Isolation and Characterization of the Soybean Sg-3 Gene that is Involved in Genetic Variation in Sugar Chain Composition at the C-3 Position in Soyasaponins

Abstract

Soyasaponins are specialized metabolites present in soybean seeds that affect the taste and quality of soy-based foods. The composition of the sugar chains attached to the aglycone moiety of soyasaponins is regulated by genetic loci such as sg-1, sg-3 and sg-4. Here, we report the cloning and characterization of the Sg-3 gene, which is responsible for conjugating the terminal (third) glucose (Glc) at the C-3 sugar chain of soyasaponins. The gene Glyma.10G104700 is disabled in the sg-3 cultivar, 'Mikuriya-ao', due to the deletion of genomic DNA that results in the absence of a terminal Glc residue on the C-3 sugar chain. Sg-3 encodes a putative glycosyltransferase (UGT91H9), and its predicted protein sequence has a high homology with that of the product of GmSGT3 (Glyma.08G181000; UGT91H4), which conjugates rhamnose (Rha) to the third position of the C-3 sugar chain in vitro. A recombinant Glyma.10G104700 protein could utilize UDP-Glc as a substrate to conjugate the third Glc to the C-3 sugar chain, and introducing a functional Glyma.10G104700 transgene into the mutant complemented the sg-3 phenotype. Conversely, induction of a premature stop codon mutation in Glyma.10G104700 (W270*) resulted in the sg-3 phenotype, suggesting that Glyma.10G104700 was Sg-3. The gmsgt3 (R339H) mutant failed to accumulate soyasaponins with the third Rha at the C-3 sugar chain, and the third Glc and Rha conjugations were both disabled in the sg-3 gmsgt3 double mutant. These results demonstrated that Sg-3 and GmSGT3 are non-redundantly involved in conjugation of the third Glc and Rha at the C-3 sugar chain of soyasaponins, respectively.

Functional specialization of UDP‐glycosyltransferase 73P12 in licorice to produce a sweet triterpenoid saponin, glycyrrhizin

“…Authentic samples, including (18β‐)glycyrrhetinic acid (Extrasynthese, Genay, France), glycyrrhetinic acid 3‐ O ‐monoglucuronide (Nacalai Tesque, Kyoto, Japan), glycyrrhizin (Wako Pure Chemical Industries, Osaka, Japan), glucoglycyrrhizin (Hayashi et al ., ), soyasapogenol B (Tokiwa Phytochemical, Chiba, Japan), soyasapogenol B 3‐ O ‐monoglucuronide (Yano et al ., ) and soyasaponin III (ChromaDex, Santa Ana, CA, USA), were used for the enzyme assay and/or LC‐MS analysis. For the initial characterization of the canonical UGT73P12 protein (WT, H29A and D131A) and the UGT73P13 protein, the reaction was initiated with the addition of 5 μl of purified protein (final concentration: 200 n m ) into 95 μl of a solution containing 50 m m Tris‐HCl (pH 7.0), 100 μ m MgCl 2 , 14 m m 2‐mercaptoethanol, 10 μ m sugar acceptors and 50 μ m UDP‐sugars.…”

Reconstruction of the Evolutionary Histories of UGT Gene Superfamily in Legumes Clarifies the Functional Divergence of Duplicates in Specialized Metabolism

“…The two members [Glyma.08G181000: UGT91H4 and Glyma.10g104700: UGT91H9 (Figure 2A)] in A02 of G. max catalyze the addition of rhamnose or glucose, respectively, at the terminal position of C-3 sugar chain of SA and SB in vitro and in vivo [28,31]. The members of A02 from 14 legumes (Table S9) formed two sister clades (i.e., A02-I and A02-II) with high bootstrap support in phylogenetic analysis (Figure 2A).…”

The unprecedented diversity of UGT94-family UDP-glycosyltransferases in Panax plants and their contribution to ginsenoside biosynthesis

“…Accordingly, sugar–sugar UGTs, which are responsible for transferring a sugar group to an existing sugar moiety in natural products, may be diverse 14 . However, only a limited number of sugar–sugar UGTs have been characterized, mainly from the UGT94, UGT73, UGT79, and UGT91 families 14 – 23 . These sugar–sugar UGTs could add a glucosyl/glucuronosyl/xylose/rhamnosyl/galactosyl molecule to an existing sugar moiety in natural products, including flavonoids and terpenoids.…”

“…These sugar–sugar UGTs could add a glucosyl/glucuronosyl/xylose/rhamnosyl/galactosyl molecule to an existing sugar moiety in natural products, including flavonoids and terpenoids. It has been reported that some UGTs from the UGT91 family (UGT91H4 and UGT91H9) can transfer a rhamnosyl or glucosyl moiety to the third position of the sugar chain 22 , 23 . In the past few decades, research has been focused on cloning and characterizing UGTs that glycosylate the OH and/or COOH group of natural products.…”