Table 1.
Primers used for cloning cDNA in this study.
Table 2.
Concentrations (μg × g FW-1) of extracted terpenes from roots, stems, and leaves of Pityopsis ruthii.
Fig 1.
Gas chromatograms of terpene volatiles emitted from open flowers and leaves of Pityopsis ruthii.
A. Compounds collected from open flowers using headspace. 1. α-pinene; 2. β-pinene; 3. myrcene; 4. limonene; 5 internal standard (1-octanol); 6. β-elemene; 7. (E)-α-bergamotene; 8. unidentified sesquiterpene 1; 9. guaia-1(10),11-diene; 10. unidentified sesquiterpene 2; 11. aromadendrene; 12. germacrene D; 13. allo-aromadendrene; 14. α-selinene; 15. pogostol; 16. unidentified diterpene 3; 17. kaur-16-ene. B. chemical structures of known compounds identified from open flowers in Pityopsis ruthii.
Table 3.
Terpene emission from Pityopsis ruthii flowers and leaves using headspace sampling (ng × h-1 × g FW-1).
Fig 2.
Terpene pathways involved in terpene biosynthesis in Pityopsis ruthii.
Heatmap of the data from flower transcriptome analyses performed in triplicate along with representative gene indicated expression level, which was calculated with fragments per kilobase of transcripts per million mapped fragments (FPKM). Abbreviations of genes: AACT, acetoacetyl-CoA thiolase; HMGS, hydroxylmethylglutaryl-CoA synthase; HMGR, hydroxymethylglutaryl-CoA reductase; MVK, mevalonate kinase; PMK, 5-phospho-mevalonate kinase, PMD, mevalonate diphosphate decarboxylase; FPPS, farnesyl pyrophosphate synthase; DXS, 1-deoxy-d-xylulose-5-phosphate synthase; DXR, 1-deoxy-d-xylulose-5-phosphate reductoisomerase; CMS, 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase; CMK, 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase; MDS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS, (E)-4-hydroxy-3-methylbut-2- enyl diphosphate synthase; HDR, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; IDI, isopentenyl-diphosphate delta-isomerase; GPPS, geranyl diphosphate synthase. Compound abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; MVA, mevalonate; DXP, 1-Deoxy-D-xylulose 5-phosphate; MEP, 2-C-Methyl-D-erythritol 4-phosphate; CDP-ME, 2-C-Methyl-d-erythritol-2,4-cyclodiphosphate; CDP-MEP, 2-Phospho-4-(cytidine 5’-diphospho)-2-C-methyl-D-erythritol; ME-CPP, 2-C-methyl-d-erythritol-2,4-cyclodiphosphate; HMBPP, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate; FPP, (E,E)-farnesyl pyrophosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethyallyl pyrophosphate; GPP, geranyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate.
Fig 3.
Phylogenetic tree of full-length TPSs from Pityopsis ruthii (PrTPSs) and TPSs from Arabidopsis thaliana (AtTPSs).
Phylogenetic tree was reconstructed by maximum likelihood method based on JTT model. The classification of TPS subfamilies was determined as previously reported [9].
Fig 4.
Monoterpene activity of PrTPS1 (A) and PrTPS3 (B). Crude protein extracts from heterologous expression in E. coli catalyzed the conversion of the substrate GPP into monoterpenes. PrTPS genes are identified. Products were identified by GC-MS: 1. α-pinene; 2. β-pinene. Peak numbers are consistent with those reported in Fig 1.
Fig 5.
Sesquiterpene synthase activity of PrTPS5 (A), PrTPS6 (B), and PrTPS7 (C). Crude proteins extracted from heterologous expression in E. coli catalyzed the conversion of the substrate FPP into sesquiterpenes. Products identified by GC-MS: 6. β-elemene; 7. (E)-α-bergamotene; 12. germacrene D; 19.δ-elemene; 20. (+)-cycloisosativene; 21. β- ylangene; 22. γ-elemene; 23. δ-cadinene. Peak numbers are consistent with those reported in Fig 1.
Fig 6.
Three-dimensional models of PrTPS6 and PrTPS7.
The models were computed by Swiss Model [34] using default parameters. Identical amino acid residues are shown in green; polymorphic amino acid chains are shown in red. The conserved motifs were identified using the Swiss Model server over web browser and marked per each respective coding frame.