Engineering high alpha-amylase levels in wheat grain
lowers falling number but improves baking properties.
Dr Jean-Philippe Ral
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CSIRO Agriculture
©CSIRO – Mark Fergus
IWC 2015 conference| Jean-Philippe Ral
α-amylase
α-amylase
IWC 2015 conference| Jean-Philippe Ral
α-amylase
α-amylase
3 |
Maltotrios
e α-Glucose
Maltose…..
Late Maturity AlphaAmylase
Currently ranked the #1 priority trait
by Australian Wheat Breeders
IWC 2015 conference| Jean-Philippe Ral
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 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.
IWC 2015 conference| Jean-Philippe Ral
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
IWC 2015 conference| Jean-Philippe Ral
Flour Falling Number Test
www.Perten.com
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IWC 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.
IWC 2015 conference| Jean-Philippe Ral
Time
Steps
Year 1
Crossing /backcrossing
Year 2
Selection of elite plants
40 000 single plants
Year 3
Seed increase
10 000 single plots
Year 4
Year 5
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Efforts
Stage 1 Yield
Year 6
Seed increase of reselection
Year7
Year 8
Stage 2 Yield
Pure seed production
(quality and disease data)
Year 9
Stage 3 Yield
Year 10
Year 11
Stage 4 Yield
Bulk seed growers
Year 12
RELEASE
IWC 2015 conference| Jean-Philippe Ral
200 crosses
Reselection
5 000 lines
200 lines
Classification
including
LMA test
40 lines
8 lines
2 lines
1 line
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Some very important dates…..
A Novel α-amylase Isoform
(AMY3) Identified in
Developing Grain
LMA (AMY1)
Consistently ranked the
#1 priority trait
by Australian Wheat
Breeders
2012
1987
Total α-amylase
Activity - Germinating
Grain
1977
Present
1970
1985
1975
I was born
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α-amylase Isoform
Specific mRNA
Identified in wheat
(AMY 1 and AMY 2)
IWC 2015 conference| Jean-Philippe Ral
2000
Over 40 publications on
Pre Harvest Sprouting
AMY 2 Affected Grain
(pubmed)
“Endosperm specific” over expression of the Alphaamylase 3 in wheat
• Seed specific AMY3 over expression constructs developed.
• Over 20 transgenic lines produced.
• Stable low copy number lines selected for characterisation.
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IWC 2015 conference| Jean-Philippe Ral
Wheat grain specific over expression of AMY3
45
Total a-amylase activity (ceralpha U/g)
40
Parent
Neg Seg
Amy3over
35
30
25
20
15
10
5
0
BW26
parent
A4.4
30 fold increase of a-amylase activity in over expressed
AMY3 lines in average.
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IWC 2015 conference| Jean-Philippe Ral
A10.4
Neg Seg Amy3over
Germination profile of high a-amylase grain
Light
Coleorhiza
Root
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IWC 2015 conference| Jean-Philippe Ral
Dark
Does AMY 3 over expression affect granule
structure?
NEG SEG
20um
AMY3 over
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IWC 2015 conference| Jean-Philippe Ral
Total a-amylase activity level throughout grain
development
7
Total alpha-amylase activity (unit/g flour)
6
5
4
3
2
1
0
10 DPA
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IWC 2015 conference| Jean-Philippe Ral
15 DPA
20 DPA
25 DPA
30 DPA
AMY3 activity level throughout grain development
Total alpha-amylase activity (unit/g flour)
12
10
8
A10.4
6
AN A4.4
4
2
0
10 DPA
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IWC 2015 conference| Jean-Philippe Ral
15 DPA
20 DPA
25 DPA
30 DPA
Impact of high a-amylase on grain composition
8
Starch Chain Length
Relative %
6
4
2
0
6 8 101214161820222426283032343638404244464850525456
DP
Flour protein content
% dry weight
20
16
12
8
4
0
A4
A4 N
A10 A10 N A17 A17 N TCC
• No significant alteration of starch content or composition.
• No significant change in total protein content.
IWC 2015 conference| Jean-Philippe Ral
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)
mg/g flour
Soluble carbohydrate
IWC 2015 conference| Jean-Philippe Ral
Glucose
Sucrose
AMY 3 over-expression affects starch viscosity
Final
Peak
breakdown
Viscosity can be restored by addition of amylase inhibitor (Silver Nitrate)
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IWC 2015 conference| Jean-Philippe Ral
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.
IWC 2015 conference| Jean-Philippe Ral
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
IWC 2015 conference| Jean-Philippe Ral
Impact on Loaf Volume
A3OE
Neg seg
A
B
C
D
E
No
baking
imp
with
baking
imp
No improver
IWC 2015 conference| Jean-Philippe Ral
+ baking improver
Impact on Loaf Color
A
E
RED
Neg
seg
B
YELLOW
C
Amy3over
D
Light/dark
No improver
IWC 2015 conference| Jean-Philippe Ral
+ baking improver
Conclusions
1. 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).
Future Work
Large Scale Study to complete the assessment of
AMY3overexpression on bread quality (Ash, Cell structure, etc…)
IWC 2015 conference| Jean-Philippe Ral
Why these results are important:
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.
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IWC 2015 conference| Jean-Philippe Ral
Acknowledgements
Project Contributors
Alex Whan
Marcus Newberry
Jos Mieog
Andrew Bowerman
Jeni Pritchard
Emmett Leyne
Oscar Larroque
Anne-Sophie Dielen
Rosemary White
Crispin Howitt
Phil Larkin
Matthew Morell
CSIRO AGRICULTURE
IWC 2015 conference| Jean-Philippe Ral
Funding:
Thank you!
CSIRO Agriculture
Dr jean-Philippe Ral
Engineering high alpha-amylase levels in wheat grain
lowers falling number but improves baking properties.
t +61 2 6246 5245
e jean.ral@csiro.au
w www.csiro.au
IWC 2015 conference| Jean-Philippe Ral