Molecular Breeding

, 39:144 | Cite as

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

  • Jagadeesh Sundaramoorthy
  • Sampathkumar Palaniswamy
  • Gyu Tae Park
  • Hae Reon Son
  • Chigen Tsukamoto
  • Jeong-Dong Lee
  • Jeong Hoe Kim
  • Hak Soo Seo
  • Jong Tae SongEmail author


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.


Soybean Saponin Sg-5 CYP72A69 Group A saponin 


Funding information

This work was carried out with the support of “Cooperative Research Program for Agricultural Science and Technology Development (Project No. PJ01327602),” Rural Development Administration, Korea.

Supplementary material

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  1. Asano M, Okubu K, Igarashi M, Yamaguchi F (1987) Effects of removing seed coat and hypocotyl and squeezing without heating on the quality of tofu. Nippon Shokuhin Kogyo Gakkaishi 34:298–304CrossRefGoogle Scholar
  2. Chae JH, Dhakal KH, Asekova S, Song JT, Lee JD (2013) Variation of fatty acid composition in soybean ‘Pungsannamul’ mutation population from EMS treatment. Curr Res Agric Life Sci 31:47–52Google Scholar
  3. Chitisankul WT, Takada Y, Takahashi Y, Ito A, Itabashi M, Varanyanond W, Kikuchi A, Ishimoto M, Tsukamoto C (2018) Saponin composition complexities in hypocotyls and cotyledons of nine soybean varieties. Food Sci Tech 89:93–103Google Scholar
  4. Fukushima EO, Seki H, Sawai S, Suzuki M, Ohyama K, Saito K, Muranaka T (2013) Combinatorial biosynthesis of legume natural and rare triterpenoids in engineered yeast. Plant Cell Physiol 54:740–749. CrossRefPubMedGoogle Scholar
  5. Graham-Lorence SE, Peterson JA (1996) Structural alignments of P450s and extrapolations to the unknown. In: Johnson EF, Waterman MR (eds) Cytochrome P450, Part B, vol 272. Methods in enzymology. Academic Press, pp 315-326.
  6. Graham-Lorence S, Amarneh B, White RE, Peterson JA, Simpson ER (1995) A three-dimensional model of aromatase cytochrome P450. Protein Sci 4:1065–1080. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Hagely KB, Palmquist D, Bilyeu KD (2013) Classification of distinct seed carbohydrate profiles in soybean. J Agric Food Chem 61:1105–1111. CrossRefPubMedGoogle Scholar
  8. Hamdi A, Jimenez-Araujo A, Rodriguez-Arcos R, Jaramillo-Carmona S, Lachaal M, Bouraoui NK, Guillen-Bejarano R (2018) Asparagus saponins: chemical characterization, bioavailability and intervention in human health. Nutrition and Food Science 7Google Scholar
  9. Hasemann CA, Kurumbail RG, Boddupalli SS, Peterson JA, Deisenhofer J (1995) Structure and function of cytochromes P450: a comparative analysis of three crystal structures. Structure 3:41–62. CrossRefPubMedGoogle Scholar
  10. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10:845–858. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Kim SL, Berhow MA, Kim JT, Chi HY, Lee SJ, Chung IM (2006) Evaluation of soyasaponin, isoflavone, protein, lipid, and free sugar accumulation in developing soybean seeds. J Agric Food Chem 54:10003–10010. CrossRefPubMedGoogle Scholar
  12. Konoshima T, Kokumai M, Kozuka M, Tokuda H, Nishino H, Iwashima A (1992) Anti-tumor-promoting activities of afromosin and soyasaponin I isolated from Wisteria brachybotrys. J Nat Prod 55:1776–1778CrossRefGoogle Scholar
  13. Krishnamurthy P, Lee JD, Ha BK, Chae JH, Song JT, Tsukamoto C, Singh RJ, Chung G (2015) Genetic characterization of group A acetylsaponin-deficient mutants from wild soybean (Glycine soja Sieb. and Zucc.). Plant Breed 134:316–321. CrossRefGoogle Scholar
  14. Kudou S, Tonomura M, Tsukamoto C, Shimoyamada M, Uchida T, Okubo K (1992) Isolation and structural elucidation of the major genuine soybean saponin. Biosci Biotechnol Biochem 56:142–143. CrossRefPubMedGoogle Scholar
  15. Moses T, Papadopoulou KK, Osbourn A (2014) Metabolic and functional diversity of saponins, biosynthetic intermediates and semi-synthetic derivatives. Crit Rev Biochem Mol Biol 49:439–462. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Okubo K, Iijima M, Kobayashi Y, Yoshikoshi M, Uchida T, Kudou S (1992) Components responsible for the undesirable taste of soybean seeds. Biosci Biotech Biochem 56:99-103.
  17. Okubo K, Kudou S, Uchida T, Yoshiki Y, Yoshikoshi M, Tonomura M (1994) Soybean saponin and isoflavonoids. In: Food phytochemicals for cancer prevention I, vol 546. ACS Symposium Series. American Chemical Society, pp 330–339.
  18. Pateraki I, Heskes AM, Hamberger B (2015) Cytochromes P450 for terpene functionalisation and metabolic engineering. In: Biotechnology of isoprenoids. Springer, pp 107–139.
  19. Patil G, Chaudhary J, Vuong TD, Jenkins B, Qiu D, Kadam S, Shannon GJ, Nguyen HT (2017) Development of SNP genotyping assays for seed composition traits in soybean. Int J Plant Genomics 2017:6572969. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Preacher KJ (2001) Calculation for the chi-square test: an interactive calculation tool for chi-square tests for goodness of fit and independence.
  21. Prosser DE, Guo Y, Jia Z, Jones G (2006) Structural motif-based homology modeling of CYP27A1 and site-directed mutational analyses affecting vitamin D hydroxylation. Biophys J 90:3389–3409. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Qiu D, Vuong T, Valliyodan B, Shi H, Guo B, Shannon JG, Nguyen HT (2015) Identification and characterization of a stachyose synthase gene controlling reduced stachyose content in soybean. Theor Appl Genet 128:2167–2176. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Rehman HM, Nawaz MA, Shah ZH, Yang SH, Chung G (2018) Functional characterization of naturally occurring wild soybean mutant (sg-5) lacking astringent saponins using whole genome sequencing approach. Plant Sci 267:148–156. CrossRefPubMedGoogle Scholar
  24. Roland WSU, Pouvreau L, Curran J, Velde F, Kok PMT (2017) Flavor aspects of pulse ingredients. Cereal Chem 94:58–65Google Scholar
  25. Sasama H, Takada Y, Ishimoto M, Kitamura K, Tsukamoto C (2010) Estimation of the mutation site of a soyasapogenol A-deficient soybean [Glycine max (L.) Merr.] by LC-MS/MS profile analysis. In: Chemistry, texture, and flavor of soy, vol 1059. ACS Symposium Series. American Chemical Society, pp 91–102.
  26. Seki H, Tamura K, Muranaka T (2015) P450s and UGTs: key players in the structural diversity of triterpenoid saponins. Plant Cell Physiol 56:1463–1471. CrossRefPubMedGoogle Scholar
  27. Shimoyamada M, Harada K, Okubo K (1991) Saponin composition in developing soybean seed (Glycine max (L.) Merrill, cv. Mikuriyaao). Agric Biol Chem 55:1403–1405. CrossRefGoogle Scholar
  28. Shin JH, Van K, Kim KD, Lee Y-H, Jun T-H, Lee S-H (2012) Molecular sequence variations of the lipoxygenase-2 gene in soybean. Theor Appl Genet 124:613–622. CrossRefPubMedGoogle Scholar
  29. Shiraiwa M, Harada K, Okubo K (1991) Composition and content of saponins in soybean seed according to variety, cultivation year and maturity. Agric Biol Chem 55:323–331. CrossRefGoogle Scholar
  30. Sirim D, Widmann M, Wagner F, Pleiss J (2010) Prediction and analysis of the modular structure of cytochrome P450 monooxygenases. BMC Struct Biol 10:34. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Suarez-Estrella D, Torri L, Pagani MA, Marti A (2018) Quinoa bitterness: cause and solutions for improving product acceptability. J Sci Food Agric 98:4033–4041CrossRefGoogle Scholar
  32. Sugano M, Goto S, Yamada Y, Yoshida K, Hashimoto Y, Matsuo T, Kimoto M (1990) Cholesterol-lowering activity of various undigested fractions of soybean protein in rats. J Nutr 120:977–985. CrossRefPubMedGoogle Scholar
  33. Sundaramoorthy J, Park GT, Mukaiyama K, Tsukamoto C, Chang JH, Lee JD, Kim JH, Seo HS, Song JT (2018) Molecular elucidation of a new allelic variation at the Sg-5 gene associated with the absence of group A saponins in wild soybean. PLoS One 13:e0192150. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Sundaramoorthy J, Park GT, Komagamine K, Tsukamoto C, Chang JH, Lee J-D, Kim JH, Seo HS, Song JT (2019) Biosynthesis of DDMP saponins in soybean is regulated by a distinct UDP-glycosyltransferase. New Phytol 222:261–274. CrossRefPubMedGoogle Scholar
  35. Takada Y, Sayama T, Kikuchi A, Kato S, Tatsuzaki N, Nakamoto Y, Suzuki A, Tsukamoto C, Ishimoto M (2010) Genetic analysis of variation in sugar chain composition at the C-22 position of group A saponins in soybean, Glycine max (L.) Merrill. Breeding Sci 60:3–8. CrossRefGoogle Scholar
  36. Takada Y, Sasama H, Sayama T, Kikuchi A, Kato S, Ishimoto M, Tsukamoto C (2013) Genetic and chemical analysis of a key biosynthetic step for soyasapogenol A, an aglycone of group A saponins that influence soymilk flavor. Theor Appl Genet 126:721–731. CrossRefPubMedGoogle Scholar
  37. Takagi K, Nishizawa K, Hirose A, Kita A, Ishimoto M (2011) Manipulation of saponin biosynthesis by RNA interference-mediated silencing of β-amyrin synthase gene expression in soybean. Plant Cell Rep 30:1835–1846CrossRefGoogle Scholar
  38. Thimmappa R, Geisler K, Louveau T, O’Maille P, Osbourn A (2014) Triterpene biosynthesis in plants. Annu Rev Plant Biol 65:225–257. CrossRefPubMedGoogle Scholar
  39. Tsukamoto C, Kikuchi A, Harada K, Kitamura K, Okubo K (1993) Genetic and chemical polymorphisms of saponins in soybean seed. Phytochemistry 34:1351–1356. CrossRefPubMedGoogle Scholar
  40. Vagadia BH, Vanga SK, Raghavan V (2017) Inactivation methods of soybean trypsin inhibitor–a review. Trends Food Sci Technol 64:115–125. CrossRefGoogle Scholar
  41. Wang WH, Takano T, Shibata D, Kitamura K, Takeda G (1994) Molecular basis of a null mutation in soybean lipoxygenase 2: substitution of glutamine for an iron-ligand histidine. Proc Natl Acad Sci U S A 91:5828–5832CrossRefGoogle Scholar
  42. Williams PA, Cosme J, Vinkovic DM, Ward A, Angove HC, Day PJ, Vonrhein C, Tickle IJ, Jhoti H (2004) Crystal structures of human cytochrome P450 3A4 bound to metyrapone and progesterone. Science 305:683–686. CrossRefPubMedGoogle Scholar
  43. Yano R, Takagi K, Takada Y, Mukaiyama K, Tsukamoto C, Sayama T, Kaga A, Anai T, Sawai S, Ohyama K, Saito K, Ishimoto M (2016) Metabolic switching of astringent and beneficial triterpenoid saponins in soybean is achieved by a loss-of-function mutation in cytochrome P450 72A69. Plant J 89:527–539. CrossRefGoogle Scholar
  44. Yoshiki Y, Kinumi M, Kahara T, Okubo K (1996) Chemiluminescence of soybean saponins in the presence of active oxygen species. Plant Sci 116:125–129. CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.School of Applied BiosciencesKyungpook National UniversityDaeguSouth Korea
  2. 2.Food Science, Bioresources Sciences, United Graduate School of Agricultural SciencesIwate UniversityMoriokaJapan
  3. 3.Department of BiologyKyungpook National UniversityDaeguSouth Korea
  4. 4.Department of Plant ScienceSeoul National UniversitySeoulSouth Korea

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