Cell Wall Fractionation and Xyloglucan Analysis

The fractionation of cell walls was conducted as previously described [62 (link)]. Separated tissues were fixed for 10 min in 15 mL of boiling methanol. The methanol-fixed tissues were rehydrated with water, then homogenized in water with a mortar and pestle. The residue obtained by centrifugation was washed with water, acetone, and a methanol:chloroform mixture (1:1, v/v) and air-dried. The washed residue was dried overnight at 40 °C and then treated with 2 units/mL α-amylase (Macklin, Shanghai, China) in 100 mM MOPS (Coolaber, Beijing, China) buffer (pH 7.3) for 0.5 h at 80 °C, then with 1 unit/mL pullulanase (Coolaber, Beijing, China) and 3 units/mL amyloglucosidase (Coolaber, Beijing, China) in sodium acetate buffer for 3 h at 50 °C, to remove starch. Hemicellulose was extracted for 18 h with 17.5% NaOH containing 0.02% NaBH4. The hemicellulosic fraction was neutralized with glacial acetic acid in an ice-cold water bath, then dialyzed against water. The dialyzed hemiceiiuiosic fraction was centrifuged for 20 min at 10,000× g, and dried. The xyloglucan content was determined using the iodine-staining method [62 (link)].