soluble 66
proteins
- The storage proteins
40
Proteomics of heat
stressed kernel
- Heat effect
26
Concluding remarks
- Regulations of WSP
22
14

14. Content Proteomics of medium thermal stressed kernel:2DE comparison for total
Introduction proteins cv Arche stressed ( 28/15) compared to control (23/11), Stage S7
What we’ve learned
from wheat proteomics IPGE NL
3 1
Proteomics of 97 5 induced spots
1
developping kernel
-The endosperm soluble
66
proteins
- The storage proteins
40
Proteomics of heat
stressed kernel
- Heat effect
26
Concluding remarks
- Regulations of WSP
22
14

15. Content Proteomics of medium thermal stressed kernel :
Introduction 2DE Mapping of the gliadins , HMW-GS and LMW-GS spots
What we’ve learned IPGE
3 11
from wheat proteomics
97
Proteomics of
developping kernel
-The endosperm soluble 66
proteins
- The storage proteins
40
Proteomics of heat
stressed kernel
- Heat effect
Concluding remarks 26
- Regulations of WSP
22
14

16. Content Proteomics of medium thermal stressed kernel :Evolution of the total
Introduction amount (sum of spots) of gliadins and glutenins in the kernel of cv Arche
What we’ve learned placed in two conditions during grain formation: Stressed 28°C/15°C;
from wheat proteomics
Control: 23°C/11°C (day/night)
Proteomics of proteins µg/grain
developping kernel
9000
-The endosperm soluble Total proteins : Control
8000
proteins
7000
- The storage proteins Total proteins : Stressed
6000
Total proteins C
5000 Total proteins S
Proteomics of heat Gliadins C
stressed kernel 4000 Gliadins S
Gliadins : Control
Glutenins C
Glutenins S
3000
- Heat effect Gliadins : S
2000
Concluding remarks Glutenins : C
1000
Glutenins : S
- Regulations of WSP 0
0 1 2 3 4 5 6 7 8
Stressed 218 288 382 stages
476 546 640 781 Cumulative average
Control 163 233 376 443 545 646 778 day temperature

17. Content Proteomics of medium thermal stressed kernel : Evolution of the ratio
Introduction gliadins/glutenins in kernel for two thermal conditions Stressed: 28°C/15°C;
What we’ve learned Control: 23°C/11°C
from wheat proteomics
Proteomics of
developping kernel
Gliadins/Glutenins
-The endosperm soluble
2.500
proteins
2.000
- The storage proteins
1.500
gliadins/glutenins C
Proteomics of heat gliadins/glutenins S
1.000
stressed kernel
0.500
- Heat effect
0.000
Concluding remarks 0 100 200 300 400 500 600 700 800 900
thermal time °C/j
- Regulations of WSP

18. Content Proteomics of medium thermal stressed kernel : Evolution of the ratio
Introduction gliadins/glutenins in kernel for two thermal conditions Stressed: 28°C/15°C;
What we’ve learned Control: 23°C/11°C
from wheat proteomics
Proteomics of
developping kernel Ratio gliadins/glutenins TAMARO
Gliadins/Glutenins
2.5
-The endosperm soluble
proteins
2.0
- The storage proteins
1.5
gliadins/glutenins C
Proteomics of heat gliadins/glutenins S
1.0
stressed kernel
0.5
- Heat effect
Concluding remarks 0.0
150 250 350 450 550 650 750 850
thermal time °C/j
- Regulations of WSP

19. Content
Introduction
What we’ve learned
from wheat proteomics
Proteomics of
developping kernel
-The endosperm
soluble proteins
- The storage proteins Some concluding remarks on Wheat storage protein regulation
Proteomics of heat from different proteomics data
stressed kernel
- Heat effect
Concluding remarks
- Regulations of WSP

20. Content Conclusion 1- on wheat storage proteins stability:
Evolution of the ratio gliadins/glutenins in kernel for two thermal conditions
Introduction
What we’ve learned
Stressed: 28°C/15°C; Control: 23°C/11°C
from wheat proteomics
Proteomics of
developping kernel
Arche : medium Quality Tamaro : Very High Quality
-The endosperm
soluble proteins
Gliadins/Glutenins cv Arche Gliadins/Glutenins cv Tamaro
Ratio gliadins/glutenins TAMARO
- The storage proteins
2.500 2.5
2.000 2.0
Proteomics of heat
1.500 1.5
gliadins/glutenins C gliadins /glutenins C
stressed kernel gliadins/glutenins S gliadins /glutenins S
1.000 1.0
- Heat effect
0.500 0.5
Concluding remarks
0.000
0 100 200 300 400 500 600 700 800 900
0.0
150 250 350 450 550 650 750 850
thermal time °C/j thermal time °C/j
- Regulations of WSP
Differences between cultivars associated to gene regulation

21. Content Conclusion 2- on wheat storage proteins response to genome variations :
ex 1: 1BL/1RS translocation vs normal 1BL
Introduction
What we’ve learned from
wheat proteomics Line 1B Line 1BL/1RS
Proteomics of
developping kernel
-The endosperm
soluble proteinsGli-B1
Glu-B3
- The storage proteins
Proteomics of heat
stressed kernel
?
LEA Gli-D1 g-gliadin Gli-R1?
a-Amyl
- Heat effect
Inhibitor with 9 cysteins
Concluding remarks
- Regulations of WSP
« Silenced gene », strongly up regulated for SP accumulation,
and revealed through selection for high quality
Gobaa S., et al. Proteomics 2007, 7 : 4349-4357.

22. Content Conclusion 2- on wheat storage proteins response to genome variations :
ex 2 : nullisomic lines vs normal lines
Introduction
What1we’ve 3 4 5 from 7
2 learned 6
IPGE
wheat proteomics
3 10
Mr Nulli1A
1 (kDa)
A * 3 2
7 5 6 100
Proteomics of 9
11 13
developping kernel 12 14 184 w1B
B 18
17
15
22 321 w1B
-The endosperm 25 24
26
soluble proteins
D * * 28
32
30
29
64
35
- The storage proteins 47 54
S 81
37
* *
69
56
89
73
52 582 g1B
D
S
87
105 648 b6B
Proteomics of heat 121 120
125
-
stressed kernel
P * 126
128
127
A 39
129
131
228 w1B
- Heat effect 41 49 40
32
G 62 20 LMW 1D
Concluding remarks
E * 71
65
72 75
79 77
85
- Regulations of WSP Compensation revealing that regulation of SP accumulation
90
97
91 92 94 141 256 a6A
166 a6A
100 104
107
involves complex interactions between homoeologous loci.
108
111
110
142 a6A
115 114
118 112
What are the cis-acting transcription factors?
113 116
20
146
62 g1B
Dumur J. et al. Proteomics, 2004; 4: 2685-2695.

23. Content Conclusion 3: Next step: Deciphering the regulation network
Introduction
What we’ve learned from
wheat proteomics Model* for Regulation of Barley Storage Protein
Proteomics of
developping kernel
-The endosperm Proximal promoters Distal promoter sequence
soluble proteins
- The storage proteins
Proteomics of heat
stressed kernel
- Heat effect
Concluding remarks
- Regulations of WSP
8 TFs capable to be linked to SP promoters
3 orthologs each 24 candidate genes
* From : Diaz et al. 2002; Rubio-Samosa et al.2006; Moreno-Risueno et al., 2008

24. Content Conclusion : 4- about the genetic and environmental factors associated to
gluten polymer size
Introduction
What we’ve learned from For developping wheat tolerant to heat stress, the expression
wheat proteomics
and regulation of some major enzymes need to be better known
Proteomics of
developping kernel - For energy and starch metabolisms
ATP synthase ; RuBisCo
-The endosperm PPase, SSS, Beta-amylase
soluble proteins
- For protein accumulation
- The storage proteins
eIF4E ; PDIs ; HSPs 90 ; HSPs 70 etc..
Proteomics of heat - For response to oxydative stress
stressed kernel
O2-, superoxide anion
- Heat effect H2O2, hydrogen peroxide
Endosperm cell apoptosis
•OH, hydroxyl radical
Concluding remarks
ROO•, peroxy radicals
- Regulations of WSP GSS, CAT, GPX, etc..
Grain Hardness impacts the gluten polymer size
Lesage, V et al., J Cereal Sci 2011, 53, 231-238.
Lesage, V. et al., J Exp Botany, 2012, 63,1001-1011.

25. Content
Introduction
The Unfolded Protein Response (UPR mammalian model)
What we’ve learned from
wheat proteomics
Proteomics of
developping kernel ROS • Increase of:
• Chaperone synthesis
-The endosperm
soluble proteins • Anti-oxidant response
ER
• ER Associated degradation
- The storage proteins
Decrease of translation
Proteomics of heat
stressed kernel
- Heat effect
Nucleus
Concluding remarks
- Regulations of WSP
Protein transport via ER
Schröder and Kaufman, 2005. Annu. Rev. Biochem.

26. ACKNOWLEDGEMENTS
Ayesha TASLEEM-TAHIR
Emmanuelle BANCEL
Catherine RAVEL
Pierre MARTRE
26

27. Thank you

29. Experiment 3
Quality variations of Arche placed in two conditions during grain formation:
Stressed 28°C/15°C; Control: 23°C/11°C (day/night)
Arche
100
Stressed
Control
P(mm)
50
0
0 50 99 149
L(mm)

30. Experiment 3
Quality variation of Tamaro placed in two conditions during grain formation: Stressed
28°C/15°C; Control: 23°C/11°C (day/night)
Tamaro
150
Stressed
Control
100
P(mm)
50
0
0 50 99 149
L(mm)