Genomic Identification and Biochemical Characterization of Methyl Jasmonate (MJ)-Inducible Terpene Synthase Genes in Lettuce (Lactuca sativa L. cv. Salinas)

Abstract

Terpenes are diverse plant metabolites with essential ecological and physiological functions, yet their biosynthetic regulation in lettuce (Lactuca sativa L.) remains poorly understood. By integrating volatile profiling, genome-wide identification, and biochemical characterization of terpene synthase (TPS) genes, we elucidated how methyl jasmonate (MJ) induces terpene formation in lettuce seedlings. Headspace analysis of 10-day-old seedlings revealed that while mock-treated tissues emitted no detectable volatiles, MJ elicitation triggered the de novo production of a terpene blend dominated by (E)-β-ocimene (9.3-14.6%), (E)-β-caryophyllene (37.2-46.9%), and caryophyllene oxide (26.2-41.4%). A genome-wide search identified 54 putative LsTPS genes, often clustered with prenyl transferases or cytochrome P450 genes. Gene expression assays revealed 17 MJ-responsive LsTPS genes; among them, LsTPS21, LsTPS23, LsTPS28, LsTPS51, and LsTPS52 showed strong (>200-fold) induction, with LsTPS52 exceeding a 20,000-fold increase. Functional characterization of six recombinant enzymes demonstrated diverse substrate specificities: LsTPS8 as an α-copaene synthase, LsTPS16 as a linalool synthase, LsTPS24 as an (E)-nerolidol synthase, LsTPS21 and LsTPS23 as (E)-β-ocimene synthases, and LsTPS10 as an (E)-β-caryophyllene synthase. Phylogenetic analyses confirmed conserved domains characteristic of the TPS-a and TPS-b subfamilies. This study presents the first comprehensive framework for MJ-induced terpene biosynthesis in lettuce, offering new insights into Asteraceae terpenoid metabolism.

Keywords: Lactuca sativa; headspace volatile; lettuce; methyl jasmonate; monoterpenes; sesquiterpenes; terpene synthase.

Figure 1

Headspace volatile terpenoids emitted from methyl jasmonate (MJ)-treated lettuce (Lactuca sativa cv. Salinas) seedlings. Only aerial part (approx. 1 g FW) was harvested from 10-day-old lettuce seedlings and enclosed in a glass vial (20 mL) at 12 h after MJ treatment with (A) 1 mM, (B) 0.5 mM, and (C) Mock (2% ethanol in water). The volatiles were analyzed by solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). Each peak was identified using authentic standards, Wiley Registry (12th edition) and the 2020 NIST library. 1, sabinene; 2, β-myrcene; 3, D-limonene; 4, β-ocimene; 5, guaia-4,11-diene; 6, copaene; 7, (E)-β-caryophyllene; 8, 10,10-dimethyl-2,6-dimethylenebicyclo[7.2.0] undecane; 9, (E)-α-farnesene; 10, caryophyllene oxide; 11, humulene epoxide; 12, 11,11-dimethyl-4,8-dimethylenebicyclo[7.2.0]undecan-3-ol; 13, hydroxycaryophyllene; and *, unknown terpenoid. Three biologically independent samples were analyzed and a representative chromatogram with similar results is shown. I.S.—Internal standard (1-bromodecane).

Figure 2

Relative abundance of volatile terpenoids released from MJ-treated lettuce seedlings. (A) Percentage distribution of the compounds detected from the shoot (approx. 1 g FW) of lettuce seedlings in response to methyl jasmonate (MJ) treatment. Each peak area of the terpenoids shown in Figure 1 was combined and employed to estimate the relative amount of a compound in percentile. A number preceding a compound name corresponds to a peak number shown in Figure 1. Sesquiterpene 1: 10,10-dimethyl-2,6-dimethylenebicyclo[7.2.0]undecane; Sesquiterpene 2: 11,11,11-dimethyl-4,8-dimethylenebicyclo[7.2.0]undecan-3-ol. (B) MJ-dose dependent production of total terpenoids collected from the shoots. The abundance of all terpene compounds at each treatment was combined. Data represent the mean ± SE from three biologically independent replicates. Statistical significance was determined by one-way ANOVA with Tukey’s HSD post hoc test. Different letters indicate significant differences at p < 0.05. (C) MJ-dose dependent production of mono- and sesquiterpenes. The amounts of terpenoids at 0.5 mM treatment was calculated as a percentage relative to the total abundance detected at 1 mM (shown in Figure 2B), which was normalized to 100%. ND, not detected.

Figure 3

Chromosomal distribution of lettuce terpene synthase genes (TPSs). The organization of TPSs and other related genes on the lettuce chromosomes. CPT, cis-prenyl transferase; CPTL, cis-prenyl transferase like; CPS, ent-copalyl diphosphate synthase; FPS, Farnesyl diphosphate synthase; GAO, germacrene A oxidase; COS, costunolide synthase; CYP, cytochrome P450. Functional TPSs include both functionally characterized genes in this work and previously reported in the studies.

Figure 4

Quantitative reverse transcription (qRT)–polymerase chain reaction (PCR) assay of MJ-inducible LsTPS genes. A LsTub was used as an endogenous reference gene. Shoot tissue of 10-day-old lettuce seedlings were foliar sprayed until saturation with methyl jasmonate at concentrations of Mock (2% ethanol in water), 0.5 mM, and 1 mM for 12 h. The expression levels were normalized to the mock treatment, which was set to 1 (fold). Data represent the mean ± SE (n = 3) in triplicate. Statistical significance was determined by the non-parametric Kruskal–Wallis test followed by Dunn’s post hoc test (n.s.: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 5

GC–MS analysis of monoterpenes catalyzed by MJ-inducible TPSs. Reaction products were identified by comparing their mass spectra and retention times with those of authentic standards, the Wiley Registry (12th edition), and the 2020 NIST Mass Spectral Library. Each TPS enzyme was reacted with (A) GPP as a substrate: 1, β-myrcene; 2, D-limonene; 3. (Z)-β-ocimene; 4, (E)-β-ocimene; 5, α-terpinolene; 6, linalool; or (B) NPP as a substrate: 1, D-limonene; 2, α-terpinolene; 3, β-myrcene; 4, (E)-β-ocimene; 5, β-thujene; 6, pseudolimonene; 7, 3-carene; 8, α-terpinene; and 9, γ-terpinene. Extracted ion chromatograms (m/z = 93) are shown. Identities, mass spectra and percentage distribution of each peak can be found in Figures S7 and S8 and Table S2.

Figure 6

GC–MS analysis of sesquiterpenes catalyzed by MJ-inducible TPSs using E,E-FPP as a substrate. The reaction products were identified by comparing their mass spectra and retention times with those of authentic standards, the Wiley Registry (12th edition), and the 2020 NIST Mass Spectral Library: 1, silphiperfol-5-ene; 2, δ-eIemene; 3, 7-epi-silphiperfol-5-ene; 4, (+)-cyclosativene; 5, α-copaene; 6, β-elemene; 7, (Z)-β-caryophyllene; 8, (E)-β-caryophyllene; 9, γ-elemene; 10, (Z)-β-copaene; 11, (E)-β-farnesene; 12, α-humulene; 13, γ-muurolene; 14, (E)-α-bergamotene; 15, (Z,E)-α-farnesene; 16, aromandendrene; 17, (-)-germacrene D; 18, (E)-α-farnesene; 19, (-)-α-muurolene; 20, (-)-β-cadinene 21, (E)-nerolidol; 22, cubebol; and 23, (+)-viridiflorol. Extracted ion chromatograms (m/z = 93) are shown. Identities, mass spectra and percentage distribution of each peak can be found in Figure S9 and Table S2.

Figure 7

GC–MS analysis of sesquiterpenes catalyzed by MJ-inducible TPSs using Z,Z-FPP as a substrate. The reaction products were identified by comparing their mass spectra and retention times with those of authentic standards, the Wiley Registry (12th edition), and the 2020 NIST Mass Spectral Library: 1, (E)-β-caryophyllene; 2, (Z)-β-farnesene; 3, (-)-α-cedrene; 4, (Z)-α-bergamotene; 5, γ-curcumene; 6, (-)-zingiberene; 7, (-)-β-bisabolene; 8, β-curcumene; 9, (Z)-γ-bisabolene; 10, β-sesquiphellandrene; 11, (E)-γ-bisabolene; and 12, (E)-α-bisabolene. Extracted ion chromatograms (m/z = 93) are shown. Identities, mass spectra and percentage distribution of each peak can be found in Figure S10 and Table S2.

Figure 8

Phylogenetic analysis between lettuce TPSs and known TPSs in plants. A maximum-likelihood phylogenetic tree was generated using MEGA11 with a bootstrap value of 1000. Each TPS subgroup is shaded in different colors. Seventeen MJ-inducible TPSs in lettuce are considered to construct the tree. A red triangle represents the six TPSs biochemically characterized in this study. Abbreviations: Ls, Lactuca sativa; Pt, Populus trichocarpa; Pa, Picea abies; Ad, Actinidia deliciosa; Ac, Aquilaria crassna; Sl, Solanum lycopersicum; Am, Aquilaria malaccensis; Cs, Cannabis sativa; Ag, Abies grandis; Tp, Thuja plicata; and At, Arabidopsis thaliana.

Figure 9

Terpene structures catalyzed by the six terpene synthases (TPSs) functionally characterized in this study. (A) GPP as a substrate: red arrows and TPS numbers, NPP as a substrate: blue arrows and TPS numbers; (B) E,E-FPP as a substrate: magenta arrows and TPS numbers; Z,Z-FPP as a substrate: black arrows and TPS numbers. β-elemene, γ-elemene, and δ-elemene are Cope-rearrangements of germacrene A, germacrene B, and germacrene C, respectively; circle in red, MJ-induced terpenes in lettuce shoot. The corresponding TPS were labeled beneath each compound in different colors.