Pierce 660 nm kit

The concentration of soluble proteins was determined with a Pierce 660 nm kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The quality of the proteins was verified by their electrophoretic separation in a polyacrylamide gel under denaturing conditions (PAGE). Then, 25–30 µg of protein were loaded on a 12% Tris–glycine gel. Electrophoresis was performed in Tris–glycine electrophoresis buffer (Serva). The gel was stained with Colloidal Coomassie Blue and washed in distilled water overnight.

Sampling was done by collecting and pooling the youngest fully expanded leaves from different branches into a 50-mL Falcon tube and immediately freezing it in liquid nitrogen. Samples were ground in liquid nitrogen using a mortar and pestle, and 400 mg of leaf powder was mixed with lysis buffer (5% SDS; 50 mM TEAB; pH = 8.5) and incubated at 70 °C for 10 min. After centrifugation at 20,000 g for 10 min, the remaining supernatant was filtered (0.45-µm filter). Total protein was quantified using the Pierce™ 660 nm kit (Thermo Fisher Scientific) and concentration adjusted to 150 µg per sample.