Genetic improvement of wheat to reduce the potential for acrylamides

Abstract

Free asparagine concentration, which is the determining factor for acrylamide-forming potential in cereals, was measured in grain from wheat grown in field trials in 2011-2012 and 2012-2013. There were 25 varieties in 2012 and 59 in 2013, with eleven present in both trials. Eight varieties were identified as having consistently low free asparagine concentration. There was a differential response of varieties to sulphur, and much higher levels of free asparagine in 2012-2013 versus 2011-2012. A key conclusion of this study was that, given the short commercial lifespan of some wheat varieties, information on free asparagine concentration should be made available when a variety is launched.   The effect of fungicide application on free asparagine accumulation in wheat grain was also investigated. Flour was analysed from 24 varieties grown in adjacent plots that were treated in identical fashion except that fungicide was applied to one and not the other. Lack of fungicide resulted in visible infection by Septoria tritici, yellow rust and brown rust.  Free asparagine concentration was much lower in the fungicide-treated wheat than the untreated wheat, resulting in less acrylamide formation in flour when it was heated. The study showed disease control by fungicide application to be an important crop management measure for mitigating the problem of acrylamide formation in wheat products. Asparagine synthesis occurs by the amidation of aspartate, catalysed by asparagine synthetase, and the expression of asparagine synthetase (TaASN) genes in wheat was therefore studied. The expression of three genes, TaASN1-3, was measured in different tissues and in response to nitrogen and sulphur supply.  The expression of TaASN2 in the grain during mid to late development was the highest of any of the genes in any tissue. Both TaASN1 and TaASN2 increased in expression through grain development, and in the grain of field-grown plants during mid-development in response to sulphur deprivation. However, only TaASN1 was affected by nitrogen or sulphur supply in pot-based experiments, showing complex responses.  A possible regulatory motif was found in the promoter of TaASN1 genes from several cereal species. As the study was completed, a fourth gene, TaASN4, was identified from recently available genome data.  Phylogenetic analysis showed that other cereal species have similar asparagine synthetase gene families to wheat. TaASN1 and TaASN2 were used to produce their encoded proteins in Escherichia coli. The proteins were shown to react with two monoclonal antibodies raised to distinct epitopes. The reaction catalysed by asparagine synthetase was modelled using publicly-available data from various species to generate a series of differential equations to describe the reaction stages.  The TaASN1 and TaASN2 proteins were purified and found to be active, synthesising asparagine and glutamate from glutamine and aspartate. Data from the reactions was entered into the model, enabling values to be determined for kinetic parameters within the differential equations. A network describing asparagine metabolism was developed and used to identify networks of genes responding to stress in wheat. The network is also being used to filter RNAseq datasets to enable the comparison of high and low asparagine genotypes.