Lignin-ferulate cross-links in grasses: active incorporation of ferulate polysaccharide esters into ryegrass lignins

Active incorporation of ferulate polysaccharide esters into ryegrass lignins has been demonstrated by NMR spectroscopy of uniformly 13C-labeled ryegrass. Observation, in the HMBC spectrum, of products of ferulate at its 8-position coupling with hydroxycinnamyl alcohols at the β-position (producing 8-β′-linked structures) is proof that ferulate-lignin radical cross-coupling reactions occur in vivo. Correlations of H-α′ (hydroxycinnamyl alcohol moiety) with guaiacyl and syringyl 1-, 2-, and 6-aromatic carbons in 8-β′ structures indicates that ferulates couple with both coniferyl and sinapyl alcohol monomers. As notable as the presence of this and other ferulate products is the absence of coupling of ferulate at its 8-position with the 5- and O-4-positions of lignin units. Such structures were significant when ferulate was biomimetically incorporated into a synthetic lignin. Since hydroxycinnamyl alcohols couple almost exclusively at their β-position in cross-coupling reactions, the 8-5′ and 8-O-4′ structures would only be formed by coupling with higher lignin oligomers (with no side-chain conjugation). Exclusive reaction of ferulates with lignin monomers is the first real evidence that ferulate polysaccharide esters in grasses are acting as initiation or nucleation sites for lignification and are critical entities in directing cell-wall cross-linking during plant growth and development.