title: Combined transcriptome and metabolite profiling reveals that IiPLR1 plays an important role in lariciresinol accumulation in Isatis indigotica.
abstract: A lignan, lariciresinol, is an important efficacious compound for the antiviral effect of Isatis indigotica, a widely used herb for the treatment of colds, fever, and influenza. Although some rate-limiting steps of the lariciresinol biosynthetic pathway are well known, the specific roles of gene family members in I. indigotica in regulating lariciresinol production are poorly understood. In the present study, a correlation analysis between the RNA sequencing (RNA-Seq) expression profile and lignan content by using I. indigotica hairy roots treated with methyl jamonate (0.5 μM) at different time points as a source implicated that I. indigotica pinoresinol/lariciresinol reductase 1 (IiPLR1), but not IiPLR2 or IiPLR3, contributed greatly to lariciresinol accumulation. Gene silencing by RNA interference (RNAi) demonstrated that IiPLR1 indeed influenced lariciresinol biosynthesis, whereas suppression of IiPLR2 or IiPLR3 did not change lariciresinol abundance significantly. IiPLR1 was thus further characterized; IiPLR1 was constitutively expressed in roots, stems, leaves, and flowers of I. indigotica, with the highest expression in roots, and it responds to different stress treatments to various degrees. Recombinant IiPLR1 reduces both (±)-pinoresinol and (±)-lariciresinol efficiently, with comparative K cat/K m values. Furthermore, overexpression of IiPLR1 significantly enhanced lariciresinol accumulation in I. indigotica hairy roots, and the best line (ovx-2) produced 353.9 μg g(-1) lariciresinol, which was ~6.3-fold more than the wild type. This study sheds light on how to increase desired metabolites effectively by more accurate or appropriate genetic engineering strategies, and also provides an effective approach for the large-scale commercial production of pharmaceutically valuable lariciresinol by using hairy root culture systems as bioreactors.
authors.affiliation: The MOE Key Laboratory of Standardization of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Xiao
authors.affiliation: Department of Pharmaceutical Botany, School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Ji
authors.affiliation: Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
authors.forename: Shouhong
authors.initials: S
authors.lastname: Gao
authors.affiliation: Department of Pharmaceutical Botany, School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Hexin
authors.initials: H
authors.lastname: Tan
authors.affiliation: Department of Pharmaceutical Botany, School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
authors.forename: Ruibing
authors.initials: R
authors.lastname: Chen
authors.affiliation: Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Li
authors.affiliation: Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
authors.forename: Junfeng
authors.initials: J
authors.lastname: Chen
authors.affiliation: The MOE Key Laboratory of Standardization of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
authors.forename: Yingbo
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Department of Pharmaceutical Botany, School of Pharmacy, Second Military Medical University, Shanghai 200433, China zhanglei@smmu.edu.cn ztwang@shutcm.edu.cn chenwansheng@smmu.edu.cn.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.affiliation: The MOE Key Laboratory of Standardization of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China zhanglei@smmu.edu.cn ztwang@shutcm.edu.cn chenwansheng@smmu.edu.cn.
authors.forename: Zhengtao
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China zhanglei@smmu.edu.cn ztwang@shutcm.edu.cn chenwansheng@smmu.edu.cn.
authors.forename: Wansheng
authors.initials: W
authors.lastname: Chen
authors.affiliation: The MOE Key Laboratory of Standardization of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
authors.forename: Zhibi
authors.initials: Z
authors.lastname: Hu
mesh_terms: D005663:Furans / Q000378:metabolism*; D031219:Isatis / Q000235:genetics* / Q000378:metabolism; D017705:Lignans / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D059467:Transcriptome
chemical_list: D005663:Furans; D017705:Lignans; D010940:Plant Proteins; C060282:lariciresinol
keywords: Correlation analysis; Isatis indigotica; RNA-Seq expression profile.; lariciresinol; lignan; pinoresinol/lariciresinol reductase