Identification of a Highly Specific Isoflavone 7-O-glucosyltransferase in the soybean (Glycine max (L.) Merr.)

Isoflavone conjugates [7-O-β-D-glucosides and 7-O-(6″-malonyl-β-D-glucosides) of daidzein and genistein] accumulate in soybean roots and serve as the stored precursors of isoflavones (aglycons), which play very important roles in the rhizobia-mediated nodulation of this plant. Thus far, the isoflavone 7-O-glucosyltransferase (GmIF7GT or GmUGT1) has been biochemically characterized and is believed to be involved in isoflavone conjugate biosynthesis. The soybean genome encodes many other glycosyltransferase homologs (GmUGTs) that are related to GmUGT1; however, their catalytic properties, substrate specificities, and role(s) in isoflavone conjugation are unknown. In this study, nine different GmUGT1-related GmUGT cDNAs were isolated; six of these cDNAs belonged to two distinct phylogenetic subgroups (A and B), and these six were functionally characterized. The results showed that GmUGT4, a representative of subgroup A, encoded a UGT that was highly specific for isoflavones showing k_cat and k_cat/K_m values for daidzein of 5.89 ± 0.65 s^-1 and 2.91 × 10⁵ s^-1M^-1, respectively. Moreover, GmUGT4 was expressed in the roots (mainly in lateral roots) of the 7-day-old seedlings and seeds, both of which contained abundant amounts of isoflavone conjugates. By contrast, GmUGT1 and GmUGT7, which were subgroup B members, encoded enzymes with broad glucosyl-acceptor specificities and were mainly expressed in the aerial portions (cotyledons and hypocotyls) of the seedlings. In the present study, we proposed that the role of isoflavone glucosylation in a soybean plant is assigned to different GmUGT members in an organ/tissue-dependent manner. We also established the functional importance of GmUGT4 in the biosynthesis of isoflavone conjugates in lateral roots that make a major contribution to overall N₂ fixation.