Figure 1. RCD1 controls tolerance of photosynthetic apparatus to ROS.

(A) MV treatment results in PSII inhibition under light, which is suppressed in the rcd1 mutant. PSII photochemical yield (Fv/Fm) was measured in rosettes pre-treated overnight in darkness with 1 μM MV and then exposed to 3 hr of continuous light (80 µmol m⁻² s⁻¹). Representative false-color image of Fv/Fm is shown. (B) Access of MV to electron-acceptor side of PSI is unaltered in rcd1. Treatment with MV led to similar changes in kinetics of PSI oxidation in Col-0 and rcd1. Oxidation of PSI reaction center (P700) was measured using DUAL-PAM. Leaves were first adapted to far-red light that is more efficiently used by PSI than PSII. In these conditions PSI is producing electrons at a faster rate than they are supplied by PSII, thus P700 is oxidized. Then a flash of orange light was provided that is efficiently absorbed by PSII (orange arrow). Electrons generated by PSII transiently reduced PSI, after which the kinetics of PSI re-oxidation was followed. Note the progressive decrease in the effect of the orange flash occurring in Col-0 at later time points, which suggests deterioration in PSII function. This was not observed in rcd1. Three leaves from three individual plants were used for each measurement. The experiment was repeated three times with similar results. (C) Redox state of the chloroplast enzyme 2-Cys peroxiredoxin (2-CP) assessed by thiol bond-specific labeling in Col-0 (left) and rcd1 (right). Total protein was isolated from leaves incubated in darkness (D), or under light (L). Free sulfhydryls were blocked with N-ethylmaleimide, then in vivo thiol bridges were reduced with DTT, and finally the newly exposed sulfhydryls were labeled with methoxypolyethylene glycol maleimide of molecular weight 5 kDa. The labeled protein extracts were separated by SDS-PAGE and immunoblotted with α2-CP antibody. DTT (-) control contained predominantly unlabeled form. Unlabeled reduced (red), singly and doubly labeled oxidized forms and the putative dimer were annotated as in Nikkanen et al. (2016). Apparent molecular weight increment after the labeling of one thiol bond appears on SDS-PAGE higher than 10 kDa because of steric hindrance exerted on branched polymers during gel separation (van Leeuwen et al., 2017). The experiment was repeated three times with similar results.

Figure 1—figure supplement 1. Inverse correlation of RCD1 abundance with tolerance to chloroplastic ROS.

(A) Several independent rcd1 complementation lines were generated in which HA-tagged RCD1 was reintroduced under the RCD1 native promoter. Immunoblotting of protein extracts from these lines with αHA antibody revealed different levels of RCD1-HA under standard light-adapted growth conditions. This was presumably due to different transgene insertion sites in the genome. Line ‘a’ was described in Jaspers et al. (2009). Rubisco large subunit (RbcL) detected by amido black staining is shown as a control for equal protein loading. (B) An antibody was raised against the full-size RCD1 protein. This allowed comparing abundance of RCD1 in independent rcd1: RCD1-HA complementation lines described in the panel (A) versus Col-0 (two rcd1: RCD1-HA lines with the lowest and two with the higher levels of RCD1-HA are shown). In the complementation lines the RCD1 signal was detected at higher molecular weight due to the triple HA tag. The rcd1: RCD1Δ7Cys-HA line will be addressed below. (C) Expression of RCD1 gene was measured by real time quantitative PCR in Col-0 and in four independent complementation lines described in the panel (A), two with the lowest and two with the higher levels of RCD1-HA. Results in panels (B) and (C) demonstrated that the levels of RCD1 protein and mRNA were about 10 times higher in the high-expressing complementation lines than in Col-0. Relative expression was calculated from three biological repeats and the data are scaled relative to Col-0. Source data are presented in Figure 6—source data 1. (D) Sensitivity of PSII to chloroplastic ROS in the rcd1 complementation lines was assessed using time-resolved analysis described in Figure 1—figure supplement 2. For that, leaf discs were pre-treated with 0.25 μM MV overnight in the darkness. PSII photochemical yield after two 1 hr light cycles was plotted against abundance of RCD1-HA in the individual lines as determined in panel (A). Line ‘a’ was described in Jaspers et al. (2009). Five individual plants were taken per each line. The experiment was repeated three times with similar results. Source data and statistics are presented in Figure 1—source data 1.

Figure 1—figure supplement 2. The Imaging PAM protocol developed to monitor kinetics of PSII inhibition by repetitive 1 hr light cycles.

Plants dark-adapted for at least 20 min were first exposed to a saturating light pulse to measure Fm. Then the blue actinic light (450 nm, 80 µmol m⁻² s⁻¹) was turned on for 1 hr, over which time chlorophyll fluorescence under light (Fs) was followed by measuring flashes given once in 2 min. Then the actinic light was turned off to allow for 20 min dark adaptation, after which Fo and Fm were measured. Following the Fm measurement, the next light cycle was initiated. Saturating light pulses to measure Fm are depicted by blue arrows, actinic light periods by blue boxes, and dark adaptation by black boxes. PSII photochemical yield was calculated as Fv/Fm = (Fm-Fo)/Fm. To study different levels of MV tolerance, different concentrations of MV were employed throughout the study, as indicated in the figures or figure legends.

Figure 1—figure supplement 3. Production rate of hydrogen peroxide in Col-0 and rcd1 during illumination of MV-pre-treated rosettes.

Col-0 and rcd1 rosettes were pre-treated with 1 μM MV overnight in the darkness. Then they were exposed to light for indicated time. After this, the rosettes were infiltrated with DAB staining solution and exposed to 20 min of light (180 µmol m⁻² s⁻¹). Similar initial increase in H₂O₂ production rate was observed in MV-pre-treated dark-adapted Col-0 and rcd1. During longer incubation under light, the production rate of H₂O₂ further increased in Col-0, but decreased in rcd1. The experiment was performed three times with similar results.

Figure 1—figure supplement 4. Altered resistance of rcd1 photosynthetic apparatus to chloroplastic ROS.

(A) Protein extracts from Col-0 and rcd1 leaves pre-treated with 1 μM MV and exposed to light for indicated time, were separated by SDS-PAGE followed by immunoblotting with antibodies against the PSII subunit D1 and the PSI subunit PsaB. No significant differences in stoichiometry of photosystems were detected. (B) Thylakoid protein complexes isolated from leaves treated as above were separated by native PAGE. Immunoblotting with αD1 antibody revealed PSII species of diverse molecular weights that were annotated as in Järvi et al. (2011). The largest of the complexes corresponds to PSII associated with its light-harvesting antennae complex (LHCII) while the smallest are the PSII monomers (top panel). Incubation under light in presence of MV led to destabilization of PSII-LHCII complexes in Col-0, but not in rcd1. At the same time, immunoblotting with αPsaB antibody showed no changes in PSI complex (bottom panel).

Figure 1—figure supplement 5. Components of photosynthetic electron transfer and chloroplast ROS scavenging; abundance and distribution of NAD⁺/NADH and NADP⁺/NADPH redox couples in Col-0 and rcd1.

(A) Abundance of proteins related to photosynthetic electron transfer or chloroplast ROS scavenging was assessed by separating Col-0 and rcd1 protein extracts (in dilution series) by SDS-PAGE and immunoblotting with specific antibodies, as indicated. 100% corresponds to 20 μg of thylakoid protein. No difference was observed between Col-0 and rcd1. (B) Abundance of nucleotides NAD⁺, NADP⁺, NADH and NADPH in total leaf extracts isolated from Col-0 and rcd1 (mean ±SE). No difference was observed between the genotypes. Source data and statistics are presented in Figure 1—source data 1. (C) Distribution of NAD⁺/NADH and NADP⁺/NADPH redox couples in various cellular compartments of Col-0 and rcd1 was assessed by non-aqueous fractionation metabolomics (mean ±SE, an asterisk indicates the value significantly different from that in the corresponding wild type, *P value < 0.05, Student’s t-test). In brief, the light-adapted rosettes were harvested in the middle of the light period, freeze-dried, homogenated and separated on non-aqueous density gradient, which allowed for enrichment in specific membrane compartments. No major difference was detected between Col-0 and rcd1. Note that the method does not allow for separation of apoplastic and vacuolar compartments or reliable definition of the mitochondria (Fettke et al., 2005). Source data and statistics are presented in Figure 1—source data 1.