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Genetic determination determination of of protein protein quality quality in in wheat wheat grain grain

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(1)

International Workshop

Modelling quality traits and their genetic variability for Wheat

18-21 July Clermont Ferrand

Genetic

Genetic determination determination of of protein protein quality quality in in wheat wheat grain grain

G érard BRANLARD rard BRANLARD INRA

INRA

Clermont Ferrand

(2)

Genetic

Genetic determination determination of of protein protein quality quality in in wheat wheat grain grain

Genetic

Genetic aspects aspects of of wheat wheat storage storage proteins proteins Wheat storage proteins and quality

Quantitative variations of wheat storage proteins

Some other proteins involved in quality

(3)

Wheat endosperm and Protein matrix

(4)

Wheat

Wheat Endosperm Endosperm Proteins Proteins

Protein type Solubility % flour Characteristics protein

Albumins Water 10 Enzymes and

cell structure

Globulins NaCl 0.5M 10 MWs 5 – 100 kDa

Soluble proteins

Gliadins Alcohol 35-45 Monomeric

MWs 30 – 80 kDa

Glutenins SDS Polymeric

HMW-GS + R. A. 10-15 MWs 75 -120 kDa

LMW-GS 20-30 MWs 25 - 45 kDa

Storage

proteins

(5)

Gluten

Gluten protein protein classification classification

Gluten Protein

Gliadins (monomeric)

Glutenins (polymeric)

ω-gliadins αβ-gliadins γ-gliadins LMW-GS HMW-GS

S poor prolamins S rich prolamins HMW prolamins

From Shewry P. et al. 1986

(6)

Genetic

Genetic determination determination of of protein protein quality quality in in wheat wheat grain grain

Genetic

Genetic aspects aspects of of wheat wheat storage storage proteins proteins

(7)

HMW-GS

LMW-GS

S D S

P a g e -

+ Glutenins

Glu-3 Glu-1

Ch 1 A B D

Ch 6 A B D

Gli-2 Gli-1

β-gliadins γ-gliadins

α-gliadins

A c

i d P a g - e +

ω-gliadins

Wheat storage protein diversity Wheat storage protein diversity

Gliadins

(8)

Main locus

Main locus involved involved in in synthesis synthesis of of wheat wheat endosperm endosperm storage

storage proteins proteins

Long arm Short arm

Glu-A1 Gli-A3 Glu-A3 Gli-A1 Gli-A5 Gli-A6

1A c

Glu-B1 Gli-B3 Glu-B3 Gli-B1 Gli-B5

1B c

Glu-D1 Glu-D3 Gli-D1

1D c

HMW GS LMW GS ω-gliadins

6A c Gli-A2

6B c Gli-B2

6D c Gli-D2

α, β, γ Gliadins

(9)

WSPs WSPs are encoded by clusters of genes are encoded by clusters of genes

Glu 1x Glu 1y

HMW-GS

Glu 3 Gli 1

LMW-GS Gliadins

(10)

1 2* 2 7 8

6

8

7

9 17

18 2*5

10 12

1 5

10 2

12

a d

/f b e

f

f g

d

d

d

d

b

b a

a

c

c

2

12 2

7 8

b e

d

a d

d

a d

d

b ’ c’

j

h i

h i

a

a b

b c

c c

c

Some Some alleles alleles of HMW- of HMW -GS and LMW GS and LMW -GS - GS

HMW-GS

LMW-GS

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

From: Branlard et al. Genetic Resources and Crop Evolution 2003, 50, 669-679

(11)

Structural

Structural diversity diversity of of the the glutenin glutenin subunits subunits

Repeat

Repeat

Repeat

(12)

Main

Main alleles alleles encoding encoding HMW- HMW -GS GS

Glu-D1 Glu-D1

Glu-B1 Glu-B1

Glu-A1

Glu-A1

(13)

Allelic

Allelic diversity diversity of of the the LMW LMW - - GS GS

Glu-A3

Glu-A3 Glu-B3 Glu-B3 Glu-D3 Glu-D3

(14)

Allelic

Allelic diversity diversity of Gliadins of Gliadins

From Metakovsky 1991, J Genet.& Breeding,45, 325-344.

(15)

Allelic diversity of wheat storage proteins Allelic diversity of wheat storage proteins

Long arm Short arm

Glu-A1 c Gli- A3 Glu-A3 Gli-A1 Gli-A5 Gli-A6

1A

Glu-B1 c Gli-B3 Glu-B3 Gli-B1 Gli-B5

1B

Glu-D1 c Glu-D3 Gli-D1

1D

Glu. HMw Glu. LMW ω-gliadins

6A c Gli-A2

6B c Gli-B2

6D c Gli- D2

α, β, γ Gliadins

Main locus for wheat stor age proteins Cultivars HMW-GS LMW-GS ω-Gliadins

Glu-A1 Glu-B1 Glu-D1 Glu-A3 Glu-B3 Glu-D3 Gli-A1 Gli-B1 Gli-D1

‘Abo’ c b d a b c k b b

‘Aboukir’ c b d a g b k f f

‘Aiglon’ c a d ef g c f e b

‘Albatros’ c b a d f c o e b

‘Alpe’ b b d a b c b b b

‘Alto’ c b d a g c k f b

‘Alvina’ c b d a c c k b b

‘Ami’ c b d a c' c o b b

‘Apexal’ c c d a g c b e b

‘Apollo’ c d a d j c o l b

‘Apostole’ c i a ef g b b f g

‘Arbon’ c d a a b c a b b

‘Arcane’ c b a a g b a f f

‘Arche’ c d a d g c o f b

‘Arcole’ c b c d g c o f b

‘Arfort’ b i d a b a b b b

‘Aristide’ b a d a c' c b b j

‘Armada’ c d a ef f c m g b

‘Arminda’ c a a a g c f f b

‘Armur’ c c d a g c k f b

‘Arsenal’ a d a a g a f e b

(16)

How these Wheat Storage Proteins are assembled ? How these Wheat Storage Proteins are assembled ?

HMW- GS

LMW- GS

S D S

P a g e -

+ Glutenin

s

β-gliadins γ-gliadins

α-gliadins

A c

i d P a g - e +

ω-gliadins

Gliadins

(17)

Covalent SS links

Covalent SS links between between wheat wheat storage storage proteins proteins

From Köhler P. Belitz H. Wieser H 1993 Z. Lebensm. Unters. Forsch 196,239-247

(18)

SDS insoluble glutenin

SDS insoluble glutenin polymer polymer formation in formation in developing developing grains grains of of wheat wheat , , ( ( cv cv : Soissons) : Soissons)

From Carceller JL, Aussenac T., Aust. J. Plant Physiol 2001, 28; 193-201 reproduced with permission

(19)

Genetic

Genetic determination determination of of protein protein quality quality in in wheat wheat grain grain

Genetic

Genetic aspects aspects of of wheat wheat storage storage proteins proteins Wheat

Wheat storage storage proteins proteins and and quality quality

(20)

Wheat

Wheat gluten proteins gluten proteins as part as part of of the the bread bread making making quality quality

(21)

Mixographe

mixing Time (minutes) 8 min

MPTi + 2 min

MRV MTxV

MPV

WS = MPV - MRV

MTxI = area under midline curve up to 8 min

consistency (%)

MPTi - 1

min peak time = MPTi

(22)

Comparison of alleles effects for phenotypic values of dough Comparison of alleles effects for phenotypic values of dough

strength strength

Locus Strength

GluA1 2* = 1 > null

GluB1 17-18 ≥ 13-16 ≥ 7-9 = 7-8 ≥ 7 = 6-8 GluD1 5-10 ≥ 3-12 = 2-12 ≥ 4-12

GluA3 a = d = f ≥ e

GluB3 b’ ≥ d = c = c’ = b = g > i > f ≥ j GluD3 a ≥ b = d = c

GliA2 t ≥ k = r = f = g = j ≥ l = b = p

GliB2 m >b ≥ r ≥ h = o = g ≥ ae = l = ac

GliD2 m = e ≥ a = h = v = g = n

(23)

Comparison of alleles effects for phenotypic values of dough Comparison of alleles effects for phenotypic values of dough

extensibility extensibility

Locus Extensibility

GluA1 nsd

GluB1 13-16 ≥ 7-8 = 7-9 = 17-18 ≥ 7 ≥ 6-8 GluD1 nsd

GluA3 d = a = f ≥ e

GluB3 i ≥ b’ ≥ c = c’ = g > b = f = d > j GluD3 nsd

GliA2 b = t ≥ k = g = l ≥ p = r = f = j

GliB2 ae ≥ m ≥ g = o = h = ac ≥ b = r = l GliD2 nsd

(nsd: not significantly different)

From: Branlard G.,Dardevet M., Saccomano R., Lagoutte F., Gourdon J.

Euphytica, 2001, 119, 59-67

(24)

Interactions

Interactions between between Glu Glu - - 1 and Glu- 1 and Glu -3 loci 3 loci

Glu-A1 x GluD3

0 50 100 150

nul 1 2*

Glu-A1

Pelsh. Glu-D3a Glu-D3b Glu-D3c

Ι : LSD

Ι

Glu-B1 x Glu-B3

0 1 2 3 4 5 6

7 6-8 7-8

Glu-B1

M P T Glu-B3b Glu-B3c' Glu-B3f Glu-B3g Ι

Glu-D1 x Glu-A3

0 10 20 30 40 50 60

2-12 3-12 4-12 5-10

Glu-D1

Elast.

Ind.

GluA3a GluA3d GluA3ef

Ι

(25)

Part (R

2

) of Protein content, G. Hardness, HMW, LMW GS and α, β, γ-gliadins in the genotypic variations of six bread wheat

quality parameters

Part (R

2

) of Protein content, G. Hardness, HMW, LMW GS and α, β, γ-gliadins in the genotypic variations of six bread wheat

quality parameters

MPT

5% 9%

41%

12%

14%

19%

MTxI

25%

2%

8% 24%

13%

28%

Pel

4.3 30.9

33.8 3.2 14.1

13.7

Prot G,H, HMW LMW Gli-2

?

Zeleny 14%

30%

19%

14%

8%

15%

L 15%

4%

15%

13% 21%

32%

W 17%

31%

19%

8%

11%

14%

From: Branlard G.,Dardevet M., Saccomano R., Lagoutte F., Gourdon J. Euphytica, 2001, 119, 59-67

(26)

Genetic

Genetic determination determination of of protein protein quality quality in in wheat wheat grain grain

Genetic

Genetic aspects aspects of of wheat wheat storage storage proteins proteins Wheat storage proteins and quality

Quantitative variations

Quantitative variations of of wheat wheat storage storage proteins proteins

(27)

Regulation

Regulation of of the the expression expression

Promoter

Promoter region region of of the the LMW LMW - - GS Glu GS Glu - - 3D 3D

TATAAA TATAAA

CCAATG

EM GLM

EM GLM

EB EB

EB

-120 -30 -15

ATG …. STOP

-236 -295

-513

-540

Glu-D3

TGTAAAGTGATACTATCTTGATAAGTGTGTGACATGTAAAGTTAATAAGGTGAGTCATA

(28)

Quantitative variation

Quantitative variation of of some some HMW HMW - - GS GS

Génial Glenlea

10

5 7 7

OE

9

2*

(29)

Regulation

Regulation of of the the expression: expression:

Duplication

Duplication of of a a sequence sequence in in promoter promoter region region

Glu-1Bx7

TTAAATATATTGTAAAATATTCCGGCAACAACTTGTGGGG

TTAAATATATTGTAAAATATTCCGGCAACAACTTGTGGGGGCCTTAAATATATTGTAAAATATTCCGGCAACAACTTGTGGGG

Glu-1Bx7

+ 20-40%

HMW-GS 7

From :Juhász, G á rdonyi, Tamás, Bedõ. 2003, XthInt Wheat Genet Symp. Paestum, Italy 1348-1350

(30)

Regulation

Regulation of of the the expression expression

(31)

Regulation of the quantitative expression of the different loci Regulation of the quantitative expression of the different loci

interactions between chromosomes (homologous and homeologous)

1 A 2 A 3 A 4 A 5 A 6 A 7 A

1 B 2 B 3 B 4 B 5 B 6 B 7 B

1 D 2 D 3 D 4 D 5 D 6 D 7 D

HMW-GS

LMW-GS α, β, γ-gliadins

ω-gliadins

(32)

Regulation of the quantitative expression of the different loci

interactions between homeologous chromosomes. cv Courtot

1

11 12

7 5 6 9 1314

17 18

26 28

32 25 24

30 29

81

47 37 54

56 89

73

87 35

52

129

146

141 131 128 127 126 125

121 120 69

22

105 2

3 IPG 10

(a)

S D S - P A G E

Mr (kDa)

64

32

20

3 100

15

114 113 116

118

41 39

49 40

71

79

75

94 90 91

100 111 110

65 62

72

77 85

92

108

97 104

112 107

115

x

x x

x x

x

x 20 1D

62 1B

184 1B

321 1B

228 1B

256 6A 166 6A 142 6A

582 1B

648 6B

Nulli1A

(33)

-

Regulation of the quantitative expression of the different loci interactions between homolgous chr and homeologous chr.

1

11 12

7 5 6 9 1314

17 18

26

28 32

25 24

30 29

81

47 37 54

56 89

73

87 35

52

129

146

141 131 128 127 126 125

121 120 69

22

105 2

3 IPG 10

(a)

S D S - P A G E

Mr (kDa)

64

32

20

3 100

15

114 113 116

118

41 39

49 40

71

79

75

94 90 91

100 111 110

65 62

72

77 85

9 2

108

97 104

112 107

11 5

Dumur J, Jahier J, Bancel E, Laurière M, Bernard M, Branlard G 2004, Proteomics, 4, 2685-2695

(34)

1

11 12

7 5 6 9 1314

17 18

26

28 32

25 24

30 29

81

47 37 54

56 89

73

87 35

52

129

146

141 131 128 127 126 125

121 120 69

22

105 2

3 IPG 10

(a)

S D S - P A G E

Mr (kDa)

64

32

20

3 100

15

114 113 116

118

41 39

49 40

71

79

75

94 90 91

100 111 110

65 62

72

77 85

9 2

108

97 104

112 107

11 5

-

Courtot Nulli1D Courtot Normal Quantitiative

Quantitiative differences differences

Courtot Nulli1D Courtot Normal Qualitative

Qualitative differences differences

Null-1A Null-1B Null-1D M-1A M-1B M-1D

up regulated 8 15 14 17 16 26

down regulated 2 10 5 17 12 19

total affected 10 25 19 34 28 45

disappeared 13 32 24 0 0 0

appeared 0 0 13 0 0 0

Regulation of the quantitative expression of the different loci interactions between homolgous chr and homeologous chr.

Dumur J, Jahier J, Bancel E, Laurière M, Bernard M, Branlard G 2004, Proteomics, 4, 2685-2695

(35)

Effect of

Effect of nullisomy nullisomy and and monosomy monosomy on the amount of wheat on the amount of wheat storage proteins as compared to the normal cultivar

storage proteins as compared to the normal cultivar Courtot Courtot

Protein Nullisomic Monosomic Courtot

Class 40 ch 41ch 42 ch

HMW-GS NS =

LMW-GS =

Gliadins NS NS =

Gliadins NS =

Glutenins

From Dumur J. et al. Proteomics, 2004, 4, 2685-2695

(36)

Effect of warm temperature on wheat proteome at kernel maturity

Effect of warm temperature on wheat proteome at kernel maturity on on cv cv Thé Th és ée e

3 IPG 10

S D S P a g e

Stressed Normal

31 32 22

Gliadins

29

33 34

Majoul T., Bancel E., Triboï E., Ben Hamida J., Branlard G. Proteomics 2003, 3, 175-183

(37)

Genetic

Genetic determination determination of of protein protein quality quality in in wheat wheat grain grain

Genetic

Genetic aspects aspects of of wheat wheat storage storage proteins proteins Wheat

Wheat storage storage proteins proteins and and quality quality Quantitative variations

Quantitative variations of of wheat wheat storage storage proteins proteins

Some Some other other proteins proteins involved involved in quality in quality

(38)

Amphiphilic

Amphiphilic proteins proteins of of Synthetic Synthetic and and Opata Opata

11

40

25 55

15

Mr (kDa)

D’après Amiour N., Merlino M;, Leroy P., Branlard G. Proteomics, 2002, 2, 632-641

6 pI

(39)

Identification of the proteins in the puroindoline zone Identification of the proteins in the puroindoline zone

Dehydrin (LTP) GSP1b

GSP1a PIN-a .

PIN-a Prec.

PIN-b.

PIN-b Prec.

PIN-a Prec.

PIN.

PIN-a Prec.

From : Branlard G., Amiour N., Igrejas G., Gaborit T., Herbette S., Dardevet M., Marion D. Proteomics 2003, 3, 168-174

(40)

Conclusion

Wheat Storage Proteins like many plant characters are inherited by families of multigenes.

A very large diversity of WSP has been described, rending more complicated the search of associations between all the different combinations of alleles and technological properties.

The use of the known alleles of glutenins and gliadins allow today to create wheats suitable for the main uses

Accumulation of the grain components and particularly WSP are continually influenced by changing environment.

Today the major criticism on the wheat varieties is not the lack of quality

but rather their environmental instability.

(41)

Question: How to find-out the key genes that govern the kernel protein content for future quality wheat ?

Micro

Micro -array provides a complex picture of the - array provides a complex picture of the numerous DNA sequences which could be

numerous DNA sequences which could be directly or not associated to the character directly or not associated to the character Micro

Micro -array is a - array is a bottom- bottom -up up approach for approach for studying gene expression

studying gene expression

Proteomic approach is an unavoidable tool for studies on:

Proteomic approach is an unavoidable tool for studies on:

- - gene expression, functional genomic, gene regulations gene expression, functional genomic, gene regulations - - relation between genotype and phenotype relation between genotype and phenotype

- - etc... etc...

Proteomic is the

Proteomic is the top top- - down down approach particularly useful for studies on plant approach particularly useful for studies on plant physiology and environmental influence

physiology and environmental influence

(42)

Acknowledgements Acknowledgements

Dr Evgueni METAKOVSKY Dr Evgueni METAKOVSKY

Dr Jé Dr J érôme DUMUR rôme DUMUR Dr Emmanuelle BANCEL Dr Emmanuelle BANCEL

Dr Pierre MARTRE Dr Pierre MARTRE

Dr Eug

Dr Eugè ène TRIBO ne TRIBOÏ Ï

(43)

Thank

Thank you you

References

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