Fig. 6

In vitro enzymatic activity of recombinant OsCYP94B5 and OsAH8. a Microsomes of OsCYP94B5-expressing yeast were prepared and incubated for 30 min with 100 μM JA-Ile (blue traces) in absence or in presence of NADPH cofactor. Expected oxidation products (red traces) were identified based on the retention time and detection in multiple reaction monitoring mode of authentic 12OH-JA-Ile and 12COOH-JA-Ile standards. b Recombinant his-tagged OsAH8 fused to Maltose Binding Protein (MBP) was affinity purified and incubated with 30 μM of either of the following amino-acid conjugates: JA-Ile, 12OH-JA-Ile or IAA-Ala. Respective cleavage products were searched for by detection based on retention time and multiple reaction monitoring mode of authentic JA, 12OH-JA or IAA standards. Analysis were performed in triplicates and one representative trace is shown