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

1. 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

2. Dough quality
 Nature of gliadins and glutenins alleles
 Polymerization
Cys: intramolecular
disulfide bonds
g-gliadin
Cys: intermolecular
disulfide bonds
PS N Repeat C LMW-Glutenin
PS N
Repeat C
HMW-GS type x
Polymers size depends on free cystine number
Repeat LMW-Glu
and also on environmental conditions
PS N C
PS N
Repeat C HMW-GS type y
PS N
Repeat C HMW-GS type y
PS N Repeat C LMW-Glu
From Köhler P. et al., 1993. Z. Lebensm. Unters. Forsch

3. Polymerization/aggregation
Formation of
insoluble polymers
during wheat kernel
Influence of puroindolines development
X 10 Da Molecular weight 6
and glutenins alleles
40 4
35
on polymers size?
30 NILs
3.5
3
25
Mw x 107 g/mol
2.5
20 2
Source Carceller JL, Aussenac T., 2001. Aust. J. Plant Physiol
hard x
PinA
15
1.5 soft PinA
10 1
5
0.5
00
1997 2008 2010
From Lesage et al., 2011, J. Cereal Science, 53(2), 231

4. Material
and
methods

5. Material and methods
Material 2009:
L1
68 bread-wheat varieties L2
2 years (L1 –L6) L3
3 locations 2010:
L4
L5
Methods / Investigated traits L6
 HMW- and LMW- Glutenin alleles (SDS-PAGE)
 Grain hardness (NIRS)
 Polymer mass (Mw) and radius of giration (Rw) AFFFF
 Polydispersity index (Mw/Mn)
 % protein fractions (SE-HPLC)
 protein content (NIRS)
 puroindolines alleles (sequencing)

6. Methods. Asymmetrical flow field-flow fractionation
AFFFF-MALLS analytical device used for molecular characterization
of storage proteins
(Multi Angular
Laser Light Scattering)

7. Methods. Asymmetrical flow field-flow fractionation
Principle

8. Methods. Asymmetrical flow field-flow fractionation
Sample preparation
15 sec.
60°C – 15 min
sonication
20 W
30 mg flour
1 ml sodium phosphate buffer 0.1M, pH 6.9
(Multi Angular
2% SDS
Laser Light Scattering
detector)
Polymers
size and
conformation

9. Methods. Asymmetrical flow field-flow fractionation
MALLS chromatogram
Monomers Polymers
Measurements:
- Mw: weight-average
- Mn: number-average
- Mw/Mn: polydispersity index
- Rw: weight-average mean square radius
(radius of giration)

10. Methods. Size Exclusion - HPLC
Sample preparation
α-Gli, 60°C – 80 min stirring
Alb,
Glo.
γ,β-
Gli 160 mg flour
Large Small F4 F5 20 ml sodium phosphate buffer 0.1M
Glu Glu Ω- 1% SDS
Pol. Pol. Gli
F1 F2 F3 SE-HPLC
filtration TSK gel G4000sw
3 min
0.2 µm Silica-based,
sonication pore size: 13-17 µm
2W
Elution Time 5 protein fractions (F1- F5)

11. Results

12. Variation of polymers characteristics
AFFFF
5.4 - 48.8 million Da
Number Number
7.1 – 82.9
Number
36.5 – 116.2 nm
60 80 40
50
60 30
40
30 40 20
20
20 10
10
0 0 0
0 1 2 3 4 5 0 20 40 60 80 100 20 40 60 80 100 120 140
(X 1,E7)
Mw 2 Mw /Mn2 Rw 2 (nm) (nm)
Mw2 Mw/Mn2 Rw2
(Polymers mass) (Polydispersity index) (Polymers radius)
SE-HPLC
0.75 - 2 million Da 90.000 – 750.000 Da 50.000 – 90.000 Da
Number Number
Number
40 30
50
25
40
30
20
30
20 15
20 10
10
10 5
0 0
0
17 19 21 23 25 27 29 5,9 6,9 7,9 8,9 9,9
10 12 14 16 18
SE-HPLC F2 % SE-HPLC F3 %
SE-HPLC F1 %
Mean F1 : 14% Mean F2 : 24% Mean F3 : 7%

13. Glutenin loci effects on quality traits
Protein Gluten Grain
Mw2 Mw/Mn2 Rw2 %F1 %F2 %F3 %F4 %F5
content Index Hardness
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

14. Glutenin loci effects on quality traits
Protein Gluten Grain
Mw2 Mw/Mn2 Rw2 %F1 %F2 %F3 %F4 %F5
content Index Hardness
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

15. - 0.16
- 0.06
0.14
0.24
0.04
-0,16
-0,06
0,04
0,14
0,24
GluA1-n
Standardized coef.
GluA1-1
Glu-A1
GluA1-2*
GluB1-68
Standardized coef.
GluB1-7
GluB1-78
GluB1-79
Glu-B1
GluB1-1316
GluB1-1415
GluB1-1718
GluB1-6.1-22
GluD1-212
B1 (7)
GluD1-312
Mw2
Glu-D1
GluD1-412
GluD1-510
GluA3-a
GluA3-ef
GluA3-d
Glu-A3 GluB3-b
GluB3-bp
GluB3-c
GluB3-cp
GluB3-d
GluB3-f
Glu-B3
GluB3-g
GluB3-j
GluD3-b
GluD3-c
Glu-D3
D3 (b)
Mw2
Effects of Glutenins alleles on polymers mass

16. 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
AA position from start: 73 75 89
PinB-D1a WPTKWWKGGC-----------L Allele of Soft varieties
PinB-D1b WPTKWWKSGC-----------L
PinB-D1d WPTKWRKGGC-----------L
PinB-D1c WPTKWWKGGC-----------P
PinB-D1b + d WPTKWRKSGC-----------L

17. 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
Grain Hardness Class
PinB
D1a
D1b
S
D1d
D1c
PinB-D1a WPTKWWKGGC-----------L D1b+ d
PinB-D1b WPTKWWKSGC-----------L M
PinB-D1d WPTKWRKGGC-----------L
H
PinB-D1c WPTKWWKGGC-----------P
0 20 40 60 80
PinB-D1b + d WPTKWRKSGC-----------L Occurence

18. 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

19. 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

20. Effects of interaction between glutenins and puroindoline B alleles
x 106 Da
Mw2
Mw2 x 106 Da
Mw2
Mw2
(X 1,E6) PinB (X 1,E6) PinB
2929 PinB
Pinb-3al
D1a
2121 PinB
Pinb-3al
D1a
2424 D1b D1b
1919
___ D1a
D1d
1919 ___ D1a
D1d
1414 ___ D1b 1717 ___ D1b
99 ___ D1d 1515
___ D1d
44
-1 1313 a d ef
7 68 78 79 1316 1718 6122
7-8Glu-B1 a Glu-A3
Glu-B1 Glu-A3
Part of environmental effects?
 Interactions Glutenins-Puroindolines had effects on polymers characteristics
 Effects were contrasted depending on each allele
 Small part of polymers characteristics variation

21. Environmental effects on quality traits
Variance analysis (68 varieties, 2 years)
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

22. Environmental effects on quality traits
Effects of sum of mean temperatures (June and July)
AFFFF gives more reliableeffect forR2 %
data native polymers characterization than SE-HPLC
that reflects more diversity of glutenins
Protein content ns 6 Mw2
x 10Mw2
Da
Grain Hardness ns (X 1,E7)
50 5
Gluten Index ns
40 4
Mw2 *** 38.5
Mw/Mn2 *** 62.7 30 3
Rw2 *** 47.5 20 2
%F1 ns 10 1
%F2 ns 00
%F3 ns 1080 1100 1120 1140 1160 1180
1080 1100 1120 Ju l (癈 ) 1160 1180
SMT-Jun 1140
%F4 ns
%F5 ns Sum of mean temperatures (°C)
(June and July)

23. Regression Mw2 on sum of daily mean temperatures of June and July
x 106 Da Mw2
35
3.50E+07
S
30
3.00E+07
p < 0.01
25
2.50E+07
**
20 H
2.00E+07 Soft
ns
15
1.50E+07
Medium
ns
Temperature had a different impact on polymers size according to grain hardness
10
Hard
1.00E+07
and this has probably a strong impact on dough quality
5
5.00E+06
0
0.00E+00
1080 1100 1120
1080 1100 1120 1140 1160 1180 1200 1220
1140 1160 1180 1200 1220
Sum mean temp. (°C)
Soft : Mw2= -203207000+196542*SumTemp R²=0.37
Medium : Mw2= -172673000+167021*SumTemp R²=0.48
Hard : Mw2= -124367000+121462*SumTemp R²=0.44

24. Conclusions

25. 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

26. Acknowledgements
Gérard Branlard Larbi Rhazi Benoî Méléard
t
François-Xavier Oury Thierry Aussenac
Private partners:
Saaten Union Research
Syngenta
Momont
Florimond-Desprez
Caussade semences

27. Thank you

28. Glutenins alleles in the experiment
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

29. Comparison of Mw2 in the 2 experiments
Falcon Hard-Soft NILs + equal Temp. 68 varieties + different Temp.
• Soft (PinA +, PinB +) • Soft (PinA +, PinB +)
Mw2 : 20.4 106 Da Average Mw2 : 17.6 106 Da
• Hard (PinA -, PinB +) • Hard (PinA +, PinB-SNPs)
Mw2 : 28.5 106 Da Average Mw2 : 13.0 106 Da
Hypothesis: Hypothesis:
PinA absence  increased SNP-PinB  decreased
aggregation polymerization
From Lesage et al., 2011. J. of Cereal Science From this experiment
and Lesage et al., 2012, J. Exp. Bot