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Plant hormone levels were quantified as de … Plant hormone levels were quantified as described in a previous report, with slight modifications. We used a 6410 Triple Quad LC-MS (Agilent Technologies, Palo Alto, CA), which includes an Agilent 1200 series rapid resolution LC system fitted with a ZORBAX Eclipse XDB-C18 column (1.8 mm, 2.1 x 50 mm). The F and FD samples were extracted with 5 volumes of 80% v/v acetonitrile containing 1% v/v acetic acid and internal standards. Extracts were centrifuged at 14 000 x g for 10 min at 4°C, and the supernatant was collected. This procedure was repeated once, and acetonitrile was removed in a SpeedVac (Thermo Fisher Scientific, Waltham, MA). Acidic water extracts were loaded onto an Oasis HLB extraction cartridge (30 mg, 1 mL; Waters, Milford, MA), and washed with 1mL of water containing 1% acetic acid to remove highly polar impurities.
Phytohormones were eluted with 2mL of 80% acetonitrile containing 1% acetic acid, and the acetonitrile in this eluent was removed in a SpeedVac. Acidic water extracts were loaded onto an Oasis MCX extraction cartridge (30 mg, 1 mL; Waters, Milford, MA). After washing with 1mL water containing 1% acetic acid, acidic and neutral compounds were eluted with 2mL of acetonitrile (acetonitrile fraction). After washing with water containing 5% v/v ammonia, basic compounds were eluted with 2mL of 60% v/v acetonitrile containing 5% v/v ammonia. After drying these basic fractions, 20 mL water containing 1% acetic acid was added, and the basic hormones (trans-zeatin and isopentenyladenine) in these fractions were analyzed by LC-MS/MS.
Subfractions (10%) of the acetonitrile fractions were collected and prepared as basic fractions to analyze for salicylic acid (SA). The remaining 90% of the acetonitrile fractions was concentrated in the SpeedVac to remove acetonitrile and loaded onto Oasis WAX extraction cartridges (30 mg, 1 mL; Waters, Milford, MA). After washing with 1mL of water containing 1% acetic acid, neutral compounds were eluted with 2mL of acetonitrile, and acidic compounds were eluted with 2mL of 80% acetonitrile containing 1% acetic acid. After drying these acidic fractions, 20 mL water containing 1% acetic acid was added and the acidic hormones (indole acetic acid, abscisic acid (ABA), jasmonic acid, jasmonic acid isoleucine
conjugate, gibberellic acid 1, and gibberellic acid 4) in these fractions were analyzed by LC-MS/MS. these fractions were analyzed by LC-MS/MS.
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