1 | engineering high alpha-amylase levels in wheat grain lowers falling number but improves baking...

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Engineering high alpha-amylase levels in wheat grain lowers falling number but improves baking properties.

Dr Jean-Philippe Ral

CSIRO Agriculture

©CSIRO – Mark Fergus

IWC 2015 conference| Jean-Philippe Ral

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Maltotriose α-Glucose

Maltose…..


α- amyl

ase

α-amylase

α-amylase

α-amylase

Late Maturity Alpha-Amylase

Currently ranked the #1 priority trait by Australian Wheat Breeders


Definition of LMALMA is defined by:

• Increased activity of a specific a-amylase isoform during the final stage of the grain development.

• Stochastic expression of a single a-amylase (AMY1) located in the aleurone layer of wheat grain.

• Increase in a-amylase activity does not have any significant detrimental effect on grain morphology, development or germination.

• Decreases starch viscosity in a Falling Number or RVA stirring number test.

• Thought to be associated with decreased end product quality.


LMA is not Pre Harvest Sprouting

• PHS is not equivalent to Late Maturity a-amylase (LMA).

• PHS involves expression of a-amylases including AMY1 but also a range of other proteolytic, cell wall degrading enzymes and other amylases.

• PHS does decrease wheat quality.

• Decreases starch viscosity in a falling number or RVA stirring number test


Flour Falling Number Test

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www.Perten.comIWC 2015 conference| Jean-Philippe Ral

LMA is important

•High starch viscosity is widely used by grain purchasers as an indicator of both grain soundness and intrinsic grain quality.

• Absence of LMA expression is considered a key classification criterion.

• Breeders are very frustrated by any inability to commercialise otherwise high performance lines due to LMA issues.


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Time Steps Efforts

Year 1

Year 2

Year 3

Year 4

Year 5

Year 10

Year 9

Year 8

Year7

Year 6

Year 12

Year 11

Stage 1 Yield

Seed increase of reselection

Crossing /backcrossing

Selection of elite plants

Seed increase

Stage 2 Yield Pure seed production

(quality and disease data)

Stage 3 Yield

Stage 4 YieldBulk seed growers

RELEASE

Reselection

Classification including LMA test

200 crosses

5 000 lines

40 000 single plants

10 000 single plots

200 lines

40 lines

8 lines

2 lines

1 line


LMA (AMY1)Consistently ranked the

#1 priority trait by Australian Wheat

Breeders

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Present 1970

Total α-amylase Activity - Germinating

Grain

1977

α-amylase Isoform Specific mRNA

Identified in wheat(AMY 1 and AMY 2)

1985

1987

A Novel α-amylase Isoform (AMY3) Identified in

Developing Grain

1975 I was born

2000

Over 40 publications on Pre Harvest Sprouting AMY 2 Affected Grain

(pubmed)

2012

Some very important dates…..


“Endosperm specific” over expression of the Alpha-amylase 3 in wheat

• Seed specific AMY3 over expression constructs developed.

• Over 20 transgenic lines produced.

• Stable low copy number lines selected for characterisation.

12 | IWC 2015 conference| Jean-Philippe Ral

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Wheat grain specific over expression of AMY3

BW26 A4.4 A10.40

5

10

15

20

25

30

35

40

Parent Neg Seg Amy3over

parent Neg Seg Amy3over

30 fold increase of a-amylase activity in over expressed AMY3 lines in average.

Tota

l a-a

myl

ase

activ

ity (c

eral

pha

U/g

)


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Germination profile of high a-amylase grain

Light Dark

Coleorhiza

Root


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20um

NEG SEG

AMY3 over

Does AMY 3 over expression affect granule structure?


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10 DPA 15 DPA 20 DPA 25 DPA 30 DPA0

1

2

3

4

5

6

7

Tota

l alp

ha-a

myl

ase

activ

ity (u

nit/

g flo

ur)

Total a-amylase activity level throughout grain development


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10 DPA 15 DPA 20 DPA 25 DPA 30 DPA0

2

4

6

8

10

12

A10.4AN A4.4

Tota

l alp

ha-a

myl

ase

activ

ity (u

nit/

g fl

our)

AMY3 activity level throughout grain development


Impact of high a-amylase on grain composition

• No significant alteration of starch content or composition.• No significant change in total protein content.

A4 A4 N A10 A10 N

A17 A17 N

TCC0

4

8

12

Flour protein content

% d

ry w

eigh

t

6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57012345678

Starch Chain Length

DP

Rela

tive

%


Impact of high a-amylase on grain composition• 10 to 30 fold increase of total amylase activity in dry grain.• No significant alteration of starch content• No significant change in total protein content• Increase in soluble carbohydrate (from 1 to 3% dry weight)

Soluble carbohydrate Glucose Sucrose

mg/

g flo

ur


AMY 3 over-expression affects starch viscosity

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Peak

breakdown

Final

Viscosity can be restored by addition of amylase inhibitor (Silver Nitrate)


Definition of LMA

LMA is defined by:

• Increased activity of a specific a-amylase isoform during the final stage of the grain development.

• Stochastic expression of a single a-amylase (AMY1) located in the aleurone layer of wheat grain.

• Increase in a-amylase activity does not have any highly significant detrimental effect on grain morphology, development or germination.

• Decreases starch viscosity ….

…..in a falling number or RVA stirring number test.


Do high levels of a-amylase impact quality?

• 10 to 30 fold increase of total a-amylase activity in dry grain.• No significant alteration of starch content, protein content.• High increase in soluble sugars (Sucrose and Glucose).

Sound

Sprouted

Severely Sprouted


A B

C D

E

No improver + baking improver

A3OENeg seg

No baking imp

with baking imp

Impact on Loaf Volume


A

B

C

D

E

No improver + baking improver

Impact on Loaf Color

YELLOW

RED

Light/dark

Neg seg

Amy3over


Conclusions1. Generated wheat lines with high level of a-amylase activity in

wheat grain that could be classified as heavily sprouted according to the Falling number test.

2. High a-amylase wheat lines generated a “self-improved flour” with increase in loaf volume and colour (in small scale baking).

Large Scale Study to complete the assessment of AMY3overexpression on bread quality (Ash, Cell structure, etc…)


Future Work

Why these results are important:

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1. Question the assumption that high a-amylase alone has detrimental effects on baking quality and end-products.

2. Reinforce the need for a thorough investigation into the effect of high a-amylase level on wheat end-product quality.


CSIRO AGRICULTURE

AcknowledgementsProject Contributors

Alex WhanMarcus NewberryJos MieogAndrew BowermanJeni PritchardEmmett LeyneOscar LarroqueAnne-Sophie DielenRosemary White

Crispin HowittPhil LarkinMatthew Morell

Funding:


CSIRO AgricultureDr jean-Philippe RalEngineering high alpha-amylase levels in wheat grain lowers falling number but improves baking properties.t +61 2 6246 5245e [email protected] www.csiro.au

Thank you!


1 | engineering high alpha-amylase levels in wheat grain lowers falling number but improves baking...

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