As05084

The leaf material was ground in liquid nitrogen. An extraction buffer (4 % SDS, 2 % β-mercaptoethanol, 2 mM PMSF, 100 mM TrisHCl, pH 8,8) was added in the proportion of 10 μl of extraction buffer per 1 mg of powder mass. The samples were vortexed vigorously, incubated at 80 °C for 3 min, centrifuged for 10 min at 16 000 g at 4 °C and supernatant was mixed with an SDS-PAGE loading buffer. The SDS-PAGE was performed according to [49 (link)] in a gel containing 12 % polyacrylamide using the Mini Protean system (Bio-Rad). After separation the proteins were either stained with Coomassie Brilliant Blue staining (for total protein visualization) or transferred to a PVDF membrane (ImmobilonP, Millipore) by the semi-dry transfer method (Trans-Blot SD Semi-Dry Transfer Cell, Bio-Rad) for Western Blot analysis. Membranes were stained with Ponceau S to ensure proper transfer, blocked with 5 % fat free dried milk in PBS with 0,5 % Tween and incubated with an anti-D1 antibody (AS05 084, Agrisera) diluted 1:10 000 for 1 h at room temperature, followed by secondary antibody incubation (Goat anti-rabbit IgG HRP conjugated, Agrisera) under the same conditions. After that a chemiluminescence substrate was added (Clarity Western ECL Substrate, Bio-Rad) and the chemiluminescence was imaged using the BioSpectrum imaging system (UVP).

All methods were conducted according to Roach et al., [13 (link)], after 4 h treatment. For HPLC analyses (glutathione and pigments), cells were first freeze-dried for 3 days and each replicate was composed of 8–10 mg dry weight. For Western blotting of GPX5 levels and LC-MS/MS analyses of RES, cells were not freeze-dried. Loading of protein extracts and normalisation of pigments was made to total chlorophyll of the extract, according to [13 (link)]. For western blotting, the GPXh antibody (AS15 2882, Agrisera, Vännäs, Sweden) at a ratio of 1:10.000 and PsbA antibody (AS05 084, Agrisera, Vännäs, Sweden) at a ratio of 1:25.000 were used.

The protein was extracted as described above. About 30 µg total proteins for each sample was separated by 12% SDS–PAGE and transferred into the PVDF membrane for Khib or Ksuc detected, followed by immunoblotting with a rabbit pan anti‐Khib/Ksuc/Kac/Kcr primary antibody (1:1000 dilution, PTM‐801/PTM‐401/PTM101/PTM502, PTM Bio) which are highly specific for the detection of Khib/Ksuc/Kac/Kcr proteins at lysine sites, and neither cross‐reacts with acetylated proteins nor binds nonspecifically with other proteins.^[61] For the PsbA (D1 protein), the C‐terminal rabbit antibody (AS05084, Agrisera) was used for immunoblotting. The specific anti‐GhPSB27K72suc antibody was customized by PTM Bio. The peroxidase conjugated goat anti‐mouse secondary antibody (1:3000, CW0103S, CW Bio) was directed against rabbit antibodies. Anti‐Actin/Histone H3 antibody serves as loading control. The SuperSignal West Femto chemiluminescence kit (NCI4106, Pierce) was used to detect the signal. The relative quantified signals were calculated using ImageJ.

Membrane protein separation was performed by SDS-PAGE (10% acrylamide gel) according to Schägger and von Jagow [25 (link)]. Proteins were stained with Coomassie Blue (Sigma-Aldrich Corp. St. Louis, MO, USA) or electroblotted to PVDF membranes (Immobilon-P, Millipore Corp. Bedford, MA, USA) at 22 V for 2.5 h. These membranes were treated with Western Blot Signal Enhancer (Pierce^®, Thermo Scientific, IL, USA) and blocked in 20 mM Tris, 150 mM sodium chloride pH 7.5 with 0.1% (v/v) Tween-20 (TBS-T) buffer with 2% defatted milk, and then successively incubated with the primary antibody and the second antibody. Antibody reacting bands were detected using alkaline phosphatase reaction (1:2500, Sigma-Aldrich, St. Louis, MO, USA) or anti-rabbit IgG horse radish peroxidase conjugated (1:20,000, Sigma-Aldrich, St. Louis, MO, USA). Antibodies used for immunoblotting were as follows: anti PIP2;1, PIP2;2, PIP2;3 (1:1000, Agrisera, Vännäs, Sweden, AS09 491), anti-Na⁺/H⁺ exchanger 1 (1:1000, Agrisera, Vännäs, Sweden, AS09 484), anti-SMT1 (1:1000, Agrisera, Vännäs, Sweden, AS07 266), anti-H⁺-ATPase (1:10,000, Agrisera, Vännäs, Sweden, AS07 260), and anti-PsbA (1:20,000, Agrisera, Vännäs, Sweden, AS05084).

Protein samples (10 µg) were separated on 4–20% Mini-PROTEAN^® TGX™ pre-cast gels (Bio-Rad). A typical example of one Coomassie Brilliant Blue-stained gel is shown in Supplementary Fig. S1. After electrophoresis, proteins were transferred to (0.45 µm) nitrocellulose membranes (Amersham 10600003). Individual membranes were then incubated with the following primary antibodies at a 1:10 000 dilution: Rubisco large subunit (RbcL) form I and form II (AS03 037, Agrisera, Sweden), the PSII D1 protein (AS05 084, Agrisera, Sweden), and PsbA (D1) and the phosphorylated form of D1 (AS13 2669, Agrisera, Sweden). A typical example of an individual Western blot is shown in Supplementary Fig. S1, together with a typical loading control gel.

Protein extraction and immunoblot analysis were performed as previously described (Wang et al., 2016 (link), 2018 (link), 2019 (link)). After quantification of total protein concentrations, samples of 50 mg protein were separated by SDS-PAGE electrophoresis. Proteins were then transferred onto nitrocellulose membranes (BioRad), which were then incubated with antibodies against PsbS (AS09533; Agrisera, Sweden) or D1 (AS05084; Agrisera, Sweden). After incubation with secondary anti-rabbit antibodies (Cell Signaling Technology 7074; Danvers, MA, United States), enhanced chemical luminescence (ECL) was performed to detect labeled proteins.

Total protein was extracted from 100 mg of plant tissues in CoIP buffer and quantified using the Pierce^TM BCA protein assay kit (Thermo Fisher Scientific). Proteins were suspended in 1x Laemmli SDS sample buffer and incubated for 10 min at 95 °C. To detect modified EX1-GFP proteins (EX1W643A/L-GFP), at least 1200 µg proteins were enriched using the chloroform-methanol enrichment method and resuspended in a final volume of 100 µL 1x Laemmli SDS sample buffer and denatured for 10 min at 95 °C.
Equal amounts of the solubilized proteins from CoIP, fractionation, and total protein extractions were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels and blotted onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad). GFP fusion proteins were detected using a mouse anti-GFP monoclonal antibody (1:5,000; Roche, 11814460001). LhcB4, D1, D2, POR, FtsH2, RbcL, and UGP proteins were immunochemically detected using rabbit anti-LhcB4 (1:7,000; Agrisera, AS04 045), rabbit anti-D1 (1:10,000; Agrisera, AS05 084), rabbit anti-D2 (1:10,000; Agrisera, AS06 146), rabbit anti-POR (1:5,000; Agrisera, AS05 067), rabbit anti-FtsH (1:10,000; Agrisera, AS11 1789), rabbit anti-RbcL (1:10,000; Agrisera, AS03 037A), and rabbit anti-UGP (1:10,000; Agrisera, AS05 086) antibodies, respectively.