The Basic Helix–Loop–Helix Transcription Factor GubHLH3 Positively Regulates Soyasaponin Biosynthetic Genes in Glycyrrhiza uralensis

Abstract Glycyrrhiza uralensis (licorice) is a widely used medicinal plant belonging to the Fabaceae. Its main active component, glycyrrhizin, is an oleanane-type triterpenoid saponin widely used as a medicine and as a natural sweetener. Licorice also produces other triterpenoids, including soyasaponins. Recent studies have revealed various oxidosqualene cyclases and cytochrome P450 monooxygenases (P450s) required for the biosynthesis of triterpenoids in licorice. Of these enzymes, β-amyrin synthase (bAS) and β-amyrin C-24 hydroxylase (CYP93E3) are involved in the biosynthesis of soyasapogenol B (an aglycone of soyasaponins) from 2,3-oxidosqualene. Although these biosynthetic enzyme genes are known to be temporally and spatially expressed in licorice, the regulatory mechanisms underlying their expression remain unknown. Here, we identified a basic helix–loop–helix (bHLH) transcription factor, GubHLH3, that positively regulates the expression of soyasaponin biosynthetic genes. GubHLH3 preferentially activates transcription from promoters of CYP93E3 and CYP72A566, the second P450 gene newly identified and shown to be responsible for C-22β hydroxylation in soyasapogenol B biosynthesis, in transient co-transfection assays of promoter–reporter constructs and transcription factors. Overexpression of GubHLH3 in transgenic hairy roots of G. uralensis enhanced the expression levels of bAS, CYP93E3 and CYP72A566. Moreover, soyasapogenol B and sophoradiol (22β-hydroxy-β-amyrin), an intermediate between β-amyrin and soyasapogenol B, were increased in transgenic hairy root lines overexpressing GubHLH3. We found that soyasaponin biosynthetic genes and GubHLH3 were co-ordinately up-regulated by methyl jasmonate (MeJA). These results suggest that GubHLH3 regulates MeJA-responsive expression of soyasaponin biosynthetic genes in G. uralensis. The regulatory mechanisms of triterpenoid biosynthesis in legumes are compared and discussed.