The existence of cellulose and lignin chemical connections in ginkgo traced by 2H-13C dual isotopes

To elucidate the covalent association between the celluloses and lignins found in gymnosperms, they were labeled with stable isotopes (deuterium and carbon-13) at specific positions and traced via mass spectroscopy and nuclear magnetic resonance (NMR). Both the 2H-labeled cellulose precursor (UDP-glucose-[6-2H2]) and the 13C-labeled lignin precursor (coniferin-[α-13C]) were added to a growing ginkgo plant, in combination with a 4-coumarate-CoA ligase inhibitor. The detection of abundance of 13C and 2H revealed that the lignin precursor and cellulose precursor deposited more actively in 300 to 1300 μm and 100 to 900 μm distance from cambium, respectively. The lignin-carbohydrate complexes (LCCs) were isolated from the newly-formed ginkgo shoot xylem and further degraded with cellulase and hemicellulase to obtain enzymatically degraded lignin-carbohydrate complexes (EDLCCs). Analysis of the solid-state cross polarization / magic angle spinning (CP/MAS) 13C-NMR of the newly-formed xylem, liquid-state 13C-NMR, and 1H-NMR of the EDLCCs confirmed that the major connection between celluloses and lignins was a benzyl ether bond (between cellulose C6 and lignin Cα). A minor ester bond was also found between the hydroxyl group (at the 6-position of cellulose) and ferulic acid (at the γ position in lignins).