Enzyme activity of DAO enzymes

For the enzyme reaction of AtDAO1 with IAA-amino acid conjugates, the reaction mixture contained 50 mM potassium phosphate buffer (pH 7.2), 0.5 mM 2-oxoglutarate, 2 μM ammonium iron(II) sulfate [(NH₄)₂Fe(SO₄)₂·6H₂O], and 5 mM ascorbic acid in a final volume of 120 µL; 5–200 μM IAA-amino acid conjugates and AtDAO1 enzymes (80 ng/120 µL for the kinetic analysis of IAA-Asp, 64 ng/120 µL for the kinetic analysis of IAA-Glu, and 100–300 ng/120 µL for the other reactions in this study) were added. The enzyme reaction was carried out at 30 °C for 3 min (kinetic analysis) or for 15-120 min (the other reactions). For the enzyme reaction of OsDAO for IAA-amino acid conjugates, the reaction mixture contained 50 mM potassium phosphate buffer (pH 7.2), 0.5 mM 2-oxoglutarate, 10 μM ammonium iron(II) sulfate [(NH₄)₂Fe(SO₄)₂·6H₂O], and 5 mM ascorbic acid in a final volume of 100 µL; 500 μM IAA-amino acid conjugates and OsDAO1 enzymes (800 ng/100 µL) were added. The enzyme reaction was carried out at 30 °C for 20 min. Then, 100 µL of the reaction mixture was added to 400 µL of methanol containing 50 mM phosphoric acid in a 1.5-mL microtube to terminate the reaction. After centrifugation at 12,000 × g for 10 min at 2 °C, the supernatant was immediately analyzed by HPLC (EXTREMA, JASCO Japan) as described in Supplementary Methods. The oxIAA-amino acids were detected as a diastereomeric mixture in the reaction mixture. One of the diastereomers of oxIAA-amino acid was readily epimerized during sample preparation (Supplementary Fig. ^4g). For the reaction conditions for IAA, the reaction mixture contained 50 mM potassium phosphate buffer (pH 7.2), 0.5 mM 2-oxoglutarate, 0.2 mM ammonium iron(II) sulfate [(NH₄)₂Fe(SO₄)₂·6H₂O], and 5 mM ascorbic acid in a final volume of 120 µL. IAA (1000 μM) and boiled or unboiled AtDAO1 enzymes (11.5 µg/120 µL) were added, and the reaction mixture was incubated in a PCR tube at 30 °C for 20 h with a thermal cycler (MJ Mini, Bio-Rad). Then, 100 µL of the reaction mixture was added to 400 µL of methanol containing 50 mM phosphoric acid in a 1.5-mL microtube to terminate the reaction. After centrifugation at 12,000 × g for 10 min at 2 °C, an aliquot of the supernatant was immediately analyzed by an HPLC system (EXTREMA, JASCO Japan) as described in Supplementary Methods. For the OsDAO reaction for IAA, OsDAO enzymes (800 ng/100 µL) were added to the reaction mixture and incubated at 30 °C for 200 min. After the enzyme reaction, 100 µL of the reaction mixture was mixed with 400 µL of methanol containing 50 mM phosphoric acid in a 1.5-mL microtube to terminate the reaction. After centrifugation at 12,000 × g for 10 min at 2 °C, the supernatant was immediately analyzed by an HPLC system (EXTREMA, JASCO Japan) as described in Supplementary Methods.