Engineering eudicot-like lignin in rice via targeted disruption of grass-specific lignin modification pathways

Summary Lignin composition exhibits substantial diversity across plant lineages. Unlike eudicot and gymnosperm lignins, grass lignin incorporates p -coumarate and tricin units alongside canonical monolignols. To elucidate their specific biochemical and physiological roles, we engineered rice ( Oryza sativa ) to produce a “eudicot-like” lignin devoid of both modifications. Using CRISPR-Cas9, we generated mutants deficient in p -coumarate and tricin by simultaneously targeting their respective biosynthetic genes. The resulting mutant cell walls underwent structural and functional characterization via wet-chemical analyses, nuclear magnetic resonance, gel permeation chromatography, and antioxidant capacity assays. The newly generated ospmt1/2 osfnsII and ospmt1/2 osa3′h/c5′h triple-knockout mutants reached maturity with minor growth penalties. Subsequent cell wall analyses demonstrated near-complete depletion of both p -coumarate and tricin units in lignins. This structural shift substantially altered overall lignin content, monomeric composition, linkage distributions, and molecular weight, highlighting the divergent and synergistic roles of these units in lignin assembly and polymerization. Functionally, the free-radical scavenging capacity of rice lignin is markedly enhanced by p -coumaroylation, but attenuated by tricin incorporation. The successful synthesis of eudicot-like lignin within a grass system underscores the inherent plasticity of lignification. These engineered rice lines offer a valuable platform to investigate the physiological functions and biotechnological potential of grass lignin modifications.