Identification of Genes Responsible for the Synthesis of Glycitein Isoflavones in Soybean Seeds.

Soybean ( Glycine max (L.) Merrill) isoflavones are among the most important secondary metabolites, with functional benefits for human health. Soybeans accumulate three aglycone forms of isoflavones: genistein, daidzein, and glycitein. Soybean landrace Kumachi-1 does not accumulate malonylglycitin at all. Gene structure analysis indicated that Glyma.11G108300 ( F6H4 ) of Kumachi-1 has a 3.8-kbp insertion, resulting in a truncated flavonoid 6-hydroxylase ( F6H ) sequence compared to the wild-type sequence in Fukuyutaka. Mapping experiments using a mutant line (MUT1246) with a phenotype similar to that of Kumachi-1, with a single-nucleotide polymorphism (SNP) in F6H4 , revealed co-segregation of this mutation and the absence of glycitein isoflavones. We also identified a mutant line (K01) that exhibited a change in the HPLC retention time of glycitein isoflavones, accumulating glycoside and malonylglycoside forms of 6-hydroxydaidzein. K01 contains an SNP that produces a premature stop codon in Glyma.01G004200 ( IOMT3 ), a novel soybean isoflavone O-methyltransferase ( IOMT ) gene. We further analyzed transgenic hairy roots of soybeans expressing Glyma.11G108300 ( F6H4 ) and Glyma.01G004200 ( IOMT3 ). Those overexpressing F6H4 accumulated malonylglycoside forms of 6-hydroxydaidzein (M_6HD), and co-expression of F6H4 and IOMT3 increased the level of malonylglycitin but not of M_6HD. These results indicate that F6H4 and IOMT3 are responsible for glycitein biosynthesis in soybean seed hypocotyl.