MetaboLights

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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14117/m_MTBLS14117_LC-MS_positive_reverse-phase_metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14117/s_MTBLS14117.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14117/a_MTBLS14117_LC-MS_positive_reverse-phase_metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14117/i_Investigation.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14117To perform a functional analysis of the metabolomics data, the original format files (.d) were transformed into the format of mzML in ProteoWizard (Chambers et al., 2012).  We conducted de novo feature annotation and compound classification based on MS/MS fragmentation patterns using the SIRIUS software (version 5.7.2; Dührkop et al., 2019). The resulting annotations were ranked according to ZODIAC scores (Ludwig et al., 2020). Molecular structures were predicted with CSI:FingerID (Dührkop et al., 2015; Hoffmann et al., 2021), and compound classes were assigned using the Natural Product Classifier (NPClassifier; Kim et al., 2021). De novo compound class prediction was performed with the CANOPUS module (Feunang et al., 2016; Dührkop et al., 2021). For SIRIUS analyses, a mass accuracy tolerance of 5 ppm was applied, the elemental composition was restricted to C, H, N, P, O, and S, and all other parameters were kept at default settings.MetaboLightsPublicLiquid Chromatography MS - positive - reverse-phaseLC-MS analysis was performed following the methods described in Weinhold et al., (2022).Chromatographic separation was performed by the injection of 3 µl of the final extracts into an UltiMate™ 3000 Standard Ultra-High-Performance Liquid Chromatography system (UHPLC, Thermo Scientific) using a C18 column (Acclaim® RSLC 120, 150 mm×2.1 mm, particle size 2.2 μm, ThermoFischer Scientific Waltham, USA) employing a stepwise water–acetonitrile gradient. An elution program employing two mobile phases consisted of mobile phase A (water:formic acid, 99.9:0.1%, v/v) and mobile phase B (acetonitrile:formic acid, 99.9/0.1%, v/v). The flow rate of solvents was set at 0.4 ml/min, and the column temperature was kept at 40°C.Inhibition of Jasmonic Acid-Isoleucine Conjugating Enzyme JAR1 Shifts the Local and Systemic Leaf Metabolic Profiles in Arabidopsis. 10.20944/preprints202511.1275.v1.Ming ZengiDivblankacetonitrileStandardleafQuality Controlmass spectrometry assayAfter four days of freeze-drying at -80℃ (FreeZone Plus 12 L Cascade Console Freeze Dry System, Labconco, Kansas City, MO, USA), the leaf samples were homogenized in 2 ml tubes with ceramic beads in a ball mill (Retsch MM400, Haan, Germany) for 5 minutes at 30 Hz. After weighing, 10 mg of leaf powdered material was extracted with 500 µl of buffer. The 1 L extraction buffer comprised 75% (v/v) methanol (LC-MS grade, VWR, Germany), 25% (v/v) acetate buffer and 50 µl of 100 mM IAA-Valin stock solution as an internal standard. The acetate buffer was prepared by mixing 2.3 ml acetic acid and 3.41 g ammonium acetate in 1 L Milli-Q water, and then pH was adjusted to 4.8. Afterwards, the samples with extraction solution were homogenized with ceramic beads in a ball mill (Retsch MM400, Haan, Germany) for 5 minutes at 30 Hz. The mixture was then centrifuged at 15,000 g for 15 minutes at a low temperature with -4℃. The supernatant was transferred into a 1.5 ml tube. The leaf material went through a second extraction with the same procedure as above. The two supernatants were mixed and then 200 µl of supernatant diluted with 800 µl extraction buffer with a ratio of 1:5. The diluted extracts were stored at -20°C overnight and centrifugated at 8,000 g for 5 minutes. The clarified supernatant was relocated to LC-MS vials.Arabidopsis thalianablankacetonitrileStandardQuality ControlThe LC-qToF-MS raw data were processed in Bruker Compass MetaboScape software (2022b; V. 9.0.1; Build 11878; Bruker Daltonics, Hamburg, Germany). Mass recalibration, peak picking, peak alignment, region complete feature extraction, and grouping of isotopes, adduct, and charge states were operated with T-ReX algorithm provided by MetaboScape program. The settings were used for the peak detection: Intensity threshold: 1000 counts; Minimum peak length: 7 spectra; Feature signal: intensity; Minimum peak length for recursive feature extraction: 7 spectra; Mass range: 90-1,600 m/z; Retention time range: 0-18 min; MS/MS import method: average, grouped by collision energy. The parameters for the ion deconvolution were set as below: EIC correlation: 0.8; Primary ion: [M+H]+; Common ions: [M+H-H2O]+; Seed ions: [M+Na]+, [M+K]+; T-ReX-Positive Recalibration: Auto-Detect. Quality checks were implemented to inspect the stability of retention time and signal intensity, examination for residue effect, and verification of group identity. The settings for feature filtering were employed as below: Minimum number of samples: present in 3 of 139, minimum for recursive feature extraction: present in 3 of 139, group filter: present in at least all samples (100%) of at least one group (1 group = all replicates in one treatment). Finally, a feature list including 6178 features was created. Features from blanks were excluded when the ratio of maximum signal of samples to blanks was ≤3. Data analysis was performed in R version 4.4.2 (R Core Team, 2024) within RStudio version 2025.09.2+418 (Posit Software, 2025) unless otherwise stated. For the principal component analysis (PCA), it was performed based on the intensity of the features at MetaboAnalyst 6.0 platform (Pang et al., 2024). The settings for the data normalization were as follows: Sample normalization: normalization by median/quantile normalization; Data transformation: square root/log2 transformation; Data scaling: pareto scaling. Chemical treatmentStudy typeLeaf numberming.zeng2@gmail.comhttps://www.ebi.ac.uk/metabolights/MTBLS14117Five to six-week-old Arabidopsis thaliana (L.) Heynh. ecotype Col-0 plants were used for all experiments. To ensure the same starting conditions, all plants used for one assay were sown on the same day and kept in the same growth chamber. After stratification for two days at 4℃, plants were grown in growth chambers in 10 cm round pots with soil substrates under short-day conditions (10/14 h light/dark period) with minor differences. For phytohormone and metabolome analyses, plants were grown at the Max Planck Institute for Chemical Ecology (MPI-CE) or iDiv. The growth chambers were adjusted to 50-60% humidity and constant 21℃ with a light intensity of 100 μmol m2 s−1. In the control group, leaf 8 was treated with the chemical DMSO or jarin-1 on the surface. There was no wounding treatment on control plants. In the local study, leaf 8 was treated with the chemical DMSO or jarin-1 on the surface. One hour and a half after chemical treatment, leaf 8 was wounded twice continuously by tweezers. Local and systemic leaves are 8 and 13, respectively. In the systemic study, leaf 8 was treated with the chemical DMSO or jarin-1 on the surface. One hour and a half after chemical treatment, leaf 13 was wounded twice continuously by tweezers. Local and systemic leaves are 13 and 8, respectively. In all experiments, leaf 8, 9, 11 and 13 were harvested separately one hour after wounding while control plants were waiting still for one hour.MetabolomicsblankArabidopsisuntargeted analysisStandardwoundingleafArabidopsis thalianasystemic signalslocal signalsThermo Scientific Dionex Ultimate 3000 UHPLC systemuntargeted metabolitesacetonitrileBruker maXis impact UHR-QTOFPlant MetabolomicsQuality Controlblankuntargeted analysisArabidopsisStandardleafwoundingArabidopsis thalianasystemic signalslocal signalsThermo Scientific Dionex Ultimate 3000 UHPLC systemuntargeted metabolitesacetonitrileBruker maXis impact UHR-QTOFPlant MetabolomicsQuality ControlMetabolite detection was conducted within a mass range of 90-1,600 m/z at a spectra rate of 5 Hz (line spectra) using an ESI-UHR-Q-ToF-MS instrument (maXis impact, Bruker Daltonics, Hamburg, Germany). The MS measurements were featured an electrospray ionization source run in positive ion mode with data-dependent collision-induced dissociation (Auto-MSMS mode). Mass calibration for each chromatogram was performed by an automated infusion at the end of the gradient using an HPC mode with a flow rate of 0.1 ml/h. The calibration was achieved by infusing a 10 mM sodium formate cluster of NaOH solution prepared in a 50:50 (v/v) mixture of isopropanol and water containing 0.2% formic acid.(-)-Perillyl alcoholLauric acid diethanolamideGlucohirsutin, desulfo fragmentKaempferol 3-O-rutinosideL-PhenylalaninePhthalide, 1(3H)-IsobenzofuranoneSucroseAsn-Pro-LysTributyl phosphate1,2-Dilinolenoyl-sn-glycero-3-phosphoethanolamineTriethylene glycol5-hydroxy-2-(4-hydroxyphenyl)-7-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxy-3-(3,4,5-trihydroxyoxan-2-yl)oxychromen-4-onePhlorizinGermacronePhloretin3,4-Ethylenedioxy-N-methylamphetamineKinetinLPC 18:3N6-Succinyladenosine; AIF; CE10; MS2Dec4-Hydroxyglucobrassicin, desulfo fragmentIndole-3-acetonitrileOctamethylcyclotetrasiloxaneKaempferol-3-O-rhamnoside-7-O-rhamnosideGlucoerucin, desulfo fragmentAvobenzone1-Palmitoyl-2-linoleoyl-sn-glycero-3-phosphateScopoletinKaempferol-3-O-galactoside-7-O-rhamnosideNarcissintrans-Cinnamic acidAvobenzone (Parsol 1789)Triethylphosphate.epsilon.-CaprolactamGlutamic acidTetraethylene glycol monomethyl etherIdebenoneHyperosideTetraethylene glycolGramine, fragment9,12-Octadecadiynoic acidGuanine4-Methylquinoline-2,7-diol4-Hydroxynonenal alkyne6-Acetyl-1,1,2,4,4,7-hexamethyltetralinCocamidopropylBetaineGentisic acid3-[4,5-dihydroxy-6-(hydroxymethyl)-3-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-2-yl]oxy-5-hydroxy-2-(4-hydroxyphenyl)-7-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one(.+/-.)7-epi-Jasmonic acidKaempferol-3-O-rhamnosideAstragalinSyringaldehydeGlucohirsutinSalicylic acidcis-Vaccenic acid9-methanesulfinylnonanenitrile (isomer of 665)Biochanin Aroseosidep-Methoxycinnamic acid ethyl esterTryptophan3,5-Di-tert-butyl-2-hydroxybenzaldehydeIberinKaempferol-7-O-rhamnosidePantothenateloliolideDibutylphthalatePentaethylene glycolOleamide5-S-Methylthioadenosine; AIF; CE10; MS2Deckaempferol-3-O-robinoside-7-O-rhamnosideLPE 18:23,5-Dimethoxy-4-hydroxycinnamic acid1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamineCoumaroyl agmatine (isomer of 1296)Quercetin-3-O-galactosideHexaethylene glycolN-Butylbenzenesulfonamide1-isothiocyanato-8-methanesulfinyloctane8-[3-oxo-2-[(E)-pent-2-enyl]cyclopenten-1-yl]octanoic acid1-Isothiocyanato-4-(methylsulfinyl)-butanePregabalinAlyssinXanthurenic acidDilinolenin (9c,12c,15c)L-Glutathione, reducedPhytosphingosineIndolineMaleic acid2,5-DimethoxyphenolStearic acidN-(4-Hydroxyphenyl)propanamideTyrosineN,N,N-Trimethyl-lysine; AIF; CE10; MS2Dec.alpha.,.beta.-ThujoneN,N-Dimethyldodecylamine N-oxideMonolinolenin (9c,12c,15c)ErucamideN-acetyl homoveratrylamineHexamethylcyclotrisiloxane3-HydroxycoumarinIndole-3-acetyl-L-valineDDAO4-(Aminomethyl)benzoic acidGlucoiberin, desulfo fragmentp-tert-Octylphenol tetraglycol etherDi-n-butyl phosphateQuercetin-3-O-rutinoside1,2-Di-(9Z,12Z,15Z-octadecatrienoyl)-sn-glycero-3-phosphocholineFlavonol base + 4O, 1MeO, O-HexNCGC00385418-01!2-(3,4-dihydroxyphenyl)-5-hydroxy-7-methoxy-8-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxychromen-4-onePalmitoyleicosapentaenoyl phosphatidylcholine6-methoxyquinolineJuglonePhthalic anhydridesalicylic acid 2-O-beta-D-glucosideLuteolin-7,3'-di-O-glucoside4-methoxy-1H-indole-3-carbaldehyde1-(1',3'-Benzodioxol-5'-yl)-2-butanamineVilazodoneGln-Leu-LysOlivetolAzelaic acidN,N-DimethylanilinePalmitamide8-methanesulfinyloctan-1-amineKaempferitrin1-Palmitoyl-sn-glycero-3-phosphocholineQuercetin 3-O-alpha-rhamnopy ranosideIsoleucineAsn-Leu-LysAdenosineGlucoraphanin, desulfo fragmentAdenine(S)-Perillic acid3,6,9,12-Tetraoxatetracosan-1-olPhosphocholine2-Hydroxy-3-methoxybenzoic acid2-Acetamido-2-deoxy-3-O-(.beta.-D-galactopyranosyl)-D-glucopyranoseGlucoraphanin, Sulforaphane glucosinolate, 4-Methylsulfinylbutyl glucosinolate, 4-MeSO-Butyl GSL, peak 11H-Indole-3-carboxylic acid1-Isothiocyanato-7-(methylsulfinyl)-heptaneMaritimeinVitexin-2''-O-rhamnoside.alpha.-IononeGlutaminefalseInhibition of jasmonic acid-isoleucine conjugating enzyme JAR1 shifts the local and systemic leaf signals and metabolic profiles in Arabidopsis• Jasmonates (JAs)-mediated pathways are central signaling hubs in plant defense responses. However, the identification of mobile and non-mobile signals involved in downstream systemic signaling is still less studied.• Here, we investigate the role of the jasmonic acid-isoleucine conjugating enzyme, JAR1, in shifting wound-induced local and systemic metabolic profiles using LC-MS/MS for untargeted metabolomics, and the mobility of jasmonic acid-isoleucine (JA-Ile) in wound-induced local and systemic defense using LC-MS/MS for targeted jasmonate analysis in Arabidopsis thaliana leaves.• The use of jarin-1, a specific inhibitor of JA-Ile biosynthesis, suggested that JA-Ile is synthesized de novo in the particular tissues, rather than being a mobile signal. In addition, inhibition of JAR1 enzyme activity affected an array of downstream metabolic pathways, locally and systemically, such as amino acids and carbohydrate metabolism.• This study suggests that the occurrence and spread of local and systemic downstream signals very likely depends on JAR1 activity, and this enzyme exclusively regulates a series of metabolic pathways under both wounding and non-wounding conditions.2026-03-312026-03-23MTBLS14117