Elucidation of terpenoid metabolism in Scoparia dulcis by RNA-seq analysis

Yoshimi Yamamura,Fumiya Kurosaki,Jung-Bum Lee

doi:10.1038/srep43311

Abstract

Scoparia dulcis biosynthesize bioactive diterpenes, such as scopadulcic acid B (SDB), which are known for their unique molecular skeleton. Although the biosynthesis of bioactive diterpenes is catalyzed by a sequence of class II and class I diterpene synthases (diTPSs), the mechanisms underlying this process are yet to be fully identified. To elucidate these biosynthetic machinery, we performed a high-throughput RNA-seq analysis, and de novo assembly of clean reads revealed 46,332 unique transcripts and 40,503 two unigenes. We found diTPSs genes including a putative syn-copalyl diphosphate synthase (SdCPS2) and two kaurene synthase-like (SdKSLs) genes. Besides them, total 79 full-length of cytochrome P450 (CYP450) genes were also discovered. The expression analyses showed selected CYP450s associated with their expression pattern of SdCPS2 and SdKSL1, suggesting that CYP450 candidates involved diterpene modification. SdCPS2 represents the first predicted gene to produce syn-copalyl diphosphate in dicots. In addition, SdKSL1 potentially contributes to the SDB biosynthetic pathway. Therefore, these identified genes associated with diterpene biosynthesis lead to the development of genetic engineering focus on diterpene metabolism in S. dulcis.

Highlights

Scoparia dulcis biosynthesize bioactive diterpenes, such as scopadulcic acid B (SDB), which are known for their unique molecular skeleton
We prepared four cDNA libraries from three tissues including leaves with or without MeJA treatment, young leaves, and roots. We choose these samples for preparation of total RNA, since it is known that SDB production in S. dulcis leaf tissue is rapidly and transiently stimulated by MeJA as an elicitor[24], and SDB contents in young leaves were higher than those in adult leaves[25]
When we phylogenetically analyzed TPSs being predicted by de novo assembly of transcripts, SdCPS2 was placed into a sub-clade consisting of (+)-CPSs and oxygenating diterpene synthases, which was relatively far from a sub-clade consisting of rice CPSs (Fig. 3)

Summary

Introduction

Scoparia dulcis biosynthesize bioactive diterpenes, such as scopadulcic acid B (SDB), which are known for their unique molecular skeleton. Phytochemical studies revealed that this plant produce various unique diterpenes its leaves: (1) labdane type: scoparic acid A; (2) scopadulane type: scopadulcic acid B (SDB) and scopadulciol; (3) aphidicolane type: scopadulin[7] Among these diterpenes, SDB was found to possess various biological activities such as antiherpetic and inhibitory effects on gastric H+, K+ -ATPase[8,9]. Due to the unique carbon skeleton and biological activities of SDB, the scopadulane type diterpenes were selected as attractive targets for chemical synthesis and their total syntheses were accomplished by several groups[15,16,17] Their synthetic route included numerous steps and produced SDB as racemic mixtures.

Methods

Results

Discussion

Conclusion