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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14515/m_MTBLS14515_GC-MS_alternating_high-polarity_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14515/s_MTBLS14515.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14515/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14515/a_MTBLS14515_GC-MS_alternating_high-polarity.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14515The raw data acquired after mass spectrometric analysis were processed using MassHunter software for qualitative and quantitative analysis.MetaboLightsPublicGas Chromatography MS - alternating - high-polarityChromatographic separation was performed using a DB-5MS capillary column (30 m ? 0.25 mm ? 0.25 ?m). The injection volume was 1 ?L, and samples were injected in split mode with a split ratio of 5:1. Helium was used as the carrier gas at a constant column flow rate of 1.2 mL/min. The oven temperature program was as follows: the initial temperature was held at 40?C for 1 min, then increased to 100?C at 20?C/min, followed by an increase to 300?C at 15?C/min, and finally held at 300?C for 5 min.Detection and Analysis of Rhizosphere Metabolites.Antwerp universityMiao JiangMicrobiomemass spectrometry assayFresh samples were weighed immediately after collection, snap-frozen in liquid nitrogen, and stored at -80 ?C until analysis. Before extraction, samples were freeze-dried and ground to a fine powder at room temperature. For metabolite extraction, 0.5 g of each sample was mixed with 1 mL methanol: isopropanol: water (3:3:2, v/v/v), vortexed for 3 min, and sonicated for 20 min. Extracts were centrifuged at 12,000 rpm for 3 min at 4 ?C, and the supernatant was transferred to a fresh vial. An internal standard solution (20 ?L, 10 ?g/mL) was added, and the extracts were evaporated under a stream of nitrogen and subsequently freeze-dried.The derivatization method was as follows: the sample was mixed with 0.1 mL solution of methoxyamine hydrochloride in pyridine (0.015 g/mL). The mixture was incubated at 37?C for 2 h. Then 0.1 mL of BSTFA (with 1% TMCS) was added into the mixture and kept at 37?C for 30 min after vortex-mixing. Pipette 0.2 mL of the derivatization solution, add n-hexane to dilute to 1 mL, filter with a 0.22 ?m organic phase syringe filter, store in a refrigerator at -20?C, and test on the machine within 24 hours.rhizospherehttps://www.ebi.ac.uk/metabolights/MTBLS14515AbdElgawad Hamada. University of Antwerp.Miao Jiang. Northwest A&F University. jiangmiao0203@163.com.Li Xiangnan. Chinese Academy of Sciences. lixiangnan@iga.ac.cn.Beemster Gerrit. University of Antwerp.Based on an in-house database, qualitative and quantitative mass spectrometric analyses of metabolites in the samples were performed. During the analysis, the sample mass spectral fingerprints were matched and compared with reference mass spectra in the database for score-based qualitative identification. Retention index information was also used to improve identification accuracy and eliminate interference from false-positive compounds. The acquired mass spectrometry files were processed using MassHunter quantitative analysis software, and quantifier ions were selected for chromatographic peak integration and correction to improve the accuracy of quantification.Nitrogen treatmentjiangmiao0203@163.com For rhizosphere sampling, whole root systems were carefully removed from the pots and gently shaken to discard loosely adhering bulk soil, and rhizosphere soil was collected from four biological replicates per treatment. Soil tightly adhering to the root surface within approximately 0-2 mm was operationally defined as rhizosphere soil. This root-adhering soil was gently brushed off with a sterile soft brush, collected into sterile tubes, and passed through a sterile 2 mm sieve after removal of visible plant debris and stones. MetabolomicsMulti-omics studyRhizosphereMetabolomicsuntargeted analysisAgilent 5977B MSDmicrobiomeAgilent 8890 GCuntargeted metabolite profilingexperimental sampleMulti-omics studyRhizosphereMetabolomicsuntargeted analysisAgilent 5977B MSDmicrobiomeAgilent 8890 GCuntargeted metabolite profilingexperimental sampleMass spectrometry Mass spectrometric detection was performed using electron ionisation with an electron energy of 70 eV. The transfer line temperature was set to 280?C, the ion source temperature was set to 230?C, and the quadrupole temperature was set to 150?C. The carrier gas was helium. falseDetection and Analysis of Rhizosphere MetabolitesTo evaluate whether maternal nitrogen histories influenced the rhizosphere metabolite profiles of offspring plants under contrasting soil N availability, seeds from different nitrogen-history maternal lineages were sown into soils established under either N0 (no additional N input) or N2 (2 g N per pot conditions, and rhizosphere metabolites of the offspring plants were subsequently detected and analyzed.2026-05-182026-05-18MTBLS14515