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Effects of HMW- & LMW-glutenins and grain hardness on size of wheat
storage proteins polymers
Véronique S. LESAGE, L. RHAZI, T. AUSSENAC, B. MELEARD, G. BRANLARD
INRA, Genetics, Diversity and Ecophysiology of Cereals, Clermont-Ferrand, France
11th International Gluten Workshop, August 12-15, 2012
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Dough quality
Nature of gliadins and glutenins alleles
Polymerization
LMW-Glutenin
g-gliadin
PS N Repeat C
From Köhler P. et al., 1993. Z. Lebensm. Unters. Forsch
LMW-Glu PS N Repeat C
HMW-GS type y PS Repeat
C N
PS N Repeat C
PS Repeat
C N HMW-GS type y
LMW-Glu
Cys: intermolecular disulfide bonds
Cys: intramolecular disulfide bonds
HMW-GS type x PS
Repeat C N
Polymers size depends on free cystine number and also on environmental conditions
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Polymerization/aggregation
Formation of insoluble polymers during wheat kernel development
Source Carceller JL, Aussenac T., 2001. Aust. J. Plant Physiol
From Lesage et al., 2011, J. Cereal Science, 53(2), 231
0
0.5
1
1.5
2
2.5
3
3.5
4
1997 2008 2010
Mw
x 1
07
g/m
ol
hard
soft
NILs
Molecular weight X 106 Da
PinA
PinA x
40 35
30
25
20
15
10
5
0
Influence of puroindolines and glutenins alleles
on polymers size?
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Material and
methods
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Material and methods
2009: L1 L2 L3
2010: L4 L5 L6
Material
Methods / Investigated traits HMW- and LMW- Glutenin alleles (SDS-PAGE) Grain hardness (NIRS) Polymer mass (Mw) and radius of giration (Rw) Polydispersity index (Mw/Mn) % protein fractions (SE-HPLC) protein content (NIRS)
puroindolines alleles (sequencing)
AFFFF
68 bread-wheat varieties 2 years 3 locations
(L1 –L6)
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Methods. Asymmetrical flow field-flow fractionation
AFFFF-MALLS analytical device used for molecular characterization of storage proteins
(Multi Angular Laser Light Scattering)
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Principle
Methods. Asymmetrical flow field-flow fractionation
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30 mg flour 1 ml sodium phosphate buffer 0.1M, pH 6.9 2% SDS
15 sec. sonication 20 W
60°C – 15 min
Sample preparation
Methods. Asymmetrical flow field-flow fractionation
(Multi Angular Laser Light Scattering detector)
Polymers size and conformation
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MALLS chromatogram
Polymers Monomers
Methods. Asymmetrical flow field-flow fractionation
Measurements: - Mw: weight-average - Mn: number-average - Mw/Mn: polydispersity index - Rw: weight-average mean square radius (radius of giration)
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Methods. Size Exclusion - HPLC
Sample preparation
160 mg flour 20 ml sodium phosphate buffer 0.1M 1% SDS
60°C – 80 min stirring
3 min sonication 2 W
filtration 0.2 µm
TSK gel G4000sw Silica-based, pore size: 13-17 µm
SE-HPLC
5 protein fractions (F1- F5)
F1 F2
F4
F3
F5
Elution Time
Large Glu Pol.
Small Glu Pol.
Ω- Gli
γ,β- Gli
α-Gli, Alb, Glo.
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Results
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Variation of polymers characteristics
AFFFF
Mw 2
Number
0 1 2 3 4 5(X 1,E7)
0
10
20
30
40
50
60
Mw2 Mw /Mn2
Number
0 20 40 60 80 100
0
20
40
60
80
Mw/Mn2 Rw 2 (nm)
Number
20 40 60 80 100 120 140
0
10
20
30
40
Rw2 (nm)
SE-HPLC
SE-HPLC F1 %
Number
10 12 14 16 18
0
10
20
30
40
50
SE-HPLC F2 %
Number
17 19 21 23 25 27 29
0
10
20
30
40
SE-HPLC F3 %
Number
5,9 6,9 7,9 8,9 9,9
0
5
10
15
20
25
30
Mean F1 : 14% Mean F2 : 24% Mean F3 : 7%
5.4 - 48.8 million Da
0.75 - 2 million Da 90.000 – 750.000 Da 50.000 – 90.000 Da
36.5 – 116.2 nm 7.1 – 82.9
(Polymers mass) (Polydispersity index) (Polymers radius)
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Glutenin loci effects on quality traits
Protein content
Gluten Index
Grain Hardness
Mw2 Mw/Mn2 Rw2 %F1 %F2 %F3 %F4 %F5
Glu-A1 ns ns ns ns ns ns ns ns ns ns ns
HMW- Glu
Glu-B1 ns ns ns * ** * * * ns ns ns
Glu-D1 ns *** *** ns ns ns *** *** * ns ns
Glu-A3 ns ns * ns ** ns *** ns ** *** ns
LMW- Glu
Glu-B3 ns ns ns ns ns ns *** *** *** *** **
Glu-D3 ns ns ns *** *** *** ns ns ns ns *
R2 0.2 17.0 19.9 9.0 15.9 11.2 47.1 58.7 16.9 45.3 10.5
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Glutenin loci effects on quality traits
Protein content
Gluten Index
Grain Hardness
Mw2 Mw/Mn2 Rw2 %F1 %F2 %F3 %F4 %F5
Glu-A1 ns ns ns ns ns ns ns ns ns ns ns
HMW- Glu
Glu-B1 ns ns ns * ** * * * ns ns ns
Glu-D1 ns *** *** ns ns ns *** *** * ns ns
Glu-A3 ns ns * ns ** ns *** ns ** *** ns
LMW- Glu
Glu-B3 ns ns ns ns ns ns *** *** *** *** **
Glu-D3 ns ns ns *** *** *** ns ns ns ns *
R2 0.2 17.0 19.9 9.0 15.9 11.2 47.1 58.7 16.9 45.3 10.5
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Effects of Glutenins alleles on polymers mass
Standardized coef.
-0,16
-0,06
0,04
0,14
0,24
Glu
A1-
nG
luA
1-1
Glu
A1-
2*
Glu
B1-
68
Glu
B1-
7G
luB
1-7
8G
luB
1-7
9G
luB
1-1
316
Glu
B1-
141
5G
luB
1-1
718
Glu
B1-
6.1
-22
Glu
D1-
212
Glu
D1-
312
Glu
D1-
412
Glu
D1-
510
Glu
A3-
aG
luA
3-e
fG
luA
3-d
Glu
B3-
bG
luB
3-b
pG
luB
3-c
Glu
B3-
cp
Glu
B3-
dG
luB
3-f
Glu
B3-
gG
luB
3-j
Glu
D3-
bG
luD
3-c
Mw2
Mw2
- 0.16
- 0.06
0.04
0.14
0.24
Standardized coef.
Glu-B1 Glu-D1 Glu-A3 Glu-B3 Glu-D3
B1 (7)
D3 (b)
Glu-A1
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Puroindoline B alleles
• 3 Grain Hardness classes: Soft < 35
35 < Medium < 75
Hard > 75
• PinA and PinB genes sequenced in all varieties
– PinA: no deletion, no SNP
– PinB : 5 alleles
PinB-D1a WPTKWWKGGC-----------L
PinB-D1b WPTKWWKSGC-----------L
PinB-D1d WPTKWRKGGC-----------L
PinB-D1c WPTKWWKGGC-----------P
PinB-D1b + d WPTKWRKSGC-----------L
AA position from start: 73 75 89
Allele of Soft varieties
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Puroindoline B alleles
• 3 Grain Hardness classes: Soft < 35
35 < Medium < 75
Hard > 75
• PinA and PinB genes sequenced in all varieties
– PinA: no deletion, no SNP
– PinB : 5 alleles
Occurence
Grain Hardness Class
0 20 40 60 80
S
M
H
PinBD1aD1bD1dD1cD1b+dPinB-D1a WPTKWWKGGC-----------L
PinB-D1b WPTKWWKSGC-----------L
PinB-D1d WPTKWRKGGC-----------L
PinB-D1c WPTKWWKGGC-----------P
PinB-D1b + d WPTKWRKSGC-----------L
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Effects of interaction between glutenins and puroindoline B alleles
Grain Hardness Mw2 Mw/Mn2 Rw2 %F3
Glu Glu x PinB Glu Glu x PinB Glu Glu x PinB Glu Glu x PinB Glu Glu x PinB
effect effect R2 effect effect R2 effect effect R2 effect effect R2 effect effect R2
A1 ns ns ** 15 ns ns ns * 15 ns ns
HMW-Glu
B1 ns * ** 14 ** ** 18 * ** 14 ns * 23
D1 *** *** 27.7 ns ns ns ns ns * 15 * ** 18
A3 * ns * 13 ** *** 22 ns ns ** *** 19
LMW-Glu
B3 ns ns ns ns ns ns ns *** *** 20
D3 ns *** ns *** * 11 *** ns ns * 18
R2 19.9 9.0 15.9 11.2 16.8
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Effects of interaction between glutenins and puroindoline B alleles
Grain Hardness Mw2 Mw/Mn2 Rw2 %F3
Glu Glu x PinB Glu Glu x PinB Glu Glu x PinB Glu Glu x PinB Glu Glu x PinB
effect effect R2 effect effect R2 effect effect R2 effect effect R2 effect effect R2
A1 ns ns ** 15 ns ns ns * 15 ns ns
HMW-Glu
B1 ns * ** 14 ** ** 18 * ** 14 ns * 23
D1 *** *** 27.7 ns ns ns ns ns * 15 * ** 18
A3 * ns * 13 ** *** 22 ns ns ** *** 19
LMW-Glu
B3 ns ns ns ns ns ns ns *** *** 20
D3 ns *** ns *** * 11 *** ns ns * 18
R2 19.9 9.0 15.9 11.2 16.8
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Effects of interaction between glutenins and puroindoline B alleles
Glu-B1
-1
4
9
14
19
24
29(X 1,E6)
Mw2
7 68 78 79 1316 1718 6122
Pinb-3alD1aD1bD1d
PinB
Mw2
Glu-B1
PinB ___ D1a ___ D1b ___ D1d
4 9
14
19 24
29 x 106 Da
7-8 Glu-A3
13
15
17
19
21(X 1,E6)
Mw2
a d ef
Pinb-3alD1aD1bD1d
PinB
Mw2
Glu-A3
PinB ___ D1a ___ D1b ___ D1d
x 106 Da
13
15
17
19
21
a
Interactions Glutenins-Puroindolines had effects on polymers characteristics Effects were contrasted depending on each allele Small part of polymers characteristics variation
Part of environmental effects?
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Environmental effects on quality traits
Year effect R2
Protein content ns
Grain Hardness ns
Gluten Index ns
Mw2 * 44.9
Mw/Mn2 * 51.0
Rw2 ** 56.4
%F1 ns
%F2 ns
%F3 ns
%F4 ns
%F5 ns
* p < 0.05
** p < 0.01
*** p < 0.001
Variance analysis (68 varieties, 2 years)
%
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Effects of sum of mean temperatures (June and July)
effect R2
Protein content ns
Grain Hardness ns
Gluten Index ns Mw2 *** 38.5 Mw/Mn2 *** 62.7
Rw2 *** 47.5 %F1 ns
%F2 ns %F3 ns
%F4 ns
%F5 ns
%
Environmental effects on quality traits
SMT-Jun Ju l (癈 )
1080 1100 1120 1140 1160 1180
0
1
2
3
4
5(X 1,E7)
Mw2 Mw2
Sum of mean temperatures (°C) (June and July)
1080 1100 1120 1140 1160 1180
0
10
20
30
40
50
x 106 Da
AFFFF gives more reliable data for native polymers characterization than SE-HPLC that reflects more diversity of glutenins
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Regression Mw2 on sum of daily mean temperatures of June and July
Soft : Mw2= -203207000+196542*SumTemp R²=0.37 Medium : Mw2= -172673000+167021*SumTemp R²=0.48 Hard : Mw2= -124367000+121462*SumTemp R²=0.44
(°C)
Mw2
Sum mean temp.
0.00E+00
5.00E+06
1.00E+07
1.50E+07
2.00E+07
2.50E+07
3.00E+07
3.50E+07
1080 1100 1120 1140 1160 1180 1200 1220
Soft
Medium
Hard
ns
ns
p < 0.01
35
x 106 Da
30
25
20
15
10
5
0
**
S
H
1080 1100 1120 1140 1160 1180 1200 1220
Temperature had a different impact on polymers size according to grain hardness and this has probably a strong impact on dough quality
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Conclusions
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Conclusions
• AFFFF provides more reliable data on polymers size than SE-HPLC
• Influence of PinB alleles on storage proteins polymers was observed in interaction with glutenins alleles
• Temperatures during the 2 last months impacted characteristics of polymers differently according to grain hardness
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Acknowledgements
Private partners: Saaten Union Research Syngenta Momont Florimond-Desprez Caussade semences
Gérard Branlard François-Xavier Oury
Larbi Rhazi Thierry Aussenac
Benoît Méléard
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Thank you
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Glu-A1 0 1 2*
HMW Glu-B1 6-8 7 7-8 7-9 13-16 14-15 17-18 6.1-22
Glu-D1 2-12 3-12 4-12 5-10
Glu-A3 a d ef
LMW Glu-B3 b b' c c' d f g j
Glu-D3 b c
Glutenins alleles in the experiment
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Falcon Hard-Soft NILs + equal Temp.
• Soft (PinA +, PinB +)
Mw2 : 20.4 106 Da
• Hard (PinA -, PinB +)
Mw2 : 28.5 106 Da
Hypothesis:
PinA absence increased aggregation
68 varieties + different Temp.
• Soft (PinA +, PinB +)
Average Mw2 : 17.6 106 Da
• Hard (PinA +, PinB-SNPs)
Average Mw2 : 13.0 106 Da
Hypothesis:
SNP-PinB decreased polymerization
Comparison of Mw2 in the 2 experiments
From Lesage et al., 2011. J. of Cereal Science and Lesage et al., 2012, J. Exp. Bot
From this experiment