Genome editing for low acrylamide wheat

Acrylamide (C3H5NO) is a food processing contaminant that has been classed as a probable (Group 2a) human carcinogen. Acrylamide forms from the reaction of free (non-protein) asparagine with reducing sugars during food processing. All major cereal products are affected and wheat products represent one of the main sources of dietary acrylamide intake in Europe. Asparagine concentration is the determining factor for acrylamide formation in cereal products. Asparagine biosynthesis is catalysed by a family of enzymes called asparagine synthetases (ASNs). The ASN genes were investigated and five ASN genes (TaASN1-4, with a double copy of TaASN3) identified in wheat (Triticum aestivum), with TaASN2 showing grain-specific expression. CRISPR/Cas9 was used to knock out the TaASN2 gene of wheat cv. Cadenza. A polycistronic gene containing four gRNAs, interspaced with tRNAs, was designed and introduced into wheat embryos by particle bombardment. The subsequent edits were characterised in the T1 and T2 generations using Next Generation Sequencing nucleotide sequence analysis. Triple (A, B, and D genome) nulls were identified, alongside an AD and an A genome null. Amino acid concentrations were measured in the T2 and T3 seed, with one triple null line showing a substantial reduction in the free asparagine concentration in the grain (90 % in the T2 seed and 50 % in the T3 seed compared with wildtype). The free asparagine also reduced as a proportion of the total free amino acid pool. Significant effects were also seen in glutamate and aspartate concentrations. Free asparagine and total free amino acid concentrations were higher in the T3 than T2 seeds, probably due to heat stress, but the concentrations in the edited plants remained substantially lower than in wildtype. Some of the edited lines showed poor germination, but this could be overcome by application of exogenous asparagine and no other phenotype was noted

Stress, nutrients and genotype: understanding and managing asparagine accumulation in wheat grain

Plant stress and poor crop management strategies compromise the foundations of food security: crop yield, nutritional quality and food safety. Accumulation of high concentrations of the amino acid asparagine in its free (soluble, non-protein) form is an example of an undesirable outcome of stress for the nutritional quality and food safety of wheat because of its role as a precursor to acrylamide, a carcinogenic processing contaminant. In this review, we cover what is known about the mechanisms and functions of free asparagine accumulation in the grain during normal development and particularly during stress in wheat. Comparisons with other plant species, yeast, and mammals are drawn in order to gain deeper insight into the conserved biology underlying asparagine accumulation. Crop management strategies and practices are discussed in the context of managing asparagine accumulation, which must be balanced against other desirable goals, such as sustainability, protein content and yield

Understanding the relationships between free asparagine in grain and other traits to breed low-asparagine wheat

Since the discovery of acrylamide in food, and the identification of free asparagine as the key determinant of acrylamide concentration in wheat products, our understanding of how grain asparagine content is regulated has improved greatly. However, the targeted reduction in grain asparagine content has not been widely implemented in breeding programmes so far. Here we summarise how free asparagine concentration relates to other quality and agronomic traits and show that these relationships are unlikely to pose major issues for the breeding of low-asparagine wheat. We also outline the strategies that are possible for the breeding of low-asparagine wheat, using both natural and induced variation

Reduced free asparagine in wheat grain resulting from a natural deletion of TaASN-B2: investigating and exploiting diversity in the asparagine synthetase gene family to improve wheat quality

Background: Understanding the determinants of free asparagine concentration in wheat grain is necessary to reduce levels of the processing contaminant acrylamide in baked and toasted wheat products. Although crop management strategies can help reduce asparagine concentrations, breeders have limited options to select for genetic variation underlying this trait. Asparagine synthetase enzymes catalyse a critical step in asparagine biosynthesis in plants and, in wheat, are encoded by five homeologous gene triads that exhibit distinct expression profiles. Within this family, TaASN2 genes are highly expressed during grain development but TaASN-B2 is absent in some varieties. Results: Natural genetic diversity in the asparagine synthetase gene family was assessed in different wheat varieties revealing instances of presence/absence variation and other polymorphisms, including some predicted to affect the function of the encoded protein. The presence and absence of TaASN-B2 was determined across a range of UK and global common wheat varieties and related species, showing that the deletion encompassing this gene was already present in some wild emmer wheat genotypes. Expression profiling confirmed that TaASN2 transcripts were only detectable in the grain, while TaASN3.1 genes were highly expressed during the early stages of grain development. TaASN-A2 was the most highly expressed TaASN2 homeologue in most assayed wheat varieties. TaASN-B2 and TaASN-D2 were expressed at similar, lower levels in varieties possessing TaASN-B2. Expression of TaASN-A2 and TaASN-D2 did not increase to compensate for the absence of TaASN-B2, so total TaASN2 expression was lower in varieties lacking TaASN-B2. Consequently, free asparagine concentrations in field-produced grain were, on average, lower in varieties lacking TaASN-B2, although the effect was lost when free asparagine accumulated to very high concentrations as a result of sulphur deficiency. Conclusions: Selecting wheat genotypes lacking the TaASN-B2 gene may be a simple and rapid way for breeders to reduce free asparagine concentrations in commercial wheat grain

Cereal asparagine synthetase genes

Asparagine synthetase catalyses the transfer of an amino group from glutamine to aspartate to form glutamate and asparagine. The accumulation of free (non-protein) asparagine in crops has implications for food safety because free asparagine is the precursor for acrylamide, a carcinogenic contaminant that forms during high-temperature cooking and processing. Here we review publicly-available genome data for asparagine synthetase genes from species of the Pooideae subfamily, including bread wheat and related wheat species (Triticum and Aegilops spp.), barley (Hordeum vulgare) and rye (Secale cereale) of the Triticeae tribe. Also from the Pooideae subfamily: brachypodium (Brachypodium dystachion) of the Brachypodiae tribe. More diverse species are also included, comprising sorghum (Sorghum bicolor) and maize (Zea mays) of the Panicoideae subfamily, and rice (Oryza sativa) of the Ehrhartoideae subfamily. The asparagine synthetase gene families of the Triticeae species each comprise five genes per genome, with the genes assigned to four groups: 1, 2, 3 (subdivided into 3.1 and 3.2) and 4. Each species has a single gene per genome in each group, except that some bread wheat varieties (genomes AABBDD) and emmer wheat (Triticum dicoccoides; genomes AABB) lack a group 2 gene in the B genome. This raises questions about the ancestry of cultivated pasta wheat and the B genome donor of bread wheat, suggesting that the hybridisation event that gave rise to hexaploid bread wheat occurred more than once. In phylogenetic analyses, genes from the other species cluster with the Triticeae genes, but brachypodium, sorghum and maize lack a group 2 gene, while rice has only two genes, one group 3 and one group 4. This means that TaASN2, the most highly expressed asparagine synthetase gene in wheat grain, has no equivalent in maize, rice, sorghum or brachypodium. An evolutionary pathway is proposed in which a series of gene duplications gave rise to the five genes found in modern Triticeae species

The Virtual Care Experience of Patients Diagnosed With COVID-19

Virtual models of care are seen as a sustainable solution to the growing demand for health care. This paper analyses the experience of virtual care among patients diagnosed with COVID-19 in home isolation or health hotel quarantine using a patient-reported experience questionnaire. Results found that patients respond well to virtual models of care during a pandemic. Lessons learned can inform future developments of virtual care models

Reducing dietary acrylamide exposure from wheat products through crop management and imaging

The nutritional safety of wheat-based food products is compromised by the presence of the processing contaminant acrylamide. Reduction of the key acrylamide precursor, free (soluble, non-protein) asparagine, in wheat grain can be achieved through crop management strategies, but such strategies have not been fully developed. We ran two field trials with 12 soft (biscuit) wheat varieties and different nitrogen, sulfur, potassium, and phosphorus fertilizer combinations. Our results indicated that a nitrogen-to-sulfur ratio of 10:1 kg/ha was sufficient to prevent large increases in free asparagine, whereas withholding potassium or phosphorus alone did not cause increases in free asparagine when sulfur was applied. Multispectral measurements of plants in the field were able to predict the free asparagine content of grain with an accuracy of 71%, while a combination of multispectral, fluorescence, and morphological measurements of seeds could distinguish high free asparagine grain from low free asparagine grain with an accuracy of 86%. The acrylamide content of biscuits correlated strongly with free asparagine content and with color measurements, indicating that agronomic strategies to decrease free asparagine would be effective and that quality control checks based on product color could eliminate high acrylamide biscuit products

Genetic control of grain amino acid composition in a UK soft wheat mapping population

Wheat (Triticum aestivum L.) is a major source of nutrients for populations across the globe, but the amino acid composition of wheat grain does not provide optimal nutrition. The nutritional value of wheat grain is limited by low concentrations of lysine (the most limiting essential amino acid) and high concentrations of free asparagine (precursor to the processing contaminant acrylamide). There are currently few available solutions for asparagine reduction and lysine biofortification through breeding. In this study, we investigated the genetic architecture controlling grain free amino acid composition and its relationship to other traits in a Robigus × Claire doubled haploid population. Multivariate analysis of amino acids and other traits showed that the two groups are largely independent of one another, with the largest effect on amino acids being from the environment. Linkage analysis of the population allowed identification of quantitative trait loci (QTL) controlling free amino acids and other traits, and this was compared against genomic prediction methods. Following identification of a QTL controlling free lysine content, wheat pangenome resources facilitated analysis of candidate genes in this region of the genome. These findings can be used to select appropriate strategies for lysine biofortification and free asparagine reduction in wheat breeding programs