For GC-MS of enzyme assay products:
Features were manually identified using comparison of retention times and spectral matches to authentic standards using Agilent MassHunter Qualititative Analysis 10.0.
For LC-MS/MS of leaf discs:
Analytes were manually identified according to (expected) accurate mass and retention time, compared to standards for cis-trans-nepetalactol, dolichodial and teucrein (all in positive mode). Dolichodial was spiked into leaf disc extracts in order to correct for retention time shifts due to the effect of the matrix. 7-deoxyloganic acid and 7-deoxyloganetic acid glucose ester were identified in negative ion mode as described previously[1]. Data analysis was performed using Xcalibur (Thermofisher Scientific, Germany).
For GC-MS of tissue samples:
Features were manually identified using comparison of retention times and spectral matches to authentic standards using Xcalibur.
For LC-MS/MS of tissue samples:
Features were picked and filtered using bespoke scripts in R 4.2.3, run in a linux environment. Briefly, xcms [2, 3] was used to pick features using the following parameters: method = 'centWaveWithPredictedIsotopeROIs', ppm = 10, snthresh = 10, peakwidth = c(3, 20), prefilter = c(3, 1000), integrate = 2, mzdiff = -0.1. Duplicate features and features with non-gaussian elution profiles were removed with custom scripts, and corresponding features across samples grouped and missing values imputed using modified versions of the xcms group() and fillPeaks() functions. Isotopes, adducts, and monoistopes were identified with CAMERA [4]. Putative MS1 hits were identified by matching (<= 5 ppm error) to theoretical [M-H]- or [M+HCOOH-H]- ions calculated from an in-house database. High-quality fragmentation spectra (i.e. those obtained nearest the MS1 feature apex) were extracted using custom R scripts and searched for neutral losses of either C6H12O6 (180.06339) or C6H12O6 - H2O (162.05282), within 20 ppm tolerances. Features that were over the blank threshold (areas > mean blank areas + 3 x blank SD), and were matches to the reference database, or exhibited glycosyl neutral losses, or were identified monoisotopic features, were retained for further manual inspection.Putative MS1 hits were identified by matching (<= 5 ppm error) to theoretical [M-H]- or [M+HCOOH-H]- ions calculated from an in-house database. High-quality fragmentation spectra (i.e. those obtained nearest the MS1 feature apex) were extracted using custom R scripts and searched for neutral losses of either C6H12O6 (180.06339) or C6H12O6 - H2O (162.05282), within 20 ppm tolerances. Features that were over the blank threshold (areas > mean blank areas + 3 x blank SD), and were matches to the reference database, or exhibited glycosyl neutral losses, or were identified monoisotopic features, were retained for further manual inspection.
Refs:
[1] Dudley, Q.M. et al. (2022). Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana. Commun. Biol. 5:949.
[2] Smith et al. (2006). XCMS: Processing Mass Spectrometry Data for Metabolite Profiling Using Nonlinear Peak Alignment, Matching, and Identification. Anal. Chem. 78:779.
[3] Tautenhahn et al. (2008). Highly sensitive feature detection for high resolution LC/MS. BMC Bioinformatics 9:504.
[4] Kuhl et al. (2012). Anal.Chem. 84:283.
"],"repository":["MetaboLights"],"study_status":["Public"],"ptm_modification":[""],"instrument_platform":["Liquid Chromatography MS - negative - reverse phase","Gas Chromatography MS - positive","Liquid Chromatography MS - positive - reverse phase"],"chromatography_protocol":["For GC-MS of enzyme assay products:
In total 2 µL of the top organic layer was analyzed using a 2:1 split through GC-MS on an Agilent 7890A gas chromatograph (Agilent Technologies) interfaced to an Agilent 5975C MSD (Agilent Technologies). Separation was performed on a Zebron ZB5-HT-INFERNO column (5% phenyl methylsiloxane; length: 30 m; diameter: 250 µm) interfaced with auxiliary transfer line (1.5 m, 150 µm) and helium as mobile phase at a constant flow of 1.2 mL/min. The inlet temperature was 220 °C with 2 different column temperature runs: Method 1 (GES and ISY/PRISE assays): 5 min at 80 °C, increased to 110 °C at 2.5 °C/min and then to 280 °C at 10 °C/min, where it was held for 4 min. Method 2 (8HGO assay): 4 min at 60 °C, increased 100 °C at 20 °C/min, then to 160 °C at 2 °C/min and finally to 280 °C at 100 °C where it was held for 4 min.
For LC-MS/MS of leaf discs:
In total 3 µL of the samples were analysed on a Thermo Vanquish Flex UPLC interfaced to a Thermo Orbitrap Fusion mass spectrometer. A Waters ACQUITY UPLC BEH C18 column (130 Å, 1.7 µm, 2.1 x 100 mm) was used at 40 °C, with the autosampler kept at 10 °C. Mobile phase A was water with 0.1% (v/v) formic acid, mobile phase B acetonitrile with 0.1% (v/v) formic acid. The gradient started at 1% B and increased to 10% B over 1 min, then 80% B at 6 min, to reach 100% B at 6.1 min where it remained until 7.6 min, to return to 1% B at 7.7 min and to re-equilibrate until 9 min; flow rate was 0.6 mL/min. Dolichodial was spiked into leaf disc extracts in order to correct for retention time shifts due to the effect of the matrix.
For GC-MS of tissue samples:
In total 2 µL of the samples were analyzed using a 5:1 split through GC-MS on an Agilent 6890 gas chromatograph (Agilent Technologies) interfaced to a Leco Pegasus IV TOF MS (LECO Analytical Instruments). Separation was performed on a Zebron ZB5-HT-INFERNO column (5% phenyl methylsiloxane; length: 35 m; diameter: 250 mm) with guard column and helium as mobile phase at a constant flow of 1.2 mL/min. The inlet temperature was 220 °C. After 5 min at 80 °C, the column temperature was increased to 110 °C at a rate of 2.5 °C/min, then to 280 °C at 120 °C/min, and kept at 280 °C for another 4 min.
For LC-MS/MS of tissue samples:
In total 4 µL of the samples were analysed on a Thermo Vanquish Flex UPLC interfaced to a Thermo Orbitrap Fusion mass spectrometer. A Waters ACQUITY UPLC BEH C18 column (130 Å, 1.7 µm, 2.1 x 100 mm) was used at 40 °C, with the autosampler kept at 10 °C. Mobile phase A was water with 0.1% (v/v) formic acid, mobile phase B acetonitrile with 0.1% (v/v) formic acid. The gradient started at 20% B and increased to 26% B at 10 min, isocratic at 26% B to 15 min, then to 95% B at 16 min, isocratic at 95% B to 17 min, then return to initial conditions at 18 min and re-equilibrate to 21 min; flow rate was 0.5 mL/min.
"],"publication":["The genomic and enzymatic basis for iridoid biosynthesis in cat thyme (Teucrium marum). 10.1111/tpj.16698. PMID:38489316"],"submitter_name":["Tony Larson"],"submitter_affiliation":["University of York"],"organism_part":["blank","leaf disc","root","Closed Flowers","Open Flowers","leaf","pure substance","stem","Whole Organism"],"technology_type":["mass spectrometry assay"],"disease":[""],"extraction_protocol":["For GC-MS of enzyme assay products:
Briefly, 2 µL of the top organic layer was analyzed using a 2:1 split through GC-MS on an Agilent 7890A gas chromatograph (Agilent Technologies) interfaced to an Agilent 5975C MSD (Agilent Technologies). Separation was performed on a Zebron ZB5-HT-INFERNO column (5% phenyl methylsiloxane; length: 30 m; diameter: 250 µm) interfaced with auxiliary transfer line (1.5 m, 150 µm) and helium as mobile phase at a constant flow of 1.2 mL/min. The inlet temperature was 220 °C with two different column temperature runs: Method 1 (GES and ISY/PRISE assays): 5 min at 80 °C, increased to 110 °C at 2.5 °C/min and then to 280 °C at 10 °C/min, where it was held for 4 min. Method 2 (8HGO assay): 4 min at 60 °C, increased 100 °C at 20 °C/min, then to 160 °C at 2 °C/min and finally to 280 °C at 100 °C where it was held for 4 min.
For LC-MS/MS of leaf discs:
Excess buffer was removed from the leaf discs using blue roll and 3 discs combined into a 1.5 mL Eppendorf tube containing 2 tungsten beads. Tubes were flash frozen in liquid nitrogen and the tissue homogenized through 90 s of grinding using a beadbeater. Then 200 µL of 70% v/v MeOH and 0.1% v/v formic acid containing 10 µM of daphylloside as internal standard was added to the tube, followed by 15 min of vortex mixing and 15 min of sonication in a water bath. Cell debris was removed through 20 min of centrifugation at maximum speed. The aqueous layer was transferred to a new tube, centrifuged again and then transferred to 96-well 1 mL round bottom collection plate (Thermofischer Scientific, Germany) that was heat sealed with foil (Thermofischer Scientific, Germany). Extracted samples were used for LC-MS/MS analysis.
For GC-MS of tissue samples:
A 150 µL volume of analytical grade methanol was added to 25 mg of frozen ground tissue, followed by vortex mixing for 10 min. Then 300 µL of hexane containing 0.2 mM of carvone as internal standard was added, followed by a further 10 min of vortexing. The samples were centrifuged at 21,000 x g for 5 min and the upper phase filtered through a 0.45 mm PTFE syringe filter. Next 80 µL of the sample was added to a 1.1 mL tapered glass vial (Thermofischer Scientific, Germany) and crimp sealed, ready for GC-MS analysis.
For LC-MS/MS of tissue samples:
Frozen tissues were homogenized using a TissueLyser II (Qiagen, UK), and 50 mg of the homogenate was suspended in 1 ml of methanol. Samples were extracted by vortex for 2 min followed by ultrasonication in a bath at room temperature for 5 min. For each tissue, three extracts were collected from the same biological tissue. The extracts were centrifuged and diluted with aqueous formic acid (0.1%, v/v and 1:5, v/v). Last, the supernatant was filtered through a hydrophilic polytetrafluoroethylene (PTFE) 0.22-µm membrane (Merck, Feltham, UK) for nontargeted LC-MS/MS analysis
"],"organism":["Nicotiana benthamiana","blank","reference compound","Escherichia coli","Teucrium marum"],"data_transformation_protocol":["For GC-MS of enzyme assay products:
Data was used as provided.
For LC-MS/MS of leaf discs:
Data was used as provided.
For GC-MS of tissue samples:
Feature areas were normalized to internal standard and then tissue weight. Data was exported from Leco .cdf to Thermo .raw format using the Thermo Xcalibur converter tool.
For LC-MS/MS of tissue samples:
Obtained Xcalibur .raw files were converted to .mzML and .mgf formats using ProteoWizard msconvert.
"],"study_factor":["Treatment","Biological replicate"],"submitter_email":["tony.larson@york.ac.uk"],"metabolights_link":["https://www.ebi.ac.uk/metabolights/MTBLS9357"],"sample_collection_protocol":["Enzyme assays:
Enzyme production was performed E. coli or N. benthamiana. For in vitro E. coli expression, enzymes were purified for assay as descibed previously[1]. For N. benthamiana transient expression, plants were infiltrated with agrobacteria containing plasmids encoding genes of interest. LBA4404 Agrobacterium tumefaciens strains expressing IO and BAHDs (benzylalcohol O-acetyl transferase, anthocyanin O-hydroxycinnamoyl transferase, N-hydroxycinnamoyl anthranilate benzoyl transferase and deacetylvindoline 4-O-acetyltransferase superfamily enzymes) in the pHREAC vector, respectively, were grown overnight at 28 °C in YEB broth (5 g/L beef extract, 1 g/L yeast extract, 5 g/L peptone, 5 g/L sucrose and 0.5 g/L MgCl2) with 50 µg/mL kanamycin and streptomycin, respectively, harvested by centrifugation (3200 x g, 10 min) and then washed with 10 mM MES buffer (Merck Life Sciences, UK). Green Fluorescent Protein (GFP) expressed in the pEAQ-HT vector served as a visual marker and empty vector control. Cell suspensions were diluted in 10 mM MES buffer (containing 100 µM acetosyringone and 10 mM MgCl2) and incubated at room temperature with gentle shaking for at least 1 h prior to transient infiltration into the abaxial side of 4-5 week old N. benthamiana leaves using a syringe. GFP and IO were infiltrated at a cell suspension OD600 of 0.25 whilst an OD600 of 0.75 was used for BAHD candidates. Wild-type and MES buffer infiltrated leaves served as negative controls. 4 days post infiltration (DPI) 19 mm diameter discs were excised from the leaves using a cork borer. Leaf discs were suspended in a 24 well Corning Falcon Multiwell Cell Culture Plate (Merck Life Sciences, UK) with 500 μL HEPES buffer (50 mM, pH 7.5) containing 50 µM cis-trans-nepetalactol (prepared as 100 mM stock in acetonitrile) and incubated for 24 h in a growth room (20 °C day on a 16/8 h day/night cycle).
Tissue samples:
Briefly, 3 biological replicates of leaves, stems, roots, open flower buds and closed flower buds were harvested from plants grown in a growth chamber (24 °C day/17 °C night on a 14/10 h day/night cycle), and flash-frozen in liquid nitrogen. Due to their small size, leaf samples included both immature and mature leaves. Altogether 3 leaf, stem and root samples were pooled for each replication; however, due to insufficient flowers, tissues were harvested on different days from 3 biological replicates.
Ref:
[1] Lichman BR, Godden GT, Hamilton JP, Palmer L, Kamileen MO, Zhao D, Vaillancourt B, Wood JC, Sun M, Kinser TJ, Henry LK. The evolutionary origins of the cat attractant nepetalactone in catnip. Science Advances. 2020 May 13;6(20):eaba0721. doi:10.1126/sciadv.aba0721.
"],"omics_type":["Metabolomics"],"study_design":["Thermo Scientific Orbitrap Fusion","gas chromatography electron ionisation mass spectrometry","Nicotiana benthamiana","blank","untargeted analysis","Escherichia coli","Closed Flowers","Comparative genomics","pure substance","Iridoids","reference compound","Agilent 7890A GC","root","Open Flowers","Teucrium marum","Agilent 5975C inert XL MSD","leaf disc","leaf","Agilent 6890N GC","untargeted metabolite profiling","LECO Pegasus IV","Whole Organism","experimental sample","Thermo Scientific Vanquish UHPLC System","liquid chromatography-tandem mass spectrometry","experimental blank","Enzymology","stem"],"curator_keywords":["Thermo Scientific Orbitrap Fusion","gas chromatography electron ionisation mass spectrometry","Nicotiana benthamiana","blank","untargeted analysis","Escherichia coli","Closed Flowers","Comparative genomics","pure substance","Iridoids","reference compound","Agilent 7890A GC","root","Open Flowers","Teucrium marum","Agilent 5975C inert XL MSD","leaf disc","leaf","Agilent 6890N GC","untargeted metabolite profiling","LECO Pegasus IV","Whole Organism","experimental sample","Thermo Scientific Vanquish UHPLC System","liquid chromatography-tandem mass spectrometry","experimental blank","Enzymology","stem"],"mass_spectrometry_protocol":["For GC-MS of enzyme assay products:
An Agilent 5975C MSD (Agilent Technologies) was used as a mass detector. A solvent delay of 5 min was allowed before collecting MS spectra at a fragmentation energy of 70 eV, at a threshold of 150 over the mass range 40-150 m/z.
For LC-MS/MS of leaf discs:
An Orbitrap Fusion mass spectrometer was operated in alternating runs in either positive (capillary voltage 3.5 kV, vaporizer temperature 350 °C, ion transfer tube temperature 350 °C) and negative (capillary voltage -2.5 kV, vaporizer temperature 350 °C, ion transfer tube temperature 350 °C) HESI, orbitrap resolution 60000, mass range 100-1000 m/z. MS2 spectra were acquired in HCD and CID mode using the ion trap as detector; cycle time was 0.4 s. MS2 spectra were acquired in stepped HCD (35, 40, 45% NCE) and CID (40% NCE) modes at the same mass resolution; cycle time was 0.4 s. EasyIC was used to achieve mass accuracy <= ~ 1 ppm.
For GC-MS of tissue samples:
An Leco Pegasus IV TOF MS (LECO Analytical Instruments) was used as a mass detector. A solvent delay of 5 min was allowed before collecting MS spectra at a fragmentation energy of 70 eV, at 20 scans / s over the mass range 40-500 m/z. CHECK
For LC-MS/MS of tissue samples:
An Orbitrap Fusion mass spectrometer was operated in negative HESI mode (capillary voltage -3.5 kV, vaporizer temperature 350 °C, ion transfer tube temperature 325 °C, sheath gas 50 units, aux gas 10 units, sweep gas 1 unit), orbitrap resolution 60000, mass range 100-1200 m/z. MS2 spectra were acquired in stepped HCD (35, 40, 45% NCE) and CID (40% NCE) modes at the same mass resolution; cycle time was 0.4 s. EasyIC was used to achieve mass accuracy <= ~ 1 ppm.
"],"metabolite_name":["8-oxogeraniol","8-hydroxygeranial","8-oxogeranial","geraniol","dolichodial","cis-trans-nepetalactol","carvone","teucrein","8-hydroxygeraniol"],"pubmed_abstract":["Iridoids are non-canonical monoterpenoids produced by both insects and plants. An example is the cat-attracting and insect-repelling volatile iridoid nepetalactone, produced by Nepeta sp. (catmint) and aphids. Recently, both nepetalactone biosynthetic pathways were elucidated, showing a remarkable convergent evolution. The iridoid, dolichodial, produced by Teucrium marum (cat thyme) and multiple insect species, has highly similar properties to nepetalactone but its biosynthetic origin remains unknown. We set out to determine the genomic, enzymatic, and evolutionary basis of iridoid biosynthesis in T. marum. First, we generated a de novo chromosome-scale genome assembly for T. marum using Oxford Nanopore Technologies long reads and proximity-by-ligation Hi-C reads. The 610.3 Mb assembly spans 15 pseudomolecules with a 32.9 Mb N50 scaffold size. This enabled identification of iridoid biosynthetic genes, whose roles were verified via activity assays. Phylogenomic analysis revealed that the evolutionary history of T. marum iridoid synthase, the iridoid scaffold-forming enzyme, is not orthologous to typical iridoid synthases but is derived from its conserved paralog. We discovered an enzymatic route from nepetalactol to diverse iridoids through the coupled activity of an iridoid oxidase cytochrome P450 and acetyltransferases, via an inferred acylated intermediate. This work provides a genomic resource for specialized metabolite research in mints and demonstration of the role of acetylation in T. marum iridoid diversity. This work will enable future biocatalytic or biosynthetic production of potent insect repellents, as well as comparative studies into iridoid biosynthesis in insects."],"pubmed_title":["The genomic and enzymatic basis for iridoid biosynthesis in cat thyme (Teucrium marum)."],"pubmed_authors":["Smit Samuel J SJ, Ayten Sefa S, Radzikowska Barbara A BA, Hamilton John P JP, Langer Swen S, Unsworth William P WP, Larson Tony R TR, Buell C Robin CR, Lichman Benjamin R BR"],"additional_accession":[]},"is_claimable":false,"name":"The genomic and enzymatic basis for iridoid biosynthesis in cat thyme (Teucrium marum)","description":"Iridoids are non-canonical monoterpenoids produced by both insects and plants. An example is the cat attracting and insect repelling volatile iridoid nepetalactone, produced by Nepeta sp. (catmint) and aphids. Recently, both nepetalactone biosynthetic pathways were elucidated, showing a remarkable convergent evolution. The iridoid, dolichodial, produced by Teucrium marum (cat thyme) and multiple insect species, has highly similar properties to nepetalactone but its biosynthetic origin remains unknown. We set out to determine the genomic, enzymatic and evolutionary basis of iridoid biosynthesis in T. marum. First, we generated a high quality de novo chromosome-scale genome assembly for T. marum using Oxford Nanopore Technologies long reads and proximity-by-ligation Hi-C reads. The 610.3 Mb assembly spans 15 pseudomolecules with a 32.9 Mb N50 scaffold size. This enabled identification of iridoid biosynthetic genes, whose roles were verified via activity assays. Phylogenomic analysis revealed that the evolutionary history of T. marum iridoid synthase, the iridoid scaffold forming enzyme, is not orthologous to typical iridoid synthases but is derived from its conserved paralog. We discovered an enzymatic route from nepetalactol to diverse iridoids through the coupled activity of an iridoid oxidase cytochrome P450 and acetyltransferases, via a cryptic acylated intermediate. This work provides a high quality genomic resource for specialized metabolite research in mints and demonstration of the role of acetylation in T. marum iridoid diversity. This work will enable future biocatalytic or biosynthetic production of potent insect repellents, as well as comparative studies into the iridoid biosynthesis in insects.","dates":{"publication":"2026-04-20","submission":"2024-02-05"},"accession":"MTBLS9357","cross_references":{"MetaboLights":["MTBLC4685","MTBLC71494","MTBLC38265","MTBLC64235","MTBLC64236","MTBLC17447","MTBLC64239","MTBLC64238"],"pubmed":["38489316"],"PubChem":["CID:590662"],"ChEBI":["CHEBI:4685","CHEBI:71494","CHEBI:38265","CHEBI:64235","CHEBI:64236","CHEBI:17447","CHEBI:64239","CHEBI:64238"]}}