Chemical profile and analysis of biosynthetic pathways and genes of volatile terpenes in Pityopsis ruthii, a rare and endangered flowering plant

Abstract

It is critical to gather biological information about rare and endangered plants to incorporate into conservation efforts. The secondary metabolism of Pityopsis ruthii, an endangered flowering plant that only occurs along limited sections of two rivers (Ocoee and Hiwassee) in Tennessee, USA was studied. Our long-term goal is to understand the mechanisms behind P. ruthii's adaptation to restricted areas in Tennessee. Here, we profiled the secondary metabolites, specifically in flowers, with a focus on terpenes, aiming to uncover the genomic and molecular basis of terpene biosynthesis in P. ruthii flowers using transcriptomic and biochemical approaches. By comparative profiling of the nonpolar portion of metabolites from various tissues, P. ruthii flowers were rich in terpenes, which included 4 monoterpenes and 10 sesquiterpenes. These terpenes were emitted from flowers as volatiles with monoterpenes and sesquiterpenes accounting for almost 68% and 32% of total emission of terpenes, respectively. These findings suggested that floral terpenes play important roles for the biology and adaptation of P. ruthii to its limited range. To investigate the biosynthesis of floral terpenes, transcriptome data for flowers were produced and analyzed. Genes involved in the terpene biosynthetic pathway were identified and their relative expressions determined. Using this approach, 67 putative terpene synthase (TPS) contigs were detected. TPSs in general are critical for terpene biosynthesis. Seven full-length TPS genes encoding putative monoterpene and sesquiterpene synthases were cloned and functionally characterized. Three catalyzed the biosynthesis of sesquiterpenes and four catalyzed the biosynthesis of monoterpenes. In conclusion, P. ruthii plants employ multiple TPS genes for the biosynthesis of a mixture of floral monoterpenes and sesquiterpenes, which probably play roles in chemical defense and attracting insect pollinators alike.

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.

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.