Fractionation and quantification of cell wall polysaccharides
were performed as previously reported [19]. Briefly, the second
internodes of culms were weighted (0.5 g) and ground into fine
powder in liquid nitrogen, then washed with water. It was treated with 80% (v/v) ethanol at 100 ◦C for 20 min and then digested
with 100 U of porcine pancreatic -amylase in 50 mM MOPS–NaOH
buffer (pH 6.5) at 37 ◦C for 4 h. After centrifugation the three parts
of carbohydrate (hot water fraction, pectin and hemicellulose)
were sequentially extracted at 100 ◦C for 10 min with hot water,
50 mM EDTA (pH 6.8) and 17.5% sodium hydroxide containing
0.04% sodium borohydride, respectively. Then the hemicellulose
fractions were neutralized with acetic acid, separated out at 4 ◦C
for 1 day and dried. At last, after washing with water, ethanol and
diethylether, the residual precipitate is cellulose fraction. Sugar
content was assayed based on the phenol–sulfuric acid method
described previously [20].