Accela series u hplc

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).

Filtrated extract (10 μL) was injected in an Accela Series UHPLC (ThermoFisher Scientific, Waltham, MA, USA) coupled to an Exactive mass spectrometer (Thermo FisherScientific, Waltham, MA, USA). A chromatographic C18 Accucore column (2.1 × 100 mm, 1.5 μm, ThermoFisher Scientific, Waltham, MA, USA) was used. Flow rate was set to 300 μL/min. Run time was 10 min, with the elution gradient starting at 20% MeOH (0–1 min), linearly increasing to 80% MeOH (1–6 min), remaining at 80% MeOH (6–8 min), then decreasing and staying at 20% MeOH for re-equilibration. Mass detection was performed in negative mode at a scan range 90–500 m/z. A heated electrospray ionisation source was used with spray voltage of 4 kV and capillary temperature of 275°C. Mass spectra were obtained using Xcalibur version 2.2 (ThermoFisher Scientific, Waltham, MA, USA). For JH and 20HE quantification, calibration curves were constructed with 1× weighted linear fitting for each component (0.01, 0.1, 1, and 10 μg l^-1). Recovery percentages (92–95%) were calculated from the internal standard methoprene.

Phytohormones were extracted and analyzed as described elsewhere [6 (link)]. Briefly, ~120 mg of frozen tissue was extracted twice with 1 ml of 80% methanol/water and centrifuged at 20,000 g for 15 min at 4°C. The supernatant was passed through a C18 cartridge, and the samples were collected in a 5-ml tube for speed-Vac evaporation to dryness. The residue was resuspended in 1 ml of 20% methanol/water. Ten μl of filtrated extract as injected in a U-HPLC-MS system consisting of an Accela Series U-HPLC (ThermoFisher Scientific, USA) coupled to an Exactive mass spectrometer (ThermoFisher Scientific) using heated electrospray ionization (HESI) interface. Mass spectra were obtained using Xcalibur software version 2.2 (ThermoFisher Scientific). The auxin homeostasis metabolites were identified according to the molecular mass and retention time values in the total ion chromatograms from the phytohormone analysis. In each compound, the area obtained was used for comparisons.

Cytokinins (trans-zeatin), gibberellins (GA1, GA4, and GA3), indole-3-acetic acid, ABA, SA, jasmonic acid, and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) were analyzed in mature leaves according to Albacete et al. (2008) (link) with some modifications (Albacete et al., 2008 (link)). Ten microliters of extracted sample was injected in a Ultra high performance liquid chromatography (UHPLC)–MS system consisting of an Accela Series U-HPLC (ThermoFisher Scientific, Waltham, MA, USA) coupled to an Exactive mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) using a heated electrospray ionization (HESI) interface. Mass spectra were obtained using the Xcalibur software version 2.2 (ThermoFisher Scientific, Waltham, MA, USA). For quantification of the phytohormones, calibration curves were constructed for each analyzed component (1, 10, 50, and 100 μg/L) and corrected for 10 μg/L deuterated internal standards. Recovery percentages ranged between 92% and 95%. To examine responses to trans-zeatin treatment, the seeds of each genotype were surface-sterilized and plated on vertical MS plate (10 g/L agar) supplemented with indicated concentration of trans-zeatin.

The second leaf of each plant under water deficit and well-watered conditions was harvested at the treatment conclusion. Samples were flash-frozen using liquid nitrogen and kept at −80 °C. Extracts of plant hormones were obtained as described by [83 (link)]. Ten microliters of each extract sample were injected into a UHPLC–MS system consisting of an Accela Series U-HPLC (ThermoFisher Scientific, Waltham, MA, USA) equipped with an Exactive mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) that uses a heated electrospray ionization (HESI) interface. Mass spectra were obtained using the Xcalibur software version 2.2 (ThermoFisher Scientific, Waltham, MA, USA). For quantification of the plant hormones (abscisic acid, 6-dymethylamino purine, gibberellic acid-1, jasmonic acid, and salicylic acid), calibration curves were constructed for each analyzed hormone (1, 10, 50, and 100 μg L⁻¹) and corrected for 10 μg L⁻¹ deuterated internal standards. Recovery percentages ranged from 92% to 95%.

Cytokinins (Z; ZR; and iP), ACC, ABA, JA, SA, and gibberellins (GA₁, GA₃, and GA₄) were analyzed according to Albacete et al. (2008 (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). Ten microliters of filtrated extract were 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, Waltham, MA, USA) using a heated electrospray ionization (HESI) interface. Mass spectra were obtained using the Xcalibur software version 2.2 (ThermoFisher Scientific, Waltham, MA, USA). For quantification of the plant hormones, calibration curves were constructed for each analyzed component (1, 10, 50, and 100 μg l⁻¹).