Isolation and Functional Characterization of the Promoters of Miltiradiene Synthase Genes, TwTPS27a and TwTPS27b, and Interaction Analysis with the Transcription Factor TwTGA1 from Tripterygium wilfordii

Abstract

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.

MYC2 Transcription Factors TwMYC2a and TwMYC2b Negatively Regulate Triptolide Biosynthesis in Tripterygium wilfordii Hairy Roots

“…For example, the promoter of SmKSL, a key tanshinone biosynthetic gene encoding miltiradiene synthase in S miltiorrhiza, containing GCC-box and E-box motifs, can be recognized by positive regulators SmERF6 [61] and SmERF128 [62], and negative regulator SmbHLH3 [63], thereby up-regulating and down-regulating the biosynthesis of tanshinone. Similarly, in T. wilfordii, the promoters of miltiradiene synthase genes TwTPS27a and TwTPS27b containing TGACG-motif, E-box, and T/G-box elements also can be regulated by positive regulator TwTGA1 upregulating triptolide biosynthesis [35,64] and negative regulators TwMYC2a and TwMYC2b reported here. These findings indicate that the regulation of triptolide biosynthesis is complex and more potential transcription factors can be involved in the biosynthesis of triptolide, which needs further study.…”

“…The bHLH TFs function by binding to the E-box (CANNTG) motif in their target gene promoters [31]. Our previous studies have isolated the promoters of key pathway genes TwTPS27a and TwTPS27b and silico analysis showed that five different types of E-box (CAN-NTG) motifs were found in their promoters [35]. It has been reported that in addition to the G-box (CACGTG) showing the strongest MYC2 binding capacity, the E-box (CACATG) and T/G-box (CACGTT) have stronger binding capacity than other E-box variants [27].…”

“…It is well known that MYC2 TFs recognize the E-box (CANNTG) and its variants G-box (CACGTG) or T/G-box (CACGTT) within the promoters of their target genes [47][48][49][50][51][52]. Our previous studies [35] showed that several different types of E-box motifs (CANNTG) were found in the promoters of miltiradiene synthase genes, TwTPS27a and TwTPS27b, which are the key genes in triptolide biosynthesis. As shown in Figure 2A, there are five different Ebox motifs present in the TwTPS27a and TwTPS27b promoters, including E1 (CAGATG), E2 (CACATG), E3 (CAAATG), E4 (CACGTT, called T/G-box), and E5 (CAATTG), indicating that these motifs may be the binding sites of TwMYC2a and TwMYC2b.…”

“…The empty vector (EV) was used as a negative control. The promoters of TwTPS27a (1494 bp upstream of the start codon) and TwTPS27b (1862 bp upstream of the start codon) were cloned into the pBI121 vector, generating the reporter vectors, 27aP::GUS and 27bP::GUS, which had been previously constructed [35]. All of these constructs were separately transformed into Agrobacterium tumefaciens GV3101 (pSoup-p19).…”

“…These findings suggest that the E-box motifs in the key pathway gene promoters play important roles in the regulation of secondary metabolism in plants. Recently, in our previous studies, five different types of E-box elements, including CAGATG, CACATG, CAAATG, CACGTT (T/G-box), and CAATTG, were found in the promoters of miltiradiene synthase genes, TwTPS27a and TwTPS27b, key genes of triptolide biosynthesis in T. wilfordii [35]. This indicated that these E-box elements in the promoters of TwTPS27a/b as the potential MYC binding sites might be recognized by MYC2 TF in T. wilfordii.…”