Characterization of a new sg-5 variant with reduced biosynthesis of group A saponins in soybean (Glycine max (L.) Merr.)

Triterpenoid saponins are major secondary metabolites in soybean; they are classified into group A and DDMP saponins. Group A saponins cause bitterness and an astringent aftertaste, whereas DDMP saponins are less bitter and more beneficial to human health. Therefore, a need exists for genetic improvement to develop bitter-free soybeans without losing other beneficial saponins. In this study, an ethyl methanesulfonate (EMS)–induced mutant, PE1327, was isolated and characterized as having a reduced level of group A saponins by chromatography. The PE1327 phenotype, which exhibits no accumulation of group A saponins, resembles that of a previously reported sg-5 wild soybean mutant whose biosynthesis is controlled by the Sg-5 (Glyma.15G243300) gene. The Sg-5 gene sequence in PE1327 revealed a single-nucleotide polymorphism (G1036A) that causes an amino acid change from Glu₃₄₆ to Lys₃₄₆ in the sg-5 protein. Multiple alignment analysis of cytochrome P450 enzymes from numerous organisms revealed that Glu₃₄₆ is highly conserved in eukaryotes, including plants and animals, but not in prokaryotes. The predicted 3D model of the Sg-5 protein showed that the Glu₃₄₆ residue is located in the J helix and is likely involved in the linkage between the J and K helices and the stabilization of the J-K loop, suggesting that the amino acid substitution from Glu₃₄₆ to Lys₃₄₆ in the PE1327 mutant results in hypofunction of Sg-5. Co-segregation analysis revealed that the Sg-5 locus is tightly linked to reduced group A saponin biosynthesis in PE1327 and that the novel sg-5 variant is recessive to Sg-5.