Fig. 5
Ammonium-sulfate induced the up-regulation of multiple CEs, which in turn improved glycosidic bond accessibility through xylan deacetylation. a HPLC results of acetate after 10-day liquid fermentation of WT and KO-ThgsfR2. The red line was WT treatments, and blue line was KO-ThgsfR2 treatments, note that the acetate mainly comes from acetyl-groups. b Peak area and concentration of acetate for WT and KO-ThgsfR2 treatments. Concentration was obtained by peak areas and standard regression equation. c Expression levels quantification of CEs genes with AS gradient (T1, T2, T3). qPCR was performed on the 7 identified CEs genes and multiple CE genes were up-regulated with the AS gradient. d HSQC spectrum of extracted xylan from WT-treated straw under T1 condition for 15 days. The signals from Xyl2Ac and Xyl3Ac could be detected. e HSQC spectrum of extracted xylan from WT-treated straw under T3 condition for 15 days, The signal peak area of Xyl2Ac was reduced and Xyl3Ac has no detectable signal. f Quantitative results of Xyl2Ac, Xyl3Ac, and XylR peak areas in the T1 and T3 NMR spectrum, which can indicate the change in acetyl content with the AS addition. The peak areas of Xyl2Ac, Xyl3Ac, and XylR were all decreased in T3 relative to T1. g T2 relaxation spectrum of T1 and T3. Low-field NMR determined the spin–spin relaxation time (T2) of straw after hyphal treated on T1 and T3 conditions for 15 days. h WSI of hyphae-treated (15 days) straw under T1 and T3 conditions, noting that the Peleg modeled WSI belonged to type II isotherm. i Degradability comparison of hyphae-treated (15 days) straw under T1 and T3 conditions. GHs (N7, A50, and XS) were used to hydrolyze hyphae-treated straw, and the affinity efficiency of GHs for polysaccharide glycosidic bonds was compared by determining the reducing sugars (40 °C and 50 °C reaction for 20 min). Note that this could compare the effects of different acetylation levels on GHs binding glycosidic bonds. Bars represent mean ± SEM, with n = 3 or 4 biological repeats; red dots resemble values from individual experiments. Student’s t-testing was conducted in (b, c, i), *significant difference to T1 at two-tailed P = 0.002 (i, T3: A50-50 ℃). ***significant difference to WT at two-tailed P = 0.000 (b, KO-ThgsfR2: peak area), 0.000 (b, KO-ThgsfR2: acetate). ***significant difference to T1 at two-tailed P = 0.000 (i, T3: XS-50 ℃). ns = no statistical difference to T1 at two-tailed P = 0.335 (i, T3: N7-40 ℃), 0.495 (i, T3: A50-40 ℃), 0.113 (i, T3: XS-40 ℃), 0.267 (i, T3: N7-50 ℃)