Molecular elucidation of a new allelic variation at the Sg-5 gene associated with the absence of group A saponins in wild soybean

Abstract

In soybean, triterpenoid saponin is one of the major secondary metabolites and is further classified into group A and DDMP saponins. Although they have known health benefits for humans and animals, acetylation of group A saponins causes bitterness and gives an astringent taste to soy products. Therefore, several studies are being conducted to eliminate acetylated group A saponins. Previous studies have isolated and characterized the Sg-5 (Glyma.15g243300) gene, which encodes the cytochrome P450 72A69 enzyme and is responsible for soyasapogenol A biosynthesis. In this study, we elucidated the molecular identity of a novel mutant of Glycine soja, ′CWS5095′. Phenotypic analysis using TLC and LC-PDA/MS/MS showed that the mutant ′CWS5095′ did not produce any group A saponins. Segregation analysis showed that the absence of group A saponins is controlled by a single recessive allele. The locus was mapped on chromosome 15 (4.3 Mb) between Affx-89193969 and Affx-89134397 where the previously identified Glyma.15g243300 gene is positioned. Sequence analysis of the coding region for the Glyma.15g243300 gene revealed the presence of four SNPs in ′CWS5095′ compared to the control lines. One of these four SNPs (G1127A) leads to the amino acid change Arg376Lys in the motif, which is invariably conserved among the CYP450 superfamily proteins. Co-segregation analysis showed that the missense mutation (Arg376Lys) was tightly linked with the absence of group A saponins in ′CWS5095′. Even though Arg and Lys have similar chemical features, the 3D modelled protein structure indicates that the replacement of Arg with Lys may cause a loss-of-function of the Sg-5 protein by inhibiting the stable binding of a heme cofactor to the CYP72A69 apoenzyme.

Biosynthesis of DDMP saponins in soybean is regulated by a distinct UDP‐glycosyltransferase

“…). Sg‐1 and Sg‐5 were used as controls: the former is involved in the attachment of a sugar chain at the C‐22 position of SS‐A (Sayama et al ., ), and the latter is involved in the conversion of SS‐B to SS‐A (Yano et al ., ; Sundaramoorthy et al ., ). In addition, β‐amyrin synthase ( BAS1 ) involved in β‐amyrin production was also used as a control.…”

“…S2; Sayama et al ., ). Recently, a hydroxylase gene, Sg‐5 , which encodes the CYP72A69 enzyme responsible for the biosynthesis of SS‐A, has been characterized in soybean (Yano et al ., ; Sundaramoorthy et al ., ). DDMP saponins contain a DDMP moiety at the C‐22 position and a single sugar chain at the C‐3 hydroxyl position of the aglycone known as soyasapogenol B (SS‐B; 3β,22β,24‐trihydroxyolean‐12‐ene) (Fig.…”

Korean Wild Soybeans (Glycine soja Sieb & Zucc.): Geographic Distribution and Germplasm Conservation

“…Moreover, the CWLGC germplasms were studied for their natural variations in saponin contents and were used to help identify different group-A acetylsaponin-deficient mutants [32,[104][105][106]. The wild soybean soyasaponin mutants from CWLGC have been used to identify and characterize the genes involved in the saponin biosynthesis pathway, e.g., Sg-1 locus (CWS2133), sg-5 locus (CWS5095), and Sg-6 locus [33,105,[107][108][109]. In addition to saponins, wild soybean accessions at the CWLGC have also been screened for the presence of Kunitz trypsin inhibitor polymorphism, alpha-linolenic acid concentration, and soyisoflavone profile diversity [110][111][112][113].…”

“…Moreover, the CWLGC germplasms were studied for their natural variations in saponin contents and were used to help identify different group-A acetylsaponin-deficient mutants [104][105][106][107]. The wild soybean soyasaponin mutants from CWLGC have been used to identify and characterize the genes involved in the saponin biosynthesis pathway, e.g., Sg-1 locus (CWS2133), sg-5 locus (CWS5095), and Sg-6 locus [33,106,[108][109][110].…”

Integrated metabolomics identifies CYP72A67 and CYP72A68 oxidases in the biosynthesis of Medicago truncatula oleanate sapogenins

“…Members of the CYP716 subfamily of P450s catalyze various oxidations on the -amyrin backbone, including the three-step oxidation of -amyrin at the C-28 position to yield oleanolic acid by CYP716A12 (Carelli et al 2011;Miettinen et al 2017), the C-16α hydroxylation of -amyrin by CYP716Y1 , and the C-3 oxidation by CYP716A14 (Moses et al 2015). In addition, CYP72A154 oxidizes -amyrin at the C-30 position (Seki et al 2011) and several P450s have been identified that further modify oxidation products of -amyrin, such as MtCYP72A61 and GmCYP72A69 (Sundaramoorthy et al 2018;Yano et al 2016) that hydroxylate 24-hydroxy--amyrin at the C-22 and C-21 positions, respectively, in soyasaponin biosynthesis, MtCYP72A68 that oxidizes C-23 of oleanolic acid, and MtCYP72A67 that catalyzes oxidation at C-2 position (Biazzi et al 2015;Fukushima et al 2013). However, several P450s and glycosyltransferases involved in saponin biosynthesis still remain uncharacterized and in planta evidence for tentatively identified genes is minimal.…”