Total lignin was measured by acetyl bromide method with slight modifications from (Foster et al., 2010a ). Briefly, 1 mL of 25% acetyl bromide (Sigma‐Aldrich) was added to 4–5 mg of protein‐free CWRs and incubated at 50 °C for 3 h; then the samples were cooled down on ice for 15 min and 2.5 mL of glacial acetic acid was added to stop the reaction. A volume of 400 μL 1.5 M NaOH and 300 μL of 0.5 M hydroxyl amine hydrochloride (Sigma‐Aldrich) were added to 300 μL of the samples and vortexed. 200 μL of the solution was pipetted into the Corning® 96‐well UV‐Transparent Microplates (Corning, Kennebunk) and the absorbance was recorded at 280 nm with Spark 20M microplate reader (Tecan, Männedorf). A blank with the reagents only was included to correct background absorbance. The extinction coefficient of 17.75 g−1/cm for grasses was used for the calculation of lignin content (Foster et al., 2010a ).
To determine the lignin monomeric composition, thioacidolysis (Rolando et al., 1992 ) was performed with 10 mg protein‐free CWRs by follow the procedure described previously (Zhao et al., 2023 (link)). For derivatization, 50 μL of pyrimidine (Sigma‐Aldrich) and N‐methyl‐N‐trimethylsilyl trifluroacteamide (Sigma‐Aldrich) was added and incubated for 5 h. Derivatized products were quantified with Agilent 7890A gas chromatography as described (Zhao et al., 2021 (link), 2023 (link)).
To determine the lignin monomeric composition, thioacidolysis (Rolando et al., 1992 ) was performed with 10 mg protein‐free CWRs by follow the procedure described previously (Zhao et al., 2023 (link)). For derivatization, 50 μL of pyrimidine (Sigma‐Aldrich) and N‐methyl‐N‐trimethylsilyl trifluroacteamide (Sigma‐Aldrich) was added and incubated for 5 h. Derivatized products were quantified with Agilent 7890A gas chromatography as described (Zhao et al., 2021 (link), 2023 (link)).