The Existence of Cellulose and Lignin Chemical Connections in Ginkgo Traced by H-2-C-13 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 H-2-labeled cellulose precursor (UDP-glucose-[6-H-2(2)]) and the C-13-labeled lignin precursor (coniferin-[alpha-C-13]) were added to a growing ginkgo plant, in combination with a 4-coumarate-CoA ligase inhibitor. The detection of abundance of C-13 and H-2 revealed that the lignin precursor and cellulose precursor deposited more actively in 300 to 1300 mu m and 100 to 900 mu m 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) C-13-NMR of the newly-formed xylem, liquid-state C-13-NMR, and H-1-NMR of the EDLCCs confirmed that the major connection between celluloses and lignins was a benzyl ether bond (between cellulose C6 and lignin C alpha). A minor ester bond was also found between the hydroxyl group (at the 6-position of cellulose) and ferulic acid (at the gamma position in lignins).