UV-Induced Camalexin Biosynthesis Analysis

For UV induction, leaves were cut at the base of the petioles and placed on wet tissue paper under a UV-lamp (Desaga UV-VIS, = 254 nm, 8 W) at a distance of 20 cm and were irradiated for 2 h (Mucha et al., 2015 (link)). Camalexin extraction and HPLC-analysis was performed essentially as previously described (Schuhegger et al., 2006 (link)). Leaves were extracted twice in 200 μl MeOH/H₂O (4:1; v/v) at 65°C for 30 min. Combined extracts were centrifuged at 17,000 g for 15 min and analyzed by reverse phase HPLC (LiChroCART 250-4, RP-18, 5 μm, Merck; 1 mL⋅min^-1; MeOH/H₂O (1:1; v/v) for 2 min, followed by a 10 min linear gradient to 100% MeOH, followed by 3 min 100% MeOH). Camalexin was quantified using a Shimadzu F-10AXL fluorescence detector (318 nm excitation, 370 nm emission) and by UV absorption at 318 nm applying a calibration curve with authentic standard. For intermediate feeding leaves were detached at the petiole after 2 h UV treatment and incubated in 400 μl 0.25 mM precursor solution or water for an additional 16 h.