Microsomal fraction and plasma membrane isolation
A pool of leaves from five different plants of the same treatment was used for four microsomal fraction extractions in order to obtain enough protein and lipid amounts for further analysis. Leaves plasma membranes were purified from microsomal fractions using the two-phase aqueous polymer technique first described by Larsson et al. , (1987) and modified by Casado-Vela et al. , (2010).
Leaves (50 g) were cut before vacuum infiltrated with 0.5 g of PVP and 160 ml of extraction buffer containing 0.5 M sucrose, 10% glycerol, 20 mM Na2EDTA, 20 mM EGTA, 50 mM NaF, 5 mM β-glycerophosphate, 1 mM 1,10-phenanthroline, 1 mM Na3VO4, 0.6% PVP, 5 mM ascorbic acid, 5 mM DTT, and 0.5 mg/L leupeptin in 50 mM Tris-MES, pH 8.0. After buffer infiltration, tissues were homogenized using a blender and filtered through a nylon mesh (pore diameter of 100 µm). The homogenate was centrifuged at 10,000 x g for 40 min at 4°C. The supernatant was collected and centrifuged at 100,000 x g for 45 min at 4°C. The pellet was suspended in buffer (FAB) containing 330 mM sucrose, 2 mM DTT, 10 mM NaF, and 5 mM phosphate buffer (pH 7.8). Plasma membranes were purified from microsomal fraction by partitioning in a two phase-system mixture with a final concentration of PEG-3350 (Sigma)/Dextran-T500 (GE Healthcare), 6.0% (w/w), in the presence of 330 mM sucrose, 5 mM KCl, 5 mM potassium phosphate (pH 7.8). The two-phase system was centrifuged for 5 min at 4000 x g at 4°C. The upper phase, enriched plasma membranes, was recollected and washed in 300 mM sucrose, 9 mM KCl, 6 mM Na2EDTA, 6 mM EGTA, 60 mM NaF, and Tris-borate, pH 8.3, and centrifuged at 100,000 x g for 45 min, 4°C. The resulting pellet was suspended in resuspension buffer (FAB). The protein concentration of both, microsomal fraction and plasma membrane-enriched fractions, was determined with Protein assay dye reagent (BioRad), with BSA as standard.