Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871 Japan.
Kihara Institute for Biological Research, Yokohama City University, 641-12 Maioka-cho, Totsuka-ku, Yokohama, Kanagawa, 244-0813 Japan.
Plant Cell Physiol. 2018 Apr 1;59(4):778-791. doi: 10.1093/pcp/pcy046.
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.
甘草是一种广泛使用的药用植物，属于豆科。其主要活性成分甘草酸是一种齐墩果烷型三萜皂苷，被广泛用作药物和天然甜味剂。甘草还产生其他三萜类化合物，包括大豆皂苷。最近的研究揭示了甘草中三萜类化合物生物合成所需的各种角鲨烯环氧化酶和细胞色素 P450 单加氧酶（P450s）。在这些酶中，β-香树脂醇合酶（bAS）和β-香树脂醇 C-24 羟化酶（CYP93E3）参与从 2,3-氧化角鲨烯合成大豆皂苷元 B（大豆皂苷的苷元）。尽管已知这些生物合成酶基因在甘草中具有时间和空间表达，但它们表达的调控机制尚不清楚。在这里，我们鉴定了一个碱性螺旋-环-螺旋（bHLH）转录因子 GubHLH3，它正向调节大豆皂苷生物合成基因的表达。GubHLH3 优先激活 CYP93E3 和 CYP72A566 启动子的转录，CYP72A566 是新鉴定的第二个 P450 基因，负责大豆皂苷元 B 生物合成中的 C-22β 羟化，在启动子-报告基因构建体和转录因子的瞬时共转染测定中。在甘草毛状根的转基因中过量表达 GubHLH3 增强了 bAS、CYP93E3 和 CYP72A566 的表达水平。此外，在过量表达 GubHLH3 的转基因毛状根系中，大豆皂苷元 B 和槐二醇（β-香树脂醇和大豆皂苷元 B 之间的 22β-羟基-β-香树脂醇）增加。我们发现大豆皂苷生物合成基因和 GubHLH3 被茉莉酸甲酯（MeJA）协调上调。这些结果表明 GubHLH3 调节甘草中大豆皂苷生物合成基因对 MeJA 的响应表达。比较和讨论了豆科植物三萜类化合物生物合成的调控机制。
