Engineering high alpha-amylase levels in wheat grain lowers falling number but improves baking properties. Good afternoon everyone, For the next 15-20 minutes I will talk about alpha-amylase in wheat. I will try to describe 2 things: First I will explain why alpha-amylase is important in the context of the wheat industry in his current situation and Second I will present some results we produced within our research group that may change the perspective around alpha-amylase. Dr Jean-Philippe Ral CSIRO Agriculture

©CSIRO – Mark Fergus IWC 2015 conference| Jean-Philippe Ral

α-amylase Maltotriose α-Glucose Maltose….. Picture wheat grains and inside starch granules α-amylase exhydrolyses α-1,4 glycosidic bonds of the starch molecules. Action breaks starch molecule producing sugar molecules – energy plant to grow. Maltotriose α-Glucose Maltose….. IWC 2015 conference| Jean-Philippe Ral

Late Maturity Alpha-Amylase 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. This is the definition of LMA as described in the literature. LMA is a stochastic expression of a unique alpha-amylase, the AMY1 in the aleurone layer of developing grain. This increase of amylase remains at maturity. the screening is very difficult that LMA is triggered by environmental condition and affected grain does not have any morphological alteration. However, the increase of alpha-amylase activity decrease starch viscosity as shown in test such as Falling number test or RVA stirring number test. Finally LMA is thought to decrease 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 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

Crossing /backcrossing 200 crosses Time Steps Efforts Year 1 Crossing /backcrossing 200 crosses Year 2 Selection of elite plants 40 000 single plants Year 3 Seed increase 10 000 single plots Year 4 Stage 1 Yield Year 5 Reselection Year 6 Seed increase of reselection Year7 Stage 2 Yield Pure seed production (quality and disease data) 5 000 lines Year 8 200 lines Year 9 Stage 3 Yield 40 lines Classification including LMA test Year 10 Stage 4 Yield Bulk seed growers 8 lines Year 11 2 lines Year 12 RELEASE 1 line IWC 2015 conference| Jean-Philippe Ral

Some very important dates….. A Novel α-amylase Isoform (AMY3) Identified in Developing Grain 1987 LMA (AMY1) Consistently ranked the #1 priority trait by Australian Wheat Breeders Total α-amylase Activity - Germinating Grain 1977 2012 Present 1970 1985 2000 α-amylase Isoform Specific mRNA Identified in wheat (AMY 1 and AMY 2) Over 40 publications on Pre Harvest Sprouting AMY 2 Affected Grain (pubmed) 1975 I was born IWC 2015 conference| Jean-Philippe Ral

“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. IWC 2015 conference| Jean-Philippe Ral

Total a-amylase activity (ceralpha U/g) Wheat grain specific over expression of AMY3 Total a-amylase activity (ceralpha U/g) Parent Neg Seg Amy3over parent Neg Seg Amy3over 30 fold increase of a-amylase activity in over expressed AMY3 lines in average. IWC 2015 conference| Jean-Philippe Ral

Germination profile of high a-amylase grain Light Dark Coleorhiza Root IWC 2015 conference| Jean-Philippe Ral

Does AMY 3 over expression affect granule structure? 20um NEG SEG AMY3 over IWC 2015 conference| Jean-Philippe Ral

Total a-amylase activity level throughout grain development IWC 2015 conference| Jean-Philippe Ral

AMY3 activity level throughout grain development IWC 2015 conference| Jean-Philippe Ral

Impact of high a-amylase on grain composition 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) Soluble carbohydrate Glucose Sucrose mg/g flour IWC 2015 conference| Jean-Philippe Ral

AMY 3 over-expression affects starch viscosity Peak breakdown Final Viscosity can be restored by addition of amylase inhibitor (Silver Nitrate) 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 A B C D E Neg seg A3OE No baking imp No improver + baking improver No baking imp with baking imp IWC 2015 conference| Jean-Philippe Ral

Impact on Loaf Color RED YELLOW Light/dark A E B C D Neg seg Amy3over No improver + baking improver RED Neg seg YELLOW Amy3over Light/dark IWC 2015 conference| Jean-Philippe Ral

Conclusions Future Work 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. 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 IWC 2015 conference| Jean-Philippe Ral

Why these results are important: Question the assumption that high a-amylase alone has detrimental effects on baking quality and end-products. Reinforce the need for a thorough investigation into the effect of high a-amylase level on wheat end-product quality. 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 Funding: CSIRO AGRICULTURE IWC 2015 conference| Jean-Philippe Ral

Thank you! IWC 2015 conference| Jean-Philippe Ral 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