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
11th Int. Wheat Gluten Workshop, Beijing August 2012
Wheat proteomics
Proteome : all the proteins expressed in a cell (an organite or a tissue) at a
given time
Wheat kernel proteomics on:
Storage proteins (since several decades)
Aneuploid and isogenic lines
2x, 4x, 6x wheats
Kernel development
Kernel response to Heat stress
Proteins associated to starch granules and plastides
Aleurone layer proteins
Specific proteins
Puroindolines, Thioredoxins, Glutenins
Amphiphilic proteins, Albumins and Globulins
Content
Introduction What we’ve learned from
wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble
proteins
- The storage proteins
Proteomics of heat
stressed kernel
- Heat effect
Concluding remarks
- Regulations of WSP
High T.: Starch synthesis
Grain Protein content
Glutenins, Gliadins
Grain filling time
Dough strength
Physiology and biochemistry Bullar and Jenner 1985 Blumenthal et al. 1990
Composition and quality variations Peterson et al. 1992 Blumenthal et al. 1993, 1998 Stone et al. 1994 Wardlaw and Wrigley 1994 Craft et al. 1996 Gibson et al. 1998 Uhlen et al. 1998 QTLs analysis Jordan et al 2007
Proteomics Skylas et al. 2000, 2002 Majoul et al. 2003, 2004 Vensel et al. 2005 Dupont et al. 2006 Hurkman et al. 2009 Laino et al. 2010
Transcriptomics Dupont and Altenbach 2003 Altenbach and Kothari 2004 Altenbach et al. 2009 Kawaura et al. 2005 Laudencia-Chingcuanco et al. 2007 McIntosh et al. 2007 Wan et al. 2008
High T.: Enzymes associated to
- Energy metabolism
- Starch synthesis
- Stress responses (HSPs)
- Protein synthesis
- Signal transduction
Content
Introduction What we’ve learned from
wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble
proteins
- The storage proteins
Proteomics of heat
stressed kernel
- Heat effect
Concluding remarks
- Regulations of WSP
Some studies of kernel responses to Heat stress
0 57 93 152 192 247 309 343 401 457 497 547 597 655 706 764 804 857 903 957 1006
°C days
Triticum aestivum L. cv Récital
Outer Pericarp & Embryo removed Wheat grain Binocular PL ESM
4
Content
Introduction
What we’ve learned
from wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins - The storage proteins Proteomics of heat stressed kernel - Heat effect Concluding remarks - Regulations of WSP
Proteomic of endosperm soluble proteins : material
Tasleem Tahir A et al., J. Proteome Res. 2012, 11, 2754−2773
152°Cd 1006°Cd
5
950 significantly varying spots 487 identified
17 biochemical functions 9 expression profiles
Proteomic of endosperm soluble proteins : some results Content
Introduction
What we’ve learned
from wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins - The storage proteins Proteomics of heat stressed kernel - Heat effect Concluding remarks - Regulations of WSP
6
Carbohydrate M.
Stress / defense
cytoskeleton
Cell division
Protein turn-over
Protein synthesis
Protein transport
Transcription / translation
Amino acid M.
Lipid M.
Protein folding
ATP Interconversion
Signal transduction
Cell wall
Folic acid M.
Storage proteins
Unknown proteins
Proteomic of endosperm soluble proteins : protein classification and expression profiles
Tasleem Tahir A et al., J. Proteome Res. 2012, 11, 2754−2773
Content
Introduction
What we’ve learned
from wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins - The storage proteins Proteomics of heat stressed kernel - Heat effect Concluding remarks - Regulations of WSP
82
63
39
21
15
4.8 6.1 7.8 9.5
kDa
pI
P7
P8
P9
7
In early stages proteins, Catalase, ascorbate reductase,
were abundant on acidic side
At later stages proteins (Serpins, alpha amylase and xylanase inhibitor)
on basic side
P1
P2
P3
P4
P5
P6
Proteomic of endosperm soluble proteins : proteome map of stress defence related proteins
Content
Introduction
What we’ve learned
from wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins - The storage proteins Proteomics of heat stressed kernel - Heat effect Concluding remarks - Regulations of WSP
Predicted Sub-cellular Localization of SD Proteins
Expression profiles
Number of identified proteins
Presence of SD proteins in these localizations (Extr, P, ER) at
grain maturity help for grain protection
0
5
10
15
20
25
30
35
40
45
1 2 3 4 5 6 7 8 9
Peroxisome
Plastid
Nucleus
Mitochondria
Extracellular
Endoplasmic Reticulum
cytosol
Proteomic of endosperm soluble proteins : predicted subcellular localization of stress defence proteins
Tasleem Tahir A et al., J. Proteome Res. 2012, 11, 2754−2773
Content
Introduction
What we’ve learned
from wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins - The storage proteins Proteomics of heat stressed kernel - Heat effect Concluding remarks - Regulations of WSP
0 57 93 152 192 247 309 343 401 457 497 547 597 655 706 764 804 857 903 957 1006
Content
Introduction
What we’ve learned
from wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins
- The storage proteins Proteomics of heat stressed kernel - Heat effect Concluding remarks - Regulations of WSP
Proteomic of storage proteins : Main protein spots and profiles
HMW 22 3 IPGE 3 11 Type Spots Profiles
Sds
Page
LMW 47 2
a- b-Gli 15 1
g- Gli 11 2
w- Gli 9 2
Proteomic of storage proteins : accumulation of major protein types
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
192 247 343 457 547 655 764 1006stages of development °Cd
alpha-beta
gamma
omega
mg protein/grain
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
192 247 343 457 547 655 764 1006
stages of development °Cd
HMW
LMW
mg protein/grain
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
192 247 343 457 547 655 764 1006stages of development °Cd
HMW/LMW
gli/glu
ratio
Content
Introduction
What we’ve learned
from wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins
- The storage proteins Proteomics of heat stressed kernel - Heat effect Concluding remarks - Regulations of WSP
Total protein extraction from whole developing seed 4-6 2DEs (IPG pH 3-10 x SDS PAGE) for each sampling
10 20 30 40 50
Day / Night
28 °C / 15 °C
23 °C / 11°C
S1 S2 S3 S4 S5 S6 S7
DAP Anthesis
Harvest
Stressed 218 288 382 476 546 640 781
Control 163 233 376 443 545 646 778Cumulative average day temperature
Content
Introduction
What we’ve learned
from wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins - The storage proteins
Proteomics of heat stressed kernel - Heat effect Concluding remarks - Regulations of WSP
Proteomics of medium thermal stressed kernel: cv Arche 28/15 vs Arche control 23/11 (°C Day/ Night)
Proteomics of medium thermal stressed kernel:2DE comparison for total proteins cv Arche stressed ( 28/15) compared to control (23/11), Stage S7
3 11 IPGE NL
14
22
26
40
66
97 57 up-regulated
spots
Content
Introduction
What we’ve learned
from wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins - The storage proteins
Proteomics of heat stressed kernel - Heat effect Concluding remarks - Regulations of WSP
14
22
26
40
66
97 25 down-regulated spots
Proteomics of medium thermal stressed kernel:2DE comparison for total proteins cv Arche stressed ( 28/15) compared to control (23/11), Stage S7
IPGE NL 3 11
Content
Introduction
What we’ve learned
from wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins - The storage proteins
Proteomics of heat stressed kernel - Heat effect Concluding remarks - Regulations of WSP
IPGE NL 3 1
1
14
22
26
40
66
97 5 induced spots
Proteomics of medium thermal stressed kernel:2DE comparison for total proteins cv Arche stressed ( 28/15) compared to control (23/11), Stage S7
Content
Introduction
What we’ve learned
from wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins - The storage proteins
Proteomics of heat stressed kernel - Heat effect Concluding remarks - Regulations of WSP
Proteomics of medium thermal stressed kernel : 2DE Mapping of the gliadins , HMW-GS and LMW-GS spots
IPGE 3 11
14
22
26
40
66
97
Content
Introduction
What we’ve learned
from wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins - The storage proteins
Proteomics of heat stressed kernel - Heat effect Concluding remarks - Regulations of WSP
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
0 2 4 6 8
stages
proteins µg/grain
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
0 2 4 6 8
stages
proteins µg/grain
Gliadins CGliadins S
Glutenins CGlutenins S
Total proteins C
Total proteins S
Gliadins CGliadins S
Glutenins CGlutenins S
Total proteins C
Total proteins S
Total proteins : Control
Gliadins : Control
Glutenins : C
7 3 5 1
Stressed 218 288 382 476 546 640 781
Control 163 233 376 443 545 646 778Cumulative average day temperature
Proteomics of medium thermal stressed kernel :Evolution of the total amount (sum of spots) of gliadins and glutenins in the kernel of cv Arche
placed in two conditions during grain formation: Stressed 28°C/15°C; Control: 23°C/11°C (day/night)
Total proteins : Stressed
Gliadins : S
Glutenins : S
Content
Introduction
What we’ve learned
from wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins - The storage proteins
Proteomics of heat stressed kernel - Heat effect Concluding remarks - Regulations of WSP
0.000
0.500
1.000
1.500
2.000
2.500
0 100 200 300 400 500 600 700 800 900
thermal time °C/j
gliadins/glutenins C
gliadins/glutenins S
Gliadins/Glutenins
Proteomics of medium thermal stressed kernel : Evolution of the ratio gliadins/glutenins in kernel for two thermal conditions Stressed: 28°C/15°C;
Control: 23°C/11°C
Content
Introduction
What we’ve learned
from wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins - The storage proteins
Proteomics of heat stressed kernel - Heat effect Concluding remarks - Regulations of WSP
Ratio gliadins/glutenins TAMARO
0.0
0.5
1.0
1.5
2.0
2.5
150 250 350 450 550 650 750 850
thermal time °C/j
gliadins/glutenins C
gliadins/glutenins S
Gliadins/Glutenins
Proteomics of medium thermal stressed kernel : Evolution of the ratio gliadins/glutenins in kernel for two thermal conditions Stressed: 28°C/15°C;
Control: 23°C/11°C
Content
Introduction
What we’ve learned
from wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins - The storage proteins
Proteomics of heat stressed kernel - Heat effect Concluding remarks - Regulations of WSP
Content
Introduction
What we’ve learned
from wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins - The storage proteins Proteomics of heat stressed kernel - Heat effect
Concluding remarks - Regulations of WSP
Some concluding remarks on Wheat storage protein regulation
from different proteomics data
Content
Introduction
What we’ve learned
from wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins - The storage proteins Proteomics of heat stressed kernel - Heat effect
Concluding remarks - Regulations of WSP
Ratio gliadins/glutenins TAMARO
0.0
0.5
1.0
1.5
2.0
2.5
150 250 350 450 550 650 750 850
thermal time °C/j
gliadins/glutenins C
gliadins/glutenins S
Gliadins/Glutenins cv Tamaro
0.000
0.500
1.000
1.500
2.000
2.500
0 100 200 300 400 500 600 700 800 900
thermal time °C/j
gliadins/glutenins C
gliadins/glutenins S
Gliadins/Glutenins cv Arche
Tamaro : Very High Quality Arche : medium Quality
Conclusion 1- on wheat storage proteins stability: Evolution of the ratio gliadins/glutenins in kernel for two thermal conditions
Stressed: 28°C/15°C; Control: 23°C/11°C
Differences between cultivars associated to gene regulation
Content
Introduction
What we’ve learned from
wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins - The storage proteins Proteomics of heat stressed kernel - Heat effect
Concluding remarks - Regulations of WSP
Line 1B Line 1BL/1RS
Gli-B1 Glu-B3
LEA a-Amyl
Inhibitor
? Gli-R1? Gli-D1 g-gliadin
with 9 cysteins
Gobaa S., et al. Proteomics 2007, 7 : 4349-4357.
Conclusion 2- on wheat storage proteins response to genome variations : ex 1: 1BL/1RS translocation vs normal 1BL
« Silenced gene », strongly up regulated for SP accumulation, and revealed through selection for high quality
Content
Introduction
What we’ve learned from
wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins - The storage proteins Proteomics of heat stressed kernel - Heat effect
Concluding remarks - Regulations of WSP
Conclusion 2- on wheat storage proteins response to genome variations : ex 2 : nullisomic lines vs normal lines
1
3
4
5
6
2
7
A
B
D
1
11 12
7 5 6
9 13 14
17
18
26 28
32
25 24
30 29
81
47 37 54
56
89 73
87
35
52
129
146
141
131
127 128
126 125
121 120
69
22
105
2
3
10 IPGE
S
D
S
-
P
A
G
E
Mr
(kDa)
64
32
20
100 3
15
114
116 113 118
41 39
49 40
71
79
75
94 91 90
100
110 111
65
62
72
77
85
92
108
97
104
112
107
115
*
* *
* *
*
*
Nulli1A
20 LMW 1D
62 g1B
184 w1B
321 w1B
228 w1B
256 a6A
166 a6A
142 a6A
582 g1B
648 b6B
Dumur J. et al. Proteomics, 2004; 4: 2685-2695.
Compensation revealing that regulation of SP accumulation involves complex interactions between homoeologous loci. What are the cis-acting transcription factors?
Conclusion 3: Next step: Deciphering the regulation network
* From : Diaz et al. 2002; Rubio-Samosa et al.2006; Moreno-Risueno et al., 2008
Proximal promoters Distal promoter sequence
8 TFs capable to be linked to SP promoters
Model* for Regulation of Barley Storage Protein
3 orthologs each 24 candidate genes
Content
Introduction
What we’ve learned from
wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins - The storage proteins Proteomics of heat stressed kernel - Heat effect
Concluding remarks - Regulations of WSP
Conclusion : 4- about the genetic and environmental factors associated to gluten polymer size
For developping wheat tolerant to heat stress, the expression
and regulation of some major enzymes need to be better known
- For energy and starch metabolisms ATP synthase ; RuBisCo PPase, SSS, Beta-amylase
- For protein accumulation
eIF4E ; PDIs ; HSPs 90 ; HSPs 70 etc..
- For response to oxydative stress
O2-, superoxide anion H2O2, hydrogen peroxide •OH, hydroxyl radical ROO•, peroxy radicals
GSS, CAT, GPX, etc..
Endosperm cell apoptosis
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.
Content
Introduction
What we’ve learned from
wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins - The storage proteins Proteomics of heat stressed kernel - Heat effect
Concluding remarks - Regulations of WSP
Content
Introduction
What we’ve learned from
wheat proteomics
Proteomics of
developping kernel
-The endosperm soluble proteins - The storage proteins Proteomics of heat stressed kernel - Heat effect
Concluding remarks - Regulations of WSP
The Unfolded Protein Response (UPR mammalian model)
• Increase of: • Chaperone synthesis • Anti-oxidant response • ER Associated degradation
Decrease of translation
Schröder and Kaufman, 2005. Annu. Rev. Biochem.
Protein transport via ER
Nucleus
ER
ROS
ACKNOWLEDGEMENTS
26
Emmanuelle BANCEL
Pierre MARTRE
Ayesha TASLEEM-TAHIR
Catherine RAVEL
Thank you
Quality variations of Arche placed in two conditions during grain formation: Stressed 28°C/15°C; Control: 23°C/11°C (day/night)
Arche
0
50
100
0 50 99 149
L(mm)
P(m
m)
Stressed
Control
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
0
50
100
150
0 50 99 149
L(mm)
P(m
m)
Stressed
Control
Experiment 3