Coomassie plus reagent

Proteins were extracted from frozen seedling tissue (50 mg) by adding 200 µL of extraction buffer (25 mM Tris–Cl pH 6.8, 1% SDS, 1% (v/v) β-mercaptoethanol, 1% (v/v) Halt protease inhibitor cocktail [Sigma-Aldrich, #78442]) and heating at 95°C for 5 min. After centrifugation for 10 min, the supernatant was collected and proteins quantified using Coomassie Plus Reagent (Sigma-Aldrich, #23236). Ten microgram total protein was mixed with 2 × Laemmli Buffer (1:1) and heated at 96°C for 10 min to dissolve AOC trimers. Proteins were separated by SDS–PAGE (12% acrylamide) and transferred to a PVDF membrane. Detection of protein bands was done by staining with Ponceau S. The membrane was blocked with 5% (w/v) BSA in TBST (20 mM Tris–Cl pH 7.8, 150 mM NaCl, 0.05% [v/v] Tween) and immuno-stained using anti-AtAOC antibody (1:5,000, Stenzel et al., 2003 (link)) and a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase (1:5,000, Sigma-Aldrich). Chemiluminescence detection was performed with Immun-Star AP Substrate (ThermoFisher Scientific, Waltham, MA, USA, #1705018) for 5 min and visualized using a Fusion FX Imaging system (Vilber, www.vilber.com).

Tryptic soybean agar (Merck KGaA, Germany), Tween 20, Bradford reagent, bovine serum albumin, sodium hydroxide, sodium carbonate, sodium dihydrogen phosphate, potassium chloride, calcium chloride, Folin-Ciocalteu phenol reagent, Coomassie plus reagent, trichloroacetic acid, sucrose, citrate buffer, glucose, disodium p-nitrophenyl phosphate (tetrahydrate), beta-glycerophosphate, ammonium molybdate, were purchased from Sigma-Aldrich (St. Louis, USA). The pollutants used in this study were: basic color (primer) (marked–FH), thinner for rinsing paint (marked–MF), thinner (marked–CN), metallic red color 108 (marked–FM), and white color 268 (marked–CP), all provided by an automotive manufacturing company (Kragujevac, Serbia).