Accessing the position-specific ¹⁸O/¹⁶O ratios of lignin monomeric units from higher plants with highly selective hydrogenolysis followed by GC/Py/IRMS analysis

The ¹⁸O/¹⁶O of lignin at bulk, molecular and positional levels can be used to extract valuable information about climate, plant growth environment, plant physiology, and plant metabolism. Access to the individual oxygen isotope compositions (δ¹⁸O) in the lignin monomeric units is, however, challenging as depolymerization of lignin to release the monomeric units may cause isotope fractionation. We have developed a novel method to measure the δ¹⁸O of the three oxygens (O-3, O-4 and O-5) attached to the aromatic ring of the monomeric units (bearing no oxygen in their side chains) releasable by highly selective W₂C/AC (tungsten carbide supported by activated carbon)-catalyzed hydrogenolysis of lignin. O-4 is obtained by measuring the δ¹⁸O of H-type monomeric unit, while O-3 and O-5 can be calculated following isotope mass balance between H, G and S-type monomeric units measurable simultaneously with GC/Py/IRMS (gas chromatography-pyrolysis-isotope ratio mass spectrometry). The measurement precisions are better than 1.15 mUr and 4.15 mUr at molecular and positional levels, respectively. It was shown that there were a δ¹⁸O_H > δ¹⁸O_G > δ¹⁸O_S isotopic order in the herbaceous plant lignin and an (inclusive) opposite order in woody plant lignin. Such differences in isotopic order is likely to be caused by the fact that both L-tyrosine, which carries an ¹⁸O-enriched leaf water signal, and L-phenylalanine, which carries mainly a molecular O₂ isotopic signal, serve as the precursors for lignin biosynthesis in herbaceous plants while only the latter serves as precursor for lignin biosynthesis in woody plants. We have highlighted the potential application of such molecular and positional levels isotopic signals in plant physiological, metabolic, lignin biosynthetic and climate studies.