Leaf Protein Extraction and Quantification

The details of leaf TSP extraction were described in He et al. (2017a) (link). The frozen samples (1 g) were powdered in liquid nitrogen and ground with 6 ml of extraction buffer [100 mM Bicine-KOH (pH 8.1), 20 mM MgCl2, 2% PVP]. The mixture was centrifuged at 35,000 rpm for 30 min at 4°C using a Beckman ultracentrifuge Optima XL-100K. 1 ml aliquot of the supernatant was mixed with 4 ml of 80% cold acetone before centrifuging at 3500 rpm for 10 min. The precipitate was dissolved in 1 ml of 1 M NaOH. The concentration of leaf TSP was determined according to Lowry et al. (1951) (link).