Fig 2: Visual phenotype and relative water content. (A) Photographs representative of WT and aox1a plants untreated with NaCl or Mannitol (Control), and after 1 day of severe NaCl or Mannitol treatments (300 mM). (B) Leaf relative water content (RWC) in leaves of WT and aox1a plants under control and stress conditions. Values are means ± SE of 10–12 biological replicates. Significant differences (P < 0.05) are denoted by different letters.

Fig 3: AOX and autophagy worked collaboratively in ethylene-induced drought tolerance. (a) Representative transmission electron microscopy (TEM) images of autophagic structures in the mesophyll cells of different treatment in transgenic plants. Autophagic bodies are indicated by red arrows. Bars: 1 µm. (b-d) MDC staining (b), autophagic activity(c) and ATG8 protein levels (d) for autophagosomes, respectively. Bars: 25 µm. (e-h) Detection of respiration rate (e, f), AOX1a expression level (g) and AOX protein level (h) in ACC-pretreated TRV-ATG8d and TRV-ATG18h plants. Ponceau S-stained membranes are shown below the blots to indicate the amount of protein loaded per lane. Means with the same letter did not significantly differ at P < 0.05 according to Duncan multiple range tests. Three independent experiments were performed with similar results.

Fig 4: Impact of ACO3-Ser91 phosphomutation on components related to mitochondrial dysfunction. A, RT-qPCR analysis of transcript abundance of ACO3, ANAC013, AOX1A, and SOT12 in WT (Col-0), aco3, aco3 expressing pACO3::ACO3-YFP (ACO3-YFP 1 and ACO3-YFP 3), pACO3::ACO3S91A-YFP (ACO3S91A-YFP 1 and ACO3S91A-YFP 2), pACO3::ACO3S91D-YFP (ACO3S91D-YFP 2 and ACO3S91D-YFP 3), or 35S::ACO3 (ACO3-OX 3 and ACO3-OX 8) in normal growth conditions. Error bars represent SE (n = 3). Letters indicate significant differences (Kruskal–Wallis test and Fisher’s least significant difference post hoc test with Bonferroni adjustment, P < 0.05). B, Protein abundance of AOXs in WT (Col-0), aco3, aco3 expressing pACO3::ACO3-YFP (ACO3-YFP 1), pACO3::ACO3S91A-YFP (ACO3S91A-YFP 1), pACO3::ACO3S91D-YFP (ACO3S91D-YFP 2, and ACO3S91D-YFP 3) or 35S::ACO3 (ACO3-OX 3) in control conditions and 24 h after UV-B treatment (UV-B; 1.5 W m-2, 45 min) as detected by immunoblotting. Coomassie staining of RBCL is shown to demonstrate the equal loading of samples. C, Protein abundance of SOT12 in WT (Col-0), aco3, aco3 expressing pACO3::ACO3-YFP (ACO3-YFP 1), pACO3::ACO3S91A-YFP (ACO3S91A-YFP 1), pACO3::ACO3S91D-YFP (ACO3S91D-YFP 2, and ACO3S91D-YFP 3) or 35S::ACO3 (ACO3-OX 3) in control conditions and 24 h after UV-B treatment (UV-B; 1.5 W m-2, 45 min) as detected by immunoblotting. Coomassie staining of RBCL is shown to demonstrate the equal loading of samples.

Fig 5: Arabidopsis aox1a and ucp1 seedlings show similar impairment of root growth as caused by the DTT thiol. Primary root length increase of WT Col-0 and transgenic seedlings (ucp1, cUCP1#9 and #14: ucp1 complemented with UCP1 under the control of the native promoter, aox1a and ero1 ero2). Seedlings were grown vertically on half-strength MS agar plates for 4 days, then transferred to fresh plates supplemented with the treatment chemicals. A, Control; C, DTT; E, dithiane. Representative images of seedlings 4 days (A and C) or 3 days (E) after transfer. Scale bars: 10 mm. B, D, and F, Root length was measured 4 days (B and D) or 3 days (F) after transfer. (B) N = 17–18, (D) N = 28–36, (F) N = 18–24. Boxplot: first and third quartiles with median and Tukey’s whiskers. Differences were tested after log-transformation of data to establish normal distribution by one-way ANOVA with Bonferroni’s multiple comparisons test (nsP > 0.05, ***P < 0.001). P-values: Supplemental Data Set 3. Note that ero1 ero2 root growth was suppressed also by dithiane (-77%). Dithiane does not possess reduced thiol groups but can pass cellular membranes. Once inside the cell it may be reduced by the cytoplasmic redox machineries to form DTT. DTT can then enter the ER, which is a likely cause of reductive stress in the ER, even in the absence of external net electron supply. Dithiane-mediated re-distribution of endogenous electrons between cell compartments leading to ER reduction is also supported by Supplemental Figure S3, D–G.