Tailoring Grain Protein Composition for Wheat Using an ... · Tailoring Grain Protein Composition...

N U T R I T I O N A G R I C U L T U R E

E N V I R O N M E N T

U M R 1 0 9 5 G D E C

A G R O 2 0 1 0 – 31 Sept. 2010

Tailoring Grain Protein Composition for Wheat Using an Ecophysiological

Modeling Approach

Pierre Martre

INRA – Blaise Pascal UniversityUMR1095 Genetic, Diversity, and Ecophysiology of Cereals

Clermont-Ferrand, France



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6

8

10

12

14

16

GrainProtein

Concentration(% DM)

CookiesCakesPastriesJapanese noodlesFlat breadsChinese noodlesCrackers

Leavened breadPastaGluten and starch extraction

The Importance of Wheat Storage ProteinsLo

afVolu

me

(cm

3)

Flour protein (%)(Finney et al., 1948)



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Storage proteins (prolamins)

Gliadins Glutenins

ω-gliadins α,β-gliadins γ-gliadins LMW-GS HMW-GS

Low Optimum High

Loaf volumeGliadin to glutenin ratio

The Importance of Wheat Storage Proteins

% Protein 17%

HMW‐GS 19%

LMW‐GS8%Gliadins

11%

Hardness31%

Unexplained 14%

Allelic polymorphism of storage proteins account for 38% of the genetic variability

of dough strength

(Branlard et al., 2001)



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49 106 250 359 393 503 746 950

Division Remplissage Maturation

°Cdays49 106 250 359 393 503 746 950

DivisionDivision Storage (filling)Storage (filling) MaturationMaturation

°

Kinetics of Grain Protein AccumulationP

rote

in fr

actio

n(m

g N

gra

in-1)

0.0

0.1

0.2

0.3

0.4

0.5

Thermal time after anthesis (°Cd)

0 200 400 600 800 1000

Rat

e of

pro

tein

frac

tion

accu

mul

atio

n(m

g N

gra

in-1 °

Cd-1

)

0.000

0.002

0.004

0.006 Albumins-GlobulinsGlutenins

Gliadines

How could we model these kinetics and E and G effects on their parameters?

Could we find stable response curves to environmental and/or endogenous plant variables?

Can response curves help us better understand the environmental (E) and genetic (G) bases of the variations of grain protein composition?



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Outdoor controlledOutdoor controlled--environment conditionsenvironment conditions

2 day time post-anthesis temperatures(19°C and 28°C)

2 post-anthesis watering regimes(100% or 15% of ETP)

Materials and Growing Conditions

3 rates of N fertilisation

(0, 70, and 100 kg N ha-1)

FieldField

2 ear halving treatments

(at anthesis or 250 °Cdays later)

Yield (Mg ha-1) 7.8 6.2 6.8 4.1

Proteins (% DM) 13.1 12.7 15.0 15.4

Grain (×103 grains m-2) 22.4 18.4 16.1 10.8

Arche Récital Renan TamaroCultivars

E and G Effects on Grain N Allocation (1/2)



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Environmental effects (cv. Récital)

Genetic effects (optimal conditions)

Pro

tein

fra

ctio

ns

(mg

N g

rain

-1)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

ArcheRécitalRenanTamaro

Non-prolamins Gliadins

Glutenins Gliadins / glutenin

Days after anthesis0 10 20 30 40 50

0.0

0.1

0.2

0.3

0.4

0.5

Glia

din

to g

lute

nin

ratio

0.0

0.2

0.4

0.6

0.8

1.0

N allocation(mature grains)

Grain N (mg N grain-1)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Non-prolamins

Gliadins

Glutenins

Prot

ein

frac

tions

(mg

N g

rain

-1)

0.0

0.1

0.2

0.3

0.4

0.5

Y = 2.99 X0.684

r2 = 0.842

Y = 0.041 X1.295

r2 = 0.931

Y = 0.2502 X1.05

r2 = 0.952

0.4 0.6 0.8 1.0 1.2 1.40.0

0.1

0.2

0.3

0.4

0.5

Pro

tein

fra

ctio

ns

(mg

N g

rain

-1)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

ControlLow NHigh TemperatureDrought

Non-prolamins Gliadins

Glutenins Gliadins / glutenin

Days after anthesis0 10 20 30 40 50 60

0

100

200

300

400

500

600

Glia

din

to g

lute

nin

ratio

0.00.20.40.60.81.01.2

E and G Effects on Grain N Allocation (2/2)



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Crop simulation model ((SiriusSirius))

N, C Fluxes

Grain Number

Environmental var.(Rg, N, θ, H20, soil,…)

Single grain massSingle grain mass

Protein quantityProtein quantity

Protein compositionProtein composition

Protein Protein concentrationconcentration

Modeling Grain N Accumulation and Allocation (1/5)

(Eur. J. Agro. 2006, 25: 138-154)



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Gra

in C

or

N(m

g gr

ain-

1 )

Thermal time (°Cdays)

1. The accumulation of structural C is a function of thermal time and grain development.

Accumulation of Structural and Storage C and N

Main Hypotheses

(Plant Physiol. 2003, 133: 1959-1967)

Dcd Der

Structural C

Total NStorage N


2. structural C : N ratio is steady.

3. The accumulation of storage C (starch) and N (gliadins and glutenins) is source driven.



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(Plant Physiol. 2003, 133: 1959-1967)


Glia

dins

(mg

N g

rain

-1)

0.00.10.20.30.40.50.6

Total grain N(mg N grain-1)

0.0 0.3 0.6 0.9 1.2 1.5

Glu

teni

ns(m

g N

gra

in-1

)

0.00.10.20.30.40.50.6

Gliadins

Glutenins

Partitioning of Grain N

Main Hypotheses

4. The accumulation of grain storage proteins scales with total grain N

1. The accumulation of structural C is a function of thermal time and grain development stage.

2. structural C : N ratio is steady during grain growth. (Dreccer et al., 1997).

3. The accumulation of storage C (starch) and N (gliadins and glutenins) is source regulated.



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Pro

tein

fra

ctio

ns

(mg

N g

rain

-1)

0.00.10.20.30.40.50.6

0 10 20 30 40 50 60

Time after anthesis (days)0 10 20 30 40 50

0.00.10.20.30.40.5

Arche Récital

Renan Tamaro


Simulation of genetic differences in grain protein fraction accumulation under optimal growing conditions

AmphiphilicGliadinGluténin

Albumin-globulin



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Genetic and environmental variability of grain protein composition is mainly due to differences in N flux per grain

y = 0.94x + 21r2 = 0.87, MEP = 0.035

y = 1.25x + 82r2 = 0.93, MEP = 0.039

Protein fractions, observed (mg N grain-1)

0.0 0.2 0.4 0.6Prot

ein

frac

tions

, sim

ulat

ed(m

g N

gra

in-1

)

0.0

0.2

0.4

0.6

0.0 0.2 0.4 0.6

GluteninsGliadins

ArcheRécitalRenanTamaro

N0 L N1 19°C/14°CIrr

19°C/14°CDry

Field semi-controled conditions

28°C/15°C Irr


Simulation of genetic differences in grain protein fraction accumulation under optimal growing conditions



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Long arm Short arm

1A

1B

1D

6A

6B

6D

c

c

c

c

c

c

Gli-A2

Gli-B2

Gli-D2

α,β,γ-Gliadins

Glu-A3

Glu-B3

LMW-GS

Glu-D3

Gli-A5Gli-A3

Gli-A1 Gli-A6

Gli-B3

Gli-B1Gli-B5

Gli-D1

ω-gliadins

Glu-A1

Glu-B1

HMW-GS

Glu-D1

Major Wheat Storage Protein Loci



U M R 1 0 9 5 G D E C

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Grain N (mg N grain-1)0.8 1.0 1.2 1.4 1.6

0.10.20.30.40.50.60.70.8

0.8 1.0 1.2 1.4 1.6 1.8

Prot

ein

frac

tions

(m

g N

gra

in-1

)

NullNullxxyGlu-D1

xyxyxxyGlu-B1

NullxyNullxGlu-A1

Null-1A1DNull-1DNull-1AParental line

(LP)

LinesHMW-GS Loci

HMW-GS Gene Dosage Effect on Grain N Allocation

Near-isogenic lines for the number of HMW-GS genes in 7 spring wheat backgrounds

LPNull-1A Null-1DNull-1A1D

Gliadins Glutenins



U M R 1 0 9 5 G D E C

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The Origins of Cultivated Wheat

Triticum monococcum Aegylops sp. Triticum tauschii

AA BB DD

X

Wild durum wheat

Triticum durum : AABB

Cultivated durum wheatCultivated durum wheat

X

Wild hexaploid wheat

Triticum aestivum : AABBDD

Cultivated bread wheatCultivated bread wheat

NaturalNaturalselectionselection



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2 Triticum monococcum(2n = 2x = 14, AA)

3 Aegilops spp.(2n = 2x = 14, BB)

2 Triticum tauschii(2n = 2x = 14, DD) P

oly

plo

ids

Dip

loid

s

Effects of Ploidy Level on Grain N Allocation (1/3)

14 Triticum aestivum(2n = 6x = 42, AABBDD)

Courtot: 1 Parental Line

4 Isohomeoallelic lines

3 Triticum durum(2n = 4x = 28, AABB)

Gliadins Glutenins

Grain N (mg N grain-1)

0.0 0.3 0.6 0.9 1.2

Pro

tein

fra

ctio

ns

(mg

N g

rain

-1)

0.00.10.20.30.40.50.6

0.0 0.3 0.6 0.9 1.2 1.5



U M R 1 0 9 5 G D E C

A G R O 2 0 1 0 – 31 Sept. 2010




oly

plo

ids

Dip

loid

s






Total gliadins (mg N grain-1)

0.0 0.1 0.2 0.3 0.4

Glia

din

su

bu

nit

s(m

g N

gra

in-1

)

0.00

0.05

0.10

0.15

0.20

0.25α-, β-Gli γ-Gli ω-Gli

0.0 0.1 0.2 0.3 0.4 0.0 0.1 0.2 0.3 0.4 0.5



U M R 1 0 9 5 G D E C

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oly

plo

ids

Dip

loid

s






Total glutenins (mg N grain-1)

0.0 0.1 0.2 0.3 0.4 0.5Glu

ten

in s

ub

un

its

(mg

N g

rain

-1)

0.0

0.1

0.2

0.3

0.4LMW-GS HMW-GS

0.0 0.1 0.2 0.3 0.4 0.5



U M R 1 0 9 5 G D E C

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ω-Gliadins α,β,γ -Gliadins Gli-A1 Gli-B1 Gli-D1 Gli-A2 Gli-B2 Gli-D2

Récital o f b j p n

Renan f b g k m e

Cultivars Quality index

HMW-GS LMW-GS

Glu-A1 Glu-B1 Glu-D1 Glu-A3 Glu-B3 Glu-D3 Récital 62 2* 6 + 8 5 + 10 d g c

Renan 78 2* 7 + 8 5 + 10 a c b

Allelic Composition of the Parental Lines

Materials194 Double haploid lines (Récital × Renan)2 sites (Clermont-Ferrand, Rennes)

Genetic Bases of the Allometric Coefficients (1/2)



U M R 1 0 9 5 G D E C

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Y = a × Nb

b (dimensionless)0.6 0.8 1.0 1.2 1.4 1.6

0

20

40

60

80

100

a (mg N grain-1)0.10 0.12 0.14 0.16

Num

ber o

f lin

es

0

10

20

30

40

Genetic Bases of the Allometric Coefficients (2/2)

Protein Parameter h2 Chromosome Collocationfraction (%)

Gliadins a 52 1A Glu-A3 Gli-A1

Glutenins a 12 1A Glu-A3 Gli-A1

Grain N (mg N grain-1)0.3 0.6 0.9 1.2 1.5

Glu

tein

s(m

g N

gra

in-1

)

0.000.020.040.060.080.100.120.14

Glutenins (F1)

Grain N (mg N grain-1)0.3 0.6 0.9 1.2 1.5

Glia

dins

(mg

N g

rain

-1)

0.00

0.05

0.10

0.15

0.20

0.25Gliadins (F4)

Clermont-FerrandRennes



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The Synthesis of Grain Storage Proteins is Regulated at the Transcriptional Level

TFs regulating the synthesis of grain storage proteins are well conserved among cereal species

916

963

556

690

711

982

738

MAIZE O2

SHORGUME O2RICE

WHEAT SPA

BARLEY BLZ2

MAIZE OHP

BARLEY BLZ1WHEAT SPA2

Arabidoposis

RICE

916

963

556

690

711

982

738

MAIZE O2

SHORGUME O2

MAIZE O2

SHORGUME O2RICE

WHEAT SPA

BARLEY BLZ2

MAIZE OHP

BARLEY BLZ1WHEAT SPA2

Arabidoposis

RICE

SPA

SPA2

(Rubio-Somoza et al., 2006)

Barley

Can the trans-regulation of grain storage proteins by TFs explain the grain N scaling laws?



U M R 1 0 9 5 G D E C

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Nucleotide Polymorphism in SPA Influences Grain Protein Allocation (1/2)

(Plant Physiol., 2009, 151, 2133-2144)

5478 bp

41 mutations - 6 indels

1 mutation/136 bp

Mean r2 (LD)= 0.73

Identification of 2 major haplotypesfor each homeologous gene

Number of base pairs fromthe transcription start site

-2000-1000 0 1000 2000 3000 400005

101520253035N

ucle

otid

e di

vers

ity, π

(x

10-3

)

05

10152025303505

101520253035

Spa-D

Spa-B

Spa-A

Spa-A



U M R 1 0 9 5 G D E C

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Gli / Glu

Haplotype 1 0.49 + 0.04

Haplotype 2 0.67 + 0.03

P-value 0.0006

Glutenins (mg N grain-1)0.1 0.2 0.3 0.4

Glia

dins

(mg

N g

rain

-1)

0.1

0.2

0.3

0.4

Dough viscoelasticityTenacity Extensibility

Haplotype 1 43.7 106.1Haplotype 2 58.9 77.0P-value 0.005 0.006

(Plant Physiol. 2009, 151: 2133-2144)

Nucleotide Polymorphism in SPA Influences Grain Protein Allocation (2/2)

Thermal time after anthesis(°Cd above 0°C)

0 100 200 300 4000

1

2

3

Nor

mal

ized

exp

ress

ion

x 10

3

0

1

2

3

0

1

2

3

Spa-D

Spa-B

Spa-A

0 100 200 300 4000.00

0.05

0.10

0.15

0.20

0.25

0 100 200 300 4000.0

0.1

0.2

0.3

0.4

0.5

Haplotype 1Haplotype 2

Prot

ein

fract

ions

(mg

N g

rain

-1)

0.0

0.1

0.2

0.3

0.4

Total grain N (mg N grain-1)0.5 0.6 0.7 0.8 0.9 1.0 1.1

0.0

0.1

0.2

0.3

0.4

0.0

0.1

0.2

0.3

0.4

0.5

Glutenins

Gliadins

Non-prolamins



U M R 1 0 9 5 G D E C

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MCB1

HMWLMW

Chimericgliadins

SAD GAMYB SPA2

SPA

MYBS3 PBF

Transcriptional Regulatory Network of Grain Storage Protein Regulations

Grain N scaling laws are emergent properties of a transcriptional regulatory network



U M R 1 0 9 5 G D E C

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Concluding Remarks

Grain protein composition shows high G x (E x M) interactions

These interactions can be explained by simple scaling laws

As a consequence of these scaling laws grain protein concentration is primarily determined by the rate and duration of grain N accumulation => determined at the whole plant (canopy) level

But we could identify both natural and induced genetic variations in the parameters of the scaling laws of grain N allocations => “decorrelate” grain protein composition from total grain N => allows manipulating grain protein composition and grain N (concentration) independently (low input systems)

Scaling laws of grain N allocation are emergent properties of a regulatory transcriptional network

Analysing and modelling these regulatory networks in the frame of the scaling laws of grain N allocation can helps us “decorrelate” grain protein composition from total grain N and develop new ideoptypewith less total grain protein by with the right balance



U M R 1 0 9 5 G D E C

A G R O 2 0 1 0 – 31 Sept. 2010

UMR1095 GDEC, Clermont-Ferrand

François BalfourierGérard BranlardGilles CharmetCatherine RavelEugène Triboi

Vitalie SamoilZhanwu DaiAnne Plessis

Sibille PerrochonMireille DardevetNathalie Duchateau

Founding

Marie Agier

Gene network modelling

TransgenesisStéphane LafargeFrançois Torney

Acknowledgements

Tailoring Grain Protein Composition for Wheat Using an ... · Tailoring Grain Protein Composition...

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