ROS Measurement in Arabidopsis Leaves

Leaves of 4‐ to 6‐wk‐old A. thaliana Col‐0 and ΔRLP23 (Albert et al., 2015 ) were used to perform reactive oxygen species (ROS) measurements. Leaf discs were obtained using a disposable biopsy punch and were placed on water in a 96‐well plate and incubated overnight. After the water was removed, discs were treated with a 50 µl assay solution containing 10 µg ml⁻¹ horseradish peroxidase (Sigma) and 50 µM luminol L‐012 (Fujifilm, Neuss, Germany). Immunity responses were triggered by the synthesized peptides nlp20 (1 μM) as described in Böhm et al. (2014 (link)), nlp27 (1 μM) from BsNep1 and BcNep1 (GIMYAWYFPKDQPAAGNVVGGHRHDWE), or with flg22 (0.1 μM) as a positive control or water as a negative control. Luminescence was measured by a microplate fluorescence reader (ClARIOstar; BMG Labtech) for 4 h.