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.

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.

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 (nonprotein) 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 dIstachyon) 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.

Wheat with greatly reduced accumulation of free asparagine in the grain, produced by CRISPR/Cas9 editing of asparagine synthetase gene TaASN2.

Free asparagine is the precursor for acrylamide, which forms during the baking, toasting and high-temperature processing of foods made from wheat. In this study, CRISPR/Cas9 was used to knock out the asparagine synthetase gene, TaASN2, of wheat (Triticum aestivum) cv. Cadenza. A 4-gRNA polycistronic gene was introduced into wheat embryos by particle bombardment and plants were regenerated. T1 plants derived from 11 of 14 T0 plants were shown to carry edits. Most edits were deletions (up to 173 base pairs), but there were also some single base pair insertions and substitutions. Editing continued beyond the T1 generation. Free asparagine concentrations in the grain of plants carrying edits in all six TaASN2 alleles (both alleles in each genome) were substantially reduced compared with wildtype, with one plant showing a more than 90 % reduction in the T2 seeds. A plant containing edits only in the A genome alleles showed a smaller reduction in free asparagine concentration in the grain, but the concentration was still lower than in wildtype. Free asparagine concentration in the edited plants was also reduced as a proportion of the free amino acid pool. Free asparagine concentration in the T3 seeds remained substantially lower in the edited lines than wildtype, although it was higher than in the T2 seeds, possibly due to stress. In contrast, the concentrations of free glutamine, glutamate and aspartate were all higher in the edited lines than wildtype. Low asparagine seeds showed poor germination but this could be overcome by exogenous application of asparagine.

The Sulphur Response in Wheat Grain and Its Implications for Acrylamide Formation and Food Safety.

Free (soluble, non-protein) asparagine concentration can increase many-fold in wheat grain in response to sulphur deficiency. This exacerbates a major food safety and regulatory compliance problem for the food industry because free asparagine may be converted to the carcinogenic contaminant, acrylamide, during baking and processing. Here, we describe the predominant route for the conversion of asparagine to acrylamide in the Maillard reaction. The effect of sulphur deficiency and its interaction with nitrogen availability is reviewed, and we reiterate our advice that sulphur should be applied to wheat being grown for human consumption at a rate of 20 kg per hectare. We describe the genetic control of free asparagine accumulation, including genes that encode metabolic enzymes (asparagine synthetase, glutamine synthetase, glutamate synthetase, and asparaginase), regulatory protein kinases (sucrose nonfermenting-1 (SNF1)-related protein kinase-1 (SnRK1) and general control nonderepressible-2 (GCN2)), and basic leucine zipper (bZIP) transcription factors, and how this genetic control responds to sulphur, highlighting the importance of asparagine synthetase-2 (ASN2) expression in the embryo. We show that expression of glutamate-cysteine ligase is reduced in response to sulphur deficiency, probably compromising glutathione synthesis. Finally, we describe unexpected effects of sulphur deficiency on carbon metabolism in the endosperm, with large increases in expression of sucrose synthase-2 (SuSy2) and starch synthases.

Acrylamide in food: Progress in and prospects for genetic and agronomic solutions.

Acrylamide is a processing contaminant and Group 2a carcinogen that was discovered in foodstuffs in 2002. Its presence in a range of popular foods has become one of the most difficult problems facing the food industry and its supply chain. Wheat, rye and potato products are major sources of dietary acrylamide, with biscuits, breakfast cereals, bread (particularly toasted), crispbread, batter, cakes, pies, French fries, crisps and snack products all affected. Here we briefly review the history of the issue, detection methods, the levels of acrylamide in popular foods and the risk that dietary acrylamide poses to human health. The pathways for acrylamide formation from free (non-protein) asparagine are described, including the role of reducing sugars such as glucose, fructose and maltose and the Maillard reaction. The evolving regulatory situation in the European Union and elsewhere is discussed, noting that food businesses and their suppliers must plan to comply not only with current regulations but with possible future regulatory scenarios. The main focus of the review is on the genetic and agronomic approaches being developed to reduce the acrylamide-forming potential of potatoes and cereals and these are described in detail, including variety selection, plant breeding, biotechnology and crop management. Obvious targets for genetic interventions include asparagine synthetase genes, and the asparagine synthetase gene families of different crop species are compared. Current knowledge on crop management best practice is described, including maintaining optimum storage conditions for potatoes and ensuring sulphur sufficiency and disease control for wheat.

Contrasting gene expression patterns in grain of high and low asparagine wheat genotypes in response to sulphur supply.

BACKGROUND: Free asparagine is the precursor for acrylamide formation during cooking and processing of grains, tubers, beans and other crop products. In wheat grain, free asparagine, free glutamine and total free amino acids accumulate to high levels in response to sulphur deficiency. In this study, RNA-seq data were acquired for the embryo and endosperm of two genotypes of bread wheat, Spark and SR3, growing under conditions of sulphur sufficiency and deficiency, and sampled at 14 and 21 days post anthesis (dpa). The aim was to provide new knowledge and understanding of the genetic control of asparagine accumulation and breakdown in wheat grain. RESULTS: There were clear differences in gene expression patterns between the genotypes. Sulphur responses were greater at 21 dpa than 14 dpa, and more evident in SR3 than Spark. TaASN2 was the most highly expressed asparagine synthetase gene in the grain, with expression in the embryo much higher than in the endosperm, and higher in Spark than SR3 during early development. There was a trend for genes encoding enzymes of nitrogen assimilation to be more highly expressed in Spark than SR3 when sulphur was supplied. TaASN2 expression in the embryo of SR3 increased in response to sulphur deficiency at 21 dpa, although this was not observed in Spark. This increase in TaASN2 expression was accompanied by an increase in glutamine synthetase gene expression and a decrease in asparaginase gene expression. Asparagine synthetase and asparaginase gene expression in the endosperm responded in the opposite way. Genes encoding regulatory protein kinases, SnRK1 and GCN2, both implicated in regulating asparagine synthetase gene expression, also responded to sulphur deficiency. Genes encoding bZIP transcription factors, including Opaque2/bZIP9, SPA/bZIP25 and BLZ1/OHP1/bZIP63, all of which contain SnRK1 target sites, were also expressed. Homeologues of many genes showed differential expression patterns and responses, including TaASN2. CONCLUSIONS: Data on the genetic control of free asparagine accumulation in wheat grain and its response to sulphur supply showed grain asparagine levels to be determined in the embryo, and identified genes encoding signalling and metabolic proteins involved in asparagine metabolism that respond to sulphur availability.

Genomic, Biochemical, and Modeling Analyses of Asparagine Synthetases from Wheat.

Asparagine synthetase activity in cereals has become an important issue with the discovery that free asparagine concentration determines the potential for formation of acrylamide, a probably carcinogenic processing contaminant, in baked cereal products. Asparagine synthetase catalyses the ATP-dependent transfer of the amino group of glutamine to a molecule of aspartate to generate glutamate and asparagine. Here, asparagine synthetase-encoding polymerase chain reaction (PCR) products were amplified from wheat (Triticum aestivum) cv. Spark cDNA. The encoded proteins were assigned the names TaASN1, TaASN2, and TaASN3 on the basis of comparisons with other wheat and cereal asparagine synthetases. Although very similar to each other they differed slightly in size, with molecular masses of 65.49, 65.06, and 66.24 kDa, respectively. Chromosomal positions and scaffold references were established for TaASN1, TaASN2, and TaASN3, and a fourth, more recently identified gene, TaASN4. TaASN1, TaASN2, and TaASN4 were all found to be single copy genes, located on chromosomes 5, 3, and 4, respectively, of each genome (A, B, and D), although variety Chinese Spring lacked a TaASN2 gene in the B genome. Two copies of TaASN3 were found on chromosome 1 of each genome, and these were given the names TaASN3.1 and TaASN3.2. The TaASN1, TaASN2, and TaASN3 PCR products were heterologously expressed in Escherichia coli (TaASN4 was not investigated in this part of the study). Western blot analysis identified two monoclonal antibodies that recognized the three proteins, but did not distinguish between them, despite being raised to epitopes SKKPRMIEVAAP and GGSNKPGVMNTV in the variable C-terminal regions of the proteins. The heterologously expressed TaASN1 and TaASN2 proteins were found to be active asparagine synthetases, producing asparagine and glutamate from glutamine and aspartate. The asparagine synthetase reaction was modeled using SNOOPY® software and information from the BRENDA database to generate differential equations to describe the reaction stages, based on mass action kinetics. Experimental data from the reactions catalyzed by TaASN1 and TaASN2 were entered into the model using Copasi, enabling values to be determined for kinetic parameters. Both the reaction data and the modeling showed that the enzymes continued to produce glutamate even when the synthesis of asparagine had ceased due to a lack of aspartate.