Figure 1. 2,4-epibrassinolide induces lateral root (LR) formation and inhibits primary root (PR) elongation in a nitric oxide-dependent process in Arabidopsis. Col-0 Arabidopsis thaliana seedlings were grown vertically on ATS plates for 5 d, treated later with 10 nM BL, 100 µM c-PTIO or 200 µM GSNO. Seedlings were analyzed after 3 d of treatment. (A) Representative images of the Arabidopsis seedlings. Bar: 1 cm. (B) For NO detection, roots were incubated with 15 µM of the fluorescent probe DAF-FMDA and examined by epi-fluorescence (excitation 490 nm; emission 525 nm) in an Eclipse E 200 microscope (Nikon). Bar: 75 min (C) The ratio of LR number/PR length was taken as a measure of LR density. LR number only included those roots that were > 1 mm in length after 3 d of treatment. (D) Seedlings were photographed, and PR length was measured using Image J software (Universal Imaging). (E) For NO quantification, DAF-FM-DA fluorescence was analyzed with the Image J 1.3 software and expressed as arbitrary units (A.U.).Values are the means ± SE of 5 independent experiments (n = 10). Asterisks indicate significant differences at p < 0.05 (t-test).