Ultrastructural analysis of engineered rice lines reveals ferulate cross-linking as a key factor mediating lignocellulose supramolecular assembly in grass cell walls

Within the secondary cell walls of vascular plants, cellulose, hemicelluloses, and lignin associate via various covalent and non-covalent linkages to form an intricate supramolecular assembly. Although the chemical structures and cross-linking levels of the lignin- hemicellulose matrix exhibit substantial diversity in planta, precisely how these structural variations affect lignocellulose supramolecular assembly and macroscopic biomass properties remains largely elusive. Here, we conducted a comparative multi-scale ultrastructural analysis across engineered rice lines with targeted modifications in lignin aromatic composition and ferulate (FA)-mediated cell wall cross-linking levels. Combined solid-state nuclear magnetic resonance and wide-angle X-ray diffraction analyses revealed that depleting FA cross-linking disrupts cellulose crystalline structure and accelerates molecular mobility markedly more severely than altering the guaiacyl-to-syringyl (G/S) lignin ratio, generating a more loosened lignocellulose network. Small-angle X-ray scattering analysis further demonstrated that specific FA-depleted lines, but none of those with an altered G/S ratio, also exhibited disruptions in the nano- to mesoscale organization of cellulose microfibrils. Furthermore, FA- depleted lines generally displayed greater improvements in saccharification efficiency and more rapid thermal softening than G/S-lignin-altered lines, suggesting that disruptions in lignocellulose molecular assembly induced by FA depletion can broadly translate into macroscopic biomass properties. These findings establish a molecular basis for the pivotal role of FA cross-linking in dictating grass cell wall architecture, offering a promising structural target for advancing grass biomass utility and crop design.