Polar Metabolite Extraction from Plant Roots

For polar metabolites analysis, a variation of the method described in Cheong et al. [70 (link)] was used. 250 µL 100% LC-MS grade MeOH (Roth, Karlsruhe, Germany), containing 4% ¹³C₆ sorbitol/valine (Sigma-Aldrich, Castle Hill, Australia) was added to approximately 25 mg of frozen root tissue from each pooled group. Tubes were shaken at 800 rpm for 15 min at 30 °C, centrifuged at 15,700× g for 15 min at room temperature and the supernatant collected. The pellet was re-extracted with 250 µL of Milli-Q H₂O as above and the supernatants combined. In case of cloudy supernatant or precipitate observed during supernatant transfer, the pooled supernatant was centrifuged at 15,700× g for 10 min at room temperature before transferring as much supernatant as possible to a new tube. From each sample, 50 µL aliquots of the supernatant were transferred into glass insert (Agilent, Santa Clara, CA, USA) and dried under vacuum at 30 °C for 90 min.