Extended Data Fig. 2: Oxidative stress and reoxygenation stabilize ERFVII and contribute to survival in wild type.
From: H2O2 repurposes plant O2 sensing to regulate post-hypoxia responses
(a) Schematic representation of N-degron pathway-controlled destabilisation of ERFVII. Methionine (M) in the N-terminal position is target of the methionine aminopeptidase enzyme (MAP), leaving an exposed cysteine which, in presence of oxygen (O2) is targeted for oxidation by plant cysteine oxidases (PCO). The resulting Cys-sulfinic acid is sequentially modified by arginyl aminotransferases 1/2 (ATE) triggering proteasomal degradation after ubiquitination by proteolysis E3 ligase (PRT6), in presence of nitric oxide (NO). Upon hypoxic conditions, ERFVII are stabilized and relocalise to the nucleus where they bind the hypoxia responsive promoter element (HRPE) triggering the hypoxic response. Additional abbreviation: M, methionine; C, cysteine; oxC, oxidized cysteine; R, arginine; Ub, ubiquitin. (b) Stabilization of 35S:RAP2.3-GFP (green) in seven-day old Arabidopsis seedlings upon 1 mM TBHP or mock treatment in normoxia (21% O2), hypoxia (1% O2) or after 3 h of reoxygenation, in the dark (scale bar, 50 µm). (c) Time-dependent stabilization of 35S:RAP2.3-GFP (green) in seven-day-old seedlings over 2 h of 1 mM TBHP treatment in air (scale bar, 50 µm), n = 1. (d) Stabilization of two independent lines of RAP2.12-FLAG (green) upon 6 h of air (21% O2) or hypoxia (1% O2), in the dark. (e) Western blot analysis of a second independent line of RAP2.12-FLAG in air, hypoxia (6 h, 1% O2), followed by 1 h or 3 h reoxygenation, or 1 mM TBHP treatment, in the dark. Loading control (LC) corresponds to a compacted image of the membrane after Ponceau staining. Unedited gel images are shown in Supplementary Fig. 1. (f) Phenotype of Col-0 and erfVII seedlings after hypoxia treatment (or air control), with or without 2 h 1 mM TBHP pre-treatment, and after 4 days reoxygenation (scale bar, 1 cm). (g) Growth rate of Col-0 and erfVII primary roots after 4 days of reoxygenation or air control, with or without 2 h 1 mM TBHP pre-treatment (Col-0 in air, n = 22; Col-0 in air + TBHP and hypoxia + TBHP, n = 28; Col-0 in hypoxia, n = 27; erfVII in air, n = 21; erfVII in air + TBHP, n = 23; erfVII in hypoxia, n = 26; erfVII in hypoxia + TBHP. n = 25). Seedlings per category are divided into damaged (dam) and not damaged depending on main root growth after reoxygenation. Statistical analyses were conducted using one-way ANOVA followed by Tukey’s HSD test (p < 0.05), where different letters indicate statistically distinct groups. Exact p-values provided in Supplementary Table 11. Experiments producing Fig. 2b-d were repeated once, the western blot shown in 2e was repeated twice.
