Induced mutations in ASPARAGINE SYNTHETASE‐A2 reduce free asparagine concentration in the wheat grain

Abstract

Acrylamide is a neurotoxin and probable carcinogen formed as a processing contaminant during baking and production of different foodstuffs, including bread products.The amino acid asparagine is the limiting substrate in the Maillard reaction that produces acrylamide, so developing wheat (Triticum aestivum L.) cultivars with low free asparagine concentrations in the grain is a promising approach to reduce dietary acrylamide exposure. A candidate gene approach was used to identify chemically induced genetic variation in ASPARAGINE SYNTHETASE 2 (ASN2) genes that exhibit a grain-specific expression profile. In field trials, durum and common wheat lines carrying asn-a2 null alleles exhibited reductions in free asparagine concentration in their grains of between 9 and 34% compared with wildtype sister lines. These plants showed no significant differences in spikelet number, grain size and weight, germination or baking quality traits. These nontransgenic variants can be deployed without regulatory oversight in elite wheat germplasm to reduce acrylamide-forming potential with no negative effects on quality or agronomic performance. INTRODUCTIONAcrylamide is a potent neurotoxin and causes cancer in rodents (Beland et al., 2013). For humans, acrylamide is classified as a probable carcinogen by the International Agency for Research on Cancer (IARC, 1994) and mutational signatures attributed to acrylamide exposure were detected in approximately one-third of assayed tumor genomes, including 19 different tumor tissues (Zhivagui et al., 2019

Reducing Acrylamide Formation Potential by Targeting Free Asparagine Accumulation in Seeds

“…16 Lastly, ethylmethanesulfonate (EMS) mutagenesis in wheat that reduced TaASN2 expression also resulted in a 34% decrease in free Asn. 29 This gene has a high similarity to TaASN1 as well as to maize ZmASN2, which is highly expressed in seeds, 30,31 and to sorghum SbASN1, which is embryospecific. 26 This makes ZmASN2 a good target for silencing or knocking down expression in maize.…”

“…However, the embryo is much smaller than the endosperm, and therefore, only In wheat, TaASN2 is expressed specifically in seeds, and knocking it out resulted in a seed-specific reduction in free Asn with no negative effects on seed quality or agronomic performance (Table 1). 29 However, this situation is not true all of the time. In potato, for example, silencing StAst1 under the Ubi3 promoter reduced both the yield and total nitrogen, and editing wheat TaASN2 resulted in greenhouse grown plants that produced seeds with low germination rates (Table 1).…”

“…Additionally, screening of a panel of wheat cultivars identified a deletion of TaASN2 in the B genome, resulting in a modest reduction in free Asn . Lastly, ethylmethanesulfonate (EMS) mutagenesis in wheat that reduced TaASN2 expression also resulted in a 34% decrease in free Asn . This gene has a high similarity to TaASN1 as well as to maize ZmASN2, which is highly expressed in seeds, , and to sorghum SbASN1, which is embryo-specific .…”

Field assessment of genome‐edited, low asparagine wheat: Europe's first CRISPR wheat field trial

“…The TILLING lines showed more variable responses when compared to Claire, with one (TILLING 3) showing an increase in free asparagine and the others a small decrease (Figure 1d). However, when compared with the TILLING control, they did show significant reductions of 20–40% ( F 1,35.7 = 26.57, P < 0.001), comparable with the reduction of 28% seen previously in A genome TILLING nulls in the Cadenza background, and 24–34% in the tetraploid Kronos background (Alarcón‐Reverte et al ., 2022). There was also an increase in free glutamine in the TILLING lines when compared with Claire ( F 1,29.3 = 47.97, P < 0.001) or the TILLING control ( F 1,37.0 = 23.71, P < 0.001) (Figure 1e).…”

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

“…In recent years, studies have investigated genetic strategies for the reduction of free asparagine content in wheat grain. Induced and natural variation in the asparagine synthetase 2 genes, for example, has been found to impact significantly on free asparagine content (Alarcon‐Reverte et al., 2022; Oddy et al., 2021; Raffan et al., 2021) and quantitative trait loci (QTLs) for grain asparagine content have been identified from previous genome‐wide association studies (GWAS) (Emebiri, 2014; Peng et al., 2018; Rapp et al., 2018). However, the small number of stable QTL available to breeders limits the progress that can be made to reduce grain asparagine content in breeding programs and no genetic strategies for soft (biscuit) wheat specifically have been investigated.…”