Fig 1.
Lycosantalonol biosynthesis in Solanum lycopersicum.
(A) The terpene gene cluster on the tip of chromosome 8. (B) The biosynthetic pathway to lycosantanolol. AOX, alcohol oxidase; TPS, terpene synthase; CPT, cis-prenyl transferase; NDPS1, neryl diphosphate synthase 1; CYP, cytochrome P450; AAT, alcohol acyltransferase; DMAPP, dimethylallyl diphosphate; IPP, isopentenyl diphosphate; NNPP, nerylneryl diphosphate. Genes that are not functional because of deletions or insertions are shown with a “ψ” symbol.
Fig 2.
qRT-PCR analyses of CPT2, TPS21 and CYP71BN1 transcripts in different tissues of S. lycopersicum.
Total RNA was isolated from various tomato tissues. Leaflets and petiolules were prepared from four different developmental compound leaf stages. Error bars represent SE. Values are from three biological and three technical replicates.
Fig 3.
qRT-PCR analysis of CPT2, TPS21, CYP71BN1 transcripts in petiolules.
RNA was isolated from whole petiolule, petiolules from which trichomes have been removed, and from the trichomes. Error bars represent SE. Values are from four biological replicates with three technical replicates of each.
Fig 4.
GC-MS analysis of diterpenes from hexane extracts of whole petiolules.
(A) Non-transformed S. lycopersicum, (B–D) three individual plants of S. lycopersicum transformed with the 35S-CPT2 gene construct. Chromatograms of the selected ion of m/z 109 are shown here. (E) Four combined chromatographs of authentic standards. 1, nerylnerol; 2, lycosantalene: 3, epoxy-lycosantalene; 4, lycosantalonol. Mass Spectra for all peaks is shown in S2 Fig. Whole petiolules were ground and extracted with hexane as described in Materials and Methods.
Table 1.
Ratios of lycosantalene levels to β-phellandrene and β-caryophyllene levels in petiolule with and without trichomes, and in trichomes of transgenic S. lycopersicum plants overexpressing CPT2.
Table 2.
NNPS activity of CPT2.
Fig 5.
Phylogenetic tree of tomato CYP71BN1 and other functionally characterized terpene-modifying P450s.
Neighbor-joining phylogenetic tree analysis using amino acid sequences was performed by MEGA 5 [40]. Bootstrap values were performed with 1000 replications (values shown next to branches). LsGAO1, Lactuca sativa germacrene A oxidase (GAO) 1 (ADF32078.1); CiGAO2, Cichorium intybus (ADF43080.1); HaGAO4, Helianthus annuus (ADF43082.1); ScGAO3, Saussurea costus (ADF43081.1); AaAMO1, Artemisia annua amorpha-4, 11-diene monooxygenase (Q1PS23.1); BsGAO5, Barnadesia spinosa (ADF43083.1); HmHPO, Hyoscyamus muticus premnaspirodiene oxygenase (HPO) (A6YIH8.1); Nt-CYP71D20, Nicotiana tabacum 5-epiaristolochene dihydroxylase (Q94FM7.2); Ms-CYP71D18, Mentha spicata (-)-(4S)-limonene-6-hydroxylase (Q9XHE8.1); Mp-CYP71D13, Mentha x piperita (-)-(4S)-limonene-3-hydroxylase (Q9XHE7.1); Mp-CYP71D15, Mentha x piperita (-)-(4S)-limonene-3-hydroxylase (Q9XHE6.1); Zz-CYP71BA1, Zingiber zerumbet α-humulene oxidase (E3W9C4.1); AtKO, Arabidopsis thaliana ent-kaurene oxidase (KO) (NM_122491); OsKO2, Oryza sativa (BAF19823); OsKO4, Oryza sativa (BAF19823).