FIGURE 4.

H₂O₂, NO, ONOO^-, and SNO quantification in Medicago truncatula roots 7 dpi. M. truncatula was cultivated on complete medium or NØ medium, and roots were harvested 7 days after inoculation with A. euteiches and used to detect H₂O₂, NO, and ONOO^- concentrations using fluorescent probes, and SNO concentrations using the Saville–Griess assay. (A) H₂O₂ quantification using 10 μM Amplex Red^® fluorophore and 0.2 U/mL of peroxidase. Catalase (1 U/μL), used as an H₂O₂ scavenger, abolished Amplex Red^® fluorescence. (B) NO quantification using the fluorophore DAF (10 μM). Root extracts were pre-incubated or not with 500 μM cPTIO as an NO scavenger. (C) ONOO^- quantification using the fluorophore APF (5 μM). Root extracts were pre-incubated or not with 1 mM of the ONOO^- scavenger epicatechin. (D) SNO quantification by the Saville–Griess assay. Error bars indicate standard errors (n = 4 for A–C; n = 14 for D), and letters indicate significant differences (p < 0.05). Data from one representative experiment out of three independent experiments for H₂O₂, NO, and ONOO^-concentrations, and data corresponding to two independent experiments pooled together for SNO concentrations. RFU, relative fluorescence units.