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+## Root metabolite profiling
+
+Approximately 25-35 sterilized *A. thaliana* Col-0 seeds were sown in a row on ArtSoil and grown for 3 weeks. After 3 weeks, both primary and lateral roots longer than 8 cm were harvested by slicing with a scalpel. The roots were segmented into 2-cm segments measuring 0-2, 2-4, and 4-6 cm from the root tip. Lateral roots that emerged from primary roots were removed, if present. The roots were rinsed in distilled water, and blotted dry on Whatman filter papers before they were weighed and kept in 1.5-ml microcentrifuge tubes, each containing 2 metal beads. The samples were flash-frozen in liquid nitrogen and stored at -80 °C until the extraction process. For metabolite extraction, 0.5 mL of chilled extraction buffer (2:5:2 ratio of ddH2O: methanol: chloroform containing 5 μM ribitol as internal standard) was added into each root sample and mixed by vortex for 20 sec. Metal beads in the tubes were removed. Sample tubes were shaken on a rotary shaker for 30 mins at 4 °C. The samples were then centrifuged at 20, 000 × g for 5 min at 4 °C. After centrifugation, 0.5 ml of supernatant was carefully aspirated and transferred to a clean 1.5-ml microcentrifuge tube. Samples were stored at -80 °C until they were subjected to GC-MS. For GC-MS analysis 30 μl of sample were dried by vacuum centrifugation in glass inlet tubes. Dried samples were derivatized according to Gu (2012) and Shim et al.(2019). Raw data files were converted to the mzXML format using ProteoWizard (Chambers, M.C. et al., 2012) and to the NetCDF format via MetAlign (Lommen, A. and Kools, H.J., 2012) using default parameters. Deconvolution of mass spectra was conducted using the free deconvolution software AMDIS (Automated Mass Spectral Deconvolution and Identification System from NIST). Deconvoluted mass spectra were matched against the NIST14 Mass Spectral Library (https://www.nist.gov/srd/nist-standard-reference-database-1a-v14). Database matches with more than 70% were further compared with an in-house chemical standard library for compound annotation. Compounds, that could not be verified by the in-house library are named according to the matched compound class and the retention time. Extracted ion peaks were integrated using MassHunter Quantitative (v b08.00, Agilent Technologies). For relative quantification, all metabolite peak areas were normalized to fresh weight and the peak area of the internal standard ribitol (Sigma-Aldrich) to correct for technical error. Here, a GC-based system was employed for metabolomics, which may detect only the fraction of metabolites that can be vaporized. It may be useful for a more in-depth analysis to use LC-based systems to identify more metabolites and improve coverage.
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