Engineering of benzoxazinoid biosynthesis in Arabidopsis thaliana: Metabolic and physiological challenges Author Abramov, Aleksej * & Chair of Plant Breeding, Technical University of Munich, Liesel-Beckman Str. 2, 85354, Freising, Germany Author Hoffmann, Thomas Author Stark, Timo D. Author Zheng, Linlin Author Lenk, Stefan Author Hammerl, Richard Author Lanzl, Tobias Author Dawid, Corinna Author Schon, Chris-Carolin Author Schwab, Wilfried Author Gierl, Alfons Author Frey, Monika text Phytochemistry 2021 112947 2021-12-31 192 1 15 http://dx.doi.org/10.1016/j.phytochem.2021.112947 journal article 10.1016/j.phytochem.2021.112947 1873-3700 5.9. Isolation of Arabidopsis microsomes and P450 assay 15 g of rosette leaves of 28 dag Arabidopsis plants were ground in 200 mL extraction buffer (100 mM ascorbic acid, 1 mM EDTA, 100 mM Tris, 20% v/v glycerol, 20% w/v Sucrose, 5 mM Dithiothreitol) with 4.5 g Polyklar AT (Merck) and sea sand. The raw extract was filtered through cloth and centrifuged twice at 15 000 g for 10 min. Microsomes were isolated from the supernatant by centrifugation at 120 000 g for 40 min and resuspended in 1 mL suspension buffer (50 mM potassium phosphate buffer pH 7.5, 20% v/v Glycerol, 1 mM Dithiothreitol). The integrity of the microsomes was tested by measuring the cytochrome C reductase activity as described by Urban et al. (1990) . The in vitro activity of the BX P450 enzymes was tested by incubation of 1 mg total microsomal protein with the respective substrate (2 mM Indole, 1 mM ION , 250 μM HION, 200 μM HBOA) in 100 mM potassium phosphate buffer pH 7.5 and 1 mM NADPH at room temperature. The reaction was stopped after 30 min by addition of 1 vol methanol and precipitated protein was pelleted by centrifugation. 2.5 vol of 100 mM acetic acid were added to the supernatant and the products were extracted three times with 2 vol of ethyl acetate. The solvent was evaporated in a vacuum centrifuge, the remaining products were resolved in methanol and analysed by HPLC.