effects of hmw- & lmw-glutenins and grain hardness on size of wheat storage proteins polymers

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

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

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?

Material and

methods

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)

Methods. Asymmetrical flow field-flow fractionation

AFFFF-MALLS analytical device used for molecular characterization of storage proteins

(Multi Angular Laser Light Scattering)

Principle


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


(Multi Angular Laser Light Scattering detector)

Polymers size and conformation

MALLS chromatogram

Polymers Monomers


Measurements: - Mw: weight-average - Mn: number-average - Mw/Mn: polydispersity index - Rw: weight-average mean square radius (radius of giration)

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.

Results

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)

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

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

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

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

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

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?

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)

%

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

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

Conclusions

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

Acknowledgements

Private partners: Saaten Union Research Syngenta Momont Florimond-Desprez Caussade semences

Gérard Branlard François-Xavier Oury

Larbi Rhazi Thierry Aussenac

Benoît Méléard

Thank you

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

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

effects of hmw- & lmw-glutenins and grain hardness on size of wheat storage proteins polymers

Technology