Functional characterization of naturally occurring wild soybean mutant (sg-5) lacking astringent saponins using whole genome sequencing approach.

Triterpenoid saponins are one of the most highly accumulated groups of functional components in soybean (Glycine max) and the oxidative reactions during their biosynthesis are required for their aglycone diversity. Natural mutants of soyasaponins in wild soybean (Glycine soja) are valuable resources for establishing the soyasaponin biosynthesis pathway and breeding new soybean varieties. In this study, we investigated the genetic mechanism behind the absence of group A saponins in a Korean wild soybean mutant, CWS5095. Whole genome sequencing (WGS) of CWS5095 identified four point mutations [Val6 → Asp, Ile231 → Thr, His294 → Gln, and Arg376 → Lys] in CYP72A69 (Glyma15g39090), which oxygenate the C-21 position of soyasapogenol B or other intermediates to produce soyasapogenol A, leading to group A saponin production. An in vitro enzyme activity assay of single-sited mutated clones indicated that the Arg376 > Lys mutation (a highly conserved mutation based on a nucleotide change from G → A at the 1,127th position) may lead to loss of gene function in the sg-5 mutant. A very high normalized expression value of 377 reads per kilo base per million (RPKM) of Glyma15g39090 in the hypocotyl axis at the early maturation seed-development stage confirmed their abundant presence in seed hypocotyls. A molecular dynamics analysis of the Arg376 > Lys mutation based on the CYP3A4 (a human CYP450) protein structure found that it was responsible for the increase in axis length toward the heme (active site), which is critically important for biological activity and ligand binding. Our results provide important information on how to eradicate bitter and astringent saponins in soybean by utilizing the reported mutation in Glyma15g39090, and its importance for seed hypocotyl development based on transcript abundance.