Dual color protein standard

For proteins with molecular weight (MW) > 200 kDa, NuPAGE™ 3–8% tris-acetate 3–8% gels (Invitrogen, Waltham, MA, USA) and HiMark™ Pre-stained Protein Standard (Invitrogen) were used. Gels were transferred to nitrocellulose membranes via ultra-low voltage overnight transfer (12 V, ~100 mA, 20 h). For proteins with MW < 200 kDa, 10% tris-glycine gels (Invitrogen) and DualColor protein standard (Bio-rad, Hercules, CA, USA) were used instead. Primary antibodies (see Supplemental List S2 for a list of antibodies used) were diluted in 5% BSA 0.1%TBST and incubated overnight at 4 °C with gentle rocking. After primary antibody incubation, membranes were washed in 0.05% TBST three times, 10min each, followed by 1h incubation in Secondary antibody diluent. After a second series of wash, membranes were incubated in Clarity ECL substrates (Bio-rad) for 5min. Images were acquired by ChemDoc MP image system (Bio-rad).

Photosynthetic protein analysis was carried out because protein pattern may be severely affected by abiotic stress, including radiation (e.g., Arena et al., 2013 (link)). Protein extraction of leaves was carried out on plants after 1-month growth using 0.3 g of plant material for each sample (Bertolde et al., 2014 (link); Wang et al., 2006 (link)). An SDS-PAGE (10%) was performed by using Dual Color Protein Standard (Bio-Rad, Hercules, CA, USA) as marker and Laemmli loading buffer added to the samples to follow protein separation. Western blot analysis on protein samples was performed using a blocking solution (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween 20, 5% BSA) and primary specific antibodies (Agrisera) to reveal the Rubisco (anti-RbcL, rabbit polyclonal serum), and the Actin (anti-ACT, rabbit polyclonal serum), used as loading control. The immunorevelation was performed using the kit for chemiluminescence (Westar Supernova; Cyanagen, Bologna, Italy) by ChemiDoc System (Bio-Rad, Hercules, CA, USA). Densitometry analysis was performed following Sorrentino et al. (2018) (link) using ImageJ software (Rasband, W.S., U.S. NIH, Bethesda, MD, USA, 1997–2012), normalizing each Rubisco band value to the corresponding actin band value. Results were expressed as percentages of the control set to 100%.