Genome-edited rice deficient in two 4-COUMARATE:COENZYME A LIGASE genes displays diverse lignin alterations.

The 4-coumarate:coenzyme A ligase (4CL) is a key enzyme that contributes to channeling metabolic flux in the cinnamate/monolignol pathway, leading to the production of monolignols, p-hydroxycinnamates, and a flavonoid tricin, the major building blocks of lignin polymer in grass cell walls. Vascular plants often contain multiple 4CL genes; however, the contribution of each 4CL isoform to lignin biosynthesis remains unclear, especially in grasses. In this study, we characterized the functions of two rice (Oryza sativa L.) 4CL isoforms (Os4CL3 and Os4CL4) primarily by analyzing the cell wall chemical structures of rice mutants generated by CRISPR/Cas9-mediated targeted mutagenesis. A series of chemical and nuclear magnetic resonance analyses revealed that loss-of-function of Os4CL3 and Os4CL4 differently altered the composition of lignin polymer units. Loss of function of Os4CL3 induced marked reductions in the major guaiacyl and syringyl lignin units derived from both the conserved non-γ-p-coumaroylated and the grass-specific γ-p-coumaroylated monolignols, with more prominent reductions in guaiacyl units than in syringyl units. In contrast, the loss-of-function mutation to Os4CL4 primarily decreased the abundance of the non-γ-p-coumaroylated guaiacyl units. Loss-of-function of Os4CL4, but not of Os4CL3, reduced the grass-specific lignin-bound tricin units, indicating that Os4CL4 plays a key role not only in monolignol biosynthesis but also in the biosynthesis of tricin used for lignification. Further, the loss-of-function of Os4CL3 and Os4CL4 notably reduced cell-wall-bound ferulates, indicating their roles in cell wall feruloylation. Overall, this study demonstrates the overlapping but divergent roles of 4CL isoforms during the coordinated production of various lignin monomers.