Fig 1.
Diterpene synthases identified in the leaf transcriptome of Isodon rubescens.
Fig 2.
Phylogeny of Lamiaceae diterpene synthases.
The illustrated maximum-likelihood tree was constructed with known diTPSs from species of the Lamiaceae family, using the ancestral bifunctional class I/II diTPS Physcomitrella patens ent-kaurene/kaurenol synthase (PpCPS/KS) as an outgroup. Different domain architecture and association with general or specialized metabolism (or both) are highlighted. Bootstrap (500 repetitions) confidence values over 80% are illustrated at branch points. Abbreviations and GenBank accession numbers are listed in S1 Table.
Fig 3.
Biochemical characterization of IrTPS5 and IrTPS3.
GC-MS analysis of extracted enzyme products resulting from Agrobacterium-mediated transient N. benthamiana co-expression assays of the class II diTPSs IrTPS5 (A) and IrTPS3 (B) with the class I diTPSs Grindelia robusta ent-kaurene synthase (GrEKS) or Marrubium vulgare epoxy-labdane/miltiradiene synthase (MvELS). Reaction products are depicted as extracted ion chromatograms (EIC, m/z 257) with corresponding mass spectra (C) for products: a, ent-copalol (i.e. dephosphorylated CPP); b, ent-kaurene; c, (+)-copalol; d, unidentified diterpene; e, miltiradiene.
Fig 4.
Biochemical characterization of IrTPS4 and IrTPS2.
Illustrated are extracted ion chromatograms (EIC, m/z 257) of products obtained in Agrobacterium-mediated transient Nicotiana benthamiana co-expression assays of IrTPS2 and IrTPS4 with IrTPS5 (A) and IrTPS3 (B), respectively. Expression of IrTPS3, IrTPS5 and the RNA silencing suppressor protein p19 served as controls. Reaction products: a, ent-copalol (i.e. dephosphorylated CPP); b, ent-kaurene; c, (+)-copalol; d, unidentified diterpene; e, miltiradiene; f, nezukol. (C) Mass spectrum of the product resulting from coupled activity of IrTPS3 and IrTPS2 with significant similarity to reference mass spectra of 8β-hydroxy-isopimar-15-ene or nezukol (product f). (D) Structure of nezukol as verified by 1D and 2D nuclear magnetic resonance (NMR) analysis.
Fig 5.
Nezukol abundance in Isodon rubescens leaf tissue.
GC-MS analysis of ethyl acetate/hexane fractionated hexane extracts of I. rubescens leaf tissue. (A) Total ion chromatogram (TIC, 1) and extracted ion chromatogram (EIC, m/z 275, 2) of a 20:80 (v/v) ethyl acetate/hexane fraction containing the compounds nezukol (product f) and manoyl oxide (product g). Compounds were identified by comparison to purified enzyme products (3–4). (B) TIC (5) and EIC (m/z 275, 6) of a 40:60 (v/v) ethyl acetate/hexane fraction containing copalol (product a or c) as compared to enzyme-produced ent-copalol (7).
Fig 6.
Relative transcript abundance of I. rubescens diTPSs in leaf tissue.
Relative transcript abundance was measured by quantitative real-time PCR (qRT-PCR) and normalized to actin as internal reference gene. Error bars represent standard errors based on duplicate measurements of three biological replicates. Reaction specificity was confirmed by melt curve analysis of each sample and sequence verification of representative amplicons.
Fig 7.
Proposed reaction pathways en route to nezukol and other I. rubescens diterpenes.
Following the common framework of labdane biosynthesis in angiosperms, pairs of monofunctional class II and class I diTPSs were identified that produce distinct diterpene scaffolds. First, IrTPS3 and IrTPS5 transform the central precursor geranylgeranyl diphosphate (GGPP) into the bicyclic prenyl diphosphate intermediates (+)-copalyl diphosphate (CPP) and ent-CPP, respectively. Second, the class I diTPSs IrTPS2 and IrTPS4 catalyze diphosphate ionization to form a common pimar-15-en-8-yl+ carbocation. IrTPS4-catalyzed rearrangement of this carbocation by 1,6 proton transfer and 1,2 methyl migration followed by deprotonation at C-13 yields miltiradiene. In contrast, IrTPS2 facilitates the direct neutralization of the carbocation by water capture at C-8 to form the hydroxylated diterpene nezukol.