Molecular Characterization of Soybean Pterocarpan 2-Dimethylallyltransferase in Glyceollin Biosynthesis: Local Gene and Whole-Genome Duplications of Prenyltransferase Genes Led to the Structural Diversity of Soybean Prenylated Isoflavonoids

Abstract

Soybean (Glycine max) accumulates several prenylated isoflavonoid phytoalexins, collectively referred to as glyceollins. Glyceollins (I, II, III, IV and V) possess modified pterocarpan skeletons with C5 moieties from dimethylallyl diphosphate, and they are commonly produced from (6aS, 11aS)-3,9,6a-trihydroxypterocarpan [(−)-glycinol]. The metabolic fate of (−)-glycinol is determined by the enzymatic introduction of a dimethylallyl group into C-4 or C-2, which is reportedly catalyzed by regiospecific prenyltransferases (PTs). 4-Dimethylallyl (−)-glycinol and 2-dimethylallyl (−)-glycinol are precursors of glyceollin I and other glyceollins, respectively. Although multiple genes encoding (−)-glycinol biosynthetic enzymes have been identified, those involved in the later steps of glyceollin formation mostly remain unidentified, except for (−)-glycinol 4-dimethylallyltransferase (G4DT), which is involved in glyceollin I biosynthesis. In this study, we identified four genes that encode isoflavonoid PTs, including (−)-glycinol 2-dimethylallyltransferase (G2DT), using homology-based in silico screening and biochemical characterization in yeast expression systems. Transcript analyses illustrated that changes in G2DT gene expression were correlated with the induction of glyceollins II, III, IV and V in elicitor-treated soybean cells and leaves, suggesting its involvement in glyceollin biosynthesis. Moreover, the genomic signatures of these PT genes revealed that G4DT and G2DT are paralogs derived from whole-genome duplications of the soybean genome, whereas other PT genes [isoflavone dimethylallyltransferase 1 (IDT1) and IDT2] were derived via local gene duplication on soybean chromosome 11.

Isoflavonoid‐specific prenyltransferase gene family in soybean: GmPT01, a pterocarpan 2‐dimethylallyltransferase involved in glyceollin biosynthesis

“…The purity of the isolated compound was measured by HPLC coupled to an evaporative light‐scattering detector, and the chemical structure of (−)‐glycinol was confirmed by NMR spectroscopy (Figure S3). 1 H and 13 C chemical shifts and coupling constants (J; Table S2) agree with those reported in the literature (Akashi et al ., ; Yoneyama et al ., ).…”

“…GmPT01 was previously reported as having no PT activity (Yoneyama et al ., ). On the contrary, when microsomal fraction containing N‐terminal truncated GmPT01 was incubated with glycinol in the presence of DMAPP and Mg 2+ , a product was formed.…”

“…Though six eliminated GmPTs either did not respond to stress or are structurally different from other UbiA PT, it does not rule out the possibility of having enzymatic activity or performing a different function. Among the five stress‐induced GmPT s, GmPT10a and GmPT20 catalyze the reactions that transfer prenyl moiety to C‐4 (Akashi et al ., ) and C‐2 (Yoneyama et al ., ) position of (−)‐glycinol, respectively, while GmPT11a is an IDT2 (Yoneyama et al ., ). Here, we report the identification of two additional GmPTs, GmPT01 as a G2DT, and GmPT10d as an IDT3.…”

“…The reaction product of GmPT10a was confirmed by LC-MS with the mass-to-charge ratio (m/z) of 323.1270 corresponding to that of 4-glyceollidin ( Figure 7c). Yoneyama et al (2016) recently found that GmPT20 prenylates glycinol on the C-2 position, thereby naming this enzyme as G2DT. Furthermore, it was reported that GmPT11a was incapable of prenylating glycinol, but instead was found to prenylate daidzein and was, thereby, named isoflavone dimethylallyltransferase 1 (IDT1).…”

“…Two isoflavonoid-specific PTs, G4DT (Akashi et al, 2009) and G2DT (Yoneyama et al, 2016), belonging to the UbiA superfamily have recently been identified in soybean. Soybean is a paleopolyploid whose genome has undergone two whole genome duplications at approximately 59 and then 13 million years ago, resulting in 75% of genes within the genome in multiple copies (Schmutz et al, 2010).…”