title: Function of AP2/ERF Transcription Factors Involved in the Regulation of Specialized Metabolism inOphiorrhiza pumilaRevealed by Transcriptomics and Metabolomics.
abstract: The hairy roots (HR) ofOphiorrhiza pumilaproduce camptothecin (CPT), a monoterpenoid indole alkaloid used as a precursor in the synthesis of chemotherapeutic drugs.O. pumilaHR culture is considered as a promising alternative source of CPT, however, the knowledge about the biosynthetic pathway and regulatory mechanism is still limited. In this study, five genes that encode AP2/ERF transcription factors, namelyOpERF1-OpERF5, were isolated from HR ofO. pumila. Phylogenetic analysis of AP2/ERF protein sequences suggested the close evolutionary relationship ofOpERF1 with stress-responsive ERF factors in Arabidopsis and ofOpERF2 with ERF factors reported to regulate alkaloid production, such as ORCA3 inCatharanthus roseus, NIC2locus ERF in tobacco, and JRE4 in tomato. We generated the transgenic HR lines ofO. pumila, ERF1i and ERF2i, in which the expression ofOpERF1andOpERF2, respectively, was suppressed using RNA interference technique. The transcriptome and metabolome of these suppressed HR were analyzed for functional characterization ofOpERF1 andOpERF2. Although significant changes were not observed in the metabolome, including CPT and related compounds, the suppression ofOpERF2resulted in reduced expression of genes in the 2-C-methyl-d-erythritol 4-phosphate and secologanin-strictosidine pathways, which supply a precursor, strictosidine, for CPT biosynthesis. Furthermore, while it was not conclusive forOpERF1, enrichment analysis of differentially expressed genes in the suppressed HR showed that the gene ontology terms for oxidation-reduction, presumably involved in secondary metabolite pathways, were enriched in the ERF2i downregulated gene set. These results suggest a positive role ofOpERF2 in regulating specialized metabolism inO. pumila.
authors.affiliation: Department of Molecular Biology and Biotechnology, Graduate School of Pharmaceutical Sciences, Chiba University Chiba, Japan.
authors.forename: Nirin
authors.initials: N
authors.lastname: Udomsom
authors.affiliation: Department of Molecular Biology and Biotechnology, Graduate School of Pharmaceutical Sciences, Chiba University Chiba, Japan.
authors.forename: Amit
authors.initials: A
authors.lastname: Rai
authors.affiliation: Department of Research and Development, Kazusa DNA Research Institute Chiba, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: Department of Molecular Biology and Biotechnology, Graduate School of Pharmaceutical Sciences, Chiba University Chiba, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Okuyama
authors.affiliation: Department of Molecular Biology and Biotechnology, Graduate School of Pharmaceutical Sciences, Chiba University Chiba, Japan.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Imai
authors.affiliation: RIKEN Center for Sustainable Resource Science Kanagawa, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Mori
authors.affiliation: RIKEN Center for Sustainable Resource Science Kanagawa, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Nakabayashi
authors.affiliation: Department of Molecular Biology and Biotechnology, Graduate School of Pharmaceutical Sciences, Chiba UniversityChiba, Japan; RIKEN Center for Sustainable Resource ScienceKanagawa, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Saito
authors.affiliation: Department of Molecular Biology and Biotechnology, Graduate School of Pharmaceutical Sciences, Chiba University Chiba, Japan.
authors.forename: Mami
authors.initials: M
authors.lastname: Yamazaki
keywords: AP/ERF; Ophiorrhiza pumila; RNA-seq; camptothecin; metabolome; secondary metabolism