Xcalibur software version 2

The peptide samples were analyzed by nano-RPLC-ESI-MS/MS with an LTQ-OrbitrapElite mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with a Dionex ultimate 3000 liquid chromatography and an ESI probe Ion Max Source with a nanospray kit. The spectrometer was controlled by Xcalibur software version 2.2 (Thermo Fisher, Waltham, MA, USA). The peptides were separated on a C18 capillary column (30 cm, 75 μm i.d./375 μm o.d.) packed with C18 silica particles (5 μm, 100 Å) with a 145 min gradient from 10 to 40% buffer B (98% ACN/0.1% FA) and analyzed on the mass spectrometer. Mass spectra were acquired in a data-dependent mode. MS1 spectra were measured at a resolution of 6 × 10⁴ and the top 10 most abundant ions with an isolation window of 2 m/z were selected for sequencing and fragmented in the data-dependent CID mode with a normalized collision energy of 35%, activation Q of 0.25, activation time of 10 ms, and one microscan. The sample was analyzed in triplicate.

Thermo Fisher Xcalibur software version 2.2 (Thermo Fisher Scientific, San Jose, CA, USA) was used for data handling and processing. The mass tolerance for peak integration was set to 5 ppm. ICIS peak integration settings were: smoothing points, 13; baseline window, 100; area noise factor, 2; and peak noise factor, 5.

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).

To assess the solvent residues of acetone, samples (100 mg tablet) were dissolved in 1000 Type I water and injected in an overhead Thermo Fisher Focus gas chromatography (GC) system. Results were analysed using xCalibur software version 2.2 (Thermo Fisher Scientific Inc., UK). The injection volume was 1 µL, the inlet and detector temperatures were 200 and 250 °C, respectively. The flow rate was 1 mL/min using helium as a carrier gas and Stabilwax GC column (Restek, Bellefonte, PA, USA).

All resulting peptide fractions were reconstituted in 20 μL of 0.1% (v/v) FA and 4 μL of each sample was analyzed by online reverse-phase LC-MS/MS platform consisting of an Eksigent NanoLC-Ultra 2D plus system (AB SCIEX) coupled with a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) via a nano-electrospray source. Peptide mixtures were separated by reverse phase chromatography using a home-packed ReproSil-Pur C18-AQ column (75 μm internal diameter × 15 cm, 1.9 μm, 200 Å pore size; Dr. Maisch GmbH, Ammerbuch, Germany) in 2 h LC gradient of 2–80% buffer B [ACN in 0.1% (v/v) FA] at a flow rate of 300 nL/min. The Q Exactive instrument was operated in the data-dependent mode to simultaneously measure survey scan MS spectra (from m/z 400–2,000) in the Orbitrap analyzer at resolution R = 70,000. Up to the 12 most intense peaks with charge state ≥2 and above a signal threshold of 500 counts were selected for fragmentation in the ion trap via higher-energy collisional dissociation. System controlling and data collection were carried out by Xcalibur software version 2.2 (Thermo Scientific).

The peptide samples were analyzed by nano-RPLC-ESI-MS/MS with an LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with a Dionex ultimate 3000 liquid chromatography and an ESI probe Ion Max Source with a nanospray kit. The spectrometer was controlled by Xcalibur software version 2.2 (Thermo Fisher, Waltham, MA, USA). The peptides were separated on a C18 capillary column (30 cm, 75 μ m i.d./375 μ m o.d.) packed with C18 silica particles (5 μ m, 100 Å) with a 145 min gradient from 10 to 40% buffer B (98% ACN/0.1% FA) and analyzed on the mass spectrometer. Mass spectra were acquired in a data-dependent mode. MS1 spectra were measured at a resolution of 6 × 10⁴ and the top 10 most abundant ions with an isolation window of 2 m/z were selected for sequencing and fragmented in the data-dependent CID mode with a normalized collision energy of 35%, activation Q of 0.25, activation time of 10 ms, and one microscan. The sample was analyzed in quadruplicate. Raw mass spectrometric data files can be downloaded from ftp://massive.ucsd.edu/MSV000079911. The evidence table containing protein data is provided as S1 Table.

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).