title: Involvement of an ent-copalyl diphosphate synthase in tissue-specific accumulation of specialized diterpenes in Andrographis paniculata.
abstract: Ent-labdane-related diterpene (ent-LRD) specialized (i.e. secondary) metabolites of the medicinal plant kalmegh (Andrographis paniculata) have long been known for several pharmacological activities. However, our understanding of the ent-LRD biosynthetic pathway has remained largely incomplete. Since ent-LRDs accumulate in leaves, we carried out a comparative transcriptional analysis using leaf and root tissues, and identified 389 differentially expressed transcripts, including 223 transcripts that were preferentially expressed in leaf tissue. Analysis of the transcripts revealed various specialized metabolic pathways, including transcripts of the ent-LRD biosynthetic pathway. Two class II diterpene synthases (ApCPS1 and ApCPS2) along with one (ApCPS1') and two (ApCPS2' and ApCPS2″) transcriptional variants that were the outcomes of alternative splicing of the precursor mRNA and alternative transcriptional termination, respectively, were identified. ApCPS1 and ApCPS2 encode for 832- and 817-amino acids proteins, respectively, and are phylogenetically related to the dicotyledons ent-copalyl diphosphate synthases (ent-CPSs). The spatio-temporal patterns of ent-LRD metabolites accumulation and gene expression suggested a likely role for ApCPS1 in general (i.e. primary) metabolism, perhaps by providing precursor for the biosynthesis of phytohormone gibberellin (GA). However, ApCPS2 is potentially involved in tissue-specific accumulation of ent-LRD specialized metabolites. Bacterially expressed recombinant ApCPS2 catalyzed the conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP), the general precursor of diterpenes to ent-copalyl diphosphate (ent-CPP), the precursor of ent-LRDs. Taken together, these results advance our understanding of the tissue-specific accumulation of specialized ent-LRDs of medicinal importance.
authors.affiliation: Biotechnology Division, Council of Scientific and Industrial Research-Central Institute of Medicinal and Aromatic Plants, Lucknow, 226015, India.
authors.forename: Rajesh Chandra
authors.initials: RC
authors.lastname: Misra
authors.affiliation: Biotechnology Division, Council of Scientific and Industrial Research-Central Institute of Medicinal and Aromatic Plants, Lucknow, 226015, India.
authors.forename: Anchal
authors.initials: A
authors.lastname: Garg
authors.affiliation: Biotechnology Division, Council of Scientific and Industrial Research-Central Institute of Medicinal and Aromatic Plants, Lucknow, 226015, India.
authors.forename: Sudeep
authors.initials: S
authors.lastname: Roy
authors.affiliation: Chemical Sciences Division, Council of Scientific and Industrial Research-Central Institute of Medicinal and Aromatic Plants, Lucknow, 226015, India.
authors.forename: Chandan Singh
authors.initials: CS
authors.lastname: Chanotiya
authors.affiliation: Metabolic and Structural Biology Division, Council of Scientific and Industrial Research-Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, India.
authors.forename: Prema G
authors.initials: PG
authors.lastname: Vasudev
authors.affiliation: Biotechnology Division, Council of Scientific and Industrial Research-Central Institute of Medicinal and Aromatic Plants, Lucknow, 226015, India. Electronic address: sumitghosh@cimap.res.in.
authors.forename: Sumit
authors.initials: S
authors.lastname: Ghosh
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics* / Q000378:metabolism*; D035685:Andrographis / Q000235:genetics* / Q000378:metabolism*; D001419:Bacteria / Q000235:genetics / Q000378:metabolism; D004224:Diterpenes / Q000378:metabolism*; D053858:Metabolic Networks and Pathways; D008969:Molecular Sequence Data; D009928:Organ Specificity; D030781:Organisms, Genetically Modified / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism*; D018517:Plant Roots / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D004224:Diterpenes; D010940:Plant Proteins; C433017:labdane; D019883:Alkyl and Aryl Transferases; C090963:ent-kaurene synthetase A
keywords: Andrographis paniculata; Ent-copalyl diphosphate synthase; Ent-labdane-related diterpene; Medicinal compound; Specialized metabolism; Transcriptional variants