Abstract
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.
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References
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 34:298–304
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 31:47–52
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 89:93–103
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. https://doi.org/10.1093/pcp/pct015
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. https://doi.org/10.1016/s0076-6879(96)72037-2
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. https://doi.org/10.1002/pro.5560040605
Hagely KB, Palmquist D, Bilyeu KD (2013) Classification of distinct seed carbohydrate profiles in soybean. J Agric Food Chem 61:1105–1111. https://doi.org/10.1021/jf303985q
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 7
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. https://doi.org/10.1016/S0969-2126(01)00134-4
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. https://doi.org/10.1038/nprot.2015.053
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. https://doi.org/10.1021/jf062275p
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 55:1776–1778
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. https://doi.org/10.1111/pbr.12269
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. https://doi.org/10.1271/bbb.56.142
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. https://doi.org/10.3109/10409238.2014.953628
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. https://doi.org/10.1271/bbb.56.99
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. https://doi.org/10.1021/bk-1994-0546.ch026
Pateraki I, Heskes AM, Hamberger B (2015) Cytochromes P450 for terpene functionalisation and metabolic engineering. In: Biotechnology of isoprenoids. Springer, pp 107–139. https://doi.org/10.1007/10_2014_301
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. https://doi.org/10.1155/2017/6572969
Preacher KJ (2001) Calculation for the chi-square test: an interactive calculation tool for chi-square tests for goodness of fit and independence. http://quantpsy.org/chisq/chisq.htm
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. https://doi.org/10.1529/biophysj.105.069369
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. https://doi.org/10.1007/s00122-015-2575-0
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. https://doi.org/10.1016/j.plantsci.2017.11.014
Roland WSU, Pouvreau L, Curran J, Velde F, Kok PMT (2017) Flavor aspects of pulse ingredients. Cereal Chem 94:58–65
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. https://doi.org/10.1021/bk-2010-1059.ch006
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. https://doi.org/10.1093/pcp/pcv062
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. https://doi.org/10.1080/00021369.1991.10870766
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. https://doi.org/10.1007/s00122-011-1733-2
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. https://doi.org/10.1080/00021369.1991.10870575
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. https://doi.org/10.1186/1472-6807-10-34
Suarez-Estrella D, Torri L, Pagani MA, Marti A (2018) Quinoa bitterness: cause and solutions for improving product acceptability. J Sci Food Agric 98:4033–4041
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. https://doi.org/10.1093/jn/120.9.977
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. https://doi.org/10.1371/journal.pone.0192150
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. https://doi.org/10.1111/nph.15588
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. https://doi.org/10.1270/jsbbs.60.3
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. https://doi.org/10.1007/s00122-012-2013-5
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 30:1835–1846
Thimmappa R, Geisler K, Louveau T, O’Maille P, Osbourn A (2014) Triterpene biosynthesis in plants. Annu Rev Plant Biol 65:225–257. https://doi.org/10.1146/annurev-arplant-050312-120229
Tsukamoto C, Kikuchi A, Harada K, Kitamura K, Okubo K (1993) Genetic and chemical polymorphisms of saponins in soybean seed. Phytochemistry 34:1351–1356. https://doi.org/10.1016/0031-9422(91)80028-Y
Vagadia BH, Vanga SK, Raghavan V (2017) Inactivation methods of soybean trypsin inhibitor–a review. Trends Food Sci Technol 64:115–125. https://doi.org/10.1016/j.tifs.2017.02.003
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 91:5828–5832
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. https://doi.org/10.1126/science.1099736
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. https://doi.org/10.1111/tpj.13403
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. https://doi.org/10.1016/0168-9452(96)04375-0
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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.
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Sundaramoorthy, J., Palaniswamy, S., Park, G.T. et al. Characterization of a new sg-5 variant with reduced biosynthesis of group A saponins in soybean (Glycine max (L.) Merr.). Mol Breeding 39, 144 (2019). https://doi.org/10.1007/s11032-019-1066-4
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DOI: https://doi.org/10.1007/s11032-019-1066-4