Pharmacological and genetical evidence supporting nitric oxide requirement for 2,4-epibrassinolide regulation of root architecture in Arabidopsis thaliana

Abstract

Brassinosteroids (BRs) regulate various physiological processes, such as tolerance to stresses and root growth. Recently, a connection was reported between BRs and nitric oxide (NO) in plant responses to abiotic stress. Here we present evidence supporting NO functions in BR signaling during root growth process. Arabidopsis seedlings treated with BR 24-epibrassinolide (BL) show increased lateral roots (LR) density, inhibition of primary root (PR) elongation and NO accumulation. Similar effects were observed adding the NO donor GSNO to BR-receptor mutant bri1-1. Furthermore, BL-induced responses in the root were abolished by the specific NO scavenger c-PTIO. The activities of nitrate reductase (NR) and nitric oxide synthase (NOS)-like, two NO generating enzymes were involved in BR signaling. These results demonstrate that BR increases the NO concentration in root cells, which is required for BR-induced changes in root architecture.

Keywords: Arabidopsis; brassinosteroids; nitric oxide; root morphology.

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).

Figure 2. Characterization of NO signaling operating downstream BL in Arabidopsis roots. Arabidopsis thaliana Col-0, nia1-2 and bri1-1 mutant lines were grown on ATS plates for 5 d, treated later with 10 nM BL, 100 µM L-NAME or 200 M 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. Bar: 75 min. LR density (C), PR length (D) and NO (E) were quantified as indicated in Figure 1. Values are the means ± SE of five independent experiments (n = 10). Asterisks indicate significant differences at p < 0.05 (t-test).