0.22 m pore size nylon membrane

In sap, IAA, ABA, SA, JA and JA-Ile were analyzed according to Albacete et al. [95 (link)], with some modifications. Briefly, xylem sap samples from eight different plants per treatment were filtered through 13 mm diameter Millex filters with a 0.22 µm pore size nylon membrane (Millipore, Bedford, MA, USA). Then, 10 µL of filtrated extract was injected in a U-HPLC-MS system consisting of an Accela Series U-HPLC (ThermoFisher Scientific, Waltham, MA, USA) coupled to an Exactive mass spectrometer (ThermoFisher Scientific), using a heated electrospray ionization (HESI) interface. Mass spectra were obtained using Xcalibur software version 2.2 (ThermoFisher Scientific). For quantification of the plant hormones, calibration curves were constructed for each analyzed component (1, 10, 50 and 100 µg L⁻¹).

ABA, IAA, SA, JA and JA-Ile were analysed in the sap collected for Lo measurement according to Albacete et al. [93 (link)] with some modifications. Briefly, xylem sap samples were filtered through 13 mm diameter Millex filters with 0.22 µm pore size nylon membrane (Millipore, Bedford, MA, USA). The deuterium-labelled internal standard used for hormones determination were the following: ²H₅-Indole-3-Acetic acid (D-IAA), ²H₆-(+)-cis,trans-Abscisic acid (D-ABA), ²H₂-N-(-)-Jasmonoyl Isoleucine (D-JA-Ile) and ²H₄-Salicylic acid (D-SA), obtained from OlChemin Ltd. (Olomouc, Czech Republic). The ²H₅-Jasmonic acid (D-JA) was obtained from CDN Isotopes (Pointe-Claire, QC, Canada). Ten µL of each internal standard was added to the filtrate. Subsequently, 10 µL of filtrate were injected in a U-HPLC-MS system consisting of an Accela U-HPLC system (ThermoFisher Scientific, Waltham, MA, USA) coupled to an Exactive^TM mass spectrometer (ThermoFisher Scientific) using a heated electrospray ionization (HESI) interface. Mass spectra were obtained using Xcalibur software version 2.2 (ThermoFisher Scientific, Waltham, MA, USA). For quantification of the plant hormones, calibration curves were constructed for each analysed component (1, 10, 50, and 100 µg L⁻¹).

Extracts from green tea hot brews were filtered through 13 mm diameter Millex filters with a 0.22 µm pore size nylon membrane (Millipore, Bedford, MA, USA). Ten microliters of filtered extract were injected using ultra-high performance liquid chromatography (UHPLC, Accela Series, ThermoFisher Scientific, Waltham, MA, USA) coupled to a high-resolution mass spectrometry (Exactive, ThermoFisher Scientific, Waltham, MA, USA) consisting of an Orbitrap detector and using a heated electrospray ionization (HESI) interface. Data processing was carried out through the Xcalibur software (version 4.3, ThermoFisher Scientific, Waltham, MA, USA), the XCMS metabolomics platform (Scripps Center for Metabolomics and Mass Spectrometry, La Jolla, CA, USA) and the KEGG, PubChem and PHENOL-EXPLORER chemical databases, amongst others. For the fine-tuning analysis method, the molecular formulas of the compounds were searched in the PubChem platform and entered into the Qual Browser package of the Xcalibur software, where mass/charge (m/z) ratios of each metabolite were identified in the negative mode, adjusting a mass tolerance of ≤2 ppm in the Processing Setup Package. Additionally, correlations between compounds of the same metabolic pathway and the LogP coefficient were used to accurately identify these metabolites.