Thioglycolic Acid Lignin Assay, Thioacidolysis, and 2D-NMR

Rice root CWR samples used for thioglycolic acid assay and thioacidolysis were prepared as described in Cui et al. (2018). For 2D NMR analysis, CWRs were further subjected to acetylation in a dimethyl sulfoxide/n-methylimidazole/acetic anhydride system as described in Tobimatsu et al. (2013). The obtained acetylated CWRs (∼15 mg) were dissolved in 600 μL of chloroform-d and subjected to NMR analysis. Thioglycolic acid lignin assay and thioacidolysis on CWRs were performed according to the methods described in Lam et al. (2017). NMR spectra were acquired on an Avance III 800US spectrometer (Bruker Biospin) fitted with a cryogenically cooled 5-mm TCI gradient probe. Adiabatic heteronuclear single quantum coherence NMR experiments on acetylated CWRs were carried out using standard implementation (hsqcetgpsp.3; Bruker Biospin) with parameters described in the literature (Wagner et al., 2011), and data processing and analysis were as described in Tobimatsu et al. (2013) and .