Pectin Methylesterase Activity Assay

Fifty of 7-day-old seedlings were ground and suspended with 100 μL extraction buffer (1 M NaCl, 0.1 M citric acid, and 0.2 M Na₂HPO₄, pH 5.0) for 5 min and centrifuged at 13,800 × g for 15 min. The supernatant was collected and cell wall protein concentrations were determined according to the method of Bradford (1976) (link). The enzymatic PME activity was quantified by a gel diffusion assay as described (Downie et al., 1998 (link); Bethke et al., 2014 (link); Lionetti, 2015 (link)) with some modification. 10 μL of 3.75 μg protein extract was loaded into the 0.3 mm well on the 20 mL gel prepared in McIlvaine buffer adjusted to pH levels of 4 to 8, which contains 2% agarose and 0.1% of high methylesterified pectin (≥85% esterified pectin from citrus fruit; P9561, Sigma-Aldrich). After 16 h incubation at 28°C, the gels were stained with 10 mL of 0.05% (w/v) ruthenium red (Sigma-Aldrich) for 1 h and de-stained with distilled water. The calibration curve of PME activity was established with a detection range from 2 to 8 μg/mL of cell-wall proteins. The PME activity was calculated by measuring the stained area (cm²) by ImageJ software⁴.