Figure 4. Mitochondrial AOXs affect energy metabolism of rcd1 and alter response to chloroplastic ROS.

Source data and statistics are presented in Figure 4—source data 1. (A) Expression of AOXs is induced in rcd1. Abundance of AOX isoforms in mitochondrial preparations was assessed by immunoblotting with αAOX antibody that recognizes AOX1a, -b, -c, -d, and AOX2 isoforms. 100% corresponds to 15 μg of mitochondrial protein. (B) Two mitochondrial respiratory pathways (red arrows) and sites of action of mitochondrial inhibitors. KCN inhibits complex IV (cytochrome c oxidase). Salicylhydroxamic acid (SHAM) inhibits AOX activity. Antimycin A (AA) and myxothiazol (myx) block electron transfer through complex III (ubiquinol-cytochrome c oxidoreductase), creating ROS-related mitochondrial retrograde signal. (C) AOX capacity is significantly increased in rcd1. Oxygen uptake by seedlings was measured in the darkness in presence of KCN and SHAM. Addition of KCN blocked respiration through complex IV, thus revealing the capacity of the alternative respiratory pathway through AOXs. Data are presented as mean ±SD, asterisks denote selected values that are significantly different (P value < 0.001, one-way ANOVA with Bonferroni post hoc correction). Each measurement was performed on 10–15 pooled seedlings and repeated at least three times. (D) Inhibitors of mitochondrial complex III increase plant tolerance to chloroplastic ROS. Effect of pre-treatment with 2.5 μM AA or 2.5 μM myx on PSII inhibition (Fv/Fm) by MV. For each experiment, leaf discs from at least four individual rosettes were used. The experiment was performed four times with similar results. Mean ±SD are shown. Asterisks indicate selected treatments that are significantly different (P value < 0.001, Bonferroni post hoc correction). AOX abundance in the leaf discs treated in the same way was quantified by immunoblotting (Figure 4—figure supplement 1). (E) AOX inhibitor SHAM decreases plant tolerance to chloroplastic ROS. 1 hr pre-treatment with 2 mM SHAM inhibited tolerance to 1 μM MV both in Col-0 and rcd1 as measured by Fv/Fm. SHAM stock solution was prepared in DMSO, thus pure DMSO was added in the SHAM-minus controls. For each experiment, leaf discs from at least four individual rosettes were used. The experiment was performed four times with similar results. Mean ±SD are shown. Asterisks indicate significant difference in the treatments of the same genotype at the selected time points (P value < 0.001, Bonferroni post hoc correction).

Figure 4—figure supplement 1. Effect of mitochondrial complex III inhibitors on expression of AOXs in Col-0 and rcd1.

(A) Changes in AOX abundance after overnight pre-treatment of leaf discs with 2.5 μM AA or 2.5 μM myx (C – control treatment with no inhibitor). Notably, rcd1 aox1a double mutant accumulated AOXs other than AOX1a, including putative AOX1d (Konert et al., 2015) (labeled with asterisk). (B) Quantification of αAOX immunoblotting signal after pre-treatment with 2.5 μM AA or myx. To avoid saturation of αAOX signal in rcd1, a dilution series of protein extracts was made. Quantification was performed using ImageJ. Mean ±SD are shown, asterisks denote selected values that are significantly different (P value < 0.001, Bonferroni post hoc correction, for source data and statistics see Figure 4—source data 1).

Figure 4—figure supplement 2. Effect of mitochondrial complex III inhibitors on abundance and redox state of the RCD1 protein.

(A) Chemical induction of mitochondrial dysfunction signaling did not alter abundance of the RCD1 protein. Leaf discs were treated with 2.5 μM AA or 2.5 μM myx overnight. Then total protein extracts were isolated and separated in SDS-PAGE. Levels of RCD1-HA and of AOXs were assessed by immunoblotting with the specific antibodies as indicated. (B) Redox state of RCD1 protein was only very mildly altered by mitochondrial complex III inhibitors or by MV in the darkness. Treatment with AA or myx was performed as in panel (A). MV, D – leaf discs after overnight pre-treatment with 1 µM MV in the darkness; MV, L – leaf discs after overnight pre-treatment with MV followed by 30 min of illumination; H₂O₂ – leaf discs after 30 min of incubation in presence of 100 mM H₂O₂ under light. Reduced (red) and oxidized (ox) forms of the protein are labelled.

Figure 4—figure supplement 3. Specificity of inhibitor treatments.

All chlorophyll fluorescence analyses are presented as mean ±SD, for source data and statistics see Figure 4—source data 1. (A) Interaction of AA with cyclic electron flow through binding to chloroplastic protein PGR5 (Sugimoto et al., 2013) is not the reason of AA-induced ROS tolerance. Possible off-target effect of AA was assessed by using the pgr5 mutant. Pre-treatment with 2.5 μM AA made both pgr5 and its background wild type gl1 equally more tolerant to chloroplastic ROS. For each experiment leaf discs from at least four individual rosettes were used. The experiment was performed three times with similar results. (B) SHAM treatment results in only slight PSII inhibition both in Col-0 and rcd1. Fv/Fm was monitored under light after 1 hr pre-treatment with 2 mM SHAM. No significant difference was detected between Col-0 and rcd1. SHAM stock solution was prepared in DMSO, thus pure DMSO was added in the SHAM-minus controls. For each experiment leaf discs from at least four individual rosettes were used. The experiment was performed three times with similar results. (C) PTOX, plastid terminal oxidase analogous to AOX, is not involved in the SHAM-induced decrease of ROS tolerance. To exclude possible involvement of PTOX in MV-induced PSII inhibition, green sectors of the ptox mutant leaves were treated with 2 mM SHAM, 1 μM MV, or both chemicals together. ptox mutant was responsive to SHAM treatment similarly to Col-0. For each experiment leaf discs from at least four individual rosettes were used. The experiment was performed twice with similar results.

Figure 4—figure supplement 4. Irrelevance of AOX1a isoform for MV tolerance.

All chlorophyll fluorescence analyses are presented as mean ±SD, for source data and statistics see Figure 4—source data 1. (A) Abundance of total AOX in the AOX1a-overexpressor line (AOX1a-OE) as assessed by immunoblotting was comparable to that in rcd1 (m – molecular weight marker; AA – overnight treatment with 2.5 μM AA). (B) Increased expression of AOX1a isoform is not sufficient to provide ROS tolerance. MV-induced PSII inhibition in the AOX1a-OE and aox1a lines was monitored by Fv/Fm. No significant difference was observed between AOX1a-OE and aox1a at any time point of the experiment. (C) AOX1a isoform is not necessary for chloroplastic ROS tolerance. MV-induced PSII inhibition in rcd1 aox1a double mutant was monitored by Fv/Fm. No significant difference was detected between rcd1 aox1a and rcd1.