Date Published: January 30, 2018
Publisher: Public Library of Science
Author(s): Jagadeesh Sundaramoorthy, Gyu Tae Park, Kyosuke Mukaiyama, Chigen Tsukamoto, Jeong Ho Chang, Jeong-Dong Lee, Jeong Hoe Kim, Hak Soo Seo, Jong Tae Song, David A Lightfoot.
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 EXXR 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.
Saponins are glycosylated compounds that are widely distributed in plants, and have different biological and pharmaceutical properties [1,2]. In addition, saponins are structurally diverse, for example triterpenoid and steroidal saponins . Triterpenoid saponins are widely distributed in higher plants [1,2]. They are composed of triterpene aglycone, with one or more sugar chains. Biosynthesis of saponins is initialized from isopentenyl pyrophosphate in the mevalonate pathway . Triterpene aglycones are derived from the 30-carbon linear 2,3-oxidosqualene precursor. In the first step of saponin synthesis, 2,3-oxidosqualene is cyclized by oxidosqualene cyclases (OSCs) to produce polycyclic triterpene [4,5]. After cyclization of the basic triterpene backbone, the backbone is oxidized to produce a hydrophobic aglycone called sapogenin. The oxidization step is catalyzed by cytochrome P450 (CYP450s) mono-oxygenases . The next step is to synthesize saponins via O-glycosylation of the aglycones. Studies on triterpenoid saponins have been undertaken on crops and medicinal plants due to their important commercial uses in cosmetic and pharmaceutical industries. However, the biological roles of triterpenoid saponins in plants remain underexplored.
Triterpenoid saponins are important secondary metabolites in soybean seeds. OSCs, CYP450s, and UGTs are the three main enzymes involved in the biosynthesis of saponins . CYP450s are mono-oxygenases that contain heme as a cofactor. In flowering plant genomes, around 300 CYP450s genes have been identified . Plant CYP450s were generally classified into two major clades: A-type and non-A-type clades . Majority of A-type and a few non-A-type CYP450s are involved in the biosynthesis of secondary metabolites such as terpenoids, flavonoids, alkaloids and phytoalexins [42, 43]. One of the non-A-type CYP450s, CYP72, were found to participate in pentacyclic triterpene modifications [43,44]. The CYP72 family consists of three subfamilies, CYP72A, CYP72B and CYP72C . The members of the CYP72A subfamily were found to be involved in the legume-specific triterpene saponin biosynthesis . To date, four CYP72As of Medicago truncatula (CYP72A61, CYP72A63, CYP72A67, and CYP72A68) and one CYP72A of Glycyrrhiza uralensis (CYP72A154) were identified to be associated in the triterpene saponin biosynthesis [32,46,47]. In soybean, CYP93E1 is first identified as an A-type CYP450 associated with the triterpenoid saponin biosynthesis, which catalyzes the hydroxylation of β-amyrin at C-24 . Recently, CYP72A69, which catalyzes the hydroxylation of C-21 during the biosynthesis of soyasapogenol A, was characterized (Glyma.15g243300) . In the present study, we obtained the new mutant accession, ′CWS5095′ from the Chung wild legume germplasm collection [33,49]. Phenotypic analyses 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 that controls group A saponin biosynthesis in soybean was mapped to 4.3 Mb between the Affx-89193969 and Affx-89134397 SNP arrays on chromosome 15 (E) where the previously identified gene Glyma.15g243300 is positioned.