As 1550

For the enzyme assays, 2 μg samples of the enriched protein fraction were mixed with assay buffer (100 mM Tris, 200 μM MnCl₂, 1 mM DTT; pH 8.0), and the IAA conjugates, IAA-Ala, IAA-Leu, IAA-Asp, IAA-Gly and IAA-Trp, were added individually at a concentration of 1 mM, as substrates. The reactions were incubated at 37 °C for 1 h and terminated by the addition of 20 μl 1 N HCl. The aqueous phases were then extracted with 250 μl of ethyl acetate, evaporated, and the reaction products were dissolved in 100 μl of methanol for ultra performance liquid chromatography (UPLC) analysis. Aliquots (0.2 μl) of the methanol extracts were then injected into a UPLC instrument (Waters H-class, Waters, Millford, MA, USA) coupled to an autosampler (Jasco AS-1550), and equipped with a 2.1 × 100-mm Cortecs C18 2.7 μm, reversed phase column. 1% aqueous acetic acid and 100% methanol were used as the solvents. The solvent program was 15% methanol for 3 min, followed by a gradient from 15 to 90% methanol for 4 min, then a decreasing gradient from 90 to 15% for 3 min with 1% acetic acid throughout. Compounds were detected using ACQUITY FLR fluorescence (excitation 278 nm, emission 350 nm) and quantitated by the peak area.