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

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.

Functional characterization of NADPH-cytochrome P450 reductase and cinnamic acid 4-hydroxylase encoding genes from Scoparia dulcis L.

“…In addition, DNA blotting analysis showed that a single copy of SdCPR was present in the S. dulcis genome (Additional file 1: Fig. S2), and the result was identical to our transcriptome analysis (Yamamura et al 2017).…”

“…It is assumed that S. dulcis has only one CPR gene from DNA blotting (Additional file 1: Fig. S2) and transcriptome analyses (Yamamura et al 2017). The mRNA of SdCPR was detected in all tissues of S. dulcis plants (Fig.…”

“…In previous reports, it was demonstrated that the biosynthesis of SDB, a tetracyclic diterpene in S. dulcis, is markedly activated by the MJ and yeast extract treatments (Nkembo et al 2005;Yamamura et al 2014). It is clear that a large number of P450s are responsible for not only SDB biosynthesis but also in the other biosynthetic reactions of secondary metabolites in S. dulcis (Yamamura et al 2017). Among P450s, C4H is a key enzyme in phenylpropanoid biosynthetic pathways such as PAL and 4CL (Additional file 1: Fig.…”

“…Hayashi et al demonstrated that the biosynthesis of SDB is markedly activated in Scoparia leaf tissues by treatment with methyl jasmonate (MJ) (Hayashi et al 1999;Nkembo et al 2006). We recently discovered novel candidate genes (encoding diterpene synthase and P450) potentially involved in SDB biosynthesis by transcriptome analysis (Yamamura et al 2017).…”

Genome-wide association study reveals candidate genes influencing lipids and diterpenes contents in Coffea arabica L

“…Terpene diversification is driven by the machinery consisting TPSs and cytochrome P450-dependent monooxygenases ( CYP ) genes. The latter is important for modifying and diversifying the terpenoid scaffolds by redox modification 42 . We identified one SNP associated with CAF (S11_29778697) that was co-localized with the gene Cc11_g12750, which encodes a cytochrome P450 704 ( CYP704 ).…”