As132640

The leaves of the transgenic Arabidopsis were harvested, flash-frozen and crushed to fine powder using liquid nitrogen. The total protein was extracted using 1 mL of extraction buffer composed of 100 mM tris-HCl (pH 7.5), 100 mM NaCl, 1 mM phenylmethyl sulfonyl fluoride (PMSF) and 5% glycerol. Since the PdMT2A is too small in size (approximately 8 kDa) and is unstable in air, it was not possible to detect this protein using the traditional Western blot assay. Therefore, the overexpressed PdMT2A protein fused to the Myc tag was detected via the dot-blot method using 0.2 µM PVDF membrane (Bio-Rad, Hercules, CA, USA), the anti-Myc-tag primary polyclonal antibody ab117499 (Abcam, Cambridge, UK) (dilution 1:1000) and the anti-mouse IgG (H&L) horseradish peroxidase (HRP) conjugated secondary antibody ab205719 (Abcam) (dilution 1:1000). As a protein loading control in the dot-blot assay, an immunoassay was carried out using the polyclonal anti-rabbit primary antibody AS13 2640 (Agrisera, Vännäs, Sweden) against the actin protein and the HRP-linked secondary anti-rabbit antibody AS09 602 (Agrisera). Immunoreactions were visualized by detecting the chemiluminescence signal on the immunoblot using Clarity enhanced chemiluminescence (ECL) substrate (Bio-Rad) and the image was visualized using the ChemiDoc™ Touch Imaging System (Bio-Rad).

To measure MAPK activation in leaves, intact leaves were immersed in 1 μm elicitor solution in a beaker and vacuum‐infiltrated for 3 min. After 15 min, four leaf discs with 8 mm diameter were collected and frozen in liquid nitrogen for further analysis. Seedlings were immersed in 1 μm elicitor solution directly for 10/15 min and then were collected and frozen immediately in liquid nitrogen for further analysis. Western blots to determine MAPK activation were performed as described (Macho et al., 2012). Blots were incubated with anti‐actin (Agrisera AS132640) to verify equal loading.

Western blotting was achieved by transferring the proteins resolved by SDS-PAGE to nitrocellulose membranes using a semi-dry blot system (BioRad). Immunodetection was carried out using either an NAD-GAPDH rabbit antiserum raised against the cytosolic recombinant wheat NAD-GAPDH, αAMPK phospho-Thr172 (Cell Signaling) or anti-actin from plants (AS13-2640, Agrisera). Primary antibodies were incubated with the membrane during 16 h at 15°C under agitation. The immunoblotting was revealed with the secondary antibody Alexa Fluor 647 goat anti-rabbit IgG (H+L; Invitrogen) and the fluorescence emissions were detected using a Typhoon 9400 scanner (GE Healthcare).

Total protein extracts were obtained from Arabidopsis lyophilized powder. The powder was resuspended on ice‐cold TKMES homogenization buffer (100 mM Tricine‐potassium hydroxide pH 7.5, 10 mM KCl, 1 mM MgCl₂, 1 mM EDTA, and 10% [w/v] Sucrose) supplemented with 0.2% (v/v) Triton X‐100, 1mM DTT, 100 µg/ml PMSF, 3 µg/ml E64, and 1X plant protease inhibitor (Sigma). The resuspended sample was centrifuged at 10,000 ×g for 10 min at 4°C and the supernatant recovered for a second step of centrifugation. Protein concentration was determined using the kit Pierce Coomassie Plus (Bradford) Protein‐Assay (Thermo Scientific). Approximately 40–50 µg of total protein was separated by SDS–PAGE, transferred to nitrocellulose membrane, and subjected to immunoblotting. The following antibodies were used anti‐CCT7 [1:1,000] (Abcam, ab170861) and anti‐Actin [1:5000] (Agrisera, AS132640).

Liquid nitrogen frozen leaf tissue (20 mg) was disrupted in a tube containing ~50 μl of lysing matrix D (MP Biomedicals) using the Fastprep 24 tissue homogenizer (MP Biomedicals) with the dry ice adapter set to 6.5 s⁻¹ for 1 min. Protein was extracted from the leaf powder by addition of 200 μl of extraction buffer (200 mM Tris, pH 6.8, 8% SDS (w/v), 40% glycerol and 200 mM dithiothreitol (DTT)) and heating to 65 °C for 10 min. Freshly isolated thylakoid membranes were treated with salts according to Karnauchov et al.³⁶ . In detail, thylakoids were incubated in 10 mM HEPES/KOH 8.0, 5 mM MgCl₂ alone or in the presence of 2 M NaBr, 0.1 M Na₂CO₃ or 2 M NaSCN for 30 min at 4 °C and then separated into soluble and membrane fractions by centrifugation at 18,500 × g for 10 min. Proteins were separated on SDS-PAGEs, blotted onto nitrocellulose membranes and immunodetected with the following specific antibodies: KEA3 and the RCT using the C-terminus specific antibody of KEA3^{6 (link)} at a 1:100 dilution, GFP (Chromotek 50430-2-AP) at 1:1000, PetA, PsaD, AtpA and Actin (Agrisera AS20 4377, AS09 461, AS08 304, AS13 2640, respectively) at 1:2000.