P9561

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.

Sorghum (Sorghum bicolor (L.) Moench) seeds were germinated at 24 + 1 °C under a 16 hour (h) light/8 h dark cycle and seedlings were then transferred to pots in glasshouse conditions. One week after anthesis, the tissues used in the analysis were collected to extract protein for PME activity analysis.
Total protein extracts were obtained using a One-Step Plant Active Protein Extraction Kit (Sangon Biotech, C510004, Taiwan, China) and calibrated with Easy Protein Quantitative Kit (Bradford; TransGen, DQ101, Beijing, China) using bovine serum albumin (BSA) standard solution and Commas Brilliant Blue solution. The same amount of protein from different tissues were used to analyze. The detailed method was followed as previously reported [12 (link)]. Protein extracts (10 μg) in a same volume (20 μL) were loaded in to the 4 mm-diameter wells in 1% agarose gels containing citrus fruit pectin (0.1% (w/v; ≥85% esterified, Sigma-Aldrich, P9561, Germany), citric acid (12.5 mM) and 50 mM Na₂HPO₄, having pH 6.5. The gels were kept overnight at 28 °C followed by staining for 1 h with 0.05% (w/v) ruthenium red and washed for 4 h in water. The stained gels were photographed and the intensity of staining quantified with ImageJ software [14 (link)]. Measurements were performed in triplicate and data were normalized with the tissue stem area set to one.