Identification, Functional Characterization, and Evolution of Terpene Synthases from a Basal Dicot

Abstract

Bay laurel (Laurus nobilis) is an agriculturally and economically important dioecious tree in the basal dicot family Lauraceae used in food and drugs and in the cosmetics industry. Bay leaves, with their abundant monoterpenes and sesquiterpenes, are used to impart flavor and aroma to food, and have also drawn attention in recent years because of their potential pharmaceutical applications. To identify terpene synthases (TPSs) involved in the production of these volatile terpenes, we performed RNA sequencing to profile the transcriptome of L. nobilis leaves. Bioinformatic analysis led to the identification of eight TPS complementary DNAs. We characterized the enzymes encoded by three of these complementary DNAs: a monoterpene synthase that belongs to the TPS-b clade catalyzes the formation of mostly 1,8-cineole; a sesquiterpene synthase belonging to the TPS-a clade catalyzes the formation of mainly cadinenes; and a diterpene synthase of the TPS-e/f clade catalyzes the formation of geranyllinalool. Comparison of the sequences of these three TPSs indicated that the TPS-a and TPS-b clades of the TPS gene family evolved early in the evolution of the angiosperm lineage, and that geranyllinalool synthase activity is the likely ancestral function in angiosperms of genes belonging to an ancient TPS-e/f subclade that diverged from the kaurene synthase gene lineages before the split of angiosperms and gymnosperms.

Figure 1.

GC analysis of volatiles present in young leaves of female and male L. nobilis trees.

Figure 2.

The levels of the five terpene compounds with the highest concentration in female organs of L. nobilis, and comparisons with the concentrations of the corresponding compounds in male organs and in organs present in both sexes. Bars labeled with different letters indicate the significant differences as determined by JMP statistical software (P ≤ 0.05; Tukey-Kramer honestly significant difference test). All analyses were performed using three biological replicates. FW, Fresh weight.

Figure 3.

An unrooted neighbor-joining tree based on protein sequences of LnTPS1, LnTPS2, and LnTPS3 and selected plant TPS sequences. The tree was generated using Phylogeny Analysis MEGA5.1 program (Tamura et al., 2013). The resulting tree was bootstrap analyzed with 1,000 replicates. The subdivisions (clades) of the TPS gene family, designated TPS-a to TPS-e/f, are according to Chen et al. (2011). The black bold underline indicates the L. nobilis LnTPS1, LnTPS2, and LnTPS3 sequences identified in this study. Ocimum basilicum (Ob) α-zingiberene synthase (syn), accession number Q5SBP4; Lavandula angustifolia (La) transα-bergamotene syn, Q2XSC4; La limonene syn, Q2XSC6; V. vinifera (Vv) α-phellandrene syn, NP_001268167; M. grandiflora (Mg) α-terpineol syn, B3TPQ7; L. cubeba (Lc) α-thujene syn, AEJ91555; Mg β-cubebene syn, B3TPQ6; Zingiber zerumbet (Zz) α-humulene syn, B1B1U3; Artemisia annua (Aa) β-caryophyllene syn, Q8SA63; Ricinus communis (Rc) α-copaene syn, JN315864; Vv germacrene A syn, ADR66821; Zea mays (Zm) β-caryophyllene syn, ABY79213; Oryza sativa (Os) β-caryophyllene/β-elemene syn, DQ872158; Abies grandis (Ag) β-phellandrene syn, Q9M7D1; Ag α-bisabolene syn, O81086; Pseudotsuga menziesii (Pm) γ-bisabolene syn, Q4QSN4; Arabidopsis (Arabidopsis thaliana; Ath) ent-copalyl diphosphate syn, NP_192187; Picea glauca (Pg) ent-copalyl diphosphate syn, ADB55707; S. lycopersicum (Sl) ent-kaurene syn, AEP82778; Ath ent-kaurene syn, NP_178064; Pg ent-kaurene syn, ADB55708; Amborella trichopada (Atr) geranyllinalool syn-like, ERN20279; P. dactylifera (Pd) geranyllinalool syn-like, NW_008248648; Ath geranyllinalool syn, At1G61120; Vv geranyllinalool syn, NP_001268004; Grindela hirsutula (Gh) geranyllinalool syn, AGN70888; Sl geranyllinalool syn, KJ755870; Nicotiana attenuate (Na) geranyllinalool syn, KJ755868; KS, kaurene synthase.

Figure 4.

GC-MS of the products generated in vitro by recombinant LnTPS1 protein. A, Analysis of products of the reaction catalyzed by recombinant LnTPS1with GPP as the substrate. B, Analysis of products of the reaction catalyzed by boiled recombinant LnTPS1with GPP as the substrate. The insert shows the structure and the mass spectrum of the enzymatic reaction products identified as 1,8-cineole by matching the retention time and the MS pattern with an authentic standard. Identification of other products was done by GC-MS according to the retention index and by comparing the compound mass spectrum to the National Institute of Standards and Technology database.

Figure 5.

GC-MS of the products generated in vitro by recombinant LnTPS2 protein. A, Analysis of products of the reaction catalyzed by recombinant LnTPS2 with eeFPP as the substrate. B, Analysis of products of the reaction catalyzed by boiled recombinant LnTPS2 with eeFPP as the substrate. The insert shows the structure and the mass spectrum of the enzymatic reaction products identified as δ-cadinene by matching the retention time and the MS pattern with an authentic standard. Identification of other products was done by GC-MS according to the retention index and by comparing the compound mass spectrum to the National Institute of Standards and Technology database.

Figure 6.

GC-MS analysis of the products from reaction assays catalyzed by recombinant LnTPS3 and containing GGPP or eeFPP as the substrate. A, Peak 1, transgeranyllinalool; peak 2, transnerolidol. B, Mass spectra comparison of peaks from enzymatic reaction products with authentic standards.

Figure 7.

qRT-PCR determination of transcript levels of LnTPS1, LnTPS2, and LnTPS3 in young and mature leaves, roots, flowers of male and female L. nobilis plants, and in green fruits of female plants. Quantification of LnTPS1, LnTPS2, and LnTPS3 transcript levels by real-time reverse transcription-PCR analysis normalized to equal levels of actin transcripts. All analyses were performed using three biological replicates.

Figure 8.

Models of the active sites of L. nobilis TPS enzymes. A, The active site of LnTPS1 with bound GPP (magenta carbon atoms). B, The active site of LnTPS1 with bound pyrophosphate and the intermediate terpineol (magenta carbon atoms). C, The active site of LnTPS2 with bound FPP (magenta carbon atoms). D, The active site of LnTPS3 with bound GGPP (magenta carbon atoms).