The in vitro activity assays of the purified recombinant protein were performed at 30°C for 30 min in 100 μl 100 mM Tris-HCl buffer (pH 7.5) containing 60 μM acyl donor (caffeoyl-CoA or feruloyl-CoA or sinapoyl-CoA) and 200 μM acyl acceptor (spermidine or spermine or putrescine) and 10 μg purified protein. The reactions were terminated by adding 20 μl ice-cold 0.5% trifluoroacetic acid and directly subjected to liquid chromatography-mass spectrometry (LC-MS) on an Agilent 1260 system (Agilent Technologies, CA), equipped with electron spray ionization mass spectrometer 6125B. The temperature of column oven is 30°C; Electron Spray Ionization (ESI) is in the positive mode; capillary voltage is at 3 Kv; and for full-scan mode, the wavelength range is from 190 to 600 nm. Samples were separated on a reverse-phase C18 column [Thermo Syncronis C18 analytical column (150 mm × 4.6 mm, 5 μm)] at a flow rate of 0.8 ml/min and a gradient mobile phase as follows: 0–5 min, 15% solvent B (0.2% acetic acid in acetonitrile) in solvent A (0.2% acetic acid in water); 5–25 min, 15–100% solvent B; 25–35 min, 100% solvent B; 35–40 min, 100 to 15% B (Grienenberger et al., 2009; Luo et al., 2009). CoA esters were synthesized according to published methods (Semler et al., 1987) and were identified and quantified by spectrophotometry (Stockigt and Zenk, 1974). All of the reactions were run for two technical replicates, and each assay was repeated for at least three independent experiments.

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