Monitoring of storage proteins: accumulation and impact of high temperature in wheat kernel using...

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

[email protected]

11th Int. Wheat Gluten Workshop, Beijing August 2012

mailto:[email protected]

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



Proteomics of

developping kernel


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


Content

Introduction



Proteomics of

developping kernel


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



Proteomics of

developping kernel


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


Content

Introduction



Proteomics of

developping kernel


0 57 93 152 192 247 309 343 401 457 497 547 597 655 706 764 804 857 903 957 1006

Content

Introduction



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



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



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



Proteomics of

developping kernel



14

22

26

40

66

97 25 down-regulated spots


IPGE NL 3 11

Content

Introduction



Proteomics of

developping kernel



IPGE NL 3 1

1

14

22

26

40

66

97 5 induced spots


Content

Introduction



Proteomics of

developping kernel



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



Proteomics of

developping kernel



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



Proteomics of

developping kernel



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



Proteomics of

developping kernel



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

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



Proteomics of

developping kernel



Content

Introduction



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



Proteomics of

developping kernel



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 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 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



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


wheat proteomics

Proteomics of

developping kernel



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


wheat proteomics

Proteomics of

developping kernel



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


wheat proteomics

Proteomics of

developping kernel



Content

Introduction


wheat proteomics

Proteomics of

developping kernel



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

Monitoring of storage proteins: accumulation and impact of high temperature in wheat kernel using...

Technology

Transcript of Monitoring of storage proteins: accumulation and impact of high temperature in wheat kernel using...