Quantifying Lignin Composition in Plant Biomass

The biomass from senesced wild‐type plants and T3 homozygous C4H::qsuB lines was used to determine lignin content and composition. Biomass was extracted sequentially by sonication (20 min) with 80% ethanol (three times), acetone (one time), chloroform–methanol (1:1, v/v, one time) and acetone (one time). The standard NREL biomass protocol was used to measure lignin content (Sluiter et al., 2008). The chemical composition of lignin was analysed by pyrolysis‐gas chromatography (GC)/mass spectrometry (MS) using a previously described method with some modifications (Del Río et al., 2012). Pyrolysis of biomass was performed with a Pyroprobe 5200 (CDS Analytical Inc., Oxford, PA, USA) connected with GC/MS (Thermo Electron Corporation with Trace GC Ultra and Polaris‐Q MS) equipped with an Agilent HP‐5MS column (30 m × 0.25 mm i.d., 0.25 μm film thickness). The pyrolysis was carried out at 550 °C. The chromatograph was programmed from 50 °C (1 min) to 300 °C at a rate of 30 °C/min; the final temperature was held for 10 min. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The mass spectrometer was operated in scan mode and the ion source was maintained at 300 °C. The compounds were identified by comparing their mass spectra with those of the NIST library and those previously reported (Del Río and Gutiérrez, 2006; Ralph and Hatfield, 1991). Peak molar areas were calculated for the lignin degradation products, and the summed areas were normalized.