title: Coupling deep transcriptome analysis with untargeted metabolic profiling in Ophiorrhiza pumila to further the understanding of the biosynthesis of the anti-cancer alkaloid camptothecin and anthraquinones.
abstract: The Rubiaceae species, Ophiorrhiza pumila, accumulates camptothecin, an anti-cancer alkaloid with a potent DNA topoisomerase I inhibitory activity, as well as anthraquinones that are derived from the combination of the isochorismate and hemiterpenoid pathways. The biosynthesis of these secondary products is active in O. pumila hairy roots yet very low in cell suspension culture. Deep transcriptome analysis was conducted in O. pumila hairy roots and cell suspension cultures using the Illumina platform, yielding a total of 2 Gb of sequence for each sample. We generated a hybrid transcriptome assembly of O. pumila using the Illumina-derived short read sequences and conventional Sanger-derived expressed sequence tag clones derived from a full-length cDNA library constructed using RNA from hairy roots. Among 35,608 non-redundant unigenes, 3,649 were preferentially expressed in hairy roots compared with cell suspension culture. Candidate genes involved in the biosynthetic pathway for the monoterpenoid indole alkaloid camptothecin were identified; specifically, genes involved in post-strictosamide biosynthetic events and genes involved in the biosynthesis of anthraquinones and chlorogenic acid. Untargeted metabolomic analysis by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) indicated that most of the proposed intermediates in the camptothecin biosynthetic pathway accumulated in hairy roots in a preferential manner compared with cell suspension culture. In addition, a number of anthraquinones and chlorogenic acid preferentially accumulated in hairy roots compared with cell suspension culture. These results suggest that deep transcriptome and metabolome data sets can facilitate the identification of genes and intermediates involved in the biosynthesis of secondary products including camptothecin in O. pumila.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chiba, 260-8675 Japan.
authors.forename: Mami
authors.initials: M
authors.lastname: Yamazaki
authors.forename: Keiichi
authors.initials: K
authors.lastname: Mochida
authors.forename: Takashi
authors.initials: T
authors.lastname: Asano
authors.forename: Ryo
authors.initials: R
authors.lastname: Nakabayashi
authors.forename: Motoaki
authors.initials: M
authors.lastname: Chiba
authors.forename: Nirin
authors.initials: N
authors.lastname: Udomson
authors.forename: Yasuyo
authors.initials: Y
authors.lastname: Yamazaki
authors.forename: Dayan B
authors.initials: DB
authors.lastname: Goodenowe
authors.forename: Ushio
authors.initials: U
authors.lastname: Sankawa
authors.forename: Takuhiro
authors.initials: T
authors.lastname: Yoshida
authors.forename: Atsushi
authors.initials: A
authors.lastname: Toyoda
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Totoki
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Sakaki
authors.forename: Elsa
authors.initials: E
authors.lastname: Góngora-Castillo
authors.forename: C Robin
authors.initials: CR
authors.lastname: Buell
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Sakurai
authors.forename: Kazuki
authors.initials: K
authors.lastname: Saito
mesh_terms: D000880:Anthraquinones / Q000737:chemistry / Q000378:metabolism*; D000972:Antineoplastic Agents, Phytogenic / Q000096:biosynthesis* / Q000737:chemistry; D002166:Camptothecin / Q000096:biosynthesis* / Q000737:chemistry; D018929:Cell Culture Techniques; D020869:Gene Expression Profiling / Q000379:methods*; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D013058:Mass Spectrometry; D055442:Metabolome / Q000235:genetics; D018517:Plant Roots / Q000235:genetics; D019666:Rubiaceae / Q000235:genetics* / Q000378:metabolism*; D064210:Secondary Metabolism / Q000235:genetics; D013535:Suspensions
chemical_list: D000880:Anthraquinones; D000972:Antineoplastic Agents, Phytogenic; D013535:Suspensions; D002166:Camptothecin
keywords: Anthraquinone; Camptothecin; Hairy root; Metabolome; Ophiorrhiza pumila; Transcriptome