Omics-based Identification of the Broader Effects of 2-hydroxyisoflavanone Synthase Gene Editing on a Gene Regulatory Network Beyond Isoflavonoid Loss in Soybean Hairy Roots.

Soybean (Glycine max) is a leguminous crop cultivated worldwide that accumulates high levels of isoflavones. Although previous research has often focused on increasing the soybean isoflavone content because of the estrogen-like activity of dietary soy in humans, the rapidly increasing demand for soybean as a plant-based meat substitute has raised concerns about excessive isoflavone intake. Therefore, the production of isoflavone-free soybean has been anticipated. However, there have been no reports of an isoflavone-free soybean until now. Here, 2-hydroxyisoflavanone synthase (IFS), which is essential for isoflavone biosynthesis, was targeted for genome editing in soybean. A novel CRISPR/Cas9 system using Staphylococcus aureus Cas9 instead of the commonly-used Streptococcus pyogenes Cas9 was established and customized. Through Agrobacterium rhizogenes-mediated transformation, IFS-edited hairy roots were generated in which all three IFS genes contained deletion mutations. Metabolome analyses of IFS-edited hairy roots revealed that isoflavone content significantly decreased, whereas levels of flavonoids, including a novel chalcone derivative, increased. A transcriptome analysis revealed changes in the expression levels of a large number of genes, including jasmonic acid-inducible genes. In addition, the functions of selected transcription factor genes (MYB14-L, GmbHLH112, and GmbHLH113), which were dramatically upregulated by IFS editing, were investigated by multi-omics analyses of their over-expressing hairy root lines. They appear to be involved in flavonoid and triterpene saponin biosynthesis, salicylic acid metabolism, and central carbon metabolism. Overall, the results indicated that editing IFS genes caused the redirection of the metabolic flux from isoflavonoid biosynthesis to flavonoid accumulation, as well as dynamic changes in gene regulatory networks.