Figure 1.
Biosynthetic pathway of tanshinone compounds.
The solid arrows denote known steps and the dashed arrows denote hypothetical steps. Modified from Ma et al. and Gao et al. [7,13]. Enzymes of 2-C-methyl-D-erythritol 4- phosphate (MEP) pathway are follows: 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (MCT), 4-diphosphocytidyl-2-Cmethyl-D-erythritol kinase (CMK), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MDS), 4-hydroxy-3-methylbut-2-enyl diphosphate synthase (HDS), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR); and enzymes of mevalonate (MVA) pathway are acetyl-CoA acetyltransferase (AACT), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), mevalonate kinase (MVK), 5-phosphomevalonate kinase (PMK) and 5-diphosphomevalonate decarboxylase (PMD). Isopentenyl diphosphate isomerase (IPPI) catalyzes the isomerisation of dimethylallyl dihosphate (DMAPP) to isopentenyl diphosphate (IPP) whereas conversion of IPP to geranylgeranyl diphosphate (GGPP) is catalysed by geranylgeranyl diphosphate synthase (GGPPS). Hypothetical tanshinones biosynthetic pathway was deduced by the consideration of identified diterpenoid natural products from S. miltiorrhiza [56,57]. These steps involve series hydroxylation, dehydrogenation and reduction reactions catalysed by cytochrome P450s, dehydrogenases and reductases.
Figure 2.
Sequence length distribution of unigenes in the S. miltiorrhiza transcriptomes of root and leaf.
Figure 3.
Functional classification of unigenes expressed in S. miltiorrhiza leaf and root.
Gene Ontology (GO) terms are summarized in three main categories of biological process (A), molecular function (B) and cellular component (C).
Figure 4.
Relative expression levels of P450 genes in S. miltiorrhiza root and leaf.
Total RNAs were extracted from root and leaf of 1-year-old S. miltiorrhiza and expression levels of P450s were analysed by quantitative real-time PCR. ACTIN was used as the internal reference gene and the relative abundance of each P450 genes in roots were compared to those in leaf, which were all set to be 1. Error bars indicate standard deviations of three biological replicates.
Figure 5.
Phylogenetic analysis of CYP450s from S. miltiorrhiza and other plants.
Amino acid sequences were aligned using CLUSTALW program, and evolutionary distances were calculated using MEGA4 software with the Poisson correction method. Nt, Nicotiana tabacum; Pg, Panax ginseng; Mt, Medicago truncatula; Vv, Vitis vinifera; Sd, Scoparia dulcis; Or, Orobanche ramose; St, Solanum tuberosum; Sm, Salvia miltiorrhiza. The GenBank/EMBL/DDBJ accession numbers of the sequences are ABC69417.1 (NtCYP72A54), AEY75218.1 (PgCYP72A129), ABC69414.1 (NtCYP72A57), ABC69422.1 (NtCYP72A58), ABC69419.1 (NtCYP72A56), ABC59075.1 (MtCYP72A67), ABC59086.1 (MtCYP98A37), XP_002283338 (VvCYP98A2), ABC69384.1 (NtCYP98A33V1), NP_189259.1 (AtCYP71B4), NP_195459.1 (AtCYP81F1), NP_182079.1 (AtCYP76C4), NP_182081.1 (AtCYP76C2), ADA70805.1 (SdCYP71D176), XP_003617706.1 (MtCYP71D10), NP_200536.3 (AtCYP71B10), NP_680107.1 (AtCYP71A25), XP_002266024.1 (VvCYP716B2), NP_850337.1 (AtCYP98A3), NP_850439.1 (AtCYP76C1), NP_172767.1 (AtCYP71B2), XP_002276576.1 (VvCYP76C4), NP_173149.1 (AtCYP72C1), XP_003592376.1 (Mt704G9), AEY75214.1 (PgCYP749A20), NP_196188.1 (AtCYP90A1), AFO63032.1 (PgCYP716A52V2), ABC59076.1 (MtCYP716A12), NP_190635.1 (AtCYP90B1), NP_182082.2 (AtCYP76C3), AFP74115.1 (OrCYP707A2), ABA55732.1 (StCYP707A1), AFP74114.1 (OrCYP707A1), ADA70806.1 (SdCYP71D177), ABC69395.1 (NtCYP71D47V2), ABC69397.1 (NtCYP71D48V1), NP_189261.1 (AtCYP71B34), NP_189264.3 (AtCYP71B37).