Miltiradiene synthase (MS) genes, TwTPS27a and TwTPS27b, are the key diterpene synthase genes in the biosynthesis of triptolide, which is an important medicinally active diterpenoid in Tripterygium wilfordii. However, the mechanism underlying the regulation of key genes TwTPS27a/b in triptolide biosynthesis remains unclear. In this study, the promoters of TwTPS27a (1496 bp) and TwTPS27b (1862 bp) were isolated and analyzed. Some hormone-/stress-responsive elements and transcription factor (TF) binding sites were predicted in both promoters, which might be responsible for the regulation mechanism of TwTPS27a/b. The β-glucuronidase (GUS) activity analysis in promoter deletion assays under normal and methyl jasmonate (MeJA) conditions showed that the sequence of −921 to −391 bp is the potential core region of the TwTPS27b promoter. And the TGACG-motif, a MeJA-responsive element found in this core region, might be responsible for MeJA-mediated stress induction of GUS activity. Moreover, the TGACG-motif is also known as the TGA TF-binding site. Yeast one-hybrid and GUS transactivation assays confirmed the interaction between the TwTPS27a/b promoters and the TwTGA1 TF (a MeJA-inducible TGA TF upregulating triptolide biosynthesis in T. wilfordii), indicating that TwTPS27a/b are two target genes regulated by TwTGA1. In conclusion, our results provide important information for elucidating the regulatory mechanism of MS genes, TwTPS27a and TwTPS27b, as two target genes of TwTGA1, in jasmonic acid (JA)-inducible triptolide biosynthesis.

In order to solve the shortage of natural Tripterygium wilfordii Hook. f. plant resource for the production of the important secondary metabolites triptolide and wilforine, hairy roots were induced from its root calli by Agrobacterium rhizogenes. Induced hairy roots not only could be maintained and grown well in hormone-free half-strength Murashige and Skoog medium but also could produce sufficient amounts of both triptolide and wilforine. Although hairy roots produced approximately 15% less triptolide than adventitious roots and 10% less wilforine than naturally grown roots, they could grow fast and could be a suitable system for producing both secondary metabolites compared with other tissues. Addition of 50 micrometer methyl jasmonate (MeJA) could slightly affect hairy root growth, but dramatically stimulated the production of both triptolide and wilforine, whereas 50 micrometer salicylic acid had no apparent effect on hairy root growth with slightly stimulatory effects on the production of both secondary metabolites. Addition of precursor nicotinic acid, isoleucine, or aspartic acid at the concentration of 500 micrometer had varying effects on hairy root growth, but none of them had stimulatory effects on triptolide production, and only the former two had slightly beneficial effects on wilforine production. The majority of triptolide produced was secreted into the medium, whereas most of the produced wilforine was retained inside of hairy roots. Our studies provide a promising way to produce triptolide and wilforine in T. wilfordii hairy root cultures combined with MeJA treatment.

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Triptolide, an important bioactive diterpenoid extracted from the plant Tripterygium wilfordii, exhibits many pharmacological activities. MYC2 transcription factor (TF) plays an important role in the regulation of various secondary metabolites in plants. However, whether MYC2 TF could regulate the biosynthesis of triptolide in T. wilfordii is still unknown. In this study, two homologous MYC2 TF genes, TwMYC2a and TwMYC2b, were isolated from T. wilfordii hairy roots and functionally characterized. The analyses of the phylogenetic tree and subcellular localization showed that they were grouped into the IIIe clade of the bHLH superfamily with other functional MYC2 proteins and localized in the nucleus. Furthermore, yeast one-hybrid and GUS transactivation assays suggested that TwMYC2a and TwMYC2b inhibited the promoter activity of the miltiradiene synthase genes, TwTPS27a and TwTPS27b, by binding to the E-box (CACATG) and T/G-box (CACGTT) motifs in their promoters. Transgenic results revealed that RNA interference of TwMYC2a/b significantly enhanced the triptolide accumulation in hairy roots and liquid medium by upregulating the expression of several key biosynthetic genes, including TwMS (TwTPS27a/b), TwCPS (TwTPS7/9), TwDXR, and TwHMGR1. In summary, our findings show that TwMYC2a and TwMYC2b act as two negative regulators of triptolide biosynthesis in T. wilfordii hairy roots and also provide new insights on metabolic engineering of triptolide in the future.

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