Hormone levels were analyzed in both xylem and leaf in four of the six plants where gas exchange and grapevine water status were measured.
Xylem was sampled from the same leaves used to measure ΨMD. Leaves were exposed to high pressure with a Scholander pressure chamber (Soilmoisture Equipment Corp., Santa Barbara, CA, USA) to collect the xylem with a micropipette in a 1.5 mL tube. The collected xylem was immediately frozen with liquid nitrogen and stored at −80 °C until analysis.
Leaves were sampled with special frozen clamps, which allow collecting and immediately froze 1.5 cm radius leaf disks. Three disks per plant, one per leaf, were collected and immediately kept in liquid nitrogen. In the laboratory, leaf disks were separately grinded in a mortar with liquid nitrogen, and 100 mg were stored in 2 mL tubes at −80 °C until hormone analysis.
Active cytokinins (trans-zeatin, TZ, zeatin riboside, ZR and isopentenyl adenine, iP), gibberellins (GA1, GA3 and GA4), indole-3-acetic acid (IAA), abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) were analysed according to Albacete et al. [86 (link)], with some modifications. Briefly, 1 mL of extraction solution (cold (−20 °C) methanol/water (80/20, v/v)) was mixed with 100 mg of plant material and centrifuged (20,000× g, 15 min) to separate solids. Then, 1 mL of the same solution was added, and the solids were re-extracted (30 min at 4 °C). Lipids and part of vegetal pigments were removed by passing the supernatants through Sep-Pak Plus †C18 cartridge (SepPak Plus, Waters, Mildford, MA, USA). Next, they were evaporated at 40 °C under vacuum. The residue was dissolved using an ultrasonic bath in 0.5 mL of extraction solution. The dissolved samples were filtered through 13 mm diameter Millex filters with 0.22 μm pore size nylon membrane (Millipore, Bedford, MA, USA).
10 μL of the resulted 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. Xcalibur 2.2 (ThermoFisher Scientific, Waltham, MA, USA, EE.UU.) was the software used to obtain the mass spectra. Phytohormones were quantified by creating calibration curves (1, 10, 50, and 100 μg·L−1), which were corrected with 10 μg·L−1 deuterated internal standards. Recovery percentages ranged between 92 and 95%.
Xylem was sampled from the same leaves used to measure ΨMD. Leaves were exposed to high pressure with a Scholander pressure chamber (Soilmoisture Equipment Corp., Santa Barbara, CA, USA) to collect the xylem with a micropipette in a 1.5 mL tube. The collected xylem was immediately frozen with liquid nitrogen and stored at −80 °C until analysis.
Leaves were sampled with special frozen clamps, which allow collecting and immediately froze 1.5 cm radius leaf disks. Three disks per plant, one per leaf, were collected and immediately kept in liquid nitrogen. In the laboratory, leaf disks were separately grinded in a mortar with liquid nitrogen, and 100 mg were stored in 2 mL tubes at −80 °C until hormone analysis.
Active cytokinins (trans-zeatin, TZ, zeatin riboside, ZR and isopentenyl adenine, iP), gibberellins (GA1, GA3 and GA4), indole-3-acetic acid (IAA), abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) were analysed according to Albacete et al. [86 (link)], with some modifications. Briefly, 1 mL of extraction solution (cold (−20 °C) methanol/water (80/20, v/v)) was mixed with 100 mg of plant material and centrifuged (20,000× g, 15 min) to separate solids. Then, 1 mL of the same solution was added, and the solids were re-extracted (30 min at 4 °C). Lipids and part of vegetal pigments were removed by passing the supernatants through Sep-Pak Plus †C18 cartridge (SepPak Plus, Waters, Mildford, MA, USA). Next, they were evaporated at 40 °C under vacuum. The residue was dissolved using an ultrasonic bath in 0.5 mL of extraction solution. The dissolved samples were filtered through 13 mm diameter Millex filters with 0.22 μm pore size nylon membrane (Millipore, Bedford, MA, USA).
10 μL of the resulted 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. Xcalibur 2.2 (ThermoFisher Scientific, Waltham, MA, USA, EE.UU.) was the software used to obtain the mass spectra. Phytohormones were quantified by creating calibration curves (1, 10, 50, and 100 μg·L−1), which were corrected with 10 μg·L−1 deuterated internal standards. Recovery percentages ranged between 92 and 95%.
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