NARO succeeds in using genome editing technology ― Barley variety forms gluten-like network similar to wheat

A research group consisting of Executive Researcher Ryozo Imai of the Institute of Agrobiological Sciences (NIAS), National Agriculture and Food Research Organization (NARO), and his colleagues has announced their success in creating a gluten-like network, similar to what is seen in wheat, in a variety of barley by using genome editing technology, with the aim of expanding barley's utility. Using this genome editing technology, the group was able to alter the primary structure of barley seed proteins. When water was added, the group observed a gluten-like network in the barley flour ground from the barley lineage they developed, confirming that its suitability for processing had changed. It is hoped that these outcomes will lead to the development of barley varieties that have excellent processing properties while retaining their functional components. Their outcomes were announced at the 142nd Meeting of the Japanese Society of Breeding, held at Obihiro University of Agriculture and Veterinary Medicine.

Both barley and wheat are grains that belong to the family Poaceae (grasses), but their properties when processed are very different. For example, wheat is an ingredient used for bread and udon, and has high suitability for processing. Barley, on the other hand, is not suitable as an ingredient for these, and in most cases is used without being ground into flour, meaning it has limited utility. Despite this, barley includes a lot of functional substances, which are present in much smaller numbers in wheat, and has the advantage of having a harvest season that is suitable for cultivation in Japan.

The difference in processing suitability between barley and wheat lies mainly in the composition of their proteins. The main protein in wheat is glutenin. Adding water to wheat flour creates gluten (a network structure), and so the substance created has an elastic texture. Conversely, the main protein in barley is hordein, and it is known that this clumps together even when water is added to barley flour. It is not possible to form a network structure with this.

In the past, the research group has worked with Kaneka to develop the iPB method, which directly introduces genes to individual plants, and have also succeeded in developing a technique that incorporates genome editing.

In this study, the research group aimed to develop a lineage with improved suitability for processing, with the goal of expanding the utility of barley.

First, they focused on the protein structures of wheat and barley. The group clarified that when it comes to the amino acid structure of high-molecular-weight glutenin, which makes up wheat protein, and D-hordein, which is a barley protein of the same ancestry, D-hordein has domains (barley-specific domains) rich in cysteine residues, which are not present in the high-molecular-weight glutenin.

Cysteine residues do exist in high-molecular-weight glutenin, and are necessary for the formation of gluten, but the numbers differed between the proteins. Believing that perhaps gluten formation is not possible because there are too many cysteine residues in D-hordein, the group investigated whether gluten-like structures could form if the cysteine residues were reduced. They used genome editing to create barley without these domains.

More specifically, they planned CRISPR/Cas9 to cut upstream and downstream of the barley specific domains. They introduced two types of CRISPR/Cas9 ribonucleoprotein using the iPB method. This method enabled them to introduce genes directly to individual plants without using cultured cells.

When this method is used, genome editing takes place in the plant's pollen, so the group ensured that the individuals obtained were able to self-pollinate and analyzed whether any genome editing occurred with the seeds obtained.

The outcomes confirmed that mutations were induced in the target D-hordein genes in three individuals, and one of these was a homozygous lineage. The group analyzed this lineage and confirmed that the barley-specific domain (252 base) was completely removed. When water was added to barley flour from this lineage and the resulting mixture was observed with an electron microscope, the group found that a gluten-like network had formed. They were also able to ascertain that this had a molecular weight similar to that of wheat.

In addition, the research group are studying techniques that do not use genome editing. They used a mutant lacking one cysteine residue and explored changes in elasticity when it was ground to barley flour and water was added. This demonstrated increased elasticity compared to regular barley lineages, and an increased gluten-like network. However, they could not obtain physical properties of the same level as those of the lineage developed through genome editing, making it apparent that further removal of cysteine residues is necessary when it comes to the acquisition of properties similar to gluten.

Imai commented, "In the future, we think it will be possible to improve the suitability of commercial varieties of barley for processing using genome editing technology, and to develop varieties that contain lots of functional substances that are suitable for noodle and bread-making. We hope that using genome editing technology will further our understanding of the mechanism behind gluten formation in barley."